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Volume 185

THE

Number 1

BIOLOGICAL
BULLETIN

AUG E 6 1993

-', M

AUGUS T, 1993

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THE

BIOLOGICAL BULLETIN

PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY

Associate Editors

I '" lne Biological

PETER A. V. ANDERSON, The Whitney Laboratory, University of Florida

DAVID EPEL, Hopkins Marine Station, Stanford University

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WILLIAM D. COHEN, Hunter College
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Institute,
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Editorial Board

AUG 2 6 1993

Hole, Mass.

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Reference: Biol. Bull. 185: 1-9. (August. 1993)

Helical Swimming in a Freshwater Oligochaete

CHARLES D. DREWES' AND CHARLES R. FOURTNER :

^Department of Zoology and Genetics, Iowa Stare University, Ames, Iowa 50011, and
-Biological Sciences, Slale University of New York-Buffalo, Buffalo, New York

Abstract. A novel pattern of undulatory swimming is
described in the freshwater oligochaete, Dew digitata (Fam-
ily Naididae). Movements are rhythmic (6-12 cycles/s),
each cycle consisting of a single, helical body wave that
passes from the worm's anterior to posterior end, thus
propelling the worm forward. Successive cycles of these
waves alternate between right-handed and left-handed
helical orientations. Helical swimming in response to
posterior tactile stimulation was commonly expressed in
all stages of asexual reproduction, including non-fissioning
"normal" worms, late prefission worms, and separated
anterior or posterior zooids. Swimming also occurred in
amputated anterior and posterior body fragments. Reyn-
olds numbers ranged from approximately 50 (in the
shortest and slowest worms) to 300 (in the longest and
fastest worms). During slow swimming, wave velocities
and forward velocities were 37 mm/s and 10 mm/s, re-
spectively; during fast swimming these values were
55 mm/s and 25 mm/s, respectively. Thus, corresponding
values of overall "slippage" were 73 and 55%, respectively.
Central conduction of swim wave excitation likely in-
volves intersegmental, non-giant fiber pathways that are
( 1 ) functionally coupled between anterior and posterior
zooids of prefission worms and (2) readily activated by
posterior mechanosensory inputs in whole worms and
isolated zooids.

Introduction

Undulatory swimming is a common mode of loco-
motion for long, narrow, and limbless animals in aquatic
environments (Gray, 1953, 1968; Trueman, 1975. 1978;
Clark, 1976). Propulsive forces during undulatory swim-
ming are achieved by retrograde passage of rhythmic
waves of body bending (Taylor. 1952). In most cases these
undulations involve sinusoidal, two-dimensional waves

Received 20 October 1992: accepted 3 June 1993.

produced by alternating lateral (i.e., left-right) body bends.
Examples include chordates, such as eels and Branchio-
stoma (Lighthill. 1969; Webb, 1976), as well as inverte-
brates, such as nematodes, polychaetes, and archiannelids
(Gray, 1 939; Gray and Lissmann, 1 964; Clark and Tritton,
1970; Seymour, 1972; Clark and Hermans, 1976).

Notable exceptions to this general pattern are leeches,
in which retrograde undulatory waves are produced by
alternating dorso-ventral, rather than lateral, bending
(Gray el al, 1938; Taylor, 1952; Sawyer, 1986). Another
apparent exception is the polychaete Glycera, in which a
spiral coiling produced by posterior segments is somehow
used to propel the animal backward (Stolte, 1932).

In the present study, we describe the undulatory swim-
ming behavior of a freshwater oligochaete. Dew digitata
(Family Naididae). Its pattern of swimming, which ap-
pears unique in comparison to all previously described
undulatory swimming in animals, involves rhythmically
alternating right-handed and left-handed helical waves
that, through retrograde passage, propel the worm for-
ward.

Our aims in this study are to ( 1 ) provide a basic de-
scription of swimming movements and frame-by-frame
analysis of forward progress and wave velocity, (2) com-
pare swim capabilities during various stages of develop-
ment associated with asexual reproduction, and (3) draw
inferences, where possible, regarding neural mechanisms
that control swimming. A preliminary report of the study
has appeared elsewhere (Drewes and Fourtner, 1990).

Materials and Methods

The freshwater oligochaete Dew digitata (Family Na-
ididae) was obtained from asexually reproducing labo-
ratory cultures, originally collected from Swan Lake, Vic-
toria, BC, Canada (for taxonomy, see Sperber, 1950).
Worms were maintained in natural sediments and fed

C. D. DREWES AND C. R. FOURTNER

fragments of wheat kernels. All cultures and experiments
were at room temperature (20-22C).

Various developmental stages were continuously avail-
able from cultures, including "normal" (i.e., non-fission-
ing) worms with resting body lengths =10 mm and late
"prefission" worms with resting lengths = 14 mm. In the
latter worms a midbody fission zone, with adjacent re-
generating head and tail segments, divides the body into
distinct anterior and posterior zooids (Drewes and Fourt-
ner, 1991). In some experiments, prefission worms were
severed at the fission plane with microdissection scissors,
and swimming capabilities of the two separated zooids
were independently examined.

To videotape swimming behavior, individual worms
were transferred from cultures to a plastic Petri dish
(35 mm D) containing 3.5-4.0 ml of artificial pond water.
The dishes were then placed on a glass stage and viewed
from below with a Panasonic PV-400 video camera. A
field of view ranging from 4.5-20.0 mm on the video
monitor was obtained using various combinations of ex-
tension tubes, a Canon 100 mm macro lens, and a Wild
2X lens extender. Frame speed was 30 frames/s and shut-
ter speed 1 X 10~ 3 s. A fiber optics illuminator, placed
above the dish at an angle of approximately 45, provided
illumination against a dark background. This arrangement
provided access to the dish for tactile stimulation of the
worm with the tip of a human hair attached to a hand-
held probe.

To measure forward progress and swim velocity, a
transparent 1 X 1 mm grid pattern was placed between
the glass stage and bottom of the Petri dish. Distance
measurements were then made directly from the video
monitor screen using frame-by-frame replay of swimming
episodes. Single-frame images were photographed directly
from the monitor with a Polaroid camera.

Results

General description of swimming behavior

In nature, Dem digitata resides in submerged or floating
organic debris along the shallow margins of lakes and
ponds, where it establishes temporary tubes or burrows,
lined with mucus and small particles. Its main mode of
locomotion in this environment is peristaltic creeping in
which bilaterally symmetrical muscle contractions (cir-
cula. and longitudinal), along with actions of ventral
chaetae, provide forward or rearward crawling move-
ments. Infrequently, however, worms may exit their tubes
and, by initiation of rhythmic swimming movements, en-
ter the surrounding water column.

When placed in a dish devoid of organic debris, worms
readily initiate swim movements (Figs. 1,2), either spon-
taneously or in response to light tactile stimulation of
posterior segments, especially the ciliated gill of the ter-

minal "segment," or pygidium (<.;/" Fig. 2 in Drewes and
Fourtner, 1991 ). Tactile-evoked swimming always begins
with a stereotyped, J-shaped, ventral flexion of the anterior
end (Fig. 1A; frame 1). If this flexion occurs while the
ventral surface of the worm is in contact with the bottom
of the dish, then the anterior end of the worm is thrust
away from the underlying substratum and into the water
column. The next phase of movement, usually evident
one or two video frames later, is a lateral bending (either
to the left or right side) that also occurs in anterior seg-
ments and accompanies ventral flexion (Fig. 1A; frame
2). This combination of ventral and lateral flexions, to-
gether with a uniform torsion of the body along its mid-
line, comprise the initial stages for forming the first helical
wave of a swimming episode.

As the wave of ventral and lateral flexion approaches
midbody segments, mainly ventral bending persists in an-
terior segments. At this time, formation of a single helical
wave is essentially achieved, with the wavelength com-
prising about 30-40% of the worm's body length (Fig.
1 B). At a slightly later time, the wave is seen in a midbody
position (Fig. 1C).

A diagram of the bending that occurs at the instant a
helical wave reaches this midbody position is shown in
Figure 1 D. Note that lateral bending occurs in segments
just posterior and anterior to the helical wave. As shown,
bending on one side (left) occurs in posterior segments
that are beginning the transition from a straight to a helical
configuration. Conversely, bending on the opposite side
(right) occurs anterior to the wave, in segments that are
reversing this transition (i.e., changing from the helical
back to straight configuration).

It should be noted that the body length of all swimming
worms appeared substantially reduced in comparison to
the body length of resting (non-swimming) worms. In a
typical swimming prefission worm (e.g.. Fig. 2B) the body
length (estimated by tracing along the helical wave formed
by the body) was 10 mm. This was about 30% less than
the resting body length in prefission worms ( 14 mm). This
reduction in length during swimming appears to involve
the entire body and probably derives from a circumfer-
ential increase in longitudinal muscle tonus.

When viewed in a line corresponding to the longitudinal
axis of head and tail segments, the helical wave in mid-
passage appears as a nearly perfect circular loop, the out-
side diameter of this circle being approximately 1.5 mm.
The outer curvature of this loop is mainly formed by the
dorsal surface of midbody segments, while the inner cur-
vature is formed by the ventral surface of these segments.
Thus, with an optimal combination of focal distance and
angle of illumination, the worm's long dorsal chaetae may
be visible on the outer curvature of the apex of the helical
loop (Fig. IB) and on the opposing dorsal surface of more
straightened anterior and posterior regions (Fig. 1C).

HELICAL SWIMMING

BENDING ON
LEFTSIDE

TAIL

BENDING ON
RIGHT SIDE

Figure 1. Body surface orientation during swimming movements. (A) Initiation of swimming, in response
to posterior touch (not shown), begins with a pronounced ventral flexion (J-shape) of the worm's anterior
end (frame Al ). The arrow shows the brush-like ventral chaetae on the first four anterior segments. Long
dorsal chaetae are evident on the outer curvature of the body bend in more posterior segments. In the next
frame (A2), continued ventral bending, in combination with lateral bending to the left side, results in formation
of the first helical body wave near the worm's anterior end. (B) Dorsal chaetae are visible at the apex ot the
helical loop (arrow) near the worm's anterior end. (C) With the helical loop in a mid-body position, dorsal
chaetae are visible in straightened anterior and posterior segments. The direction of swimming is from left
to right in B and C. The scale bar in C is 1 mm and applies to panels A-C. (D) Diagram of bending
movements and body surface orientation during passage of a helical wave. The dorsal surface of the worm,
labeled as a dotted line, faces away from the central axis of the helix (indicated by the solid arrow). The
ventral surface, in black, faces toward the central axis of the helix.

These images indicate that bending movements associated
with the formation of each wave are also accompanied
by a unidirectional torsion along the worm's longitudinal
axis.

Depending on the direction of the initial lateral bending
in anterior segments, the first helical loop may be either
right-handed or left-handed in orientation. Thus, in the
case of initial bending to the worm's right, helical wave

orientation along the worm's longitudinal axis is right-
handed. Conversely, with initial bending to the left, the
helical wave orientation is left-handed (e.g., see Fig. ID).
In all cases, movements associated with each subse-
quent helical wave also begin in anterior segments. How-
ever, two important differences should be noted: (1) in
contrast to the mechanics of the very first wave, initial
movements during the second wave (and all subsequent

C. D. DREWES AND C. R. FOURTNER

X ^1 '

"

Figure 2. Frame-by-frame analysis of swimming movements. (A) Frames 1 and 2 show passage of a
left-handed helical wave through middle and posterior segments of a normal worm. No wave is evident in
frame 3. In frames 4 and 5, the next helical wave is nght-handed in orientation. (B) A right-handed, helical
wave is seen in posterior segments of a prefission worm (frame I ), but the next wave (frame 3) is left-handed
(frame 4). The midbody fission zone is shown in frame 3 (arrow). The direction of swimming is from left
to right (;.'.. anterior end of worm positioned to the right) for all five frames in A and B. Scale bar
= 2 mm).

waves in a single swim episode) involve nearly synchro-
nous ventral and lateral bending in anterior segments,
rather than strong ventral bending followed by lateral
bending; (2) the orientation of the second and each suc-
cessive helical wave alternates with the preceding wave.
Consequently, swimming in these animals involves
rhythmic waves of alternating right-handed and left-

handed helical loops, with each loop appearing in lateral
view to form a plane that is roughly perpendicular to, and
moving retrograde with respect to, the direction of forward
swimming (Fig. 2A).

Essentially the same pattern of swimming was seen
throughout all stages of asexual segmental regeneration
in late prefission worms (if., Drewes and Fourtner, 1991).

HELICAL SWIMMING

Thus, alternating helical waves progressed without obvious
delay or interruption through anterior zooids, across the
fission plane, and into posterior zooids (Fig. 2B). In both
normal and prefission worms, the number of cycles in
each swimming episode appeared highly variable, ranging
from two or three to more than 50. One slight difference
between swim patterns in prefission and normal worms
was that a second helical wave frequently began in anterior
segments of late prefission stages before the preceding wave
was completed in extreme posterior segments, a situation
that seldom occurred in normal worms. This concurrence
of two spatially separated waves correlated with the fact
that worms at the late prefission stage were about 30%
longer than most normal worms.

Wave frequency and forward velocity

Frame-by-frame video analysis was used to determine
the wave frequency and forward velocity of worms during
repeated swimming episodes. Although frequencies in
normal worms varied significantly among different swim
episodes, wave frequency within a single episode was rel-
atively constant. The mean wave frequency during each
episode was determined by dividing the number of con-
secutive waves (usually 4-10 waves were counted in the
field of view) by the number of elapsed video frames. The
resulting value was then multiplied by 30 frames/s. Wave
frequencies ranged from approximately 6 to 12 waves/s
(Fig. 3A).

Forward velocity was determined by measuring the lin-
ear distance between the position of the worm's head at
the beginning and end of a swimming sequence. This dis-
tance was divided by the number of elapsed frames and
multiplied by 30 frames/s. Figure 3A shows that forward
velocity was directly related to wave frequency. The slope
(regression coefficient) of this relationship was 1.72 mm/
wave, a distance of about one-third the body length of a
normal worm during swimming.

Wave frequency and forward velocity were also deter-
mined for prefission worms. Frequencies ranged from ap-
proximately 5.5 to 13 waves/s. As in normal worms, a
direct relationship was evident between forward velocity
and mean frequency (Fig. 3B). However, the slope of this
relationship was 2.34 mm/wave, a value significantly
greater (P < 0.05) than that in normal worms (Fig. 3A).
The greater forward progress per cycle may be related to
the proportionately longer body length of prefission
worms; that is, propulsive forces generated by each wave
may act over a longer distance and greater time in prefis-
sion worms.

To study whether anterior and posterior zooids were
capable of independent swimming, worms in late prefis-
sion stages were severed at the fission zone. Swim behavior
in anterior and posterior zooids was readily initiated by

30

| 26

> 20

5

I "

10

I

30
| 2S

> 20

IS

10

i

10 11 12 13

B

12

13

WAVE FREQUENCY (cycles/e)

Figure 3. Relationship between forward velocity and wave frequency
during swimming in normal and prefission worms. (A) Each point shows
the mean velocity from one swim episode in a normal worm (total = 40
episodes from six worms: r = 0.73; slope = 1.72). (B) Each point shows
the mean swim velocity from one episode in a prefission worm (total
= 43 episodes from six worms; r = 0.89; slope = 2.34).

touch to the most posterior segments of each zooid.
Ranges of wave frequencies in the two zooids were nearly
the same as normal worms (Fig. 4A, B). However, the
slopes (approximately 1 .0 mm/wave in both anterior and
posterior zooids) were significantly less (P < 0.001 ) than
the slope for intact, prefission worms (Fig. 3B).

Capabilities for swimming were also examined in pos-
terior body halves, obtained by transecting normal worms
(n = 4) at a midbody position. Each posterior half initiated
and sustained independent helical swimming in response
to posterior tactile stimulation, just as in posterior zooids
from prefission worms. However, forward progress in the
posterior halves of normal worms was erratic. This was
apparently due to a biomechanical inability of segments
just posterior to the transection site to consistently
straighten and project in a forward direction immediately
after the passage of each propulsive wave. Such straight-
ening occurred in anterior segments of intact normal and
prefission worms (see frame 1 in Fig. 2A, B). Nevertheless,
the wave frequency in transected posterior halves was

C. D. DREWES AND C. R. FOURTNER

o

o

I

or
O

u.

30

25

20

15

10

S -

12 13

> 20

i

Q

H 10

B

10

WAVE FREQUENCY (cycles/8)

12

Figure 4. Relationship between forward velocity and wave frequency
during swimming in anterior and posterior zooids. (A) Data from 25
swim episodes in five anterior zooids (r = 0.74; slope = 1. 07). (B) Data
from 30 swim episodes in five posterior zooids (r = 0.63: slope = 0.9 1).

comparable to normal worms, varying from 5.5 to 9.5
waves/s.

H 'ave velocity and slippage

Estimates of retrograde wave velocity were obtained by
measuring the change in wave position from two succes-
sive video frames (Fig. 5A). All wave velocity measure-
ments were (a) made along a line parallel to the longitu-
dinal axis of the worm's body, (b) referenced to the worm's
anterior end, and (c) obtained as the wave was in a mid-
body position. Such measurements were seldom possible
in short, normal worms (or recently fissioned zooids) be-
cause the frame rate of the video camera was too slow in
relation to wave frequency to capture at least two succes-
sive frames showing clear-cut wave positions along the
body. However, in long prefission worms, these measure-
ments were possible during some swim episodes. Figure
5B shows the direct relationship between retrograde wave
velocity and frequency (c/s) in these worms. Wave veloc-
ities ranged from 35 to 55 mm/s, values substantially
greater than forward velocities of swimming.

Specific values of V wavc and V rorwar d were then used to
calculate "slippage" according to the following equation
(Gray and Lissmann, 1 964):

% slippage =

V V

* VA . I \ I > *

forward

X 100

From the linear relationship shown in Figure 3B, seven
values of V forward were interpolated for each unitary value
of frequency over the range of 6- 1 2 waves/s. Seven values
of V wave were similarly interpolated from Figure 5B. These
values were then used to calculate "percent slippage" (as
above) and "swim efficiency," the latter defined as: (100%
- percent slippage), or (V fonvard /V wavc ).

As shown in Figure 6, slippage ranged from 55 to 73%
over the normal range of wave frequencies and was ap-
proximately \ 5% less during fast swimming ( 1 2 waves/s)
than slow swimming (6 waves/s). However, these results
assume that V, onvard was uniform throughout a swimming
episode. This assumption was tested using frame-by-frame
tracking of forward progress of the worm's head during
swim episodes (Fig. 7).

60

SWIM FREQUENCY (cycles/s)

Figure 5. Determination of wave velocity in pretission worms. (A)
The change in position of the apex of the helical wave, relative to the
worm, is measured in two consecutive video frames (fl and (2). The
worm's anterior end is to the right. (B) Data from eight swim episodes
in four pretission worms indicate that wave velocity is directly related
to wave frequency (r = 0.97; slope = 3.24).

UJ

o

<

CL

a

_i
to

100

80

60

40

20

HKLICAL SWIMMING
12

20

40

60

80

100

>
O

UJ

o

u.

u.

LJ

to

5 6 7 8 9 10111213

WAVE FREQUENCY

Figure 6. Slippage and swim efficiency at different wave frequencies
in prefission worms. Points are derived from the linear slopes shown in
Figs. 3 and 5. Filled dots show values of overall slippage. Swim efficiency
values for fast and slow swimming are much greater when considering
slippage during only the propulsive phase of swimming ( + ).

In prefission worms, each cycle of swimming showed
two distinct phases. One phase, hereafter termed the pro-
pulsive phase, was characterized by a rapid and relatively
uniform forward progression of the worm's head. The
propulsive phase corresponded to video frames in which
the helical body wave was in retrograde passage through
middle and posterior segments. The second (recovery)
phase was characterized by head retrogression and cor-
responded to the video frame in which the lateral and
ventral bending movements required for reformation of
the next swim wave occurred in anterior segments.

Forward velocity during the propulsive phase was es-
timated by tracking head movements in both slow (6
waves/s) and fast (12 waves/s) swimming worms. The re-
sults, plotted in Figure 6, suggest that at least half of the
overall slippage occurred during the recovery phase and,
therefore, may be attributed to movements of head retro-
gression required for reformation of the swim wave in
anterior segments. The remainder of slippage, ranging
from approximately 25 to 40% in fast and slow swimming
worms, respectively, presumably occurred during the ac-
tual propulsive phase of swimming.

Discussion

Undulatory swimming in Dem involves retrograde
passage of helical body waves and, thus, appears funda-
mentally different from any other previously described
patterns of undulatory locomotion. Unlike two-dimen-
sional, sinusoidal waves that typically involve waves of
antagonistic muscle contractions (i.e.. either dorsal-lateral
or left-right), the propulsive waves for swimming in Dem

10

V)

<n

UJ

rr
O

o
or.

CL

Q

or.

I

or.
O
u.

I

;

2 4 6 8 10 12 14 16

FRAME NUMBER

Figure 7. Tracking of forward progress of the head in a prefission
worm. Each point shows the linear forward progress of the worm's anterior
end. as determined from a video frame. Posterior passage of the helical
wave along the body during each cycle of swimming is accompanied by
a steep increase in the rate of forward progress (e.g., frames 4-6. 7-9).
Reformation of the helical wave at the anterior end (e.g.. frames 7, 10,
13) results in head retrogression between each swim cycle.

are three-dimensional and helical in conformation. Based
on direct observations of the spatial orientations of body
surfaces during helical bending (Fig. 1 ), as well as estimates
of the relative timing and duration of various bending
components (Fig. 2), the sequence and phase relationships
of requisite bending movements were reconstructed as
shown in Figure 8. The illustration applies to any two
successive swim cycles (excluding the first cycle) in a typ-
ical midbody segment. It should be noted (though not
illustrated) that the combination of bending movements
for each wave is accompanied by a unidirectional torsion

RH

LH

--HI

ill--

time

Figure 8. Relative timing of bending movements for a typical mid-
body segment during two consecutive cycles of helical swimming. A
right-handed helical wave (RH) is formed by a combination of right (R),
ventral (V). and left (L) bending. Reversal of the bending sequence in
the next wave results in a left-handed helical wave (LH).

8

C. D. DREWES AND C. R. FOURTNER

of the body about the longitudinal axis. The direction of
this torsion reverses with each successively alternating
helical wave.

Several key features of this sequence are evident: (a)
the phase of ventral flexion represents the longest active
phase of bending during each cycle and persists through
both the left and right flexions that act to straighten the
worm; (b) each cycle in a swim episode, beginning with
the second cycle, starts with the same direction of lateral
bending that terminated the preceding cycle; (c) during
formation of each helical wave, lateral and ventral bending
begin nearly synchronously, except when anterior seg-
ments form the very first wave of a swim episode; and (d)
every cycle includes an "inactive" period during which
no substantial bending is evident. During this period we
assume the worm's body passively, rather than actively,
returns to a straightened position. Transition to this
position may be enhanced by the maintained increase in
longitudinal muscle tonus throughout the body, as pre-
viously described.

One behavioral consequence of this pattern is that the
orientation of successive waves alternates between right-
handed and left-handed helices. The advantages of this
alternation are not entirely clear. One likely possibility is
that repeated passage of waves in the same direction could
result in excessive yaw or pitch, because vectors of the
propulsive forces generated by each element of the body
wave are, undoubtedly, neither perfectly parallel to the
longitudinal axis of the helical coil nor symmetrically dis-
tributed in a circumferential pattern around its axis. Al-
ternating orientation of successive helical waves may tend
to counterbalance yaw or pitch. Thus, when such effects
are averaged over numerous cycles, the result may be rel-
atively straight swimming.

Another possibility relates to the amount of forward
progress achieved by passage of each cycle, which repre-
sents about 30-40% of the worm's length. Assuming that
each helical wave causes a corresponding, though very
weak, helical vortex of water displacement during passage,
then some stage of the next wave may encounter residual
fluid movements associated with the passage of the pre-
ceding wave. If such fluid movements are in the same
direction as those produced by the next wave, propulsive
forces of the moving body wave on the surrounding water
may be attenuated. Conversely, greater propulsive force
may occur if the next helical wave acts against water that
is residually moving in a direction of greater opposition
to the body wave.

Helical swimming in Dero involves an overall slippage
of approximately 50% at maximal swim velocity within
the observation arena. These values are comparable to
those of larger marine polychaetes that move by lateral
undulations, with presumably little or no assistance from
parapodial movements (Clark and Hermans, 1976).

Overall slippage in Dero, however, appears less than in a
wide variety of other animals (nematodes, insect larvae,
leeches, and snakes), all using two-dimensional undulatory
swimming (Gray and Lissmann, 1964; Taylor. 1952).
Nevertheless, several considerations suggest that our cal-
culations of slippage in Dero probably represent overes-
timations.

First, all values of forward progress used to calculate
swim velocity assumed straight-line swimming. However,
frame-by-frame tracking of head and tail segments showed
that, due to periodic yaw and pitch, this assumption was
never perfectly met during any swim episode; thus the
actual swimming distance was underestimated.

Second, to help ensure that swimming movements oc-
curred in the focal plane of the camera, worms were con-
fined to a 3-4 mm layer of water during all swim episodes.
Because swimming was not straight and because the loop
formed by helical bending was approximately 1.5 mm in
diameter, it is likely that some portions of the worm were
briefly near, or actually contacted, the bottom of the dish
or water surface during some swim cycles. Thus, there
were potential "wall effects" (Vogel, 1981). Such effects
could influence forward velocity in two opposing ways:
(a) if the helical loop (i.e., that portion of the body gen-
erating propulsive force) approached or contacted a rigid
surface, then forward velocity would be expected to in-
crease because resistance coefficients are larger as a wall
is approached by the loop (Katz and Blake, 1975); (b) if
a non-propulsive portion of the body came close to these
surfaces, then forward velocity may decrease due to en-
hanced drag (Taylor, 1972). If the latter effect (which is
inversely related to Reynolds number) predominates, then
the net result is retardation of the worm's forward progress
and an overestimation of slippage for assumed open-water
conditions. If the former effect predominates, then the
result is enhancement of forward progress and an under-
estimation of slippage for assumed open-water conditions.

We have recently analyzed forward velocity versus wave
frequency in normal (non-fissioning) worms under open-
water conditions (Drewes and Fourtner, unpub.). The
slope of this relationship was less than (though not statis-
tically different from) that of normal worms s\ 'imming
in shallow water (Fig. 3A). This suggests that the two op-
posing influences on forward velocity generated by "wall
effects" were either negligible or essentially counterbal-
anced one another when worms were swimming in a shal-
low dish.

For swimming in the fastest and longest prefission
worms the calculated Reynolds number was 300; for this
calculation a forward velocity of 30 mm/s and worm body
length of 10 mm (during swimming) were used. In com-
parison, for the slowest and shortest worms (normal) the
value was 50; for this calculation a forward velocity of
9 mm/s and worm body length of 6 mm (during swim-

HELICAL SWIMMING

ming) were used. These are similar to Reynolds numbers
for swimming in copepods or flight in small insects
(Strickler, 1975; Vogel, 1988).

Our results indicated that, in the absence of sediments
in which to retreat, helical swimming was readily induced
by posterior tactile stimulation. Such swimming was
evoked in normal and prefission worms, anterior and
posterior zooids, or anterior and posterior fragments of
normal worms. This suggests that sensory pathways ca-
pable of activating swimming are distributed throughout
the posterior and middle regions of the body. Previously,
electrophysiological studies of Dem (Drewes and Fourtner.
1991) showed that tactile stimulation in these same regions
triggered lateral giant nerve fiber spikes in worms placed
on recording grids and covered by a thin film of water. It
would be helpful to determine whether touch-evoked lat-
eral giant fiber spiking necessarily precedes or is somehow
causally linked to swim initiation in open-water condi-
tions.

Because helical swimming occurs in posterior zooids
or isolated posterior fragments, it appears that segmental
capabilities for integrating tactile sensory inputs and ini-
tiating resultant motor outputs for swimming are widely
distributed throughout the body. Normally, however,
expression of these outputs in middle and posterior body
segments is contingent upon integration of inputs and
prior initiation of outputs in cephalic segments. The au-
tonomous expression of a locomotor rhythm at subce-
phalic levels in body fragments is not unique to oligo-
chaete swimming and appears similar to capabilities for
peristaltic crawling in body fragments of earthworms and
polychaetes (Friedlander, 1894; Lawry, 1970).

Calculations of retrograde wave velocity (Fig. 5) provide
insight into functional properties of intersegmental path-
ways that coordinate swimming. Clearly, the velocity is
variable and correlates directly with wave frequency
(c/s). Maximal wave velocity was 55 mm/s, or approxi-
mately 1/20 the velocity of the lateral giant nerve fibers
and about 1/30 the velocity of the medial giant nerve
fibers in this species (Drewes and Fourtner, 1 99 1 ). There-
fore it seems highly improbable that either of the two
giant fiber pathways directly mediates the retrograde pas-
sage of excitation required for helical wave formation.

Future experiments will be directed at examining the
adaptive significance of swimming behavior. Because Dero
appears to maintain a predominantly sessile lifestyle, dis-
covery of intrinsic or extrinsic factors that trigger and
modulate swimming may be of special interest.

Acknowledgments

We thank Drs. B. Munson and J. Redmond for helpful
discussions.

Literature Cited

Clark, R. B. 1976. LJndulatory swimming in polychaetes. Pp. 437-446

in Perspectives in Experimental Biology. I '<>/ 1. Zoology. P. S. Davies,

ed. Pergammon Press, Oxford.
Clark, R. B., and D. J. Tritton. 1970. Swimming mechanisms in nere-

idiform polychaetes. J. Zoo/. Land. 161: 257-271.
Clark, R. B., and C. O. Hermans. 1976. Kinetics of swimming in some

smooth-bodied polychaetes. J. Zoo/. Land. 178: 147-159.
Drewes, C. D., and C. R. Fourtner. 1990. Corkscrew swimming in an

aquatic oligochaete. Am. Zoo/. 30: 1 ISA.
Drewes, C. D., and C. R. Fourtner. 1991. Reorganization of escape

reflexes during asexual fission in an aquatic oligochaete, Dero digitata.

J. Exp. Zoo/ 260: 170-180.
Friedlander, B. 1894. Beitrage zur Physiologic des Centralnervensys-

tems und des Bewegungsmechamsmus der Regenwurmer. Pfliig. Arch.

Ges. Physiol. 58: 168-206.
Gray, J. 1939. Studies in animal locomotion. VIII. The kinetics of

locomotion of Nereis diversicolor. J. Exp Bio/. 16: 9-17.
Gray, J. 1953. Undulatory propulsion. Q J Microsc. Sci. 94: 551-

578.
Gray, J. 1968. Pp. 435-453 in Animal Locomotion. Weidenfeld and

Nicolson, London.
Gray, J., and H. \V. Lissmann. 1964. The locomotion of nematodes.

J. Exp. Biol. 41: 135-154.
Gray, J., H. W. Lissmann, and R. J. Humphrey. 1938. The mechanism

of locomotion in the leech (Hirndo medicina/is Ray). J. Exp. Biol.

15: 408-430.
Katz, D. F., and J. R. Blake. 1975. Flagellar motions near walls. Pp.

173-184 in Swimming and Flying in Nature. Vol. 1, T. Y.-T. Wu,

C. J. Brokaw and C. Brennan. eds. Plenum Press, New York.
Lawry, J. V. 1970. Mechanisms oflocomotion in the polychaete, Har-

mothoe. Comp. Biochem Physiol. 37: 167-179.
Lighthill, M. J. 1969. Hydrodynamics of aquatic animal propulsion.

Ann. Rev. Fluid Mech. 1: 413-445.
Sawyer, R. T. 1986. Pp. 375-384 in Leech Biology and Behavior. ! ol. 1

Clarendon Press, Oxford.
Seymour, M. K. 1972. Swimming in Arenicola marina (L.). Comp.

Biochem. Physiol. 41A: 285-288.
Sperber, C. 1950. A taxonomical study of the Naididae. Zoo/. Bidr

Lppsala 28: 3-296.
Stolte, H. A. 1932. Analyse aussergewohnlicher Formen der Bewegung

bei einigen bodenbewohnenden Polychaten. Jen. Z. Naturwiss. 67:

199-220.
Strickler, J. R. 1975. Swimming of planktonic Cyclops species (Co-

pepoda, Crustacea): pattern, movements, and their control. Pp. 599-

6 1 3 in Symposium on Swimming and Flying in Nature. Vol. 2, T.

Y.-T. Wu, C. J. Brokaw and C. Brennan. eds. Plenum Press, New

York.
Taylor, G. 1952. Analysis of the swimming of long and narrow animals.

Proc. Roy. Soc. (A) 214: 158-183.
Taylor, G. 1972. Low-Reynolds-number flows. Pp. 47-54 in Illustrated

Experiments in Fluid Mechanics. National Committee for Fluid Me-
chanics Film Notes. MIT Press, Cambridge.
Trueman, E. R. 1975. Pp. 107-128 in The Locomotion of Soft Bodied

Animals. Edward Arnold. London.

Trueman, E. R. 1978. Locomotion. Pp. 261-269 in Physiology of An-
nelids. P. J. Mill, ed. Academic Press, New York.
Vogel, S. 1981. Pp. 249-250 in Life in Moving Fluids. Willard Grant

Press, Boston.
Vogel, S. 1988. Pp. 105-129 in Life's Devices. Princeton University

Press, Princeton.
Webb, J. E. 1976. A review of swimming in Amphioxus. Pp. 447-454

in Perspectives in Experimental Biology. I'ol. 1. Zoology. P. S. Davies.

ed. Pergammon Press. Oxford.

Reference: Biol. Bull 185: 10-19. (August, 1993)

Sag-Mediated Modulated Tension in Terebellid
Tentacles Exposed to Flow

AMY S. JOHNSON
Department of Biology, Bowdoin College, Brunswick. Maine 0401 1

Abstract. The long, compliant feeding tentacles of the
terebellid polychaete Eupo/ymnia heterobranchia not only
stretch out over a sandflat substratum but also extend
into flow. Tentacles suspended perpendicular to flow re-
sponded to increasing velocity by increasing their sag. An
analysis of tension in these tentacles, mathematically
analogous to that applicable to suspension bridges, shows
that sagging permits the tentacles to avoid increases in
tension that would otherwise occur as flow increases. Force
modulation was achieved by active muscular control
rather than by passive material properties. Although these
tentacles would certainly break in the experimental flows
if they did not sag, the low tension achieved suggests that
some other reason, such as limitations on the adherence
of cilia and mucus, accounts for the level of tension ob-
served. Because drag is maximum on tentacles oriented
perpendicular to flow, reorientation of tentacles, either
by sagging or by dangling parallel to flow, additionally
reduces tension by reducing drag. Theoretical estimates
of drag on tentacles oriented parallel to flow show that
they are never in danger of being broken. Drag is sufficient,
however, to assist in passive extension of tentacles. While
reorientation is a common mode of drag reduction among
marine organisms, sagging represents a novel mechanism
of mediating structural forces resulting from flow.

Introduction

Reliance upon a paniculate food resource is common
among marine invertebrates, and tentacles are among the
most versatile of structures used to capture such food.
The mechanical demands on tentacles during feeding vary
widely depending on the way that tentacles are used, and
the mechanical characteristics of the tentacles constrain

Received 10 March 1993; accepted 27 May 1993.

the ways that tentacles can be used by animals during
feeding.

The mechanical demands on the tentacles used to cap-
ture suspended food consist largely of resisting the drag
forces exerted upon them by flow. Although there is a
range in mechanical characteristics of tentacles specialized
for capturing suspended food, typically the tentacles that
are used exclusively for suspension feeding are relatively
inextensible. Bryozoans, for example, extend a short, stiff
crown of tentacles into flow to capture suspended particles.

In contrast, the deposit feeding terebellid polychaetes
feed off the surface of the substratum surrounding their
tubes by means of numerous, long compliant tentacles.
Most terebellids remain within their tubes, so that the
area of substratum over which they can feed is limited by
the length of their tentacles. During the process of feeding,
force is required to pull the tentacles out, to keep them
out, and to pull the tentacles back in. Terebellids exert
the force to extend their tentacles, at least partially, by
means of cilia located in a ventral ciliated food groove.
To do this, they flatten the food groove against the sub-
stratum and use the cilia to crawl. Mucus in the food
groove, in addition to its role in particle adhesion and
transport, helps the tentacles to adhere to surfaces. The
existence of circular muscles surrounding a fluid-filled
tentacular coelom indicates that terebellids may also use
internal hydrostatic pressures to help generate the forces
required to extend their tentacles.

The present study examines a terebellid polychaete,
Eupo/ymnia heterobranchia, that extends tentacles not
only over the substratum, but also into flow. E. hetero-
branchia individuals found on False Bay, San Juan Island,
Washington suspend their tentacles between layers in
dense mats of the sheet-like green alga Ulva feneslrata
(Fig. 1 ). The forces exerted on these tentacles include those
required to extend the tentacles over the surface of the
substratum and flow-induced drag on suspended tentacles.

10

DRAG AND SAG IN COMPLIANT TENTACLES

II

Figure 1. Schematic drawing (not to scale) of the terehellid Eupo-
IYIIUIUI heterobranchia in its natural habitat. Arrow indicates direction
of flow. /:' helerobranchia (I) extends tentacles out over substrata. (2)
dangles the ends of tentacles parallel to How, and (3) suspends tentacles
at an angle to flow b\ attachment either to sheet-like fronds of L'lva
ti'iii"*triiiii or to the water surface.

Tentacles exposed to flow between two points of at-
tachment sag in the direction of flow (Fig. 2). much as
the cables of suspension bridges sag in the direction of
gravitational forces. The similarity is such that the equa-
tions used to analyze tension in the cables of suspension
bridges are appropriate for analyzing tension in the ten-
tacles of E. heterobranchia oriented perpendicular to flow.
Such an analysis is used to address the specific question:
how do the behavior and mechanical characteristics of
the tentacles of the terebellid polychaete Eupolymnia het-
erobranchia allow them to remain suspended in flow? The
results of these experiments represent one step in under-
standing the association between the mechanical char-
acteristics of feeding tentacles and the way that they are
used for feeding.

Materials and Methods

Field site and collection ofterebellids

Eupolymnia heterobranchia specimens were collected
from the False Bay tidal flat on San Juan Island, Wash-
ington (48 29' N: 1 23 04' W) at about 0.0 m mean lower
low water within areas covered by extensive mats of Viva
fenestrata. These layered mats occur annually and persist
from May through October (Price and Hylleberg, 1982).
The unattached thalli of U. fenestrata float above the sub-
stratum when the flat is submerged. E. helerobranchia
commonly construct tubes within folds in these floating
algal sheets rather than within the mud of the flat. Whether
they construct their tubes within the mud or the algal
mats, these terebeilids suspend their tentacles between the
layers of algal thalli and into flow (Fig. 1 ).

False Bay is characterized by tidally dominated, uni-
directional flow. Flow averaged over five days in a slightly-
more exposed area of False Bay (east side, near site C in

Price and Hylleberg, 1982) was 0.043 m s '; daily peak
velocities ranged between 0. 10 and 0.20 m s ' (Pentcheff,
unpubl. data: measured in the field by a Marsh-McBirney
electromagnetic flow probe at 6 cm above a rock substra-
tum). Flow over the relatively protected area where E.
heterobranchia was collected (west side, site D in Price
and Hylleberg, 1982) and particularly between the layers
of I' fenestrata. should be comparable, although some-
what slower.

Once collected, E. helerobranchia individuals were
maintained at the University of Washington's Friday

(a)

tentacle

(c)

sag

Figure 2. Schematic drawing (not to scale) of Eupolymnia hetero-
branfliia in a flow tank, (a) Animals were allowed to self-attach a tentacle
to the probe (using cilia and mucus), (b) Raising the probe oriented the
tentacles perpendicular to flow. Span was the distance between points
of attachment, (c) Arrow indicates direction of flow. Sag was the distance
between the vertical line drawn between points of attachment and the
point of maximum sag.

12

A. S. JOHNSON

Harbor Laboratory in circulating seawater tables at around
15C. When possible, worms were kept in their original
tubes. Those specimens of E. heterobranchia that lost their
original tubes were placed on muddy sediment where they
constructed new (flimsier) tubes.

The parabolic

The tension (force) in suspended tentacles that results
from drag can be estimated with engineering formulas
used to calculate tension in the cables of suspension
bridges. Tension in the cables of suspension bridges is
calculated using the force acting on the cables, the distance
(or span) between points of attachment of the cable, and
the amount of sag in the cable. Below, I review briefly the
standard engineering equations for tension in parabolic
cables (those that carry a uniform distributed load: see
Steinman, 1942).

The horizontal tension at the points of attachment of
a cable is exactly the tension in the cable at the point of
maximum sag. At this point, there is only a horizontal
component to the tension, and no vertical component.
The horizontal tension, H, is:

H =

8f

I)

where uj for a parabolic cable is the uniform distributed
load (a force) per horizontal linear unit, L is the span
distance between points of attachment of the cable, and
f is the sag of the cable. Sag of a cable is denned as the
distance between the lowest point and a horizontal line
drawn between the points of attachment of the cable.

The maximum tension, T, in the cable occurs at the
points of attachment, where there is the greatest summed
contribution from the vertical component of tension. The
maximum tension. T. can be calculated from the hori-
zontal tension, H, as follows (Steinman, 1942):

(2)

Maximum tension can also be expressed as a function
of the ratio of span to sag, (L/f). by substituting Eqn ( 1 )
into Eqn (2) for H:

T = W

fH(f)(f

(3)

Similar to the parabolic cable of suspension bridges,
where the cable carries a uniformly distributed gravita-
tional load, drag exerts a force that acts along the hori-
zontal distance (projected area) of a tentacle oriented per-
pendicular to flow and suspended between two points of
attachment. Therefore, force, which is o>L for the parabolic-
cable, is drag, D, in the tentacle-cable equation. The de-

termination of drag on the tentacles of E. heterobranchia
and its use in the cable equation are described below.

Drag calculations

Drag is greatest on a tentacle oriented perpendicular to
flow (Vogel, 1981 ). To orient tentacles perpendicular to
flow, specimens of E. heterobranchia. intact within their
tubes, were buried in mud in glass dishes that were placed
in the bottom of a recirculating seawater flow tank (similar
in design to that of Vogel and LaBarbera, 1978) (Fig. 2a).
A micromanipulator was used to lower a probe to the
mouth of the worm's tube and, subsequently, to raise the
probe after the worm had attached a tentacle using cilia
and mucus. In this way, tentacles were oriented perpen-
dicular to the direction of flow, with two points of at-
tachment: one on the probe, the other on the body of the
worm (Fig. 2b). The tip ends of these suspended tentacles
usually dangled downstream from the point of attachment
to the probe (Fig. 2c).

Behavior of the tentacles in response to flow was quan-
tified by photographing each tentacle several times at each
velocity with a camera oriented perpendicular to the sag.
A known distance on the probe was used to determine
scale in all photographs. Span (L), sag (f), and tentacle
length (1) were measured off photographs; where span was
the distance between points of attachment of a tentacle
(Fig. 2b), sag was the distance between the maximum sag
(parallel to flow) in that tentacle and a line defined by the
two points of attachment (Fig. 2c) and tentacle length was
the real length of the tentacle between the points of at-
tachment. The span was set at a minimum by this method
(no lower than the tip of the probe), but a tentacle could
increase or decrease its span by changing attachment
points along the length of the probe.

Drag on tentacles was determined using the standard
equation for the drag on a cylinder perpendicular to flow
(Vogel. 1981):

D = '/2C D pSU :

(4)

where D is drag. C D is the drag coefficient, p is the density
of seawater. S is the projected area of each tentacle per-
pendicular to flow and U is the velocity. The projected
area, S, of each tentacle was calculated as L x d: where
d was the tentacle diameter. Tentacle diameter, d, was
measured on 31 living, unstretched tentacles under a
compound microscope (mean = 0.10; minimum = 0.05
mm; maximum =0.13 mm; SE = 0.003 mm). Because
diameter could not be measured during experiments in
the flow tank, this mean tentacle diameter was used in all
calculations of tension, except as described below. A
propagation of error analysis using the minimum and
maximum diameters measured indicated that this resulted
in a maximum error of 15% in the tension estimated

DRAG AND SAG IN COMPLIANT TENTACLES

13

for any particular tentacle. The average error from this
source was unbiased.

The conventional drag coefficient, C D . was estimated
from an empirical formula (for Reynolds numbers from
unity to 10 5 ) for a cylinder oriented perpendicular to flow
(White. 1974 cited in Vogel. 1981):

C D = 1 + 10 Re" 2/3

where Re is the Reynolds number:

Re =

(5)

(6)

where d is the diameter of the tentacle and ^ is the dynamic
viscosity of seawater. Unless otherwise specified, all ref-
erences to drag in this paper refer to the calculation of
drag using the variables as described above.

All experimental velocities in the flow tank were de-
termined by timing neutrally buoyant particles. Experi-
ments were conducted at velocities ranging between 0.5
and 7.0cm s~': these velocities corresponded to Reynolds
numbers for the tentacles between 1 and 7. Tentacles did
not remain attached to the probe at velocities greater than
7.0 cm s~ '. These velocities fell within the range measured
in the field.

Use of l lie cable equation for the tentacles

Drag was used to calculate the maximum tension in
the tentacle by substituting D for u)L in Eqn (3):

T-W'

DVL
f

(7)

From Eqn 7 it can be seen that tension is a function
of drag as well as of the ratio between span and sag

(L/f).

Material tests

The material properties of the tentacles of E. hetero-
branchia were determined by performing force-extension
tests on an Instron tensometer (University of British Co-
lumbia, Vancouver). Lengths of tentacles, freshly cut off
live animals, were fastened to grips, and relaxed by im-
mersion in a solution of 7.5% MgQ 2 . After relaxation,
grips were moved slowly apart until the tentacle was put
nearly into tension. The original length of the tentacles
between the grips (1 ) was determined by reading the value
off the Instron to the nearest 0.1 mm. Tentacles were
pulled at constant speed of 50 mm min ' (= a strain rate
of between 0.04 and 0.15 s' 1 ; mean = 0.09, SE = 0.006)
until they broke. Breaking force was measured off the
chart paper to the nearest ^N; breaking length (1) was
measured off the chart paper to the nearest 0.1 mm. Ex-
tension ratio (X) was calculated as (1/1 ). Extension ratio
is useful as an intuitive description of length changes be-

cause X = 2 corresponds to a doubling of length, X = 3
corresponds to a tripling of length, etc. True strain can
be determined from the extension ratio as In X (Vincent.
1990). Linear regression analysis revealed that, within the
range of strain rates used in these experiments, both
breaking force and breaking X were independent of strain
rate [breaking X: f (] 2 4> = 2.39, P (that the slope is zero)
= 0.14; breaking force: f ( ,. 24) = 0.22, P = 0.64].

Sag- related drag reduction

It is assumed above that the drag of a sagging tentacle
is equal to that of a non-sagging tentacle of equal span
oriented perpendicular to flow. I used drag on a tentacle
perpendicular to flow to isolate the effects of tension-re-
duction due to the geometry of sagging (the cable equa-
tion) from the drag reducing effects of sagging described
below. In fact, C D (Eqn 5) will be an overestimate of the
coefficient of drag for a sagging tentacle because portions
of the tentacle are oriented at some smaller (lower drag)
angle to flow. The greater the sag of the tentacles, the
greater the degree to which the coefficient of drag is over-
estimated by C D . Furthermore, in a constant volume ten-
tacle, diameter will decrease with increasing sag (increasing
stretch). The magnitude of these two sag-related mecha-
nisms of drag reduction can be calculated as follows.

The coefficient of drag for a cylinder at some angle to
flow. C Dfl , can be determined from C D (for Reynolds
numbers to 10\ Hoerner, 1965) by:

= C D cos 3 !?

(8)

where is the smallest angle between a line perpendicular
to flow and the surface of the tentacle. The angle 6 was
obtained at each point on the tentacle by:

d /4fx :

= arctangent -
dx \ L-

(9)

where x is the distance along the span from the origin (at
the point of maximum sag) and (4f x 2 /L 2 ) is the parabolic
equation describing the sagging tentacles (Steinman,
1942). An estimate of the corrected coefficient of drag for
the entire tentacle, C Dc , was obtained by substituting Eqn
9 into Eqn 8 for 6. integrating Eqn 8 over the span of the
tentacle (i.e.. adding up the coefficient of drag for each
infinitesimal piece of tentacle) and dividing by the span
of the tentacle:

r L/:

2C D

Jo

cos 3 i9dx

(10)

This is similar to the method used to determine the drag
coefficient of a wing from the sum of the local drag coef-
ficients of infinitesimal sections of the wing (Abbott and
Von Doenhoff, 1959).

14

A. S. JOHNSON

60 -

0.01

0.02 0.03 0.04 0.05 0.06 0.07

VELOCITY (rns' 1 )

Figure 3. Tension (>iN) of suspended tentacles of Eupdlymnia hci-
erobranchia as a function of velocity (m s ' ). Points indicate data. Tension
in these tentacles was independent of velocity. Lines indicate tension
calculated from Eqn 7 using the mean span and (a) the highest value for
L/f. (h) the lowest value for L/f. and (c) the lowest value of L/f and the
drag corrected for either the coefficient of drag or both the coefficient of
drag and the tentacle diameter. These lines indicate that tension in the
tentacles is reduced by sagging, mostly due to geometry (compare b to
a) but also partly due to drag reduction (compare b to c).

A corrected diameter, d c , was estimated by assuming
that the tentacles maintain a constant volume as they
stretch:

d c = d(X) 5 (ID

where X was determined from the ratio of the total length
of the tentacle. I (measured from the photographs), and
the span of the tentacle (L = 1 ).

Results

Tentacles, a\ cuh/c\

Calculated drag on suspended tentacles of Eupolymnia
heterobranchia increased with increasing velocity (F (1 76 ,
= 77. 1, P = 0.000 1, R : = 0.50). Despite this relationship,
linear regression analysis indicates that maximum tension
(at the points of attachment) was independent of velocity
(Fig. 3, F u 76) = 2.67, P = O.I I). Maximum tension in the
tentacles was between 0.34 X I0~ 5 N and 5.2 X I0~ 5 N
(mean = 1 .6 X I0~ 5 N, n = 78, SE = O.IO X KT 5 N).
The results of linear regression analysis using data cor-
rected for just C Dc (F,i. 76) = 0.72, P = 0.40) as well as both
C Dc and d c (F (l 76) = 2.38, P = O.I 3) are consistent with
the results of linear regression analysis on the uncorrected
data: maximum tension (at the points of attachment) re-
mained independent of velocity.

Tension is a function not only of drag, but also of the
ratio between the span and sag of the tentacle (Eqn 7).
The independence of tension and velocity indicate that
the ratio of span to sag (L/f) should decrease as a function

of increasing velocity. The relationship between velocity
and (L/f) is shown in Figure 4. where curve (a) on Figure
4 represents the linear regression of In (L/f) as a function
of In (velocity) plotted on linear axes. Linear regression
analysis of In (L/f) with In (velocity) demonstrates that
(L/f) decreased with increasing velocity (F n 7f)) = 90.1, P
= 0.0001, R 2 = 0.54).

This relationship could be created by some combination
of increasing sag and decreasing span with increasing ve-
locity. Although minimum span was set by the end of the
probe, span deviated from this minimum as a terebellid
behaviorally moved the point of attachment relative to
the end of the probe. Accordingly, span varied between
13.3 and 47.2 mm (mean span = 29.4 mm, n = 78,
SE = 1.10) and sag varied between 0.160 and 10.3 mm
(mean sag = 3.32 mm. n = 78, SE = 0.29). Linear regres-
sion analysis on the In-ln relationship between variables
reveals that while sag did increase significantly with ve-
locity (F, ,. 76) = 52.9, P = 0.000 1 , R- = 0.4 1 ; for examples,
see Fig. 5 and Fig. 6), span was independent of velocity
(F (1>76) = 1.18, />= 0.28).

Individual tentacles initially responded to increases in
velocity by increasing sag over a number of seconds (<30

150

125

% 100-

Z

<

c.

75

50

25

I

0.01

0.02

0.03

0.04

0.05

0.06

0.07

VELOCITY (m s - 1

Figure 4. The ratio of span-to-sag of suspended tentacles of Eitpo-
Ivinnui heterobranchia as a function of velocity (m s '). Points indicate
data. Line (a) indicates the regression from a In-ln plot of this data; linear
regression analysis on In-ln transformed data indicates that span-to-sag
decreased as a function of velocity. Line (b) indicates the theoretical
relationship between (L/f) and velocity when constant values of tension
and span were used in Eqn 12 (means for the data set: 1.57 x 1CT 5 N
and 0.0295 m. respectively).

DRAG AND SAG IN COMPLIANT TENTACLES

15

Figure 5. Photograph of a tentacle ofEupolymnia helerobranchia suspended perpendicular to flow. The
tip of the tentacle dangles in the direction of flow, which is from right to left. For scale, span of the tentacle
in both photographs is 2.5 cm. The photograph on the left is taken just as the velocity in the flow tank is
increased from 1.5 to 3 cm s"'. The photograph on the right is of the same tentacle taken several minutes
after the increase in velocity. Tentacles respond to increasing velocity by increasing sag.

seconds; n = 13 tentacles). After the initial increase in
sag, relatively small increases and decreases in sag occurred
while velocity remained constant (Fig. 6).

Mechanical response oj tentacles

A typical force-extension curve for a tentacle of E. hct-
erobranclua is shown in Figure 7 (obtained by tracing the
result of one force-extension trial directly off the chart
paper). A force of 4.6 X 1(T 4 N more than tripled the
length of this tentacle (X = 3.5) before breakage. Tentacles
broke at forces between 0.68 X 1(T 4 N and 7.6 x 10 4 N
(mean = 3.5 X 10 4 N, n = 29, SE = 3.1 >< 10 5 N).
Tentacles broke at As ranging from 1.79 to 5.35 (mean
= 3.34. n = 29, SE = 0.17).

The slope of this curve is a measure of the relative stiff-
ness of the tentacle, or its resistance to being extended.
The tentacles of E. heterobranchia are least stiff at low Xs,
requiring relatively little force to extend (Fig. 7a); than at

high X's (Fig. 7b). The transition in stiffness (i.e., where
the curve deviated from a line tangent to the lower portion
of the curve; Fig. 7a) began at Xs in the range 1.66 to 2.48
(mean X = 1.93. n = 5, SE = 0. 15) and at forces between
0.18 X 10" 4 and 0.64 X 10 4 N (mean =0.36 X 10~ 4 N,
n = 5, SE = 0.08 X 10 4 N). Although these tentacles do
exhibit strain rate dependent stiffness and stress-relaxation
(sensu Vincent, 1990) at high Xs, these effects are not seen
at low Xs (Johnson, unpubl. data).

The mean maximum tension in suspended tentacles
was significantly less than the mean force at which stiffness
increased (uncorrected data. ANOVA: F ( I X:) = 22.4. P
= 0.0001).

Discussion

Tentacles of the terebellid polychaete Eitjwlyinnia het-
erobranchia demonstrate a new mechanism of coping with
flow forces. Sagging reduces flow-induced tension in ten-

16

A. S. JOHNSON

3-

35

.(5)

,(6)
(3). (4)

(1)

.(1)

0.01 0.02 0.03

VELOCITY (ms' 1 )

0.04

Figured. Sag(mm) as a function of velocity (m s ') for one tentacle
of Eitpolvmnia heterobranchia that maintained a constant span (mean
= 2.8 cm. n = 9. SE = 1.15) at 0.005 m s~' 0.023 m s ', and 0.034
m s"'. Small numbers indicate the order in which the tentacle was pho-
tographed at a specific velocity. Photographs were taken approximately
5 s apart. Tentacles respond to velocity by increasing sag over a number
of seconds. Within a velocity sag may also decrease (e.g., 5 -* 6 at 0.034
m s~ ') suggesting that muscles are actively involved in regulating sag in
the tentacles.

tacles; the largest contributor to this reduction in tension
was achieved by closer alignment of force vectors that
resist flow (parallel with the length of the tentacle) with
force vectors imposed by flow (parallel with the direction
of flow). This mechanism has the same ultimate effect as
drag reduction by flexibility, streamlining, and reorien-
tation: by modifying the effects of flow forces an organism
can function in a wider range of flows (Wainwright and
Dillon, 1969; Wainwright et al., 1976;Koehl, 1977, 1984;
Vogel, 1984; Denny et al., 1985; Harvell and LaBarbera,
1985; Carrington, 1990). The discussion below analyzes
the contribution of sagging and reorientation to tension
experienced in the tentacles of E. heterobranchia.

The versatile, extensible, and flexible tentacles of ter-
ebellid polychaetes commonly occur adjacent to a sub-
stratum in low flow, mud, or sand flat environments (re-
viewed in Fauchald, 1977; Fauchald and Jumars, 1979).
Such proximity to a substratum reduces the relative drag
experienced for a given freestream flow because of the
effect of the boundary layer (slower flows occur adjacent
to the substratum). In contrast, the tentacles of the tere-
bellid E. heterobranchia, extend not only over a substra-
tum but also out into flow. Although extending tentacles
into flow and over fronds of floating algae might increase
access to potential food resources, this behavior also in-
creases the exposure of tentacles to drag. Drag will have

conflicting mechanical consequences to tentacles, both
positive, including facilitation of tentacle extension, as
well as negative, including breakage or dislodgment.

Mechanical analysis, in which tentacles are modelled
as the cables of suspension bridges (Eqn 7), reveals the
behavioral and mechanical response of these tentacles to
drag. For example. Eqn 7 shows that maximum tension
in these tentacles (at the points of attachment) was a func-
tion not only of drag but also of the ratio between span
and sag (L/f). Maximum tension was statistically inde-
pendent of drag, because increasing sag (shown by Figs.
5 and 6) decreased the ratio of span to sag and thus main-
tained a constant tension despite increasing velocity and,
therefore, increasing drag.

Maximum tensions experienced by suspended tentacles
were an order of magnitude less than the forces required
to break the tentacles, indicating that suspended tentacles
that sag are probably never in danger of being broken
over the range of velocities at which they remained sus-
pended. One can then ask: would tentacles in this ori-
entation and in these flows break if they didn't sag? To
examine this question, tension was calculated using Eqn
(7) for tentacles of mean span, keeping (L/f) constant over
the range of velocities examined. Two extremes, the high-

500

1.0

2.0

3.0

EXTENSION RATIO

Figure 7. Force (^N) as a function of extension ratio (A) fora relaxed
tentacle of Eupolymnia heterobranchia. The curve shown in this figure
was obtained by tracing the result of one force-extension trial off the
chart paper. The slope of this curve is a measure of the relative stiffness
of the tentacle, (a) These tentacles are least stiff at the lowest strains,
requiring relatively little force to extend; (b) tentacles are most stiffjust
prior to breakage. Comparison of this curve with tensions and Xs of
sagging tentacles indicates that sagging involves active muscles and is
not merely a consequence of the passive material properties of the ten-
tacles.

DRAG AND SAG IN COMPLIANT TENTACLES

17

est (150.75) and the lowest (4.54) obtained from this data
set, were chosen for (L/C). The curves (a) and (b), obtained
from the highest and lowest L/f respectively, were super-
imposed on the experimental data (Fig. 3; the spans of
the tentacles whose data points fall below line b were
shorter than the mean tentacle span. Because span con-
tributes not only to this ratio but also to drag, the con-
sequent tension was lower than that obtained from the
line calculated from mean span). It can be seen from these
calculations that all values for tension obtained from the
data are lower than they would be if the tentacles were
allowed only a small sag (as in curve 3a). These low-sag-
ging tentacles would be subjected to their mean breaking
force of 3.5 X 10 4 N at 0.053 m s '. well below the highest
velocity at which tentacles remained suspended perpen-
dicular to flow (0.07 m s~'). Thus, sagging allows tentacles
to remain suspended without breakage at higher flows
than would otherwise be possible.

Although sagging potentially avoids breakage, the force
in suspended tentacles was an order of magnitude less
than that which would break them, suggesting that some
other explanation accounts for the particularly low level
of tension in suspended tentacles. Perhaps the tentacles
sagged passively until they reached an equilibrium be-
tween the force imposed on the tentacles and the force
with which the material of the tentacles could resist further
extension. This mechanism assumes that muscles played
no active role in determining the extension of the tentacles.
If the tentacles were sagging passively, than a given cal-
culated tension should have produced an extension ratio
similar to that produced by a given force for the relaxed
tentacle in Figure 7. In fact, although calculated tensions
for sagging tentacles were as high as 5.2 X 10 5 N, cor-
responding to X ^ 2.5 in Figure 7, the greatest X for sagging
tentacles was only X = 1.12. Thus, tentacles extended too
little to be sagging passively: active muscular control must
have been involved in controlling sag in the tentacles.
Further, suspended tentacles sometimes decreased their
sag by as much as 1 5% when experiencing a constant flow
(Fig. 6), also indicating that there is an active muscular
contribution controlling sag.

Given that muscles actively control sag in the tentacles,
could the observed mean maximum tension correspond
to the peak isometric tension of the muscle? Although
some invertebrate muscles are somewhat stronger, vir-
tually all muscle exerts a maximum stress of 0.5 MN m :
(Schmidt-Neilsen, 1983). One can estimate whether the
stress in the tentacles exceeds the ability of muscles to
hold tentacles in tension by dividing the mean maximum
tension ( 1 .6 X 10~ 5 N) by an estimate of the cross-sectional
area of the muscle in the tentacles (3.9 X 10" 8 m : : n
= 28, SE = 0.9 X 10~ 8 m : ; Johnson, 1992). The resulting
estimate of stress in the muscles of suspended tentacles

(4.1 X 10~ 4 MN m : ) is several orders of magnitude less
than that which the muscles can maximally exert.

Thus, E. heterobranchia are modulating tension at such
low levels in tentacles neither ( 1 ) to prevent breakage per
sc. nor (2) as a result of passive material properties of the
tentacles, nor (3) because of the limits of peak isometric
tension in their muscles. One question arises from these
results: is there a functional significance to the level of
tension actively maintained in suspended tentacles? The
next obvious hypothesis is that E. heterobranchia maintain
tension in their tentacles below the detachment strength
of the mucus and cilia. This hypothesis remains to be
tested.

L/f as a function of velocity

The shape of the relationship between (L/f) and veloc-
ity, described by the curve in Figure 4, suggests that rel-
atively large changes in sag are required to modulate ten-
sion at low velocities: whereas relatively small changes in
sag result in constant tension at higher velocities. What
accounts for the shape of this relationship between (L/f)
and velocity? To address this question, the theoretical re-
lationship between (L/f) and velocity was determined us-
ing constant tension and span by rearranging Eqn (7) to
solve for a theoretical (L/f) as indicated below:

64| -16

L
f

Mean values of tension and span (used in the calcu-
lation of D) for the data set were used to determine these
constants (1.57 X 10~ 5 N and 0.0295 m, respectively).
The result of this calculation is shown by curve (b) in
Figure 4. Not surprisingly, comparison of curve (b) with
curve (a) in Figure 4 illustrates the earlier statistical results:
that these curves represent the sag that is necessary to
mediate drag so that tension is modulated under condi-
tions of constant span.

Effects oj tentacle orientation on drag

The above theoretical calculations assumed a constant
coefficient of drag and a constant diameter, independent
of sag. The above results are thus independent of changes
in C D and d that will occur in a sagging tentacle. The
following section examines the effects of changes in C D
and d to drag reduction (Fig. 3).

Drag on sagging tentacles should be lower than that of
a non-sagging tentacle of equal span because tentacles
thin as they sag and because portions of the tentacles are
oriented at angles less than perpendicular to flow. To ex-
amine the potential contribution of sagging to drag re-
duction, tension was calculated, again using Eqn (7) and
the highest and lowest L/f, but including C Dc as the coef-
ficient of drag and d c as the diameter of these tentacles.

18

A. S. JOHNSON

Calculations using the highest L/f, C Dc and d c resulted in
a line identical to (a): there was no significant drag re-
duction in a tentacle of such low sag. Calculations using
the lowest L/f with either just C Dc or both C Dc and d c
resulted in curve (c). There are several points that are
illustrated by these results: (1) sagging results in drag re-
duction, especially at high velocities and high sags, (2)
drag reduction due to decrease in diameter with increasing
sag is negligible, and (3) even at high sags, drag reduction
by sag-related reduction in the coefficient of drag con-
tributes much less to reduction of tension than structural
alignment of force vectors, especially at low velocities.

An extreme case of orientation relative to flow occurs
when tentacles are dangling (i.e., the danglers shown in
Fig. 2 and Fig. 5) and thus are oriented parallel to flow.
Could a tentacle become long enough and flow fast enough
that drag would be sufficient to extend, or even to break,
tentacles dangling out into flow? Drag on tentacles ori-
ented parallel to flow can be estimated as approximately
half that of tentacles oriented perpendicular to flow ( Vogel.
1981). Actual drag should be slightly higher than given
by this estimate because tentacles are of finite length. For
this reason, I call this estimate "theoretical minimum
drag." Figure 8 shows the theoretical minimum drag as
a function of velocity for danglers that are 1 cm, 10 cm,
and 30 cm long (a reasonable range of lengths for the
tentacles of E. heterobranchid). Danglers in the low-flow
environment of an intertidal flat are never in danger of
being broken by drag. When terebellids relax the longi-
tudinal muscles in their tentacles, however, drag is suffi-
cient to contribute to passive tentacle extension. For ex-
ample. Figure 8 indicates that danglers that are 10 cm
long in flow of 0.10 m s ' (the lowest peak speed mea-
sured) experience forces (1.3 x 10~ ? N) that, while still
within the low stiffness region of the force-extension curve,
are sufficient to nearly double the length of the average
relaxed tentacle (Fig. 7). This mechanism of passive ten-
tacle extension is most effective for longer tentacles in
faster flows.

Ecological consequences

Quite apart from the mechanical results of this study,
the observation that /:. heterobranchia individuals suspend
their tentacles into flow suggests that they are using ten-
tacles to supplement deposit feeding with the capture of
suspended food particles. While I have not observed sus-
pension feeding in /:. heterohranchia. it seems unlikely
that it would reject suspended food particles that intercept
its suspended tentacles. Although most terebellids are en-
tirely selective deposit feeders (Fauchald and Jumars,
1979), suspension feeding does contribute to the diet of
the terebellid Lanicc conchilega (Buhr, 1976; Buhr and
Winter, 1977; Fauchald, 1977). Furthermore, other ter-

iso -

0.05 0.10 0.15

VELOCITY (m s 1 )

0.20

Kigurc 8. Theoretical minimum drag (^N) as a function of velocity
(m s ') for 1 cm, 10 cm, and 30 cm long tentacles oriented parallel to
flow (danglers). Maximum velocity of 0.2 m s" 1 represents the maximum
speed measured at False Bay (Pentcherf, unpuh. data). Drag on danglers
was always well below the maximum breaking force of tentacles (7.6
II)" 4 N). but often within the range that would assist in passive extension
of tentacles.

ebellids. such as Loimia medusa (Filers, pers. comm.) also
extend their tentacles into flow in a manner similar to
that which I have described for E. heterobranchia.

Flow over the relatively protected area where E. het-
erobranchia is found, and particularly between the layers
of U. jenestrata. will be slower than that measured by
Pentcheff (see Materials and Methods). Furthermore, the
angle of tentacles to flow will often be less than perpen-
dicular. Thus, E. hetcrtthranchia would be able to suspend
tentacles into flow during most of the tidal cycle.

Sagging allows the tentacles to remain suspended in
higher flows than would otherwise be possible, presumably
increasing the amount of food these tentacles can gather.
Feeding would be enhanced by the ability to sag not only
because of increased access to suspended material but also
because of increased access to food resources deposited
upon the prodigious surface area of the stacked thalli of
U. jenestrata. Thus, sagging increases the ecological range
over which these terebellid polychaetes can function.

Acknowledgments

The author is deeply appreciative to M. Koehl for her
support, advice, and encouragement. Thanks also to J.
Gosline, O. Filers, and R. Emlet for helpful discussions
and advice; A. O. D. Willows for providing space at Friday
Harbor Marine Laboratory; K. Durante, G. Shinn, S.
Walker, and W. H. Wilson for field assistance; B. Hale
for assistance with data analysis; and D. Pentcheff for use
of his unpublished flow data. Special thanks to M. La-
Barbera for suggesting the use of the parabolic cable equa-
tion and to both him and S. Vogel for being wonderful
teachers in the 1981 Biomechanics course at Friday Har-

DRAG AND SAG IN COMPLIANT TENTACLES

19

bor. The quality of this manuscript was improved by two
anonymous reviewers. This research was supported, in
part, by a Libbie Hyman Memorial Field Scholarship.
Northeastern University, and NATO post-doctoral fel-
lowships, N.S.F. Research Planning Grant OCE 90-09763
to A. Johnson and N.S.F. Grants OCE-8352459 and OCE-
85 10834 to M. Koehl.

Literature Cited

Abbott, I. H., and A. E. Von Doenhotf. 1959. Theory o/'H'ing Sections.

Dover Publications. Inc., New York. 693 pp.
Buhr, K.-J. 1976. Suspension-feeding and assimilation efficiency in

Lattice conchilega (Polychaeta). Mar. Biol. 38: 373-383.
Buhr, K.-J.. and J. E. \\ inter. 1977. Distribution and maintenance of

a Lattice conchilega association in the Weser Estuary (FRG). with

special reference to the suspension-feeding behaviour of La nice con-

chilega. Biology oj Benthic Organisms. I llh European Marine Bio/ogv

Symposium. Galway, Ireland 1976: 101-113.
Carringlon, K. 1990. Drag and dislodgment of an intertidal macroalga:

consequences of morphological variation in Mastocarpus papillatus

Kutzing. J E.\p Mar Biol Ecol 139: 185-200.
Denny, M. \V., T. L. Daniel, and M. A. R. Koehl. 1985. Mechanical

limits to size in wave-swept organisms. Ecol. Monogr. 55: 69-102.
Fauchald. K. 1977. The Polychaete Worms. Definitions and Keys to

ihe Orders. Families and Genera. Natural History Museum of Los

Angeles County. Los Angeles. 188 pp.
Fauchald, K., and P. A. Jumars. 1979. The diet of worms: a study of

polychaete feeding guilds. Oceanogr. Mar. Biol. Ann. Rev 17: 193-

284.

Harvell, C. D., and M. I.aBarbera. 1985. Flexibility: a mechanism for

control of local velocities in hydroid colonies. Biol. Bull 168: 312-

320.
Hoerner, S. F. 1965. Fluid-Dynamic Drag Hoerner Fluid Dynamics,

Bncktown, NJ.
Johnson, A. S. 1992. Morphology, mechanics and behavior in feeding

with stretchy worm tentacles. Am. Zooi 32: 1 16A.
Koehl, M. A. R. 1977. Effects of sea anemones on the flow forces they

encounter. / E.\p Biol. 69: 87-105.
Koehl, M. A. R. 1984. How do benthic organism withstand moving

water? Am Zoo/. 24: 57-70.
Price, I,. H., and J. Hylleberg. 1982. Algal-faunal interactions in a

mat of Viva tenesirata in False Bay, Washington. Ophelia 21: 75-

88.

Schmidt-Neilsen, K. 1983. Animal Physiology. Adaptation and Envi-
ronment (3rd ed.). Cambndge University Press. Cambridge. 619 pp.
Steinman, D. B. 1942. Suspension bridges. Pp. 289-358 in Movable

and Long-span Steel Bridges. G. A. Hool and W. S. Kinne, eds.

McGraw-Hill, New York.
Vincent, J. 1990. Structural Biomaterials. Princeton University Press,

Princeton. NJ. 244 pp.
Vogel, S. 1981. Life in Moving Fluids. Willard Grant Press, Boston,

MA. 352 pp.
Vogel, S. 1984. Drag and flexibility in sessile organisms. Am. Zool. 24:

37-44.
Vogel, S., and M. LaBarbera. 1978. Simple flow tanks for research

and teaching. BioScience 28: 638-643.
Wainwright, S. A., W. D. Biggs, J. D. Currey, and J. W. Gosline.

1976. Mechanical Design in Organisms. Princeton University Press,

Princeton. 423 pp.
Wainwright, S. A., and J. R. Dillon. 1969. On the orientation of sea

fans. Biol. Bull 136: 130-139.

Reference: Biol. Bull 185: 20-27. (August, 1993)

Flow Velocity Induces a Switch From Active to Passive

Suspension Feeding in the Porcelain Crab

Petrolisthes leptocheles (Heller)

GEOFF TRACER 1 * AND AMATZIA GENIN 2

1 Bar-Han University, Ramat-Gan, Israel, and -The H. Steiniti Marine Biology Laboratory.

The Interuniversity of Eilat. Israel

Abstract. A flow-induced switch in suspension-feeding
behavior of the porcelain crab Petrolisthes leptocheles was
investigated in a laboratory flow tank. Crabs were exposed
to two types of experimental water flow to stimulate them
to switch from active to passive suspension feeding. In
the first experiment, feeding crabs were exposed to a uni-
directional accelerating water current, and they switched
from active to passive suspension feeding at a mean water
velocity of 3.49 cm s '. In the second experiment, crabs
were exposed to flow that was fixed at a constant velocity
for at least 10 min, and their feeding behavior in this
steady flow was observed. This procedure was repeated,
using a range of constant- velocity flows that were succes-
sively adjusted to increased velocity levels. Crabs exposed
to these different constant-velocity flows fed exclusively
actively at flows below 1 .5 cm s" ' and exclusively passively
at those above 4.5 cm s" 1 . Switches from active to passive
feeding occurred throughout the range of constant-velocity
flows from 1.5 to 4.5 cm s" 1 .

Changes in feeding activity rate induced by an increase
in water velocity were measured. The mean activity rate
of active feeding ( 1 .05 Hz) was 3.4 times higher than that
of passive feeding (0.31 Hz).

The porcelain crab's ability to switch feeding modes in
response to increased water velocity probably enhances
energetic feeding efficiency in two ways. First, the passive
feeding activity rate is lower than the active one and should
reduce energetic expenditure. Additionally, the flux of

Received 6 October 1992; accepted 6 May 1993.

* Author to whom repnnt requests should be addressed. Correspon-
dence address: Dr. Geoffrey C. Trager, The H. Steinitz Marine Biology
Laboratory, The Interuniversity of Eilat, P.O. Box 469, Eilat 88103,
Israel.

suspended food increases with water flow velocity, so pas-
sive feeders are likely to catch more food per unit time
than active feeders do. The ability to switch feeding modes
is quite similar to that already described for balanomorph
barnacles and appears to represent convergent evolution
of flexible feeding behavior in response to variable water
flow environment.

Introduction

Many benthic marine invertebrates can switch their
mode of feeding in response to changes in environmental
conditions. For example, in the grapsid crab Gaetice de-
pressus. Depledge (1989) observed several feeding modes
including scavenging, predation, deposit feeding, and sus-
pension feeding, noting that the particular mode observed
depended on the availability of different food types.
Turner and Miller (1991) showed that a sufficient water
movement would induce deposit-feeding chaetopterid
polychaetes to extend their tentacles into the current and
switch to suspension feeding. Okamura (1987) inferred a
switch from ciliary to tentacular suspension feeding that
was induced in bryozoans by particle size and flow ve-
locity. According to foraging theory, such behavioral
switches tend to enhance feeding efficiency (as measured
in some energy currency, such as net energy gained per
unit time, or the ratio of energy gained to energy spent)
and thus increase fitness (Schoener, 1971; Pyke, 1984).

In aquatic habitats suspension feeding is a widespread
mechanism for obtaining energy and materials from the
water column, and representative suspension feeders are
found in most major animal phyla (Jorgensen, 1966).
Suspension feeders can be generally categorized as either
active or passive. Active feeders spend their own metabolic
energy to pump water past feeding structures, whereas

20

FLOW AND PORCELAIN CRAB FEEDING

21

passive feeders rely on the external source of energy in
ambient currents for delivery of food particles to feeding
structures (LaBarhera, 1984). Some animals, such as bar-
nacles and tunicates, can suspension feed both actively
and passively (LaBarbera. 1977).

The passive feeding mode in porcelain crabs has re-
ceived only brief mention in the literature (e.g.. Wicksten,
1973; Kropp, 1981), probably because the velocities of
incidental flow induced by aeration devices are typically
too low to produce active feeding in laboratory aquaria.

Here, we quantitatively describe a flow-induced switch
from active to passive suspension feeding in the porcelain
crab Petrolisthes leptocheles. A behavior indexing tech-
nique was employed to provide both qualitative infor-
mation on feeding behavior components and quantitative
data on feeding activity rates. These data were amenable
to standard time-series analysis (fast Fourier transforms)
for quantitative differentiation of active and passive feed-
ing behaviors on the basis of differences in their activity
rates (frequencies). Our results supplement the list of spe-
cies that are known to switch from active to passive sus-
pension feeding when water currents are sufficiently high.

Materials and Methods

P. leptocheles was collected in the Gulf of Eilat, Red
Sea, Israel, from under stones in the shallow subtidal (to
0.5 m depth) off the H. Steinitz Marine Biology Labora-
tory, the Interuniversity of Eilat, Israel. Specimens were
kept in unfiltered. flowing seawater. Experiments were
carried out at the Laboratory from January through Feb-
ruary 1992 and in February 1993. Throughout all experi-
ments, the water temperature was 25-26C.

To control water flow precisely, a flow-pattern generator
was used. It consisted of a recirculating flow tank (Vogel
and LaBarbera, 1978) modified by the addition of com-
puter-controlled water flow. Water movement was pro-
duced by computer programs that were interfaced,
through a digital-to-analog circuit, with a motor-driven
propeller immersed in the flow tank (Fig. la). To record
behavior and water flow simultaneously, laser optics
(Strickler, 1985), fiber optic lamps, close-up lenses, and
video equipment were used (Fig. Ib). This video-optical
system allowed close-up observation (image magnification
from 10 to 30X) of animal motions, as well as visualization
of water flow as indicated by the movements of brightly
illuminated particles naturally suspended in unfiltered
seawater. For videorecording during experiments, all an-
imals were placed on a gravel-filled petri dish which was
then positioned on the floor of the flow tank so that the
anteroposterior axis of the crab was perpendicular to the
axis of flow direction while its mouth faced towards the
camera.

Two computer programs were used to produce two
types of experimental water flow change. In the first ex-

periment, water was continually accelerated
a suspension feeding crab at a rate of 0.15 cm s
program that produced the repeating pattern of alternating
accelerating and decelerating flows shown in Figure 2.
The suspension-feeding behavior of one crab at a time
was videorecorded during one acceleration period. During
the following deceleration period, the crab was removed
from the flow tank, and the flow tank motor was shut off.
Another crab was placed in the tank, and when feeding
began, the motor was turned on to expose the crab to the
same accelerating flow stimulus that the previously tested
crab experienced. This protocol was repeated until 19
crabs had been tested. The computer program assured
that the water flow acceleration stimulus was the same
for each crab.

In the second experiment, a different computer program
was used to maintain a constant water flow velocity (ac-
celeration = 0) for an extended period. Here, a crab was
placed in the flow tank, first in nearly still water (less than
2 mm s" 1 , flow tank motor off), and when feeding began,
its behavior was videorecorded for 10 min. Then the crab
was removed, the motor was turned on with the computer
program at a fixed setting, and flow in the tank was allowed
to stabilize (for 1 min) at a steady flow velocity of 0.5 cm
s" 1 . The crab was then put back into the tank, and when
feeding began, its behavior was videorecorded for another
10 min with flow constantly maintained at 0.5 cm s"'
(i.e.. no change in velocity) throughout the recording pe-
riod. This procedure was repeated with the same crab, at
successively increased, constant velocity flows (1.0, 1.5.
2.5, 3.5,4.5, 5. 5. and 6. 5 cm s '). Each velocity was main-
tained for 10 min while feeding behavior was videore-
corded. The entire protocol was repeated until 1 1 crabs
had been tested.

Flow velocities local to the animal (from 2 to 6 mm
above the distal edges of extended feeding fans) were
measured by frame-by-frame tracking of the movements
of back-lit particles suspended in flowing water as de-
scribed by Trager et al. (1990).

Time-series data on feeding-motion cycles were ob-
tained by playing back the videorecordings. Components
of cyclical feeding behavior were identified, and a nu-
merical value was assigned to each one, producing a be-
havioral index. For example, crab feeding-fan movements
during active suspension feeding consist of three easily
identified components (transfer of food particles from the
fan to the mouth, upward extension of the fan, and a
forward and downward capture stroke) that occur over
and over again in the same sequence. These three com-
ponents were assigned index values 1, 2, and 3, respec-
tively. Behavioral time-series data were taken every
0.04 s (every video frame), over a total period of 81.9 s
(more than 50 behavior cycles), by assigning the appro-
priate index value to the behavioral component observed

22

G. C. TRACER AND A. GENIN

a.

16cm

fiber optics
lamp

video camera

computer control

\ signal \

D/A
circuit

reflected light \^ II

scattered laser

motor

white light ^^

~T
*

^ "" and whitf

flow

specimen

t

*

water
level

white light

\ collimator

-V. -V-M--

propeller

expanded collimated
laser beam (740 nm)

expander-collimator
lens system

laser

Figure 1. Apparatus for controlling water flow, (a) Side view of computer-controlled flume. Computer
programs generate digital signals and send them to the digital-to-analog (D/A) circuit, where they are converted
to analog signals to the 1 2 V DC flume motor. This system allows precise repetition of experimental flow
regimes, (b) Top view of flow tank showing positioning of video camera, lens system, light sources, optical
paths, and specimen. Backlighting was provided, either by a laser in line with the specimen and the video
camera or by a fiber optics lamp at an angle of about 25 to a line through the specimen and video camera.
The dark-field lens system was used only with the laser. The two backlighting sources were not used simul-
taneously. Both techniques provide a similar dark-field image in which naturally suspended particles in
flowing seawater appear as bright points of light against a dark background, for flow visualization. Frontlighting
by fiber optics was also provided for bright-field illumination of moving feeding appendages.

in each consecutive video frame. Plotting the indexed be-
havioral data against time produces a wave form, or cy-
clical curve, that illustrates the changes in feeding com-

~ 6

to

5
8s

en

% '
lot

\

- <

50 100 150 200

TIME (s)

Figure 2. Repeating pattern of alternating accelerating and deceler-
ating flows producing replicable, linear, water flow acceleration inducing
porcelain crabs to switch from active to passive feeding. Feeding behavior
was monitored only while flow accelerated. Flow deceleration simply
reset the water velocity back to its original starting level, so that the next
animal tested could be exposed to the same flow acceleration stimulus
that the previously tested crab experienced. This flow pattern was gen-
erated by a triangle-wave signal from a computer program. Each point
is the mean velocity ot 20 suspended particles tracked from 0.5 s before
to 0.5 seconds after the time indicated on the abscissa. Vertical bars show
standard errors.

ponents and activity rhythms that concur with changes
in flow velocity. The cyclically fluctuating numerical data
produced by behavioral indexing are also suitable for
analysis with fast Fourier transforms in the frequency do-
main, so that the distinct activity rates (frequencies of
cyclical feeding-fan motions) distinguishing active versus
passive feeding could be identified.

Results

Although active and passive feeding were easy to dis-
tinguish on the basis of activity rate and orientation of
feeding structures, there was a period of transition during
the acceleration experiments when, as water was gradually
accelerating, the crabs exhibited intermediate feeding be-
haviors with characteristics of both modes. These flow-
induced behavioral changes, from active to intermediate
to passive suspension feeding, are described below.

In very calm water (<\ cm s~'), the crabs always sus-
pension feed actively, by simultaneously extending both
of their setose third maxillipeds to form two extensive
spoon-shaped feeding fans (Fig. 3a). Both fully extended
fans are then flexed, sweeping rapidly forward, downward,
and laterally toward the mouth through a volume of water

FLOW AND PORCELAIN CRAB FEEDING

23

ACTIVE SUSPENSION FEEDING (1.05 Hz)

rmx Imx

U.

extension

flexion

PASSIVE SUSPENSION FEEDING (0.31 Hz)
C . rmx

flow

stationary holding in current

Figure 3. Diagram of porcelain crah (front view) carrying out active
suspension feeding, which occurs only in slow ambient flow (on average,
at flow velocities less than about 3.5 cm s~'), and passive suspension
feeding, which occurs only in fast flow (on average, at flow velocities
greater than about 3.5 cm s~'). (a) During active feeding, the third max-
illipeds are spread laterally and then unflexed so that the setae spread
out to form a spoon-shaped fan. Abbreviations: ca. carapace; ey, eye;
Imx, left third maxilliped; rmx, right third maxilliped. (b) The next com-
ponent of active feeding consists of the fan being flexed and sweeping
forward, downward, and laterally towards the mouth for removal and
transfer of trapped food particles to the mouth, (c) During passive feeding,
both feeding fans are held stationary with the concave sides facing into
the current. Arrow indicates water flow direction.

(Fig. 3b). Next, the fans contact the second maxillipeds,
which remove trapped food particles that are passed to
the mouth. This entire sequence is performed repeatedly
for as long as active feeding lasts. This feeding behavior
is similar to that described by Nicole (1932) for the por-
celain crab Porcellana longicomis, except that P. longi-
cornis usually sweeps its left and right fans alternately,
whereas in this study, P. leptocheles always swept both
fans simultaneously.

When the accelerating water reaches a velocity of about
1.5 to 2 cm s ', intermediate behaviors begin with char-
acteristics of both active and passive feeding. The crabs
still sweep both fans rapidly and rhythmically, but begin
to angle the concave side of the left fan towards the current
so that the capture stroke begins in the upstream direction.
This is not possible for the right fan because the structure
of the third maxillipeds does not permit a sweep (flexion)
laterally away from the animal (see Fig. 3). Thus, the right
fan, unlike the left fan, continued to repeatedly extend
and flex, as in calmer water.

As the water continues to accelerate, the frequency of
the cvclical motions for both fans decreases because a

new behavioral component appears. Immediately after
fan extension, there is a pause, during which the fan is
held fully extended and stationary, with the concave side
facing upstream, positioned for passive suspension feeding
in the current (Fig. 3c). The duration of this stationary
fan-holding behavior steadily increases, up to a point, as
the water velocity continues to increase, and the sweep
into the current of the left fan is quickly phased out, so
that stationary holding is followed by a twisting, forward,
downward, and proximolateral flexion similar to that of
active feeding. Extension of the fans into the current,
holding in a stationary concave-upstream position for a
time, and then flexion for particle removal is the typical
cycle of passive feeding behavior.

In a few cases, the water was accidentally accelerated
very rapidly (e.g., around 10 cm s~ : ) when the flow system
was turned on with the motor already at a high-speed
setting. In these cases, the crabs switched from active to
passive feeding almost instantaneously, with no evident
intermediate behavior. But in the controlled experimental
flow that was gradually accelerated (at a rate of 0.15 cm
s -), there was always a period of intermediate behavior.
Thus, switch velocity, which is denned here as the ambient
water velocity in accelerating flow at which a suspension-
feeding animal switches from active to passive feeding,
was determined as the point at which the stationary hold-
ing component of passive feeding (which is the primary
distinguishing characteristic of the passive feeding mode)
is longer than 1 s.

The flow-induced behavioral changes from active to
intermediate to passive suspension feeding are depicted
graphically in an indexed-behavior time-series plot (Fig.
4) that displays both qualitative and quantitative (fre-
quency) information.

The mean switch velocity for all crabs tested in accel-
erating flow was 3.49 cm s ' (Fig. 5). The mean cycle
frequencies of active (1.03 Hz) and passive (0.31 Hz)
feeding motions are compared in Table I. The passive
feeding rate was much more variable than the active feed-
ing rate, and the mean activity rate of actively feeding
crabs was 3.4 times greater than that of passively feeding
crabs.

In the constant-velocity experiments, all 1 1 crabs tested
fed exclusively in the active mode at velocities less than
1 .5 cm s^ ' and exclusively in the passive mode at velocities
greater than 3.5 cm s~'. Switches from active to passive
feeding occurred from 1.5 to 3.5 cm s"' (Fig. 6). In Figure
6, a crab was considered to have switched to passive feed-
ing if it was observed to perform any passive feeding at
all during a 10-min period. Five crabs switched in the
middle of an observation period, and two of these switched
back and forth several times during that period. For these
five crabs that performed both active and passive feeding
during an observation period, the mean percent of time

24

G. C. TRACER AND A. GENIN

a.

(/)
E

8

si

> o

b - hold left s

sweep left 4

fan down 3

fan up 2

clean off 1

c.

hold left s

4

fan down 3

fan up 2

clean off 1

10 20 30 40 50

TIME (sec)

Figure 4. Behavioral change from active to intermediate to passive
suspension feeding induced by linearly accelerated unidirectional water
flow, (a) Linear change in flow velocity inducing the behavioral changes
depicted graphically in (b) and (c), which show numerically indexed
components of cyclical feeding behavior plotted against time. Each be-
havioral component and its assigned index value is listed on the ordinate,
and the occurrence of any component is indicated by a peak or plateau
in the curve at the appropriate level. The length of each horizontal portion
of the indexed behavior curve indicates the duration (seconds) of a be-
havioral component. Steeply inclined rises or drops of the curve show
when, from one video frame to the next, a behavioral component has
changed. Thus, changes in activity rate (frequency) are indicated by
changes in the horizontal distance between curve peaks, and changes in
behavioral components are seen as changes in the height of the curve
peaks, (b) Indexed behavior time series for the left feeding fan. Active
feeding is rapid (0.95 Hz), occurs in slowly moving water, and consists
of only 3 components (clean off, fan up, and fan down). The beginning
of intermediate behavior, with characteristics of both active and passive
feeding, first appears at a velocity of about 1 .5 cm s"', when the behavior
curve suddenly jumps up to an index value of 4 (sweep left). The time
and velocity at which this new component appears is indicated by (I) on
the velocity curve. The activity rate also slows down somewhat during
this transition period, with the appearance of the "hold left" component
[stationary holding of the fan into the current (index value 5); its first
appearance is indicated by II on the velocity curve]. Strict passive feeding
begins at a velocity of about 3.2 cm s" 1 (see III), when the length of the
hold left component is greater than 1 s. (c) The right fan shows changes
in activity rate that are very similar to those of the left fan, but there is
no sweep left (index value 4) component. The activity rate of the right
Ian during passive feeding is also considerably lower than that of strictly
active feeding.

spent in passive feeding was 40.2% (SD = 13.2). The re-
maining six crabs fed only actively during the entire
10-min observation periods at low velocities, and then

rr

O
UL

O
rr

UJ

m

X = 3.49 cm s
n = 19 crabs

1 15 2 25 3 35 4 45 5

SWITCH VELOCITY (cm s 1 )

Figure 5. Switch velocities (i.e.. the ambient water velocities at which
crabs switched from active to passive feeding) in unidirectional accelerated
flow. Frequency histogram of the number of porcelain crabs in different
switch-velocity classes. Each class is the median water velocity indicated
on the .v axis 0.25 cm s" 1 . Water velocities at the behavioral switch
point were measured by calculating the mean velocity of 20 suspended
particles video-tracked from 0.5 s before, to 0.5 s after the switch. A
switch-velocity value for an individual crab is the average of the left and
right fan values.

switched to feeding entirely in the passive mode through-
out the observation periods at higher velocities.

Because the flow-induced changes exhibited by all crabs
were similar, only two characteristic time series (2048 data
points for each feeding fan of one animal) were chosen
from the flow acceleration experiments for spectral anal-
ysis. Fast Fourier transforms of indexed behavior time
series (portions of which are plotted in Fig. 4b and c)
show that active and passive feeding behavior can be dis-
tinguished by their respective frequencies (Fig. 7). The
power spectrum curve in Figure 7, produced by plotting
the results of the fast Fourier transforms, indicates the
relative importance of different component frequencies

Table I

Activity rates measured for active and passive suspension feeding
in 1 9 porcelain crabs

Mean cycle

Variability

Feeding
mode

frequency
(Hz)

Within individuals

Between individuals

Active
Passive

1.05
0.31

6.4

27.0

12.5
19.4

* Calculated by measuring length (in seconds) of 10 consecutive cycles
of active and passive feeding that were recorded on videotape.

FLOW AND PORCELAIN CRAB FEEDING

25

100 -

0.5 1.0 1.5 2.5 3.5 4.5 5.5 6.5

FLOW VELOCITY INCREMENTS

Figure 6. Percentage of crabs feeding passively over a range of con-
stant velocity flows. Each constant flow velocity indicated on the x axis
was maintained for 10 min. All crabs fed only actively from to 1.0 cm
s~'. An increasing percentage of crabs switched from active to passive
feeding over the range of 1.5 to 4.5 cm s~'. At flows of 4.5 cm s~' and
above, all crabs fed exclusively in the passive mode.

of feeding-fan movements. Thus, a distinct peak on the
power spectrum curve indicates a dominant activity rate,
or frequency (on the A axis directly below the peak), that
characterizes a distinct feeding mode. For both the left
and right fans, two dominant power spectrum peaks rep-
resent the distinct activity rates of active and passive feed-
ing. Several peaks between the active and passive peaks
represent intermediate frequency behaviors that occurred
during the transition from active to passive feeding.

Discussion

Not all suspension feeders are able to switch between
active and passive modes. The switch may increase feeding
efficiency in at least several ways. First, passively feeding
crabs are able to depend on an external ambient current
to deliver suspended food particles to feeding structures;
they therefore do not spend their own metabolic energy
to pump water past food-capturing structures. They are
also able to orient their feeding appendages optimally with
respect to current direction. Baumiller (1988) demon-
strated that fluid flux through a concave model filter (a
concave-shaped mesh) oriented perpendicular to the flow
with the concave side facing upstream was greater than
fluid flux through a similarly positioned planar mesh.
Spielman and Goren (1968) showed that particle capture
efficiency depends on the orientation of filter fibers. These
facts together offer an explanation for the consistent pas-
sive-feeding orientation of crab feeding fans perpendicular
to flow direction, with the concave side facing upstream
(see Fig. 3c).

Particle capture rates are also predicted to be higher
during passive feeding than during active feeding because
passive feeding occurs only in relatively high-velocity flow,
when fluid flux and thus food flux to capture struc-
tures should be greater than in calm water in which ac-
tive feeding occurs.

Passive feeding is likely to be more efficient than active
feeding for another reason. The proportion of total feeding
time during which a fan is collecting particles is much
greater for a passive feeder than an active feeder. The
proportion of total feeding activity time that a fan was

a.

o

C 200
CD

<T

"~ 150

o

C 100

<r

LU

passive (0.36 Hz)
', intermediate (0.4 to 0.83 Hz)

active (1.0 Hz)

b.

1 2

passive (0.30 Hz)
250 |- intermediate (0 29 to 0.9 Hz)

0)
3
CT

CD

*= 150

X

03
T3

DC

LU

O

D_

FREQUENCY (Hz)

Figure 7. Active and passive suspension feeding are distinguished
by their respective activity rates, or frequencies: (a) right fan; (b) left fan.
Plotting the results of fast Founer transforms ( Press el a/.. 1 986) of indexed
time-series data on feeding motion cycles (portions of which are plotted
in Figure 4b and 4c) yields two power spectra that indicate the relative
importance of the different frequency components of active, intermediate,
and passive feeding behavior. In both the right and left fan plots, two
distinct power spectrum peaks represent the distinct frequencies that
characterize active and passive feeding. Several peaks between the active
and passive peaks represent intermediate-frequency transition behaviors
with characteristics of both active and passive feeding.

26

G. C. TRACER AND A. GENIN

held stationary into the current during passive feeding
was 87% (the other 13% of time was occupied by fan flex-
ion, clean off, and extension). During active feeding,
however, the fan is trapping particles primarily when a
spread-open fan is swept through the water, and this sweep
occupied only about 20% of feeding activity time (the
other 80% was occupied by folding setae, cleaning them
off, and extending the fan for the next sweep). Thus, the
ratio of food catching time to food handling time (a re-
flection of energetic feeding efficiency) is greater for a pas-
sive feeder than an active feeder.

Active feeding should be more efficient than passive
feeding only when ambient flow is so low that the bulk
of the water, and thus the suspended food particles, will
move around rather than through a stationary extended
fan. This effect has been demonstrated for the barnacle
Semibalanus balanoides by Trager et al. (1990), who
showed that a dye stream aimed slightly (2 mm) above
the geometrical center of an extended barnacle feeding
fan flowed between the filter elements near the fan's center
at an ambient flow speed greater than 3.5 cm s ', but was
diverted completely around the entire fan at a lower am-
bient flow speed of 0.5 cm s '. This effect is also predicted
by the "leakiness" model of Cheer and Koehl (1987) which
describes, as a function of flow speed, bristle spacing, and
bristle diameter, how much fluid leaks between the ele-
ments of bristled appendages of small organisms, as op-
posed to how much goes around the entire appendage.
When passive feeding becomes less efficient than active
feeding (at some ambient flow velocity that can be pre-
dicted to be less than, but near, the crab's mean switch
velocity of 3.49 cm s '), a crab must create the relative
flow velocity past the filter needed to increase leakiness
by actively sweeping its fan rapidly through the water.
Therefore, it is the effect of flow velocity on fluid flux (and
thus suspended food flux) to the filter that appears to de-
termine the ambient flow velocity at which an animal
switches from active to passive feeding.

The factors responsible for the large difference in ac-
tivity rate between active and passive suspension feeding
of porcelain crabs remain obscure. For effective active
feeding, the animal must sweep its fans rapidly to pump
new water within reach of feeding structures. Thus, a fan
not only needs some degree of leakiness for particle cap-
ture, but also must have some "non-leakiness" so it can
act as a paddle that pushes water past the animal. There
are physical and energetic limits on how fast the animal
can pump water. Even if these limits are not reached,
pumping too fast may result in inefficient processing of
the water moving past the animal. This inefficiency might
be due not only to the increase in energy cost at a high
pumping rate but also to the reduction in particle retention
as drag forces increase and allow particles that contact
the filter to be carried away by the current. The active

feeding activity rate may represent a trade-off between
maximizing flow rate (and thus food flux to the vicinity
of the filter) and maximizing food particle capture rate.

The passive feeding activity rate is determined primarily
by the length of time a feeding fan is held stationary into
the current. In the passive mode, a feeding fan can capture
particles without rapid sweeping if it is simply held in
position (concave-upstream) for extended periods. Thus,
muscular activity rate (and presumably metabolic energy
expenditure) is considerably reduced by switching from
active to passive feeding when ambient water currents are
strong enough.

For the porcelain crab P. leptocheles and the barnacle
S. balanoides, both of which inhabit wave-swept environ-
ments, flow velocities are commonly well above the switch
velocities of around 3 to 3.5 cm s" 1 measured in the lab-
oratory. In this study, field measurements taken every
0.5 s over 24 hours with an S4 Interocean current meter,
in shallow water (0.5 m) in the crabs' habitat in the Gulf
of Eilat, gave a mean flow speed of 15.5 cm s" 1 (SD = 8.2,
maximum = 50.5 cm s" 1 ). Moreover, when first taken
into captivity, both species began to feed passively if cur-
rents were slightly more than 3 cm s ', but did not feed
at all for 1 or 2 days if kept in calm water (<1 cm s" 1 ),
then began to feed actively. This suggests that the animals
are accustomed to feeding mainly in the passive mode,
and a period of starvation is required to induce feeding
in the presumably more energy-demanding active mode.
In nature, passive feeding may be the dominant mode for
these animals because currents are usually sufficiently
high. Active feeding in the natural habitat probably occurs
only in restricted flow situations, such as tide pools at low
tide or very quiet bays and estuaries when tidal currents
are weak or absent. In the laboratory, active feeding has
appeared to be the dominant mode only because flow
velocities in typical observation aquaria are usually lower
than the behavioral active-to-passive switch threshold.

Porcelain crabs and balanomorph barnacles demon-
strate striking similarities in suspension-feeding structures
and behavior. Although the animals are from different
major crustacean taxa (Malacostraca and Cirripedia, re-
spectively; Bowman and Abele, 1982), both possess
moveable, scoop-shaped, setal feeding nets that can be
oriented concave-upstream to current direction. Both are
capable of tracking oscillating flow with high precision
(Trager et al., 1992), and both can switch from active to
passive feeding in the presence of sufficiently rapid water
currents. This suggests that other benthic crustacean spe-
cies with jointed moveable suspension-feeding apparatus
(e.g., Hippid sand crabs and suspension-feeding hermit
crabs) may also exhibit comparable convergent structural
and behavioral adaptations for suspension feeding in
variable flow regimes.

FLOW AND PORCELAIN CRAB FEEDING

27

Acknowledgments

We thank David Coughlin for writing the computer
programs used to control water flow. Programs for per-
forming fast Fourier transforms were written by Emanuel
Boz. A helium-neon laser was provided by J. R. Strickler.
We also thank Yair Achituv and the staff" at the Bar-Ilan
University mechanics and physics workshop for con-
structing the flow tank and the digital-to-analog electronic
circuit. This work was supported by a Wolfson Founda-
tion grant administrated by the Israel Academy of Sciences
to A. Genin. and a Nussbaum fellowship to G. Trager,
through Bar-Ilan University. Ramat-Gan. Israel.

Literature Cited

Baumiller. T. K. 1988. Effects of filler porosity and shape on fluid flux:

Implications for the biology and evolutionary history of stalked cri-

noids. Pp. 786 in Proc. 6tli Int. Echinoderm Biol. Conf.. R. D. Burke,

P. Mladenov. P. Lambert and R. L. Parsley, eds. Victoria, British

Columbia.
Bowman. I. E., and L. G. Abele. 1982. Classification of the recent

Crustacea. Pp. 1-27 in The Biology of Crustacea, Vol. 1. L. G. Abele.

Academic Press. New York.
Cheer, A. Y. L., and M. A. R. Koehl. 1987. Paddles and rakes: Fluid

flow through bristled appendages of small organisms. / Theor. Biol.

129: 17-39.
Depledge, 1). H. 1989. Observations on the feeding behavior ofGaetice

depressus (Grapsidae: Varuninae) with special reference to suspension

feeding. Mar Biol. 100: 253-259.
Jorgensen. C. B. 1966. Biology of Suspension Feeding. Pergamon Press,

Oxford. 357 pp.
Kropp, R. K. 1981. Additional porcelain crab feeding methods (De-

capoda. Porcellanidae). C'rusiaccana 40: 307-310.

I.aBarbera, M. 1977. Brachiopod orientation to water movement. I.

Theory, laboratory behavior, and field orientations. Paleobiologv 3:

270-287.
LaBarbera, M. 1984. Feeding currents and particle capture mechanisms

in suspension feeding animals. Amer. /.no/. 24: 71-84.
Nicol, E. A. T. 1932. The feeding habits of the Galatheidae. J Mar.

Binl. Assoc. L'.K. 18: 87-106.
Okamura. B. 1987. Particle size and flow velocity induce an inferred

switch in bryozoan suspension-feeding behavior. Biol. Bull. 173: 222-

229.
Press, VV. H., B. P. Flannery, S. A. Teukolsky, and VV. T. Vetterling.

1986. Numerical Recipes. Cambridge University Press. Cambridge.

818 pp.
Pyke, G. H. 1984. Optimal foraging theory: a critical review. Annu.

Rev Ecol. Sysl 15: 523-575.
Schoener, T. W. 1971. Theory of feeding strategies. Annu. Rev. Ecol.

Syst. 2: 369-404.
Spielman, L., and S. L. Goren. 1968. Model for predicting pressure

drop and filtration efficiency in fibrous media. Environ. Sci. Technol

2: 279-287.
Strickler, J. R. 1985. Feeding currents in calanoid copepods: two new

hypotheses. Symp. Soc. Exp. Biol. 39: 459-485.
Trager, G. C., D. Coughlin, A. Genin, Y. Achituv, and A. Gangopadhyay.

1992. Foraging to the rhythm of ocean waves: Porcelain crabs and

barnacles synchronize feeding motions with flow oscillations. J. Mar

Biol. Ecol. 164: 73-86.

Trager, G. C., J.-S. Hwang, and J. R. Strickler. 1990. Barnacle sus-
pension feeding in variable flow. Mar Biol. 105: 1 17-127.
Turner, E. J.. and E. C. Miller. 1991. Behavior of a passive suspension

feeder (Phiochaetopterus oculatus (Webster)) under oscillatory flow.

J. Exp Mar Biol. Ecol. 149: 123-137.
Vogel, S., and M. LaBarbera. 1978. Simple flow tanks for teaching

and research. Bioscience 28: 638-643.
VVicksten, M. K. 1973. Feeding in the porcelain crab. Pelrolisthes cinc-

lipes. ( Randall MAnomura: Porcellanidae). Bull. So. California Acad.

Sci. 72: 161-162.

Reference: Biol. Bull 185: 28-41. (August, 1993)

Effects of Flow Speed on Growth of Benthic
Suspension Feeders

JAMES E. ECKMAN 1 AND DAVID O. DUGGINS 2

1 Skidaway Institute of Oceanography, P.O. Box 13687. Savannah, Georgia 31416, and -Friday
Harbor Laboratories. 620 University Rd., Friday Harbor, Washington 98250

Abstract. In separate experiments in 1991 and 1992,
the sensitivities of growth rates of six species of benthic
suspension feeder to flow speed were tested in a series of
turbulent pipe flows. Species examined were the cheilo-
stome bryozoan Membranipora membranacea ( 1 99 1 ); the
serpulid polychaete Pseudochitinopoma occidentalis
(1991); and the barnacles Balanus glandula (1991),
B. crenatus (1992), Semibalanus cariosus (1992), and
Pollicipes polymerus (1992), In both experiments, animals
were exposed to one of five constant, narrow ranges of
speed that varied from about 2-15 cm s~'. Growth rates
of Membranipora and Pseudochitinopoma in 1991 de-
clined significantly and monotonically with increasing
flow speed, despite evidence that at faster flows there were
greater concentrations of suspended food available and
higher paniculate fluxes. In contrast, there was no de-
tectable relationship between speed and growth of
B. glandula over the same range of flow speeds in 1991.
Results of the 1992 experiment indicated variability in
growth responses among three species of barnacle. Growth
rates of S. cariosus and P. polymerus were insensitive to
flow speed, whereas growth rates of B. crenatus increased
from low speeds to a maximum at an intermediate speed
of about 8 cm s" 1 , and then tended to decrease at higher
speeds. Combined results of the two experiments indicate
that the growth response of animals to flow was most
obviously related to the relative flow energy of the animal's
natural habitat. Growth rates of animals that typi-
cally experience relatively weak flows (P. occidentalis,
M. membranacea, and B. crenatus) were affected signifi-
cantly by flow speed, whereas growth rates of animals
from comparatively high-energy environments (B. glan-

Received 13 November 1992; accepted 3 June 1993.

dula, S. cariosus, and P. polymerus) were relatively in-
sensitive to flow speed. In contrast, animal morphology
and behavior were not obviously related to growth re-
sponses to flow. A key to understanding the impact of
flow on a suspension-feeder's growth may be the animal's
ability to handle and process particles that have impacted
the feeding apparatus and to deliver them to the point of
ingestion.

Introduction

One clear trend that has emerged from many studies
of effects of flow speed on both passive and active sus-
pension feeders is that rates of particle capture or growth
are lower on both sides of a narrow range of intermediate
speeds that appear to be most beneficial. This pattern has
been demonstrated for growth of a scallop (Kirby-Smith,
1972) and particle capture by an alcyonacean coral
(McFadden, 1986), a sea pen (Best, 1988), a crinoid
(Leonard el a/., 1988), and a gorgonian coral (Sponaugle
and LaBarbera, 1 99 1 ). There are several causes for this
trend. As flow speed increases from near-zero levels, rates
of particle encounter with passively deployed filter ele-
ments increase (Shimeta and Jumars, 199 1 ), and increased
turbulent mixing makes depletion of food particles within
the animal's feeding ambit less likely (Wildish and Krist-
manson, 1979; Patterson, 1984; Frechette and Bourget,
1985; Frechette et ai. 1989). At relatively high flow speeds,
deformation of filtering structures may reduce the total
surface area available for particle capture (Patterson, 1984;
Harvell and LaBarbera, 1985; Best, 1988; Shimeta and
Jumars, 1991 ), particles impacting filter elements may be
handled or processed with greatly reduced efficiency due
to drag effects (Patterson, 1991; Shimeta and Jumars,
1991 ), or adverse pressure gradients may inhibit process-

28

FLOW AND SUSPENSION-FEEDER GROWTH

29

ing of water containing suspended paniculate food (Wil-
dish iY /.. 1987).

The trend of maximum particle capture or growth at
intermediate flow speeds has not been exhibited in all
studies that have addressed the relationship, however. By
restricting study to a comparatively narrow range of flow
speed on either side of the intermediate speed of maxi-
mum growth (nearly always unknown a priori), a simpler
direct or inverse relationship between speed and particle
capture or growth would appear. Thus, a monotonic in-
crease in particle capture with flow speed was noted for
a coral (Sebens and Johnson, 199 1 ), but inverse relation-
ships were reported between flow speed and scallop growth
(Wildish ct ul.. 1987; Eckman el a/.. 1989; Wildish and
Saulnier, 1992), barnacle growth (Smith. 1946), and par-
ticle capture or growth by bryozoans (Okamura, 1984.
1985. 1992).

Despite the attention devoted to this topic of research,
some important gaps remain in our understanding of the
influence of flow on growth of suspension feeders. First,
because prior studies have focused on individual species,
it is not yet clear how different species, which have dif-
ferent behaviors and employ different mechanisms of sus-
pension feeding, will respond to the same range of
conditions. This knowledge would be useful in determin-
ing how assemblages of suspension feeders might become
partitioned in space according to flow microhabitat, or in
predicting the relative competitive success among species
that exhibit differential sensitivities to flow. The relative
competitive abilities of suspension-feeding species may
well vary among sites depending on their flow energies.
Second, for tentaculate suspension feeders (e.g., hydro-
zoans, bryozoans. gorgonians), there is little evidence that
the relationship between flow speed and particle capture
(the dependent variable typically evaluated for this diverse
guild) translates into identical effects on growth. This result
may seem logical, but it has been demonstrated only by
Okamura (1992). Moreover, the translation of particle
capture to growth may not always be straightforward. For
example, for two bryozoans there was a change in direc-
tion of the dependence of particle capture rate on flow
speed as food-particle size changed over a comparatively
narrow range (Okamura, 1987). Consequently, for ten-
taculate suspension feeders that enjoy a comparatively
diverse diet, relationships between flow speed and capture
rates of one particle type may not translate faithfully into
growth responses.

We therefore carried out two experiments testing effects
of flow speed on growth rates of several species of sessile,
benthic suspension feeder. Our first experiment, carried
out in 1991, involved the cheilostome bryozoan Mem-
branipora membranacea. the barnacle Balanus glantlula,
and the serpulid polychaete Pseudochitinopoma occiden-

talis. These species all feed via a structure that is extended
into the flow, beyond the calcareous zoecium, test, or tube
that encloses the main body of the colony or animal.
However, the behavior and the mechanism for operation
of the feeding organ vary among these species, which may
affect their relative growth responses to flow. In the first
experiment, only the barnacle grew at rates independent
of flow speed. Based on this result, we conducted a second
experiment in 1992 to determine whether other barnacles
from a wide range of habitats showed growth responses
similar to that observed in B. glandula. This experiment
involved the balanoids B. crenatus and Semibalanus car-
iosus and the pedunculate species Pollicipes polymerus.
Finally, to complement these experiments, we also ex-
amined the feeding behavior of most of these species as
a function of flow speed.

Materials and Methods

Experiments were conducted at the Friday Harbor
Laboratories of the University of Washington. In both
experiments, effects of flow speed on growth were deter-
mined using animals suspended in pipes characterized by
steady, turbulent flows. Relationships describing turbulent
flow through a pipe are well known (e.g., Schlichting,
1979; Vogel, 1981), making it comparatively simple to
establish, monitor, and describe flow velocities to which
animals were exposed.

Experimental apparatus 1 991

Five narrow ranges of flow speed were established
within 10 PVC pipes (two replicates per speed). Each pipe
was 3-m long and either 2.5 cm or 3.8 cm in radius. All
pipes were submerged in a seawater bath within a single
tank that was 3.66-m long, 1.22-m wide, and 1.22-m deep.
Pipes were aligned in parallel in a single row along the
bottom of the tank, and were assigned randomly to dif-
ferent flow speeds. The pipes passed through a water-tight
partition inside the tank. 1.22 m from one end. A dia-
phragm pump continuously supplied the smaller com-
partment of the tank (the head) with fresh seawater from
a depth of 4 m. An overflow pipe in the head tank kept
the seawater at a constant depth. The high rate of discharge
of the pump (about 31s') mixed water in the head tank
thoroughly. An overflow pipe in the other section of the
tank (the tail) kept seawater at a constant, lower depth.
The resulting constant pressure gradient between the head
and tail ends drove steady flows through the pipes. The
rate of flow through each pipe was regulated with an ad-
justable valve located at the discharge end of each pipe.

Six windows (each about 2.5 X 4 cm) were cut in the
wall of each pipe through which were inserted thin strips
of plexiglass containing animals used in growth studies

30

J.E. ECKMAN AND D.O. DUGGINS

machine screw

hose clamp

plexiglass strip

collar

Figure 1. Cross-sectional view of the apparatus used to position sus-
pension feeders within pipe flows. The hose clamp created a nearly wa-
tertight seal between the cut-out section of pipe mounted on the collar
and the main body of the pipe.

(Fig. 1 ). The first window was cut 110 cm downstream of
the pipe's entrance, and the other five windows were
spaced 20-cm apart. The remaining downstream section
of each pipe was used in a larval settlement study that
will be described elsewhere. Each cut-out section of pipe
was glued onto a larger PVC collar that could be clamped
onto the pipe to make a nearly watertight seal. This entire
assembly was removable. A machine screw passed through
a hole in the collar and the section of pipe from which
the window was cut (Fig. 1 ). The screw was used to sus-
pend plexiglass strips containing animals across the di-
ameter of each pipe. Plexiglass strips were 0. 16-cm thick,
spanning 2 cm in the along-stream dimension and either
5 or 7.6 cm across-stream, depending on the pipe's di-
ameter. Strips were mounted level within pipes (i.e.. nor-
mal to gravity) such that all animals were facing down.

The growth experiment ran from 6 June-22 November
1991. During this period discharge rates of the pipes (Q)
were measured on 1 7 separate dates. Pertinent character-
istics of turbulent pipe flows were calculated from pipe
discharge rate and radius (R) using relationships either
given by Schlichting ( 1979) and Vogel (1981) or derived
from equations they presented. The mean flow speed
within each pipe ((/) was

V = Q/(*R 2 )
Pipe Reynolds number (Re) was

Re = U(2R)/v

(1)

(2)

where v is the kinematic viscosity of seawater (0.0 1 crrr
s ' ). The flow speed ( U v ) at any distance r from the pipe's
wall (r = 0) was

where L' max is the maximum flow speed (along the pipe's
center line) and

= 6 + 0.6((Re- 4000)/19,000) (4)

Eq. (4) was derived from Eq. (20.6) and Fig. (20.3) in
Schlichting (1979, p. 599). From Eqs. (3, 4) one can cal-
culate the maximum and minimum (U mn ) speeds to
which feeding organs of animals in any pipe were exposed,
assuming that they were a distance at least 10% of the
radius away from the pipe wall:

f/max= U(\

(5)
(6)

As seawater entered the pipe from the head tank, a
boundary layer developed along the pipe's wall. Eqs. (3-
6) are valid only for a fully developed flow, which existed
at all locations downstream of the distance (A' cq ) required
for the boundary layer to grow to a thickness equal to the
pipe's radius. For a turbulent boundary layer, this distance
was approximately

.V eq = (/?/0.37) 1 - 25 ([/ max /V)

(7)

U y =

\l/

(3)

as derived from Schlichting (1979, his Eq. 21.8).

Table I lists flow properties for each of the 10 pipes in
the experiment, based on the mean of the 1 7 separate
measurements of discharge rate. In all cases, flow was
fully developed before the first animal was encountered
1 10-cm downstream of each pipe's entrance (i.e., X eq
< 1 10 cm). There were five levels of mean speed (tO that
ranged from about 2-15 cm s '. Within each pipe there
was little temporal variability throughout the experiment
(Table I); the coefficient of variation (standard deviation/
mean) of U averaged 6.45%. However, due to the radial
velocity gradient (Eq. 3), the range of flow speeds expe-
rienced by animals within a pipe could have been as great
as 10%- 15% of U.

It is important to note that a boundary layer also de-
veloped along each plexiglass strip to which animals were
attached. Calculations (based on Eq. 21.8 in Schlichting
[ 1 979]) indicate that this boundary layer could have been
no more than a few millimeters thick, even at the down-
stream end of each 2-cm-long strip, given the flow veloc-
ities in Table I. Because the feeding structures of animals
studied extended even greater distances above the sub-
stratum (Eckman and Duggins, 1991; personal observa-
tions), the effect of this boundary layer on flow speeds
experienced by animals can be ignored, and animals were
exposed to speeds reported in Table I.

Experimental protocols 7997

Colonies of Membranipora membranacea were ob-
tained after larvae settled onto large strips of plexiglass

FLOW AND SUSPENSION-FEEDER GROWTH

31

Table I

Average properties of pipe flows, arranged in order of increasing mean flow speed, in the 1991 and 1992 experiments

Pipe No.

R (cm)

Re

I' (cm s"

t' mal (cm s' 1 )

t/ mm (cms"')

A',, (cm)

1991 Experiment

7

3.

! 85.7

1433

1.9

(.04)

2.2

1.5

71

1

3.

5 93.8

1567

2.1

(.03)

2.4

1.6

73

8

2

) 84.8

2124

4.2

(.11)

4.9

3.3

52

3

2

i 92.5

2317

4.6

(.22)

5.3

3.6

53

10

2

> 141

3527

6.9

(.24)

8.1

5.5

59

4

2

j 142

3555

7.0

(.17)

8.2

5.6

59

5

2

j 227

5692

11.2

(.31)

13.0

8.9

67

9

2

i 228

5706

11.2

(.30)

13.1

8.9

67

2

2

i 297

7446

14.7

(.30)

17.1

11.7

71

6

2

i 299

7482

14.7

(.35)

17.1

11.8

71

1992 Experiment

5

5.

2652

2.6

3.0

(.12)

2.3

111

10

5.

2805

2.8

3.2

(.10)

2.4

112

9

5.

5457

5.4

6.2

(.10)

4.8

133

4

5.

5457

5.4

6.2

(.10)

4.8

133

3

5.

8160

8.0

9.2

(.10)

7.2

147

8

5.

8160

8.0

9.2

(.06)

7.2

147

2

S.

10812

10.6

12.3

(.06)

9.5

157

7

5.

10914

10.7

12.4

(.15)

9.6

157

6

5.

13158

12.9

14.9

(.29)

11.6

165

1

5.

1 3464

13.2

15.3

(.05)

11.8

166

Pipe No. refers to the relative position of pipes in the tank. R is inner radius. Q is discharge rate (not measured in the 1992 experiment). Re is
Reynolds number. I', {/, and U mm refer, respectively, to mean flow speed, maximum speed along the centerline, and minimum speed experienced
by animals. Standard error (in parentheses) is given for L' in the 1991 experiment and ', in the 1992 experiment. A',,, is the distance downstream
from the entrance required to achieve fully developed flow.

attached to racks suspended at a depth of about 2 m.
Strips for deployment in pipes were cut from these larger
strips such that each contained two or more colonies < 2
mm in diameter. Colonies on strips were photographed,
using a video camera, just before they were placed in pipes
on 6 June. Strips were removed from pipes on 25 June,
and colonies were rephotographed.

Colonies that had grown to contact other colonies were
not analyzed because intraspecific contact interferes with
growth (Ellison and Harvell. 1989). Although most of the
remaining colonies were circular on 25 June, many others
contained invaginations, probably as a result of partial
predation (Harvell el ai. 1990) inflicted by gastropods
introduced into pipes by the pump. To minimize effects
of these invaginations on estimates of growth, a "potential
growth rate" of each colony was calculated as in Eckman
and Duggins ( 1991 ). Potential growth was denned as the
difference in areas of circles with radii equal to the maxi-
mum radius of each living colony (i.e.. the farthest distance
from ancestrula to colony edge) on 6 and 25 June.
Maximum radii were estimated from photographs made
on 6 June and measured directly using calipers on 25
June. Use of this method, rather than simple differences
in total colonv area, allowed us to minimize the influence

of factors such as predation, which would have reduced
the size of the colony only at certain points along its edge.
Use of this method is further justified since estimates of
potential growth rate are strongly correlated with absolute
measures of growth rate based on increase in colony area
(see Results).

One assumption implicit in the use of this method is
that colony growth was isotropic, and not sensitive to How
direction in pipes. To evaluate this assumption quanti-
tatively, we measured maximum dimensions (on 25 June)
of 45 haphazardly selected colonies in both across-stream
and along-stream directions. The ratio of these dimen-
sions should be 1 .0 if growth was insensitive to flow di-
rection.

Balanus glandula was obtained after cyprids settled
onto large strips of plexiglass attached to rocks in the lower
intertidal. Strips for deployment in pipes were cut from
these larger strips such that each contained two or more
small individuals. Animals on strips were photographed
just before being placed in pipes on 1 August. At that time
the mean basal diameter of all barnacles was 14.3 mirr;
the range was 5-3 1 mm 2 . Strips were removed from pipes
on 24 September, and animals were rephotographed.
Growth was defined as the increase in basal area of animals

32

J.E. ECKMAN AND D.O. DUGGINS

still alive on 24 September. Barnacles that had grown
against neighbors were not considered.

Pseudochitinopoma occidentalis was obtained after lar-
vae settled onto large strips of plexiglass attached to racks
mounted about 20 cm above the bottom at 10-m depth.
Strips for deployment in pipes were cut from these larger
strips such that each contained two or more small indi-
viduals. Animals on strips were photographed just before
being placed in pipes on 26 August. At that time the mean
length of the calcareous tube of all individuals was
7.1 mm; the range was 2.5-13 mm. Strips were removed
from pipes on 22 November, and animals were repho-
tographed. Growth was denned as the increase in length
of the tube of individuals still alive on 22 November.

By using a small head tank and a pump with a high
discharge rate, we hoped to ensure that water supplied to
pipes was well mixed and that each pipe received the same
initial concentration and composition of suspended par-
ticulates as food for suspension feeders. However, the
possibility of inhomogeneous supply existed. Moreover,
because flows varied in strength among pipes, the rates at
which particulates settled out of suspension might have
varied among pipes, causing differences in food supply.
To assess these effects, we twice sampled seawater that
exited each pipe and one time each tested for differences
in concentrations of chlorophyll, paniculate organic car-
bon (POC), and paniculate organic nitrogen (PON).

For chlorophyll, three replicate samples of 1000 ml were
collected from each pipe. One replicate was collected from
each of the 10 pipes before collecting the next replicate
sample. Each sample was filtered through a 0.22-^m glass-
fiber filter. Pigments collected on the filter were extracted
in acetone, and chlorophyll was measured spectropho-
tometrically according to Parsons ct al. ( 1984).

The number of replicates and the sampling protocol
for POC and PON were the same as for chlorophyll. A
subsample of each 500-ml seawater sample (volume varied
according to the amount of particulates in the sample)
was filtered through a 0.22-j/m glass-fiber filter that had
been baked at 550C; all glassware was acid-washed. Fil-
ters were immediately dried at 65C and then frozen.
Treatment of the filter to remove carbonates was judged
unnecessary because the local phytoplankton and zoo-
plankton assemblages contain few individuals with cal-
careous tests. Masses of C and N on filters were determined
using a Perkin-Elmer 2400 Elemental Analyzer and con-
verted to POC and PON concentrations.

Experimental apparatus 1 992

Results of the 1991 experiment prompted us to run a
similar experiment in 1992 to evaluate growth responses
of three other species of barnacle (Semihalaints cariosm.

Balanus crenatus, and Pollicipes polymenis) to flow speed.
Because our original device had been dismantled, we used
a different pipe flow apparatus that had been built for
another experiment.

Ten straight sections of pipe were used, as in 1991.
Each pipe (5.1 -cm inner radius) was 307 cm long. The
10 pipes were submerged in a large, round seawater tank
(400 cm in diameter, 55 cm deep) that was supplied con-
tinuously with fresh, unfiltered seawater. A standpipe in
the tank allowed overflow. All pipes were connected di-
rectly to a centrifugal pump that supplied each with sea-
water (at a constant pressure) drawn directly from the
tank in which the pipes were submerged. Flow through
each pipe was regulated by a valve at the end of each pipe,
as in the 1991 experiment. The connecting pipes that led
from the pump to the straight sections used in the exper-
iment also had a 5.1-cm inner radius. These connectors
varied in length among the 10 pipes from 20-150 cm. To
break down the jet of water that exited the pump, two
turns were built into the connectors.

Eight windows were cut in each 307-cm-long straight
section of pipe. Animals were introduced into pipes
through these windows as in the 1991 experiment (Fig. 1).
The first window in each pipe was located 46 cm down-
stream of the beginning of the straight section. The spacing
between windows was 30 cm.

We attempted to duplicate the five flow levels used in
the 1991 experiment, again with two replicate pipes as-
signed to each flow level. Flow speeds were not calculated
from pipe discharge (Q) as in 199 1 , but instead were mea-
sured directly using a 2-axis Marsh-McBirney electro-
magnetic current meter that was placed on the centerline
of each pipe through a window 30 cm from the discharge
end. Trials that timed injected dye indicated that the walls
of the pipe did not interfere with the magnetic field about
the probe enough to affect measurements of current speed,
and that measurements obtained with the probe were ac-
curate. Speeds measured with the current meter approx-
imated t/max. since speed was measured along each pipe's
centerline. Other flow parameters were calculated from
t' max using equations presented above, with one exception.
The minimum flow speed to which animals were exposed
('min) was calculated assuming that all individuals were
at least 20% of the radius away from the pipe wall (in the
1991 experiment a minimum distance of 10% of R was
assumed). This change was justified since barnacles were
attached to plexiglass strips by hand in 1992 (see below),
and care was taken to place them farther from the pipe
wall.

The growth experiment ran from 22 May-30 July 1992.
Flows were measured six times during this 69-day period.
Table I lists flow properties for each of the 10 pipes, based
on the mean L' max . The coefficient of variation in t/ max

FLOW AND SUSPENSION-FEEDER GROWTH

33

was < 10% for all 10 pipes. Values of A cg in Table I indicate
a problem with the experimental apparatus used in 1992.
Depending on flow level, two to four of the eight plexiglass
strips containing animals were upstream of the minimum
distance required to achieve fully developed flow. Animals
on these strips (far from the pipe wall) may have experi-
enced flow speeds somewhat slower on average than an-
imals located further downstream. However, calculations
indicate that speeds experienced by animals on these up-
stream strips would have exceeded U mn shown in Table
I, so the range of speeds between t/ max and U min is still an
accurate indicator of flow speeds experienced by all ani-
mals.

Experimental protocols 7 992

Small individuals of Pollicipes polymerus and Serni-
balanus cariosus were collected from a comparatively
high-energy, mid-intertidal zone in the Straits of Juan de
Fuca near Clallam Bay, Washington. We collected indi-
viduals of both species that had settled onto shells of mus-
sels (Mytilus edulis and M. californianus), in addition to
P. polymerus that were attached to plates of larger S. cari-
osus. Relatively small Balanits crenatus on discarded alu-
minum cans were collected from a depth of about 5 m
below the public docks of the town of Friday Harbor,
Washington. Animals of all three species were returned
to the Friday Harbor Laboratories where they were held
in seawater tables for several days.

Individuals of all species were glued by hand onto the
plexiglass strips that were subsequently placed in pipes.
We prepared pieces of mussel shell, aluminum can, or
calcareous plate that were about 1 cm 2 and contained one
or two individuals of one of the three species. Two or
three pieces of each substrate were attached to each plexi-
glass strip using a submarine adhesive. We positioned each
barnacle such that the extended cirral net would be ori-
ented approximately concave into the flow. In addition,
B. crenatus and 5. cariosus can rotate the cirrus into this
apparently "preferred" orientation (see below). Species
were not segregated among strips, and strips were assigned
randomly to pipes. We did not use animals that were in
contact with other individuals.

Each animal was measured within 2 days of the start
of the experiment. The basal area of each individual of
B. crenatus or S. cariosus was calculated from the average
of two orthogonal measurements of basal diameter ob-
tained using calipers. At the start of the experiment, the
mean basal diameter of B. crenatus was 62.8 mm 2 ; the
range was 17.7-130.5 mm 2 . The mean basal diameter of
S. cariosus was 52. 1 mm 2 ; the range was 7.4- 1 39.8 mm 2 .
For P. polymerus we used calipers to measure the height
of the capitulum (distance from the top of the peduncle

to the furthest extension of the plates). At the start of the
experiment the mean capitular height was 5.45 mm; the
range was 1.6-1 1.5 mm.

Plexiglass strips were mounted in pipes as in 1 99 1 , and
animals were exposed to one of the five steady flows from
22 May-30 July. Thereafter, plexiglass strips were re-
turned to seawater tables, and animals were remeasured
within several days. Growth is denned as the increase in
basal area (B. crenatus or S. cariosus) or capitular height
(P. polymerus).

As in the 1991 experiment, pipes were supplied with
seawater from a common, presumably well-mixed source
so that there would be little variability among pipes in
the concentration or composition of suspended particu-
lates supplied to suspension feeders. To assess this vari-
ability, on one occasion we sampled the seawater exiting
the pipes and tested for differences in concentrations of
chlorophyll. Sampling and analytical protocols followed
those in the 1991 experiment.

Flume observations 1992

To aid in interpreting results of growth experiments,
we observed the feeding behavior of all species (except
Pseudochitinopoma occidentalis) in a range of flows.
Qualitative observations of responses of feeding structures
were made in a simple recirculating flume of the type
described by Vogel and LaBarbera (1978). The main
channel of the flume was 200 cm long and 30 cm wide.
A flow depth of about 1 5 cm was used.

Each species was observed separately. Approximately
20-30 individuals or colonies were placed in the center
of the flume approximately midway down the channel.
We used barnacles attached to small rocks or aluminum
cans to elevate them above the flume's developing bottom
boundary layer and to expose them to near free-stream
flow speeds. For a similar reason, we cut out small pieces
of kelp that were encrusted with colonies of Membrani-
pora membranacea, used a cyanoacrylate glue to attach
them to strips of plexiglass ( = 3 cm X 5 cm X 0.3 cm
thick), and suspended them in the free-stream flow.

Each species was observed at four to five levels of flow,
with flow held constant for at least 10 min before the
observations. For any given speed, no differences in feed-
ing behavior were apparent between accelerating and de-
celerating flows. Because flow in this crude flume was
three-dimensional and not fully developed, we measured
horizontal flow speeds as close as possible to the animals
being observed. To obtain these measurements we used
a Marsh-McBirney current meter and also timed the pas-
sage of suspended particulates. We consider measured flow
speeds to be only approximations.

34

J.E. ECKMAN AND D.O. DUGGINS

Statistical analysis

Regression was used to examine relationships between
the mean growth rate of all individuals (or the mean con-
centration of chlorophyll, POC, or PON) within a pipe
and the mean flow speed in the pipe. Mean growth rate
was used because individual growth rates within each pipe
were not independent, and it would have inflated the de-
grees of freedom in the regression had individuals been
considered separately. The issue addressed by regression,
therefore, was whether or not the mean growth rate of a
population varied predictably as a function of flow speed.

Prior research on particle capture by suspension feeders
suggests that one of two relationships between flow speed
and growth would be expected a priori (see Introduction):
(1) a convex upward relationship in which growth rates
are lower on both sides of a narrow range of intermediate
speeds that appear to support the highest growth; (2) a
monotonically increasing or decreasing relationship. We
had no a priori reason to expect that any monotonic re-
lationship between growth rate (or concentration) and
speed would be linear. Therefore, we searched for a
monotonic relationship of the form

G = aU h + c

where G is growth rate (or concentration), U is mean ve-
locity, and a, b, and c are constants. We selected constants
that produced the maximum r with the constraint that
0.5 < b < 3. This range was imposed because higher or
lower exponents would in some instances produce strongly
curved functions especially sensitive to flow speeds near
the end of the range; also, growth rates and concentrations
predicted from these functions could have been non-sen-

70-

f 60-

50-

B

^ 40-

>.

. 30-

e

o 20-

10-

10

14

16

Speed (cm s ~ 1 )

Figure 2. Concentrations of chlorophyll as a function of flow speed
measured in each pipe in 1991. Each point and error bar represents the
mean (1 SE) of three replicate samples obtained from each pipe. Also
shown is the best-fit, significant regression line obtained using the 10
mean values.

250-
200-

2> 150-

co
O

100-
50-

0-

i i r~

10 12 14 16

0)
O)

o

35-

30-

25-

20-

15-

10-

5-

T~

10

12 14 16

Speed (cms" 1 )

Figure 3. Concentrations of POC (top) and PON (bottom) as a func-
tion of flow speed measured in each pipe in 1991. Other interpretations
as in Figure 2.

sical (e.g.. negative growth of calcareous structures or
negative concentrations at near-zero flow speeds).

To evaluate whether growth rate was maximal at some
intermediate flow speed, we used a 2nd-degree polynomial
to regress growth rate against flow speed:

G = aU 2 + bU + c

with terms as denned above. A 2nd-degree polynomial
defines a parabola, and this functional relationship be-
tween growth and speed can assume a convex-upward
shape as expected a priori.

Results

7997 experiment

The concentration of chlorophyll in pipes increased with
flow speed by a factor of more than 2 from 2-15 cm s" 1
(Fig. 2; b = 0.5, P = 0.03, r = 0.46). The horizontal flux
(the product of concentration and velocity) of chlorophyll
varied among pipes by a factor of approximately 18 over
the 7.5 X range of flow speeds.

FLOW AND SUSPENSION-FEEDER GROWTH

35

Table II

M i'l animals per pipe providing measures o/ growth rale

Table III

Analysis o/i sol ropy in shapes <>/ Membranipora colonies

in bolh e\penment-:

(.'(cms" 1 ) DJD a . n P

Pipe

1991 Experiment
t'(cm s ') M.m.

E.g.

P.O.

2 0.967(0.013) 14 *

7
1
8

1.9

2.1
4.2

8
8
2

12
14

7

12
14
6

4 1.010 (0.026) NS
7 1.063 (0.016) 7 *
11 1.004 (0.029) 6 NS
15 0.989 (0.023) 11 NS

3

10
4

4.6
6.9
7.0

8
4
6

9
5
9

5
2

5

[' is the mean flow speed for each of the five treatments. DJD^ gives
the mean ratio (1 SE) of colony diameter measured cross-stream to

5

11.2

7

6

2

colony diameter measured along-stream. n is the number of colonies.

9

11.2

1

8

6

P is the probability that the observed ratio differs from 1.0 (Student's I

2

14.7

4

10

6

test): NS = not significant (P > 0.05); * = 0.01 < P < 0.05.

d

14.7

4

8

7

Pipe

1992 Experiment
f(cms~') S.c. P.p.

B.C.

5

2.6

7

7

7

10

2.8

3

7

5

9

5.4

7

14

6

4

5.4

6

11

7

3

8.0

7

11

6

8

8.0

3

9

8

2

10.6

6

13

9

7

10.7

6

8

8

6

12.9

4

9

9

1

13.2

6

7

5

M.m.. Membranipora membranacea: E.g.. Balanus glandula; P.O.,
Pseudochitinopoma occidentalis; S.c., Semibalanus cariosits: P.p., Pol-
licipes polymerus: B.C.. Balanus crenatus.

Concentrations of POC and PON showed nearly iden-
tical patterns (Fig. 3), and resembled the variation in
chlorophyll with flow speed. Concentrations of POC and
PON both doubled as flow speed increased by 7.5 X, pro-
ducing about 1 5-fold differences in C and N fluxes between
the weakest and strongest flows. The regression of POC
with speed was significant (b = 0.5. P = 0.05, r = 0.39),
and that for PON was nearly significant (b = 0.5, P = 0.06,
r = 0.38).

Table II lists numbers of animals of all species from
which mean growth rate per pipe was estimated. Growth
of Membranipora was effectively isotropic at all flow
speeds. The ratio of cross-stream to along-stream diameter
of colonies was indistinguishable from 1.0 at three of five
flow speeds (Table III); diverged slightly from unity, but
in different directions, at the other two speeds; and at all
speeds was within 6%. of 1 .0. Consequently, there was
effectively no anisotropic growth that would have biased
calculations of potential growth rates. In addition, poten-
tial growth rates were strongly correlated with growth rates
based on changes in total colony area (r =0.81,
P < 0.0001, n = 50). Potential growth rates of Membran-

ipora colonies declined monotonically with flow speed
(Fig. 4), and the regression was significant (b = 1.2,
P= 0.04, r = 0.42).

The large difference in mean colony growth rate be-
tween the two replicate pipes at the slowest ("optimal")
flow speed (Fig. 4) suggested that conditions in one pipe
were anomalously poor. To investigate this possibility we
compared probabilities of colony survival among pipes.
In both slowest flow pipes, percent colony survival was
high (100% and 94%) over the 19-day experiment. The
average percent colony survival for all pipes was 84.2%
1 1.3% and was not related to flow speed (r = 0.08,
P = 0.44).

Growth rates ofBalanus glanditla individuals were not
detectably sensitive to flow speed over the range studied
(Fig. 5). There appeared to be a weak (perhaps 10%), but

Membranipora membranacea

^ 30'

TJ

25-

CM

E

.. 20-

o>

03

E 15H

CJ
15

o

Q-

5-

68 10

Speed (ems' 1 )

14 16

Figure 4. Potential growth rates of Membranipora membranacea as
a function of the average flow speed measured in each pipe during the
19-day deployment in 1991. Each point and error bar represents the
mean growth rate ( 1 SE). Also shown is the best-fit, significant regression
line obtained using the 10 mean values.

36

J.E. ECKMAN AND D.O. DUGGINS

Balanus glandula

l.U

i

T> 0.8
"
in

"g 0.6-
o

0>

d 0.4

f

1 * ? , s f

0.0

t

1 1 1 1 1 1 1

2 46 8 10 12 14 1!

Speed (cm s" 1 )

Figure 5. Growth rates of Balanus glandula as a function of flow
speed during the 54-day deployment in 199 1. Other interpretations as
in Figure 4. There was no significant best-fit regression.

0.7-

0.6-

t

1

ilorophyll (ug I" 1 )

O O CD CD

ro co *- cn

I I I I

i * ;

* ]

n % i

f

0.1-

00 I I I II

02 468 10 12

1

14 1f

Speed (ems' 1 )

Figure 7. Concentrations of chlorophyll as a function of flow speed
in 1992. Other interpretations as in Figure 2. There was no significant
best-fit regression.

nonsignificant, monotonic decline in growth rate as speed
increased (for all b: minimum P = 0.10, maximum
r = 0.30).

Growth rates of Pseudochitinopoma individuals de-
clined monotonically and significantly with flow speed
(Fig. 6;b= 1.1, P = 0.009, r = 0.59).

1 992 experiment

The concentration of chlorophyll in pipes did not de-
pend on flow speed (Fig. 7). The monotonic regression
was not significant (for all b: minimum P = 0.39, maxi-
mum r = 0.095), nor was the polynomial regression.

Therefore, the horizontal flux of chlorophyll varied by a
factor of only about 5 over the 5 X range of flow speeds.

Growth rates of Semibalanus cariosus did not depend
on flow speed (Fig. 8). The monotonic regression was not
significant (for all b: minimum P = 0.17, maximum
r = 0.22), nor was the polynomial regression.

Growth rates of Pollicipes polymerus also did not de-
pend on flow speed (Fig. 9). The monotonic regression
was not significant (for all b: minimum P = 0.38, maxi-
mum r = 0.10), nor was the polynomial regression.

Growth rates of Balanus crenatus appeared to be min-
imal at the two lowest flow speeds, to increase sharply to
a maximum growth rate at U = 8 cm s~ ', and to decline

2.2-

Pseudochitinopoma occidentalis

Semibalanus cariosus

E 1.6-

o

O)

TO 1.4-
OC

1.2-

1.0-

0.8

I
12

14 16

Speed (ems' 1 )

Figure 6. Growth rates of Pseudochitinopoma occidentalis as a func-
tion of flow speed during the 88-day deployment in 199 1. Other inter-
pretations as in Figure 4.

ouu

^j 250-

o>

II

<>

T

CM" 200-

1

i

<

> T A

E

O

f'50-

(

|

(

i

T

15

o

oc

^ 100-

-i-

5

~^

o

50-

c

I

2 '

6

f

1 1

1 10 12 14 1f

Speed (cm s' 1 )

Figure 8. Growth rates of Semibalanus cariosus as a function of
flow speed during the 69-day deployment in 1 992. Other interpretations
as in Figure 4. There was no significant best-fit regression.

FLOW AND SUSPENSION-FEEDER GROWTH

37

Pollicipes polymerus

*Z- 5-

E

0)

I

I

O

3-

10

12

14

16

Speed (cms -1 )

Figure 9. Growth rates of Pollicipes polymerus as a function of flow
speed during the 69-day deployment in 1 992. Other interpretations as
in Figure 4. There was no significant best-fit regression.

at higher speeds (Fig. 10). Polynomial regression indicates
that this convex-upward trend was weak and not signifi-
cant at the conventional level of P = 0.05 (r = 0.49,
P = 0.09). However, further evidence of a speed-depen-
dent growth response is supported by results of ANOVA.
which indicated that replicate pipe effects were not sig-
nificant (F 5 . 60 = 0.63, P = 0.68), but that effects of flow
level on growth were highly significant (F 4t>5 = 3.66,
P = 0.0095). A posteriori multiple comparison tests (T-
method Sokal and Rohlf, 1981. p. 246) indicated that
growth rates at U = 8 cm s~ ' significantly exceeded those
at U = 2.7 and 5.3 cm s ' (a = 0.05).

Friday Harbor Laboratories can be quite strong (Eckman
et al.. 1989), this species also thrives in areas of compar-
atively weak flow energy. Animals used in this study were
collected from a quiescent environment where even free-
stream flows typically are <10 cm s ' (personal obser-
vations).

In contrast to the balanoid barnacles, the pedunculate
barnacle Pollicipes polymerus never swept the cirral net
through the water. This species curled the cirrus inward
when exposed to stronger flows (a behavior and not a
passive deflection of the cirrus by flow) and retracted the
cirrus completely at the highest speeds examined. This
dissimilarity from the balanoid barnacles held despite the
fact that P. polymerus lives sympatrically with S. cariosus
in comparatively high-energy intertidal regions.

The bryozoan Membranipora membranacea was able
to extend and use the lophophore at flow speeds < 10 cm
s '. However, in the face of stronger flows, the lophophore
bent and shook noticeably, and was retracted upon contact
with moving particles. At the highest speed examined,
tentacles were not able to open from the lophophore.
M. membranacea typically encrusts thalli of kelps. It lives
both on the surface-canopy species Nereocystis luetkeana
and on several understory species whose thalli extend only
tens of centimeters above the bottom. Although abundant
on plants in high-current regions (personal observations),
these essentially flat colonies are submerged deep in the
boundary' layer that develops on a thallus that typically
is aligned somewhat parallel to flow direction. Thus even
in high-flow regions, M. membranacea on thalli of surface-
canopy plants would experience significantly lower flow
speeds.

Flume observations of feeding behavior

A detailed description of feeding behaviors as a function
of flow speed is given in Table IV. The three balanoid
barnacles (Balanus glandula. Semibalamts cariosus. and
B. crenatus) all were able to control the cirrus (i.e., actively
sweep or hold the cirrus still and erect) over the entire
range of flow speeds examined, with little or no defor-
mation of the cirrus by flow. This similarity held despite
the wide range of habitats in which these species typically
occur. Balanus glandula and 5. cariosus are found in
moderate to comparatively high-energy intertidal regions
in the Pacific Northwest. These species typically experi-
ence oscillatory flows that often have instantaneous speeds
far higher than those examined in this study. In contrast.
B. crenatus lives predominantly in relatively low-energy,
subtidal environments. It more typically experiences tidal
flows that are not often impacted by wind-generated sur-
face waves. Although tidal currents in the region of the

300-

Balanus crenatus

250-

CM

E.

o>
re

cc

200-

150-

100 -I

50-

10

16

Speed (ems' 1 )

Figure 10. Growth rates of Balanus crenatus as a function of flow
speed during the 69-day deployment in 1992. Other interpretations as
in Figure 4. Also shown is the best-fit, 2nd-degree polynomial regression
line obtained using the 10 mean values.

38 J.E. ECKMAN AND D.O. DUGGINS

Table IV

Summary oj feeding behaviors as a function of flow speed

Flow

speed

Membranipora

(cm s~')

Balamis glandula

Pollicipes polymerus

Semibalanus cariosus Balamis cre/ia/us

membranacea

2-3 Cirri of all animals

sweep continuously
( = 2 Hz)

4.5-6 Most animals sweep
continuously;
= 10% pause
between sweeps to
hold cirrus erect
and still for = 1 s

8-10 Continuous sweeping
predominates;
= 20% pause
briefly between
sweeps

14-18 =50% sweep

constantly; =50%
pause briefly
between sweeps; no
deformation of
cirral net

19-21 Most animals pause
between sweeps; no
deformation of
cirral net

(N.O.)

Cirri of all animals held
erect and still; no
deformation of cirral
net

Cirri held erect and still;
distal portions curled
inward slightly
(apparently by
muscular action)

Increased inward curl of
cirri

(N.O.)

(N.O.)

Cirri of all animals sweeping;
cirrus retracts fully into
mantle with each beat

Many animals pause
between sweeps to hold
cirrus erect and still for
2-4 s; some animals
rotate cirrus up to 90 to
orient it into flow

Most animals pause between
sweeps; cirral net not
deformed by flow;
rotation of cirrus up to
90 into flow is common

(N.O.)

(N.O.)

Cirri of most animals
sweeping; no cirral
deformation by
flow; rotation of
cirrus up to 90
into flow is
common

As at 4.5-6 cm s"'

Lophophores extended;
no deformation of
tentacles by flow

Lophophores extended;
some weak shaking of
tentacles by flow

As at 4.5-6 cm s

As at 4.5-6 cm s

(N.O.)

Pronounced bending of
tentacles in flow;
lophophore retracts
on contact with
particles in transport

Lophophores bent up to

45

>25 (N.O.)

Cirri almost totally As at 14-18 cm s '
retracted

As at 4.5-6 cm s ',
but some deflection
of cirrus by flow at
peak of extension

Tentacles unable to open
from lophophore

(N.O.) = Not Observed at this speed.

The behavior of Pseudochitinopoma occidentalis was
not observed in flow. This species typically lives in cryptic,
subtidal habitats such as the crevices in rocks and the
undersides of rock ledges. Consequently, animals of this
species normally would experience comparatively slow
flow speeds.

Discussion

Results from the 1991 experiment provide clear evi-
dence that growth rates of Membranipora and Pseudochi-
tinopoma declined significantly with increasing flow speed.
In contrast, over the same range of flow speeds, there was
no detectable relationship between speed and growth of
Balanus glandula. though there was qualitative evidence
of a weak inverse relationship. Higher growth rates of the
bryozoan and serpulid occurred at weaker flows despite
evidence that concentrations of suspended paniculate

food may have been lower under these conditions
(Figs. 2. 3). By the end of the 1991 experiment, we noted
a substantial amount of material deposited on the bottoms
of the pipes with weaker flows. Therefore, it is probable
that the reduction in concentrations of suspended particles
observed twice in the slowest flow pipes was consistent
over time, and was caused by gravitational settlement of
particles onto the bottom of a pipe, rather than removal
of suspended particles by the small number of suspension
feeders in each pipe. It is important to recognize that data
presented in Figures 2 and 3 may overestimate differences
among pipes in concentrations of food available to sus-
pension feeders. Concentrations reported here character-
ized water exiting the ends of pipes, and gravitational
settling would have produced less marked differences ap-
proximately halfway down the pipes, where animals were
located. However, some differences would have occurred,
and growth rates of the bryozoan and serpulid apparently

FLOW AND SUSPENSION-FEEDER GROWTH

39

were greatest under low-flow conditions where food con-
centrations were also lowest. Had concentrations of sus-
pended food been constant among treatments, we might
have seen even stronger effects of flow on growth of these
two species.

The 1992 experiment was prompted by the observation
that growth of Balanus glandula was comparatively
insensitive to flow speed, over a moderate range, in con-
trast to the pronounced sensitivities shown by the bry-
ozoan and serpulid. More specifically, we were interested
in determining the extent to which this difference related
to the barnacle's behavior, its adaptation to a typically
more energetic habitat, and its mechanical and morpho-
logical design. Regarding the latter, it seemed possible that
barnacles might be generally more capable of feeding ef-
ficiently and growing well in a wider range of flows because
the cirral net is composed of a rigid exoskeleton operated
by a network of internal muscles (though each cirrus is
extended hydraulically). and because they use the smaller
(1st and 2nd) cirri to handle and process captured parti-
cles. These properties distinguish barnacles from many
other suspension-feeding invertebrates, like the bryozoan
and serpulid, that utilize feeding structures composed of
soft tissue, that operate feeding structures mainly hydro-
statically. and that process particles adhering to feeding
structures primarily using cilia. These properties may
render feeding more susceptible to fluid drag forces.

Combined results of the 1991 and 1992 experiments
indicate significant differences in growth responses among
barnacle species. There was no taxon-wide insensitivity
of barnacle growth to flow speed, over even a moderate
range of speeds. The interspecific variability relates most
obviously to the range of flows that animals typically ex-
perience. Barnacles that inhabit comparatively high-
energy intertidal environments (Balanus glandula. Semi-
balanm cariosits. and Pollicipes polymerns) grew well at
a wide range of flow speeds (Figs. 5, 8, and 9). In contrast,
growth rates of B. crenatus. which typically inhabits lower
energy, subtidal environments, were significantly affected
by flow speed and appeared to be maximal at a fairly
weak, intermediate speed (about 8 cm s~'; Fig. 10). This
pattern extends beyond the four species of barnacles stud-
ied. The bryozoan and serpulid. whose growth rates were
lowest in the strongest flows (Figs. 4, 6), also typically
inhabit subtidal microhabitats characterized by weaker
flow (see above).

The interspecific variability in growth responses among
barnacles was not obviously related to feeding behavior.
Like the other balanoid barnacles, Balanus crenatus was
apparently able to sweep with its cirrus over a wide range
of flow speeds (Table IV), yet its growth but not that of
the others was highly sensitive to flow speed.

We therefore conclude that, for each of the six species
studied here, growth responses to flow speed were most
clearly coupled to the relative flow energy of the animals'
natural habitat. Growth response to flow did not reflect
the clear similarities in morphology and behavior among
some species. The mechanism(s) responsible for the ob-
served differences among species are not obvious from
our observations, and warrant further consideration.

Shimeta and Jumars ( 199 1 ) extended the work of Rub-
enstein and Koehl (1977) to predict how features of a
suspension-feeder's environment and structural aspects
of its filtration apparatus combine to determine rates of
particle contact with the filter. Their analysis indicates
that rates of particle contact with an individual filter ele-
ment should increase both with flow speed and with the
concentration of food particles in suspension. In our ex-
periments, flow speeds increased about 7.5 X, while food
concentrations either remained constant (1992 experi-
ment. Fig. 7) or simultaneously increased by about
2 X (1991 experiment. Figs. 2, 3). Thus, rates of particle
contact with an individual filter element should have in-
creased with speed. Despite this prediction, we observed
no monotonic increase in growth rate with increasing flow
speed for any of the six species studied, and for two species
(Membranipora membranacca, Pseudochitinopoma oc-
cidentalis) we noted an inverse relationship. The most
reasonable explanation for this apparent contradiction is
that, at speeds studied here, growth rates were governed
by a strong inverse relationship between flow speed and
efficiency at which particles are retained on the filter ap-
paratus and passed to the point of ingestion. Among these
species, the particle-handling abilities of the bryozoan and
the serpulid were apparently more sensitive to speed than
were those of B glandula. S. cariosux. and P. polymerus.
This heightened sensitivity of particle processing to flow
may relate to the way that bryozoans and serpulids use
cilia to process particles. An additional factor that may
have contributed to the poor growth of the bryozoan and
the serpulid in stronger flow is that their feeding apparatus
(composed of soft tissue) may have experienced greater
deformation in flows than the cirral nets of the barnacles
(cf. Patterson, 1984; Harvell and LaBarbera, 1985; Best,
1988: Shimeta and Jumars, 1991; Sponaugle and La-
Barbera, 1991).

Some results from our growth study are consistent with
many results from prior, shorter term studies that ex-
amined effects of flow on particle capture by "tentaculate"
suspension feeders (Okamura, 1984, 1985; McFadden.
1986; Best, 1988; Leonard el al. 1988; Sponaugle and
LaBarbera. 1 99 1 ). These studies all noted a maximum
feeding rate at a low speed (though not necessarily at the
lowest examined) and a strong decline in feeding rate at
higher speeds. Our results confirm that at least two other

40

J.E. ECKMAN AND D.O. DUGGINS

tentaculate suspension feeders (M. membranacea and
P. occidentalis), and one species of barnacle (B. crenatus),
exhibit a similar negative performance in strong flows,
and that relationships noted previously between flow and
particle capture probably translate into similar effects on
somatic growth (see also Okamura, 1992).

In contrast to results of many previous studies of par-
ticle capture, for five of the six species studied we noted
no reduction in growth rate at the lowest flow speeds (i.e.,
no intermediate speed associated with a seemingly max-
imal growth rate). We suspect that this difference between
our current and many previous results may in part reflect
our experimental apparatus and design. The minimum
flow speed examined was about 2 cm s ', and our pipe
flows were all turbulent. In the absence of flows strong
enough to interfere with particle processing, the supply
of food to animals at 2 cm s" ' may have been high enough
to ensure maximum growth rates. Moreover, the com-
paratively efficient mixing of particles in turbulent flows
may have prevented an animal's feeding ambit from be-
coming depleted of food, even at the lowest flow speed.
Had we included a flow speed slow enough to establish
laminar, and not turbulent, conditions in a pipe, a marked
reduction in growth rate might have resulted. At least two
of the species studied here (M. membranacea and P. oc-
cidentalis) have no obvious mechanism for avoiding re-
filtration of water already depleted of particles in extremely
weak flows. A reduction in growth rates of M. membran-
acea was noted when colonies were exposed to laminar
flows at speeds < 0.5 cm s" 1 (Grunbaum. 1992).

Our results help to confirm a conclusion we have
drawn previously (Eckman and Duggins, 1991) about
the factors that affect growth of Pseitdochitinopoma
occidentalis in situ. This serpulid grew faster in weaker
flows found beneath canopies of understory kelps than
within more energetic clearings in the canopy. Results
of our manipulative experiments implicated flow as one
of several factors responsible for this pattern. Our current
results confirm that this species of tentaculate suspension
feeder is poorly adapted for life in stronger flow envi-
ronments.

In conclusion, we have shown that there is wide vari-
ability among six species of benthic suspension feeder
in the sensitivity of growth rate to a fixed range of com-
paratively moderate flow speeds between about 2 and
about 1 5 cm s" ' . The growth response of animals to flow
related most obviously to the range of flows typically
experienced by the animal. Animals that inhabited
comparatively weak flow environments showed a strong
sensitivity to flow speed, whereas animals from com-
paratively high-energy environments were relatively in-
sensitive to flow speed. Growth responses to flow did not
reflect the clear similarities in morphology and behavior

among some species. We suspect that a key to under-
standing the impact of flow on a suspension feeder's
growth is the animal's ability to handle and process par-
ticles that have impacted the feeding structure, and to
deliver them to the point of ingestion. Further study of
this phenomenon may significantly increase our under-
standing of the influence of hydrodynamic processes on
benthic suspension feeders.

Acknowledgments

The considerable efforts of Amy Sewell were instru-
mental in the completion of this research. Helpful com-
ments on earlier versions of the manuscript were provided
by Drs. Carl Andre, Drew Harvell, Roberta Marinelli,
and Don Webb, and by an anonymous reviewer. Dr. Dan
Grunbaum assisted with observations on the feeding be-
havior of Membranipora in flow. Dr. Jon Grant provided
access to the CHN analyzer at Dalhousie University. Fig-
ures were prepared by Anna Boyette and Suzanne Mc-
Intosh. We thank A.O.D. Willows, Director of the Friday
Harbor Laboratories, and the staff of the Labs for their
critical support and assistance. We also thank the De-
partment of Oceanography at Dalhousie University for
providing office space for one of us (JEE) during a sab-
batical leave. This research was supported by NSF grant
OCE-8911116.

Literature Cited

Best, B. A. 1988. Passive suspension feeding in a sea pen: effects of

ambient flow on volume flow rate and filtering efficiency. Biol Bull

175: 332-342.
Eckman, J . E., and D. O. Duggins. 1 99 1 . Life and death beneath mac-

rophyte canopies: effects of understory kelps on growth rates and

survival of marine, benthic suspension feeders. Oecologia (Bert.) 87:

473-487.
Eckman, J. E., C. H. Peterson, and J. A. Cahalan. 1989. Effects of

flow speed, turbulence, and orientation on growth of juvenile bay

scallops Argopecien inactions concentricus (Say). J. Exp. Mar Biol.

Ecol. 132: 123-140.
Ellison, A. M., and C. D. Harvell. 1989. Size hierarchies in Mem-

hrampora membranacea: Do colonial animals follow the same rules

as plants? Oikos 55: 349-355.
Frechette, M., and E. Bourget. 1985. Food-limited growth of Mytilus

eJulix (I..) in relation to the benthic boundary layer. Can. J. Fish.

Aquat Sa. 42: 1166-1170.
Frechette, M., C. A. Butman, and W. R. Geyer. 1989. The importance

of boundary-layer flows in supplying phytoplankton to the benthic

suspension feeder, Mytiltis cdulis L. Limnol. Oceanogr 34: 19-36.
Grunbaum, D. 1992. Local processes and global patterns: Biomathe-

matical models of bryozoan feeding currents and density dependent

aggregations in Antarctic knll. Ph.D. Thesis, Cornell University.
Harvell, C. D., and M. LaBarbera. 1985. Flexibility: A mechanism for

control of local velocities in hydroid colonies. Biol. Bull 168: 312-

320.
Harvell, C. D., H. Caswell, and P. Simpson. 1990. Density effects in

a colonial monoculture: experimental studies with a marine bryozoan

(Membranipora membranacea). Oecologia (Berl.) 82: 227-237.

FLOW AND SUSPENSION-FEEDER GROWTH

41

Kirby-Smith, \V. \V. 1972. Growth of the hay scallop: the influence

of experimental water currents. J Exp. Mar. Biol. Ecol. 8: 7- IS.
Leonard, A. B., J. R. Strickler, and N. D. Holland. 1988. Effects of

current speed on nitration during suspension feeding in Oligometra

serripinnu (Echinodermata: Crinoidea). Mar. Biol. 97: 1 1 1-126.
McFadden, 0. S. 1986. Colony fission increases particle capture rates

of a soft coral: advantages of being a small colony. J. Exp. Mar. Biol.

Ecol 103: 1-20.
Okamura. B. 198-4. The effects of ambient flow velocity, colony size,

and upstream colonies on the feeding success of bryozoa. I. Bugula

stolonilera Ryland, an arborescent species. J. E.\p. Mar. Biol Ecol.

83: 179-193.
Okamura, B. 1985. The effects of ambient flow velocity, colony size.

and upstream colonies on the feeding success of bryozoa. II. Cono-

peuin iclicii/nin (Linnaeus), an encrusting species. / Exp. Mar. Biol.

Ecol. 89: 69-80.
Okamura, B. 1987. Particle size and flow velocity induce an interred switch

in bryozoan suspension-feeding behavior. Biol Bull. 173: 222-229.
Okamura, B. 1992. Microhabitat variation and patterns of colony

growth and feeding in a marine bryozoan. Ecology 73: 1502-1513.
Parsons, T. R., V. Malta, and L. M. l.alli. 1984. A Manual of Chemical

anil Biological Methods for Seawalcr Analy.ii.'i Pergamon Press, New

York.
Patterson, M. R. 1984. Patterns of whole colony prey capture in the

octocoral, Alcyonium siderinin. Biol. Bull 167:613-629.
Patterson, M. R. 1991. The effects of flow on polyp-level prey capture

in an octocoral, Alcyonium siderinin. Biol. Bull. 180: 93-102.
Rubenstein, D. 1., and M. A. R. Koehl. 1977. The mechanisms of filter

feeding: some theoretical considerations. Amer. Xalitr. Ill: 981-994.

Sdilii-hting. H. 1979. Boundary-Layer Theory. 7th ed., McGraw-Hill

Book Co., New York. 817 pp.
Sebcns, K. P., and A. S. Johnson. 1991. Effects of water movement on

prey capture and distribution of reef corals. Hydrobiol. 226: 91-101.
Shimeta, J., and P. A. Jumars. 1991. Physical mechanisms and rates

of particle capture by suspension feeders. Oceanogr. Mar. Biol. Annii.

Rev. 29: 191-257.
Smith, F. G. \V. 1946. Effect of water currents upon the attachment

and growth of barnacles. Biol. Bull. 90: 51-70.
Sokal, R. R., and F. J. Rohlf. 1981. Biometry. 2nd ed., W.H. Freeman

& Co., San Francisco. 859 pp.
Sponaugle, S., and M. l.aBarbera. 1991. Drag-induced deformation:

a functional feeding strategy in two species of gorgonians. J. Exp

Mar. Biol. Ecol. 148: 121-134.
Vogel, S. 1981. Life in Moving Fluids. Princeton University Press,

Princeton, NJ. 352 pp.
Vogel, S., and M. LaBarbera. 1978. Simple flow tanks for research

and teaching. Bioscicnce 28: 638-643.

\\ildish, D. J., and D. D. Kristmanson. 1979. Tidal energy and sub-
littoral macrobenthic animals in estuaries. J. Fish. Res. Board Can.

36: 1197-1206.
\\ildish, D. J., and A. M. Saulnier. 1992. The effect of velocity and

flow direction on the growth of juvenile and adult giant scallops.

J. Exp. Mar Biol. Ecol. 133: 133-143.
Wildish, D. J., D. D. Kristmanson, R. L. Hoar, A. M. DeCoste, S. D.

McCormick, and A. \V. White. 1987. Giant scallop feeding and

growth responses to flow. / Exp Mar. Biol. Ecol. 113: 207-220.

Reference: Biol. Bull. 185: 42-55. (August, 1993)

Ontogenic Changes in Microhabitat Distribution of
Juvenile Bay Scallops, Argopecten ir radians ir radians
(L.), in Eelgrass Beds, and Their Potential Significance

to Early Recruitment

ZAUL GARCIA-ESQUIVEL AND V. MONICA BRICELJ 1

Marine Sciences Research Center, State University of New York, Stony Brook. New York 11794-5000

Abstract. Ontogenetic changes in the vertical distribu-
tion of a cohort of juvenile bay scallops, Argopecten
irradians. on eelgrass, Zostera marina, were followed
throughout the summer and early fall in two Long Island
embayments (New York, USA). Despite site-specific dif-
ferences in eelgrass height and density, more than 95% of
post-settlement scallops remained attached above the
bottom until they reached a shell height of about 1 1 mm.
Over a 5-week period, scallops gradually relocated until,
at a mean size of 3 1 mm, all occurred on the bottom. The
decline in percent attachment coincided with a 5-fold in-
crease (from 16 to 84 ^moles min ' g muscle dry wt~')
in the activity of octopine dehydrogenase (proposed here
as an index of the scallops' capacity for burst swimming
activity), and in maximum rate of increase in the shell
aspect ratio. While attached to eelgrass, scallops were
nonuniformly distributed, with greatest concentration at
mid-canopy. Following disturbance, they rapidly regained
above-ground position, attaining asymptotic heights
within 3-10 h. This and prior studies suggest that the
climbing behavior of the bay scallop is an adaptive re-
sponse to high predation pressure at small sizes. Enhanced
scope for activity (predator avoidance) may enhance sur-
vival of scallops at intermediate sizes, when they become
too heavy to maintain elevation but have not yet attained
effective refuge in size.

Introduction

Bay scallops, Argopecten irradians. which commonly
inhabit shallow, sheltered bays along the east coast of the

Received 17 May 1992; accepted 10 May 1993.

1 Author to whom reprint requests should he addressed.

United States, are closely associated with seagrasses, par-
ticularly during their early life history. Planktonic larvae
of this species settle and attach by byssus threads, primarily
but not exclusively to submerged vegetation such as eel-
grass, Zostera marina L.; adults occupy a wider range of
habitats, including bare, sandy substrata. Thus, depen-
dence on vegetation and habitat restriction appear to de-
crease with age and size, as has been shown for a variety
of marine and freshwater fish that extend their foraging
grounds beyond areas with plant cover once they achieve
size refuge from predators (Ebeling and Laur, 1985; Wer-
ner and Hall. 1988).

In common with other pectinids such as the sea scallop,
Placopecten magellanicus, bay scallops can attach byssally
throughout life, but seldom do so as adults ( Belding, 1910;
Stanley, 1970). Attachment of juveniles is reversible and
dynamic, because Z. marina blades have a high turnover
rate and elongate rapidly, at a rate of up to 2-5 cm day"'
in the summer (Kemp el a/.. 1987). In pectinids, byssal
attachment and swimming represent antagonistic behav-
iors (Caddy, 1972). Ontogenetic changes in attachment
and swimming capacity of pectinids have been related to
morphological, hydrodynamic features of their shells, such
as aspect ratio (shell length to height ratio), umbonal angle,
and degree of auricle asymmetry (Stanley, 1970; Dadswell,
1990). Burst swimming, which provides scallops with a
mechanism to avoid predators, is associated with the pro-
duction of octopine in rapidly contracting adductor mus-
cle tissue. This end reaction of anaerobic glycolysis, which
serves an important role in replenishing NAD* and thus
maintaining glycolytic flux during functional anaerobiosis
in highly mobile molluscs such as scallops and cephalo-

42

MICROHABITAT OF JUVENILE BAY SCALLOPS

43

pods, is catalyzed by octopine dehydrogcnase (ODH), an
enzyme functionally analogous to lactate dehydrogenase
(LDH) (Gade and Grieshaber, 1986). Ontogenetic changes
in the activities of glycolytic enzymes such as LDH and
pyruvate kinase and their relation to swimming perfor-
mance have been described in several fish species (e.g.,
Somero and Childress, 1980), but changes in ODH activity
throughout the life cycle of pectinids have not been pre-
viously documented.

As reviewed by Brodie ct ul. (1991), prey have evolved
two types of defense mechanisms to curtail predation.
The first type, predator avoidance mechanisms, involves
spatial (e.g.. Palmer. 1983; Main, 1987; Werner and Hall,
1988) or temporal (e.g.. Kitting, 1985) segregation from
predators to minimize the probability of predator-prey
encounter. The second type, antipredator mechanisms,
increases the probability of survival upon encounter with
a predator. Antipredator mechanisms include morpho-
logical adaptations such as large size (Crowl, 1990), in-
creased ornamentation or thickening of the shell ( Vermeij,
1987), distastefulness, or behavioral responses (e.g., escape
response or immobility; reviewed by Main, 1987).

Bay scallops are epifaunal, incapable of complete or
prolonged valve closure, and less protected by morpho-
logical defenses than other molluscs with heavier, thicker
shells; thus they require alternate mechanisms to reduce
their vulnerability to predators. Using tethering tech-
niques, Pohle el al. (1991) demonstrated that above-
ground attachment to the eelgrass canopy gives juvenile
bay scallops a significant refuge from benthic predators.
Yet the laboratory experiments these investigators con-
ducted using artificial grass suggested that this refuge may
be ephemeral because the ability to attach above the bot-
tom decreased markedly over a narrow range of scallop
sizes (about 10 to 20 mm). The distributional pattern of
the epiphytic, above-bottom habit for juvenile A. irradians
and the transition to the adult epibenthic habit have not
been adequately described in the natural environment and
are the focus of the present study.

Information about the rate at which scallops can regain
an elevated position and the timing and size at which they
lose this ability is important for several reasons. First, it
is necessary for determining the relative profitability of
different elevations within the eelgrass canopy in terms
of growth and survival. Second, it is a prerequisite for
predicting the relative vulnerability of scallops to predators
during early ontogeny. Third, it is an important consid-
eration in rehabilitating stocks of this commercially ex-
ploited species because, in several states on the east coast
of the United States, rehabilitation efforts mainly involve
planting juveniles in suitable nursery habitat.

This work has three objectives: ( 1 ) to describe temporal/
ontogenetic patterns in the vertical distribution of bay

scallops in two bays in the Peconic-Gardiners estuary
(Long Island, New York, USA) which differ in eelgrass
structure; (2) to determine the rate of relocation and the
height attained by individual scallops in the eelgrass can-
opy following disturbance, as well as the effect of scallop
density on their climbing behavior in the laboratory; and
(3) to relate distributional changes observed in the field
to the swimming performance of scallops, as assessed in-
directly from shell morphometrics (aspect ratio) and from
the activity of octopine dehydrogenase in the adductor
muscle.

Materials and Methods

Laboratory studies

Juvenile scallops obtained from a local hatchery on
2 May and 1 June 1990 were maintained in a flow-through
upweller system at SUNY's Flax Pond Marine Laboratory
until ready for use in experiments. These were carried out
in rectangular plexiglass tanks (basal area = 31.5 X 78.5
cm) provided with recirculating, filtered ( 1 to 5 /urn) sea-
water introduced immediately below the water surface
(see Pohle el al., 1991, for a detailed description of the
experimental system). Eelgrass shoots were simulated with
artificial mimics constructed of buoyant, green polypro-
pylene ribbon (Synthetic Fibers Inc., Newton, PA) 0.5 cm
in width, woven into plastic VEXAR mesh at a density
of 500 shoots irT : , and buried under about 5 cm of clean
sand. Seawater was kept at 19 to 23C and 26 to 28 ppt
salinity with a cooling system and freshwater dilutions,
respectively. White fluorescent lamps provided artificial
12 h:12 h (lightidark) photoperiod.

A first set of experiments tested the effect of scallop
stocking density on the overall success of attachment to
vertical substrates. Three experiments were conducted
using scallops averaging 9.3 mm in shell height (H, greatest
distance from the umbo to the ventral margin), each with
density treatments of 300, 100 and 50 scallops per tank
(1213, 404, and 202 scallops m 2 , respectively) on 1 1 May
(H SE = 8.6 mm 0.09), 28 June (H SE = 10.0 mm
0.05), and 1 July (H SE = 9.2 mm 0.06). Shell
height was measured for a subsample of individuals, and
scallops were then randomly distributed on the bottom
of each tank (water depth = 30 cm; height of eelgrass
mimics = 25 cm). The number of scallops attached to
blade mimics and tank walls was recorded every 30 min
during the first 3 h, every hour during the following 3 h,
and at the end of 24 h.

A second set of experiments investigated individual,
size-specific climbing behavior. Individually marked scal-
lops were followed over time for 46-49 h. Their vertical
position was recorded hourly during the first 5-6 h and
at less frequent intervals thereafter. Individuals were

44

Z. GARCIA-ESQUIVEL AND V. M. BRICELJ

.NO

10 20KM

CONNECTICUT

Hal lock Bay

LONG ISLAND SOUND

Napeague Harbor

LONG ISLAND

Northwest Harbor

Peconic Bays

Figure 1 . Location of field study sites in eastern Long Island, New York.

identified with numbered, plastic-coated, miniature wire
markers glued with Krazy Glue to the upper valve at
least 1 day before running experiments. For each trial,
20-22 scallops of a given size class were released into each
of three experimental tanks (density = 89 scallops nT 2 )
containing eelgrass shoot mimics 50 cm in length. Three
scallop size classes were tested on the following dates: 1 3.2
mm (SE = 0.07) scallops on 8 and 10 June, 5.7 mm (SE
= 0.04) scallops on 1 1 and 13 June, and 7.2 mm (SE
= 0.04) scallops on 14 and 16 June. The coefficient of
variation in scallop sizes within any given experimental
tank never exceeded 13%. A preliminary experiment
showed that the height attained by 7-mm scallops did not
differ significantly between one tank containing eelgrass

mimics, canopy height = 50 mm and density = 500 shoots
m 2 , and two tanks containing natural, transplanted eel-
grass, canopy height = 50 cm and density = 224 shoots
irT 2 (ANOVAat48h, F= 1.104,df= 2, 61, .P = 0.338).

Field studies

I 'ertical distribution of natural set. The vertical distri-
bution of naturally occurring juvenile bay scallops within
the Z. marina canopy was characterized throughout the
summer and early fall of 1990 in two bays in eastern Long
Island, New York, which contrasted in eelgrass shoot
density and canopy height: Napeague Harbor (NAPH,
4101' N. 7203' W) and Northwest Harbor (NWH,

MICROHABITAT OF JUVENILE BAY SCALLOPS

45

4 1 1 ' N, 72 1 5' W) (Fig. 1 ). Both study sites are shallow
(about 1 m deep in NAPH and 2-3 m in NWH), well-
mixed, and characterized by gentle slopes, sandy substrate,
and fairly extensive eelgrass (Z. marina) beds, which sup-
ported productive bay scallop populations prior to the
occurrence of "brown tides" in the region (Hickey, 1977;
Eckman, 1987; Bricelj el al, 1987). They have also been
the target of scallop reseeding efforts in recent years (C.
Smith, pers. comm.. Cornell Sea Grant Coop. Extension,
NY; Tettelbach and Wenczel, 1991).

Eelgrass densities at the study sites were estimated in
the second week of September by counting the number
of shoots contained in 25-cm : quadrats randomly de-
ployed within the survey area. Surface water temperatures
were recorded with a hand-held thermometer ( 0.5C).

Although scallop settlement was first observed in NWH
in mid-July, sampling for determination of growth rate,
vertical position, and percent attachment of the scallop
population on eelgrass did not begin until 26 July, when
scallops averaged 4.5 mm in shell height and could thus
be readily sampled by divers. Sampling continued until
10 October at NWH and extended between 16 August
and 19 September at NAPH. In each harbor, an area well
within the eelgrass meadow was sampled weekly. The size
of the area was about 200 m : in NWH and about 50 m 2
in NAPH.

At each sampling, divers collected 100 to 150 juvenile
scallops by swimming along set transects. Using rulers,
the divers obtained in situ measurements ( 0.5 cm) of
the vertical position (height of attachment above bottom)
of each individual on the eelgrass blade, and the total
length of the blade. Scallops found on the bottom were
assigned a position of cm. The organisms were placed
into numbered, perforated plastic boxes and brought to
shore, where their individual shell height was measured
with digital calipers ( 0.01 mm).

Subsamples of 30 to 100 scallops (depending on size)
were transported live in coolers to the laboratory, where
they were immediately frozen and stored at -70C until
further analysis. These scallops were individually weighed
(total wet body weight) using an analytical balance ( 0.1
mg), and lyophylized. Soft tissues were then dissected and
weighed using a Cahn electrobalance ( 1 jug) or analytical
balance, depending on scallop size. Shell height and length
(greatest anteroposterior dimension) were determined
prior to dissection in order to calculate the aspect ratio.

Octopine dehydrogenase activity. Lyophylized tissues
were stored with dessicant at -70C until used for enzyme
assays. The adductor muscle of individual scallops (in-
cluding both catch and phasic portions) was dissected out,
weighed, and used for determination of octopine dehy-
drogenase (ODH. EC 1.5.1.11) activity, because more than

97% of the total activity in whole scallop homogenates is
found in this tissue (Baldwin and Opie, 1978).

Powdered muscle samples (ca. 1 mg) were homogenized
with a sonicator probe (Bronwill, Biosonik III) in 1 ml of
100 mM Tris-HCl buffer (pH 7.5) containing Triton
x-100 (1% v/v). Homogenates were cooled in ice/water
(0-2C) during, and for a 30-min incubation period fol-
lowing, sonication. They were then centrifuged for 30 min
at 1C and 16,000 X g. The supernatant was decanted
and assayed for ODH activity at 25 C by following
changes in absorbance at 340 nm due to the oxidation of
NADH, using a Milton Roy Spectronic 1201 spectropho-
tometer equipped with a thermal cell controlled by an
external, recirculating water bath. Activity was determined
by dividing the rate of change in absorbance by the ex-
tinction coefficient (f 340 = 6.23 mM~ [ cm" 1 ) as described
by Fersht (1985). All determinations were made in du-
plicate, using 50 n\ of tissue extract in a total extraction
volume of 1 ml, and were completed within several hours
of tissue preparation. The composition of the reaction
mixture and the concentration of the reactants were those
reported to yield maximum enzyme activities (Baldwin
and Opie, 1978): 1 mM sodium pyruvate, 0. 1 mMNADH,
10 mM L-arginine and 100 mM tris-maleate buffer (pH
7.0). No controls were run for nonspecific activity because
previous studies have demonstrated that in scallops, in-
cluding A. irradians, the contribution of lactate dehydro-
genase to the oxidation of NADH is negligible (Baldwin
and Opie, 1978; Grieshaber, 1978;deZwaan elal. 1980;
and Chih and Ellington. 1983).

Relocation experiments. Experiments designed to test
the ability of juvenile scallops to relocate (climb and re-
attach) to eelgrass blades following dislodgement were
carried out in Northwest Harbor and within sandy habitat
in Hallock Bay (4102' N 75 15' W; mean depth at low
tide = 0.5 m; tidal range = 0.75 m) (Fig. 1).

The first relocation experiment was carried out in NWH
on 9 August using scallops collected from natural popu-
lations at this site. Divers collected 1 50 scallops (H = 8.6,
SE = 0. 1 3) after determining their individual position on
eelgrass as described earlier. Scallops were measured at
the shore, individually numbered, and held in ambient
bay seawater until released (within about 1 h of collection).
Scallops were freely broadcast on the bottom of a previ-
ously marked plot within the eelgrass bed, where no pred-
ators were present. A diving survey around and inside the
plot was carried out the following day (24 h after release)
and the vertical position of each recovered individual was
recorded.

A second relocation experiment was conducted using
hatchery-reared scallops ( 10 mm), which were transported
from the Flax Pond Laboratory to Hallock Bay ( 1 .5 h) in
coolers containing ice packs layered with wet newspaper.

46

Z. GARCIA-ESQUIVEL AND V. M. BRICELJ

Unmarked scallops were released by divers on 28 August
within a 30-cm 2 area at the center of sandy plots ( 1 m
X 2 m) within an eelgrass meadow averaging 32 cm in
canopy height and 249 shoots m~ 2 in density. No natural
scallop set was observed at this site in the year of the
study. The perimeter of the plots was delimited by a gal-
vanized chain (4.8 mm = 3/16 in. diam.), and plot lo-
cation was marked with bright fluorescent subsurface
buoys (Fig. 2 in Pohleet at, 1991). Three hundred scallops
were released in each of four experimental plots, and the
percent attachment and vertical position of recaptured
scallops were recorded after 3 h (plot 1 ), 5 h (plot 2) and
24 h (plots 3 and 4). Diver surveys covered a total area
of 12 m 2 (a 2-m 2 plot plus a 10-irr-peri meter area located
1 m around each plot). Plots were thus sampled destruc-
tively, rather than repeatedly over time, to avoid distur-
bance by divers.

Statistical analysis

Except where otherwise indicated, statistical analyses
followed standard procedures described by Sokal and
Rohlf(1981). Percent attachment data obtained from the
three stocking density trials were pooled for each time
interval and scallop density (50, 100, and 300 scallops
per tank). Differences in arcsine-transformed percent at-
tachment values obtained every hour between 1 and 6 h
and at the end of 24 h were analyzed with a repeated one-
way ANOVA (Wilkinson, 1990).

Differences in height attained on eelgrass mimics with
scallop size were analyzed by a two-step procedure. The
first step consisted of a posteriori multiple comparisons
of the mean height attained by scallops (three trials pooled
for each size) at 1,3, 5, 10, 24, and 46-49 h. A Tukey-
Kramer test was used on scallop sizes that had homoge-
neous variances (13.2-mm scallops), and the Games and
Howell test was used for those with heterogeneous vari-
ances (5.7- and 7.2-mm scallops). In the second step, the
position of smaller scallops (5.7 and 7.2 mm) was (1 n
+ 1 (-transformed to correct for heterogeneity of variances,
and the mean height of the scallops on eelgrass was com-
pared using a repeated two-way ANOVA (Wilkinson,
1990) with scallop size and aquaria as factors. Time treat-
ments were selected on the basis of results of the multiple
comparisons tests and fulfillment of criteria for homo-
scedasticity among samples (F max test).

The degree of association between shell height and ele-
vation of scallops attached to eelgrass blades in the field
was measured with the Pearson product-moment corre-
lation coefficient. All data gathered throughout the sum-
mer were included in this analysis, but excluding scallops
found on the bottom, i.e.. at height = 0. The height at-
tained, calculated as a fraction of total blade length to

normalize data for differences in canopy height between
bays, was used to test for differences between the relative
vertical distribution of scallops from NWH and NAPH,
using the Mann-Whitney test for two independent sam-
ples.

Data from the relocation experiment conducted in
Hallock Bay were analyzed by the a posteriori Games and
Howell approximate test for equality of means, with height
attained at each sampling time as the dependent variable.
For the relocation experiment carried out in NWH, the
nonparametric Wilcoxon signed-rank test for paired
comparisons was used to test for differences in the mean
position of scallops at the time of collection and 24 h
following release.

Results

Laboratory studies

Scallop stocking density, over the range tested in this
study, had no significant effect on percent attachment to
eelgrass mimics (P = 0.293, repeated one-way ANOVA).
Percent attachment averaged 80% 2 h after release and
85% by the end of 24 h, irrespective of stocking density.

Although scallops swam actively during the first 1 5 min
following release into the experimental tanks, very few
(< 1%) attached to the blades by swimming onto them.
Swimming generally resulted in vertical rather than hori-
zontal displacement. Scallops primarily gained an elevated
position by crawling, as reported by Pohle et at (1991).
Downward crawling was never observed, suggesting that
scallops may display negative geotaxis at this stage of their
life cycle. Tracking of individual trajectories showed that,
until they attained their final attachment position, scallops
occasionally fell off the blades and had to re-initiate their
ascent; but on average, over all trials, only 13% (range
= 5 to 29%-) fell to the bottom during ascent. Climbing
behavior consistently showed two phases: rapid crawling
during the first 4-5 h after broadcasting on the bottom,
followed by a slowing or complete cessation of crawling.
Mean crawling rates, calculated over the first 4 h for an-
imals that did not fall during ascent, were 4.5 cm h" 1 (SE
= 0.8, /; = 156) for small scallops (5.7- and 7.2-mm size
classes) and 1.2 cm h ' for 13-mm scallops (SE = 0.2,
= 41).

Laboratory experiments showed that the vertical dis-
tribution of scallops on eelgrass mimics was markedly
affected by size. Within a 50-cm canopy, small scallops
(H = 5.7 and 7.2 mm) reached a near-average asymptotic
elevation of 20.4 and 18.8 cm respectively, 10 h after being
released into the tanks (Fig. 2); larger scallops ( 1 3.2 mm)
reached a near-asymptotic height of 6.0 cm after 1 1 h.
Mean height attained by small scallops after 1 h was sig-
nificantly different from that achieved after 24 h and

MICROHABITAT OF JUVENILE BAY SCALLOPS

47

40

E
o

CD

c

30 -

20 -

10 -

H = 5.7 mm (SE = 0.04)

H - 7.2 mm (SE = 0.04)

* H - 13.2 mm (SE = 0.07)

30 -

20 -

10 -

4 8 12 16 20 24 28 32 36 40 44 48

Time (hours)

Figure 2. Temporal changes in the mean height above bottom at-
tained by Argopecien irradians of three different size classes on eelgrass
mimics in the laboratory (mean blade height = 50 cm). Data points are
fitted to a rectangular hyperbolic function (Y - aX/b + X). Vertical
bars represent 95% confidence intervals.

46-48 h (for 7.2- and 5.7-mm scallops, respectively), but
not before (P < 0.01, Games and Howell approximate
test for equality of means with unequal variances. Table
IB). A Tukey-Kramer test of multiple comparisons also
indicated significant differences (P < 0.01) in the mean
position of larger (13 mm) scallops after 1, 3, or 24 h
(Table IB). No significant size effects (P = 0.428) were
found in the mean height attained by 5.7- and 7.2-mm
scallops, or in that attained by scallops of the same size
class located in different aquaria (P = 0.376, Table IA).
Mean elevation of small scallops (< 7.2 mm) coincided
with mid-canopy height (0.57 of blade height; SD = 0.286,
= 126), whereas 22% of the scallop population was found
near the top (upper 1/10) of the canopy, and none at the
base of shoot mimics. In general, however, scallops were
distributed throughout most of the length of eelgrass
mimics at the end of 48 h (from 2.5 to 48 cm above
bottom).

Field studies

Vertical distribution of natural set. A single cohort of
first-year A. irradians occurred in both embayments
throughout this study (July-October), as indicated by
unimodal size-frequency distributions obtained in NWH
and NAPH over time. No significant new recruitment of
post-settlement scallops was observed during late summer
and early fall at either study site, except for a few new
recruits (< 4% of the total) observed in NAPH in early
September, although spawning in these embayments is
known to extend throughout June and July (Bricelj et al,
1987). Growth parameters (changes in total body weight

Table I

Results of repeated two-wa\ analysis of variance for comparison of
mean heights attained by Argopecten irradians on eelgrass mimics

A.
Source of
variation

Degrees of
freedom

MS

F

Significance

Size

Between subjects

1

2.043

0.632

NS

Aquaria
Size x aquaria
Error

2
2

124

3.185
8.321

0.985

2.573

NS
NS

Time

Within subjects
5

21.173

62.309

* * *

Time x size

5

0.139

0.409

NS

Time x aquaria
Time x size

10

0.327

0.963

NS

x aquaria
Error

10
620

0.513
0.340

1.509

NS

B.

A posteriori multiple comparisons

5.7-mm scallops

Time(h) 1 3 5 10 24 46

Height attained (mm) 10.7 16.1 18.5 20.4 22.4 29.9

7.2-mm scallops

Time(h) 1 3 5 10 24 48

Height attained (mm) 9.5 13.4 15.7 18.8 21.2 26.4

13.2-mm scallops

Time(h) 1359 25 49

Height attained (mm) 1.3 3.1 3.8 4.7 6.9 8.4

A. Scallop size (5.7 and 7.2 mm) and experimental aquaria (3 per
size), with n = 20-23 scallops each) were used as testing factors. Runs
were carried out on separate dates for each size under identical
laboratory conditions. Comparisons were based on records for the
same individuals at times = 1,3, 5, 10, 24, and 46-48 h. B. Results of
the Games and Howell approximate test for equality of means are
shown for 5.7- and 7.2-mm scallops at selected sampling times. The
Tukey-Kramer test was used for 13.2-mm scallops. *** = P < 0.001.
NS = not significant at P > 0.05.

48

Z. GARCIA-ESQUIVEL AND V. M. BRICELJ

and dry tissue weight) of scallops collected in NWH are
shown in Table II. Surface water temperatures at this site
averaged 27C in August and 23C in September, attain-
ing maxima during the second week of August. Temper-
atures at NAPH were within 1C of those recorded at
NWH. Shell growth rate throughout the study period av-
eraged 13.3 and 12.5 mm month ' at NWH and NAPH
respectively. Based on these measured growth rates for
juvenile scallops and a duration of 7 days for metamor-
phosis of dissoconch larvae into 1.5-mm plicated juveniles
in NWH (Eckman, 1987), we estimate that initial settle-
ment of this cohort occurred in the second week of July.
Densities of juvenile scallops were determined quan-
titatively only at NWH on 16 August and 20 September,
when they averaged 16 and 14 scallops m : respectively.
Although eelgrass shoot density and canopy height were
1.5 times higher in NAPH (mean density SE = 704
35 shoots m* 2 ; mean canopy height SE = 38 0.5
cm ) than in NWH (density = 464 29 shoots m 2 ; canopy
height = 23.5 0.3 cm), temporal patterns in the per-
centage of scallops attached to eelgrass were very similar
at both sites (Fig. 3), and therefore did not appear to be
strongly influenced by differences in eelgrass structure. In
NWH, 100% of the population remained attached to eel-
grass blades until the second week of August, when scal-
lops reached 11.2 mm in mean shell height. In NAPH.
100% attachment was also observed until scallops reached
1 1.3 mm, one week later than in NWH (Fig. 3). A 5-week
transitional period followed, during which scallops relo-
cated from their elevated position on eelgrass blades to
the bottom. At both sites, more than 90% of new recruits
were found on the bottom by the time they reached a
mean size of 26-29 mm. Scallops found on the bottom

(h = 0) are included in the calculation of mean heights
plotted in Figure 4, showing clearly that in both bays the
entire population had relocated to the bottom by the time
scallops reached 31 mm.

At NWH, shell growth rate increased from 1 .9-2.8 mm
week" 1 between 26 July and 30 August, when most of the
scallops remained attached to the eelgrass canopy, to 3. 1-
4.6 mm week" 1 during the period of relocation to the
bottom (30 August to 23 September) (Table II), when
attachment dropped sharply from 75% to 1%. Maximum
shell growth during the first week of September coincided
with a reduced rate of growth for soft tissues, which was
equal to 30 mg week" 1 , compared to values of 52 and 59
mg week ' during the preceding and following weeks re-
spectively (Table II). At NAPH, maximum rate of shell
growth (4.5 mm week' 1 ) was recorded at the same time
as in NWH. Allometric changes in shell shape with growth
are evidenced by the sharp increase in the degree of shell
elongation, as measured by the aspect ratio, between sizes
of 10 and 25 mm, when scallops are gradually shifting
from a byssate to a free-living habit (Fig. 5). Near-asymp-
totic values in this parameter (> 1 .05) are attained at larger
sizes.

Diver observations indicated that scallops exhibited a
marked increase in swimming activity (diver avoidance
response) during transition from an elevated position to
the bottom, especially during the last week of August and
the second week of September. This habitat shift was ac-
companied by qualitative changes in vegetation charac-
teristics, notably an increase in the incidence of senescent
(brown or discolored) Z. marina blades. Furthermore, in
July, the drift red alga (Gracilaria vermcosa, was mostly
restricted to the subtidal zone delimited by the lower eel-

Table H

Mean tola! bodv weigh! (TBW), mean dry soft /issue might (DTW), and shell growth rates of juvenile bay scallops collected from natural
populations in Northwest Harbor. New York, between -6 July and 10 October

Date

Temp. (C)

TBW (mg)

DTW (mg)

Shell growth rate
(mm wk~')

Mean

(SE; n)

Mean

(SE; H)

26 July

25

_

_

2 Aug.

27

83.3

(53.6; 49)

5.7

(0.4; 50)

2.2

9 Aug.

30

228.6

(15.0; 50)

15.7

(1.1; 48)

1.9

16 Aug.

29

389.4

(27.6; 51)

28.4

(2. 2; 51)

2.6

23 Aug.

22

753.8

(53.0; 50)

57.7

(4.2; 48)

2.8

30 Aug.

27

1459.4

(82.8; 48)

109.3

(6.5; 48)

2.2

7 Sept.

25

1954.4

(128.6; 40)

143.8

(8. 7; 48)

4.6

13 Sept.

25

2587.2

(177.6; 37)

193.2

(14.4; 37)

3.5

23 Sept.

20

4742.9

(276.2; 34)

342.4

(21.4; 33)

3.1

29 Sept.

22

5858.4

(423.6; 17)

424.8

(33.0; 17)

1.9

10 Oct.

21

8960.4

(538.6; 19)

608.8

(45.6; 16)

3.6

Standard error and sample size are indicated in parentheses.

MICROHABITAT OF JUVENILE BAY SCALLOPS

49

n
a
o

~o
u
(rt

o
u
.c
u
o

1990

Figure 3. Temporal changes in mean shell height and percent at-
tachment to eelgrass blades of a first-year cohort ofArgopecten irradians
in two eastern Long Island bays, during the summer and early fall of
1990. All standard errors are smaller than the symbol denoting mean
shell height. Mean eelgrass (Zoslera marina) shoot densities within the
scallop distributional area are indicated for each harbor.

leptokurtic (g, = 0.547 and 0.151, and g 2 = -0.101 and
-0.718 at NWH and NAPH respectively. Skewness was
significant only at NWH (/ = 5.24, P < 0.00 1 ), but kurtosis
was significant at NAPH (/ = 2.95, P < 0.01). The size-
specific distribution of scallops, expressed as a fraction of
total blade length, differed significantly between the two
bays (P < 0.001, nonparametric Mann-Whitney test for
two independent samples). In general, a larger proportion
of the scallop population was located in the upper half of
the canopy in NAPH than in NWH (about 50 and 30%
respectively; Fig. 6).

Octopine dehydrogenase activity. Octopine dehydro-
genase activity was used as an instantaneous index of the
scallops' capacity for burst swimming activity. Within-
individual variability in ODH activity, determined by as-
saying two to three replicate subsamples of ground ad-
ductor muscle from each scallop collected on 10 October
(mean adductor dry weight = 266 mg). averaged 7%.
Lowest mean enzymatic activity (16 ^moles min"' g~'
adductor dry wt~ ' ) and lowest variation in activity among
similar-sized individuals were measured in early August
(Fig. 7), when the entire NWH scallop population
(H = 6.7 mm) remained attached to the eelgrass canopy
(see Fig. 3). Mean ODH activity increased markedly be-
tween 30 August and 23 September, during relocation to
the bottom, when attachment incidence dropped from

grass boundary, outside the scallop's main distributional
area. However, by the first week of September, G. verni-
cosa was conspicuous throughout the entire scallop zone,
intermingled with Z. marina or in irregular patches of
1 to 2 nr.

Contrary to laboratory results, no significant correlation
was found between scallop attachment height (excluding
individuals found on the bottom) and scallop size in either
NWH (r = 0.003, P = 0.176) or NAPH (r = 0.001,
P = 0.538). This also differs from the laboratory results
of Pohle el al. (1991), who found that the relative pro-
portion of scallops attached to the upper vs. lower half of
the canopy decreased monotonically between scallop sizes
of 6 and 20 mm.

Although scallops were found distributed throughout
the eelgrass canopy at both study sites, they clustered pri-
marily at or around mid-blade height, between 0.3 and
0.5 of the canopy height, as indicated by the modes of
the frequency distributions shown in Figure 6. This ob-
servation is in general agreement with laboratory results.
The frequency distributions observed in the field departed
significantly from normality (Kolmogorov-Smirnov in-
trinsic test for goodness of fit, Z) max = 0.048 and 0.053 for
NWH and NAPH respectively, P = 0.01). These distri-
butions were skewed towards the upper canopy and were

^_^

o o Napeague Harbor

f

NW Harbor

o

I i

(n 25 -

V)

\

O

\

rji 20 -
~0)

HH

\ T

LU

c 15 '

v\

C 10 -

'-M

o 5 "

^H\

^z\

Q_
n _

vi

10

20

30

40

Scallop Shell Height (mm)

Figure 4. Vertical position (mean height above bottom SE) of
Argopei'len irradians on eelgrass (Znstera marina) blades as a function
of shell height, in two eastern Long Island bays. Scallops found on the
bottom (height = 0) are included in calculation of the means; eelgrass
canopy height = 38 and 23 cm in Napeague and Northwest Harbors
respectively.

50

Z. GARCIA-ESQUIVEL AND V. M. BRICELJ

1.05 -

v

.C

1.00 -

0)

.C
00

0.95 -

46

28

10 20 30 40

Mean Shell Height (mm)

Figure 5. Aspect ratio (AR = mean shell length:height (H) ratio
SE) with increasing shell height of a natural population ofArgopecten
irradians collected in Northwest Harbor. Sample size is indicated for
each mean value. Fitted curve is described by the equation AR
= (a- d)/[l - (H/21.242) """ + d], where d = 0.944.

75% to 1%. Maximum mean values of 84 ^moles min '
g~' were recorded on 23 September, when scallops had
reached a mean size of 29 mm, and appeared to decline
thereafter.

Over the scallop size range sampled in this study, there
was an overall, significant, positive relationship between
octopine dehydrogenase activity (A) and muscle dry
weight (W in milligrams), as described by the equation:
A = 9.87 (SE = 3.52) w' U5ISE= "" 7 > (r = 0.81; n = 57),
determined using SYSTAT iterative nonlinear curve fit-
ting (Wilkinson, 1990). It remains to be determined
whether adult scallops exhibit a decline in ODH activities.
High individual variability in ODH activity on any given
sampling date may be partly attributable to the large vari-
ation in scallop sizes. A comparable three-fold range in
the weight of the adductor muscles was obtained within
any given sampling date throughout the study period.

Relocation experiments. Results of the relocation ex-
periment carried out in NWH using individually marked
natural set are shown in Figure 8. Forty-three percent of
the scallops initially released (n { = 150. H = 8.6 mm)
were recovered alive and 7% were found dead (crushed
or with empty intact valves) by the end of 24 h. There
was no significant difference between the mean position
at time zero (collection) and 24 h following dislodgement
(P = 0.063, Wilcoxon signed-rank test for paired com-
parisons; Fig. 8), although final height was lower than

initial height in 72% of scallops recovered on eelgrass,
suggesting that 24 h may have been insufficient to achieve
maximum, asymptotic height in this field experiment.

Relocation experiments carried out in Hallock Bay with
hatchery-reared scallops (H = 10 mm) resulted in rates
of attachment similar to those found in the laboratory
with scallops of comparable size. Three hours after being
released in the field, about 60% of the scallops recovered
were attached to eelgrass blades, at a mean height of 8.7 cm
(0.3 of canopy height), and these values remained relatively
constant thereafter (Fig. 9). Maximum percent attachment
and maximum height attained (when expressed as a fraction

D

->->

O

en

CL

"5
o

CO

o

C
0)

0)

20 -

10 -

20 -

10 -

Napeague Harbor

n - 407

X - 0.54

SD - 0.274

Relative Elevation

Figure 6. Distribution of juvenile Argopcctcn irradians attached in
the eelgrass canopy. Relative elevation is expressed as a fraction of total
blade length. All individuals collected above ground during the study
are included, since there was no relationship between elevation and size
of attached scallops.

MICROHABITAT OF JUVENILE BAY SCALLOPS

51

100-

DATE

Figure 7. Octopine dehydrogenase activity (in ^moles of substrate
min~ ' g freeze-dned weight of adductor muscle" ' ) of juvenile Argopecten
irradians collected from a natural population in Northwest Harbor, be-
tween 2 August and 10 October. Error bars represent 95% confidence
intervals around the mean for each sampling date. Total number of
assayed individuals is indicated in parentheses; the 10 October mean
includes three values obtained for pooled rather than individual samples.

of total canopy height), were thus lower than those of natural
set in NWH and NAPH, and also somewhat lower than
those obtained in laboratory trials (Fig. 2). No significant
difference was detected between the mean height attained
after 3, 5, and 24 h (F = 0.178).

Discussion

Laboratory and field results demonstrate that bay scal-
lops can rapidly gain and maintain above-bottom position
in the eelgrass canopy at sizes below ca. 10-15 mm. The
adaptive significance of this behavior, which allows spatial
segregation of juveniles from benthic predators and con-
specific adults, may involve (a) enhanced survival through
avoidance of predators and burial in unconsolidated sed-
iments; (b) enhanced growth by positioning scallops in
an optimum hydrodynamic regime that minimizes ex-
posure to resuspended bottom sediments and maximizes
food capture; or (c) a combination of these factors. Sea-
grasses are known to markedly reduce near-bottom cur-
rent velocities and water flux (e.g., Fonseca et al., 1983;
Eckman, 1987; Irlandi and Peterson, 1991) while gener-
ating increased turbulence at the water-canopy interface
(Fonseca et al., 1982; Gambi et al., 1990), thus creating
steep vertical gradients in the flow regime. Significant

E
o

en
OT
o

0)

LJ

C
O

c
o

'-*^

'OT
O
Q.

Scallop Shell Height (mm)

Figure 8. Above-bottom attachment height of individually marked,
juvenile Argopecten irradians from a natural population in Northwest
Harbor, before and after dislodgement from eelgrass blades. Open circles
indicate scallop position at the time of initial sampling, and closed circles
indicate position 24 h after dislodgement. Mean initial and final heights
(9.9 and 8.0 cm) are indicated by solid and dashed horizontal lines re-
spectively. The arrow marks the mean eelgrass canopy height at the
study site.

variation in scallop growth rates with elevation within the
seagrass bed was found by Ambrose and Irlandi (1992)
and Borrero and Bricelj (in prep.), an effect that may be
related to vertical gradients in food quality and quantity,
as well as to flow.

The predator-refuge value of the eelgrass canopy for
juvenile bay scallops has been well established (Pohle et
al., 1991; Ambrose and Irlandi, 1992). Refuge in elevation
is effective in the presence of both nonswimming predators
such as green crabs (Carcinus maenas), mud crabs (Dis-
panopeus sayii), and spider crabs (Libinia spp.) (Pohle et

E *'

Shell Ht. = 10 mm

" 80 s?

o

Eelgrass Ht. = 31.8 cm (SE = 1.1)

y> 15 -

a--""*

- 60 -

o

,

O>

/

"5

Ld 10 -

'LI

o

- +0 CO

C

l/li

-a

O

0}

1 /

.c

C 5 -

1

- 20

O

U Hallock Bay

O

M

/

-

16

24

Time (hours)

Figure 9. Time-dependent changes in percent attachment and at-
tachment position on eelgrass blades (mean height SE) of hatchery-
reared juvenile Argopecten irradians released in Hallock Bay, Long Island.
(Total number of scallops recovered = 50 and 66 at 3 and 5 h respectively,
and 95 and 60 from each of two plots at 24 h.)

52

Z. GARCIA-ESQUIVEL AND V. M. BRICELJ

ai. 1991) and swimming, portunid crabs (Bauer and Bri-
celj, unpublished). The existence of a spatial, off-bottom
refuge from predators in vegetated habitats was also de-
scribed for the caridean shrimp Tozeuma carolinense
(Main, 1987), and several gastropod molluscs. For ex-
ample, juveniles of the freshwater snail Planowella
trivolvis and all life history stages ofPhysella virgata (both
characteristically thin-shelled species) are known to crawl
above the water line to avoid predation by crayfish, Pro-
cambrus simulans (Alexander and Covich, 1991). A sim-
ilar predator-avoidance strategy was reported for the in-
tertidal marsh periwinkle Littorina irrorata (Vaughn and
Fisher, 1988).

During the period of attachment to eelgrass, most scal-
lops were found concentrated at about mid-canopy height,
with fewer individuals at the tips and bases of eelgrass
shoots. In the field, this vertical distribution could reflect
preferential aggregation of scallops at mid-height, or it
could result from secondary effects such as differential
predation pressure or physical disturbance with varying
height. The reduction in scallop numbers near-bottom
can be readily ascribed to the effects of benthic predation
(Pohle el ai, 1991), but selective depletion in the upper
canopy is harder to explain. Although this region is char-
acterized by higher current velocities and turbulence in-
tensity (Gambi ct al., 1990), free-stream velocities at the
two study sites were relatively weak, typically attaining
maxima < 10 cm s" 1 (Borrero and Bricelj, unpublished),
and thus unlikely to cause selective dislodgement of scal-
lops in the upper canopy. Eckman et al. (1989) found
that juvenile bay scallops commonly experience current
speeds > 17 cm s" 1 in seagrass beds in North Carolina,
and remain attached to Zostera blades at flume velocities
of at least 15 cm s '. We cannot, however, rule out the
possibility that selective removal of scallops occurs during
storm events. The northern puffer, Sphoeroides macula-
tus, a transient predator in some Long Island bays, can
preferentially forage for scallops in the upper canopy
(Tanikawa and Bricelj, unpublished). Greatest concen-
tration of scallops around mid-canopy, however, was ob-
served in both NWH and NAPH, even though S. macu-
latits was present during the summer of 1990 only in the
former embay ment.

Eckman et al. (1989) found that 5- to 15-mm scallops
attained a mean elevation of 3.5 cm within an 8-cm mixed
seagrass canopy in Core Sound, North Carolina. In our
study, rates of relocation and patterns of microhabitat
distribution (attainment of a near-asymptotic mean ele-
vation at about mid-canopy height within 5 to 8 h) were
similar for small scallops in the field and in the laboratory.
This was the case despite the absence of food, predators,
wind and wave action, and epiphytic cover in the labo-
ratory, although fewer scallops (<3%) were found in the

top 1 / 1 of the canopy in the field than in the laboratory
(22%). This similarity suggests that the scallops' distri-
bution on eelgrass results from an adaptive behavior,
rather than solely from mortality or disturbance operating
after settlement. The findings of Pohle et al. ( 1 99 1 ) further
suggest that predator avoidance is the most likely selection
factor for climbing behavior in juvenile scallops, although
attachment height may also reflect preference within a
vertically heterogeneous flow microenvironment.

The present study demonstrates that scallops between
the sizes of about 14 and 29 mm undergo a gradual on-
togenetic shift in habitat, from the emergent canopy to
the bottom, over a 5-week period. The timing of this tran-
sition, which was remarkably similar in two nonadjacent
bays, may be influenced by physical constraints that pre-
vent larger scallops from maintaining elevation. Because
bay scallops have no buoyancy mechanism and gravita-
tional force scales in proportion to mass, larger scallops
are expected to exhibit increasing difficulty in generating
sufficient lift and/or strength of byssal adhesion to over-
come the force of gravity and support their own weight.
Irreversible relocation to the bottom, where predation risk
is highest, is observed at a size of about 30 mm and a
total body mass of about 5 g (weight in water was not
determined in this study). At this size, however, scallops
can attain complete refuge from the mud crab, Dyspan-
opeus sayi, a numerically dominant predator in these bays
(Strieb, 1992), and partial refuge from other common crab
predators (Tettelbach, 1986).

A sharp drop in percent attachment with scallop size
is consistent with laboratory results obtained by Pohle et
al. ( 1991 ) at a constant temperature, using eelgrass mim-
ics. Percent attachment of scallops < 10 mm was consis-
tently high in both studies, ranging from 97 to 100% in
NWH and NAPH, and from 83 to 98% in the study by
Pohle ct al. (1991). High percent attachment was also
obtained in our laboratory stocking-density experiments
(e.g., 85% for 9-mm scallops), although maximum at-
tachment was only 65% for 10-mm hatchery-reared ani-
mals broadcast in Hallock Bay. Variable attachment suc-
cess may result from differences in condition and scope
for activity between hatchery-reared animals and natural
set, from selective predatory loss of unattached scallops
in the field, or from a combination of both factors. This
variability may have important consequences for the sur-
vival of cultured juveniles used in reseeding efforts aimed
at stock enhancement. An inverse relationship between
percent byssal attachment and scallop size (between 40
and 150 mm) was also described, both in situ and in the
laboratory, in the sea scallop, Placopecten magellanicus,
a species that attaches primarily to bottom features such
as shell and gravel (Caddy, 1972).

MICROHABITAT OF JUVENILE BAY SCALLOPS

53

Our qualitative observations suggest that swimming
activity (escape response) of Argopccten increases at in-
termediate sizes, during the period of transition to the
bottom. This corroborates observations made by Tettel-
bach (1991), who reported that adults and 2- to 7-mm
spat of .-1. irradians swam infrequently compared to 15-
to 35-mm juveniles. Dadswell (1990) identified three
stages in the life history of the longer lived species Pla-
copeclen magellanicus. including a stage of highest mo-
tility at intermediate sizes (30 to 100 mm). Manuel (1992)
also observed that juvenile sea scallops (< 10-12 mm)
generally remain attached and are more reluctant to swim
than larger juveniles. This author identified a discontinuity
in the relationship between relative swimming speed and
Reynolds number at scallop sizes of 12-16 mm: she at-
tributed reduced swimming capacity below this size to
increased drag and greater energy cost associated with
swimming below this hydrodynamic threshold. Small ju-
veniles were reported to swim primarily vertically,
achieving little horizontal displacement (Manuel, 1992).
This type of swimming behavior (vertical ascent), which
was also characteristic of small (< 10 mm) Argopecten in
our study, is expected to be relatively ineffective in avoid-
ing predators, unless elevation allows scallops to be more
readily advected away from predators by bottom currents.

In general, scallop species that are active and frequently
swim have higher aspect ratios than those that rarely swim
and remain byssate throughout life (Stanley, 1970). Fur-
thermore, several scallop species that occur as free-living
adults and byssate juveniles display an ontogenetic in-
crease in both umbonal angle and aspect ratio (Stanley,
1970; Gould, 1971). This was confirmed for Argopecten
irradians irradians in this study, where aspect ratios in-
creased from about 0.95 to 1.07 at sizes between 5 and
43 mm. We are aware of no prior estimates of aspect
ratios for juveniles of this subspecies. Our values are com-
parable to those reported for Placopecten magellanicus.
a more proficient swimmer than Argopecten, over its life
cycle (0.90 to 1.05 in the range 15 to 160 mm; Dadswell,
1990). An increase in aspect ratio with growth leads to a
decrease in frictional drag and an increase in the lift coef-
ficient of scallops, thus providing an advantage in swim-
ming by partially counteracting the mechanical difficulties
associated with increased weight (Gould, 1971).

The increase in swimming activity divers observed while
the bay scallops were relocating to the bottom is presum-
ably related to an increase in predator avoidance or dis-
persal potential and coincided in this study with a marked
increase in adductor muscle ODH activity. A. irradians
relies to a greater extent on anaerobic glycolysis via the
octopine pathway to supply energy during burst activity
(escape response) than do other scallops such as Placo-
pecten magellanicus (de Zwaan et ai, \ 980) and Chlamys

opercularis (Grieshaber, 1978). In these species, energy is
primarily generated through breakdown of arginine phos-
phate, and octopine production is largely restricted to the
recovery phase following exhaustion. Thus, in A. i. con-
centricus, glycolysis contributes up to 25-88% of ATP
production during exhaustive swimming (Chih and El-
lington, 1983). Therefore, in this species, ODH per gram
of muscle can be interpreted as an index of weight-specific
potential for anaerobic metabolism. Enzymatic activity
of field-collected scallops showed a 5-fold increase be-
tween sizes of 6 and 29 mm, thus following a scaling
pattern inverse to that of weight-specific aerobic metab-
olism, which typically decreases with increasing body
size.

Future work should extend measurements of ODH ac-
tivity to both smaller and larger scallop sizes and increase
the sample size, given the large individual variability ob-
served in this study. Ontogenetic changes in ODH activity
and differences in scaling with body size between aerobic
and anaerobic metabolism have not been previously
studied in swimming molluscs. However, increased reli-
ance on anaerobic metabolism for the maintenance of
burst-swimming performance with increasing body mass,
based on activities per gram of muscle of other glycolytic
enzymes, has been described in finfish (Somero and Chil-
dress, 1980; Goolish, 1991).

In the present study, mean ODH activity in whole
adductor muscle homogenates of Argopecten irradians
attained a maximum of 84 ^moles min~ ' g freeze-dried
weight' 1 , a value equivalent to about 17 /umoles min~'
g adductor wet wt"', assuming 80% water content of
the adductor muscle. This estimate is lower than the
value of 98 ^moles min~' g wet wt~' reported for A. i.
concentricus of unspecified size (Chih and Ellington,
1983). and somewhat lower than values reported for
other pectinids i.e.. 30 ^moles min~' g wet wt" 1 in
Pecten maximus and Chlamys varhts (Zammit and
Newsholme. 1976), 58 ;umolesmin~' g" 1 in Pecten alba
(Baldwin and Opie, 1978), and 26 ^moles min~' g" 1 in
Placopecten magellanicus (de Zwann et ai, 1980). All
literature values cited were measured at 25 C for ho-
mogenates of the phasic (striated) adductor muscle,
which typically comprises the bulk (about 80%) of total
adductor weight (de Zwann et ai, 1980). whereas whole
muscle homogenates (catch+ phasic portions) were used
in the present study.

In conclusion, the present study, in concert with prior
related studies (Pohle et ai. 1991; Strieb, 1992), supports
the existence of three distinct phases during the bay scal-
lop's early life history. During these phases, juveniles em-
ploy three different tactics to increase their survival: ( 1 )
development of upward crawling behavior by plicated and
early juveniles (< about 1 1 mm in shell height), enabling

54

Z. GARCIA-ESQUIVEL AND V. M. BRICELJ

them to rapidly relocate above a minimum threshold
height on eelgrass blades to achieve spatial refuge from
predators; (2) active escape response, coincident with
substantial energy allocation towards shell growth and
gradual loss of vertical refuge at intermediate sizes; and
(3) attainment of a partial size refuge at sizes exceeding
about 30 mm. Mortality rates of natural scallop popula-
tions before, during, and after relocation to the bottom
are needed to determine the relative value of these suc-
cessive refugia.

Acknowledgments

We thank Shino Tanikawa-Ogleswby, Max Strieb, and
Jim Christie for their assistance in the field; Chris Smith
(Cornell Cooperative Extension) for alerting us to the
presence of natural scallop set in NWH; the Shinnnecock
Indian Reservation Hatchery, Southampton, NY, for
providing juvenile scallops: Jonathan Zehrand Pat Hassett
for their advice on enzyme assays; Francisco Borrero for
reviewing this manuscript; and J. K. Winfree of Synthetic
Fibers Inc. for donating material for the construction of
eelgrass mimics. This project was supported by the NOAA
Office of Sea Grant, U.S. Department of Commerce, un-
der grant No. NA90AA-DSG078 to the New York Sea
Grant Institute, and by the Living Marine Resources In-
stitute, SUNY at Stony Brook. One of the authors (ZGE)
is also indebted to the Consejo Nacional de Ciencia y
Tecnologia (CONACYT), Mexico, for support while
writing this work. This is Contribution No. 909 from the
Marine Sciences Research Center, SUNY Stony Brook.

Literature Cited

Alexander, J. E., Jr., and A. P. Covich. 1991. Predation risk and
avoidance behavior in two freshwater snails. Biol. Bull. 180: 387-
393.

Ambrose, W. G., Jr., and E. A. Irlandi. 1992. Height of attachment
on seagrass leads to trade-off between growth and survival in the bay
scallop Argopecten irradians. Mar. Ecol. Prog. Ser. 90: 45-51.

Baldwin, J., and A. M. Opie. 1978. On the role of octopine dehydro-
genase in the adductor muscles of bivalve molluscs. Comp. Biochem.
Physiol. 61 B: 85-93.

Belding, D. L. 1910. The scallop fishery of Massachusetts: including
an account of the natural history of the common scallop. Common-
wealth of Massachusetts Mar. Fish. Ser. No. 3: 51 pp.

Bricelj, V. M., J. Epp, and R. E. Malouf. 1987. Intraspecific variation
in reproductive and somatic growth cycles of bay scallops Argopecten
irradians. Mar. Ecol. Prog. Ser. 36: 123-137.

Brodie, E. D., Jr., D. R. Formanowicz, and E. D. Brodie III.
1991. Predator avoidance and antipredator mechanisms: distinct
pathways to survival. Elliot Ecol. & Evot 3: 73-77.

Caddy, J. F. 1972. Progressive loss of byssus attachment with size in
the sea scallop. Placopecten magellanicux (Gmelin). J. Exp. Mar.
Biol. Ecol. 9: 179-190.

Chin, C. P., and \V. R. Ellington. 1983. Energy metabolism during
contractile activity and environmental hypoxia in the phasic adductor

muscle of the bay scallop Argopecten irradiates concentricus. Physiol.

Zoo/. 56:623-631.
Crowl, T. A. 1990. Life-history strategies of a freshwater snail in response

to stream permanence and predation: balancing conflicting demands.

Oecologia 84: 238-243.
Dadswell, M. J. 1990. Size-related hydrodynamic characteristics of

the giant scallop, Placopecten mugel/anicus (Bivalvia: Pectinidae).

Can. J. Zoo/. 68: 778-785.
de Zwann, A., R. J. Thompson, and D. R. Livingstone.

1980. Physiological and biochemical aspects of valve snap and valve

closure responses in the giant scallop Placopecten magellanicus. II.

Biochemistry. J. Comp. Physiol. 137: 105-1 14.
Ebeling, A. W., and D. R. Laur. 1985. The influence of plant cover

on surfperch abundance at an offshore temperate reef. Environ Biol.

Fishes 12(3): 169-179.
Eckman, J. E. 1987. The role of hydrodynamics in recruitment, growth

and survival of Argopecten irradians (L.) and Anomia simplex

(D'Orbigny) within eelgrass meadows. J. Exp. Mar. Biol. Ecol. 106:

165-191.
Eckman, J. E., C. H. Peterson, and J. A. Cahalan. 1989. Effects of

flow speed, turbulence, and orientation on growth of juvenile bay

scallops Argopecien irradians concentricus (Say). J. Exp. Afar. Biol.

Ecol. 132: 123-140.
Fersht, A. 1985. Pp. 176-177 in Enzyme Structure and Mechanism.

W. H. Freeman, New York.
Fonseca, M. S., J. S. Fisher, J. C. Zieman, and G. W. Thayer.

1982. Influence of the seagrass. Zostera marina L., on current flow.

Estuarine Coastal Shelf Sci. 15: 351-364.
Fonseca, M. S., J. C. Zieman, G. VV. Thayer, and J. S. Fisher. 1983. The

role of current velocity in structuring eelgrass (Zostera marina L.)

meadows. Estuarine Coasial Shell Sci. 11: 367-380.
Gade, G., and M. K. Grieshaber. 1986. Pyruvate reductases catalyze

the formation of lactate and opines in anaerobic invertebrates. Comp.

Biochem. Physiol. 83B: 255-272.

Gambi, M. C., A. R. Nowell, and P. A. Jumars. 1990. Flume obser-
vations on flow dynamics in Zosiera marina (eelgrass) beds. Mar.

Ecol. Prog. Ser 61: 159-169.
Goolish, E. M. 1991. Aerobic and anaerobic scaling in fish. Biol. Rev.

66: 33-56.
Gould, S. J. 1971. Muscular mechanics and the ontogeny of swimming

in scallops. Paleontology 14: 61-94.
Grieshaber, M. K. 1978. Breakdown and formation of high energy

phosphates and octopine in the adductor muscle of the scallop, Chla-

mys operciilaris ( L. ) during escape swimming and recovery. / Comp.

Physiol. 126:269-276.
Mickey, M. T. 1977. Age. growth, reproduction and distribution of

the bay scallop, Aeauipecten irradians irradians (Lamarck), in three

embayments in eastern Long Island, New York, as related to the

fishery. M.S. Thesis, C.W. Post College, Long Island University, New

York, 101 pp.
Irlandi, E. A., and C. II. Peterson. 1991. Modification of animal habitat

by large plants: mechanisms by which seagrasses influence clam

growth. Oecologia 87: 307-318.
Kemp, W. M., L. Murray, J. Borum, and K. Sand-Jensen. 1987. Diel

growth in eelgrass Zostera marina. Mar. Ecol. Prog. Ser. 41: 79-86.
Kitting, C. I.. 1985. Adaptive significance of short-range diel migration

differences in a seagrass-meadow snail population. Pp. 227-243 in

Migration: Mechanisms and Adaptive Significance. M.A. Rankin.

ed. Contrih. Mar. Sci. 27.

Main, K. L. 1987. Predator avoidance in seagrass meadows: prey be-
havior, microhabitat selection, and cryptic coloration. Ecology 68:

170-180.

MICROHABITAT OF JUVENILE BAY SCALLOPS

55

Manuel, .1. 1992. The effect of size and hydrodynamics on the ability
of juvenile scallops (Placopeclen magellaniais) to use jet propulsion
for swimming. M.S. Thesis. Acadia University. Canada.

Palmer, A. R. 1983. Growth rate as a measure of food value in lliaidid
gastropods: assumptions and implications for prey morphology and
distribution. ./ E\r Mai Biol Ecol. 73: 95-124.

Pohlc, D. G., V. M. Bricelj, and Z. Garcia-Esquivel. 1991 . The eelgrass
canopy: an above-bottom refuge from benthic predators for juvenile
bay scallops Argofiecten irradians. Mar. Ecol. PrK. Ser 14: 47-59.

Sokal, R. R., and F. J. Rohlf. 1981. Biometry. 2nd ed. W.H. Freeman,
San Francisco. 859 pp.

Somcro, G. N., and J. J. Childress. 1980. A violation ofthe metabolism-
size scaling paradigm: activities of glycolytic enzymes in muscle in-
crease in larger-size fish. Physiol. Zoo/. 53: 322-337.

Stanley, S. M. 1970. Relation of shell form to life habits in the Bivalvia.
Geol. Soc. Am Mem 125: 1-296.

Strieb, M. D. 1992. The effects of prey size, prey density and eelgrass
habitat characteristics on predation of post-settlement bay scallops,
Argopeclen irradians M.S. Thesis. Marine Sciences Research Center.
State University of New York, Stony Brook. NY. 145 pp.

Tettelbach, S. T. 1986. Dynamics of crustacean predation on the
northern bay scallop. Argopeclen irradians irradians. Ph.D. Disser-
tation, University of Connecticut. 229 pp.

Tettelbach, S. T. 1991. Seasonal changes in a population of northern
bay scallops, Argopecten irradians irradians (Lamarck. 1819). Pp.

164-175 in An International Compendium of Scallop Biology and
Culture. S.E. Shumway and P.A. Sandifer, eds.. World Aquaculture
Workshops. I . Baton Rouge, LA.

Tettelbach, S. T., and P. VVenczel. 1991. Reseeding efforts and the
status of bay scallop populations in New York following the ap-
pearance of brown tide. Natl. Shellfish. Assoc. Annual Meeting, June
23-27, 1991, Portland, Maine, p. 273 (Abstract).

Vaughn, C. C., and F. M. Fisher. 1988. Vertical migration as a refuge
from predation in intertidal marsh snails: a field test. J. E.\p. Mar.
Biol. Ecol. 123: 163-176.

Vermeij, G. J. 1987. Armor and locomotion in bivalved animals.
Chapter 1 1 in Evolution and Escalation: An Ecological History of
Life. Princeton University Press, New Jersey.

Werner, E.E., and D.J. Hall. 1988. Ontogenetic habitat shifts in blue-
gill: the foraging rate-predation risk trade-off. >/<>gv69: 1352-1366.

Wilkinson, L. 1990. SYSTA T: The System far Statistics. SYSTAT Inc.,
Evanston, IL.

Zammit, V. A., and E. A. Newsholme. 1976. The maximum activities
of hexokinase, phosphorylase, phosphofructokinase, glycerol phos-
phate dehydrogenases, lactate dehydrogenase, octopine dehydroge-
nase, phosphoenolpyruvate carboxykinase, nucleoside diohosphate-
kinase, glutamate-oxalacetate transaminase and argmine kinase in
relation to carbohydrate utilization in muscles from marine inver-
tebrates. Biochem. J. 160: 447-462.

Reference: Biol. Bull. 185: 56-76. (August, 1993)

Highly Derived Coelomic and Water- Vascular

Morphogenesis in a Starfish with Pelagic

Direct Development

DANIEL A. JANIES AND LARRY R. McEDWARD

University of Florida, Department of Zoology. Gainesville, Florida 32611

Abstract. The coelomic development of the starfish
Pterasler lesselatus (order Velatida, family Pterasteridae)
is fundamentally different from that reported for all other
asteroids. Coeloms arise from seven separate enterocoels
that evaginate from different regions of the archenteron.
The water-vascular coelomic system develops from the
first five enterocoels (homologous to hydrocoel lobes)
which extend radially, in a transverse orientation, from
the central region of the archenteron. All other coelomic
compartments derive from two enterocoels that evaginate
later in development from posterior regions of the arch-
enteron. This mode of coelom formation in P. tesselatus
leads directly to the adult organization. We hypothesize
that this altered pattern of coelomogenesis evolved from
the pattern that occurs in the larvae of other spinulosacean
asteroids, by a rotation in the site of origin of the anterior
enterocoels relative to the archenteron. The altered pattern
of coelomogenesis accounts for most of the unusual fea-
tures of development in P. tesselatus: parallel embryonic
and adult axes of symmetry, transverse orientation of the
juvenile disk, absence of bilateral symmetry, absence of
purely larval structures, and the lack of a metamorphosis.
We conclude, contrary to previous interpretations, that
P. tesselatus does not have a larval stage and thus repre-
sents the only described case of truly direct development
in the asteroids.

Introduction

The development of Pterasler tesselatus is morpholog-
ically different from that of all other starfish (McEdward,
1992). P. tesselatus has pelagic development but does not
pass through the typical asteroid larval forms, the bipin-

Received 27 January 1993; accepted 28 May 1993.

naria or the brachiolaria. Important features that distin-
guish P. tesselatus from other asteroids include: absence
of specialized larval attachment structures (brachiolar
arms and adhesive disc); accelerated development of the
water-vascular system and the use of podia for attachment
to the substratum at settlement; radial rather than bilateral
symmetry; parallel rather than orthogonal embryonic and
adult axes of symmetry; a transverse orientation of the
juvenile disc; and complex morphogenesis of a supradorsal
membrane.

McEdward (1992) concluded that this set of unusual
developmental features in P. tesselatus characterized a
novel type of pelagic larva in the Asteroidea. Subsequent
observations led to an intriguing, but more radical, alter-
native interpretation: P. tesselatus completely lacks a lar-
val stage and undergoes direct development (see text box:
Definitions). Direct development, in which the embryo
develops progressively into the juvenile, with no inter-
vening larval features, has not been reported previously
in the asteroids. Documentation of direct development
would greatly expand the range of developmental diversity
among starfish and would have important implications
for the study of the evolution of echinoderm life cycles
(McEdward and Janies, 1993).

Evaluation of the hypothesis of direct development re-
quires additional information about the morphology of
the developmental stages. Is the asteroid larval body plan
present during development in P. tesselatus'' Specifically,
do internal structures develop in a bilaterally symmetrical
arrangement, as is typical of starfish larvae, or is the body
radially symmetrical, as is suggested by external mor-
phology? Are there independent morphogenetic axes for
early (larval) development and later (juvenile) develop-
ment? Do the coeloms arise and develop in the pattern
that is typical among asteroid larvae? Are there any purely

56

INTERNAL DEVELOPMENT OF PTER.4STER

57

larval (transitory) structures in the development of P. tes-
selatus?

Definitions (alter McEdward and Janies, 1993)

K.mbryo the stages of development between fertilization and
the completion of gastrulation.

l.arva the intermediate stages in development, produced by
post-gastrulation morphogenesis, and eliminated by
metamorphosis to the juvenile: these intermediate stages
must possess transitory structures that are not involved in,
and are not necessary for, morphogenesis of the juvenile.

Mesogen the intermediate stages, transitional between the
embryo and the juvenile, in the direct type of development;
characterized by a complete absence of larval structures.

Juvenile the developmental stages subsequent to the

attainment of the definitive (adult) body plan, but prior to
reproductive maturity.

Metamorphosis the morphological transition from the larval
body plan to the adult body plan.

Indirect development development that involves a larval
stage and a metamorphosis.

Direct development development that lacks a larval stage and
a metamorphosis; the juvenile develops progressively
(directly) from the embryo, through a series of intermediate
stages that are transitional towards the juvenile and do not
involve the morphogenesis of any larval structures.

Developmental pattern a set of characters (e.g., development
type, habitat, nutrition), each with discrete, mutually
exclusive states (t'.j?.. indirect or direct; pelagic or benthic;
feeding or nonfeeding) that describes features of the life
cycle.

This paper describes some aspects of internal devel-
opment in P. tesselatux. with emphasis on coelom for-
mation and morphogenesis of the water-vascular system.
Comparison with the typical pattern of indirect devel-
opment via pelagic, feeding bipinnarian and brachiolarian
larvae, as well as with development via modified pelagic,
nonfeeding (lecithotrophic) brachiolarian larvae, reveals
the coelomic and water-vascular development in P. tes-
selatus to be completely novel among asteroids. These
findings, together with unusual external features of de-
velopment (McEdward, 1992), lead us to conclude that
P. tesse/atus does not develop through a bilateral larval
stage, nor does it undergo a metamorphosis. P. tesse/atus
represents the only known case of truly direct development
in the asteroids.

Materials and Methods

Adults of the starfish Pteraster tesse/atus Ives, 1888
(Order Velatida. Family Pterasteridae) were collected us-
ing SCUBA, from subtidal populations (5 to 20 m) at
several sites near the Bamfield Marine Station (4849'N.
12508"W) in Barkley Sound, Vancouver Island, British
Columbia, Canada, and from depths of 1 5 to 30 m near
the Friday Harbor Laboratories (4832'N, 1230'W) in
the San Juan Archipelago, Washington. Adults of P. tes-

selatus were induced to spawn by intracoelomic injection
of 2 to 5 ml ( 10 4 M) of the hormone 1-methyl adenine.
Eggs (about 1000-1400 /urn in diameter) were released
within 1 to 3 h after injection. The eggs developed without
artificial insemination and were cultured as described by
McEdward (1992).

Microscopy and 3-D reconstruction

Specimens were fixed for scanning electron microscopy
(SEM) in cold osmium tetroxide (2% for 1 h) in 0.45 yum
filtered seawater, rinsed twice in distilled water, dehydrated
through a graded ethanol series (30%, 50%, 70%, 15 min
each), and stored in 70% ethanol. In preparation for
drying, specimens were dehydrated stepwise to absolute
ethanol (90%, 100%, 15 min. each), then infiltrated with
hexamethyldisilazane (HMDS, Sigma Chemical Co.) for
several hours. Specimens were air-dried at room temper-
ature (Nation, 1983) in a dust-free chamber, sputter-
coated with gold-palladium, and stored under desiccation.

Specimens were fixed for serial histological sectioning
in Bouin's fluid (24 h), dehydrated through a graded
ethanol series for 15 min in each concentration (30%,
50%, 70%), and stored in 70% ethanol. Later, specimens
were dehydrated to absolute ethanol (90%, 100% 15 min
each, 100% overnight), transferred to absolute ethanol
with eosin y for 30 min, transferred to xylene for 30 min,
infiltrated with a graded series of paraplast-xylene mixtures
(at 56C under vacuum), then embedded in paraplast.
Embedded specimens were serially sectioned at 7 or 12
/urn, and stained in hematoxylin-eosin. Some specimens
were partially sectioned. The tissue remaining in the block
was prepared for SEM by dissolving the paraplast in xylene
and drying the tissue with HMDS, as described above.

Serial sections (about 80 per mesogen) were examined
and photographed with a compound light microscope.
Three-dimensional reconstruction was achieved as fol-
lows. Sequential sections were aligned visually for tracing.
The outer edges of the body wall, coelomic compartments,
and developing gut were traced as color-coded contours
onto paper with the aid of a camera lucida drawing tube.
The tracings were marked with a set of fiduciary points
for alignment during digitization. The x, y, and z coor-
dinates of points along the contours were entered in a
computer with a digitizing tablet and stored as ASCII files.
These coordinate data were then plotted as a graphic image
of each section and stored as a bitmapped file of raw eight-
bit color pixel data. Digitization and data conversion were
done with programs written for this purpose (commented
Pascal source code for DOS systems can be obtained from
McEdward). Entire series of sections (bitmapped files)
were imported into the program NIH Image 1 .44 (a public
domain program for Apple Macintosh computers avail-
able over Internet by anonymous ftp from zippy.nimh.

58

D. A. JANIES AND L. R. McEDWARD

Figure 1. Early development of the Plerasler tessi'latus embryo. A.
SEM of early (Id) blastula showing irregular pattern of cleavages and
blastomeres of various sizes. B. Histological section of Id 12h wrinkled
blastula showing deep folds in blastular wall and blastocoel. C. Longi-

nih.gov [128.231.98.32]). The regions bounded by en-
dodermal and mesodermal contours in each section were
filled with color to allow production of solid (rather than
"wire-frame") reconstructions. These filled images were
saved as a series of files in TIFF format. Stacking and
projecting routines (based on a brightest point algorithm)
in NIH Image were used to create 3-D reconstructions.
Selected layers, such as the ectoderm could be removed
by adjustment of the transparency bounds allowing vi-
sualization of internal structures.

Results

Overview of development in Pteraster tesselatus

External features of development were described by
McEdward (1992). Here we summarize that description
and add some additional observations. The pattern of early
cleavages was irregular and resulted in blastomeres of var-
ious sizes without a regular arrangement (Fig. 1A). As
cleavage progressed the blastomeres became smaller, and
the surface of the embryo acquired a smooth appearance.
The wall of the blastula was thrown into deep folds re-
sulting in a wrinkled blastula stage (Fig. IB). McEdward
(1992) had reported that, during gastrulation, initiation
of archenteron formation in P. tesselatus was correlated
with the loss of folding of the blastular wall in the vegetal
hemisphere of the embryo. Our study confirmed that sub-
sequent enlargement of the archenteron was accompanied
by a progressive loss of folding of the blastular wall, from
the equatorial region to the animal pole of the embryo.
These observations suggest that the formation of the arch-
enteron may be due primarily to involution and not
ingression. The gastrula elongated along the animal-veg-
etal axis and acquired an ovoid body form, just before
hatching at 3 days. Within 1 to 2 days of hatching, an
ectodermal depression produced a groove completely
around the circumference that divided the body into an-
terior and posterior regions (Fig. 3A).

The oral surface of the juvenile corresponded to the
anterior region of the mesogen (= animal pole of the em-
bryo), and the aboral surface of the juvenile corresponded
to the posterior end of the mesogen ( = embryonic vegetal
pole and blastopore) (Fig. 2). Consequently, the juvenile
disc developed in a transverse orientation with respect to
the anterior-posterior and animal-vegetal axes. All of the
stages of development were characterized by radial, rather
than bilateral, symmetry. Morphogenesis of the supra-

tudmal section of 2d 5h gastrula showing the large archenteron and small
blastocoelic space. The posterior end, as indicated by the blastopore is
oriented to the left. D. Transverse section, perpendicular to the anterior
posterior axis of a 4d 1 6h elongate mesogen. showing five lateral pouches
evagmating simultaneously from the equatorial region of the archenteron.

INTERNAL DEVELOPMENT OF PTER.4STKR

59

animal

vegetal

posterior

oral

aboral

Figure 2. Diagram illustrating the orientation of the axes of symmetry
ot'developmental stages of Pteraster lesselalus. Top, animal-vegetal axis
of the embryonic stages, zygote to gastrula: middle, anterior-posterior
axis of the mesogen; bottom, oral-aboral axis of the juvenile and adult
starfish.

dorsal membrane began at 5 to 8 days with the formation
of five broad marginal bulges around the circumference
of the body, immediately posterior to the groove (Fig. 3A,
4A). The marginal bulges became bilobed at 8 to 10 days
(Fig. 4A, O) and eventually divided to produce a total of
10 distinct marginal lobes at 16 days. Five additional lobes
developed at the aboral pole of the body at 1 1 to 1 3 days.
All 1 5 lobes fused at 1 7 to 19 days, resulting in a complete
supradorsal membrane above the aboral body wall
(McEdward, 1992. pp. 181-183). In the adult, the supra-
dordal membrane encloses a space, the nidamental
chamber, that protects, ventilates, and perhaps nourishes
young in pterasterids that brood (McClary and Mladenov,
1990). Functional podia, arranged in five clusters, emerged
from the circumferential groove at 9 days, long before the
development of juvenile arms (at 2.5 months). Early de-
velopment of the podia is important because the mesogen
of P. tesselatus lacks the brachiolar arms and adhesive
disk of other pelagic nonfeeding larvae (Fig. 3 A, 4A). Po-
dia of P. tesselatus are used for attachment to the sub-
stratum at settlement at 10 to 12 days. Each podial cluster
initially consisted of a pair of podia and a terminal po-
dium. Three to four additional pairs of podia were added
by 28 days. The juvenile mouth did not form until the
second month, and distinct arms were not present in most

P. tesselatus juveniles until the third month (McEdward,
1992).

Internal development of Pteraster tesselatus

Gastrulation. At 2 to 3 days of development in P. tes-
selatus. the archenteron widened within the gastrula. The
interior of the ovoid stage contained a large archenteron
that extended from the blastopore at the posterior (= veg-
etal) end all the way to the anterior (= animal) end. The
archenteron nearly filled the interior of the body, causing
the ectodermal and mesendodermal cell layers to lie close
together and greatly reduced the blastocoelic space (Fig.
1C). The blastopore closed between days 3 and 4 resulting
in a completely closed archenteron sac. The archenteron
did not join with the animal ectoderm to form a stomodeal
opening. P. tesselatus produces large, yolky mesogens that
do not feed on particulate food.

Morphogenesis of the water-vascular system. At 4 days,
five lateral coelomic pouches (enterocoels) evaginated si-
multaneously from the equatorial region of the archen-
teron (Fig. ID, 3E-H). The five enterocoels were arranged
symmetrically around the circumference of the archen-
teron in a transverse plane (i.e.. perpendicular to the an-
terior-posterior axis of the mesogen; Fig. 2, 7IIIB). These
five enterocoels were hydrocoelic in nature because they
became the coelomic lining of the water-vascular system
(i.e., radial canals, podia, ampullae, and circumoral ring
canal). Initially, these hydrocoel lobes were broad, simple
evaginations from the archenteron (Fig. ID). The hydro-
coel lobes elongated as they extended radially towards the
ectodermal body wall. The hydrocoel lobes remained
connected to the archenteron throughout much of the
development of the water- vascular system. The hydrocoel
lobes and their relationship to the archenteron are evident
in transverse section (Fig. 3E-H) and in 3-D reconstruc-
tion (Fig. 5 A, B). A diagram of the typical adult asteroid
water-vascular coelomic system, perihemal coelomic sys-
tem, hemal system, and axial complex (Fig. 6) and a table
of terminology used in this paper, cross referenced to that
of Hyman (1955), (Fig. 8) are provided to aid in visualizing
the morphogenesis of P. tesselatus.

At 5 days, the distal ends of the hydrocoel lobes began
to contact the overlying ectoderm (Fig. 3E, F). At the
time and location of this contact, the ectoderm began to
fold inward to produce the circumferential groove. The
groove formed just anterior to the hydrocoel, so that the
contact with the hydrocoel lobes occurred along the pos-
terior wall of the groove (Fig. 3C-J).

The proximal and central portions of each hydrocoel
lobe developed into a radial canal. The distal part of each
hydrocoel lobe widened and then bifurcated to form the
coelomic lining of the first pair of podia (Fig. 3G, H). The
coelomic lining of each terminal (unpaired) podium de-

H

60

INTERNAL DEVELOPMENT OF PTER.4STER

61

Figure 3. (Conlinncd)

62

D. A. JANIES AND L. R. McEDWARD

r.c.

o.pv.c

Figure 3. Internal morphogenesis of the early (about 6-8d) mesogen of Ptcraxter lesselatus. Magnification
is the same in all panels and scale bar equals 0.2 mm. In all lateral views the posterior of the mesogen is
oriented to the left. A. SEM, lateral view of 6d mesogen. showing circumferential groove dividing anterior
and posterior body regions. Podia are visible in the circumferential groove. B. Drawing of lateral view of
mesogen in panel A showing location and orientation of planes of section for the following panels of this
figure. C-T. Paired light micrographs and interpretive diagrams of histological sections. See Table I for
abbreviations. C, D. Longitudinal section of 6d mesogen showing origin of the enterocoels from the archenteron
and relationship of circumferential groove to the hydrocoels. E. F. Transverse section of 5d 8h mesogen
showing the first five evaginations (hydrocoels) in pentaradial symmetry around the circumference of the
archenteron and their early contact with the overlying ectoderm. G, H. Transverse section of 6d mesogen
showing the bifurcation of the distal part of each hydrocoel lobe that forms the coelomic lining of the first
pair of podia. I, J. Oblique section of 7d mesogen showing the coelomic lining of the terminal unpaired
podium forming as an extension from the cleft of the original bifurcation and the coelomic lining second
pair of podia evaginating between the terminal podium and the first pair. K, L. Transverse section through
a 5d 8h mesogen showing the large posterior enterocoel evaginating as a large crescent shape from the
extreme posterior region of the archenteron. M, N. Transverse section of 6d mesogen showing the large
posterior enterocoel encircling the gut and enveloping the posterior side of the hydrocoel lobes. O. P. Transverse
section of 6d mesogen showing four of the five initial small pouches growing orally from the oral perivisceral
coelom between the hydrocoel lobes to originate the outer oral penhemal ring coelom. Q. R. Longitudinal
section of 6d 16h mesogen showing the enterocoels, especially the small posterior enterocoel and its mixed
set of fates of the (i.e.. axocoelic and somatocoelic). S, T. Longitudinal section of 6d I6h mesogen (same
mesogen as section in Q, R) showing the somatocoelic derivative (the aboral pemisceral coelom) separating
from the distal region of the small posterior enterocoel and moving to the extreme posterior of the body.
The proximal region of the small posterior enterocoel develops into the inner oral perihemal ring coelom
and likely contributes to the coelomic and hemal axial complex, such as the axial coelom and madreporic
vesicle.

INTERNAL DEVELOPMENT OF PTER-iSTER

63

Table I

. \hhrcrian<'ii\ </ in /'/.t.'ii't'v 3 and 4

Abbreviation

Description

lp. coelomic lining of the primary 1 podia of the

water-vascular system

2p. coelomic lining of the secondary podia of the

water-vascular system

a. ampulla of the podia of the water-vascular system

a.c. anterior compartment of the archenteron

a.pv.c. aboral perivisceral coelom

a.r. anterior region of the mesogen

ax.c. axial coelom

e.g. circumferential groove

co.r.c. circumoral ring canal ot the water-vascular system

g. gut

h.l. hydrocoel lobe of the water-vascular system

hp.c. hydropore canal

i.ph.c. inner oral perihemal ring coelom

I.e. lateral canal of the water-vascular system

l.p.e. large posterior enterocoel

m.b. marginal bulge of the mesogen

o.ph.c. outer oral perihemal ring coelom

o.pv.c. oral penvisceral coelom

p. ectodermal coven ng of the podia

r.c. radial canal of the water-vascular system

s.p.e. small posterior enterocoel

t.p. coelomic lining oi the terminal podium of the

water-vascular system

veloped as an extension from the cleft of the original bi-
furcation. At 7 days, the coelomic lining of the second
pair of podia evaginated between the terminal podium
and the first pair (Fig. 31, J). In the region of the hydrocoel
lobes, the ectoderm of the circumferential groove thick-
ened, enveloped the developing coelomic linings, and
produced the epidermal covering of the podia. In subse-
quent development, additional pairs of podia were added
immediately proximal to the terminal podium as the ra-
dial canals elongated (Fig. 4M, N).

The five hydrocoel lobes remained connected to the
gut but were otherwise independent of each other during
the early development of the radial canals and podia.
Later, these live independent evaginations were connected
by a coelomic tube (ring canal) that encircled the gut. At
8 to 9 days, each radial canal separated from the arch-
enteron by a constriction of the oral side of its proximal
end. Concurrently, the aboral portion of the proximal
end of each radial canal produced small lateral evagina-
tions in the transverse plane of the body. These evagi-
nations grew around the circumference of the archenteron,
met in the interadii, and fused to complete the circumoral
ring canal (Fig. 4E-N, 5B).

The ampullae developed by an expansion of the coe-
lomic lining on the aboral side of each podium. By 8 days,
the ampullae of the first two pairs of podia had greatly

enlarged and extended up against the oral perivisceral
coelom (Fig. 4C, D). At 8 to 9 days, the hydropore canal
developed as a long tubular invagination from the aboral
ectoderm in the extreme posterior region of the body. It
extended orally and joined with the developing circumoral
ring canal (Fig. 4E, F, I, J, K, L, S, T, and 5B). The hy-
dropore canal was an ectodermal structure that was con-
spicuous in early development because it stained very
deeply. We assumed the hydropore canal gave rise to the
stone canal of the adult as is typical of asteroids although
we did not trace its development beyond 12 days.

The completion of the major water-vascular compo-
nents (circumoral ring canal, radial canals, podia with
expanded ampullae, and hydropore canal) at 8 to 9 days
corresponded to the time at which a mesogen possessed
functional podia that could be extended from the body
and used to adhere to the substratum. In laboratory cul-
tures, settlement occurred as early as 1 to 2 days after the
completion of a functional water-vascular system (Mc-
Edward, 1992).

Formation of the perivisceral coeloms and axial coni-
ple.\. Soon after the evagination of the hydrocoel lobes
from the archenteron, two additional enterocoelic evag-
inations appeared (Fig. 3C, D, K, L, Q-T, 5 A, B): ( 1 ) a
large posterior enterocoel ( = 6th of 7 enterocoels) whose
fate was to become the oral perivisceral coelom which in
turn produced the outer oral perihemal ring coelom in
the juvenile; and (2) a small posterior enterocoel (= 7th
of 7 enterocoels) that formed the axial and inner oral peri-
hemal coeloms and the aboral perivisceral coelom.

The large posterior enterocoel developed from the ex-
treme posterior region of the archenteron at 4 to 5 days.
Initially, this enterocoel had the shape of a very large cres-
cent lying in a plane transverse to the anterior-posterior
axis of the mesogen (Fig. 3K, L. 5 A). By 5 days, this large
posterior enterocoel assumed the adult location of the
oral perivisceral coelom: it encircled the gut and enveloped
the posterior side of the hydrocoel (especially the ampullae
of the podia) (Fig. 4C, D; 3M, N; 5A). At 6 days, the oral
perivisceral coelom developed five small pouches that grew
orally (= anteriorly) between the hydrocoel lobes in in-
teradial positions (Fig. 3O, P). These processes then grew
laterally and fused in the radii to form the outer oral peri-
hemal ring coelom (Fig. 4O, P).

At 4 to 6 days, the small posterior enterocoel evaginated
from the archenteron posterior to the hydrocoel and
slightly anterior to site of evagination of the large posterior
enterocoel (Fig. 3C, D, M, N; 5A). The small posterior
enterocoel subdivided into a complex set of coeloms. At
6 days, the aboral perivisceral coelom separated from the
distal region of the small posterior enterocoel and moved
to the extreme posterior of the body (Fig. 3Q-T; 5A). The
proximal region of the small posterior enterocoel formed
a crescentic coelom near the aboral, inner surface of the

tp

a.

.ph.c.

hp.c.

Figure -4.

64

INTERNAL DEVELOPMENT OF PTER.4STER

65

H

o.pv.c

o.pv.c

Figure 4. (Continued)

66

D. A. JANIES AND L. R. McEDWARD

2 p.

Q

o.pv.c

Figure 4. (Continued)

co.r.c.

o.phx.

a.pv.c

o.pv.c

o.ph.c.

Figure 4. Morphogenesis of the late ( = 9-1 Id) mesogen of Plcrasler lesselalus. Magnification is equal
in all panels (except G, H) and all scale bars equal 0.2 mm. In all lateral views the posterior of the mesogen
is oriented to the left. See Table I for abbreviations. A. SEM. lateral view of 9d mesogen showing five
marginal bulges and well developed podia. B. Drawing of lateral view of mesogen in panel A showing location
and orientation of planes of section for the following panels of this figure. C, D. SEM and interpretive
diagram oi 9d mesogen sectioned in two planes and oriented obliquely to the viewer. Transverse face revealing
the oral perivisceral coelom and elements of the hemal and water-vascular system in the oral region of the
mesogen, especially the ampullae of the podia. Longitudinal face revealing an ampulla and the ectodermal
covering of a cluster of podia. E-V. Paired light micrographs and interpretive diagrams of histological
sections. E, F. Slightly oblique section of 9d mesogen showing the proximal lateral evaginations of the radial
canals that fuse to form the circumoral ring canal of the water-vascular system. Also, the proximity of the
hydropore canal to the small posterior enterocoel is visible. G. H. Transverse section at high magnification
of an 8d ambulacrum revealing the proximal lateral evaginations of the radial canals that form the circumoral
ring canal and showing the early development of a portion of the outer oral perihemal ring coelom. 1, J, K,
L. Two longitudinal sections from the same lOd mesogen. These illustrate the ectodermal origin of the
hydropore canal, it's confluence with the middle region of the small posterior enterocoel to form part of the
axial complex, and the connection of the hydropore canal to the circumoral ring canal. M. N. Transverse
section though the completed water-vascular system of an I Id mesogen. O, P. Transverse section through
the same 1 Id mesogen as in panels M, N. This section is slightly aboral to M. N and hence reveals the
outpockets of the oral perivisceral coelom that form the outer oral perihemal ring coelom. Q, R. Oblique
section through 9d mesogen showing formation of the crescentic shaped inner oral perihemal ring coelom
from the proximal region of the small posterior enterocoel. This view illustrates the proximity of the inner
oral perihemal ring coelom with the circumoral ring canal of the water-vascular system. S, T. Oblique section
of 7d 16h mesogen showing the formation of the axial complex from the confluence between the inner oral
penhemal ring coelom and the hydropore canal. U. V. Longitudinal section of 1 Id mesogen showing an
adult internal organization. At this stage the anterior compartment is in the process of transferring the
contents of the anterior region of the mesogen to the gut in order to fuel development.

67

68

D. A. JANIES AND L. R. McEDWARD

Figure 5. Two 3-D reconstructions of internal features (mesoderm and endoderm) of two stages (a. 6d,
b. 9d) of a Pteraster tesselatus mesogen. Each half page panel contains a montage of 1 2 views of a single
specimen as a series of 30 rotations through a total of 360. The rotational sequence begins in the upper
left and progresses across the top row to the right. Subsequent rows are also read left to right. The initial
(upper left) and final (lower right) views are oriented with the posterior of the mesogen up. The identity of
each structure is color keyed as follows: red = water-vascular coelomic lining, green = gut and anterior
compartment, yellow = small posterior enterocoel. blue = large posterior enterocoel, and purple = hydropore.
Scale bar = 0.2 mm.

circumoral ring canal of the water-vascular system (Fig.
4Q, R). This crescentic coelom developed into the inner
oral perihemal ring coelom. The middle region of the
small posterior enterocoel was confluent with the hydro-
pore canal (Fig. 3Q-T; 4E, F, I-L, S. T). The region of
contact between these coeloms likely formed some sub-
structures of the coelomic and hemal axial complex, such
as the axial coelom and madreporic vesicle. However, a
comprehensive and definitive description of the devel-
opment of the axial complex will require substantial ad-
ditional study.

Development of the gut and anterior compartment. A
great deal of coelomic morphogenesis occurred in P. tes-
selatus while the seven enterocoels remained confluent
via the central portion of the archenteron. Masterman

( 1902) described a similar arrangement of connected en-
terocoels and archenteron as a "mesenteron" in Hcnricia
sanguinolenta (formerly Crihella oculata). This term is
useful in describing the internal development of P. tes-
selatus. At 4 to 5 days, in P. tesselatus, despite the con-
fluence among all seven of the enterocoels, the gut (en-
doderm) was histologically differentiated from the coe-
lomic (mesodermal) regions of the mesenteron. The gut
was delineated as a deeply staining, thickened epithelial
region located posterior to the five hydrocoel lobes and
in between the two posterior enterocoels (Fig. 3C, D, Q-
T; 5A, B).

The region of the mesogen anterior to the circumfer-
ential groove was filled peripherally with yolk and fibrous
material, but also contained an epithelial layer that en-

INTERNAL DEVELOPMENT OF PTER.4STER

69

Lateral canal

Axial & inner oral
perihemal coeloms (cut

rcumoral ring canal

Radial canal (cut)

Radial & outer oral
perihemal coeloms (cut)

Hemal space

Figure 6. Drawing of the internal anatomy of a typical adult asteroid
showing the arrangement of water-vascular and perihemal coelomic sys-
tems as well as components of the hemal system. See Figure 8 for an
interpretation of the stippling.

closed a large, central internal compartment (Fig. 3C, D,
Q-T: 4I-L). The epithelial compartment was not the
product of enterocoely but was simply the anterior of the
archenteron produced by gastrulation. Hence we do not
believe it is properly termed a coelom but rather an an-
terior compartment of the gut. The anterior compartment
remained confluent with the gut region of the archenteron
throughout juvenile development. Identifiable structures
did not develop in the anterior region of the mesogen and
its blastocoelic contents were depleted during develop-
ment (Fig. 4U, V).

Discussion

P. tesselatus is radially symmetrical throughout devel-
opment, lacks all larval structures, and does not undergo
a metamorphosis. In addition, the coeloms arise from
seven separate enterocoels that evaginate from different
regions of the archenteron. The water-vascular coelomic
system develops from the first five enterocoels (homolo-
gous to hydrocoel lobes) that extend radially, in a trans-
verse orientation, from the central region of the archen-
teron. All other coelomic compartments develop from
two enterocoels that evaginate later in development from
posterior regions of the archenteron. This mode of coelom
formation in P. tesselatus leads directly to the adult or-
ganization. From these results, we conclude that P. tes-
selatus represents the first case of truly direct development
described in the asteroids.

Did direct development evolve through modification
of the typical pattern of asteroid larval development? The
considerable morphological and ecological diversity of
asteroid larvae seems to argue against the idea of a typical
larva or typical pattern of development. However, only a

single component of development, coelomogenesis, un-
derlies all of the important features characterizing direct
development in P. lessclatitx: radial symmetry, transverse
disc, parallel embryonic and adult axes, and lack of larval
organization. If there is a general pattern of coelom de-
velopment among asteroids, or if the starfish most closely
related to P. tesselatus share common features of coelom-
ogenesis, then there would be a basis from which to explore
the morphogenetic changes that could lead to the evolu-
tion of direct development. In the following sections, we
analyze the literature on asteroid larvae and conclude that
there are extremely conservative features of coelomic de-
velopment. Then we present a hypothesis to explain the
evolution of the highly modified coelomogenesis that we
have described in P. tesselatus.

Patterns of asteroid coelomogenesis

Asteroids with indirect development and pelagic feeding
larvae. Development from small eggs (about 100-200 /urn
in diameter) via feeding bipinnarian and feeding brachio-
larian larvae is the ancestral pattern in asteroids (Strath-
mann. 1974, 1978; McEdward and Janies, 1993). In spe-
cies with feeding bipinnarian larvae, the coeloms develop
from a pair of enterocoels that evaginate from the tip of
the archenteron (Fig. 7IA-B) (e.g., Asterias rubens. Gem-
mill, 1914; see also reviews by Horstadius, 1939;Hyman,
1955; Chia and Walker, 1991). The enterocoels develop
into three pairs of coeloms (from anterior to posterior:
axocoels, hydrocoels, and somatocoels) (Fig. 7IC). The
somatocoels pinch off from the enterocoels as separate
coeloms early in larval development, but the axocoels and
hydrocoels are often confluent via small coelomic tubes
and hence are considered axohydrocoels by some (Hyman,
1955). However, each coelomic sac or region can be con-
sidered to have a separate role in organogenesis (Fig. 7,
8). By the brachiolarian stage, the left and right axocoels
fuse in the anterior region of the larva to form a single
coelom (axocoel) in the shape and orientation of an in-
verted U (Fig. 7ID) (Gemmill, 1914). The axocoel extends
anteriorly into the preoral lobe, which is defined by its
location anterior to the larval mouth. This portion of the
axocoel is termed the preoral coelom (or anterior coelom).
The preoral coelom extends into the lumen of the bra-
chiolar arms (Barker, 1978), if present.

Paxillosids with indirect development and pelagic feed-
ing larvae. Asteroids in the order Paxillosida with pelagic
feeding development have only a bipinnarian stage. These
are termed, "non-brachiolarian larvae" because they lack
brachiolar arms and the associated coelomic projections
(Oguro el ul. 1976). Despite this, these larvae undergo
the typical pattern of coelomogenesis (described above
and in Fig. 7IA-D) and have a well-developed preoral
coelom that extends anteriorly into the preoral lobe of

70

D. A. JAMES AND L. R. McEDWARD

Posterior / Aboral

Figure 7. Comparison of coelomic origin and fates during development (A through D) in different
asteroids (I, II, and III). All drawings are oriented as dorsal views of longitudinal sections with the anterior
end up. A key to the stippling indicating coelomic fates is provided in figure 8. I A-D. Ancestral pattern of
coelomogenesis via feeding bipinnarian and feeding brachiolarian larval stages. II A-D. Coelomogenesis in
the nonfeeding brachiolarian larvae of the superorder Spinulosacea (e.K the families Solasteridae and Echi-
nasteridae). Ill A-D. Coelomogenesis in Plerasler tesxelatux

the bipinnaria (e.g., Astropecten scoparius, Oguro el al..
1976, p. 561, 566; Lnidia clathrata Komatsu et al. 1991,
p. 497).

Paxillosids with indirect development and pelagic non-
feeding larvae. In several asteroid clades, feeding larvae
have been replaced by nonfeeding larvae (Strathmann,
1974, 1978). Some species of the order Paxillosida develop
from moderately large (about 300-500 /urn in diameter)
yolky eggs via pelagic nonfeeding larvae. These are termed
barrel-shaped larvae because they have a highly simplified
external morphology. Barrel-shaped larvae lack brachiolar
apparatus and are likely derived from paxillosids with
feeding bipinnaria only (Komatsu et al., 1988; McEdward
and Janies, 1993). Although few details of internal de-
velopment are known, coelom formation in barrel-shaped

larvae is similar to the ancestral pattern because it includes
the formation of a U -shape and an anterior coelom (Ko-
matsu, 1975, p. 54; 1982, p. 202; Komatsu and Nojima,
1985, p. 276).

Asteroids with indirect development and benthic non-
feeding larvae. Asterina gibbosa (MacBride, 1896) and
Leptasterias hexacti.s (Chia. 1968) develop from large
yolky eggs (about 500 ^m and 800 fim, respectively) via
benthic, simplified, nonfeeding brachiolaria. Coelomo-
genesis in these species is only slightly modified from the
ancestral pattern. A single, large enterocoel evaginates
from the anterior tip of the archenteron. This unpaired
coelomic sac expands anteriorly and laterally, then extends
backwards with a pair (left and right) of short, posterior
projections, thus producing the characteristic U-shaped

INTERNAL DEVELOPMENT OF PTER.1STER

71

Pattern Cavity

Adult fate in the
terminology of this paper

Adult fate in the
terminology of Hyman, 1955

Resorbed

Inner oral perihemal coelom
Axial perihemal coelom

Water-vascular system

Oral perivisceral coelom
Outer oral and radial { erihemal coeloms

Aboral perivisceral coelom
Gut

Resorbed

Inner oral hyponeural sinus
Axial sinus

Water-vascular system

Hypogastric coelom
Outer oral and radial hyponeural sinuses

Epigastric coelom
Gut

Preoral coelom

jjvjj; Axocoel

Hft Hydrocoel
Somatocoel

vv Somatocoel

v^

t& Archenteron

Figure 8. Key to the stippling patterns used in the coding of internal cavities and their adult fates in
figures 3. 4, 6, 7. and 10. This information is cross referenced to the terminology of Hyman ( 1955).

coelom. The lateral projections eventually give rise to
separate coelomic regions (primarily a sagitally oriented
hydrocoel on the left side of the larval body and a so-
matocoel on each side of the larva). The anterior region
of the unpaired U-shaped coelomic sac extends into the
lumen of the brachiolar arms and is considered homol-
ogous to the preoral coelom of feeding larvae (Erber, 1985;
Strathmann. 1988).

Asteroids with indirect development and pelagic non-
feeding lan'ae. Several asteroids develop from large eggs
(about 1000 urn in diameter) via pelagic nonfeeding bra-
chiolarian larvae (distributed in the orders: Forcipulata.
Valvatida, Spinulosida, and Velatida: see Fig. 9) (Mc-
Edward and Janies, 1993). There are several studies of
internal structure in these larvae (e.g., Fromia ghardaq-
ana. Mortensen. 1938; Crossaster papposus Gemmill,
1920; Henricia sanguinolenta, Masterman, 1902; and So-
laster endeca Gemmill, 1912). The early pattern of an-
terior and hydrocoelic coelomogenesis of these brachio-
laria is identical to that of the benthic brachiolaria.

Features oj coelomogenesis in taxa closely related to
Pteraster tesselatus. In an echinasterid, Henricia sanguin-
olenta (Masterman. 1902). and two solasterids, Solaster
endeca (Gemmill, 1912) and Crossaster pappossus (Gem-
mill 1916, 1920), for which there is detailed information
on internal features of development, the archenteron
constricts into unpaired anterior, middle, and posterior
portions (Fig. 7IIA, B). The anterior portion and the pos-
terior portion of the archenteron are presumptive coelom,
whereas the middle portion is presumptive gut (Master-
man, 1902: Gemmill. 1912, 1920; Hyman, 1955. p. 298).
The axocoels, hydrocoels, and right somatocoel develop
from the unpaired anterior coelom. as is typical in non-
feeding larval development (Fig. 7IIB-D), but the left so-

matocoel develops from the posterior enterocoel (Fig.
7IIB. C) in proximity to its definitive larval location. The
anterior, unpaired enterocoel develops from the anterior
portion of the archenteron (Fig. 7IIA-IIB). Two long lat-
eral projections extend posteriorly from the anterior en-
terocoel, producing a U-shaped coelom. In spite of these
modifications, many characteristics of the ancestral pat-
tern of asteroid development occur in these species. ( 1 )
A hydrocoel pinches off from the left lateral region of the
U-shaped coelom, (2) a somatocoel (adult aboral perivis-

Brisingida

Forcipulatida
Valvatida

Notomyotida
Paxillosida

Velatida
(includes families
Pterasteridae and
Solasteridae)

Spinulosida
(one family:
Echinasteridae)

Superorder
Spinulosacea

Figure 9. A cladogram of asteroid orders based on Blake (1987). The
taxonomic location of the families of the superorder Spinulosacea. This
group includes Pterasler tesselatus and close relatives.

72

D. A. JANIES AND L. R. McEDWARD

ceral coelom) forms from the posterior portion of the right
side of the U-shaped coelom, (3) the left anterior region
of the U-shaped coelom forms the axocoel (adult perihe-
mal coelom of the axial complex and the inner oral peri-
hemal ring coelom), and (4) a preoral coelom lines the
brachiolar arms (Fig. 7IIB-D).

One striking difference in the pattern of coelomogenesis
in solasterids and echinasterids is a radical departure from
the ancestral pattern of coelom development: there has
been a change in the site of origin of the left somatocoel.
This heterotopic modification, which produces multiple
(i.e.. anterior and posterior) enterocoels. is a synapomor-
phy of the asteroids of the superorder Spinulosacea (O.
Spinulosida = family Echinasteridae and O. Velatida in-
cludes families Solasteridae, Pterasteridae; see Blake,
1987) (Fig. 9).

The evolution of posterior enterocoely dissociated the
origin of the left somatocoel from the anterior region of
the archenteron, in the lineage leading to P. tesselatus.
We believe that this modification of coelomogenesis was
a necessary precursor to the radical rearrangements of
coelomogenesis that occurred in P. tesselatus to produce
direct development. This was an important preadaptation
for the novel positioning and acceleration of water-vas-
cular morphogenesis that occurred in the evolution of
pterasterids. Because P. tesselatus lacks the bilateral pat-
tern of enterocoely and has instead a radial and transverse
pattern, the terms "left" and "right" are inappropriate.
Although Gemmill (1912, 1916. 1920) and Masterman
( 1902) referred to the posterior coelom of the spinulosa-
ceans as the "left posterior coelom" because of homology
with the left somatocoel of other asteroid larvae, we refer
to this cavity, which is also found in P. tesselatus, as the
large posterior coelom.

Unity and diversity in asteroid coelomic development.
In spite of considerable diversity in external larval mor-
phology, the pattern of coelomic development among as-
teroids is very conservative, regardless of shifts from feed-
ing to nonfeeding, or from pelagic to benthic development.
On the basis of the similarity in anterior coelomic devel-
opment, Erber (1985) argued that a preoral coelom de-
velops in the anterior region of the larva in all asteroids
and that, therefore, a homologous brachiolaria stage oc-
curs throughout the Asteroidea, including species in which
brachiolar arms are absent (e.g.. paxillosids and P. tes-
selatus). Erber's criterion for brachiolarian development
is sufficiently general to include all known asteroids, yet
it also leads to at least two conclusions that we believe
are false. First, Erber (1985) concluded that paxillosids
have a "brachiolarian" stage of development. However,
as stated earlier, paxillosids develop as feeding bipinnaria
or nonfeeding simplifications thereof they are not bra-
chiolaria nor are they derived from (i.e., homologous with)
brachiolaria (Komatsu el al., 1988). Second, Erber (1985)

assumed that the mode of coelom formation in P. tesse-
latus would be identical to that of its close relatives, the
echinasterids and solasterids. This assumption was based,
in part, on the prevailing interpretation of the evolution
of the "larva" of P. tesselatus. Fell ( 1 967, p. S7 1 ) consid-
ered the absence of brachiolar structures in P. tesselatus
to be the result of minor simplifications of the external
structures of a typical pelagic nonfeeding brachiolarian
larva.

In all larval types, most of the anterior of the larva
(including the preoral coelomic structures) is resorbed at
metamorphosis (Fig. 8). This resorption is accomplished
by translocation of the larval structures to the oral side
of the juvenile rudiment, followed by histolysis and trans-
fer to the juvenile digestive tract (Chia and Burke, 1978).
The postmetamorphic (juvenile and adult) fates of the
larval coeloms are highly conservative among asteroids,
as illustrated by a comparison of figures 71 and 711. The
left side of the axocoel forms the perihemal coelom of the
axial complex and the inner oral perihemal ring coelom
(Fig. 7, 8) (Hyman, 1955; Dawydoff, 1948). The left hy-
drocoel forms the entire coelomic lining of the water-vas-
cular system. The left somatocoel. by growing dorsal and
ventral extensions medial to the hydrocoel, forms the oral
perivisceral coelom. The outer oral perihemal ring coelom
forms from small interadial evaginations of the oral peri-
visceral coelom. The right axohydrocoel is mostly resorbed
at metamorphosis yet becomes the madreporic vesicle (an
aboral component of the axial complex) in some species
(Hyman, 1955). The right somatocoel lies on the side of
the gut opposite the hydrocoel (i.e., on the aboral side of
the juvenile) and forms the aboral perivisceral coelom.

Given the conservative nature of asteroid coelomoge-
nesis, analysis of the evolution of direct development can
be focused on the transition from the larval pattern of
coelomogenesis in the nonfeeding brachiolaria of taxa
closely related to P. tesselatus (e.g.. Solasterand Henricia)
to the highly derived pattern of P. tesselatus.

What evolutionary changes in morphogenesis occurred
to transform a brachiolarian lan'a into a mesogen?

Direct development is characterized by the morpho-
genesis of the juvenile from the embryonic stages and by
the complete absence of larval structures in development
(McEdward and Janies, 1993). The prevailing view is that
evolution of direct development must involve the loss of
larval features from the life cycle by the suppression or
elimination of larval programs in development (Fell, 1945;
Raff, 1987). This hypothesis accurately describes the re-
sultant differences between solasterid or echinasterid bra-
chiolarian larvae and the mesogen of P. tesselatus. How-
ever, this hypothesis offers little insight into the processes
by which the ancestral ontogeny has been modified to

INTERNAL DEVELOPMENT OF PTER.4STER

73

yield the derived ontogeny. One result of eliminating larval
features is that juvenile features arise earlier in ontogeny.
Direct development cannot evolve simply by accelerating
developmental processes. Direct development requires
spatial, as well as temporal, changes in morphogenesis
because the juvenile must develop from an embryonic
rather than a larval organization. We propose that direct
development evolved in P. tesselatus largely via a series
of topological changes in coelomogenesis. These hetero-
topic changes allowed the embryo (gastrula) to develop
into a juvenile coelomic organization, thereby eliminating
the larval stage and producing a "recession of metamor-
phosis" (sensu Fell. 1945, see also Raff, 1987).

A hypothesis of coelomic rearrangement. The altered
pattern of coelomogenesis underlies all of the important
features that characterize direct development in P. tes-
selatus. A relatively simple change in the topology of early
coelomic development can account for the loss of bilateral
symmetry, loss of the preoral lobe, and the development
of the coelomic systems in their adult orientation. The
arrangement of hydrocoelic lobes from the archenteron
establishes radial symmetry and accounts for the trans-
verse orientation of the juvenile disc and the parallel em-
bryonic and adult axes.

We believe that the radical rearrangement of coeloms
in P. tesselatus evolved by means of a 90 rotation of the
anterior enterocoels from the spinulosacean pattern, rel-
ative to the animal-vegetal axis of the archenteron (Fig.
10). This rotation shifted the hydrocoel from its ancestral
location on the left side of the body (Fig. 10A) to a central
location on the longitudinal (animal-vegetal) axis of the
body (Fig. 10B). Consequently, the hydrocoel lobes (radial
canal precursors) develop in a transverse plane around
the archenteron in P. tesselatus (Fig. IOC) rather than in
the sagittal orientation typical of all asteroid larvae (Fig.
71, II).

In conjunction with this hypothesized rotation, the an-
cestral axocoels fused with the ancestral right somatocoel,
and the preoral coelom was lost (Fig. 10B). The site of
e vagi nation of the large posterior enterocoel in the spi-
nulosacean pattern was unaffected by the relocation of
the anterior enterocoels (Figs. 7. 10).

Our hypothesis has several points in its favor. First, it
is consistent with the interpretation of the small posterior
enterocoel of P. tesselatus as homologous to fused left
and right axocoels and right somatocoel of the solasterids
and echinasterids (Figs. 711, III; IOC). The mixed coelomic
fates of the small posterior enterocoel in P. tesselatus (axial
and inner oral perihemal ring coeloms and aboral peri-
visceral coelom) are the basis for this interpretation
(Fig. 8).

Second, the rotation hypothesis is supported by the ob-
servation that a 90 rotation to the right (flexion sensu
Gemmill, 1912) occurs at metamorphosis in all asteroids

with indirect development. Metamorphic flexion shifts
the coeloms from the larval arrangement into the juvenile
arrangement. We argue that an analogous rotation of the
positional information specifying sites of enterocoel for-
mation occurred in the lineage leading to P. tesselatus.
This effectively shifted the specification of the post-meta-
morphic coelomic arrangement into the very early stages
of development. The result was direct development of the
coeloms in the juvenile orientation from the archenteron
of the embryo (Figs. 7III; 10). It is not surprising that
conservative and fundamental features of asteroid meta-
morphosis, such as the transformation of coeloms from
the larval to the adult organization, should be co-opted
into a derived pattern of development in which coeloms
arise in their adult orientation.

Third, the rotation hypothesis is attractive in its sim-
plicity; a single alteration of the map for enterocoel for-
mation can explain the derivation of a highly modified
pattern of coelomogenesis in the evolution of direct (non-
larval) development.

Differences in the site of the origin of enterocoels have
occurred in other asteroids. In addition to the solasterids
and the echinasterids that were discussed above, there are
other documented cases of the heterotopic origin of a pos-
terior coelom: Patiriella regularis (Byrne and Barker,
1991, p. 334, 341) and Marthasterias glacialis (Gemmill,
1916). Although heterotopy seems to be a rare event, on
the basis of the limited developmental diversity that has
been documented in asteroids, its occurrence illustrates
that the presumptive fates of the regions of the archenteron
can be modified evolutionarily.

Our rotation hypothesis leads to a new interpretation
of some of the features of P. tesselatus. The yolky anterior
region of the mesogen has traditionally been interpreted
as a preoral lobe (e.g.. Chia, 1966; Fell, 1967 p. S71). If
rotation of the coeloms occurred as we have suggested,
then the anterior region of P. tesselatus is not homologous
to the preoral lobe of a brachiolarian larva, because it
lacks the only defining characteristics of a preoral lobe,
namely the preoral extension of the anterior coelom into
brachiolar apparatus. We believe that, in P. tesselatus, the
evolution of fused coelomic regions from opposite sides
of the solasterid or echinasterid larva involved the loss of
the intervening preoral coelom (Fig. 10B). In our inter-
pretation, the site of origin of the former preoral coelom
would correspond to the lateral region of the archenteron,
posterior to the hydrocoelic lobes and anterior to the site
of formation of the small posterior coelom (this point is
denoted by the * on Fig. 10B). The yolky anterior region
of the developing P. tesselatus is located on the oral surface
of the juvenile; it probably functions only as a nutritional
store. The extension of the archenteron into the anterior
region of the mesogen likely serves as a conduit to deliver
nutrients to the gut and is not a preoral coelom.

74

D. A. JANIES AND L. R. McEDWARD

B

1

Figure 10. A hypothesized evolutionary sequence of coelomic rearrangements (dorsal view) from the
spinulosacean pattern of development (A) to that of Pteraster lesselatus. Step 1 involves a simple 90
rotation of the ancestral anterior enterocoels relative to the animal-vegetal axis of the archenteron. This
shifted the hydrocoel from its ancestral location on the left side of the spinulosacean embryo (A) to a central
location in a hypothesized intermediate form (B). Also in step 1. the ancestral axocoel shifted from an
anterior-left-central position (A) to an antenor-right-central location where it fused with the ancestral right
somatocoel (B) and became the small posterior enterocoel of P. tesselatus (C). This fusion was likely facilitated
by the loss of the preoral coelom as noted by the * in stages B and C. Step 2 shows the transition from the
hypothesized intermediate form (B) to P. tesse/alus (C). This could be accomplished by a heterotopic change
in the point of origin of the enterocoels from the anterior of the archenteron to the central and posterior
locations seen in P. tesselatus (C). In this hypothesis the anterior of the archenteron of P. tesselatus is a
novel feature and not a vestige of a preoral coelom. The posterior site of evagination of the spinulosacean
large posterior enterocoel was unaffected by the relocation of the anterior enterocoels.

Comparative morphogenesis in asteroids as a system Jor
studying the evolution of development

P tesselatus provides an important system for analysis
of major evolutionary changes in development, particu-
larly morphogenesis and early pattern formation (e.g., es-
tablishment of body axes and symmetry). The alterations
in developmental mechanisms associated with the evo-
lution of direct development have recently been ap-
proached through a combination of cellular methods (e.g.,
cell lineage specific gene-expression, cell lineage tracing)
and species comparisons (e.g., Jeffrey and Swalla, 1991;
Wray and Raff, 1990). Raff ( 1992) proposed that remod-
eling of development via changes in the primordial mor-
phogenetic axes of the egg is important in shifts from in-
direct to direct development. We postulate that just such
changes have occurred in the evolution of direct devel-
opment in P. tesselatus. We have documented radical to-
pological changes associated with morphogenesis of ju-
venile coeloms directly from the embryonic archenteron,
rather than from the bilaterally arranged series of larval
coeloms. An important component of those changes in-
volved the elimination of larval bilateral symmetry. In
solasterid and echinasterid asteroids, larval morphogenesis
involves the establishment of a bilateral symmetry that
replaces the embryonic radial symmetry. At metamor-

phosis, a new axis of radial symmetry is established for
the morphogenesis of the juvenile body. In P. tesselatus,
the axis of primary embryonic symmetry (radial) is re-
tained as the axis of radial symmetry in the juvenile
throughout the development of the mesogen. We postulate
that the loss of the larval phase of development and mor-
phogenesis of the juvenile from the embryonic organi-
zation may be due. in part, to the elimination of the system
for specifying larval left-right and larval dorsal-ventral
polarity in the egg and early embryo. Loss of such polarity
information would result in a radially symmetrical mor-
phogenetic framework on which to build juvenile coe-
lomic systems and other structures. The relationship be-
tween the first cleavage plane and the dorsal-ventral axis
is variable among echinoids( Henry et at, 1992), and this
has had a mechanistic role in evolutionary changes in
development (Henry and Raff, 1990; Henry et at, 1990).
These recent findings invite the investigation of changes
of morphogenetic axes among asteroids. A comparison
of the polarity characteristics of solasterid, echinasterid,
and pterasterid eggs and embryos might yield important
insights into structural patterning in early development
and the mechanisms underlying its evolutionary modi-
fication.

Some starfish do not free spawn, but rather brood large
yolky nonfeeding young on the benthos. These young de-

INTERNAL. DEVELOPMENT OF PTKR.4STKR

75

velop through larval stages with brachiolarian arms and
an adhesive disc, but are not released from the mother
until after metamorphosis. Morphological simplification
of young might be expected in species with benthic
brooding because larvae may be released from selection
pressures for effective settlement structures. The morpho-
genesis of internal structures in brooded asteroids is un-
known, so the diversity of developmental modifications
that may occur as a result of brooding are not appreciated.
An understanding of this diversity is important for as-
sessing whether the evolution of brooding results in spe-
cializations and simplifications of morphology and de-
velopment that are effectively irreversible (as is the case
with the loss of feeding larvae, Strathmann. 1978). Alter-
natively, the evolution of benthic development could
eliminate some of the selective pressures on larvae, pro-
viding the evolutionary flexibility to generate novel pat-
terns of morphogenesis and thus to allow the re-evolution
of pelagic development in new ways despite the loss of
larval structures.

Most species of the family Pterasteridae that have been
studied hold their eggs under the supradorsal membrane
and brood young long into juvenile development. Larval
attachment structures were likely lost in the pterasterids
after the lineage became very specialized brooders in the
deep-sea. Direct development was likely the result of se-
lection for general developmental efficiency, through the
elimination of nonfunctional larval features, during a very
long history of brooding (see McEdward and Janies. 1993:
McEdward, unpubl.). Pelagic development is likely a de-
rived trait in this family.

The comparative study of development among spi-
nulosacean asteroids can directly address the ecological
context of evolutionary shifts from indirect to direct de-
velopment, as well as transitions between pelagic and
benthic development. Specifically, comparisons of devel-
opment among the pelagic brachiolarian larvae from the
outgroups Solasteridae and Echinasteridae. the young of
a brooding pterasterid, and the pelagic mesogen of P. tes-
selatus would provide a rare opportunity to investigate
the role of the evolution of development in the important
ecological transition from a nondispersive to a dispersive
life history.

Changes in development in conjunction with paedo-
morphic shifts in the life cycle have potential for the evo-
lution of new body plans. The newly discovered deep-sea
concentricycloid echinoderms are characterized by a
unique water-vascular geometry of dual circumoral rings
(Baker ct <//., 1986: Rowe ct al. 1988). The novel pattern
of morphogenesis of the circumoral ring canal in P. tes-
selatm, combined with progenesis, may have provided a
mechanism for the evolution of the unique water- vascular
geometry of the concentricycloids (Janies and McEdward.
in press). We postulate that proximal lateral evaginations

of the radial canal could be duplicated at an early devel-
opmental stage to produce dual circumoral ring canals.
The duplicated formation of circumoral rings was a likely
a key innovation in the evolution of the concentricycloids
(Janies and McEdward, in press).

Acknowledgments

SEM facilities were provided by the University of Flor-
ida. Interdisciplinary Center for Biotechnology Research
and the Department of Zoology. Histology facilities were
provided by L. Guillette. R. Shammami, T. George, and
L. Becht entered sectional data for the reconstructions.
A. Griffin and S. McWeeney assisted with preparation of
some specimens for SEM. Photographic printing and the
illustration in Figure 6 were done by D. Harrison.

Funding was provided by the University of Florida Di-
vision of Sponsored Research (#89100245, #90012443,
and DSR-B) to LRM, The Aylesworth Foundation for
the Advancement of Marine Science Scholarship, to DAJ,
and the National Science Foundation (OCE 9115549)
to LRM.

A. O. D. Willows, Director, provided space and facilities
at the Friday Harbor Laboratories and J. Mclhnerney.
Director, provided space and facilities at the Bamfield
Marine Station. D. Duggins, C. Staude, S. Carson, and
G. Gibson helped collect starfish.

Literature Cited

Barker, M. F. 1978. Structure of the organs of attachment of brachio-
laria larvae of Snclui.\tcr anstrali.t (Verrill) and Coscinasterias cala-
maria (Gray) (Echinodermata: Asteroidea). J. E\p. Afar. Bio/ l-'col.
33: 1-36.

Baker, A. N., F. W. E. Rowe, and H. E. S. Clark. 1986. A new class
of Echinodermata from New Zealand. Nature 321: 862-864.

Blake, D. B. 1987. A classification and phylogeny of post-Paleozoic
sea stars (Asteroidea: Echinodermata). J. Nat. Hist. 21: 481-528.

Byrne, M., and M. F. Barker. 1991. Emhryogenesis and larval devel-
opment of the asteroid Putiriella regularis viewed by light and scan-
ning electron microscopy. Biol Bull 180: 332-345.

Ohia, F.-S. 1966. Development of a deep-sea cushion star, Plerasler
tesselaiuf. Proc. Cat. Acail. Sci. 34: 505-510.

Chia, F.-S. 1968. The embryology of a brooding starfish. Leplasterias
/;cA<((7/.s (Stimpson). Ada Zoo/. 49: 321-364.

Chia, F.-S., and R. D. Burke. 1978. Echinoderm metamorphosis: fate
of larval structures. Pp. 219-234 in Settlement and Metamorphosis
of Marine Invertebrate Larvae. F.-S. Chia and M. E. Rice eds. Elsevier.
New York.

Chia, F.-S., and C. \V. Walker. 1991. Echinodermata: Asteroidea. Pp.
301-340 in Reprudiielian nf Marine Invertebrates. A. Giese. J. S.
Pearse, and V. B. Pearse. eds. Boxwood, Pacific Grove, CA.

I)a)doff, C. 1948. Embryologie des echinoderms. Pp. 278-363 in
I'raite de Zoo/ogle. P.-P. Grasse, ed. Masson. Paris.

Frber, \V. 1985. The larval coelom as a significant feature of bipinnaria
and brachiolana in asteroid ontogeny. A critical approach. Zool. An:.
215: 329-337.

Fell, 11. B. 1945. A revision of the current theory of echmoderm em-
bryology. Trans ROY Sin: N. Z 75: 73-101.

76

D. A. JANIES AND L. R. McEDWARD

Fell, H. B. 1967. Echinoderm ontogeny. Pp. S60-S85 in Treatise on
Invertebrate Paleontology. Pan S. Echinodermaia. R. C. Moore, ed.
Geological Society of America and Kansas University Press, Law-
rence.

Gemmill, J. F. 1912. The development of the starfish Solaster endeca
Forbes. Trans. Zoo/. Soc. Land. 20: 1-71.

Gemmill, J. F. 1914. The development and certain points in the adult
structure of the starfish Asterin\ n/'vi.v, L. Phil. Trans. Roy. Soc.
Lond. (B) 205:213-394.

Gemmill, J. F. 1916. Notes on the development of the starfishes Asterias
glacialis O. F. M.; Cribella ocnlata (Link) Forbes; So/aster endeca
(Retzius) Forbes; Slichasier roseus (O. F. M.) Sars. Proc. Zoo/. Soc.
Lond. 39: 553-565.

Gemmill, J. F. 1920. The development of the starfish Crossaster pap-
posus. Mullerand Troschel. Q. J. Microsc. Sci. 64: 155-190.

Henry, J. J., and R. A. Ratf. 1990. Evolutionary change in the process
of dorsalventral axis determination in the direct developing sea urchin.
Heliocidaris erythrogramma. Dev Biol. 141: 55-69.

Henry, J. J., G. A. \\ray, and R. A. Raff. 1990. The dorsalventral axis
is specified prior to first cleavage in the direct developing sea urchin
Heliocidaris erythrogramma. Development 110: 875-884.

Henry, J. J., K. M. Klueg, and R. A. Raff. 1992. Evolutionary disso-
ciation between cleavage, cell lineage and embryonic axes in sea urchin
embryos. Development 114: 931-938.

Horstadius, S. 1939. L'ber die Entwicklung von Aslropecten aranciacus
L. Pubbl. Sla:. Zoo/. Napoli 17: 221-312.

I hni HI. L. H. 1955. The Invertebrates. \'oi 4. Echinodermata.
McGraw-Hill. New York. 763 pp.

Janies, D. A., and L. R. McEdward. 1994. A hypothesis for the evo-
lution of the concentricycloid water-vascular system. In Reproduction
and Development of Marine Invertebrates. W. H. Wilson, Jr.. S. A.
Strieker and G. L. Shinn, eds. Johns Hopkins Press, Baltimore. MD.
(In Press).

Jeffery, W. R., and B. J. Swalla. 1991. An evolutionary change in
muscle lineage of an anural ascidian embryo is restored by interspecific
hybridization with a urodele ascidian. Dev Biol. 145: 328-337.

Komatsu, M. 1975. On the development of the sea-star, Aslropeclen
latespinosiis Meissner. Biol. Bull. 148: 49-59.

Komatsu, M. 1982. Development of the sea-star Clcnopleiira fisheri.
Mar. Biol. 66: 199-205.

Komatsu, M., and S. Nojima. 1985. Development of the sea-star, As-
tmpecten gisselbrechti Doderlein. Pac Sci. 39: 274-282.

Komatsu, M., M. Murase, and C. Oguro. 1988. Morphology of the
barrel-shaped larva of the sea-star, Aslropeclen lutexpinosux. Pp. 267-
272 in Echinoderm Biology. R. D. Burke. P. V. Mladenov. P. Lambert,
and R. L. Parsley, eds. A. A. Balkema, Rotterdam.

Komatsu, M., C. Oguro, and J. M. Lawrence. 1991. A comparison of
development in three species of the genus. Luidia (Echinodermata:

Asteroidea) From Florida. Pp. 489-498 in Biology of Echinodermata,
T. Yanagisawa. I. Yasumasu. C. Oguro. N. Suzuki, and T. Motokawa,
ed. A. A. Balkema, Rotterdam.

MacBride, E. \V. 1896. The development of Asterina gibhosa. Q. J
Microsc. Sci. 38: 339-41 I.

Masterman, A. T. 1902. The early development of Cnbrella oculata
(Forbes) with remarks on echinoderm development. Trans. R. Soc.
Eihn. 40:373-418.

McClary, D. J., and P. V. Mladenov. 1990. Brooding biology of the
sea-star Pterasler millions (O. F. Muller): energetic and histological
evidence for nutrient translocation to brooded juveniles. / Exp. Mar.
Bio. Ecol. 142: 183-199.

McEdward, L. R. 1992. Morphology and development of a unique
type of pelagic larva in the starfish Pleruster tesselaliis (Echinoder-
mata: Asteroidea). Biol Bull 182: 177-187.

McEdward, L. R., and D. A. Janies. 1993. Life cycle evolution in as-
teroids: What is a larva 1 .' Biol. Bull 184: 255-268.

Mortensen, T. 1938. Contributions to the study of the development
and larval forms of echinoderms IV. D. Kgl. Danske Videnske Selsk.
Skrifter, Naturv. og Math. Afd.. 9 R;ckke, VII. 3.

Nation, J. L. 1983. A new method using hexamethyldisilazane for
preparation of soft insect tissue for scanning electron microscopy.
Stain Technol. 58: 347-351.

Oguro, C., M. Komatsu, and V. Kano. 1976. Development and meta-
morphosis of the sea-star Aslropecten scopanus Valenciennes. Biol.
Bull. 151: 560-573.

Raff, R. A. 1987. Constraint, flexibility and phylogenetic history in the
evolution of direct development in sea urchins. Dev Biol. 119: 6-
19.

Raff, R. A. 1992. Direct-developing sea urchins and the evolutionary
reorganization of early development. BioEssays 14: 21 1-218.

Rowe, F. W. E., A. N. Baker, and H. E. S. Clark. 1988. The mor-
phology, development and taxonomic status ofXyloplux Baker. Rowe,
and Clark (1986) (Echinodermata: Concentricycloidea). with the de-
scription of a new species. Proc. R. Soc. Lond. B. 223: 431-439.

Strathmann, R. R. 1974. Introduction to function and adaptation in
Echinoderm larvae. Thalassia Jugoslav. 10:321-339.

Strathmann, R. R. 1978. The evolution and loss of feeding larval stages
of marine invertebrates. Evolution 32: 894-906.

Strathmann, R. R. 1988. Functional requirements and the evolution
of developmental patterns. Pp. 55-61 in Echinoderm Biology. R. D.
Burke, P. V. Mladenov, P. Lambert, and R. L. Parsley, eds. A. A.
Balkema. Rotterdam.

Wray, G. A., and R. A. Raff. 1990. Novel origins of lineage founder
cells in the direct-developing sea urchin Heliocidaris erythrogramma.
Dev Biol. 141: 41-54.

Reference: Hiol. Hull. 185: 77-85. (August.

Larval Development (with Observations on Spawning)

of the Pencil Urchin Phyllacanthus imperialis: a New

Intermediate Larval Form?

RICHARD RANDOLPH OLSON 121 , J. LANE CAMERON 24 , AND CRAIG M. YOUNG 2

^Australian Institute of Marine Science. P.M.B. 3, Townsville. Queensland 4810. Australia. 2 Harbor

Branch Oceanographic Institution. 5600 Old Dixie Hwy.. Ft. Pierce. FL 33450, 3 Department of

Zoology. University of New Hampshire. Durham. NH03S01. and ^DAMES & MOORE. 500 Market

Place Tower, 2025 First Avenue, Seattle. WA 98121.

Abstract. Information for understanding the evolution-
ary shift from feeding to nonfeeding in echinoderm larvae
can be gained from species whose larval development
pattern appears to be intermediate between these ex-
tremes. In this paper we report the development of one
such species.

The pencil urchin Phyllacanthus imperialis spawned
synchronously with the mass spawning of scleractinian
corals at Lizard Island, Australia, in two consecutive years.
Their large yolky eggs (507 jim diameter) developed into
nonfeeding echinopluteus larvae with two pairs of larval
arms. The arms were identified as postoral and postero-
dorsal, which are the first and third pairs in typical echi-
noplutei. A larval skeleton was present, with skeletal rods
extending the length of the arms. Five primary podia of
the juvenile rudiment appeared at 2 days of age. Meta-
morphosis of the larvae and settlement began 4 days after
fertilization. Histological examination of 2-day-old larvae
revealed the presence of a developing gut, but no mouth
opened in what would be the oral region of a typical
echinopluteus, or the oral surface of the juvenile rudiment
in older larvae. Like other cidaroid larvae, this species
showed no evidence of an amniotic invagination.

The larva of P. imperialis appears to be a transitional
form between the morphology of feeding and nonfeeding
echinoid larvae. Traces of the ciliary band in the oral re-
gion and the presence of arms typical of the echinopluteus
larva indicate its evolutionary past, whereas the large egg
size and absence of a mouth hint at its future. This larval
form provides insights into developmental changes that

Received 1 1 January 1993; accepted 17 May 1993.

occur during the shift from planktotrophy to lecithotrophy
in echinoid larvae.

Introduction

The causes and consequences of the evolutionary shift
from feeding to nonfeeding larvae has become a popular
topic in recent years (Raff, 1987; Parks et al.. 1988; Wray
and Raff, 1991; Amemiya and Emlet, 1992; McMillan et
al.. 1992). Among echinoids, feeding larvae (plankto-
trophs) are considered to be the ancestral form, and non-
feeding larvae (lecithotrophs) are thought to be more de-
rived (Strathmann, 1974). Within this group of echino-
derms, the shift from planktotrophy to lecithotrophy is
believed to have evolved independently as many as 14
times (Emlet, 1990; cf. Strathmann, 1974). Although the
potential costs and benefits of direct development have
been well discussed (Vance, 1973; Christiansen and Fen-
chel, 1979; Obrebski. 1979), the reasons for shifts in de-
velopmental mode are not completely understood.

Planktotrophy among echinoid larvae is known to be
correlated with egg size (Emlet et al.. 1987). Of the 276
echinoids reviewed by Emlet (1990), 66% have plankto-
trophic development and develop from small eggs (<350
urn), whereas 20% have lecithotrophic development and
develop from larger eggs (>500 ^m).

Before our study, only three echinoids had been re-
ported with planktonic larvae that do not fall into one of
these two groups; Brisaster latifrons, which has a feeding
echinopluteus that develops from an egg 345 pm in di-
ameter (Strathmann, 1979); Clypeaster rosaceus (egg di-
ameter = 280 Mm)- whose larvae are facultative plank-
totrophs, having the echinopluteus form (Emlet, 1986);

77

78

R. R. OLSON ET AL

and Peronella japonica (egg diameter = 276 /um), which
produces a lecithotrophic "pluteus" with only two arms,
but shows normal development of the echinus rudiment
(Okazaki and Dan, 1954). Here we report a fourth "in-
termediate" larval form.

The pencil urchin Phyllacanthus imperialis is a member
of the order Cidaroida, which is believed to be the most
primitive extant echinoid order (Paul and Smith, 1984).
It is common on coral reefs throughout the Indo-west
Pacific but, being nocturnal and cryptic, is rarely seen
even at night. Mortensen (1938), who collected P. imper-
ialis in the Red Sea, reported its egg to be 500 /urn in
diameter, but was unsuccessful in attempting in vitro fer-
tilization. In this paper we provide the first description of
the larva and development of P. imperialis, which has the
echinopluteus form but is lecithotrophic.

Materials and Methods

Gametes of P. imperialis were obtained from adults
collected at Lizard Island, Australia, during the summers
of 1986 and 1987. Urchins were spawned in the days fol-
lowing collection by injecting 5 to 10 ml of 0.55 M KC1
into the coelomic cavity. Eggs were fertilized within 1 h
of spawning. Embryos and larvae were maintained in an
open 3-1 beaker at room temperature (29-3 1C) without
stirring. For histological and morphological examination,
embryos and larvae were preserved in Bouin's solution at
20 h and 2, 3, and 4 days after fertilization. To preserve
the larval skeleton, a few larvae were preserved in buffered
10% formalin. Embryos and larvae were prepared for
scanning electron microscopy (SEM) by dehydration in
a graded series of ethyl alcohol through amyl acetate, dried
at the critical point (CO : ), gold coated, then examined
and photographed using a Novascan 30 scanning electron
microscope. Larvae for histological examination were de-
hydrated to 50% ethyl alcohol, then embedded in JB-4
embedding medium (Polysciences, Inc.). Sequential sec-
tions 2-3 ^m thick were cut with glass knives and stained
with Richardson's stain. Larval skeletal pieces were ex-
posed by corrosion of the soft tissues with 5% sodium
hypochlorite (household bleach) from 4-day larvae fixed
in buffered formalin.

Results

Spawning

On the Great Barrier Reef of Australia, adult P. im-
perialis are usually cryptic and nocturnal and are rarely
encountered even on night dives. However, on the night
of 20 November 1986, approximately 20 P. imperialis
were collected by divers in an area less than 10 m in di-
ameter. This was 4 days after the full moon and the same
night as the mass spawning of acroporid scleractinian cor-

als ((.;/." Babcock et a/., 1986). Numerous P. imperialis were
observed "perched" on the tops of coral heads or mounds
of rubble; one male P. imperialis was observed spawning.
In the days following collection, spawning was induced
in these urchins by injecting them with 0.55 M KC1.
Freshly spawned eggs were yellow-tan and embedded in
a clear gelatinous outer coating that was viscous and sticky.
Spawned eggs were held together by this coating to form
a strand of eggs that floated directly to the surface as it
was released. Mean egg diameter was 507 ^m (SD = 3 1 .9,
n = 10).

Additional urchins were collected on 9 December (five),
and 1 3 December (three). None of these urchins spawned
when injected with KC1; examination of their excised go-
nads revealed that all were spent.

The following year (1987) a few adult P. imperialis were
observed early in November shortly after the full moon.
The density of urchins was considerably lower, and the
urchins were not as exposed as had been noted the pre-
vious year. Approximately 10 urchins were collected over
several nights, but only males produced viable gametes
upon injection with KC1. The few females that did respond
to KC1 injection released masses of what appeared to be
undifferentiated yolk material. Urchins collected in late
November also were not ripe. No male P. imperialis
collected at this time spawned when injected, and those
females that did spawn produced egg-like irregularly
shaped masses of yolk. Urchins collected on 2, 3, 4, and
8 December and injected with KG all responded similarly.
Full moon occurred on 5 December in 1987. On the
morning of 9 December, two females injected with K.C1
released large numbers of eggs, the majority of which were
spherical and uniform in size. About a third of these eggs
were successfully fertilized. The following day (10 De-
cember) two remaining females were spawned, and vir-
tually all of their eggs were uniform in size and shape.
Fertilization of these eggs was nearly 100%.

Development

Oocytes were opaque (yellow-tan in color) and floated
at the surface of the water in their culture beakers. A dis-
tinct fertilization membrane was noted 15 min after the
introduction of sperm. First cleavage was observed 15 min
later i.e., 30 min after fertilization (Table I). Ten hours
after fertilization the embryos were ciliated blastulae ro-
tating within the fertilization membrane and were still
floating. Twenty hours after fertilization the embryos were
hatched swimming gastrulae, still opaque yellow-tan in
color, and floating (Figs. 1. 5). It was not noted whether
gastrulation occurred before hatching. Twenty-four hours
after fertilization there was considerable variation in the
morphology of the larvae, ranging from embryos that were
nearly rectangular with one pair of arm rudiments (Fig.

URCHIN LARVAL DEVELOPMENT

79

Table I

Development schedule o/Phyllacanthus imperialis

Time since fertilization

Stage

30 mm

10 h

:4h

48 h

72 h

96 h
100 h
116 h

First cleavage

Ciliated blastula

Swimming gastrula, some 2 arms

4 short arms

Arms elongating, rudiment visible

Attachment

Arms break off

Fully formed juvenile

2) to larvae that were beginning to develop four equal-
length arms (as seen at 2 days old; Fig. 3). A few of these
larvae were swimming just below the surface of the water.
The first evidence of development of the echinus rudiment
was apparent within the oral field of these early four-armed
plutei (Fig. 3). A band of cilia, extending along the margins
of the arms and coursing up onto the preoral lobe, can
be seen to be just beginning to form on these larvae (Fig.
3). Three days after fertilization four-armed plutei with
well-developed echinus rudiments (Fig. 4) were distributed
throughout the culture vessel. Purple pigment granules
appeared around the preoral lobe of the larvae and along
the arms. Many larvae 3 days old had begun to settle on
the bottom and sides of the culture beaker. At this point
virtually no larvae remained at the surface of the water.
Six days after fertilization all larvae had settled and meta-
morphosed into juvenile urchins. These juveniles were
maintained for another 3 weeks in the beaker without
further care (changing of water or feeding).

Description of embryology

Details of the first few hours of development were not
observed, but observations on fixed embryos revealed that
there was no wrinkling of the blastula or gastrula. Ap-
proximately 15 h after fertilization a typical smooth gas-
trula was formed by invagination of the blastular wall at
the vegetal pole (Fig. 5). Histological sections of gastrulae,
20 h after fertilization, are reminiscent of the typical gas-
trula of feeding echinoid larvae. The ectodermal wall is
relatively thin and there is a distinct blastocoel surround-
ing the archenteron. At this time the archenteron extends
approximately one-third the length of the blastocoel with
the distal tip bending toward the ventral surface of the
embryo at an angle of approximately 45 (Fig. 5). The
timing of mesenchyme migration into the blastocoel was
not observed, but in embryos 20 h old, a large clump of
mesenchyme cells was aggregated around the tip of arch-
enteron (Figs. 1, 5) and around its base (Fig. 5). Though
the archenteron bends toward the ventral surface of the

larva, a stomodeal invagination (larval mouth) never
formed. In histological sections of gastrulae, large numbers
of intracellular yolk-like granular inclusions can be seen
within the mesenchymal, endodermal, and ectodermal
cells of the embryo (Figs. 5, 6). The cells of the embryonic
wall and the archenteron are distinctly columnar, with
their nuclei arranged distally within the cells. The re-
mainder of the cellular inclusions other than yolk-like
bodies are also located near the nucleus; the yolk-like
bodies are stacked into columns that are generally directed
toward the center of the gastrula (Figs. 5, 6). The blasto-
pore is unusually wide, with the opening ( 1 10 ^m) being
almost one-quarter the diameter of the entire embryo
(Fig. 5).

Histological cross sections through the preoral lobe of
early plutei show the hydropore opening on the dorsal
surface of the larva, and that the hydrocoel, juvenile gut,
and perivisceral coelom have all formed (Fig. 7; compare
Fig. 15a). Cross sections taken near the posterior of these
larvae show the extent of development of the water vas-
cular system with the lumens of the primordial tube feet
having formed. Except for the perivisceral coelom, all
coelomic spaces are filled with cellular and yolky-vesicle
inclusions (Figs. 7, 8, 9, 10). The epidermis of the larva
is a single layer of cells nearly cuboidal in form.

Three- and four-day-old lan>ae

At 3 days the larva is fully formed (Fig. 4), and the
juvenile rudiment is very conspicuous. A single contin-
uous ciliary band runs up and down the length of each
arm. At the juncture of the posterodorsal arms the ciliary
band courses up onto what appears to be the vestige of
the preoral lobe (Fig. 1 1 ). The preoral lobe has undergone
a torsion of nearly 45 to the left, as evidenced by the
twisting of the ciliary band on the preoral lobe (Fig. 1 1),
and the ciliary band disappears along the distal edge of
the preoral lobe (Fig. 1 1). Oblique sections though 3-day
plutei show an extensive convoluted juvenile gut and fully
formed tube feet (Figs. 9, 10). Juvenile spines and pedi-
cellaria are also present (Figs. 4, 13, and 14), giving the
impression that 3-day-old larvae are in reality juvenile
rudiments with somewhat shortened larval arms. This is
similar to the larvae of Eucidaris thoursi as described by
Emlet(1988).

The juvenile rudiment, with five primary podia, de-
velops out of the left side of the larva (Figs. 4, 11, 12, 13).
There is no evidence of a juvenile mouth on the oral sur-
face of the rudiment (Fig. 12). The posterior of the larva
shows a striking bilateral symmetry (Fig. 14). With cross-
polarized light, the larval skeleton was observed to extend
to the tips of the arms (Fig. 16). The extent of development
of the larval skeleton within the body of the larva was
obscured by the development of juvenile skeletal struc-

80

R. R. OLSON ET AL.

Figure 1. Scanning electron micrograph of a fractured gastrula of Phyllacanlhus imperialis showing
mesenchyme cells (ME) aggregated around the base of the archenteron. Note the small hlastocoel (BC).

Figure 2. One-day-old embryo showing rudiments of post-oral arms (POA) and blastopore (B).

Figure 3. Two-day-old larva. PDA, posterodorsal arms; TF, tube foot; POL. preoral lobe.

Figure 4. Anterior view of 3- to 4-day-old larva. The preoral lobe has undergone torsion nearly 45 to
the left from its orientation in a typical pluteus. CB, ciliary band.

tures and the general opacity of the preoral lobe. Scanning
electron micrographs of arm rods showed them to be fen-
estrated approximately 1 mm in length with a lattice-like
plate at the base (Fig. 17). The larvae of P. imperial 'is
swim in a typical echinopluteus fashion with the anterior-
posterior axis oriented vertically and the posterior directed
downwards much like the orientation of a falling bad-
minton shuttlecock (Fig. 13).

Metamorphosis and description oj juvenile urchin

Metamorphosis of the echinopluteus includes the for-
mation of juvenile structures such as tube feet, spines,
and pedicellaria, and the resorption of larval tissues. In
the majority of echinoids with an echinopluteus larvae,
the first appearance of juvenile structures is within the

vestibule, which is an invagination that forms on the left
side of the larva. In cidaroids, there is no vestibule (Emlet,
1988). but juvenile structures develop on the left side of
the echinopluteus in the same general location as on other
echinoid larvae. The onset of metamorphosis can be ob-
served when juvenile structures are visible on the exterior
of an echinopluteus larva. These juvenile structures are
called the "echinus rudiment."

Settlement and metamorphosis of P. imperialis larvae
began 4 days after fertilization. Larvae settled by attaching
themselves to the side or bottom of the culture vessel with
their tube feet splaying their arms out radially. Within
4 h the tissue at the tips of the arms began to retract,
exposing the ends of the skeletal rods. Six hours after at-
tachment the tissue on all arms pulled back to the main
body of the larva. Over the next 4 h the four arm rods

URCHIN LARVAL DEVELOPMENT

81

broke oft' at their bases. Periodic upward jerking move-
ment of the spines may have facilitated this breakage.
Twenty hours after attachment all resemblance to the lar-
val form was lost and small juvenile urchins remained.

Perhaps because the 4-day-old larva was virtually a fully
formed juvenile urchin, little morphological change ap-
pears to have occurred either internally or externally at
this point. Although numerous juveniles survived and
formed large numbers of spines, there was still no evidence
of a mouth on urchins 21 days old. The nutritional needs
of the young urchins are probably met from remaining
stored nutrients or possibly through uptake of dissolved
organic matter; this is similar to the early development
reported for Heliocidaris erythrogramma (Williams and
Anderson. 1975).

Discussion

The larva of P. imperialis is unlike any previously de-
scribed echinoid larva. Its external form is surprisingly
similar to a feeding echinopluteus, yet it is completely
lecithotrophic. The larva most similar to P. imperialis is
that of the Japanese sand dollar, Peronellajaponica, which
develops from an egg of 276-jum diameter into an echin-
opluteus-like larva with two, three, or four arms (Okazaki
and Dan, 1954). The larva off. japonica lacks a preoral
region and does not retain pluteal bilateral symmetry
(Okazaki and Dan, 1954; Mortensen, 1921). Phyllacan-
thus parvispinus, a congener also from Australia, has larger
eggs (700 j/m) that develop into little more than opaque
spheres on which five primary podia appear just before
settlement (Parks el a!., 1989).

In a review of the evolution of direct development in
sea urchins. Raff ( 1987) identified four patterns of devel-
opment that could describe the transition from the feeding
pluteus to complete direct development (as in brooded
embryos). These patterns, which he loosely correlated with
egg size, are ( 1 ) typical feeding pluteus (100 ^m egg), (2)
partial pluteus, non-feeding (300 ^m egg), (3) direct de-
velopment with a floating larva (500 ^m egg), and (4)
brooded by mother, complete direct development ( 1 300
Mm egg).

The larva of P. imperialis has features of both groups
2 and 3. It still has the echinopluteus form, placing it in
group 2. but the buoyancy of the larva and size of the egg
place it closer to group 3. Overall, it seems to be a more
reduced larval form than Peronellajaponica (Okazaki and
Dan, 1954) in that it is more yolky; however, it is not as
reduced as Asthenosoma ijimai (Amemiya and Emlet.
1992). which shows only the slightest of echinopluteus
traits (i.e.. little more than primary tube feet and two pairs
of "para-arms").

Raff ( 1988) suggested that three major developmental
changes occur in the shift to lecithotrophic direct devel-

opment in echinoderms. First, egg size increases; second,
typical pluteus structures are lost; and third, the appear-
ance of juvenile features is accelerated.

The pattern of development observed in P. imperialis
is consistent with all three of these criteria. The eggs of
P. imperialis are larger than those of planktotrophs, and
some pluteal structures (such as the second and fourth
pairs of arms and the mouth) do not develop. Finally
there is the rapid (when compared to planktotrophic
forms) appearance of juvenile features. At 48 h after fer-
tilization, the juvenile gut is forming (Fig. 7), the primary
podia are visible externally (Figs. 11. 12, 13) even though
the larval arms have only just begun to develop, and ju-
venile spines and pedicellaria are well formed.

An important question in considering the apparent shift
from planktotrophy to direct development in echinoids
is whether the loss of pluteus features occurs before, at
the same time as, or after an increase in egg size and a
loss of feeding ability. In P. imperialis many larval features
have been retained despite the increase in egg size and
loss of feeding ability. However, there is clear evidence
for the disappearance of other pluteus features. The ciliary-
band, which is well developed along the arms, is greatly
reduced on the preoral lobe (Fig. 1 1 ), and not all pluteus
arms form. With the loss of feeding, the generation of
water currents around the oral region is presumably no
longer necessary; these currents are known to facilitate
particle capture. It would be interesting to examine the
effectiveness and structure of the preoral cilia in Clypeaster
rosaceus, which is a yolky facultative planktotroph that
arises from 280-^m eggs. Since it has no obligatory need
for paniculate food material (although it does gain from
feeding), are its preoral cilia reduced?

It is unlikely that the pluteus form in P. imperialis is
secondarily derived from a brooded embryo (Strathmann,
1974; however, see McEdward (1992) for an argument of
re-evolution of pelagic larval development in an asteroid).
The retention of pluteus-like features in the larvae of
P. imperialis may be insignificant from the perspective of
the evolution of a form of lecithotrophic direct develop-
ment. More likely, the pattern of development displayed
by P. imperialis provides insight into the transition from
a feeding larva to a nonfeeding larva and into the relative
importance of certain larval characters in making such a
transition.

In addition to its unique development pattern. P. im-
perialis also shows an interesting pattern of spawning be-
havior. At least at Lizard Island, this species spawns and
the larvae develop coincidentally with the scleractinian
corals of the Great Barrier Reef. In this region, more than
100 species of scleractinian corals mass spawn over the
course of three nights between the full and last-quarter
moons in late spring (Babcock et ai. 1986). These are
joined by many species of other taxa such as polychaetes

82

R. R. OLSON ET AL.

TF

Figure 5. Light micrograph (LM) section of the gastrula of P. imperialix. Note blastopore (BP) and
blastocoel (BP).

Figure 6. LM close-up of the tip of the archenteron (AE), showing migration of yolk-filled mesenchyme
cells into the blastocoel (BC).

URCHIN LARVAL DEVELOPMENT

83

Figure 11. Dorsal view of 3- to 4-day-old larva. Note ciliary band which diminishes as it extends up
onto the preoral lobe.

Figure 12. View of the "oral region" of the juvenile rudiment from a 3-day-old larva showing absence
of mouth.

Figure 13. Swimming orientation of 3- and 4-day-old larva.

Figure 14. Posterior view of a larva that is ready to settle. Note the presence of juvenile spines and
pediccllaria (PD).

(P. Hutchings, pers. comm.) and holothurians (RRO pers.
observation). In the two years that we observed P. im-
perialis, its spawning coincided with that of the corals,
and similar to corals, it settled out of the water column
4 to 6 days after fertilization (Babcock and Heyward,
1986). Additionally, both P. imperialis larvae and coral

larvae are initially quite buoyant and develop near the
surface of the water.

It would be easy to speculate that direct development
as observed in P. imperialis could be a response to selective
pressures that have caused this species and corals to con-
verge on a similar pattern of development. However, direct

Figure 7. LM cross section through the preoral lobe of 2-day-old larva. HY, hydropore; HC, hydrocoel;
G, gut. See Figure 1 5a for reference.

Figure 8. LM cross section through the body of a 2-day-old larva. POA. preoral arm; G. gut; TF, tube
foot. See Figure 1 5a for reference.

Figure 9. LM sagittal section through the preoral lobe of a 3-day-old larva. See Figure 1 5b for reference.

Figure 10. LM sagittal section through the preoral lobe and arms of a 3-day-old larva. See Figure 15b
for reference.

84

R R. OLSON ET AL.
B

Left

Figure 15. Diagrammatic representation oflocations of light micrograph sections shown in Figure 7
(a:a'(. Figure 8 (b:b'). Figure 9 (c:c') and Figure 10 (d:d').

lecithotrophic development in echinoids is not limited to
the Great Barrier Reef, or even to tropical regions. Phyl-
lacanthus parvispinus (Raff, 1987) and Heliocidaris ery-

Figure 16. Light micrograph of the larval skeleton of P. imperialis
highlighted with cross-polarized light.

Figure 17. S.E.M. of a skeletal rod from one arm of a 4-day-old
larva of P. imperialis.

throgramma (Williams and Anderson, 1975) produce lar-
vae that are ecologically similar to P. imperialis, yet these
species live in temperate habitats where the corals are not
present.

It is difficult to determine exactly why a species un-
dergoes mass spawning (Babcock et ai, 1986); however,
further examination of the spawning behavior of P. im-
perially across a broader geographic region might show
patterns that point more clearly to the exact environ-
mental cues that trigger such behavior. This type of study
might also show whether the pattern of development ex-
hibits any geographic variation that could provide insights
into the evolutionary reasons for an invertebrate to shift
its pattern of development from feeding to nonfeeding.

Acknowledgments

This project was supported in part by a Harbor Branch
postdoctoral fellowship. We thank the Lizard Island Re-
search Station for providing field facilities and the Aus-
tralian Institute of Marine Science for logistical support.
Field assistance was provided by A. R. Davis, E. M. Ley-
decker, R. Z. McPherson, M. B. Olson, K. Osborne, L.
Sullivan, and P. Watts. Special thanks to P. Dixon for
videotaping metamorphosis and to P. Linley for assisting
with SEM. This paper has benefited from discussions with
R. Emlet and R. Raff. Harbor Branch Oceanographic In-
stitute Contribution #965. University of New Hampshire,
Center for Marine Biology Contribution #28 1 .

Literature Cited

Amemiya, S., and R. B. Emlet. 1992. The development and larval
form of an echinothunoid echinoid, Astheno.wma ijimai. revisited.
Biol Bull- 182: 15-30.

Babcock, R. C, G. D. Bull. P. L. Harrison, A. J. Heyward, J. K. Oliver,
C. C. Wallace, and B. L. Willis. 1986. Synchronous spawnings of
105 scleractinian coral species on the Great Barrier Reef. Afar. Biol.
90: 379-394.

Babcock, R. C., and A. J. Heyward. 1986. Larval development of certain
gamete-spawning scleractinian corals. Coral Reefs 5: 1 1 1-1 16.

URCHIN LARVAL DEVELOPMENT

85

Christiansen, F. B., and T. M. Kenohel. 1979. Evolution of marine
invertebrate reproductive patterns. Theor. Popiil. Bid/. 16: 267-282.

1 ink-i. R. B. 1986. Facultative planktotrophy in the tropical echinoid
Clypeaster rosaceus (Linneaus) and a comparison with obligate
planktotrophy in Clypeaster subdepressus (Gray) (Clypeasteroida:
Echinoidea). ./ l-:\p Mar. Biol Ecol. 95: 183-202.

Kmlct, R. B. 1988. Larval form and metamorphosis of a "primitive"
sea urchin. Kucidans iliouarst (Echinodermata:Echinoidea:Cida-
roida). with implications for developmental and phylogenetic studies.
Biol. Bull 174:4-19.

Kmlet. R. B. 1990. World patterns of developmental mode in echinoid
echinoderms. Pp. 329-335 in Advances in Invertebrate Reproduction,
Vol. 5, M. Itoshi and O. Yamashita. eds. Elsevier Science. Amsterdam.

F.mlet, R. B., L. R. McF.dward, and R. R. Strathmann. 1987.
Echinoderm larval ecology viewed from the egg. Pp. 55-136 in Echi-
noderm Studies. Vol. 2, M. Jangoux and J. M. Lawrence, eds. Bal-
kema. Rotterdam.

McFdward. I.. R. 1992. Morphology and development of a unique
type of pelagic lar\a in the starfish Pleraxler lesselalus (Echinoder-
mata: Asteroidea). Biol. Bull. 182: 177-187.

McMillan, \V. O., R. A. Raff, and S. R. Palumbi. 1992. Population
genetic consequences of developmental evolution in sea urchins (genus
Heliocidaris). Evolution 46: 1299-1312.

Mortensen, T. 1921. Studies of the Development and Larval Forms of
Echinoderms. G.E.C. Gad., Copenhagen.

Mortensen, T. 1938. Contributions to the study of the development
and larval forms of echinoderms IV. Kgl. Dan. Vidensk. Selsk.. Skr.
Naturvid. Math. Afd. (9). 7(3): 1-59.

Obrebski. S. 1979. Larval colonizing strategies in marine benthic in-
vertebrates. Mar. Ecol. Prog. Ser. 1: 293-300.

Okazaki, K., and K. Dan. 1954. The metamorphosis of partial larvae
of Peronella japonica Mortensen. a sand dollar. Biol. Bull 106: 83-
99.

Parks, A. L., B. A. Parr, J. Chin, D. S. Leaf, and R. A. Raff.
1 988. Molecular analysis of heterochronic changes in the evolution
of direct developing sea urchins. J. Evol. Biol 1: 27-44.

Parks, A. L., B. VV . Bisgrove, G. A. Wray, and R. A. Raff. 1989. Direct
development in the sea urchin Phyllacanlhus parvispinus (Qdzaoidea):
Phylogenetic history and functional modification. Biol. Bull 111:
96-109.

Paul, C. R. C, and A. B. Smith. 1984. The early radiation and phylogeny
of echinoderms. Biol. Rev 59: 443-481.

Raff, R. A. 1987. Constraint, flexibility, and phylogenetic history in
the evolution of direct development in sea urchins. Dev. Biol 119:
6-19.

Raff, R. A. 1988. Direct developing sea urchins: a system for the study
of developmental processes in evolution. Pp. 63-69 in Echinoderm
Biology. R. Burke, P. Mlandenov. P. Lambert, R. Parsley, eds. Bal-
kema Press.

Sfrathmann, R. R. 1974. Introduction to function and adaptation in
echinoderm larvae. Thalassia Jugos. 10: 321-339.

Strathmann, R. R. 1979. Echinoid larvae of the Pacific northeast (with
a key and comment on an unusual type of planktotrophic develop-
ment). Can. J. '/MO!. 57: 610-616.

Vance, R. R. 1973. On reproductive strategies in marine benthic in-
vertebrates. Am. Nat. 107: 339-352.

Williams, D. H. C., and D. T. Anderson. 1975. The reproductive system,
embryonic development, larval development, and metamorphosis of
the sea urchin Heliocidaris erythrogramma (Val.) (Echinoidea: Echi-
nometndae). Aitxl. J Zoo! 23: 371-403.

Wray, G. A., and R. A. Raff. 1991. The evolution of developmental
strategy in marine invertebrates. Trends Res. Ecol. Evol. 6: 45-50.

Reference: Biol. Bull. 185: 86-96. (August, 1993)

Ultrastructure of the Coeloms of Auricularia Larvae

(Holothuroidea: Echinodermata): Evidence for the

Presence of an Axocoel

ELIZABETH J. BALSER 1 . EDWARD E. RUPPERT 1 , AND WILLIAM B. JAECKLE 2

1 Department of Biological Sciences, 132 Long Hull, Cleniwn University, Clemson,

South Carolina 29634-1903, and 2 Smithsonian Environmental Research Center,

PO Box 28, Edxewater, Maryland 21037-0028

Abstract. A hallmark feature of echinoderm larvae is
the development of the left anterior coelom. This coelom,
called the axohydrocoel, consists of the morphologically
distinct, but undivided, left axocoel and hydrocoel. The
axocoelic portion forms a duct that opens to the exterior
via a pore on the dorsal surface of the animal. Holothuroid
larvae are thought to lack an axocoel, but develop an an-
terior coelom, duct, and pore that are regarded as parts
of the hydrocoel. New ultrastructural data, however, show
that holothuroid auricularia larvae possess an axocoel and
hydrocoel united together into an axohydrocoel. During
development the anterior coelom consists of an intercon-
nected left somatocoel, hydrocoel, and axocoel. 'The left
somatocoel separates from the axohydrocoel and subdi-
vides into left and right somatocoels. The somatocoels
and hydrocoel region of the axohydrocoel are lined by a
monociliated mesothelium having characteristics of
transporting epithelia. The axocoel epithelium, like that
of asteroid larvae, is composed of mesothelial podocytes.
A duct connects the axocoel directly to the open dorsal
pore and is lined with a columnar transporting epithelium.
The occurrence of a specialized podocyte-lined cavity be-
tween the surface pore and the hydrocoel in echinoderm
larvae is indicative of an axocoel. That similar structures
occur in auricularia larvae supports the identification of
an axocoel in holothuroids.

Introduction

Holothuroids with indirect development typically have
a planktonic feeding auricularia larva that undergoes

Received 2 December 1992; accepted 10 May 1993.
Smithsonian Marine Station at Link Port Contribution #325.

metamorphosis to produce a pentactula. or pelagic ju-
venile. In most holothuroids, the juvenile settles shortly
after metamorphosis and assumes an adult lifestyle. The
auricularia larva is restricted to the family Synaptidae in
the order Apodida and to the families Holothuriidae and
Stichopodidae in the order Aspidochirotida (Smiley el ai.
1991).

Auricularia larvae have a large blastocoel that houses
the J-shaped gut, the paired body coeloms, and the left
anterior coelom. In most larval asteroids, ophiuroids, and
echinoids, a right anterior coelom is also present, but its
developmental fate varies among groups. It reportedly ei-
ther fuses with the right somatocoel, becomes the pulsatile
vesicle (dorsal sac, madreporic vesicle) of the heart, or
disappears at metamorphosis (Hyman. 1955; Hendler,
1 99 1 ; Pearse and Cameron, 1 99 1 ). The right anterior coe-
lom does not develop in holothuroids and crinoids, which
are believed to lack a pulsatile vesicle (Hyman, 1955;
Holland, 1991; Smiley el at., 1991).

The left anterior coelom is a unifying feature of echi-
noderm larvae and, despite differences in specific details
of morphogenesis, is positionally and anatomically similar
in five of the six extant classes (Hyman, 1955; Dan, 1968;
Ruppert and Balser. 1986; for review see chapters 4-8 in
Giese et ai. 1991); no information exists on the larval
development of the Concentricycloidea. In echinoderm
larvae, with the reported exception of holothuroids (Smi-
ley et ai. 1 99 1 ), the left anterior coelom represents a fusion
of the left axocoel and hydrocoel (Bury, 1 885, 1 889; Rup-
pert and Balser, 1986; Giese et ai. 1991). Hyman (1955),
drawing principally on the work of earlier researchers,
emphasized this connection and retained the term axohy-

86

AURICULARIA LARVAL COELOMS

87

drocoel. The axohydrocoel is open, if only transiently, to
the larval somatocoels and to the exterior via a duct and
dorsal pore.

Holothuroid auricularia larvae develop an anterior
coelom, duct, and pore, but are believed to lack any vestige
of an axocoel (Hyman, 1955; Smiley. 1986, 1989). If ho-
lothuroids do not have an axocoel, then the anterior coe-
lom. unlike that of other echinoderms. consists only of
the hydrocoel and the somatocoel primoridium. The left
somatocoel arises from the anterior coelom and later di-
vides forming the right and left larval somatocoels. A re-
cent study of the development of the aspidochirotid Sti-
chopus californicus (Smiley, 1986) suggests that the left
anterior coelom and duct consists solely of the hydrocoel
and the somatocoel primoridium. Based primarily on the
ultrastructure of the madreporic vesicle and the hypothesis
that adult holothuroids lack any axoelic derivatives (Smi-
ley, 1986, 1989). Smiley et al. ( 1991 ) conclude that holo-
thuroid auriculariae have no "identifiable axocoel as part
of their complement of coelomic primordia at any stage
of development."

Several ultrastructural examinations of axocoelic de-
rivatives in adult echinoderms (Bachmann and Gold-
schmid, 1978; Welsch and Rehkamper, 1987; Balser,
1990; Balser et al., 1993, unpubl. data) and one of the
axohydrocoel of a larval asteroid (Ruppert and Balser,
1986) indicate that podocytes typify the lining of the axo-
coel. This study was undertaken to search for podocytes
and other evidence of an axocoel in holothuroid larvae
and is part of a larger investigation that attempts to re-
construct the phylogeny of extant echinoderms using a
distinctive echinoderm organ, the axial gland, as a sys-
tematic character. Specific objectives of this investigation
include examination of the ultrastructure of the larval
coeloms of Holot/utria grisea and different species of field-
collected auricularia larvae. A comparison of the coeloms
of these larvae with those, particularly the axohydrocoel,
of other echinoderms will be used to test the hypothesis
that an axocoel is present in holothuroid auricularia
larvae.

Methods and Materials

Auricularia larvae of the holothuriid Holothuria grisea
were obtained from fertilization of freely spawned ga-
metes. Adults were collected in June 1992 from rock rub-
ble beneath the Ft. Pierce South Seaway Bridge and trans-
ported to the Smithsonian Marine Station at Link Port.
Ft. Pierce, Florida. Specimens were kept in buckets of
unaerated seawater until spawning occurred. Fertilized
eggs were washed and transferred to culture dishes with
clean seawater. Cultures were maintained at 26C and
provided daily with clean water and the marine alga Iso-
chrysis galbana (Tahitian strain) as food.

During the spring and summer of 1 992, auricularia lar-
vae were collected from the waters off Grand Bahama
Island (approx. 23N 79.8W) and from the Gulf Stream
offthe coast of Florida (approx. 26.5N 78. 8 W). Auricu-
lariae were hand-sorted from plankton tows conducted
aboard the R/V Sunburst of the Smithsonian Marine Sta-
tion at Link Port and aboard the R/V Seadiver during
expeditions 3 (4/27/92) and 4 (6/22/92) headed by Dr.
Tammy Frank, Harbor Branch Oceanographic Institu-
tion. Ft. Pierce, Florida. Larvae were identified as apodids
or aspidochirotids based on the presence or absence of
wheel ossicles. Within the order Apodida, only synaptids
have an auricularia larva, and only larvae in this family
possess wheel ossicles (e.g.. Smiley et al., 1 99 1 ). Based on
larval characters, further classification of the aspidochi-
rotids was not possible.

Morphological data for H. grisea and field-collected
auriculariae were acquired from living larvae and from
light and electron microscopy of plastic-embedded spec-
imens. Live auriculariae were photographed with a Zeiss
Photomicroscope II loaded with T-max (Kodak) black
and white film. Serial developmental stages of//, grisea
larvae were fixed for microscopy using 2.5% glutaralde-
hyde in Millonig's phosphate buffer adjusted to 1080 mil-
liosmoles with NaCl. Post fixation in 1 .0% OsO 4 in 0.2 M
Millonig's buffer was followed by alcohol dehydration,
propylene oxide and Epon 812 infiltration, and embed-
ment in Epon 812 (Electron Microscopy Sciences). Field-
collected auricularia larvae were prepared for microscopy
by the same method.

Serial thick ( 1 /urn) and representative thin sections were
cut with a Porter-Blum Sorvall or Reichert-Jung Ultracut
E ultramicrotome. Thick sections were stained with
aqueous 1% toulidine blue in 0.5% borax and were ob-
served and photographed with a Zeiss Photomicroscope
I. Thin sections were stained with aqueous uranyl acetate
followed by lead citrate and examined either with a Zeiss
EM 9S or a Hitachi 600 electron microscope.

Results

Coelomic organization

The development from fertilized egg to auricularia larva
of //. grisea follows that described for other indirect-de-
veloping holothuroids (e.g.. Smiley et al., 1991). The first
24 h postfertilization are marked by the formation of a
uniformly ciliated blastula that hatches from the egg en-
velope and further develops into a gastrula. Gastrulation
is followed by differentiation of the archenteron into a
gut and a single unpaired anterior coelom. This anterior
coelom arises from the apex of the archenteron and is the
primordium of all other larval coeloms. Between the sec-
ond and third day of development, the archenteron bends

88

E. J. BALSER ET AL

toward and fuses with the ventral epidermis to form the
mouth. At the same time, the anterior coelom produces
a duct, which grows towards the dorsal surface and even-
tually opens at a pore just to the left of the larval midline.
By day 4, the now recognizable auricularia larva has an
unbroken epidermal ciliary band and a complete func-
tional gut (Fig. 1). The gut is C-shaped and consists of a
ventral mouth and anus separated by an esophagus,
stomach, and intestine. In addition to the gut, the blas-
tocoel is occupied by mesenchymal cells and by the an-
terior coelom (Figs. 1, 2).

Data from a developmental series of//, grisea auricu-
laria larvae and from field-collected larvae show that the
anterior coelom forms an undivided cavity consisting ini-
tially of three morphologically distinct regions (Figs. 1,
3). These regions are the primordia of the hydrocoel, the
axocoel, and the paired somatocoels. The somatocoel pri-
mordium grows posteriorly along the left side of the large
bulbous stomach and eventually separates from the orig-
inal anterior coelom (Figs. 1. 3, 6). This cavity later sub-
divides (Fig. 6) to form both the left and right somatocoels
flanking the larval stomach (Dan, 1968; Smiley el al.,
1991).

The remaining two regions of the anterior coelom, lo-
cated dorsal to the gut, correspond to the axohydrocoel
found in other echinoderm larvae (Fig. 3). The medial
lobe, or hydrocoel, extends toward the middle of the larva
and grows to encircle the esophagus. This lobe gives rise
to the water vascular system of the pentactula and adult.

The third lobe, or axocoel, is situated between the hy-
drocoel and the dorsal pore and includes the duct con-
necting the axohydrocoel to the exterior (Figs. 3, 4, 5).
The axocoel varies in size among examined species. In
H. grisea, the axocoel appears to be restricted to the duct
and a few cells at the proximal inner end of the duct. In
field-collected aspidochirotids and apodids, the axocoel
cavity, which is largest in the synapids, is oval and extends
either anteriorly or concentrically from the inner part of
the duct (Figs. 4, 5). Dissimilarity in the size of the axocoel
may be a reflection of differences in the size of the auricu-

laria of each species. H. grisea auriculariae are consider-
ably smaller (0.75 mm in length 1 5 days postfertilization)
than field-collected aspidochirotids (1-3 mm in length)
and synaptids (up to 5 mm in length).

Coelomic ultrastructure

Following morphogenesis of the duct and its connection
to the exterior, further growth and differentiation of the
anterior coelom results in the three ultrastructurally dis-
tinct, but continuous cavities representing the somatocoel
primordium, the hydrocoel, and the axocoel. The meso-
thelium lining all three lobes of the anterior coelom is
composed of monociliated cells that rest on a continuous
basal lamina and are interconnected by cellular junctions.
Ultrastructural dissimilarities in the lining of each cavity
are principally differences in cellular junctions, apical mi-
crovilli, and basal modifications. Although differentiation
is evident, at the latest developmental stage of auricularia
examined, the epithelium of each region had not yet ac-
quired all the characteristics of juvenile mesothelia. For
example, the hydrocoel and the somatocoel primordial
cells lack basal myofilaments typical of cells lining the
juvenile water vascular system and body cavity.

After the somatocoel primordium loses its connection
to the anterior coelom, the initially oval cavity becomes
elliptical and asymmetrical as it grows posteriorly along
the length of the stomach. The cellular lining of the medial
surface of the coelom closest to the stomach becomes flat
and squamous, while that of the lateral surface remains
columnar (Fig. 7). Regardless of shape, somatocoel cells
are characterized by apical adhering and septate junctions,
scattered apical microvilli, extensive paracellular spaces,
and basal, vertebrate-like, tight junctions (Figs. 8, 9, 10).
Not all somatocoel cells are coupled by basal tight junc-
tions. In some cases, only basal adhering junctions were
observed or basal junctions were absent (Fig. 9). The par-
acellular spaces often extend to the basal junctions or to
the basal lamina. The lateral membranes lining these
spaces possess coated pits, endocytic pits, and invagina-

Figure 1. Ventral view of a field-collected aspidochirotid auricularia. The axohydrocoel consists of the
axocoel (ax) and hydrocoel (hy). This coelom is situated on the left dorsal side of the animal at the level of
the junction between the esophagus (es) and the stomach (st). The somatocoels (so), which are initially
connected to the axohydrocoel, lie lateral to the stomach. Scale bar = 0.1 mm; be. blastocoel; cb, ciliated
band; in, intestine; mo mouth.

Figure 2. Lateral view of a 7-day-old larva of//, grisea- At this stage of development, the anterior coelom
(ac) consists of the duct (du) and the undivided and undifferentiated left axocoel. hydrocoel, and somatocoel
primordium. The duct provides an open connection between the anterior coelom and the dorsal pore (dp).
Scale bar = 0.05 mm; an, anus; es. esophagus; in, intestine; st, stomach.

Figure 3. Dorsal view of the coeloms of a field-collected aspidochirotid auricularia larva showing the
interconnected left axocoel (ax), hydrocoel (hy), and somatocoel (so). Scale bar = 0.05 mm; dp, dorsal pore.

Figure 4. Dorsal view of the axocoel (ax), duct (du), and pore (dp) of a field-collected synaptid auncularia
larva. Scale bar = 0.05 mm; cb, ciliary band; co, coelom (hydrocoel?); ws, wheel ossicle.

Al'RICULARIA LARVAL COELOMS

89

','//*

M A . i-

i^vllE

\ - - \>

V *

Figures 1-4. Light micrographs of living auricularia larvae.

90

E. J. BALSER ET AL

Figures 5-6. Light micrographs of tangential sections through the larval coeloms of a field-collected
aspidochirotid auricularia.

Figure 5. Shows axocoel (ax) and hydrocoel (hy). The axocoel consists of a thin-walled cavity and a
ciliated axocoelic duct (du), which opens at the dorsal pore (dp) on the left dorsal side of the animal. Scale
bar = 0.01 mm; he, blastocoel; ep, epidermis, lu. lumen of the stomach; me, vesiculated mesenchyme cell.

Figure 6. The somatocoels (so) lie lateral to the stomach (st) and the upper intestine (in). Scale bar
= 0.025 mm; be, blastocoel; me, vesiculated mesenchyme cell.

tions suggestive of transfer tubules (Fig. 8). Somatocoel
cells, like those of the other coelomic mesothelia, are
monociliated (Fig. 7). The cytoplasm contains coated
vesicles, numerous other vesicles, mitochondria, and a
large nucleus.

The hydrocoel lobe of the axohydrocoel is lined by
squamous and cubodial epithelial cells joined by apical
adhering junctions followed by septate junctions (Figs.
11, 13). Basally, the lateral membranes are extensively
interdigitated and are interconnected by tight junctions
(Figs. 12, 14). The cytoplasm is replete with basal mito-
chondria, putative lysosomes, and vesicles. The nucleus
is basal and is elongated. Apical microvilli were infre-
quently observed.

The defining feature of the axocoel mesothelium is the
presence of podocytes (Figs. 15-19). Podocytes are epi-
thelial cells that exhibit basal modifications forming foot
processes, or pedicels (Figs. 16, 18, 19). Pedicels rest on
the underlying basal lamina and provide breaks, or fen-
estrations, in an otherwise continuous epithelium. Pedicels
are bridged by the extracellular matrix of the underlying
basal lamina (Figs. 18, 19). In addition to the basal lamina,
some pedicels are bridged by diaphragms similar to fil-
tration-slit membranes found across the pores of the fen-

estrated epithelia associated with the vertebrate nephron
(Bulger. 1983). The cell bodies of podocytes are generally
interconnected only by adhering junctions, but short septate
junctions were occasionally observed connecting podocytes
to hydrocoel or somatocoel cells or to axocoelic duct cells
(Fig. 17). In addition to scattered microvilli (Fig. 17), the
apical membrane possesses a single cilium and endocytic
and coated pits. The cytoplasm contains vesicles, mito-
chondria, lysosomes, and a large circular nucleus (Figs.
16, 17).

Columnar monociliated epithelial cells line the lumen
of the axocoelic duct which directly connects the axocoel
lobe of the axohydrocoel to the dorsal pore (Fig. 20). The
apices of these cells have a single cilium, many microvilli
(Figs. 20, 21 ), and coated pits. Coated vesicles, numerous
other vesicles, mitochondria, putative lysosomes, and a
basal nucleus were observed (Fig. 21). Apical adhering
and subapical septate and basal tight junctions are typical
of this epithelium (Figs. 22, 23).

Discussion

The left axohydrocoel develops similarly in all echi-
noderm larvae except crinoids (Hyman, 1955; Balser and

AURICULARIA LARVAL COELOMS

91

Figures 7-10. Transmission electron micrographs of sections through the somatocoels ol'aspidochirotid
auriculariae.

Figure 7. The epithelium lining the lumen (lu) of the somatocoel is composed of squamous and columnar
monociliated cells. Scale bar = 1.0 pm; be, blastocoel; ci, cilium; nu, nucleus; pc, paracellular space.

Figure 8. Large paracellular spaces (pc) lead to channels suggestive of transfer tubules (arrowhead). Scale
bar = 0.5 MTU lu, lumen of somatocoel; mi, mitochondrion; nu, nucleus; se, grazing section through a
subapical septate junction; ve, vesicle; arrow indicates apical coated pit.

Figure 9. Somatocoel epithelial cells are joined by apical adhering junctions followed by subapical septate
junctions (arrowheads). Basal junctions are absent between some cells (arrow). Scale bar = 0.5 ^m; be,
blastocoel; lu. lumen of somatocoel. mi. mitochondrion; pc, paracellular space.

Figure 10. The basal portion of some somatocoel cells are interconnected by tight junctions. Arrowheads
indicate points of membrane contact. Scale bar = 0. 1 Mm: be. blastocoel; pc, paracellular space.

92

I I BALSER ET AL.

. , :

SA >,-- j. 1 :

K # ^##-** '

Figures 1 1-14. Transmission electron micrographs of tangenital sections of the hydrocoel region of the
axohydrocoel of field-collected synaptid auriculariae.

Figure 11. Hydrocoel epithelial cells have elongated nuclei (nu). numerous putative small (500-750 nm
in diameter) lysosomes (arrowheads), and larger lysosomes (ly). Scale bar = 1 pm; lu. lumen of hydrocoel.

Figure 12. Hydrocoel epithelial cells show extensive basal interdigitation. Basally located mitochondria
(mi) are often associated with these dighations. Scale bar = 0.5 pm; be, blastocoel: lu. lumen; ve, vesicle;
arrowhead indicates putative lysosome.

Figure 13. Apical adhering junctions (ad) are followed by septate junctions (se). Scale bar = 0.5 jim; lu,
lumen; ve, vesicle; arrowhead indicates putative lysosome.

Figure 14. Basal junctions are similar to vertebrate tight junctions. Arrows indicate definitive points of
membrane contact. Scale bar = 0.5 ^m.

Ruppert, 1993). Crinoids differ from other echinoderms
because the archenteron separates into anterior and pos-
terior cavities. The anterior cavity gives rise to the enteric
sac and the axohydrocoel. The posterior cavity gives rise
to the left and right somatocoels. A connection, which is
maintained in the adult, is secondarily established between
the somatocoels and the axohydrocoel.

In noncrinoid larvae, the left anterior coelom separates
from the archenteron and is the primordium of the paired
somatocoel, hydrocoel, and axocoel. The somatocoel pri-
mordium eventually loses its connection to the anterior
coelom, but the axocoel and hydrocoel remain united.

The axohydrocoel establishes a duct that opens to the
exterior via the dorsal pore. In crinoids, asteroids, ophiu-
roids, and echinoids, the undivided axohydrocoel and its
union with the exterior are retained through metamor-
phosis via the stone canal and madreporic pores. The adult
stone canal, which may originate from the axocoel, pro-
vides a link between the axial gland coelom and the hy-
drocoel-derived water vascular coelom. The madreporite
opens internally into the ampulla (an axocoelic deriva-
tive), which joins the axial coelom to the stone canal. A
stone canal and madreporite and, according to Erber
(1983), a rudimentary ampulla all develop in juvenile

\l Kl( ULAR1A LARVAL COELOMS

93

Figures 15-19. Transmission electron micrographs of podocytes lining the axocoelic region of the axo-
hydrocoel of several different auricularia larvae.

Figure 15. Longitudinal section of the axocoel of a field-collected aspidochirotid larva shows the transition
of the cuboidal mesothelium of the duct (du) to the mesothelial podocytes (po) lining the lumen (lu) of the
axocoel. Scale bar = 1 ^m; be. blastocoel; go, Golgi body.

Figure 16. Field-collected synaptid auricularia podocyle. Podocytes are defined by the presence of foot
processes or pedicels (arrows). Scale bar = 0.5 iim; bb. ciliary basal body; be. blastocoel; lu, axocoel lumen.

Figure 17. Apical region of podocytes in a field-collected aspidochirotid auricularia showing apical junc-
tions (arrows) and microvilli (mv). Scale bar = 0.5 ^m; lu. lumen; ly, lysosome; mi, mitochondrion; nu.
nucleus; ve, vesicle.

Figures 18-19. Basal region of field-collected aspidochirotid auricularia podocytes (po) showing pedicels
that provide gaps or fenestrations (arrows) in an otherwise continuous epithelium. Pedicels that are not
joined by filtration slit membranes are bridged only by the basal lamina (bl) supporting this mesothelium.
Between pedicels the lumen (lu) of the axocoel and the blastocoel (be) are separated only by these extracellular
connective tissue layers.

Figure 18. Scale bar = 0.5 i^m.

Figure 19. Scale bar = 0.25 ^m.

94

E. J. BALSER ET AL

lu

Figures 20-23. Transmission electron micrographs of the axocoelic duct of an //. grisea larva (20) and
a field-collected synaptid auricularia larva (21-13).

Figure 20. Longitudinal section through the duct that opens to the exterior via the dorsal pore (dp).
Duct cells are cuboidal monociliated epithelial cells that have basal nuclei (nu) and apical microvilli (mv).
Scale bar = 2 nm: be. blastocoel; ep. epidermis; lu. lumen of duct.

Figure 21. Cells lining the duct lumen (lu) possess numerous apical cellular vesicles (ve) and putative
lysosomes (ly). Scale bar = 2 ^m; be. blastocoel; ci. cilium; nu. nucleus.

Figure 22. Shows apical adhering (ad) and septate (se) junctions. Scale bar = 0.25 ^m; lu. lumen; mi.
mitochondrion.

Figure 23. Shows basal tight junctions. Arrowheads indicate areas of membrane contact. Scale bar
= 0.25 ^m; mi. mitochondrion; nu, nucleus.

AURIC't'LARIA LARVAL COELOMS

95

holothuroids, hut the fate of the larval axocoel reported
here is unknown at present.

The ultrastructure of holothuroid larval somatocoels is
similar to that of asteroid larvae (unpuhl. data) and sug-
gests a transportive function for this epithelium. The
presence of paracellular spaces extending from the basal
lamina towards the cell apex and the absence of basal
junctions in some areas suggest that this epithelium is
transporting substances from the blastocoel into the cell
or indirectly into the coelom (Berridge and Oschman,
1972). This hypothesis is supported by the presence of
apical septate junctions, putative transfer tubules, endo-
cytic and coated pits on the lateral membranes, and nu-
merous vesicles.

The ultrastructure of the hydrocoel also indicates a
transporting function. Deeply folded lateral and basal
membranes and associated basal mitochondria are typical
of osmoregulatory epithelia (Berridge and Oschman,
1972). The presence of occluding apical septate junctions
and basal tight junctions and the paucity of apical micro-
villi suggest that the presumed transport is basally located.

Like that of asteroid larvae, (Ruppert and Balser, 1986),
the holothuroid axocoel mesothelium is composed, in
part, of podocytes. The presence of podocytes and of the
open ciliated duct to the exterior indicates a pressure-
driven nitration system and suggests an excretory function
(Ruppert and Smith, 1988). Although, at present, no
physiological data are available, we predict that blasto-
coelic fluid is filtered across the basal lamina underlying
the podocytes, through the fenestrations between pedicels,
and into the coelomic cavity of the axohydrocoel. Mod-
ification of the primary urine could be accomplished by
the axohydrocoel and duct mesothelium. The occurrence
of apical microvilli. endocytic and coated pits, and nu-
merous apical vesicles suggests that the duct epithelium
is transporting substances from the lumen.

Results presented in this paper indicate that holothuroid
auricularia larvae possess a left axohydrocoel morpholog-
ically similar to that of other echinoderm larvae. In con-
trast. Smiley el al. ( 1991 ) argue that holothuroids do not
have an axocoel and, thus, the anterior coelom is com-
posed of the hydrocoel and the somatocoel primordia
only. They base this hypothesis principally on the unusual
ultrastructure of the madreporic vesicle and the presumed
lack of axocoelic derivatives in the adult (i.e.. the axial
gland). Unlike the madreporic vesicle (dorsal sac, pulsatile
vesicle), which is a muscular sac in other echinoderms,
the madreporic vesicle of holothuroids, as described by
Smiley ( 1986), is a syncytium and probably functions in
the secretion of the madreporite ossicles.

Smiley (1989) speculates that axocoelic functions such
as larval attachment are assumed by the hydrocoel and
that the axocoel of other echinoderms arose from differ-

entiation of the epithelium forming the buccal podia. He
supports this idea with the fact that the pentactula (as well
as juveniles of direct developers) use the buccal podia as
attachment organs. The undivided, undifferentiated ho-
lothuroid axohydrocoel is regarded as a hydrocoel only.
In his view, the axocoel of other echinoderms evolved
later from the plesiomorphic holothuroid "hydrocoel."

The discovery of a morphologically distinct region of
the holothuroid larval axohydrocoel, combined with the
positional and ultrastructural agreement of that region
with the axocoelic portion of the left axohydrocoel of other
echinoderms, reopens the question of an axocoel in holo-
thuroids. Data presented here show that holothuroid au-
ricularia larvae develop an axohydrocoel, axocoelic duct,
and open dorsal pore. The position of the axocoel and
duct between the dorsal pore and hydrocoel, the connec-
tion of the axocoel to other larval coeloms, and the pres-
ence of podocytes indicate the existence of an axocoel in
holothuroid larvae.

Acknowledgments

We thank Dr. Tammy Frank for the opportunity to
collect holothuroid larvae during her Harbor Branch
Oceanographic Institution (Ft. Pierce, Florida) trawling
expeditions. We are grateful to Dr. Mary Rice and the
staff at the Smithsonian Marine Laboratory at Link Port,
Ft. Pierce, Florida, for providing the laboratory space,
equipment, and plankton samples that made much of
this work possible. Supported by NSF Grant #9006599
to E. E. Ruppert.

Literature Cited

Bachmann, S., and A. Goldschmid. 1978. Fine structure of the axial

gland of Spluiereclunus granularix. Cell Tiss. Res. 193: 107-123.
Balser, E. J. 1990. The fine structure of the axial complex in the brit-

tlestars Ophknhrix angulata and Ophiactis savignyi. Am. Zool. 30:

1I4A.
Balser, E. J., and K. E. Ruppert. 1993. Ultrastructure of axial vascular

and coelomic organs in comasterid feather stars (Crinoidea: Echi-

nodermata). Ada Zoo! 74:87-101.
Berridge, M. J., and J. L. Oschman. 1972. Pp. 1-91 in Transporting

Epithelia. Academic Press. New York.
Bulger, R. E. 1983. The urinary system. Pp. 869-912 in Histology Cell

ami Tissue Biology. L. Weiss, ed. Elsevier Science Publ. Co., New

York.
Bury, H. 1885. The metamorphosis of echinoderms. Q J. Microsc.

Set. 38: 45- 131.
Bury, H. 1889. Studies in the embryology of echinoderms. Q J Mt-

cruxc. Set. 29: 407-449.
Dan, K. 1968. Echmodermata. Pp. 280-315 in Invertebrate Embrvol-

ni>\; M. Kume and K. Dan. eds. Prosveta Press, Belgrade.
Erber, \V. 1983. Zum Nachweis des Axialkomplexes bei Holothunen.

'/Mil. Scripta. 12: 305-313.
Giese, A. C, J. S. Pearse, and V. B. Pearse, eds. 1991. Reproduction

of Marine Invertebrates: Echinoderms and Lophopharuies. Vol. 6.

The Boxwood Press. California. 808 pp.

96

E. J. BALSER ET AL.

Hendler, G. 1991. Echinodermata: Ophiuroidea. Pp. 356-479 in Re-
production of Marine Invertebrates: Echinoderms and Lophophora/es.
Vol. 6. A. C. Giese. J. S. Pearse. and V. B. Pearse. eds. The Boxwood
Press. California.

Holland, N. D. 1991. Echinodermata: Crinoidea. Pp. 247-292 in Re-
production of Marine Invertebrates: Echinoderms and Lophophorates.
Vol. 6. A. C. Giese. J. S. Pearse, and V. B. Pearse. eds. The Boxwood
Press, California.

Hyman, L. H. 1955. The Invertebrates: Echinodermata. Vol. 4.
MacGraw-Hill Book Co., New York. 763 pp.

Pearse, J. S., and R. A. Cameron. 1991. Echinodermata: Echinoidea.
Pp. 514-624 in Reproduction oj Marine Invertebrates: Echinoderms
and Lophophorates. Vol. 6. A. C. Giese, J. S. Pearse, and V. B. Pearse,
eds. The Boxwood Press, California.

Ruppert, E. E., and E. J. Balser. 1986. Nephndia in the larvae of
hemichordates and echinoderms. Bio/. Bull 171: 188-196.

Ruppert, E. E., and P. R. Smith. 1988. The functional organization of
nitration nephndia. Bio/. Rev 63: 231-258.

Smiley, S. 1986. Metamorphosis of Slichopus californicus (Echino-
dermata: Holothuroidea) and its phylogenetic implications. Bio/ Bull.
171:611-631.

Smiley, S. 1989. The phylogenetic relationships of holothurians: a
cladistic analysis of the extant echinoderm classes. Pp. 69-84 in
Echinoderm Phytogeny and Evolutionary Biology. C. R. C. Paul and
A. B. Smith, eds. Oxford Science Publ.. England.

Smiley, S., F. S. McEuen, C. Chafee, and S. Krishnan.
1991 . Echinodermata: Holothuroidea. Pp. 664-732 in Reproduction
of Marine Invertebrates: Echinoderms and Lophophorates, Vol. 6.
A. C. Giese, J. S. Pearse, and V. B. Pearse, eds. The Boxwood Press,
California.

Welsch, U., and G. Rehkamper. 1987. Podocytes in the axial gland of
echinoderms. J. Zool. 213: 45-50.

Reference: Biol. Bull. 185: 97-108. (August, 1993)

Lime-Twig Glands*: A Unique Invention of an
Antarctic Entoproct**

PETER EMSCHERMANN

Fakultdt fiir Biologie der Universitat Freiburg, Biologie fiir Mediziner,
Schanzlestr. 1, D 7800 Freiburg i.Br. BRD

Abstract. Specialized glands that release formed secre-
tions of a complex structure are known from several in-
vertebrate phyla. A novel type of such an extrusive organ
has been detected in the newly described Antarctic en-
toproct Loxosomella brochobola Emschermann, 1993 and
is reported here. The specialized extrusive organs known
from other invertebrates are generally unicellular, but
these entoproctan glands are multicellular organs. The
structured secretion of these glands is an extracellular
product homologous to the body cuticle and is discharged
in long sticky, hollow threads. In evolutionary conver-
gence to the glutinant spirocysts of the Anthozoa, these
threads are assumed like set out single lime-twigs to
trap larger prey organisms inaccessible to the ciliary feed-
ing current of the entoproct. Specialized glands of this
kind have not been known previously in Entoprocta. This
"invention" by a nanoplankton feeder must be seen as a
specific adaptation to life in an environment that is poor
in nanoplankton. L. brochobola was found exclusively on
the inner, abfrontal surface of the tube-shaped, calcareous
colonies of the bryozoon Pore/la malouinensis and shares
this microhabitat only with some smaller predators, such
as the hydrozoan Halecium sp.; no other ciliary feeders
are present.

Introduction

Based on the structure and function of their products,
three major types of glands that extrude formed secretions

* Lime-twigs, used even today in some countries to catch song-hirds,
are foot-long twigs or willow rods smeared with hird-lime (sticky plant
extracts e.g.. from mistletoe berries). Such lime-twigs, singly or in groups.
are suspended in trees, fixed in hedges or simply stuck into the earth,
and small birds adhere to them by their plumage.

** These investigations have been supported by the Deutsche For-
schungsgemeinschaft.

Received 1 1 January 1993; accepted 6 May 1993.

have been described in invertebrate animals. In all cases,
these glands are unicellular. First, the well-known stinging
and gluing cells (or nemato- and spirocysts) are the char-
acteristic weapons of cnidarian polyps and medusae, es-
pecially arranged in batteries along their tentacles. These
unicellular glands produce the most complicated glandular
products in the animal kingdom: i.e., intracellular capsules
containing an invaginated, heavily convoluted, ejectible
tubule. Upon irritation, the capsule discharges a harpoon-
like thread poisonous or sticky within a few millisec-
onds (Holstein, 1981; Tardent and Holstein, 1982; Hoi-
stein and Tardent, 1984).

The second type of such highly specialized gland cells
are the colloblasts of the Ctenophora. The fishing tentacles
of these predatory animals are laden with these glutinous
cells that release sticky secretion granules. These, after
secretion, remain fixed to the resilient cytoskeleton by
bundles of microtubules, and so hold the stuck prey fast
(Franc, 1978).

Finally, the so-called rhabdite and rhabdoid-forming
gland cells, less spectacular in structure, occur in a va-
riety of worm-like animal phyla, such as the Platyhel-
minthes (especially the Turbellaria) (Smith cl ai. 1982),
Gnathostomulida (Rieger and Meinitz, 1977), Gastro-
tricha (Rieger et ai, 1974), Nemertinea (Jennings and
Gibson, 1969), and Archiannelida among annelids
(Martin, 1978). Rhabdite cells are usually scattered over
the epithelium covering the body surface or, in nem-
ertineans, the proboscis. They release bundles of rhab-
dites, and are presumably under nervous control.
Rhabdites are rod-like, membrane-bound bodies of la-
mellar ultrastructure. Upon extrusion by exocytosis,
they contact the water and swell, either forming a pro-
tective gelatinous sheath around the animal, or en-
hancing the animal's adhesion to the substratum, or as
in nemertineans, aiding the adhesion of prey to the pro-

97

98

P. EMSCHERMANN

Figure 1. Luxmomella brochobola n.sp. seen from the oral side. The
four lime-twig glands (arrows) are visible, and the pair at the left have
discharged. Four zooid buds (*) of different ages are seen at either side
on the anterior body wall (Scale bar: 100 ^m}.

boscis epithelium. Generally- animals with rhabdite
cells are small, freely mobile predators that can readily
benefit from such extrusive glands.

The newly detected extrusive organs of the Antarctic
entoproct Loxosomella brochobola Emschermann, 1993
differ considerably from the above types in their genesis,
as well as in their dimensions, structure, extruding mech-
anism, and function.

Materials and Methods

The entoproct species bearing lime twig glands is a
newly described species of Loxosomatidae discovered
during the 1989/90 German Antarctic Expedition to the
Weddell Sea (Emschermann, 1993). The animals were
found exclusively at two locations in the northeastern
Weddell Sea, at depths between 250 and 300 m, occurring
in small groups of individuals only at the inner, abfrontal
side of the tube-shaped colonies of the bryozoan Porella
imi/ouincnsis. About 500 specimens were collected. Small
numbers of specimens were kept alive on their original
substratum and were examined under the stereo micro-
scope, but these animals could not survive for longer than
about 5-8 days under the conditions available aboard ship.

Fixation ami narcotization

Most of the specimens were fixed. For general mor-
phological investigation, 4% formalin in seawater was
used. For histological purposes, fixation was at room tem-

Figurt 2. a: Specimen with its tentacles withdrawn and the lime-
twig threads ejected (arrow). A large bud (*) is seen at the base of the
calyx; b: Isolated lime-twig gland with its invaginated, heavily coiled,
dischargeable thread. At the bottom of the gland, in the marginal cyto-
plasm, one of the four nuclei is in focus. Inset: Optical cross section of
the invaginated, hollow cuticular thread (diameter 3 ^m) with its cru-
ciform lumen visible (Scale bars: a: 100 ^m: b: 10 ^m; Inset: 1 Mm).

LIME-TWIG GLANDS

99

B

i ; fc

\ . '?%_. '' -' ' ^ f*!> . -* . ' H~A ~- ^-^^ ~. *. *

i-t. - ,.. . ,

^H v.-O'y--- '?*" -/./->;.'>:.,<;'

Figure 3. Electron micrograph of a mature lime-twig capsule in longitudinal section. The coiled, invag-
inated extrudible tubule (arrows) is visible in the bottle-shaped gland, and a mucous cell (*) is seen in the
epidermis. The circled area is shown in higher magnification in Figures 4a and c (Scale bar: 10 j/m).

perature in a 2.5% buffered glutaraldehyde solution. Most
of the animals were narcotized before fixation, which pre-
served them in an expanded state. A two-step narcotiza-
tion gave the best results. First, several grains of the local
anesthetic amyleine hydrochloride [Stovaine R Rhone
Poukmc 1 methyl- l-(dimethylaminomethyl)-propyl-
benzoate (hydrochloride)] were added to the jar containing
the loxosomatids in about 3 ml of seawater. As soon as
the animals were fully expanded, the seawater was re-
placed by an isotonic solution of MgCN. Amyleine blocks
nervous conduction, whereas Mg* + ions prevent auton-
omous muscular contraction.

The glutaraldehyde fixation was unsatisfactory because,
after narcotization, it caused a precipitation, and an ad-
equate postfixation and embedding of the material was
impossible aboard ship. Narcotized specimens fixed in
formalin were excellently preserved for the general mor-
phological and light microscopical investigation that was
carried out in the laboratory at Freiburg. Fortuitously, the
formalin-fixed samples turned out to be usable even for
electron microscopy. But the preservation of most intra-
cellular membranes, especially of ER-membranes and
mitochondria, was not satisfactory, and most of the con-
tents of vesicular compartments of the cells were washed

100

P. EMSCHERMANN

Figure 4. a: Cross section of the extrudible hollow thread; the cutieular core (arrows) is traversed by
epithelial microvilli (arrowheads), and the cruciform luminal surface of the cutieular tube is decorated by
the blister-like microvilli tips (double arrowheads); secretion vesicles (*) are seen attached to the cytoplasmic
side of the cutieular core. At the four edges of the cutieular core the membranes of the four adjacent capsular
cells, connected by desmosomes, can be seen, b: Edge of one arm of the x-shaped cross section shown in a.
demonstrating the subcuticular membranes ot two adjacent capsular cells connected by desmosomes (arrows),
c: Sectioned subepidermal nerve libers (*) beside the capsular neck, partly filled with synaptic vesicles; ca:
capsular cell; ep: epidermal cell, d: Longitudinal section through the neck of a lime-twig capsule; at either
side (circled area) desmosome-stabilized junctions between capsular cells (ca) and adjacent epidermal cells
(ep) are seen; mi: microvillous border of capsular cells in the mouth funnel, e: Circled area of d in higher
magnification showing interdigitations and desmosomes between capsular cell (ca) and epidermal cell (ep)
(Scale bars: 1 ^m).

out, presumably because the material had been stored
aboard ship in the fixative for several months before post-
fixation and embedding.

Microscopy

Microscopical investigations were carried out in
Freiburg because shipboard microscopical work at

higher magnification turned out to be impossible. The
light microscopical evaluation of the material was car-
ried out with Zeiss-Nomarski interference contrast
equipment. For ultrastructural examination, the spec-
imens, after postfixation in buffered 2% OsO 4 , were
embedded in Epon, sectioned with a diamond knife,
and examined under a Zeiss EM 9A electron micro-
scope.

LIME-TWIG GLANDS

101

<S^^ ' ' P ^3^ ,,-r^

'.SlE^^ Tr>

Figure 5. Scheme of an immature lime-twig capsule (reconstructed
from serial sections). The already slightly coiled, invaginated cuticular
tuhule and, in cross sections of the latter, at the edges of the arms parts
of the twisted membranes of the capsular cells (double arrow) can be
seen; at the bottom, the borders of three of the four capsular cells and
their nuclei are visible, bl: thickened basal lamina; cp: sectioned para-
capsular nervous processes (shown at higher magnification in Fig. 4c);
dg: remains of a degenerating discharged capsule; ed: epidermis covered
by cuticle; ip: invagination pit with microvillous border, not covered by
the tough cuticle (rupture zone); mg: epidermal mucous cell (Scale bar:

Observations

In their normal expanded posture, living zooids of
Loxosomella brochobola have the peduncle slightly
curved, with the oral side of the calyx tilted upward. Seen
from above in this position, four large whitish-blue opaque
blister-like structures are conspicuous, two on either side
of the mouth between the bases of the second and third,
and the third and fourth oral tentacles (Fig. 1). Sponta-
neously, or sometimes upon irritation, long (300-400 ^m),
delicate, helically twisted threads can be ejected from these
enigmatic blisters (Figs. 1, 2a). These stretchable sticky
threads, hardly visible under the stereo microscope, are
as long, or somewhat longer, than the expanded tentacles.

Floating outside the tentacular crown, they remain an-
chored with their proximal ends in the cells from which
they originate. Under the light microscope, the mature
extrusive organs are comparatively large, ovoid epithelial
capsules, about 80 /j.m long and 45 urn wide (Fig. 2b),
situated in the outer edge of the peritentacular epidermal
fold (Fig. 1). At higher magnification, each capsule appears
to be tightly filled by a highly coiled hollow thread or tube
(Fig. 2b), the narrow lumen of which is cruciform in op-
tical cross section (Fig. 2b, inset). The capsular cytoplasm
is reduced to a narrow marginal layer with four nuclei in
its basal portion (Figs. 2b, 5).

Cellular structure of the lime twig capsule

In electron microscopic sections, the capsule appears
bottle shaped (Fig. 3). In its central part, embedded in
mainly vesicular contents, numerous sections of the coiled
extrudible thread can be seen. The thread is roughly rec-
tangular to X-shaped in cross section, about 3 pm thick,
and consists of an invaginated cuticular tube (Figs. 4a, 5).
Its wall structure is exactly the same as that of the ento-
proctan epidermal cuticle: a tough glycoprotein layer tra-
versed by epithelial microvilli (Emschermann, 1982). The
inner surface of this cuticular tube, facing its cruciform
lumen, is decorated by the densely staining, knob-like tips
of the microvilli. On its cytoplasmic side, the cuticular
tubule, below the subcuticular cell membranes, is densely
coated with vesicles (Fig. 4a) probably Golgi vesicles, as
can be inferred by the presence of Golgi complexes in
young, differentiating capsular cells (see Fig. 10). These
vesicles are partly filled with electron-dense contents (Figs.
4a, 5, 9, 10). The membranes of four adjacent capsular
cells connected by the remains of desmosomes (Fig. 4a,
b) can be seen at the four edges of the cross-sectioned
cuticular core of the invaginated tube. These cellular bor-
ders usually cannot be followed for a longer distance, as
they are lost in the confusion of vesicle membranes around
the tubule (Fig. 5).

In serial longitudinal sections, the four nuclei visible
with the light microscope can be demonstrated in the cap-
sular base (Fig. 5). The marginal cytoplasmic rim of the
capsular cells has a fine granular structure and is rich in
lumpy electron-dense material, possibly degenerating
rough ER and storage vacuoles; in contrast, the density
of secretion vesicles in this peripheral area is considerably
decreased (Figs. 3, 5). Sometimes single multilamellar
bodies, probably degenerating membrane complexes or
fixation artifacts, can be observed. Because the formal-
dehyde fixation was inadequate, the remains of mito-
chondria could only occasionally be identified.

Apically, the capsule constricts like a bottle-neck (Figs.
3. 5) and. in this narrow zone of junction, the capsular

P. EMSCHERMANN

Figure 6. a: Electron micrograph of a discharged, empty lime-twig capsule: remnants of the marginal
cytoplasm are seen in the capsular lumen (arrows); the invagination pit (arrowhead) is flanked at either side
by the adjacent electron-dense residue of a degenerating capsule; the thickened basal lamina (double arrow
heads) and a mucous cell in the body epidermis (*) are also visible, b: Longitudinal sections through two
degenerating, residual bodies of lime-twig capsules (*) at higher magnification; with their upper ends they
take part in the formation of the mouth funnel (arrows) of a younger lime-twig capsule. (Scale bars: a: 10
^m; b: 1 ^m).

cells are connected to the adjacent, flattened epidermal
cells by interdigitations and desmosomes (Fig. 4d, e). Spe-
cialized supporting cells comparable, for example, to the
supporting cells of cnidarian nematocysts are absent, as
are sensory receptors at the capsular apex. The invagi-
nation area of the extrusive tubule is a narrow funnel-
shaped pit in the epidermal surface. At the pit's outer
margin, the tough body cuticle transforms abruptly into
a loose mucous layer (a prospective rupture zone: cf.. Ex-
trusion Mechanism). Apical membranes of the capsular

cells in this zone are lined by dense, branched microvilli.
At the bottom of this pit, at the beginning of the invagi-
nated tubule, the mucous layer is again replaced by the
normal cuticular structure (Figs. 4d, 5, 7).

Towards the body cavity, each capsule is surrounded
by a robust two-layered basal lamina (Figs. 5. 6, 7) con-
sisting of an electron-dense inner layer about 30 nm thick,
the normal subepidermal basement membrane, and a
considerably thicker (300-400 nm) outer layer, which in
tangential sections seems to have a filamentous structure.

LIME-TWIG GLANDS

103

et

-

bt

Figure 7. Scheme of a discharged lime-twig capsule: remnants of the
marginal cytoplasm of the capsular cells (arrow headsl and, at the bottom,
two of the nuclei are visible; bl: thickened basal lamina; ed: body epidermis
covered by cuticle; et: extruded lime twig thread, discharged bv a rupture
of the apical cell membrane (at the right); ip: invagination pit with mi-
crovillous border, not covered b> the tough cuticle (rupture zone) (Scale
bar: 10 fim).

In shrunken, discharged capsules this outer layer is about
twice as thick (700-800 nm) and shows an increased elec-
tron density (Fig. 6a).

In several sections, beside the capsular neck, trans-
versely sectioned single subepithelial nerve fibers can be
seen (cf. Emschermann. 1982, 1985). some of which con-
tain small vesicles resembling presynaptic vesicles (Figs.
4c. 5). However, additional indications of a synaptic con-
nection between these nerve processes and the capsular
cells, as well as any evidence of a nervous control of the
lime-twig glands, are lacking.

Discharged capsules appear somewhat shrunken, but
not collapsed (Figs. 6. 7). They are empty except for re-
mains of the marginal cytoplasm and, basally, the degen-
erating nuclei. The extruded cuticular tubule is not evagi-
nated (i.e.. everted like a glove finger as in the cnidarian
spiro- and nematocysts), or released by exocytosis, as in
rhabdocysts, rather it is ejected through a rupture of the
apical cell membrane in the funnel area not covered by
cuticle (Figs. 7. 8). After its ejection, the unfolded thread
remains covered by a dense coat of the secretion vesicles
described above (Figs. 7. 8). Gradually, the latter swell
and give off their contents, forming a sticky mucous cover
around the unfolding thread (Fig. 8).

Discharged lime-twig capsules are not reloaded. Some-
times, especially in older specimens, large electron-dense
complexes of degenerating cells occur; usually from one
to three of them at either side of the capsular neck (Figs.
5, 6). They look like extremely shrunken capsules. With
their apical ends, these cell complexes take part in the
formation of the mouth funnel of the adjacent capsule.
These complexes are characterized by pycnotic nuclei and
are surrounded by the same robust, but heavily folded
basal lamina (Fig. 6b) as the mature capsules. Because
these pycnotic complexes contain no trace of an invagi-
nated thread, they are interpreted as degenerating residual
bodies of older discharged capsules.

The extrusion mechanism

I observed neither nervous synaptic connections nor
receptor structures associated with the capsular cells
themselves (such as the cnidocil of the cnidarian nema-
tocyst). nor specialized adjacent receptor cells. Therefore,
the lime-twig thread probably is discharged automati-
cally for example through a rapid swelling of the mucous
contents of the capsule as soon as the latter is mature
rather than by an external stimulus. Presumably triggered
by an increased internal pressure, the apical cell mem-
brane ruptures at its point of minimal resistance, i.e.. in
the invagination funnel where it is not stabilized by an
overlying cuticle (Figs. 5, 6b, 7). From the increased
thickness and density of the basal lamina in the discharged
capsule (Fig. 6a). one might infer that the outer filamen-
tous layer of the lamina acts as an elastic coat and a but-
tress against the internal pressure.

Development and replacement of lime-twig capsules

Extrusive capsules in Loxosomella brochobola are al-
ready present in young, undetached buds. Later, in the
adult, discharged capsules are replaced by new ones. In
the vicinity of a capsule, sometimes enlarged, bulging ep-

104

P. EMSCHERMANN

*^ T

UA Q

* -* 'f '1

K'l;^

Figure 8. a: Lime-twig thread, fixed during discharge hut before its total unfolding, hidden under adhering
masses of secretion vesicles except at the top right. Visible at the bottom (arrow head) is the rim of the
capsular extrusion opening; at the top right (*), between the secretion masses, the distal-most coiled part of
the lime-twig thread is visible, b: Electron microscopic section through a loop of an ejected lime-twig thread;
the cuticular core (arrows) is still covered by partly empty secretion vesicles (*): inset: Part of an extruded
lime-twig thread at higher light microscopic magnification (a and inset in b Nomarski contrast; scale bars:
a: 100 urn; b and inset: 10 ^m).

ithelial cells can be found (Figs. 3, 5, 6, 1 Ib). Like the
mucous cells of unknown function that occur in all lox-
osomatids(Emschermann, 1982; 1994) and are scattered
between the normal, uniform epidermal cells in the ten-
tacles and the periatrial fold (Fig. 1 la), these paracapsular
cells in L. brochobola are characterized by large, tightly
packed vacuoles full of an electron-dense, granular mu-
copolysaccharide material and by well-developed rough
ER and Golgi complexes in the narrow plasmatic domains
between them. Such epidermal mucous cell clusters in
the vicinity of discharged capsules quite probably give rise
to replacement lime-twig organs.

I have found different developmental stages of lime-
twig capsules in most of the specimens examined. Capsule
differentiation starts from a rosette of four enlarged epi-
dermal cells (Fig. 9c, inset) bulging out slightly below the
underside of the epidermal layer. They contain the same

large electron-dense vacuoles as the mucous cells men-
tioned above (Fig. 9a). Judging from their arrangement,
these clusters each seem to arise from single "mother-
cells." probably the above epidermal mucous cells.

The ejectible thread initially differentiates as a central
cuticular pit which, step by step, invaginates centrally be-
tween the four prospective capsular cells. By the time a
short finger-like cuticular tube has invaginated, increas-
ingly small vesicles with electron-dense, non-granular
contents are deposited around this growing tube, attached
to the subcuticular cell membrane. Simultaneously the
initial large vacuoles with their granular contents disap-
pear (Figs. 9, 10). As the tubule increases in length, the
four capsular cells twist around each other on their lon-
gitudinal axis, in the process curling the tubule into spiral
loops (Fig. 5). In a later stage, the membranes between
the four capsular cells seem to disappear, thus forming a

LIME-TWIG GLANDS

105

Figure 9. Electron microscopic cross sections of developing lime-twig glands, a: Early stage; in three of
the capsular cells the nuclei are visible: in the fourth capsular cell are large storage vacuoles (vc). At the top
right, bordered by the dotted line, are extremely flattened epidermal cells covered by the cuticle, b: Enlarged
central part of a, with the developing extrudible tubule already surrounded by a few secretion vesicles and
its luminal surface decorated by microvillous tips, c: Older stage of a developing capsule, the storage mu-
copolysacchande vacuoles (vc) gradually disappearing and the electron-dense secretion vesicles increasing
in number; d; enlarged central part of c. the cuticular tube surrounded by numerous secretion vesicles; inset
in c a developing capsule in optical cross section appearing under light microscopy as a rosette of four
enlarged epidermal cells (Scale bars: 1 pm).

tetranuclear syncytium that surrounds the ever more
coiling extrusive thread.

Functions, Origin, and Phylogenetic
Relevance of the Lime-Twig Glands

The lime-twig capsules have been observed to extrude
their threads, but the actual functioning of these sticky

threads has not yet been seen in living specimens of Lox-
osomelta brocliobola. Because these glands are arranged
at either side of the mouth, they are most likely to be
associated with feeding. As possible alternative or addi-
tional functions of these entoproctan extrusive glands,
defense or locomotion might be taken into consideration.
But this last role can be excluded in a sessile species that

106

Figure 10. Schematic cross section through the basal region of a
developing lime-twig gland; the cell at the right contains some early
storage vacuoles. The arrows point to Golgi complexes (Scale bar: 1 ^m).

lives irreversibly fixed to its substratum, and the limited
number and localized arrangement of the lime twig cap-
sules in a restricted body area speaks at least against their
major defensive function. Most probably, the sticky
threads act as lime-twigs, trapping larger planktonic food
particles or sedentary organisms, both these being inac-
cessible to the entoproctan ciliary feeding apparatus. From
time to time the threads with any adhering matter might
be swallowed.

Indeed, Loxosomella hrochohola was found living
within bryozoan tubes in an environment that is extremely
poor in nanoplankton. the normal food of Lo.xosomatids.
This microhabitat is shared only with some smaller sessile
predators such as the hydrozoan Haleciitm sp.; other cil-
iary feeders were absent. The stomach contents of about
100 specimens examined was consistent with the as-
sumption that L. hrochohola. at least facultatively, lives
as a predator. In 50% of these specimens the stomachs
were empty; the stomachs of the rest contained: a very
few small, planktonic pennate diatoms; some larger cells,
possibly ciliates and the eggs of other small invertebrates;
occasional batches of conglomerated small (diameter
about 10-12 ^m), brownish-green corpuscules looking like
plastids; a great variety of unidentifiable large skeleton
fragments of diatoms; and single frustules of larger, non-
plan ktonic pennate and central diatoms up to a size of
90 Mm.

Another observation is consistent with Loxosomella
brochobola catching single larger food particles with its

lime-twig threads rather than being a true ciliary feeder:
the tentacular cilia of this species, on average 30-40 /urn
long, are conspicuously shorter than in most other loxo-
somatids of comparable size. For example, in Loxosomella
antarctica, a species living at the same locations but in
other, more exposed habitats, the tentacular cilia have a
length of 50-60 pm on average. A comparison of the
stomach contents of these two species revealed that the
latter species feeds predominantly on microcellular ma-
terial, such as bacteria and small algae. Even where larger
sedentary diatoms were abundant and settled on the lox-
osomatid stalks, they were found only occasionally in its
digestive tract.

On the other hand, one would expect to find remains
of the swallowed lime-twig threads in the gut of the lime-
twig-fishing species; but tangled filamentous material (di-
ameter about 2 yum) could be detected in the digestive
tract of only a few specimens (30% of those with a filled
gut), and these tangled threads could not be reliably iden-
tified as lime-twig threads, because they did not show the
characteristic X-shaped cross section in the light micro-
scope. Of course, the swallowed lime twigs may have al-
ready lost their original structure through digestion.

The multicellular or syncytial lime-twig cysts represent
a new type of extrusive organ, unique in the animal king-
dom. As evidenced by their genesis and structure, they
are not homologous to other extruding glands such as the
cnidarian spirocysts or the rhabdite-forming cells, and
probably represent an isolated apomorphic character of
this particular entoproctan species.

These highly specialized organs are most likely derived
from mucous glands that occur in great variety in the
epidermis of most Loxosomatidae, the most primitive
group of Entoprocta. Mucous glands are seen in the calyx
epithelium of the adult (Nielsen, 1966a, b; Emschermann.
1982, 1985, 1994), as well as in the larval episphere epi-
thelium (Jagersten, 1964); but they seem to be generally
lacking in higher, colonial entoproctan families. Mostly
these entoproctan mucous glands are unicellular (Fig. 1 la,
b). and appear dispersed between normal epidermal cells.
In some species, such unicellular glands aggregate to plu-
ricellular. stratified plaques (Fig. 1 Ic), which can second-
arily invaginate to form subepithelial mucus alveoli, e.g.,
in Loxosomella thethyae, where conspicuous pearl-like
alveolar glands (Fig. 1 2a) are arranged all around the ten-
tacular crown (Salensky, 1877; Nielsen, 1988b). This type
of invaginated mucous gland presumably presents the
evolutionary basis for the formation of highly specialized
glandular organs such as the enigmatic giant, bell-shaped
aboral gland of Loxosomnella vivipara (Nielsen 1966b;
Emschermann, 1994) (Fig. 12b) and the lime twig capsules
of Loxosomella brochohola. described here.

I IME-TW1G GLANDS

107

Figure 1 1 . Epidermal mucous cells of Loxosomatidae. Single mucous cells of (a) Loxosomella crassicauda
Salensky, 1877, and (b) Loxosoinclla brochobola. (c) Stratified mucous plaque of Loxosomella ihelhyae
Salensky 1877 (Scale bar: 1 M m).

Acknowledgments

I thank Mrs. S. Collatz for her skillful technical assis-
tance and for taking most of the electron micrographs.

Literature Cited

Emschermann, P. 1982. Les Kamptozoaires. Etat actuel de nos con-
naissances sur leur anatomie. leur developpement. leur biologie et
leur position phylogenetique. Bull. Sot'. Zoo/. France 107: 317-344.

Emschermann. P. 1985. Kamptozoa. In Lehrhuch der Zoologie. vol.
2: Sysiematik. R. Slewing, ed. Gustav Fischer. Stuttgart.

Emschermann, P. 1993. On Antarctic Entoprocta nematocyst-like

organs in a loxosomatid, adaptive developmental strategies, host

specificity, and bipolar occurrence of species. Biol. Bull. 184: 153-

185.
Emschermann, P. 1994. Kamptozoa. In Lehrhuch der speziellen Zool-

><vie. W. Westheide und R. Rieger. eds. Gustav Fischer. Stuttgart (in

press).
Franc, J. M. 1978. Organization and function of ctenophore colloblasts:

an ultrastructural study. Biol. Bull 155: 527-541.
Holstein, Th. 1981. The morphogenesis of nematocytes in hydra and

forskalia. J. I'ltruslruci. Rc\ 75:276-290.
llolstein, Th., and P. Tardenl. 1984. An ultrahigh-speed analysis of

exocytosis: nematocyst discharge. Science 223: 830-833.

108

P. EMSCHERMANN

Figure 12. a Alveolar, subepithelial mucous gland (*) from Loxosomella thethyaeSalensky, 1877 (inset,
arrows point to the glands); b highly specialized, giant, bell-shaped mucous gland from Loxosomella vivipara
Nielsen. 1964. This enigmatic, double-walled gland, situated in the aboral part of the peritentacular rim (see
Inset, arrow), consists of an outer nng of about 16 cells and an inner one of 10-20 cells: its glandular lumen
is filled by a mucous plug (Scale bars: a: 10 ^m; b and Insets: 100 jim).

Jagersten, G. 1964. On the morphology and reproduction of entoproct

larvae. Zoo/. Biclrag (Lppxalal 36: 295-314.
Jennings, J. B., and R. Gibson. 1969. Observations on the nutrition

of seven species of rhynchocoelan worms. Bio/. Bull. 136: 405-433.
Martin, G. G. 1978. A new finding of rhabdites: mucus production

for ciliary gliding. Zoomorphologie 91: 235-248.
Nielsen, 0. 1966a. On the life cycle of some loxosomatidae (Ento-

procta). Ophelia 3: 221-247.
Nielsen, C. I966b. Some Loxosomatidae ( Entoprocta) from the Atlantic

coast of the United States. Ophelia 3: 249-275.
Rieger, R. M., E. Ruppert, G. E. Ricger, and C. Schoepfer-Sterrer.

197-4. On the fine structure of gastrotrichs with description of

Chordodaxys unlennaliix. sp.n. Zoo/ Scr. 3: 219-237.

Rieger, R. M., and M. Mainit/.. 1977. Comparative fine structure study
of the body wall in Gnathostomulida and their phylogenetic position
between platyhelminthes and aschelminthes. ./. Zoo/. Syst. Evoliu.-
lunch 5: 9-34.

Salensky, M. 1877. Etudes sur les Bryozoaires entoproctes. Ann. Sci.
Nat. xer. 6, /Ml 5: 1-60.

Smith III, J., S. Tyler, M. B. Thomas, and R. M. Rieger. 1982. The

morphology of turbellarian rhabdites: phylogenetic implications.
Trunx. Am Micnnc Soc 101: 209-228.

Tardent, P. and Th. Ilolslein. 1982. Morphology and morphodynamics

of the stenotele nematocyst ofHyilra attenuata Pall. ( Hydrozoa, Cni-
daria). Cell T/.V.VIH- Rex. 224: 269-290.

Reference: #/'/ Bull. 185: 109-1 14. (August.

Antho-RFamide Immunoreactivity in Neuronal
Synaptic and Nonsynaptic Vesicles of Sea Anemones

JANE A. WESTFALL 1 * AND CORNELIS J. P. GRIMMELIKHUIJZEN 2

1 Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506,

and 2 Zentrum Fiir Molekulare Neurobiologie, Universitdt Hamburg,

Martinistr. 52. 2000 Hamburg 20, Germany

Abstract. Antho-RFamide is a neuropeptide isolated
from the sea anemone Anthopleura elegantissima. Antho-
RFamide immunoreactivity was localized in four different
populations of neuronal vesicles in the tentacle nerve
plexus of Anthopleura. Small, opaque, neuronal vesicles,
averaging 49 nm in diameter, were gold-labeled at two-
way synapses. Heterogranular vesicles, averaging 184 nm
in diameter, were gold-labeled in a neuronal swelling ad-
jacent to a muscle cell process. These vesicles were similar
in size to a third class of gold-labeled dense-cored vesicles.
A fourth class of immunogold-labeled vesicles observed
in neuronal swellings had light cores and averaged 1 29 nm
in diameter. Using 5-nm gold particles, we observed a
heavy labeling of the granular cores of the dense-cored
vesicles, suggesting that the immunoreactivity is specific
to the vesicle core. The ultrastructural demonstration of
Antho-RFamide immunoreactivity in interneuronal syn-
aptic vesicles, together with the immunofluorescence and
electrophysiological studies of other investigators, suggest
that Antho-RFamide plays a role in neurotransmission
in sea anemones.

Introduction

Phe-Met-Arg-Phe-NH: (FMRFamide) and Arg-Phe-
NH^ (RFamide) immunoreactivity is present in the ner-
vous system of many invertebrate species, including coe-
lenterates. suggesting a neurotransmitter or neuromod-
ulatory function for peptides related to FMRFamide (Boer
etal.. 1980; Grimmelikhuijzen f/fl/.. 1992). A FMRFam-

Received 10 March 1993: accepted 12 May 1993.
* To whom correspondence should be sent.

Abbreviations: BSA. bovine serum albumin: ddH 2 O. double distilled
water: PBS, phosphate buttered saline.

ide-like peptide, Antho-RFamide (<Glu-Gly-Arg-Phe-
NH : ), isolated from the sea anemone Anthopleura ele-
gantissima (Grimmelikhuijzen and Graff. 1986) and the
sea pansy Renilla kollikeri (Grimmelikhuijzen and Groe-
ger, 1987), has been demonstrated to play a role in neu-
rotransmission or neuromodulation in sea anemones
(McFarlane et ill.. 1987, 1991; McFarlane and Grim-
melikhuijzen. 1991) and sea pansies (Anctil and Grim-
melikhuijzen. 1989). Antho-RFamide immunoreactivity
has been found in neurons in the ectoderm of tentacles,
in the oral disk, and in neurons located near the endo-
dermal muscles of the sea anemone Calliactis parasitiea
(Grimmelikhuijzen et a/.. 1988, 1992), but the subcellular
location of the peptide is unknown.

Using the immunogold-labeling technique and an
antiserum against RFamide, we have located RFamide-
immunoreactive material in dense-cored vesicles in both
neurites and nerve terminals of the freshwater coelenterate
Hydra littoralis (Koizumi et al. 1989). The chemical na-
ture of this immunoreactive material is, however, un-
known, except that it contains a C-terminal dipeptide
amide similar to Arg-Phe-NH : . The aim of the present
study was to determine the ultrastructural localization of
a known sea anemone neuropeptide, Antho-RFamide, in
neurons of Anthopleura elegantissima. Synaptic and non-
synaptic release sites have been demonstrated previously
in sea anemones (Westfall, 1970. 1973; Peteya, 1973a, b;
Quaglia, 1976; Quaglia and Grasso, 1986). FMRFamide-
like immunoreactivity has been demonstrated in large
dense-cored vesicles in the hydromedusa Aglantha digi-
tate, but is not present in small clear vesicles at synapses
(Singla and Mackie, 1991). In this study, we provide
evidence for the presence of Antho-RFamide-like material
in both synaptic and nonsynaptic granular vesicles.

109

110

J. A. WESTFALL AND C. J. P. GRIMMELIK.HUIJZEN

Materials and Methods

The tentacles ofAnthopleura elegantissinui (Bio-Marine
Laboratories, Venice, California) were extirpated and ex-
tended with succinyl choline chloride, as suggested by
C. DellaCorte, or placed directly in cold fixative for 1 or
2 h. A solution of 4% paraformaldehyde and 0.1% glu-
taraldehyde in 0. 1 M phosphate-buffered saline (PBS; pH
7.6, 0.3 M NaCl) was used to fix various lengths of ten-
tacles, which were rinsed in the same buffer, postfixed for
1 h in 1% OsO 4 , dehydrated in ethanol, rinsed in acetone,
and embedded in a mixture of Epon and Araldite.

Thin sections of tentacles were cut with a diamond
knife and mounted on Formvar-coated 100-mesh nickel
grids. The sections were rinsed in doubly distilled water
(ddH : O), then exposed to saturated sodium metaperiodate
for 30 min to open antigenic sites. After a ddH 2 O rinse,
the sections were exposed to normal goat serum diluted
1:20 with PBS-Tween-BSA buffer to block nonspecific
antigenic sites. They were incubated for 1 h with rabbit
antiserum # 177IV or 177IVA against Antho-RFamide
diluted 1:500 with buffer. After rinsing in buffer, the sec-
tions were immunogold stained for 1 h in goat anti-rabbit
IgG conjugated to 5-, 10-, or 15-nm gold particles, which
were diluted in buffer 1 :40. After rinsing in buffer with
BSA, then in PBS, they were postfixed for 1 5 min in 2%
glutaraldehyde in PBS and rinsed in ddH 2 O. The sections
were further stained in 7% uranyl acetate in 70% ethanol,
then in Reynold's lead citrate. The grids with sections
were lightly coated with carbon and examined in a trans-
mission electron microscope.

Antiserum 177 was raised in a rabbit against the se-
quence Tyr-Gln-Gly-Arg-Phe-NHi (Bachem, Bubendorf.
Switzerland), which was coupled by carbodiimide to bo-
vine thyroglobulin (Grimmelikhuijzen and Graff, 1986).
Antiserum 177IVA was obtained by affinity chromatog-
raphy of antiserum 1 77IV over a column containing Arg-
Trp-NH : (Bachem) coupled to Sepharose. Cyanobromide-
activated Sepharose was obtained from Pharmacia, and
the coupling reaction was carried out according to a pro-
tocol supplied by the manufacturer. Antiserum 177IVA
did not cross react with Antho-RWamide I (<Glu-Ser-
Leu-Arg-Trp-NH 2 ) or Antho-RWamide II Glu-Gly-
Leu-Arg-Trp-NH 2 ), as suggested by light microscopic ex-
amination of sphincter muscle sections of the sea anemone
Calliactis parasitica. which is rich in Antho-RWamide
immunoreactive neurons. Although Antho-RFamide
antisera 177IV and 177IVA labeled the same type of small
opaque vesicles at interneuronal synapses in the tentacles

.

M

Figure 1. Antho-RFamide immunoreactivity in opaque vesicles on
both sides of a two-way synapse in the tentacle nerve plexus of Antho-
pleura elegantissima. Note transverse filaments (arrow) in the synaptic
cleft and 15-nm gold particles associated with the granular content of
the neuronal vesicles. An adjacent muscle cell (M) lacks gold label. Scale
bar = 0.1 Mm.

of Anthopleura elegantissima, most of the experiments
were done with 177IV.

Control sections of the tentacle were exposed to Antho-
RFamide antiserum (1:500) that had been incubated
overnight in 10 /ug/ml of Antho-RFamide (Bachem, Bub-
endorf, Switzerland). Vesicle comparisons were made by
measuring 10 or more vesicles of each type and analyzing
the data with ANOVA.

Results

Immunogold labeling of thin sections incubated with
antisera against Antho-RFamide revealed gold particles
in a variety of granular vesicles in the tentacle nerve plexus
of the sea anemone Anthopleura elegantissima. Two-way
(symmetrical) synapses containing labeled opaque vesicles
were observed between neurons in two different tentacles.
The paired synaptic membranes were characterized by
paramembranous densities extending approximately 0.27
//m in length and by an 18-nm-wide cleft containing a
series of transverse filaments. In one synapse, gold-labeled
opaque vesicles with an average diameter of 49. 1 5 2.33
nm were found in close contact with both sides of the
synaptic cleft (Fig. 1 ). Vesicles in two other symmetrical

Figure 2. Heterogranular vesicles (arrowheads) with 1-8 or more gold particles label consistently in six
serial sections of a single nerve process (a-f). Note absence of gold label in adjacent muscle cell (M) contacted
by at least one labeled vesicle (e). Scale bar = 0.5 nm.

ANTHO-RFAMIDE IN SEA ANEMONE NEURONS

111

ujr. -r '* ,* '<?

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< a:. !

M

'
^

M

.

.
e

f

12

J. A. WESTFALL AND C. J. P. GRIMMELIKHUIJZEN

synapses were labeled on only one side of the synaptic
complex. The 15-nm gold particles were located mainly
over the opaque centers of the vesicles, but occasionally
they were present on the synaptic and vesicular mem-
branes.

Serial thin sections revealed immunogold labeling in
six successive profiles of a nerve cell process with granular
vesicles averaging 184.43 5.11 nm in diameter (Fig. 2a-
f). The contents of these vesicles were heterogeneous.
Some of the vesicles contained pale vacuolate spheres
associated with granular crescents. We have termed such
vesicles heterogranular to distinguish them from the more
homogeneously granular, dense-cored vesicles described
below. The successive profiles of the nerve process that
was traced serially were in close contact with a cytoplasmic
extension of a muscle cell, but no paired membrane
thickenings indicative of a synaptic complex were present.
In one section, a labeled vesicle appeared in close prox-
imity to the muscle cell membrane (arrowhead. Fig. 2e),
suggesting a possible paracrine release site. Although we
have counted as many as eleven 15-nm gold particles in
a single heterogranular vesicle, many vesicles had fewer
or no gold particles.

A third population of gold-labeled neuronal vesicles
contained a homogeneous dense core and a prominent
halo between core and membrane (Fig. 3). The cores were
approximately 120 nm in diameter and had a 20-nm halo
in the nonflared region of the membrane. Often, the ve-
sicular membrane was irregularly flared, probably as a
result of fixation, making measurements difficult. The 10
dense-cored vesicles that we measured were 1 8 1 .20 6. 1 1
nm in diameter and were similar in size to the hetero-
granular vesicles. One to five 15-nm gold particles were
present in some core profiles, whereas other particles oc-
casionally were located on the vesicular membrane.
Therefore, we used 5-nm gold particles to localize the
labeled area more specifically. With the smaller sized gold,
we counted 30 or more particles in several granular cores
and only an occasional particle on the membrane (Fig.
4). This demonstrated that the Antho-RFamide-like ma-
terial was confined to the granular cores.

Some neuronal swellings contained light-cored vesicles
with irregular halos (Fig. 5). These vesicles tended to be
smaller than the dark dense-cored vesicles and had an
average diameter of 129 4.47 nm. We have termed this
fourth population of vesicles light-cored vesicles.

Not all neurites containing granular vesicles were la-
beled, but this was to be expected because sea anemone
neurons produce at least 16 different neuropeptides
(Grimmelikhuijzen et al., 1992).

We did not observe immunogold staining of granular
vesicles in the control sections, which were exposed to
primary antibody that had been incubated overnight with

Antho-RFamide (Fig. 6). In order to validate this control,
we compared granular vesicles in serial sections of a nerve
process (Figs. 5, 6).

Discussion

In the sea pansy Renilla kollikeri, Antho-RFamide
causes tonic rachidial contractions in a dose-dependent
fashion and hence is believed to play a role in neuro-
muscular transmission (Anctil and Grimmelikhuijzen,
1989). On the other hand, in the sea anemone Calliactis
parasitica, Antho-RFamide increases the frequency and
amplitude of spontaneous contractions in endodermal
muscles and also excites the slow conduction systems 1
and 2, which are believed to be neuronal, suggesting that
the peptide may act at neuro-neuronal synapses (Mc-
Farlane et al., 1987, 199 1 ). Antho-RFamide also increases
spontaneous contractions of isolated tentacles of the sea
anemone Actinia eqitina (McFarlane and Grimmelikhu-
ijzen, 1991). In the present ultrastructural study on the
tentacle nerve plexus of the sea anemone Anthopleura
elegantissima, Antho-RFamide immunoreactivity was
present in small opaque vesicles associated with both sides
of a two-way synapse. Our demonstration of gold-labeled
interneuronal synapses therefore adds morphological
support to the hypothesis that Antho-RFamide acts as a
neurotransmitter at neuro-neuronal synapses in sea ane-
mones.

FMRFamide-like immunoreactivity has been demon-
strated in neurons of the gastropod Helix pomatia, where
granular vesicles of different sizes and homogeneity were
selectively labeled by gold particles (Elekes and Ude,
1993). This variability in size and content of gold-labeled
granular vesicles was also observed in the sea anemone
Anthopleura elegantissima when antisera to Antho-
RFamide were used. Based on granular size and appear-
ance, neuronal vesicles containing Antho-RFamide ma-
terial in the tentacle nerve plexus can be divided into at
least four distinct subpopulations.

In Helix, many immunogold-labeled granular vesicles
are present a short distance from the synaptic contact,
which is usually aligned with clear vesicles that lack the
gold marker and therefore lack peptides (Elekes and Ude,
1993). In contrast, in the sea anemone, gold-labeled
opaque vesicles were present at interneuronal synaptic
loci, supporting the concept that peptidergic neurotrans-
mission is phylogenetically primitive (Grimmelikhuijzen
et al. 1992).

Symmetrical synapses with vesicles that are 50-100 nm
in diameter and contain occasional electron-dense centers
have been observed in the sea anemone Metridium
(Westfall, 1970). The 49-nm opaque vesicles we found at
symmetrical synapses are similar to those observed pre-

ANTHO-RFAMIDE IN SEA ANEMONE NEURONS

113

Figures 3 and 4. Comparison of dense-cored vesicles (arrowheads) labeled with 1 5-nm gold (Fig. 3) and
5-nm gold (Fig. 4) provides evidence that Antho-RFamide immunoreactivity is specifically located in the
granular cores. Scale bar = 0.2 ^m.

viously in Metridium. Symmetrical synapses in coelen-
terates were first reported in the rhopalia of the jellyfish
Cyanea (Horridge et ai, 1962; Horridge and Mackay,
1962) and were thought to transmit neuronal activity in
two directions. The bidirectional nature of these chemical
synapses in Cyanea motor neurons has been demonstrated
electrophysiologically by Anderson (1985). The synaptic
vesicles in the scyphozoan bidirectional synapses are rel-
atively translucent with only a slight graininess and are
90-125 nm in diameter (Anderson and Griinert, 1988).
Variations in size and core densities may be a result of

the coelenterate species studied and the fixation method
used.

Not all granular vesicles in the nerve plexus of sea ane-
mone tentacles are gold-labeled with antisera to Antho-
RFamide, suggesting that additional putative transmitter
substances may also be present. Each profile of a neurite
contains only one type of granular vesicle. Electrophysi-
ological experimentation has shown that a variety of other
known sea anemone neuropeptides excite or inhibit
tentacle contractions (Grimmelikhuijzen et al, 1992;
Carstensen el al.. 1992, 1993). Moreover, most of these

1 '

I/:

Figures 5 and 6. Comparison of experimental (Fig. 5) and control (Fig. 6) serial thin sections of a
neuronal process with light-cored vesicles (arrowheads) provides further evidence of the specificity of antisera
to Antho-RFamide for some neuronal granular vesicles. Scale bar = 0.5 Mm.

114

J. A. WESTFALL AND C. J. P. GR1MMELIKHUIJZEN

neuropeptides are produced by different sets of tentacle
neurons (Grimmelikhuijzen, unpub.)- Thus, the number
of types of neuronal vesicles present in sea anemone ten-
tacles remains unresolved, as does the number of types
of putative transmitter substances, suggesting that neu-
rotransmission in sea anemones may be highly complex.

Acknowledgments

Contribution No. 93-300- J of the Kansas Agricultural
Experiment Station. We thank Dr. C-Z. Yu and K. L.
Sayyar for technical help and Dr. S. ThyagaRajan for sta-
tistical analysis. This study was funded by NSF grant IBN-
9120161 and NIH grant NS- 10264 to J. A. Westfall and
by the Bundesministerium fur Forschung und Technol-
ogic and the Deutsche Forschungsgemeinschaft to C.J.P.
Grimmelikhuijzen.

Literature Cited

Anctil. M., and C. J. P. Grimmelikhuijzen. 1989. Excitatory action of
the native neuropeptide Antho-RFamide on muscles in the pennatulid
Renilla kollikeri. Gen Phurmacol. 20: 381-384.

Anderson, P. A. V. 1985. Physiology of a bidirectional, excitatory,
chemical synapse. J. Neurophysiol. 53: 821-835.

Anderson, P. A. V., and I'. Griinert. 1988. Three-dimensional structure
of bidirectional, excitatory chemical synapses in the jellyfish Cyanca
capillaia. Synapse 2: 606-613.

Boer, H. H., L. P. C. Schot, J. A. Veenstra, and D. Reichelt.
1980. Immunocytochemical identification of neural elements in
the central nervous systems of a snail, some insects, a fish, and a
mammal with an antiserum to the molluscan cardio-excitatory tet-
rapeptide FMRF-amide. Cell Tissue Res. 213: 21-27.

Carstensen, K., K. L. Rinehart, I. D. McFarlane, and C. J. P. Grim-
melikhuijzen. 1992. Isolation of Leu-Pro-Pro-Gly-Pro-Leu-Pro-Arg-
Pro-NH : (Antho-RPamide). an N-terminally protected, biologically
active neuropeptide from sea anemones. Peptides 13: 851-857.

Carstensen, K., I. D. McFarlane, K. L. Rinehart, D. lludman, F. Sun,
and C. J. P. Grimmelikhuijzen. 1993. Isolation of <Gln-Asn-Phe-
His-Leu-Arg-Pro-NH 2 (Antho-RPamide II), a novel, biologically ac-
tive neuropeptide from sea anemones. Peptides 14: 131-135.

Elekes, K., and J. Ude. 1993. An immunogold electron microscopic
analysis of FMRFamide-like immunoreactive neurons in the CNS
of Helix pomatia: ultrastructure and synaptic connections. / Nen-
rocvtol 22: 1-13.

Grimmelikhuijzen, C. J. P., and D. Graff. 1986. Isolation of pGlu-Gly-
Arg-Phe-NH, (Antho-RFamide). a neuropeptide from sea anemones.
Proc. Natl. Acad. Set. USA 83: 9817-9821.

Grimmelikhuijzen, C. J. P., and A. Groeger. 1987. Isolation of the
neuropeptide pGlu-Gly-Arg-Phe-amide from the pennatulid Renilla
kollikeri. FEBS Lett. 211: 105-108.

Grimmelikhuijzen, C. J. P., D. Graff, and A. N. Spencer.

1988. Structure, location and possible actions of Arg-Phe-amide
peptides in coelenterates. Pp. 199-217 in Neurohormones in Inver-
tebrates, M. C. Thorndyke and G. J. Goldsworthy. eds. Cambridge
University Press, Cambridge.

Grimmelikhuijzen, C. J. P., K. Carslensen, D. Darmer, A. Moosler,
H.-P. Nothacker, R. K. Reinscheid, C. Schmutzler, H. Vollert, I. D.
McFarlane, and K. 1,. Rinehart. 1992. Coelenterate neuropeptides:
structure, action and biosynthesis. Am. Zoo/. 32: 1-12.

Horridge, G. A., and B. Mackey. 1962. Naked axons and symmetrical
synapses in coelenterates. Q J. Microsc. Sei. 103: 531-541.

Horridge, G. A., D. M. Chapman, and B. Mackey. 1962. Naked axons
and symmetrical synapses in an elementary nervous system. Nature
193: 899-900.

Koizumi, O., J. D. Wilson, C. J. P. Grimmelikhuijzen, and J. A. Westfall.

1989. Ultras! ructural localization of RFamide-like peptides in
neuronal dense-cored vesicles in the peduncle of Hydra. J E.\p. Zoo/.
249: 17-22.

McFarlane, I. D., and C. J. P. Grimmelikhuijzen. 1991. Three antho-
zoan neuropeptides, Antho-RFamide and Antho-RWamides I and
II. modulate spontaneous tentacle contractions in sea anemones.
J. Exp. Biol 155: 669-673.

McFarlane, I. D., D. Graff, and C. J. P. Grimmelikhuijzen.
1987. Excitatory actions of Antho-RFamide. an anthozoan neu-
ropeptide on muscles and conducting systems in the sea anemone
Calliaetis parasitica. J. E.\p. Biol. 133: 157-168.

McFarlane, I. D., P. A. V. Anderson, and C. J. P. Grimmelikhuijzen.
1991. Effects of three anthozoan neuropeptides, Antho-RWamide I,
Antho-RWamide II and Antho-RFamide on slow muscles from sea
anemones. J. Exp. Biol. 156: 419-431.

Peteya, D. J. 1973a. A light and electron microscope study of the ner-
vous system of Ceriantheopsis americanit.i (Cnidaria. Ceriantharia).
Z. Zellforsch. 141: 301-317.

Peteya, D. J. 1973b. A possible propnoceptor in Cerianthoeopsis
uinerieanux (Cnidara. Cerianthana) Z Zellforsch. 144: 1-10.

Quaglia, A. 1976. Osservazioni sul sistema nervoso degh antozoi. Boll.
Zoo/ 43: 397-398.

Quaglia, A., and M. Grasso. 1986. Ultrastructural evidence for a pep-
tidergic-like neurosecretory cell in a sea anemone. Oebalia 13: 147-
156.

Singla, C. L., and G. O. Mackie. 1991. Immunogold labelling of
FMRFamide-like neuropeptide in neurons of Aglanlha digitate (Hy-
dromedusae: Trachylina). Can. J Zoo/. 69: 800-802.

Westfall, J. A. 1970. Synapses in a sea anemone Metridiitm ( Anthozoa).
Electron Microscopy Proc. Int. Congr. 7th, Societe Francaise de Mi-
croscopie Electronique. Paris 3: 717-718.

Westfall, J. A. 1973. Ultrastructural evidence for neuromuscular sys- I
terns in coelenterates. Am. Zoo/. 13: 237-246.

Reference: Bio/. Bull. 185: 115-122. (August.

Hemoglobin Structure and Function in the Rat-Tailed
Sea Cucumber, Paracaudina chilensis

SHIRLEY M. BAKER* AND NORA B. TERWILLIGER

Oregon Institute of Marine Biology. University of Oregon, Charleston, Oregon 97420 and
Department of Biology Eugene, Oregon 97403

Abstract. The rat-tailed sea cucumber. Paracaudina
chilensis. has abundant hemoglobin-filled hemocytes in
its perivisceral coelom. water vascular system, and hemal
system. The perivisceral ox\ hemoglobin consisted of 34
kDa dimers and molecules with an apparent molecular
weight of ft;. 50 kDa. The perivisceral hemoglobin had a
high oxygen affinity with a A,, of 1.5 mm Hg at 15C. It
exhibited cooperative oxygen binding with a Hill coeffi-
cient of 1.26 to 1.86. Oxygen affinity appeared to be pH
dependent, but the effect was not significant. The heat of
oxygenation was -1 1.2 kcal mol '. At high hemoglobin
concentrations, the perivisceral hemoglobin oxygen affin-
ity was lower and the apparent pH effect and cooperativity
were increased. Perivisceral and water vascular hemoglo-
bins had spectral characteristics similar to those of other
invertebrate and vertebrate hemoglobins. The perivisceral
hemoglobin appeared to be electrophoretically heteroge-
neous and was structurally distinguishable from water
vascular hemoglobin. The oxygen affinity of water vas-
cular hemoglobin was not different from that of the peri-
visceral hemoglobin in spite of the difference in structure
and location in the animal. The exceptionally high oxygen
affinity hemoglobin of P. chilensis, a burrowing sea cu-
cumber, may be adaptive to this animal's oxygen-limited
habitat.

Introduction

Two orders of Holothuroidea. Dendrochirotida and
Molpadiida. possess hemoglobin (Hb) in nucleated he-
Received 23 October 1992; accepted 4 May 1993.
* Present address: The College of William and Mary. Virginia Institute
of Marine Science. School of Marine Science. Gloucester Point. VA
23062.

Abbreviations: Hb. hemoglobin; kDa. kiloDaltons: n. number of de-
terminations; n H . Hill coefficient of cooperativity; P 50 . pressure of half
saturation; P = calculated level of significance.

mocytes (Howel. 1886: Hyman. 1955). Hemocytes may
be present in one or more of the three holothurian body
cavity systems: the perivisceral coelom, the water vascular
system, and the hemal system. The perivisceral coelom.
largest in volume, extends the length of the animal be-
tween the body wall and the digestive tract. The water
vascular system includes the ring canal encircling the
pharynx, the buccal podia ampullae, the polian vesicle,
and the stone canal, which terminates in the coelom as a
perforated madreporic plate. A mesh of contractile sinuses
in the gut mesenteries and a ring around the pharynx
constitute the hemal system (Hyman, 1955). The rat-tailed
sea cucumber, Paracaudina chilensis, order Molpadiida.
has abundant Hb in all three body cavities. The Hb is
contained in nucleated hemocytes, 10-12 ^m in diameter
and 4-6 nm in thickness (Kawamoto. 1927).

Hyman (1955) considered the three body cavities in
holothurians morphologically continuous, allowing ex-
change of blood cells and fluid. However, the existence
of holothurian species with Hb in only one system suggests
that the compartments are not continuous, at least with
respect to the passage of hemocytes (Manwell and Baker.
1963: Roberts et ai. 1984). In holothurians with hemo-
cytes in more than one system, the Hbs may or may not
be electrophoretically distinguishable (Manwell and
Baker. 1963; Terwilliger and Terwilliger, 1988). While all
three body cavities of P. chilensis contain hemocytes, dif-
ferences in hemocyte cell counts and the presence of other
cell types specific to one compartment (Kawamoto, 1927)
indicate that the body cavities may not be continuous in
this species as well. The presence of dissimilar Hbs in
distinct areas of an animal provides the possibility of
complementary oxygen affinities and thus an oxygen
transfer system (Manwell, 1960: Weber, 1980). Such ox-
ygen transfer systems have been proposed to occur in some
polychaetesf Terwilliger, 1974: Mangum et a/.. 1975)and

115

16

S. M. BAKER AND N. B. TERWILLIGER

sipunculans (Manwell, 1960; Terwilliger et a!.. 1985;
Mangum and Burnett, 1987). Oxygen affinities of Hbs
from separate compartments in holothurians have not
been compared.

Molpadiid Hbs generally have relatively high oxygen
affinities (/>,<, = 3.5 to 4.0 mm Hg at pH 7.5 and 20C)
and show cooperative oxygen binding (Hill coefficients,
n H = 1.45 to 1.60) (Terwilliger and Read, 1972; Bonaven-
tura and Kitto, 1973). The Hb properties may be corre-
lated with the oxygen-limited habitat of these burrowing
sea cucumbers. The only information on P. chilensis Hb
function is a 1928 investigation by Kawamoto, who found
that the Hb had a relatively lower oxygen affinity (P 50
= 8.0 mm Hg at 20C) and negative cooperativity (n H
= 0.82). Hemoglobin concentration, pH, and other con-
ditions used in the study were not reported. The absorp-
tion spectra reported for P. chilensis Hbs are similar to
those of other Hbs, but the peaks were interpreted as
shifted 4 nm towards the red with respect to vertebrate
and other invertebrate Hbs (Kobayashi, 1932).

The structures of holothurian Hbs have been exten-
sively examined and show interesting features compared
to other Hbs. Holothurian Hbs are usually 34 kDa dimers
that reversibly form tetramers or higher aggregates (6S)
upon deoxygenation (Terwilliger and Read, 1970, 1972;
Bonaventura and Kitto, 1973; Terwilliger, 1974; Bona-
ventura el ai, 1976). Aggregation states of holothurian
Hbs may also vary with Hb concentration, association
occurring at high Hb concentrations and dissociation at
low concentrations (Bonaventura and Kitto. 1973; Ter-
williger, 1975). Perivisceral HbofP. chilensis has recently
been separated into three globin fractions and the com-
plete amino acid sequence of globin I determined (Suzuki,
1989). The sequence shows 59% homology with a major
globin from another molpadiid sea cucumber. Molpadiu
arenicola (Mauri, 1985). Like other holothurian Hbs, P.
chilensis Hb has a blocked n-terminus and an n-terminal
extension of 9-10 residues relative to vertebrate globins.

Little more has been reported on the function of P.
chilensis Hb since the studies by Kawamoto (1928) and
Kobayashi ( 1932). One objective of this study, therefore,
was to examine in detail the oxygen binding properties
of the perivisceral Hb, including a determination of
whether it is sensitive to homotropic or heterotropic ef-
fectors. We also compared the structure and function of
Hbs from the perivisceral coelom and water vascular
system.

Materials and Methods

Specimens of Parac audina chilensis (Miiller) ( 10-40 g
wet weight) were collected from the low intertidal zone
of the protected muddy-sand beach at Sunset Bay, Coos
Bay, Oregon. Animals were dissected immediately or kept

in sand filled aquaria under running seawater at the Or-
egon Institute of Marine Biology until use.

Perivisceral fluid was collected by slicing the animals
along an interambulacral region and collecting the fluid
in an ice-cold dish. Water vascular fluid was collected
from the bright red polian vesicle which had been first
rinsed to remove perivisceral fluid and then carefully re-
moved from the animal.

Hematocrits were determined on perivisceral fluid ac-
cording to Davidsohn (1962), and colorimetric determi-
nations of total Hb content were performed according to
the cyanmethemoglobin method (Sigma Procedure No.
525). Hematocrit and total Hb results were used to cal-
culate the mean corpuscular Hb concentration (MCHC)
(Davidsohn, 1962).

The fluids were centrifuged in a Sorvall RC2B refrig-
erated centrifuge at 121 X g for 10 min at 4C to pellet
the hemocytes. The hemocytes were washed three times
in a saline buffer based on the ionic composition of sea-
water; 50 mM Mg ++ , 10 mA/ Ca ++ , 10 mM K + , 540 mM
CT, 418 mMNa + , 29 mMSO 4 = , 0.01 ionic strength Tris-
HC1, pH 7.6. The hemocytes were gently lysed with a
sintered glass homogenizer in a 1:100 dilution of saline
buffer. Cell debris was pelleted by centrifugation at 1 3,300
X g for 10 min.

Hemoglobin was separated from small molecules and
potential organic modulators by gel filtration on a Seph-
adex G-100 column (24.0 X 1.8 cm) in equilibrium with
saline buffer. Changes of buffer for studies of the pH effect
were accomplished by dialyzing the supernatant against
saline buffer of the desired pH. Hemoglobin concentration
was determined using a millimolar extinction coefficient
at 578 nm of 14.2 (Terwilliger and Read, 1970).

Water vascular Hb was prepared essentially as described
for the perivisceral sample except that the small volume
of supernatant was not chromatographed in order to avoid
diluting the sample. Perivisceral Hb, when used in com-
parisons with water vascular Hb. was prepared in the same
manner.

Oxygen equilibrium experiments were performed as in
Benesch et ui ( 1965) using tonometers equipped with 1
cm path length cuvettes and a Beckman DU 70 spectro-
photometer. For concentrated samples (0.318 mM heme)
and water vascular samples, tonometers equipped with 1
mm path length cuvettes, requiring only 0.3 ml of sample,
were employed.

Oxygen binding characteristics of stripped and un-
stripped perivisceral Hb were compared. Stripped Hbwas
prepared as described above but was not dialyzed. Un-
stripped Hb was not separated from other molecules by
gel filtration. Instead, unstripped Hb was prepared by di-
luting the supernatant of lysed cells with saline buffer, to
the same Hb concentration as that of the stripped Hb.
Stripped Hb was also examined in the presence of 0.15

SEA CUCUMBER HEMOGLOBIN

117

mA/ organic phosphates: adenosine 5'-triphosphate (ATP),
disodium salt; 2.3-diphospho-D-glyceric acid (2,3-DPG),
pentasodium salt: and inositol hexaphosphoric acid (IHP),
dodecasodium salt (Sigma Chem. Co.). When IHP was
tested, the Hb sample was first dialyzed against a Tris-
HC1 buffer containing 550 mA/ Cl and 500 mM Na + ,
because the addition of IHP to saline buffer resulted in
precipitation.

For structural studies, hemocytes were obtained as de-
scribed above. After centrifugation, washing, and lysing
of the cells, the red supernatant was chromatographed on
a Sephadex G-100 column ( 100 X 1.8 cm) in equilibrium
with a 0.1 M sodium chloride, 0.1 M sodium phosphate
buffer, pH 7.4. Spectra of elution fractions were measured
with either a Zeiss PMQ-II or a Beckman DU-70 spec-
trophotometer.

Hemoglobins were analyzed by non-denaturing poly-
acrylamide gel electrophoresis (PAGE) and sodium do-
decyl sulfate polyacrylamide gel electrophoresis (SDS
PAGE). PAGE was carried out on oxy-, carboxy-, cyan-
met-, and met-Hb samples on 1.5 mm slab gels, 7.5%
acrylamide. with a discontinuous buffer system, pH 8.9
(Tris-glycine) as a cathodal buffer, and pH 8. 1 (Tris-HCl)
as the anodal buffer (Davis, 1964). Hemoglobins were
prepared for SDS PAGE by incubation in 2% SDS, 5%
mercaptoethanol, and 1 mA/ phenylmethylsulfonyl flu-
oride (PMSF) at 100C for 1.5 min. Samples were elec-
trophoresed on 1.5 mm slab gels, 12.5% acrylamide, with
a continuous buffer system of pH 8.5 (Laemmli, 1970).

Analyses of variance were performed to test the signif-
icance of regressions of log P 50 and n H \ersus pH. The
slopes of none of the regressions were significantly different
from zero (P = 0.099-0.989). Therefore, for comparisons
between treatments, oxygen binding data obtained at dif-
ferent pH were pooled. Results are reported as mean
standard deviation. Analyses of variance and Student's
/-tests were performed to test the null hypotheses that
treatment means were equal. For those analyses of vari-
ance in which the null hypothesis was rejected, Ryan's Q
multiple comparison test was performed to determine be-
tween which treatment means differences existed. Statis-
tical analyses were performed using Minitab (Minitab,
Inc.).

Results

Hematocrits of perivisceral fluid from 14 individuals
of Paracaudina chilensis ranged from 0.6 to 3.5% with a
mean of 1.5%. Hemoglobin concentration of the perivis-
ceral fluid ranged from 0.04 to 0.74 g Hb dl ' with a mean
of 0.32 g Hb dr '. The mean corpuscular Hb concentration
(MCHC) was 20.1 g dl ' with a range from 4.5 to 34.0 g
dl ' . The means and ranges of these values are comparable
to those of other holothurians (Roberts et ai, 1984).

The perivisceral Hb of P. chilensis has spectral char-
acteristics similar to those of other Hbs, with no shift in
wavelength observed. Spectral characteristics of water
vascular Hb were indistinguishable from those of perivis-
ceral Hb.

Oxygen binding properties of the perivisceral Hb at
three temperatures and three heme concentrations are
shown in Figures 1 and 2. The perivisceral Hb had a high
oxygen affinity with a P i0 of 1.5 0.3 mm Hg at 15C.
The oxygen binding was cooperative, with a Hill coeffi-
cient, n H , greater than one under all experimental con-
ditions examined (n H = 1.26-1.86). Although the slopes
of the regressions of log P 50 versus pH were not signifi-
cantly different from zero, there was a trend towards a
slight normal pH dependence between pH 7.0 and pH
8.1; A log Pso/A pH ranged from -0.08 to -0.44 as a
function of temperature and heme concentration. The
oxygen affinity of perivisceral Hb was significantly differ-
ent at all three temperatures examined (P = 0.000) while
the cooperativity was not (P = 0.683). Oxygen affinity
decreased with increasing temperature. The heat of oxy-
genation, AH, calculated using the Van't Hoff equation,
and including the heat of solution of oxygen in water,
was -1 1.2 kcal mol' 1 (Fig. 3). Both oxygen affinity and
cooperativity of perivisceral Hb were significantly different
at all three heme concentrations examined (P = 0.000

06

o

a."

03
O

0.4-

0.2

0.0

22

1.4

1.0

B

7,0

7,2

7.4

7.6

78

80

82

PH

Figure I. Oxygen affinity (A) and cooperativity (B) of perivisceral
Hb of Paracaudina chilensis as a function of pH. Hemoglobin concen-
tration, 0.057 mM heme. . !5C; A, 20C; x. 25C.

118

S. M. BAKER AND N. B. TERWILLIGER

cn
O

B

7 7.2 7.4 7.6 78 8 8.2

pH

Figure 2. Oxygen affinity (A) and cooperativity (B) of perivisceral
Hb of Paracaudina chilensis as a function of pH. Temperature, 20C.
0.018 mM heme; A. 0.060 mM heme; X, 0.318 mM heme.

and 0.001, respectively). Oxygen affinity was lowest (P x
= 3.8 1.3 mm Hg) and cooperativity most pronounced
(n H = 1.86 0.34) at 0.318 mM heme, the highest Hb
concentration examined (Fig. 4).

The oxygen affinities of stripped perivisceral Hb. un-
stripped Hb, and Hb to which ATP or 2,3 DPG had been
added, were not significantly different (P = 0.165) (Table
I). The oxygen affinity of Hb to which IHP had been added
was not significantly different from that of the control (P
= 0.790). The results of this experiment, in which a buffer
free of calcium and magnesium was used, suggest that
either IHP and divalent cations have opposite effects or
divalent cations do not affect P. chilensis Hb oxygen af-
finity. Given the insensitivity of P- chilensis Hb to ATP
and DPG, the latter is the most likely possibility.

Water vascular Hb had oxygen affinity and cooperativ-
ity values which were not significantly different from ox-
ygen binding data obtained from perivisceral Hb samples
treated in the same way (P = 0.690 and 0.600 for P 50 and
n H . respectively) (Table I).

The result of gel chromatography on Sephadex G-100
of perivisceral oxyHb is shown in Figure 5. The main Hb
peak. Hbll, corresponded to a protein with an apparent
molecular mass of 34 to 36 kDa. The curve was asym-
metric with a leading shoulder peak, Hbl, that had an
apparent molecular mass of about 50 kDa. Cucitmaria
miniata oxyHb chromatographed as a 34-36 kDa sym-

metncal peak on the same column. Chromatography of
Hb samples from 12 animals indicated that different in-
dividuals had slightly different ratios of Hbl and Hbll.
There were no apparent spectral differences between the
two peaks. When dilute Hbl and Hbll were rechromato-
graphed separately on the same column, each maintained
its elution position, suggesting the peaks were neither as-
sociating nor dissociating.

Electrophoresis of perivisceral Hbl and Hbll on PAGE
at pH 8.9 showed differences in banding patterns in both
met and cyanmet ligand states (Fig. 6). Although we are
unable at present to interpret the bands as subunits or
multiples thereof, the two fractions are different under
these conditions. Each fraction had an apparent subunit
molecular weight, as determined by SDS-PAGE. of about
17 kDa. Only one band was resolved on SDS-PAGE for
each peak as well as for a mixture of the two samples.
Electrophoretic properties of unpurified perivisceral and
water vascular carboxyHbs were compared by PAGE (Fig.
7, top). Both samples appeared to have several Hb bands
and one non-heme containing band in common. One ad-
ditional Hb band was evident in the water vascular Hb
sample. Heme-containing bands were red prior to staining
with Coomassie Brilliant Blue. After staining, an addi-
tional band, #5, was present which was equal in Coomassie
staining intensity to the heme-containing bands (Fig. 7,

0.61

or

o

oo

1.8

1.4

1.0

B

336 3.40 3.44

1/T x 10 3 (K)

348

Figure 3. The effect of temperature on oxygen affinity (A) and coop-
erativity (B) of perivisceral Hb of Paracaudina chilensis expressed as a
van't Hoff plot. Hemoglobin concentration, 0.057 m.M heme.

SEA CUCUMBER HEMOGLOBIN

19

0.8

Q."

TO

9 0.2

00

-02

22

B

-16 -1.2 -08 -04

Log mM heme

Figure 4. Concentration effect. Oxygen affinity (A) and cooperativity
(B) of Paracaudina chilensis perivisceral Hb in relation to Hb concen-
tration. Temperature, 20C.

05

O

C
(0
.Q

6

1.2

1.0

0.6

02

100 120 140

ml effluent

160

Figure 5. Paracaudina chilensis penvisceral Hb chromatographed
on Sephadex G-IOO column. Buffer, O.I M sodium phosphate, pH 7.4.
Absorbance at 280 nm () and 540 nm (A). Peak Hb-I rechromato-
graphed, absorbance at 418 nm (CD); Peak Hb-II rechromatographed,
absorbance at 418 nm (O). Void volume: A, Blue dextran. Calibrants:
B, Bovine serum albumin; C, Cucumaria miniata oxyHb; D. a-chy-
motrypsinogen; E, Sperm whale myoglobin.

top). When the heme-containing protein bands from
PAGE were cut out and re-electrophoresed on SDS-
PAGE, only one 17 kDa band was observed (Fig. 7, bot-
tom). The non-heme-containing bands in both perivisceral
and water vascular samples electrophoresed in SDS-PAGE
as 16 kDa components.

Discussion

One novel feature of the perivisceral Hb of Paracaudina
c/ulensis is that the molecular weight is heterogeneous in
the oxy-state. While other holothurian oxyHbs consist
entirely of 34 to 36 kDa dimers in the oxy-state (Roberts
el ai, 1984), P. chilensis oxyHb consisted of 34 kDa di-

Table I

Oxygen affinity and cooperativity ofHb o/'Paracaudina chilensis

Condition

n H

Penvisceral Hb in saline buffer 6

unstnpped

stripped

stripped, 0. 1 5 mM ATP

stnpped, 0.15 mM 2,3-DPG
Perivisceral Hb in buffer free of Ca*
and Mg** c

stripped

stnpped, 0.15 mM IMP
Hb in saline buffer* 1

penvisceral

water vascular

4 3.0 + 0.54 1.86 0.24

4 2.1 0.37 1.66 0.28

4 3.5 1.36 1.71 0.17

2 2.5 1.78

3.1 0.70

3.2 0.28

2.3 0.14
2.2 0.55

" Mean standard deviation.
b pH 7.5-7.7, 20C, 0.061 mA/heme.
c pH 7.5, 20C, 0.059 mM heme.
" pH 7.3-7.7, 20C, 0.060 mM heme.

1.85 0.22
1.80 0.09

2.01 0.19
1.89 0.50

B

Figure 6. PAGE of Paracaudina chilensis penvisceral Hbs. pH 8.9.
A, cyanmet Hbl; B. cyanmet Hbll; C, met Hbl; D, met Hbll.

120

S. M. BAKER AND N. B. TERWILLIGER

ipvl wv

-1

-2
- 3

-4

-5

PV

WV

1

3 4

1

2345

Figure 7. Electrophoretic comparison of Paracaudina chilensis per-
ivisceral and water vascular Hbs. Upper, perivisceral (PV) and water
vascular (WV) Hbs on pH 8.9 PAGE. Lower. SDS PAGE. PV 1.2. 3.

4, heme-containing slices from PV PAGE; PV 5 non-heme band from
PV PAGE; WV 1,2, 3, 4, heme-containing slices from WV PAGE; WV

5. non-heme band from WV PAGE.

mers as well as molecules with an apparent molecular
weight of 50 kDa (see also Terwilliger and Terwilliger,
1988). The 34 kDa (Hb II) and the 50 kDa (Hb I) fractions
were electrophoretically distinguishable. Several holothu-
rian Hbs aggregate to tetramers in the deoxy-state. For
example, Ciicnmaria miniata Hb is a dimer of 36 kDa in
the oxy-state and aggregates to a tetramer of 55 kDa in
the deoxy-state (Bonaventura and Kitto, 1973; Terwilliger,
1975). In our study, C miniata Hb, which has an oxygen
affinity about five times lower (Terwilliger, 1975) than
that of P. chilensis Hb, showed no evidence of aggregation
under the column conditions used. This indicates that the
column was sufficiently oxygenated to prevent aggregation
due to a deoxy-state. Therefore, the apparent 50 kDa Hb
in P. chilensis is not the result of aggregation in the deoxy-
state. The presence of a possible tetrameric oxyHb in a
holothurian echinoderm is especially interesting in light
of the predominance of the tetrameric aggregation state
in the vertebrate Hbs. The subunit molecular weight is
similar to that calculated by Suzuki (1989) from the amino
acid sequence data.

We found that P. chilensis Hb had a very high oxygen
affinity with a P 50 of 1 .5 0.29 mmHg (pH 7.0-7.7, 1 5C,
0.058 mA/ heme). In our studies the Hill coefficient was
always greater than one (n H = 1.26-1.86). Our data agree
with others: almost all holothurian Hbs, even those with
multiple components (e.g.. Molpadia arenicola and M.
oolitica), have Hill coefficients greater than one (Terwil-
liger and Read, 1972; Bonaventura and Kitto, 1973). The
Hill coefficient that we found for P. chilensis Hb is con-
sistent with homotropic interactions within a dimer
(Chiancone el ai, 1981; Royer el ai. 1985; Terwilliger
and Terwilliger, 1988).

The increase in oxygen affinity upon dilution for P.
chilensis Hb has also been observed for several vertebrate
Hbs. This phenomenon has been explained by a rapid
association-dissociation equilibrium between dimer and
tetramer (Rossi-Fanelli et ai, 1964; Antonini and Brunori,
1971). The concentration effect is particularly noticeable
in lamprey Hb, which has a ligand-linked association from
monomer to tetramer upon deoxygenation (Briehl, 1968).
Hemoglobins from the polychaete Glycera and several
other holothurians are examples of invertebrate Hbs dis-
playing a concentration effect (Mizukami and Vinogra-
dov, 1972; Terwilliger, 1975). The concentration effect
exhibited by P. chilensis Hb is of similar magnitude as
that of other Hbs, at least at low Hb concentrations. Bo-
naventura and Kitto (1973) report that dilution of M.
arenicola Hb to low pigment concentrations tends to dis-
sociate the oxy-dimers to oxy-monomers. If P . chilensis
Hb dissociates to monomers upon dilution as well, the
monomer may have a higher affinity than the dimer. A
physiological implication of the concentration effect is
that, because the concentration of Hb in P. chilensis hem-
ocytes (MCHC) is higher than that used in our oxygen
binding studies, the oxygen affinity of Hb in the hemocytes
is probably lower than that determined in vitro.

It would be interesting to learn whether the apparent
pH dependence of oxygen affinity in P. chilensis becomes
significant at physiological Hb concentrations. Only one
other holothurian Hb, that of Molpadia arenicola, is re-
ported to exhibit pH sensitivity in the kinetics of ligand
binding (Bonaventura et ai, 1976). Cooperativity of P.
c/ii/ensis Hb is more pronounced at the highest Hb con-
centration, and may be greater in vivo than in vitro.

Invertebrate Hbs are generally insensitive to organic
phosphates (Terwilliger and Terwilliger, 1988; Scholnick
and Mangum, 1991). Nonetheless, because of the possi-
bility of a tetrameric oxyHb in P. chilensis and the phy-
logenetic relationship between echinoderms and verte-
brates, both deuterostomes, we looked for possible effects
of organic modulators known to influence vertebrate tet-
rameric Hb. There were no observable effects of the or-
ganic phosphates, ATP, 2,3-DPG, or IHP, on oxygen
binding. This insensitivity may be due to blocking of the

SEA CUCUMBER HEMOGLOBIN

121

n-termini of the Hh chain of P. chilensis, and as seen in
several other holothurian Hbs (Kitto cl at.. 1976; Suzuki,
1989).

The heat of oxygenation of P. chilensis Hb is similar
to that of most other invertebrate and vertebrate Hbs.
Hemoglobins from the perivisceral coelom and the water
vascular system of P. chilensis were not distinguishable
by absorbance spectra. However, these two compartments
appear to contain different Hbs whose subunits differ in
charge but not size as determined electrophoretically. This
information suggests that the compartments are discon-
tinuous, at least with respect to hemocytes. At the same
Hb concentration, the oxygen affinities of Hbs from the
two compartments are indistinguishable, indicating that
an oxygen transfer system is unlikely in this animal. Al-
though Hb concentration parameters were not measured
for water vascular fluid, it was darker red than the peri-
visceral fluid, suggesting a higher Hb concentration. If the
Hb concentration in water vascular hemocytes is higher
than that in perivisceral hemocytes, then the water vas-
cular Hb may have a lower oxygen affinity //; vivo due to
the concentration effect on oxygen affinity.

Paracaiuiina chilensis lives buried up to 50 cm deep
in direct contact with black, muddy sand smelling of hy-
drogen sulphide. The posterior "tail" is extended to the
surface of the substrate and oxygen is obtained via oxy-
genated water inspired through the anus into the respi-
ratory trees. The body wall of the animal is so thin that
the internal organs and dark red coelomic fluid are visible
through the skin (Kawamoto, 1928: present authors, pers.
obs.). It appears then that P. chilensis lives in an oxygen-
limited habitat that could potentially draw oxygen away
from the sea cucumber.

The oxygen affinity of P. chilensis Hb (/% = 2. 1 mmHg
at 20C) is higher than that of most other holothurian
Hbs (P 50 = 3.5-10.0 mmHg at 20C) (Terwilliger and
Terwilliger. 1988). The oxygen affinity is comparable,
however, to those of many other invertebrates that inhabit
intertidal, microxic substrates. For example the poly-
chaetes, Enoplobranchus sanguineus (Mangum el aL
1975) and Notomastus latericeus (Wells and Warren,
1975), and the phoronids, Phoronopsis viridis (Garlick el
a/.. 1979). Phomnis mulleri (Weber. 1980), and Phoronis
archiiecta (Vandergon and Colacino, 1989), have PSO val-
ues from 1.3 to 3.0 mmHg. The fact that many inverte-
brates living intertidally in microxic substrates have Hbs
with exceptionally high oxygen affinities suggests that the
high oxygen affinity is adaptive to the habitat. One func-
tion of the high oxygen affinity of Hb of these burrowing
animals may be to minimize the loss of oxygen to an
oxygen-sink habitat. Such a function is proposed for the
Hb of the polychaete Cirntonnia lentacitlata which has
Hb oxygen affinity and concentration similar to that of
P. chilensis (Dales and Warren, 1980). The Hb of P. chi-

lensis may be a storage molecule, the high oxygen affinity
serving to retain oxygen even though the thin body wall
is surrounded by sand of low oxygen tension.

Acknowledgments

The authors wish to acknowledge the contribution of
Dr. Robert C. Terwilliger to this study. This study was
supported by NSF grant 85111 50 to RCT and NBT and
by an American Heart Association, Oregon Affiliate,
summer science research fellowship and an International
Women's Fishing Association Scholarship Trust grant to
SMB. We would like to thank Dr. L. Burnett, University
of Charleston, for the use of his small tonometers. C. P.
Mangum and two anonymous reviewers made helpful
comments on the manuscript. This is Oregon Institute of
Marine Biology Contribution Number 92-02.

Literature Cited

Antonini, F.., and M. Brunori. 1971. Frontiers ol Biology. I'olume 21.
Hemoglobin and Myoglobin in Their Reactions with Ligands. Amer-
ican Elsevier, New York.

Bcnesch, R., G. MacDutf. and R. E. Benesch. 1965. Determination of
oxygen equilibria with a versatile new tonometer. Anal Biochem.
11:81-87.

Bonaventura. C., J. Bonaventura. B. Kitlo, M. Brunori, and E. Antonini.
1976. Functional consequences of ligand-linked dissociation in he-
moglobin from the sea cucumber Motpadia arenicola. Biochem. Bio-
phvs. .lew 428: 779-786.

BonaM'titura. J.. and G. B. Kitto. 1973. Ligand-linked dissociation of
some invertebrate hemoglobins. Pp. 493-506 in Comparative Phys-
iology. L. Bolis. K. Schmidt-Nielson. and S. H. P. Maddress. eds.
North-Holland Publishing Company. Amsterdam.

Brirhl. R. W. 1968. The relation between the oxygen equilibrium and
aggregation of subunits in lamprey hemoglobin. ./. Biol. Chem. 238:
2361-2366.

Chiancone, E., P. Vecchini, D. Verzili, F. Ascoli, and E. Antonini.
1981. Dimeric and tetrameric hemoglobins from the mollusc Sca-
phurca inaequivalvis: structural and functional properties. ./ Mol
Biol. 152: 577-592.

Dales, R. P., and I.. M. Warren. 1980. Survival of hypoxic conditions
by the polychaete Cirriformia teniaeitlatu. J. Mar. Biol. Assoc. L'. A'.
60: 509-516.

Davidsohn, I. 1962. The blood. Pp. 61-262 in Clinical Diagnosis by
Laboratory Methods. I. Davidsohn and B. B. Wells, eds. W. B. Saun-
dersCompan>, Philadelphia.

Davis, B. 1964. Disc electrophoresis II. Method and application to
human serum proteins. Ann. \. }'. Acad. Set. 121: 404-427.

Garlick, R. L., B. J. Williams, and A. F. Riggs. 1979. The hemoglobins
of Phoronopsis viridis. of the primitive invertebrate phylum Phoron-
ida: characterization and subunit structure. Arch Biochem. Biophvs
194: 13-23.

Honel, W. II. 1886. Presence of haemoglobin in the echinoderms.
Studies Biol Lah- Johns Hopkins L'niv. 3: 289-291.

Flyman. I.. H. 1955. The Invertebrates. I 'olume 11. Echmodcrmata.
McGraw-Hill Book Company, New York.

Kawamoto, N. 1927. The anatomy of Caudina chi/ensis- (J. Miiller)
with especial reference to the perivisceral cavity, the blood and water
vascular systems in their relation to the blood circulation. Sci. Rep.
Tohoku L'niv. h'ourlh Ser. (Biol.) 2: 239-264.

122

S. M. BAKER AND N. B. TERWILLIGER

Kawamoto, N. 1928. Oxygen capacity of the blood of certain inver-
tebrates which contains haemoglobin. Sci. Rep Tohoku Univ. Fourth
Ser. (Biol.) 3: 561-515.

Kitto, G. B., D. Erwin, R. West, and J. Omnaas. 1976. N-terminal
substitution of some sea cucumbei hemoglobins. Comp. Biochem.
Physiol 55: 105-107.

Kobayashi, S. 1932. The spectral properties of haemoglobin in the
holothurians. Caudina chilcnsis (J. Miiller) and Molpadia roret:ii
(v. Marenzeller). Sd. Rep Tohoku Univ. Fourth Ser. (Biol.) 7: 21 1-
227.

I ni'ininli. H. 1970. Cleavage of structural proteins during assembly of
the head of bacteriophage T4. Nature 227: 680-685.

Mangum, C. P., and L. E. Burnett. 1987. Response of sipunculan hem-
erythrins to inorganic ions and CO. J. Exp. Zoo/. 244: 59-65.

Mangum, C. P., B. R. Woodin, C. Bonaventura, B. Sullivan, and J. Bona-
ventura. 1975. The role of coelomic and water vascular hemoglobin
in the annelid family Terebellidae. Comp. Biochem. Physiol. 51: 281-
294.

Manwell, C. 1960. Histological specificity of respiratory pigments
II. Oxygen transfer systems involving hemerythrins in sipunculid
worms of different ecologies. Comp. Biochem Physiol. 1: 277-285.

Manwell, C., and C. M. A. Baker. 1963. A sibling species of sea cu-
cumber discovered by starch gel electrophoresis. Comp. Biochem
Physiol. 10: 39-53.

Mauri, F. C. 1985. The structural and functional studies of an echi-
noderm hemoglobin. PhD dissertation. University of Texas at Austin.

Mizukami, H., and S. N. Vinogradov. 1972. Oxygen association equi-
libria of Glycera hemoglobins. Biochem Biophys. Ada 285: 314-
319.

Roberts, M. S., R. C. Terwilliger, and N. B. Terwilliger. 1984. Com-
parison of sea cucumber hemoglobin structures. Comp. Biochem.
Physiol. 77: 237-243.

Rossi-Kanelli, A., E. Antonini, and A. Caputo. 1964. Hemoglobin and
myoglobin. Adv. Protein Client 19: 73-222.

Royer, W., W. Love, and F. F. Fenderson. 1985. Cooperative dimenc
and tetrameric clam haemoglobins are novel assemblages of myo-
globin folds. Nature 316: 277-280.

Scholnick, D. A., and C. P. Mangum. 1991. Sensitivity of hemoglobins
to intracellular effectors: primitive and derived features. J. Exp. Zool.
259: 32-42.

Suzuki, T. 1989. Amino acid sequence of a major globin from the sea
cucumber Paracaudina chilensis. Biochim. Biophys. Ada 998: 292-
296.

Terwilliger, N. B., R. C. Terwilliger, and E. Schabtach. 1985. Intra-
cellular respiratory proteins of Sipuncula, Echiura, and Annelida.
Pp. 193-225 in Blood Cells of Marine Invertebrates: Experimental
Systems in Cell Biology and Comparative Physio/of;y. W. D. Cohen,
ed. Alan R. Liss, Inc., New York.

Terwilliger, R. C. 1974. Oxygen equilibria of the vascular and coelomic
hemoglobins of the terebellid polychaete, Piata padftca. Evidence
for an oxygen transfer system. Comp. Biochem. Physiol. 48: 745-
755.

Terwilliger, R. C. 1975. Oxygen equilibrium and subunit aggregation
of a holothunan hemoglobin. Biochem Biophys. Ac/a 386: 62-68.

Terwilliger, R. and K. Read. 1970. The hemoglobins of the holothunan
echinoderms, Ciiaunana mimata Brant. Ciicitmaria piperala Stimson
and Molpadia intermedia Ludwig. Comp. Biochem. Physiol. 36: 339-
351.

Terwilliger, R. C., and K. Read. 1972. The hemoglobin of the holoth-
urian echinoderm, Molpadia oo/itica Pourtales. Comp. Biochem.
Physiol. 42: 65-72.

Terwilliger, R. C., and N. B. Terwilliger. 1988. Structure and function
of holothunan hemoglobins. Pp. 589-595 in Echinoderm Biology,
Proceedings of the Sixth International Echinoderm Conference. R.
D. Burke. P. V. Mladenov, P. Lambert, and R. L. Parsley, eds. A.
A. Balkema, Rotterdam

Vandergon, T. L., and J. M. Colacino. 1989. Characterization of he-
moglobin from Phoronis architect a (Phoronida). Comp. Biochem.
Physiol 94B: 31-39.

Weber, R. E. 1980. Functions of invertebrate hemoglobins with special
reference to adaptations to environmental hypoxia. Am. Zool. 20:
79-101.

Wells, R. M. G., and L. M. Warren. 1975. The function of the cellular
haemoglobins in Capitella capitata (fahridux) and Notomastus la-
/mei(Capitellidae: polychaeta). Comp. Biochem Physiol. 52: 737-
740.

Reference: Biol. Bull 185: 123-139. (August, 1993)

Effect of Dietary Protein Content on Growth of
Juvenile Mussels, Mytilus trossulus (Gould 1850)

D. A. KREEGER' AND C. J. LANGDON

I fat field Marine Science Center, Department of Fisheries and Wildlife. Oregon State University,

Newport, Oregon 97365

Abstract. Juvenile mussels, Mytilus trossulus, were fed
for 3 weeks on either low-protein (LP) algae, high-protein
(HP) algae, or a combination of LP algae and protein
microcapsules (PM). Growth rates of mussels fed a satia-
tion ration of 27.5% body weight (bw; ash-free dry weight
of algae/ash-free dry tissue weight of mussels) per day of
LP algae (28% protein percent weight per weight) were
significantly (P < 0.05) lower than growth rates of mussels
fed a satiation ration (27.5%. bw d~') of HP algae (43%
protein weight per weight). However, growth rates of
mussels fed LP algae (27.5% bw d~') supplemented with
one of three different rations (6, 12 and 18% bw d~') of
PM increased proportionally to PM ration size. Mussels
fed a diet containing LP algae with the highest level of
PM supplementation grew at rates that were not signifi-
cantly different from those of mussels fed a diet of HP
algae alone. Growth rates of mussels fed LP algae alone
were not improved if the ration of LP algae was increased
(34.1% bw d '), indicating that the positive growth re-
sponse of mussels fed PM supplements was due to an
increase in dietary protein content and not simply due to
an overall increase in food (energy) availability. In addi-
tion, mussels fed LP algae had O/N ratios > 1 8, indicating
that they were conserving dietary protein from catabolism;
whereas mussels fed protein-rich diets had O/N ratios <10,
indicating that they were catabolizing dietary protein.
These results suggest that dietary protein contents below
40% w/w and dietary C/N ratios above 10 can qualitatively
limit growth rates of juvenile M. trossulus.

Introduction

The availability of nitrogen frequently affects produc-
tivity in marine systems (Dugdale, 1967; Riley, 1972;

Received 15 January 1993; accepted 4 May 1993.
1 Present address: Plymouth Manne Laboratory, Prospect Place, The
Hoe, Plymouth, Devon PL1 3DH. United Kingdom.

Mann, 1982; Roman, 1983; Tenore and Chesney, 1985;
Asmus, 1986; Rice el al, 1986). A large portion of nitrogen
in marine habitats can cycle through populations of sus-
pension-feeding animals, such as bivalve molluscs (Jordan
and Valiela, 1982; Dame et al., 1984, 1985; Kautsky and
Evans, 1987; Dame and Dankers, 1988; Asmus and
Asmus, 1991). The bioavailability of nitrogen and protein
for suspension-feeding bivalves in natural habitats is not
well documented and may be especially low for many
species that use suspended detritus as a food source. New-
ell (1965) first reported that the bivalve Macoma balthica
utilizes the microbial coating of detrital particles as a
source of dietary protein, and this potential protein source
for macroinvertebrates has been cited repeatedly (for re-
views, see Fenchel, 1972; Sieburth, 1976; Tenore, 1977),
but most of the nitrogen associated with detritus is now
known to consist of nonmicrobial humic geopolymers,
which are indigestible (Rice, 1982; Crosby et al.. 1990;
Hicks et al.. 1991). Crosby et al. (1990) reported, for ex-
ample, that oysters, Crassostrea virginica, were able to
assimilate nitrogen of bacteria with an efficiency of 57.2%,
but the oyster's assimilation efficiency for detritus-
associated nitrogen was only 3.4%.

The importance of nitrogen limitation in the nutrition
of suspension-feeding bivalves has not been clearly estab-
lished. Hawkins and Bayne (for reviews, see Hawkins and
Bayne, 1991, 1992) determined nitrogen and carbon bud-
gets ofAfytilus edit/ is fed 15 N- and 14 C-labeled Phaeodac-
irlum tricormttum and concluded that mussels in their
natural environment may be more limited by carbon than
nitrogen, in part because mussels were found to be very
efficient in recycling nitrogen within their tissues. In sup-
port of this conclusion, Flaak and Epifanio (1978) reported
that growth rates of oysters, C. virginica. increased when
they were fed algal diets containing a greater proportion
of carbohydrate than protein. In contrast, the dietary pro-
tein content of experimental diets has been positively cor-

123

124

D. A. KREEGER AND C. J. LANGDON

related with growth of juvenile Manila clams. Tapes ja-
ponica (Langton el al. 1977; Gallager and Mann, 1981);
C. virginica (Webb and Chu, 1982); and Ostrea edulis
(Enright ft al.. 1986b). Settlement of larval C. gigas was
also improved by increasing the protein content of algal
diets (Utting, 1986).

Most research on bivalve nutrition has focused on
quantitative requirements for energy and nitrogen rather
than on qualitative requirements for specific amino acids
(for reviews, see Newell, 1979; Bayne and Newell, 1983;
Hawkins and Bayne, 1991, 1992; Langdon and Newell,
1992). Few attempts have been made to identify the spe-
cific nutritional requirements of suspension-feeders, pri-
marily because it is technically difficult to define and
manipulate the biochemical composition of their diet.
Unlike macroconsumers, which are commonly fed pel-
letized artificial diets, suspension-feeders require micro-
scopic particles that are more difficult to prepare. Con-
sequently, dietary protein requirements of bivalve sus-
pension-feeders have had to be inferred by correlating
growth and survival rates with dietary protein content of
algal diets, even though it is often uncertain whether the
observed response was caused by protein content per se
or by variation in undetermined, nonprotein constituents
of algal diets.

In this study, protein microcapsules were used to ma-
nipulate dietary protein content, thus allowing unequiv-
ocal determination of the effect of dietary protein content
on growth of a suspension-feeding bivalve, the mussel
Alytiliis trossulus. The advantage of using microencap-
sulated diets is that their biochemical composition can be
accurately measured and controlled, unlike that of algal
diets, which is dependent on culture age and conditions
(Sakshaug and Holm-Hansen, 1977; Webb and Chu,
1982; Fabregas el al., 1985, 1986; Utting, 1985; Martin-
Jezequel el al.. 1988; Fernandez-Reiriz et al.. 1989;
Thompson et al.. 1989, 1990). PM have been shown to
be filtered and digested by oysters, C. virginica (Chu et
al.. 1982) and C. gigas (Langdon, 1989; Langdon and
DeBevoise, 1990), and mussels. M. trossulus (Kreeger,
1992), but the ability of bivalves to utilize PM for growth
has not been previously reported.

Materials and Methods

Growth rates were first measured and compared among
groups of juvenile mussels, Mytilus trossulus. which were
fed diets of algae that were isocaloric but different in pro-
tein content, to determine whether mussel growth could
be correlated with algal protein content, as has been shown
for other bivalve species (Langton el al.. 1977; Gallager
and Mann, 1981; Webb and Chu, 1982; Enright et al..
1986b). Second, direct evidence for the effect of dietary
protein content on mussel growth was obtained by com-
paring growth of mussels fed on high-protein (HP) algae
to growth of mussels fed on an equivalent ration of low-

protein (LP) algae, which was supplemented with protein
microcapsules (PM). The effect of differences in energy
availability due to different supplementary rations of PM
was examined in a separate control treatment in which
the ration of LP algae was increased to make it isocaloric
with the highest PM-supplemented ration.

Preparation of microalgae

The alga Isochrysis galbana (clone T-ISO) is widely
cultured as a nutritious food for suspension-feeding bi-
valves (Epifanio, 1979; Ewart and Epifanio, 1981; Webb
and Chu, 1982; Enright et al., 1986a;Whyte, 1987; Whyte
et al., 1 989) and was fed to mussels in this study. Cultures
were nonsterile but monospecific. Algae were initially
cultured at 18C, aerated (no CO 2 enrichment) with f/2
nutrients (Guillard and Ryther, 1962), in 1.5-1 flasks that
were then used to inoculate 20-1 carboys. The nitrogen
content of the nutrient medium of 20-1 carboy cultures
was adjusted to provide either 200% (nonlimited nitrogen)
or 40% (limited nitrogen) of the nitrogen off/2 medium.
High-nitrogen and low-nitrogen cultures contained 150
and 30 ppm (1.765 and 0.353 mg-at N 1 ') NaNO 3 , re-
spectively.

Biochemical and physical characteristics of both LP
(from low-nitrogen cultures) and HP (from high-nitrogen
cultures) algae were measured throughout the culture pe-
riod; measurements included cell concentration, volume,
ash-free dry weight ( AFDW), and proximate biochemical
composition. Algal cell concentrations (number per mil-
liliter) were measured in triplicate using a Coulter Counter
(Coulter Electronics, Model ZB 1 ). A Channelyzer (Coulter
Electronics, Model 256) was used to examine the spectrum
of cell volumes of each algal sample. In healthy cultures,
cell sizes were normally distributed, allowing determi-
nation of modal cell volume (in cubic micrometers) by
finding the volumetric channel with the greatest number
of particles (precision approximately 0.4 ^m 3 ). Algal
AFDW was routinely measured by filtering four samples,
containing known numbers of cells, through preweighed
and pre-ashed (450C, 2 d) Whatman GF/C filters, rinsing
each with 10 ml 0.5 M ammonium formate to remove
seawater, and conducting weight-on-ignition analyses
(dried 60C, 2 d; ashed 450C, 2 d).

The volume and AFDW of algal cells was found to
vary with culture age and conditions and, because diets
used for the growth experiment required careful control
of energy content, algae could not be fed to mussels simply
on the basis of cell concentration. AFDW is more closely
related to caloric content than either live or dry weight
(Brey et al.. 1988), and so algal diets were rationed daily
according to AFDW using a linear regression to predict
algal AFDW from measured cell volume. Each day of the
experiment, both cell concentrations (cell number ml" 1 )
and modal cell volumes (^m 3 cell"') of 5- to 7-day-old

EFFECT OF DIETARY PROTEIN CONTENT ON MUSSELS

125

LP and HP algal cultures were determined and used to
calculate volumetric cell concentrations (units = ^m 3
X 10 7 ml '). Volumetric cell concentrations were then
used to predict concentrations of algal AFDW, based on
a highly significant (P < 0.0001 ) linear regression between
cell volume (units = ^m 3 ceir 1 ) and AFDW (units = mg
X 10~ 8 cell'), as follows:

AFDW = (0.258 X cell volume) - 0.146. ( 1 )

Appropriate volumes of LP or HP algal culture were dis-
pensed into stock flasks of the feeding system and diluted
to a total volume of 3 1 with filtered (0.7 ^m) seawater.

Algal protein content was routinely measured in trip-
licate. A known volume of algal culture containing 10 s
cells was centrifuged (800 X g, 5 min) to form a pellet,
rinsed with 10 ml 0.5 M ammonium formate to remove
seawater, and dried at 60C for 2 days. The pellet was
resuspended in 2 ml 0.5 M NaOH, sonicated (Braun-
Sonic, Model 2000) for 10s to disrupt cell walls, heated
at 90C for 30 min. and left to stand overnight at room
temperature. The suspension was then centrifuged ( 1 500
X g, 15 min), and 100^1 of the supernatant was transferred
to an acid-washed ( 10% HNO 3 ) and baked (450C, 24 h)
5-ml test tube. The protein content was analyzed spec-
trophotometrically using a test kit (Pierce, BCA 23225)
based on the procedure of Lowry el al. (1951), and stan-
dardized with purified algal protein (see Kreeger, 1992.
for purification method). Color development in this assay
was rapid and continuous over at least a 24-h period;
therefore, the absorbance was measured with a spectro-
photometer twice, and a linear correction factor was
calculated for each sample to correct absorbances for dif-
ferences in time intervals between analysis of samples and
standards. The carbohydrate content of centrifuged and
dried (as described above) LP and HP algae was measured
using the procedure described by Dubois el al (1956),
which was standardized with oyster glycogen (Sigma G-
8751, Type II). The lipid content of LP and HP algae was
determined gravimetrically after extracting dried algal
samples twice with 7.5 ml 1:2 chloroform/methanol, fol-
lowed by treatment of the combined extract with 5 ml
0.7% weight per volume (w/v) NaCl (Folch et al., 1957;
and see "proximate analysis" below). Tripalmitin (Sigma
T-5888) was used as a standard in lipid determinations.

The caloric content of LP and HP algae was determined
by microbomb calorimetry (L. Wootton, Center for
Estuarine and Environmental Studies, Horn Point Lab-
oratory, University of Maryland, Cambridge, MD) of four
replicate, freeze-dried samples of each type. LP and HP
algae were not statistically (Student's / test, P> 0.05) dif-
ferent in caloric content. Six replicate samples each of LP
and HP algae were also analyzed for their elemental car-
bon, hydrogen, and nitrogen (CHN) composition (R. L.
Petty, Marine Science Institute Analytical Laboratory,
University of California, Santa Barbara, CA).

Preparation of protein microcapsules

Approximately 10 g dry weight of PM was produced
from purified crab (Cancer irroratus) protein using the
technique described by Langdon (1989). The proximate
biochemical composition of PM was determined using
methods described by Kreeger (1992). The elemental
CHN composition and caloric content of PM were also
analyzed, as described above for microalgae. The amino
acid composition of PM was analyzed to assess the nu-
tritional quality of encapsulated protein and to ensure
that the encapsulation process did not selectively destroy
amino acids, based on comparison with the amino acid
composition of nonencapsulated crab protein. The amino
acid compositions of purified protein from /. galbana and
from tissues of M. trossulns were also analyzed for com-
parisons.

Amino acids were analyzed by B. Robbins at the Central
Services Laboratory. Center for Gene Research and Bio-
technology, Oregon State University, Corvallis, OR. Pro-
teins were hydrolyzed in either 1% phenol dissolved in
6 N HC1 for 24 h at 1 10C, or in 0.2% 3-(2-aminoethyl)
indole HC1 in 4 N methane sulphonic acid (MSA) for
20 h at 1 10C under vacuum. After hydrolysis, samples
were freeze-dried. Hydrolysates were reconstituted in So-
dium DiLuent (Beckman, PN 239440), separated on a
Spherogel ion exchange column (Beckman 126), and de-
rivatized with ninhydrin reagent (Beckman, Trione). The
absorbances were then detected at 570 nm with a tungsten
lamp (Beckman, 166). MSA hydrolysis resulted in little
loss of tryptophan (tryptophan was lost from samples hy-
drolyzed with HC1). Amino acid analyses were standard-
ized with Pierce Standard H (Pierce, 20089); Pierce Stan-
dard B (Pierce, 20087) was used as a standard for tryp-
tophan analysis.

Description of experimental diets

Growth of juvenile M. trossulns over a 3-week period
was compared among groups of mussels fed eight different
diets: ( 1 ) LP algae delivered at a ration of 27.5% bw (body
weight, ash-free dry algal weight/ash-free dry mussel tissue
weight) d ', (2) LP algae (34.1% bw d~'). (3) LP algae
(27.5% bw d" 1 ) and PM (6.07% bw d" 1 ); (4) LP algae
(27.5% bw d" 1 ) and PM (12.1% bw d" 1 ); (5) LP algae
(27.5% bw d ') and PM (18.2% bw d" 1 ); (6) HP algae
(27.5% bw d" 1 ); (7) HP algae (27.5% bw d" 1 ) and PM
(6.07% bw d" 1 ); and (8) no food (starved control). The
standard algal ration of 27.5% bw d~' (except Diets 2 and
8) was chosen based on results from a preliminary growth
experiment in which juvenile mussels were fed for 3 weeks
on either LP or HP algae, each of which was delivered to
separate groups of mussels at 10 different rations (from
to 55% bw d" 1 ). Mussels fed HP algae in the preliminary
experiment grew significantly (a = 0.05) faster than mus-
sels fed LP algae at all rations: however, greatest differences

126

D. A. KREEGER AND C. J. LANGDON

in growth rates between mussels fed LP and HP algae
were observed when algae were delivered to mussels at a
ration of 27.5% bw d '. A higher ration of either LP or
HP algae resulted in the production of pseudofeces by
mussels and a small decrease in mussel growth rate. The
"satiation" ration of 27.5% bw d" ' of algae was therefore
used as a standard algal ration in the present experiment.
Dietary rations were adjusted weekly according to esti-
mated changes in mussel AFDTWs, which were predicted
from mussel live weights by regression analysis. Starved
mussels (Diet 8) were used as a control for uptake of nat-
urally occurring particles or dissolved organic material.

LP and HP algae were fed to mussels in Diets 1 and 6,
respectively, to verify preliminary findings that mussels
grew better when fed HP algae than when fed LP algae.
PM were fed to mussels in combination with LP algae in
Diets 3, 4, and 5 to determine whether differences in
growth rates of mussels fed LP and HP algae resulted
from differences in the protein content of the algae. The
PM ration of 12.1% bw d" 1 delivered to mussels in com-
bination with LP algae (Diet 4) was estimated to balance
the difference in protein content between LP and HP algae
(Diets 1 and 6), so that mussels fed Diet 4 were provided
with the same overall amount of protein for potential
assimilation (bioavailable protein) as mussels receiving
only HP algae (Diet 6). Rations of PM were calculated
based on their potential assimilation, because mussels
typically assimilate PM with lower efficiency (e.g., 30%;
Kreeger, 1993) than microalgae (e.g.. 75%; Bayne and
Newell, 1983). Rations of bioavailable protein associated
with delivered rations of LP and HP algae were calculated
to be 5.73 and 8.78% bw d ', respectively, by multiplying
the delivered ration of 27.5% bw d" 1 by the expected as-
similation efficiency for mussels fed microalgae (75%;
Bayne and Newell, 1983) and by the respective protein
contents of LP and HP algae (27.8 and 42.6% w/w; see
Results). The difference between these estimated values
for bioavailable protein (3.05% bw d~') was termed the
replacement protein ration (RPR). To calculate the ration
of PM that would need to be delivered to mussels to equal
RPR (12.1%, bw d" 1 . Diet 4), it was necessary to divide
RPR by both the assimilation efficiency for PM deter-
mined for M. trossiilus during spring (30%; Kreeger, 1993)
and the protein content of PM (84%, see Results). Esti-
mated rations of bioavailable protein in Diets 1-7 are
calculated in Table I and summarized in Figure la. Sup-
plements of PM in Diets 3 and 5 were calculated to equal
0.5 RPR and 1.5 RPR, respectively (Table I; Fig. 1, top).
PM were also added at 0.5 RPR to HP algae in Diet 7 to
determine whether capsule supplements could further in-
crease mussel growth, even when the algal diet was high
in protein content (Fig. 1, top).

This experimental design was chosen so that growth
differences among mussels fed diets containing PM sup-
plements could be unequivocally attributed to dietary

Table I

Estimation of the ration of dietary protein measured as percentage of
body weight (bw) per day, w/w ash-free dry, potentially assimilated by
('i.e., bioavailable to) Mytilus trossulus fed seven experimental diets

Ration delivered

Protein potentially
assimilated bv mussels

to mussels

from ration

Diet

Algae

PM a

Total

Algae b

PM C

Total d

1.

LP

27.5

_

27.5

5.73

_

5.73

9

LP

+ LP

34.1

34.1

7.11

7.11

3.

LP

+ 0.5

RPR

27.5

6.07

33.6

5.73

1.53

7.26

4.

LP

+ 1.0

RPR

27.5

12.1

39.6

5.73

3.05

8.78

5.

LP

+ 1.5

RPR

27.5

18.2

45.7

5.73

4.58

10.31

6.

HP

27.5

27.5

8.78

8.78

7.

HP

+ 0.5

RPR

27.5

6.07

33.6

8.78

1.53

10.31

Diets were composed of low-protein Isoclirysis galbana (LP) or high-
protein /. galbana (HP), with or without protein microcapsules (PM).
The ration of PM required to balance the difference in bioavailable protein
between diets of LP and HP algae is the replacement protein ration
(RPR).

a Rations of PM were calculated to balance the difference in protein
content between LP and HP A galbana when fed at 27.5% bw d~ '.

b Protein that was potentially assimilated from /. galbana by mussels
was estimated by multiplying the delivered algal ration by both a typical
assimilation efficiency of mussels fed on microalgae (75%; Bayne and
Newell, 1983) and measured algal protein contents (different for LP and
HP/ galbana, see Table IV).

c Protein that was potentially assimilated from PM by mussels was
estimated by multiplying the delivered PM ration by both the assimilation
efficiency of juvenile mussels fed on PM (assumed to be 30%, based on
data for adult mussels during spring; Kreeger, 1993) and the measured
protein content of PM (84% w/w. Table IV).

d The total potentially assimilated protein ration in each diet was cal-
culated by summing potentially assimilated protein from algae and PM
components of each diet.

protein content and not to qualitative variation among
nonprotein components of the diet. As a control treatment
to assess whether mussel growth was affected by an in-
crease in dietary caloric content in PM-supp!emented
diets, the ration of LP algae in Diet 2 (34.1%- bw d" 1 ) was
increased above the standard algal ration (27.5% bw d" 1 ).
This caloric supplement was calculated to approximately
equal the bioavailable (potentially assimilated) energy in
the highest ration of PM (18.2% bw d" 1 of PM, Diet 5)
and was termed the replacement energy ration (RER).
RER was calculated to be 108 J mg~' d~' by multiplying
the delivered ration of PM in Diet 5 (18.2%. bw d ') by
both the measured capsule energy content (19.7 J mg~',
see Results) and the assimilation efficiency for PM by
mussels during spring (30%; Kreeger, 1993). RER was
then divided by both the energy content of LP algae (21.8
J mg ', see Results) and a typical assimilation efficiency
for mussels fed on algae (75%; Bayne and Newell, 1983)
to calculate the ration of LP algae (6.6% bwd ') required
to provide the same bioavailable energy as the capsule

EFFECT OF DIETARY PROTEIN CONTENT ON MUSSELS

127

400 -

Diet

Figure 1 . Protein content, energy content, and C/N ratio of the pro-
portions of Diets 1-7 estimated to have been assimilated by (i.e., bio-
available for) Mvlilus trossulus Diets were composed of low-protein
I whrvsis galbana (LP) or high-protein / galbana (HP), with or without
protein microcapsules (PM). RPR refers to the replacement protein ration,
and RER refers to the replacement energy ration.

supplement in Diet 5. Bioavailable energy contents of ex-
perimental diets are calculated in Table II and summa-
rized in Figure 1, middle. Ratios for bioavailable C/N of
diets were calculated using results from elemental analysis
of PM and both LP and HP algae (Table III; Fig. 1
bottom).

Measurement of mussel growth rates

One week prior to the experiment, which was conducted
during May 1 992, six hundred juvenile M. trossulus (shell
height = 9-16 mm, wet weight = 100-450 mg) were col-
lected from a genetically characterized (Kreeger, 1992)
population in Yaquina Bay, Oregon. Juvenile mussels
rather than adults were used in this study so that all tissue
production would be somatic and not influenced by
changes in mussel reproductive condition, which could
affect dietary protein utilization in adults (Kreeger, 1993).
Mussels were acclimated in a continuous flow of sand-
filtered (approximately 50 urn) seawater at ambient tem-
perature (13-16C). During acclimation, mussels were
fed 10 4 cells ml~' (approximate final concentration) of

HP /. galbana (T-ISO) until 24 h before experimentation;
no food was delivered after that time. At the start of the
experiment, 480 mussels were removed from the accli-
mation system, cleaned of epiphytes and byssus threads,
and randomly divided into 24 groups of 20 individuals.
The shell of each mussel was dried and numbered using
a diamond-tipped etching pen. Individual shell heights
(anteroposterior axis) and live weights were measured.

Mussels were reared for 3 weeks at 16-18C in a series
of 24 (three replicates per diet treatment) 4-1 polycarbonate
beakers, with each beaker containing a group of 20 mussels
in 3 1 of seawater. The position of tanks on the laboratory
bench was assigned randomly. Sand-filtered ambient sea-
water was cartridge-filtered to 0.7 nm and delivered under
constant pressure to a 200-1 reservoir tank, which had a
float valve at its inflow to maintain a constant head pres-
sure (Fig. 2). Seawater was then gravity delivered to each
beaker at a constant rate of 1 1 h" 1 beaker' 1 , controlled
with a needle valve at the inflow of each beaker. Water
exited each beaker through an outflow port to waste.

Table II

Estimation of the ration of dietary energy measured in joules (per
milligram of ash-free mussel tissue) per day. potentially assimilated by
(i.e., bioavailable to) Mytilus trossulus fed on seven experimental diets

Ration delivered

Energy potentially
assimilated by mussels

to mussels

from ration

Diet

Algae

PM a

Total

Algae"

PM C

Total"

1.

LP

27.5

_

27.5

449

449

2

LP

+ LP

34.1

34.1

557

557

3.

LP

+ 0.5

RPR

27.5

6.07

33.6

449

35.9

485

4.

LP

+ 1.0

RPR

27.5

12.1

39.6

449

71.5

521

5.

LP

+ 1.5

RPR

27.5

18.2

45.7

449

108

557

6.

HP

27.5

27.5

501

501

7.

HP

+ 0.5

RPR

27.5

6.07

33.6

501

35.9

537

Diets were composed of low-protein Isochrysis galbana (LP) or high-
protein / galbana (HP), with or without protein microcapsules (PM).
The ration of PM required to balance the difference in bioavailable protein
between diets of LP and HP algae is the replacement protein ration
(RPR).

a Rations of PM were calculated.to balance the difference in protein
content between LP and HP /. galbana when fed at 27.5% bw d" 1 .

" Energy that was potentially assimilated from / galbana by mussels
was estimated by multiplying the delivered algal ration by both a typical
assimilation efficiency of mussels fed on microalgae (75%; Bayne and
Newell, 1983) and measured algal energy contents (21.8 and 24.3 J mg" '
for LP and HP /. galbana. respectively. Table IV).

c Energy that was potentially assimilated from PM by mussels was
estimated by multiplying the delivered PM ration by both the assimilation
efficiency of juvenile mussels fed on PM (assumed to be 30%, based on
data for adult mussels during spring; Kreeger, 1993) and the measured
energy content of PM (19.7 J mg" 1 . Table IV).

d The total potentially assimilated energy in each diet was calculated
by summing potentially assimilated energy from algae and PM com-
ponents of each diet.

128

D. A KREEGER AND C. J. LANGDON

I able III

Estimates of the C/N ratios ol the portion o/ /)nv.\ 1-7 potentially
assimilated by (i.e., hioavailable tot M ::;us trossulus (C/N as , ratios)
fed on seven experimental </;</ \

Potentially assimilated ration (f

bwd-')

Algae

PM

Total diet

Diet

C a

N a

C"

N"

C

N'

ratio

1.

LP

12.0

0.801

_

12

0.801

15.0

2.

LP +

LP

14.9

0.995

14.9 0.995

15.0

3.

LP +

0.5

RPR

12.0

0.801

0.836

0.224

12

8

.03

12.4

4.

LP +

1.0

RPR

12.0

0.801

1.67

0.446

13

7

.25

11.0

5.

LP +

1.5

RPR

12.0

0.801

2.51

0.672

14.5

.47

9.86

6.

HP

1 1.7

1.55

1 1

7

.55

7.55

7.

HP +

0.5

RPR

11.7

1.55

0.836

0.224

12

5

.77

7.06

Diets were composed of low-protein /.vnr/;m/.v galhana (LP) or high-
protein / galhana (HP), with or without protein microcapsules (PM).
The ration of PM required to balance the difference in bioavailable protein
between diets of LP and HP algae is the replacement protein ration
(RPR).

J The elemental composition of the potentially assimilated (PA) ration
of algae was calculated by multiplying the PA algal ration by the elemental
carbon (C) and nitrogen (N) weight percent composition of the algae
(Table IV).

b The elemental composition of the PA ration of PM was calculated
by multiplying the PA ration of PM by the measured C and N weight
percentage content (Table IV).

c Total dietary C and N contents were calculated by summation of
algal and PM values.

Beakers were aerated to facilitate mixing and suspension
of food panicles. Diets 1-7 were delivered continuously
at 125 ml h ' from a 3-1 flask to the inflow of each beaker
using one of 21 separate peristaltic-pump channels. A
stock suspension of each diet was prepared in each 3-1
tlask and continuously mixed with a magnetic stir-bar.

At the start of the experiment. 50 additional juvenile
mussels were removed from the acclimation system, and
their live weights and shell heights were determined. Each
mussel's tissue was dissected into an individual preweighed
and pre-ashed (450C) vial for analysis of its ash-free dry
tissue weight (AFDTW), using standard weight-on-igni-
tion procedures (dried at 60C, 2 d; ashed at 450C,
2 days). Since AFDTW is a better measure of organic
content than live or dry weight (Brey el ai, 1988), linear
regression was then used to derive predictive formulae for
mussel AFDTW from measures of live weight and shell
height, and these formulae were used to estimate the initial
AFDTW of each mussel used in the experiment.

Live weights and shell heights of individual mussels
were measured weekly. After these measurements were
recorded for the final week, tissues were individually dis-
sected from one-half of the surviving mussels of each
treatment and analyzed for their AFDTW using weight-
on-ignition analysis (as described previously). The proxi-

mate biochemical composition of the remaining mussels
in each treatment was analyzed to determine whether
protein ration affected the protein content of mussel tis-
sues (see below). Changes in shell height, live weight, and
AFDTW over the 3-week period were then calculated for
individual mussels within each treatment. For both live
weight and AFDTW data, each mussel's instantaneous
growth rate (IGR) was calculated as follows:

IGR = [(In W r - In W,)]/21 d.

(2)

where W, and W, refer to final and initial mussel weight,
respectively. Growth in shell height was expressed as a
percentage change in height. Individual changes in shell
height, live weight, and AFDTW during the 3-week ex-
periment were compared statistically among replicates
(n = 3) and diet treatments (n = 8) using nested ANOVA
procedures (Sokal and Rohlf, 1969). Percentage increases
in shell height were arcsine square root transformed (Sokal
and Rohlf, 1969). No significant variation among repli-
cates was found in any analysis, so the replicates were
pooled and treatments were analyzed by one-way ANOVA
and Tukey's multiple range procedures (Sokal and Rohlf.
1969).

Dclcniunation of O/N ratios for mussels

Rates of oxygen consumption and nitrogen excretion
were measured (breach group of mussels, and O/N ratios
were calculated. O/N ratios are useful indicators of the
relative catabolism of dietary protein (ratio <10) versus
carbohydrate (ratio >20). After mussels were added to
experimental beakers on the first day of the experiment,
they were allowed to purge their guts for 24 h, during

Replicated
n = 24)

Figure 2. The continuous-flow rearing system used in the mussel
growth experiment. S = seawater, C = cartridge niters, F = float valve,
R = reservoir tank. D = experimental diet, M = magnetic mixer.
P = peristaltic pump, V = needle valve, A = air, B = bivalves, and E
= effluent.

EFFECT OF DIETARY PROTEIN CONTENT ON MUSSELS

129

which they received only filtered (0.7 jtm) seawater. For
each group of 20 mussels, routine rates of oxygen con-
sumption and ammonia nitrogen excretion were then
measured during the same day. The same procedure was
repeated on the final day of the experiment.

Rates of oxygen consumption were measured for each
group by transferring the mussels from their experimental
beaker to a 400-ml respirometer chamber (Strathkelvin,
RC 400) which contained filtered (0.7 ^m) seawater. The
respirometer chamber was partially immersed in a recir-
culating water bath (Forma Scientific Bath and Circulator,
Model 2067) that maintained a constant water tempera-
ture of 15C. After adding mussels to the chamber, the
chamber was sealed, all air bubbles were removed, and
an oxygen probe from a Strathkelvin oxygen meter (Model
78 1 ) was used to monitor internal O : concentration. The
oxygen meter was calibrated with air-saturated water, and
zero oxygen concentration was determined with a freshly
prepared solution of 200 mg sodium sulfite (anhydrous)
in 100 ml 0.01 A/disodium tetraborate. Mussels rapidly
opened their shell valves after being placed in the chamber
and, after all appeared active, both oxygen concentration
(milliliters per liter) and time were recorded, and recorded
again after 15-30 min. This procedure was repeated for
each group of animals. In addition, at five different times
during the measurement period, oxygen consumption
rates of microorganisms (present in respiration chambers)
were measured, using a group of "dummy" mussels
(empty shells that had been glued together with silicone)
that had previously been held in the acclimation tank.
Rates of oxygen consumption (milliliters per hour) were
calculated using standard procedures, corrected for mi-
crobial activity, and expressed in gram-atomic units per
AFDTW <Mg-at O 2 h~' g~') for each group of mussels.
Seawater in the respirometer chamber was replaced be-
tween each oxygen measurement to prevent accumulation
of excreta and to keep dissolved oxygen concentrations
from falling below 60% saturation.

Excretion of ammonia-nitrogen was measured by
transferring mussels to sealed 1-1 Nalgene jars containing
500 ml of filtered (0.7 nm) seawater for an incubation
period of 2-4 h. Initial and final times were recorded to
calculate total elapsed time for each measurement. Three
additional chambers, without mussels, received NH,SO 4
standards of known concentration. Three chambers also
contained dummy mussels. At the start of the incubation
period, 50 ml was removed from each of the six control
chambers and fixed with 2 ml 10% w/v phenol in 95%
ethanol. The pH of each sample was lowered to <8 by
addition of 500 ^1 of 1 A/ HC1 to facilitate retention of
ammonia in solution. At the end of the incubation period,
a 100-ml sample was removed from each chamber and
pH-adjusted with 1 ml of 1 A/ HC1. Each sample was then
vacuum filtered (5 psi) through a 0.45-^m membrane filter
to remove paniculate material (a small amount of feces

was occasionally produced). Fifty milliliters of the filtrate
was removed from each sample and fixed with phenol as
described above. The concentration of ammonia in each
sample was analyzed using the method of Solorzano
(1969) and standardized with samples that contained
known levels of NH,SO 4 . Increases in the gram-atomic
weight of ammonia in chambers with living mussels were
then corrected for background concentrations of ammonia
and for microbial activity, using ammonia concentrations
measured in chambers containing dummy mussels. Rates
of ammonia excretion of each group of mussels were then
calculated and expressed relative to total group AFDTW
(Mg-at NH 4 + -N h ' g ').

Gram-atomic and weight-specific rates of oxygen con-
sumption and ammonia-nitrogen excretion were directly
compared among groups of mussels. At the start of the
experiment, oxygen consumption rates, nitrogen excretion
rates, and O/N ratios were averaged among the three rep-
licate groups of mussels from each dietary treatment and
compared statistically among treatments by one-way
ANOVA to determine whether there were any initial dif-
ferences in physiological parameters among treatments.
This analysis was repeated at the conclusion of the ex-
periment to determine whether the protein content of diets
fed to mussels over the 3-week experimental period af-
fected the manner in which mussels catabolized protein.

Proximate analysis

At the end of the experiment, approximately five ran-
domly selected tissue samples from each group of mussels
were combined and freeze-dried. After drying, tissue sam-
ples were ground into a powder with a mortar and pestle,
and the powder was weighed. Each combined tissue sam-
ple was then resuspended in 4.5 ml (final volume) distilled
H 2 O and homogenized for 1 5 s at maximum speed (Ultra
Turrax, Model SDT 1810). From each homogenate, 2 ml
was transferred to a second vial for lipid extraction and
analysis, using a method modified from Folch cl al. ( 1956).
Lipids were twice extracted with 7.5 ml 1:2 chloroform/
methanol and 2-min bath sonication (Braun-Sonic, Model
52). Supernatants were collected by centrifugation (800
X g. 10 min) and combined, and 5 ml 0.7% NaCl was
added. The mixture was then vortexed at maximum speed
for 30 s and allowed to stand overnight at 4C. After
settlement, samples were centrifuged (800 x g. 15 min),
and 4 ml of the lower organic phase was carefully with-
drawn by pipet and added to a baked (450C, 2 d) and
preweighed 7-ml vial. The samples were then dried for
2 days at 60C to remove organic solvents. Vials were
weighed after drying and amounts of lipid in original
samples were calculated, after correction for losses deter-
mined with tripalmitin (Sigma, T-5888) standards that
underwent the same process of lipid extraction and drying
as tissue samples.

130

D. A. KREEGER AND C J. LANGDON

The remainder of each tissue homogenate (2.5 ml) was
treated with 4 ml of 5% trichloroacetic acid (TCA), vor-
texed for 30 s (Vortex-Genie, Model 12-812), heated at
90C for 30 min, cooled in an ice bath for 30 min, and
centrifuged at 1 500 X g for 20 min. The supernatant was
withdrawn and 1 ml used for spectrophotometric deter-
mination of carbohydrate using the procedure described
by Dubois et al. (1956), standardized with oyster glycogen
(Sigma, Type II, G-8751) that had been subjected to the
same extraction process as the tissue samples. The pellet
was further treated with 3 ml 0.5 M NaOH, vortexed for
10 s, heated at 90C for 30 min, and allowed to stand
overnight at room temperature. Samples were then cen-
trifuged ( 1 500 X g, 10 min), and the protein concentration
of the supernatant was determined spectrophotometrically
using a test kit based on the procedure of Lowry el al.
(1951) (Pierce, BCA 23225). The protein content of the
initial tissue homogenate was calculated from standard
absorbances of purified mussel protein (for procedure, see
Kreeger, 1992) that had been treated identically to the
samples. Average protein, lipid, and carbohydrate contents
in mussel tissues from each beaker were calculated from
arcsine square root transformed data and compared sta-
tistically among treatments using ANOVA and Tukey's
HSD multiple range tests (Sokal and Rohlf, 1969).

Results

Characteristics ofmicroalgae

The alga /. galbana (clone T-ISO) was successfully cul-
tured in low-nitrogen (40% of the nitrogen off/2 medium,
30 mg NaNO 3 1~') and high-nitrogen (200% of the nitro-
gen off/2 medium, 1 50 mg NaNO 3 1~ ' ) enriched seawater.
Further reductions in nitrogen concentration below 30 mg
NaNO 3 r' caused algal cultures to collapse after 6 days
of growth. Temporal changes in cell concentrations of
low-nitrogen and high-nitrogen cultures are shown in
Figure 3a. Both types of algae grew exponentially during
the first week of culture (Fig. 3, top). After 7 days, LP
(low-nitrogen cultures) algae reached stationary phase;
whereas HP (high-nitrogen cultures) algae grew exponen-
tially until about day 10, when stationary phase was
reached (Fig. 3, top). Cell concentrations of LP algal
cultures were consistently lower than those of HP algal
cultures (Fig. 3, top). Both the age and the nitrogen
concentration of the culture were highly significant
(P < 0.0001) predictors of cell concentration, as deter-
mined by multiple regression (adjusted R 2 = 0.38).

During the culture period, both LP and HP algae in-
creased in cell size, although LP algal cells were usually
larger than HP algal cells of similar culture age (Fig. 3,
middle). A multiple regression (adjusted R 2 = 0.51) for
predicting cell volume indicated that both nitrogen con-
centration and culture age were significant (P < 0.0001)
variables affecting cell volume. Cell volumes of LP and

5 -

|

* 50 -

30 -

20 -

12

Figure 3. Temporal changes in the mean (95% confidence interval)
concentration (top), volume (middle), and protein content (bottom) of
Isochrysis ga/bana (T-ISO) cells cultured in low-nitrogen (LP) or high-
nitrogen (HP) media.

HP algae were averaged (SE) for the entire culture period
and were 18.0 2.3 jum 3 ( = 127) and 17.6 1.5 ^m 3
(n = 93), respectively. As with cell volume, cell AFDW
also increased with culture age and nitrogen concentration
and averaged (SE) 28.0 13.1 pg cell ' (n = 33) and
29.3 12.9 pg ceir 1 (n = 29) for LP and HP algae, re-
spectively. Equal weight-specific rations of LP and HP
algae to be fed to mussels in the growth experiment were
calculated from a highly significant (P < 0.0001) linear
regression equation (R 2 = 0.94) for prediction of cell
AFDW from cell volume measurements (see Methods).

The biochemical composition and energy content of
algae cultured on low-nitrogen and high-nitrogen media
are summarized in Table IV. No significant differences
(P > 0.05) were found between LP and HP algae in their
percentage (dry w/w) ash composition; however, LP algae
had a significantly (P < 0.0001) lower protein content
(27.8% w/w) than HP algae (42.6% w/w). Carbohydrate
and lipid contents were significantly (P = 0.002 and
P = 0.006, respectively) greater in LP algae (25.1% and
22.0%, respectively) than in HP algae (20.2% and 17.1%,

EFFECT OF DIETARY PROTEIN CONTENT ON MUSSELS

131

Table IV

Biochemical composition (percentage lota/ dry weight) and energy
content (per unit dry weight) of protein microcapsules (PM). low-
protein (LP) Isochrysis galbana (T-ISO). and high-protein (HP)
I. galbana

Percentage content* (Mean SE)

Student's 1 test
P value
(LP algae vs.
HP algae)

Parameter

PM

LP algae

HP algae

Ash

4.181 1.49

23.8 1.1

19.7 1.4

NS

( = 3)

(" = 5)

(n = 6)

Carbohydrate

0.49 0.15

25.1 0.6

20.2 0.3

P = 0.002

(n = 11)

(n = 7)

(n = 7)

Lipid

ND

22.0 0.6

17.1 0.6

P = 0.006

(n = 5)

(n = 7)

(n = 7)

Protei n

83.7 3.9

27.8 + 0.8

42.6 1.1

P< 0.0001

(n = 10)

(n = 25)

(n = 26)

Carbon

45.9 1.8

58.4 3.5

56.5 5.9

NS

(n = 5)

(n = 6)

(ii = 6)

Nitrogen

12.3 0.2

3.89 0.27

7.51 0.22

P< 0.0001

(n = 5)

(n = 6)

(n = 6)

C/N ratio

3.73 0.05

15.0 1.5

7.55 0.87

P< 0.0001

( = 5)

(n = 6)

(n = 6)

(J

mg~') (Mean

SE)

Energy content

19.7 0.3

21.8 2.3

24.3 0.6

NS

(n = 5)

(n = 3)

(n = 4)

Statistical differences between algae types were assessed with (-tests on arcsine
square root transformed percentages.

NS = not significant (a = 0.05); ND = not detected.

* Except for C/N ratio, which is the percentage carbon composition divided by
the percentage nitrogen composition, and energy content, which has units of joules
per milligram.

respectively). Interestingly, LP and HP algae did not differ
significantly (P > 0.05) in carbon content; whereas HP
algae contained significantly (P < 0.0001 ) greater nitrogen
(7.51%) than LP algae (3.89%), which resulted in a sig-
nificantly (P< 0.0001) lower C/N ratio in HP algae (7. 55)
than in LP algae (15.0). The energy content of LP and
HP algae did not differ significantly (Table IV).

Cell protein content is plotted as a function of time in
the bottom portion of Figure 3 for LP and HP algae. A
multiple regression analysis (R 2 = 0.53) with culture age
and nitrogen concentration as variables suggested that al-
gal protein content was significantly (P = 0.003) correlated
with culture nitrogen concentration, but not significantly
(P > 0.05) correlated with culture age. Separate linear
regressions for LP and HP algae, however, indicated that
protein content of HP algae significantly (P = 0.0002)
increased with culture age; whereas the protein content
of LP algae was not significantly (P > 0.05) correlated
with culture age. Only 5- to 7-day-old cultures of LP and
HP algae were harvested for mussel growth experiments.

Characteristics of protein microcapsules

The biochemical composition and energy content of
PM used in this study are summarized in Table IV. The

average protein content (percent weight per weight) was
83.7%. Carbohydrate content was less than 1% w/w, and
no measurable lipid was recovered gravimetrically. Ash
accounted for 4.2% of the capsule weight. The nitrogen
content of PM was 12.3%, which, if multiplied by the
standard protein/nitrogen conversion factor of 6.25, gives
a substantially lower estimate of capsule protein content
(76.9%) than that measured analytically (83.7%). The rea-
son for this discrepancy is unclear; however, the test kit
for protein (Pierce BCA) may have overestimated capsule
protein content (for a discussion of error associated with
this method, see Zamer el al, 1989). The carbon content
of PM was measured to be 45.9%. (Table IV), which, when
expressed relative to nitrogen content, resulted in a C/N
ratio of 3.73. The energy content of PM was 19.7 J mg~',
which was not significantly (ANOVA, P> 0.05 (different
from the energy content of either LP or HP algae (21.8
and 24.3 J mg '. respectively).

Amino acid compositions of mussel protein, algal pro-
tein, crab protein, and crab PM are given in Table V.
With the exception of cysteine, all amino acids were pres-
ent in all protein samples. Cysteine was poorly represented
in both purified crab protein and crab PM, but it is not
thought to be essential for mussels (see Discussion). To
facilitate a comparison of protein quality between PM

Table V

Percent relative concentration of amino acids in protein hydrolysates
from different sources

Percent relative concentration
in protein hydrolysate

Percent relative
to mussel protein

Amino
acid

Algal
protein

Crab

protein

Crab
PM

Mussel
protein

In algal
protein

In crab
PM

THR*

7.13

8.23

7.34

8.16

87.4

90.0

VAL*

4.88

5.07

4.65

4.30

>100

>100

MET*

2.40

1.68

2.03

2.01

>100

>100

ILE*

4.20

4.59

6.90

4.17

>100

>100

LEU*

9.26

6.19

7.17

6.98

>100

>100

PHE*

4.53

3.98

3.80

3.32

>100

>100

LYS*

5.21

6.85

10.3

7.82

66.6

>100

HIS*

1.96

2.23

2.06

1.74

>100

>100

TRP*

0.761

0.544

0.567

0.467

>100

>100

ARG*

4.38

4.75

4.47

5.11

85.7

87.5

ASP

13.0

15.9

15.3

15.7

82.8

97.5

SER

6.78

6.72

6.34

8.53

79.5

74.3

GLU

9.62

14.7

12.0

11.8

81.5

>100

PRO*

4.74

3.14

2.24

4.20

>100

53.3

GLY

8.67

6.03

5.72

5.68

>100

>100

ALA

9.05

6.51

6.09

5.81

>100

>100

CYS

0.697

0.270

ND

1.39

50.2 a

o a

TYR

2.77

3.14

2.99

2.85

97.2

>100

PM = protein microcapsules; ND = not detected.
* Potentially essential amino acids.

a Cysteine was suspected to have been lost during purification of algal
and crab protein.

132

D. A. K.REEGER AND C. J. LANGDON

and algal protein, the amount of each amino acid in each
protein type was expressed relative to the amount mea-
sured in mussel tissue protein (Table V). In both algal
protein and PM, the relative a .icentration of all poten-
tially essential amino acids except lysine was estimated
to be greater than 85% of the relative concentration in
mussel protein. Lysine was present in algal protein at a
relative concentration equivalent to only 67% of that in
mussel protein.

Growth of mussels fed experimental diets

Shell heights and live weights of juvenile M. trossulux
did not differ significantly (ANOVA, P > 0.05) among
the 24 experimental groups at the beginning of the ex-
periment; however, within 1 week, significant differences
in mussel growth rates were measured among mussels fed
on the eight different diet types. Only growth rates cal-
culated for the total 3-week period are given (Table VI).
Nested ANOVAs indicated that mussel growth rates were
statistically similar among the three replicates in each di-
etary treatment; therefore, shell height, live weight, and
AFDTW measures of growth were combined for each set
of three replicates and analyzed among diet treatments
with one-way ANOVAs and Tukey's HSD multiple range
tests.

Mussels fed LP /. galbana (T-ISO) (Diet 1) grew at
significantly (P < 0.05) lower rates than mussels fed HP
/. galbana (Diet 6), when delivered at satiation rations of
27.5% bw d~'. Based on shell height, live weight, and
AFDTW, growth rates of mussels fed LP algae (Diet 1 )
were 51, 56. and 53%-, respectively, of corresponding
growth rates of mussels fed HP algae (Diet 6). Starved
control mussels (Diet 8) failed to grow, demonstrating
that mussels were unable to derive sufficient nutrients
from their culture medium (e.g.. dissolved organic ma-
terial, "background" particulate material) to exceed
maintenance requirements. Mortality did not vary sig-
nificantly (ANOVA, P > 0.05) among dietary treatments.

Growth rates of mussels fed LP algae were significantly
improved when the ration of LP algae was supplemented
with PM (Table VI). Furthermore, there was a propor-
tional increase in mussel growth rate with progressively
higher supplements of PM to the LP algal diet. The in-
stantaneous growth (live weight) rate of mussels fed LP
algae with the highest supplement of PM (Diet 5; 17.0
X 10~ 3 d~') was statistically equal to that of mussels fed
only HP algae (Diet 6; 17.5 X 10~ 3 d~'). Similarly, in-
stantaneous AFDTW growth rates were not significantly
(P > 0.05) different among groups of mussels fed either
LP algae with a 1 .0 RPR capsule supplement (Diet 4; 48.0
X 10~ 3 d~'), LP algae with a 1.5 RPR capsule supplement
(DietS; 47.1 X 10~ 3 d~'), or HP algae alone (Diet 6; 53.3
X 10~ 3 d' 1 ) (Table VI). Supplements of PM added to HP
algae (Diet 7) did not improve mussel growth rates corn-

Table VI

Increase in shell height, live weight, and ash-free dry tissue weight
(Af-'DTH'i of /iivcni/e Mytilus trossulus fed eight experimental diets

Increase in

Instantaneous growth rate
(X10- 3 d-')

Diet

Mortality

shei

1 height

Live weight

AFDTW

1.

LP

9.6

3.6

5.81

0.27 C

9.73

0.59

28.0

1.95 C

2.

LP + LP

(RER)

12.2

6.4

5.90

0.37 C

10.3

0.71 CD

29.9

2.23 C

3.

LP + 0.5

RPR

6.5

2.3

7.92

().31 BC

13.4

0.74 C

36.8

2.86^

4.

LP+ 1.0

RPR

17.8

9.7

7.30

0.38 C

13.8

l.ll) BC

48.0

4.85 AB

5.

LP + 1.5

RPR

0.6

5.0

10.8

0.46 AB

17.0

0.87 AB

47.1

3.58*"

6.

HP

12.6

5.1

11.5

0.42 A

17.5

0.86*

53.3

3.23*

7.

HP + 0.5

RPR

4.2

8.1

11.4

0.52 A

17.6

1.00*

54.7

4.35*

8.

Starved

5.6

8.6

0.02

0.01 D

1.49

0.46 E

-15.5

2.49 D

LP and HP refer to low-protein algae and high-protein algae, respectively. RER
refers to the replacement energy ration, and RPR refers to the replacement protein
ration. For each growth parameter, values having common superscripted letters
among dietary treatments are not statistically different (ANOVA, P < 0.05). Values
reported are means SE (n = 3 per value).

pared to those of mussels fed only HP algae (Diet 6: Table
VI). Growth of mussels that were fed LP algae at a ration
of 34. 1% bw d ' (LP + LP; Diet 2) did not grow signifi-
cantly ( > 0.05) faster than mussels fed LP algae at 27.5%
bw d ' (Diet 1; see Table VI). This result confirms the
findings of a preliminary experiment in which growth of
mussels fed on algal rations reached an asymptote at a
ration of 27.5% bw d ', and growth rates did not increase
at higher rations. These latter results clearly indicate that
growth of mussels fed on LP algae was not limited by
ration size.

The instantaneous growth (live weight) rate (IGR, W ) of
mussels is plotted versus the estimated C/N ratio of the
assimilated (bioavailable) portion of each diet (C/N ass ra-
tio) in Figure 4. C/N ass ratios were slightly different than
the C/N ratios of the diets actually delivered to mussels
(C/N tot ratio) because PM were assumed to be assimilated
with lower efficiency (e.g., 30%; Kreeger, 1993) than mi-
croalgae (e.g., 75%, Bayne and Newell, 1983). C/N Ull ratios
were 15.0, 15.0, 10.4, 8.44, 7.39, 7.55, and 6.51 for Diets
1-7, respectively; whereas estimated C/N ass ratios were
15.0. 15.0, 12.4. 11.0, 9.86, 7.55, and 7.06 for Diets 1-7.
respectively (Table III). Mussel growth rates were inversely
related to estimated C/N ass ratios between 10 and 15;
whereas growth rates appeared to be independent of
C/N ass ratios less than 10 (Fig. 4). A linear regression of
IGR 1W as a function of C/N ass ratio was highly significant
(P < 0.0001 ) over the range of C/N ass ratios between 7. 1
and 15.0. Additional regressions indicated that IGR IW was
also significantly (P < 0.01 ) correlated with C/N 101 as well
as with the estimated total and bioavailable protein ration
in the mussel's diet.

EFFECT OF DIETARY PROTEIN CONTENT ON MUSSELS

133

ce
o

LP

V LP + LP

V LP + 0.5 RPR

D LP + 1.0 RPR

LP + 1.5 RPR
A HP

A HP + 0.5 RPR

15 14 13 12 11 10 9 8

C/N Ratio of Assimilated Diet

Figure 4. Mean (95% CI) instantaneous live weight growth rate
(1GR, W ; x I0~ 3 d~') of juvenile Myiilns tmssulus as a function of the
estimated C/N ratio of the assimilated portion of Diets 1-7. LP and HP
refer to low-protein algae and high-protein algae, respectively. RPR refers
to the replacement protein ration, and RER refers to the replacement
energ\ ration.

Despite highly significant differences in growth rates of
mussels fed different diets, mussel tissue biochemical
composition was not appreciably altered by ration com-
position. Initially, the protein content of all mussels was
53% (Table VII). After 3 weeks, the protein content of
mussel tissues varied significantly (ANOVA, P < 0.05),
ranging from 34% to 56%, among dietary treatments;
however, tissue protein content was not clearly correlated
with dietary protein content, and differences were only
marginally significant. The lowest tissue protein contents
were measured in mussels that were fed LP algae (Diet
1 ). and the highest tissue protein contents were measured
in mussels fed LP algae with either 0.5 RPR or 1.0 RPR
protein capsules (Diets 3 and 4). Lipid content was sig-
nificantly (ANOVA, P < 0.05) greater in tissues of mussels
fed LP algae alone (Diet 1) than in any other group of
mussels, including the starved controls (Table VII) a re-
sult that is questionable given the high variability asso-
ciated with lipid values for mussels fed Diet 1. No signif-
icant (ANOVA, P > 0.05) differences were found in car-
bohydrate contents among mussels fed different diets
(Table VII).

Oxygen consumption rates of mussels at the start of
the experiment did not differ significantly (ANOVA,
P> 0.05) among the 24 groups and averaged 1 16 /ug-at
O, h ' [g AFDTW] ' (Table VIII). After the 3-week rear-
ing period, however, rates of oxygen consumption were
lower in all groups (9.3-75.3 ^g-at O 2 h~' g~'). Mussels
fed LP diets generally had higher rates of oxygen con-
sumption than mussels fed HP diets but, due to high vari-
ability in rates, differences were not significant (P > 0.05)
among groups fed Diets 1-7 (Table VIII). Starved controls

Table VII

Proximate biochemical composition of the tissues of juvenile Mytilus
trossulus after being fed for 3 weeks on eight experimental diets and al
the start (initial) of the growth experiment

Percentage composition
(Mean + SE; n = 3)

Diet

Protein

Lipid

Carbohydrate

Initial

53.3 + 4.5

NM

8.00 + 0.20

1 LP

34.1 2.7"

29.7 + 3.2 A

10.86 0.20

2. LP + LP(RER)

44.3 2.6 AB

17.1 +0.6"

11.36 1.30

3. LP + 0.5 RPR

50.0 1.6*

19.2 0.4 B

10.12 +0.17

4. LP + 1.0 RPR

55.7 3.1*

17.1 +0.7"

10.23 + 0.06

5. LP+ 1.5 RPR

43.6 0.7 AB

16.8 0.3"

8.39 +0.84

6. HP

41.1 0.9 AB

16.8 0.2 B

8.08 0.92

7. HP + 0.5 RPR

47.5 + 1.4 AB

16.30.4 B

9.56+0.34

8. Starved

43.7 + 2.I AB

16.8 + 0.1"

9.03 + 0.32

LP and HP refer to low-protein algae and high-protein algae, respec-
tively. RER refers to the replacement energy ration, and RPR refers to
the replacement protein ration. For each of the protein, lipid, or car-
bohydrate contents (percent dry weight), values having common super-
scripted letters among dietary treatments are not statistically different
(ANOVA, F<0.05).

NM = not measured; samples lost.

consumed only 9.3 Mg-at O : h ' g ', which was signifi-
cantly (P < 0.05) lower than rates of oxygen consumption
by mussels fed Diets 1 (72.8 ng-ai O 2 FT 1 g" 1 ) and 3 (75.3
Mg-at O; IT 1 g"'). Rates of ammonia nitrogen excretion
were greatest in mussels fed LP algae with the greatest
supplementation of PM (Diet 5; 7.01 ^g-at NH^-N h~'
g" 1 ) (Table VIII). The only significant (ANOVA, P< 0.05)

Table VTII

Rates of oxygen consumption and nitrogen excretion and O/N ratios of
juvenile Mytilus trossulus at the beginning and end of the growth
experiment

Oxygen

Nitrogen

consumption

excretion

(Mg-atO : h-'

Oig-atNH 4 *-Nh-'

Diet

[g AFDTW]-')

[g AFDTW]-')

O/N ratio

Initial

116 9.8

6. 14 0.39

18.9

.0

1 LP

72.8 11.7*

3.44 + 0.38"

21.2 5.5*

2. LP + LP(RER)

66.8 15.4*"

3.60 0.08 AB

18.64.8 A

3. LP + 0.5 RPR

75.3 25.7 A

6.68 1.30 AB

11.3 4.3**

4 LP + 1.0 RPR

41.8 3.7 AB

6.06 1.02 AB

6.90

.36*"

5. LP+ 1.5 RPR

51.2 9.4*"

7.01 0.91 A

7.30 +

.53*"

6. HP

31.5 8.6 AB

6.09 0.50 AB

5.17 +

.81"

7 HP + 0.5 RPR

54.5 + 7.I* B

5.43 0.09 AB

10.0

.4*"

8. Starved

9.30 7.38*

4.97 0.38 AB

1.87 +

.37 C

LP and HP refer to low-protein algae and high-protein algae, respectively. RER
refers to the replacement energy ration, and RPR refers to the replacement protein
ration. For each parameter, values having common superscripted letters among
dietary treatments are not statistically different (ANOVA, P < 0.05). Values reported
are mean SE (n = 3 per measurement, except n = 24 for initial readings).

134

D. A. KREEGER AND C. J. LANGDON

difference in nitrogen excretion among treatment groups
was between mussels fed Diet 5 and mussels fed on LP
algae alone (Diet 1; 3.44 ^g-at NHj-N h ' g~').

Even though rates of oxygen consumption and nitrogen
excretion did not vary substantially among treatments,
differences in O/N ratios among treatments were more
discernible (Table VIII, Fig. 5). At the beginning of the
experiment, no significant differences were detected in
O/N ratios among the 24 groups of mussels, and the initial
mean O/N ratio was 18.9, indicating that carbohydrates
and perhaps lipids were being catabolized preferentially
compared to protein. In contrast, after the 3-week exper-
imental period, O/N ratios of mussels fed diets rich in
protein (e.g.. Diets 4-6) were reduced to less than 8, re-
flecting greater catabolism of protein than carbohydrates
(Fig. 5). Due to high variability among replicates (statis-
tical resolution of differences in O/N ratios among treat-
ments was poor, see Table VIII), however, O/N ratios of
mussels fed LP algae alone (Diets 1 and 2) were signifi-
cantly (P < 0.05) greater than those of mussels fed on HP
algae alone (Diet 6). Due to very low rates of oxygen con-
sumption, starved mussels (Diet 8) had an O/N ratio of
1.9, which was statistically (P < 0.05) lower than that of
fed mussels.

Discussion

Much is now known about quantitative and caloric
dietary effects on feeding, assimilation efficiency, and
growth of bivalve molluscs because quantitative aspects
of bivalve diets (e.g., algal cell concentration) are easily
manipulated (for reviews, see Bayne and Newell, 1983;
Hawkins and Bayne, 1992; Langdon and Newell, 1992;
Newell and Langdon, 1992). Previous findings (Gallager
and Mann, 1981; Langdon and Waldock, 1981; Langdon.
1982; Webb and Chu, 1982; Enright et ai. 1986b; Utting.
1986) indicated, however, that qualitative aspects of diets
are also important in determining growth of bivalves. For
example, it has commonly been observed that bivalves
grow more quickly when fed mixtures of several algal spe-
cies than when fed monospecific diets (Epifanio, 1979,
1982; Webb and Chu, 1982), presumably because the
quality of mixed-species diets is more balanced. The
qualitative nutritional requirements of suspension-feeding
animals are poorly known compared to those of macro-
phagic marine animals, because there are technical diffi-
culties in manipulating the composition of microparti-
culate diets.

The effect of diet quality on suspension-feeding bivalves
has previously been studied primarily by comparison of
algal diets that vary in biochemical composition. Two
related approaches have been used. First, bivalves have
been fed different algal species that vary in biochemical
composition, and bivalve assimilation or growth has then
been correlated with qualitative attributes of the diets

10 -

Diet

LP +
LP

(RER)
2

LP +

05

RPR

3

LP +

1

RPR

4

LP +

1.5

RPR

5

HP

HP + Starved

0.5 Controls
RPR
7 8

Figure 5. O/N ratios (mean 95% CI) of juvenile Mytilus trossiihis
after being fed for 3 weeks on Diets 1-7 composed of low-protein (LP)
or high-protein (HP) algae, with or without protein microcapsules. Mus-
sels in dietary treatment 8 were starved. RER refers to the replacement
energy ration, and RPR refers to the replacement protein ration. Values
having common letters are not significantly (ANOVA, P < 0.05) different.

(Flaak and Epifanio, 1978; Webb and Chu, 1982; Enright
el al.. 1986a; Whyte et ai, 1989). Alternatively, growth
rates have been compared among bivalves fed monospe-
cific algal diets that vary in biochemical composition due
to manipulation of algal culture conditions such as (1)
availability of inorganic carbon (Pruder and Bolton, 1979),

(2) light intensity or wavelength (Flaak and Epifanio, 1978;
Gallager and Mann, 1981; Thompson et al., 1989. 1990),

(3) age at which cells are harvested (Sakshaug and Holm-
Hansen. 1977; Fabregas et al.. 1985, 1986; Utting, 1985;
Whyte, 1987; Fernandez-Reirez et al.. 1989), and (4) nu-
trient concentrations in culture media (Sakshaug and
Holm-Hansen, 1977; Gallager and Mann, 1981; Utting,
1985; Enright el al., 1986b). The latter approach was used
in this study: nitrogen concentrations of cultures of the
alga /. galhanu (clone T-ISO) were manipulated, and the
consequent nutritional value of nitrogen-limited algae was
determined for the mussel M. trossulus.

Reduction of the nitrogen concentration of the culture
medium has been shown to lower cellular protein content
during both exponential and stationary growth phases of
algae (Utting. 1985; Enright et al., 1986b). In this study,
nitrogen limitation in cultures of /. galbami effectively
reduced cell protein content regardless of the age of the
culture (Fig. 3, bottom). Algae cultured in f/2 medium
(Guillard and Ryther, 1962) in which the nitrogen con-
centration was reduced by 60%, to 0.353 mg-at NaNO,-
N 1 ', contained only 65% of the protein of algae grown
in a medium having twice the standard nitrogen concen-
tration (1.765 mg-at NaNO 3 -N 1~'). Similarly, Utting
( 1985) reported a 30% reduction in the cell protein content

EFFECT OF DIETARY PROTEIN CONTENT ON MUSSELS

135

of/, galbana when the nitrogen concentration of the cul-
ture medium was lowered by 94%'.

Other researchers have reported that the biochemical
composition of algae can vary with culture age (Sakshaug
and Holm-Hansen, 1977; Fabregas et til.. 1985, 1986;
Utting, 1985;Whyte. 1987; Fernandez-Reiriz el ai. 1989).
The protein content of algal cells can either decrease or
increase with culture age, depending on species and con-
ditions (Utting. 1985; Whyte, 1987; Fernandez-Reiriz et
at.. 1989). In this study, the cellular protein content of
HP /. galbana significantly increased during the first 12
days of culture. HP algae did not reach stationary phase
until about the 10th day of culture, and the protein content
remained high during this phase. The protein content of
LP /. galbana tended to decline after stationary phase was
reached on day 7, but this decrease was not significant.

Algae were routinely harvested for delivery to mussels
between days 5 and 7 of culture because during this period
LP and HP cultures consistently appeared healthy, as
judged visually by the normality of their cell-size distri-
butions. Cell volumes (14-21 urn*) of I. galbana were
generally smaller than those reported by other workers
(e.g.. 46-74 nm\ Enright et ai. 1986a: 14-34 M m\ Riis-
gard, 1988). although cell volumes of both LP and HP
algae increased significantly with age. Furthermore, algal
cells cultured under low-nitrogen conditions were always
slightly larger than those of the same age cultured under
high-nitrogen conditions. The purpose of measuring cell
volume in this study was to allow accurate prediction of
cell AFDW. Cell volume was measured with high preci-
sion (low coefficients of variation), and the linear regres-
sion equation relating algal volume to AFDW was highly
significant. The range of cell volume, 14 to 21 /jm\ cor-
responded to a range in cell AFDW of between 35 and
53 pg cell ', which was greater than the cell dry weight
of 29.7 pg cell 1 for /. galbana (clone T-ISO) reported by
Brown ( 199 1 ). Because cell volume (and therefore AFDW)
varied with both time and protein content, it was impor-
tant to dispense algal rations for mussels based on cell
volume (and therefore AFDW) and not simply on cell
concentration: otherwise the diets might have differed by
up to 50% in energy content. No significant (Student's
t test, P > 0.05) differences were measured in the energy
content (per unit dry weight) of LP and HP algae in this
study, which confirms a similar report for /. galbana by
Utting (1985).

Growth rates of juvenile M. trossulus were significantly
greater when mussels were fed HP /. galbana rather than
an equal ration of the LP algae. For example, mussels fed
LP algae at a ration of 27.5% bw d ' grew in shell height,
live weight, and AFDTW at rates (5.8, 9.7, 28.0. respec-
tively) that were only 50-55%> of those of mussels fed an
equivalent ration of HP algae (1 1.5, 17.5. 53.3. respec-
tively). These results supported findings from a prelimi-
nary experiment in which growth rates of mussels fed LP

algae at 10 different rations were approximately 50% of
growth rates of mussels fed equivalent rations of HP algae.

A standard algal ration of 27.5%- bw d" 1 was used in
this study because a preliminary experiment showed that
this ration supported the highest growth rates of mussels,
and that rations above 27.5% bw d"' caused mussels to
produce pseudofeces. An increase in the ration of LP algae
from the standard ration (Diet I)to34.1%bwd ' (Diet 2)
did not improve mussel growth rates. This finding, to-
gether with results from the preliminary experiment (see
above), indicate that the difference in growth rates between
mussels fed LP and HP algae was a result of differences
in diet quality rather than diet quantity or ration size.
This correlation between the growth rate of juvenile
M. trossulus and the protein content of algal diets agrees
with previous findings for other bivalve species (Langton
et ai. 1977; Gallager and Mann, 1981; Webb and Chu,
1982; Enright et ai. 1986b). Gallager and Mann (1981),
for example, reported that algae with low protein content
were inferior to algae of higher protein content in sup-
porting growth of juvenile Manila clams. Tapes japonica.
Inreases in the protein content of algal diets have also
been shown to improve settlement of larval Crassostrea
gigas (Utting, 1986).

As with all previous studies in which bivalve growth
has been correlated with algal composition, it is not pos-
sible to conclude, based only on data from LP and HP
diets (Diets 1, 2, and 6), that algal protein content was
directly responsible for variation in mussel growth rates
among experimental diets, because nonprotein nutritional
constituents of these diets may have co-varied with protein
content. In Diets 3. 4. and 5, however, the ration of dietary
protein for M. tmssulus was manipulated independently
of algal composition by addition of supplements of mi-
croencapsulated protein to LP and HP algal diets. There-
fore, the effect of dietary protein content on mussel growth
was directly examined without altering algal biochemical
composition. Supplementation of LP algae with PM sig-
nificantly improved the growth rate of juvenile mussels
compared to that of mussels fed LP algae alone, which
clearly demonstrates that juvenile M. trossulus used PM
for growth. PM delivered at the highest ration in com-
bination with LP algae supported mussel growth equiv-
alent to that of mussels fed HP algae, and so completely
compensated for the lower growth rates of mussels fed LP
algae alone. Similar experiments have been used to ex-
amine the nutritional requirements of macrophagous
marine animals (Kanazawa, 1982; Wilson, 1989). Few
researchers, however, have conducted this type of exper-
iment with bivalve suspension-feeders (Gabbott et ai.
1976; Langdon and Waldock, 1981; Chu et ai. 1982).

PM were composed of approximately 84%. protein
(based on biochemical analysis). PM have been shown to
be filtered, digested, and assimilated by mussels (Kreeger,
1992. 1993). To be of value as a growth supplement.

136

D. A. KREEGER AND C. J. LANGDON

encapsulated protein must not only be bioavailable, but
must also have a balance of essential amino acids that
meets the qualitative requirements of the consumer. For
many animals, the essential amino acids have been iden-
tified as threonine (THR), valine (VAL), methionine
(MET), isoleucine (ILE), leucine (LEU), phenylalanine
(PHE), lysine (LYS), histidine (HIS), tryptophan (TRP),
andarginine(ARG)(Lehninger, 1976; Deshimaru, 1982;
Bishop el al. 1983). Unfortunately, the essential amino
acid requirements of bivalve molluscs have not been fully
ascertained, primarily due to problems in developing a
defined diet for suspension-feeders, but are generally be-
lieved to be similar to those of other animals (Bishop el
al, 1983). Harrison (197 5), for example, reported that the
California mussel, M. californianus, had a dietary re-
quirement for the same 10 amino acids listed above, and
perhaps also for proline (PRO).

All of the amino acids considered to be essential for
bivalves were substantially present in both algal protein
and PM made from crab protein, with two exceptions.
The concentration of LYS in protein from /. galbana was
only 67% of the relative concentration measured in protein
from M. trossulus. Brown (1991) similarly reported that
LYS concentrations in protein from the same clone of
/. galbana, as well as from 1 5 other species of microalgae,
were lower than the relative concentrations of LYS in
protein derived from larval C. gigas. The only amino acid
for which PM apparently had a deficiency was cysteine
(CYS). CYS may have been oxidized during TCA puri-
fication of the crab protein (Allen, 1981) because the CYS
concentration in purified crab protein was only one-sixth
that found before purification. This loss was not consid-
ered crucial because M. ednlis is reported to be capable
of converting MET into CYS (Allen and Awapara, 1960).

A nutritious protein has an amino acid profile similar
to that in the tissues of the consumer organism (Phillips
and Brockway. 1956). Both algal protein and crab PM
contained relative concentrations of essential amino acids
that were comparable to the amino acid profile of protein
purified from M. trossulus. Relative concentrations of es-
sential amino acids have been quantitatively compared
between bivalve suspension-feeders and their algal diets
by calculation of an essential amino acid index (EAAI)
(Brown and Jeffrey, 1992). If the quality of a dietary pro-
tein is high, the EAAI will be greater than 90; EAAI values
less than 70 indicate that the dietary protein is qualitatively
poor (Brown and Jeffrey, 1992). Assuming that M. tros-
sulus requires 1 1 essential amino acids (including PRO),
then the EAAI calculated for /. galbana protein and crab
PM were 109 and 105, respectively, which indicates that
the quality of protein in these dietary constituents was
indeed sufficient for supporting the growth of mussels.

One difficulty in conducting bivalve growth experi-
ments with artificial diets is that bacteria commonly mul-
tiply rapidly in the culture system. Thus, it can be unclear

whether bivalve growth is significantly affected by the uti-
lization of bacteria associated with the artificial diets (e.g.,
Langdon and Bolton, 1984; Langdon and Siegfried, 1984).
Considerable recent evidence indicates that some suspen-
sion-feeding bivalves are able to derive nutrition from
free-living bacteria in the water column or bacteria at-
tached to suspended particulate material (Birkbeck and
McHenery, 1982; Wright el al., 1982; Crosby et al.. 1990;
Langdon and Newell, 1990; Baldwin and Newell, 1991;
Douillet, 1991). Leakage rates of PM in this study were
low (e.g.. 0.5-2% cT 1 , Kreeger, 1992) compared to rates
previously reported from PM (e.g.. 5-40% cT 1 ; Langdon,
1989: Langdon and DeBevoise, 1990). Therefore, mussels
probably did not obtain additional nutrients either by
taking up the leaked nutrients directly or by ingesting
capsule-derived nutrients indirectly in the form of micro-
organisms. Furthermore, mussels were reared in a con-
tinuous flow of filtered (0.7 ;um) seawater with eight water
volume changes per day, which probably helped to keep
the concentrations of microorganisms low. Kreeger and
Langdon ( 1993) reported, for example, that bacteria did
not contribute to I4 CO : respiration in a similar continu-
ous-How system in which Al. trossulus were fed 14 C-labeled
PM. Starved control mussels either did not grow or lost
weight, which suggests that fed mussels derived food pri-
marily from their experimental diets. Because bacteria
apparently did not contribute to mussel nutrition in this
study, we concluded that dietary protein content was in-
deed the qualitative dietary factor of LP algae that was
limiting the growth of mussels fed only LP algal diets.

Compared to mussels fed LP algae, mussels fed diets
rich in protein for 3 weeks tended to have more protein
and less lipid and carbohydrate in their tissues, although
the differences were only marginally significant. O/N ra-
tios of mussels fed on LP algae at either 27.5 or 34.1%
bw d ' were 21.2 and 18.6, respectively, suggesting that
these mussels were primarily catabolizing carbohydrates
and conserving protein for anabolism. In contrast, mussels
fed diets with greater protein content (e.g., HP algae or
LP algae with PM supplements) had significantly lower
O/N ratios (e.g., 5-10), which suggests that dietary protein
was being preferentially catabolized. Starved mussels had
O/N ratios below theoretical minimums. probably because
their oxygen consumption had decreased to standard rates
while nitrogen excretion continued at higher rates during
stress (Bayne, 1973a, b; Gabbott and Bayne. 1973). If di-
etary protein content led to both significantly lower growth
rates and higher O/N ratios in mussels fed LP algae com-
pared with HP algae, then the high O/N ratios (e.g., 19)
of mussels at the beginning of the experiment indicate
that growth rates of juvenile M. trossulus in Yaquina Bay,
Oregon, may have been limited by dietary protein bio-
availability at the time they were sampled (May 1992).

C/N ratios of the bioavailable portion of diets (C/N ass
ratios; based on estimated assimilation by mussels) varied

EFFECT OF DIETARY PROTEIN CONTENT ON MUSSELS

137

from 7.1 to 15.0 and were similar to overall C/N ratios
of diets delivered to mussels (C/N Ul , ratios; 6.5 to 15.0).
Hawkins and Bayne (1992) reported that M. edulis re-
quires an average dietary C/N 10 , ratio of 16 in order to
meet its maintenance requirements for carbon and nitro-
gen, and they suggested that C/N, , ratios below 1 6, such
as those typically recorded during algal blooms, may cause
transient nutritional limitation by utilizable carbon. In
this study, however, the growth rates of juvenile M. tros-
sulus were highest at the lowest C/N 101 ratios (e.g., 6.5-
7.5 for Diets 6 and 7) and became progressively lower as
dietary C/N ass ratios increased above 9.9. Similarly, Gal-
lager and Mann (1981) reported that growth of juvenile
clams. Tapes japonica, was greatest when diets had
C/N, ot ratios between 8.4 and 10.5 and that ratios above
10.5 negatively affected growth. Thus, high dietary C/N, ot
ratios (e.g.. > 10- 16) could cause mussel growth to be lim-
ited by available nitrogen and protein in the diet.

Little is known about C/N ratios of natural diets for
suspension-feeding bivalves. In natural seston, these ratios
are likely to vary widely during the year in association
with phytoplankton blooms, storm events, or variation
in allochthonous food sources. Seasonal changes in con-
centrations of paniculate organic material (POM) and
particulate "bioavailable" protein in the seston of Yaquina
Bay, Oregon, were reported by Kreeger (1993), and are
used to estimate dietary C/N ratios for M. trossitliis in
situ. Assuming that 16% of protein is nitrogen and 50%
of POM is carbon, then C/N ratios of seston in Yaquina
Bay are estimated to vary from 6 (during summer) to 17
(during winter), which is about equal to the experimental
range of dietary C/N lot ratios used in the growth experi-
ment. Dietary protein availability could, therefore, affect
growth rates of juvenile M. trossit/its in Yaquina Bay at
certain times of the year, such as during winter when con-
centrations of bioavailable protein are lowest (Kreeger,
1993).

In summary, growth of juvenile M. trossulns can be
significantly affected by variation in dietary protein con-
tent at rations at which mussels are satiated, and possibly
at lower rations as well. Growth rates of mussels in this
study were directly related to dietary protein content (and
the estimated bioavailable, assimilated portion) over the
range 28 to 43% dry w/w, and inversely related to dietary
C/N ratios (and C/N ratios of estimated bioavailable, as-
similated portions) over the range 6.5 to 1 5.0. Cross-linked
walled protein microcapsules were found to be beneficial
dietary supplements for mussels fed on nitrogen-limited,
cultured algae, and they were capable of fully compen-
sating for algal protein deficiencies. Future studies of this
kind will be necessary to ascertain the overall nutritional
requirements of bivalves. This knowledge will contribute
to a better understanding of both the trophic ecology of
natural populations of these animals and the ingredients

necessary to produce balanced, inexpensive diets for bi-
valve aquaculture.

Acknowledgments

We thank A. J. S. Hawkins, K. Y. Kreeger, and B. L.
Bayne for comments on this manuscript. This research
was supported by Oregon Sea Grant, National Oceano-
graphic and Atmospheric Administration, Office of Sea
Grant, Department of Commerce, Grant No. NA 85AA-
D-SG095 under Project R/AQ-56 to C. J. Langdon. Ad-
ditional support was provided to D. A. Kreeger by a
Graduate School Fellowship from Oregon State Univer-
sity, a Brucefield-Reynolds Scholarship from the Hatfield
Marine Science Center, and a Mastin Grant from the De-
partment of Fisheries and Wildlife, Oregon State Uni-
versity.

Literature Cited

Allen, G. 1981. Conversion of cysteine residues to stable derivatives.
Pp. 28-29 in Sequencing oj Proteins and Peplides. North-Holland
Publishing Company, New York.

Allen, K., and .J. Awapara. 1960. Metabolism of sulfur amino acids in
Mytilus edulis and Rangia cuneata. Bio/ Bull 118: 173-182.

Asmus, R. 1986. Nutrient flux in short-term enclosures of intertidal
sand communities. Ophelia 26: 1-18.

Asmus, R. M., and H. Asmus. 1991. Mussel beds: limiting or promoting
phytoplankton?/ Exp. Mar. Biol. Ecoi 148: 215-232.

Baldwin, B. S., and R. I. E. Newell. 1991. Omnivorous feeding by
planktotrophic larvae of the eastern oyster Crassoslrea virginica. Mar.
Ecol Prog. Scr. 78:285-301.

Bayne, B. L. 1973a. Aspects of the metabolism of Mytilus edulis during
starvation. Ni'th. J Sea Res. 7: 399-410.

Bayne, B. L. 1973b. Physiological changes in Mytilus edulis L. induced
by temperature and nutritive stress. /. Mar. Biol. Ass. U. K. 53: 39-
58.

Bayne, B. L., and R. C. Newell. 1983. Physiological energetics of marine
Mollusca. Pp. 407-515 in The Mollusca. Vol. 4, A. S. M. Saleuddin,
and K. M. Wilbur, eds. Academic Press, New York.

Birkbeck, T. H., and J. G. McHenery. 1982. Degradation of bacteria
by Mynlitx edulis Mar Biol 72: 7-15.

Bishop, S. H., I,. L. Ellis, and J. M. Burcham. 1983. Pp. 243-327 in
The Mollusca. Vol. I, P. W. Hochachka. ed. Academic Press, New
York.

Brey, T., H. Rumohr, and S. Ankar. 1988. Energy content of macro-
benthic invertebrates: general conversion factors from weight to en-
ergy. J Exp. Mar. Biol. Ecol. 117: 271-278.

Brown, M. R. 1991. The amino-acid and sugar composition of 16 spe-
cies of microalgae used in mariculture. J. Exp. Mar. Biol. Ecol. 145:
79-99.

Brown, M. R., and S. \V. Jeffrey. 1992. Biochemical composition of
microalgae from the green algal classes Chlorophyceae and Prasi-
nophyceae. I . Ammo acids, sugars and pigments. J. Exp. Mar Biol
Ecol 161: 91-113.

Chu, F., K. L. Webb, D. Hepworth, and M. Roberts. 1982. The ac-
ceptability and digestibility of microcapsules by larvae of Crassostrea
virginica. J Shellfish Res 2: 29-34.

Crosby, M. P., R. I. E. Newell, and C. J. Langdon. 1990. Bacterial
mediation in the utilization of carbon and nitrogen from detntal
complexes by Crassoslrea virginica. Lnnnol. Oceanogr 35(3): 625-
639.

138

D. A. KREEGER AND C. J. LANGDON

Dame, R. F., and N. Dankers. 1988. Uptake and release of materials
by a Wadden Sea mussel bed. / Exr Mur Bio/. Ecol. 118: 207-
216.

Dame, R. F., R. G. Zingmark, and E Haskin. 1984. Oyster reefs as
processors of estuarine materials. ./ Exp Mar- Bio/. Ecol. 83: 239-
247.

Dame, R. F., T. G. Wolaver, and S. M. Libes. 1985. The summer
uptake of nitrogen by an mtertidal oyster reef. Nelh. J. Sea Res 19:
265-268.

Deshimaru, O. 1982. Protein and amino acid nutrition of the prawn
Penaeus japonicus. Pp. 106-123 in Proceedings of the 2nd Interna-
tional Conference on Aauacultttre Nutrition: Biochemical and Phys-
iological Approaches to Shellfish Nutrition, G. Pruder, C. Langdon.
and D. Conklin, eds. Louisiana State University, Baton Rouge, LA.

Douillet, P. A. 1991. Beneficial effects of bacteria on the culture of
larvae of the Pacific oyster Crassostrea gigas (Thunberg). Ph.D. Dis-
sertation, Oregon State University. 185 pp.

Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith.
1956. A colorimetric method for the determination of sugars and
related substances. Anal. Chem. 28: 350-356.

Dugdale, R. C. 1967. Nutrient limitation in the sea: dynamics, iden-
tification, and significance. Limnol. Oceanogr. 12: 685-695.

Enright, C. T., G. F. Newkirk, J. S. Craigie, and J. D. Castell.
1986a. Evaluation of phytoplankton as diets for juvenile Ostrea
eiln/is L. J. Exp. Mar Biol. Ecol. 96: 1-13.

Enright, C. T., G. F. Newkirk, J. S. Craigie, and J. D. Castell.
1986b. Growth of juvenile Ostrea edulis L. fed Chaetoceros gracilus
Schutt of varied chemical composition. J. Exp. Mar. Biol. Ecol 96:
15-26.

Epifanio, C. K. 1979. Growth in bivalve molluscs: nutritional effects
of two or more species of algae in diets fed to the American oyster
Crassostrea virginica (Gmelin) and the hard clam Mercenaria (L.).
A(/iiucullurc 18: 1-12.

Epifanio, C. E. 1982. Phytoplankton and yeast as foods for juvenile
bivalves: a review of research at the University of Delaware. Pp. 292-
304 in Proceedings of the 2nd International Conference on Aquaculture
Nutrition: Biochemical and Physiological Approaches to Shellfish
Nutrition. G. Pruder, C. Langdon, and D. Conklin, eds. Louisiana
State University, Baton Rouge, LA.

Ewart, J. W., and C. E. Epifanio. 1981. A tropical flagellate food for
larval and juvenile oysters, Crassostrea virginica Gmelin. Aquaculture
22: 297-300.

Fabregas, J.,C. Herrero, B.Cabezas,and J. Abalde. 1985. Massculture
and biochemical variability of the marine microalga Tetraselmis
suecica Kylin (Butch.) with high nutrient concentration. Aquaculture
49: 231-244.

Fabregas, J., C. Herrero, B. Cabezas, and J. Abalde. 1986. Biomass
production and biochemical composition in mass cultures of the
marine microaiga Isochrysis galbana Parke at varying nutrient con-
centrations. Aquaculture 53: 101-1 13.

Fenchel, T. 1972. Aspects of decomposer food chains in marine benthos.
Sond. I<T/) Deulx Zool Ges. 65: 14-22.

Fernandez-Reiriz, M. J., A. Perez-Camacho, M. J. Ferreiro, J. Blanco,
M. Planas, M. J. Campos, and I', Labarta. 1989. Biomass pro-
duction and variation in the biochemical profile (total protein, car-
bohydrates. RNA, lipids and fatty acids) of seven species of marine
microalgae. Aquaculture 83: 17-37.

Flaak, A. R., and C. E. Epifanio. 1978. Dietary' protein levels and growth
ol the oyster Crassostrea virginica. Mar. Biol. 45: 157-163.

Folch, J., M. Lees, and G. H. Sloane-Slanley. 1957. A simple method
for the isolation and purification of total lipids from animal tissue.
J. Biol. Chem 226: 497-509.

Gabbott, P. A., and B. L. Bayne. 1973. Biochemical effects of tempera-
ture and nutritive stress on Mvlilus edulis L. / Mar. Biol. Ass. U.K.
53: 269-286.

Gabbott, P. A., D. A. Jones, and D. H. Nicols. 1976. Studies on the
design and acceptability olmicroencapsulated diets tor marine particle
feeders. II. Bivalve molluscs. Pp. 127-141 in Proceedings of the 10th
European Marine Biology Symposium, G. Personne and E. Jaspers,
eds. Universa Press, Wetteren.

Gallager, S. M., and R. Mann. 1981. The effect of varying carbon/
nitrogen ratio in the phytoplankter Thallassiosira pseudonana (3H)
on its food value to the bivalve Tapes japonica. Aquaculture 26: 95-
105.

Guillard, R. R. L., and J. Ryther. 1962. Studies of marine planktonic
diatoms. I. Cyclotella nana and Delonula confervacea. Can. J. Micro-
biol. 8: 229-239.

Harrison, C. 1975. The essential amino acids ofMytilus californianus.
I'eligerlS: 189-193.

Hawkins, A. J. S., and B. L. Bayne. 1991. Nutrition of marine mussels:
factors influencing the relative utilizations of protein and energy.
A(iitaciillitrc94: 177-196.

Hawkins, A. J. S., and B. L. Bayne. 1992. Physiological interrelations,
and the regulation of production. Pp. 171 -222 in The Mussel Mytilus:
Ecology. Physiology. Genetics and Culture. E. Gosling, ed. Elsevier,
London.

Hicks, R. E., C. I.ee, and A. C. Marinucci. 1991. Loss and recycling
of amino acids and protein from smooth cordgrass (Spartina aller-
ni/lnra) litter. Estuaries 14: 430-439.

Jordan, T. E., and I. Yaliela. 1982. A nitrogen budget of the ribbed
mussel, Geukensia demissa, and its significance in nitrogen flow in
a New England salt marsh. Limnol. Occanogr. 27: 75-90.

Kanazawa, A. 1982. Penaeid nutrition. Pp. 87-105 in Proceedings of
the 2nd International Conference on Aquaculture Nutrition: Bio-
chemical and Physiological Approaches to Shellfish Nutrition. G.
Pruder, C. Langdon, and D. Conklin, eds. Louisiana State University,
Baton Rouge. LA.

Kautsk\ N., and S. Evans. 1987. Role of biodeposition by Mytilus
edulis in the circulation of matter and nutrients in a Baltic coastal
ecosystem. Mar. Ecol. Prog Ser. 38: 201-212.

Kreeger, D. A. 1992. Utilization of dietary protein by the mussel, My-
lilus edulis trossiilus Ph.D. Dissertation, Oregon State University.
209 pp.

Kreeger, D. A. 1993. Seasonal patterns in the utilization of dietary
protein by the mussel Mytilus trossuliis. Mar. Ecol. Prog. Ser. (in
press).

Langdon, C. J. 1982. New techniques and their application to studies
of bivalve nutrition. Pp. 305-320 in Proceedings of the 2nd Inter-
national Conference on Aquaculture Nutrition: Biochemical and
Physiological Approaches to Shellfish Nutrition. G. Pruder, C. Lang-
don, and D. Conklin, eds. Louisiana State University, Baton Rouge,
LA.

Langdon, C. J. 1989. Preparation and evaluation of protein microcap-
sules for a marine suspension-feeder, the Pacific oyster Crassostrea
gigas Mar. Biol 102: 217-224.

Langdon, C. J., and E. T. Bolton. 1984. A microparticulate diet for a
suspension-feeding bivalve mollusc. Crassostrea virginica. J Exp.
Mar. Biol. Ecol 353: 1-21.

Langdon, C. J., and E. A. DeBevoise. 1990. Effect of microcapsule
type on delivery of dietary protein to a marine suspension-feeder,
the oyster Crassoslrea gigas. Mar. Biol. 104: 437-443.

Langdon, C. J., and R. I. E. Newell. 1990. Utilization of detritus and
bacteria as food sources by two bivalve suspension-feeders, the oyster

EFFECT OF DIETARY PROTEIN CONTENT ON MUSSELS

139

Cru\\o\irt-a virgiinca and the mussel Geukensiu demissa. Mar, Ecol.
. Ser. 58:299-310.

ii. ('. J.. and R. I. E. Newell. 1992. Digestion and nutrition.
Chapter h in The Eastern Oyster. Crassostrea virginica. A. Eble.
V. S. Kennedy, and R. I. E. Newell, eds. Maryland Sea Grant Pub-
lication (in press).

I.angdon. C. J., and C. A. Siegfried. 198-1. Progress in the development
of artificial diets for bivalve suspension-feeders. . Iquacullure 39: 1 35-
153.

Langdon, C. J., and M. J. Waldock. 1981. The effect of algal and
artificial diets on the growth and fatty acid composition of Crassostrea
gigasspm. / Mar. Biol Ass. L'.K. 61: 431-448.

Langton, R. \\ '., J. E. Winter, and O. A. Roels. 1977. The effect of
ration size on growth and growth efficiency of the bivalve mollusc
Tapes laponica Aquaculture 12: 283-292.
Lehninger, A. L. 1976. Biochemistry, 2nd ed. Worth. New York.

1104 pp.

Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. .1. Randall.
1951. Protein measurement with the folin phenol reagent. J. Biol.
Chem 193: 265-275.
Mann, K. H. 1982. Ecology of Coastal Waters, a Systems Approach

Blackwell. Oxford.

Martin-Jezequel, \ ., S. A. Poulet, R. P. Harris, J. Moal, and J. F.
Samain. 1988. Interspecific and intraspecific composition and
variation of free amino acids in marine phytoplankton. Mar. Ecol.
Prog Ser 44: 303-313.

Newell, R. 1965. The role of detritus in the nutrition of two marine
deposit feeders, the prosobranch Hydrobia u/vaeand the bivalve Ma-
coma balthica. Proc, Zool. Soc. Land. 144: 25-45.
Newell, R. C. 1979. Biology of Intertidal Animals. Marine Ecological

Surveys, Faversham, U. K., 781 pp.

Newell, R. I. E., and C. J. Langdon. 1992. Mechanism and physiology
of larval and adult feeding. Chapter 5 in The Eastern Oyster, Cras-
sostrea virginica. A. Eble, V. S. Kennedy, and R. I. E. Newell, eds.
Maryland Sea Grant Publication, (in press).
Phillips, A. M., and D. R. Brockway. 1956. The nutrition of trout. II.

Protein and carbohydrate. Prog. Fish-Cult 18: 159-164.
Pruder, G. D., and E. T. Bolton. 1979. The role of CO : enrichment of
aerating gas in the growth of an estuarine diatom. Aquaculture 17:
1-15.

Rice. D. L. 1982. The detritus nitrogen problem: new observations and
perspectives from organic geochemistry. Mar. Ecol. Prog. Ser. 9: 153-
162.

Rice, D. L., T. S. Bianchi. and E. H. Roper. 1986. Uptake and internal
distribution of exogenously supplied amino acids in the Pacific oyster,
Crassosirea gigas (Thunberg). Aquaculture 66: 19-3 1 .
Riisgard, H. U. 1988. Efficiency of particle retention and filtration rate
in 6 species of northeast American bivalves. Mar. Ecol. Prog. Ser.
45:217-223.

Riley, G. A. 1972. Patterns of production in marine ecosystems. Pp.
9 1 - 1 1 2 in Ecosystem Structure and Function, J. A. Wiens. ed. Oregon
State University Press. Corvallis, OR.

Roman, M. R. 1983. Nitrogenous nutrition of marine invertebrates.
Pp. 347-384 in Mlmgen in the Marine Environment, E. J. Carpenter
and D. G. Capone. eds. Academic Press, New York.

Sakshaug, E., and O. Ilolni-IIansen. 1977. Chemical composition of
Skeletonema cositinim (Grev.) Cleve and Pavlova (Monochrysis)
lutheri (Droop) Green as a function of nitrate-, phosphate-, and iron-
limited growth. J E\p. Mar. Biol. Ecol. 29: 1-34.

Sieberth, J. M. 1976. Bacterial substrates and productivity in marine
ecosystems. Ann. Rev Ecol. Syst. 1: 259-285.

Sokal, R. R., and E. J. Rohlf. 1969. Biometry. W. H. Freeman, San
Francisco.

Solorzano, L. 1969. Determination of ammonia in natural waters by
the phenolhypochlorite method. Linmal. Oceanogr. 14: 799.

Tenore, K. R. 1977. Food chain pathways in detrital feeding benthic
communities: a review, with new observations on sediment resus-
pension and detrital recycling. Pp. 37-53 in Ecology of Marine Ben-
thos. B. C. Coull, ed. University of South Carolina Press.

Tenore, K. R., and E. J. Chesney, Jr. 1985. The effects of interaction
of rate of food supply and population density on the bioenergetics
of the opportunistic polychaete, Capitella capilata (type 1). Limnol.
Oceanogr. 30: 1188-1195.

Thompson, P. A., M. E. Levasseur, and P. J. Harrison. 1989. Light-
limited growth on ammonium vs. nitrate: what is the advantage for
marine phytoplankton? Limnol. Oceanogr. 34: 1014-1024.

Thompson, P. A., P. J. Harrison, and J. N. C. Whyte. 1990. The in-
fluence of irradiance on the fatty acid composition of phytoplankton.
J. Phycol. 26: 278-288.

Utting, S. D. 1985. Influence of nitrogen availability on the biochemical
composition of three unicellular marine algae of commercial im-
portance. Aquacult. Eng. 4: 175-190.

Utting, S. D. 1986. A preliminary study on growth of Crassostrea gigas
larvae and spat in relation to dietary protein. Aquaculture 56: 123-
138.

Webb, K. L., and F-L. E. Chu. 1982. Phytoplankton as a food source
for bivalve larvae. Pp. 272-291 in Proceedings of the 2nd International
Conference on Aquaculture Nutrition: Biochemical and Physiological
Approaches to Shellfish Nutrition. G. Pruder, C. Langdon. and
D. Conklm, eds. Louisiana State University. Baton Rouge. LA.
Whyte, J. N. C. 1987. Biochemical composition and energy content
of six species of phytoplankton used in manculture of bivalves.
Aauaeitlture60: 231-241.

Whyte, J. N. C., N. Bourne, and C. A. Hodgson. 1989. Influence of
algal diets on biochemical composition and energy reserves in Pa-
nnopecten yessoensis (Jay) larvae. Aquaculture 78: 333-347.
Wilson, R. P. 1989. Amino acids and proteins. Pp. 1 12-148 in Fish

Nutrition. J. E. Halver. ed. Academic Press. New York.
Wright, R. T., R. B. Coffin, C. P. Ersing, and D. Pearson. 1982. Field
and laboratory measurements of bivalve filtration of natural marine
bacterioplankton. Limnol. Oceanogr 27: 91-98.
Zamer, W. E., J. M. Shick, and D. W. Tapley. 1989. Protein mea-
surement and energetic considerations: comparisons of biochemical
and stoichiometric methods using bovine serum albumin and protein
isolated from sea anemones. Limnol. Oceanogr. 34: 256-263.

Reference: Biol. Bull 185: 140-148. (August, 1993)

Hemolymph Insulin-Like Peptides (ILP) Titers and

the Influence of ILP and Mammalian Insulin on the

Amino Acid Incorporation in the Mantle Collar

In Vitro in Helisoma (Mollusca)

V. M. SEVALA, V. L. SEVALA, AND A. S. M. SALEUDDIN
Department of Biology, York University, North York, Ontario, Canada M3J 1P3

Abstract. In Helisoma ditryi. the periostracum is the
outermost organic layer of the shell and it is secreted by
the mantle collar. Addition of porcine insulin (0. 1 Mg/ m O
to the incubation medium increases the incorporation of
labeled amino acids in the mantle collar in vitro. The
immunoblotting technique revealed two immunoreactive
insulin bands with a molecular weight of 16 and 7 kDa
in the hemolymph. Partial purification of insulin-like
peptides from the hemolymph by gel filtration chroma-
tography showed that only one fraction containing ap-
proximately 7 kDa polypeptide stimulated the incorpo-
ration of amino acids into the mantle collar as well as
into the periostracum in a dose-dependent manner. In
laboratory populations of Helisoma, snails with two dif-
ferent shell growth rates can be recognized: fast and slow.
Hemolymph titers of insulin-like peptide are low in fast-
growing snails (2.3 0.25 mlU/ml) and higher in slow-
growing snails (7.8 0.46 mlU/ml). When a piece of
shell at the edge is removed, a structurally identical new
piece is formed within either two days (fast regeneration)
or a longer period of seven days (slow regeneration). He-
molymph titers of insulin-like peptide undergo fluctua-
tions during the period of shell regeneration, but a general
pattern can be recognized. The titers are low when the
shell deposition rate is high and vice versa. We suggest
that the insulin-like peptide in the hemolymph is involved
in shell growth or shell regeneration.

Introduction

Insulin-like peptides (ILP) are found in a number of
invertebrate species from different phyla (Thorpe and

Received 18 May 1992; accepted 4 May 1993.

Duve, 1984). In several members of the phylum Mollusca,
ILP have been detected in the cells belonging to the diges-
tive system and in the central nervous system (CNS).
Many similarities exist between molluscan ILP and
mammalian insulin at both the structural and functional
levels (Ebberink et ai, 1989).

In the freshwater pulmonate Lymnaea stagnalis, three
genes encoding molluscan insulin-related peptides MIP I,
MIP II, and MIP V have been sequenced (Smit et ai,
1988. 1991, 1992). The in situ hybridization technique
using cDNA has shown that the neurosecretory light green
cells (LGC) contained the mRNA encoding these peptides
(van Minnen and Schallig, 1990; Smit et al, 1991). In
this species, LGC regulate shell and body growth (Geraerts,
1976).

The neurosecretory mediodorsal cells (MDC) of fresh-
water snails Helisoma spp. (H. duryi, H. tenne, and
H. thvolvis) are homologous to the LGC of Lymnaea,
and are also involved in shell growth (Saleuddin and Ku-
nigelis, 1984). Using porcine and human insulin antisera,
immunoreactive ILP have been detected in the MDC and
hemolymph of Helisoma (Saleuddin et ai. 1991; Khan
et ai. 1992). Fine structural studies suggest that the syn-
thetic activity of the MDC, and the rate of release profiles
of neurosecretory granules of the MDC axon terminals,
reflect the growth rates of the animal (Khan and Saleud-
din, 1992).

Among laboratory populations of Helisoma, snails with
two distinct shell growth rates can be found: fast and slow.
Also present are the non-growers whose shell increments
are extremely small. A brain extract from a fast-growing
snail contains a factor that will stimulate shell growth in
slow-growing snails (Kunigelis and Saleuddin, 1978).

140

INSULIN-LIKE PEPTIDE TITERS IN HEL1SOMA

141

The organic periostracum is the pliable outermost layer
of the molluscan calcareous shell. Its formation must pre-
cede the formation of the calcareous layer. The mantle
edge, which secretes the periostracum, can be cultured in
vitro. A brain-derived factor increases incorporation of
labeled amino acids into the periostracum in vitro (Ku-
nigelis and Saleuddin, 1985).

In Helisoma when a piece of shell is removed from the
edge, a new piece is secreted filling the damaged area
(Wong and Saleuddin, 1972; Kunigelis and Saleuddin,
1983). Snails undergoing shell regeneration, where rapid
transport of calcium and proteins is involved, are suitable
for monitoring the hormones involved in shell formation
and shell regeneration.

In the present experiments, ILP liters in the hemolymph
in snails of known growth rates and in shell-regenerating
snails were measured by a radioimmunoassay using hu-
man insulin antiserum. The effects of porcine insulin and
Helisoma ILP on the amino acid incorporation into peri-
ostracum by the mantle collar in vitro were also studied.

Materials and Methods

Animals

Specimens of H. duryi were raised in 70-1 glass aquaria
in dechlorinated tap water at 22C under a 1 2L: 1 2D pho-
toperiod. Boiled lettuce, fish-food, and blackboard chalk
(source of calcium) were supplied as food ad libitum. Adult
snails, 6-8-month-old and 10-12 mm shell diameter, were
used for studies.

Shell growth monitoring

Adult snails were marked individually with an identi-
fying number, and a reference line parallel to the growing
edge was painted on the shell using a water-soluble poly-
mer acrylic paint with a very fine artist's paint brush. The
number and reference line were dried and coated with
lacquer containing nylon (nail polish). Shell growth was
measured optically as the distance between the reference
line and the growing edge using a calibrated micrometer
on a Wild M5 stereomicroscope. The shell growth was
monitored daily. Based on daily growth increments, the
rate of shell deposition was calculated, representing the
"net daily increase" in the "mean shell area" deposited
and expressed as mean daily linear shell deposition rate
(MDLSDR). These snails were grouped into fast growers,
slow growers, and non-growers based on the growth rates
as outlined by Kunigelis and Saleuddin, 1978.

Shell damage

Snails with known shell growth rates were used. A piece
of shell about 4X4 mm was carefully removed at the
growing edge of a snail, using a fine rotating blade (Dremel

Tools, Wisconsin), without injuring the underlying mantle
tissue. After the shell was damaged, shell regeneration was
monitored every day for two weeks. Shell growth ceased
until shell regeneration was completed. Previous studies
showed that the mantle collars of Helisoma cultured in
vitro synthesized the periostracum. In this experiment,
the incorporation of tritium-labeled amino acids into the
mantle collar and periostracal proteins was measured both
in the presence and absence of insulin.

Periostracum synthesis in vitro

The mantle collars were dissected from snails of known
growth rates under aseptic conditions in physiological sa-
line (40 mM NaCl, 3 mM KC1, 3 mM CaCl : , 1 mM
MgCU, 14 mM NaHCO,, 0.2 mM NaH 2 PO 4 ), pH 7.2,
osmolality 1 15 mOsm/1, containing antibiotics and an
antimycotic [100 ILJ/ml penicillin and streptomycin and
0.25 Mg/ml fungizone (GIBCO, Canada)] and cultured in
vitro according to the procedure of Kunigelis and Saleud-
din (1985). The culture medium contained 55 parts sterile
distilled water and 45 parts Medium 199 (GIBCO) con-
taining 25 mM Hepes buffer, Hank's salts, and L-gluta-
mine, osmolality 140 mOsm/1 and pH 7.1 A tritiated
amino acid mixture (leucine, lysine, phenylalanine, pro-
line, and tyrosine; specific activity 57 Ci/mmol) (Amer-
sham, Canada) was used at a concentration of 1 nCi/m\
culture medium. The culture medium was filtered through
0.2 nm sterile disposable filter units and subsequently 100
lU/ml penicillin and streptomycin and 0.25 Mg/ml fun-
gizone were added. A mantle collar was placed dorsal sur-
face uppermost in 1 .0 ml medium in each well of a sterile
24-well dish (Costar, Cambridge, Massachusetts) at 22C.
In some experiments porcine insulin (0.01-2.0 Mg/ml) was
added to the culture medium. After 48 hours of incuba-
tion, the mantle collars formed periostracum which was
collected with a glass pipette. The mantle collar and peri-
ostracum were homogenized separately in 100 n\ and
200 n\ distilled water, respectively. Aliquots of 25 n\ of
homogenate were spotted onto squares of filter paper
(2X2 cm; Whatman No. 42) and allowed to dry at room
temperature. The amount of label incorporation into
proteins was determined by the method of Bramhall et
al. (1969). Briefly, proteins were precipitated by soaking
in 7.5% chilled trichloroacetic acid and heated to 80C
for 30 min to remove non-proteinaceous material. The
TCA was removed by rinsing in 50% ethanol in diethyl
ether followed by diethyl ether, and the papers were air
dried before transferring to a vial containing 10 ml of
scintillation cocktail (ACS) (Amersham, Canada). The
vials were dark-adapted and counted in a Packard A3000
Scintillation counter. Subsequently, the papers were
washed in acetone, air dried, and the protein present on
these filter papers determined by using the method of

142

V. M. SEVALA ET AL.

Bramhall et at. (1969). Incorporation of amino acid was
expressed as DPM per microgram protein.

Hemolymph collection and e\ action

Snails were wiped and al! traces of mucus were removed
using absorbent tissue. They were then bled on a piece of
parafilm by puncturing the heart with a No. 10 surgical
blade. About 100 /ul hemolymph was collected in 1.5 ml
microcentrifuge tubes from each snail. Hemolymph sam-
ples were centrifuged at 10,000 X g for 10 min and su-
pernatants were stored at -80C until use.

Gel filtration

Hemolymph was subjected to gel nitration G-75 (Phar-
macia; Particle size 40-120 /urn) column of 75 X 2 cm
equilibrated with 0.06 M phosphate buffer (pH 7) at a
flow rate of 0.3 ml/min. The column was calibrated with
proteins of known molecular weight. Approximately 5 ml
of hemolymph from snails of various growth rates was
lyophilized and reconstituted with 2 ml of buffer, then
loaded onto the column. The elution of protein was mon-
itored at 280 nm. Fractions (3.5 ml) were collected, 500 /zl
of each fraction was vacuum dried and subjected to RIA
for the presence of insulin-like peptides.

Bioassay

The partially purified hemolymph fractions from the
gel filtration column containing ILPs (approximately 17,
7, and 2 kDa) were lyophilized and tested in vitro for
possible stimulation of incorporation of labeled amino
acids into TCA precipitable mantle collar and periostracal
proteins. The mantle collars were cultured in vitro with
or without insulin-like peptides (250 yuIU equivalents/ml).
After 48 h of incubation, mantle collars and periostra-
cum were removed, homogenized separately in 200 /ul and
100 /il of distilled water, respectively. Aliquots were spot-
ted on filter papers and processed according to the pro-
cedure of Bramhall et al. ( 1 969), as described earlier. Fur-
thermore, various doses (0, 100, 250, 500 ^IU equivalents/
ml) of the 7-kDa insulin immunoreactive fraction were
also tested /// vitro for the stimulation of 3 [H]-labeled
amino acid incorporation into mantle and periostracal
proteins.

Radioiinmunoassay

The hemolymph tilers of ILP were determined with a
commercially available kit that uses antiserum against
human insulin (Diagnostic Products, Los Angeles, Cali-
fornia). The assay is based on competitive binding of ILP
present in the samples and I25 l-labeled insulin. Immobi-
lized insulin antibody is supplied in polypropylene tubes.
In each of these tubes, 100 /ul hemolymph sample and

500 n\ labeled insulin (0.03 ^Ci/ml) were incubated for
18 h at 22C. After being carefully decanted, the tubes
were counted for 1 min in 10 ml ACS in a Packard A300
Scintillation Counter. The amount otTLP present in sam-
ples was determined by comparing the observed counts
to a standard curve. The limit of detection of insulin was
1 /uIU using this procedure. A series of hemolymph and
tissue extracts were prepared to compare the competition
curve of human insulin with that of the Helisoma ILP.
The dilution curves for snail ILP ran parallel to the human
insulin standard curve (Fig. 1 ). Thus the relative concen-
tration of Helisoma ILP can be determined with this assay.
Because we do not know the absolute amount of Helisoma
ILP in any sample, the tilers are expressed as mlU
equivalents of insulin.

Immunoblotting

The proleins presenl in Ihe hemolymph and brain ex-
Iracls were analyzed by sodium dodecyl sulphale gradienl
(1 1-23%) polyacrylamide gel eleclrophoresis employing
Ihe "phorcasl" gel syslem (Amersham, Canada). Al Ihe
end of Ihe eleclrophorelic run, Ihe proteins were trans-
ferred lo nilrocellulose (0.2 nm) (Bio-Rad, Canada) by
using Bio-Rad mini Irans blol eleclrophoresis cell al 4C
for 16 h wilh a constant current of 40 mA. The transfer
buffer consisled of 25 mA/Tris pH 8.3, 192 mA/glycine,
20% melhanol. Immedialely following the transfer, the
blots were placed in a phosphale buffer saline, pH 7.4,
conlaining 5% bovine serum albumin and 0.05% Tween
20 (PBST) for 45 min al 22C. The membranes were Ihen

Human insulir
Hemolymph

Micro units immuno reactive insulin/ml

Figure 1. Standard curve showing competitive inhibition of I25 l-in-
sulm binding to anti-insulin with unlabeled human insulin (5, 15, 50,
100, 200, and 400 //III) and with samples of hemolymph (5, 10, 25, 50,
100, and 200 /jl) collected from fast-growing snails and adjusted to 0.1
ml prior to the assay. The amount of insulin present in the hemolymph
samples was determined from the insulin standard curve.

INSULIN-LIKE PEPT1DE TITERS IN HELISOMA

143

incubated with bovine insulin antibody raised in guinea
pigs (Sigma Chemical Co.) diluted 1: 1000 for 1 h, rinsed
three times in PBST, followed by incubation with a sec-
ondary antibody (anti-guinea pig IgG) conjugated to al-
kaline phosphatase (1:1000 dilution). The membranes
were washed three times in PBST. Finally, the enzyme
was localized by 0.033% nitro blue tetrazolium with
0.0165% 5-bromo-4-chloro-3-indolyl-phosphate (Pro-
mega. Wisconsin) as a substrate. The presence of an in-
sulin-like molecule was indicated by blue bands on the
membrane. The specificity of the antisera used was verified
by appropriate controls involving ( 1 ) the omission of the
primary antiserum. (2) the omission of the secondary
antiserum, (3) preincubation of the primary antiserum
with bovine insulin, and (4) replacement of the primary
antiserum with pre-immune rabbit serum. Under all these
control treatments no immunoreactive material was de-
tected on the nitrocellulose membrane.

Statistical analysis

The results were tested for significance using ANOVA
and the difference between means was also tested by Stu-
dents Mest as described by Sokal and Rohlf ( 1973).

Results

Effect of insulin on protein synthesis in mantle

The influence of porcine insulin on periostracum for-
mation by the mantle collar in vitro was determined by
assaying the rate of incorporation of a mixture of tritiated

O MDLSDR
H ILP

Insulin ug/ml

Figure 2. Dose response curve showing the in vitro incorporation of
'[H]-labeled amino acids into TCA precipitahle protein of the mantle
collar of Helisoma. The mantle collars from snails of known growth
rates were dissected and cultured in the medium with and without the
indicated dose of insulin for 48 h. The mantle collars were processed
(see Methods) to measure the amount of label incorporation into mantle
collar protein and the results shown are pooled data (mean SEM) from
three experiments. For each experiment at least 9-1 1 controls and 9-1 1
treated mantle collars per insulin dose were assayed.

a:
a
oo

T

a.

_>,

o

_

E

Q.

Non growers

Slow

Fast

Figure 3. A comparison of ILP tilers and mean daily linear shell
deposition rates (MDLSDR) in non-growing, slow, and fast growing snails.
The snails were monitored for shell deposition over a 3-4 week period.
Based on daily growth increments, the rate of shell deposition was cal-
culated, which represents the "net daily increase" in the "mean shell
area" deposited and expressed as mean daily linear shell deposition rate
(MDLSDR) (mm/day). The hemolymph samples were collected from
snails after determining their shell growth rates and were used for assaying
the ILP tilers. The ILP tilers are significantly lower in fast growing snails
than in non-growing and slow-growing snails (**. P < 0.01). Columns
indicate the mean of n = 15, bars the SEM from three separate experi-
ments.

amino acids in the mantle collar. At a concentration of
0.1 Mg/ml of insulin, the rate of amino acid incorporation
was two-fold higher than at 0.01 Mg/ml (Fig. 2).

ILP liters and shell growth

Daily shell deposition rates have been compared with
ILP liters of the hemolymph (Fig. 3). ILP liters are sig-
nificantly lower in fast-growing snails than in nongrowing
and slow-growing snails (P < 0.01). However, the ILP
tilers of slow and nongrowing snails did not differ signif-
icantly (P > 0.5).

Rate of shell regeneration

When a piece of shell edge is removed the damaged
area is rapidly replaced by a piece of regenerated shell
which is first visible 6-8 h following shell damage. Shell
regeneration is restricted to the damaged area only. The
rale of shell regeneration varied among regenerating snails.
Snails thai had repaired more lhan 50% of the damaged
shell within 24 h were classified as fast-regeneraling snails,
while Ihose lhal took longer than 24 h were slow regen-

144

V. M. SEVALA ET AL

erating snails. Normally, fast regenerating snails com-
pleted shell repair in two days, while slow regenerating
snails took up to seven days to complete shell regeneration
(Fig. 4).

ILP liters during shell regeneration

ILP liters in the hemolymph of both fast and slow re-
generating snails fluctuated considerably not only during
the period of shell regeneration but also over the 1 4-day
study period. In fast regenerating snails, the ILP liter dur-
ing the initial phase of regeneration (first 17 h) was about
3 mlU/ml of hemolymph. This level was mainlained unlil
day 3 when Ihe liter dropped lo aboul 2 mlU/ml of he-
molymph. This decrease was followed laler (after day 9)
by a significant increase to 6 mlU/ml al day 14 after the
shell growlh had virtually stopped (Fig. 5).

In slow regeneraling snails, ILP lilers showed a differenl
profile. In Ihese snails al Ihe inilial phase of regeneralion
(17 h), the ILP tiler was significantly higher than in fast
regeneraling snails (P < 0.01 ). However, Ihe ILP liler de-
creased as Ihe rale of shell regeneralion acceleraled. The
ILP tilers Ihen increased, coincidenlally wilh Ihe fall of
shell regeneralion rales (Fig. 5). The ILP tilers did not
significantly differ belween slow and fasl regenerating
snails from 1 1 days after injury, and al day 14 Ihe ILP

.c

Q.

_>,

o

V

E

Q.

10 11 12 13 14

Days after injury

Figure 5. ILP liters following shell injury in the snails with known
shell growth rates. The snails were monitored over a 3-4 week period
and MDLSDR were calculated. A shell area of approximately 4X4 mm
was carefully removed from these snails at time zero. Following this
removal, shell regeneration and the resumption of normal shell growth
were monitored daily for 2 weeks. Hemolymph samples from these snails
were collected at different times following shell removal and assayed by
RIA to determine the ILP tilers. Each individual was sampled only once
during the experiment and results shown indicate the mean of n = 16,
bars indicate the SEM from three experiments. The liter in slow-regen-
erating animals is significantly higher lhan in fasl-regeneraling animals
(**, P < 0.01 ) excepl on days 2 and 1 3 after shell removal.

Growth al new edge
(Fasti

Days after shell injury

Figure 4. MDLSDR in fasl- and slow-growing snails. The shell growth
in snails was monitored for 3-4 weeks to establish shell deposilion rales.
Following shell growth rate delermination, the shell was carefully injured
at lime zero. These snails were further monilored for 2 weeks to determine
shell regeneration of injured area and the resumplion of normal growth
al Ihe edge. Based on daily growlh incremenls, Ihe rale of shell deposilion
was calculated, which represents Ihe "nel daily increase" in the "mean
shell area" deposiled and expressed as mean daily linear shell deposilion
rate (MDLSDR). Results indicate the mean of n = 16 from three ex-
periments. Arrows indicale Ihe lime of complelion of shell regeneralion.
Shell growlh al the new edge resumes beyond ihis poinl. Nole lhal for
slow-growing snails Ihe rate of shell regeneration is nol only slower, bul
no new shell growlh occurred following the completion of regeneralion.

liler relurned lo Ihe inilial level of aboul 7 mlU/ml of
hemolymph.

Immunoblotting

Immunoblols prepared by SDS-PAGE from the he-
molymph samples using bovine anli-insulin showed Iwo
immunoreaclive ILP bands whose apparenl molecular
weighls were 1 6 and 7 kDa, as delermined by comparison
wilh Ihe mobilily of known molecular weighl markers
(Fig. 6).

Gel filtration

Three peaks of insulin-like immunoreaclive-peplide-
conlaining fraclions were idenlified from Ihe eluales of
gel fillration as detected by RIA. The apparent molecular
weights of Ihe peptides were estimated by calibrating the
column with slandards of known molecular weighls and
were found lo be 17, 7, and 2 kDa from Ihe hemolymph
(Fig. 7). The fraclions conlaining Ihese peplides were
pooled and lested for Iheir abilily lo slimulale Ihe incor-
poration of Irilialed amino acids inlo TCA precipilable
proteins of the mantle collar and periostracum. The dala
presented in Figures 8 and 9 show lhal only one fraclion
conlaining ILP of 7 kDa significanlly slimulaled Ihe

INSULIN-LIKE PEPTIDE TITERS IN HEL1SOMA

145

46K

30 K

21-5K

14-3 K

- 6-5K

- 3-4K

Figure 6. Immunoblot of SDS-PAGE of hemolymph from snails
(left lane). The hemolymph samples from a mixed population of snails
of known growth rates were subjected to SDS-gel electrophoresis and
the fractionated proteins were subsequently transferred to nitrocellulose
membrane. The membranes were incubated with the primary antiserum
(anti-bovine insulin, 1 : 1000 dilution) for I h, rinsed in phosphate buffered
saline, pH 7.4, containing 5% bovine serum albumin and 0.05% Tween
20 (PBST) followed by secondary antiserum conjugated to alkaline
phosphatasc (l:IOOO dilution). The membranes were washed and the
sites of IgG binding was visualized with nitro blue tetrazolium and
5-bromo-4-chloro-3-indolyl phosphate. Arrows indicate ILPs (16 and
7 kDa) in llcli.wma hemolymph. The molecular weights of the insulin
immunoreactive bands were estimated by running the following known
molecular weight protein standards (Amersham) on the same gel (right
lane): Ovalbumin 46000, carbonic anhydrase 30000, trypsin inhibitor
21500, lysozyme 14300, aprotinin 6500. and insulin (b) chain 3400.

incorporation of labeled amino acids into mantle collar
and periostrical proteins (P < 0.05; P < 0.0 1 , respectively).
Furthermore, Figures 10 and 1 1 demonstrate that addition

of the 7-kDa-containing fraction to the culture medium
increases incorporation of tritiated amino acids into TCA
precipitable proteins of mantle collar and periostracum
in a dose-dependent manner up to a dose of 250 ^ILI/ml.

Discussion

In many mollusks, shell growth is incremental and is
influenced by various internal and external factors (Lutz
and Rhoads, 1980). The molluscan shell, which includes
an organic outer periostracum, is secreted by the mantle
collar. Saleuddin and Kunigelis (1984) showed that when
the mantle collar was maintained in organ culture, the
periostracum formed in vitro was structurally similar to
that formed in vivo. A crude extract of CNS from a fast
growing snail stimulated in vitro periostracum formation
and increased amino acid incorporation into the mantle
collar of a slow-growing snail. In this study, addition of
up to 0. 1 /ig/ml porcine insulin to the incubation medium
stimulated amino acid incorporation into the mantle col-
lar. Higher concentrations of insulin were not stimulatory,
however; inhibitory effects of higher doses of insulin on
protein synthesis are not uncommon (de Pablo et ai,
1985). Because this study did not specifically include the
effect of insulin either on the incorporation of amino acid
in the periostracum or on the periostracum formation

o

Ve/Vo

Figure 7. Molecular weight determinations of ILP by gel nitration.
A mixture of standard proteins was applied to G-75 column (2 x 75
cm) and eluted at 0.3 ml/min with 0.06 A/ phosphate buffer. Five ml of
hemolymph collected from a mixed population of (80) snails of known
growth rates was extracted, lyophilized (see Methods), and reconstituted
with 2 ml of buffer, and applied onto the column. ILPs were eluted with
phosphate buffer. Fractions (3.5 ml) were collected and 500 n\ of each
fraction was vacuum dried, reconstituted with 100 n\ of distilled water,
and then subjected to RIA for the presence of insulin-like peptides. The
following standard proteins were used to calibrate the column: ovalbumin
46000. myoglobin 17000, aprotinin 6500, and vitamin Bl 2 UOOdalton.
The arrows indicate the Ve/Vo (elution volume/void volume) ratios for
ILPs in the hemolymph of Helisoma. The fractions containing the ILPs
were further tested in vitro for the stimulation of incorporation of '[H]-
labeled amino acids into TCA precipitable mantle collar and periostracal
proteins of Helisoma.

146

V. M. SEVALA ET AL

Q.

1500

Q

T

EZ] Control
S 17 kDa
[SJ 7kDa
CD 2 kDa

Fractions

Figure 8. The effect of partially purified hemolymph insulin im-
munoreactive fractions on amino acid incorporation into the TCA pre-
cipitahle mantle collar protein of Helisoma. Three peaks of immuno-
reactivity ( 1 7 kDa, 7 kDa. and 2 kDa) were found in the chromatographic
fractions collected from hemolymph eluate as detected by RIA. All of
these fractions were tested in vitro for the stimulation of incorporation
of 3 [H]-labeled ammo acids into TCA precipitable protein of the mantle
collar of Helisoma. The mantle collars from different snails of known
growth rates were dissected and cultured in vitro with and without the
immunoreactive fractions (250 n\U/m\) obtained from the gel filtration
column. The mantle collars were processed to measure the label incor-
poration into mantle collar proteins (see Methods). The 7 kDa ILP sig-
nificantly stimulated amino acid incorporation into mantle collar protein
(*, P < 0.05). Columns in the figure indicate the mean of n = 12, bars
the SEM from three experiments.

c

0)

o

Q.

Q.
Q

7kda
2kDa

Fractions

Figure 9. The effect of partially purified hemolymph insulin im-
munoreactive fractions on the stimulation of incorporation of 3 [H]-labeled
amino acids into TCA precipitable periostracal protein of Helisoma.
The mantle collars were cultured in vitro (similar to Fig. 8) for 48 h and
the periostracum produced was collected with a glass pipette and processed
(see Methods) for assaying the 3 H amino acid incorporation into perios-
tracal protein. The 7 kDa ILP significantly stimulated the amino acid
incorporation into periostracal protein (**, P < 0.01 ). Columns indicate
the mean of n = 12, bars the SEM from three experiments in triplicate.

release of the ILP at the median lip nerve (neurohemal
area) correlate well with shell-growth periods (Khan and
Saleuddin, 1992).

itself, the definitive role of insulin on periostracum for-
mation can only be conjectured. Thus it is possible that
insulin/ILP could be acting on the mantle collar as a gen-
eral metabolic stimulator rather than as a specific shell
growth factor. Saleuddin ct al. (1991) showed that injec-
tions of microgram quantities of porcine insulin-stimu-
lated shell growth, whereas injection of porcine anti-in-
sulin inhibited shell growth in Helisoma.

Geraerts (1976) suggested that a factor from the neu-
rosecretory light green cells (LGC) in Lymnaea stagnalis
is involved in body and shell growth. It is now known
that LGC produce three insulin-related peptides MIP I,
MIP II, and MIP V (Smit ft al., 1988, 1991, 1992). MIP
I has been implicated as a growth hormone. However,
direct evidence of the involvement of MIPs as stimulators
of shell growth has not been documented. In Helisoma.
the neurosecretory mediodorsal cells (MDC), which are
homologous to LGC of Lymnaea, contain immunoreac-
tive insulin-like material that is released into the medium
when the CNS is treated with high potassium or 4-ami-
nopyridine (Khan el al.. 1992; Khan and Saleuddin,
1992). The synthetic activity of the MDC and the observed

0)

o

Q.

O> 3000

Q.
Q

ILP ulU/ ml

Figure 10. The dose-response curve for the stimulation of incor-
poration of 3 [H]-labeled amino acids into TCA precipitable mantle collar
protein of Helisoma by the partially purified 7 kDa insulin immuno-
reactive fraction. The mantle collars were cultured in vitro with various
doses of 7 kDa ILP (0. 100, 250, 500 ^lU/ml) and amino acid incor-
poration into mantle collar proteins was determined (see Methods). Re-
sults shown are pooled data from two separate experiments in triplicate
and for each experiment at least 12-15 controls and 10-15 treated mantle
collars per dose were assayed.

INSULIN-L1K.E PEPTIDE TITERS IN HELISOMA

147

V

o

Q.
O)

ILP ulU/ml

Figure 11. The dose-response curve for the stimulation of incor-
poration of '[H]-labeled amino acids into TCA precipitable periostracal
proteins ofHelisoma by the partialh punned 7 kDa hemolymph insulin-
like immunoreactive fraction. The mantle collars were cultured in vitro
with various doses of 7-kDa ILP (0. 100. 250. and 500 ^lU/ml) and the
periostraca formed were processed for assaying the '[H]-labeled ammo
acid incorporation into periostracal proteins (see Methods). Results shown
are pooled data from two separate experiments in triplicate and in each
experiment at least 12-15 controls and 10-15 treated penostraca per
dose were assayed.

We have identified ILP in the hemolymph in Helisoma.
Furthermore we have measured the tilers of ILP in the
hemolymph of snails with different shell-growth rates and
also during the period of shell regeneration. The difference
in tilers during shell growth or shell regeneration suggesls
lhal ILP is involved in Ihese Iwo processes. Allhough ILP
lilers were measured by an RIA using anli-human insulin,
Ihe liters reflect Ihe relalive concenlralions of ILP, because
Ihe compelilion curve for Helisoma ILP showed a linear
relalionship lo human insulin slandards (Fig. 1). In an-
other terreslrial pulmonale, Otala lactea, we were able lo
detecl ILP in Ihe hemolymph, hepalopancreas, and CNS,
bul did nol invesligale ils physiological function (unpub.
dala). Gomol et al. ( 1 992) found insulin immunoreaclive
neurosecrelory cells in Ihe CNS of Helix aspersa. Caulery
of Ihese cells causes a reduclion in body weighl. Gomot
(pers. comm.) reports lhat injections of microgram quan-
tities of bovine insulin cause juvenile Helix aspersa lo
grow significantly larger than controls over a six-week pe-
riod.

In Ihis paper, ILP concentralions in Ihe hemolymph
were measured by an immunoassay procedure. This lech-
nique is based upon recognilion of an anligenic deler-
minanl of Ihe hormone molecule wilh specific antibodies
raised against Ihe hormone. Furthermore, Ihis lechnique
does nol take inlo accounl Ihe biological effecls of Ihe
hormone on largel cells or lissues and Ihus is unable lo

distinguish belween biologically aclive and inactive forms
of Ihe antigen. Equally significanl is Ihe facl lhal immu-
noassay techniques fail lo distinguish belween isohor-
mones wilh differenl functions working on separale largel
lissues (Robertson and Sidney, 1990). For example, Ihe
immunoassay used in Ihis sludy identified Ihe lolal ILP
in Ihe hemolymph wilhoul discriminating belween ILP
from various sources, such as Ihe hepalopancreas or Ihe
CNS. The ILP from Ihe hepalopancreas could be involved
in sugar metabolism, whereas we believe the ILP from
the CNS is a shell-growth factor.

Many mollusks show a daily rhythm of shell deposition.
Wilhin laboratory populations ofHelisoma. fasl and slow
shell regeneration rales can be recognized over a 14-day
period of monitoring. Interestingly, Ihe shell regeneration
rates and the hemolymph ILP tilers are inversely relaled.
The high liters of ILP associaled wilh slow shell regen-
erations rales may be due lo ILP, which is immunoreactive
bul lacks bioaclivily. A similar silualion has been reported
in Ihe case of paralhyroid hormone (Martin et al., 1977).
Il is equally possible lhal Ihe anliserum used in Ihis sludy
recognized degraded hormone fragmenls wilh anligenic
delerminanls lhal are intact.

Partial purification of Helisoma hemolymph ILPs by
gel filtration showed thai only one insulin immunoreaclive
fraction conlaining 7-kDa polypeplide slimulaled amino
acid incorporation in Ihe manlle collar as well as in Ihe
perioslracum. The presence of high and low molecular
weighl ILPs in the hemolymph ofHelisoma may represent
the precursor molecule and the degraded fragments of
ILP, respectively. It will be inleresling lo know whelher
Ihe bioaclive ILP fraction is high in fasl-growing snails
bul low or nol detectable in slow-growing snails. Both low
and high molecular weight immunoreactive insulin ma-
lerials were present in the hepatopancreas of oyslers (de
Martinez et al., 1973) and in Ihe gul, hepalopancreas and
hemolymph of lobslers (Sanders, 1983).

In Helisoma. sludies using bioassay, RIA, partial pu-
rification, and immunoblolling confirm lhal ILP (7 kDa)
can slimulale periostracum formation by stimulating
protein synthesis in the mantle collar in vitro and that it
is involved in shell growth. In vertebrates il is well eslab-
lished lhal insulin and relaled peplides (IGFs) nol only
regulate glycogen, lipid and protein synthesis but also
growlh and differentiation (Kahn, 1985; Froesch et al.,
1985; Rosen, 1987; de Pablo et al.. 1990). The prolho-
racicolropic hormone (PTTH) in insecls, which occurs in
multiple forms and which shows some slruclural similarity
with vertebrate insulin, is involved in growlh (Mizoguchi
et al.. 1990). Il remains lo be seen whelher ILP in Heli-
soma is slruclurally similar lo MIPs of Lvmnaea or lo
olher ILP or to vertebrale insulin. Experiments are cur-
renlly under way lo elucidale Ihe gene slruclure of ILP
in Helisoma using cDNA recombinanl lechniques.

148

V. M. SEVALA ET AL

Acknowledgments

This work was supported by Natural Sciences and En-
gineering Research Council of Canada. We thank Dr.
B. G. Loughton for critically reading the manuscript.

Literature Cited

Bramhall, S., N. Noack, M. Wu, and J. R. Loewenberg. 1969. A simple

colorimetric method for determination of protein. Anal. Biochem.

31: 146-148.
Ebberink, R. H. M., A. B. Smit, and J. Van Minnen. 1989. The insulin

family: evolution of structure and function in vertebrates and inver-
tebrates. Biol. Bull. Ill: 176-182.
Froesh, E. R., Chr. Si timid, J. Schwander, and J. Zapf. 1985. Actions

of insulin-like growth factors. Ann. Rev. Physiol. 47: 443-467.
Geraerts, W. P. M. 1976. Control of growth by the neurosecretory

hormone of the light green cells of the fresh water snail Lymnaea

stagnalis. Gen. Comp. Endocrinol 29: 61-71.
Gomot, A., L. (.omul. C. R. Marchand, C. Colard, and J. Bride.

1992. Immunocytochemical localization of insulin-related pep-

tide(s) in the central nervous system of the snail Helix aspersa Muller:

involvement in growth control. Cell Moi Newobiol. 12: 21-31.
Kahn, C. R. 1985. Current concepts of the molecular mechanism of

insulin action. Ann. Rev Med, 36: 429-45.
Khan, H. R., B. Griffond, and A. S. M. Saleuddin. 1992. Insulin-like

peptide(s) in the central nervous system of the snail Helisoma duryi.

Brain Res 580: 111-114.
Khan, H. R., and A. S. M. Saleuddin. 1992. Neurosecretion of the

mediodorsal cells of the central nervous system of the snail Helisoma

duryi. Cell Tissue Res 268: 131-139.
Kunigelis, S. C., and A. S. M. Saleuddin. 1978. Regulation of shell

growth in the pulmonate gastropod Helisoma duryi. Can. J Zoo/.

56: 1975-1980.
Kunigelis, S. C., and A. S. M. Saleuddin. 1983. Shell repair rates and

carbonic anhydrase activity during shell repair in Helisoma duryi

(Mollusca). Can. J Zoo/. 61: 597-602.
Kunigelis, S. C., and A. S. M. Saleuddin. 1985. Studies on the in vitro

formation of periostracum in Helisoma duryi: the influence of brain.

/ Comp. Physiol. 13: 177-183.
Lutz, R. A., and D. C. Rhoads. 1980. Growth patterns within the mol-

luscan shell. Pp. 203-254 in Skeletal Growth of Aquatic Organisms,

D. C. Rhoads and R. A. Lutz, eds. Plenum Press, New York,
de Martinez, N. R., M. C. Garcia, M. Salas, and J. L. R. Candela.

1973. Proteins with insulin like activity isolated from oyster (Ostrea

edulis L.) hepatopancreas. Gen. Comp. Endocrinol. 20: 305-31 1.
Martin, K. J., K. A. Hruska, J. Lewis, C. Anderson, and E. Slatopolsky.

1977. The renal handling of parathyroid hormone role of pentubular

uptake and glomerular filtration. J din. Invest. 60: 808-814.

Minnen, J. V., and H. Schallig. 1990. Demonstration of insulin-related
substances in the central nervous system of pulmonates and Aplysia
caltformca. Cell. Tissue Res. 260: 381-386.

Mizoguchi, A., M. Hatta, S. Sato, H. Nagasawa, A. Suzuki, and H.
Ishizaki. 1990. Developmental change of bombyxin content in the
brain of the silk moth Bambyx mori. J. Insect. Physiol. 36: 655-664.

de Pablo, F., E. Hernandez, F. Collia, and J. A. Gomez. 1985. Untoward
effects of pharmacological doses of insulin in early chick embryos:
through which receptors are they mediated? Diabetologica 28: 308-
318.

de Pablo, F., L. A. Scott, and J. Roth. 1990. Insulin and insulin-like
growth factor I in early development: peptides, receptors, and bio-
logical events. Endocrine Rev. 4: 558-577.

Robertson, W. R., and S. P. Bidney. 1990. The in vitro bioassay of
peptide hormones. Pp. 121-157 in Peptide Hormone Secretion. A
Practical Approach. J. C. Hutton and K.. Siddle, eds. Oxford University
Press, Oxford, New York, Tokyo.

Rosen, O. M. 1987. After insulin binds. Science 237: 1452.

Saleuddin, A. S. M., and S. C. Kunigelis. 1984. Neuroendocrine control
mechanisms in shell formation. Am. Zoo/. 24: 91 1-916.

Saleuddin, A. S. M., H. R. Khan, M. Sevala, and V. L. Sevala.
1991. Hormonal control of confirmed shell growth in the snail
Helisoma duryi (Mollusca: Gastropoda). Pp. 161-165 in Mechanisms
and Phytogeny of Mineralization in Biological Systems, S. Suga and
H. Nakahara, eds. Sponger- Verlag, Tokyo.

Sanders, B. 1983. Insulin-like peptides in the lobster Homarus amer-
icanus 1. Insulin immunoreactivity. Gen. Comp. Endocrinol. 50: 366-
373.

Smit, A. B., E. Vreugdenhil, R. H. M. Ebberink, W. P. M. Geraerts, J.
Klootwijk, and J. Joosse. 1988. Growth-controlling molluscan
neurons produce the precursor ot an insulin-related peptide. Nature
33: 535-538.

Smit, A. B., W. P. M. Geraerts, I. Meester, H. V. Heerikhuizen, and J.
Joosse. 1991. Characterization of a cDNA clone encoding mol-
luscan insulin-related peptide II of Lymnaea slagnalis. Eur. J
Biochem. 199: 699-703.

Smit, A. B., S. F. T. Thijsen, W. P. M. Geraerts, I. Meester, H. V.
Heerikhuizen, and J. Joosse. 1992. Characterization of a cDNA
clone encoding molluscan insulin-related peptide V of Lymnaea
stagnalis. Mol. Brain Res 14: 7-12.

Sokal, R. R., and F. J. Rohlf. 1973. Introduction to Biostatistics.
W. H. Freeman, San Francisco.

Thorpe, A., and H. Duve. 1984. Insulin- and glucagon-like peptides in
insects and molluscs. Mol. Physiol. 5: 235-260.

Wong, V., and A. S. M. Saleuddin. 1972. Fine structure of normal and
regenerated shell of Helisoma duryi. Can. J Zoo/. 50: 1563-1568.

Reference: Biol. Bull. 185: 149-151. (August, 1993)

A Light-Independent Magnetic Compass in the
Leatherback Sea Turtle

KENNETH J. LOHMANN AND CATHERINE M. FITTINGHOFF LOHMANN

Department of Biology, Coker Hall. CB-3280, University of North Carolina, Chapel Hill,

North Carolina 27599

Diverse animals can orient to the earth 's magnetic field
(1-6), but the mechanism or mechanisms underlying
magnetic field detection have not been determined. Be-
havioral (7-9) and neuwphysiological (10-12) results
suggest that the transduction process underlying magnetic
compass orientation in vertebrates is light-dependent, a
finding consistent with theoretical models proposing that
magneloreceplion involves a modulation of the response
of retinal photoreceptors to light (13, 14). We report, how-
ever, that leatherback sea turtle (Dermochelys coriacea)
hatchlings orient to the geomagnetic field in complete
darkness. Thus, light-dependence is not a universal feature
of vertebrate magnetic compasses.

Immediately after emerging from underground nests
on oceanic beaches, sea turtle hatchlings enter the sea and
swim toward the open ocean in a migration lasting several
days. Hatchlings leaving the east coast of Florida quickly
establish easterly courses that lead them away from land
and toward the Gulf Stream current (15-17). Previous
laboratory experiments have demonstrated that hatchling
loggerhead turtles (Caret t a caretta) will orient to the earth's
magnetic field (2). To determine whether leatherbacks
have a similar ability and whether the transduction mech-
anism underlying magnetic compass orientation in sea
turtles is dependent on light, we investigated the orien-
tation of hatchling leatherbacks swimming in darkness.
Hatchling leatherback sea turtles were obtained from
nests deposited on beaches in the vicinity of Fort Pierce,
Florida. Nests were examined daily. When a depression
formed in the sand above a nest (indicating that the eggs
had hatched and that emergence would probably occur
that night), several hatchlings were removed, placed into
a darkened styrofoam cooler, and transported to the lab-
Received 18 February 1993; accepted 27 May 1993.

oratory. Orientation was assessed in a circular water-filled
arena surrounded by a Rubens cube coil (18) (Fig. 1) that
could be used to reverse the direction of the horizontal
component of the ambient magnetic field. Experiments
were conducted in a light-tight room between sundown
and sunrise, the time when most hatchlings normally enter
the sea (19, 20). To eliminate the light emanating from
computers and power supplies, all electronic equipment
was removed from the room. As an additional precaution
against unexpected light sources (e.g., bioluminescence),
an observer periodically sat silently beside the coil while
experiments were in progress. Following dark-adaptation
of one hour or longer, three different observers were un-
able to perceive any light in the room despite systematic
searches and efforts to elicit bioluminescent flashes by
stirring the water.

While in darkness, hatchling leatherbacks tested in the
earth's magnetic field were significantly oriented in an
eastward direction (Fig. 2a). In contrast, hatchlings tested
in darkness under reversed field conditions oriented in
approximately the opposite direction (Fig. 2b). The two
distributions are significantly different, indicating that the
ambient magnetic field influenced the orientation of
hatchling leatherbacks swimming in darkness.

These results demonstrate that leatherbacks are able to
detect the geomagnetic field in the absence of visible light.
We conclude that the transduction mechanism underlying
magnetic compass orientation is not light-dependent in
all vertebrate species.

The magnetic compass of sea turtles could rely on a
mechanism different from that used by other vertebrates.
The functional characteristics of the loggerhead turtle
compass and those of magnetic compasses in two other
vertebrate classes, however, appear identical. Like the
magnetic compass of birds (5) and of shoreward-orienting
newts (4), the loggerhead compass is axial and based on

149

150

K. J. LOHMANN AND C. M. F. LOHMANN

D

Figure 1. The orientation arena was an inverted fiberglass satellite
dish ( 1 .02 m diameter) filled with water. The arena was surrounded by
a Rubens cube coil 130 cm on a side. Prior to testing, each hatchling
was placed into a nylon-Lycra harness that encircled the turtle's carapace
without impeding swimming (15). The harness was connected by a short
monofilament line to a lever arm mounted on a 360 rheostat. The
rheostat was positioned on a post in the center of the orientation arena
(2). The lever arm was free to rotate within the horizontal plane and
could easily be pulled clockwise or counterclockwise by a swimming
turtle. The arm thus tracked the direction toward which the hatchling
swam. The central rheostat was wired to a computer in an adjacent
room, which recorded the orientation of the turtle every 30 s with an
accuracy of 2. Thus, the orientation of turtles swimming in darkness
could be tracked.

Methods: Detailed descriptions of methods are provided in ref #2.
Each hatchling was tested once on either its first, second, or third night
of captivity. Each trial began in the earth's field (coil off) with a dim
light hanging in magnetic east so that hatchlings quickly established a
course toward the light (2). The light was provided because hatchlings
emerging from their nests at night under natural conditions find the sea
using light cues associated with the ocean surface (25); light reflected
from the ocean may also provide a directional cue necessary for hatchlings
to initiate a seaward course (2, 26). After one hour, the light was turned
off and the turtles were permitted to swim in darkness either in the
unaltered magnetic field (i.e.. the coil remained off) or in a reversed field
(i.e.. the coil was turned on 10-20 s after the light was turned off). Ten
minutes after the light was turned off, the computer began recording the
orientation of each hatchling at 30-s intervals. Thus, orientation data
were collected only while the turtles were swimming in darkness. Between
trials, we periodically altered the position of the power supply relative
to the arena to reduce the chance that subtle sounds or vibrations could
serve as an orientation cue; such positional changes, however, had no
discernible effect on orientation.

field line inclination, rather than on field polarity (21).
The possibility therefore exists that all three compasses
are based on a common underlying mechanism.

One hypothesis for magnetic field detection in verte-
brates proposes that particles of the mineral magnetite

transduce geomagnetic stimuli to the nervous system (22).
Although magnetite particles have been detected in ce-
phalic tissues of sea turtles (23) and in numerous other
animals known to orient magnetically (22), no direct neu-
rophysiological evidence has been obtained demonstrating
a link between magnetite and magnetic field detection in
any multicellular organism.

A second hypothesis of magnetoreception has been
proposed for elasmobranch fishes. These animals possess
sensitive electroreceptors that may endow them with a
magnetic compass sense based on electromagnetic induc-
tion (24). Because electroreceptors have not been found
in reptiles, however, an induction-based mechanism ap-
pears unlikely for sea turtles. Moreover, the elasmobranch
induction hypothesis requires consistent movement
through the earth's magnetic field, yet hatchlings suc-
cessfully oriented magnetically while tethered and nearly
stationary (Fig. 1 ).

A third hypothesis proposes that magnetoreception oc-
curs in photoreceptors through a transduction process re-
quiring light (7, 1 3, 14). Our results demonstrate that light
is not necessary for magnetic orientation in marine turtles.
The results are therefore not consistent with current mod-
els of light-dependent magnetoreception. Further research
will be required to determine whether the light-indepen-
dent magnetic compass of sea turtles relies on different

270'

180

Earth's Magnetic Field

Reversed Field

Figure 2. Results of magnetic orientation experiments with hatchling
leatherbacks tested in darkness. (A) Mean angles of hatchlings tested in
the earth's magnetic field. The group was significantly oriented with a
mean angle of 70 (Rayleigh test: r = 0.55, Z = 3.33, P < 0.05). (B)
Mean angles of hatchling leatherbacks tested in darkness in a reversed
field. The group was significantly oriented with a mean angle of 255
(Rayleigh test: r = 0.53, Z = 3.09, P < 0.05). The arrow between the
two distributions indicates they are significantly different (Watson test:
\J 2 = 0.300, P < 0.005). Arrows in the center of each circle indicate the
mean angle of the group; the arrow length is proportional to the magnitude
of the mean vector r. with the radius of the circle corresponding to r = 1 .
Dashed lines indicate the 95% confidence interval for the mean angle
(27).

Methods: Procedures used in analyzing orientation data are described
in detail in ref #2, except that: (i) the accuracy of measurements in this
study was improved to 2. and (ii) to avoid tiring leatherback hatchlings
(an endangered species) before release, we terminated experiments as
soon as a turtle completed its first oriented swimming period; the mean
angle of this period (2) was the hatchling's orientation angle.

SEA TURTLE MAGNETIC ORIENTATION

151

receptors than the apparently light-dependent magnetic
compasses of birds (5. 9) and newts (7). or whether all
three compasses in fact share a common underlying
mechanism.

Acknowledgments

We thank Jay Callaway for developing the data acqui-
sition software, Mike Salmon and Jeanette Wyneken for
discussions of experimental protocols, and Erik Martin
and Robert Ernest for assistance in locating leatherback
turtle nests. The work was supported by NSF grants IBN-
9120338 and BNS-87-07173. Endangered species research
was authorized under Florida DNR special permit
TP 073.

Literature Cited

1. Lohmann, K. J., and A. O. D. Willows. 1987. Lunar-modulated
geomagnetic orientation by a marine mollusk. Science 235: 331-
334.

2. Lohmann, K.J. 1991. Magnetic orientation by hatchling loggerhead
sea turtles (Carella carelta). J. Exp. Bioi 155: 37-49.

3. Quinn, T. P. 1980. Evidence for celestial and magnetic compass
orientation in lake migrating sockeye salmon fry. / Camp. Plmial.
A 137: 243-248.

4. Phillips. J. B. 1986. Two magnetoreception pathways in a migra-
tory salamander. Science 233: 765-767.

5. \\illschko, \V., and R. Wiltschko. 1988. Magnetic orientation in
birds. Pp. 67-121 in Current Ornithology, vol. 5. Johnston, R. F..
ed. Plenum Press. New York.

6. Burda, H.. S. Marhold, T. Westenberger, R. Wiltschko, and W.
Wiltschko. 1990. Magnetic compass orientation in the subterra-
nean rodent Cryplomys hottenloliis Expcricnlia 46: 528-530.

7. Phillips, J. B., and S. C. Borland. 1992. Behavioural evidence for
use of a light-dependent magnetoreception mechanism by a verte-
brate. Nature 359: 1 42- 1 44.

8. Phillips, J. B., and S. C. Borland. 1992. Wavelength specific effects
of light on magnetic compass orientation of the eastern red-spotted
newt Nolophtlta/mus viridescens. Elhol. Ecal. Evol. 4: 33-42.

9. Wiltschko, W., and R. Wiltschko. 1981. Disorientation of inex-
perienced young pigeons after transportation in total darkness. Nature
291:433-434.

10. Olcese, J., S. Reuss, and P. Semm. 1988. Geomagnetic field de-
tection in rodents. Li/eSci. 42: 605-613.

1 1 . Semm, P., D. Nohr, C. Demaine, and W. Wiltschko. 1984. Neural
basis of the magnetic compass: interactions of visual, magnetic and

vestibular inputs in the pigeon's brain. J. Camp. Phvsiol. A. 155:
283-288.

12. Semm, P., and C. Demaine. 1986. Neurophysiological properties
of magnetic cells in the pigeon's visual system. J. Camp. Phyuol I
159: 619-625.

1 3. Leask, M. J. M. 1977. A physicochemical mechanism for magnetic
field detection by migrating birds and homing pigeons. Nature 267:
144-145.

14. Schulten, K., and A. \\ indemuth. 1986. Model for a physiological
magnetic compass. Pp. 99-106 in Biophysical Effects of Steady
Magnetic Fields. G. Maret. N. Boccara, and J. Kiepenheuer. eds.
Springer-Verlag. Berlin.

15. Salmon, M., and J. Wyneken. 1987. Orientation and swimming
behavior of hatchling loggerhead turtles (Caretta caret/a L.) during
their offshore migration. J. I-:\p. Mar. Biol. Ecol. 109: 137-153.

16. Carr, A. 1986. Rips. FADS, and little loggerheads. BioScience 36:
92-100.

17. Carr, A. 1986. New perspectives on the pelagic stage of sea turtle
development. .V0.-l.-l Tech. Memorandum NMFS-SEFC 190: 1-36.

18. Rubens, S. M. 1945. Cube-surface coil for producing a uniform
magnetic field, Rev. Sci. lustrum. 16: 243-245.

19. \\itherington, B. E., K. A. Bjorndal, and C. M. McCabe.
1990. Temporal pattern of nocturnal emergence of loggerhead
turtle hatchiings from natural nests. Capeia 4: 1 165-1 168.

20. Bustard, H. R. 1967. Mechanism of nocturnal emergence from
the nest in green turtle hatchiings. Nature 214: 317-318.

21. Light, P., M. Salmon, and K. J. Lohmann. 1993. Geomagnetic
orientation of loggerhead sea turtles: evidence for an inclination
compass. J Exp. Biol. (in press).

22. Kirschvink, J. L., D. S. Jones, and B. J. MacFadden.
1985. Magnetite Biomineralization and Magnetoreception in Or-
ganisms. Plenum Press, New York.

23. Perry, A., G. B. Bauer, and A. E. Dizon. 1985. Magnetoreception
and biomineralization of magnetite in amphibians and reptiles. Pp.
439-453 in Magnetite Biomineralization and Magnetoreception in
Organisms. J. L. Kirschvink. D. S. Jones and B. J. MacFadden. eds.
Plenum Press, New York.

24. kalmijn, A. J. 1978. Experimental evidence of geomagnetic ori-
entation in elasmobranch fishes. Pp. 347-353 in Animal Migration.
Orientation, and Homing. K. Schmidt-Koenig and W. T. Keeton,
eds. Springer-Verlag. Berlin.

25. Mrosovsky, N., and S. F. Kingsmill. 1985. How turtles find the
sea. 7. Tierpsychal 67: 237-265.

26. Mrosovsky, N. 1978. Orientation mechanisms of marine turtles.
Pp. 413-419 in Animal Migration. Navigation, and Homing. K.
Schmidt-Koenig and W. T. Keeton, eds. Springer-Verlag. Berlin.

27. Batschelet, E. 1981. Circular Statistics in Biology: Academic Press,
London.

The Marine

Biological

Laboratory

Woods Hole

Massachusetts

Ninety-Fifth Report

for the Year 1992

One-Hundred and Fourth Year

Officers of the Corporation

Denis M. Robinson, Honorary Chairman of the Board

of Trustees

Sheldon J. Segal, Chairman of the Board of Trustees
Robert E. Mainer, I 'ice Chairman of the Board of

Trustees

James D. Ebert, President of the Corporation
John E. Burns, Director and Chief Executive Officer
Robert D. Manz, Treasurer
Neil Jacobs, Clerk of the Corporation

Contents

Report of the Chairman Rl

Report of the Director R2

Report of the Treasurer R6

Financial Statements R8

Report of the Library Director R 1 8

Educational Programs

Summer Courses R2 1

Short Courses R25

Summer Research Programs

Principal Investigators R30

Other Research Personnel R3 1

Library Readers R33

Institutions Represented R34

Year-Round Research Programs R38

Honors R45

Board of Trustees and Committees R48

Laboratory Support Staff R5 1

Members of the Corporation

Life Members R53

Regular Members R54

Associate Members R65

Certificate of Organization R68

Articles of Amendment R68

Bylaws R68

Report of the Chairman

The year 1992 was one of historic and exciting
change for the Marine Biological Laboratory. In
August, the MBL's Board of Trustees voted to adopt
major changes in the governance of the Laboratory,
and in September we welcomed the Laboratory's
newest director and CEO, John E. Burris.

New Governance Structure

The decision to modify the governance of the MBL
came after discussions at a Trustees retreat in 1991 and
a year of careful study and work by members of an Ad
Hoc Committee on Governance, in consultation with
the MBL community. When fully constituted, the
MBL's new Board of Trustees will be composed of 18
members drawn from a wide community of
distinguished, influential, and experienced individuals.
The Board will be a fiduciary body, the guardian of the
MBL mission. It will have strong fundraising
capabilities, be independent and self regenerating, set
policies, and appoint and evaluate the CEO. Board
members provide to the laboratory a broad range of
valuable experience and will be able to address the
institution's challenges with wisdom and objectivity.

A Nominating Committee of the Board will seek
eligible candidates for Board membership and the
candidates will be elected by the full Board. Six of the
Trustees will be selected by the Board from a list of
nominees all scientists and MBL Corporation
members compiled by the MBL Corporation. Other
members of the Board may also be chosen from the
scientific community at large.

The Corporation will continue to play the crucial
role of providing governance on the scientific direction
of the MBL. Drawing from its membership, the
Corporation will establish a Science Council that will
provide advice and counsel on scientific affairs to the
MBL's director and to the Board of Trustees. Members

of the Science Council will represent all aspects of
scientific life at the MBL.

Finally, an independent Board of Overseers will be
established. This group will consist of distinguished
scientists not necessarily members of the MBL
Corporation who will serve as advisors to the Director
and to the Board.

New Director Appointed

After a year-long search, I was pleased to announce
in July the appointment of John E. Burris to serve as
Director and CEO of the Marine Biological Laboratory.
Dr. Burris received his Ph.D. in marine biology from
Scripps Institution of Oceanography in 1976. He came
to the MBL from the National Research Council of the
National Academy of Sciences where he served most
recently as Executive Director of the Commission on
Life Sciences. He brings to his new role as MBL's
Director a broad range of experience in science policy
and administration, and as a university faculty member
and researcher in marine biology and physiological
ecology.

While welcoming John to his new position, I'd also
like to pay tribute and offer thanks on behalf of the
Laboratory to John's predecessor, Harlyn Halvorson.
He served with distinction as Director of the MBL from
September 1987 to July 1992. Under Harlyn's
leadership the laboratory celebrated a milestone event
of June 20, 1992, with the opening of the new Marine
Resources Center. Harlyn's dedication and
commitment to the Marine Resources project never
faltered. He worked all over the country, indeed around
the world, garnering support for this effort. We owe
Harlyn great thanks as our Director and as the
visionary who made this new building possible.

Sheldon J. Segal

Rl

Report of the Director
and Chief Executive Officer

It is with great pleasure that I recount the past year at
the Marine Biological Laboratory and contemplate its
future, for in my first nine months at the Laboratory I
have come to appreciate its many resources and its
potential. The greatest of these resources is the human
one the staff, the scientists, the students, and the
community. Next is the environment of the MBL an
institution composed of a variety of well-run research
laboratories surrounded by waters rich in biological
diversity.

What did 1992 bring to the Marine Biological
Laboratory?

Marine Resources Center

Our most visible achievement of 1992 was the June
20th opening of the MBL's new Marine Resources
Center. Senators Edward M. Kennedy and John Kerry,
Congressman Gerry Studds, and NIH Deputy Director
Jay Moskowitz participated in the opening ceremonies,
which were attended by more than 500 members of the
MBL and Woods Hole communities. We owe a great
debt of gratitude to Harlyn Halvorson, for his tireless
efforts towards making this milestone project a reality.

The new Marine Resources Center provides state-of-
the-art engineering systems and advanced laboratory
and mariculture facilities, as well as a collaborative
veterinary program through the University of
Pennsylvania's Laboratory for Marine Animal Health,
directed by Donald Abt. Dr. Alan M. Kuzirian has
been appointed Acting Director of the new Center. The
MRC is reporting early successes in maintaining a

healthier and more reliable supply of marine organisms.
For example, a new generation of dogfish has been born
and is thriving in our circulating seawater tanks. Species
requiring cold seawater are now available to researchers
into the summer months as a result of improved chilled
seawater holding facilities. Warmer-water species such
as toadfish and scup are being maintained in the facility
well past the time when they would have departed
Woods Hole waters for warmer climes. A colony of sea
worms is also being raised under uniform conditions to
produce reliable, standardized bioassays in
environmental monitoring.

The challenge facing the MBL is to maximize the use
of this wonderful facility. We need to use it not only to

Conjjressman Gerry Studds, Senators John Kerry and Edward M.
Kennedy, and MBL Director llarlyn Halvorson at the Opening of the
MBI.'s Marine Resources Center.

R2

Report of the Director and CEO R3

maintain organisms caught locally, hut also to culture
organisms from egg to egg to assure a reliable and
genetically and nutritionally denned set of experimental
organisms. The facility has extensive capabilities for
mariculture and biomedical research, providing exciting
potential for expanding the Laboratory's research and
educational programs.

Year- Round Research

The MBL's year-round community remains a strong
group of investigators, as evidenced by their continued
ability to obtain funding through peer-reviewed grants
even in these tight times. Some examples of these
successes include:

The Ecosystems Center, jointly directed by John
Hobbie and Jerry Melillo, won a large, multi-year
award from the National Science Foundation to
support a Land-Margin Ecosystems Research program.
This research measures the effects of human habitation
on fragile coastal zone ecosystems by studying changes
in food webs and water quality. The Center is also
continuing its research in tropical forests to evaluate the
effects of deforestation and air pollution on these
rapidly changing environments with grants from the
Texaco and Exxon corporations.

Investigators in the Center for Architectural
Dynamics in Living Cells continue to explore the
mechanisms and structural dynamics of cell division,
differentiation, and motility in living cells. Center
scientists are developing an automated polarized light
microscope that will document cell architecture on the
molecular level. Dr. Shinya Inoue, the Director of the
Center, was awarded the E.B. Wilson Award in
November, the American Society for Cell Biology's
highest honor. As this report goes to press, we have
learned that Dr. Inoue was also elected to membership
in the National Academy of Sciences.

The Center for Molecular Evolution, directed by
Mitchell Sogin. received national media coverage
recently for a dramatic discovery made there in 1992:
that the fungi are more closely related to animals than
they are to plants. This is a radical departure from
accepted classification systems where the fungi have
either been grouped with plants or given separate
kingdom status. Work at the Center is funded by the
NIH and the G. Unger Vetlesen Foundation.

Lionel Jaffe and his colleagues Peter Smith and
Andrew Miller have expanded and diversified funding
for the National Vibrating Probe Facility and the
Calcium Aequorin Imaging Laboratory. Recent exciting
results in these two facilities have provided new insights
on the blood-brain barrier, as well as the movement of
calcium in newly fertilized eggs.

Summer Research at the MEL

In 1992, more than 300 scientists from around the
world came to the MBL to perform research and
collaborate with their peers. Using the latest biomedical
technologies, their work has implications on studies of
aging, diabetes, cognition and memory, mariculture,
pollution, vision, and tissue bioadhesives. A series of
evening lectures, including the traditional Friday
Evening Lecture Series, symposia, and brown bag
lunches facilitated the exchange of ideas among our
eminent researchers.

Of particular note, summer researchers Sergei
Kuznetzov, George Langford, and Dieter Weiss found
evidence that a myosin-like motor is responsible for the
actin-dependent movement of organelles in squid giant
axons. These investigators propose a "dual filament
system" to account for the movement of organelles
during fast axonal transport. According to their
hypothesis, both actin filament-based and microtubule-
based motility systems work together to produce and
regulate the movement of organelles within the axon.
The breakdown of these kinds of transport systems in
nerve cells is thought to be involved in various
degenerative diseases such as amyotrophic lateral
sclerosis.

Educational Program

The MBL's exceptional educational program
continued its strong tradition of providing graduate
students and postdoctoral fellows with innovative
courses in the life sciences. In 1992, 347 students and
200 faculty and lecturers from more than 200
universities and research institutions participated.

In addition to the wide range of core courses at the
MBL, 1992 marked the return of the Mariculture
course. The course now uses a basic science approach
to address the problems of maintaining and culturing
marine organisms used in biological research.

The MBL's Information Systems Division teamed up
with the National Library of Medicine to provide a
one-week course for 30 students in Medical
Informatics. Workshops were also designed to teach the
MBL community how to use the NLM software. These
programs will continue for the next three years.

The MBL was also chosen to host the first course in
Fundamental Issues in Vision Research: Molecular and
Biological Approaches. This two-week laboratory and
lecture course, sponsored by the NIH's National Eye
Institute, demonstrated the challenging problems in
vision research available to graduate students. Several

R4 Annual Report

MBL summer investigators participated in planning
and teaching this course.

Fellowship Programs

The MBL's Fellowship and Scholarship programs
received strong support in 1992. More than $103,000
was available, enabling 16 young investigators and
students to participate in the MBL's research and
educational programs. The fellowship program was
enhanced again this year by a weekly luncheon seminar
series in which our young scholars shared their research
with the MBL community. Our fellows were also
honored at the annual Chamber Music Concert, which
in 1992 featured the renowned Tokyo String Quartet.

Ten journalists participated in the 7th annual Science
Writing Fellowships Program at the MBL. The one-
week hands-on laboratory in cell and molecular biology
techniques was again a great success. For the second
year, a journalist was sent to the Toolik Lake research
site on the north slope of Alaska's Brooks Range, to
work with scientists from the MBL's Ecosystems
Center.

Library^

The MBL/WHOI Library has made significant
progress towards its goal of serving as a conduit for,
rather than being exclusively a repository of, scientific
knowledge, thanks largely to support from the Andrew
Mellon Foundation and the Howard Hughes Medical
Institute. A fiber optic data network has been installed,
which will provide access to the Internet, electronic
mail and bulletin boards, and limited access to our
journal collection to scientists working around the
world. The U.S. Department of Education included the
MBL/WHOI Library in its constellation of officially
designated research libraries.

Construction

As I write this report, work has begun on a number
of construction and renovation projects at the
Laboratory. The Lillie research laboratory is in the
early stages of being renovated, the result of a $575,000
matching grant from the National Science Foundation.
Although major components of the project won't begin
until the fall of 1993, the first phases of the project the
removal of asbestos from some of the research
laboratories is underway. Included in the renovations
are a central air conditioning system, forced ventilation

into the labs, a new freight elevator, and a larger-
capacity standby electrical power generator.

The Brick Apartment Dormitory is undergoing its
first major renovations since it was constructed in 1923.
When completed in the summer of 1993, the building
will be more energy-efficient and modern. Asbestos will
have been removed and a new boiler and bathroom
facilities in the dorm wing installed.

December 22, 1992 marked the end of an era for the
MBL's Supply Department, when the old shingled
supply building was razed to make room for the Lab's
new Collection Support Facility. The CSF will occupy
basically the same footprint as the old supply building.
It will contain a small boat maintenance facility and
a diving support facility on the first floor, and a
500 kWatt emergency generator for the new MRC on
the second.

Special Events and Public Programs

In 1992, the MBL continued to offer outreach
programs to the general public. Three weeks of
ELDERHOSTEL programming was offered, and the
sessions, which included courses on evolution,
ichthyology, biochemistry, and genetics, were met with
great enthusiasm by the students. The MBL also
continued its acclaimed Falmouth Forum winter series
of lectures, presentations, and concerts. Among our
1992 Forum participants was Pulitzer Prize winning
author, David McCullough.

The Future

The preceding chronicles the recent concrete
achievements of the Laboratory, but it doesn't capture
adequately the excitement we all feel about the future.
As the times change in biomedical and biological
research, the MBL is well-positioned to take advantage
of the changes. In education, for instance, where
everyone is bemoaning the expense of equipment and
laboratories, and where institutions are closing down
laboratory courses, we continue to be able to offer
modern courses with state-of-the-art equipment. Our
rotating faculty and the generous loans of equipment
from scores of different vendors are crucial in our
ability to do this, as is the generous support we receive
from the federal government and private organizations
such as the Howard Hughes Medical Institute, the
MacArthur Foundation, and the Burroughs Wellcome
Fund. We know that our courses are filling a need, as
indicated by the increased number of applications for
all of them in the summer of 1993.

Report of the Director and CEO R5

In an era when young scientists are finding it scientists may pursue a variety of new research

progressively more difficult to get started and to obtain opportunities.

funding, we have expanded our fellowship program. I feel fortunate to have assumed the directorship of

Again we see the demand for such programs; the Marine Biological Laboratory, long recognized as

applications for fellowships more than doubled in 1993 one of the world's most important biological

from 1992. laboratories, at this propitious moment. I look forward

The future of the MBL is indeed bright for our to working will all of you in the years to come,
researchers as well. Our new Marine Resources Center
and the soon-to-be renovated Lillie research laboratory
will provide an improved physical setting where our John E. Burris

Report of the Treasurer

Financially, 1992 will be remembered as the year of
transition. With the "changing of the guard" in
leadership and the change in governance comes the
challenge and opportunity for strengthening the
financial future of the MBL.

We finished 1992 with an excess of support and
revenues over expenses in the unrestricted current
funds of $275,968. After mandatory and non-
mandatory transfers, the net result was a decrease in
our unrestricted current fund balance of $8,451.

This modestly successful result meant that for the
first time in a number of years we did not have to use
unrestricted quasi-endowment funds to support current
operations.

This is a heartening result, but it must be tempered
by the observation that this is before a depreciation
expense of $722.959. Like most other not-for-profit
organizations, the MBL does not charge depreciation
expense to operations. Until the MBL funds

TOTAL ENDOWMENT
FUND BALANCE

$20

$15 -

MILLIONS

$10

II

SO

1984 1985 1986 1987 1988 1989 1990 1991 1992

MBL ENDOWMENT FUNDS
1992

MILLIONS

Figure 1

RESTRICTED

UNRESTRICTED

TRUE ENDOWMENT OUASI-ENDOWMENT

D1-004

Figure 2

depreciation of operations, there can be no use of the
word "surplus."

1992 saw the completion of the Marine Resources
Center, and the growth of our plant fund balances from
about $10 million in at the end of 1991 to almost $20
million at the end of 1992.

Our endowment funds (Figs. 1 and 2) increased
slightly from $15.9 million to $16.3 million, almost all
of this increase resulting from gifts. The total return
before fees on our portfolios in 1992 ranged between
5% and 5.8%; clearly, we need to improve on this
performance, and we shall endeavor to do so through
the efforts of the Investment Committee.

Support and Revenues increased from $20.8 million
in 1991 to $21.1 milion in 1992, due primarily to
increasing grant support of research and education.
Private gift support decreased due primarily to a lull in
receipt of major multi-year grants. This decrease masks

R6

Report of the Treasurer R7

MBL ANNUAL CAMPAIGN

$300

$250

$200

$160

(000)

Figure 3

the robust success of the Annual Campaign (Fig. 3).
and other unrestricted gift support. These grew by
$1 10,000 in 1992, an increase of 34% over the previous
year. Revenue from deterred support drawdowns on
previously received gifts increased by $436,000 from
1991. This reflects the continuing spend-down of
monies received primarily from the Howard Hughes
Medical Institute to support the educational and library
programs.

Our expenditures increased by $900,000 with most of
the increase coming in the research and education
programs as well as administration.

Housing and Dining operations continue to do well,
contributing $158,000 of depreciation expense to the
Housing Renewals and Replacements fund. This has
allowed us to undertake major upgrades of our housing
plant, both through capital expenditures from our
Repairs and Replacement fund, and through
borrowing.

In 1992 the MBL borrowed $2,600,000 at favorable
tax-exempt rates through the Massachusetts Industrial
Finance Agency (MIFA). We use this money to
refinance existing debt on our cottages, to renovate the
Brick Apartments, and to modernize research facilities
in the Crane wing of the Lillie/Crane building. Through
this borrowing, and the generous support of donors, we
were able to match a $575,000 award from the
National Science Foundation to undertake a
$ 1 ,800,000 project for the renovation of scientific
facilities.

Looking to 1993 we have a realistically balanced
budget that reflects the continued determination to
maintain the current level of services at the Laboratory
in the most cost-effective manner possible. We can
expect the financial stabilization witnessed in 1992 to
continue while we take advantage of new funding
opportunities. The MBL's long tradition of innovation
in fostering the development of scientists and the
stimulation of intellectual breakthroughs means that an
investment here will yield superior returns.

Robert D. Manz

Financial Statements

Coopers
&Lybrand

certified public accounianls

REPORT OF INDEPENDENT ACCOUNTANTS

To the Trustees of

Marine Biological Laboratory

Woods Hole, Massachusetts

We have audited the accompanying balance sheet of Marine Biological Laboratory as of December 31,
1992 and the related statement of support, revenues, expenses and changes in fund balances for the year
then ended. We previously examined and reported upon the financial statements of the Laboratory for the
year ended December 31, 1991, for which condensed statements are presented for comparative purposes
only. These financial statements are the responsibility of the Laboratory's management. Our responsibility
is to express an opinion on these financial statements based on our audit.

We conducted our audit in accordance with generally accepted auditing standards. Those standards
require that we plan and perform the audit to obtain reasonable assurance about whether the financial
statements are free of material misstatement. An audit includes examining, on a test basis, evidence
supporting the amounts and disclosures in the financial statements. An audit also includes assessing the
accounting principles used and significant estimates made by management, as well as evaluating the
overall financial statement presentation. We believe that our audit provides a reasonable basis for our
opinion.

In our opinion, the financial statements referred to above present fairly, in all material respects, the
financial position of Marine Biological Laboratory at December 31, 1992, and its support, revenues,
expenses and changes in fund balances for the year then ended in conformity with generally accepted
accounting principles.

Our audit was conducted for the purpose of forming an opinion on the basic financial statements taken
as a whole. The supplemental schedules of support, revenues, expenses and changes in fund balances for
current funds (Schedule I), endowment funds (Schedule II) and plant funds (Schedule III) as of December
31. 1992 are presented for purposes of additional analysis and are not a required part of the basic financial
statements. Such information has been subjected to the auditing procedures applied in the audit of the
basic financial statements and, in our opinion, is fairly stated, in all material respects, in relation to the
basic financial statements taken as a whole.

Boston, Massachusetts v jQOOtHO . <Av^t>nOrnd

April 16, 1993 I

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XPENSES:
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K10

Financial Statements Rl 1

Marine Biological Laboratory

Notes to Financial Statements

A. I'urpose nl 1 lie Laboratory:

The purpose of Manne Biological Laboratory (the "Laboratory") is to establish and maintain a laboratory or station for scientific study and
investigations, and a school lor instruction in biology and natural history.

B. Significant Accounting Policies:

Basis of Presentation Fund Accounting

In order to ensure observance of limitations and restrictions placed on the use of resources available to the Laboratory, the accounts of the
Laboratory are maintained in accordance with the principles of fund accounting. This is the procedure by which resources are classified into
separate funds in accordance with specified activities or objectives. Separate accounts are maintained for each fund; however, in the accompanying
financial statements, funds that have similar characteristics have been combined into fund groups. Accordingly, all financial transactions have
been recorded and reported by fund group.

Externally restricted funds may only be utilized in accordance with the purposes established by the donor or grantor of such funds. However,
the Laboratory has full control over the utilization of unrestricted funds. Restricted gifts, grants, and other restricted support are accounted for
in the appropriate restricted funds. Restricted current funds are reported as revenue as the related costs are incurred (see Note G).

Endowment funds are subject to restrictions which require that the principal be invested in perpetuity. Related investment income is available
for use for restricted or unrestricted purposes by the Laboratory depending on donor restrictions. Quasi-endowment funds have been established
by the Laboratory for the same purposes as endowment funds; however, the principal of these funds may be expended for various restricted and
unrestricted purposes.

Fixed Assets

Land, buildings and equipment purchased by the Laboratory are recorded at cost. Donated fixed assets are recorded at fair market value at the
date of the gift. Depreciation is computed using the straight-line method, beginning the month after the asset is placed in service, over the asset's
estimated useful life. Estimated useful lives are generally three to five years for equipment and 20 to 40 years for buildings and improvements.
When assets are sold or retired, the cost and accumulated depreciation are removed from the accounts and any resulting gain or loss is included
in income for the period.

Contracts and Grants

Revenues associated with contracts and grants are recognized in the statement of support, revenues, expenses and changes in fund balances as
the related costs are incurred (see Note G). Reimbursement of indirect costs relating to government contracts and grants is based on negotiated
indirect cost rates. Any over or underrecovery of indirect costs is recognized through future adjustments of indirect cost rates.

Investments

Investments purchased by the Laboratory are carried at market value. Money market securities are carried at cost plus accrued interest, which
approximates market value. Donated investments are recorded at fair market value at the date of the gift. Land held for sale included in
investments is carried at the initially recorded market value of $330.000. For determination of gain or loss upon disposal of investments, cost is
determined based on the first-in, first-out method.

The Laboratory is the beneficiary of certain investments reported in the endowment funds which are held in trust by others. The Laboratory's
continuing right to these funds is subject to review every ten years by an independent committee. The market values of such investments are
$4,743.257 and $4.717.315 at December 31, 1992 and 1991, respectively.

Investment Income and Distribution

The Laboratory follows the accrual basis of accounting except that investment income is recorded on a cash basis. The difference between such
basis and the accrual basis does not have a material effect on the determination of investment income earned on a year-to-year basis.

Investment income includes income from a pooled investment account, which income is allocated to the participating funds on the market value
unit basis (Note D).

Annuities Payable

Amounts due to donors in connection with gift annuities are determined based on remainder value calculations which at December 31, 1992
assumed a rate of return of 10%. maximum payout terms of 18 years, and an interest payout rate of 8%.

Tux-Exempt Status

The Laboratory is exempt from federal income tax under Section 501(c)3 of the Internal Revenue Code.

R12 Annual Report

C. Investments:

The following is a summary of the cost and market value of investments at December 31, 1992 and 1991:

Market

Cost

Certili' ueposit

Mone\ ' ket securities
U.S. < ' ei nment securities
Corporate fixed income
Common stocks
Real estate

Total investments

1992

1991

$ 47,700

$ 258,351

1,952,867

1,767,300

1,788,066

1,456,944

8,968,745

8,387,547

6,764,488

7,864,060

343,247

343,247

$19,865,113

$20,077,449

7992

1991

$ 46.745

$ 258.351

1,952.867

1.767.300

1,771,308

1,423.722

8.533,859

7,718,034

4,173,965

4,589,520

343,247

343,247

$16,821,991

$16,100,174

Investments by fund group and related portfolios for the years ended December 31. 1992 and 1991 are as follows:

Current Funds
Certificates of deposit
Money market securities
Library funds
Total

47,700

1.400,000

180.570

1.628,270

258.351

1.200.000

320,419

1.778,770

46.745

1,400,000

183.548

1,630,293

$ 258.351

1,200.000

317.557

1,775,908

Market

Cost

Endowment and Quasi-Endowment
General endowment trust fund
Library endowment trust fund
Ecosystem funds
Pooled funds
Instruction fund
Real estate

Total

Total investments

3,755.124

3,735,252

988,133

982,063

4,507,857

4,488,315

6,910,904

6,608,044

1.731,578

2,141,758

343,247

343,247

18,236.843

18,298,679

$19.865,113

$20.077,449

2,897.718

2,707,869

753,904

635,203

3,756.446

3.411,559

5,806,114

5,253,616

1,634,269

1,972,772

343,247

343.247

15,191,698

14,324.266

$16,821.991

$16,100.174

D. Accounting for Pooled Investments

Certain endowment fund assets are pooled for investment purposes. Investment income from the pooled investment account is allocated on the
market value unit basis, and each endowment fund subscribes to or disposes of units on the basis of the market value per unit at the beginning
of the calendar quarter within which the transaction takes place. The unit participation of the funds at December 31. 1992 and 1991 is as follows:

Quasi-endowment unrestricted
Quasi-endowment restricted
Endowment, income for restricted purposes
Endowment, income for unrestricted purposes

Total

4,260

8.717

40,345

79

53,401

1991

4,618

8,644

39.458

52.720

Pooled investment activity on a per-unit basis was as follows:

Unit value at beginning of year
Unit value at end of year

Increase in realized and unrealized appreciation
Net income earned on pooled investments

Total return on pooled investments

1992

$128.42
128.66

.24
5.71

$ 5.95

1991

$108.90

128.42

19.52
5.15

$ 24.67

Financial Statements R13

E. Deposits and Commitments for Construction Programs:

As of December 31, 1992, the Laboratory has $285,094 in restricted cash for the construction of the new Collection Support Facility and
$1.548,810 in Deposit with Trustees for the renovations of laboratories and housing facilities. On December 31, 1992, the Laboratory was
contractually obligated for approximately $1.616,288 of additional expenditures in connection with its current building program. The expenditures
are covered by funding commitments.

F. Long-Term Debt:

Long-term debt at December 31, 1992 amounted to $2,600,000. The aggregate amount of principal due for each of the next five fiscal years is
as follows:

1993
1994
1995
1996
1997
Thereafter

Less current portion
Total

$ 65,000
65,000
75.000
75,000
80,000
2.240,000

2,600,000
65.000

$2,535,000

In 1992, the Laboratory issued $1,100,000 Massachusetts Industrial Finance Authority (MIFA) Series 1992A Bonds and $1,500,000 MIFA Series
1992B. These bonds pay varying annual interest rates and Series 1992 A and B Bonds mature on December 1, 2012. The Series 1992 A and B Bonds
are collateralized by a first mortgage on certain Laboratory property.

A portion of Series 1992B Bonds were issued for the purpose of refunding the $1,330,000 MIFA Series 1989 Bonds, which pay varying annual
interest rates and mature on October 31, 2011. The amount of Series 1989 principal outstanding at December 31. 1992 was $1,140,000. The
Laboratory has on deposit investments of $1,306,172 with Shawmut Bank N.A., as trustee for the MIFA Series 1989 Bonds, for redemption February
1993.

In compliance with the 1992 MIFA bond indentures, the Laboratory has on deposit with State Street Bank and Trust, as trustee for Series 1992
Bonds, investments for construction projects in the amount of $1,548,810. In 1991, the Laboratory was required to have on deposit $126,1 16 for a
debt service reserve fund.

Under the most restrictive covenant of long-term debt, the Laboratory's operating surplus (before transfers), interest, expense and transfers from the
quasi-endowment for debt service must equal or exceed all debt service payments.

G. Reslneled Current Funds Deferred Support:

The Laboratory' defers revenue on current restricted funds until the related costs are incurred. Amounts received in excess of expenses are recorded
as deferred support. The following summarizes the activity of the deferred support account:

1992

1W1

Balance at beginning of year
Additions:

Gifts, endowment income and grants received

Net unrealized gains (losses)

Net realized gains

Transfers
Deductions:

Funds expended under gifts and grants

Transfers

Balance at end of year

$4,437,140

7,997,702
(77,518)
78,636

8,722,851
49.411

$3,663,698

$4,774,618

7,832,555

121.308

5,102

36.777

$4.437.140

Deferred restricted gifts of $453,970 and $475,874 were expended in 1992 and 1991, respectively, for the support of indirect costs attributable
to the Laboratory's instruction programs.

H. Retirement Plan:

The Laboratory participates in the denned contribution pension plan of TIAA-CREF (the "Plan"). The Plan is available to permanent employees
that have completed two years of service. Under the Plan, the Laboratory contributes 10% of total compensation for each participant. Contributions
amounted to $502,215 in 1992 and $495,848 in 1991.

R14 Annual Report

I. Pledges:

As of December 31, 1992, the Laboratory has outstanding pledges of $737,146 of which $681,728 is restricted (unaudited). Pledges are not
included in the financial statements since it is not practicable to estimate the net realizable value of such pledges. These pledges are scheduled to
be paid over the next three years in the amounts of $509,969, $169,177, and $58.000, respectively.

J. Interfund Borrowings:

Current unrestricted fund mterfund borrowings at December 31 are as follows:

1992 1991

Due to restricted endowment fund $( 1 1 8,755) $ (4,1 50)

Due to restricted quasi-endowment funds (50) (150.000)

Total $(118.805) $(154,150)

K. Financial Accounting Standard No 106:

In December 1990, the Financial Accounting Standards Board (FASB) released Statement No. 106, "Employers' Accounting for Postretirement
Benefits Other Than Pensions." This new standard requires employers to accrue, during the years that the employee renders the necessary service,
the expected cost of benefits to be provided during retirement, and will apply to years beginning after December 15, 1994. The Laboratory is
currently analyzing and interpreting the provisions of the Statement as it relates to its current and planned benefits program and its funding
options. It is anticipated that adoption of this accounting standard will result in the Laboratory recording a significant liability.

SCHEDULE I

MARINE BIOLOGICAL LABORATORY

STATEMENT OF SUPPORT. REVENUES, EXPENSES AND CHANGES IN FUND BALANCES

CURRENT FUNDS
for the year ended December 31, 1992

SUPPORT AND REVENUES:

Grant reimbursements of direct costs

Recovers of indirect costs

Tuition

Fees for ser\ ices:

Dormitories

Dining hall

Library

BiolnKiail BU//CIIII

Research services

Marine resources
Investment income

Gifts

Change in deterred support

Miscellaneous revenue

Total support and revenues

EXPENSES:
Research

Instruction

Scholarships, fellowships and stipends

Services:

Dormitories

Dining hall

Libra ry

Biological Bulletin

Research services

Marine resources
Administration:

Administration

Sponsored projects administration
Plant operations
Other

Total expenses

Excess of support and revenues over
expenses

Net unrealized gain on investments
Net realized (loss) on investments

Net gain on investments

TRANSFERS AMONG FUNDS:
Debt service

Acquisition of fixed assets
Repairs and replacement
Endowment transfer
Capitalization of income
Other

Total transfers among funds
Net change in fund balances
Fund balances, beginning of year
Fund balances, end of vear

Current I'nretlricled Fund\

Auxiliary

Current

Operating

Enterprises

Restricted

Fund

Fund Total

Fund

Total

$5.982.265

$5.982.265

$3.530.621

$3,530,621

3.530.621

485.247

485.247

$ 974.076 974.076

974.076

824.327 824.327

824,327

439.822

439,822

439.822

223.81!

223,811

223.811

459.233

459.233

173.733

632.966

179.929

179.929

179.929

406.212

406,212

521,698

927,910

5,239.628

1,798.403 7.038.031

7.162.943

14.200.974

428.711

428.711

954,248

1.382.959

453.970

453.970

319,472

773.442

882.681

882,681

1,273.720

2.156,401

97.429

97,429

334.481

431.910

6.219.738

1.798.403 8.018.141

8.771.144

16,789.285

6.019,891

6.019.891

1.526,154

1,526,154

394.544

394.544

745.787 745.787

745.787

676.949 676.949

676.949

736.347

736.347

302.135

1.038.482

189.098

189.098

189.098

622.731

622.731

179.904

802.635

433,825

433.825

5.847

439.672

2.254,935

145.641 2.400.576

10.000

2.410.576

351.177

351.177

351.177

1.585.953

1.585.953

40.805

1,626,758

243.571

243.571

6.174.066

1.568.377 7.742.443

8.722.851

16.465.294

45,672

230.026 275.698

48.293

323.991

78.636

78.636

(77.518)

(77.518)

1.118

1,118

(60.000) (60.000)

(60.000)

(91.313)

(12.026) (103.339)

(103.339)

(158.000) (158.000)

(158.000)

200,000

200.000

(219.539)

(219.539)

37.100

37.100

(29.872)

7.228

(54.213)

(230.026) (284.239)

(49,41 1)

(333.650)

(8,541)

(8,541)

(8.541)

21,398

21.398

21.398

$ 12.857

$ 12.857

$ 12,857

SCHEDULE II

MARINE BIOLOGICAL LABORATORY
STATEMENT OF SUPPORT, REVENUES, EXPENSES AND CHANGES IN FUND BALANCES

ENDOWMENT FUNDS
for the year ended December 31, 1992

SUPPORT AND REVENUES:
Gifts

Total support and revenues

Net realized gain on investments
Net unrealized (loss) on investments

Net gain (loss) on investments

TRANSFERS AMONG FUNDS:
Capitalization of income

Endowment transfers

Total transfers among funds

Net change in fund balances
Fund balances, beginning of year
Fund balances, end of year

Unrestricted

Restricted

Income for
Unrestricted
Purposes

Income for
Restricted
Purposes

Quasi-

Endowmcnt

Total
Restricted

Total

Qtiuxi-
Endowmeni

$ 23,038
(23,216)

$ 128,755

$ 151,140

$ 425

$ 280,320

$ 280,320

128,755

151,140

425

280,320

280,320

1X9.911
(169.785)

323.933
(304,847)

384,875
(360,770)

898.719
(835.402)

921,757
(858,618)

(178)

20.126

19.086

24,105

63,317

63,139

5,190

214,349
(200,000)

214,349
(200.000)

219.539
(200,000)

5,190

14,349

14.349

19,539

5,012

148,881

170,226

38,879

357,986

362,998

543,049

3,735,253

6.049.1)07

5,590.516

15,374.776

15.917.825

$ 548,061

$ 3,884,134

$ 6.219,233

$ 5.629,395

$15,732,762

$16,280,823

RI6

SCHEDULE III

MARINE BIOLOGICAL LABORATORY
STATEMENT OF SUPPORT, REVENUES, EXPENSES AND CHANGES IN FUND BALANCES

PLANT FUNDS
for the year ended December 31, 1992

SUPPORT AND REVENUES:
Grant tor capital additions
Gifts
Other revenue

Total support and revenues

EXPENSES:
Depreciation
Plant operations
Other

Total expenses

Excess (deficit) of support and revenues over expenses

TRANSFERS AMONG FUNDS:
Debt service

Acquisition of fixed assets
Capital additions, net of disposals
Repairs and replacements
Other transfers

Total transfers among funds
Net change in fund balances
Fund balances, beginning of year
Fund balances, end of year

( nicslncled

\ 722,959

3.410
726.369
(726.369)

60.000

107.495

10.232.950

10.400.445
9.674.076
10.034.642

Unrestricted

Repairs and

Replacements

Reserve

$ 158,901

158.901
(158,901)

Total
Unrestricted

722.959

158,901

3.410

885.270
(885.270)

Restricted

$4,032.660
15,000

14.473

4,062,133

4.062.133

60.000

107,495

10,232,950

(10,232.950)

158,000

158,000

9.082

9,082

(20,466)

167.082

10,567,527

(10.253,416)

8.181

9,682,257

(6,191.283)

188.289

10.222.931

6,214.181

Total

$ 4.032,660
15,000

14.473

4.062,133

722,959

158.901

3.410

885.270
3,176.863

60.000
107.495

158,000
(11.384)

314.111

3.490.974

16.437,112

$19.708.718 $ 196.470 $19.905.188 $ 22.898 $19.928.086

R17

Report of the Library Director

The Libraiy

The Library's work during 1992 concentrated on the
journal collection, the book collection, the rare book
room, cataloging, and cooperative relationships.

The journal collection

The journal collection is the heart of the Library's
service. The MBL/WHOI Library, in common with all
academic research libraries, is working to sustain the
collection as far as possible, despite a 12% annual
escalation in scientific journal prices over the past
decade. Given present national trends in research
funding, sustaining the collection calls for innovative
cooperation with the publishing industry. Working with
Elsevier publishers, the Library was successful in
restoring an important journal. Brain Research, that
was discontinued in 1991.

However, the spiraling cost of the journal collection
is a long-term problem, and each year the Library is
faced with difficult choices. After extensive review by a
panel of 50 scientists, the journal collection was
reduced by 32 titles in 1992. Most of the discontinued
journals were in clinical and medical areas. These
reductions were partially offset by the addition of seven
other journals: Molecular Microbiology, Physical
Review E. H 'ate r Environment Research, the Canadian
Journal of Fisheries and Ai/uatic Sciences. Perspectives
on Science. Issues in Science and Technology, and
Serial Sources for the Biosis Previews.

The book collection

The MBL/WHOI book collection was reviewed to
strengthen it and to increase its utility. The Joint MBL/
WHOI Library Advisory Committee defined the role of
the collection as follows: ". . . to collect, for general
use, those volumes of seminal and enduring value
supporting the major scientific fields in which the
scientific community is active. The collection should
provide a scientist who is proficient in a given field with
entry to other disciplines and with broad reviews of
techniques and current directions supporting his or her
own work."

To meet this purpose, the book collection should be
developed with care and should become more available.
Some 19% of the collection 7000 books or roughly 30
for every scientist in Woods Hole are in circulation,
including half of the books acquired in the last 3 years.
The books are withdrawn for an average of two and
a half years, and one book has been taken out for
twenty-one.

To remedy this situation, the Joint MBL/WHOI
Library Advisory Committee has approved a one-year
checkout policy to improve the accessibility of the book
collection. A project was started in the fall of 1992 to
recall all Library books and check out those that
scientists still needed for a one-year period. This project
has allowed us to locate and catalog many books that
had been held indefinitely in laboratories and offices.
The recall project has a long way to go, but it is
progressing steadily and will be completed by the end of
1993.

R18

Report of the I.ibrar> Director R19

The early winter months bought severe storms to
Woods Hole. Strong winds drove rain under the roof of
the Lillie building, causing leaks onto the book
collection. The wet books, 100 volumes, were frozen
and dried: all but two were returned in good condition
to the shelves. All books have been moved from the
area under the leaks, and a capital project has been
approved, subject to funding, for repair of the roof.

The Rare Book Room

The Library's Rare Book Room is an MBL treasure.
This year the collection has been enhanced by the
donation of a number of old and rare volumes.
Through a gift from Cambridge Scientific Abstracts, we
have photographed over 100 Leuckart charts, which
have also been made into 35-mm slides that can be
useful teaching aids. The slides are currently being
digitized for easy transfer in electronic format. Our
complete collection of these rare wall charts is now
maintained in a specially constructed cabinet that
preserves them in a acid-free environment in the Rare
Book Room.

The rare book collection is currently being placed in
the Library catalog. The audio tapes of Friday Evening
Lectures, Falmouth Forum, and other lectures have
been cataloged and filed. Furniture in the Agassiz
Room has been restored, and the framed photographic
collection has been identified, covered with LJV paper,
and hung in related groups in the Agassiz Room and
the Rare Book Room. A new cherry filing cabinet has
been added to the Agassiz Room to house the
biographical files of scientists who have worked at the
MBL. The off-season displays in the Lillie Building
lobby are maintained by the staff of the Rare Book
Room.

Cataloging

The Library worked to make electronically available
information more complete and accurate. The catalogs
in the Library's computer, the CLAMS system, have
been brought up-to-date. The currently subscribed-to
serials have been added to the serials catalog, and those
journals acquired through the exchange of The
Biological Bulletin with other institutions are being
added. The book collection has been entered into the
system, with the exception of expeditions and a few
difficult monographic series that require specialized
cataloging. The Library began automation of the book
circulation process in the fall. Year-round patrons now
have a book card, and circulation has been transferred
to the automated system.

( 'imperative relationships

The Library's membership in the Boston Library
Consortium flourished during the year. Among other
benefits, this membership provides us with no-cost
access to the journal collections of 12 academic libraries
in the Boston area. This access privilege is in active use,
saving Woods Hole scientists $20,000 annually in direct
charges for articles obtained from other libraries. The
addition of a networked document scanning station
(ARIEL), financed in part by a grant, has added
interlibrary document delivery over the Internet to our
services. This station makes it possible electronically to
obtain high resolution document images from Boston
libraries.

The lowered cost and improved response of these
agreements and systems have made it possible to
fashion a reciprocal agreement with Brandeis University
to develop our collections in concert. Under this
agreement, the institutions each agree to retain certain
journals. Tables of contents of these journals will be
sent monthly to the partner library, and documents
from these journals will be delivered with the goal of
24-hour response time.

Other Library activities included the successful
September open house, publication of a handy booklet
for users of electronic services, and a new program of
presentations by Woods Hole scientists that give the
Library staff insight into research in the institutions
thev serve.

The Information Systems Division

The Information Systems Division (ISO) has been
extremely productive over the past year, completing
projects in Library systems, data networks, classroom
and laboratory support, and instruction in Medical
Informatics.

Lihrury systems

The bibliographic search system, based on CD-ROM
technology, was upgraded and made reliable. The
system now makes five bibliographical data bases
available, including Aquatic Sciences and Fisheries
Abstracts; Life Sciences Abstracts; National Library of
Medicine's Medline Service: and The Wilson Disc
General Science Index. The fifth and latest is GeoRef, a
bibliographic compendium of geological literature from
1785 to the present. The system can now send selected
abstracts to scientists' desks by e-mail, or it can directly
download the abstracts onto floppy discs on office

R20 Annual Report

computers. All of these services are available over the
Internet to Woods Hole scientists -. tiere in the
world as part of the Library's b :. no-cost service. The
majority of bibliographic se;i' now come to the
Library's systems over th .net rather from the local
terminals in the Librar oy.

The Information ' .s Division has gone beyond
these services to de. p an electronic menu presenting
a variety of sen - . at MBL and at other institutions. It
is possible to . > specimens, equipment, or Chemistry
Room supplies; to directly access grant programs at
NSF, NIH. NASA, and DOD; to access catalogs of
libraries around the world; or to learn about MBL's
course offerings, services, and rates through this Library
"front end." All Library patrons can have access to this
service by talking with the Librarian.

The data network

The MBL data network has been extended to all
campus buildings, including the Marine Resources
Center. A new computer was procured to handle
increased electronic mail traffic. Equipment to isolate
data transmissions is being installed so that laboratories
and classrooms do not load the MBL network with
their internal traffic. The network was available with
99.8% reliability during 1992.

Classroom and laboratory support

In 1992, the Information Systems Division became
responsible for MBL computing support to courses,
wherever that was needed. This involved the
installation, and subsequent removal, of 80 networked
computers for four courses. As part of the grant from

the National Library of Medicine (NLM) for Medical
Informatics training, ISD was called on for selection
and installation of 30 personal computers, and for full
technical support of a one-week course taught by NLM
in early June. More recently the Ecosystems Center has
asked the ISD to take care of the computer network in
their laboratories.

Instruction in medical informatics

This year was the first of a grant from the National
Library of Medicine to support instruction in Medical
Informatics. Following the formal course given by the
NLM, the Information Systems Division continued to
teach medical informatics to summer course sessions
and to audiences at special short courses. These
Medical Informatics sessions attracted 150 individuals.

Loeb 308 is being prepared for use as a computer-
ready classroom. The computers and software, network
connections, and special air handling equipment will
assist several courses during the summer as well as the
Medical Informatics program.

The Coming Year

The Library is grateful for the support of the MBL
science community and for the help of its many friends.
Our goals for the coming year include sustaining the
journal collection, improving its housing, and
developing electronic delivery of articles from the
collection directly to scientists' desks. We look forward
to 1993-1994 as another active and productive year.

David L. Stonehill

Educational Programs

Summer Courses

Biology of Parasitism: Modern Approaches
(June 14-August 15)

Course Director
John Boothroyd. Stanford University School of Medicine

Associate Director

Richard Komuniecki, University of Toledo

Faculty

Jean-Francois Dubremetz. INSERM. France

Alan Fairlamh, London School of Hygiene and Tropical Medicine,

UK

Fred Finkelman, USUHS/Herhert School of Medicine
Kasturi Haldar, Stanford University School of Medicine
Joe Urban. United States Department of Agriculture

Instructor

Richard E. Davis. San Francisco State University

Teaching Assistants

Patricia Dorn. Stanford University School of Medicine

Erniho Duran. University of Toledo

Ashraf El Meanawy. University of Cairo Medical School. Egypt

Heidi Elmendorf. Stanford University School of Medicine

Suzanne Morns. USUHS/Herbert School of Medicine

Steven L. Reiner. University of California. San Francisco

Keith Smith. London School of Hygiene & Tropical Medicine, UK.

Marline Socle. INSERM. France

Course Assistants

Michele Klingbeil. University of Toledo
Michelle Rathman, Stanford University

Students

Thomas Allen. Oregon Health Science University
Prasanta Chakraborty. Washington Llnixersity
Claire Chougnet. INSERM, France
Johanna Daily, Beth Isreal Hospital

Hans Hagen. Keele University. UK

Norton Heise, Escola Paulista de Medicina. Brazil

Hans-Juergen Hoppe. Oxford University, UK

Kuo-Yuan Hwa, Johns Hopkins University

Aslog Jansson, Uppsala University, Sweden

Assan .lave. ILRAD. Kenya

Adrian Lawrence. Albert Einstein College of Medicine

Jose Lima Filho, Harvard School of Public Health

Ingrid Loeffler. Michigan State University

Michael Mdntosh. Hahnemann University

Fernando Monroy, University of New Mexico

Carlos Moreno. New York University Medical Center

Sharon Moshitch. Weizmann Institute of Science. Israel

Hagir Suliman, Virginia Tech

Lakshmi Venkatakrishnaiah. Medical University of South Carolina

Ying-zi Yang. CUNY Medical School

Embryology: Cell Differentiation and Gene

Expression in Early Development

(June 20-July 31)

Course Directors

Eric H. Davidson, California Institute of Technology
Michael Levine, University of California, San Diego
David R. McClay, Duke University

Faculty

Marianne Bronner-Fraser, University of California, Irvine

Andrew R. Cameron. California Institute of Technology

Scott E. Fraser, California Institute of Technology

Janet Heasman. Wellcome/CRC Institute, UK

Steven L. McKnight. Carnegie Institution of Washington

Noriyuki Saloh. K\oto University, Japan

Paul Sternberg. California Institute of Technology

Christopher C. Wylic. Wellcome/CRC Institute, UK

Faculty Adjunct

David Epel. Stanford University

Teaching Assistants

Mary K. Anderson. Johns Hopkins LIniversity/Carnegie Institute of
Washington

K2I

R22 Annual Report

Helen Chamberlin, California Institute of Technology
Michael Dunn. Massachusetts General Hospital
Michael Figdor, California Institute of Technology
Suresh J. Jesuthasan, Oxford University, UK
Carole LaBonne. Harvard University
Jeffrey R. Miller. Duke University
Talma Y. Scherson, University of California, Irvine
John Shih, California Institute of Technology
Stephen Small. University of California, San Diego
Sergei Sokol, Harvard University
Tanya Whitfield, Wellcome/CRC Institute. UK
Robert W. Zeller. California Institute of Technology

Lecturers

William McGmnis. Yale University
Randy Moon. University of Washington

Speakers

Don Fischman, Cornell University Medical College
Nancy Hopkins. Massachusetts Institute of Technology
Alexander Johnson. University of California. San Francisco
Andrew P. McMahon, Roche Institute
James Posakony. University of California, San Diego
Claudio Stern, Oxford University, UK

Course Administrator

Jane Rigg, California Institute of Technology

Course Coordinator
Linda Hufl'er. Marine Biological Laboratory

Course Assistants
Cheryl Booth, University of Wisconsin
Alex Goldberg, Washington University

Students

Yupmg Cai, Michigan State University

Isabelle Desjeux. Edinburgh University. Scotland

Corrella Detweiler, Max-Planck Institute. Germany

Carmen Domingo. University of California, Berkeley

Mane-Anne Felix, Institut Jacques Monod, France

Gabor Forgacs, Clarkson University

Rudiger Fntsch, Max-Planck Institute. Germany

Tony Frudakis, University of California, Berkeley

Eleonore Fusco, Massachusetts Institute of Technology

Maureen Gannon, Cornell University Medical College

Uta Gneshammer, Boston University

Catriona Logan, Duke University

Lily Lou, Yale University

John Matese, Duke University

Anna Myat, Imperial Cancer Research Fund. UK

Lesley Narburgh, St. George's Hospital Medical School, UK.

Zoe Pettway, University of California, Irvine

David Pleasure, Children's Hospital of Philadelphia

Paola Polosa, Llniversity of Ban, Italy

Remhard Schroder, Freiburg University. Germany

Mark Van Doren, Llniversity of California, San Diego

Paul Vrana. American Museum of Natural History

Daniel Wagner, University of Texas Anderson Cancer Center

Cindy Wilson, University of California, Irvine

Stella Zannini. Stazione Zoologica "A Dohrn". Italy

Hong Zhang, Case Western Reserve University

Microbial Diversity (June 14-July 30)

Course Directors
John Breznak, Michigan State Llniversity
Martin Dworkin. University of Minnesota

Course Coordinator

Richard M. Behmlander, University of Minnesota

Course Assistant

Pamela Contag. Stanford University Medical School

Teaching Assistants
Joseph P. Calabrese. West Virginia University
S. Courtney Frasch, University of Minnesota
Jorg Overmann, University of British Columbia. Canada
Daniel R. Smith, University of Minnesota

Instructor

Howard Gest, Indiana University

Lecturers

Steve Block, Rowland Institute for Science
Colleen Cavanaugh, Harvard University
Paul Dunlap. Woods Hole Oceanographic Institution
Dale Gehnnger, Woods Hole Oceanographic Institution
Steve Goodwin, University of Massachusetts, Amherst
Brian Howe, Woods Hole Oceanographic Institution
Holger Jannasch. Woods Hole Oceanographic Institution
Shadid Khan, Albert Einstein School of Medicine
Edward Leadbetter, University of Connecticut, Storrs
Michael Madigan. University of Southern Illinois
Sandra Nierzwicki-Bauer, Rensselaer Polytechnic Institute
Bernhard Schink, University of Konstanz, Germany
Mitchell Sogin, Marine Biological Laboratory
Karl Stetter. University of Regensburg, Germany
Sidney Tamm, Boston University Marine Program, MBL
John Waterbury, Woods Hole Oceanographic Institution
Carl Woese, University of Illinois

Students

Valerie Bernan. Lederle Labs

Jennifer Byrnes. Harvard University

Francesco Canganella, Llniversity ofTuscia. Italy

Susan Childers, University of Connecticut. Storrs

Maria Ganeva. Sofia Llniversity, Bulgaria

John Gibson, Florida State University

Antje Hofmeister, Philipps-L'niversity Marburg, Germany

Scott Kroken, University of Wisconsin

Ariel Kusmaro, Tel Aviv University. Israel

Edouard Miambi, Center ORSTOM/DGRST. Congo

Marganta Miroshnichenko. Moscow State Llniversity. Russia

Tommy Nielson. Aarhus University, Denmark

Lorraine Olendzenski, University of Massachusetts, Amherst

Martin Polz, Harvard University

Michael Renner, Michigan State University

Karl Rusterholtz, Merck Sharp & Dohme Research Lab

Heinnch Sandmeier, University of Basel, Switzerland

Dirk Schuler. Max-Planck Institute. Germany

Angelica Seitz, University of Connecticut, Storrs

Robert Shannon. Indiana Llniversity

Educational Programs R23

Neural Systems & Behavior (June 14-August 7)

Course Directors
Ronald L. Calabrese, Emory University
Martha Constantme-Paton. Yale University

Faculty

Thomas Abrams. University of Pennsylvania

Robert Douglas. University of British Columbia. Canada

Scholars-in-Residence

Larry Abbott, Brandeis University

Man E. Hatten. College of Physicians & Surgeons at Columbia

University
William T. Newsome. Stanford University

Instructors

Alexander Borst. Max-Planck-Institiit fur Biologische Kybernetik.

Germany

Holly Cline. University of Iowa
Sally Hoskins. City College of New York
Alan Kay. University of Iowa
John Koester. Columbia University
Richard B. Levine. University of Arizona
Eduardo Macagno. Columbia University
Robert Malinow, University of Iowa
Michael Nusbaum, University of Alabama, Birmingham
Mu-Ming Poo. Columbia University
Leslie Stevens. Albert Einstein College of Medicine
Janis C. Weeks. University of Oregon
Angela Wenning, Universitat Konstanz. Germany

Lecturers

Gwendal Le Masson. Brandeis University
William M. Roberts. University of Oregon
Edgar T. Walters, University of Texas Medical School

Guest Lecturers

Robert Barlow, Jr.. Syracuse University
John Dowling. Harvard University

Teaching Assistants
Yolonda Alston, Benedict College

Melissa J. Coleman. University of Alabama. Birmingham
Yang Dan. Columbia University
Lise Eliot. Baylor College of Medicine
Cole Gilbert. Cornell University
Juergen Haag. Max-Planck-lnstitiit fur Biologische Rybernetik.

Germany

Dawn Lewis. Albert Einstein College of Medicine
Ann Lohof. Columbia University
Brian J. Norris. University of Alabama. Birmingham
Andrea Novicki, University of Oregon
Glen Prusky. Yale University
David J. Sandstrom, University of Oregon
Laura Wolszon, Columbia University

Course Coordinator

Miriam Ashley, University of California, Irvine

Course Assistant

Kyle Lennon, Atlanta, GA

Students

Lisa Boulanger, Wesleyan University

Mary Boyle, University of California, San Diego

Beatrice Casasnovas. University of Bordeaux. France

Peter Dayan. Salk Institute

Joseph Erlichman. Dartmouth Medical School

Daniel Feldman, Stanford University School of Medicine

Maribel Feliciano, University of Connecticut, Storrs

Maria Feller, AT&T Bell Laboratories

Lisa Foa, Deakin University, Australia

Timothy Gershon, Columbia University

Erin Jacobs, University of California, Los Angeles

Juan Jorge-Rivera, Brandeis University

Lisa Kelly. University of Ottawa, Canada

Carole Landisman. Rockefeller University

Stephen Macknik, Harvard Medical School

Zachary Mamen, University of California, San Diego

David Raizen. University of Texas Southwestern Medical School

Jeffrey Reznic, New York University

Christine Rose. University of Kaiserslautern, Germany

Stephan Wurden, University of Konstanz, Germany

Neurobiology (June 14-August 15)

Course Directors

Leonard K. Kaczmarek, Yale University School of Medicine
Irwin B. Levitan, Brandeis University

Faculty

Hana Asmussen. University of Virginia Medical School

Gary Banker, University of Virginia

Arlene Chiu, Beckman Research Institute

Judith A. Drazba. NIH/NINDS

Keith Elmslie. Case Western Reserve University

Steve Goldstein, HHMI/Brandeis University

Richard Horn. Jefferson Medical College

Stephen Jones. Case Western Reserve University

Bechara Kachar. NIH/N1DCD

Julie A. Kauer. Duke University Medical School

Richard Kramer. Columbia University

Lonny Levin, Johns Hopkins University

John Marshall, Yale University School of Medicine

Andrew 1. Matus, Friedrich Miescher Institute. Switzerland

Sally Moody. University of Virginia School of Medicine

Angus C. Nairn. Rockefeller University

Marina Picciotto, The Pasteur Institute. France

Randall Reed, HHMI/Johns Hopkins Medical School

Thomas Reese, NIH/NINDS

Peter H. Remhart, Duke University Medical Center

Talvinder Sihra, University of Dundee. Scotland

Carolyn Smith, NIH/NINDS

Lecturers

George Augustine, University of Southern California

David Brautigan. Brown University

Xandra Breakeneld, Massachusetts General Hospital

William Catterall, University of Washington

Pietro DeCamilli. Yale University

Michael Greenberg. Harvard University

R24 Annual Report

Lloyd Greene, New York University

Michael R. Hanley, University of California, Davis

Ed Havvrot, Brown University

Robert Horvitz, Massachusetts Institute of Technology

Richard Huganir, Johns Hopkins School of Medicine

Christopher Miller. Brandeis University

Linda Nowak, Cornell University

Dale Purves, Duke University

Edward D. Salmon, University of North Carolina, Chapel Hill

Chris Walsh. Harvard University

Course Assistants

Ethan Treistman, Llniversity of North Carolina, Chapel Hill
Cecilia Armstrong. University of Pennsylvania

SllK/C/ltS

Pavle Andjus, University of Belgrade. Yugoslavia
Cynthia Cowden, University of Wisconsin
Ann Marie Craig, University of Virginia
Matthew Dalva. Duke University
Atsushi Miyawaki. University of Tokyo, Japan
Klaus Raming, Hohenheim Llniversity. Germany
Enrique Saldana, University of Salamanca, Spain
Hilary Smith, University of North Carolina. Chapel Hill
Camilla Tornoe. Cambridge University, UK
Ferdinand Vilim, Columbia University
Elisabeth Walcott, Llniversity of California, Irvine
Kevin Wickman, Mayo Foundation

Physiology: Cellular and Molecular Biology
(June 13- July 25)

Course Director
Thomas D. Pollard, Johns Hopkins Medical School

Faculty

Robert Jensen. Johns Hopkins Medical School

Michael E. Mendelsohn, Harvard Medical School/Brigham &

Women's Hospital

Andrew Murray, University of California. San Francisco
Edward D. Salmon, University of North Carolina, Chapel Hill
Cynthia Stauffacher, Purdue Llniversity
Murray Stewart. Medical Research Council, UK
Katherinc Swenson, Duke University Medical School
Edwin Taylor, University of Chicago
Ron Vale, Llniversity of California, San Francisco
Katherine L. Wilson, Johns Hopkins Llniversity School of Medicine

Instructors

William B. Busa. Johns Hopkins University
Margaret A. Titus, Duke University Medical Center

Teaching Assistants

Michael Glot/er, University of California. San Francisco
John R. Jordan, Llniversity of Utah. Salt Lake City
Jenmter Kalish, Johns Hopkins Medical School
Helen Kent. Medical Research Council, UK
C. Martin Lawrence. Purdue University
Sarah O'Neill, Brigham & Women's Hospital/Harvard Medical

School
Stephen F. Parsons. Llniversity of North Carolina. Chapel Hill

Homero L. Rey, University of California. Berkeley

Yan Zhu, Brigham & Women's Hospital/Harvard Medical School

Lecturers

Paula Fitzgerald, Merck Corporation

William Garrard. University of Texas, Dallas

Don Gill, University of Maryland, College Park

Reid Gilmore, University of Massachusetts Medical School

Pascal Goldschmitd-Clermon. Johns Hopkins University

Jonathan Horowit/. Duke Llniversity

Laurinda Jaffe. Llniversity of Connecticut, Storrs

Jack Johnson. Purdue University

Paul Lazarow, Mt. Sinai Medical

Jennifer Lippencott-Schwartz, National Institutes of Health

Lee Makowski. Boston University

Peter Novak. Yale University

Robert Palazzo. Manne Biological Laboratory

Howard Schachman, University of California. Berkeley

Pam Silver, Princeton Llniversity

Rick Stemhardt. Llniversity of California, Berkeley

Course Assistants

Daniel Pollard, Baltimore, MD
Katie Pollard. Baltimore, MD
Michael Salmon. Chapel Hill. NC

Students

Carol Bascom. Tufts University

Ellen Brisch, Llniversity of Kansas, Lawrence

Darien Cohen. Dartmouth College

Hugh Crenshaw, Duke University

Mario Delmar. SUNY. Syracuse

Haiyan Deng, Harvard Llniversity

Matthew Frerking, University of California. Davis

Vicki Goodman. Duke Llniversity

Robin Hammell. Robert Wood Medical School

Edward Hinchclifte. Llniversity of Minnesota. Minneapolis

Joseph Kclleher. Johns Hopkins Medical School

Errol Kolen. University of Missouri. Columbia

Bodo Lange. University of Manchester, LJK

Annick Le Gall, Cornell University Medical College

Edward Leonard. University of Pittsburgh Medical School

Eileen Luque, SUNY, Syracuse

Deborah Miller. University of Massachusetts, Amherst

John Murray. Albert Einstein Medical College

Dana Nojima. University of Minnesota. Minneapolis

Valerie Pierce, University of Chicago

Frances Reis, University of Idaho. Moscow

Normand Richard, University of California. Riverside

William Robinson, New England Aquarium

Baerbel Rohrer, Llniversity of Calgary, Canada

Beth Schomer. Stanford University

.ling Shang, Yale University

Sidney Shaw, University of North Carolina. Chapel Hill

Elspeth Stewart. ICRF Clare Hall Laboratories, UK

Douglas Swank, University of Pennsylvania

Penny Tavormina. Llniversity of Virginia

Kenan Turnacioglu. University of Pennsylvania

James Whaley. Llniversity of Illinois. Urbana

Ben Whitlock. Ohio State University

I iK Wong. Llniversity of Virginia

I in Wu, Scripps Research Institute

Jane Ye. Dartmouth College

Kducational Programs R25

Short Courses

Advanced H 'orkshop on Recombinant DNA
Methodology (July 6- July 10)

Instructors

Robert E. Farrell. Jr.. Exon-Intron. Inc.
Greg Leppert. Exon-Intron. Inc.
Charles Vaslet. CAV Consulting

Students

Isabel Baanantc. University of Barcelona. Spam
Thomas Borgese. City University of New York
Meredith Hullar. Marine Biological Laboratory
Robert Lauzon, Albany Medical College
Tim Mukoda. United States Air Force
\nnie Pardo. Unhersidad Nacional Autonoma. Mexico
Karen Ridge. Humana Hospital-Michael Reese
Gary Schneider. Loyola University Medical School
Earl Weidner. Louisiana State University. Baton Rouge
Zhaohui Yang, Marine Biological Laboratory

Advances in Mariculture: Techniques and
Future Directions for Providing Marine
Organisms for Biological Research (May 17-29)

Course Director

Roger Hanlon. Marine Biomedical Institute

Course Manager

Philip Alatalo. Woods Hole Oceanographic Institution

Faculty

Jelle Atema. Boston University Marine Program. MBL

David Bengtson. University of Rhode Island. Kingston

Patricia Bubucis. Sea Research Foundation

Robert Bullis. Marine Biological Laboratory

Elizabeth Clarke. University of Miami

Linda Da\is. Woods Hole Oceanographic Institution

Michael Feldgarden. Yale University

Patrick Gaffnev. University of Delaware. Lewes

Scott M. Gallagher. Woods Hole Oceanographic Institution

Dian Gifford. University of Rhode Island. Narragansett

Robert Guillard. Bigelow Laboratory for Ocean Sciences

Herb Hidu, Wiscasset, ME

Holger Jannasch. Woods Hole Oeeanographic Institution

Rick Karney, Martha Vineyard Shellhsh Group. Inc.

Alan Kuzinan. Marine Biological Laboratory

Donal Manahan. University of Southern California. Los Angeles

Jud\ McDowell. Woods Hole Oceanographic Institution

Phil Presley. Carl Zeiss. Inc.

Stephen Spotte. Sea Research Foundation

Michael Syslo. State Lobster Hatchers & Research Station

Stephen Ward, U.S. Fish and Wildlife Service

Students

Imad Ghossoub, University of Southern Mississippi

Dana Krueger. Harvard University

Carole Lanteigne. Aquarium & Marine Center, Canada

Amoy Lum Kong, Institute of Marine Affairs, Trinidad and Tobago

Darlene Manning. Dalhousie University, Canada

Joan Manuel. Dalhousie University. Canada

David Remsen, Marine Biological Laboratory

Nicholas Roden. Exxon Biomedical Sciences. Inc.

Mark Rosenqvist. Aquatic Research Organisms

Tina Schappach, MCI Telecommunications

Benediktc Vercaemer. Dalhousie University, Canada

Analytical and Quantitative Light Microscopy
in Biology, Medicine, and Materials Science
(May 14-22)

Course Directors

Edward D. Salmon, University of North Carolina. Chapel Hill
Greenfield Sluder, Worcester Foundation for Experimental Biology
Da\id E. Wolf, Worcester Foundation for Experimental Biology

Faculty

Brad Amos, Medical Research Council. UK

Steven M. Block, Rowland Institute for Science

Richard Cardullo, University of California, Riverside

Gordon Ellis, University of Pennsylvania

Harvey Florman. Worcester Foundation for Experimental Biology

Jeff Gelles, Brandeis LIniversity

Anthony Moss, Worcester Foundation for Experimental Biology

Rudolf Oldenbourg. Marine Biological Laboratory

Kenneth R. Spring, National Institutes of Health

Lecturer

Shinya Inoue. Marine Biological Laboratory

Teaching Assistants

Neil Gliksman. University of North Carolina, Chapel Hill
Chnstine McKinnon. Worcester Foundation for Experimental
Biology

Course Coordinator

Frederick Miller, Worcester Foundation for Experimental Biology

Course Assistants

Robert Knudson, Marine Biological Laboratory
Phong Tran, LIniversity of North Carolina, Chapel Hill

Students

John Axelson, Holy Cross College
Sandy Chang. Rockefeller LIniversity
Jean-Yves Chatton. National Institutes of Health
Ronald Cohn. Syntex Research
William Crowe, University of Texas, Galveston
Fernando Delasille. Thomas Jefferson LIniversity
Dimiter Dimitros, National Institutes of Health
Stephen Doty. Hospital for Special Surgery
Deborah Fygenson, Princeton University
Peder Gasbjerg. Panum Institute. Denmark
Craig Giroux, Wayne State University
Norman Harris. Louisiana State University
Ulrich Kersting. National Institutes of Health
Kimberly Kotz. Mayo Clinic
Donald O'Malley, SUNY. Stonv Brook

R26 Annual Report

Joseph Neary. VA Medical Center

Charles Pak. National Institutes of Health

Harold Payne, Case Western Reserve University

Robert Prusch. Gonzaga University

Andreas Stemmer, MRC, UK

Kath\ Suprenant. University of Kansas. Lawrence

Ole Thastrup, Zymogenetics

Joseph Unthank, Indiana Llniversiu

Janice Voltzow. Harvard University

Hanry Yu. Duke University

Basic H 'orkshop on Recombinant DNA
Methodology (June 29-July 3)

Instructors

Robert E. Farrell. Jr., Exon-Intron. Inc.
Greg Leppert, Exon-Intron, Inc.
Charles Vaslet, CAV Consulting

Ehud Kaplan, Rockfeller University

Jerry R. Kuszak, Rush Presbyterian St. Luke's Medical Center

Wen Hwa Lee, Institute of Biotechnology

Ellen Liberman, NEI/NIH

Thomas F. Linsenmayer, Tufts University Medical School

Robert Paul Malchow, University of Illinois College of Medicine

Richard H. Masland, Massachusetts General Hospital

Robert S. Molday, University of British Columbia, Canada

Jeremy Nathans, Johns Hopkins University School of Medicine

Harry A. Quigley, Johns Hopkins Hospital

James Rae, Mayo Foundation

Robert R. Rando, Harvard Medical School

Elio Raviola, Harvard Medical School

Harris Ripps, LIniversity of Illinois College of Medicine

Paul Russell, NEI/NIH

Barbara G. Schneider, University of Texas Health Science Center

Abe Spector. Columbia University

Tung-Tien Sun, New York University Medical Center

Guo-Ming Wang, Columbia University

Charles Zuker. University of California. San Diego

Students

Isabel Baanante. University of Barcelona, Spain

David Beggs. Northern Ireland Horticultural and Plant Breeding

Station. Armagh

Thomas Borgese. City University of New York
Lotta Chi, CP Li Biomedical Research Corp.
Judith Grassle, Rutgers University
Connie Hart, Woods Hole Oceanographic Institution
Meredith Hullar, Marine Biological Laboratory
Daniel Johnson, Bowman Gray School of Medicine
Alan Kuzirian, Marine Biological Laboratory
Tim Mukoda, LInited States Air Force
Annie Pardo, Universidad Nacional Autonoma, Mexico
Gary Schneider, Loyola University Medical School
Dean Schraufnagel, University of Illinois, Chicago
Jacob Sznajder. Humana Hospital-Michael Reese
Lewis Tilney, Marine Biological Laboratory
Michael Tytell, Bowman Gray School of Medicine
Earl Weidner, Louisiana State University
Zhaohui Yang. Marine Biological Laboratory

Fundamental Issues in Vision Research
(August 16-29)

Course Directors

David S. Papermaster. LIniversity of Texas Health Science Center
John N. Dowling. Harvard University

Faculty

Robert Barlow, Syracuse LIniversity

Robert W. Baughman. Harvard Medical School

George B. Benedek. Massachusetts Institute of Technology

Eliot L. Berson. Massachusetts Eye and Ear Infirmary

Martin Breitman. Ml. Sinai Hospital. Canada

Debra Carper. NEI/NIH

Judah Folkman. Children's Hospital Medical Center

Ilene K. Gipson. Schepens Eye Research Institute

Paul A. Hargrave, University of Florida. Gainesville

John R. Hassell, The Eye & Ear Institute of Pittsburgh

Fielding Hejtmancik. National Institutes of Health

Paul N. Hoffman, The Johns Hopkins Hospital

Students

Jacqueline Biscardi, University of North Carolina. Chapel Hill

Julie Brown. Oregon Regional Primate Research Center

Suzanne Bruhn, Harvard Medical School

Qian Chen. Tufts University

Heather Fabry, University of California

John Gardner. Fox Chase Cancer Center

Kenneth Giuhano, Carnegie Mellon University

Brian Jiang. New York University

Amy Johnson, Columbia LIniversity

Diana Kania, SUNY, Buffalo

Francis LaRosa, Boston University

Richard LeBaron, La Jolla Cancer Research Foundation

Wan-Cheng Li, University of Washington

Mingyao Liu, University of Maryland

Qiang Lu. Brandeis LIniversity

Tomoko Nakayama, Whitehead Institute

Ron Pelton. Vanderbilt University

Elizabeth Roquemore, Johns Hopkins University

Mary Woo, Scripps Research Institute

Yimin Yan. University of Pittsburgh

Medical Informatics (May 31 -June 6)

Course Director

Homer Warner, LIniversity of Utah

Faculty

Paul Clayton, Columbia University

Peter Haug, University of Utah

David J. Lipman. National Library of Medicine

Donald A. B. Lindberg. National Library of Medicine

Daniel R. Masys. National Library of Medicine

Robert Sideli, Columbia University

Course Assistant

Sylvia Jessen, LIniversity of Utah

Students

James Baggott, Hahnemann University

Gerald Bashein, University of Washington, Seattle

Educational Programs R27

Gary 1 Berman, Albert Einstein College of Medicine
Janine Bluckman. University of Maryland, Baltimore
Athos Bousvaros. Children's Hospital
Mona Couts, University of North Carolina, Chapel Hill
Stephen Duhin, Drexel University
Peter Ellis, Brown University-
Michael Fisher, University of Maryland. Baltimore
Peter Fleming, Cleveland Clinic Foundation
Cathy Harbert. Howard Hughes Medical Institute
Linda Jacknowitz. West Virginia University
John Kelly. America! Medical Association
Michael Kessler, American Board of Quality Assurance
Michael Krall. Oregon Health Sciences University
Lisa Kregel, Case Western Reserve LIniversity
Rosanne Labree, McLean Hospital
Carol Lelonek. University of Buffalo
Saul Malozowski. Food & Drug Administration
Karen Martinez. Catawba Memorial Hospital
Peter Mathews. Kaiser Permanente
Julie McGowan. LIniversity of Vermont, Burlington
Ana Nunez. Hahnemann University
Michael Rissinger, New York University
Barbara Schultz, VA Medical Center
Anthony So, University of California. San Francisco
Zoe Stavri. Massachusetts General Hospital
Bryan Thompson, Lovelace Medical Foundation
Monica Linger, Northeastern Ohio University
George Wesley. Office of the Inspector General, Washington. DC

Methods in Computational Neuroscience

(August 2-29)

Course Directors

James M. Bower. California Institute of Technology
Christof Koch, California Institute of Technology

Computer Managers

Maneesh Sahani. California Institute of Technology

Charles F. Stevens. Salk Institute

John Uhley, California Institute of Technology

Students

Erik Cook, Baylor LIniversity

Adelle Coster. University of New South Wales. Australia

Sharon Crook, University of Maryland, College Park

Winnch Freiwald. Tubingen LIniversity, Germany

Alberto Herrera-Becerra, Universidad Nacional Autonoma, Mexico

Martin Huber, Phillips-University-Marburg. Germany

Michael Irizarry, Massachusetts General Hospital

Ranu Jung. Case Western Reserve University

Brandt Kehoe, California State University

Han Lampl. Hebrew University. Israel

Mitchell Maltenfort, Rehabilitation Institute of Chicago

Ference Mechler, New York LIniversity

Jill Nicolaus, University of Chicago

Harmon Nine, University of Michigan. Ann Arbor

Monica Paolini. University of California, San Diego

Yifat Prut, Hebrew University, Israel

Ins Reuveni, Ben Gunon LJniversity of the Negev. Israel

Emilio Salinas, National University. Mexico

Eyal Seidemann. Tel Aviv University, Israel

Nangiavaram Sekar, University of Iowa, Iowa City

Micah Siegel. Yale University

Mark Tommerdahl, University of North Carolina, Chapel Hill

Yi-Xiong Zhou, McGill Vision Research Center. Canada

Microinjection Techniques in Cell Biology
(May 26-June 1)

Course Director

Robert Silver, Cornell University

Faculty

Paul R. Adams, HHMI. SUNY, Stony Brook
Richard Andersen. Massachusetts Institute of Technology
Joseph J. Atick, Rockefeller University
William Bialek, NEC Research Institute
Avis Cohen. University of Maryland, College Park
Rodney James Douglas, MRC Anatomical Neuropharmacology

Unit. UK

Nancy Kopell. Boston University

Rodolfo R. Llinas, New York University Medical Center
Eve Marder, Brandeis University

Michael V. Mascagni, Supercomputing Research Center
Kenneth D. Miller, California Institute of Technology
John Rinzel. National Institutes of Health
Idan Segev. Hebrew University, Israel
Terrence Sejnowski. The Salk Institute

Teaching Assistants

David Beeman. LJniversity of Colorado, Boulder
David Berkowicz. Yale University Medical School
Ojvmd Bernander, California Institute of Technology
Dieter Jaeger, California Institute of Technology
Maurice Lee. California Institute of Technology

Faculty

Suzanne Chandler, Cornell University

Donald Chang. Hong Kong LIniversity of Science & Technology.

Hong Kong

Douglas Kline, Kent State LIniversity
Joanne Kline. Kent State University
Patricia Wadsworth, LIniversity of Massachusetts, Amherst

Students

David Brauer, United States Department of Agriculture

Roger Buchanan, National Institutes of Health

Thomas Burke, Ohio State University

Hattie Gresham. University of Missouri, Columbia

Gupta Kalpana. University of Ottawa, Canada

Karen Hedberg, University of Oregon. Eugene

Lynne Lucher. Illinois State University

Katnna Marsh. Queen's Medical Centre, UK

Thomas Martin, LIniversity of Wisconsin, Madison

James McGill. Duke LIniversity

Thomas Reese, National Institutes of Health

Frieda Reichsman. University of Massachusetts. Amherst

Paulo Serodio. New York University

Donald Siwek, VA Hospital

R28 Annual Report

Steve Scrota, Columbia University
James Swanson, Old Dominion University
Johanna Talavera, Boston University
Suresh Tiwari, University of Kansas. Lawrence

Rapid Measurement of Neurotransmitter
Signals in the Central Nervous System Using
In Vivo Electrochemistry (August 19-24)

Course Directors

Greg Gerhardt, University of Colorado, Denver
Paul Moore, University of Colorado, Denver

Faculty

Alain Gratton, Douglas Research Hospital, Canada
Michael Palmer, University of Colorado, Denver
William Proctor, University of Colorado. Denver

Michael Donoghue. University of Arizona, Tucson

Doug Ernisse. University of Michigan. Ann Arbor

Joseph Felsenstein, University of Washington, Seattle

Walter Gilbert, Harvard University

Martin Kreitman. University of Chicago

Laura Landweber, Harvard University

Bcrnd Franz Lang, University of Montreal, Canada

David Maddison, University of Arizona. Tucson

Marcella McClure. University of California. Irvine

Roger Milkman, University of Iowa. Iowa City

Gary Olsen. University of Illinois, Urbana

Monica Riley, Marine Biological Laboratory

Terry Speed, University of California. Berkeley

David Swofford. Smithsonian Museum Support Center

Bruce Walsh, University of Arizona, Tucson

Course Assistant

Brendan Reilly. Software Editing Corporation

Technicians

Paula Bickford, University of Colorado, Denver
Scot Brock. University of Colorado. Denver
Mike Doherty, McGill University. Canada
Marilyn Friedemann. University of Colorado. Denser
Ron Maloney. University of Colorado, Denver
Mike Parrish, University of Colorado. Denver
Steve Robinson. University of Colorado, Denver
Scott Robinson, University of Colorado, Denver

Students

Abdel Abdel-Rahman, East Carolina University

Juan Advis, Rutgers University

Kurt Batsche. SUNY, Stony Brook

Thomas Clark, U.S. Army Aeromedical Research

Dennis Dahl. University of Texas, Dallas

Audrey Davis, George Washington University

Matthew Davidson, University of Oregon, Eugene

Michele Dwyer, Smith College

Siegward Elsas. University of California. Berkeley

Michael Horner, I. Zoologisches Institut. Germany

Robert Huber, Karl-Franzens-Universitat Graz, Austria

Robert Leipheimer, Youngstown State University

Tamala Mallett, Meharry Medical College

Dorothy Pocock, Concordia University, Canada

David Rothblat, Hahnemann University

Claude Rouillard, Hospital de I'Enfant-Jesus, Canada

Charles Stewart, Franklin & Marshall College

James Suojanen, New England Deaconess Hospital

Tina Thompson, University of Texas Southwestern

Ruth Weissenborn, University of St. Andrews, Scotland

Workshop on Molecular Evolution

(August 2-14)

Director

Mitchell L. Sogin, Marine Biological Laboratory

Faculty

Marlene Belfort, New York State Department of Health
Daniel Davison, University of Houston

Students

Rashid Aman, National Museums of Kenya. Nairobi
Linda Amaral. Woods Hole Oceanographic Institution
Wendy Bailey. Yale University
Charles Baker. Kewalo Marine Laboratory
Ulrike Beemelmanns, University of Cologne, Germany
Joy Bergelson, Washington University
David Bermudes, Yale University-
Nancy Bowers, Pennsylvania State University
Barry Campbell, Queen's University, Canada
David Carmean, University of California, Davis
Carlos Cerpa, University of Montreal. Canada
Belinda Chang. Harvard University
Bernard Cohen. L'niversity of Glasgow, Scotland
Julio Collado-Vides. Massachusetts Institute of Technology
Jan Conn. University of Florida. Gainesville
Eric Delvvart, Stanford University Medical Center
Floyd Dew hirst. Forsyth Dental Center
Megan Eskey, NASA Ames Research Center
David Eaguy, Queen's University. Canada
Thomas Friedl, University of Bayreuth, Germany
Manohar Furtado. Northwestern University Cancer Center
Steven Gagnon. Laval University. France
Anne Gerber. Washington University
Manuel Glynias, Cleveland Clinic Research Institute
John Gosink, University of Washington, Seattle
Jotun Hein, Aarhus University, Denmark
Stephen Hinton, Exxon Corporate Research Laboratories
Wen-Yen Kao, University of Wisconsin. Milwaukee
Laura Katz. Cornell University

Michelle Kelly-Borges, Harbor Branch Oceanographic Institute
Hans-Peter Klenk, Max-Planck-Institut, Germany
Richard Kliman. Rutgers University-
Stuart Kuhstoss. Lilly Research Laboratories
Bernard Labedan. University of Pans-Sud. France
Damian Labuda. University of Montreal, Canada
Benedicte I afa\. Station Zoologique, France
Joyce Lewis. Colorado State University
Ee Lin Lim. Woods Hole Oceanographic Institution
Francois Lutzoni, Duke University
Kersti Maclnnes. Los Alamos National Laboratory
Katrina Mangin. Llmversity of California, Santa Cruz
Gerogiana May, University of Minnesota, St. Paul
James McLaughlin, Massachusetts General Hospital

Educational Programs R29

Helen McVeigh, Natural History' Museum, UK

Annabel Miles. James Cook University of North Queensland,

Australia

Christine Miller, University of Cincinnati
Yue Ming. Michigan State University
Sharon Mitchell, USDA/ARS
Miklos Muller, Rockefeller University
Jan Pawlowski, University ot Geneva, Switzerland
Marian Peris, University of California, Los Angeles
William Piel, Harvard University
Norman Pieniazek, Centers for Disease Control
James Pierce, Dupont Merck Pharmaceutical Company

Frank Robert, Idaho National Engineering Laboratory

Andres Ruiz-Linares, Stanford University

Robert Setlerquist, University of Houston

Robert Sheehy, University of Arizona, Tucson

Emmanuel Skoufos, University of Minnesota, Minneapolis

Ralph Tanner, University of Oklahoma, Norman

Steven Thompson, Washington State University

Moira van Staaden, Karl-Franzens-University Graz, Austria

Todd Wareham, Memorial University of Newfoundland, Canada

Lee Weigt, Smithsonian Tropical Research Institute, Panama

Lisa White, University of Houston

Hong Xie. Smith College

Summer Research Programs

Principal Investigators

Adams. James A., University of Maryland Eastern Shore

Akeson, R., University of Cincinnati

Alkon, Daniel L. National Institutes of Health

Allen. Nina S.. Wake Forest University

Armstrong, Clay, University of Pennsylvania

Armstrong, Peter B.. University of California, Davis

Arnold, John M.. University of Hawaii

Augustine, George J., Duke University Medical Center

Baker, Robert. New York University Medical Center

Baldridge, William H.. McMaster University Medical School. Canada

Barlow, Jr.. Robert B.. Syracuse University

Bearer, Elaine, Brown University

Beauge, Luis. Instituto M. y M. Ferreyra, Argentina

Bennett. M. V. L., Albert Einstein College of Medicine

Bezanilla. Francisco, University of California. Los Angeles

Bezprozvanny, Ilya. University of Connecticut Health Center

Bingham. Eula. University of Cincinnati

Bloom. George S., The University of Texas Southwestern Medical

Center. Dallas

Bodznick, David. Wesleyan University

Borgese. Thomas A.. Lehman College. City University of New York
Boron, Walter, Yale University School of Medicine
Borst, David, Illinois State Llniversity
Bowlby, Mark R., Harvard Medical School
Boyer, Barbara C., Union College
Brady. Scott T., The University of Texas Southwestern Medical

Center, Dallas

Brown, Joel, Albert Einstein College of Medicine
Buchanan, Roger A.. National Institutes of Health
Burdick. Carolyn J.. Brooklyn College of the City Llniversity of New

York
Burger. Max M., Friedrich Miescher Institut. Switzerland

Chaet, A. B.. University of West Florida

Chang. Donald C.. Baylor College of Medicine

Chanson. Marc, Albert Einstein College of Medicine

Chappell, Richard L., Hunter College of the City University of New

York

Charlton, Milton. University of Toronto, Canada
Clay. John, National Institutes of Health
Cohen, Lawrence B.. Yale University School of Medicine

Cohen. William D.. Hunter College of the City University of New

York

Cooperstein. Sherwin J.. The Llniversity of Connecticut Health Center
Cuppoletti, John. Llniversity of Cincinnati College of Medicine

D'Avanzo. Charlene, Hampshire College
De Weer. Paul, Llniversity of Pennsylvania
Di Polo, Reinaldo, IVIC, Venezuela
Doroshenko, Peter, Duke Medical Center
Dowling, John E., Harvard University

Eckberg, William R.. Howard University

Fhrlich. Barbara E.. University of Connecticut Health Center

Eisthen, Heather L.. Indiana University

Farbman, Albert. Northwestern University

Fein, Alan. Llniversity of Connecticut Health Center

Fink, Rachel. Mount Holyoke College

Fishman. Harvey M., The University of Texas Medical Branch.

Galveston
Flonn-Christensen. Jorge, University of Cincinnati

Gadsby, David. The Rockefeller University

Gainer. Harold. National Institutes of Health

Garber, Sarah S., University of Alabama, Birmingham

Garnck, Rita Anne, Fordham University College at Lincoln Center

Gesteland, Robert C., University of Cincinnati College of Medicine

Gilland, Edwin H.. Harvard University

Giuditta, Antonio. University of Naples, Italy

Goldman. Robert D., Northwestern University Medical School

Grant. Philip. National Institutes of Health

GrifT. Edwin R., Llniversity of Cincinnati

Gyoeva. Fatima, Institute of Protein Research. Russia

Haimo, Leah, University of California. Riverside

Hans. Michael. Max-Planck-Institiit fur Biophysikalische Chemie.

Germany

Hegde. Ashok N.. Columbia University
Helluy. Simone. Wellesley College
Hernandez-Cruz. Arturo. Instituto de Fisologia Celular, UNAM.

Mexico

Highstem. Steven M.. Washington Llniversity School of Medicine
Hill, Susan D., Michigan State Llniversity

R30

Summer Research Programs R31

Holz. George G., IV, Howard Hughes Medical Institution.

Massachusetts General Hospital
Hoskin. Francis C. G., Illinois Institute of Technology

Ip. Wallace. University of Cincinnati College of Medicine

Johnston, Daniel. Baylor College of Medicine
Josephson. Robert K.. University of California, Irvine

Kaneshiro. Edna, University ol Cincinnati

Kaplan. Barn, B.. Western Psychiatric Institute and Clinic

Kaplan, llene M.. Union College

Khan. Shahid, Albert Einstein College of Medicine/National Institutes

of Health

Khan. Sohaib, University of Cincinnati College of Medicine
Knowlton. Robert. Jefferson Medical College
Koide. Samuel S.. The Population Council
Krasitz, Edward. Harvard Medical School
Kremer, James N., University of Southern California
Kumar. Ajit. George Washington University School of Medicine
Kuznetsov, Sergei. Moscow State University, Russia

Landowne. David, University of Miami

Langford. George, Dartmouth College

Laufer. Hans. The University of Connecticut

Lauzon. Robert J.. Albany Medical College

Liman. Emily R., Harvard Medical School

Lipick>. Raymond J.. Food & Drug Administration

Lisman. John. Brandeis University

Llinas. Rodolfo R., New York University Medical Center

Malchow. Robert Paul, University of Illinois at Chicago College of

Medicine

Martin. Ramer, University of Ulm, Germany
Metuzals. Janis. University of Ottawa. Canada
Misevic. Gradimir, University Hospital of Basel. Switzerland

Nagle, Ronald L.. Albert Einstein College of Medicine
Nasi. Enrico. Boston University School of Medicine
Nelson. Leonard, Medical College of Ohio
Noe. Bryan D.. Emory University School of Medicine

Obaid. Ana Lia. University of Pennsylvania School of Medicine

Pant. Harish. NTNDS, National Institutes of Health
Parysek. Linda. University of Cincinnati Medical School
Patterson. David J.. University of Bristol, UK

Quiglev. James P.. SUNY, Stony Brook

Segal, Sheldon, The Population Council

Severin, Fedor F., Institute of Protein Research. Russia

Shipley. Michael T.. University of Cincinnati College of Medicine

Silver. Robert B., Cornell University, N.Y. State College of Veterinary

Medicine

Siwicki. Kathleen K.. Swarthmore College
Sloboda. Roger D.. Darmouth College

Smith. David V.. University of Cincinnati College of Medicine
Sperelakis. Nicholas. University of Cincinnati College of Medicine
Spirin, Alexander S., Academy of Sciences of Russia
Spray, David C.. Albert Einstein College of Medicine
Steinacker. A., Washington University School of Medicine
Stemmer. Andreas, Medical Research Council, UK
Sweeney. H. Lee. University of Pennsylvania School of Medicine

Tanguy, Joelle. Northwestern University

Telzer, Bruce, Pomona College

Treistman, Steven N.. Worcester Foundation for Experimental

Biology

Trejo-Borowski. Amy V., Northwestern University
Trinkaus, John P., Yale University
Troll. Walter, New York University Medical Center
Tytell, Michael, Bowman Gray School of Medicine of Wake Forest

University

Ueno, Hiroshi. Osaka Medical College. Japan

Vallee. Richard. Worcester Foundation for Experimental Biology

Wadsworth, Patricia. University of Massachusetts

Watson, Win, University of New Hampshire

Wehner, Rudiger, University of Zurich, Switzerland

Weidner, Earl. Louisiana State University

Weiss, Dieter, Technical University. Munich, Germany

Wonderlin, William F., West Virginia University

Wood, Emma R.. University of British Columbia. Canada

Yen. Jay Z., Northwestern University Medical School
Yoshioka, Tohru, Waseda University. Japan

Zigman, Seymour, University of Rochester School of Medicine &

Dentistry

Zottoli. Steven J., Williams College
Zou, Dong-Jing, Biocenter. Basel University, Switzerland
Zuazaga de Ortiz. Conchita, University of Puerto Rico
Zukm. R. Suzanne, Albert Einstein College of Medicine

Other Research Personnel

Rakowski. Robert F.. University ol Health Sciences/The Chicago

Medical School

Ratner, Nancy, University of Cincinnati
Reese. Thomas S.. NINDS. National Institutes of Health
Render, JoAnn, University of Illinois

Rieder. Conly L.. Wadsworth Center for Laboratories & Research
Ripps, Harris. University of Illinois College of Medicine
Rome. Lawrence. University ol Pennsylvania
Ross. William. New York Medical College
Ruderman, Joan V.. Harvard Medical School
Russell, John M., University of Texas Medical Branch

Salzberg. Brian M.. University of Pennsylvania School of Medicine
Schmidt. Joachim. Emorv University

Ahl, Jonna, University of Hartford
Alston. Yolonda. Benedict College
Altamirano. Anibal A., University of Texas Medical Branch,

Galveston

Andrews. S. Brian, National Institutes of Health
Araneda. Ricardo. Albert Einstein College of Medicine
Ascher. Phillipe, Ecole Normale Supeneure, France

Bartley. Annette. Hunter College of CUNY

Benech, Juan Claudio, Institute de Investigaciones Biologicas

Clemente Estable, Uruguay

Bezprozvannaya, Svetlana. University of Connecticut Health Center
Bhattacharyya, Anita, University of Cincinnati
Bittner. George D., University of Texas. Austin

R32 Annual Report

Bommert, Kurt, Max-Planck-Institute for Brain Research, Germany
Bouhassira, Eric. Albert Einstein College of Medicine
Brackenbury, Robert. University of Cincinnati Medical Center
Breitwieser, Gerda E.. Johns Hopkins University School of Medicine
Buelow. Neal, Syracuse University
Bullock, Luce, University of Roc hi

Callaway. Joseph C. New York Medical College

Chludzmski, John, NINDS. National Institutes of Health

Chun, Jong Tai, Western Inslitute and Psychiatric Clinic

Clare, Everton, City College of New York

Cohen, Avrum, Yale University School of Medicine

Cohen, Darien L.. Dartmouth College

Cohen. Matthew. Yale University

Collin, Carlos, NINDS, National Institutes of Health

Corda, David. University of Pennsylvania

Correa, Ana H.. University of California, Los Angeles

Couch, Ernest, Texas Christian University

Cox, Daniel, Wake Forest University

Crispino, Mananna, University of Naples, Italy

Crutcher. Keith. University of Cincinnati

Cruz, Rena, Lehman College, CUNY

Danae, Hadi, University of Connecticut

Davis, Adam, Yale University

DeBello, William. Duke University Medical Center

Deffenbaugh. Max. Massachusetts Institute of Technology

Dermietzel, Rolf. Universitat Regensburg, Germany

Drazba, Judith, NINDS, National Institutes of Health

Dudley, Nathaniel, Hampshire College

Edwards. M. Kaye, Haverford College
Ehrlich, Michelle. Princeton University
Engman. James A., University of Cincinnati

Falk, Chun Xiao, Yale University School of Medicine

Felle. Hubert. Botanical Institute I. University Giessen. Germany

Florin-Christensen, Monica. University of Cincinnati

Flucher, Bernhard E., National Institutes of Health

Folwell. Mary Grace, Swarthmore College

French, Robert J., University of Calgary, Canada

Frenkel, Kryslyna, New York University Medical Center

Gallant, Paul, NINDS, National Institutes of Health
Gerosa, Daniela, Fnedrich Miescher-Institut, Switzerland
Gill-Kumar, Pritam, Food & Drug Administration
Goldman, Anne E.. Northwestern University
Gomez, Maria, Boston University School of Medicine
Gomez Lagunas, Froylan, University of Pennsylvania
Gould, Robert. New York State Institute of Basic Research
Grassi, Daniel, Ft. Lauderdale, Florida
Greenblatt, Daniel, Brandeis University

Hammar, Kassia, NINDS. National Institutes of Health

Harper. David, University of British Columbia, Canada

Hershko, Avram, Technion. Israel

Herzog, Erik, Syracuse University

Hitt. Austin, University of West Florida

Hogan. Emilia, Yale University School of Medicine

Holmgren, Miguel. University of Health Sciences/The Chicago

Medical School
Hunt, James. Duke Medical Center

Ito, Etsuro. NINDS, National Institutes of Health

Johnson, Michelle R., Howard University
Johnston. Jennifer, Dartmouth College
Juneja. Renu. The Population Council

Kaftan, Edward, University of Connecticut Health Center

Kammerer, Richard, Friedrich Miescher-Institut. Switzerland

Kehoe, Jacsue, Ecole Normale Supeneure, France

Kelly, Mary E., Syracuse University

Kirino, Yutaka, Kyushu University, Japan

Klein, Kathryn, Emory University School of Medicine

Knudsen, Knud D.. Food & Drug Administration

Konnerth, Arthur, Max-Planck-Institute, Germany

Krause, Todd L., University of Texas, Austin

Kudo. Yoshihisa, Mitsubishi Kasei Life Sciences Institute, Japan

Kuhns, William, Hospital for Sick Children, Canada

Lasser-Ross, Nechama, New York Medical College

Lauzon, Cindy, Albany Medical College

Leidigh. Christopher. Albert Einstein College of Medicine

Leopold. Philip L., University of Texas Southwestern Medical Center

Leung, Doreen Siu Yi. Hong Kong University of Science &

Technology. Hong Kong

Lim, Jong. New York University Medical Center
Lin, Jen-Wei, New York University Medical Center
Liu, Lei, University of Connecticut

Locke, Rachel. Washington University School of Medicine
Lorenzoni, Patrizia, Fnedrich Miescher Institut. Switzerland
Lu, Jin, University of Texas Medical Branch
Luca, Frank, Harvard Medical School

Mangel, Stuart, University of Alabama School of Medicine

McCartney. Brooke, Duke University

McPhie, Donna. National Institutes of Health/Georgetown University

Moir, Robert, Northwestern University

Monterrubio, Jose, University of Puerto Rico

Moreira, Jorge, NINDS, National Institutes of Health

Morrell, Candy M.. University of Maryland Eastern Shore

Morrison, Paul. Llniversity of Glasgow. Scotland

Noe, Jennifer R.. Emory University

O'Neil, Peggy, Illinois State University

Oka, Kotaro, NINDS, National Institutes of Health

Olds. James L.. National Institutes of Health

Perez, Reynaldo, University of Puerto Rico

Perozo, Eduardo, Jules Stem Eye Institute, University of California,

Los Angeles

Pethig, Ronald, University of Wales, UK
Petri, Victoria, Albert Einstein College of Medicine
Plotner, Robert. University of Texas Medical Branch, Galveston
Powers, Maureen. Vanderbilt University
Pumphn, David W., Llniversity of Maryland, Baltimore

Rayos, Nancy, Hunter College

Reese, Barbara. NINDS, National Institutes of Health

Regehr, Wade, University of Pennsylvania

Richards, Kathryn S., Emory University

Rook. Martin B., Albert Einstein College of Medicine

Rothenherg. Mark, Emory University

Rusciano, Dario. Fnedrich Miescher Institut, Switzerland

Sakakibara, Manabu, Toyohashi University of Technology
Salyapongse. Aimee, Wesleyan University

Summer Research Pnjyrams R33

Sanchez. Ivelisse. Hunter College of CUNY

Sartain. Julie Ann. Illinois State University

Schitfmann. Dietmar. University of Wurzburg, Germany

Shelter. Rebecca. University of Texas

Shibuya. Ellen. Harvard Medical School

Shner. Alvin. McGill University. Canada

Sigg. Daniel. University of California. Los Angeles

Slater. N. Tra\is. Northwestern University

Stanley. Elis. NINDS. National Institutes of Health

Slockbridge. Lisa. National Institutes of Health

Stockbridge, Norman, Food & Drug Administration

Stokes. Darrell R.. Emory University

Stoyano\sky. Delcho. University of Connecticut Health Center

Sugimon. Mutsuyuki. New York University Medical Center

Sumanovski, Lazar. University Hospital of Basel. Switzerland

Suandulla. Dieter. Ma\-Planek-Institut. Germany

Swank. Douglas. University of Pennsylvania

Takagi. Hiroshi. VVaseda University, Japan
Tang. Akaysha. Yale University School of Medicine
Terasaki. Mark. NINDS, National Institutes of Health
Tsau. Yang. Yale University School of Medicine
Tseng, Daniel. Northwestern University
Tsukimura. Brian. Illinois State University
Tucker. Meryl Y., Albany Medical College

Vargas. Fernando. Food & Drug Administration

YVeiler. Reto. University of Oldenburg. Germany

Werman. Robert. Hebrew University, Israel

YVu. Jian- Young. Yale University School of Medicine

Yang, Zhaohui. University of Pennsylvania School of Medicine

Zake\ icius. Jane. University of Illinois at Chicago College of Medicine
Z.mlowitz. Joseph. Albert Einstein College of Medicine
Zigman. Bunnie R.. University of Rochester School of Medicine &
Dentistrv

Library Readers: General

Adelberg. Edward A.. Yale Medical School

Bahitsky. Sle\en. Falmouth. MA
Barrett, Dennis. University of Denver
Barry. Susan F.. University Hospital
Browne. Robert. Wake Forest University

Candeles. Graciela C.. University of Puerto Rico

Carriere. Rita M.. Brooklyn. New York

Clarkson. Kenneth L.. AT&T Bell Labs

Cobb. Jewel Plummer. California State University

Cohen. Ira S.. SUNY, Stony Brook

Cohen. Leonard A.. American Health Foundation

Dixon. Keith E.. Hinders University

Dube, Francois. University of Quebec at Rimousky, Canada

DuBrul. Ernest F.. University of Toledo

Duncan. Thomas K.. Nichols College

Eglington. Aislmg. New England Fisheries, World Trade Center
Eisen. Herman N., Massachusetts Institute of Technology
Epstein. Herman T.. Brandeis University

Farmanfarmaian, A., Rutgers University

Frenkel, Krystyna. New York University Medical Center

Fnedler. Gladys, Bunting Institute

Fussell. Catherine. University of Pennsylvania

Gabriel. Mordezai L.. Brooklyn College
German. James. The New York Blood Center
Gilbert. Daniel L.. National Institutes of Health
Goldfarb. Ronald H.. Pittsburgh Cancer Institute
Goldstein. Moise H.. The Johns Hopkins University
Goward. Samuel N., University of Maryland
Grossman, Albert, New York University Medical Center
Guttenplan. Joseph, New York LIniversity Dental Center

Harrington. John P.. University of South Alabama
Hill, Richard W., Michigan State University
Humphreys. Tom. University of Hawaii

Inoue. Sadyuki, McGill University. Canada

Kaltenbaeh. Jane, Mount Holyoke College

Kammer. Benjamin. Boston University School of Medicine

Klemow. Kenneth. Wilkes University

Kline. Richard Paul. Columbia University

Lee, John J.. City College of CUNY
Levitz, Mortimer. NYU Medical Center

Marine Biocontrol Co.. Sandwich. MA

Marine Research, Falmouth, MA

Martin. Donald Creagh. Conrad Jobst Tower

McCoy, Floyd, Associated Scientists of Woods Hole

Michaelson. James. MGH Cancer Center

Mooseker, Mark S., Yale University

Morrell. Leyla DeToledo. Rush Medical College

Ohns. Ada L., University of Tennessee
Olins, Donald E., University of Tennessee
Ostrer, Harry. NYU Medical Center

Prosser. C. Ladd. University of Chicago
Prusch. Robert D., Gonzaga University

Ramamurthy. Baskar, Indian Institute of Science

Robinson. Dems. Marine Biological Laboratory

Rose, Birgit, University of Miami School of Medicine

Rosenbluth. Jack, NYU School of Medicine

Rosenfeld. Allan, Columbia University School of Medicine

Roth, Lorraine, Brookline. MA

Russell-Hunter. W. D.. Syracuse University

Schippers. Jay. New York. NY

Schweitzer. Nicola. Imperial College, UK

Scott-Connor. Harry, Madison. MS

Selby. Cecily Cannan, NYU

Shepro, David. Boston University

Shnflman, Mollie Starr, N. Nassau Health Center

Silva. Robert, Marine Research

Spiegel, Evelyn. Dartmouth College

Spotte, Stephen. Sea Research Foundation & Marine Sci. Inst.

Stephenson, William K., Earlham College

Sweet, Frederick. Washington University School of Medicine

Szent-Gyorgyi. Andrew. Brandeis University

R34 Annual Report

Trager, William, The Rockefeller University
Van Holde, Kensal E., Oregon State I 'r '

Warren, Leonard, Wistar Institir

Weir, Gray E., Naval Historic

Wilhur, Charles G., Colorad' Diversity

Wittenberg. Beatrice, Alb- m College of Medicine

Wittenberg, Jonathan. ' jiistein College of Medicine

Library Riders: Desks

Anderson, Everett, Harvard Medical School
Boyer, John. Union College

Chambers, Edward L., University of Miami
Collier, Marjorie McCann, Saint Peters College
Clark, Arnold M.. Woods Hole, MA
Copeland, Eugene, Woods Hole, MA
Cohen, Seymour, Woods Hole, MA

Edds, Louise L.. Ohio University

Ellington, Athleen, University of Massachusetts

Fussell, Catherine P., Pennsylvania School of Medicine

Gehrke, Lee, Massachusetts Institute of Technology
Gray, Richard A., Baylor College of Medicine

Haubrich, Robert, Denison University
Herskovits, Theodore T., Fordham University

Johnston, Dan, Baylor College of Medicine

Kelly, Robert E., University of Illinois, Chicago

King. Kenneth. Woods Hole. MA

Korf, Bruce R.. Boston. MA

Krane, Stephen. Massachusetts General Hospital

Laderman. Aimlee D., Yale University

Leighton, Joseph. Peralta Cancer Research Institute

Lorand, Laszlo, Northwestern University

Mauzerall, David, Rockefeller University
Mizell, Merle, Tulane LIniversity
Morrell, Frank, Rush Medieal Center

Narahashi. Toshio, Northwestern University
Nickerson, Peter A., SUNY, Buffalo

Pappas. George D.. University of Illinois, Chicago
Person, Philip, Flushing. NY

Rao, T. S., Donapaula, Gao-India

Rickles. Frederick R., University of Connecticut Health

Rosati, Floriana, Siena. Italy

Roth. Jay. Woods Hole. MA

Shepard. Frank, Falmouth, MA
Sonnenblick, Benjamin, Rutgers University
Spector, Abraham, Columbia L'niversity
Spiegel, Evelyn, Dartmouth College
Spiegel, Melvin, Dartmouth College
Sundquist, Eric, United States Geological Survey
Sydlik, Mary Ann, SUNY, Geneseo

Tilney, Louis, University of Pennsylvania
Tilney, Molly, University of Pennsylvania
Tsuji, Frederick, Scripps Institute
Tweedell, Kenyon S., University of Notre Dame

Webb, H. Marguerite, Woods Hole, MA
Weir, Gary E., U.S. Naval Historical Center

Library Readers: Rooms

Canello. Lucio. Stazione Zoologica, Italy

D'Alessio, Giuseppe, Via Mezzocannone, Italy

Filley, O. D., Filley & Co.

Goldman. Robert, Northwestern University Medical School

Hines, Michael, Duke L'niversity

Ilan, Joseph, Case Western Reserve University

Ilan. Judith, Case Western Reserve University

Moore, John W.. Duke University

Patterson, David, University of Sydney, Australia

Rabinowitz. Michael, MBL/Harvard Medical School

Reynolds, George T.. Princeton University

Sheetz, Michael P., Duke University

Speck, William. Case Western Reserve University

Spirin, Alexander, Academy of Sciences. Russia

Stuart. Ann E., University of North Carolina. Chapel Hill

Tykocinski, Mark L.. Case Western Reserve University

Weissmann, Gerald, NYU Medical Center

Yoshioka, Tohru, Waseda University. Japan

Zweig. Ron, Ecologic

Domestic Institutions Represented

Alabama, University of, Birmingham
Alabama, University of. School of Medicine
Albany Medical College
Albert Einstein College of Medicine
American Board of Quality Assurance
American Medical Association
American Museum of Natural History
Aquatic Research Organisms
Arizona State University
Arizona, University of

AT&T Bell Laboratories
Axon Instruments, Inc.

Barry Controls

Baylor College of Medicine

Beckman Instruments, Inc.

Becton Dickinson IS

Benedict College

Bigelow Laboratories for Ocean Studies

Bio-Rad Laboratories

Boston University
Boston University Marine Program
Boston University School of Medicine
Bowman Gray School of Medicine of Wake

Forest University
Brandeis University
Brandeis University, HHMI
Brinkmann Instruments, Inc.
Brooklyn College of the City University of

New York

Summer Research Programs R35

Brown University

Brown University School of Medicine

BTX

Buffalo. University of

C. P. Li Biomedical Research Corp.
California Institute of Lechnolog\
California Institute of Technology, Beckman

Institute

California State University. Fresno
California. University of. Berkeley
California, University of, Davis
California. University of. Irvine
California, University of. Los Angeles
California, University of. Riverside
California. University of. San Diego. HHM1
California. University of. San Francisco
Cambridge Technology
Carnegie Mellon University
Case Western Reserve University
Catawba Memorial Hospital
CAV Consulting
Center for Disease Control
Chicago Medical School. University of

Health Sciences

Chicago. Rehabilitation Institute of
Chicago. University of
Children's Hospital. Boston
Children's Hospital, Philadelphia
Children's Hospital Medical Center
Cincinnati, University of
Cincinnati. University of. College of

Medicine

Cincinnati. University of. Medical Center
City College of New York
Clarkson University
Cleveland Clinic Foundation
Cleveland Clinic Research Institute
Codonics

Colorado. University of. Boulder
Colorado, University of. Health Science

Center
Columbia Graduate School of Arts &

Sciences

Columbia University
Columbia University, College of Physicians &

Surgeons

Columbia University for Medical Informatics
Connecticut, University of. Health Center
Connecticut, University of. Storrs
Cornell University
Cornell University Medical College
Cornell University. N.Y. State College of

Veterinary Medicine
Costar Corporation
Coy Laboratory Products
CUNY Medical School

Dage MTI. Inc.
Dartmouth College
Dartmouth Medical School
David Kopf Instruments
Delaware, University of
Digital Equipment Corporation

Drexel University

Dummond Scientific

Duke University

Duke University Medical Center

Dupont Merck Pharmaceutical Company

E. I. duPont de Nemours & Co. (Inc.)

East Carolina University

Eastman Kodak Company

EG & G Instruments

Emory University

Emory University School of Medicine

ENRM VA Hospital

Eppendorf, Inc.

Ericomp. Inc.

Exon-Intron, Inc.

Exxon Biomedical Sciences. Inc.

Fisher Scientific

Florida Medical Entomology Lab, Gainesville

Florida State University

Florida, LIniversity of. College of Medicine

Food & Drug Administration

Fordham LIniversity College at Lincoln

Center

Forsyth Dental Center
Fox Chase Cancer Center
Franklin & Marshall College
Frederick Haer & Company

General Valve Corporation

George Washington University

George Washington University School of

Medicine

GIBCO/BRL Life Technologies, Inc.
Gilson Medical Electronics. Inc.
Gonzaga LIniversity
Grass Instrument Company

Hahnemann University

Hahnemann LIniversity School of Medicine

Hamamatsu Photonic Systems

Hampshire College

Harbor Branch Oceanographic Institution

Harvard Community Health Plan

Harvard Medical School

Harvard Medical School/Brigham &

Women's Hospital
Harvard School of Public Health
Harvard University
Haverford College
Hawaii. University of
Hitachi

Hoefer Scientific
Hoffman-La Roche, Inc.
Holy Cross College
Honeywell Corporation
Hospital for Special Surgery
Houston. University of
Howard Hughes Medical Institution,

Massachusetts General Hospital
Howard University

Humana Hospital
Hunter College

Hunter College of the City University of New
York

ICN Radiochemicals (Division of ICN

Biomedicals, Inc.)

Idaho National Engineering Laboratory
Idaho, University of
Illinois Institute of Technology
Illinois State University
Illinois, University of
Illinois, University of. College of Medicine
Indec Systems Corporation
Indiana LIniversity
Indiana University Medical Center
Inovision Corporation
Institute of Biotechnology
International Equipment Company
Iowa, University of
1SCO, Inc.

James Madison University
Jefferson Medical College
JEOL

Johns Hopkins Hospital
Johns Hopkins University
Jouan, Inc.

Jules Stein Eye Institute, University of
California, Los Angeles

Kansas, University of
Kent State University
Kewalo Marine Laboratories
Kramer Scientific Corporation

Lab Line Instruments, Inc.

Lab Products

La Jolla Cancer Research Foundation

Laser Science

Lederle Laboratories

Lehman College. City University of New

York

Leica, Inc.

Lilly Research Laboratories
Lister Hill National Center for Biomedical

Communications
Los Alamos National Laboratory
Louisiana State University
Louisiana State University Medical College
Lovelace Medical Foundation
Loyola LIniversity Medical School
Ludlum Measurements. Inc.

M. D. Anderson Cancer Center
MCI Telecommunications
Maryland, LIniversity of, Baltimore
Maryland. University of. Eastern Shore
Maryland, LIniversity of. Medical School
Massachusetts Eye and Ear Infirmary,

Berman-Gund Laboratory
Massachusetts General Hospital

R36 Annual Report

Massachusetts Institute of Technology
Massachusetts, University of, Amherst
Mayo Clinic
Mayo Foundation
Medical College of Ohio
Medical Systems Corporation
McLean Hospital
Meharry Medical College
Merck Sharp & Dohme Re-

Laboratories
Meridian Instrument-
Miami, University of
Michigan State University
Michigan, Llmversily of
Micro Video Instruments
Miles Inc., Diagnostics Division
Millipore Corporation
Minnesota, University of
Missouri, University of. School of Medicine
Missouri, University of, Columbia
MJ Research
Molecular Probes
Mount Holyoke College

NACVIS Systems

NAOS Marine Laboratory

NAPA Permanente Medical Group

Nanshige USA, Inc.

NASA Ames Research Center

National Institutes of Health

National Institutes of Health/NCI

National Institutes of Health/NEI

National Institutes of Health, NHLBI

National Institutes of Health/NIDCD

National Institutes of Health/NINDS

National Library of Medicine

Nebraska. University of

NEC Research Institute

Neuro Data Instrument Corporation

New Brunswick Scientific Company, Inc.

New England Aquarium

New England Deaconess Hospital

New Hampshire, University of

New Mexico, University of

New York Medical College

New York State Department of Health

New York State Institute of Basic Research

New York. State University of

New York, State University of. Albany

New York, State LIniversity of, Buffalo

New York. State University of. Stony Brook

New York, State University of, Syracuse

New York University

New York University Medical Center

Newport Corporation

Nikon, Inc.

Nikon Instruments

North Carolina, University of

North Carolina, University of. Chapel Hill

Northeast Ohio University College of

Medicine

Northwestern University
Northwestern University Cancer Center
Northwestern Universitv Medical School

Office of the Inspector General
Ohio State University
Oklahoma, University of
Old Dominion University
Olympus Corporation
Opti-Quip

Oregon Health Sciences University-
Oregon Regional Primate Research Center
Oregon. University of
Owl Scientific

Pennsylvania State University
Pennsylvania, University of
Pennsylvania. University of. School of

Medicine

Perceptics Corporation
Perkm-Elmer Corporation
Pharmacia, Inc.
Photometries. Ltd.
Photon Technology International
Physitemp Instruments, Inc.
Pittsburgh Eye and Ear Institute
Pittsburgh. University of
Pittsburgh. University of. School of Medicine
Pomona College
Population Council. The
Princeton University
Puerto Rico, University of
Purdue University

R & M Biometrics, Inc.
Radiomatic Instruments & Chemical

Company, Inc.

Research Precision Instruments
Rhode Island, Universitv of
RMC

Rohbins Scientific Corp.
Rochester. University of
Rochester. University of. School of Medicine

Dentistry
Rockefeller University
Rowland Institute for Science
Rush Presbyterian St. Luke's Medical Center
Rutgers University

Sulk Institute, The

San Francisco State University

Savant Instruments, Inc.

Schepens Eye Research Institute. Inc.

Scientific Systems

Scripps Research Institute

Shandon-Lipshavv

Shimadzu Scientific Instruments. Inc.

Smith College

Smithsonian Institution Museum

Sony Medical Electronics

South Carolina. University of. Medical

School

Southern California, University of
Southern Mississippi, University of
Stanford University-
Stanford University School of Medicine,

Beckman Center
Stoelting Companv

Strategene

Supercomputing Research Center

Sutler Instrument Company

Swarthmore College

Syntex Research

Syracuse University

Technical Manufacturing Corporation

Technical Products Internationa, Inc.

Technical Video. Ltd.

Texas Christian University

Texas. University of

Texas. University of, Austin

Texas, University of. Health Science Center

Texas, University of. Medical Branch,

Galveston
Texas, University of. Medical School.

Houston
Texas, University of. Southwestern Medical

Center. Dallas

Thomas Jefferson University
Toledo, University of
Tufts University-
Tufts University Medical School
Tufts University School of Veterinary

Medicine
Turner Designs

UMDNJ, Robert Wood Medical School

USDA, ARS, ERRC

Uniformed Services University of the Health

Sciences, Hebert School of Medicine
Union College
United States Air Force
United States Department of Veteran

Administration, Affairs Medical Center
United States Fish & Wildlife Service
Universal Imaging Corporation
Utah, University of
Utah, University of. School of Medicine

Vanderbilt LIniversity

Vanderhilt LIniversity School ol Medicine

Vermont, University of

Video Scope International, Ltd.

Virginia/Maryland Regional College

Virginia, University of

Virginia. Universitv of. Medical School

Vital Images

Wadsworth Center for Laboratories &

Research

Wake Forest University
Warner Instrument Corporation
Washington State University
Washington. University of
Washington University
Washington University School of Medicine
Waters Chromatography Division
Wayne State LIniversity
Wellesley College
Wesleyan University-
West Florida. University of
West Virginia University

Summer Research Programs R37

West Virginia University Health Sciences

Center

Western Psychiatric Institute and Clinic
Whitehead Institute
Williams College
Wisconsin. University of. Madison

Wisconsin. University of, Milwaukee
Woods Hole Oceanographic Institution
Worcester Foundation for Experimental
Biology

Xybion Corporation

Yale University

Yale University School of Medicine

Youngstown State University

Carl Zeiss. Ine.
Zymogenetics Corporation

Foreign Institutions Represented

Aarhus University. Denmark
Academy of Sciences. Russia
Academy of Sciences. Institute of Protein

Research. Russia
Aquarium & Marine Center. Canada

Barcelona. University of. Spain

Bari. Universita di. Italy

Basel. University of. Switzerland

Basel. University of. Biocenter. Switzerland

Buyreuth. Universitaet. Germany

Belgrade. University of. Yugoslavia

Ben Gurion. University of. Israel

Bordeaux. University of. France

Botanical Institute I. University Giessen,

Germany

Bristol. University of. United Kingdom
British Columbia. University of. Canada

Cairo. University of. Medical School. Egypt
Calgary. University of. Canada
CINEVESTAV-IPN. Mexico City. Mexico
CNRS. Station Zoologique, France
Concordia University. Canada

Dalhousie University. Canada
Deakin University. Australia
Douglas Research Hospital. Canada

Ecole Normale Supeneure. France
Edinburgh University. United Kingdom
Escola Paulista de Medicina. Brasil

Freiburg Institut fur Biologie, Germany
Fnedrich Miescher Institut. Switzerland

Geneve. Universite de, Switzerland
Glasgow, University of. Scotland

Hebrew University. Israel

Hong Kong University of Science &

Technology. Hong Kong
Hospital de I'Enfant-Jesus. Canada
Hospital for Sick Children. Canada

1LRAD. Kenya

Imperial Cancer Research Fund. United

Kingdom
INSERM. France
Institut fuer Zoologie. Germany
Institut J. Monod. France

Institute of Marine Affairs, West Indies
Institute of Zoophysiology. Germany
Institute de Fisologia Celular, UNAM.

Mexico
Instituto de Investigaciones Biologicas

Clemente Estable. Uruguay
Instituto M.y.M. Ferreyra. Argentina
I.V.I.C.. Venezuela
I. Zoologishes Institut. Germany

James Cook University of North Queensland.
Australia

Kaiserlautern, University of, Germany

Karl-Franzens-Universitat, Austria

Keele University. UK Institut for Tropical

Medicine. Germany
Koln. University of, Germany
Konstanz. University of. Germany
Kyoto University. Japan

Laval, Universite dekl. Canada

Manchester, University of. United Kingdom
Max-Planck-Institut, Germany
Max-Planck-Institut fur Biologische

Kyhernetick, Germany
Max-Planck-Institut fur Biophysikalische

Chemie. Nikolausberg. Germany
Max-Planck-Institut fur Brain Research.

Germany
Max-Planck-Institut fur

Entwicklungsbiologie, Germany
McGill University, Canada
McGill Vision Research Center, Canada
McMaster University Medical School,

Canada

Medical Research Council. United Kingdom
Mexico. University of. Mexico
Montreal. University of, Canada
Moscow State University, Russia
MRC Anatomical Neuropharmacology Unit
MRC Laboratory of Molecular Biology.

United Kingdom

Naples, University of. Italy
National Museums. Kenya
National University. Mexico
Newfoundland, Memorial University of,
Canada

New South Wales. University of. Australia
Northern Ireland Department of Agriculture.
Ireland

Oldenburg. University of. Germany
ORSTOM/DGRST Center. Congo
Osaka Medical College. Japan
Ottawa. University of. Canada
Oxford University, United Kingdom

Panum Institute, Denmark
Paris-Sud. L'niversite. France
Pasteur Institute. France
Pembroke College, United Kingdom
Philipps-Universitat. Germany

Queen's Medical Centre. L'nited Kingdom
Queens University, Canada

Regensburg. LJniversitat. Germany

St. Andrews University. Scotland

St. George's Hospital Medical School, United

Kingdom

Salamanca. University of. Spain
Sofia University, Bulgaria
Stazione Zoologica. Italy
Sydney. University of, Australia

Technion. Israel
Tel Aviv University, Israel
Tokyo, University of. Japan
Toronto. University of. Canada
Toyohashi University of Technology
Tubingen. University of. Germany
Tuscia. LJniversity of. Italy

Ulm, University of. Germany
University Hospital of Basel. Switzerland
Uppsala Biomedical Center, Sweden

Wales. University of. United Kingdom
Waseda University. Japan
Weizmann Institute of Science, Israel
Wellcome Research Laboratories, LInited

Kingdom
Wursburg, University of. Germany

Zurich. L'niversitv of, Switzerland

Year-Round Research Programs

Architectural Dynamics in Living Cells
Program

Established in 1992 this program focuses on architectural dynamics
in living cells the timely and coordinated assembly and disassembly
of macromolecular structures essential for the proper functioning,
division, motility. and differentiation of cells: the spatial and temporal
organization of these structures', and their physiological and genetic
control. The program is also devoted to the development and
application of powerful new imaging and manipulation devices that
permit such studies directly in living cells and functional cell-free
extracts. The Architectural Dynamics in Living Cells Program
promotes interdisciplinary research and consists of resident core
investigators and a cadre of adjunct members.

Staff

Inoue. Shinya, Distinguished Scientist
Oldenbourg. Rudolf. Associate Scientist
Stemmer. Andreas, Visiting Assistant Scientist

Boston University Marine Program

Faculty

Atema. Jelle, Professor of Biology, Program Director
Humes, Arthur G., Professor of Biology Emeritus
Tamm, Sidney L., Professor of Biology
Valiela, Ivan, Professor of Biology

Staff

Hahn, Dorothy, Senior Administrative Secretary
Kean. Knsten, Program Assistant
Schillizzi. Cynthia. Program Manager

I 'isiling f-'uciiliv and Investigators

Collette. Bruce. NMFS National Museum of Natural History,

Washington, DC

Cuomo, Carmela, Yale University
D'Avanzo. Charlene, Hampshire College

Hinga. Kenneth. University of Rhode Island

Kaufman. Les. Edgerton Research Lab, New England Aquarium

Kremer, James, University of Southern California

McFall-Ngai. Margaret, Scnpps Institute of Oceanography

McPhee. Linda, postgraduate WHMS student from Ontario

Muscatine. Leonard, University of California, Los Angeles

Peckol, Paulette. Smith College

Rietsma, Carol, SUNY, New Paltz

Sardet, Christian, Villefranche-sur-Mer, France

Seeler, Jacob. University of Texas Southwestern Medical Center

Simmons, William, Visiting Lecturer, Boston University

Wainright, Sam, Rutgers University

Ward, Nathalie. Center for Coastal Studies

Research Staff

Basil. Jennifer. Postdoctoral Investigator
Breithaupt. Thomas. Postdoctoral Investigator
Chang, Patnque, Visiting Investigator
Dudley, Judy. Visiting Research Assistant
Eisthen, Heather. Grass Fellow
Demer, Michael. Visiting Research Assistant
Foreman. Kenneth, Research Associate
Hammes, Michelle, Research Assistant
Langton. Lori, Research Assistant
MacDonald, Robin, Research Assistant
Miller, Caroline, Visiting Lab Assistant
Seely. Brad, Visiting Research Assistant
Tamm, Signhild. Senior Research Associate
Voigt. Rainer. Research Associate

Teaching Assistants

Asmutis. Regina. Course Assistant

Bushman. Paul. Woods Hole Marine Semester Coordinator

Farley. Lynda. Course Assistant

Gomez, George, Course Assistant

Karavanich, Christy, Course Assistant

Lowe. Brian, Course Assistant

O'Brien, Todd, Course Assistant

Schlezmger, David. Course Assistant

R38

Year-Round Research Programs R39

Graduate Student*

Alber, Merry!

Anderson, John

Bohaehevsky, Bons

Bryden. Cynthia

Bushmann. Paul

Cowan, Diane

Farley, Lynda

Gomez, George

Hersh, Douglas

Joy, Jennifer

Karavanich. Christy

LaMontagne, Michael

Lavalli, Kari

Lindner. Kate

Lowe. Brian

McPhee, Linda (postgrad, Ontano)

Mosiach. Simon

O'Bnen, Todd

Portno>. John

Schlezinger. David

Tamse, Armando

Usup, Gires

White. David

Undergraduate Students Fall 1992

Alterman. Randy

Bay ha. Keith

Bechtel. Jamla

Benning. Linda

Brown. Timothy (Wesleyan)

Bowman. Liza

Castro, Natalia

Conlon, Jeffrey

Crooks. Wendy

Davidson. Stacie

DeSantis, Krystal

DePalma. James

Ellison, Rob

Emerson. Lyndal

Esham, Kristina

Ettinger, Brian

Fox. Ellen

Harding. Jennifer

Harmer, Tara

Hoffner. Jude (Wesleyan)

Morgan. Edward

Kachra. Tasleem (Brandeis)

Keyser, Alisa

Kim, Hong

Kotwas. Kristin

Lentine. Maria

Lombard. Benjamin

Maron, Christopher

Pedersen. Jennifer

Peralta. Michelle

Pimental, Helenia

Pretto. Christopher

Rader. Lauren

Rulison. Steve

Suarez. Mark

Szczepankiewicz. Peter

Vanmarke. Knstien

Vavpetic, Lisa
Wanger, Jolle
Wingertner. Scott
Yang, Grace

Summer Undergraduate Interns

Barak, Jen
Berg, Kathleen
Collins, Glynnis
Cordray, Diane
Rick. Kevin
Guilfoyle, Kern Jo
Harrison, John
Herr, Barbara
Kirkendall, Ellen
Owen, Jennifer
Rollenhagen, Julianne
Watt. Melissa

Laboratory ofJelle Atema

Organisms use chemical signals as their main channel of
information about the environment. These signals are transported in
the marine environment by turbulent currents, viscous flow, and
molecular diffusion. Receptor cells extract signals through various
filtering processes. Currently, the lobster with its exquisite sense of
taste and smell, is our major model to study the signal filtering
capabilities of the whole animal and its narrowly tuned receptor cells.
Research focuses on amino acids (food signals) and pheromones
(courtship and dominance), neurophysiology of receptor cells,
behavior guided or modulated by chemical signals, and computational
models ol odor plumes and neural filters.

Laboratory of Arthur G. Humes

Research interests include systematics. development, host
specificity, and geographical distribution of copepods associated with
marine invertebrates. Current research is on taxonomic studies of
copepods from invertebrates in the tropical Indo-Pacific area, and
poecilostomatoid and siphonostomatoid copepods from deep-sea
hydrothermal vents and cold seeps.

Laboratory of Sidney Tanvn

Research interests include cell physiology and motility,
cytoskeleton, ciliary and flagellar motion, and trophic ecology of
gelatinous zooplankton. Current research is on neural and ionic
control of ciliary feeding and escape behaviors of manne invertebrates,
distribution of calcium channels and calcium sensors in ctenophore
cilia, geotactic mechanisms and sensory receptors in ctenophores,
jellies with jaws (macrociliary teeth and actin bundles in Bcroe), and
rotary motors and fluid membranes in symbiotic protozoa.

Laboratory of Ivan Valiela

Our major research activity involves the Waquoit Bay Land Margin
Ecosystems Research Project. This work examines how human
activity in coastal watersheds (including landscape use and
urbanization) increases nutrient loading to groundwater and streams.
Nutrients in groundwater are transported to the sea, and, after
biogeochemical transformation, enter coastal waters. There, increased
nutrients bring about a series of changes. The Waquoit Bay LMER is
designed to help us to understand and model the coupling of land use

R40 Annual Report

and consequences to receiving waters, and to study the processes
involved.

A second long-term research topu IK- structure and function of
salt marsh ecosystems, including tl . lo-sses of predation, herbivory,
decomposition, and nutrient eye

Center for lolecular Evolution

The major research dtort of this laboratory is the structure analysis
of ribosomal RNA. Similarities between small subunit ribosomal
RNA sequences arc used to infer the evolutionary history of
eukaryotic microorganisms and to design molecular probes for studies
in marine ecology.

Staff

Sogm. Mitchell L., Director and Senior Scientist

Gunderson, John. Research Associate

Hinkle, Greg, Postdoctoral Fellow

Leipe, Detlev, Postdoctoral Fellow

Morrison, Hillary, Research Associate/ Postdoctoral fellow

The Ecosystems Center

The Center was established in 1975 to promote research and
education in ecosystems ecology. Twelve senior scientific staff and 43
research assistants and support staff study the terrestrial and aquatic
ecology of a wide variety of ecosystems ranging from Brazil (carbon
cycling and trace gas emissions from tropical forests and pastures) to
the Alaskan Arctic (long-term studies of the response of tundra, lake,
and stream biota to change) to the Harvard Forest (long-term studies
of the effects of disturbance in forest ecosystems) to Massachusetts Bay
(rates of denitnfication). Many projects, such as those dealing with
sulfur transformations in lakes and nitrogen cycling in the forest floor,
investigate the movements of nutrients and make use of the Center's
mass spectrometry laboratory (directed by Brian Fry) to measure the
stable isotopes of carbon, nitrogen, and sulfur. The research results are
applied wherever possible to questions of the successful management
of the natural resources of the earth. In addition, the ecological
expertise of the staff is made available to public affairs groups and
government agencies who deal with such problems in acid rain,
ground water contamination, and possible carbon dioxide-caused
climate change.

Scanlon, Deborah
Schwamb, Carol
Shaver, Gaius

Postdoctorals

Johnson, Loretta
McKane, Robert

Staff

Hobbie. John E., Co-Director

Melillo. Jerry M., Co-Director

Bahr, Michele

Castro, Mark

Chapman. Jonathan

Deegan, Linda

Donovan. Suzanne

Dornblaser. Mark

Downs. Martha

Drummey. Todd

Fry, Brian

Garritt, Robert

Geyer, Heidi

Giblin. Anne

Griffin, Elisabeth

Helfrich, John

Hopkinson. Charles

Hullar. Meredith

Jesse. Martha

Jones, David
Ricklighter. David
Laundre, James
Martin. Daniel
McGuire, A. David
Miliefsky. Michele
Murray. Georgia
Nadclhoffer. Knute
Newkirk. Kathleen
O'Hara, Patricia
Padien. Daniel
Pallant. Julie
Parmcntier, Nancy
Peterson, Bruce
Rastetter, Edward
Redmond. Leslie
Regan. Kathleen
Repert, Deborah
Ricca. Andrea

Steudler, Paul
Tholke, Kristin
Tucker, Jane

Neill, Christopher
Peterjohn. William

Normann. Bosse, University of Umea, Sweden

Consultants

Bowles, Francis
Bowles, Margaret
Schwarzman, Elisabeth

Laboratory for Marine Animal Health

The laboratory provides diagnostic, consultative research, and
educational services to the institutions and scientists of the Woods
Hole community concerned with marine animal health. Diseases of
wild, captive, and cultured animals are investigated.

Staff

Abt. Donald A., Director and The Robert R. Marshak Term Professor

of Aquatic Animal Medicine and Pathology, School of Veterinary

Medicine. University of Pennsylvania
Bullis, Robert A.. Research Assistant Professor of Microbiology,

University of Pennsylvania
Lawrence, Wade B.. Research Assistant Professor of Pathology.

University of Pennsylvania
Leibovitz, Louis, Director Emeritus
McCafferty. Michelle, Histology Technician, L'niversity of

Pennsylvania

Moniz, Priscilla C, Secretary
Smolowitz, Ro\anna M., Research Associate in Pathology. University

of Pennsylvania
Wadman. Elizabeth A.. Microbiology Technician, University of

Pennsylvania

Laboratory of Aquatic Biomedicine

This laboratory investigates leukemias of soft shell clams.
Monoclonal antibodies developed by this laboratory and techniques in
molecular biology are used to investigate the differences between
normal and leukemic cells and their ontogeny. The impact of
pollutants on leukemogenesis is currently being studied with an
emphasis on regional superfund sites.

Staff

Reimsch. Carol L., Investigator, MBL. and Chairperson Department
of Comparative Medicine. Tufts University School of Veterinary
Medicine

Leland. Christian. Laboratory Assistant

Laboratory of Cell Biochemistry

This laboratory uses cell and molecular biological methods to study
the regulation of gene expression in marine fish. Current emphasis is
on gene products involved in hepatic heme biosynthesis and

Year-Round Research Programs R41

utilization. These processes are affected by hormonal, nutritional, and
pharmacological agents as well as xenobiotics. and carcinogens. In
addition, free heme is a feedback regulator of its biosynthesis. The
principal site at which these agents act to control the rate of heme
production is 5-aminolevulinate synthase (ALS), the first enzyme of
the pathway. However, it has not been possible to define the
mechanisms that lead to enzyme induction or repression. Marine fish
are attractive for that purpose because they have similar regulatory
features for heme biosynthesis but lack some of the hepatic processes
that have confounded studies in mammals. Evidence to date strongly
indicates that expression of fish ALS is regulated at a post-
transcnptional stage. Cloned cDNAs have been isolated for both the
housekeeping and erythroid forms of ALS. and the sequence of the e-
type cDNA encodes an iron regulatory element that controls the rate
of mRNA translation. It is expected that these studies of the fish ALS
system will give new insights into the control of heme biosynthesis in
vertebrate organisms, including man. Primary cultures offish
hepatocytes provide the experimental material for this work, and an
additional interest of this laboratory' is in establishing these cell
cultures as a nonmammalian model for biomedical research.

Staff

Cornell. Neal W.. Senior Scientist
Abilock, Rigele. Research Assistant
Bruning. Grace. Research Assistant
Stukey. Jetley, Laboratory Assistant

I 'isiting Scientist

Fox, T. O., Harvard Medical School

Laboratory of Developmental Genetics

This research group studies the early gene control of cellular
differentiation pathways (cell lineage determination) in the embryos of
tunicates and other marine invertebrate species.

Staff

Whittaker. J. Richard, Senior Scientist
Crowther. Robert, Research Associate
Loescher, Jane L., Research Assistant
Meedel, Thomas H.. Assistant Scientist

I 'isiting investigators

Collier, J. R., Brooklyn College

Lee, James J.. Columbia University. College of Physicians & Surgeons

Laboratory' of Judith P. Grassle

Studies on the population genetics and ecology of marine
invertebrates living in disturbed environments, especially of sibling
species in the genus Capiti'llu (Polychaeta). Flume studies on bivalve
and polychaete larval habitat selection.

Staff

Grassle. Judith P., Senior Scientist
Mills. Susan W., Research Assistant

Laboratory of Harlyn O. Halvorson

Over the past year, we have isolated a large number of
actinomycetes and sporeformers from various marine environments

like deep sea cores and sediments. Our intention is to characterize
these bacteria at the molecular level and to look for biologically active
components. Protocols based on DNA fingerprinting and quantitative
hybridizations have been developed to differentiate marine
sporeformers from one another, as well as from terrestrial
sporeformers. The hybridization data has shown that the bacterial
isolates are not closely related to one another.

Numerical taxonomic methods are also being used to cluster the
various isolates. The physiologically interesting sporeformers will also
be characterized by physical mapping using rare-cutting restriction
endonucleases.

Staff

Halvorson. Harlyn O.. Principal Investigator
Chikarmane. Hemant. Assistant Scientist
VanLooy, Lori, Research Assistant

I 'isiting Investigators

Anderson, Porter, University of Rochester
Keynan, Alex, Hebrew University, Jerusalem. Israel
Rornberg. Hans, Christ's College, Cambridge, UK
Vincent, Walter, University of Delaware
Yashphe. Jacob, Hebrew University, Jerusalem, Israel
Wainwright, Norman

Laboratory of Shinya Inoue

Study of the molecular mechanism and control of mitosis, cell
division, cell motility, and cell morphogenesis, with emphasis on
biophysical studies made directly on single living cells, especially
developing eggs in marine invertebrates. Development of biophysical
instrumentation and methodology, such as polarization optical and
video microscopy and digital image processing techniques, and
exploration of their underlying theory are an integral part of the
laboratory's effort.

Staff

Inoue. Shinya, Distinguished Scientist

Knudson, Robert, Instrument Development Engineer

Oldenbourg, Rudolf. Assistant Scientist

Stemmer, Andreas. Visiting Assistant Scientist

Stukey. Jelly, Research Assistant

Woodward. Bertha M., Laboratory Manager

I 'isiting investigators

Bajer. Andrew, University of Oregon
Burgos, Mario, Universidad Nacional de Cuyo Conicet
Febvre, Colette, Station Zoologique, Villefranche-sur-Mer, France
Febvre, Jean, Station Zoologique. Villefranche-sur-Mer, France
Sardet, Christian, Station Zoologique. Villefranche-sur-Mer, France

Laboratory of Alan M. Kuzirian

Research in this laboratory' explores the functional morphology and
ultrastructure of various organ systems present in opisthobranch
mollusks. The program includes mariculture of the nudibranch,
Hermissenda crassicurnis, with emphasis on developing reliable
culture methods for rearing and maintaining this animal as a research
resource. Studies include optimization of adult and larval nutrition,
control of facultative pathogens and disease, development of
morphologic criteria for staging larvae and juveniles, and

R42 Annual Report

metamorphic induction. Morphologic studies stress the ontogeny of
neural and sensory structures, and neurochemicals associated with the
photic and vestibular systems that have been used as models systems
in learning and memory studies.

Concurrent with these studies is ti-.c development of a new
technique to obtain and reconstruct serial block face images (SBFI) of
epoxy-embedded or cryoprepared tissues sectioned or freeze-fractured/
freeze-etched inside an SEM b\ an in silu miniature ultramicrotome.

Additional collaborative research includes histochemical
investigations on strontium's role in initiating calcification in
molluscan embryos (shell and statoliths), as well as
immunocytochemical labeling of cell-surface and secretory product
antigens using monoclonal and polyclonal antibodies on Hermissenda
sensory and neurosecretory neurons in cell culture. Systematic and
taxonomic studies of nudibranch mollusks are also of interest.

Staff

Kuzirian, Alan M., Associate Scientist
Tamse. Catherine T., Research Assistant

Laboratory of Andrew L. Miller and
Lionel F. Jaffe

This laboratory investigates the role played by calcium ions in a
wide range of fundamental cell processes; in developing eggs, in
differentiated tissues, and in cell extracts. This is possible through the
use of aequorin, a bioluminescent protein complex. Aequorin can
either be microinjected into cells or transgenically expressed without
disturbing function or development. The pattern of luminescence that
is emitted by an aequorin-loaded cell reveals changing patterns and
levels of free calcium within the cell (or its progeny). Photons are
collected and correlated with dynamic cellular events by an imaging
system developed in our laboratory. This technique has some
substantial advantages over other methods of imaging intracellular
calcium and as a result supports an extensive collaborative research
effort. The laboratory is currently studying cytokinesis in frog and fish
eggs; cell cycle control in sea urchin and surf clam eggs; polarity
expression in frog eggs; tip growth in pollen tubes; injury and
degeneration in neurons; mechanisms of fertilization in sea urchins;
differentiation in slime molds; and calcium release in cell extracts
from frog eggs. The laboratory is supported by the NSF to both pursue
biological questions and to develop the aequorin-based imaging
technique.

Staff

Miller, Andrew L., Assistant Scientist
Karplus, Eric, Design Engineer
Jaffe. Lionel F., Senior Scientist

I 'inning Investigators

Alexander, Steve, Wadsworth Center

Bearer. Elaine, Brown University

Browne. Carole, Wake Forest University

Felle. Hubert, University of Giessen

Fishman, Harvey M., University of Texas Medical Branch

Fluck. Richard A., Franklin and Marshall College

Hepler, Peter, University of Massachusetts

Krause, Todd L., University of Texas

Metuzals, Janis, University of Ottowa, Canada

Sardet. Christian. Ville-Franche-sur-Mer. France

Swenson, Katherine, Duke University Medical School

Woodruff, Richard. Westchester University

Laboratory of Rudolf Oldenbourg

We study physical optics relevant to microscopic imaging and
develop advanced instrumentation in light microscopy for the study of
structural dynamics in cells and cell components. The current focus of
this new laboratory is the development of a novel polarized light
microscope that combines polarization optics with new electro-optical
components, video, and digital image processing for a fast analysis of
specimen anisotropies over the entire viewing field at the highest
resolution of the light microscope. Biological mechanisms to be
explored with this new instrument range from the emergence and
functional role of filamentous structures in living cells, to the
generation of ordered domains in liquid crystals and polymer
solutions. The laboratory currently investigates the fine structure of
myofibrils and the mechano-elastic properties of virus liquid crystals.

Staff

Oldenbourg. Rudolf, Associate Scientist

Laboratory of Robert E. Palazzo

This laboratory studies the biochemical regulation of cellular events
during meiosis and mitosis. An integral part of the research effort is
the design of reconstitution systems that faithfully execute cell cycle-
dependent events under defined conditions. Current cell biological,
immunochemical, biochemical, and microscopic methodologies are
employed. Using marine eggs as a material source, assays have been
developed that allow the study of germinal vesicle breakdown, aster
formation, and reactivation of isolated mitotic apparatus in vilni.
Current focus of the laboratory is on the identification of cell cycle-
dependent regulatory events with major emphasis on protein
phosphorylation and other post-translational modifications. The
ultimate goal is the identification of key enzymes and target substrates
that are involved in the regulation of cell division and are highly
conserved during evolution.

Staff

Palazzo, Robert E., Assistant Scientist

Dawson, Tim, Undergraduate Research Assistant

Peng, Gang, Postdoctoral Associate

Przybyla. Beata, Graduate Research Assistant

Sun. Yong, Postdoctoral Associate

Vogel. Jacalyn, Research Assistant

1 'ixitint' investigator*

Eckberg, William, R., Howard University

Heins, Susanne, Maurice E. Muller Institute, Basel, Switzerland

Rieder, Conly, L., Wadsworth Center for Labs and Research

Laboratory of Nancy Rafferty

This laboratory investigates the role of the lens cytoskeleton and its
associated proteins in maintenance of lens shape, in lens
accommodation and development of cataract when the cytoskeleton is
disrupted. Studies include an assessment of the role of cytosolic free
calcium on homeostasis of the lens cytoskeleton, the localization of
various cytoskeletal proteins in lens epithelium, and determination of
the relative amounts of soluble actin to filamentous actin in lens cells
during aging. Most of these studies employ a fish model using primary
cultures ot lens epithelium and electron and immunofluorescence
microscopy.

Year-Round Research Programs R43

Staff

RaflerU. Nancy S., Scientist. Northwestern University
Rafferty, Keen A.. Research Associate

Laboratory of Monica Riley

Research in this laboratory focuses on the molecular evolution and
gene expression in the bacterium Exclwrii-liui cn/i In a collaborative
effort, a database containing information on the intermediary
metabolism and biochemical pathways of /;' culi is being developed.
When completed, this database is expected to contain information on
each metabolic reaction, the enzyme, the reactants. products.
cofactors, activators, inhibitors, kinetics, equilibrium constants.
binding constants, etc.

Related research is on the evolution of the /:. cull DNA and
organization of the genes in the chromosome. Comparative nucleotide
and amino acid sequence data provide information on the
evolutionary relationships of E. coli genes to other genes in the E. coli
genome and to homologous genes in related bacteria.

Staff

Riley, Monica. Senior Scientist
Farquhar. Karyn. Research Assistant

Laboratory of Sensory Physiology

Since 1973. the laboratory has conducted research on various
aspects of vision. Current studies focus on photoreceptor cells, on
their light-absorbing pigments, and on their biochemical reactions
initiated by light stimulation. Microspectrophotometric and
biochemical techniques are used to study the receptors ot both
vertebrates (amphibia, fish, and mammals) and invertebrates
(horseshoe crab and squid).

Staff

Harosi. Ferenc. Director, Associate Scientist. MBL, and Boston

University School of Medicine
Szuts, Ete, Associate Scientist. MBL. and Boston University School of

Medicine

I 'isiting /

Erickson, Martha. Brandeis University

Evans. Barbara I.. University of Oregon. Eugene

Greenblatt, Daniel. Brandeis University

Hawryshyn. Craig W., University of Victoria, B. C., Canada

Kleinschmidt, Jochen, NYU Medical Center

Singarajah, Kandar, V., Federal University of Paraiba, Brazil

Laboratory of Neuroendocrinology

This laboratory studies the molecular and cellular bases of two
neural programs that regulate different important behaviors in the
model mollusk Aplvuu Research is conducted on the mechanisms of
the neuronal circadian oscillators located in the eyes. These circadian
oscillators drive the circadian activity rhythm of the animal, which is
concerned with the daily timing of food gathering and of prolonged
rest. Additional research is conducted on a group of neuroendocrine
cells that produce a peptide. "egg-laying hormone." that initiates egg
laying and associated behaviors. The laboratory is interested in how
the three-dimensional shape of this peptide hormone allows a highly
specific interaction with its receptor and the intracellular processes

that are triggered by it. In another project, the laboratory has
discovered and is continuing research on a novel second messenger
en/vine, an NADase. in the oocyles of . l/i/r.vio. that generates cyclic
ADPR. a Ca : *-mobilizing product.

Staff

Strumwasser, Felix, Director
Cox, Rachel L., Senior Research Assistant
Elder, Peggy, Laboratory Assistant
Groelle, Holly, Postdoctoral Fellow
Heisermann, Gary. Postdoctoral Fellow
Hellmich, Mark, Postdoctoral Fellow
Lewis. Karen, Laboratory Assistant
Vogel, Jackie, Research Assistant

Laboratory ofOsamu Shimomura

Biochemical studies of the various types of bioluminescent systems.
Preparation of the improved forms of aequorin for measuring
intracellular free calcium.

Staff

Shimomura. Osamu, Senior Scientist. MBL. and Boston University

School of Medicine

Shimomura. Akemi, Research Assistant
Nakamura, Hideshi, Harvard University

Laboratory of Raquel Sussman

We investigate the molecular mechanism of DNA damage-inducible
functions in E. coll- Present studies deal with novel genes that affect
radiation-induced mutagenesis and analysis of RecA functions.

Staff

Sussman, Raquel, Associate Scientist

National Vibrating Probe Facility

The vibrating probe is an instrument that enables an investigator to
explore, map. measure, and analyze the patterns of natural ionic
currents through living cells, embryos, and even adult organisms. This
is done non-invasively by measuring the very minute electrical
voltage gradients or specific ion gradients generated by those currents
within the external medium. Among the current collaborative projects
are studies of currents through epithelial cells, Ai>ly.\ui bag cells,
cockroach nervous system, injured squid axons, growing pollen tubes,
root hairs, and fungi. Investigators are welcome to conduct
exploratory studies on their own systems during the summer months.
Extensive investigations may be carried out at other times throughout
the year. This facility is supported by the Biomedical Research
Technology Program, National Center for Research Resources, NIH.
Applications for research time should be made to P. Smith.

Staff

Jaffe, Lionel, Senior Scientist and Facility Co-Director
McLaughlin, Jane, Research Assistant
Sanger. Richard, Senior Electronics Technician
Shipley, Alan. Research Associate
Smith, Peter J. S.. Co-Director

R44 Annual Report

I isiting investigators

Allen. Nina. Wake Forest University

Backie, Iain. Robert Gordon University, .otland

Bittner. George. University of Texas Gal ;->n

Cox, Daniel, Wake Forest University

Demarest, Jeff. University of Ark

Fay, Frederic, University of Ma^nrhusetts Medical School

Feijo, Jose, University of Lisbon, Portugal

Felle, Hubert, University of Giessen, Germany

Fishman, Harvey, University of Texas, Galveston

Ford, Timothy, Harvard University

Giblin, Anne, Ecosystems. MBL

Hill. Susan, Michigan State University

Hoch, Harvey, Cornell University

Karplus, Eric, MBL

Krause. Todd. University of Texas. Galveston

Leech, Colin, Howard Hughes Institute, Massachusetts General

Hospital

Mitton. Bryce, Massachusetts Institute of Technology
Morgan. Jim. University of Arkansas
Nagel. Wolfram, University of Munich. Germany
Pierson. Elisabeth, University of Siena, Italy
Wright, Jonathan, McMaster University, Ontario. Canada

Other Year- Round Investigators and Staff

Stephens. Raymond E., Principal Investigator
Szent-Gyorgyi, Gwen, Research Assistant
Tilney, Lewis G., University of Pennsylvania
Tilney, Mollv S.. University of Pennsylvania

Honors

Friday Evening Lectures

Carl Woese, University of Illinois, 26 June "The Revolution in

Evolution: Microbiology Comes Into us Own"
Eric Lander. Whitehead Institute for Biomedical Research.

Massachusetts Institute of Technology. 3 July "Dissecting Iliiiih/n

Heredity"
Mary Woolley. Research! America, 10 Julv "The Problem isn't

Science- . The Problem is Silence 'When the People Lead, the

Leaders U'ill Follow
Rudiger Wehner. Universitat Zurich. 16, 17 July (Forbes Lectures)

"Phi 'ii 'receptor Twist: A .Solution to the False Color Problem" ( 16

July) "Ant Xavixdtion. How a Small Brain Solves a Complex Task"

(17 July)
Michael Bennett. Albert Einstein College of Medicine. 24 July "From

Electric Fishes to Electric Synapses: dap Junctions Into the Modern

Era"
Stephen Heinemann. The Salk Institute. 31 July "Molecular Biology

ol the (jlulamate Receptors Structure ami Function"
William Catterall. University of Washington, 7 August (Monsanto

Lecture) "From Ionic Currents to Molecules The Molecular Basis

of Electrical Excitability in the Brain"
Peter Narins. University of California, Los Angeles. 14 August (Lang

Lecture) " Biostruclural Adaptations lor Iconstic ami Seismic

Communication in Amphibians: Lessons From the Forest"
Carolyn Cohen. Brandeis University. 21 August "A'cit Twists on an

Old Protein Fold The ^-helical Coiled ( 'oil"
Richard Rowe, The Faxon Company, 28 August "The Evolution of

Scholarlv Communications"

Fellowships

Robert Day Allen Fellowship

Sergei A. Kuznetsov. Technische Universitat. German), and Moscow
Universits. Russia

Frederik B. Bang Fellowship Fund

Robert J. Lauzon, Albany Medical College

Bakalar Fellowship

Fatima Gyoeva. Russian Academy of Sciences, Institute of Protein
Research

Frank A. Brown Memorial Readership

Gary E. Weir. Naval Historical Center. Washington, DC

Bernard Davis Fellowship

David J. Patterson. University of Sydney. Australia

Frank R. Lillie Fellowship

Peter Doroshenko. Duke University Medical Center

William Randolph Hearst Fellowship

Eleanore Fusco. Massachusetts Institute of Technology

Stephen VV. Kuffler Fellowship

Arturo Hernandez-Cruz, Universidad Nacional Atonoma de Mexico,
Institute de Fisiologia Celular. Mexico

Jacques Loeb Fellowship

Christine R. Rose, University of Kaiserslautern. Germany

Nikon, Inc. Fellowship

Andreas Stemmer. MRC Laboratory of Molecular Biology. UK

R45

R46 Annual Report

H. Burr Steinbach Memorial Fellowship

Ilya Bezprozvanny, University of Connecticut Health Center

M. G. F. Fuortes Fellowship

Arturo Hernandez-Cruz. Institute de Fisiologia Celular, UNAM.
Mexico

MBL Summer Fellows

Roger Buchanan, NINDS, National Institutes of Health

Peter Doroshenko, Duke University Medical Center

Fedor Severin. Institute of Protein Research, Russia

Joelle Tanguy, Northwestern University

Amy V. Trejo, Northwestern University

Patricia Wadsworth, University of Massachusetts, Amherst

William Wonderlin, West Virginia University

Herbert W. Rand Fellowship

Alexander S. Spinn, Institute of Protein Research, Russia

Science Writing Fellowships

Stephen Braun. Freelance

Robert Cooke. Newsday

Elizabeth Culotta, Freelance

Heather Dewar, The Miami Herald

Donald Frederick, National Geographic Society News Service

David Graham, San Diego Union-Tribune

Jeff Hecht, New Sciential

Rebecca Perl, Atlanta Jourmil-C 'on\ti/iiini

John Schieszer, KPLR-TV, St. Louis, MO

Cindy Schreuder, Chicago Trihitnc

Joannie Schrof, U.S. News & H'orlil Report

Steps Toward Independence MBL Summer
Fellowships

Ilya Bezprozvanny, University of Connecticut Health Center
Roger Buchanan, NINDS, National Institutes of Health
Fatima Gyoeva. Institute of Protein Research, Russia
Arturo Hernandez-Cruz. Institute de Fisologia Celular, UNAM,

Mexico

Sergei Kuznetsov, Technische University, Germany
Patricia Wadsworth, University of Massachusetts, Amherst

Scholarships

Alberto Monroy Fellow

Paola Loguercio Polosa, Universita di Bari. Italy

Bernard Davis Scholarship

Maria Ganeva. Sofia University. Bulgaria

Ariel Kusmaro, Tel Aviv University. Israel

Edouard Miambi. Center ORSTOM/DGRST, Congo

Margarita L. Miroshnichenko, Moscow State University, Russia

Biology Club of CUNY

Stefan Wurden, Universitat Konstanz, Germany

Father Arsenius Boyer Scholarship Fund

Atsushi Miyawaki, University of Tokyo. Japan

C. Lalor Burdick Scholarship

Beatrice Casasnovas, University of Bordeaux, France

Gary N. Calkins Memorial Scholarship

Lesley J. Narburgh, St. George's Hospital Medical School, UK

Frances S. Claff Memorial Scholarship

Atsushi Miyawaki, University of Tokyo. Japan

Edwin Grant Conklin Memorial Scholarship

Lisa C. Foa. Deakin University, Australia

Lucretia Crocker Endowment Fund

Isabelle M. Desjeux, Edinburgh University, UK

Marie-Anne Felix, Institut J Monod, France

Anna M. Myat, Imperial Cancer Research Fund. UK

William F. and Irene Diller Scholarship Fund

Stefan Wurden, Universitat Konstanz. Germany

Caswell Grave Scholarship

Beatrice Casasnovas, University of Bordeaux, France
Peter S. Dayan, CNL, Salk Institute

Aline D. Gross Scholarship

Cindy A. Wilson, University of California, Irvine

Merke! H. Jacobs Scholarship

Lisa C. Foa, Deakin University, Australia

Christine R. Rose, University of Kaiserslautern, Germany

Arthur Klorfein Fund Scholarship

Pavle R. Andjus, University of Belgrade, Yugoslavia
Atsushi Miyawaki, University of Tokyo, Japan
Enrique Saldana, University of Salamanca, Spam
Camilla Tornoe, Pembroke College, UK
Elisabeth C. Walcott, University of California, Irvine

S. O. Mast Founders Scholarship

Lisa A. Kelly, University of Ottawa. Canada

Michigan State Scholarship Center for Microbial
Ecology Fellow

Jennifer L. Byrnes, Harvard University

Faith Miller Scholarship

Sergei Kuznetsov, Technische University, Germany, and Moscow
University, Russia

Honors R47

James S. Mountain Memorial Fund Scholarship

Joseph F. Kelleher. Johns Hopkins University School of Medicine
Anni'ck H. Le Gall. Cornell University Medical College
Valene A. Pierce, University of Chicago
Ben B. Whitlock, Ohio State University
Lin Wu, Scripps Research Institute

Planetary Biology Internship

Dirk Schuler. Max-Planck-lnstitut. Germany
Angelica P. Seitz. University of" Connecticut, Storrs

NVilliam Townsend Porter Foundation Fellowship

Danen L. Cohen. Dartmouth College

Nathaniel Dudlex. Hampshire College

Juan C. Jorge-Rivera. Brandeis University

Errol R. Rolen. University of Missouri. Columbia

Edward E. Leonard. University of Pittsburgh School of Medicine

Herbert \\. Rand Scholarship

Stefan Wurden. Universitat Ronstanz, Germany

Society for Developmental Biology Scholarship

Maureen A. Gannon. Cornell University Medical College
John C. Matese. Duke University

Marjorie W. Stetten Fund

Lisa A. Relly. University of Ottawa, Canada

Surdna Foundation Scholarship

Isabelle M. Desjeux. Edinburgh University, UR
Christine R. Rose, University of Raiserslautern. Germany

\Yilliam Morton \\ heeler Family Founders'
Scholarship

Beatrice Casasnovas, University of Bordeaux, France

American Society for Cell Biology

Carmen R. Domingo, University of California. Berkeley

Darien L. Cohen. Dartmouth College

Errol R. Rolen, University of Missouri, Columbia

Adrian C. Lawrence. Albert Einstein College of Medicine

Edward E. Leonard, University of Pittsburgh School of Medicine

Zoe Y. Pettway, University of California, Irvine

American Psychological Association

Manbel Feliciano. L'niversity of Connecticut

Society for General Physiologists Scholarships

Mane-Anne Felix. Institut J. Monod. France

Maria B. Feller. AT&T Bell Laboratories

Pavle Andius, Institute of General & Physical Chemistry, Serbia.

Yugoslavia
Matthew Frerkmg. University of California. Davis

Awards

Lewis Thomas Award

Richard Harris. National Public Radio
Larry Thompson, Freelance

Board of Trustees and Committees

Corporation Officers and Trustees

Ex officio

Honorary Chairman of the Board of Trustees, Denis M. Robinson,

Key Biscayne. FL
Chairman of the BoaiJ ot Trustees, Sheldon J. Segal. The Population

Council, New York, NY
Vice Chairman of the Board of Trustees, Robert E. Mainer, The

Boston Company. Boston, MA
President of the Corporation, James D. Ebert, Chesapeake Bay

Institute. Baltimore. MD
Director of the Corporation, Harlyn O. HaKorson. Marine Biological

Laboratory, Woods Hole. MA 1
Director and Chief Executive Officer. John E. Burns. Marine

Biological Laboratory. Woods Hole, MA :

Treasurer, Robert D. Man/. Helmer & Associates. Waltham, MA
Clerk of the Corporation, Kathleen Dunlap, Tufts University School

of Medicine. Boston. MA

Class of 1996

Eloise E. Clark. Bowling Green State University, Bowling Green. OH
Norman Bernstein. Diane and Norman Bernstein Foundation,

Washington. DC
Martha W. Cox, Nantucket, MA
John E. Dowling. Harvard University. Boston, MA
Gerald Fischbach. Harvard University, Boston, MA
John G. Hildebrand, University of Arizona, Tucson, AR
Shinya Inoue. Marine Biological Laboratory. Woods Hole, MA
Neil Jacobs, Hale & Dorr, Boston, MA
Gerald Weissmann. New York University Medical Center, New York,

NY

Class a/1995

Clay M. Armstrong, University of Pennsylvania Medical School.

Philadelphia. PA

Dieter Blennemann, Carl Zeiss, Inc., Thornwood, NY
Dick Grace, The Brain Center, New Seabury, MA
Eric H. Davidson, California Institute of Technology. Pasadena, CA
Judith P. Grassle. Institute of Marine & Coastal Sciences, Rutgers

University, New Brunswick, NJ
Mary J. Greer, Cambridge, MA
Franklin M. Loew. Tufts University School of Veterinary Medicine.

North Grafton. MA

Brian M. Salzberg, University of Philadelphia School of Medicine, PA
Robert B. Silver, Cornell University, Ithaca. NY
J. Philip Tnnkaus, Yale University. New Haven, CT

Class of 1994

Frederick Bay. The Bay Foundation, New York. NY

Mary-Ellen Cunningham, Grosse Pointe Farms. MI

Robert D. Goldman, Northwestern University Medical School,

Chicago, II
Rodolfo R. I hn.r .> York University Medical Center,

New York, NY

Robert W. Pierce, BIK;. II

Thomas D. Pollard. Johns Hopkins University. Baltimore, MD
Irving W. Rabb, University I", A ,it Harvard Square. Cambridge. MA

1 to August 31. 1992

2 from September I, 1992

Joan V. Ruderman. Harvard University School of Medicine,

Boston, MA
Joseph Sanger. University of Pennsylvania School of Medicine.

Philadelphia, PA
Ann E. Stuart. University of North Carolina. Chapel Hill, NC

Class of 1993

Garland E. Allen, Washington University, St. Louis. MO

Jelle Atema, Marine Biological Laboratory, Woods Hole, MA

William L. Brown. Weston. MA

Alexander W. Clowes, University of Washington School of Medicine.

Seattle, WA

Barbara E. Ehrlich. University of Connecticut. Farmington. CT
Richard E. Kendall. East Falmouth. MA
Edward A. Kravitz, Harvard Medical School. Boston. MA
Jerry M. Melillo, Marine Biological Laboratory. Woods Hole. MA
Henry H. Schmidek. Neurosurgeon, Marion, MA
Roger D. Sloboda, Dartmouth College. Hanover, NH

Emeriti

Edward A. Adelberg, Yale University. New Haven. CT

John B. Buck. Sykesville. MD

Seymour S. Cohen, Woods Hole. MA

Arthur L. Colwin, Ke> Biscayne, FL

Laura Hunter Colwin. Key Biscayne, FL

D. Eugene Copeland. Marine Biological Laboratory, Woods Hole, MA

Sears Crowell. Indiana University, Bloomington. IN

Alexander T. Daignault, Boston, MA

William T. Golden. New York. NY

Teru Hayashi. Woods Hole. MA

Ruth Hubbard. Cambridge. MA

Lewis Kleinholz. Reed College. Portland. OR

Maurice E. Krahl, Tucson. AZ

Charles B. Metz, Miami, FL

Keith R. Porter, L'niversity of Pennsylvania. Philadelphia, PA

C. Ladd Prosser, University of Illinois. Urbana. II.
S. Meryl Rose. Waquoil, MA

W. D. Russell-Hunter. Syracuse University. Syracuse, NY

John W. Saunders. Jr., Waquoit. MA

Mary Sears. Woods Hole. MA

David Shepro. Boston University. Boston. MA

Homer P. Smith. Woods Hole. MA

D. Thomas Tngg, Wellesley, MA
Walter S. Vincent. Woods Hole. MA
George Wald, Cambridge, MA

Executive Committee of the Board of Trustees

Sheldon J. Segal, Chairman

Frederick Ba\. 1994

John E. Bums* (effective 9/1/92)

Mary-Ellen Cunningham. 1994

James D. Ebert*

Ray L. Epstein*

Robert D. Goldman. 1994

Harlyn O. Halvorson* (through 8/31/92)

Robert E. Mainer, Vice Chairman

Robert Manz*

Jerry M. Melillo, 1993

Joseph W. Sanger. 1994

Roger D. Sloboda. 1993

R48

Trustees and Committees R49

Trustee Committees

Audit

Robert E. Mainer. Chairman

Ray L. Epstein*

Pamela Ghetti*

Robert D. Manz*

Joan V. Ruderman

Gaius R. Shaver

John Speer*

Andrew Szent-Gyorgyi

D. Thomas Trigg

Stanley W. Watson

Compensation

Sheldon J. Segal, Chairman
Robert E. Mainer
Robert D. Manz

Development

Frederick Bay

Robert B. Barlow. Jr.

John E. Burns* (effective 9/1/92)

James D. Ebert*

Harlyn O. Halvorson* (through 8/31/92)

Rodolfo R. Llinas

Luigi Mastroianm

Robert Pierce

Sheldon J. Segal*

In vestment

William L. Brown, Chairman
Pamela Ghetti*
William T. Golden

Maurice Lazarus
Werner R. Lowenstein
Robert D. Manz
Irving W. Rabb
John Speer*
W. Nicholas Thorndike
D. Thomas Trigg

Long-Range Planning

Robert D. Manz. Chairman

Dieter Blennemann

Ray Epstein*

Rodolfo R. Llinas

Robert Mainer

John Speer*

Andrew Szent-Gyorgyi

Standing Committees

Buildings & Grounds

Kenyon S. Tweedell. Chairman

Barbara C. Boyer

Alfred B. Chaet

Lawrence B. Cohen

Richard D. Cutler*

William R. Eckberg

Alan Fein

Ferenc Harosi

Donald B. Lehy*

Thomas Meedel

Evelyn Spiegel

Fellowships

Thoru Pederson, Chairman
Martha Constantine-Paton
Ra\ L. Epstein*
Leslie D. Garrick*
Anne E. Giblin
George M. Langford
Jose Lemos
Eduardo R. Macagno
Carol L. Reimsch
J. Richard Whittaker

Housing, Food Service,
and Child Care

Thomas S. Reese. Chairman
Susan R. Barry
Milton Charlton
Richard Cutler*
Robert Michael Gould
Stephen M. Highstein
LouAnn King*
Darrell R. Stokes

ex officio

Institutional Animal Care and Use Marine Resources

Leslie D. Garrick. Chairman
Robert A. Bullis
Alfred B. Chaet
Ray L. Epstein
Alan M. Kuzinan
Andrew Mattox

Instruction

Roger D. Sloboda, Chairman

George Augustine, Jr.

Ray L. Epstein*

Rachel D. Fink

Leslie D. Garrick*

Leah T. Haimo

Susan Hill

Ronald R. Hoy

Hans Laufer

Joan V. Ruderman

Robert B. Silver

Raymond Stephens

John B. Waterbury

Library Joint Management

John E. Burris. MBL

Robert Gagosian. WHOI

Larry Ladd. WHOI

John Speer, MBL*

David Stonehill. MBL/WHOI

Library Joint Advisory

David Shepro. Chairman. MBL
John E. Hobbie. MBL
Gerald Weissmann, MBL
Henry Dick. WHOI
Werner Deuser, WHOI
Page Valentine. USGS
Kevin Friedland, NMFS

Robert D. Goldman, Chairman

Donald A. Abt

William D. Cohen

Richard Cutler*

Donald B. Lehy*

Toshio Narahashi

George D. Pappas

Roger D. Sloboda

Melvin Spiegel

Antoinette Steinacher

Radiation Safety

Ete Z. Szutz. Chairman
David W. Borst
Richard L. Chappell
Sherwin J. Cooperstein
Louis M. Kerr
Andrew Mattox*
Robert Rakowski
Walter S. Vincent

Research Services

Peter B. Armstrong, Chairman

Neal W. Cornell

Richard Cutler*

Barbara E. Ehrlich

Kenneth H. Foreman

Joseph Ilan

Ehud Kaplan

Samuel S. Koide

Aimlee D. Laderman

Jack Levin

Andrew Mattox*

Robert D. Palazzo

James P. Quigley

Peter J. S. Smith

Paul A. Steudler

Mark L. Tvkocinski

R50 Annual Report

Research Space Jerry M. Melillo Richard Cutler*

Joan V. Ruderman Edward Enos*

Joseph W. Sanger, Chairman
Paul J De Weer Robert B. Silver Susan Goux

Steven N. Treistman Louis M. Kerr

i\a\ u. tpsiein

Leslie D. Gamck' Ivan Vahda Alan Kuzman

David Landowne Rlchard Vallee Donald B Leh ^

Hans Laufer v aft , tv Andrew MattOX *

D , . bajety Pau i A

Eduardo R. Macagno

John E. Hobbie. Chairman
* ex officio Lee Bourgoin

Laboratory Support Staff"

Biological Bulletin

Clapp. Pamela L. Managing Editor

McCaffrey. Karen

Ready. Beth

Showalter. Christine M.

Controller 's Office
Speer. John W.. Controller

Accounting Services
Afonso. Janis E.
Binda. Ellen F.
Campbell. Ruth B.
Davis. Doris C.
Ghetti. Pamela M.
Gilmore. Mary F.
Hobhs. Roger W., Jr.
Poravas. Maria

Chem Room
Miller, Lisa A.
Schorer. Timothy M.
Mancevice. Denise M.

Purchasing

Hall. Lionel E.. Jr.
Mancevice. Denise M.
Schorer. Timothy M.

Director's Office

Burris, John E.. Director and CEO

Halvorson. Harlyn O.. Director

Epstein. Ra\ L.. Associate Director

Burrhus. I. Elaine

Catania. Didia

External Affairs
Carotenuto. Frank C.. Director

Aspinwall. Duncan P.

Berthel. Dorothy

Faxon. Wendy P.

Lessard. Kelley J.

"Including per\on\ \vho joined or kit the
Matt during 1W2

t\\ni'iutes Program
Armstrong. Ellen P., Liaison
Dilorio. Anne E.
Price. F. Carol
Scanlon, Deborah

Communications Office
Clapp, Pamela L.. Director
Rave-Peterson, Amy
Liles, George
Readv. Beth

Gray Museum

Backus. Richard H.. Curator

Armstrong. Ellen P

Montiero. Eva

Housing

King, LouAnn D., Conference Center and

Housing Manager
Johnson. Frances N.

Telephone Office
Baker. Ida M.
Geggatt. Agnes L.
Ridlev. Alberta W.

Human Resources
Goux. Susan P.. Manager
Donovan. Marcia H.

MBL/\VHOI Library
Stonehill. David L.. Director. MBL/WHOI
Library Center

Ashmore, Judith A.

Costa, Marguerite E.

Mirra, Anthony J.

Monahan, A. Jean

Nelson, Heidi

\ickerson. Ruth L

Pratson. Patricia G.

deVeer. Joseph M.

Copy Service Center
Mounttord. Rebecca J., Supervisor
Jackson, Jacquelyn F.
Mancini. Mary
Ridley. Sherie

Information Systems Division
Norton. Catherine N.. Director
Hamre. Lynne
Kogelnik. Andreas
Remsen. David
Space. David B.
Tollios, Constantine D.

Safety Services

Mattox. Andrew H.. Safety Officer

. Ipparatus
Barnes, Franklin D.
Haskins, William A.
Martin. Lowell V.
Nichols, Francis H., Jr.

Shipping and Receiving
Geggatt. Richard E.
Illgen. Robert F.

Services. Projects, and Facilities
Cutler. Richard D., Manager

Enos, Joyce B.

Kurland. Charles I.

Building Service^ unit Grounds

Hayes. Joseph N.. Superintendent

Allen. Wayne D.

Anderson. Lewis B.

Barnes. Susan M.

Beaudom. Helen

Boucher, Richard L.

Bowin, Dara

Collins. Paul J.

Conlin. Henry P.

Doms. John J.

Dutra, Roger S.. Jr.

Gibbons, Roberto G.

R51

R52 Annual Report

Gonsalves, Walter W., Jr.

P/ioto/ab

Fiset, Christopher

Krajewski, Viola I.

Colder, Linda M.

Funkhouser. Margaret

Lynch, Henry L.

Colder, Robert J.

Galvao, Anne Marie

Mancevice, Denise M.

Hainfeld. David

Mancini, Mary

Hammond, Jeramie

McNamara, Noreen

Sponsored Programs

Hibbitt. Karen

Rattacasa, Frank D.

Garrick, Leslie D., Administrator

Hoerner. Pauline

Sabo. Linda P.

Chrysler. Dorianne

Johnson. Paul C.

Dwane. Florence

Just. Thomas

Plant Operations and Maintcnaiiic

Huffer, Linda

Kessler. Anne P.

Lehy. Donald B., Supenntendent

Lynch, Kathleen F.

Kilpatnck, Bnan

Baldic, David P

Price, F. Carol

Kociemba. David

Blunt, Hugh F.

Kovac, Marc

Bourgoin, Lee E.
Carini. Robert J.

Electron Microscopy Lab
Kerr, Louis M.

Krajewski, Chester
Langton, Lori

Fish, David L., Jr.

Lovell, Lynne

Gonsalves, Paul J.

Mansfield. Darren

Gonsalves, Walter W., Jr.
Hathaway. Peter J.
Justason, C. Scott
Lochhead. William M.
Lunn. Alan G.
McAdams, Herbert Mill
Mills, Stephen A.
Olive, Charles W., Jr.

Temporan ' Ei > ip/i >] ves

McCartney, Tegan
McDonald, Brian
McLeish, Elizabeth
McNeil!, Jeffrey
Moorhouse, Laura
Neeley, Maire
Nelson, Beth
Northern, Marc

Cardoza, Laurie A.
Cserny, Mary
Elder, Peggy
Kaufmann. Sandra J.
Laurencot, Colette
Lyons. Elaine D.

Schoepf, Claude

O'Connor, Patricia M.

deVeer, Robert L.

Summer Support Staff

Regan. John F.
Remsen, Andrew S.

Amon, Tyler C.

Rickles, Andrew

Instrument Development Lab

Andrews. Ethan

Riekles, Jason

Knudson. Robert A.

Andrews, Mark

Santos, Marcelina

Balmer, Ethan

Shenandoah, Denise

Machine Shop

Boyer, Cynthia

Shephard, Jennifer

Sylvia, Frank E.

Cadieux, C. Bnan

Sloboda, Aaron

Cardoza, Laurie A.

Smith, Kelli M.

Marine Resources Center

Carpenter, Mark

Swan, Elizabeth

Enos, Edward G.. Jr., Superintendent

Chin, Hong

Torres. Sophie J.

Cipoletta, Charles D.

Clark, Martina

Towle. Jennifer

Fisher. H. Thomas. Jr.

Cloherty, Sean

Ulbrich, Ciona

Hanley, Janice S.

Connor, John H.

Varao, John

Moniz. Priscilla C.

Cutler, Laura

Vernaglia. David

Monteiro. Dana

DeLinks, Audrey

Vogel, Augustus

Savers, Scott

DeLinks, Elizabeth

Welenc, Karen

Sullivan. Daniel A.

Diachun, Peter

Wetzel, Ernest D.

Tassinari, Eugene

Donovan, Jason P.

Zakaria, Pauzi

\

t

Members of the Corporation'

Life Members

\delberg, Edward A.. Provost's Office. Yale University, 1 15 Hall of

Graduate Studies. New Haven. CT 06520
\matniek, Krncsl, 4797 Boston Post Road. Pelham Manor, NY 10803

Bang. Betsy G., 76 F.R. Lillie Road. Woods Hole. MA 02543
Barllett. James H.. Department of Physics. University of Alabama.

Box 870324. Tuscaloosa. AL 35487-0324
Beams. Harold \\ ., Department of Biology. University of Iowa. Iowa

Ch>. IA 52242
Bernheimer, Alan \\ ., Department of Microbiology. New York

University Medical Center, 550 First Ave.. New York. NY 10016
Bertholf. Lloyd M., Westminster Village #2114. 2025 E. Lincoln St..

Bloomington. IL 61701

Bodian, Da\id, 4100 North Charles St.. #913. Baltimore. MD2I218
Bridgman, A. Josephine, 715 Kirk Rd.. Decatur. GA 30030
Buck. John B.. 7200 Third Ave.. #C020. Sykesville. MD 21784
Burbanck. Madeline P., Box 15134. Atlanta. GA 30333
Burbanck, \\illiam D., Box 15134. Atlanta. GA 30333

Clark, Arnold M., 53 Wilson Rd.. Woods Hole. MA 02543
Cohen, Adolph I.. Department of Ophthalmology. Washington

University School of Medicine. St. Louis. MO 631 10 (resigned)
Cohen, Seymour S., 10 Carrot Hill Rd.. Woods Hole. MA 02543-1206
Colwin, Arthur, 320 Woodcrest Rd.. Key Biscayne. FL 33149
C'olwin, Laura Hunter, 320 Woodcrest. Key Biscayne, FL 33149
Copeland, D. K... 41 Fern Lane, Woods Hole. MA 02543
Corliss, John O., P. O. Box 53008. Albuquerque. NM 87153
Costello, Helen M., Carolina Meadows. Villa 137, Chapel Hill, NC

27514
Crouse, Helen, Address unknown

Dudley. Patricia L., Department of Biological Sciences, Barnard
College. Columbia University, 3009 Broadway. New York. NY
10027

Edwards, Charles, 2244 Harbour Court Drive. Longboat Key. FL
34228

Failla, Patricia M., 2149 Loblolly Lane. Johns Island, SC 29455
Ferguson, James K. \\ ., 56 Clarkehaven St.. Thornhill. Ontario I 4J
2B4 Canada

* Including action of the 1992 Annual Meeting.

Glusman, Murray, 50 E. 72nd St.. New York. NY 10021
Goldman, David, 63 Loop Rd.. Falmouth, MA 02540
Graham. Herbert, 36 Wilson Rd., Woods Hole. MA 02543
Green, James \V., 409 Grant Ave.. Highland Park, NJ 08904
Grosch, Daniel S., 1222 Duplin Road. Raleigh, NC 27607

Hamburger, Viktor, Department of Biology. Washington University,

St. Louis. MO 63 130
Hamilton, Howard L., Department of Biology, University of Virginia,

238 Gilmer Hall, Charlottesville, VA 22901
Harding, Clifford V., Jr., Wayne State University School of Medicine,

Department of Ophthalmology, Detroit, Ml 48201
Haschemeyer, Audrey E. V'., 21 Glendon Road, Woods Hole, MA

02543

Hauschka, Theodore S., FDI, Box 781. Damanscotta. ME 04543
Hisaw, F. L., 5925 SW Plymouth Drive, Corvallis. OR 97330
Hubbard. Ruth, 21 Lakeview Avenue. Cambridge, MA 02138
Humes, Arthur G., Marine Biological Laboratory. Boston L'niversity

Marine Program. Woods Hole, MA 02543
Hum it/, Charles, Veterans Administration Hospital, Basic Science

Research Laboratory. Albany. NY 12208

Jones, Meredith I,., Division of Worms. Museum of Natural History.
Smithsonian Institution, Washington, DC 20560

karush, Fred, Department of Microbiology. University of

Pennsylvania School ofMedicine, Philadelphia, PA 19104-6076

Kille, Frank R., 1 I I 1 S. Lakemont Ave. #444, Winter Park. FL 32792

Kingsbun, John M., Department of Plant Biology. Cornell
University, Ithaca. NY 14853

kleinholz, Lewis, Department of Biology, Reed College. 3203 SE
Woodstock Blvd.. Portland. OR 97202

I .uli i m. ui. E/ra, Yale University, School of Music, New Haven, CT

06520

I.autfer, Max A., Address unknown

LeFevre, Paul G., 15 Agassiz Road, Woods Hole. MA 02543
Levine. Rachmiel. 2024 Canyon Rd., Arcadia, CA 91006
Lochhead, John H., 49 Woudlawn Rd., London SW6 6PS, England,

UK
Loewus, Frank A., Washington State University. Institute of

Biological Chemistry. Pullman. WA 99164
Loftheld, Robert B.. Department of Chemistry, University of New

Mexico School ofMedicine. Albuquerque. NM 87131

R53

R54 Annual Report

Magruder, Samuel R., 270 Cedar Lane, Paducah, K.Y 42001
Malkiel, Saul, Allergic Diseases, Inc.. 130 Lincoln St., Worcester, MA

01609

Mathews, Rita \\ ., Box 131. Southfield, MA 01259
Miller, James A., 307 Shorewood Dnve, E. Falmouth, MA 02536
Moore, John A., Department of Biology, University of California.

Riverside, C A 92521
Moscona, Arthur A., University of Chicago, Department of Molecular

Genetics and Cell Biology, 920 East 58th Street. Chicago, IL 60637
Mullins, Lorin J., University of Maryland School of Medicine.

Department of Biophysics, Baltimore, MD 21201

Nasatir, Maimon, P. O. Box 379, Ojai, CA 93024-0379

Pollister, A. W., 8 Euclid Ave.. Belle Mead. NJ 08502

Prosser, C. Ladd, Department of Physiology and Biophysics, Burrill

Hall 524. University of Illinois, Urbana, IL 61801
Provasoli, Luigi, Via Stazione 43, 21025 Comerio (VA). Italy
Prytz, Margaret McDonald, Address unknown

Ratner. Sarah, Department of Biochemistry. Public Health Research

Institute, 455 First Ave., New York, NY 10016
Renn, Charles E., Address unknown
Reynolds, George, Department of Physics, Princeton University,

Jadwin Hall, Princeton. NJ 08544
Rice, Robert V., 30 Burnham Dr.. Falmouth, MA 02540
Richards, A. Glenn, 942 Cromwell Ave.. St. Paul, MN 551 14
Rockstein, Morris, 600 Biltmore Way, Apt. 805. Coral Gables. FL

33134

Ronkin, Raphael R., 3212 McKinley St., NW, Washington, DC 20015
Rose, S. Meryl, 32 Crosby Ln.. E. Falmouth. MA 02536

Sanders, Howard, Woods Hole Oceanographic Institution. Woods

Hole, MA 02543
Sato, Hidemi, Faculty of Social Science, Nagano University.

Shiminogo, Ueda, Nagano 386-12, Japan
Scharrer, Berta, Department of Anatomy, Albert Einstein College of

Medicine. 1300 Morns Park Avenue. Bronx, NY 10461
Schlesinger, R. Walter, University of Medicine and Dentistry of New

Jersey. Department of Molecular Genetics and Microbiology,

Robert Wood Johnson Medical School, Piscataway, NJ 08854-5635
Schmitt, F. O., Room 16-512, Massachusetts Institute of Technology,

Cambridge. MA 02 139

Scott, Allan C., 1 Nudd St., Waterville, ME 04901
Silverstcin, Arthur M., The Johns Hopkins Hospital Wilmer Institute.

Baltimore, MD 21205

Smith, Homer P., 8 Quissett Ave.. Woods Hole, MA 02543
Smith, Paul F., P. O. Box 264, Woods Hole, MA 02543
Sonnenblick, B. P., 515A Heritage Hill, Southbury, CT 06488
Steinhardt, Jacinlo, I 508 Spruce St., Berkeley. CA 94709
Stephens, Grover C., Department of Ecology & Evolutionary Biology,

School of Biological Sciences, LIniversity of California, Irvine, CA

92717

Taylor, Robert E., 20 Harbor Hill Rd., Woods Hole. MA 02543
Trager, William, The Rockefeller University, 1230 York Ave., New
York. NY 10021

Villee, Claude A., Harvard Medical School, Parcel B/Room 122. 25

Shattuck Street. Boston. MA 021 15
Vincent, Walter S., 16 F.R. Lillie Rd.. Woods Hole, MA 02543

\\'ald, George, 21 Lakeview Ave., Cambridge, MA 02138
Waterman, T. H., Yale University, Biology Department, Box 6666,
New Haven, CT065I 1

\\ichterman, Ralph, 31 Buzzards Bay Ave., Woods Hole, MA 02543
Wiercinski, Floyd J., 21 Glenview Road. Glenview, IL 60025
Wigle), Roland L., 35 Wilson Rd., Woods Hole, MA 02543
\\ilber, Charles G., Department of Biology, Colorado State
University, Fort Collins, CO 80523

/.inn, Donald J., Department of Zoology. University of Rhode Island,

Kingston, RI 02881

Zorzoli, Anita, 18 Wilbur Blvd., Poughkeepsie. NY 12603
/weifach, Benjamin W., 881 1 Nottingham Place. La Jolla, CA 92037

Regular Members

Abt, Donald A., Marine Biological Laboratory, Laboratory for Marine

Animal Health. Woods Hole, MA 02543

Acheson, George H., 25 Quissett Ave.. Woods Hole. MA 02543
Adams, James A., Department of Natural Sciences. LIniversity of

Maryland. Eastern Shore. Princess Anne. MD 21853
Adelberg, Edward A., Provost's Office. 1 15 Hall of Graduate Studies,

Yale University. New Haven, CT 06520

Adelman. William J., Jr., 160 Locust St.. Falmouth, MA 02540
Afzclius, Bjorn, Address unknown
Alherte, Randall S., Department of Molecular Genetics and Cell

Biology. University of Chicago, 1 103 E. 57th Street, Chicago, IL

60637
Alkon, Daniel, NINCDS/NIH, Dept. LMNC. Bldg. Park, Rm. 431,

Bethesda, MD 20852
Allen, Garland K., Department of Biology, Washington University,

Box I 137, One Brookings Drive, St. Louis, MO 63130-4899
Allen, Nina S., Department of Biology, Wake Forest University, Box

7325, Winston-Salem. NC 27109

Amatniek, Ernest, 4797 Boston Post Rd.. Pelham Manor. NY 10803
Anderson, Everett, Department of Anatomy & Cell Biology, LHRRB.

Harvard Medical School, 45 Shattuck St.. Boston. MA 021 15
Anderson, J. M., 1 10 Roat St.. Ithaca. NY 14850
Anderson, Porter \\ ., Department of Pediatrics. University of

Rochester Medical Center. Box 690, 601 Elmwood Ave.. Rochester.

NY 14642
Armett-Kinel, Christine, Dean of Science Faculty. University of

Massachusetts. Boston, MA 02125
Armstrong, Clay M., Department of Physiology. University of

Pennsylvania Medical School. Philadelphia. PA 19104-8725
Armstrong, Peter B., Department of Zoology, University of

California, Davis, CA 95616
Arnold, John M., Pacific Biomedical Research Center, 209A Snyder

Hall, University of Hawaii. Honolulu. HI 96822
Arnold, William A., 102 Balsam Rd., Oak Ridge. TN 37830
Ashton, Robert W., Esq.. Bay Foundation, Eikenbeiry and

Schoolman. 99 Wall St.. New York, NY 10005
Atema, Jelle, Boston LIniversity Marine Program, Marine Biological

Laboratory. Woods Hole. MA 02543
Atwood, Kimhall C'., Ill, P O. Box 673. Woods Hole, MA 02543

(deceased)
Augustine Jr., George J., Department of Neurobiology. Duke

University Medical Center, Durham, NC 27710
Ayers, Donald E., 4607 1/2 MacArthur Blvd.. NW #B. Washington.

DC 20007-2533

Baccetti, Baccio, Institute of Zoology. University of Siena, 53100

Siena, Italy
Baker, Robert G., Department of Physiology and Biophysics, New

York University Medical Center, 550 First Ave., New York. NY

10016

Members of the Corporation R55

Baldwin. Thomas O., Department of Biochemistry and Biophysics,

Texas A&M University. College Station. TX 77843
Barlow, Robert B., Jr., Institute for Sensory Research. Syracuse

University, Merrill Lane, Syracuse, NY 13244-5290
Barry, Daniel T., 1014 Barren Ridge Drive. Seabrook. TX 77586-

4002
Barn, Susan R.. Department of Physical Medicine and

Rehabilitation. ID204, University of Michigan Hospital. Ann

Arbor. MI 48109-0042

Bartoll, C lelmer K., 2000 Lake Shore Dnve. New Orleans. LA 70122
Bass, Andrew H., Seely Mudd Hall. Department of Neurobiology,

Cornell University. Ithaca. NY 14853
Battelle. Barbara- Anne, Whitney Laboratory. University of Florida,

9505 Ocean Shore Blvd.. St. Augustine. FL 32086
Bauer, G. Eric, Department of Anatomy. University of Minnesota,

Minneapolis. MN 55455
Bay, Frederick, Bay Foundation. 99 Wall St.. 18th FL. New York. NY

10005

BaUor. Edward R., P. O. Box 93. Woods Hole, MA 02543
Baylor, Martha B., P. O. Box 93, Woods Hole, MA 02543
Beams. Harold \\ '., Department of Biology, University of Iowa. Iowa

City. IA 52242 (deceased)
Bearer, Elaine L., Division of Biology & Medicine. Department of

Pathology. Brown University. Box G. Providence, RI 02912
Beauge, Luis Alberto, Department of Biophysics. Institute M.y.M.

Ferreyra. Casilla de Correo 389, 5000 Cordoba, Argentina
Beck, Lyle V., 2455 Tamarack Trail. Apt. 8. Bloomington. IN 47408
Begenisich, Ted, Department of Physiology. University of Rochester,

Medical Center. Box 642, 601 Elmwood Ave., Rochester, NY

14642
Begg, David A., Department of Anatomy & Cell Biology. University

of Alberta. Edmonton. Alberta T6G 2H7. Canada
Bell, Eugene, Marine Biological Laboratory. Tissue Engineering. Inc.,

Woods Hole, MA 02543
Benjamin, Thomas L., Department of Pathology, Harvard Medical

School, 25 Shattuck St.. Boston. MA 02 1 1 5
Bennett, M. V. L., Albert Einstein College of Medicine. Department

of Neuroscience. 1410 Pelham Pkwy. S.. Bronx, NY 10461
Bennett, Miriam F., Department of Biology, Colby College,

Waterville, ME 04901

Berg, Carl J., Jr., P. O. Box 769. Kilauea. Kauai. HI 96754-0769
Berlin. Suzanne T., 5 Highland St.. Gloucester. MA 01930
Berne, Robert M., Department of Physiology, University of Virginia.

School of Medicine. Charlottesville. VA 22908
Bernstein, Norman, Diane and Norman Bernstein Foundation. Inc..

5301 Wisconsin Ave.. #600. Washington. DC 20015-2015
Bezanilla, Francisco, Department of Physiology. University of

California. Los Angeles. CA 90024
Biggers. John D.. Department of Physiology, Harvard Medical

School, Boston. MA 02115
Bishop, Stephen !!., Department of Zoology. Iowa State University.

Ames. I A 50010
Blaustein, Mordecai P., Department of Physiology. School of

Medicine. University of Maryland, 655 W. Baltimore Street.

Baltimore. MD 21201
Blennemann, Dieter, Carl Zeiss. Inc.. One Zeiss Drive. Thornwood.

NY 10594
Bloom, George S., Department of Cell Biology and Neuroscience. The

University of Texas Southwestern Medical Center. 5223 Harry

Hines Blvd., Dallas. TX 75235-9039
Bloom, Kerry S., Department of Biology. University of North

Carolina. Wilson Hall. CB#3280. Chapel Hill, NC 27599-3280
Bod/nick. David A., Department of Biology. Wesleyan University,

Lawn Avenue. Middletown. CT 06457
Boettigcr, Edward G., 29 Juniper Point. Woods Hole. MA 02543

Boolootian, Richard A., Science Software Systems, Inc., 357

Woodcliff Rd.. Sherman Oaks, CA 91403
Borgese, Thomas A., Department of Biology, Lehman College.

CUNY, Bedford Park Blvd.. West. Bronx, NY 10468
Boris), Gary G., Laboratory of Molecular Biology, University of

Wisconsin. Madison. WI 53706
Borst, David \\ ., Jr., Department of Biological Sciences, Illinois State

University. Normal, IL 61761-6901
Bosch. Herman F., Box 617. Woods Hole. MA 02543
Bowles, Francis P., P. O. Box 674. Woods Hole, MA 02543
Boyer, Barbara C., Department of Biology. Union College.

Schenectady. NY 12308
Brandhorst, Bruce P., Department of Biological Sciences, Simon

Fraser University. Barnaby, BC V5A 156, Canada
Brinley, F. J., Neurological Disorders Program. NINCDS, NIH, 812

Federal Building. Bethesda, MD 20892
Brown, Joel E., Albert Einstein College of Medicine. 506 Kennedy

Center. 1400 Pelham Parkway. Bronx. NY 10461
Brown, Stephen C., Department of Biological Sciences. SUNY,

Albany, NY 12222
Brown, William L., Retired Chairman. Bank of Boston (01-23-1 1 ).

100 Federal St., Boston, MA 02106-2016
Browne, Carole L., Department of Biology. Wake Forest University.

Winston-Salem. NC 27109
Browne, Robert A., Department of Biology. Wake Forest University,

Box 7325, Winston-Salem. NC 27109
Bryant, Shirley H., Department of Pharmacology and Cell Biophysics.

ML 575, University of Cincinnati. Cincinnati. OH 45267
Bucklin, Anne C., Manne Biological Laboratory. Woods Hole, MA

02543
Bullis, Robert A., Marine Biological Laboratory. Woods Hole. MA

02543
Burd, Gail Deerin, Department of Molecular and Cell Biology. Life

Sciences South. Rm 444, University of Arizona, Tucson, AZ 85721
Burdick. Carolyn J., Department of Biology. Brooklyn College.

Bedford Avenue & Avenue H, Brooklyn, NY 11210
Burger, Max, Freidrich Miesner Institut Bau 1060 Postfach 2543.

Basel 4002. Switzerland
Burgos, Mario, IHEM Medical School, UNC Conicet, Casilla de

Correo 56. 5500 Mendoza. Argentina
Burk>, Albert, Department of Biology, University of Dayton, Dayton.

OH 45469
Burris, John E., Manne Biological Laboratory. Woods Hole, MA

02543
Burstyn. Harold L., Morrison Law Firm, The Morrison Building, 145

North Fifth Avenue, Mt. Vernon. NY 10550
Bursztajn, Sherry, Harvard Medical School. Mailman Research

Center. 1 1 5 Mill St.. Belmont. MA 02 1 78
Busa. William, Department of Biology. Johns Hopkins University.

3400 N. Charles St.. Baltimore, MD 21218

Calabrese, Ronald I,., Department of Biology. Emory University,

1555 Pierce Drive, Atlanta, GA 30322
Callaway, Joseph C., Department of Physiology. New York Medical

College, Basic Sciences Bldg.. Valhalla. NY 10595
Cameron, Andrew, Department of Biology. California Institute of

Technology. Pasadena. CA 91 125
Campbell, Richard II., Bang-Campbell Associates. Box 47. Woods

Hole. MA 02543
Candelas, Graciela C., Department of Biology, University of Puerto

Rico. Rio Piedras. PR 0093 1
Carew, 'Thomas J., Department of Psychology. Yale University, P. O.

Box I I A. Yale Station. New Haven. CT 06520
Cariello, Lucio, Biochemistry Department. Stazione Zoologica. Villa

Comunale. 80121 Naples, Italy

R56 Annual Report

Carlson, Francis D., Biophysics Department, The John Hopkins

University. N. Charles St., Baltimore, MD 2 1 ?
Carrierc, Rita M., Department of Anatomy a: l! ""gv. Box 5,

SUNY Health Science Center, 450 ' I iiooklyn, NY

11203
Case, James, University of California. e Chancellor of

Research. Santa Barbara. CA 93 !('.
Cassidy, Rev. J. D., Providence ("i ! of St. Thomas

Aquinas, Providence. RI v
Caanaugh, Colleen M., Harvj ity, Biological Laboratories.

16 Divinity Ave.. Canilu ;)2138

Cebra, John J., Department of Biology, Leidy Labs, G-6, University

of Pennsylvania, Philadelphia. PA 19174

Chaet, Alfred B., University of West Florida, Pensacola. FL 32504
Chambers, Edward L., Department of Physiology and Biophysics,

University of Miami. School of Medicine, P. O. Box 016430,

Miami. FL 33101
Chang, Donald C., Department of Physiology. Baylor College of

Medicine. One Baylor Plaza, Houston. TX 77030
Chappell, Richard I.., Department of Biological Sciences, Hunter

College, Box 210, 695 Park Ave.. New York, NY 10021
Chauncey, Howard H., 30 Falmouth St., Wellesley Hills, MA 02181

(deceased)
Chen, Thomas T., Center for Marine Biotechnology. University of

Maryland, 600 E. Lombard St.. Baltimore. MD 21202
Chikarmane, Hemant M., Marine Biological Laboratory. Woods Hole.

MA 02543
Child, Frank M., HI, Department of Biology, Trinity College,

Hartford, CT06106
Chisholm, Rex L., Department of Cell Biology, Northwestern

University Medical School, Chicago, IL 6061 I
Citkowitz, Elena, 410 Livingston St., New Haven, CT 0651 I
Clark, Eloise E., Vice President, Bowling Green State University,

Bowling Green, OH 43403

Clark, Hays, 26 Deer Park Drive, Greenwich. CT 06830
Clark, James M., 210 Emerald Lane, Palm Beach, FL 33480
Clark, Wallis H., Jr., Bodega Marine Laboratory, P. O. Box 247.

Bodega Bay, CA 94923

C'laude, IMiilippa. Pnmate Center, Capitol Court, Madison, WI 53706
Clay, John R., Laboratory of Biophysics. N1H, Building 9. Room 1E-

124. Bethesda. MD 20S92
Clowes, Alexander W., Department of Surgery RF-25, University of

Washington School of Medicine. Seattle, WA 98195
C'lutter, Mary, Office of the Director. Room 518, National Science

Foundation. Washington, DC 20550
Cobb, Jewel Plummer, California State University, 5151 State

University Drive, Los Angeles, CA 90032-8500
Cohen, Avis H., Section of Neurobiology and Behavior, Mudd Hall,

Cornell University, Ithaca, NY 14X53-2702 (resigned)
Cohen, Carolyn, Rosenstiel Basic Medical Sciences Research Center.

Brandeis University, Waltham, MA 02254
Cohen, Lawrence B., Department of Physiology. Yale University

School of Medicine. 333 Cedar Street, New Haven, CT 06510-8026
Cohen, Maynard, Department of Neurological Sciences. Rush Medical

College. 600 South Paulina. Chicago. I L 60612
Cohen, Rochelle S.. Department of Anatomy. University of Illinois,

808 W. Wood Street. Chicago, IL 60612
Cohen, William D., Department of Biological Sciences, Hunter

College, 695 Park Ave., Box 79. New York, NY 10021
C'oleman, Annette W., Division of Biology and Medicine, Brown

University, Providence. RI 01412
Collier, Jack R., Department of Biology, Brooklyn College. Bedford &

Avenue H, Brooklyn, NY II 2 10
Collier, Marjorie McCann, Biology Department, Saint Peter's College,

2641 Kennedy Boulevard. Jersey City, NJ 07306

Cook, Joseph A., The Edna McConnell Clark Foundation, 250 Park

Ave.. New York, NY 10017
Cooperstein, S. J., University of Connecticut Health Center.

Department of Anatomy, Farmington Ave.. Farmington. CT 06032
Cornell, Neal \\ '., Marine Biological Laboratory. Woods Hole. MA

02543
Cornwall, Melvin C., Jr., Department of Physiology L7I4. Boston

University School of Medicine. 80 E. Concord St.. Boston, MA

021 18
Corson, David Wesley, Jr., 516 Rice Hope Dr.. Ml. Pleasant, SC

29464-9296
Corwin, Jeffrey T., Department of Otolaryngology, University of

Virginia Medical Center, Box 430, Charlottesville, VA 22908
Costello, Walter J., Department of Zoology Z/BS, College of

Medicine, Ohio University, Athens, OH 45701
Couch, Ernest F., Department of Biology, Texas Christian University.

Fort Worth. TX 76129
Cox, Martha, William C. Cox Foundation, 190 South Beach Road,

Hobe Sound, FL 33455

Crane, Sylvia E., 438 Wendover Drive. Princeton. NJ 08540
Cremer-Bartels, Gertrud, Universitats Augenklinik. 44 Munster.

Germany
Crow, Terry J., Department of Neurobiology and Anatomy,

University of Texas Medical School, Houston. TX 77225
Crowell, Sears, Department of Biology, Indiana University,

Bloomington, IN 47405
C'rowther, Robert, Department of Biology, University of New

Brunswick. BS 451 1 1. Fredericton. NB, Canada E3B 6E1
Cunningham, Mary-Ellen, 62 Cloverly Road, Grosse Pointe Farms.

MI 48236

C'urrier, David I,., P. O. Box 2476, Vineyard Haven, MA 02568
C'utler, Richard, Marine Biological Laboratory, Woods Hole, MA

02543

D'Alessio, Giuseppe. Department of Organic & Biological Chemistry.

University ol Naples, Via Mezzocannone 16, Naples. Italy 80134
D'A van/o, Charlene, School of Natural Science. Hampshire College,

Amherst, MA 01002
Daignault, Alexander T., 29 Quisset Harbor Rd., Falmouth, MA

02540
Dan, katsuma, Tokyo Metropolitan Union, 1-1 Minami-Osawa.

Huchioji City 192-03, Japan
David, John R., Tropical Public Health. Harvard School of Public

Health, 665 Huntington Ave., Boston. MA 021 15
Davidson, Eric 11., Division of Biology, 156-29, California Institute of

Technology. Pasadena, CA 91 125
Davis, Bernard I)., Bacterial Physiology Unit, Harvard Medical

School, Boston, MA 02 1 I 5

Davis, Joel P., Seapuit, Inc., P. O. Box G, Osterville, MA 02655
Daw, Nigel W., 5 Old Pawson Rd., Branford, CT 06405
Deegan, I.inda A., The Ecosystems Center, Marine Biological

Laboratory. Woods Hole. MA 02543

DeGroof, Robert ('., 145 Water Crest Dr., Doylestown, PA 18901
Dellaan, Robert L., Department ol Anatomy and Cell Biology.

Emory University School of Medicine. Atlanta, GA 30322
DeLanney, Louis E., Institute for Medical Research, 2260 Clove

Drive, San Jose, CA 95128
Denkla, Marth B.. Kennedy-Krieger Institute. Johns Hopkins School

of Medicine. 707 North Broadway, Baltimore, MD 21205
Dentler, William I,., Department of Physiology & Cell Biology.

University of Kansas. 401 1 Haworth Hall, Lawrence, KS 66044
DePhillips, llenrv A., Jr., Department of Chemistry, Trinity College,

300 Summit Street, Hartford, CT 06106
DeSimone, Douglas W., Department of Anatomy and Cell Biology,

Box 439, Health Sciences Center, University of Virginia,

Charlottesville, VA 22908

Members of the Corporation R57

DeToledo-Morrell, I.cyla, Department of Neurologieal Seiences, Rush

Medical College. Chicago, IL 60612
Dotlharn, \\olf-Dietrich, Department of Pharmacology, School of

Medicine. Vanderbilt University, Nashville. TN 37127
DC \Veer, Paul J., Department of Physiology, University of

Pennsylvania School of Medicine, Philadelphia, PA 19104-6085
Dixon, Keith E., School of Biological Sciences, Flinders University,

Bedford Park, 5042, South Australia, Australia
Don ling. John E., The Biological Laboratories. Harvard University.

16 Divinity St.. Cambridge, MA 02138
DuBois, Arthur Brooks. John B. Pierce Foundation Laboratory. 290

Congress Ave.. New Haven. CT 06519
Dunean, Thomas K., Department of Environmental Sciences. Nichols

College. Dudley, MA 01570
Dunham. Philip B.. Department of Biology. Syracuse University.

Syracuse. NY 13244
Dunlap, Kathleen, Department of Physiology. Tufts University

Medical School. Boston. MA 02 1 1 1
Dunlap, Paul V., Department of Biology, Woods Hole Oceanographic

Institution. Redfield 316. Woods Hole, MA 02543
Dorkin, Martin, Department of Microbiology, University of

Minnesota. 1460 Mayo Bldg., Box 196 UMHC. Minneapolis. MN

55455-0312

Ebert, James D., Department of Biology. The Johns Hopkins

University, 213 Macaulay Hall, Baltimore, MD 21218
Eckberg. William R., Department of Zoology. Howard University,

Washington. DC 20059
Edds, Kenneth T., Department of Anatomical Sciences. SUNY,

Buffalo. NY 14214
Eder, Howard A., Albert Einstein College of Medicine. 1300 Morris

Park Ave.. Bronx. NY 10461

Edstrom, Joan, 2515 Milton Hills Dr., Charlottesville. VA 22901
Egyud, Laszlo G., 18 Skyview. Newton. MA 02150
Ehrlich, Barbara E., Division of Cardiology, University of

Connecticut Health Center, 263 Farmington Avenue, Farmington,

CT 06030
Eisen, Arthur Z., Division of Dermatology, Washington University,

St. Louis, MO 63 110
Eisen, Herman N'., Massachusetts Institute of Technology. El 7- 1 28.

77 Massachusetts Ave., Cambridge, MA 02139
Elder, Hugh Young, Institute of Physiology, University of Glasgow,

Glasgow, Scotland G12 8QQ
Elliott, Gerald K., The Open University Research Unit. Foxcombe

Hall. Berkeley Rd.. Boars Hill. Oxford. England OX I 5HR
Englund, Paul T., Department of Biological Chemistry. Johns

Hopkins School of Medicine. Baltimore. MD 21205
Epcl. Dai id, Hopkins Marine Station, Pacific Grove, CA 93950
Epstein. Herman T., 18 Lawrence Farm Road. Woods Hole. MA

02543

Epstein, Ray L., 30 Coonamessett Circle. Falmouth. MA 02540
Erulkar, Solomon D., 318 Kent Rd., Bala Cynwyd. PA 19004
Essner, Edward S., Kresge Eye Institute, Wayne State University, 540

E. Canfield Ave., Detroit, MI 48201 (resigned)

Farb, David H., Department of Pharmacology L603. Boston

University School of Medicine, Boston, MA 021 18
Farmunfarmaian, A., Department of Biological Sciences, Nelson

Biological Laboratory, Rutgers University, Piscataway, NJ 08855
Fein, Alan, Department of Physiology, University of Connecticut

Health Center, Farmington. CT 06032
Feinman, Richard D., Box 8. Department of Biochemistry, SUNY

Health Science Center. 450 Clarkson Avenue. Brooklyn. NY 1 1 203

Feldman, Susan C., Department ol Anatomy, University of Medicine

and Dentistry of New Jersey, New Jersey Medical School, 100

Bergen St., Newark. NJ 07103

Fessenden, Jane, 225 Lakeview Ave., Falmouth, MA 02540
FestorT, Barry \V., Neurology Service (127), Veterans Administration

Medical Center, 4801 Lmwood Blvd.. Kansas City. MO 64128
Fink, Rachel D., Department of Biological Sciences. Clapp

Laboratory, Mount Holyoke College, South Hadley, MA 01075
Finkelstein, Alan, Albert Einstein College of Medicine. 1300 Morris

Park Ave.. Bronx, NY 10461
Fischbach, Gerald, Department of Neurobiology. Harvard Medical

School. 220 Longwood Ave., Boston, MA 021 15
I isluii.iii. Harvey M., Department of Physiology and Biophysics.

University of Texas Medical Branch, Galveston, TX 77550
Flanagan, Dennis, 12 Gay St., New York, NY 10014
Fluek, Richard Allen, Department of Biology, Franklin & Marshall

College, Box 3003. Lancaster, PA 17604-3003
Foreman, K. H., Boston University Marine Program. Marine

Biological Laboratory, Woods Hole. MA 02543
Fox, Thomas Oren, Division of Medical Sciences. Harvard Medical

School, 260 Longwood Ave., Boston. MA 02 1 1 5
Franzini-Armstrong. Clara, School of Medicine, University of

Pennsylvania, 330 S. 46th Street, Philadelphia, PA 19143
Frazier, Donald T., Department of Physiology and Biophysics.

University of Kentucky Medical Center, Lexington. KY 40536
French, Robert J., Health Sciences Center. University of Calgary.

Calgary, Alberta, T2N 4N1, Canada
Freygang, Walter J., Jr., 6247 29th St.. NW, Washington. DC 20015

(resigned)
Friedler, Gladys, Boston LIniversity School of Medicine. 80 East

Concord Street, Boston, MA 021 18

Fry, Brian, Manne Biological Laboratory. Woods Hole, MA 02543
Fulton, Chandler M., Department of Biology, Brandeis University.

Waltham. MA 02254
Furshpan. Edwin J., Department of Neurophysiology. Harvard

Medical School, Boston, MA 021 15
Futrelle. Robert P., College of Computer Science, Northeastern

University. 360 Huntington Avenue. Boston. MA 021 15

Gabriel, Mordecai, Department of Biology. Brooklyn College,

Brooklyn, NY 11210
Gadsby, David C., Laboratory of Cardiac Physiology, The Rockefeller

University. 1230 York Avenue. New York. NY 10021
Gainer, Harold. Lab of Functional Neurochemistry. NIH. Bldg. 36,

Room 4D-20. Bethesda. MD 20892
Galatzer-Levy, Robert M., 180 N. Michigan Avenue. Chicago. IL

60601
Gall, Joseph G., Carnegie Institution. 1 15 West University Parkway.

Baltimore. MD 21210

Gallant, Paul E., NIH, Bldg. 36. Rm. 2A-29. Bethesda, MD 20892
Garber, Sarah S., Department of Physiology. Medical College of

Pennsylvania. 2900 Queen Ln.. Philadelphia. PA 19129
Gascoyne, Peter, Box 85E. University of Texas, M. D. Anderson

Hospital and Tumor Institute. 6723 Bertner Avenue. Houston. TX

77030
Gelperin, Alan, Department of Biophysics, AT&T Bell Labs, Room

IC464, 600 Mountain Avenue. Murray Hill, NJ 07974
German, James L., HI, Lab of Human Genetics. The New York

Blood Center. 310 East 67th St., New York, NY 10021
Gibbs, Martin, Institute for Photobiology of Cells and Organelles,

Brandeis University. Waltham. MA 02254
Giblin, Anne E., Ecosystems Center. Marine Biological Laboratory,

Woods Hole. MA 02543

R58 Annual Report

Gibson, A. Jane, Department of Biochemistry, Cornel! University,

Ithaca. NY 14850

Gifford, Prosser, 540 N Street, SW, S-903, WashinjMn DC 20024
Gilbert, Daniel L., Clinical Neurosciencc B. xINDS. Bldg.

9. Room IE- 124. Bethesda. MD 20892
Giudice, Giovanni, Dipartimento di B re e Dello

Sviluppo. 1-90123. Via Archirafi ' .a di Palermo.

Palermo. Italy
Giuditta, Antonio, Department ..i Physiology, University of

Naples. Via Mezzocaiv s, Italy 80134

Glynn, Paul, 2770 Beechux .. Pittsburgh. PA 15217

Golden, William T., Amr- iuseum of Natural History, 40 Wall

St., Room 4201. N \Y 10005

Goldman, Robert D . Dep cut of Cell, Molecular and Structural

Biology. Northwestern University, 303 E. Chicago Ave., Chicago, IL

60611
Goldsmith, Paul K., NIH. Bldg. 10, Room 9C-IOI. Bethesda, MD

20892
Goldsmith, Timothy H., Department of Biology, Yale University, New

Haven. CT 065 10
Goldstein, Moise H., Jr., ECE Department. Barton Hall. Johns

Hopkins University, Baltimore. MD 21218
Goodman, Lesley Jean, Department of Biological Sciences. Queen

Man College. Mile End Road, London. El 4NS, England, UK
Gould, Robert Michael, Institute for Basic Research in Developmental

Disabilities. 1050 Forest Hill Rd.. Staten Island. NY 10314
Gould, Stephen J., Museum of Comparative Zoology, Harvard

University. Cambridge. MA 02138
Govind, C. K., Life Sciences Division. University of Toronto. 1265

Military Trail. West Hill, Ontario. MIC IA4. Canada
Graf, Werner, Rockefeller University, 1230 York Ave., New York.

NY 10021
Grant, Philip, 2939 Van Ness Street. N.W., Apt. 302. Washington.

DC 20008
Grass, Ellen R., The Grass Foundation. 77 Reservoir Rd.. Quincy.

MA 02 170
Grassle, Judith, Institute of Marine & Coastal Studies, Rutgers

University. Box 231. New Brunswick. NJ 08903
Graubard. Katherine, Department of Zoology, NJ-15. University of

Washington, Seattle. WA 98195
Greenberg, Everett Peter, Department of Microbiology. College of

Medicine. University of Iowa. Iowa City. IA 52242
Greenberg, Michael J., Whitney Laboratory. 9505 Ocean Shore Blvd..

St. Augustine. FL 32086-8623

Greer, Mary J., 16 Hillside Ave.. Cambridge, MA 02140
Griffin, Donald R., Concord Field Station, Harvard University, Old

Causeway Road, Bedford. MA 01730
Gross, Paul R., Center for Advanced Studies, University of Virginia,

444 Cabell Hall, Charlottesville, VA 22903
Grossman, Albert, New York University Medical Center, 550 First

Ave.. New York, NY 10016
Grossman, Lawrence, Department of Biochemistry. Johns Hopkins

University. 615 North Wolfe Street, Baltimore, MD 21205
Gruner, John, Cephalon. Inc.. 145 Brandywine Parkway. W. Chester.

PA 19380-4245

Gunning, A. Robert, P. O. Box 165. Falmouth. MA 02541
(, illiam. G. P., Department of Biology. Reed College. Portland, OR

97202

Haimo, Leah, Department of Biology. University of California.

Riverside. CA 92521
Hall, Linda M., Department of Biochemistry and Pharmacology,

SUNY. 317 Hochstetter. Buffalo, NY 14260
Hall, Zack W., Department of Physiology, University of California,

San Francisco, CA 94143

Halvorson, Harlyn O., 26 Fa> Road. Woods Hole, MA 02543
llamlett, Nancy V., Department of Biology. Harvey Mudd College,

301 E. 12th St.. Claremont. CA 9171 1
I fin. i L Tatsuji, Chiba University Medical School. 1-8-1. Inohana.

Chiba, 280. Japan
Hanna, Robert B., College of Environmental Science and Forestry,

SUNY, Syracuse, NY 1 32 10
Harosi, Ferenc L, Laboratory of Sensory Physiology. Marine

Biological Laboratory. Woods Hole, MA 02543
Harrigan. June K., 7415 Makaa Place, Honolulu, HI 96825
Harrington, Glenn W., Division of Cell Biology and Biophysics. 403

Biological Sciences Building. University of Missouri, Kansas City,

MO 641 10
Harris, Andrew L., Department of Biophysics, Johns Hopkins

University. 34th & Charles Sts., Baltimore, MD 21218
Hastings, J. VV., The Biological Laboratories, Harvard University. 16

Divinity Street. Cambridge. MA 02138
Hayashi, Teru, 7105 SW 1 12 Place. Miami. FL 33173
llaydon-Baillie, \\ensley G., Porton Int.. 2 Lowndes Place. London,

SW1X 8DD. England. UK
Hayes, Raymond L., Jr., Department of Anatomy. Howard

University, College of Medicine. 520 W St.. NW. Washington, DC

20059
Hepler, Peter K., Department of Botany, University of Massachusetts.

Amherst. MA 01003
Herndon, Walter R., University of Tennessee, Department of Botany,

Knoxville. TN 37996-1100
Herskovits, Theodore T., Department of Chemistry, Fordham

University, John Mulcahy Hall, Room 638. Bronx, NY 10458
Hiatt, Howard H., Department of Medicine, Brigham and Women's

Hospital, 75 Francis Street, Boston, MA 021 15
Highstein, Stephen M., Department of Otolaryngology, Washington

University School of Medicine. St. Louis. MO 63110
Ilildebrand, John G., Arizona Research Laboratories. Division of

Neurobiology. 603 Gould-Simpson Science Building, University of

Arizona, Tucson, AZ 85721
Hill, Richard \\ .. Department of Zoology. Michigan Slate University,

E. Lansing. MI 48824
Hill. Susan D.. Department of Zoology. Michigan State University. E.

Lansing. MI 48824
Hillis Llewellya, Smithsonian Tropical Research Institute. Unit 0948

APO-AA. Miami, FL 34002-0948
Hillman. Peter, Department of Biology, Life Sciences &

Neurobiology. Hebrew University. Jerusalem 91904. Israel
Hinegardner, Ralph T., Division of Natural Sciences. University of

California, Santa Cruz, CA 95064
Hines, Michael, Department of Neurobiology, Duke University

Medical Center, Box 3209, Durham, NC 27710
Hinsch, Gertrude, \\ '., Department of Biology. University of South

Florida, Tampa. FL 33620
Hobbie, John E., Ecosystems Center. Marine Biological Laboratory,

Woods Hole, MA 02543

Hodge, Alan J., 3843 Ml. Blackburn Ave., San Diego, CA 92 I 1 1
Hoffman. Joseph, Department of Physiology, School of Medicine.

Yale University. New Haven, CT 06515
Hollyheld, Joe G., Baylor School of Medicine, Texas Medical Center.

Houston. TX 77030
Holt/man, Eric, Department of Biological Sciences, Columbia

University. New York, NY 10027
Hopkinson. Charles S., Jr., Marine Biological Laboratory, Woods

Hole. MA 02543

Hoskin, Erancis C. G., 33 Hyatt Road, Woods Hole, MA 02543
lloughton, Richard A., Ill, Woods Hole Research Center, P. O. Box

296, Woods Hole. MA 02543

Members of the Corporation R59

Ho), Knnald R., Section of Neurobiology and Behavior, Cornell

lini\ersit>. Ilhaea, NY I4S53
Hufnagel. Linda A., Department of Microbiology, University of

Rhode Island, Kingston. RI 02881
lhiiiiiii.il]. \\ Mli. mi D., Department ol Zoology, Ohio Universilv.

\thens. OH 45701
Humphreys, Susie IL, Food and Drug Administration. HFF-156.

Switzer. 200 C Street. SW. Washington. DC 20204-0001
Humphreys. Tom D., University of Hawaii. PBRC. 41 Ahui St..

Honolulu. HI 96813
Hunt. Richard I'., ICRF, Clare Hall Laboratories. South Mimms

Potter's Bar. Herb EN6-3LD. England
Hunter. Robert D.. Department ol Biological Sciences. Oakland

University. Rochester. MI 48309-4401
Hunter. \\ . Bruce, Bo\ 321. Lincoln Center. MA 01773
Hum it A .lerard. Memorial Sloan Kettering Institute for Cancer

Research. 1275 York Avenue. New York. NY I 1021
Huxley. Hugh E., Department of Biology, Rosenstiel Center, Brandeis

University. Waltham. MA 02154
Hynes, Thomas J.. Jr., Meredith and Grew. Inc.. 160 Federal Street.

Boston. MA 021 10-1701

llan. Joseph, Department of Developmental Genetics and Anatomy,

Case Western Reserve University School of Medicine, Cleveland.

OH 44106
Ingoglia. Nicholas. Department of Physiology, New Jersey Medical

School. 100 Bergen St.. Newark. NJ 07103
Inoue, Saduvki, Department of Anatomy, McGill University Cancer

C'entre. 3640 University St.. Montreal. PQ H3A 2B2. Canada
Inoue, Shinya, Marine Biological Laboratory, Woods Hole, MA 02543
Isselbacher, Kurt J.. Massachusetts General Hospital Cancer Center,

149 13th Street. Charlestown. MA 02129
Issidorides, Marietta, R., Department of Psychiatry. University of

Athens. Monis Petraki 8, Athens. 140 Greece
Izzard, Colin S.. Department of Biological Sciences. SUNY. 1400

Washington Ave.. Albany. NY 12222

Jacobs, Neil. Hale & Dorr. 60 State St.. Boston. MA 02109

Jaffe, Lionel, Marine Biological Laboratory. Woods Hole. MA 02543

Jannasch, Holger \V., Department of Biology. Woods Hole

Oceanographic Institution. Woods Hole, MA 02543
Jeffery, William R.. Bodega Marine Laboratory, Box 247, Bodega

Bay. CA 94923
Johnston, Daniel, Division of Neuroscience. Baylor College of

Medicine. Baylor Plaza. Houston, TX 77030
Josephson, Robert K., Department of Biological Sciences, University

of California. Irvine. CA 92717

Kabat, E. A., Department of Microbiology, College of Physicians and

Surgeons. Columbia University, 630 West 168th St., New York.

NY 10032 (resigned)
kaczmarek, Leonard K., Department of Pharmacology. Yale

University School of Medicine. 333 Cedar St., New Haven. CT

06510
Kale>, Gabor, Department of Physiology. Basic Sciences Building,

New York Medical College. Valhalla. NY 10595
kaltenbach, Jane, Department of Biological Sciences. Mount Holyoke

College. South Hadley, MA 01075
Kaminer, Benjamin, Department of Physiology. School of Medicine.

Boston University, 80 East Concord St.. Boston. MA 021 18
Kane, Robert K., PBRC, University of Hawaii. 41 Ahui St.. Honolulu.

HI 96813
Kaneshiro, Kdna S., Department of Biological Sciences. University of

Cincinnati. JL 006, Cincinnati. OH 45221

Kao, Chien-yuan, Department of Pharmacology. Box 29. SUNY.

Downstate Medical Center. 450 Clarkson Avenue. Brooklyn. NY

11203
Kaplan. I Imil. Department of Biophysics. The Rockefeller University.

1230 York Ave.. New York. NY 10024
Karakashian, Stephen J., Apt. 16-F. 165 West 91st St.. New York.

NY 10024
Karlin, Arthur, Department of Biochemistry and Neurology,

Columbia University. 630 West 168th St.. New York. NY 10032
Katz, George M., Fundamental and Experimental Research Labs,

Merck Sharp and Dohme, P. O. Box 2000, Rahway, NJ 07065
kelly, Robert E., Department of Anatomy, College of Medicine,

University of Illinois. P. O. Box 6998, Chicago, IL 60680
Kemp, Norman E., Department of Biology, University of Michigan.

Ann Arbor. MI 48109
Kendall. John P., Faneuil Hall Associates, 176 Federal Street. 2nd

Floor, Boston, MA 021 10
Kendall, Richard E., 26 Green Harbor Road, East Falmouth, MA

02536
Kerr, Louis M., Marine Biological Laboratory. Woods Hole. MA

02543
Keynan, Alexander. Laboratory for Developmental and Molecular

Biology, Department of Biochemistry. Hebrew University of

Jerusalem, Givat-Ram. Jerusalem. Israel
Khan. Shahid M. M., Department of Anatomy & Structural Biology,

Albert Einstein College of Medicine. 1300 Morris Park Ave.. Bronx.

NY 10461
K i, II.HI. Daniel P., Department of Cellular Biology. Duke Medical

Center. Box 3709, 307 Nanaline Duke Bldg.. Durham, NC 27710
Kirk, Mark D., Division of Biological Sciences, University of

Missouri. Columbia. MO 652 1 1
Klotz, Irving M., Department of Chemistry. Northwestern University.

Evanston. IL 60201
Knudson, Robert A., Marine Biological Laboratory, Instrument

Development Lab, Woods Hole, MA 02543
Koide, Samuel S., Population Council, The Rockefeller University,

1230 York Avenue. New York. NY 10021
Kornberg, Sir Hans, The Master's Lodge, Christ's College, Cambridge

CB2 3BU. England, UK
Kosower, Edward M., address unknown
Krahl, M. E.. 2783 W. Casas Circle. Tucson, AZ 85741
Krane, Stephen M., Arthritis Unit. Massachusetts General Hospital.

Fruit Street, Boston, MA 02 1 1 4

Krauss, Robert, FASEB, 9650 Rockville Pike. Bethesda. MD 20814
Kratitz, Edward A., Department of Neurobiology, Harvard Medical

School. 220 Longwood Ave.. Boston. MA 021 15
Kriebel, Mahlon E., Department of Physiology. SUNY Health Science

Center. Syracuse. NY 13210
Kristan. William B., Jr., Department of Biology B-022, University of

California San Diego, La Jolla, CA 92093
Kropinski, Andrew M. B., Department of Microbiology/Immunology,

Queen's University, Kingston. Ontario K7L 3N6, Canada
Kuhns. William J., Hospital for Sick Children. Department of

Biochemistry Research, Toronto, Ontario M5G 1X8, Canada
Kuhtreiber, Willem M., Marine Biological Laboratory, Woods Hole,

MA 02543 (resigned)

Kusano, Kiyoshi, NIH, Bldg. 36. Room 4D-20. Bethesda. MD 20892
Ku/irian. Alan M., Marine Biological Laboratory. Woods Hole, MA

02543

Laderman, Aimlee, Yale University School of Forestry, New Haven,

CT065I1
LaMarche, Paul H., Eastern Maine Medical Center, 489 State St.,

Bangor, ME 04401

R60 Annual Report

Landis, Dennis M. D., Department of Developmental netics and

Anatomy. Case Western Reserve University Sclir -i of Medicine,

Cleveland, OH 44106
Landowne, David, Department of Physiol . . U 16430,

University of Miami School of Medi 33101

Langford, George M., Department ct Viences, Dartmouth

College. 6044 Oilman Laborator, . NH 03755

Lasser-Ross, Nechama, Departn ology. New York

Medical College, Valhalla. s -
Laster. Leonard, University iiisetts Medical School, 55 Lake

Avenue. North, Worcester it>55

Laufer, Hans, Departir -logical Science, Molecular and Cell

Biology. Group U- 1 - ' i> of Connecticut. Storrs, CT 06268

Lazarow, Paul B.. I.V|> rl tit ol Cell Biology and Anatomy. Mount

Sinai Medical Sclioni - . 1007, 5th Avenue & 100th Street, New

York. NY 10021
Lazarus, Maurice, federated Department Stores, Inc., Sears Cresent.

City Hall Plaza. Boston, MA 02108
Leadbeltcr, Edward R., Department of Molecular and Cell Biology.

U-131, University of Connecticut, Storrs, CT 06268
Lederberg, Joshua, The Rockefeller University, 1230 York Ave., New

York, NY 10021
Lee, John J., Department of Biology. City College of CUNY.

Convent Ave. and 138th St., New York, NY 10031
Lehy, Don. ild B., Marine Biological Laboratory. Woods Hole, MA

02543

Leibovitz, Louis, 3 Kettle Hole Road. Falmouth, MA 02540
Leighton, Joseph, 2324 Lakeshore Avenue, #2, Oakland, CA 94606
Leighton, Stephen, NIH. Bldg. 13 3WI3. Bethesda. MD 20892
Leinwand, Leslie Ann, Department of Microbiology and Immunology,

Albert Einstein College of Medicine, 1300 Morns Park Ave., Bronx.

NY 10461
Lerman, Sidney, Eye Research Lab, Room 41. New Y'ork Medical

College, 100 Grasslands Ave., Valhalla, NY 10595
Lerner, Aaron B., Yale University. School of Medicine, New Haven,

CT06510
Lester, Henry A., California Institute of Technology, 156-29,

Pasadena. CA 91125
Levin, Jack, Veterans Administration Medical Center. 1 13A. 4150

Clement St., San Francisco, CA 94 1 2 1
Levine, Richard B., ARL, Division of Neurobiology, University of

Arizona, 61 1 Gould-Simpson Bldg.. Tucson, AZ 85721
Levinthal, Krancoise, 435 Riverside Dr., New York. NY 10025
Levitan, Herbert, Department of Zoology, University of Maryland,

College Park, MD 20742
Levitan, Irwin B., Department of Biochemistry, Brandeis University,

Waltham, MA 02254
Linck, Richard \V., Department of Anatomy, Jackson Hall, University

of Minnesota. 321 Church Street, S. E., Minneapolis. MN 55455
Lipicky, Raymond J.. Department of Cardio-Renal/Drug Prod. Div.,

FDA. Rm. 16B-45, 5M>0 Fishers Lane, Rockville, MD 20857
Lisman, John E., Department of Biology, Brandeis University,

Waltham, MA 02254

Liuzzi, Anthony, 320 Beacon St., Boston. MA 021 16
Llinas, Rodolfo R., Department of Physiology and Biophysics, New

York University Medical Center. 550 First Ave.. New York, NY

10016
Loew, Franklin M., Tufts University School of Veterinary Medicine,

200 Westboro Rd.. N. Grafton, MA 01536
Loewenslein, Birgit R., Department of Physiology and Biophysics. R-

430, University of Miami School of Medicine. Miami, FL 33101
Loewenslein, Werner R., Department of Physiology and Biophysics,

University of Miami. P. O. Box 016430. Miami, FL 33101
London, Irving M., Massachusetts Institute of Technology. Harvard-
MIT Division. E-25-55 I. Cambridge. MA 02139

Longo, Erank J., Department of Anatomy, University of Iowa. Iowa

City, IA 52442
Lorand, Laszlo, Department of Biochemistry and Molecular Biology,

Northwestern University, 2153 Shendan Road. Evanston, IL 60208
l.uckenbill-Edds, Louise, 155 Columbia Ave., Athens. OH 45701

Macagno, Eduardo R., 1003B Fairchild. Department of Biosciences,

Columbia University, New York, NY 10027
MacNichol, E. F., Jr., Department of Physiology, Boston University

School of Medicine, 80 E. Concord St., Boston, MA 021 18
Maglott-Duffield, Donna R., American Type Culture Collection.

12301 Parklawn Drive, Rockville, MD 20852-1776
Maienschein, Jane Ann, Department of Philosophy. Arizona State

University. Tempe. AZ 85287-2004
Mainer, Robert, The Boston Company, One Boston Place. OBP- 1 5-D,

Boston, MA 02 1 08
Malbon, Craig Curtis, Department of Pharmacology, Health Sciences

Center. SUNY, Stony Brook, NY 1 1794-8651
Manalis, Richard S., Department of Biological Sciences. Indiana

University Purdue University at Fort Wayne, 2101 Coliseum

Blvd., E.. Fort Wayne, IN 46805
Mangum, Charlotte P., Department of Biology, College of William

and Mary, Williamsburg, VA 23185-8795
Manz, Robert D., Helmer and Associates. Suite 1310. 950 Winter St..

Walthan, MA 02 154
Margulis, Lynn, Botany Department. University of Massachusetts.

Morrill Science Center. Amherst. MA 01003
Marinucci, Andrew C, 102 Nancy Drive, Mercerville, NJ 08619
Marsh, Julian B., Department of Biochemistry' and Physiology.

Medical College of Pennsylvania. 3300 Henry Ave., Philadelphia,

PA 19129
Martin, Lowell V., 10 Buzzards Bay Ave.. Woods Hole. MA 02543

02543
Martinez, Jr., Joe L., Department of Psychology. University of

California, Berkeley, 3210 Tolman Hall, Berkeley. CA 94720
Martinez-l'alomo, Adolfo, Seccion de Patologia Experimental,

Cinvesav-ipn. 07000 Mexico, D.F. A. P.. 140740, Mexico
Maser, Morton, Woods Hole Education Assoc., P. O. Box EM,

Woods Hole, MA 02543
Mastroianni, Luigi, Jr., Department of Obstetrics and Gynecology.

Hospital of the University of Pennsylvania, 106 Dulles, 3400 Spruce

Street. Philadelphia. PA 19104-4283
Matteson, Donald R., Department of Biophysics, University of

Maryland School of Medicine. 660 West Redwood Street.

Baltimore. MD 21201
Mautner, Henry G., Department of Biochemistry. Tufts University

School of Medicine. 136 Harrison Ave.. Boston. MA 021 1 1
Mauzerall, David. The Rockefeller University, 1230 York Ave., New

York, NY 10021
McC'ann, Frances, Department of Physiology, Dartmouth Medical

School, Hanover. NH 03755
McLaughlin, Jane A., Marine Biological Laboratory. Woods Hole,

MA 02543
McMahon, Robert K., Department of Biology. Box 19498, University

of Texas. Arlington, TX 76019
Meedel, Thomas, Biology Department, Rhode Island College, 600 Mt.

Pleasant Ave.. Providence. RI 02908
Meinertzhagen, Ian A., Department of Psychology. Lite Sciences

Center, Dalhousie University, Halifax. Nova Scotia B3H 451,

Canada
Meiss, Dennis E., Immunodiagnostic Laboratories. 488 McCormick

St., San Leandro, CA 94577
Melillo. Jerry M., Ecosystems Center, Marine Biological Laboratory.

Woods Hole, MA 02543

Members of the Corporation R61

Mellon, DeForest, Jr., Department of Biology, Gilmer Hall,

University of Virginia. Charlottesville. VA 22903
Mellon, Richard P., P. O. Box 187. Laughlimown. PA 15655
Mendelsohn, Michael K., Cardiovascular Division. Harvard Medical

School, 75 Francis Street. Boston, MA 021 15
Meluzals. Janis, Department of Pathology. University of Ottawa.

Ottawa. Ontario K1H 8M5. Canada
Metz, Charles B.. 7220 SW 124th St.. Miami. FL 33156
Miledi, Kicardo. Department of Psychobiology. University of

California. Irvine, CA 92717
Milkman, Roger, Department of Biology, University of Iowa, Iowa

City, 1A 52242
Miller, Andrew I.., Marine Biological Laboratory. Woods Hole, MA

02543
Mills. Robert. 10315 44th Avenue, W 12 H Street, Bradenton. FL

33507-1535
Misevic, Gradimir, Department of Research. University Hospital of

Basel. Mebelstrasse 20. CH-4031. Basel. Switzerland
Mitchell. Ralph. DAS. Harvard University. 29 Oxford Street.

Cambridge. MA 02138
Mi\akaa, Hiroyoshi, Department of Physiology, Yamagata

University School of Medicine. Yamagata 990-23. 7990-23 Japan
Miyamoto, Datid M., Department of Biology, Drew University,

Madison, NJ 07940
Mi/.ell. Merle, Department of Cell & Molecular Biology. Tulane

University. New Orleans. LA 701 18
Moore, John \\ ., Department of Neurobiology. Box 3209. Duke

University Medical Center. Durham. NC 27710
Moore. Lee E.. Department of Physiology and Biophysics. LJniversity

of Texas Medical Branch. Galveston. TX 77550
Morin. James G.. Department of Biology. University of California.

Los Angeles. CA 90024
Morrell. Frank, Department of Neurological Science, Rush Medical

Center. 1753 W. Congress Parkway, Chicago. IL 60612
Morse, Patricia M., University of Washington Marine Labs. 620

University Rd.. Friday Harbor, WA 98250
Morse, Stephen Scott, The Rockefeller University, 1230 York Ave.,

Box 2. New York. NY 10021-6399
Mote, Michael I., Department of Biology. Temple University.

Philadelphia. PA 19122
Mountain, Isabel, Vinson Hall #112. 6251 Old Dominion Drive.

McLean. VA 22101-4804
Muller, Kenneth J.. Department of Physiology and Biophysics,

University of Miami School of Medicine, Miami, FL 33101
Murray, Andre \V., Department of Physiology, University of

California. Box 0444, Parnassus Ave.. San Francisco. CA 94143-

0444
Murrav, Sandra Ann, Department of Neurology. Anatomy and Cell

Science, University of Pittsburgh School of Medicine, Pittsburgh,

PA 15261
Musacchia. \avier J., P.O. Box 5054. Delia Vista, AR 72714-0054

Nabrit, S. M., 686 Beckvvith St., SW. Atlanta. GA 30314
NadelhorTer, knute. Marine Biological Laboratory. Woods Hole, MA

02543

Naka, Ken-iehi, 2-9-2 Tatsumi Higashi. Okazaki. Japan 444
Nakajima, Shigehiro, Department of Pharmacology and Cell Biology,

University of Illinois College of Medicine at Chicago. 835 S.

Wolcott Ave.. Chicago, IL 60612
Nakajima, Yasuko, Department of Anatomy and Cell Biology.

University of Illinois College of Medicine at Chicago. M/C 5 1 2.

Chicago. IL 606 12
Narahashi, Toshio, Department of Pharmacology. Northwestern

University Medical School. 303 East Chicago Ave.. Chicago. IL

60611

Nasi, Knrico, Department of Physiology. Boston University v.hool of

Medicine. R-406. 80 E. Concord St.. Boston, MA 021 IS
Nealson, Kenneth II., Great Lakes Research Center, University of

Milwaukee. 600 E. Greenfield Ave., Milwaukee. WI 53204
Nelson, Leonard, Department of Physiology. CS10008. Medical

College of Ohio. Toledo. OH 43699
Nelson, Margaret C., Section of Neurobiology and Behavior, Cornell

University. Ithaca, NY 14850
Nicholls, John G., Biocenter. Klingelbergstrasse 70, Basel 4056,

Switzerland
Nickerson, Peter A., Department of Pathology, SUNY. Buffalo, NY

14214
Nicosia, Santo V., Department of Pathology. University of South

Florida, College of Medicine, Box 1 1. 12901 North 30th St..

Tampa. FL 336 1 2
Noe, Bryan D., Department of Anatomy and Cell Biology. Emory

University School of Medicine, Atlanta. GA 30322
Northcutt, R. Glenn, Department of Neuroscience. A-OOI. Scripps

Institution of Oceanography. La Jolla, CA 92093-0201
Norton, Catherine N., Marine Biological Laboratory, Woods Hole.

MA 02543
Nusbaum. Michael P., Neurobiology Research Center. University of

Alabama, Birmingham. Volker Hall. G878S, Birmingham, AL

35294

O'Herron, Jonathan, Jonathan & Shirley O'Herron Foundation, One

Rockefeller Plaza, New York, NY 10020

O'Melia, Anne F., 16 Evergreen Lane, Chappaqua, New York 10514
Obaid, Ana Lia, Department of Neuroscience. University of

Pennsylvania School of Medicine, 234 Stemmler Hall. Philadelphia.

PA 19104-6074
Oertel, Donata, Department of Neurophysiology, University of

Wisconsin. 281 Medical Science Bldg.. Madison, WI 53706
Ohki, Shinpei. Department of Biophysical Sciences. SUNY at Buffalo.

224 Can Hall, Buffalo, NY 14214
Oldenbourg, Rudolf, Marine Biological Laboratory. Woods Hole. MA

02543

Olds, James L., NIH, 9/1W125, Bldg. 9, Bethesda, MD 20892
Olins, Ada L., University of Tennessee-Oak Ridge, Graduate School

of Biomedical Sciences, Biology Division ORNL, P. O. Box 2009,

Oak Ridge, TN 37831-8077
Olins, Donald E., University of Tennessee-Oak Ridge. Graduate

School of Biomedical Sciences, Biology Division ORNL. P. O. Box

2009. Oak Ridge. TN 37831-8077
Oschman, James L., 31 Whittier Street, Dover. NH 03820

Palazzo, Robert E., Department of Physiology & Cell Biology,

L'mversity of Kansas, Lawrence. KS 66045
Palmer, John D., Department of Zoology, University of

Massachusetts, Amherst. MA 01002
Palti, Yoram, Rappaport Institution, Techmon, POB 9697. Haifa,

31096 Israel
Pant, Harish C., N1NCDS/NIH. Laboratory of Neurochemistry, Bldg.

36. Room 4D-20, Bethesda, MD 20892
Pappas, George D., Department of Anatomy, College of Medicine,

University of Illinois, 808 South Wolcott St., Chicago, IL 60612
Pardee. Arthur B., Department of Pharmacology, Harvard Medical

School, Boston, MA 02 1 1 5
Pardy, Roosevelt L., School of Life Sciences, University of Nebraska,

Lincoln, NE 68588
Parmentier, James L., Cato Research, Ltd.. 4364 South Alston Ave.,

Durham. NC27713
Passano, Leonard M., Department of Zoology, Birge Hall. University

of Wisconsin. Madison. WI 53706

R62 Annual Report

Pearlman. Alan L., Department of Physiology. Schc <ne,

Washington University, St. Louis. MO 631 10
Pederson, Thoru, Worcester Foundation tbi > Oology.

Shrewsbury. MA 01 54?
Perkins, C. D., 400 Hilltop Terrace, Alevi
Person, Philip, Research Testing Labs. '

Huntington Station. NY 1 1746
Peterson, Bruce J., Ecosystem^ < .cal Laboratory.

Woods Hole. MA 02543
Pethig, Ronald, School of 1 ring Science. University

College of N. Wales. IX d. \ynedd, LL57 IUT. UK

Pfohl, Ronald J., DepannKi' ingy. Miami University, Oxford.

OH 45056
Pierce, Robert W., 4S> I ire Lane, P. O. Box 1404, Boca Grande,

FL 33921 (dea
Pierce, Sidney K., Jr., Department of Zoology, University of

Maryland, College Park, MD 20742
Poindexter, Jeanne S., Barnard University, Columbia University,

3009 Broadway, New York, NY 10027-6598
Pollard, Harvey B.. NIH, NIDDKD, Lab of Cell Biology & Genetics.

Bldg. 8, Rm. 401. Bethesda. MD 20892
Pollard, Thomas D., Department of Cell Biology and Anatomy. Johns

Hopkins University, 725 North Wolfe St.. Baltimore. MD 21205
Poole, Alan F., Academy of Natural Sciences of Philadelphia. 19th

and the Parkway, Philadelphia, PA 19103
Porter, Beverly H., 5542 Windysun Ct., Columbia. MD 21045
Porter, Keith R., Department of Biology. Leidy Laboratories, Rm.

303, University of Pennsylvania, Philadelphia, PA 19104-6018
Porter, Mary E., Department of Cell Biology and Neurology,

University of Minnesota, 4-147 Jackson Hall, Minneapolis, MN

55455
Potter, David, Department of Neurobiology, Harvard Medical School.

Longwood Avenue, Boston, MA 021 15
Potts, William T., Department of Biology, University of Lancaster.

Lancaster. England, UK.
Powers, Dennis A., Hopkins Marine Station. Stanford University.

Pacific Grove, CA 93950
Powers, Maureen K., Department of Psychology, Vanderbilt

University, Nashville, TN 37240
Pratt, Melanie M., Department of Anatomy and Cell Biology,

University of Miami School of Medicine (R124), P. O. Box 016960,

Miami, FL 33101
Prendergast, Robert A., Wilmer Institute. Johns Hopkins Hospital,

601 N. Broadway, Baltimore, MD 21287-9142
Presley, Phillip H., Carl Zeiss, Inc.. I Zeiss Drive. Thornwood. NY

10594
Price, Carl A., Waksman Institute of Microbiology. Rutgers

University. P. O. Box 759. Piscataway, NJ 08854
Prior, David J., Department of Biological Sciences, NAU Box 5640,

Northern Arizona Llniversity, Flagstaff, AZ 8601 1
Prusch, Robert D., Department of Life Sciences, Gonzaga University,

Spokane, WA 99258
Purves, Dale, Department of Neurobiology, Duke University Medical

School, Box 3209, Durham. NC 27710

Quigley, James, Department of Pathology. SUNY Health Science
Center. BHS Tower 9. Rm. 140, Stony Brook, NY 1 1794

Rabb, Irving W., University Place at Harvard Square, 124 Mt.

Auburn St., Suite 200, Cambridge. MA 02138
Rabin, Harvey, DuPonl Merck Pharmaceutical, R&D Division, Exp.

Station 328/358, Wilmington, DE 19880
Rabinowitz, Michael B., Marine Biological Laboratory, Woods Hole,

MA 02543

Ratferty, Nancy S., Department of Anatomy, Northwestern

University Medical School, 303 E. Chicago Avenue, Chicago. IL

60611
Rakowski, Robert F., Department of Physiology and Biophysics.

UHS/The Chicago Medical School. 3333 Greenbay Rd., N.

Chicago. IL 60064
Ramon, Fidel, CIN VESTA V. Depto Fisiologia, Apto Postal 14-740.

Mexico, D.F., 07000
Ranzi, Silvio, Sez Zoologia Sc Nat, Via Coloria 26. 120133. Milano.

Italy
Rastetter, Edward B., Ecosystems Center, Marine Biological

Laboratory. Woods Hole, MA 02543
Rebhun, Lionel I., Department of Biology, Gilmer Hall, University of

Virginia, Charlottesville, VA 22901
Reddan, John R., Department of Biological Sciences, Oakland

University, Rochester, MI 48309-4401
Reese, Barbara F., NINCDS/NIH. Bldg. 36, Room 3B26. 9000

Rockville Pike, Bethesda, MD 20892
Reese, Thomas S., NINCDS/NIH, Bldg. 36, Room 2A27, 9000

Rockville Pike, Bethesda, MD 20892
Reiner, John M., 1 1 1 Emerson St.. Apt. 623. Denver. CO 80218

(deceased)
Reinisch, Carol L., Department of Comparative Medicine. Tufts

University School of Veterinary Medicine. 200 Westboro Rd.. Bldg.

20. North Grafton, MA 01536
Reynolds, George T., Department of Physics, Jadwin Hall, Princeton

University, Princeton, NJ 08544
Rich, Alexander, Department of Biology, Massachusetts Institute of

Technology. Cambridge, MA 02139
Rickles, Frederick R., Department of Medicine. Division of

Hematology-Oncology. University of Connecticut Health Center,

Farmington, CT 06032
Riley, Monica, Marine Biological Laboratory, Woods Hole, MA

02543
Ripps. Harris, Department of Ophthalmology, University of Illinois,

1855 W. Taylor Street. Chicago. IL 6061 1
Ritchie, Murdoch, Department of Pharmacology. Yale University

School of Medicine, 333 Cedar St., New Haven. CT 065 10
Robinson, Denis M., 200 Ocean Lane Drive #908. Key Biscayne. FL

33149
Rome, Lawrence C., Department of Biology, University of

Pennsylvania, Philadelphia. PA 19104
Rosenbaum. Joel L., Department of Biology, Kline Biology Tower,

Yale University, New Haven. CT 06520
Rosenbluth, Jack, Department of Physiology, New York University

School of Medicine. 550 First Ave., New York, NY 10016
Rosenbluth, Raja, Department of Biological Sciences, Simon Fraser

University, Burnaby, BC, V5A 1S6, Canada
Roslansky, John, Box 208. 26 Bar Neck Road. Woods Hole, MA

02543
Roslansky, Prisdlla F., 57 Buzzards Bay Ave.. Woods Hole. MA

02543
Ross, William N., Department of Physiology. New York Medical

College. Valhalla, NY 10595
Roth, Jay S., 18 Millneld Street. P. O. Box 285. Woods Hole. MA

02543
Rowland, Lewis P., Neurological Institute. 710 West 168th St.. New

York, NY 10032
Ruderman, Joan V., Department of Anatomy and Cell Biology.

Harvard University School of Medicine, 220 Longwood Ave.,

Boston, MA 02 1 1 5
Rushforth, Norman B., Department of Biology. Case Western Reserve

University. Cleveland, OH 44106
Russell-Hunter, \V. D., Department of Biology, Lyman Hall 012,

Syracuse University. Syracuse, NY 13244

Members of the Corporation R63

Satfo. Mar) Beth, Institute of Marine Sciences, 272 Applied Sciences,

University of California, Santa Cruz, CA 95064
Sager, Ruth, Dana Farher Cancer Institute. 44 Binney St.. Boston.

MA 021 15
Sagi, Amir, Department of Life Sciences. Ben-Gurion University of

the Negev, P.O. Box 653. Bee-Sheva. Israel. 84105
Salama, Guy, Department of Physiology. University of Pittsburgh,

Pittsburgh, PA 15261
Salmon. Edward D., Department of Biology. Wilson Hall. CB3280.

University of North Carolina, Chapel Hill, NC 27599
Salzberg, Brian M., Department of Neuroscience. University of

Pennsylvania. 234 Stemmler Hall. Philadelphia. PA 19104-6074
Sanborn. Richard C., 1 I Oak Ridge Road, Teatieket. MA 02536

(deceased)
Sanger, Jean M., Department of Anatomy. School of Medicine.

University of Pennsylvania. 36th and Hamilton Walk, Philadelphia.

PA 19174
Sanger, Joseph, Department of Anatomy, School of Medicine,

University of Pennsylvania. 36th and Hamilton Walk, Philadelphia.

PA 19174
Sattelle, David B., AFRC Unit-Department of Zoology, University of

Cambridge, Downing St., Cambridge CB2 3EJ. England. UK

(resigned)
Saunders, John \\., Jr., P. O. Box 381, Waquoit Station. Waquoit,

MA 02536
Saz, Arthur K., Department of Immunology. Georgetown University

Medical School. Washington, DC 20007
Schachman, Howard K., Department of Molecular Biology, University

of California. Berkeley, CA 94720
Schatten, Gerald P., Integrated Microscopy Facility for Biomedical

Research. University of Wisconsin, 1 1 17 W. Johnson St., Madison,

WI 53706
Schatten, Heide, Department of Zoology, University of Wisconsin,

Madison, WI 53706
Schirf, Jerome A., Institute for Photohiology of Cells and Organelles,

Brandeis University, Waltham. MA 02254
Schmeer, Arline C., Mercenene Cancer Research Institute, Hospital of

Saint Raphael. New Haven. CT 065 I 1
Schmidek, Henry H., Department of Neurosurgery. St. Luke's

Hospital. 102 Page St.. New Bedford. MA 02740
Schnapp, Bruce J., Department of Cellular & Molecular Physiology,

Harvard Medical School. 25 Shattuck St., Boston, MA 021 15
Schuel, Herbert, Department of Anatomical Sciences. SUNY. Buffalo.

Buffalo, NY 14214
Schwartz, James II., Center for Neurobiology and Behavior, New

York State Psychiatric Institute Research Annex, 722 W. 168th

St.. 7th Floor, New York, NY 10032
Schweitzer, A. Nicola, School of Medicine. Section of

Immunobiology. Yale University. New Haven, CT 06510
Scofield, Virginia Lee, Department of Microbiology and Immunology,

UCLA School of Medicine. Los Angeles. CA 90024
Sears, Mary, P. O. Box 152. Woods Hole, MA 02543
Segal, Sheldon J., The Population Council. One Dag Hammarskjold

Plaza, New York, NY 10036
Selman, Kelly, Department of Anatomy, College of Medicine,

University of Florida. Gainesville, FL 32601
Shanklin, Douglas R., Department of Pathology, Room 584,

University of Tennessee College of Medicine, 800 Madison Avenue,

Memphis, TN 38163
Shapiro, Herbert, 6025 North 13th St.. Philadelphia. PA 19 14 1

(deceased)
Shashoua, Victor E., Ralph Lowell Labs. Harvard Medical School.

McLean Hospital. 115 Mill St.. Belmont. MA 02178
Shaver. Gaius R., Ecosystems Center. Marine Biological Laboratory.

Woods Hole, MA 02543

Shaver, John R., Department of Zoology. Michigan State I 'diversity,

East Lansing, MI 48824
Sheet/., Michael P., Department of Cell Biology, Duke University

Medical Center, Box 3709, 385 Nanaline Duke Bldg., Durham, NC

27710

Shepard, David C., P. O. Box 44, Woods Hole, MA 02543
Shcpro, David, Department of Microvascular Research, Boston

University. 5 Cummington St., Boston. MA 02215
Sheridan, William F., Biology Department. University of North

Dakota. Box 8238, University Station. Grand Forks, ND 58202-

8238
Sherman, I. \V., Department of Biology. University of California.

Riverside, CA 9252 1
Shimomura, Osamu, Marine Biological Laboratory, Woods Hole, MA

02543
Shipley. Alan M., Marine Biological Laboratory, Woods Hole, MA

02543
Siegel, Irwin M., Department ol Ophthalmology, New York

University Medical Center, 550 First Avenue, New York, NY

10016
Siegelman, Harold \V., Department of Biology. Brookhaven National

Laboratory. Upton. NY 1 1973
Silver, Robert B., Department of Physiology, Cornell University, 822

Veterinary Research Tower. Ithaca. NY 14853-6401
Siwicki, Kathleen K., Biology Department. Swarthmore College, 500

College Ave., Swarthmore, PA 19081
Sjodin, Raymond A., Department of Biophysics, LIniversity of

Maryland, Baltimore, MD 21201
Skinner, Dorothy M., Oak Ridge National Laboratory, P. O. Box

2009. Biology Division. Oak Ridge, TN 3783 1
Sloboda, Roger D., Department of Biological Sciences, 306 Oilman,

Dartmouth College. Hanover. NH 03755
Sluder, Greenfield, Worcester Foundation for Experimental Biology,

222 Maple Ave.. Shrewsbury, MA 01545
Smith, Peter J. S.. Marine Biological Laboratory, Woods Hole, MA

02543
Smith, Ralph I., Department of Integrative Biology. University of

California. Berkeley, CA 94720
Smith, Stephen J., Department of Molecular & Cellular Physiology.

Beckman Center, Stanford University School of Medicine. Stanford,

CA 94305-5426
Smolowitz, Rovanne M., Laboratory of Marine Animal Health,

Marine Biological Laboratory, Woods Hole, MA 02543
Sogin, Mitchell, Marine Biological Laboratory, Woods Hole, MA

02543
Sorenson, Martha M., Cicade Universitaria-RFRJ. Department de

Bioquimica-ICB/CCS. Rio de Janeiro. RJ 21910. Brasil
Speck, William T., The Presbyterian Hospital in the City of New

York. New York, NY 10032-3784
Spector, Abraham, Department of Ophthalmology. Columbia

University. 630 West 168th Street, New York, NY 10032
Speer, John W., Marine Biological Laboratory, Woods Hole, MA

02543
Speksnijder, Johanna E., Hubrecht Laboratory, LIppsalalaan 8, 3584

CT Utrecht, The Netherlands
Sperelakis, Nicholas, Department of Physiology & Biophysics,

University of Cincinnati, Cincinnati, OH 45267-0576
Spiegel, Evelyn, Department of Biological Sciences, Dartmouth

College, Hanover. NH 03755
Spiegel, Melvin, Department of Biological Sciences. Dartmouth

College, Hanover. NH 03755
Spray, David C., Albert Einstein College of Medicine, Department of

Neurosciences, 1300 Morris Park Avenue, Bronx, NY 10461
Steele, John Hyslop, Woods Hole Oceanographic Institution, Woods

Hole. MA 02543

R64 Annual Report

Steinacker, Antoinette, Dept. of Otolaryngology, W:;

University. School of Medicine. Box 8115. 4566 ^ .venue. St.

Louis. MO 63 1 10
Steinberg, Malcolm, Department of Bin! niversity,

Princeton, NJ 08544- 10 14
Stemmer, Andreas C., Marine Biologn Woods Hole.

MA 02543
Stelten, Jane Lazarow, 4701 Willar.i use. MD 20815-

4635
Steudler, Paul A., Ecosystcn. Biological Laboratory.

Woods Hole. MA 02543
Stokes. Darrell R.. Department of !3iology. Emory University,

Atlanta, GA 30322
Stommel, Elijah W ., Section of Neurology, Dartmouth-Hitchcock

Medical Center, 2 Maynard St., Hanover, NH 03756
Stracher, Alfred, Department of Biochemistry, SUNY Health Science

Center. 450 Clarkson Ave.. Brooklyn. NY 1 1203
Strehler, Bernard I,., 2310 Laguna Circle Dr.. Agoura. CA 91301-

2884
Strickler, J. Rudi, Center for Great Lakes Studies. 600 East Greenfield

Ave.. Milwaukee. WI 53204 (resigned)
Strumwasser, Felix, USUHS, Department of Psychiatry. 4301 Jones

Bridge Rd.. Bethesda, MD 20814-4799
Stuart, Ann E., Department of Physiology, Medical Sciences Research

Bldg. 206H. University of North Carolina. Chapel Hill. NC 27599-

7545
Sugimori, Mufsuyuki, Department of Physiology and Biophysics. New

York University Medical Center, 550 First Avenue, New York, NY

10016
Summers, William C., Huxley College of Environmental Studies.

Western Washington University, Bellingham, WA 98225
Suprenant, Kathy A., Department of Physiology and Cell Biology.

4010 Haworth Hall, University of Kansas, Lawrence. KS 66045
Sussman, Maurice, 72 Carey Lane, Falmouth, MA 02540
Sussman, Raquel B., Marine Biological Laboratory, Woods Hole, MA

02543
Sweet, Frederick, Department of OB & GYN, Box 8064. Washington

University School of Medicine, 499 South Euclid. St. Louis. MO

63110
Sydlik. Mary Anne, Department of Biology. Westfield State College.

Westfield. MA 01086
Szent-Gyorgyi, Andrew. Department of Biology, Brandeis University.

Bassme 244. 415 South Street. Waltham. MA 02254
Szent-Gyorgyi, Gwen P., Marine Biological Laboratory, Woods Hole,

MA 02543
Szuts, Ete Z., 12 Hamlin Ave., Falmouth, M \ 02540

Tabares, Lucia, AVDA, Department of Physiology, Sanchez, Pizjuan

4,411009 Seville, Spain
Tamm, Sidney L., Boston University Marine Program. Marine

Biological Laboratory, Woods Hole. MA 02543 (reinstated)
Tanzer, Marvin L., Department of Biostructure & Function. Medical

School, University of Connecticut, Farmington, CT 06030-3705
Tasaki, Ichiji, Laboratory of Neurobiology, NIMH/NIH. Bldg. 3d.

Rm. 2B-lh. Bethesda. MD 20892
Taylor, Douglass I,., Center for Fluorescence Research. Carnegie

Mellon University. 4400 Fifth Avenue, Pittsburgh. PA 15213
Teal, John M., Department of Biology, Woods Hole Oceanographic

Institution. Woods Hole, MA 02543
Teller, William II., Department of Biology, University of

Pennsylvania, Philadelphia, PA 14104
Telzer, Bruce, Thille Building. Pomona College. 175 W. 6th Street.

Claremont. CA 9 1 7 1 I

Thorndike, W. Nicholas, Wellington Management Company. 28 State

St.. Boston. MA 02109
Townsel. James G., Department of Physiology, Meharry Medical

College. Nashville, TN 37208

Travis, David M., 223 Newell Road, Holden, MA 01520-1442
Treistman, Steven N., Worcester Foundation for Experimental

Biology. 222 Maple Avenue, Shrewsbury. MA 01545
Trigg, D. Thomas, One Federal Street. 9th Floor. Boston. MA 022 1 1
I rinkaus, J. P., Department of Biology. Yale University. New Haven,

CT06511
I roll, Walter, Department of Environmental Medicine, College of

Medicine, New York University, New York, NY 10016
Troxler, Robert E., Department of Biochemistry, School of Medicine,

Boston University, 80 East Concord St., Boston, MA 021 18
Tucker, Edward B.. Department of Natural Sciences, Baruch College,

CUNY. 17 Lexington Ave.. New York. NY 10010
Turner, Ruth D., Mollusk Department. Museum of Comparative

Zoology. Harvard LIniversity, Cambridge, MA 02138
Tweedell, kenvon S., Department of Biological Sciences. University of

Notre Dame, Notre Dame, IN 46656
Tykocinski, Mark I.., Institute of Pathology, Case Western Reserve

University, 2085 Adelbert Rd.. Cleveland. OH 44106
Tvtell. Michael, Department of Anatomy. Bowman Gray School of

Medicine, Wake Forest University, Winston-Salem, NC 27103

Ueno, Ilirnshi, Department of Medical Chemistry, Osaka Medical
College, 2-7 Daigaku-machi, Takatsuki, Osaka 569, Japan

Valiela, Ivan, Boston University Marine Program, Marine Biological

Laboratory, Woods Hole, MA 02543
Vallei 1 , Richard, Cell Biology Group. Worcester Foundation for

Experimental Biology. Shrewsbury, MA 01545

Valois, John, Marine Biological Laboratory, Woods Hole. MA 02543
Van llolde, kensal. Department of Biochemistry and Biophysics,

Oregon State University. Corvallis. OR 97331-6503
Vogel. Steven S., LBM. NIDDK/NIH. Bldg. 10. Rm. 9604. Bethesda,

MD 20892

Waksman, Bvmn, Foundation for Microbiology. 300 East 54th St..

New York, NY 10022

Wall, Betty, 9 George St., Woods Hole. MA 02543
Wallace, Robin A., Whitney Laboratory. 9505 Ocean Shore Blvd.. St.

Augustine. FL. 320X6
Wang, C'hing Chung, Department of Pharmaceutical Chemistry,

University of California, San Francisco, CA 94143
Wang, llsien-yu. Department of Biochemistry, National Defense

Medical Center, Taipei. Taiwan. Republic of China
Wangh, Lawrence J., Department of Biology, Brandeis University.

415 South St.. Waltham, MA 02254
Warner. Robert C., Department of Molecular Biology and

Biochemistry. University of California, Irvine, CA 92717
\\arren, Kenneth S., Maxwell Communications Corp.. 866 Third

Avenue. New York. NV 10022
\\arren, Leonard. Wistar Institute. 36th and Spruce Streets.

Philadelphia. PA 19104
\\ aterbury, John B., Department of Biology. Woods Hole

Oceanographic Institution, Woods Hole, MA 02543
Watson, Stanley, Associates of Cape Cod, Inc., P. O. Box 224. Woods

Hole, MA 02543
\\a\man, Stephen G., Department of Neurology, LCI 708. Yale

School of Medicine. 333 Cedar Street. New Haven. CT 065 10
Webb, II. Marguerite, Marine Biological Laboratory, Woods Hole,

MA 02543

Members of the Corporation R65

Weber, Annemarie, Department of Biochemistry and Biophysics.

School of Medicine, University of Pennsylvania, Philadelphia, PA

19066
Weidner, Earl, Department of Zoology and Physiology, Louisiana

State University, Baton Rouge, LA 70803
Weiss, Dieter G., Institut fur Zoologie. Technische Universitat

Munchen, 8046 Garching. FRG
Weiss, I.eon P., Department of Animal Biology. School of Veterinary

Medicine, University of Pennsylvania, Philadelphia, PA 19104
Weissmann, Gerald, New York University Medical Center, 550 First

Avenue, New York. NY 10016
Werman, Robert, Neurobiology Unit, The Hebrew University,

Jerusalem. Israel
\\esterfield, R. Monte, The Institute of Neuroscience, University of

Oregon, Eugene. OR 97403
\\hiltaker, J. Richard, Department of Biology, Bag Service #451 I 1.

University of New Brunswick, Fredericton. NB E3B 6E1, Canada
Wilson, Darcy B., San Diego Regional Cancer Center, 3099 Science

Park Road, San Diego, CA 92 1 2 1
Wilson, T. Hastings, Department of Physiology. Harvard Medical

School. Boston. MA 02 1 15
Witkotsk), Paul, Department of Ophthalmology. New York

University Medical Center. 550 First Ave., New York. NY 10016
Wittenberg, Beatrice, Department of Physiology & Biophysics. Albert

Einstein College of Medicine, Bronx, NY 10461
Wittenberg, Jonathan B., Department of Physiology and Biophysics,

Albert Einstein College, 1300 Morris Park Ave., Bronx, NY 01461
\Volken. Jerome J., Department of Biological Sciences, Carnegie

Mellon University, 440 Fifth Ave.. Pittsburgh, PA 15213

Wonderlin, William p.. Department of Pharmacology & Toxicology.

West Virginia University, Morgantown. WV 26506
\\orden, Mary Kate, Department of Neurobiology. Harvard Mi il

School, 220 Longwood Ave.. Boston, MA 021 15
Worgul, Basil V., Department of Ophthalmology. Columbia

University. 630 West 168th St.. New York, NY 10032
Wu, Chau Hsiung, Department of Pharmacology. Northwestern

University Medical School. Chicago, IL 6061 I
Wyttenbach, Charles R., Department of Physiology and Cell Biology,

University of Kansas, Lawrence, KS 66045

Yashphe, Jacob, Hebrew University, Hadassah Medical School,

Jerusalem, Israel, 91010
Yen, Jay Z., Department of Pharmacology, Northwestern University

Medical School, Chicago, IL 6061 1

Zigman, Seymour, School of Medicine and Dentistry, University of

Rochester. 260 Crittenden Blvd., Rochester, NY 14620
Zimmerberg, Joshua J., NIH. Bldg. 12A, Room 2007, Bethesda, MD

20892
Zottoli, Steven J., Department of Biology, Williams College,

Williamstown, MA 01267
Zucker, Robert S., Neurobiology Division. Department of Molecular

and Cellular Biology, University of California, Berkeley, CA 94720
Zukin, Ruth Suzanne. Department of Neuroscience, Albert Einstein

College of Medicine, 1410 Pelham Parkway South, Bronx, NY

10461

Associate Members

Alfano, Dr. Louis
Allen. Mr. and Mrs. Wayne
Allison, Mr. and Mrs. Douglas F.
Anderson. Mr. and Mrs. Seneca
Andrews. Dr. Edwin J.
Aristide. Ms. Tracy
Armstrong, Dr. and Mrs.

Richard A.

Aspinwall. Mr. and Mrs. Duncan
Atwood. Mrs. Kimball
Bagley. Mr. Everett E.
Bakalar, Mr. and Mrs. David
Ballantme. Mrs. Elizabeth E.
Bang. Mrs. Betsy G.
Bang, Ms. Molly
Banks. Ms. Jamie
Banks. Mr. and Mrs. William L
Barlow. Mr. and Mrs. R.

Chaning

Barnes. Mr. John
Benthos. Inc.

Berg. Mr. and Mrs. C. John
Berg. Ms. Linnea
Bernheimer, Drs. Alan W. and

Harriet P.

Bigelow. Mr. and Mrs. Robert O.
Bihrle. Dr. William
Bleck, Dr. Thomas P.
Blumenfeld, Dr. Olga
Boche, Mr. Robert D.
Bolton. Mr. and Mrs. Thomas C.

Borg. Dr. and Mrs. Alfred F.
Borgese. Dr. and Mrs. Thomas
Bowles, Dr. and Mrs. Francis P.
Bran r. Dr. and Mrs. Mark
Bnana, Anthony
Brown, Mrs. Jennie P.
Brown. Mrs. Tomas A.
Brown. Dr. and Mrs. Thornton
Buck, Dr. and Mrs. John B.
Burghauser, Dr. Alan H.
Burns, Dr. and Mrs. John E.
Buxton, Mr. and Mrs. Bruce E.
Canney. Ms. Paula
Carlson, Dr. and Mrs. Francis
Carlton. Mr. and Mrs. Winslow

G.

Case. Mrs. Patricia A.
Chaet. Mr. and Mrs. Alfred
Chandler, Mr. Robert
Child, Dr. and Mrs. Frank M..

Ill

Chisholm, Dr. Sallie W.
Clark. Dr. and Mrs. Arnold M.
Clark. Mr. and Mrs. Leroy. Jr.
Clement, Mrs. Octavia
Cloud. Dr. Laurence P.
Clowes Fund, Inc.
Clowes. Dr. and Mrs. Alexander

W.

Clowes, Mr. Allen W.
Clowes. Mrs. Margaret

Cobb. Dr. Jewel P.
Copeland, Dr. and Mrs. D.

Eugene

Cornell, Dr. and Mrs. Neal
Cowan, Ms. Stacy
Cowling. Mr. John
Cowling. Dr. Vincent
Crabb. Mr. and Mrs. David L.
Crain. Mr. and Mrs. Melvin C.
Cross, Mr. and Mrs. Norman C.
Crossley. Miss Dorothy
Crossley, Miss Helen
Crowell, Mrs. Villa
Davis, Mr. and Mrs. Joel P.
DiBerardmo, Dr. Marie A.
Donnette, Mr. and Mrs. Joseph
Donovan, Mr. and Mrs. David

L.

Douglas. Ms. Jean
Drohan. Ms. Suzanne
Drummey. Mr. and Mrs. Todd

A.

Dugan, Mr. and Mrs. William P.
Duplaix, Dr. Nicole
Ebert, Dr. and Mrs. James D.
Egloff, Mrs. F. R. L.
Eliott, Mr. Raymond
Ellis, Dr. and Mrs. David
Engles, Mr. and Mrs. George
Esswein. Dr. Arthur
Estabrooks. Mr. Gordon C.

Eustis. Mr. and Mrs. Jack
Farnham, Ms. Elizabeth
Fausch, Mr. and Mrs. David
Fisher, Mr. and Mrs. Frederick

S.. Ill

Folino, Mr. John W., Jr.
Freeman, Mr. and Mrs. Howard
Fribourgh, Dr. James H.
Friendship Fund
Frosch, Dr. and Mrs. Robert A.
Fye, Mrs. Paul M.
Gartield, Ms. Eleanor
Garrett, Dr. Patricia
Gault, Ms. Christine
Gellis. Dr. and Mrs. Sydney
Glazebrook. Mrs. Rebeckah D.
Glenn. Mr. Gary
Goldstein. Dr. and Mrs. Moise

H.. Jr.

Goodwin, Mr. and Mrs. Charles
Grant, Mrs. Rose
Greer, Mr. and Mrs. W. H., Jr.
Griffith, Dr. and Mrs. B. Herold
Grossman, Barbara
Haakonsen, Dr. Harry O.
Hadamard, Dr. Antoine F.
Halvorson, Dr. and Mrs. Harlyn

O.

Hamstrom. Ms. Mary Elizabeth
Harrington, Mr. Robert B.
Harrington. Mr. Robert D., Jr.

R66 Annual Report

Harvey, Mrs. Janet
Hastings, Dr. and Mrs. J.

Woodland

Haubrich. Dr. Robert R
Hays, Dr. David S.
Hiatt, Dr. and Mrs. Howard H.
Hibbitt, Mrs. H. D.
Hichar, Dr. Barbara
Hirschfeld, Mrs. Eleanor M.
Hodge. Dr. and Mrs. Stuart
Hodosh. Mrs. Helen
Holmes, Mrs. George
Hoskin, Dr. Francis
Hough, Mr. John T.
Howard, Mrs. Man Jean
Huettner, Dr. and Mrs. Robert J.
Huettner, Ms. Susan A.
Inoue. Dr. and Mrs. Shinya
Jackson, Miss Elizabeth B.
Jewett. G. F., Foundation
Jewett. Mr. and Mrs. G. F., Jr.
Jewett. Mr. and Mrs. Raymond

L.
Jones, Mr. and Mrs. DeWitt C.,

Ill

Jones. Mr. and Mrs. Frederick. II
Jones, Mr. Frederick S., Ill
Jordan. Dr. and Mrs. Edwin P
Kahn, Dr. Harry S.
Karush, Mrs. Sally
Katz, Mrs. Marcella
Keoaghan, Ms. Patricia E.
Kivy, Dr. and Mrs. Peter
Knoble, Ms. Mary
Knowles, Mr. and Mrs. Sidney

A.

Korgen. Dr. Ben J.
Kraco, Ms. Karen
Kravitz. Dr. and Mrs. Edward A.
KufFler. Mrs. Phyllis
Laderman, Mr. Ezra and Dr.

Aimlee

Lakian, Mr. and Mrs. John
Lash, Ms. Rebecca
Laster, Dr. and Mrs. Leonard
Laufer, Dr. and Mrs. Hans
Laufer, Ms. Jessica, and Weiss,

Mr. Malcolm
Lauter, Dr. Marc R.
Lawrence, Mr. and Mrs. William
Leach, Dr. and Mrs. Berton J.
Leahy, Michael

LeBlond, Mr. and Mrs. Arthur
Leeson, Mr. and Mrs. A. Dix
LeFevre, Dr. Marian E.
Leffler, Dr. Charles W.
Levitz. Dr. Mortimer
Light, Mr. and Mrs. Donald W.
Lindberg, Mr. Lennart
Livingstone. Jr., Mr. and Mrs.

Robert

Lloyd, Mr. and Mrs. James E.
Loessel, Mrs. Sarah

Low, Miss Don

Mackey, Mr. :< .. William

K.

MacLci'.l! i:etM.

Map

d '.Irs. Philip B.
\. Madeline
and Mrs. Julian B.
. ir. and Mrs. Joseph

I

Masari. Dr. Mansa
Mason, Mr. and Mrs. Appleton
Mavor, Mr. and Mrs. James
Mastroianni, Dr. and Mrs. Luigi,

Jr.

Mauzerall, Mrs. Miriam J.
McElroy, Mrs. Nella W.
McGonigle, Mr. Paul
McKoan. Ms. Mary W.
McMahon, Mr. John J.
McMurtrie, Mrs. Cornelia

Hanna

Meigs. Mr. and Mrs. Arthur V.
Meigs, Dr. and Mrs. J. Wister
Mehllo, Dr. and Mrs. Jerry M.
Mellon, Mr. and Mrs. Richard P.
Mendelson, Dr. Martin
Metz, Dr. and Mrs. Charles B.
Meyers, Mr. and Mrs. Richard
Mills, Mrs. Margaret A.
Mixer, Mrs. Florence E.
Monroy. Mrs. Anna
Montgomery, Dr. and Mrs.

Charles H.

Montgomery, Mrs. Mary E.
Morse, Dr. M. Patricia
Morse, Jr., Mr. and Mrs. Richard
Munson, Mr. William
Murchelano, Dr. Robert
Nace, Mr. Paul F.. Jr.
Naugle. Mr. John E.
Neall, Mr. William G.
Nelson, Dr. Pamela
Netsky, Dr. Martin
O'Connell, Dr. and Mrs. Clifford
Olszowka, Dr. Janice S.
O'Neil, Mr. Thomas
Ott, Dr. Karen
Palmer, Mr. and Mrs. David
Pappas. Dr. and Mrs. George D.
Parmenter. Dr. Charles
Pearce, Dr. John B.
Pearson, Mrs. Helen M.
Pederson, Dr. and Mrs. Thoru
Peri, Mr. and Mrs. John B.
Person. Dr. and Mrs. Philip
Plough, Ms. Frances
Plough, Mr. and Mrs. George H.
Plymouth Savings Bank
Porter. Dr. and Mrs. Keith R

Press. Dr. Frank

Price, Ms. Carol

Price. Mr. John S.

Prosser. Dr. and Mrs. C. Ladd

Putnam, Mr. and Mrs. Allan Ray

Putnam. Mr. and Mrs. William

A., Ill

Quezada, Dr. Fernando
Rankm. Mrs. Julia S.
Regan. Reverend Msgr. John J.
Righter, Mr. and Mrs. Harold
Riina, Mr. John R.
Riley, Dr. Monica
Ripple, Mr. and Mrs. John
Robbms, Ms. Ann
Robertson, Mrs. Lola E.
Robinson, Dr. Denis M.
Robinson, Mr. John G.
Robinson, Mr. and Mrs. Marius

A.

Roose, Ms. Elayne
Root, Mrs. Pauline
Rosenthal, Ms. Hilde
Rosett. Mrs. Atholie K.
Roslansky, Drs. John and

Pnscilla

Ross, Dr. Robert
Ross, Dr. Virginia S.
Rowe, Dr. Don
Rubinow, Mrs. Shirley
Sallet, Mrs. Grace W.
Sallop, Ms. Linda and Fenlon,

Mr. Michael

Sanidas, Dr. and Mrs. Dennis J.
Saunders, Dr. and Mrs. John W.
Sawyer, Mr. and Mrs. John E.
Scheffler, Ms. Astnd
Schlesmger, Mrs. R. Walter
Schwamb, Mr. and Mrs. Peter
Scott, Mrs. Elsie M.
Seder, Mr. John
Selby. Dr. Cecily
Seliger-Egelson, Ms. Pauline
Senft. Mrs. Deborah G.
Shanklin. Dr. and Mrs. D. R.
Shapiro, Mrs. Harriet
Sharp, Mr. and Mrs. Robert W.
Shaver, Dr. John R
Sheehy, Mr. David
Shemin. Mrs. Charlotte
Shepro, Dr. and Mrs. David
Silver, Mr. and Mrs. Bertram R.
Simon, Mr. and Mrs. Stephen A.
Simonds, Mr. and Mrs. Jonathon

O.

Singer. Mr. and Mrs. Daniel M.
Smith. Drs. Frederick E. and

Marguerite A.

Smith. Mr. and Mrs. Homer P.
Smith. Ms. Stacy Cowan
Solomon, Dr. and Mrs. A. K.
Speck, Dr. William T.

Spiegel, Drs. Melvin and Evelyn
Steele. Mrs. M. Evelyn
Steele, Dr. Robert E.
Steinbach, Mrs. Eleanor
Stephenson, Dr. and Mrs. Wm.

K.

Stetson. Mrs. Judith G.
Stetten, Mrs. Jane Lazarow
Stump, Mr. Robert
Swain, Mr. Albert H.
Swanson, Dr. and Mrs. Carl P.
Swift, Mr. and Mrs. E. Kent
Swift. Mr. and Mrs. Robert
Swope. Mr. and Mrs. Gerard L.
Swope, Mrs. Marjorie P.
Taylor, Mr. James K.
Taylor, Mrs. Jean G.
Tebbetts, Mr. and Mrs. Edwin H.
Thier, Dr. and Mrs. Samuel
Timmins. Mrs. Linda L.
Todd, Mr. and Mrs. Gordon F.
Trager. Mrs. Ida
Trigg. Mr. and Mrs. D. Thomas
Troll. Dr. and Mrs. Walter
Trousof, Miss Natalie
Ulbnch. Ms. Ciona
Ulbrich, Mr. and Mrs. Volker
Valois, Mr. and Mrs. John
Van Buren, Mrs. Alice H.
Vincent. Dr. and Mrs. Walter S.
Vonderhaar, Dr. William
Voorhis, Mr. Arthur
Wagner. Mr. Mark
Waite. Mrs. Charles
Waksman. Mrs. Joyce
Walter, Mr. and Mrs. Henry
Warren. Mrs. Eve
Weeks, Mr. and Mrs. John T.
Weiffenbach. Dr. and Mrs.

George

Weissmann. Dr. and Mrs. Gerald
Wendorff. Ms. Lillian
Wessel, Dr. Gary
Wheeler, Dr. and Mrs. Paul S.
Wheeler, Dr. William M.
Whitehead. Mrs. Barbara
Wickersham, Mrs. Joan
Wigley, Mrs. Roland
Wigley, Ms. Susan
Wilber, Mrs. Clare M.
Willis, Mr. Herbert F.
Wilson, Mr. and Mrs. Leslie J.
Winn, Dr. William M.
Wittenberg. Dr. Beatrice
Woitkoski, Miss Nancy
Wolfinsohn. Mrs. Sarah A.
Wolfinsohn. Mr. and Mrs. Wolfe
Woodwell, Dr. and Mrs. George

M.

Zacks, Dr. and Mrs. Sumner
Zimmerli. Mr. and Mrs. Bruce
Zinn. Dr. and Mrs. Donald J.

Members of the Corporation R67

Gift Shop Volunteers

Barbara \twood

Helen Hodosh

Virginia Reynolds

Patricia Barlow

Polly Hyde

Erika Righter

Harriet Bernheimer

Sally Karush

Jean Ripps

Glorie Borgese

Brookie Ketchum

Lola Robinson

Jennie Brown

Ruth Ann Laster

Lilyan Saunders

Shannon Brown

Evelyn Laufer

Elsie Scott

Elizabeth Buck

Barbara Little

Marilyn Shepro

Man Buckley

Sally Loessell

Fran Silverstein

Shirle> Chact

Vinnie Mackey

Marcia Simmons

Vera Clark

Connie Martyna

Cynthia Smith

Peggy Clowes

Mariam Mauzerall

Peggy Smith

Jewell Cobb

Phyllis Myers

Louise Specht

Villa Crowcll

Florence Mixer

Susie Steinbach

Janet Daniels

Lorraine Mizell

Dorothy Stracher

Alma Ebert

Eleanor Nace

Natalie Trousof

Ellie Gabriel

Arlene Park

Mary Ulbrich

Vi Gifford

Bertha Person

Barbara Van Holde

Rose Grant

Dottie Phinney

Alice Veeder

Edie Grosch

Elizabeth Price

Dorothy Ville

Barbara Grossman

Kathryn Price

Clare Wilber

Jean Halvorson

Julia Rankin

MEL Jour Guides

Betsy Bang

Teru Hayashi

Mary Ulbrich

John Buck

Julie Rankin

Donald Zinn

Sears Crowell

Lola Robertson

Margery Zinn

Certificate of Org >a

Articles of Amer
Bylaws

Certificate of Organization

Articles of Amendment

(On File in the Office of Ihe Secretary of the Commonwealth)
No. 3 1 70

We, Alpheus Hyatt. President, William Stanford Stevens. Treasurer, and William
T. Sedgwick. Edward G. Gardiner, Susan Mims and Charles Sedgwick Mmot being
a majority ot the Trustees of the Marine Biological Laboratory' in compliance with
the requirements of the fourth section of chapter one hundred and fifteen of the
Public Statutes do hereby certify that the following is a true copy of the agreement
of association to constitute said Corporation, with the names of the subscribers
thereto:

We. whose names are hereto subscribed, do. by this agreement, associate ourselves
with the intention to constitute a Corporation according to the provisions of the
one hundred and fifteenth chapter of the Public Statutes of the Commonwealth of
Massachusetts, and the Acts in amendment thereof and in addition thereto.

The name by which the Corporation shall be known is
THE MARINE BIOLOGICAL LABORATORY.

(On File in the Office of the Secretary of the Commonwealth)

We, James D. Ebert, President, and David Shepro. Clerk of the Marine Biological
Laboratory, located at Woods Hole, Massachusetts 02543, do hereby certify that
the following amendment to the Articles of Organization of the Corporation was
duly adopted at a meeting held on August 15. 1975, as adjourned to August 29.
1975. by vote of 444 members, being at least two-thirds of its members legally
qualified to vote in the meeting of the corporation:

Voted: That the Certificate of Organization of this corporation be and it hereby is

amended by the addition of the following provisions:
"No Officer, Trustee or Corporate Member of the corporation shall be personally

liable for the payment or satisfaction of any obligation or liabilities incurred

as a result of. or otherwise in connection with, any commitments, agreements,

activities or affairs of the corporation.
"Except as otherwise specifically provided by the Bylaws of the corporation, meetings

of the Corporate Members of the corporation may be held anywhere in the

United States.
"The Trustees of the corporation may make, amend or repeal the Bylaws of the

corporation in whole or in part, except with respect to any provisions thereof

which shall by law, this Certificate or the bylaws of the corporation, require

action by the Corporate Members."

The purpose lor which the Corporation is constituted is to establish and maintain
a laboratory or station for scientific study and investigations, and a school for
instruction in biology and natural history.

The place within which the Corporation is established or located is the city of
Boston within said Commonwealth
The amount of its capital stock is none.

In Witness Whereof, we have hereunto set our hands, this twenty seventh day of
February in the year eighteen hundred and eighty-eight. Alpheus Hyatt. Samuel
Mills. William T. Sedgwick, Edward G. Gardiner, Charles Sedgwick Mmot, William
G. Farlow, William Stanford Stevens. Anna D. Phillips. Susan Mims, B. H. Van
Vleck.

That the first meeting of the subscribers to said agreement was held on the thirteenth
day ot March in the year eighteen hundred and eighty-eight.
In Witness Whereof, we have hereunto signed our names, this thirteenth day of
March in the year eighteen hundred and eighty-eight, Alpheus Hyatt, President.
William Stanford Stevens, Treasurer, Edward G. Gardiner, William T. Sedgwick.
Susan Mims, Charles Sedgwick Mmot.
(Approved on March 20. 1988 as follows:

I hereby certify that it appears upon an examination o; the within written certificate
and the records of the corporation duly submitted to my inspection, that the re-
quirements of sections one, two and three of chapter one hundred and fifteen, and
sections eighteen, twenty and twenty-one of chapter one hundred and six, of the
Public Statutes, have been complied with and I hereby approve said certificate this
twentieth day of March A.D. eighteen hundred and eighty-eight.

Charles Endicott
Commissioner of Corporations)

The foregoing amendment will become effective when these articles of amendment
are filed in accordance with Chapter 180. Section 7 of the General Laws unless
these articles specify, in accordance with the vote adopting the amendment, a later
effective date not more than thirty days after such filing, in which event the amend-
ment will become effective on such later date.

In Witness whereof and Under the Penalties of Perjury, we have hereto signed our

names this 2nd day of September, in the year 1975. James D. Ebert. President:

David Shepro. Clerk.

(Approved on October 24, 1975, as follows:

I hereby approve the within articles of amendment and. the filing fee in the amount

ot $10 having been paid, said articles are deemed to have been filed with me this

24th day of October. 1975.

Paul Guzzi

Secretary of the Commonwealth)

Bylaws

(Revised August 7. 1992 and December 10, 1992)
ARTICLE 1 THE CORPORATION

A. Name and Purpose The name of the Corporation shall be The Marine Bio-
logical Laboratory. The Corporation's purpose shall be to establish and maintain
a laboratory or station for scientific study and investigation and a school for in-
struction in biology and natural history.

K68

Bylaws of the Corporation R69

B Nondiscrimination The Corporation shall not discriminate on the basis of
age, religion, color, race, national or ethnic origin, sex or sexual preference in its
policies on employment and administration or in its educational and other programs

ARTICLE II MEMBERSHIP

V Memlvrs The Members of the Corporation ("Members") shall consist of
persons elected by Ihe Board of Trustees (the "Board"), upon such terms and
conditions and in accordance with such procedures, not inconsistent with law or
these Bylaws, as may be determined by the Board. At any regular or special meeting
ol the Board, the Board may elect new Members. Members shall have no voting
or other rights with respect to the Corporation or us activities except as specified
in these Bylaws, and an\ Member may vote at any meeting of the Members in
person only and not by proxy. Members shall serve until their death or resignation
unless earlier removed with or without cause by the affirmative vote of two-thirds
ol the Trustees then in office. Any Member who has retired from his or her home
institution may. upon written request to the Corporation, be designated a Life
Member Life Members shall not have the right to vote and shall not be assessed
for dues.

B. Meetings The annual meeting of the Members shall be held on the Friday
following the hrst Tuesday in August of each year, at the Laboratory of the Cor-
poration in Woods Hole. Massachusetts, at 9:30 a.m. The Chairperson of the Board
shall preside at meetings of the Corporation. If no annual meeting is held in ac-
cordance with the foregoing provision, a special meeting may be held in lieu thereof
with the same effect as the annual meeting, and in such case all references in these
Bylaws, except in this Article II. B., to the annual meeting of the Members shall be
deemed to refer to such special meeting. Members shall transact business as may
properly come before the meeting. Special meetings of the Members may be called
by the Chairperson or the Trustees, and shall be called by the Clerk, or in the case
of the death, absence, incapacity or refusal by the Clerk, by any other officer, upon
written application of Members representing at least ten percent of the smallest
quorum of Members required for a vote upon any matter at the annual meeting
of the Members, to be held at such time and place as may be designated.

C. Quorum. One hundred (100) Members shall constitute a quorum at any
meeting. Except as otherwise required by law or these Bylaws, the affirmative vote
ot a majority of the Members voting in person at a meeting attended by a quorum
shall constitute action on behalf of the Members.

D. \otit-c nl Meetings. Notice of any annual meeting or special meeting of
Members, if necessary, shall be given by the Clerk by mailing notice of the time
and place and purpose of such meeting at least 15 days before such meeting to
each Member at his or her address as shown on the records of the Corporation.

E. H'avier ol \uiice. Whenever notice of a meeting is required to be given a
Member, under any provision of the Articles or Organization or Bylaws of the
Corporation, a written waiver thereof, executed before or after the Meeting by such
Member, or his or her duh authorized attorney, shall be deemed equivalent to
such notice.

F. Adjournments. Any meeting of the Members ma\ be adjourned to any other
time and place by the vote of a majority of those Members present at the meeting,
whether or not such Members constitute a quorum, or by any officer entitled to
preside at or to act as Clerk of such meeting, if no Member is present or represented
It shall not be necessary to notify any Members of any adjournment unless no
Member is present or represented at the meeting which is adjourned, in which case.
notice of the adjournment shall be given in accordance with Article II. D. Any
business which could have been transacted at any meeting of the Members as
originally called may be transacted at an adjournment thereof.

ARTICLE III ASSOCIATES OF THE CORPORATION

Associates ol the Corporation. The Associates of the Marine Biological Laboratory
shall be an unincorporated group ofpersons (including associations and corporations)
interested in the Laboratory and shall be organized and operated under the general
supervision and authority of the Trustees. The Associates of the Marine Biological
Laboratory shall have no voting rights.

ARTICLE IV BOARD OF TRUSTEES

A. Powers. The Board of Trustees shall have the control and management of
the affairs of the Corporation. The Trustees shall elect a Chairperson of the Board
who shall serve until his or her successor is elected and qualified. They shall annually
elect a President of the Corporation. They shall annually elect a Vice Chairperson
of the Board who shall be Vice Chairperson of the meetings of the Corporation
They shall annually elect a Treasurer. They shall annually elect a Clerk, who shall

be a resident of Massachusetts. They shall elect I rustecs-at-Large as specified in
this Article IV. They shall appoint a Director of the Laboratory fora term not to
exceed five years, provided the term shall not exceed one year if the candidate has
attained the age of 65 years pnor to the dale of the appointment. They shall choose
such other officers and agents as they shall think best. They may fix the compensation
of all officers and agents of the Corporation and may remove them at any time.
They may fill vacancies occurring in any of the offices. The Board shall have the
power to choose an Executive Committee from their own number as provided in
Article V. and to delegate to such Committee such of their own powers as they
may deem expedient in addition to those powers conferred by Article V. They
shall, from time to time, elect Members to the Corporation upon such terms and
conditions as they shall have determined, not inconsistent with law or these Bylaws.
B ( 'i>m/'f>w//<w anil I:lallt>n

(1) The Board shall include 24 Trustees elected by the Board as provided
below:

(a) At least six Trustees ("Corporate Trustees") shall be Members who are
scientists, and the other Trustees ("Trustees-at-Large") shall be individuals who
need not he Members or otherwise affiliated with the Corporation.

(b) The 24 elected Trustees shall be divided into four classes of six Trustees
each, with one class to be elected each year to serve for a term of four years, and
with each such class to include at least one Corporate Trustee. Such classes of
Trustees shall be designated by the year of expiration of their respective terms.

(2) The Board shall also include the Chief Executive Officer. Treasurer and
the Chairperson of the Science Council, who shall be ex qtficio voting members of
the Board.

(3) Although Members or Trustees may recommend individuals for nomi-
nation as Trustees, nominations for Trustee elections shall be made by the Nom-
inating Committee in its sole discretion. The Board may also elect Trustees who
have not been nominated by the Nominating Committee

C. Eligibility A Corporate Trustee or a Trustee-at-Large who has been elected
to an initial four-year term or remaining portion thereof, of which he/she has
served at least two years, shall be eligible for re-election to a second four-year term,
but shall he ineligible for re-election to any subsequent term until one year has
elapsed after he/she has last served as a Trustee.

D. Remmal Any Trustee may be removed from office at any time with or
without cause, by vote of a majority of the Members entitled to vote in the election
of Trustees: or for cause, by vote of two-thirds of the Trustees then in office. A
Trustee may be removed for cause only if notice of such action shall have been
given to all of Ihe Trustees or Members entitled to vote, as the case may be. prior
to the meeting at which such action is to be taken and if the Trustee to be so
removed shall have been given reasonable notice and opportunity to be heard
before the body proposing to remove him or her.

E. I'acaneie.s. Any vacancy in the Board may be filled by vote of a majority of
the remaining Trustees present at a meeting of Trustees at which a quorum is
present. Any vacancy in the Board resulting from the resignation or removal of a
Corporate Trustee shall be filled by a Member who is a scientist.

F. Meetings Meetings of the Board shall be held from time to time, not less
frequently than twice annually, as determined by the Board. Special meetings of
Trustees may be called by the Chairperson, or by any seven Trustees, to be held
at such time and place as may be designated. The Chairperson of the Board, when
present, shall preside over all meetings of the Trustees. Written notice shall be sent
to a Trustee's usual or last known place of residence at least two weeks before the
meeting. Notice of a meeting need not be given to any Trustee if a written waiver
of notice executed by such Trustee before or after the meeting is filed with the
records of the meeting, or if such Trustee shall attend the meeting without protesting
prior thereto or at its commencement the lack of notice given to him or her.

G. Quorum untl .Iftion />i Trustees A maiorm ot all Trustees then in office
shall constitute a quorum. Any meeting of Trustees may be adjourned by vote of
a majority of Trustees present, whether or not a quorum is present, and the meeting
may be held as adjourned without further notice. When a quorum is present at
any meeting of the Trustees, a majority of the Trustees present and voting (excluding
abstentions) shall decide any question, including the election of officers, unless
otherwise required by law. the Articles of Organization or these Bylaws.

M liiinsii-i-s :il Interests in Land There shall be no transfer of title nor long-
term lease ol real property held by the Corporation without pnor approval of not
less than two-thirds of the Trustees. Such real property transactions shall be finally
acted upon at a meeting of the Board only if presented and discussed at a pnor
meeting of the Board. Either meeting may be a special meeting and no less than
lour weeks shall elapse between the two meetings. Any property acquired by the
Corporation after December I. 1989 may be sold, any mortgage or pledge of real

R70 Annual Report

property (regardless of when acquired) to secure borrowings b he Corporation
may be granted, and any transfer of title or interest in real pro: iv pursuant to
the foreclosure or endorsement of any such mortgapc "i real property

may be effected by any holder of a mortgage 01 property of the

Corporation, with the prior approval of not lev '- of the Trustees

(other than any Trustee or Trustees with a direi . .incial interest in

the transaction being considered for approv nt at a regular or

special meeting of the Board at which ih. .

ARTK > is

A. Executive Committee Ih' i utive Committee of the Board
of Trustees which shall consi-.i <>i n re than eleven (1 1) Trustees, including ex
officio Trustees, elected by the Boar-.l.

The Chairperson of the Board shall act as Chairperson of the Executive Committee
and the Vice Chairperson as Vice Chairperson. The Executive Committee shall
meet at such times and places and upon such notice and appoint such subcommittees
as the Committee shall determine.

The Executive Committee shall have and may exercise all the powers of the
Board during the intervals between meetings of the Board except those powers
specifically withheld, from time to time, by vote of the Board or by law. The Executive
Committee may also appoint such committees, including persons who are not
Trustees, as it may. from time to time, approve to make recommendations with
respect to matters to be acted upon by the Executive Committee or the Board.

The Executive Committee shall keep appropriate minutes of its meetings, which
shall be reported to the Board. Any actions taken by the Executive Committee
shall also be reported to the Board.

B. Nominating Committee There shall be a Nominating Committee which shall
consist of not fewer than four nor more than six Trustees appointed by the Board
in a manner which shall reflect the balance between Corporate Trustees and Trustees-
at-Large on the Board, The Nominating Committee shall nominate persons for
election as Corporate Trustees and Trustees-at-Large, Chairperson of the Board,
Vice Chairperson of the Board. President, Treasurer, Clerk, Director of the Lab-
oratory and such other officers, if any, as needed, in accordance with the requirements
of these Bylaws. The Nominating Committee shall also be responsible for overseeing
the training of new Trustees. The Chairperson of the Board of Trustees shall appoint
the Chairperson of the Nominating Committee. The Chairperson of the Science
Council shall be an ex officio voting member of the Nominating Committee.

C. Science Council There shall be a Science Council (the "Council") which shall
consist of Members of the Corporation elected to the Council by vote of the Members
of the Corporation, and which shall advise the Board with respect to matters con-
cerning the Corporation's mission, its scientihc and instructional endeavors, and
the appointment and promotions of persons or committees with responsibility for
matters requiring scientific expertise. Unless otherwise approved by a majority of
the members of the Council, the Chairperson of the Council shall be elected annually
by the Council. The chief executive officer of the Corporation shall be an C-\ ollicin
voting member of the Council.

D. Board of Ovt'rscm There shall be a Board of Overseers which shall consist
ot not fewer than hve nor more than eight scientists who have expertise concerning
matters with which the Corporation is involved. Members of the Board of Overseers
may or may not be Members of the Corporation and may be appointed by the
Board of Trustees on the basis of recommendations submitted from scientists and
scientific organizations or societies. The Board of Overseers shall be available to
review and offer recommendations to the officers. Trustees and Science Council
regarding scientihc activities conducted or proposed by the Corporation and shall
meet from time to time, not less frequently than annually, as determined by the
Board of Trustees.

E. Board Conimiiici'\ Generally The Trustees may elect or appoint one or more
other committees (including. Inil not limited to, an Investment Committee, a De-
velopment Committee, an Audii Commiiu ' , i facilities and Capital Equipment
Committee and a Long-Range Planning Committee) and may delegate to any such
committee or committees any or all of iheir powers, except those which by law,
the Articles of Organization or these B\lj\vs the Trustees are prohibited from del-
egating; provided that any committee to which the powers of the Trustees are
delegated shall consist solely of Trustees. The members of any such committee
shall have such tenure and duties as the Trustees shall determine. The Investment
Committee, which shall oversee the management ot "the Corporation's endowment
funds and marketable secunties shall include as o i ilium members, the Chairperson
of the Board, the Treasurer and the Chairperson of the Audit Committee, together
with such Trustees as may be required for not less than two-thirds of the Investment

Committee to consist of Trustees. Except as otherwise provided by these Bylaws
or determined by the Trustees, any such committee may make rules for the conduct
of its business, but. unless otherwise provided by the Trustees or in such rules, its
business shall be conducted as nearly as possible in the same manner as is provided
by these Bylaws for the Trustees.

F. Aclinn\ H'lthiiiil a Meeting Any action required or permitted to be taken at
any meeting of the Executive Committee or any other committee elected by the
Trustees may be taken without a meeting if all members of such committees consent
to the action in writing and such written consents are filed with the records of
meetings. Members of the Executive Committee or any other committee elected
by the Trustees may also participate in any meeting by means of a telephone con-
ference call, or otherwise take action in such a manner as may, from time to time,
be permitted by law.

G. Manual of Procedures The Board of Trustees, on the recommendation of
the Executive Committee, shall establish guidelines and modifications thereof to
be recorded in a Manual of Procedures. Guidelines shall establish procedures for:
(I) Nomination and election of members of the Corporation, Board of Trustees
and Executive Committee; (2) Election of Officers; (3) Formation and Function of
Standing Committees.

ARTICLE VI OFFICERS

A. Enumeration The officers of the Corporation shall consist of a President, a
Treasurer and a Clerk, and such other officers having the powers of President,
Treasurer and Clerk as the Board may determine, and a Director of the Laboratory.
The Corporation may have such other officers and assistant officers as the Board
may determine, including (without limitation) a Chairperson of the Board, Vice
Chairperson and one or more Vice Presidents, Assistant Treasurers or Assistant
Clerks. Any two or more offices may be held by the same person. The Chairperson
and Vice Chairperson of the Board shall be elected by and from the Trustees, but
other officers of the Corporation need not be Trustees or Members. If required by
the Trustees, any officer shall give the Corporation a bond for the faithlul performance
of his or her duties in such amount and with such surety or sureties as shall be
satisfactory to the Trustees.

B Tenure Except as otherwise provided by law. by the Articles of Organization
or by these Bylaws, the President, Treasurer, and all other officers shall hold office
until the first meeting of the Board following the annual meeting of Members and
thereafter, until his or her successor is chosen and qualified.

C. Reuxnalion Any officer may resign by delivering his or her written resignation
to the Corporation at its principal office or to the President or Clerk and such
resignation shall be effective upon receipt unless it is specified to be effective at
some other time or upon the happening of some other event.

D. Removal The Board may remove any officer with or without cause by a vote
of a majority of the entire number of Trustees then in office, at a meeting ol the
Board called for that purpose and for which notice of the purpose thereof has been
given, provided that an officer may be removed for cause only after having an
opportunity to be heard by the Board at a meeting of the Board at which a quorum
is personally present and voting.

E. I 'acancv A vacancy in any office may be filled for the unexpired balance of
the term by vole of a majority of the Trustees present at any meeting of Trustees
at which a quorum is present or by written consent of all of the Trustees, if less
than a quorum of Trustees shall remain in office.

F. Chairpcnum The Chairperson shall have such powers and duties as may be
determined by the Board and, unless otherwise determined by the Board, shall
serve in that capacity for a term coterminous with his or her term as Trustee.

G. I ice Chairperson. The Vice Chairperson shall perform the duties and exercise
the powers of the Chairperson in the absence or disability of the Chairperson, and
shall perform such other duties and possess such other powers as may be determined
by the Board. Unless otherwise determined by the Board, the Vice Chairperson
shall serve for a one-year term.

H Dim-tor The Director shall be the chief operating officer and. unless otherwise
voted by the Trustees, the chief executive officer of the Corporation. The Director
shall, subject to the direction of the Trustees, have general supervision of the Lab-
oratory and control of the business of the Corporation. At the annual meeting, the
Director shall submit a report of the operations of the Corporation for such year
and a statement of its affairs, and shall, from time to time, report to the Board all
matters within his or her knowledge which the interests of the Corporation may
require to be brought to its notice.

I. Deputy Director The Deputy Director, if any, or if there shall be more than
one, the Deputy Directors in the order determined by the Trustees, shall, in the

Bylaws of the Corporation R71

absence or disability of the Director, perform the duties and exercise the powers
of the Director and shall perform such other duties and shall have such other
powers as the Trustees may, from time to time, prescribe.

J. President The President shall have the powers and duties as ma> be vested
in him or her bv the Board.

Iv / rcauirer and Assistant Treasurer The Treasurer shall, subject to the direction
of the Trustees, have general charge of the financial affairs of the Corporation,
including its long-range financial planning, and shall cause to be kept accurate
books of account. The Treasurer shall prepare a yearly report on the financial status
of the Corporation to be delivered at the annual meeting. The Treasurer shall also
prepare or oversee all filings required by the Commonwealth of Massachusetts, the
Internal Revenue Service, or other Federal and State Agencies. The account of the
Treasurer shall be audited annually b\ a certified public accountant.

The Assistant Treasurer, if any. or if there shall be more than one, the Assistant
Treasurers in the order determined by the Trustees, shall, in the absence or disability
of the Treasurer, perform the duties and exercise the powers of the Treasurer, shall
perform such other duties and shall have such other powers as the Trustees may,
from time to time, prescribe.

L. Clerk and Assistant Clerk The Clerk shall be a resident of the Commonwealth
of Massachusetts, unless the Corporation has designated a resident agent in the
manner provided by law. The minutes or records of all meetings of the Trustees
and Members shall be kept by the Clerk who shall record, upon the record books
of the Corporation, minutes of the proceedings at such meetings. He or she shall
have custody of the record books of the Corporation and shall have such other
powers and shall perform such other duties as the Trustees may. from time to time,
prescribe.

The Assistant Clerk, if any, or if there shall be more than one. the Assistant
Clerks in the order determined by the Trustees, shall, in the absence or disability
of the Clerk, perform the duties and exercise the powers of the Clerk and shall
perform such other duties and shall have such other powers as the Trustees may,
from time to time, prescribe.

In the absence of the Clerk and an Assistant Clerk from any meeting, a temporary
Clerk shall be appointed at the meeting.

M. Other Powers and Duties. Each officer shall have in addition to the duties
and powers specifically set forth in these Bylaws, such duties and powers as are
customarily incident to his or her office, and such duties and powers as the Trustees
may. from time to time, designate.

ARTICLE VII AMENDMENTS

These Bylaws may be amended by the affirmative vote of the Members at any
meeting, provided that notice of the substance ot the proposed amendment is stated
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No later than the time of giving notice of meeting of Members next following
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Any Bylaw adopted by the Trustees may be amended or repealed by the Members
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ARTICLE VIII INDEMNITY

Except as otherwise provided below, the Corporation shall, to the extent legally
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or officer of the Corporation or who is serving, or shall have served at the request
of the Corporation as a Trustee, director or officer of another organization in which
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The Corporation shall provide no indemnification with respect to any matter as
to which any such Trustee, director or officer shall be finally adjudicated in such
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shall provide no indemnification with respect to any matter settled or composed
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As used in the Article VIII. the terms "Trustee." "director," and "officer" include
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the Corporation which is governed by the Act of Congress entitled "Employee
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ARTICLE IX DISSOLUTION

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R72 Annual Report

ration's Clerk or resident agent. Said copies and reconK ; t in the

same office. They shall be available at all reasonable i (ion by any

Member for any proper purpose, but not to secure a list foi a purpose

other lhan in the interest of the applicant, as a "I " 'he affairs of

the Corporation.

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Organization shall be deemed to refer i> .'.nm of the Cor-
poration, as amended and in effect '

F. Transactions with linen vr,/ 1 ' >l fraud, no contract or
other transaction between this i .ner corporation or any firm,
association, partnership or p or imalidated by the fact that
any Trustee or officer ol lit " umarily or otherwise interested in
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to such contract or transaction must be adopted by a majority of the Trustees then
in office who have no interest in such contract or transaction.

CONTENTS

BEHAVIOR AND BIOMECHANICS

Drewes, Charles D., and Charles R. Fourtner

Helical swimming in a freshwater oligochaete ... 1

Johnson, Amy S.

Sag-mediated modulated tension in terebellid ten-
tacles exposed to flow 10

Trager, Geoff, and Amatzia Genin

Flow velocity induces a switch from active to passive
suspension feeding in the porcelain crab Petrolisthes
leptocheles (Heller) 20

ECOLOGY AND EVOLUTION

Eckman, James E., and David O. Duggins

Effects of flow speed on growth of benthic suspen-
sion feeders 28

Garcia-Esquivel, Zaul, and V. Monica Bricelj

Ontogenic changes in microhabitat distribution of
juvenile bay scallops, Argopecten irradians irradians
(L.), in eelgrass beds, and their potential significance
to early recruitment 42

DEVELOPMENT AND REPRODUCTION

Janies, Daniel A., and Larry R. McEdward

Highly derived coelomic and water-vascular mor-
phogenesis in a starfish with pelagic direct devel-
opment 56

Olson, Richard Randolph, J. Lane Cameron, and

Craig M. Young

Larval development (with observations on spawn-
ing) of the pencil urchin Phyllacanthus imperialis: a
new intermediate larval form? 77

INVERTEBRATE MORPHOLOGY AND PHYLOGENY

Balser, Elizabeth J., Edward E. Ruppert, and William
B. Jaeckle

Ultrastructure of the coeloms of auricularia larvae
(Holothuroidea: Echinodermata): evidence for the

presence of an axocoel 86

Emschermann, Peter

Lime-twig glands: a unique invention of an Antarc-
tic entoproct 97

NEUROBIOLOGY

Westfall, Jane A., and Cornelis J. P. Grimmelik-
huijzen

Antho-RFamide immunoreactivity in neuronal
synaptic and nonsynaptic vesicles of sea anemones 109

PHYSIOLOGY

Baker, Shirley M., and Nora B. Terwilliger

Hemoglobin structure and function in the rat-tailed

sea cucumber, Paracaudina chilensis 115

Kreeger, D. A., and C. J. Langdon

Effect of dietary protein content on growth of ju-
venile mussels, Mytilus trossulus (Gould 1850) ... 123

Sevala, V. M., V. L. Sevala, and A. S. M. Saleuddin
Hemolymph insulin-like peptides (ILP) liters and
the influence of ILP and mammalian insulin on the
amino acid incorporation in the mantle collar in vitro
in Helisoma (Mollusca) 140

RESEARCH NOTE

Lohmann, Kenneth J., and Catherine M. Fittinghoff
Lohmann

A light-independent magnetic compass in the leath-

erback sea turtle 1 49

Annual Report of the Marine Biological Laboratory R 1

Volume 185

THE

Number 2

BIOLOGICAL
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OCTOBER, 1993

iViarine Gioiogical Laboratory
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Published by the Marine Biological Laboratory

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THE

BIOLOGICAL BULLETIN

PUBLISHED BY
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Editorial Board

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Stanford University University of Washington

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Institute,
University of Texas Medical Branch SARAH ANN WOODIN, University of South Carolina

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OCTOBER, 1993

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ERRATA

The Biological Bulletin Volume 184, Number 3, pages 322-329

The following corrections should be noted in the paper by Gabriele Gaus et al. titled "The sequences of five neuropeptides
isolated from Limitlus using antisera to FMRFamide."

Table I

Representatives of four classes of FMRFamide-related peptides present in dipteran insects*

Class Sequence Reference

FaGRPs 3 Asp-Pro-Lys-Gln-Asp-Phe-Met-Arg-Phe-NH 2 (Schneider and Taghert, 1988)

Myosuppressins" Thr-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-NH 2 (Nichols, 1992a)

Sulfakinms c Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe-NH, (Nichols, 1992b)

Head peptides d pGlu-Arg-Pro-Pro-Ser-Leu-Lys-Thr-Arg-Phe-NH 2 (Lea and Brown, 1990)

The first three peptides in the list were isolated from Drosophila melanogaster; the fourth is from Aedes aegypii.
' FMRFamide-gene-reiated peptides; multiple, variable copies processed from a precursor.

b Similar C-terminal tetrapeptide, but not encoded on any known FMRFamide gene: peptide shown is Drosophila myosuppressin;
type of the class is leucomyosuppressin from Leucophaea maderae.

c Only C-terminal tnpeptide is analogous to FaGRPs: peptide shown is drosulfakinin; type of the class is leucosulfakinin.
d Only C-terminal dipeptide is analogous to FaGRPs.

The Table shown above replaces the original Table I in the paper. Its significance is unchanged; i.e.. it illustrates, with
selected examples, the four distinct classes of FaRPs that seem to occur in all insect species. The errors are as follows:

The sulfakinin sequence shown in the original Table I was feucosulfakinin (pGlu-Ser-Asp-Asp-), rather than drosul-
fakinin (Phe-Asp-Asp-). Moreover, the original reference (Nichols et al.. 1988) is to the DNA sequence of drosulfakinin;
but the processed peptide has also been sequenced (Nichols and Conkright, 1990; Nichols, 1992). The last of these is the
most appropriate citation.

In the original Table I, the reference to the head peptide is Matsumoto ct al.. 1989. Indeed, as Lea and Brown (1990)
state: "Dr. Shogo Matsumoto, working in our laboratory, developed a purification protocol that yielded three related
peptides from heads ofAeilex acgypti. These were purified to homogeneity and sequenced." Two of these sequences were
published in Matsumoto ct al. ( 1989), but the head peptide shown in Table I was not. The correct citation, now in the
revised Table I, is therefore Lea and Brown (1990). To complete the story, the two sequences presented in Matsumoto
et al. (1989) are: pGlu-Arg-Pro-Hyp-Ser-Leu-Lys-Thr-Arg-Phe-NH 2 and Thr-Arg-Phe-NH,. Note that the first of these
is the same as the head peptide in Table I, except that the proline at position 4 is hydroxylated.

Literature Cited

Lea, A. O., and M. R. Brown. 1990. Neuropeptides of mosquitoes. Pp. 181-188 in Molecular Insect Science. H. H.
Hagedorn, J. E. Hildebrand, M. G. Kidwell, and J. H. Law, eds. Plenum Press, New York.

Matsumoto, S., M. R. Brown, J. W. Crim, S. R. Vigna, and A. O. Lea. 1989. Isolation and primary structure of neu-
ropeptides from the mosquito, Aedes aegypti. immunoreactive to FMRFamide antiserum. Insect Biochfm. 19: 277-
283.

Nichols, R. 1992a. Isolation and structural characterization of Drosophila TDVDHVFLRFamide and FMRFamide
containing neural peptides. J. Mol. Neurosci. 3: 213-218.

Nichols, R. 1992b. Isolation and expression of the Drosophila drosulfakinin neural peptide gene-product, DSK-I. Mol.
Cell Neurosci. 3: 342-347.

Nichols, R., and M. Conkright. 1990. Isolation and characterization of Drosophila peptides containing an -ArgPheNH 2
C-terminus. Soc. Nctirosci. Ahstr, 16: 1031.

The Biological Bulletin, Volume 185, Number 1, page 42

The following correction should be noted in the paper by Zaul Garcia-Esquival and V. Monica Bricelj titled "Ontogenic
Changes in microhabitat distribution of juvenile bay scallops, Argopecten irradians irradians (L.). in eelgrass beds, and
tlieir potential significance to early recruitment."

The first word of the title should be replaced with the word "Ontogenetic" so that the corrected title now reads:
"Ontogenetic changes in microhabitat distribution of juvenile bay scallops. Argopecten irradians irradians (L.). in eelgrass
beds, and their potential significance to early recruitment."

Reference: Binl. Bull 185: 153-167. (October, 1993)

A Comparative Study of Reproduction and
Development in the Polychaete Family Terebellidae

DAMHNAIT McHUGH*

Department of Biology and Institute of Marine Sciences. University of California,

Santa Cruz, California 95064

Abstract. The reproduction and development of four
species of terebellid polychaetes from the west coast of
North America were studied and compared with several
other terebellid species to reveal the covariation of life
history traits in the group, and assess any limitations on
terebellid life history evolution that may be imposed by
ancestry or body design. The four species in the present
study span the range of reproductive and developmental
modes known for the family Terebellidae. Eupolymnia
crescentis and Neoamphitrite robusta are both free
spawners that reproduce during discrete 3-month breeding
periods. In E. crescentis. oogenesis takes from 5 to 8
months and spawning occurs from July to September,
maximum oocyte diameter is 210 jum, and fecundity
reaches ~ 128, 500 during a single breeding period. The
E. crescentis larva develops near the bottom for about 7
days before settling as a five-setiger juvenile. Neoamphi-
trite robusta reproduces from April to July after a 12-
month oogenic cycle; oocytes in this species measure up
to 180 Mm, and fecundity reaches ~ 830,000. The two
brooders in the study, Ramex californiensis and Tlielepus
crispus, brood their larvae in the maternal tube. T. crispus
reproduces continuously for at least 6 months, and has
up to 5 1 ,500 larvae in a single brood. The oocytes in this
species (400 /jm) give rise to larvae that are brooded to
the one-setiger stage and then emerge to undergo a one-
day planktonic period before the larvae settle and become
juveniles at eight setigers. Ramex californiensis reproduces
continuously year round; larvae are brooded in cocoons
that are laid sequentially in the tube, with up to 44 larvae
in a single cocoon. Development from the 410 /urn oocytes

is direct, and juveniles have 1 1 setigers. Unlike E. cres-
centis and N. robusta, in which oogenesis is synchronized
within individuals to produce a peak of large oocytes dur-
ing the discrete spawning period, R. californiensis and T.
crispus females have a wide range of oocyte sizes through-
out the year.

Correlation analysis and analysis of variance of repro-
ductive and developmental traits of these and several other
terebellid species revealed some expected trends. For ex-
ample, egg size varies according to the mode of repro-
duction (free spawning, extratubular brooding, or intra-
tubular brooding), and is also correlated with juvenile size.
However, egg size does not predict fecundity in terebellids
when body size is held constant, and brooding is not re-
stricted to small-bodied species. Indeed, the largest and
smallest species in the study brood their larvae intratu-
bularly, suggesting that allometric constraints may not be
important in determining mode of reproduction in these
polychaetes. The Terebellidae is a diverse family found
in all marine habitats, yet all known terebellid larvae are
non-feeding; this contrasts with the occurrence of both
planktotrophy and lecithotrophy in other polychaete
families, and leads to the proposal that larval development
in terebellids has been constrained during the evolution
of the lineage. The results of this study demonstrate that
generalizations regarding complex relationships among
life history traits are often inappropriate. The need for
more comparative studies of marine invertebrate repro-
duction and development, and the integration of phylo-
genetic analyses into the study of life history evolution in
marine invertebrates is highlighted.

Received 18 December 1992; accepted 14 July 1993.

* Present address: Department of Invertebrate Zoology. NHB-163.
National Museum of Natural History. Smithsonian Institution, Wash-
ington. DC 20560.

Introduction

The great diversity of reproductive and developmental
modes exhibited by marine invertebrates presents a chal-

153

154

D. MrHUGH

lenge to evolutionary biologists who seek patterns and
trends in life histories. Responses have included models
of optimal combinations of life history traits (e.g., Vance,
1973; Christiansen and Fenchel, 1979; Roughgarden,
1989), and theoretical arguments and experimental tests
on the adaptive significance of certain traits (e.g., Thorson,
1950; Chia. 1974; Pechenik, 1979; Doyle and Hunte,
1981; Sinervo and McEdward, 1988; Strathmann, 1985;
Grant, 1990; Hart, 1992). Several hypotheses regarding
the associations between traits have also been investigated
using specific cases (e.g., Strathmann el ai, 1984; Rabat,
1985; Hess, 1993). However, only a few studies have un-
dertaken statistical analyses of comparative data to assess
the covariation of life history traits in marine invertebrate
groups (Hines, 1982; Buroker, 1985; Olive, 1985; Emlet
et ai. 1987; McEdward and Chia, 1991). This contrasts
with the numerous investigations into life history evolu-
tion of vertebrates, in which the comparative approach
has clearly identified relationships among life history traits,
and has documented biological constraints and phylo-
genetic effects on the evolution of life histories (e.g.. Tren-
dall, 1982; Stearns, 1983, 1984a, b; Dunham and Miles,
1985; Harvey and Clutton-Brock, 1985; Gittleman, 1986;
Saether. 1988; Miles and Dunham, 1992).

While the range of reproductive modes in most marine
invertebrate phyla is well known, life history data are
scattered and in many cases incomplete, so that compar-
ative data sets are rare. In this study, I combine data from
autecological investigations of terebellid polychaetes with
my own results for four terebellid species from the west
coast of North America to construct a data matrix of life
history traits for the family Terebellidae. Despite large
variations in size, the body organization is relatively ho-
mogenous among all the genera in the family. This feature,
as well as the fact that no feeding larvae have been reported
in this group, means that the comparisons made in the
present study will not be complicated by the covariation
of traits with different body forms or different larval nu-
trition mechanisms. Nonetheless, it is obvious from pre-
vious studies of terebellids [Nicolea zoslehcola (Eckel-
barger, 1974, 1975, 1976), Neoleprea streptochaeta
(Duchene, 1979), T/iele/nis setoMis (Duchene, 1980,
1991), Eupolymnia nebulosa (Gremare, 1986; Bhaud et
ai. 1987; Bhaud and Gremare, 1988; Bhaud, 1991), Lan-
ice amchilegu (\ic\m\er, 1981; Bhaud. 1988, 1991; Smith,
1989a, b), and Rame.\ calijorniensis (Blake, 1991)], that
the Terebellidae, like many other polychaete families, ex-
hibit a high degree of heterogeneity in terms of reproduc-
tion and development (Wilson, 1991 ).

The following life history traits were investigated in
Eupolymnia crescentis Chaiabernn, 1919. Neoamphitrite
robusta (Johnson, 1901), Thelepus crispus Johnson, 1901,
and Rame.\ calijorniensis Hartman, 1944, and gathered
from previously published accounts of several other ter-

ebellid species: ( 1 ) mode of reproduction, i.e., free spawn-
ing, extratubular brooding or intratubular brooding; (2)
body size, i.e., maximum body length; (3) maximum oo-
cyte size; (4) maximum fecundity, i.e.. maximum number
of full-grown oocytes in free spawning species, or maxi-
mum number of larvae in a single brood in brooding spe-
cies; (5) mode of development, i.e., planktonic, mixed,
or direct development; (6) stage at juvenile, i.e.. the num-
ber of setigers present when larvae begin to feed on the
adult diet; (7) duration of the breeding season; and (8)
breeding strategy, i.e., iteroparous or semelparous. These
data were analyzed to reveal the covariation of life history
traits in the Terebellidae, and comparisons were made
with other polychaetes and marine invertebrates in gen-
eral. The possible limitations on life history evolution in
the Terebellidae, imposed either by ancestry or design,
were also examined, and the need fora phylogenetic anal-
ysis of the group discussed.

Materials and Methods
Species

Eupolymnia creseentis. which ranges from Alaska to
western Mexico, is common in sandy mud sediments of
bays and estuaries of the California coast. Neoamphitrite
robustu, which is distributed from Alaska to California,
is also found in abundance in mud and under rocks of
the intertidal zone of the central California coast. T/ielepus
crispus is found in tubes of coarse sand and gravel attached
to the undersides of rocks in the intertidal from Alaska
to southern California. Apart from the observation that
T. crispus spawns yellow-orange oocytes onto the sedi-
ment surface in July and August on San Juan Island,
Washington (Strathmann. 1987), the reproduction and
development of these three common species have re-
mained undescribed. The larval development of the fourth
species, Rame.\ calijorniensis. which is restricted in its
distribution to the central California coast, is known
(Blake, 1991). I have supplemented the observations of
Blake ( 1 99 1 ) to provide more information about the re-
productive biology of this species.

Reproductive mode

The reproductive mode refers to the degree of parental
care provided to fertilized eggs. Eggs may be free spawned
to give rise to planktonic larvae, or larvae may be brooded
either within the maternal tube or in a gelatinous mass
outside the tube. Each species was assigned a reproductive
mode according to observations made in the field or in
the lab.

Body size

Maximum body length of each species was recorded
from personal observations or from the literature. Length

TEREBELL1D REPRODUCTION AND DEVELOPMENT

155

was chosen as a measure of body size because of its cor-
relation with body volume (e.g., T. crispus: r = 0.88, //
= 17, P < 0.001). and because it is a parameter that is
often reported for terebellids in the literature.

Length of breeding season

Between April 1990 and May 1992, E. crescentis and
A', robusla were collected intertidally from Bodega Harbor.
California. Monthly samples were fixed in 10% seawater-
buffered formalin and preserved in 70% ethanol. The coe-
lomic contents from up to 10 females from each sample
were examined, and the maximum diameters of 80 oo-
cytes from each female were measured at 100X magni-
fication. A year-long time series of oocyte size frequency
histograms was constructed for each species to show the
pattern of oocyte development and likely breeding season
for the population. Oocyte diameter data were log trans-
formed before analysis of variance (ANOVA) to examine
the degree of synchrony among samples throughout the
year, and among females in each sample; Scheffes post
hoc test was used to clarify the reasons for any significant
differences (Sokal and Rolilf. 1981). The reproductive pe-
riod of each species was confirmed by spawning obser-
vations in the lab. and by qualitative observations of coe-
lomic contents during a second reproductive season.

R. califomiensis was sampled intertidally from Dillon
Beach. California, almost every month from August 1990
to February 1992. The worms were fixed and preserved
as described above, and oocyte size frequency histograms
were constructed for bimonthly intervals from August
1990 to June 1991. The number of oocytes in each female
was small, therefore all the oocytes found in each female
were measured. The presence of broods in tubes from the
monthly collections was also noted. Thelepus crispus was
observed in the field and collected from a variety of lo-
cations on San Juan Island. Washington, from July to
December. 1991. The presence of egg masses in the field
and in the lab was recorded during this time to estimate
the length of the reproductive period.

Maximum oocyte si:e and fecundity

Maximum oocyte size was recorded from coelomic
samples of mature females of each species, and estimates
of fecundity for E. crescentis and A', robusla were made
as follows. The number of oocytes in three 2-ml subsam-
ples from a 250-ml suspension of all oocytes removed
from weighed, mature females were counted. An estimate
of the total number of oocyles in each worm was then
extrapolated from the mean number in the subsamples.
To estimate fecundity in R. califomiensis. the number of
larvae were counted in all cocoons taken from five tubes
from each of three months (April. May. and June. 1991 );
as the cocoons of R. califomiensis are deposited as se-

quential broods rather than simultaneously, maximum
fecundity was recorded as the highest number of larvae
in a single cocoon, i.e.. the maximum number of larvae
in one brood. In T. crispus. a single brood consists of
paired, elongated egg masses that are attached to the ma-
ternal tube. Entire broods collected in the field in July
and October 1991 were blotted and weighed, and the
number of embryos in a weighed subsample of each brood
was counted. This number was extrapolated to give an
estimate of the number of larvae in an entire brood.

Mode of development

Several adults of E. crescentis spawned spontaneously
in seawater tables at Bodega Marine Laboratory, Califor-
nia, following collection on August 9, 1991. On August
13, other adults were placed in individual finger bowls in
0.2 ^m filtered seawater and induced to spawn by increas-
ing the seawater temperature by about 8C over one hour
in direct sunlight, followed by flushing with ambient tem-
perature seawater (~ 16C). Fertilizations were made in
0.2 /jm filtered seawater and larvae were maintained at
Long Marine Laboratory. California, at about 16C with
regular water changes. Settled larvae were transferred to
100 ^m filtered seawater. and raised in dishes that were
covered with microbial films.

Although individuals of A', robusla spawned occasion-
ally in the lab during May 1991. attempts to induce syn-
chronous spawning of males and females during two re-
productive periods (May. 1991: April and May, 1992)
failed. Development in R. califomiensis has been de-
scribed by Blake ( 1 99 1 ), and was also monitored in Oc-
tober and November. 1990. in this study. Embryos were
collected in the field, or from cocoons laid in the lab.
Worms and cocoons were maintained in small petri dishes
with some sand and nitex mesh in 5 ^m filtered seawater
at about 14C. For T. crispus. development was recorded
in October and November, 1991, from broods collected
on San Juan Island and raised at Friday Harbor Labo-
ratories, Washington, in 64 ^m filtered seawater at
about 11C.

Comparative data

Data on the reproduction and development of seven
other terebellids were collected from the literature. When
available, the following life history traits were recorded
for each species: (1) mode of reproduction, i.e., free
spawning, extratubular brooding or intratubular brooding;
(2) maximum body length (mm); (3) maximum diameter
(j/m) of coelomic oocytes; (4) maximum fecundity, i.e.,
maximum number of full grown oocytes in free spawning
species, or maximum number of larvae in a single brood
in brooding species: these data vary over several orders
of magnitude and therefore were log transformed for sta-

156

D. McHUGH

tistical analyses; (5) number of days larvae spend in the
plankton; (6) stage at juvenile, i.e.. the number ofsetigers
present on larvae when they begin to feed on the adult
diet; (7) duration of the breeding season (months); and
(8) breeding strategy, i.e.. iteroparous or semelparous. The
relationships between these traits in the family Terebel-
lidae were examined using a correlation matrix. ANOVA
was used to determine whether traits differed significantly
among the reproductive modes, and multiple regression
analysis was used to examine the covariation of some traits
when body size is held constant.

APR 90

Results

Present studv

Reproductive mode. Eiipolymnia crescent is and
Neoamphitrite robusta are both broadcast spawners, as
shown by their free-spawning behavior in the lab during
July and August, 1991, and May, 1991, respectively.
Moreover, no brooded larvae were ever found in the field.
Thelepus crispus and Rame.\ californiensis, on the other
hand, are both brooders. Females of T. crispus were col-
lected in the field with several elongated egg masses at-
tached to the interior of the tube. Ramex californiensis
sequentially lays small cocoons along the length of the
inner tube wall.

Body size. The maximum body length of the four spe-
cies ranges from 25 mm in R. californiensis to 280 mm
in T. crispus: E. crescentis measures up to 1 30 mm, and
N. robusta has a maximum length of 250 mm (Hartman,
1969; pers. obs.). Using the volume of a cone ('/airrh) as
an estimate of body volume, with body length = h and
body width = 2r, the order of body sizes among the four
species remains the same (R. californiensis: ~26 mm 3 ;
E. crescentis: ~3400 mm 3 ; N. robusta: ~1 1,000 mm 3 ;
T. crispus: ~~ 12,400 mm 3 ).

Length of breeding season. Figure 1 shows the oocyte
size frequency histogram for E. crescentis from April 1 990
to March 1991. Oocytes grow rapidly from about 50 ^trn
in April to begin to accumulate as full-grown oocytes of
about 180 //m in diameter from late May, until there is
a single peak around 185 ^m in August (Fig. 1). The an-
nual spawning period of E. crescentis at Bodega Harbor
is from July through September, as is indicated from the
histograms and confirmed by observations of spawning
in the lab; although no sample is available for September
1990, mature females were present in a sample taken in
September 1991. No coelomic oocytes were observed in
the post-spawning population in October 1990, and it is
not until January 1991 that proliferation of primary oo-
cytes begins again. Development from small primary oo-
cytes released into the coelom to full grown oocytes takes
from five to eight months in this species. ANOVA tests
show significant differences in mean oocyte diameter

10

20-

OCT90

10-

(

100

200 300
NOV90

20-

10-

o
c

100

200 300
DEC 90

20-

10-

o
c

50-
40
30
20

10

100

1

200 300

JAN 91
N= 10

100

200 300

50-

FEB91

40-

N = 10

30-
20-

10-

n .

1

100

200

300

100 200 300

MAR 91

20-

10-

100

200

300

OOCYTE DIAMETER (urn)

Figure 1. Monthly size-frequency histograms of maximum oocyte
diameter for Eiipolymnia crescentis from Bodega Harbor. California,
from April 1 990 to March 1 99 1 . No sample was available for September
1 990; 1 5 worms were examined in each sample from October, November,
and December, 1 990. but no gametes were seen. N, number of females
in each sample.

among females in monthly samples (e.g.. June, 1990: F
= 6.295, df = 8, P < 0.0001; July, 1990: F = 1 1.50, df
= 9, P < 0.0001; August, 1990: F = 12.34, df = 9. P
< 0.0001 ). However, multiple comparison tests reveal that
in August, 1990. the month in which the highest propor-
tion of full-grown oocytes are present in the population,
nine of the ten females did not differ significantly in their
mean oocyte diameter.

Neoamphitrite robusta shows a different pattern of re-
production in which oogenesis takes a full year, from May
to the following April, and mature females with large,
full-grown oocytes are present in the population from

II REBELL1D REPRODUCTION AND DEVELOPMENT

157

o

LLJ

tr

50-
40-
30-
20-

10-

50 100 150 200

AUG 90

100 150 200

fc OCT 90

'$, N = 5

- -^ 1 1 1

50 100 150 200

50 100 150 200 50 100 150 200

OOCYTE DIAMETER (urn)

Figure 2. Monthly size-frequency histograms of maximum oocyte
diameter for Neoamphilrite mhnsla from Bodega Harbor, California,
from July 1990 to June 1991. The bimodal peaks for May and June,
1991, and July 1990, reflect differences in oocyte size-frequency distri-
butions among worms in the samples, not within worms. No sample
was available for September 1990. N, number of females in each sample.

April through July (Fig. 2). This breeding season coincides
with the spawning of some individuals in the lab during
May 1991. In the oocyte size frequency histograms for
July 1990 through June 1991, July 1990 represents the
end of the spawning period. In that sample, a single mature
female accounts for the peak of large oocytes, and three
other worms in the sample contain a few small oocytes.
By August 1990, only two worms out of nine examined
contained any gametes, and they each had only very early

stage oocytes. Proliferation of primary oocytes continues
in the population from May to March, with steady growth
from October into full grown oocytes and spawning the
following April through July (Fig. 2). ANOVA tests show
significant differences in mean oocyte diameter among
females in the monthly samples (e.g.. October 1990: F
= 1 3.70, df = 4, P < 0.000 1 ; February 1 99 1 : F = 1 2.24,
df = 9, P < 0.0001; June 1991: F = 109.80, df = 6, P
< 0.0001), and multiple comparison tests confirm that
oogenesis is not tightly synchronized among females at
any time throughout the year. Bimodal distributions of
oocytes appear in the May and June 1991 samples, and
also in the July 1990 sample (Fig. 2). However, in each
case, the peak of smaller oocyte sizes is fully accounted
for by two or three females that have only 10-30 ^m
diameter oocytes in the coelom.

In R. californiensis. bimonthly samples of females from
Dillon Beach all show a wide range of oocyte sizes
throughout the year; in each sample there is skewing to-
wards smaller oocytes, with just a small proportion of
oocytes > 300 ^m in diameter present (Fig. 3). Intratu-
bular cocoons of larvae of R. californiensis were found in
samples from every month sampled between June 1990
and February 1992; in many cases females were still with
the broods, and they always contained oocytes of various
sizes in their coeloms. While the larvae within each cocoon
were at the same development stage, the stages of larval
development differed among cocoons in a tube, indicating
that the cocoons were laid sequentially.

AUG 90

FEB91
N = 5

100 200 300 400

15i

o

LU

rr

15-1

; JL

l_jiPfc*fc^^

100 200 300 400

100 200 300 400

APR 91

DEC 90
N = 5

15-

10
5 -

100 200 300 400

JUN91
N = 5

100 200 300 400

100 200 300 400

OOCYTE DIAMETER (urn)

Figure 3. Bimonthly size-frequency histograms of maximum oocyte
diameter for Ranwx californiensis from Dillon Beach, California, from
August 1990 to June 1991. N, number of females in each sample.

158

D. McHUGH

Table I

Maximum caelomic oocyle diameter and maximum fecundity
recorded for Eupolymnia crescentis, Neoamphitnte robusta, Ramex
californiensis, and Thelepus crispus

Maximum Maximum fecundity*

oocyte size (^m) (n; X S.E.)

Eupolymnia crescentis

210

128,500

(6; 105,430 12,951)

Neoamphitnte robusta

180

829,833

(5; 404.799 109,338)

Ramex californiensis

410

44

(15:29 2.4)

Thelepus crispus

400

51,555

(4; 28,582 11,796)

* Fecundity in E. crescentis and N. robusta is the total number of
oocytes in gravid females; fecundity in R. californiensis and T. crispus
is the number of larvae in a single brood, i.e.. a single cocoon in R
californiensis. or a single mass of elongated egg sacs in T. crispus: n,
sample size; X S.E., mean standard error.

Intratubular egg masses of T. crispus were observed in
the field in July, October, and November of 1991, and
have also been reported in August (Strathmann, 1987).
Egg masses were also laid in the lab in November and
December of 1 99 1 . It appears then that T. crispus breeds
for at least six months from July to December, and per-
haps longer.

Maximum oocyte size and fecundity. In all four species,
oocytes change from a spherical to a discoid shape as they
develop in the coelom, and they round out again when
spawned. Table I shows the maximum oocyte diameter
recorded from coelomic samples of each species. Ramex
californiensis and T. crispus have the largest oocyte sizes,
at 410 and 400 ^m. respectively, while N. robusta has the
smallest (180 ^m); the maximum oocyte size in E. cres-
centis is 210 /urn. The maximum fecundity ranges from
44 in a single cocoon of R. californiensis. to 829,833 full-

grown oocytes in a mature N. robusta female, with T.
crispus and E. crescentis having maximum fecundities of
51,555 and 128,500, respectively (Table I). The high de-
gree of variation in fecundity of each species is due to
body size differences among females in each sample. In
Table II total spawn or brood volume is expressed as a
percentage of total body volume for the four species. This
crude estimate shows that reproductive output (percent
of total body volume given to a single brood or spawn)
in the worms ranges from about 2.37% in R. californiensis
to about 8.60%- in N. robusta. E. crescentis and T. crispus
have values of about 6.67% and 5.88%, respectively.

Mode of development. Table III summarizes the larval
development of E. crescentis at about 16C. Twenty-four
hours after fertilization, the larva is about 150 ^m long,
fully ciliated and free swimming, and has two red eyespots.
By about 48 h, the ~200 /im-long trochophore stage is
reached; the larva possesses an apical tuft, a wide proto-
trochal band, a telotroch, and a neurotroch. During the
third day of development, surface constrictions indicate
the onset of segmentation, and the first pair of hooded
setae appear. By the end of the third day, larvae possess
a pair of hooded setae on each of two segments. Setae and
setigers are continually added as outlined in Table III.
The ciliated mouth appears when the larvae are about 5
days old and approximately 350 fj.m long. At this stage
the gut outline is already visible, but yolk granules are
still found in the gut and body cavity, and it is not until
the larva is about 7 days old that the mouth and gut be-
come fully functional. At this stage the larva possesses
five setigers, the posterior four of which bear a pair of
uncini. The telotroch is lost and the neurotroch is reduced
to a ciliary patch behind the mouth. Up to this point in
their development the larvae have been mainly near the
bottom, and they are not very active swimmers. After this
stage, the juveniles crawl around, feeding on microbial
films on the bottom of the culture dishes. By 21 days the
first tentacle bud is 100 ^m long, there are eight setigers,
and the first pair of nephridia has begun to develop.

Table II

Estimates of reproductive output for Eupolymnia crescentis, Neoamphitnte robusta, Thelepus crispus. and Ramex californiensis

Single oocyte
volume 1 " (mm 3 )

Total oocyte
volume 11 (mm 3 )

Total body
volume' (mm 3 )

Total oocyte volume

Total body volume
(%)

Eupolymnia crescentis
.Yiv >amphitrite robusta
Runicx californiensis
Thelepus crispus

1.766 x 10~ 3
1.150 x 10~ 3
14.137 X 10~ 3
14.137 X 10"-'

2.270 x 10-
9.544 ; 10 :
0.622
7.288 x 10 2

3.403 x 10 3
1 1.062 .- 10 J
26.180
12.389 x 10 3

6.67
8.60

2.37
5.88

event

w

Reproductive output is expressed as the percentage of total body volume that is given to free-spawned or brooded eggs in a single reproductive
ent. a = 4 / 3 jrr 3 , where 2r is the diameter of a spawned, spherical oocyte in each species; b = a * fecundity in each species (see Table I); c = '/jir'k.

. rj , __ _ r _ ------ r ------

here 2r is the maximum width, and h is the maximum length ot each species.

TEREBELLID REPRODUCTION AND DEVELOPMENT

159

Table HI

Development ol EupoKmnia crescentis larvae raised at approximately 16C in August 1991

Days alter
spawning

Stage of development

1 150 Mm ciliated larva: 2 red eyespols appear.

2 200 jim long trochophore larva with apical tuft, a wide prototroch and telotroch: neurotroch also present, and intersecting the

telotroch; first segmental constriction appears.

3 250 ftm long larva with 1 or 2 setigers. each with a single pair of capillary setae.

4 ~300 ftm long larva with 2 setigers, the first with 2 pairs capillary setae and the second with a single pair.

350 Mm long larva with 3 setigers. the first 2 with 2 pairs of capillary and the third with a single pair of capillary setae; prototroch and

telotroch are reduced.
6 350 urn long larva with 4 setigers. the first 3 with 2 pairs of capillary setae, and a single pair of uncini on setigers 2 through 4:

neurotroch beginning to disappear posteriorly.
~-500 yum long juvenile with 5 setigers (4 with uneini) and a fully functional gut; telotroch almost gone and neurotroch further

reduced to ciliary patch behind mouth; animals stick to the bottom or sides of the container and slowly sink passively if disturbed.
1 1 Six setigers. all but the first with a single pair or uncini; all larval ciliation gone.

21 -~950 fim long juvenile with 7 to 8 setigers. and the first tentacle bud ( 100 /jm long); heavily ciliated lips everted often in feeding;

first pair of nephridia visible in the first asetigerous segment.

The development of T. crispus is summarized in Table
IV. In this species, larvae hatch from the intratubular egg
mass as 350 ,um long, one-setiger larvae. While hatching
from an egg mass is not tightly synchronized among all
larvae, newly hatched larvae are usually at this stage of
development. The prototroch. telotroch, and neurotroch
are well developed, and there are two eyespots; there is
no apical tuft and segmental ciliary bands are absent. Up
to the second day post-hatching, larvae have limited
swimming abilities, and thereafter they remain on the
bottom of the container. The second setiger develops on
the third day, and by day four there are three setigers. At
7 days, the prototroch and telotroch have been lost from
the 4-setiger larvae, and the neurotroch begins to recede
from the posterior end leaving only segmental patches of
cilia by day 12. At this stage there are five setigers, each
with two pairs of capillary setae. In eight-setiger stages the
gut is fully functional and the juveniles form mucus tubes
to which sediment particles adhere.

Observations of development in R. californiensis in the
present study are generally similar to those reported by
Blake (1991); larvae undergo direct development, and
emerge as eight-setiger, crawl away larvae with three to
five tentacles. The mouth and gut become functional at
the 11- to 12-setiger stage, approximately 15 days after
hatching.

Comparative data

Data on reproductive and developmental traits of seven
other terebellid species were available in the literature.
Combined with the data from the present study, the traits
of three intratubular brooders, three extratubular brood-
ers, and six broadcast spawners are represented in Table
V [one species, Eupolymnia nebulosa, is reported as an
extratubular brooder in the Mediterranean and as a
broadcast spawner in the English Channel (Gremare,
1986; Bhaud ct a/.. 1987)]. There are 1 1 species from 9
genera represented in the matrix.

Table IV

Development <>/ Thelepus cnspus larvae raised at approximately 14 C in October and November 1991

Days after

hatching

Stage of development

1 ~350 /um long larva with a well-developed prototroch, telotroch, and neurotroch; 2 red eyespots; the first segmental constriction and

the first pair of hooded setae develop.
3 Larvae remain on the bottom of the container, the second segmental constrictions appear and the second pair of capillary setae

develop on the first setiger.

~450 Mm long larvae with 3 setigers; tentacle bud beginning to develop.
~500 /jm long larva with 4 setigers. the first 2 with 2 pairs of capillary setae and a single pair of setae on the third and fourth setigers;

prototroch and telotroch are gone, and the neurotroch is receding from the posterior end.

12 Five setigers. each with 2 pairs of capillary setae; neurotroch reduced to segmental patches; first tentacle increasing in length.

26 ~800 Mm long juvenile with functional gut; 8 setigers. 5 with a pair of uncini: tentacle now ~ 100 Mm long, but food ingested by

evening lips.

160

D. McHUGH

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TEREBEl LID REPRODUCTION AND DEVELOPMENT

161

400

I 300-

0)

<7> 200-

m 100-

Intratubular Extratubular Broadcast
brooders brooders spawners

Figure 4. The reproductive modes of terebellids graphed against body
size (length in mm).

Size ranges from 23 to 380 mm among all species, with
no significant difference in body size among reproductive
modes (F = 0.51 8, df= 1 1, P < 0.612). Indeed, as Figure
4 illustrates, each of the three reproductive modes are
represented in species of vastly differing body sizes.
ANOVA reveals a statistically significant difference in the
maximum oocyte size among the three reproductive
modes (F == 19.33, df = 1 1. P < 0.0006). The smallest
oocyte sizes are associated with the six broadcast spawners,
while the largest ones are those of species that brood their
larvae in the maternal tube. Fecundity ranges over several
orders of magnitude among the species in the study, with
the two extremes being R. califomiensis (44) and A', m-
busta (829.833). While log of fecundity does not differ
significantly among the three reproductive modes (F
= 3.039, df = 9. P < 0. 1 1 2). it does differ between brooders
with direct development and those with a planktonic stage
during development (F = 108.03. df = 5, P < 0.009 1 ).

Three of the species in the study undergo direct devel-
opment, and in the five species for which planktonic de-
velopment is known, the larvae remain in the plankton
from 1 to about 7.5 days. The length of the planktonic
period does not differ according to reproductive mode (F
= 3.07. df = 7, P < 0.134). but the stage at which the
larval gut becomes fully functional and exogenous food

supplies are taken in (i.e., number of setigers at initial
juvenile stage) does (F = 6.97, df = 8, P < 0.027). Larvae
from intratubular brooders become independent from
yolk supplies at a more developed stage (up to 11 setigers
in R. califarniensis) than either extratubular brooders (5-
10 setigers) or broadcast spawners (2-5 setigers).

Free-spawning species and extratubular brooders gen-
erally have short, discrete breeding seasons, while two of
the intratubular brooding species reproduce continuously
for extended periods. However, the length of the breeding
season does not differ significantly according to repro-
ductive mode (F = 2.25, df = 10. P < 0.167). Only two
species, Nicolea lostericola andAmaeana occidentalis, are
reported to be semelparous; six others are iteroparous.

The matrix of correlation coefficients for the life history
traits is shown in Table VI. It shows that fecundity covaries
directly with body size, and inversely with egg size. How-
ever, there is no significant relationship between body size
and oocyte size, and multiple regression analysis shows
that egg size does not predict fecundity if body size is held
constant (F ( 2. 5) = 3.390, P < 0. 1 1 7). Juvenile stage covaries
directly with egg size, and inversely with the length of the
planktonic period during development. While the rela-
tionship is not significant (P < 0.054), there is a definite
trend towards decreasing planktonic period with increas-
ing egg size. No other correlations among traits are sta-
tistically significant.

Discussion

Present slut/]'

The four species in the present study span the range of
reproductive and developmental modes recorded for the
family Terebellidae. Eupo/ymnia crescentis and Neoam-
philrite robust a are both free-spawning terebellids with
reproductive periods lasting approximately three months.
Interestingly, these two co-occurring species differ in their
timing of primary oocyte release, the pattern of oocyte
growth, and the timing of spawning. Environmental
stimuli control primary oocyte release and rate of oocyte

Table VI

Correlations between reproductive and developmental trans among v/)cr/i"> of the family TcrcMlulue

Body size

Maximum
oocyte size

Maximum
fecundity

Days in
plankton

Stage at
juvenile

Breeding season

Bods size
Maximum oocyte size
Maximum fecundity
Days in plankton
Stage at juvenile
Breeding season

1
-0.449
0.747

1
-0.743

1
0.671
-0.581
-0.523

1
-0.778
-0.389

1
0.570

1

0.106
0.053
-0.177

-0.699
0.698

0.312

P < 0.05 for underlined values.

162

D. McHUGH

growth in some polychaetes (see Olive, 1984). However,
without further study, it is not possible to speculate on
how the same environmental stimuli affect E. crescentis
and N. robusta so differently. While reproduction in E.
crescentis and N. robusta populations is not tightly syn-
chronized, fertilization efficiency in these species could
potentially be enhanced by the stimulation of conspecifics
by spawning individuals, as apparently happened with E.
crescentis in the lab in August, 1 99 1 . Such an effect would
presumably be greater in E. crescentis, which reaches
densities of 3 animals per 10 cm 2 at Bodega Harbor; N.
robusta is patchy in its distribution, usually occurring in
small groups of several adults.

The maximum egg sizes in E. crescentis and N. robusta,
210 and 180 nm, respectively, are within the range as-
sociated with lecithotrophy in polychaetes generally
(Schroeder and Hermans, 1975), and are very similar to
those reported for other free spawning terebellids, Eu-
polymnia nebulosa (Gremare, 1986) and Amaeana occi-
dentalis (Hannan el at., 1977). The number of eggs pro-
duced by E. crescentis in a single reproductive period is
substantially larger than that of A. occidentalis. which has
a similar body size and egg size (Fauchald, 1983). Fecun-
dity of N. robusta is very high compared to reports for
other terebellids, and other lecithotrophic polychaetes
(Gremare and Olive, 1986), although it should be noted
that fecundity is rarely reported for large, free-spawning
species.

Larval development in E. crescentis is planktonic and
non-feeding, and corresponds generally with the primary
planktonic larval development of Lanice conchilega
(Heimler, 1981). Like L. conchilega, E. crescentis has a
demersal metatrochophore, but E. crescentis larvae are
not good swimmers. Although the dispersal of E. crescentis
larvae over the seven-day planktonic period observed in
this study might be expected to be substantial, it may be
restricted due to the habitat of the species. Like many
terebellids (Day, 1967; Bhaud, 1991), E. crescentis is found
in quiet bays where fine sediments are deposited, areas in
which offshore/alongshore currents may not exert great
influences on larval dispersal. The common high popu-
lation densities of E. crescentis (e.g.. Bodega Harbor, Cal-
ifornia, and South Slough, Oregon) could result from the
retention of larvae in the parental habitat. While attempts
to raise larvae of TV. robusta during the present study were
unsuccessful, it is predicted, with a maximum egg size of
180 ^m and broadcast spawning, that N. robusta under-
goes development similar to E. crescentis, with a com-
parable planktonic period.

The two brooders in the study, Ramex californiensis
and Thelepus crispus, both brood their larvae in the ma-
ternal tube. These two species represent the extremes in
body size in this study, 25 and 280 mm, respectively, and
the presence of brooding in a worm as large as T. crispus

is unexpected (see discussion below). Despite the great
differences in body size, these two species share a number
of life history traits. Both reproduce continuously, R. cal-
iforniensis year round and T. crispus for at least six
months. Continuous year-round breeding has never been
reported in a terebellid before, although it is known in
other brooding polychaetes, for example, the ampharetid
Hypaniola kowalewskii (Marinescu, 1964). The extratu-
bular brooding terebellids, Nicolea zoslericola. Eitpolym-
nui nebulosa, and Thelepus seto.sus breed over three to
four months, but only a single batch of oocytes is produced
annually in these species (Eckelbarger, 1975; Bhaud et al.,
1987; Duchene. 199 1 ). This contrasts with R. californien-
sis in which females with broods of larvae have oocytes
of all developmental stages present in the coelom at all
times of the year. While oogenesis in T. crispus was not
monitored from month to month, those females found
with broods also had oocytes of all sizes in the coelom.

Ramex californiensis and T. crispus have very similar
maximum oocyte sizes, 410 and 400 ^m, respectively.
Such large eggs are typical of brooding polychaetes
(Schroeder and Hermans, 1975) and of some brooding
terebellids; however, they are substantially smaller than
the maximum egg size of 600 /urn reported by Duchene
( 1980) for the terebellid, Neoleprea streptochaeta. Fecun-
dity in R. californiensis, 44, is very small. Nicolea zoster-
icola. which is approximately the same size as R. califor-
niensis but produces eggs of 300 /urn, has a maximum
fecundity of 665 for its single, discrete spawning period
(Eckelbarger, 1974). Nonetheless, the number of larvae
produced by these two species in a lifetime may be com-
parable, because R. californiensis can have up to 1 1 co-
coons at any one time, and apparently lays additional
cocoons over time. The fecundity of T. crispus, 51,555,
is very high compared to other large intratubular brooding
polychaetes, like some sabellids (G. Rouse, pers. comm.).
It is much smaller than the fecundity of large free-spawn-
ing species in the study, TV. robusta, but the reproductive
output of T. crispus is closer to the two free-spawning
species in the study than to R. californiensis (see Table
II). Pairing of males and females prior to reproduction,
as has been described in N. lostericola (Eckelbarger, 1974),
has not been observed in either R. californiensis or T.
crispus. However, some special mode of sperm transfer
seems necessary in these two species to accommodate fer-
tilization of eggs before cocoon or egg mass formation
within the tube. In R. californiensis, this speculation is
supported by the presence of elongated sperm (~ 10 ^m:
pers. obs.).

Despite their similar egg sizes, R. californiensis and T.
crispus differ substantially in their dispersal potentials.
Ramex cu/i/orniensis undergoes direct development with
no planktonic stage, whereas T. crispus has a mixed mode
of development with brooded larvae that undergo a short-

TEREBELLID REPRODUCTION AND DEVELOPMENT

163

lived planktonic stage. Development in R califomiensis
most closely resembles that of N. streptochaeta, in which
juveniles of 10 setigers emerge from the maternal tube
after a lengthy period of intratubular development
(Duchene, 1980). The 1 1-setiger juveniles of R. ca/ifar-
niensis can build mucus tubes and feed on detrital matter;
they avoid whatever mortality risks of planktonic devel-
opment there might be. The rate of post-settlement mor-
tality in either species is unknown, however, in R. cali-
fomiensis at least, it is unlikely that there are any negative
intraspecific interactions between adults and juveniles as
seen in E. nebulosa (Bhaud, 1990, 1991), because of the
small adult size in this species and the tubicolous habit
of newly emerged juveniles.

The mixed development of T. crispus is similar to that
of T. setosus and E. nebulosa (Bhaud and Gremare, 1 988;
Duchene, 1991). In both species of Thelepus, the plank-
tonic period of development is short ( ~ 1 day), and T.
crispus larvae, at least, are poor swimmers. This may re-
strict the dispersal of the larvae, effectively isolating dif-
ferent populations of the species. The population genetics
of T. crispus, R. califomiensis, and E. crescentis, species
with larval planktonic periods ranging from to 7 days,
are being investigated to reveal whether the degree of ge-
netic exchange among populations of each species cor-
responds with their differing dispersal potentials.

Comparative data

Correlation analyses of the matrix of reproductive and
developmental traits for the 1 1 species in the study reveal
several expected trends, but also a few that are contrary
to widely accepted generalizations regarding the covari-
ation of life history traits. Egg size is an important life
history trait; it is an indicator of maternal investment per
offspring, is easily measured, and is correlated with several
other life history traits in marine invertebrates. Thorson
( 1950) reported that egg size is correlated with fecundity
and development mode in several marine invertebrate
groups, and these trends have also been noted for poly-
chaetes in general (Schroeder and Hermans, 1975), and
some polychaete families in particular (e.g., Sabellidae:
McEuen et ai, 1983). Egg volume ranges over two orders
of magnitude among the Terebellidae, and the relationship
between egg size and mode of reproduction has been noted
for several terebellid species in the past (Blake, 1991;
Bhaud, 1988). However, this is the first study in which a
significant difference in egg size among reproductive
modes has been demonstrated for this or any polychaete
family. Egg size increases with the degree of parental care
provided to the larvae, with intratubular brooders having
the largest eggs and free-spawning species the smallest. In
echinoderms, egg size is generally correlated with energetic
content (Emlet et ai, 1987), however, no studies on the

energetic content of terebellid eggs have been published.
Nonetheless, from the observations of this study it is ex-
pected that egg size (i.e., oocyte diameter) is also correlated
with energetic content among terebellid species.

It is usually assumed that for organisms of comparable
size, smaller eggs are associated with increased fecundity,
and vice versa (Olive et al., 1984). While egg size is a
commonly reported variable in studies on reproduction
and development, data on fecundity in many marine in-
vertebrate groups are not available, and the trade off be-
tween these two life history traits has not often been in-
vestigated. The present study is the first in which fecundity
of different polychaete species is compared with other re-
productive traits. Initial correlation analysis indicates that
the trade off between egg size and fecundity is as expected
among terebellids; however, multiple regression analysis
shows that egg size does not predict fecundity if body size
is held constant. In a regression analysis of life history
traits in oysters, Buroker (1985) found no significant re-
lationship between egg size and fecundity. These results
demonstrate that generalizations regarding the relation-
ship between these two traits are not appropriate until
further comparative investigations in other groups are
made; the relationship is obviously complex, and may be
linked to reproductive effort or larval survivorship, neither
of which are well known for polychaetes.

All terebellids in the study undergo external fertiliza-
tion, i.e., copulation is absent, and there is no evidence
of self fertilization in any terebellid species, although rarely
hermaphrodites have been observed (e.g., Ramex califor-
niensis. pers. obs.; Pista pacifica. R. I. Smith, pers.
comm.). In Nicolea zostericola, which broods its embryos
in an extratubular cocoon, males and females pair before
spawning and sperm is gathered in the short oral tentacles
of the female before mature eggs are passed over the ten-
tacles for fertilization (Eckelbarger, 1974). This is the case
also in the intratubular brooder, Neoleprea streptochaeta
(Duchene, 1980), but aot-Eupolymnia nebulosa. in which
eggs in an extratubular gelatinous mass are fertilized by
free sperm in the water (Smith, 1989a). Unfortunately,
sperm ultrastructure, which is generally indicative of fer-
tilization mechanism in polychaetes (see Jamieson and
Rouse, 1989), has been reported for only one terebellid
species, N. zostericola (Eckelbarger, 1975). Additional
studies of this aspect of terebellid reproduction would
provide more insights into the fertilization biology of this
group.

While terebellids display a great variety of reproductive
modes, one feature shared by the species in this compar-
ative study is the use of endogenous nutrient supplies dur-
ing larval development. Two terebellid species feed in the
plankton, but this occurs during a secondary planktonic
stage after metamorphosis (aulophore stage) (Wilson,
1928; Heimler, 1981; Bhaud. 1988). Newly metamor-

164

D. McHUGH

phosed juveniles of Lanice conchilega and Loimia medusa
engage in this activity, which is functionally equivalent
to larval feeding, i.e., it involves ingestion of plankton
and potential increased dispersal during the planktonic
feeding period, but morphologically it is a very different
process; larval cilia are not used in feeding or in loco-
motion during the planktonic stage (Heimler. 1981;
Bhaud. 1988). Therefore, the planktonic stage must be
considered analogous rather than homologous to plank-
totrophic larva (Bhaud, 1988).

In marine invertebrates generally, planktotrophic larval
development has been proposed as the common ancestral
condition (Jagersten, 1972; Strathmann, 1978). The evo-
lutionary loss of larval feeding involves the loss of ciliary
feeding bands and sometimes the whole larval gut, and
the probability of regaining these feeding structures is
thought to be so unlikely that a biased transition from
planktotrophy to lecithotrophy is expected (Strathmann,
1985). The descendents from a lineage from which larval
feeding forms have been lost will be restricted in their
options for development modes (Strathmann, 1978,
1985). Once development is constrained, the evolution
of other life history traits like egg size and fecundity may
also be affected. The present study emphasizes the absence
of larval feeding in terebellids, a well-defined polychaete
family that is found in all marine habitats. Assuming the
monophyly of terebellids, lecithotrophy apparently rep-
resents a fixed trait in this group. This contrasts with the
occurrence of both planktotrophy and lecithotrophy in
other polychaete families (e.g., Spionidae, Cirratulidae,
Nereidae, Onuphidae, Dorvilleidae, and Serpulidae)
(Wilson, 1991), and suggests that development has been
constrained during the evolution of the terebellid lineage.
Alternatively, lecithotrophy may be selectively advanta-
geous in terebellids; planktotrophy may be an option that
has never been favored. This hypothesis seems implausible
given the diversity of habitats occupied by members of
the family Terebellidae.

No studies on field mortality rates of terebellid larvae
have been published, although Bhaud ( 1991 ) reported low
mortality rates ofEupolymnia nehulosa larvae under lab
conditions. Terebellid larvae lack defensive structures seen
in some polychaete larvae (Bhaud and Cazaux, 1982;
Pennington and Chia, 1 984), and they are poor swimmers,
in which buoyancy appears to play a major role in the
maintenance of a planktonic existence (Nyholm, 1951;
pers. obs.). These features of terebellid larvae suggest that
they are probably vulnerable to predation in the plankton,
with increasing size during development being their only
protection. Brooding of larvae is presumably less hazard-
ous than planktonic development, and in terebellids the
fertilization efficiency among brooders is thought to be
substantially higher than in broadcast spawning species
(Eckelbarger, 1974; Duchene, 1980). Mixed development

involves brooding followed by a planktonic larval period
(Pechenik, 1979). Initial brooding likely increases fertil-
ization efficiency and provides protection for the early
stages. The maintenance of a planktonic stage, despite the
possible increased mortality risks, suggests some selective
advantage associated with dispersal or some disadvantage
associated with no dispersal; alternatively, functional
limits (e.g., space, oxygen requirements) may prohibit
brooding of larvae beyond certain stages. In any case, the
significantly higher fecundities among terebellids with
mixed development compared with those with direct de-
velopment may compensate for any increased mortality
risks associated with planktonic stages.

Juvenile stage (number of setigers), which differs sig-
nificantly among reproductive modes, is significantly cor-
related with egg size in terebellids. As larval development
in terebellids is apparently dependent on yolk supplies,
this relationship between egg size and juvenile stage is not
surprising; in the absence of planktonic feeding, larger
eggs will give rise to larger, more developed juveniles
(Strathmann, 1985). Interestingly, there is no correlation
between the number of days during development spent
in the plankton and the initial egg size. This is also the
case in some cirripedes (Barnes and Barnes, 1965), and
it follows the prediction of Strathmann (1977) that the
larval period will be independent of egg size, if size at
metamorphosis increases with increasing egg size.

If, as previously mentioned, brooding of larvae is the
safest mode of development, then all other things being
equal, all marine invertebrates would brood their larvae
(Emlet el ai, 1987). However, among related species, if
any of them brood it is usually only the smaller ones
(Strathmann and Strathmann, 1982; Strathmann et at.,
1984). Numerous studies have confirmed this association
of brooding with small body size in a wide variety of
groups, including echinoderms (e.g., Menge, 1975), mol-
lusks (Pearse, 1979; Sastry, 1979), and some polychaetes
(Knight-Jones and Bowden, 1984). Various hypotheses
have been invoked to explain this pattern, and the one
based on allometric constraints has been supported by
several studies (Strathmann and Chaffee, 1984; Strath-
mann et a!.. 1984; McClary and Mladenov, 1989). In or-
ganisms of increasing size, fecundity increases dispropor-
tionately with the surface area available for brooding,
therefore larger animals are less capable of retaining and
ventilating all the offspring that they can produce; small
animals are less likely to encounter this problem (Strath-
mann and Strathmann, 1982). Terebellids, however, do
not support this hypothesis. Indeed, the terebellids in the
present study provide a clear exception to the generaliza-
tion that large body size may be incompatible with brood-
ing in marine invertebrates. One of the largest species in
the study, Thelepm crixpus, broods its embryos in its tube,
as does one of the smallest species in the study, Ramex

TEREBELL1D REPRODUCTION AND DEVELOPMENT

165

californiensis. While reproductive output is expected to
be limited by space in large, tubicolous polychaetes (Hines.
1986), there does not appear to be a substantial limitation
in terebellids when large broadcast spawners are compared
with a large intratubular brooder in the present study (see
Table II). It is possible that large terebellids, and perhaps
other sedentary polychaetes, can overcome the suggested
constraints of brooding by (i) having a tube that has greater
surface area than the body (i.e., longer, wider), or (ii) by
ventilating the tube well with peristalitic contractions of
the body, as has been described by Dales ( 196 1 ) for several
terebellid species. In some other marine invertebrates,
scaling constraints do not provide adequate explanation
for the association of brooding with small size; Hess (1993)
found no evidence of allometric contraints on brood size
in spirorbid polychaetes (1-3 mm in length), and Rabat
(1985) showed that brood area does not constrain repro-
ductive output in the small bivalve, Transenella tantilla
(6 mm shell length).

From the matrix of reproductive and developmental
traits, it is clear that brooders show wider ranges of values
for all traits than broadcasting species (Table V). This
probably reflects the grouping of species with functionally
similar reproductive modes that are of different evolu-
tionary origins. Only with a phylogenetic analysis of the
family will it be possible to address such issues. With such
an analysis we can also begin to evaluate how much of
the variation in life history traits is due to phylogenetic
effects, and how much is the result of adaptation to dif-
ferent selective environments, as has already been done
for some vertebrate groups (e.g., Harvey and Clutton-
Brock, 1985;Gittleman, 1986; Miles and Dunham, 1992).
Although biases in the data are likely, due to the nonran-
dom way in which subjects were chosen, and the phylo-
genetic effects on life history traits can only be speculated
upon for now, the results of this study nonetheless show
that generalizations about the covariation of life history
traits in marine invertebrates need to be viewed with cau-
tion. The exceptions to some commonly held assumptions
provided by the Terebellidae show that more comparative
studies of marine invertebrate reproduction and devel-
opment are needed to broaden our base for life history
theories.

Acknowledgments

This paper is dedicated to the memory of Dr. Ralph I.
Smith, who introduced me to the terebellids of the central
California coast. I am grateful to him, and also to Dr.
John S. Pearse for their support, advice, and encourage-
ment throughout this study. I thank them, Drs. G. W.
Rouse and P. D. Reynolds, and two anonymous reviewers
for critical reviews of the manuscript. Dr. G. Griggs, Di-
rector, Long Marine Laboratory and Institute of Marine

Sciences, University of California, Santa Cruz; Dr. J.
Clegg, Director, Bodega Marine Laboratory; and Dr.
A. O. D. Willows, Director, Friday Harbor Laboratories,
graciously provided the use of those facilities. I thank J.
Kurpius for assistance with oocyte measurements, B.
Steele for help with larval culture and maintenance, G.
Pierce for advice on statistics, and S. Edmands, P. Fong,
J. Kurpius, E. McHugh, C. E. Mills, P. D. Reynolds,
R. I. Smith, and P. Wolfe for accompanying me on field
trips. Some collection of data and revisions of the manu-
script were made while the author was a Scholar-in-Res-
idence at Hamilton College. Financial support from the
following sources is gratefully acknowledged; Biology In-
dependent Study Funds, University of California, Santa
Cruz; the Society for Sigma Xi; the Dr. E. H. and E. M.
Myers Oceanographic and Marine Biology Trust; the
Friends of Long Marine Laboratory; and the Lerner-Gray
Fund for Marine Research (American Museum of Natural
History).

Literature Cited

Barnes, H., and M. Barnes. 1965. Egg size, nauplius size, and their
variation with local, geographical, and specific factors in some com-
mon cirripedes. J. Anim. Ecol. 34: 391-402.

Bhaud, M. 1988. The two planktonic larval periods of Lanke conchilega
(Pallas. 1766) Annelida Polychaeta, a peculiar example of the irre-
versibility of evolution. Ophelia 29(2): 141-152.

Bhaud, M. 1990. Conditions d'establissement des larves de Eupolymnia
nebulosa: acquis experimentaux et observations en milieu natural;
utilite d'une confrontation. Oceanis 16(3): 181-189.

Bhaud, M. R. 1991. Larval release from the egg mass and settlement
of Eupolymnia nebulosa (Polychaeta, Terebellidae). Bull. Mar. Sci.
48(2): 420-431.

Bhaud, M., and C. Cazaux. 1982. Les larves de polychetes des cotes
de France. Oceanis S: 57-160.

Bhaud, M., and A. Gremare. 1988. Larval development of the terebellid
polychaete Eupolymnia nebulosa (Montagu) in the Mediterranean
Sea. Zoo/. Scripta 17(4): 347-356.

Bhaud, M., A. Gremare, F. Lang, and C. Retiere. 1987. Etude com-
parees des caracteres reproductifs du terebellien Eupolymnia nebulosa
(Montagu) (Annelide Polychete) en deux points de son aire geogra-
phique. C. R Acad. Sci. Pans Serie 111 304(5): 1 19-122.

Blake, J. A. 1991. Larval development of Polychaeta from the northern
California coast V. Ramcx calijorniensis Hartman (Polychaeta: Ter-
ebellidae). Bull. Mai: Sa 48(2): 448-460.

Buroker, N. E. 1985. Evolutionary patterns in the family Ostreidae:
larviparity v.v. oviparity. J. Exp. Mar. Biol Ecol. 90: 233-247.

Chia, F-S. 1974. Classification and adaptive significance of develop-
mental patterns in marine invertebrates. Thallassia Jugoslav. 10: 121-
120.

Christiansen, F. B., and T. M. Fenchel. 1979. Evolution of marine
invertebrate reproductive patterns. Theor. Pop. Biol. 16: 267-282.

Dales, R. P. 1961. Oxygen uptake and irrigation of the burrow by
three polychaetes: Eupolymnia, Tlielepus and Neoamphilrite. Physiol.
7.ool. 34: 306-311.

Day, J. II. 1967. A Monograph on ihc Polychaeia of Southern Africa.
British Museum of Natural History Publications. London.

Doyle. R. \\ '., and \V. I hum 1981. Genetic changes in "fitness" and
yield of a crustacean population in a controlled environment. J Exp.
Mar. Bio. Ecol. 52: 147-156.

166

D. McHUGH

Duchene, J. C. 1979. Premieres donnees sur la reproduction et la

croissance de la polychete Thelepus setosus (Terebellidae) en province

subantarctique. Ann. Inst. Oceanogr. Paris 55(2): 145-154.
Duchene, J. C. 1980. Premieres donnees sur la reproduction at la

croissance de la polychete Neoleprea streptochaeta (Terebellidae) en

province subantarctique. Ann Inst. Oceanogr. Paris 56(2): 109-1 15.
Duchene, J. C. 1991. Growth rate, fecundity and spawning in two

subantarctic populations of Thelepus setosus (Quatrefages) (Poly-

chaeta: Terebellidae). Ophelia Suppl. 5: 313-320.
Dunham, A. E., and D. B. Miles. 1985. Patterns of covariation in life

history traits of squamate reptiles: the effect of size and phylogeny

reconsidered. Am. Nat. 126: 231-257.
Eckelbarger. K. J. 1974. Population biology and larval development

of the terebellid polychaete Nicolea zostericola. Mar. Biol. 27: 101-

113.
Eckelbarger, K. J. 1975. A light and electron microscope investigation

of gametogenesis in Nicolea zostericola (Polychaeta: Terebellidae).

Mar. Biol. 30: 353-370.
Eckelbarger, K. J. 1976. Origin and development of the amoebocytes

of Nicolea :ostericola (Polychaeta: Terebellidae) with a discussion of

their possible role in oogenesis. Mar Biol. 36: 169-182.
Emlet, R. B., L. R. McEdward, and R. R. Strathmann.

1987. Echinoderm larval ecology viewed from the egg. Echinodenn

Studies 2: 55-136.
Fauchald, K. 1983. Life diagram patterns in benthic polychaetes. Proc.

Biol Soc. Hash. 96(1): 160-177.
Gittleman, J. L. 1986. Carnivore life history' patterns: allometnc, phy-

logenetic. and ecological associations. Am. Nat. 127: 744-771.
Grant, A. 1990. Mode of development and reproductive effort in marine

invertebrates: should there be any relationship? Ftinc. Ecol. 4(1): 128-

129.
Gremare, A. 1986. A comparative study of reproductive energetics in

two populations of the terebellid polychaete Eupolymnia nebulosa

Montagu with different reproductive modes. J E.\p. Mar. Biol. Ecol.

96: 287-302.
Gremare, A., and P. J. W. Olive. 1986. A preliminary study of fecundity

and reproductive effort in two polychaetous annelids with contrasting

reproductive strategies. Int J Invert. Rcpro. Dcv 9: 1-16.
Hannan, C. A., L. W. Hulberg, K. M. Mawn, and J. W. Nybakkcn.

1977. A Study to Develop Standard Procedures for Life History

Analyses of Benthic Invertebrates for Biological Monitoring in Marine

and Estuarine Environments. Moss Landing Marine Laboratories.

California State University Consortium. 217 pp.
Hart, M. W. 1992. Larval feeding performance and egg size evolution

in echmoids. Am. Zoo! 32(5): 1 I4A.
Ilartman, O. 1969. Atlas of Sedentanatc Polychaetous Annelids from

California. Allan Hancock Foundation, Llniversity of Southern Cal-
ifornia, Los Angeles. 812 pp.
Harvey, P. H., and T. H. Clutton-Brock. 1985. Life history variation

in primates. Evolution 39(3): 559-581.
Heimler, \V. 1981. Untersuchungen zur Larvalentwicklung von Lanice

cintchilega (Pallas) 1766 (Polychaeta, Terebellomorpha) Teil I: En-

twicklungsablauf. Zool. Jb Anal 106: 12-45.
Hess, H.C. 1993. The evolution of parental care in brooding spirorbid

polychaetes: the effect of scaling constraints. Am Nat. 141: 577-596.
Hines, A. H. 1982. Allometnc constraints and variables of reproductive

effort in brachyuran crabs. Mar Biol 69: 309-320.
Hines, A. H. 1986. Larval problems and perspectives in life histories

of marine invertebrates. Bull Afar. Sci. 39(2): 506-525.
Jagersten, G. 1972. Evolution l the Meta:oan Life Cycle. Academic

Press, London and New York.
Jamieson, B. G. M., and G. \V. Rouse. 1989. The spermatozoa of the

Polychaeta (Annelida): an ultrastructural review. Biol. Rev. 64: 93-

157.

Kabat, A. R. 1985. The allometry of brooding in Transenella luntilla
(Gould) (Mollusca: Bivalvia). J. E\p Mar Biol. Ecol. 91: 271-279.

knight-Jones, P., and N. Bowden. 1984. Incubation and scissiparity
in Sabellidae (Polychaeta). J. Mar Biol. Assoc. U.K.. 64: 809-818.

Marinescu, \'. P. 1964. La reproduction et le develooppement des po-
lychetes reliques ponto-caspiens du Danube: Hypaniola kowalewskii
(Grimm) et Manayunkia caspica. Ann Rev. Roum. Biol. (serie Zool.)
9:87-100.

McClary, D. J., and P. V. Mladenov. 1989. Reproductive pattern in
the brooding and broadcasting sea star Pleraster mi/itaris. Afar. Biol.
103: 531-540.

McEdward, L. R., and F.-S. Chia. 1991. Size and energy content of
eggs from echinoderms with pelagic lecithotrophic development. J.
Exp. Mar. Biol. Ecol. 147: 95-102.

McEuen, F. S., B. L. Wu, and F.-S. Chia. 1983. Reproduction and
development otSabe/la media, a sabellid polychaete with extratubular
brooding. Mar. Biol 76: 301-309.

Mead, A. D. 1897. The early development of marine annelids. / Mor-
phol. 3(2): 227-326.

Menge, B. 1975. Brood or broadcast? The adaptive significance of dif-
ferent reproductive strategies in the two intertidal sea-stars Leptas-
lerias hexactis and Pisaster ochraeeus. Mar. Biol 31: 87-100.

Miles, D. B., and A. E. Dunham. 1992. Comparative analyses of phy-
logenetic effects in the life-history patterns of iguanid reptiles. Am
Nat. 139(4): 848-869.

Nyholm, K.-G. 1951. Contributions to the life-history of the amphar-
ctid, Meltnna cnslala. Zool. Bidr. Uppsala 29: 79-93.

Olive, P. J. \V. 1984. Environmental control of reproduction in Poly-
chaeta. I-'ortschr. Zool 29: 17-38.

Olive, P. J. W. 1985. Covariability of reproductive traits in marine
invertebrates: implications for the phylogeny of the lower inverte-
brates. Pp. 42-59 in The Origin and Relationships of Lower Inver-
tebrates. Conway Morris, S., D. George. R. Gibson, and H. M. Platt,
eds. Oxford University Press.

Olive, P. J. \V., P. J. Morgan, N. H. Wright, and S. L. Zhang.
1984. Variable reproductive output in Polychaeta: options and de-
sign constraints. Pp. 399-408 in Advances in Invertebrate Reproduc-
tion. Vol. 3, W. Engels, ed. Elsevier Science Publications, New York.

Pearse, J. S. 1979. Polyplacophora. Pp. 27-86 in Reproduction oj Ma-
rine Invertebrates, Vol. 5, A. C. Giese and J. S. Pearse, eds. Academic
Press, New York.

Pechenik, J. A. 1979. Role of encapsulation in invertebrate life histories.
Am \ai 114: 859-870.

Pennington, J. I ., and F-S. Chia. 1984. Morphological and behavioral
defenses of trochophore larvae of Sabellaria ceinenlariiim (Poly-
chaeta) against four planktonic predators. Biol. Bull. 167: 168-175.

Roughgarden, J. 1989. The evolution of marine life cycles. Pp. 270-
300 in Mathematical Evolutionary Theory. M. Feldman, ed. Princeton
University Press, Princeton. New Jersey.

Saelher, B.-E. 1988. Pattern of covariation between life-history traits
of European birds. Nature 331: 616-617.

Sastry, A. N. 1979. Pelecypoda (excluding Ostreidae). Pp. I 1 3-292 in
Reproduction of Marine Invertebrates. Vol. 5, A. C. Giese and J. S.
Pearse, eds. Academic Press, New York.

Schroeder, P. C., and C. O. Hermans. 1975. Annelida: Polychaeta. Pp.
1-214 in Reproduction ol .Marine Invertebrates. Vol. 3, A. C. Giese
and J. S. Pearse, eds. Academic Press, New York.

Scott,,). \V. 19(19. Some egg-laying hablls of Amphitrite ornata Verrill.
Biol. Hull 17: 327-340.

Scott, J. \V. 1911). Further experiments on the methods of egg-laying
in Amphilnte. Biol Bull 20: 252-265.

Sinervo, B. R., and L. R. McEdward. 1988. Developmental conse-
quences of an evolutionary change in egg size: an experimental test.
Evolution 42(5): 885-899.

TEREBEI L ID REPRODUCTION AND DEVELOPMENT

167

Smith, R. I. I989a. Observations on spawning behavior of Eupolymnia

nehii/osa. and eomparisons with Lanice conchilega (Annelida. Poly-

ehaeta. Terebellidae). Hull Mar Sci 45(2): 406-414.
Smith, R. I. I989b. Notes on gamete production in Lanice conchilega

(Annelida. Polychaeta. Terebellidae). Invert. Rep Dev. 15: 7-12.
Sokal, R. R.. and F. J. Rohlf. 19X1. Buum-ln: Freeman, New York.

324 pp.
Spight, T. M., C. Birkeland. and A. Lyons. 1974. Lite histories of large

and small murexes (Prosobranchia: Muricidae). Mar. Biol 24: 229-

242.
Stearns, S. C. 1983. The influence of size and phylogeny on patterns

of covariation among life-history traits in mammals. Oikox4l: 173-

187.
Stearns. S. C. 1984a. The effects of size and plylogeny on patterns of

covariation in the life history traits of li/ards and snakes. Am. Nat.

123: 56-72.
Stearns. S. C. 1984b. The tension between adaptation and constraint

in the evolution of reproductive patterns. Pp. 387-398 in Advances

in Invertebrate Reproduction. Vol. 3. W. Engels, ed. Elsevier Science

Publishers, New York.
Slrathmann. M. F. 1987. Reproduction and Development ot .Marine

Invertehrales of I he Northern Pacific Coast. Dal a and Methods for

the Study of Eggs, Embryos, anil Larvae University of Washington

Press. Seattle and London. 670 pp.
Strathmann, R. R. 1977. Egg size, larval development, and juvenile

size in benthic marine invertebrates. Am. Nat 108: 29-44.
Strathmann, R. R. 1978. The evolution and loss of feeding larval stages

of marine invertebrates. Evolution 32: 894-906.

Strathmann, R. R. 1985. Feeding and nonfeeding larval development
and lite-history evolution in marine invertebrates. Ann. Rev. Ecol.
.SpY. 16: 339-361.

Slralhmann, R. R. 1986. What controls the type of larval development?
Summary statement for the evolution session. Bull Mar. Sci 39(2):
616-622.

Slrathmann, R. R., and C. Chaffec. 1984. Constraints of e