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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.

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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.

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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.

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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.

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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 .

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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

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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.

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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
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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.
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Tardent, P. and Th. Ilolslein. 1982. Morphology and morphodynamics

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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.

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the native neuropeptide Antho-RFamide on muscles in the pennatulid
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Anderson, P. A. V. 1985. Physiology of a bidirectional, excitatory,
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Pro-NH : (Antho-RPamide). an N-terminally protected, biologically
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Carstensen, K., I. D. McFarlane, K. L. Rinehart, D. lludman, F. Sun,
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Elekes, K., and J. Ude. 1993. An immunogold electron microscopic
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Grimmelikhuijzen, C. J. P., and D. Graff. 1986. Isolation of pGlu-Gly-
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Grimmelikhuijzen, C. J. P., and A. Groeger. 1987. Isolation of the
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Grimmelikhuijzen, C. J. P., D. Graff, and A. N. Spencer.

1988. Structure, location and possible actions of Arg-Phe-amide
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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:
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Horridge, G. A., and B. Mackey. 1962. Naked axons and symmetrical
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Horridge, G. A., D. M. Chapman, and B. Mackey. 1962. Naked axons
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Koizumi, O., J. D. Wilson, C. J. P. Grimmelikhuijzen, and J. A. Westfall.

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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.
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Antho-RWamide II and Antho-RFamide on slow muscles from sea
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Peteya, D. J. 1973a. A light and electron microscope study of the ner-
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Peteya, D. J. 1973b. A possible propnoceptor in Cerianthoeopsis
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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-
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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.

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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.

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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). Ce