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Volume 180

THE

Number 1

BIOLOGICAL
BULLETIN

FEI

FEBRUARY, 1991

Published by the Marine Biological Laboratory

THE

BIOLOGICAL BULLETIN

PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY

GEORGE J. AUGUSTINE, University of Southern

LOUIS LEIBOVITZ, Marine Biological Laboratory

Editorial Board

RUDOLK A. RAI I . h
brnia
Ki NSAL VAN HOLDI

STEVEN VOGEL, Du

-MU^-U^—

Marine Biological Laboratory i
LIBRARY

FEB 11 1991

diana Llniversity
, Oregon Stale University .

Woods Hole; Mass.

t University

Associate Editors

Peter A. V. Anderson, The Whitney Laboratory, University of Florida

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FEBRUARY, 1991

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Chemical Mediation of Larval Release Behaviors
in the Crab Neopanope sayi

M. C. DE VRIES, D. RITTSCHOF. AND R. B. FORWARD JR.

Duke University Marine Laboratory, Beaufort, North Carolina 28516 and Duke University
Zoologv Department. Durham. North Carolina 27706

Abstract. Control of egg hatching was investigated in
ovigerous females of the crab Neopanope sayi. Larval re-
lease is a brief event, generally lasting less than 15 min,
during which females perform stereotypic behaviors in-
volving vigorous abdomen pumping. Substances released
by hatching eggs (pumping factors) of TV. sayi. Rhithro-
panopeus harrisii, and Uea pugilator, but not Sesarma
cinereiim, evoked these stereotypic behaviors (pumping
response) in ovigerous N. sayi. Spontaneous pumping and
responsiveness to pumping factors varied with the age of
the embryos. These results indicate that the eggs release
pheromones around the time of hatching, which supports
the general model for egg-hatching control described for
R. harrisii (Forward and Lohmann, 1983). The chemistry
of N. sayi pumping factors was investigated, and the
pumping response was used as a bioassay in this study.
Pumping factors adsorbed to Amberlite XAD-7 resin and
could be eluted from it with methanol. Size fractionation
by cascade pressure dialysis showed that the active mol-
ecules were <1000 daltons. Acid hydrolysis followed by
reverse-phase HPLC amino acid analysis showed that the
biologically active fraction contained peptides. Cysteine,
glycine, methionine, and isoleucine were the four most
common amino acids in these peptides. The responsive-
ness of N. sayi to hatch water from R harrisii. the general
similarity of adsorptive characteristics of hatch waters
from the two species toward XAD-7 resin, and the amino
acid compositional analysis suggest that the pumping fac-
tors from both species are similar. This supports the hy-
pothesis that N. sayi pumping factors are also small pep-
tides, as was suggested for those of R. harrisii (Rittschof
etal.. 1985, 1989).

Introduction

Rhythms in larval release corresponding to lunar, diel,
and tidal cycles have been observed for numerous species

Received 15 December 1989; accepted 28 November 1990.

of Brachyura (see DeCoursey, 1983; Forward, 1987, for
reviews). In species showing rhythms, egg hatching gen-
erally occurs during the dark phase of the diel cycle, and
often near the high tide of a tidal cycle (Saigusa and Hi-
daka, 1978; Saigusa, 1981, 1982; Forward et ai. 1982;
Wolcott and Wolcott, 1982; Christy, 1986; Salmon et ai,
1986; De Vries and Forward, 1989). For most warm-water
species, such as the xanthid Neopanope sayi, larval release
is a brief event, usually lasting less than 15 min for an
individual (DeCoursey, 1979; Forward el ai. 1982; De
Vries, 1990). During this time, in general, all of a female's
eggs hatch while the female vigorously pumps her abdo-
men. Occasionally, larvae are released in more than one
short burst at the time of consecutive tidal phases or nights
(Forward el ai. 1982; Christy, 1986; De Vries and For-
ward. 1989).

The control of egg-hatching time in decapods seems to
vary with species. Hatching time has been reported to be
controlled by the female (Branford, 1978; DeCoursey,
1979) and alternatively by the developing embryos (Pan-
dian, 1970; Ennis, 1973; Forward and Lohmann, 1983)
in crabs and lobsters. The site of egg-hatching control may
also be related to adult habitat (De Vries and Forward,
1 99 1 a). Control of egg-hatching time has been well studied
only in the subtidal xanthid crab Rhithropanopeus harrisii
(Forward and Lohmann, 1983; Rittschof et ai. 1985;
Forward et ai, 1987; Rittschof etal.. 1989). In this species,
the developing embryos control the exact timing, while
the female controls the synchrony of hatching. Substances
associated with hatching eggs are released near the time
of hatching and induce the ovigerous female to perform
stereotyped larval release behaviors that synchronize
hatching. These pheromones are collectively called
"pumping factors" and are a heterogeneous group of pep-
tides mostly of <500 daltons.

The present study was performed to investigate the
generality of the model for hatching-time control de-

M. C. DE VRIES ET AL.

scribed for R. harrisii (Forward and Lohmann, 1983). In
particular, we aimed to determine whether active sub-
stances from another xanthid, Neopanope sayi, are similar
to active components from R. harrisii. If active molecules
from N. sayi are similar to those from R harrisii, they
should cross react biologically, and produce similar results
upon chemical purification and analysis, as done for
R. harrisii pumping (actors (Rittschof ct a/.. 1985).

Neopanope sayi is a subtidal xanthid crab, occurring
in coastal and estuarine areas, from the low littoral to the
sublittoral zones (Williams, 1984). Experiments were de-
signed to determine whether hatching eggs of A', sayi and
other crab species produce substances that stimulate ovi-
gerous N. sayi to perform larval release behaviors. Pump-
ing factors were indicated, and some of their chemical
characteristics were investigated, the crabs' stereotyped
larval release behavior serving as an assay for biological
activity. Our results suggest that pumping factors from N.
sayi are similar in composition, but not identical, to those
from R. harrisii.

Materials and Methods

General collection and maintenance of animals

Ovigerous females of Neopanope sayi (Smith) were col-
lected from among the subtidal hard substrate community
near the Duke University Marine Laboratory in Beaufort,
North Carolina. Crabs were brought into the laboratory
and placed into individually numbered culture bowls (di-
ameter, 10.4 cm) containing approximately 160 ml of 5
^m filtered ambient salinity seawater (approximately 32-
35%o). Crabs were located in a controlled-environment
room (27°C ± 1°C), under a 14 h light: 10 h dark cycle,
with lights-out at 2000 h. This LD cycle corresponded to
the cycle in the field at the time of collection.

The water in each crab's bowl was changed daily be-
tween 0900 and 1200 h. At this time, the presence of
larvae in the bowls was noted and the date of larval release
recorded for each crab. Experiments were performed on
ovigerous females and the data were examined in relation
to the age of the embryo. Embryonic age at the time of
experimentation (expressed as days until hatching) was
determined by counting backwards from the subsequent
time of hatching. For individuals that released larvae in
more than one burst, the release date was considered to
be on the day that the first group of eggs hatched. Crabs
were not fed while in the laboratory.

Spontaneous levels of pumping

Until they released their larvae, the crabs were placed
once each day for 2 min into filtered (0.45 /urn) seawater,
and the number of spontaneous pumps counted. The
spontaneous pumping activities of 62 N. savi carrying
embryos of various developmental stages were recorded

in this way. These data were collected to determine
whether the percentage of crabs that pumped sponta-
neously, and the absolute frequency of spontaneous
pumping varied with embryo age.

Because crabs were brought into the laboratory carrying
embryos of all ages, and because their pumping activities
were measured repeatedly, the effects on spontaneous
pumping activity of length of time in the laboratory and
of embryo age might be confounded. To separate the ef-
fects of the two variables, plots of frequency of sponta-
neous abdomen pumping versus length of time in the
laboratory were made for three groups of crabs (i.e.. those
carrying embryos that would hatch in 0-1, 2-3, and 4-5
days, respectively). These plots showed no relationship
between pumping and time in the laboratory (De Vries,
unpub. data). Thus, when embryo age was held constant,
the frequency of spontaneous abdomen pumping ap-
peared to be independent of time in the laboratory. Plots
were not made for crabs carrying embryos in earlier stages
because of the small numbers and extremely low pumping
rate of such crabs. N. sayi release their larvae within at
most 10 days of egg deposition at the laboratory mainte-
nance temperature of 27°C.

Biological assays

An abdomen-pumping bioassay was used to detect
chemicals that stimulated larval release behaviors in ovi-
gerous crabs. The assay, a modification of that described
in Forward and Lohmann (1983), proceeded as follows.
A crab was placed in a bowl (diameter, 7.9 cm) containing
80 ml of filtered, (0.45 ^m) ambient salinity seawater, and
the frequency of abdomen pumps was counted for 2 min.
The crab was then transferred to a second bowl containing
80 ml of test solution, and the count was repeated. If a
crab pumped at least five more times in the second bowl
than in the first, this was counted as a positive signal or
a "response" to that test solution. For any given test so-
lution, 20-60 (but usually 30) animals were assayed. The
percentage of crabs tested that responded to each test so-
lution was defined as the % response, and is considered a
measure of the biological activity of that solution. Most
test solutions were derived from water in which crab larvae
were released by females. We used the number of larvae
released per ml of water as an indication of the concen-
tration of active substances in that solution. The results
of pumping-response assays are shown as dose-response
curves (i.e., larvae/ml vs. % response), such as that in Fig-
ure 2.

The two-bowl protocol, described above, was used to
allow for variability in spontaneous pumping activity
among individuals, as well as for changes in this parameter
within an individual that might occur between 1000 and
1700 h, the interval during which these assays were per-
formed. An assay was also performed in which both bowls

CONTROL OF CRAB LARVAL RELEASE

contained filtered seawater — a control of the effects of the
experimental procedure upon spontaneous pumping rates.
Although pumps were usually vigorous, they were some-
times subtle, and could be unseen if crabs suddenly moved
their abdomens out of view. The criterion of a five pump
difference to define a positive response was therefore used
to preclude potential observational errors that could occur
with differences of less than five pumps. A simple pro-
portional increase in pumping between two bowls to de-
fine a response was inappropriate, because many crabs
pumped 0 times in the first bowl.

Water in the control and test bowls was replaced after
every 10 crabs to ensure a minimal change in water com-
position between crabs (Forward et ai, 1987). Individuals
were assayed in each concentration of a test solution only
once, and were not retested with another concentration
within 30 min. Individuals were generally tested 3-5
times/day. Crabs were returned to their home bowls con-
taining filtered seawater between tests. Substances were
tested from the lowest to the highest concentrations to
reduce adaptation. Significant differences between test and
control response levels were established by the use of a
Z-statistic for testing differences between two proportions
at a = 0.05 (Walpole, 1974).

Preparation oj hatch water

We collected water into which N. sari had released lar-
vae and determined whether it would stimulate larval re-
lease behaviors (i.e., abdominal pumping) in ovigerous
crabs. About 1-2 h before the predicted time of larval
release (generally evening high tide for N. sayi: De Vries
and Forward, 1989), ovigerous crabs were placed into in-
dividual culture bowls. The bowls were 10.4 or 7.9 cm in
diameter (depending upon crab size) and contained 100
or 50 ml (respectively) of 0.45 ^m filtered ambient salinity
seawater. Just after a female had released her larvae, she
was removed from the bowl, and the water was passed
through 100 nm plankton netting to remove the larvae.
This filtered hatch water was kept on ice only briefly and
was then frozen (-20°C) for later use. The liter of pump-
ing factors in a hatch water sample was estimated by
counting subsamples of the larvae contributing to it, and
is expressed as larvae/ml. Hatch water was collected from
three additional crab species, Sesarma cinerewn (Grap-
sidae), Uca pugilator (Ocypodidae), and Rhithropanopeus
harrisii (Xanthidae), as described above, except that R.
harrisii hatch water was collected in 10%o water, as ne-
cessitated by their upper estuarine habitat. For testing in
biological assays with N. sayi, the hatch water from R.
harrisiivtas raised to ambient salinity with Instant Ocean.

The response to hatch water from A', sayi was assayed
with crabs carrying embryos of different ages to determine
whether sensitivity changed with the stage of embryonic
development. Crabs with early (>5 days until hatching)

embryos and crabs with late-stage (<3 days until hatching)
embryos were tested, and control levels (i.e., percent re-
sponse) were established for them. For all other assays,
however, only crabs with late-stage embryos were used.

Isolation and purification of pumping factors

Adsorption chromatography. The molecular character-
istics of N. sayi pumping factors were determined by a
modification of the adsorption chromatography and size
fractionation procedures of Rittschof et ai (1985), with
the pumping bioassay being used to monitor the process.
The pumping factors from hatch water were first concen-
trated on Amberlite XAD-7 resin. The column of resin
(24 cm X 1 cm bed volume) was stored in 100% HPLC-
grade methanol (Fisher Chemical Co.), and immediately
before being loaded with pumping factors was rinsed with
hexane, then back flushed and rinsed with at least 200 ml
of deionized water. Loading was done by gravity-feed at
approximately 16 ml/min. The passage of hatch water
through the column was stopped before the resin was ex-
posed. Methanol was carefully overlaid upon the hatch
water. At the first signs of methanol breakthrough in the
eluate (decrease in drop size and effervescence), the next
1 3 ml of solution was collected. The methanol in this
sample was then evaporated under a stream of N2 until
approximately 1-2 ml of solution remained. This con-
centrated pumping factor was stored at — 20°C until it
was bioassayed or size fractionated.

For bioassays, concentrated pumping factor was diluted
with filtered (0.45 urn) seawater to the desired test con-
centration. The calculation of larval concentrations in the
test solutions was based upon that estimated in the original
hatch water samples, assuming 100% recovery of pumping
factors from the resin.

Cascade pressure dialysis. Hatch water from N. sayi
that had been concentrated on XAD-7 resin was brought
to a volume of about 100 ml with deionized water. This
solution was subjected to cascade pressure dialysis (4°C,
40 psi); Amicon YM 10 and YM2 Diaflo membranes with
nominal cutoffs at 10 and 1 kDa, respectively, were used.
The membranes were stored and rinsed according to the
manufacturer's instructions. Two additional rinses with
50 ml deionized water were carried out under pressure to
insure that all preservatives were washed from the mem-
branes. A sample was first passed through the 10 kDa
cutoff membrane. Part of this filtrate was bioassayed, and
the remainder was fractionated with the 1 kDa cutoff
membrane. When about 10 ml of sample remained above
each membrane, three successive 40 ml rinses with dis-
tilled water were done. This procedure effectively elimi-
nated small molecules (<10 or <1 kDa) in the original
solution that might have been passively retained above
the membranes. Rinse water passing through the mem-
branes was discarded. Those solutions that passed through

M. C. DE VRIES /:/ I/

the membranes, as well as those that were retained, were
kept on ice and bioassayed immediately.

For bioassays, size fractionated pumping factor was di-
luted with filtered (0.45 nm) seawater to the desired test
concentration. Larval concentrations in the test solutions
were calculated based on those estimated in the original
hatch water sample, assuming 100% recovery of pumping
factors from size fractionation procedures.

Control solutions

To be certain that substances with biological activity
were directly related to the hatching process, two control
solutions were subjected to the hatch water purification
procedure described above. One solution was 0.45 ^m
filtered seawater. The other was seawater in which ovi-
gerous crabs had been incubated under conditions similar
to those used for crabs releasing larvae (ovigerous crab
essence). The filtered seawater was processed to ensure
that no stimulatory effects were produced by the adsorp-
tion chromatography or cascade pressure dialysis. A vol-
ume of seawater equal to the average volume of hatch
water processed in each batch was used. The number of
larval equivalents in the filtered seawater control was based
on the average concentration of all N. sayi hatch water
processed during the present experiments.

Ovigerous crab essence was included as a control to
ensure that substances associated with hatching eggs, and
not substances secreted by ovigerous crabs or their em-
bryos at other times, were responsible for the observed
pumping activity. Ovigerous crab essence was prepared
by placing 20 ovigerous N. sayi with embryos of various
developmental stages into 2 1 of 0.45 /urn filtered seawater.
After 2 h, the crabs were removed and the water treated
as described above for the filtered seawater control. Each
crab was assumed to carry 2500 eggs (based on unpub-
lished estimates of egg-mass sizes for N. sayi), from which
the larval concentrations of this control were calculated.
Aliquots of the seawater and ovigerous crab essence con-
trols were diluted such that they contained concentrations
equivalent to 20 and 50 larvae/ml and were assayed: these
concentrations of crude and size-fractionated hatch water
produced strong pumping responses.

To be certain that larger active molecules were not de-
natured on the column matrix upon elution, hatch water
not passed through the column was filtered through a 10
kDa membrane. Dilution series of the < 10 kDa and > 10
kDa fractions were assayed.

Amino acid analysis

The amino acid composition of N. sari pumping factors
was analyzed by Dr. Dano Fiorio at Florida State Uni-
versity. A < 1 kDa sample from the release of approxi-
mately 19,000 larvae was analyzed. Reverse-phase high-
performance liquid chromatography (HPLC) and pre-

column derivatization with phenylisothiocyanate were
performed using a modification of the method in Hen-
rickson and Meredith (1984). Unhydrolyzed and hydro-
lyzed (in 6 N HC1 for 24 h at 1 10°C) samples were ana-
lyzed to determine the initial composition of free amino
acids and the composition of the peptides (<1 kDa), re-
spectively. Phenylthiocarbomyl derivatives were separated
on an octadecasilyl reverse-phase column and detected
spectrophotometrically at 254 nm. Identification and
quantification of amino acids was by comparison of de-
rivatized standard amino acids with those in the samples.

Amino acid experiment*

Results of the above compositional analysis provided
the basis for testing the biological activity of mixtures of
pure amino acids (Sigma Chemicals). Pumping assays
were performed using a mixture of the four most abundant
amino acids in hydrolyzed pumping factor, L-cysteine,
glycine. L-isoleucine, and L-methionine. These amino
acids were combined in equimolar amounts (as in hydro-
lyzed factor) and tested at concentrations bracketing those
for which hatch water was active. In addition, pumping
assays were performed using a combination of glycine
and arginine, the most abundant amino acids in Rhith-
ropanopeux harrisii pumping factor, in proportions and
concentrations bracketing their level in hydrolyzed
pumping factor from this species (Rittschof et at., 1985).

Results

Spontaneous pumping rates

Frequency of spontaneous abdomen pumping generally
increased with the age of the embryos (Fig. 1A). For crabs
with embryos of all ages, at least 25% did not pump at
all. However, the percentage of crabs which pumped in-
creased sharply with increasing embryo age (Fig. IB).

Response to hatch water

The percent pumping response of ovigerous N. sayi
individuals increased when the animals were exposed to
hatch water (Fig. 2A). Responsiveness varied with con-
centration for crabs with both early- and late-stage em-
bryos. At concentrations lower than 2.5 larvae/ml for
crabs with late embryos, and lower than 5.0 larvae/ml for
crabs with early embryos, the percentages of crabs re-
sponding were not significantly different from controls.
At these concentrations and higher, however, the per-
centages of crabs responding were significantly greater
than controls.

For each concentration tested, the percentages of crabs
with early embryos that responded were consistently lower
than those of crabs with late embryos. This reflects, in
part, the greater inclination of crabs with older embryos
to spontaneously pump their abdomens (as evidenced by

CONTROL OF CRAB LARVAL RELEASE

the increased control levels), but probably also indicates
that crabs with older embryos are more sensitive or re-
sponsive to pumping factors. The latter is evidenced by
the higher concentration of hatch water necessary to elicit
a significant percent response for crabs with early embryos
compared to those with late embryos. Because crabs with
late stage embryos were more responsive to pumping fac-
tors, they were used in all subsequent experiments.

N. sayi with late embryos also had significantly higher
percent pumping responses upon exposure to hatch water
from Rhithropanopeus harrixii and Uca pugilator at con-
centrations > 20 larvae/ml (Fig. 3). Exposure of N. sayi
to hatch water from Sesarnw cincrcnm at concentrations
from 1 to 60 larvae/ml, however, produced levels of re-
sponse not significantly different from the control (Fig.
3). These results indicate some, but not complete, cross-
reactivity of hatch waters among species, and suggest that
active substances from some species are similar in com-
position.

Adsorption chromatography and
cascade pressure dialysis

Hatch water was fractionated into substances with and
without affinity for Amberlite XAD-7 resin, and the frac-
tions were bioassayed with crabs bearing late-stage em-
bryos. A concentration of 10 larvae/ml was tested because
it produced maximum response (Fig. 2A). When exposed
to untreated hatch water, 69% of the crabs responded (Ta-
ble I), which was significantly greater than the control
level (23%; n = 158). After passage of hatch water through
the resin however, activity was lost. Only 33% of the crabs
responded, which was not significantly different from the
control level.

To recover adsorbed activity, the resin was eluted with
methanol, which removes lipophilic and proteinaceous
substances. Bioassays showed a modest increase in re-
sponse when tested with the methanol eluate, but at con-
centrations up to 20 larvae/ml, these responses were not
significantly different from those of the control (Fig. 2B,
before size fractionation). After passage of the methanol
eluate through the 10 kDa and 1 kDa membranes, activity
reappeared (Fig. 2B), presumably due to the removal of
an inhibitor introduced by, or concentrated by, the resin
(see Discussion). In these two fractions, an increase in the
percentage of response with concentration was observed,
with concentrations > 10 larvae/ml producing levels of
response significantly different from control. The fractions
from these two nitrations, which were retained above the
membranes (the >10 kDa and the <10 kDa but >1 kDa
fractions), produced levels of response no different from
controls at concentrations up to 20 larvae/ml.

When untreated hatch water was passed through the
10 kDa cutoff membrane, the retained > 10 kDa fraction
lacked biological activity (Fig. 4). Response levels in the

8765432 I
Days until Hatching

Figure 1. Frequency of spontaneous abdomen pumping (A), and
percentage of crabs which pumped (B), as a function of embryo age in
ovigerous female Neopamtpc sari Means and 95% confidence limits of
spontaneous pumping frequency are shown for crabs that pumped at
least once. The numbers beside the points are the sample sizes.

<10 kDa fraction were significantly greater than control
levels at > 10 larvae/ml. The absence of biological activity
in the > 10 kDa fraction of the untreated hatch water sug-
gests that precipitation of large (>10 kDa), biologically
active molecules onto the resin upon methanol elution
was unimportant. In summary, pumping factors were ad-
sorbed to XAD-7 resin, eluted with methanol, and were
<1 kDa.

Controls demonstrated that biologically active sub-
stances originate from hatching eggs. Filtered seawater
and ovigerous crab essence were subjected to the adsorp-
tion chromatograph and fractionation procedures. At
concentrations equivalent to 20 and 50 larvae/ml, neither
filtered seawater nor crab essence produced responses sig-
nificantly greater than the filtered seawater control, either
before or after passage of the two solutions through the
resin and membranes (Table II). These results show that
passage of seawater through the resin and membranes did
not add excitatory substances to the water, and that sub-
stances associated with ovigerous females, in the absence
of egg hatching, did not produce a significant level of
pumping response.

M. C. DE VRIES KT AL

70
60

50-
40
30
20
10-

70-
60
50-
40-
30
20-
10-

A Neoponope soy/
«59

B. Neoponope sayi

« <l KD n=30

<IOkD n=23-30

„ - -* before size f ro>
n = 60

\'J' "'•-.,....-•> 10 kD n =23-25

''•'-+-—-" "'"• <IOkDbut>l kD n =

23-29

.Control
n = !58

15 20 25 30 35

Concentration (larvae/ml)

40 60

Figure 2. Percentage of ovigerous female Neopanope sari that responded to N. sari hatch water (A) and
to various size fractions of a methanol eluate of hatch water after concentration on XAD-7 resin (B). Con-
centration of active substances (abscissa) was calculated from estimates of larval concentration in the untreated
hatch water solutions. Numbers by points are specific sample sizes (A) or a range of sample sizes (B). Asterisks
indicate the first concentration at which pumping response was significantly different from controls. Control
levels for pumping response were established in filtered seawater. Crabs with late embryos were 0-3 days
from larval release, and those with early embrvos were >5 davs from larval release.

Amino acid analysis

Reverse-phase HPLC analysis of free and hydrolyzable
amino acids showed picomolar concentrations of 15
amino acids in the biologically active (<1 kDa) fraction
of hatch water concentrated on the resin [calculated to a
liter of 160 larvae/ml for comparison with results in
Rittschof et al. (1985); Table III]. The most abundant
amino acids after hydrolysis — cysteine, glycine, isoleucine,
and methionine — accounted for 47% of the total free, and
57% of the total hydrolyzable amino acids. These four
amino acids were approximately equimolar, at > 100 pA/
after acid hydrolysis. Free amino acids before hydrolysis
represented 23% of the total amino acids in the sample
after hydrolysis. N. sayi pumping factors of this liter thus

conlain picomolar amounls of amino acids, mosl of which
appear lo be bound in peplides.

Amino acid experiments

When presenled wilh mixlures of Ihe amino acids mosl
abundant in partially purified halch waler from N. sayi
and R. hanisii, ovigerous N. sayi individuals significantly
increased Iheir levels of pumping over Ihose of controls,
al concentralions > 10~4 M (Fig. 5). Concenlralions al
which nalive pumping factors were effeclive (10~7-10~9
M), produced pumping responses no differenl from con-
Irols. These effeclive concenlralions are based on those
of Ihe four major amino acids (Table III) in halch waler
of tiler > 2.5 larvae/ml (i.e.. Ihe Ihreshold for crabs car-
rying lale embryos; Fig. 2). In conlrasl, amino acid mix-

CONTROL OF CRAB LARVAL RELEASE

Neopanope sayi
n = 27-3l

Rhtfhroponopeus narrtsit
hotch water .

Uca pugitator
hotch water

- •— _^ Sesarma cinereum

-"---_ hatch water

Control
n=!58

30 50

Concentration (larvae/ml)

Figure 3. Percentage of ovigerous female A Vi >/><(/;<>/>(• xayi with late
embryos that responded to hatch water from three other brachyurans:
Rhillm tpanopent liarrisii. T. </ pux/lutor, and Scsamui cincrcuni Asterisks
indicate the first concentration at which the pumping response was sig-
nificantly different from controls. Control levels for pumping response
were established in filtered seawater.

tures first produced significant responses at concentrations
corresponding to approximately 100,000 larvae/ml (2.5
X 10~4 A/) for the N. sayi mixture, and approximately
7,000 larvae/ml (8.3 X 10~4 M) for the R. liarrisii mixture.
These results suggest that simple mixtures of the amino
acids most abundant in pumping factors are not the mol-
ecules most active in producing abdomen pumping at the
time of larval release.

Discussion

Substances associated with hatching eggs evoked ste-
reotyped larval release behaviors in ovigerous females of
the crab Neopanope sayi. Responsiveness to these pump-
ing factors varied with embryo age. as did the spontaneous
pumping activity of ovigerous crabs. The pumping factors
adsorbed to Amberlite XAD-7 resin and could be eluted
with methanol, but biological activity did not appear in
the methanol eluate until after size fractionation. The
presence of small peptides was inferred from the size frac-
tionation and amino acid analysis of a partially purified
preparation of hatch water.

For N. sayi, a clear variation was observed in the fre-
quency of spontaneous abdomen pumping with embryo
age. A possible physiological explanation for this phe-
nomenon is that, as nonliving yolk is converted into em-
bryo, metabolic rate increases, causing increased Oi de-
mand and waste production. Abdomen pumping by crabs
is thought to facilitate O2 transport to, and waste removal
from, developing embryos (Templeman, 1937; Ennis,
1973), hence older embryos with higher metabolic rates
would require more water pumped around them.

In contrast to those of N. sayi, the spontaneous abdo-
men pumping rates of R. harrisii were independent of

Table I

Effect til IHISMHK lunch miler ilmiitgh Anjher/ile \AD-7 resin

Test

concentration

Percentage

Fraction

larvae/ml

responding

Hatch water before resin

Hatch water after resin

Control

—

158

embryo age (Forward and Lohmann, 1983). This species
difference may be due in part to the smaller size of R
harrisii egg masses (average size, about 1000 eggs; For-
ward, unpub. data) compared with N. sayi (generally
2000-4000 eggs; De Vries unpub. data). Eggs of the two
species are of approximately the same diameter [about
400 urn on the day of hatching (De Vries and Forward,
1990b; De Vries, unpub. data)]. Therefore, the egg masses
of N. sayi are 2-4 times larger in volume than those of
R liarrisii. and the former would have a greater total
metabolic demand and slower diffusion rate of water
through the egg mass. These effects may compound one
another, leading to a much greater need for water transport
around A', sayi eggs, and thus a more pronounced increase
in pumping rate as the embryos mature. The increase in
egg mass size (De Vries and Forward, 1990b) with embryo
age may cause the increase in spontaneous pumping rate
by stimulating stretch receptors.

The response of N. sayi to hatch water of conspecifics
is similar to that observed previously for R. harrisii (For-

50-

30-

10-

Neopanope sayi
n= 30-60

/< 10 kd N. sayi hatch water

.Control

n=l58

>IOkd N. sayi hatch water

10 20

Concentration (larvae/ml)

Figure -4. Percentage of ovigerous females of Neopanope sayi with
late embryos that responded to the retained and non-retained fractions
o(N. suvi hatch water after ultrafiltration with a 10 kDa membrane. The
control is for responses in filtered seawater.

M. C. DE VRIES ET AL
Table II

Controls performed for Neopanope sayi pumpiiiK factor cx

Solution

Test concentration
(larvae/ml)

Percentage
responding

1. 0.45 Mm filtered seawater (FSW) (untreated) (control)

—

158

2. 10 kDa fraction MeOH eluate FSW

2(1

10 kDa fraction MeOH eluate FSW

3. 1 kDa fraction MeOH eluate FSW

I kDa fraction MeOH eluate FSW

4. Ovigerous crab essence (OCE) (untreated)

5 10 kDa fraction MeOH eluate OCE
1 0 kDa fraction MeOH eluate OCE

6. 1 kDa fraction MeOH eluate OCE
1 kDa fraction MeOH eluate OCE

20
50

35
35

26
17

23
6

In all tests, crabs cam-ing embryos within three days of hatching were used. Test concentrations were calculated as described in the text. Solutions
2-6 produced response rates not significantly greater than control (Solution 1 ). Ovigerous crab essence was seawater in which ovigerous crabs had
been incubated for several hours (details in text).

ward and Lohmann, 1983), which suggests that the model
for hatching-time control described for R. harrisii also
applies for N. sayi. In this model, synchronized devel-
opment of the eggs is believed to result from an unknown
interaction between the embryos and the females, but ac-
tual hatching time is controlled by the embryos. When
the eggs become ready to hatch, several eggs hatch spon-
taneously, releasing substances into the water that stim-
ulate additional abdomen pumping. This action breaks
open the remaining eggs, and the result is the synchronous
release of larvae. The release of pheromones by embryos
to communicate their readiness to hatch may have adap-
tive significance. During larval release, female crabs are
presumably exposed and therefore at greater risk to pre-
dation than at other times (Forward and Lohmann, 1983).
In addition, abdominal pumping during larval release is
a vigorous, hence an energetically costly activity. Con-
centration of larval release behaviors to a time when the
embryos are ready to hatch may thus decrease the risk of
predation and energetic costs to the female.

Substances (pumping factors) that evoke larval release
behavior (pumping response) were released at the time of
egg hatching and were not released from eggs prior to this
time. The effects of different treatments with these pump-
ing factors can be determined by comparing the effective
concentrations that evoked a 50% pumping response
(EC50; Table IV). When exposed to untreated hatch water,
the liter for the EC50 was 6 larvae/ml. If this water was
passed through a 10 kDa cutoff membrane, the EC50 in-
creased to 10 larvae/ml. Because activity could not be
detected in the water above the 10 kDa membrane, this
decrease in activity did not result from the removal of
large active molecules. Two possible explanations are ad-

sorption of active molecules to the membrane during the
filtration process, or degradation during the filtration in-
terval by enzymes released by lysed microorganisms dur-
ing thawing and pressure dialysis of hatch water. Crabs
were unresponsive to hatch water that had been passed
through the X AD- 7 resin, suggesting that active substances

Table III

Amino acid composition of hydrolyzed and unhydrolyied Neopanope
sayi pumping factor of < 1 000 daltons

Picomolar amount

Amino Acid

Unhydrolyzed
factor

Hydrolyzed
factor

Difference

Pro

Cys

134

Asp

—

Thr

Ser

Glu

—

Gly

125

119

Ala

Val

Met

137

134

lie

123

Leu

Tyr

—

Phe

His

—

Lys

Arg

—

Total

2?7

945

723

Concentrations are calculated for a sample with a titer of 160 larvae/

ml.

CONTROL OF CRAB LARVAL RLLF.ASE

were removed by the column. The methanol eluate was
also inactive. Pressure dialysis of the methanol eluate re-
sulted in a titer of activity comparable to size fractionated
(10 kd filtered) hatch water that had not been passed
through the resin. This result suggests high recovery of
pumping factors from the column, as previously obtained
for R harrisii factors (Rittschof ct ul.. 1985).

The apparent absence of activity in the methanol eluate
may have resulted because the resin: (1) removed an im-
portant component of the pumping factors that was not
eluted with methanol; (2) concentrated high molecular
weight inhibitory substances in the hatch water; or (3)
added inhibitory substances dc novo. The column did not
add inhibitory molecules because XAD-7 resin leaches
low molecular weight compounds ( Jolley el ai, 1981) that
would not be removed by size fractionation. Because ac-
tivity reappeared after passage through the 10 kDa mem-
brane (Fig. 4), the active factors and components were
recovered from the resin. Thus, the most likely explana-
tion is that inhibition resulted from concentration of high
molecular weight inhibitory substances present in the
hatch water.

The EC5o for the methanol eluate after passage through
the 10 kDa membrane was 13 larvae/ml (Table IV). Be-
cause this value is very close to that for untreated hatch
water (10 larvae/ml) after passage through the 10 kDa

Effective ciineenlruli(iii\ lor a 5
carrying lalc-slage embryos

Table IV

0% pumping response (E(.'SII) in cnihs

Hatch water treatment

EC50
(larvae/ml)

Data source

Untreated

Fig. 2

10 kDa filtered

Fig. 4

XAD-7 resin

none

Fig. 2

XAD-7 resin. 10 kDa

filtered

Fig. 2

XAD-7 resin, 10 kDa.

1 kDa filtered

Fig. 2

Rhilhropanopeus hamsn

hatch water

Fig. 3

Veil pugilator hatch water

Fig. 3

membrane, recovery of active molecules from the XAD-
7 column was close to 100%. Rittschof el ai (1985) had
similar success rates in recovery of active molecules in R.
harrisii hatch water from an XAD-7 column. The activity
of N. xayi hatch water remained the same (EC50 = 12
larvae/ml) after it had been passed through the 1 kDa
membrane, indicating that activity can be attributed solely
to molecules that are less than 1 kDa in size.

N. sari responded to hatch water from R. harrisii and
Uca piigilalor, but the EC50 values were high at 1 5 and

<1>
(f>
c 40

CL
0>

01 30

Neopanope say/
n=29-34

N soy/ mixture
cystglytile + met
(1=1=1=1

Control
n = !58

Rhithroponopeus harrisii mixture

glytorg

(LI)

.-9

ICT

'-7

10" 10 10 10"

Concentration (M)

10'

,-3

10'

Figure 5. Percentage of ovigerous Neopanope sayi with late embryos that responded to different con-
centrations of mixtures ot amino acids. Mixtures represent the main components of hydrolyzed pumping
factors of N. sayi (equimolar amounts of L-cysteine, glycine. L-isoleucine, and L-metlnonine) and Rhilh-
ropanopeus harrisii (equimolar amounts of glycine and L-arginine; Rittschof el ul., 1985). Asterisks indicate
the first concentration at which the pumping response was significantly different from controls. Control
levels for the response were established in filtered seawater.

M. C. DE VRIES ET AL

50 larvae/ml, respectively (Table IV). This result, and the
lack of response to S. cinerewn hatch water, show that
cross-reactivity among species occurs, but is incomplete,
implying that hatch waters from different species may be
similar, but are probably not identical.

Three lines of evidence suggest that the active molecules
in hatch water of R. harrisii are peptides. First, activity
is lost if hatch water is treated with a protease. Second,
compositional analysis of partially purified active mole-
cules suggests that they may consist of di- or tripeptides
with a neutral amino acid at the amino-terminus and an
arginine carboxy-terminus (Rittschof et at., 1985). Third,
pure peptides having this structure induce pumping re-
sponses (Forward et a/.. 1987; Rittschof et ai, 1989).

Results from the present study support the hypothesis
that active substances in hatch water of N. sayi are also
peptides, though their exact chemical nature is unknown.
Evidence for this includes: (1) responsiveness of N. sayi
to R. harrisii hatch water; (2) general similarity in the
adsorptive characteristics of N. sayi and R. harrisii
pumping factors toward XAD-7 resin; (3) suggestion of
peptides in a partially purified hatch water preparation;
and (4) similarity in the response of N. sayi and R. harrisii
to mixtures of amino acids.

The amino acid analysis of Neopanope sayi pumping
factors indicates that the four main amino acids in the
proposed peptides are cysteine, glycine, isoleucine, and
methionine. With the exception of glycine, these amino
acids have neutral side chains and are hydrophobic. In
contrast, the proposed active peptides of Rhithropanopeus
harrisii contain arginine (present only in low amounts in
N. sayi factor), which is strongly charged and hydrophilic.
Thus the foregoing analysis suggests that the pumping
factors of N. sayi are more hydrophobic than those of
R. harrisii.

Bioassays of amino acids suggest that, for both species,
simple mixtures of amino acids are not the active com-
ponents of pumping factors (Rittschof et ai. 1985). Mix-
tures of the amino acids most abundant in the pumping
factors produced significant levels of pumping only at
concentrations much higher than those present in native
hatch waters. The threshold concentrations for responses
of N. sayi to amino acids were about 10~4 M. while free
amino acid levels based on compositional analysis (Table
III) at the threshold concentration of hatch water (2.5
larvae/ml; Fig. 2) were about 3.0 X 10~12 M (calculated
from Table III). For TV. sayi, small peptides of undeter-
mined sequence are hypothesized to be the active com-
ponents of pumping factors as concluded for R. harrisii
(Rittschof et al., 1985). The source of these peptides is as
yet unknown. However we have postulated elsewhere that
enzymatic degradation of the egg membranes (De Vries
and Forward, 1 99 Ib; Rittschof el al.. 1990a) produces the
pumping factors.

The chemical mediation of a diversity of behaviors has
been described in virtually all phyla and may be partic-
ularly important in aquatic environments (e.g., Knight-
Jones, 1953; Collins, 1975; Trott and Dimock. 1978;
Derby and Atema, 1980; Tierney and Dunham. 1982;
Rittschof el al.. 1983, 1984). In particular, proteins and
peptides elicit behaviors in various taxa, including feeding
behavior in the snail Ilyanassa (=Nassarius) obsoleta
(Carr et al., 1974), creeping in predatory snails (Rittschof
et a/., 1984), and metamorphosis in the sand dollar Den-
drastcr excentricus (Burke, 1984) and the abalone Haliotis
(Morse, 1988). Among crustaceans, serine protease gen-
erated peptides are implicated in hermit crab shell ac-
quisition behavior (Rittschof, 1980; Lepore and Gilchrist,
1988; Rittschof et al.. 1990b), barnacle attachment be-
havior (Rittschof, 1985) and metamorphosis (Tegtmeyer
and Rittschof, 1989), and crustacean larval release (Ritts-
chof et al.. 1990a). Rittschof and Bonaventura (1986) ar-
gue that distinct advantages are inherent in the use of
peptides as chemical cues in aquatic systems, including:
( 1 ) increased complexity of primary structure (compared
to amino acids), allowing opportunity for increased re-
sponse specificity; (2) background concentrations in ma-
rine systems are low (Mopper and Lindroth, 1982), al-
lowing for high signal to noise ratios; and (3) metabolic
inexpensiveness, because they need not be synthesized de
novo. but may be broken down from existing structural
and metabolic components.

Acknowledgments

We thank Dr. D. Fiorio for performing the amino acid
analysis of TV. sayi pumping factors. We are grateful to C.
Buswell, K. Eisenman, E. Herzog, S. Posey, M. Wach-
owiak and C. Wellins for technical assistance. This ma-
terial is based on research supported by the National Sci-
ence Foundation under Grants No. OCE-8603945 and
DCB-8701544.

Literature Cited

Branford, J. R. 1978. The influence of daylength, temperature and
season on the hatching rhythm of Homanis gamntanis. ./. Mar. Biol.
ASXM: L'.K. 58: 639-658.

Burke, R. D. 1984. Pheromonal control of metamorphosis in the Pacific
sand dollar. Dendraster excentricus. Science 225: 442-443.

Carr, W. E. S., E. R. Hall, and S. Gurin. 1974. Chemoreception and
the role of proteins, a comparative study. Comp. Biochem. Physio!
47A: 559-566.

Christy, J. H. 1986. Timing of larval release by intertidal crabs on an
exposed shore. Bull. Mar. Sci. 39: 176-191.

Collins, A. R. S. 1975. Biochemical investigation of two responses in-
volved in the feeding behavior of Acanthaster planci (L.) II. Isolation
and characterization of chemical stimuli. J Exp. Mar. Biol Ecol.
17: 69-86.

DeCoursey, P. J. 1979. Egg hatching rhythms in three species of fiddler
crabs. Pp. 399-406 in Cyclic Phenomena in Marine Plants and An-
imals. E. Naylor and R. G. Hartnoll, eds. Pergamon Press. Oxford.

CONTROL OF CRAB LARVAL RELEASE

1 I

DeCoursey, P. J. 1983. Biological timing. Pp. 107-162 in The Biology
of Crustacea, Vol. 7, F. J. Vernherg and W. B. Vernberg, eds. Aca-
demic Press, New York.

Derby, C. D., and J. Atema. 1980. Induced host odor attraction in the
pea crab Pinnotheres maeulaliis. Biol. Bull. 158: 26-33.

De Vries, M. C. 1990. Control of egg hatching in crabs: a comparative
study of species from different tidal heights. Ph.D. dissertation. Duke
Univ., Durham, NC.

De Vries, M. O, and R. B. Forward Jr. 1989. Rhythms in larval release
of the sublittoral crab Neopanope sayi and the supralittoral crab Se-
sarma cincrcum Mar. Bio/. 100: 241-248.

De Vries, M. C., and R. B. Forward Jr. 1991a. Control of egg-hatching
time in crabs from different tidal heights. J Crust. Biol. In press.

De Vries, M. C., and R. B. Forward Jr. 1991b. Mechanisms of crus-
tacean egg hatching: evidence for enzyme release by crab embryos.
Mar. Biol.

Ennis, G. P. 1973. Endogenous rhythmicity associated with larval
hatching in the lobster Homarus gammartts. J. Mar. Biol. Assoc.
I '.A.'. 53: 531-538.

Forward, R. B., Jr. 1987. Larval release rhythms of decapod crusta-
ceans: an overview. Bull. Mar. Sci 41: 165-176.

Forward, R. B., Jr., and K. J. Lohmann. 1983. Control of egg hatching
in the crab Rhithropanopeus harrisii Biol. Bull 165: 154-166.

Forward, R. B., Jr., K. J. Lohmann. and T. \V. Cronin. 1982. Rhythms
in larval release by an estuarine crab (Rhithropanopeus harrisii). Biol.
Bull 163: 2X7-300.

Forward, R. B., Jr., D. Rittschof. and M. C. De Vries. 1987. Peptide
pheromones synchronize crustacean egg hatching and larval release.
Chem. Seines 12: 491-498.

Henrikson, R. L., and S. C. Meredith. 1984. Amino acid analysis by
reverse-phase high-performance liquid chromatography: precolumn
derivatization with phenyhsothiocyanate. Anal Biochem, 136: 65-
74.

Jolley, R. L. 1981 . Concentrating organics in water for biological testing.
Environ. Sci. Technol. 158: 874-880.

Lepore, M., and S. Gilchrist. 1988. Hermit crab attraction to gastropod
predation sites. Am. Zoo/. 28(4): 93A.

Knight-Jones, G. W. 1953. Laboratory experiments on greganousness
during setting in Balamis balanoidcs and other barnacles. J Exp.
Biol. 30: 584-598.

Mopper, K., and P. Lindroth. 1982. Diel and depth variations in dis-
solved free amino acids and ammonium in the Baltic Sea determined
by shipboard HPLC analysis. Limnol. Oeeanogr. 27: 336-337.

Morse, A. 1988. The role of algal metabolites in the recruitment process.
Pp. 463-475 in Marine Biodeterioration: Advanced Technique* Ap-
plicable to the Indian Ocean. M. Thompson and R. S. Nagahhu-
shanam. eds. Oxford & IBW. Bombay.

Pandian, T. J. 1970. Ecophysiological studies on the developing eggs
and embryos of the European lobster Homarus gammarus. Afar.
Biol 5: 154-167.

Rittschof, D. 1980. Enzymatic production of small molecules attracting
hermit crabs to simulated gastropod predation sites. J. Chem. Ecol.
6: 665-675.

Rittschof, D. 1985. Oyster drills and the frontiers of chemical ecology:
unsettling ideas. Am. Malucolog. Bull . Spec. Ed. 1: I I 1-1 16.

Rittschof, D., and J. Bonaventura. 1986. Macromolecular cues in ma-
rine systems. / Chem. Ecol. 12: 1013-1023.

Rittschof, D., L. G. Williams, B. Brown, and M. R. Carriker.
1983. Chemical attraction of newly hatched oyster drills. Biol. Bull.
164: 493-505.

Rittschof, D., D. keiber, and C. Merrill. 1984. Modification of responses
of newly hatched snails by exposure to odors during development.
Chem. Senses 9: 181-192.

Rittschof, D., R. B. Forward Jr., and D. D. Mott. 1985. Larval release
in the crab Rlulhropunopeus harrisii (Gould): chemical cues from
hatching eggs. Chem. Senses 10: 567-577.

Rittschof, D., R. B. Forward Jr., D. A. Simons, P. Amanlha Reddy, and
B. W. Erickson. 1989. Peptide analogs of the mud crab pumping
pheromone. Chem Senses 14: 137-148.

Rittschof, D., R. B. Forward Jr., and B. W. Erickson. 1990a. Larval
release in brachyuran crustaceans: functional similarity of the peptide
receptor and the catalytic site of trypsm. J. Chem. Ecol 16: 1359-
1370.

Rittschof, D., C. M. Kratt, and A. S. Clare. 1990b. Gastropod predation
sites: the role of predator and prey in chemical attraction of the hermit
crab Clibananus villains (Bosc). JMBA 70: 583-596.

Saigusa. M. 1981. Adaptive significance of semilunar rhythm in the
terrestrial crab Sesarma. Biol Bull 160: 31 1-321.

Saigusa, M. 1982. Larval release rhythm coinciding with solar day and
tidal cycles in the terrestrial crab Sesarma — harmony with the semi-
lunar timing and its adaptive significance. Biol. Bull. 162: 371-386.

Saigusa, M., and T. llidaka. 1978. Semilunar rhythms in zoeae-release
activity of the land crab Sesarma. Oecologia 37: 163-176.

Salmon, M., W. H. Seiple, and S. G. Morgan. 1986. Hatching rhythms
of fiddler crabs and associated species at Beaufort, North Carolina.
J. Crust. Biol 6: 24-36.

Tegtmeyer, K., and D. Rittschof. 1989. Synthetic peptide analogs to
barnacle settlement pheromone. Peptides9: 1403-1406.

Templeman, W. 1937. Egg-laying and hatching postures and habits of
the American lobster (Homarus americanus). J Eish Res. Bd. Can-
ada 3: 339-342.

Tierney, A. J., and D. VV. Dunham. 1982. Chemical communication
in the reproductive isolation of the crayfishes Orconecles propinqus
and Orconectes vinlis (Decapoda. Cambaridae). / Crust. Biol. 2:
544_548.

Trott, T. J., and R. V. Dimock. 1978. Intraspecific trail following by
the mud snail Ilvanassu ohsuleta. .Mar. Behav Physiol, 5: 91-101.

VValpole, R. E. 1974. Introduction to Statistics. Macmillan. New York.

Williams, A. B. 1984. Shrimps. Lobsters, and Crabs of the Atlantic
Coast of the Eastern United States, Maine to Florida. Smithsonian
Institution Press, Washington DC.

W'olcott, T. W., and D. L. Wolcott. 1982. Larval loss and spawning
behavior in the land crab Gecarcinus lateralis (Fremenville). J. Crust.
Biol. 2: 477-485.

Reference: Biol. Bull 180: 12-27. (February. 1991)

Particle Captures and the Method of Suspension
Feeding by Echinoderm Larvae

MICHAEL W. HART

Department of Zoology. NJ-15. University of Washington, Scuttle. Washington. 98195 and
Friday Harbor Laboratories, 620 University Road, Friday Harbor. Washington. 98250

Abstract. Motivated by discrepancies between two re-
cent descriptions of the suspension-feeding mechanism
employed by echinoderm larvae, I describe particle cap-
tures by the larvae of seven species of temperate eastern
Pacific echinoderms from four classes. When videotape
recordings of free-swimming larvae clearing plastic spheres
from suspension were analyzed, two modes of particle
capture were observed to operate. The majority of cap-
tured spheres were caught at the peripheral ciliated band
and then transported to the mouth, often by repeated
capture on portions of the band progressively nearer to
the mouth. This description is consistent with the ciliary
reversal model of suspension feeding described by R. R.
Strathmann. A small minority of captured spheres fol-
lowed broad, curving paths directly into the larval mouth
without interception at the ciliated band. These particle
paths resemble those described by T. H. J. Gilmour. The
videotape recordings also permitted a quantitative com-
parison of suspension feeding by these larvae. Several as-
pects of this behavior varied among developmental stages
or among types of larvae, including: the distribution of
particle captures among different segments of the ciliated
band, the number of captures for single particles en route
to the mouth, and the frequency of particles lost after
initial capture. This variation raises a number of questions
regarding the feeding performance of different larval spe-
cies and the efficacy of these different larvae as elements
of a reproductive strategy.

Introduction

The form and function of suspension-feeding aquatic
animals is of wide interest, in part because they face a

Received 27 November 1989; accepted 30 November 1990.

formidable challenge: concentrating materials and energy
from a pool of resources that is both patchily distributed
and highly dilute. Many different structures for concen-
trating food from suspension have evolved. These struc-
tures range from the relatively simple collar-cell filters of
sponges to the morphologically and geometrically com-
plex ciliated gills of bivalves and setose appendages of
many crustaceans (J0rgensen, 1966). The method of cap-
turing and concentrating food particles from suspension
undoubtedly affects the effectiveness of particle capture
and aspects of the growth and metabolism of suspension
feeders (Conover, 1968). Inefficient suspension feeding
may even limit the range of alternative strategies for
growth and reproduction (McEdward and Strathmann.
1987). Thus, even if we were not generally curious about
how organic particle filters work, there are particular rea-
sons (the diversity of filters, and the physiological and
evolutionary consequences of this diversity) for investi-
gating the nature of different kinds of filters that remove
food particles from suspension.

Suspension feeding by the planktonic larvae of echi-
noderms has been described by a number of authors
(Gemmill, 1914, 1916; MacBride, 1914; Runnstrom,
1918; Meeks, 1927; Tattersall and Sheppard, 1934; Gar-
stang, 1939; Strathmann, 1971, 1975; Strathmann el al..
1972; Gilmour. 1985. 1986, 1988a, b). These larvae de-
velop a band of tightly packed ciliated columnar epithelial
cells (the ciliated band) that circumscribes the mouth, di-
viding the surface of the larva into circumoral and aboral
fields (see Strathmann, 1971. 1975). Early workers offered
divergent interpretations of the method by which these
larvae concentrate suspended particles from seawater.
They variously attributed larval feeding abilities to the
actions of: (/') cilia on the circumoral field, (//(water cur-
rents generated by the ciliated band, (///) cilia surrounding

FEEDING BY ECHINODERM LARVAE

0.00

0.31

0.51

0.60

0.88

0.94

1.01

1.10

1.16

Figure 1. A collage of videotape frames showing the capture of a 20 ^m diameter sphere by a six-armed
echinopluteus (Dendrasler e\centricus). The number in the upper right of each panel is elapsed time in
seconds (starting arbitrarily at 0 s). The arrow in the first panel shows the initial position of the particle. For
scale, the arrow is about 135 ^m long. The larva is shown in anterior ventral view, moving forward toward
the top of each panel. The sphere moved toward the right postoral arm (0.00-0.51 s). was captured on the
ciliated band and changed direction toward the base of the same arm (0.60-0.94 s), then was captured a
second time near the base of the arm and moved toward the larval midline and mouth ( 1.01-1.16 s).

the mouth, and (;V) mucus secreted between opposed parts
of the ciliated band. The more recent studies of Strath-
mann (1971 ) and Strathmann et al. (1972 (resolved many
of these conflicting descriptions: these studies suggest that
echinoderm larvae remove particles from dilute suspen-
sions by the brief reversal of the direction of the beat of
cilia on the ciliated band. Particles are retained on the
circumoral field, at the upstream side of the ciliated band,
and then are transported toward the larval mouth. How-
ever, Gilmour (1985, 1986, 1988a, b) has disputed this
interpretation of larval feeding and has suggested two
completely different methods of particle capture.

Studies of suspension feeding by marine invertebrates
often suffer from the inherent difficulty of relating rates
of feeding to mechanisms of particle capture. For example,
there is no general agreement on how the naplius larvae
of copepods and barnacles capture particles, even though
these are among the best-studied suspension feeders (re-
viewed by R. Strathmann, 1987). The feeding mechanism
of nauplii is difficult to study because the movements of
the feeding appendages and food particles are swift and
complex. Measures of feeding rates of these animals are
therefore restricted to indirect observations, such as the
depletion of food particles (Paftenhoffer, 197 1 ) or the in-

M. W. HART

Figure 2. A cartoon of the particle capture sequence shown in Figure
1. The positions of the sphere in each panel of Figure 1 are indicated by
the dots, and the particle path between these positions is interpolated by
the solid line. The ciliated band ot the larva is shown by the heavy lines;
the mouth is shown in outline.

corporation of radioactivity from radiolabelled com-
pounds in food particles (Marshall and Orr, 1956). How-
ever, without direct observations of feeding, it is difficult
to relate variation in feeding rate U'.,s,'.. among different
naupliar stages) to variation in the morphological features
(e.g., the size and number of setae) that determine the
feeding mechanism.

Unlike nauplii, the feeding larvae of echinoderms lend
themselves to direct observation of particle capture. These
larvae are relatively transparent, they swim with slow and
continuous movement, and particle captures are suffi-
ciently slow events that they can be counted and described
with some precision. Given an accurate description of
particle capture by these larvae, one can then interpret
quantitative variation in feeding in terms of the particle
capture mechanism. Echinoderm larvae are therefore ex-
cellent model organisms for comparative studies of form
and function in suspension feeding.

In this report, I describe particle captures and suspen-
sion feeding by the larvae of seven species from four dif-
ferent echinoderm classes. A qualitative analysis of vid-
eotape recording (including still video images of particle
captures) of free-swimming larvae clearing a dilute sus-
pension of particles is generally consistent with Strath-
mann's description of the ciliary reversal suspension feed-
ing mechanism. My observations also refute Gilmour's
interpretation of the predominant method of particle
capture by echinoderm larvae. However, a quantitative
analysis of these recordings (which is difficult without a
permanent record of larval behavior) leads to several novel
inferences about larval feeding. First, these larvae appear
to have two modes of particle capture: most particles are
caught by apparent ciliary reversal at the ciliated band,
but a small proportion of particles are captured without
contacting the peripheral band, and this proportion does
not vary among the different larvae examined. Second,
changes in the distribution of particle captures on the cil-

iated bands of larvae do not correspond to changes in the
lengths of particular segments of the band as larvae grow;
some parts of the band appear to be more effective than
others, and this discrepancy changes during larval devel-
opment. Third, the number of independent ciliary rever-
sals involved in a single particle capture (from the pe-
ripheral band to the mouth) varies among segments of
the band, among developmental stages, and among species
of echinoderms. These analyses also serve as a basis for
quantitative comparisons of feeding performance among
echinoderm larvae of different size, shape, and develop-
mental stage that will be presented elsewhere.

Materials and Methods

Colled// in of adults

The sea urchin Strongylocentrotus pitrpiiratm (Stimp-
son, 1857) (O. Echinoida) was collected from tidepools
at Botanical Beach, Renfrew County, British Columbia.
Canada. All other adults were collected from intertidal or
shallow subtidal locations off the San Juan Islands. San
Juan County, Washington, USA. Strongylocentrotus
droebachiensis (O. F. Miiller, 1776) and the sea star Sty-
lasterias forreri (de Loriol, 1887) (O. Forcipulatida) were
collected by dredge from San Juan Channel. The sea star
Dermasterias imbricata (Grube, 1857) (O. Valvatida) was
collected at 10 m depth from a rock wall off Turn Island.
The sand dollar Dendraster excentricus (Eschscholtz,
1831) (O. Clypeasteroida) was collected from an intertidal
bed in East Sound, Orcas Island. The brittle star Ophio-
pholis aculeata (L., 1767) (O. Ophiurida) was collected
from a low intertidal cobble beach in Mitchell Bay on
San Juan Island. The sea cucumber Parastichopiis cali-
fornk'iis (Stimpson. 1857) (O. Aspidochirotida) was col-
lected at 15 m depth from a silt bottom off" Brown Island.

Cull tire of embryos and lan'ae

Gametes, embryos, and larvae were treated according
to methods described by M. Strathmann ( 1 987). Gametes
of echinoids were obtained by intracoelomic injection of
0.5 M KC1. Asteroid gonads were obtained by dissection;
oocytes were induced to mature by incubation in 10 6A/1-
methyladenine in seawater. Parastichopus gonads were
also obtained by dissection: oocytes were matured in a 1
g-r1 solution oflyophilized radial nerve in seawater; and
sperm were activated in 10 mA/ NH4C1 in seawater. The
radial nerves were obtained from the asteroid Pycnopodia
helianthoides (Brandt, 1835). Ophiopholis females, in
separate glass bowls filled with seawater, were allowed to
warm on the benchtop for several hours and were thus
induced to spawn; sperm were obtained by dissection.

FEEDING BY ECHINODERM LARVAE

0.00

0.52

0.20

0.71

0.57

0.41

0.84

Figure 3. A collage of videotape frames showing the capture of a 20 /jm diameter sphere by an eight-
armed ophiopluteus (Ophiopholis acn/ea/a) (the short postoral arms are not visible in this view). Numbers
and arrow as in Figure 1. For scale, the arrow is 87 jjm long. The larva is shown in anterior ventral view,
moving forward toward the lower left of each panel. The sphere moved past the tips of the right anterolateral
and posterodorsal arms (0.00-0.52 s), was captured on the right posterolateral arm (0.57-0.60 s), and changed
direction back toward the larval mouth (0.71-0.94 s).

0.60

0.94

For all species, eggs were washed in 5-^m filtered seawater,
fertilized with a few drops of a dilute sperm suspension,
then washed again and transferred in groups of a few
thousand to 3-1 glass jars filled with filtered seawater. The
jars were immersed in a flowing seawater bath at tem-
peratures of 9-l3°C (near local ambient sea temperature),
stirred gently by paddles. Feeding larval stages were fed
2-3 ml per jar from dense cultures of each of three algae
(Dunaliella tertiolecia Butcher, Isochrysisgalba.no. Parke,
and Rhodomonas sp.) at intervals of five to ten days coin-
cident with water changes. These combinations of algae
produced initial algal concentrations of about 10 cells ^r'
in the jars. Over five to ten days, groups of several hundred

or thousand larvae, clearing I -2 yul ' min (averaged over
time), probably captured most of this food.

Observing larval feeding

Larvae selected at random from the culture jar were
placed singly, by pipette, into the bottom of a 63 ml cy-
lindrical glass observation chamber (4.8 cm diameter by
3.5 cm deep) containing a suspension of 20 /xm diameter
polystyrene divinylbenzene microspheres (Duke Scien-
tific) at a concentration of 2.4 n\~ ' in filtered seawater.
The concentration of spheres was reduced to 1 M'~' for a
few very large Dennasterias larvae with very high clear-
ance rates. In those cases where the mouths of larvae were

M. W. HART

Figure 4. A cartoon of the particle capture sequence shown in Figure
3. The positions of the sphere in each panel of Figure 3 are indicated by
the dots, and the particle path between these positions is interpolated by
the solid line. The ciliated band of the larva is shown by the heavy lines;
the mouth is shown in outline.

too small to ingest 20 ^m spheres, or where small larvae
were unable to capture these particles, 10 nm diameter
spheres were used (these cases include all of the smaller
Opliioplwlis larvae, and several of the smallest Dendraster.
Parastichopus. and Strongylocentrotus purpiiratus larvae).
The larger spheres were used whenever possible, because
they were easier to identify and follow on videotape. Lar-
vae of a wide range of sizes and developmental stages
were used for all seven species. Temperatures inside the
observation chamber could be held within 0.5-1.0°C of
ambient seawater temperature because the chamber was
equipped with a circulating seawater jacket. The top of
the chamber was sealed with a clear plastic lid, eliminating
trapped air and preventing image distortion by surface
waves. As the larva swam from the bottom to the top of
the chamber, several minutes of feeding were observed.
For most larvae, several such feeding periods were ob-
served. After each feeding period, the larva was returned
to the bottom of the chamber by pipette and observed as
it again swam upward.

Some larvae did not swim or capture spheres at high
rates. These individuals were not used in subsequent
analyses. Slow swimming, frequent stops, infrequent par-
ticle captures, or rejection of captured spheres by these
larvae were probably the result of disturbance during
transfer from the jar to the observation chamber. Under
the conditions described, many larvae swam rapidly and
had high clearance rates, but readers should not assume

that larvae exhibit such behavior continuously, or that all
larvae will do so under any conditions of observation.

I tried to get larvae to capture and ingest a number of
other kinds of artificial particles, including Sephadex
spheres of various sizes, other types of plastic spheres, and
ragweed pollen, with variable success. I also used various
unicellular algae. Some of these algae [e.g.. Ixochrysis gal-
bana, Pavlova lutlwri (Droop)] are small or non-refractile;
others (e.g., Diimiliella lertiolecta) are larger but tend to
clump in suspension. The most promising cultured uni-
cellular organism was the dinoflagellate Prorocentrum
micans Ehrenb., which is large and highly visible, does
not clump, and keeps itself suspended in water by flagellar
movements. Unfortunately, many larvae refused to cap-
ture or ingest these cells. Strathmann ( 197 1 ) used the di-
noflagellate Amphidiniwn carteri Hulburt, which I did
not have in culture. Polystyrene spheres are useful for
observations of suspension feeding because they are highly
retractile, are available in a range of sizes, do not readily
form clumps, settle from suspension slowly, and are
readily captured and ingested by echinoderm larvae. An
added advantage of indigestible particles is that larvae are
unlikely to become quickly satiated as they clear particles
from suspension.

Videotape recordings of larvae feeding were made with
transmitted light at 30 frames s~' with a videocamera
mounted on the trinocular head of a dissecting microscope.
I controlled both the focus and field of view manually. Thir-
teen to forty-four individuals were videotaped for each spe-
cies, and I made some observations of feeding by larvae that
were not taped. For illustrations of particle captures, single
video frames were captured from the videotape by a frame
grabber. The size and contrast of the sphere were increased
in each of these images, and much of the background con-
trast was removed. These computer-enhanced images were
then laser-printed and assembled into collages.

I calculated a clearance rate (volume of water cleared of
particles per unit time, in ^1 min"1) for each larva by
counting particle captures and dividing the total number
of captures by the length of the observation period, then
dividing this capture rate (number min~') by the concen-
tration of spheres in suspension (number ^L~')- Only pe-
riods of continuous swimming and feeding were used,
therefore the calculated clearance rates represent maximum
feeding performance over several minutes. A number of
laboratory artifacts, including handling and transfer, high
light intensity, and novel food particles, may affect the rate
of feeding and the method of particle capture (Strathmann,
1971). Therefore, interpretations of the method of particle
capture must be based on observations of larvae clearing
particles from suspension at near maximal rates. High
clearance rates indicate that the behavior of larvae in the
laboratory has not been strongly altered by any of these

FEEDING BY ECHINODERM LARVAE

0.00

0.30

0.50

0.66

0.76

1.03

1.20

1.66

Figure 5. A collage of videotape frames showing the capture of a 20 nm diameter sphere by a bipinnaria
(Dermasterias imbricata). Numbers (in the upper left) and arrow as in Figure 1. For scale, the arrow is 79
^m long. The larva is shown in ventral view, moving forward toward the upper right of each panel. The
sphere approached the ciliated band on the right side lateral to the larva! mouth (0.00-0.50 s), was captured
there (0.66 s). and changed direction back toward the circumoral field (0.76-0.90 s). The sphere was captured
a second time, on the preoral transverse ciliated band ( 1 .03 s) and then swept into the mouth ( 1 .20- 1 .66 s).

artifacts. Rates of growth and development of larvae in
nature are probably often limited by low phytoplankton
concentrations (Paulay et al, 1985; but see Olson and Ol-
son, 1989). High clearance rates are probably typical of
larvae feeding on these dilute phytoplankton suspensions.

Measuring ciliated band lengths

Larvae were removed from the observation chamber,
killed in a dilute solution of formalin in seawater, then
mounted in a drop of seawater beneath a raised coverglass.
Ciliated band length was estimated by summing the dis-
tances between sequential landmark points on the band

(such as the tips and bases of the larval arms of plutei).
The planar location of each landmark was determined by
digitizing a camera lucida tracing of the band for each
mounted larva; the location of each landmark in the third
dimension, when in focus under the microscope, was de-
termined from the vertical displacement of the microscope
stage (McEdward, 1985).

Results

Particle captures

All larvae typically swam with the anterior end upper-
most, from the bottom of the observation chamber, up

M. W. HART

Figure 6. A cartoon of the particle capture sequence shown in Figure
5. The positions of the sphere in each panel of Figure 5 are indicated by
the dots, and the particle path between these positions is interpolated by
the solid line. The ciliated band of the larva is shown by the heavy lines;
the mouth and stomach are shown in outline.

toward the observer and videocamera, capturing spheres
as they swam. Runnstrom (1918) described this and a
variety of other swimming postures; I observed some of
them (most notably a lateral swimming direction, usually
with the ventral side uppermost, as the larva swam slowly
along the bottom of the chamber). These alternative
swimming patterns were usually associated with low rates
of feeding and frequent general ciliary arrests during which
the larva came to a halt on the chamber bottom. I am
not sure whether these behaviors are likely to be common
in the plankton.

The aborally directed beat of cilia on the ciliated band
produces water currents with a net posterior component
that drives the larva forward while moving water laden
with particles toward the ciliated band. Polystyrene
spheres entrained in these currents approached the ciliated
band on the upstream side of the band (usually on the
arms of plutei, or on the loops of band between the bases
of the arms, and on the anterior, posterior, and lateral
portions of the band on bipinnariae and auriculariae). In
cases where the proximity of the particle to the ciliated
band could be judged, spheres appeared to approach
within about one diameter of the surface of the larva ( 10-
20 ^m), less than the length of the cilia on most parts of
the ciliated band (20-30 ^m; Strathmann, 1971; Mc-
Edward, 1984). For larvae that were actively feeding,
spheres approached the ciliated band, then abruptly
changed direction at the band, and moved back toward
the circumoral field rather than passing over the band
toward the aboral field. On nearby portions of the band.

water continued to pass over the band, while spheres were
retained on the circumoral field (thus they were concen-
trated from suspension). Subsequent to this initial capture,
spheres caught near the mouth often were swept imme-
diately into the suboral pocket, probably aided by the
beat of cilia on the circumoral field (Runnstrom. 1918)
and by water currents generated by the aboral beat of cilia
on the transverse portions of the ciliated band directly
anterior and posterior to the mouth (the preoral and pos-
toral transverse bands, respectively; see Strathmann,
197 1 ). Spheres captured at any great distance (more than
50-100 fjm) anterior or posterior to the mouth were often
captured repeatedly on portions of the ciliated band pro-
gressively closer to the mouth; they were then transported
to the mouth, probably by the same two mechanisms de-
scribed above. I observed hundreds of such captures for
each species examined; the specific descriptions that follow
are for four particular species (one for each larval type),
but they apply equivalently to other larvae of the same
type.

Figures 1. 3, 5, and 7 show sequences of frames, from
videotapes of particle captures like those described above,
for an echinopluteus (Dendraster excentricus. Fig. 1), an
ophiopluteus (Ophiopholis aculeata. Fig. 3), a bipinnaria
(Dcrmasterias imbricalu. Fig. 5), and an auricularia
(Parastichopus califomicns. Fig. 7). The accompanying
line drawings (Figs. 2, 4, 6, and 8) depict the paths of
spheres shown in the photocollages. These four pictorial
accounts of particle captures are representative of almost
all of the several thousand captures that I observed. Figures
1 and 2 show the abrupt change in direction of a sphere
at the ciliated band of a six-armed echinopluteus, on the
right postoral arm (the larva is shown in ventral view).
The sphere was captured twice enroute to the mouth, once
near the arm tip, and once nearer the base of the arm. A
similar pluteus capture, on the right posterolateral arm
of an advanced ophiopluteus, is shown in Figures 3 and
4. In this sequence, the sphere was held briefly on the
ciliated band on the leading edge of the arm, then moved
back toward the circumoral field (between the opposed
bands on the arm) and the mouth. Because the oral hood
above the mouths of these larvae is opaque, the end of
the particle path cannot be followed into the mouth and
esophagus. Figures 5 and 6 illustrate the capture of a
sphere by a large bipinnaria: the sphere first approached
the ciliated band on the right side of the larva, lateral to
the suboral pocket and mouth. The sphere crossed the
circumoral field, was arrested at the band, and moved
back toward the mouth; it was captured again on the an-
terior transverse ciliated band (near the mouth) and was
then swept into the mouth. A similar capture by an au-
ricularia is shown in Figures 7 and 8; the sphere was cap-
tured first on the dorsal part of the ciliated band anterior

FEEDING BY ECHINODERM LARVAE

0.00

0.42

0.67

1.69

Figure 7. A collage of videotape frames showing the capture of a 20 ^m diameter sphere by an auricularia
(Parastichopus californicus). Numbers and arrow as in Figure I. For scale, the arrow is 1 28 ^m long. The
larva is shown in ventral view, moving forward toward the upper left ot each panel. The sphere approached
the dorsal ciliated band on the right side anterior to the larval mouth (0.00-0.42 s), was captured there
(0.067 s). and changed direction posteriorly along the circumoral field toward the right lateral portion of
the band (1.09-1.39 s). The sphere was captured a second time, lateral to the mouth (1. 69 s), and then
moved toward the larval midline and into the mouth ( 1. 83-2. 69 s).

to the mouth, then was recaptured on the lateral ciliated
band before entering the suboral pocket and mouth.

Larvae of all species occasionally captured spheres
without close approach of the sphere to the ciliated
band, and without abrupt change in the direction of
movement of the sphere at the band. Such a particle
capture (by the same Dennasterias larva illustrated in
Figs. 5 and 6) is shown in Figures 9 and 10. These few
spheres followed broad, curving paths into the suboral
pocket of the larva, where they were swept into the larval
mouth (probably by the current generated by the cir-
cumoral cilia). These particle paths resembled those

described by GUmour (1985, 1986, 1988b). Strathmann
(1971) also depicted such particle captures, but did not
emphasize their frequency or importance. I observed
44 individuals of Strongylocentrotus droebachiensis
capture 1594 spheres; of these, only 80 (5.2%) were
caught without an approach and a change of direction
at the ciliated band. Similar proportions obtained for
1 3 Parastichopus (23 of 438 captures without ciliary
reversals, (5.3%) and 17 Dennasterias (24 of 504 cap-
tures, 4.8%). These proportions do not vary significantly
among species (compared by contingency table analysis,
X2 = 0.118, P> 0.90).

M. W. HART

Figure 8. A cartoon of the particle capture sequence shown in Figure
7. The positions of the sphere in each panel of Figure 7 are indicated b>
the dots, and the particle path between these positions is interpolated by
the solid line. The ciliated band of the larva is shown by the heavy lines;
the mouth and stomach are shown in outline.

Some readers may be unconvinced that collages of still
video frames can accurately represent the dynamic events
involved in particle capture by these echinoderm larvae.
I encourage such readers to photocopy the collages (en-
larging them, if possible), to cut the frames of each collage
out of the photocopy, and then to view the frames, as a
stack of flip pictures, thus simulating the particle move-
ment that occurs during the capture of spheres. Especially
skeptical readers, who will be persuaded by nothing else,
can contact me about receiving a copy of a short videotape
sequence that demonstrates these particle captures.

The distribution oj particle captures on ciliated bands

Spheres were caught on all parts of the ciliated bands
of larvae, including the most anterior and posterior por-
tions of the bands of auriculariae and bipinnariae and the
tips of the arms of echinoplutei and ophioplutei. For
Parastichopus larvae, 169 spheres (41.0%) were captured
by ciliary reversal on the anterior portions of the ciliated
band, 118 (28.5%) on the band lateral to the suboral
pocket and mouth, and 127 (30.7%) on the portions of
the band posterior to the mouth; for Dermasterias larvae,
the same distribution was 242 (50.4%) anterior, 122
(25.4%) lateral, and 1 16 (24.2%) posterior captures (Table
I). These distributions vary significantly between species
(compared by contingency table analysis, X2 = 8.471, P
= 0.015), perhaps because the lengths of the different seg-
ments of the band vary as well. This is a difficult com-
parison (between the lengths of segments of the band and
the proportion of captures by those segments) for bipin-
nariae and auriculariae, because the same landmarks that

can be used to identify the locations of captures on vid-
eotape cannot always be precisely identified on the draw-
ings of ciliated bands used to measure band lengths.

A similar comparison is more easily made among dif-
ferent developmental stages of echinoplutei, because such
landmarks (the tips and bases of the larval arms) are
readily identifiable on these larvae from all aspects. The
growth of early pluteus stages involves the addition of
ciliated band to only a few portions of the band (especially
the postoral and anterolateral arms), whereas larger plutei
grow by elongating other arm pairs, as well as that part
of the band carried on the body of the larva (see Strath-
mann, 1971, 1975). All segments of the ciliated band (four
arm pairs and the larval body) grew as Strongylocentrotus
droehachiensis larvae progressed from four- to six- to
eight-armed stages (Fig. 1 1 ); most of Ihe post hoc pairwise
contrasts (four- v.v. six-armed, or six- vs. eight-armed)
among these mean band lengths were significant (Table
II). But in three cases, these size increases led to no mea-
surable increase in the maximum clearance rate of the
same segment (determined by counting particle captures
on each segment). Eight-armed larvae had longer postoral
and anterolateral arms, and longer ciliated bands on the
larval body, than did six-armed larvae, but mean clearance
rates for these segments of the ciliated band were no greater
for the more advanced larval stage (Fig. 1 1, Table II). In
a fourth case, feeding performance for one segment of the
band declined: the length of the ciliated band borne on
the larval body was similar for four-armed and six-armed
stages, but the mean clearance rate for that portion of the
band was significantly lower for the later larval stage. The
lack of correspondence between size and performance of
various parts of the ciliated bands of plutei suggests that
some segments of the band are more effective at particle
capture than other segments, and that this variation
among segments changes as larvae develop.

Repeated capture of particles

One striking aspect of particle capture by echinoderm
larvae was the repeated capture of individual spheres on
the ciliated band. Figures 1 and 7 show good examples
of such events. In many cases, these repeated captures
produced a sort of pinball effect as spheres "bounced"
from peripheral portions of the band to segments of the
band nearer the mouth. I counted as many as 1 1 distinct
capture events for single spheres caught by Dermasterias
and Parastichopus larvae (Table I), though most spheres
were captured 1-4 times, and even spheres captured near
the most anterior or posterior ends of the band could be
transported directly to the mouth after a single capture
on the band. The mean number of captures varied among
segments of the band (anterior, lateral, and posterior to

FEEDING BY ECHINODERM LARVAE

0.00

0.93

/•

•>''.- '

••'•• /

••'

^ ?''*-
t,-r

1.92

2.32

2.62

Figure 9. A collage of videotape frames showing the capture of a 20 ^m diameter sphere by a bipinnaria
(Dermasterias imbricala). Numbers and arrow as in Figure 5. For scale, the arrow is 89 ^m long. The larva
is shown in ventral view, moving forward toward the top of each panel. The sphere approached the left
anterior side of the larva (0.00-1.74 s) and was swept directly into the larval mouth (1.92-2.62 s) without
close approach to any part of the ciliated band and without changing direction at the band.

the mouth) for both species. Spheres initially caught lateral
to the mouth were captured fewer times before ingestion
than were spheres caught either anterior, or posterior, to
the mouth (comparison of mean capture numbers by
analysis of variance and post hoc contrasts for Parasti-
chopiis, ¥ = 39.60; for Dermasterias, F = 69.84; for both
comparisons, P < 0.001). Spheres caught initially on the
anterior part of the ciliated band were also captured more
times than those caught initially on the posterior end of
the larva (for Parastichopus, F = 16.96, P < 0.001; for
Dermasterias, F = 5.60; P = 0.018). The mean (± one
standard deviation) number of captures for all spheres
was also greater for Parastichopus (2.123 ± 1.254) than

for Dermasterias (1.944 ± 0.890) (compared by /-test, /
= 2.488, P = 0.013). These observations support the
probable role of cilia on the circumoral field in trans-
porting captured particles to the mouth. Spheres captured
several hundred micrometers posterior to the mouth could
be moved swiftly to the suboral pocket, in spite of the
anterior direction of movement of the whole larva. In
similar captures, larvae of Parastichopus, which lack cir-
cumoral ciliation (Strathmann, 1971), retained captured
spheres more often en route to the mouth (see above)
than did asteroid larvae, whicrfjiave abundant circumoral
cilia (Gemmill, 1914, 1916; Tattersall and Sheppard,
1934; Strathmann, 1971).

M. W. HART

Figure 10. A cartoon of the particle capture sequence shown in Figure
9. The positions of the sphere in each panel of Figure 9 are indicated by
the dots, and the particle path between these panels is interpolated by
the solid line. The ciliated band of the larva is shown by the heavy lines;
the mouth and stomach are shown in outline.

Most spheres caught by echinoplutei were captured just
once on the ciliated band, but the incidence of multiple
captures of spheres increased for Strongylocentrotus droe-
bachiensis as these larvae developed more arms: for four-
armed larvae (n = 9), 10.8 ± 2.7% (mean ± S.E.) of spheres
captured were retained at more than one location on the
ciliated band before entering the mouth; for six-armed
larvae (n = 18), 16.1 ± 2.5%; for eight-armed larvae (n
= 17). 21.6 ± 2.0%. Analysis of variance ofarcsine-trans-
formed proportions suggests that this is a significant in-
crease in the incidence of multiple captures of spheres (F
= 4. 1 1, P = 0.023). Thus the complexity of particle paths
to the mouth increases as plutei increase in size and change
shape.

Retention of captured particles

Larvae of all species rarely failed to move to the mouth
particles that had been removed from suspension at the
ciliated band. For example, of 443 spheres captured by
Parastichopus larvae at the ciliated band (where the site
and number of captures for each sphere could be deter-
mined), only 29 (6.5%) were lost before reaching the
mouth (Table I); Dermasterias larvae lost only 11 of 49 1
such spheres (2.2%). The frequency of loss did not vary
significantly among segments of the band (anterior, lateral,
and posterior to the mouth) for Dermasterias larvae
(compared by contingency table analysis, \2 = 0.71, P
> 0.25). The same proportions varied significantly for
Parastichopus (X2 = 12.33, P < 0.001), mainly because I

observed no spheres lost from the lateral portions of the
ciliated bands of these larvae. The certainty of retention
and transport from the initial site of capture to the mouth,
often a distance of hundreds of micrometers, was re-
markable. The exceptions to this generalization include
a few small echinoplutei and bipinnariae that were unable
to retain the larger spheres at the ciliated band, and some
ophiuroid larvae that occasionally captured spheres with-
out ingesting them. In these cases, some spheres ap-
proached the ciliated band on the upstream side, changed
direction toward the circumoral field, then subsequently
passes over the band and were lost. Thus, under some
circumstances, some larvae may reject particles before
they reach the mouth. Control over particle captures at
the ciliated band may allow the collection of food to be
inhibited even as the larva continues to swim forward

Table I

Main niunher of captures lor single spheres caught hy larvae of (A)
Parastichopus caiifornicus and (B) Dermasterias imbncata

A. Parastichopus caiifornicus

Ciliated band segment

Lateral

Posterior

x (range)
SD

(Spheres ingested)

2.592(1-11)
1.510
169

1.517 (1-7)
0.855
118

2.039(1-5)
0.858

127

X (range)
SD
n

(Spheres not ingested)

2.643(1-5)
1.277
14

2.733(1-10)
2.314
15

B. Dcnnaslenas nnhriciiui

Ciliated band segment

Lateral

Posterior

(Spheres ingested)

X (range)

2.21 1 (1-10)

1.369(1-5)

1.991 (1-4)

0.947

0.619

0.761

242

122

116

(Spheres not ingested)

X (range)

1.200(1-2)
0.447

1.250(1-2}

0.500

1 .000 ( I )
0

Observations are tabulated by ciliated band segment (anterior, lateral,
or posterior to the mouth of the larva) and by capture success (ingested
or not ingested). SD = standard deviation; n = number of spheres.

FEEDING BY ECHINODERM LARVAE

under conditions where the mouth is jammed with par-
ticles, or the particles are not desirable, or the larva is
attempting to reject particles from its buccal cavity
(Strathmann, 1971).

Clearance rates

Maximum clearance rates ranged from 1-2 n\ min~'
for early larval stages (four-armed plutei and the simple
bipinnaria-shaped larvae of asteroids and holothuroids)
with short ciliated bands, to 6-10 v\ min~' for late larval
stages (the large eight-armed plutei and the bipinnariae
and auriculariae with large loops and folds of the ciliated
band) with longer bands. Maximum clearance rate in-
creases with the length of the ciliated band in all of these
larvae (Strathmann, 1971; M. Hart, unpub. data).

These clearance rates are similar to those of other larvae
of comparable size and type, but measured by very dif-
ferent techniques. Strathmann ( 197 1 ) measured clearance
rates for larvae by two methods: counting algal cells en-
tering the mouths of swimming larvae, or counting cells
in the guts of larvae left briefly in algal suspensions. Lucas
( 1982) measured clearance rates for groups of larvae by
estimating the depletion of algal cells from suspension in
prolonged feeding trials (of about 24 h duration). The
similar range of clearance rates estimated for larvae of
similar types clearing algal cells or polystyrene spheres
from suspension suggests that the use of artificial sus-
pended particles can give accurate estimates of clearance
rates. Flavoring particles with some transferable factor
from algal cells may enhance the rate of ingestion of poly-
styrene spheres (Fenaux el al., 1985), but larvae capturing
unflavored spheres, in my study, ingested almost all of

Table II

pruhahiliiY value-- /nrposi hoc paired comparisons «t
mean lengths aiui <>/ mean maximum clearance rales among lan'al
stages of Strongylocentrotus droebachiensis. for di/t'emil st'xmail*
of the ciliated hand

Larval stage comparison

Ciliated hand segment

Four-armed (9)
v.v. six-armed (18)

Six-armed vs.
eight-armed (17)

Postoral arms

length

F = 34.762"*

F= 26.461***

maximum clearance rate

11.826***

3.030M

Anterolateral arms

length

50.647***

38.756***

maximum clearance rale

12.149***

0.003M

Posterodorsal arms

length

—

48.607***

maximum clearance rate

—

6.384*

Body

length

0.9S4"5

12.290***

maximum clearance rate

4.227*

l.617n!

Note that only one set of comparisons for poslerodorsal arms is made
(four-armed larvae lack these arms). Numbers in parentheses indicate
sample sizes for each larval stage. ***. P < 0.001; *, P < 0.05; ns, P
> 0.05.

the spheres captured on the ciliated band (see below).
Larvae may respond to particle flavor by altering the rate
of forward swimming and water processing.

Although these different measuring techniques produce
similar clearance rates, the techniques are not necessarily
equivalent. Strathmann (1971) found that maximum
clearance rates measured by counting algal cells captured

<u .
4

468
number of arms

4 6
number of arms

D preoral arms

1 posterodorsal arms

u anterolateral arms

@ postoral arms

• body

Figure 11. A bar graph showing the (A) length and (B) maximum clearance rate of different segments
ot the ciliated bands (borne on the larval body and on the postoral. anterolateral, posterodorsal, and preoral
arms) for four-, six-, and eight-armed larvae of Strongylocentrotus droebachiensis. The total height of each
bar indicates the mean ciliated band length or mean maximum clearance rate for whole larvae; for each
bar. the height of segments with different shading indicates the same measures for particular segments of
the ciliated band. Cartoons of larvae above each bar indicate the approximate changes in size and shape
from one stage to the next.

M. W. HART

during periods of 1-3 min were generally higher and less
variable than those measured by counting algal cells in
the guts of larvae left in algal suspensions for 5-13 min,
presumably because the latter periods include some in-
tervals when larvae are not feeding rapidly. Lucas' ( 1982)
highest clearance rate for Acanthaster larvae (5.8 ^1 min" '
for early brachiolaria larvae) is much lower than the high-
est maximum rate that I measured for Dermasterias larvae
of similar stage ( 10.0 ^1 min"1). However, because of the
large variance in clearance rates measured for different
individuals, it is difficult to make precise contrasts among
these three studies. Maximum clearance rates measured
by watching larvae for a few minutes should usually be
greater than rates measured by allowing larvae to feed for
many minutes or hours, but other factors may obscure
this effect.

One study is not consistent with the above prediction.
Rivkin et al. (1986) found exceptionally high clearance
rates (measured as the incorporation of radiolabel) for
echinoderm larvae capturing pHlthymidine-labelled
bacteria. For example, in feeding trials of ~4 h, the mean
clearance rate for larvae of Sterechinus neumayeri (an
echinoid) was 13.8 ^1 min"1. The largest clearance rates
(which were time-integrated averages) in their study must
have been substantially higher: the mean + 1 SD clearance
rate for Sterechinus was 18.5 ^' rnin '. The largest niu.\-
imum clearance rate I measured for an echinopluteus was
5.4 ^1 min"1 for a large Dendraster e.xcentricits. This is a
substantial difference. The thymidine-incorporation
technique appears sound. Unless these Antarctic larvae
are exceptionally large, these clearance rates may reflect
a dramatic adaptation for the rapid capture of very small
(<2 jim) particles. Measures of maximum clearance rates
by direct observation of these larvae would be of consid-
erable interest.

Ingestion of particles

Most larvae ingested captured spheres by accumulating
a bolus of spheres in the middle and lower esophagus.
They then swallowed the bolus into the stomach by a
rapid peristaltic contraction accompanied by opening of
the cardiac sphincter. Most other workers have observed
the same process. Other individuals, at times, did not
readily ingest spheres, but instead accumulated them in
a whirling mass that rotated within the buccal cavity under
the influence of water currents directed into the mouth
by the adoral cilia, and out of the suboral pocket by the
transverse ciliated bands. If this mass of spheres was not
ingested, it was eventually rejected from the buccal cavity,
probably by reversal of the direction of beat of the adoral
or other cilia of the buccal cavity ( MacBride, 1914; Gem-
mill, 1914, 1916;Runnstrom, 1918; Strathmann, 1971),

and then moved out of the suboral pocket over the pos-
toral transverse band. Rejection of a mass of spheres was
not accompanied by a general arrest or reversal of beat
of the cilia on the ciliated band (i.e., the larvae did not
stop swimming or swim backward), and the rejected mass
was not captured again at the postoral transverse band.
These events indicate an impressive subtlety of control
over ciliary beat that is probably modulated by the larval
nervous system (Burke, 1978, 1983).

Discussion

Methods of suspension feeding by echinoderm larvae

My observations of particle capture by echinoderm lar-
vae suggest a resolution of the conflicting accounts of sus-
pension feeding by these larvae. The majority of particle
captures (by all of the stages and species of larvae that I
examined) were similar to those described by Strathmann
( 197 1 ). The retention of particles on the upstream side of
the ciliated band of larvae, accompanied by a change in
the direction of particle movement toward the circumoral
field, supports the hypothesis that echinoderm larvae re-
move particles from suspension mainly by a brief, local-
ized reversal in the direction of beat of cilia on the ciliated
band (Strathmann et al.. 1972). However, about 5% of
all particle captures appeared to occur without the close
approach of the particle to the ciliated band and without
an abrupt change in the direction of particle movement
at the band. This proportion was similar among the three
species 1 examined; larvae of a fourth species (Stylasterias
forreri) also captured about 5% of the particles that they
encountered when prevented from generating ciliary re-
versals (Hart, 1990). The paths of particles caught by this
second method were reminiscent of those described by
Gilmour (1985, 1986, 1988b) for echinoplutei and bip-
innariae.

The resolution of these conflicting descriptions depends
on two factors: the availability of videotape as a permanent
record of behavior suitable for quantitative analysis; and
high rates of partical clearance, indicating normal larval
behavior uncompromised by laboratory artifacts. Lacking
any permanent record of larval feeding, Strathmann
probably described only the most common mode of par-
ticle capture that he observed for free-swimming larvae
in relatively large volumes of seawater. For his part, Gil-
mour has principally described particle captures by larvae
attached to suction pipettes or trapped between glass sur-
faces, and such methods of manipulating and orienting
larvae for observation may disrupt normal swimming and
feeding behaviors, due to the disturbing effects of strong
suction by the pipette, or to the close proximity of surfaces
and their large effect on flow patterns at low Reynolds
numbers ( Vogel, 198 1 ). Larvae may respond to these dis-

FEEDING BY ECHINODERM LARVAE

turbances with reduced clearance rates. At low clearance
rates, a few particles may enter the mouths of echinoderm
larvae without apparent change of direction at the ciliated
band, but this is not the method of particle capture that
is most common when larvae are processing water at high
rates (Strathmann, 1971, 1982; Hart, 1990). The particle
paths described by Gilmour (1985) also occur in free-
swimming larvae, but at a lower frequency than his studies
suggest. Because he has not reported clearance rates in
any of his studies, it is difficult to interpret Gilmour's
observations. Gilmour has probably observed larvae that
are not actively feeding. To the extent that larvae exhibit
such behavior in nature (perhaps in dense phytoplankton
patches, or in response to other disturbances), these ob-
servations may indicate the lower limit of the capacity of
larvae to reduce clearance rate in situations where feeding
is actively suppressed. Gilmour's methods are useful for
some kinds of observations, and larvae may feed at high
rates under these conditions if care is taken, but the in-
terpretation of observations on methods of suspension
feeding made under such conditions also requires careful
consideration.

I cannot account for the differences between Gilmour's
(1988a) description of particle capture by the auricularia
of Parastichopus califarnicus and my own observations
of feeding by these larvae. Parastichopus larvae in my
study removed large numbers of spheres from suspension
in a manner identical with that of plutei and bipinnariae.
I could not confirm Gilmour's (1988a) observation that
an encounter between an auricularia and a particle results
in a brief reversal in the direction of rotation of the larva
and entry of the particle to the suboral pocket. The ro-
tation of these larvae was not disturbed by particle capture,
and they cleared spheres from suspension at rates com-
parable to those for other larvae of similar size and de-
velopmental stage.

The kinds of descriptions I have presented are crucial
for the interpretation of quantitative aspects of suspension
feeding. For example, the observation that echinoderm
larvae retain captured particles at the ciliated band leads
to the prediction that the clearance rates of these larvae
should increase as their ciliated bands grow longer during
development (Strathmann. 1971). Such explicit predic-
tions are more difficult to derive for larvae (or other sus-
pension feeders) where feeding rates cannot be determined
by direct observation. For echinoderm larvae, one can
now try to interpret ontogenetic and phylogenetic varia-
tion in feeding rates as a consequence of the variation
in the length and arrangement of the ciliated band (see
below).

Larval shapes and the development of ciliated bands

The forms of echinoderm larvae vary among classes,
among species within classes, and among developmental

stages of single species. Suspension feeding by these larvae
covaries in several ways with these form differences. For
example, the number of capture events for single particles
varied among parts of the ciliated bands of both bipin-
nariae and auriculariae, and the same measure (averaged
over all segments) varied between these two larval forms.
The most significant of these differences, I think, are the
distribution of particle captures among segments of the
ciliated bands of echinoplutei and the change in this dis-
tribution during larval development. For Strongylocen-
trotus droebachiensis. the clearance rate of a single seg-
ment of the band was not necessarily reflected in the
growth of that segment as the larva grows and adds new
larval arms. The surprising implication of this result is
that some ciliated bands (on a single larva) are more ef-
fective suspension-feeding devices than are other bands.
LaBarbera (1981) made a similar observation for adult
articulate brachiopods. The ciliated lophophore of these
animals consists of a pair of lateral arms and a median
coil. The area-specific pumping rate (which would be pro-
portional to a clearance rate if LaBarbera had observed
particle captures instead of dye stream movement) of the
median coil was only about 60% of the rate for the lateral
arms. LaBarbera ascribed this difference to the geometrical
arrangement of the different parts of the lophophore and
the consequences of this geometry for shear stress and
viscous energy loss (resulting in lower fluid flow rates)
over the median coil.

This inference (of shape effects on feeding performance)
could clearly be extended to variations on the pluteus
form among echinoid species, or to variation among the
basic larval forms of different echinoderm classes. Emlet
(1991) has predicted that such effects could arise from
ontogenetic changes in larval shape or from phylogenetic
variation in ciliated band arrangement. Using scaled
models of whole larvae with different shapes, or of isolated
ciliated bands with different orientation, Emlet showed
that changes in both the gross morphology of larvae and
the arrangement of ciliated bands could enhance particle
capture rates (by increasing velocity gradients and fluid
flow rates over the band). My direct measurements of the
feeding performance of different ciliated bands confirm
that performance differences among larvae of different
development stages do manifest themselves, possibly due
to the fluid-mechanical effects described by Emlet. Other
observations (M. Hart, unpub. data) suggest that these
effects may also extend to comparisons among different
types of echinoderm larvae. If the geometrical develop-
ment of a ciliated band affects the functional performance
of that band, then there may be taxonomic biases in per-
formance associated with evolutionarily conserved differ-
ences in patterns of larval development.

M. W. HART

The evolution of larval form ami reproductive strategies

Two general conclusions derive from the previous dis-
cussion: all feeding echinoderm larvae employ the same
mechanisms to concentrate food particles from suspen-
sion; and quantitative aspects of feeding by these larvae
change during larval development. These conclusions in-
vite some interesting corollaries. First, the method of par-
ticle capture by echinoderm larvae has remained similar
among different classes in spite of considerable evolution
of larval form. The four types of echinoderm larvae are
not necessarily related phylogenetically in a manner ob-
vious from their gross organization. Raff et al. (1988),
Smiley ( 1 988), Smith ( 1 988), and Strathmann ( 1 988) have
all recently proposed phylogenies for the extant echino-
derm classes based on different combinations of morpho-
logical, embryological, and molecular information. In
spite of the apparent similarities in elaboration and or-
ganization of the ciliated band between ophiuroid and
echinoid larvae, and between holothuroid and asteroid
larvae, few of these phylogenies group the pairs of classes
together in this way. There are relatively few points of
agreement among the different phylogenies or among their
authors. One is left to conclude that there may have been
both convergent and divergent evolution of larval form
in echinoderms. However, the method of suspension
feeding by echinoderm larvae has apparently been strongly
conserved throughout the evolutionary history of the
phylum (though numerous groups have lost the means
and requirement to feed during larval development).

Second, quantitative variation in feeding among echi-
noderm larvae may imply variation in the effectiveness
of these different larvae as elements of a reproductive
strategy. Echinoderm larvae (and other feeding larval
forms) can be thought of as devices for turning small eggs
into large juveniles (by concentrating materials and energy
from the plankton). The effectiveness of these devices
turns on the relative rates of development and mortality
during larval life. The availability of food to larvae affects
the development of larval and juvenile structures and the
duration of the larval period (Fenaux el a!.. 1985; Paulay
et al.. 1985; Hart and Scheibling, 1988). Larval duration
figures prominently in several theoretical and comparative
treatments of life history evolution in marine invertebrates
(Vance, 1973; Christiansen and Fenchel, 1979; Strath-
mann, 1985; Emlet et al., 1987). Although all of the larvae
that I have observed use the same methods to remove
particles from suspension, they vary considerably in the
organization and development of the ciliated band (see
Figs. 2, 4, 6, 8). Some quantitative aspects of larval feeding
vary as larvae change shape, or vary among larvae of dif-
ferent classes. This variation may be reflected in measures
of clearance rates for different larvae. In this case, we could

reject the tacit assumption that all larvae are equivalent
solutions to the problem of building a large juvenile from
a small egg. The functional and life-historical conse-
quences of such a result are the subject of a second paper.

Acknowledgments

The Director and staff of the Friday Harbor Labora-
tories provided space, facilities, and assistance for which
I am grateful. Larry McEdward, Joe Pawlik, Richard
Strathmann, Malcolm Telford and the editors of the jour-
nal provided encouragement and helpful comments on
the manuscript. Larry McEdward generously loaned the
equipment and software for measurement of ciliated band
lengths. Richard Strathmann provided me with his trans-
lation of Runnstrom ( 1918). I was supported by NSF grant
OCE 8606850 and by an award from the Graduate School
Research Fund of the University of Washington, both to
Richard Strathmann.

Literature Cited

Burke, R. D. 1978. The structure of the nervous system of the pluteus

larfaofStrongylocentrotuspurpuratus. Cell Tisx. Rex 191: 233-247.
Burke, R. D. 1983. The structure of the larval nervous system of Pisaxler

nclmiceiix (Echinodermata: Asteroidea). J. Morphol. 178: 23-35.
Christiansen, F. B., and T. M. Fenchel. 1979. Evolution of marine

invertebrate reproductive patterns. Theor. Pop. Biol. 16: 267-282.
Conover, R. J. 1968. Zooplankton— life in a nutritionally dilute en-
vironment. Am. '/Mill. 8: 107-1 18.
Emlet, R. B. 1991. Functional constraints on the evolution of larval

forms of marine invertebrates: experimental and comparative evi-
dence. Am. /an/ in press.
Emlet, R. B., L. R. McEdward, and R. R. Strathmann.

1987. Echinoderm larval ecology viewed from the egg. Pp. 55-1 36

in Eclwwdcrm Siiulies. I '<>/. 2, M. Jangoux and J. M. Lawrence, eds.

A. A. Balkema, Rotterdam.
Fenaux, L., C. Cellario, and M. Etienne. 1985. Croissance de la larve

de 1'oursin Parucentmtus lividus. Afar. Bid. 86: 151-157.
Garstang, \V. 1939. Spolia Bermudiana. I. On a remarkable new type

of Auncularia larva (A. herinudenxix. n. sp.). Q. J Microsc. Sci.. N.

S 81: 321-345.
Gemmill, J. F. 19M. The development and certain points in the adult

structure of the starfish Axlerias rubens, L. Philos. Trans. R Soc

LoiHl. B Bin/ Sci. 205:213-294.
Gemmill, J. F. 1916. The larva of the starfish Porania pulvillux (O. F.

M.). Q. J. Microst: Sci.. N. S. 61: 27-50.
Gilmour, T. H. J. 1985. An analysis of videotape recordings of larval

feeding in the sea urchin Lyiechimis pictus (Verril). Can. J Zoo/. 63:

1354-1359.
Gilmour, T. H. J. 1986. Streamlines and particle paths in the feeding

mechanisms of larvae of the sea urchin Lytt'cliinux piclux Verril. J.

Exp. Mm Bin/. Kail 95: 27-36.
Gilmour, T. H. J. 1988a. Feeding behaviour of holothurian larvae.

Am. /Mill. 28: I67A.
Gilmour, T. H. J. 1988b. Particle paths and streamlines in the feeding

behaviour of echinoderm larvae. Pp. 253-258 in Echinoderm Biology.

R. D. Burke. P. V. Mladenov. P. Lambert, and R. L. Parsley, eds.

A. A. Balkema, Rotterdam.

FEEDING BY ECHINODERM LARVAE

Hart, M. \V. 1990. Manipulating external Ca:+ inhibits particle capture

b\ planktotrophic echmoderm larvae. Can J /no/ . in press.
Harl, M. \V., and R. E. Scheibling. 1988. Heat waves, baby booms.

and the destruction ot'kelp beds b\ sea urchins. Mar. Butt 99: 167-

176.
Jurnensen, C. B. 1966. Biology ol'Suspenvon Feeding. Pergamon Press,

Oxford.
LaBarbera, M. 1981. Water flow patterns in and around three species

of articulate brachiopods. J E.\p. Mar. Biol Ecol. 55: 185-206.
Lucas, J. S. 1982. Quantitative studies of feeding and nutrition during

larval development of the coral reel asteroid Acanthaster planci (L.),

J Exp. Mar. Biol Ecol. 65: 173-193.
MacBride, E. \V. 1914. Text-book oj Embryology \'ol. I. [nvertebrata.

Macmillan and Co.. London.
Marshall, S. M., and A. P. Orr. 1956. On the biology ofCalanusfin-

marchicus IX. Feeding and digestion in the young stages. J. Mar

Biol Ax.wc. C K 35: 587-603.
Mi-Edward, L. R. 1984. Morphometric and metabolic analyses of the

growth and form of an echinopluteus. J. E.\p. Mar. Biol Ecol 82:

259-287.
McEdward, I.. R. 1985. An apparatus for measuring and recording

the depth dimension of microscopic organisms. Trans Am Micros

Soc 104: 194-200.
McEdward, L. R., and R. R. Strathmann. 1987. The body plan of the

cyphonautes larva of bryozoans prevents high clearance rates: com-
parison with the pluteus and a growth model. Biol. Bull 172: 30-

45.
Meeks, A. 1927. Bipinnaria astengera (Echinodermata). from the

Northumberland plankton. Proc. Zool. Soc. Lond . I'l 1 1927: 159-

171.
Olson, R. R., and M. H. Olson. 1989. Food limitation of planktotrophic

marine invertebrate larvae: does it control recruitment success?. Inn

Rev. Ecol. Sysl. 20: 225-247.
PafTenhofer, G. A. 1971. Grazing and ingestion rates of nauplii, cope-

podids. and adults of the marine planktonic copepod Calami* hcl-

golandicus. Mar. Bio/. 11: 286-298.
Paulay, G., I,. Boring, and R. R. Strathmann. 1985. Food limited growth

and development of larvae: experiments with natural sea water. J

l-:.\p Mar Biol Ecol. 93: 1-10.
Raff. R. A., K. G. Field, M. T. Ghiselin, D. J. Lane, G. J. Olsen, N. R.

Pace, A. L. Parks, B. A. Parr, and E. C. Raff. 1988. Molecular

analysis of distant phylogenetic relationships in echinoderms. Pp.

29-41 in Echinodcrni Phytogeny and Evolutionary Biology. C. R. C.

Paul and A. B. Smith, eds. Clarendon Press, Oxford.

Rivkin, R. B., I. Bosch, J. S. Pearse, and E. J. Lessard.

1986. Bactenovory: a novel feeding mode for asteroid larvae. Science

233: 1311-1314.
Runnstrbm, J. 1918. Zur Biologic und Physiologic der Seeigellarve.

Bergen* MiiM-iini Aarhek, Naturvid Ruceke. ,\'r 1 . 60 pp.
Smiley, S. 1988. The phylogenetic relationships of holothurians: a

cladistic analysis of the extant echmoderm classes. Pp. 69-84 in

Eclunoderm Phytogeny and Evolutionary Biology. C. R. C. Paul and

A. B Smith, eds. Clarendon Press, Oxford.
Smith, A. B. 1988. Fossil evidence for the relationships of extant echi-

noderm classes and their times of divergence. Pp. 85-97 in Echino-
dcrni Phytogeny and Evolutionary Biology. C. R. C. Paul and A. B.

Smith, eds. Clarendon Press, Oxford.
Strathmann, M. F. 1987. Reproduction and Development oj Mamie

Invertebrate.* of the Northern Pacific Coast. University of Washington

Press. Seattle.
Strathmann, R. R. 1971. The feeding behavior of planktotrophic echi-

noderm larvae: mechanisms, regulation, and rates of suspension

feeding./ E.\p. Mar Biol Ecol 6: 109-160.
Strathmann. R. R. 1975. Larval feeding in echinoderms. Am. Zool

15: 717-730.
Strathmann, R. R. 1982. Comment on Dr. Gilmour's views on feeding

by hcmichordates and lophophorates. Can J Zool. 60: 3466-3468.
Strathmann, R. R. 1985. Feeding and nonfeeding larval development

and life-history evolution in marine invertebrates. Annu. Rev. Ecol.

Sysi. 16: 339-361.
Strathmann, R. R. 1987. Larval feeding. Pp. 465-550 in Reproduction

of Marine Invertebrates Vol. V. A. C. Giese. J. S. Pearse, and V. B.

Pearse. eds. Blackwell Scientific Publications and The Boxwood Press,

Palo Alto, CA.
Strathmann, R. R. 1988. Larvae, phylogeny. and von Baer's Law. Pp.

53-68 in Echmoderm Phylogeny and Evolutionary Biology. C. R. C.

Paul and A. B. Smith, eds. Clarendon Press, Oxford.
Slrathmann, R. R., 1. L. Jahn, and J. R. C. Fonseca. 1972. Suspension

feeding by marine invertebrate larvae: clearance of particles by ciliated

bands of a rotifer, pluteus, and trochophore. Biol. Butt. 142: 505-

519.
Taltersall, \V. M., and E. M. Sheppard. 1934. Observations on the

bipinnaria of the asteroid genus Luidia Pp. 35-61 in James Johnstone

Memorial I 'olume. LIniversity of Liverpool Press, Liverpool.
Vance, R. R. 1973. On reproductive strategies in marine benthic in-
vertebrates. Am Sat. 107: 339-352.
Vogel, S. 1981. Life in Moving Fluids. Willard Grant Press, Boston.

Reference: Bioi. Bull 180: 28-33. (February, 1991)

Retarded and Mosaic Phenotype in Regenerated Claw
Closer Muscles of Juvenile Lobsters

C. K. GOVIND. CHRISTINE GEE, AND JOANNE PEARCE

Life Sciences Division. Scarborough Campus, University of Toronto. 1265 Military Trail.
Scarborough. Ontario. Canada MIC 1A4

Abstract. The closer muscle in the paired claws of the
lobster Homanis americanus become determined into
their asymmetric form of a cutter and crusher type claw
during the 4th and 5th juvenile stages and differentiate
their fiber composition accordingly in subsequent juvenile
stages. Our aim was to study the effects of claw loss during
this critical juvenile period on muscle regeneration. Hence
the fiber composition of the paired closer muscles in newly
regenerated claws was examined histochemically following
removal of both claws either in the 4th and 5th stages or
in the 4th through 7th stages. The newly regenerated
muscle was retarded compared to its original counterpart
in both cases. In the former case, however, the retardation
was temporary as the muscle composition in later stages
resembled the original. Recovery in the latter was not
apparent in later stages, suggesting that retardation is more
permanent. Also in both protocols the newly regenerated
closer muscle occasionally displayed a mosaic distribution,
with slow fibers interspersed among fast fibers in a central
band that is normally homogenously fast. Therefore, loss
of the paired claws during a developmentally sensitive
period affects the phenotype of the regenerated muscle
with the change persisting for shorter or longer periods
depending on how often the claws are lost.

Introduction

Crustaceans have an amazing ability of dropping an
entrapped or endangered limb by breaking it off at a pre-
formed fracture plane, thus allowing the animal to escape.
Because such limb autotomy involves little loss of blood,
the animal usually lives to regenerate a new limb. The
ability to autotomize a limb varies not only among species,
or within a species, but also within an individual, in that

Received 7 August 1990; accepted 6 November 1990.

the chelipeds autotomize more readily than the walking
legs. This is the case in lobsters (Homanis americanus)
and particularly in their juvenile forms when a gentle
pinch to the cheliped will result in autotomy whereas the
walking limbs will need greater provocation. Indeed, lob-
sters with their solitary life-style and aggressive nature of-
ten lose claws in the wild and often lose them more than
once.

Following the loss of a claw, a new one is regenerated
which, in structure and function, resembles its predeces-
sor. Although smaller in size initially, the regenerate limb
grows over several molt cycles to assume pristine pro-
portion at which time there is little to distinguish it from
the original limb. A similar degree of fidelity applies in-
ternally, at least with muscles that regenerate the same
fiber types as the original in the claw closer muscles in
lobsters (Kent el ai. 1989). as well as in snapping shrimps
(Govind el ai. 1986) and crayfish (Govind and Pearce,
1985).

A variation seen consistently in the newly regenerated
closer muscle of the claw in crayfish and occasionally in
the major claw of snapping shrimps was the appearance
of a central band of fast fibers in a muscle that otherwise
comprises 100% slow fibers. Because this regional distri-
bution of fast and slow fibers is reminiscent of an early
developmental stage in the closer muscle of crayfish and
snapping shrimps, it was assumed that some aspects of
ontogeny were recapitulated during regeneration. Such a
variation in the phenotype did not persist, and the muscle
assumed its pristine character over the next few molt cy-
cles. These regenerative events were recorded in adult
crayfish and shrimps where the muscle is fully differen-
tiated. What would be the condition of regenerate muscles
that had not yet differentiated their adult phenotype? We
studied this question in the lobster Homanis americanus

REGENERATED LOBSTER MUSCLE

because we were familiar with the development of its closer
muscle (Govind, 1984, 1989).

The paired claws and closer muscles in the lobster,
Homarus americamts, become determined into a major
and minor type early in juvenile development and sub-
sequently differentiate their claw morphology and muscle
fiber composition into their final form. While loss of both
claws in the critical juvenile stages delays the determi-
nation of claw asymmetry to a later stage, more prolonged
loss suppresses asymmetry altogether (Govind and Pearce,
1989). With such clear-cut effects of claw loss on the de-
termination of asymmetry, it seemed likely that muscle
regeneration might also be affected. The present experi-
ments record the phenotypic variations in the paired claw
closer muscles of juvenile lobsters following regeneration.

Materials and Methods

Larval lobsters (Homarus americamts} were obtained
from the Massachusetts State Lobster Hatchery on Mar-
tha's Vineyard and reared communally at the Marine
Biological Laboratory, Woods Hole, Massachusetts, by
methods described previously (Govind and Kent, 1982).
Upon molting to the first post-larval or 4th stage, lobsters
were reared individually in plastic trays containing pieces
of oyster shells as substrate (Lang, 1975). On a daily basis,
the animals were fed frozen brine shrimp and checked for
molts to record their juvenile development.

Claws were removed by a gentle pinch, which elicited
a reflex autotomy, resulting in the claw breaking off at a
preformed fracture plane without much loss of blood.
Both claws were so removed within 24 h after the animal
had molted.

At the appropriate stages the regenerated paired claws
were autotomized and prepared for histochemical ex-
amination of their muscles based on the stability of the
myofibrillar ATPase enzyme to the pH of the incubating
medium (Ogonowski and Lang, 1979). Thus, at pH 8.
the enzyme is relatively stable in fast crustacean muscle,
and hence these fibers stain more intensely in frozen cross-
sections of the claw compared to slow muscle. The his-
tochemically treated cross-sections of the claws were pho-
tographed, and the resulting photographs were used to
calculate the percentage of fast and slow fibers. These cal-
culations were made from the medial region of the claw,
which provides the largest surface area, and hence is most
representative of the entire muscle.

Results

Regenerated phenotype is retarded

We have previously shown that juvenile 4th and 5th
stage lobsters reared with a substrate of oyster chips de-
velop paired asymmetric (cutter/crusher) claws, while their

counterparts reared without a substrate develop paired
symmetric (cutter/cutter) claws (Lang el at.. 1978). Both
rearing conditions were adopted for the present experi-
ments. Thus, in the first experiment with oyster chips as
a substrate, the development into asymmetric cutter and
crusher type muscles is shown by plotting the percent of
fast fibers in the paired original muscles (Fig. 1A). One
of the muscles rapidly accumulates fast fibers to make up
90% of its mass and thus becomes a cutter type closer
muscle. The slow fibers persist in a small (10%) ventral
band (Fig. 2b). The contralateral muscle, which is the
putative crusher, shows a more gradual loss of fast fibers,
making up 10-20% by the 8th or 9th stage (Fig. 1 A) and
becoming zero by the 1 3th to 20th stage. The fast muscle
in the putative crusher is restricted to a narrow central
region (Figs. 2a).

Following autotomy of the paired claws in the 4th and
5th stages, the regenerated muscles in the 6th stage have
a phenotype that is intermediate to the normal asymmetric
condition (Fig. 1A). The fast muscle composition of the
paired regenerated muscles is 57%. and 44%, while that of
the paired original muscles is 75% and 28%. The regional
distribution of fast and slow fibers, however, is similar
between original and regenerated claws in that the fast
muscle is restricted to a central band while the slow muscle
appears on either side (Fig. 2c, d). The regenerated muscle
therefore appears to be retarded in its development. This
retardation is temporary because the paired muscles show
a normal phenotype by the 8th or 9th stage, despite loss
of the paired claws in the 4th and 5th stages. In other
words, recovery of the muscle phenotype following claw
loss in the 4th and 5th stages occurs within 3 to 4 molts.
Loss of the paired claws for more prolonged periods,
such as from the 4th to the 7th stage, successively results
in the regenerated muscles in the 8th stage showing a re-
tarded phenotype (Fig. 1A). The percent fast muscle in
these regenerated muscles is 52% and 44% compared to
the 90% of a normal cutter muscle. In both retarded mus-
cles, the fast fibers are restricted to a central band (Fig.
3c, d) as compared to the normal cutter muscle in which
the fast fibers occur over the entire area except for a small
ventral band (Fig. 3b). The retardation in this case appears
to be more permanent because paired muscles examined
in the 10th stage still showed subnormal amounts of fast
fibers, between 60-70%.. Both muscles remained as pu-
tative cutter types as loss of the paired claws successively
from the 4th to the 7th stage prevents the determination
of bilateral asymmetry (Govind and Pearce, 1989). Re-
covery to 80-90%. fast fiber composition was still not seen
by the 13th to 15th juvenile stages, indicating that retar-
dation of the muscle phenotype may be more permanent
in these animals.

In the second experiment in which lobsters were reared
without a substrate of oyster chips, the development of

C. K. GOVIND /. / AL

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JUVENILE STAGES

Figure 1. Percent composition of fast fibers in the paired claw closer muscles of original (circles) and
regenerated (triangles) claws of juvenile lobsters reared with a substrate of oyster chips (A) and without a
graspable substrate (B). For the regenerated condition, the paired claws were removed in all of the previous
juvenile stages. Each point represents the mean and standard deviation of five animals. Curves fitting the
points for each of the paired claw muscles is drawn by eye. The two curves in ( B) were generated by arbitrarily-
assigning the muscle with the higher percentage of fast fibers to one group (upper curve) while its counterpart
was assigned to the second group (lower curve).

the paired closer muscles into symmetric cutter types was
followed by plotting the percent fast fibers in the paired
original muscles (Fig. 1 B). The paired muscles develop in
a parallel fashion, accumulating fast fibers until these
make up 80-90% of the total mass, and the remainder
are slow fibers restricted to a ventral band. In other words,
the paired muscles develop as typical cutter type muscles.
In comparison, the regenerated phenotype in lobsters that
had successively lost their claws from the 4th to the 8th
stage, is distinctly retarded (Fig. IB). The fast fibers in
these regenerated muscles is between 40-50% compared
to 80-90% in the original muscles. Moreover, as in the
first experiment, the retarded condition persists for several
subsequent stages at least until the 1 3th stage, which is as
far as we proceeded in this experiment.

Regenerated phenotype shows mosaic pattern

As described above, the distribution of fast fibers in the
paired closer muscles is restricted to a distinct central
band. In the putative cutter muscle, this fast band during
juvenile development rapidly enlarges to occupy almost
the entire cross-sectional face, except for a slim ventral
band (Figs. 2b, 3b). In the putative crusher, on the other
hand, this central fast band gradually diminishes in size
until it completely disappears (Figs. 2a, 3a). Throughout
these developmental changes, the central band of fast fi-
bers is homogenous and sharply delineated from the ad-
jacent slow fibers.

The homogeneity of the fast band, however, was dis-
rupted to various degrees in some of the regenerated mus-

cles following autotomy of the paired claws. The least
disruptive case was where slow fibers were occasionally
interspersed among the fast fibers, especially along the
lateral edges of the fast band (Fig. 2c, d; 3c, d). This gave
the fast band a ragged edge, which was in contrast to its
usual sharp edge. Much more disruptive cases involved
considerable interspersing of slow fibers in the fast band
(Fig. 4a, b), resulting in a distinct mosaic pattern.

Discussion

In a previous study (Kent el ai. 1989), we examined
the phenotype of the regenerated closer muscle following
claw loss in late juveniles and adults, when the claws and
closer muscles were well differentiated into cutter and
crusher types. In these cases, the regenerated claws and
closer muscles resembled their predecessors with consid-
erable fidelity. The present report examines the effect on
the regenerate muscle phenotype following the loss of both
claws in early juvenile stages when claw type is being de-
termined (Emmel, 1908; Lang el a/., 1978) and fiber typ-
ing in the closer muscle is being expressed (Govind and
Lang. 1978; Ogonowski el til.. 1980). Thus removal of
paired claws successively either in the 4th and 5th stages
or in the 4th through 7th stages resulted in a regenerated
phenotype that resembled the undifferentiated condition
in the normal 4th stage lobster. In the case where claw
loss encompassed only the 4th and 5th stages, the regen-
erate muscle completes its differentiation into crusher and
cutter types in subsequent stages. In the animals subjected
to more prolonged claw loss (i.e., from the 4th to the 7th

REGENERATED LOBSTER MUSCLE

Figure 2. Cross-sections through the paired original (a, hi claws of
a juvenile 6th stage lobster and through the paired regenerated (c. d)
claws of another 6th stage lobster in which the claws had been removed
in the 4th and 5th stages. Histochemical detection of myofibrillar ATPase
activity shows fast fibers staining more intensely than slow, and hence
the small, dorsally located opener muscle (arrow) is entirely slow while
the large closer muscle occupying most of the cross-sectional area has a
central band of fast fibers sandwiched dorsally and ventrally by slow
fibers. The fast band varies considerably in size between the paired original
muscles being narrow in the putative crusher muscle (a) and broad in
the putative cutter muscle (b). In the paired regenerate muscles, however,
the fast band is similar in size. Magnification 25X.

stage), however, the regenerate muscles have not com-
pletely differentiated into cutter types in the subsequent
3-5 stages. Thus the absence of the muscle during the
critical juvenile stages results in regenerate phenotype
being retarded. How long the muscle is retarded appears
to depend on how often the claws are lost; when lost for
two successive stages, the retardation is temporary but
when lost over several successive stages, the retardation
is more permanent.

A few of the regenerate muscles had slow fibers inter-
spersed in the fast muscle band, giving rise to a mosaic
distribution of these two types of fibers. This is an unusual
distribution of fast and slow fibers in the closer muscle of
lobsters as well as other decapod crustaceans. Thus, in
the claw closer muscle of lobsters (Ogonowski el ill., 1980),
crayfish (Govind and Pearce, 1985), snapping shrimps
(O'Connor el til. 1984), and hermit crabs (Stephens el

al. 1984), fast and slow muscle is regionally distributed;
the fast fibers are restricted to a band in the central region.
The closer muscle in the more anterior walking limbs in
lobsters (Mearow and Govind, 1986) and hermit crabs
(Stephens el ill. 1984) have a similar pattern. In no in-
stance has a mosaic distribution of fast and slow fibers in
the closer muscle been reported in the above mentioned
species.

Apart from the closer muscles listed above containing
discrete populations of fast and slow fibers, other muscles
that have been examined are composed of a single fiber
type, e.g., the abdominal extensor and flexor systems that
have separate fast and slow muscles in tailed crustaceans
(Govind and Atwood, 1982). Consequently, the appear-
ance of a mosaic distribution of fiber types is an uncom-
mon finding among decapod crustaceans. That such a
mosaic pattern occurs only in regenerated closer muscles
and not in the originals suggests that the instructions for
differentiating an entire muscle are not as robust as those

Figure 3. Cross-sections through the paired original (a, b) claws of
a juvenile 8th stage lobster and through the paired regenerated (c, d)
claws of another 8th stage lobster in which the claws had been removed
in the 4th, 5th, 6th, and 7th stages. The proportion of fast fibers is highly
asymmetric in the paired original closer muscles being restricted to a
narrow central band in the crusher claw (a) but widespread in the cutter
claw (b). In the paired regenerate muscles, however, the band of fast
fibers is symmetric. Magnification 15X.

C. K. GOVIND ET AL

Figure 4. Cross-sections through the claws of two juvenile 8th stage lobsters (a. h) showing different
degrees of interspersion of slow fibers (light staining) in the hand of fast fibers (dark-staining), resulting in a
mosaic appearance in the closer muscle. Magnification 35x.

for differentiating individual fiber types. Perhaps along
similar lines is our observation that in the regenerated
chelipeds of adult lobsters, the main limb nerve will often
travel in a scattered, diffuse fashion rather than in discrete
bundles. Although haphazard in appearance, the regen-
erated nerve contains the requisite motor and sensory
neurons.

While a mosaic distribution of fiber types within a
muscle occurs rarely in crustaceans, it is commonplace
among vertebrates where individual limb muscles are in-
nervated by a large number of motor neurons (Burke,
1981). Despite being randomly distributed within the
muscle, fibers comprising a motor unit are of the same
type. This has led to the suggestion that the innervating
neuron regulates muscle fiber properties. Such neuro-
trophic regulation in the lobster claw closer muscle is un-
likely as there are only two excitor neurons (Wiersma,
1961), both of which distribute to most of the muscle
fibers (Govind and Lang, 1974).

Our findings also underscore the very robust nature of
the regenerative capacity among juvenile lobsters. Apart
from the slowing down in muscle differentiation and the
occasional appearance of a mosaic distribution of fiber
types, conditions that may be ameliorated, the regenerate
muscle otherwise resembles its original counterpart. Thus,
the loss of claws seen particularly in the early juvenile
stages does not appear, in the long term, to impede the
differentiation of a typical phenotype in the closer muscle.

Acknowledgments

We thank Michael Syslow and Kevin Johnson for gen-
erous supplies of larval lobsters and the Natural Sciences
and Engineering Research Council of Canada for financial
support.

Literature Cited

Burke, R. K. 1981. Motor units: anatomy, physiology and functional
units. Pp. 345-422 in Handbook oj Physiology: The Nen'ous System.
Vol. II. J. M. Brookhart and V. B. Mountcastle, eds. Williams and
Wilkens. Baltimore.

Kmmel, V. E. 1908. The experimental control of asymmetry at different
stages in the development of the lobster. J. Exp. Zoo/- 5: 471-484.

Govind, C. K. 1984. Development of asymmetry in the neuromuscular
system of lobster claws. Bwl Bull. 167: 94-1 19.

Govind, C. K. 1989. Asymmetry in lobster claws. Am. Sci. 77: 468-
474.

Govind, C. K., and H. L. Atwood. 1982. Organization of the neuro-
muscular system. Pp. 63-103 in The Biology of Crustacea I '<>/. 3..
Neurobiology: Structure and Function, D. E. Bliss, H. L. Atwood,
and D. C. Sandeman. eds. Academic Press. New York.

Govind. C. K., and K. S. Kent. 1982. Transformation of fast fibres to
slow presented by lack of activity in developing lobster muscle. Nature
298: 755-757.

Govind, C. K., and F. Lang. 1974. Neuromuscular analysis of closing
in the dimorphic claws of the lobster. Homams americanus. J Exp.
Zoo/. 190: 281-288.

Govind, C. K., and F. Lang. 1978. Development of the dimorphic claw
closer muscles of the lobster llomarus americanus. III. Transfor-
mation to dimorphic muscles in juveniles. Biol. Bull 154: 55-67.

REGENERATED LOBSTER MUSCLE

Govind. C. K., and J. Pearce. 1985. Enhanced reappearance of fast

fibers in regenerating crayfish claw closer muscle. Dc\: Biol 107:

206-212.
Govind, C. K., and J. Pearce. 1989. Critical period for determining

claw asymmetry in juvenile lobsters. J. Exp. Znol 249: 31-35.
Govind. C. K., K. M. Mearow, and A. \\ong. 1986. Regeneration of

fiber types in paired asymmetric closer muscle of the snapping shrimp,

Alpheus heierochelis. J Exp. Biol. 123: 55-71.
Kent, K. S., J. Pearce, C. Gee, and C. K. Govind. 1989. Regenerative

fidelity in the paired claw closer muscles of lobsters. Can. J. Zoo/.

67: 1573-1577.

Lang, F. 1975. A simple culture system for juvenile lobsters. At/iiu-
ciiltnre 6: 389-343.

Lang, F., C. K. Govind. and \V. J. Costello. 1978. Experimental trans-
formation of fiber properties in lobster muscle. Science 201: 1037-
1039.

Mearow, K. M., and C. K. Govind. 1986. Neuromuscular properties

in the serially homologous lobster limbs. J. Exp Zoo/. 239: 197-

204.
O'Connor, K., P. J. Stephens, and J. M. Leferovich. 1982. Regional

distribution of muscle fiber types in the asymmetric claws of Cali-

fornian snapping shnmp. Biol. Bull. 163: 329-336.
Ogonowski, M. M., and F. Lang. 1979. Histochemical evidence for

enzyme differences in crustacean fast and slow muscle. J. Exp. Zoo/.

207: 143-151.
Ogonowski, M. M., F. Lang, and C. K. Govind. 1980. Histochemistry

of lobster claw closer muscles during development. / Exp. /.ool.

213: 359-367.

Stephens, P. J., L. M. Lofton, and P. klainer. 1984. The dimorphic-
claws of the hermit crab, Pagiimx pollicaris: properties of the closer

muscle. Biol. Bull 167: 713-721.
Wiersma, C. A. G. 1961. The neuromuscular system. Pp. 191-240 in

The Physiology ol 'Crustacea. Vol. 2, T. H. Waterman, ed. Academic

Press, New York.

Reference: Bio/. Bull. 180: 34-55. (February. 1991)

Gastropod Egg Capsules and Their Contents From
Deep-Sea Hydrothermal Vent Environments

R. G. GUSTAFSON, D. T. J. LITTLEWOOD, AND R. A. LUTZ

Institute of Marine and Coastal Sciences, Rutgers University, New Brunswick, New Jersey 08903

Abstract. Egg capsules from three different prosobranch
gastropods were retrieved from the Galapagos Rift and
Juan de Fuca Ridge deep-sea hydrothermal vent fields.
The morphology of these capsules and their excapsulated
embryos and larvae are described and illustrated. Based
on their capsule type and the protoconch morphology of
their contained larvae, 29 lenticular capsules from the
Galapagos Rift could be attributed to a provisionally de-
scribed neogastropod turrid, Phymorhynchus sp. But 3
inflated, triangular capsules from the Galapagos Rift, and
56 different egg capsules from the Juan de Fuca Ridge,
each shaped like an inflated pouch, could not be unam-
biguously assigned to a member of the known vent gas-
tropod fauna. The mode of development and potential
for dispersal is inferred from egg capsule type, the number
of embryos per capsule, and protoconch characters com-
parable to those of confamilial shallow-water gastropods
for which the type of development is known. These criteria
and a comparison to the known juvenile shell morphology
of Phymorhynchus sp., suggest that, after encapsulation,
this species develops planktotrophically and is capable of
long-range dispersal. Similar evidence suggests that the
larvae contained in the inflated triangular capsules from
the Galapagos Rift may also develop planktotrophically
after hatching; but the larvae in the pouch-like egg capsules
from the Juan de Fuca Ridge probably develop non-
planktotrophically without a dispersal stage. These de-
velopmental patterns are characteristic of shallow-water
members of the systematic groups to which these species
belong, indicating, as previous studies have shown, that
vent gastropods can persist in these patchy, ephemeral
environments in the absence of unique adaptations al-
lowing dispersal between active hydrothermal sites.

Received 14 August 1990; accepted 30 November 1990.

Introduction

Active hydrothermal vent systems accompanied by
dense benthic fauna occur at several widely separated sites
along active oceanic ridges in the eastern Pacific, from
48°N along the Juan de Fuca Ridge, to 22°S along the
East Pacific Rise. Known hydrothermal fields on the Juan
de Fuca Ridge are separated by as much as 100 km,
whereas separation along transform faults on the East Pa-
cific Rise indicates that vent fields are at least 100 km
apart in this region (Crane, 1985; Grassle, 1986). Local
vent habitats appear to be transient, with populations
being susceptible to intermittent establishment and ex-
tinction (Lulzeial., 1985; J. F. Grassle. 1985; Lutz, 1988).
Despite their apparent geographic isolation and ephemeral
nature, vent areas are characterized by the remarkable
similarity of their faunal assemblages (Lutz, 1988). Fun-
damental biological questions remain regarding both the
manner in which these ephemeral habitats are colonized,
and the mechanisms of organism dispersal and rates of
gene flow between discrete areas of hydrothermal activity
associated with contiguous and non-contiguous oceanic
ridge systems.

Because laboratory culture of deep-sea organisms is dif-
ficult (Turner et ui, 1985), many of our perceptions about
the development and larval dispersal of vent biota have
been, by necessity, inferred from analyses of egg size, fe-
cundity, and morphology of larval structures retained on
juvenile and adult specimens. Gastropod mollusks have
been widely used for such studies, because a record of the
larval developmental pattern can be inferred from the
morphology of the initial shell, comprising the Protoconch

I in non-planktotrophic species and, also, the Protoconch

II shell stages, in planktotrophic species (Powell, 1942;
Thorson, 1950;Shuto, 1974; Robertson, 1976: Jablonski
and Lutz, 1980).

The mode of larval development in recent (Rodriguez
Babio and Thiriot-Quievreux, 1974; Bandel, 1975a, b, c,

HYDROTHERMAL VENT EGG CAPSULES

1982; Bouchet, 1976a, b; Scheltema. 1978; Bouchet and
Waren, 1979b; Rex and Waren. 1982; Scheltema and
Williams. 1983; Lutz ctal., 1984, 1 986; Turner and Lutz,
1984; Turner ct al.. 1985;Colman ctal., 1986; Lutz, 1988;
Lima and Lutz, 1990) and fossil (Jung, 1975; Scheltema,
1978, 1981; Jablonski and Lutz, 1980, 1983; Bouchet,
1981;Hansen. 1982, 1983; Jablonski, 1986)prosobranch
gastropods has been classified as either planktotrophic or
non-planktotrophic based on criteria of larval shell mor-
phology formulated by Kesteven (1912). Dall (1924),
Powell (1942), Thorson (1950), Robertson ( 1971, 1976),
Rodriguez Babio and Thiriot-Quievreux (1974), Shuto
(1974), Sohl (1977), and Jablonski and Lutz (1980, 1983).

Prosobranch species with larval shells having 1.5 to 9
whorls, a distinct fine sculpture, a brown coloration in
contrast to a white or gray adult shell, a narrow high spire,
a clear difference between the Protoconch I and Proto-
conch II, and possibly a projection on the outer lip of the
larval shell which interdigitates with the velum [("sinu-
sigera" larvae in terminology of Robertson (1976)] are
categorized as planktotrophic. Species with larval shells
having 0.5 to 1.5 whorls, simple or no ornamentation,
the same coloration as the teleoconch, a large bulbous
apex, and no evidence of separation between the Proto-
conch I and Protoconch II are categorized as non-plank-
totrophic. In the general terminology of Thorson's (1950)
"apex theory." shells of the planktotrophic type are termed
multispiral or polygyrate and shells of the non-plankto-
trophic type are termed paucispiral.

Although these criteria allow differentiation between
planktotrophic and non-planktotrophic larvae, recent
culturing of trochoidean archeogastropods demonstrates
that the presence of a paucispiral protoconch is insufficient
evidence on which to discriminate between a planktonic
and a non-planktonic larval existence (Hadfield and
Strathmann, 1990). Of four trochoideans cultured, Had-
field and Strathmann (1990) found two with pelagic de-
velopment of 7 d or more and two with entirely benthic
life histories, although all four produced veliger larvae
and had similar inflated paucispiral protoconchs. Al-
though the mode of larval development in shelled opis-
thobranchs may also be reflected in the larval shell mor-
phology, this relationship has not been demonstrated
throughout the group (Rex and Waren, 1982).

Ockelmann (1965) formulated criteria distinguishing
between planktotrophic and non-planktotrophic devel-
opment in a wide range of bivalves based on relatively
precise dimensions of the prodissoconch I and II, but the
only effort to establish similar criteria for gastropod pro-
toconchs was based on data from comparatively few spe-
cies (Lima and Lutz, 1990). Nevertheless, Shuto (1974)
has shown that, given a complete Protoconch I and II,
the ratio of the maximum diameter (D; in mm) of the
whole protoconch to the number of whorls or volutions

(Vol) provides an index to the developmental type of a
marine prosobranch gastropod. A species with more than
three whorls and a D/Vol value less than 0.3 suggests
planktotrophic development. A D/Vol value between 0.3
and 1.0 with less than three volutions indicates a species
with either planktotrophic or non-planktotrophic devel-
opment, whereas a D/Vol value between 0.3 and 1.0 and
less than 2.25 volutions suggests a species with a non-
planktotrophic larval type. A D/Vol value higher than 1 .0
would suggest a species with direct development (Shuto,
1974). However, Pawlik ct al. (1988) have shown that the
criteria of Shuto ( 1974) cannot accurately predict the ac-
tual mode of development in a majority of cancellariid
gastropods.

The type of sculpture or ornamentation on the proto-
conch has been widely used to infer the mode of devel-
opment in prosobranch gastropods (Thorson, 1950;
Shuto, 1974; Bandel, 1975a, b, c, 1982; Lima and Lutz,
1990). Planktotrophy has been indicated for those larvae
with protoconchs possessing a fine reticulate or cancellate
pattern, oblique radial ribs or both, whereas a smooth or
simply sculptured protoconch suggests that the larvae are
non-planktotrophic (Thorson, 1950; Shuto. 1974; Bandel.
1975a, b, c, 1982). A well developed protoconch orna-
mentation is thought to strengthen the shell, a benefit to
planktotrophic larvae spending lengthy periods in the
plankton (Bandel, 1975a; Jablonski and Lutz, 1980). Two
recent reviews of poecilogony, or intraspecific variation
in the mode of larval development, found no evidence
for the occurrence of this phenomenon in prosobranch
gastropods, indicating that the form of the protoconch is
a species-specific character (Hoagland and Robertson,
1988; Bouchet, 1989). Nevertheless, the species variability
of protoconch and teleoconch morphologies of cultured
meso- and neogastropods led Lima and Lutz (1990) to
stress the need for caution when inferring type of devel-
opment from shell morphology alone.

The most reliable method for determining develop-
mental mode from protoconch morphologies is to com-
pare confamilial or congeneric species with known de-
velopmental histories (Scheltema, 1978; Jablonski and
Lutz, 1980, 1983). In the case of deep-sea prosobranchs,
the comparison must be made with taxonomically related
shallow-water species, the assumption being that similar
protoconch morphologies result from similar life history
patterns in shallow and deep seas (Colman et a/.. 1986).

Based on the above larval shell criteria, the majority of
vent gastropods are believed to have non-planktotrophic
development and to have limited larval dispersal capa-
bility (Lutz et a/., 1984, 1986; Turner et al, 1985), al-
though low temperatures encountered in the deep sea may
extend the period available for dispersal of swimming but
non-feeding veligers (Turner et al., 1985). This abundance
of non-planktotrophy may, in part, be due to the fact that

36 R. G. GUSTAFSON ET AL

Table I

DS\ ' "Alvin " Jive number, dale, location, latitude/longitude, and depth oj dive* in which gastropod egg capsules were retrieved

Dive*

Date

Location

Latitude; Longitude

Depth (m)

1418

24 July 1984

Juan de Fuca Ridge

47°57.0'N: 129=04.0^

2212

Endeavour Segment

1419

25 July 1984

Juan de Fuca Ridge

47°57.0'N; 129°04.0'W

2208

Endeavour Segment

1523

11 March 1985

Galapagos Rift

0°48.3'N;86013.5'W

2450

Rose Garden Vent

1527

16 March 1985

Galapagos Rift

0°48.3'N;86°13.5'W

2450

Rose Garden Vent

1528

17 March 1985

Galapagos Rift

0°48.3'N; 86°13.5'W

2450

Rose Garden Vent

1529

18 March 1985

Galapagos Rift

0°48.3'N;86°13.5'W

2450

Rose Garden Vent

1531

20 March 1985

Galapagos Rift

0°48.3'N;86°13.5'W

2450

Rose Garden Vent

2031

3 May 1988

Galapagos Rift

0°48.3'N;86013.5'W

2450

Rose Garden Vent

the majority of gastropods found at the vents are limpet-
like or coiled archeogastropods. When found in shallow
seas, these gastropods appear to be phylogenetically con-
strained to non-planktotrophy (Anderson, 1960; Heslinga,
1981;Strathmann, 1978a, b; Rex and Waren, 1982;Lutz
etui.. 1984;Jablonski, 1985; Waren and Bouchet, 1989).

Analysis of developmental stages contained in benthic
egg capsules also provides information about the life his-
tory of bottom-dwelling gastropods. Although many re-
searchers have described the egg capsules and encapsulated
embryos of shallow-water (see reviews in Fretter and Gra-
ham, 1962; Robertson, 1976; Webber, 1977; Fretter, 1984;
Pechenik, 1986; Soliman, 1987; M. F. Strathmann, 1987)
and deep-sea (Thorson, 1940b; Bouchet and Waren,
1979a, 1980, 1985a, 1985b; Colman and Tyler, 1988)
marine prosobranch gastropods, only brief mention has
been made of the rarely collected egg capsules from hy-
drothermal vent habitats and their contents (Turner et
al,, 1985; Berg, 1985). "Lens-shaped" egg capsules mea-
suring 10-12 mm (Turner et al., 1985) or 17.7 ± 3.8 mm
(Berg, 1985) in diameter have been reported at the Gal-
apagos Rift, while Berg (1985) has briefly described four
small prosobranch egg capsules 4.9 mm in length by 1.7
mm wide and shaped like an "inflated triangle" from
Garden of Eden vent on the Galapagos Rift.

When specimens of the previously reported egg capsules
from the Galapagos Rift (Turner et al, 1985; Berg, 1985)
and numerous egg capsules from the Endeavour Segment
of the Juan de Fuca Ridge, came into our possession, it
was evident that a more detailed study of these capsules
and their contents might yield insights into the life his-
tories and the means of dispersal of these species. This
paper is a description of the morphology of three different
egg capsules from hydrothermal vents and the embryos

and larvae contained in those capsules. Inferences are also
made about the dispersal capabilities of the contained lar-
vae, and an attempt is made to predict which of the known
hydrothermal vent gastropod species produced each cap-
sule type.

Materials and Methods

Specimens were retrieved with the assistance of DSV
"Alvin" during the dives summarized in Table I. Egg cap-
sules were collected: ( 1 ) from the surfaces of geological
and biological samples brought up in the "Alvin" basket
or in insulated retrieval boxes; (2) from sampling gear or
markers that had been left at the vents and later retrieved;
and (3) from sorted material collected with a "slurp gun"
attached to "Alvin."

On board ship, specimens were fixed for 24-48 h in
10% formalin buffered with borax, thoroughly rinsed,
transferred to 70% ethanol, and finally to 95% ethanol to
prevent corrosion of larval protoconchs. Terminology
used in egg capsule descriptions follows that of D'Asaro
(1970a). Egg capsule length is the distance between the
lateral edges at the widest point parallel to the apical su-
ture; width is the distance between the two sides at the
widest point perpendicular to the apical suture; and height
is the distance from the apex to the basal membrane
through the capsule's central axis.

Photographs, drawings, and measurements were made
of pertinent views of the capsules. Capsules containing
embryos were then dissected into two equal halves and
the embryos were removed. Both capsules and free em-
bryos were critical point dried, placed on stubs, coated
with approximately 400 A of gold-palladium, and ex-
amined on an Hitachi S-450 scanning electron micro-
scope (SEM).

HVDROTHERMAL VENT EGG CAPSULES

table II

.IvcniKc dimensions (mean ± standard deviation) i>l hydrothermal vei
gastropod egg capsules collected on specific dives o/ AST ". ilvin"
Number oj specimens collected on each dive are in parentheses

Galapagos Rift Lenlicular Egg Capsules

Length
"Alvin" Dive # (mm)

Width
(mm)

Height
(mm)

1528(2) 14.9 ±0.8
1529(1) 17.4
1531 (4) 16.4 ± 1.3
2031 (22) 14.4 ± 1.7

1 3.4 ± 0.0
15.9
14.0 ±0.7
13.9 ± 1.8

Galapagos Rift Inflated-Triangular

Egg Capsules

Length
"Alvin" Dive # (mm)

Width
(mm)

Height
(mm)

1523(1) 4.1
1527(1) 3.2
1528(1) 5.0

1.1
1.1
1.6

2.4
3.2
3.4

Juan de Fuca Ridge Egg Capsules

Length
"Alvin" Dive # (mm)

Width
(mm)

Height
(mm)

1418(8) 3. 3 ±0.6
1419(48) 3.7+0.5

1.2 ±0.1
1.4 ±0.3

2.7 ±0.7
3.8 ± 0.6

Values for the maximum dimension of the protoconch
and the number of volutions of the larval shell were mea-
sured directly from scanning electron micrographs by the
methods of Shuto (1974). Maximum diameter of the pro-
toconch was denned as the straight-line distance from the
protoconch-teleoconch boundary to the opposite side of
the protoconch in the region of greatest width. Maximum
diameter of the Protoconch I was denned as ( 1 ) the greatest

Figures 1-2. Lenticular egg capsules from Rose Garden Vent on the
Galapagos Rift. Figure 1. Photograph of egg capsules attached to location
marker retrieved on "Alvin" Dive 2031. Scale bar = 50 mm. Figure 2.
Light micrograph of apical view of egg capsule removed from substrate.
Arrow marks the escape aperture. Scale bar = 5 mm. bm, basal mem-
brane.

straight-line distance from the Protoconch I-Protoconch
II boundary in species with planktotrophic development,
or (2) from the Protoconch I-teleoconch boundary in non-
planktotrophic species lacking a Protoconch II, to the op-
posite side of the Protoconch I in the region of greatest
width [see Lima and Lutz (1990), their figure IB, for a
diagrammatic depiction of these dimensions]. Maximum
diameter measurements of the protoconch should not be
confused with Robertson's ( 197 1 ) "first whorl diameter,"
which is a measurement of the straight-line distance tan-
gent to the straight beginning of the suture and extended
in both directions to where it intersects the nearest suture
(Lima and Lutz, 1990, their figure 1A). All these values
are most accurately determined when the shell is viewed
with an apical orientation. Taxonomic terminology and
categories are in agreement with those outlined in Vaught
(1989).

Results

The gastropod egg capsules described in this report
consist of three distinct groups: (1) lenticular capsules,
with a flattened oval or circular base and a convex upper
surface, from the Galapagos Rift; (2) inflated triangular-
shaped capsules, from the Galapagos Rift; and (3) inflated
oval or pouch-like capsules attached to the substratum by
a flattened basal membrane, from the Endeavour Segment
of the Juan de Fuca Ridge. Dimensions of each collected
capsule are summarized in Table II.

Galapagos Rift lenticular egg capsules

Twenty-nine round to oval, lenticular egg capsules av-
eraging 14.8 ± 1.8 mm in length by 14.0 ± 1.7 mm in
width were collected during four separate dives at Rose
Garden Vent on the Galapagos Rift (Figs. 1-3; Tables I,
II). Height could not be measured due to unequal defor-
mation of the capsules during fixation and dehydration.
Twenty-two capsules were found attached to a gray poly-
ethylene marker retrieved on "Alvin" Dive 2031 (Fig. 2),
while the remaining specimens were found attached to
basaltic rocks by a thin basal membrane that extends be-
yond the limits of the capsule chamber (Figs. 2, 3).

The outer surface of the whitish to transparent capsules
was smooth; there were no apparent ridges (Figs. 2, 3). A
transparent elongated escape aperture, centered about the
long axis of each capsule, blended into an indistinct apical
suture that effectively separated each low capsule into two
equal halves (Figs. 2, 3). The capsule wall had three layers
consisting of a compact, dense inner layer, a spongy-fi-
brous middle layer, and a compact, dense outer layer.
(Figs. 4-5). The elongated escape aperture was derived
from a hollow chamber within the middle spongy-fibrous
layer; this chamber caused the capsule wall to bulge out-
ward above the level of the capsule surface (Fig. 5).

R. G. GUSTAFSON ET AL

••'

Figure 3. ( A ) Apical view oflenticular egg capsule from the Galapagos
Rift with individual embryos visible through transparent capsule wall. Ar-
row marks the escape aperture. Scale bar = 5 mm. bm, basal membrane.
(B) Lateral view of lenticular egg capsule with peripheral extension of basal
membrane. Arrow marks the escape aperture. Scale bar = 5 mm. (C)
Group of four lenticular capsules drawn as they appeared attached to lo-
cation marker in Figure 1. prior to fixation. Scale bar = 5 mm.

Early trochophore and veliger larvae in various stages
of development were present in capsules collected during
"Alvin" Dives 1528, 1529, and 1531 (Figs. 6-11): one
capsule collected during Dive 2031 contained 1052 veliger
larvae, all with a fully formed Protoconch I (Figs. 12-14).
All other lenticular capsules collected during Dive 2031
were empty. No nurse eggs were observed in lenticular
egg capsules.

The following is a chronological reconstruction of de-
velopmental stages found in a number oflenticular cap-
sules from the Galapagos Rift. The earliest stage encoun-
tered, a late prototroch, was approximately 175 ^m in
length by 100 /um in width (as measured from electron
micrographs), with a prominent apical plate, short pre-
trochal region, prototroch. long post-trochal region,
mouth, and very early larval shell (Figs. 6-10). The apical
plate lacked an apical ciliary tuft while the prototrochal
cilia appeared to be of the compound type and 15-19 //m
in length (Fig. 7). The posterior-dorsal shell field (see Eys-
terand Morse, 1984, for terminology) had already invag-
inated in the earliest specimens obtained, and some shell
secretion had commenced (Figs. 8-10).

The next observed stage of development was a veliger
larva, which had a bi-lobed velum, a mouth leading into
the stomadeum, a foot primordium — a protruding knob
located immediately posterior to the mouth — an oper-
culum. and a more developed larval shell (Fig. 1 1). This
was a very early veliger because the body was still much
too large to be withdrawn into the shell.

One capsule collected during Dive 2031 (Figs. 12-14)
contained late Protoconch I larvae that were almost ready
to hatch. The embryonic shells of these specimens had a
maximum diameter of 234 ^m (as measured from electron
micrographs). The larval shells of these larvae had a fine
reticulate sculpture formed of spiral raised ridges running
in the direction of growth, and crossed by regularly spaced
perpendicular riblets (Figs. 12-14). An uncalcified oper-
culum was present at this stage (Fig. 13).

Galapagos Rift inflated triangular egg capsules

Three specimens of an egg capsule 4. 1 ± 0.9 mm in
length by 1.3 ± 0.3 mm in width by 3.0 ± 0.5 mm in
height and shaped like an inflated triangle were found
attached to basaltic substrates during a series of "Alvin"
dives at the Galapagos Rift in 1985 (Figs. 15, 16; Table
II). Capsules were attached by a basal membrane that
barely extends beyond the limits of the capsule chamber
(Fig. 16). A lateral ridge extended up from either end of
the long axis of these capsules to meet at the capsule's
slightly off-center apex (Figs. 15, 16). Except for the
prominent lateral ridge, the surfaces of these capsules were
smooth. Capsules fixed in 10% buttered formalin and
subsequently stored in ethanol, ranged in color from white

HYDROTHERMAL VENT EGG CAPSULES

Figures 4-5. Scanning electron micrographs of lenticular egg capsule wall from Galapagos Rit't. Figure
4. Cross-section of capsule wall. Scale bar = 10 nm. ow. outer capsule wall; iw. inner capsule wall; si. spongy
layer. Figure 5. Cross-section through the escape aperture chamber. The outer surface of the capsule is
towards the top. Scale bar = 100 /im.

to yellowish-white to almost orange. The capsule wall was
composed of what appeared to be one spongy-fibrous layer
(Fig. 17).

Each capsule contained several hundred early veliger
larvae approximately 165 /urn in length by 98 /urn in width,
as measured from electron micrographs. Larvae in all
three capsules were at the same relative stage of devel-
opment and were characterized by a bi-lobed velum, an
apical sensory region with cephalic cilia, a mouth, a foot
primordium with attached operculum, and an early Pro-
toconch I (Figs. 18, 19). Velar compound cilia were ap-
proximately 30 urn long. The early protoconch was over-
laid by a membrane that obscured a sculpture of radially
arranged rows of short tubercules intersected by weak
concentric raised ridges or lines (Figs. 19-21). Distal to
this membrane, the sculpture consisted of parallel raised
ridges running in the direction of growth, crossed by radial
riblets, and forming a cancellate or net-like pattern (Fig.
19). Nurse eggs were not present in the three inflated tri-
angular egg capsules.

Juan de Fuca Ridge egg capsules

Fifty-six orange egg capsules, each shaped like an in-
flated oval or pouch and measuring 3.6 ± 0.5 mm in
length, 1.3 ± 0.3 mm in width, and 3.6 ± 0.7 mm in
height were collected during "Alvin" Dives 14 18 and 1419
on the Endeavour Segment of the Juan de Fuca Ridge in
1984 (Tables I, II). Each capsule was attached to the sub-
strate by a flattened basal membrane (Figs. 22, 23). A
lateral ridge rose abruptly from the thin basal membrane
at either end of the capsule. About 2 mm above the sub-
stratum, the ridges at either end of the capsule split into

two wing-like extensions forming a saddle-shaped struc-
ture around the central oval escape aperture (Fig. 23B).
In most cases an amorphous, poorly fixed, orange em-
bryonic mass, containing an indeterminate number of
embryos, occupied the capsule chamber (Figs. 22, 23C).
In other cases, from one to six, but most often five, larvae
were observed through the capsule walls. The Juan de
Fuca Ridge egg capsule wall consisted of two compact
dense layers: an outer and an inner layer separated by a
sharp boundary (Fig. 24).

Examination of the amorphous yolk mass present in
most capsules revealed that some larval shell had been
secreted, but structural details were indeterminable. Nurse
eggs may have been present, but fixation was too poor
for this to be determined. However, more advanced larvae
were present in a few capsules, which revealed a paucispi-
ral protoconch that was large and bulbous and lacked
ornamentation other than that due to weak growth lines
(Figs. 25, 26).

Discussion

Although some archeogastropods embed their eggs in
a benthic gelatinous mass or ribbon, the majority of shal-
low-water archeogastropods do not produce benthic egg
capsules (Fretter and Graham, 1962;Hyman, 1967; Rob-
ertson, 1976; Webber. 1977; Bandel, 1982; Fretter, 1984;
Soliman, 1987; M. F. Strathmann, 1987). Therefore, egg
capsules described in this paper from hydrothermal vents
are most likely the spawn of prosobranchs of the higher
orders Mesogastropoda or Neogastropoda. Various au-
thors (Anderson, 1960; Amio, 1963; Bandel, 1976a, b;
Soliman, 1987) have stressed that the general form of the

Figures 6-10. Scanning electron micrographs of early trochophore larvae removed from Galapagos Rifl
lenticular egg capsules. Figure 6. Ventral view showing apical plate (ap). prototroch (pt), and mouth (m).
Scale bar = 25 nm. Figure 7. Apical view showing apical plate (ap) and prototroch (pt). Scale bar = 20 /im.
Figure 8. Early protoconch at extreme posterior end. Scale bar = 10 /im. Figure 9. Right lateral aspect
showing apical plate (ap). prototroch (pt), and protoconch (pc). Scale bar = 25 urn. Figure 10. Left lateral
aspect of different specimen to that shown in Figure 9. Scale as in Figure 9. ap. apical plate; pt, prototroch:

pc, protoconch.

Figures 1 1-14. Scanning electron micrographs of early and late veliger larvae extracted from lenticular
egg capsules from the Galapagos Rift. Figure II. Early veliger larva showing apical plate (ap), velum (v),
mouth (m). foot primordium (f), operculum (o). and protoconch (pc). Scale bar = 25 p.m. Figure 12. Apical
view of Protoconch I in larva near hatching. Scale bar = 50 urn. Figure 13. Apertural view of Protoconch
1 in larva near hatching. Scale bar = 25 jim. o. operculum. Figure 14. Ventral view of Protoconch I in larva
near hatching. Scale bar = 50 MHI.

R. G. GUSTAFSON KT A I.

Figure 15. Light micrograph of convex side of Galapagos Rift inflated
triangular egg capsule with embryos visible through the transparent cap-
sule wall. Arrows mark the lateral ridges. Scale bar = 1 mm. bm. basal
membrane.

oothecae in different gastropod taxa is characteristic of
the species, and in some cases, of higher orders of clas-
sification, and may be valuable in taxonomy. It should
be noted, however, that similar capsules may be produced
by taxonomically diverse species, while in other cases in-
terspecific variation in capsule morphology is insufficient
to differentiate closely related species (Kohn. 1961 ).

Figure 16. (A) View of the convex side of Galapagos Rift inflated
triangular egg capsule with individual embryos visible through transparent
membrane. Arrows mark the lateral ridges. Scale bar = 1 mm. (B) Apical
view of Galapagos Rift inflated triangular egg capsule. Arrows mark the
lateral ridges. Scale bar = 1 mm. bm. basal membrane.

Galapagos Rift lenticular egg capsules

Flattened lenticular egg capsules with a centrally located
escape aperture are known from the neogastropod families
Muricidae, Fasciolariidae, and Turridae. Dimensions and
other statistics pertaining to selected lenticular egg capsules
from these families are presented in Table III. The only
member of these families known to occur at the Galapagos
Rift hydrothermal vents is a large turrid, provisionally
described as Phymorhynchus sp. (Waren and Bouchet,
1 989). A similar species occurs at 1 3°N and 2 1 °N on the
East Pacific Rise (Turner el a!., 1985; Waren and Bouchet,
1989). Both the six egg capsules described by Turner el
al. (1985) and the five "lens-shaped" egg cases described
by Berg (1985) as characteristic of turrids, as well as, the
lenticular egg capsules described in this paper, may all
belong to Phymorhynchus sp. from the Galapagos Rift.
Differences in reported average size between these three
groups of capsules is not unexpected, because capsule size
in neogastropods is proportional to adult size. Capsule
size is also correlated with female foot width; the capsule
is formed and manipulated by the foot during deposition
(Robertson, 1976; Shimek, 1986).

Berg ( 1985) estimated that "lens-shaped" oothecae from
the Garden of Eden and Mussel Bed hydrothermal vent
sites along the Galapagos Rift contained from 500-1000

eggs with a mean size of 192.1 ± 13.5 nm by 136.2 ±10.1
nm. This agrees well with our count of 1052 larvae in one
capsule from Dive 203 1 and with the size of larvae both
from this capsule (234 ^m maximum diameter) and from

Figure 17. Scanning electron micrograph of cross-section of single-
layered spongy capsule wall of inflated triangular egg capsule from Gal-
apagos Rift. The outer surface is towards the top. Scale bar = 5 ^m.

HYDROTHERMAL VENT EGG CAPSULES

Figures 18-21. Scanning electron micrographs of veliger larvae extracted from inflated triangular egg
capsules from Galapagos Rift. Figure 18. Lateral ventral view showing velum (v). apical plate (ap). mouth
(m). foot primordium (f). and early protoconch (pc). Scale bar = 25 urn. Figure 19. Lateral view showing
cancellate or net-like early protoconch (pc) sculpture and obscuring membrane (me). Scale bar = 25 urn.
o. operculum: v. velum. Figure 20. Apical view of early protoconch. Scale bar = 20 urn. Figure 21. Dorsal
view of early protoconch. Scale bar = 20 ^m.

capsules with larvae in earlier stages of development ( 1 75
/um in length by 100 ^m in width). The absence of nurse
eggs further suggests these capsules were laid by a turrid,
because nurse eggs are unknown in the Turridae (Table III).

Although the basal diameter of lenticular egg capsules
described herein (14.8 X 14 mm) is larger than the 2-6
mm of normal turrid egg capsules, it is not unprecedented.
Egg capsules of the turrid Mangelia plicosa are 30-33

R. G. GUSTAFSON ET AL.

Figure 22. View of the convex side of Juan de Fuca Ridge pouch-
like egg capsule containing amorphous embryonic mass. Scale bar = 1
mm. bm, basal membrane.

mm in diameter, while those ofPolystira harretli measure
up to 10.7 mm in basal diameter (Table III). Furthermore,
the large size of the Galapagos Rift turrid (up to 74 mm
in height, pers. obs., RGG) is consistent with the large
size of the egg capsules. Although turrid egg capsule wall
structure has not been previously studied, the three-lay-
ered capsule wall (Fig. 4) is similar to that described for
the closely related Conidae (D'Asaro, 1988). This is con-
sistent with the designation of these capsules as belonging
to a turrid, because lenticular capsules of the Muricoidea
have four layers (D'Asaro, 1988). Some degree of repro-
ductive synchrony may occur in the population of the
Galapagos Rift turrid provisionally described as Pliymo-
rhvnchus sp., because eggs, embryos, and larvae contained
in different lenticular capsules collected during March
1985 and May 1988 in the present study (Tables I, II),
and by Berg (1985), were at the same relative stage of
development on each collection date but were at different
stages of development between collection dates.

Galapagos Rift inflated triangular capsules

Berg's (1985) account of four egg capsules, each "shaped
like a small inflated triangle," retrieved from a larval trap
in 1979 at the Garden of Eden vent on the Galapagos
Rift, agrees in every respect with the description found
here of the inflated triangular capsules from Rose Garden
vent. Although Berg (1985) does not give a measure of
the egg capsule's height, the average length of 4.9 mm
and width of 1.7 mm of his capsules is similar to the
average length of 4.1 mm and width of 1.3 mm for the
three inflated triangular capsules described in this report.

Inflated triangular capsules from the Galapagos Rift
vent fields are similar in morphology, but not in size, to
those from 5480 m in the Kermadec Trench illustrated
by Bouchet and Waren ( 1 985a) and attributed to the buc-
cinid Calliloconcha knudseni Bouchet and Waren. These
1 5 mm long by 1 2 mm high C. knudseni capsules were
empty and had been drilled by a predator. Certain small
capsules similar to the inflated triangular capsules found
at the Galapagos Rift, but without a prominent lateral
and apical ridge, are produced by members of the neo-
gastropod family Columbellidae (Petit and Risbec, 1929;
Thorson, 1 940a; Bacci, 1947; Amio. 1955; Bandel, 1974a).
Capsules of this group contain no more than 60 eggs,
which are usually reduced in number through oophagy
(Bandel. 1974a). Small capsules attributed to the buccinid
Tacita danielsseni (Friele) and to the turrid Oenopota
ovalis (Friele) from abyssal parts of the Norwegian Sea
resemble the inflated triangular capsules in size but appear
to lack the lateral ridge and strong off-center apex (Bouchet
and Waren. 1 979a). The egg capsule attributed to O. ovalis
in Bouchet and Waren (1979a, Fig. 15) is unlike any
known for the genus Oenopota (Thorson, 1935; Shimek.
1983b, 1986) or for any other turrid. Capsules belonging
to T. danielsseni contained thousands of small eggs, al-
though only one large embryo (4 mm in maximum di-
mension) developed in each capsule. A large protoconch
(840-880 /urn) is also present in O ovalis, which is indic-
ative of direct development (Bouchet and Waren, 1979a:
Rex and Waren, 1982). By comparison to the above spe-
cies, several hundred veligers were present in each of the
triangular capsules from the Galapagos Rift examined in
this study. Neither a buccinid nor a columbellid has as
yet been reported from the Galapagos Rift hydrothermal
vents.

Gastropods that are large enough to have laid these
capsules at the Galapagos Rift vent sites include Phy-
nuirhvnchus sp., Provanna ios Waren and Bouchet and
P. muricata Waren and Bouchet (Waren and Bouchet,
1986, 1989). The turrid Phymorhynchus is an unlikely
candidate because the capsule type for this species has
been provisionally assigned in the present study. Although
the inflated triangular capsules from the Galapagos Rift
are unlike the typical lenticular capsules of turrids, a sim-
ilar capsule from the deep-sea has been attributed to the
turrid Oenopota ovalis (Bouchet and Waren, 1979a; see
discussion above). Either species of Provanna is also an
unlikely choice, because the recent placement of Provanna
within the Littorinoidea (Waren and Bouchet, 1989) sug-
gests that the production of such an elaborate egg capsule
is unlikely, because all known littorinoids spawn either a
benthic, amorphous gelatinous mass, a single pelagic cap-
sule containing a single egg, or release veligers or fully
formed juveniles from an internal brood pouch (Thorson,
1946; Anderson. 1960, 1962; Amio, 1963; Pilkington,

HYDROTHERMAL VENT EGG CAPSULES

Figure 23. (A) View of the convex side of Juan de Fuca Ridge egg
capsule. Scale bar = 1 mm. bm. basal membrane. (B) Lateral view of
Juan de Fuca capsule showing lateral ridge running into two apical wing-

1974; Robertson. 1976; Bandel, 1974b, 1975b, 1982; So-
liman, 1987; M. F. Strathmann, 1987). Based on egg cap-
sule morphology, we cannot assign the inflated triangular
egg capsules from the Galapagos Rift to a particular spe-
cies. A more definitive statement on the taxonomic affil-
iation of the inflated triangular capsule must await ad-
ditional collections of organisms from the Galapagos Rift
vent fields and further taxonomic examination of existing
material.

The wall structure of the inflated triangular egg capsules
from the Galapagos Rift is similar to that seen in several
species within the Muricoidea (Roller and Stickle, 1988;
D'Asaro, 1988). although too little is known of egg capsule
wall structures at this time to make a definitive statement
as to this capsule's affinity. Because all three inflated tri-
angular capsules collected from Galapagos Rift on three
separate dives in 1985 contained early veligers at the same
stage of development, it is possible that some degree of
reproductive synchrony occurs in this population.

Juan dc Fuca Ridge egg capsules

Egg capsules strikingly similar in size and shape to the
Juan de Fuca Ridge capsules described in the present study
are produced by the cancellariid neogastropod, Admele
viridula (Fabricius) [Thorson, 1935: fig. 71 (mistakenly
attributed to I 'eliitina undala Brown, see Thorson, 1944:
108); Bouchet and Waren, 1985b: fig. 687]. The capsules
described herein and those of A. virudula both possess
parallel wing-like extensions, a flattened base, and an api-
cal escape aperture. Species ofAdmete have been described
from 6700 m deep in the Kermadec Trench, whereas A.
viridula, which is circumpolar, has a depth range of 4-
2,295 m, according to Clarke (1962). The small number
(1-6) of large larvae present in Juan de Fuca capsules is
consistent with the 6-7 larvae per capsule found in Adnielc
viridula by Thorson (1935) and the 6 larvae per capsule
found in Admele sp. by MacGinitie (1955). However, a
species of Admele has not been collected from the Juan
de Fuca Ridge.

Other pouch-like egg capsules, with or without a flat-
tened base, but without wing-like extensions, are found
in the neogastropod families Muricidae and Buccinidae
(Thorson, 1935, 1940b; Anderson. 1960; Golikov, 1961:
Cowan, 1964; Radwin and Chamberlin, 1973; Macintosh,
1979, 1986; D'Asaro, 1986). However, buccinids typically
form their capsules into clusters (Thorson, 1935; Cowan,
1964), in contrast to the pouch-like capsules from Juan

like extensions (arrows) forming a saddle-like structure. Scale bar = 1
mm. (C) View of the convex side of Juan de Fuca egg capsule with
amorphous embryonic mass visible through transparent capsule wall.
Scale bar = 1 mm. bm, basal membrane.

R. G. GUSTAFSON ET AL

Figure 24. Scanning electron micrograph of cross-section of double-
layered capsule wall of Juan de Fuca Ridge egg capsule. Scale bar = 5
^m. ow, outer wall; iw, inner wall.

de Fuca, which are individually attached to the substrate.
Capsules secreted by members of the neogastropod family
Columbellidae frequently possess apical collars that sur-

round a central escape aperture somewhat similar to the
wing-like extensions seen in capsules from the Juan de
Fuca Ridge described in the present study (Thorson,
1940a; Perry and Schwengel. 1955; Amio, 1955, 1963;
Marcus and Marcus, 1962; Scheltema, 1969; D'Asaro,
1970b; Bandel, 1974a).

Only two gastropods, Buccinum viridum Dall and Pro-
vanna variabilis Waren and Bouchet, which are large
enough to have secreted these capsules, have been re-
corded from the Juan de Fuca Ridge system (Waren and
Bouchet, 1986; Tunnicliffe el a/., 1985; Tunnicliffe and
Fontaine, 1987; Tunnicliffe, 1988). Dall (1890) described
B viridum as having a maximum shell height of 46 mm,
while P. variabilis reaches a maximum height of 8.7 mm
(Waren and Bouchet. 1986). Waren and Bouchet (1989)
have recently placed P. variabilis within the Superfamily
Littorinoidea. No members of the Littorinoidea have as
yet been shown to produce complex egg capsules (see dis-
cussion above of inflated triangular capsules), which sug-
gests that P. variabilis is not the source of the egg capsule
from the Juan de Fuca Ridge. The Buccinidae produce
egg capsules either singly or in clusters, each containing
many nurse eggs; only a few of these survive (Thorson,
1935, 1946;Lebour, 1937; Anderson. 1960). Encapsulated

Figures 25-26. Scanning electron micrographs ol larvae extracted from Juan de Fuca Ridge egg capsules.
Figure 25. Lateral view of protoconch showing height of the first whorl and weak concentric growth lines.
Scale bar = 200 ^m. Figure 26. Apical view showing unsculptured appearance of protoconch. Scale bar
= 200 urn.

HYDROTHERMAL VENT EGG CAPSULES

Table III

Taxonomic affiliation, dimension.'!, number of eggs, egg si:e. and number of veligers present at hatchint>
for lenticular egg cases as reported in selected references

Size (mm)

Number of
veligers at
hatching

Post-hatching
development
type

Reference

Species

Basal
diameter

Number
Height of eggs

Egg size

(Mil)

Order Neogastropoda

Muricoidea

Family Muricidae

Bedeva hanleyi (Angas)

3.0

50-70

250

Anderson, 1965

Bedevina (Lataxiena) biri/effi

(Lischke)

3.0

0.85 60-90

190

—

Amio, 1963

Ergalalax constractus (Reeve)

2.5-3.0

130

—

Habe. 1960

Ergalatax calcareus (Dunker)

3.5-4.5

130

—

Habe, 1960

Trophon clalhrants (L.)

6-7

— —

—

9-18

Thorson. 1940b

Trophon muricaliis (Montagu)

2.5

— —

—

2-9

Lebour. 1936

Trophon tnincalus (Strom.)

1.8-3.1

— —

—

6-11

Thorson, 1946

Zeairophon (\ymene) amhiguiis

(Philippi)

6-10

1.0-1.5

—

600

N-P

Pilkington, 1974

Family Fasciolariidae

Glaphyrina vulpia >/< >r

(Sowerby)

— —

—

Pilkington, 1974

Conoidea

Family Turridae

Clavus japonicus (Lischke)

2.5

1.8 2-4

650

2-4

Amio, 1963

Crassispira sp.

1.5

— 2-5

—

2-5

Bandel. 1976b

Drillia crenu/aris (Lamarck)

6-7

150-170

230-300

150-170

Thorson, 1940a

Drillia so/ida (C. B. Adams)

1 2-7

—

2-7

Bandel, 1976b

Kurliiella plumbea (Hinds)

2.3 ± 0.3

180 ±43

137 ± 8

180 + 43

Shimek. 1983c

Mangelia nebula (Montagu)

1.6

Lebour, 1914, 1916. 1917

Mungelia plicosa (C. B. Adams)

30.1-33.0

0.32 60

160

Perry and Schwengel, 1955

Oenopoia simplex (Midd.)

2-3

— —

—

5-6

Thorson. 1935

Oenopota exarata (Moller)

3.0-4.5

— —

—

5-21

Thorson, 1935

Oenopota bicarinata (Couth.)

2.25-3.25

— —

—

3-11

Thorson. 1935

Oenopota pyramidialis (Strom.)

3.5-6

— —

—

4-20

Thorson, 1935

Oenopota nohilis (Moller)

4.5-4.75

— —

—

3-7

Thorson. 1935

Oenopota trevelyana (Tunon)

3.1-3.3

— —

—

25-3 1

N-P

Thorson, 1946

Oenopota turricola (Montagu)

2.5

100-150

—

100-150

Vestergaard. 1935

Oenopoia elegans (Moller)

3.02

250

150

250

Shimek. I983b

Oenopoia excurvata (Carpenter)

2.08

1 .08 30

212

—

Shimek, 1983b

Oenopota jtdiciila (Gould)

2.24

0.98 20

371

—

Shimek, I983b

Oenopoia levidensis (Carpenter)

5.30

1.38 175 ±85

286

175 ±85

Shimek, 1983b

Ophiodermella inermis (Hinds)

4.68 ± 0.99

1.44 ±0.29 208 ±61

222 ± 15

208 ± 61

Shimek, 1983a

Philberlia gracilis (Montagu)

3.4

40-80

Lebour 1914 1916 1917

Philbertia lineari.s (Montagu)

1.5-2.0

60-80

140-150

Lebour. 1934. 1936, 1937

—

51-114

150

—

Thorson, 1946

Philberlia purpurea (Montagu)

5.3

0.6 350-400

100

—

Franc. 1950

Polystira barrelti (Guppy)

5.0-10.7

32-126

438

32-126

Penchaszadeh, 1982

Raphitoma (Teretia) amoena

(Sars)

1.25-1.5

— —

—

Thorson. 1935

Unknown turrid

2-3.5

— —

—

Arnaud and Zibrowius, 1973

Unknown turrid

2.6

— —

—

Bouchet and Waren. 1980

Unknown turrid

4.0-4.2

— —

—

Bouchet and Waren. 1980

direct development is universal within this group (Lebour.
1937; Robertson, 1976; Colman et at.. 1986; Waren and
Bouchet, 1989), although planktotrophic buccinids are
known from the Early Tertiary (Hansen, 1982). Capsule

wall morphology ofthe Juan de Fuca egg capsule described
in the present study is similar to that seen in certain Buc-
cinidae and M uricidae ( D' Asaro, 1988). although a defin-
itive statement is not possible until the micromorphology

R. G. GUSTAFSON ET AL

of a wider taxonomic grouping of capsules is known. On
the basis of egg capsule morphology we are unable to as-
sign the pouch-like egg capsules from the Juan de Fuca
Ridge to any species that has as yet been collected from
this site. However, the similarity of the Juan de Fuca Ridge
capsules to those produced by species of Admclc (Thorson,
1935; Bouchet and Waren, 1985b) is so striking that it
leads us to predict that a member of this genus may soon
be found associated with this hydrothermal vent.

Egg size, fecundity, and protoconch morphology

The size of the ovum in those prosobranch gastropods
that do not provide nurse eggs or albumen in the egg
capsule regulates the amount of nutrition supplied to the
larva and has a close relationship with the type of larval
development [Thorson 1946, 1950:Shuto, 1974; Bandel,
1975a; Lima and Lutz. 1990; but see discussion on this
topic in Vance (1973, 1974), Underwood (1974), Steele
(1977), Strathmann (1977), Perron (1981), Todd and
Doyle (1981), and Hines, (1986)]. Planktotrophic pro-
sobranch gastropod larvae typically have a small pointed
apex, often with delicate sculpture, reflecting an originally
small ovum. However, non-planktotrophic larvae typi-
cally have a large rounded apex reflecting a large ovum
with plenty of yolk available for the larva to grow to a
large size (Ockelmann, 1965; Shuto, 1974). This relation-
ship may become obscured in those non-planktotrophs
that feed on nurse eggs or other forms of extraembryonic
nutrition and emerge as large juveniles, because they often
have egg diameters no larger than those of free-swimming,
non-planktotrophs (Bandel. 1975a, c; Jablonski, 1986).
Similarly, Hadfield and Strathmann (1990) have shown
that egg size is not a reliable indicator for differentiating
between a pelagic and a benthic mode of development in
non-planktotrophic trochoidean archeogastropods. In
addition, although archeogastropods are purported to be
exclusively non-planktotrophic (Anderson, 1960; Hes-
linga, 1981; Strathmann, 1978a, b; Rex and Waren, 1982;
Lutz et al., 1984; Jablonski, 1985; Waren and Bouchet,
1989), many species develop from relatively small eggs
between 1 10 and 230 ^m in diameter (Amio, 1963), and
as small as 80 p.m in some cases (Bandel, 1982). Unfor-
tunately, egg size criteria could not be applied in the pres-
ent study, because ova were not encountered.

In the absence of nurse eggs, the number of eggs pro-
duced by a gastropod species is also indicative of its mode
of larval development (Thorson, 1950; Crisp, 1978; Shuto,
1974; Bandel, 1975a). A large number of eggs suggests
planktotrophic development, because many free-swim-
ming embryos and larvae are assumed to be lost to pre-
dation, whereas few eggs suggest non-planktotrophic de-
velopment (Thorson, 1950; Jablonski and Lutz, 1983).
Estimates of fecundity in the present study are compli-

cated by the fact that we have no way of knowing how
many capsules were produced by each laying female.
However, the over 1000 larvae contained in the lenticular
capsules from the Galapagos Rift, when compared to in-
formation on the number of eggs per capsule, total fe-
cundity, and development type in other species with len-
ticular egg capsules (Table III), suggests that this species
develops planktotrophically following hatching. The ap-
parent absence of nurse eggs and the presence of several
hundred embryos in the inflated triangular capsules from
the Galapagos Rift also suggests the potential for plank-
totrophic development after hatching; while the small
number of embryos (1-6) in pouch-like capsules from the
Juan de Fuca Ridge is strongly indicative of non-plank-
totrophic development.

Non-planktotrophic development is the most common
strategy among prosobranchs in the deep-sea, soft-sedi-
ment environment of the western North Atlantic (Rex
and Waren, 1982). However, an examination of bathyal
and abyssal prosobranch larval shells reveals that roughly
30% may develop planktotrophically below 1000 m in
the north-eastern Atlantic (Bouchet, 1976a, b; Bouchet
and Waren, 1979b) and that the incidence of plankto-
trophic development in this group increases with depth
below the continental shelf (Rex and Waren, 1982). About
50% of mesogastropod and neogastropod prosobranch
species on the abyssal plain may have planktotrophic de-
velopment based on their protoconch morphologies (Rex
and Waren, 1982). Bouchet (1976a, b) and Bouchet and
Waren ( 1979b) have proposed that deep-sea prosobranch
larvae with protoconchs, indicative of planktotrophy, mi-
grate to feed and undergo development in surface waters.
In addition, 18O and I3C isotope analyses of larval shells
from deep-sea gastropods have suggested that at least some
larvae of abyssal species may migrate upwards into
warmer waters during development (Bouchet and Fontes,
1981; Killingley and Rex, 1985).

Protoconch I sculpture of the type seen in larvae from
lenticular egg capsules at the Galapagos Rift (Figs. 12-
14) is characteristic of many turrid species with putative
planktotrophic development in both shallow waters
[Philhertia linearis (Montagu) (Rodriguez Babio and
Thiriot-Quievreux, 1974), Ruphitoma spp. (Richter and
Thorson, 1975)] and in the deep sea [Pleurotomella spp..
Teretia spp., Xanlliodaphne spp., Phymorhynchus spp..
Gymnobela spp., Thetu spp. (Bouchet and Waren. 1980),
Gymnobela sitbaraneosa (Dautzenberg and Fischer)
(Colman et al., 1986)]. The Turridae includes species with
larval shell morphologies that are indicative of both
planktotrophic and non-planktotrophic development
(Rex and Waren, 1982; Shimek, 1983a, b, c, 1986). The
maximum diameter of the Protoconch I in certain plank-
totrophic-type turrid species can be roughly estimated
from published micrographs, including the species Phil-

HYDROTHERMAL VENT EGG CAPSULES

hen in linearis (260 nm) (Rodriguez Babio and Thiriot-
Quievreux, 1974), Raphitoma reticn/ata (Renier) (220
^m). R (P/iilhenia) piirpurea (Montagu) (245 ^m). R
(Cirilla) linearis (Montagu) (245 ^m), R. (Leufroyi) leuf-
royi (Michaud) (225 ^m) (Richter and Thorson, 1975).
Pleurotomella coeloraphe (Dautzenberg and Fischer) (265
nm), Pleitrotomella demosia (Dautzenberg and Fischer)
(355 ^m), Pleurotomella megalembryon (Dautzenberg and
Fischer) (280 ^m), Pleurotomella hureaui (Dautzenberg
and Fischer) (220 nm), Pleurotomella sandersoni Verrill
(195 ^m), Teretia teres (Forbes) (260 ^m), Xanllunlaphne
dalmasi (Dautzenberg and Fischer) (215 ^m), Thela
chariessa (Watson) (240 /urn) (Bouchet and Waren, 1980),
and Gymnobela siibaraneosa (Dautzenberg and Fischer)
(180 Mm) (Colman el al. 1986).

Assuming that the larvae in lenticular egg capsules from
the Galapagos Rift retrieved on "Alvin" Dive 203 1 were
near hatching and represent the complete Protoconch I
stage, then measurements made from electron micro-
graphs reveal that this species has a Protoconch I maxi-
mum diameter of approximately 235 /im (Figs. 12-14).
This compares favorably with the Protoconch I maximum
diameters above, estimated for various turrid species ( 195-
280 ^m), with the exception of the large Protoconch I in
Pleurotomella demosia (355 nm) and the small Proto-
conch I in Gymnobela sitbaraneosa (180 nm). The max-
imum diameter of the Protoconch I in larvae from the
lenticular capsules also agrees well with the size of the
Protoconch I (as estimated from published micrographs)
in both the unnamed, newly settled turrid from the Gal-
apagos Rift (205-225 ^m) and the unnamed newly settled
turrid from 21°N on the East Pacific Rise (260 /jm)
(Turner el ai. 1985; their figs. 27a-e and 26a-e, respec-
tively). Unfortunately, pre-juvenile development is in-
complete for larvae in lenticular capsules in the present
study, and the final Protoconch II maximum diameter
and number of whorls cannot be determined, which pre-
cludes the estimation of D/Vol values for these samples.
However, as estimated from Fig. 27a in Turner el al.
(1985), the unnamed newly settled turrid from the Gal-
apagos Rift has a Protoconch II with approximately 4
whorls and a maximum diameter of 950 ^m (giving a D/
Vol value of 0.19). Similarly, the unnamed newly settled
turrid from 21°N (Turner el ai, 1985) has a Protoconch
II maximum diameter of approximately 775 ^m with
about 4 whorls (giving a D/Vol value of 0.19), as estimated
from Fig. 26d in Turner et al. (1985). The number of
volutions, D/Vol values, clear demarcation between Pro-
toconch I and II, and the protoconch ornamentation of
spiral threads crossed by axial riblets of both unnamed
newly settled turrids from the Galapagos Rift and 21°N,
depicted in Turner et al. (1985), are all indicative of
planktotrophic development.

Although the Protoconch I sculpture of the unnamed
newly settled turrid from the Galapagos Rift, depicted in
Turner et al. ( 1985. their figs. 27a-e), and Protoconch I
larvae from lenticular egg capsules in the present study,
also from the Galapagos Rift (Figs. 12-14), cannot be
directly compared due to corrosion of the former, the
similarly sized Protoconchs I (205-225 ^m and 235 ^m,
respectively) suggest that these specimens are taxonomi-
cally related. Because the only turrid to be collected at
the Galapagos Rift is the provisionally classified Phy-
morhynchus sp. (Waren and Bouchet, 1989), it is likely
that both the turrid juvenile from the Galapagos Rift de-
scribed by Turner el al. (1985), and the lenticular egg
capsules and larvae within, belong to this species. How-
ever, adult or juvenile Phymorhynchus sp. with intact,
non-corroded protoconchs, characteristics that would
verify this identification, have not been collected at the
Galapagos Rift.

Although it is not possible to determine the maximum
diameter of the Protoconch I stage or the D/Vol value for
larvae from the inflated triangular egg capsules from the
Galapagos Rift due to the incomplete development of the
larval shell, the initial reticulate sculpture (Figs. 19-21)
is indicative of planktotrophic development, following the
encapsulated phase. This type of sculpture is similar to
that seen on the Protoconch I of some members of the
mesogastropod families Rissoidae (Lebour, 1936, 1937;
Amio, 1963) and Cypraeidae (Richter and Thorson,
1975), as well as the neogastropod families Columbellidae
(Colman el al., 1986) and Turridae (Lebour, 1934; Amio,
1963; Rodriguez Babio and Thiriot-Quievreux, 1974;
Richter and Thorson, 1975; Bouchet, 1976a; Bouchet and
Waren. 1980). Members of the Cypraeidae have not been
collected in the deep-sea (Clarke, 1962), and no member
of this family or of the Rissoidae has been collected at the
Galapagos Rift. The columbellid Anachis haliaeeti (Jef-
freys) from the Rockall Trough has an ornately sculptured
Protoconch I and has been designated a planktotrophic
developer (Colman el al., 1986). Shallow-water species of
this genus produce small egg capsules similar to the in-
flated triangular type from the Galapagos Rift but with a
circular collar around the capsule's apex and containing
only 10-30 embryos (Scheltema, 1969) in contrast to the
several hundred larvae seen in capsules from the Gala-
pagos Rift. Likewise, the morphology of the inflated tri-
angular egg capsule is unlike that seen in most Turridae.
However, the egg capsule attributed to the turrid Oenopota
ovalis •( Bouchet and Waren, 1979a) resembles the inflated
triangular capsules, but contains only one embryo with
an unsculptured protoconch, in contrast to the several
hundred highly ornamented larvae encountered in the
inflated triangular capsules from the Galapagos Rift.
Given the incomplete formation of the larval shell and
unique structure of the egg capsule, it has proved impos-

R. G. GUSTAFSON ET AL.

sible to assign the inflated triangular capsules from the
Galapagos Rift to any known gastropod species from this
site.

Larvae from the Juan de Fuca Ridge egg capsules have
a large, paucispiral protoconch devoid of sculpture (Figs.
25, 26), which suggests that this species develops non-
planktotrophically. Assuming that these larvae have a
nearly fully developed protoconch, with a maximum di-
ameter of approximately 1.3 mm and 1.5 whorls (as es-
timate from Fig. 26), their calculated D/Vol value of 0.87
in concert with the small number of whorls is also indic-
ative of non-planktotrophic development. This type of
protoconch, lacking ornamentation, is similar to that
produced by members of the neogastropod superfamilies
Muricoidea and Cancellaroidea (Thorson, 1935; Radwin
and Chamberlin, 1973: Bandel. 1975a, b, c; Bouchet and
Waren, 1985a; Colman et a/., 1986; Colman and Tyler,
1988), however taxonomic placement of these larvae is
uncertain using protoconch morphology alone. The pro-
toconch of the cancellaroid Admete viridu/a (Thorson,
1935: fig. 72), the species with the most similar egg capsule
morphology to the specimens described in this study from
the Juan de Fuca Ridge, has a maximum diameter of
about 0.88 mm [as estimated from Thorson (1935: fig.
72)] and 1.25 whorls, for a D/Vol value of 0.77, which
also compares favorably with that calculated for encap-
sulated larvae from the Juan de Fuca Ridge (0.87).
Bouchet and Waren (1985a) give the protoconch maxi-
mum diameters and number of whorls for the deep-sea
buccinids Eosipho thorybopux Bouchet and Waren (0.7
mm, 1 whorl, D/Vol = 0.7), Manaria lirata Kuroda and
Habe (0.8 mm, 1+ whorl, D/Vol = 0.8), and M. clan-
destina Bouchet and Waren (0.7 mm, 1+ whorl, D/Vol
= 0.7). Colman et al. (1986) also provide protoconch
maximum diameters, whorl number, and D/Vol values
for the deep-sea buccinids Tacit a abyssorum (Locard)
(0.75 mm, 1.2 whorls, D/Vol = 0.63), Coins jeffreysianus
(Fischer) (2.5 mm, 1.25 whorls, D/Vol = 2), and the
muricid Trophon sp. ( 1 . 1 mm, 1 .5 whorls, D/Vol = 0.73).
All of these species lack protoconch ornamentation. Or-
namentation is also lacking on some members of the me-
sogastropod families Rissoidae (Richter and Thorson,
1975) and Cerithiidae (Rodriguez Babio and Thiriot-
Quievreux, 1974), as well as the neogastropod families
Nassariidae (Richter and Thorson, 1975) and Turridae
(Bouchet and Waren. 1980; Colman et al., 1986).

Protoconchs retained on adult specimens of the two
known gastropod species from the Juan de Fuca Ridge,
large enough to have laid the capsules from this site [Buc-
cinum viridum (pers. obs.) and Provanna variabilis( Waren
and Bouchet, 1986)], were badly corroded, precluding
comparison with larvae extracted from the Juan de Fuca
Ridge egg capsules. The potential for either of these two

species to be the source of the pouch-like capsules from
the Juan de Fuca Ridge is discussed above.

Molluscan larval dispersal at hydrothermal vents

Despite the ephemeral nature and patchy distribution
of hydrothermal vent environments, an analysis of the
developmental mode of 30 species of mollusks (gastropods
and bivalves) present at three deep-sea hydrothermal vents
(13°N, 21 °N and Galapagos Rift; Lutz el al.. 1980. 1984,
1986; Turner and Lutz, 1984; Turner et al., 1985; Lutz,
1988) suggested that only three species (two turrids and
a mytilid) have larvae capable of long-range dispersal. The
other 27 species have larval shell morphologies indicative
of non-planktotrophic, low-dispersal modes of develop-
ment. These developmental patterns are all typical of the
shallow-water members of the systematic group to which
these vent species belong. Although some unusual ad-
aptations may be present among larvae of vent organisms,
such as prolonged delay of metamorphosis in response to
low deep-sea temperatures (Lutz et al., 1980, 1984, 1986;
Turner and Lutz, 1984; Turner et al.. 1985; Lutz, 1988),
inferences made from egg size, fecundity, and larval mor-
phology suggest that unique adaptations to ensure suc-
cessful larval dispersal between vent habitats have not
evolved in hydrothermal vent mollusks (Turner et al.,
1985; Waren and Bouchet, 1989). However, many of the
molluscan morpho-species described from vents in the
eastern Pacific are present at more than one vent field (9
are shared by Galapagos Rift and 13°N; 10 are shared by
Galapagos Rift and 21°N; 18 are shared by 13°N and
2 1 °N; 2 are shared by Juan de Fuca and Explorer Ridges;
and 7 are shared by Galapagos Rift, 13°N and 21°N)
(Boss and Turner, 1980: Kenk and Wilson, 1985;Schein-
Fatton, 1985: McLean and Haszprunar, 1987; McLean,
1988. 1989a. b: Waren and Bouchet, 1986, 1989). This
paradox may be partially explained by recent findings of
vent fauna on or near whale carcasses (Smith et al., 1989;
but see Tunnicliffe and Juniper, 1990) and at cold meth-
ane and sulfide seeps (Paull et al., 1984; Kennicutt et al.,
1985, 1989; Juniper and Sibuet, 1987; Mayer et al.. 1988),
which may serve as stepping-stone habitats for dispersal
between vents. In addition, Johannesson (1988), and to
a lesser degree others (Palmer and Strathmann, 1981;
Burton, 1983; Highsmith, 1985; Hedgecock, 1986; Jack-
son, 1986; R. R. Strathmann, 1987; Safriel and Hadfield,
1988; O'Foighil, 1989), question the effective dispersal
benefits of the planktotrophic versus the non-plankto-
trophic mode of development, over long distances. It is
suggested that a small founder group of direct developers
or hermaphroditic individuals, passively transported to a
new site as adults or in a drifted egg mass, would have an
advantage over planktonic developers in establishing a
new colony, because their offspring would remain in the

HYDROTHERMAL VENT EGG CAPSULES

immediate vicinity of the founder group where the en-
counter rate with mates would be high. On the other hand,
the offspring of planktonic developers are free-swimming
for weeks and may settle far from the founder group, and
from each other, where the encounter rate with mates is
low (Johannesson. 1988).

Similar, if not identical, species of Phymorhynchus oc-
cur at the Galapagos Rift and at 1 3°N and 2 1 °N on the
East Pacific Rise (Waren and Bouchet, 1989). The plank-
totrophic-type larvae found in turrid egg capsules from
the Galapagos Rift, putatively identified as the spawn of
Phymorhynchus sp., suggests that this species has a great
potential for disperal and may explain its apparent wide
distribution (but see discussion above). Shimek (I983a,
b, 1986) has cultured three shallow-water turrids,
Ophiodermclla imrmis (Hinds). Ocnopolu levidensis
(Carpenter), and Oenopota elegans (Moller). with encap-
sulated periods of 50 days, 50 days, and 42-49 days, re-
spectively, and free-swimming periods of 35 days. 7-10
days, and 42-49 days, respectively. In addition, Oenopota
levidensis assumes a benthic existence and develops for a
further 25 days prior to metamorphosis as a "demersal-
planktotrophic larva," using the terminology of Shimek
(1986). If parallel conditions obtain in deep-sea turrids
the potential for dispersal of larvae during a 1-7 week
planktonic phase, given known bottom currents on the
East Pacific Rise (Lonsdale, 1977), would be on the order
of hundreds of kilometers (Lutz et a/.. 1980). A maximum
current speed of 18 cm s~' was recorded at a site 50 m
above the crest of the East Pacific Rise within 350 m of
a suspected hydrothermal plume (Lonsdale, 1977). In ad-
dition, a decrease in developmental rate in response to
cold ambient bottom waters away from the vents may
increase the length of larval life and further enhance dis-
peral (Lutz et ai, 1980, 1984, 1986; Turner and Lutz,
1984; Turner et ai. 1985; Lutz, 1988). Based on com-
parison of the stable isotope compositions (18O:'6O ratios)
of adult and larval shells, Killingley and Rex (1985) re-
ported that three deep-sea turrids with similar protoconch
sculpture to that seen in Phymorhynchus (Theta lyronu-
clea, T. chariessa and Pleurotomella sandersoni ), migrate
vertically, as larvae, to develop in warm surface waters.
At present, it is indeterminable whether Phymorhynchus
larvae complete their development as demersal feeders,
ascend to feed on plankton in surface waters, or undergo
some combination of these developmental modes. The
fact that Phymorhynchus is either a predator or scavenger
at the vents (Waren and Bouchet, 1989) indicates that it
may not be restricted to vent habitats, although it is able
to tolerate the extreme vent environment and exploit the
abundant food energy available at these sites. Adult Phy-
morhynchus are mobile and the extent to which move-
ments of adults aid in dispersal is unknown.

Although it has not been possible to unambiguously
identify the species to which the inflated triangular egg
capsules from the Galapagos Rift or the pouch-like egg
capsules from the Juan de Fuca Ridge belong, we can
infer something about the mode of development of these
two organisms. The presence of several hundred veliger
larvae in inflated triangular capsules from the Galapagos
Rift and the intricate sculpture on the early protoconch
both suggest that this species may develop planktotroph-
ically. On the other hand, the small number of larvae (1-
6) in capsules from the Juan de Fuca Ridge and the pro-
toconch's large size, inferred value of D/Vol, and un-
sculptured appearance all suggest that this species has a
non-planktotrophic mode of development.

How relatively sedentary organisms at deep-sea hydro-
thermal vents locate and colonize these geographically
isolated environments remains an open question. With
the exception of a few preliminary population genetic
studies (J. P. Grassle, 1985; Bucklin, 1988). our knowledge
of colonization, gene flow and dispersal of organisms be-
tween hydrothermal vents has been obtained from infer-
ences drawn from egg capsule type, egg size, fecundity,
and larval morphologies retained on adults (Lutz, 1988).
Further zoogeographic data and systematic descriptions
are needed before we can provide more rigorous answers
to questions involving the mechanisms of dispersal and
rates of gene flow between isolated areas of deep-sea hy-
drothermal vent activity. Laboratory culture of these un-
usual deep-sea molluscan taxa is also necessary to confirm
the link between larval shell characteristics and the mode
of development. If we assume that the majority of the
vent fauna is endemic to the hydrothermal vent habitat
[(but see contrasting opinion of Clarke (1986)], and that
larval dispersal in non-planktotrophic species is a step-
wise process, then each ridge axis should be a discrete
dispersal corridor. Given these assumptions, genetic re-
latedness of the most widely separated non-planktotrophic
species' populations along a single ridge axis should be
more homogeneous than among populations that are
equally separated but belong to two different ridge axes.
On the other hand, genetic relatedness of species with
planktotrophic development should be more homogenous
in the prevailing direction of bottom currents and less
reliant on the configuration of ridge systems. The studies
of J. P. Grassle (1985) and Bucklin (1988) provide highly
paradoxical results. In Bathymodiolus thermophilus, a
species reported to have a lengthy dispersal stage (Lutz et
ai, 1980), populations from the Galapagos Rift and 13°N
(separated by 2200 km) are genetically distinct. Yet in
Riftiapachyptila, a non-molluscan species that is believed
to have lecithotrophic, demersal larvae with limited dis-
persal abilities, more widely separated populations from
the Galapagos Rift and 2 1 °N along the East Pacific Rise
(separated by 3300 km) are genetically similar (Bucklin,

R. G. GUSTAFSON ET .-)/_

1988). Clearly, an expanded research effort using electro-
phoretic and molecular techniques to ascertain population
structure within species and genetic relatedness among
species, coupled with analyses of molluscan larval shell
morphology, will be needed to answer questions concern-
ing the rates of gene flow between discrete areas of hy-
drothermal activity associated with contiguous and non-
contiguous oceanic ridge systems, as well as the validity
of using larval shell morphology to ascertain dispersal ca-
pability in the deep sea.

Acknowledgments

We thank the pilots and crew of "Atlantis II"/"Alvin"
for invaluable technical assistance with the retrieval of
specimens; Robert Hessler of Scripps Institution of
Oceanography for his extraordinary powers of observation
which led to retrieval of marker H9 and its attached egg
capsules from the Galapagos Rift; John Grazul of the
Electron Microscopy Facility, Nelson Biological Labo-
ratories, Rutgers University, for assistance with SEM;
Meridith L. Jones of the Smithsonian Institution, for
samples from Juan de Fuca Ridge; and Lowell Fritz for
critical comments throughout this study and for critically
reading early versions of the manuscript. This is Contri-
bution #90-30 of the Institute of Marine and Coastal Sci-
ences, Rutgers University and New Jersey Agricultural
Experiment Station Publication No. D-32402-1-90, sup-
ported by State funds and by NSF Grants OCE-87- 16591
and OCE-89- 17311.

Literature Cited

Amio, M. 1955. On the eggs and early life histories of Pyrenidae (Col-
umbellidae) in marine gastropods. J. Shimonoseki Co/I. Fish. 4: 231-
238.

Amio, M. 1963. A comparative embryology of marine gastropods, with
ecological considerations. J. Shimonoseki Coll. Fish. 12: 229-358.

Anderson, D. T. 1960. The life histories of marine prosobranch gas-
tropods. J. Malawi. Soc. Ansl. 4: 16-29.

Anderson, D. T. 1962. The reproduction and early life histories ot the
gastropods Bembicium aiirantin (Quoy and Gaimard) (Fam. Litto-
rinidae). Ccllana tramoserica (Sower.) (Fam. Patellidae) and Mela-
nerita melanotragus (Smith) (Fam. Neritidae). Proc. Linn. Soc. N.
S. ir. 87:62-68.

Anderson, D. T. 1965. Further observations on the life histories of
littoral gastropods in New South Wales. Proc. Linn. Soc. N. S. W.
90:242-251.

Arnaud, P. M., and H. Zibrowius. 1973. Capsules ovigeres de gastero-
podes Turridae et corrosion du squelette des scleractinaires bathyaux
des Acores. Rev. Fac. Cienc. Univ. Lisb. Ser. C Cienc Nat. 17: 581-
597.

Bacci, G. 1947. Le capsule ovigere di Columbella ntslica (L.) e di /•'<«-
ciolaria lignaria (L.) (Prosobranchia. Stenoglossa). Boll. Zool. 14:
75-81.

Bandel, K. 1974a. Spawning and development of some Columbellidae
from the Caribbean Sea of Colombia (South America). I'eliger 16:
271-282.

Bandel, K. 1974b. Studies on Littorinidae from the Atlantic. I'eliger

17:92-114.
Bandel, K. 1975a. Embryonalgehause karibischer Meso- und Neogas-

tropoden (Mollusca). Abhandlungen der Mathematisch-Naturwis-

\cnschajilichen Masse. Akademic tier Wissenshaften und tier Liter-
aim: Maim. Jahrgang IV7\ A> /.. 133 pp.
Bandel. K. 1975b. Embryonale und larvale Schale einiger Prosobranchier

(Gastropoda, Mollusca) der Oosterschelde (Nordsee). Hydrohiol. Bull

9: 3-22.
Bandel, K. 1975c. Das Embryonalgehause mariner Prosobranchier der

Region von Banyuls-sur-Mer. I 7c Milieu Ser. A Biol. Mar. 25: 83-

1 18.
Bandel, K. 1976a. Observations on spawn, embryonic development

and ecology of some Caribbean lower Mesogastropoda. Veliger 18:

249-270.
Bandel, K. 1976b. Spawning, development and ecology of some higher

Neogastropoda from the Caribbean Sea of Colombia (South America).

Veliger 19: 176-193.
Bandel, K. 1982. Morphologic und Bildung der friihontogenetischen

Gehause bei conchiferan Mollusken. Fades 1: 1-198.
Berg, C. J., Jr. 1985. Reproductive strategies of mollusks from abyssal

hydrothermal vent communities. In The Hydrothermal Vents of the

Eastern Pacific: An Overview. M. L. Jones, ed. Biol. Soc. ll'ash. Bull.

6: 185-197.
Boss, K. J., and R. D. Turner. 1980. The giant white clam from the

Galapagos Rift. Calyptogena magnitica. species novum. Ma/acologia

20: 161-194.
Bouchel, P. 1976a. Mise en evidence de stades larvaires planctoniques

chez des Gasteropodes Prosobranches des etages bathyal et abyssal.

Bull. Mits. Nail. Hist. Nat. Zool. 277: 947-971.
Bouchet, P. 1976b. Mise en evidence d'une migration de larves veligeres

entre I'etage abyssal et la surface. C. R. Hebd. Seances Acad. Sd.

Ser. D Set. Nal. 283: 821-824.
Bouchel, P. 1981. Evolution of larval development in Eastern Atlantic

Terebridae (Gastropoda), Neogene to Recent. Malacologia 21: 363-

369.
Bouchet, P. 1989. A review of poecilogony in gastropods. J. Moll. Stud.

55: 67-78.
Bouchet, P., and J-C. Fontes. 1981. Migrations verticales des larves de

Gasteropodes abyssaux: arguments nouveaux dus a 1'analyse isoto-

pique de la coquille larvaire et postlarvaire. C R Hebd. Seances

Acad. Sd. Ser. Ill 292: 1005-1008.
Bouchet, P., and A. \\aren. I979a. The abysal molluscan fauna of the

Norwegian Sea and its relation to other faunas. Sarsia 64: 21 1-243.
Bouchet, P., and A. \\aren. 1979b. Planktotrophic larval development

in deep-water gastropods. Sarsia 64: 37-40.
Bouchet, P., and A. \\aren. 1980. Revision of the north-east Atlantic

bathyal and abyssal Turndae (Mollusca, Gastropoda). J. Moll. Stud

Suppl. 8: 1-119.
Bouchet, P., and A. \\aren. 1985a. Mollusca Gastropoda: Taxonomical

notes on tropical deep water Buccinidae with descriptions of new

taxa. Mem. Mus. Nail. Hist Nat. Ser. A Zool. 133: 457-499.
Bouchet, P., and A. Waren. I985b. Revision of the north-east Atlantic

bathyal and abyssal Neogastropoda, excluding Tumdae. Boll. Ma-

lacol. Suppl. 1: 121-296.
Hiii-Hin. A. 1988. Allozymic variability of Riftia pachypnla populations

from the Galapagos Rift and 21°N hydrothermal vents. Deep-Sea

Res 35: 1759-1768.
Burton, R. S. 1983. Protein polymorphisms and genetic differentiation

of marine invertebrate populations. Mar. Biol. Lett. 4: 193-206.
Clarke, A. H. 1962. Annotated list and bibliography of the abyssal

marine molluscs of the world. Bull. Nail. Mils. Canada 181: 1-1 14.
Clarke, A. H. 1986. Some alternative views about deep-sea mollusks

from the Arctic and from hydrothermal vents. Malacology Data Net

1:49-57.

HYDROTHERMAL VENT EGG CAPSULES

Colman, J. G., and P. A. Tyler. 1988. The egg capsules of ( 'O/H.V /(•/•
freysianus (Fischer, 1868) (Prosobranchia: Neogastropoda) from the
Rockall Trough, Nonh East Atlantic. Sarsia 73: 139-145.

Colman, J. G., P. A. Tyler, and J. D. Gage. 1986. Larval development
of deep-sea gastropods (Prosobranchia: Neogastropoda) from the
Rockall Trough. / Mar. Biol. Assoc. IK 66: 951-965.

Cowan, I. M. 196-4. The egg capsule and young of Beringius everdanu
Smith. I 'cli.wr 1: 43-44.

Crane, K. 1985. The distribution of geothermal fields along the mid-
ocean ridge: an overview. In The Hydrothermal I 'ems of tin- Eastern
Pacific An Ovcn'ic»; M. L. Jones, ed. Biol. Sue. II 'ash Bull 6: 3-
18.

Crisp, D. J. 1978. Genetic consequences of different reproductive
strategies in marine invertebrates. Pp. 257-273 in Marine Organisms:
Genetics. Ecology, and Evolution. B. Battaglia and J. A. Beardmore,
eds. Plenum Press, New York.

Dall, \V. H. 1890. Scientific results of explorations by the U.S. Fish
Commission steamer "Albatross." VII. Preliminary report on the
collection of Mollusca and Brachiopoda. Proi: US Nail. Mus 12:
219-362.

Dall, \V. H. 192-4. On the value of nuclear characters in the classification
of marine gastropods. J \Vash.Acad Sci 1-4: 177-180.

D'Asaro, C. N. 1970a. Egg capsules of prosobranch mollusks from
South Florida and the Bahamas and notes on spawning in the lab-
oratory. Bull. Mar Sci. 20: 414-440.

D'Asaro, C. N. 1970b. Egg capsules of some prosobranchs from the
Pacific coast of Panama. I'cliger 13: 37-43.

D'Asaro, C. N. 1986. Egg capsules of eleven marine prosobranchs from
northwest Florida. Bull Afar. Sci. 39: 76-9 1 .

D'Asaro, C. N. 1988. Micromorphology of neogastropod egg capsules.
Nautilus 102: 134-148.

Eyster, L. S., and M. P. Morse. 1984. Early shell formation during
molluscan embryogenesis, with new studies on the surf clam, Spisula
soluhssima Am. 7.ool. 24: 871-882.

Franc, A. 1950. Ponte et larves planctoniques de Philbcrtia purpurea
(Montagu). Bull Lab Maril. Dinard 33: 23-25.

Fretter, V. 1984. Prosobranchs. Pp. 1-45 in The Mollusca. Vol. 7.
Reproduction. A. S. Tompa, N. H. Verdonk and J. A. M. van den
Biggelaar. eds. Academic Press. New York.

Fretter, \ ., and A. Graham. 1962. British Prosobranch .\to/liisc\ I heir
Functional Anatomy and Ecology. Bartholomew Press. London. 755
pp.

Golikov, A. N. 1961. Ecology of reproduction and the nature of egg
capsules in some gastropod molluscs of the genus Neptimca (Bolten).
Zoo/. Z/i. 40:997-1008.

Grassle, J. F. 1985. Hydrothermal vent animals: distribution and bi-
ology. Science 229: 713-725.

Grassle, J. F. 1986. The ecology of deep-sea hydrothermal vent com-
munities. Adv. Mar Biol. 23: 301-362.

Grassle, J. P. 1985. Genetic differentiation in populations of hydro-
thermal vent mussels (Bathymodiolus thermophilus) from the Gal-
apagos Rift and 1 3°N on the East Pacific Rise. In The Hydrothermal
I 'ems of the Eastern Pacific: An Overview. M. L. Jones, ed. Biol. Soc.
Wash. Bull 6:429-442.

llabe, I. 1960. Egg masses and egg capsules of some Japanese marine
prosobranchiate gastropod. Bull Mar Biol. Station Asamushi 10:
121-126.

Hadfield, M. G., and M. F. Strathmann. 1990. Heterostrophic shells
and pelagic development in trochoideans: implications for classifi-
cation, phylogeny and palaeoecology. / Moll. Stud. 56: 239-256.

Hansen, T. A. 1982. Modes of larval development in Early Tertiary
neogastropods. Paleobiology 8: 367-377.

Hansen, T. A. 1983. Modes of larval development and rates of specia-
tion in Early Tertiary neogastropods. Science 220: 501-502.

Hedgecock, D. 1986. Is gene flow from pelagic larval dispersal important
in the adaptation and evolution of marine invertebrates? Bull. Mar.
Sci. 39: 550-564.

Heslinga, G. A. 1981. Larval development, settlement and metamor-
phosis of the tropical gastropod Troclnis niloticus. Malacologia 20:
349-357.

Highsmith, R. C. 1985. Floating and algal rafting as potential dispersal
mechanisms in brooding invertebrates. Mar. Ecol. Prog. Ser. 25: 169-
179.

Hines, A. H. 1986. Larval problems and perspectives in life histories
of marine invertebrates. Bull. Mar. Sci. 39: 506-525.

Hoagland, K. E., and R. Robertson. 1988. An assessment of poecilogony
in marine invertebrates: phenomenon or fantasy? Biol. Bull. 174:
109-125.

llyman, L. H. 1967. The Invertebrates, ^'ol 1 7. Mollusca I McGraw-
Hill, New York. 790 pp.

Jablonski. D. 1985. Molluscan development. Pp. 33-49 in Mollusks.
Notes For a Shon Course. T. W. Broadhead. ed. LJniversity of Ten-
nessee Studies in Geology 13, University of Tennessee Press, Knox-
ville.

Jablonski, D. 1986. Larval ecology and macroevolution in marine in-
vertebrates. Bull. Mar Sci. 39: 565-587.

Jablonski. D., and R. A. Lutz. 1980. Molluscan larval shell morphology:
ecological and paleontological applications. Pp. 323-377 in Skeletal
Growth of Aquatic Organisms. D C. Rhoads and R. A. Lutz, eds.
Plenum Press, New York.

Jablonski, D., and R. A. l.utz. 1983. Larval ecology of marine benthic
invertebrates: paleobiological implications. Biol. Rev 58: 21-89.

Jackson, J. B. C. 1986. Modes ot dispersal of clonal benthic inverte-
brates: consequences for species' distributions and genetic structure
of local populations. Bull. Mai: Sci. 39: 588-606.

Johannesson, K. 1988. The paradox of Rockall: why is a brooding
gastropod (Lillorina sa.\alilis) more widespread than one having a
planktonic dispersal stage (L. litloreaY! Mar Biol 99: 507-513.

Jung, P. 1975. Quaternary larval gastropods from leg 15. site 147, deep
sea drilling project. Preliminary report. I'eliger 18: 109-126.

Juniper, S. K., and M. Sibuet. 1987. Cold seep benthic communities
in Japan subduction zones: spatial organization, trophic strategies
and evidence for temporal evolution. Mar. Ecol. Prog. Ser. 40: 1 15-
126.

Kenk, V. C., and B. R. Wilson. 1985. A new mussel (Bivalvia. Mytilidae)
from hydrothermal vents in the Galapagos Rift zone. Malacologia
26:253-271.

Kennicutt, M. C., II, J. M. Brooks, R. R. Bidigare, T. L. Wade, and
R. R. Faye. 1985. Vent type taxa in a hydrocarbon seep region on
the Louisiana slope. Nature 317: 351-353.

Kennicutt, M. C., II, J. M. Brooks, R. R. Bidigare, S. J. McDonald, and
D. L. Adkison. 1989. An upper slope "cold" seep community:
northern California. Limnol. Oceanogr. 34: 635-640.

Kesteven, H. L. 1912. The constitution of the gastropod protoconch:
its value as a taxonomic feature and the significance of some of its
forms. Proc. Linn. Soc. New South M 'ales 37: 49-82.

killingley, J. S., and M. A. Rex. 1985. Mode of larval development
in some deep-sea gastropods indicated by oxygen- 1 8 values of their
carbonate shells. Deep-Sea Res. 32: 809-818.

kohn. A. J. 1961. Studies on spawning behavior, egg masses, and larval
development in the gastropod genus Comix. II. Observations in the
Indian Ocean during the Yale Seychelles Expedition. Bull. Bingham
Oceanogr. Collect. Yale Univ. 17: 3-51.

Lebour, M. V. 1934. The eggs and larvae of some British Turridae. J.
Mar. Biol. Assoc. U.K. 19: 541-554.

Lebour, M. V. 1936. Notes on the eggs and larvae of some Plymouth
prosobranchs. / Mar. Biol. Assoc. U.K 20: 547-564.

R. G. GUSTAFSON ET AL.

Lebour, M. \r. 1937. The eggs and larvae of the British prosobranchs
with special reference to those living in the plankton. J Mar Biol.
Assoc i'.K 22: 105-166.

Lima, G. M., and R. A. Lutz. 1990. The relationship of larval shell
morphology to mode of development in marine prosohranch gastro-
pods. J Mar. Bid/ Assoc I A 70: 61 1-637.

Lonsdale, P. 1977. Clustering of suspension-feeding macrohenthos near
abyssal hydrothermal vents at oceanic spreading centers. Deep-Sea
Res. 24: 857-863.

Lutz, R. A. 1988. Dispersal of organisms at deep-sea hydrothermal
vents. Pp. 23-29 in Proceedings of the Hydrothermalism, Biology
and Ecology Symposium, Pans. 4-7 November 1985. Oceanol Ada
Special Vol. No. 8.

Lutz, R. A., P. Bouchet, D. Jablonski, R. D. Turner, and A. Waren.
1986. Larval ecology of mollusks at deep-sea hydrothermal vents.
Am. Malacol. Bull 4: 49-54.

Lutz, R. A., L. W. Fritz, and D. C. Rhoads. 1985. Molluscan growth
at deep-sea hydrothermal vents. In The Hydrothermal I'ents of the
Eastern Pacific': An Overview. M. L. Jones, ed. Biol. Soc. Wash. Bull.
6: 199-210.

Lutz, R. A., D. Jablonski, D. C. Rhoads, and R. D. Turner. 1980. Larval
dispersal of a deep-sea hydrothermal vent bivalve from the Galapagos
Rid. Alar. Biol. 57: 127-133.

Lutz, R. A., D. Jablonski, and R. D. Turner. 1984. Larval development
and dispersal at deep-sea hydrothermal vents. Science 226: 1451-
1454.

MacGinitie, G. E. 1955. Distribution and ecology of the marine in-
vertebrates of Point Barrow, Alaska. Smilhson. Misc. Colled. 128(9):
1-201.

Macintosh, R. A. 1979. Egg capsule and young of the gastropod Ber-
ingius bermgii (MiddendorfT) (Neptuneidae). I'eliger 21: 439-441.

Macintosh, R. A. 1986. Egg capsule and young of the gastropod Ber-
nigins (Neoberingius) Inelei (Dall) (Neptuneidae). }'eliger 28: 426-
428.

Marcus, E. B.-R., and E. Marcus. 1962. Studies on Columbellidae.
Bui. Fac Filos dene. Lei L'niv. Sao Paulo Ser Zool 24: 335-402.

Mayer, L. A., A. N. Shor, J. Hughes Clarke, and D. J. \V. Piper.
1988. Dense biological communities at 3850 m on the Laurentian
Fan and their relationship to the deposits of the 1929 Grand Banks
earthquake. Deep-Sea Res. 35: 1235-1246.

McLean, J. H. 1988. New archaeogastropod limpets from hydrother-
mal vents. Superfamily Lepetodnlacea. Part 1: Systematic descrip-
tions. Philos. Trans. R Soc. Land. B Biol Sfi. 319: 1-32.

McLean, J. H. 1989a. New archaeogastropod limpets from hydro-
thermal vents: New family Peltospindae, new superfamily Peltospi-
racea. Zool. Scr. 18: 49-66.

Mcl^ean, J.ll. 1989b. New slit limpets (Scissurellacea and Fissurellacea)
from hydrothermal vents. Part 1 . Systematic descriptions and com-
parisons based on shell and radular characters. Contrib. Sci. (Los
Angel.) 407: 1-29.

McLean, J. H., and G. Haszprunar. 1987. Pyropeltidae, a new family
of cocculiniform limpets from hydrothermal vents. I'eliger 30: 196-
205.

Ockelmann, K. VV. 1965. Developmental types in marine bivalves and
their distribution along the Atlantic coast of Europe. Pp. 25-35 in
Proceedings of the First European Malacological Congress. London.
1962. L. R. Cox and J. F. Peake, eds. Conchological Society of Great
Britain and Ireland and the Malacological Society of London. London.

O'Foighil, D. 1989. Planktotrophic larval development is associated
with a restricted geographic range in Lasaea, a genus of brooding.
hermaphroditic bivalves. Alar. Biol. 103: 349-358.

Palmer, A. R., and R. R. Strathmann. 1981. Scale ol dispersal in varying
environments and its implications for life histories of marine inver-
tebrates. Oecologia 48: 308-318.

Paull, C. K., B. 1 lecker, R. C'ommeau. R. P. Kreeman-Lynde, C. Neumann,
W. P. Corso, S. Golubic. J. E. Hook, E. Sikes, and J. Curray.
1984. Biological communities at the Florida Escarpment resemble
hydrothermal vent taxa. Science 226: 965-967.

Pa«lik, J. R., J. B. O'SulIivan, and M. G. Harasewych. 1988. The egg
capsules, embryos, and larvae of Cancellaria cooperi (Gastropoda:
Cancellariidae). Nautilus 102: 47-53.

Pechenik, J. A. 1986. The encapsulation of eggs and embryos by mol-
luscs: an overview. Am. Malacol. Bull. 4: 165-172.

Penchaszadeh, P. E. 1982. Reproductive aspects of Polystira barretli
(Guppy. 1866) (Gastropoda, Turridae) from Golfo Triste, Venezuela.
1'eliger 25: 160-162.

Perron, F. E. 1981. Larval biology of six species of the genus Conns
(Gastropoda: Toxoglossa) in Hawaii. USA. Afar. Biol. 61: 215-220.

Perry, L. M., and J. S. Schwengel. 1955. Marine shells of the western
coast of Florida. Bull Am Paleonlol 26: 1-318.

Petit, G., and J. Risbec. 1929. Sur la ponte de quelques Gasteropodes
Prosobranches. Bull. Soc. Zool Fr 54: 564-570.

Pilkington, M. C. 1974. The eggs and hatching stages of some New
Zealand prosobranch molluscs. / R Soc. N.Z. 4: 41 1-431.

Powell, A. \V. B. 1942. The New Zealand recent and fossil Mollusca
of the family Turndae. Bull. Aiickl Insl. Alus. No. 2: 1-188.

Radwin, G. E., and J. L. Chamberlin. 1973. Patterns of larval devel-
opment in stenoglossan gastropods. Traits. San Diego Soc. Nat. Hist
17: 107-1 18.

Rex, M. A., and A. Waren. 1982. Planktotrophic development in deep-
sea prosobranch snails from the western North Atlantic. Deep-Sea
Res 29: 171-184.

Richter. G., and G. Thorson. 1975. Pelagische prosobranchier-larven
des Golfes von Neapel. Ophelia 13: 109-185.

Robertson, R. 1971. Scanning electron microscopy of planktonic larval
marine gastropod shells. Veliger\4: 1-12.

Robertson. R. 1976. Marine prosobranch gastropods: larval studies and
systematics. Tlialass. Jugoslav 10: 213-238.

Rodriguez Babio, C., and C. Thiriot-Quievreux. 1974. Gasteropodes
de la Region de Roscoff. Etude particuliere de la protoconque. Cah.
Biol .Mar 15: 531-549.

Roller, R. A., and W. B. Stickle. 1988. Intracapsular development of
Thais haemastoma canaliculata (Gray) (Prosobranchia; Muricidae)
under laboratory conditions. Am. Malacol. Bull. 6: 189-197.

Safriel, U. N., and M. G. Hadfield. 1988. Sibling speciation by life-
history divergence in Dendropoma (Gastropoda; Vermetidae). Biol.
J Linn. Soc. 35: 1-13.

Schein-Fatton, E. 1985. Decouverte sur la ride du Pacifique oriental
a 13°N d'un Pectinidae (Bivalvia. Pteromorpha) d'affinites paleo-
zoiques. C R Hehd Seances Acad. Sci. Ser. Ill 301: 491-496.

Scheltema, A. H. 1969. Pelagic larvae of New England gastropods. IV.
Anachis translirala and Anacllis avara (Columbellidae, Prosobran-
chia). I'ie Milieu Ser. A Biol Mar. 20: 94-104.

Scheltema, R. S. 1978. On the relationship between dispersal of pelagic
veliger larvae and the evolution of marine prosobranch gastropods.
Pp. 303-322 in Marine Organisms. B. Battaglia and J. A. Beardmore.
eds. Plenum, New York.

Scheltema, R. S. 1981. The significance of larval dispersal to the evo-
lution ot benthic marine species. Pp. 130-145 in Genetics and Re-
production of Marine Organisms. V. L. Kasyanov and A. I. Poduvkin.
eds. Proc. XIV Pacific Sci. Congr. Khabarovsk. Aug. 1979, Section
Marine Biology, Issue 2, Acad. Sci. USSR.

Scheltema, R. S., and I. P. Williams. 1983. Long-distance dispersal of
planktonic larvae and the biogeography and evolution of some Poly-
nesian and western Pacific mollusks. Bull. Mar. Sci. 33: 545-565.

Shimek, R. L. 1983a. Biology of the Northeastern Pacific Tumdae. I.
Opluodermella. Alalacologia 23: 281-312.

HVDROTHERMAL VENT EGG CAPSULES

Shimek, R. L. I983b. Biology of the Northeastern Pacific Turndae. II.

Oenopoui ./ Moll Stud 49: 146-163.
Shimek, R. L. I983c. Biology of the Northeastern Pacific Turridae.

111. The habit and diet of Kurtziella plumbea (Hinds. 1843). I'eliger

26: 10-17.
Shimek, R. I,. 1986. Biology of the Northeastern Pacific Turridae. IV.

Demersal development, synchronous settlement and other aspects

of the larval biology of Oenopota /evidensis Int ./ I men Reprod

10: 313-333.

Shuto, T. 1974. Larval ecology of prosobranch gastropods and its bear-
ing on biogeography and paleontology. Lethaia 7: 239-256.
Smith, C. R., H. Kukert, R. A. \\heatcroft, P. A. Jumars. and J. W.

Drilling. 1989. Vent fauna on whale remains. Nature 341: 27-28.
Sohl, N. F. 1977. Utility of gastropods in biostratigraphy. Pp. 519-

539 in Concepts and Methods in Biostratigraphy. E. G. KaulTman

and J. E. Hazel, eds. Dowden, Hutchmson & Ross. Stroudsburg,

Pennsylvania.
Soliman, G. N. 1987. A scheme for classifying gastropod egg masses

with special reference to those from the northwestern Red Sea. /

Moll Slttii. 53: 1-12.
Steele, D. H. 1977. Correlation between egg size and developmental

period. Am. \ai. 111: 371-372.
Strathmann. M. F. 1987. Phylum Mollusca. Class Gastropoda. Subclass

Prosobranchia. Pp. 220-267 in Reproduction and Development of

Marine Invertebrates of the Northern Pacific Const Univ. of Wash-
ington Press, Seattle and London.
Strathmann, R. R. 1977. Egg size, larval development, and juvenile

size in benthic marine invertebrates. Am. Nat. Ill: 373-376.
Strathmann, R. R. 1978a. The evolution and loss of feeding larval stages

of marine invertebrates. Evolution 32: 894-906.
Strathmann, R. R. I978b. Progressive vacating of adaptive types during

the Phanerozoic. Evolution 32: 907-914.
Stralhmann, R. R. 1987. Larval feeding. Pp. 465-550 in Reproduction

of Marine Invertebrates, I'oL IX. Genera/ Aspects Seeking L'nity in

Diversity. Blackwell. Palo Alto. California and Boxwood Press. Pacific

Grove. California.
Thorson, G. 1935. Studies on the egg-capsules and development of

arctic marine prosobranchs. Medd. Grant. 100: 1-71.
Thorson, G. 1940a. Studies on the egg masses and larval development

of Gastropoda from the Iranian Gulf. Dan Invest Iran 2: 159-238.
Thorson, G. 1940b. Notes on the egg capsules of some North-Atlantic

Prosobranchs of the genus Troschelia. Chrysodomus, \'olutopsis. Si-

phoand Trophon. I'idensk. Medd. Dan. Naturhisl. Foren. 104: 251-

266.
Thorson, G. 1944. The zoology of east Greenland: Marine Gastropoda

Prosobranchiata. Medd. Grtml 121: 1-181.

Thorson, G. 1946. Reproduction and larval development of Danish
marine bottom invertebrates, with special reference to the planktonic
larvae in the Sound (Oresund). Medd Dan Eisk Havunders Scnc
Plankton 4: 1-523.

Thorson, G. 1950. Reproduction and larval ecology of marine bottom
invertebrates. Bio/ Rev 25: 1-45.

Todd, C. D., and R. \V. Doyle. 1981 . Reproductive strategies of marine
benthic invertebrates: a settling-timing hypothesis. Afar. Ecol. Prog
Ser 4: 75-83.

Tunnicliffe, V. 1988. Biogeography and evolution of hydrothermal-
vent fauna in the eastern Pacific Ocean. Proe. R Soc. Loud. B 233:
347-366.

Tunnicliffe, V., and S. K. Juniper. 1990. Cosmopolitan underwater
fauna. Nature 344: 300.

Tunnicliffe, V., and A. R. Fontaine. 1987. Faunal composition and
organic surface encrustations at hydrothermal vents on the southern
Juan de Fuca Ridge. ./. Geophvs Res 92(B1 1): 1 1.303-1 1,314.

Tunnicliffe, \ ., S. K. Juniper, and M. F. de Burgh. 1985. The hydro-
thermal vent community on Axial Seamount, Juan de Fuca Ridge.
In The Ilvtlroihennal I iwv ot the Eastern Pacific: An Overview.
M. L. Jones, ed. Biol. Soc. Hash Bull. 6: 199-210.

Turner, R. D., and R. A. l.utz. 1984. Growth and distribution of mol-
lusks at deep-sea vents and seeps. Oceanus 27: 55-62.

Turner, R. D., R. A. Lutz, and D. Jablonski. 1985. Modes of molluscan
larval development at deep-sea hydrothermal vents. In The Hydro-
thermal I cuts o/ the Eastern I'acilic An Oven-ie\\: M. L. Jones, ed.
Biol. Soc Hash Hull 6: 167-184.

Underwood, A. J. 1974. On models for reproductive strategy in marine
benthic invertebrates. Am Hal 108: 874-878.

Vance, R. R. 1973. On reproductive strategies in marine benthic in-
vertebrates. Am. Nat. 107: 339-352.

Vance, R. R. 1974. Reply to Underwood. Am. Nat 108: 879-880.

Vaught.K.C. 1989. .1 Classification of the Living Mollusca. Melbourne.
Florida. American Malacologists. 189 pp.

Vestergaard, K. 1935. Uber den Laich und die Larven von Sea/aria
cominuius (Lam.), h'assanus pygmaeus (Lam.) und Be/a turricola
(Mont.). Zool An:. 109: 217-222.

\Varen, A., and P. Bouchet. 1986. Four new species of Provanna Dall
(Prosobranchia. Centhiacea?) from East Pacific hydrothermal sites.
/ool. Scr 15: 157-164.

\Varen. A., and P. Bouchet. 1989. New gastropods from East Pacific
hydrothermal vents. Zool. Scr 18: 67-102.

Webber, H. H. 1977. Gastropoda: Prosobranchia. Pp. 1-97 in Repro-
duction of Marine Invertebrates. I'ol. 4. Molluscs: Gastropods and
Cephalopods. A. C. Giese and J. S. Pearse. eds. Academic Press. New
York.

Reference: Biol. Bull- 180: 56-64. (February. 1'i'H I

Expansion of the Sperm Nucleus and Association

of the Maternal and Paternal Genomes in

Fertilized Mulinia lateralis Eggs

FRANK J. LONGO1 AND JOHN SCARPA2

* Department of Anatomy. The University of Iowa. Iowa City. Iowa 52242 and 2 Rutgers Shellfish
Research Laboratory. Port Norris, New Jersey 08349

Abstract. Sperm nuclear expansion, meiotic maturation
of the maternal chromatin, and events involving the as-
sociation of the male and female pronuclei leading to the
two-cell stage were observed in Mulinia zygotes using the
fluorochromes DAPI and Hoechst. The effects of ultra-
violet irradiation on the fertilizing sperm were also ex-
amined. Incorporated sperm nuclei underwent changes
in diameter that were temporally correlated with meiotic
processes of the maternal chromatin. Following its entry,
the sperm nucleus underwent a rapid, initial enlargement,
which was correlated with germinal vesicle breakdown.
Sperm nuclear expansion ceased during the period in
which the egg was engaged in polar body formation and
was re-initiated with formation and enlargement of the
female pronucleus. The rates of enlargement of the male
and female pronuclei were 0.59 and 0.65 ^m/min, re-
spectively. Following their migration into apposition with
one another, the male and female pronuclei synchro-
nously underwent events characteristic of prophase as
separate structures; i.e.. chromosome condensation, and
nuclear envelope breakdown. The two groups of chro-
mosomes that formed became organized on the metaphase
plate in preparation of the first cleavage division; hence,
there was no fusion of pronuclei. Ultraviolet irradiation
of fertilizing sperm had no apparent affect on sperm nu-
clear transformations leading to the development of a male
pronucleus or on female pronuclear development. How-
ever, events subsequent to the apposition of the pronuclei
were affected and included asynchrony of prophase and
the nondisjunction of chromosomes at anaphase. These
observations are discussed in relationship to events reg-

Received 17 September 1990; accepted 29 November 1990.

ulating transformations of the sperm nucleus and exper-
iments to generate gynogenetic bivalve embryos.

Introduction

For the eggs of most animals insemination occurs at
an arrested stage of meiosis; i.e., meiotic prophase (the
germinal vesicle stage), metaphase I, or metaphase II
(Longo, 1987a). Representatives of these three stages in-
clude the eggs of annelids, mollusks, and chordates, re-
spectively. In comparison, the eggs of relatively few or-
ganisms are fertilized following the completion of meiotic
maturation (the pronuclear stage). The most notable ex-
ample of the latter group are eggs of echinoids. Although
processes of fertilization are fundamentally the same in
eggs inseminated at different stages of meiotic maturation,
there are prominent differences, particularly during the
transformation of the sperm nucleus into a male pronu-
cleus and pronuclear association (Wilson, 1925; Longo.
1985).

In eggs inseminated at the completion of meiotic mat-
uration, the female pronucleus is already present and
"waiting" for the entry and transformation of the sperm
nucleus into a pronucleus. In contrast, in eggs inseminated
at an arrested stage of meiotic maturation, the sperm nu-
cleus, following its entry into the egg cytoplasm, must
"wait" for the maternal chromatin to complete its meiotic
maturation. Observations carried out with the gametes of
a variety of organisms (sea urchin, surf clam, mussel,
hamster, rabbit, and mouse) have shown that both the
kinetics of sperm nuclear enlargement into a male pro-
nucleus and events attending pronuclear association are
correlated with the stage of meiosis at which the egg is
inseminated and the length of time the sperm nucleus
spends in the egg cytoplasm before pronuclear association

FERTILIZATION EVENTS IN MULINIA

(Wilson, 1925; Longo, 1985). For example, in eggs in-
seminated at the pronuclear stage, the rate of sperm nu-
clear expansion is uniform, whereas in eggs inseminated
at an arrested stage of meiosis, the rate of expansion is
much more complex and shows different phases that are
correlated with stages of meiotic maturation of the ma-
ternal chromatin(Luttmer and Longo, 1987. 1988; Wright
and Longo, 1988; Longo, 1989).

In eggs inseminated at an arrested stage of meiosis,
pronuclear fusion does not occur as in eggs fertilized at
the pronuclear stage. The paternally and maternally de-
rived chromatin do not become associated with one an-
other until prophase of the first cleavage division when
chromosomes derived from the male and female pronuclei
intermix and become aligned on the metaphase plate of
the mitotic spindle (Longo, 1985). Evidence suggests that
differences in the kinetics of sperm nuclear expansion and
pronuclear association are related to cell cycle events as-
sociated with meiotic maturation and mitosis of the first
mitotic division (Luttmer and Longo, 1988; Wright and
Longo, 1988; Longo, 1989).

Because analyses of sperm nuclear expansion and its
relationship to meiotic events of the maternal chromatin
have been carried out in relatively few organisms (see
Longo, 1989), and to further explore possible relationships
between these processes of fertilization and cell cycle phe-
nomena, we have initiated studies with a variety of or-
ganisms, the eggs of which are inseminated at an arrested
stage of meiosis. Here we describe the course of meiotic
maturation of the maternal chromatin, corresponding
events of sperm nuclear enlargement, and association of
the male and female pronuclei in Mulinia lateralis (dwarf
surf clam or coot clam) eggs, which are inseminated at
meiotic prophase. The effects of ultraviolet irradiation on
sperm nuclear transformations and events involving and
subsequent to male and female pronuclear association
are also presented.

Material and Methods

Sexually mature individuals of Mulinia lateralis. col-
lected from Massey's Landing, Delaware, were kept at
15°C in a recirculating seawater system. Spawning was
induced by placing individual animals into 100 ml beakers
containing seawater at 30°C. Eggs from 1 to 3 spawned
females were pooled, washed in fresh seawater, insemi-
nated and permitted to develop at 20°C. Unfertilized eggs
and samples of fertilized ova, taken at 5 min intervals up
to 1 to 1.5 h after the addition of sperm, were fixed in 1%
formalin in seawater. In some experiments, eggs and
sperm were incubated with 10 ^M Hoechst 33342
(Hoechst) in seawater, washed in fresh seawater, and used
for insemination (Luttmer and Longo, 1986).

To examine the effects of ultraviolet irradiation on
sperm nuclear transformations leading to pronuclear de-

velopment, sperm were irradiated with ultraviolet light as
previously described (Nace el a!., 1970; Chourrout and
Quillet, 1982;ScarpaandBolton, 1988). Sperm suspended
in a plastic Petri dish were exposed for 20 min, at a dis-
tance of 20 cm, to a 15-watt tube generating ultraviolet
light ranging from 200 nm to 295 nm, with 60% of the
ultraviolet light concentrated at 254 nm. Irradiated sperm
were mixed with a suspension of eggs; samples were taken
and fixed as described above for non-irradiated specimens.

Fixed specimens were washed in seawater and stained
with one of the following DNA intercalating fluoro-
chromes: 1 Mg/mL 4',6-diamidino-2-phenylindole (DAPI)
in seawater, or 10 ^Af Hoechst in seawater. Stained spec-
imens were washed once in seawater, placed into a droplet
of glycerol on a glass slide, and covered with a glass cov-
erslip. To improve microscopic observation, some spec-
imens were compressed. Specimens were observed with
a Nikon Diaphot microscope equipped with epifluorosc-
ence. Photographs were taken of specimens using 40X or
100X objectives and Kodak T-Max film.

Because Mulinia sperm nuclei are spheroid, and their
transformations leading to male pronuclei produced a
symmetrical distribution of chromatin (i.e., spheroid),
changes in the size of incorporated sperm nuclei were
measured throughout the period of fertilization. To mea-
sure incorporated sperm nuclei at different periods after
insemination, as well as the developing spheroid female
pronucleus, stained specimens were placed into droplets
of glycerol as described above. A coverslip, bearing a thin
layer of Vaseline along its edges, was lowered over the
droplets such that the eggs or zygotes were suspended be-
tween the slide and coverslip. Images of the cross-sectional
diameters of transforming sperm nuclei and male and
female pronuclei were projected onto the screen of a video
monitor, checked for linearity, and traced onto plastic
sheets with a felt tip pen. The traced images were analyzed
with a Micro-plan II Image Analysis System (Laboratory
Computer Systems, Cambridge, Massachusetts). Diame-
ters and maximum cross-sectional areas of transforming
sperm nuclei and male and female pronuclei were mea-
sured (mean ± standard deviation) at 5-min intervals fol-
lowing insemination and temporally correlated with the
progression of meiotic maturation, female pronuclear de-
velopment, and first mitosis. Twenty to forty specimens
were measured at each time point.

Results

Structure of the unfertilized egg and spermatozoon

Unfertilized Mulinia eggs measured 46.4 ± 0.4 /*m in
diameter. When viewed with phase or Nomarski optics
they were seen to possess a large, meiotic prophase nucleus
(29.7 ± 1.4 ^m in diameter), the germinal vesicle, which
usually contained a single, spheroid nucleolus ( 10.4 ± 0.8

F. J. LONGO AND J. SCARPA

Figures I and 2. Nomarski (Fig. 1) and fluorescent (Fig. 2) preparations of unfertilized Mulima eggs
showing germinal vesicles, nucleoli (Nu) and meiotic chromosomes. Figure I, X760; Figure 2, X960.

Figures 3 and 4. Fertilized Mulima eggs depicting incorporated sperm nuclei (S) and meiotic chromosomes
which are distributed throughout the germinal vesicle (5 min pi). Figure 3. •960; Figure 4. x 1500.

Figure 5. Zygote ( 10 min pi) in which the meiotic chromosomes are condensing and the sperm nucleus
(S) is dispersing. XI 800.

Figures 6-8. Zygotes (15 min pi) in which the meiotic chromosomes have become condensed and
organized on the same optical plane (Fig. 7). The chromosomes move as a group to the cortex in preparation
for polar body formation (Fig. 8). Figure 6 is a Nomarski preparation in which the meiotic chromosomes
and incorporated sperm nucleus are difficult to discern; these structures are intensely stained in Hoechst-
or DAPI-prepared specimens. Figure 6. X760; Figures 7 and 8, X960.

FERTILIZATION EVENTS IN MULINIA

fim in diameter) suspended in a nucleoplasm (Fig. 1).
Occasionally, specimens containing two large nucleoli
were observed. Unfertilized eggs prepared with DAPI
(fixed eggs) or Hoechst (fixed or unfixed eggs) observed
with epi-fluorescence were essentially identical. Two fea-
tures were apparent with both methods: ( 1 ) a low back-
ground staining of the cytoplasm, and (2) a relatively in-
tense staining of the maternal tetrad chromosomes. Tet-
rads were distributed throughout the interior of the
germinal vesicle such that chromosome number and in-
dividual chromosomal features (e.g., chiasma) could be
ascertained (Fig. 2). Examination of whole mounts and
compressed specimens revealed that the number of
meiotic chromosomes in Mulinia eggs; i.e.. the haploid
number, was 19 (see also Menzel, 1968; Scarpa and Bol-
ton, 1988; Wadatva/., 1990).

The structure of Mulinia sperm as examined by light
microscopy was similar to that of other pelecypods (Fran-
zen, 1955). The sperm nucleus was spheroidal, 1.7 ±0.1 5
j/m in diameter, and contained a uniform distribution of
DNA as determined in fluorochrome stained preparations.

As was found for the surf clam, Spisula (Luttmer and
Longo, 1986), living Mulinia sperm or eggs treated with
Hoechst 33342 could inseminate and develop with no
apparent ill-effects. In living Mulinia zygotes in which
only one of the gametes was treated with Hoechst dye
prior to insemination, staining of both the maternal and
paternal genomes was found consistently after fertiliza-
tion, indicating that the dye was not remaining confined
to the nucleus of one gamete. Unlike the situation in Spi-
sula (Luttmer and Longo, 1986), we were unable to
achieve exclusive staining of only one genome in Mulinia
zygotes. The following account is based on experiments
employing fixed and unfixed, stained specimens.

Meiotic maturation of the maternal chromatin leading
to development of the female pronucleus

The interaction of the sperm with the egg initiated the
resumption of meiotic maturation and development of
the female pronucleus in Mulinia. Resumption of meiotic
maturation was heralded by the breakdown of the nuclear
envelope of the germinal vesicle and the disappearance
of the nucleolus (Figs. 3-6). These characteristic features

of germinal vesicle breakdown were readily apparent with
Nomarski and phase contrast optics (Fig. 6), but changes
in the structure and location of the tetrads were much
more difficult to ascertain. Meiotic events of the maternal
chromosomes and transformations of the sperm nucleus
were readily apparent with fluorochrome stained Mulinia
preparations and epi-fluorescence microscopy (Figs. 3, 4,
5. 7). Concomitant with germinal vesicle breakdown was
the condensation of the tetrads (Figs. 5, 7). The tetrads
formed a cluster within the center of the egg; eventually
they were organized on the metaphase plate of the first
meiotic spindle (Figs. 7, 8). The spindle and tetrads then
moved to one pole of the egg where completion of meiosis
and polar body formation occurred (Fig. 9). In almost all
cases examined, more than 90% of the specimens were in
synchrony and had developed to metaphase I by 15 min
postinsemination (pi).

Anaphase I followed localization of the meiotic spindle
to the egg cortex and was seen as the separation of two
fluorescent masses of chromosomes (Figs. 9, 10). With
the completion of anaphase I, the chromosomes emitted
within the first polar body formed a compact mass; those
within the egg became reorganized on a metaphase plate
in preparation for second polar body formation (Figs.
11, 12).

Anaphase II quickly followed formation of the first po-
lar body (Fig. 13) and appeared as the separation of two
fluorescent masses that were of less intensity than the
chromosomal masses that formed at anaphase I, reflecting
the decrease in DNA. After anaphase II, chromosomes
within the second polar body formed a densely stained
cluster (Fig. 14). The first and the second polar bodies
became positioned side by side and remained at the pole
of the egg where the meiotic divisions took place.

By 35 min pi, more than 95% of the specimens ex-
amined had completed polar body formation and were
engaged in the formation of a female pronucleus (Figs.
14-16). The maternal chromosomes remaining in the egg
dispersed, forming an irregularly shaped nucleus that
eventually expanded to become a spheroidal female pro-
nucleus. Measurements of the female pronucleus at dif-
ferent times following its formation (35 to 45 min pi)
indicated that its rate of expansion was 0.65 ^m/min (Fig.
23). Its average maximal size, measured at 45 min pi.

Figures 9a, b. Zygote (25 min pi) at two optical planes depicting anaphase I (Fig. 9a) and the incorporated
sperm chromatin (S). Note that the latter has ceased dispersion and is smaller than the sperm nucleus
depicted in Figure 7. X960.

Figures 10a,b and I la, b. Zygotes (30 min pi) at two optical planes in which the first polar body (P) has
formed and the chromosomes remaining in the zygote are preparing for the second meiotic division (Figs.
lOa, 1 la). Figures lOb and 1 Ib are at the level of the incorporated sperm nucleus which has ceased its
enlargement. <960.

Figures 12 and 13. Zygotes (35 min pi) at metaphase II (Fig. 12) and anaphase II (Fig. 13). S, sperm
nucleus. X820.

14"

01©

Figures 14a, b. Zygote (35 min pi) at two optical planes depicting the maternal chromosomes (arrow)
that have just completed their second meiotic division and are dispersing to form the female pronucleus
(Fig. I4a). Figure 14b shows the sperm nucleus (S). which is enlarging P. first and second polar bodies.
XI 500.

Figure 15. Zygote (40 min pi) at a slightly later stage of pronuclear development than the egg depicted
in Figure 14 in which the male (M land female (F) pronuclei are expanding. P, first and second polar bodies.

xl 900.

FERTILIZATION EVENTS IN MULINIA

was 9.8 ± 1.4 //m. By 50 min pi the female pronucleus
had decreased in diameter (9.4 ± 1.1 ^m ) and was engaged
in prophase of the first mitotic (cleavage) division (Figs.

17,23).

Transformations of incorporated sperm nuclei leading
to the development of male pronuc/ei

Upon its incorporation into the egg cytoplasm, the
sperm nucleus underwent an expansion from 1.7 ±0.15
to 3.7 ± 0.28 M"i in diameter (Figs. 3-5, 7, 23). This
initial expansion occurred symmetrically while the ma-
ternal tetrads were condensing and becoming aligned on
the metaphase plate of the first meiotic spindle (Figs. 5,
7). From 20 to 35 min pi, coincident with the period in
which the maternal chromosomes were engaged in polar
body formation, the incorporated sperm nucleus did not
expand and, in fact, decreased slightly in size to 3.5 ± 0.28
l/m in diameter (Figs. 9-12, 23). With the completion of
meiosis and the development of the female pronucleus
there was a dramatic enlargement in the sperm nucleus
(rate = 0.59 yum/min; Figs. 14-16, 23). At the completion
of its expansion (45 min pi), the male pronucleus mea-
sured 9.7 ± 1.4 ^m in diameter. Subsequent changes in
the male pronucleus included its reduction in size (9.6
± 0.8 nm) as a part of prophase of the first cleavage di-
vision.

Morphogenesis of the male and female pronuclei
leading to the first cleavage division

By 45 min pi, both the male and female pronuclei had
reached their maximal sizes (Fig. 23) and individually
and synchronously undergone prophase events leading to
the first cleavage division (Fig. 17). By 50 min pi con-
densing chromosomes appeared in the two pronuclei. As
the chromosomes condensed, the nuclear envelopes broke
down, forming two distinct groups of chromosomes in

the midregion of the zygote, one derived from the female
pronucleus and the other from the male (Fig. 17). The
two groups of chromosomes moved together, intermixed,
and became positioned on the metaphase plate of the first
mitotic spindle (Fig. 17). Subsequent morphogenesis of
the maternally and paternally derived chromosomes in-
volved their participation in the first cleavage division,
which was asymmetric with respect to cytokinesis (Fig.
18). That is, the metaphase plate was displaced from the
center of the zygote and, as a consequence, two unequally
sized blastomeres formed upon cleavage.

Effects of ultraviolet irradiation on male pronuc/ear
development and morphogenesis

Effects of ultraviolet irradiation were not apparent dur-
ing transformation of the sperm nucleus into a male pro-
nucleus, nor was there any apparent effect on meiotic
maturation and development of the female pronucleus.
Irradiated sperm nuclei expanded into pronuclei of a size
comparable to those of control preparations. Effects of
ultraviolet irradiation on sperm nuclei were not observed
until the male pronucleus was engaged in prophase events
of the first cleavage division. In eggs inseminated with
ultraviolet irradiated sperm, mitotic prophase events in
the two pronuclei were asynchronous (Fig. 19). The ma-
ternally and paternally derived chromosomes eventually
became aligned on a metaphase plate, but anaphase of
mitosis was abnormal as evidenced by chromosomal
nondisjunction (Figs. 20, 21). The number of chromo-
somes that failed to move to the spindle poles was not
constant. Consequently, material of varying fluorescent
intensity was seen between the spindle poles at telophase,
and between interconnecting blastomere nuclei at sub-
sequent stages of development (Fig. 22).

Discussion

The results presented here demonstrate nuclear changes
that occur in fertilized Mnlinia eggs and lead to the two-

Figure 16. Expanded male and female pronuclei that have become associated with one another in the
center ot a zygote (45 min pi). P, polar bodies. X820.

Figures 17a-e. Morphogensis of the male and female pronuclei following their apposition. Initiation of
prophase in each pronucleus is evident by chromosome condensation (Fig. 17a, b). Two groups of chro-
mosomes are produced (arrows. Fig. 17c, d) which become closely associated and positioned on the metaphase
plate of the first mitotic spindle (Fig. 17e). P, polar bodies. Figure 17a, c, and d. X2000; Figure 17b, XI 830;
Figure 17e, X870.

Figures 18a-c. First cleavage division of Mnlinia leading to unequal size blastomers (Fig. I8c). Figures
18a and b depict early and late anaphase. P. polar bodies; B, developing blastomere nuclei. X870.

Figure 19. Asynchronous pronuclear morphogenesis in an egg fertilized with an ultraviolet irradiated
sperm. x750.

Figure 20. Nondisjunction of mitotic chromosomes in an egg inseminated with an ultraviolet irradiated
sperm. K1400.

Figure 21. Cleaving egg which was inseminated with an ultraviolet irradiated sperm. Chromatin is
spread between the two developing blastomere nuclei. > 1400.

Figure 22. Cleaved zygote that was fertilized by an ultraviolet irradiated sperm. DAPI staining material
(i.e., DNA) connects the two blastomere nuclei (arrows), x 1700.

F. J. LONGO AND J. SCARPA

10 -

8 •

a.
ui

2 -

1 0

— I —
20

— i —
30

— i —
40

— I —
50

TIME (min.)

Figure 23. Expansion (mean ± S.D.) of incorporated sperm nuclei (•) and female pronuclei (A) of
Mulinia zygotes. The sperm nucleus shows three periods of transformation: 0 to 15, 15 to 35, and 35 to 45
min pi corresponding to periods encompassing germinal vesicle breakdown, polar body formation, and
female pronuclear development, respectively. The decrease in size of the male and female pronuclei from
45 to 50 min pi is correlated with the onset of mitotic prophase in both pronuclei.

cell stage. Meiosis of the maternal chromatin, transfor-
mations of the sperm nucleus, and pronuclear develop-
ment and association are readily amenable to analysis in
specimens prepared with the DNA intercalating dyes
DAPI and Hoechst. This suitability is due to a combi-
nation of factors, such as low background of the egg cy-
toplasm, and chromosome size, number and structure
(Wadae/fl/., 1990).

Meiotic maturation of the maternal chromatin of Mu-
linia eggs is similar to that previously described for other
mollusks (Longo, 1983; Luttmer and Longo, 1988). In-
teraction of the sperm with the egg induces germinal ves-
icle breakdown. The chromosomes become organized on
the metaphase plate of the first meiotic spindle apparatus
which then moves to, and becomes positioned within, the
egg's cortex. The mechanism by which this movement
takes place has not been established, although investiga-
tions demonstrating that cytochalasin B inhibits the cor-
tical localization of the meiotic spindle suggests that it
may be an actin-mediated process (Longo, 1987b).

Anaphase I and II, as well as the formation of the first
and second polar bodies, followed in quick succession, as
occurs in the surf clam Spisula (see Longo, 1983). For-

mation of the female pronucleus was evident subsequent
to the formation of the second polar body by the formation
of an expanding mass of material staining with either
DAPI or Hoechst. The rate of expansion of the forming
female pronucleus was comparable to that of the male
pronucleus, suggesting that the two chromatin masses may
be regulated by similar mechanisms. A corresponding re-
lationship has also been demonstrated in polygynic and
polyspermic Spisula zygotes (Luttmer and Longo, 1988).
The kinetics of sperm nuclear expansion in fertilized
Mulinia eggs is in agreement with previous studies dem-
onstrating that sperm nuclear transformations share a
temporal relationship with changes of the maternal
chromatin (Das and Barker, 1976; Da-Yuan and Longo,
1983;Yamashita, 1985; Luttmer and Longo, 1987, 1988;
Wright and Longo, 1988; Longo, 1989). Measurements
of sperm nuclear expansion in Mulinia zygotes indicates
that this process takes place in three distinct phases tem-
porally correlated with meiotic maturation of the ma-
ternal chromatin. In previous studies, as well as in the
one reported here, the incorporated sperm nucleus un-
dergoes a period of rapid expansion followed by one of
no enlargement or condensation. This is succeeded by a

FERTILIZATION EVENTS IN MVLINIA

dramatic expansion of the sperm nucleus leading to a
male pronucleus similar in size to that of the female.
The three phases of sperm nuclear enlargement in Mu-
linia correlate with germinal vesicle breakdown, polar
body formation, and female pronuclear development,
respectively. The kinetics of sperm nuclear expansion is
similar to that described in the surf clam, Spisula so/i-
disima, where four phases were observed based on closer
sampling times than those taken during the course of
the present study (Luttmer and Longo, 1988). In Spisula,
the sperm nucleus, upon incorporation, underwent little
change in size until germinal vesicle breakdown. Addi-
tionally, during the period of polar body formation, the
expanded sperm nucleus of Spisula underwent a signif-
icant reduction in size; i.e., it condensed. A reduction
(one time point) in size of the expanded sperm nucleus
of Mulinia zygotes was observed during polar body for-
mation. We suspect that with closer sampling times, this
reduction, as well as the status of the incorporated sperm
nucleus prior to germinal vesicle breakdown, would be-
come apparent in Mulinia zygotes.

Expansion of the sperm nucleus following germinal
vesicle breakdown is consistent with other studies dem-
onstrating that mixing of germinal vesicle substances with
the cytoplasm precedes sperm nuclear changes (Masui and
Clarke, 1979; Longo, 1981; Schuetz and Longo, 1981;
Hirait'/d/.. 1981; Yamada and Hirai, 1984). This change
in the sperm nucleus may be a manifestation of sperm
basic protein replacement by histones present in the oocyte
cytoplasm. Histone changes that occur with the early onset
of sperm chromatin dispersion have been demonstrated
(Poccia el al., 1978, 1981; see Poccia, 1986). Because
agents affecting meiotic maturation of the maternal chro-
matin also affect the kinetics of sperm nuclear expansion
(Luttmer and Longo, 1988; Wright and Longo, 1988),
factors regulating the status of the maternal chromatin
during polar body formation probably act on the trans-
formed sperm nucleus such that it ceases expansion and
in some instances condenses; e.g , surf clam, hamster, and
starfish (Luttmer and Longo, 1988; Wright and Longo,
1988; Longo, 1989).

The second expansion of the sperm nucleus, which is
correlated with enlargement of the maternal chromatin
and female pronuclear formation (Zirkin el al., 1989), is
set into motion as a result of cell cycle changes within the
fertilized egg that affect both the maternally and paternally
derived chromation (Longo, 1989). In the case of Mulinia,
as well as other species that have been studied to date,
both chromatin masses undergo dramatic rates of expan-
sion to form enlarged pronuclei (Luttmer and Longo,
1988; Wright and Longo, 1 988). Unlike the situation seen
in mammalian zygotes (Wright and Longo, 1988), ex-
pansion of the maternally and paternally derived chro-

matin resulted in pronuclei of nearly equal size (see also
Luttmer and Longo, 1989).

Results presented here demonstrate that pronuclear fu-
sion in Mulinia does not occur as in sea urchins (fertilized
at the completion of meiotic maturation) or as in other
cellular systems (Longo and Anderson, 1968). Rather,
both the male and female pronuclei, as separate bodies,
synchronously undergo prophase events in preparation
for first mitosis. The chromosomes from each pronucleus,
which replicated during the period following polar body
formation, become aligned on the metaphase plate of the
mitotic spindle and separate at anaphase into two masses
consisting of both maternally and paternally derived
chromosomes. Hence, maternal and paternally derived
chromosomes do not become enclosed within the same
nucleus until formation of the two-cell stage.

The effects of ultraviolet irradiation on sperm trans-
formations that lead to the development of male pronuclei
in Mulinia are consistent with what has been shown in
other systems (Onozato and Yamaha, 1983; Arai el al.,
1 984). Ultraviolet irradiation disrupts the DNA helix and
thus interferes with the proper duplication of chromo-
somes prior to first cleavage (Strickberger, 1976). We an-
ticipated that the effects of ultraviolet irradiation might
be manifested at two periods during fertilization: ( 1 ) dur-
ing transformation of the sperm nucleus into a male pro-
nucleus, indicative of gross DNA disruption; and (2) sub-
sequent to DNA replication, during the period in which
the paternally derived chromosomes were engaged in mi-
tosis. Alterations were not apparent during any of the
stages leading to a male pronucleus, possibly due to an
insensitivity of the method of analysis, or more likely to
an inability to achieve concomitant high levels of irradia-
tion and fertilization. (Higher doses of ultraviolet irradia-
tion were tested but resulted in an inhibition of fertiliza-
tion.) Radiation effects were seen only after pronuclear
association — i.e.. during prophase and anaphase of the
first cleavage division — and involved variable numbers of
chromosomes. The manner in which the male and female
pronuclei become associated in Mulinia and other mol-
luscan eggs (see Longo, 1983), as well as parameters af-
fecting both the quantity and quality of ultraviolet irra-
diation, call into question the effectiveness of using ultra-
violet irradiation to form gynogenetic molluscan embryos.
Induction of gynogenesis with variable results has been
achieved by a variety of techniques, including irradiation
of sperm with ultraviolet light (Chourrout, 1980; Streis-
inger el al., 1981; Onozato and Yamaha, 1983; Lou and
Purdom, 1984; Onozato. 1984; Suzuki el al., 1985). Vari-
ability in cases employing ultraviolet irradiation (Chour-
rout, 1980; Onozato and Yamaha, 1983; Arai el al.. 1984)
appeared to be due to difficulties in controlling parameters
associated with the exposure of sperm to ultraviolet rays

F. J. LONGO AND J. SCARPA

and an inability to uniformly and effectively destroy all
of the paternally derived DNA.

Acknowledgments

The assistance of Tena Perry and Lori Mathews is
greatfully appreciated. Portions of the study presented here
were supported by funds from the NIH. Support for John
Scarpa was provided by the New Jersey Agricultural Ex-
periment Station grant to Standish K. Allen, Jr.

Literature Cited

Aral, K., F. Naito, H. Sasaki, and K. Fujino. 1984. Gynogenesis with
ultraviolet ray irradiated sperm in the Pacific abalone. Bull Jpn. Sue.
Sci. Fish. 50:2019-2023.

Das, N. K., and C. Barker. 1976. Mitotic chromosome condensation
in the sperm nucleus during postfertilization maturation division in
t'w/i/.veggs. J. Cell BioL 68: 155-159.

Da-Yuan, C., and F. J. Longo. 1983. Sperm nuclear dispersion coor-
dinate with meiotic maturation in fertilized Spisti/a solidixsima eggs.
Dev. Bml 99: 217-244.

Chourrout, D. 1980. Thermal induction of diploid gynogenesis and
tnploidy in the eggs of the rainbow trout (Salmo gairdneri). Reprod.
Nutr. Dev. 20: 727-733.

Chourrout, D., and F.. Quillet. 1982. Induced gynogenesis in the rainbow
trout: sex and survival of progenies production of all-triploid popu-
lations. Them. Appl. Genel. 63: 201-205.

Franzen, A. 1955. Comparative morphological investigations into the
spermiogenesis among Mollusca. Zool. Buin.it;. 30: 399-456.

Hirai, S., Y. Nagahama, and M. Kanatani. 1981. Cytoplasmic maturity
revealed by the structural changes in incorporated spermatozoan
during the course of starfish oocyte maturation. Dev. Growth Differ.
23: 465-478.

Longo, F. J. 1981. Regulation of pronuclear development. Pp. 529-
557 in Bioregulalon of Reproduction, G. Jagiello and C. Vogel. eds.
Academic Press, New York.

Longo, F. J. 1983. Meiotic maturation and fertilization. Pp. 49-89 in
The Mollusca, Vol. 3, N. H. Verdonk and J. A. M. van den Biggelaar.
eds. Academic Press. New York.

Longo, F. J. 1985. Pronuclear events. Pp. 25 1-298 in Biology of Fer-
tilization. Vol. 3, C. B. Metz and A. Monroy, eds. Academic Press.
NY.

Longo, F. J. 1987a. Fertilization. Chapman and Hall. New York.

Longo, F. J. 1987b. Egg cortical architecture. Pp. 108-138 in The Cell
Biology of Fertilization, G. Schatten and H. Schatten, eds. Academic
Press. Inc.

Longo, F. J. 1989. Dynamics of sperm nuclear transformations at fer-
tilization. Pp. 297-307 in fertili:ation in Mammals. B. D. Bavister,
J. Cummins and E. R. S. Roldan. eds. Serono Symposia. Norwell,
MA.

Longo, F. J., and E. Anderson. 1968. The fine structure of pronuclear
development and fusion in the sea urchin Arhacia puneiulata. J. Cell
Bio/. 39: 335-368.

Lou, Y. D., and C. E. Purdom. 1984. Diploid gynogenesis induced by
hydrostatic pressure in the rainbow trout, Salmo gairdneri. J. Fish
Biol. 24: 665-670.

Luttmer, S. J. and F. J. Longo. 1986. Examination of living and fixed
gametes and early embryos stained with supervital Quorochromes

(Hoechst 33342 and 3,3'-dihexyloxacarboxyanine iodide). Gamete
Res 15:267-283.

I mum i, S. J., and F. J. Longo. 1987. Rates of male pronuclear en-
largement in sea urchin zygotes. J. Exp Zoul 243: 289-298.

Luttmer, S. J., and F. J. Longo. 1988. Sperm nuclear transformations
consist of enlargement and condensation coordinate with stages of
meiotic maturation in fertilized Spisu/a solidissima oocytes. Dev.
BioL 128: 86-96.

Masui, Y., and H. Clarke. 1979. Oocyte maturation. Inl Rev Cvtol.
57: 185-282.

Menzel, R. W. 1968. Chromosome number in nine families of marine
pelecypod mollusks. Nautilus 82: 45-58.

Nace, G. W., C. M. Richards, and J. H. Asher Jr. 1970. Parthenogenesis
and genetic variability. I. Linkage and inbreeding estimations in the
frog. Rana pipiens Genetics 66: 349-368.

Onozato, II. 1984. Diploidization of gynogenetically activated salmonid
eggs using hydrostatic pressure. Aquaculture 43: 91-97.

Onozato, H., and E. Yamaha. 1983. Induction of gynogenesis with
ultraviolet rays in four species of salmoniformes. Bull. Jpn. Soc. Sci.
Fish. 49: 693-699.

Poccia, D. 1986. Remodeling of nucleoproteins during gametogenesis.
fertilization and early development. Int. Rev Cytol. 105: 1-65.

Poccia, D., G. Krystal, D. Nishioka, and J. Salik. 1978. Controls of
sperm chromatin structure by egg cytoplasm in the sea urchin. 1CN-
UCLA Symp. Mol. Cell. Biol. 12: 197-206.

Poccia, D., J. Salik, and G. Krystal. 1981. Transitions in histone vari-
ants of the male pronucleus following fertilization and evidence lor
a maternal stage of cleavage-stage histones in sea urchin eggs. Dev.
Biol. 82: 287-296.

Scarpa, J., and E. T. Bolton. 1988. Experimental production of gyn-
ogenetic and parthenogenetic Mulinia lateralis (Say). / Shellfish Res.
7: 132.

Sehuetz, A., and F. J. Longo. 1981. Hormone-cytoplasmic interaction
controlling sperm nuclear decondensation and male pronuclear de-
velopment in starfish oocytes. J. Exp. Zoo/. 215: 107-1 1 1.

Streisinger, G., C. Walker, N. Dower, C. Dnauber, and F. Singer.
1981. Production of clones of homozygous diploid zebra fish
Brachydanio reno. Nature 291: 293-296.

Strickberger, M. W. 1976. Genetics. Macmillan Pub. Co.. New York.

Suzuki, R.. I . Oshiro, and T. Nakanishi. 1985. Survival, growth and
fertility of gynogenetic diploids induced in the cypnmd loach, Afis-
giirnus anguillicaudatus. Aquaculture 48: 45-55.

\\ada, K. T., J. Scarpa, and S. K. Allen Jr. 1990. Karyotype of the
dwarf surf clam Mulinia lateralis (Mactndne. Bivalvia). / Shellfish
Res 9: in press.

Wilson, E. B. 1925. The Cell in Development and Heredity. McMillian,
New York.

Wright, S. J., and F. J. Longo. 1988. Sperm nuclear enlargement in
fertilized hamster eggs is related to meiotic maturation of the maternal
chromatin./ Exp. Zoo/ 247: 155-165.

Yamada, II., and S. Hirai. 1984. Role of contents of the germinal vesicle
in male pronuclear development and cleavage of starfish oocytes.
Dev. Growth Difier 26: 479-487.

Yamashita, M. 1985. Electron microscopic analysis of the sperm nu-
clear changes in meiosis inhibited eggs of the brittle star, Amphipholis
kochii. J Exp. Zool. 235: 105-1 17.

Zirkin, B., S. D. Perrault, and S. J. Naish. 1989. Formation and func-
tion of the male pronucleus during mammalian fertilization. Pp. 91-
1 14 in The Molecular Biology of Fertilization, H. Schatten and G.
Schatten. eds. Academic Press. New York.

Reference: Btol. Bull. 180: 65-71. (February.

Putative Molt-Inhibiting Hormone in Larvae

of the Shore Crab Carcinus maenas L.:

An Immunocytochemical Approach

S. G. WEBSTER' AND H. DIRCKSEN*

School of Biological Sciences, University College of North Wales, Bangor. Gwynedd LL57 2UW, UK,

an d *Institut fiir Zoophysiologie. Universitdt Bonn, Endenicher Allee 11-13.
D-5300 Bonn 1. Germany.

Abstract. Immunocytochemical investigations of the
eyestalk of Carcinus maenas zoeal larval stages, using an
antiserum directed against putative Carcinus molt-inhib-
iting hormone (M1H), revealed immunopositive neuronal
structures. These structures included perikarya associated
with the medulla terminalis X-organ, parts of the sinus
gland tract, and the neurohemal organ — the sinus gland.
Apart from an increase in volume of the sinus gland be-
tween zoeal stage I and II, no striking changes in the to-
pography or morphology of the MIH neurosecretory sys-
tem were observed. Immunopositive structures were
found in similar locations to those seen in adult crabs.
Our results suggest that the control of molting by MIH
in crustacean larvae may be similar to the currently ac-
cepted model of molt control in adult decapod crusta-
ceans.

Introduction

A current model of molt control in decapod crustaceans
involves regulation of ecdysteroid synthesis by a molt-
inhibiting hormone (MIH), released by neurosecretory
neurons in the eyestalk. Much evidence has now accu-
mulated suggesting that increased synthesis and liters of
circulating ecdysteroids necessary for induction of premolt
are directly repressed by this neuropeptide, thus inhibiting
proecdysis and molting. Nevertheless, alternative hy-
potheses have implicated processes such as metabolism
and excretion of ecdysteroids in molt regulation (see
Skinner, 1985; Webster and Keller, 1988; Watson el ai.

Received 23 May 1990; accepted 6 November 1990.
1 To whom correspondence should be sent.

1989 for recent reviews). Despite recent advances in our
knowledge concerning mechanisms of molt control in
adult decapod crustaceans, little is known about the reg-
ulation of molting in larval crustaceans. This deficiency
has been reiterated in a recent review by Christiansen
(1988).

Evidence for molt regulation by MIH in crustacean
larvae has, until recently, been obtained by eyestalk abla-
tion experiments (for references see Charmantier et ai,
1988; Christiansen 1988), which have given equivocal re-
sults, suggesting that in some instances, the larval molt is
not regulated by MIH until shortly before metamorphosis.
However, with regard to morphological correlates of neu-
rosecretory structures in larval eyestalks, several reports
(Orlamunder, 1942; Pyle, 1943; Hubschman. 1953; Dahl,
1957; Matsumoto, 1958; Little, 1969; Zielhorst and Van
Herp, 1976; Bellon-Humbert et a/., 1978; Gorgels-Kallen
and Meij, 1985) detail the ontogeny of larval neurosecre-
tory systems in a wide variety of crustaceans. With the
exception of studies by Gorgels-Kallen and Meij (1985),
Beltz and Kravitz (1987), and Beltz et a/., (1990), there
are no other studies in which neurosecretory systems con-
taining immunocytochemically defined neuropeptides
have been described in crustacean larvae.

Recently, we have characterized a neuropeptide from
the sinus gland of Carcinus maenas, which, by virtue of
its ability to repress ecdysteroidogenesis by Y-organs cul-
tured in vitro, could be described as a putative MIH
(Webster, 1986; Webster and Keller, 1986). It should be
stressed that the precise significance and function of this
neuropeptide as a molt-inhibitor in vivo has not yet been
elucidated, and until suitable in vivo bioassays are devel-
oped, the status of MIH must remain "putative." Re-

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Figure 1. Characteristic structures of MIH-immunoreactive (IR) neurons in prezoeal (a, c), stage I zoea
(h. d-f. left eye), and stage II zoea (g-l) eyes of Carcinus maenas larvae. Phase contrast micrographs of
immunostained semithin ( 1 nm) transverse sections. (Orientation of dorsal parts of larvae to the tops of
micrographs.)

MOLT-INHIBITING HORMONE IN CRAB LARVAE

cently, we demonstrated that the neurosecretory system
produced putative MIH in the eyestalk ganglia of several
adult brachyuran crustaceans (Dircksen el at.. 1988). Be-
cause these studies provide compelling evidence to suggest
that MIH is a secretable neuropeptide, and in view of our
earlier observations on the nature and mode of action of
this neuropeptide on ecdysteroid synthesis in Carcinus
(Webster and Keller. 1986; Lachaise el al. 1989), it
seemed opportune to examine the larval eyestalk neu-
rosecretory system immunocytochemically, using anti-
bodies raised against Carcinus MIH. Evidence presented
here suggests that a functional MIH-like neurosecretory
system exists in all larval stages of Carcinus.

Materials and Methods

Laboratory rearing of larvae

Ovigerous Carcinus maenas L. females were collected
from the Menai Strait, North Wales, between May and
July, and maintained in the laboratory until larvae were
released. Only positively phototropic, rapidly swimming
larvae were collected. Rearing techniques were initially
based upon those of Rice and Ingle (1975), but were found
to be inadequate. Successful rearing to first crab with a
high survival was achieved using a mixed diet of (A) phy-
toplankton (Tetraselmis clniii), (B) rotifers (Brachionus
plicatilis), (C) barnacle nauplii (Ehninius modeslus), and
(D) brine shrimp nauplii (Anemia salina). During each
larval stage, prey ratios were supplied as follows: Zoea I

III (C):l, (D):l. Zoea IV, Megalopa and First crab (D):l.
With the exception of phytoplankton (culture density ca.
106 cells mr ': 1 part = 1 5 ml), the total prey concentration
was around 25-50 items per ml. Larvae were reared in
50-ml plastic containers in constantly aerated, filtered
seawater (33%o) under ambient temperature (15-18°C)
and photoperiod (L 15-18 h: D9-6 h). Maximum density
of larvae was 1 per 5 ml. Water and food were changed
every two days, at which time instars were staged accord-

ing to Rice and Ingle (1975). Under these maintenance
conditions, survival was good (80%), and instar durations
were approximately: Z I: 7, Z II: 5, Z III: 6, Z IV: 7, M:
8, days. Samples of larvae were taken at the middle of
each instar, which was considered to be during intermoult.

Tissue processing and immunocytochemistry

Fixations were carried out in a mixture of 2% parafor-
maldehyde, 2% glutaraldehyde. and 0. 1% saturated picric
acid in 0. 1 M sodium cacodylate buffer, pH 7.4, supple-
mented with 0.5 M sucrose and 5 mM CaCl2 for 2-4 h
at 4°C according to Dircksen et al. (1987). Tissues were
washed extensively in the same buffer, dehydrated, and
embedded in low viscosity resin (Spurr, 1969). Semithin
frontal cross-sections ( 1 ^m) through the whole animal
were cut on a LKB Ultrotome III or a Reichert Ultracut
E, and processed for immunocytochemistry using a rabbit
antiserum (code R1TB) directed against HPLC-purified
MIH of Carcinus (Dircksen el al., 1988), diluted 1:4000
in 0.0 1 M phosphate buffered saline (PBS) and PAP stain-
ing techniques (Dircksen et al., 1987). Micrographs were
taken with a Zeiss Axioskop using phase contrast optics
and documented on Agfapan 25 film.

Results

Despite several attempts to improve the penetration of
fixative into the eyestalks (for example, by piercing the
exoskeleton behind the eyestalks, using other fixatives or
fixation times), adequate fixation of megalopae and first
crab stages was impossible. Thus, by necessity, this study
is restricted to the zoeal stages of Carcinus, and in later
zoeal stages problems with fixation and tissue shrinkage
were encountered. A sometimes confusing feature of the
zoeal eyestalk was the presence of a pigmented perineural
sheath (Fig. 2c, 2f), which could have been identified as
an immunopositive structure. This problem was resolved
by using normal bright field optics, under which immu-
nopositive material appears brownish, or by higher mag-

fa) MIH-IR axon profiles within the sinus gland (center of rectangle) of a prezoea. Note ommatidial pnmordia.
brain (*) and yolk droplets (arrowhead), (b) MIH-IR axon profiles within the sinus gland (rectangle) of a
stage I zoea. Note dense pigmentation at the base of the ommatidia, and well-developed neuropiles of the
lamina ganglionaris (LG). medulla externa (ME), and the brain (*). (c, d) Higher magnifications of sinus
glands corresponding to rectangles in a. b. (e) Cross-sectioned MIH-IR axons (inset enlarged from the rectangle).
(f) Two MIH-IR perikarya in an anterior dorsal cell group of the left eyestalk ganglia (inset enlarged from
the rectangle), (g) MIH-IR axon profiles in the sinus gland (rectangle) of a stage II zoea adjacent to the ME
and large hemolymph spaces. Note stalk formation of the eye at this stage, (i) Cross-sectioned MIH-IR axons
in the medulla terminalis. (k) Three clustered MIH-IR penkarya in an anterior dorsal position of the pre-
sumptive X-organ cell group. Note well-developed ganglia and neuropiles in the eye. (h, j, I) Higher mag-
nifications of rectangles outlined in g, i, k. Note axon profiles and putative terminals abutting on the surface
of the sinus gland (h) and dark PAP reaction products restricted to the cytoplasm of the penkarya (1) of
MIH-IR neurons.

Scale bars: 50 ^m in a. b, e. f, g, i. k. 10 /jm in c. d, h. j, 1, and insets in e, f.

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Figure 2. Characteristic structures of MIH-immunoreactive (IR) neurons in stage III zoea (a-f, left eye)
and stage IV zoea (g-1, right eye) eyes ofCarcimis macnas larvae. Phase contrast micrographs of immunostained
semithin ( 1 ^m) transverse sections. (Orientation of dorsal parts of the larvae to the tops of the micrographs.)

MOLT-INHIB1TING HORMONE IN CRAB LARVAE

nification (Fig. 2f) when the black pigment granules could
be clearly resolved by phase contrast optics.

M1H immunoreactivity was found in all zoeal stages
examined, including the so-called prezoeal stage, which,
in view of its brevity (ca. 30 min), and association with
hatching, could well be described as an embryonic molt.
(Fig. la, c). In general, MIH immunoreactivity was found
in structures similar to those found in the adult, including
perikarya in a position similar to the X-organ in adults,
an X-organ sinus gland tract, and a sinus gland (Figs. 1,
2). In several preparations the sinus gland appeared to be
in close proximity to a large hemolymph vessel (Figs. Ih,
2a, d). By serially sectioning through the entire eyestalk,
a maximum of four immunopositive perikarya of about
8-10 ^m in diameter were observed in all zoeal stages
localized in a cluster of neuroblasts in an anterior dorsal
position of the eyestalk, with large nuclei and scarce cy-
toplasm (Figs. If, k, 1. 2c, f, i). Axonal projections were
found in the medulla terminalis of the well-developed
eyestalk ganglia in a typical circular arrangement of four
cross-sectioned axons (Figs. Ij. 2e, k), reminiscent of the
axonal arrangement in the adult crab. This pattern was
found in all zoeal stages. Despite exhaustive investigation,
the only discernable change in the morphology of the
neurosecretory structures was the size of the sinus gland,
which appeared to increase in volume between zoea I and
II, when the eye became stalked and mobile. Indeed, it
was frequently difficult to observe the sinus gland in zoea
I due to its small size, but in zoea II, the sinus gland was
often the most striking immunopositive structure (Fig.
Ib, d, g, h). In control incubations, preabsorbtion of the
antiserum with 2 nmoles of MIH per jul of crude antiserum
completely abolished immunostaining, thus proving the
specificity of the immunocytochemical detection (results
not shown).

Discussion

In the present study, the location of perikarya, axons,
and sinus gland terminals immunopositive for MIH have

been demonstrated in all zoeal instars ofCarcinus larvae.
Surprisingly, larval immunopositive structures were to-
pographically and morphologically similar to those found
in the adult crab. However, very few (maximum 4) MIH-
immunoreactive perikarya were observed in any larval
stage, compared to the adult crab where there are 32-36
MIH-immunoreactive perikarya (Dircksen et ai, 1988).
It is likely that the increase in number of immunopositive
cells during larval to juvenile/adult development is due
to increased MIH gene expression rather than by cell di-
vision because neuroblasts are generally considered to be
too highly differentiated to undergo further division. A
striking similarity of the larval MIH immunopositive
structures to those of the adult concerns the morphology
of the X-organ sinus gland tract. In the adult, MIH im-
munoreactive axons form a peripheral tract around the
central axon bundle containing crustacean hyperglycemic
hormone (CHH) immunopositive axons (Dircksen et ai,
1988). Although we did not determine CHH in the present
study, the similarity in the arrangement of the four MIH-
immunoreactive axons around a central tract was clearly
suggestive of the adult morphology.

Several studies have reported the general development of
neural systems in the crustacean eyestalk. Cells correspond-
ing to the X-organ have been found in the first larval stages
of all species examined (Birgits. Orlamiinder, 1942; Horn-
arm. Pinnotheres. Pyle, 1943: Crangon. Dahl, 1957; Pota-
mon. Matsumoto, 1958; Palaemonetes, Hubschman, 1963;
Palaemon. Little, 1969, Bellon-Humbert et ai, 1978; As-
tacus, Zielhorst and Van Herp, 1976, Gorgels-Kallen and
Meij, 1985). With regard to the development of the sinus
gland, for freshwater crustaceans, which hatch at an ad-
vanced developmental stage, the sinus gland is present in
the first larval stage (Matsumoto, 1958; Gorgels-Kallen and
Meij, 1985). In marine crustaceans, which hatch at a rela-
tively early stage of development, and which often undergo
a lengthy planktonic existence prior to a dramatic meta-
morphosis, all studies suggest that the sinus gland develops
(or can first be observed) late in larval life, at about the time

(a) MIH-IR axon profiles in the sinus gland (rectangle) adjacent to the large hemolymph vessel (*) of the
eyestalk. (b) Section slightly anterior to (a) showing the sinus gland (arrowhead) and cross-sectioned MIH-
IR axons (rectangle) in the medulla terminalis. (c) Four MIH-IR penkarya (rectangle) are found in an
anterior dorsal position of the presumptive X-organ cell group, (d, e. I") Higher magnifications of rectangles
outlined in a. b, c. MIH-IR putative axon terminals adjacent to the hemolymph vessel (*) are found in the
sinus gland (d). Note also cross-sectioned MIH-IR axons (e) in the medulla terminalis and strong immu-
noreactivity of three perikarya (f, arrowheads). Arrows in (f) point to dark pigments usually found in perineural
sheaths of eyestalk ganglia, (g) MIH-IR axon profiles in the sinus gland (rectangle) adjacent to the large
hemolymph vessel (*) of the eyestalk. (h) Cross-sectioned axons of the presumptive X-organ sinus gland
(XO-SG) tract in the medulla terminalis. (i) Two MIH-IR perikarya in the presumptive X-organ cell group
in a dorsal anterior position of the proximal eyestalk ganglia, (j, k, 1) Higher magnification of rectangles
outlined in g, h, i, MIH-IR axon profiles and putative axon terminals abutting on the surface of the sinus
gland, (*) indicates hemolymph vessel, (j), MIH-IR axons in the XO-SG tract (k) and two strongly immu-
nopositive XO perikarya (I). Note unstained axons in the center of the XO-SG tract (k).
Scale bars: 50 ^m in a-c, g-i. 10 j/m in d-f, j-l.

S. G. WEBSTER AND H. DIRCKSON

of metamorphosis (stage V Palaemonetes, Hubschman,
1963, Palaemon, Bellon-Humbert et a/., 1978; stage III
Homarns. Pyle 1943). Apart from a report by Jaques ( 1975)
demonstrating the presence of a sinus gland in stage I Squilla
mantis larvae, this paper reports the first demonstration of
a sinus gland in first stage larvae of a marine decapod crus-
tacean, and is undoubtedly due to the great resolving power
of immunocytochemical techniques compared to conven-
tional histochemical staining methods. To our knowledge,
the only other reports using immunocytochemical tech-
niques to identify larval neurosecretory structures are those
by Gorgels-Kallen and Meij (1985), demonstrating the neu-
rosecretory structures containing CHH immunoreactivity
in Astaais leptodactylns larvae, and Beltz and Kravitz (1987)
and Beltz et al. (1990), demonstrating proctolin-like im-
munoreactivity in the CNS of larval Homarns americanus.
While immunocytochemical evidence indicates that
Carcinus zoeae possess a M1H neurosecretory system,
which may participate in the control of larval molting,
experiments involving eyestalk ablation in several species
of crustacean larvae (see specific examples in Charmantier
et al.. 1988; Christiansen, 1988) have demonstrated that,
in general, eyestalk ablation is only effective in accelerating
proecdysis and molting when performed during the last
instar before metamorphosis. Although the deficiencies
of these experiments have been commented upon by
Freeman and Costlow ( 1980), particularly with regard to
difficulties in determining the precise duration of instars
and the time of initiation of proecdysis in rapidly moulting
larvae, it has been suggested (Freeman et al., 1983) that
the larval molt cycle is not regulated by MIH until meta-
morphosis. However, studies demonstrating that larval
ecdysteroid liters cycle in a molt-stage-dependent manner
in much the same way as adults (Chang and Bruce, 1981;
Spindler and Anger, 1986), and a report by Snyder and
Chang (1986), demonstrating that increases in proecdysial
ecdysteroid titer induced by eyestalk removal of Stage II
Homarns zoeae can be repressed by the injection of adult
sinus gland extracts, strongly support the hypothesis that
larval molting (or at least, initiation of proecdysis) is reg-
ulated by MIH, and the results presented here would also
support this hypothesis. However, it should be stressed
that no firm inferences as to the function of the immu-
noreactive MIH can yet be made; it is not known whether
larval MIH-immunoreactive material is identical to that
in adults, although the antiserum used displays a very
high specificity in immunodot assays (Dircksen el al..
1988), RIA, and ELISA (Webster, unpub.), or whether it
is released during the zoeal stages. Although in vivo ex-
periments involving injection of MIH or sinus gland ex-
tracts into zoeal larvae and subsequent monitoring of
proecdysis or instar length would undoubtedly strengthen
hypotheses concerning larval molt control, the small size
of most crab zoeae argues against the success of such ex-

periments in crab larvae. A further problem, which re-
mains unresolved, concerns the increase in number of
immunoreactive perikarya between the last zoeal stage
and the adult. It is possible that this transition occurs
during metamorphosis (a phenomenon we could not elu-
cidate due to difficulties in achieving adequate fixation of
megalopae and first crab stages). If the MIH secretory
system became synthetically active at this time, and stored
MIH was released, then previous observations regarding
the failure to accelerate molting in zoeal larvae, and the
appearance of the sinus gland as a structure stainable by
conventional histochemical methods prior to metamor-
phosis, could be reconciled with the model of molt control
suggested by Freeman et al. (1983).

Acknowledgments

We are grateful to Mr. M. Budd, School of Ocean Sci-
ences, Menai Bridge, UK, for culturing the phytoplankton
and rotifers used in this study, and for much useful advice
concerning larval rearing techniques. This work was sup-
ported by a Royal Society University Research Fellowship
(S.G.W.). Financial support from the British Council for
travel to Bangor (H.D.) is gratefully acknowledged.

Literature Cited

Bellon-llumbert, C, M. J. P. Thijssen, and F. Van Herp.

1978. Development, location and relocation of sensory and neu-
rosecretory sites in the eyestalks during the larval and postlarval life
of Palaemon .terrains (Pennant). J. Mar. Biol. Assoc. U.K. 58: 851-
868.

Beltz, B. S., and E. A. Kravitz. 1987. Physiological identification, mor-
phological analysis and development of identified serotonin-proctolin
containing neurons in the lobster ventral nerve cord. J. Neurosci. 7:
533-546.

Beltz, B. S., M. Pontes, S. M. Helluy, and E. A. Kravitz. 1990. Patterns
of appearance of serotonin and proctolin immunoreactivities in the
developing nervous system of the American lobster. / Neurobiol.
21:521-542.

C'hang, E. S., and M. J. Bruce. 1981. Ecdysteroid liters of larval lobsters.
Com/1 Biochein. Physiol. 70A: 239-241.

Charmantier, G., M. Charmantier-Daures, and D. E. Aiken.
1988. Larval development and metamorphosis of the American
lobster Homarus americanus (Crustacea, Decapoda): effect of eyestalk
ablation and juvenile hormone injection. Gen. Comp. Endocrinol.
70: 319-333.

Christiansen, M. E. 1988. Hormonal processes in decapod crustacean
larvae. Symp. Zool. Soc. Loud. 59: 46-68.

Dahl, E. 1957. Embryology of X-organs in Crangon allmanni. Nature
179: 482.

Dircksen, H., C. A. Zahnow, G. Gaus, R. Keller, K. R. Rao, and J. P.
Riehm. 1987. The ultrastructure of nerve endings containing pig-
ment dispersing hormone (PDH) in crustacean sinus glands: iden-
tification by an antiserum against synthetic PDH. Cell Tiss. Res.
250: 377-387.

Dircksen, H., S. G. Webster, and R. Keller. 1988. Immunocytochemical
demonstration of the neurosecretory systems containing putative

MOLT-INHIBITING HORMONE IN CRAB LARVAE

molt-inhihiting hormone and hyperglycemic hormone in the eyestalk

of hrachyuran crustaceans. Cell Tiss. Res 251: 3-12.
Freeman, J. A., and J. D. Costlow. 1980. The molt cycle and its hor-
monal control in Rhithmpanopeus liarnsi larvae. Dev Biol 74: 479-

485.
Freeman, J. A.,T. L. West, and J. D.Costkm. 1983. Postlarval growth

in juvenile Rlulhropanopeus harnsii. Biol Bull 165:409-415.
Gorgels-Kallen, J. L., and J. T. A. Meij. 1985. Immunocytochemical

study of the hyperglycemic hormone (CHH)-producing system in

the eyestalk of the crayfish Astacus leptodactylus during larval and

postlarval development. J Morphol. 185: 155-163.
Hubschman, J. II. 1963. Development and function of neurosecretory

sites in the eyestalks of larval Palaemoneles (Decapoda, Natantia).

Biol. Bull. 125:96-113.
Jaques, F. 1975. Decouverte de la glande du sinus chez la larve de

Si/iii/lu mantis (stade I) (Crustace, Stomatopodes). Ultrastructure.

C. R Acad. Sa. (D) (Paris) 280: 1575-1577.
Lachaise, F., M. Hubert, S. G. Webster, and R. Lafont. 1988. Effect

of molt-inhibiting hormone on ketodiol conversion by crab Y-organs.

J Insect Physiol. 34: 557-562.
Little, G. 1969. The larval development of the shrimp. Palaemon mac-

rodaaylus Rathbun, reared in the laboratory, and the effect of eyestalk

extirpation on development. Crustaceana 17: 69-87.
Matsumoto, K. 1958. Morphological studies on the neurosecretion in

crabs. Biol J. Okayama L'nir. (Japan) 4: 103-176.
Orlamunder, J. 1942. Zur Entwicklung und Formbildung des Birgiix

lalro L. mil besonderer Beriacksichtigung des X-Organs. Z Wiss.

Zool. 155:280-316.
Pyle, R. W. 1943. The histogenesis and cyclic phenomena of the sinus

gland and X-organ in Crustacea. Biol. Bull 85: 87-102.
Rice, A. L., and R. W. Ingle. 1975. The larval development ofCarcinus

maenax (L.) and C mediterraneus Czerniavsky (Crustacea. Brachyura.

Portumdae) reared in the laboratory. Bull Bril Mus. (Not. Hist.) 28:
103-119.

Skinner, D. M. 1985. Interacting factors in the control of the crustacean
molt cycle. Am. Zool 25: 275-284.

Snyder, M. J., and E. S. Chang. 1986. Effects of sinus gland extracts
on larval molting and ecdysteroid liters of the American lobster.
Homants amcricanus. Biol Bull 170: 244-254.

Spindler, K. D., and K. Anger. 1986. Ecdysteroid levels during the
larval development of the spider crab Hya.i araneus. Gen. Camp.
Endoa-moL 64: 122-128.

Spurr, A. R. 1969. A low-viscosity epoxy resin embedding medium
for electron microscopy. J. Ultrastruc. Res. 26: 31-43.

Watson, R. D., E. Spaziani, and W. E. Bollenbacher. 1989. Regulation
of ecdysone synthesis in insects and crustaceans: a comparison. Pp.
188-203 in Ecdysone. From Chemistry 10 Mode of Action, J. Kool-
man. ed. Georg Thieme Verlag. Stuttgart.

Webster, S. G. 1986. Neurohormonal control of ecdysteroid biosyn-
thesis by Carcinus maenas Y-organs in vitro and preliminary char-
acterization of the putative moult-inhibiting hormone (MIH). Gen.
Comp Endocrinol. 61: 237-247.

Webster. S. G., and R. Keller. 1986. Purification, characterization and
amino acid composition of the putative moult-inhibiting hormone
(MIH) ofCarcinin >iiiicna\ (Crustacea, Decapoda). / Comp. Physiol
B 156: 617-624.

Webster, S. G., and R. Keller. 1988. Physiology and biochemistry of
crustacean neurohormonai peptides. Pp. 173-196 in Neurohormones
in Invertebrates, M. C. Thorndyke and G. J. Goldsworthy. eds. Cam-
bridge LJniversity Press.

Zielhorst, A. J. A. G., and F. Van llerp. 1976. Developpement du
systeme neurosecreteur du pedoncle oculaire des larves d' Astacus
leptodactylus salinus Nordmann (Crustacea, Decapoda, Reptantia):
Microscopie photonique. C R Acad. Sci (D) (Paris) 283: 1755-1758.

Reference: Bin/. Bull. 180: 72-80. (February, 1991)

The First Historical Extinction of a Marine

Invertebrate in an Ocean Basin: The Demise

of the Eelgrass Limpet Lottia alveus

JAMES T. CARLTON1, GEERAT J. VERMEIJ2, DAVID R. LINDBERG3,
DEBBY A. CARLTON1, AND ELIZABETH C. DUDLEY4

^Maritime Studies Program, Williams College — Mystic Seaport, Mystic, Connecticut 06355;

^Department of Geology, University of California, Davis, California 95616; 3 'Museum of

Paleontology, University of California, Berkeley, California 94720; and4 Department of Zoology,

University of Maryland, College Park. Maryland 20742

Abstract. Lottia alveus, a gastropod limpet once found
only on the blades of the eelgrass Zostera marina from
Labrador to New York in the western Atlantic Ocean, is
the first marine invertebrate known to have become ex-
tinct in an ocean basin in historical time. The last known
specimens were collected in 1929, immediately prior to
the catastrophic decline of Zostera in the early 1930s in
the North Atlantic Ocean. The brackish water refugium
ofZoslera throughout the decline was apparently outside
of this gastropod's physiological range, and the limpet
became extinct. Few marine invertebrates have habits as
specialized and ranges and tolerances as narrow as did L.
alveus. The fact that most marine invertebrates have large
effective population sizes may account for their relative
invulnerability to extinction.

Introduction

There are no reports of the post-Pleistocene extinction
of any marine invertebrate, in spite of the fact that
hundreds of terrestrial and freshwater species of animals
and plants have become extinct as human activity has
increased around the world (Martin and Klein, 1984:
Vermeij, 1986; McNeely et a/.. 1990). This is perhaps
even more remarkable given the widespread perception
that many marine invertebrate species have suffered ex-
tensive decimation and that a number of them are on
endangered species lists (for example, Gee and Wilson,
1981; Franz, 1982; Wells et ai, 1983; Wicksten, 1984).

Received 18 July 1990; accepted 24 November 1990.

We report here the first historical extinction of a marine
invertebrate from an ocean basin. The limpet Lottia alveus
(Fig. 1), a once abundant stenotopic species that ranged
from southern Labrador to Long Island Sound and lived
only on the blades of the eelgrass Zostera marina (Conrad,
1831;Couthouy, 1839; Gould and Binney, 1870), is now
extinct in the Atlantic Ocean. Here we consider the evi-
dence for this conclusion and suggest why this extinction
occurred.

Materials and Methods

Field studies

Eelgrass populations were searched specifically for lim-
pets in the following locations: Cape Cod, Massachusetts,
between 1979 and 1982; along the eastern Connecticut
shore (Fishers Island and Long Island Sounds) between
1982-1987 and 1989-1990, and at Vinalhaven (25 km
east of Rockland). central Maine in 1984 (J.T.C. and
D.A.C.); at Boothbay Harbor (45 km southwest of Rock-
land), central Maine in 1971, and in Newfoundland (Come
by Chance, in Placentia Bay, and at Norris Point, Bonne
Bay, in the Gulf of St. Lawrence) in 1990 (G.J.V.). We
contacted biologists who are familiar with the common
Atlantic limpet Tectura testudinalis (= Acmaea testudi-
nalis) and who have sampled Zostera epiphytes in Quebec
(Rimouski), Nova Scotia (Halifax), Maine, Massachusetts,
Rhode Island, and Connecticut. Since 1965, L. alveus has
been searched for without success in south-central Nova
Scotia, and in Labrador and Newfoundland (D. Davis and
R. Noseworthy, pers. comm., respectively). We examined

EXTINCT MARINE INVERTEBRATE

Figure I . Dorsal and lateral views of the extinct Atlantic limpet Loltia
alreiis (a pre-1900 specimen from Massachusetts. ANSP 39044, 1.6X).
The laterally compressed shell of this limpet precisely fitted the narrow
blade of the eelgrass Zostera marina.

all published records(from 1831 to 1989) of shallow-water
marine mollusks and eelgrass biota from the Arctic Ocean
to the central Atlantic coast of the United States.

Museum studies

We examined 14 museum malacological collections in
search of specimens of L. alveus. For systematic purposes
and for trophic analyses, we studied radulae of alcohol
preserved and rehydrated specimens of L. alveus. as well
as radulae of L. alveus parallela and illustrations of the
radula of L. alveus angusta.

These collections are located in the following museums
(abbreviations are given for museums cited later in the
text): Academy of Natural Sciences, Philadelphia (ANSP);
American Museum of Natural History, New York; British
Columbia Provincial Museum, Victoria (BCPM); Cali-
fornia Academy of Sciences, San Francisco; Los Angeles
County Museum of Natural History; Museum of Com-
parative Zoology, Harvard University (MCZ); Museum
of Paleontology, University of California, Berkeley; Na-
tional Museums of Canada, Ottawa; New York State Mu-
seum, Albany (NYSM); Natural History Museum, Lon-
don [formerly British Museum (Natural History)]; Nova
Scotia Museum, Halifax; Santa Barbara Museum of Nat-
ural History. Santa Barbara, California; United States
Museum of Natural History, Smithsonian Institution.
Washington, DC (USNM); University of Alaska Museum,
Fairbanks (U AM).

In addition, a number of major United States herbar-
ium collections of the eelgrass Zostera marina from North
America were examined in an independent study on eel-
grass wasting diseases by F. Short, who has provided us
with his records of dried limpets found on herbarium
sheets.

Results

Systematics and biogeography

The limpet Lot tin alveus (Conrad) was described in
1831 from Massachusetts. It is more commonly known
as Acmaea alveus or Co/lisella alveus. We follow the no-
menclatural revision of Lindberg (1986) in referring this
species to Lottia. Two situations led to the previously
overlooked history of this limpet in the North Atlantic
Ocean. First, there was a persistent belief that L. alveus
was an ecotype of the rocky intertidal limpet Tectura tes-
tui/ina/is(Mu\\cT, 1776) (Dall, 1871; Johnson, 1928; Ab-
bott, 1974), and that it was thus not a separate species.
Second, there are continued reports of its presumed pres-
ence on the Atlantic coast in molluscan checklists and
books (for example, Abbott, 1954, 1974; Emerson and
Jacobson, 1976).

However, as Jackson (1907) and Morse (1910, 1921)
clearly demonstrated, L. alveus is distinct from T. testu-
dinalis in anatomy, behavior, shell shape, sculpture, and
color. Morse (1910) noted that shells of the two species
could be distinguished at "a millimeter or more" in length,
by sculpture, apex shape, and color. McLean (1966) fur-
ther noted that L. a/veux was not a form of T. tesludinalis
that had settled on eelgrass blades, as both T. testudina/is
and the eelgrass Zostera marina are common in European
waters, where L. alveus does not occur. William Healey
Dall, whose opinion was widely regarded by contemporary
malacologists, also concluded, in a reversal of his earlier
belief (Dall, 1871), that L. alveus was "a good species"
(Sumner et a/.. 1913). He was apparently influenced by
the findings of Jackson (and perhaps Morse), but his
opinion apparently did not reach the general malacologi-
cal community.

McLean (1966) and Lindberg (1986) have shown that
L. alveus and T. testudinalis are properly placed in dif-
ferent genera. The genus Lottia possesses a single pair of
reduced marginal teeth (uncini) that are present at the
posterior end of the ribbon segment. The genus Tectura
lacks these marginal teeth on the radula. Lindberg (1981,
1 986, 1 988) discusses the phylogenetic importance of these
radular characters in diagnosing limpet genera. Jackson
( 1907) detailed other differences between the radulae of
the two species, although he failed to illustrate the uncini.

Lottia alveus originated in the North Pacific Ocean
from an ancestral lineage represented in the Mio-Pliocene
of Japan by Lottia august itesta (Yokoyama, 1926) (Yo-
koyama, 1926; Kotaka and Ogasawara, 1974; D.R.L., in
prep.). The Western North Pacific Ocean is also consid-
ered the center of origin in the Tertiary of Zostera
(McRoy, 1968; den Hartog, 1970). Both L. alveus and
Zostera invaded the North Atlantic Ocean through the
Bering Strait and the Arctic Ocean in the late Tertiary,

J. T. CARLTON ET AL.

as did numerous other marine organisms (Durham and
MacNeil, 1967; G.J.V., in prep.).

Pleistocene glaciation subsequently created three allo-
patric subspecies: Lotlia alveus parallela (Dall, 1914) in
the Northeast Pacific, Lottia alveus angusta (Moskalev,
1967) in the Northwest Pacific, and Lottia alveus alveus
(hereafter. L alveus) in the Northwest Atlantic. The three
subspecies are distinguished on the basis of external mor-
phology and radulae (D.R.L., in prep.). In addition, the
Atlantic subspecies had markedly less variation in color
and shell pattern than Pacific populations, and also pos-
sessed a widespread radular abnormality (an extra first
lateral tooth on the left side of the radula) absent in Pacific
individuals. These characteristics in the Atlantic subspe-
cies suggest a founder effect. Mitochondria! DNA analysis
(of the extant North Pacific populations and of preserved
material of the North Atlantic populations) may aid in
resolving whether these three taxa should be treated as
full species.

Lottia alveus parallela occurs only on Zostera between
Kazuna Bay, Cook Inlet (60° North Latitude) in southern
Alaska (UAM, N. Foster collections, 1975) and Smith's
Inlet in Queen Charlotte Sound, British Columbia (51°
North Latitude) (BCPM, late nineteenth century speci-
mens). Dall (1921) cites a southern Pacific coast limit of
L. a. parallela as Victoria, British Columbia (48° North
Latitude), but the specimen lot in BCPM upon which this
record is apparently based indicates that the material may
also have been collected at Skidegate Inlet, on the east
coast of Queen Charlotte Island. Burch (1946) cites what
appears to be an independent Victoria record, but without
data, and we have been unable to locate supporting ma-
terial. We know of no formal searches in Alaska or British
Columbia that have attempted to establish the exact dis-
tribution of L. a. parallela. Lottia a. angusta has been
recorded only from Sakhalin Island, Sea of Japan (46°
North Latitude), on Zostera (Moskalev, 1967).

Lottia alveus was known as far west (south) on the At-
lantic coast as Long Island Sound, where it was recorded
from New York by De Kay ( 1 843) and Letson ( 1 905) (see
also Table I, herein) and from Stratford, Connecticut by
Linsley ( 1 845). It occurred as far east (north) as Egg Har-
bor, Labrador (USNM, O. Bryant collections, 1908)
(Fig. 2).

The last known populations

No eelgrass limpets have been collected in the Atlantic
Ocean since 1929 (Table I). The previous known range
of this limpet (Labrador to New York) has been searched
thoroughly by us and others. Given the planktotrophic
larva that lottiid limpets possess (Lindberg, 1981), and
the now widespread occurrence and availability of Zostera
as a habitat, we do not believe that there are refugial.

isolated "pockets" of this limpet in remote coves, offshore
islands, or similar sites.

Two live collected USNM specimens ( 1 3.0 and 9.3 mm
in length) of L. alveus bear a label indicating the place of
collection as Cape Ann. Massachusetts (50 km northeast
of Boston) and a date of 14 July 1953. We have excluded
this record from Table I for the following reasons. In con-
trast to the records listed in Table I, we have been unable
to verify that this is the date of collection (for example,
by other species collected at the same time and place by
the same collector, by knowledge of the collector's specific
activities at the time and place of collection, and so forth).
The collector (J. A. Weber) specialized (as a hobby) in
collecting gastropod radulae, and obtained material from
many sources. Thus he may. for example, have obtained
preserved or dried material of this limpet from another
shell collector (Weber made a long trip up the coast in
1953, visiting shell collectors and collecting specimens).
The specimens were received at the Smithsonian Insti-
tution in 1966; while the Latin name and location are
part of the original writing, the date has been added in
black ink at a later time. Dexter (1968) systematically
sampled the mollusks at five widely separated stations at
Cape Ann from 1933 to 1937 and from 1956 to 1961,
and in many intervening years through 1967. While find-
ing many uncommon and rare species, he never found
L. alveus (R. Dexter, pers. comm., 1990). Dexter specif-
ically examined the mollusks on eelgrass blades at Cape
Ann in 1949 (Dexter, 1950). again without finding L. al-
veus. Dexter was also at Cape Ann in July 1953, where
he did not find L. alveus in informal surveys of the eel-
grass, nor did he meet Weber there (R. Dexter, pers.
comm.. 1990).

We do not discount this record because it occurs after
1929, nor because it does not fit our view of the timing
of the extinction of this mollusk. The possible persistence
of L. alveus until the early 1950s does not alter our con-
clusion that this limpet is extinct. Many extinctions are
characterized by a lengthy and slow decline of a species,
rather than by the precipitous disappearance documented
here. Thus, one scenario for the demise of L. alveiis would
have been a catastrophic bottleneck followed by the even-
tual disappearance of the last remnant populations over
subsequent decades. Rather, we reject this record because
decades of sampling and collecting mollusks specifically
at Cape Ann, and in the Boston area in general, before
and after 1953 have failed to discover this limpet. It is
not infrequent to find on museum labels transmittal dates,
exchange dates, and cataloging dates, and we thus suggest,
pending other confirmation, that "1953" is one of these
dates-of-record.

The last verifiable report of living eelgrass limpets in
the Atlantic Ocean is that of Proctor (1933). Collecting
in 1 929 ( fide Johnson, 1 929) at Bar Harbor on Mt. Desert

EXTINCT MARINE INVERTEBRATE

Long lilond
Sound

Figure 2. Former populations (dots) of the limpet Lotlia alveus in the Northwest Atlantic Ocean. Triangles
represent other localities mentioned in text.

Island on the northeastern Maine coast. Proctor reported
that "One may go to the Narrows [near Bar Harbor] at
low tide today and find . . . thousands of individuals
readily accessible . . ." Proctor believed (evidently on the
basis of shell color and shape) that L. alveus and T. tes-
tudinalis were identical species. Their abundance may
have been a source of his confusion. It is possible that he
found dislodged L. alveus individuals upon rocks and er-
rant T. testudinalis individuals on eelgrass blades. There
are reports ofL. alveus from rocks (Stimpson, 1851; Jack-
son, 1907; Morse, 1910) that Morse (19 10) believed to be
the result of specimens detached by waves and storms.
Lottia alveus was on occasion also found on other sub-
strates. There is, for example, a specimen (MCZ) collected
in 1 897 at Isle au Haul, Maine, attached to the periwinkle
Littorina liltorea (Linnaeus, 1758), bearing the label,
"living thus on this specimen of L. lillorea which was on

(a) float . . . in bed of eelgrass." The typically rock-dwell-
ing limpet Lottia pelta (Rathke, 1833) can be found oc-
casionally in California on the blades of the surfgrass
Phyllospadix when dense stands of the latter overlap in-
tertidal rocks (J.T.C., pers. observ.)

Reconstruction of the biology o/"Lottia alveus

The morphology, anatomy, habitat, and collection rec-
ords of Lottia alveus permit a partial reconstruction of
the biology and natural history of this extinct Atlantic
species. There are no studies of the extant subspecies in
the North Pacific Ocean.

Abundance

As with many now uncommon animals and plants re-
ported as "common" or "abundant" in the nineteenth

J. T. CARLTON ET AL

century, there are no quantitative analyses of the popu-
lation size or structure of Lottia alveus. However, a sense
of the abundance of this eelgrass limpet can be gleaned
from the literature (Table II). It is clear that this limpet
was sufficiently common throughout much of northern
New England that it could be collected "on demand" be-
tween the 1860s and the late 1920s. While workers con-
tinued to refer to L. alveus in later years [for example.
Miner's (1950) statement, "found abundantly on eel-
grass"], it is clear that these are references to older liter-
ature and collections.

Trophic ecology

The radula of L. alveus was illustrated by Jackson
(1907). We find it to be an accurate figure, with the ex-
ception of the missing uncini. Analysis of the radular
morphology of L. alveus indicates that it was a trophic
specialist, feeding upon the epithelial cells of the eelgrass,
rather than upon epiphytic diatoms and algae. The radula
of all alveus subspecies has broad, straight cutting edges
on its first and second lateral teeth. It is analogous to the
radula of the Northeast Pacific Ocean stenotopic surfgrass
(Phyllospadix) limpet Tectwa paleacea (Gould, 1853),
which eats only the epithelial cells of that grass (Fishlyn
and Phillips, 1980). With the exceptions of specimens that
presumably wandered offor were dislodged from eelgrass,
all reliable literature reports and museum material indicate
that Lottia alveus was restricted to, and by our analysis
ate only, the eelgrass Zostera marina. We predict that the
extant subspecies in the North Pacific feed upon the ep-
ithelial cells of Zostera.

Distributional ecology

We have studied all reported localities (including con-
sideration of their probable nineteenth century shoreline

Table I

Final records of the limpet Lottia alveus in the Atlantic Ocean

Table II

Record*, i if ihf abundance of the limpet Lottia alveus on the Atlantic
coast of North America

Locality

Last known
collection

Reference

New York: Long Island:

Noyack Bay

1926

(1)

Massachusetts: Boston region

1921

(2)

Maine: Rockland

1922

(3)

Maine: Mt. Desert Island

1929

(4)

New Brunswick: Bay of

Fundy: Grand Manan

1920

(5)

Quebec: Saguenay County:

Sept-lies

1925

(6)

References: ( 1 ) NYSM, R. C. Latham, collector; (2) Thompson, 1921;
(3) Lermond, 1922; (4) Johnson, 1929; (5) ANSP. H. S. Colton, collector;
(6) ANSP, on Zostera herbarium sheet.

Locality and date

Remarks

Reference

"New England",

"Found abundantly

Gould and Binney.

1860s

on the eel-grass"

1870

Grand Manan

"very abundant on

Ganong, 1890

Island. Bay of

eel-grass at low

Fundy. 1890

water"

Isle au Haul, Maine.

[> 1000 specimens in

MCZ

1893-1847

many lots]

North Haven, Maine

"very common on

Jackson, 1908

[25 km east of

Zostera marina"

Rockland], 1908

"Maine", 1909

"very common all

Lermond. 1909

Boston region, 1910

Rockland, Maine,
1922

Mt. Desert Island,
Maine, 1929

along the coast,

on eel grass and

occasionally on

rocks"
"in certain places

hundreds may be

collected in a

short time"
75 specimens taken

on eel grass in one

afternoon,

incidental to other

collections
"thousands of

individuals readily

accessible" at low

tide

Morse. 1910

Lermond, 1922

Proctor, 1933

configurations) for Lottia alveus from Long Island Sound
to Labrador to reconstruct aspects of the distributional
ecology of this limpet.

Although no authors reported the salinity of the water
in which they collected, it appears that all localities in
which L. alveus was collected were and are characteristic
of fully marine (32-33%o or greater), rather than estuarine,
habitats. Of course it is difficult to establish the salinity
of a locality without actual records, but no collections
indicate that populations of L. alveus were maintained
on eelgrass in low salinity (brackish water) sites. Further
evidence may be sought in the associated biota: the mol-
lusks reported to have been collected with or in the im-
mediate vicinity of L. alveus (for example, Rathbun, 1 88 1 ;
Jackson, 1908; Winkley, 1909; Thompson. 1921; Ler-
mond, 1922) include strictly marine species, as well as
euryhaline species, but never was the co-occurring mol-
luscan fauna (nor authors' site descriptions) characteristic
of strictly brackish water.

We conclude that L. alveus was probably a stenohaline
species of open coastal waters. We predict that extant sub-

EXTINCT MARINE INVERTEBRATE

species of L. alveus in the North Pacific will he found to
be stenohaline.

Discussion

An extinction scenario

What factors led to the extinction of this limpet? We
suggest a scenario that focuses upon a combination of the
stenotopic habitat of this species and its apparently narrow
physiological range.

Between 1930 and 1933, Zostera precipitously disap-
peared from both the eastern and western North Atlantic
Ocean on a scale and in geographic breadth far exceeding
any previous historical declines (Rasmussen, 1973, 1977).
The dramatic decline of this eelgrass led to extensive dis-
ruptions in neritic ecosystems, including large reductions
in migratory waterfowl populations, loss of commercial
scallop fisheries, and alterations for decades of nearshore
soft sediment habitats (Rasmussen, 1977; Short et ai,
1987). Until now, however, no extinctions have been at-
tributed to this decline. The primary cause of this decline
was probably a "wasting disease" caused by the slime mold
Labyrinthula (Muehlstein et ai, 1988; Short et ai. 1986,
1987, 1988). More than 90% of the standing stock of Zos-
tera was eliminated with concomitant and often striking
changes in associated biota (Stauffer, 1937; Dreyer and
Castle, 1941).

Populations of Zostera marina survived, however, in
low-salinity refugia (Short et ai, 1986). As argued above,
we suggest that Lottia alveus was probably a stenohaline
species; collection records indicate that it did not, unlike
Zostera. extend into brackish waters. We speculate that
the presumably narrow salinity range of this limpet may
have prevented it from surviving on refugial eelgrass pop-
ulations in lower salinity waters.

In contrast, the sacoglossan opisthobranch Elysia ca-
tiilns Gould, 1 870, similarly restricted to and feeding solely
upon eelgrass (Clark, 1975), did not become extinct. This
small sea slug ranges from Boston, Massachusetts (John-
son, 1915) to Virginia (Clark, 1975), and probably south
to the southern limit of Zostera in the Carolinas (Jensen
and Clark, 1983). Eelgrass populations were similarly
eliminated throughout Elysia's range, except, as noted,
in brackish water. We suggest that Elysia did not become
extinct because it lives in salinities at least as low as 17%o
(Marcus, 1972), and thus survived the eelgrass blight in
the estuarine eelgrass refugia.

It remains possible, of course, that factors other than
the putative osmoregulatory abilities (which cannot now
be experimentally determined for Atlantic populations)
prevented L. alveus from extending into brackish waters.
These factors could include respiratory intolerance of the
clay-silt loads typical of estuarine environments, or the
build-up of sediments or epiphytes in brackish water on

eelgrass blades that may have inhibited the limpet's feed-
ing. For whatever reasons, the evidence suggests that L.
alveus did not occur in the upper bay environments in
which Zostera survived.

Further evidence for this scenario is gained by the ob-
servation that other eelgrass-associated gastropods also
found refugia in other habitats or on Zostera in lower
salinity waters. Snails typically found on eelgrass in New
England and the middle Atlantic coast include the pro-
sobranchs Lacuna vincta (Montagu, 1803), Bittium al-
ternatum (Say, 1822), Bittium varium (Pfeiffer, 1840),
Crepidula convexa Say, 1822, and Afitrclla htnata (Say,
1826) (Nagle, 1968; Marsh, 1973). None of these is re-
stricted to Zostera, and none became extinct, although
there are reports of changes in microhabitat and abun-
dance following the eelgrass decline (Dexter, 1962;
O'Connor, 1972). Russell-Hunter and Tashiro( 1985) have
similarly noted the decline of the Z(W/t>ra-associated in-
faunal bivalve Cumingia tellinoides following the disap-
pearance of eelgrass beds.

The survival of Lottia alveus parallela and Lottia alveus
angusta may result from the fact that no extensive areas
of eelgrass were eliminated in the North Pacific Ocean
(den Hartog, 1987).

Other reported marine mollusk extinctions

Other marine mollusks have been reported as possibly
extinct. We have found no records of any other docu-
mented historical marine invertebrate extinctions.

A single living specimen of the limpet "Col/isella" ed-
mitchelli was collected in the early 1 860s in southern Cal-
ifornia (Lindberg, 1984). Nothing is known further of the
Holocene history or habitat of this otherwise Pleistocene
species. The Caribbean bivalve Pholadomya Candida was
believed extinct (Runnegar, 1979), but it is extant in waters
off Venezuela (Gibson-Smith and Gibson-Smith, 1981).
The nudibranch sea slug Doridella batava, once believed
to be endemic to the Netherlands, is reported as possibly
extinct (Wells et ai, 1983), but has been found living in
France (Platts, 1985). Moreover, D. batava may represent
an introduction of a previously described species from
elsewhere in the world (Wells et ai, 1983; T. Gosliner,
pers. comm., 1990).

Six to eight species of brackish water hydrobiid snails
were reported as possibly extinct on the United States
Atlantic coast by Morrison (1970). The distinction of these
undescribed species from still living and closely related
taxa has not been demonstrated (F. Thompson, pers.
comm., 1986), nor is it clear that searches were made for
still extant populations.

The most intriguing record that we have found is that
of the Californian potamidid estuarine snail Cerithidea
fuscata, which Taylor (1981) reported as "possibly ex-

J. T. CARLTON ET AL.

tinct." This high intertidal, mudflat-dwelling horn snail
is known only from San Diego Bay in southern California;
it was last collected in 1935. Taylor ( 198 1 ) suggested that
threats to its existence were "pollution, dredging, and land
fill." Taylor (1981) treated C. fuscata Gould, 1857, as a
distinct species, with Cerithidea sacrata hyporhyssa Berry,
1 906, in synonymy. Grant and Gale (1931) and Bequaert
(1942) considered the latter a synonym of Cerithidea cal-
ifornica (Haldeman, 1840). The status of C. fuscata as a
species distinct from Cerithidea californica. rather than
either an ecophenotype or subspecies, has not been clar-
ified (J. McLean, pers. comm., 1986). Cerithidea fuscata
differs from other populations of Cerithidea by virtue of
its smooth, tapered shell with flat whorls (Berry, 1906).
While C. californica is common and widespread both to
the north and south, the smooth-shell population has long
been considered to occur only in San Diego Bay (Burch,
1945). There are no details of population declines or dis-
appearances of C. fuscata as yet documented, nor is there
published evidence that searches have been made for ex-
tant populations. Nevertheless, that populations of Cer-
ithidea. whose life history is characterized by non-plank-
tonic larvae (Race, 1981), are susceptible to bay-wide ex-
tinctions has been documented elsewhere (Carlton, 1976).
It is clear from these and other reports that there are
historical records of marine and estuarine mollusks with
small and geographically limited populations, and that
some of these populations are believed to have disap-
peared. There are also many species of crustaceans, an-
nelids, flatworms, hydroids, and other invertebrates that
have never been reported since the nineteenth century.
Some of these are from coastal localities that have been
obliterated during the course of human population ex-
pansion and concomitant littoral urbanization. Finally,
at both local (state) and international levels, various ma-
rine invertebrates have been reported as "endangered."
Listings for marine mollusks have been achieved in part
due to the collecting activities of shell collectors. We dis-
tinguish all of these records from demonstrably extinct
taxa.

Conclusions

With its specialized feeding habits and narrow habitat
range, Lottia alveus conforms well to the profile of species
that are believed to be highly susceptible to extinction
(Martin and Klein, 1984; Vermeij, 1986). The limited
geographic range (a consequence of Pleistocene glacia-
tions), the limited trophic range (an adaptation dating
from the Mio-Pliocene), and the presumably limited
physiological range (a phylogenetic constraint shared by
almost all lottiid limpets and perhaps dating from the
Paleozoic origin of the group) were interwoven and cas-
cading attributes that set the stage to make this species

vulnerable to extinction. We suggest that the refugia of
Lotlia's sole food source during a period of catastrophic
decline were outside of this limpet's habitat range, and
the limpet became extinct.

Most marine invertebrates whose biology and distri-
bution are well-known do not have habits as specialized
and ranges as narrow as did Lottia alveus. (There are, of
course, a great many species described from only one lo-
cality, but whose actual ranges are not known). Most of
those taxa that are known from one or a few host species
have much wider geographical ranges than did this limpet.
The fact that most marine invertebrates have large effec-
tive population sizes, often over broad ranges, may ac-
count further for their relative invulnerability to extinction
in historical time. In contrast, small and geographically
restricted populations of species (short-range endemics,
for example) may be particularly vulnerable to extinction.
Those species whose life history combines non-planktonic
larvae with juvenile or adult stages not likely to be asso-
ciated with drifting algae or wood may be specifically sus-
ceptible to extinction.

While our records to date indicate that marine inver-
tebrates in general have escaped historical extinctions by
the end of the twentieth century, human activities have
been and are clearly capable of severely reducing and
completely eliminating populations of marine inverte-
brates from extensive parts of their ranges. These actions
have and will continue to fundamentally alter the structure
of natural communities.

Acknowledgments

We thank numerous museum curators and correspon-
dents for aid in establishing the status of this small snail.
Frederick Short (University of New Hampshire) provided
the herbarium-based record from Quebec. Ralph Dexter
(Kent State University) provided valuable information
and references on his mollusk collections at Cape Ann,
Massachusetts. Terrence Gosliner (California Academy
of Sciences). James McLean (Los Angeles County Mu-
seum of Natural History), and Fred Thompson (Florida
State Museum, Gainesville) provided valuable comments
on the taxonomic status of certain mollusks. Peter Frank
(University of Oregon), Peter Petraitis (University of
Pennsylvania), and Janie Wulff (Williams College) com-
mented upon various versions of the manuscript.

Literature Cited

Abbott, R. T. 1954. American Seashells. D. Van Nostrand Company,
Inc., Princeton. NJ.

Abbott, R. T. 1974. .-lincnain Seashells. 2nd ed. Van Nostrand Rein-
hold Company. New York City, NY.

Bequaert, J. 1942. Random notes on American Potamididae. Nautilus
56: 20-30.

EXTINCT MARINE INVERTEBRATE

7')

Berry, S. S. 1906. Note on a new variety of Centhidea sacrala Gld.,

from San Diego, Cal. Nautilus 19: 133.
Burch, J. Q. 1945. Ccrilhidea California] hyporhyssa. Mm Concho-

logical Club So. Calif. 54: 34-35.
Burch, J. Q. 1946. Family Acmaeidae. Mm. Conchological Club So

Calif. 57: 5-16.
Carlton, J. T. 1976. Extinct and endangered populations ofthe endemic

mudsnail Cerithidea californica in northern California. Bull Am

Malacol L'nioii 1975: 65.
Clark, K. B. 1975. Nudibranch life cycles in the Northwest Atlantic

and their relationship to the ecology of fouling communities, He/go/.

Wiss. Meercsuntcrs. 27: 28-69.
Conrad, T. 1831. Descriptions of fifteen new species of Recent, three

of fossil shells, chiefly from the coast ofthe United States. J Acad

Nat. Sci. Philadelphia 6: 256-268.
Couthouy, J. P. 1839. Monograph on the family Osteodesmacea of

Deshayes, with remarks on two species of Patelloidea. and descriptions

of new species of marine shells, a species ofAnculotus, and one of

£0/1.5. Boston J. Nat. Hist. 2: 129-189.
Dall, VV. H. 1871. On the limpets; with special reference to the species

ofthe west coast of America, and to a more natural classification of

the group. Am. J Conch. 6: 228-282.
Dall, VV. H. 1914. Notes on some northwest coast acmaeas. Nautilus

28: 13-15.
Dall, VV . H. 1921. Summary ofthe marine shell-hearing mollusks of

the northwest coast of Alaska. Bull. U. S Nail. Mils. 112: 1-217.
De Kay, J. E. 1843. Zoology of New York. Part \' Mollusca. Carroll

and Cook, Albany.
den Hartog, C. 1970. The Sea Grasses of the H'orld North-Holland

Publishing Company. Amsterdam.
den Hartog, C. 1987. "Wasting disease" and other dynamic phenomena

in eelgrass beds. Ai/ual. Bo/ 27: 3-14.
Dexter, R. \V . 1950. Restoration ofthe Zostera faciation at Cape Ann,

Massachusetts. Ecology 31: 286-288.
Dexter, R. W. 1962. Further studies on the marine mollusks of Cape

Ann. Massachusetts. Nautilus 76: 63-70.
Dexter, R. VV . 1968. Distribution ofthe marine molluscs at Cape Ann.

Massachusetts. Proc. Symp. Mollusca (Mar. Biol. Assoc. India,

Cochin. Symp. Ser. 3) I: 214-222.
Dreyer, W. A., and W. A. Castle. 1941. Occurrence ofthe bay scallop,

Pecten irradians. Ecology 22: 425-427.

Durham, J. VV., and F. S. MacNeil. 1967. Cenozoic migrations of ma-
rine invertebrates through the Bering Strait region. Pp. 326-349 in

The Bering Land Bridge. D. M. Hopkins, ed. Stanford University

Press, Stanford, CA.
Emerson, VV. K., and M. K. Jacobson. 1976. The American Museum

of Natural History Guide to Shells. Alfred A. Knopf, New York.
Fishlyn, D. A., and D. VV. Phillips. 1980. Chemical camouflaging and

behavioral defenses against a predatory seastar by three species of

gastropods from the surfgrass Phyllospadix community. Biol. Bull

158: 34-48.
Franz, D. R. 1982. An historical perspective on molluscs in lower New

York harbor, with emphasis on oysters. Pp. 181-197 in Ecological

Stress and the New York Bight: Science and Management, G. F.

Mayer, ed. Estuarine Research Federation, Columbia. SC.
Ganong, W. F. 1890. Article III. Zoological notes. Report ofthe Com-
mittee on Marine Invertebrate Zoology. I. Mollusca. Bull Nat. Hist.

Soc. New Brunswick 9: 1-14.

Gee, J. H. R., and K. Wilson. 1981. The littoral Mollusca of Green-
island, Co Antrim: 40 years of change in Belfast Lough. Ir. Nat. J

20: 343-345.
Gibson-Smith, J., and W. Gibson-Smith. 1981. The status of Pholu-

domya Candida G. B. Sowerby. I. 1823. I'cliger 23: 355-356.

Gould. A. A., and VV . G. Binney. 1870. Report on the Invcrtchrata nf

Massachusetts. Wright and Potter, Boston, MA.
Grant, U. S., and H. R. Gale. 1931. Catalogue ofthe marine Pliocene

and Pleistocene Mollusca of California and adjacent regions. Mem.

San Diego Soc. Nat. /list. 1: 1-1036.

Jackson, H. 1907. The differences between the two New England spe-
cies of Acmaea Nautilus 21: 1-5,24.
Jackson, H. 1908. The Mollusca of North Haven. Maine. Nautilus

21: 142-144.
Jensen, K., and K. B. Clark. 1983. Annotated checklist of Florida as-

coglossan Opisthobranchia. Nautilus 91: 1-13.
Johnson, C. W. 1915. Fauna of New England List of Mollusca. Occ

Pap. Boston Soc. Nat Hist. 13: 1-231.
Johnson, C. W. 1928. A review ofthe New England limpets. Nautilus

41: 109-117.

Johnson, C. W. 1929. Acmaea lesludinalis (Mull.). Nautilus 42: 103.
Kotaka, T., and K. Ogasawara. 1974. A new abalone from the Miocene

of Aomon Prefecture, northeast Honshu, Japan. I 'enus33: 1 17-128.
I minimi. N. VV. 1909. Shells of Maine. A catalogue ofthe land, fresh-
water and marine Mollusca of Maine. Fourth Ann. Rep/ Stale En-
tomologist, Augusta, ME. Pp. 217-262.
Lermond, N. VV. 1922. One haul ofthe dredge. The Maine Naturalist.

Journal o/ the Knox Academy of Arts and Sciences 2: 1 38-140.
Letson, E. 1905. Check list ofthe Mollusca of New York. Bull New

York S/a/e Museum 88, Zoology I I . Pp. 1-112.
Lindberg, D. R. 1981. Acmaeidae Gastropoda Mollusca. Invertebrates

ofthe San Francisco Bay Estuary System. The Boxwood Press, Pacific

Grove, CA.
I jndberg, D. R. 1984. A recent specimen otCollise/la edmilche/li from

San Pedro, California (Mollusca: Acmaeidae). Bull. So. Calif. Acad.

Sci. S3: 148-151.
Lindberg, D. R. 1986. Name changes in the "Acmaeidae." t'eliger 29:

142-148.
Lindberg, D. R. 1988. The Patellogastropoda. Malacol. Rev, Suppl. 4:

35-63.
Linsley, J. H. 1845. Catalogue ofthe shells of Connecticut. Am. J Sci.

48:271-286.
Marcus, E. d. B.-R. 1972. Notes on some opisthobranch gastropods

from the Chesapeake Bay. Ches. Sci. 13: 300-317.
Marsh, G. A. 1973. The Zostera epifaunal community in the York River,

Virginia. Ches. Sci 14: 87-97.
Martin, P. S., and R. G. Klein, eds. 1984. Quarternary Extinctions \

Prehistoric Revolution. University of Arizona Press, Tucson, AZ.
McLean, J. H. 1966. West American prosobranch Gastropoda: su-

perfamilies Patellacea, Pleurotomariacea, and Fissurellacea. Ph.D.

dissertation, Stanford University, Stanford, CA.
McNeely, J. A., K. R. Miller, W. V. Reid, R. A. Mittermeier, and T. B.

Werner. 1990. Conserving the World's Biological Diversity. Inter-
national Union for Conservation of Nature and Natural Resources,

Gland, Switzerland, and Washington, D. C.
McRoy, C. P. 1968. The distribution and biogeography of Zostera

manna (eelgrass) in Alaska. Pac. Sci. 22: 507-513.
Miner, R. W. 1 950. Field Book of Seashore Life. G. P. Putnam's Sons,

New York City. NY.
Morrison, J. P. E. 1970. Brackish water mollusks. Malacologia 10:

55-56.
Morse, E. S. 1910. An early stage of Acmaea. Proc. Bos. Soc. Natl.

Hist. 34: 313-323.
Morse, E. S. 1921. Observations on the Living Gastempods of New

England. Peabody Museum, Salem, Massachusetts.
Moskalev, L. I. 1967. Collisella angusla, n. sp. Pp. 18-19 in A. N.

Golikov and O. A. Scarlato. Molluscs ofthe Possiet Bay (the Sea of

Japan) and their ecology. Trudy Zool. Insl. Leningrad 42: 5-154 (in

Russian).

J. T. CARLTON ET AL

Muehlstein, L. K., D. Porter, and K. T. Short. 1988. Labyrinthula sp.,

a marine slime mold producing the symptoms of wasting disease in

eelgrass, Zostera marina. Mar. Bin/. 99: 465-472
Nagle, J. S. 1968. Distribution of the epibiota of macroepibenthic

plants. Contrib. Mai Sci 13: 105-144.
O'Connor, J. S. 1972. The benthic macrofauna of Moriches Bay, New

York. Biol. Bull 142: 84-102.
Platts, E. 1985. An annotated list of the North Atlantic Opisthobran-

chia. excluding Thecosomata and Gymnosomata. Ophelia Supple-
ment 2: 150-170.
Proctor, \V. 1933. Biological survey of the Mount Desert region. Part

V. Wistar Institute of Anatomy and Biology. Philadelphia, PA.
Race, M. S. 1981. Field ecology and natural history ofCerilliidea cal-

ifornica (Gastropoda: Prosobranchia) in San Francisco Bay. I'eliger

24: 18-27.
Rasmussen, E. 1973. Systematics and ecology of the Isefjord marine

fauna (Denmark). (With a survey of the eelgrass ( Zostera) vegetation

and its communities). Ophelia II: 1-495
Rasmussen, E. 1977. The wasting disease of eelgrass (Zoslera marina)

and its effects on environmental factors and fauna. Pp. 1-52 in Sea-
grass Ecosystems: A Scientific Perspectives. C. P. McRoy and C.

Helfferich. eds. Marcel Dekker. New York.
Rathbun, R. 1881. The littoral marine fauna of Provincetown, Cape

Cod, Massachusetts. Proc. U. S Nail. Mils. 3: 1 16-133.
Runnegar, B. 1979. Pholutlomya Candida Sowerby: the last cadaver

unearthed. I 'eliger 22: 171-172.
Russell-Hunter, W. D., and J. S. Tashiro. 1985. Life-habits and infaunal

posture ofCitmingia telltnoiiles (Tellinacea, Semelidae): an example

of evolutionary parallelism. I'digcr 27: 253-260.
Short, F. T., A. C. Mathieson, and J. I. Nelson. 1986. Recurrence of

the eelgrass wasting disease at the border of New Hampshire and

Maine, USA. Mar. Ecol. Prog. Set: 29: 89-92.

Short, F. T., L. K. Muehlstein, and D. Porter. 1987. Eelgrass wasting
disease: cause and recurrence of a marine epidemic. Biol. Bull 173:
557-562.

Short, F. T., B. \V. Ibelings, and C. den Hartog. 1988. Comparison of
a current eelgrass disease to the wasting disease of the 1930s. Auiuii
But. 30: 295-304.

Stauffer, R. C. 1937. Changes in the invertebrate community of a lagoon
after disappearance of the eelgrass. Ecology 18: 427-431.

Stimpson, \V. 1851. Shells of New England. A Revision of the Synonymy
nl the Testaceous Mo/lusks of New England. Phillips. Sampson, and
Co.. Boston.

Sumner, F. B., R. C. Osburn, and L. J. Cole. 1913. A biological survey
of the waters of Woods Hole and vicinity. Section III. A catalogue
of the marine fauna. Bull. Bur. Fisheries 31: 545-794.

Taylor, D. \V. 1981. Freshwater mollusks of California: a distributional
checklist. Calif /•'/.*•/; Game 67: 140-163.

Thompson, L. D. 1921. Collecting at Nahant Beach, Mass. Nautilus
34: 100- 10 L

\ ermeij, G. J. 1986. The biology of human-caused extinction. Pp. 28-
49 in The Preservation of Species. B. G. Norton, ed. Princeton Uni-
versity Press, Princeton, NJ.

Hells, S., R. Pyle, and N. Collins. 1983. The IUCN Invertebrate Red
Data Book International Union for Conservation of Nature, Swit-
zerland.

\\icksten, M. K. 1984. Early twentieth century records of marine decapod
crustaceans from Los Angeles and Orange counties, California. Bull.
So. Calif. Acad. Sci. 83: 12-42.

Winkley, H. VV. 1909. Essex County notes. Nautilus 23: 86-87.

Yokoyama, M. 1926. Fossil shells from Sado. J Fac. Sci. Imp. Univ.
Tokyo. Sec. 2. 1:249-312.

Reference: Bio/ Bull 180: 81-92. (February. 1991)

Passive Suspension Feeding by an Octocoral

in Plankton Patches: Empirical Test

of a Mathematical Model

MARK R. PATTERSON

Division of Environmental Studies, University of California, Davis, California 95676

Abstract. Feeding rate in the octocoral, Alcyonium sid-
eriitin, was investigated as a function of colony size, flow
speed, and prey concentration. The feeding rate decreases
with time in high prey concentrations. A model of passive
suspension feeding is formulated that successfully predicts
feeding behavior. At low prey concentrations, the model
predicts a linear feeding response as particle flux or colony
size increases. The dominant constraint on feeding is the
"handling time" required to transfer prey from tentacle
to pharynx and to re-extend the tentacle. The time con-
stant of prey capture shows no relation to particle flux,
in agreement with the model. Another constraint, the
"nitration time," is inversely related to colony size and
flow speed. Filtration time becomes important only during
feeding in sparse prey concentrations, when feeding rate
is proportional to flow speed, colony size, and prey con-
centration. In the field, Alcyonium colonies reduce nitra-
tion time by orienting at right angles to the dominant
flow direction. Feeding efficiency on prey patches is low
and inversely related to flow speed, colony size, and prey
concentration. Feeding in patches is not a simple process
for this octocoral, because colonies will "saturate" with
prey before all polyps have successfully captured a single
prey item.

Introduction

Suspension feeding occurs in nearly all animal groups
(J0rgensen, 1966), and virtually every body of water pos-
sesses a guild of organisms making a living by filtering
the soup in which they live. Groups that have received
the greatest amount of attention in the literature are active
suspension feeders, i.e., those organisms that generate their

Received 15 September 1989; accepted 6 November 1990.

own feeding currents. Organisms that rely exclusively on
environmentally produced currents to bring them food
are termed passive suspension feeders.

Experimental studies on active suspension feeders led
to the formulation of the first mathematical models of
suspension feeding. Decreases in the concentration of
particles in closed systems containing these animals could
be easily monitored: use of a decreasing exponential model
of filtration allowed calculation of pumping rate (J0rgen-
sen. 1943). Coughlan (1969) reviews the use of the ex-
ponential model in calculating pumping rates (sometimes
erroneously called filtration rates) for active suspension
feeders. Filtration efficiency was assumed to be 100% in
his treatment. Williams (1982) showed that if this as-
sumption is seriously violated, the decline in cell concen-
tration will be a double exponential, and measured de-
clines cannot be easily converted into a filtration or
pumping rate. His formulation of suspension feeding also
predicts that the apparent filtration rate will be a function
of time as physical limitations of the system with respect
to filtration efficiency become important. Thus, apparent
variations in filtration rate may be nothing more than
manifestations of how sieving and other means of particle
capture (Rubenstein and Koehl, 1977) interact with the
population of cells of different sizes available for capture.
Behavioral modifications of pumping rate need not be
invoked to explain variation in pumping rate. Williams
(1982) provides a prescription for measuring pumping
rate accurately and testing for any behavioral modifica-
tions; this involves finding a particle that is filtered with
100% efficiency by the organism under investigation. Most
active suspension feeders such as bivalves (Jorgensen,
1975; Mohlenberg and Riisgard, 1978; Palmer and Wil-
liams, 1980) and ascidians (Fiala-Medioni, 1973, 1978a,
b, c, d) attain remarkable capture efficiencies for the small

M R PATTERSON

particles on which they feed (bacterio- and phytoplank-
ton). Efficiencies can often reach 100% for particles on
the order of 10 j*m in diameter, and thus pumping rates
can be easily measured following the recommendations
of Williams (1982).

Predictions of mathematical models

Mathematical models have also been used to clarify the
control of suspension feeding. Two complementary and
not entirely separable approaches have been: ( 1 ) to predict
how an organism's feeding rate should relate to the density
or quality of the food it encounters (Holling, 1965; Emlen,
1973; Doyle, 1979; Scale, 1982) and, (2) to see whether
suspension feeding organisms maximize the rate of energy
gain (Lehman, 1976; Lam and Frost, 1976).

Both Holling (1965; functional response type I) and
Lehman (1976) predict that ingestion or filtering rate
should show a linear dependence on prey availability or
density up to some saturation value in organisms such as
cnidarians, where encounter rate with the prey is deter-
mined by organism size and environment (in this case,
flow speed). The saturation level is presumably set by the
digestive physiology of the organism, e.g.. the "packed
gut" assumption of Townsend and Hughes (1981). An
implicit assumption is that all prey encountered, or at
least some constant fraction of them, are retained by the

organism (constant efficiency); symbolically, - - = K,

where N = number of prey caught, and K is a constant.
K can be further decomposed: K = U X V X SA, where
U = flow speed, V = prey concentration, and SA is the
surface area of the organism available for prey capture. I
term this hypothesis the "linear" model of passive sus-
pension feeding, which is typically used in analyzing pas-
sive suspension feeding.

The "linear" model predicts that for a given prey density
below the saturation level, feeding rate should be constant.
Figure 1 A gives the solution to the linear model and shows
how doubling the prey concentration, flow speed, or pro-
jected surface area (size) of the organism should affect the
feeding "response." Note that this "filling" curve gives
the cumulative number of prey caught as a function of
time; it assumes that prey density is not changing as the
organisms feeds. The "filling" curve is mathematically
isomorphic with the functional response type I of Holling
(1965) at a given prey concentration. The curve is also
conceptually equivalent to viewing filtration as a Poisson
process, i.e., the probability (P) of capture during a small
increment of time (At) is constant, and the magnitude of
P is the product of U, V, and SA (Fig. IB). Furthermore,
the interval between capture events is large at low prey
concentrations, for reasons to be discussed below, and
hence capture events are rare.

saturation level

3
O.

Time

Poisson simulation of prey capture

500
400
300
200
100
0

P = 06

0 200 400 600 800

Time Units (At)

Figure 1. The classical view of passive suspension feeding. (A) The
"linear" deterministic model of passive suspension feeding, which assumes
prey encounter rate is proportional to the projected surface area normal
to the flow (SA), the flow speed (U), and concentration of prey present
(V,). The feeding rate is constant until some saturating level of prey
inside the organism is attained. This curve is implicit in Holhng's( 1965)
type I functional response curve for predator-prey systems similar to
passive suspension feeding. (B) The previous model is functionally
equivalent to a process governed by the Poisson interval distribution.
i.e., a process where the probability of capture (P) during a small interval
of time (At) is constant. The filling curves were generated by computer
simulation for three levels of P, corresponding to increasing levels of
flow, colony size, or particle concentration. Note the linear dependence
between the number of particles captured and the time the suspension
feeder has been exposed to a current carrying prey items.

Energy maximization arguments (cf. Townsend and
Hughes, 1981) argue that filter feeders should feed pref-
erentially on particles with the higher nutritional value,
unless the cost of sorting and rejection are too high. The
few tests in the literature (Doyle, 1979, amphipod; Scale,
1982, anuran tadpole larvae) indicate that these suspen-
sion feeders do behave in a manner consistent with energy
maximization. Some work has addressed whether models
formulated for other organisms make predictions com-
patible with observations of feeding rate as a function of
prey density for passive suspension feeding in cnidarians
(Clayton and Lasker, 1982). Sebens (1979, 1984) for-
mulated a cost/benefit model for cnidarians using energy
maximization as a means of predicting optimum organism
size in a given habitat. The model has had good success

MODEL OF PASSIVE SUSPENSION FEEDING

in predicting maximum organism sizes observed in the
field. However, little attention has been paid to modeling
the response of passive suspension feeders to dense prey
concentrations, which may change suddenly in time, i.e.,
what happens when a plankton patch sweeps by a cni-
darian colony?

A dynamic mathematical model of passive
suspension feeding

The limitations of the "linear" model are those imposed
by its assumptions. Feeding rates may not be constant
overtime, especially if the handling of individual particles,
or digestive or neurally mediated behavior becomes im-
portant. A more robust model of suspension feeding for
cnidarian colonies was formulated and tested against real
feeding in "patch" concentrations in the laboratory. The
model takes a systems analysis view of passive suspension
feeding: the input to the system (colony) is prey in the
water column, the output is prey inside the organism.
The model allows sudden changes in prey concentration
and predicts the time course of feeding using two param-
eters. Congruence between the observed and predicted
parameters of the model implies the assumptions used in
the formulation are not too far from reality, i.e., identi-
fication has been made of the salient features of the fil-
tration system that determine feeding performance.

There are three model assumptions. ( 1 ) The colony
"fills" up with prey at a rate proportional to the difference
between the ambient plankton concentration (V,) and the
amount of prey already in the colony (V0); symbolically,
(V, - V0). (2) The colony fills at a rate inversely propor-
tional to the time necessary to handle the particles caught
during filtering (R,) and the time needed to filter the
water containing the particles (R2); symbolically.

— — . Operationally, R, is the time taken to transfer,
(R, + R:)

from tentacle to polyp mouth, the particle caught from a
unit volume of water and to re-extend the tentacle; R2 is
the inverse of the filtration rate, which depends on the
projected area of the organism perpendicular to the flow,
and the flow speed. (3) A sudden jump in the plankton
concentration results in a jump in the number of particles
caught. The size of the jump is directly proportional to

the jump in the particle concentration, .the colony

volume (C). and the proportion of time spent filtering

particles during feeding, - ; symbolically, C -

(Ri ~1~ R->) dt

(R, + R2) '

The first and second assumptions address the steady-
state behavior of the passive suspension feeder, while the
third deals with the dynamic aspect of prey capture. Ex-

pressed as a differential equation, passive suspension
feeding may obey:

dV0 _ (V, ~ Vo)

. 0*1)

dt (R, + R2) (R, + R2) dt

dV0

where C - - is the time change in the total number of
dt

particles caught by the organism.

Dividing by the size of the colony yields:

dV0 (V, - V0)

dt (R, + R:)C (R, + R2) dt
Eq. (2) can be rearranged algebraically to:

^. (Eq.2)

where r = (R, + R:) C. and « =

(Eq.3)

is the time

(R, + R2)

constant of colony "filling," while a is the measure of
how many prey are caught as the edge of the patch sweeps
by the colony.

The solution to Eq. (3) depends on the nature of the
change in the plankton concentration in the water column
or laboratory flume. An electrical circuit that mimics ex-
actly the behavior of this mathematical feeding model is
called a lag-lead network (Milsum, 1966) and is shown
in Figure 2. Formulation of the resistive-capacitive analog
is motivated by the observation that prey filtration and
prey handling are discrete processes. They are modeled
as "resistances" through which the "current" of prey must
pass to fill the organism's "capacity" (the etymological
root of capacitance). This circuit can be easily wired up
with variable resistors, Rj 2 and variable capacitor, C, al-
lowing exploration of the model's qualitative behavior. A
change in plankton concentration would be simulated by

o vw

t Rl t

I CT I

Figure 2. Electrical analog to the differential equations used to model
the process of passive suspension feeding. The behavior of this circuit
exactly mimics the model. R, is the handling time "resistance" and R2
is the filtration time "resistance." The concentration of particles in the
water column is a "voltage" (V,) that may cause a "current" of particles
to enter and reside inside the organism (V0); this is controlled by the
resistances, R, : and the volume "capacitance" (C) of the animal.

M. R. PATTERSON

a change in the input voltage (V,); simulation of colony
feeding response predicted by the model can be seen by
watching the behavior of V0 as a function of time on an
oscilloscope.

Applying KirchofFs law to this circuit. I obtain:

iR, +

E/«

: = Vj(t)

and

c/'

idt + iR: = V0(t),

(Eq.4)

(Eq.5)

where i is the "current" of particles.

Taking the Laplace transform of Eqs. (4) and (5) yields:

R, + R: +

= V,(s) (Eq.6)

and

R: + — li(s) = V0(s), (Eq.7)

where s is the frequency-domain variable. Some algebra
then results in:

V0(s)
V,(s)

RI + R2

a(s + b)
b(s + a)

(Eq.8)

-, and b =

in i p \ /- p /- '

( ix i T~ K-2/ *- rS-2*-

Eq. (8) is the Laplace transform of Eq. (3). It can be
rearranged to:

V,(s)

V0(s) = s

Vj(s) +

(Eq.9)

(rs + 1) (S + 1)

To solve Eq. (9), the nature of the input change in
plankton concentration must by specified. For a step
increase in the plankton availability to level V,, caused
by a patch of plankton flowing past the colony, V,(s)

V,
- . Substituting, I obtain:

V0(s) = s -

(rs -r i

which can be rearranged to:
R2CV,

V,(s)
(TS+ 1)

(Eq. 10)

u,-, -, . -• (Eq. 11)

(rs + 1) s(rs + 1)

Taking the inverse Laplace transform, I obtain:

V0(t) = V,j 1 - (1 - «)e(-t/r)}, (Eq. 12)
the solution in the time domain.

Since « and T can be computed from known quantities,
it is possible to compare predicted with observed values
of these two model parameters. In particular, T will be an
important descriptor of how quickly a colony can use a
change in plankton concentration. Figure 3A shows the
"filling" curve for colonies of different size (C), while Fig-
ure 3B shows identically sized colonies as the ratio between
"handling" time (R,) and "nitration" time (R:) changes.
The implications of the behavior of this model, its de-
composition into the "linear" model (Type I functional
response) under certain conditions, and the extent of its
congruence with reality will be more fully developed in
the Discussion section.

I experimentally tested this model by measuring feeding
rates for a colonial cnidarian in the laboratory. Alcyonium
siderium, an octocoral, is a dominant zooplanktivore on
subtidal hard rock substrates in New England (Sebens and
Koehl, 1984; Sebens, 1986). Colonies assume a variety of
shapes varying from fingers to globose forms to com-
pressed ellipsoids (Patterson, 1980). In plankton-rich

200 400 600 800
Time Units (At)

Equal C

a 0.8 '

O.
•o

-I
a

i-
o

200 400 600 800
Time Units (At)

1000

Figure 3. The time course of organism feeding as predicted by the
model. (A) Colonies differing in size by a factor of five (C. 5C) and with
handling time ( R i ) much greater than filtration time (R2), as occurs during
feeding in plankton patches. (B) Colonies of identical size where the
handling time (R,) is much greater than filtration time (R2) and vice
versa. The ordinates for both graphs are normalized for the effects of
colony size. Note that if R: becomes much larger than R,, i.e.. the particle
flux drops, then the filling curve tends toward a step function. Since
particle flux is low, each passing particle is caught, and the model de-
composes to the "linear" model of Figure 1.

MODEL OF PASSIVE SUSPENSION FEEDING

habitats, fully expanded globose colonies can reach 10cm
in diameter. Previous work with Alcyonium colonies
feeding in a closed system has shown that they readily
accept prey particles (Patterson, 1984). This octocoral
might be expected to follow the "linear" filling curve if
prey concentration is held constant, and obey the changes
predicted in Figure 1 as size and flow speed are varied.
The aim of this laboratory feeding study was to test the
"linear" model and the proposed alternative model for
feeding in plankton patches. An intensive study of the
diet of this species over a diel cycle (Sebens and Koehl,
1984) provides information useful in analyzing the results
of this study.

Materials and Methods

Colony collection, maintenance, and flow generation
and measurement

Feeding rate experiments were conducted at the Marine
Science Center (MSC), Northeastern University, Nahant,
Massachusetts, and in the biomechanics laboratory at the
University of California, Davis. Colonies of Alcyonium
siderium were collected by SCUBA diving and maintained
in flowing seawater tables or recirculating chilled aquaria.
Feeding observations were made in a recirculating flume
described in Patterson (1984). All experiments were per-
formed with the flow straighteners installed, which re-
moved turbulence of length scales greater than 1 cm. Flow
speeds and turbulence intensities were measured with a
two channel thermistor flowmeter circuit modified from
LaBarbera and Vogel (1976). The voltage output of the
flowmeter was either connected to an eight-bit successive
approximation A/D converter (Mountain Computer)
connected to an Apple He, or to a MacADIOS A/D con-
verter (GW Instruments) connected to an Apple Macin-
tosh Plus. The sampling rate was 10 Hz.

Octocoral colonies attached to horse mussels (Modiolus
modiolus) were collected subtidally from 1 5-23 m depth.
Mussel shell fragments bearing Alcyonium colonies were
mounted in the flow tank working section. The prey of-
fered to the colonies were cysts of the brine shrimp, Ar-
temia salina. Characteristics of the cysts are described in
Patterson (1984). Capture of the cysts on individual ten-
tacles of this species is readily observed. At the end of
each feeding bout, three 60-ml samples were withdrawn
isokinetically using a Cole-Parmer peristaltic pump
(model no. 7568) smoothed with hydraulic capacitors.
Samples were filtered onto gridded Millipore filters, the
number of cysts was counted, and a mean concentration
of particles present in the flow was calculated. The con-
centration of cysts offered (0.056-0.40 part./ml) was of
the order of plankton concentrations seen in the field (Se-
bens and Koehl, 1984). However, even greater concen-
trations may be typical of dense patches of plankton that

are seasonally and spatially abundant (Fasham, 1978;
Grosberg, 1982).

Documenting the time course ofprcv capture

Alcyonium colonies were introduced individually into
the working section of the flume and allowed to acclimate
to the flow. Prey were not introduced until the polyps
were fully expanded. A standard volume concentration
(0.45 g dry cysts/1) of Anemia cysts was added all at once
to the flume. Observations of capture events were made
at a magnification of 35X through a dissecting microscope
suspended over the flume. A watch glass floating on the
water and anchored over the colony prevented blurring
ot the image from capillary waves at the air/water inter-
face. An interval timer program (0.05 s resolution) running
on an Apple He microcomputer measured the time be-
tween capture events. The time required for a tentacle to
transfer a captured particle to the pharynx (JR, [ in the
above model) was timed with a stop watch during separate
experiments.

Filtration time for an individual particle was calculated
using the projected surface area of the organism, and the
flow speed measured 4 cm upstream of the top of the
colony. Specimen volume was measured by volumetric
displacement of water in a graduated cylinder. The num-
ber of prey caught as a function of time was plotted for
each specimen; the observed values for the model param-
eters T- and a were obtained using a least squares algorithm,
and then compared with the values predicted by the model
calculations through linear regression.

Feeding efficiency

Efficiency of prey capture at the colony level was com-
puted as follows: the number of particles caught by a col-
ony during a standard feeding bout of 10 min was divided
by the number of particles that would pass through the
cross-sectional area occupied by the colony if the colony
were not there. This is the standard engineering definition
of efficiency of particle capture (Dorman, 1966). Because
feeding rate at dense concentrations of prey is non-linear
(Fig. 4), efficiency will be a function of time. Hence, for
purposes of comparison, efficiency is computed over the
time necessary to reach "saturation." Saturation is defined
as the point at which capture events drop to less than one
prey item caught per 5 min period per colony.

Field measurements of flow and orientation to flow

Field observations of orientation to flow m Alcyonium
colonies and flow regime were made at the following sites
(depths) in the subtidal of Massachusetts Bay: ( 1 ) Dive
Beach site (8m), located near Nahant, Massachusetts
(42°25'N: 70°54'W), (2) Shag Rocks inner wall (7 m)

M. R PATTERSON

Step response of Alcyonium siderium

Figure 4. Typical feeding response of an Alcyonium colony to a step
increase in the plankton concentration (Anemia cysts). The time axis
(abscissa) is expressed in units of T, a model parameter denned as the
time necessary for the cumulative prey capture to reach (I - e~') = 63%
ol the saturating value. The ordinate. V., is the cumulative number of
prey captured normalized to the saturation level. Note that the response
is curvilinear and can be characterized by two parameters, the time con-
stant, 7, and a, the initial jump in plankton caught as the concentration
changes.

located near Dive Beach, (3) Shag Rocks outer wall (9
m), and (4) Halfway Rock (14m) (42°30'N: 70°46'W).
Orientation to the direction of current flow by colonies
on subtidal rock walls was measured with a protractor
and plumb line. The direction of current flow was deter-
mined with a filament of dye, and was parallel to the
bottom and the wall. Flow measurements were made in
situ at 1.0 cm and 10.0 cm height over Alcyonium colonies
using a submersible thermistor flowmeter recording a dig-
ital signal on magnetic tape. Flow measurements were
made over a three year period in all kinds of weather
throughout the year. The sampling rate was 3 Hz.

Results

Feeding response to plankton patch concentrations

When Alcyonium colonies were subjected to sharp (step)
increases in the plankton concentration, the "filling" curve
was markedly curvilinear and showed an asymptote (see
Fig. 4 for a typical example). Similar results were obtained
with the sea anemone Metridium .v<w/t'(unpub. data). At
these high prey concentrations, doubling the flow speed
and hence the particle flux typically had little effect on
the feeding curve for a given colony (Fig. 5), providing
evidence that the linear model of passive suspension feed-
ing doesn't apply very well in patch concentrations.

Figure 4 gives the graphical interpretation of T and a.
Figure 6 shows how closely the model formulated in the
Introduction predicts T, the time constant, [time needed
to reach (l-e~') of saturation], and a, the proportion of
prey caught as the edge of the patch sweeps past the colony
at the start of feeding bout. Model I linear regression was
used to test the ability of the predicted (calculated) model
parameters to forecast the observed values. This type of

100 150 200 250 300 350 400 450
Time (sec)

Figure 5. Feeding response of the same colony of Alcyonium to Ar-
lemia cysts offered at very different flux rates (flow speed j LI | X particle
concentration |V, ]). This effect is not predicted by the linear model
(Fig. 1 ).

regression analysis is appropriate since the x values (the
computed model parameters) were known precisely and
fixed by the choice of colony (Sokal and Rohlf, 1981).
Alpha values were log transformed before calculations of
the regression to eliminate problems with non-normality.
The aim of the model was to predict feeding behavior in
dense suspensions to within a factor of two. The model
achieves this goal in predicting T and a. Linear regressions
are robs = 6.9+1 .04 In (rpred) and In «obs = - 1 .93 + 0.28
In («pred). R2 values for these regressions for « and T are
0.33 (P = 0.05) and 0.70 (P = 0.0006), respectively.

t - lime constant of colony feeding

4UO

300

' *A

200

*fr

100

0 100 200 300 40

Predicted Value (s)

a - initial patch capture fraction

1.2
1.0
0.8
0.6
0.4
0.2

0.0 0.2 0.4 0.6 0.8 1.0 1.2
Predicted Value (X 10''%)

Figure 6. Predicted and observed values of (A) the time constant, T,
and ( B) the patch edge capture traction. «. for particle nitration by colonies
ofAlcyanium siderium. Predicted values of the model parameters were
computed from the handling time (R,), nitration time (R2), and the
colony volume (C).

MODEL OF PASSIVE SUSPENSION FEEDING

300

•= 200

01234 5 6789 10 11
Particle Flux (particles/Jem 2 • s})

Figure 7. Plol of the time constant of colony tilling, T, as a function
of the particle flux (flow speed JUJ X particle concentration ]V,[). The
data do not have a slope significantly different from zero (P < 0.001)
showing the lack of dependence of?- on particle flux at high flux rates.

Because R, > R2 at the prey concentrations used, and
T = (R, + R:) C, R: will have little effect on T. Thus a
corollary to the model is that particle flux past the colony
for high prey densities will have no correlation with the
time constant (T) or organism filling. This indeed was the
case (Fig. 7). The model has slightly lower success in pre-
dicting the magnitude of «, which measures the degree to
which a colony can "grab" the edge of a plankton patch
as it sweeps by. Alpha is consistently overestimated; it is
probably sensitive to colony shape and the precise patterns
of flow obtained for a particular shape, and these aspects
of passive suspension feeding were not part of the model
formulation.

Colony size and feeding efficiency

Figure 9 demonstrates an inverse relationship between
efficiency of capture (as defined in the Materials and
Methods) and colony size. Smaller colonies are more ef-
ficient filters, although all sizes have very low efficiencies
when feeding in dense concentrations. Figure 10 shows
that there is also an inverse relationship between efficiency
and flow speed, and hence particle flux, for a given particle
concentration, for feeding by colonies.

20-

mean = 105 =
N = 210

0 20 40 60 80 100 120 140 160 180
Colony long-axis orientation (°)

Figure 8. Orientation of the longest dimension ofAlcyonium colonies
to the local direction of current flow at four subtidal sites in Massachusetts
Bay. Angles were measured with protractor and plumb line; current di-
rection was determined with a filament of sodium fluorescein dye.

0 5 10 15 20 25 30 35 40 45

Colony Size, S (cnr* )

Figure 9. Efficiency of particle capture per colony (E) in Alcyonium
as a function of colony size (S). Efficiency is defined as the number of
particles caught by the colony in the time interval to saturation of the
colony divided by the number of particles that would have passed through
the space occupied by the colony. The regression is given by the equation:
E = 12.8 - 0.68S + (9.62 x \0~3)S2(P < 0.05; R2 = 0.55; df = II).

Discussion

The dynamics of cnidarian passive suspension feeding

Most cnidarians use passive suspension feeding, even
though many forms such as scleractinian corals also pos-
sess symbiotic dinoflagellates that supply them with some
large fraction of their nutrition (Muscatine and Porter,
1977). While the independence of zooplankton capture
from autotrophy has been questioned (Clayton and Las-
ker, 1982), there is no doubt that for most boreal cnidar-
ians lacking zooxanthellae, capture of paniculate prey
from the water column is of prime importance in their
biology. Hence, modeling of the passive suspension feed-
ing process is worthwhile because (1) it is ubiquitous in
marine systems, (2) the particles filtered from the water
column are patchy (Wiebe, 1970, 1971; Ortner et a/.,
1984), i.e.. discontinuously distributed in space and time.

S 10
x

£ 4

. 2

"S o-

£ o

5 10 15 20 25 30 35
Flow Speed, U (cm/s)

Figure 10. Efficiency (E) of particle capture per colony in Alcyonium
as a function of flow speed (U). Efficiency is defined as the number of
particles caught by the colony in the time interval to saturation of the
colony divided by the number of particles that would have passed through
the space occupied by the colony. The regression is given by the equation:
E = 32.30U-|"(/) < 0.05; R: = 0.81; df = 11). These flow speeds cor-
respond to a Reynolds number (Re) range of 800-12.000 calculated
using the greatest dimension of each colony.

88 M. R. PATTERSON

Table I

/•'/cM measurement <>l tl<>» v/xr</ / II cm and In II cm above colonies <>/ Alcyonium al lour \uhiulal Mies in Massachusetts Bay
(December 1981-Sepiemher 1984)

Site

Depth (m)

1 .0 cm

10.0cm

Dives

Flow speed (cm/s)

Re x 102

Dives

Row speed (cm/s)

Re x 102

Halfway Rock
Shag Rocks Inner
Shag Rocks Outer
Dive Beach

14
7
9
8

10
18
20
23

18.8(9.8)
8.7(7.2)
10.5(8.5)
9.3(7.1)

90.8 (47.3)
42.0 (34.8)
50.7(41.11
44.9(34.3)

11
18
21
24

18.4(10.6)
10.2(9.7)
11.3(8.2)
9.3(7.6)

88.9(51.2)
49.3 (46.9)
54.6 (39.6)
44.9 (36.7)

Flow was sampled at 3 Hz for 6 minutes per date.

Reynolds number (Re) was calculated for a colony 5 cm in greatest dimension.

Values are mean (standard deviation).

and the feeding response of these organisms to patches
may have important effects on growth and metabolism
(Szmant-Froelich and Pilson, 1984), or competition for
food and space (Okamura. 1984), and (3) any predictive
model must make assumptions that will offer insight into
which features of the system are the most important.

Patchiness of particles is a common phenomenon in
aquatic systems; the causes can be both biological (nutrient
tracking, mass spawning) and abiotic (eddy entrainment)
in origin (reviewed in Okubo. 1980). Passive suspension
feeders respond to patchiness in interesting ways. Leversee
(1976) found that an octocoral could alter its feeding rate
in response to jumps in prey concentration. Crowell( 1957)
observed that a hydroid grew better on a single large daily
ration of food than more frequent feedings, implicating
more efficient digestion in a packed gut (coelenteron).
Lasker el al. (1982) discovered that Hydra interrupts its
feeding to digest its prey; therefore a single large feeding
(dense prey) is just as good as a continuous supply. The
response of Alcyoniitm to patch concentrations is inter-
esting, especially when contrasted with the capture of prey
in the field under more dilute conditions (Sebens and
Koehl, 1984).

Lasker el al. ( 1982) used prey concentrations more than
an order of magnitude greater than those used in this
study. Plankton densities over coral reefs (Alldredge and
King, 1977) and near boreal subtidal rock walls (K. Se-
bens, Northeastern University, pers. comm.) can often be
greater than those in the water column nearby. Densities
of plankton in freshwater aquatic systems are typically
on the order of tens of plankters per liter (Wetzel, 1975);
comparable densities are found in New England inshore
waters (Sebens, 1984). The concentrations used in this
study were on the order of 100/1. By comparison, oligo-
trophic oceanic waters usually have less than one zoo-
plankter per liter (Ortner, et al.. 1981). Feeding on high
densities of prey resulted in a curvilinear feeding response
for the octocoral species investigated in this study. Al-

cyonium did not feed markedly faster at enhanced levels
of prey density, in contrast to previous work with scler-
actinians (Lasker, 1976; Clayton and Lasker, 1982).

Handling time, filtration time, and flow in the field

Part of the error in the predictions of the parameter
values of the model (Fig. 6) can be attributed to the vari-
ation in handling time (R,). The mean handling time for
a particle was 8.0 s (SD = 3.0; n = 20 colonies). It was
not possible to measure handling time and capture events
simultaneously, so the mean handling time was used to
calculate the model parameters. The filtration resistance
(R;), is not subject to as wide a variation within a colony
unless the organism changes its size by pumping water
into its gastrovascular spaces. Size change is usually a re-
sponse to severe hydromechanical stress (cf. Patterson,
1980); these organisms do not appear to regulate feeding
rate through size changes except to turn feeding on and
oft". Robbins and Shick (1980) found similar behavior in
the sea anemone Metridium senile. Colonies did not
change size during the course of these feeding experiments.
During ontogeny, the potential exists for Alcyoniuni col-
onies to reduce the value of the filtration time, R2, by
growing in an oriented fashion to the predominant direc-
tion of flow. This indeed seems the case (Fig. 8).

The flow regime experienced by these colonies over a
three year period at the four sites demonstrates a wide
range of flow speeds (Table I), with mean flows on the
order of 10-20 cm/s. This corresponds to a whole colony
Reynolds number of ca. 5,000 to 10,000. The flow at these
depths is tidally driven and is often dominated by wave-
induced oscillations (Patterson and Sebens, 1989). Col-
onies generally do not feed as velocities approach 50 cm/
s (unpubl. obs.) and instead begin contraction.

The time constant of organism "filling" (T) depends on
colony size, handling time, and filtration time. Handling
time was so much larger than filtration time for the particle

MODEL OF PASSIVE SUSPENSION FEEDING

fluxes tested, that it dominated the time constant. For
example, at the lowest particle flux tested |0.25 part./
(cm2 • s)}. the handling time was three orders of magnitude
greater than the nitration time for a typical colony with
a projected surface area of 10 cm2. At what particle flux
would the handling time and the nitration time become
comparable in magnitude, i.e., at what particle concen-
tration and flow speed would passive suspension feeding
be expected to be responsive to the changes in particle
flux? For the same size colony considered above, the han-
dling time will equal the nitration time at a particle flux
of 0.01 part./(cm2 • s). What are particle fluxes like in the
field?

Comparison with field data: do colonies become more
efficient at lower particle fluxes?

Using the data of Sebens (1984) and Sebens and Koehl
(1984), it is possible to calculate how many particles are
caught by Alcyonium in the field, and make some order
of magnitude calculations of the particle flux they are
experiencing. Knowing the prey caught and the particle
flux, we can calculate efficiency of capture. Particle flux
is the product of prey concentration and flow speed. Flow
speeds have been measured (Sebens, 1984; Table I) and
Sebens (1984) reports plankton concentrations averaging
about 3500 zooplankters/m\ or 3.5 particles/1, in the
warmer months of the year at the Nahant, Massachusetts,
sites. Flow speeds are on the order of about 10-20 cm/s
measured 1.0 cm above the tops of Alcyonium colonies.
The integrated flow over the colony will show a lower
mean value, since the flow speed is reduced as one ap-
proaches the substrate through the logarithmic boundary
layer (Denny, 1988).

Calculations show that a mean particle flux of 0.04
particles/fern2 • s) occurs around these colonies in the field,
not far from the value necessary for equality of the han-
dling time and filtration time for Alcyonium [0.01 parti-
cles/(cnr • s)]. If the mean flow speed is ca. 1 cm/s, the
particle flux will be reduced another order of magnitude.
Now the filtration time will be much greater than the
handling time. Under such conditions, increases in the
flow speed or prey concentration will cause an increase
in the feeding rate, and a quasi-linear response will be
found, similar to that predicted by the linear model! The
model described in the Introduction decomposes to the
"linear" model of passive suspension feeding described
above when the particle flux past the organism is low.
When R2 (filtration time) is large compared to R, (han-
dling time), a becomes almost one. The second term of
Eq. (12) goes to zero; hence prey in the water becomes
prey in the organism. In essence, as the particle flux be-
comes lower (through slower flow or lower prey concen-
trations), the model predicts instantaneous step responses

(capture) of single plankters or 100% efficiency. Are field
data on feeding consistent with this prediction of high
feeding efficiency?

Sebens and Koehl (1984) sampled gut contents of the
sea anemone, Melridium and Alcyonium over a diel cycle.
Using Sebens (1984), the plankton concentration for the
site averages about 4 plankters/1 during the warmer
months. Assume the prey inside the organisms were
caught during the previous two hours as per Sebens and
Koehl ( 1 984). Their data give a mean number of prey per
colony of Alcyonium (n = 90). Assume each Alcyonium
colony had a projected surface area normal to the flow
capable of capturing prey of 10 cm2. Given the above
plankton density, and an efficiency of 100%, a current of
2. 1 cm/s would be needed to account for the gut contents.
This flow speed is within the typical range of speeds seen
above these organisms (Table I). Of course, these calcu-
lations are crude estimates because ( 1 ) different sizes and
types of plankton are lumped in particle counts, and (2)
both species prefer certain types of plankton over others.
But high efficiencies for prey capture in Alcyonium seem
reasonable for field values of flow and prey concentration
(non-patch conditions).

Efficiencies measured under very high particle concen-
trations in the flume were an order of magnitude lower
than these field estimates. This dichotomy is predicted hy
the model: at very high plankton concentrations, feeding
becomes uncoupled from particle flux; under field con-
ditions, efficiencies skyrocket, presumably due to the lower
particle flux and hence favorable (R:/R,) ratio. Why
couldn't these feeding experiments be repeated in the lab-
oratory using particle fluxes representative of non-patch
concentrations? In the flume, feeding was studied at high
concentrations over short period of time for two reasons:
( 1 ) concentration and hence particle flux remained con-
stant in the flume only over a period of 30 min; after that
time, gravitational settlement significantly affects con-
centration, and (2) at realistic concentrations, capture
events are on the order of minutes to large fractions of
an hour apart, and would be tedious to document, even
if concentration could be kept constant. Using SCUBA,
I did spend several hours observing colonies of Alcyonium
feeding in situ at the four sites sampled for flow speed.
Because the particles on which they feed are only a few
hundred micra in length, this requires approaching within
30 cm of the colony to observe capture events; this nec-
essarily alters the flow around the colony. Only rarely in
the field did I see "rapid" capture of prey at a rate com-
parable to that seen in the flume (seconds between cap-
tures); during these rare events there was an easily dis-
cerned "cloud" of copepods near the colonies. However,
most of the time, the interval between prey capture events
(visible particle adhesion followed by movement of the
tentacle towards the pharynx) was several minutes in

M R PATTERSON

length, with occasional mind (and body) numbing pauses
of up to 10 min between capture events.

An examination of the data of Sebens and Koehl (1984)
shows that even under the best conditions, the interval
between capture events must be over a minute for Al-
cyonium and 2 min for Metridlum. Barange and Gili
( 1988) sampled the coelenteron contents of a benthic hy-
droid over a diel cycle. From their data [mean prey items
captured per (polyp • day), number of polyps per colony],
I calculated the average interval between capture events
to be about 1.3 min. Thus, passive suspension feeding for
these organisms is a slow process for non-patch concen-
trations of prey. Cnidarian colonies snag particles slowly
from the water when a patch isn't around, unlike some
vertebrate suspension feeders that capture enormous
quantities of particles in the same period of time (Sand-
erson and Wassersug, 1990).

Saturation of colonies remains a [muling phenomenon

The utility of this model is that it points out some new
directions for work with passive suspension feeding cni-
darians. An unanswered question is why are these filters
not adapted for high efficiency nitration under high par-
ticle fluxes? Is there a biological constraint on the system
that limits feeding? Constraints found in other suspension
feeding systems include saturation of the filter (Parker,
1975; Real, 1977) or gut-filling (Doyle, 1979). Neither of
these constraints appears likely for this species. Alcyonium
colonies began slowing their feeding rate long before most
polyps had successfully fed once. They are also capable
of packing many prey items into a single polyp (Patterson,
1984). Lasker el al. (1982) showed that in single-polyped
Hydra, the ingestion of prey was controlled by previous
feeding events, i.e.. prey captured later in a feeding bout
were less likely to be ingested than prey caught near the
beginning. Burnett el al. ( 1 960) and Hand ( 1 96 1 ) showed
that nematocyst discharge in Hydra is inhibited by food
in the gastrovascular cavity, and Lasker et al. ( 1 982) spec-
ulate that this may be important in limiting ingestion rate.
But Ruch and Cook (1984) have demonstrated inacti-
vation of nematocyst discharge even in the absence of
food in the gut. This startling observation was explored
further by Clark and Cook (1986) using a colonial hydroid.
They provide evidence from lab feeding experiments that
the accumulation of discharge products from the stenotele
nematocysts used by this hydroid in prey capture is suf-
ficient to inhibit further feeding, and that it is not necessary
to invoke waste product accumulation from digestion, or
depletion of nematocysts, to explain the phenomenon.
For those cnidarians exhibiting this interesting feedback,
the second assumption of the model (see Introduction)
could easily be reformulated to incorporate a term spec-
ifying the diffusion time of the nematocyst discharge

products. It is unknown whether nematocyst discharge
products affect Alcyonium in a similar fashion.

The nerve net is also probably involved in the process
of modulating prey capture in cnidarians (McFarlane,
1978). Deformation of the tentacle by repeated particle
impactions may be important in producing inhibition of
nematocyst discharge during feeding in plankton patches.
On a larger scale, flow induced deformation of the entire
colony may be important in regulating the rate process
of prey capture. Best ( 1988) found that feeding rate in a
sea pen, Ptilosarcus, increased then decreased with flow
speed and attributed this behavior to changes in volume
flow rate that occurred as the filtering surfaces changed
their orientation. A similar phenomenon was noted in a
crinoid (Leonard et al.. 1988).

Some experiments that would help solve the mystery
of why colonies saturate long before all filtering units
(polyps) have fed would include ( 1 ) stealing particles from
the tentacles after capture but before transfer to the mouth,
while monitoring frequency of capture and attempted
ingestion events, (2) eliciting repeated nematocyst dis-
charge by micromolar diffusion clouds of amino acids
from a micropipette near tentacle tips or mechanical
stimulation of tentacles while the cnidarian colony is si-
multaneously feeding, (3) separating a cnidarian colony
into two halves except for a strip of tissue and examining
feeding rates in the two halves before and after the con-
nection is severed [Clark and Cook ( 1 986) found no effect
for a hydroid], and (4) offering digestible and non-digest-
ible prey to a species that will ingest both types of particles
(cf. Lasker ct al.. 1983) and measuring feeding rates on
both types of particles separately and together while ne-
matocyst discharge products are monitored.

It is very intriguing that this colonial octocoral saturates
after a few minutes of feeding in high prey densities at
about the same number of prey that would be caught over
a 2-4 h period in the field (Sebens and Koehl, 1984).
Digestion of prey items renders them unidentifiable after
4-6 h (Sebens and Koehl, 1984). Have these colonial sus-
pension feeders evolved to "charge their capacitance" on
a time scale of approximately two hours because they are
limited by the activity of their digestive enzymes? For
boreal cnidarians in the Atlantic, the strongest tidal flows
will be obtained for a 2-4 h period between slack tides.
Because plankton patches are the exception rather than
the rule, the feeding response may have evolved to cope
with sparse prey moving past the colony over a 2-4 h
period. During periods of flow dominated by wave-driven
oscillations, e.g., slack tides, colonies will re-filter water
already low in prey. Feeding in a bi-directional flow can
actually increase feeding success in a hydroid exposed to
high (patch) concentration of prey (Hunter, 1989). At
present, it is unknown for this species whether feeding

MODEL OF PASSIVE SUSPENSION FEEDING

effectiveness is higher in bi-directional flow at low prey
concentrations.

Application of this model to other passive suspension
feeders will test its generality and provide evidence for
whether the dichotomy in feeding behavior characteristic
of this species when feeding in low and high prey con-
centrations is a widespread phenomenon. Future devel-
opments in the measurement and description of plankton
patchiness on a small scale in nearshore waters (Pieper
and Holliday, 1985) and description of the benthic
boundary layer in which these organisms live (Jumars
and Nowell, 1984) will improve our ability to model and
understand this fascinating process.

Acknowledgments

Discussions with P. Basser, B. Best, R. Etter, T. Givnish,
R. Grosberg, S. Kleinhaus, W. McFarland, T. McMahon,
R. Olson, S. L. Sanderson, K. Sebens, R. Turner, the
HUMP seminar group, and the Aquatic Sciences group
at M. I. T., have improved the paper greatly. P. Rudy,
Acting Director, Marine Science Center, Northeastern
University. Nahant, Massachusetts, kindly provided lab-
oratory space. C. Alexander and B. Otteson provided
technical assistance. Financial support for this project was
provided by the Richmond Fund of Harvard University,
the Lerner-Gray Fund for Marine Research of the Amer-
ican Museum of Natural History, NSF OCE-8308958 to
K. Sebens of Harvard University, a Faculty Research
Award from UC Davis, and NSF OCE-87 16427 to the
author.

Literature Cited

Alldredge, A. A., and J. M. King. 1977. Distribution, abundance, and

substrate preferences of demersal reef zooplankton at Lizard Is., Great

Barrier Reef. Mar. Biol. 41: 317-334.
Barange, M., and J. M. Gili, 1988. Feeding cycles and prey capture

in Eudendniim racemosum (Cavolini, 1785). J. Exp. Mar. Biol. Ecol.

115: 281-293.
Best, B. A. 1988. Passive suspension feeding in a sea pen: effects of

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

175: 332-342.
Burnett, A. L., T. Lenlz, and M. Warren. 1960. The question of control

of the nematocyst discharge reaction by fully fed hydra. Ann. Soc..

Roy Zoo/ Big. 90: 247-268.
Clark, S. D., and C. B. Cook. 1986. Inhibition of nematocyst discharge

during feeding in the colonial hydroid Halocordy/e disticha

(=Pennaria tiarella): the role of previous prey killing. Bin!. Bull 171:

405-416.
Clayton. VV. S., Jr., and H. R. Lasker, 1982. Effects of light and dark

treatments on feeding by the reef coral Pocilloporu damicornts (Lin-
naeus). J E\p. Mar Biol. Ecol. 63: 269-279.
Coughlan, J. 1969. The estimation of filtering rate from the clearance

of suspensions. Mar. Biol. 2: 356-358.
Crowell, S. 1957. Differential responses of growth zones to nutritive

level, age and temperature in the colonial hydroid, Campanularia.

J Exp. Zoo/. 134: 63-90.

Denny, M. 1988. Biology ami I he Mechanics of I he Wave-Swept En-
vironment. Princeton University Press, Princeton.
Dorman, R. G. 1966. Filtration. Pp. 195-222 in Aerosol Science.

C. N. Davies, ed. Academic Press, New York.

Doyle, R. \V. 1979. Ingestion rate of a selective deposit feeder in a
complex mixture of particles: testing the energy-optimization hy-
pothesis. Limnol. Oceanogr. 24: 867-874.
Emlen, J. M. 1973. Ecology .In Evolutionary Approach. Addison-

Wesley, Reading. Massachusetts.
Kasham, M. J. R. 1978. The statistical and mathematical analysis of

plankton patchiness. Oceanogr. Mar. Biol. Ann. Rev. 16: 43-79.
Fiala-Medioni, A. 1973. Ethologie alimentaire d'invertebres benthiques
filtreurs (ascidies). I. Dispotif experimental. Taux de filtration et de
digestion chez Phallusni mammillata Mar. Biol. 23: 137-145.
Fiala-Medioni, A. I978a. Filter feeding ethology of benthic invertebrates
(ascidians). III. Recording of water current in stilt-rate and rhythm
of pumping. Mar Biol 45: 185-140.

Fiala-Medioni, A. I978b. Filter feeding ethology of benthic inverte-
brates (ascidians). IV. Pumping rate, filtration rate, filtration efficiency.
Mar. Biol. 48: 243-249.

Fiala-Medioni, A. 1978c. Filter feeding ethology of benthic invertebrates
(ascidians). V. Influence of temperature on pumping, filtration and
digestion rates and rhythms in Phallusia mammillata. Mar Biol 48:
251-259.

Fiala-Medioni, A. 1978d. A scanning electron microscope study of the
branchial sac of benthic filter-feeding invertebrates (ascidians). Ada
Zoo/. (Stockholm) 59: 1-10.

Grosberg, R. K. 1982. Intertidal zonation of barnacles: the influence
of planktonic zonation of larvae on vertical distribution of adults.
Ecology 63(4): 894-899.

Hand, C. 1961. Present state of nematocyst research types, structure,
and function. Pp. 187-202 in The Biology of Hydra. H. M. LenhofT
and W. F. Loomis, eds. Univ. of Miami Press, Miami.
1 lolling, C. S. 1965. The functional response of predators to prey density
and its role in mimicry and population regulation. Mem Enlontol
Soc. Canada No. 45, 60 pp.

Hunter, T. 1989. Suspension feeding in oscillating flow: the effect of
colony morphology and flow regime on plankton capture by the hy-
droid Obelta longissima Biol Bull 176: 41-49.
Jergensen, C. B. 1943. On the water transport through the gills of

bivalves. Ada Physiol. Scam/ 5: 277-304.
Jergensen, C. B. 1966. Biology o/ Suspension Feeding. Pergamon Press,

Oxford.
Jorgensen, C. B. 1975. On gill function in the mussel Mvtilus edulix

L. Ophelia 13: 187-232.

Jumars, P. A., and A. R. M. Nowell. 1984. Fluid and sediment dynamic
effects on marine benthic community structure. Am. Zool. 24: 45-
55.
La Bar her a. M., and S. Vogel. 1976. An inexpensive thermistor flowmeter

for aquatic biology. Limnol. Oceanogr. 21: 750-756.
Lam, R. K., and B. VV. Frost. 1976. Model of copepod filtering response
to changes in size and concentration of food. Limnol. Oceanogr 21:
490-500.

Lasker, H. R. 1976. Intraspecific variability of zooplankton feeding in
the hermatypic coral Montastrea cavernosa. Pp. 101-109 in Coel-
en/erate Ecology and Behavior. C. O. Mackie, ed. Plenum, New York.
Lasker, H. R., J. A. Syron, and W. S. Clayton, Jr. 1982. The feeding
response of Hydra vindis effects of prey density on capture rates.
Biol. Bull 162: 290-298.

Lasker, H. R., M. D. Gottfried, and M. A. Coffroth. 1983. Effects of
depth on the feeding capabilities of two octocorals. Mar. Biol. 73:
73-78.

Lehman, J. T. 1976. The filter-feeder as an optimal forager, and the
predicted shapes of feeding curves. Limnol. Oceanogr. 21 : 50 1 -5 1 6.

M. R. PATTERSON

Leonard, A. B., J. R. Strickler, and N. D. Holland. 1988. Effects of
current speed on filtration during suspension feeding in Oligometra
serripinna (Echinodermala: Crinoidea). Mar. Biol. 97: 1 1 1-122.

Leversee, G. J. 1976. Flow and feeding in fan-shaped colonies of a
gorgonian coral. l.eptogorgia. Bio/. Bull 151: 344-356.

McFarlane, I. D. 1978. Multiple conducting systems and the control
of behaviour in the brain coral Mcandnna meandriles (L.). Proc.
Roy. Sac. Lond B. 200: 193-216.

MiKinii. J. II. 1966. Biological Control Systems Analysis. McGraw-
Hill, New York.

Mohlenberg, F., and H. U. Riisgard. 1978. Efficiency of particle re-
tention in 13 species of suspension feeding bivalves. Ophelia 17: 239-
246.

Muscatine. L., and J. W. Porter. 1977. Reef corals: mutualistic sym-
bioses adapted to nutrient-poor environments. BioScience 27(7): 454-
460.

Okamura, B. 1984. The effects of ambient flow velocity, colony size,
and upstream colonies on the feeding sucess of Bryozoa., I. Buguhi
slolonifera Ryland, an arborescent species. / E.\p. Mar. Biol. Ecol.
83: 179-193.

Okubo, A. 1980. Diffusion and Ecological Problems: Mathematical
Models. (Biomathematics, Vol. 10). Springer Verlag, New York.

Ortner, P. B., L. C. Hill, and H. E. Edgerton. 1981. In-sitii silhouette
photography of Gulf Stream zooplankton. Deep-Sea Res. 28A(12):
1569-1576.

Ortner. P. B., R. L. Ferguson, S. R. Piotrowicz, L. Chesal, G. Berberian,
and A. V. Palumbo. 1984. Biological consequences of hydrographic
and atmospheric advection within the Gulf Loop Intrusion. Deep-
Sea Res. 31(9): 1 101-1 120.

Palmer, R., and L. Williams. 1980. Effect of particle concentration on
filtration efficiency of the Bay Scallop Argopeclen irradians and the
oyster Crassoslrea virginica. Ophelia 19: 163-174.

Parker, M. 1975. Similarities between the uptake of nutrients and the
ingestion of prey. \'crh. Internal, herein. Limnol 19: 56-59.

Patterson, M. R. 1980. Hydromechanical adaptations in Alcyonium
sideriinn (Octocorallia). Pp. 183-201 in Biojluid Mechanics 2, D. J.
Schneck. ed. Plenum, New York.

Patterson, M. R. 1984. Patterns of whole colony prey capture in the
octocoral, Alcyonium sidenum. Biol. Bull. 167: 613-629.

Patterson, M. R., and K. P. Sebens. 1989. Forced convection modulates
gas exchange in cnidarians. Proc. Nat. Acad. Sci. (USA) 86: 8833-
8836.

Pieper, R. E., and D. V. Holliday. 1985. Acoustic measurements of
zooplankton distributions in the sea. J. Cons. Cons. Int. Explor. Mer
41(3): 226-238.

Real, L. A. 1977. The kinetics of functional response. Am. Nalur 111:
284-300.

Robbins, R. E., and J. M. Snick, 1980. Expansion-contraction behavior
in the sea anemone Meiridium senile: environmental cues and ener-
getic consequences. Pp. 101-1 16 in Nutrition in the Lower Metazoa,
D. C. Smith and Y. Tiffon. eds. Pergamon, New York.

Rubenstein, D. J.. and M. A. R. Koehl. 1977. The mechanism of filter
feeding: some theoretical considerations. Am. Nalur. Ill: 981-994.

Ruch, R. J., and C. B. Cook. 1984. Nematocyst inactivation during
feeding in Hydra liltoralis J E.\p. Biol. Ill: 31-42.

Sanderson, S. L., and R. Wassersug. 1990. Suspension-feeding verte-
brates. Sci. Am. 262: 96-101.

Seale, D. B. 1982. Obligate and facultative suspension feeding in anuran
larvae: feeding regulation in Xenoptis and Rana. Biol Bull 162: 214-
231.

Sebens, K. P. 1979. The energetics of asexual reproduction and colony
formation in benthic marine invertebrates. Am. Zoo/. 19: 683-697.

Sebens, K. P. 1984. Water flow and coral colony size: interhabitat
comparisons of the octocoral Alcyonium sidenum. Proc Natl. Aead.
Sci (USA) 81: 5473-5477.

Sebens, K. P., and M. A. R. Koehl. 1984. Predation on zooplankton
by the benthic anthozoans, Alcyonium sidenum (Alcyonacea) and
Metridiwn senile (Actiniaria) in the New England subtidal. Mar.
Biol 81:255-271.

Sebens, K. P. 1986. Spatial relationships among encrusting manne
organisms in the New England subtidal zone. Ecol. Monogr. 56: 73-
96.

Sokal, R. R., and F. J. Rohlf. 1981. Biometry. W. H. Freeman, San
Francisco.

Szmant-Froelich, A., and M. E. Q. Pilson. 1984. Effects of feeding
frequency and symbiosis with zooxanthellae on nitrogen metabolism
and respiration of the coral Astrangia danae. Alar. Biol. 81: 153-
162.

Townsend, C. R., and R. N. Hughes. 1981. Maximizing net energy
returns from foraging. Pp. 86-108 in Physiological Ecology: An Evo-
lutionary Approach. C. R. Townsend and P. Calow. eds. Sinauer
Associates. Sunderland, Massachusetts.

Wetzel, R. G. 1975. Limnology W. B. Saunders. Philadelphia.

Wiebe, P. H. 1970. Small-scale spatial distribution in oceanic zoo-
plankton. Limnol. Oceanogr. 15: 205-217.

Wiebe, P. H. 1971. A computer model study of zooplankton patchiness
and its effects on sampling error. Limnol. Oceanogr. 16: 29-38.

Williams, L. G. 1982. Mathematical analysis of the effects of particle
retention efficiency on determination of filtration rate. Mar. Biol.
66: 171-177.

Reference: Bwl Bull 180: 93-102. (February, 1991)

The Effects of Flow on Polyp-Level Prey Capture in an
Octocoral, Alcyonium siderium

MARK R. PATTERSON

Division of Environmental Studies, University of California, Davis. California 95616

Abstract. Particle capture by individual polyps and ten-
tacles of the octocoral, Alcyonium siderium, was investi-
gated in flows of different speed and turbulence intensity.
In low flow (Umean = 2.1 cm/s; u' = 1.2 cm/s. where u' is
the root mean square of the fluctuations from Umean), ten-
tacles on the upstream side of a polyp capture the most
prey. In intermediate flow (Umean = 12.2 cm/s; u' = 6.0
cm/s). downstream tentacles within a polyp catch the most
prey. In high flow (Umcan = 19.8 cm/s; u' = 4.0 cm/s),
polyps are bent downstream, eddies form over the ten-
tacular surfaces, and the capture distribution over tentacles
becomes radially symmetric. At all flow speeds tested,
particles are caught with increasing frequency nearer the
tip of the tentacle relative to locations near the pharynx.
At the highest flow speed tested, no particles are caught
on the segment of each tentacle closest to the pharynx.
The per polyp capture efficiency is low and drops mark-
edly with increasing Reynolds number. The capture
mechanism for this species appears to be direct intercep-
tion; inertial impaction is shown to be unimportant. Flow
modulation of particle capture by polyps is probably a
general phenomenon among octocorals.

Introduction

The application of modern engineering theory to the
analysis of particle capture by suspension feeding organ-
isms began with a review of the engineering and fluid
mechanics literature by Rubenstein and Koehl (1977).
The medical community has long been interested in the
related problem of how particles are deposited in the tra-
cheobronchial tree of mammalian lungs (Findeisen, 1935;
Landahl, 1950, 1963). The theory invoked was the same
(McMahon el a/.. 1977), and importance of flow pattern
in affecting the location of particle capture inside lungs

Received 30 August 1989; accepted 6 November 1990.

was recognized (Bell, 1974). Particle capture mechanisms
used by biological niters include direct interception (the
particle comes within one particle radius of a filtering sur-
face), inertial impaction (the particle contacts the filter
because particle inertia causes it to deviate from a fluid
streamline around the filter), diffusive deposition (both
Brownian and eddy-enhanced — the motion of the particle
across streamlines leads to contact with the filter), sieving
(the particle is too large to pass through gaps in the filter
geometry), electrostatic attraction (requires net surface
charges of opposite sign on filter and particle), and grav-
itational deposition (a particle denser than the fluid sinks
and contacts the filter). Aerosol filtration theory allows
estimation of the relative importance of these mechanisms
by calculating the ratios of relevant forces acting on par-
ticles of different charge, size, and density, in flows of
different velocity and viscosity (LaBarbera, 1984).

Recent work in organisms as diverse as conifers (Niklas,
1982a.b), dipteran larvae (Ross and Craig, 1980), poly-
chaetes (Taghon el a/.. 1980; Merz, 1984), veliger larvae
of mollusks (Strathmann and Liese, 1979), ophiuroids
(LaBarbera, 1978), bryozoans (Okamura, 1984, 1987),
hydroids (Harvell and LaBarbera, 1985; Hunter, 1989),
and crinoids (Holland el al. 1987) has extended our
knowledge of how biological filters work and how flow
speed can affect the geometric pattern of particle capture
at the level of the colony or individual. An important
generalization from these works is that the coupling be-
tween an organism's filter morphology and the resulting
flow field, whether generated actively or passively, is ex-
tremely important in the particle capture process.

Few studies have examined the mechanics of zoo-
plankton capture by passive suspension feeding cnidarians
at the level of the filtering elements themselves, the polyps
composed of tentacles. Lewis and Price (1976) investigated
mucus feeding in corals. Hunter (1989) demonstrated that
feeding effectiveness of hydroid polyps was greater in os-

M. R. PATTERSON

dilatory flow compared to steady, unidirectional flow.
Harvell and LaBarbera (1985) examined how flexibility
affects local flow speeds over polyps in hydroid colonies.
Best (1988) has investigated prey capture in the sea pen,
Ptilosarcus gumeyi, a species that has the polyps arranged
at the ends of cantilevered support tissue through which
flowing seawater passes. She found that the deformability
of the organism strongly affected nitration efficiency and
volume of water filtered; in effect the organism could tune
its feeding performance by maintaining a variable porosity
filter.

While some workers have investigated the location of
prey capture on the surface of cnidarian colonies (Lev-
ersee, 1976; Lasker, 1981; Patterson, 1984), no studies
have addressed the location of prey capture within indi-
vidual polyps or tentacles. Alcyonium siderium Verrill is
a planktivorous colonial octocoral common on hard rock
substrate in the New England subtidal (Sebens, 1986). Its
diet consists largely of small zooplankton; invertebrate
eggs, foraminiferans, ascidian larvae, nematodes, harpac-
ticoid and calanoid copepods, barnacle nauplii and cy-
prids, ostracods. and crustacean fragments were common,
although algal material is often present (Sebens and Koehl,
1984). It readily captures and eats live zooplankton and
Artemia cysts in a laboratory flume (Patterson, 1984). Prey
capture events by individual tentacles are easily observed
by the naked eye. The subtidal habitats it occupies can
differ greatly in water motion (Sebens, 1984; Patterson,
1985), and there is a change in colony morphology of
local populations that is correlated with flow regime, with
finger-like colonies found in slower flow areas, and lobate
ellipsoids and roughly spherical forms found in higher
flows (Patterson, 1980). The present study tested whether
the location of prey capture within individual polyps and
tentacles of the boreal octocoral, Alcyonium siderium. is
affected by flow patterns over the colony.

Materials and Methods

Colony collection, maintenance, and flow generation
and measurement

Polyp feeding experiments were conducted at the Ma-
rine Science Center (MSC), Northeastern University, Na-
hant, Massachusetts, and at the University of California,
Davis. Colonies of A. siderium were collected and main-
tained in flowing seawater tables or recirculating chilled
aquaria. A recirculating Plexiglas flume (98 liters; 15 cm
X 15 cm working section; 1.9 m long) patterned after a
design published by Vogel and LaBarbera (1978) and
equipped with a chiller (Aquanetics) enabled colonies to
feed in flows of various speeds and turbulence intensities
at 15°C. Patterson (1984) gives a quantitative description
of flow in this particular flume. All experiments were per-
formed with the flow straighteners removed, resulting in

higher turbulence in the flow tank and a closer approxi-
mation to the flow seen over the colonies in the field (Pat-
terson and Sebens, 1989). Seawater for the flume was ob-
tained from the MSC seawater system and was filtered
twice (sand, cotton mesh) to remove particles greater than
20 jim diameter or was made fresh using InstantOcean,
and adjusted to a salinity of 34%o.

Flow speeds and turbulence intensities were measured
with a two channel thermistor flowmeter circuit modified
from LaBarbera and Vogel (1976). The frequency re-
sponse of the probe plus circuit is 5 Hz at —6 dB down
from maximum response. The turbulence intensities may
underestimate the true turbulence in the flume, if there
is significant energy at frequencies above 5 Hz; this aspect
of flume flow regime was not evaluated. The velocity signal
was converted into an FM signal for transmission over a
distance of several meters to a frequency-to-voltage (f/v)
converter. The output of the f/v converter was sent to a
signal conditioner (custom-made) and eight bit successive
approximation A/D converter (Mountain Computer)
connected to an Apple He microcomputer. The sampling
rate was 10 Hz.

Octocoral colonies attached to mussels (Modiolus mo-
diolus) were collected subtidally. Mussel shell fragments
bearing Alcyonium were mounted securely in the flow
tank working section. The prey offered to the colonies
were cysts of the brine shrimp, Artemia. Characteristics
of the cysts are described in Patterson (1984). The cysts
are about the same size as the mean prey size taken by
Alcyonium in the field (Sebens and Koehl, 1984).

Location oj prey capture

The spatial location of prey capture on individual po-
lyps of Alcyonium was studied in flows of high turbulence
levels and differing mean flow speeds. Colonies used
ranged from 2 to 8 cm in greatest dimension when ex-
panded. Colonies were all roughly spherical. Prior to an
observation period, a single colony was introduced to the
flow tank and allowed to acclimate to the flow regime
and expand its polyps. A standard volume of seawater
(11) and weight of hydrated cysts (0.45 g) were added to
the flow tank at the beginning of the observation period.
The flow 1 cm above the observed polyp was adjusted to
have a mean value of 2.7, 12.2, or 19.8 cm/s (4 s average
achieved through an electronic integrator) by adjusting
the speed control on the flume motor. Five minutes into
the experiment, three 60-ml samples were withdrawn iso-
kinetically (Brodkey and Hershey, 1988) using a Cole-
Parmer peristaltic pump (model no. 7568) smoothed with
hydraulic capacitors. Each sample was filtered onto grid-
ded Millipore filters, the cysts counted, and the mean
concentration of particles calculated. The range of particle
concentrations encountered by the colony among exper-
iments ranged from 0.13 to 0.53 cysts/ml.

POLYP-LEVEL PREY CAPTURE EVENTS

Figure 1A shows a typical top view of a polyp after a
feeding bout and the coordinate system used to assess
location of capture around the polyp's circumference.
Note that the coordinate system for the top projections
paired tentacles from the bilaterally symmetric halves of
the polyp (Fig. 1A). The coordinate variable (x) used for
describing capture along individual tentacles is shown in
Figure IB; note that the distance along the tentacle (x) is
normalized to the tentacle length (L). Capture events on
individual polyps were observed at a magnification of 35 X
through a dissecting microscope suspended over the flume.
A watch glass floating on the water and anchored over
the colony prevented blurring of the image from capillary
waves at the air/water interface. As a feeding bout pro-
gressed, composite maps of capture sites of cysts from
individual polyps were made (Fig. 1A). All polyps cen-
sused were located near the top of the colony; polyps cho-
sen for observation had their tentacles oriented to the flow
(Fig. 1A).

An ocular micrometer permitted measurement of rel-
ative position of capture along the length of a tentacle.
Only particles caught on the oral side of the tentacles were
noted; particles were very rarely captured on the aboral
side of the tentacles. The tentacle capture maps were di-
vided into five non-dimensional length sectors. Particle
counts in the tentacle length sectors were normalized
within a tentacle to the projected area available for particle
capture. Surface areas of polyps were computed using a
camera lucida and an interactive digitizing tablet (Apple
Graphics Tablet). The area of the pinnules was not mea-
sured in calculating areas available for capture. Projected
surface area of the entire colony was measured similarly.

Feeding efficiency

A dimensionless measure of feeding effectiveness, the
efficiency of prey capture at the polyp level was computed
as follows: the number of particles caught per polyp during
a standard feeding bout was divided by the number of
particles passing through the cross-sectional area occupied
by the colony (divided by the number of polyps), if the
colony were not there. This result is the standard definition
of filtration efficiency from engineering theory (Dorman,
1966). The number passing through the cross-sectional
area was calculated by integrating the flow over the height
and width of the colony and multiplying by the sampled
particle concentration. Feeding rate in the dense concen-
trations of prey used in the flume is not constant, but
decreases with time (Patterson, 1991). These concentra-
tions were similar to zooplankton patch concentrations
in the field. Hence, efficiency is a function of time. For
purposes of comparison, efficiency was computed over
the time necessary to reach saturation. Saturation is de-
fined as the point at which capture events drop to less
than one prey item caught per 5-min period per colony.

Results

Prey capture around the periphery of a polyp

Figure 1C shows the distribution of particle capture
around the polyps on the different tentacles as one moves
in the downstream direction; the histograms have been
adjusted for the amount of surface area available for prey
capture. At low flow speeds (Umean = 2.7 cm/s), prey cap-
ture is remarkably asymmetric, with upstream tentacles
capturing the most prey [Kolmogorov-Smirnov (K-S) test;
P < 0.001 ; df = 207]. At intermediate flow speeds (Umean
= 12.2 cm/s), the distribution is again asymmetric, but
in the opposite direction, with downstream tentacles fa-
vored in particle capture (K-S test; P < 0.001; df = 205).
At the highest flow speed tested (Umean = 19.8 cm/s), the
distribution is indistinguishable from an even distribution
of prey capture around the tentacles (K-S test; P > 0.5;
df = 70). The distribution of capture events is thus mark-
edly affected by flow speed. Only at the highest speeds
can capture be modeled by a Poisson process.

Prey capture by tentacles

When prey capture events are examined over the length
of individual tentacles, an additional pattern emerges (Fig.
2). At low speeds (Umcan = 2.7 cm/s), the distribution is
significantly asymmetric, with the outer segments of the
tentacles capturing the most prey (K-S test; P < 0.001; df
= 207). The same pattern is found at intermediate flow
speeds (Umcan = 12.2 cm/s), with the asymmetry shifted
even further in the direction away from the pharynx (K-
S test; P < 0.00 1 ; df = 205). Finally, at the highest speed
used in the flume (Umt.an = 19.8 cm/s), the distribution is
still asymmetric with a bias toward the tentacle tips (K-S
test; P < 0.001 ; df = 70), but with some differences. Cap-
ture in the innermost 20% of the tentacle's length has
disappeared, and the outermost 20% experiences much
more variable prey capture success.

Feeding efficiency

When the efficiency of filtration for individual polyps
is examined with respect to Reynolds number (Re) com-
puted for flow around a polyp, a significant inverse rela-
tionship is found (P < 0.05; df =11), and the efficiency
is remarkably low at all speeds tested for feeding in dense
concentrations of prey (Fig. 3). Re = Umeand/i', where d
= polyp oral disk diameter, and v = kinematic viscosity
of seawater. Because polyp size is relatively constant. Re
can be viewed as a dimensionless flow speed.

Discussion

Mechanisms of particle impact ion

Rubenstein and Koehl (1977) presented dimensionless
indices for various mechanisms of particle capture by sus-

M. R. PATTERSON

flow

45
40-
35
30
25-
20-
15-
JO-
S'
0

Umean=2-7cm/s

n = 207

Umean=12-2cm/s

L
x

Hnean = 19'8

Upstream Tentacles — > Downstream Tentacles

Figure 1. Coordinate system used to quantify prey capture in individual polyps in colonies ofAliyonium.
(A) Quantification in the circumferential direction around the polyp. Note the pairing of tentacles from the
bilaterally symmetric halves of the polyp, ;.t'.. prey caught on tentacles with the same identification number
were paired. The five concentric rings delineate the dimensionless length coordinate used for assessing capture
by individual tentacles. (B) The distance from the pharynx toward the tentacle tip (X) is divided by the
overall length of the tentacle (L) to generate the dimensionless distance (X/L) used as the independent
variable (abscissa) in Figure 2. (C) Circumferential position of prey capture events in individual polyps of
Alcyonium at three different flow speeds. Data from the bilateral halves of the colony are pooled; see Figure
1 A for interpretation of abscissa. Capture frequencies are normalized relative to the amount of tentacle area
assigned to the numbers one through five. Data were arc-sine transformed and then back-transformed for
graphical portrayal. Vertical bars are 95% confidence intervals. For flow speeds of 2.7, 12.2. and 19.8 cm/
s, the total numbers of cysts caught were 207, 205, and 70. respectively.

pension feeding organisms. Table I gives these values for
polyps of Alcyonium feeding on Anemia cysts. Note that
direct interception or "geometric" interception (Chang,
1973) has the highest value and is thus most likely to be
the dominant mechanism of particle capture. For a rigid
filter, the efficiency of capture by direct interception
should be independent of flow speed (Fuchs, 1964): this

is contrary to the pattern observed. A formulation of the
model more appropriate to aqueous suspensions invokes
the importance of short-range (London- van der Waals)
forces and does not ignore the hydrodynamic resistance
that occurs as the particle squeezes fluid from the space
between itself and the filter element (Chang, 1973; La-
Barbera, 1984). This more refined theory predicts an in-

POLYP-LEVEL PREY CAPTURE EVENTS

Umean = 2-7 cm/s

60 •
50 '
40 •
1 30 '

u
01

ft, 20 •
10 '

n = 207

==£±1

mean

= 12.2cm/s

n = 205

50 •

*- 40

| 30"

ft. 20 '

10 '

= 19'8

80 •
70 '
60 '
£ 50 '

£ 40 '
£ 30 *
20 '
10 '

n = 70

0 '

0.0 0.2 0.4 0.6 0.8 1.0

Pharynx > Tentacle Tip (X/L)

Figure 2. Location of prey capture events along the length of tentacles of polyps in the octocoral Alcyonium
at three different flow speeds. See Figure I A for interpretation of abscissa. Capture frequencies are normali/ed
relative to the amount of tentacle area available for prey capture. Data were arc-sine transformed and then
back-transformed for graphical portrayal. Vertical bars are 95"i confidence intervals. For flow speeds of 2.7,
12.2, and 19.8 cm/s, the total numbers of cysts caught were 207, 205, and 70, respectively.

verse relationship between flow speed at the surface of
the filter and efficiency of particle capture and is here
observed in Alcyonium (Fig. 3). Rubenstein and Koehl
( 1977) originally predicted that direct interception would
be the dominant mode of feeding in aquatic suspension
feeders and subsequent work with a diversity of organisms
seems to be bearing out their hypothesis (LaBarbera,
1984).

Alcyonium could sieve particles larger than the spacing
between pinnules on the tentacles (200-280 /urn) as sug-
gested by Sebens and Koehl (1984). However, Anemia
cysts sieved on the aboral side of a tentacle were almost
always dislodged subsequently by the flow and lost to the
polyp (pers. obs.). Anemia cysts were occasionally sieved
by tentacles on the downstream side of the polyp. For
larger prey items, sieving may be an important capture
mechanism, but probably only for tentacles on the down-
stream side of a polyp.

The parameter for diffusive deposition assumes the
particle has a diffusion coefficient (D) predicted by
Brownian motion for a particle of a certain size in a given
liquid of a certain temperature and viscosity. If eddy-en-
hanced diffusion is allowed, the value for D is no longer
a constant, but will be a property of the flow speed and
eddy size (Richardson, 1926; Okubo, 1971); it can be as
much as 104 larger than the diffusion coefficient for lam-
inar or "Fickian" diffusion (Okubo, 1980). Turbulent dif-
fusive deposition would increase by a similar factor and
would be comparatively more important as the size of
the particle of interest decreased, perhaps becoming very
important as a mechanism for suspension feeders eating

phyto- and bacterioplankton. Schrij ver el ul. (1981) stud-
ied particle collection efficiencies by small glass fibers of
about the same diameter as the tentacles in Alcyonium.
For small particles (ca. 5 Mm), diffusive deposition was a
very significant mechanism. More work is needed in this
area, which is technically difficult, because eddy diffusiv-
ities near surfaces must be measured (Denny, 1988), and

>•»

.Si
°3

DC
C

"•5

100 200 300 400
Polyp Re

500

Figure 3. Efficiency (E) of particle capture per polyp in colonies of
Alcyonium as a function of polyp Reynolds number (Re). Efficiency is
denned as the number of particles caught by the polyp in the time interval
to saturation of the colony, divided by the number of particles that would
have passed through the space occupied by the polyp. Reynolds number
was calculated using the oral disk diameter of the polyp and flow speed
measured 1.0 cm above the oral disk. Whole colony Re for the specimens
used was 10-40 x greater than polyp Re. The regression is given by the
equation: E = 2066 Re'1 55; P < 0.05; r = 0.92; df = 11.

M. R. PATTERSON

the convection of unfiltered water to the vicinity of the
filter should be considered. Methods of carefully releasing
dye and studying its motion near filter feeders using image
processing techniques are being developed to allow better
investigation of microscale turbulent diffusive deposition
(unpub. obs.).

Rates of particle encounter and possible capture by
gravitational deposition are independent of flow speed
but directly proportional to settling velocity. The settling
velocity for Anemia cysts is absolutely low, so the total
flux of particles to the polyps via this mechanism is much
lower than the contribution provided by direct intercep-
tion. It is unlikely that natural food particles have settling
velocities appreciably greater than Anemia cysts. The
contribution of inertial impaction to the capture of par-
ticles at higher Reynolds number could be a potentially
important mechanism if particle inertia is appreciable.
The upstream side of an individual tentacle will have a
stagnation point and the flow will split at this point (Fig.
4) and flow around the tentacle at low flow speeds or up
and over the tentacular crown at higher Re (Patterson,
1984). A calculation in the Appendix demonstrates that
this mechanism is highly unlikely to be an important
mode of particle capture by polyps in this octocoral.

Feeding efficiency of cnidarian filters

The filtration efficiency as calculated per Dorman
( 1 966) is very low for individual polyps (Fig. 3); a possible
explanation involves partitioning capture efficiency into
collection efficiency and adhesion efficiency (Weber et ai.
1983). Many particles that appear to strike the surface of

Figure 4. Streamlines of fluid How near the upstream stagnation
point of a tiller (Bird el al.. 1960), where inertial impaction is most likely
to occur. X and Y are directional coordinates. The upstream plane (in-
dicated by X0) marks where particles earned by the flow have not yet
deviated from the streamlines due to their inertia. See the Appendix for
a derivation showing how inertial impaction is not likely to be an im-
portant capture mechanism for cnidanans that use passive suspension
feeding.

A/croniiim tentacles are not trapped; relatively few are
lost if they initially adhere, although J. Miles (Northeastern
Univ. pers. comm.) found that adhesion and loss was sig-
nificantly affected by flow speed in the sea anemone, Me-
tridium senile, and Leonard et al. (1988) found that flow
speed affected capture probabilities in a crinoid. My study
of capture at the level of the polyp addresses successful
prey capture events only.

Future work on the mechanisms of particle capture by
cnidarians should investigate the role of unsuccessful
adhesions. In particular, the importance of London-van
der Waals forces versus nematocyst firing should be ex-

Table I

\ 'allies of ditnensionless parameters for four potential modes oj particle capture (cf. Rubenstein and Koehl. 1977) in the octocoral Alcyonium
siderium feeding on Artemia cysts al two di/lerenl flow speeds commonly encountered in nature

Gravitational
deposition

Direct
interception

Inertial impaction

Diffusive
deposition

«" II
L1!)

di .

1 8 ^df

dfLJo

U0 = 3.0 cm/s 0.027
U0 = 30.0 cm/s 0.003

0.67
0.67

4.0 x 10"6
4.0 x 10~s

8.2 X 10""
8.2 X 10~12

dp = particle diameter = 0.02 cm (Patterson, 1984).

dr = filter element diameter = 0.03 cm (Sebens and Koehl, 1984).

Ug = Stokes" settling velocity = 0.08 cm/s (Gibbs, 1985).

PP = density of particle = 1.05 g/ml (Gibbs. 1985).

pm = density of seawater = 1.02 g/ml at 10°C (Zerbe and Taylor. 1953).

>i = dynamic viscosity of seawater = 1.4 X 10~2 g/(cm -s) at IO°C (Sverdrup el a!., 1942).

U0 = flow near the tentacle (two values used in table above; cf. Patterson, 1984; Patterson and Sebens, 1989).

K.T

D = diffusion coefficient of an Artemia cyst = = 7.4 X 10~12 cm2/s.

6!T^dp

KT = energy of thermal fluctuation = 3.9 x 1C)-'4 (g-cm2)/s2.

POLYP-LEVEL PREY CAPTURE EVENTS

plored, as current data are insufficient to address this issue.
The subject bears further attention because Best (1988)
also found that feeding efficiency was an inverse function
of flow speed (read Reynolds number) in the sea pen,
Ptilosarcus gumeyi. She attributed this decline to the pre-
dicted behavior of particles that are caught by direct in-
terception (Spielman, 1977), perhaps aided by a defor-
mation of the filter elements as the hydrodynamic drag
increased. Alcyonium polyps on the upstream side of col-
onies deform in strong flows while polyps in the wake
undergo little deformation (Patterson, 1984). The octo-
corals she studied are more efficient than Alcyonium by
a factor of five at a similar flow speed. This study used
only polyps located near the top of the colony. Increasing
mechanical deformation of this subpopulation of polyps
occurred at higher flows, resulting in less surface area
available for prey capture, but the reduction was detected
in the measurements of projected colony surface area.
The reasons for this interspecific difference in feeding ef-
fectiveness are presently unknown.

Panicle capture locations

The patterns of prey capture seen (Figs. 1C, 2) are in
agreement with a direct interception model of prey cap-
ture. Polyps resemble inverted umbrellas (see photograph
in Sebens and Koehl, 1984); they do not hold their ten-
tacles in a flat canopy as the top view in Figure 1 A might
imply. At low speeds, the first tentacles to encounter par-
ticles are the upstream ones, and here capture is more
likely. As the flow speed increases, the polyps are bent by
the flow (cf. Patterson, 1984). and the aboral side of the
upstream polyps is presented to the flow. For prey particles
the size of Anemia cysts (ca. 200 ^m; close in value to
the mean size of natural zooplankton prey, Sebens and
Koehl, 1984), few particles are caught on these upstream
tentacles because particles do not adhere to the aboral
side. The downstream tentacles then begin feeding. In
strong flows, the polyp is severely bent downstream, and
a small eddy forms over the tentacular disk (pers. obs.);
the distribution becomes roughly symmetric again. The
relative roles that attached eddy formulation and turbulent
diffusion play in this smoothing out of the particle capture
distribution are unknown.

These experiments were conducted under turbulent but
steady flow conditions. Alcyonium occurs over a range of
depths and habitats in the shallow subtidal; it is exposed
to both oscillatory flow from wind-driven waves and to
steady tidal flows (Patterson, 1984, 1985). The particle
capture behavior of individual polyps and tentacles might
be quite different in an oscillating flow. Hunter (1989)
found that the feeding effectiveness of the hydroid Obelia
longissima was much greater in an oscillating flow relative
to a steady current. Alcyonium colonies are inherently

more rigid than those of Ohelia. and hence it is not clear
without further experimentation whether oscillatory flow
would result in enhanced feeding in Alcyonium. Thus
these results should be applied only to feeding in the field
under steady flow conditions when wind-driven oscilla-
tions have a small contribution to the flow field.

The distribution of particle capture over the length of
an individual tentacle is also in agreement with a geo-
metric (direct) interception model. Parts of the tentacle
furthest out of the boundary layer of the polyp intercept
the most prey. Patterns of prey capture discerned through
flume experiments using non-motile particles such asAr-
lemia cysts may be different from those measured using
live prey, but only if the loss rate of captured particles
differs between the two types of food or the motility of
live prey causes the diffusive deposition mechanism to
increase capture preferentially at a location different from
direct interception. Loss of captured particles is most likely
caused by hydrodynamic drag forces exceeding the break-
ing strength of the attachment between particle and ten-
tacle. Live zooplankton prey and Anemia cysts will ex-
perience very similar amounts of drag because size and
shape are similar. The motility of live zooplankton should
result in an increase in the diffusive particle flux relative
to Anemia cysts, but it should not affect the geometric
location of capture on tentacles if movement is random
in all directions.

This study has shown how flow regime can dramatically
affect patterns of particle capture at the level of the filtering
elements in an octocoral. Variation in feeding ability at
the level of the polyp caused by hydrodynamics may help
explain the variation Lasker (1981) observed in prey cap-
ture between polyps and branches in colonies of tropical
gorgonians. Capture events in the three species Lasker
studied did not fit a Poisson distribution, and he invoked
differential feeding ability of the polyps as the cause of
the variation. He offered no explanation for the differential
feeding ability other than to note that other authors had
also seen asymmetric patterns in prey capture by cnidar-
ians (e.g., Leversee, 1976). I have demonstrated that mo-
mentum transport (fluid flow) directly affects mass trans-
port (particle capture) at the level of the individual feeding
elements, polyps. Upon closer inspection, other passive
suspension feeding cnidarians may exhibit similar pat-
terns.

Acknowledgments

This paper has benefited from discussions with P. Bas-
ser, T. Givnish, M. Koehl, M. LaBarbera, T. McMahon,
R. Olson, S. L. Sanderson, K. Sebens, and R. Turner. T.
McMahon provided inspiration and insight into the me-
chanics of particle deposition through his graduate course
on fluid flow in the human body. P. Rudy, Acting Di-

100

M. R. PATTERSON

rector. Marine Science Center, Northeastern University,
Nahant, Massachusetts, kindly provided laboratory space.
C. Alexander provided technical assistance. Financial
support for this project was provided by the Richmond
Fund of Harvard University, the Lerner-Gray Fund for
Marine Research of the American Museum of Natural
History, a Faculty Research Award from UC Davis, and
NSFOCE87- 16427.

Literature Cited

Bell, K. 1974. Aerosol deposition in models of a human lung bifur-
cation. Ph.D. Dissertation. California Institute of Technology. Pas-
adena.

Berg, H. C. 1983. Random Walks in Biology. Princeton University
Press. Princeton.

Best, B. A. 1988. Passive suspension feeding in a sea pen: effects of
ambient flow on volume flow rate and filtering efficiency. Dial. Bull
175: 332-342.

Bird, R. B., VV. E. Stewart, and E. N. Lightfoot. I960. Transport Phe-
nomena. John Wiley. New York.

Brodkey, R. S., and H. C. Hershey. 1988. Transport Phenomena: A
Unified Approach McGraw-Hill, New York.

Chang, D. P. Y. 1973. Particle collection from aqueous suspensions
by solid and hollow single fibers. Ph.D. Dissertation. California In-
stitute of Technology. Pasadena.

Denny, M. 1988. Biology and the Mechanics of the Ware-Swept En-
vironment. Princeton University Press, Princeton.

Dorman, R. G. 1966. Filtration. Pp. 195-222 in Aerosol Science.
C. N. Davies, ed. Academic Press. New York.

Kindeisen, VV. 1935. Uber das Abstetzen kleiner. in der Luft suspen-
dierter Teilchen in der menschlichen Lunge bei der Atmung. PJhiger
Arch. 6>.v. Physiol. 236: 367-379.

Fuchs, N. A. 1964. The Mechanics of Aerosols. Pergamon Press. New
York.

Gibbs, R. J. 1985. Estuarine floes: their size, settling velocity, and den-
sity. / Geophys. Res 90(C2): 3249-3251.

Glauert, M. 1940. A method of constructing the paths of raindrops of
different diameters moving in the neighbourhood of (Da circular
cylinder. (2) an aerofoil, placed in a uniform stream of air: and a
determination of the rate of deposit of the drops on the surface and
the percentage of drops caught. Aeronaut. Res Council. Reports and
Memoranda No. 2025. London.

Harvell, D., and M. LaBarbera. 1985. Flexibility: a mechanism for
control of local velocity in hydroid colonies. Biol. Bull 168: 312-
320.

Holland, N. D., A. B. Leonard, and J. R. Strickler. 1987. Upstream
and downstream capture during suspension feeding by Oligometra
serripinna (Echinodermata: Crinoideal under surge conditions. Biol.
Bull. 173: 552-556.

Hunter, T. 1989. Suspension feeding in oscillating flow: the effect of
colony morphology and flow regime on plankton capture by the hy-
droid Obelia longissima. Biol. Bull. 176: 41-49.

Koehl, M. A. R., and J. R. Strickler. 1981. Copepod feeding currents:
food capture at low Reynolds number. Limnol. Oceanogr 26: 1062-
1093.

LaBarbera, M. 1978. Particle capture by a Pacific brittle star: experi-
mental test of the aerosol suspension feeding model. Science 201:
1147-1149.

LaBarbera, M. 1984. Feeding currents and particle capture mechanisms
in suspension feeding animals. Am. Zool. 24: 71-84.

LaBarbera, M., and S. Vogel. 1976. An inexpensive thermistor flow-
meter for aquatic biology. Limnol. Oceanogr. 21: 750-756.

Landahl, H. D. 1950. On the removal of air-borne droplets by the

human respiratory tract: 1. The lung. Bull. Math. Biophys. 12: 43-

56.
Landahl, H. D. 1963. Particle removal by the respiratory system: note

on the removal of airborne particulates by the human respiratory

tract with particular reference to the role of diffusion. A M. A. Arch.

Ind. Hyg Occup. Med. 25: 29-39.
Lasker, H. R. 1981. A comparison of the paniculate feeding abilities

of three species of gorgonian soft coral. Mar. Ecol. Prog. Ser. 5: 61-

67.
Leversee, G. J. 1976. Flow and feeding in fan-shaped colonies of a

gorgonian coral. Leptogorgia. Biol. Bull. 151: 344-356.
Leonard, A. B., J. R. Strickler, and N. D. Holland. 1988. Effects of

current speed on filtration during suspension feeding in Oligometra

serripinna (Echinodermata: Crinoidea). Mar. Biol 97: 1 1 1-122.
Lewis, J. B., and \V. S. Price. 1976. Patterns of ciliary currents in

Atlantic reef corals and their functional significance. J. Zool 178:

77-89.

McMahon, T. A., J. D. Brain, and S. Lemott. 1977. Species differ-
ences in aerosol deposition. Pp. 23-33 in Inhaled Particles IT.

W. H. Walton, ed. Pergamon Press. Oxford.
Merz, R. 1984. Self-generated versus environmentally produced feeding

currents: a comparison for the Sabellid polychaete Eudislylia van-

couven Biol. Bull. 167: 200-209.
Niklas, K. J. 1982a. Pollination and airflow patterns around conifer

ovulate cones. Science 217: 442-444.
Niklas, K. J. 1982b. Simulated and empiric wind pollination patterns

of conifer ovulate cones. Proc. Nat. Acad. Sci. USA 79: 510-514.
Okamura, B. 1984. The effects of ambient flow velocity, colony size.

and upstream colonies on the feeding success of Bryozoa.. I. Bugida

stolonijera Ryland. an arborescent species. J. Exp. Mar. Biol. Ecol.

83: 179-193.
Okamura, B. 1987. Particle size and flow velocity induce an inferred

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

229.
Okubo, A. 1971. Oceanic diffusion diagrams. Deep-Sea Res. 18: 789-

802.
Okubo, A. 1980. Di/fusion ami Ecological Problems: Mathematical

Models. (Biomathematics, Vol. 10). Springer Verlag. New York.
Patterson, M. R. 1980. Hydromechanical adaptations in Alcyonium

.sulerium (Octocorallia). Pp. 183-201 in Biofluid Mechanics 2. D. J.

Schneck, ed. Plenum, New York.
Patterson, M. R. 1984. Patterns of whole colony prey capture in the

octocoral. Alcyonium siderium. Biol. Bull. 167: 613-629.
Patterson, M. R. 1985. The effects of flow on the biology of passive

suspension feeders: prey capture, feeding rate, and gas exchange in

selected cnidarians. Ph.D. dissertation. Harvard University. Cam-
bridge. Massachusetts.
Patterson, M. R., and K. P. Sebens. 1989. Forced convection modulates

gas exchange in cnidarians. Proc. Nat. Acad. Sci. USA 86: 8833-

8836.
Patterson, M. R. 1991. Passive suspension feeding by an octocoral in

plankton patches: empirical test of a mathematical model. Biol. Bull

180:81-92.

Richardson, L. F. 1926. Atmospheric diffusion shown on a distance-
neighbor graph. Proc. Roy. Soc. London A 110: 709-727.
Ross, D. H., and D. A. Craig. 1980. Mechanisms of fine particle capture

by larval black flies (Diptera: Simuliidae). Can J Zool. 58: 1 186-

1192.
Rubenstein, D. J., and M. A. R. Koehl. 1977. The mechanism of filter

feeding: some theoretical considerations. Am. Nat. Ill: 981-994.
Schrijver, J. H. M., C. Vreeker, and J. A. Wesselingh. 1981 . Deposition

of particles on a cylindrical collector. J. Coll. Inter/. Sci. 81: 249-

256.

POLYP-LEVEL PREY CAPTURE EVENTS

101

Sebens, K. P. 1984. Water flow and coral colony size: mterhahilal

comparisons of the octocoral Alcyoniwn sidenum Proc. Nat. Acad.

Sci (i'SA)Sl: 5473-5477.
Sebens, K. P. 1986. Spatial relationships among encrusting marine

organisms in the New England subtidal zone. Ecu!. Monogr. 56: 73-

96.
Sebens, K. P., and M. A. R. Koehl. 1984. Predation on zooplankton

by the benthic anthozoans. Alcyoniwn sideriuni (Alcyonacea) and

Metridium senile (Actiniaria) in the New England subtidal. Mar

Biol 81: 255-271.
Spielman. I.. A. 1977. Particle capture from low-speed laminar Hows.

Anna Re\ l-'liiu/ Mec/L 9: 297-319.
Strathmann, R. R., and E. Liese. 1979. On feeding mechanisms and

clearance rates of molluscan veligers. Biol. Bull- 157: 524-535.
Sverdrup, H. U., M. W. Johnson, and R. H. Fleming. 1942. The Oceans:

Their Physics, Chemistry, and Biology Prentice-Hall, New York.
Taghon, G. I,., A. R. M. Nowell, and P. A. Jumars. 1980. Induction

of suspension feeding in spionid polychaetes by high particle fluxes.

Science 210: 562-564.
Taylor, C. I. 1940. Notes on possible equipment and technique for

experiments on icing on aircraft. Aeronaut Res Council. Report \

and Memoranda No. 2024. London.
Vogel, S., and M. LaBarbera. 1978. Simple flow tanks for research

and teaching. BioScience 10: 638-643.

Weber, M. E., D. C. Blanchard, and L. D. Syzdek. 1983. The mech-
anism ot scavenging of waterborne bacteria by a rising bubble, l.tmnol

Oceanogr. 28(1): 101-105.
Zerbe, VV. B., and C. B. Taylor. 1953. Sea water density reduction

tables. Pp. 18-19 in Coast and Geodetic Survey. Special f'uhl no

298. U. S. Dept. of Commerce, Washington. DC.

Appendix

The following derivation, developed from the work of
Glauert (1940) on raindrop capture by airfoils and from
Taylor ( 1940) on aircraft icing, shows how unlikely it is
that inertial impaction will be an important mechanism
for passive suspension feeding cnidarians for the range of
velocities normally encountered in the field.

Consider the motion of a solid particle (e.g.. a plankter),
moving with a velocity relative to the seawater, as it ap-
proaches the upstream end of a filtering organism (Fig.
4). In other words, the particle does not follow the stream-
lines perfectly. If the Reynolds number (Re) of the particle
is on the order of one or less, as it would be for plankton-
size particles (Koehl and Strickler, 1 98 1 ), the forces acting
on the particle are due solely to the Stokes' drag (Berg,
1983). The equation governing the motion of the particle
in the x-direction is:

p _

= 67rrM(U - UD)

(Eq. 1)

where r = particle radius. ps = particle density, n = dy-
namic viscosity, Up = velocity component of the particle
in the x-direction, and U = velocity component of the
seawater in the x-direction.

The velocity components in the y- and z-directions are
V and W, respectively, and the equations of motion are
similar. If I define:

(Eq.2)

then (Eq. 1) becomes:

ko-^ -Up (Eq.3)

and other directions can be transformed similarly.

The equations of motion must be solved subject to the
initial conditions. Let me introduce a scaled time variable.

and the differential operator,

£ = l

Now (Eq. 3) becomes
dUn

(Eq.4)

(Eq.5)

(Eq.6)

and similarly for the other directions.

Flow near the upstream stagnation point of the filter
will look like Figure 4 (Bird c/ a/.. 1960). The velocity
field near the stagnation point is given by:

U = -ex

and

V = cy
Substituting into (Eq. 6), I obtain:

d:x dx

^3 + - + cM =

(Eq.7)

(Eq.8)

(Eq.9)

It is reasonable to assume that upstream of the tentacle
a certain distance, X0, the flow field is not distorted by
the presence of the tentacle and the particle is following
the streamlines of the moving seawater (see Fig. 4). If the
time at which the particle starts to deviate from the
streamlines of flow is called T = 0, I obtain:

x(0) = - X0
dt

(Eq. 10)
(Eq.ll)

A reasonable guess to the solution of (Eq. 1 1 ) is one of
the form x(t) = Aepl, where A = constant. The charac-
teristic equation is thus.

p: + p +

= 0

(Eq.12)

102

M. R. PATTERSON

The roots of the equation are:

PL: =

- 1 ± VI -

(Eq. 13)

When one of the roots is imaginary, the solution to the
equation of motion of the passing particle will be oscil-
latory, i.e.. the particle position and the filter position will
eventually coincide (x = 0). This condition will occur
when:

4ck<,

(Eq. 14)

Glauert (1940) showed for a cylindrical geometry that
a negligible number of particles will impact if k^c^s 0.125,
where c = 2 and the definition of ko is as follows:

2psrRU
9pR2v

(Eq. 15)

where R = radius of the filter, and v = n/p, the kinematic
viscosity of the seawater.

Using reasonable values for flow around an Alcyonium
colony, I obtain k<jC = 0.003, for p = 1.024 g/cm\ (Zerbe
and Taylor, 1953), p, = 1.049 g/cm3, (Gibbs, 1985), r
= 100 X 10~4cm, R = 5cm, U = 5 cm/s, v= 1.36X 10~:
cnr/s for seawater at 10°C (calculated from Sverdrup
el til.. 1942). If the flow speed increases by an order of mag-
nitude to U = 50 cm/s, then koC = 0.034. Appreciable im-
paction will not occur until U = 185 cm/s, far above the
range of speeds normally encountered near this species (Pat-
terson and Sebens, 1989). Such a flow would only be found
under stormy conditions in the subtidal or in tidal currents
in fjords. Alcyonium contracts its prey-capturing surfaces
long before this flow speed is obtained (Patterson. 1980).

Reference: Biol. Bull 180: 103-1 1 1. (February, 1991)

Differential Ingestion and Digestion of Bivalve Larvae

by the Scyphozoan Chrysaora quinquedrrha

and the Ctenophore Mnemiopsis leidyi

JENNIFER E. PURCELL1. FRANCES P. CRESSWELL1. DAVID G. CARGO2,

AND VICTOR S. KENNEDY1

The University of Maryland

Abstract. We investigated predation on bivalve veligers
by the scyphozoan Chrysaora quinquedrrha and the
Ctenophore Mnemiopsis leidyi. We found that the medusa
stage of C. quinquedrrha captures, but does not digest,
veliger larvae: 99% of oyster veligers (Crassostrea virgin-
tea) caught by medusae were egested alive within 7 h of
capture, and 98% survived for 24 h after egestion; 98% of
oyster, mussel (Afylilus edulis), and clam (Mulinia later-
alis) veligers placed on the oral arms of medusae were
rejected; all bivalve veligers in field-collected medusae
were closed and full of tissue. Our laboratory evidence
suggests that the shell of larval bivalves probably offers
protection from medusae: 23%) of dead, open veligers were
ingested by medusae compared with 0.7% of live, closed
veligers; open veligers were retained longer than closed
veligers; and tissue excised from recently settled oyster
larvae was ingested and digested. Freeswimming C quin-
quedrrha ephyrae ingested but did not digest veligers. By
contrast, the benthic scyphistoma stage ingested 69% of
veligers that contacted their tentacles and digested 48%
of those ingested. Each scyphistoma consumed an average
of 1 veliger/day at densities of 0.3 veligers ml" ' . However,
larval settlement was not reduced on oyster shells bearing
scyphistomae. By contrast to the results on C. quinque-
drrha, ctenophores egested only 4% of veligers alive, and
25% of the veligers in their gut contents were digested.
Predation on veligers by ctenophores was estimated to be
0.2 to 1.7%/day in Chesapeake Bay. We conclude that C.

Received 14 August 1990; accepted 6 November 1990.

1 Horn Point Environmental Laboratories, P. O. Box 775, Cambridge,
Maryland 2 161 3.

2 Chesapeake Biological Laboratory, Box 38, Solomons, Maryland

20688-0038.

quinquedrrha medusae are not important predators of
bivalve veligers, but rather may reduce their mortality by
consuming ctenophores, which do eat veligers.

Introduction

Predation on planktonic larvae is one of the least un-
derstood factors affecting abundance of adult benthic in-
vertebrates! YoungandChia, 1987). Early studies reported
that the scyphomedusan Chrysaora quinquedrrha (DeSor)
and the Ctenophore Mnemiopsis leidyi A. Agassiz may
prey heavily upon the larvae of the eastern oyster Cras-
sostrea virginica (Gmelin) (Truitt and Mook, 1925; and
Nelson, 1925, 1953, respectively). Both species are sea-
sonally abundant in Atlantic coast estuaries, and co-occur
with oyster larvae. Their effects on survival of oyster larvae
have not been documented.

In several Atlantic coast estuaries, M. leidyi has been
shown to be an important predator of crustacean zoo-
plankton (e.g.. Cronin et ai. 1962; Cargo and Schultz,
1967; Bishop, 1967; Burrell, 1968; Herman et ai. 1968;
Kremer, 1979; Deason and Smayda, 1982; Feigenbaum
and Kelly, 1984; Olson, 1987) and bivalve veliger larvae
(Nelson, 1925; Truitt and Mook, 1925; Burrell and Van
Engel, 1976). Bivalve veligers were 75% of the prey of M.
leidyi in New Jersey waters, and high larval settlement of
three bivalve species, including oysters, occurred in years
when Ctenophore densities were low (Nelson, 1925). In
the York River, Virginia, bivalve larvae were inversely
related to the biomass of ctenophores (Burrell and Van
Engel, 1976).

Studies on the feeding of scyphomedusae have shown
them to eat a variety of zooplankton (reviewed in Larson,
1978; Clifford and Cargo, 1978; Feigenbaum and Kelly,
1984; Larson, 1987; Fancett, 1988; Brewer, 1989). Al-

103

104

J. E. PURCELL ET AL

though C. quinquecirrha medusae were reported to feed
on oyster larvae (Truitt and Mook, 1925; Loosanoff,
1974). high numbers of oyster larvae and medusae often
co-occurred (Truitt and Mook, 1925). This apparent par-
adox may be due to the fact that C. quinquecirrha medusae
prey heavily upon ctenophores (Cargo and Schultz, 1967;
Burrell, 1968; Miller, 1974; Feigenbaum and Kelly. 1984;
Larson, 1986), thus decreasing ctenophore predation on
oyster larvae.

Nothing is known of the trophic ecology of the incon-
spicuous benthic scyphistoma or early free-swimming
ephyra stages of scyphozoans. Large numbers of C. quin-
quecirrha scyphistomae are found on oyster shell (Cargo
and Schultz, 1966, 1967), which is a preferred settling
substrate for oyster larvae (Kennedy and Breisch, 1981 ).
Therefore, these scyphistomae may be predators of oyster
pediveliger larvae that are preparing to settle upon oyster
shells.

To test the potential importance of C. quinquecirrha
and M. leidyi as predators of bivalve larvae, we compare

( 1 ) medusa and ctenophore digestion of oyster veligers.

(2) rejection or ingestion of oyster, blue mussel (Alytilus
edulis L.), and coot clam [Mulinia lateralis (Say)] veligers
by medusae, and (3) rejection, or ingestion and digestion
of oyster trochophores and veligers by the ephyra and
scyphistoma stages of C. quinquecirrha. We also present
data on bivalve veligers in gut contents of medusae and
ctenophores, and in situ densities of those predators and
veligers, to estimate the importance of predation by ge-
latinous zooplankton on bivalve larvae in the mesohaline
region of Chesapeake Bay.

Materials and Methods

During June through August, 1987, 1988, and 1989,
C. quinquecirrha medusae and M. leidyi were collected
in jars from the boat basin of the Horn Point Environ-
mental Laboratories (HPEL) on the Choptank River. In
the laboratory, we used 30 nm filtered Choptank River
water at ambient salinity (11-1 2%o) and temperature (20-
27°C). After collection, medusae and ctenophores were
held in 20-1 plastic containers of water, and fed on Anemia
salina nauplii for at least 1 2 h to clear their guts of natural
zooplankton. Oyster larvae from trochophore (60 ^m
long) to pediveliger (270 ^m) stages, and clam veligers
(100-260 ^m) were obtained from the HPEL hatchery.
For the following experiments, veligers were separated into
size fractions on screens of different mesh sizes. Mussel
veligers ( 1 80 ^m) were supplied by the University of Del-
aware, College of Marine Studies in Lewes, DE.

Digestion and survival of oyster veligers after capture
by medusae and ctenophores

Individual medusae and ctenophores were exposed for
10 min either to high densities of oyster veligers alone (2-
9 ml"1), or to oyster veligers (0.1 ml"1) with copepods

(Acartiu tonsa) as alternative prey in 4-1 containers. The
predators then were gently transferred twice with sieves
(1 mm mesh) at 5-min intervals to 4-1 containers with
filtered water to remove prey adhering to their external
surfaces and to dilute swimming zooplankton possibly
transferred with the predators. Each predator was subse-
quently transferred at hourly intervals to new containers
of filtered water. After the predator was removed from
each container, the water was poured through a 60-^m
screen, and live oyster veligers, larval shells, live copepods,
and copepod exoskeletons were counted with a dissecting
microscope, thus recording all prey egested each hour.
Egestion times were calculated from the midpoint of each
interval, so the accuracy is ±0.5 h. Living veligers that
were retrieved after egestion by the medusae were put in
beakers of water with food (phytoplankton Isochrysis gal-
bana) to determine their survival after 24 h.

Rejection and ingestion oj bivalve veligers by medusae

To examine the feeding reactions of C. quinquecirrha
medusae to bivalve veligers and copepods, we placed me-
dusae ( 15-90 mm in bell diameter) exumbrellar surface
down in fingerbowls with less than 100 ml water. In this
position, medusae continued to take food, and were easily
examined with a dissecting microscope. Individual prey
were placed by pipette on the oral arms, where prey are
captured and transferred to the gastric pouches (Larson,
1986). The length of time it took prey to reach a gastric
pouch (ingestion) or to be rejected from the oral arm was
measured during continuous observation.

Prey in this experiment included live (closed) and
freshly killed (gaping) oyster veligers, live clam and mussel
veligers, live and heat-killed copepods (Acartia tonsa). and
tissue removed from 2- to 3-day-old oyster spat (recently
settled larvae). Gaping veligers were used to determine
whether the larval shell caused the rejection of veligers by
medusae. To obtain gaping veligers, we anaesthetized
them by gradually adding seltzer water (CO2) until the
shells opened, and then rapidly heating the water to kill
them. To ensure that the medusae were feeding well, live
copepods, which were readily accepted, were alternated
with other prey.

C. quinquecirrha ephyrae 2 to 3 mm in diameter, bud-
ded from scyphistomae in the laboratory, were placed
singly in a depression slide with 0.5 ml of water and a few
live oyster trochophores or live oyster or clam veligers;
the process of rejection or ingestion was timed after con-
tact occurred. Scyphistomae attached to plastic slides in
the laboratory were offered live oyster trochophores or
veligers in 25-ml dishes, and rejection or ingestion was
timed after contact.

Effect of scyphistomae on veliger settlement

To determine if C. quinquecirrha scyphistomae reduced
oyster settlement, field-collected oyster shells containing

PREDATION ON BIVALVE LARVAE

105

scyphistomae were cut into 5 to 8 crrr pieces and cleaned
of other epifauna. Seven pieces of shell with scyphistomae
(9.3 ± 3.7 individuals per shell for all experiments) and
seven without were placed in 3 1 of 1 \%« water at 24° to
27 °C in dishes of 143 cm: bottom area. Shell pieces were
oriented so that scyphistomae were on the underside,
which is their preferred location in nature (Cargo and
Schultz. 1966. 1967). About 500 oyster pediveligers (179-
250 ^m long) were added to the dishes, plus algae (Iso-
chrysis galbana) as food for the larvae and Anemia salina
nauplii as alternate prey for the scyphistomae. The dishes
were gently aerated and were covered with black plastic,
because oyster veligers prefer low light levels for settlement
(Ritchie and Menzel, 1969). The shell pieces were checked
at 24 and 48 h for newly settled larvae. Six trials, each
with two replicates, were run with different pieces of shell.
There were 4 controls, each with 14 shell pieces without
scyphistomae.

Scyphistoma predation and digestion rales on veligers

Predation by scyphistomae on oyster veligers was de-
termined at the end of each trial (24 or 48 h) by counting
the empty larval shells retrieved from the experimental
containers. In additional predation experiments at the
Chesapeake Biological Laboratory (CBL), containers were
filled with 150 ml of estuary (Patuxent River) water. Each
container had one plastic slide that was raised off the bot-
tom by fishing weights so that the 3 to 20 attached scy-
phistomae were on the lower surface. Fifty oyster veligers
( 179 to 250 jum long) and algal food were added to each
container. After 24 and 48 h, larvae inside scyphistomae
and clear shells were counted. There were 159 trials, and
26 controls without scyphistomae to check for veliger
death due to experimental manipulations. In combination
with the preceding experiment. 171 predation measure-
ments were taken.

The length of time required by scyphistomae for diges-
tion of both closed (live) and gaping (anaesthetized and
killed) oyster larvae was determined by pipetting the larvae
into the tentacles and mouth region of the scyphistomae.
The times of ingestion were recorded, then containers were
checked at intervals for empty larval shells.

Field studies on medusae and ctenophores

In 1987, we sampled medusae, ctenophores, and bivalve
veligers weekly from May to September in two tributaries
of Chesapeake Bay [Broad Creek (38° 40', 76°15'W) and
Tred Avon River (38°40'N, 76°05'W)], and on three dates
in both May and August, and on one day in both June
and July at five stations across the Bay at the same latitude.
At each station, we collected individual medusae and
ctenophores by dip net and immediately preserved them
in 5% formalin for dietary analysis with a dissecting mi-

croscope. All bivalve veligers in these samples were
counted. Empty and open larval shells were counted sep-
arately from closed shells that contained tissue.

Densities of C. quinquecirrha and M. leidyi were mea-
sured with a 1 m diameter, 1.6-mm mesh net with flow-
meter towed at 1 m depth in the tributaries (bottom depth
< 4 m), and above the pycnocline in the Bay (<1 1 m).
Medusae and ctenophores were counted from samples
preserved in 5% formalin (Purcell, 1988). Densities of bi-
valve larvae were determined from plankton samples
taken at the same times as the net tows at 1 m depth in
the tributaries with a portable bilge pump, and at 1-m
intervals above 1 1 m depth in the Bay with a submersible
pump. Pump samples were filtered through a 64 /urn
plankton net in the field, then preserved in 5% formalin,
and veligers were counted in the laboratory from whole
samples or subsamples taken with a Hensen Stempel pi-
pette.

Rates of ctenophores feeding on bivalve veligers in sirit
were estimated from individual clearance rates (Kremer,
1979) times the numbers of ctenophores per cubic meter.

Statistics

Our results are presented as the mean ± one standard
deviation. Comparisons on the numbers of prey rejected
or ingested were by contingency tables and Chi-square
tests, and comparisons of the retention times of different
prey species were by one-way analysis of variance. In re-
sults reported here as significantly different, the statistical
probability is less than 0.001. unless stated otherwise.

Results

Digestion and survival of oyster veligers after capture
by medusae and ctenophores

Chrysaora quinquecirrha medusae captured copepods
and oyster veligers (80-270 ^m long). Ninety-three percent
of the copepods were digested, compared with only 1%
of the veligers (Table I). Medusae egested copepod remains
in less than 5 h. and the few undigested copepods were

Table I

Numbers of copepods and oyster ve/igers digested after capture by
Chrysaora quinquecirrha medusae and Mnemiopsis leidyi

Predators

Species

Captured

Digested

tested

C. quinquecirrha

copepods

12,143

1 1.276(93%)

110

oyster veligers

4.800

48(1%)

100

M. leidvi

oyster veligers

333

316(96%)

106

J. E. PURCELL ET AL

Table II

Percentages «! oyster vcliKcr.s o/ different si:es sun-iving for 24 h after
cgestion by Chrysaora quinquecirrha medusae. Numbers of egested
veligers are in parentheses

Table III

Numbers of oyster, mussel, and clam veligers. copepods. and oyster
spat tissue rejected, ingested, and digested by Chrysaora quinquecirrha
medusae, ephyrae. and scyphislomae

Time inside

medusa (h)

Veliger size

1-2

2-3

3-4

4-5

5-6

<100^m

96.3

88.9

75.0

72.7

100

(164)

(18)

(12)

(ID

(0)

(2)

1 00-200 urn

99.6

95.2

97.1

94.1

71.4

91.7

(1559)

(272)

(102)

(51)

(14)

(12)

>200 fim

99.4

100

93.9

81.8

100

66.7

(335)

(65)

(33)

(11)

(6)

(3)

dead. Undigested veligers were egested in less than 7 h,
with over 90% egested in less than 2 h. Medusae egested
shells of the 48 veligers that were digested in 3.4 ± 1.8 h
at 22 to 27°C. Digested veligers included 31 small (<100
j/m) and 17 medium (100-200 ^m), but no large (>200
veligers. These numbers represent 0.03%, 0.006%>,

and 0% of the numbers of veligers ingested in each size
class. Medusae digested veligers less than 100 ^m long
significantly more frequently than those in both larger
size classes. More medium sized veligers were digested
than large ones (P < 0.05). Ctenophores digested signifi-
cantly more oyster veligers (96%) than did medusae (1%)
(Table I), and egested 333 empty larval shells in 2.0 ± 1.0
hat 19.5 to20.5°C.

Many veligers egested by medusae were alive. Overall,
98.4% of 2670 veligers that we retrieved after egestion by
medusae survived for 24 h afterward. Veligers smaller than
100 /im long showed significantly lower survival than
larger veligers (Table II). Veligers retained for more than
2 h showed significantly lower survival than those retained
for less than 2 h (Table II). Differences between the <1 h
and the 1-2 h groups, and among the groups >2 h were
not significant (P > 0.2 for all comparisons).

Rejection and ingestion of bivalve veligers by medusae

Prey placed on an oral arm of C. quinquecirrha medusae
were immediately rejected, or they were taken briefly in-
side the oral arm by the medusae before rejection, or they
were transported inside the oral arm and then to a gastric
pouch (ingestion). Medusae rejected significantly more
live oyster veligers (99.3%) from the oral arms than live
copepods (1.5%) (Table III). The numbers of live oyster,
mussel, and clam veligers rejected were not significantly
different (P = 0.2 to 0.8).

The closed shell protected veligers from ingestion and
digestion by medusae. Open oyster veligers were rejected
significantly less than closed, live ones, but the difference
between closed and open mussel larvae was not significant

Prey

Rejected

Ingested

Digested

Specimens
tested

Medusae

Veligers

Oyster — live

134

Oyster — dead

—

Oyster— shells

Mussel — live

—

Mussel — dead

Clam — live

Oyster spat tissue

>10

Copepods — live

451

— dead

137

—

Ephyrae

Veligers

Oyster — live

Clam — live

Trochophores

117

2:105

Scyphistomae

Veligers

Oyster — live

Clam — live

= Not quantified because we were unable to track the prey.

(Table III). Open oyster veligers also were retained sig-
nificantly longer in the oral arms than were closed veligers
(Table IV). Empty larval shells were never ingested (Table
III). Oyster spat tissue was ingested significantly more fre-
quently than either open or closed oyster veligers (Table
III). Dead copepods were rejected significantly more often

Table IV

Percentages of bivalve veligers that were retained for five time intervals
in l/ie oral arms of Chrysaora quinquecirrha medusae. The numbers
of veligers tested are in the "Rejected" column in Table III

Time inside oral arm (min)

Maximum time

Prey

<1 1-2

2-4

4-10

>10

(min)

Oysters

live

16 31

dead

12 24

156

Mussels

live

9 31

Clams

live

2 10

PREDATION ON BIVALVE LARVAE

107

than live ones (Table III), but most dead ones were still
accepted as food.

Although nearly all veligers were eventually rejected
from the oral arms of C. quinquecirrha medusae, differ-
ences in retention time existed among the three bivalve
species tested (Table IV). Most live veligers were rejected
in less than 10 min. Live mussel veligers were retained
somewhat longer than live oysters, but the difference was
not significant (P = 0.2). Clams were retained significantly
longer before rejection than were oysters and mussels.

Comparisons among life history stages of C. quinque-
cirrha showed that ephyrae and scyphistomae ingested
proportionately more oyster and clam veligers than did
the medusae (Table III). Ingestion of oyster veligers dif-
fered significantly between medusae and ephyrae, and be-
tween medusae and scyphistomae; however, differences
between ephyrae and scyphistomae were not significant
(P = 0. 1 ). Ingestion of clam veligers differed significantly
between scyphistomae and medusae, and between scy-
phistomae and ephyrae; however the difference between
medusae and ephyrae was not significant (P = 0. 1 ).

Of the ingested veligers, scyphistomae digested signif-
icantly more oysters than did ephyrae (Table III), but not
clams (P = 0.9). Thus, ephyrae behaved more like me-
dusae than scyphistomae in that they digested few oyster
veligers. Ephyrae digested five clam veligers in 1 .8 to 20.6
hfmean 10.6 ± 8.3 h).

Comparisons between types of veligers showed that
ephyrae ingested significantly more oyster than clam ve-
ligers (Table III), but digested significantly more clams
than oysters. In contrast, scyphistomae ingested signifi-
cantly more clam than oyster veligers (P < 0.05), and
digested significantly more clams than oysters (P < 0.05).
These results suggest that clam and oyster veligers are
captured with different success by ephyrae and scyphis-
tomae, and that oyster veligers show greater resistance to
digestion than do clam veligers once captured.

Because individual oyster trochophore larvae were dif-
ficult to observe due to their small size (<60 ^m), we were
successful at offering them only to ephyrae, which ingested
and digested significantly more trochophores than veligers
(Table III).

Effect of scyphistomae on veliger settlement

No settlement of oyster veligers occurred in three of
six experiments. Veligers in three experiments and one
control settled preferentially on the lower surfaces of the
shell pieces, even those with C. quinquecirrha scyphisto-
mae. Numbers of spat on the upper/lower shell surfaces
were: shells with scyphistomae 19/69; without scyphis-
tomae 22/49; control 22/77. No significant differences in
spat settlement were seen among shell pieces with or
without scyphistomae. which were on the lower surfaces

(P > 0.2 for all comparisons). Total settlement was greater
in the control container (average of seven veligers settled
per shell), where there were no scyphistomae, as compared
with the experimental containers (average settlement of
two per shell), probably because predation by scyphisto-
mae reduced the numbers of veligers.

Scyphistoma predation and digestion rates on veligers

A total of 4409 oyster veligers were consumed by Chrv-
saora quinquecirrha scyphistomae in 171 predation ex-
periments, as evidenced by the presence of empty shells.
In contrast, only 9 empty shells were retrieved from 27
controls without scyphistomae. No significant differences
existed between the ingestion rates measured at 24 and
48 h, therefore the results were pooled. The initial densities
of larvae in the experimental and control containers av-
eraged 0.31 ± 0.06 veligers ml"1. Over the range of prey
density (0.1-0.7 veligers ml1), the number of larvae con-
sumed per scyphistoma per day (range 0-13) was posi-
tively correlated with larval density (r = 0.26, P < 0.01).
On average, each scyphistoma consumed 0.9 ± 0.6 ve-
ligers/day. As many as 15 larvae were observed within a
single scyphistoma. These results indicate that scyphis-
tomae are more effective predators on oyster veligers than
are medusae. However, we observed that after a few hours,
scyphistomae sometimes expelled ingested larvae, which
began swimming again. These larvae then were available
for recapture.

Closed bivalve veligers were very resistant to digestion
by scyphistomae. Closed D-stage clam veligers were di-
gested in 37.5 to 41 h (mean 39.2 ± 1.2 h, n = 34), and
clam pedi veligers were digested in 4 to 47 h (mean 30.6
± 15.6 h, n = 6). Scyphistomae that had ingested one or
two closed oyster pediveligers egested empty shells in 24
to 67 h (mean 34.6 ± 12.9 h, n = 13). Three pediveligers
removed from scyphistomae after 18.5 h appeared to be
healthy. In contrast, open oyster pediveligers were digested
in only 1.3 to 5.1 h (mean 3.7 ± 0.8 h, n = 32).

Field studies on medusae and ctenophores

Field-collected M. leidyi and C. quinquecirrha medusae
both contained bivalve veligers. In 67 medusae, the shells
of all 77 veligers were closed and full of tissue, indicating
that they had not been digested. By contrast, 19 of 76
(25%) of the shells in 9 ctenophores were open and empty,
indicating complete digestion. The proportions of open
and closed shells in medusae and ctenophores were sig-
nificantly different. Ctenophores contained more veligers
(an average of six each) than did medusae (about one
each). This may be because the ctenophores were collected
in Chesapeake Bay, where veliger densities were much
greater than in the tributaries, which was where the me-
dusae were collected for diet studies (Table V).

108

J. E. PURCELL ET AL.

Table V

Densities (numbers m'3) ol Chrysaora quinquecirrha medusae. Mncmiopsis leidyi. and bivalve veligers in Chesapeake Bay and the Broad Creek
and Trcd Avon River tributaries from May in August. I9S7, and ihe percentages of veligers consumed per day by Mnemiopsis

Chesapeake Bay

Tributaries

Veligers*
consumed

per day

Month

Medusae

Ctenophores

Veligers

(%)

Medusae

Ctenophores

Veligers

(%)

May

0.3 ± 0.5

13,826 ± 11.491

0.2 ± 0.2

0-0.3

0.2-33.1

June

2.7 ± 1.6

14.210 ± 8,145

1.0 ±0.6

5.4 ± 5.8

1786 ± 1550

July

O.I ±0.1

0.1 ±0.1

60,032 ± 97,380

0.2 ± 0.2

9.6 ± 4.2

419 ± 279

August

0.6 ± 0.7

0.7 ±0.8

1 2,284 ± 15,234

1.7 ± 1.9

7.2 ± 3.7

1421 ± 1060

* Percentage daily consumption estimated from ctenophore filtering rates (Kremer. 1979).
— = No data.

To estimate the importance of predation on veligers by
medusae and Ctenophores in nature, we measured /// situ
densities of M. leidyi, C. quinquecirrha, and bivalve ve-
ligers in May through August, 1987 (Table V). Cteno-
phores occurred in the Bay throughout this period, but
they were excluded from the tributaries by high densities
of medusae that fed on them from June through August.
Medusae were much less abundant in the Bay than in the
tributaries. Sampled densities of bivalve veligers were
much greater in the Bay than in the tributaries, possibly
due to different efficiencies of the pumps used to collect
them. If we assume that only Ctenophores ate the bivalve
veligers, then 0.2 to 1.7% of the veligers were consumed
daily in the main Bay, and none were eaten in the trib-
utaries during that period (Table V).

Discussion

A surprising result of this study is that Chrysaora quin-
quecirrha medusae do not ingest or digest bivalve veliger
larvae. Three lines of evidence lead to this conclusion. ( 1 )
Medusae that caught swimming veligers egested them
alive. (2) Veligers placed on oral arms were subsequently
rejected. (3) Veligers in the gut contents of field-collected
medusae were closed and full of tissue. The ephyra stage
ingested oyster veligers but did not digest them. By con-
trast, scyphistomae egested some living veligers, but many
were retained and eventually digested.

The larval shell may protect bivalve veligers from
ingestion by C. quinquecirrha medusae. The rapid rejec-
tion of veligers from the oral arms suggests that medusae
either do not recognize veligers as food items because of
the shell, or that veligers provide a "distasteful" stimulus.
Larvae of an echinoderm (Acanthaster planci) and an as-
cidian (Ecteinascidia turbinata) contain chemicals that
make them unpalatable to planktivorous fishes (Lucas et
a/.. 1979; Young and Bingham, 1987).

The sensing and recognition of food must take place
in the oral arms of the medusae, as indicated by the dif-
ferences in ingestion of copepods and veliger larvae. This
recognition may involve a mechanical stimulus from ac-
tive prey, as suggested by the facts that more living, active
copepods were ingested than dead ones, and that immobile
veligers nearly always were rejected. Recognition also may
be due to chemical stimuli, because more open oyster
veligers. which presumably leaked body fluids, were in-
gested than closed ones. The various bivalve species also
may present different stimuli, as suggested by the different
retention times of oyster, mussel, and clam veligers in the
medusae.

The larval shell probably protects veligers from diges-
tion as long as they remain closed within the predators.
Veligers were retained for up to 7 h in medusae, and then
egested alive. Veligers were removed alive from scyphis-
tomae after 1 8 h, but closed oyster pediveligers eventually
were digested in over 24 h. By contrast, newly killed ve-
ligers with open shells were digested by scyphistomae in
3 to 5 h. Therefore, open veligers apparently are more
susceptible to digestion than closed ones. Digestion of
some veligers may be due to their injury by the scyphis-
tomae's nematocysts at capture, causing the shells to open.
Presumably, this also could explain why a few veligers
were digested by the medusae.

Suspension-feeding benthic invertebrates can be im-
portant predators of pelagic larvae (Thorson, 1946). Bi-
valve larvae have been found in the stomach contents of
their own and other bivalve species (summarized in Mil-
eikovsky, 1974; Young and Chia, 1987). However, oyster
larvae taken into the mantle cavities of six mollusk species
were rejected in the pseudofeces, from which they may
be able to escape (MacKenzie, 1981). A few veligers were
ingested and eliminated in the feces of these mollusks,
from which they could not escape (MacKenzie, 1981).

PREDATION ON BIVALVE LARVA!

109

Oyster veligers also were rejected unharmed by a barnacle
(Balanus eburneus) and a polychaete (Polydora ligni) (in
MacKenzie, 1981), but the common barnacle (Balanus
improvisus) ate oyster veligers in Chesapeake Bay (Stein-
berg and Kennedy, 1979).

From earlier studies, Mileikovsky ( 1 974) concluded that
bivalve veligers often could pass alive through the guts of
primarily herbivorous feeders. However, no larvae were
known to pass alive through primarily carnivorous feeders,
although protectively coated gametes of a polychaete
(Melinna palnmta) passed through fish (Acipenser stella-
tus) feeding on the adult worms (in Mileikovsky, 1974).
Numerous examples exist of benthic cnidarians feeding
on bivalve veligers (Young and Chia, 1987). Also, oyster
veligers were eaten by the common sea anemone Dia-
dumene leucolena in Chesapeake Bay (Steinberg and
Kennedy, 1979). To our knowledge, our study presents
the first evidence of bivalve veligers passing alive through
a carnivorous predator, the medusa stage of Chrysaora
quinquecirrha.

The diets of several species of pelagic cnidarians are
reported to include bivalve veligers, but the numbers of
veligers in siphonophores (Purcell, 1981) and hydrome-
dusae (reviewed in Purcell and Mills, 1988) usually were
less than 1% of the prey items. Similarly, the scyphome-
dusae Aurclia aurita and Stomolophux meleagris in the
Gulf of Mexico, and Mastigias sp. in Jellyfish Lake. Palau,
contained small numbers of bivalve veligers (Purcell, un-
pub. data). However, bivalve veligers were 25 to 67% of
the prey in the hydromedusan Proboscidactylaflavicirrata
(Purcell and Mills, 1988), and 40 to 80% of the prey in
the scyphomedusan Cyanea sp. (Brewer, 1989). None of
the above studies distinguished between digested or un-
digested veligers.

The importance of predation on oyster larvae by scy-
phistomae in nature is difficult to predict because there
are few density estimates for scyphistomae or for oyster
veligers near the estuary bottom. Only 2.8 ± 3.1% of oyster
shells had scyphistomae in the York River, Virginia
(Cones and Haven, 1969). One third of those shells had
an average of more than 10 scyphistomae per shell (max-
imum 2 1 ), and densities were <1 to 53 scyphistomae m"2
of bottom. However, 53.4 ± 25.3% of oyster shells con-
tained scyphistomae in eleven tributaries of the Chesa-
peake Bay in Maryland, and 70% of those shells had more
than 10 individuals (maximum 200; Cargo, unpub. data).
Predation by scyphistomae on oyster veligers in those
tributaries probably would be higher than in the York
River.

The predation rate of one oyster veliger scyphistoma"1
day"1 from our laboratory experiments should be applied
to field conditions with caution, because the experimental
larval densities (100-700 1~', mean 300 I"1) were generally
high in comparison with densities of pediveligers in bot-

tom waters. Oyster veliger densities were generally less
than 14 T ' near the bottom in Broad Creek and the Tred
Avon River, but one sample had 134 1' (Seliger el a!..
1982). Densities of oyster veligers > 200 nm long were
23 to 2 1 5 r ' near the bottom in the James River, Virginia
(Andrews, 1983). Mortality in our laboratory experiments
could be higher than in the field because veligers that were
expelled undigested by scyphistomae in our experiments
could have been repeatedly ingested, eventually resulting
in death, while veligers in nature might have escaped.

Molluscan trochophore larvae lack a shell, and are
probably vulnerable to predation by all life history stages
of C. quinquecirrha. We could only follow the fate of
trochophores offered to ephyrae, which did ingest and di-
gest them. In nature, trochophores may be distributed
throughout the water column, and may seldom encounter
benthic scyphistomae. Although medusae do consume
some copepod nauplii and rotifers of the same size as
trochophores (about 60 ^m), such small animals were only
a few percent of the prey items (Purcell, unpub. data).
Therefore, medusae probably do not capture many
trochophores in nature. Depending on temperature, the
trochophore stage lasts only 24 to 30 h, so this period of
vulnerability to predators is short, compared with the 6
to 1 8 day veliger stage of various bivalve species (Loosanoft"
and Davis, 1963). Ctenophores readily ingested and di-
gested veligers, and they probably also eat trochophores,
because they consume many copepod nauplii (Purcell,
unpub. data) and ciliates (Stoecker et a/.. 1987) of the
same size.

Quaglietta ( 1987) studied potential predation by Alne-
miopsis leidyi on larvae of the hard clam Mercenaria
mercenaria in Great South Bay, New York. Clam veligers
and Ctenophores co-occurred in July through December,
and were most abundant in August through September.
Ctenophore feeding reached a maximum in September,
with an average of 1 1 and 36% of the water cleared of
prey per day in 1985 and 1986, respectively. Both the
biomass of Ctenophores and their estimated predation
on veligers were greater during Quaglietta's (1987) study
in Great South Bay than during our study in Chesa-
peake Bay.

Predation on bivalve veligers by M. leidyi during our
study was apparently limited to Chesapeake Bay, because
the Ctenophores were not found in Broad Creek and Tred
Avon River after the appearance of C quinquecirrha me-
dusae in June. Predation by medusae on M. leidyi also
may have reduced ctenophore densities in the main Bay.
We conclude that not only do C. quinquecirrha medusae
not consume bivalve veligers. but the medusae may reduce
other predation on them by feeding on Ctenophores.

In the mesohaline region of Chesapeake Bay, C. quin-
quecirrha medusae are present during June through Sep-
tember or October (Cargo and Schultz, 1966). Therefore,

J. E. PURCELL /•/ II

medusae could reduce ctenophore predation on veligers
ofCrassostrea virginica, as well as other bivalves such as
Ischadium recumtm Raftnesque, Macoma nuic/ielli Dall.
Mulinia lalcralis. Mytilopsis congeria (Conrad), and Ta-
gflux plebeius (Lightfoot) which spawn throughout the
summer (Shaw, 1965; Kennedy, pers. obs.). However, bi-
valve species that spawn only in the spring and autumn
in Chesapeake Bay, e.g.. Macoma balthica (L.) and Mya
arenaria (L.) (Shaw, 1965), would be most vulnerable to
predation by M. Icidyi.

Acknowledgments

We thank T. Dean, C. Densmore, C. Kalafus, L. Hill,
and V. Steele-Perkins for their excellent assistance in the
laboratory, and C. A. Miller and J. R. White for their
comments on the manuscript. We are also grateful to Drs.
R. I. E. Newell, G. S. Alspach, and T. C. Malone for
allowing us to sample during their cruises, to Dr. J. H.
Waite of the College of Marine Studies of the University
of Delaware for providing mussel veligers, and to Dr.
M. R. Roman and J. R. White, who provided data on
bivalve veliger densities from Chesapeake Bay. This re-
search was partially funded by the Maryland Department
of Natural Resources, and the NSF Research Experiences
for Undergraduates Grant OCE-8900707 to the University
of Maryland Sea Grant College. UMCEES Contribution
No. 2175.

Literature Cited

Andrews, J. D. 1983. Transport of bivalve larvae in James River, Vir-
ginia. / Shellfish Res. 3: 29-40.

Bishop, J. \V. 1967. Feeding rates of the ctenophore, Mnemiopsis Icidyi.
Chesapeake Sci. 8: 259-261.

Brewer, R. H. 1989. The annual pattern of feeding, growth, and sexual
reproduction in Cyanea (CnidariaiScyphozoa) in the Niantic River
Estuary, Connecticut. Biol. Bull. 176: 272-281.

Burrell, V. G., Jr. 1968. The ecological significance of the ctenophore
(Mnemiopsis leiityi A. Agassiz) in a fish nursery grounds. M. S. Thesis.
College of William and Mary. Williamsburg. VA. 61 pp.

Burrell, V. B., Jr., and W. A. Van Engel. 1976. Predation by and dis-
tribution of a ctenophore, Mnemiopsis Icidyi A. Agassiz, in the York
Estuary. Estuarine Coastal Mar. Sci. 4: 235-242.

Cargo, D. G., and I.. P. Schultz. 1966. Notes on the biology of the sea
nettle. Chrysaora quinquecirrha, in Chesapeake Bay. Chesapeake Sci.
7:95-100.

Cargo, D. G., and I.. P. Schultz. 1967. Further observations on the
biology of the sea nettle and jellylishes in Chesapeake Bay. Chesapeake
Sci. 8: 209-220.

Clifford, H. C., and D. G. Cargo. 1978. Feeding rates of the sea nettle
Chrysaora quinquecirrha, under laboratory conditions. Estuaries I:
58-61.

Cones, H. N., Jr., and D. S. Haven. 1969. Distribution of Chrysaora
quinquecirrha in the York River. Chesapeake Sci. 10: 75-84.

Cronin, L. E., J. C. Daiber, and E. M. Ilulbert. 1962. Quantitative
seasonal aspects of zooplankton in the Delaware River estuary. Ches-
apeake Sci. 3: 63-93.

Deason, E. E., andT. J. Smayda. 1982. Ctenophore-zooplankton-phy-

toplankton interactions in Narragansett Bay, Rhode Island, USA,

during 1972-1977. Plankton Res. 2: 203-218.
Fancett. M. S. 1988. Diet and prey selectivity of scyphomedusae from

Port Phillip Bay, Australia. Mar. Biol. 98: 503-509.
Feigcnbaum, D., and M. Kell). 1984. Changes in the lower Chesapeake

Bay food chain in presence of the sea nettle, Chrysaora quinquecirrha

(Scyphomedusa). Mar. Ecol. Prog. Ser. 19: 39-47.
Herman, S. S., J. A. Mihursky, and A. M. McErlean. 1968.

Zooplankton and environmental characteristics of the Patuxent River

estuary 1963-1965. Chesapeake Sci. 9: 67-82.
Kremcr, P. 1979. Predation by the ctenophore Mnemiopsis Icidyi in

Narragansett Bay. R. I. Estuaries 2: 97-105.
Kenned), V. S., and L. L. Breisch. 1981. Mary/anil's Oysters: Research

and Management. Univ. of MD Sea Grant Program. College Park.

MD Publ. No. UM-SG-TS-81-04.
Larson, R. J. 1978. Aspects of feeding and functional morphology of

scyphomedusae. M. S. Thesis, University of Puerto Rico. 132 pp.
Larson, R. J. 1986. The feeding and growth of the sea nettle. Chrysaora

quinquecirrha (Desor), in the laboratory. Estuaries 9: 376-379.
Larson, R. J. 1987. A note on the feeding, growth, and reproduction

of the epipelagic scyphomedusa Pelagia nocliluca (Foskal). Biol.

Oceanogr 4: 447-454.
Loosanoff. V. L. 1974. Factors responsible for the mass mortalities of

molluscan larvae in nature: a review. Proc. Ann. Mlg. World Mari-

cultitre Soc. 5: 297-309.
LoosanofT, V. L., and II. C. Davis. 1963. Rearing of bivalve mollusks.

,\d\ Mar. Biol. \. 1-135.
Lucas, J. S., R. J. Hart, M. E. Howden, and R. Salathe. 1979. Saponins

in eggs and larvae of Acanthasler planci (Asteroidea) as chemical

defenses against planktivorous fish. / Exp. Afar. Biol. Ecol. 40: 155-

165.

Mackenzie, C. L., Jr. 1981. Biotic potential and environmental resis-
tance in the American oyster (Crassostrea virginica) in Long Island

Sound. Aatiacttliitre 22: 229-268.
Mileikovsky, S. A. 1974. On predation of pelagic larvae and early

juveniles of marine bottom invertebrates by adult benthic inverte-
brates and their passing alive through their predators. Mar. Biol 26:

303-311.
Miller, R. J. 1974. Distribution and biomass of an estuarine ctenophore

population, Mnemiopsis leidyi (A. Agassiz). Chesapeake Sci. 15: 1-8.
Nelson, T. C. 1925. On the occurrence and food habits of ctenophores

in New Jersey inland coastal waters. Biol. Bull 48: 92-1 1 1.
Nelson, T. C. 1953. Some observations on the migrations and setting

of oyster larvae. Proi: .\'all. Shellfish. Assoc. (1952): 99-104.
Olson, M. M. 1987. Zooplankton. Pp. 38-8 1 in Ecological Studies in

the Middle Reach ol Chesapeake Bay. K. L. Heck. Jr., ed. Springer

Verlag. Berlin.
Purcell, J. E. 1981. Dietary composition and diel feeding patterns of

epipelagic siphonophores. Mar. Biol. 65: 83-90.
Purcell, J. E. 1988. Quantification of Mnemiopsis leidyi (Ctenophora,

Lobata) from formalin preserved plankton samples. Mar. Ecol. Prog.

Ser. 45: 197-200.
Purcell, J. E., and C. E. Mills. 1988. The correlation between nema-

tocyst types and diets in pelagic Hydrozoa. Pp. 463-485 in The Bi-
ology of Nematocyst.s. D. A. Hessinger and H. M. Lenhoff. eds. Ac-
ademic Press, Inc.. San Diego.
Quaglietla, C. E. 1987. Predation by Mnemiopsis Icidyi on hard clam

larvae and other natural zooplankton in Great South Bay. N.Y.

M. S. Thesis, State University of New York, Stony Brook. 66 pp.
Ritchie, T. P., and R. W. Menzel. 1969. Influence of light on larval

settlement of American oyster. Proc. Nail. Shellfish. Assoc. 59: 1 16-

120.

PREDATION ON BIVALVE LARVAE

Seliger, H. H., J. A. Boggs, R. B. Rivkin, W. H. Biggley, and K. R. II.

Aspden. 1982. The transpon of oyster larvae in an estuary. Mar.

Biol 71: 57-72,
Shaw, \V. N. 1965. Seasonal setting patterns of five species of bivalves

in the Tred Avon River. Maryland. Chesapeake Set. 6: 33-37.
Steinberg, P. D., and V. S. Kennedy. 1979. Predation upon Crassostrea

virginicu (Gmelin) larvae by two invertebrate species common to

Chesapeake Bay oyster bars. I 'eliger 22: 78-84.
Sloecker, D. K., P. G. Verity, A. E. Michaels, and I.. H. Davis.

1987. Feeding by larval and post-larval ctenophores on microzoo-

plankton. / Plankton Res 9: 667-683.
Thorson, G. 1946. Reproduction and larval development of Danish

marine bottom invertebrates with special reference to planktomc lar-

vae in the Sound (Oresund). Meddr Damn l-'isk. -og Havunders.
(Ser. Plankton) 4: 1-523.

I ruitt, R. V., and P. V. Mook. 1925. Oyster problem inquiry of Ches-
apeake Bay. Third Ann Rc;> Consent. Dept oj MD Pp. 25-55.

Young, C. M., and B. 1,. Bingham. 1987. Chemical defense and apo-
sematic coloration in larvae of the ascidian Ecteinascidin turbinata
Mar. Bio/ 96: 539-544.

Young, C. M., and K.-S. C'hia. 1987. Abundance and distribution of
pelagic larvae as influenced by predation, behavior, and hydrographic
factors. Pp. 385-463 in Reproduction of Marine Invertebrates,
\'ol. IX General aspects: seeking unity in diversity. A. C.
Giese, J. S. Pearse, and V. B. Pearse. eds. Boxwood Press, Pacific
Grove, CA.

Reference: Biol. Bull 180: 112-118. (February, 1991)

Settlement, Refuges, and Adult Body Form in Colonial
Marine Invertebrates: A Field Experiment

LINDA J. WALTERS' AND DAVID S. WETHEY12

1 Department of Biological Sciences and -Marine Science Program, University of South Carolina.

Columbia, South Carolina 29208

Abstract. We examine the relationship between adult
body form (sheet vs. arborescent) and larval settlement
in colonial animals. Because thin sheet forms are more
susceptible to overgrowth than arborescent forms, we
predict that larvae of sheet forms should preferentially
settle in refuges from competitors. On both natural and
artificial substrata, the larvae of the sheet form (Mem-
branipora membranacea) settled more often on high spots,
which could serve as refuges from competition. The ar-
borescent forms (Bitgula ncrilina and Distaplia occiden-
talis) settled around the bases of bumps more frequently
than would be expected by chance. For many arborescent
forms, their most vulnerable periods are the days im-
mediately following settlement, when individuals can be
consumed easily by predators or dislodged by physical
disturbances. Settlement in a crevice (base of a bump)
would provide protection from the bulky mouthparts of
predators. Moreover, dislodgment would be less likely
than if settlement had occurred on flat locations, such as
the tops of bumps or the areas between bumps.

Introduction

Striking patterns of spatial distribution are characteristic
of many marine invertebrates sessile on algae, rocks, and
other hard surfaces. Individuals are often found in aggre-
gations relative to each other (e.g., Knight-Jones, 1951;
Crisp, 1961; Wethey, 1984), relative to topographic fea-
tures of the substrata (e.g., Crisp and Barnes, 1954; Ry-
land, 1959; Crisp, 1961; Wisely, 1960; Hayward and
Harvey, 1974;Keoughand Downes, 1982; Wethey, 1986;
LeTourneux and Bourget, 1988), or relative to microflora
(e.g.. Crisp and Ryland, 1960; Brancato and Woollacott,
1982; Strathmann et ai. 1981). These patterns may arise

Received 27 July 1990; accepted 29 November 1990.

at the time of larval settlement or develop later as a result
of differential mortality. The distribution of individuals
at the time of larval settlement has a strong influence on
their future success. Individuals that settle near dominant
competitors are more likely to die quickly, as are those
that settle within the range of predators or where distur-
bance events frequently occur.

There are a number of potential escapes from sources
of biotic mortality, including simple avoidance of settle-
ment near enemies (e.g.. Grosberg, 1981; Young and Chia,
1981) and recruitment to spatial refuges (e.g., Connell,
1961; Dayton. 1971; Paine, 1974; Wethey, 1983; Walters
and Wethey, 1986). Organisms located in spatial refuges
increase their chances of survival against competitors,
predators, and disturbance events. Size can also be pro-
tective to colonies once they have grown to certain di-
mensions unaffected by competitors; this is the size refuge.
Potential morphological escapes may also exist. Among
colonial organisms attached to hard substrata, one can
distinguish a number of morphological types, including
sheet and tree forms (Jackson, 1979). The outcomes of
competitive interactions can be strongly influenced by the
morphologies of the competitors. Tree forms are relatively
isolated from the substratum-associated competitors
(Jackson, 1979; Grosberg, 1981), whereas sheet forms en-
crust the substratum and may suffer competitive inter-
actions along their edges. Thin sheets tend to lose to
thicker forms (Buss, 1980; Seed and O'Connor, 1981;
Russ, 1982; Sebens, 1985, 1986; Walters and Wethey,
1986) unless they have a height advantage in the zone of
contact (Walters and Wethey, 1986). Therefore, one would
predict that animals with thin, sheet-like growth forms
should preferentially settle on or near locations where they
have a height advantage (Walters and Wethey, 1986).

Although tree forms are less likely to be overgrown by
competitors, they can be more visible to predators and

112

INVERTEBRATE SETTLEMENT REFUGES

113

are more susceptible to total colony mortality than sheet
forms. On irregular substrata, a potential settlement refuge
location would be found around the bases of bumps. Here,
certain predators may not be able to reach newly settled
individuals. Here they are also protected from more dis-
turbance events than they would be if they were located
on a flat surface or on the top of a bump.

We examined the patterns of larval settlement in three
species of encrusting colonial animals with different
growth forms. We asked whether the settlement patterns
were consistent with our prediction that species with thin
sheet morphologies should choose spatial refuges from
competitors, whereas species with tree morphologies
should choose refuge locations that would reduce the risk
of predation and disturbance. The encrusting cheilostome
bryozoan Membranipora membranacea was our example
of a thin sheet morphology, and the arborescent bryozoan
Bugiila neritina and the pedunculate ascidian Distaplia
occidentalis were our examples of tree morphologies. We
examined two kinds of substrata. The kelp Laminaria
saccharina is a substratum commonly colonized by all
three species. Settlement plates cast from bumps on Lego
toy building blocks and pits created from bubble plastic
served as model topographies of the same spatial scale as
those found on Laminaria. Our analysis was carried out
in two phases: ( 1 ) we examined the extent to which set-
tlement on our model substrata mimicked that on natural
surfaces; and (2) we examined in detail the spatial pattern
of settlement on the model substrata.

Materials and Methods

Sludy organisms

The bryozoans Membranipora membranacea and
Bugiila neritina have small, ciliated larvae (Membrani-
pora: 750 urn; Bugiila: 200 ^m, from Reed, 1987) that
have limited swimming abilities in the ocean (Chia et ai,
1984). However, these larvae can choose their settlement
locations. When competent, they move closely over the
substrata and test it (Woollacott and Zimmer, 1978, for
Bugiila: Atkins, 1955, for Membranipora). During this
phase, Bugit/a larvae form temporary attachments using
adhesives that are sufficiently strong to prevent the indi-
vidual from being mechanically dislodged (Loeb and
Walker, 1977). Bugiila can quickly dissolve the adhesive
or change its viscosity to detach from, or reject the surface
(Reed and Woollacott, 1982).

In the plankton, Distaplia occidentalis larvae are much
larger than those of the other two species, measuring up
to 3.2 mm in length (Cloney and Torrence, 1984). Most
encounter a number of surface locations before meta-
morphosing on one of them (R. A. Cloney, pers. comm.).
Torrence and Cloney (1988) suggest that sensory neurons
in the adhesive papillae may be common in ascidians. In

the laboratory, adhesion in Distaplia occurs within 30 s
at 15°C (Cloney, 1978). Tail resorption reduces the size
of the newly settled individual to approximately 650 ^m
within 7 min (Cloney, 1978).

For the purposes of this study, it was important to dis-
tinguish between newly settled and metamorphosed in-
dividuals. Newly metamorphosed Membranipora colonies
have only the twin ancestrula skeleton fully formed, and
Bugiila has only the first zooid skeleton completed. Dis-
laplia colonies were considered new individuals if they
occupied less than 1 mm2.

Experimental procedure

To study larval settlement on natural substrata, we ex-
amined the alga Laminaria saccharina. Plants were col-
lected on the floating docks at the Friday Harbor Labo-
ratories, San Juan Island, Washington state (48° 32' 42"
N; 123° 0' 39" W) and on the floating public docks at
Fisherman's Bay on Lopez Island, Washington state (48°
30'30"N; 122° 54'51"W). Entire blades were either placed
in running seawater tables and a census taken within 48
h, or frozen immediately for a later census. Random pieces
of the alga (20 X 20 cm) were cut from the central portion
of large ( 1 .0-2.0 m in length) Laminaria fronds. All new
settlers were recorded on each algal square. As the topog-
raphies of the blades are quite variable, we could not dis-
tinguish a pit from a bump. Instead, each topographical
feature on the blade was defined as a continuous slope
extending from a lowest to a highest point (Fig. 1). The
lowest point on one side of an algal blade is the highest
point on the reverse side. The diameter (base) and the
height of each topographic feature were recorded with
vernier calipers. The slopes ranged in length from 1 to 20
mm. The location of each animal was determined by cre-

Hiqhest point

Height of topogrophic
feoture (h)

Distonce from lowest point to animal

Diameter of topographic feature (d)

Figure I. Each colony was mapped in relation to the nearest topo-
graphic high and low point. The dimensions of the topographic feature
were measured.

L J. WALTERS AND D. S. WETHEY

ating a right triangle with the animal location and the
lowest point as two of the points (Fig. 1 ). The distance
from the lowest point to the animal and the animal height
above the lowest point were measured (Fig. 1 ).

Using the diameter (d) and height (h) of each topo-
graphic feature, we calculated:
( 1 ) the radius of curvature (re) of the topographic feature:

re =

h2 + (d/2)

2*(1 + d)*(d/(2*h))

(2) the vertical position (vp) of the animal, which we use
to determine the location (top, side or base) of the organ-
ism on the topographic feature:

vp = (animal height/h).

Wilcoxon rank sum tests were used to determine if there
were differences in locations occupied by larvae of the
three common species. We examined the effects of size
and shape of topographic features (height, diameter, and
radius of curvature) as well as larval position (animal
height and vertical position). When differences were
found, pairwise Wilcoxon rank sum tests were run to de-
termine which species were significantly different. Data
from the Friday Harbor Laboratories and Lopez Island
were pooled after Wilcoxon rank sum tests showed that
there were no differences between the two sites.

To model the kinds and size scales of topographic fea-
tures found on natural substrata, such as the alga Lami-
naria saccharina, we constructed three types of plastic
plates 8.9 cm in diameter: (1) small Lego (Lego Systems
Inc.) building block bumps (cylindrical, 2 mm high, 5
mm diameter) simulated small algal bumps; (2) large Lego
building block bumps (cylindrical, 5 mm high, 9 mm di-
ameter) simulated large algal bumps; and (3) bubble plastic
pits (hemispherical, 2 mm deep, 5 mm diameter) simu-
lated small algal pits. These materials were used because
their topographic features were of the appropriate spatial
scale and were uniformly spaced. We produced settlement
plates by pouring polyester resin into silicone rubber
molds (Sylgard 184 Silicone Elastomer, Dow Corning
Corp.). Black resin pigment (Titan Corp.) was added to
the uncatalyzed resin to make newly settled larvae more
visible on the plates.

The settlement plates were attached to wooden boards
with stainless steel screws. These were hung beneath the
floating docks with polypropylene rope. The plates were
oriented face down to prevent algal colonization. Six rep-
licates of each surface were submerged in each trial. Plates
were arranged in a Latin square design, with one replicate
of each type of plate on each board. Six trials were run
during the summers of 1987 and 1989.

Photographs were taken every two days at the Friday
Harbor Laboratories and once or twice a week at Lopez
Island during 1987. Additional data were collected by di-

rect observation at Lopez Island in 1989. Flash-lit pho-
tographs were taken underwater using Kodak Technical
Pan 2415 film and a Nikonos 5 camera equipped with a
5:1 extension tube and focal framer. Negatives were ob-
served under a dissecting microscope equipped with an
ocular micrometer to determine the specific locations of
newly settled individuals. We distinguished among four
kinds of locations on the plates with bumps: (1) top of
bump; (2) side of bump; (3) touching the base of the bump;
and (4) on the flat surface not touching the base of the
bump. On the pitted surface, we distinguished among
three kinds of locations: ( 1 ) in the pit; (2) touching the
edge of the pit, and (3) on the flat surface not touching
the pit. Individual larvae were scored as touching a to-
pographic feature if they were within 250 ^m of the fea-
ture. This distance represents approximately one body
length of the settled larvae (200 to 750 urn in length).

To determine whether larvae settled preferentially in
relation to topographic features, we compared our obser-
vations to a random distribution. For example, if larvae
settled randomly, then the proportion of larvae settling
in pits should be equal to the proportion of space ac-
counted for by pits. In this way we calculated the number
of larvae expected to settle in each of our classes of lo-
cations (on or in pits or bumps, touching pits or bumps,
away from pits or bumps). Paired simultaneous /-tests
were used to compare the observed versus expected num-
ber of individuals in each location on a settlement plate.
The simultaneous /-tests were weighted because the esti-
mates of proportions of larvae were all based on samples
of different sizes. The estimate p of a proportion has a
gaussian distribution with a variance p( 1 - p)/N, where
N is the sample size (Snedecor and Cochran, 1967: p.
208). We weighted our estimates by the reciprocal of this
variance because we have higher confidence in estimates
with the lowest variance. Plates with less than two indi-
viduals were not included. We used the Bonferroni in-
equality to make the tests simultaneous (Miller, 1966).
For example, when we compared three settlement loca-
tions, to maintain an overall error rate of 0.05, we used
an error rate of 0.05/3 = 0.016 in each individual com-
parison.

To determine whether settlement preference changed
as space became occupied, we examined the relationship
between the proportion of larvae settling in the feature
and the proportion of unoccupied space accounted for by
that feature. On all dates we calculated the space available
for settlement by subtracting from the total the area oc-
cupied by settled individuals. We assumed that all newly
metamorphosed larvae occupied 1 mm2. We compared
settlement in samples with more than the average amount
of free space, to settlement in samples with less than the
average amount of free space.

INVERTEBRATE SETTLEMENT REFUGES

115

Stoloniferous hydrozoan (primarily Obelia dichotoma
and Obelia geniculata) and entoproct (Barentxia hcncdcni)
colonies were present on all of the plates within 10 days,
and at least a few stolons rapidly covered the entire surface
of most plates. To determine whether the stolons affected
settlement of Bugula, Distaplia, and Membranipora, the
tops of the Lego bumps were divided into ten pie-shaped
wedges. Similarly, the bases of the Lego bumps were di-
vided into ten equal sections. If settlement was random
with respect to stolons, then the ratio of wedges where
stolons and larvae co-occur, to wedges with larvae, should
equal the ratio of wedges with stolons to total wedges.
Paired simultaneous /-tests were used to determine
whether the observed and expected ratios were equal.

Very few individuals of other species settled on our
experimental plates. Approximately 75% of the plates of
each type had no other species settling on them. The re-
maining 25% had an average of two individuals of other
species on them. These other species included: the bryo-
zoans Tegella armifera and Schizoporella itnicornis, the
ascidian Diplosoma macdonaldi, the barnacle Balanus
crenatus, the serpulid polychaete worm Pseudochitono-
ponui occidentalis, and spirorbid polychaetes.

Results

Natural alga substrata

Bugula nentina, Distaplia occidentalis, and Membran-
ipora membranacea settled in locations with similar di-
ameters and radii of curvature (Table I). Bugula and Dis-
taplia settled in significantly lower elevations relative to
topographic features than did Membranipora (Table I:
Vertical Position). Bugula settled on topographic features
that were significantly taller than those on which the other
two species settled (Table I).

Settlement plate experiments

On the Lego settlement plates, settlement was non-ran-
dom for all species (Table II). Distaplia and Bugula were
found most often around the bases of bumps (Table II).
These locations covered less than 5% of the total surface
area of the settlement plates, yet more than 50% of the
larvae of Distaplia and Bugula settled there.

Both arborescent forms, Distaplia and Bugula, were
found significantly less often than expected on flat surfaces
of the large and small Legos and the flat surfaces of plates
with small pits (Table II). Distaplia settled more than ex-
pected by chance in the pits. In contrast, Bugula signifi-
cantly avoided pits (Table II). The sheet form, Membran-
ipora, was found more than expected on the tops of bumps
and on the flat surfaces away from the topographic features
in the large Lego treatment, but less than expected around
the bases of bumps (Table II). On the pitted surfaces.

Table I

Settlement location* o/Membranipora membranacea. Bugula neritina
and Distaplia occidentalis mi the alt;a Laminaria sacchanna

Species

Mean

Group

Height of Topographic Feature

Bugula

64 9.22

Distaplia

95 7. XI

Membranipora

147 7.80

Bugula

Distaplia

Mcmhranipora

Diameter of Topographic Feature
64

147

26.30
28.13
27.12

Radius of. Curvature of the Topographic Feature

Bugula

Dislaplia

Membranipora

Biiguta

Distaplia

Membranipora

Bugula

Distaplia

Membranipora

64
95
147

213.62
446.45
453.29

Animal Height Above Lowest Point

64
95
147

1.80

1.44
5.32

Vertical Position of the Animal

147

0.23
0.18

0.71

A
A
A

A
A
B

N = the number of individuals. Mean = the mean in millimeters, and
Group = the results of Wilcoxon rank sum tests. Different letters refer
to significant differences (P < 0.05). For explanation of the measured
values, see Figure 1 and the text.

Membranipora settled significantly less than expected in
the pits and more than expected around the edges of the
pits. Bugula and Distaplia settled preferentially around
the bases of bumps, while Membranipora appeared to
avoid this location. To estimate whether there was pre-
emption of space by Bugula and Distaplia, we compared
Membranipora settlement in samples with more than the
average percent free space to settlement in samples with
less than the average. Free space around the bumps de-
creased during the settlement season from 2.0% to 1.5%
on the small Legos and from 4.3% to 3.6% on large Legos.
Membranipora settlement was independent of availability
of free space on both large Lego plates (F = 0.24; d.f. = 1,
23: P = 0.63). and small Lego plates (F = 0.16; d.f. = 1,
19; P = 0.69).

The settling larvae were not affected by the presence of
hydrozoan or entoproct stolons (Table III). The larvae
neither preferentially settled in locations where stolons
were present nor did they significantly avoid these loca-
tions.

116

L. J. WALTERS AND D. S. WETHEY

Table II

Test oi f randomness of settlement locations: the results of simultaneous
paired l-lests comparing the i:\peclcel versus
the observed number ot settlers

Species

Location

N Difference

S.E.

Sign.

Large Lego

Bugula

Top

-2.14

0.33

Less

Base

5.28

0.64

Flat

-2.60

0.96

Less

Di.itaplia

Top

-2.60

0.36

Less

Base

11.05

1.33

Flat

1.18

0.46

Less

Membranipora

Top

5.47

1.16

Base

-0.65

0. 1 3

Less

Rat

3.92

0.57

Small Lego

Bugula

Top

-2.75

0.43

Less

Base

7.01

1.30

Flat

-2.57

0.77

Less

Distaplia

Top

-4.05

0.49

Less

Base

15.78

1.99

Rat

-5.67

0.90

Less

Membranipora

Top

1.21

0.88

n.s.

Base

-0.20

0.08

n.s.

Rat

1.26

0.84

n.s.

Small Pits

Bugula

Pit

1 1

-1.86

0.60

Less

Edge

3.22

0.96

Flat

1 1

-1.51

0.43

Less

Distaplia

Pit

3.99

0.82

Edge

1.38

0.29

Flat

-4.57

0.85

Less

Membranipora

Pit

-3.23

0.78

Less

Edge

1.64

0.52

Flat

2.15

0.95

n.s.

N = the number ot plates on which at least two larvae settled; Difference
= the mean for N plates of the observed - expected values; S.E. = the
standard error of the Difference; and Sign. = the direction of the signif-
icance value with n.s. = not significant (P > 0.05). A Bonferonni com-
parisonwise error rate of 0.016 was used to keep the expenmentwise
error rate = 0.05.

Discussion

In this study we examined the relationship between
larval settlement pattern and adult growth form in colonial
epifauna on hard substrata. We asked whether larvae of
species with thin sheet morphologies chose different set-
tlement locations from those of larvae of species with ar-
borescent morphologies. We argued that species with thin
sheet growth forms should be more susceptible to over-
growth by competitors than species with tree morpholo-
gies. Because topographic high spots may serve as spatial

refuges from competitors (Walters and Wethey 1986), we
expected species with thin sheet morphologies to settle
preferentially on topographic high spots.

In the present study, the thin sheet species, Membran-
ipora membranacea. preferentially settled on the highest
available locations on topographically complex surfaces
(tops of bumps and flat areas between pits: Table II). This
is consistent with our predictions, because the tops of
bumps and the flat areas on a pitted surface are both lo-
cations where a colony has a height advantage over com-
petitors, and thus has a potential refuge from competition.
This result indicates that physical cues may allow larvae
to escape from competitors, much as biogenic cues (e.g.,
Grosberg, 1981; Young and Chia, 1981) allow larvae to
avoid recruitment near enemies.

We argued that species with arborescent growth forms
should be relatively immune to competitors, but that they
might suffer damage from mobile predators like fish. In
North Carolina, for example, filefish feed voraciously on
newly settled colonies of Bugula stolonifera growing on
flat surfaces (L.J.W., pers. obs.). Thus, tree forms might
be expected to settle in cracks and crevices. In the present
study, the arborescent forms, Distaplia occidentalis and
Bugula neritina. settled preferentially around the bases of
topographic irregularities (Table II). This result is consis-
tent with our predictions, because the bases of bumps on
our experimental plates are the locations most like crev-
ices.

Similar spatial partitioning occurred on the alga Lam-
inaria saccharina (Table I). Arborescent Distaplia and
Bugula were found low on the slopes of the alga, while
the thin sheet Membranipora was found significantly
higher (Table I: Animal Height). The algal low spots.

Table III
Test of response of larvae to hydroid and enioproct stolons

Species

Location

Diff.

S.E.

P
Value

Sign.

Bugula

Top

0.00

N.A.

Bugula

Base

-0.53

0.50

0.3293

n.s.

Distaplia

Top

2.50

0.3910

n.s.

Distaplia

Base

-1.10

3.11

0.7243

n.s.

Membranipora

Top

3.25

2.82

0.2553

n.s.

Membranipora

Base

-2.14

8.86

0.8144

n.s.

If settlement is random with respect to stolons, then the ratio of wedges
with stolons and larvae, to wedges with larvae, should be equal to the
ratio of wedges with stolons to total wedges. N = the number of indi-
viduals; Diff. = the mean of the difference: [(wedges with stolons)/! total
wedges)] - [(wedges with larvae + stolons)/! wedges with larvae)]; S.E.
= the standard error of the Difference; and Sign. = the sign of the sig-
nificance value if a < 0.05. A negative difference denotes bumps that
had more larvae settling than it had stolons, and N.A. = not applicable.

INVERTEBRATE SETTLEMENT REFUGES

where Bitgula and Distaplia settled, are functionally
equivalent to the bases of Lego bumps and the pits in the
artificial settlement surfaces (Table II). Similarly, the high
positions on algal slopes where Membranipora settled are
functionally equivalent to the elevated locations where
they settled on the settlement plates (Table II). However,
topography does not fully control settlement pattern, be-
cause neither previously settled individuals, nor the stolon
mats of hydrozoans and entoprocts, affected settlement
by the larvae (Table III), even though the presence of any
organisms on the substratum alters the local microtopog-
raphy.

An alternative mechanism that could account for the
settlement patterns is passive transport of larvae by hy-
drodynamic forces. Because of their limited swimming
abilities (Chia el ai, 1981). larvae are often passively
transported in boundary layer flows (e.g., Butman, 1987).
One can model passive larval transport as analogous to
sediment transport (e.g., Middleton and Southward,
1984). The patterns of transport are influenced by the
turbulent motion of the water and by the topography of
the substratum. When the surface topography protrudes
beyond the 'viscous sublayer' into the turbulent overlying
water, turbulent eddies can cause erosion. The roughness
Reynolds number. Re*, is a measure of the degree to
which roughness elements protrude above the viscous
sublayer:

Re* = u*Lp/M

where u* is the shear velocity of the fluid flow regime, L
is the height of the roughness element, p is the density of
seawater, and fj. is the dynamic viscosity of seawater.

In a wave-influenced environment, u* is approximately
10% of the maximum water velocity (Denny, 1988; Denny
and Shibata, 1989; Svenden, 1987). We estimate u* to be
in the range of 1.6 to 2.4 cm/s, yielding Re* values of
30-50 for the small Legos and 75-120 for the large Legos.
If the roughness Reynolds number is less than 5, the
bumps lie within the viscous sublayer. Thus, in all cases,
the bumps on our settlement plates are in a potentially
erosional regime. Larvae differ from sediment particles in
their ability to adhere to surfaces. In tlume experiments
with our settlement plates, sediment never accumulated
on the tops of the Lego bumps, presumably because the
erosional forces are very high in these locations. Therefore,
if the pattern were passive, larvae would not have accu-
mulated on the tops of bumps. However, the tops of the
bumps are the locations where Membranipora larvae did
accumulate. Thus, we believe that the passive model can-
not explain our patterns.

Competitive interactions were infrequent on these set-
tlement plates, because recruitment rates were low and
space did not become limiting during our experiments.
The only common encounters were between the entoproct

and hydrozoan stolons and the three species, with the
later arrival always growing over the previously established
colony. Neither colony appeared to be affected by these
interactions. Although space was not filled on our settle-
ment plates during the time course of this study, little
bare space existed on the docks from which the plates
were suspended. Because so little free space existed on
the persistent hard substrata, we believe that competition
could act as a selective agent on larval behavior.

The results of these studies are consistent with our pre-
diction that adult body form should be correlated with
larval settlement pattern. The arborescent forms (Bugnla
and Distaplia) settled preferentially in the small amount
of space touching the bases of the bumps, potentially hid-
den from predators and disturbance events. The thin sheet
form (Alembranipora) settled most frequently on the
highest available locations on topographically complex
surfaces. Thus Membranipora, the adult growth form of
which is most susceptible to overgrowth, had larvae that
settled in potential refuges from competitors. Adult com-
petitive ability and susceptibility to predation and distur-
bance may be an important influence on selection for
larval settlement behavior.

Acknowledgments

This study was supported by the University of South
Carolina, grants from the Office of Naval Research (Con-
tract N00014-82-K-0645) and the National Science
Foundation (Grant OCE86-00531) to D. Wethey and
grants from the Lerner-Gray Fund for Marine Research,
Sigma Xi, and the International Women's Fishing As-
sociation to L. Walters. We are grateful to all at the Friday
Harbor Laboratories for providing us with space and fa-
cilities. D. Padilla, D. Pencheff, L. Muehlstein, A. Kettle,
S. Cohen, A. Sewell, and countless others assisted with
the field work. J. Sutherland and A. Underwood assisted
with the statistical analyses. S. Woodin, J. Sutherland, R.
Showman, and two anonymous reviewers made helpful
comments on the manuscript.

Literature Cited

Atkins, 1955. The cyphonautes larvae of the Plymouth Area and the
metamorphosis of Membranipora membranacea. J. Mar. Biol. Axsiv.
L' K 34:441-449.

Brancato, M. S., and R. S. VVoollacott. 1982. Effect of microbial films
on settlement of bryozoan larvae (Bugida simplex. B. slolonifera and
B n/rriui). .Mar, Bml. 71: 51-56.

Buss, L. W. 1980. Bryozoan overgrowth interactions — the interde-
pendence of competition for space and food. Nature 281: 475-477.

Butman, C. A. 1987. Larval settlement of soft-sediment invertebrates:
the spatial scales of pattern explained by active habitat selection and
the emerging role of hydrodynamic processes. Oceanogr. Mar Biol
Ann. Rev. 25: 113-165.

Chia, F-S., J. Buckland-Nicks, and C. M. Young. 1984. Locomotion
of marine invertebrate larvae: a review. Can. J Zoo/. 62: 1205-1222.

118

L. J. WALTERS AND D. S. WETHEY

Clone), R. A. 1978. Ascidian metamorphosis: review and analysis. Pp.
255-282 in Settlement ami Metamorphosis <>l Mamie Invertebrate
Larvae, F-S. Chia and M. E. Rice. eds. Elsevier. New York.

Cloney, R. A., and S. A. Torrence. 1984. Ascidian larvae: structure
and settlement. Pp. 103-126 in Marine Biodeterioration, J. D. Costlow
and R. C. Tipper, eds. Naval Institute Press. Annapolis.

Connell, J. H. 1961. The effects of competition, predation by Than
lapillus. and other factors on natural populations of the barnacle,
Balanus balanoides. Ecol. Monogr 31: 61-104.

Crisp, D. J. 1961. Territorial behavior in barnacle settlement. J Exp
Biol 38: 569-590.

Crisp, D. J., and H. Barnes. 1954. The orientation and distribution of
barnacles at settlement with particular reference to surface contour.
J. Anim. Eco/. 23: 142-162.

Crisp, D. J., and J.S. Ryland. I960. Influence of filming and of surface
texture on the settlement of marine organisms. Xalnre 185: 1 19.

Dayton, P. 1971. Competition, disturbance, and community organi-
zation: the provision and subsequent utilization of space in the rocky
intertidal community. Ecol. Monogr. 41: 351-389.

Denny, M. \V. 1988. Biology and Mechanics of the Have-Swept En-
vironment Princeton University Press, Princeton, NJ.

Denny, M. VV., and M. F. Shibata. 1989. Consequences of surf-zone
turbulence for settlement and external fertilization. Am. .\al. 134:
859-889.

Grosberg, R. K. 1981. Competitive ability influences habitat choice in
marine environments. Nature 290: 700-702.

Hayward, P. J., and P. H. Harvey. 1974. The distribution of settled
larvae of the bryozoans Alcynidium hirsutum (Fleming) and Alcyn-
idium polyoum (Hassall). J Mar Bio/. Assoc I' K 54: 665-676.

Jackson, J. B. C. 1979. Morphological strategies of sessile animals.
Pp. 499-555 in Biology and Systematics of Colonial Organisms, G.
Larwood and B. R. Rosen, eds. Academic Press, New York.

keough, M. J., and B. J. Downes. 1982. Recruitment of marine in-
vertebrates: the role of active larval choices and early mortality. Oec-
ologia 54: 348-352.

Knight-Jones. E. VV. 1951. Gregariousness and some other aspects of
the settling behavior of Spirorbis. J. Mar. Biol. Assoc. U. A". 30: 201-
222.

LeTourneux, F., and E. Bourget. 1988. Importance of physical and
biological settlement cues used at different spatial scales by the larvae
of Semibalanus balanoides. Mar. Biol. 97: 57-66.

Loeb, M. J., and G. Walker. 1977. Origin, composition and function
of secretions from pynform organs and internal sacs of four settling
cheilo-ctenostome bryozoan larvae. Mar. Biol. 42: 37-46.

Middletnn, G. \ '., and J. B. Southward. 1984. Mechanics of Sediment
Movement. 2d ed. Society of Economic Paleontologists and Miner-
alogists. Tulsa, Okla.

Miller, R. G. 1966. Simultaneous Statistical Inference. McGraw-Hill.
Inc., New York.

Paine, R. T. 1974. Intertidal community structure: experimental studies
on the relationship between a dominant competitor and its principal
predator. Oeailogia 15: 93-120.

Reed, C. G. 1987. Phylum Bryozoa. Pp. 494-5 10 in Reproduction and

Development ol Marine Invertebrates ot the Northern Pacific (.'oast.

M. F. Strathmann. ed. University of Washington Press, Seattle.
Reed. C. G., and R. M. \\oollacott. 1982. Mechanisms of rapid mor-

phogenetic movements in the metamorphosis of the bryozoan Bugiila

neritina. J Morphol. 172: 335-348.
Ryland, J. S. 1959. Experiments on the selection of algal substrates by

polyzoan larvae. J E.\p. Biol. 36: 613-631.
Russ, G. R. 1982. Overgrowth in a marine epifaunal community:

competitive hierarchies and competitive networks. Oecologia: 53:

12-19.
Sebens, K. P. 1985. Community ecology of vertical rock walls in the

Gulf of Maine, U.S.A.: small scale processes and alternative stable

states. Pp. 346-371 in The Ecology oj Rocky Coasts, P. G. Moore

and R. Seed. eds. Hodder and Stroughton, London.
Sebens, K. P. 1986. Spatial relationships among encrusting marine

organisms in the New England subtidal zone. Ecol. Monogr. 56: 73-

96.
Seed. R.. and R. J. O'Connor. 1981. Community organization in marine

algal epifaunas. Ann. Rev Ecol Sysl. 12: 49-74.
Snedecor, G. VV., and VV. G. Cochran. 1967. Statistical Methods. Iowa

State University Press. Ames. Iowa.
Strathmann, R. R.. K.S. Branscomb, and K. V'edder. 1981. Fatal errors

in set as a cost of dispersal and the influence of intertidal flora on set

of barnacles. Oecologia 48: 13-18.
Svenden, I. A. 1987. Analysis of surf zone turbulence. J. Geophys. Res

92: 51 15-5124.
Torrence, S. A., and R. A. Cloney. 1988. Larval sensory organs of as-

cidians. Pp. 151-163 in Marine Biodeterioration, Advanced Tech-
niques Applicable to the Indian Ocean, M-F. Thompson. R. Sarogini.

and R. Nagabhushanam, eds. Oxford and IBH Publishing Company

Pvt. Ltd.. New Dehli.
Walters, I.. .].. and D. S. Wethey. 1986. Surface topography influences

competitive hierarchies on marine hard substrata: a field experiment.

Biol. Bull. 170:441-449.
VV ethey, D. S. 1983. Geographic limits and local zonation: the barnacles

Semibalanus (Balanusl and Chthamalus in New England. Biol. Bull.

165: 330-341.
VV ethe>, D. S. 1984. Spatial pattern in barnacle settlement: day to day

changes during the settlement season. / Mar. Biol. Assoc ( A 64:

687-698.
Wethey, D. S. 1986. Ranking of settlement cues by barnacle larvae:

influence of surface contour. Bull Mar. Sci. 39: 393-400.
Wisely, B. 1960. Observations on the settling behavior oflarvae of the

tubeworm Spirorbis borealts Daudlin (Polychaete). Atixt. J. Mar.

Freshwat. Res. II: 55.
Woollacott, R. M., and R. L. Zimmer. 1978. Metamorphosis of cel-

lularioid bryozoans. Pp. 49-64 in Settlement and Metamorphosis of

Marine Invertebrate Larvae. F-S. Chia and M. E. Rice, eds. Elsevier.

New York.
Young, C. M., and F. S. Chia. 1981. Laboratory evidence for delay of

larval settlement in response to a dominant competitor. Int. J Invert.

Reprod 3:221-226.

Reference: Biol. Bull 180: 114-124. (February. 1991)

GABA-Like Immunoreactivity in the Nervous System
of Oikopleura dioica (Appendicularia)

TOMAS BOLLNER' *. JON STORM-MATHISEN2. AND OLE FETTER OTTERSEN:

* Department of Zoology. Stockholm University, 5-706 91. Sweden, and Anatomical Institute.

University of Oslo, Karl Johans gt. 47. N-0162 Oslo 1. Norway

Abstract. The cellular localization of -y-aminobutyric
acid (GABA) has been visualized immunocytochemically
in the nervous system of Oikopleura dioica by using an
antiserum to glutaraldehyde fixation complexes of GABA.
The results show GABA-like immunoreactivity in neurons
of the brain, in cells of the sensory vesicle, in the caudal
ganglion, and in the nerve cord. Positive reactions were
also found at the neuromuscular terminals in the tail.

Introduction

Amino acids are considered to be important neuro-
transmitters in the vertebrate central nervous system (re-
view: Ottersen and Storm-Mathisen, 1984a). Among
these, 7-aminobutyric acid (GABA) is a dominant inhib-
itory neurotransmitter in the brain and spinal cord, and
even occurs in the peripheral nervous system (Jessen et
ai. 1986). GABA has also been reported to be a major
inhibitory neurotransmitter in a wide variety of the in-
vertebrate phyla (Gerschenfeld, 1973; Meyer et ai. 1986;
Vitellaro-Zuccarello and De Biasi, 1988). But no infor-
mation is available concerning this amino acid in the
Urochordata, a group that is often considered to be a phy-
logenetic link between invertebrates and vertebrates.

The organization of the nervous system of Oikopleura
dioica has been investigated by several authors (Gait and
Mackie. 1971; Holmberg, 1984; Bollner et ai. 1986), and
the presence of acetylecholinesterase in this species has
been reported (Durante, 1959; Flood, 1973). The aim of
this investigation was to establish whether Oikopleura
dioica exhibits GABA immunoreactivity in its central or
peripheral neurons.

Received 22 January 1990; accepted 6 November 1990.

* Present address: Department of Biology. University of London. Royal
Holloway and Bedford New College. Egham Hill. Egham, Surrey TW20
OEX, UK.

Materials and Methods

Specimens of 0. dioica Fol, 1872. were collected at the
Kristineberg Marine Biological Station and at the Tjarno
Marine Biological Laboratory, both on the west coast of
Sweden. For immunocytochemistry the following fixatives
were used: (1) 5% glutaraldehyde, (2) 3% glutaraldehyde
and 1% paraformaldehyde, or (3) 1% glutaraldehyde and
1% paraformaldehyde, all in 0.1 A/ sodium-phosphate
buffer at pH 7.4. After 1 h fixation at room temperature,
animals were kept and transported in cold sodium-phos-
phate buffer with 0.5% glutaraldehyde added. Free floating
whole tissues were processed for immunocytochemistry
as described by Storm-Mathisen et ai (1983) and Dale et
ai, (1986); the primary anti-serum was diluted 1:300 be-
fore processing according to the peroxidase-anti peroxi-
dase technique (Sternberger, 1979).

After fixation in 3% glutaraldehyde and 1% paraform-
aldehyde or 1% glutaraldehyde alone, both in 0.1 M so-
dium-phosphate buffer at pH 7.4. animals were embedded
in Epon resin and cut with a glass knife. One-^m sections
were processed on glass slides previously coated with either
chrome alum gelatin or poly-L-lysine, and processed by
the immunogold-silver (IGS) method, as follows.

The sections were etched for 45 min in sodium-etha-
nolate, washed 3X5 min in absolute alcohol, followed
by 2 X 5 min in distilled water, and rinsed briefly in 20
mM Tris buffer at pH 7. 4 containing 155 mMNaCl, 0.1%
BSA, and 20 mA/ NaN3 . The same medium was also used
for subsequent rinses and for diluting sera. The sections
were then incubated with a droplet of 5% normal goat
serum in a moist chamber for 20 min. Thereafter they
were incubated overnight in 50 n\ primary anti-serum
diluted 1 : 100. After a rinse in buffer, followed by washes
3 X 10 min in buffer at pH 8.2, the sections were incubated
for 60 min with GAR G5 (goat anti-rabbit immunoglob-

119

120

T. BOLLNER ET AL

ulin adsorbed to 5 nm colloidal gold. Janssen. Belgium)
diluted 1:80 in the same buffer. Finally the sections were
washed with the same medium as after primary serum,
rinsed in distilled water several times (5 min each at least),
developed in 100 ^1 silver enhancer kit (Janssen), washed
in water, and coverslipped.

Indirect immunofluorescense as well as the peroxidase-
antiperoxidase (PAP) method of Sternberger (1979) were
also tried; the same primary antibodies were visualized
by either a second layer of FITC-conjugated sheep anti-
rabbit serum (SIGMA), or by unlabeled sheep anti-rabbit
serum (Statens Bakteriologiska Laboratorium, Stockholm)
followed by PAP complex (Dakopatts).

The antiserum against glutaraldehyde-conjugated 7-
aminobutyric acid (GABA antiserum 26) was raised, pu-
rified, and characterized as described previously (Ottersen
and Storm-Mathisen, 1984b; Ottersen el ai. 1986). For
all methods used, the controls included absorption of
GABA antiserum with GABA-glutaraldehyde complexes
(GABA-G) and glutamate complexes (Glu-G) at final
concentrations of 300 ^Af. or replacement of the primary
antiserum with normal rabbit serum. Furthermore, the
immunoreactivity of the antiserum used was tested ac-
cording to the filter disc method described by Ottersen
and Storm-Mathisen (1984b). The fixation conjugates
spotted on the discs were made from macromolecules ex-
tracted from rat brain homogenate and from homogenate
of the neural complex from the ascidian Ciona intestinalis.

Results

The central nervous system of Oikopleiira dioica con-
sists of an anterior ganglion (brain) and tail ganglia. The
anterior part of the brain is extended into paired bulbs,
and in the mid-region it has a sensory vesicle. The brain
is connected to several ganglia in the tail by a solid nerve
cord. The largest of these tail ganglia is referred to as the
caudal ganglion (Figs. 1, 2).

Although the brain is hard to see in whole mounts due
to the thick oikoplast epithelium, GABA-like immuno-
reaction was observed in the paired anterior bulbs de-
scribed by Bollner el a/., (1986). In semithin sections, a
positive reaction is easily seen in some of the neurons in
the bulbs (Fig. 3). In the rest of the brain, staining with
the GABA anti-serum were found: in one cell located
ventrally in the mid region, in one of the most caudal
cells (Figs. 2, 4), and in a dorsal cell close to the sensory
vesicle (Fig. 5). Furthermore, immunopositive staining
was seen in the epithelial cells referred to as the "brain
vesicle cells" by Holmberg (1984) (Fig. 6).

Two immunostained cell bodies situated in the caudal
part of the caudal ganglion were observed in semithin
sections (Fig. 7) and in the whole mount preparations
(Fig. 8). Immunoreactive fibers were seen in both semithin

Figure 1. Schematic drawing of Oikopleiira dioica showing the po-
sition of the nervous system with the immune-positive neurons indicated,
the position ot the sensory' vesicle is indicated by the dotted circle, ab.
anterior bulb: br, brain; eg, caudal ganglion: g. gonads: i. intestine: m,
mouth: n. nerve cord; nm. neuromuscular junction. Bar = 200 Mm.

sections (Fig. 7) and in whole mounts. Transverse semithin
sections through the tail showed GABA-like immuno-
reactivity in the large nerve terminals (Fig. 9) innervating
the muscles (cf. Flood, 1973, 1975) and in fibers of the
dorsal nerve cord. Furthermore, positive staining was of-
ten seen in the epithelial cells of the notochord. Because
the tail of the animal is twisted 90° counter-clockwise,
the dorsal nerve cord is seen to the right of the notochord
in a frontal view of a transverse section of tail.

The semithin sections from animals fixed with 1% glu-
taraldehyde processed using the immunogold technique
gave a stronger reaction than any other method. The PAP-
method showed weak staining in the anterior bulbs and
in the caudal ganglion, whereas the FITC-incubated sec-
tions showed clear label in the same regions as did the
immunogold method. However, all methods showed
similar patterns of immunoreactivity.

The anti GABA serum, either absorbed with glutamate-
glutaraldehyde complex (Glu-G) or not, produced selec-
tive staining of the GABA conjugates on the filter discs,
but no significant staining could be seen after pretreatment
of the GABA antiserum with GABA-glutaraldehyde
complex (GABA-G) (Fig. 8). Similar results were obtained
with spots of amino acids conjugated to macromolecules
from rat and Ciona, suggesting that the previously dem-
onstrated specificity is valid also for urochordates. In
whole mounts as well as in semithin sections, the reaction
was virtually abolished when antisera treated with GABA-
G or normal rabbit serum were used instead of GABA
antiserum. Treatment of the anti serum with Glu-G did
not have this effect.

Discussion

Many investigations have established the presence of
either GABA or glutamic acid decarboxylase (GAD),

GABA IN OIKOPLKL'RA NERVOUS SYSTEM

121

•<^

IL br

Y|Ai-4Q*ij '
,'•'•

Figure 2. Sagittal section through the whole animal showing the localization and GABA-like immu-
noreactivity (arrowheads) of the brain (br), one of the anterior bulbs (ab). the nerve cord (n), the caudal
ganglion (eg) and in a neuromuscular junction of the tail (arrow). Staining can also be seen in the gonads
(g), at the apical surface of some of the intestinal cells (i). and in the rectum (r). No staining could be seen
in an adjacent section treated with anti-GABA/GABA-G. 1GS method. Differential interference contrast.
Bar = 100 Aim.

which catalyzes the synthesis of GABA from glutamate,
in lower chordates and in invertebrates (Osborne, 1972;
Osborne ct at., 1979; De Biasi, 1986). More recently, the
use of specific antibodies (Storm-Mathisen ct ai. 1983)
has led to a more precise knowledge about the cellular
localization of GABA in both vertebrates (Ottersen and
Storm-Mathisen 1984, a, b; Roberts et ai. 1987) and in-
vertebrates (Bicker el ai. 1985; Meyer et ai. 1986; Hom-
berg et ai. 1987). This is, to our knowledge, the first im-
munocytochemical study on the occurrence of amino ac-
ids in the nervous system of a protochordate. Using
biochemical analyses, Osborne et ai (1979) found GABA
and several other putative amino acid neurotransmitters
in homogenates of the cerebral ganglion of another pro-
tochordate, the tunicate Ciona intestinalis.

The present investigation shows the cellular localization
of a GABA-like substance in the nervous tissue of O.
dioica. The GABA-positive cells in the anterior bulbs and
in the rest of the brain are thought to be neurons judging
from their location and their ultrastructural appearance
previously described by Bollner et ai. (1986, unpubl.).
Also, most of the cells in the caudal ganglion are consid-
ered to be neurons. However, one of its anterior cells, a
large ependymal cell, produces the Reissner's fiber
(Holmberg and Olsson, 1984). The significance of GABA-
like immunoreactivity in the neurons of the central ner-
vous system is difficult to evaluate. Although they may

be neurons with inhibitory functions, it should be re-
membered that GABA may also have depolarizing effects
(Alger and Nicoll, 1982). In addition to the neural local-
ization, GABA was clearly present in epithelial cells. This
agrees with the situation in vertebrates, where GABA has
been demonstrated in non-neural epithelial cells (Oren-
sanz el ai, 1986; Davanger et ai, 1989). Amino acids in
general are also known to modulate osmoregulation (see
Gilles, 1979, for review). Synthesis of GABA has been
reported to occur in fish erythrocytes where it may par-
ticipate in the maintenance of a constant cell volume (Fu-
gelli et ai, 1970). O. dioica is an isosmotic animal, and
the vesicle and the chorda are the only internal structures
not totally surrounded by hemolymph, and therefore
might use GABA for regulating the intracellular osmo-
larity.

The muscles in the tail are innervated both by fibers
branching directly from the nerve cord and from perikarya
along the cord (Flood, 1973). These nerves have elaborate
end-arborizations on the surface of the muscle cells and
are thought to be cholinergic (Flood, 1975; Bone and
Mackie. 1982). Cholinergic neuromuscular transmission
is widely distributed throughout the animal kingdom, but
GABA-ergic inhibition of muscles is only known in in-
vertebrate phyla (Gerschenfeld, 1973). GABA-like im-
munoreactivity has been demonstrated in inhibitory
nerves in insect muscle (Bicker et ai, 1988; Robertson

122

T. BOLLNER ET AL

••• it

7 nc k

anti-GABA

anti-GABA
+Glu-G

null

GABA

Gly

Gin

anti-GABA
+GABA-G

Figure 3. Transverse section through the anterior bulbs with staining in neurons (arrowheads) and in
some of the fibers of the neuropile (arrows), m, mouth. IGS method. Differential interference contrast. Bar
= 15 Aim.

Figure 4. Sagittal section showing positive staining in one cell in the mid part and in one cell in the rear
part of the brain (arrowheads) and also in a fiber of the nerve cord (arrow). IGS method. Differential
interference contrast. Bar = 15 ^m.

Figure 5. Transverse section through the brain in the region of the sensory vesicle (sv). with staining in
one cell body (arrowhead). IGS method. Differential interference contrast. Bar = 15 ^m.

Figure 6. Section through the sensory vesicle (sv) showing positive staining in one of the brain vesicle
cells (arrowhead) and in the epithelial cells forming the vesicle wall. IGS method. Differential interference
contrast. Bar = 15 ^m.

Figure 7. Transverse section through the caudal ganglion showing positive staining in cell somata (ar-
rowheads) and in neuropile fibers (arrow); nc, notochord. IGS method. Differential interference contrast.
Bar = 10 jjm.

Figure 8. Whole-mount preparations of the caudal ganglion showing positive reaction in neurons (arrows)
after treatment with anti-GABA (a) and anti-GABA/Glu-G (b) and no reaction after treatment with anti-
GABA/GABA-G (c), corresponding control filter discs with amino acids conjugated to macromolecules
from dona neural tissue by glutaraldehyde are shown at the right. Gin. glutamine; Gly. glycine; null,
glutaraldehyde-treated protein with no amino acid added. PAP method. Bar = 15 ^m.

GABA IN OlKOPLEUR.-\ NERVOUS SYSTEM

123

Figure 9. Transverse section through the tail near the trunk, frontal view. GABA-like immunoreaction
can be seen in a neuromuscular terminal (arrowhead! and in the nerve cord (n). This section also shows
staining in a part of the fiber between the nerve cord and the neuromuscular terminal (arrow), m, muscle;
nc, notochord. IGS method. Differential interference contrast. Bar = 2(1 j/m

and Wisniowski, 1988). Our rinding that neuromuscular
synapses of O. dioica show GABA-like immunoreactivity
is the first indication that GABA may also act as neuro-
muscular inhibitory substance in some chordates.

More work is needed to establish whether GABA is a
neurotransmitter in O. dioica. Its possible role in osmo-
regulation in the vesicle should also be subject to further
investigation.

Acknowledgments

We are grateful to A. T. Bore and Y. Lilliemark for
technical assistance. This investigation was supported by
the Hierta-Retzius and Lars Hiertas Minne foundations.

Literature Cited

Alger, B. E.. and R. A. Nicoll. 1982. Pharmacological evidence for two
kinds of GABA receptor on rat hippocampal pyramidal cells studied
in vilro. J Physio! 328: 125-141.

Bicker, G., S. Schafer, and T. G. Kingan. 1985. Mushroom body feed-
back inter neurons in the honeybee show GABA-like immunoreac-
tivity. Brain Res. 360: 394-397.

Bicker, G., S. Schafer, O. P. Ottersen, and J. Storm-Mathisen.
1988. Glutamate-like immunoreactivity in identified neuronal
populations of insect nervous systems. J. Neurosci. 8: 2108-2122.

Bollner, T., K. Holmberg, and R. Olsson. 1986. A rostral sensory
mechanism in Oikopleura dioica (Appcndiculana). Acta Zool 67:
235-241.

Bone, Q., and G. O. Mackie, 1982. Urochordata. Pp. 473-535 in Klcc-
irical Conduction and Behavior in "Simple" Invertebrates. G. A. B.
Shelton, ed. Oxford University Press, Oxford.

Dale, N., O. P. Ottersen, A. Roberts, and J. Storm-Mathisen.
1986. Inhibitory neurons of a motor pattern generator in Xenopus
revealed by antibodies to glycine. Nature 324: 255-257.

Davanger, S., O. P. Ottersen, and J. Storm-Mathisen. 1989. GABA
immunoreactive cells in the rat gastrointestinal epithelium. Anal.
Embryo! 179: 221-226.

I)e Biasi, S., C. Frassoni, and I,. Vitellaro Zuccarello. 1986. Glutamic
acid decarboxylase (GAD)-like immunoreactivity in the pedal gan-
glion of Myiiliis galloprovincialis. Cell Tissue Res 244: 591-593.

Durantc, M. 1959. Sulla localizzazione istochimica della acetilcolines-
terasi lungo lo sviluppo di alcune ascidie e in appendicularie. Acta
Lmbryol. Morphol Exp 2: 234-243.

Flood, P. R. 1973. Ultrastructural and cytochemical studies on the
muscle innervation in Appendiculana. Tunicata. J Microsc. 18: 317-
326.

Flood, P. R. 1975. Scanning electron microscope observations on the
muscle innervation of Oikopleura dioica Fol (Appendiculana, Tun-
icata) with notes on the arrangement of connective tissue fibers. Cell
Tissue Res 164: 357-369.

Fol, II. 1872. Etudes sur les appendiculaires du detroit de Messine.
Mem. Soc. Phys. Hist. Nat. Geneve 21: 445-449.

Fugelli, K., J. Storm-Mathisen, and F. Fonnum. 1970. Synthesis of
vaminobutyric acid in fish erythrocytes. Nature 228: 1001.

Gait, C. P., and G. O. Mackie. 1971. Electrical correlates of ciliary
reversal in Oikopleura. J. Exp. Biol. 55: 205-212.

Gerschenfeld, II. M. 1973. Chemical transmission in invertebrate cen-
tral nervous system and neuromuscular junctions. Physiol. Rev 53:
1-1 19.

Gilles, R. 1979. Intracellular organic osmotic effectors. Pp. 1 1-154 in
Mechanisms ofOsmoregulation in Animals. R. Gilles, ed. Wiley Chi-
chester. New York.

Holmberg, K. 1984. A transmission electron microscopic investigation
of the sensory vesicle in the brain of Oikopleura dioica (Appendi-
cularia). Zoomorpliology 104: 298-303.

Holmberg, K., and R. Olsson. 1984. The origin of Reissner's fibre in
the appendicularian. Oikopleura dioica. 1'iddensk. Aledd. Dan. Na-
lurlusl. Foren. 145: 43-52.

Homberg, U., T. G. Kingan, and J. G. Ilildebrand.
1987. Immunocytochemistry of GABA in the brain and sub-
oesophageal ganglion of Manduea sexta. Cell Tissue Res. 248: 1-24.

Jessen, K. R., J. M. Hills, and J. Saffrey. 1986. Immunohistochemical
demonstration of GABA ergeic neurons in the enteric nervous system.
J. Neurosci 6: 1628-1634.

Meyer, E. P., C. Matute, P. Streit, and D. R. Nassel. 1986. Insect
optic lobe neurons identifiable with monoclonal antibodies to GABA.
Htslochemistrv 84: 207-2 16.

124

T. BOLLNER KT AL

Orensanz, L. M., I. Fernandez, R. Martin del Rio, and J. Storm-Malhisen.
1986. Gamma-aminohutync acid in the rat oviduct. Adv. Biocliem.
Psychophamacol. 42: 265-274.

Osborne, N. N. 1972. Occurrence of glycine and glutamic acid in the
nervous system of two fish species and some invertebrates. Coinp.
Biocliem I'hyvol 43B: 579-585.

Osborne, N. N., V. Neuhoff, E. Ewers, and H. A. Robertson.
1979. Putative neurotransmitters in the cerebral ganglia of the tu-
nicate Ciiina intestinalis. Coinp. Biocliem. Physiol. 63C: 209-213.

Ottersen, O. P., and J. Storm-Mathisen. 1984a. Neurons containing
or accumulating transmitter amino acids. Pp. 141-146 in Classical
Transmitters ami Transmitter Receptors in the CMS Purl II. A.
Bjorklund, T. Hokfelt, and M. J. Kuhar, eds. Handbook of Chemical
Neuroanatomy. Vol. 3 Elsevier, Amsterdam.

Ottersen, O. P., and J. Storm-Mathisen. 1984b. Glutamate- and
GABA-contaming neurons in the mouse and rat brain, as demon-
strated with a new immunocytochemical technique. J. Coinp. Neural.
229: 374-392.

Ottersen, O. P., J. Storm-Mathisen, S. Madsen, S. Skumlien, and J.
Stremhaug. 1986. Evaluation of the immunocytochemical method
for amino acids. Med. Biol. 64: 147-158.

Roberts, A., N. Dale, O. P. Ottersen, and J. Storm-Mathisen. 1987. The
early development of neurons with GABA immunoreactivity in CNS
of X'enopus laevis embryos. J Coinp. N enrol. 261: 435-449.

Robertson, R. M., and I., \\isniowski. 1988. GABA-like immuno-
reactivity of identified inter neurons in the flight system of the locust.
Locusta migraloria. Cell Tissue Res 254: 331-340.

Sternberger, I,. A. 1979. Immune/cytochemistry, Second Edition. John
Wiley and Sons, New York. Chichester.

Storm-Mathisen, J., A. K. Leknes, A. T. Bore, J. L. Vaaland, P. Ed-
minson. F-M. S. Haug, and O. P. Ottersen. 1983. First visualization
of glutamate and GABA in neurons by immunocytochemistry. Nature
301: 517-520.

Vitcllaro Zuccarello, I,., and S. De Biasi. 1988. GABA-like immu-
noreactivity in the pedal ganglia of Mytilus galloprovmciulis- light
and electron microscopic study. J. Coinp. Neural. 267: 516-524.

Reference: Biol Bull 180: 125-134. (February,

Ontogeny of Osmoregulation and Salinity Tolerance in

Cancer irroratus; Elements of Comparison with

C. borealis (Crustacea, Decapoda)

G. CHARMANTIER AND M. CHARMANTIER-DAURES

Laboratoire d'Ecop/iysiologie des Invertebres, Universite des Sciences. Montpellier 2.
Place E. Bataillon. 3409? Montpellier tedex 05. France

Abstract. Osmoregulation and salinity tolerance were
studied in zoeae, megalopae, first crab stage (osmoregu-
lation only), and adults of Cancer irroratus, and in zoeae
and adults of C. borealis.

In C. irroratus. salinity tolerance was moderate in zoeae,
decreased in late zoeae 5, was at a minimum in megalopae.
and increased in adults. The lower and upper lethal sa-
linities for 50% of the animals (48 h LS 50) at 15°C were
about 13-17%o/42-50%o in zoeae, 24%o/37%o in mega-
lopae, and 8.5%o/65%o in adults.

In C. borealis. the corresponding values of LS 50s were
16-20%o/46-50%o in zoeae and 12%«/65%o in adults.

In both species, zoeae were hyper-osmoconformers;
adults were isosmotic in high salinities and slightly hyper-
regulators in low salinities. In C. irroratus. the change
from larval to adult type of regulation occurred from
megalopa (hyper-osmoconformer) to first crab stage (hy-
per-regulator in dilute media), i.e.. after the completion
of metamorphosis.

Osmoregulation and salinity tolerance appear corre-
lated and are modified at metamorphosis. These results
are discussed with an emphasis on the effects of meta-
morphosis on Osmoregulation of developing decapods.

Introduction

While Osmoregulation has been extensively studied in
adult crustaceans (review in Mantel and Farmer, 1983),
relatively few data are available on larval or post-larval
Osmoregulation, in the following species: Rhilhropanopeus
harrisii (Kalber and Costlow, 1966), Cardisoma guan-
humi (Kalber and Costlow, 1968), Callinectes sapidus.

Received 1 1 June 1990; accepted 27 November 1990.

Hepalus ep/ie/iticiis. Lihinia cinarginata (Kalber, 1970),
Sesaniui reticii/atnm (Foskett, 1977), Clibanarim vittatus
(Young, 1979), Callianassa Jamaica (Felderc/ a/., 1986),
Alacrohrac/iiuni pelersi (Read, 1984), Uca snhcylindrica
(Rabalais and Cameron, 1985), Homarus americanus, and
Penaeus japonicm (Charmantier, 1986; Charmantier et
a/.. 1984a, 1988).

For different reasons, including culture difficulties in
late larval stages, Osmoregulation was frequently studied
in larval stages only, particularly in brachyuran crabs.
However, in the four latter species, Osmoregulation was
studied throughout the post-embryonic development in-
cluding post-metamorphic stages, thus Osmoregulation in
larvae, postlarvae, and adults could be compared. The
ability to osmoregulate did not change along the devel-
opment in some species. In other species, metamorphosis
marked the appearance of the adult type of regulation;
among brachyurans, this case has been so far documented
in only one species, U. snhcylindrica, the adults of which
are strong hyper-hypo-regulators (Rabalais and Cameron,
1985).

Consequently, one of the objectives of this study was
to determine whether comparable changes in Osmoregu-
lation at metamorphosis exist in brachyurans with lower
abilities to osmoregulate, particularly in species of the ge-
nus Cancer, which slightly hyper-regulate in low salinities.
Experiments designed to study the ontogeny of Osmoreg-
ulation were performed on the most abundant Cancer
species of the Canadian East coast, the rock crab Cancer
irroratus Say 1817. Comparative data were also obtained
from some developmental stages of a sympatric species,
the Jonah crab Cancer borealis Stimpson, 1859.

Numerous studies have dealt with the tolerance to sa-
linity of crustacean larvae and postlarvae, but the corre-

125

126

G. CHARMANTIER AND M. CHARMANTIER-DAURES

lation between the salinity tolerance of different devel-
opmental stages and their corresponding osmoregulatory
capabilities has only been experimentally investigated in
a few species, U. subcylindrica (Rabalais and Cameron.
1985), H. americann.s and P. japonicus (Charmantier et
al., 1988).

Thus, the second objective of this study was to deter-
mine the salinity tolerance of larval, postlarval, and adult
stages of C. irroratus (and, for the purpose of comparison,
of C. borealis). and to attempt to correlate their osmo-
regulatory abilities and their salinity tolerance. This is of
particular interest in Cancer species, the larvae of which
are submitted to different patterns of salinity changes: in
C. irroratus, zoeae hatch offshore and, as development
proceeds, late larvae and postlarvae are found nearer to
shore (Sandifer, 1973).

In both species, the early post-embryonic development
comprises one prezoea, five zoeal stages, one megalopal
stage, and several early crab stages (Sastry, 1977a, b). Fol-
lowing a wide acceptance, zoeae are larval stages, and the
following stages, beginning with the megalopa, are con-
sidered postlarvae (Felder et al., 1985).

In C. irroratus, osmoregulation has been studied in
adults and large juveniles (Thurbergrf al., 1973; Cantelmo
etai. 1975;Neufeldand Pritchard, 1979), and preliminary
data on osmoregulation are also available in the early post-
embryonic stages (Charmantier et al., 1989).

Materials and Methods

Animals

Adult C. irroratus and C. borealis were caught by
SCUBA diving in summer and autumn in Passama-
quoddy Bay and transferred to the culture facility at the
Biological Station, St Andrews, New Brunswick, Canada.
They were kept in tanks supplied with running seawater
(S ^ 29-32%o; T s 12°C) under natural photoperiod, and
were fed cod, squid, and shrimp (Pandalus borealis). In
September, two weeks before the experiments, four groups
of crabs were selected: large and small C. irroratus [ceph-
alothoracic width (CTW): 91 to 115 mm and 45 to 70
mm, respectively], and large and small C. borealis (CTW:
80-110 mm and 57-71 mm). Males and females were
equally represented in each group. They were transferred
to 250-1 tanks with charcoal-nitrated recirculated seawater
kept at 15°C. Only animals in molt stages C or Do (ac-
cording to the nomenclature of Drach, 1939) were retained
for survival and osmoregulatory experiments.

Larvae of both species were obtained in spring and
summer from some of the aforementioned crabs. After
hatching, larvae were transferred to 40-1 planktonkreisels
(Hughes et al., 1974) supplied with flow-through seawater
at a salinity of 29-32%o under natural photoperiod. The
planktonkreisels, normally used for culturing lobster lar-

vae, were modified for the culture of crab larvae. Seawater
was filtered to 50 ^m during the zoea development then
suppressed; flow-rate was set at 2.5-3 1 min"' from zoeae
1 to early megalops, then at 1.-1.5 1 min"1; a 280 ^m
mesh screen was used around the overflow system. Water
temperature was set at 15°C during the zoeal develop-
ment, then at 19°C. Cephalothoracic length was about
0.56 mm in zoeae 1, 1.5 mm in zoeae 5, 2.2 mm in mega-
lopae, and CTW was 2.3 mm in first crab stage (Char-
mantier-Daures and Charmantier, 199 1 ). Crab larvae were
fed three times a day with live Anemia nauplii. Larvae
of C. irroratus were cultured to the second crab stage, and
those of C. borealis to the third zoea. As each larval stage
lasts several days, molting stages were obtained according
to the time elapsed from the preceeding molt, and three
groups of animals, postmolt stage A. stage C, and premolt
stage D. were selected.

Preparatit >n < if media

Experimental media were prepared in compartmented
250-1 tanks for adults and 0.5-1 plastic containers for larvae
and young crabs. Dilute media were prepared by adding
tap water to seawater, and high salinity media were pre-
pared by adding "Instant Ocean Synthetic Sea Salts"
(Aquarium Systems, Inc.) to seawater. All experiments
were conducted at 15°C. Salinities were expressed ac-
cording to the osmotic pressure in mosm-kg ', and to
the salt content in the medium in %o. A value of 3.4%o is
equivalent to 100 mosm-kg"1. Osmotic pressure was
measured with an Advanced Instruments 3 1 LA or Wes-
cor 5000 osmometer, and salinity on a YSI 33 salinometer.

Survival bioassays

Due to the small number of available animals, salinity
tolerance in adults was evaluated only from the number
of surviving and dead animals in media of different salin-
ities. Adult crabs were progressively adapted from seawater
to diluted or concentrated media by adding freshwater or
Instant Ocean salts to the original medium; each change
of 100 mosm-kg"1 in the salinity required about 24 h.
Between two changes of salinity, they were kept for two
days at constant salinity in each test medium, which dif-
fered from one another by increments of 1 00 mosm • kg" '
(=3.4%o).

Acute static 48-96 h bioassays were conducted with
zoeae and megalopae held in test media ranging from 100
mosm • kg" ' to seawater ( ^900- 1 000 mosm • kg" ' ) and to
1600 mosm-kg"1, and differing by increments of 100
mosm -kg '. Each bioassay was run on a group of 10
individuals and replicated. Animals were counted and
dead animals removed at 0.5, 1, 3, 6, 12, 24, 36, 48, 72,
96 h according to the prescriptions of Sprague (1969) in
toxicity studies. The criteria for death were total lack of

ONTOGENY OF CANCER OSMOREGULAT1ON

127

600
20

1000
30 40

1800 mosm. kg
60 n/«

Medium

Figure 1. Salinity tolerance in adult Cancer irn»atu,\ and C horciilis at 15°C. Percent mortality of
animals according to the salinity of the medium. Number of animals at the start of the experiments in
seawater (SW): C' irnmiltis: 16 (to low salinity media) and 8 (to high salinity media); C borealis: 19 and 7.

movement, immobility of appendages and heart, and lack
of response after repeated touches with a probe. Median
lethal salinities (LS 50) and 95% confidence intervals were
calculated by techniques of probit analysis (Lichtfield and
Wilcoxon, 1949; Finney, 1962) computerized on the Let-
cur program (Zitko, 1982; Lieberman, 1983). LS 50s were
calculated at 24, 48, and 96 h. Survival bioassays were
not run in first and second crab stages due to the small
number of available animals.

Osmoregulation

The hemolymph was collected from adult crabs via a
hypodermic needle inserted through the articulation
membrane at the basis of the fourth or fifth pereiopods.
At least seven days elapsed between hemolymph samples
were taken from the same animal.

Zoeae, megalopae, and young crabs were quickly dried
on filter paper and immersed in mineral oil to avoid evap-
oration and desiccation. The hemolymph was then sam-
pled with a glass micropipette inserted in the heart.

Osmotic pressure of hemolymph was measured on an
Advanced Instruments 31 LA or Wescor 5000 osmometer
(adults) or on a Kalber-Clifton micro-osmometer, with
reference to the osmotic pressure of the medium (young
stages). Student / tests were used for statistical compari-
sons.

Results

Salinity tolerance

The ability of C. irroratus and C. borealis to tolerate
low and high salinities varied with post-embryonic de-
velopment.

Adults of C. irroratus survived without mortality in
media ranging from 500 mosm • kg" ' to 1 300 mosm • kg" '

(=17%» to 44%o). The LS 50 s were about 250 and 1900
mosm -kg"1 (s8.5 and 65%o). In adult C. borealis. no
mortality was observed between 600 and 1300
mosm -kg"1 (^20.4 and 44%o), and LS 50s were about
350 and 1900 mosm -kg"' (12 and 65%o) (Fig. 1). No
difference in salinity tolerance was detected between large
and small crabs of either species.

In larvae and postlarvae of C. irroratus the 48 h LS 50
in low salinity media varied around 450 ± 60 mosm • kg~'
(^15 ± 2%o) in zoeal stages 1 to 4 and early 5, then in-
creased from the end of stage zoea 5 through early mega-
lopae (^600 mosm -kg"1, 20%o) to a highly significant
maximum value (corresponding to a minimum tolerance)
of 700 mosm • kg"1 (=24%o) in intermolt megalopae. The
24 h and 96 h LS 50s were, respectively, generally lower
and higher than the 48 h value but followed the same
pattern of variation. Maximum LS 50s at 24, 48, and 96
h occurred in megalopae with respective values of 520,
700, and 820 mosm -kg"1 (18, 24, and 28%o). differing
significantly from one another.

In high salinity media, the 48 h LS 50 of C. irroratus
young stages varied around 1350± 120 mosm -kg"' (^46
± 4%o) in zoeal stages 1 to 5, then decreased in early meg-
alopae (^1240 mosm • kg"1, ^42%o) to a highly significant
minimum value of 1 100 mosm • kg"1 (^37%o) in intermolt
megalopae. The 24 h and 96 h LS 50s were respectively
higher and lower than the 48 h value; they followed the
same pattern of variation, decreasing to minima of 1 150
mosm -kg"1 (24 h: 39%o) and 1000 mosm -kg"1 (96 h:
34%o) in megalopae (Fig. 2).

In zoeae of C. borealis. the 48 h LS 50 varied around
530 ± 60 mosm -kg"1 (^18 ± 2%») in low salinities. The
24 h and 96 h LS 50s were markedly lower and higher
than the 48 h LS 50. The differences between 96 h and
24 h LS 50s were more important than in C. irroratus: in

128

G. CHARMANTIER AND M. CHARMANTIER-DAURES

700 6

A C
1 Zl -

DA C DA C DA C DA
— ' — Z2— ' — Z3— •— Z4-

C
-Z5-

-Mgl —

300

Molt stages
L. stages
Days

Figure 2. Salinity tolerance in zoeae 1-5 and megalopae of Cancer irrorulti.i at 15°C. Variations in LS
50 in %o and mosm • kg"' according to larval and molt stages and to days of development, in high and low
salinities (upper and lower traces). Each point represents the mean value of at least two determinations from
10 animals, with 95% confidence interval. Closed triangles: 24 h LS 50. Closed circles: 48 h LS 50; open
circles: 96 h LS 50.

some instances (early zoea 1 and zoea 2), the 24 LS 50
was about 300 mosm • kg"' ( 10%o), the 96 h LS 50 reaching
about 740 mosm • kg ' (25%). In high salinities, the 48 h
LS 50 varied around 1420 ± 60 mosm • kg" ' (^48 ± 2%o);
24 h and 96 h LS 50s were respectively higher and lower
(Fig. 3).

Osmoregulation

Adaptation time. The time of adaptation after a
change in the environmental salinity was evaluated in
stage C zoeae 1 and 5 and in adults of C. irroratus. After
a rapid transfer from seawater at 920 mosm • kg"1 (31%o)

to a dilute medium of 500 mosm -kg ' (17%o), the he-
molymph osmotic pressure stabilized within 1 to 2 h in
zoeae 1 and 5. In adults transferred from seawater to a
dilute medium of 677 mosm -kg"' (23%o), the corre-
sponding time was 24 h (Fig. 4). The time of osmotic
adaptation to concentrated media was not tested; in other
species, it was shorter than the time of adaptation to dilute
media (Charmantier ct ai. 1988). In all subsequent ex-
periments and in both species, we kept the young stages
6-24 h and the adults 3-4 days in each medium before
sampling.

Osmoregulation. Adults of C. irroratus and C. bo-
realis were almost osmoconformers in high salinities and

ONTOGENY OF CANCKR OSMOREGULATION

129

700 ~
O

-zi

C
-22-

C Molt stages

L stages

25 Days

Figure3. Salinity tolerance in zoeae [-3 of Cancer borealis at 15°C.
Variations in LS 50 in %» and mosm • kg"' according to larval and molt
stages and to days of development, in high and low salinities (upper and
lower traces). Each point represents the mean value of two determinations
from 10 animals, with 95% confidence interval. Closed triangles: 24 h
LS 50; closed circles: 48 h LS 50; open circles: 96 h LS 50.

seawater, and their regulation was slightly hyper-osmotic
in dilute media (Fig. 5). No difference of hyper-regulation
was detected between large and small adults in C. bomilis.
In C. irroratus large crabs were significantly stronger reg-
ulators than small crabs (hemolymph-medium differences
of 7 1 ± 7 and 55 ± 1 3 mosm • kg"1 respectively, P < 0.005,
in a 500 mosm • kg"1; 17%o, medium). The ability to hy-
per-regulate in dilute media was significantly higher in C.
irroratus than in C. borealis (in large crabs, hemolymph-
medium differences of 71 ±7 and 37 ± 5 mosm-kg ', P
< 0.001, in a 500 mosm -kg"1, 17%o, medium).

Zoeae 1 to 5 of C. irroratus in molting stage C hyper-
osmoconformed at almost all tested salinities, i.e., their
hemolymph osmotic pressure varied as a function of ex-
ternal osmotic pressure but remained above external by
about 1 5-50 mosm • kg"1; at the lowest tested salinity (300
mosm • kg"1), zoeae were isosmotic. In some zoeal stages.

regulation in dilute media was slightly more hyper-osmotic
in premolt ( zoeae 1 ,4 at 500 mosm • kg" ' : P < 0.05 ) and
less hyper-osmotic in post-molt (zoeae 2, 3, 4 at 500 and
900 mosm • kg" ': P < 0.0 1 ). Megalopae were also hyper-
osmoconformers. The pattern of osmoregulation seemed
to change after the completion of metamorphosis. First
crab stages were almost osmoconformers in seawater and
their regulation was slightly hyper-osmotic in a dilute me-
dium of 500 mosm • kg '. 1 1%« (hemolymph-medium dif-
ference of 55 ± 16 mosm-kg"') (Fig. 6).

Zoeae 1 to 3 of C. borealis had the same pattern of
hyperosmocon form ing regulation as zoeae of C. irroratus
(Fig. 7).

Discussion

Sa/initv tolerance

In Cancer uroraliis, the interval of tolerable salinities
tends to decrease at the end of the larval development
and is minimum in megalopae. At this stage, the 96 h LS
50s are about 28%o and 34%o, which means that megalopae
are almost restricted to seawater. In adults, approximate
48 h LS 50s are 8.5%o and 65%o. The wide euryhalinity
demonstrated by adults could be partly related to the rel-
atively short time of exposure to the different media and
to the progressive adaptation to changing salinities. Long-
term exposure to extreme salinities could yield more re-
strictive results.

These results are in agreement with previous data. Sas-
try (1970) found that at 15°C, only 5.5% of megalopae
of C. irroratus molted to the first crab stage in a medium
of 15%o, while this molt was successful in a higher per-
centage of megalopae in media ranging from 20 to 35%o,
with a maximum rate of 76% in a medium of 30%o. Com-
plete development from zoea 1 to first crab stage was
found possible at 15°C between 20 and 35%o (Sastry and
McCarthy, 1973) or 25 and 35%o, but survival exceeded
50% only in 30-35%. (Johns, 1981). In adults, McCluskey
(1975, cited in Bigford, 1979) found survival was possible
for three days at 5-8°C in salinities ranging from 10 to
20%o (the upper limit was not tested).

Compared to the larvae of C. irroratus, zoeae of C.
borealis were less tolerant to prolonged exposure to low
salinities. In C. borealis, Sastry and McCarthy (1973)
found that complete larval development was only possible
in a medium of 30%» at 20°C. These and our results dem-
onstrate that C. borealis is more stenohaline than C ir-
roratus, and, in particular, less tolerant to low salinities
during the larval development and in adults.

Adaptation time

In C. irroratus. the time of osmotic equilibration in a
dilute medium is about 1 to 2 h in larvae and 24 h in

130

G. CHARMANTIER AND M. CHARMANTIER-DAURES

Figure 4. Change in hemolymph osmotic pressure in stages zoeae 1 and 5 of Cancer irroratus after rapid
transfer from seawater (920 mosm • kg"', 31%») to a dilute medium (500 mosm • kg"', 17%o), and in adults
of C. irroratus after rapid transfer from seawater to 677 mosm • kg~', 23%o. at 1 5°C. Each point represents
the mean value of determinations from 3 to 5 zoeae or 5 adults, with 95% confidence interval.

adults. Adaptation time is thus size-dependent, which
could be related to differences in the volumes of water
and ion exchanges and to differences in tegument per-
meability between development stages. These times of
adaptation are similar to those of the corresponding stages
of other species (see Charmantier el ai, 1988).

Osmoregulation

In C. irroratus and C. borealis, adults osmoconform in
high salinities and seawater and slightly hyper-regulate in
dilute media. The ability to hyper-regulate is higher in C.
irroratus.

A similar pattern of osmoregulation has been described
in C. irroratus and other species of Cancer, but the ability
to hyper-regulate varies with the species, although other
factors such as size and temperature can affect this pa-
rameter. In media of approximately 500 mosm -kg"',
17%o, at temperatures of =15-20°C, the difference be-
tween the osmotic pressures of hemolymph and medium
expressed in mosm -kg"' is about 15 in C. antennarius
(Jones, 1941), 30-40 in C borealis (this study), 50 in C.
pagurus (Wanson et ai, 1983), 50-120 in C. irroratus
(Thurberg et al.. 1973; Cantelmo et ai, 1975; Neufeld
and Pritchard, 1979; this study), 150-250 in C. nwgister

(Jones, 1941; Engelhardt and Dehnel, 1973; Hunter and
Rudy. 1975).

Zoeae of C. irroratus hyper-osmoconform in all tested
salinities. Most decapod larvae that have been studied

Medium (mosm. kg )

Figure 5. Variations in the difference between the osmotic pressures
of hemolymph and medium according to the osmotic pressure of the
medium in large and small adults of Cancer irroralus and C. borealis at
I5°C. Each point represents the mean value of determinations from 7
to 10 animals (exception in lowest salinity: 4-6 animals) with 95% con-
fidence interval.

ONTOGENY OF CANCER OSMOREGULATION

131

E 60

T)
«

0 I

300 500 700 900 1100 1300

Medium (moim.kg-1)

^ 40
E
1 20

-c
a.

I 20

40 .

o L

300 500 700 900 1100 1300

Medium (mosm.kg-1)

Figure 6. Variations in the difference between the osmotic pressures of hemolymph and medium according
to the osmotic pressure ol the medium in zoeae 1-5, megalopae. and first crab stage of Cancer irroratiis at
15°C. Each point represents the mean value of determinations from 9-15 animals (5- 10 animals in extreme
salinities) with 95% confidence interval. O O: post-molt; • •: stage C; A - - A: premolt.

hyper-osmoconform in salinities that they normally en-
counter in their environment (Charmantier ct ui, 1988).
This could be considered an adaptation of small organisms
to planktonic or pelagic life. The slight positive difference
in osmotic pressure between hemolymph and medium
maintains an osmotic influx of water, which in turn favors
the turgescence of the body and particularly of the ex-
tended appendages and exopodites involved in the buoy-
ancy of the larvae. In a few zoeal stages of C. irroratiis,
the osmotic pressure of hemolymph is affected by the
molting stage, increasing in premolt and decreasing in
postmolt but much less regularly than in other species
like Rhithropanopeus harrisii (Kalber and Costlow, 1966),
Cardisoma giianhumi (Kalber and Costlow, 1968), Hom-
arus americanus and Penaeus japonicus (Charmantier ct
a/.. 1988). Like zoeae, megalopae of C. irroratiis hyper-
osmoconform in all media. First crab stages osmoconform
in seawater, but they hyper-regulate in a dilute medium.
Thus, the adult type of osmoregulation seems to be ac-
quired at the first crab stage. However, the ability to hyper-

regulate, evaluated by the difference between the osmotic
pressures of hemolymph and medium in a dilute medium,
increases with size in adults: in a medium of 500
mosm-kg"1, 17%», this difference was 55 ± 16
mosm-kg"' in first crab stage, 55 ± 13 mosm-kg"1 in
small adults, and 71 ± 7 mosm-kg"1 in large adults. As
in C. irroratiis, zoeae of C. borealis hyper-osmoconform,
while adults have a slight hyper-isoregulation. In a pre-
liminary study. Brown and Terwilliger (1989) found that
megalopae and first crabs of C. magister were weaker os-
moregulators than adults, after 8 h exposure to dilute me-
dia. A comparison with our results is difficult due to the
lack of numerical data. Additionally, it is possible that
the short time of exposure did not allow for complete
osmotic equilibration in adults.

In a recent study, we reviewed the evolution of os-
moregulatory abilities that have been described for
the post-embryonic development of decapod crustaceans
(Charmantier el ai. 1988). Most decapod larvae that have
been studied, including zoeae of C. irroratiis and C. bo-

132

G. CHARMANTIER AND M. CHARMANTIER-DAURES

E
o

D
•O

300 500 700 900 1100 1300

Medium (mosm.kg"1 )

Figure 7. Variations in the difference between the osmotic pressures
of hemolymph and medium according to the osmotic pressure of the
medium in zoeae 1-3 of Cancer borealis at I5°C. Each point represents
the mean value of determinations from 10 animals with 95% confidence
interval. O O: post-molt; • •: stage C; A - - A: premolt.

realis, are hyper-osmoconformers or weak regulators, an
exception being the larvae of Macrobrachium petersi
(Read, 1 984), which are confronted with very low salinities
in their natural environment and which can efficiently
regulate the osmotic concentration of their hemolymph.
During or after the larval phase, different patterns of on-
togeny of osmoregulation have been described, which we
proposed to separate into three groups (Charmantier et
al., 1988). In one group of species, osmoregulation varies
little with developmental stage; the adults of these species
are often weak regulators or osmoconformers, like He-
patus epheliticus and Libinia emarginuta (Kalber, 1970).
In M. petersi (Read, 1 984), which lives in variable salinities
due to its migration, the adult type of regulation is estab-
lished as early as the first larval stage. In a third group of
species, which includes Uca subcylindrica (Rabalais and
Cameron, 1 985), Homarus americanus, Penaeus japon-
icus (Charmantier et at., \ 988), and Cancer irroratus (this
study), metamorphosis marks the appearance of the adult
type of regulation. In other species that do not still fit in
those three categories, in which "no clear trend toward
development of adult osmoregulatory patterns toward the
end of larval life" was found (Foskett, 1 977), this could
be due to the lack of information about the osmoregu-

latory capacity of early post-metamorphic stages. For ex-
ample, in Sesarmu reticulatum studied by Foskett, 1977,
zoeae and megalopae were hyper-osmoconformers and
adults were hyper-hyporegulators, but early crab stages
were not studied. Foskett stated: ". . . even by late mega-
lops the adult osmoregulatory response is still not attained.
Examination of osmoregulation in early juvenile crab
stages may reveal osmoregulatory responses that are tran-
sitional between the larval and adult forms."

The few studies that were conducted in decapods
throughout the post-embryonic development, including
post-metamorphic stages, confirm this statement. The
transition from larval to adult type of osmoregulation may
occur rapidly when metamorphosis itself is sudden, as in
//. americanus, or may be progressive when metamor-
phosis is spread over several post-larval stages as in P.
japonicus (Charmantier et al.. 1 988). There is a lack of
agreement on the exact timing of metamorphosis in
brachyurans: it has been located either at the molt from
megalopa to first crab stage (Costlow. 1 968), or at the
molt from last zoea to megalopa (Felder et al.. 1985).
Actually, most morphological changes result from the
molt separating the last zoea and the megalopa and, in
U. subcylindrica, the type of osmoregulation also changes
at this molt ( Rabalais and Cameron, 1 985 ). However, this
physiological modification occurs only after the molt sep-
arating the megalopa and the first crab stage in C. irroratus
(this study), and possibly in 5. reticulatum (Foskett, 1 977).
Additionally, as stated by Felder et al.. (1985), "in almost
all the Decapoda, some ontogenic changes in locomotion,
feeding, and habitat coincide with early postlarval
growth." Thus, in our opinion, metamorphosis in brach-
yurans requires two molts to be completed and the mega-
lopa, while clearly postlarval, is a transitional stage be-
tween the larvae and the postmetamorphic stages starting
with the first crab stage.

In summary, studies conducted on the species of the
third group of decapods cited above demonstrate that the
completion of metamorphosis yields a change to the adult
type of osmoregulation. We propose the hypothesis that,
in most species in which larvae are hyper-osmoconformers
or weak regulators and adults are efficient osmoregulators,
the transition from the larval type to the adult type of
osmoregulation occurs at metamorphosis. More generally,
metamorphosis can be considered a combination of mor-
phological, ecological, behavioral, and physiological
changes (Costlow, 1 968; Charmantier et al.. I984b).

Relation between osmoregulation and salinity tolerance

In C. irroratus and C. borealis. zoeae are weak regu-
lators and their salinity tolerance is comparatively mod-
erate or low. In megalopae of C. irroratus, salinity toler-
ance is minimum just before the pattern of osmoregula-

ONTOGENY OF CANCER OSMOREGULATION

133

tion changes. Under natural conditions, developing
brachyurans are known to settle on the bottom during
the megalopal stage, usually near the shores (Sandifer,
1973), i.e.. in an environment subjected to possible vari-
ations of salinity. The limited salinity tolerance of mega-
lopae could cause high rates of mortality, which have been
noted in different species of Cancer, at least under culture
conditions (Charmantier-Daures and Charmantier, 1991).
Unsufficient number of available animals prevented us
from determining the salinity tolerance of early crab
stages, in which the pattern of osmoregulation has
changed. We may suppose that their salinity tolerance
has correlatively increased. In adults of both species, the
ability to osmoregulate is higher and so are their salinity
tolerances. Compared to C. borealis, adult C. irroratm
are stronger hyper-regulators in dilute media and are more
tolerant to low salinity. Thus, as previously noted in Mac-
robrachiwn petersi (Read, 1984), Uca subcylindriai (Ra-
balais and Cameron, 1985), Homarus americanus and
Penaeus japonicus (Charmantier et til., 1988), there is a
strong correlation between increased ability to osmoregu-
late and improved salinity tolerance.

Acknowledgments

Part of this study was supported by a grant from NATO.
We thank Dr. D. E. Aiken for providing lab space and
facilities, and Ross Chandler, Jay Parsons, David Robi-
chaud, and Wilfred Young-Lai for their help in capturing
and rearing crabs.

Literature Cited

Bigford, T. E. 1979. Synopsis of biological data on the Rock Crah.
Cancer irroralns Say. NOAA Techn Rep NMFS 426. FAO Synopsis
n° 123, 26 pp.

Brown, A. C., and N. B. Terwilliger. 1989. Developmental changes in
osmotic and ionic regulation in the Dungeness crab, Cancer magister.
Am /<«)/. 29: 156A.

Cantelmo, A. C., F. R. Cantelmo, and D. M. Langsam.
1975. Osmoregulatory ability of the rock crab. Cancer irroralus.
under osmotic stress. Comp. Biochem Physiol. 51A: 537-542.

Charmantier, G. 1986. Variation des capacites osmoregulatnces au
cours du developpement post-embryonnaire de Penaeus laponicus
Bate, 1888 (Crustacea Decapoda). C R AcaiL Sci. Paris 303: 217-
222

Charmantier, G., M. Charmantier-Daures, and D. E. Aiken.
1984a. Variation des capacites osmoregulatrices des larves et post-
larves de Homarus americanus H. Milne-Edwards. 1 837 (Crustacea,
Decapoda). C. R Acad. Sci. Pans 299: 863-866.

Charmantier, G., M. Charmantier-Daures, and D. E. Aiken.
1984b. Neuroendocnne control ol'hydromineral regulation in the
American lobster Homarus americanus H. Milne-Edwards, 1837
(Crustacea, Decapoda). 2. Larval and post-larval stages. Gen. Comp
Endocrmol. 54: 20-34.

Charmantier, G., M. Charmantier-Daures, N. Bouaricha, P. Thuet,
D. E. Aiken, and J.-P. Trilles. 1988. Ontogeny of osmoregulation
and salinity tolerance in two decapod crustaceans: Homarus amer-
icanus and Penaeus japonicus. Biol Bull 175: 102-110.

Charmantier, G., M. Charmantier-Daures, N. Bouaricha, P. I hurt.
J.-P. Trilles, and D. K. Aiken. 1989. Ontogeny of osmoregulation
and salinity tolerance in Homarus americanus, Penaeus laponicus
and Cancer irroratus Am. /<><>/. 29: 155A.

Charmantier-Daures, M., and G. Charmantier. 1991. Mass-culture of
Cancer irroralus larvae (Crustacea, Decapoda): adaptation of a flow-
through sea water system. Aquaculture.

Costlow, J. D., Jr. 1968. Metamorphosis in crustaceans. Pp. 3-41 in
Metamorphosis. W. Etkin and L. I. Gilbert, eds. Appleton-Century-
Crofts. New York.

Drach, P. 1939. Mue et cycle d'intermue chez les Crustaces Decapodes.
Ann lust Oceanogr.. Monaco \9: 103-341.

Engelhardt, F. R., and P. A. Dehnel. 1973. Ionic regulation in the
Pacific edible crab. Cancer magister (Dana). Can. J 7.ool. 51: 735-
743.

Felder. J. M., D. L. Felder, and S. C. Hand. 1986. Ontogeny of os-
moregulation in the estuarine ghost shrimp Callumassa iamaicen.se
var. louisianensis Schmitt (Decapoda. Thalassinidea). J Exp. Mar
Biol Ecol. 99:91-105.

Felder, D. L., J. \V. Martin, and J. \V. Goy. 1985. Patterns in early
postlarval development of decapods. Pp. 163-225 in Larval Growth,
A. M. Wenner, ed. A. A. Balkema. Rotterdam. Boston.

Finney, D. J. 1962. Probit Analysis 1 Statistical Treatment of the
Sigmoid Response Curve Second Edition. Cambridge Univ. Press,
London.

Foskett, J. K. 1977. Osmoregulation in the larvae and adults of the
grapsid crab Sesarma rclicutalum Say. Biol Bull 153: 505-526.

Hughes, J. T., R. A. Shleser, and G. Tchobanoglous. 1974. A rearing
tank for lobster larvae and other aquatic species. Progr. Fish. Cult.
36: 129-132.

Hunter, K. C., and P. P. Rudy Jr. 1975. Osmotic and ionic regulation
in the Dungeness crab. Cancer magisler Dana. Comp Biochem
Physiol. 51 A: 439-447.

Johns, D. M. 1981. Physiological studies on Cancer irroralus larvae.
I. Effects of temperature and salinity on survival, development rate
and size. Mar. Ecol. Progr. Ser. 5: 75-83.

Jones, L. L. 1941. Osmotic regulation in several crabs of the Pacific
coast of North America. J Cell. Comp. Physiol. 18: 79-92.

Kalber, F. A. 1970. Osmoregulation in decapod larvae as a consider-
ation in culture techniques. Helgoi. M'I.V.V. Mcercsuntcrs. 20: 697-
706.

kalber, F. A., and J. D. Costlow. 1966. The ontogeny of osmoregulation
and its neurosecretory control in the decapod crustacean, Rhithro-
panopeus harrisii. Am. Zool. 6: 221-229.

Kalber, F. A., and J. D. Costlow. 1968. Osmoregulation in larvae of
the land crab, Cardisoma guanhumi Latreille. Am. Zool. 8: 4 1 1 -4 1 6.

I it lulu Id. J. T., and F. Wilcoxon. 1949. The reliability of graphic es-
timates of relative potency from dose-percent effect curves. J Phar-
macol E.\p Tlieor. 108: 18-25.

Lieberman, H. R. 1983. Estimating LD 50 using the probit technique:
a basic computer program. Drug Chem Toxicol. 6: 111-116.

Mantel, L. H., and L. L. Farmer. 1983. Osmotic and ionic regulation.
Pp 53-161 in The Biology of Crustacea. I'ol. 5. Internal Anatomy
and Physiological Regulation, L. H. Mantel, ed. Academic Press,
New York.

McCluskey, W. J. 1975. The effects of hyposaline shocks on two species
of Cancer Unpubl. manuscr., 15 p. Narragansett Marine Laboratory,
University of Rhode Island, Narragansett, RI 02882.

Neufeld, G. J., and J. B. Pritchard. 1979. Osmoregulation and gill
Na+, K1 ATPase in the rock crab. Cancer irroratus: response to DDT.
Comp. Biochem Physiol. 62C: 165-172.

Rabalais, N. N., and J. N. Cameron. 1985. The effects of factors im-
portant in semi-arid environments on the early development of Uca
subcylindrica Biol. Bull. 168: 147-160.

134

G. CHARMANTIER AND M. CHARMANTIER-DAURES

Read, CJ. II. L. 1984. Intraspecific variation in the osmoregulatory ca-
pacity of larval, post-larval, juvenile and adult Macrnhrachiiim petersi
(Hilgendorf). Comp. Biochem. Physiol. 78A: 501-506.

Sandifer, P. A. 1973. Distribution and abundance of decapod crusta-
cean larvae in the York River estuary and adjacent Chesapeake Bay,
Virginia. 1968-1969. Che.ianciike Sci. 14: 235-257.

Sastry, A. N. 1970. Culture of brachyuran crab larvae using a re-cir-
culating sea water system in the laboratory, llelgol II V.v.v Meeresun-
lers, 20:406-416.

Sastry, A. N. I977a. The larval development of the rock crab. Cancer
irruralus Say, 1817. under laboratory' conditions (Decapoda Brachy-
ura). Crustaceana 32: 155-168.

Sastry1- A. N. 1977b. The larval development of the Jonah crab. Cancer
borealis Stimpson. 1859. under laboratory conditions (Decapoda
Brachyura). Crustaceana 32: 290-303.

Sastry, A. N., and J. F. McCarthy. 1973. Diversity in metabolic ad-
aptation of pelagic larval stages of two sympatnc species of brachyuran
crabs. Nelherl. J Sea Res 7: 434-446.

Sprague, J. B. 1969. Measurement of pollutant toxicity to fish. I.
Bioassay methods for acute toxicity. Water Res 3: 793-821.

Thurberg, F. P., M. A. Dawson, and R. S. Collier. 1973. Effects of
copper and cadmium on osmoregulation and oxygen consumption
in two species of estuarine crabs. Mar. Bio/. 23: 171-175.

\\ .HIM >n. S.. A. Pequeux, and R. Gilles. 1983. Osmoregulation in the
stone crab Cancer pagurtix. Mar. Bio/. Lett. 4: 321-330.

Young, A. M. 1979. Osmoregulation in larvae of the striped hermit
crab Clibanarius villains (Bosc) (Decapoda: Anomoura, Diogenidae).
Est. Coast. Mar Sci. 9: 595-601.

/itko, Y. 1982. Letcur, the lethality curve program. Can. Tech. Rep
Fish. A(/tial. Sci. 1134: 10 pp.

Reference: Biol Bull 180: L 35- 153. (February,

Sulfide-Driven Autotrophic Balance in the Bacterial
Symbiont-Containing Hydrothermal Vent Tubeworm,

Riftia pachyptila Jones

J. J. CHILDRESS1. C. R. FISHER-. J. A. FAVUZZI1, R. E. KOCHEVAR1.
N. K. SANDERS3, AND A. M. ALAYSE4

* Department of Biological Sciences and Marine Science Institute. University of California, Santa

Barbara, California 93106, ^Department of Biological Sciences. Pennsylvania State University.

University Park. Pennsylvania 16802. 'Bam/ielil Marine Station, Bamfield. British Coiimhia.

Canada I OR I BO. and4 Department Environment Profond. 1FREMER.

Centre de Brest, B. P. 70-29263 Plouiane. France

Abstract. Hydrothermal vent tubeworms, Riftia pa-
chyptila Jones, were maintained alive and studied on
board ship using flow-through pressure aquaria. Simul-
taneous measurements of O2, SCO:, -H2S fluxes showed
that the intact symbioses reach maximum rates of uptake
of 2CO2 ( > 2 Mmole g~ ' h~ ' ) at about 90 \iM 2H2S. Mea-
surements were made of hemolymph and coelomic fluid
2CO2. 2H2S, thiosulfate, pH, and hemoglobin concen-
trations in worms kept under various conditions of O2
and 2H2S. Normal hemolymph pH appears to be about
7.5 and is not affected by 2H2S and 2CO2 concentrations
within the ranges observed. We conclude that Riftia is
specialized to provide sulfide to its symbionts with min-
imal interaction of sulfide with the animal metabolism.
The uptake of sulfide is apparently by diffusion into the
hemolymph, facilitated by the sulfide-binding properties
of the hemoglobins. Both 2CO2 and PCo, are elevated in
the hemolymph above their levels in the medium, al-
though they are reduced under autotrophic conditions.
Thus inorganic carbon is apparently concentrated from
the medium into the hemolymph by an unknown mech-
anism.

Introduction

The giant hydrothermal vent tubeworm, Riftia pa-
chyptila Jones, is perhaps the most distinctive of the an-
imals living around the deep-sea hydrothermal vents. Like

Received 10 September 1990; accepted 24 November 1990.

all vestimentiferan tubeworms, adults of this species lack
a mouth and a gut (Jones, 1981, 1988; Jones and Gardiner,
1988; Southward, 1988). The adult worms appear to de-
rive their nutritional needs from the large population of
sulfur-oxidizing chemolithoautotrophic bacterial sym-
bionts that live in cells within a specialized organ — the
trophosome — in their trunk (Cavanaugh el al.. 1981; Fel-
beck, 198 1 ). The trophosome is a highly vascularized or-
gan lying between two coelomic cavities that contain a
hemoglobin-rich fluid (Jones, 1988). This anatomy re-
quires that the animal supply the needs of the symbionts
through its circulatory system (Arp et al.. 1985; Felbeck
and Childress. 1988). Because these symbionts are sulfide-
oxidizing autotrophs (Felbeck, 1981; Belkin et al.. 1986;
Fisher et al.. 1989), the worm must take up sulfide, oxygen,
and carbon dioxide from the medium and transport them
to the symbionts. These substances can be taken up from
the water by the large obturacular plume, a highly vas-
cularized organ that has a large surface area and brings
the hemolymph very close to the surrounding water (Arp
et al.. 1985; Jones, 1988). The hemolymph and the coe-
lomic fluid both have abundant extracellular hemoglobins
which are believed to play a key role in the transport of
all three of these metabolites (Childress et al.. 1984; Arp
et al.. 1985).

Two hemoglobins are found in the extracellular fluids
of these worms. One has a molecular weight of about 1.7
X 106 Mrand is found primarily in the hemolymph, while
the second is smaller (0.4 X 106 Mr) and is found in both
the coelomic and vascular compartments (Terwilliger et

135

136

J J CHILDRESS ET AL

a/.. 1980; Arp and Childress, 1981; Terwilliger and Ter-
williger, 1985; Arp el ai. 1987). Both hemoglobins bind
oxygen and sulfide reversibly with a high affinity (Arp and
Childress, 1981, 1983; Childress et ai, 1984; Arp el ai.
1987; Fisher et ai, 1988a). The sulfide binding does not
affect the simultaneous binding of oxygen, and appears
to occur at a site removed from the heme (Childress et
ai. 1984; Arp et ai. 1987). When sulfide and oxygen are
below saturation in the hemolymph, their normally rapid,
spontaneous reaction is suppressed (Fisher and Childress,
1984). Further, the hemoglobin can protect the animal
tissues from sulfide toxicity by binding the sulfide with a
higher affinity than does the site of toxic effects, cyto-
chrome-c-oxidase (Powell and Somero, 1983, 1986). The
hemoglobin does, however, release sulfide to the sym-
bionts while simultaneously protecting them from sulfide
toxicity by holding free sulfide concentrations down
(Fisher and Childress, 1984; Fisher el ai. 1988a, 1989).
The hemoglobins also buffer the hemolymph for carbon
dioxide transport (Childress et ai, 1984). Thus, the he-
molymph apparently has the properties required to take
oxygen, carbon dioxide, and sulfide from the medium
and to transport them to the endosymbionts.

Most of the experiments described in this paper were
performed to test the role of the hemolymph in gas uptake
and transport in intact, living Riftiapachyptila individuals.
In particular, we were concerned with demonstrating the
continuous uptake and oxidation of sulfide by the intact
organisms, evaluating the role of the hemoglobins in con-
centrating sulfide from the medium, looking for the pos-
sible roles of other forms of sulfur in the symbiosis, ex-
amining the impact of sulfide and symbiont autotrophy
on internal CO2 pools and pH, and observing the pattern
of exchange of gases between the coelomic fluid and the
hemolymph.

A consistent chemical terminology will be used
throughout this paper. Sulfide and inorganic carbon refer
to these substances without specifying the chemical species
involved. 2H:S and 2CO2 refer to the amounts of these
gases analyzed from acidified samples using the analytical
methods described below. They are measures of the sum
of the various chemical forms in which these substances
are found. H:S, HS~, S2~, S°, CO2, HCO, and any other
chemical formulae refer only to the chemical species
symbolized. "Free" refers to that fraction of a substance
in the body fluids that is not bound to the hemoglobins.

Materials and Methods

The tubeworms used in these studies were collected
from depths of about 2600 m at sites on the Galapagos
Rift (00°48.247'N, 86° 1 3.478^) and the East Pacific Rise
(12°48'N, 108°57'W) by deep submersibles (Alvin at the
Galapagos Rift site and Nautile at the East Pacific Rise

site). Both submersibles pulled the worms off the rocks
using their manipulators, placed them in thermally in-
sulated containers, and brought them to the surface about
2 to 8 h after capture. Once at the surface the worms were
quickly transferred to cold seawater (7°C) where undam-
aged worms were set apart for the whole animal experi-
ments described here. The worms chosen were then care-
fully removed from their natural tubes and placed in
straight plastic tubes of appropriate size so they could be
fitted into the pressure vessels necessary for their mainte-
nance. They were then quickly placed in pressure aquaria.

The worms were routinely maintained in flowing-water
pressure aquaria (Quetin and Childress, 1980) at 200 atm
pressure, 8°C, and more than 100 nAI O2. Water was
pumped through these stainless steel pressure vessels at
about 12 1/h. Previous studies (Childress et ai. 1984) and
preliminary observations during this study indicate that
although the worms live at a hydrostatic pressure of about
260 atm at these sites, they are able to survive and display
apparently "normal" behavior at pressures as low as about
100 atm. The symbionts themselves do not show signif-
icant effects of pressure on carbon fixation rates within
the pressure range used here (Fisher et ai, 1989). In the
present study pressures as low as 120 atm were used in
some experiments, but the experience cited above suggests
that these lower pressures should have little effect on the
results.

Studies involving the maintenance of the worms at
known sulfide concentrations were carried out in flowing-
water aquaria (120 atm, about 4 1/h) using transparent
acrylic pressure vessels (Quetin and Childress, 1980), al-
lowing the activity of the worms to be observed during
the experiments. Anaerobic sulfide stock solution (5 or
10 mM sodium sulfide in seawater at ph 7.0 or 7.5) was
added continuously at the intakes of the pressure pumps
with low pressure metering pumps to achieve stable sulfide
concentrations in the pressure vessels. The effluent water
from the vessels was periodically sampled with a 0.5 ml
glass syringe, and the gases were analyzed by gas chro-
matography (Childress et ai. 1984). The pH of the effluent
water was measured with a double junction electrode and
was between 7. and 8.1, depending on the experiment.

Metabolism measurements

Measurements of whole animal metabolism were made
in a flowing water system similar to that used by Anderson
et ai. (1987), but adapted for use at the high pressures
required for the survival of the worms. The system
pumped seawater through the respirometer chambers us-
ing HPLC pressure pumps with small acrylic pressure
vessels as respirometer chambers. The water in this system
was first passed through a series of filters (5.0 and 0.2 yum)
and a UV sterilizer. It was then continuously mixed by

AUTOTROPHIC FUNCTION IN RlH'l.l

137

means of metering pumps with an antibiotic solution to
achieve a final concentration of 150 mg penicillin-G per
liter and with a sulfide solution (pH 7.5 in seawater) to
achieve the desired sulfide concentration. It then went to
a vertically oriented column measuring 1 X 0. 1 m, with
the seawater entering at the top and exiting near the bot-
tom. The pH of the water in the column was maintained
at 7.5 by a pH controller that pumped 1 Af acid (HCL)
or base (NaOH) into the column. Oxygen and N2 bubbled
via the bottom of the column mixed the water in the
column while maintaining the desired O2 concentration.
The water was then pumped through the respirometer
chambers to a gas chromatograph for analysis. Two res-
pirometer streams were continuously used in these mea-
surements, one with animals in the respirometer chamber
and the other an identical system without animals, which
served as a control for spontaneous oxidation of sulfide.
Fluxes of the measured gases due to the animals were
calculated from the differences in gas concentrations in
the water exiting the experimental and control chambers.
These experiments were carried out at 1 30 atm hydrostatic
pressure.

Ammonium flux was measured for several worms while
they were in the respirometer system described above.
The ammonium concentrations in the effluents from the
two chambers were measured by flow injection analysis
(Willason and Johnson, 1986).

Dissection procedure

Worms were dissected so that samples of hemolymph,
coelomic fluid, and trophosome could be obtained for
further analysis. Worms to be sacrificed were quickly re-
moved from the pressure aquaria and the plastic tubes
and then stretched out in a dissecting tray. The body wall
below the vestimentum was carefully slit for a few cen-
timeters parallel to the main axis of the worm on the
ventral side. A sample of coelomic fluid (1-5 ml) was
quickly drawn, with a blunt needle, from the pool of this
fluid in the coelomic space and placed on ice. Subsamples
for the various analyses were quickly taken. The remaining
coelomic fluid was then drained from the worm, and a
1-ml syringe with a 30-ga needle was used to remove he-
molymph from the major dorsal vessel leading from the
trophosome to the plume of the worm. Aliquots of this
post-trophosome (pre-branchial) hemolymph sample were
quickly taken for the various analyses. Samples of tro-
phosome tissue were also frozen for later analysis of ele-
mental sulfur. If the trophosome appeared "unhealthy"
[the pinkish appearance correlated with lack of CO: fix-
ation in trophosome preparations (Fisher et a/., 1989),
occurred in 7 of the 50 animals used] for an individual
worm, the data from that worm were excluded from fur-
ther consideration. These unhealthy worms were always
characterized by low (<7.0) hemolymph pH values.

Analytical methods

Gas chromatographic methods similar to those de-
scribed by Childress el al. (1984) were used to analyze
gases in body fluids and seawater. Briefly, water samples
were acidified with phosphoric acid, and gases were
stripped from them using a glass and teflon extractor, in-
line with a thermal conductivity gas chromatograph. This
system allowed the analysis of the O:, CO:, H^S, N-,,
CH4, and CO concentrations in fluid samples of 0.2 to
1 .0 ml. The limit of sensitivity for these gases was between
1 and 20 nM. depending on the gas and the sample size.
Throughout this paper, the terms 2H;S and SCO, refer
to the amounts measured using this analytical method
without regard for the chemical species present at the very
different pH values and conditions in the worms.

To measure pH, a sample of hemolymph or coelomic
fluid was drawn from an animal with a syringe. The dead
space of the syringe was filled with blood by drawing a
small amount of sample into the syringe and then expel-
ling the air and excess blood before drawing the sample
for analysis. Without air exposure, the sample was im-
mediately injected into a Radiometer glass capillary elec-
trode (Radiometer America G298A) used in conjunction
with a reference electrode (Radiometer K171) in a water
jacketed chamber. Precision buffers (Radiometer SI 500
& S1510) were used to calibrate the electrode.

The abundances of the two hemoglobins in the he-
molymph were quantified by separating them by HPLC
gel filtration and measuring the absorbance as they eluted
from the column (Arp et al., 1987). A TSK-50 column,
7.5 mm in diameter and 300 mm long, was used with a
TSK guard column (7.5 mm by 75 mm). The eluent was
a citric acid/phosphate buffer ( 1 .63 g citric acid and 26. 1 7
g KH2PO4/1) at pH 7.5, pumped at 0.3 ml/min at 5°C.
The run time was about 40 min, and an undiluted 1-yul
sample was used. The absorbance was measured at 4 1 5
nm as the eluent left the column.

Determinations of thiosulfate and other unbound thiols
in the body fluids were made by HPLC analysis of samples
derivatized by monobromobimane using the methods of
Newton et al. (1981) and Fahey et al. (1983) as modified
by Vetter et al. (1989). Derivatives were separated on a
15 cm C- 18 reversed phase column and detected using a
235 nm filter for excitation and a 442 nm filter for detec-
tion of fluorescence. The eluent flow rate was 1.5 ml per
min, using an increasing hydrophobic gradient of HPLC
grade methanol and 2% acetic acid, starting at 10% meth-
anol and increasing to 100% during the run.

Elemental sulfur in the extracts was quantified by gas
chromatography according to the method of Richard et
al. (1977) as modified by Fisher et al. (1988b). Pieces of
tissue (0.5-2.0 g wet weight) were dried for 18 h in a 100°C
drying oven, and then extracted for 24 h with cyclohexane

138

J. J. CHILDRESS ET AL.

in a micro-Soxhlet apparatus. The extracts were "cleaned
up" by passing them through a fluorosil column to remove
lipids, and concentrated by evaporation. The injector
temperature was 240°C, and the initial column temper-
ature was 150°C, programmed to 220°C during the sep-
aration. A six foot (1.8 m) glass column with a 2 mm
bore, packed with 5% SP2401 on 100/120 mesh Supel-
coport, was used to separate sulfur. The sulfur was detected
and quantified using a thermal conductivity detector. The
detection limit for elemental sulfur was ca. 0.001% of the
dry weight of the sample (depending somewhat on sample
size). The identity of the separated sulfur was confirmed
by the distinctive smell of sulfur vapor coming out of the
gas chromatograph detector at the time of the putative
sulfur peak.

Estimation of free ~ZH:S and H}S

Because 2H2S, pH, and hemoglobin contents were
measured, it was possible, using previously published
data, to estimate the concentration of free (unbound)
2H2S as well as the various species of sulfide. Free 2H2S
was estimated by using the Hill equation describing the
relationship between fractional saturation and free sul-
fide measured at 6°C, pH 7.5 in a mixture of coelomic
fluid and hemolymph (Fisher et ill., 1988a): In [% sat-
uration/(100 - % saturation)] = 0.737(ln free 2H2S
fiM)- 1.778.

To use this equation, the capacity of each fluid sample
to bind sulfide was estimated by multiplying the small
hemoglobin aggregate concentration by one sulnde/heme
and the large aggregate concentration by three sulndes/
heme. These estimates were derived from a multiple
regression of the sulfide concentrations in nine coelomic
fluid samples dialyzed at saturating sulfide concentrations
against the concentrations of the two aggregates in those
samples (data from Arp, 1987). This regression had an r2
of 0.97 and gave coefficients of 0.90 ± 0.27 (95% C. I.)
and 2.97 ± 0.86, respectively, for the two hemoglobins.
From the estimated capacity for binding sulfide and the
measured ZH2S in the fluid, the percent saturation was
approximated, and the above equation was solved for free
sulfide. This approximation of free sulfide was then used
with the estimated sulfide binding capacity [% saturation
= 100 (bound sulfide/binding capacity)] in the Hill equa-
tion to estimate the sulfide bound to the hemoglobin. This
procedure was then carried through several iterations until
the estimates converged on a single value for free sulfide.
This value was then used to calculate the percentage sat-
uration of the fluid.

The free H2S in each sample was calculated from the
free 2H2S using a pK, value (8°C, 35%o and 120 atm) of

6.784 (Millero. 1986; Millero et ai. 1988) and the pH
measured in that particular sample.

Estimation ofPCo:

Because 2CO:. pH, and hemoglobin contents were
measured, it was possible, using previously published data,
to estimate the PCo, in the fluids. The data relating pH.
2CO:, and PCO; in Rifiia coelomic fluid at 10°C (Childress
et ai. 1984) were used as the basis of a family of curves
that predict Pco, from pH and 2CO2 . However, although
the coelomic fluid and hemolymph are quite similar in
ionic composition, the hemolymph often has much higher
hemoglobin content. Because hemoglobin is the only
protein in any concentration in the hemolymph (Arp et
ai, 1987), we used the concentration of heme as an in-
dicator of protein content in these fluids. An approximate
correction factor for hemoglobin concentration was de-
veloped by equilibrating subsamples, brought to different
concentrations in Riftia saline, of the same hemolymph
sample with gases of known Pco, and then measuring the
SCO2 in these subsamples using the gas chromatographic
method. These subsamples (0.909 and 3.554 mM heme)
were equilibrated with 2.09 torr Pco, and a final pH of
7.70 (Arp el ai, 1987). These measurements indicated
that the effect of heme concentration on 2CO2 in this
range was 0.50 mmole 2CO2/mmole heme. This was
added to the final equation used to calculate PCo; as a
factor that changed the slope of the relationship between
Pco, and SCO-, at different pH values. The equation was:
PCO* = (4.199 - 0.537 pH) + SCO2[(eA(- 1.845 pH
+ 13.396)] + 0.0667(heme - 0.79). PCO: in the medium
was estimated from the medium pH and 2CO2 with the
pKapp estimated from the equation given by Heisler
(1984), and «CO2 (0.06345) at 8°C (Skirrow. 1975).

Statistical methods

Statistical analyses were carried out using Statview SE+
and SuperANOVA (Abacus Concepts) and Fastat (Systat
Inc.). The Kendall rank correlation was used to test for a
relationship between two parameters without any as-
sumptions about the form or linearity of the relationship.
Testing for differences in the medians in paired data sets
employed the Wilcoxon signed rank test. The Mann-
Whitney U test was used to test for differences in medians
between unpaired datasets. Simple and multiple linear
regressions of raw and in transformed data were used to
describe the relationships between parameters.

Results

H 'hole animal metabolism

Due to a variety of equipment problems, only one such
experiment was successfully conducted. In this expert-

AUTOTROPHIC FUNCTION IN Rll-TIA

139

ment, two worms (8.7 and 5.0 g) were run in their natural
tubes in one chamber for 68 h. This experiment was
started at 13.5°C without sulfide. After 6 h, sulfide was
added continuously and 14 h later the animals showed
net 2CO2 uptake (autotrophy). For the next 20 h the ef-
fects of different sulfide concentrations on the fluxes of
O2, 2H2S and 2CO2 were measured while maintaining
O2 between 105 and 209 ^M (Fig. IB). After that time
the temperature of the system was lowered to 8.4°C over
2 h, and a similar set of measurements repeated at O2
concentrations between 72 and 2 1 1 nM during the next
24 h (Fig. 1A). The set of observations at 8.4°C started
at 92 nAt 2H2S, decreased in steps to 0.0 nM 2H2S, and
then was then raised to 41-49 pM 2H:S for 6 h. As can
be seen in Figure 1A, the lower 2H2S concentrations re-
sulted in less uptake of 2CO2, and without added sulfide
the 2CO2 balance was fully heterotrophic ( + 3.05 /umoles
2CO2 g~'h~'). For the first three hours after the reintro-
duction of sulfide, this balance remained heterotrophic
(+1.93 Mmoles 2CO2 g~'h~'. high point at 43 nM 2H2S
in Fig. 1A), but autotrophy was reached in the next 3 h
(-0.64Mmoles2CO2g ' h~', at 49 ^/2H2S in Fig. 1A).
The worms were then removed and their tubes replaced
in the vessels. The tubes alone did not show significant
2CO2 flux (<0. 1 /umole 2CO2 g ' worm h" ') in the pres-
ence of 130 ju/U 2H2S. When the worms were dissected
after the experiment, S° was visible in their trophosomes.

These data demonstrate that these worms were depen-
dent on 2H2S levels greater than about 50 nM to break
even on carbon flux and more than 90 nM was required
for maximum uptake of 2CO2. They also show that the
lag-time for changes in 2CO2 flux when 2H2S was re-
moved was short, suggesting that use of stored S° was not
quantitatively very important. In contrast, when sulfide
was introduced after an absence, the lag time was relatively
long (3- 14 h).

Both O2 and 2CO2 flux were significantly dependent
on the 2H2S flux (Fig. 1C). The slope of the line relating
2CO2 flux to 2H2S flux was 0.92 ±0.18 (95% C. I.) in-
dicating that 0.92 mole CO2 was fixed for each mole H2S
consumed. The slope of the line relating O2 flux to 2H2S
flux (Fig. 1C) was 1.14 ± 0.17 (95% C. 1.), indicating that
1.14 mole O2 was consumed for each mole 2H2S con-
sumed. The lines relating 2CO: and O2 fluxes to 2H2S
flux both intercept the y-axis at virtually the fluxes found
for the worms in the absence of sulfide. This indicates
that sulfide does not interact with the metabolism of car-
bon or O2 by the animal tissues. The R. Q. in the absence
of sulfide is 0.83 suggesting a metabolism based on a mix-
ture of the major substrates.

The autotrophic Riftia experiment described above
failed to show net uptake of N2, supporting other negative
data that this species' symbionts do not fix N2. A prelim-
inary study has also been carried out on ammonia flux

CD
_<D
O

CD
_O
O

+ ICC^ - - y = 1 96 • 0 046x. R= 0 83

0 -i

X IhLS y = -0 48 - 0 040x. R= 0.91

-2 -

X . x+ A

X '-.+"*• o ,-r
X x °A °

-4 -
-6 -

-8 -
-10 :

•^•V* x

•*V.

2 -

+ ICC^ - - y = 1 42 0 027x, R= 0.49

0 -H

+ X It-LS y = -0 76 - 0 035x. R= 0 62

-2 -

X + ++x - B
+ 13.5-C

-4 -
-6 -
-8 -

.1 n

\-*x^ .x' x

X« x

. . •

•

50 100 150 200 250 300

Figure 1 . Riftia pac/iypnla metabolic fluxes in a flowing water, pres-
sure respirometer system. Closed circles are oxygen fluxes, x symbols
are sulfide fluxes, and crosses are CO; fluxes. (A) Fluxes presented as
functions of the ambient sulnde concentrations measured at 8.4°C. (B)
Fluxes presented as functions of the ambient sulnde concentrations mea-
sured at 13.5°C. (C) Fluxes at both temperatures combined, presented
as functions of sulnde consumption rate as manipulated by controlling
the sulfide concentration around the worms.

in Riftia. Three different animals (3. 3, 6.1, and 17.2 g wet
weight) in flowing water pressure respirometers in heter-
otrophic carbon balance showed appreciable rates of am-
monia excretion (0.07, 0.19, and 0.27 ^mol g"1 h~', re-
spectively).

Hemolymph parameters after capture and
maintenance without sulfide

In these experiments, several properties related to au-
totrophic metabolism in Riftia pachyptila were followed
over time, after capture and recovery of the tubeworms.

140

J. J. CH1LDRESS /;/ AL

Five worms were sacrificed immediately after capture, and
their hemolymph and coelomic fluid pH, 2CO2, 2H2S,
and S2O32 as well as trophosome S° concentrations were
measured. Nine other R. pachyptila were placed in high-
pressure, flowing-water aquaria immediately after recovery
and maintained, under pressure (120 atm), in seawater
without added sulfide for varying periods of time before
sacrifice and analysis. The initial values found (Table I)
were comparable to those found previously for this species
(Childress el at.. 1984) with 2CO2 being quite elevated
and pH values being quite low. This indicated that the
worms were probably withdrawn into their tubes and an-
aerobic while they were being brought to the surface. Data
following recovery in the aquarium system supports the
same conclusion. Hemolymph and coelomic pH rose and
2CO2 concentration declined (from very high levels found
immediately after capture) after the animals were main-
tained for one or more days under pressure (Table I).
Hemolymph 2H2S concentrations in the freshly collected
animals were substantial, ranging up to 1.75 mA/(mean
of 0.71 mA/ Table I), decreased rapidly in animals main-
tained under pressure in the absence of added sulfide, and
was undetectable after three and five days (Table I). Tro-
phosome S° declined significantly with time as well, ap-
proaching zero after 3 to 5 days (Table I). Thiosulfate
concentrations in the hemolymph of R. pachyptila were

always very low (less than 36 pM, average = 24 ^Af) and
did not decline during the five days in captivity (Table I).
To examine the hypothesis that the pattern of high
2CO: and low pH found in the hemolymph of freshly
recovered worms resulted from oxygen deprivation, we
maintained two individuals for 24 h in the flowing water
aquarium system at 14 juA/ O2 and 15 nM 2H2S. Prior
to this experiment these worms had been kept in the
aquarium system for 2 days with no sulfide and more
than 100 nAIO2. The hemolymph pH was depressed (6.48
and 6.82), supporting the suggestion that depressed pH
values after recovery are the result of anaerobic metabo-
lism (Childress cl a/.. 1984). The 2CO2 values were low
(3.357 and 3.280 mAl), but at the low pH values these
represent high Pco, values (13.9 and 8.0 torr). The failure
of these worms to accumulate the higher 2CO2 concen-
trations found in freshly recovered worms (Table I) prob-
ably resulted from their plumes remaining extended and
thus continuing to exchange CO2 with the medium during
the experiment. In contrast, during recovery from the
bottom, worms were constrained in a box and could not
extend their plumes to exchange gases. This is consistent
with observations that Riftia pachyptila individuals release
substantial amounts of 2CO2 to the medium under hyp-
oxic conditions (Childress et a/., 1984). The hemolymph
2H2S contents were substantial (5.497 and 5.013 mAl)

Table I

Riftia pachyptila hemolymph, coelomic fluid, and trophosome parameters immediately after capture and alter maintenance

in the ah\ence <>/ sulfide inflowing water, pressure (120 atm) aquaria

Days after

SCO,

2H:S

s2o3:-

S°

capture

Tissue

(m moles/1)

(mmoles/l)

( mmoles/l)

(%wet wt.)

Hemolymph

7.07 ± 0.07

10.37 ± 1.05

0.714 ± 0.332

0.024 ± 0.004

Coelomic

7.14 ± 0.78

11.56 ± 2.81

0.089 ± 0.087

0.013 ±0.017

Trophosome

2.76 ± 1.38

Hemolymph

7.39

7.78

0.066

0.000

Coelomic

7.48

8.67

0.000

Trophosome

1.94

Hemolymph

7.38, 7.47

5.48. 9.45

0.000. 0.000

0.000. 0.0 1 3

Coelomic

7.42, 7.39

6.39. 9.17

0.000. 0.000

Trophosome

1.75,0.03

Hemolymph

7.49 ± 0.40

5.91 ±0.25

0.000 ± 0.000

0.014 ±0.013

Coelomic

7.59 ±0.12

5.91 ±0.56

0.000 ± 0.000

0.003 ± 0.006

Trophosome

0.092 ±0.21

Test of change over time

in captivity (Kendall

rank correlation

tan. P =)

Hemolymph

0.67,0.0014

-0.77, 0.0005

-0.70, 0.0009

-0.29.0.19

Coelomic

0.71,0.0007

-0.77, 0.0005

-0.55, 0.0085

-0.20, 0.35

Trophosome

-0.54, 0.0097

"n" indicates the number of worms and samples at each time period and the parameter values are shown as mean ± standard error of the mean.
-CO; and 2H:S indicate the total concentration ot all forms of these substances, released by acidification of the samples in the process of analysis.
The Kendall rank correlation tests the significance of changes over time in captivity (underlined tail values indicate P < 0.05) and are listed beneath
each parameter tested.

AUTOTROPHIC FUNCTION IN Rll-'TlA

141

Table II

Coelomic /hud hemoglobin concentrations as /'unctions of heinolymph hemoglobin concent rations in Riftia pachyptila alter maintenance (24 h) in
high-pressure (120 aim), flowing-water aquaria ill various fixed ~2<H:S concentrations >0.0 and <S(W ^M.
Data on freshly collected worms from Arp et al. (1987)

Wilcoxon signed-rank

[coelomic] - a + b[hemolymph]

Coel:Hemo

+ , =, -

Parameter

n b ± 95% CI

a r

All Worms
Heme (mAf)
Hemoglobin FI (mAf)
Hemoglobin FI1 (m.l/)
Data from Arp et al (1987)
Hemoglobin FI (mAf)
Hemoglobin FI1 (m.l/)

34 0.304 ± 0.204

0.873 0.47
0.04
0.02

0.05
0.633 0.39

0.005
0.145
0.187

0.70
0.0059

1.0. 33
0, 0, 34

<0.0001
<0.0001
0.228

0.0002
0.013

34
34

18
18 0.413 ±0.276

18,0. 16
0.0. 18

11.0. 7

"n" indicates the number of worms and samples and the regression coefficients are shown ±95% confidence intervals (CI ). FI is the large hemoglobin
aggregate (1.700.000 Mr) and FII is the smaller aggregate (400.000 Mr) described by Terwilliger et al (1980) and Arp el al. (1987). Underlined
regression coefficients and Wilcoxon distributions are significant at the level of at least P < 0.05. Not all analyses were completed on all specimens.
Regressions are given only when they are significant at the level of at least P < 0.05.

and apparently in equilibrium (0.52 and 0.57 fractional
sulnde saturation) with the external 2H2S (Fig. 5A, B).
This is consistent with uptake being due solely to the
binding of sulnde by the hemoglobins.

Functioning of titbeworms exposed to various
concentrations of oxygen and sulfide

To test the existing hypotheses concerning carbon
dioxide and sulnde transport in the hemolymph (Rau and
Hedges, 1979; Arp and Childress, 1983; Childress et a/..
1984; Arp et al.. 1985; Felbeck, 1985; Fisher et al.. 1989;
Fisher et al.. 1990) and to examine responses of this species
to different external sulnde concentrations, a series of ex-
periments were conducted in which individual tubeworms
were maintained under different conditions before dis-
section and analysis. In these experiments, R. pachyptila
individuals were maintained in the high-pressure flowing-
seawater aquaria in the absence of sulnde for two days
after capture. This allowed the worms to recover from
capture and to metabolize most of their internal stores of
inorganic sulfur compounds. These worms were then ex-
posed continuously to constant concentrations of sulfide
(0-805 nAl 2H2S) for 24 to 36 h while in the high-pressure
( 120 atm) flowing-seawater aquaria at 8°C. Oxygen con-
centrations in the seawater during these experiments were
between 0 and 276 \iM. After the sulfide exposure, the
worms were dissected and samples taken. The hemolymph
and coelomic fluid samples were analyzed for pH, 2CO2,
2H2S, S2O3: , and the two hemoglobin fractions. Tro-
phosome samples were analyzed for S°. Upon dissection,
seven of the worms were found to have substantial
amounts of trophosome that appeared to be in poor con-

dition (see Materials and Methods), and these were
dropped from further consideration, leaving 43 worms in
the study. Extensive exploration of the data with scatter-
plots suggested that the data could best be presented in
two groups; one of these consisted of 28 animals kept at
O2 concentrations greater than 42 juA/ and whose he-
molymph pH was greater than 7.2. These worms showed
evidence of autotrophy and blood circulation (to be dis-
cussed below). The other group of 1 5 worms consisted of
individuals kept at O2 concentrations of 42 \iM or less (8
worms) and individuals whose hemolymph pH was less
than or equal to 7.2(11 worms). The low O2 apparently
limited sulfide oxidation while the low pH values appar-
ently indicated anaerobic metabolism in the worms due
to behavioral (remaining contracted in the tubes) or un-
detected physiological constraints. In the figures and tables
to be presented, the numbers for each analysis are often
less than the total number of individuals, because not all
analyses were successfully executed on all specimens.

The heme contents of the hemolymph and coelomic
fluid samples of the worms were significantly correlated,
but the hemolymph samples had much higher heme con-
centrations than did the coelomic fluid ones (Table II).
There was no significant correlation between the concen-
trations of the large hemoglobin (FI) in the two com-
partments, but the concentration in the hemolymph was
always much higher (Table II, Fig. 2). In contrast, there
was no significant difference in the concentrations of the
small hemoglobin (F II) between the two compartments
(Table II). However, because there was no significant cor-
relation between the concentrations in the two compart-
ments, it is apparent that they are not confluent. Arp et
al. (1987) suggested that these two compartments may be

142

J J. CHILDRESS ET AL

Hemoglobin Fraction I (mM)

Figure 2. Frequency distributions of the concentrations of the large
hemoglobin [FI, 1.7 X 10" Mr, (Terwilliger el al. 1980; Arp el at .. 1987)]
in coelomic fluid and hemolymph of Ri/tia pachvptila kept for 24 h at
different external sultide and d concentrations.

1.2 -

O °8 H

° 0.4 H
01

- y = 00133*0 176x . R= 090

4 6 8 10

Hemolymph XH2S (mM)

Figure 3. Coelomic fluid -H,S as a function of hemolymph iH:S
in Rifia pachypltla kept for 24 h at different external sulfide concentrations
and>42

confluent at the size of the smaller hemoglobin, because
the concentrations in the two compartments were signif-
icantly correlated. However, re-analysis showed that while
the concentrations were significantly correlated in their
data, the coelomic fluid had higher concentrations pre-
cluding confluence for this size molecule (Table II).

Although the two compartments do not exchange he-
moglobin molecules, it is apparent that small dissolved
molecules are readily exchanged, because the higher O2,
higher pH group had highly significant correlations be-
tween the values of all of the measured parameters be-
tween the two compartments (Table III). In addition, there
were significant differences in the values of all of these

parameters, except thiosulfate, between the compart-
ments, apparently resulting from their interactions with
the hemoglobins. 2H2S was in much higher concentra-
tions in the hemolymph (Fig. 3) than in the coelomic
fluid because of the much higher concentrations of he-
moglobin FI, which binds 3 moles of sulfide per mole of
heme, although this binding is not to the heme group
itself (Arp el al., 1987), versus 1 mole of sulfide per mole
of heme for F II (Fig. 2). These correlations and distri-
butions indicate that these worms were circulating their
blood effectively. In contrast, the low O2, low pH group
had no significant correlations between the two com-
partments for these parameters, and the values were sig-

Table III

Riftia pachyptila coelomic fluid parameters as a function of the same parameters in liemolymph after maintmancc (24 h) of the worms in flowing
water, pressure (120 atml aquaria at various fixed ^.H;S concentrations between 0.0 and 600 ^M
with external O2 concentrations > 42 ^M ami hemolymph pH > 7.2

Wilcoxon signed-rank

[coelomicj - a + blhemolymphj

Coel:Hemo

Parameter

b ± 95% CI

+, =, -

0.841 ±0.394

1.223

0.45

0.0002

16. 2, 8

0.042

£CO2 (mM)

0.990 ±0.105

0.520

0.94

<0.0001

25, 0. 2

<O.OOOI

PCOJ (torr)

0.744 ±0.171

0.638

0.82

<0.0001

6, 0, 14

0.04

2H2S(mA/)

0. 1 76 ± 0.034

0.013

0.81

<O.OOOI

0, 7, 20

<O.OOOI

°>c- Hb sulfide saturation

0.759 ± 0.123

-0.019

0.90

<0.0001

0, 7, 13

0.0015

Free 2H,S (mA/)

0.1 40 ±0.031

0.002

0.84

<0.0001

0.6, 13

0.0015

FreeH2S(mA/)

0. 1 33 ± 0.024

0.0003

0.89

<0.0001

1,6, 12

0.0024

S2O32~ (mA/)

1.54 ±0.25

-0.021

0.89

<0.0001

7,3. 12

0.41

"n" indicates the number of worms and samples and the regression coefficients are shown ±95% confidence intervals (CI). -CO; and -HiS indicate
the total concentration ot all forms of these substances, bound and free, released by acidification of the samples in the process of the analyses. Free
-H,S is an estimate of the free sulfide of all molecular species. Free H2S is an estimate of the free (i.e.. unbound) concentration of this molecular
species. Underlined Wilcoxon distributions are significant at the level of at least P < 0.05. These data include observations for all parameters for
seven animals that were not exposed to sulfide during the experiment and had internal sulfide concentrations of zero. Not all analyses were completed
on all individuals studied.

AUTOTROPH1C FUNCTION IN R1FTIA

143

nificantly different for only three of the parameters (Table
IV). Such lack of equilibration indicates a lack of oppor-
tunity for exchange between the two fluids, suggesting
that circulation was impaired in this group of worms.

Because the hemolymph and coelomic fluid parameters
were always parallel and closely correlated, and because
the low O2 , low pH worms do not, for the most part, show
signs of autotrophy and effective circulation, the hemo-
lymph parameters from the higher O:, higher pH worms
will be emphasized in considering the responses of the
internal parameters to external sulfide (Table V). The low
O2, low pH group (Table VI) will be considered primarily
in contrast to the other group.

In the higher O2 group, hemolymph and coelomic 2H2S
were correlated with external 2H2S. They were at least
one order of magnitude higher than the external concen-
tration in all cases (Table V, VII, Fig. 4A), clearly dem-
onstrating the ability of this worm to concentrate sulfide
from its environment. However, their hemoglobin was
maintained well below sulfide saturation at all external
sulfide concentrations tested (Fig. 5A) showing 50% sat-
uration at an external 2H2S of 122 ^M as compared to
an /'/; vitro affinity of 50% saturation at 1 1.2 fj.Af 2H:S
(Fisher el <//.. 1988a). The hemolymph free 2H2S and free
H2S also increased with external 2H2S, but remained
about an order of magnitude lower than the external con-
centrations (Table V, Fig. 5B, C). Thus, although the 2H:S
concentration in the hemolymph was much higher than
outside the worm, there was a significant gradient from
the outside to the inside for the free chemical species. The
latter gradient could only be maintained by the con-
sumption of sulfide within the worm, presumably by the
symbionts.

In contrast, the low O2. low pH group shows only three
barely significant correlations between external SH2S and
any of the hemolymph sulfide parameters (Table VI).
Further, the hemolymph sulfide saturation is close to that
expected in vitro, and 50%. saturation is close to the //;
vitro value (3.3 and 1 1 .2 pM 2H2S, respectively, Fig. 5A).
Thus, the ability of these worms to concentrate 2H2S in
their hemolymph appears to be explained entirely by the
binding of sulfide by the hemoglobins. In addition, the
hemolymph free 2H2S and free H2S were essentially in
equilibrium distributions with the corresponding external
parameters (Fig. 5B, C) indicating that no gradient for
passive uptake exists in these worms. Symbiont sulfide
oxidation had apparently essentially ceased under these
conditions so the hemoglobins could no longer function
to depress free sulfide concentrations.

It is also apparent from the data that sulfide is the only
sulfur compound of importance in the hemolymph. Al-
though thiosulfate was found in both groups, and increases
significantly in the presence of external sulfide (Table V),
it is typically less than hemolymph 2H2S by more than
one order of magnitude (Fig. 4).

The hemolymph 2CO2 and Pco, values are both re-
duced at higher external 2H:S concentrations in the
higher O2 group (Table V, VII, Fig. 6A, B), suggesting the
removal of inorganic carbon by the autotrophic sym-
bionts. There were no significant relations between these
parameters for the low O2, low pH group (Table V, Fig.
6A. B). In addition, the internal PCo, was higher than the
external under virtually all conditions (Fig. 6B, Table V,
VI) precluding uptake by passive diffusion into the he-
molymph.

Table IV

Coelomic fluid parameters ax a Junction oj the same parameters in hemolymph oj Riftia pachyptila after maintenance {24 h) in high-pressure (120
aim), flowing-water aquaria at various fixed sulfide concentralicinx between 0 13 and SOI) ^M
with O: concentrations < 42 /iM or hemolymph pH < 7.2

[coelomic] = a + b[hemolymph]

Parameter

b ± 95% CI

Wilcoxon signed-rank

Coel:Vasc

0.19

0.105

11.0.4

0.125

SCO; (mM)

0.45

0.054

10.0,3

0.022

PCOl (torr)

0.05

0.47

0. 0, 1 3

0.0015

2H2S(mAf)

0.00

0.96

2,0, 12

0.0019

% Hb sulfide saturation

0.03

0.56

5.0. 8

0.34

Free2H,S(m.U)

0.04

0.49

5.0, 8

0.60

Free[H2S](mM)

0.03

0.55

4.0.9

0.34

S2O32~ (mM)

0.04

0.691

5,0,2

0.13

"n" indicates the number of worms and samples and the regression coefficients are shown ±95% confidence intervals (CI). 2CO2 and SH:S indicate
the total concentration of all forms of these substances, released by acidification of the samples in the process ol the analyses. Free 2H2S is an estimate
of the unbound sulfide of all molecular species. Free [H2S] is an estimate of the concentration of this molecular species. Underlined Wilcoxon
distributions are significant at the P < 0.05 level. No regressions were significant at the P < 0.05 level and therefore none are listed.

144

J. J. CHILDRESS ET Al.
Table V

Riftia pachyptila liemolvmph parameters and S° in trophosome as Junctions of external conditions after maintenance (24 h) in high-pressure 1 1 2/1
aim), flowing-water aquaria in various fixed 2//,5 concentrations greater than 0.0 and less than 600 /jM (external O: concentrations > 42 ^M and
hcniolvmph pH > 7.2). kcndall correlations, but not the regressions or Wilcoxon tests, include seven individuals at 0.0 S//i

X Vanable

Kendall correlation

[hemolymph parameter] = aXb

Wilcoxon signed-rank

External:Hemo
+, =, - P

Hemolymph
Parameter

lau

P =

n b ± 95% CI a

X = External ZH2S

-0.165

0.24

0.05

0.38

SCO2 (mA/)

-0.629

<O.OOOI

20 -0.224 ±0.1 24 1.565

0.44

0.0014

Pco, (torr)

-0.529

<0.0001

19 -0.1 70 ±0.1 50 1.525

0.25

0.031

2H-,S(mA/)

0.826

<0.0001

20 0.448 + 0. PI P. 53

0.76

<0.0001

0.0,20 <0.0001

Sulfide saturation

0.772

<o.ooo i

18 0.417 ± 0.138 1.057

0.72

<0.0001

Free 2H,S (mA/)

0.861

<0.000 1

18 1.05 +0.41 0.105

0.65

<0.0001

17,0. 1 0.0002

Free H,S

0.832

<0.0001

18 1.086 ±0.438 0.0219

0.63

<0.0001

S2032^ (mA/)

0.487

0.0015

0.11

0.13

S° (% wet wt.)

0.504

0.0006

0.017

0.66

X = External H^S

Free H2S

0.694

<0.0001

18 1053+0591 0.179

0.47

0.016

17,0. 1 0.0004

A' = External PCO!

Pco,(mA/)

-0.361

0.011

0.007

0.74

1.0,17 0.0002

Af = External pi I

0.123

0.38

0.018

0.68

14, OJ> 0.0079

"n" indicates the number of worms and samples and the regression coefficients are shown ±95% confidence intervals (CI). 2CO2 and -H2S indicate
the total concentration of all forms of these substances, released by acidification of the samples in the process of the analyses. H,S indicates only this
chemical species itself. "Free" indicates an estimate of quantity present in a fluid but not bound to the hemoglobin. Only regressions that were
significant at the P < 0.05 level are shown.

Hemolymph pH appeared to be unaffected by external
or internal sulfide or external pH (Table V, VI, VII, Fig.
6C), although it is somewhat variable. This lack of inter-
action suggests that pH is not apt to be a significant factor
in the uptake and distribution of sulfide or inorganic car-
bon from the environment into the hemolymph. The
strong effect of low O: on hemolymph pH is probably
due to the accumulation of acidic endproducts of anaer-
obic metabolism.

These various parameters followed the same trends in
the coelomic fluid. However, the multiple regression
analyses consistently showed that the strongest predictor
of a chemical parameter in the coelomic fluid is not an
external parameter but the corresponding parameter in
the hemolymph. This indicates that the route of transport
to the coelomic fluid is via the hemolymph and not the
body wall.

Discussion

A utotrophy

Riftia pachyptila, like all vestimentiferan tubeworms,
lacks a mouth and a gut as an adult, and as a result, its
nutrition has been a matter of considerable investigation

and speculation. While net 2CO2 uptake (autotrophy) by
the intact symbioses between sulfur-oxidizing, autotrophic
bacteria and marine invertebrates living around deep-sea
hydrothermal vents has been widely assumed (Cavanaugh
el a/.. 1981; Felbeck. 1981, 1985; Felbeck el a/.. 1981;
Cavanaugh, 1985; Southward, 1987), the data presented
here are the first actually to demonstrate this in a vesti-
mentiferan tubeworm or any hydrothermal vent animal.
Actual autotrophic balance for the tubeworm symbiosis
of course depends on the reasonable assumptions that
much of the fixed carbon is available to the animal tissues,
and the production of mucus and loss of small organic
molecules are not large compared to the rate of carbon
fixation.

The only previous demonstration of autotrophic bal-
ance in an animal/bacterial symbiosis was for the shallow-
living, gutless protobranch bivalve, Solemya reidi (An-
derson et ai. 1987). In that case, the clams showed a max-
imum net 2CO2 uptake of 0.89 ^mole g~' h"1 which
equals about 0.24% of the clam's total organic carbon per
day. In contrast, Riftia pachyptila apparently has a con-
siderably higher maximum rate of net SCCK uptake (2.74
/jmole g ' h ' maximum in this study). This high rate,
combined with the relatively low carbon content of 5.5%

AUTOTROPHIC FUNCTION IN RII-T1A
Table VI

145

Hemolymph parameters anil S° in trophosome us Junctions oj external stil/ule in Riftia pachyptila alter maintenance (24 li) in high-pressure (120
aim). Jhrning-water aquaria al various fixed I//,5 concentrations between 0.013 ant/ 8I><> ^M
(external O; concentrations < 42 ^M or hemolymph pH < 7.2)

Wilcoxon signed-rank

Kendall correlation

[hemolymph parameter] - aX

External:Hemo

+ , . - P

Hemolymph
Parameter

tau

P =

b ± 95% CI a r

,V = External -//,S

-0.154

0.42

0.00

0.97

2CO2(mA/)

-0.174

0.37

0.10

0.26

PCO, (torr)

-0.223

0.91

0.02

0.61

2H,S(mA/l

0.385

0.045

0.08

0.30

0, 0, 1 5 0.0007

Sulfide saturation

0.425

0.034

0.098 ± 0.088 0.885 0.33

0.033

Free SH,S(mA/)

0.438

0.029

0.646 + 0.523 0.270 0.38

0.020

8, 0, 6 0.55

Free H2S

0.402

0.040

0.669 ±0.562 0.100 0.36

0.023

S2O,2~ (mA/)

-0.410

0.20

0.56

0.053

S° (% wet wt.)

1 1

-0.185

0.94

1 1

0.028

0.62

A" = External H,S

Free H,S

0.291

0.148

0.20

0.106

4,0, 10 0.177

.V = External t>co.

Pc02(niA/)

-0.205

0.31

0.005

0.82

0, 0, 14 0.001

X = External />//

-0.216

0.26

0.021

0.87

13,0,2 0.002

"n" indicates the number of worms and samples and the regression coefficients are shown ±95'" confidence intervals (CI). 2CO2 and 2H2S indicate
the total concentration of all forms of these substances, released by acidification of the samples in the process of the analyses. H2S indicates only this
chemical species itself. "Free" indicates an estimate of quantity present in a fluid hut not hound to the hemoglobin.

of wet weight of this species (Fisher et al., 1988b), results
in a much higher estimate of 1 .4% of the tubeworm's total
organic carbon per day. This suggests a high potential
growth rate in this species, which is supported by some
evidence of growth in length in situ (Roux et al., 1989).
Very rapid growth has been hypothesized to be important
in this species' apparent domination of young vent sites
(Childress, 1988; Fustec el al.. 1988; Hessler et al., 1988).
The maximum rate of carbon fixation (2.74 /umole
2CO: g worrrr'tT1) for the intact symbiosis corresponds
to a rate of 17.9 ^mole 2CO2 g trophosome" 'h"1, assum-
ing that trophosome accounts for 15.3% of the wet weight
of the intact symbiosis (Childress et al., 1984). This rate
is in good agreement with the maximum rates of fixation
of H14CO3 (13 to 28 Mmol SCO2 g trophosome' V)
observed in preparations of trophosome tissue from Riftia
pachyptila containing viable endosymbiotic bacteria using
sulfide as a substrate (Fisher el al., 1989).

Substrates used by the symbiosis

Studies of the isolated symbionts of Riftia pachyptila
have indicated that these symbionts use only sulfide and
not thiosulfate as a source of externally derived reducing
power (Belkin et al., 1986; Fisher et al., 1989; Wilmot
and Vetter, 1990). The data presented in this paper sup-

port the view that vestimentiferan tubeworms are spe-
cialized to supply only sulfide to their symbionts (Childress
et al.. 1984), because sulfide is concentrated from the me-
dium and is quickly used by the symbionts (Fig. 4, 5,
Table I). In contrast, thiosulfate, an endproduct of animal
sulfide oxidation (Vetter et al.. 1987; O'Brien and Vetter,
1990), is always at a very low concentration in the he-
molymph (Fig. 4) and is not quickly used by the symbiosis
(Table I). The lack of interaction (either inhibition or uti-
lization as substrate) of sulfide with the animal metabolism
is also shown by the fact that the regressions of O-. and
2CO2 fluxes versus 2H2S flux pass essentially through the
values of O2 and CO2 flux measured in the absence of
SH2S (Fig. 1C). The low levels of sulfide oxidase activity
reported from the body wall of R. pachyptila (Powell and
Somero, 1986) are apparently of little significance in the
overall metabolism of this species because so little thio-
sulfate is found in the body fluids. Thus, the animal me-
tabolism has little interaction with sulfide, delivering it
intact to the symbionts.

This arrangement is quite different from that of sym-
biont-containing bivalves, which appear to oxidize sulfide
to thiosulfate and to supply this to the symbionts. Solernya
reidi mitochondria can produce ATP from the oxidation
of sulfide to thiosulfate (Powell and Somero, 1985;
O'Brien and Vetter, 1990), which can then be supplied

146

J J. CHILDRESS ET AL

E,
CO

C\J

c\
CO

0.2 -

External SH2S (mM)

Figure 4. 3H2S and thiosulfate concentrations in hemolymph and
coelomic fluid in Riflia pachyptila kept for 24 h at different external
sulfide concentrations and >42 jjA/CK. (A) The broad solid line represents
equal concentrations of 2H2S outside the worm and in its fluids. The
narrow solid line and the closed circles apply to the hemolymph while
the dashed line and crosses apply to the coelomic fluid. (B) The closed
circles represent the concentrations of thiosulfate in the hemolymph in
these same worms.

to the symbionts to support their metabolism (Anderson
et at.. 1987; Vetter el ai. 1989). In this species, the oxi-
dation of sulfide apparently has substantial effects on the
animal metabolism, reducing the animal carbon oxida-
tion, which indicates that the sulfide oxidation is of met-
abolic significance to the animal tissues (Anderson et al.,
1987). Similarly, the symbionts of Balhymodiolus ther-
mophilus, the vent mussel, and Calyplogena magnified,
the vent clam, both appear to be able to use thiosulfate
to drive carbon-fixation (Belkin et al., 1986; Childress et
al., 1991), and the animals involved accumulate substan-
tial thiosulfate in their body fluids (Fisher et ai, 1988c;
Childress et ai, 1991). Whether these other animals can
obtain energy from the oxidation of sulfide remains to be
tested.

Rift i a pachyptila symbionts are dependent on the im-
mediate availability of sulfide to drive significant rates of
autotrophy. While the symbionts do store S° at concen-
trations up to 3200 /ig atoms/g fresh weight (Fisher et ai,
1988b) and can oxidize the stored S° in the absence of
external 2H2S (Table I), the metabolism experiment re-
ported here demonstrated that both O2 and autotrophic
2CO2 fluxes were dependent upon an external supply of
2H2S. The stored S° did not support a detectable rate of

2CO: uptake for even a few hours. This is the same sit-
uation found in the bivalve Solemya reidi (Anderson et
u/.. 1987). Thus, it appears that while the substantial S°
stores often found associated with sulfur-oxidizing sym-
bionts (Vetter. 1985; Somero et ai. 1989) can be used by
the symbionts, these rates are only a small fraction of the
rates of oxidation of sulfide or thiosulfate. This may well
be the case for free-living sulfur-oxidizing bacteria as well
(Nelson et ai. 1986). S° sulfur stores may be of signifi-
cance for the survival of the symbionts during times of
sulfide deprivation, but apparently do not represent a sig-
nificant store to support the symbiosis.

Respiratory fluxes in response to sulfide

Oxygen and 2CO2 fluxes in Riflia pachyptila are de-
pendent upon the sulfide flux. Net 2CO2 uptake requires
the presence of both O2 and sulfide. About 90 ^M 2H2S
was necessary to reach the maximum 2CO2 uptake rate
(Fig. 1), and external O2 concentrations greater than 42
nM appeared to be necessary for the use of sulfide by the
symbionts (Fig. 5). These concentrations are similar to
those that stimulate maximal autotrophy in 5. reidi (An-
derson el ai, 1987). However, autotrophic 2CO2 uptake
by the R. pachyptila symbionts in the intact symbiosis
did not appear to be inhibited by external sulfide concen-
trations up to 600 pM (Fig. 5B), unlike that of the sym-
bionts of S. reidi, which are inhibited in the intact sym-
biosis at external 2H2S concentrations of about 250 nAI
(Anderson et al., 1987).

These environmental requirements of R. pachyptila
appear to match closely the environmental conditions
where the species is found. Where Riflia is in abundance,
the flow of vent water is high, 2H2S can approach 350
fj.M in the vent water and O2 in the ambient water is
around 110 ^M (Fisher et ai. 1988b; Johnson et al.,
1988b). In situ measurements of sulfide and O2 distri-
butions around the tubeworms have suggested that they
take up sulfide from concentrations above about 60 nAf
and O2 from concentrations above 70 fiAf, with maximal
uptake rates from concentrations around 100 ^M in both
cases (Johnson et ai. 1988b). The worms gain access to
both substrates at high concentrations because the water
around them is not well mixed, and they are therefore
exposed to conditions that fluctuate between vent water
( 1 5°C, 350 nM 2H2S, 0 pM O2) and ambient water (2°C,
0 fiM SH2S, 1 10 |«A/ O2) on time scales of fractions of a
second and longer (Johnson et ai. 1988a). This species
then appears to be specialized for high rates of autotrophic
function, and, as a result, it requires the high concentration
and supply of sulfide associated with rapid venting. These
stringent habitat requirements make Rijtia pachyptila
vulnerable to either natural reduction in vent flow over
time or diversion of vent flow by mussels. R. pachyptila

AUTOTROPHIC FUNCTION IN Rll-11 A

147

13
(jy

_fZ
Q.

0.8 -

(n 0.6 -

~03 .O

§2 0.4

'tsa

03 03

.>- rn 0.2 -

c\j

0.1 --.

0.01

0.001 -

10'4

0.1

0.01 i

0.001 -

ul 10

H 1 1 I I I I

K External water •

0.01

i 1 1 — I — i — i i | 1 r~

0.1

External IH2S (mM)

Figure 5. Rillui pachyplila hemolymph sullide parameters as a function ofexternal £H:S concentrations
in worms kept for 24 h at fixed SH;S concentrations. Closed circles and narrow solid lines represent values
from worms that were kept at >42 nM O2 and had hemolymph pH values > 7.2. Open circles and dashed
lines represent values from worms that were kept at <42 pM O2 or had hemolymph pH values <7.2.
Regression equations for the plotted lines may be found in Tables V and VI. (A) The fractional saturation
of the hemoglobins in vivo with sulfide as estimated from the hemoglobin concentrations, hemolymph 3H2S
and in vitro sulfide binding properties. The broad solid line is the saturation versus 2H;S relationship
determined in vitro (Fisher el a/.. 1988a). (B) The relationship between free (unbound) iH:S and external
-H;S in hemolymph. The broad solid line represents equal concentrations in the hemolymph and outside.
(C) The relationship between free (unbound) H:S and external 3H2S. The x symbols and broad solid line
represent the external H:S concentrations in these same experiments.

numbers might therefore decline at a vent site long before
venting ceased, as has been observed at the Galapagos
Rift Rose Garden site (Hessler et ai. 1988).

Molar ^CO::O::^H,S ratios

The maximal measured uptake rates of O2, 2H2S, and
2CO2 by Riftia pacliyptila are high (Fig. 1 ); they are about

twice those of 5. reidi for O2 and 2H2S and three times
that of S. reidi for 2CO: (Anderson et a!., 1987). The
relationships between the O2 and 2CO2 fluxes and the
2H2S flux provide quantitative estimates of the depen-
dences of the former fluxes upon the latter (Fig. 1C). As
noted earlier, these relationships suggest that there is little
direct interaction of the animal metabolism and the SH2S
flux, and thus they apparently reflect the symbiont me-

148 J J CHILDRESS ET AL

Table VII

(.'omi\in\on\ ol hemolymph and coelomic fluid parameters in Riftia pachyptila alter maintenance (24 h) in high pressure (120 aim), flowing-water
tu/itiirui cither in ihe absence or presence (0.016 to 0.593 mM. mean = 0.19 mM ~H,S) of sulfide

Hemolymph

Coelomic lluid

Wilcoxon Coel:Hemo

+. =, -

Parameter

0 sulfide

sulfide

0 sulfide

sulfide

0 sulfide

sulfide

pH
2CO2 (mM)
PCO! (torr)
2H,S(m;U)

7.47 (+0.32.7)
5.76(+0.66. 7)

7.40 (±0.26, 19)
2.78 (±0.25. 20)

7.54 (0.05. 7)
6.44 (±0.049. 7)

7.44 (0.03, 20)
2.78(0.26, 21)

5,0. 2
6, 0, 1

14, 0. 5

19.0. 1

5.81 (+0.78,7)

3.16 (+0.28, 18)

4.95 (+0.00. 7)

2.92 (+0.00, 19)

1,0,6

1.0. 17

0.0 (7)

5.38 (+0.61. 20)

0.0 (7)

0.95 (±0.12, 21)

0. 7,0
0. 3. 4

0. 0. 20

0.01 (+0,01, 7)

0.06 (+0.01. 15)

0.00 (±0.00, 7)

0.06 (±0.02, 17)

7.0. 8

Parameter values are shown as means with the standard errors of the means and the number of observations in parentheses. For the hemolymph
and coelomic fluid comparisons with and without external sulfide. the data sets were compared using the Mann-Whitney U test. Single underlined
pairs of means are from groups that have null hypothesis P values < 0.05. Double underlining indicates lvalues < 0.005. The relative concentrations
of each substance were compared between the coelomic and vascular compartments using the Wilcoxon signed-rank test. Single and double underlining
have the same meanings for this test as for the Mann-Whitney.

tabolism. The regression analyses of the metabolism ex-
periment (Fig. 1C) indicate that these symbionts fix 0.92
mole 2CO: using 1.14 mole O2 and 1 mole 2H2S. Using
the calculation methods of Kelly (1982), the thermody-
namic efficiencies implicit in these ratios can be deter-
mined. Given a requirement of 496 kJ to reduce CO2 to
hexose, and a AG = — 716 kJ for the oxidation of sulfide
to sulfate, the resulting efficiency is 63%. The molar ratio
observed for 5. reidi, 0.38 ZCO2:0.92 O2:l 2H2S, gives
an efficiency of 40% if one assumes that the bacteria are
using thiosulfate (AG = -936 kJ for two S atoms) (An-
derson el a!.. 1987). In contrast, studies of the thermo-
dynamic efficiencies of free-living bacteria have been done
using very different methods, and they have shown lower
efficiencies. The studies of free-living bacteria have used
the ymax or "true growth yields" to estimate fixation in-
dependent of maintenance metabolism (Kelly, 1982).
Thermothrix thiopara has the highest ratios yet deter-
mined for aerobic sulfur-oxidizers (0.58 2CO2:1 thiosul-
fate at 72°C), corresponding to a thermodynamic effi-
ciency of 29% (Mason et al, 1987). While the ratios and
efficiencies for the 5. reidi and R. pachyptila symbionts
seem unusually high, this may be a result of the symbiotic
lifestyle.

Measurements of the ymax in free-living bacteria are
generally made in a chemostat that maintains constant,
optimal conditions for the growth of the bacteria. The
production of bacterial biomass is then measured. This
situation is very different from that in a symbiosis in that
microbial growth involves the synthesis of a variety of
complex compounds, not primarily the production of
small organic molecules as is typical of animal/algal sym-
bioses and is probably typical of most animal/bacterial

symbioses as well. In .S. reidi. the symbionts "leak" newly
fixed carbon within seconds and are apparently held at a
very low rate of growth (Fisher and Childress, 1986). While
much less is known about this aspect of the R pachyptila
symbiosis, it too is believed to operate primarily by the
"leakage" of small organic compounds from the bacteria
to the animal with the bacteria being held in a state of
slowed reproduction (Felbeck. 1985). Under the condi-
tions present in these symbioses (low microbial growth
rates, synthesis of small organic compounds, and mainte-
nance in an environment controlled by the host) it may
be possible for bacteria to achieve unusually high effi-
ciencies for CO2 fixation.

The internal consistency of the molar ratios for Riftia
pachyptila can also be evaluated from the O2:2H2S ratios.
The ratio of 1.14:1 falls well short of the expected 2:1 if
all of the sulfide is oxidized to sulfate in the absence of
other reductive processes. However, because carbon fix-
ation is a reductive process, the reducing equivalents used
in carbon fixation must also be taken into account. Fol-
lowing the reasoning of Kelly (1982), each CO2 fixed to
the level of CH2O via the Calvin-Benson cycle requires
4e~ and 4H+. For our ratio of 0.92 2CO2:1 2H2S, CO2
fixation requires 0.92 X 4(H) = 3.68 of the 8(H) available
from complete oxidation of sulfide. Thus, 8 -- 3.68
= 4.32(H) remain for the reduction of O2, and the pre-
dicted O2 uptake would be 4.32/8 X 2 = 1.08 O2, com-
pared with our value of 1.14. This agreement supports
the validity of the observed ratios.

In contrast, the ratios determined for S. reidi showed
a considerable discrepancy (0.92 O2 : 1 2H2S observed ver-
sus 1.62:1 calculated as above) with insufficient O2 con-
sumption seemingly to account for the observed fixation

AUTOTROPHIC FUNCTION IN R11-T1A

149

_
OJ

E
co

CO
D_

JZ
Q_

E
^

o
E

O
O

c\j
O

10 -

1 -

0.1
7.6

5 -

74 -

73 -

H 1 — I I I I

-t * ( — I I I

001

0 1

External XH0S (mM)

Figure 6. Rillui pacliypnla hemolymph parameters as a function of external -H;S in worms kept for
24 h at a given 2H2S. Closed circles and solid lines represent values from worms which were kept at >42
nM Oi and had hemolymph pH values >7.2. Open circles represent values from worms which were kept
at <42 n\l O2 or had hemolymph pH values <7.2. Regression equations for the plotted lines may be found
in Table V. Where no line is plotted, the relationship was not significant (Tables V and VI). (A) Hemolymph
SCO2 as a function of external —HiS. (B) Pco, as a function of external iH:S in hemolymph and in the
external water (x symbols). (C) Hemolymph pH as a function ol external 2H;S.

of carbon and oxidation of sulfide (Anderson el at., 1987).
This discrepancy was attributed to the interactions be-
tween the animal sulfide and carbon metabolism. There-
fore, the agreement observed for R. pachyptila may be
yet another indicator of the degree to which the animal
metabolism is isolated from the sulfur metabolism of the
symbiosis.

Uptake and transport processes

The data presented here provide much new information
on the processes for the uptake and transport of sulfide
and carbon dioxide that are operative in these worms.

The central role of the sulfide-binding hemoglobins in
sulfide uptake, transport, and toxicity control (Arp and
Childress, 1983; Powell and Somero, 1983; Childress et
ai, 1984; Powell and Somero, 1986; Fisher et a/.. 1988a,
1989) is fully supported by these data. In particular, the
worms can bind sulfide reversibly in vivo (Table I); they
can concentrate 2H2S from the medium by a factor of 1
to 2 orders of magnitude (Fig. 4A); and when the worms
are not in autotrophic balance (low O2, low pH group),
the hemolymph hemoglobins approach equilibrium sul-
fide binding (half saturation near 1 1 ^M 2H2S) at all sul-
fide concentrations tested (Fig. 5A). In addition, free 2H2S
and free H2S in the hemolymph of these worms is in equi-

150

J. J. CHILDRESS 1:1 II

librium with the same parameters in the external medium
(Fig. 5B, C). Because the endpoint, in the absence of au-
totrophy, appears to be sulfide equilibrium between the
hemolymph and the medium, no mechanism other than
diffusion appears to be functioning to bring sulfide into
the hemolymph.

When the worms are in autotrophic balance, the sym-
bionts remove sulfide from the hemolymph at a sufficient
rate to keep the hemoglobins below equilibrium binding
of sulfide, resulting in an apparent in vivo affinity of 122
nM ZH2S for half saturation of the hemoglobins (Fig.
5A). This uptake by the symbionts is sufficient to maintain
the hemolymph free 2H2S and free H2S about an order
of magnitude below the values of those parameters in the
external medium (Fig. 5B, C). This provides a gradient
to drive the diffusion of sulfide into the hemolymph. The
available evidence indicates that this gradient is sufficient
for the uptake of sulfide by the worms. Once the sulfide
is transported to the trophosome, it presumably diffuses
from the hemolymph into the bacteriocytes and subse-
quently to the bacteria. A sulfide binding factor found in
the trophosome may also be important in this process
(Childress et a/.. 1984).

The cooperative role of the hemoglobin sulnde-binding
and the symbiont sulfide consumption in controlling he-
molymph free 2H2S concentrations to prevent sulfide
toxicity to either the host tissues or the symbionts can be
appreciated from Figure 4 and Figure 5. For example, at
an external 2H2S of 100 fiM, the internal 2H2S is 4.5
mM, however, the hemoglobins are only 47% saturated,
and, as a result, the free 2H2S is only 9.3 nAI and the free
H2S is 1 .8 nM. Above 300 /uA/ external SH2S, the internal
free sulfide rises rapidly due to the increasing saturation
of the hemoglobins. Because 50% inhibition of R. pa-
chyptila cytochrome c oxidase activity occurs at about 25
nM 2H2S in vitro at pH 7.0 (Powell and Somero, 1986),
the observed hemolymph free 2H2S concentrations in-
dicate a significant degree of protection for this critical
enzyme at the usual external 2H2S concentrations found
in this species' environment, with 50% inhibition being
reached at about 250 \i.M external 2H2S concentration
(Fig. 5B). While the sensitivity of the R. pachyptila sym-
bionts to sulfide in vitro has not been precisely defined,
the onset of inhibition of carbon fixation at pH 7.5 appears
to occur at about 300 \iM free 2H2S (Fisher el al. 1989).
In autotrophic worms, such concentrations would not be
reached until the hemoglobin was more than 90% satu-
rated, which would not be expected until external wH2S
concentrations reached more than 900 ^Af 2H2S (Fig. 5).
Thus, the proposed protective role of the hemoglobin sul-
fide binding activity, both for the tubeworm tissues and
the symbionts, is supported by these observations of in
vivo sulfide concentrations.

The uptake and transport of inorganic carbon appear
to be very different from those for sulfide. The environ-
mental pH around the worms ranges from 7.0 in vent
water to 7.9 in ambient water, and the 2CO2 is about the
same in both (K. Johnson, pers. comm.). While O2 must
be taken up primarily from ambient water and 2H2S from
vent water, inorganic carbon could be taken up from either
or both, although the Pco, values would be higher in the
vent water. The hemolymph pH of the worms appears to
be somewhat variable, with typical values being between
7.4 and 7.55. The hemolymph pH does not appear to be
affected even by the large 2H2S concentrations that it
carries at high external sulfide concentrations. This is very
different from the situation described for nonsymbiotic
organisms where H2S diffuses into the organism and then
dissociates causing a drop in pH (Jaques, 1936; Groenen-
daal, 198 1 ). It suggests that either the uptake mechanism
is different in Riftiu or the binding mechanism does not
result in the release of H* from H2S. Hemolymph pH is
apparently also not affected by the 2CO2 concentration
variations or the FT produced by sulfide oxidation. He-
molymph 2CO2 and Pco, values in R. pachyptila were
unusually high for a worm under oxic conditions (pH
= 7.47, 2CO2 = 5.76 mM, Pco, = 5.81 torr at 8°C in the
absence of sulfide and 7.4, 2.78, and 3.16, respectively,
in the presence of sulfide. Table VII). For comparison,
Arenicola marina has a hemolymph pH of about 7.53, a
2CO2 of about 2.5 mM. and a PCo: of about 1.1 torr at
7.5°C (Toulmond, 1977). Thus, it appears that, in spite
of its apparently large respiratory surface and effective
circulation (Arp et al., 1985; Jones, 1981), R pachyptila
has unusually high internal 2CO2 and PCo: levels. When
the worms were in apparent autotrophic balance, the he-
molymph SCO2 and PCo, were decreased significantly as
a function of external 2H2S, with minimal values ap-
proaching 1 mM and 1 torr, respectively, but were still
above the environmental values (Table VII, Fig. 6A, B).
While we believe that the observed distributions represent
conditions in the worms under net autotrophy, it is pos-
sible that the observed distribution could result from the
experimental worms not being in net autotrophic balance.
The decrease in 2CO2 under autotrophic conditions is
most likely due to the demand of the symbionts, because
they have been shown to readily fix inorganic carbon
(Belkin et al.. 1986; Fisher et al.. 1989). The implication
of the observed distributions is that these tubeworms con-
centrate 2CO2 to relatively high PCo, values in the he-
molymph and then depend on diffusion through the bac-
teriocytes to supply the symbionts.

This hypothesis is supported by the unusual 6L1C values
of Riftia pachyptila of between -9 and -15.6%o (Rau,
1981; Fisher?/ al.. 1988b, 1 990). These workers have sug-
gested that these low values of isotope discrimination re-
sult from carbon fixation in this species operating under

AUTOTROPHIC FUNCTION IN RIFTI.l

151

conditions approaching carbon limitation, as can happen
in marine plankton (Degens el a/.. 1968). Because the Km
for the carbon fixation by the Riftia symbionts is between
400 and 700 ^M 2CO2 at pH 7.5 (Fisher el ai. 1988d),
the hemolymph carbon dioxide values (as low as 1100
tiM SCO; in the presence of sulfide) might well be low
enough to limit carbon isotope discrimination under con-
ditions of active autotrophy.

Relationship between coelomic fluid and hemolymph

The data presented here support the view that there is
free exchange of small molecules between the coelomic
fluid and hemolymph (Childress el ai, 1984), although
this exchange does not extend to molecules as large as the
hemoglobins. The much higher hemoglobin concentration
in the hemolymph is clearly responsible for the much
higher 2H2S in that fluid, and may well be responsible
for differences in pH, 2CO2, and Pco, as well. However,
the cause of the consistently lower percent sulfide satu-
ration, free 2H2S, and free H2S in the coelomic fluid is
not apparent at this time. The new data reported here
support the concept that the coelomic fluid is a reservoir
of O2, 2CO2, and 2H2S which the worms can use to
buffer the effects of brief fluctuations in vent flow (Arp
and Childress, 1981; Childress et ai. 1984).

Model of the functioning of the intact symbiosis

Rift in puc/iyplila appears to have the greatest auto-
trophic potential and as a result the fastest growth rate of
any of the sulfur-oxidizing symbioses investigated to date.
It, and probably all vestimentiferans, appears to be unique
among the studied species in that the animal is specialized
to minimize the interaction of the animal metabolism
with sulfide and to provide only sulfide to symbionts that
are only capable of using sulfide. Central to the ability of
the vestimentiferan symbioses to use sulfide are the he-
moglobins, which reversibly bind both sulfide and oxygen
to different sites simultaneously. These hemoglobins en-
able the worms to concentrate sulfide from the medium
and by almost two orders of magnitude. Yet, because of
the high affinity of the hemoglobins for sulfide as well as
the consumption of sulfide by the symbionts, which holds
the hemoglobins well below sulfide saturation, the worms
can maintain their hemolymph free 2H2S concentrations
an order of magnitude lower than external 2H2S concen-
trations. The high capacitance of the hemolymph for sul-
fide is essential for the transport of sufficient quantities
of sulfide to the symbionts via the circulatory system. The
low free sulfide concentrations are essential for preventing
the inhibition of animal metabolism or symbiont carbon
fixation by sulfide. Diffusion of sulfide across the plume
into the hemolymph appears sufficient to explain the
movement of sulfide into the worms. Because the sym-

bionts can take sulfide from the hemoglobins, diffusion
from the hemolymph into the bacteriocytes in the highly
vascularized trophosome may well be sufficient to supply
the needs of the symbionts.

The uptake and supply of O2 to both the symbionts
and the worm tissues is apparently accounted for by the
high affinity of the hemoglobins for oxygen and the ability
of the symbionts and the tissues to use O2 at low P02
values. The combination of the high O2 affinity and the
high sulfide affinity is responsible for the ability of these
hemoglobins to suppress the spontaneous oxidation of
sulfide by O: (Fisher and Childress. 1984).

About half the inorganic carbon fixed by the symbionts
is potentially derived from the heterotrophic metabolism
of the symbiosis, while the remaining half requires the
uptake of inorganic carbon from the medium. The he-
molymph 2CO2 and Pco, are apparently elevated above
the medium by some mechanism, other than a pH-based
one, which concentrates carbon dioxide in the hemo-
lymph. This elevated hemolymph inorganic carbon can
then diffuse into the bacteriocytes and to the bacteria,
although the available evidence indicates that, at maximal
rates of autotrophy, this supply may approach values lim-
iting the rate of carbon fixation. The supply of fixed carbon
from the symbionts to the host is presumably predomi-
nantly via small organic molecules transported in the he-
molymph.

Using the available data, one can evaluate this model
by creating a hypothetical 100-g worm that has 5 ml of
hemolymph and 15 g of trophosome (Childress el ai,
1984). At 200 juM external 2H2S, one would expect 5.9
mM 2H2S, 5 mM O2, and 2 mM SCO2 in the hemo-
lymph. At an uptake rate of 5 ^mole 2H2S g~'rr', the
O2 uptake rate would be 8 j/mole g~ 'h~ ' and the net 2CO2
uptake would be 2 j/mole g~' h~'. If one assumes that the
hemolymph makes one circuit per minute, one can cal-
culate that 27% of the 2H2S, 53% of the O2, and 33% of
the 2CO2 must be exchanged on each circuit. These
numbers are not unreasonable, while at the same time
the similarity of the percentages provides some confidence
that the values used for the hemolymph concentrations
are approximately correct.

Acknowledgments

This work was supported by NSF grants OCE-8609202
and OCE-90 12076 to J.J.C. and OCE-8610514 to J.J.C.
and C.R.F. and funding from IFREMER to A.-M. Alayse.
Some travel support was provided by NATO grant
D.880423 to H. Felbeck. We would like to thank R. Van
Buskirk, V. Vanderveer, and D. Gage for technical assis-
tance during the cruises. Thanks are also due to the cap-
tains and crews of the R}' Melville, R V Thomas Thomp-
son, and N/O Nadir, as well as the sub crews and pilots

152

J. J. CH1LDRESS ET AL.

of the submersibles Alvin and Nautile, without whom this
work would not have been possible. This manuscript has
benefited from discussions with and comments by A. An-
derson, H. Felbeck, R. Trench, and J. Feigenbaum.

Literature Cited

Anderson, A. E., J. J. Childress, and J. Favuzzi. 1987. Net uptake of
CO2 driven by sulfide and thiosulfate oxidation in the bacterial sym-
biont-containing clam Solcmya reidi. J. Exp. Biol. 133: 1-31.

Arp, A. J., and J. J. Childress. 1981. Blood function in the hydro-
thermal vent vestimentiferan tube worm. Science 213: 342-344.

Arp, A. J., and J. J. Childress. 1983. Sulfide binding by the blood of
the hydrothermal vent tube worm Ri/iia pachvplila Science 219:
295-297.

Arp, A. J., J. J. Childress, and C. R. Fisher Jr. 1985. Blood gas transport
in Riftia pachyptila. Bull. Biol. Sue. Hash. 6: 289-300.

Arp, A. J., J. J. Childress, and R. D. Vetter. 1987. The sulfide binding
protein in the blood of Ri/iia pachvplila is the extracellular hemo-
globin. J. Exp. Biol. 128: 139-158.

Belkin, S., D. C. Nelson, and H. \V . Jannasch. 1986. Symbiotic assim-
ilation of CO2 in two hydrothermal vent animals, the mussel Bathy-
modiolus thermophilus and the tube worm Riftia pachvptila. Biol.
Bull 170: 110-121.

Ca»anaugh, C. M. 1985. Symbiosis of chemoautotrophic bacteria and
marine invertebrates from hydrothermal vents and reducing sedi-
ments. Bull. Biol. Sot: Wash. 6: 373-388.

Cavanaugh, C. M., S. L. Gardiner, M. L. Jones, H. \V. Jannasch, and
J. B. Waterbury. 1981 . Prokaryotic cells in the hydrothermal vent
tube worm Riftia pachvptila: possible chemoautotrophic symbionts.
Science 213: 340-342.

Childress, J. J. 1988. Biology and chemistry of a deep-sea hydrothermal
vent on the Galapagos Rift; the Rose Garden in 1985, an introduction.
Deep-Sea Res 35: 1677-1680.

Childress, J. J., A. J. Arp, and C. R. Fisher Jr. 1984. Metabolic and
blood characteristics of the hydrothermal vent tube worm Rijiia pa-
chvptila. Mar. Biol. 83: 109-124.

Childress, J. J., C. R. Fisher, J. A. Favuzzi, and N. K. Sanders.
1991 . Factors affecting the uptake of sulfide and carbon dioxide by
the hydrothermal vent clam, Calyptitgena magnijica. Phvsiol. Zool.
In press.

Degens, E. T., R. R. L. Guillard, W. M. Sackett, and A. Hellebust.
1968. Metabolic fractionation of carbon isotopes in marine plank-
ton— I. Temperature and respiration experiments. Deep-Sea Res
15: 1-9.

Fahey, R. C, R. D. Dorian, G. L. Newton, and J. Utley.
1983. Determination of intracellular thiol levels using monobro-
mobimane fluorescent labeling: applications involving radioprotective
drugs. Pp. 103-120 in Radioproleclors and Carcinogens. O. F. Ny-
gaard, and M. G. Simie, ed. Academic Press, New York.

Felbeck, H. 1981. Chemoautotrophic potential of the hydrothermal
vent tube worm, Riftia pachyptila Jones (Vestimentifera). Science
213: 336-338.

Felbeck, H. 1985. CO2 fixation in the hydrothermal vent tube worm
Riftia pachyptila (Jones). Physiol. Zool. 53: 272-281.

Felbeck, H., and J. J. Childress. 1988. Rijiia pachyptila: a highly in-
tegrated symbiosis. Oceanol. Ada Special I'ol. 8: 131-138.

Felbeck, H., G. N. Somero, and J. J. Childress. 1981. Calvin-Benson
cycle sulphide oxidation enzymes in animals from sulphide rich hab-
itats. Nature 293: 291-293.

Fisher, C. R., Jr., and J. J. Childress. 1984. Substrate oxidation by
trophosome tissue from Riftia pachyptila Jones (Phylum Pogono-
phora). Mar. Biol. Lett 5: 171-184.

Fisher, C. R., and J. J. Childress. 1986. Translocation of fixed carbon
from symbiotic bacteria to host tissues in the gutless bivalve, Solemva
reidi. Mar. Biol 93: 59-68.

Fisher, C. R., J. J. Childress, and N. K. Sanders. I988a. The role of
vestimentiferan hemoglobin in providing an environment suitable
for chemoautotrophic sulfide-oxidizing endosymbionts. Symbiosis
5: 229-246.

Fisher, C. R., J. J. Childress, A. J. Arp. J. M. Brooks, D. Distel, J. A.
Favuzzi, S. A. Macko, A. Newton, M. A. Powell, G. N. Somero, and
T. Soto. I988b. Physiology, morphology, and composition of Ri/iia
pachyptila at Rose Garden in 1985. Deep-Sea Res 35: 1745-1758.
Fisher, C. R., J. J. Childress, A. J. Arp, J. M. Brooks, D. Distel, J. A.
Favuzzi, H. Felbeck, R. Messier, K. S. Johnson, M. C. Kennicutt II,
S. A. Macko, A. Newton, M. A. Powell, G. N. Somero, and T. Soto.
1988c. Microhabitat variation in the hydrothermal vent mussel
Bathymodiolus thermophilus, at Rose Garden vent on the Galapagos
rift. Deep-Sea Res. 35: 1769-1792.

Fisher, C. R., J. J. Childress, and J. M. Brooks. 1988d. Are hydro-
thermal-vent vestimentifera carbon-limited? Am. Zool. 28: 128A.
Fisher, C. R., J. J. Childress, and E. Minnich. 1989. Autotrophic carbon
fixation by the chemoautotrophic symbionts of Riftia pachvptila.
Biol. Bull. 177: 372-385.

Fisher, C. R., M. C. Kennicutt II, and J. M. Brooks. 1990. Stable
carbon isotopic evidence for carbon limitation in hydrothermal vent
vestimentiferans. Science 247: 1094-1096.

Fustec, A., D. Desbruyeres, and L. Laubier. 1988. Biomass estimation
of animal communities associated with deep-sea hydrothermal vents
near 13°N/EPR. Oceanol. Ada Spec. I'ol. 8: 15-22.
Groenendaal, M. 1981. The adaptation ofArenico/a marina to sulphide

solutions. Neth. J. Sea Res. 15: 65-77.

Heisler. N. 1984. Acid-base regulation in fishes. Pp. 315-392 in Fish
Physiology. W. S. Hoar, and D. J. Randall, ed. Academic Press, Or-
lando.

Hessler, R. R., W. M. Smithey, M. A. Boudrias, C. H. Keller, R. A.

Lutz, and J. J. Childress. 1988. Temporal change in megafauna at

the Rose Garden hydrothermal vent. Deep-Sea Res. 35: 1681-1710.

Jaques, A. G. 1936. The kinetics of penetration: XII. Hydrogen sulfide

J. Gen. Physiol. 19:397-418.

Johnson, K. S., J. J. Childress, and C. L. Beehler. 1988a. Short term
temperature variability in the Rose Garden hydrothermal vent field.
Deep-Sea Res 35: 171 1-1722.

Johnson, K. S., C. M. Sakamoto-Arnold, J. J. Childress, and C. L. Beeh-
ler. 1988b. The biogeochemistry of hydrothermal vent communities
in the Galapagos Rift. Eos 69: 1271.

Jones, M. L. 1981. Ri/iia pachyptila, new genus, new species, the ves-
timentiferan tubeworm from the Galapagos Rift geothermal vents.
Pmc. Biol. Sot: Hash. 93: 1295-1313.
Jones, M. L. 1988. The Vestimentifera. their biology, systematic and

evolutionary patterns. Oceanol Ada Spec Vol. 8: 69-82.
Jones, M. L., and S. L. Gardiner. 1988. Evidence for a transient diges-
tive tract in Vestimentifera. Prot: Biol. Sot: Wash. 101: 423-433.
Kelly, D. P. 1982. Biochemistry of the chemolithotrophic oxidation
of inorganic sulphur. Phil. Trans. ROY. Sot: Loud. Ser. B 298: 499-
528.

Mason, J., D. P. Kelly, and A. P. Wood. 1987. Chemolithotrophic and
autotrophic growth of Thermothrix thiopara and some thiobacilli on
thiosulphate and polythionates, and a reassessment of the growth
yields of T thiopara in chemostat culture. / (Jen. Microbiol. 133:
1249-1256.
Millero, F. J. 1986. The thermodynamics and kinetics of the hydrogen

sulfide system in natural waters. Mar. Chem. 18: 121-147.
Millero, F. J., T. Plese, and M. Fernandez. 1988. The dissociation of
hydrogen sulfide in seawater. Limnol. Oceanogr 33: 269-274.

AUTOTROPH1C FUNCTION IN Rlb'TIA

153

Nelson, D. C, B. B. Jorgensen, and N. P. Rcvsbach. 1986. Growth
pattern and yield of a chemoautotrophic Beggiatoa sp. in oxygen-
sulfide microgradients. Appl Environ Murohiol 52: 225-233.

Newton, G. L., R. Dorian, and R. C. Fahey. 1981. Analysis of biological
thiols by derivatization with monobromobimane and separation by
reverse-phase high performance liquid chromatography. Amil
Biochem. 114: 383-387.

O'Brien, J.. and R. D. Vetter. 1990. Production of thiosulfate during
sulphide oxidation by mitochondria of the symbiom-containing bi-
valve Sulemya reidi. J Exp. Biol. 149: 133-148.

Powell, M. A., and G. N. Somero. 1983. Blood components prevent
sulfide poisoning of respiration of the hydrothermal vent tubeworm
Riftiii paehyptila. Science 219: 297-299.

Powell, M. A., and G. N. Somero. 1985. Sulfide oxidation occurs in
the animal tissue of the gutless clam. Solemya reidi. Binl. Bull 169:
164-181.

Powell, M. A., and G. N. Somero. 1986. Adaptations to sulfide by
hydrothermal vent animals: sites and mechanisms of detoxification
and metabolism. Biol. Bull 171: 274-290.

Quetin, I.. B., and J. J. Childress. 1980. Observations on the swimming
activity of two bathypelagic mysid species maintained at high hy-
drostatic pressures. Deep-Sea Res 27 A: 383-391.

Rau, G. H. 1981. Hydrothermal vent clam and tubeworm I3C/I2C.
Further evidence of nonphotosynthetic food sources. Science 213:
338_340.

Rau, G. H., and J. I. Hedges. 1979. Carbon-13 depletion in a hydro-
thermal vent mussel: suggestion of a chemosynthetic food source.
Science 203: 648-649.

Richard, J. J., R. D. Vick, and G. A. Junk. 1977. Determination of
elemental sulfur by gas chromatography. Env Sci. and Techno!. 11:
1084-1086.

Roux, M., M. Rio, E. Schein, R. Lutz, L. Fritz, and L. Ragone.
1989. Mesures in situ de la croissance des bivalves et des vesti-
mentiferes et de la corrosion des coquilles au site hydrothermal de
13°N (dorsale du Pacinque oriental). C R Acad. Sci. Paris. Ser III
308: 121-127.

Skirrow, G. 1975. The dissolved gases— carbon dioxide. P. 1 in Chem-
ical Oceanography. J. P. Riley. and G. Skirrow, ed. Academic Press,
New York.

Somero, G. N., J. J. Childress, and A. E. Anderson. 1989. Transport,
metabolism and detoxification of hydrogen sulfide in animals from
sulfide nch marine environments. Crit. Rev Aqual. Sci. 1: 591-614.

Southward, E. C. 1987. Contribution of symbiotic chemoautotrophs
to the nutrition of benthic invertebrates. Pp. 83-1 18 in Microbes in
the Sea. M A. Sleigh, ed. Horwood Ltd., Chichester.

Southward, E. C. 1988. Development of the gut and segmentation of
newly settled stages of Ridgeiu (Vestimentifera): implications for re-
lationship between Vestimentifera and Pogonophora. /. Mar. Biol.
As.wc. U. A 68: 465-487.

Terwilliger, R. C., and N. B. Terwilliger. 1985. Respiratory proteins
of hydrothermal vent animals. Bull Biol. Soc. H'axh. 6: 273-288.

Terwilliger, R. C., N. B. Terwilliger, and E. Schabtach. 1980. The
structure of hemoglobin from an unusual deep-sea worm (Vesti-
mentifera). Camp. Biochcm. Physiol. 65B: 531-535.

Toulmond, A. 1977. Temperature-induced variations of blood acid-
base status in the lugworm. Arenicola marina (L.): I. In vitro study.
Rcspir. P/n-siol 31: 139-149.

Vetter, R. D. 1985. Elemental sulfur in the gills of three species of
clams containing chemoautrophic symbiotic bacteria: a possible in-
organic energy storage compound. Mar Biol 88: 33-42.

Vetter, R. D., P. A. Matrai, B. Javor, and J. O'Brien. 1989. Reduced
sulfur compounds in the marine environment: analysis by HPLC.
Pp. 244-26 1 in Biogenic Siillin in the Environment. E. S. Saltzman.
and W. J. Cooper, ed. American Chemical Society, Washington.

Vetter, R. D., M. E. Wells, A. L. Kurtsman, and G. N. Somero.
1987. Sulfide detoxification by the hydrothermal vent crab Bytho-
graea ihermvdron and other decapod crustaceans. Physiol. Zool. 60:
121-137.

VVillason, S. VV., and K. S. Johnson. 1986. A rapid, highly sensitive
technique for the determination of ammonia in seawater. Afar. Biol
91: 285-290.

\\ ilmot, D. B. J., and R. D. Vetter. 1990. The bacterial symbiont from
the hydrothermal vent tubeworm Riliia paehyptila is a sulfide spe-
cialist. Alar. Biol. 106: 273-283.

Reference: Bin! Bull 180: 154-166. (February. ll»91)

Distribution and Characterization of Ion Transporting

and Respiratory Filaments in the Gills

of Procambarus clarkii

JOHN S. DICKSON, RICHARD M. DILLAMAN,
ROBERT D. ROER, AND DAVID B. ROYE

Center for Marine Science Research, University of North Carolina at Wilmington,
7205 n'rightsvi/le Ave., Wilmington, North Carolina 28403

Abstract. Individual gill filaments of the freshwater
crayfish Procambarus clarkii were determined to be either
predominantly respiratory or transporting. Silver staining
revealed that the filaments within the central bed of the
gills formed silver deposits whereas filaments at the mar-
gins and the entire sixth pleurobranch formed no deposits.
Designation of the silver staining gills as predominantly
transporting and unstained filaments as predominantly
respiratory was substantiated by ultrastructural analyses
and measurements of ATPase and transepithelial poten-
tials. Presumptive transporting filaments had an epithe-
lium subjacent to the cuticle that was relatively thick and
dominated by abundant mitochondria. Lacunae were de-
lineated by pillar structures and served as collateral path-
ways for the movement of blood from the afferent to ef-
ferent blood channels, which were separated by a thin
septum. Presumptive respiratory filaments had an ex-
tremely thin epithelium with few organelles, but a rela-
tively thick septum. Present in both types of filaments
were nerves and podocytes. The values for Na, K-ATPase
were significantly higher in the transporting filaments than
in those designated as respiratory. The measurement of
transepithelial potentials showed both filaments to be cat-
ion selective with the respiratory filaments slightly more
positive and the transporting filaments slightly more neg-
ative than the diffusion potential for Na.

Introduction

Gills are a site for exchange between the blood of the
organism and the external medium and, as such, are in-
Received 28 June 1990; accepted 9 November 1990.

volved in the separate but interrelated functions of gas
exchange, acid/base balance, and ion regulation. Those
substances exchanged may include oxygen, electrolytes,
carbon dioxide, and ammonia. To produce a large surface
area for such exchange, gills are often branched along one
or more axes. In the crabs (Crustacea, Decapoda) lamellar
gills are present (Copeland, 1963, 1968; Copeland and
Fitzjarrell. 1968; Taylor and Greenaway, 1979; Finol and
Croghan, 1983) and consist of a parallel afferent and ef-
ferent blood vessel between which are broad flattened
sheets. In branchipod crustaceans, such Artemia salina
and Daphnia nuigna (Copeland, 1967 and Kikuchi, 1983),
amphipods and isopods (Milne and Ellis, 1973; Bubel and
Jones, 1974) gills are flat, oval, sac-like extensions of tho-
racic appendages or modified pleopods. In crayfish and
shrimp the gills are filamentous (Morse el a/.. 1970; Bie-
lawski, 1971; Fisher, 1972; Burggren el ai. 1974; Foster
and Howse, 1 978). In crayfish the gills are trichobranchiate
and have a central stalk from which hundreds of tiny
finger-like filaments arise. The podobranchia (outermost
gills) are attached to the coxae of the appendages. A mem-
branous lamina, devoid of filaments, extends from the
inner side of the podobranch and lies against the thoracic
body wall; its distal tip is flattened into a plate bearing a
few filaments. The arthrobranchia, which are attached to
the articular membrane between the body wall and the
appendages, and the pleurobranchia, which are attached
to the pleural wall of each somite, lie beneath the podob-
ranchia and have no distal plate structure (Huxley, 1986;
Burggren et ai, 1974).

Despite variations in gill anatomy, the ultrastructure
of the gill epithelium in those species that are capable of
osmoregulation is similar, and can be placed into one of

154

GILL FILAMENTS IN P Cl.ARkll

155

two categories. Both categories consist of a single layered
epithelium that separates blood spaces from the uncal-
cified cuticle. One type of epithelium is relatively thick
and has the characteristics of an ion transporting epithe-
lium (seeCioffi. 1984, for a review of arthropod ion trans-
porting epithelia). Briefly, it consists of cells with highly
folded apical and basal processes, a prominent basal lam-
ina and cytoplasm containing abundant mitochondria as
well as golgi apparati, smooth and rough endoplasmic
reticulum and glycogen. The second type of epithelium
consists of very thin squamous cells subjacent to the cu-
ticle. The cells are only one tenth the thickness of the
previously described type, and the cytoplasm contains very
few organelles (Copeland and Fitzjarrell, 1968). This ul-
trastructure would appear to favor diffusion of gases be-
tween the external and internal media and consequently
this epithelium has been categorized as being respiratory
in function.

Studies on a variety of species of decapod crustaceans
have demonstrated the existence of a partitioning of ion
regulatory and respiratory functions among different gills
or within different regions of gills. Both morphological
and biochemical (Na, K-ATPase distribution) studies have
provided evidence that the anterior gills are dedicated to
gas exchange while the posterior gills are involved in ion
regulation in the euryhaline brachyurans Eriocheir xinen-
sis (Pequeux and Gilles, 1978, 1981), Uca pugnax (Hol-
liday, 1985), Uca /n/miv (Wanson etal.. \984),Callinectes
sapidus (Copeland and Fitzjarrell, 1968; Neufeld et al.,
1980; Towle, 1984), and Carcinus maenas (Mantel and
Landesman, 1977). Although we and other investigators
realize that gill tissues possessing transport epithelia are
also engaged in gas exchange, and that ions move across
respiratory surfaces, we still use. for simplicity, the pre-
dominant function to designate that of the entire filament.
Henceforth, we will refer to those gill filaments that possess
a typical transporting epithelium as "transport" types and
those gill filaments with a squamous epithelium as "re-
spiratory" types.

Crayfish, like the euryhaline brachyurans in dilute me-
dia, exist in a hypoosmotic medium, and must maintain
their ion balance by actively transporting electrolytes
across the gills (Maluf, 1940; Bryan, 1959; Shaw, 1960a.
b; Ehrenfeld, 1974). Unlike the euryhaline brachyurans
studied, however, most crayfish are stenohaline freshwater
crustaceans and possess filamentous, rather than lamellate,
gills. The partitioning of respiratory and ion transporting
function in the gills of crayfish is still in question. While
Wheatly and Henry (1987) have used biochemical anal-
yses of pooled gill sets to show that the distribution of Na,
K-ATPase was similar among gills of Pacifastacus len-
iusculus, Morse et al. (1970) used silver staining and re-
ported a partitioning of respiratory and transport functions
among the gill filaments of the same species. Dunel-Erb

el al. ( 1982). on the other hand, observed only respiratory
epithelial tissues in the filaments and ion transporting ep-
ithelial tissues in the podobranch lamina of Astacus pal-
lipes and therefore assigned the appropriate function to
the two structures. The only ultrastructural observations
of filaments have been of transporting epithelia (Morse
et al.. 1970; Burggren et al.. 1974; Bielawski, 1971; Fisher,
1972).

This investigation was therefore directed at determin-
ing— within a single filament, within a single gill, and
among gills — the distribution and frequency of the two
types of epithelia of the freshwater crayfish Procambarus
chirkii. The methods employed in this study were silver
staining (Maluf, 1940), an indicator of ion transporting
tissue; transmission electron microscopy (TEM), which
reveals the striking difference between the morphology of
the two types of epithelia; analysis for Na, K-ATPase, an
effector of ion movement; and measurement of transep-
ithelial potentials in individual filaments, which reflects
possible differences in transport or permeability of the
combined epithelial and cuticular layers.

Materials and Methods

The crayfish (Procambarus clarkii), obtained from
Carolina Biological Supply, were kept in aquaria of arti-
ficial pond water (Roer and Shelton, 1982), maintained
between 20°C and 25°C, and fed 2-3 times a week. Only
active crayfish in the intermolt stage, as determined by
the criteria of Drach and Tchernigovtzeff ( 1967) and Ste-
venson ( 1972), were used in this study.

Silver staining

Crayfish subjected to silver staining were first rinsed in
deionized water and placed in a 0.05% AgNO3 solution
at 25°C for 30 min. The volume of the solution was suf-
ficient to completely submerge the crayfish. Crayfish were
then rinsed with deionized water. The branchiostegites
were removed to expose the gills, and the crayfish were
placed in Kodak Microdol-X developer (diluted 3:1 to
lower the osmolarity to 630 mOsm) for one hour with
frequent agitation. After the gills were thoroughly washed
and placed in distilled water, one or two drops of NH4S
were added to intensify the stain.

Preparation oj gill filament tissues
for electron microscopy

Silver treated filaments were fixed in 3% glutaraldehyde
in 0. 1 M cacodylate buffer (pH 7.2) containing 5% sucrose
(osmolarity = 640 mOsm) at 25 °C for at least 1 h. To
facilitate penetration of the various solutions, the tips were
cut off" the filaments and the tissue was gently agitated at
each step of the preparation. The filaments were then

156

J. S. DICKSON ET AL

rinsed in buffer and postfixed with 2% osmium tetroxide
in 0.1 M cacodylate buffer (pH 7.2) plus 5% sucrose at
25°C for 2 h. After another rinse with buffer, tissues were
dehydrated with a graded series of ethanol and propylene
oxide, and embedded in Spurr low viscosity embedding
medium (Spurr, 1969).

Filaments not silver-treated were fixed in 2% glutaral-
dehyde in 0.15 M cacodylate buffer, pH 7.4, with 5 m.M
CaCl2 for 1 h, rinsed in buffer, and post-fixed in fresh
0.5% OsO4 with 0.8% K4Fe(CN)6 in 0.1 M cacodylate
buffer, pH 7.4, with 5 mA/CaC!2 at 25°C for 1 h. After
rinsing again in cacodylate buffer, the tissues were placed
in 0. 1 5% tannic acid in 0. 1 5 M cacodylate buffer, pH 7.4,
with 5 mA/ CaCl2 for 5 min. Following a short rinse in
buffer and distilled water, the tissues were stained en bloc
with 2% aqueous uranyl acetate for 2 h. After a distilled
water rinse, tissues were dehydrated through a graded se-
ries of acetone and embedded in Spurr.

Cross-sections were cut in the basal, medial, and distal
regions of the filaments. Sections were post-stained with
4% uranyl acetate in 50% ethanol and Reynolds lead ci-
trate (Reynolds, 1963), and were viewed with a Zeiss EM
9S transmission electron microscope operated at 60 kV.

Na, K-A TPase assay of filaments

Na, K-ATPase assays were done according to the
method of Horiuchi (1977). After washing excised gills in
0.25 M sucrose buffered to pH 7.5 with 100 mAl Tris-
HC1, predicted transporting or respiratory filaments were
removed from the gills, pooled by type, and homogenized
at 4°C in 2 volumes of the buffered sucrose solution during
35 passes in a glass tissue homogenizer. The homogenate
was centrifuged at 3000 X g for 15 min and the super-
natant was assayed for activity.

The reaction mixture consisted of 60 mA/ NaCl. 20
mA/ KC1, 2 mA/ MgCl2 and 30 mA/ Tris-HCl, pH 7.5,
in 0.8 ml. This reaction mixture was preincubated at 32°C
before 0. 1 ml of the enzyme solution and 0. 1 ml of the
3.0 mA/ Tris-ATP were added. The enzymatic reaction
was allowed to proceed for 45 min, being terminated by
the addition of cold 3% trichloroacetic acid. The amount
of inorganic phosphate, hydrolyzed from ATP, present in
the supernatant was measured according to Wheeler
( 1975). Mg-ATPase activity was determined as above with
the substitution of 10 mA/ ouabain for KC1 in the reaction
mixture to inhibit Na, K-ATPase. Na, K-ATPase activity
was calculated as the difference between the total ATPase
and Mg-ATPase values. The amount of protein was de-
termined according to Peterson's (1977) modified Lowry
protein assay using a Bausch and Lomb Spectronic 710
spectrophotometer and bovine serum albumin as a stan-
dard. The specific activities were recorded as micromoles
of inorganic phosphate per milligram of protein per hour.

Transepithetial potential measurements

Crayfish were submerged in 'Xi strength Van Harreveld's
Ringer solution (final concentrations: NaCl = 51.3 mA/;
KC1 = 1.3 mA/; CaCl: = 3.4 mA/; MgCl2 = 0.3 mA/) in
a finger bowl, and restrained by rubber bands attached to
a notched plexiglass plate. Diluted Ringer's solution was
used to provide sufficient electrolytes for electrode con-
duction. Potentials measured with pond water as the ex-
ternal medium were unreliable. The branchial region of
the carapace was cut away from one side of the animal
to expose the gills. In this position, individual gill filaments
could easily be impaled by glass microelectrodes using a
micromanipulator.

Microelectrodes were drawn from glass capillary tubing
and filled with 3 A/ KC1. A Ag-AgCl reference electrode
was immersed in the bathing medium. Both electrodes
were connected by shielded cables to a WPI model 701
microprobe system and a Tectronix 5111 storage oscil-
loscope.

Only filaments of the podobranchs were impaled and
the assignment of putative transport and respiratory fil-
aments was based upon the results of the silver staining
experiments. Transepithelial potentials (TEP's) and the
position of the electrode tip within the filament were re-
corded each time a gill was impaled. Sodium concentra-
tions of the medium and crayfish hemolymph were mea-
sured by flame photometry (Turner model 510).

Results

Silver staining

In six crayfish subjected to silver staining all showed
some staining in all gills except pleurobranch 6. Staining
occurred only in the filaments. That is, no staining was
observed in the central gill stalk, in the lamina, in the
distal plate of the podobranchia, nor at the base of the
gills. Figure 1 is representative of the filament staining
patterns. The podobranchs had the largest proportion of
stained filaments, whereas the pleurobranchs had the least
for each set of gills. All gills showed the same basic pattern,
a population of stained filaments in the central area of
the filament bed surrounded by lateral rows of unstained
filaments, with the exception of the pleurobranch of the
sixth gill set, which showed no silver staining. Silver
stained filaments had precipitate distributed evenly over
the length of the filament.

Transmission electron microscopy of silver stained fil-
aments showed that most of the precipitate was within
the gill cuticle, although lesser amounts were observed
outside or beneath the cuticle (Fig. 2). Precipitate within
the cuticle was localized almost exclusively in a layer cor-
responding to the exocuticle, rarely being found in the
epicuticle or endocuticle. While the treatment employed

GILL FILAMENTS IN P. CLIRKII

157

Podob ranch

Arthrobranch

Pleurobranch

Gill Set

Gill Set #2

GUI Set #3

Gill Set

Gill Set #6

Figure I. Light micrographs of six sets of gills from the right side of a crayfish after silver staining. Each
gill is oriented with the base to the right. Dark staining indicates silver deposition.

in the silver staining disrupted the soft tissues, it was pos-
sible to note that the epithelium underlying the cuticle
was relatively thick and had numerous basal and apical
infoldings. Filaments containing no precipitate after silver
treatment (Fig. 3) had an epithelium subjacent to the cu-
ticle that was markedly thinner than that of the silver
stained filaments and had few, if any, basal and apical
infoldings.

Ultrastructure of the gill filaments

Choice of filaments for ultrastructural analysis of pre-
sumptive ion-transporting and respiratory tissue was based

upon the distribution of the two types as indicated by
silver staining (Fig. 1 ) and proved to be accurate in all 20
of the filaments examined. No differences were observed
among sections from proximal, medial, or distal regions
of a filament. Diagrammatic representations of cross sec-
tions through the two types of filaments are seen in Figures
4 and 5. The transporting filaments, regardless of the gill
set, had a diameter of 0.1 mm to 0.2 mm, while the re-
spiratory filaments were 0.2 mm to 0.3 mm in diameter.
Both types of filaments contained an afferent and an ef-
ferent blood channel and lateral lacunae. The afferent
channel was on the side of the filament that faced toward
the gill stalk, and the efferent channel on the side that

158

J. S. DICKSON ET AL

Figure 2. TEM of transporting gill filament after silver staining. Note crystals within and on the cuticle
(arrows). Bar = 5 nm.

Figure 3. TEM of respiratory gill filament after silver staining. Bar = 1.0 pm.

faced away from the stalk. A septum and the surrounding
connective tissue separated the two major blood spaces.
An epithelium, 80-90% of which occurred in the lacunar
blood space, lined the noncalcified cuticle of the filaments.
Epithelial cells extended across the lacunae forming pillar
structures with their basal portion embedded in loose
connective tissue. Also depicted is the marked difference
in epithelial thickness within and between the filament
types.

In the transporting filaments, the cuticle epithelium
bordering the afferent channel was the thickest (approx-
imately 6 ^m) (Fig. 4). Numerous mitochondria, generally
oriented perpendicular to the cuticle, were observed and
were most abundant in the apical portion of the cells (Figs.
6, 7, 8). Apical microvillar processes were approximately
80 nm wide and varied in length from 0.4 ^m to 1.2 pm
(Fig. 8). Electron-dense material was observed between
the tips of the microvilli and the overlying cuticle (Fig.
9). Numerous infoldings and interdigitations occurred in
the basal region of the epithelium (Figs. 6, 7). The micro-
tubules observed within the cytoplasm were oriented
nearly perpendicular to the cuticle and were often closely
apposed to mitochondria (Fig. 8). Occasional golgi ap-
parati were observed in the epithelium lining the outer
margin of the afferent canal (Fig. 6), as well as a thin basal
lamina (Figs. 6, 9). Adhering junctions occurred apically
on lateral cell membranes, below which were septate
junctions. Gap junctions were observed below the septate
junctions (Fig. 6).

The portion of the epithelium lateral to the septum and
on the efferent side of the filament was thinner (approx-
imately 1.5 nm) than that portion bordering the afferent
canal, but also contained numerous mitochondria and
microvillar processes (Fig. 9).

The epithelial cells forming the pillar structures had a
highly convoluted basal nucleus (Fig. 7) and were usually
rich in mitochondria, rough endoplasmic reticulum, and
golgi apparati (Fig. 8).

The cuticle of the transporting gill filaments was of uni-
form thickness (approximately 1 .8 j/m) around their entire
circumference. An outer thin epicuticle, the thin lamellae
of the exocuticle, and the thicker lamellae of the endo-
cuticle were easily differentiated in most sections (Figs. 6,
9). The approximate thicknesses of the exocuticle and the
endocuticle were 0.8 ^m and 0.6 ^m, respectively.

The septum traversed the central blood space, joining
the loose connective tissue on either side of the filament,
thereby forming a partition between the afferent and ef-
ferent blood channels (Figs. 4, 5, 10, 16). Along the efferent
channel the septum was relatively smooth, whereas the
side facing the afferent channel possessed processes that
extended into the channel (Figs. 10, 16). In the trans-
porting filaments the septum was relatively thin (approx-
imately 1.1 ^m) (Fig. 10) and became shorter as the fil-
ament tapered in its distal portions. Numerous mem-
branes were observed along the length of the septum, some
of which constituted the cell membranes of loose con-
nective tissue cells on either side of the filament. A thin
basal lamina was observed on the efferent side of the sep-
tum, and a thicker fibrous basement membrane covered
the afferent septal process (Figs. 10, 16).

The epithelium of the respiratory filament varied in
thickness, ranging from 0.7 ^m opposite the septum to
3.0 nm in regions adjacent to pillar structures (Figs. 5,
1 1 ). A few scattered round or oval mitochondria were
observed in the granular cytoplasm whereas thicker re-
gions of the epithelium adjacent to the pillar cells also
had some glycogen granules (Figs. 12. 13). The regions of

GILL FILAMENTS IN P CLARkll

159

Figures -4 and 5. Diagrammatic representations of cross sections
through a transporting I Fig. 4) and respiratory (Fig. 5) gill filament. Note
the afferent blood channel (Ab). efferent blood channel (Eb), epithelium
(E), cuticle (c), septum (s). podocyte (P), hemocyte (h). pillar structure
(p), and lacuna (1).

the epithelium lateral to the septum and on the efferent
side of the filament were much thinner, having a width
as small as 0.08 ^m (Figs. 11, 14). The epithelium was
lined by a thin basal lamina and had a sparse cytoplasm
containing microtubules. Fibrous material was also seen
between the basal lamina and the epithelium. No apical
microvillar processes or basal infoldings were observed in
the respiratory filament epithelia.

The most prominent feature of the cells in the pillar
regions was the bundles of microtubules observed in their
cytoplasm (Figs. 12, 13). These bundles were generally
oriented perpendicular to the cuticle, apparently attached

to extensions of the cuticle that penetrated into the epi-
thelial cells (Fig. 13). The pillar cytoplasm contained some
mitochondria, rough endoplasmic reticulum, and golgi,
but noticeably fewer than in the transporting filament tis-
sues (Fig. 12).

The cuticle of the respiratory filaments was thickest on
the side of the afferent channel (approximately 2.0 ^m)
(Fig. 14) and thinnest on the efferent side of the filament
(approximately 0.5 fim) (Fig. 15). The attenuation of the
cuticle from the afferent to the efferent side was observed
lateral to the septum of the filament. It appeared as if no
endocuticle had been deposited on the afferent side of the
filament, the side facing the carapace.

The septum of the respiratory filament was thicker than
the transporting filament septum, possessing afferent sep-
tal projections, a thick afferent basement membrane, and
a thin efferent basal lamina. The septum had a width of
about 5 ^m, with an abundance of cell membrane inter-
digitations (Figs. 5, 16). A thick layer of fibrous material
frequently lined the inside of the cell membrane on the
afferent side of the septum (Fig. 16). Thicker septa ob-
served in these filaments usually had loose connective
tissue extending across the septum on the afferent side
(Fig. 5).

Neural tissue was often observed in both filament types
(Fig. 17). The small nerves were located within one of the
small blood spaces of the connective tissue lateral to the
septum. Usually there was one nerve per filament, though
a few filaments were observed that contained two or three
nerves, each in a separate blood space. The nerve was
continuous throughout the length of a filament. Nerves
ranged from 1 .0 ^m to 2.7 jum in diameter and contained
two to six neurons. The neurons, which ranged from 0. 16
fim to 1.5 Mm in diameter, contained numerous, evenly
spaced neurotubules. No cell bodies, neurosecretory
granules, or synapses were observed in any section.

Also occurring in both types of filaments were cells
resembling "podocytes" (Morse et ai, 1970). They were
observed near the septum, attached to the loose connective
tissue bordering the afferent channel (Figs. 4, 5, 18) and
were surrounded by a basal lamina. These cells possessed
an interdigitating cell membrane, or pedicels, which
formed extracellular spaces between the pedicels and the
cell body. Much of the cytoplasm contained an abundance
of smooth endoplasmic reticulum. Coated pits and vesicles
were often observed along or near the cell membrane and
numerous dense granules, varying in size and number,
occurred within the cytoplasm along with golgi apparati
consisting of thin, curved cisternae (Fig. 19).

A TPase determinations

As shown in Figure 20, the mean for total ATPase ac-
tivity was higher in the transporting than in the respiratory

160

J. S. DICKSON ET AL

Figures 6-9. TEM ot'lransporting filaments. Figure 6. Section showing the cuticle with its three layers,
the epicuticle (arrow), the exocuticle (Ex) and the endocuticle (En). Also note a Golgi apparatus (g). the
basal lamina (b). and a junctional complex (arrowhead). Bar = 1.0 pm. Figure 7. Section showing a pillar
structure (p) separating two lacunar spaces (1) from an efferent blood channel (Eb). Note heterochromatic
nucleus (n). Bar = 5.0 ^m. Figure 8. Section of epithelium under cuticle showing microtubules (arrowheads)
and rough endoplasmic reticulum (r) in the cytoplasm. Also note the apical microvillar structures (arrow)
immediately below the cuticle. Bar = 1 .0 ^m. Figure 9. Section of epithelium between cuticle and lacuna
showing the electron dense material at the tip of the microvillar processes (arrowhead) and the thin basal
lamina (b). Bar = 1.0 pm.

GILL FILAMENTS IN P CLIRK1I

161

Figures 10-16. TEM of transporting (10) and respiratory (11-16) filaments. Figure 10. Section of a
septum in a transporting filament that separates the afferent (Ah) from the efferent (Eh) hlood channel. Bar
= 2.0^11. Figure 11. Section of a respiratory filament showing the thin epithelium underlying the cuticle.
Also note the pillar structure (p) and a hemocyte (h). Bar = 5.0 ^m. Figure 12. Pillar structure of a
respiratory filament showing the bundles of microtubules running perpendicular to the cuticle (arrowheads).
Bar = 1.0 nim. Figure 13. Section showing relationship of microtubules (m) to extensions of the cuticle
(arrowheads). Bar = 0.2 /jm. Figure 14. Cuticle on side of afferent channel. Bar = I.O/im. Figure 15. Cuticle
on side of efferent channel. Bar = 1.0 ^m. Figure 16. Septum from respiratory filament separating the
afferent blood channel (Ah) from the efferent blood channel (Eb). Note the thin basal lamina on the efferent
side (arrowhead) as compared with the thick layer on the afferent side (arrow). Bar = 1.0 /im.

162

J. S. DICKSON AT AL

Figures 17-19. TEM of filaments. Figure 17. Cross section of nerve showing individual neurons (n)
having evenly spaced neurotuhules (arrowheads). Bar = 0.5 /jm. Figure 18. Podocyte (P) in loose connective
tissue (Ct). Note electron-dense inclusions (d). Bar = 5.0 ^m. Figure 19. Podocyte cytoplasm showing
pedicels (arrowheads) golgi (g) and basal lamina (arrow). Bar = 1.0 ^m.

filaments; however, the difference between the means was
not significant. Mean values for Mg-ATPase were very
similar between respiratory and transporting filaments and
were likewise not significantly different. The means for
Na, K-ATPase, in contrast, were significantly different (P
< 0.001, Mann-Whitney U test), with the transporting
filaments having more than five times as much mean ac-
tivity as the respiratory filaments.

Transepithelial potentials

The values of the TEP's in the filaments were indepen-
dent of the region (proximal, medial, or distal) of the fil-

ament impaled and of the podobranch upon which the
filament was located. The TEP's of filaments that were
presumed to be respiratory (based upon the silver staining
results) ranged from -6 to -18 mV (-1 1.9 ± 3.2 mV,
mean ± S.D., n = 19). The TEP's measured in presump-
tive transport filaments ranged from -21 to -36 mV
(-28.1 ± 4.7 mV, mean ± S.D.; n = 21). There was no
overlap in the frequency distributions of the ranges of
TEP's (Fig. 2 1 ) and the difference between the means was
highly significant (P < 0.00 1 , two-tailed t test). The sodium
concentration of the medium was 51 mA/and that of the
hemolymph was 107.5 ± 15.0 mA/ (mean ± S.D.,
n = 11).

GILL FILAMENTS IN P. CLARKII

163

/^.rnol P
mg protein*hr

Total

Na.K

Figure 20. Mean (± standard deviation) ATPase activity comparing
pooled transporting (open bars) and respiratory (solid bars) filaments.
** = P< 0.001

Discussion

The results of silver staining, ultrastructural analyses,
ATPase measurements, and measurements of TEP all in-
dicate that the gills of P. clarkii are not homogeneous in
structure, but contain filaments dedicated to ion transport
and filaments dedicated to respiration. No filaments had
both characteristics; rather individual filaments were
uniquely respiratory or transporting. Respiratory filaments
occurred on the lateral rows of most gills, whereas the
transporting filaments predominated in the central bed
of the gills. This distribution of the two filament types in
discrete regions of the gill placed the respiratory filaments
in the path of the most rapid water flow (Burggren el ai,
1974).

The respiratory filaments had a very thin, squamous
epithelium, whereas the ion transporting filaments pos-
sessed a markedly thicker epithelium. The ultrastructure
of the ion transporting epithelia observed in P. clarkii gill
filaments was similar to ion transporting epithelia de-
scribed in other crayfish (Morse el a/., 1970; Bielawski.
1971; Fisher, 1972), in isopods (Bubel and Jones, 1974),
in crabs (Copeland and Fitzjarrell, 1968; Taylor and
Greenaway, 1979; Finol and Croghan, 1983), in shrimp
(Foster and Howse, 1978), and in Dapluua (Kikuchi,
1983). Apical microvillar processes were present to in-
crease the surface area over which ions could be trans-
ported as well as numerous basal infoldings with associated
mitochondria. Such basal infoldings are reported to be
the major site of Na, K-ATPase (Diamond and Bossert,
1968; Ernst, 1972; Ernst et ai. 1981; Towle, 1985;Towle
and Kays, 1986). This would be consistent with the higher
Na, K-ATPase activity measured in transporting fila-
ments, because few basal infoldings or mitochondria were
observed in the epithelia of the respiratory filaments.

The structure of the thin squamous epithelia in P. clar-
kii respiratory filaments was similar to the respiratory ep-
ithelia described in other crayfish (Dunel-Erb et ai, 1982)
in crabs (Copeland and Fitzjarrell, 1968; Taylor and
Greenaway. 1979), and in shrimp (Foster and Howse,
1978). The paucity of organelles observed in the epithe-
lium and pillar structures indicates that little cellular ac-
tivity occurs in these cells other than maintenance of cell
components. The thin epithelial layer may serve as a per-
meability barrier to the diffusive loss of ions and blood
proteins, while the thin cytoplasm would allow efficient
gas exchange to occur.

Large bundles of microtubules were observed in the
pillar structures of the respiratory filaments, whereas single
microtubules were the rule in the transporting epithelia
of this crayfish and in most gill epithelia of other crusta-
ceans (Copeland and Fitzjarrell, 1968; Bielawski, 1971;
Foster and Howse, 1978; Taylor and Greenaway, 1979;
Finol and Croghan, 1983; Compere et ai, 1989). These
microtubules appear to anchor the epithelium to the cu-
ticle. Finol and Croghan (1983) have proposed that the
microtubules function to stabilize the gill epithelium
against the hydrostatic pressure of the blood. Because the
respiratory epithelium contains very little cytoplasm, ad-
ditional microtubules, in the form of bundles near the
periphery of the pillar structures, may give the additional
support needed to withstand the shear forces of the blood
flow in these filaments.

The loose connective tissue in the gills of P. clarkii is
similar to that observed in Callinectes sapidus (Johnson,
1980). The cells within the loose connective tissue, with
their abundant glycogen rosettes and granules, may reg-
ulate the blood glucose levels as suggested by Finol and
Croghan ( 1983) for Uca more/a.

Frequency

-16 -24

T.E.P (mV)

-32

-40

Figure 21. Transepithelial potentials (TEP) for respiratory (solid bars,
n = 19) and transporting (open bars, n = 21) filaments.

164

J. S. DICKSON

The observed difference in the size and structure of the
septum in the two types of filaments suggests its function
may likewise differ. The thicker septum in the respiratory
filaments may make the vascular canals more rigid, or it
may also be a more substantial barrier to diffusion between
the two compartments.

The neurons observed in the filaments of P. clarkii were
similar to those constituting the branchial nerve observed
in A. pallipes and A. leptodactylus(Dune\-Erb el al, 1 982).
While those authors have described the nerve cell bodies
and described structures resembling neurosecretory gran-
ules, the role of the nerve is uncertain. Massabuau el al.
( 1 980) and Ishii el al. ( 1 989) have suggested that the nerve
may serve peripheral sensory elements involved in oxygen
sensing. While the presence of the nerve in both the re-
spiratory and transporting filaments would seem to ques-
tion this function, the absence of any description of a
transducing element leaves this question unresolved.

The "podocytes" observed in the filament tissues pos-
sessed the same ultrastructure as "podocytes" observed
in other crustacean gills (Wright, 1964; Strangeways-
Dixon and Smith, 1970: Doughtie and Rao, 1981). The
presence of coated vesicles and large storage vacuoles gives
support to the hypothesis that these cells take up toxic
substances and blood components from the hemolymph
for degradation or storage (Strangeways-Dixon and Smith,
1970; Doughtie and Rao, 1981).

ATPase measurements supported the silver staining and
morphological observations in that values were highest in
the transporting filaments. These data represent an ap-
parent contradiction to the conclusion of Wheatly and
Henry (1987) who reported that enzyme activity was ho-
mogeneously distributed throughout the branchial tissue.
However, Wheatly and Henry (1987) pooled tissue from
entire gill sets, and, therefore, could not have detected
differences at the level of individual filaments. In fact,
their data would represent a mean of respiratory and
transporting filaments, assuming that Pacifastacm has a
distribution of filament types similar to Procambarus. Our
values for Na, K-ATPase activity in respiratory filaments
are comparable to theirs, but the values we obtained for
transport filaments are fivefold higher, reflecting the en-
richment of transport tissue resulting from analyses of
selected filaments.

While the in situ measurements of TEP's in the gill
filaments were not intended to supply extensive data on
the mechanisms of transport, a few interesting conclusions
may be made. The data clearly suggest two functionally
different populations of filaments. These data are quali-
tatively similar to those of Pequeux and Gilles (1988) who
found positive TEP's in isolated, anterior, respiratory gills
of Eriocheir, but negative potentials in the posterior,
transporting gills.

The fact that the TEP's were negative with respect to
the dilute medium, suggests that under the experimental
circumstances the integumental barrier is preferentially
cation conductive. The TEP's of the respiratory filaments
were slightly more positive and those of the transport fil-
aments slightly more negative than the diffusion potential
for Na (-19 mV) calculated from the measured concen-
tration difference. Cation selectivity is consistent with the
data from other gill potential studies (Austropotamobius
- Astacus pallipes, Croghan et al., 1965; Callinectes,
Mantel, 1967) and a study on the isolated gill cuticle of
Carcinus (Lignon, 1987). Only a study by Avenet and
Lignon (1985) presented data showing anion selectivity
in the isolated cuticle from the gill lamina (plate) of As-
tacus leptodactylus. Whether the TEP's arise from diffu-
sion potentials or from electrogenic transport is unknown,
however, thecontributionoftransport-generated potentials
is generally small in gills in the presence (Siebers et al..
1985) or in the absence of concentration gradients (Siebers
et al.. 1985; Drews and Graszynski, 1987). Avenet and
Lignon (1985) and Lignon (1987) suggested that the ion
selectivity of the integumental barrier resided in the epi-
cuticle. Such cation selectivity of the epicuticle of the gill
of Procambarus would be consistent with the location of
the silver precipitate within the exocuticle. Presumably,
the divalent silver cations could easily cross the outer cu-
ticular barrier to precipitate with higher internal concen-
trations of chloride or, more likely, bicarbonate. If the
observed TEP's are the result of diffusion potentials, the
differences in TEP's between the respiratory and transport
filaments would suggest either different ion permeabilities
or different local ion concentrations within the two types
of filaments.

The circulation of hemolymph within the gill filament
has been described by Bock (1925), Fisher (1972), and
Burggren et al. (1974). They describe hemolymph as
flowing toward the tip of the filament in the afferent chan-
nel and down the filament toward the gill stalk in the
efferent channel. Hemolymph may also be shunted from
the afferent to the efferent channels via the lateral lacunae.
The thick septum of connective tissue in respiratory fil-
aments may act as a permeability barrier to counter-cur-
rent gas exchange between the deoxygenated afferent he-
molymph and the oxygenated efferent hemolymph, be-
cause this type of exchange would result in deoxygenated
blood being returned to the body. The thin septum of the
transporting filaments, on the other hand, may promote
counter-current diffusion of ions from the afferent to the
efferent channel, preventing further diffusive ion loss from
the lacunae.

The observations in the present study indicate that there
is a precise partitioning of structural and functional epi-
thelium types within the gills of Procambarus clarkii, a
thin squamous epithelium functioning in respiration and

GILL FILAMENTS IN P CLARKI1

165

a thick, mitochondria-rich epithelium functioning in ion
transport. It is important to remember, however, that the
integument consists not only of the underlying epithelium,
but also of the overlying cuticle. Because this cuticle must
periodically be shed to permit growth, the question arises
as to whether the specialized functions of the filaments
are disrupted during the period surrounding ecdysis. Dur-
ing premolt the hypodermis is normally engaged in cu-
ticular synthesis, and prior to ecdysis the preexuvial cuticle
exists as an additional barrier to the movement of sub-
stances. A more complete understanding of gill function
must await examination of gills not only during intermolt,
but also during the molt.

Literature Cited

Avenet, P., and J. M. Lignon. 1985. Ionic permeabilities of the gill
lamina of the crayfish. Astacus leptodacly/us (E). ./ Physiol. 363:
377-401.

Bielawski, J. 1971. Ultrastructure and ion transport in gill epithelium
of the crayfish, Astacus leplodaclylus Esch. Protuplasma 73: 177
190.

Bock, F. 1925. Die Respirationsorgane von Potamolmis astacin, Leach.
(Astacus flimatilis Fabr.) Ein Beitrag zur Morphologic der Decapoden.
Z. H'iss. Zool. 124: 51-117.
Bryan, G. \V. 1959. Sodium regulation in the crayfish Astacusfluviatilis.

I. The normal animal. J. E.\p. Biol. 37: 83-99.

Bubel, A., and M. B. Jones. 1974. Fine structure of the gills of Jacra
«o«/wuwi/(Rathke) [Crustacea, Isopoda]. J. Mar Biol Assoc L' K.
54: 737-743.

Burggren, W. VV., B. R. McMahon. and J. \V. Costerton.

1974. Branchial water- and blood-flow patterns and the structure

of the gill of the crayfish Pmcambariis clarkit. Can- J. tool. 52: 1511-

1518.

Cioffi, M. 1984. Comparative ultrastructure of arthropod transporting

epithelia. Am. Zool. 24: 139-156.

Compere, P., S. Wanson, A. Pequeux, R. Gilles, and G. Goffmet.

1989. Ultrastructural changes in the gill epithelium of the green

crab Cardmts maenas in relation to the external salinity. Tissue Cell

21:299-318.

Copeland, D. E. 1963. Possible osmoregulatory cells in crab gills. J

Cell Biol. 19: 16A.
Copeland, D. E. 1967. A study of salt secreting cells in the bnne shrimp

(Anemia sa/ina). Protoplasma 63: 363-384.
Copeland, D. E. 1968. Fine structure of salt and water uptake in the

land-crab, (Jecarcmus lateralis. Am. Zool. 8: 417-432.
Copeland, D. E., and A. T. Fitzjarrell. 1968. The salt absorbing cells
in the gills of the blue crab (Callinectes sapiiiiis Rathbun) with notes
on modified mitochondria. Z. Zelljorscli. 92: 1-22.
Croghan, P. C, R. A. Curra, and A. P. M. Lockwood. 1965. The elec-
trical potential difference across the isolated gills of the crayfish Aus-
tropotamobiiis pallipes (Lereboullet). / Exp. Biol. 42: 463-474.
Diamond, J. M., and W. H. Bossert. 1968. Functional consequences
of ultrastructural geometry in "backwards" fluid-transporting epi-
thelia. J. Cell Bio/ 37: 694-702.

Doughtie, D. G. and K. R. Rao. 1981. The syncytial nature and phago-
cytic activity of the branchial podocytes in the grass shrimp, Palae-
monetes pugiu. Tissue Cell 13: 93-104.

Drach, P., and C. Tchernigovtzeff. 1967. Sur la methode de determi-
nation des stades d'intermue et son application generate aux crustaces.
Vie Milieu 18: 595-610.

Drews, G., and K. Graszynski. 1987. The transepithelial potential dif-
ference in the gills of the fiddler crab, L'ca lanxcri influence of some
inhibitors. J. Comp Physiol 157B: 345-353.

Dunel-Erb, S., J.-C. Massabuau, and P. C. Laurent. 1982. Organisation
fonctionelle de la branchie d'ecrevisse. C R Seances Soc Biol. 176:
248-258.
F.hrenfeld, J. 1974. Aspects of ionic transport mechanisms in crayfish

Abacus leptodacly/us J. Exp. Biol. 61: 57-70.

Ernst, S. A. 1972. Transport adenosine triphosphatase cytochemistry.
II. Cytochemical localization of ouabain-sensitive. potassium-depen-
dent phosphatase activity in the secretory epithelium of the avian
salt gland. J. Histoehem. Cytnehem. 20: 23-28.

Ernst, S. A., C. V. Riddle, and K. J. Karnaky, Jr. 1981. Relationship
between localization of Na+-K+-ATPase, cellular fine structure, and
reabsorptive and secretory electrolyte transport. Pp. 355-385 in Cur-
rent Topics in Membranes and Transport. V. 30. F. Bronner and A.
Kleinzeller. eds. Academic Press. New York.

Finol, H. J., and P. C. Croghan. 1983. Ultrastructure of the branchial
epithelium of an amphibious brackish-water crab. Tissue Cell 15:
63-75.

Fisher, J. M. 1972. Fine-structural observations on the gill filaments
of the freshwater crayfish. Astacus pallipes Lereboullet. Tissue Cell
4: 287-299.
Foster, C. A., and H. D. Howse. 1978. A morphological study on gills

of the brown shrimp, Penaeus a:tecus. Tissue Cell 10: 77-92.
Holliday, C. \V. 1985. Salinity-induced changes in gill Na, K-ATPase
activity in the mud fiddler crab, Ucapugnax. J Exp. Zool. 233: 199-
208.

Horiuchi, S. 1977. Characterization of gill Na, K-ATPase in the fresh-
water crayfish, Procambarus clarkii (Girard). Comp Biochem. Phys-
nil. 56B: 135-138.

Huxley, T. H. 1896. The Crayfish— An Introduction to the Study of
Zoo/ogv The Internal. Scientific Series. Paul, Trench and Trubner.
London.

Johnson, P. T. 1 980. The III stology of the Blue Crab. Callinectes sap-
idus. A Model for the Decapoda. Praeger. New York. Pp. 34-40.
Ishii, K., K. Ishii, J.-C. Massabuau, and P. Dejours. 1989. Oxygen-
sensitive chemoreceptors in the branchio-cardiac veins of the crayfish,
Astacus leptodactylns. Resp. Physiol. 78:73-81.

Kikuchi, S. 1983. The fine structure of the gill epithelium of a fresh-
water flea. Daphma magna (Crustacea: Phyllopoda) and changes as-
sociated with acclimation to various salinities. I. Normal fine structure.
Cell Tissue Rex 229: 253-268.
Lignon, J. M. 1987. Ionic permeabilities of the isolated gill cuticle of

the shore crab Curcinus maenas. J Exp. Biol 131: 159-174.
Maluf, N. S. R. 1940. The uptake of inorganic electrolytes by the cray-
fish. J. Gen Physiol. 24: 151-167.

Mantel, L. H. 1967. Asymmetry potentials, metabolism and sodium
fluxes in gills of the blue crab, Callinectes sapidus. Comp. Biochem.
Physiol. 20: 743-753.

Mantel, L. H., and J. Landesman. 1977. Osmotic regulation and Na-
K.-activated ATPase in the green crab. Carcinus maenas and the
spider crab Lihinia emarginata. Biol. Bull. 153: 437-438.
Massabuau, J.-C., B. Elancher, and P. Dejours. 1980. Ventilatory reflex
response to hypoxia in the crayfish, Astacus pallipes. J. Comp Physiol.
140: 193-198.

Milne, D. J., and R. A. Ellis. 1973. The effect of salinity acclimation
on the ultrastructure of the gills ofGammarus oceamcus (Segerstrale.
1947) (Crustacea: Amphipoda). Z. Zelljorscli. 139: 311-318.
Morse, H. C., P. J. Harris, and E. J. Dornheld. 1970. Pacifastacus
leniusculus: fine structure of the arthrobranch with reference to active
ion uptake. Trans. Am. Micr. Soc. 89: 12-27.

Neufeld, G. J., C. W. Holliday, and J. B. Pritchard. 1980. Salinity
adaptation of gill Na. K-ATPase in the blue crab, Callinectes sapidus.
J. Exp. Zool. 211: 215-224.

166

J. S. DICK.SON ET AL

Pequeux, A., and R. Gilles. 1978. Osmoregulation of the euryhaline
Chinese crab Enochen xiiienstx. Ionic transports across isolated per-
fused gills as related to the salinity of the environment. Pp. 105-1 1 1
in Pnif. I2lh EMBS, Stirling, Scotland. Pergamon Press. Oxford.

Pequeux, A., and R. Gilles. 1981. Na* fluxes across isolated perfused
gills of the Chinese crab Eriocheir smensis. J Exp. Biot 92: 173-
186.

Pequeux, A., and R. Gilles. 1988. The transepithelial potential difference
of isolated perfused gills of the Chinese crab Eriocheir sinensis ac-
climated to fresh water. Comp. Biochem. Physiol. 89A: 163-172.

Peterson, G. L. 1977. A simplification of the protein assay method of
Lowry el a/, which is more generally applicable. Anal. Biocliem. 83:
346-356.

Reynolds, E. S. 1963. The use of lead citrate at high pH as an electron
opaque stain in electron microscopy. J Cell Biol 17: 208-212.

Roer, R. D., and M. G. Shelton. 1982. The effects of hydrostatic pressure
on sodium transport in the crayfish, Procamharnx clarkii. Coin/'
Biochem. Physiol. 71A: 271-276.

Shaw, J. 1960a. The absorption of sodium ions by the crayfish. Asiiicif,
pallipes Lereboullet. 11. The effect of the external anion. J. Exp. Biol.
37: 534-547.

Shaw, J. I960b. The absorption of sodium ions by the crayfish Astaais
pallipes Lereboullet. 111. The effect of other cations in the external
solution. J. Exp Biol 37: 548-556.

Siebers, D., A. \Vinkler, C. Lucu, G. Thedens and D. VVeichart.
1985. Na-K-ATPase generates an active transport potential in the
gills of the hyperregulating shore crab Carcinus maenas. Mar. Biol.
87: 185-142.

Spurr, A. R. 1969. A low-viscosity epoxy resin embedding medium
for electron microscopy. / Ultraxlnicl. Res 26: 31-43.

Stevenson,.!. R. 1972. Changing activities of the crustacean epidermis
during the molt cycle. Am. Znol. 12: 373-380.

Strangeways-Dixon, J., and D. S. Smith. 1970. The fine structure of
the gill "podocytes" in Panulirux argtis (Crustacea). Tissue Cell 2:
61 1-624.

Taylor, H. H., and P. Greenaway. 1979. The structure of the gills and
lungs of the arid-zone crab, Holthuixana (Auslrolhelphusa) Iransversa
(Brachyura: Sundathelphusidae) including observation on arterial
vessels within the gills. J /.mil. 189: 359-384.

Towle, D. \V. 1984. Membrane-bound ATPases in arthropod ion-
transporting tissues. Am /no/ 24: 177-185.

Towle, D. VV. 1985. Localization of Na+ + K+-ATPase in basolateral
membranes of crab (Carcinus maenas) gill epithelium. Bull MD1BL
25: 80-83.

Towle, D. VV., and VV. T. Kays. 1986. Basolateral localization of Na*
+ K*-ATPase in gill epithelium of two osmoregulating crabs. Cal-
linectcs sapidus and Carcinus maenas. J. Exp. Zool. 239: 311-318.

Wanson, S. A., A. J. R. Pequeux, and R. D. Roer. 1984. Na+ regulation
and the (Na+ + K+)ATPase activity in the euryhaline fiddler crab
Vca mmax (Le Conte). Comp. Biochem Physiol. 79A: 673-678.

VVheatly, M. G., and R. P. Henry. 1987. Branchial and antennal gland
Na+/K+-dependent ATPase and carbonic anhydrase activity during
salinity acclimation of the euryhaline crayfish Pacifastacus /ennixcii/us.
J Exp. Biol 133: 73-86.

Wheeler, A. P. 1975. Oyster mantle carbonic anhydrase: evidence for
plasma membrane bound activity and for a role in bicarbonate trans-
port. Ph.D. Dissertation, Duke LIniversity. Durham. North Carolina.

Wright. K. A. 1964. The fine structure of the nephrocyte of the gills
of two marine decapods. J I'llraslructiire Res 10: 1-13.

Reference: Biol Bull 180: 167-184. (February, 1991)

Nutrient Translocation during Early Disc Regeneration

in the Brittlestar Microphiopholis gracillima

(Stimpson) (Echinodermata: Ophiuroidea)

WILLIAM E. DOBSON1*, STEPHEN E. STANCYK, LEE ANN CLEMENTS2,
AND RICHARD M. SHOWMAN

Department of Biology, University of South Carolina, Columbia, South Carolina, 29208;

] Department of Biology. Cla/lin College. Orangeburg, South Carolina 291 15; and -Division of

Science and Mathematics, Jacksonville University. Jacksonville, Florida 3221 1

Abstract. Microphiopholis gracillima can autotomize
and then regenerate the autotomized central disc, includ-
ing integument, gut, and gonads. Experiments were car-
ried out to determine the relative importance of internal
nutrient reserve translocation and exogenous nutrient
uptake during the regeneration process. Approximately
60% of the dry body weight of M. gracillima is organic
material. Intact animals held for three weeks in natural
seawater did not change significantly in weight, caloric
content, or relative concentration of protein, carbohy-
drates, or lipids. Intact animals held for three weeks in
artificial seawater devoid of nutrients lost weight and ca-
loric content. The rate of loss was rapid initially, but
slowed after about eight days. Animals regenerated in
natural seawater lost weight initially, then regained the
lost weight. Animals regenerated in artificial seawater lost
weight constantly and at a higher rate than either the ar-
tificial seawater control or natural seawater regenerated
animals. All weight losses were attributable to significant
changes in the protein and carbohydrate fractions of the
organic body component. The lipid fraction and ash
components did not change significantly in any treatment.
M. gracillima appears to be adapted to regenerate the lost
disk rapidly, even under conditions of food deprivation.

Introduction

Autotomy (self-mutilation by casting off body parts),
followed by regeneration of the lost parts, is widespread

Received 26 February 1990; accepted 26 November 1990.
Contribution #830 from the Belle W. Baruch Institute for Marine
Biology and Coastal Research.
* To whom all correspondence and reprint requests should be sent.

among the echinoderms, and these animals are known to
have superb wound-healing and regenerative capacities
(see review by Emson and Wilkie, 1980; Brown, 1982).
The few published studies of echinoderm regeneration
have dealt almost exclusively with the capacity of an in-
dividual species to regenerate, descriptions of the appear-
ance of new structures, or measurements of regeneration
rates (Gibson and Burke, 1983). Although two recent
studies have estimated the environmental energy produc-
tion represented by regenerating brittlestar arms (Dui-
neveld and Van Noort, 1986; O'Conner el al., 1986), the
energetic costs to the regenerating animal of autotomy,
and the sources of nutrition for regeneration in echino-
derms, have not been evaluated.

Members of at least five families of ophiuroid echino-
derms (brittlestars) autotomize arms or the aboral disc
(including digestive tract, gonads, and disc epithelium)
when disturbed. They can regenerate these tissues within
a few weeks in the laboratory (Emson and Wilkie, 1980;
pers. obs.). The rate of tissue replacement must be related
to the amount of stored nutrients available and the rate
at which the animal can accumulate and allocate addi-
tional nutrients for tissue regeneration. The ophiuroid disc
begins to regenerate before the gut has been replaced, but
the sources of the nutrients that support that process are
uncertain. To date, no specific nutrient storage organ other
than the disc has been found, although the interstices of
the arm ossicles may be repositories for nutrients (Turner
and Murdoch, 1976). The ability of many echinoderms
to translocate nutrients during gametogenesis (Lawrence,
1987) suggests that the same mechanism could be used
during regeneration. Echinoderms may also take up dis-

167

168

W. E. DOBSON ET .11.

solved organic matter (DOM) from the surrounding sea-
water, or process exogenous paniculate nutrients by ex-
ternal digestion (Lawrence, 1987; Clements, 1988).

Theoretically, use of either DOM alone or external
digestion alone would result in a net gain of organic matter
by regenerating animals with no concomitant tissue loss
from non-regenerating portions of the body. In contrast,
internal nutrient translocation would result in the decrease
of tissue in non-regenerating portions of the animal, with
no net gain of organic matter during regeneration. There
should even be a net loss of tissue due to catabolism of
tissue constituents for respiration during regeneration. But
translocation of stored nutrients, external digestion, and
DOM uptake are not mutually exclusive and may operate
sequentially or simultaneously. Other echinoderms lack-
ing special storage organs resorb body parts under star-
vation conditions (Ebert, 1967; Feral, 1985; Lawrence,
1987). We hypothesize that early disc regeneration (prior
to reformation of the functional gut) relies heavily on
translocation of internal nutrient stores that are mobilized
from the non-regenerating somatic tissues. The purpose
of this paper is to estimate the relative contribution of
internal nutrient translocation to disc regeneration.

Material and Methods

Individuals of Microphiopholis gracillima were col-
lected from intertidal mud flats in the North Inlet Estuary
just north of Georgetown, South Carolina (37°20rN,
79° 10'W). After collection, animals were taken to the lab-
oratory and sorted to eliminate all but individuals with
complete (or almost completely regenerated) discs. Ani-
mals were then placed in autoclaved all-glass aquaria in
an environmental chamber held at 25 °C with a 12:12
light:dark cycle. The aquaria contained Millipore-filtered
(0.45 /urn) natural seawater (30%o). All aquaria were con-
stantly aerated. The seawater was changed daily to control
bacterial contamination (Clements et ai, 1988). Field-
collected animals may have large differences in their nu-
tritional states; therefore all animals were allowed to ac-
climate to the above conditions for seven days before the
start of the experiments to help equalize nutritional dif-
ferences. Animals were then randomly assigned to exper-
imental groups.

The amount of nutrients translocated during regener-
ation was estimated in the following treatments. Intact
and autotomized individuals were held in autoclaved all-
glass aquaria containing either Millipore-filtered (0.45 urn)
natural seawater without sediment; artificial seawater
alone (Cavanaugh, 1956; trace minerals formula 5); or
artificial seawater with approximately 125 ^mol/1 glucose,
125 /imol/1 palmitic acid, and 12.5 /umol/1 of each of 21
amino acids (alanine, arginine, asparagine, aspartic acid,
cysteine, cystine, glutamic acid, glutamine, glycine, his-

tidine, isoleucine, leucine. lysine. methionine, phenylala-
nine, proline, serine, threonine, tryptophan, tyrosine, and
valine) added, for a total DOM concentration of approx-
imately 513 ^mol/1. This represents about a five-fold in-
crease over natural DOM levels. (Clements, 1988; Wil-
liams, 1975). All aquaria were constantly aerated and kept
in an environmental chamber at 25°C with a 12:12 light:
dark lighting regimen. All media were changed daily. At
4-day intervals, 10 animals were removed from each
treatment and dissected into the following body fractions:
proximal, medial, and distal thirds of the arms, the oral
frame, and the regenerated (or intact) discs. All of these
fractions were dried to constant weight in vacua over an-
hydrous calcium carbonate. About half of each fraction
was ashed at 400°C for 6 h and its ash-free dry weight
determined. The remaining three parts were subjected to
biochemical analyses for protein, carbohydrate, and lipid,
respectively. Before biochemical analysis, each part was
split into three replicates, weighed, and ground to a dry
powder in a hand-held all-glass homogenizer. In this way,
replicated estimates of protein, carbohydrate, and lipid
were obtained for each body fraction, as well as estimates
of ash-free dry weight and caloric content. Because the
tissue samples were small, colorimetric techniques were
used in the biochemical analyses.

Total carbohydrate levels (as reducing sugars) were es-
timated by the phenol-sulfuric acid method of Dubois et
ul. (1956). with a 1:1 glucose:maltose solution as the stan-
dard. Total proteins were quantified by the Bio-Rad
(Richmond, CA) modification of the Bradford (1976)
method, with a 1 : 1 mixture of bovine serum albumin and
purified mollusk protein (Sigma) as the standard. Lipids
were extracted from the fraction homogenate with a 2:1
(v:v) chloroform:methanol solution. The extract was pro-
cessed according to the sulphophosphovanillin method of
Barnes and Blackstock (1973), with purified Microphio-
pholis gracillima lipid as the standard. The purified stan-
dard was prepared according to the procedures outlined
in Barnes and Blackstock (1973). To compensate for the
possibility of significant variation in total weights of the
specimens used in this study, and to control for the in-
evitable loss of tissue during the fractioning of the samples
for the different biochemical assays, all biochemical mea-
sures are reported as units per gram dry weight of specimen
rather than as absolute quantities per body part.

To determine the relative translocation rate of proteins,
carbohydrates, and lipids during regeneration, 300 ani-
mals were incubated in artificial seawater with either I4C-
leucine (specific activity 348.0 mCi/mmol), 14C-glucose
(3.5 mCi/mmol), or MC-palmitic acid (850.0 mCi/mmol)
added at concentrations of 0.03 ^Ci/ml, 0.04 ^Ci/ml, and
0.04 yuCi/ml, respectively. After 48 h, the animals were
removed from the medium and rinsed in several changes
of artificial seawater. One half of the animals in each nu-

NUTRIENT TRANSLOCATION DURING OPHIURO1D DISC REGENERATION

169

trient treatment were induced to autotomize following
the procedure of Dobson ( 1984, 1985). Five animals from
each of the six treatments were immediately processed
(see below). The remaining specimens were held in au-
toclaved all-glass aquaria containing constantly aerated
artificial seawater in an environmental chamber at 25°C
with a 12:12 light:dark cycle. The seawater was changed
daily. At 4-day intervals, for 20 days. 8 animals were re-
moved from each treatment and dissected to separate the
distal half of the arms, the proximal half of the arms (in-
cluding the oral frame), and the regenerated (or intact)
disc tissue. Each fraction of five individuals was dried to
constant weight at 80°C and placed in a separate glass
scintillation vial containing 1 ml of 1:1 ProtoSol (New
England Nuclear) tissue solublizenethanol. Five ml of
AquaSol liquid scintillation cocktail (New England Nu-
clear) was added to each solublized specimen vial, and
the samples were counted with a Beckman liquid scintil-
lation counter with internal quench correction. The other
three individuals were dried to constant weight at 80°C,
ashed at 400°C for 6 h, and weighed again. We normalized
all counts by computing the counts per minute (CPM)
per gram dry weight and per gram ash-free dry weight of
the tissue.

All data were analyzed by one-way or two-way AN-
OVA, Tukey's multiple comparison procedure (Ostle and
Mensing, 1975; Sokal and Rohlf, 1981), or least-squares
linear regression using the General Linear Models Pro-
cedure of the Statistical Analysis System (Carey, North
Carolina). The probability of making any type I error at
all in the entire series of tests was held at a = 0.05 or less
[= Experimentwise error rate (Sokal and Rohlf, 1981,
pg. 241)].

Results

Biochemical composition of the intact hri/t/es/ar

Normal values for organic and inorganic constituents
of whole and individual regions of Microphiopholis gra-
ci/lima were obtained by pooling all of the initial (time
= 0) biochemical measurements from each experiment.
The results are summarized in Table I. About 60% of the
total dry body weight is organic tissue (as ash-free dry
weight), and most of it is located in the arms. The central
disc has the highest organic content (74%) relative to in-
organic material, but this represents only 7% of the total
dry body weight and 10% of the total organic tissue weight.
The proximal, medial, and distal arm parts and the oral
frame region contain 50 to 60% organic material, which
accounts for 90%. of the total organic material. The arms
have a higher percentage of organic material at their bases
than at their tips.

The central disc and oral frame have higher concen-
trations (per gram dry weight) of all organic components

than do any of the arm regions. The disc has the highest
concentration (per gram dry weight) of protein and lipid,
whereas the oral frame has the highest concentration of
carbohydrates. All arm fractions are similar in their pro-
tein, carbohydrate, and lipid concentrations. Interestingly,
the assayed total protein, carbohydrate, and lipid content
of the body accounts for only 30% of the total ash-free
dry weight (= organic content) of the brittlestar. The rel-
ative underrepresentation of organic material is constant
between body fractions with the exception of the oral
frame, which has a relatively lower underrepresentation.
Most of the total missing organic material is located in
the arm parts. Although colorimetric assays commonly
underestimate the actual amount of material present
(Dubois eta/., 1956; Barnes and Blackstock, 1973; Davis,
1988), the magnitude of the underrepresentation in this
case is unusual. We assume that, as has been reported for
other echinoderms (Geise, 1966: Feral, 1985), the majority
of the missing material represents insoluble organic ma-
terial (such as connective tissue), organics tied up in the
stromal spaces of the ossicles, complexed biochemicals
(e.g.. glycoproteins and lipoproteins) that were not de-
tected by the assays, and nucleic acids.

Change in biochemical composition of tissues
during regeneration

Bodv weight changes. The changes in total dry weight
(DW). total organic weight (= ash-free dry weight, AFDW)
and total inorganic material weight (=ASH) fractions with
time in individuals in the natural seawater control (NC),
artificial seawater control (AC), natural seawater regen-
erated (NR), and artificial seawater regenerated (AR)
treatments are shown in Figure 1. Animals in artificial
seawater with added organics did not survive the experi-
ment and thus were not analyzed. Animals in the NC
group did not exhibit any significant change in total DW
(P = 0.3919), ASH weight (P = 0.9406), or AFDW (P
= 0.4805) during the course of the experiment (Fig. 1 A).
AC animals showed a rapid initial drop in both total DW
(P = 0.0466) and AFDW (P = 0.0002) until day eight,
after which both weight measures remained relatively
constant. Total ASH weight did not change significantly
at any time in the AC group (P = 0.0828) (Fig. IB). NR
animals did not lose significant amounts of total DW (P
= 0.0546) or ASH weight (P = 0.4458) with time, but
gradually lost AFDW (P = 0.0022) until about day 12,
after which AFDW gradually increased through day 20
(Fig. 1C). The NR and AC groups lost as much as 40%
of their initial AFDW values at some point during the 20-
day experiment. AR animals displayed a rapid initial drop
in total DW (P < 0.000 1 ) and AFDW (P < 0.000 1 ) from
day 0 to day 4, followed by a slower constant decrease in
these values. The maximum loss of DW during the ex-

170

W. E. DOBSON ET AL

Table I

Normal hiachcmical iv>»i/"w"""' "/ Microphiopholis gracillima

Constituent

Whole

Disc

Body

Part

Distal arms

Oral frame

Proximal
arms

Medial arms

DRY WEIGHT

91.32 ± 10.87

8.85 ± 1.44

29.27 ± 3.57

27.50 ± 4.76

22.32 ± 4.71

2.26 ± 0.26

(% total body part DW)

100

(% whole DW)

100

9.7

30.0

24.5

2.4

ASH-FREE DRY WEIGHT

52.47 ± 7.78

6.52 ± 1.05

15.96 ± 2.64

16.32 ± 2.87

12.35 ± 2.58

1.35 ±0.22

(% total body part DW)

100

54.5

59.3

55.3

59.7

(% whole DW)

7.1

17.5

17.8

13.5

1.47

(% whole AFDW)

100

30.4

31.1

23.5

2.5

ASH WEIGHT

37.70 ± 4.81

2.33 ± 0.42

13.31 ± 1.30

11.19 + 3.72

9.97 ± 2.14

0.91 ±0.14

(% total body part DW)

100

45.5

40.6

44.6

40.3

(% whole DW)

2.6

14.6

12.3

10.9

0.9

(% whole Ash weight)

100

6.2

35.4

29.7

26.4

2.4

PROTEIN

6.53 ± 0.35

1.59 ±0.18

2.04 ± 0.05

1 .44 ± 0.03

1.17 ±0.09

0.21 ±0.01

(% total body part DW)

7.15

18.02

7.00

5.27

9.60

(% whole DW)

7.15

1.74

2.23

1.57

1.28

0.23

(% total protein)

100

24.35

31.24

22.05

17.91

3.21

CARBOHYDR.4TES

2.88 ± 0.26

0.74 ± 0.07

0.9 1 ± 0. 1 3

0.65 ±0.14

0.33 ± 0.04

0.11 ± 0.01

(% total body part DW)

3.15

8.42

3.08

2.37

1.48

8.80

(% whole DW)

3.15

0.81

0.99

0.71

0.36

0.12

(% total carbohydrates)

100

25.69

31.59

22.56

11.45

3.81

LIPIDS

3.46 ± 0.33

0.74 ± 0.05

1.05 ±0.24

0.94 ± 0.08

0.60 ±0.1 7

0.10 ±0.01

(% total body part DW)

3.79

8.36

3.58

3.41

2.68

4.50

(% whole DW)

3.79

0.81

1.14

1.02

0.65

0.11

(% total lipids)

100

21.40

30.60

27.10

17.60

2.89

UNACCOUNTED

ORGANICS

39.60 ± 3.89

3.45 ± 0.09

11.96 ±0.96

13.29 ± 1.13

10.25 ±0.76

0.42 ± 0.02

(% total body part DW)

43.36

38.90

40.86

48.30

45.93

18.58

(% whole DW)

43.36

3.77

13.09

14.50

11.22

0.46

(% whole AFDW)

65.47

6.57

22.79

25.32

19.53

0.80

(% total UO)

100

8.71

30.20

33.56

25.88

1.06

CALORIC CONTENT (calc.)

0.93 ± 0.04

2.40 ± 0.06

0.83 + 0.08

0.70 ± 0.03

0.58 ± 0.08

1.08 ±0.07

(kCal/gdry weight)

All values are averages ± one standard deviation. All units are milligrams unless otherwise noted.
DW = Dry Weight, AFDW = Ash-Free Dry Weight, UO = Unaccounted Organics.

periment occurred in this group, which lost as much as
40% of the initial DW and 50% of the initial AFDW. Ash
weight did not change significantly in the AR group (P
= 0.4893) (Fig. ID).

When the total DW measurements were broken down
by body part, the following trends were observed. In the
NC group, no significant DW change occurred in any
body part with time (P > 0.05 in all fractions) (Fig. 2A).
In the AC group, the DW of the medial (P = 0.4247) and
proximal (P = 0.4928) regions of the arms remained rel-
atively constant, but the disc, distal arm regions, and oral
frame lost DW until about day eight, after which their
dry weights remained constant (P = 0.0003, P = 0.0029,
P = 0.0025, respectively) (Fig. 2B). Animals in the NR
group exhibited no overall change in DW in any body
part (P > 0.05) after first appearance of the disc tissue,
although the weight of the oral frame on day 16 was sig-

nificantly different from all other days. Animals in the
AR group lost DW throughout the experiment in all non-
regenerating body parts (P < 0.05). This loss was rapid
until approximately day eight, after which the decline
proceeded at a slower rate.

Ash-free dry weight and ASH weight measurements by
body part with time indicate that the loss in DW is due
to loss exclusively from the organic fraction (Fig. 3). There
were no significant changes in the ASH weights of any
body parts in any experimental treatment over the 20-day
period with the exception of first appearance of the discs
(between days zero and four) in the regenerating groups
(Fig. 4). There was no significant change in AFDW in
anybody part with time (P > 0.05) in the NC group (Fig.
3A). In the AC group, all body parts with the exception
of the proximal arm fractions lost AFDW until approx-
imately day eight, after which AFDW remained relatively

NUTRIENT TRANSLOCATION DURING OPHIUROID DISC REGENERATION

171

01
OOP
0.08
0.07
0.06
005
004
0.03
0.02
0.01
0

(A) Natural Seawater - Control

(B) Artificial Seawaler ~ Control

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0,01 -
0

34 8 12 16

TIME (Days)

-I

8 12

TIME (Days)

ID) Ajtitical Seawater - Regenerating

4 8 12

TIME (Days)

Figure 1 . Total body weight changes during early disc regeneration. (A) Natural Seawater control group.
(B) Artificial seawater control group. (C) Natural seawater regenerating group. (D) Artificial seawater regen-
erating group. Error bars represent 95% confidence intervals. Error bars and points offset slightly for graphical
clarity.

constant in all body fractions (Fig. 3B). The most rapid
drop in AFDW occurred between day zero and day four.
Although the proximal arm fractions did lose AFDW over
the course of the experiment, the loss was not significant
at any time (P = 0.1443). Animals in the NR group lost
AFDW from all non-regenerating body fractions until ap-
proximately day 12, after which AFDW increased (Fig.
3C). Because of the high variability in the data, the changes
in AFDW of the proximal and medial arm fractions were
not statistically significant from day zero at any other time
(P = 0.0566 and P = 0.0853, respectively). The AFDW
of all non-regenerating body part fractions in the AR group
declined continuously until day 16 of the experiment (P
< 0.05) (Fig. 3D). The most rapid decrease occurred be-

tween day zero and day four, except in the proximal arm
regions, where tissue was lost at a constant rate. The disc
tissue in both the NR and AR groups did not increase in
AFDW content significantly after first appearing.

Protein content changes. The changes in total body
protein concentration over time are shown in Figure 5A.
The natural seawater control group did not change in
total protein concentration over the course of the exper-
iment (P = 0.4717). The artificial seawater control group
exhibited a slight decline in protein concentration with
time (P = 0.0 1 2 1 ), but the only day that was significantly
different from the others in this group was day eight. The
groups regenerating in natural seawater and in artificial
seawater both changed slightly in total protein concen-

172

W. E. DOBSON El AL

0.03

(A) Natural Seawaler - Control

D DISTAL" DISC O PROXIMAL

0.03

(B) Artificial Seawater - Control

0.026 -

_ O-022
O 0.018

O 0.014

1 i 1 I 1 Q

0.00

0.03

0.026

0.018

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0

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TIME (Days)

0006

0,002
0

0.03

•t-

4 8 12 16 20

TIME (Days)

(D) Artifical Seawater - Regenerating

0 4 8 12 16 20

TIME (Days)

Figure 2. Changes in dry weights by body parts during early disc regeneration. (A) Natural seawater
control group. (B) Artificial seawater control group. (C) Natural seawater regenerating group. (D) Artificial
seawater regenerating group. Error bars represent 95% confidence intervals. Error bars and points offset
slightly for graphical clarity.

tration over the course of the experiment (P < 0.001 in
both). The protein concentration increased at the same
rate in both the NR and AR groups until day eight, after
which the NR group continued to gradually increase while
the AR group began to decline. By the end of 20 days,
the protein concentration in the AR group was the same
as its initial (day zero) protein concentration.

The change in protein concentration over time by
treatment group and body part is shown in Figure 6. There
was no change in protein concentration in any body part
in the NC group over the course of the experiment (P
> 0.05) (Fig. 6 A). The AC group lost protein in significant
amounts from the disc (P = 0.0012), distal arm fractions
(P < 0.0001 ), and oral frame (P < 0.0001 ). The protein

concentration of the medial and proximal arm fractions
did not change (P = 0.0675, P = 0.7822. respectively)
(Fig. 6B). There was no change in the protein concentra-
tion of any arm fractions in the NR group (P > 0.05), but
the oral frame lost significant amounts of protein relative
to its dry weight (P = 0.0003), while the disc rapidly in-
creased in protein concentration (P < 0.0001) (Fig. 6C).
The AR treatment group lost protein from all non-regen-
erating body parts (P < 0.05) (Fig. 6D). The protein con-
centration of the disc in the AR group increased rapidly
until day 12 (P < 0.0001), then fell off rapidly through
day 20 (P < 0.0001). The rate of increase to day 12 was
the same as in the NR treatment (P > 0.05). The rate of
protein loss from the oral frame was slower in the AR

NUTRIENT TRANSLOCATION DURING OPHIUROID DISC REGENERATION

173

0.017

(A) Natural Seawater - Control

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Figure 3. Changes in ash-free dry weights by body parts during early disc regeneration. (A) Natural
seawater control group. (B) Artificial seawater control group. (C) Natural seawater regenerating group. (D)
Artificial seawater regenerating group. Error bars represent 95% confidence intervals. Error bars and points
offset slightly for graphical clarity.

and NR groups than in the AC group (P < 0.0001), but
loss occurred throughout the experiment, whereas the AC
group stopped losing protein from the oral frame at about
day 8. The AR and NR treatment groups lost protein
from the oral frame at the same rate throughout the ex-
periment (P = 0.0931). The AR treatment lost protein
from the distal arms at a higher rate than did the AC
treatment group (P < 0.0001 ).

Carbohydrate content changes. The results of the total
body carbohydrate assays are graphed by day in Figure
5B. The NC and NR groups did not exhibit any significant
change in total carbohydrate concentration with time (P
= 0.0877, P = 0.4784). The AC and AR groups did exhibit
changes in total carbohydrate concentration (P = 0.0063,

P < 0.0001 ) over the course of the experiment. There was
no difference in the rate of loss between the AC and AR
groups (P = 0.5675).

The changes in carbohydrate concentration of the var-
ious body parts with time in the different treatments is
graphed in Figure 7. The NC, AC, and NR groups lost
significant amounts of carbohydrates only from the oral
frame (P = 0.01 73, P = 0.0002. P = 0.0075, respectively).
Although there were fluctuations in the carbohydrate
concentration of the other non-regenerating body parts
in each of these groups, they did not represent significant
changes in concentration with time (Fig. 7 A, B, C). The
AR group lost significant amounts of carbohydrates from
the distal and medial arm parts and the oral frame (P

174

W. E. DOBSON ET AL.
(A) Natural Seawaler - Conlrol (Bl Artificial Seawaler - Conlrol

0.016-

0.014-

0012

I °°'

£ 0.008

uuio-
0.014-

0.012
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4 8 12 16 2

4 8 12 16 2(

TIME (Days) TIME (Days)

0.016

(D) Artiftcal Seawaler- Regenerating

8 12

TIME (Days)

Figure 4. Changes in ash weights by body parts during early disc regeneration. (A) Natural seawater
control group. (B) Artificial seawater control group. (C) Natural seawater regenerating group. (D) Artificial
seawater regenerating group. Error bars represent 95"i confidence intervals. Error bars and points offset
slightly tor graphical clarity.

< 0.05). The proximal arm parts did not exhibit a signif-
icant change, although there appeared to be a gradual
decline in carbohydrate concentration (P = 0.0623, Fig.
7D). Both the NR and AR groups exhibited a rapid in-
crease in the carbohydrate content of the regenerating disc,
with rate of increase being the same in both groups through
day 12. After day 12, the disc continued to increase in
carbohydrate concentration in the NR group, while the
carbohydrate concentration in the disc tissue of the AR
group began to decline. The rate of decline in oral frame
carbohydrate concentration was identical across all treat-
ments (P > 0.05) until day 20, when the NC treatment
was different from the AC, NR, and AR treatments, which
were still the same (P < 0.05).

Lipid content changes. The changes in total body lipid
concentration over time are shown in Figure 5C. There
were no significant changes in the total lipid concentration
within any of the treatments with time (P > 0.05). Between
treatments, the NC and AC treatments were identical on
all days. In addition, the NR and AR treatments were
identical through day 12. The NR treatment was different
from the AR treatment and the same as the NC and AC
treatments at day 16. All treatments had the same lipid
concentrations by day 20.

The changes in lipid concentration by day and body
part are illustrated in Figure 8. There was no significant
change in lipid concentration in any non-regenerating
body part in the NC, AC, and NR groups (P > 0.05) (Fig.

NUTRIENT TRANSLOCATION DURING OPHIUROID DISC REGENERATION

175

(A) TOTAL BODY PROTEIN
(All Treatments)

o
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60-

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30
20
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(B) TOTAL BODY CARBOHYDRATE
(All Treatments)

40-

1(1

4 8 12 16 20

TIME (Days)

(All Treatments)

4=44

8 12

TIME (Days)

4 8 12

TIME (Days)

(D) TOTAL BODY CALORIC CONTENT
(All Treatments - Averages)

Figure 5. Total body biochemical concentration changes during early disc regeneration. (A) Total body
protein by treatment group. (B) Total body carbohydrate by treatment group. (C) Total body lipid by
treatment group. (D) Total body caloric content (calculated) by treatment group. Error bars represent 95%
confidence intervals. Error bars and points offset slightly for graphical clanty.

8A, B, C). Although the AC and NR groups showed a
constant decline in lipid concentration in all body parts
except the regenerating disc of the NR treatment, the
overall changes were not statistically significant. The AR
group showed a significant decrease in lipid concentration
in the medial arm fraction (P = 0.0235) as well as the
same non-significant concentration decline in all other
non-regenerating body parts shown by the AC and NR
groups. The NR and AR groups exhibited rapid increases
in lipid concentration in the disc tissue fragment, which
were the same through day 16 (P> 0.05). The NR group
had a higher lipid concentration in the disc fraction by
day 20 (P = 0.2430).

Caloric content changes. Caloric values presented
here were calculated from the biochemical data using
caloric-conversion values (protein, 5.65 kcal/g; carbo-
hydrate, 4.10 kcal/g; lipid, 9.45 kcal/g;) (Brody, 1964;
Ekert and Randall, 1978). Although the current trend
in physiological research is to use the SI unit of energy
(joules), we determined energy content as calories and
present the data here in calories for ease of comparison
with previous literature. However, one calorie equals
4.184 joules (Crisp, 1984), so direct conversion between
units is relatively simple. Although there is potential
for error in using calculated values instead of real values
for caloric content (Giese, 1966; Cummins and Wuy-

176

W. E. DOBSON £T .•)!

(A) Natural Seawater - Control

(B) Artificial Seawaler • Control

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0 4 8 12 16 20

0 4 8 12 16 2

TIME (Days)

(D) Artificial Seawater - Regenerating

180-

160

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Figure 6. Protein content changes by body part during early disc regeneration. (A) Natural seawater
control group. (B) Artificial seawater control group. (C) Natural seawater regenerating group. (D) Artificial
seawater regenerating group. Error bars represent 95% confidence intervals. Error bars and points offset
slightly for graphical clarity.

check, 1971; Feral, 1985). the calculated caloric values
are probably closer to the "true" values than the actual
calorimetry data due to procedural errors in obtaining
the micro-bomb calorimetry data and the resulting wide
variations in the actual caloric values. The total cal-
culated caloric content of the body in the different
treatments is illustrated in Figure 5D. With the excep-
tion of day four in the AC group (which was only dif-
ferent from day 20), there were no statistically signifi-
cant differences in caloric content with time in either
of the NCand AC groups (P> 0.05). The caloric content
of the NR and AR groups declined constantly (P
< 0.0001), with the AR group losing caloric content
faster than the NR group (P < 0.0001).

The caloric content changes with time by body part are
diagrammed in Figure 9. The natural seawater control
group lost calories only in the disc fraction (P = 0.0310)
(Fig. 9A). All other body parts maintained their caloric
levels (P > 0.05). The artificial seawater control group
lost calories only from the disc and oral frame (P = 0.0045,
P = 0.0050). not the arm fractions (P > 0.05) (Fig. 9B).
The NR treatment group lost calories from the oral frame
and distal arm fractions (P = 0.001 7, P = 0.0064) but not
the medial and proximal arm fractions (P > 0.05) (Fig.
9C). The AR group lost calories from every non-regen-
erating body part (P < 0.0001) (Fig. 9D). The NR and
AR groups both increased the caloric content of their disc
tissue until day 16. By day 20. the AR group had begun

NUTRIENT TRANSLOCATION DURING OPHIUROID DISC REGENERATION

177

(A) Natural Seawaler - Control

(B) Artificial Seawaler - Control

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0 4 8 12 16 20

TIME (Days)

(D) Artificial Seawater - Regenerating

4 8 12 14 20

TIME (Days)

4 8

TIME (Days)

Figure 7. Carbohydrate content changes by body part during early disc regeneration. (A) Natural seawater
control group. (B) Artificial seawater control group. (C) Natural seawater regenerating group. (D) Artificial
seawater regenerating group. Error bars represent 95% confidence intervals. Error bars and points oftset
slightly for graphical clarity.

to lose calories from the disc tissue, whereas the NR group
continued to add calories to the disc tissue. The rate of
increase in caloric content was the same in the NR and
AR groups through day 12.

Rate of nutrient translocation

All brittlestars took up statistically significant amounts
of l4C-leucine and '4C-glucose during the pulse portion
of the experiment (Fig. 10, 1 1). Counts of the individual
body parts indicated that all body parts absorbed label in
approximately the same quantities per gram of dry body
weight (P = 0.5740). However, the animals only accu-
mulated significant amounts of '4C-palmitic acid in the

disc region of the body. This result was somewhat unex-
pected, because other echinoderms are known to take up
lipids, especially exogenous palmitic acid, from their en-
vironment across their dermal surfaces (Beijnink and
Voogt, 1984).

During the post-absorption portion of the experiment,
l4C-leucine and l4C-glucose label counts decreased rapidly
and in approximately linear fashion in all the experimental
treatments (Fig. 10, 11). 14C-palmitic acid concentration
changes were not followed because the animals failed to
take up the material in non-regenerating body parts.
Counts of the individual body parts indicated that 14C-
leucine and l4C-glucose labels were lost in approximately
the same proportions from all non-regenerating fractions

178

W. E. DOBSON ET 11.

(A) Natural Seawaler - Control

(B) Artificial Seawaler - Control

I
t

90-
80

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50-
40-

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D DISTAL • DISC O PROXIMAI
• ORAL A MEDIAL

0 4 8 12 16 20

TIME (Days)

4 8 12 16 20

TIME (Days)

(D) Artificial Seawater - Regenerating

4 8 12

TIME (Days)

4 8 12

TIME (Days)

Figure 8. Lipid content changes by body part during early disc regeneration. (A) Natural seawater control
group. (B) Artificial seawater control group. (C) Natural seawater regenerating group. (D) Artificial seawater
regenerating group. Error bars represent 95% confidence intervals. Error bars and points offset slightly for
graphical clarity.

of the body, including the disc of intact specimens. How-
ever, little of the label lost from the non-regenerating tis-
sues of regenerating animals was incorporated into the
regenerating disc tissue. Although counts of the regener-
ating disc tissue showed that some radiolabel was incor-
porated into the disc tissue, the levels were not significantly
different from background counts throughout the course
of the experiment (P > 0.05).

Discussion

The experimental treatments used to study the
amount of nutrients translocated during disc regener-
ation can be described in terms of nutrient availability.

The NC group represented control animals that were
given access to dissolved organic material (DOM), but
not paniculate food, to determine the effect of mainte-
nance metabolism on the body's biochemical compo-
sition when both stored nutrient catabolism and DOM
uptake were available as energy sources. The AC group
represented control animals that had to rely on stored
nutrients alone to supply energy for maintenance. The
NR group were animals that had to supply energy for
both maintenance metabolism and regeneration, as well
as building materials for regeneration. These animals
had access to both stored nutrients and DOM uptake
sources of nutrients. The AR group represented animals
that had to both maintain metabolism and regenerate

NUTRIENT TRANSLOCATION DURING OPHIUROID DISC REGENERATION

179

(A) Natural Seawater - Control

(B) Anificial Seawarer - Conirol

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TIME (Days)

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0 •

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TIME (Days) TIME (Days!

Figure 9. Tissue caloric content changes by body part during early disc regeneration. (A) Natural seawater
control group. (B) Anificial seawater control group. (C) Natural seawatei regenerating group. (D) Artificial
seawater regenerating group. Error bars represent 95% confidence intervals. Error bars and points offset
slightly for graphical clarity.

in the absence of any external nutrient source (i.e.. only
stored nutrients were available).

Under natural seawater control conditions, animals
survived for at least four weeks (one week of acclimation
plus three weeks of experimentation) with no significant
change in the overall biochemical composition of the
body. The only localized changes in body constituents
occurred in the oral frame region, which lost small
amounts of carbohydrates during the experiment. Al-
though the animals probably lost stored nutrients from
all body parts under these conditions, the losses were below
the limits of detection. The total energy content of the
animal did not change. The only localized caloric content
change occurred in the disc region and could not be at-

tributed to changes in any measured biochemical com-
ponent. This indicates that, although animals deprived of
particulate food may be stressed, they probably are not
starving (i.e., they are obtaining nutrients by direct uptake
from the environment). This result is consistent with pre-
vious studies showing that echinoderms, including brit-
tlestars, can obtain up to 58% of their energetic require-
ments from DOM (Ferguson, 1982a, b; Feral, 1985; Law-
rence, 1987; Clements, 1988).

When deprived of all exogenous food (artificial seawater
treatments), control animals initially lost stored material
at a rapid rate. The material was lost from the disc, oral
frame, and distal arm regions of the body, and was at-
tributable to losses of protein and carbohydrates, but not

180

W. E. DOBSON ET AL

(A) Natural Seawater Experiment

(B) Artificial Seawater Experiment

• AR-D1ST
*• AR-PROX

• AC-DIST
O AC-PROX
D> AC-DISC
O AR-DISC

NR-DIST
NR-PROX
• NC-DIST

n NC-PROX

NC-DISC
O MR-DISC

TIME (Days) TIME (Days)

Figure 10. 14C-Leucine tracer content of tissues during early disc regeneration. (A) Natural seawater
experiment. (B) Artificial seawater experiment. AC = Artificial seawater control, AR = Artificial seawater
regenerating, NC = Natural seawater control, NR = Natural seawater regenerating, DIST = Distal arm,
PROX = proximal arm and oral frame. Error bars represent 95% confidence intervals. Error bars and points
offset slightly for graphical clarity.

lipids. The loss of total caloric content in the various body
parts followed the same pattern, with no significant loss
in any other body parts. After four days, these animals
appeared to acclimate to the lack of food such that the
rate of overall materials loss was reduced; (i.e., they ap-
parently adjusted to food deprivation by reducing their
consumption of stored material). The temporal pattern
of material loss may also represent a rapid initial use of
stored resources followed by a breakdown of essential body
tissues to maintain metabolism. As tissue mass decreased,
the metabolic load due to those tissues decreased, and the
rate of tissue loss declined. Because mass-specific meta-
bolic rates were not obtained during this experiment, these
observations could not be empirically verified.

There are two possible explanations for the spatial pat-
tern of material loss in the artificial seawater control group.
The first is that the disc, oral frame, and arm tips are
preferentially resorbed when the animal is forced to ca-
tabolize tissue for maintenance. Turner and Murdoch

(1976) described such a pattern of arm tissue loss during
regeneration of the disc in Ophiophragmus filograneus.
This mechanism would leave the majority of the arm tis-
sue undisturbed so that normal feeding activity would not
be impaired when feeding conditions improved. The sec-
ond possibility is that the absolute rate of loss is the same
from all body parts, but there is less material in the disc,
oral frame, and arm tips to begin with, so the material
available within them is exhausted sooner than that in
other body parts. The latter possibility is the more likely,
because the medial and proximal arms have the highest
total amounts of all biochemical constituents (Table I).

Animals regenerating in natural seawater showed an
initial decrease in organic mass followed by a gradual in-
crease. This indicates that the use of material during early
regeneration exceeded the rate at which DOM uptake
from the medium could compensate for it, and thus must
have been at least partially independent of external nu-
trient availability. Loss of organic material occurred in

NUTRIENT TRANSLOCATION DURING OPHIUROID DISC REGENERATION

181

(A) Natural Seawater Experiment

(B) Artificial Seawater Experiment

Q i

.1
I

50-

40-

30-

20-

10-

40-
30-

20-
10-

1 NR-DIST

> NR-PROX
• NC-DIST
n NC-PROX

> NC-DISC
O MR-DISC

TIME (Days)

£ ii

Q
-S"

cu
U

TIME (Days)

Figure 11. '4C-Glucose tracer content of tissues during early disc regeneration. (A) Natural seawater
experiment. (B) Artificial seawater experiment. AC = Artificial seawater control, AR = Artificial seawater
regenerating, NC = Natural seawater control. NR = Natural seawater regenerating, DIST = Distal arm,
PROX = proximal arm and oral frame. Error bars represent 95% confidence intervals. Error bars and points
offset slightly for graphical clarity.

all non-regenerating body parts, but the overall trend was
similar to that exhibited by the artificial seawater control
group in that most of the loss was from the oral frame
and arm tips. The subsequent increase in organic material
appeared to be localized in the arms. The relative protein
content of the body increased constantly during regen-
eration, indicating either a net gain of protein during re-
generation, or a loss of minerals as tissue breakdown oc-
curred. This gain could be due to a net uptake of proteins
(or amino acids) from the medium, or a combination of
uptake and overall loss of other body biochemical con-
stituents during regeneration. Although the carbohydrate
and lipid content of the body did not change significantly
over the same period, the latter explanation is more likely,
because the animals lost total caloric content constantly
during regeneration. The increase in organic material in
the non-regenerating portions of the body after day 12 is
problematical, because no corresponding increase in bio-
chemical constituents in those parts could be demon-

strated. This increase might be explained as the summa-
tion of non-significant increases in each biochemical con-
stituent to make a significant increase in total organics.

Animals regenerating in the absence of exogenous nu-
trients constantly lost organic material from non-regen-
erating body parts. The rate of loss was relatively rapid
through day 8, and slower from day 12 through day 20.
This change was related to the constant decrease in pro-
tein, carbohydrate, and lipid content of the non-regen-
erating tissues. Although lipid content loss was statistically
significant only in the medical portions of the arms, all
non-regenerating body parts showed a trend toward lipid
loss. The regenerating disc tissue increased in protein,
carbohydrate, and lipid content through day 12, after
which protein and carbohydrate content dropped dra-
matically, while lipid content remained the same or
slightly increased (the continued proportional lipid in-
crease was probably due to the loss of protein and car-
bohydrates). The caloric content of these animals dropped

182

W. E. DOBSON ET AL

constantly, and consistently faster than that in the exper-
imental group regenerating in natural seawater. All non-
regenerating parts of the body lost calories throughout the
experiment. The caloric content of the disc increased
through day 16, then dropped dramatically.

The data on the consumption of hiochemicals and disc
tissue production in the regeneration experimental groups,
especially the artificial seawater regeneration group, seems
to indicate that the process of regeneration runs at a set
rate, and may be independent of the nutritional state of
the animal (at least for the first two weeks of disc regen-
eration). A similar phenomenon has recently been re-
ported in crinoids under field conditions (Meyer, 1988).
These observations imply that early replacement of initial
disc tissues and structures has priority over the mainte-
nance of body mass. Since these observations coincide
temporally with appearance of the functional gut (Dobson
and Stancyk, in prep), one can conclude that the animal
tries to replace the gut so it can feed again regardless of
its initial nutritional state. Only when resources drop be-
low some critical level (i.e., the actual onset of starvation)
do they stop regenerating the disc. This experiment should
be repeated with animals that have been held without
food sources for varying lengths of time to determine
whether regeneration is even initiated after the critical
point in the food withdrawal period has passed.

Regeneration appears to require a set amount of nu-
trients, which are transported from the deep tissues of all
the non-regenerating body parts. If food is present (as
DOM in this case) the loss of material due to translocation
may be offset by uptake. Further, after the gut lining is
reformed and becomes functional, ingestion of particu-
lates, including small bacteria, may ameliorate the loss of
stored nutrients.

A previous attempt to verify and quantify nutrient
translocation into the disc from somatic body parts during
disc regeneration in Al. gracillima was unsuccessful
(Clements, 1988). That study relied on the assumption
that loss of organic material from the arms would result
in a decrease in total arm size. This assumption was based
on the results of Turner and Murdoch (1976) and the
observation that echinoid test diameter decreases during
starvation (Ebert, 1967). However, a loss of arm tissue
without a reduction in overall arm size has been dem-
onstrated in starving asteroids (Lawrence et al.. 1986).
Thus, the internal soft tissues of asteroid arms are scav-
enged while leaving the calcified structures in place. In-
deed, the arms of asteroids have been implicated as general
nutrient storage organs (Beijnink and Voogt, 1984; Law-
rence, 1987). If the non-regenerating body parts of M.
gracillima are fulfilling a similar role, then translocation
of organic material from the non-regenerating body parts
should occur without an overall decrease in body part size
or inorganic ( = ASH) weight. The calcification of tissues

in marine invertebrates is also a relatively expensive pro-
cess compared to the production of soft tissues, due to
the energetics of mineralization and the cost of producing
the skeletal matrix (Simkiss, 1976; Palmer, 1983; Law-
rence, 1987). Consequently, we would expect the calcified
structures of M. gracillima to be conserved even as its
soft tissues are degraded to supply catabolic and regen-
erative nutrients. Because the entire external surface of
M. gracillima is covered with plate ossicles and spines,
the shape and size of body parts would not change much
as the soft tissues are degraded inside the structures. The
absence of change in the ash weight of all the body parts
of all animals in the current study supports this hypothesis.

Abnormal regeneration and death of specimens in the
nutrient-enriched experimental groups is perplexing, but
has been verified by repeated experimentation (Clements.
1988; K. Fielman, pers. comm.). Because preliminary ex-
periments indicated that these conditions promoted bac-
terial growth (Clements, 1988), we took care to inhibit
such growth by completely changing the medium each
day. Several researchers have proposed that echinoderm
regeneration requires the presence of functional nerve fi-
bers that produce recognition and regulatory molecules
(Bisgrove et al.. 1988; P. Mladenov, pers. comm.). Ab-
normally high ambient concentrations of nutrients (es-
pecially amino acids, which can act as neurotransmitters)
may have directly affected the regeneration process by
interfering with the actions of these recognition molecules.

Uptake of 14C-leucine and l4C-glucose indicated that
dissolved organic material is taken up in statistically sig-
nificant amounts in all treatments. The results agree
closely with those of Clements (1988) for net uptake of
the amino acids leucine and glycine by M. gracillima.
However, her study showed significant retention of the
labeled compound over time. The current results indicate
that the initially retained labeled molecules are rapidly
turned over or leaked back into the medium, with little
permanent incorporation of the labeled molecules into
the tissues and no translocation of the labeled material to
the active regeneration site. The labeled compounds may
have been transported in quantities below the detected
threshold of the assay method. We do not know whether
the loss of label from non-regenerating tissues is due to
leakage or respiration, because the experiment was not
designed to test for respired I4CO2 or for an increase in
the label content of the medium with time. We would
understand this process better if the '4C-leucine, '4C-glu-
cose, and I4CO: evolved in the medium during the post-
absorption portion of the experiment had been assayed
to determine what fraction of the material taken up by
the animals was catabolized or leaked out.

The absence of detectable translocation of radiolabeled
material into regenerating tissue indicates that, if the la-
beled material is not simply leaking out of the body [which

NUTRIENT TRANSLOCATION DURING OPHIUROID DISC REGENERATION

183

is not expected to be the case based on the results obtained
by Clements (1988)], then the material may have been
absorbed only into the surface tissue layers of the body,
and not subsequently transported into the deeper tissues.
Several investigators have proposed such DOM absorption
as a mechanism by which echinoderms, which have poor
circulatory systems, maintain their external tissues (Fer-
guson, 1982b; Bamford, 1982). In these models, DOM
feeds the external tissues, but is not transported into the
deep tissues, whereas material ingested and digested is not
transported to the surface layers but supplies nutrients
only to the internal tissues. Because the current results,
and the results of previous work on regeneration (Dobson
and Stancyk, in prep), indicate that nutrients are trans-
located from the deep tissues of the non-regenerating body
parts — probably by coelomocytes of the water-vascular
system — the lack of label in the regenerating disc may be
ascribed to its inability to migrate into the deep tissues
and thus to be available for regeneration.

Disc autotomy is probably a predator avoidance mech-
anism (Turner et a/., 1981). Because the disc (or at least
the gut) is needed for feeding, some mechanism should
be available to replace it after escape-response disc au-
totomy, irrespective of the nutritional state of the animal.
Such an effect has been demonstrated in this and a pre-
vious set of experiments (Dobson, Stancyk. and Clements,
in prep). In addition, because M. gracillima is a seasonal
spawner (pers. obs.), selection for rapid replacement of
the disc structures to facilitate replacement of gonads and
gametes would be expected. Because these animals lose
up to one-fourth of their available body organic mass dur-
ing early regeneration, a massive amount of body reserves
must enter the process. However, a significant amount of
the reserves must be used for maintenance metabolism.
This study shows that, although these animals do have
some energy storage resources (because the starving and
regenerating animals still produce disc tissue), there is still
no specific nutrient storage organ or tissue. Without ad-
ditional exogenous nutrient input, these stores are depleted
within about two weeks, a sufficient time for replacement
of the gut and initiation of feeding, even when paniculate
and dissolved exogenous organic material is absent.

Acknowledgments

This work would not have been possible without the
facilities of the Biology Department of U.S.C. and the
Belle W. Baruch Institute. This work was supported in
part by a Grant from the Slocum-Lunz Foundation of
South Carolina to W. E. Dobson, and in part from a Na-
tional Institute of Health Biomedical Research Support
Grant (grant #BSR-85 14326) to S. E. Stancyk, W. E.
Dobson, R. M. Showman, and L. A. Clements. Field as-
sistance was provided by K. Zimmerman and K. Fielman.
Special thanks to L. F. Dobson for gestalt support.

Literature Cited

Bamford, D. 1982. Epithelial absorption. Pp. 317-330 in Echinoderm
\ntrinoH. M. Jangoux and J. M. Lawrence, eds. A. A. Balkema.
Rotterdam.

Barnes, H., and J. Blackstock. 1973. Estimation of lipids in marine
animals and tissues: detailed investigation of the sulphophospho-
vamllin method for "total" lipids. J. E\p. Mar. Biol. Ecol. 12: 103-
118.

Beijnink, F. B., and P. A. Voogt. 1984. Nutrient translocation in the
sea star: whole-body and microautoradiography after ingestion of
radiolabeled leucine and palmitic acid. Biol. Bull. 167: 669-682.

Bisgrove, B. W., P. V. Mladenov, S. K. Asotra, and R. D. Burke.
1988. Wound healing and regeneration following arm-tip ampu-
tation in the sea star Lefiiaxieruix hcxactis. P. 788 in Echinoderm
Biology. Proceedings ol the Sixth International Echinoderm Confer-
ence. R. D. Burke. P. V Mladenov, P. Lambert, and R. L. Parsley,
eds. Victoria. 23-28 August.

Bradford, M. M. 1976. A rapid and sensitive method for the quanti-
fication of protein utilizing the principle of protein dye-binding. Anal
Biochem 72: 248-254.

Brody, S. 1964. Bioenergetics and Growth. Hafner. New York. 1023
pp.

Brown, B. K. 1982. Gut replacement during disc regeneration of the
autotomized disc of Ophiopliruginux filograneus (Echmodermata:
Ophiuroidea). Unpublished master's thesis, Florida Institute of
Technology. Melbourne. 90 pp.

Cavanaugh.G. M. 1965. Formulae and Methods I', of the Marine Bio-
logical Laboratory Chemical Room Mar. Biol. Lab., Woods Hole.
Mass. 87 pp.

Clements, L. A. J. 1988. Uptake and utilization of dissolved free amino
acids by the brittlestar Microphiopholis gracillima (Say, 1852) (Echi-
nodermata: Ophiuroidea). Unpublished Ph.D. dissertation. University
of South Carolina. 1 16 pp.

Clements, L. A. J., Fielman, K. T., and S. E. Stancyk.
1988. Regeneration by an amphiund bnttlestar exposed to different
concentrations of dissolved organic material. J Exp. Mar Biol. Ecol.
122: 47-61.

Crisp, D. J. 1984. Energy flow measurements. Pp. 284-372 in Methods
for the Stiuly of Marine Benthos. N. A. Holme and A. D. Mclntyre.
eds. Blackwell Scientific Publications, Oxford.

Cummins. K. \V., and Wuycheck, J. C. 1971. Caloric equivalents for
investigations in ecological energetics. Int. Ass. Theor. App. Limnol.
Vol 18. 162 pp.

Davis, E. M. 1988. Protein assays: a review of common techniques.
Am. Biotech Lab 5: 28-37.

Dobson, \V. E. 1984. Nervous mediation of disc autotomy in Ophio-
phragmus filograneus (Echinodermata: Ophiuroidea). M.S. Thesis.
Florida Institute of Technology, Melbourne. 68 pp.

Dobson, W. E. 1985. A pharmacological study of neural mediation of
disc autotomy in Ophiophragmus filograneus (Lyman) (Echino-
dermata: Ophiuroidea). / Exp. Mar Biol. Ecol. 94: 223-232.

Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith.
1956. Colonmetnc method for determination of sugars and related
substances. Anal Chcm. 28: 350-356.

Duineveld, G. C. A., and G. J. Van Noort. 1986. Observations on the
population dynamics of Amphnira ftliformis (Ophiuroidea: Echi-
nodermata) in the southern North Sea and its exploitation by the
Dab. Limanda limanda. Neth. J Sea Res. 20: 85-94.
Ebert, T. A. 1967. Negative growth and longevity in the purple sea
urchin Slrongylocenlrolus piirpuratux (Stimpson). Science 157: 557-
558.

Ekert, R.. and D. Randall. 1978. Animal Physiology. Freeman, San
Francisco. 558 pp.

184

W. E. DOBSON ET .41.

Emson, R. H., and I. C. Wilkie. 1980. Fission and autotomy in echi-
noderms. Oceiuwgr. Mar. Bid. Aim Review 18: 155-250.

Feral, J.-P. 1985. Effect of short-term starvation on the biochemical
composition of the apodous holothurian Lenlosynapla gallicnnei
(Echinodermata): possible role of dissolved organic material as an
energy source. Mar. BH//. 86: 297-306.

Ferguson, J. C. 1982a. A comparative study of the net metabolic ben-
efits derived from the uptake and release of free amino acids by marine
invertebrates. Biol. Bull. 162: 1-17.

Ferguson, J. C. 1982b. Support of metabolism of superficial structures
through direct uptake of dissolved primary amines in echinoderms.
Pp. 345-351 in Echinoderm Biology: Proceedings of the International
Echinoderm Conference, J. M. Lawrence, ed. Tampa Bay, 14-17
September.

Gibson, A. W., and R. D. Burke. 1983. Gut regeneration by mor-
phallaxis in the sea cucumber Leptosynapla clarki (Heding. 1928).
Can.J. Zoo/. 61:2720-2732.

Giese, A. C. 1966. On the biochemical constitution of some echino-
derms. Pp. 757-796 in Physiology oj Echinodermata, R. A. Booloo-
tian, ed. John Wiley, New York.

Lawrence, J. 1987. A Functional Biology ol Echinoderms. Johns Hop-
kins University Press, Baltimore. 340 pp.

Lawrence, J., T. S. Klinger, J. B. McClintock, S. A. Watts, C. P. Chen,
A. March, and L. Smith. 1986. Allocation of nutrient resources to
body components by regenerating Lindia clalhrala (Say) (Echino-
dermata: Asteroidea). J E.\p. Mar. Biol. Ecol. 102: 47-53.

Myer, D. L. 1988. Crinoids as renewable resources: rapid regeneration
of the visceral mass in a tropical reef-dwelling crinoid from Australia.

Pp. 519-522 in Echinoderm Biology: Proceedings of the Sixth Inter-
national Eclunoderm Conference, R. D. Burke. P. V. Mladenov, P.
Lambert, and R. L. Parsley, eds. Victoria, 23-28 August. 818 pp.

O'Conner, B., T. Bowmer, D. McGrath, and R. Raine. 1986. Energy
flow through an Amphmra /i/i/ormis (Ophiuroidea: Echinodermata)
population in Galway Bay, west coast of Ireland: a preliminary in-
vestigation. Ophelia 26: 351-357.

Ostle, B. and R. \V. Mensing. 1975. Statistics in Research, 3rd ed.
Iowa State University Press. 596 pp.

Palmer, A. R. 1983. Relative cost of producing skeletal organic matrix
versus calcification: evidence from marine gastropods. Mar Biol. 75:
287-292.

Simkiss, K. 1976. Cellular aspects of calcification. Pp 1-32 in The
Mechanisms ol Mineralization in the Invertebrates and Plants, N.
Watabe and K. M. Wilbur, eds. Univ. of South Carolina Press.

Sokal, R. R., and F. J. Rohlf. 1981. Biometry Freeman. New York.
859 pp.

Turner, R. I,., and J.D. Murdoch. 1976. Potential of arms as a nutrient
source for disc regeneration in bnttlestars. Am. Zoo/. 16: 288 (Ab-
stract).

Turner, R. L., D. VV. Heatwole, and S. E. Stancyk. 1981. Ophiuroid
discs in stingray stomachs: evasive autotomy or partial consumption
of prey? Pp. 331-335 in Echinoderm Biology: Proceedings of the
International Echinoderm Conference, J. M. Lawrence, ed. Tampa
Bay, 14-17 September.

Williams, P. J. LeB. 1975. Biological and chemical aspects of dissolved
organic material in seawater. Pp. 301-363 in Chemical Oceanography,
Vol. 2, Rilev, J. P. and G. Skirrow, eds. Academic Press, London.

Reference: Biol. Bull 180: 185-195. (February,

Calcium-Proton Exchange During Algal Calcification

TED A. McCONNAUGHEY1* AND RICHARD H. FALK2

1 Marine Biological Laboratory, Woods Hole, Massachusetts 02543, and 2 Botany Department,
University of California, Davis, California 95616

Abstract. Extracellular calcification by the giant celled
alga Chara coral Una may involve active Ca2^ extrusion
from the cell in exchange for protons. The following ev-
idence is presented: CaCO, incrustations accrete largely
along the inside, facing the cell, as revealed by X-ray mi-
croanalysis using Sr:+ and Mn2+ as tracers for new min-
eralization. Inward proton currents are inhibited by the
Ca:+ transport antagonists Gdu and La34 . Low Ca2+ con-
centrations inhibit pH banding and photosynthesis, and
solutions of low Ca:+ activity support more photosynthesis
in the presence of additional buffered calcium. The ratio
of calcification to photosynthesis in moderately alkaline
solutions containing sufficient calcium remains stable at
about 1.0 independent of solution Ca24 concentration.
Ion specific microelectrodes placed close to the calcified
surface sometimes detect increases in Ca:+ activity coin-
cident with decreases in proton activity. As the pCa of
solution increases, the maximum pH observed at the al-
kaline surface increases, as does the maximum solution
pH which supports electrochemical currents by the cell.
Combinations of extracellular pH and pCa approach the
calculated thermodynamic limits for ATP driven 2H+/
Ca2+ exchange against the cytosol.

Introduction

Ca2+ ATPase appears to be associated with calcification
in various animals and plants (e.g., Klaveness, 1976;
Okazaki, 1 97 7; Okazaki eM/., 1984;Kingsley and Watabe,
1 985). This report explores the possibility that extracellular

Received 3 April 1990; accepted 6 November 1990.

*Address communications to: Dr. Ted McConnaughey. U.S. Geolog-
ical Survey, Box 25046 MS 413, Lakewood, CO 80225.

Abbreviations: CAPS = 3-(cyclohexylamino)propanesulfbnate; CHES

= 2-(N-cyclohexylamino)ethanesulfonate; MOPS = 2-(N-morpho-

linolpropanesulfonate; PIPES = l,4-piperazinediethane-sulfonate;TAPS

tris(hydroxymethyl)methylaminopropane sult'onate; TRIS

= tris(hydroxymethyl)aminomethane.

calcification in characean algae involves active calcium-
proton exchange.

Characeans calcify as a by-product of bicarbonate as-
similation from alkaline waters (Spear et al., 1969; Raven
etal.. 1986; Okazaki and Tokita, 1988). The plants extract
proton equivalents from the medium along parts of their
giant cells, forming alkaline patches or bands that may
become heavily calcified. The proton equivalents are ex-
truded elsewhere, forming acidic patches or bands. There,
HCOr is apparently protonated to form CO:, which the
plant absorbs (Walker el al., 1980; Smith and Walker,
1980; Price and Badger, 198?). Pericellular carbonic an-
hydrase and complicated invaginations of the plasma
membrane within the acid zones may facilitate CO2 gen-
eration and absorbtion (Price el al., 1985).

Characeans can be more than half CaCO3 by dry weight,
and as will be shown here, calcification is often stoichio-
metric to photosynthesis. Nevertheless, calcification
physiology has been largely neglected, and calcification is
generally assumed to be independent of active Ca2+ trans-
port (e.g., Raven et al.. 1986). Various evidence neverthe-
less suggests that active Ca2+ transport might be involved.
First, Ca2+ ATPases apparently catalyze Ca2+ extrusion
from cells in exchange for protons (Niggli et al.. 1982;
Villalobo and Roufogalis, 1986; Rasi-Caldogno et al.,
1987; Dixon and Haynes, 1989). Ca2+ ATPase could
therefore catalyze proton uptake at the site of calcification
in Chara. Second, characeans are functionally analogous
to coccolithophorid algae, which also calcify in an ap-
proximate ratio of 1 : 1 to photosynthesis, but do so intra-
cellularly (e.g.. Sikes et al.. 1980). Ca2+ and carbon pre-
sumably traverse the cytoplasm to reach the vesicular site
of calcification, and Ca2+ ATPase seems to be involved
(Klaveness, 1976; Okazaki et al., 1984). Third, molecular
CO2 apparently provides most of the precipitating carbon
during calcification by various plants and animals
(McConnaughey, I989a, b, c), including Chara (Mc-

185

186

T. A. McCONNAUGHEY AND R. H. FALK

Connaughey, in prep). Since HCO3 is more abundant
in alkaline solutions, its unimportance in calcification
suggests that the calcifying region can be fairly isolated
from solution. The calcifying cell must therefore supply
calcium, and remove the protons generated by the reaction
Ca2+ + CO2 + H2O = CaCO3 + 2H+. And finally, Ca2+
is well known to affect characean photosynthesis and
membrane properties associated with pH banding (e.g.,
Lucas, 1976; Wiesenseel and Ruppert, 1977; Luhring and
Tazawa, 1985; Bisson, 1984; Tazawa el a/.. 1987).

A Ca:+ ATPase model and a more conventional proton
channel model for characean calcification are illustrated
in Figure 1 . Both models are elaborated to fit the available
data. Ca2+ influx into the cell, in the Ca:+ ATPase model,
occurs within the alkaline band (Fig. Ib) to produce the
observed electrogenic character of pH banding (Walker
and Smith, 1977). Figure Ic, e shows the use of molecular
COi from the plant as the major carbon source for cal-
cification (McConnaughey, in prep.) and the accretion of
extracellular calcium deposits from the inside (demon-
strated here). Figures Id and Ifdepict non-calcifying con-
ditions, such as when Ca2+ or carbon levels are too low
to sustain much CaCO^ precipitation. The non-calcifying
condition can be experimentally useful, because the H+

fluxes measured extracellularly then reflect cellular H +
transport most closely.

The energy (E) required for proton uptake under both
models is given by:

E = FV(aZCa - bZH) + RT In (Ca,/Cat,)a/(H,/H0)b ( 1 )

The terms on the right represent work done against the
membrane electrical potential V, and against the mem-
brane chemical gradients. F is the Faraday constant, "a"
and "b" are the numbers of Ca2i and H+ ions transported
per cycle, Z is ionic charge. R is the gas constant, and T
is Kelvin temperature. Cytoplasmic and external Ca2+ and
H+ activities are subscripted "i" and "o," respectively.

For the proton channel model, protons are drawn into
the cell by the membrane electrical potential. The ther-
modynamic limit for passive (E = 0) proton uptake occurs
when the membrane electrical and chemical gradient
energies balance, yielding a proton Nernst equation:

0 = FV + 2.3 RT(pH0 - pH,) (2)

For an illuminated cell in alkaline solution, the membrane
potential might be around —200 mV and cytoplasmic
pH, might be about 7.5-8.0 (Smith and Raven, 1979;
Spanswick and Miller, 1977; Mimura and Kirino, 1984;

Ca"

Alkaline
Band

CaCOj

H Channel

CALCIFYING NON-CALCIFYING

• Ca"

H* Channel

Ca ATPase

2 OH

Figure 1. Models of extracellular calcification and its coupling to bicarbonate utilization in Chora. Left:
schematic of a cell, showing alkaline band at top (with trapezoidal CaCO3 incrustations), and acid band
below (with plasmalemmasomes, participating in bicarbonate use). "P" represents photosynthesis, (a) Proton
channel model. HCO3~ diffuses to the alkaline surface and donates a proton, becoming converted to CO3",
which precipitates with Ca2+. (b) Ca2+ ATPase model. ATP driven 2H+/Ca2+ exchange alkalimzes the external
medium and locally increases its Ca2+ concentration. CO2 diffuses from the cell and reacts with water to
yield the protons needed for exchange with Ca;+, and the CO5" which precipitates as CaCOj. A 1:1 ration
of calcification to photosynthesis is shown for both models. Right: elaborations on the H* channel and Ca2+
ATPase models for the alkaline band, incorporating inward accretion ofCaCO, incrustations, using CO2 as
the carbon source. Proton channel (e, d) and Ca:+ ATPase models (e, I) showing the alkaline band under
calcifying (c. e) and non-calcifying conditions (d. f). The ion fluxes detectable externally are highlighted.

ALGAL CALCIFICATION

187

Smith. 1984a, b). The maximum pH in the alkaline band
would then be about 10.9-1 1.4, independent of solution
Ca2+ activity.

For ATP-driven 2H+/Ca:+ exchange, the energy of ATP
hydrolysis (E) is about -50 to -55 KJ/mol (e.g.. Hashi-
moto el a/.. 1984). The electrical term in eq. (1) drops
out, leaving

E/2.3RT =

- pCa,) - 2(pH0 - pH,) (3)

Cytoplasmic pCa( = -log{Ca2+,} is about 6.9 (Miller and
Sanders, 1987). Equation 3 describes a line in (pH(l, pCa,^
space having a slope of 1/2. and displaced from the com-
position ofthecytosol, (pH,, pCa,), by 4.4 to 4. 8 pH units.
The present experiments look for evidence that CaCO3
incrustations accrete from the inside, as would be expected
if the plant supplies the precipitating calcium and carbon.
Proton and calcium specific microelectrodes search for
regions of elevated Ca2+ and depressed FT activities along
the calcifying surface. The combinations of calcium and
proton activities are compared with the thermodynamic
constraints of ATP driven 2H+/Ca2+ exchange against the
cytosol. Calcium transport antagonists are used to inhibit
proton uptake. And the stoichiometry of calcification to
photosynthesis is examined to see if calcium merely dif-
fuses to the calcification site. In the end, the Ca2' ATPase
model offers some advantages, but presents some inter-
esting difficulties. The discussion touches on how the plant
uses calcification as a photosynthetic adaptation.

Materials and Methods

The present experiments used male plants of Cham
corallina from South Australia, provided by Bill Lucas.
Plants were maintained in the laboratory, in aquaria ini-
tially containing "CPW/B" solution (in mA/, CaCl2 0.2,
NaHCO, 1, NaCl 1, KG 0.2) overlying 5-20 cm mud.
Nutrients and additional calcium and carbon were some-
times added to stimulate growth and calcification. Cool
white fluorescent lights provided illumination.

Regions of new mineralization were identified by X-
ray microanalysis. Plants first accumulated CaCO, in a
medium containing (in mA/) CaCl2 2, NaHCO3 2, CaSO4
0.2, KC1 0.2, and NaCl 1, and were then transferred to
media containing additional SrCli 1, and MnSO4 0.1. to
label regions of new mineralization. Cells showing heavy
calcification and good cytoplasmic streaming were rapidly
frozen in liquid nitrogen slush, fractured, and given a thin
coating of aluminum by vacuum evaporation (Emscope
SP2000) at -196°C, to increase surface conductivity.
Frozen hydrated specimens were transferred under vac-
uum to the cryostage of a scanning electron microscope
(Hitachi S800) equipped with a solid state X-ray spec-
trometer (Kevex 8000 series). Secondary electron mode
images provided details of surface morphology. X-ray

maps, line scans, and area scans made with an accelerat-
ing voltage of 15 KeV revealed distributions of Ca, Sr,
and Mn.

Rates of calcification and photosynthesis were estimated
from changes in the alkalinity and total dissolved inor-
ganic carbon content of solution. Alkalinity was measured
by acidometric titration using the Gran method (Stumm
and Morgan, 1970). Heavily calcified plants were incu-
bated in stoppered flasks at 25°C under a mixture of flu-
orescent and incandescent lights for 6-8 h. Solutions ini-
tially contained (in m/l//l) NaHCO, 1, NaCl 1, KC1 .2.
and 0-50 mA/ CaCl,, pH 8 to 8.2, adjusted with NaOH.
Calcification was calculated as half the change in alkalin-
ity, and photosynthesis was calculated as the change in
total carbon minus calcification.

In experiments designed to see whether buffered Ca2+
stimulated photosynthesis in solutions of low Ca2+ activ-
ity, photosynthesis was monitored using an oxygen elec-
trode (Orion 97-08). Wide mouth jars (500 ml) containing
about 5 g of algae were filled with solutions prepared from
partially degassed, deionized water, and capped under-
water to exclude air bubbles. Control solutions contained
(in mA/) CaCl: 0.05, KC1 0. 1 , NaCl 1 , NaHCO, 1 .8. and
Na2CO3 0.2. Test solutions contained an additional 0.8
mA/ CaCl: and sodium citrate ( 1 .5 mA/). These solutions
exhibited the same Ca2' activity, using a Ca2+ specific
microelectrode. A relatively high pH (9.1) ensured that
the plants obtained most of their carbon through the
physiology associated with pH banding. Half of the plants
had been mostly decalcified before the experiment by
soaking them for 2 days in a solution containing 10 mA/
MES buffer, initial pH 5.2. Cool white fluorescent lights
provided illumination during 2-3 h incubations at about
25°C.

The effect of calcium transport antagonists on inward
proton currents were investigated by exposing an illu-
minated cell to LaCl3 or GdCl3, while measuring proton
uptake with an extracellular vibrating H' specific micro-
electrode (Kuhtrieber and Jaffe, 1990). The cell was
mounted in an open Petri dish (solution volume about 3
ml) and perfused at a rate of 0.08 ml/s with a solution
containing, in mA/. CaCl: 0.2, KC1 0.2, NaCl 1 .0, TRIS
5, pH adjusted to 8.3 with NaOH. Fiberoptic lights pro-
vided illumination. The proton electrode vibrated per-
pendicularly to the cell over an excursion of 10 ji, at a
frequency of 0.5 Hz, at a distance of about 10 ^m from
the cell. 4 ^Moles of the lanthanide was added to the
input stream without changing flow rate. The signal here
is the voltage difference registered by the electrode as it
moves back and forth near the cell. A proton gradient of
one pH unit within the sampled region ideally yields a
signal of about 58 mV, although in practice the signal is
smaller. Fluxes of proton equivalents carried by H+, OH~,
and protonated TRIS buffer were calculated from Pick's

188

T. A. McCONNAUGHEY AND R. H. FALK.

first law, using diffusion coefficients 93, 53, and 7 X 10 6
cnr/s, respectively, and concentrations calculated from
the measured pH. In the case illustrated, the pH at the
electrode was about 9 before adding the lanthanides. The
voltage field arising from net charge uptake by the alkaline
band introduces only a small bias to the pH signal; relative
to background solution, the alkaline band might show a
voltage differential of about -4 m V, while the pH gradient
of around 2 units produces a voltage signal of about
-120 mV.

Ca2+ and FT activities at the alkaline surface of the cell
were measured using stationary ion specific microelec-
trodes, constructed as described by Borelli et al. (1985).
Electrodes were connected to a high impedance amplifier
(World Precision Instruments FD223), with output to a
chart recorder. Additional potential sensing electrodes
were sometimes used as well. The pericellular electrical
field (about -4 mV relative to background) biased pH
and pCa measurements by about +.07 and 0. 14 pCa unit,
respectively. The cells were exposed to buffered solutions
of various calcium concentrations, usually lacking dis-
solved inorganic carbon to discourage calcification. Fiber
optic lights provided illumination.

In experiments comparing pericellular pH against a
thermodynamic model for 2H+/Ca2+ exchange, the pH
data represent the highest values observed during electrode
scans of the cell surface, and during observations of several
minutes duration at particularly alkaline locations. So-
lution pCa was calculated from solution Ca2+ concentra-
tions and ionic strength, using Davies' individual ion ac-
tivity coefficient (see Stum m and Morgan, 1970), or mea-
sured using an Orion 93-20 electrode for solutions
containing citrate. In experiments comparing simulta-
neous variations in pericellular pH and pCa, H+ and Ca:+
electrodes were placed close together near the alkaline
surface of the cell, and the intensity of pH banding either
fluctuated spontaneously or was modulated by turning
the light off and on. In the examples shown, the medium
contained MOPS (5 mA/) and citrate (2 mM), plus NaOH
and CaCl2 to produce pH 7.98, pCa 3.89.

Extracellular electrical currents were measured using a
vibrating probe electrometer (Jaffe and Nuccitelli, 1974).
The probe vibrated perpendicular to the cell surface, ap-
proximately 30-50 Mm from the cell while the cell moved
by on a motorized stage. Fiberoptic lights provided illu-
mination. Most experiments used nominally carbon-free
solutions containing (in mAf) KC1 0.2, NaCl 1, and a
zwitterionic buffer (MOPS, PIPES, EPPS, CHES, or
CAPS, 5 mM), pH adjusted to the desired value using
NaOH. At the chosen concentration of CaCl2 (0.1-50
mM), the cell was repeatedly scanned along its length for
electrical activity while solutions of progressively higher
pH or Ca2+ concentration were added. The cell was al-
lowed to adjust in each solution for at least 30 min. Elec-

trical currents were calculated from the electrical con-
ductivity of solution, using Ohm's law. The example
shown used a divided chamber, so that opposite halves
of the cell were exposed to different solutions. Cytoplasmic
streaming between the two halves was uninterrupted. The
"control" half was bathed in CPW/B, while the "test"
half went from CPW/B to carbon-free solutions containing
zwitterionic buffers and 20 mM CaCl2 at progressively
higher pH.

Results

Mineralization patterns

Calcified cells exposed to solutions enriched in Sr and
Mn accumulate significant Sr and Mn mainly along the
inward surface of CaCO3 incrustations (Figs. 2. 3. 4). This
distribution suggests metal transport from the cell to the
extracellular site of deposition, although diffusion along
the cell wall is also possible. Mn/Sr ratios are spatially
variable, suggesting some elemental segregation during
transport or precipitation. This is indicated by variations
in the relative intensities of their X-ray peaks observed in
area scans. Some of this variability is visible in the X-ray
maps presented in Figure 3.

Figure 2. Scanning electron micrograph of frozen, hydrated cell la-
beled with Sr:+ and Mn2+. showing extracellular CaCO, incrustations,
with inward dimpling of the cell and apparent duplication of the cell
wall. Magnification: top 162x, bottom 830x. Scale bar = 30 ».

ALGAL CALCIFICATION 189

CALCIFICATION TO PHOTOSYNTHESIS RATIO

15KV X1.00K 30UM

Figure 3. Distributions of Ca (yellow), Sr (blue), and Mn (red) in an
extracellular CaCO3 deposit, visualized by X-ray mapping of a frozen.
hydrated cell exposed to Sr:+ and Mn:+ after first accumulating significant
CaCO3.

Sr and Mn accumulations presumably consist of di-
valent metal carbonates and MnO2, the latter inferred
from its dark color. Manganese oxidation, Mn2+ + H2O
+ '/2O2 = MnO2 + 2H+, is favored in the alkaline, oxygen-
rich environment of the plant surface. More or less pure
Mn accumulations, based on relative X-ray counts for

120

4 6

ENERGY (KeV)

Figure 4. X-ray spectra taken at points "A" and "B" of cell shown
in Figure 2. Spectra correspond to materials deposited after (A) and
before (B) addition of Sr2+ and Mn2+ to the medium. X-ray counts are
scaled relative to the Ca peak (100%); spectrum A has been shifted up-
wards by 20% for clarity.

1 0 -

D
D ,' *-,

a'n

' ° 10 -

° ~~^j

0.5

PHOTOSYNTHESIS

f :

1 05

1 /'

0 -

-j

-4

DIC = 2mM

pH - 8 o •

CALCIUM (mM)
1234

20 30

CALCIUM (mM)

Figure 5. Ratio of calcification to photosynthesis near pH 8 as a
function of Ca2* concentration. Inset: inhibition of photosynthesis by
low Ca2+ concentrations. Error bars: 1 S.D.

Mn, Sr, and Ca, sometimes occur beneath CaCO, in-
crustations, even when the incubating medium contains
considerably more Ca2+ and Sr2+. Mn enrichment may
reflect kinetics of transport or precipitation, and was
probably assisted by oxidation of Mn:+ to Mn4+, thus
producing a less soluble, non-transportable cation.

Indentations of the cell and apparent duplications of
the cell wall sometimes occur underneath CaCO3 incrus-
tations (Fig. 2). Non-calcified regions of the cell lack such
features. Calcification within the cell wall may force the
plasma membrane inward, followed by the secretion of a
new wall. This scenario again suggests CaCO, accretion
to the inward side of CaCO3 incrustations, and CaCO,
adhesion to the cell wall.

Physiological stoichiometry

The molar ratio of calcification to photosynthesis (C/
P), determined using the pH-alkalinity method, is rela-
tively constant at about 1.0 for Ca2+ concentrations be-
tween 2 and 50 mM (pH 8, 1 mM NaHCO3) (Fig. 5).
Controls (dark, no algae, or boiled algae) show little cal-
cification, even at 50 mM CaCl2 .

The Ca2+ ATPase model (Fig. 1 ) correctly predicts the
1:1 C/P ratio, provided the calcifying region is fairly iso-
lated from bulk solution. Each CO2 precipitated at the
alkaline band yields 2H+, which the plant uses to generate
2CO2 at the acid band. Calcification uses one CO2, leaving
one for photosynthesis, yielding a 1 : 1 C/P ratio. The pro-
ton channel model would predict lower C/P ratios, in-
creasing with Ca2+ concentration, because a diffusion
pathway must exist to the calcifying region. OH and
CO3= can therefore diffuse away. These results conse-
quently favor the Ca2+ ATPase model.

190

T. A. McCONNAUGHEY AND R. H. FALK.

Proton cycling involves calcium

Low Ca2+ concentrations inhibit photosynthesis (Fig.
5, inset). This inhibition appears to involve Ca2+ fluxes,
because plants incubated at the same low Ca2+ activity
show more photosynthesis if additional buffered Ca2+ is
added to solution (Table I). The rate of photosynthesis
and the stimulation by buffered Ca2+ are greater with cal-
cified than with decalcified plants (two way ANOVA, both
factors and interactions significant at P < 0.05).

Proton uptake at the alkaline band, measured using a
vibrating FT specific electrode, is inhibited by the Ca2 +
transport antagonists Gd3+ and La3+ (Fig. 6). In this ex-
ample, the electrode was positioned over a point showing
particularly strong alkalinization. Gd3+ reduced the signal
registered by the vibrating electrode by about half, but
the cell soon recovered about 80-90% of its former signal.
Subsequent treatment with La3+ reduced the signal more
strongly, and H+ uptake did not recover for over an hour.
Before adding the lanthanides, the voltage difference signal
registered by the vibrating electrode (about 7 mV at pH
9) corresponds ideally to a flux of proton equivalents
around 2 nMoles cirT2/s, carried mostly by OH" and
TRIS buffer (calculation. Fig. 6 inset).

The alkaline bands of C/iara can turn on and oft in-
dependently, sometimes without obvious provocation, so
reductions in the pH gradient are not necessarily propor-
tional to pathology. The pH gradient is also affected by
CaCO} dissolution at the plant surface, and by ion pairing
and precipitation of the introduced lanthanides. The ef-
fects here appear to be mostly physiological, however.
Perfusion of the chamber should have brought solution
pH back to normal within a few minutes (theoretical di-
lution time about 38 s).

An approximately 2-3 min oscillation in apparent H +
influx is observed in this experiment (Fig. 6). Such oscil-
lations are detected using various techniques (Fisahn et
ai. 1989), andean sometimes be induced by adding Ca2+
to the medium.

Electrochemical detection of calcium efflux

Both the Ca2+ ATPase and proton channel models pre-
dict Ca2+ diffusion toward the alkaline surface under cal-

Table I

Stimulation of photosynthesis by buffered calcium, at low solution
calcium activity. Photosynthesis estimated by oxygen evolution,
in micromoles O; per gram wet weight per hour,
with standard deviation (n = 10)

U 3 -
UJ

|.H

Solution

Unbuffered

Buffered

Change

Calcified
Decalcified

6.43 ± 0.67
4.48 ±0.10

8.11 ±0.68
5.29 ±0.41

+26%
+ 18%

KILOSECONDS

Figure 6. Inhibition of proton influx by La3t and Gd'*, measured
with a vibrating proton specific electrode. Ordinate: voltage difference
registered by the electrode between the extremes of its 10 micron excursion
perpendicular to the cell. Inset: apparent proton influx calculated from
a diffusion model, as a function of pH at the probe, for a signal of 1 mV
over an excursion of 10 microns. Fluxes scale almost linearly with ex-
cursion and voltage.

diving conditions. The Ca2+ ATPase model also predicts
localized Ca:+ efflux, which, in principle, should be de-
tectable with Ca2+ specific microelectrodes. This efflux
might be difficult to detect, however. It may occur un-
derneath CaCO3 crystals or within an endomembrane
system, and calcification may consume it before it is de-
tected externally. More importantly, Ca2+ influx and efflux
must both occur within the alkaline band to produce its
electrogenic character (Fig. Ib), regionally cancelling the
Ca2+ efflux signal. Therefore, detection requires a local
asymmetry between Ca2+ influx and efflux under non-
calcifying conditions (Fig. If).

Such conditions encourage CaCO, dissolution and Ca2+
leaching from the cell wall. The resulting increase in peri-
cellular Ca2+ concentration may be confused with the ef-
fects of 2H+/Ca2+ exchange. The former effect will be most
pronounced at low pH, while the latter will be associated
with high pH. Simultaneous pH observations are therefore
needed to distinguish these two cases.

Increases in Ca2+ activity (pCa decreases) are often ob-
served coincident with pH decreases (Fig. 7a), suggesting
Ca2+ leaching or CaCO3 dissolution. Small drops in pCa
are also observed coincident with pH increases (Fig. 7b),
suggesting 2H+/Ca2+ exchange. At one point in the case
illustrated, the apparent pericellular Ca2+ activity increases
about 30% as the pH rises from 8.2 to 9.8. The actual
Ca2+ activity presumably increased even more, because
the alkaline band develops a pericellular electrical field of
around —4 mV when it turns on. This biases the Ca2+
electrode toward higher apparent pCa by about — 4/— 28
= 0.14 pCa unit. The increase in pericellular Ca2+ due to

ALGAL CALCIFICATION

191

40 60

TIME (MIN)

Figure 7. Extracellular pH and pCa measured with stationary ion
specific electrodes placed close to the calcined surface of a cell under
non-calcifying conditions. (A) Positive correlation between pH and pCa,
probably caused by increased CaCO, dissolution or leaching at low pH.
(B) Anticorrelation between pH and pCa. suggesting calcium-proton ex-
change.

2H+/Ca2+ exchange must also be sufficient to overcome
the decrease in pericellular Ca2+ at high pH. caused by
reductions in CaCO, dissolution and Ca2+ leaching from
the cell wall.

Thermodynamics

The maximum pH observed at the alkaline surface us-
ing microelectrodes approaches the thermodynamic limit
for ATP driven 2H+/Ca:+ exchange, calculated using eq.
3 (Fig. 8). The approach is closest at high solution Ca2+
activities (low pCa). As pCa:+ increases, the maximum
pH also increases, although not as much as allowed by
thermodynamics. At pCa >4, higher pH readings are ob-
tained in the presence of the weak Ca2+ buffer citrate,
suggesting that the rate of Ca2+ supply to the cell may
limit proton uptake. All pericellular pH, pCa observations
fall within the thermodynamic constraints for ATP driven
2H+/Ca2+ exchange, and the Ca2+ dependence for peri-
cellular pH provides some support for the Ca2+ ATPase
model.

The pH and pCa in large culture vessels containing
Cham also approach the calculated thermodynamic limits
for 2H+/Ca:+ exchange (Fig. 8). The most extreme con-
ditions observed (pH 10.78, pCa 4.30) are close to the
most extreme conditions observed at the cell surface with
microelectrodes. Rather high pericellular pH (about 10.7)
is observed transiently in Ca2+ free solutions, but pH
banding eventually collapses, consistent with a Ca2+ re-
quirement for banding. Internal Ca24 stores, perhaps sup-
plemented by CaCO, dissolution and Ca2+ leaching from
the cell wall, may support banding for awhile.

Extracellular electrical currents

The ion fluxes associated with pH banding create ex-
tracellular current loops which can be measured with a
vibrating probe electrometer. These currents persist until
solution pH is raised above a critical value, at which point
the currents cease or may reverse with much diminished
amplitude. The solution pH at which current cessation
occurs varies with solution Ca2+ activity in more or less
the same way as the extracellular pH data in Figure 8.
The Ca2+ dependence suggests that proton uptake is cou-
pled to Ca2+ expulsion.

Presumably, as 2H+/Ca:+ exchange becomes impos-
sible, cytosolic Ca2+ rises and inhibits Ca2+ influx (see
Eckert and Chad, 1984). The proton ATPase of the acid
band shuts down as the cytoplasm becomes alkalinized
(due to cessation of proton uptake) and the membrane
potential increases (due to cessation of Ca2+ uptake).
Consequently, even though 2H+/Ca2+ exchange is elec-
trically silent, preventing this exchange can stop extra-
cellular electrical activity.

Figure 9 illustrates an experiment in which a cell is
placed in a divided chamber, and increasingly alkaline
solutions containing 20 mAI CaCN (pCa = 2. 1 ) are applied
to the right (test) side. The left (control) side remains at
pH 8.2, pCa = 3.8. Cytoplasmic streaming between the
two sides is uninterrupted. As the test side approaches the
calculated thermodynamic limits for ATP driven 2H+/
Ca2+ exchange, its currents diminish, but currents on the
control side are unaffected. In the last test solution (pH
10.0, pCa 2. 1 ). banding is strongly suppressed and an ap-

10-

t
I

4 »

CYTOSOL O

pH AT PLANT SURFACE

-- WITH CITRATE
CULTURE SOLUTIONS

345

pCa of solution

Figure 8. pH observations at the alkaline surface as a function of
solution Ca2+ activity. Diagonal line: calculated thermodynamic limits
for ATP driven 2H+/Ca:+ exchange between the cytosol and external
solution, assuming E( ATP) = 50 KJ/mol. Symbols: (Diamond) assumed
cytosolic composition, pH = 8, pCa = 6.9. (Squares) maximum pH
observed at alkaline surface under experimental conditions, without ci-
trate. ( + ) Same, with citrate. (A) Combinations of solution pH and pCa
observed in large vat cultures.

192

T. A. McCONNAUGHEY AND R. H. FALK.

LU
O

a:
o

-1.8

-20

-10

DISTANCE FROM DIVIDER (mm)

Figure 9. Extracellular currents measured using a vibrating voltage probe. Cell was placed in a divided
chamber, and solution on the right side was replaced with solutions having higher Ca2* activity and pro-
gressively higher pH. Duplicate scans are shown in each medium, and cell responses to different media are
offset by -500 /iA/cm2. Positive currents denote regions of positive current influx to cell (alkaline bands).
Solutions contained (in rruV/) NaCl I, KG 0.2. plus the following additions: (a) CaSO4 0.2, NaHCO3 1. pH
8.2. (b) CaCI2 20, CHES 5, pH 9.0. (c) CaCl, 20. CAPS 5, pH 9.8. (d) CaCl, 20, CAPS 5. pH 10.0).

parent efflux of positive charge prevails over the test side
of the cell. The control side does not appear to compen-
sate, so if real, this current efflux should hyperpolarize
the cell.

Discussion

Antecedents to the Ca2+ ATPase model for Chara ex-
tend back at least to 1829, when Bishoff (cited in Pring-
sheim, 1 888) suggested that characean lime deposits grow
from the inside. Classical works on bicarbonate use also
favored calcium and carbon movement through the po-
larized leaves of calcareous aquatic angiosperms and
characeans to reach the site of mineralization (Arens,
1933, 1938, 1939). Kishimoto et al. (1984) suggested that
proton uptake in Chara might occur through an electro-
neutral proton cotransport or countertransport system.
Many aspects of the Ca2+ ATPase model have therefore
been discussed.

The Ca2+ ATPase model correctly predicts the data
presented here. Sr2+ and Mn2+ accumulate largely along
the inner surface of CaCO3 incrustations, facing the cell.
Increasing the Ca2+ concentration in solution (from 2 to

50 mA/) has a minimal effect on the ratio of calcification
to photosynthesis, suggesting that diffusion to the calci-
fication site can be minimal. Ca2+ transport antagonists
interfere with H+ uptake. In solutions of low Ca2+ activity,
additional "buffered" Ca:+ enhances photosynthesis and
proton uptake. Pericellular Ca2+ activities sometimes in-
crease simultaneously with stronger alkalization. Com-
binations of extracellular Ca2+ and H+ activities are ther-
modynamically compatible with ATP driven 2H+/Ca2~f
exchange, and the maximum pH at the alkaline band
increases with pCa, as would be expected if Ca2+ extrusion
accompanies proton uptake.

Most of the precipitating carbon also appears to be sup-
plied by the cell as CO2 (McConnaughey, in prep.). This
further implies that the calcifying region can become iso-
lated from bulk solution. Consequently, the cell must
supply Ca2+, and remove protons in 1:2 stoichiometry,
as indicated by the reaction Ca2+ + CO2 + H2O = CaCO3
+ 2H+.

The data are less supportive of the proton channel
model, which offers no explanation for the Ca2+ depen-
dence of photosynthesis, or the elevations of pericellular
Ca2+ coincident with H+ depletion. The diffusion pathway

ALGAL CALCIFICATION

193

to the site of calcification creates additional conceptual
problems. Why won't it accept OH" and CO?=, which
should diffuse away from the cell, reducing the ratio of
calcification to photosynthesis to values below 1 .0. and
making it dependent on the Ca2+ concentration, or phos-
phate, which fails to precipitate where Sr and Mn do
(McConnaughey, in prep.)?

Most of the published data appears compatible with
the Ca2+ ATPase model. Both models attribute the extra-
cellular current influx in the alkaline band largely to Ca:+
under calcifying conditions, and to H+ equivalents under
non-calcifying conditions (Fig. 1). Membrane hyperpo-
larizations are caused by electrogenic H+ extrusion in the
acid band under either model. Increased membrane con-
ductivity at high pH (Bisson and Walker, 1980, 1982)
might result from more favorable thermodynamics for
the proton ATPase of the acid band, reversibility of 2H+/
Ca2+ exchange, and perhaps opening of additional ion
channels (e.g.. Kikuyama et a/., 1984).

Calcium transport

Certain caveats apply to the thermodynamic analysis
of Ca:+ transport attempted here. Pericellular pCa varies
locally, and depressions relative to solution values are
likely at high pCa (Fig. 7). Cytoplasmic pH and pCa may
also vary. Extracellular and intracellular activity scales
may be offset with respect to each other. The slope of the
extracellular pH data is closer to 1/3 than 1/2, but pH is
too high for ATP driven 3H+/Ca2+ exchange. 4H+/2Ca:+
exchange is likewise excluded. Several factors may con-
tribute to the fall-off from the limits calculated for 2H+/
Ca2+ exchange at high pCa. As noted above, Ca2+ extru-
sion may locally depress pCa below ambient values. Ca2+
diffusion toward the cell may limit the rate of Ca2+ and
H+ cycling, as suggested by the higher pH values and pho-
tosynthetic rates obtained with buffered Ca2+. Finally, the
diffusion of alkalinity from the plant surface increases
enormously as pericellular pH increases (see Fig. 6 inset),
so if the proton flux remains constant, diffusion should
reduce pericellular pH most strongly at high pCa. In sum-
mary, there are many reasons why the data might fall
short of the thermodynamic limit, even if the plant op-
erates close to the limit. The more interesting feature is
that the plant apparently approaches the thermodynamic
limit.

Why are extracellular H+ fluxes so much easier to detect
than Ca2+ fluxes? The difference, presumably, is that Ca2+
influx and efflux occur close together, while proton fluxes
must be separated to create the acid bands needed for
bicarbonate assimilation. Proton electrodes are also twice
as sensitive as Ca2+ electrodes, and proton fluxes should
be twice as large.

The Ca2+ ATPase model postulates high rates of Ca2+
cycling through the cell, around 100 pMol cm"2 s"1 within

the alkaline band of Chara. 45Ca2+ exchange rates are
generally less than 3 pMol cm"2 s"1, measured over the
whole cell (Spanswick and Williams, 1965, Hayama et
at. 1979; MacRobbie and Banfield, 1988). Some exper-
iments employed conditions unfavorable to pH banding,
but the disparity between inferred and published steady
state 45Ca2+ fluxes nevertheless requires further study. Low
Ca2+ exchange rates may be caused by containment of
fluxes to the cortical cytoplasm of the alkaline band, with
little exchange into major cellular Ca2+ reservoirs such as
the vacuole, chloroplasts, or mitochondria. The available
evidence supports this possibility. Extracellular electrical
currents presumably reflect Ca2+ uptake mainly within
the alkaline bands. When cells are placed in a divided
chamber with Mn:+ on one side, Mn precipitation is vis-
ible only on that side, suggesting minimal transport along
the cell. 45Ca2+ fluxes measured during repeated electrical
stimulation yield values around 60 pMol cm"2 s"1, mea-
sured over the whole cell, at 1 mAl external Ca2+ (Hayama
el a!.. 1979). The metabolic machinery needed for large
fluxes therefore appears to be present.

An analogy to coccolithophorid algae is instructive.
Calcification in these algae occurs within intracellular
vesicles, so both calcium and carbon presumably traverse
the cytoplasm to reach the calcification site. Ca2+ ATPase
apparently participates in calcification (Klaveness, 1976;
Okazaki el a!., 1984). Sufficient data are sometimes avail-
able to estimate Ca2+ fluxes. For example, Emiliania hux-
leyi calcifies at a rate of around 5-7 X 10"18 moles/s, and
the surface area of the finished coccolith is around 1-1.5
X 10"7 cm2 (Paasche, 1964; Klaveness, 1976; Sikes et at.
1980). Therefore, the trans-membrane Ca2+ flux may be
30-50 pMol cm"2 s"1. This is of the same magnitude as
estimated for Chara.

Because cytosolic "free" Ca2+ concentrations are uni-
formly rather low, large trans-cellular Ca2+ fluxes pre-
sumably involve Ca2+ rich vesicles, vacuoles, reticula, etc.
Total Ca2+ concentrations in characean cytoplasm and
vacuoles is in the millimolar range (Okihara and Kiyo-
sawa, 1988). To the extent that cytosolic free ion concen-
trations pose a transport problem, the issue may be more
acute with protons. The proton fluxes are presumably
twice as large and involve longer distances.

Applicability to other organisms

If Ca2+ ATPase underlies extracellular calcification in
Chara and intracellular calcification in coccolithophorids,
it might contribute similarly elsewhere. For example, large
Ca2+ dependent proton influxes, sensitive to lanthanides,
also occur at the calcified rhizoid of the siphonaceous
marine alga Acetabularia (McConnaughey, in prep.).

Coupling calcification to photosynthesis

Although a photosynthetic organism may loose CO2 to
calcification, it gains two protons for each carbon lost. In

194

T. A. McCONNAUGHEY AND R. H. FALK

mildly alkaline waters, these 2H+ potentially enable it to
convert 2HCO3 to 2CO2, yielding a net gain of one CO2
for photosynthesis. An approximately 1:1 ratio of calci-
fication to photosynthesis is observed not only in Chara,
but sometimes also in coccolithophorid algae (Paasche,
1964; Sikes el a/., 1980), calcareous seaweeds (Pentecost,
1978), and invertebrate-algae symbioses (Goreau, 1963;
Barnes and Taylor, 1973; Duguay and Taylor, 1978; Kuile
et ai, 1989). In the symbioses, the animal calcifies while
the algae use the CO;.. Rapid and massive calcification
may exceed structural or defensive uses for CaCO3, and
coccolithophorids, for example, discard excess scales to
remain suspended in the water. Corals build up huge skel-
etal mounds but occupy only the top few millimeters. No
structural or defensive use of CaCO3 is obvious in Chara.

Proton cycling theoretically allows organisms to gen-
erate pericellular CO2 concentrations well above ambient
(Walker et ai. 1980). Photosynthesis generally saturates
at CO2 concentrations higher than the atmospheric equi-
librium value (e.g.. Smith and Walker, 1980), and far
higher than present in many natural waters subjected to
strong photosynthesis. Elevating CO2 concentrations (by
protonating HCO3 ) therefore increases carboxylation
rate. Many aquatic plants and invertebrate-algae appar-
ently promote photosynthesis through proton cycling. In
this context, protons, rather than CaCO3, may be the
principle product of calcification.

From a geochemical perspective, biologically precipi-
tated carbonates comprise one of the more abundant
crustal materials, and represent the principle biogeo-
chemical reservoir for carbon (Garrels el ai. 1976). Be-
cause organisms often calcify much faster than the am-
bient media in which they live, biological calcification
may provide an important brake on the photosynthetic
alkalinization of natural waters, and thereby affect such
processes as the partitioning of CO2 between the oceans
and the atmosphere.

Acknowledgments

Primary funding was provided through NSF fellowship
DCB-88076 1 3 to Ted McConnaughey. W. J. Lucas and
L. F. Jaffe contributed laboratory facilities. The authors
are grateful to J. Fisahn, W. Kiihtreiber, A. Miller, and
A. Shipley, for their assistance, and to D. McCorkle, A.
Kuzerian, C. Barr, and others for their enthusiasm and
helpful comments.

Literature Cited

Arens, K. 1933. Physiologisch polarisierter Massenaustausch und Pho-
tosynthese bei submersen Wasserpflanzen. I. Planla 20: 621-658.

Arens, K. 1938. Manganablagerungen bei Wasserpflanzen als Folge
des Physiologisch polarisierten Massenaustausches. Proloplasma 30:
104-129.

Arens, K. 1939. Physiologische Multipolantat der zelle von Nitella
wahrend der Photosynthese. Proloplasma 33: 295-300.

Barnes, D. J., and D. L. Taylor. 1973. In situ studies of calcification
and photosynthetic carbon fixation in the coral Monlastrea anniilaris.
llelgol. li'iss. Meeresunlers. 24: 284-291.

Bisson, M. A. 1984. Calcium effects on electrogenic pump and passive
permeability of the plasma membrane of Chara corallina. J Mem-
brane Biol 81: 59-67.

Bisson, M. A., and N. A. Walker. 1980. The Chara plasmalemma at
high pH. Electrical measurements show rapid specific passive uniport
of H + or OH'. J Membrane Biol. 56: 1-7.

Bisson, M. A., and N. A. \\alker. 1982. Control of passive permeability
in the Chara plasmalemma. J E\p Hot 33: 520-532.

Borelli, M. M., W. G. Carlini, W. C. Dewey, and R. R. Ransom. 1985. A
simple method tor making ion-selective microelectrodes suitable for
intracellular recording in vertebrate cells. / Neurosci. Methods 15:
141-154.

Dixon, D. A., and D. H. Haynes. 1989. Ca:+ pumping ATPase of cardiac
sarcolemma is insensitive to membrane potential produced by K+
and Cr gradients but requires a source of counter-transportable HT.
J Membr Biol 112: 169-183.

Duguay, L. E., and D. L. Taylor. 1978. Primary production and cal-
cification by the soritid foraminiferan Archats angnlatus (Fichtel &
Moll). J. Proloiool. 25: 356-361.

Eckert, R., and J. E. Chad. 1984. Inactivation of Ca channels. Prog.
Biophys Molec. Biol. 44: 215-267.

Fisahn, J., T. McConnaughey, and \V. J. Lucas. 1989. Oscillations in
extracellular current, external pH, and membrane potential and con-
ductance in the alkaline bands of Nitella and Chara. J. E.\p. Bol. 40:
1185-1193.

Garrels, R. M., A. Lerman, and F. T. Mackenzie. 1976. Controls of
atmospheric CO2 and O2: past, present, and future. Am. Sci. 64: 306-
315.

Goreau, T. F. 1963. Calcium carbonate deposition by coralline algae
and corals in relation to their roles as reef-builders. Ann. N. Y Acad
Sci. 107: 127-167.

Hayama, T., T. Shimmen, and M. Tazawa. 1979. Participation of Ca:+
in cessation of cytoplasmic streaming induced by membrane exci-
tation in Characeae internodal cells. Proloplasma 99: 305-321.

Jaffe, L. F., and R. Nuccitelli. 1974. An ultrasensitive vibrating probe
for measuring extracellular currents. J. Cell. Biol. 63: 614-628.

Kingsley, R. J., and N. \\atabe. 1985. Ca-ATPase localization and
inhibition in the gorgonian Leplogorgia virgulata (Lamarck) (Coe-
lenterata: Gorgonacea). J Exp. Mar. Biol. Ecol 93: 157-167.

Kishimoto, U., N. Kami-iki, Y. Takeuchi, and T. Ohdawa. 1984. A
kinetic analysis of the electrogenic pump of Chara corallina: I. In-
hibition of the pump by DCCD. J. Membrane Biol. 80: 175-183.

Klaveness, D. 1976. Emiliania Intxleyi (Lohman) Hay and Mohler. 3.
Mineral deposition and the origin of the matrix during coccolith
formation. Protistologica 12: 217-224.

Kuhtreiber, \V. M., and L. F. Jaffe. 1990. Detection of extracellular
calcium gradients with a calcium specific vibrating electrode. J. Cell
Biol 110: 1565-1573.

Kuile, B. ter, J. Erez, and E. Padan. 1989. Mechanisms for the uptake
of inorganic carbon by two species of symbiont-beanng foraminifera.
Mar. Biol 103: 241-251.

Lucas, \V. J. 1976. The influence of Ca:+ and K/ on HI4CO3 influx
in internodal cells of Chara corallina. J. Exp. Bol. 27: 32-42.

Luhring, H. and M. Tazawa. 1985. Effect of cytoplasmic Ca2+ on the
membrane potential and membrane resistance of Chara plasma-
lemma. Plant Cell Physiol. 26: 635-646.

MacRobbie, E. A. C., and J. Banfield. 1988. Calcium influx at the
plasmalemma of Chara corallina. Planla 176: 98-108.

ALGAL CALCIFICATION

195

McConnaughey, T. A. 1989a. Biominerahzation mechanisms. Pp. 57-

73 in The Origin. Evolution, and Modem Aspects of Biomineralization

in Animals ami Plants. R. E. Crick, ed. Plenum Press, New York.
McConnaughey, T. A. 1989b. 13C and I8O isotopic disequilibrium in

biological carbonates. I. Patterns. Geoclum. Cosiuochnn Ada 53:

151-162.
McConnaughey, T. A. I989c. "C and I8O isotopic disequilibrium in

biological carbonates. II. In vitro simulation of kinetic isotope effects.

Geochim. Cosmoclum. Ada 53: 163-171.
Miller, A. J., and D. Sanders. 1987. Depletion of cytosolic free calcium

induced by photosynthesis. Nature 326: 397-400.
Mimura, I ., and Y. Kirino. 1984. Changes in cytoplasmic pH measured

by 31P-NMR in cells of Nilellopxix ohlusa. Plant Cell Physiol. 25:

813-820.
Niggli, V., E. Sigel, and E. Carafoli. 1982. The punned Ca2+ pump of

human erythrocyte membranes catalyzes an electroneutral Ca2+-H+

exchange in reconstituted liposomal systems. J. Biol. Chem 257:

2350-2356.
Okazaki. M. 1977. Some enzymatic properties of Ca2+-dependent

adenosine tnphosphatase from a calcareous marine alga, Scrralicardia

maxima and its distribution in marine algae. But Mar 20: 347-354.
Okazaki, M., M. Fujii, Y. Usuda, and K. Furuya. 1984. Soluble Ca2+

activated ATPase and its possible role in calcification in the cocco-

lithophorid Cricosphaera roscqffensis var. Haptonemofera (Hapto-

phyta). But Mar 27: 363-369.
Okazaki, M., and M. Tokita. 1988. Calcification of C/iara hrau/in

(Charophyta) caused by alkaline band formation coupled with pho-
tosynthesis. Jpn J Phycol. 36: 193-201.
Okihara, K., and K. Kiyosawa. 1988. Ion composition of the Chara

mternode. Plant Cell Physiol. 29: 21-25.
Paasche, E. 1964. A tracer study of the inorganic carbon uptake during

coccolith formation and photosynthesis in the coccolithophond Coc-

co/ilhus huxleyi. Physiol, Plant Supp. III: 5-82.
Pentecost, A. 1978. Calcification and photosynthesis in corallina ol-

hcinalis L. using the 14CO2 method. Br Phycol J 13: 383-390.
Price, G. D., and M. R. Badger. 1985. Inhibition by proton buffers of

photosynthetic utilization of bicarbonate in Chara corallina. Au.il.

J Plant Physio/. 12: 257-267.
Price, G. D., M. R. Badger, M. E. Bassett, and M. 1. \\hitecross.

1985. Involvement of plasmalemmasomes and carbonic anhydrase

in photosynthelic utilization of bicarbonate in Chara corallina. Au.il

J Plant Physiol 12:241-256.
Pringsheim, N. 1888. Uber die Entstehung der Kalkencrustationen an

Susswasserpflanzen. Jahrh. H'ixxensch. Bol 19: 138-154.

Rasi-Caldogno, F., M. C. Pugliarello. and M. I. De Michelis. 1987. The

Ca:+-transport ATPase of plant plasma membrane catalyzes a nH+/
Ca2+ exchange. Plant Physiol 83: 994-1000.

Raven, J. A., F. A. Smith, and N. A. Walker. 1986. Biommeralization
in the Charophyceae sensu lain Pp. 125-139 in Biomineralization
in Lower Plants and Animals. B. S. C. Leadbeater and R. Riding,
eds. Clarendon Press, Oxford.

Sikes, C. S., R. D. Roer, and K. M. Wilbur. 1980. Photosynthesis and
coccolith formation: inorganic carbon sources and net inorganic re-
action of deposition. Lnnnol. Oceanogr. 25: 248-261.

Smith, F. A. 1984a. Regulation of the cytoplasmic pH of Chara cor-
allina: response to changes in external pH. J. Ii.\p. Bol 35: 43-50.

Smith, F. A. 1984b. Regulation of the cytoplasmic pH ol 'Chara cor-
allina in the absence of external Ca2*: its significance in relation to
activity and control of the H+ pump. J. Exp. Bol 35: 1525-1536.

Smith, F. A., and J. A. Raven. 1979. Intracellular pH and its regulation.
Ann Re\: Plant Physiol. 30: 289-31 1.

Smith, F. A., and N. A. Walker. 1980. Photosynthesis by aquatic plants:
effects of unstirred layers in relation to assimilation of CO: and HCOr
and to carbon isotope discrimination. AVir Pliytol. 86: 245-259.

Spanswick. R. M., and A. G. Miller. 1977. Measurement of the cy-
toplasmic pH in \itella translucens. Plant Physiol. 59: 664-666.

Spanswick, R. M., and E. J. Williams. 1965. Ca fluxes and membrane
potentials in Nilella translucens J Exp. Bol 16: 463-473.

Spear, D. G., J. K. Barr, and C. E. Barr. 1969. Localization of hydrogen
ion and chloride ion fluxes in Nilella. J. Gen. Physiol. 54: 397-414.

Stumm, \\ ., and J. J. Morgan. 1970. At/italic Chemistry. Wiley-Inter-
science. New York. 583 pp.

Tazawa, M., T. Shimmen, and I . Mimura. 1987. Membrane control
in the characeae. Ann Re\' Plant Physiol. 38: 95-1 17.

Villalobo, A., and B. D. Roufogalis. 1986. Proton countertransport by
the reconstituted erythrocyte Ca2+-translocating ATPase: evidence
using lonophoretic compounds. / Mcmbr. Biol. 93: 249-258.

Walker, N. A., and F. A. Smith. 1977. Circulating electrical currents
between acid and alkaline zones associated with HCO, assimilation
in Chara. J Exp Bol 28: I 190-1206.

Walker, N. A., F. A. Smith, and I. R. Cathers. 1980. Bicarbonate as-
similation by fresh-water charophytes and higher plants: I. Membrane
transport of bicarbonate ions is not proven. J. Membr. Biol. 57: 51-
58.

W iesenseel, M. H., and H. K. Ruppert. 1977. Phytochrome and calcium
ions are involved in light-induced membrane depolarization in Nilella.
Planta 137: 225-229.

CONTENTS

BEHAVIOR

De Vries, M. C., D. Rittschof, and R. B. Forward Jr.

Chemical mediation of larval release behaviors in

the crab Neopanope sa\i 1

Hart, Michael W.

Particle captures and the method of suspension
feeding by echinoderm larvae 12

DEVELOPMENT AND REPRODUCTION

Patterson, Mark R.

The effects of flow on polyp-level prey capture in

an octocoral, Alcyonium siderium 93

Purcell, Jennifer E., Frances P. Cresswell, David G.

Cargo, and Victor S. Kennedy

Differential ingest ion and digestion of bivalve larvae
by the scyphozoan Chrysaora quinquecirrha and the

ctenophore Mneiniopu.'* leidyi 103

"Walters, Linda J., and David S. Wethey

Settlement, refuges, and adult body form in colonial
marine invertebrates: a field experiment 112

Govind, C. K., Christine Gee, and Joanne Pearce

Retarded and mosaic phenotype in regenerated claw
closer muscles of juvenile lobsters 28

Gustafson, R. G., D. T. J. Littlewood, and R. A. Lutz
Gastropod egg capsules and their contents from
deep-sea hydrothermal vent environments 34

Longo, Frank J., and John Scarpa

Expansion of the sperm nucleus and association of
the maternal and paternal genomes in fertilized
Miilnua laterals eggs 56

Webster, S. G., and H. Dircksen

Putative molt-inhibiting hormone in larvae of the
shore crab Cumnm mamas L.: an immunocyto-
chemical approach 65

ECOLOGY AND EVOLUTION

Carlton, James T., Geerat J. Vermeij, David R. Lind-
berg, Debby A. Carlton, and Elizabeth C. Dudley

The first historical extinction of a marine inverte-
brate in an ocean basin: the demise of the eelgrass

limpet Lotlia alveus s 72

Patterson, Mark R.

Passive suspension feeding by an octocoral in plank-
ton patches: empirical test of a mathematical model 8 1

PHYSIOLOGY

Bollner, Tomas, Jon Storm-Mathisen, and Ole Petter
Ottersen

GABA-like immunoreactivity in the nervous system
ofOikopleura diuiin (Appendicularia) 119

Charmantier, G., and M. Charmantier-Daures

Ontogeny of osmoregulation and salinity tolerance
in Cancer irroratus; elements of comparison with C.
h< 1 1 <•(/ /is (Crustacea, Decapoda) 125

Childress, J. J., C. R. Fisher, J. A. Favuzzi, R. E. Ko-

chevar, N. K. Sanders, and A. M. Alayse

Sulfide-di iven autotrophic balance in the bacterial
symbiont-containing hydrothermal vent tubeworm,
Riftiu (inchyptilti Jones 135

Dickson, John S., Richard M. Dillaman, Robert D.

Roer, and David B. Roye

Distribution and characterization of ion transporting
and respiratory filaments in the gills of Procambarus
flurkii 154

Dobson, William E., Stephen E. Stancyk, Lee Ann

Clements, and Richard M. Showman

Nutrient translocation during early disc regenera-
tion in the brittlestar Microfihiopholis gracilliina
(Stimpson) (Echinodermata: Ophiuroidea) 167

McConnaughey, Ted A., and Richard H. Falk

Calcium-proton exchange during algal calcification 185

Volume 180

THE

Number 2

BIOLOGICAL
BULLETIN

LIB

APR 171991 «

Hole,

APRIL, 1991

Published by the Marine Biological Laboratory

THE

BIOLOGICAL BULLETIN

PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY

Associate Editors

Marine Biological Laboratory
LIBRARY

APR 1? 1991

Woods Hole, Mass.

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

DAVID EPEL, Hopkins Marine Station, Stanford University

J. MALCOLM SHICK., University of Maine, Orono

Editorial Board

GEORGE J. AUGUSTINE, University of Southern RUDOLF A. RAFF, Indiana University

California

KENSAL VAN HOLDE, Oregon State LJniversity

Louis LEIBOVITZ, Marine Biological Laboratory STEVEN VOGEL, Duke University

Eclitur: MICHAEL J. GREENBERG, The Whitney Laboratory, University of Florida
Managing Editor. PAMELA L. CLAPP, Marine Biological Laboratory

APRIL, 1991

Printed and Issued by
LANCASTER PRESS, Inc.

PRINCE & LEMON STS.
LANCASTER, PA

Erratum

The Biological Bulletin, Volume 179, Number 3, pages 358 and 363

The following corrections should be made in the article by William J. Kuhns et al. titled, "Biochemical
and functional effects of sulfate restriction in the marine sponge, Microciona prolifera" (Biol. Bull. 179:
358-365). Due to a printing error, the last two lines of the abstract on page 358 were transposed from the
first column (abstract) to the second column (introduction). We apologize for the error.

On page 363, the sentence beginning on line 7 of Figure 5 should now read "The upper two lines depict
uptake of 35SO4 by cells pretreated in MBL-SO4." The words "upper two lines" replace the words "solid
lines."

Reference: Biol. Bull 180: 197-199. (April, 1991)

Integrative Neurobiology and Behavior of Mollusks

Symposium: Introduction, Perspectives, and

Round-Table Discussion

ROGER T. HANLON

The Marine Biomedical Institute. University of Texas Medical Branch,
Galveston, Texas 77550

The objective of this symposium was to bring together
molluscan researchers from a wide variety of disciplines
to consider the behavior of mollusks, particularly in re-
lation to their evolutionary history and to their neural
structure and function. For that reason, the symposium
was convened during the 56th Annual Meeting of the
American Malacological Union (AMU), attended by 280
persons and held at the Marine Biological Laboratory
(MBL) in Woods Hole from 3-7 June 1990. The AMU
membership includes many researchers interested pri-
marily in systematics, phylogeny. and evolution: the MBL
enjoys a long and distinguished history as a global center
for molluscan neurobiological research. Thirty-two papers
and seven posters were presented at this symposium,
which was held concurrently with The Behavior of Mol-
lusks symposium in which 26 papers and 1 2 posters were
presented; selected papers from the latter symposium will
be published in the American Malacological Bulletin dur-
ing 1991. Support for the symposia was provided by the
National Science Foundation (BNS 9007661) and the
AMU Symposium Endowment Fund. The organizers and
participants greatly appreciate this funding as well as the
assistance of The Biological Bulletin and the hospitality
of the MBL.

Perspectives

The Phylum Mollusca is large (about 100,000 liv-
ing species), diverse, well represented in the fossil rec-
ord (approx. 35,000 species dating to the Cambrian), and
richly studied (cf., Tasch, 1973; Wilbur, 1983-1988;
Barnes, 1987). It is also important to man; its uses range
from food to models in biomedicine. In the latter instance

it has been mainly species with unusually large or easily
identified neurons that have been studied so intensively.
These species were well represented in the symposium:
e.g.. Aplysia, Hermissenda, Navanax, Pleurobranchaea.
Tritonia. Lymnaea, and Loligo among several others.

Molluscan diversity offers fertile grounds for thought
among evolutionary biologists, ethologists, and neuro-
biologists. In what other phylum can you find organisms
as different as chitons, with their simple nervous system
and behavior, and cephalopods, with their immensely
complex nervous system and correspondingly complex
and varied behavior that rival vertebrates? In the middle
of this continuum, consider the marine opisthobranchiate
gastropod Aplysia and some of its cousins, whose relatively
simple behaviors are being studied even at molecular
levels by thousands of researchers worldwide. The chal-
lenge, of course, is to make some sense of this dazzling
diversity.

The scope and goals of the relatively new field of
Neuroethology have been well reviewed by Hoyle
(1984) and Bullock (1990). As Bullock (1990) explains
eloquently, although neurobiologists mainly study prox-
imate mechanisms of neural function, the implications
of their findings are basic to the philosophy of science and
to evolutionary biology because it is likely that the nervous
system and behavior represent the system most responsible
for large evolutionary leaps in the grade of complexity
among higher taxa. Notwithstanding this provocative as-
sertion, most neurobiologists do not spend much time
mingling with evolutionary biologists, especially those who
study the same phylum, because their professional orga-
nizations and journals are usually quite distinct. Con-
versely, evolutionary biologists do not often consider

197

198

R. T. HANLON

proximate mechanisms of neural control of behavior or
its implications in evolution. This meeting and its round-
table discussion were organized to help bridge this gap
among molluscan researchers.

Round-Table Discussion

Are round-table discussions worthwhile? While con-
troversial, they are worthwhile in forcing the consider-
ation of differing views and alternative hypotheses
in a less binding way than a paper or poster presentation.
I include here the briefest synopsis of our recorded 3-h
session.

Can the comparative method bridge studies involving
neurobiology, behavior, and evolution?

There was some consensus that proper use of homol-
ogies could provide common ground to test hypotheses
across these disciplines. However, gaps in our knowledge
in each broad discipline are considerable, and future
progress was anticipated to be slow unless workers were
willing to take evidence from many disciplines (including
molecular and neural biology, behavior and ecology) and
integrate it into analyses of convergence and parallelism
in the Mollusca. This was one of the few points of general
consensus!

Are there any uniquely molluscan behaviors?

As a matter of perspective, only a dozen or so species
are being studied in detail, so generalizations about mol-
luscan behavior seem inappropriate; no unique behaviors
were mentioned. It was noted that some groups of mol-
lusks have similar nervous system organization but ap-
parently very different behaviors.

What is the role of behavior in evolution?

Some researchers consider behavior to be the phenotype
upon which selection occurs, and the corollary is that
the neural, endocrine, and related systems exist to
produce behavior. Perhaps, then, homologies at differ-
ent levels of organization can be used to study the role
of behavior in evolution. However, several concerns
were voiced about the seemingly endless variability of
molluscan behavior that would make it difficult to discern
homologs in any concrete fashion. How, for example, do
we account for phenotypic plasticity, and what is the role
of ecological constraint? One related subject of interest to
many in attendance was the future need for analyses of
the evolution of behavior in mollusks, taking advantage
of what has been learned from paleobiology and taphon-
omy (Tasch, 1973) as well as recent behavioral and eco-
logical studies of extant molluscs.

How did the nervous system evolve?

The action potential is a common feature throughout
the animal phyla, from the simplest organisms to the most
complex. However, pharmacological sensitivities and
other properties are different in various groups and it was
suggested that molecular analysis of specific differences
in the relevant ion channels may eventually lead to mean-
ingful phylogenetic relationships among different phyla.
The role of peptides and other neurotransmitters in pro-
ducing behavior (e.g., egg laying in many mollusks) may
lead to complementary findings (see recent papers in Bio-
logical Bulletin Vol. 177, 1989).

\\'hv do we find large neurons in some mollusks, and
what is their functional and evolutionary significance?

One obvious function for large cells is the rapid trans-
mission of a signal; additionally, cells must be large if they
have to spread over large distances to communicate with
many places. If both needs must be satisfied, giant cells
like the squid axon can obviously evolve. Perhaps many
of these cells are associated with relatively "simple" be-
haviors. There does seem to be an emerging trend that
small neuron size is a requirement for complex infor-
mation processing. For example, consider the cephalopod
CNS and its complex integrative abilities in comparison
with the vertebrate, and especially the mammalian, CNS.
It is apparent that existing and future neurobiological
techniques will dictate to some extent what can be studied
and learned from small versus large neurons. There will
probably continue to be philosophical disagreement about
the reductionist versus the integrative approach to un-
derstanding how behavior is produced by the nervous
system.

A thread throughout the discussion was the amazing
diversity of mollusks and the difficulty in agreeing on
many generalizations about neural organization and how
it relates to behavior. It was suggested several times that
we should appreciate this diversity and not rush to apply
our findings to evolutionary principles. As Bullock ( 1 990)
has pointed out "Must we assume everything is adaptive?"
Various participants echoed a recent thought that evo-
lution does not necessarily work with the logic of engi-
neering, but perhaps more like a tinkerer. The papers in
this volume include many examples of neurobiologists
evaluating evolutionary considerations of the systems that
they study. Perhaps the discussions facilitated this in some
small way.

Dedication

We were honored to have Professor J. Z. Young, F.R.S.,
of Oxford University, enrich our meeting. In the 1930s,

INTRODUCTION

199

Figure 1 . Professor J. Z. Young presenting his review ol the cellular
basis of learning in eephalopods.

Professor Young rediscovered the squid giant axon and
performed the critical experiments demonstrating that
these neurons transmitted an electrical signal; this pi-
oneering work (performed partly at the MBL) led to the
Nobel Prize by Hodgkin and Huxley in 1962 and made
the squid Loligo a preferred research organism for thou-
sands of neuroscientists, a trend that continues apace to-
day. In addition to recounting this story at the banquet.
Professor Young gave a rousing lecture on learning in
eephalopods (Fig. 1 ). Delivered with panache and his usual
scientific fervor, he earned a standing ovation by a packed
audience in Whitman Auditorium. For his innumerable
contributions to molluscan biology. Professor Young
was awarded Honorary Life Membership in the AMU,
a distinction bestowed upon only five members in
the 56-year history of the organization. We hereby dedi-
cate this volume to him and thank him for inspiring so
many students and researchers throughout his brilliant
career.

Literature Cited

Barnes, Robert D. 1987. Invcrlchralc /.oology. Fifth Edition. CBS Col-
lege Publishing/Holt. Rinehart and Winston, Philadelphia, PA.

Bullock. Theodore Holmes. 1990. Goals of neuroethology. Bioscience
40(4): 244-248.

Hoyle, Graham. 198-4. The scope of neuroethology. Behav. Brain Sci.
7:367-412.

Tasch, Paul. 197.1. Palenbiiilugy of the Invertebrates: Data Retrieval
from Ike Fossil Record. Wiley and Sons. New York.

Wilbur, Karl M. 1983-1988. The Mollusca. Volumes 1-12. Academic
Press. New York.

Reference: Bio/. Bull. 180: 200-208. (April,

Computation in the Learning System of Cephalopods

J. Z. YOUNG

Department of Experimental Psychology, University of Oxford,
South Parks Road, Oxford OX1 3UD, United Kingdom

Abstract. The memory mechanisms of cephalopods
consist of a series of matrices of intersecting axes, which
find associations between the signals of input events and
their consequences. The tactile memory is distributed
among eight such matrices, and there is also some suboe-
sophageal learning capacity. The visual memory lies in
the optic lobe and four matrices, with some re-exciting
pathways. In both systems, damage to any part reduces
proportionally the effectiveness of the whole memory.
These matrices are somewhat like those in mammals, for
instance those in the hippocampus.

The first matrix in both visual and tactile systems re-
ceives signals of vision and taste, and its output serves to
increase the tendency to attack or to take with the arms.
The second matrix provides for the correlation of groups
of signals on its neurons, which pass signals to the third
matrix. Here large cells find clusters in the sets of signals.
Their output re-excites those of the first lobe, unless pain
occurs. In that case, this set of cells provides a record that
ensures retreat.

There is experimental evidence that these distributed
memory systems allow for the identification of categories
of visual and tactile inputs, for generalization, and for
decision on appropriate behavior in the light of experience.

The evidence suggests that learning in cephalopods is
not localized to certain layers or "grandmother cells" but
is distributed with high redundance in serial networks,
with recurrent circuits.

Introduction

Responding appropriately in a complex environment
depends upon the categorization of events and a decision
of what to do. Animals with good brains have the ability
to learn the useful responses to particular events that they
encounter. They may not be born with receptors tuned

Received 7 August 1990; accepted 18 January 1991.

to identify objects or situations, say a rock or a tree or a
fish, but learn the classification of particular sets of stimuli
by virtue of the large number of their neurons. It has been
claimed that this involves simply "the spontaneous emer-
gence of new computational capabilities from the collec-
tive behaviour of large numbers of simple processing ele-
ments" (Hopfield. 1982). Biologists will probably suspect
that a genetic component is involved in the organization.

Cephalopods have such nervous systems with numerous
neurons, and there is sufficient information about their
arrangement to suggest how they function. Formerly, I
have emphasized that the circuits in their brains must
allow for the outputs from feature detectors to produce
alternative effects after learning (Fig. 1). I proposed that
the feature detectors must become restricted during
learning to establish units of memory or mnemons. This
view is correct in that it emphasizes the possibility of al-
ternative outputs from feature detectors, but it is much
too restrictive. Emphasis on units obscures the essential
fact that these are systems with numerous parallel, inter-
acting channels. It is now evident that the various lobes
of the brain provide sequences of matrices of intersecting
axes, with feedback. They enable the identification of cat-
egories of input and storage of records of the probable
value of each, in the form of bias to particular directions
of action to each set of input signals.

The principle of the matrices is to provide for selection
of paths that are used and inhibition of those that are not
used. This is accomplished by various means that allow
interaction between pathways. One of the best analyzed
systems is in the mammalian hippocampus (Fig. 2) (Rolls,
1990). In a competitive learning matrix such as the dentate
gyrus, "different input patterns on the horizontal axons
will tend to activate different output neurons. The ten-
dency for each pattern to select different neurons can be
enhanced by providing inhibition between the output
neurons. . . . Synaptic modification then occurs . . . and

200

COMPUTATION IN CEPHALOPOD LEARNING SYSTEM

201

Feature
detectors

Basal
lobes

Attack
Taste Medsupfr

Vertical

Subvert ic al

Magno
cellular

Retreat

Figure 1. Scheme to show alternative pathways from the visual fea-
ture detectors of an octopus. There are output pathways for attack or
retreat. A third pathway leads to the four matrices of the vertical lobe
system. Here particular patterns of visual signals are combined with those
of taste to increase the future tendency to attack, or with signals of pain
to reduce it.

the response of the system as a categorizer climbs over
repeated iterations" (Rolls, 1990).

In autocorrelation networks, such as the CAi cells
(Fig. 2), the preferred pathways are reinforced by mutu-
ally strengthening each other. In this case, the cells of
the matrix have collaterals that feed back to their own
inputs. These recurrent synapses follow the Hebb rule
so that "any strongly activated cell or set of cells

becomes linked by strengthened synapses with any other
conjunctively activated cell or set of cells" (Rolls, 1990).
As a result, during recall "presentation of even part of the
original pattern . . . comes to elicit the firing of the
whole set of cells that were originally conjunctively
activated."

In the hippocampus, this result is achieved by the col-
laterals of the CA3 cells, which reactivate the dendrites of
their own and a large number of other CA, cells. It may
be that in cephalopods a similar effect is achieved by pass-
ing the signals through a series of lobes, each serving as a
matrix whose output may be returned to a previous
member of the series (Fig. 3). The functioning of any such
matrix system depends on the particular anatomical ar-
rangements and details of synaptic functioning and its
alteration with use. We do not know enough about such
factors in cephalopods to be able to specify precisely how
they operate. However, the system is simple enough to
allow us to follow the whole sequence through these lobes,
from sense organs to motor output, and at least to spec-
ulate about its functioning.

Matrix systems of this sort have the properties that we
associate with complex animal behavior. They ensure that
there is generalization: presentation of even a part of the
original figure, or one like it, activates the firing of the
whole set of cells that were conjunctively activated
("completion""). Moreover, the system continues to operate
even if some of the cells fail to operate or are removed
("fault tolerance"). We can show that these essential

FDgc

CA1

Neoc or te x

Parahippocarripal
gyrus

to septum
mammillary bodies

Parahippccampal
gyrus

1 1

Neocorte x

Figure 2. Schematic representation of the scheme of matrices and connections within the primate hip-
pocampus and with the neocortex. The competitive matrix in the dentate gyrus leads to an auto association
matrix formed by the CA3 cells, which in turn lead to a competitive matrix on the CA, cells (From Rolls,
1990, with permission).

202

J. Z. YOUNG

median

inferior frontal
>2

sub
frontal
3 ,v

trauma

'sucker

Figure 3. Diagram of the connections of the tactile memory system
ofOclopus. The successive matrices are labelled 1 to 8. In addition, there
is some learning capacity in the suboesophageal centers.

properties of memory systems are present in the nervous
system of octopuses.

The nervous system of an octopus provides for cate-
gorization and setting up of memories both for vision and
touch. There are two distinct systems, each made up of
four lobes, with elements whose arrangement can now be
seen to constitute sequences of matrices (Fig. 3, 4). It was
thought at one time that these were independent visual
and tactile systems, but it is now clear that in tactile learn-
ing all eight lobes are involved (Young, 1983). This,
therefore, constitutes a remarkable example of a distrib-
uted memory system using a series of networks. This
model is rather similar to that suggested by Wells (1978).
It does not depend on detailed preformed connections
and so avoids problems of complex morphogenesis.

The Chemo-Tactile Memory System

Octopuses readily recognize differences in the chemical
nature and texture of objects by touch, although they can-
not discriminate between shapes (Wells, 1978). The re-
ceptors for touch are in the rims of the suckers (see Gra-
ziadei in Young, 1971). Their axons proceed through
synapses in the arm, but no details are known of the coding
signals that are sent to the brain.

In our experiments. Wells and I train octopuses to dis-
tinguish between plastic balls, either smooth or with up
to thirteen incised rings (Wells, 1978; Young, 1983). We
train an animal by giving it food when it takes one ball
(say a smooth one) and by giving it no reward or a small
electric shock for taking the other (rough) (Fig. 5). In crit-
ical experiments, the optic nerves are cut to avoid possible

visual discrimination. Many of the experiments are done
after the whole supraoesophageal lobe has been bisected.
The arms of the two sides then learn independently and
can even be trained in opposite directions.

Afferent fibers from the arms and also taste fibers from
the lips cross the dendrite systems of the first tactile lobe,
the lateral inferior frontal (Fig. 6, 7). The axons of the
cells of this first lobe pass partly to the fourth lobe, the
posterior buccal, and partly to the lateral superior frontal
and so to the vertical lobe system (below). The fibers from
the arms and lips then pass on to the second matrix, in
the median inferior frontal, where they interweave and
cross the trunks of a large sample of the 10" cells, this
allows maximum opportunity for any cell of the lobe to
receive signals from a variety of input fibers (Fig. 7). These
median inferior frontal cells then send their axons to the
third matrix, the subfrontal lobe, which contains relatively
few large cells with twisted trunks and many bushy den-
drites and, in addition, a great number (5 X 10b) of very
small amacrine cells. The subfrontal also receives nu-
merous fibers from below, presumed to signal trauma.
The large subfrontal cells send their axons to the fourth
lobe, the posterior buccal, from which, in turn, large axons
pass directly to the arms and cause them either to draw
in or reject the object touched (Budelmann and Young,
1985). These cells must be of two sets, some causing the
object touched to be drawn in, the others to reject it.

subv

med^upfr medinf.fr

ant has

sub.fr

sup. buc

Figure 4. Sagittal section of the supraoesophageal lobe of Octopus
mlgaris stained with Cajal's silver method. Abbreviations for all figures:
ant. bas., anterior basal; b. med., median basal; buc. p., posterior buccal;
cer. br. con., cerebrobrachial connective; cer. tr.. cerebral tract; lat. int.
fr, lateral inferior frontal; lat. sup. fr., lateral superior frontal; mag.,
magnocellular; med. inf. fr.. median inferior frontal; med. sup. fr., median
superior frontal; op., optic; ped., peduncle; post, buc., posterior buccal;
plex., plexiform layer, prec., precommissural: pv., palliovisceral; ret.,
retina; subfr.. subfrontal; sup. buc.. superior buccal; subv.. subvertical;
vert., vertical.

COMPUTATION IN CEPHALOPOD LEARNING SYSTEM

203

4 tests tests -,

smooth + fish 1 smooth + fish 1

i£

73
»_*

*™

v».-'*'^

rough, "~\

v
M

no reward

5 2

— V-*

12 _^

-5

.-*-, rough
x-,+ shock

V-*.,

'v_

11+12 21+2'_

1 t

! ! I 1 1 1 1 1 1 II 1 1 ! 1 1 1 1 ! II

1 5 10 13 15 20

day

Figure 5. Sequence oflearning by 129 control half brains to take a
smooth ball and reject a rough. Four trials daily. On days 1 1 and 1 2.
and 21 and 22, the figure shows mean takes out of 24 unrewarded tests
with each ball (Young, 1983).

The basic action of the system is that an arm cautiously
and slowly draws in an unfamiliar object that it touches.
If this proves to provide food, the taste signals from the
lips activate cells of the lateral inferior frontal, which in-
crease the tendency to take and perhaps operate as a com-
petitive learning matrix (like the dentate gyrus). The pat-
tern of input and taste signals is then passed on to the
median inferior frontal where that proportion of cells that
receive this pattern of signals of touch and taste is acti-
vated. This can be considered a re-coding of the input
pattern on to a more sparse set of cells.

The axons of these cells then proceed through the inter-
weaving bundles to the subfrontal, where they make con-
nection with a still smaller set of large cells with complex
dendritic fields, having also an input of fibers indicating
pain. If trauma occurs and these pain fibers are also ac-
tivated, then the large cells of the subfrontal operate the
rejection neurons of the posterior buccal lobe. The syn-
apses activated by this particular pattern of input become
consolidated, presumably by the action of the large num-
ber of amacrine cells whose short axons end among the
dendrites of the larger cells of the subfrontal lobe (Fig. 6).

The basic operation of the system is thus to take objects
touched unless signals of pain arrive. Signals of taste set
up a greater tendency to take by competitive learning in
the lateral inferior frontal lobe. Signals of pain set up a
tendency to reject that pattern of touch by modification
of synapses in the subfrontal lobe. As good evidence of
this it was found that, after lesions destroyed all the small

amacnne cells, an octopus tailed to learn not to take ob-
jects from which shocks were received (see this paper and
Wells. 1978).

The Vertical Lobe System and Touch Learning

The inferior frontal system contains the major tactile
memory, but the vertical lobe also contributes. Experi-
ments show that removal of the vertical lobe impairs the
tactile memory, but removal of the median inferior frontal
has no effect on visual learning.

The tactile signals enter the vertical lobe circuit through
fibers from the lateral inferior frontal that enter the outer
plexus of the lateral superior frontal lobe (Fig. 3). The
vertical lobe system contains four lobes precisely similar
to those we have described in the inferior frontal. The
lateral superior frontal sends fibers to the subvertical lobe
and from there fibers pass down the cerebral tract to the
posterior buccal lobe (Fig. 6). This circuit through the
lateral superior frontal is thus in a position to increase
still further the tendency to take objects that have been
associated with taste reward.

The signals for touch are then passed on from the lateral
to the median superior frontal. Here the bundles are again
interwoven, exactly as in the median inferior frontal. The
1.8 X 106 cells thus receive varied combinations of signals
of touch and taste, and these are passed on again through

medinf f-

certr

Figure 6. Diagram of connections in the inferior frontal system of
an octopus (Young, 197 1 ).

204

J. Z. YOUNG

Figure 7. Transverse section of the inferior frontal system of Octopus
vii/garis stained with Cajal's silver method. Abbreviations as Figure 4.
cr. tr.. cerebral tract (from subvertical lobe): p. tract of probably pain
fibers from hind end of bodv.

a complex plexus to the vertical lobe. Here there are rel-
atively few large cells (65,000), with complex dendrites,
exactly like those of the subfrontal, and no less than 25
million amacrine cells.

The large cells send their axons down to the subvertical
lobe and so to the posterior buccal, but also back to the
lateral superior frontal (Fig. 3). This circuit evidently plays
some part in re-enforcing the conjunctions, possibly by
maintaining particular patterns by re-excitation.

The Distributed Tactile Learning System

The system for touch learning thus includes no less
than eight distinct lobes with matrix structure (Fig. 3).
The relative parts played by the various lobes was studied
over a number of years in a large number of animals with
divided brains. Lesions were made on one side, and the
other was left as a control. In many of the experiments,
discrimination was between completely smooth balls (0
rings) and those with 13 incisions. The sequence of train-
ing for 129 normal sides is shown in Figure 5.

A useful measure of the extent and reliability of dis-
crimination is to give a series of 24 extinction tests with
balls of differing roughness, shown at short intervals (1-
3 min) without any reward. Such tests are arduous to
give, but they show that habituation proceeds more slowly
in proportion to similarity of each ball to the one for
which reward was previously given (Fig. 8). The capacity
for discrimination was also tested by using more nearly
similar balls, with 4 and 7 rings. With long training, oc-

topuses could probably make some discrimination even
between a difference of one ring.

By such tests we can compare the discrimination by
animals after various lesions. Without the median inferior
frontal there is still discrimination, but it is much less
accurate than in control animals (Fig. 9). Removal of the
vertical lobe also reduces accuracy, although to a lesser
extent. Clearly each of the lobes through which the in-
formation passes adds something to the effectiveness of
the representations that are formed, as would be expected
from a system of matrices.

Animals without vertical lobes show errors largely when
they take the negative ball, showing again that this lobe
serves to increase the effectiveness of shocks. In normal
octopuses, learning is possible even if rewards are delayed
for up to 30 s after the ball has been removed. In animals
without vertical lobes, such delay is no longer possible
(Wells and Young, 1968). The re-excitation within the
vertical lobe system serves to maintain the necessary ex-
citability for a Hebb type of learning.

We have used this technique to make a large number
of experiments, leaving one side as a control. With this
technique it is possible to remove the subfrontal lobe,
which cannot be approached laterally. The effect is to
produce a complete inability to learn on that side: there
is a strong and irreversible preference for the rougher balls
(Young, 1983). The lobe evidently has some specific effect
on the coding and discrimination process.

After cutting the cerebro-brachial tract (Fig. 6), the
whole influence of the inferior frontal and vertical systems
is removed. Nevertheless, there is still a slight capacity for
learned discrimination (Fig. 10), which must lie in the
suboesophageal ganglia, or in the arms themselves. This
residual learning is difficult to study. Animals with these

CONTROL HALF-BRAINS

0 4 6 9 13
number of rings

Figure 8. Tests after training in three different directions. Means
and standard errors of ratios of takes of each ball to total takes, with 24
trials with each ball. The bars show standard errors.

COMPUTATION IN CEPHALOPOD LEARNING SYSTEM

205

~\NV

"0469 13 0469 13 0469 13

number nf rings

Figure 9. Tests after training. Comparison of results (a): 1 1 animals after cutting the tract between
superior frontal and vertical lobes on one side (NSFV) and removing the vertical lobe (NV) on the other
side; 36 controls for comparison (b): 36 animals after removing the median interior frontal (NMIF); controls
and NV added for comparison. In a & b, all training was with smooth balls positive and 13 rings negative.
In (c) balls with six rings were positive and smooth balls negative. The figure shows the results for nine
animals with the median inferior frontal removed on one side three animals with no vertical lobe on one
side added for comparison. Note that here (and Fig. 8) the balls with 9 and 13 rings were seldom taken,
although they had not been associated with shock.

large operations do not feed well and tend to hold pieces
of food and other objects close to the mouth. Nevertheless,
the differences in numbers of takes of the balls during
tests are significant and show that some learning has oc-
curred.

Some measure of the accuracy of the memory after the
various lesions is given by the difference between the takes
of the rough and smooth balls in the tests (Table I). Using
the difference in controls as a standard (100%), we can
judge that animals without vertical lobes are rather less
than half as efficient, without the median inferior frontal
are one third as efficient, and that the suboesophageal
contribution is about one sixth. Damage to the subfrontal
produces a perverse effect.

The Visual Memory System

The vertical lobe system, which plays a part in tactile
learning, forms, with the optic lobes, the main and only

control

control trained 13-t-_,
trained 0-*- n~L6

n=36

I 20

NCB.13+

component of the visual learning system. The existence
of this double capacity is a striking demonstration of the
power of such a distributed matrix system to store a variety
of inputs. It will be very interesting to investigate whether
individual cells of the vertical lobe system play a part in
both systems. In a study of combined visual and tactile
training, no mutual interactions between the two modal-
ities was seen (Allen ct at., 1986).

The study of visual memory is made difficult by the
complexity of the connections in the optic lobes, which
are not fully understood. Cells with large tangential den-
drites in the plexiform layer probably act as feature de-
tectors (Fig. 1 1 ). Their axons form columns proceeding
to the center of the lobe, where they interact in an inter-
weaving matrix of cells and fibers. Second or third order
visual neurons then send axons to the central nervous

Table I

Mean takes in final tests after various lesions

04 6 9 13
number of rings

Figure 10. Tests after training. Comparison of control sides with
those with the cerebrobrachial tracts cut (NCB).

Smooth

Rough

Percent

ball

accuracy

Lobe removed

(positive)

(negative)

Difference

remaining

None (controls)

20.22

2.44

17.78

100

Vertical

17.68

10.55

7.13

Median inferior

frontal

16.32

10.69

5.63

Cerebro-brachial

tract

13.78

10.94

2.84

Subfrontal

7.89

8.42

-0.53

—

The last column indicates the capacity for learned discrimination that
remains after the lesions, estimated as a percentage of the differences in
the controls.

206

J. Z. YOUNG

vert

med sup f r
I at sup fr

buc

op.

ret

Figure 11. Diagram of the connections of the visual and tactile
learning system of an octopus. 1, retina; 2, second order visual cells
(feature detectors): 3, centrifugal cells; 4, amacrine cells; 5, tangential
cells; 6, optic-peduncle; 7. optic-magnocellular; 8, optic-anterior basal;
9. optic-median basal; 10. optic-lateral superior frontal; 1 1, optic-median
superior frontal; 12. taste fibers-lateral inferior frontal; 13, taste fibers
median inferior frontal; 14, lateral inferior frontal-lateral superior frontal;
15. lateral inferior frontal-median superior frontal; 16, median superior
frontal-vertical; 17, vertical-lateral superior frontal; 18, lateral superior
frontal-subvertical; 19, pain fibers-vertical: 20, pain fibers-subfrontal; 2 1 ,
vertical-subvertical: 22, subvertical-optic; 23, subvertical-precommissural:
24, precommissural-palliovisceral; 25, precommissural-magnocellular;
26, subvertical-posterior buccal; 27, chemo-tactile libers-lateral inferior
frontal; 28, chemo-tactile fibers-median inferior frontal; 29, median in-
ferior frontal-subfrontal; 30. subfrontal-postenor buccal; 31, lateral in-
ferior frontal-posterior buccal; 32. motor fibers from posterior buccal to
arms.

system (Fig. 1 1). Some pass to the magnocellular lobe,
and this is probably a pathway for rapid escape reactions.
Other fibers pass to the peduncle and basal lobes, which
together regulate movement, including attack. A third
pathway leads to the superior frontal, and so to the vertical
lobe, and is responsible for learned behavior.

The system is organized exactly as we have seen for
tactile learning. In the lateral superior frontal, the visual
fibers interact with those of taste, and this is a pathway
that promotes attack. After removing this lobe from one
side, an octopus will no longer attack when that eye has
been used to see a crab, for instance, at a distance (Boycott
and Young, 1955). The median superior frontal and ver-
tical lobes provide a system that prevents visual attack
when trauma occurs. After removal of these lobes or an

interruption of the circuit, an octopus will continue to
make attacks, even at crabs, in spite of receiving shocks,
unless these shocks are given at intervals of five minutes
or less. "The setting-up of a memory representing asso-
ciation of a given situation with a shock is therefore a
property of the optic and basal lobes but persistence of
the representation depends upon the presence of the ver-
tical lobe" (Boycott and Young, 1955).

Many other experiments have confirmed that learning
of visual discrimination is impaired by lesions of the ver-
tical lobe system (Young, 1961. 1965). If part of the ver-
tical lobe is removed, the accuracy of the memory is pro-
portionately reduced. This "graceful degradation" is a
property to be expected in such a distributed system. In-
cidentally, Boycott and I were able to show that the same
is true of the optic lobes. Memories are retained after re-
moval of at least 50% of the lobe or after making lesions
in several places with a cataract knife.

Discussion

The two memory systems of an octopus thus work on
precisely similar principles. The input signals are passed
through a series of matrices of intersecting axes allowing
for particular groupings of signals to interact and to be
directed to the pathways for attack or retreat. The systems
are tuned to produce exploratory investigation of novel
situations. If the results are favorable, the particular set
of connections in the lateral frontal lobes are re-enforced
by signals of taste, and this set later produces more rapid
attacks or takes by the arms. The inputs are given further
opportunity for interaction in the matrices of the median
frontal lobes. In the vertical and subfrontal lobes, partic-
ular sets are then concentrated into rather few large cells.
The recurrent output from these to the lateral superior
frontal lobe presumably re-enforces the tendency to pos-
itive action, unless pain occurs. In that case, the other
outputs from these large cells of the vertical or subfrontal
prevent further investigation of that configuration of in-
puts. The numerous amacrine cells in these lobes are ev-
idently concerned with establishing the conjunction be-
tween particular sets of input signals and the pathways of
retreat.

The organization of these lobes, and the effects of re-
moving them, suggests that learning in these animals is
not localized to one or two "hidden layers" or to a few
essential "grandmother cells," but is distributed with high
redundancy in a series of matrices networks, with recur-
rent circuitry, up to a late stage where funneling to a few
cells occurs.

We can gain some insight into how this process has
evolved by considering the differences between octopods
and decapods. Cuttlefishes and squids have a system of
matrices for visual learning similar to that of octopods

COMPUTATION IN CEPHALOPOD LEARNING SYSTEM

vert B

207

Figure 12. Sagittal section of the brain of Sepia. Note that there is
no median interior frontal or subfrontal. The superior frontal has a matrix
structure like that of Octopus. The vertical lobe has a rather different
structure. Cajal silver stain, b.a., anterior basal; h. med., median basal;
fr. ].. interior frontal; fr. s., superior frontal; prec., precommissural; subv.,
subvertical; v.. vertical.

(Fig. 12). In an early experiment it was shown that inter-
ruption of the vertical lobe circuit damages the visual
memory system of Sepia (Young, 1938; Sanders and
Young. 1940). This was the first suggestion that the cir-
culation of impulses around a circuit provides a basis for
memory (Fig. 13). There has been little further progress
because the experiments are more difficult than in octo-
pods. In decapods, the inferior frontal system is much
simpler than in octopods: there is no median inferior
frontal or subfrontal lobe. These animals detect the prey
visually and often seize by ejection of the tentacles. It
seems likely that they have, at best, only a small capacity
for learned tactile discrimination; the operations of ma-
nipulating and eating the prey are complex, but are prob-
ably largely reflex. Nevertheless, there must be a mecha-
nism for release of any object that gives pain when it is
held. Probably all reflex systems have some method of
inhibition, especially if they involve muscles acting recip-
rocally, such as flexors and extensors in mammals, where
the inhibition is produced by Golgi type II cells in the
spinal cord. In cephalopods, reciprocal inhibition is prob-
ably produced by the smaller amacrine neurons that are
common among the larger motorneurons of the superior
buccal and suboesophageal centers (Fig. 14). These mi-

Figure 13. An early suggestion of re-excitation as the basis of memory.
Diagram of Scpiu to show how circulation between the lateral superior
frontal (lat. sup. fr.) and vertical (vert.) might facilitate the firing of a
motorneuron (M) by conjunctive excitation from the optic lobe (O) and
taste fibers (V)( Young.

croneurons have processes restricted to a limited field,
where they may serve to repress activity in the larger cells.
In this context, it is especially interesting that we found
some simple capacity for tactile memory in the suboe-
sophageal lobes.

It is suggested that the amacrine cells of the subfrontal
and vertical lobes of octopods have evolved from inhib-
itors of the reciprocal feeding reflexes. The inferior frontal
and vertical lobe systems are backward extensions of the
superior buccal lobes (see Fig. 4). The matrices that are
responsible for learning have evolved by the modification
of these simpler reflex centers. The incoming afferent fibers
have become marshalled into rows crossing the axons of
cells of the lobe, allowing the formation of conjunctive
response to the incoming patterns of stimuli. The ama-
crine cells became collected together in distinct lobes,
serving to prolong the effects, perhaps especially of inhib-

Figure 14. Drawing ofa single large cell from the pedal lobe of Loligo.
accompanied by very small cells with branches in the neighborhood.

208

J. Z. YOUNG

itory inputs. The details are far from clear, but this pro-
vides a possible scenario for the evolution of memory
mechanisms, at least among cephalopods.

Acknowledgments

The work reported here has been helped by many col-
leagues. I am especially grateful to Brian Boycott, Martin
Wells, Marion Nixon, and Pamela Stephens. The Stazione
Zoologica at Naples provided excellent conditions for ex-
periment over many years. Recently, work has been
helped by grants from The Wellcome Trust and University
College, London. I am grateful to Professor L. Weiskrantz,
F.R.S., for accommodation in the Psychology Department
at Oxford and to my wife Raye for her secretarial help
and typing. Dr. M. J. Wells kindly commented on an
early draft of the paper.

Literature Cited

Allen, A., J. Michels, and J. Z. Young. 1986. Possible interactions
between visual and tactile memories in octopus. Mar. Behav. Physiol.
12:81-97.

Boycott, B. B., and J. Z. Young. 1955. A memory system in Octopus
vulgaris Lamark. Prof. R Soc. B 143: 449-480.

Budelmann, B.-l ., and J. Z. Young. 1985. Central nervous pathways
for the arms and mantle of Octopus. Phil. Trans. R Soc. B 310:
109-122.

Hopfield.J. J. 1982. Neural networks and physical systems with emer-
gent collective computational abilities. Proc Natl. Acad. Sci. USA
79: 2554-2558.

Rolls, E. T. 1990. The representation and storage of information in
neural networks in the primate cerebral cortex and hippocampus. In
The Computing Neuron. R. Durbin. C. Miall, and G. Mitchison, eds.
Addison-Wesley, Wokingham.

Sanders. F. K., and J. Z. Young. 1940. Learning and other functions
of the higher nervous centres of Sepia. J. Neurophysiol. 3: 501-526.

Wells, M. J. 1978. Octopus. In Physiology and Behaviour of an Ad-
vanced Invertebrate. Chapman and Hall. London.

Wells, M. J., and J. Z. Young. 1968. Learning with delayed rewards
in Octopus. Z I'ergl. Physiol. 61: 103-128.

Young, J. Z. 1938. The evolution of the nervous system and of the
relationship of organism and environment. Pp. 1 79-204 in Evolution.
essays presented to E. S. Goodrich, G. R. de Beer. ed. Clarendon
Press, Oxford.

Young, J. Z. 1961. Learning and discrimination in the octopus. Biol
Rev 36: 32-96.

Young, J. Z. 1965. The organization of a memory system. The Crooman
Lecture. Proc. R Soc. B 163: 285-320.

Young, J. Z. 1971. The Anatomy of the Nervous System of Octopus
vulgaris. Clarendon Press. Oxford.

Young, J. Z. 1983. The distributed tactile memory system of Octopus.
Proc. R Soc. B 218: 135-176.

Reference: Biol Bull 180: 209-220. (April. 1991)

Development of Giant Motor Axons and

Neural Control of Escape Responses

in Squid Embryos and Hatchlings

W. F. GILLY, BRUCE HOPKINS, AND G. O. MACKIE*

Hopkins Marine Station, Department of Biological Sciences,
Slanf'onl University, Pacific Grove, California 93950

Abstract. Anatomical development of the third-order
giant axons was studied in conjunction with ontogeny of
the escape response and the underlying neural control.
Stimulated escape jetting appears at stage 26 (Segawa el
a/.. 1988): such responses are driven solely by a small
axon motor system. Giant axons become morphologically
identifiable in the more posterior stellar nerves that effect
jetting by stage 28, and electrical activity in the stellate
ganglia associated with the giant axons is first recordable
at this time. Maturation of the giant axons is accompanied
by a marked improvement in temporal aspects of escape
behavior up to the time of hatching. In embryonic and
hatchling Loligo. all escape responses, regardless of the
mode of stimulation, are fast-start responses with latencies
less than the minimum value displayed by adults (50 ms).
Giant axon activity recorded in the stellate ganglion always
precedes small axon motor activity; this is not true for
adults which display two distinct modes of giant axon
use. Both giant and non-giant motor systems are thus
functional in embryonic and hatchling squid, and both
contribute to escape jetting. However, these animals do
not yet display the concerted interplay of the two motor
systems characteristic of adults.

Introduction

Lolliginid squid possess giant neurons with very large
axons that are important components of the motor path-
ways mediating jet-propelled escape responses (Young,
1938). Anatomical details of the giant fiber pathway were
beautifully described at the light microscope level over 50
years ago by Young ( 1939). In brief, two bilaterally sym-

Received 15 August 1990; accepted 6 November 1990.
* Permanent Address: Department of Biology, University of Victoria,
Victoria. B.C., Canada.

metrical first order interneurons lie in the magnocellular
lobe of the brain and receive massive sensory inputs from
many sources, including the statocysts, optic lobes, and
mechanoreceptors in the tentacles. These cells are unusual
in that their short axons are fused via a cytoplasmic bridge.
Each first-order giant axon contacts seven second-order
giant cells in the palliovisceral lobe. The largest of these
cells are interneurons, and each projects a giant axon via
the pallial nerve to the ipsilateral stellate ganglion where
it contacts the third-order giant motor axons of the stellar
nerves. The six other second-order giant cells are moto-
neurons that innervate the musculature associated with
head retraction, siphon aiming, and ink ejection.

This basic plan is straightforward, but oversimplified.
Each of the above giant cells receives numerous other
synaptic inputs, about which little is known (Boyle, 1986).
The first-order giants also receive major inputs from
higher-order centers such as the cerebral ganglia. Inte-
grated outputs from these regions must influence activity
of the first-order giants, which are likely to be an important
decision-making element, given the commanding ana-
tomical position they hold. Complexity in the giant fiber
pathway and the biological necessity to strictly control
giant fiber excitation was clearly recognized by Young
(1939), who pointed out that lack of such control ". . .
would lead to behavior by the squid even more 'nervous'
than that for which the animals have a reputation."

Despite the wealth of anatomical data, physiological
studies of squid escape behavior have lagged far behind
work on many other preparations (Eaton, 1984; Mackie,
1990). The pioneering work of Young [1938; Prosserand
Young (1937)], in which reflex activation of escape jetting
was inferred from studies of nerve-mantle preparations,
was followed up only much later by Wilson (1960), who
studied control of mantle contractions by a small axon

209

210

W. F. GILLY ET AL

system as well as by the giant axons. Since that time it
has become widely accepted that ( 1 ) high-pressure escape
jetting is mediated solely by the giant axon pathway es-
sentially as a reflex and that (2) low-pressure respiratory
'jetting' is the primary, if not only, function of the small
axon (non-giant) motor system.

Reinvestigation of these ideas has revealed a more
complex picture (Otis and Gilly. 1990). Recordings of
stellar nerve activity during escape responses //; vivo show
that strong escape jets can be driven by the small axon
system acting independently or in concert with the giant
axons. Moreover, the giant axon pathway can be used in
two distinct modes. One produces a short latency startle
response, whereas the second leads to a complex delayed
escape response. In the latter case, critically timed exci-
tation of the giants provides a potent, but secondary boost
to the jet.

The present study investigates the neural control of es-
cape jetting in embryonic and hatchling squid. All three
neural elements of the giant fiber pathway are highly de-
veloped at the time of hatching (Young, 1939), and escape
jetting capabilities are respectable (Packard, 1969). Em-
bryonic development of the first-order giant neurons has
been studied in detail (Martin. 1965, 1969, 1977), but
much less is known about the second- and third-order
giants (Marthy, 1987). Behavioral and neurophysiological
studies concerning development of the giant fiber pathway
are completely lacking.

This paper describes the ontogeny of the escape re-
sponse in relation to development of the giant fiber system,
with emphasis on the role of the third-order motor axons.
Several questions are addressed, answers to which provide
the framework for future studies. When do the giant motor
axons develop morphologically, and when do they begin
to mediate escape responses'? What functional conse-
quences are manifested in parallel with growth of the giant
axons? How does the pattern of giant axons use compare
to that in the adult animal? Finally, when does controlled
interplay between the giant and non-giant motor systems
develop?

Materials and Methods

Animals

Loligo opalescens was collected from Monterey Bay
and maintained in holding tanks plumbed with flow-
through seawater (~ 13°C). Spawning occurred in these
tanks, and clusters of fertilized eggs were maintained in
small mesh enclosures in gently flowing seawater until
natural hatching occurred ( ~30 days). Staging of animals
was carried out following criteria of Segawa et ai ( 1988)
with only minor modifications (see Results and Table I).

Sepioteuthis lessoniana was supplied by the Marine
Biomedical Institute, University of Texas Medical Branch,
Galveston, where the animals had been reared. Animals

ranging from 5 to 2 1 days post-hatching were shipped
overnight and studied the following day.

Behavioral experiments

LoHgo from stage 24 through post-hatching was used
for studying development of escape-jetting behavior. To
obtain animals for each experiment, a "finger" of eggs
was disrupted, the eggs were dispersed in seawater, and
the chorionic membranes were ruptured to release the
embryos. Individual embryos were chosen from this pool
and staged under a stereomicroscope.

After staging, an animal was placed in a 35 mm culture
dish lined with Sylgard (Dow-Corning, Midland, Michi-
gan) and filled with seawater at room temperature ( 16-
20°C). The animal was lightly restrained (ventral surface
up) with a wire yoke, which was formed to fit over the
base of the arms and inserted into the Sylgard. Electrical
stimuli (20-60 V. 6 ms duration) were delivered via a
seawater-filled micropipette positioned near the ventral
midline in the area of the brachial ganglion (see Fig. 2A).

Behavioral responses were recorded on conventional
videotape. The stimulating pulse also triggered a small
light source beneath the experimental chamber, which
served as a timing marker. Although the exact frame when
the stimulus occurred is identifiable with this method,
stimuli were not synchronized with the video framing and
uncertainty therefore exists about precisely when in the
frame the pulse occurred (each video frame spans 33 ms).
Data were later sampled in blocks of 16 real-time frames
with an image-analysis system (Megavision 1024 XM.
Santa Barbara. California) and analyzed on a frame-by-
frame basis to determine the time course of the change
in mantle diameter following stimulation (see Fig. 2B).

Electrophysiologiccd experiments: adult squid

Adult specimens of Loligo were lightly anesthetized in
0.4% urethane in aerated seawater at 15°C. The mantle
was slit ventrally along the midline, exposing the gills,
and the squid was pinned ventral side up through the
outspread mantle flaps to the bottom of a Sylgard-filled
glass dish. A pin through the region of the mouth pre-
vented head retraction. The left pallial nerve was cut
proximal to the stellate ganglion, thereby immobilizing
the mantle musculature on that side. On the right side,
the stellate ganglion was exposed by removing the thin
layer of skin that covers it. The larger, more posterior
stellar nerves were severed close to their emergence from
the ganglion, but the pallial nerve was left intact. This
paralyzed the right side of the animal but retained synaptic
transmission at the giant synapses and motor outputs in
the stellar nerve stumps. Throughout these operations,
chilled seawater was kept flowing over the gills. Upon
completion of surgery, the urethane-seawater was ex-

DEVELOPMENT OF SQUID ESCAPE RESPONSE

211

changed for O:-saturated seawater ( 12-15°C) which was
thereafter perfused continuously over the gills.

Conventional extracellular recording techniques were
used. One polyethylene suction electrode was attached to
the pallial nerve for en passant recording. A second elec-
trode was used to record from a stellar nerve by sucking
the proximal stump into the lumen of the electrode. Volt-
age was measured differentially between Ag:AgCl wires,
one in the electrode's lumen and the other wrapped
around the tip. Polarity of the recordings was arranged
so that the initial phase of activity in the third-order giant
axon was positive-going. AC-coupled amplifiers served to
amplify and low-pass filter (3-10 KHz) the signals, which
were displayed on a digital oscilloscope or recorded onto
videotape via a digital audio processor (sampling at 44
KHz; Unitrade, Philadelphia, Pennsylvania) for subse-
quent analysis using a laboratory computer.

Electrophysiological experiments: embryonic and
hatchling squill

Recording techniques were adapted from those used
with the adult animals. Hatchlings and embryos (removed
from their chorions and staged as in the behavioral ex-
periments) were pinned out with fine cactus spines, ventral
side up, after slitting the ventral mantle wall to allow access
to the stellate ganglia. No nerves were cut, and recordings
from the stellate ganglion were made with a suction elec-
trode applied directly over the ganglion through the over-
lying tissue. Recordings from the magnocellular lobe (site
of the first-order giant neurons) required removal of the
skin and cartilaginous material just anterior to the stato-
cysts, and the recording electrode was applied directly to
the nervous tissue thereby exposed. It was also necessary
to remove some of the mass of small cells, presumably
undifferentiated neuroblasts (Martin, 1965), which overlie
the first-order giant cells by sucking or blowing water jets
through the electrode tip before attaching it.

For all neurophysiological recordings, electrical stimuli
were delivered through a fine coaxial metal electrode, and
a triggered strobe light was used for delivering photic
stimuli. Shocks of 1-3 ms. 20-80 V were most effective
in stimulating adults and 0.1-0.4 ms, 10-50 V in hatch-
lings and embryos. All experiments were carried out at
12-15°C in the case ofLoligo and 2 1 °C with Sepioteutliis.

Electron microscopy

Staged embryos were fixed in a 0.965 osmolar solution
of 1% glutaraldehyde, 0.1% tannic acid. 0.2 M sodium
cacodylate and sucrose (pH 7.4) and post-fixed in 2% os-
mium tetroxide, 0.8% potassium ferrocyanide, and 0.2 M
sodium cacodylate (pH 6.8). Fixed material was dehy-
drated in graded hexalene glycol and embedded in Spurr's
resin. Animals were sectioned both perpendicular and
oblique to the long body axis to obtain transverse sections

of the posterior stellar nerves. En bloc staining was carried
out in saturated uranyl acetate at 60°C for 3 h. Thin sec-
tions were examined in a Philips 30 1 transmission electron
microscope operating at 60 kV.

Results

Developmental timetable of giant motor a.\ons and
jetting behavior

Loligo opalescens develops embryologically in close
correspondence to L. forbesi, and staging criteria for the
latter species (Segawa et al., 1988) can be directly applied.
Stages 24 through hatching (30), representing approxi-
mately half of the total developmental period, are relevant
to the present study, and selected characteristics applicable
to our staging of L. opalescens are summarized in Table
I. Based on our work with hundreds of embryos in this
study, ontogeny of jetting behavior can be related to these
anatomical stages with a high degree of confidence, and
this information is included in Table I and covered in
detail below.

Electron microscopic examination of conventionally
fixed and embedded material reveals that axons in stellar
nerves which can be labeled 'giant' (i.e., distinctly larger
than any other processes) first occur at stage 26, but only
in the more anterior nerves emanating from the stellate
ganglion (Fig. 1A). The more posterior stellar nerves do

Table I

Staging characteristics employed and development of escape behavior
in embryonic Loligo opalescens. Developmental staging follows
the criteria described by Segawa et al. (1988) for Loligo forbesi.
except in our mirk \\e lumped stages 27 and 27+ (into 27)
and 28 and 2S+ (into 28)

Stage 25: 1. Spontaneous, symmetrical mantle contractions begin.

Stage 26: 1. Eyes are brilliant red.

2. Ink sac is visible.

3. Chromatophores are on dorsal side of head.

4. Electrical and mechanical stimulation of escape response
is possible.

Stage 27: 1. Ink is barely visible in ink sac.

2. Eyes are dark, but not black.

3. Edge of primary lid covers half of optic vesicle.

Stage 28: 1. Eye is completely covered by primary lid.

2. Mid-gut gland is visible.

3. Yellow chromatophores are on arms.

4. Head retraction occurs during escape responses.

Stage 29: 1. Olfactory organ is visible as thickened disk.
2. Light flashes stimulate escape responses.

Stage 29+: 1. External yolk sac is equal to arm length.
Stage 29+ + : 1. Spontaneous vigorous jetting occurs.
Stage 30: 1. Natural hatching occurs; no external yolk sac.

212

W. F. GILLY ET AL.

C Stage 28

Stage 30

Figure 1. Anatomical development ot the giant axons in stellar nerves of embryonic Loligo opalescens.
Each panel shows a cross-section of a stellar nerve at the indicated developmental stage. (A) A single large
axonal process is first identifiable in an anterior stellar nerve at stage 26 (*), although other processes are

DEVELOPMENT OF SQUID ESCAPE RESPONSE

213

not display a singularly large axon leading to the mantle
muscle at this time (Fig. IB), but clearly do so by stage
28 (Fig. 1C). As discussed below, stage 28 is the first time
at which giant axon activity could be recorded from the
stellate ganglion. Well-developed giant axons exist in all
stellar nerves at the time of hatching, and an example of
a hind-most nerve is shown in Figure ID.

We have not characterized the apparent anterior to
posterior wave of giant axon maturation in detail, nor do
we at this time have a complete picture of where, when,
and how the axons of giant fiber lobe motoneurons ac-
tually fuse to form the giant axons prior to stage 28. Giant
processes can be identified in all stellar nerves at stage 27,
but (in every case examined) only proximal to the first
branch point of a stellar nerve shortly after it enters the
mantle tissue.

Development of escape response: behavioral studies

Escape responses in embryonic and hatchling squid
were stimulated with electrical shocks, strobe light flashes,
or mechanical stimuli and videotaped for analysis. An
example of such data from a stage 29 embryo is shown
in Figure 2A. Sequential video frames, photographed from
the display monitor of the image analysis system, are
numbered — 3 through 4. A brief electrical stimulus was
applied during frame 0, and the timing is identifiable by
a light flash marker (*). Mantle diameter is indicated by
arrowheads in each panel, and Figure 2B illustrates the
time course of mantle contraction ( 1 frame equals 33 ms).
After a delay of one frame, mantle diameter decreases to
40% of its original (time 0) value over the subsequent four
frames.

Peak response, delay, and time to peak (as indicated
in Fig. 2B) were measured for electrically stimulated jets
in 3-4 animals of every developmental stage from 25
through several days post-hatching. Mean data are sum-
marized in Figures 3A-B, along with values obtained in
similar experiments on adult animals in a previous study
(Otis and Gilly, 1990).

Escape jetting increases in strength (Fig. 3A) smoothly
up to hatching and then begins to slowly decline. Electrical
stimuli (•) can elicit relatively strong responses at stages
26-27, a time when there is no anatomical sign of giant
axons in the more posterior stellar nerves that innervate
the mantle area in which diameter was monitored. Pre-
sumably these responses are mediated by the small motor
axon system, which also can drive strong escape jets in
adults. There is no dramatic sign of increased strength in

the response accompanying the appearance of functional
posterior giant axons by stage 28. Mechanical stimulation
(A; taps with a fine probe) also leads to strong escape jets,
and light flashes (O) become effective at stage 29.

A functional correlate of the development of the giant
axons in the posterior stellar nerves is suggested in Figure
3B. Both time-to-peak response (•) and delay (•) decrease
dramatically between stage 29 and hatching. As described
below, giant axon activity can be first recorded in the
stellate ganglion at stage 28, and the developmental de-
crease in behavioral delay (Fig. 3B) is also evident in the
electrical recordings (Fig. 10A).

Development of giant axons thus improves temporal
aspects of jetting performance. Acceleration of a squid
through water is a function of both intra-mantle pressure
and the rate of change in pressure (O'Dor, 1988). Rate of
change in mantle diameter is thus an important deter-
minant of escape performance. Figure 3C plots the max-
imum change in mantle diameter (data in Fig. 3A) divided
by the frames to peak change (data in Fig. 3B) for each
developmental stage. A sharp rise in the rate of mantle
contraction occursjust before hatching, and performance
then declines towards the adult level.

Recordings of motor activity in adult Loligo

In a previous study examining the functional role of
the giant motor axons (third-order) in escape jetting (Otis
and Gilly, 1990), adult squid were attached to a plastic
support platform by their dorsal mantle surface but were
otherwise free to make unrestrained respiratory and
swimming movements. Under these relatively natural
conditions, it was demonstrated that squid show appar-
ently normal behavioral responses in regard to escape-
jetting when compared with free-swimming animals in
large tanks. The present study goes a step further and
shows that similar neural responses can be obtained with
dissected, pinned, and inverted animals.

Electrical stimulation directly over the magnocellular
lobe in an adult squid leads to a large, short latency
action potential in the pallial nerve (2) followed by a
spike in an ipsilateral stellar nerve (3) as illustrated in
Figure 4A. These 'directly' evoked events are assumed
to result from direct activation of the first-order giant
cells and represent the sequential firing of the second-
and third-order giant axons, respectively, as described
by Bryant (1959).

The electrode on the stellar nerve also picks up a small
potential just before the third-order spike, and this pre-

almost as large. (B) No singularly large axon exists in the hind-most stellar nerve at stage 26. (C) By stage
28, a distinctly large 'giant' axon is present in the hind-most stellar nerve (*). The fin nerve lies to the left
of the stellar nerve and is composed entirely of small axons. (D) The hind-most stellar nerve at the time of
hatching shows a well-developed giant axon (*).

214

W. F. GILLY ET AL.

(1)

(D
O)

.c
o

-40 -i

-20 -

Time to peak

^ ^

, 4

•

* •

Delay
• * t • t

Peak r

t t T T " i i ' i '

-20246

response

*
Video

Frame Number

Figure 2. Escape behavior of a stage 29 Loligo embryo in response to an electric shock. (A) Sequential
video frames are illustrated; the stimulus was applied via the pipette during frame 0 (*). Arrows indicate
mantle diameter (measured at the widest point). See text for additional details. (B) Data from the experiment
in Figure 2A is plotted in graphical form, and the parameters measured are indicated.

sumably represents the arrival of the second-order wave
in the stellate ganglion. The delay between its peak and
the start of the third-order event is approximately 1 ms
and represents synaptic delay at the giant synapse. This
characteristic short-latency pattern of activity in the stel-
late ganglion was consistently observed in adults (Fig. 4A),

hatchlings (Fig. 4B), and embryos (Fig. 6A) only when
stimuli were applied directly over the magnocellular lobe.
When the squid is stimulated by a light flash (Fig. 5A, B)
or by electrical shocks in regions other than the immediate
vicinity of the first-order giant cells, e.g., the tentacles (Fig.
5C, D), 'indirect' responses are obtained. These occur after

DEVELOPMENT OF SQUID ESCAPE RESPONSE

215

Peak Response

(% change)

o •-•

"•-•-

25 26 27 28 29 29+ 29t30
••-Embryonic Stage *-°-*-

i — i

Days post Hatching

Frames to Peak

Frames Delay

,
•

-f— i — i — r

26 28

% Change
Frames to Peak

1 1 1 1 1 1 1 1 1 1 1 1 I 1 I

26 28 29» 30 2 4 6 8

Figure 3. Ontogeny of escape jetting in Loligo. Three or four animals
at each developmental stage were studied as described in conjunction
with Figure 2. and mean values are plotted. Adult values are from data
obtained in a previous study (Otis and Gilly, 1440). Electrical (•, •).
tactile (A), and photic (O, D) stimuli are individually plotted. (A) Peak
mantle contraction rises smoothly from stage 25 until hatching. Each of
the stimulus modes yields strong escape jets. (B) Temporal aspects of
escape performance [frames to peak (•) and delay • D)] improve
markedly between stages 28 and 30. The adult value for delay with an
electrical stimulus (• over arrow) has a minimum value of 7-8 frames
(Otis and Gilly, 1990). (C) Rate of mantle contraction, approximated as
maximum diameter change divided by frames to peak, shows a sharp
increase before hatching (stage 30) and a post-hatching decline towards
the adult level.

sizable delays and represent activation through more
physiological pathways.

General features of indirectly stimulated motor activity
due to giant and non-giant (small) axon pathways ob-
served in the present study closely resemble those previ-
ously reported for tethered, intact squid. In escape re-
sponses evoked by light flashes, the giant axons fire either
after a 50 ms delay at the start of a burst of small axon
activity (Figs. 5A, B) or not at all. In an indirect response
stimulated by an electric shock, the giant axons fire only
after a much longer delay (several hundred ms) and always
during a burst of small-axon activity (Figs. 5C, D). Giant
axons do not fire in every jet cycle, and the small-axon

Figure 4. 'Direct' responses to electrical stimulation (•) applied over
the magnocellular lobe in an adult Loligo (A) and a 7-day post-hatching
.Viyd'rViiV/H.s (B). (A) The electrode on the pallial nerve (lower trace)
records the second-order giant axon spike (2) en passant, while a second
electrode on a posterior stellar nerve stump (upper trace) records the
third-order spike (3) of the giant motor axon. (B) A single electrode
placed over the stellate ganglion records both second- and third-order
events. See text for additional details.

system acting alone can generate intense episodes of motor
activity (second cycle in Fig. 5C). In all these respects our
findings agree with Otis and Gilly ( 1990).

Recordings of motor outputs in hatchling
and embryonic Loligo

Recordings from the stellate ganglion of late-stage em-
bryos and hatchlings after electrical stimulation of the
head show activity associated with both giant and non-

—^

400 uV
1000 uV

5 ms i , 3

400 uV
600 uV

Figure 5. Bursts of motor activity associated with escape jetting in
adult Loligo. In each panel (A-D) the upper trace is from the pallial
nerve, and the lower trace is from a stellar nerve. (A) A light flash stimulus
(*) results in firing of the giant axons (2 and 3) after a delay of ~50 ms
and before the onset of the non-giant motor burst. (B) Portions ot the
records in (A) are displayed on an expanded time scale to illustrate the
time course of the giant axon spikes. (C) An electrical stimulus (•) leads
to giant axon activation after a long delay (>500 ms). In this case, the
burst of small axon motor activity commences before the giant spike. A
second escape cycle at the end of the record is driven by non-giant axons
acting alone and shows no giant spike. (D) The first cycle in (C) is dis-
played at an expanded time base.

216

W. F. GILLY ET AL.

giant motor systems. Figure 6A illustrates such recordings
from a stage 29+ embryo. Giant fiber excitation is in-
dicated by an initial small, negative-going spike (2) fol-
lowed by a larger positive event (3). As discussed above,
the first component (2) represents arrival of the impulse
in the second-order giant fiber entering the stellate gan-
glion via the pallial nerve, whereas the second component
(3) reflects the summed action potentials from proximal
parts of the third-order giants lying within the ganglion.
Thus, moving the recording electrode anteriorly along the
pallial nerve amplifies the second-order event and elim-
inates the third (Fig. 6B), whereas moving the electrode
posteriorly along the larger stellar nerves isolates activity
in the third-order giant fiber (Fig. 6C). Moving the elec-
trode peripherally along one of the smaller (anterior) stellar
nerves out into the muscle field shows the third-order spike
followed by a large muscle potential (m. Fig. 6D).

Following stimulation over the brachial ganglion, ac-
tivity can be recorded at all three stages in the giant fiber
pathway by placing one recording electrode over the mag-
nocellular lobe and a second on the stellate ganglion. The
first electrode records the first-order giant spike ( 1 ) while
the other records the second- and third-order events in
sequence (Figs. 7A, B).

Firing of the first-order cell is invariably followed by
activation of the other two elements, and the delay from
the peak of the first-order spike to the start of the third-
order event is only 2.0-2.5 ms. The delay that precedes
firing of the first-order spike during an indirect response
is much longer (e.g.. 26 ms in Fig. 7A or 13 ms in Fig.

Figure 6. Electrical activity in the second- and third-order giant axons
in a stage 29+ Loligo embryo. Direct responses of the giant fiber pathway
were generated by stimulation over the magnocellular lobe. (A) The re-
cording electrode was placed directly over the stellate ganglion, and the
incoming second-order spike (2) and the out-going third-order wave (3)
are recorded. (B) The electrode was positioned on the pallial nerve just
proximal to the stellate ganglion, and the second-order spike is thereby
isolated. (C) The electrode was placed on the emergence of the posterior
stellar nerves from the ganglion; this isolates activity in the third-order
giant axons. (D) The electrode was placed over an anterior stellar nerve
in the muscle field of the mantle. This reveals the third-order giant axon
spike and a large muscle potential (m).

Figure 7. Timing of electrical activity from all three stages of the
giant fiber pathway following indirect electrical stimulation over the bra-
chial ganglion in a stage 29++ (A) or 30 (B) Loligo. In each panel the
lower trace is recorded with an electrode on the magnocellular lobe, and
a composite event including the first-order giant spike is obtained (1).
Upper traces are recorded from the stellate ganglion, where second- (2)
and third-order (3) events are detected.

7B) and presumably reflects 'processing' time in the central
nervous system. Small potentials precede the initiation of
the first-order spike in Figure 7B and must represent
summed activity in pathways leading into the magnocel-
lular lobe. There is also generally an outburst of small-
unit activity coincident with and following the first-order
giant spike, which itself appears difficult to resolve except
for the rising phase ( 1 in Fig. 7B).

Indirectly stimulated escape responses in hatchlings
produce the pattern of second- and third-order giant spikes
discussed above (Figs. 8 A, B), and the subsequent activity
in the small motor axons takes the form of flurries of
irregular, compound action potentials (Fig. 8 A). Unit ac-
tivity is generally difficult to resolve, and these potentials
probably represent the firing of dozens of axons in rough
synchrony. Although these compound events may ap-
proach third-order giant spikes in amplitude, they are
readily distinguished from the latter by their irregular and
variable waveforms and slower rise times. Bursts of small
unit activity can be elicited by weak stimulation without
excitation of the giant fiber system (upper trace in Fig.
8A) and would be associated in nature with non-giant
escape jetting, as occurs in adults.

Two consistent and striking features characterize neural
recordings of indirectly stimulated escape responses in
late-stage embryos and hatchlings and clearly differentiate
them from analogous records obtained in adults. First,
the minimum latency for excitation of the third-order
giant axons in hatchlings by photic stimulation is consid-
erably shorter than that in adults (~15 ms in Figs. 8B,
9F vs. 50 ms for adults in Fig. 5A), and the latency fol-
lowing electrical stimulation in hatchlings can be nearly
as brief (Fig. 8A). In adult animals, this latency is at least
several hundred ms (Fig. 5C; c.f. Otis and Gilly, 1990).

A second and more pronounced difference is that when
the third-order giants fire in hatchlings, their impulses
arise in the stellate ganglion several ms before those of
the small motor axons. This is true regardless of whether
stimulation is via electric shocks (Fig. 8A) or light flash
(Fig. 8B). This pattern of giant versus non-giant activity

DEVELOPMENT OF SQUID ESCAPE RESPONSE

217

"^ "' '!•" *'\«A.v~w>'"

A B

Figure 8. Companson of motor outputs from a stage 30 Loligo in
response to an electrical shock at the base of the tentacles (•. A) and to
a light flash (*, B). (A) Upper trace shows activity of small axon system
acting alone; stimulus was sub-threshold for giant fiber activation. Lower
trace was recorded with a slightly stronger shock, which led to indirect
excitation of the giant fiber pathway. The giant axon spike arrives in the
stellate ganglion before the small axon activity. ( B) A light flash produces
a similar pattern of motor activity in which the small axon wave follows
the giant fiber response.

for electrically stimulated responses is thus temporally in-
verted in comparison to the picture in adults, where giant
axon spikes always fire 50-75 ms after the onset of the
burst of non-giant activity (Fig. 5C, D: Otis and Gilly,
1990).

Development of motor patterns prior to hatching

Small axon control of jetting behavior is demonstrable
at stage 26 (the earliest examined). Intermittent bouts of
spontaneous rhythmic activity occur and represent normal
respiratory cycles. Electrical stimulation evokes bursts of
small unit activity resembling these respiratory bursts, but
stimulated activity is generally more intense and long-
lasting (Fig. 9A). Latency for small axon excitation appears
to be brief at this stage, but this may reflect direct stim-
ulation of these pathways in the small embryos at this
early stage. Small axon responses continue with little
change in the above pattern throughout subsequent de-
velopmental stages, except for an apparent increase in
latency to 30-50 ms by stages 27-28 (Fig. 9C).

Light flashes are ineffective at triggering escape jets or
neural activity in the stellar nerves at stages 26 (Fig. 9B)
or 27 (not illustrated). Photic stimulation does produce
bursts of small axon activity in the stellate ganglion by
stage 28 (Fig. 9D). coincident with anatomical maturation
of the eye (Segawa et ai, 1988).

Giant axon responses can first be evoked by electrical
stimuli at stage 28 in embryonic squid (Fig. 9E) and by
light flashes around the time of hatching (Fig. 9F). Gen-
erally, the picture of functional development of the giant
fiber system from stage 28 through hatching is one of
progressive maturation, in terms of speeding of the action
potential waveform, reduced response latencies, and de-
creased synaptic delay. The immaturity of the giant fiber
pathway at stages 28-29 is suggested by: (i) the small am-
plitudes and long durations of both second- and third-
order giant spikes; (ii) the relatively long response la-
tency— the second-order spike takes more than 20 ms to
arrive in the stellate ganglion compared with a delay of

-12 ms at stages 29++ or 30 (Fig. 10A), and (iii) the
progressive decrease for second- to third-order synaptic
transmission during development (Fig. 10B). These
changes parallel the improvement in behavioral perfor-
mance as indicated by the square symbols and dashed
curve in Figure 10A. which are behavioral data replotted
from Figure 3B.

Post-hatching development of giant and non-giant
motor patterns

Although the embryonic pattern of giant axon use is
evident at the time of hatching, several distinctive features
of the adult motor patterns (Otis and Gilly, 1990) may
emerge shortly thereafter. Figure 1 1 A shows stellar nerve
discharge of a 2-week-old Sepioteuthis in response to an
electric shock applied to the tentacles. A burst of small
unit activity follows the stimulus at short latency; this is
a pattern typical of embryonic and hatchling Loligo. The
lower trace is plotted at an expanded time base. A stronger
shock produced the response pictured in Figure 1 1 B. A
short latency, non-giant burst again occurs, but in this
case it is followed by a giant axon spike after 80 ms. Tim-
ing of giant axon activation in relation to that of the small
axons is adult-like (Fig. 5D), although the overall latency

?00 ^jV

Stage 26

IJ^T ^~w-^

Slage 28

100 200 jjV

Slage 30

Figure 9. Functional maturation of the giant fiber pathway in em-
bryonic Loligo. Indirect electrical (•) or light flash (*) stimuli were used
to elicit escape responses at various stages of development; all recordings
illustrated were obtained from the stellate ganglion. (A) At stage 26.
electrical stimulation elicits only small axon activity. (B) A light flash is
ineffective at stimulating escape responses or producing any detectable
motor outputs at stage 26. (C) Giant axon activity is still not produced
by electrical stimuli at stage 27. (D) Photic stimulation produces small
axon activity but no giant spikes at stage 28. (E) Giant axon responses
are first detectable at stage 28 with electrical stimuli. (F) A light flash
stimulus at stage 30 produces a giant axon response. See text for additional
details.

218

W. F. GILLY I.I I/

50 —I

Latency
(ms)

> 2,

\ ~.

i — rn — i i i i — i — r~r

26 27 28 29 29+ 29++ 30 1 2 3

Embryonic
Stage

Days post
Hatching

20 —

Synaptic
Delay

(ms) 1.0-

I I I I I I I I I I

26 27 28 29 29+ 29++ 30 1 2 3

Embryonic
Stage

Days post
Hatching

Figure 10. Improvement in performance of the giant fiber pathway
during late embryogenesis in / oligo. (A) Response latency (time to sec-
ond-order giant spike) decreases between stage 28 (time of first detectable
giant axon activity) and 30 (hatching). Filled circles were obtained with
indirect electrical stimuli; open symbol was obtained with light flashes.
Mean values and the number of experiments are given; standard error
of the mean is smaller than the symbols. Squares and dashed curve are
behavioral data which has been replotted from Figure 3B. (B) Synaptic
delay between the second- and third-order giants (across the giant synapse
in the stellate ganglion) also decreases between stages 28 and 30. Means,
number of experiments, and standard error are indicated.

is much less than that in adults (Fig. 5C). The motor pat-
tern underlying the escape response in Figure 1 IB thus
shows both embryonic and adult qualities.

More complex escape responses with the long delays
characteristic of the adult can also be generated shortly
after hatching in some animals. Results from another Se-
pioteuthis (~5 days post-hatching) are shown in Figure
12. Delayed escape jets driven by only the small axon
system (Fig. 1 2A) and by both small and giant axons acting
in concert (Fig. 12B) are well developed. Adult-like mul-
tiple cycle responses showing both of the above types of
motor patterns are also evident (Fig. 1 2C). When the giant
axons are used during such delayed escape responses, their
firing is timed to occur after the non-giant system initiates
the jet cycle. In every respect, the motor patterns in Figure
12 are adult-like. We do not yet know precisely when the
adult-like motor patterns appear in Sepioteuthis after
hatching, but stage 29-29++ embryos display only the
fast-start embryonic pattern seen in Loligo (data not il-
lustrated).

In addition to the delayed bouts of motor activity in
Figure 12, an initial brief burst of activity occurs with a
delay of ~20 ms in every case illustrated. The origin of

this activity is presently unknown, and recordings in adult
squid also reveal similar short-latency activity in stellar
nerves following an electrical stimulus (cf. Fig. 3 in Otis
and Gilly, 1990). In the latter case, there is no detectable
short-latency mantle contraction. Comparable behavioral
experiments have not been carried out with juvenile Se-
piotheuthis.

Discussion

Motor control of escape behavior in late-stage embryos
and hatchlings shows important similarities to the cor-
responding situation in adult squid. In both cases, escape
jetting is under the influence of two parallel motor path-
ways: the giant fiber system and a small axon (non-giant)
system. Strong escape jets can be driven by the small-
axon system acting alone, with no giant fiber involvement
whatsoever (Figs. 9C, 8 A). The small axon pathway is the
first to develop anatomically and become functional dur-
ing embryonic development, and vigorous escape jets are
possible before the giant axons appear.

Giant axon excitation provides a potent boost to the
small axon system, however, and this greatly improves
escape jetting performance. This was directly demon-
strated in adult animals (Otis and Gilly, 1990). It is also
evident in embryonic development (stages 28-30) as a
marked decrease in response latency [seen behaviorally

Figure II. Partial development of adult-like use of the giant fiber
pathway in a 14-day post-hatching Sepioteuthis. (A) Upper trace was
recorded from the stellate ganglion following an electrical stimulus de-
livered to the base of the tentacles (•). Small unit activity only is gen-
erated, and the latency is brief. Lower trace shows the initial portion of
the response following the shock displayed at an expanded time scale.
(B) A stronger shock to the same site as in (A) produces a stronger burst
of small axon activity at a short latency and a single giant axon spike in
the middle of this burst. Lower trace shows the expanded version to
identify the giant fiber spike. The occurrence of the giant spike well alter
the onset of the small unit burst is thus adult-like, but the brief latency
(<20 ms) for the non-giant burst is characteristic of embryos.

DEVELOPMENT OF SQUID ESCAPE RESPONSE

219

7*jl^v«X>W,

Figure 12. Fully developed adult-like interplay of giant and non-
giant motor systems in escape responses of a 5-day post-hatching Sc/n-
Kti'iilhis. (A) An electrical stimulus (•) to the side of the head produces
an escape jet at a latency of —500 ms that is driven only by small motor
axons. (B) A stronger stimulus to the same site produces a delayed escape
jet driven by both small axons and giant axons acting in concert. Non-
giant activity precedes giant axon activation. Lower trace shows an ex-
pansion of the record around the giant fiber spike. (C) Stimulation on
the ventral surface of the head just anterior to the eye produces a multiple
cycle escape response at a long latency. The first cycle is driven by the
small axon motor system acting alone, whereas the second also involves
the giant axon pathway.

(Fig. 3B) or neurophysiologically (Fig. 10A)], time to peak
mantle contraction (Fig. 3B), and rate of contraction (Fig.
3C). The time course of this improvement, beginning at
stage 28-29, coincides closely with the anatomical ap-
pearance and maturation of the third-order giant motor
axons in the more posterior stellar nerves that control
jetting. Performance peaks at hatching. Presumably this
is a valuable capability, because the embryonic squid must
jet vigorously to escape from the confines of the egg mass
in order to hatch.

At present it is not known how the neural portions of
these giant and non-giant motor systems are associated
with the two types of circular muscle fibers in adult squid
mantle (Bone el at.. 1981; Mommsen el ai, 1981). De-
veloping embryos and hatchlings would provide a valuable
system in which to pursue this question, because the two
motor systems do not develop in perfect synchrony. We
have not carried out an anatomical analysis of the muscle
fiber types in these young animals.

Although the overall picture of dual motor control of

escape jetting is similar in embryos, hatchlings, and adults,
details of how the two motor systems are employed when
they act together are strikingly different. In embryos and
hatchlings, activity in the third-order giant axons always
precedes the burst of small unit motor activity by several
ms. This is true regardless of whether stimulation is by
electric shock or light flash. Latency to firing for the giant
axon varies from ~40 to 10 ms, depending on the exact
stage of development (Fig. 10 A). This delay basically rep-
resents the time required to excite the first-order giant cell
(Fig. 7). Once this cell fires, the second- and third-order
giants follow within 2-3 ms. In embryos and hatchlings,
all escape responses are thus of the fast-start variety, al-
though the latency is never so brief as with artificial direct
stimulation of the first-order giant (Fig. 4).

In our experiments on adult squid, only sudden visual
stimuli (e.g. light flash) have been effective in producing
a fast-start pattern of giant axon excitation (Fig. 5A) like
that seen in embryos or hatchlings, where giant axon firing
precedes the burst of non-giant activity. Latency for giant
axon activation, even in this fastest case, is much longer
than that in hatchlings (50 ms versus 10 ms) and presum-
ably again represents the time before the first-order giant
fires.

Electrical stimuli in adults produce a second pattern of
giant axon use that is not seen in the embryo. This delayed-
escape mode shows a minimum latency of several hundred
ms and, more significantly, a burst of non-giant motor
output that commences 50-100 ms before the giant axons
fire (Fig. 5C). The small-axon system thus appears to be
the primary effecter of this type of escape jet, and the
giant axons are booster elements that are optionally re-
cruited during any given cycle of a delayed-type response.

Otis and Gilly (1990) have argued that a great deal of
complex processing in the central nervous system under-
lies effective use of the giant fiber pathway in this delayed-
escape mode. This idea is in consonance with results on
embryonic and hatchling Loligo presented in this paper.
Much of the brain at these stages is not yet differentiated
(Young, 1939; Martin, 1965), and these animals do not
yet have the capability of orchestrating complex escape
behavior, despite the functional presence of all the nec-
essary peripheral motor components. Presumably, devel-
opment of higher order neural centers is necessary to co-
ordinate the interplay of giant and non-giant motor sys-
tems.

When and how does the strictly fast-start embryonic
pattern in escape jetting evolve into the complex capa-
bilities of the adult? This is not yet clear in Loligo. We
have seen no indication of delayed escape responses in
either behavioral or neurophysiological experiments for
up to 6 days post-hatching. Preliminary work on post-
hatching Sepioteuthis is also described in this paper. In
this case, perfectly normal, adult-like firing patterns of the
giant and non-giant motor systems were observed in an-

220

W. F. GILLY ET AL.

imals only 5 days old (Fig. 1 2 ), and other animals showed
a curious mixture of embryonic and adult-like character-
istics (Fig. 1 1).

The specimens of Sepi< >teuth is that we studied differed
from our Loligo subjects in two important respects. First.
Sepioteuthis is a much larger and possibly more developed
(i.e., neurologically) animal at birth. Second, the Sepi-
oteuthis had been actively feeding and growing for the
entire time after hatching (5-21 days), whereas Loligo
was provided with no (for the neurophysiological work)
or minimal food (for the behavioral work) and was main-
tained for no more than 6 days. Either or both of these
factors could be relevant to the early attainment of co-
ordinated delayed-escape jetting in young Sepioteuthis.
In the first case, Sepioteuthis may simply develop the
adult-like motor patterns more quickly after birth than
does Loligo. The second possibility is more intriguing,
however.

Hatchling squid have limited energy reserves sufficient
only for several days (O'Dor el ai. 1986) during which
time the animals must learn to capture prey or face star-
vation (Hurley. 1976; Yang et ai. 1983. 1986). Because
prey items consist largely of fast-moving copepods. and
high speed pursuit is involved in prey-capture, it would
seem advantageous to employ the giant fiber pathway in
this activity. To do this effectively, however, strict control
over excitation of the first order giants must be necessary
to provide the giant axon-mediated boost precisely at the
correct moment. This capability — critically timed exci-
tation of the giant fiber pathway — is the basic feature of
the adult delayed-escape response that differentiates it
from the fast-start response. The possibility that such a
profound change-over in the pattern of giant axon use in
hatchling squid might be associated with the perfection
of feeding behavior is a prospect that we are currently
pursuing.

Acknowledgments

We thank Natasha Fraley and Patricia Gosling for per-
forming the behavioral experiments, and Dr. Roger Han-
Ion's group at U.T.M.B., Galveston, Texas, for providing
living Sepioteuthis. This work was supported by grants
from the Whitehall Foundation (J86-1 10) the Office of
Naval Research (N00014-89-J-1744) and the N.I.H. (NS-
17510).

Literature Cited

Bone, Q., A. Pulsford, and A. D. Chubb. 1981. Squid mantle muscle.
J. Mar. Bio/. Assoc. L'.A'. 61: 327-342.

Boyle, P. R. 1986. Neural control of cephalopod behavior. Pp. 1-97
in The Mullusca. vol. 9, pan 2. A. O. D. Willows, ed. Academic
Press, New York.
Bryant, S. H. 1959. The function of the proximal synapses of the squid

stellate ganglion. J. Gen. Physio/. 42: 609-616.
Eaton, R. C. 1984. ed. h'eural Mechanisms ol Startle Behavior. Plenum.

New York.

Hurley, A. C. 1976. Feeding behavior, food consumption, growth, and
respiration of the squid Loligo opalescens raised in the laboratory.
Fish. Bull 74: 176-182.
Mackie, G. O. 1990. Giant axons and control of jetting in the squid

Loligo and the jellyfish Ag/anlha. Can. J Zoo/. 68: 1421-1431.
Marthy, H.-J. 1987. Ontogenesis of the nervous system in cephalopods.
Pp. 443-459 in Nervous Systems in Invertebrates, M. A. Ali, ed.
Plenum. New York.

Martin, R. 1965. On the structure and embryonic development of the
giant fibre system of the squid Loligo vulgaris. Z Zellforsch. 67: 77-
85.
Martin, R. 1969. The structural organization of the intracerebral giant

fiber system of cephalopods. Z Zellforsch. 97: 50-68.
Martin, R. K. 1977. The giant nerve fibre system of cephalopds. Recent

structural findings. Symp. Zoo/. Soc Loud. 38: 261-275.443.
Messenger, J. B. 1983. Multimodal convergence and the regulation

of motor programs in cephalopods. Fortschr. Zoo/ 28: 77-97.
Mommesen, I. P., J. Ballantyne, D. MacDonald, J. Gosline, and P. \V.
Hochachka. 1981. Analogues of red and white muscle in squid
mantle. Proc. Nail. Acad. Sci. L'SA 78: 3274-3278.
O'Dor, R. K. 1988. The forces acting on swimming squid. J. E\p. Biol.

137:421-442.

O'Dor, R. K., E. A. Foy, P. A. Helm, and N. Balch. 1986. The loco-
motion and energetics of hatchling squid. Ilex illecebrosus. Am. Ma-
lawi Bull. 4: 55-60.

Otis, T. S., and W. F. Gilly. 1990. Jet-propelled escape in the squid
Loligo opalescent: concerted control by giant and non-giant motor
axon pathways. Proc. Nad. Acad. Sci. USA 87: 291 1-2915.
Packard, A. 1969. Jet propulsion and the giant fibre response of Loligo.

Nature 221: 875-877.
Prosser, C. L., and J. Z. Young. 1937. Responses of muscles of the

squid to repetitive stimulation. Biol. Bull 73: 237-241.
Segawa, S., \V. T. Yang, H.-J. Marthy, and R. I. Hanlon.
1988. Illustrated embryonic stages of the eastern Atlantic squid Lo-
ligo forbesi. I'eligeriQ: 230-243.
Wilson, D. 1960. Nervous control of movement in cephalopods. J.

Exp. Biol. 37: 57-72.

Yang, \V. T., R. F. Hixon, P. E. Turk, M. E. Krejci, W. H. Hulet, and
R. T. Hanlon. 1986. Growth, behavior, and sexual maturation of
the market squid, Loligo opalescens, cultured through the life cycle.
Fish. fiii/A 84:771-798.

Yang, W. T., R. T. Hanlon, M. E. Krejci, R. F. Hixon, and \V. H. Hulet.
1983. Laboratory rearing of Loligo opalescens. the market squid
of California. Aquacullure 31: 77-88.

Young, J. Z. 1939. Fused neurons and synaptic contacts in the giant
nerve fibres of cephalopods. Philas. Trans. R. Soc. Loud. Ser. B 229:
465-503.

Young, J. Z. 1938. The functioning of the giant nerve fibres of the
squid../. E.\p. Biol. 15: 170-185.

Reference: Bid. Bull 180: 221-227. (April. 1991)

Factors Affecting the Sensory Response Characteristics

of the Cephalopod Statocyst and their Relevance

in Predicting Swimming Performance

RODDY WILLIAMSON

The Marine Biological Association, Citadel Hill, Plymouth PL I 2PB, England

Abstract. The statocyst in cephalopods is the main organ
of balance and operates in a manner similar to the ves-
tihular system of vertebrates. This paper reviews the prin-
cipal factors affecting the sensitivity and frequency re-
sponse of the statocyst. These include morphological fea-
tures, such as the size and shape of the statocyst, its canal
structure, and the size of the cupulae and maculae, as well
as physiological features, such as the electrotonic coupling
of sensory cells, the impact of the efterents, and the mo-
tility of some cells. The use of statocyst characteristics in
predicting the locomotory performance of different ceph-
alopod species is discussed.

Introduction

For spatial control of locomotion, an animal needs in-
formation about its orientation with respect to gravity
and its motion relative to its surroundings. This infor-
mation could be derived from a variety of sensory systems,
ranging from vision to electroreception, but most animals
have developed specific sense organs responding to linear
and angular accelerations; e.g., the vestibular system in
vertebrates and the statocysts of cephalopods. It has been
proposed that the sensory response characteristics of these
receptor systems are matched to their likely inputs; i.e..
that the frequency response range and sensitivity of the
system reflect the accelerations imposed by an animal's
own movements (Jones and Spells, 1963; Jones, 1984).
Thus the vestibular system of a small agile animal (e.g..
a bird) is more sensitive to higher frequencies of move-
ment than that of a slower moving animal living in a
denser medium (e.g., a fish) (Correia et a/.. 1981). This
idea can also be applied to cephalopods where, by looking
at the statocysts, we can try to predict what kind of lo-

Received 22 August 1990; accepted 27 November 1990.

comotion is used by the animal (Maddock and Young.
1984; Morris, 1988; Young, 1989). This is a particularly
valuable approach to animals with unstudied lifestyles.
This paper reviews some of the evidence for this propo-
sition and identifies some of the features that are likely
to influence the sensitivity and response characteristics of
cephalopod statocysts.

The parameters affecting statocyst sensitivity can be
divided into two main areas, morphological features and
physiological features. For convenience these are consid-
ered separately, but of course, they act in concert within
the living animal.

Morphological Features

The general morphology of each of the paired right and
left statocysts is a fluid-filled cavity within the cranial car-
tilage (Fig. 1). The statocyst itself varies considerably in
shape in different cephalopods; in Octopus (Fig. 1A) it is
almost spherical, whereas in \ 'ampyrotenthis it is short,
wide, and shallow (Young, 1960, 1989). Many statocysts
have sac-like protrusions into the surrounding cartilage
(Stephens and Young, 1982), or cartilaginous pegs or
hooks (the anticristae and hamuli) that project into the
statocyst interior (Fig. IB); these projections presumably
constrict or direct the flow of the endolymph. Again, this
can vary from the single anticrista in Octopus, to the 38
anticristae and 5 hamuli in Egea (Young, 1984).

Each statocyst has two main areas of receptor epithe-
lium (Fig. 1 ). The first is a macula or plate of sensory hair
cells with an overlying statolith. All coleoids have a macula
carrying a single compact statolith, but decapods have
two additional maculae carrying numerous small stato-
conia. Where three maculae are present, they are set in
different planes, thus being able to resolve linear accel-
erations in any direction.

221

222

R. WILLIAMSON

Dorsal

Forward view Rear view

Ventral

Figure 1. (A) Diagram of the statocyst of Octopus viewed from the
side. The statocyst sac is suspended within the statocyst cavity by fibrous
strands. There are two areas of receptor epithelium: a single, oval shaped
macula with an attached statolith, and a crista strip that passes around
the inside of the sac. such that it covers all three planes. The crista strip
is divided into 9 segments, each segment carries a cupula (not shown).
After Budelmann. 1980. (B) A forward and rear view of the cut open
statocyst of Sepia officinalix. There are 3 maculae, arranged in 3 different
planes, and the crista strip is divided into 4 segments. Anticristae and
hamuli project into the cavity of the statocyst. After Budelmann. 1980.

The second area of receptor epithelium consists of a
narrow strip of sensory hair cells that runs around the
inside of the statocyst such that it covers all three planes
(Fig. 1). This strip is usually divided into segments: the
crista segments, each carrying a cupula attached along the
length of the crista segment. Octopods (excluding cirroc-
topods) have nine crista segments, whereas decapods have
four, each with its own cupula. Rotational movements of
the animal cause a flow of endolymph relative to the sta-
tocyst wall; this flow in turn deflects the cupula and stim-
ulates the underlying hair cells. A transverse section
through a crista segment (Fig. 2) reveals three main types
of cells in the sensory epithelium: primary sensory hair
cells, secondary sensory hair cells, and afferent neurons.
This combination of primary sensory hair cells and sec-
ondary sensory hair cells in a single epithelium is unique
to cephalopods (Budelmann el al.. 1987). Although the
crista/cupula system responds principally to angular ac-
celerations, it may also respond to linear accelerations
(Budelmann and Wolff, 1973; Williamson and Budel-
mann, 1 985a). Because the crista/cupula system is crucial
for signalling most of the animal's movements, and be-
cause this system is dependent upon the physical param-

eters of the statocyst, we will concentrate on the responses
of the crista.

Statocyst size and shape

The idea that the size and shape of the statocyst are
correlated with its likely response characteristics, and
hence with the animal's locomotory performance, arises
from the physical models of the operation of the vertebrate
semicircular canal system (Steinhausen, 1933; Wilson and
Jones, 1979) and from comparisons of canal dimensions
in different animals (Jones and Spells, 1963; Jones, 1984;
Gauldie and Radtke. 1990) and in animals of different
sizes (Curthoys, 1983). The Steinhausen torsion pendulum
model (Steinhausen, 1933; Oman et al.. 1987) identifies
the radius of curvature of the canal, the bore radius of
the canal duct, the viscosity and density of the endolymph.
and the stiffness of the cupula as being important factors
determining the vestibular response characteristics. Al-
though, in vertebrates, there is a good correlation between
the frequency sensitivity predicted from measurements
of the radius of curvature of the canal and the bore radius
of canal duct, and the actual response characteristics
(Correia et al.. 1981), the statocyst position is much less
clear.

The use of such a model in cephalopods is supported
by the relatively large size of the statocysts in newly
hatched coleoids. Those statocysts are more than a quarter
of the mantle length, but grow at a much lower rate than
the animal; i.e.. they increase in size by about 29 times
while the mantle length is increasing by 390 times (Mad-
dock and Young, 1984). This relative conservation of
statocyst size fits well with the idea that statocyst size is
constrained by the physical principles under which the
organ operates, and that the dimensions of the system are
adjusted to the speed at which the animal turns. In ad-
dition, Maddock and Young (Maddock and Young, 1984;
Young, 1984. 1989) have described a number of corre-
lations between statocyst morphology and probable
swimming performance, including data showing that the
faster moving squids tend to have a narrower canal, thus

Ventral

Figure 2. Diagram of a cross-section through the crista strip of the
squid. Alfali'iithis xiibulala. Three main cell types are present: the primary
sensory hair cells (lightly stippled), the secondary sensory hair cells (darkly
stippled), and the afferent neurons (unstippled). After Williamson, I989a.

STATOCYST RESPONSE CHARACTERISTICS

223

presumably improving the high frequency response,
whereas slow moving cephalopods tend to have relatively
large statocysts, thus increasing their low frequency sen-
sitivity.

As pointed out by Young ( 1984), the main difficulties
in applying this idea to cephalopod statocysts is that the
radius of curvature can only be approximated as the cross-
sectional diameter of the statocyst, and there is only rarely
a canal-like structure in the statocyst formed by the an-
ticrista and hamuli. In addition, although there are rec-
ognizable patterns of anticristae and hamuli in different
groups of cephalopods, it is unclear how these projections
affect the flow of endolymph. Clearly, we need a more
realistic model of how the endolymph flows within the
statocyst. and how this is influenced by the various mor-
phological features of the statocyst.

Cupula parameters

Other morphological features likely to effect the fre-
quency response and sensitivity of the statocyst angular
acceleration receptor system are the size, shape, and at-
tachment of the cupulae. The cupulae are gelatinous, flap-
like structures, projecting towards the middle of the stato-
cyst, and attached to the crista ridge along the whole length
of a segment. The cupulae however, appear to be irregular
in shape, often being much taller in the center of the crista
segment than at the edges; this is particularly prominent
in the squid, Allolcul/iis (Fig. 3a). The center of the cupula
will therefore present a much greater area of resistance to
endolymph flow than the edges and hence, unless the cu-
pula is very rigid, will more easily stimulate the underlying
hair cells. This likely differential sensitivity in different
parts of a single crista segment may be a method of frac-
tionating the sensitivity range of the system. In Octopus
this is even more pronounced (Fig. 3b.c). Here, the nine
crista segments have alternating large and small cupulae.
with the tall cupulae having narrower bases than the small
ones (Budelmann el ai, 1987). This, again, is likely to
fractionate the range over which the system operates and,
indeed, recordings from the afferent neurons in represen-
tatives of these two different segments indicate that the
segment with the large cupula is up to 10 times more
sensitive than that with the small cupula (Williamson and
Budelmann, 1985a,b). The increase in sensitivity means,
however, that the afferents from the large segment can be
driven into response saturation at a much lower stimulus
intensity than those from the small cupula segment. This
arrangement could be correlated with Octopus ' two forms
of locomotion, the high sensitivity, large cupulae being
needed during slow crawling movements, and the low
sensitivity, small cupulae during jet propelled movements.

Like the statoliths, anticristae, and hamuli, the cupulae
are also likely to have an effect on the pattern of endo-
lymph flow within the statocyst. Although Young (Mad-

100pm

5O>um

50 /urn

Figure 3. (A) Crista cupula from the squid, Alloteuthis subulala.
The cupula has been fixed in osmium and then detached from the crista
segment. Note that it has a large central mass and is much shorter at the
edges. From Williamson I990a. (B and C) Transverse sections through
two different crista segments in the statocyst of Octopus showing a small,
wide-based cupula type and a large, narrow-based cupula type. From
Williamson and Budelmann, I985b.

dock and Young, 1 984; Young, 1 989) has used a verte-
brate semicircular canal model to predict endolymph flow,
and hence sensory response characteristics, this is unlikely
to be adequate. Recent vertebrate models have shown that
even a good canal structure, with the three canals or-
thogonally arranged, is likely to have a complicated pat-
tern of endolymph flow with crosstalk between the canals
(Omanetal.. 1987; Mullerand Verhagen, 1988). In ceph-
alopods, which rarely have a single canal structure, en-
dolymph flow patterns are extraordinarily difficult to pre-
dict (Govardovskii, 1971; Muller, pers. comm.). Even the
manner of movement of the cupula is unknown; i.e..
whether it pivots like a lever, or slides like a piston, or
flexes like a diaphram, although recent modelling work
has suggested that the cupula does not operate as a simple
pivot (Morris, 1 988).

Another unknown with respect to cupula movement
is the strength of its attachment to the crista and the re-
storing force it develops when displaced. This will have a

224

R. WILLIAMSON

major impact on the frequency response characteristics
of the crista/cupula system, and any variation between
crista segments, or between different animals, would have
to be taken into account in a model describing statocyst
response characteristics.

The presence or absence of a perilymphatic space may
also affect the sensitivity of the statocyst. The octopods,
cirroctopods, and I 'ampyroteuthis all have a lymph-filled
space between the cartilaginous wall of the statocyst cavity
and the statocyst sac containing the sensory epithelia. An-
liker and van Buskirk (1971), dealing with the vertebrate
semicircular canal system, have argued that the movement
of perilymph may have a major effect on the dynamic
response characteristics of the system. Although any peri-
lymph flow in the statocyst would be restricted by the
fibers supporting the statocyst sac, there may well be an
effect in cephalopods from this source.

Physiological Features

Extracellular recordings from statocyst afferents have
shown that the crista/cupula system in Octopus acts as a
velocity transducer over a middle range of frequencies
and has response characteristics similar to those of the
vertebrate semicircular canal system (Williamson and
Budelmann, 1985a). There is as yet no data on afferent
response characteristics from decapod statocysts. Recent
intracellular recordings from hair cells in the statocyst of
the squid. Alloteut/iis subulata. have provided the first
measurements of the sensitivities of cephalopod hair cells
(Williamson. 199 la). This work (Fig. 4) has shown that
the secondary sensory hair cells in the crista have sensi-
tivities of at least 0.5 mV per degree of cilia deflection.
This compares with sensitivities of about 3 m V per degree
for frog saccular hair cells (Hudspeth and Corey, 1977),
10 mV per degree for turtle basilar papillar hair cells
(Crawford and Fettiplace, 1985), and 30 mV per degree
for mouse cochlear hair cells (Russell et ai, 1986). This
work has also confirmed morphological studies (Budel-
mann el a/., 1987) showing that at least some of the sec-
ondary hair cells are physiologically polarized in the op-
posite direction to the primary hair cells (Fig. 4). This
bipolar sensitivity does not occur in vertebrate vestibular
cristae and, although it may be more energy efficient
(Williamson, 199 la), it is not clear if it will have any
effect on the sensitivity or frequency bandwidth of the
system.

Differences in hair cell sensitivity

There may well be differences in the intrinsic sensitiv-
ities of the individual crista hair cells. In Octopus, there
are at least three different morphological types of crista
hair cells: the primary sensory hair cells, the small sec-
ondary sensory hair cells, and the large secondary sensory
hair cells. In addition, there are different types of afferent

50ms

5mV

3Mm

1mV

6|.im

Figure 4. Intracellular recordings from a primary sensory hair cell
(A) and a secondary sensory hair cell (B) in the crista of the squid statocyst.
showing their responses to small mechanical displacements of the over-
lying cupula (displacements shown in lower traces). Note that the primary
and secondary hair cell depolarizations are caused by cupula displace-
ments of opposite directions, indicating that the cells are polarized in
opposing directions, and that only the primary hair cell carries action
potentials. From Williamson, 1 99 la.

neurons, and there may also be subdivisions of the hair
cells types (Budelmann et a/., 1987). These morphological
differences are likely to be reflected in physiological cell
parameters, such as input impedance and cell conduc-
tance, and therefore result in differences in the sensitivities
of the various cell types (Williamson and Budelmann,
1985a).

Electrical coupling

At least some of the secondary sensory hair cells in the
squid statocyst cristae are known to be electrically coupled
along the length of the crista segment (Fig. 5) (Williamson,
1989a). It has been argued that this coupling will lead to
an improvement in the signal to noise ratio of the system
and hence enhance its overall sensitivity. However, such
coupling is also likely to lower the high frequency response
of the system. Clearly, if the coupling could be varied
under direct nervous control, this would be a powerful
mechanism for changing the sensitivity and frequency re-
sponse of the system. A comparable sensory system with

STATOCYST RESPONSE CHARACTERISTICS

Electrotonic coupling

CelM

Cell 2

5mV

Current

50ms

|4nA

Figure 5. Intracellular recordings from two nearby secondary sensory
hair cells in the statocyst crista of the cuttlefish. Sepia officinalis, showing
their electrotonic coupling. A small current (bottom trace) is injected
into Cell 1 (top tracel, producing a depolarization, and this causes a
simultaneous, but smaller, depolarization in the neighboring cells (Cell
2, middle trace). This provides evidence that the secondary sensory hair
cells in a crista segment are electrotonically coupled along the segment.
From Williamson, 1991K

neurally controlled electrical coupling is in the vertebrate
retina, where the neurotransmitter dopamine alters the
coupling ratio between retinal horizontal cells (Knapp and
Dowling, 1987). Dopamine has been located in the retinal
efferents in Octopus (Suzuki and Tasaki, 1983) and has
also been tentatively identified in the statocyst efferents
(Budelmann and Bonn, 1982; Williamson, 1989b). It
would be an astonishing example of parallel evolution if
these two disparate sense organs, the eye and the statocyst,
used dopaminergic control of electrical coupling to reg-
ulate their sensory input.

Efferent system

The statocysts have an exceptionally large efferent in-
nervation; of the axons in the Octopus statocyst crista
nerves, 75% are efferent fibers travelling from the brain
to the statocyst (Budelmann el ai, 1987). In contrast,
about 8% of axons in a vertebrate vestibular nerve are
efferents (Goldberg and Fernandez, 1980). This efferent
innervation forms a plexus running beneath the crista
epithelium and makes synaptic contact with primary and
secondary sensory hair cells, as well as with the afferent
and other efferent neurons (Budelmann et ai, 1987). The
efferent fibers are active during movements of the animal's
head (Williamson, 1986) and can depress or enhance (Fig.
6) the afferent output from the statocyst (Williamson,
1985). These effects are due to direct synaptic hyperpo-
larization, or to depolarization, of the secondary sensory
hair cells, their first-order afferent neurons, and possibly,
the primary sensory hair cells (Williamson, 1989c). The
inhibitory response is probably due to cholinergic synapses
(Auerbach and Budelmann, 1986; Williamson, 1989b),
and the excitatory response to catecholaminergic synapses
(Budelmann and Bonn, 1982; Williamson, 1989b).

Such a widespread and complex efferent innervation
provides the animal with direct and independent control
of both the hair cell receptor potential and the level of
activity of the afferent neurons. Thus, not only can the
gain of the overall system be increased or decreased, but
the responses of individual elements can also be varied.
This permits an extension of the dynamic range of the
system by allowing adjustments to the membrane poten-
tials of the hair cells and afferent neurons, so that the
cells' responses are maintained within their operating
ranges and at their maximum sensitivities.

Motile cilia and cells

Another feature that may have an impact on the sen-
sitivity of the statocyst hair cells is the presence of motile
cilia. Ciliated cells are distributed all over the inner surface
of the statocyst. as well as in Kolliker's canal (Young,
1960); these cells have beating cilia that set up minute
endolymph currents within the statocyst (Budelmann,
1990). The biological significance of these cells is not clear,
but the fluid flow that they produce may be sufficient to
increase the background noise within the system and thus
reduce the overall sensitivity of the receptor system.

In addition to these ciliated cells, which are motile,
some of the sensory hair cells within the crista or macula
epithelia may also have a motor capability. Sensory cells
with motile beating cilia are present in the statocysts of
other mollusks (Stommel et ai. 1980), and some circum-

m
>- o

V)
Q)

I60

Time (sec)

Figure 6. Peristimulus time histograms showing the effect of efferent
activity on the statocyst afferent activity. Extracellular recordings were
obtained from afferent neurons from the Octopus crista and then efferents
to this segment activated by electrical stimulation (duration and time
indicated by heavy bar on time axes). This caused an inhibition of the
activity of unit A, but an increase in the activity of unit B. This provides
evidence that there are both inhibitory and excitatory efferents innervating
the statocyst crista. Bin width, 400 ms; stimulus, 50 Hz pulses for 6 s.
From Williamson. 1985.

226

R. WILLIAMSON

stantial evidence suggests that part of the membrane po-
tential noise in recordings from some secondary sensory
hair cells in squid crista may be due to ciliary movement
(Williamson, 1991a). If some of the hair cells in the cnsta
or macula do have a motor capability, this could have a
large impact on the responses of the system. Recent work
on vertebrate hair cells has shown that motility in the
outer hair cells of the cochlea can change the response
characteristics of the sensory system by altering the mi-
cromechanics of the basilar membrane responses (Hud-
speth, 1989). This is thought to be due to changes in the
length of the cells rather than an active beating of their
cilia. Such a system, operating under efferent control,
could also be present in the cephalopod statocyst.

Central processing

A final feature that can influence the characteristics of
the statocyst input is the central processing of the statocyst
information. This has two major functions: first, the cen-
tral control of the statocyst efferents, and second, the an-
alytical processing of the statocyst afferent information.
As has already been discussed, the efferents can have a
major impact on the response characteristics of the stato-
cyst. This can operate through a variety of mechanisms.
In a feed-forward system, for example, where the animal
makes a voluntary movement such as a jet propelled es-
cape, the efferents can be used to suppress, peripherally,
the massive input from the statocysts that may saturate
the afferent system. This could also be achieved centrally
by an efference copy mechanism, as has been proposed
for fish electroreception (Bell, 1981). Additionally, the
system may operate in a feedback mode, whereby the
afferent input feeds back through the efferents to dynam-
ically adjust the sensitivity of the system (Williamson,
1986). This may be important in sustained swimming or
in movements imposed by external water currents. Where
the efferents are acting at the periphery, the frequency
response of the system may well be limited by the con-
duction velocities of the efferents. The efferent axons are
small, unmyelinated fibers (Budelmann el al, 1987) and
are likely to have much slower conduction velocities than
the larger afferent fibers.

The statocyst afferents project to the ipsi- and contra-
lateral lateral pedal, pedal, and ventral magnocellular lobes
within the suboesophageal mass of the octopus brain (Bu-
delmann and Young, 1984; Plan, 1987). Probably, the
sensitivity of the sensory system can be improved centrally
by summing multiple afferent inputs. For example, where
ipsi- and contralateral statocyst inputs are from receptors
responding to the same direction of movement, then these
multiple channel inputs could be combined to improve
the sensitivity of the system or to reduce the noise in the
system (Aidley, 1971). This could also occur at the pe-
riphery, where some afferent neurons, in both crista and

macula, receive multiple inputs from a number of nearby
hair cells (Colmers, 1981; Budelmann el al. 1987).

Future research

The idea of being able to predict the locomotory per-
formance of a cephalopod solely from the morphology of
its statocysts is very attractive, especially because all but
a few species are unavailable for free swimming studies
or for physiological testing. However, although such pre-
diction based on a study of the vestibular system is now
feasible for vertebrates, only generalized statements can
be made about cephalopods.

There are two main reasons for this. First, there is no
hydrodynamic model of endolymph flow within the
statocyst that takes into account the special features of
the statocysts. Although vertebrate semicircular canal
models are a good starting point, we can have only limited
confidence in the accuracy of predictions transported di-
rectly into the cephalopod domain. Second, there is no
base of physiological work on cephalopods to provide the
constants needed fora mathematical description of stato-
cyst performance, or to test and refine any model predic-
tions. For example, even the best model based on mor-
phological studies, could not predict the effects of electrical
coupling or motile cilia on the afferent response charac-
teristics.

Future work, therefore, should be concentrated on de-
veloping an adequate model of statocyst endolymph flow,
including a description of cupula movement. This should
be complemented by an investigation of the afferent re-
sponse characteristics of representatives of the different
cephalopod groups. These data, together with a description
of the swimming styles and the likely accelerations pro-
duced in a few species of cephalopods. should give us
sufficient information to predict with some confidence
the probable locomotory performance of an animal, based
only on the morphology of its statocyst.

Acknowledgments

1 would like to thank Prof. J. Z. Young for his unending
stimulation, enthusiasm, and encouragement. Much of
this work was supported by the Alexander von Humboldt
Stiftung and the Wellcome Trust.

Literature Cited

Aidley, D. J. 1971 . The Physiology of Excitable Cells Cambridge Uni-
versity Press, London. Pp. 306-333.

Anliker, M., and \V. van Buskirk. 1971. The role of perilymph in the
responses of the semicircular canals to angular accelerations. Ada
Ololaryngol. 72:93-100.

Auerbach, B., and B. U. Budelmann. 1986. Evidence for acetylcholine
as a neurotransmitter in the statocyst of Octopus vulgaris. Cell Tissue
Res 243: 429-436.

Bell, C. C. 1981. An efference copy which is modified by reafference
input. Science 214: 450-453.

STATOCYST RESPONSE CHARACTERISTICS

227

Budelmann, B. I'. 1980. Equilibrium and orientation in cephalopods.
Oceania 23: 34-43.

Budelmann, B. V. 1990. The statocysts of squid. In Squid ax Experi-
mental Animals. D. L. Gilbert. W. J. Adelman and J. M. Arnold,
eds. Plenum Press. New York.

Itiuli Iniaiin. B. t '., and I1. Bonn. 1982. Histochemical evidence for
catecholamines as neurotransmitters in the statocyst of Octopus vul-
garis. Cell Tissue Res 227: 475-483.

Budelmann, B. U., M. Sachse, and M. Staudigl. 1987. The angular
acceleration receptor system of the statocyst of Octopus vu/garis:
morphometry. ultrastructure, and neuronal and synaptic organization.
Phil Trans. R Soc. Loud. B 315: 305-343.

Budelmann, B. II., and H. G. Wolff. 1973. Gravity response from an-
gular acceleration receptors in Octopus vulgaris. J. Comp. Physio!.
85: 283-290.

Budelmann, B. I'., and J. Z. Young. 1984. The statocyst-oculomotor
system of Octopus vulgaris: extraocular eye muscles, eye muscle
nerves, statocyst nerves and the oculomotor centre in the central
nervous system. Phil. Trans. R Soc. Land. B 306: 159-189.

Colmers, \V. F. 1981. Afferent synaptic connections between hair cells
and the somata of intramacular neurons in the gravity receptor system
of the statocyst of Octopus vu/garis. J Comp Neurol. 197: 385-394.

Correia, M. J.. J. P. Landolt, M.-D. Ni. A. R. Eden, and J. L. Rae.
1981. A species comparison of linear and nonlinear transfer char-
acteristics of primary afferents innervating the semicircular canal. In
The Veslibular System: Function and Morphology, T. Gualtierotti.
ed. Springer Verlag, New York.

Crawford, A. C., and R. Feltiplace. 1985. The mechanical properties
of ciliary bundles of turtle cochlear hair cells. / Phyxiol. 364: 359-
380.

Curthoys, I.S. 1983. The development of function of primary vestibular
neurons. Pp. 425-461 In Development ol Auditory and Vestibular
Systems. E. Romand, ed. Academic Press, New York.

Gauldie, R. W., and R. L. Radtke. 1990. Using the physical dimensions
of the semicircular canal as a probe to evaluate inner ear function
in fishes. Comp. Biochem. Physiol. 96A: 199-203.

Goldberg, J. M., and C. Fernandez. 1980. Efferent vestibular system
in the squirrel monkey: anatomical location and influence on afferent
activity. J. \europhysiol. 43: 986-1025.

Govardovskii, V. I. 1971. Some characteristics and dynamics of the
endolymph in cephalopod statocysts. J. Evol. Biochem Physiol. 1:
347-351.

Hudspeth, A.J. 1989. How the ear's ear works. Science 341: 397-404.

Hudspeth, A. J., and D. P. Corey. 1977. Sensitivity, polarity and con-
ductance change in the response of vertebrate hair cells to controlled
mechanical stimuli. Proc. Nat/. Acad. Sci. USA. 74: 2407-241 1.

Jones, G. M. 1984. The functional significance of semicircular canal
size. In Handbook of Sensory Physiology. Vol. 6 (part 1), H. H.
Kornhuber, ed. Springer- Verlag, New York.

Jones, G. M., and K. E. Spells. 1963. A theoretical and comparative
study of the functional dependence of the semicircular canal upon
its physical dimensions. Proc. R Soc. Land. B. 157: 403-419.

knapp, A.G., and J. E. Dowling. 1987. Dopamine enhances excitatory
amino acid-gated conductances in cultured retinal horizontal cells.
Nature 325: 437-439.

Maddock. L., and J. 7.. Y'oung. 1984. Some dimensions of the angular
acceleration receptor systems of cephalopods. J. Afar. Biol. Assoc.
U.K. 64: 55-79.

Morris, C. C. 1988. Statolith growth lines and statocyst function in

the cephalopoda. Ph.D. thesis. University of Cambridge. U.K.
Muller, M., and J. H. G. Verhagen. 1988. A new quantitative model

of total endolymph flow in the system of semicircular ducts. ./ Theor

Bio/. 134:473-501.
Oman, C. M., E. N. Marcus, and 1. S. Curthoys. 1987. The influence

of semicircular canal morphology on endolymph flow dynamics. Acta

Otolaryngol. (Slockli.) 103: 1-13.
Plan, T. 1987. Functional neuroanatomy of the sensory-motor lobes

of the brain of Octopus vulgaris. Ph.D. thesis. University of Regens-

burg. West Germany.
Russell, I. J., A. R. Cody, and G. P. Richardson. 1986. The responses

of inner and outer hair cells in the basal turn of the guinea-pig cochlea

and in the mouse cochlea grown in vitro. Hearing Res. 22: 199-2 16.
Steinhausen, W. 1933. Uber die Beobachtung der Cupula in den Bo-

gengangsampullen des Labyrinths des lebenden Hechts. P/liigers Arch.

Ges. Physio/. 232: 500-512.
Stephens, P. R., and J. Z. Young. 1982. The statocysts of the squid

Loligo. J Zoo/. Loud. 197: 241-266.

Stommel, E. W., R. E. Stephens, and D. L. Alkon. 1980. Motile stato-
cyst cilia transmit rather than directly transduce mechanical stimuli.

/ Cell Bio/ 87: 652-662.
Suzuki, H., and K. Tasaki. 1983. Inhibitory retinal efferents from do-

paminergic cells in the optic lobe of the Octopus. I 'ision Res. 23:

451-457.
Williamson, R. 1985. Efferent influences on the afferent activity from

the octopus angular acceleration receptor system. / E.\p. Biol 1 19:

251-254.
Williamson, R. 1986. Efferent activity in the Octopus statocyst nerves.

/ Comp. Physiol A. 158: 125-132.
Williamson, R. 1989a. Electrical coupling between secondary hair cells

in the statocyst of the squid. Alloteiitliis subulata Brain Res 486:

67-72.
Williamson, R. I989b. Electrophysiological evidence for cholinergic

and catecholaminergic efferent transmitters in the statocyst of Oc-
topus. Comp. Biochem. Physiol. 93C: 23-27.
Williamson. R. 1989c. Secondary hair cells and afferent neurones of

the squid statocyst receive both inhibitory and excitatory efferent

inputs. J. Comp. Physiol. A. 165: 847-860.
Williamson, R. 1991a. The responses of primary and secondary sensory

hair cells in the squid statocyst to mechanical stimulation. J. Comp.

Physio/. A. 167: 655-664.
Williamson, R. 1991b. The responses of the sensory hair cells in the

statocyst of Sepia. In The Cuttlefish, E. Boucaud-Camou, ed. Uni-
versity de Caen Press, Caen, France.
Williamson, R., and B. U. Budelmann. 1985a. The responses of the

Octopus angular acceleration receptor system to sinusoidal stimu-
lation. / Comp. Physiol. A. 156: 403-412.
Williamson, R., and B. U. Budelmann. I985b. An angular acceleration

receptor system of dual sensitivity in the statocyst of Octopus vulgaris.

Experientia 41: 1321-1323.

Wilson, V. J., and G. M. Jones. 1979. Mammalian \'eslibular Phys-
iology. Plenum Press, New York.
Y'oung, J. Z. 1960. The statocyst of Octopus vulgaris. Proc. R Soc

Land. B 152: 3-29.
Y'oung, J. Z. 1984. The statocysts of cranchiid squids (Cephalopoda).

J. Zool. Land. 203: 1-21.
Y'oung, J. Z. 1989. The angular acceleration receptor system of diverse

cephalopods. Phil. Trans. R Soc. Land. 325: 1 89-238.

Reference: Biol. Bull 180: 228-233. (April. 1991)

Neural Control of Speed Changes in an
Opisthobranch Locomotory System

RICHARD A. SATTERLIE

Department of Zoology, Arizona Stale University, Tempe, Arizona 85287-1501
and Friday Harbor Laboratories, Friday Harbor, Washington 98250

Abstract. Three forms of forward locomotion have been
described in the pteropod mollusk C/ione limaeina, in-
cluding slow, fast, and escape swimming. The neuro-
muscular organization of the swimming system suggests
that a two-geared system operates for slow and fast swim-
ming, while the escape response is superimposed on fast
swimming. In addition to escape, changes in locomotory
speed can occur through a dramatic "change-of-gears,"
or through a more subtle change of speed within gears.
The former involves reconfiguration of the central pattern
generator and recruitment of previously inactive motor
units. The latter can be due to: changes in tonic inputs to
the central neurons, central modulation that is not suf-
ficient to "change gears," endogenous properties of muscle
cells, and peripheral modulation of muscle contractility.
The initial ballistic phase of escape swimming is believed
to be triggered by activity in a newly identified pair of
swim motor neurons that neither receive information
from, nor provide input to, the central pattern generator.
These neurons appear to produce a startle response. Ev-
idence presented suggests that most, if not all, of these
variables help produce locomotory plasticity in Clione.

Introduction

Locomotory speed is a function of several factors, most
notably the frequency of movements of locomotory ap-
pendages and the force of appendage movements. A
change in either of these factors can directly trigger a
change in locomotory speed. The former is the provence
of the central pattern generator circuitry, whereas the latter
can be linked to modifications of the neuromuscular sys-
tem, and can conceivably include purely peripheral plas-
ticity. Furthermore, activity of central and peripheral

Received 17 August 1990; accepted 23 January 1991.

modulators can serve to increase the richness of loco-
motory variability.

Few preparations are conducive to simultaneous elec-
trophysiological monitoring of both central and peripheral
activity during both dramatic and subtle changes in pro-
pulsive activity. One preparation that combines similar
behavioral variability with the typical advantages of the
molluscan nervous system — relatively simple neural or-
ganization coupled with large cell size — is the locomotory
system of the pteropod mollusk Clione limacina. Thus
far, the majority of work on Clione has centered on the
central generation of rhythmic locomotory activity (Ar-
shavsky et al, 1985a. b. c. d, 1986, 1989; Satterlie. 1985,
1989; Satterlie and Spencer, 1985; Satterlie et al.. 1985),
although recent work has focussed on peripheral neuro-
muscular physiology (Satterlie, 1987, 1988; Satterlie et
al., 1990). The purpose of this review is to summarize
current work and present new data that relate to the neu-
robiological basis of locomotory plasticity in the Clione
swimming system.

Results and Discussion

Locomotory movements of Clione include relatively
simple two-phase flapping movements of wing-like para-
podia (wings). Three forms of locomotion have been de-
scribed including slow, fast, and escape swimming (Ar-
shavskyt'M/., 1985a; Satterlie et al.. 1985, 1990; Satterlie,
1989). The predominant form is slow swimming, which
allows the animal to maintain position in the water col-
umn or to move forward (upward) slowly. Wing beat fre-
quencies observed during slow swimming ranged from 1
to 4 Hz. Changes in the rate of forward movement within
the slow speed occur both with and without a change in
the frequency of wing movements. The latter cases pre-
sumably involve changes in wing contractility, as sug-

228

LOCOMOTORV SPEED CHANGES

229

gested by behavioral observations in which noticeable
changes in the vigor of wing movements have been ob-
served in the absence of a change in wing beat frequency.
The change to fast swimming is a triggered, typically dra-
matic change in the frequency (range: 3-8 Hz) and force
of wing movements. In addition to these two basic forms
of swimming, a ballistic escape response can be triggered
following vigorous stimulation of the tail (Satterlie et ai.
1990). The initial phase of escape swimming involves one
or two wing cycles characterized by massive contractions
of the swim musculature. This "startle" phase is followed
by a variable period of enhanced fast swimming. While
fast swimming can be triggered without an escape response
being activated, escape is always followed by fast swim-
ming.

Despite the three-phase swimming behavior, both the
central and peripheral organization of the swimming sys-
tem appears to be based on two speeds. Evidence presented
later suggests that escape swimming is merely superim-
posed on the fast swimming system. Centrally, the change
from slow to fast swimming involves a "change-of-gears."
defined here as a change in pattern generator output that
results in recruitment (or dropping out) of motor units
that have significantly different biochemical and contrac-
tile properties than those that were previously (or contin-
uously) active. Peripherally, Clione has two types of
striated swim muscle fibers: slow-twitch fatigue-resistant
and fast-twitch fatigable fibers (Satterlie. 1987; Satterlie
et ai. 1990). To complement the peripheral organization,
two types of swim motor neurons have been described:
one associated with slow-twitch muscle activity (and slow
swimming), and the other associated with both types of
muscle fibers. The latter motor units, which include two
large swim motor neurons in each pedal ganglion, are
recruited into activity during fast swimming (Satterlie.
1987, 1988, 1989).

With the two-geared arrangement of the Clione swim-
ming system before us, three categories of locomotory
speed changes will be described, with evidence presented
to suggest neurobiological mechanisms for each. Cate-
gories of speed change mechanisms include: ( 1 ) change-
of-gears, (2) change of speed within gears, and (3) escape
swimming.

c; Satterlie, 1985. 1989). One group of interneurons (V-
phase interneurons) produces a single action potential
during ventral bending of the wings, whereas the other
group (D-phase interneurons) spikes during dorsal bend-
ing of the wings. Alternating activity of these two groups
of interneurons continues during fast swimming, but two
additional interneuron types become active (Arshavsky
et nl.. 1985d, 1989). Delayed V-phase interneurons, which
receive only inhibitory input from D-phase interneurons
during slow swimming, produce slightly delayed (with re-
spect to normal V-phase interneurons) V-phase spikes
during fast swimming. Spikes in the delayed V-phase in-
terneurons trigger activity in the second type of interneu-
ron, called interneurons 12 (Arshavsky et ai. 1985d.
1989). Each interneuron 12 produces a plateau potential
that is turned on by excitatory input from delayed V-
phase interneurons, and is turned off by inhibitory input
from D-phase interneurons. Plateau potentials of inter-
neurons 12 inhibit V-phase interneurons and excite D-
phase interneurons. Addition of the delayed V-phase and
type 12 interneurons to the swim pattern generator thus
produces an early termination of V-phase activity coupled
with onset of the next D-phase. This change increases the
cycle frequency of pattern generator output (Arshavsky
t'/ ai. 1985a) and is associated with a recruitment of pre-
viously inactive large motor neurons (Fig. 1). As men-
tioned previously, recruitment of these motor neurons is
associated with the activation of the fast-twitch muscu-
lature of the wings. The change-of-gears is also associated
with a 5- 1 5 m V tonic depolarization in "normal" D- and
V-phase interneurons of the swim pattern generator (Fig.
1). The combination of increased cycle frequency and
increased force of wing contractions through recruitment
of "fast-twitch" motor units produces a dramatic increase
in forward propulsion speed.

Speed changes due to a "change-of-gears"

Centrally, the change-of-gears from slow to fast swim-
ming involves reconfiguration of the central pattern gen-
erator (Arshavsky et ai. 1985d. 1989). as previously in-
active pedal interneurons become active elements of the
swim pattern generator. During slow swimming, a two-
phase motor drive is produced by activity in two antag-
onistic groups of pedal interneurons that interact through
reciprocal inhibitory connections (Arshavsky et ai. 1985b,

Figure 1 . Intracellular recording from a pattern generator interneuron
of Clione (top trace) with a simultaneous extracellular recording from
the wing nerve (bottom trace). The record shows a change-of-gears (arrow)
involving an increase in cycle frequency and a tonic depolarization in
the interneuron. The change recruits large spikes in the wing nerve re-
cording. These large spikes have been shown to reflect activity in large
swim motor neurons. Recording by A. N. Spencer. University of Alberta.

230

R. A. SATTERLIE

Rewiring of a central pattern generator is certainly not
a new concept. The pyloric central pattern generator of
the lobster stomatogastric system can exhibit at least four
distinct functional circuits and thus four distinct motor
activities (Flamm and Harris- Warrick. 1986a. b; Harris-
Warrick el al, 1989). In addition to the unmodulated
circuit, the amine modulators dopamine. octopamine, and
serotonin can each produce a dramatically distinct func-
tional circuit (see Harris- Warrick el al., 1 989, for a review).
In the opisthobranch mollusk Tritonia. variable output
in the body wall motor systems can be produced by vary-
ing the types and intensities of triggering sensory inputs.
According to the polymorphic network concept (Getting
and Dekin, 1985). different inputs can activate different
configurations of motor control systems to produce
unique motor outputs as distinctive as body wall with-
drawal and swimming movements. These two examples
demonstrate that a motor control system, or part of it,
can be used for more than one behavior. In comparison,
reconfiguration of the Clioneswim pattern generator ap-
pears to involve exclusively frequency modulation rather
than changes in the phase relationships or functional wir-
ing of the pattern generator.

The change from slow to fast swimming in Clione is
induced in both intact and reduced preparations when
the preparations are bathed in 10~- to 1CT6 M serotonin
(Arshavsky el al., 1985a. d: Satterlie 1989). Under these
conditions, fast swimming continues as long as serotonin
remains in the bath. At the level of individual pattern
generator interneurons, serotonin produces a 5-10 mV
tonic depolarization similar to that seen during sponta-
neous fast swimming. The source of these tonic depolar-
izations is not known. Serotonin has also been implicated
in the initiation of swimming activity in the leech (Kristan
and Weeks, 1983; Nusbaum and Kristan 1986; Nusbaum.
1986) and of Aplysia brasiliana (Parsons and Pinsker.
1989), as well as pedal locomotion in non-swimming
Aplysia (Mackey and Carew, 1983). Serotonin also mod-
ulates ongoing rhythmic activity in a number of prepa-
rations, including lamprey swimming (Harris- Warrick and
Cohen, 1985), feeding in Aplysia (Kupfermann and Weiss,
1982), insect flight (Claassen and Kammer, 1986), and
the pyloric rhythm of the lobster (Flamm and Harris-
Warrick, 1986a, b). Serotonin can also have system-wide
behavioral effects, as in the regulation of posture in lobsters
(Kravitz el al., 1985).

Changes of swimming speed within gears

Although the possibilities for changes of speed within
gears are numerous, four possibilities will be considered
here: ( 1 ) changes in tonic input to swim interneurons and
motor neurons, (2) central modulation of the pattern gen-
erator (e.g., with serotonergic inputs) at a level not suffi-

cient to change gears, (3) the role of endogenous properties
of muscle cells, and (4) peripheral modulation of muscle
contractility. The first two involve central modifications
while the last two modify peripheral activity.

Changes in tonic input to swim neurons

Despite the description of pedal neurons that show
variable tonic activity associated with changes in pattern
generator activity in Clione (Arshavsky et al., 1984), little
is known about the variety and sources of tonic influences
over pattern generator activity. Inasmuch as tonic depo-
larization of isolated pattern generator interneurons is re-
lated to spontaneous firing frequency (Arshavsky et al.,
1986), then tonic inputs can presumably modify the fre-
quency of pattern generator output. Provided that the in-
puts do not cause pattern generator reconfiguration, the
change in cycle frequency will be translated into a change
of locomotory speed within the appropriate "gear." Tonic
input could exert this influence in either slow or fast
swimming gears.

Central modulation not sufficient to change gears

The source of central serotonergic inputs to the pattern
generator that are responsible for reconfiguration and gear
change have not yet been identified. But circumstantial
evidence now in hand has led us to investigate descending
serotonergic inputs from the cerebral ganglia. Serotonin-
immunoreactive neurons have been found in the medial
posterior and medial anterior regions of the cerebral gan-
glia. Axons from some of these cells run from the cerebral
ganglia to the pedal ganglia via the cerebro-pedal con-
nectives. Focal extracellular stimulation of the medial
posterior region of a pedal ganglion results in acceleration
of pattern generator activity, or with strong stimuli,
changes in pattern generator activity identical to changes
associated with activation of fast swimming activity.
Transection of the cerebro-pedal connective greatly re-
duces these responses. Assuming that the central modu-
lation does not operate in an all-or-none manner, sub-
threshold levels of modulation (subthreshold for change
of gears) might trigger a change of swimming speed within
the slow gear, and different levels of supra-threshold
modulation might produce variable pattern generator ac-
tivity in the fast gear. Such changes of swimming speed
should be expressed as a change in cycle frequency, unless
swim motor neurons are also affected by the central mod-
ulatory subsystem. In the latter case, changes in both cycle
frequency and force of wing movements will be seen. A
further, purely speculative possibility allows for separate
modulation of pattern generator interneurons and swim
motor neurons, a condition that would add greatly to the
complexity of the behavioral output. Potential central

LOCOMOTORY SPEED CHANGES

231

modulators other than serotonin are not being considered
here, but should not be discounted.

Intrinsic properties of muscle cells

Intrinsic properties of muscle cells, particularly related
to repetitive firing activity, can influence the force of swim
muscle contractions. Such intrinsic properties could be
synaptic or non-synaptic, the latter including changes in
passive or active membrane properties, or in excitation-
contraction coupling. Both slow-twitch and fast-twitch fi-
bers of the Clione swimming system exhibited non-syn-
aptic facilitation of the amplitude of spike-like responses
with repetitive, direct depolarization of individual muscle
cells (Satterlie, 1988). The facilitation was strongly fre-
quency-dependent, so that both overall amplitude of
spike-like responses and initial rate of change of spike-
like response amplitude showed a positive correlation with
frequency of induced activity over the range of frequencies
normally encountered during slow and fast swimming (in
prep.). Provided that the contractile force of whole muscles
is related to changes in spike-like response amplitude re-
corded from individual cells, overall muscle force should
change in parallel with changes in pattern generator fre-
quency.

Peripheral modulation of contractile force

A cluster of 7-10 serotonin-immunoreactive neurons
have been found in the medial margin of each pedal gan-
glion of Clione (Fig. 2). At least two neurons from this
cluster send axons to the ipsilateral wing via the wing
nerve. Induced activity in these two neurons produced
no direct motor response: but when activity was triggered
during ongoing swimming activity, muscle contractions

Figure 2. Schematic diagram of the dorsal surface of the left pedal
ganglion of Clione. The two large motor neurons (major landmarks of
the ganglion) are indicated by cells 1 and 2. Cells 3 and 4 represent motor
neurons that initiate escape swimming. The remaining cells represent
serotonin-immunoreactive cells. The two cells marked with an asterisk
have been electrophysiologically identified', they send axons into the ip-
silateral wing via the wing nerve (wn). These cells enhance muscle con-
tractility as shown in Figure 3. pc-pedal-pedal commissure.

Figure 3. Dual recording from a serotonin-immunoreactive neuron
(bottom trace) and a wing force transducer (top trace — not calibrated).
Following a burst of action potentials in the neuron, muscle contractions
are enhanced. The latency of the response is approximately one second,
and the duration is 5 s. The neuron was hyperpolarized by a -1 nA
current during the recording to prevent spiking. The burst was triggered
by switching to a + 1 nA current.

were enhanced (Fig. 3). The response latency was ap-
proximately one second from the initiation of the induced
burst, and the effect lasted from 3-10 s. Preliminary ev-
idence suggests that this enhancement was due to an in-
creased amplitude of the spike-like response in some, but
not all, of the muscle cells.

Peripheral modulation, including both pre- and post-
synaptic effects, have been noted in numerous prepara-
tions (e.g.. Kravitz et at.. 1985; Kobayashi and Hasimoto,
1982; Maranto and Calabrese, 1984; Weiss et ai, 1978).
Induced bursts in the pedal serotonin-immunoreactive
neurons of Clione produced no apparent synaptic activity
in either pattern generator or motor neurons, and pro-
duced no changes in frequency or intensity of spike ac-
tivity in either neuron type. This suggests an interesting
dichotomy in serotonin modulation of swimming in
Clione: i.e.. pedal serotonin-immunoreactive neurons
modulate muscle activity, whereas proposed cerebral se-
rotonergic neurons modulate pattern generator activity.
A similar separation of central and peripheral modulation
is seen in the leech heartbeat system (Calabrese and Arbas,

Table I

Summary of four possible modulatory states in the swimming
M'Wi'iH "/ Clione limacina based on separate central
and peripheral modulatory subsystems

Modulatory state

No modulation

Peripheral modulation only

Central modulation only

Central and peripheral
modulation

Swimming activity

Slow swimming, normal muscle
contractility

Slow swimming, enhanced muscle
contractility

Fast swimming, normal muscle
contractility

Fast swimming, enhanced muscle
contractility

232

R. A. SATTERLIE

*f f f

ImV

0.5s

Figure 4. Dual recording from a "startle" motor neuron (bottom
trace) and an uncalibrated wing force transducer (top trace). Note the
absence of pattern generator input to the neuron despite the ongoing
swimming activity. Bursts of action potentials were triggered in the neuron
with +12 nA injected currents (through the recording electrode). The
resultant bursts of activity induced strong contractions of the wing.

1985). Assuming the simplest case of supra-threshold
modulation in both central and peripheral subsystems of
Clione. the separation of pattern generator and muscle
modulatory subsystems allows four possible states with
respect to serotonin modulation of swimming activity
(Table I). As mentioned previously, central modulation
will primarily affect cycle frequency, whereas peripheral
modulation will affect contractile force.

This discussion takes into account only one peripheral
modulatory system. The possibility of other modulatory
inputs, as well as the release of multiple transmitters or
modulators from the serotonin-immunoreactive neurons,
could add further complexity to the swimming system.

Escape swimming

An interesting pair of motor neurons have recently been
identified from each pedal ganglion of Clione (Fig. 4).
These motor neurons activate both slow-twitch and fast-
twitch fibers of the swim musculature, but do not receive
input from the swim pattern generator. The neurons were
originally overlooked, because they are electrically silent
during normal swimming activity and have extremely high
firing thresholds. In some preparations, it is very difficult
to stimulate electrical activity from these cells with intra-
cellular current injection. Induced bursts of spikes in the
motor neurons produce massive contractions of the ip-
silateral wing. Despite this strong peripheral input, the
cells have no inputs to, or influence over, the activity of
interneurons of the pattern generator or swim motor neu-
rons. Their powerful effect on swim musculature, their
total independence from the swim pattern generator, and
their high firing threshold suggest that these motor neurons
may participate in the primary phase of escape swimming
by triggering the initial ballistic movement; indeed, the
ballistic movement may function as a startle response.
The maintenance of escape swimming, involving the
variable period of enhanced fast swimming, could rep-
resent activation of both central and peripheral seroton-
ergic modulatory subsystems (see Table I). Multiple re-
cordings from "startle" neurons and serotonin-immu-

noreactive neurons following tail stimulation in intact
preparations have not yet been completed due to technical
difficulties, but should help clarify this relationship.

The foregoing discussion introduces several levels at
which changes of locomotory speed can occur in the
swimming system of Clione. Some of the results are pre-
liminary, while a few are purely speculative. It is clear,
however, that both central and peripheral modulatory in-
fluences are operating, and that significant changes in both
frequency and strength of wing movements can contribute
to locomotory speed changes. With this information, we
are beginning to gain an appreciation for the neurobio-
logical complexity involved in locomotory plasticity in
this relatively "simple" swimming system. Our compre-
hension of the neuronal bases of speed changes involves
changes of gears, changes of speed within gears, and su-
perimposed inputs, such as escape. This understanding is
providing a good starting point for further investigation
of other forms of input and modulation, as well as detailed
descriptions of the intrinsic properties of all cells involved
in swimming behavior.

Acknowledgments

I thank Lou and Alison Satterlie for help collecting
experimental animals. Dr. A. O. D. Willows for providing
space and facilities at Friday Harbor Laboratories, and
Dr. A. N. Spencer for the use of Figure 1 . Research covered
in this paper was supported by a National Science Foun-
dation grant (BNS85-1 1692) and a research grant from
the Whitehall Foundation.

Literature Cited

Arshavsky, Yu.I., I. N. Beloozerova, G. N. Orlovsky, G. A. Pavlova, and
Vu.V. Panchin. 1984. Neurons of pedal ganglia of pteropodial mol-
lusc controlling activity of the locomotor generator. Nejrofiziologia
16: 543-546.

Arshavsky, Yu.I., I. N. Beloozerova, G. N. Orlovsky, Yu.V. Panchin,
and G. A. Pavlova. I985a. Control of locomotion in marine mollusc
Clume limacina. I. Efferent activity during actual and fictitious
swimming. Exp. Brain Res 58: 255-262.

Arshavsky, Yu.I., I. N. Beloozerova, G. N. Orlovsky, Yu.V. Panchin,
and G. A. Pavlova. 1985b. Control of locomotion in marine mollusc
Clione limacina. II. Rhythmic neurons of pedal ganglia. Exp. Brain
Rex. 58: 263-272.

Arshavsky, Yu.I., I. N. Beloozerova, G. N. Orlovsky, Yu.V. Panchin,
and G. A. Pavlova. I985c. Control of locomotion in marine mollusc
Clione limacina. III. On the origin of locomotory rhythm. Exp Brain
Res 58: 273-284.

Arshavsky, Yu.I., I. N. Beloozerova, G. N. Orlovsky, Yu.V. Panchin,
and G. A. Pavlova. 1985d. Control of locomotion in marine mollusc
Clione I i mac i mi. IV. Role of type 12 interneurons. Exp. Brain Res.
58: 285-293.

Arshavsky, Yu.I., T. G. Beliagina, G. N. Orlovsky, Yu.V. Panchin,
G. A. Pavlova, and L. B. Popova. 1986. Control of locomotion in
marine mollusc Clione limacina. VI. Activity of isolated neurons of
pedal ganglia. Exp. Brain Res. 63: 106-1 12.

LOCOMOTORY SPEED CHANGES

233

Arshavsky, Yu.l., 0. N. Orlovsky, Yu.V. Panchin, and G. A. Pavlova.
1989. Control of locomotion in marine mollusc Clione liniacina.
VII. Rcexamination of type 12 interneurons. /L".Y/> Brain Res 78:
398-406.

Calabrese, R. L., and E. A. Arbas. 1985. Modulation of central and
peripheral rhythmicity in the heartbeat system of the leech. Pp. 69-
85 in Model Neural Networks and Behavior. A. I. Selverston, ed.
Plenum. New York.

Claassen, D. E., and A. E. Kammer. 1986. Effects of octopamine. do-
pamine and serotonin on production of flight motor output by tho-
racic ganglia of Manduca sexta. J Neurobiol. 17: 1-14.

Flamm, R. E., and R. INI. Harris-Warrick. 1986a. Aminergic modu-
lation in the lobster stomatogastnc ganglion. I. Effects on the motor
pattern and individual neurons within the pyloric circuit. ./, Neiiro-
physiol. 55: 847-865.

Flamm, R. E., and R. M. Harris- \\arrick. 1986b. Aminergic modu-
lation in the lobster stomatogastnc ganglion. II. Target neurons of
dopamine. octopamine and serotonin within the pyloric circuit. ./
Neurophysiol. 55: 866-881.

Getting, P. A., and M. S. Dekin. 1985. Tritn/iui swimming. A model
system for integration within rhythmic motor systems. Pp. 3-20 in
Model \eural Networks and Behavior. A. I. Selverston. ed. Plenum,
New York.

Harris-Warrick. R. M., and A. H. Cohen. 1985. Serotonin modulates
the central pattern generator for locomotion in the isolated lamprey
spinal cord. J Exp Biol. 116: 27-46.

Harris-Warrick, R. M., R. E. Flamm, B. R. Johnson, and P. S. Katz.
1989. Modulation of neural circuits in Crustacea. Am tool 29:
1305-1320.

Robayashi, M., and T. Hasimoto. 1982. Antagonistic responses of the
radular protractor and retractor to the same putative neurotrans-
mitters. (.'oiup Hun-hem, t'hysiol. 72C: 343-348.

Kravitz, E. A., B. Beltz, S. Glusman, M. Goy, R. Harris- Warrick, M.
Johnston, M. Livingstone, T. Schwarz, and K. K. Siwicki. 1985. The
well-modulated lobster. The roles of serotonin, octopamine and
proctolin in the lobster nervous systems. Pp. 339-360 in Model Neural
Networks and Behavior. A. I. Selverston, ed. Plenum, New York.

Kristan. \V. B., Jr., and J. C. Weeks. 1983. Neurons controlling the
initiation, generation, and modulation of leech swimming. Pp. 243-
260 in Neural Origin oJ'Rhylhmie Movements. A. Roberts and B. L.
Roberts, eds. Cambridge University Press, Cambridge.

Kupfermann, I., and k. R. Weiss. 1982. Activity of an identified se-
rotonergic neuron in free moving. -Iplysia correlates with behavioral
arousal. Brain Res 241: 334-337.

Mackey, S., and I . J. Carew. 1983. Locomotion in Aplysia: triggering
by serotonin and modulation by bag cell extract. / Neumsci. 3: 1469-
1477.

Maranto, A. R., and R. L. Calabrese. 1984. Neural control of the hearts
in the leech, Hirudo medicinalis II. Myogenic activity and its control
by heart motor neurons. ./. Comp. I'hysiol. 154: 381-391.

Nusbaum, M. P. 1986. Synaptic basis of swim initiation in the leech.
III. Synaptic effects of serotonin-containing interneurones (cells 21
and 6 1 ) on swim CPG neurones (cells 1 8 and 208). / Exp. Bio/. 1 22:
303-321.

Nusbaum, M. P., and W. B. Kristan. 1986. Swim initiation in the leech
by serotonin-containing interneurones, cells 2 1 and 6 1 . J. Exp. Biol.
122:277-302.

Parsons, D. W., and H. M. Pinsker. 1989. Swimming in Aplysia bras-
i liana: behavioral and cellular effects of serotonin. /. Neurophysiol.
62: I 163-1176.

Satterlie, R. A. 1985. Reciprocal inhibition and postinhibitory rebound
produce reverberation in a locomotor pattern generator. Seience 229:
402-404.

Satterlie, R. A. 1987. Neuromuscular organization for two-speed
swimming in a pteropod mollusc. Six. Neumsci. Abslr. 13: 1061.

Satterlie, R. A. 1988. Peripheral organization of the swimming system
in a pteropod mollusc. Soc Neurosci Ahslr 14: 1001.

Satterlie, R. A. 1989. Reciprocal inhibition and rhythmicity: swimming
in a pteropod mollusc. Pp. 151-171 in Neuronal and Cellular Os-
cillators. J. W. Jacklet, ed. Dekker. New York.

Satterlie, R. A., and A. N. Spencer. 1985. Swimming in the pteropod
mollusc. Clione linuicinu II. Physiology./ Exp. Biol. 116:205-222.

Satterlie, R. A., M. LaBarbera, and A. N. Spencer. 1985. Swimming
in the pteropod mollusc C/ione liinaeinu. I. Behaviour and mor-
phology. / Exp Biol. 116: 184-204.

Satterlie, R. A., G. E. Goslow Jr., and A. Reyes. 1990. Two types of
striated muscle suggest two-geared swimming in the pteropod mollusc
Clione lunaana. J E.v/>. /<«•/. 255: 131-140.

Weiss, K. R., J. L. Cohen, and I. Kupfermann. 1978. Modulatory con-
trol of buccal musculature by a serotonergic neuron (metacerebral
cell) tn Aplysia. J Neurophysiol 41: 181-203.

Reference: Bwi Bull. 180: 234-240. (April, 1991)

On the Significance of Neuronal Giantism

in Gastropods

RHANOR GILLETTE

Department of Physiology & Biophysics and The Neuroscience Program, 524 Burrill Hall,
407 S. Goodwin Ave., University of Illinois, Urbana, Illinois 61801

Abstract. Neurons of the central ganglia of opistho-
branch and pulmonate gastropods increase in size as the
animals grow, some becoming veritable giants. The origins
and functions of neuronal giantism are considered here
from a comparative viewpoint. A review of the properties
of identified neurons in a variety of opisthobranch and
pulmonate species indicates that neuronal size is directly
related to the extent of postsynaptic innervation. DNA
endoreplication, resulting in partial or complete poly-
ploidy, supports giantism in molluscan neurons as it does
in eukaryotic cells elsewhere. Apparently, the functional
significance of giantism is enhanced synthesis and trans-
port of materials to serve an expanded presynaptic func-
tion.

Giant neurons are found in larger snails where they
innervate large areas of the periphery; interneurons and
sensory neurons are enlarged to a lesser degree, probably
to that which enables load-matching to the peripheral ef-
fectors. Neuronal giantism may be an adaptation for the
innervation of the periphery in large animals with simple
behaviors and uncomplex sensoria, this adaptation en-
abling growth of body and CNS without a proportionate
increase in neuronal number. A more complete under-
standing of the evolutionary and adaptive significance of
neuronal giantism should be sought in comparative studies
of the cellular properties of simple and complex molluscan
brains.

Introduction

The condition of neuronal giantism in the pulmonate
and opisthobranch gastropods has been a point of marvel
at least since Buchholz' observations in 1863 (reviewed
by Bullock, 1965). The conveniences offered by giant

Received 7 August 1990; accepted 25 January 1991.

nerve cells to experimenters have also invited numerous
biophysical and neuroethological studies; these have con-
tributed greatly to our knowledge of nerve cell function
and behavioral mechanisms. Even so, the significance of
neuronal giants to the animals in which they are found
has not been satisfactorily understood.

The question of neuronal giantism is particularly open
to the methods of comparative analysis. The physiology,
anatomy, and behavioral roles of giant neurons have been
analyzed from a wide variety of species, and homologous
neurons have been identified across species. The following
paragraphs marshal evidence that supports several hy-
potheses for the origin and functional significance of neu-
ronal giantism.

The Molluscan Neuron

The typical molluscan neuron is a monopolar or bipolar
cell with its soma lying in the ganglion periphery (Fig. 1 ).
An axon enters the neuropil in the core of the ganglion
where it branches off neurites that both receive and make
synaptic contacts. Neurites generally sprout close to the
cell body and even originate from it in opisthobranch and
pulmonate neurons. Action potentials are initiated in the
axon and regulated by synaptic inputs to the neurites; the
region of spike initiation and synaptic activity is referred
to here as the integrating region.

Neuronal Giantism: The Condition

The condition of "giantism" is one of degree. The cen-
tral ganglia of opisthobranch and pulmonate snails com-
monly possess 10-20 distinct and identifiable nerve cells
with cell bodies so large that they stand out from their
neighbors as relative giants. Aside from the obvious giant
neurons, the entire central nervous system of such animals
contains only several tens of thousands of neurons, several

234

GIANT NEURONS IN SNAILS

235

cbc

Figure 1. Typical morphology of giant neurons of pulmonates and
opisthobranchs, as exemplified by this drawing of the serotonergic giant
of the cerebral ganglion of Tntuniu hombcrgi (from Dorset!. 1986). The
large excitable soma is close to the integrating region of axon and fine
neuntes, where synaptic potentials occur and spikes are initiated. The
large axons, with high specific membrane resistances, favor current spread
from integrating region to soma.

hundred of which may be identified on the basis of po-
sition, color, synaptic and axonal connections (<.;/. Bullock.
1965; Coggeshall. 1967; Frazier ct ai. 1967). In the larger
pulmonates, the biggest neurons have somata approaching
100 /urn in diameter, whereas in the larger sea slugs, certain
neuronal somata reach over 700-800 j/m. Moreover, as
the animals increase in size, all of their identifiable neurons
also grow in diameter.

Neuronal Size is Related to Postsynaptic Innervation

In approaching the nature of neuronal giantism, the
first relevant observation is that neuron giants must in-
nervate larger postsynaptic target areas than non-giants.
The evidence that neuronal size is directly related to the
extent of postsynaptic innervation comes from the liter-
ature characterizing a variety of identified neurons in
opisthobranch and pulmonate snails. The largest neurons
of the central ganglia act as effectors that innervate large
areas of the periphery.

Prominent examples are a bilateral pair of giant sero-
tonergic neurons identified in many opisthobranch and
pulmonate snail species. These neurons are commonly
the largest neuronal somata of the cerebral ganglion
(Senseman and Gelperin, 1973; Berry and Pentreath,
1976; Weiss and Kupfermann, 1976; Gillette and Davis,
1977; Granzow and Kater, 1977). Approaching 400-500
jim in size in the larger opisthobranchs, these giant effec-
tors send large axons down the cerebrobuccal connectives;
the axons ramify within the buccal ganglion so that an
axonal branch is sent out in each nerve. These axons in-
nervate large areas of the muscular buccal mass and the
esophagus; the neurons also send branches out the lip or
mouth nerves of the cerebral ganglion to innervate the
oral region (Fig. 2). In addition, the giant serotonergic
neurons have some synaptic output in the buccal ganglia
(ibid.).

Other well-studied giants are two of the largest neurons
known, the neurons R2 and LP11 of the anaspid opis-

thobranch Aplysia California. R2 and LP1 1 are bilaterally
homologous and cholinergic, attaining soma diameters
nearly 1000 nm in large animals. Due to assymetrical
ganglionic fusion in the embryo, R2 is found in the ab-
dominal ganglion, and LP11 in the left parietal ganglion.
The cell bodies give off giant axons that send branches to
most ganglia and out many nerves thence innervating ex-
tensive areas of the skin (Hughes and Tauc, 1963; Cobbs
and Pinsker, 1979). Their electrical activity stimulates
mucus secretion (Rayport el ai. 1983).

Among the motorneurons innervating the gills of nu-
dibranchs and notaspids are some of the largest neurons
of the pedal, pleural, and cerebral ganglia (Blackshaw and
Dorsett, 1976; Dickinson, 1979, 1980).

The well-studied buccal ganglia provide more examples
of giantism. The largest neurons of opisthobranch buccal

Figure 2. Extensive innervation of the periphery by the serotonergic
cerebral giant neurons of Helix pomalia (from Berry and Pentreath.
1974). Aside from some interneuronal function in the CNS, these giants
send many branches to the buccal ganglion and out the nerves to innervate
the musculature of the buccal mass and esophagus. Other branches leave
anterior nerves to innervate the feeding musculature of the oral region.
This general plan is found in the homologous giant cells of many pul-
monate and opisthobranch species.

236

R. GILLETTE

ganglia are typically motorneurons: sensory neurons are,
on the average, much smaller (Byrne el a/., 1974; Siegler,
1977; Spray el a/., 1980; Dorsett and Sigger, 1981). The
largest known buccal cells may be those of the buccal
ganglion of the cephalaspid Nuvanax. These neurons in-
nervate the musculature of the large pharynx, driving its
expansion during prey-capture (Spira and Bennett, 1972).
In aeolid and doridacean buccal ganglia, the largest neu-
rons are often a bilateral pair called the Dorsal White
Cells (Bulloch and Dorsett, 1979). The Dorsal White Cells
are peptidergic neurons that send axons out the gastro-
esophageal nerve to ramify over, and innervate, the large
esophagus (Masinovsky and Lloyd, 1985).

Interneurons with only central synaptic outputs tend
to be smaller than interneurons of dual function, i.e.. with
both CNS output and peripheral axons innervating mus-
cle. For instance, both the identified VWC and B3I neu-
rons of Pleurobranchaea can drive intense cyclic motor
output in the buccal oscillator network; but the VWC also
innervates the muscular esophagus, and the diameter of
its soma is nearly three times that of the B3I soma (Gillette
elal. 1980). Identified neurons with purely central outputs
also may differ in size according to the extent of their
postsynaptic output. The paired SO interneurons of the
buccal ganglion of the pulmonate Lymnaea have a large
dendritic field, and their somata are three times the size
of the interneurons of the Nl, N2, and N3 populations,
which have collectively rather similar functions as oscil-
lator elements, but smaller dendritic fields in the ganglion
and weaker effects, individually, on the network (Elliot
and Benjamin, 1985a, b).

Sensory neurons can innervate large peripheral areas,
but their presynaptic function is largely confined to central
ganglia, and they tend to be small. Sensory neurons of
the buccal ganglia of Pleurobranchaea tend to have smaller
somata than motorneurons innervating the same muscles
of the buccal mass (Siegler, 1977). Similarly, the buccal
ganglia of Navanax contain mechanosensory neurons that
serve the pharynx and are much smaller than their post-
synaptic giant motorneurons that drive the pharyngeal
musculature (Spray el ai, 1980a, b). Sensory neurons car-
rying mechanosensory information from the skin of Tri-
tonia are quite smaller than the interneurons and motor-
neurons they drive (Getting, 1977). The abdominal gan-
glion ofApIysia contains sensory neurons that innervate
the gill and siphon and that are much smaller than the
gill and siphon motorneurons they drive (Byrne el al.,
1974). In each case, the sensory neurons have smaller
dendritic fields, and thus may make fewer synaptic con-
tacts, than the larger motorneurons and interneurons.

A direct relationship between the field of postsynaptic
innervation of a neuron and its soma size has been pre-
viously recognized by some workers in arthropod neu-
robiology. Mittenthal and Wine (1978) showed that the

soma diameter of serially homologous motorneurons in
the segmental nervous system of crayfish is roughly pro-
portional to the area of the serially homologous muscle
they innervate. Mellon el al. (1981) showed that ampu-
tation of the specialized snapping claw of the snapping
shrimp Alpheus causes the contralateral claw and its mus-
culature to enlarge into a larger snapping claw at subse-
quent molts; the soma of the claw opener motorneuron
enlarges with the size of its target organ.

Finally, the peripheral effector neurons of the opis-
thobranch central nervous system increase in size with
the growth of their target organs. The size of identified
neurons, in soma diameter, axon diameter, and dendritic
field, increases with the size of the animal during growth
(Coggeshall, 1967; Frazier el al., 1967). Accordingly, sen-
sory interneurons monitoring the peripheral effectors and
the smaller interneurons also increase in size; this is a
form of load matching. All of these observations argue
for a trophic relationship between the area of the inner-
vated structure and the size of the presynaptic neuron. It
is assumed here, notwithstanding the lack of direct evi-
dence, that increases in innervated area and extent of pre-
synaptic branching are accompanied by increases in syn-
aptic contact area, number of synaptic sites, or both.
Therefore, the beginning of the answer to the question:
"Why do some neurons become giants?" is probably that
their size is related to the actual total area of synaptic
contact.

The Mechanism of Giantism: DNA Endoreplication

For certain cell types in many animals, an increase in
cell size is generally accompanied by an increase in the
actual mass of the genomic DNA and of RNA (Mirsky
andOsawa, 1 96 1 ; r/. Cavalier-Smith, 1978); this is effected
either through polyploidy or polyteny. An increase in
polyploidy with neuronal size has been demonstrated in
molluscan neurons. The nuclei of the largest neurons of
mature Aplysia (e.g.. R2) contain >0.2 n% of DNA — more
than 200,000 times the haploid amount (Lasek and
Dower, 1971). Neurons of the terrestrial pulmonate
Achatina, with soma diameters of >9 nm (nuclear di-
ameter > 7 ^m), were found to be polyploid (Chase and
Tolloczko, 1987). The frequency distribution of the DNA
content in Achatina (Chase and Tollockzo, 1987) and
Planorbis (Lombardo et al.. 1980) neurons indicates that
endoreplication during growth probably represents selec-
tive gene amplification, rather than simple sequential
doubling. However, sequential doubling may occur during
growth in Aplysia (Coggeshall et al.. 1970; Lasek and
Dower, 1971). Giantism in molluscan neurons is thus
like giantism in other metazoan cells, and is simply based
on increased amounts of nucleic acids and proteins.

Polyploid neurons of varying sizes may be common to
the nervous systems of molluscs in general; i.e.. increasing

GIANT NEURONS IN SNAILS

237

neuron size and ploidy may be a usual feature of growth
within all of the molluscan classes; one that is, perhaps,
carried to the extreme in the pulmonates and opistho-
branchs.

The Functions of Giantism: Synthesis and Transport

Neuronal giants apparently innervate larger postsyn-
aptic target areas than non-giants. Neuronal giantism,
therefore, may allow an increase in animal size without
a proportional increase in the number of central neurons.
Giant cells in most tissues are more metabolically active
than smaller cells and are frequently associated with
transport and secretory processes. Familiar examples are
the giant polytene cells of dipteran salivary glands, mal-
pighian tubules, and gut, all of which are notably active
in ion and peptide transport and exocytotic secretion.
Thus, elaboration of DNA, RNA. and protein in many
giant cells is indicative of enhanced synthetic capacity,
presumably to serve the needs of increased cell activity.
In giant neurons, these needs are likely to be connected
with increased axon transport and secretion processes at
their extensively distributed synaptic terminals.

Thus, the picture of the giant neuron becomes one
where the size, synthetic capacity, and axonal transport
traffic is adapted to the extent of postsynaptic innervation.
The giant cells do the work of many smaller cells in other
nervous systems.

The Evolutionary Origin and Integrative Significance
of Neuronal Giantism in Gastropods

The occurrence of giant neurons in snails is explained
in one sense by the observation that the giant neurons
must innervate large postsynaptic areas. The imposing
question that looms is: why do the pulmonates and opis-
thobranchs display such pronounced neuron giantism
whereas other gastropod taxa do not? The best answer
will probably rest on future comparative observations on
species chosen for particular nervous system characters,
but the context for such comparative observations can be
set here. The approach is to enumerate the specific set of
behavioral and neurophysiological characteristics that
may place the opisthobranch/pulmonate line apart from
other gastropods: in the process, perhaps, a few useful
speculations may be generated.

Those gastropods that are distinguished by possession
of a score or more of large neurons are also distinguished
by the combination of the following characteristics:

1 . relatively large body size;

2. motile, foraging lifestyles sustained by relatively simple
behavior;

3. simple nervous systems lacking, for the most part,
complex sensoria;

4. a fairly high degree of centralization within the CNS;
and

5. excitable neuron cell bodies.

Although one or more of these characteristics may appear
in various gastropod taxa, the appearance of all five may
be relatively specific to the opisthobranch/pulmonate line.

The gastropods crept into the fossil record around 580
million years ago as minute animals 1-2 mm in shell
diameter, and today most are still smaller than 5 mm.
The larger modern gastropods are thus truly somato-
morphic giants; their greater body size demands enhanced
innervation of the periphery. In most large species, this
need is met largely by an increase in brain size and neuron
number: even in the opisthobranch/pulmonate line, the
number of neurons (and the number of peripheral axons)
increases with body size, in parallel with the striking in-
crease in size of identified neurons (Coggeshall, 1967).
But if, as has been argued, giant neurons are an adaptation
for increased area of innervation, then during evolution
these snails have made a trade of neuron size for neuron
number in the innervation of an enlarging periphery. This
trade has apparently not been made by the other larger
gastropods belonging to the prosobranchs.

Large body size in gastropods is associated with a mo-
tile, foraging lifestyle, as opposed to the sedentary life of
a parasite or filter feeder. Motile foragers are generally
expected to exhibit a certain complexity in their behavior,
complexity that would emerge from corresponding com-
plexity in the nervous system. However. I suggest that the
behavior of the opisthobranchs and pulmonates, relative
to that of the larger advanced prosobranchs, is both sim-
pler and underlain by a simpler nervous system.

CNS development is directly associated with sensory
and behavioral ability. The behavior of opisthobranchs
and pulmonates, like their nervous systems, probably lacks
the complexity shown by the larger prosobranch snails;
the number of behavioral sub-routines they use in daily
living is obviously smaller than those of animals living in
more complex ecological niches. Larger, more complex
brains, with large numbers of small neurons, are associated
with the development of sense organs for high-resolution
analysis of the environment and greater complexity of
behavior. In the predatory prosobranch whelks, the many
tiny neurons, relatively large ganglia, and eyes are likely
to mediate similarly complex behaviors. The whelk Fu-
sitriton oregonensis devotes considerable behavioral
strategy to reproduction. Mating pairs form seasonally
and persist for as long as 4 months. Subsequently, a parent
attaches its clutch of eggs to a rock surface and patrols
them against predators (Eaton, 1972). Potential predators
may be sensed in part by the whelk's well-developed eyes;
the whelk, with twisting movements of its shell, attempts
to attack and dislodge the preditor; failing that, the whelk

238

R. GILLETTE

may directionally squirt an aversive acid secretion. The
opisthobranchs and pulmonates, with their rudimentary-
at-best vision and small numbers of CNS neurons, come
nowhere near such complexity of behavior. Indeed, the
behavior of the opisthobranchs and pulmonates really
seems simple.

The relative lack of complex sensoria and their atten-
dant complex central processing may allow the opistho-
branch/pulmonate lines to live successfully with a highly
reduced CNS. Their eyes are very small and quite limited
in both the number and resolution of photoreceptors; in
many opisthobranch species, the eyes are even internal-
ized. Their function may be largely limited to setting the
circadian rhythms of animal activity (Jacklet, 1969). High
resolution eyes in the cephalopods are associated with
comparably high resolution, visually directed motor be-
havior (cf. Wells, 1978). High resolution in sensory-motor
systems requires larger numbers of neurons, as are found
in the cephalopod optic lobes. The opisthobranchs get
along mostly with the environmental information pro-
vided by chemosensory and tactile abilities. The opis-
thobranchs and pulmonates do have specialized chemo-
sensory sites for detecting food: the rhinophores. and the
tentacles and other regions about the oral area. These sites
appear to be served by peripheral ganglia that may take
the burden of a great deal of sensory-motor processing
(cf., Mpitsos and Lukowiak, 1986). leaving the central
nervous system to process simple tactile information and
to integrate motivational and learning processes with the
expression of behavior.

Contrasting examples support this interpretation. Some
pulmonates and prosobranchs have developed accessory
CNS ganglionic lobes; these structures are associated with
chemosensation and are composed of many smaller neu-
rons (cf. Bullock, 1965; Chase and Tolloczko, 1989). In
the terrestrial slug Umax, the structure is the procerebral
lobe, and it shows oscillating electrical field potentials
characteristic of rather complex sensory feature extraction
systems in vertebrates (Gelperin and Tank, 1990). Outside
of the gastropods, the obvious example is the complexity
of sensoria and sensory processing in the complex brains
of cephalopods. In the opisthobranchs and pulmonates,
the lack of complexity in sensoria and underlying neural
processing underscores their simplicity of brain and life-
style.

Finally, the opisthobranch and pulmonate nervous
systems show a relatively high degree of centralization
into a few discrete ganglia. Although centralization and
cephalization have not proceeded as far as in the cepha-
lopods, these characteristics still distinguish them from
the mostly sessile bivalves and the parasitic or filter-feeding
gastropods that rely heavily on peripheral control of re-
flexes and show generally less centralization. It also dis-
tinguishes them from large, motile mollusks like the giant

chitons, from which giant neurons are not reported. The
chitons attain large size in some cases, and are slowly
motile, but their nervous system is only poorly centralized
relative to that of the opisthobranchs and pulmonates.
Amphineuran ganglia are simply formed nodes lying on
a major nerve ring, and many neuronal somata are simply
dispersed along the nerves and connectives in a primitive
medullary condition (cf. Bullock, 1965).

Thus, snails with giant neurons constitute a group that
has grown large in body size, but has retained an uncom-
plicated behavioral repertory and sensory-motor capaci-
ties. To serve the innervation needs of the enlarged body,
the nervous system has favored an increase in neuron size
relative to neuron number.

The above characters provide the context within which
I think we must seek the evolutionary reasons that some
snails chose the architecture of neuronal giantism in their
nervous systems. Why didn't they choose instead to in-
nervate their enlarged periphery with many smaller central
neurons, like their large prosobranch cousins? The de-
velopmental simplicity of innervating a large area with
one neuron rather than many may be a useful consider-
ation. Another is that neuronal giantism allows increased
animal size without a proportional increase in central
neuron number.

A potential answer lies in the electrophysiological
properties of neurons of simple and complex gastropod
brains. In molluscan ganglia, the neurons lie peripherally
and send axons into a central neuropil to make synaptic
connections. For the opisthobranch and pulmonate snails
the neuronal somata are excitable and are placed spatially
and electrically quite close to the integrating region (Gor-
man and Mirolli, 1972; Graubard, 1975); they are thus
able to follow almost synchronously the spike activity of
the integrating region (cf. Fig. 1). In larger ganglia, the
distances from the synaptic integrating region in the neu-
ropil, where spikes are initiated, to soma also become
longer. Thus, for a neuron that grows in pace with the
whole ganglion, the larger axon diameter would enhance
the synchrony of soma and integrating region. In this
manner, the continuing enlargement of neuronal somata
with the growth of the organism may not only adapt the
cells to the innervation of an enlarged periphery, but also
to an increased separation of the soma from the integrating
region.

Some data suggest that the very small neuronal somata
of advanced cephalopod ganglia are inexcitable (Gilly and
Brismar, 1989; Williamson and Budelmann, 1991; Rob-
ertson et a/.. 1990), like those of arthropods. Many of
these somata lie rather distant from the neuropil inte-
grating regions, because they are packed on top of many
intervening cells in the cell body layer (cf. Young, 1971).
For a small neuron, this longer distance could cause a
disadvantageous desynchronization of the action potential

GIANT NEURONS IN SNAILS

239

currents between the soma and integrating region, with
the result that the late somatic currents would interfere
with ongoing integration (in the worst case, by reflecting
spikes). Thus, for the nervous systems having a high pro-
portion of such small neurons, it might be functionally
advantageous if the cellular mechanisms of somatic ex-
citability were turned off. We would then wonder, in gen-
eral, whether large brains with many small neurons have
inexcitable somata. Do gastropods, such as the larger
whelks with large central ganglia and many small neuron
somata distant from the integrating neuropil, have spiking
or non-spiking somata? Few intracellular recordings have
been made in the complex nervous systems of the ad-
vanced giant prosobranchs, but if their small neuron so-
mata were also inexcitable, a strong case could be made
that the simplicity and small neuronal numbers of the
opisthobranch/pulmonate central nervous system permits
the retention, in evolution, of excitable somata with an
increase in the size of ganglia.

Conclusion

The opisthobranch and pulmonate gastropods consti-
tute large and successful taxa. In the picture drawn here,
selection during evolution has tightly interwoven neuronal
giantism in the CNS with the physiology and behavior of
the animal. A number of testable hypotheses have been
proposed, and each can be verified or falsified by more
detailed, quantitative observations. The hoped-for result,
a more complete resolution of the adaptive significance
of neuronal giantism, may one day make a useful con-
tribution to our understanding of how the nervous system
has evolved in tandem with behavior.

Acknowledgment

The observations leading to this paper were made while
the author was supported by NSF grant BNS 86-038 16.

Literature Cited

Berry, M. S., and V. W. Pentreath. 1976. Properties of a symmetric

pair of serotonin-containing neurones in the cerebral ganglia ofP/iui-

orbis. J. Exp. Biol. 80: 1 19-135.
Blackshaw, S. E., and D. A. Dorset!. 1976. Behavioural correlates of

activity in the giant cerebral neurons of Archidoris. Proc. R. Soc.

Land. (Biol.) 192: 393-419.
KMI hln 1 1/. R. 1863. Bemerkungen uber den histologischen Bau des

Zentralnervensystems der Susswassermollusken. Arch. Anal. Physio/.

LPZ 1863: 234-264.
Bulloch, A. G. M., and D. A. Dorsett. 1979. The integration of the

patterned output of buccal motorneurones during feeding in Tritonia

homhergi. J. Exp. Biol. 79: 491-508.
Bullock, T. H. 1965. The Mollusca. Pp. 1273-1515 in Structure and

Function in the Nervous Systems of Invertebrates, \. 2., T. H. Bullock

and G. A. Horridge. eds. Freeman Press. San Francisco.
Byrne, J. H., V. Castellucci, and E. R. Kandel. 1974. Receptive fields

and response properties of mechanoreceptor neurons innervating si-

phon skin and mantle shelf in Aplysia J Neurophysiol. 37: 1041-
1064.

Cavalier-Smith, T. 1978. Nuclear volume control by nucleoskeletal
DNA, selection for cell volume and cell growth rate, and the solution
of the DNA C-value paradox. J Cell Sci. 34: 247-279.

Chase, R., and B. Tolloczko. 1987. Evidence for differential DNA en-
doreplication during the development of a molluscan brain. / Neti-
rohiol. 18: 395-406.

Chase, R., and B. Tolloczko. 1989. Interganglionic dendrites constitute
an output pathway from the procerebrum of the snail Achalina fulica.
J Comp Neural. 283: 143-152.

Cobbs, J. S., and H. M. Pinsker. 1979. //; vivo responses of paired
giant mechanoreceptor neurons in Aply.iia abdominal ganglion. /
Nftirohiol. 9: 121-141.

Coggeshall, R. E. 1967. A light and electron microscope study of the
abdominal ganglion of Aplysia califomica. J Neurophysiol. 30: 1263-
12X7.

Coggeshall, R. E., B. A. Yaksta, and F. J. Swartz. 1970. A cytopho-
tometric analysis of the DNA in the nucleus of the giant cell, R-2.
in Aplysia. Chromosoma 32: 205-212.

Dickinson, P. S. 1979. Homologous neurons control movements of
diverse gill types in nudibranch molluscs. / Comp. Physiol. 131:
277-283.

Dickinson, P. S. 1980. Gill control in the notaspidean Pleurobranchaea
and possible homologies with nudibranchs. / Comp. Physiol. 139:
1 1-16.

Dorsett, D. A. 1986. Brains to cells: the neuroanatomy of selected gas-
tropod species. Pp. 101-187 in The Mollusca. K. M. Wilbur, ed., v.
9, Neufobiology ami Behavior, part .. A. O. D. Willows, ed.. Academic
Press. New York.

Dorsetl, D. A., and J. N. Sigger. 1981. Sensory fields and properties
of the oesophageal propnoceptors in the mollusc, Pliiline. J Exp.
Biol 94: 77-93.

Eaton, C. M. 1972. The reproductive and feeding biology of the pro-
sobranch gastropod Fusitriton oregonensis (Redfield) (Fam. Cyma-
tiidae). University of Washington M.S. thesis. 40 pp.

Elliot, C. J. H., and P. R. Benjamin. 1985a. Interactions of pattern-
generating interneurons controlling feeding in Lymnaea stagnalis.
J Neurophysiol. 54: 1396-141 I.

Elliot, C. J. H., and P. R. Benjamin. 1985b. Interactions of the slow
oscillator interneuron with feeding pattern-generating interneurons
in Lymnaea stagnalis. J Neurophysiol. 54: 1412-1421.

Frazier, W. T., E. R. Kandel, I. Kupfermann, R. VVaziri, and R. E. Cog-
geshall. 1967. Morphological and functional properties of identified
neurons in the abdominal ganglion of Aplysia califomica. J. Neu-
rophysiol. 30: 1288-1351.

Gelperin, A., and D. \V. Tank. 1990. Odour-modulated collective net-
work oscillations of olfactory interneurons in a terrestrial mollusc.
Nature 345: 437-440.

Getting, P. A. 1977. Afferent neurons mediating escape swimming of
the marine mollusc Tritonia. J. Comp. Physiol. 110: 271-286.

Gillette, R., and W. J. Davis. 1977. The role of the metacerebral giant
neurone in the feeding behaviour of Pleurobranchaea. J Comp.
Physiol 116: 129-159.

Gillette, R., M. U. Gillette, and W. J. Davis. 1980. Action potential
broadening and endogenously sustained bursting are substrates of
command ability in a feeding neuron of Pleurobranchaea. J. Neu-
rophysiol. 43: 669-685.

Gilly, W. F., and T. Brismar. 1989. Properties of appropriately and
inappropriately expressed sodium channels in squid giant axon and
its somata. / Neurosci. 9: 1362-1374.

Gorman, A. L. F., and M. Mirolli. 1972. The passive electrical properties
of the membrane of a molluscan neurone. J. Physiol. (Loud.) 227:
35-49.

240

R. GILLETTE

Granzow, B., and S. B. Kater. 1977. Identified higher-order neurones

controlling the feeding motor program ofHelisoma. Neuroscience 2:

1049-1063.
Graubard, K. 1975. Voltage attenuation within Ap/ysia neurons: the

effect of branching pattern. Brain Rex 88: 325-332.
Hughes, G. M., and L. Tauc. 1963. An electrophysiological study of

the anatomical relations of two giant nerve cells in Ap/ysia depilans.

J. Exp. Biol. 40: 469-486.
Jacklet, J. \V. 1969. Circadian rhythm of optic nerve impulses recorded

in darkness from isolated eye of Aplysia. Science 164: 562-563.
Lasek, R. J., and W. J. Dower. 1971. Aplysia californica: analysis of

nuclear DNA in individual nuclei of giant neurons. Science 172:

278-280.
Lombardo, F., O. Sonelti, and K. Baraldi. 1980. Differential staining

and fluorescence of chromatin in populations of neuronal nuclei from

Planorbis. Nucleus 23: 30-36.
Masinovsky, B., and P. V.. Lloyd. 1985. Morphology of two pairs of

identified peptidergic neurons in the buccal ganglia of the mollusc

Tnloma diomedea. J. Neurobiol 16: 27-39.
Mellon, DeF., Jr., J. A. Wilson, and C. E. Phillips. 1981 . Modification

of motor neuron size and position in the central nervous system ot

adult snapping shrimps. Brain Res 223: 134-140.
Mittenlhal, J. E., and J. J. Wine. 1978. Segmental homology and

variation in flexor motoneurons of the crayfish abdomen. / Comp.

Neural. Ill: 311-334.
Mirsky, A. E., and S. Osawa. 1961. The mterphase nucleus. Pp. 677

770 in The Cell. 3. Brachet and A. E. Mirsky. eds. Academic Press.

New York.
Mpitsos, G. J., and K. Lukowiak. 1986. Learning in gastropod molluscs.

Pp. 95-267 in The Mollusca. K. M. Wilbur, ed., v. 8, Neurobiology

and Behavior, part 1. A. O. D. Willows, ed.. Academic Press, New

York.

Rayport, S. G., R. T. Ambron, and J. Babiarz. 1983. Identified cholin-
ergic neurons R2 and LP1, control mucus release in Aplysia J Neu-
mpliysiol 49: 864-876.

Robertson, J. D., R. Gillette, P. Lee, S. Meadows, and J. Zitz. 1990.

CNS neuronal somata of octopus are mexcitable and label retrogradely
with carbocyanine dyes. Sot: Neurosd. Abs. 16: 249.7.

Senseman, D., and A. Gelperin. 1973. Comparative aspects of the
morphology and physiology of a single identifiable neurone in Helix
aspersa, Limax maxunus. and Ariolimax californica. Malacol. Rev
1: 51-52.

Siegler, M. V. S. 1977. Motor neurone coordination and sensory
modulation in the feeding system of the mollusc Pleurobranchaea
californica J Exp. Biol. 71: 27-48.

Spray, D. C., M. E. Spira, and M. V. L. Bennett. 1980a. Peripheral
fields and branching patterns of buccal mechanosensory neurons n
the opisthobranch mollusc Naranax inermis. Brain Res. 182: 253-
270.

Spray, D. C, M. E. Spira, and M. V. L. Bennett. 1980b. Synaptic
connections of buccal mechanosensory neurons in the opisthobranch
mollusc .\avanax inermis. Brain Res. 182: 271-286.

Spira, M. E., and M. V. L. Bennett. 1972. Synaptic control of electrotonic
coupling between neurons. Brain Res. 37: 294-300.

Weiss, K. R., and I. Kupfermann. 1976. Homology of the giant seroton-
ergic neurones (metacerebral cells) in Aplysia and pulmonate mol-
luscs. Brain Res 117: 33-49.

Wells, M. J. 1978. Octopus: Physiology and Behaviour of an Advanced
Invertebrate. Chapman and Hall, London.

Williamson, R., and B. U. Budelmann. 1991. Convergent inputs to
octopus oculomotor neurones demonstrated in a brain slice prepa-
ration. Neurosci. Lett. 121: 215-218.

Young, J. Z. 1971. The Anatomy of the Nervous System (/Octopus
vulgaris. Oxford University Press, London.

Reference: Butt Bull 180: 241-251. (April. 1991)

A Functional, Cellular, and Evolutionary Model of
Nociceptive Plasticity in Aplysia

EDGAR T. WALTERS

Department of Physiology and Cell Biology, University of Texas
Medical School at Houston. Houston. Texas 7722?

Abstract. Nociceptive plasticity is defined as behavioral
and cellular modification produced by activation of no-
ciceptors. A brief survey of nociceptive plasticity in Aplysia
reveals a puzzling mixture of behavioral modifications of
opposite sign and widely varying durations. These include
general sensitization. site-specific sensitization. response-
specific facilitation, and inhibition of defensive responses.
This behavioral complexity is more than matched by the
complexity of cellular correlates reported for the behav-
ioral modifications. A functional model is proposed link-
ing complex patterns of behavioral and neural plasticity
in Aplysia to potentially general principles of nociceptive
function. This model is centered around three overlapping
but functionally distinct phases: injury detection, escape,
and recuperation. A hypothesis about the early origin of
nociceptive plasticity in primitive mechanosensory neu-
rons is then developed, based on similarities in the or-
ganization and modinability of nociceptive systems in
evolutionarily divergent groups (primarily mollusks and
mammals) and on inferences about the early adaptiveness
of postinjury behavioral plasticity. Preliminary evidence
suggests that aspects of nociceptive plasticity, and perhaps
other forms of memory, may have been derived from cel-
lular repair and signal compensation mechanisms.

Introduction

Neuronal mechanisms controlling withdrawal re-
sponses in the opisthobranch mollusc, Aplysia californica,
have been studied extensively by neurobiologists for over
two decades. Investigators have been attracted to the gill.

siphon, and tail withdrawal responses of Aplysia, in part
because the CNS can be readily analyzed in this animal,
but largely because these responses and their underlying
neurophysiology display a remarkable degree of modin-
ability. The rare opportunity, provided by Aplysia and
several other gastropod mollusks, to link behavioral and
cellular alterations has been used to advantage by several
laboratories and has led to the discovery of various mech-
anisms contributing to learning and memory in this spe-
cies.

Although we presume that some mechanisms from
gastropod "model systems" are general, the possibility that
a given mechanism will be common to groups as evolu-
tionarily divergent as are the mollusks and mammals re-
quires serious scrutiny. As a first step in examining the
potential generality of mechanisms contributing to learn-
ing and memory in Aplysia, I will discuss some nociceptive
functions of these mechanisms, propose a three-phase
model of nociceptive plasticity, and consider the possi-
bility that mechanisms of nociceptive plasticity evolved
in primitive mechanosensory neurons and have been
conserved in diverse phyla. Central to this discussion is
that most cellular mechanisms of behavioral modification
revealed to date in Aplysia have been produced either by
noxious stimulation, or by manipulations that mimic ef-
fects of noxious stimulation. The behavioral and cellular
modifications are thus examples of nociceptive plasticity,
which I define as modifications induced by the activation
of nociceptors. Nociceptors are defined as sensory neurons
that are activated maximally by stimuli that, if sufficiently
prolonged, cause tissue damage (Sherrington, 1906).

Received 14 August 1990; accepted 22 January 1991.

Abbreviations: Activity-dependent extrinsic modulation, ADEM;
central nervous system, CNS; Excitatory postsynaptic potential. EPSP;
Phe-Met-Arg-Phe-NH;, FMRFamide; serotonin, 5-HT.

Forms of Nociceptive Plasticity

Noxious stimuli, such as strong pinch or shock, were
at first assumed to have only two major effects on Aplysia:

241

242

E. T. WALTERS

to trigger vigorous defensive responses, and to cause gen-
eral sensitization of the animal for several minutes. The
term "sensitization" has been used independently by psy-
chologists and physiologists to describe an increase in
sensitivity or magnitude of. respectively, a behavioral or
physiological response. I define nociceptive sensitization
as sensitization produced by noxious stimulation, where
sensitization is defined physiologically as an increase in
sensitivity or responsiveness of the organism to a constant
test stimulus. Such hypersensitivity after noxious stimu-
lation need not be expressed as overt behavior, but is often
expressed as a decrease in threshold and an increase in
the magnitude of defensive responses evoked by a test
stimulus. By this definition, sensitization may also be ex-
pressed as inhibition of ongoing behavior (usually non-
defensive) by the test stimulus. Nociceptive sensitization
can be general (expressed by changes in response to a
broad range of test stimuli and stimulation sites) or, as
discussed below, specific to a warning signal or to a re-
stricted site on the body. The apparent function of general
nociceptive sensitization is to prime the animal for con-
tinued defense, so that it responds rapidly and energeti-
cally to a wide range of stimuli that might presage an
attack.

The first clue that nociceptive plasticity involves more
than a brief, general sensitization came from the obser-
vation that repeated application of noxious stimuli over
hours or days causes general sensitization of siphon with-
drawal that can last for weeks (Pinsker et al.. 1973; Frost
el al., 1985). It was then discovered that sensitization can
be conditioned to a warning signal; a variety of defensive
responses are selectively facilitated by a chemosensory cue
(e.g., shrimp extract) if it is repeatedly paired with noxious
shock (Walters et al., 1981; Colwill et al.. 1988). Further
links between sensitization and associative processes were
indicated by behavioral data (Carew el al., 1981, 1983;
Hawkins et al., 1983) and neuronal data (see next section),
suggesting considerable overlap of sensitization and pu-
tative classical conditioning mechanisms within individual
sensory neurons.

The next discovery, site-specific sensitization, is crucial
to the functional and evolutionary arguments of this pa-
per. Noxious stimulation enhances siphon and tail with-
drawal test responses; but responses evoked by test stimuli
applied near the site of noxious stimulation are more dra-
matic than those evoked by test stimuli applied at other
sites on the body (Walters, 1987a). The site-specific be-
havioral plasticity is particularly potent; a single 45 s nox-
ious stimulation sequence that is insufficient to cause long-
term general sensitization produces site-specific sensiti-
zation lasting a week or more (Fig. 1 ).

The complexity of nociceptive plasticity was under-
scored recently when several groups found that noxious
stimulation can inhibit, as well as enhance, defensive re-

Siphon

Mantle Cavity

Rhinophores

Tail

Foot

Tentacles

80
60
40
20

Training

/ \ 1

Pre 2 hr

Days

Figure 1. Site-specific sensitization following noxious tail stimulation
in freely moving Aplyxia. (A) Diagram of unrestrained animal used for
testing and training. Before and after site-specific sensitization training,
weak test stimuli were applied with a hand-held electrode to a site on
each side of the tail that had been marked with a suture (not shown).
Training consisted of a 45 s sequence of strong shocks to one of the test
sites. During each test, the duration of siphon withdrawal was timed,
and the magnitude of tail withdrawal was estimated. (B) Site-specific
sensitization of siphon withdrawal. Siphon withdrawal was significantly
greater when tested at the trained site than the contralateral control site.
Similar differences were seen in tail withdrawal (not shown) and when
other parts of the body were trained and tested (Walters,

sponses (Krontiris-Litowitz et a!.. 1987; Mackey et al..
1987; Marcus et al., 1988). The most complete behavioral
study of nociceptive inhibition was reported by Marcus
et al. (1988), who showed that inhibition of siphon with-
drawal occurs following noxious but not innocuous stim-
uli, and that net inhibition has a brief duration.

These various forms of nociceptive plasticity differ in
the sign, duration, and stimulus specificity of behavioral
modulation. Yet another dimension of plasticity was re-
vealed by the discovery that particular siphon responses
are modulated selectively by noxious stimulation of dif-

NOCICEPTIVE PLASTICITY IN APLYSIA

243

Intact Animal

Reduced Preparation

Relaxed

siphon

mantle

shell ink gland

parapodium

gill

Flaring, Tail-type Response

posterior stimulation

Constricting, Head-type Response

anterior stimulation

Response Transformation

Anterior US

Posterior US 10 sec

Figure 2. Transformation of siphon responses following noxious
stimulation. The left column shows a cutaway view of the siphon and
mantle organs in the intact animal (compare Fig. 1 A). The right column
shows the mantle organs and CNS in a reduced preparation. The photocell
monitors the breadth but not the length of the siphon. (A) Relaxed siphon.
Weak test stimuli were applied to a midbody nerve at 1 min intervals.
A noxious unconditioned stimulus (US), a 1 5 s sequence of strong shock,
was delivered to either a tentacle nerve or a tail nerve. (B) Flanng response,
typical of posterior stimulation. The photocell shows a negative deflection.
(C) Constricting response typical of anterior stimulation. The photocell
shows a positive deflection. (D) Examples of transformed responses.
Top— flaring responses are converted to constricting responses after
noxious anterior stimulation. Bottom— constricting responses are con-
verted to flaring responses after noxious posterior stimulation (Erickson
and Walters, 1988).

ferent regions of the body. This response-specific noci-
ceptive plasticity is expressed most clearly when noxious
stimulation causes the animal to respond to a test stimulus
with a qualitatively different response than it did before
noxious stimulation (Erickson and Walters, 1988). Figure
2 shows examples of siphon responses being transformed
into opposite responses following intense stimulation of
nerves from the head or tail. Like sensitization, response
transformation can be enhanced by associative training.
The incidence and degree of transformation of motor re-
sponses to particular test stimuli are preferentially in-
creased if the test stimulus is repeatedly paired with a
noxious stimulus (Walters, 1989; Hawkins el til.. 1989).

Mechanisms of Nociceptive Plasticity

Mechanisms of general sensitization in Aplysia have
received detailed analysis. Here I briefly describe selected
aspects of cellular mechanisms, focusing on those that
have been closely linked to changes in defensive behavior.
For reviews of subcellular mechanisms of sensitization.
see Kandel and Schwartz (1982) and Byrne (1987).

In principle, sensitization might involve alterations in
any of various classes of neurons known to contribute to
defensive behavior in Aplysia (Fig. 3). Although some in-
terneurons and motor neurons show alterations during
general sensitization (e.g.. Frost el til.. 1988), analysis has
centered on mechanosensory neurons: the LE cluster,
which innervates the siphon (Byrne et a/.. 1974); and the
VC clusters, which innervate most of the rest of the body
(Walters et al.. 1 983a). No major differences between these
sensory clusters have been described in their response
properties or plasticity. Cells in both clusters show a wide
dynamic range, responding weakly to stimuli of moderate
intensity and more strongly as stimulus intensity is in-

General Neural Organization
Underlying Nociceptive Behavior in Aplysia

Figure 3. General pattern of neural organization controlling noci-
ceptive behavior in Aplysiu. Each indicated population of cells may in-
clude hundreds of neurons distributed throughout the nervous system.
Wide-dynamic range nociceptive sensory neurons (S) innervate the entire
body surface. Each cell connects to peripheral motor neurons (P), to
central motor neurons (M) innervating the same region, and to inhibitory,
excitatory, and facilitatory interneurons. Sensory neurons also make
connections (largely polysynaptic) to complex pattern generating networks
responsible for rhythmic defensive behaviors such as mantle pumping
(used to eject ink and to increase respiration and blood circulation) and
escape locomotion. Relatively little is known about interconnections
among the various types of interneurons. A further complication is that
some interneurons are multifunctional U'.#., having both excitatory and
facilitatory effects on the same follower neuron). Based on data from
Bailey et al. (1979), Byrne (1980, 1983), Hawkins et al. (1981), Frost et
al. (1988), and Hickie and Walters (unpub. obs.).

244

E. T. WALTERS

creased (Byrne el a/.. 1978; Walters el al.. 1983a). Both
clusters have nociceptive functions, because they respond
maximally to noxious pinching stimuli (Walters el at..
1983a: Walters and Clatworthy, unpub. obs.). and they
are therefore indicated, in Figure 3, within the circle la-
beled "nociceptors".

Sensory neurons in the LE cluster (Bailey el al.. 1979),
and probably in other central nociceptive clusters (e.g.,
Walters, 1987b), make some synaptic connections to pe-
ripheral motor neurons (Fig. 3). But with few exceptions
(see Clark and Kandel, 1984), analysis of synaptic plas-
ticity in these sensory populations has focused on their
strong monosynaptic connections to identified motor
neurons within the CNS. Because of these connections
and others to excitatory, facilitatory. and inhibitory in-
terneurons involved in defensive responses (Fig. 3),
changes in the signalling properties of LE and VC sensory
neurons should have potent effects on behavioral re-
sponses elicited by moderate to strong cutaneous stimuli.
Short-term behavioral sensitization is correlated with
general facilitation of synapses from sensory neurons to
motor and interneurons (Kandel and Schwartz, 1982;
Walters el al.. 1983b) and with increased excitability of
peripheral branches of the sensory neuron (Clatworthy
and Walters, 1990). The presynaptic facilitation is me-
diated, at least in part, by 5-HT (Glanzman et al.. 1989),
which can also enhance excitability of the central and
peripheral parts of the sensory neuron (Walters et al.,
1983b; Klein et al.. 1986; Billy and Walters, 1989b). Many
of the effects of 5-HT are mediated by cyclic AMP-de-
pendent protein kinase (Kandel and Schwartz, 1982), and
some are mediated by protein kinase C (Braha el a/..
1990). The most notable effects involve the depression of
K+ conductances (Klein et ul., 1982; Baxter and Byrne,
1989; Walsh and Byrne, 1989). which increase transmitter
release and excitability by broadening spikes and decreas-
ing spike accomodation (Kandel and Schwartz, 1982;
Walters et al., 1983b; Klein et al.. 1986). Noxious stim-
ulation also appears to enhance a Ca2+ conductance (Ed-
monds et al.. 1990).

The expression of long-term sensitization in sensory
neurons involves some of the same mechanisms as short-
term sensitization: depressed K ' conductances, increased
transmitter release, and increased excitability (Frost et al.,
1985; Scholz and Byrne, 1987; Walters, 1987b). Specific
morphological changes also occur in the sensory neuron,
including the growth of new synaptic varicosities and ac-
tive zones within the CNS (Bailey and Chen, 1983, 1988;
Nazif et al.. 1989), and possibly the growth of peripheral
processes that expand the size of the receptive field (Billy
and Walters, 1989a). Considerable effort is being made
to identify molecular mechanisms involved in inducing
and maintaining long-term changes in these sensory neu-
rons (e.g., Barzilai et al., 1989: Eskin el al., 1989).

Associative enhancement of withdrawal responses to
mechanosensory cues and site-specific sensitization ap-
pears to involve the same basic mechanism: activity-de-
pendent enhancement of the mechanisms of general sen-
sitization. as described above (Walters and Byrne, 1983a;
Hawkins et al.. 1983: Walters. 1987b). Figure 4 illustrates
two of the sensory neuron alterations contributing to long-
term site-specific sensitization: synaptic facilitation and
increased soma excitability; the latter is expressed dra-
matically as a prolonged afterdischarge to brief depolar-
ization. During the induction of site-specific sensitization.
the sensory neuron is activated by the noxious stimulus.
In associative conditioning, the sensory neuron is activated
by a cue presented immediately before the noxious stim-
ulus. In both cases, the activity enhances the effects of
extrinsic chemical modulators (e.g., 5-HT) on the sensory
neuron, and thus this general class of plasticity is termed
activity-dependent extrinsic modulation (ADEM). Acti-
vation of sensory neurons opens Ca:+ channels (Walters
and Byrne, 1983b; Edmonds et al., 1990). The resulting
Ca+ influx enhances adenylate cyclase activity, increasing
the rate of cyclic AMP synthesis, and thus amplifying the
degree and duration of plasticity induced by neuromodu-
lators released during noxious stimulation (Abrams and
Kandel. 1988). Ca:+ might also enhance plasticity in other
ways; for example, Ca24 -dependent kinases may directly
phosphorylate transcription factors (cf. Dash et a/.. 1990).

The cellular mechanisms of nociceptive inhibition have
also been studied. Mackey et al. (1987) reported that tail
shock causes presynaptic inhibition and spike narrowing
in siphon sensory neurons, and that these effects are partly
mediated by an identified interneuron containing the
neuropeptide FMRFamide. Application of FMRFamide
to the sensory cell soma and synaptic region in the CNS
causes hyperpolarization, spike narrowing, and presyn-

A Control side B Trained side

Sensory
Neuron

5mV
30mV

Figure 4. Example of synaptic facilitation and afterdischarge in a
sensory neuron after site-specific sensitization. The intact animal was
trained with strong noxious shock delivered to one side of the body. One
day later, the CNS was removed and sensory and motor neurons inner-
vating the trained side and corresponding regions on the contralateral
side were examined. (A) Typical connection between a tail sensory neuron
and tail motor neuron innervating the untrained side of the tail. The
connection was tested by activating the sensory neuron with a 10 ms
depolarizing pulse injected into the soma. (B) The same test procedure
on the trained side elicits a larger synaptic potential and an afterdischarge
of 20 spikes (Walters, 1987b).

NOCICEPTIVE PLASTICITY IN APLYSIA

245

Table I

I'lircc-i>lhi\c model of the functions and general meehani\ni\ «l nociceptive pluMiaiy in Aplysia

Phase 1 . Injury detection

Phase 2. Escape

Phase 3. Recuperation

Period:

Functions:

0.1 s-10 mm
• Severity appraisal

1 s-30 min
• Flight

10 min-1 month
• General inactivity

Mechanisms:

> Localization

• Compensation for destruction

of nociceptive channels

• Defensive response triggering

> Anticipation

> Nociceptor activation

a. Frequency code

b. Wide dynamic range

> Activation of defensive circuits

(Somatotopic organization)

> Nociceptor facilitation
a. Afterdischarge

h. PTP

c. HSF

d. Hyperexcitability

e. ADEM

• Motor facilitation

1 Inhibition of competing
responses

• Activity in circuits generating

escape behavior

• Nociceptor inhibition

a. Presynaptic inhibition

(neuromodulation)

b. Activity-dependent reduction

in excitability
> Inhibition of motor and
interneurons controlling
other behaviors

(healing)

• Defensive readiness

(sensitization)

a. General

b. Wound specific

c. Cue specific

• Inhibition of circuits

controlling feeding,
reproduction, etc.

• Nociceptor facilitation

a. ADEM

b. Axon injury signals

c. Lower threshold

d. Less accomodation

e. Afterdischarge

f. Synaptic facilitation

g. Sprouting

• Motor facilitation

The times for each phase indicate the approximate beginning and end of the phase relative to the beginning of the noxious stimulus. ADEM —
activity-dependent extrinsic modulation; HSF — heterosynaptic facilitation; PTP — posttetanic potentiation.

aptic inhibition in sensory neurons (Belardetti ct til.. 1987),
whereas peripheral application increases mechanosensory
threshold (Billy and Walters, 1989b). Extrapolation from
studies in other mollusks suggests that other neuromodu-
lators may also contribute to nociceptive inhibition of
defensive responses in Aplysia. For example, pharmaco-
logical evidence suggests that inhibition of a defensive
response in another gastropod, the snail Cepaea, may in-
volve opiate-like modulators (Kavaliers, 1987). In Aplysia.
noxious stimulation also produces activity-dependent re-
duction in the excitability of sensory neuron axons and
receptive fields (Clatworthy and Walters, 1990), and can
sometimes block afferent spikes (Clatworthy and Wal-
ters, 1989). The presynaptic inhibition produced by
FMRFamide is also activity-dependent (Small el at..
1989). Recently, Wright el al (1989) suggested that in-
terneurons may be more important loci than sensory
neurons for inhibition in the siphon withdrawal system.
They found that tail shock suppressed polysynaptic (in-
terneuronal) components of a complex test EPSP in si-
phon motor neurons under conditions in which no in-
hibition of the monosynaptic EPSP from the sensory neu-
rons was detected.

Mechanisms of response-specific nociceptive plasticity
are not yet known. However, tests of several potential
mechanisms have recently begun in identified neurons
within siphon control circuits (Erickson and Walters,

1988; Frost et al. 1988: Hickie and Walters, 1990; Fang
and Clark, 1990).

A Functional Model of Nociceptive Plasticity

These findings show that noxious stimulation causes
highly complex behavioral and neuronal alterations in
Aplvsia. Two issues have not been clear: the functional
significance of this complexity, and the integration of ap-
parently opposing forms of plasticity to produce adaptive
behavior. General similarities between the patterns of no-
ciceptive behavior observed in Aplysia and in other species
(primarily rats and humans) suggest that forms of noci-
ceptive plasticity in Aplysia might represent common be-
havioral adaptations to ubiquitous selection pressures;
namely, escape from a source of bodily injury, and op-
timization of recuperation. This possibility encouraged
the formulation of a model linking potentially general
principles of nociceptive function to patterns of behavioral
and neural plasticity that have been described in Aplysia.
In a functional model of pain and fear in mammals, Bolles
and Fanselow (1980) divided nociceptive responses into
perceptual, defensive, and recuperative phases. Somewhat
similar phases can be used in a functional model to explain
much of the complexity of behavioral and neuronal plas-
ticity observed in Aplysia following noxious stimulation
(Table I). These overlapping phases of injury detection.

246

E. T. WALTERS

escape, and recuperation correspond to periods of im-
mediate facilitation, short-term inhibition, and long-term
facilitation of defensive responses.

Phase 1 — injury detection

How does an animal know it is injured, or about to be
injured? False negative answers mean an animal will fail
to initiate escape and recuperative behavior, jeopardizing
its life. False positive answers also reduce biological fitness
by committing the animal unnecessarily to energy-con-
suming escape behavior, and possibly to a long period of
recuperative behavior during which important activities
such as reproduction and feeding are inhibited. One way
for a CNS to decide whether an injury has occurred is to
interpret any activity on nociceptive labeled lines from
the body as proof of injury. However, because an animal
needs to match its responses to the severity and location
of its injuries, nociceptive signals should also carry inten-
sity and spatial information. In addition, if the severity
of an injury is represented by the number of active no-
ciceptive fibers and by their degree of activity, there should
be some way of compensating for the loss of signal strength
during and after injury severe enough to destroy or damage
nociceptive fibers from the injured region. Finally, a no-
ciceptive system would be highly adaptive if it could rec-
ognize noxious stimuli prior to actual injury.

These general functional considerations are reflected
in the organization of the nociceptive system of Aplysia
and in the alterations of this system that immediately fol-
low moderate intensity or noxious cutaneous stimulation
(Table I). LE and VC sensory neurons trigger defensive
withdrawal responses, and the magnitude of the responses
thus evoked depends upon the number of LE or VC neu-
rons activated, upon the number and frequency of spikes
generated, and upon the amount of transmitter released
per spike ( By rne el al. , 1978; Walters eta I.. 1 9 8 3a; Walters,
unpub. obs.). Therefore, the likelihood and severity of
body wall injury in Aplysia appear to be coded, at least
in part, by the total level of activity in these nociceptive
channels and by the strength of nociceptive connections
to interneurons and motor neurons. As in mammals, the
relatively small size of nociceptive receptive fields and the
somatotopic organization of sensory and motor pathways
in Aplysia (Walters el al., 1983a) contribute to the local-
ization of noxious stimuli.

How does this system compensate for the destruction
of nociceptive axons during severe injury? The VC and
LE sensory neurons provide labeled lines to the CNS from
the periphery, but their wide dynamic range and response
properties also make possible a frequency code for the
severity of injury. A brief, punctate, moderately intense
stimulus to the tail, which does not cause injury unless
greatly prolonged, typically evokes one to five spikes in

each of three to five VC neurons, the receptive fields of
which are estimated to overlap any given point on the tail
(Walters el al., 1983a; Billy and Walters, 1989a). The total
activity in these sensory neurons ( 10-20 spikes over about
0.5 s) leads to a relatively brief withdrawal of the tail and
siphon, and perhaps to escape locomotion. A strong,
punctate, pinching stimulus of the same duration, which
may cause some cutaneous damage but does not destroy
major axons of VC sensory neurons, causes high frequency
activation of each sensory neuron, but the activation rarely
outlasts a moderately noxious stimulus. Thus, a 0.5 s
stimulus might lead to a 0.5 s barrage of perhaps 75 spikes
across the same 5 VC neurons, leading to strong, long-
lasting withdrawal of the tail and siphon, as well as to
inking and vigorous escape locomotion. Finally, a severe,
crushing stimulus of the same duration will probably de-
stroy some of the sensory axons innervating the region.
Assuming, for illustrative purposes, that axons from three
of the five sensory neurons are destroyed, how is the CNS
informed of the severity of the injury? First, the remaining
fibers will fire at maximal frequency (about 50 Hz) during
the stimulus. Second, the crushed axons will produce an
injury discharge of high frequency spikes. Third, very
strong stimuli produce an afterdischarge in VC sensory
neurons (see Fig. 4) that can last 0.1 to 3 s (Clatworthy
and Walters, 1988, and unpub. obs.). The afterdischarge
is generated, at least in part, within the CNS (Clatworthy
and Walters, 1988), raising the possibility that both the
intact VC neurons, and the VC neurons with injury-de-
stroyed axons, fire at high frequency during the noxious
stimulus and for 1 to 2 s afterwards. Thus, even with a
majority of the sensory fibers from the injured region dis-
connected from the CNS by the injury, a barrage of 100-
200 high frequency sensory spikes may reach central syn-
aptic terminals onto defensive motor and interneurons.
The mechanism of afterdischarge is not yet known, but
it appears to depend upon both the initial spike activity
and the extracellular release of chemical modulators, i.e.,
ADEM (Clatworthy and Walters, unpub. obs.). Finally,
under natural conditions, more severe stimuli usually af-
fect larger areas of body wall and thus activate more no-
ciceptors.

How does the system anticipate injury during moderate
intensity cutaneous stimulation that threatens but does
not immediately produce tissue damage? Stimuli suffi-
ciently intense to activate LE and VC neurons, but not
severe enough to cause immediate body wall injury, have
transient facilitatory effects upon defensive responses. This
facilitation involves brief (seconds to minutes) heterosy-
naptic facilitation (Carew el al., 1971; Walters el al.,
1983b), post-tetanic potentiation (Walters and Byrne,
1984; Clark and Kandel, 1984), and enhanced peripheral
excitability (Clatworthy and Walters, 1990) in the sensory
neurons. The facilitation of peripheral excitability, as well

NOCICEPTIVE PLASTICITY IN A/'LYS/A

247

as facilitation of synaptic transmission (Hawkins el ai,
1983; Walters and Byrne, 1983a), is greatest in sensory
neurons activated by the noxious stimulus. As a conse-
quence, continued or repeated application of a moderately
noxious stimulus to the same region should cause in-
creasing activation of the nociceptors, increasing synaptic
facilitation (Walters ct a/.. 1983b), and increasingly effec-
tive sensory input to the CNS. Temporal summation of
excitatory and facilitatory inputs to defensive interneurons
and motor neurons occurs (Carew and Kandel, 1977;
Walters, unpub. obs.), facilitating motor responsiveness.
These effects also increase the spontaneous firing rates of
some motor neurons, which can lead to neuromuscular
facilitation (e.g.. Frost ct a/.. 1988). All of these sensory
and motor facilitation mechanisms produce "windup" of
neural responses to repeated or prolonged stimulation that
is intense enough to be threatening. Windup has two con-
sequences: withdrawal and escape responses are triggered
before a prolonged, moderately noxious stimulus injures
the animal; and the animal is prepared to respond max-
imally if more severe stimulation follows. Windup of re-
sponses to noxious stimuli in mammals appears to involve
some of the same mechanisms (Woolf and Walters, 1991).
Brief habituating and inhibitory effects, reported for rel-
atively weak cutaneous stimuli in Aplysia (e.g.. Kupfer-
mann et ai. 1970; Mackey ct ai. 1987). should oppose
and delay these facilitatory effects, reducing the chances
of overreaction to innocuous stimuli.

Phase 2 — escape

When the CNS interprets a stimulus as injurious or
potentially injurious, escape behavior is initiated, and the
second phase of nociceptive plasticity begins. It has long
been observed that animals in the act of fleeing or fighting
ignore their injuries. Nociceptive responses are inhibited,
and this inhibition prevents less urgent behavior patterns
from interfering with emergency responses critical for es-
caping from, or repelling, mortal threats (Wall, 1979;
Bolles and Fanselow, 1980). In Aplysia, strong shock or
pinching stimuli inhibit withdrawal reflexes and associated
neural activity in an intensity-dependent manner, and the
inhibitory effects generally last for 1 to 15 min (Marcus
et ai. 1988; Walters, Erickson, and Clatworthy, unpub.
obs.). This time course and intensity dependence roughly
parallel those of escape locomotion (Walters and Erickson,
1986). Because massive withdrawal of any region of the
body interferes with escape locomotion, a major function
of nociceptive inhibition in this animal is probably to
prevent the disruption of escape behavior that would occur
if strong withdrawal responses were triggered during flight.
As described above, inhibition of defensive responses ap-
pears to involve neuromodulation of sensory neurons, in-
terneurons, and perhaps motor neurons, with some of the
inhibition being activity-dependent (Table I).

Phase 3 — rcciiperal it >n

After several minutes of escape locomotion, Aplysia
stop (in a crevice if available), contract into a tight spher-
ical shape, and remain motionless. If the injury is severe,
an animal may show little sign of activity for up to several
days. If the animal is touched during this time, it will
show exaggerated withdrawal responses and a low thresh-
old for escape locomotion, especially if contact is made
near the wound (Walters, 1987a, and unpub. obs.). In-
activity during wound healing presumably involves in-
hibition of circuits controlling active behaviors, such as
feeding and mating, which are not immediately essential
and which would subject the wound to further stress. In-
hibitory signals may include neuroendocrine substances
and factors released into the blood from ruptured cells at
the site of trauma (Krontiris-Litowitz et ai. 1989).

While little is known about mechanisms underlying
inhibition of nonessential behaviors during the recuper-
ative phase, a great deal has been learned about the en-
hancement of defensive responses during this phase. The
various mechanisms of sensitization reviewed earlier in
this article serve to increase the animal's readiness for
defensive action while the wound heals (Table I). This
sensitization is functionally equivalent to long-term hy-
peralgesia in mammals. Hypersensitivity is especially im-
portant around the region of injury because a wound may
leak substances that can invite further attack from pred-
ators or parasites, and because a wounded region is likely
to be weakened and vulnerable to further disturbance.
Persistent general sensitization in Aplysia is mediated, at
least in part, by long-term heterosynaptic facilitation of
wide-dynamic range nociceptors (Frost et ai. 1985; Wal-
ters, 1987b). Wound-specific sensitization involves at least
three basic mechanisms. First, long-term site-specific sen-
sitization is produced by ADEM of nociceptors activated
during wounding. This selectively decreases peripheral
mechanosensory threshold (Billy and Walters, 1989a),
enhances nociceptor afterdischarge, and produces synaptic
facilitation in sensory neurons innervating the wounded
region (Walters, 1987b). Second, signals generated at a
site of axonal injury may be carried by retrograde axonal
transport to the soma and synapses, where they induce
the same set of hyperexcitability and facilitatory effects
as are triggered by ADEM (Walters, Alizadeh, and Castro,
unpub. obs.). The generation of signals at sites of axonal
injury may involve interactions with extracellular factors
associated with immunocytes aggregating at damaged tis-
sue (Alizadeh et ai, 1990). In each case, persistent sen-
sitization may involve growth of new synapses and
sprouting of new branches from central and peripheral
sensory arbors (Bailey and Chen, 1988; Billy and Walters,
1989a). A third basic mechanism of long-term wound-
specific sensitization has been implicated by behavioral

248

E. T. WALTERS

experiments (Fig. 2), but has not yet been demonstrated
within the nervous system — selective enhancement of the
responsiveness of elements within motor control circuits
controlling specific defensive responses appropriate for
the wounded region (motor facilitation; Erickson and
Walters, 1988).

An interesting feature of nociceptive systems in Aplysia
and in mammals is the prominence of wide-dynamic
range neurons. Results from Aplysia suggest that this fea-
ture may be important for the induction of nociceptive
behavior by innocuous stimuli during nociceptive sensi-
tization (an effect functionally equivalent to allodynia in
humans — pain evoked by innocuous stimuli). In Aplysia
the severity of an injury is partially encoded by the total
output of nociceptors representing the injured region (i.e.,
the number of cells activated X firing rate per cell X trans-
mitter release per spike). Thus, a moderately intense
stimulus that would normally be innocuous will be in-
terpreted by the CNS as noxious if transmitter release or
spike frequency are enhanced in wide-dynamic range no-
ciceptors after an injury. Presumably, innocuous tactile
stimulation near a serious wound is often sufficiently
threatening to evoke nociceptive behavior during recu-
peration in both Aplysia and mammals.

The ADEM mechanism in LE and VC sensory neurons
may contribute, not only to site-specific sensitization
around a wound, but also to classical conditioning of a
cutaneous warning cue distant from a wound, provided
that the warning cue is at least moderately intense and is
delivered shortly before wounding (Walters and Byrne.
1983; Hawkins ct at.. 1983). However, this cue-specific
sensitization mechanism would only be useful if the cue
were subsequently to contact the same receptive field.
Given the small size of these cells' receptive fields (de-
creasing the chances of repeated contact), conditioned
enhancement of their signals would seem to provide an
undependable warning cue (see Walters, 1987b). Cue-
specific sensitization mechanisms should be more effective
in sensory neurons that have global receptive fields and
that can detect a threat at a distance (before contact),
allowing more time for avoidance of a threatening situ-
ation. Chemosensory neurons have these properties, and
chemical stimuli may thus be more effective cues for
aversive conditioning than tactile cues. Aversive condi-
tioning with chemosensory cues occurs readily in Aplysia
(Walters el ai. 1981; Colwill et ai. 1988), but whether
such conditioning is more rapid or potent than condi-
tioning with tactile cues is not yet known (e.g., Carew et
ai. 1981).

A Hypothesis About the Evolution
of Nociceptive Plasticity

Although nociceptive neurons and nociceptive re-
sponses have been examined in a variety of species (e.g..

Nicholls and Baylor. 1968; Kavaliers, 1988), investigations
of behavioral and neuronal plasticity following noxious
stimulation have largely been restricted to mammalian
and molluscan preparations. Nociceptive plasticity in
these two groups shows a number of interesting similarities
(reviewed by Walters. 1987a,b; Kavaliers, 1988; Woolf
and Walters, 1991). In both groups, facilitatory and in-
hibitory alterations occur in the first stages of nociceptive
processing — within wide dynamic range nociceptors in
Aplysia. and in primary nociceptors and secondary wide-
dynamic range spinal interneurons in mammals. Simi-
larities include: intensity-dependent enhancement and
inhibition of central excitability, enhanced peripheral
sensitivity, enlargement of nociceptive receptive fields, and
activity-dependent plasticity. Furthermore, preliminary
evidence suggests that aspects of the underlying subcellular
mechanisms (e.g., depressed K+ conductances, mediation
by common protein kinases, and activation of "imme-
diate-early" genes) might also be shared (see Woolf and
Walters, 1991). In principle, the similarity of any given
feature may be due to either convergent evolution of in-
dependent mechanisms in response to common environ-
mental pressures (analogy), or to conservation of primitive
mechanisms that had evolved in ancestors common to
both mollusks and mammals (homology). The ancestors
of mollusks and mammals diverged very early in the his-
tory of the animal kingdom, before the protostome and
deuterostome lineages split during the Precambrian era.
Thus, homologous features in mollusks and mammals
must be very primitive, having descended from small,
soft-bodied animals that lived more than 600 million years
ago, before the hard shells or skeletons that would leave
a fossil record had evolved (Avers, 1989).

To what extent are mechanisms of nociceptive plasticity
homologous in mollusks and mammals? Undoubtedly
many similarities are due to analogous adaptations de-
veloped independently by these groups in response to a
ubiquitous pressure — the dangers that follow sublethal
injury in a hostile environment. On the other hand, two
arguments suggest that this same pressure existed during
the evolution of primitive common ancestors of mollusks
and mammals, and could have supported the early evo-
lution of adaptations to optimize defensive behavior fol-
lowing noxious stimulation. First, the presence of pred-
ators during early periods of animal evolution is suggested
by the occurrence of withdrawal and escape responses in
virtually all existing animal groups, including protozoans
(Kavaliers, 1988). Unfortunately, almost nothing is known
about predators in the Precambrian world except that
they, like their prey, would have been small and soft-bod-
ied, and probably lacked specialized feeding appendages
(Vermeij, 1987). Nevertheless, Precambrian predators
probably existed (Hickman el ai. 1984) and would not
have needed specialized appendages. For example, prey

NOCICEPTIVE PLASTICITY IN APLYSIA

249

may have been captured with nets of mucus, and eaten
with simple grasping and extracellular digestion methods,
similar to those used by some flatworms today. Second,
injury could have also been produced by random assaults
from the environment, such as wave action. An injured
animal would have been more vulnerable to further phys-
ical disturbance, and to detection and attack by predators.

If early mechanisms of nociceptive plasticity did, in
fact, originate in primitive animals having very simple
nervous systems, an attractive possibility is that some of
these mechanisms first appeared in primary mechanosen-
sory neurons. These cells were among the earliest neurons
(e.g.. Bullock and Horridge, 1965). Because they are di-
rectly exposed to surface trauma, they provide a single
locus for both recognizing noxious stimulation and alter-
ing the responses of an animal to subsequent mechanical
stimulation. Exposure of the peripheral branches of
mechanosensory neurons to surface trauma suggests a
specific cellular hypothesis for the origin of some mech-
anisms of nociceptive plasticity. Trauma to the body sur-
face (or even wear on soft body parts in a turbulent en-
vironment) can damage peripheral sensory branches. The
ubiquity of cellular repair processes in modern cells sug-
gests that such processes appeared very early in the evo-
lution of life and were available to repair damaged sensory
branches in primitive animals. For an organism to take
mechanisms that had evolved to regenerate and maintain
excitability in damaged neuronal branches, and use these
mechanisms in undamaged neurons where they could
amplify the neurons' normal signalling effectiveness,
seems a small step. For example, mechanisms of cellular
repair, growth, and signal compensation triggered by in-
tracellular signals of cellular injury might become induc-
ible by extracellular neuromodulators released during
noxious stimulation, or by interactions of such neuro-
modulators with spike activity in the neuron (i.e., ADEM).

Recently we tested whether a close relationship exists
between mechanisms of general and site-specific sensiti-
zation on the one hand (i.e., involving neuromodulation
and ADEM), and responses to axonal injury on the other
(Walters, Alizadeh, and Castro, unpub. obs.). We studied
the effects of axonal injury by crushing nerves containing
sensory neuron axons under conditions in which synaptic
release of neuromodulators (such as 5-HT) and ADEM
were blocked. Tests of the sensory neurons after nerve
crush revealed profound hyperexcitability and synaptic
facilitation, lasting weeks, that were specific to cells with
axons in the crushed nerves. The long latency of the effects
(1-2 days) suggested that signals from axonal damage were
conveyed back to the soma and synapses by axonal trans-
port. Of particular interest was the qualitative identity of
the set of changes produced by axon crush with the al-
terations produced by neuromodulation and ADEM. One
interpretation of these results is that a common set of

mechanisms underlying long-term hyperexcitability and
synaptic facilitation can be triggered by two signalling
pathways from the periphery to the soma. The more
primitive pathway would be provided by intracellular sig-
nals conveyed slowly by axonal transport from damaged
axons. ADEM (conjoint electrical activation and extrinsic
neuromodulation) of the soma during injury would pro-
vide a much more rapid signal of peripheral injury in
nociceptors activated by injurious stimuli (see Billy and
Walters, 1989a). ADEM may have evolved later, when
the increased size of animals and resulting distances be-
tween nociceptor somata and their receptive processes se-
lected for faster signals, so that regulation of protein syn-
thesis necessary for injury-induced plasticity would not
be delayed.

Although highly speculative, this hypothesis and our
preliminary indications of links between cellular injury
responses and long-term sensitization in Aplysia raise the
possibility that some of the earliest forms of memory may
have evolved from cellular repair and signal compensation
mechanisms in primitive mechanosensory neurons. If. as
brains became increasingly complex, these cells served as
evolutionary precursors of other neuronal types, then
primitive mechanisms of nociceptive plasticity might have
been important for the evolution of other forms of mem-
ory as well. Indeed, both axon damage (e.g.. Janig, 1988;
Kelly el a/., 1988) and learning (e.g., Disterhoft el al.
1986) have been associated with long-term hyperexcita-
bility in mammalian neurons.

Evolutionary arguments have obvious weaknesses, es-
pecially when they deal with eras that left almost no fossil
record. Nevertheless, such arguments can lead to fresh
perspectives and novel physiological and molecular pre-
dictions. The present hypothesis makes three testable pre-
dictions: ( 1 ) that common cellular mechanisms (involving
homologous molecules) contribute to nociceptive sensi-
tization in a broad range of species; (2) that critical mo-
lecular steps are shared with mechanisms involved in cel-
lular repair and signal compensation following axonal
damage; and (3) that some of these molecular steps are
shared with mechanisms involved in traditional forms of
learning and memory. Systematic comparison of learning-
related mechanisms and injury-related mechanisms in di-
verse animals will put these predictions to the test, and
may provide insight into the evolution of some forms of
memory.

Acknowledgments

I am grateful to Dr. Andrea Clatworthy and Mr. Chris
Hickie for their comments and for allowing me to discuss
some of their unpublished data. I also thank Dr. Bill Frost
for his comments and Mr. Jim Pastore and Ms. Linda
Eshelman for preparing the illustrations. Supported by

250

E. T. WALTERS

National Institute of Mental Health grant MH38726 and
National Science Foundation grant BNS901 1907.

Literature Cited

Abrams, T. \V., and E. R. Kandel. 1988. Is contiguity detection in
classical conditioning a system or a cellular property? Learning in
Aplvsia suggests a possible molecular site. Trends Neurosd. 11:1 28-
135.

Alizadeh, H., Clatworthy, A. L., Castro, G. A., and E. T. Walters.
1990. Induction of an immune reaction in Aplysia is accompanied
by long-term enhancement of sensory neuron excitability. Soc. Neu-
msci. Abslr 15: 597.
Avers, C. J., 1989. Process and Pattern in Evolution. Oxford University

Press, New York. 590 pp.

Bailey, C. H., V. F. Castellucci, J. Koester, and E. R. Kandel.
1979. Cellular studies of peripheral neurons in siphon skin of Aplysia
ca/ifornica. J Neurophysiol. 42: 530-557.
Bailey, C. H., and M. Chen. 1983. Morphological basis of long-term

habituation and sensitization in Aplysia. Science 220: 91-93.
Bailey, C. H., and M. Chen. 1988. Long-term memory' in Aplysia
modulates the total number of varicosities of single identified sensory
neurons. Proc. Nat. Acad. Sci. USA 85: 2373-2377
Barzilai, A., T. E. Kennedy, J. D. Sweatt, and E. R. Kandel. 1989. 5-
HT modulates protein synthesis and the expression of specific proteins
during long-term facilitation in Aplysia sensory neurons. Neuron 2:
1577-1586.

Baxter, D. A., and J. H. Byrne. 1989. Serotonergic modulation of two
potassium currents in the pleural sensory neurons of Aplysia. J. Neu-
ropliysiol. 62: 665-679.

Belardetti, F., E. R. Kandel, and S. A. Siegelbaum. 1987. Neuronal
inhibition by the peptide FMRFamide involves opening of S K+
channels. Nature 325: 153-156.

Billy, A. J., and E. T. Walters. I989a. Long-term expansion and sen-
sitization of mechanosensory receptive fields in Aplysia support an
activity-dependent model of whole-cell sensory plasticity. J. Neurosd.
9: 1254-1262.

Billy, A. J., and E. T. Walters. 1989b. Modulation of mechanosensory

threshold in Aplysia by 5-HT, small cardioactive peptideB (SCPB).

FMRFamide, acetylcholine, and dopamine. Neurosci Lett 105: 200-

204.

Bolles, R. C, and M. S. Fanselow. 1980. A perceptual-defensive-re-

cuperative model of fear and pain. Behav. Brain Sd. 3: 291-323.
Braha, O., N. Dale, B. Hochner, M. Klein, T. W. Abrams, and E. R.
Kandel. 1990. Second messengers involved in the two processes of
presynaptic facilitation that contribute to sensitization and dishabit-
uation in Aplysia sensory neurons. Proc. Nat. Acad. Sd. USA 87:
2040-2044.

Bullock, T. H., and G. A. Horridge. 1965. Structure and Function in
the Nemnis Systems of Invertebrates. Vol. 1. W. H. Freeman and
Co., San Francisco. 798 pp.
Byrne, J. H., 1980. Identification of neurons contributing to presynaptic

inhibition in Aplysia ca/ifornica. Brain Res 199: 235-239.
Byrne, J. H., 1983. Identification and initial characterization of a cluster
of command and pattern-generating neurons underlying respiratory
pumping in Aplvsia cali/ornica. J. Neurophysiol. 49: 491-508.
Byrne, J. H., 1987. Cellular analysis of associative learning. Physio/.

Re\: 67: 329-439.

Byrne, J. H., V. Castellucci, and E. R. Kandel. 1974. Receptive fields
and response properties of mechanoreceptor neurons innervating si-
phon skin and mantle shelf in Aplysia. J. Neurophysiol. 37: 1041-
1064.

Byrne, J. H., V. E. Castellucci, T. J. Carew, and E. R. Kandel.
1978. Stimulus-response relations and stability of mechanoreceptor

and motor neurons mediating defensive gill-withdrawal reflex in
Aplvsia. J. Neurophysiol. 41: 402-417.

Carew, T. J., V. F. Castellucci, and E. R. Kandel. 1971. An analysis
of dishabituation and sensitization of the gill-withdrawal reflex in
Aplvsia. Intern. J Neurosci. 2: 79-98.

Carew, T. J., R. D. Hawkins, and E. R. Kandel. 1983. Differential
classical conditioning of a defensive withdrawal reflex in Aplysia cal-
ijornica. Science 219: 397-400.

Carew, T. J., and E. R. Kandel. 1977. Inking in Aplysia calitormca.
III. Two different synaptic conductance mechanisms for triggering
central program for inking. /. Neurophysiol 40: 721-734.
Carew, T. J., E. T. Walters, and E. R. Kandel. 1981. Classical condi-
tioning in a simple withdrawal reflex in Aplysia calilornica. J. Neu-
rosci. 1: 1426-1437.

Clark, G. A., and E. R. Kandel. 1984. Branch-specific heterosynaptic
facilitation in Aplysia siphon sensory cells. Proc. Nat. Acad. Sd. USA
81: 2577-2581.

Clatworthy, A. L., and E. T. Walters. 1988. Sensitization and sensory
signals in Aplysia II. Modulation of central bursting and spike con-
duction. Soc. Neurosci. .Ihslr 14: 609.

Clatworthy, A. L., and E. T. Walters. 1989. Activity-dependent block
of central sensory conduction during inhibition of tail withdrawal
reflex in Aplysia. Sue Neurosci- Abxtr. 15: 1263.
Clatworthy, A. L., and E. T. Walters. 1990. Modulation of the excit-
ability of peripheral receptive fields and axons in sensory neurons of
Aplvsia. Soc. Neurosci. Abstr. 16: 20.
Colwill, R. M., R. A. Absher, and M. L. Roberts. 1988. Context-US

learning in Aplysia ca/itornica J Neurosci. 8: 4434-4439.
Dash, P. K., B. Hochner. and E. R. Kandel. 1990. Injection of the
cAMP-responsive element into the nucleus of Aplysia sensory neurons
blocks long-term facilitation. Nature 345: 718-721.
Disterhoft, J. F., D. A. Coulter, and D. L. Alkon. 1986. Conditioning-
specific membrane change of hippocampal neurons in vitro. Proc.
Nat. Acad. Sci. USA 83: 2733-2737.

Edmonds, B., M. Klein, N. Dale, and E. R. Kandel. 1990. Contributions
of two types of calcium channels to synaptic transmission and plas-
ticity. Science 250: 1142-1147.

Erickson, M. T., and E. T. Walters. 1988. Differential expression of
pseudoconditioning and sensitization by siphon responses in Aplysia:
novel response selection after training. J. Neurosd. 8: 3000-3010.
Eskin, A., K. S. Garcia, and .1. H. Byrne. 1989. Information storage
in the nervous system of Aplysia: specific proteins affected by 5-HT
and cAMP. Proc. Nat. Acad. Sci. USA 86: 2458-2462.
Fang, X., and G. A. Clark. 1990. Neural mechanisms of response spec-
ificity. I. Tail and mantle nerve shock produce differential effects on
the siphon-withdrawal neuronal circuit in Aplysia. Soc. Neurosci.
Abstr. 16: 596.

Frost, W . N., V. F. Castellucci, R. D. Hawkins, and E. R. Kandel.
1985. Monosynaptic connections made by the sensory neurons of
the gill- and siphon-withdrawal reflex in Aplysia participate in the
storage of long-term memory for sensitization. Proc. Nat. Acad. Sci.
USA 82: 8266-8269.

Frost, W. N., G. A. Clark, and E. R. Kandel. 1988. Parallel processing
of short-term memory' for sensitization in Aplysia. J. Neurobiol. 19:
297-334.

Glanzman, D. L., S. L. Mackey, R. D. Hawkins, A. M. Dyke, P. E.
Lloyd, and E. R. Kandel. 1989. Depletion of 5-HT in the nervous
system of Aplysia reduces the behavioral enhancement of gill with-
drawal as well as the heterosynaptic facilitation produced by tail shock.
J. Neurosd. 9: 4200-4213.

Hawkins, R. D., T. W . Abrams, T. J. Carew, and E. R. Kandel. 1983. A
cellular mechanism of classical conditioning in Aplysia: activity-de-
pendent amplification of presynaptic facilitation. Science 219: 400-
404.

NOC1CEPT1VE PLASTICITY IN APLYSIA

251

Hawkins, R. D., V. F. Castellucci, and E. R. Kandel. 1981. Interneurons
involved in mediation and modulation of gill- withdrawal reflex in
Aplyxia I. Identification and characterization. ./ Neurophysiol, 45:
304-314.

Hawkins, R. D., N. Ealevic, G. A. Clark, and E. R. Kandel.
1989. Classical conditioning of the Aplyxia siphon-withdrawal reflex
exhibits response specificity. Pwc. Nat. Acacl. Sci. USA 86: 7620-
7624.

Hickie, C., and E. T. Walters. 1990. Identified central motor neurons
are necessary for directional siphon responses in Aplysia. Soc. Neu-
rosci. Absir. 16: 19.

I In km. in. C. P., L. S. Roberts, and F. M. Hickman. 1984. Integrated
Principles of Zoology. Mosby. St. Louis, Missouri. 1065 pp.

Janig. \V. 1988. Pathophysiology of nerve following mechanical injury.
Pp. 84-108 in Proceedings ot the I'lli World Congress on Pain R.
Dubner. G. F. Gebhart, and M. R. Bond. eds. Elsevier, Amsterdam.

Kandel, E. R., and J. H. Schwartz. 1982. Molecular biology of learning:
modulation of transmitter release. Science 218: 433-444.

Kavaliers, M. 1987. Evidence for opioid and non-opioid forms of stress-
induced analgesia in the snail. Cepaea nemoralis Brain Rex. 410:
111-115.

Kavaliers, M. 1988. Evolutionary and comparative aspects of noci-
ception. Bruin Res Bull 21:923-931.

Kelly, M. E., M. A. Bisby, and K. Eukowiak. 1988. Regeneration re-
stores some of the altered electrical properties of axotomized bullfrog
B-cells. J. .\enrohiol. 19: 357-372.

Klein, M., J. Camardo, and E. R. Kandel. 1982. Serotonin modulates
a specific potassium current in the sensory neurons that show pre-
synaptic facilitation in Aplysia. Proe. Nat. Acad. Set. VSA 79: 57 1 3-
5717.

Klein, M., B. Hoclmer, and E. R. Kandel. 1986. Facilitatory transmitters
and cAMP can modulate accomodation as well as transmitter release
in Aplvxta sensory neurons: evidence for parallel processing in a single
cell. Proe. Nal. Acutl. Sci. VSA 83: 7994-7998.

Krontiris-Litowitz, J. K., B. F. Cooper, and E. T. Walters.
1989. Humoral factors released during trauma of Aplyxia. I. Body
wall contraction, cardiac modulation, and central reflex suppression.
/ Com/' Phyxiol. B 159: 21 1-223.

Krontiris-Eitowitz, J. K., M. T. Erickson, and E. T. Walters.

1987. Central suppression of defensive reflexes in Aplyxia by noxious
stimulation and by factors released from body wall. Soc. Neuroxci.
Ahxlr 13: 815.

Kupfermann, I., V. Castellucci, H. Pinsker, and E. R. Kandel.
1970. Neuronal correlates of habituation and dishabituation of the
gill-withdrawal reflex in Aplyxia. Science 167: 1743-1745.

Mackey, S. L., D. L. Glanzman, S. A. Small, A. M. Dyke, E. R. Kandel,
and R. D. Hawkins. 1987. Tail shock produces inhibition as well
as sensitization of the siphon-withdrawal reflex of Aplysia: possible
behavioral role for presynaptic inhibition mediated by the peptide
Phe-Met-Arg-Phe-NH2. Proe. Nat. Acad. Sci. USA 84: 8730-8734.

Marcus, E. A., T. G. Nolen, C. H. Rankin, and T. J. Carew.

1988. Behavioral dissociation of dishabituation. sensitization. and
inhibition in Aplyxia. Science 241: 210-212.

Nazif, F., J. H. Byrne, and L. J. Cleary. 1989. Intracellular injection
of cAMP produces a long-term (24 hr) increase in the number of

vancosities in pleural sensory neurons of Aplyxia. Soc. Neurosci. Ahstr.
15: 1283.

Nicholls, J. G., and D. A. Baylor. 1968. Specific modalities and re-
ceptive fields of sensory neurons in the CNS of the leech. / Neuro-
physiol. 31: 740-756.

Pinsker, H. M., W. A. Hening, T. J. Carew, and E. R. Kandel.
1973. Long-term sensitization of a defensive withdrawal reflex in
Aplysia. Science 182: 1039-1042.

Scholz, K. P., and J. H. Byrne. 1987. Long-term sensitization in Aplysia:
biophysical correlates in tail sensory neurons. Science 235: 685-687.

Sherrington, S. C. 1906. The Integrative Action of the Nervous System
2nd Ed. 1947. University Press, Cambridge. LI. K. 433 pp.

Small, S. A., E. R. Kandel, and R. D. Hawkins. 1989. Activity-depen-
dent enhancement of presynaptic inhibition in Aplysia sensory neu-
rons. Science 243: 1603-1606.

Vermeij, G. J. 1987. Evolution and Escalation: An Ecological History
oj Lite. Princeton Llniversity Press, New Jersey.

Wall, P. D., 1979. On the relation of injury to pain. Pain 6: 253-264.

Walsh, J. P., and J. H. Byrne. 1989. Modulation of a steady-state
Ca2+-activated, K+ current in tail sensory neurons of Aplysia: role
of 5-HT and cAMP. J. Neurophysiol. 61: 32-44.

Walters, E. T. 1987a. Site-specific sensitization of defensive reflexes
in Aplrsia: a simple model of long-term hyperalgesia. J. Netirose:. 7:
400-407.

Walters, E. T. I987b. Multiple sensory neuronal correlates of site-
specific sensitization in Aplyxia. ./. Neurosci. 7: 408-417.

Walters, E. T. 1989. Transformation of siphon responses during con-
ditioning of . Iplvsia suggests a model of primitive stimulus-response
association. Proe. Nal. Acad. Sci USA 86: 7616-7619.

Walters, E. T., and J. H. Byrne. 1983a. Associative conditioning of
single sensory neurons suggests a cellular mechanism for learning.
Science 219: 405-408.

Walters, E. T., and J. H. Byrne. I983b. Slow depolarization produced
by associative conditioning of Aplysia sensory neurons may enhance
Ca2+ entry. Brain Res 280: 165-168.

Walters, E. t., and J. H. Byrne. 1984. Post-tetanic potentiation in
Iplvxia sensory neurons. Brain Res 293: 377-380.

Walters, E. T., J. H. Byrne, T. J. Carew, and E. R. Kandel.
1983a. Mechanoafferent neurons innervating tail of Aplysia. I. Re-
sponse properties and synaptic connections. J Neurophysiol. 50:
1522-1542.

Walters, E. T., J. H. Byrne, T. J. Carew, and E. R. Kandel.
1983b. Mechanoafferent neurons innervating tail of Aplysia. II.
Modulation by sensitizing stimulation. J. Neurophysiol. 50: 1543-
1559.

Walters, E. T., T. J. Carew, and E. R. Kandel. 1981. Associative learning
in Aplysia: evidence for conditioned fear in an invertebrate. Science
211: 504-506.

Walters, E. T., and M. T. Erickson. 1986. Directional control and the
functional organization of defensive responses in Aplysia. J. Comp.
Physiol. A 159: 339-351.

Woolf, C. J., and E. T. Walters. 1991 . Common patterns of plasticity
contributing to nociceptive sensitization in mammals and Aplysia.
Trends In Neurosci. 14: 74-78.

Wright, W. G., E. A. Marcus, and T. J. Carew. 1989. Dissociation of
monosynaptic and polysynaptic contributions to dishabituation.
sensitization and inhibition in Aplysia. Soc. Neurosci. Abstr. 15: 1265.

Reference: Biol. Bull. 180: 252-261. (April, 1991)

Neural Mechanisms Underlying Sensitization
of a Defensive Reflex in Aplysia

L. J. CLEARY, D. A. BAXTER, F. NAZIF, AND J. H. BYRNE

Department of Neurobiology and Anatomy. University of Texas Medical School,
P. O. Box 20708, Houston, Texas 77225

Introduction

One of the last frontiers in modern biology is under-
standing the neural basis of behavior and its modification
by processes such as learning. While the ultimate goal for
many is to understand human behavior, other model sys-
tems are more commonly used in the laboratory with the
expectation that general principles of neuronal function
will be conserved across phyla. Mollusks in particular have
proven to be valuable model systems because of their rel-
atively simple and accessible nervous systems. Aplysia
califomica is a gastropod mollusk with a simple behavioral
repertoire and a relatively simple nervous system (see
Kandel, 1979). Nevertheless, a detailed understanding of
more complex phenomena, such as the modification of
behaviors by learning (Kandel and Schwartz, 1982) and
arousal (Weiss et ai, 1982) has also emerged from study
of this animal. One class of behaviors that has been studied
extensively in Aplysia is that of defensive withdrawal re-
flexes. We have focused on the tail-siphon withdrawal re-
flex, which is elicited by mechanical stimulation of the
tail. The features of this reflex are similar to those of the
siphon-gill withdrawal reflex, which is elicited by stimu-
lation of the siphon. Moreover, both of these reflexes, or
their in vitro analogues, can be modulated in several ways,
including habituation, sensitization, and classical condi-
tioning (Pinsker et a!., 1970; Carew et ai, 1983; Walters
and Byrne, 1983; Walters et at.. 1983b). In this paper, the
focus will be on sensitization of the tail-siphon withdrawal
reflex.

Tail-Siphon Withdrawal Reflex

Weak mechanical stimulation of the tail elicits a co-
ordinated contraction of the tail, siphon, and also the gill

Received 18 October 1990: accepted 24 January 1991.

(Fig. 1 ; Walters et a/., 1983a; Walters and Erickson, 1986).
A more intense stimulus may also elicit the release of ink
from glands in the mantle cavity. Weak electrical stim-
ulation through implanted electrodes elicits contraction
of the tail and siphon (Fig. 2). The strength of the reflex
response is estimated by measuring the duration of siphon
withdrawal. When weak test stimuli are delivered at 5
min intervals, the strength of the response is fairly constant
at approximately 7 s. After delivery of a strong stimulus
through a hand-held electrode over the tail and lateral
body wall, the strength of the response to subsequent weak
stimuli is enhanced significantly. This enhancement is
called sensitization. In general, the time course of sensi-
tization depends on the training protocol and can last for
a relatively short time, such as 15 minutes to an hour
(Pinsker a ai, 1970), or for a relatively long time, greater
than 24 hours (Pinsker et ai, 1973; Frost et ai, 1985;
Scholzand Byrne, 1987).

Neural Circuit for Tail-Siphon Withdrawal

To approach this phenomenon at the cellular level, the
neurons that mediate the behavior must be identified (Fig.
3). Sensory neurons innervating the tail (TSN) are located
in the pleural ganglion. The main axon from these neurons
projects to the pedal ganglion, but there are numerous
fine branches within the pleural ganglion as well. The tail
component of the withdrawal response is mediated by a
monosynaptic circuit. Tail motor neurons (TMN) are lo-
cated in the pedal ganglion (Walters et ai, 1983a). The
monosynaptic circuit is sufficiently strong to elicit tail
withdrawal (Walters et ai. 1983a), but other neurons also
contribute. For example, interneurons (IN) in the pleural
ganglion receive excitatory input from sensory neurons
and, in turn, project to the pedal ganglion where they

252

NEUROMODULATION IN APLYSIA

253

Siphon

Tail

Stimulus

Figure 1. Dorsal view of Aplysiu illustrating the tail-siphon with-
drawal reflex. (A) Relaxed. (B) Stimulation of the tail elicits a coordinated
set of defensive responses including reflex withdrawal of the tail, siphon,
and gill.

excite motor neurons, forming a second, parallel pathway
for tail withdrawal (Cleary and Byrne, 1985). The siphon
withdrawal component of the reflex is mediated by a
polysynaptic pathway. The population of interneurons in
the pleural ganglion that receives input from tail sensory
neurons also projects to the abdominal and cerebral gan-
glia. The siphon component of the response is mediated
by motor neurons (SMN) in the abdominal ganglion
(Perlman, 1979; Frost et a/., 1988). Motor neurons for
other mantle organs such as the gill and the ink gland are
located in this ganglion as well (Kupfermann ft a!., 1974;
Carew and Kandel, 1977). When stimulated, the pleural
interneurons excite siphon (LSF), gill (LDG:), and ink
(L14) motor neurons. In addition, they may also trigger
a burst in the L25 neurons. L25 is a group of pattern-
generating neurons that appears to control respiratory
pumping, a behavior that also involves contraction of the
gill and siphon (Byrne, 1983; Koester, 1989). L25 neurons
have extensive connections throughout the abdominal
ganglion. Therefore, interneurons in the pleural ganglion
appear to integrate sensory input from tail stimulation
and coordinate the total behavioral response.

One characteristic of the connection between pleural
interneurons and tail motor neurons is a biphasic exci-
tation of the tail motor neurons. While a single spike fre-
quently produces a fast excitatory postsynaptic potential
(EPSP). a short burst in the interneuron produces a long-
lasting excitation that appears to have two components
(Fig. 4). The first component is the fast EPSP and lasts
for the duration of the interneuron burst, whereas the

B TESTING AND TRAINING PROTOCOL

Sensitiang .
Stimuli

Siphon

Figure 2. Sensitization of the tail-siphon withdrawal reflex. (A) Sites
for delivering test and sensitizing stimuli. (B| Testing and training pro-
tocol. (C) Behavioral results. A single brief train (10 s) of sensitizing
stimuli leads to an enhancement of siphon withdrawal elicited by stimuli
to the tail. (From McClendon. Goldsmith and Byrne, in prep.)

second component is a slow EPSP that can last over one
minute. This slow EPSP is sufficient in some preparations
to prolong the burst elicited by the first component. In
addition, the slow EPSP increases the effectiveness of syn-
aptic input produced by subsequent stimulation of pe-
ripheral nerves. Thus, this population of pleural inter-
neurons plays a modulatory role by using a conventional
postsynaptic mechanism, temporal summation of excit-
atory synaptic inputs.

Delivery of a sensitizing stimulus activates conventional
interneurons, but, in addition, a separate modulatory cir-
cuit must be recruited. This circuit has not been identified

Figure 3. Simplified schematic diagram of the neural circuit con-
trolling the tail-siphon withdrawal reflex.

254

L. J. CLEARY ET AL.

5mV

]40mV

10 sec

Figure 4. Simultaneous intracellular recordings from isolated pleural-
pedal ganglia. A bnef (1 s) depolarization of a pleural interneuron (IN)
elicits a long-lasting depolarization in a tail motor neuron (MN) in the
pedal ganglion. The slow EPSP was of sufficient amplitude to prolong
the high-frequency burst of action potentials produced directly by the
interneuron. (From Geary and Byrne, in prep.)

for the tail-siphon withdrawal reflex. The pleural inter-
neurons, however, provide a link to interneurons in the
abdominal ganglion that modulate the neural circuit me-
diating the gill-siphon withdrawal reflex. The strength of
the synapse between siphon sensory and motor neurons
is enhanced by the activation of at least three cells in the
abdominal ganglion: L22, L28, and L29 (Hawkins el al.,
1981). One of these, L29, is excited by pleural interneurons
(Cleary and Byrne, 1986). Moreover, the pleural inter-
neuron to L29 connections are reciprocal. Thus, a positive
feedback loop exists that could prolong and enhance the
effects of a sensitizing tail stimulus.

Serotonin as a Facilitatory Transmitter

Modification of the tail-siphon withdrawal reflex by
sensitization is due to changes in the properties of neurons
that mediate the reflex. The tail sensory neurons in par-
ticular have been characterized as a site of plasticity (Wal-
ters el al, 1983b; Scholz and Byrne, 1987). Sensitizing
stimuli increase synaptic efficacy and alter membrane
properties of the sensory neurons, such as resting mem-
brane potential, excitability, and action potential kinetics
(Walters el al., 1983b). These changes are similar to those
occurring in siphon sensory neurons as a result of sensi-
tization (Castellucci and Kandel, 1976; Kandel and
Schwartz, 1982).

Interneurons whose activity modifies the properties of
the tail-siphon withdrawal circuit have not been identified.
Several lines of evidence indicate that serotonin is one of
the modulatory transmitters released by sensitizing stim-
uli. Serotonin mimics many of the short- and long-term
effects of sensitization. Serotonin is necessary for sensi-
tization to occur. In addition, serotonergic circuitry is
available in the nervous system to mediate sensitization.

Serotonin mimics several of the biochemical and elec-
trophysiological correlates of sensitization. In a semi-intact
preparation, the selective application of serotonin to the
pleural-pedal ganglia increases the intensity of the tail

withdrawal reflex, just as the application of a sensitizing
stimulus to the tail does (Walters et al., 1983b). At the
cellular level, serotonin enhances the size of the mono-
synaptic EPSP elicited in a follower motor neuron by the
sensory neuron. A slow depolarization is produced in the
sensory neuron, and this depolarization is associated with
an increase in membrane input resistance (Ocorr and
Byrne, 1985). Membrane excitability is increased as well
(Baxter and Byrne, 1990). Finally, serotonin increases the
amplitude and duration of the sensory neuron action po-
tential (Baxter and Byrne, 1990).

The mechanism by which serotonin exerts these phys-
iological effects appears to be due to its ability to mimic
at least one of the biochemical correlates of sensitization.
Using a symmetrical experimental design, the level of
cAMP is higher in the cell bodies of tail sensory neurons
that innervate the body wall exposed to sensitizing stimuli
than in cell bodies of sensory neurons innervating the
contralateral unstimulated side (Ocorr et al., 1986). Sim-
ilarly, application of serotonin to isolated clusters of sen-
sory neurons increases the cAMP content compared to
contralateral controls (Ocorr and Byrne, 1985; Pollock et
al.. 1985; Sweatt el al., 1989). The dose-response rela-
tionship indicates that the half-maximal concentration of
serotonin is about 1 5 nM.

To test the hypothesis that serotonin acts by activating
adenylyl cyclase, the effects of serotonin on two membrane
properties, excitability and action potential duration, were
examined and compared with the effects of an analogue
of cAMP (Baxter and Byrne, 1990). Neuronal excitability
was measured as the number of action potentials elicited
by injecting constant depolarizing current pulses of 1 s
duration and of increasing magnitude. For example, in
artificial seawater, a test pulse of 2 nA elicited an average
of three action potentials. Application of serotonin in-
creased the average number of spikes during the depolar-
izing pulse by a factor of 1 .8. Application of a membrane-
permeable, phosphodiesterase-resistant analogue ofcAMP
(8-4-parachlorophenylthio-cAMP, 8-pcpt-cAMP) in-
creased the excitability of the sensory neuron and doubled
the number of action potentials elicited by an identical
current pulse (Fig. 5A). Addition of serotonin to the bath,
which still contained the cAMP analogue, failed to pro-
duce a further increase in excitability. Thus, cAMP has
potent effects on excitability, and these cAMP-mediated
effects are sufficient to fully account for the effects of se-
rotonin on neuronal excitability.

The effects of 8-pcpt-cAMP on action potential dura-
tion did not parallel the effects of serotonin, however.
Action potential duration was measured as the time be-
tween the peak of the action potential and its repolariza-
tion to the resting membrane potential. Application of
the analogue of cAMP increased the duration of the action

A Excitability (2 nA)
A1 ASW

NEUROMODULATION IN APLYS1A

B Action Potential

255

-45 mV *•

A2 +CAMP

0 mV - -I -

-45 mV J

A3 (ASW + cAMP) + 5-HT

-45 mV

(ASW + cAMP) + 5-HT

5 ms

-45 mV

l/V

200 ms

50 mV

Figure 5. Differential effects of an analogue ofcAMP and 5-HT on the duration of the action potential
and excitability in tail sensory neurons. Recordings were made from somata that had been isolated surgically
from the pleural ganglion. (A) A 1 s. 2 nA depolarizing current pulse elicited three action potentials in
artifical seawater ( A 1 ). An identical current pulse elicited twice as many action potentials alter hath application
of 8-pcpt-cAMP (A2). The subsequent addition of serotonin to the bath, which still contained 8-pcpt-cAMP,
did not increase further the number of spikes during the pulse (A3). (B) In the same sensory neuron, bath
application of 8-pcpt-cAMP (50 pM) increased the duration of the somatic action potential by a modest
amount. The subsequent addition of serotonin (50 pM) to the bath, which still contained 8-pcpt-cAMP.
dramatically increased the duration of the action potential. (From Baxter and Byrne. 1990).

potential by an average of 17% (Fig. 5B). The subsequent
addition of serotonin to the bath, which still contained
the cAMP analogue, further increased the duration of the
action potential by an additional 230%, on average. Thus,
cAMP has modest effects on spike broadening that are
not sufficient to occlude the effects of serotonin.

The differential effects of serotonin and cAMP can be
accounted for by the actions of these compounds on spe-
cific membrane currents. Use of voltage-clamp and com-
puter-subtraction techniques has shown that cAMP re-
duces a potassium current, the S-current, that is activated
at relatively negative membrane potentials, does not in-
activate and is relatively insensitive to block by TEA
(Klein et al, 1982; Pollock el ai. 1985; Shuster and Sie-
gelbaum, 1987; Baxter and Byrne, 1989; Walsh and Byrne,
1989). cAMP activates a protein kinase that phosphory-
lates the S-channel or a protein associated with it (Sie-
gelbaum et al., 1982; Shuster et al.. 1985). This phos-

phorylation results in an all-or-nothing closure of the
S-channel and thus a suppression of the macroscopic S-
current. The kinetics of the outward currents reduced by
cAMP are qualitatively similar at all levels of depolariza-
tion. At low levels of depolarization (i.e.. —20 mV or be-
low), the subsequent addition of serotonin to the bath,
which still contained the cAMP analogue, did not produce
any further reduction in membrane current (Fig. 6A).
Thus these results and others indicate that the suppression
of the S-current by 5-HT is mediated by cAMP.

In addition, serotonin appears to modulate a second cur-
rent in tail sensory neurons that is not affected by cAMP
and is activated only at depolarized levels (Baxter and Byrne,
1989, 1990). The addition of cAMP changes the currents
produced by voltage-clamp steps to +20 mV by decreasing
the outward current at the end of the pulse (Fig. 6B). Sub-
sequent addition of serotonin to the bath results in a reduc-
tion in outward current at the beginning of the pulse and

256

L. J. CLEARY KT AL

B1 Step to +20 mV

A1 Step to -20 mV

a ASW

b +CAMP

c (ASW+cAMP) + 5-HT

2nA

a ASW

c (ASW+cAMP)+5-HT

b +CAMP

30 nA

B2 Difference Currents

A2 Difference Currents

(I CAMP)

(IS.HT)

1 nA

- b (I CAMP)

(IS.HT)

15nA
20 ms

Figure 6. Differential effects of an analogue of cAMP and serotonin on membrane current in tail sensory
neurons. In all panels, the label for an individual trace is aligned with the current level at the end of the
voltage-clamp pulse. (Al ) Current responses were elicited by voltage-clamp pulses from —70 to -20 mV in
artificial seawater ( inn c <;). after application of 8-pcpt-eAMP (50 n\l) (mice b). and after addition of serotonin
(50 iiM) to the bath, which still contained the analogue (trace c). Note that the membrane currents do not
return to the preclamp level because the cell was clamped from a holding potential of -70 to -20 mV and
then back to -50 mV. (A2) The cAMP difference current (IcAMP) was isolated by subtracting trace b from
trace a. The cAMP-independent component of the serotonin difference current (IS.HT) was isolated by sub-
tracting /race c from trace b. (Bl ) Current responses from the same cell were elicited by voltage-clamp pulses
from -70 to +20 mV in artificial seawater (trace a), after bath application of 8-pcpt-cAMP (trace />) and
after adding serotonin to the bath, which still contained the analogue (trace c). (B2) The cAMP difference
current (IcAMP) was isolated by subtracting trace b from trace a. The cAMP-independent component of the
serotonin difference current (I5.HT) was isolated by subtracting (race c from trace />. The qualitative features
of the cAMP difference currents (lc^Mp) that were isolated from voltage-clamp pulses to -20 mV (A2) and
+20 mv (B2) were similar (note the change in scale). In contrast, the qualitative features of the cAMP-
independent component of the serotonin difference current (km) were vei7 different at the two potentials.
At -20 mV, the presence of 8-pcpt-cAMP completely occluded further modulation of membrane current
by serotonin (A2). At +20 mV, however, serotonin modulated an additional component of membrane
current (B2). This additional component probably represents modulation of the kinetics of the delayed
potassium current. (From Baxter and Byrne, 1990).

an increase in outward current at the end of the pulse. The
voltage-dependence and sensitivity to potassium channel
blockers of the cAMP-independent effects of serotonin sug-
gest that the current affected by serotonin under these con-

ditions is the delayed potassium current. The effects of se-
rotonin appear to be produced by a mechanism in which
the kinetics of activation and inactivation are slowed, rather
than one in which the conductance of channels is blocked.

NEUROMODULATION IN APLYSIA

257

Figure 7. Electron micrograph through the cell hod> of a pleural
sensory neuron illustrating a direct contact between a serotonergic process
and the plasma membrane ol the sensory neuron. The straight pre- and
postsynaptic membrane (arrowheads) and the widened synaptic cleft
suggest that this section is through an active zone, a possible site of
transmitter release. Numerous glial processes (G I ) invagmate the sensory
neuron membrane. Pleural ganglia were fixed and incubated in primary
antisera to serotonin (Incstar. Inc.) for 1 week. Distribution of the antibody-
was revealed by an avidin-peroxidase technique (Vectastain ABC, Vector.
Inc.). The tissue was then osmicated. embedded in plastic, and cut into
thin (100 nm) sections. The scale bar represents 250 nm. (From Zhang.
Cleary, Marshak and Byrne, in prep.)

Thus, the differential effects of serotonin and cAMP
suggest that the cAMP-mediated modulation of the
membrane current is primarily responsible for the effects
of serotonin on neuronal excitability. cAMP-independent
modulation of the delayed potassium current appears to
be primarily responsible for the effects of serotonin on
action potential duration. The mechanism underlying this
component of the serotonin response is not yet known.
Other second messenger pathways may be involved, how-
ever. For example, protein kinase C appears to contribute
to presynaptic facilitation of the siphon sensory-motor
synapse after it has been depressed (Hochner et al.. 1986;
Braha el al.. 1990; Sacktor and Schwartz, 1990).

For sensitization to occur, intact serotonergic neurons
must be present in the nervous system. When serotonin
is selectively depleted from the nervous system by injec-
tion of 5,7-dihydroxytryptamine (5,7-DHT). the effects
of subsequent sensitizing stimuli on the siphon-gill with-
drawal reflex are blocked (Glanzman et ai. 1989). While
the average amplitude of the EPSP evoked in a follower
neuron by the sensory neuron is not affected by 5,7-DHT
treatment, facilitation of PSPs by sensitizing stimuli is
drastically reduced. These experiments do not reveal,
however, whether the endogenous serotonin acts directly
or indirectly on the sensory neurons.

Evidence supporting a direct action of serotonin in the
tail-siphon withdrawal reflex comes from the observation
that serotonergic fibers are in close proximity to pleural
sensory neurons (Lo et al.. 1987; Zhang et al.. 1988). Al-
though there are no serotonergic neurons in the pleural
ganglion itself (Tritt et a/.. 1983; Ono and McCaman.
1984; Longley and Longley, 1986), there are many sero-
tonin-containing axons within the neuropil that originate
from neurons in other ganglia. Some of these serotonergic
axons send fine processes up to surround the cell bodies
of the pleural sensory neurons. Subsequent examination
using electron microscopic immunocytochemistry has
shown that these serotonergic axons contain varicosities
that are in direct contact with the plasma membrane of
sensory neurons (Fig. 7). Moreover, serotonergic contacts
may occur on either the cell body or the axon hillock. We
have not yet performed double-labeling experiments to
examine the distribution of serotonergic contacts along
the axons and processes of sensory neurons in the neuropil
of the pleural and pedal ganglia.

Although neurons that give rise to serotonergic pro-
cesses in the pleural ganglion have not been identified,
there are several candidates. Most promising among these
are the serotonergic cells in the cerebral B cluster (Mackey
el cil-. 1989; Cleary and Byrne, unpub.). These cells appear
to send axons that project through the cerebral-pleural
connective into the pleural ganglion and continue to the
abdominal ganglion through the pleural-abdominal con-
nective. Stimulation of the serotonergic B cell produces
both spike broadening in siphon sensory cells in the ab-
dominal ganglion and facilitation of the PSP between si-
phon sensory and follower motor neurons.

We have focused on the role of serotonin, but other
modulatory transmitters may also be involved. For ex-
ample, the peptide SCPh mimics many of the effects of
serotonin, although it binds to a different receptor
(Abramsria/.. 1984; Ocorr et al.. 1986). Moreover, some
identified neurons that produce facilitation of the synapse
between siphon sensory and motor neurons do not contain
serotonin (Kistler et al.. 1985; Hawkins and Schacher,
1989). Future research will be necessary to elaborate the
roles of other transmitters in sensitization.

Role of the Cell Body in Modulation

Activation of interneurons that provide serotonergic
input to the cell bodies of tail sensory neurons may have
multiple modulatory effects. For example, changes in so-
matic membrane potential would propagate passively to
proximal axon branches within the pleural ganglion. In
addition, elevation of cAMP levels in cell bodies may be
sufficient to elevate cAMP in proximal axonal branches,
producing local changes in membrane properties that

258

L. J. CLEARY ET AL.

could contribute to enhanced release of transmitter as
observed in siphon sensory neurons (Brunelli et a!.. 1976;
Castellucci ct ai. 1982). Because of the limits imposed
by diffusion, however, somatic alterations may not be suf-
ficient to account for the effects of sensitizing stimuli at
distal sites of transmitter release. Enhancement by sero-
tonin of synaptic transmission at the sensory to tail motor
neuron synapse in the pedal ganglion does not require an
intact connection between the axon of the sensory neuron
in the pedal ganglion and the cell body in the pleural
ganglion (Hammer et ai, 1989). Similarly, application of
serotonin to a peripheral synapse in the siphon selectively
enhances transmission from that synapse (Clark and
Kandel, 1984). Therefore, facilitation at sites distant from
the cell body is probably due to the local action of the
modulatory transmitter.

That the cell body is functionally independent from
some regions of the neuron suggests that serotonergic in-
put to the cell body is specialized to activate mechanisms
that are localized there. Mechanisms that might be reg-
ulated by somatic serotonin receptors include mRNA
synthesis, which is restricted to the nucleus, and protein
synthesis, which is restricted to the somatic cytoplasm.
These mechanisms are of particular interest because of
their role in long-term forms of facilitation and sensiti-
zation (Montarolo et ai, 1986; Castellucci et ai. 1989).

Some features of long-term sensitization are similar to
those of short-term; the major difference is in the duration
of the behavioral change. To alter the time course from
1 to 24 h or more, however, a longer training period is
required (Pinsker et ai. 1973; Frost et ai. 1985; Scholz
and Byrne, 1987). Nevertheless, the cellular correlates of
long-term sensitization are nearly identical to those of
short-term. The amplitude of EPSPs at synapses between
siphon sensory and motor neurons is enhanced (Frost et
ai. 1985). and membrane properties of tail sensory neu-
rons are altered (Scholz and Byrne, 1987). The similarities
between short- and long-term modifications of sensory
neurons suggest that correlates of long-term sensitization
are due to the action of a common mediator. Several lines
of evidence suggest that cAMP can by itself produce long-
term changes in the properties of sensory neurons. Tran-
sient elevation of the intracellular level of cAMP leads to
altered membrane currents (Scholz and Byrne, 1988) and
enhanced synaptic transmission (Schacher et ai. 1988)
that each persist for at least 24 h.

An additional cellular correlate of long-term sensiti-
zation in siphon sensory neurons is a change in the mor-
phology of sensory neuron axons and synapses (Bailey
and Chen, 1983; Bailey and Chen, 1988). To examine its
role in producing these changes, cAMP was injected ion-
tophoretically, over a period of 1 5 min, into the cell bodies
of tail sensory neurons. These neurons were subsequently

cAMP

5' AMP

50pm

Figure 8. Camera lucida drawings of two HRP-filled sensory neurons
from the same animal. The cell in the top panel was injected with cAMP
approximately 24 h before fixation; the cell in the lower panel was injected
with 5'-AMP. In each cell, a single large axon extends from the cell body
to the pleural-pedal connective. Because we analyzed only their number,
varicosities (arrows) were drawn slightly larger than scale to enhance
visibility. Varicosities counted in the cAMP-filled cell were 78 and in
the 5'-AMP-nlled cell 46. Branch points (arrowheads) counted in the
cAMP-filled cell were 32 and in the 5'-AMP-filled cell 24. Not all the
branch crossings in these drawings are branch points since these are
three-dimensional objects drawn in two dimensions. Some branches ap-
pear disconnected as a consequence of the tissue processing procedure.
(From Nazif rtu/.. 1991.)

labeled with HRP and fixed 24 h after cAMP injection
(Fig. 8). Close analysis of the structure of these neurons
revealed that both the number of varicosities was doubled
and the number of branch points was increased by 50%
compared to neurons that had been injected with 5'-AMP,
the inactive metabolite of cAMP. The significance of these
morphological changes lies in the hypothesis that sensory
neurons make more synapses with follower neurons in
the pleural ganglion as a result of sensitization. This im-
plies that connections between sensory neurons and their
followers are strengthened. Additional followers might also
be recruited by the formation of new connections.
Changes in both of these morphological features could
contribute to the enhanced strength of the reflex. In future
experiments, it will be interesting to test the role of cAMP
in producing other morphological changes such as in-
creases in the number, size, and vesicle complement of
synaptic active zones (Bailey and Chen, 1983).

NEUROMODULATION IN APLYSIA

259

VARICOSITIES
BRANCH POINTS

Figure 9. Simplified diagram illustrating the activation of parallel
cAMP-dependent pathways by neurites that release serotonin (5-HT)
onto the cell bodies of sensory neurons. Transient elevation of cAMP
in the soma produces rapid but short-lasting effects on membrane con-
ductance (\i>lul lines) via activation of protein kinase (PK). In addition,
it produces persistent effects on membrane conductance and neuronal
structure (dashed lines). The mechanism underlying these long-term ef-
fects is not known, but regulation of gene expression and protein synthesis
appears to be involved.

Because cAMP in sensory neurons is broken down rap-
idly (Bernier et a/., 1982; Schwartz ct ai. 1983), long-
term changes must depend on a more persistent cellular
process. A likely model, then, is one in which the transient
cAMP signal activates at least two pathways (Fig. 9).
cAMP produces a rapid but short-lasting modulation of
membrane currents, decreasing membrane conductance.
In addition, a slower but more enduring mechanism is
also activated, producing long-term changes in membrane
conductance and cell structure. The mechanisms under-
lying these long-term alterations are not known, but reg-
ulatory pathways that alter protein synthesis or gene
expression are presumably involved. Indeed, long-term
enhancement of synaptic transmission and membrane
excitability are affected by inhibitors of protein and RNA
synthesis (Montarolo et ai, 1986; Dale et ai. 1987;
Schacher et ai, 1988). In addition, serotonin appears to
activate regulatory proteins that bind to DNA (Dash et
ai, 1990). As a consequence, messenger RNA is synthe-
sized (Zwartjes et ai, 1990), and the incorporation of
amino acids into proteins of sensory neurons is altered
(Barzilai et ai, 1989; Eskin et ai, 1989). Other mecha-
nisms, such as post-translational modification of proteins
(Greenberg et ai. 1987), may be used as a result of sen-
sitization, providing an intermediate time course.

Acknowledgments

Supported by Texas Higher Education Co-ordination
Board Grant 1945 (L.J.C.), National Research Service Award
F31 MH09956 (F.A.N.), NIMH Award KO2 MH00649
(J.H.B.), and NIH Grant RO1 NS19895 (J.H.B.).

Literature Cited

Abrams, T. W., V. F. Caslellucci, J. S. Camardo, E. R. Kandel, and
P. E. Lloyd. 1984. Two endogenous neuropeptides modulate the
gill and siphon withdrawal reflex in Aplysia by presynaptic facilitation
involving cAMP-dependent closure of a serotonin-sensitive potassium
channel. Pruc Null Acad. Sci. USA 81: 7956-7960.

Bailey, C. H., and M. Chen. 1983. Morphological basis of long-term
habituation and sensitization in Aplyxia. Science 220: 91-93.

Bailey, C. H., and M. Chen. 1988. Long-term memory in Aplysia
modulates the total number of varicosities of single identified sensory
neurons. Pruc. Nail. Acacl. Sci. USA 85: 2373-2377.

Barzilai, A., T. E. Kennedy, J. D. Sweatt, and F. R. Kandel. 1989. 5-
HT modulates protein synthesis and the expression of specific proteins
during long-term facilitation in Aplysia sensory neurons. Neunm 2:
1577-1586.

Baxter, D. A., and J. H. Byrne. 1989. Serotonergic modulation of two
potassium currents in the pleural sensory neurons of Aplysia. J. Neii-
rophysiol. 62: 665-679.

Baxter, D. A., and J. H. Byrne. 1990. Differential effects of cAMP and
serotonin on membrane current, action potential duration, and ex-
citability in somata of pleural sensory neurons of Aplysia. J. Neu-
mphyswl. 64: 978-990.

Bernier, L., V. F. Castellucci, E. R. Kandel, and J. H. Schwartz.
1982. Facilitatory transmitter causes a selective and prolonged in-
crease in adenosine 3':5'-monophosphate in sensory neurons me-
diating the gill and siphon withdrawal reflex in Aplysia. J. Neurosci.
2: 1682-1691.

Braha, O., N. Dale, B. Hochner, M. Klein, T. W. Abrams, and E. R.
Kandel. 1990. Second messengers involved in two processes of pre-
synaptic facilitation that contribute to sensitization and dishabituation
in Aplvsia sensory neurons. Pruc. Nat. Acad. Sci. USA 87: 2040-
2044.

Brunelli, M., V. F. Castellucci, and E. R. Kandel. 1976. Synaptic fa-
cilitation and behavioral sensitization in Aplysia: possible role for
serotonin and cAMP. Science 194: 1 178-1 181.

Byrne, J. H. 1983. Identification and initial characterization of a cluster
of command and pattern-generating neurons underlying respiratory'
pumping in Aplvsia califomica, J Neurophysiol. 49: 491-508.

Carew, T. J., R. D. Hawkins, and E. R. Kandel. 1983. Differential
classical conditioning of a defensive withdrawal reflex in Aplysia cal-
ifomica. Science 219: 397-400.

Carew, T. J., and E. R. Kandel. 1977. Inking in Aplysia califomica:
neural circuit of an all-or-none behavioral response. / Neurophysiol.
40: 692-707.

Castellucci, V. F., H. Blumenfeld, P. Goelet, and E. R. Kandel.
1989. Inhibitor of protein synthesis blocks long-term behavioral
sensitization in isolated gill-withdrawal reflex of Aplysia. J. Neurobiol.
20: 1-9.

Castellucci, V. F., and E. R. Kandel. 1976. Presynaptic facilitation as
a mechanism for behavioral sensitization in Aplysia. Science 194:
1176-1178.

Castellucci, V. F., A. Nairn, P. Greengard, J. H. Schwartz, and E. R.
Kandel. 1982. Inhibitor of adenosine 3':5'-monophosphate-depen-
dent protein kinase blocks presynaptic facilitation in Aplysia. J. Neu-
rosci. 2: 1673-1681.

Clark, G. A., and E. R. Kandel. 1984. Branch-specific heterosynaptic
facilitation in Aplysia siphon sensory cells. Proc. Nat. Acad. Sci. USA
81:2577-2581.

Cleary, L. J., and J. H. Byrne. 1985. Interneurons contributing to the
mediation and modulation of the tail withdrawal reflex in Aplysia.
Soc. Neurosci. Abstr. 11: 692.

260

L. J. CLEARV KT AL.

deary, I,. J., and J. II. Byrne. 1986. Associative learning of the gill
and siphon withdrawal reflex in . lpl\'\ia: interneurons mediating the
unconditioned reflex. Soc. Neurosci. Ab.t. 12: 397.

Dale, N., E. R. Kandel, and S. Schachcr. 1987. Serotonin produces
long-term changes in the excitability ofAplysia sensory neurons in
culture that depend on new protein synthesis. J. Neurosci. 1: 2232-
2238.

Dash, P. K., B. lloechner, and E. R. kandel. 1990. Injection of the
cyclic AMP responsive element into the nucleus ofAplysia sensory
neurons blocks long-term facilitation. Nature 345: 718-721.

I skin. A., K. S. Garcia, and J. H. Byrne. 1989. Information storage
in the nervous system ofAplysia: specific proteins affected by serotonin
and cAMP. Proc. Nal. Aaiii. Sci. USA 86: 2458-2462.

Frost, W. N., V. F. Castellucci, R. D. Hawkins, and E. R. Kandel.
1985. Monosynaptic connections made by the sensory neurons of
the gill- and siphon-withdrawal reflex in Aplysia participate in the
storage of long-term memory for sensitization. Proc. Nat. Acad. Sci.
USA 82: 8266-8269.

Frost, W. N., G. A. Clark, and E. R. Kandel. 1988. Parallel processing
of short-term memory for sensitization in Aplysia. J. Neurobiol 19:
297-334.

Glanzman, D. I... S. L. Mackey, R. D. Hawkins, A. M. Dyke, P. E.
Lloyd, and E. R. Kandel. 1989. Depletion of serotonin in the nervous
system ofAplysia reduces the behavioral enhancement of gill with-
drawal as well as the heterosynaptic facilitation produced by tail shock.
J. Neurosci. 9: 4200-42 1 3.

Greenberg, S. M., V. F. Castellucci, H. Bayley, and J. H. Schwartz.
1987. A molecular mechanism for long-term sensitization in Aply-
sia. Nature 329: 62-65.

Hammer, M., L. J. Cleary, and J. II. Byrne. 1989. Serotonin acts in
the synaptic region of sensory neurons in Aplysia to enhance trans-
mitter release. Neurosci. Lett. 104: 235-240.

Hawkins, R. D., V. F. Castellucci, and E. R. Kandel. 1981. Interneurons
involved in mediation and modulation of gill-withdrawal reflex in
Aplysia II. Identified neurons produce heterosynaptic facilitation
contributing to behavioral sensitization. / Neurophysiol. 45: 315-
326.

Hawkins, R. D., and S. Schacher. 1989. Identified facilitator neurons
L29 and L28 are excited by cutaneous stimuli used in dishabituation,
sensitization. and classical conditioning of Aplysia. ./. Neurosci. 9:
4236-4245

Hochner, B., M. Klein, S. Schacher, and E. R. Kandel. 1986. Action-
potential duration and the modulation of transmitter release from
the sensory neurons of Aplysia in presynaptic facilitation and be-
havioral sensitization. Proc. Nat. Acad. Sci. i'SA 83: 8410-8414.

Kandel, E. R. 1979. Behavioral biology of Aplysia. W. H. Freeman,
San Francisco.

Kandel, E. R., and J. H. Schwartz. 1982. Molecular biology of learning:
modulation of transmitter release. Science 218: 433-443.

Kistler, H. B., Jr., R. D. Hawkins, J. Koester, H. W. Steinbusch, E. R.
Kandel and J. H. Schwartz. 1985. Distribution of serotonin-im-
munoreactive cell bodies and processes in the abdominal ganglion
of mature Aplysia. ./. Neurosci. 5: 72-80.

Klein. M., J. S. Camardo, and E. R. Kandel. 1982. Serotonin modulates
a new potassium current in the sensory neurons that show presynaptic
facilitation in Aplysia. Proc. Nat. Acad. Sci. USA 79: 5713-5717.

Koester, J. 1989. Chemically and electrically coupled interneurons
mediate respiratory pumping in Aplysia. J Neurophvsiol. 62: 1113-
1126.

Kupfermann, I., T. J. Carew, and E. R. Kandel. 1974. Local, reflex and
central commands controlling gill and siphon movements in Aplysia.
J Neurophysiol. 37: 996-1019.

Lo, L. T., J. H. Byrne, and L. J. Cleary. 1987. Distribution of three
modulatory transmitters within the pleural ganglion ofAplysia. Soc.
Neurosci. Abstr. 13: 1073.

I.ongley, R. D., and A. J. Longley. 1986. Serotonin immunoreactivity
of neurons in the gastropod Aplysia californica. J. Neurobiol. 17:
339-358.

Mackey, S. L., E. R. Kandel, and R. D. Hawkins. 1989. Identified
serotonergic neurons LCB1 and RCB1 in the cerebral ganglia of
Aplysia produce presynaptic facilitation of siphon sensory neurons.
J. Neurosci 9: 4227-4235.

Montarolo, P. G., P. Goelet, V. F. Castellucci, J. Morgan, E. R. Kandel,
and S. Schacher. 1986. A critical period for macromolecular syn-
thesis in long-term heterosynaptic facilitation in Aplysia. Science 234:
1249-1254.

Nazif, F. A., J. H. Byrne, and L. J. Cleary. 1991. cAMP induces long-
term morphological changes in sensory neurons of Aplysia. Brain
Res. 539: 324-327.

Ocorr, K. A., and J. II. Byrne. 1985. Membrane responses and changes
in cAMP levels in Aplysia sensory neurons produced by serotonin,
tryptamine, FMRFamide and small cardioactive peptide B (SCPB).
Neurosci. Lett 55: 1 13-1 IS.

Ocorr, K. A., and J. II. Byrne. 1986. Evidence for separate receptors
that mediate parallel effects of serotonin and small cardioactive pep-
tide B (SCPB) on adenylate cyclase in Aplysia californica. Neurosci.
Lett. 70: 283-288.

Ocorr, K. A., M. Tabata, and J. II. Byrne. 1986. Stimuli that produce
sensitization lead to elevation of cyclic AMP levels in tail sensory
neurons of Aplysia. Brain. Res. 371: 190-192.

Ono, J. K., and R. E. McCaman. 1984. Immunocytochemical local-
ization and direct assays of serotonin-containing neurons in Aplysia.
Newoscicncc 11: 549-560.

Perlman, A. J. 1979. Central and peripheral control of siphon-with-
drawal reflex in Aplysia californica. J. Neurophysiol. 42: 510-529.

Pinsker, H., I. Kupfermann, V. F. Castellucci, and E. R. Kandel.
1970. Habituation and dishabituation of the gill-withdrawal reflex
in Ap/ysia. Science 167: 1740-1742.

Pinsker, H. M., W. A. Hening, T. J. Carew, and E. R. Kandel.
1973. Long-term sensitization of a defensive withdrawal reflex in
Aplysia. Science 182: 1039-1042.

Pollock, J. D., L. Bernier, and J. S. Camardo. 1985. Serotonin and
cyclic adenosine 3':5'-monophosphate modulate the potassium current
in tail sensory neurons in the pleural ganglion ofAplysia. J. Neurosci.
5: 1862-1871.

Sacktor, T., and J. H. Schwartz. 1990. Sensitizing stimuli cause trans-
location of protein kinase C in Aplysia sensory neurons. Proc. Nat.
Acad. Sci. USA 87: 2036-2039.

Schacher, S., V. F. Castellucci, and E. R. Kandel. 1988. cAMP evokes
long-term facilitation in Aplysia sensory neurons that requires new
protein synthesis. Science 240: 1667-1669.

Scholz, K. P., and J. H. Byrne. 1987. Long-term sensitization in Aplysia:
biophysical correlates in tail sensory neurons. Science 235: 685-687.

Scholz, K. P., and J. H. Byrne. 1988. Intracellular injection of cAMP
induces a long-term reduction of neuronal K' currents. Science 240:
1664-1666.

Schwartz, J. H., L. Bernier, V. F. Castellucci, M. Palazzolo, T. Saitoh,
A. Stapleton, and E. R. Kandel. 1983. What molecular steps de-
termine the time course of the memory for short-term sensitization
in Aplysia? Cold. Spring. Harbor. Symp. Quant. Biol. 48: 8 1 1 -8 1 9.

Shuster, M. J., J. S. Camardo, S. A. Siegelbaum, and E. R. Kandel.
1985. Cyclic AMP-dependent protein kinase closes the serotonin-
sensitive K+ channels of Aplysia sensory neurons in cell-free mem-
brane patches. Naiure 313: 392-395.

NEUROMODULATION IN APLYSIA

261

Shuster, M. J.. and 8. A. Siegelbaum. 1987. Pharmacological char-
acterization of the serotonin-sensitive potassium channel of .-l/>/y.v/d
sensory neurons. J. Gen. Physiol. 90: 587-608.

Siegelbaum, S. A., J. S. Camardo, and E. R. Kandcl. 1982. Serotonin
and cAMP close single K+ channels in Aply.siu sensory neurons. Na-
ture 299:413-417.

Sweatt, J. D., A. Volterra, B. Edmonds, K. A. Karl, S. A. Siegelbaum,
and K. R. kandel. 1989. FMRFamide reverses protein phosphor-
ylation produced by 5-HT and cAMP in Aplysia sensory neurons.
Nalurc 342: 275-278.

Tritt, S. H., I. P. Lowe, and J. H. Byrne. 1983. A modification of the
glyoxylic acid induced histofluorescence technique for demonstration
of catecholamines and serotonin in tissues of Aplysia califomica.
Brain Re". 259: 159-162.

Walsh, J. P. and J. H. Byrne. 1989. Modulation of a steady-state Ca2+-
activated. K+ current in tail sensory neurons of Aplysitr role of se-
rotonin and cAMP. / Neurophysiol. 61: 32-44.

Walters, E. T., and J. H. Byrne. 1983. Associative conditioning of
single sensory neurons suggests a cellular mechanism for learning.
Science 219: 405-408.

Walters, E. T., J. H. Byrne, T. J. Carew, and E. R. Kandel.
I983a. Mechanoafferent neurons innervating tail of. -Iplysia I. Re-

sponse properties and synaptic connections. J Neurophysiol 50:
1522-1542.
Walters, E. T., J. II. Byrne, T. J. Carew, and E. R. Kandel.

1983b. Mechanoafferent neurons innervating tail of Afily.ua. II. Mod-
ulation by sensitizing stimulation. ./ Nciirophysml. 50: 1543-1559.

Walters, E. T., and M. I . Eriekson. 1986. Directional control and the
functional organization of defensive responses in Aplysia. J Comp
Physiol. A 159:339-351.

Weiss, K. R., U. T. Koch, J. Koester, S. C. Rosen, and I. Kupfermann.
1982. The role of arousal in modulating feeding behavior ot'Aplysin:
neural and behavioral studies. Pp. 25-57 in The Neural BUMS <>/
{•'ceiling and Reward. B. G. Hoebel and D. Novin, ed. Haer Institute,
New Brunswick, ME.

Zhang, Z. S., L. J. Cleary, 1). M. Marshak, and J. H. Byrne.
1988. Serotonergic varicosities make apparent synaptic contacts
with pleural sensory neurons of Aplysia. Soc. Neurosci. Ahxir 14:
841.

/wartjes, R. E., M. I . Crow, J. II. Byrne, and A. Eskin. 1990. Serotonin
regulates the expression of mRNA in the pleural-pedal ganglia of
Aplyxia. as determined by in viirn translation. Soc. Neurosci. Ahslr.
16: 596.

Reference: Biol. Bull. 180: 262-268. (April,

Studies of Behavioral State in Aplysia

IRVING KUPFERMANN, THOMAS TEYKE, STEVEN C. ROSEN,
AND KLAUDIUSZ R. WEISS*

Center for Neurobialogy and Behavior, Columbia University College of Physicians and Surgeons:

New York State Psychiatric Institute: and * Department of Physiology and Biophysics.

Aft. Sinai School of Medicine, New York, New York

Abstract. This paper reviews a series of studies on the
neural organization and the cellular mechanisms under-
lying behavioral states; in these studies, feeding behavior
in Aplysia was used as a model system. Feeding in Aplysia
has similarities to motivated behaviors in other animals
and is modulated by a number of interesting state vari-
ables, including arousal. Food-induced arousal manifests
itself in two categories of feeding behavior: ( 1 ) appetitive
responses (e.g.. head-up feeding posture and directed head
turning), which orient the animal to potential goal objects
such as food; and (2) consummatory responses (biting,
swallowing), which obtain the goal object. The consum-
matory responses are rhythmic and relatively stereotyped,
whereas the appetitive responses are highly variable. Our
evidence suggests that one consummatory response, bit-
ing, appears to be controlled by command elements in
the cerebral-ganglion, such as neuron CBI-2, which are
capable of driving the behavior. One component of the
appetitive behavior, head lifting, may be controlled (at
least in part) by another cerebral neuron, C-PR. C-PR,
however, affects numerous systems in the animal, but all
the systems affected seem to be involved in the food-in-
duced arousal state of the animal. We postulate that C-
PR is, in some ways, analogous to command neurons that
evoke behaviors. The C-PR, however, not only evokes a
behavior, but also evokes a central motive state which
aids in insuring that behavior is efficiently expressed.

Introduction

Mollusks have long been used for studies that are de-
signed to investigate general neurobiological principles
rather than the details of a single species. One important
advantage of mollusks is the large size of their neurons.

Received 7 August 1990; accepted 6 November 1990.

For many years, studies that were difficult or impossible
in vertebrates could be approached by investigating the
squid giant axon and the large somata of gastropod neu-
rons. In recent years, the use of cell culture, brain slices,
and other methodologies has made it possible to do many
types of cellular studies on vertebrate neurons that could
previously be done only in mollusks. However, it is still
very difficult to study the integrative functions of the ver-
tebrate nervous system and to relate cellular processes to
behavior. For this reason, the presence of a relatively few
neurons in gastropod mollusks has assumed increased
importance.

We have been studying the marine mollusk Aplysia in
order to understand the neural organization and the cel-
lular mechanisms underlying behavioral states. We have
concentrated on feeding behavior because our early studies
indicated that the feeding responses of these animals are
modulated by a number of interesting state variables, in-
cluding arousal and satiation (Kupfermann el a/., 1982;
Susswein et ai, 1978). This paper is a review of our work.
It emphasizes studies of the appetitive aspects of feeding,
and is not meant to be a general review of feeding in
Aplysia.

Feeding Behavior in Aplysia has Similarities to
Motivated Behaviors in Other Animals

To provide themselves with adequate nutrients, Aplysia
has many of the same problems faced by most other an-
imals. They must detect and locate appropriate food
sources. They must approach the food and orient it to the
buccal orifice. They must then bite and swallow the food.
Finally, when a sufficient amount has been consumed,
they need to stop feeding. These operations must all be
carried out in a manner that is efficient in time and energy
expenditure. One of the means by which the animals im-

262

STUDIES OF BEHAVIORAL STATE IN APLYSIA

263

Figure 1. Aplysia in the feeding posture. In this position the animal
shows directed turning responses to seaweed applied to the head.

prove the efficiency of their behavior is by regulating it
according to particular internal states. These internal states
are modulated by external and internal stimuli and by an
internal endogenous process associated with a circadian
activity rhythm. In higher animals, the constellation of
state variables that regulate feeding are termed "hunger,"
and by analogy, a hunger-like state also appears to regulate
feeding in Aplysia. As in higher animals, feeding in Aplysia
is greatly potentiated by pre-exposing the animals to food;
i.e.. the animal exhibits incentive motivation. When a
quiescent Aplysia is first stimulated with seaweed, it be-
comes activated after a relatively long delay (up to a min-

Figure 2. Vectors indicating the magnitude and direction that the
head turns in response to tactile stimuli briefly presented (open loop) to
different points on the rhinophores and tentacles. The movements turn
the head in the direction of the stimulus. In the open loop condition the
animal greatly overshoots the stimulus. If, however, the stimulus is
maintained in place (closed loop), when the animal begins the response,
the movement is represented by the indicated vectors, but as the animal
turns, the response progressively decreases in magnitude so that the mouth
comes to be accurately centered over the stimulus. Data from Teyke
el al (1990b)

too-

80-

*\ °\i Y \i
\ H ' °\l

60-
40-

\T
'\I *

20-
n .

?^\_, °-°

seaweed
tactile

01 23456789 10
STIMULUS NUMBER

Figure 3. Turning angle evoked by repeated seaweed (open circles)
or tactile (filled circles) stimuli. The animals (n = 5) were first induced
into the feeding posture by means of seaweed. They were then stimulated
at a locus 10° from the mouth, either with a purely tactile stimulus, or
with seaweed. The stimulus was repeated every 10 s ( 10 successive stimuli;
3 series each). Final turning angles of the responses are shown as the
percent of the final angle of the first response (means ± S.E.M.). Note
the marked decline in the magnitude of the turning response evoked by
repeated tactile stimulation and the relatively steady response magnitude
upon repeated seaweed stimulation. Data from Teyke et al. (1990b).

ute). We refer to this activated state as "food-induced
arousal."

Food-induced arousal in Aplysia manifests itself in at
least two stages. First, appetitive behaviors (the orienting
phase of motivated behaviors) are affected; second, con-
summatory responses are modified. Initial contact with
food evokes a defensive withdrawal reflex of the head.
The fast phase of this reflex appears to be controlled by
the cerebral Bn neurons (Teyke el al, 1989), which receive
powerful tactile input, and which evoke withdrawal
movements of the head and tentacles. After the initial
defensive response, the animal ceases to withdraw. The
response appears to be habituated, but unlike other forms
of habituation in Aplysia (Castellucci et al.. 1 970, see also
Fig. 3), the response decrement occurs very rapidly, typ-
ically following just a single application of the stimulus.
A subsequent brief food stimulus elicits an orienting re-
sponse, instead of eliciting withdrawal. The animal gets
into a characteristic feeding posture in which the posterior
part of the foot is attached to the substrate, and the neck,
head, and anterior part of the foot are lifted (Fig. 1). In
addition, there are signs of "autonomic" arousal, such as
an increase in blood pressure and heart rate (Koch et al..
1984). The feeding posture is maintained even when the
food is removed, indicating that the appetitive arousal
has a "memory" component. From the feeding posture
the animal can readily move its head toward a source of
food. When the tentacles of the food-aroused animal make
physical contact with food (seaweed), the animal moves
its head so as to direct its mouth towards the stimulus
(Fig. 2). For a brief (open loop) stimulus within the re-
ceptive field, the animal greatly overshoots the food, and
the amount of overshoot is proportional to the angular

264
BMN

B16

C12

I. KUPFERMANN F.T Al.

U ILL IlILllliLL
JJJIJJJJIIJUJU

Figure 4. Example of the motor program dnven by CBI-2. CBI-2 was fired by a constant depolarizing
current (dark horizontal line). The rhythmic program incorporated neurons in the cerebral and buccal
ganglia. The buccal program is reflected in the activity of an identified ARC muscle motor neuron. B16.
Another buccal motor neuron. BMN. illustrates that the program is present in numerous other buccal
neurons. CI2 is a cerebral ganglion neuron that controls movements of the lips, and it is one of several
cerebral neurons that is recruited by the buccal program that is driven by CBI-2. CBI-2 also shows periodic
synaptic input driven by the buccal program. Note that when CBI-2 stops firing, the program briefly persists
and then terminates. The data are from Rosen el al. ( 1 987. 1 988).

10 i

JlO mV

distance of the stimulus from the mouth. If, however, the
stimulus is maintained in position so that it provides con-
tinuous feedback during the movement (closed loop), the
food is accurately centered over the mouth (Teyke et al.,
1990b). Seaweed provides the animal with two distinct
types of stimuli: tactile and chemical. Surprisingly, the
stimulus that results in the animal turning toward the
food is the tactile component. A purely chemical stimulus,
provided by an aqueous extract of seaweed, is not very
effective in eliciting turning. On the other hand, if the
animal is first aroused with a chemical stimulus, a purely
tactile stimulus (provided by a glass rod) very effectively
evokes a turning response. If, however, the tactile stimulus
is repeated without intermittent chemical stimulation, the
turning response habituates until no response at all is
evoked (Fig. 3). Thus, the chemical component of the
seaweed maintains the arousal level of the animal, while
the tactile component directs the response.

When the animal turns toward the stimulus, contact
with food to the region immediately around the mouth
(perioral zone) initiates consummatory behaviors and a
new set of arousal responses. The consummatory arousal
is characterized by a progressive build-up of the rate and
magnitude of the rhythmic biting response that occurs
when food touches the perioral zone ( Kupfermann, 1974;
Weiss et al., 1982). Whereas the appetitive feeding re-
sponses are highly variable, the consummatory biting re-
sponse is more stereotyped, although it consists of several
components (Kupfermann, 1974; Weiss et al.. 1986): (1)
There is a forward movement (cocking) of the whole buc-

cal mass. The forward position is maintained during the
whole meal. (2) The whole buccal mass undergoes forward
and backward movements. These movements occur on
a background of the maintained forward movement. (3)
The radula rotates forward and backward. (4) The radula
halves open and close. The latter two movements cause
the food to be grasped and deposited into the buccal cavity.
The relatively small backward movement, which deposits
the food in the buccal cavity during biting behavior, can
be distinguished from a larger backward movement
(swallowing) that is triggered by the presence of food in
the buccal cavity, and which results in the food being
moved into the esophagus (Kupfermann. 1974). Biting
movements, which are elicited by food contacting the
perioral zone, thus consist of a large forward component
of the radula. followed by a relatively small backward
movement. Swallowing, which is elicited by food in the
buccal cavity, consists of a relatively small forward move-
ment and a large backward movement. The swallowing
movements are associated with an inhibition of the biting
movements; i.e.. as long as food is present in the buccal
cavity, stimulation of the perioral zone never elicits a large
forward movement of the radula.

Biting Responses are Elicited by the Activity of
Individual Neurons Located in the Cerebral Ganglion

In a number of species, including gastropod mollusks,
stereotyped responses are elicited by the activity of indi-
vidual cells or small groups of cells (Kupfermann and

STUDIES OF BEHAVIORAL STATE IN APLYSIA

265

A Postural System

B Consummatory System

CBI-2

MCC

CPR

Cardiovascular System D Defensive Systems

L10

Bncell

OPR

CPR

Figure 5. The various effects of firing C-PR on different systems
associated with food-induced arousal. For each experiment, C-PR was
intracellularly stimulated at 20 Hz for 5 s. For illustrative purposes, mul-
tiple follower cells of the C-PR are shown for each part of the figure, but
the data for each trace were obtained in separate experiments. (A) Ex-
amples of the effects of firing C-PR on different pedal ganglion neurons,
that may be part of the postural control system. (B) Effects of C-PR on
cerebral ganglion neurons that control consummatory feeding responses
(biting command element, CBI-2, and the modulatory neuron, meta-
cerebral cell, MCC). (C) Effects of C-PR on abdominal ganglion neurons
that control the cardiovascular system (command element LIO. heart
exciter RBHE, and vasoconstrictor LBVC. (D) Effects of C-PR on various
neurons that participate in defensive responses [head withdrawal neuron,
Bn cell (Teyke el al.. 1989); gill withdrawal motor neurons, L7; defensive
secretion neurons, R2 and PL1], Calibration: 2 s, 20 mV, except 5 mV
for cells R2 and PL1 in part D. Data are from Teyke cl al. (1990a).

Weiss, 1978; Gillette el al, 1982; McClellan, 1986; Ben-
jamin and Elliott, 1989; McCrohan and Kyriakides, 1989;
Delaney and Gelperin, 1990). To determine the critical
control elements for the consummatory phase of feeding
in Aplyxia, we back-filled the cerebral-buccal connectives
and located a population of cerebral neurons that send
their axons to the buccal ganglion. Several of these cells
had been previously identified, including the serotonergic
metacerebral cells (MCCs) and ICBM mechanosensory
cells (Rosen et al.. 1989a,b). In addition, two small pop-
ulations of cells were found in anterior and lateral posi-
tions. Firing of one of the cells (cerebral to buccal inter-
neuron two, or CBI-2) within the anterior cluster, pro-

duced a robust and reliable rhythmic motor program of
the buccal ganglion (Fig. 4) (Rosen el al.. 1987). CBI-2
receives chemosensory input from the perioral zone. In
addition, when it elicits a buccal motor program it receives
rhythmic synaptic input from the buccal ganglion, and
thus it fires in phase with the buccal motor program. If,
however, the synaptic feedback from the buccal ganglion
is blocked by placing the cerebral ganglion in seawater
containing cobalt ions, the firing of CBI-2 still evokes
rhythmic activity in the buccal ganglion, in the absence
of rhythmic activity in CBI-2 (Rosen et al., 1988). For a
discussion of recent work on the central pattern generating
circuitry intrinsic to the buccal ganglion of Aplysia, see
Kirk (1989), Nagahama and Takata (1989), and Susswein
and Byrne (1988).

Using a semi-intact preparation, we found that the firing
of CBI-2 can evoke rhythmic movements of the buccal
mass and radula, and the movements are similar to the
repetitive biting responses seen in the intact animal. The
responses do not resemble swallowing or rejection. The
firing of two other cerebral to buccal interneurons also
evokes coordinated buccal ganglion activity, but the motor
programs are different for each of the CBIs. Thus we hy-
pothesize that the CBIs in Aplysia, as in other gastropods
(Gillette et al., 1982; Benjamin and Elliott, 1989; Mc-
Crohan and Kyriakides, 1989; Delaney and Gelperin,
1990), may constitute a command system, the conjoint
activity of which drives consummatory feeding responses.

Activity of an Identified Cerebral Neuron Appears to
Elicit Elements of Appetitive Arousal

Although stereotyped consummatory responses are
driven by a relatively few command-like elements, it is
difficult to imagine how the highly variable responses that
constitute appetitive behavior could be similarly driven
by a small number of neurons. Nevertheless, we set out
to determine whether the nervous system contains neu-
rons that can evoke appetitive feeding behavior. Backfills
of the cerebral-pedal connectives revealed a small subset
of cerebral ganglion neurons that send their axons to the
pedal or pleural ganglia (Teyke et al., 1990a). The firing
of these neurons revealed a single (bilateral) cerebral neu-
ron that can influence the activity of numerous neurons
in the abdominal, pedal, and cerebral ganglia. We termed
this neuron the cerebral to pedal regulator [to avoid con-
fusion with the caudal photoreceptor (CPR) interneuron
of crayfish, we abbreviate this neuron C-PR, although
previously we did not use the hyphen].

The pedal ganglion in particular contains a large num-
ber of neurons that are excited by C-PR (Fig. 5). A smaller
number of pedal ganglion neurons are inhibited by C-
PR. Each neuron that is affected by C-PR activity receives
input following the firing of either the left or right C-PR,

266

I. KUPFERMANN ET AL

OPR

MCC

Figure 6. Example of a prolonged excitatory response in C-PR and the MCC to a brief seaweed stimulus.
Calibration: 5 s, 20 mV. Data from Teyke et al. (1990a).

suggesting that C-PR is probably not directly involved in
the directed head turning response, which is very strongly
lateralized. Nevertheless, head turning does not occur un-
less the animal is first aroused, so that C-PR activity may
enable head turning. Some of the effects of C-PR are
monosynaptic, whereas others are mediated by interneu-
rons. Firing of many of the pedal cells that are affected
by the activity of C-PR causes the muscles of the anterior-
dorsal region of the neck to contract (Teyke et al.. 1990a),
which suggests that C-PR may evoke movements that
cause the head to be lifted into the feeding posture. Con-
sistent with a role of C-PR in eliciting head-lifting in re-
sponse to food, we found that seaweed applied to the ten-
tacles evokes strong activity in C-PR (Fig. 6). Furthermore,
preliminary studies involving extracellular recordings
from the cerebral-pedal connectives, support the idea that
C-PR is active just before and during the time that the

TIME (SEC)

Figure 7. Sequence of mouth openings and closings during seven
repetitive biting responses, in two animals that have had bilateral chronic
lesions (protease injected) of the MCCs. The sequence is based on a
videotaped analysis of biting. Width of the mouth opening is expressed
as a percentage of the maximum. A. An example, illustrating "stuck"
radula in a MCC lesioned animal, in which the radula stays protracted
for an abnormally long duration. B. Responses of a normal animal (Bl ),
and of the same animal (B2) following lesion of the MCCs. Data from
Rosen et al. (I989a).

animal lifts its head into the feeding posture (Teyke et al.,
1990c). Thus, the total complex of appetitive feeding re-
sponses may consist of two components: a stereotyped
postural head-lifting response and a more varied directed
turning response.

We have formulated a simple neural model (Teyke et
a/.. 1990b) whose input-output functions are similar to
the behavioral results concerning the directed head turning
component of appetitive feeding behavior. The model is
based on reflex circuits and does not contain command
elements. By contrast, the head lifting response may be
importantly controlled by a small number of neurons,
such as C-PR, that have command-like properties. C-PR,
however, affects responses other than head lifting. In fact,

Feeding 1
Latency 1

3osture
sees)

ter-Bite Interval
(sees)

f^n K
&JL

1CC 30
esion

T W

I Cell
.esion

.A.

FT% K
i&JS

ICC 2.0
ihom

• • • •

i • ••

• • •<
• • • i

•

1.0

™

tr&a
y££j
i

-10

LiJ -I

-1.0

-20

-2.0

Figure 8. MCC lesion (n = 6), B cell control lesion (n = 7), and dye
injection MCC control (n = 6) group mean difference scores (postop-
erative overall mean, minus preoperative scores, ± SEMs) for latency to
assume feeding posture (A) and for interbite interval (B). Bites were
elicited by continually stimulating the lips with seaweed, without allowing
the animal to obtain the food. Data from Rosen et al. (1989a).

STUDIES OF BEHAVIORAL STATE IN APLYSIA

267

LJ
l/l

20--

15--

3 10
O

5--

MCC Lesion, Pre-Op
* • MCC Lesion, Post-Op

D D Control, Pre-Op

Q Q Control, Post-Op

012345678910111213
SWALLOW NUMBER

Figure 9. MCC lesion and control groups mean interswallow inter-
vals, measured dunng ingestion of strips of seaweed. No significant group
differences were found. Data from Rosen ct ill ( 1989a).

we found that C-PR activity affects neurons involved in
three other types of responses: defensive withdrawal re-
flexes (Fig. 5D), consummatory biting (Fig. 5B), and car-
diovascular responses (Fig. 5C). The neurons involved in
defensive responses were inhibited by the firing of C-PR.
and in semi-intact preparations we showed that the firing
of head withdrawal neurons in response to a strong tactile
stimulus to the head was reduced when the C-PR neuron
was permitted to fire. The rapid depression of withdrawal
responses following contact with seaweed may therefore
be due either in part, or wholly, to an active inhibition,
rather than to low frequency depression, as appears to be
the case for habituation of the gill and siphon reflex to
tactile stimulation (Castellucci el al, 1970).

Firing of C-PR evokes complex mixtures of excitatory
and inhibitory synaptic responses in abdominal ganglion
neurons controlling the heart and blood vessels (Fig. 5C).
These effects could contribute to aspects of cardiovascular
responses that occur during food-induced arousal.

The cerebral ganglion neurons involved in consum-
matory behaviors generally receive pure excitation when
C-PR is fired. These neurons include command-like ele-
ments for biting (CBI-2) and the metacerebral cells
(MCCs). The MCCs modulate the muscles and neurons
that effectuate biting and account, in part, for the build-
up of the speed and magnitude of successive bites, which
occurs during consummatory arousal (Rosen el ai,
1989a). In contrast to the C-PR, the modulatory effects
of the MCC are very restricted. It only modulates con-
summatory responses, and only the bite component. For
example, if the MCCs are destroyed, there is no change
in the capacity to elicit the feeding posture (Fig. 8) (Rosen
et al., 1989a), but there is an increase in the bite latency
and inter-bite intervals (Fig. 7, 8B). Inter-swallow intervals
are unchanged (Fig. 9).

When C-PR is fired at physiological rates, its excitatory
effect on neurons involved in biting responses is never
strong enough to drive the neurons at a rate sufficient to
evoke biting. C-PR appears to function to increase the
excitability of these neurons without directly driving con-
summatory responses.

By cutting various connectives we could localize the
ganglia that contain the interneurons that produce the
effects of C-PR on the various non-postural systems. We
found that all of these effects are mediated by the activity
of the pedal-pleural ganglia. It may be significant that
these ganglia mediate the postural responses associated
with food arousal. Thus appetitive arousal may involve a
primary effect on a postural system, which, in turn, mod-
ulates the activity of the numerous other systems that will
eventually come into play during feeding. In the vertebrate
brain, indeed, neurons thought to be concerned with reg-
ulation of consciousness and arousal are concentrated in
the brain stem in regions intimately involved with postural
regulation (Hobson and Brazier, 1980). Because virtually
all behaviors require a particular posture for their exe-
cution, the postural neural system may serve a primary
role in arousal in highly diverse species.

Some of the effects of C-PR, such as those on the ele-
ments of consummatory responses, could enhance these
responses. Other effects, such as those on Bn neurons,
may suppress responses that are incompatible with feeding
behavior. We postulate that C-PR is, in some ways, anal-
ogous to command neurons, which evoke behaviors. The
C-PR, however, not only evokes a behavior (head lifting),
but also evokes a central motive state that aids in insuring
that behavior is efficiently expressed. A behavioral action
such as feeding is made up of a number of different be-
havioral acts (e.g., head lifting, biting, swallowing). Thus,
a consideration of the ways in which behavioral efficiency
is maximized raises two fundamental questions. First, how
are multiple responses of the organism coordinated with
one another, and second, how are the individual behav-
ioral acts which make up a behavioral action modulated
so as to optimize their speed and minimize energy ex-
penditure? Our evidence suggests that one means of co-
ordinating diverse responses directed toward a single goal
is to affect diverse neuronal systems through the activity
of a relatively few neuronal elements. Data presented
elsewhere indicate that maximization of the efficiency of
individual responses is accomplished, in part, by the ac-
tivity of subordinate specialized neurons such as the MCC
(Weiss et al.. 1978; Rosen et al.. 1989a). In addition, in-
dividual responses may be regulated by neuromodulators
that occur as cotransmitters in motorneurons innervating
the muscles that effectuate feeding responses (Lloyd et a!.,
1985; Cropper et al., 1987a,b, 1988, 1990). The motor
neurons are subordinate to the modulatory effects of the
MCC, which, in turn, is modulated by C-PR. Thus the

268

I KUPFERMANN ET AL.

final motor activity appears to be regulated by modulatory
neurons of progressively higher order. We are beginning
to reduce the elusive concept of motivational state to ex-
planations in terms of the actions of ordinary neural
mechanisms, operating in networks of appropriately in-
terconnected neurons.

Literature Cited

Benjamin, P. R., and C. J. H. Elliott. 1989. Snail feeding oscillator:
the central pattern generator and its control by modulatory inter-
neurons. Pp. 173-214 in Neuronal and Cellular Oscillators. J. W.
Jacklet, ed. Marcel Dekker. Inc., New York and Basel.

Castellucci, V., H. Pinsker, I. Kupfermann, and E. R. kandel.
1970. Neuronal mechanisms of habituation and dishabituation of
the gill-withdrawal reflex in Aplysia. Science 167: 1745-1748.

Cropper, E. C., R. Tenenbaum, M. A. Gawinowicz Kolks, I. Kupfermann,
and K. R. Weiss. 1987a. Myomodulin: a bioactive neuropeptide
present in an identified cholinergic buccal motor neuron ofAplysia.
Proc. Nat/. Acad. Sci. USA 84: 5483-5486.

Cropper, E. C., P. E. Lloyd, VV. Reed, R. Tenenbaum, I. Kupfermann,
and K. R. Weiss. 1987b. Multiple neuropeptides in cholinergic
motor neurons of Aplysia: evidence for modulation intrinsic to the
motor circuit. Proc. Nail Acad. Sci. USA 84: 3486-3490.

Cropper, E. C., M. W. Miller, R. Tenenbaum, M. A. Gawinowicz Kolks,
I. Kupfermann, and K. R. Weiss. 1988. Structure and action of
buccalin: a modulatory neuropeptide localized to an identified small
cardioactive peptide-containing cholinergic motor neuron ofAplysia
californica. Proc. Nail. Acad. Sci. USA 85: 6 1 77-6 181.

Cropper, E. C., M. W. Miller, F. S. Vilim, R. Tenenbaum, I. Kupfermann,
and K. R. Weiss. 1990. Buccalin is present in the cholinergic motor
neuron B16 ofAplysia and it depresses accessory radula closer muscle
contractions evoked by stimulation of B16. Brain Res 512: 175-
179.

Delaney, K., and A. Gelperin. 1990. Cerebral interneurons controlling
fictive feeding in Umax maximus. I. Anatomy and criteria for re-
identification. J. Comp. Physiol. [A] 166: 297-310.

Gillette, R., M. P. Kovac, and VV. J. Davis. 1982. Control of feeding
motor output by paracerebral neurons in brain of Pleurobranchaea
californica. J Neurophysiol. 47: 885-908.

Hobson, J. A., and M. A. B. Brazier, eds. 1980. The Reticular For-
mation Revisited. Specifying Functionjbr a Nonspecific Svstein. Raven
Press, New York. 552 pp.

Kirk, M. D. 1989. Premotor neurons in the feeding system of Aplvsia
californica. J. Neurobiol. 20: 497-512.

Koch, U. T., J. Koester, and K. R. Weiss. 1984. Neuronal mediation
of cardiovascular effects of food arousal in Aplvsia. J Neurophvsiol.
51: 126-135.

Kupfermann, I. 1974. Feeding behavior in Aplysia: a simple system for
the study of motivation. Behav. Biol. 10: 1-26.

Kupfermann, I., L. Shkolnik, and K. R. Weiss. 1982. Modulatory syn-
aptic actions of neurotransmitters in circuits controlling feeding in
Aplysia. Pp. 517-531 in Cytochemical Methods in Neuroanatomy.
Alan R. Liss. Inc., New York.

Kupfermann, I., and K. R. Weiss. 1978. The command neuron concept.
Behav. Brain Sci. 1: 3-10.

Lloyd, P. E., A. C. Mahon, I. Kupfermann, J. L. Cohen, R. H. Scheller,
and K. R. Weiss. 1985. Biochemical and immunocytological lo-

calization of molluscan small cardioactive peptides in the nervous
system ofAplysia californica. J. Neurosci. 5: 1851-1861.

McClellan, A. D. 1986. Command systems for initiating locomotion
in fish and amphibians: parallels to initiation systems in mammals.
Pp. 3-20 in Neurohiology of I 'ertehrale Locomotion. S. Grillner, R.
Herman, P. Stein, D. Stuart, eds. MacMillan Press, London.

McCrohan, C. R., and M. A. Kyriakides. 1989. Cerebral interneurones
controlling feeding motor output in the snail Lvmnaea stagnalis. J.
Exp. Biol. 147: 361-374.

Nagahama, T., and M. Takata. 1990. Neural mechanism generating
firing patterns in jaw motoneurons during the food-induced response
in Aplysia kurodai II. Functional role of premotor neurons on gen-
eration of firing patterns in motoneurons. ./. Comp. Plivsiol. [A] 166:
277-286.

Rosen, S. C., M. W. Miller, K. R. Weiss, and I. Kupfermann.

1987. Control of buccal motor programs in Aplysia by identified
neurons in the cerebral ganglion. Soc. Neurosci. Abs. 13: 1060.

Rosen, S. C., M. W. Miller, K. R. Weiss, and I. Kupfermann.

1988. Activity of CBI-2 ofAplysia elicits biting-like responses. Soc.
Neurosci. Abs. 14: 608.

Rosen, S. C., K. R. Weiss, R. S. Goldstein, and I. Kupfermann.
1989a. The role of a modulatory neuron in feeding and satiation
in Aplysia: effects of lesioning of the serotonergic metacerebral cells.
/ Neurosci. 9: 1562-1578.

Rosen, S. C., A. J. Susswein, E. C. Cropper, K. R. Weiss, and I. Kup-
fermann. 1989b. Selective modulation of spike duration by serotonin
and the neuropeptides. FMRFamide. SCPB. buccalin and myomod-
ulin in different classes of mechanoafferent neurons in the cerebral
ganglion ofAplysia. J. Neurosci. 9: 390-402.

Susswein, A. J., K. R. Weiss, and I. Kupfermann. 1978. The effects of
food arousal on the latency of biting in Aplysia. J. Comp. Psvchol.
123: 31-41.

Susswein, A. J., and J. H. Byrne. 1988. Identification and character-
ization of neurons initiating patterned neural activity in the buccal
ganglion ofAplysia. J. Neurosci. 8: 2049-2061.

Teyke, T., K. R. Weiss, and I. Kupfermann. 1989. A subpopulation of
cerebral B cluster neurones ofAplysia californica is involved in de-
fensive head withdrawal but not appetitive head movements. J. Exp.
Biol. 147: 1-20.

Teyke, T., K. R. Weiss, and 1. Kupfermann. 1990a. In vivo firing pattern
of the food arousal neuron CPR in Aplvsia. Soc. Neurosci. Abs. 16:
627.

Teyke, T., K. R. Weiss, and I. Kupfermann. 1990b. Appetitive feeding
behavior ofAplysia: behavioral and neural analysis of directed head
turning. J. Neurosci. 10: 3922-3934.

Teyke, T., K. R. Weiss, and I. Kupfermann. 1990c. An identified neuron
(CPR) evokes neuronal responses reflecting food arousal in Aplysia.
Science 247: 85-87.

Weiss, K. R., J. L. Cohen, and I. Kupfermann. 1978. Modulatory con-
trol of buccal musculature by a serotonergic neuron (metacerebral
cell) in Aplysia. J Neurophysiol. 41: 181-203.

Weiss, K. R., II. T. Koch, J. Koester, S. C. Rosen, and I. Kupfermann.
1982. The role of arousal in modulating feeding behavior in Aplysia:
neural and behavioral studies. Pp. 25-57 in The Neural Basis of
Feeding and Reward. B. G. Hoebel. and D. Novin. eds. Haer Institute.
Brunswick, ME.

Weiss, K. R., H. J. Chiel, U. Koch, and I. Kupfermann. 1986. Activity
of an identified histaminergic neuron, and its possible role in arousal
of feeding behavior in semi-intact Aplysia. J Neurosci. 6: 2403-2415.

Reference: Bin! Bull 180: 269-275. (April,

A Comparison of Bursting Neurons in Aplysia

A. ALEVIZOS1 \ M. SKELTON1, K. R. WEISS1-, AND J. KOESTER1 2

1 ] Center for Newobiology and Behavior, - Department of Psychiatry, and ' Department of Physiology

and Cellular Biophysics, College of Physicians and Surgeons, Columbia University.

'l22 \V. 16S St.. New York. New York 10032

Abstract. Five types of bursting neurons have been de-
scribed in Aplysia: three types of individual bursters — the
LUQ cells, L10, and R15, plus two types of population
bursters — the bag cells and the R25/L25 cells. Individual
bursters can burst without any synaptic input, while bursts
generated by the population bursters are shaped largely
by their synaptic interactions. In this paper we review
what is known about the burst mechanisms of these five
classes of neurons and attempt to relate them to the roles
of the five cell types in the control of autonomic function.

Introduction

Molluscan neurons that have endogenous burst gen-
erating capabilities are useful experimental preparations
for the study of burst generation and modulation. This is
particularly true for Aplysia. Because the neural circuitry
of Aplysia has been studied extensively, one can attempt
to relate the functional properties of bursting neurons to
their roles in the control of behavior, and to begin a com-
parative study of different types of bursters within the
same organism. In this paper we will compare and contrast
the burst mechanisms and functional roles of five different
types of bursting neurons, all of which are found in the
abdominal ganglion, and all of which have unique prop-
erties: the bag cells, the R25/L25 cells, cell L10, the LUQ
cells, and cell R15. Particular attention is given to R15,
which has been the focus of our recent studies. The in-
teractions between these five classes of bursting cells, as
well as some of their outputs, are shown schematically in
Figure 1.

The Bag Cells

The neuroendocrine bag cells consist of two symmet-
rical clusters of about 400 neurosecretory cells each, which

Received 22 August 1990; accepted 6 November 1990.

are strongly coupled to one another electrically. They have
only one mode of firing — a synchronous population burst
in all 800 cells that lasts approximately 1 5-30 min (Kup-
fermann and Kandel, 1970). This burst is necessary and
sufficient to trigger normal egg laying behavior (Pinsker
and Dudek, 1977; Dudek et a/.. 1979). Oviposition is a
complex, stereotyped behavior that lasts from one to a
few hours and typically occurs at an interval of one or
more days (Cobbs and Pinsker, 1982; Ferguson et al.,
1989). The physiological stimulus that triggers a popu-
lation burst in the bag cells is unknown, but when the bag
cells are excited experimentally, the intraburst firing fre-
quency and the duration of the burst are independent of
the intensity of the triggering stimulus (Kupfermann and
Kandel, 1970). This all-or-none burst triggers the release
of a dose of egg-laying hormone into the circulatory sys-
tem. This hormone then initiates the release of mature
oocytes from the ovotestis (Dudek et al.. 1980; Rothman
et al.. 1983b). The population burst has an exceptionally
long refractory period, lasting on the order of 18-24
h. which limits the rate of occurrence of egg laying
(Kaczmarek and Kauer, 1983).

The all-or-none nature of the bag cell burst results from
a positive feedback, reverberatory interaction within the
population. In addition to releasing egg laying hormone,
the bag cells also release three neuropeptides, a-, £-, and
7-bag cell peptides, which are autoexcitatory (Rothman
et al.. 1983a; Brown and Mayeri, 1989). Even weak ex-
citation of the bag cells can lead to an all-or-none burst,
as the cells excite one-another by these slow, chemically
mediated interactions. Not only do these peptides depo-
larize the bag cells, they also down-regulate voltage-sen-
sitive K+ channels and up-regulate Ca++ channels, ren-
dering the cells more excitable and resulting in enhanced
spike duration. These effects contribute to prolonging the
burst, thus ensuring that a suprathreshold dose of egg-

269

270

A. ALEVIZOS ET AL

Figure 1. Summary of the interactions between bursting cells in the
abdominal ganglion ofAplysia. The dashed lines represent indirect con-
nections. The bag cells. L10, and the R25/L25 cells are known to make
several other connections to neurons that are not shown here for simplicity
(Koester and Kandel, 1977; Mayen and Rothman. 1985; Segal and
Koester. 1982).

laying hormone is released into the blood. These effects
on ion channels are apparently mediated in part by ac-
tivation of A-kinase and C-kinase (Strong and Kaczmarek,
1986; Strong et al, 1987; De Riemer et ai. 1985; Conn
et al., 1988). An initial decrease in the levels of cyclic
AMP, followed at a longer latency by a decrease in sen-
sitivity to cyclic AMP, contribute to termination of the
burst as well as to the refractory period that follows the
burst (Kauer and Kaczmarek, 1985).

The R25/L25 Neurons

The R25 and L25 cells are two interconnected clusters
of approximately 15 cells each, which act as trigger cells
for respiratory pumping. They connect directly to the
motoneurons that drive the behavior. A population burst
in the R25/L25 network is necessary and probably suffi-
cient for triggering the complete behavior (Byrne, 1983;
Koester, 1989). Like egg laying, respiratory pumping often
occurs episodically and in an all-or-none fashion (Pinsker
etal.. 1 970; Eberly and Pinsker, 1984). Unlike egg laying,
each episode of respiratory pumping is brief, consisting
of synchronous contractions of the mantle organs accom-
panied by heart inhibition (Pinsker et al.. 1970; Byrne
and Koester, 1978). The motor effects typically last only
5-10 s. Individual episodes can occur spontaneously or
in response to tactile or noxious stimuli (Pinsker et al.,
1970; Walters and Erickson, 1986). Respiratory pumping
can also occur repetitively — either in a stationary rhythm
with a period of a few minutes (unpub. obs.) or in a de-
celerating "seizure" pattern (Kanz and Quast, 1990).
These repetitive episodes of respiratory pumping can oc-

cur spontaneously or in response to various environmental
stimuli (Eberly et al.. 1981;Croll, 1985; Kanz and Quast.
1990). The functional significance of respiratory pumping
appears to vary with the context in which it occurs. It has
been hypothesized that respiratory pumping may function
to enhance defensive withdrawal (Pinsker et al., 1970), to
expel defensive secretions or debris from the mantle cavity
(Kupfermann and Kandel, 1969), to increase respiratory
exchange (Byrne and Koester, 1978), or to contribute to
the systemic circulation of hormones (Kanz and Quast.
1990).

The basic mechanism of burst generation in the R25/
L25 network resembles that of the bag cells. Low fre-
quency firing leads to a regenerative, all-or-none stereo-
typed burst that results from positive feedback interactions
between cells in the R25/L25 network. Conventional fa-
cilitating chemical EPSPs, as well as electrical coupling,
mediate these mutually excitatory connections. This pos-
itive feedback state can be accessed by two separate path-
ways— slow pacemaker potentials that are endogenous to
the R25/L25 cells or excitatory chemical EPSPs that are
generated by afferent input. Termination of the all-or-
none population burst in these cells is mediated largely
by synaptic interactions — slowly developing mutual syn-
aptic inhibition and heterosynaptic depression of the mu-
tually excitatory chemical connections (Byrne, 1983;
Koester, 1989).

The LUQ Neurons

The left upper quadrant (LUQ) cells are a cluster of
five similar neurons (Frazier et ai, 1967). A subset of the
LUQ cells project to the kidney, where they ramify ex-
tensively. On the basis of their axonal projections they
are thought to have extensive effects on kidney function.
The only effects of these cells that have been examined
in detail are on the renal pore, which they cause to close.
The synaptic actions of the LUQ cells on this pore have
very slow onsets and offsets, on the order of several seconds
(Koester and Alevizos, 1989).

Unlike the bag cells and the R25/L25 cells, individual
LUQ cells burst independently of one another. Their en-
dogenous burst properties have been analyzed in detail
(Kramer and Zucker, 1985a,b; Thompson et al., 1986).
The depolarizing pacemaker potential of each burst is ini-
tiated by the activation of voltage-dependent Ca++ chan-
nels. When the cell reaches action potential threshold, the
Ca++ influx during each action potential causes a buildup
of cytoplasmic free Ca++, which has three effects. The
initial effect is to activate Ca++-dependent, non-specific
cation-selective channels, which contribute to burst ac-
celeration. Eventually the two slower effects of intracel-
lular Ca* +-buildup predominate: (1) Ca++-dependent in-
activation of the Ca++ channels that initiated the depo-

BURSTING NEURONS IN APLYSIA

271

larizing pacemaker potential leads to a phase of
regenerative repolarization. (2) Activation of Catf-de-
pendent K* channels also contributes to the repolariza-
tion, particularly at low temperatures.

Neuron L10

L 10 also bursts endogenously (Kandel, 1976; Kleinfeld
el ai, 1990). It is a multiaction interneuron and moto-
neuron that is thought to play a major role in integrating
various aspects of renal function. It makes direct and in-
direct connections to the renal pore that oppose the syn-
aptic actions of the LUQ cells — i.e., it causes the pore to
open. In vitro these openings occur at a rate of about one
per minute. This peripheral antagonism is complemented
by direct inhibitory projections from L10 to the LUQ
cells. L10 also ramifies extensively in the kidney and is
presumed to modulate other aspects of renal function
(Koesterand Alevizos, 1989). One way in which L10 may
modulate renal excretion is by its excitatory connection
to the heart excitatory motoneuron RBHt (Koester et al,
1974). The bulk filtration that gives rise to renal fluid is
thought to occur within a specialized structure, the cristae
aorta, which lies in series with the heart in the pericardia!
sac (Andrews, 1988). Therefore the increase in heart rate
caused indirectly by L10 activity may increase renal fil-
tration.

The mechanism that underlies spontaneous bursting
in L10 has not been studied in detail. However, prelim-
inary results suggest that many of the spikes that occur
during a spontaneous burst are generated in peripheral
axonal processes, far outside the ganglion (unpub. obs.).

Neuron R15

R15 is an endogenously bursting peptidergic neuron
that is thought to play a role in integrating various aspects
of egg laying. It was observed several years ago that spon-
taneous burst generation by R15 is enhanced by the bag
cells when they fire in their population burst (Branton et
al.. 1978). More recently, using an in vitro preparation,
it has been found that R15 has several synaptic actions
that may contribute to efficient egg laying behavior. ( 1 )
When R15 bursts spontaneously, it increases the fre-
quency of respiratory pumping via its excitatory connec-
tions to the R25/L25 cells (Alevizos etal.. 1991a).(2)R15
causes contraction of the pleuroabdominal connectives
by its excitatory connection to motoneuron L7 (Alevizos
et ai, 1991b). (3) R15 increases the rate of anterograde
peristalsis of the large hermaphroditic duct via its periph-
eral axonal processes ( Alevizos et al., 199 Ic). (4) R15 also
sends processes to the left pedal-parapodial artery, by
which it causes local vasoconstriction of this branch of
the arterial tree (Skelton, in prep.).

It has been postulated that R 1 5 integrates five different
aspects of egg laying behavior: (1) The increase in respi-
ratory pumping rate may enhance respiratory exchange
(Alevizos et ai, 199 la). Alternatively, the vigorous pres-
sure surges that occur in the arterial system as the result
of gill contractions may assist in circulating egg laying
hormone throughout the body (Kanz and Quast, 1990).
(2) L7 is a multiaction excitatory neuron that connects
to muscle in a variety of organs, as well as to neurons in
the peripheral nervous system (reviewed by Umitsu et al.,
1987; Alevizos et ai, 1989). At the low rates of L7 firing
elicited by R15 bursting in vitro, the only synaptic action
that L7 expresses is excitation of the sheath muscle of the
paired pleuroabdominal connectives. Each connective
consists of a central axonal core surrounded by a con-
nective tissue sheath that contains vascular channels into
which the bag cells release their peptides and hormones.
The accordion-like folding of the connectives in response
to L7 activity may increase the fluid resistance of their
vascular channels, thereby delaying the washout of the
autoexcitatory peptides and ensuring that mutual exci-
tation of the bag cells is maximally expressed (Alevizos
et ai, 1991b). (3) The increase in peristalsis of the her-
maphroditic duct presumably contributes to the mixing
of the eggs with the secretory products of the duct, as well
as assisting the cilia within the duct in moving the eggs
to the caudal end of the genital groove (Alevizos et ai,
1991c). (4) The constriction of the left pedal/parapodial
artery shunts arterial blood to the right pedal/parapodial
artery, which perfuses the genital groove. Such an effect
could help support the metabolic activity of the cilia lining
the groove, which move the eggs several cm up the groove
to its anterior orifice, from which they are deposited on
the substrate. (5) In addition to its direct synaptic actions,
R15 is also thought to have a neurosecretory action that
influences water balance. R 1 5 synthesizes R 1 5« 1 peptide,
a 38 amino acid neuropeptide that causes an increase in
net water retention when injected into the animal (Weiss
et ai. 1989). R15 has numerous varicosities that appear
to release into systemic vascular spaces (Rittenhouse and
Price, 1985), leading to the suggestion that R15 may in-
crease net water uptake when it is excited by the bag cells
(Alevizos et ai, 1991c). Such an effect may be required
to counter the water lost in egg formation, for the eggs
are fertilized and packaged into gelatinous egg capsules
on demand — i.e., in response to the bag cell burst
(Thompson, 1976). It will be necessary to record RIS's
firing pattern during spontaneous egg laying in the intact
animal to determine the actual contributions of these dif-
ferent effects of R 1 5 activity to egg laying behavior.

Each of the four direct synaptic actions of R15 can be
mimicked by R15«l peptide, and the peptide probably
mediates them when R 1 5 bursts. These synaptic actions
are unusual in that they decay quite rapidly with repeated

272

A. ALEVIZOS ET AL.

activation of R15. An example of this synaptic decrement
is shown in Figure 2, for the R15-R25/L25 connections.
Prolonging the R15 burst period to greater than 10 min
has no added effect on the excitation of the R25/L25 cells.
The fact that the response of the R25/L25 cells to direct
application of R15al peptide decreases in a similar fash-
ion argues against depression of release being critical for
the decrement in synaptic transmission. In addition, the
R25/L25 cells respond normally to another excitatory
transmitter when the response to R 1 5 is depressed, ruling
out non-specific refractoriness or postsynaptic inhibition
as contributing to this synaptic decrement. Thus, post-
synaptic desensitization appears to be the most likely ex-
planation for the decay of R15's direct synaptic actions
on the R25/L25 network. A similar conclusion is drawn
from the actions of R15 on L7, on the hermaphroditic
duct and on the arterial muscle. However, in the case of
the two peripheral tissues, one cannot rule out muscular
fatigue as a contributor to response decrement (Alevizos
el al.. 199 Ic).

The direct synaptic actions of R15 are difficult to ob-
serve in vitro without taking special precautions. They are
normally chronically depressed by the profound desen-
sitization that results from the fact that R15 fires spon-
taneously at a high rate in vitro. Only if R15 is silenced
by injecting hyperpolarizing current for 1-2 h does the
desensitization decay, unmasking the four synaptic actions
described above (Alevizos et al.. 1991a,b,c). This obser-
vation has two important implications. First, the synaptic
actions of other spontaneously active neurons may be
masked if they undergo profound depression. It may be
necessary to silence such cells for a long time to restore
their synaptic connections to a level where they can be
detected. Second, the observation that R15's synaptic ac-
tions rapidly become completely depressed results in a
paradox. How can these actions ever be expressed, given
that R15 bursts continuously in //; vitro experiments? Are
its synaptic connections constantly desensitized? This
question was addressed in chronic recording experiments,
in which the axon of R15 was recorded from in intact,
freely moving animals (Alevizos et al., 199 la). It was
found that R 1 5 does not burst spontaneously in the intact
animal (Fig. 3). Given that R15 is inactive in the intact
animal and is excited by the bag cells in vitro, it has been
suggested that R 1 5 is a conditional burster that is switched
to the bursting mode by the bag cell burst that triggers
egg laying (Alevizos et al.. 199 la). Although preliminary
results support this hypothesis, it has not yet been deter-
mined whether R 1 5 fires during a spontaneous egg laying
episode in the intact animal.

The mechanism that generates spontaneous bursts in
R15 has been studied extensively (Adams, 1985; Adams
andLevitan, 1985; Lewis, 1988; Thompson et al.. 1986).
In its broad details it resembles the burst generating

- 5 MIN N=10
K30 MIN N=6

10 -|

CO
EC

LT>

in
c\i

-10 MIN N=12
-60 MIN N = 10

R15 FIRING

50
TIME (MIN)

Figure 2. Modulation of the frequency of respiratory- pumping by
R15 decays during prolonged R15 activity. When R15 was allowed to
burst spontaneously for various amounts of time after a 2-h period of
hyperpolanzation, it produced a long-lasting increase in the frequency
of respirator, pumping. The amplitudes of the maximum effect and the
time courses of decay of these increases were not significantly different
for the 10-. 30-, and 60-min firing periods, while the effect produced by
the 5-min firing was significantly smaller in both amplitude and duration.
These data indicate that the maximum effect of R 1 5 bursting is exerted
within the first 5-10 min of R15 bursting, beyond which the response
is independent of R 1 5 activity. There was no trend for the firing rate of
R 1 5 to slow down over the course of the long burst periods (Alevizos et
al.. 199 la).

mechanism described above for the LUQ cells. Kramer
and Levitan (1990) have demonstrated that modulation
of R 1 5 bursting by egg laying hormone is most effective
when R 1 5 is inactive, consistent with the observation that
R15 is silent in the intact animal.

Conclusions

It is interesting to see whether a comparison of these
five types of bursting neurons leads to any conclusions
about how the properties of each class relates to its func-
tional role. Even with this relatively small sample of cell
types, a few generalizations do emerge from such a com-
parison.

Is there a difference between individual bursters, which
are not coupled to other bursting cells (L10. the LUQ
cells, and R 1 5 ) and population bursters, which fire as part
of a population burst (the bag cells and the R25/L25 cells)?
The two classes are alike in one respect — both individual
bursters and population bursters can have endogenous
pacemaker mechanisms (the LUQ cells, L10, R15 and
the R25/L25 cells). They differ, however, in their ability
to generate episodic bursts. In the in vitro preparations
that have been examined so far. individual bursters do
not seem to fire in isolated bursts. The population bursters
(the bag cells and the R25/L25 cells), however, by virtue
of the positive feedback chemical and electrical connec-

BURSTING NEURONS IN APLYSIA

273

NERVE
BRANCH

10uV

6sec

NERVE
BRANCH

R15

20mV

6sec

Figure 3. R15 does not hurst spontaneously in the intact animal. An extracellular electrode chronically
implanted in the subject was used to record activity from a small branch of the pericardial nerve that contains
R 1 5 processes. (A) In the intact animal there was no bursting activity of R 1 5 recorded from the nerve
branch. Three random 1.5-min samples are shown from a 2-h recording. (B) At the end of the experiment
the animal was sacrificed, and the abdominal ganglion was dissected from the animal, along with the electrode
still attached to the nerve branch. Bursting activity appeared in the nerve of the isolated ganglion. Intracellular
recording from the R15 soma confirmed that the bursting activity in the nerve was due to R15 (n = 9)
(Alevizosrtu/.. 199 la).

tions within each population, are well adapted to generate
single bursts in response to brief volleys of excitatory syn-
aptic input.

The excitatory and inhibitory chemical synaptic con-
nections between members of a population of bursting
cells also appear to extend the range of possible burst du-
rations and intensities. While typical burst durations for
the individual bursters are about 5-30 s (at 15°C), the
bag cell bursts last 1 5-30 min. At the other extreme, al-
though the burst duration of the R25/L25 network is quite
variable, the high frequency terminal phase of the burst,
which actually drives the motoneurons, lasts only 1-2 s.
The excitatory synaptic connections between the R25/
L25 cells also contribute significantly to the high firing
frequencies that these cells attain during a burst — as high
as 25-40 Hz. In contrast, individual bursters generally
reach peak firing frequencies of only 1-3 Hz during a
burst. The role of chemical connections in shaping the
burst can also be extended to controlling the refractory
period by the activation of second messenger systems that
generate long-lasting effects. In the case of the bag cells.

the refractory period can be made to last as long as 18-
24 h in this way.

The shaping of the duration of the population bursts
of the bag cells and the R25/L25 cells seems to have clear
functional consequences. In the case of the bag cells, the
long-lasting bursts with the gradual increase in spike width
appear to provide a large safety margin for release of an
effective dose of egg laying hormone into the circulation.
For the R25/L25 cells, the very brief, high frequency burst
is well suited for driving intense, synchronous contractions
of the mantle organs, thereby optimizing the pumping
action.

It is more problematic to understand the significance
of the bursting patterns of the individual bursters. The
bursts generated by one of them, L10, does have an ob-
vious function. Each burst elicits a phasic opening of the
renal pore. But whether the pore actually opens this way
in vivo remains to be determined. In addition, it is not
clear why the other two classes of individual bursters fire
in a bursting mode. The synaptic actions generated by
R15 and the LUQ cells are so slow that their follower

274

A. ALEVIZOS ET AL.

cells effectively integrate their firing patterns. That is, there
is no reflection of the phasic bursting patterns of R15 and
the LUQ cells on any of the nerve or muscle cells on
which they synapse. This raises the question of why R15
and the LUQ cells burst, rather than firing in steady trains.
Three possible explanations come to mind: ( 1 ) They may
have other, more phasic synaptic actions. For example,
R 1 5 has transient synaptic actions on other neurons in
the abdominal ganglion. These effects are not observed
in all preparations, however, suggesting that they may be
gated by some undetermined physiological variable (Ale-
vizos and Koester, 1986; Brown and Mayeri; 1987). (2)
The release properties of these cells may be such that brief
bursts of activity are the most efficient for optimizing re-
lease. (3) Synaptic release in Aplysia is strongly influenced
by the level of membrane potential immediately preceding
initiation of an action potential. Hyperpolarization de-
presses release and depolarization enhances release (Shi-
mahara and Peretz, 1978; Shapiro el ai, 1980). Perhaps
the slow depolarizing waves of membrane potential that
generate the bursts in these cells are conducted electro-
tonically to the terminals, where they may modulate spike-
evoked release. However, it seems unlikely that such
changes in resting potential would be conducted to ter-
minals in the periphery. Therefore, a cell like R 1 5, which
makes both central and peripheral synapses, may have
quite different release properties at its synapses within the
ganglion compared to those in the periphery. If there does
exist a difference between central and peripheral release
sites, it may be amplified by modulatory inputs to R15
such as the one from the bag cells. When they fire in a
population burst, the bag cells increase the depth of the
depolarizing pacemaker waves recorded from the soma
of R15 (Mayeri et ai. 1979). Thus, within the ganglion,
the bag cells may influence release of peptides from R15
by two mechanisms: an increase in spike frequency and
modulation of the slower membrane potential trajectory
between bursts. The terminals in the periphery, however,
are likely to experience only the increase in spike fre-
quency.

Acknowledgments

This work was supported by NIH grant NS 14385.

Literature Cited

Adams, W . B. 1985. Slow depolarizing and hyperpolanzing currents
which mediate bursting in Aplysia neurone R15. / Physio! 360: 51-
68.

Adams, W. B., and I. B. Levitan. 1985. Voltage and ion dependence
of the slow currents which mediate bursting in Aplysia neurone Rl 5.
J. PhysioL 360: 69-93.

Alevizos, A., and J. Koester. 1986. Identified gill motoneuron L7 of
Aplysia is a multimodal motoneuron that also innervates the cardio-
vascular system. Soc. Neurosci. Abslr. 12: 499.

Alevizos, A., C. H. Bailey, M. Chen, and J. Koester. 1989. Innervation
of vascular and cardiac muscle of Aplysia by multimodal motoneuron
L7. J. Neiirophysuil. 61: 1053-1063,

Alevizos, A., K. R. Weiss, and J. KoesCer. 199la. Synaptic actions of
identified peptidergic neuron R15 in Aplysia: I. Activation ot respi-
ratory pumping. J. Neurosci. (in press).

Alevizos, A., K. R. Weiss, and J. Koesler. 1991b. Synaptic actions of
identified peptidergic neuron R 1 5 in Aplysia: II. Contraction of pleu-
roabdominal connectives mediated by motoneuron L7. J. Neurosci.
(in press).

Alevizos, A., K. R. Weiss, and J. Koester. 199lc. Synaptic actions of
identified peptidergic neuron RI5 in Aplysia: III. Activation of the
large hermaphroditic duct. J. Neurosci. (in press).

Andrews, E. B. 1988. Excretory systems of molluscs. Pp. 381-448 in
The Mollusca. Vol. 1 1. A. S. M. Saleuddin and K. M. Wilbur, eds.
Academic Press. Inc., New York.

Branton, D. VV., E. Mayeri, P. Browne!!, and S. S. Simon. 1978.
Evidence for local hormonal communication between neurones in
Aplysia. Nature 274: 70-72.

Brown, R. O., and E. Mayeri. 1987. Central actions of R 15. a putative
peptidergic neuron in Aplysia. J. Neurobiol. 18: 3-13.

Brown, R. O., and E. Mayeri. 1989. Positive feedback by autoexcitatory
neuroendocrine bag cells of Aplysia. J \enrwci. 9: 1443-1451.

Byrne, J. 1983. Identification and initial characterization of a cluster
of command and pattern-generating neurons underlying respiratory
pumping in Aplysia californica. J. Nenropliysiol. 49: 491-508.

Byrne, J., and J. Koester. 1978. Respiratory pumping: neuronal control
of a centrally commanded behavior in Aplysia. Brain Res. 143: 87-
105.

Cobbs, J. S.. and H. M. Pinsker. 1982. Role of bag cells in egg de-
position of Aplysia hrasiliana. I. Comparison of normal and elicited
behaviors. J. Comp. Physiol. 147: 523-535.

Conn, P. J., J. S. Strong, E. M. Azhderian, A. C. Nairn, P. Greengard,
and L. K. Kaczmarek. 1988. Protein kinase inhibitors selectively
block phorbol ester or forskolin-induced changes in excitability of
Aplysia neurons. / Neurosci. 9: 480-487.

Croll, R. P. 1985. Sensory control of respiratory pumping in Aplysia
californica. J E\p. Biol. 117: 15-27.

De Riemer, S. A., J. A. Strong, K. A. Albert, P. Greengard. and L. K.
Kaczmarek. 1985. Enhancement of calcium current in Aplysia
neurons by phorbol ester and protein kinase C. Nature 313: 313-
316.

Dudek, F.E..J.S. Cobbs, and H.M. Pinsker. 1979. Bag cell electrical
activity underlying spontaneous egg laying in freely behaving Aplysia
brasiliana. J. Neurophysiol. 42: 804-817.

Dudek, F. E., G. Weir, J. Acosta-Urquidi, and S. S. Tobe. 1980. A
secretion from neuroendocrine bag cells evokes release in vitro from
ovotestis of Aplysia californica. Gen. Comp. Endocrinol. 40: 241-
244.

Eberly, L., J. Kanz, C. Taylor, and H. Pinsker. 1981. Environmental
modulation of a central pattern generator in freely behaving Aplysia.
Behav Neural Biol. 32: 21-34.

Eberly, L. B., and H. M. Pinsker. 1984. Neuroethological studies of
reflex plasticity in intact Aplysia. Behav. Neurosci. 98: 609-630.

Ferguson, G. P., A. Ter Maat, D. W. Parsons, and H. M. Pinsker.
1989. Egg laying in Aplysia: I. Behavioral patterns and muscle ac-
tivity of freely behaving animals after selectively elicited bag cell dis-
charges. ./ Comp. PhysioL A 164: 835-847.

Frazier, W. T., E. R. Kandel. I. Kupfermann. R. Waziri, and R. E. Cog-
geshall. 1967. Morphological and functional properties of identified
neurons in the abdominal ganglion of Aplysia californica. J. Neu-
rophysiol. 30: 1288-1351.

Kaczmarek, L. K., and J. A. Kauer. 1983. Calcium entry causes a pro-
longed refractory period in peptidergic neurons of Aplysia. J. Neurosci.
3: 2230-2239.

BURSTING NEURONS IN APLYSIA

275

Kandel, E. R. 1976. The Cellular Basis of Behavior Freeman. San

Francisco.
Kan/. J. E., and W. D. Quasi. 1990. Respiratory pumping seizure: a

newly discovered spontaneous stereotyped behavior pattern in the

opisthobranch mollusc Aplysia californica. J Comp. Physio! A 166:

619-627.
Kauer, J. A., and L. K. Kac/marek. 1985. Peptidergic neurons of Aplysia

lose their response to cAMP during a prolonged refractory period.

J. Neurosci. 5: 1339-1345.
Kleinfeld. D., T. D. Parsons, F. Raccuia-Behling, B. M. Salzberg, and

A. L. Obaid. 1990. Foreign connections are formed in vitro by

Aplvsia californica interneurone L10 and its in vivo followers and

non-followers. J. E.\p. BioL 154: 237-255.
Koester, J. 1989. Chemically and electrically coupled intcrneurons

mediate respiratory pumping in Aplysia. J. Neurophysiol. 62: I 1 1 3-

1126.
Koester, J., and A. Alevizos. 1989. Innervation of the kidney of Aplysia

by L10, the LUQ cells, and an identified peripheral motoneuron. J.

Neurosci. 9: 4078-4088.
Koesler, J., and E. R. Kandel. 1977. Further identification of neurons

in the abdominal ganglion of Aplysia using behavioral criteria. Brain

Res 121: 1-20.
Koester, J., E. Mayeri, G. Liebeswar, and E. R. Kandel. 1974. Neural

control of circulation in Aplysia. II. Interneurons. / Neurophysiol.

37: 476-496.
Kramer, R. H., and R. S. Zucker. 1985a. Calcium-dependent inward

current in Aplysia bursting pacemaker neurones. J Physiol 362:

107-130.
Kramer, R. H., and R. S. Zucker. 1985b. Calcium-induced inactivation

of calcium current causes the inter-burst hyperpolanzation of Aply.siu

bursting neurones. J. Physiol 362: 131-160.
Kramer, R. H., and I. B. Levitan. 1990. Activity-dependent neuro-

modulation in Aplysia neuron Rl 5: intracellular calcium antagonizes

neurotransmitter responses mediated by cAMP. ./. Neurophysiol. 63:

1075-1088.

Kupfermann, 1., and E. R. Kandel. 1969. Neuronal controls of a be-
havioral response mediated by the abdominal ganglion of Aplysia

Science 164: 847-850.
Kupfermann, I., and E. R. Kandel. 1970. Electrophysiological properties

and functional interconnections of two symmetrical neurosecretory

clusters (bag cells) in abdominal ganglion of Aplysia. J. Neurophysiol

33: 865-876.
Lewis, D. V. 1988. Calcium-activated inward spike after-currents in

bursting neurone R15 of Aplysia. J Physiol. 396: 285-302.
Mayeri, E., P. Brownell, W. D. Branton, and S. B. Simon. 1979.

Multiple, prolonged actions of neuroendocrine bag cells on neurons

in Aplysia. I. Effects on bursting pacemaker neurons. J. Neurophysiol.

42: 1165-1184.

Mayeri, E., and B. S. Rothman. 1985. Neuropeptides and the control
of egg-laying behavior in Aplysia. Pp. 285-301 in Model Neural Net-
works and Behavior. A. I. Selverston. ed. Plenum Press. New York.

Pinsker, M. H., and F. E. Dudek. 1977. Bag cell control of egg laying
in freely behaving Aplysia. Science 197: 490-493.

Pinsker, H., I. Kupfermann, V. Castellucci, and E. R. Kandel.
1970. Habhuation and dishabituation of the gill-withdrawal reflex
in Aplysia. Science 167: 1740-1742.

Rittenhouse. A. R., and C. H. Price. 1985. Peripheral axons of the
parabolic burster neuron R15. Brain Res. 333: 330-335.

Rothman, B. S., E. Mayeri, R. O. Brown, P.-M. Yuan, and J. E. Shively.
1983a. Primary structure and neuronal effects of alpha-bag cell
peptide. a second candidate neurotransmitter encoded by a single
gene in bag cell neurons of Aplysia. Proc. Nat. Acad. Sci. USA 80:
5753-5757.

Rothman, B. S., G. Weir, and F. E. Dudek. 1983b. Egg laying hormone:
direct action on the ovotestis of Aplysia. Gen. Comp. Endocrinol. 52:
131-141.

Segal, M. M., and J. Koester. 1982. Convergent cholinergic neurons
produce similar postsynaptic actions in Aplysia: implications for
neural organization. J. Neurophysiol. 47: 742-759.

Shapiro, E., V. F. Castellucci, and E. R. Kandel. 1980. Presynaptic
membrane potential affects transmitter release in an identified neuron
in Aplvsia by modulating theCa** and K.* currents. Proc. Nat. Acad.
Sci. USA 77: 629-633.

Shimahara. T., and B. Peret/. 1978. Some potential of an interneurone
controls transmitter release in a monosynoptic pathway in Aplysia.
Nature 273: 158-160.

Strong, J. A., and L. K. Kaczmarek. 1986. Multiple components of
delayed potassium current in peptidergic neurons of Aplysia: mod-
ulation by an activator of adenylate cyclase. /. Neurosci. 6: 8 14-822.

Strong, J. A., A. P. Fox, R. W. Tsien, and L. K. Kaczmarek.
1987. Stimulation of protein kinase C recruits covert calcium
channels in Aplysia bag cell neurons. Nature 325: 714-717.

Thompson, T. E. 1976. Biology of Opisthobranch Molluscs. Vol. I. The
Ray Society. London.

Thompson, S., S. J. Smith, and J. \V. Johnson. 1986. Slow outward
currents in molluscan bursting pacemaker neurons: two components
differing in temperature selectivity. J. Neurosci. 6: 3169-3176.

I'mitsu, Y., H. Matsumoto, and H. Koike. 1987. Active contraction
of nerve bundle and identification of a nerve-contractor motoneuron
in Aplysia. J Neurophysiol. 58: 1016-1034.

Walters, E. T., and M. T. Erickson. 1986. Directional control and the
functional organization of defensive responses in Aplysia. J Comp.
Physio/. A 159: 339-351.

Weiss, K. R., H. Bayley, P. E. Lloyd, R. Tenenbaum, M. A. Gawinowicz
Kolks, L. Buck, E. C. Cropper, S. C. Rosen, and I. Kupfermann.
1989. Purification and sequencing of neuropeptides contained in
neuron R 1 5 of Aplysia californica. Proc. Nat. Acad. Sci. USA 86:
2913-2917.

Reference: Biol. Bull. 180: 276-283. (April, 1991)

Contraction, Serotonin-Elicited Modulation, and

Membrane Currents of Dissociated Fibers

of Aplysia Buccal Muscle

JEFFREY L. RAM, FENG ZHANG, AND LI-XIN LIU
Department of Physiology, Wayne State University, Detroit, Michigan 48201

Abstract. Feeding muscles of the buccal mass of Aplysia
are innervated by cholinergic and serotonergic neurons.
Buccal muscle 15 contracts in response to acetylcholine
(ACh). During feeding arousal, ACh-elicited contraction
of muscle 15 is potentiated by serotonin (5-HT). This paper
demonstrates a dissociated cell preparation of muscle 15
in which cellular mechanisms regulating contraction can
be investigated.

Dissociated muscle fibers contracted in response to both
KC1 and ACh. Serotonin (10~6 M) significantly poten-
tiated the shortening caused by both KC1 and ACh. Po-
tentiation lasted at least 4 min, similar to potentiation in
intact muscles.

Four types of currents recorded by patch clamp meth-
ods are illustrated. With 540 m/U KG in the patch elec-
trode, stretch-activated channels having a chord conduc-
tance of 150 pS are observed in on-cell patches. In whole
cell configuration, ACh elicits inward current at a holding
potential of -60 mV. With high potassium in the elec-
trode, depolarization elicits an outward current. The volt-
age-dependent outward current is blocked with cesium in
the electrode and 4-aminopyridine and tetraethylam-
monium outside the cells. The remaining voltage-depen-
dent inward current is calcium dependent. The voltage-
dependent inward and outward currents are activated
within the range of depolarization produced by ACh and
may therefore play roles in regulating contractile responses
elicited by ACh.

Introduction

Neurotransmitters can cause both direct and modula-
tory effects on target tissues. A "modulatory effect" of a
neurotransmitter is one in which the transmitter has no

Received 9 August 1990: accepted 6 November 1990.

immediate effect, but rather, it modifies the influence of
other effectors. These modulatory influences appear to
fine-tune the nervous system and the muscles it controls
for switching between different behavioral tasks. The same
circuits can subserve several different behaviors; the be-
havioral output depends on the relative weighting of dif-
ferent synapses and the excitability of circuit elements.
Modulation of muscle is part of this integrated scheme.
Although the relative strength of contractions appropriate
for different behaviors could be manipulated by discrete
relative changes in motoneuron activity, an additional,
possibly more efficient method may be to give different
"global commands" that change the relative strengths of
contractile responses in ways appropriate for particular
behaviors. An example of a modulatory effect of a neu-
rotransmitter mediating a change in behavior is the po-
tentiating effect of serotonin (5-HT) on buccal mass mus-
cles of Aplysia, which is believed to mediate, in part, feed-
ing arousal.

The buccal mass muscles of Aplysia are smooth muscles
used in voluntary feeding movements. These muscles are
innervated by cholinergic, peptidergic, and serotonergic
neurons. Acetylcholine (ACh) is the direct effector of these
muscles, as exemplified by the contractile response to ACh
of buccal muscle 15. Muscle 15, also known as the acces-
sory radular closer muscle (Cohen et al., 1978), is the
most intensively studied Aplysia muscle. 15 is innervated
by buccal ganglion neurons B15 and B16 (Cohen et al.,
1978; Ram, 1983), both of which synthesize ACh (Cohen
c/ al., 1978) as well as several peptides (Cropper et at.,
1987, 1988). In addition, 15 is innervated by the seroto-
nergic metacerebral giant cell (MCG). Activity of MCG
causes no direct response of the muscle; however, it po-
tentiates subsequent contractile responses to B15 and B16.
This modulation is achieved largely through post-synaptic
actions on the muscle (Weiss el a/., 1978). In isolated 15

276

DISSOCIATED MUSCLE FIBERS OF APLYSIA

111

buccal muscles were dissected from the animal, teased
into thin strips, and incubated for 2-4 h at 28°C in Instant
Ocean containing 10 mAI HEPES (pH 7.0, adjusted with
NaOH), 0. 1 5% collagenase (Sigma Type I). 0. 1% soybean
trypsin inhibitor (Sigma Type I-S), and 1 //g/ml leupeptin

Figure 1. Typical dissociated buccal muscle fiber. At the right is a
patch electrode pointing to the fiber. Scale: calibration marks are 7.8
apart.

muscles 5-HT produces no contractile response itself but
does potentiate ACh-elicited contractions (Ram el a/..
198 1 ). MCGs are active during feeding (Weiss et a/.. 1978).
Lesion of serotonergic neurons changes (although does
not completely block) feeding motor activity (Rosen et
a/.. 1983, 1989). Thus, the modulatory effect of the se-
rotonergic MCG neurons on feeding muscles appears to
have an important role in feeding arousal.

Although previous experiments on mechanisms me-
diating the modulatory effect of 5-HT on buccal muscles
have suggested roles for cyclic AMP (Mandelbaum, 1980:
Rametal.. 1983, 1 984a) and calcium ( Ram etal.. 1984b;
Ram and Parti. 1985), the cellular targets of these me-
diators have not been determined. For example, it is un-
known whether cyclic AMP or calcium modify membrane
mechanisms such as ion channels or change the sensitivity
or activity of contractile proteins. For studying effects on
contractile proteins, this laboratory developed a skinned
muscle preparation, which is described elsewhere (Ram
and Patel, 1989). To study membrane mechanisms, we
developed a dissociated muscle fiber preparation. This
paper describes the contractile properties of this disso-
ciated muscle preparation, including its modulatory re-
sponse to 5-HT, and demonstrates its suitability for patch
clamp analysis of single channel and whole cell ionic cur-
rents. Preliminary descriptions of some of these data have
appeared previously (Ram and Liu, 1990; Zhang and
Ram. 1990).

Materials and Methods

Individuals ofAplysia californica (200-400 g) were ob-
tained from Marinus (Long Beach, California) and main-
tained at 18°C in Instant Ocean with a 12:12 L:D light
cycle. To obtain dissociated muscle fibers, a modification
of the methods of Ishii et al. (1986), previously used to
dissociate muscle fibers in Mytilus, was used. Both 15

Figure 2. Serotonin (5-HT) potentiates high potassium-elicited con-
traction. Dissociated muscle fibers in a small chamber (0.4 ml volume)
were constantly superfused with artificial seawater ( ASW) at 5.5 ml/min.
(A) Fibers at rest. (B) Contractile response to a 3-s pulse of ASW con-
taining 100 mAI KG. All but the fiber in the upper right contracted.
Fibers returned to rest length at the end of the pulse of KG. (C) Fibers
at the end of 1 mm superfusion with ASW containing 10~6 M 5-HT.
Fibers remained at rest length. (D) Contractile response to a 3-s pulse
of 100 mAI KG ASW. identical to that given in (B), immediately after
1 min superfusion with 10~" M 5-HT ASW. All contracting fibers short-
ened more after 5-HT treatment. Scale: calibration marks are 12 pm
apart.

278

J. L. RAM /:/ .11.

150T

100

KCI contractions

mean resting length = 248 ± 28 fj.m. n=9

* , p<0.05, paired t compared to pro — 5HT ( — 2 min)

-2024
Time post — 5HT (min)

Figure 3. Magnitude and time course of 5-HT potentiation of K.C1-
elicited contractions. Fibers were measured in images similar to those
illustrated in Figure 2. High potassium (100 m.U KCI ASW) pulses were
given every two min. Fibers were exposed to 5-HT (10~6 M in ASW)
for 1 min immediately prior to the 0 time point.

(Sigma). When cells began appearing in the medium, fibers
in remaining muscle pieces were dispersed by gentle trit-
uration. The resultant dissociated cells were washed by
centrifuging and resuspending them in wash medium
containing all ingredients of the dissociating medium ex-
cept collagenase. Washed cells were plated onto glass cov-
erslips in 30-mm plastic petri dishes and stored at 4°C in
a humidified chamber. Cells were usually used within 1-
4 days, although viable, contractile cells have survived for
as long as 10 days under these conditions. Experiments
were done at room temperature (20-24°C) after allowing
the cells to warm gradually for at least 30 min.

A plexiglass insert having a central hole approximately
1 cm in diameter was clamped into the 30-mm petri dish.
The insert formed a small chamber, approximately 0.4
ml in volume. Medium was constantly pumped into the
chamber (5.5 ml/min) and removed by suction from a
surface wick opposite the inflow. The dish was mounted
on a movable stage of an inverted microscope. The shape
and movement of muscle fibers were recorded by a VHS
camcorder (RCA CC310). A videotape demonstrating
many of the contractile and electrophysiological responses
reported in this paper ("Dissociated Muscle Fibers of
Aplysia," by J. L. Ram) is available from the authors upon
request. Morphometric analysis was done by measuring
still-images on the tape playback. Photographs were made
by oscilloscope camera directly off the TV monitor.

A Dagan 8900 Patch Clamp-Whole Cell Clamp was
used for single channel and whole cell recording. Data
were filtered by the 1 kHz low-pass filter in the Dagan
amplifier. Electrodes were fabricated from Fisher non-
heparinized hematocrit glass, polished to bubble number
3-4 (Corey and Stevens, 1985), and coated with Sylgard.
Pipet solutions are described in relevant figure captions.

Electrical stimuli and digital recording of currents were
controlled by pCLAMP software (Axon Instruments,
Burlingame, California).

Results

.Morphology and contraction

The typical appearance of a dissociated fiber from buc-
cal muscle 15 is illustrated in Figure 1 . Dissociated muscle
fibers were spindle-shaped and ranged from 5 to 25 nm
in diameter and up to a mm in length. The widest diameter
usually occurred near the middle of the fiber, adjacent to
the nucleus, and averaged 13.6 ± 0.9 ^m (mean ± S.E.,
n = 19). The average diameter of the fibers, measured
every 20 ^m along the length of the fiber, was 10.8 ± 0.7
urn. The average length of fibers at rest was 270 ± 10 ^m
(n = 37).

Dissociated muscle fibers contracted in response to KG.
In response to a 2- or 3-s pulse of ASW containing 100
mM KCI, 22 fibers that had average resting lengths of 262
± 13 iim shortened to 218 ± 12 ^m. An illustration of a
subset of these fibers is shown in Figure 2. in which Figure
2 A shows the fibers at rest and Figure 2B shows the max-

Figure 4. Serotonin (5-HT) potentiates ACh-elicited contractions.
The procedure is identical to that of Figure 2 except that contraction
was elicited by a 3-s pulse of 10"4 M ACh. (A) Fibers at rest. (B) Contractile
response to ACh. (C) Contractile response to identical pulse of ACh as
in (B) immediately after 1 min superfusion with 10~6 A/ 5-HT ASW.
Scale: calibration marks are 20 ^m apart.

DISSOCIATED MUSCLE FIBERS OF APLYS1A

279

ACh contractions

mean resting length = 264 ± 20 ^zm, n = 6

* , p<0.05. paired t compared to pre-5HT (-2 mln)

1SU-

*
1

| "S too

C -t-

to o

r °

o £

-C C

VI O

^ o

O i

° ' 50

c 01

3 C

o ^i

E S

n .

— J—

— •

—

- H —

-2024
Time post-5HT (min)

Figure 5. Magnitude and time course of 5-HT potentiation of ACh-
elicited contractions. Fibers were measured on the field in Figure 4 and
subsequent images. ACh pulses were given even1 - rnin. Fibers were
exposed to 5-HT ( ICT6 M in ASW) for 1 min immediately prior to the
0 time point.

imal contraction produced by a 3-s pulse of KC1. Follow-
ing the KC1 pulse, fibers relaxed to their resting lengths
within a few seconds.

Serotonin potentiated the contractile response to KC1
(Fig. 2D). Figure 3 summarizes data from nine fibers that
were exposed to 1(T6 M 5-HT for 1 min. The amount of
shortening produced by KC1 pulses was almost doubled
following 5-HT, and the effect lasted at least 4 min, similar
to the long-lasting potentiation in intact muscles produced
by 5-HT (Ram et ai, 1981). The fibers remained relaxed
during the 5-HT application (Fig. 2C).

Similarly, ACh caused contraction of dissociated fibers,
which could be potentiated by 5-HT. Figure 4 shows ACh-
elicited contractions prior to 5-HT and immediately fol-
lowing one min 1(T6 M 5-HT. Data from six fibers, sum-
marized in Figure 5, show the significant increase in
shortening caused by 5-HT and the similar time course
of recovery from the effects of 5-HT to KCl-elicited con-
tractions.

— vr-n — icr-n-TwryV| -i'|-| inrrv

suction

|10 pA

1 sec

Figure 6. Channel activity in on-cell patches. The electrode contained
(in roA/ ) 406 KC1, 20 NaCl, 2 MgCl, , 1 0 ATP, 0. 1 GTP. 1 0 glutathione,
and 100 HEPES. pH 7.0 with KOFI. Electrode potential was identical
to the bath potential, and the cell was at resting potential (not measured
for this cell). Suction increased the opening of at least one population
of large channels, conducting approximately 10 pA per unitary channel
opening.

150

| —

120

g
<

LJ
Q

25 ms

Figure 7. Current through stretch-activated channels in an on-cell
patch, measured over a range of membrane potentials. The electrode
contained 540 m.U K.C1. Stretch sensitivity of these channels was dem-
onstrated during another part of the experiment (not illustrated here).
Membrane potential was varied by changing the potential of the patch
electrode, and the membrane potential is given as the change from resting
potential. Pipet potential was held at each potential for at least 20 s.
Channel current reversed at approximately 87 mV; chord conductance
was approximately 150 pS; and channel opening probability was inde-
pendent of membrane potential.

Single channel and whole cell patch clamp recording

Dissociated buccal muscle fibers were suitable for
forming gigaseals for single channel and whole cell re-
cording. With high potassium in the patch electrode, on-
cell patches revealed the presence of a variety of channels
conducting inward current at resting potential, including
at least one prominent channel that could be activated
by increased suction on the electrode (Fig. 6). The fre-
quency of the opening of suction-activated channels in-
creased with negative pressures of 50-100 cm HiO (ap-
proximately 40-80 mm Hg), as measured by a water ma-
nometer (n = 3; see also Ram et ai, 1990). The current
through unitary channel openings of the most prominent
channel activated by suction, with the fiber at resting po-
tential and the electrode at bath potential, averaged 12
± 1 pA (n = 7 patches). The chord conductance of this

280

J. L. RAM ET AL

10~5 M ACh

0.5 nA

-Vm=-60 mV
Vm=-80 mV

10 15 20 25 30
TIME (s)

Figure 8. Response to ACh in whole-cell recording configuration.
Pipet solution contained (in mA/) 20 NaCl. 406 K.C1, 2 MgCU, 100
HEPES. 5 EGTA, 5 MgATP, 0.1 NaGTP. 10 glutathione, pH 7.0 (ad-
justed with KOH). Fibers were constantly superfused with artificial sea-
water. Holding potential was -80 mV and was moved to -65 mV every
3s. A 1 5-s pulse of 10~5 M ACh elicited inward current at both membrane
potentials.

suction-activated channel, determined by eliciting channel
activity at several different holding potentials, averaged
140 ± 20 pS (n = 3), as exemplified by the patch illustrated
in Figure 7.

After gigaseal formation, whole cell configuration could
be achieved by applying greater suction than is necessary
to activate suction-activated channels. With a pipet so-
lution containing high potassium and other ingredients
meant to mimic the normal intracellular milieu of the
fibers (complete composition is given in the caption to
Fig. 8), ACh elicited an inward current (Fig. 8). The peak
current elicited by 10 5 M ACh at a holding potential of
-80 mV was -2.4 ± 0.5 nA (n = 20); at a holding po-
tential of -60 mV. the peak current averaged - 1 .4 ± 0.4
nA (n = 20). Under the same ionic conditions, depolar-
ization activated outward current (Fig. 9). In observations
of more than 20 cells, a net voltage dependent inward
current was never seen under conditions of having high
potassium in the pipet and normal sea water outside. Oc-
casionally, there was a slight delay in activation of outward
current (not seen in Fig. 9), possibly indicating an initial
counterbalancing inward current.

Voltage-dependent inward current can, however, be
seen under conditions that block potassium channels.
With cesium in the electrode and 4-aminopyridine and
tetraethylammonium in the extracellular solution, de-
polarization elicited an inward current. The peak inward
current averaged 2.4 ± .4 nA (n = 11 fibers). As illustrated
in Figure 10, the voltage dependent inward current was
dependent upon calcium in the extracellular medium.

Discussion

This paper demonstrates that smooth muscle fibers dis-
sociated from buccal muscles of Aplysia are a suitable

preparation for studying mechanisms regulating contrac-
tion and its modulation. First, isolated fibers have appro-
priate contractile responses: They contract in response to
both high potassium and ACh. and the contractions to
both are potentiated by 5-HT. Second, the dissociated
fibers are suitable for patch clamp analysis of single chan-
nel and whole cell currents.

Previous studies have used indirect methods for inves-
tigating the roles of specific ion channels in regulating
contraction of molluscan muscles. One set of questions
that arise concerns the sources of activator calcium in the
physiological responses to neurotransmitters. Is contrac-
tion dependent upon the influx of extracellular calcium?
If so, are the channels receptor operated or voltage de-
pendent, and are they specific for calcium? Many mol-
luscan muscles are highly dependent upon extracellular
calcium to trigger contraction. For example, ACh-elicited
contractions of buccal muscle El (another muscle of the
Aplvsui buccal musculature whose contraction is poten-
tiated by 5-HT) fail within two minutes of removal of
extracellular calcium (Ram et ai, 1984b). Similarly, cal-
cium-dependence of ACh-elicited contractions have also
been demonstrated in a non-spiking muscle of Aplysia
gill (Reilly and Peretz, 1987) and in four different pro-

15 nA

Membrane Potential, mV
- 100 -50

5
0

L -5 nA
Current

Figure 9. Voltage-dependent outward current in whole-cell config-
uration. Pipet solution and external medium were the same as in Figure
8. Holding potential was -80 mV. Currents were elicited by 60-ms pulses
to various potentials, from -100 mV to +20 mV. given at intervals of
1 .2 s. Linear leak and capacitative transients have been subtracted. (Up-
per) Typical response to —30 mV. (Lower) Current voltage relationship
for peak current during the pulse.

DISSOCIATED MUSCLE FIBERS OF APLYSIA

281

VH=-80 mV

1 nA

10 ms

normal
calcium

Membrane potential (mV)

-T 1 nA

Figure 10. Voltage-dependent calcium current in whole-cell config-
uration. Pipet solution contained (in mA/) 20 NaCl, 406 CsCI. 2 MgCl:.
100 HEPES, 5 EGTA, 5 MgATP. 0.1 NaGTP. 10 glutathione. pH 7.0
(adjusted with CsOH). External medium was artificial seawater containing
(in m.U) 50 tetraethylammonium, 5 4-ammopyndine. 485 NaCl. 25
MgSO4, 25 MgCl:, 10 KC1, 10 CaCI,, and 10 HEPES, pH 7.8 (adjusted
with NaOH). For 0 calcium medium, the CaCl: was left out and all other
ingredients increased in concentration by 1%. Holding potential was
-80 mV. Currents were elicited by 60 ms pulses to various potentials,
from - 100 mV to + 10 mV at intervals of 1.2 s. Linear leak and capac-
itative transients have been subtracted. (Upper) Typical responses to
depolarization to -30 mV in the presence and absence of calcium.
(Lower) Current-voltage relationships for peak current in the presence
and absence of calcium.

boscis muscles of the marine snail Busycon (Huddart and
Hill, 1988; Huddart el ai. 1990a, b; Hill and McDonald-
Ordzie. 1979; Hill el ai, 1970). Another molluscan mus-
cle, the anterior byssus retractor muscle (ABRM) seems
less dependent upon extracellular calcium because ACh-
elicited contractions of ABRM are not abolished by the
removal of extracellular calcium for up to 10 min (Sugi
and Yamaguchi, 1976). Recent investigations of intra-
cellular calcium levels in ABRM using the calcium-sen-
sitive fluorescent indicator FURA-2 have shown that al-
though extracellular calcium may account for the majority
of the rise in intracellular calcium in response to cholin-

ergic activation, about 30% of the increase in intracellular
free calcium may be attributed to the release of stored
calcium (Ishii tf ai. 1988).

Studies on the influx of calcium-45 in response to ACh
have been used to determine whether calcium dependence
of contraction involves physical movement of calcium
into the cell. ACh stimulates influx of calcium-45 into
Aplysia buccal muscles El and 15 (Ram and Parti, 1985;
Gole el ai. 1987); however, ACh does not significantly
increase calcium-45 influx into ABRM (Tameyasu and
Sugi. 1976). The lack of significant ACh-stimulated cal-
cium-45 influx in ABRM not only contrasts with the ob-
servations in Aplysia muscles but also stands in apparent
contradiction with FURA-2 measurements showing a
significant extracellular dependence of the ACh-stimu-
lated rise in intracellular calcium in ABRM (see above).
Calcium-45 influx measurements are inherently more
variable than FURA-2 measurements. Therefore, the lack
of significant effect of ACh on calcium-45 influx in ABRM
probably reflects a relatively lower importance of calcium
influx in ABRM compared to Aplysia buccal muscles
rather than a complete absence of ACh-stimulated influx.

A possible route for calcium entry into muscle cells
during the response to ACh is via voltage-dependent cal-
cium channels. As discussed below, ACh causes depolar-
ization of molluscan muscles. Previous evidence that de-
polarization could activate voltage dependent calcium
channels included demonstrating that another depolar-
izing stimulus, a high potassium medium, causes con-
traction. High potassium induces contractions of Busycon
proboscis retractor muscles (e.g.. Huddart el ai. 1990a.
b), ABRM (e.g., TwarogandMuneoka, 1972), and. Aplysia
buccal muscles (Ram, unpub. data). Furthermore, high
potassium elicits contraction of isolated fibers, as described
in Aplysia buccal muscle (Figs. 2 and 3, this paper) and
in ABRM (Ishii el ai. 1986), unambiguously proving that
contraction elicited by high potassium is a direct effect
on single fibers and is not dependent upon either release
of neurotransmitters from nerve endings in the muscle
or mechanical or electrical coupling between fibers. Ishii
el ai (1988) has also used FURA-2 to show that high
potassium causes an increase in intracellular calcium that
is completely dependent upon extracellular calcium.

Blockers of voltage-dependent calcium channels reduce
the contractile responses produced by both ACh and high
potassium. Thus, Huddart el ai ( 1990b) found that dilti-
azam, verapamil, and nifedipine all decrease ACh-elicited
contractions of Busycon proboscis muscles. Similarly, ni-
fedipine reduces ACh-elicited contraction of Aplysia buc-
cal muscle (Ram and Liu, 1990).

The above indirect evidence for the existence of voltage-
dependent calcium channels is now supported in the
present paper by voltage clamp recordings of a voltage-
dependent inward current that is dependent on extracel-
lular calcium (Fig. 10). As described in a preliminary re-

282

J. L. RAM ET AL.

port (Ram and Liu, 1990). we have also demonstrated
that this current is partially inhibited by nifedipine and
completely blocked by lanthanum.

For voltage-dependent calcium channels to play a role
in mediating ACh responses, it must also be shown that
the range of membrane potentials at which a voltage-de-
pendent calcium current can be activated is within the
range of membrane potentials caused by ACh. As illus-
trated in Figure 10, voltage-dependent calcium current
begins to activate with depolarizations to —40 mV. In
other cells (data not shown), voltage-dependent calcium
current has been activated with depolarization to as little
as -50 mV. As discussed below, ACh can depolarize cells
to approximately -35 mV. Thus, the membrane potential
required to activate voltage-dependent calcium channels
is clearly within the range of depolarization produced by
ACh in Aplysia buccal muscles. This paper provides the
strongest evidence yet that activator calcium enters mol-
luscan muscle fibers during cholinergic stimulation by
voltage-dependent calcium channels.

This paper also initiates the analysis of receptor-oper-
ated channels activated by ACh. ACh causes depolariza-
tion of molluscan muscle fibers in clam heart (Wilkens
and Greenberg, 1973), ABRM (Twarog, 1954), Bmycon
proboscis muscles (Huddart ct ai, 1990b; Hill and Licis,
1985; Hill and McDonald-Ordzie, 1979), Aplysia gill
muscle (Reilly and Peretz, 1987) and Aplysia buccal mus-
cles (Ram ct ai. 1990). Detailed quantitative studies of
Aplysia buccal muscle revealed (a) an average resting po-
tential of -65 mV (Gole et ai. 1987), (b) no contraction
elicited with depolarizations less than approximately 10
mV above rest, (c) a non-linear relationship between con-
traction and depolarization in which increasing ACh be-
yond a certain concentration was accompanied by in-
creasing contraction with little or no further increase in
depolarization, and (d) maximal ACh-elicited depolariza-
tion of approximately 30 mV above rest (Ram et ai,
1990).

The limit on depolarization produced by ACh to only
30 mV above rest may result from several mechanisms.
One possibility is that the reversal potential for ACh-ac-
tivated channels is only 30 mV above rest. An alternative
explanation is that depolarization activates voltage-de-
pendent potassium channels that act as an effective brake
on further depolarization even in the face of activation
of more ACh-activated channels. Data in this paper show
that analysis of this question is feasible. As expected for
a depolarizing stimulus, ACh activates inward current
(Fig. 8). The reversal potential of the ACh response is
under investigation (Ram and Liu, 1990). Furthermore,
Figure 9 demonstrates that the voltage-dependent outward
current is activated within the voltage range elicited by
ACh. The voltage-dependent outward current is un-
doubtedly potassium because it is blocked by TEA and
4-AP outside the cell and Cs in the electrode (Fig. 10).

This paper also demonstrates that buccal muscle fibers
contain stretch-activated channels. Because these channels
were observed with on-cell patch electrodes containing
only KC1, the inward currents illustrated are almost cer-
tainly due to potassium current. Similarly, stretch-acti-
vated channels conducting primarily potassium ions have
been reported previously in molluscan neurons (Morris
and Sigurdson, 1989; Sigurdson and Morris, 1989) and
cardiac muscle (Brezden and Gardner, 1986; Sigurdson
ct ai. 1987). Stretch-activated channels that are somewhat
less selective for potassium have been reported in various
mammalian tissues, including skeletal muscle (Guharay
and Sachs, 1984) and smooth muscle (Kirberrt ai. 1988).

In future experiments it should be possible to determine
whether 5-HT potentiates contraction of dissociated
muscle fibers by modifying the ionic currents illustrated
here. One indication in a molluscan muscle that 5-HT
may change membrane currents of molluscan muscles is
that, in ABRM. 5-HT potentiates the rise in intracellular
calcium caused by 100 mA/KCl (Ishii et ai. 1989). The
rise in intracellular calcium in response to KC1 is com-
pletely dependent upon extracellular calcium (Ishii et ai,
1988) and is presumed to be due to voltage-dependent
calcium current, similar to the current described in this
paper in Figure 10. In addition, 5-HT might also be mod-
ifying potassium channels, receptor-operated channels,
and stretch-activated channels.

Acknowledgments

This work was supported by the Muscular Dystrophy
Association and NIH grant RR-08167. I am indebted to
R. B. Hill for his critical comments on the manuscript.

Literature Cited

Brezden, B. L., and D. R. Gardner. 1986. A potassium selective channel
in isolated Lymnaca stagnalis heart muscle cells. J. Exp. Bio/. 123:
175-189.

Cohen, J. L., K. R. Weiss, and I. Kupfermann. 1978. Motor control
of buccal muscles in Aplysia. J. Neitmphys. 41: 157-180.

Corey, D., and C. S. Stevens. 1985. Science and technology of patch-
recording electrodes. Pp. 53-68 'm Single Channel Recording, B. Sak-
mann and E. Neher, eds. Plenum Press, New York.

Cropper, E. C, M. \V. Miller, R. Tenenbaum, M. A. G. Kolks, I. Kup-
fermann, and K. R. Weiss. 1988. Structure and action of buccalin:
a modulator, neuropeptide localized to an identified small cardioac-
tive peptide-containing cholinergic motor neuron of Aplysia califor-
nicii. Proc. Nal. Acad. Sci. 85: 6177-6181.

Cropper, E. C., R. Tenenbaum, M. A. G. Kolks, I. Kupfermann, K. R.
Weiss. 1987. Myomodulin: a bioactive neuropeptide present in an
identified cholinergic buccal motor neuron of Aplysia. Proc. Nal.
Acad. Sci. 84: 5483-5486.

Gole, D.. I.. Munday, M. Kreiman, and J. L. Ram. 1987. Caffeine
effects on buccal muscles of Aplysia. Camp. Biochciu. Physial. 88C:
313-318.

Guharay, F., and K. Sachs. 1984. Stretch-activated single ion channel
currents in tissue-cultured embryonic chick skeletal muscle. / Physiol.
352:685-701.

DISSOCIATED MUSCLE FIBERS OF APLYSIA

283

Hill, R. B., M. J. Greenberg, H. Irisawa, and II. Nomura.
1970. Electromechanical coupling in a molluscan muscle, the radula
protractor of Busyci >n canaliciilatum J Exp Zoo/ 174:331-348.

Hill, R. B., and P. Licis. 1985. Lanthanum and caffeine affect both
membrane responses and force in a molluscan muscle, the radular
protractor of Busycon cana/iculatum. Comp. Biochem. Physio/ 82C:
363-376.

Hill, R. B., and P. K. McDonald-Ordzie. 1979. Ionic dependence of
the response to acetylcholine of a molluscan buccal muscle: the radula
protractor of Busycon cana/iculatum Comp Biochcm Physiol 62C:
19-30.

Huddart, H., D. D. Brooks, R. B. Hill, and R. I.ennard. 1990a. Diversity
of mechanical responses and their possible underlying mechanisms
in the proboscis muscles of Busycon canaliciilatum. J Comp. Physiol.
B 159: 717-725.

Huddart, H., D. D. Brooks, R. Lennard, and R. B. Hill. I990b. Unusual
responses of proboscis muscles of Busycon canaliculatum to some
calcium antagonist agents. J Comp. Physiol. B 159: 727-738.

Huddart, H., and R. B. Hill. 1988. Electrochemical uncoupling in a
molluscan muscle examined b> the sucrose gap technique. The effect
of calcium antagonist and agonist agents. J Comp. Physiol. B 158:
501-512.

Ishii, N., T. Takakuua, and K. Takahashi. 1986. Isolation of acetyl-
choline-sensitive smooth muscle cells from a molluscan catch muscle.
Comp. Biochem. Physiol. 84C: 1-6.

Ishii, N., A. W. M. Simpson, and C. C. Ashley. 1988. Carbachol and
KCl-induced changes in intracellular free calcium concentration in
isolated, fura-2 loaded smooth-muscle cells from the anterior byssus
retractor muscle of Mylilus edu/is Biochcm. Bmphys. Re*. Cumin.
153: 683-689.

Ishii, N., A. W. M. Simpson, and C. C. Ashley. 1989. Effects of 5-
hydroxytryptamine (serotonin) and forskolin on intracellular tree
calcium in isolated and fura-2 loaded smooth-muscle cells from the
anterior byssus retractor (catch) muscle of Mytilns din/is. Pflugers
Archiv4\4: 162-170.

Kirber. M. T., J. V. Walsh, Jr., J. J. Singer. 1988. Stretch-activated
ion channels in smooth muscle: a mechanism for the initiation of
stretch-induced contraction. Pflugers Arc/uv 412: 339-345.

Mandelbaum. D. E. 1980. Studies of the role of cyclic AMP and protein
phosphor} lation in the mediation of food arousal in Aplysia. Ph.D.
thesis. Columbia University. 154 pp.

Morris, C. E., and W. J. Sigurdson. 1989. Stretch-inactivated ion
channels coexist with stretch-activated ion channels. Science 243:
807-809.

Ram, J. I.. 1983. Neuropeptide activation of an identifiable buccal
ganglion motoneuron in Aplysia. Brain Res 288: 177-186.

Ram, J. L., G. S. Ajimal, D. Gole, K. A. Haller, and A. Williams.
1984a. Serotonin and forskolin enhance both magnitude of con-
traction and relaxation rate of Aplysia dorsal extrinsic muscle in-
dependently of acetylcholine receptor. Comp. Biochem. Physio/. 79C:
455-459.

Ram, J. L., S. Barmatoski, L.-X. Liu, and S. C. Mahaffey.
1990. Structure, contraction, and electrophysiological properties of
intact and dissociated smooth muscle fibers of Aplysia. Pp. 65 1-658
in Frontiers in Smooth Muscle Research Progress in Clinical and
Biological Research. Vol. 327. N. Sperelakis and J. D. Wood. eds.
Wilev-Liss. New York.

Ram, J. L., D. Gole, V. Shukla. and I.. Greenberg. 1983. Serotonin-
actuated adenylate cyclase and the possible role of cyclic AMP in
modulation of buccal muscle contraction in Aplysia. J Neurobiol.
14: 113-121.

Ram, J. L., and L.-X. Liu. 1990. Ion channels in Aplysia smooth muscle:
currents activated by voltage and acetylcholine (ACh). Soc. Netirosci.
Abstr. 16: 675.

Ram, J. L., and R. Parti. 1985. Regulation of calcium influx into buccal
muscles of Aplysia by acetylcholine and serotonin. /. Neurobiol. 16:
57-68.

Ram. J. L., and S. Patel. 1989. Skinned muscle fibers of Aplysia: po-
tentiation of contraction by "background" calcium and lack of effect
of cyclic AMP. Comp. Biochcm Physiol. 93A: 745-749.

Ram, J. L., U. A. Shukla, and G. S. Ajimal. 1981. Serotonin has both
excitatory and inhibitory modulatory effects on feeding muscles in
Aplysia J Neurobiol. 12:61 3-62 1 .

Ram, J. L., U. A. Shukla, R. Parti, and R. L. Goines.
1984b. Extracellular calcium dependence of contracture and mod-
ulation by serotonin in buccal muscle E 1 of Aplysia. J. Neurobiol.
15: 197-206.

Reilly, W. M., and B. Peretz. 1987. Excitation-contraction coupling
in a non-spiking smooth muscle in the gill of Aplysia. J Comp. Phys-
iol. 157B: 659-666.

Rosen, S. C., I. kupfermann, R. S. Goldstein, and K. R. Weiss.
1983. Lesion of a serotonergic modulatory neuron in Aplysia pro-
duces a specific defect in feeding behavior. Brain Res. 260: 1 50- 155.

Rosen, S. C., K. R. Weiss, R. S. Goldstein, and I. Kupfermann.
1989. The role of a modulatory neuron in feeding and satiation in
Aplysia. effects of lesioning of the serotonergic metacerebral cells. J.
Neurosci 9: 1562-1578.

Sigurdson, W . J., and C. E. Morris. 1989. Stretch-activated ion channels
in growth cones of snail neurons. / Neurosci 9: 2801-2808.

Sigurdson, W. J., C. E. Morris, B. L. Brezden, and D. R. Gardner.
1987. Stretch activation of a K+ channel in molluscan heart cells.
J Exp. Bu>l. 127: 191-209.

Sugi, H., and T. Yamaguchi. 1976. Activation of the contractile mech-
anism in the anterior byssal retractor muscle of Mytilns edulis. J.
Physiol.. Land. 257: 531-547.

Tameyasu, T.. and H. Sugi. 1976. Effect of acetylcholine and high ex-
ternal potassium ions on 4SCa movements in molluscan smooth
muscle. Comp. Biochem Physiol. 53C: 101-103.

Twarog, B. M., and Y. Muneoka. 1972. Calcium and the control of
contraction and relaxation in a molluscan catch muscle. Cold Spring
Harbor Symp. Quant Biol. 37: 489-503.

Twarog, B. M. 1954. Responses of a molluscan smooth muscle to ace-
tylcholine and 5-hydroxytryptamine. / Cell. Comp. Physiol. 44: 141-
164.

Weiss, K. R., J. L. Cohen, and I. Kupfermann. 1978. Modulatory con-
trol of buccal musculature by a serotonergic neuron (metacerebral
cell) in Aplysia. J. Neurophysiol. 41: 181-203.

Wilkens, L. A., and M. J. Greenberg. 1973. Effects of acetylcholine
and 5-hydroxytryptamine and their ionic mechanisms of action on
the electrical and mechanical activity of molluscan heart smooth
muscle. Comp. Biochem. Physio/. 45A: 637-651.

Zhang, F., and J. L. Ram. 1990. Serotonin (5-HT) potentiates con-
traction of dissociated fibers of Aplysia smooth muscle. Soc. Neurosci.
Abstr. 16: 1227.

Reference: Biol. Bull 180: 284-244. (April, 1941)

Photoresponsiveness of Aplysia Eye is Modulated by
the Ocular Circadian Pacemaker and Serotonin

JON W. JACKLET

Department of Biological Sciences, Neurobiology Research Center, State University
of New York at Albany, Albanv. New York 12222

Abstract. The eye of the sea hare, Aplysia, contains a
circadian pacemaker that controls rhythmic behaviors of
the animal. This report shows that the pacemaker controls
the photoresponsiveness of the eye as well. The electro-
retinogram (ERG) of the isolated eye-optic nerve prepa-
ration, evoked by brief green light pulses in otherwise dark
conditions, was recorded regularly, while the circadian
rhythm of compound action potential activity was con-
tinuously recorded from the optic nerve. The waveform
of the ERG changed systematically and rhythmically dur-
ing the circadian cycle. One wave component of the ERG
was prominent during the subjective night phase of the
rhythm when the compound action potential frequency
was minimal; and it was inconspicuous during the sub-
jective day phase of the rhythm when the compound ac-
tion potential frequency was maximal. Because eyes at-
tached to the central nervous system and isolated eyes
both exhibited the same rhythmic ERG changes, the cir-
cadian pacemaker in the eye is responsible for modulation
of the ERG. Addition of serotonin, a putative efferent
transmitter, to the bathing saline induced the ERG wave
component characteristic of the subjective night phase of
the rhythm. The threshold serotonin concentration was
10 7 AI. and serotonin had a long lasting effect.

Introduction

Each eye of Aplysia contains a circadian pacemaker
(Jacklet, 1969a) that produces a circadian rhythm in the
frequency of optic nerve (ON) autonomous compound
action potentials (CAPs). The CAP activity is produced

Received 9 August 1990; accepted 28 December 1990.

Non-standard abbreviations: CAP, compound action potential: ON,
optic nerve: CT, circadian time; ERG, electroretinogram; CM. culture
medium.

by the synchronous firing of a population of retinal pace-
maker neurons (Jacklet et ai, 1982), the axons of which
enter the optic nerve and project to the central ganglia
(Olson and Jacklet, 1985). This rhythm of CAP activity
is known to control rhythmic behaviors, such as loco-
motor activity-rest, because eyeless animals lack the well
defined circadian rhythm of activity-rest that normal an-
imals display (Strumwasser et at., 1979; Lickey et ai,
1977). Feeding behavior also exhibits a circadian rhythm
(Kupfermann, 1974). but the contribution of the ocular
circadian pacemaker to that rhythm has not been tested.
The ocular circadian pacemaker probably controls
rhythmic behavior by neural connections to central motor
control centers, or by affecting physiological processes in
the eye itself, although the mechanisms are not yet known.

An eye contains the photoreceptors needed to entrain
the ocular circadian rhythm to the solar day light-dark
cycles, because the rhythm is entrained by light-dark cycles
(Eskin. 1971), and the phase of the CAP frequency rhythm
is shifted by light pulses given during the subjective night,
yielding a phase response curve (Jacklet, 1974). The spe-
cific ocular photoreceptor responsible for the phase shifts
have not been identified. The eyes and other cephalic
photoreceptors also mediate simple phototactic behaviors
(see Jacklet, 1980).

The electroretinogram (ERG) has been recorded from
the eye by an extracellular pipette placed in the retina
(Jacklet, 1969b) and by a suction electrode placed on the
cornea (Eskin, 1977; Eskin and Maresh, 1982). The ERG
amplitude is increased by serotonin treatment or by ON
stimulation, which presumably releases serotonin from
the terminal in the eye of the efferent neurons (Eskin and
Maresh, 1982). Eyes contain several types of photorecep-
tors, in addition to the pacemaker neurons involved in
the circadian rhythm. The largest and most numerous
type is the R photoreceptor (Jacklet and Rolerson, 1982)

284

ORCADIAN CONTROL OF PHOTORESPONSIVENESS

285

that responds to light with a graded prolonged depolar-
ization. The light response of this receptor is largely re-
sponsible for the ERG (Jacklet, 1969b).

Rhythmic changes in the ERG ofAplysia. or any other
gastropod, have not been reported, to my knowledge, even
though ERG circadian rhythms of other animals are well
known. For example, the ERG amplitude rhythm of the
compound eye of Limit/us has been intensively studied
(Barlow. 1983). Photosensitivity increases by 20-100 fold
during the night, and the rhythm, driven by a central
nervous system circadian pacemaker, is mediated by ef-
ferent innervation. The efferent transmitter appears to be
octopamine (Battelle el ai, 1989; Kass and Barlow, 1984).

I report here that the ERG waveform recorded from
the isolated Aplysia eye changes rhythmically, and the
rhythm maintains a stable phase relationship with the cir-
cadian rhythm in CAP frequency. Thus, the ocular cir-
cadian pacemaker affects physiological processes within
the eye itself. The waveform of the ERG that is charac-
teristic of subjective night is induced by the addition of
serotonin to the bathing saline during subjective day,
mimicking the influence of the circadian pacemaker dur-
ing subjective night.

Materials and Methods

Individuals of Aplysia californica were obtained from
Marinus, Inc., Long Beach. California, and kept in Instant
Ocean tanks maintained at 16°C under light-dark cycles
of 12:12. Two isolated preparations were used: the eye-
ON, and the eye attached to the cerebral ganglion by the
ON. They were placed in a recording dish (100 ml)
equipped with tubing (polyethylene, PE) electrodes
embedded in the RTV silicone rubber base. Preparations
were maintained in a dark box at 18°C for several days
of recording. The ON was drawn, by negative pressure
applied by a syringe, into one electrode (PE 10) that was
used to record CAPs, and the eye was drawn into another
electrode (PE 50) for ERG recording. The negative pres-
sure was released, and the eye and ON remained in place
for recording. The eye was drawn completely into the
electrode so that the activity of all retinal cells could be
recorded.

The eye is spherical and about 0.7 mm in diameter. It
has a central lens, a poorly developed cornea, and a com-
plex retina containing photoreceptors and neurons (see
Jacklet el ai, 1982; Herman and Strumwasser, 1984).
ERG electrode recordings routinely picked up small CAPs.
Activity was amplified with an A-M Systems model 1700
(gain, XI 000; bandpass 0.1-1000 Hz.) and displayed on
a Tektronix 5300 oscilloscope. ERGs and CAPs were re-
corded and stored with Asystant+ software in an IBM
AT computer, and the data were plotted with a Hewlett-
Packard model 7470A plotter. The latencies of the ERGs

and CAPs were measured from computer generated plots
of the recordings.

Pairs of eyes from eight animals were used in the cir-
cadian rhythm study. Four eyes attached to the cerebral
ganglion yielded 1 1 circadian cycles of data, and 7 isolated
eyes yielded 1 2 circadian cycles of data. Eyes produced
three to four cycles of CAP rhythm data routinely, but
complete ERG data were not collected from all eyes. Pairs
of eyes from seven animals were used in the serotonin
experiments.

Light pulses were produced by driving an Archer green
LED with 1 -2 s ( 1 5, 30 or 70V) pulses from a Grass model
S88 stimulator. Pulses were given at regular intervals of
10 min, 30 min, or 1 h in otherwise constant darkness.
The LED was positioned 3 cm away and directly over the
eye drawn into the PE tubing. The intensity incident on
the eye was measured by placing the sensor of a radi-
ometer/photometer (United Detector Corp., Model 40X)
3 cm from the LED. Light intensities for voltage pulses
used to drive the LED were 0.6 ^W/cm2 at 1 5 V, 0.8 ^ W/
cm2 at 30 V, and 3 ^W/cm2 at 70 V. The Aplysia eye has
high sensitivity to green light and the threshold intensity
is about 0.06 ^W/cm2 at 500 nm (Jacklet, 1980).

Artificial seawater ( ASW) was made up of the following
salts in millimoles/liter: NaCl, 425; KC1, 10; CaCl:, 10;
MgCl:, 22; MgSO4, 26; NaHCO,, 2.5; adjusted to pH
7.8. Culture medium (CM) was composed of ASW, 20%
Aplysia blood, and 100 U/ml penicillin, 0. 1 mg/ml strep-
tomycin. Serotonin (Sigma, creatinine sulfate) was added
to the CM to final concentrations of 10 7, 10", or 10 -
M. A 10-ml chamber fitted with polyethylene tubing for
changing solutions inside the dark box was used for the
serotonin experiments. The CM was removed entirely by
applying suction to the polyethylene tubing and was re-
placed within a few seconds with the serotonin solution.
The statistically significant differences between average
latencies were determined using a two tailed / test. The
level of significance used was a = .05.

Results

Rhythmic changes in the ERG

The basic ERG waveform recorded in these experi-
ments was triphasic, even though the entire eye was pulled
into the tubing electrode. The waveform consisted of a
sharply rising wave, followed by a slower wave of opposite
polarity and a weak slow third phase. There was no ob-
vious "off" response. This triphasic ERG waveform is
very similar to the ERG recorded by Eskin (1977) on a
Grass polygraph using a suction electrode applied on or
near the cornea.

The latency of the ERG in the present study was about
0.9 s in response to a 1 ^W/cm2 green light pulse. This
compares well with the latency of about 0.9 s obtained

286

J. W. JACKLET

3 sec.

1 -,

a
E
to -

hr.

50O-,

hr.

ORCADIAN CONTROL OF PHOTORESPONS1VENESS

287

earlier in response to about 6 lux (<1 /jW/cm:) white
light (Jacklet, 1969b). This is a rather long latency, but
similar to those of other gastropod eyes under similar
conditions. Latency is 1-3 s for Otala, a land snail (Ciliary
and Wolbarsht, 1967), and 0.2-0.5 s for the well-formed
eye of Stronibu.s, another marine gastropod (Gillary.
1974). The Aplysia eye latency is about 0.4 s in response
to 600 lux white light (Jacklet, 1969b).

The ERG waveform was usually smooth during the
subjective day, as shown in Figure 1 A at CT2.5. CT refers
to circadian time, which is measured from the actual pe-
riod of the free-running circadian rhythm of interest, in
this case the CAP frequency rhythm shown in Figure ID.
The period is divided into 24 equal units. Circadian time
0-12 is subjective day, and CT 12-24 is subjective night.
The phase point in the CAP rhythm corresponding to
subjective dawn (CT 0) is the CAP frequency at '/: max-
imum marked in Figure ID. During subjective night, the
waveform developed a notch following the initial wave,
as shown in Figure 1 A at CT 20.5. For convenience, the
waveform has been labeled in the figure. The initial wave
is A, followed by the B-C wave or notch, followed by the
slower D wave. The B-C wave has not previously been
reported. This waveform is typical of the ERGs recorded
during subjective night in these experiments.

The ERGs shown in Figure 1 A were recorded from the
same isolated eye at different phases (CT 20.5 and CT
2.5) in the circadian cycle. Similar ERGs were recorded
from the other eye of the same animal at the same phases,
even though the eye was attached to the cerebral ganglion
by the ON (Fig. IB). Being attached to the cerebral gan-
glion made no apparent difference in the waveform of the
ERG or in the rhythmic changes in the waveform. In
general, eye pairs exhibited very similar ERG waveforms,
whether or not the eye was attached to the cerebral gan-
glion.

The ERG B-C wave changed rhythmically during the
circadian cycle. It virtually disappeared during subjective
day and reappeared during subjective night. The relative
amplitude of the B-C wave, measured from the peak of
the B to the peak of the C wave, cycled continuously, as
shown in Figure 1C for the B-C wave of the isolated eye

used in Figure 1 A. When the cycling of the relative am-
plitude of the B-C wave is compared to the CAP frequency
rhythm plotted in Figure ID, the maximum B-C wave
amplitude seems to occur at about CT 20 and to coincide
with minimal CAP frequency during subjective night. The
period of both rhythms is about 24 hours.

A few eyes exhibited weak rhythmic changes in the A
wave of the ERG, but either they did not persist over two
cycles, or they were not sufficiently robust to be considered
true rhythms. The D wave was very stable and showed
no rhythmicity.

The ERG waveform recorded from eyes of different
animals varied somewhat, but the basic waveform could
always be observed. One of the most extreme waveforms
is shown in Figure 2A, B. This eye was attached to the
cerebral ganglion, but the paired isolated eye exhibited
the same ERG waveform and changed rhythmically. The
A, B, C. and D waves are readily apparent. But the A
wave is relatively small and the B-C wave is huge. The
relative amplitude of the B-C wave changed rhythmically,
but it never completely disappeared. It remained in the
appropriate phase relationship with the CAP frequency
rhythm for many cycles (Fig. 2C, D). The period of both
rhythms is about 23 hours.

In most preparations, the latency (time from stimulus
onset to '/: peak of A wave) of the ERG A wave was shorter
during the subjective night, when the B-C wave was
prominent, than during the subjective day. For example,
in Figure 1A and B the latency is about 100 ms shorter
during subjective night. The ERG latencies for light pulses
given at CT 19-22, during subjective night, were com-
pared to latencies obtained at CT 1-4, during subjective
day, for 1 2 circadian cycles for both isolated eyes and eyes
attached to the cerebral ganglion. Mean latency and the
standard error of the mean (SEM) were calculated, and /
tests were performed to determine whether mean differ-
ences were statistically significant. The average latency
for isolated eyes was 1000 ms (SEM, 20; N, 34) during
CT 19-22, and 1050 ms (SEM, 10; N, 39) during CT 1-
4. The means were significantly different at the .05 level.
The average latency for eyes attached to the cerebral gan-
glion was 920 ms (SEM, 20; N, 36) during CT 19-22, and

Figure 1. Rhythmic changes in the ERG. ERG waveforms recorded from the same isolated eye during
subjective night (CT 20.5) and during subjective day (CT 2.5) are shown in A. The A. B. C. and D waves
are labeled. The 2-s light pulse (3 /jW/cm2) is indicated by the black bar. The other eye from the same
animal, but attached to the cerebral ganglion, exhibited the ERG waveform shown in B taken at CT 20.5
and CT 2.5 as in A. Vertical scales in A and B are 50 /jV and 25 ^V per division. The changes in relative
amplitude of the B-C wave of the ERGs recorded from the isolated eye are plotted in C using the same time
scale as the CAP frequency rhythm shown in D. Arrows identify the relative amplitude points of the B-C
wave in C, and the CAP frequency in D corresponding to the ERGs in panel A taken at CT 20.5 and CT
2.5. Time reference for CT 0 is the thin labeled line in D that occurs at 'A the maximum CAP frequency.
The projected light-dark cycle experienced by the animal before dissection is shown by the white/crosshatched
bar.

288

J. W. JACK.LET

CT 15

sec.

400

ORCADIAN CONTROL OF PHOTORESPONSIVENESS

289

970 ms (SEM, 20; N, 34) during CT 1 -4. The means were
significantly different at the .05 level. Although the mean
A wave latencies are shorter for attached eyes than for
isolated eyes, both show similar shifts in ERG latency
during the circadian cycle.

To test for the involvement of chemical synapses in
the circadian pacemaker modulation of the ERG wave-
form, two eyes were subjected to ASW containing 10 4
M Ca++ and 10 ' M Mg++. This treatment drastically
reduced the ERG as expected (Eskin, 1977). Thus, a re-
liable test for the involvement of chemical synapses was
not possible.

Removal of the ON from the isolated eye did not in-
terrupt cycling of the ERG waveform, but it did reduce
the number of cycles that an eye exhibited. The ON was
cut away from four eyes at the bases of their retinas, and
ERGs were recorded as usual. Small CAPs recorded with
the ERG electrode verified that the CAP circadian rhythm
continued. Two of the eyes remained active for two cycles,
and both exhibited cycling of the B-C wave, suggesting
that the ON itself is not necessary for circadian cycling
of the ERG.

Serotonin induces the ERG B-C wave

Serotonin induced the B-C wave in eyes at circadian
times when it was not normally expressed. The induced
B-C wave closely resembled the wave characteristic of
subjective night. As shown in the example of Figure 3,
the B-C wave was well developed at CT 20, as expected,
and it became inconspicuous later, at CT 0.5, during sub-
jective day. A short time later at CT 2.0. and just 1 3 min
after the addition of 10~6 M serotonin to the bathing so-
lution by perfusion, the induced B-C wave (Fig. 3C) was
nearly identical to the B-C wave recorded at CT 20 (Fig.
3A). Continued exposure to serotonin enhanced the B-C
wave (Fig. 3D) beyond the amplitude of the subjective
night B-C wave. The effects of serotonin were long lasting
and reached a maximum in about 1 h. Once induced, the
B-C wave required several hours of washout before it re-
turned to normal. Serotonin also enhanced the B-C wave
of eyes tested during subjective night when the wave was
already present.

During exposure to serotonin, the autogenous CAP
frequency was reduced as expected from previous work

(CorrenU'M/.. 1978; Eskin and Maresh, 1982). The num-
ber of CAPs evoked by the light pulse, especially those
evoked several seconds after the pulse, were also reduced
as shown in Figure 4B. However, compared to the re-
sponse just before the addition of 10~6 M serotonin, there
was no change in the latency of the initial CAP produced
by a light pulse. The mean latency for the initial CAP in
the light response was 1.40 s (SEM, 0.14; N, 7) before
serotonin treatment, and 1 .36 s (SEM, 0.05; N, 7) 1 5 min
after serotonin treatment. A / test showed that these means
were not significantly different. The initial CAP occurs at
about the same time as the B-C wave, and small deflections
on the ERG waveform caused by CAPs are clearly visible
(Fig. 4A, B). During serotonin treatment, the CAP de-
flections on the ERG waveform, as well as the size and
timing of the initial CAP during the light response, re-
mained the same. Thus, changes in the light-evoked ac-
tivity of the pacemaker neurons that produce the CAPs
are not likely to account for the B-C wave induced by
serotonin.

Serotonin shortened the latency of the ERG and oc-
casionally increased the amplitude of the A wave. Both
Figures 3 and 4 show a substantial decrease in the latency
and increase in the A wave. The average ERG latency
before serotonin was 960 ms (SEM, 20; N, 9); 15 min
after the addition of 10 6 M serotonin it was 900 ms (SEM,
30; N, 9), and 45 min after an addition of serotonin it
was 860 ms (SEM 40; N, 7). The difference in mean la-
tency was not significant at 1 5 min, but at 45 min it was
significantly different at the .05 level. The average am-
plitude of the A wave increased only 1.1 times at 45 min.
At the threshold concentration (10 7 M) for ERG B-C
wave induction, the average latency decreased by only 25
ms (N, 4), and the A wave amplitude did not change.

Discussion

ERG waveform changes

A major finding of our study is that the ERG changes
systematically and rhythmically during the circadian cycle
of CAP frequency. One component of the ERG, the B-C
wave, is prominent during the subjective night phase of
the rhythm when the CAP rate is minimal, and incon-
spicuous during the subjective day phase when the CAP

Figure 2. Rhythmic changes in the ERG of an eye attached to the cerebral ganglion. Waveforms char-
acteristic of subjective night (CT 15) and subjective day (CT 3) for the same eye attached to the cerebral
ganglion by the ON are shown respectively in A and B. The 1-s light pulse (3 /jW/cm2) is indicated by the
black bar. Vertical scales in A and B are 50 j/V per division. The B-C wave became very prominent in the
eyes of this animal after several days of recording. C shows the changes in relative amplitude of the B-C
wave plotted on the time scale of the CAP frequency rhythms in D. Arrows in C and D mark the times at
which the ERGs shown in A and B were taken. Time reference for CT 0 is the thin labeled line in D at '/:
the maximum CAP frequency. The projected light-dark cycle experienced by the animal before dissection
is shown by the white/crosshatched bar. Light pulse is indicated by black bar in panels A, B.

290

J. W. JACKLET

•ec.

Figure 3. Changes in the ERG of an isolated eye induced by serotonin. The ERG waveform recorded
at CT 20 with a characteristic B-C wave is shown in A. At CT 0.5 the ERG had changed to the subjective
day waveform, lacking the B-C wave. Thirteen min after the addition of 10~6 M serotonin, the ERG shown
in C with a prominent B-C wave was recorded. The B-C wave progressively increased until the maximum
response in D was observed at 43 min after the addition of serotonin. At that time the latency of the A wave
had decreased about 100 ms, and the A wave amplitude had increased 140%. Vertical scale is 20 »V per
division. The black bar on the time scale marks the 1-s light (.8 nW/cnr) pulse.

rate is maximal. Thus, the maximal B-C wave occurs dur-
ing minimal CAP rate in an antiphase relationship. The
B-C wave decreases in size sharply during the increase in
CAP rate at subjective dawn, suggesting that a high CAP
rate might suppress the B-C wave. However, the causal
relationship between these events has not been deter-

mined. Isolated eyes, and eyes attached to the cerebral
ganglion, exhibited similar ERG rhythms suggesting that
the B-C wave pacemaker resides within the eye and that
it is likely to be the CAP circadian pacemaker. The in-
duction of the B-C wave by serotonin is an intriguing
observation that will be addressed later in this discussion.

ORCADIAN CONTROL OF PHOTORESPONSIVENESS

291

sec.

Figure 4. Changes in the ERG and CAP frequency induced by serotonin. Simultaneous recordings were
made of CAPs (upper traces) and the ERG (lower trace) in response to 1-s light pulses (dark bar). Responses
at CT 1.5 appear in A. CAPs appear on the ON trace and as small deflections on the ERG trace as well. At
CT 2.5, 43 min after addition of 10~6 M serotonin, the B-C wave seen in B was prominent, the latency had
decreased about 100 ms, and the number of CAPs evoked decreased. Black bars on time scale mark the 1-
s light (.8 MW/cnr) pulse. Vertical scales are 50 /W per division.

292

J. W. JACKLET

The ERG B-C wave characteristic of subjective night
has not been previously reported. Several years ago, I
looked for rhythmic changes in the ERG A wave ampli-
tude because ERG amplitude changes are well known in
other animals (see Barlow, 1983), but did not find con-
sistent rhythms. In the present study, the use of computer
assisted recording to compare ERG waveforms soon re-
vealed that the B-C wave occurs and changes rhythmically.
It also revealed that the A wave latency changes rhyth-
mically, but that the A wave amplitude does not. Average
latency differences of 50 ms during the cycle were found
for both isolated and attached eyes.

The ERG recordings were made from eyes completely
pulled into the tubing electrode, so signals could be re-
corded from any of the responding cell types in the eye,
including cells in the basal retina. Previous work had
shown that the largest photoresponses, recorded with a
glass pipette in the retina, were corneal negative and were
obtained near the distal segments of the large photore-
ceptors (Jacklet. 1969b). In the present study, light from
the LED reached all surfaces of the isolated eye and was
not restricted to the pathway through the cornea and lens.

The photoreceptor organization of the Aplysia retina
was investigated with localized illumination by Block and
McMahon (1983). They illuminated (100 lux, white light)
the distal segments of the photoreceptors surrounding the
lens and, as a result, unitary ON activity without CAPs
was evoked in the ON. Illumination of the basal retina
produced CAPs. Block and McMahon concluded that
chemical synaptic inhibition, especially the inhibitory ac-
tion of the receptor layer onto the CAP generating neu-
rons, shapes in part the light responses of the isolated eye.
Other evidence of synaptic inhibition in the retina is pro-
vided below.

Retinal cells that may contribute to the ERG B-C wave

The ERG consists of a sharply rising A wave, a rhyth-
mically changing B-C wave, and a stable D wave. The A
wave is likely to be caused by the R type photoreceptors
with microvillous distal segments adjacent to the lens
(Jacklet. 1969b; Jacklet and Rolerson, 1982). Intracellular
recordings show that dark adapted R photoreceptors re-
spond to white light pulses of 600 lux after a latency of
400 ms, comparable to the ERG latency of 400 ms at that
intensity (Jacklet, 1969b); the response is a prolonged de-
polarization of 60-70 mV (Jacklet and Rolerson, 1982).
Light adapted, but not dark adapted, R photoreceptors
have a notch on the rising phase of the depolarization.
The notch is probably not responsible for the B-C wave
because it occurs early during the A wave, and because
ERGs were recorded at interstimulus intervals of up to 1
h when the R photoreceptor are dark adapted and not
expected to have a notch. Some R photoreceptors display

prolonged hyperpolarization following the initial depo-
larization (see Fig. 3 in Jacklet and Rolerson. 1982).
However this hyperpolarization is also unlikely to cause
the B-C wave because it continues much longer than the
B-C wave. Responses from R photoreceptors have not
been studied throughout the circadian cycle, especially
not during the subjective night when the B-C wave occurs,
so changes that might account for the B-C wave have not
been observed.

Light responses of R photoreceptors are not completely
blocked by low Ca++ and high Mg* * ASW, but the resting
potential is decreased, and the light-evoked depolarization
is reduced and prolonged (Jacklet and Rolerson, 1982).
This should account for the reduction of the ERG in low
Ca+4 and high Mg++ ASW previously observed (Eskin,
1977) and confirmed in this study.

The pacemaker neurons (or secondary neurones. Jack-
let et a/., 1982) responsible for the CAPs also respond to
light. They depolarize and fire synchronous action poten-
tials that are correlated 1 : 1 with the ON CAPs. As shown
in this study. CAPs produce small but observable deflec-
tions on the ERG waveform at all phases of the circadian
cycle, but they are tiny compared to the B-C wave, and
none are synchronized with the B-C wave. The light re-
sponses of the pacemaker neurons themselves seem un-
likely to contribute to the B-C wave. However, the B-C
wave is most conspicuous during the phase of the circadian
cycle when the autogenous CAP frequency is low or ab-
sent. Perhaps low autogenous CAP activity creates the
conditions necessary for the B-C wave to occur. The re-
lationship between autogenous CAP activity and expres-
sion of the B-C wave has not yet been tested directly.

A retinal cell type that may contribute to the ERG B-
C wave is the H photoreceptor (Jacklet and Rolerson,
1982). Its typical light response is a volley of action po-
tentials followed by brisk hyperpolarization, and then de-
polarization accompanied by action potentials. The hy-
perpolarization occurs just after the initial photoresponse,
at about the time that the B-C wave of the ERG is oc-
curring. The H cell hyperpolarization appears to be syn-
aptically evoked, because electrical stimulation of the ON
evokes a similar sharp hyperpolarization (Jacklet and
Rolerson, 1982). This cell type may be involved in shaping
the light responses observed by Block and McMahon
(1983) during selective illumination. The ERG B-C wave
might be produced by enhanced inhibitory synaptic in-
teractions within the retina controlled by the circadian
pacemaker. Such enhanced interactions might improve
the visual performance of the eye.

A determination of the retinal cell types in Aplysia that
contribute to the rhythmic B-C wave must await a sys-
tematic intracellular study of cellular light responses
throughout the circadian cycle, preferably with simulta-
neous ERG recordings.

ORCADIAN CONTROL OF PHOTORESPONSIVENESS

293

The eye of a marine gastropod. Stwmhux, may share
some of the features of the Aplysia eye photoresponses,
including the B-C wave. The ERG exhibits two peaks of
negativity that are separable under certain conditions of
light and temperature (Gillary, 1974). The second peak
resembles the B-C wave. This eye is 3 times the diameter,
and contains about 100 times as many cells, as the Aplysia
eye. It appears to lack circadian pacemaker neurons, and
changes in the photoresponse during the circadian cycle
have not been explored to my knowledge. This retina
contains two types of depolarizing cells and one hyper-
polarizing type (Quandt and Gillary, 1979), similar to the
Aplysia retina. One depolarizing cell type (Type II) exhibits
two peaks of depolarization that are similar, but of op-
posite polarity, to the ERG waveform (Quandt and Gil-
lary, 1980).

Role of serotonin

Serotonin has been shown by Eskin and Maresh (1982)
to increase the first wave (A wave in this study) of the
Aplysia ERG when it is recorded with a suction electrode
applied to the cornea. They did not see a B-C wave. That
may be due to differences in recording methods, because
they made polygraph recordings and did not report laten-
cies. They found an average increase in the ERG ampli-
tude of 63% after 20 min in 10~6 M serotonin, and a
threshold concentration near 10~7 M. Dopamine, acetyl-
choline, and octopamine were tested but did not produce
consistent ERG changes. They also reported a 20% in-
crease in the ERG in response to ON stimulation and
proposed that the stimulation might cause the release of
serotonin from efferent terminals in the eye. Terminals
have been identified by serotonin antisera (Goldstein el
al. 1984; Takahashi el ai, 1989).

The effect of serotonin on the ERG suggests that cyclic
nucleotide second messengers may be involved. Cyclic
AMP mediates many of the serotonin effects on short-
and long-term central synapses in Aplysia (Kandel and
Schwartz, 1982), and serotonin phase shifts the CAP
rhythm (Corrent et al, 1978) by a mechanism involving
cAMP (Eskin et ai, 1982). In addition, cGMP mimics
the effect of light on the circadian pacemaker by inducing
phase shifts of the CAP rhythm (Eskin et ai, 1984). During
the cGMP treatment, the membrane potentials of R pho-
toreceptors were not altered, but changes in photorespon-
siveness were not explored. A ten minute exposure to light
increased the cGMP level by 50%.. Cyclic GMP may be
elevated, either as a consequence of photoresponses, or
because it is involved in the phototransduction process
(Eskin el ai, 1984). If any rhythmic changes in the levels
of cyclic nucleotides occur, they might be involved in the
B-C wave and the A wave latency changes.

Serotonin does not appear to induce the B-C wave by
a direct effect on the initial light response of pacemaker

neurons, because the initial CAP that most nearly coin-
cides temporally with the B-C wave is unaffected by se-
rotonin. Thus, serotonin does not allow expression of the
B-C wave by suppressing the light-induced pacemaker
neuron activity. However, the phase of the circadian cycle
during low or zero CAP frequency is associated with
expression of the B-C wave, and serotonin does suppress
autogenous CAP activity. Serotonin may create the nec-
essary conditions for expression of the B-C wave, in part,
by suppressing autogenous CAP activity.

Because serotonin induces the ERG B-C wave and the
reduction of the A wave latency, one may ask how it might
be involved in circadian control of the ERG. Serotonin
may just be mimicking a natural process, but because
there are efferent synaptic terminals containing serotonin
in the eye, they may well be involved. Because isolated
eyes show circadian rhythms in the B-C wave, the control
of serotonin release from the efferent terminals by central
neurons is eliminated. But how then might serotonin be
released by activity within the eye? Could processes con-
trolled by the circadian pacemaker in the eye release se-
rotonin? Because the appearance of the B-C wave is as-
sociated with minimal CAP frequency, release cannot be
a direct effect of CAP activity. To produce the appropriate
response, autogenous CAP activity would have to suppress
serotonin release, and inactivity would have to promote
release. Otherwise another process controlled by the cir-
cadian pacemaker must be involved.

Acknowledgments

I thank Mark Goldberg for excellent technical assis-
tance. Research supported by NSF grant BNS 88-19773
toJ.W.J.

Literature Cited

Barlow, R. B., Jr. 1983. Circadian rhythms in the Limulus visual sys-
tem. J. Neumsci. 3: 856-870.

Battelle, B-A., J. A. Evans, and S. C. Chamberlain. 1982. Efferent
fibers to Limulus eyes synthesize and release octopamine. Science
216: 1250-1252.

Block, G., and D. McMahon. 1983. Localized illumination of the
Aplysia and Bulla eye reveals new relationships between retinal layers.
Brain Res 265: 134-137.

Corrent, G., D. McAdoo, and A. Eskin. 1978. Serotonin shifts the phase
of the circadian rhythm from the Aplysia eye. Science 202: 977-979.

Eskin, A. 1971. Properties of the Aplysia visual system: in vitro en-
trainment of the circadian rhythm and centrifugal regulation of the
eye. Z vergl. Physiologic 74: 353-371.

Eskin, A. 1977. Neurophysiological Mechanisms involved in photo-
entrainment of the circadian rhythm from the Aplysia eye. J. Neu-
robtoi 8: 273-299.

Eskin, A., and R. Maresh. 1982. Serotonin or electrical optic nerve
stimulation increases the photosensitivity of the Aplysia eye. Comp.
Biochem. Physiol. 73C: 27-31.

Eskin, A., J. S. Takahashi, M. Zatz, and G. D. Block. 1984. Cyclic
guanosine 3':5'-monophosphate mimics the effects of light on a cir-
cadian pacemaker in the eye of Aplysia. J Neurosci. 4: 2466-2471.

294

J. W. JACKLET

Eskin, A., G. Corrent, C. Y. Lin, and J. McAdoo. 1982. Mechanism
for shifting the phase of a circadian rhythm by serotonin: involvement
of cAMP. Proc. Nat. Acad, Sci. USA 79: 660-664.

Ciliary, H. L. 1974. Light-evoked electrical potentials from the eye
and optic nerve of Stromhus: response waveform and spectral sen-
sitivity. J. Exp. Bioi 60: 383-396.

Ciliary, H. L., and M. L. Wolbarsht. 1967. Electrical responses from
the eye of a land snail. Rev. Can. Biol 26: 125-134.

Goldstein, R., H. B. Kistler, H. W. M. Steinbusch, and J. H. Schwartz.
1984. Distribution of serotonin-immunoreactivity in juvenile
Aplysia. J. Neurosci. 11: 535-547.

Herman, K. G., and F. Strumwasser. 1984. Regional specializations in
the eye of Aplysia. a neuronal circadian oscillator. J. Camp. Neural.
230: 593-613.

Jacklet, J. W. 1969a. Circadian rhythm of optic nerve impulses re-
corded in darkness from isolated eye of Aplysia. Science 164: 562-
563.

Jacklet, J. W. 1969b. Electrophysiological organization of the eye of
Aplysia. J. Gen. Physio/. 53: 21-42.

Jacklet, J. \V. 1974. The effect of constant light and light pulses on
the circadian rhythm in the eye of Aplysia. J Comp. Physio/ 90:
33-45.

Jacklet, J. \V. 1980. Light sensitivity of the rhinophores and eyes of
Aplysia. J. Comp. Physiol. 136: 257-262.

Jacklet, J. W., and C. Rolerson. 1982. Electrical activity and structure
of retinal cells of the Aplysia eye. II. Photoreceptors. J. Exp. Bio/
99:381-395.

Jacklet, J. W., L. Schuster, and C. Rolerson. 1982. Electrical activity

and structure of retinal cells of the Aplysia eye. I. Secondary neurones.

/ Exp. Biol. 99: 369-380.
kandel, E. R., and J. H. Schwartz. 1982. Molecular biology of learning:

Modulation of transmitter release. Science 218: 433-443.
Kass, L., and R. B. Barlow, Jr. 1984. Efferent neurotransmission of

circadian rhythms in Limulus lateral eye. J Neurosci. 4(4): 908-9 1 7.
kupfermann. I. 1974. Feeding behavior of Aplysia: A simple system

for the study of motivation. Behav. Biol 10: 1026.
Lickey, M., J. Wozniak, G. Block, D. Hudson, and G. Augter.

1977. The consequences of eye removal for the circadian rhythm

of behavioral activity in Aplysia. J. Comp. Physiol 118: 121-143.
Olson, L., and J. W. Jacklet. 1985. The circadian pacemaker in the

eye of Aplysia sends axons throughout the central nervous system.

J. Neurosci. 5(12): 3214-3227.
Quandt, F. N., and H. L. Gillarj. 1979. Classes of light-evoked response

in the retina of Stmmhus. J. Exp. Biol. 80: 287-297.
Quandt, F. N., and H. L. Ciliary. 1980. Separable phases of light-evoked

depolarizations in the retina of Slrombus. J E.\p Biol 84: 137-148.
Strumwasser, F., R. Alvarez, D. Viele, and J. Woolum. 1979. Structure

and function of a circadian oscillator system. Pp. 5 1 -56 in Biological

Rhythms and their Central Mechanisms. M. Suda, O. Hayaishi. and

H. Nakagawa. eds. Elsevier/North Holland Biomedical Press.
Takahashi, J. S., D. E. Nelson, and A. Eskin. 1989. Immuno-

cytochemical localization of serotonergic fibers innervating the ocular

circadian system of Aplysia. Neuroscience 28: 139-147.

Reference: Bwl Bull. 180: 295-300. (April, 1991)

Control of Central and Peripheral Targets by a
Multifunctional Peptidergic Interneuron

DAVID J. PRIOR

Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona 86011

Abstract. In the terrestrial slug. Limax maximus. feed-
ing activity and cardiovascular function have been shown
to be correlated. For example, in intact animals, both
feeding responsiveness and heart activity are suppressed
during dehydration (Grega and Prior, 1986). The paired
peptidergic buccal ganglion neurons RBI and LB1 have
dramatic modulatory effects on both the feeding motor
program (FMP) and the force of heart contraction (Wels-
ford and Prior, 1991). The Bl neurons appear to contain
the small cardioactive peptides (SCPs). Observations have
a frequency dependent excitation of both the FMP and
the heart demonstrated by intracellular stimulation of B 1 .
Thus, interneuron Bl may serve to mediate the coincident
modulation of multiple responses to physiological stresses.

Introduction

Environmental stress or a change in the physiological
state of an organism very often results in a concerted array
of regulatory responses. Such responses usually include
modification of behavioral patterns, or the level of be-
havioral responsiveness, as well as changes in physiological
functions such as cardiac output and respiratory activity.
With the use of certain invertebrate organisms, recent re-
search has addressed the question of the control of such
concerted response patterns (Prior, 1989; Teyke et al,
1990; Frugal and Brownell, 1987).

Terrestrial gastropods, such as Limax maximns, are
remarkably susceptible to environmental stresses such as
dehydration. In a drying environment, they can lose 30-
40% of their body weight within a few hours (see Prior et
al., 1983; Riddle, 1983; Prior, 1985, for reviews). Among
the array of regulatory responses displayed by dehydrating
slugs are contact-rehydration (Prior, 1984; Prior and Ug-
lem, 1984), modifications in respiratory function (Dick-
Received 14 December 1990; accepted 30 January 1991.

inson et al., 1988). alterations in feeding responsiveness
(Prior, 1983; Phifer and Prior. 1985) and modifications
in cardiovascular function (Grega and Prior, 1986; Wels-
ford and Prior, 199 1 ). As such, Umax represents a useful
model for the analysis of the integration of multiple reg-
ulatory responses.

The concerted control of feeding behavior and cardio-
vascular function in Umax has been a focus of recent
work (see Grega and Prior, 1985; Prior and Welsford,
1989). Rhythmic feeding behavior in this organism in-
volves alternating protraction and retraction of the toothed
radula against a food source. Feeding bouts often last
many minutes and can involve hundreds of bite cycles
(see Gelperin et al.. 1978). In semi-intact or isolated prep-
arations of the central nervous system (CNS: Fig. 1),
chemical stimuli applied to the lips or electrical stimu-
lation of the lip nerves can elicit a prolonged pattern of
efferent neural activity that underlies the feeding move-
ments. This feeding motor program (FMP; Prior and Gel-
perin, 1977; Gelperin et al.. 1978) consists of alternating
bursts of activity in protractor and retractor motoneurons
(Fig. 1, 2). In addition to activation of the major buccal
musculature, the FMP involves synchronized activation
of the accessory salivary system. During feeding, the ac-
tivity of the fast salivary burster neurons (FSBs), which
are the motoneurons to the salivary ducts, becomes phase-
locked with protraction (Fig. 2).

SCPB Modulation of Feeding and Heart Function

In gastropods, the small cardioactive peptides (SCPs)
have an excitatory effect upon both the musculature (see
Lloyd and Willows, 1988; Lloyd, 1989) and the neural
networks underlying patterned feeding activity (see Wil-
lows et al.. 1988). In several species, SCPB can initiate
patterned efferent activity in isolated CNS preparations
(e.g.. Helisoma. Murphy et al.. 1985; Tritonia, Willows

295

296

D J PRIOR

.SN

BUCCAL
GANGLION

TENTACULAR
NERVE -

CEREBRAL
GANGLION

ABDOMINAL
GANGLION

o o

MCG MCG

BODY WALL

Figure 1 . A diagram of the isolated central nervous system of Limax
including: the paired huccal ganglia and the fused cerebral and abdominal
ganglia; the pneumostome region; abdominal nerves N8-N12 and the pos-
terior pedal nerves, PPN; buccal nerves N 1-N3; gastric nerve, GN; salivary
nerve. SN; buccal protractor motoneuron B7; fast salivary burster neuron,
SB; cerebrobuccal connective, CBC; metaccrebral giant cell. MCG.

ci nl., 1988). In Limax, however, SCPB has a modulatory
role, increasing the responsiveness of the central pattern
generator to stimuli (Prior and Watson, 1987). In the
presence of 10 7 to 10 6 A/ SCPB, otherwise ineffective
stimuli can initiate full expression of the feeding motor
program.

Among the neurons in Limax that are responsive to
SCPB are the paired fast salivary bursters. The rate of en-
dogenous burst activity in these motoneurons is enhanced
by application of SCPB in a concentration-dependent
manner (Fig. 3. 4). Short-term application ofSCPB results
in a slow increase in FSB burst frequency and an even
slower decrement of the effect follows initiation of a saline
wash. In addition, continuous perfusion of a preparation
for 20-30 min reveals no indication of desensitization of
the effect. In 10 6 M SCPB. the burst frequency was sus-
tained at 14 bursts/min compared with a control fre-
quency of 1 burst/min (see Prior and Watson. 1987). It
has been determined that this excitatory effect is mediated
by an increase in the rate of the interburst depolarization
rather than a general decrease in resting potential (Hess
and Prior, 1989). Thus the effects of SCPB on the Limax
feeding system include modulation of the responsiveness
of the FMP in addition to direct excitation of specific
motoneurons.

To assess further the potential role of an SCPB-like pep-
tide in the regulation of feeding responsiveness, exogenous

LSN

*••••• I.I I . , .

, lu| ,, ^u|

I i-i

i U i

II 11, 11

yP» ' 'PI1

1 Ik

. 1 Ik

Figure 2. Activation of the feeding motor program (FMP) in an isolated buccal ganglia-brain preparation by
electrical stimulation of an external lip nerve (artifacts at beginning at A). The FMP is characterized by alternation
of efferent bursts correlated with protraction (buccal nerve 1: RBR1; and the right and left salivary nerves: RSN.
LSN), and retraction (buccal nerve 2: RBR2). The nonfeeding endogenous bursts of the right FSB are noted with
dots. The upper calibration trace indicates one mark/second. (From Prior and Watson, 1987)

MULTIFUNCTIONAL PEPTIDERGIC INTERNEURON

297

I I I I I

I I I UNI

SCPB ON

MINIMUM I I Ml I I M

SCP_ OFF

3. I I M I I I I ( I I I I I I I I I I I I.

LSB

SCP ON

Figure 3. (A) A continuous extracellular recording from the left salivary nerve (LSN) of an isolated
buccal ganglia-brain preparation is shown in 1-3. The prominent bursting unit in this record is the fast
salivary burster (FSB; each burst consists of 12-15 spikes). Within 20 s of the application of 2 x 10~6 M
SCPB to the preparation (first arrow), the burst frequency of the FSB increases. Following removal of SCPB
from the superfusion medium (second arrow), burst frequency of the FSB returns to the pretreatment level.
(B) an intracellular recording from the fast salivary burster neuron (FSB) showing the increase in burst
frequency and. in this case, progressive depolarization, in response to 2 • I(T6 M SCPB (the dashed line
indicates the level of the interburst hyperpolarization before exposure to SCPB). Bar = 30 s (A) and 20 mV
(B). (From Prior and Watson. 1988)

SCPB was injected into intact animals and their feeding
responsiveness measured. As shown in Table I., SCPB can
initiate the apetetive phases of feeding behavior including:
(1) cessation of locomotion, (2) tentacular retraction, (3)
lip eversion, and (4) lip movement. That the consumatory
phase of feeding was not regularly initiated was not un-
expected, in that in isolated CNS preparations SCPB did
not initiate feeding, but rather, increased responsiveness
to stimuli. Nevertheless, this would appear to be the first
demonstration of an orderly effect of injected SCPB in an
intact organism. This result certainly supports the notion
that an SCPB-like peptidergic system is involved in the
control of the feeding system in Limax.

The small cardioactive peptides have been shown to
have an excitatory effect on the musculature of numerous
systems, including Helix heart (Lloyd 1978, 1982), Aplysia
and Tritonia buccal mass and gut (Lloyd ct a/., 1984;
Lloyd and Willows, 1988), and Limax ventricle (Welsford
and Prior, 1991; Lloyd, 1979; 1989). In Limax, both SCPB

and SCPA cause a concentration-dependent increase in
the force of ventricular contraction (Welsford and Prior,
1991). At a concentration of 10 6 M, SCPB can cause a
150% increase in the force of ventricular contractions.
Although lower concentrations of SCPB ( 10~9 to 10"7 M)
can cause a slight increase in heart rate, there does not
appear to be a consistent effect (Prior and Welsford, 1 989).
The excitatory effects of SCPB on heart and the feeding
system of Limax, together with the stress-induced coin-
cident changes in feeding and cardiovascular function ob-
served in intact animals (Grega and Prior, 1985), are in-
dicative of the possibility of coincident control of these
two systems.

Multifunctional Modulatory Interneuron Bl

In that exogenous SCPB can simultaneously modify
feeding and cardiovascular function, immunohistochem-
ical techniques were used in an effort to identify central

298

D. J. PRIOR

•— •2XIO~*M
O — O 2XIO"7M
A— £ 2XIO"*

I.OX SALINE

456

TIME(min)

Figure 4. The responses of the fast salivary burster neuron (FSB) to
van ing concentrations of SCPB are presented by plotting burst frequency
as a function of time during the experiment. In each case, SCPB was
superfused over an isolated buccal ganglion-brain preparation between
minutes 2 and 4. The preparation was superfused with saline for 20 min
between each trial. (A) The responses obtained in three trials with the
same preparation using various concentrations of SCPB are shown. Each
point represents the burst frequency of the FSB in the preceding 60 s.
(B) The responses of a second preparation to SCPB. In this case four
different concentrations of SCPB were used as well as a control saline
trial. (C) The extent of the variability between preparations is illustrated
by plotting the mean (±SD) burst frequency at each time point for 29
trials in 12 preparations during exposure to 2 x 10'6 A/ SCPB. (From
Prior and Watson, 1987)

neurons containing SCPB-like-immunoreactive-material
(SLIM) that might be involved. Among the most prom-
inent SLIM-reactive neurons were the right and left Bl
buccal neurons (Prior and Watson, 1987). In addition to
those neurons that clearly contain SCPB immunoreactive
material, there are numerous cell bodies that are enmeshed
by networks of immunoreactive fibers (e.g., B7, FSB),
which is suggestive of peptidergic endings near the target
feeding neurons.

The morphology of Bl was examined by intracellular
injection of horseradish peroxidase (Fig. 5a). There are

Figure 5a. A photomicrograph of a preparation of paired buccal
ganglia (outlined) showing the morphology of the left Bl neuron injected
with horseradish peroxidase. Cerebrobuccal connective. CBC; salivary
nerve. SN; this preparation was made by K. Delaney)

two major axonal projections and an extensive dendritic
arborization in the lateral lobe of the buccal ganglion. A
small axon projects across the buccal-buccal commissure.

Figure 5b. Panel 1 is a camera lucida drawing of the soma of LB1
following injection of Co** showing the major axon exiting the buccal
ganglion in the ipsilateral cerebrobuccal connective. Panel 2 is a camera
lucida drawing of the abdominal ganglion showing the continued axonal
projection of the injected LB1, with one axonal branch in abdominal
ganglion nerve 9 and two axonal branches in nerve 1 1 . Panel 3 illustrates
antidromic activation of Bl in response to repetitive stimulation of the
cardiac branch of nerve 9. Panel 4 shows repetitive intracellular stimu-
lation of Bl causing in a constant-latency axonal impulse recorded in
nerve 9.

MULTIFUNCTIONAL PEPTIDERGIC INTERNEURON

299

lahlc I

Behavioral effects iifSCPg injeclums in Limax maximus

Behavioral observations

Treatment

Tentacular locomotion

Lip retraction

Lip eversion

Lip movement

Saline

83.0%

8.0%

0.0%

ICT'moir1 SCPB

83.0%

25.0%

17.0%

8.0%

lO-'moll'1 SCPB*

92.0%

33.0%

42.0%

25.0%

ID"5 moir1 SCPB"

17.0%

83.0%

100%

58.0%-

The percentage of animals that displayed each behavior is presented in each case.
Each animal received injections of each concentration of SCPB and the saline control.

The 0.05 probability level was accepted as significant (determined by a Friedman's test and a non-parametric multiple comparisons procedure).
All concentrations of SCPB are the calculated final hemolymph concentrations.

The results with 1 5 and 10"" moll"' SCPB injection were significantly different from those with injection of control saline and 10~7 moll~' SCPB.
Furthermore, the results with 10~5 moll'1 SCPB injection were significantly greater than those observed with injection of 10~6 moll"' SCPB.
* P < 0.05, ** P < 0.01. n = 12.
(From Schagene el ai. 1989)

Although the dendritic arborizations occur primarily in
the lateral lobe, they do span into the medial lobe, in-
cluding the region containing both retractor and protractor
feeding motoneurons. The major axon projects out the
ipsilateral cerebrocuccal connective, through the cerebral
ganglion, into the abdominal ganglion and out abdominal

^ 350
o

o:
u.

i- 250-

<s>

m 150
oo

50-

-50

01 23456789
B1 SPIKE FREQUENCY (Hz)

Figure 6. A summary of the change in the burst frequency of the
fast salivary burster neuron initiated by intracellular stimulation ot the
ipsilateral Bl neuron at different impulse frequencies. The apparent
threshold frequency for Bl is 2-4 Hz with the maximal effect occurring
at about 7 Hz. Changes in the FSB burst frequency were normalized as
a percentage of the pre-stimulation level of activity in each preparation.
Each bar represents the mean (±SD) response of five buccal ganglion-
brain preparations. (From Prior and Welsford. 1989)

nerves 9 and 1 1, which innervate the heart and kidney
complex, respectively (Fig. 5b). Rapid stimulation (5 Hz)
of a cardiac branch of nerve 9 resulted in antidromic ac-
tivation of the soma of Bl. Correspondingly, repetitive
activation of the soma of Bl by intracellular current in-
jection was followed by a constant latency impulse in the
cardiac branch of nerve 9 (Fig. 5a).

The immunohistochemical results together with the
basic morphology of Bl. including significant arboriza-
tions in the region of the feeding neurons, and, remark-
ably, a major axonal projection to the heart, were sugges-
tive of a role for Bl in the concerted control of feeding
and cardiovascular functions.

Intracellular stimulation of Bl at quite low frequencies
results in a progressive increase in the activity of the fast

o
cc
o

z
o

z
o
u

o
z

I
o

75-

65-

55-
45-
35-
25-
15-
5-

2345678
B1 SPIKE FREQUENCY (Hz)

Figure 7. The effect of unilateral stimulation of Bl on the force of
ventricular contraction. The bars represent the mean (±SD) response
from 56 trials. Ten action potentials were elicited at each frequency. The
a. b, c notation refers to significant differences (e.g.. C's are significantly.
P < 0.001, different from a's and b's). (Redrawn from data of Welsford
and Prior. 1991)

300

D. J. PRIOR

salivary bursters. As shown in Figure 6, driving Bl at 5
impulses/s can result in a 50% increase in FSB burst fre-
quency. Even two to three impulses at low frequencies
are sufficient to elicit a transient increase in FSB burst
frequency. These effects are sustained in high Mg+ + . high
Ca++ saline indicating the possibility of monosynaptic
connection.

To assess the potential role of Bl in the control of heart
function, semi-intact preparations of the CNS and inner-
vated heart were used, which allowed intracellular stim-
ulation of Bl and measurement of ventricular activity.
Stimulation of Bl at low frequencies resulted in an in-
crease in the force of contraction of the heart (Fig. 7). It
is of interest that Bl frequencies of 5 to 7 impulse/s were
the most effective in the activation of both the FSB and
the heart.

When this experiment was repeated with the CNS
bathed in high Mgff, high Ca++ saline, there was no
change in the effectiveness of Bl to increase heart function.
This suggests that B 1 has a direct effect on peripheral tar-
gets rather than acting via additional CNS neuronal path-
ways.

Thus, it would appear that buccal neuron Bl may be
a multifunction peptidergic interneuron capable of si-
multaneously modulating the central feeding motor pro-
gram and cardiovascular function. As such, Bl, along with
other similar neurons, is positioned to control the syn-
chrony of multiple behavioral responses normally ob-
served in response to environmental stress and changes
in the physiological state of an organism.

Acknowledgments

The work described in this paper was supported, in
part, by grants from the Arizona Disease Control Research
Commission (#82-0698) and The National Institutes of
Health (M.B.R.S.* 2 SO3 RR03401-03).

Literature Cited

Dickinson, P. S., D. J. Prior, and C. Avery. 1988. The pneumostome
closure rhythm in slugs: a response to dehydration controlled by
hemolymph osmolality and peptide hormones. Comp. Biochcm.
Physiol. 89A(4): 579-586.

Furgal, S. M., and P. H. Brownell. 1987. Ganglionic circulation and
its effects on neurons controlling cardiovascular functions in the
Aplysia calilonuca J Exp. Zool. 244: 347-364.

Gelperin, A., J. J. Chang, and S. Reingold. 1978. Feeding motor pro-
gram in Limax maximus: neuromuscular correlates and control by
chemosensory input. J. Neurobiol. 9: 285-300.

Grega, D. S., and D. J. Prior. 1985. The effects of feeding on heart
activity in the terrestrial slug. Limax maximus: central and peripheral
control. J. Comp. Physio/. 156A: 539-545.

Grega, D. S., and D. J. Prior. 1986. Modification of heart activity in
response to dehydration in the terrestrial slug. Limax maximus. J
Exp. Biol. 237: 185-190.

Hess, S. D., and D. J. Prior. 1989. Small cardioactive peptide B mod-
ulates feeding motoneurons in Limax maximus J Exp. Biol. 142:
473-478.

Lloyd, P. E. 1978. Distribution and molecular characteristics ot car-
dioactive peptides in the snail. Helix aspersa. J. Comp. Physiol. 128:
269-276.

Lloyd, P. E. 1979. Central peptide containing neurons modulate gut
activity in Trilona. Soc. Neurosci. Abstr. 5: 252.

Lloyd, P. E. 1982. Cardioactive neuropeptides in gastropods. Fed. Proc.
41:2948-2852.

Lloyd, P. E. 1989. Peripheral actions of the SCPs in Aplysia and other
gastropod molluscs. Am. /.ool. 29: 1265-1274.

Lloyd, P. E., I. Kiiplri m. urn, and K. R. Weiss. 1984. Evidence for
parallel actions of a molluscan neuropeptide (SCPA) and serotonin
in mediating arousal in Aplysia Proc. Nat. Acad. Sci. L'SA 81: 2934-
2937.

Lloyd, P. E., and A. O. D. Willows. 1988. Multiple transmitter neurons
in Tritimia II. Control of gut motility. / \eurohml. 19(1): 55-67.

Murphy, A. D., K. Lukowiak, and \V. K. Stell. 1985. Peptide modu-
lation of patterned motor activity in identified neurons of Hclisoma.
Proc Nat. Acad Sci. USA 82: 7140-7144.

Phifer, C. B., and D. J. Prior. 1985. Body hydration and haemolymph
osmolality affect feeding and its neural correlate in the terrestrial
gastropod, Limax maximus. J. Exp. Biol. 1 18: 405-426.

Prior, D. J. 1983. Hydration-induced modulation of feeding respon-
siveness in terrestrial slugs. J. Exp Zool. 227: 15-22.

Prior, D. J. 1984. Analysis of contact-rehydration in terrestrial gastro-
pods: osmotic control of drinking behaviour. J Exp. Biol 111: 63-
73.

Prior, D. J. 1985. Water-regulatory behaviour in terrestrial gastropods.
Biol. Rer. 60: 403-424.

Prior, D. J., and G. L. Uglem. 1984. Analysis of contact-rehydration
in terrestrial gastropods: absorption of l4C-inulin through the epi-
thelium of the foot. ./. Exp. Biol. Ill: 73-80.

Prior, D. J. 1989. Neuronal control of osmoregulatory responses in
gastropods. Pp. 1-24 in Advances in Comparative Environmental
Physiology. R. Gilles, ed. 5. Springer- Verlag, New York.

Prior, D. J., and A. Gelperin. 1977. Autoactive molluscan neuron:
reflex function and synaptic modulation during feeding in the ter-
restrial slug, Limax maximus J. Comp. Physiol. 114: 217-232.

Prior. D. J., and W. H. Walson. 1987. The molluscan neuropeptide,
SCPB, increases the responsiveness of the feeding motor program of
Limax maximus. J. Neurobiol. 19(1): 87-105.

Prior, D. J., and I. G. Welsford. 1989. The role of small cardioactive
peptide. SCPB. in the regulatory responses of terrestrial slugs. Am.
Zool. 29: 1255-1263.

Prior, D. J., M. Hume, D. Varga, and S. D. Hess. 1983. Physiological
and behavioural aspects of water balance and respiratory function
in the terrestrial slug, Limax maximus. J. Exp. Biol. 104: 1 1 1-127.

Riddle, W. A. 1983. Physiological ecology of land snails and slugs. Pp.
431-461 in The Mollusca, W. D. Russell-Hunter, ed. Ecology. 6.
Academic Press, New York.

Schagene, K. A., I. G. Welsford, D. J. Prior, and P. A. Banta.
1989. Behavioral effects of injection of small cardioactive peptide,
SCPB, on the slug Limax maximus. J Exp. Biol. 143: 553-557.

Teyke, T., K. R. Weiss, and I. kupfermann. 1990. An identified neuron
(CPR) evokes neuronal responses reflecting food arousal in Aplysia.
Science 247: 85-87.

Welsford, I. G., and D. J. Prior. 1991. Modulation of heart activity in
the terrestrial slug Limax maximus by the feeding motor program,
small cardioactive peptides and stimulation of buccal neurone Bl.
./. Exp. Biol. 155: 1-19.

Willows, A. O. D., P. E. Lloyd, and B. P. Masinovsky. 1988. Multiple
transmitter neurons in Trilonia III. Modulation of central pattern
generator controlling pattern. / Neurobiol. 19( 1 ): 69-86.

Reference: Binl. Bull 180: 301-309. (April, 1991)

Opioid Systems and Magnetic Field Effects
in the Land Snail, Cepaea nemomlis

MARTIN KAVALIERS AND KLAUS-PETER OSSENKOPP

Division of Oral Biology, Faculty of Dentistry, and Department of Psychology,
University of Western Ontario, London. Ontario, Canada N6A 5C1

Abstract. Accumulating evidence shows that magnetic
fields can affect a variety of opioid-mediated behavioral
and physiological functions. The idea that endogenous
opioids are involved in the mediation of fundamental be-
havioral responses in invertebrates is also gaining support.
Evidence exists for opioid involvement in the mediation
of nociceptive and antinociceptive ("analgesic") responses
of the land snail, Cepaea neinoralis. and other mollusks.
in a manner comparable to that in vertebrates. Exposure
to various magnetic stimuli, including weak 60 Hz mag-
netic fields, has significant inhibitory effects on exogenous
opiate-induced analgesia and endogenous opioid-me-
diated nociceptive responses of Cepaea in a manner anal-
ogous to that described for vertebrates. These effects of
the magnetic stimuli are evident under both laboratory
and natural conditions and include disruptions of the day-
night rhythms of opioid-mediated nociception. These
similar effects in Cepaea and rodents raise the possibility
of a phylogenetic continuity in the effects of magnetic
fields on basic opioid-mediated biological responses.

Introduction

Results of field and laboratory studies show that the
behavioral, cellular, and physiological functions of ani-
mals can be affected by magnetic stimuli (see reviews in
Adey, 1981; Gould, 1984; Ossenkopp and Kavaliers,
1988). These diverse actions have led to speculation on
the possible modes of action of magnetic fields on bio-
logical systems (Leask, 1977; Semm et al., 1980; Adey,
1 98 l;Kirschvink and Gould, 1981; Liburdy et al.. 1987;
Liboff and McLeod, 1988; Blackman et al.. 1989).

Evidence has accumulated that endogenous opioid sys-
tems and opioid peptides, which are involved in the mod-
Received 19 July 1990; accepted 6 November 1990.

ulation of a broad range of basic functions (Akil et at..
1984), can be affected by magnetic stimuli. Substantial
data now indicates that time-varying magnetic fields, es-
pecially those in the extremely low frequency (ELF) range
(0.10-100 Hz), affect endogenous opioid systems and the
actions of exogenous opiates such as morphine (Kavaliers
and Ossenkopp, 1984, 1986. 1987; Miller el al., 1985;
Ossenkopp and Kavaliers. 1987; Praloetal., 1987). Opioid
systems may, thus, be an integral part of the mechanism(s)
whereby magnetic fields exert their diverse behavioral and
physiological effects (Ossenkopp and Kavaliers, 1988).

Although interest has primarily focused on vertebrates,
there is evidence that magnetic fields affect a variety of
behavioral physiological processes in invertebrates (Gould,
1984). Recently, opioid-mediated behaviors that are sen-
sitive to magnetic stimuli have been demonstrated in a
gastropod mollusk, the land snail Cepaea neinoralis
(Kavaliers et al.. 1983; Kavaliers and Ossenkopp, 1989).
This paper briefly describes (i) opioid modulation of be-
havioral responses in mollusks and (ii) the effects of mag-
netic fields on opioid mediated responses and their day-
night rhythms in the snail, Cepaea.

Opioid Systems and Molluscs

General aspects

In vertebrates, endogenous opioid peptides co-exist with
diverse hormones in endocrine glands and with classical
or peptide transmitters in peripheral autonomic and sen-
sory neurones. In addition, opioid peptides are widely
distributed in the central nervous system where they
function as transmitters or neuromodulators. Three fam-
ilies of endogenous opioid peptides derived from three
precursor peptides are known to date: the pro-opiomela-
nocortin (POMC), the pro-enkephalin, and the pro-dy-
norphin system. These precursors undergo differential

301

302

M. K.AVALIERS AND K..-P. OSSENKOPP

processing in various regions of the central and peripheral
nervous systems, and the major cleavage products have
different affinities to the three major types of opioid re-
ceptors: n, 5, and K (Hollt, 1986).

These opioid peptides and receptors have now been
identified in a variety of invertebrate taxa, strongly sug-
gesting a phylogenetic conservation of opioid peptide
structure and function (Kream el a/., 1980; Leung and
Stefano, 1984, 1987; Scharrer el a/.. 1988; Zisper el al..
1988; Leung et al.. 1990; Santoro el al.. 1990). Results of
behavioral, electrophysiological, immunological, and
pharmacological studies have shown that endogenous
opioid peptides and exogenous opiate agonists and an-
tagonists have behavioral and physiological actions in in-
vertebrates resembling those induced in mammals (Ste-
fano, 1982, 1989; Leung and Stefano, 1987; Stefano el
al.. 1989).

Behavioral aspects

Nociception. One of the primary roles of vertebrate
opioid systems is the modulation of nociception and be-
havioral responses to aversive and stressful stimuli (Besson
and Chaouch, 1987; Kavaliers, 1989a). In nature, animals
commonly encounter aversive stimuli that can influence
their survival. To effectively respond to these stimuli, or-
ganisms require: (i) a mechanism for recognizing aversive
stimuli, (ii) a set of effectors that can react to the noxious
stimulus, and (iii) a system for producing coordinated
and directed movements and behavior in response to the
stimuli. The ability of animals to recognize and physically
react to aversive or noxious stimuli that can compromise
their integrity is embodied in the term "nociception"
(Sherrington, 1906). Nociceptors are preferentially sen-
sitive to either a noxious stimulus or to an aversive stim-
ulus that would become noxious if prolonged, and they
code the intensity of the stimulus (Besson and Chaouch,
1987). In addition, the responses from the effectors are
appropriate to the input from the receptors. Nociception
can be used to provide an index of an animal's sensitivity
to aversive environmental conditions and, thus, can allow
for the determination of the capacity to execute adaptive
behavior. Measurements of alterations in nociceptive-re-
lated responses (decreases in sensitivity-antinociception
or analgesia when considered in terms of pain) are widely
used to determine the behavioral and physiological status
of animals following exposure to aversive, or potentially
aversive, stimuli. In rodents, laboratory measures of no-
ciception include recording of limb flexion or withdrawal
(lifting a foot off an aversive, usually thermal surface);
active avoidance (flinch jump, jumping, or moving from
an aversive situation); and removal of the tail away from
a thermal stimulus (tail-flick) (Kavaliers, 1989a).

Assays for invertebrate nociception have been devel-
oped, and nociceptive responses have been observed in

invertebrates as well as vertebrates (Kavaliers, 1989a). For
example, within a few seconds after Cepaea is placed on
a surface warmed to 40°C, the snail lifts the anterior por-
tion of its fully extended foot away from the aversive sur-
face (Fig. 1). The behavioral end point used is the time
at which the foot reaches its readily discernible maximum
elevation. This "foot-lifting" behavior is not observed in
snails that are exposed to temperatures normally present
in their natural habitats, but becomes increasingly evident
as the temperature is raised towards 40°C. This nocicep-
tive response is comparable to the foot-lifting response
exhibited by rodents when placed on a warmed surface.
Similar, thermally induced nociceptive responses have
also been reported for the snail. Helix aspersa. and the
slug, Arion alter (Leung and Stefano, 1987; Dalton and
Widdowson, 1989). A nociceptive function is also indi-
cated for specific mechanoafferent neurons innervating
the tail, parapodia, and much of the foot and body wall
of the marine mollusk Aplysia califarnica (Walters and
Erickson, 1986). These neurons display increasing dis-
charge frequency in response to progressively increasing
pressure, with maximal responses occurring to stimuli that
could cause tissue damage (Walters, 1987). A similar
graded pattern of response has been used to define the
activity of classical mammalian nociceptors (Besson and
Chaouch, 1987).

Opioid mediation of nociception and antinociception.
Antinociception has been widely documented in experi-
mental animals following exposure to diverse environ-
mental stimuli, with both opioid and non-opioid mech-
anisms being implicated (Rodgers and Randall, 1988).
Furthermore, it now seems clear that environmentally in-
duced pain inhibition is an important component of an
organism's defensive repertoire and hence has high adap-

Figure 1. Thermal 'nociceptive' response of a hydrated individual
Cepaea nemoralis placed on a 40°C surface. The behavioral end point
used is the maximum elevation of the anterior portion of the fully ex-
tended foot.

MAGNETIC FIELDS AND OPIOID SYSTEMS

303

live value (Amit and Galina, 1986). In vertebrates, the
tonic activity of endogenous opioid systems can be in-
creased by a range of environmental stimuli. In laboratory
rodents, this "stress" — or environmentally induced an-
algesia (Amit and Galina, 1984) — can be recorded as an
increased latency of a foot-lift or tail-flick response. Ad-
ministration of either endogenous opioid peptides, such
as enkephalin or exogenous opiate antagonists, such as
the prototypic n opiate agonist, morphine, produces sim-
ilar analgesic effects. Prototypic exogenous opiate antag-
onists, such as naloxone or naltrexone, can reverse or at-
tenuate these analgesic effects, and, in certain cases, can
reduce nociceptive responses and induce hyperanalgesia
(Martin, 1984).

Similar evidence for opioid involvement in the media-
tion of antinociception or analgesia and nociception is
present for mollusks. Morphine, as well as the endogenous
opioids /i-endorphin and methionine-enkephalin. en-
hance, in a dose-dependent manner, the latency of the
nociceptive responses ofCepaea and the slug. Arion. to a
warmed surface ( Dalton and Widdowson, 1989; Kavaliers
ct ul., 1983, 1985). As in mammals, maximum antino-
ciceptive effects of morphine in Cepaea are seen 15-30
min after injection, with a decline to basal thermal re-
sponse latencies by 60-120 min (Kavaliers ct ai. 1983).
These antinociceptive effects occur without any evident
effects on the spontaneous locomotor activity or motor
abilities of the animals. The antinociceptive effect of mor-
phine is also produced by the benzomorphan levorphanol.
but not by the stereoisomer dextrophan, suggesting that
the receptor that interacts with these opiates has stereo-
specific requirements (Hirst and Kavaliers. 1987). Nal-
oxone suppresses, and dose-dependently reverses, the an-
algesic effects of morphine in Cepaea, and reduces the
response times (hyperalgesia) of particular morphological
types of Cepaea that display elevated nociceptive re-
sponses (Kavaliers et al., 1983; Kavaliers, 1989b). This
further supports opioid involvement in the mediation of
antinociception and nociception in Cepaea.

The specific n and 5 opioid agonists, (D-Ala:-Me-Phe5,
Gly-ol)-enkephalin (DAMGO) and (D-Ala2, D-Leu4) en-
kephalin (DADLE), respectively, also have significant an-
tinociceptive effects in Cepaea and Arion, suggesting the
presence of M and b opioid receptors (Dalton and Wid-
dowson, 1989; Kavaliers et ai, 1985). In addition, the
specific K opiate agonist U-50.488H, has significant an-
tinociceptive effects in Cepaea (Kavaliers and Ossenkopp.
1989). As in mammals, the duration of effect of U-
50.488H is longer than that of morphine, and there is a
low sensitivity to reversal by naloxone. Taken together
with the demonstrations of K opioid binding sites and the
immunocytochemical localization of the endogenous K
ligand dynorphin, in invertebrates (Ford el al.. 1986), these

antinociceptive effects raise the possibility of a K opioid-
mediated antinociceptive system in Cepaea.

Day-night rhythms of nociception. Significant day-night
rhythms are exhibited in the nociceptive responses and
analgesic effects of morphine in Cepaea. These noctumally
and crepuscularly active snails display elevated night-time
levels of nociception and morphine-induced analgesia
under both field and laboratory conditions (Kavaliers et
al.. 1990). The elevated nocturnal response latencies to a
thermal stimulus are reduced by naloxone and the diel
rhythm of nociception can be disrupted by pretreatment
with the irreversible /u opioid receptor alkylating agent,
fJ-funaltrexamine (/3-FNA) (Kavaliers and Ossenkopp,
1991). This suggests that endogenous n opioid systems
may be involved in the generation or expression of the
day-night rhythm of this measure of nociception in
Cepaea.

Stress-induced analgesia. In rodents, diverse stimuli
have been shown to increase endogenous opioid activity
and induce integrated adaptive behavioral responses, in-
cluding analgesia (Amit and Galina, 1986). Similar en-
vironmentally induced opioid activation and analgesia is
also evident in mollusks and other invertebrates (Kava-
liers, 1987; Maldonado and Miralto, 1987; Dalton and
Widdowson, 1989; Valeggia et al., 1989). Exposure to ei-
ther heat, centrifugal rotation, or novel chemical stimuli
has been shown to increase thermal nociceptive thresholds
of Cepaea (Kavaliers, 1987, 1989a). The warm-stress-in-
duced analgesia is blocked by naloxone and the 6 opioid
antagonist ICI 154,129, and is suppressed by a 24-h pre-
treatment with 0-FNA (Kavaliers, 1987). Brief body (tail)
pinch stress of the slugs Arion and Umax also resulted in
significant increases in their response latencies (Kavaliers
and Hirst, 1986; Dalton and Widdowson. 1988). The an-
algesic response ofLimax was blocked by naloxone, while
that of Arion was reduced in a dose-dependent manner
by naltrexone and the d opiate antagonist ICI 148,164.
Moreover, the duration of the stress-induced analgesia in
Arion could be prolonged by the injection of enkephalin-
ase inhibitors (Dalton and Widdowson, 1988). This fur-
ther supports the involvement of endogenous opioid pep-
tides in the mediation of a number of forms of stress-
induced analgesia in gastropod mollusks. It should be
noted, however, that although these antinociceptive re-
sponses are opioid-mediated, it would be desirable to
demonstrate cross-tolerance to exogenous opiate-induced
analgesia, as well as to show changes in endogenous opioid
peptide levels and receptor binding.

Mechanisms. At a biochemical and cellular level, there
is evidence to suggest that the antinociceptive effects of
opiates, in both Cepaea and rodents, are associated with
alterations in calcium channel activity. Calcium channels
are reported to be involved in the regulation of neuronal
functions in mollusks in a manner similar, but not nee-

304

M. KAVALIERS AND K..-P. OSSENKOPP

essarily identical, to that in vertebrates (Akaike el a/.. 1 98 1 ;
Gerschenfeld et a/.. 1986; Hammond el at, 1987; Miller.
1987). In vertebrates, the activation of n or o opioid re-
ceptor types increases potassium channel conductance and
indirectly reduces calcium channel conductance, while
activation of K receptors causes a direct reduction in volt-
age dependent calcium conductance (North, 1986). In
both cases, the net result is a reduction in neuronal dis-
charge frequency and the amount of transmitter released.
In both rodents and Cepaea, the dihydropyridine (DHP)
and non-DHP calcium channel antagonists diltiazem.
verapamil, and nifedipine can reduce exogenous opiate
and stress-induced opioid analgesia (Kavaliers and Os-
senkopp, 1987, 1989). This suggests similar roles for cal-
cium-channel-related mechanisms in the mediation of
opiate-induced analgesia in mammals and mollusks. In
addition, pharmacological reductions of G protein activity
by pertussis toxin pretreatment have similar inhibitory
effects on morphine-induced analgesia in Cepaea and ro-
dents ( Yu and Kavaliers, 1991). This suggests that similar
intermediary messenger systems are involved in the me-
diation of opiate effects in Cepaea and rodents. Moreover,
data also indicate that opiates have similar inhibitory ef-
fects on dopamine and possibly other monoamine systems
in rodents and mollusks (Stefano, 1982). These observa-
tions suggest similar modes of action and sensitivities of
opioid systems in vertebrates and mollusks.

Magnetic Fields and Opioid Systems

General aspects-
Research on the roles of geomagnetic information in
avian and invertebrate orientation and migration has
provided some of the most convincing results on the bio-
logical effects of magnetic fields (Ossenkopp and Barbeito,
1978; Gould, 1984; Wiltschko and Wiltschko, 1990). A
variety of other biological effects produced by exposure
to magnetic fields have also been documented in both
invertebrates and vertebrates (reviews in Adey, 1981;
Gould, 1984; Ossenkopp and Kavaliers, 1988). In mol-
lusks, these effects of magnetic fields include alterations
in neuronal activity and orientation behaviors ( Brown and
Webb, 1960; Brown et at, 1960 a,b; Brown, 1971; Loh-
mann and Willows, 1987; Azanza, 1989; Balaban et at,
1990).

As previously indicated, among the more dramatic ac-
tions of magnetic stimuli in mammals are reversible
modifications in the effects of exogenous opiates and en-
dogenous opioids. Natural geomagnetic disturbances
arising from intense solar activity, earth strength, 0.5-1.5
gauss 60 Hz magnetic fields, relatively weak rotating mag-
netic fields, and stronger magnetic fields associated with
diagnostic magnetic resonance imaging have all been
shown to reduce the analgesic effects of morphine in mice

(Kavaliers and Ossenkopp. 1984. 1986; Miller et at, 1985;
Ossenkopp et at. 1983; Prato et at. 1987).

Results of recent investigations with the snail Cepaea,
have extended these inhibitory effects of magnetic stimuli
on opioid systems to mollusks ( Kavaliers and Ossenkopp,
1989). These, and additional findings from in vitro prep-
arations (Golding et at. 1985). avian orientation (Papi
and Luschi, 1991), and spatial learning in rodents (Ka-
valiers et at. 199 la), which relate the effects of magnetic
fields to alterations in opioid activity, suggest that a broad
range of fundamental opioid-mediated functions may be
sensitive to magnetic stimuli.

Magnetic fields and opioid-mediated nociception in
Cepaea

Rotating magnetic fields. Results of investigations of
the effects of exposure to a 0.5 Hz rotating magnetic field
(RMF) on morphine-induced antinociception in Cepaea
provided the first direct evidence that magnetic stimuli
could affect opioid systems in an invertebrate (Kavaliers
and Ossenkopp, 1989). As in rodents (Kavaliers and Os-
senkopp, 1986, 1987), exposure for 15-30 min to a het-
erogenous time-varying magnetic field (0.15-9.0 mT or
1.5-90 gauss, produced by two rotating horseshoe mag-
nets) of about 0.5 Hz significantly reduced day-time mor-
phine-induced analgesia in Cepaea without any evident
effects on the basal nociceptive responses of saline-treated
control animals. The rotating magnetic fields also atten-
uated the analgesic effects of the K opiate agonist U-
50,488H. In addition, and as in rodents, exposure to the
rotating magnetic fields reduced stress-induced opioid
analgesia in Cepaea. These findings show that time-vary-
ing magnetic fields can significantly alter both exogenous
opiate- (n and * ) and endogenous opioid-induced analgesia
in an invertebrate. These observations also raise the pos-
sibility that exposure to magnetic stimuli may compro-
mise the expression of adaptive opioid-mediated behav-
ioral and physiological responses to environmental
stresses. It should be noted that in control sham exposure
conditions, where dummy weights rather than horseshoe
magnets were used, there were no effects on opioid-me-
diated antinociception. In these studies there was an
equivalent electric field in the sham and magnetic field
exposure conditions. This minimizes the potential in-
volvement of electric fields in the inhibition of opioid
analgesia.

60 Hz magnetic fields. Increasing concerns about the
possible health effects due to exposure to high-voltage
transmission lines and electrical appliances in the home
have been expressed (Ahlboom, 1988). There have been
numerous reports documenting biological effects in ver-
tebrates following exposure to 50 or 60 Hz magnetic fields,
although relatively little is known about the possible effects

MAGNETIC FIELDS AND OPIOID SYSTEMS

305

in invertebrates. The effects in vertebrates have included
retardation in embryological development, changes in
behavioral activity levels and inhibition of chemically and
electrically kindled seizures. These effects are compatible
with alterations in the functioning of endogenous opioid
systems (review in Ossenkopp and Kavaliers, 1988). This
speculation of opioid involvement is encouraged by the
observation that acute (30-min) exposure to low intensity
60 Hz magnetic fields markedly reduces morphine-in-
duced analgesia levels in mice, with a functional relation-
ship between magnetic field intensity and the degree of
inhibition of analgesia (Ossenkopp and Kavaliers, 1987).

Recently, it was observed that exposure of C'cpaca to
low intensity ( 1 .0 gauss, rms) 60 Hz magnetic fields in a
Helmholtz coil apparatus, as shown in Figure 2, also re-
sulted in an attenuation of morphine-induced analgesia
(Kavaliers et til.. 1990). Various durations of exposure
(0.50, 2, 12, 48, or 120 h) to the 60 Hz fields reduced the
levels of morphine-induced analgesia in both the light and
dark periods of a 12 h light: 12 h dark cycle, with the
magnetic stimuli having significantly greater inhibitory
effects in the dark period. The inhibitory effects of the
magnetic fields were reversible. Twenty-four hours after
exposure, the levels of morphine-induced analgesia were
not significantly different from pre-exposure levels (Kav-
aliers et al., 1990). These effects in C'cpaca are consistent
with the day-night rhythms in the inhibitory effects of
naloxone and 60 Hz and rotating magnetic fields on mor-
phine-induced analgesia in nocturnal rodents (Kavaliers
and Ossenkopp, 1984; Ossenkopp and Kavaliers, 1987).
The weak 60 Hz magnetic fields also significantly reduced
the levels of the elevated naloxone-sensitive dark period
nociceptive response latencies in Cepaea, while not af-
fecting the lower level light period responses. Moreover,
the degree of attenuation of the analgesic and nociceptive
response latencies was related to the duration of exposure
to the 60 Hz magnetic fields.

Determinations were also made of the effects of 60 Hz
magnetic fields on opioid-mediated responses outside the
laboratory under natural conditions. Exposure to 1 .0 gauss
60 Hz magnetic fields under field conditions significantly
attenuated morphine-induced antinociception and noci-
ceptive responses of Cepaea, with the degree of attenuation
being related to the duration of exposure to the magnetic
fields ( Tysdale e I al , 1 99 1 ). The 60 Hz fields also disrupted
the day-night rhythm of nociception, with particularly
marked alterations in responses occurring during the rap-
idly changing light levels of the twilight periods as shown
in Figure 3. These field observations suggest a possible
relation between light reception or changes and magnetic
field reception in Cepaea. A connection between magnetic
field and light reception has been previously theoretically
postulated (Leask, 1977) and experimentally indicated in
several species of arthropods and vertebrates (Leucht,

Figure 2. Helmholtz coil apparatus used for generation of 60 Hz
magnetic fields to which the Ccpuea nemoralis were exposed. Snails were
held individually in translucent polypropylene 50 ml centrifuge tubes
(10 X 2.5) containing a saturated atmosphere and natural vegetation.
The tubes were placed upright on a platform (exposure volume) in the
Helmholtz coil apparatus. The Helmholtz coils consisted of 100 turns
of no. 24 motor enamel wire with a resistance of about 25 Q per coil.
The coils were 100 cm in diameter spaced 103 cm apart on the Z-axis
and attached to the outside of a plywood frame. The coils were covered
with a resin coating which immobilized the wires in the coils and pre-
vented them from vibrating when they were carrying current. Line current
(60 Hz) from standard outlets was applied to the coils and regulated
with two variable autotransformers. The experimental exposure volume
(30 x 30 x 330 cm) in which the snails in the tubes were placed was
centered between the energized coils on the Z-axis. By altering the voltage
input to the two coils, 60 Hz fields with linear polarity and field intensities
up to 1.5 gauss (rms) could be generated [a field intensity of 1.0 gauss
(rms) was used in the studies described in the text]. A sham field exposure
condition was produced by turning of) the current to the coils and placing
test animals in the same exposure volume.

1984, 1990; Olcese et al, 1985. 1988; Phillips, 1987).
However, it has not been established whether light itself
is a prerequisite for reception of the magnetic field.

These observations with Cepaea show that exposure to
weak 60 Hz magnetic fields significantly affects the diel
rhythms of opioid-mediated responses in both the labo-
ratory and under natural environmental conditions. They
also show that the degree of inhibition of opioid-mediated
responses is affected by both the duration and timing (day-
night variations) of exposure to 60 Hz magnetic fields.
These latter findings are particularly significant in view
of the growing reports of associations between prolonged
exposures to low intensity 50 and 60 Hz magnetic fields
and the occurrence of various types of neoplasms (e.g.,
Savitz et al.. 1988) and the evidence that opioid systems
can modulate tumorigenesis (Zagon and McLaughlin,
1987). In this regard, it is of interest that the snails exposed
to the 60 Hz magnetic fields showed, over a two-week
period following exposures, increased levels of mortality
relative to control sham-field exposed animals, and that
night-time exposures resulted in greater mortality levels
than day-time exposures (Ossenkopp et al., 1990). This

306

M. (CAVALIERS AND K..-P. OSSENKOPP

1830

2200

Time [hr]

Figure 3. Examples of daytime, nighttime and twilight period thermal (40°C) response latencies (no-
ciceptive responses) of Cepaea nemoralis held under natural summer (August) outdoor (Environmental
Sciences Center. London. Ontario. 43° 4' 30" and 8P 18' 30" W) light conditions and exposed to either a
60 Hz magnetic field (1.0 gauss, rms. as described in Fig. 2) or a sham magnetic field (sham). Each point
shown represents the mean nociceptive response of 18-24 snails. Different groups of snails were tested on
each of the four days shown. For ease ot presentation standard errors are excluded.

Exposure to the 60 Hz magnetic fields had no significant effects on the daytime (pre-sunset) nociceptive
responses, but significantly (P < 0.01. repeated measures analysis of variance for 2200 h) reduced the night-
time response latencies as compared to the sham exposed snails and other control animals (not shown).
Exposure to the magnetic fields also significantly (P < 0.05, for 2000 and 2100 h) attenuated the marked
increases in thermal response latencies that occurred during the decreasing light levels of the twilight periods
[civil (c), nautical (n) and astronomical (a) twilights; defined by the sun at -6°, -12° and -18°, respectively,
from the horizon]. The greatest effects of the 60 Hz magnetic field on nociceptive responses occurred during
the nautical and astronomical portions of the twilight period.

The temperatures and light intensities that the snails were exposed to ranged from 22 to 28°C and 100
to 200 /jw/cm2 (20-40 Mw/cm2 in the tubes) in the daytime, and from 14 to 22°C and 0.01 to 10 Mw/cm2
(0.00 1 - 1 .0 ^w/cnr in the tubes) in the twilight transitions and nighttime. These light (tubes) and temperature
values were similar to the conditions present in the natural habitat of the snails (Kavaliers, 1989b). The
background geomagnetic field had a daytime horizontal (H) intensity of 0.48 gauss, a vertical (Z) intensity
of 0.24 gauss, and inclination (I) of 75. The Helmholtz coils were oriented with the x-axis oriented almost
directly towards magnetic north.

is of relevance in view of the suggestions of synergistic
effects between exposure to magnetic fields and environ-
mental pollutants in the induction of neoplasms (Adey,
1987, 1990).

Mechanisms of action of magnetic stimuli on opioid
systems

The inhibitory effects of the magnetic stimuli observed
in both the day- and night-time may arise from the in-
creased levels of the magnetic field as compared to earth
strength fields or fluctuations in field strength. Although
data has been presented to suggest that both of these com-
ponents can influence biological systems (Adey, 1981;
Cremer-Bartels et at, 1984), evidence is accumulating that
the biological effects of magnetic fields are primarily due
to fluctuations in field strength (Blackman et ai, 1985,
1989; Prato et at.. 1987). Furthermore, data indicate that

the extent of the biological effects of weak magnetic fields
are dependent on the relative intensity and orientations
of both the steady state [local geomagnetic field, which
varies on a day-night basis (Cremer-Bartels et a/.. 1984)
and oscillating field (Blackman et ai, 1985; Prato et at,
1987)]. However, it should be noted that many behavioral
and physiological responses show no evidence of sensitiv-
ity to fluctuating magnetic fields (Ossenkopp and Kava-
liers. 1988).

Magnetic fields have been proposed to alter the prop-
erties and stability of biological membranes, their trans-
port characteristics, and the intra- and extra-cellular dis-
tributions and flux of calcium ions (Bawin and Adey,
1976; Adey, 1981, 1989; Liboflf et at. 1 987; Carson et at.
1990). Blackman et at (1985, 1989) indicated that ex-
posure to various combinations of time-varying and local
geomagnetic fields caused significant changes in the efflux
of calcium ions from in vitro preparations of chick brain

MAGNETIC FIELDS AND OPIOID SYSTEMS

307

tissue. They speculated that this effect of magnetic fields
on calcium ion efflux might involve a general property of
biological tissue.

There is evidence that the inhibitory effects of the mag-
netic fields on opioid analgesia also involve changes in
the levels, flux, and distribution of calcium ions, altera-
tions in the functioning of calcium channels, along with
modifications in the coupling between opioid receptors
and calcium channels. This is supported by the findings
that the DHP and non-DHP calcium channel antagonists
diltiazem, nifedipine, and verapamil significantly reduce,
while the DHP calcium channel agonist BAY K.8644, sig-
nificantly enhances the inhibitory effects of rotating mag-
netic fields on morphine-induced analgesia in Cepaea and
mice (Kavaliers and Ossenkopp, 1987, 1989). In addition,
the inhibitory effects of rotating magnetic fields on murine
morphine-induced analgesia are reduced by the calcium
chelator EGTA, and potentiated by the ionophore A2 1 387
(Kavaliers and Ossenkopp, 1986).

Magnetic stimuli could affect calcium channel activa-
tion and conductance either directly or indirectly through
alterations of intermediary effector or messenger systems.
The second messenger system most commonly associated
with opioid receptors and changes in ion transport in-
volves inhibition of adenyl cyclase through G proteins
(North, 1 986; Stryer and Bourne, 1986). Administrations
of pertussis toxin, which deactivates G proteins, reduce
opiate-induced analgesia in both rodents and Cepaea
(Parenti i>t al.. 1986; Przewlocki et ai, 1987; Yu and Ka-
valiers, 1990). Whether magnetic fields affect G protein
activity is not known.

Calcium-activated, phospholipid-dependent protein
kinase (protein kinase C; PKC) also plays an important
role in relaying transmembrane signalling in diverse cal-
cium-dependent cellular processes (Kaczmarek, 1987).
Results of studies with PKC activators and inhibitors have
shown that modulation of ion channel activity is an im-
portant function of PKC (DeRiemer et ai, 1985; Kacz-
marek, 1987; Strong et al., 1987; Conn et al., 1989). Rel-
atively little is known about the relations between PKC
and opioid receptor activity, although results of a recent
study indicate that stimulation of PKC with phorbol esters
attenuates opioid activity through a decrease in G protein
activity (Louie et al.. 1990).

There is, however, accumulating evidence linking
magnetic fields and PKC activity. Magnetic stimuli have
been reported to augment the effects of phorbol esters
(PKC activators) and increase PKC activity in a number
of cell culture preparations (Byus et al., 1987; Adey, 1987,
1990). In Cepaea, the isoqinoline sulfonamides H-7 and
H-9, which are specific inhibitors of PKC, reduce the in-
hibitory effects of 60 Hz magnetic fields on morphine-
induced analgesia, whereas administration of the PKC
activator SC-9 augments the effects of the magnetic fields

(Kavaliers et ai, 1991b). This suggests that the inhibitory
effects of magnetic fields on opiate-induced analgesia in
Cepaea may include increases in PKC activity. Whether
this involves effects on G proteins remains to be deter-
mined.

These mechanisms of action encompass a broader range
of effects than just that of the opioid systems. However,
in view of the broad range and phylogentic conservation
of fundamental processes in which opioid systems are in-
volved, these findings suggest that some of the biological
effects of magnetic fields may arise through alterations of
opioid activity.

Acknowledgments

We thank Susan Lipa and Donna Tysdale for their
technical assistance and twilight determinations. The re-
search described here was supported by Natural Science
and Engineering and Research Council of Canada grants
to M.K. and K.P.O.

Literature Cited

Adey, \V. R. 1981. Tissue interactions with non-ionizing electromag-
netic fields. Physiol Rev 61: 435-513.

Adey, W . R. 1987. Cell membranes: the electromagnetic environment
and cancer promotion. Neurochem. Res. 13:671-677.

Adey, W. R. 1990. Joint actions of environmental nonionizing elec-
tromagnetic fields and chemical pollution in cancer promotion. En-
viron. Health Perxpec. 86: 297-305.

Ahlboom, A. 1988. A review of the epidemiologic literature on magnetic
fields and cancer. Scan J \fork limiro. Health 14: 337-343.

Akaike, N., A. M. Brown, K. Nishi, and V. Tsuda. 1981. Actions of
verapamil, diltiazem and other divalent cations on the calcium current
of Helix neurons. Br J Phurmacul. 74: 87-95.

Akil, H., S. J. Watson, M. E. I^ewis, II. Khachaturian, and M. J. Walker.
1984. Endogenous opioids: biology and function. Ann. Rev. Neu-
rosci. 1: 22-256.

Amil, Z., and G. H. Galina. 1986. Stress-induced analgesia: adaptive
pain suppression. Physiol. Rev 66: 1091-1 120.

Azanza, M. J. 1989. Steady magnetic fields mimic the effect of caffeine
on neurons. Brain Res. 489: 195-198.

Balaban, P. M., N. I. Bravarenko, and A. N. Kuznetzov. 1990. Influence
of a stationary magnetic field on bioelectric properties of snail neurons.
Bioelectromagnetics 11: 13-25.

Bawin, S. M., and W. R. Adey. 1976. Sensitivity of calcium binding
in cerebral tissue to weak environmental electric fields oscillating at
low frequency. Proc. Nat. Acad. Set. USA 73: 1999-2003.

Besson, J.-M., and A. Chaouch. 1987. Peripheral and spinal mecha-
nisms of nociception. Physiol Rev 67: 68-186.

Blackman, C. F., S. G. Beane, R. J. Rabinoowicz, D. R. House, and
W. T. Joines. 1985. A role for the magnetic field in the radiation-
induced efflux of calcium ions from brain tissue in vivo. Bioelec-
iromagnetics 6: 327-339.

Blackman, C. F., L. S. Kinney, D. E. House, and W. T. Joines.
1989. Multiple power-density windows and their possible origin.
Bioelectromagnetics 10: 1 15-128.

Brown, F. A., Jr. 1971. Some orientational influences of non-visual
terrestrial electromagnetic fields. Ann. N.Y. Acad. Sci- 188: 224-241.

Brown, F. A., Jr., and H. M. Webb. 1960. A "compass-direction"
effect for snails in constant conditions and its lunar modulation. Bio!
Bull. 119:65-74.

308

M. KAVALIERS AND K..-P. OSSENKOPP

Brown, F. A., Jr., M. F. Bennett, and II. M. \\ ebb. 1960s. A magnetic
compass response of an organism. Biol. Bull. 119: 65-74.

Brown, F. A., Jr., VV. J. Brett, M. F. Bennett, and F. H. Barnwell.
1960b. Magnetic response of an organism and its solar relationships.
Bio/ Bull. 118: 367-381.

Byus, C. V., S. E. Pieper. and NV. R. Adey. 1987. The effects of low-
energy 60 Hz environmental electromagnetic fields upon the growth-
related enzyme ornithine decarboxylase. Carcinogenesis 8: 1385-
1389.

Carson, J. J. L., F. S. Prato, D. J. Prost, L. D. Diesbourg, and S. J.
Dixon. 1990. Time-varying magnetic fields increase cytosolic free
Ca:+ in HL60 cells. Am. J Physiol 28: C687-C692.

Chiles, C., E. Hawrot, J. Gore, and R. Byck. 1989. Magnetic field
modulation of receptor binding. Magnetic Resonance in Medicine
10:241-245.

Conn, J. P., J. A. Strong, and L. K. Kaczmarek. 1989. Inhibitors of
protein kinase C prevent enhancement of calcium current and action
potentials in peptidergic neuron ofAplysia. J. Neitrosci. 9: 480-487.

Cremer-Bartel, G., K. krause, G. Mitoskas, and D. Brodersen.

1984. Magnetic fields of the earth as additional zeitgeber for en-
dogenous rhythms. Naturwissenschaften 71: 567-574.

Dalton, L. M., and P. S. \\iddowson. 1989. The involvement of opioid
peptides in stress-induced analgesia in the slug Arion ater. Peplides
10:9-13.

De Riemer, S. A., J. A. Strong, K. A. Albert, P. Greengard, and L. K.
Kaczmarek. 1985. Enhancement of calcium current in Aplysia
neurons by phorbol ester and kinase C. Nature 313: 313-316.

Ford, R., D. M. Jackson, L. Tetrault, J. C. Torres, P. Assanh, J. Harper,
M. K. Leung, and G. B. Stefano. 1986. A behavioral role for en-
kephalins in regulating locomotor activity in the insect Leucophaea
maderae: evidence for high affinity kappa-like opioid binding sites.
Comp. Biochem. Physiol. 85C: 61-66.

Gerschenfeld, H. M., C. Hammond, and D. Paupardin-Tritsch. 1986.
Modulation of the calcium current of molluscan neurones by neu-
rotransmitters. J. Exp. Biol. 124: 73-91.

Golding. G. P., L. Newboult, J. M. H. Rees, and B. R. Varlow.

1985. The effects of 50 Hz magnetic fields on opioid peptide me-
diated inhibition of guinea pig ileum. Neuropeptides 5: 357-358.

Gould, J. 1984. Magnetic field sensitivity in animals. Ann. Re\'. Physiol.
4: 585-598.

Hammond, C., D. Paupardin-Tritsch, A. C. Narin, P. Greengard, and
H. M. Gerschenfeld. 1987. Cholecystokinin induces a decrease in
Ca++ current in snail neurons that appears to be mediated by protein
kinase C. Nature 325: 809-8 1 1 .

Hirst, M., and M. Kavaliers. 1987. Levorphanol but not dextrophan
suppresses the foot-lifting response to an aversive stimulus in the
terrestrial snail (Cepaea nemoralis). Neuropharmaeology 26: 121-
123.

Hollt, V. 1986. Opioid peptide processing and receptor selectivity. Ann.
Rev. Pharmacol. Tmicol. 26: 59-77.

Kaczmarek, L. K. 1987. The role of protein kinase C in the regulation
of ion channels and neurotransmitter release. Trends Neurosci. 10:
30-34.

Kavaliers, M. 1987. Evidence for opioid and non-opioid forms of stress-
induced analgesia in the snail. Cepaea nemoralis. Bruin Rex 410:
111-115.

Kavaliers, M. 1989a. Evolutionary and comparative aspect of noci-
ception. Brain Rex. Bull. 6: 923-931.

Kavaliers, M. 1989b. Polymorphism in opioid modulation of the ther-
mal responses of the land snail Cepaea nemoralis Can. J. Zoo/. 67:
2721-2724.

Kavaliers, M., and M. Hirst. 1986. Naloxone-reversible stress-induced
feeding and analgesia in the slug (Limax maximus). Life Sci. 38:
203-209.

Kavaliers, M., M. Hirst, and G. C. Teskey. 1983. A functional role
for an opiate system in snail thermal behavior. Science 220: 99-101.

Kavaliers, M., M. Hirst, and G. C. Teskey. 1985. The effects of opioid
and FMRF-amide peptides on thermal behavior in the snail. Neu-
ropharmacology 24: 621-626.

Kavaliers, M., and K.-P. Ossenkopp. 1984. Exposure to rotating mag-
netic fields alters morphine-induced behavioural responses in two
strains of mice. Neuropharmaeology 89: 440-443.

Kavaliers, M., and K.-P. Ossenkopp. 1986. Magnetic field inhibition
of morphine-induced analgesia and behavioral activity in mice: ev-
idence for involvement of calcium ions. Brain Res 379: 830-838.

Kavaliers, M., and K.-P. Ossenkopp. 1987. Calcium channel involve-
ment in magnetic field inhibition of morphine-induced analgesia.
Naunyn-Schmiedebergs Arch. Pharmakol. 336: 308-315.

Kavaliers, M., and K.-P. Ossenkopp. 1989. Magnetic fields inhibit
opioid-mediated 'analgesic' behaviours of the terrestrial snail, Cepaea
nemoralis. J. Comp. Physio/. A. 162: 551-558.

Kavaliers, M., and K.-P. Ossenkopp. 1991. Magnetic fields, opioid
systems and day-night rhythms of behavior. In Electromagnetic Fields
and Circadian Rhythms. \'oi I. Advances in Circadian Physiology.
M. Moore-Ede and S. Campbell, eds. Binkhauser, Boston, (in press).

Kavaliers, M., K.-P. Ossenkopp, and S. Lipa. 1990. Day-night rhythms
in the inhibitory effects of 60 Hz magnetic fields on opiate-mediated
'analgesic' behaviors of the land snail. Cepaea nemoralis. Brain Res.
517: 276-282.

Kavaliers, M., L. Eckel, K.-P. Ossenkopp, and N. Vu. 1991a. Enhanced
learning of a sexually dimorphic spatial task in meadow voles fol-
lowing brief exposure to 60 Hz magnetic fields. Soc. Neurosci. Abstr.
21 (in press).

Kavaliers, M., K.-P. Ossenkopp, and D. M. Tysdale. I991b. Evidence
for the involvement of protein kinase C in the modulation of mor-
phine-induced 'analgesia' and the inhibitory effects of exposure to
60Hz magnetic fields in the land snail, Cepaea nemoralis. Brain Res.
(in press).

Kirschvink, J. I.., and J. L.Gould. 1981. Biogenic magnetite as a basis
for magnetic field detection in animals. Biosystems 13: 181-201.

Kream, R. M., R. S. /ukin, and G. B. Stefano. 1980. Demonstration
of two classes of opiate binding sites in the nervous tissue of the
marine mollusc, Mytilim edulis. J. Biol. Chem. 225: 9218-9224.

Leask, M. J. M. 1977. A physiochemical mechanism for magnetic
field detection by migratory birds and homing pigeons. Nature 267:
144-145.

Leucht, T. 1984. Responses to light under varying magnetic conditions
in the honey bee. Apis mellificia. J. Comp. Physio/. A. 154: 865-870.

Leucht, T. 1990. Ineractions of light and gravity reception with magnetic
fields in \enopit.s lacvis. J. Exp. Biol. 148: 325-334.

Leung, M. K., and G. B. Stefano. 1984. Isolation and identification of
enkephalins in pedal ganglia of Mytilus edulis (Mollusc) Proc. Nat.
Acad. Sci. USA 81: 955-958.

Leung, M. K., and G. B. Stefano. 1987. Comparative neurobiology of
opioids in invertebrates with special attention to senescent alterations.
Prog. Neurobiol. 28: 131-159.

Leung, M. K., H. H. Boer, J. van Minnen, J. Lundy, and G. B Stefano.
1990. Evidence for an enkephalmergic system in the nervous system
of the pond snail, Lymnaea slagna/is. Brain Res. 531: 66-71.

Liboff, A. R., R. J. Rozek, M. L. Sherman, B. R. McLeod, and S. D.
Smith. 1987. 45Ca cyclotron resonance in human lymphocytes. J.
Bioe/ecl 6: 13-22.

Liboff, A. R., and B. R. McLeod. 1988. Kinetics of channelised mem-
brane ions in magnetic fields. Bioelectromagnetics 9: 39-5 1 .

Liburdy, R. P., R. Tenforde, and R. L. Magin. 1986. Magnetic-field
induced drug permeability in liposome vesicles. Radial. Res. 108:
102-111.

MAGNETIC FIELDS AND OPIOID SYSTEMS

309

Lohmann, K. J., and A. O. D. Willows. 1987. Lunar-modulated geo-
magnetic orientation by a marine mollusk. Science 235: 231-235.

Louie, A. K., E. S. Bass, J. Zahn, P. E. Law, and H. H. Loh.
1990. Attenuation of opioid receptor activity by phorbol esters in
neuroblastoma X Glioma NG 108-15 Hybrid cells. J Pharmacol
E\p Therp 253:401-407.

Maldonado. H., and A. Miralto. 1987. Effects of morphine and nal-
oxone on a defensive response of the mantis shnmp, Si/iu/lti mantis
J Cinnp. Physio/. A147: 455-459.

Martin, \V. R. 198-4. Pharmacology of opioids. Pharmncol Rev 35:
283-323.

Miller, R. J. 1987. Multiple calcium channels and neuronal function.
Science 325: 46-52.

Miller, D. B., C. F. Blackman, and J. S. Alie. 1985. Behavioral responses
of morphine-treated mice to ELF magnetic fields. BioelectrrJrnagn.
Soc. Meet Ahstr 1985: 54.

North, R. A. 1986. Opioid receptor types and membrane ion channels.
TINS 9: 114-117.

Olcesc, J., S. Reuss, and L. Vollrath. 1985. Evidence for the involve-
ment of the visual system in mediating magnetic field effects on pineal
melatonin synthesis in the rat. Brain Res 333: 382-384.

Olcese, J., S. Reuss, J. Stehle, S. Steinlecher, and L. Vollrath.
1988. Response of the mammalian retina to experimental alteration
of the ambient magnetic field. Brain Rex. 448: 325-330.

Ossenkopp, K.-P., and R. Barbeito. 1978. Bird orientation and the
geomagnetic field: a review. Neurosd. Bioheltav. Rev 2: 255-270.

Ossenkopp, K.-P., and M. Kavaliers. 1987. Morphine-induced anal-
gesia and exposure to 60 Hz magnetic fields: inhibition of nocturnal
analgesia is a function of magnetic field intensity. Brain Res 418:
356-360.

Ossenkopp, K.-P., and M. Kavaliers. 1988. Clinical and applied aspects
of magnetic field exposure: a possible role for the endogenous opioid
systems. J. Bioelect. 7: 189-208.

Ossenkopp. K.-P., M. Kavaliers, and M. Hirst. 1983. Reduced mor-
phine analgesia in mice following a geomagnetic disturbance. Neu-
rosci. Lett. 40: 321-325.

Ossenkopp, K.-P., M. Kavaliers, and S. M. Lipa. 1990. Increased
mortality in land snails (Cepaea nemoralis) exposed to powerline
(60-Hz) magnetic fields and effects of the light-dark cycle. Neurosd.
Lett 114: 89-94.

Papi, F., and P. Luschi. 1991. Pigeon navigation: naloxone injection
and magnetic disturbance have a similar effect on initial orientation.
Rend. Ace. Na:. Lincei (in press).

Parent!, M., F. Tirone, G. Giagnoni, N. Pecora, and D. Parolaro.
1986. Pertussis toxin inhibits the antinociceptive action of morphine
in the rat. Eur. J Pharmacol. 124: 357-359.

Phillips, J. B. 1987. Specialized visual receptors respond to magnetic
field alignment in the bowfly (Calliphora vicina). Soc. Neurosd. Ahstr.
13: 397.

Prato, F. S., K.-P. Ossenkopp, and M. Kavaliers. 1987. Nuclear mag-
netic resonance inhibition of morphine-induced analgesia in mice:
differential effects of the static, radio-frequency and time-varying
magnetic field components. Mag. Reson. Imag. 5: 9-14.

Przewlocki, R., T. Costa, J. Lang, and A. Herz. 1987. Pertussis toxin
abolishes the antmociception mediated by opioid receptors in rat
spinal cord. Eur. J Pharmacol. 144: 91-93.

Rodgers, R. J., and J. I. Randall. 1988. Environmentally-induced an-

algesia: situational factors, mechanisms, and significance. Pp 107-
147 in Endorphins, Opiates ami Behavioural Processes. R. J. Rodgers
and S. J. Cooper, eds. Wiley, Chichester.

Santoro, C., L. M. Hall, and R. S. Zukin. 1990. Characterization of
two classes of opioid binding sites in Drosoplnla melanogasler head
membranes./ Neurochem. 54: 154-170.

Savitz, D. A., H. Watchel, F. A. Barnes, E. M. John, and J. G. Tvrdik.
1988. Case-control study of childhood cancer and exposure to 60-
Hz magnetic fields. Am J Epulemiol 128: 21-38.

Scharrer, B., G. B. Stefano, and M. K. Leung. 1988. Opioid mechanisms
in insects, with special attention to Leucophaea maderae. Cell. Mol.
Neurobiol. 8: 269-284.

Semm, P., T. Schneider, and L. Vollrath. 1980. The effects of an earth-
strength magnetic field on the electrical activity of pineal cells. Nature
288: 607-608.

Sherrington, C. S. 1906. The Intcxrutive Action of the Nervous System.
Yale University Press, New Haven.

Stefano, G. B. 1982. Comparative aspects of opioid-dopamine inter-
action. Cell. Mol. Neurohiol 2: 167-182.

Stefano, G. B. 1989. Role of opioid neuropeptides in immunoregu-
lation. Pr»t>. Neurohiol. 33: 149-153.

Stefano, G. B., M. K. Leung, X. /hao, and B. Scharrer. 1989. Evidence
for the involvement of opioid neuropeptides in the adherence and
migration of immunocompetent invertebrate hemocytes. Proc. Nat.
Acad. Sci. USA 86: 626-630.

Strong, J. A., A. P. Fox, R. W. Tsien, and L. K. Kaczmarek.
1987. Stimulation of protein kinase C recruits covert calcium
channels in Ap/ysui hag cell neurons. Nature 325: 714-717.

Stryer, L., and H. R. Bourne. 1986. G proteins: a family of signal
transducers. . I nn. Rev Cell liiol 2:391-419.

Tysdale, D., S. M. Lipa, K.-P. Ossenkopp, and M. Kavaliers.
1991. Inhibitory effects of 60Hz magnetic fields on opiate-induced
'analgesia' in the land snail, Cepaea nemoralis. under natural con-
ditions. Physiol Behiiv 49: 53-56.

Valeggia, C., E. Ferandez-Duque, and II. Maldonado. 1989. Danger
stimulus-induced analgesia in the crab Chasmagnethus granulatus.
Brain Res 481: 304-308.

Walters. E. T. 1987. Site-specific sensitization of defensive reflexes in
Aplysia: a simple model of long-term hyperalgesia. J Neurosd. 1:
400-407.

Walters, E. T., and M. T. Erickson. 1986. Directional control and the
functional organization of defensive responses in Aplysia. J. Comp.
Physio/. A 159: 339-351.

Wiltschko, W., and R. Wiltschko. 1990. Magnetic orientation and
celestial cues in migratory orientation. Experentia 46: 342-352.

Yu, N., and M. Kavaliers. 1991. Pertussis toxin reduces the day-night
rhythm of nociception and opioid mediated antinociception in the
land snail Cepaea nemoralis. Peptides (in press).

Zagon, I. S., and P. J. McLaughlin. 1989. Opioid antagonist modu-
lation of murine neuroblastoma: a profile of cell proliferation and
opioid peptides and receptors. Brain Res 480: 16-28.

Zispcr, B., M. R. Ruff, J. B. O'Neill, C. C. Smith, W. J. Higgins, and
C. B. Pert. 1988. The opiate receptor: a single 1 10 kDa recognition
molecule appears to be conserved in Tetrahymena leech and rat.
Brain Res 463: 296-304.

Reference: Biol. Bull 180: 310-317. (April, 1991)

Oxidative Breakdown Products of Catecholamines and

Hydrogen Peroxide Induce Partial Metamorphosis

in the Nudibranch Phestilla sibogae Bergh

(Gastropoda: Opisthobranchia)

ANTHONY PIRES AND MICHAEL G. HADFIELD

Kewalo Marine Laboratory. P.B.R.C.. University of Hawaii, 41 Afwi St., Honolulu. Hawaii 96813

Abstract. Veliger larvae of the aeolid nudibranch Phes-
tilla sibogae metamorphose in response to a soluble factor
from their prey coral, Porites compressa. Metamorphosis
begins with destruction of the velum, a ciliated structure
used for swimming and feeding. Previous investigation
had shown that P. sibogae larvae exposed to certain cat-
echolamines lost the velum, but then failed to complete
any subsequent steps characteristic of natural coral-in-
duced metamorphosis. Because catecholamines oxidize
rapidly in seawater, we have re-examined morphogenic
effects of catecholamines using superfusion chambers that
allow periodic replacement of test solutions. We report
that fresh, unoxidized catecholamines do not induce velar
loss, but that this morphogenic activity develops in aged,
oxidized solutions of a variety of catecholamines and other
catechol compounds. Evidence is presented that this ac-
tivity is attributable to hydrogen peroxide, a byproduct
of catechol autoxidation. Hydrogen peroxide induces velar
loss at 10~4 M. The possible relationship of peroxide-in-
duced velar loss to natural coral-induced metamorphosis
is discussed.

Introduction

Chemical and neural mechanisms governing meta-
morphosis in marine invertebrates have long been of in-
terest both to ecologists seeking to understand recruitment

Received 14 August 1990: accepted 6 November 1990.

Abbreviations: CI: natural coral-derived metamorphic inducer; ASW:
artificial seawater. FSW: filtered seawater: EP: (-)epmephrine; NE:
(-)norepinephnne; IP: (-)isoproterenol; DA: dopamine; DOPA: L-B-
3, 4-dihydroxyphenylalanine; DOPAC: 3.4-dihydroxyphenylacetic acid:
DOM A: 3,4-dihydroxymandelic acid; DOB: 1,2-dihydroxybenzene;
HVA: homovanillic acid; OCT: octopamine.

of larvae into adult populations, and to more reductionist
biologists who view invertebrate larvae as excellent model
systems for exploring the regulation of development
(Hadneld, 1986). Larvae of the aeolid nudibranch Phes-
tilla sibogae metamorphose upon exposure to a water-
soluble factor derived from the stony coral Porites com-
pressa, P. sibogae's adult prey. Efforts to isolate and iden-
tify the coral-derived metamorphic inducer (CI, Hadneld
and Pennington, 1990) have been accompanied by the
screening of a wide range of chemical species for their
capacity to induce metamorphosis (Hadneld, 1984; Hirata
and Hadneld, 1986; Yool et al, 1986: Pennington and
Hadneld. 1989). Any such morphogens discovered by this
second approach can be evaluated as possible structural
analogues of CI, or as molecules involved in internal
transduction of the CI signal, or as regulators of devel-
opmental mechanisms that normally unfold as a conse-
quence of metamorphic induction by CI. Known neu-
rotransmitters and neurohormones are among the plau-
sible candidates for all three of these roles (D. E. Morse,
this symposium; Bonar et al., 1990). If a morphogenic
response can be induced by application of a known neu-
roactive compound, certain plausible hypotheses may be
made about what sorts of receptors and internal trans-
duction systems might mediate natural metamorphosis,
and appropriate experiments designed to test the impli-
cated mechanisms.

Hadneld (1984) reported that larvae exposed to the cat-
echolamines epinephrine (10~4 A/) or norepinephrine
(10~3 At) would often undergo a partial metamorphosis
restricted to loss of the velum (a ciliated larval swimming
and feeding organ), not followed by any of the subsequent
steps in the morphogenic sequence characteristic of nat-

310

MORPHOGENIC EFFECT OF

311

ural metamorphosis (described in Bonar and Hadfield,
1974; Hadfield. 1978). A difficulty in the interpretation
of this result was that catechols autoxidize rapidly to qui-
nones in alkaline aqueous solutions, in a multi-step re-
action that generates hydrogen peroxide (H:O;.). Figure 1
shows the net result of this reaction; its mechanism, the
nature of intermediate products and regulation by pH
and various catalysts have been explored for several cat-
echolamines and are the subject of an extensive literature
(Heacock, 1959; Hawley et al.. 1967; Misra and Fridovich,
1972; Graham, 1978; Cohen, 1983). At 25°C, a 1CT4 M
solution ofepinephrine in MBL artificial seawater (ASW;
Cavanaugh. 1956) Tris-buffered to pH 8.2 begins to turn
visibly pink within 15 min due to the appearance of the
quinone oxidation intermediate, adrenochrome. Maxi-
mum adrenochrome concentration, measured spectro-
photometrically, is attained within 3 h (Piresand Hadfield,
unpub. data). We therefore decided to re-examine mor-
phogenic effects of catecholamines and related compounds
using superfusion chambers that permit rapid periodic
replacement of test solutions to control for catechol au-
toxidation. Our goal was to determine whether the pre-
viously reported partial metamorphosis was indeed due
to catecholamines, or to some product of catecholamine
oxidation.

We report that partial metamorphosis (velar loss) is
induced in P. sibogae by solutions of any of several cat-
echol compounds aged in ASW or by H:O2 but not by
fresh catecholamines. We also provide evidence that the
morphogenic potency of aged catechols is due to H2O2 or
a derivative oxygen species generated as a consequence
of catechol autoxidation. These results emphasize the need
for caution in interpreting biological effects of bath-applied
catecholamines and other unstable chemical species. Our
results also suggest testable hypotheses concerning possible
roles of H2O2 and oxygen radicals in natural coral-induced
metamorphosis.

Materials and Methods

Larval culture

All larvae used in these experiments were taken from
our laboratory culture system. Adult P. sibogae were kept
together with field-collected heads of their prey coral P.
compressa in outdoor sea tables supplied with running
unfiltered seawater (~25°C). Egg masses deposited on
the coral were collected daily and transferred to .22 /im
filtered seawater (FSW). Eggs developed at 25°C in aerated
glass beakers in an incubator and were mechanically
hatched at day 6 post-fertilization. Subsequent culture
procedures were as previously described (Miller and Had-
field, 1 986) except that larval culture chambers continued
to be maintained in an incubator at 25 °C after hatching.

Figure 1. Generalized autoxidation of a catechol to a quinone, with
the production of hydrogen peroxide (H:O:). For catecholamines. this
reaction may also involve cyclization of the side chain R.

Experiments were conducted on 10-day-old (post-fer-
tilization), unfed larvae. These larvae are facultative
planktotrophs; under our culture conditions nearly all 10-
day-old veligers are competent for metamorphosis without
having to feed (Kempf and Hadfield, 1985; Miller and
Hadfield, 1986).

Preparation of test solutions

(-)Epinephrine (EP), (-)norepinephrine (NE), (-)iso-
proterenol (IP), dopamine (DA), L-B-3, 4-dihydroxyphe-
nylalanine (DOPA), 3,4-dihydroxyphenylacetic acid
(DOPAC), 3,4-dihydroxymandelic acid (DOMA), cate-
chol (1,2-dihydroxybenzene, DOB), homovanillic acid
(HVA), octopamine (OCT). acetylsalicylic acid (aspirin),
and thymol-free bovine catalase were purchased from
Sigma Chemical Co. (St. Louis, Missouri). The first eight
compounds above, all of which contain a catechol group,
are sometimes referred to genetically in the text as "cat-
echol compounds" or "catechols." To avoid confusion,
catechol itself is referred to as 1,2-dihydroxybenzene
(DOB). All monoamines were obtained as hydrochloride
salts except for EP (bitartrate) and DOPA (free acid).
Pharmaceutical 3% H2O2 (Parke-Davis) was purchased
locally. Stock solutions of test compounds were made fresh
daily in deionized water at 10 times the desired final con-
centration and then diluted into l.lx normal strength
MBL ASW (Cavanaugh, 1956) buffered to pH 8.1-8.2
with 10 mMTrizma® (Sigma). This practice yielded ASW
solutions of test compounds that were ionically equivalent
to normal strength (l.OX) MBL ASW.

Many of the experiments presented in this paper were
conducted to assay morphogenic potencies of fresh versus
aged (oxidized) solutions of various catechol compounds.
Aged solutions of test compounds in ASW were prepared
as above and allowed to stand 10-14 h at 23-25°C. Fresh
solutions were prepared in the same way from test com-
pound stocks and 1.1X MBL ASW but were mixed im-
mediately before use, as were ASW solutions of H2O2 for
experiments on morphogenic effects of H2O2.

Experimental chambers

Experimental chambers (Fig. 2) were designed to permit
rapid, frequent replacement of test solutions during ex-

312

A. PIRES AND M. G. HADFIELD

periments. Each chamber is assembled from two sym-
metrical halves, constructed as follows. The body of each
half-chamber is made from the base of a disposable plastic
spectrophotometer cuvette cut to a volume of 1 cm1. An
18-gauge hypodermic needle, cut to a length of 1-2 mm,
is inserted through a small hole in the base of the half-
chamber and cemented in place with silicone rubber
aquarium sealant (Dow-Corning or equivalent). This fea-
ture allows replacement of test solutions with a syringe.
Silicone rubber gaskets on the open rim of each half-
chamber seal a mesh barrier (100 jum Nitex®) that sepa-
rates the two halves. The halves are clamped together with
rubber bands.

Our method for making the gaskets is generally useful
for the construction of small watertight apparatuses. First,
silicone rubber aquarium sealant is applied to the gasket-
bearing surface to a slightly greater depth than the desired
thickness of the finished gasket. Then a microscope slide
(or other piece of smooth flat glass) smeared with a very
thin film of silicone stopcock grease is laid down on the
wet sealant and lightly pressed down until good contact
is visible between sealant and glass along the entire surface
of the gasket. After the sealant has cured, the glass can be
"popped" off the finished gasket with a razor blade.

Comparisons of morphogenic potencies of fresh and
aged test solutions (Figs. 4 and 6) were carried out in the
superfusion chambers according to the following protocol.
Each chamber was loaded with 17-1 12 larvae using a pi-
pet. After the chamber halves were assembled, each
chamber was flushed with 10 ml ( = 5X chamber volume)
of test solution. Thereafter, test solutions were replaced
in the same manner every 30 min until a total exposure
time of 7 h had elapsed. Then the chambers were flushed
with FSW and larvae washed out into Slender dishes.
Larvae were scored for velar loss 16-24 h later (criteria
below). In these experiments one set of 3 or 4 chambers
was run with fresh test solutions (prepared as above for
each solution change), while another set was run simul-
taneously with aged solutions (prepared as above the night
before the experiment). The solution changing procedure,
as well as other aspects of physical manipulation of the
larvae, was identical in fresh solution and aged solution
treatments.

Experiments on dose-dependent morphogenic effects
of H2O: and of aged DA (Figs. 5 and 7) were performed
in 6 cm Stender dishes. Larvae (53-220 per dish) were
exposed to several concentrations of H:O: in ASW for 7
h without solution changes, and then washed into FSW
for scoring as above.

Scoring oj velar loss

Different degrees of velar loss are described and illus-
trated below. For scoring purposes, larvae able to swim

OSF

,SG

I SF

Figure 2. Superfusion chamber used in experiments comparing
morphogenic potencies of fresh versus aged solutions of catechols. Larvae
are retained in the lower half-chamber (HC) by a Nitex mesh barrier
(NM). held in place by silicone rubber gaskets (SG). Inlet and outlet
syringe fittings (1SF and OSF) allow periodic flushing with a syringe
containing test solution. Total chamber volume is 2 cm1. Half-chambers
are held together during use by rubber bands (not shown).

freely and climb through the water column were consid-
ered to be intact. Such individuals never showed any ev-
idence of velar reduction when examined at SOX mag-
nification. Larvae lying on the bottom of the dish or
swimming, but failing to clear the bottom, were scored
as "velum lost" if any of the large ciliated cells at the velar
margin were missing, or if the velar lobes were noticeably
shortened. Instances of partial (Fig. 3B) and complete (Fig.
3C) velar loss were combined and divided by the total
number of larvae to obtain a frequency of velar loss for
each trial.

Results

Induction of velar loss by aged catechols and H:O:

Loss of the velum, one of the major morphological
transformations occurring during metamorphosis, can be
considered a partial metamorphosis (Bonar and Hadfield,
1974; Hadfield, 1984). We consistently found that high
proportions of larvae lost some or all of the velum (except
for small remnant clumps of cephalic supportive cells)
when exposed for 7 h to aged solutions of any of the cat-

MORPHOGENIC EFFECT OF H,O,

B C

313

Figure 3. Velar loss in Phestilla sibogae. A. Lateral view of 11-day-old, untreated larva. Animal was
photographed with intact velum (arrow) partly withdrawn into the larval shell to keep velum in same plane
of focus as the rest of the animal: foot (f) is partly extended. Outlines of large ciliated cells are visible along
the velar margin. B. Partial velar loss in an 1 1-day-old larva treated with 10~4 Kf H:O: on day 10. C.
Complete velar loss in an 1 1-day-old larva treated with 2 • 10"1 M H:O; on day 10. Differences between
larvae in shape of foot represent a range of movement independent of velar loss. Scale bar = 100 pm.

echol compounds tested, or to H2O2, but not when ex-
posed to fresh catecholamines. Figure 3 illustrates velar
loss in response to H2O2 but it could just as well indicate
the results obtained with aged solutions of catechols. An
1 1 day-old untreated larva with intact velum is shown in
Figure 3A. Velar loss induced by H2O2 or by aged cate-
chols (or by CI) begins with the detachment of the large
ciliated cells at the velar margin. These cells are cast oft"
intact, and their cilia continue to beat for some time after
detachment. After separation of these cells begins, regres-
sion of non-ciliated supportive cells of the velar lobes be-
comes apparent. This state of partial velar loss is depicted
in Figure 3B. This state is stable in that velar loss will
proceed no further in larvae washed out of the H:O2 or
aged catechol treatment into FSW. If the above treatments
are applied in sufficiently high concentration (see below),
7-h treatment results in detachment of all the large ciliated
velar cells and detachment or regression of remaining tis-
sues, leaving small cephalic mounds of supportive cells
where the velar lobes had been (Fig. 3C). Velar loss in
response to these treatments, like that seen in natural
coral-induced metamorphosis, is highly tissue-specific.
Other ciliated epithelia (of the foot, for example) remain
intact. When velar loss is induced with H2O2 or aged cat-
echols, metamorphosis does not proceed beyond this
point. However, if such larvae are then exposed to CI,
many will right themselves on the foot, take up the settled
posture characteristic of natural metamorphosis (Hadneld,
1978), and complete metamorphosis in an apparently
normal fashion.

Comparison of effects of fresh and aged catechols

Morphogenic effects of fresh and aged solutions of the
catecholamines EP, NE, and IP (2 X 1(T4 M) and DA
(10~4 M) were quantitatively compared (Fig. 4). Dra-
matically different results were obtained in parallel trials
using aged and fresh solutions of the same compounds,
replaced every 30 min during the 7-h exposure period.
Most larvae lost some or all of the velum after exposure
to aged catecholamines. After exposure to fresh cate-
cholamines. larvae rarely showed any indication of velar

1.0-

0.9-
0.8-
0.7-
0.6-
0.5-
0.4-
0.3-
0.2-
0.1-
0.0

Dopamme Norepmephrme Epmephrme Isoproterenol

Figure 4. Frequencies of velar loss after 7-h exposure to fresh (open
bars) or aged (hatched bars) solutions of catecholamines. Concentrations
are 2 x 10~4 M except for dopamine (I0~4 M). Ordinate values and
error bars are means and standard deviations, respectively, calculated
from arcsine transformed data. Numbers above error bars indicate the
number of replicate trials, each involving a chamber containing 17-96
larvae. Trials for each compound were conducted on at least two different
batches of larvae.

314

A. PIRES AND M. G. HADFIELD

0.2 0.3 0.4 05 06 0.7 08 09 1.0
(Aged Dopamme] x 1 0 ^ (M)

Figure 5. Frequencies of velar loss after 7-h exposure to varying
concentrations of aged dopamine solutions. Triangles and circles represent
two assays conducted in triplicate on two different batches of larvae.
Each symbol represents a trial involving 53-220 larvae. Lines connect
grand means of velar loss at each concentration, calculated from arcsine
transformed data.

loss and were generally indistinguishable in morphology
and behavior from untreated animals. Larvae exposed to
a higher concentration of fresh DA (2 X 1(T4 M) according
to this protocol did sometimes metamorphose completely
by the time the experiment was scored, but at low fre-
quency (0-.25, typically .10-. 15). as suggested by earlier
experiments conducted without solution replacement
(Hadfield, 1984). However, in the current work, we used
1CT4 M DA for the aged versus fresh comparison because
aged solutions at higher concentrations proved somewhat
toxic under these conditions. Complete metamorphosis
was observed at very low frequency (<.05) after exposure
to fresh 1(T4MDA.

The frequency of velar loss in response to varying con-
centrations of aged DA is given in Figure 5. Concentration
threshold for velar loss after 7-h exposure to fresh DA
appears to lie between .25 and .5 X 10~4 M.

Experiments to test morphogenic effects of other cat-
echol compounds yielded similar results (Fig. 6). Aged
solutions of the deaminated catecholamine metabolites
DOPAC or DOM A (both 2 X 1(T4 A/) as well as of DOB
(10~4 M) consistently yielded high frequencies of velar
loss. Fresh solutions of DOPAC and DOMA were rela-
tively ineffective. Fresh DOB caused most larvae to show
some evidence of velar loss, but this was invariably con-
fined to the loss of a few large ciliated cells at the velar
margin. Aged DOB, in contrast, nearly always resulted in
detachment of all the ciliated velar cells and substantial
regression of the velar lobes. DOPA, a catechol amino
acid precursor of the catecholamine neurotransmitters,
also caused a high mean frequency of velar loss (.67) in
aged 10~4 M solutions, but a quantitative comparison
could not be made with fresh solutions. Larvae treated
with fresh DOPA tended to withdraw completely into the
shell, and although vela appeared to be intact, accurate
scoring of velar condition was not possible. OCT (NE

1.0-
0.9-
0.8-

07-

0.6

0.5

0.3

0.2

0.1

6
T

DOPAC

DOMA

DOB

Figure 6. Frequencies of velar loss after 7-h exposure to fresh (open
bars) or aged (hatched bars) solutions of the catechol compounds dihy-
droxyphenylacetic acid (DOPAC). dihydroxymandelic acid (DOMA).
and dihydroxybenzene (DOB). Concentrations are 2 X 1CT4 At except
for DOB ( 10~4 A/). Ordinate values and error bars are means and standard
deviations, respectively, calculated from arcsine transformed data.
Numbers above error bars indicate the number of replicate trials, each
involving a chamber containing 18-112 larvae. Trials for each compound
were conducted on at least two different batches of larvae.

minus one ring hydroxyl group) and HVA (DOPAC with
one ring hydroxyl group methylated) do not oxidize as
easily as their related catechol compounds, and had no
morphogenic effects in aged or fresh 2 X 1CT4 M solutions.

Quantification of H2O2-induced velar loss and abolition
of effects ofH2O: and aged catechols by catalase

Because autoxidation of catechols in water yields H2O2
(Graham ct at.. 1978). we tested the ability of H2O2 in
ASW solutions to induce velar loss. Exposure to H2O2 for
7 h reliably induced velar loss at a concentration threshold
in the range of .2S-.5 X 1(T4 A/ (Fig. 7). Solutions of .25
X 10~4 M H2O2 were never sufficient to cause observable
velar loss; these animals were indistinguishable from un-
treated individuals (Fig. 3A). A large but variable fraction

1 On

Figure 7. Frequencies of velar loss after 7-h exposure to varying
concentrations of H2O2 . Triangles, circles and squares represent three
assays conducted on three different batches of larvae. Each symbol rep-
resents a trial involving 54-123 larvae. Lines connect grand means of
velar loss at each concentration, calculated from arcsine transformed
data.

MORPHOGENIC EFFECT OF

315

of larvae tested at .5 X 10 4 M H2O2 showed clear indi-
cations of partial velar loss. In ICT4 M H2O2 nearly all
larvae experienced at least partial velar loss; a typical in-
stance is shown in Figure 3B. In 2 X ICT4 M H2O2, most
larvae lost the entire velum except for small mounds of
cephalic supportive cells (Fig. 3C).

Morphogenic potencies of H2O2 and aged solutions of
all of the above catechol compounds were completely-
abolished by 10 min incubation with purified bovine cat-
alase (5 ^g/ml), prior to addition of larvae. The presence
of H2O2 in aged solutions of catechols was confirmed by
measuring an increase in dissolved oxygen concentration
upon catalase treatment, with a Clark-type oxygen meter.
(Catalase catalyzes the decomposition of H2O2 to water
and molecular oxygen.) Hydrogen peroxide-induced velar
loss was not inhibited by acetylsalicylic acid (aspirin) in
any concentration between 10~h and 10~3 M. [Aspirin.
an inhibitor of prostaglandin endoperoxide synthetase.
inhibits H:O;-induced spawning in the abalone Haliotix
nifescens (Morse et a/.. 1977).]

Discussion

Relationship oj velar loss induced by H:O: and hy aged
catechols to velar loss in natural metamorphosis

Velar loss in larvae of P. sibogae can be induced by
application of H2O: or aged solutions of catechols in the
tenth-millimolar concentration range (Figs. 3-7). The
stoichiometry of catechol autoxidation (Fig. 1), together
with our observation that similar concentrations of H2O2
or aged catechols are required to induce velar loss, suggests
the hypothesis that morphogenic activity of aged catechols
is due to H2O2 produced upon autoxidation or to some
other reactive species derived from H2O2 such as the hy-
droxyl radical HO' (for discussions of H2O2 metabolism
and oxygen radical biochemistry see Fridovich, 1978; Im-
lay and Linn, 1988; Cadenas, 1989; Kontos, 1989; Gut-
teridge el ul., 1990). Direct evidence for this hypothesis
is the fact that morphogenic activity of aged catechol so-
lutions is lost on incubation with catalase. an enzyme that
selectively degrades H2O2 to molecular oxygen and water.
However, we have not yet rigorously excluded cooperative
effects of quinone oxidation products of catechols in velar
destruction.

Natural coral-induced metamorphosis is preceded by
settlement behavior in which the larva takes up a char-
acteristic posture, attached by the foot to the substratum
(BonarandHadfield, 1974; Hadfield, 1978). This behavior
is not elicited by H2O2 or aged catechols. In natural meta-
morphosis, velar loss ensues in this settled position. Larvae
treated with H2O2 or aged catechols begin to lose the
velum while swimming; after enough large ciliated velar
cells have been lost, larvae sink to the bottom of the ex-

perimental chamber and typically lie on a side of the shell
in an extended posture.

Although the behavioral contexts for natural and ar-
tificially induced velar loss are different, the morphological
phenomena share several common features. Both begin
with the detachment of the large ciliated cells at the velar
margin. Natural and artificially induced velar loss are both
highly tissue-specific in that cell separation and tissue
regression are confined to the velum and are not mani-
fested in other ciliated epithelia. Following loss of the cil-
iated velar cells, clumps of nonciliated supportive cells
remain as cephalic mounds (Fig. 3C). Partial metamor-
phosis induced by H2O2 or aged catechols does not pro-
ceed beyond this point. However, no loss of metamorphic
competence has occurred, because such larvae can resume
metamorphosis once exposed to CI.

Further experiments are required to test the hypothesis
that H2O2 or derivative oxygen radicals mediate velar loss
in natural metamorphosis. Several techniques exist that
are potentially applicable to detection of H2O2 or oxygen
radical production in metamorphosing tissues (Freeman
and Crapo, 1981; Radzik el ai. 1983; Ruch et ai. 1983;
Kontos, 1989). Chemical scavengers of free radicals and
of H2O2 can also be used to interfere with radical-depen-
dent mechanisms (Cadenas, 1989; Kontos, 1989). We
were unable to inhibit coral-induced metamorphosis with
catalase, but that result is difficult to interpret because
catalase is not expected to penetrate cells to reach potential
sites of endogenous H2O2 generation or action. Future
experiments will include other more penetrant scavengers.
It is important to note that even if H2O2 or oxygen radicals
are implicated in the mechanism of natural metamor-
phosis, there are many biological sources of these oxygen
species other than oxidation of catechols (Cohen, 1983;
Kontos, 1989).

Possible modes of action ofH:O:

Hydrogen peroxide is well-known for its cytotoxic
properties, which render it useful as a topical disinfectant.
Mechanisms of cell damage by H2O2 and oxygen radicals
have been subjects of several recent discussions (Imlay
and Linn, 1988; Kontos, 1989; Gutteridge and Halliwell,
1990). Indeed, significant lysis of certain cultured verte-
brate epithelial cells occurs upon exposure to H2O2 con-
centrations only slightly higher than those demonstrated
to cause velar loss in the present study (Hayden et ai,
1990; Polansky et ai, 1990). However, the question of
whether H2O2-induced velar disintegration in Phestilla
larvae is a cytotoxic response remains. The facts that cil-
iated cells appear to be shed intact with cilia still beating
and that other epithelial tissues show no evidence of injury
at morphogenically active concentrations, might argue
otherwise. Close structural examination of shed velar cells

316

A. PIRES AND M. G. HADFIELD

in both H:O:-induced and coral-induced velar loss will
be instructive in this regard. Non-toxic regulatory effects
of H:O: mimic the effects of insulin on glucose, carbo-
hydrate, and lipid metabolism in vertebrate cells, possibly
by stimulating phosphorylation of the insulin receptor
(Heffetz el a/.. 1990). This mechanism of action may war-
rant investigation in Phestilla. particularly in light of the
recent discovery of a preproinsulin-related peptide in
growth-regulating neuroendocrine cells of the gastropod
Lymnaea stagnalis (Smit et ai, 1988) and the growing
appreciation of insulin's role in cellular differentiation
during embryogenesis (Alemany et a/., 1990). In another
gastropod, the abalone H. ntfescens. H:O2 induces
spawning, probably by activating prostaglandin endoper-
oxide synthetase (Morse et ai, 1977). This particular
pathway is unlikely to be involved in velar loss in Phestilla
because H^Oi induction of velar loss is not blocked by
aspirin, a potent inhibitor of that enzyme. One might
speculate that FLO: could also regulate the activity of a
factor involved in epithelial cell adhesive interactions,
perhaps akin to the "scatter factor" recently described as
a promoter of cell-cell separation in cultured mammalian
epithelia (Stoker and Gherardi, 1989).

Implications for other taxa

Our study points out the need for caution in the inter-
pretation of behavioral and morphogenic effects of bath-
applied catecholamines on marine animals. In the bivalve
Crassostrea gigas. sufficient physical controls and cor-
roborating pharmacological evidence have been mar-
shalled in support of the hypothesis that dopaminergic
and adrenergic neural pathways, mediate settlement and
morphogenesis, respectively (Coon and Bonar, 1987;
Bonar et a/.. 1990). There are also brief reports of meta-
morphic induction by DOPA in the mussel Mytilits eclulis
(Cooper, 1982), and by DA in the mud snail Ilyanassa
obsoleta (Levantine and Bonar, 1986), but details of the
methods are not given. Certain catecholamines have been
reported to induce metamorphosis in the scallops Patin-
opecten yessoensis (Kingzett et a/.. 1990) and Pecten
maximus (Cochard el al., 1 989), but in both of these stud-
ies the problem of catechol oxidation was not thoroughly
resolved. DOPA induces a low frequency of metamor-
phosis in the polychaete Phragmatopoma ealifornica
(Jensen, 1987), but that work has implicated cross-linked
quinoid derivatives of DOPA residues in proteins, rather
than catecholamine neurotransmitters, in the inductive
pathway (Jensen and Morse, 1990). To our knowledge
the only other published example of catecholamine in-
duction of larval metamorphosis is in the echinoid Den-
draster excentricits (Burke, 1983). Whole larvae and ex-
cised larval arms metamorphosed in response to DA, but
not to EP or NE. The response specificity suggests that

DA did not act as a source of H:O: in those experiments,
but the protocols are not detailed enough to permit clear
resolution of this issue.

Our results should not be interpreted to mean that cat-
echolamines do not act as neurotransmitters mediating
metamorphosis in P. sibogae. Bath-applied substances
might not reach their target tissues in the proper concen-
tration, or the co-activation of receptors on many cells
throughout the nervous system may result in net inhibi-
tion of circuits that effect metamorphosis. We do hope
that this study sounds a cautionary note to others inves-
tigating chemical control of metamorphosis, and prompts
consideration of a possible morphogenic role for H2O;.

Acknowledgments

We thank Esther Leise for critically reading the manu-
script, and Leonard Deal for competent and reliable tech-
nical assistance. Supported by NSF grant DCB89-03800
to M.G.H.

Literature Cited

Alemany, J., M. Girbau. and F. de Pablo. 1990. Insulin action and
insulin receptors in embryogenesis of vertebrates and invertebrates.
Pp. 198-204 in Progress in Comparative Endocrinology: Proceedings
of the Eleventh International Symposium on Comparative Endocri-
nology, A. Epple, C. G. Scanes, and M. H. Stetson, eds. Wiley-Liss.
New York.

Bonar, D. B., S. L. Coon, M. Walch, R. M. Weiner, and \V. Fitt.
1990. Control of oyster settlement and metamorphosis by endog-
enous and exogenous chemical cues. Bull. Mar Sci. 46: 484-498.

Bonar, D. B., and M. G. Hadfield. 1974. Metamorphosis of the marine
gastropod Phextilla sibogae Bergh ( Nudibranchia: Aeolidacea). I. Light
and electron microscopic analysis of larval and metamorphic stages.
J. Exp. Mar. Biol. Ecol. 16: 227-255.

Burke, R. D. 1983. Neural control of metamorphosis in Dendraster
exccninciis Biol Bull. 164: 176-188.

Cadenas, F. 1989. Biochemistry of oxygen toxicity. Ann. Rev Biochem.
58:79-110.

Cavanaugh, G. M. 1956. Formulae and Methods 17 of I he Marine
Biological Laboratory Chemical Room Marine Biological Laboratory.
Woods Hole. Massachusetts.

Cochard, J. C., L. Chevolot, J. C. Yvin, and A. M. Chevolot-Mageur.
1989. Induction de la metamorphose de la coquille Saint Jacques
Pecten maximus L. par des derives de la tyrosine extraits de 1'algue
Delesseria sanguinea Lamouroux ou synthetiques. Hahtotis 19: 259-
274.

Cohen, G. 1983. The pathobiology of Parkinson's disease: biochemical
aspects of dopamine neuron senescence. / Neural Transmission,
Suppl. 19:89-103.

Coon, S. L., and D. B. Bonar. 1987. Pharmacological evidence that
alpha- 1 adrenoceptors mediate metamorphosis ol the Pacific oyster,
Crassostrea gigas. Neuroscience 23: 1 169-1 174.

Cooper, K. 1982. A model to explain the induction of settlement and
metamorphosis of planktonic eyed-pediveligers of the blue mussel
Mytilus edulis L. by chemical and tactile cues. J. Shellfish Res. 2:
117.

Freeman, B. A., and J. D. Crapo. 1981. Hyperoxia increases oxygen
radical production in rat lungs and lung mitochondria. ./ Biol. Chem.
256: 10986-10992.

MORPHOGEN1C EFFECT OF H:O,

317

Fridovich, I. 1978. The biology of oxygen radicals. Science 201: 875-

880.
Graham, D. G. 1978. Oxidative pathways for catecholamines in the

genesis of neuromelanin and cytotoxic quinones. Mol Pharmacol

14: 633-643.
Graham, D. G., S. M. Tiffany, VV. R. J. Bell, and \V. F. Gutknechl.

1978. Autoxidation versus covalent binding of quinones as the

mechanism of toxicity of dopamine. 6-hydroxydopamine. and related

compounds toward C1300 neuroblastoma cells in vitro Mol f'/nu-

nnicol 14: 644-653.
Gutteridge, J. M. C., and B. Halliwell. 1990. The measurement and

mechanism of lipid peroxidation in biological systems. TIBS 15:

129-135.
Gutteridge, J. M. C., I. Zs.-Nagy, L. Maidt, and R. A. Floyd.

1990. ADP-iron as a Fenton reactant: radical reactions detected by

spin trapping, hydrogen abstraction, and aromatic hydroxylation.

Arch Biochem Biophys. Ill: 422-428.
Hadtield, M. G. 1978. Metamorphosis in marine molluscan larvae: an

analysis of stimulus and response. Pp. 165-175 in Settlement and

Metamorphosis ol Marine Invertebrate Larvae. F.-S. Chia and M.

Rice. eds. Elsevier/North-Holland, New York
Itadfield, M. G. 1984. Settlement requirements of molluscan larvae:

new data on chemical and genetic roles. Atinuciilture 39: 283-298.
Hadfield, M. G. 1986. Settlement and recruitment of marine inver-
tebrates: a perspective and some proposals. Bull Mar Sci 39: 4 1 8-

425.
Hadfield, M. G., and J. T. Pennington. 1990. Nature of the metamor-

phic signal and its internal transduction in larvae of the nudibranch

Phestilla sihogtie Bull Mar Set. 46: 455-464.
Hawley, M. D., S. V. Tatawawadi, S. Pickarski, and R. N. Adams.

1967. Electrochemical studies of the oxidation pathways of cate-
cholamines. J Am Chem Soc 89: 447-450.
Haydcn, B. J., L. Zhu, D. Sens, M. J. Tapert, and R. K. Crouch.

1990. Cytolysis of corneal epithelial cells by hydrogen peroxide.

E.\p. Eye Res. 50: 11-16.
Heacock, R. A. 1959. The chemistry of adrenochrome and related

compounds. Chem. Rev 59: 181-237.
Heffetz, D.. I. Bushkin, R. Dror, and V. Zick. 1990. The insulinomi-

metic agents HiCK and vanadate stimulate protein tyrosine phos-

phorylation in intact cells. J. Biol. Chem. 265: 2896-2902.
Mirata, K. Y., and M. G. Hadfield. 1986. The role of choline in meta-

morphic induction of Phestilla (Gastropoda: Nudibranchia). J Comp.

Biochem. Physiol. 84C: 15-21.
Imlay, J. A., and S. Linn. 1988. DNA damage and oxygen radical

toxicity. Seienee 240: 1302-1309.
Jensen, R. A. 1987. Factors affecting the settlement, metamorphosis

and distribution of larvae of the marine polychaete Phragmalopoithi

ciilifarnica (Fewkes). Ph.D. dissertation. University of California,
Santa Barbara, California. 175 pp.

Jensen, R. A., and D. E. Morse. 1990. Chemically induced metamor-
phosis of polychaete larvae in both the laboratory and ocean envi-
ronment. / Chem Ecol 16:911-430.

Kempf, S. C., and M. G. Hadfield. 1985. Planktotrophy by the lecitho-
trophic larvae of a nudibranch, Phe\lilla sibogae (Gastropoda). Biol
Bull. 169: 119-130.

Kingzetl, B. C., N. Bourne, and K. l.eask. 1990. Induction of meta-
morphosis of the Japanese scallop Potinopecten yessoensis Jay. J.
Shellfish Res. 9: 119-124.

Kontos, H. A. 1989. Oxygen radicals in CNS damage. Chem. -Biol
Interactions 72: 229-255.

Levantine, P. L., and D. B. Bonar. 1986. Metamorphosis ofllyanassa
obso/ela: natural and artihcial inducers. Am. Zoo/. 26: 14A.

Miller, S. E., and M. G. Hadfield. 1986. Ontogeny of phototaxis and
metamorphic competence in larvae of the nudibranch Phestilla si-
bogae Bergh (Gastropoda: Opisthobranchia). J Exp. Mar. Biol. Ecol.
97:95-112.

Misra, H. P., and I. Fridovich. 1972. The role of superoxide anion in
the autoxidation of epinephrine and a simple assay for superoxide
dismutase. J. Biol Chem 247: 3170-3175.

Morse, D. E., H. Duncan, N. Hooker, and A. Morse. 1977. Hydrogen
peroxide induces spawning in mollusks, with activation of prosta-
glandin endoperoxide synthetase. Science 196: 298-300.

Pennington, J. T., and M. G. Hadfield. 1989. Larvae of a nudibranch
mollusc ( Pheslilla sihogac) metamorphose when exposed to common
organic solvents. Biol. Bull. 177: 350-355.

Polansky, J. R., D. J. Fauss, T. Hydorn, and E. Bloom. 1990. Cellular
injury from sustained r.v. acute hydrogen peroxide exposure in cul-
tured human corneal endothelium and human lens epithelium.
C LAO J. 16(1 Suppl.): S23-S28.

Radzik, D. M., D. A. Roston, and P. I . Kissinger. 1983. Determination
of hydro.xylated aromatic compounds produced via superoxide-de-
pendent formation of hydroxyl radicals by liquid chromatography/
electrochemistry. Anal. Biochem. 131: 458-464.

Ruch, W., P. H. Cooper, and M. Baggiolini. 1983. Assay of H:O2 pro-
duction by macrophages and neutrophils with homovanillic acid and
horse-radish peroxidase. / Immunol Methods 63: 347-357.

Smit, A. B., E. Vrenghdenhil. R. H. M. Ebberink, W. P. M. Geraerts,
J. RIootHijk, and J. Joose. 1988. Growth-controlling molluscan
neurons produce the precursor of an insulin-related peptide. Nature
331: 535-538.

Stoker, M., and E. Gherardi. 1989. Scatter factor and other regulators
of cell mobility. Br Med. Bull. 45: 481-491.

Yool, A. J., S. M. Grau, M. G. Hadfield, R. A. Jensen, D. A. Markell,
and D. E. Morse. 1986. Excess potassium induces larval meta-
morphosis in four marine invertebrate species. Biol. Bull. 170: 255-
266.

Reference: Biol. Bid! 180: 318-327. (April, 1991)

cDNA Sequences Reveal mRNAs for Two Ga Signal
Transducing Proteins from Larval Cilia

LISA M. WODICKA AND DANIEL E. MORSE

Department of Biological Sciences and the Marine Biotechnology Center,
University of California, Santa Barbara, California 93106

Abstract. In planktonic larvae of the gastropod mollusk,
H allot is nifescens (red abalone), settlement behavior and
subsequent metamorphosis are controlled by two con-
vergent chemosensory pathways that report unique pep-
tide and amino acid signals from the environment. The
integration of signals from these two sensory pathways
provides for variable amplification, or fine-tuning, of larval
responsiveness to the inducers of settlement and meta-
morphosis. These pathways may be analogous to the neu-
ronal and molecular mechanisms of facilitation and long-
term potentiation characterized in other (adult) molluscan
systems. Recently, the chemosensory receptors and signal
transducers apparently belonging to the regulatory path-
way (including a G protein and protein kinase C) have
been identified in cilia purified from H. nifescens larvae.
These elements retain their sequential receptor-dependent
regulation in the isolated cilia in vitro. As a first step toward
the molecular genetic dissection of the receptors, trans-
ducers, and the mechanisms of their control of settlement
behavior and metamorphosis, we present evidence that
the cilia purified from these larvae contain polyadenylated
mRNA corresponding to unique signal transducers. Pu-
rification of this mRNA, enzymatic synthesis of the cor-
responding cDNAs, amplification by the polymerase chain
reaction, cloning, and sequence analysis reveal that the
ciliary mRNA includes sequences that apparently code
for two Ga signal transducing proteins. One of these is
highly homologous to members of the Gq family, recently

Received 9 July 1990; accepted 28 January 1991.

1 Abbreviations: GITC. guanidinium isothiocyanate: SDS. sodium
dodecyl sulfate; IPTG. isopropyl-fMhiogalactoside; X-gal, bromo-, chloro-
indolylgalactoside: AMV, avian myeloblastosis virus; Tris, tris-hydroxy-
methylaminomethane; EDTA. ethylenediamine-tetra-acetic acid; TE,
Tris-EDTA; TBE, Tris-borate-EDTA; TAE. Tris-acetate-EDTA; bp, base-
pair(s). The standard one-letter amino acid code is used.

shown in other systems to control the activity of phos-
pholipase C; the other is more closely related to G, and
G0. These results extend the tractability of the Haliotis
system to analyses of cDNA and protein sequences of
chemosensory elements from isolated cilia. This is the
first time that mRNA has been purified from isolated cilia,
and the corresponding cDNA synthesized and character-
ized.

Introduction

Larvae of the gastropod mollusk, Haliotis nifescens (red
abalone), undergo a dramatic behavioral change when
they encounter a specific chemical cue at the surfaces of
crustose red algae: the planktonic larvae cease swimming,
attach to the algal surface, and commence metamorphosis
and plantigrade locomotion and feeding. This behavioral
transition is controlled by the integration of two conver-
gent chemosensory pathways that respond to chemical
signals from the environment: a morphogenetic pathway
activated by a GABA-mimetic morphogen encountered
by the larvae on surfaces of recruiting algae, and a regu-
latory or amplifier pathway stimulated by lysine in sea-
water (Morse et al., 1984; Trapido-Rosenthal and Morse,
1985, 1986a, b; Baxter and Morse, 1987; Morse 1990a).
Activation of the morphogenetic pathway receptors is
thought to trigger an efflux of chloride or other anions
across the membrane of the primary chemosensory cell,
apparently resulting in excitatory depolarization (Baloun
and Morse. 1984). This transduction of the exogenous
chemical signal to one that can be propagated by the larval
nervous system is evidently sufficient to induce the change
in larval behavior culminating in settlement, attachment,
and the start of metamorphosis. Activation of the amplifier
pathway receptors increases the sensitivity or output of
the morphogenetic pathway by as much as 100-fold. The

318

Ga CDNAS FROM LARVAL CILIA

319

receptors of the amplifier pathway are activated when they
bind lysine, lysine polymers, or certain lysine analogs
(Trapido-Rosenthal and Morse, 1985. 1986b). Experi-
ments //; vivo demonstrated that the amplifier pathway is
controlled by chemosensory receptors and signal trans-
ducers distinct from those of the morphogenetic pathway,
and that the lysine receptors of the amplifier pathway ac-
tivate a sequential G protein-(phospholipase C) diacyl-
glycerol-protein kinase C signal transduction cascade
(Baxter and Morse, 1987). This system of dual control,
in which the integration of two different kinds of che-
mosensory signals from the environment modulates the
settlement behavior of the Haliotis larvae, fine-tunes larval
responsiveness to exogenous settlement cues. The result
of this integration may enhance the site-specificity of larval
settlement and metamorphosis in potentially favorable
habitats (Trapido-Rosenthal and Morse, 1985, 1986b:
Morse, 1990a, b).

Because both the morphogenetic and amplifier path-
ways can be activated by macromolecular (protein-asso-
ciated or polypeptide) ligands that are presumably im-
permeant (Morse ct a/.. 1984; Trapido-Rosenthal and
Morse. 1985. 1986b), it was suspected that the chemo-
sensory receptors controlling these two pathways might
be located on externally accessible epithelia (Morse, 1985,
1990a, b). Epithelial cilia are known to carry chemosen-
sory receptors in a wide variety of systems, including the
well-characterized olfactory epithelia of frogs, fish, and
mammals (e.g., Rhein and Cagan, 1980; Chen and Lancet
1984; Pace el at., 1985; Pace and Lancet, 1986; Lancet
and Pace, 1987; Anholt, 1987; Anholt el ai. 1987). Epi-
thelial cilia also have long been suspected to carry the
chemosensory structures that mediate substratum rec-
ognition and thereby control settlement behavior and
metamorphosis in various molluscan larvae (Raven, 1958:
Fretter and Graham, 1962; Bonar, 1978a, b; Chia and
Ross. 1984; Yool, 1985).

Recently, epithelial cilia isolated from H. rufescens lar-
vae were shown to contain the lysine receptors and signal
transducers that may control the amplifier pathway in
vivo (Baxter and Morse, in prep.). These elements retain
their functional coupling in the isolated cilia in vitro: i.e.,
the specific and saturable binding of lysine to sodium-
independent lysine receptors activates sequentially a G
protein and diacylglycerol-stimulated protein kinase C
(Baxter, 1991; Baxter and Morse, in prep.). The lysine-
binding receptor was found to be reciprocally regulated
by its tightly coupled G protein in the cilia in vitro (Baxter
and Morse, in prep.); similar behavior is exhibited by other
members of the rhodopsin and /3-adrenergic G protein-
coupled transmembrane receptor superfamily.

The tools of molecular genetics are required to further
resolve the mechanisms by which the chemosensory and
neuronal receptors, transducers, and pathways are inte-

grated to control behavior in these small larvae (Morse,
1990a). As a first step toward that objective, we report
here the amplification, cloning, and partial sequence
analysis of cDNAs apparently corresponding to two Ga
signal transducing proteins, from mRNA purified from
the isolated cilia.

Materials and Methods

Cilia isolation

Larvae of Haliotis rufescens were produced in the lab-
oratory by hydrogen peroxide-induced spawning of gravid
adults (Morse et ai. 1977). Larvae were maintained at
15°C in 5 /urn-filtered, UV-sterilized running seawater
until 7 days post-fertilization; at this time they become
developmental^ competent to metamorphose in response
to inducer (Morse et ai. 1979, 1980). Cilia were purified
by differential centrifugation, after abscision induced by
exposure of the larvae to a mild calcium-ethanol shock
(Baxter and Morse, in prep.). This method is a modifi-
cation of that used for the purification of functional re-
ceptor-bearing cilia from olfactory epithelia (Rhein and
Cagan, 1980; Chen and Lancet, 1984) and other sources
(Watson and Hopkins, 1962; Linck, 1973). Electron mi-
crograph examination reveals the purified cilia to be intact
and completely free of cell bodies and debris; the cilia are
heterogeneous, and include short (ca. 0.5 ^m) spatulate
cilia similar to the sensory cilia found in other invertebrate
systems, and long (> 10 ^m) propulsive cilia from the lar-
val velum (Baxter and Morse, in prep.).

RNA isolation

We isolated total RNA from cilia freshly purified from
Haliotis rufescens larvae, using a single-step extraction
with an acid guanidinium isothiocyanate (GITC1 (-phenol-
chloroform mixture (Chomczynski and Sacchi, 1987).
Poly A+ mRNA was purified using oligo (dT) cellulose
columns either centrifuged (Clontech, Palo Alto, Califor-
nia) or used with a syringe (Stratagene, La Jolla, Califor-
nia) in a modification of the technique described by Aviv
and Leder( 1972).

RNA was purified from bovine retina to provide a pos-
itive control enriched for G protein (transducin) mRNA.
For this purpose, fresh bovine eyes were obtained from
Federal Meat Market (Vernon, California). Retinas were
immediately dissected on ice in Tris-buffered saline
(Maniatis et ai, 1982) and placed on dry ice for transport
to a nearby laboratory. There, half the samples were frozen
in liquid nitrogen, and half were homogenized in GITC;
these samples then were transported to Santa Barbara in
GITC or on dry ice. RNA was isolated by the GITC-
cesium chloride centrifugation method (Chirgwin et ai,
1979), and mRNA was then purified as described above.

320

L M. WODICRA AND D E. MORSE

Total RNA from both sources was analyzed by elec-
trophoresis on formaldehyde gels (Maniatis el at. 1982)
with ethidium bromide added to the sample buffer, or
was denatured at 65 °C for 1 5 min. quickly chilled on ice.
electrophoresed on 1% agarose/TBE gels (Han el at. 1987)
and visualized by U V-excited fluorescence after ethidium
bromide staining (Maniatis el at. 1982). Purity of total
and poly A+ RNA was confirmed by the ratio of absor-
bances at 260 and 280 nm and concentrations estimated
from the A2W> •

Synthesis ofcDNA and oligonucleotide primers

Reverse transcriptase from avian myeloblastosis virus
( AMV) (Invitrogen. San Diego, CA) was used to synthesize
first strand cDNA. Cilia mRNA (100-500 ng) or bovine
mRNA ( 1 Mg) was used for each 50 jul reaction.

For polymerase chain reaction (PCR) amplifications,
two kinds of oligonucleotide primers were made: degen-
erate primers (D) were used for the first amplifications of
the cDNA, and (once the exact sequence of the Ga cDNA
was determined) specific primers (S) were used to amplify
the genomic sequences from sperm DNA. Oligonucleo-
tides used as primers for PCR were synthesized (as the
trityl-derivatives) by an automated oligonucleotide syn-
thesizer (Applied Biosystems Inc., Foster City, California).
To reduce the degeneracy of the primers, Haliolis rufes-
cens condon usage frequencies (Groppe and Morse, 1989)
were taken into consideration, and two separate pools of
the downstream primer were synthesized (D2 and D, ).
Degenerate oligonucleotide primer sequences corre-
sponding to the conserved G and G' domains (Lochrie
and Simon, 1988) of G protein a subunits are: D,: 5'
GAAGGATCCAAGTGGATCCA(GC)TG(CT)TTT 3':
D,: 5' CTCAAGCTTTCCT(TG)CTT(AG)TT(TG)AG-
(AG)AA 3': D3: 5' CTCAAGCTTTCTT(TG)CTT(AG)-
TT(CA)AG(AG)AA 3'. D, corresponds to the conserved
G' domain amino acid sequence, KWI(HQ)CF; D2 and
D, correspond to the conserved G domain sequence
FLNK(KQ)D. Amino acid sequences chosen for these
domains were based on the findings of Strathmann el at
(1989), with inclusion of a degeneracy representing ad-
ditional sequences determined for yeast. In addition, these
primers include oligonucleotide sequences (indicated by
underlining) corresponding to the BamH I (D, ) and Hind
III (D2 and D, ) restriction enzyme targets; these sequences
were added as linkers to facilitate cloning of the amplified
products. A shorter variant of D, also was produced with-
out the 9-nucleotide linker. Specific (non-degenerate)
primer sequences based on the cDNA sequence subse-
quently determined for the Haliolis cilia G«l (see below)
are: S, : 5' GCAGGATCCACGTCCATCATGTTCTTA
3'; and S2: 5' CTCAAGCTTCGGGTAGGTGA-
TAATCGT 3'. These primers also have 9-nucleotide long
5'-linkers with a BamH 1 site (S, ) or a Hind III site (S2).

After synthesis, oligonucleotides were deprotected at
55°C in ammonia overnight; they were then purified and
detritylated by reverse-phase chromatography (Oligonu-
cleotide Purification Cartridges from Applied Biosystems).
The oligonucleotides were dried down, resuspended in
sterile water, and concentration was estimated by absor-
bance at 260 nm.

PCR amplification

For amplification of cDNA, 50 pmol of each primer
and 40% (20 ^D of the reverse transcription reaction were
added directly to 100 n\ PCR reactions. All other reaction
components, including Taq DNA polymerase, were pur-
chased from Perkin Elmer-Cetus Corp. (Norwalk, Con-
necticut) and used as suggested by that manufacturer. The
number of amplification cycles was varied from 25 to 45
with the following parameters: denaturation at 94°C, 1
min; annealing at 37°C, 1 min; extension at 72°C. 3 min.
For amplification of genomic DNA using specific primers,
0.3 Mg Haliolis rufescens sperm DNA. 0.75 ng Telrahy-
mena ihermophila DNA, 0.5 Mg Vibrio harveyi DNA, or
1 Mg salmon sperm DNA were added to otherwise identical
amplification reactions. (The Haliolis. Telraliymena. and
I 'ibrio genomic DNA samples were generously provided
by Jay Groppe. Jennifer Ortiz, and Richard Showalter,
respectively.) All DNA samples were tested for amplifi-
cation at amounts equal to or greater than the number
of genome equivalents of the Haliolis DNA. Because DNA
samples were in TE buffer, additions were adjusted such
that the total amount of TE (and thus the concentration
of EDTA) in all PCR reactions was equal. Optimum
magnesium concentration, primer concentration, and cy-
cling parameters were determined empirically. For am-
plification of genomic DNA, 25 pmol of each specific
primer was used; the final concentration of magnesium
was increased from the standard 1.5 mA/ to 2 mAI: and
a total of 30 amplification cycles was performed as before,
except that the annealing temperature was raised to 45°C
for the 2nd cycle and to 55°C for the 3rd-30th cycles. A
5 min extension step (72°C) was added after the last cycle.

Gel elect rophoresis for analysis and purification of PCR
reaction products

PCR reaction products were analyzed by electrophoresis
on agarose gels (3% Nuseive agarose plus 1% Seaplaque
agarose in TBE for cDNA-PCR reactions and 1.5% aga-
rose/TAE buffer for genomic reactions), run at 2-6 v/cm.
and stained with ethidium bromide. For analysis on the
higher percentage gels, samples were loaded into wells
cast from 0.7% agarose. One ^g of restriction enzyme-
digested plasmid (PBR322-BstN 1. from New England
Biolabs Inc., Beverly, Massachusetts) was included for
molecular weight markers.

G« cDNAs FROM LARVAL CILIA

321

For purification, amplified cDNA was electrophoresed
on 3% low melting temperature agarose (Mermaid, from
Bio 101 Corp., La Jolla), and DNA bands were excised
while visualized on a 365 nm light box. DNA then was
removed from the agarose by binding to glass beads (Glass
Fog, from Bio 101 Corp.). Genomic products were run
on 1.5% agarose gels as described above; bands were ex-
cised, and DNA was purified by binding to glass beads
(Geneclean, from Bio 101 Corp.).

DNA cloning

Purified PCR products and a recombinant plasmid
vector (/7Bluescript K.SII+, from Stratagene Corp.) were
digested at 37°C for 1 h in 20 n\ volumes with Hindlll
followed by BamHI (enzymes from New England Bio-
labs). Digested products were purified by gel electropho-
resis as described above. The purified, linearized vector
( 100 ng) then was ligated to an approximately equimolar
amount of PCR product insert; this reaction was catalyzed
by T4 DNA ligase overnight at 4°C. Controls with no
added insert were treated identically. Transformation of
recipient bacteria (Epicurian Coli XL-1 Blue, from Stra-
tagene Corp.) was performed by the method of Hanahan
(1983), with modifications recommended by Stratagene
Corp. After transformation, colonies with recombinant
plasmids were identified on the antibiotic-containing agar
medium with the chromogenetic substrate, X-gal. Each
clone was then subcultured in 5 ml of LB containing am-
picillin and tetracycline (37°C, overnight). Plasmid DNA
was purified from these cultures after lysis with alkalai,
using the miniprep procedure (Maniatis el al., 1982).

Cloned plasmid DNA was digested as above with BamH

I and Hind III simultaneously, and separately with BssH

II (for which the plasmid has two sites, flanking the BamH
I and Hind III sites). Restriction digests were analyzed by
agarose gel electrophoresis. Plasmid DNA was purified by
centrifugation chromatography (Sephacryl S-400 Mini-
prep Spun Columns, from Pharmacia, Piscataway, New
Jersey).

DNA sequence analysis

Di-deoxy sequencing reactions were performed with
modified T7 DNA polymerase (Sequenase II; United
States Biochemical, Cleveland, Ohio) and 5' [«-35S]dATP,
1 100 Ci/mmol (Amersham) by procedures modified from
Sanger el al. (1977). Primers used for sequencing were
plasmid primers T7, T3, KS, and SK (purchased from
Stratagene) and the specific primers S, and S:, described
above. Reactions were analyzed by electrophoresis on 8%
polyacrylamide-50% urea wedge sequencing gels. Gels
were washed in 10% acetic acid- 10% methanol, dried with
vacuum at 80°C, and subjected to autoradiography. The
autoradiograms were read with a sonic digitizer, and the

resulting sequences analyzed with the aid of the Pustell
Sequence Analysis software (IBI Macintosh).

Results

PCR amplification of cilia Ga cDNA

Poly A+ mRNA was isolated from the purified cilia of
7-9-day-old, competent larvae of Haliotis rufescens. as
described in the Methods. Starting with about 10" larvae,
typical yields were 300-400 mg (wet weight) cilia, 50 ng
total RNA, and 1 ^g of poly A+ mRNA ( = 2% of total
RNA). AMV reverse transcriptase was used to catalyze
random-hexamer primed synthesis of first strand cDNA
from the purified mRNA, and the resulting mRNA-cDNA
duplex was then used as template for PCR amplification
with the degenerate primers corresponding to the con-
served G and G' domains, as described in the Methods.

The primers used clearly directed the amplification of
a 196 bp product from the cDNA templates prepared from
the larval cilia (Fig. la). The size of this product is within
the range predicted for a G« cDNA domain lying between
the highly conserved G and G' domains. This result sug-
gests that the cilia purified from Haliotis rufescens larvae
may contain mRNA coding for a G« protein.

The positive result shown in Figure la allowed us to
further optimize the primers used for PCR-amplification.
The upstream primer used in the first PCR amplifications
was relatively short, consisting of a degenerate pool of 17-
mers. This primer only weakly amplified the positive con-
trol template, cDNA from bovine retina, a tissue highly
enriched for the G« known as transducin (results not
shown). The addition of nine nucleotides containing the
sequence of the BamHI restriction endonuclease site to
the 5'-end of the 1 7-mers. to generate the D, primers (see
Materials and Methods), makes efficient amplification of
the control Ga sequence from bovine retina cDNA pos-
sible (Fig. Ib). [Similarly, we had found earlier that ad-
dition of the nine nucleotides containing the sequence of
the Hind III restriction site to the 5'-end of short down-
stream primers, to generate the 26-mer D2 and D, pools,
also significantly enhanced the efficiency of these oligo-
nucleotides as primers. These non-matching nucleotides
do not reduce primer specificity. Similar observations have
been reported by others (e.g.. Mack and Sninsky, 1988).]
The results in Figure Ib show the downstream primers
in the degenerate pool D; are more effective than those
in D3 for detecting and amplifying Go cDNA sequences
from both bovine retina and Haliotis rufescens larval cilia.
Primer D, differs from D: by two nucleotides and appar-
ently fails to hybridize efficiently to the target G protein
cDNA sequence. Agarose gel electrophoresis of PCR
products amplified with the optimized 26-mer primers
(D, and D2) reveals the expected 205 bp product from

322

L. M. WODICKA AND D. E. MORSE

cDNAs from both the larval cilia and bovine retina. The
product is nine nucleotides longer than that seen in Figure
la, as expected because the upstream primer (D, ) is nine
nucleotides longer than that used in the first experiment.
The cilia cDNA required more cycles of amplification
(35) than did the bovine retina cDNA (25) before the
product on an ethidium bromide stained gel could be
visualized. Thus, there may be greater primer-template
mismatch, or the target mRNA may be less abundant, in
the larval cilia.

A control PCR reaction with no added DNA template,
a test for DNA contamination of reagents, yielded no de-
tectable PCR products amplified after 45 cycles (Fig. Ib).
In addition, no amplified PCR products were observed
in the following control reactions (not shown): (a) no
primers in the PCR reaction; (b) no Taq polymerase in
the PCR reaction; and (c) no reverse transcriptase in the
cDNA reaction.

Cloning and sequence analysis of Ga cDNA

The 205 bp PCR product from the cilia cDNA was
cloned in the plasmid vector as described in Materials
and Methods. Twelve transformant colonies were picked
and subcultured; gel electrophoresis of the restriction en-
zyme-digested plasmid DNA showed that 1 1 of these
clones contained inserts of the correct size.

Sequence analysis of three of the cloned cilia PCR
products revealed two unique cDNA sequences (Fig. 2).
As shown, both of these share a number of the highly
conserved residues of other G« proteins in the G-G' re-
gion. Two out of the three clones proved to have identical

Cilia

DNA

(S) A. rRet.

-Cilia-

nNone

196 bp —

2nd Primer - (X) D2 D3 D2 D2 D2 D3 D2 D2
Cycles - 30 30 30 30 35 40 40 45 45

121 —

9 10

Figure l(b). PCR amplification of larval cilia cDNA using degenerate
primers D, and either D, or D,. Lanes: ( 1 ) molecular weight standards;
(2) \ DNA and primers as PCR control; (3) bovine retina cDNA. primers
D| and D2. 30 cycles; (4) bovine retina cDNA, primers D, and D3, 30
cycles; (5) cilia cDNA, primers D, and D,, 30 cycles: (6) cilia cDNA,
primers D, and D,, 35 cycles; (7) cilia cDNA, primers D, and D2, 40
cycles; (8) cilia cDNA, primers D, and D3, 40 cycles (9) cilia cDNA.
primers D, and D2, 45 cycles; (10) no DNA template, primers D, and
D:, 45 cycles.

nucleotide (and deduced protein) sequences over the 51
amino acid region between the primers. This Haliotis G
protein « subunit (G«l ) differs in the region analyzed by
only one amino acid from the sequence of mouse brain
G« 1 1 , a member of the newly discovered Gq class of a
subunits (Strathmann el ai, 1989; Strathmann and Si-
mon, 1990). This sequence differs significantly in the re-
gion analyzed from all other known classes of G protein
a subunits (Gs, G,, G0n, G0, G,, GJ from mammals.
Drosophila, and yeast. The second G protein sequence
from the larval cilia (Haliotis G«2) is most homologous
to G0 and GJ from Drosophila,

Figure l(a). G protein a subunit cDNA from cilia, amplified by
PCR. Product from 50 cycles of amplification; primers were a 17-mer
variant of D, (without the BamHI site) and D2. Number of base-pairs
in product is indicated. Details in Materials and Methods.

Amplification, cloning, and sequence analysis oj Got
from Haliotis genomic DNA

The nucleotide sequence from the Haliotis ntfescens
larval cilia Gal clone was used to design specific (i.e..
non-degenerate) primers to amplify the corresponding re-
gion of G« from H. rufescens sperm genomic DNA. The
specific primers (S} and S:) were based on regions of the
Haliotis sequence that differed significantly from other
G« protein sequences (Fig. 5; cf. Fig. 2).

Ga cDNAs FROM LARVAL CILIA

323

Inlron

HaliotisGal
Haliotis Gtt2
Mouse Gq
Drosoph. Gj
Drosoph. G0
Rat G0
RatGx
Bov. Transd.
Yeast GP1
Yeast GP 2
Drosoph. Gs
RatGolf

Ident/51
Gal Ga2

ENVTSIMFLVALSEYDQVLVESDSENRMEESKALFRTII TYPWFQNSSVIL

EG VTA I 1 FI VAMSEYDLTLAEDQEMNRMMESMKLFDS ICNMCWFTDTSI 1L 26

ENVTS1MFLVALSEYDQVLVESDNENRMEESKALFRTI I TYPWFQNSSVIL SO 26

EGVTAI 1 FCVALSGYDLVLAEDEEMNRMIESLKLFDS ICNSKWFVETSI IL 27 41

EDVTAI 1 FCVAMSEYDQVLHEDETTNRMQESLKLFDS ICNNKWFTDTSi 1L 28 41

EDVTAI I FCVALSGYDQVLHE0ETTNRMHESLMLFDS rCNNKFF I DTSI I L 27 36

EGVTAI IFCVELSGYDLKLYE0NQT SRMAESLRLFDS ICNNNWFI NTSLIL 25 33

EGVTC1 I FI AALSAYDMVLVE0DEVNRMHESLHLFNS ICNHRYFATTSIVL 26 32

EGITAVLFVLAMSEYDQMLFEDERVNRMHESIMLFDTLLNSKWFKDTPFIL 23 30

DNVTLV I FCVS LSEYDQTLMEDKNQNR FQESLVLFDN I VNS RWFARTSVVL 25 27

NDVTAI I FVTACSSYNMVLREDPTQNRLRESLDLFKS IWNN RWLR T I S I IL 21 27

NDVTAI IYVAACSSYNMVI REDNN T NR L RESLDLF E S IWNNRWLR T I S I I L 18 25

:•:•:.:•:•:•:•:•:•:•:-:•: :•:•:•:•:•:•:•:•.•:•.•.•.•.-.•.-.

Figure 2. Ga sequences from cilia. Deduced amino acid sequences of the cloned PCR products (Gal
and G«2) from Haliolis rufescens cilia cDNA are compared with the corresponding regions of other Ga
subunits. Residues highly homologous in several Ga proteins are indicated by shading. Region shown is
between (not including) degenerate primers D, and D;. The number of amino acids identical to Haliotis
Gal and Ga2 (of 5 1 total) is shown for each G«. Sequences used to design specific primers S, and S2 are
shown by arrows: position of the intron in genomic DNA is indicated. Standard (IUPAC) one-letter amino
acid code is used.

Two distinct DNA products were seen when Haliotis
genomic DNA was amplified by PCR reactions with the
S, and S: primers, and the products were analyzed on an
agarose gel (Fig. 3). The specificity of these primers for
Haliotis DNA is evident by their failure to direct ampli-
fication of genomic DNA sequences from Tetrahymena,
Vibrio, or salmon sperm in otherwise identical PCR re-
actions.

The 1.25 kb and 1.45 kb Haliolis genomic PCR prod-
ucts were electrophoretically purified, separately amplified
again, and analyzed electrophoretically (Fig. 4). The suc-
cessful purification of the two genomic PCR products is
shown by the lack of visible contamination after this sec-
ond round of amplification and gel electrophoresis. Each
purified PCR product was cloned as described above, and
the cloned inserts were then sequenced using primers
matched to the vector.

During the cloning step we discovered that the larger
of the two genomic PCR products had an internal Hind
III site not found in the smaller product. This difference
apparently resides in an intron, a non-coding sequence
not present in the cDNA (see below). Although the re-
sulting Hind Ill-Hind III fragment was not cloned, a par-
tial sequence for this region was obtained from the direct
sequencing of the uncloned PCR products using S, and

S: as primers for the sequencing reaction. While this
method of direct sequencing proved useful in this case,
the quality of the sequencing reactions was highly variable
(t/McCabe, 1989).

The genomic PCR product sequences were identical to
the G«l cDNA sequence from the cilia, and to one an-
other, in the putative coding regions. But both of the
cloned genomic sequences contain an intron that inter-
rupts the coding sequence. The exact position of this in-
tron between exons 6 and 7 is conserved in the genomic
sequences corresponding to the Haliotis Gal and the G0
and G, of mammals and Drosophila (Figs. 2, 5). The two
Haliotis genomic sequences prove to be highly homolo-
gous to one another at both ends of the introns that are
adjacent to the coding regions (corresponding to exons 6
and 7 in other species; cf. Fig. 5); however, the two introns
differ in length by about 200 bp.

Discussion

The larvae of Haliotis rufescens provide a uniquely
tractable model system for resolving and analyzing the
chemosensory receptors and signal transducers, and the
mechanisms of their functional integration, controlling
behavior and development in response to chemical signals

324

L. M. WODICKA AND D. E. MORSE

1857 —

1450 —

1250 —

1060 —

929 —

Figure 3. PCR amplification of genomic DNA using Haliotis-sped&c
pnmers S, and S: (30 cycles). Lanes: 1 1 ) molecular weight standards; (2)
and (3) 0.3 Mg Haliolis sperm DNA; (4) 0.75 ^g Tetrahymena DNA; (5)
0.5 Mg Vibrio DNA; (6) 1 Mg salmon sperm DNA; (7) no DNA. Other
details in Materials and Methods.

from the environment (Morse, 1990a, b). Purification of
cilia in milligram quantities from the cultured larvae has
allowed us to analyze the chemosensory receptors and
signal transducers /// vitro (Baxter, 1991; Baxter and
Morse, in prep.), and (as shown here) to isolate mRNAs
encoding some of these elements and conduct analyses at
the cDNA sequence level. We have shown here that cilia
purified from //. rufescens larvae contain polyadenylated
mRNA, and that this mRNA includes sequences corre-
sponding to two Ga signal transducing proteins.

The central role of the G proteins as chemosensory
signal transducers was confirmed by /// vitro studies of
olfactory cilia isolated from frog (Pace et ai, 1985). Jones
and Reed (1989) recently have identified a unique G0|f
from the ciliated sensory epithelium of the rat olfactory
mucosa; the sequence of this G protein a subunit was
determined by analysis of its cDNA. G protein also acts
as the primary chemosensory signal transducer in taste
receptor cells in the frog; this G protein controls the ac-
tivation of adenyl cylase, with the resulting sequential ac-
tivation of a protein kinase and membrane depolarization
(Avenet et ai, 1988). G protein transduction of chemo-
sensory stimulation in catfish controls an inositol tri-
phosphate (protein kinase) cascade (Huque et ai. 1987).

Although distal localization of mRNA has been ob-
served in other systems (Merlie and Sanes, 1985; Garner
et ai. 1988; Kosik et ai. 1989), the results reported here
are the first of which we are aware in which mRNA has
been purified, and the corresponding cDNA synthesized,
amplified, cloned, and sequenced from purified cilia. Be-

1857 —

1450 —

1250 —

1060 —

929 —

Figure 4. Haliolia genomic PCR products after gel purification and
30 additional cycles of amplification with specific primers S, and Si.
Lanes: (I) molecular weight standards; (2) ca. 1.45 kb genomic PCR
product; (3) ca. 1.25 kb genomic PCR product; (4) molecular weight
standards.

cause the mRNA from which the G protein sequences
were identified was extracted from cilia that had been
purified and washed by four sequential cycles of differ-
ential centrifugation, it was probably not contaminated
by free RNA released from the larvae. Electron micros-
copy shows no other cell fragments or cells contaminating
the purified cilia (Baxter, 1991; Baxter and Morse, in
prep.). //; situ hybridization will be required, however, to
verify the intraciliary localization of the Get mRNA. Three
independent lines of evidence confirm that the source of

1 2 3

4567 8

G R

9 10 11

1 1 1

III 1

1 2 3

456

7 8

G,a/Goa I III III I

HaliotisGa
PCR Product

S2 D2

Figure 5. Genomic organization of mammalian G protein « subunits
(after Kaziro ef a/., 1 990). Exons (coding regions) are numbered; vertical
lines indicate introns. Conserved domains P (GTP hydrolysis), G' and
G (GTP binding) and R are shown above. [In the convention of Halliday
( 1 984). domain G also is designated G, but the other domains are des-
ignated differently.]. The region of the Haliotis G protein corresponding
to mammalian G0 and G, exons 6 and 7 is expanded to show relative
positions and orientations of PCR primers. D,, D^.and D, are degenerate
primers used for cDNA amplification; S, and S: are the Haliotis-specific
pnmers used lor genomic amplification.

G« cDNAs FROM LARVAL CILIA

325

the G protein sequences characterized could not have
come from bacterial contamination: (1) the mRNA se-
lected was poly A+; (2) the sequence of Gal determined
corresponds exactly to a sequence in the genomic DNA
(from sperm) of Haliotis rufescens: and (3) that genomic
sequence contains an intron (at the expected position)
that would be lacking from bacterial DNA. That these
sequences were from Haliotis mRNA, and not from pos-
sible contaminants, was further confirmed by the failure
of the unique ciliary sequences to direct amplification of
any homologous sequences in control samples of bacterial,
protozoan, or fish DNA, under conditions in which the
perfectly homologous sequences were detected and am-
plified from Haliotis genomic (sperm) DNA.

The cDNA sequences that we obtained from mRNA
purified from the cilia isolated from Haliotis rufescens
larvae reveal two apparent G protein « subunit sequences.
Although definitive characterization must await comple-
tion of the entire translated sequences, our identification
of these sequences as members of the G protein family is
strengthened by the finding that Gal is virtually identical
(50/51 residues) to Gqa in the region sequenced. and the
observation that the most highly conserved amino acids
in this region of the other known Ga proteins also are
conserved in the Gal and Ga2 sequences from the Hal-
iotis larvae (Fig. 2). The genomic sequences that we thus
far have characterized correspond exactly to their cDNAs,
and contain the intron at the same position between the
G and G' domains as those found in mammalian G pro-
tein genes.

The two Ga sequences obtained from the cilia of Hal-
iotis larvae are clearly related to the Go sequences from
other species (Fig. 2) (and unrelated to tubulin, for ex-
ample). Yet the two sequences differ significantly from
one another. The larval cilia Gal sequence is highly ho-
mologous to that of the corresponding domain of the alpha
subunit of the mammalian Gq. This is of particular in-
terest, in light of the finding that Gq is a pertussis toxin-
insensitive regulator of phospholipase C (Strathmann and
Simon, 1990; Smrcka et al, 1991), and our observation
that the lysine-dependent regulatory pathway in Haliotis
larvae is mediated by a pertussis toxin-insensitive G pro-
tein-phospholipase C-protein kinase cascade (Baxter and
Morse, 1987; Baxter, 1991; Baxter and Morse, in prep.).
Gal is markedly different from G,, Gs, G0, Gx, and G0ir
characterized from other systems, whereas the larval cilia
Go2 sequence is significantly more closely related to G,
and G0 (from Drosophila and rat) than it is to the Gs and
G0|f from these species. We are now in the process of iden-
tifying the Ga sequence corresponding to the transducing
protein specifically activated by the lysine receptors in the
isolated cilia. These experiments are facilitated by the ob-
servation that lysine binding to the ciliary receptors ac-
tivates the associated Ga protein, increasing its radioactive

labeling with ADP-ribose catalyzed by cholera toxin
(Baxter and Morse, in prep.).

In molluscan larvae, the cilia of the cephalic apical tuft
and of the propodium have been suggested to mediate
chemosensory substratum-recognition and the resulting
control of metamorphosis (Raven, 1958; Fretter and Gra-
ham, 1962;Bonar, 1978a, b; Chiaand Koss, 1984; Yool,
1985). Ciliary receptors have been implicated in this pro-
cess in other invertebrate larvae as well (e.g., Laverack,
1968; Carthy and Newell, 1968; Chia and Spaulding,
1972; Siebert, 1974; Zimmer and Wollacott, 1977; Eck-
elbarger, 1978; Reed and Cloney, 1982; Reed, 1987). The
ciliated cells of the apical tufts of Haliotis larvae are stellate
neurons that appear anatomically to be primary chemo-
sensory receptor cells (Yool, 1985). These neurons project
axons to the cephalic ganglia; they also send lateral den-
drites to a pair of adjacent mucus gland cells that are
stimulated to secrete their contents in one of the first cel-
lular changes observed in metamorphosis (Morse et al.,
1980a; Yool. 1985). These neurons and their cilia dis-
appear from the organism after induction of metamor-
phosis; the time of this loss coincides with the time of loss
of the labeled morphogenic receptors (Trapido-Rosenthal
and Morse, 1986a), supporting the suggestion that some
of the biochemically labeled chemosensory receptors
controlling metamorphosis may be located on these cells
or their cilia.

Our finding that sufficient mRNA can be obtained from
the cilia of Haliotis larvae to establish a cDNA library
opens this system to analyses, similar to that reported
here, of the cDNAs for the receptors and other signal
transducers of the morphogenetic and amplifier pathways
that control metamorphosis. These analyses should pro-
vide insights into the mechanisms of action, functional
integration, and evolution of the chemoreceptors and their
associated signal-transducers in the molluscan larvae.

Acknowledgments

This research was supported by grants from the Na-
tional Science Foundation (DCB-87- 18224), the Office of
Naval Research Molecular Biology Program (NOOO 14-87-
K-0762), and the University of California at Santa Barbara
Training Grant in Marine Biotechnology. We especially
appreciate the expert advice, and time and effort provided
by Jay C. Groppe. We also gratefully acknowledge the
helpful suggestions provided by Mel Simon, Thomas T.
Amatruda, and John Sninsky; gifts of genomic DNA
samples provided by Richard Showalter, Jennifer Ortiz,
and Jay Groppe; and expert instruction and assistance in
the dissection of retinas, provided by Page Erickson.

Literature Cited

Anholt, R. R. H. 1987. Primary events in olfaction. Trends Biochem.
Sd. 12: 58-62.

326

L. M. WODICK.A AND D. E. MORSE

Anholt, R. R. H., S. M. Mumby, D. A. Stoffers, P. R. Girard, J. F. Kuo,

and S. H. Snyder. 1987. Transduction proteins of olfactory receptor

cells: identification of guanine nucleotide binding proteins and protein

kinase C. Biochemistry 26: 788-795.
Avenet, P., F. Mofmann, and B. Lindemann. 1988. Transduction in

taste receptor cells requires cAMP-dependent protein kinase. Nature

331: 351-354.
Aviv, H., and P. Leder. 1972. Purification of biologically active globin

messenger RNA by chromatography on oligothymidylic acid-cellu-
lose. Proc. Nat. Acad. Sd. USA 69: 1408.
Baloun, A. J., and D. E. Morse. 1984. Ionic control of settlement and

metamorphosis in larval Haliotis rufescens (gastropoda). Dial. Bull.

167: 124-138.
Baxter, G. 1991. Chemosensory signal transduction in larvae of the

mollusc, Haliotis rujescens. Doctoral Dissertation, University of Cal-
ifornia, Santa Barbara. 94 pp.
Baxter, G., and D. E. Morse. 1987. G protein and diacylglycerol regulate

metamorphosis of planktonic molluscan larvae. Proc. Nat. Acad. Sci.

USAM: 1867-1870.
Bonar, D. B. I978a. Infrastructure of a cephalic sensory organ in larvae

of the gastropod Phestilla sibogae (Aeohdacea: Nudibranchia). Tissue

Cell 10: 153-165.
Bonar, D. B. 1978b. Morphogenesis at metamorphosis in opistobranch

molluscs. Pp. 1 77- 1 96 in Settlement and Metamorphosis of Marine

Invertebrate Larvae, F.-S. Chia and M. E. Rice, eds. Elsevier/North

Holland Biomedical Press. New York.
Carthy, J. D., and G. E. Newell, eds. 1968. Invertebrate Receptors

Academic Press, London.
Chen, 'L., and D. Lancet. 1984. Membrane proteins unique to vertebrate

olfactory cilia: candidates for sensory receptor molecules. Proc. Nat.

Acad. Sci. USA 81: 1859-1863.
Chia, F.-S., and R. Koss. 1984. Fine structure of the cephalic sensory

organ in the larva of the nudibranch Rtistunga pulchra (Mollusca:

Opistobranchia. Nudibranchia). Zoomorphology 104: 131-139.
Chia, F.-S.. and J. G. Spaulding. 1972. Development and juvenile

growth of the sea anemone, Tealia erassicornis. Bin/. Bull 142: 206-

218.
Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, and \V. J. Ruder.

1979. Isolation of biologically active ribonucleic acid from sources

enriched in nbonuclease. Biochemistry 18: 5294.
Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA

isolation by acid guanidinium thiocyanate-phenol-chloroform ex-
traction. Anal. Biochem. 162: 156-159.
Eckelbarger, K. J. 1978. Metamorphosis and settlement in the Sabel-

lariidae. Pp. 145-164 in Settlement and Metamorphosis of Marine

Invertebrate Larvae. F.-S. Chia and M. E. Rice. eds. Elsevier, New

York.
Fretter, V., and A. Graham, eds. 1962. British Prosobranch Molluscs.

Ray Society, London.
Garner, C. C., R. P. Tucker, and A. Matus. 1988. Selective localization

of messenger RNA for cytoskeletal protein MAP2 in dendrites. Nature

336: 674-677.
Groppe, J., and D. E. Morse. 1989. Molecular cloning of novel serine

protease cDNAs from abalone. Pp. 285-288 in Current Topics in

Marine Biotechnology. S. Miyachi, I. Karube, and Y. Ishida, eds.

Japan Soc. Mar. Biotechnol., Tokyo.
Halliday, K. R. 1984. Regional homology in GTP-binding proto-on-

cogene products and elongation factors. J. Cyclic Nucleolide Res. 9:

435_44g.

Han, J. H., C. Stratowa, and W. J. Rutter. 1987. Isolation of full-
length putative rat lysophospholipase cDNA using improved methods
for mRNA isolation and cDNA cloning. Biochemistry 26: 1617-
1625.

Hanahan, D. 1983. Studies on transformation of Eschenchia coli with
plasmids. / Mol. Biol. 166: 557-580.

Huque, I . J. G. Brand, J. L. Rabinowitz, and D. L. Bailey.

1987. Phosphohpid turnover in catfish barbel (taste) epithelium with

special reference to phosphatidylinositol-4,5-bisphosphate. Chem.

Senses 12: 666-667.
Jones. D. T., and R. R. Reed. 1989. G0if: an olfactory neuron-specific

G protein involved in odorant signal transduction. Science 244: 790-

795.
kaziro Y., H. Itoh, and M. Nakafuku. 1990. Organization of genes

coding for G-protem « subumts in higher and lower eukaryotes. Pp.

63-76 in G Proteins. R. lyengar and L. Birnbaumer, eds. Academic

Press, San Diego.

Kosik, K. S., J. E. Crandall, E. J. Mufson, and R. L. Neve. 1989. Tau
//; situ hybridization in normal and Alzheimer brain: localization in
the somatodenditic compartment. Ann. Neurol. 26: 352-361.

Lancet, D., and V. Pace. 1987. The molecular basis of odor recognition.
Trends Biochem Sci. 12: 63-66.

Laverack, M. S. 1968. On superficial receptors. Symp. Zoo/. Sac. Land.
23: 299-326.

Linck, R. VV. 1973. Comparative isolation Of cilia and flagella from
the lamellibranch mollusc, Aequipecten irradians. J. Cell Sci. 12:
345-367.

Lochrie, M. A., and M. J. Simon. 1988. G protein multiplicity in eu-
karyotic signal transduction systems. Biochemistry 27: 4957-4965.

Mack, D. H., and J. J. Sninsky. 1988. A sensitive method for iden-
tification of uncharacterized viruses related to known virus groups:
hepadnavirus model system. Proc. Nat. Acad. Sci. USA 85( 18): 6977-
6981.

Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular Cloning:
A Laboratory M annul . Cold Spring Harbor Laboratory. Cold Spring
Harbor, New York.

McCabe, P. C. 1989. Asymmetric polymerase chain reaction. Ampli-
fications 3: 1-3.

Merlie, J. P., and J. R. Sanes. 1985. Concentrations of acetylcholine
receptor mRNA in synaptic regions of adult muscle fibres. Nature.
317: 66-68.

Morse, A. N. C, C. Froyd, and D. E. Morse. 1984. Molecules from
cyanobacteria and red algae that induce larval settlement and meta-
morphosis in the mollusc Haliotis rufescens. Mar. Bio/ 81: 293-298.

Morse, D. E. 1985. Neurotransmitter-mimetic inducers of settlement
and metamorphosis of marine planktonic larvae. Bull. Mar. Sci. 37:
697-706.

Morse, D. E. 1990a. Recent progress in larval settlement and meta-
morphosis: closing the gaps between molecular biology and ecology.
Bull. Mar. Sci. 46: 465-483.

Morse, D. E. 1990b. Molecular mechanisms controlling metamorphosis
in abalone larvae. In Abalone. M. Tegner and S. Shepherd, eds.
Blackwell (in press).

Morse, D. E., H. Duncan, N. Hooker, and A. Morse. 1977. Hydrogen
peroxide induces spawning in molluscs, with activation of prosta-
glandin endoperoxide synthetase. Science 196: 298-300.

Morse, D. E., N. Hooker, H. Dunan, and L. Jensen. 1979. -y-amino-
butyric acid, a neurotransmitter. induces planktonic abalone larvae
to settle and begin metamorphosis. Science 204: 407-410.

Morse, D. E., H. Duncan. N. Hooker, A. Baloun, and G. Young.
1980. GABA induces behavioral and developmental metamorphosis
in planktonic molluscan larvae. Fed. Proc. 39: 3237-3241.

Pace, U., and D. Lancet. 1986. Olfactory GTP-binding protein: signal-
transducing polypeptide of vertebrate chemosensory neurons. Proc.
Nat. Acad. Sci. USA 83: 4947-4951.

Pace, II., E. Hanski, Y. Saloman. and D. Lancet. 1985. Odorant-sen-
sitive adenylate cyclase may mediate olfactory reception. Nature 316:
255-258.

G« cDNAs FROM LARVAL CILIA

327

Raven, C. P. 1958. Morphogenesis: the Analysis ofMolluscan Devel-
opment Pergamon Press, New York.

Reed, C. G. 1987. The organization and isolation of the ciliary loco-
motory and sensory organs of marine bryozoan larvae. Pp. 397-408
in Marine Biodeterioration, R. Turner and R. Nagahhushanam, eds.,
Oxford Press. New Delhi.

Reed, C., and R. A. Cloney. 1982. The larval morphology of the marine
bryozoan Bowerbankia graci/is (Ctenostomata: Vosiculariodea).
Zoomorpliiilogy 100: 23-54.

Rhein, L. D., and R. H. Cagan. 1980. Biochemical studies of olfaction:
isolation, characterization, and odorant binding activity of isolated
cilia from rainbow trout olfactory rosettes. Prnc. Nat. Aaul. Sci. USA
77: 4412-4416.

Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing
with chain-terminating inhibitors. Pritc. Nat Acad. Sci. USA 74:
5463-5467.

Siebert, A. E. 1974. A description of the embryology, larval develop-
ment and feeding of the sea anemones Anthopleura elgantissima and
A. xanthogrammica. Can. J. Zoo/. 52: 1383-1388.

Smrcka, V., J. R. Hepler, K. O. Brown, and P. C. Sternweis.
1991. Regulation of polyphosphoinositide-specific phospholipase
C activity by purified Gq. Science 251: 804-807.

Strathmann, M., and M. I. Simon. 1990. G protein diversity: a distinct
class of « subunits is present in vertebrates and invertebrates. Proc
Nat. Acad. Sci. USA 87: 91 13-9117.

Stralhmann, M., T. M. Wilkie, and M. 1. Simon. 1989. Diversity of
the G-protein family: sequences from five additional « subunits in
the mouse. Proc Nat. Acad. Sci. USA 86: 7407-7409.

Trapido-Rosenthal, H. G., and D. E. Morse. 1985. L-«, w-Diamino
acids facilitate induction of settlement and metamorphosis in larvae
of a gastropod mottusc(Haliotis mfescens). J Comp. Physiol. B 155:
403-414.

Trapido-Rosenthal, H. G., and D. E. Morse. 1986a. Availability of
chemosensory receptors is down-regulated by habituation of larve
to a morphogenetic signal. Prtu: Nat. Acad. Sci. USA 83: 7658-
7662.

Trapido-Rosenthal, 11. G., and D. E. Morse. 1986b. Regulation of
receptor-mediated settlement and metamorphosis in larvae of
a gastropod mollusc (llaliolis mfescens). Bull. Mar. Sci. 39:
383-392.

Watson, M. R., and J. M. Hopkins. 1962. Isolated cilia from Tetra-
hymena pyriformis. E\p. Cell. Res. 28: 280-295.

Yool, A. J. 1985. Physiological analysis of the induction of metamor-
phosis in larvae of Haiiolis rnlesccnx (Gastropoda: Mollusca). Ph.D.
Thesis. University of California. Santa Barbara. Ill pp.

Zimmer, R. L., and R. M. \\oolacott. 1977. Metamorphosis, ances-
trulae. and coloniality in bryozoan life cycles. Pp. 91-142 in Biology
of Brvoioans. R. M. Woolacott and R. L. Zimmer, eds.. Academic
Press, New York.

CONTENTS

Hanlon, Roger T.

Integrative neurobiology and behavior of mollusks
symposium: introduction, perspectives, and round-
table discussion 197

Young, J. Z.

Computation in the learning system of cephalopods
200

Gilly, W. F., Bruce Hopkins, and G. O. Mackie
Development of giant motor axons and neural con-
trol of escape responses in squid embryos and
hatchlings 209

Williamson, Roddy

Factors affecting the sensory response characteristics
of the cephalopod statocyst and their relevance in
predicting swimming performance 221

Satterlie, Richard A.

Neural control of speed changes in an opisthobranch
locomotory system 228

Gillette, Rhanor

On the significance of neuronal giantism in gastro-
pods 234

Walters, Edgar T.

A functional, cellular, and evolutionary model of
nociceptive plasticity in Aplysia 241

Cleary, L. J., D. A. Baxter, F. Nazif, andj. H. Byrne
Neural mechanisms underlying sensitization of a
defensive reflex in Aphsia 252

Kiip term a M ii, Irving, Thomas Teyke, Steven C. Ro-
sen, and Klaudiusz R. Weiss

Studies of behavioral state in Apl\sia 262

Alevizos, A., M. Skelton, K. R. Weiss, and J. Koester

A comparison of bursting neurons in Aphsia .... 269

Ram, Jeffrey L., Feng Zhang, and Li-Xin Liu

Contraction, serotonin-elicited modulation, and
membrane currents of dissociated fibers of Af)l\iia
buccal muscle 276

Jacklet, Jon W.

Photoresponsiveness of Apl\sia eye is modulated by

the ocular circadian pacemaker and serotonin . . . 284

Prior, David J.

Control of central and peripheral targets by a mul-
tifunctional peptidergic interneuron 295

Kavaliers, Martin, and Klaus-Peter Ossenkopp
Opiod systems and magnetic field effects in the land
snail, Cepaea nemoralis 301

Pires, Anthony, and Michael G. Hadfield

Oxidative breakdown products of catecholamines
and hydrogen peroxide induce partial metamor-
phosis in the nudibranch Phestilta sibogae Bergh
(Gastropoda: Opisthobranchia) 310

Wodicka, Lisa M., and Daniel E. Morse

cDNA sequences reveal mRNAs for two Ga signal
transducing proteins from larval cilia 318

Volume 180

THE

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iii

CONTENTS

No. 1. FEBRUARY 1991

BEHAVIOR

De Vries, M. C., D. Rittschof, and R. B. Forward Jr.
Chemical mediation of larval release behaviors in
the crab Neopcmope \n\i

Hart, Michael W.

Particle captures and the method of suspension
feeding by echinoderm larvae

DEVELOPMENT AND REPRODUCTION

Patterson, Mark R.

The effects of flow on polyp-level prey capture in

an octocoral, Alcyonium nderium 93

Purcell, Jennifer E., Frances P. Cresswell, David G.

Cargo, and Victor S. Kennedy

Differential ingestion and digestion of bivalve larvae
by the scyphozoan Chiysaora quinquecirrha and the
ctenophore A/HCMD'H/MM li'nl\i 103

Walters, Linda J., and David S. Wethey

Settlement, refuges, and adult body form in colonial
marine invertebrates: a field experiment 112

Govind, C. K., Christine Gee, and Joanne Pearce

Retarded and mosaic phenotype in regenerated claw
closer muscles of juvenile lobsters 28

Gustafson, R. G., D. T. J. Littlewood, and R. A. Lutz
Gastropod egg capsules and their contents from
deep-sea hydrothermal vent environments 34

Longo, Frank J., and John Scarpa

Expansion of the sperm nucleus and association of
the maternal and paternal genomes in fertilized
Mill/inn laterals eggs 56

Webster, S. G., and H. Dircksen

Putative molt-inhibiting hormone in larvae of the
shore crab (.'.arcinu* maenas L.: an immunocyto-
chemical approach 65

ECOLOGY AND EVOLUTION

Carlton, James T., Geerat J. Vermeij, David R. Lind-
berg, Debby A. Carlton, and Elizabeth C. Dudley

The first historical extinction of a marine inverte-
brate in an ocean basin: the demise of the eelgrass

limpet Lottia alivus 72

Patterson, Mark R.

Passive suspension feeding by an octocoral in plank-
ton patches: empirical test of a mathematical model 8 1

PHYSIOLOGY

Bollner, Tomas, Jon Storm-Mathisen, and Ole Petter
Ottersen

GABA-like immunoreactivitv in the nervous system

of Oikopleura i/imiu (Appendicularia) 119

Charmantier, G., and M. Charmantier-Daures

Ontogeny of osmoregulation and salinity tolerance
in ('./inter immitm; elements of comparison with C.
burealia (Crustacea, Decapoda) 125

Childress, J. J., C. R. Fisher, J. A. Favuzzi, R. E. Ko-

chevar, N. K. Sanders, and A. M. Alayse

Sulfide-driven autotrophic balance in the bacterial
symbiont-containing hydrothermal vent tubeworm,
Rift in pafh\ptil(i (ones 135

Dickson, John S., Richard M. Dillaman, Robert D.

Roer, and David B. Roye

Distribution and characterization of ion transporting
and respiratory filaments in the gills of Procambarus
eld ikii 154

Dobson, William E., Stephen E. Stancyk, Lee Ann

Clements, and Richard M. Showman

Nutrient translocation during early disc regenera-
tion in the brittlestar Microphiopholis gradllima
(Stimpson) (Echinodermata: Ophiuroidea) 167

McConnaughey, Ted A., and Richard H. Falk

Calcium-proton exchange during algal calcification 1 85

No. 2, APRIL 1991

Hanlon, Roger T.

Integrative neurobiology and behavior of mollusks
symposium: introduction, perspectives, and round-
table discussion .... 197

Young, J. Z.

Computation in the learning system of cephalopods
Gilly, W. F., Bruce Hopkins, and G. O. Mackie

Development of giant motor axons and neural con-

CONTENTS

trol of escape responses in squid embryos and
hatchlings "2W

Williamson, Roddy

Factors affecting the sensory response characteristics
of the cephalopod statocyst and their relevance in
predicting swimming performance 221

Satterlie, Richard A.

Neural control of speed changes in .111 opisthobranch
locomotory system 22<S

Gillette, Rhanor

On the significance of neuronal giantism in gastro-
pods 234

Walters, Edgar T.

A functional, cellular, and evolutionary model of
nociceptive plasticity in Apl\sia 241

Cleary, L. J., D. A. Baxter, F. Nazif, andj. H. Byrne
Neural mechanisms underlying sensitization of a
defensive reflex in Aplwia 252

Kupfermann, Irving, Thomas Teyke, Steven C. Ro-
sen, and Klaudiusz R. Weiss

Studies of behavioral state in Aply.wi 262

Alevizos, A., M. Skelton, K. R. Weiss, and J. Koester

A comparison of "bursting neurons in .\/</vw</ .... 269

Ram, Jeffrey L., Feng Zhang, and Li-Xin Liu

Contraction, serotonin-elicited modulation, and
membrane currents of dissociated fibers of Aplysia
buccal muscle 276

Jacklet, Jon W.

Photoresponsiveness of Aphsia eye is modulated bv

the ocular circadian pacemaker and serotonin . . . 284

Prior, David J.

Control of central and peripheral targets by a mul-
tifunctional peptidergic interneuron 295

Kavaliers, Martin, and Klaus-Peter Ossenkopp
Opiod systems and magnetic field effects in the land
snail, Gt'paeii nemoralis 301

Pires, Anthony, and Michael G. Hadfield

Oxidative breakdown products of catecholamines
and hydrogen peroxide induce partial metamor-
phosis in the nudibranch Pheitillu \ihagni' Bergh
(Gastropoda: Opisthobranchia) 310

Wodicka, Lisa M., and Daniel E. Morse

cDNA sequences reveal mRNAs for two Got signal
transducing proteins from larval cilia 3 1 IS

No. 3, JUNE 1991

Kravitz, Edward A.

The rime of the ancient scientist

329

DEVELOPMENT AND REPRODUCTION

Byrne, M., and M. F. Barker

Embryogenesis and larval development of the as-
teroid Patiriella regularis viewed by light and scan-
ning electron microscopy 332

Cheng, Sou-De, Patricia S. Glas, and Jeffrey D. Green
Abnormal sea urchin fertilization envelope assembly
in low sodium seawater 346

Helluy, S. M., and B. S. Beltz

Embryonic development of the American lobster
(Homants americanus): quantitative staging and
characterization of an embryonic molt cycle .... 355

Zimmerman, Kerry M., and Jan A. Pechenik

How do temperature and salinity affect relative rates
of growth, morphological differentiation, and time
to metamorphic competence in larvae of the marine
gastropod Crfpirlula plana? 372

ECOLOGY AND EVOLUTION

Alexander, James E., Jr., and Alan P. Covich

Predation risk and avoidance behavior in two fresh-
water snails ... 387

Blackstone, Neil W., and Leo W. Buss

Shape variation in hydractiniid hydroids 394

Miles, J. S.

Inducible agonistic structures in the tropical coral-
limorpharian, DI\«>\UIIKI sanctithomae 406

Smith, L. David, and Anson H. Mines

Autotomy in blue crab (Callmectes sapidus, Rathbun)
populations: geographic, temporal, and ontogenetic
variation 416

ENVIRONMENTAL PHYSIOLOGY

Drinkwater, Laurie E., and John H. Crowe

Hydration state, metabolism, and hatching of Mono
Lake Artemia cycts 432

PHYSIOLOGY

Bowlby, Mark R., and James F. Case

Ultrastructure and neuronal control of luminous
cells in the copepod Ginissia princepa 440

Engel, David W., and Marius Brouwer

Short-term metallothionein and copper changes in
blue crabs at ecdvsis 447

Gardiner, David B., Harold Silverman, and Thomas

H. Dietz

Musculature associated with the water canals in

CONTENTS

freshwater mussels and response to monoamines in

I'itrn 453

Short, Graham, and Sidney L. Tamm

On the nature of paddle cilia and discocilia 466

Snyder, Mark J., and Ernest S. Chang

Metabolism and excretion of injected ['H]-ecdysone

bv female lobsters, Hnmanif. iinu'rinniu.^ 475

Takei, Y., A. Takahashi, T. X. Watanabe, K. Naka-

jima, S. Sakakibara, Y. Sasayama, N. Suzuki, and C.

Oguro

New calcitonin isolated from the ray, Du\\titi\ nkuji-i 485

ter Kuile, B. H., and J. Erez

Carbon budgets for two species of benthonic sym-
biont-bearing Foraminifera 489

Weis, Virginia M.

The induction of carbonic anhvdrase in the sym-
biotic sea anemone Aiptasia piili'hi'lla 496

RESEARCH NOTE

Ellington, W. Ross, and Amy C. Hines

Mitochondria! activities of phosphagen kinases are

not widely distributed in the invertebrates 505

Index to Volume 180

r.ns

Reference: Biol. Bull 180: 329-331. (June. 1991)

The Rime of the Ancient Scientist

EDWARD A. KRAVITZ*

(With apologies to Coleridge. Tennyson, Gray, Blake, and others too numerous to mention)

Part the First

//; which the ancient Scientist detaineth and praiseth his
beloved.

It was an ancient Scientist,

And he choseth one of three.

"By thy horned rim glass and balding pate.

Now wherefore choos't thou me?

Yon lobster pot is opened wide
And stuffed full with kin:
Choose mollusk, moth or fruity fly,
Blood-sucking leech or fishy fin."

He holds him with his skinny hand,
"Biochemist trained was't I;
Thou art the best of all the rest,
I'll give thee reasons why.

Hail to thee most noble of crustaceans!
Worthy of Homaric ode,
Behemoth of a briny deep.
Traveler, of a wet, sandy road.

Phalo h\ Rnben Huher

* The author is the George Packer Berry Professor of Neurobiology at
Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115.

No predator dare match thy might;
Who choose with thee to fight?
No spindly jaws or furry paws
Dare tamper with thy mega-claws.

Along the way, limbs that are lost
Can be replaced, no extra cost.
How practical, how devine;
How cute thou art, how truly fine.

Inventor of contraception! Thou scoff? Thou doubt?
What other creature neatly packs all its sperm cells up in

sacs?

Thou matest but once a year, tis true.
But that one time is quite a time!

If after all, one's going to breed.
Why one? Why two? Like lowly man.
Fifty thousand is the plan.
Cute little larval brood
(of course, to mom they're mostly food),
Which also makes good sense, thou see'st.
If mom didn't help by munching some.
The sea woulds't be a lobster slum
With millions and millions of the beasts;
No room for fish, crabs or other feasts.

Is't thy color really red?
Only when thou'rt really dead.

This poem was recited for the first time on November 2, 1989, at the
annual meeting of the Society for Neurosciences. in Phoenix, Arizona.
The occasion was an Invertebrate Neurobiology social which had as its
theme: "Toasts to the Invertebrates." The organizer (M. J. Greenberg)
had invited W. B. ICristan, I. Kupfermann. J. G. Hildebrand. and E. A.
Kravitz to extol the advantages, utility, and other virtues of their favorite
experimental animals, respectively, the leech, the sea slug, the moth, and
the lobster. Stirring words were spoken that evening, and emotions ran
high. Not one to take a challenge lightly, nor to miss an opportunity to
discuss the virtues of other animals (and with a little help from Alice
and a college textbook of literature) E. A. Kravitz produced "The Rime."

329

330

E. A. KRAVITZ

Greenish brown is more thy hue:

Not a bit like me or you.

Lobster, lobster, cooking red.

Boiling water, seaweed bed;

What happy hand or eye

Dare'st match drawn butter, lemon juice, and lobster pie?

Boiled Aplysia, tastes like rubber;
Sauteed leech, makes me shudder!
Marinated Manditca. scales for free.
Did he who cooked the lobster, cook thee?

Half a leg, half a leg,

Half a leg onward.

Back to the lobster tank each time

Till 8 or 10 appendages goneward

Thoughtful economical friend!

Nerve cells here, the muscles there,
The hormones all around;
Big cells thou art, and pluckable.
Over and over can'st be found.

Transmitters peptides and the like
For studies fundamental.

0 joyous day! Callooh! Callay!
The data're transcendental."

"I fear thee, ancient Scientist!

1 fear thy skinny hand."

"Fear not! Fear not! Thou noble beast!
List, while on others, I expand."

Part the Second

Fig. at. [

From Hemck. F H. 1911. Natural history of the American lobster Bull. V. S. Eur. Fish. 1909.
}9: 149-40&

In which the ancient Scientist compareth lowly beasties
to precious lobster.

"First there's Aplysia: sea hare 'tis called.

Sea hare? Hare indeed! Some jokester must have been in

need

Of immortality for selecting the beast least like the bunny.
Who's really cute and very funny (can'st imagine Bugs

Aplysia?).

Molluscan mass of pulpy flesh!
Slimy inking shell-less blob!
Learner of Kandelian puzzles that aren't hard;
'Cause what's to train, in a slug without a brain?
But I digress and must continue.
Flies can fly, tis true. So what?
It really doesn't mean a lot:
So can frisbees, jets and kites:
Mostly what flies does is bites
They're things to trap, and squash, and shoo
With sticky paper, swatters, and fancy kung-fu.
Their names are inelegant, crude and lewd.
Like houseflies, blowflies, horseflies and gnats;
I'd almost rather work with cats.
Their genes are cloned, they are well bred,
I'll grant thee that. But when that's said,
What good are they, these beasts so small?
Their whole brain can set
In one of thy glorious neurons, pet.
Guesseth whom I describeth next!
Eat n'eat n'eat n'eat n'eat n'eat n'eat n'eat,
n'eat n'eat n'eat n'eat n'eat n'eat n'eat n'eat n'molt.
Can'st yet tell? Nay? Then I go on!
Eat n'eat n'eat n'eat n'eat n'eat n'eat n'eat,
n'eat n'eat n'eat n'eat n'eat n'eat n'eat n'eat n'molt.

Art bored yet? Can'st bear more?
I'll spare thee that!

Now twice or thrice more say the same;
It doesn't matter which thee claim.
For now a change! O frabjous day!
Can't hardly wait! Can't barely stay!
Eat, etcetera and molt. And find a hole,
lie around, and turn to goo;
fly away and live a day,
Make 500 more like you.

Most interesting,

Ah, well.

They art a lovely shade of green;

Whilst dull, they're pretty to be seen.

Leeches, locusts, bees and ants

THE RIME OF THE ANCIENT SCIENTIST

331

Have their precious sycophants.
Praising that ungainly lot
of vampires, swarms, and crop destroyers.
And fat queens of interest but to voyeurs.

Can'st not thou see thou art the best?
Ne'er another passeth test
Of beauty, wit, charm, intellect, and learning.
Accepteth me, for thee I'm yearning.

Part the Third

The noble beast agreeeth: the ancient Scientist getteth
tenure.

"I see thy point old craggy beak;
I fear thee not, no more.
I am so good, its understood
All others but me foreswore.

Collect from my nerves;

Find all my cells;

Inject hormones by the score.

I'll behave for thee, and fight, not flee.

To please thee even more.

I warn thee, though, to leave me not.
Though funding turneth lean.
For if thou doth my chelipeds
Will teareth thee to tiny shreds.
My gastric mill will grind thy bones
Till nothing doth remain.
I'll chomp! I'll chew! I'll eat thee up!
Thou'll never be the same."

"Fear not, fear not, my precious pet,
I'll never leave thee cold.
I am so happy I could dance.
If I could be so bold."

In Xanadu did E A K

A stately lobster-palace plan,

Where Homar, with his next of kin.

In burrows with two entrances in.
Hides from his nemesis, man.

Twilight tolls the knell of parting day.
The blowing winds wind slowly o'er the sea.
The lobstermen homeward plod their weary way,
And leave the world to darkness, and to thee.

Reference: Biol. Bull, 180: 332-345. (June. 1991)

Embryogenesis and Larval Development of the

Asteroid Patiriella regularis Viewed by Light

and Scanning Electron Microscopy

M. BYRNE1 AND M. F. BARKER

School of Biological Sciences, Zoology A08, University of Sydney, N.S.W. 2006, Australia:
and Department of Zoology, University of Otago, Dunedin, New Zealand

Abstract. The sea star Patiriella regiilaris (Verrill, 1 867)
has indirect development through bipinnaria and bra-
chiolaria larvae. Development of this species is typical of
asteroids with planktotrophic larvae and takes 9-10 weeks.
The embryos develop through a wrinkled blastula and
hatch as early gastrulae. In contrast to most asteroids, a
third enterocoel forms on the left side of the stomach of
the bipinnaria. This structure gives rise to the left posterior
coelom; its significance is discussed. We suggest that this
coelom is homologous to the trunk coelom in entero-
pneust embryology. The surface features of the larvae were
examined by scanning electron microscopy. Newly
hatched gastrulae are covered by cilia, and the bipinnaria
have bands of cilia that follow the contours of the larval
processes. A previously undescribed plug-like structure
positioned on the post-oral surface appears to function as
a seal for the mouth. Brachiolaria larvae have three bra-
chiolar arms and a centrally located adhesive disc. Each
arm is covered by adhesive papillae. Raised epithelial cells
that dot the surface of the papillae and adhesive disc may
be batteries of secretory cells. The brachiolar arms have
an extracellular coat that may serve as a protective cover
for the adhesive surfaces. Competent brachiolaria swim
along the substratum and exhibit searching behavior with
flexure of the median brachium. They settle on the un-
dersides of natural shell substrata and do not respond to
a primary algal film. Shade appears to be an important
factor in settlement and metamorphosis in P. regularis.
Metamorphosis takes 5-6 days, and the post-larvae take
up a free existence at a diameter of 450-500 ^m. The

Received 20 November 1990; accepted 8 March 1991.
1 Present address: Department of Histology and Embryology, F-13,
University of Sydney, N.S.W. 2006.

indirect development of P. regularis contrasts with the
lecithotrophic and viviparous modes of development of
other Patiriella species and provides the comparative basis
to determine the ontogenic changes involved with evo-
lution of direct development in the genus. The use of the
divergent life histories of Patiriella as a model system for
the study of evolutionary change in development is dis-
cussed.

Introduction

The spinulosan sea star Patiriella regularis (Verrill,
1867) is common in New Zealand waters, ranging from
the intertidal zone to 100 m depth (Mortensen, 1921;
Crump, 1971). This species is a member of the Patiriella
group of which there are eleven species in the Australia-
New Zealand region (Dartnall, 1971; Keough and Dart-
nail, 1978). A remarkable feature of these asteroids is the
diversity of life histories that they exhibit, ranging along
a continuum from indirect to direct development (Dart-
nall, 1971; Lawson-Kerr and Anderson, 1978; Byrne,
1991; Table I). P. regularis spawns small eggs and develops
indirectly through planktotrophic bipinnaria and bra-
chiolaria larvae (Mortensen, 1921; Crump, 1971). These
feeding larvae are typical of the Asteroidea and are con-
sidered to be of great antiquity (Strathmann, 1978a). In
contrast, all the Australian species examined thus far are
direct developers. P. calcar, P. pseudoexigua, and P. gunnii
have large yolky eggs and develop directly through a non-
feeding planktonic brachiolaria (Lawson-Kerr and An-
derson, 1978; Grice and Lethbridge, 1989; Byrne, 1991;
Chen and Chen, 1991). P. exigua oviposits large eggs that
develop through a modified benthic brachiolaria (Lawson-
Kerr and Anderson, 1978; Byrne, 1991). At the end of
the indirect-direct continuum of development exhibited

332

ASTEROID DEVELOPMENT

333

Table I

Life history /raits of Patiriella species from Australia
and New Zealand*

Oocyte
diameter
Species (mm) Developmental pattern

Larvae

P. regitlaris 150 Indirect/planktotrophic Bipinnaria and

brachiolaria
P. gunnii 360 Direct/lecithotrophic Planktonic

brachiolaria
P. calcar 400 Direct/lecitholrophic Planktonic

brachiolaria
P. pseudoexigua Direct/lecithotrophic Planktonic

brachiolaria
P. exigua 400 Direct/lecithotrophic Benthic

brachiolaria
P. vivipara 120 Direct/viviparous Intraovarian

brooder
No larva
P. parvivipara 100 Direct/viviparous Intraovanan

brooder
No larva

* Data from: Dartnall (1969); Crump (1971); Keough and Dartnall
(1978); Lawson-Kerr and Anderson (1978); Byrne (1991); Chen and
Chen (1991).

by Patiriella. are the intraovarian brooders, P. vivipara
and P. parvivipara, which give birth to crawl-away juve-
niles (Dartnall, 1969; Chia, 1976; Keough and Dartnall,
1978; Byrne, 1991).

Several nomenclatural systems have been suggested for
the diverse developmental patterns in the Asteroidea
(Chia, 1968. 1974; Oguro et al.. 1976, 1988). In one sys-
tem, development through a bipinnaria and brachiolaria
larvae is termed indirect, whereas development only
through a brachiolaria is termed direct (for review, Oguro
et al., 1988). This system is most appropriate for Patiriella.
Other systems make the distinction between indirect-
planktotrophic larvae with a functional gut and direct-
lecithotrophic larvae without a functional gut (Chia,
1968). The recent finding, however, of an intermediate
pattern of asteroid development, through a larva that has
both planktotrophic and lecithotrophic features, obscures
this distinction (Bosch, 1989).

Comparative embryology of closely related species is a
powerful tool for the investigation of developmental pro-
cesses in evolution because homologous characters can
be compared (Raff, 1987). This approach has attracted
renewed interest, particularly with respect to echinoids,
where recent studies have revealed that direct development
arose through heterochronies in the appearance of adult
features (Raff, 1987; Wray and Raff, 1989). Heterochro-
nies, changes in the relative timing or rate of ontogenic
events, are considered to be an important means of ef-

fecting evolutionary change (Anderson, 1987). The range
of life histories in Patiriella listed in Table I presents an
ideal system with which to investigate the modifications
involved with the shift to direct development within a
monogeneric group. In P. vivipara, direct development is
achieved by heterochrony in suppression of larval char-
acters and accelerated development of adult features
(Byrne, 1991).

In the evolutionary sequence of developmental change
in Patiriella, the planktotrophic development of P. reg-
ularis represents the ancestral mode of development in
the genus, and, in this investigation, is described in detail.
Particular attention is paid to the pattern of larval ciliation
and the structure of the larval arms, features often mod-
ified in lecithotrophic larvae (Strathmann, 1978a). Settle-
ment behavior and metamorphosis are also described. The
ontogeny of P. regularis will provide the chronological

Table II

Chronology of development of Patiriella regularis at I8-22°C

Time

Stage

0 Fertilization

15-60 s Elevation of fertilization membrane

40-60 mm First cleavage

1-1.5 h Second cleavage

2-2.5 h Third cleavage

3 h Fourth cleavage

3.5-5 h Early blastula

6-9 h Wrinkled blastula

15-17.5 h Late blastula/early gastrula

25 h Hatching, gastrula with elongating archenteron

30-35 h Advanced gastrula, budding of mesenchyme cells from

terminal expansion of archenteron

45-55 h Early bipinnaria, enterocoel and stomodeum formation,

archenteron bent towards oral surface to complete
gut, the posterior enterocoel starts as a thickening of
the left side of the archenteron

55-60 h Bipinnaria. ciliary bands distinct, gut regions

differentiate

65-75 h Bipinnarial arms well-developed, posterior enterocoel is

vesicular, posterior elongation of right and left
enterocoels, hydropore open, fusion of left enterocoel
with posterior enterocoel

4-5 days Anterior growth of enterocoels into oral hood and
fusion to form the axohydrocoel

6 days Extension of axohydrocoel into oral hood, right and left

enterocoels continue to grow posteriorly, posterior
enterocoel forms part of the left posterior coelom,
formation of the ventral horn

10-14 days Advanced bipinnaria, fusion of the ventral horn with
the right enterocoel, the axohydrocoel with two
lateral extensions for brachia

4-6 weeks Early brachiolaria, growing brachiolar arms

8 weeks Advanced brachiolaria, arms and adhesive disc well-

developed, five lobes of the hydrocoel present,
formation of adult primordium and skeleton

9-10 weeks Larvae competent to metamorphose

334

M BYRNE AND M. F. BARKER

Figure 1 . Development through the bipinnaria stage, a. Egg shortly after fertilization with an elevated
fertilization membrane and one polar body (arrowhead), b. One hour, first cleavage, two polar bodies are
evident (arrowhead), c. One and a half hours, second cleavage, d. Four hours, early blastula. e. Eight
hours.wrinkled blastula. f. Sixteen hours, late coeloblastula rotating in membrane, g. Twenty-five hours.

ASTEROID DEVELOPMENT

335

basis required to determine the morphological and het-
erochronic changes underlying the evolution of direct de-
velopment within the genus.

Materials and Methods

Specimens of Patiriella regularis were collected during
slack water at 5-10 m depth from Otago Harbour, New
Zealand (45 °49.7'S; 170° 38.4'E), near the Portobello Ma-
rine Laboratory, in January 1990. This species is gono-
choric, and ovaries and testes were dissected from mature
specimens. The testes were stored dry at 4°C, and the
ovaries were placed in a 10~5 M solution of 1-methylad-
enine in filtered seawater (Kanatani, 1969). Following
ovulation, the ova were transferred to beakers of 1 .0 ^m
filtered seawater and washed gently through several
changes of filtered seawater. For fertilization, the eggs were
placed approximately one cell deep in 250-ml beakers,
and a few drops of diluted sperm were added. After 5-10
min, the eggs were washed in fresh seawater to remove
excess sperm, and the fertilized eggs were placed in 5-1
beakers of filtered seawater. The cultures were stirred
gently by motor-driven paddles. When the embryos at-
tained the swimming gastrula stage, the cultures were fil-
tered and placed in fresh seawater. Thereafter, the seawater
in the cultures was changed once a week. Just prior to the
early bipinnaria stage, feeding of the larvae commenced.
The larvae were fed from unialgal cultures of the flagellate
Dunaliella primolecta Butcher. The temperature of the
culture room ranged from 18° to 22°C.

Embryogenesis and larval development of Patiriella
regularis were documented by light and scanning electron
microscopy (SEM). For SEM, the embryos and larvae
were fixed in 2.5% glutaraldehyde in 0.45 ^m filtered sea-
water for 1 h at room temperature. Once the bipinnaria
stage was attained, larvae fixed by this method were first
relaxed in 6.8% MgCl2 in distilled water before being
placed in the primary fixative. Following primary fixation,
the specimens were washed in 2.5% sodium bicarbonate
(pH 7.2) and post-fixed in 2% OsO4 in 1.25% sodium
bicarbonate for 1 h at room temperature. The specimens
were then washed in distilled water and dehydrated in

ethanol. After dehydration, the specimens were critical
point dried, sputter coated, and viewed with a Joel JSM-
35C scanning electron microscope. In addition, the fix-
ation method of Barker ( 1978a) was also used. According
to this method, larvae placed in a small drop of seawater
were initially fixed by the addition of Bouin's fluid. The
larvae were then transferred to 3% glutaraldehyde in 0.2
Mcacodylate buffer for 1 h at room temperature. Follow-
ing a rinse in the same buffer, the specimens were post-
fixed in 2% OsO4 in cacodylate buffer. Although the in-
troduction of Bouin's caused the larvae to contract slightly,
this method resulted in good preservation of the extra-
cellular coat of the larvae. After fixation, the larvae were
rinsed in distilled water and processed as described above.

Results

Spawning

Spawning of Patiriella regularis was observed in situ
on 25 January 1990. Approximately 20 individuals, both
males and females, were observed releasing gametes. The
sperm exited from the gonopores as a narrow plume that
dissipated 5-10 cm above the spawning individual. For
the females, the eggs rolled on to the aboral surface after
exiting from the gonopore. The shortest distance between
spawning individuals ranged from 0.5 to 1 .0 m, while the
longest distance ranged from 4 to 5 m. These observations
were recorded during the day under sunlit conditions and
coincided with slack water.

In the laboratory, the ovaries of Patiriella regularis ex-
hibited a long hormone-dependent period. It took 3-5 h
before oocyte maturation; ovulation and spawning was
induced by 1-methyladenine. The spawned ova were green
and 150 /tm in diameter (±9 jim; n = 20).

Embryogenesis

The chronology of the development of Patiriella reg-
ularis is outlined in Table II. A fertilization membrane
forms 1 5-60 s after the introduction of sperm into the
beakers containing ova, and the two polar bodies are given
off within 20 min (Fig. la, b). Cleavage is radial and ho-

hatching gastrulae. B, blastopore: F. fertilization membrane, h. Twenty-five hours, swimming gastrula. i.
Thirty-five hours, advanced gastrula, mesenchyme cells (M) are budding off into the blastocoel. A, archenteron. j.
Fourty-five hours, early bipinnaria, right and left enterocoels are starting to form (C). A, archenteron. k.
Fourty-seven hours, bipinnaria, enterocoels are forming (C), archenteron is complete. A small bulge on the
archenteron is the beginning of the posterior enterocoel (P). I. Fifty-five hours, the right (RC) and left
enterocoels grow posteriorly. On the left side of the achenteron, a small group of cells form the posterior
enterocoel (P). m. Seventy hours, dorsal view. LC, left enterocoels; M, mouth; P, posterior enterocoel; O,
oesophagus; S, stomach, n. Seventy hours, bipinnaria in side view, gut regions and hydropore (H) are evident.
LC, left anterior enterocoel; O, oesophagus; P, posterior enterocoel; S, stomach, o. Seventy-five hours, ventral
view. The arrow points to the fusing left anterior (LC) and posterior (P) enterocoels. The right and left
anterior enterocoels have started to grow into the oral hood. Scale bars = 100 Mm.

336

M BYRNE AND M. F BARKER

; o-.^X

SiSfcSi/ •

Figure 2. Development through metamorphosis, a. Eighty-eight hours, dorsal view. Growth of the right
enterocoel (RC) into the oral hood. At the position where the two left enterocoels have met, tissue derived
from fusion of their end walls is evident (arrow), h. Four and a half days, side view of a bipinnaria showing

ASTEROID DEVELOPMENT

337

loblastic (Fig. Ib-d). The first cell division occurs 40-60
min after fertilization, and the early blastula stage is
reached witnin 4 h. By 4 h, asynchrony was evident in all
cultures, with some embryos at a more advanced stage
than others. Five hours post-fertilization, the embryos are
well-developed blastulae 164 ^m in diameter (±7 ^m; n
= 10) (Fig. Id). The blastulae rotate within their close-
fitting fertilization membranes, propelled by their ciliary
covering. Blastular wrinkling starts 6 h after fertilization
with folding of the blastoderm into the blastocoel. The
furrows of the wrinkled blastulae are most apparent in
8-h larvae (Fig. le). Subsequently, the furrows smooth
out and 1 8-h cultures contain late blastulae with a smooth
surface and early gastrulae (Fig. If, g). Hatching ensues
through rupture of the fertilization membrane, and the
gastrulae become free-swimming larvae (Fig. Ig, h). At
hatching, the larvae are round to elongate and have a
shallow blastopore. They continue to elongate with growth
of the archenteron into the blastocoel. The blind end of
the archenteron expands and mesenchyme cells detach
from its tip, moving into the blastocoel (Fig. li). At this
stage, the larvae are 197 ^m long (±1.2 ^m, n = 10).

Early bipinnaria are present by the end of the second
day. The right and left enterocoels form as pouches off
the expanded tip of the archenteron (Fig. Ij). A shallow
stomodeum is present, and the blind end of the archen-
teron bends towards the oral surface. During this stage,
the posterior region bends ventrally, and from this time
the blastopore can be regarded as the larval anus. By 55
h, the archenteron fuses with the stomodeal invagination,
thereby completing the larval gut (Fig. Ik). With devel-
opment of the ciliated bands, algal food was introduced
into the cultures. The larvae now have a distinct peroral
hood region. In addition, a shallow evagination, destined
to form the posterior enterocoel, is evident on the left-
hand wall of the archenteron; this soon grows to form a
small thickening of cells (Fig. Ik, 1).

By the end of the third day, the bipinnaria are feeding
and have well-defined pre- and postoral ciliary bands. At
this stage, the bipinnarial processes — lateral and anterior

projections of the larval body wall — start to form. The
regions of the gut differentiate with the expansion of the
stomach and the separation of the stomach from the oe-
sophagus by the cardiac sphincter (Fig. 1m). In three-day-
old bipinnaria, the right and left enterocoels increase in
length as they grow posteriorly, and the hydropore exits
on the dorsal surface (Fig. In). During the fourth day of
development, the small thickening on the archenteron
wall grows to form a solid ball of cells attached to the
stomach. A central cavity forms in this structure, thereby
forming a posterior enterocoel on the left side of the larvae
(Fig. In, o). This posterior enterocoel increases in size
and is a conspicuous feature of all the larvae examined
from five different cultures. When the advancing left an-
terior enterocoel reaches the posterior enterocoel, the two
enterocoels fuse (Figs, lo, 2a). In some larvae, fusion of
the two left coelomic pouches was complete 75 h after
fertilization. With subsequent development, it was evident
that the posterior enterocoel forms part, if not all, of the
left posterior coelom. Where the two enterocoels meet, a
partition derived from fusion of their tissues forms (Fig.
2e, g). During the fourth day of development, the right
and left enterocoels extend anteriorly into the oral hood
(Figs, lo, 2a). The larval length is now 630 ^m (±5.8 ^/m;
n = 10).

In 4.5 day larvae, the anterior extensions of the right
and left enterocoels fuse to form the axohydrocoel in the
oral hood (Fig. 2b). This anterior coelom grows to form
an extension into the hood where the median-dorsal pro-
cess develops (Fig. 2c-e). Five-day-old larvae are well-
developed bipinnaria and the ciliary tracts increase in
length following the edges of the bipinnarial processes.
The bipinnaria exhibit muscular movements including
contraction of the cardiac sphincter and dorsal and ventral
flexure of the oral hood, which results in broadening and
closure of the oral cavity (Fig. 2c). Internally, the fused
left enterocoels extend below the gut, while growth of the
right enterocoel is slower. The partition derived from fu-
sion of the two left enterocoels divides the left enterocoel
into anterior and posterior regions (Fig. 2e, g). Partition

the anterior coelom (arrow) in the oral hood formed through fusion of the right and left enterocoels. H.
hydropore. c. Six days, bipmnaria from the side, exhibiting dorsal flexure, the anterior coelom has grown
into the oral hood (arrow). I. intestine. O, oesophagus, S, stomach, d. Four-week-old culture containing late
bipinnaria. e. Ten days, dorsal view, late bipinnaria/early brachiolaria. The anterior coelom (AC) has grown
to form the lumen of the future median brachium, two small lateral branches at the base of this coelom (L)
are destined to be the coelomic lumina of the posterior brachia. The left posterior enterocoel (P) has grown
below the gut. A septum-like structure (arrow), partitions the left coelom into anterior and posterior sections,
f. Four weeks, ventral view, early brachiolaria, the ventral horn (V) of the left posterior coelom has grown
around the gut and fused with the right enterocoel. M, mouth, g. Four weeks, detail of the septum (arrow)
dividing the left anterior (LC) and posterior coelom (P). RC, right enterocoel, S. stomach, h. Eight weeks,
ventral view, late brachiolaria, the median (MB) and posterior (PB) brachia and the adhesive disc (AD) are
well-developed. The lobes of the hydrocoel (He) are evident, i. Eight weeks, ventral view, adult pnmordium
of a late brachiolana, primary spicules (S) lie along the lobes of the hydrocoel. j. Nine weeks, metamorphosing
larva from the aboral surface. S, skeleton. Scale bars: a,b,c,g,i,j = 100 ^m. Scale bars: d.e.f.h; = 150 ^m.

Figure 3. SEM of development through the bipinnaria stage, a. Late blastula within the fertilization
membrane, b. Hatching and newly hatched gastrulae. B. blastopore; F. fertilization membrane, c. Gastrula
starting to elongate, d. Elongate gastrula. e. Early bipinnaria forming the stomodeal invagination (S). f.
Bipinnanal shape starting to develop, the stomodeum (S) has enlarged and the blaslopore has moved to a
ventral position to form the anus (A). O, forming oral hood. g. Bipinnaria side view with a distinct oral
hood (O) and ciliary tracts (C). h. Late bipinnaria ventral view with oral- (OC) and postoral (PC) ciliary
tracts. M, mouth, i. Ciliary field covering gastrula. j. Detail of bipinnaria in Figure 3h, showing the ciliary
tracts around mouth and scattered cilia covering the larva, k. Ciliary tract (C) of a bipinnaria. 1. Bipinnaria
fixed in the dorsally-llexed position showing the plug-like structure on the post-oral surface (arrow). A. anus;
M. mouth; C, ciliary tract. Scales: Fig. 3a-h. I = 50 ^m; Fig. 3i-k = 20 ^m.

338

ASTEROID DEVELOPMENT

339

of the right enterocoel was not observed. By day 6, the
ventral horn of the left posterior coelom forms and extends
between the stomach and the intestine. Eight-day-old lar-
vae are 790 ^m in length (±85 /^m; n = 20).

Ten-day-old larvae are advanced bipinnaria (length, 990
± 1 50 /*m; n = 20). By day 1 4, the ventral horn completes
its growth fusing with the right enterocoel (Fig. 2f ). In the
preoral hood, the coelom extends anteriorly beyond the
median-dorsal process, forming the lumen of the future
median brachiolar arm. At the base of the median-dorsal
process, the anterior coelom gives rise to two lateral ex-
tensions destined to be the coelomic lumina of the pos-
terior brachiolar arms (Fig. 2e).

The larvae grow as advanced bipinnaria through the
first month of development. By week five, early brachio-
laria are present with three brachiolar arms or brachia.
The longest brachium extends from the median-dorsal
process and contains the main branch of the anterior coe-
lom. On either side are two small brachia into which the
lateral coelomic extensions grow. Each of these brachiolar
arms are contractile. Advanced brachiolaria were present
in eight-week-old cultures (Fig. 2h). These larvae have a
well-developed brachiolar complex comprised of the three
brachia and a centrally located adhesive disc. The adult
primordium develops the posterior region of the brachio-
laria (Fig. 2h, i). On the left side of the larvae, the five
lobes of the hydrocoel are evident (Fig. 2h). The first adult
spicules form as small rods positioned along each lobe of
the hydrocoel (Fig. 2i). By week nine, the larvae were
competent to metamorphose at a length of 1 430 nm (± 1 94
^m; n = 20). This appears to be the upper growth limit
of the larvae, as three-month-old brachiolaria were similar
in length.

Metamorphosis

Advanced brachiolaria extend their arms and attach
them to the bottom of the culture dishes in what appears
to be searching behavior. The large median brachium
bends at a 90° angle to the larval body, bringing the two
posterior brachia and the adhesive disc into contact with
the substratum. The larva then adhere temporarily to the
bottom of the dish by means of the arm and then detach
and continue swimming. To induce metamorphosis, glass
slides with a primary algal film or natural shell substrata
were placed in finger bowls, and competent larvae were
introduced. The brachiolaria did not respond to the slides,
but attached to the undersurfaces of the shells within a
few hours of introduction. During temporary attachment,
larvae moved over the surface of the substratum and ex-
hibited searching behavior, with the brachiolar arms at-
taching and detaching as the larvae "walked" over the
substratum. Following this exploratory phase, the larvae
ceased to move, attached permanently with their brachia

and adhesive disc, and started to metamorphose. During
metamorphosis, the larval body is shortened and resorbed
to a thin stalk. The adult primordium develops with for-
mation of a pentamerous shape. The hydrocoel expands,
and the first adult tube feet form on the oral surface. These
tube feet are used for attachment and locomotion. Even-
tually, the post-larvae break free of their attachment stalks
taking up an independent existence at a diameter of 450-
500 /urn five to six days after settlement (Fig. 2j). Devel-
opment continues with completion of the adult digestive
tract. Newly detached post-larvae do not have a mouth
or an anus.

Scanning electron microscopy

Examination of the surface of hatching gastrulae ofPa-
tiriella regiilaris shows that they are covered by a uniform
field of cilia (Fig. 3b). The wrinkled appearance of the
fertilization membrane is probably due to the collapse of
the membrane during fixation and drying (Fig. 3a, b). On
hatching, the gastrulae start to elongate (Fig. 3b-d, i). With
the development of the bipinnaria, pre- and postoral por-
tions of the larvae are evident with a slight depression
between them where the stomodeal invagination arises
(Fig. 3e, f ). As the larvae grow, the bipinnarial processes
and the pre- and postoral ciliary tracts form (Fig. 3g, h).
These tracts, a conspicuous feature of the larvae, are sin-
uous ridges of dense cilia that follow the contours of the
bipinnarial processes (Fig. 3j, k). In addition to the ciliary
tracts, the bipinnaria are also covered by a uniform field
of cilia (Fig. 3j, k). Bipinnaria preserved in the dorsally
flexed position reveal the presence of a plug-like structure
on the postoral surface (Fig. 31). On contraction of the
larva, this structure would function as a seal over the
mouth.

Formation of the brachiolar complex is evident with
the appearance of the median brachiolar arm and two
small lateral projections (Fig. 4a). Ridges on the median
arm are developing papillae (Fig. 4a). In advanced bra-
chiolaria, the arms take on their distinctive shape and are
covered by adhesive papillae (Fig. 4b, c). An adhesive disc
is positioned at the base of the arms (Fig. 5a). Like the
bipinnaria, the brachiolaria has ciliary tracts and is covered
by cilia (Fig. 4b, c). In addition to the preoral ciliary tract
on the median-dorsal process, a lateral ciliary tract is
present along the median brachium (Fig. 4b, i, j).

Preservation of the brachiolaria larvae differed with the
two fixation methods used. An external coat covers the
adhesive surface of the arms of brachiolaria fixed with the
Bouin's method (Fig. 4a-j), whereas this coat is not present
in larvae fixed with the glutaraldehyde-seawater method
(Fig. 5a-h). The coat is a thin mesh-like material on the
surface of the brachia that gives the arms a smooth ap-
pearance (Fig. 4b-j). Due to contraction of the larvae, it

340

M. BYRNE AND M. F. BARKER

Figure 4. SEM of development through the brachiolana stage of larvae fixed initially with Bouin's. a.
Early brachiolana. The two posterior arms (PB) are starting to form at the base of the median brachium
(MB). Ridges (arrow) on the surface of the median brachium are developing papillae, b. Brachiolana side
view. The median brachium (MB) emerges from the median-dorsal process (M) and the posterior brachia
(PB). A, developing adult primordium; C. ciliary tract, c. Late brachiolana ventral view. Note the smooth
surface of the median brachium (MB), which has two rows of papillae (P), and the cilia covering of the
larva. The adult primordial (A) region is evident posteriorly. PB. posterior brachium. d. Detail of the bra-
chiolaria shown in Figure 2b. The posterior brachium has a smooth extracellular coat through which cilia
(C) emerge, e. Detail of the advanced brachiolana shown in Figure 2c showing the posterior brachium and

ASTEROID DEVELOPMENT

341

is not possible to determine whether the adhesive disc has
an extracellular covering. The rest of the brachiolar surface
does not have this coat (Fig. 4b-j).

The removal of the extracellular coat by the glutaral-
dehyde-seawater method reveals the underlying structure
of the brachiolar complex (Fig. 5a-g). Papillae cover the
brachia down to their bases and surround the adhesive
disc (Fig. 5a, b). The median brachium is considerably
longer than the other two and has 9-16 papillae arranged
in two rows. A cluster of papillae covers the surface of
the posterior arms (Fig. 5c). Nodular arrays of raised ep-
ithelial cells dot the surface of the papillae (Fig. 5b,d-f).
In side-view, these nodules are raised structures that have
a fuzzy tip, probably comprised of microvilli (Fig. 5e). In
brachiolaria with an intact extracellular coat, small ele-
vations of the coat indicate the position of the underlying
nodules (Fig. 4g, h). Cilia on and around the papillae oc-
casionally protrude through the glycocalyx (Figs. 4d, h:
5d, e). The adhesive disc is a round, flat structure with
raised epithelial cells similar to those seen on the papillae
(Fig. 5b, g). In larvae fixed with the glutaraldehyde-sea-
water method, smooth patches of material apparently se-
creted by the papillae are evident on the surface of the
brachia (Fig. 5c).

Competent brachiolaria have a distinct adult rudiment
at the posterior end of the larvae and scattered cilia cover
the future aboral surface (Fig. 5h). The reduction of the
larval body to a thin attachment stalk is shown in the
whispy tissue attached to the metamorphosing larva in
Figure 5i. Post-larvae have two pairs of tube feet per arm,
and cilia are present on the epidermis (Fig. 5j).

Discussion

Development of Patiriella regularis is similar to other
asteroids that develop indirectly through planktotrophic
bipinnaria and brachiolaria larvae (Dan, 1968; Strath-
mann, 1987). The bipinnarial processes of P. regularis
larvae, characteristic of spinulosan asteroids, are relatively
short in comparison with those of forcipulate larvae, which
develop into long and slender extensions of the larval body
(Gemmill, 1914; Strathmann, 1971; Barker, 1978b).

The wrinkled blastula has been widely reported in as-
teroid embryology for both indirect and direct developers
(Mortensen, 1921; Chia, 1968; Komatsu, 1972, 1976;

Oguro el a!.. 1976; Byrne, 1991). A wrinkled blastula oc-
curs in Patiriella regularis. which has small eggs, and it
also occurs in the Australian species, P. exigua. P. ca/car,
and P. gunnii. which have large ova 350-400 fjm in di-
ameter (Lawson-Kerr and Anderson, 1978; Byrne, 1991).
Blastular wrinkling in each of these Patiriella species re-
sults from the folding of the blastoderm into the blastocoel
with subsequent smoothing out at the advanced blastula
stage (Lawson-Kerr and Anderson, 1978; Byrne, 1991).
In echinoids, wrinkled blastulae are only reported in spe-
cies with large eggs (Williams and Anderson, 1975; Ame-
miya and Tsuchiya, 1979; Raff, 1987; Parks el al. 1989),
and the wrinkled blastula may be a consequence of the
shift from indirect to direct development (Raff, 1987;
Parks el ui. 1989). There is no evidence of a relationship
between blastular wrinkling and egg size in asteroids, and
infolding of the blastoderm may be associated with the
mechanics of cleavage (Anderson, pers. comm.). Up to
the early blastula stage, cleavage gives rise to large cuboidal
blastomeres held within a close-fitting fertilization mem-
brane. As development continues, the embryo may not
be able to accommodate additional cuboidal cells in a
spherical shape due to insufficient space within the fertil-
ization membrane, resulting in the onset of wrinkling. In
asteroids and echinoids that have a wrinkled blastula in
their development, smoothing of advanced blastula cor-
responds with the transition from a cuboidal to a colum-
nar blastomere organization (Parks el al., 1989; Byrne,
pers. obs.). Compared with cuboidal blastomeres, this co-
lumnar organization may be more readily accommodated
in a spherical shape. But not all asteroids have a wrinkled
blastula (Dan, 1968; Strathmann, 1987), and for these
species, the spatial relationship between the blastular sur-
face and the fertilization membrane during the cuboidal-
columnar transition should be documented. Recent work
suggests that wrinkling of lecithotrophic echinoid embryos
may also be a mechanical phenomenon (Henry, pers.
comm.).

The posterior enterocoel that forms on the left side of
the archenteron in the early bipinnaria of Patiriella regu-
laris is not a general feature of asteroid embryology (Dan,
1968; Strathmann, 1987). Homologous structures are re-
ported in the bipinnaria ofAsterias rubens and Marthas-
terias glacialis, where similar masses of cells may arise
on the right or left side of the archenteron (Gemmill.

papillae (P).The arm has an extracellular coat that gives it a smooth appearance, f. Median brachium and
papillae (P). The arm has an extracellular coat that gives it a smooth appearance, g. Detail of the papillae
of the medium brachium and the smooth mesh-like extracellular coat. Raised bumps on the papillae (arrow)
indicate the position of underlying raised epithelial cells (see Fig. 5b). h. Papillae at the tip of the median
brachium with cilia (C) emerging through the surface coat. i. Anterior portion of brachiolaria shown in
Figure 2b, showing the pre-oral ciliary tract on the median dorsal process (arrow) and the ciliary tract (C)
on the median brachium. j. Detail of the ciliary tract (C) along the median brachium. Scales: Fig. 4a, d-j
= 20 Mm; Fig. 4b. c = 100 ^m.

Figure 5. SEM of development through metamorphosis of larvae fixed by the glutaraldehyde-seawater
method, a. Brachiolana ventral view. Note the median (MB) and posterior brachia (PB) and the adhesive
disc (AD). Cilia cover the larva, and the adult primordium is evident posteriorly (A), b. Median brachium
(MB) with two rows of papillae. Note the absence of an extracellular coat. The papillae and adhesive disc
(AD) are dotted by raised epithelial cells, c. Posterior brachium with a patch of secreted material on its
surface (arrow). AD, Adhesive disc. d. Detail of the brachiolaria shown in Figure 5a. Cilia cover the larva
and are also present on and around the papillae (C). Note the absence of an extracellular coat on the posterior

342

ASTEROID DEVELOPMENT

343

1914). In these species, this cell mass detaches from the
gut and either breaks up into mesoderm or fuses with the
advancing right or left enterocoel; in M. glacialis a central
cavity occasionally forms (Gemmill, 1914). A similar sit-
uation to that seen in P. regularis occurs in the other
asterinid species Asterina miniata and A. pectinifera
(Heath. 1917; Newman, 1925; Komatsu, pers. comm.).
As in P. regularis, a posterior enterocoelic growth arises
on the left side of the archenteron of A. miniata and A.
pectinifera, forming a third enterocoel that fuses with the
anterior left enterocoel (Heath, 1917;Newman, 1925). In
P. regularis and ,-1. miniata, the posterior enterocoel is a
functionally important structure that grows during de-
velopment and gives rise to the posterior coelom (New-
man, 1925). This contrasts with typical asteroid devel-
opment, where the posterior coelom is derived from par-
tition of the left anterior enterocoel (Gemmill, 1914; Dan,
1968; Strathmann, 1987). There is speculation as to the
significance of the presence of a posterior enterocoelic
growth and a third enterocoel (Gemmill, 1914; Heath,
1917; Newman, 1925). In the development of A. miniata,
Newman ( 1925) considered the posterior enterocoel to be
a vestigial feature. Gemmill (1914) and Heath (1917)
considered the thickening of the archenteron wall in some
asteroids, and the third enterocoel in others, to be rudi-
ments of a posterior coelom present in the larvae of a
common enteropneust-echinoderm ancestor. Thus, as
suggested by Gemmill (1914), the posterior enterocoel of
P. regularis may be homologous to the trunk coelom in
enteropneust embryology.

The plug-like structure on the post-oral surface of the
bipinnaria of Patiriella regularis has not been described
before. This structure is evident only in bipinnaria fixed
in the dorsally flexed posture and appears to serve as a
seal for the mouth on ventral contraction of the larvae.
In this manner it may function as a mechanism to prevent
undesirable particles from entering the mouth. It was not
seen in live specimens examined with the light microscope.
Although this mouth seal has not been reported in the
bipinnaria of other asteroids, its presence may be revealed
by scanning electron microscopy.

The ultrastructure of the brachiolar complex has been
described for the forcipulate asteroids Stichaster australis
and Coscinasterias calamaria (Barker, 1978a). In com-
parison with these species, the median brachiolar arm of
Patiriella regularis is well-supplied with adhesive papillae.

In S. australis and C. calamaria, adhesive papillae are
limited to the tip of the brachia, and the stem of the me-
dian brachium is smooth (Barker, 1978a). Like the bra-
chiolaria of these species, cilia are also present on the
papillae of P. regularis and may have a sensory role in
the location of suitable substrata for settlement (Barker,
1978a). The raised epithelial nodules on the brachial pa-
pillae and adhesive disc of P. regularis appear to corre-
spond to the batteries of secretory cells revealed by trans-
mission electron microscopy of the brachiolar complex
of S. australis and C. calamaria (Barker, 1978a).

Temporary attachment of P. regularis brachiolaria is
achieved by the median brachium as it extends over sub-
stratum, assisted by adhesion of the posterior arms. The
patches of smooth material on the brachia may be used
for adhesion. For P. regularis, as reported for S. australis
and C. calamaria (Barker, 1978a), it appears that per-
manent attachment is achieved by secretion of a cement-
like material by the adhesive disc. Early descriptions of
brachiolaria refer to the attachment disc as a 'sucker' be-
cause it was thought to effect attachment by means of
suction with the edge of the disc forming a seal (Gemmill,
1914; Mortensen, 1921).

The difference in preservation of the brachiolaria by
the two fixation methods is striking. Brachiolaria fixed
initially with Bouin's fluid have a glycocalyx-like material
covering their brachia, whereas larvae fixed with the glu-
taraldehyde-seawater method do not. Removal of surface
coats by conventional fixation methods is reported for
several echinoderms (Cameron and Holland, 1983;
McKenzie, 1987). An extracellular coat similar to that on
the brachiolar complex of P. regularis is present on the
larvae of Asterina miniata (Cameron and Holland, 1983).
In A. miniata, however, this coat covers the entire surface
of the larva (Cameron and Holland, 1983). The preser-
vation of a glycocalyx on the brachia, but not on the rest
of the larval surface of P. regularis, suggests that it may
function in association with the brachiolar complex as a
protective covering for the attachment surface.

The searching behavior, settlement, and metamorphosis
of Patiriella regularis is characteristic of asteroid brachio-
laria (Gemmill, 1914; Barker, 1977). In contrast to that
reported for Coscinasterias calamaria, the presence of a
primary algal film is not sufficient to induce metamor-
phosis of P. regularis (Barker, 1977). The attachment and
metamorphosis of the brachiolaria on the undersides of

brachium. e. Detail of the median brachium showing raised epithelial cells (arrows) on the papillar surface.
Cilia are present on and around the papillae (C). f. Papillae (P) at the tip of the median brachium. g. Adhesive
disc (AD), the arrows point to raised epithelial cells, h. Late brachiolaria ventral view. The larva is in the
exploratory/attachment posture with the median brachium extended 90° to the larval body (arrow). A, adult
primordium. i. Metamorphosing larva detached from its stalk, which appears as wispy material (arrow).
Two pairs of tube feet (T) are present in each radius, j. Detail of the metamorphosing larva. The future
adult surface is covered by cilia (C). Scales: Fig. 5a, h, i = 50 mm; Fig. 5b-g, j = 20 mm.

344

M. BYRNE AND M. F BARKER

shells and not on the film-covered slides, suggests that
shade and a rough-textured surface may be an important
factor in selecting a site for metamorphosis in this species.
Crump (1969) also reported attachment and metamor-
phosis of P. regularis brachiolaria on the undersides of
introduced substrata.

The observation of simultaneous spawning of male and
female Putiriella regularis in the field is similar to that
reported for several asteroids (Minchin, 1987; Pearse el
ai. 1988). The nearest distance between spawning P. reg-
ularis, however, is considerably longer than for Marthas-
terias glacialis, which gathers in spawning assemblages
prior to gamete release (Minchin, 1987). Although some
of the P. regidaris releasing gametes were 0.5 m from an
adjacent spawner, several individuals appeared to be
spawning in isolation, as reported by Pearse ct al. (1988).
At this distance, and particularly for those individuals
spawning in isolation, gamete dilution would reduce the
chance of fertilization. Echinoid zygote production in the
field decreases dramatically if females are more than 20
cm apart from spawning males (Pennington, 1985). There
is evidence, however, that asteroid sperm and sperm of
other echinoderms are attracted to conspecific ova, and
this, to some extent, may ameliorate the problem of ga-
mete dilution (Miller, 1989; Byrne, 1990). The collection
site is subject to strong currents, and it seems that the
chances of fertilization would be enhanced by the slack
water conditions that coincided with spawning, as noted
for breeding in holothuroids (McEuen, 1988).

From laboratory culture at 18-22°C, the pelagic period
of Paliriella regularis has a duration of 9-10 weeks; it
may be longer in the field, where ambient sea surface
temperatures of 16-18°C in Otago Harbour coincide with
the planktonic period of this species. This duration of
larval life is similar to that of other temperature plank-
totrophic asteroids, although it is somewhat shorter than
that of asteroids from the northern Pacific, where ambient
temperatures range from 7 to 1 3°C (Strathmann, 1978b).
Latitudinal differences in larval life undoubtedly reflect
differences in ambient temperature, with the longest
planktonic period of 22 weeks recorded for the antarctic
asteroid Odontaster validus at sea temperatures of — 2-
- 1 °C (Pearse and Bosch, 1986).

Development of Patiriella regularis through feeding
bipinnaria and brachiolaria larvae is typical of asteroid
embryogenesis and contrasts with the development of the
Australian Patiriella. P. e.xigua, P. pseudoexigua, P. cal-
car, and P. gimnii have completely lost the bipinnarial
stage and develop directly through a non-feeding bra-
chiolaria ( Mortensen, 1921; Lawson-Kerr and Anderson,
1978; Byrne, 1991; Chen and Chen, 1991). Also in con-
trast to P. regularis, these species have large ova, the evo-
lution of which is considered to be a pre-adaptive trait
for the shift to direct development (Chia, 1968). Larvae

derived from such eggs would no longer be obligate
planktotrophs. resulting in the loss of structures required
for feeding (Strathmann. 1978a). The development of P.
regularis provides the basic reference for comparison with
the direct developers. Features that are particularly im-
portant for comparison include the mode and timing of
archenteron and coelom formation in P. regularis. and
the morphology of larval feeding structures. As docu-
mented for echinoids (Raff, 1987), the evolution of direct
development in Patiriella may involve heterochronic
changes in these features. Together with the developmen-
tal chronologies of the other Patiriella species, the ontog-
eny of P. regularis presented here will be used to determine
the changes underlying the shift to direct development
within the genus and to assess the pathways by which
feeding larvae were lost. The use of Patiriella as a tool
with which to examine developmental processes in evo-
lution is the subject of ongoing research.

Acknowledgments

We thank Professor John Jillett, Director of the Por-
tobello Marine Laboratory (PML), for the use of facilities.
The staff of the PML also provided technical assistance.
In particular we thank Mr. Michael Stuart. Thanks also
to Ms. Pia Laegdsgaard for technical assistance. Special
thanks to Dr. V. B. Morris for reading the manuscript.
This work was supported by a grant from the Australian
Research Council.

Literature Cited

Anderson, D. T. 1987. Developmental pathways and evolutionary rates.
Pp. 143-155 in Rales of Evolution, K. S. W. Campbell and M. F.
Day, eds. Allen and Unwin, Sydney.

Amemiya, S., and T. Tsuchiya. 1979. Development of the echinothund
sea urchin Alhenosoma ijimai. Mar Biol 52: 93-96.

Barker, M. F. 1977. Observations on the settlement of the brachiolaria
larvae ofStichaster australis (Verrill) and Coscinasterias calamaria
(Gray) (Echinodermata: Asteroidea) in the laboratory and on the
shore. J. Exp. Mar. Biol. Ecol. 30: 95-108.

Barker, M. F. I978a. Structure of the organs of attachment of the
brachiolaria larvae of Stichuster australis (Verrill) and Coscinasterias
calamaria (Gray) (Echinodermata: Asteroidea). / Exp. Mar. Biol.
Ecol. 33: 1-36.

Barker, M. F. 1978b. Descriptions of the larvae ofStichaster australis
(Verrill) and Coscinasterias calamaria (Gray) (Echinodermata: As-
teroidea) from New Zealand, obtained from laboratory culture. Biol
Bull. 154: 32-46.

Bosch, 1. 1989. Contrasting modes of reproduction in two antarctic
asteroids of the genus Porania, with a description of unusual feeding
and non-feeding larval types. Biol. Bull ill: 77-82.

Byrne, M. 1990. Annual reproductive cycles of the commercial sea
urchin Paracentrotus lividits from an exposed intertidal and a sheltered
subtidal habitat on the west coast of Ireland. Mar. Biol. 104: 275-
289.

Byrne, M. 1991 . Developmental diversity in the starfish genus Patiriella.
In Proceedings 7;h International Echinoderm Conference. N. Suzuki.
ed. Balkema. Rotterdam.

ASTEROID DEVELOPMENT

345

Cameron, R. A., and N. D. Holland. 1983. Electron microscopy of
extracellular materials during the development of a sea star, Patina
miniala (Echinodermata: Asteroidea). Cell Tissue Res 234: 193-
200.

Chen, B., and C. Chen. 1991 . Reproductive cycle, larval development,
juvenile growth and size distribution of Patiriella pscmlocxigua
Dartnall (Echinodermata: Asteroidea). In Proceedings "ill Interna-
tional Ki'luncilerm Conference. N. Suzuki, ed. Balkema. Rotterdam.

Chia. K. S. 1968. The embryology of a brooding starfish, I.eplasterias
he.\ticti.\ (Stimpson). Ada. Zool. 49: 321-364.

Chia, F. S. 1974. Classification and adaptive significance of develop-
mental patterns in marine invertebates. Thalassia Jugoslav. 10: 121-
130.

Chia, F. S. 1976. Reproductive biology of an intraovarian brooding
starfish Patiriella vivipera Dartnall. 1969. Am. Zoo/. 16: 181.

Crump, R. G. 1969. Aspects of the biology of some New Zealand echi-
noderms. Ph.D. thesis. University of Otago, Dunedin, New Zealand.

Crump, R. G. 1971. Annual reproductive cycles in three geographically
separated populations of Patiriella regtilaris ( Verrill), a common New
Zealand asteroid. / Exp. Mar. Biol. Ecol. 7: 137-162.

Dan, K. 1968. Asteroidea. Pp. 303-308 in Invertebrate Embryology.
H H. Kume and K.. Dan, eds. NOLIT Publishing House. Belgrade.
Yugoslavia.

Dartnall. A. J. 1969. A viviparous species of Paliriella (Asteroidea,
Astenmdae) from Tasmania. Pmc. Linn. Soc. NSW 93: 294-296.

Dartnall. A. J. 1971. Australian sea stars of the genus Patiriella (As-
teroidea. Asterimdae). Proc. Linn. Soc. NSW 96: 39-51.

Gemmill, J. F. 1914. The development and certain points in the adult
structure of the starfish Asterias ruhens. L. Plnlos. Trail* R Soc.
Loud Ser B 205: 2 1 3-294.

Grice, A. J., and R. C. Lethbridge. 1989. Reproductive studies on Pa-
tiriella gimnii (Asteroidea: Asterinidae) in south-western Australia.
Auxt. J. Mar Freshwater Res, 39: 399-407.

Heath, H. 1917. The early development of a starfish Pateria lA.sterina)
Mmeala. J. Morphol. 29: 461-469.

kanatani, II. 1969. Induction of spawning and oocyte maturation by
1-methyladenine in starfishes. Exp. Cell Res. 57: 333-337.

Keough, M. J., and A. J. Dartnall. 1978. A new species of viviparous
asterinid asteroid from Eyre Peninsula, South Australia. Ree S. Aust.
Mils 17: 407-416.

Komatsu, M. 1972. On the wrinkled blastula of the sea-star A.ster/iiti
pectnnfera Zoo/. Mag. 81: 227-231.

Komatsu, M. 1976. Wrinkled blastula of the sea-star A.sterina minor
Hayashi. Dev. Growth Differ. 18: 435-438.

Lawson-Kerr, C., and D. T. Anderson. 1978. Reproduction, spawning
and development of the starfish Patiriella exigua (Lamarck) (Aster-
oidea. Asterinidae) and some comparisons with P. ca/car( Lamarck).
Aust. J. Mar. Freshwater Res. 29: 45-53.

McEuen, F. S. 1988. Spawning behaviors of northeast Pacific sea cu-
cumbers (Holothuroidea: Echinodermata). Mar. Biol 98: 565-585.

McKenzie, J. D. 1987. The ultrastructure of the tentacles of eleven
species of dendrochirote holothurians studied with special reference
to the surface coats and papillae. Cell Tissue Res. 248: 187-199.

Miller, R. L. 1989. Evidence for the presence of sexual pheromones
in free-spawning starfish. ./ .Exp Mar Biol Ecol. 130:205-221.

Minchin, D. 1987. Sea-water temperature and spawning behaviour in
the seastar Marthastenas glaeialis. .Mar. Biol. 95: 139-143.

Mortensen, T. 1921. Studies on the Development and Larval Forms
oj Echinoderms G.E.C. Gad.. Copenhagen. 216 pp.

Newman, H. H. 1925. An experimental analysis of asymmetry in the
starfish. Paliria mmiata Biol. Hull 49: 1 1 1-138.

Oguro, C., M. Komatsu, and V. T. Kano. 1976. Development and
metamorphosis of the sea-star Asteropeden scoparius Valenciennes.
Biol. Bull. 151: 560-573.

Oguro, C.. M. Komatsu, and Y. T. Kano. 1988. Significance of the
nonbrachiolanan type of development in sea-stars. Pp. 241-246 in
Echinoderm Biology. R. Burke, P. Lambert, and R. Parsley, eds.
Balkema, Rotterdam.

Parks, A. L., B. W. Bisgrove, G. A. Wray, and R. R. Raff. 1989. Direct
development in the sea urchin Phyllacanthiis panispimis (Cidaroidea):
phylogenetic history and functional modification. Biol. Bull. 177:
96-109.

Pearse, J. S., and I. Bosch. 1986. Are the feeding larvae of the com-
monest antarctic asteroid Odonlaster validus really demersal? Bull.
Mar Sci 39: 477-484.

Pearse, J. S., D. J. McClary, M. A. Sewell, \V. C. Austin, A. Perez-
Ruzafa, and M. Byrne, 1988. Simultaneous spawning of six species
of echinoderms in Barkley Sound, British Columbia. Im. Reprod.
Dev 14: 279-288.

Pennington, J. T. 1985. The ecology of fertilization of echinoid eggs:
the consequences of sperm dilution, adult aggregation, and synchro-
nous spawning. Biol Bull 169:417-430.

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

Strathmann, M. F. 1987. Reproduction and Development ot Marine
Invertebrates of the Northern Pacific Coast. University of Washington
Press, Seattle. Pp. 535-555.

Strathmann, R. R. 1971. The feeding behaviour of planktotrophic
echinoderm larvae: mechanisms, regulation, and rates of suspension-
feeding. J. Exp. Mar. Biol Ecol. 6: 109-160.

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

Strathmann, R. R. 1978b. Length of pelagic period of echinoderms
with feeding larvae from the northeast Pacific. J. Exp. Mar. Biol.
Ecol. 34: 23-27.

Williams, D. H. C., and D. T. Anderson. 1975. The reproductive system,
embryonic development, larval development and metamorphosis of
the sea urchin Heliocidans erythrogramma (Val.) (Echinodermata:
Echinometridae). Aust. J. Zool. 23: 371-403.

Wray, G. A., and R. A. Raff. 1989. Evolutionary modification of cell
lineage in the direct-developing sea urchin Heliocidaris erythro-
gramma. Dev. Biol. 132: 458-470.

Reference: Uinl Bull 180: 346-354. (June, 1991)

Abnormal Sea Urchin Fertilization Envelope Assembly

in Low Sodium Seawater

SOU-DE CHENG1, PATRICIA S. GLAS2, AND JEFFREY D. GREEN*

Department of Anatomy, Louisiana State University Medical Center, New Orleans, Louisiana 70112

Abstract. The structuralization of the sea urchin fertil-
ization envelope (FE), a model for extracellular macro-
molecular assembly, was found to require sodium ions,
the predominant cation of seawater. Eggs from Strongy-
locentrotus purpuratits activated in sea waters with sodium
chloride substitutes (choline or Tris chloride) elevated
incomplete FEs. In addition, the conversion of the mi-
crovillar casts of the FE from blunt (I-form) to angular
(T-form) did not occur. The permeability of the abnormal
FEs was also compromised, as approximately eight times
more protein than normal was released into the ambient
seawater. There were also significant increases in the es-
cape of two cortical granule (CG) enzymes, 0-1,3-gluca-
nase and ovoperoxidase. Furthermore. FEs elevated in
choline chloride (ChCl) seawater appeared to be deficient
in the incorporation of ovoperoxidase, an enzyme that is
normally bound to the FE and that cross-links structural
proteins in the nascent FE. The morphology of FEs ele-
vated in potassium chloride-substituted seawater was
similar to those in normal sodium seawater. Thus, it ap-
pears that sodium, or at least a similar ion, is necessary
for the proper functioning of ovoperoxidase and structural
proteins in the elevation and normal assembly of the sea
urchin FE.

Introduction

Sea urchin fertilization has been intensively studied for
the dramatic intra- and extracellular events concomitant

Received 29 August 1990: accepted 24 January 1991.

' Genetics Division. Children's Hospital, 300 Longwood Avenue.
Boston, MA 02 11 5.

2 Department of Zoology and Physiology. Louisiana State University.
Baton Rouge. LA 70803.

Abbreviations: SW (artificial seawater); CG (cortical granule); ChCl
(choline chloride); FE (fertilization envelope); FP (fertilization product);
VL (vitelline layer).

* To whom correspondence should be sent.

with the change from egg to embryo. The irreversible
transformation of the relatively thin, soft vitelline layer
(VL = glycocalyx), investing the unfertilized egg, into a
hardened, insoluble fertilization envelope (FE), elevated
from the egg surface, is a critical step for the protection
of the developing embryo. Furthermore, this process has
been the subject of numerous investigations as an example
of regulated extracellular matrix assembly. Several recent
reviews (Kay and Shapiro. 1985; Shapiro et al., 1989;
Somers and Shapiro, 1989) contain the details of such
investigations: therefore, the process will be summarized
only briefly here.

Transglutaminase has an important role in the earliest
stages of VL modification (Battaglia and Shapiro, 1988).
It is located on the egg surface and catalyzes the incor-
poration of primary amines into the nascent FE during
the first 4 min of egg activation. This process is apparently
related to the I-T transition of the microvillar projections
of the VL (in 5. purpuratiis). because the transition does
not occur in the presence of transglutaminase inhibitors.
During the next few minutes, ovoperoxidase secreted from
the cortical granules (CG) catalyzes the insertion of struc-
tural proteins from the CGs into the VL by cross-linking
tyrosyl residues between polypeptides (Foerder and Sha-
piro, 1977; Hall, 1978). Besides this catalytic reaction,
ovoperoxidase itself is incorporated into the nascent FE
via a specific interaction with another CG protein, pro-
teoliaisin (Weidman et al., 1985). These enzyme activities
result in a hardened, insoluble, fully formed FE within
the first 10 min following egg activation.

Several recent reports have been concerned with ionic
requirements for FE formation. Carroll and Endress
(1982) demonstrated that formation of mature FEs re-
quires the normal 9 mM [Ca2+] and 48 mM [Mg2+] in
the seawater. By omitting these ions they could produce
an "intermediate envelope" that was much thinner than
the normal FE and did not incorporate structural proteins.

346

FERTILIZATION ENVELOPE ASSEMBLY

347

Deficiency of Cl , the most abundant anion in seawater,
not only interferes with the normal I-T transformation,
but also increases the permeability of the FE (Lynn ct ai,
1988: Green et ai. 1990). Furthermore, the normal ex-
ternal Na+ concentration (419 mA/) is not only essential
for preventing polyspermy and hardening the FE, but also
for the normal embryonic development of the sea urchins
Arbacia pnnctiilata and Strongylocentrotus purpwatiis
(Schuel ct ai. 1982).

To better understand the mechanism of FE elevation—
an important early event of embryonic development—
we concentrated on the effect of Na+ deficiency on the
formation of the FE. In the present study, the elevation
and morphology of FEs from normal and low sodium (2
mA/) seawaters were investigated. Envelopes were ob-
served with phase, scanning, and transmission electron
microscopy. Total soluble secreted protein and the en-
zymatic activities of ovoperoxidase (Foerder and Shapiro.
1977; Hall, 1 978) and ^-l,3-glucanase( Schuel et ai. 1972;
Wessel et ai. 1987) in the fertilization products (FP) were
compared. Portions of this investigation have been pre-
sented in a preliminary form (Cheng et ai. 1989).

Materials and Methods

Handling of gametes

Sea urchin (5. piirpuratits) eggs were collected and acid
dejellied as described previously (Green et ai. 1990). Eggs
were divided into four equal portions, the three low Na+
groups: KC1-, Tris-, and ChCl-substituted seawaters (SW)
and the control: normal Na+-SW group. They were
washed and incubated in the appropriate SW for 1 5-40
min. All seawater formulations were based on Cavanaugh
(1956). For the low Na+-SWs, equimolar concentrations
of KG, Tris HC1, or ChCl (Sigma) replaced NaCl, yielding
a calculated residual [Na+] of approximately 2 mA/. Nor-
mal and low Na+ SWs were buffered with 10 mA/ TAPS
(Sigma) and adjusted to pH 8.3 with NaOH or KOH,
respectively. To avoid contamination from sperm proteins
and secretions, eggs were activated by adding the Ca'+
ionophore A23187 (Chambers et ai, 1974; Steinhardt and
Epel, 1974) to a final concentration of 38 ^Af in 1% di-
methyl sulfoxide (DMSO). Experiments were performed
at 20°C.

Light microscopy

Eggs activated in normal and low Na+-SWs were ob-
served continuously on a 1 X 3 inch microscope slide
under a coverslip supported by a ring of petroleum jelly.
Photographs were taken with phase contrast optics on
Kodak Technical Pan Film 2415.

Ovoperoxidase localization

Activated eggs of the control group (normal Na4-SW)
were rinsed at 75 min postactivation with 0.45 A/ NaCl-

0.1 AI Tris HC1 (KlebanoftV/ ai. 1979). Ten percent egg
suspensions (v/v) were made and incubated in 5.6 mA/
3,3-diaminobenzidine (DAB; Sigma) in 0.45 A/NaCl-0. 1
M Tris HC1 in an ice bath for 10 min. The experimental
eggs (low Na+) were handled identically except that NaCl
was replaced with ChCl. Activated eggs were then fixed
and prepared for TEM as described below.

Transmission electron microscopy

Eggs were fixed with 2% glutaraldehyde in the appro-
priate seawaters for 1 h at room temperature and washed
with 0. 1 A/ sodium cacodylate (pH 7.4). Postfixation with
1% osmium tetroxide (OsO4) was performed for 30 min.
Eggs were washed with double distilled water and dehy-
drated in ascending concentrations of ethanol. Ethanol
was replaced with propylene oxide and eggs were infil-
trated with EM bed-812 (Electron Microscopy Sciences).
The blocks were cured at 58-60°C for 3 days. Sections
were cut with glass knives on a Reichert-Jung Ultracut E;
mounted on copper grids; stained with lead citrate (Reyn-
olds, 1963) and uranyl acetate; and observed with a Philips
301 transmission electron microscope at 60 kV.

Scanning electron microscopy

Fixations were accomplished at 20°C by mixing equal
volumes of eggs with 4% glutaraldehyde in seawater (with
10 mMTAPS pH 8.3) before activation and at 1, 3, 10,
30, and 60 min postactivation. Eggs were fixed for 1 h
and washed in 0.1 M sodium cacodylate (pH 7.4). Post-
fixation took place in 1% OsO4 in 0.1 M sodium caco-
dylate (pH 7.4) for 30 min. They were then washed with
double distilled water and dehydrated in an ascending
series of ethanol. Absolute ethanol was replaced gradually
by acetone. Eggs were transferred to porous containers
(Bio-Rad) and processed for CO: critical point drying
(Samdri-790, Tousimis Research Corp.). The eggs were
then attached to aluminum mounts coated with colloidal
silver liquid (Ted Pella, Inc.) for sputter coating with gold:
palladium (60:40; Electron Microscopy Sciences) for 3
min in a Hummer VI (Technics). Eggs were observed with
a JEOL JSM-35CF scanning electron microscope at 25
kV and a condenser lens setting of 3. The photos were
taken with Polaroid type 55 (4 X 5 in.) positive/negative
instant sheet film.

Total protein assay

lonophore-activated eggs were allowed to settle for ap-
proximately 10 min and the supernatant (secreted FP)
was collected and centrifuged by hand to remove the few
remaining eggs. Ionophore activation resulted in at least
95% elevated FEs. For protein determination the proteins
in 1 .0 ml of FP (5% egg suspension, v/v) were precipitated

348

S.-D. CHENG ET AL

by adding 1 10 n\ ice-cold 50% trichloroacetic acid (TCA)
and centrifuged for 20 min at 8800 X g (Eppendorf cen-
trifuge 5413) at 10°C. Tubes were drained by inversion
and the protein pellets air dried. Bovine serum albumin
(BSA; Sigma) was the standard. All the precipitates were
assayed according to Lowry el til. (1951). As a control,
protein determinations were performed with BSA in each
of the seawaters to ascertain their influences, if any, on
the Lowry procedure.

0-1,3-glucanase assay

Glucanase activity was measured according to Green
and Summers ( 1980). The FP was collected as described
above. FP (0.2 ml; 5% egg suspension, v/v). normal Na+-
or ChCl-substituted SW (0.2 ml), and laminarin (0.2 ml
of a 2.5 mg/ml SW solution) were incubated for 1 h at
37°C. Then 0.2 ml of this mixture, 0.4 ml enzyme solution
(0.4 ml glucose oxidase and 3 mg horseradish peroxidase
in 50 ml of 25 mM phosphate bufTer, pH 6) and 0.4 ml
o-dianisidine (40 mg in 50 ml double distilled water) were
incubated for 10 min at 37 °C. The reaction was stopped
by the addition with rapid vortexing of 0.8 ml 4 A'sulfuric
acid. Spectrophotometric readings were taken at 530 nm.
Controls lacking the substrate laminarin were used to
check for the presence of glucose (the product of the glu-
canase-laminarin reaction) in the FP. Glucose was gen-
erated only in the presence of both the FP and laminarin.
A glucose solution was the standard. Control glucose de-
terminations were performed in normal Na+-SW and
ChCl-substituted-SW to ascertain the effects, if any, of
ChCl substitution on the glucose oxidase-peroxidase re-
action. All the chemicals for this assay were purchased
from Sigma.

Ovoperoxidase assay

Ovoperoxidase assays were performed in 1 ml contain-
ing 18 mA/guaiacol (Sigma), 0.3 mA/ H2O: (Sigma), and
10 mM TAPS at pH 8.0 and 20°C (Deits et a/., 1984).
The reaction was started by adding enzyme (in the FP),
and the increase in absorbency at 436 nm was recorded
spectrophotometrically with a strip chart recorder. All re-
ported values are initial rates, because the reaction slows
after 15-30 s. A unit of Ovoperoxidase was defined as that
which is required to oxidize 1 ^mole of guaiacol per min
in a 1-ml assay volume (Deits et ai, 1984).

FP including the Ovoperoxidase was collected from a
5% (v/v) suspension of activated eggs. Ten minutes after
activation, eggs were settled by low speed centrifugation
and the supernatant (FP) was removed for the assay.

Statistics

Statistical analyses for total protein, ft- 1 ,3-glucanase and
Ovoperoxidase assays were performed using the Student's
I test.

Results

Observations with light microscopy

The elevation of FEs in normal and low Na+-SWs are
compared in Figure 1. Figure 1A-D depicts eggs at 1 min
postactivation. Although difficult to quantify from the
photomicrographs in the normal Na+-SW control group
(Fig. 1 A, E), the FE appeared relatively thin at the end of
the first minute and thickened with increasing time.
However, it appeared thicker (more refractive) than the
low Na+ groups, especially Tris and ChCl (Fig. 1C, D).
At later time points, the FE of the control group remained
more refractive than those of the low Na+ groups. These
differences were more striking at 3 min postactivation
when the FE of ChCl eggs began to shrink and some col-
lapsed, while those of the control remained spherical. By
30 min postactivation, the FEs remained robust in the
normal and K+-substituted SWs (Fig. IE, F), while those
of the Tris- and choline-substituted SWs had collapsed
back nearer to the egg surface (Fig. 1G, H).

An interesting attribute of the activated eggs is that of
their increased stickiness in the Tris and ChCl groups. In
the first 3 min there was no apparent difference among
the 4 groups. At approximately 4 min postactivation,
however, the eggs of the Tris and ChCl groups formed
extensive clumps.

Ultrastructural changes

Unactivated eggs incubated in normal Na+- or ChCl-
substituted-SWs and observed by SEM displayed similar
surface morphology (Fig. 2A, E). At 1 min postactivation,
the FEs in both SWs were elevated with rounded (I-form)
microvillar projections (Fig. 2B, F). In contrast to FEs in
ChCl-substituted SW (Fig. 2G), the typical I-T ("Igloo-
Tent") transformation of S. purpuratus FEs in normal
Na+-SW was completed by 3 min (Fig. 2C) and resembled
those of later time points (Fig. 2D). However, ChCl FEs
did not undergo the transformation even by 30 min
(Fig. 2H).

As judged with TEM (Fig. 3), FEs that elevated in nor-
mal Na ' -SW resulted in the well-defined, angular T-form
projections (Fig. 3A) characteristic of this species. How-
ever, those in K'-substituted-SW appeared to be inter-
mediate in form (Fig. 3B), compared to those in Tris- and
ChCl-substituted SWs, which were similar to each other
in retaining rounded projections (Fig. 3C, D).

The above observations of the "soft" FEs (incomplete
formation) suggested that their permeability, as well as
their morphology, might be altered. Therefore, several
measurements of permeability were undertaken.

Total protein secretion

Soluble secreted protein that leaked through the FEs
of eggs activated in normal or low Na+ SWs was measured

FERTILIZATION ENVELOPE ASSEMBLY

349

Figure 1. Phase microscopy of Strongylocentrotus piirpiiranix eggs activated in normal (A. E) and Na*
depleted SWs (B-D and F-H). A. E: Normal Na+-SW. B, F: KCl-substituted-SW. C, G: Tns-substituted-
SW. D, H: ChCl-substituted-SW. A-D: 1 min postactivation. E-H: 30 min. These micrographs were taken
focusing on the fertilization envelopes, fe = fertilization envelope; PVS = perivitelline space. Scale bar
= 50 ^m.

(Fig. 4). FPs were collected at 10 min postactivation from
eggs pooled from several females. The FPs of the normal
and K+ -substituted SW eggs had 22.7 ± 1.8 Mg and 22.2
± 5.7 Mg (Mean ± S.E.M.) of protein/ml FP, respectively.
They were not significantly different. However, the FPs
of Tris- and ChCl-substituted SWs contained 162. 1 ± 25.9
Mg and 168.7 ± 3.4 ^g- respectively. This 7- to 8-fold in-
crease over normal and KC1 was highly significant (P
< 0.0001).

Control assays demonstrated no significant difference
(95% confidence level) in TCA-precipitable BSA between
normal and low Na+ SWs. Therefore, the various SWs
had no adverse effects on the Lowry assay. In addition,
supernatant protein from unactivated eggs in DMSO was
measured and found to contribute little to the total ( ~ 1 .4
h see also Green et ai, 1990).

ft- 1 ,3-glucanase secretion

Glucanase activity was measured (in normal and ChCl
SWs) by the amount of glucose hydrolyzed from the /8-
1,3-glucan polysaccharide laminarin by egg-derived-glu-
canase in the FP (Fig. 5). Aliquots of FP of experimental
and control groups were taken at 10 min postactivation.
The glucose measurements from ChCl and normal SW
were 1.62 ± 0.19 and 0.93 ±0.19 ^moles glucose per ml
of FP, respectively. Approximately 75% more glucanase

activity was found in the FP from the ChCl eggs. This
difference was significant (P < 0.05).

Control incubations of glucose were assayed in normal
and ChCl-substituted SWs, and no significant differences
(95% confidence level) were observed. Therefore, it is un-
likely that the ChCl interfered with the glucose determi-
nation.

Ovoperoxidase secretion

Ovoperoxidase released from ChCl-SW eggs (3.72
± 0.78 ^moles guaiacol oxidized/min/ml of FP) had sig-
nificantly higher activity (see Fig. 6) than that released
from normal SW eggs (0.82 ± 0.21 jumole/min/ml). Per-
oxidase activities in KC1-SW ( 1 .09 ± 0.30 /Ljmoles/min/
ml) and Tris-SW ( 1 .68 ± 0.30 ^moles/min/ml) FPs were
intermediate between control and ChCl groups. There
were significant differences between normal and ChCl (P
< 0.001), and Tris (P < 0.05). However, Ovoperoxidase
release was not significantly different between normal and
KC1(P>0.35).

Ovoperoxidase localization

Because Ovoperoxidase is incorporated into the FE
(Somers et ai, 1989) and more enzyme activity was ob-
served in the FP of the low Na+ treatments (see above),
it is possible that the higher activity was not only due to

350

S.-D. CHENG ET AL.

Figure 2. SEM of Strongylocentrotus purpwatus egg surfaces. A. VL ofegg(unactivatedl in normal Na*-
SW. B-D. FEs of normal Na+-SW eggs at 1,3, and 60 min postactivation. E. VL of egg (unactivated) in
ChCl-substituted-SW. F-H. FEs of ChCl-suhstituted-SW eggs at 1,3, and 30 min. The microvillar projections
of the ChCl-suhstituted-SW eggs did not undergo the I-T transformation. Scale bar = 1 ^m.

FE permeability, but that the ovoperoxidase was not in-
corporated efficiently into the structure of the FE. There-
fore, DAB localization of ovoperoxidase was performed.
Comparing the TEM micrographs (Fig. 7) of FEs after
DAB incubation, the normal SW FE (Fig. 7C) is con-
spicuously darker than that of the ChCl FE (Fig. 7D).
Although both normal and ChCl FEs stained more inten-
sively than the controls (Fig. 7A, B), the intensity of stain-
ing was higher in the normal FEs. Presumably, there was
more ovoperoxidase incorporated into the FEs in normal
Na+-SW than in ChCl-substituted SW.

Discussion

Sea urchin eggs are excellent material for many bio-
logical studies because they can be harvested in large
numbers, cultured in a well-defined medium (artificial
seawater), and they develop synchronously. They are well
suited for the study of extracellular self-assembly (Kay
and Shapiro, 1985; Somers and Shapiro, 1989; Shapiro
el al., 1989). The complexity of the transition of the vi-
telline layer (VL) glycoprotein to the FE tempts one to
try to dissect the myriad of sequential processes involved.
The VL is not merely an inert cell coat, but it serves as a
template or scaffolding upon which other proteins are as-

sembled and intercalated under the influence of several
enzymes. A requisite for proper structuralization is the
presence of several ions in the seawater, e.g.. Ca2+, Mg:4
(Carroll and Endress, 1982), Cl~ (Lynn et al. 1988; Green
et al.. 1990), and Na+ (Schuel et al. 1982). In the present
study, we focused on the effects of Na+ -depletion on the
elevation and structuralization of the FE.

There is some information on the sea urchin egg during
fertilization in Na+-depleted seawater. It was found that
the fast block to polyspermy decayed concurrently with
the retardation of the depolarization of the egg plasma
membrane, a Na+-dependent process (Jaffe, 1980; Schuel
and Schuel, 1981). Additional investigations have shown
that Na+ accounts for the release of acid from the egg,
resulting in an increased intracellular pH. This increase
is necessary for increased protein synthesis, DNA synthe-
sis, and cell division (Nishioka and Cross, 1978). In re-
lation to the assembly of the FE, Schuel et al. (1982) dem-
onstrated that low Na+ (19 mM), ChCl-substituted sea-
water resulted in FEs that collapsed and failed to undergo
normal structuralization. including the I to T transfor-
mation of the microvillar casts. The inhibition of the nor-
mal hardening process was attributed to the failure of CG
structural proteins to impregnate or insert into the VL.
However, this impairment of the hardening process is dis-

FERTILIZATION ENVELOPE ASSEMBLY

2.00-1

1.5O-

1.OO-

O.5O-

351

Figure 3. TEM of Strongylocentrotus piirpuraius FEs. Eggs were fixed
15 min postactivation in the following SWs: A. normal Na+. B. K.C1. C.
Tris. D. ChCl. Scale bar = 0.5 urn.

tinct from the phenomenon of "cross-linking," in that the
latter is assayed by the disruption of FEs in urea. In their
experiments, cross-linking was not affected. Furthermore,
K+ and Li+ substituted for Na+ in normal structuraliza-
tion, while ChCl and Tris did not.

o.oo

MBL ChCl

sea water treatment

Mean +/- S.EM

Figure 5. 0-1,3-glucanase secretion. Glucanase activity released
through FEs was assayed as described in Materials and Methods. The
Mean + S.E.M. are shown for four trials. The * denotes a statistically
significant difference from the control, normal SW.

In the present study we lowered the Na+ concentration
to approximately 2 m.M and observed an earlier collapse
of the FE, 3 min as opposed to 30 min. It is not surprising
that our FEs collapsed earlier than those of Schuel et al.
( 1982). because our Na+ concentration was ten-fold less.
Additional evidence of the failure of the sodium-deficient
FEs to harden is that initially they expanded more than
the normal FEs. This greater distension may also be re-
lated to the failure of proteins to insert into the FE, thereby
raising the hydrostatic pressure in the perivitelline space
(Schuel et al., 1974; Green and Summers, 1980). However,
within a few minutes, the FE began to shrink, suggesting
that the FE was permeable to the secreted proteins, and
this allowed for a decrease in the hydrostatic pressure

200-

150-

<U 1OO

5O-

MBL KCI Tris ChCl

sea water treatment

Mean +/- S.EM.

Figure 4. Protein release through FEs. Protein concentrations were
determined by the Lowry assay. Each measurement represents the Mean
± S.E.M. of three trials. The * denotes a statistically significant difference
from the control, normal SW.

5-1

MBL KCI Tris ChCl

sea water treatment

Mean +/- S.E.M.

Figure 6. Ovoperoxidase secretion. Ovoperoxidase activity released
through FEs was measured as described in Materials and Methods. Mean
± S.E.M. are shown (n = 3-7) and * denotes statistically significant
differences from the control, normal SW.

352

S.-D. CHENG AT AL.

^»;

. . - - >,V-

L.- . • *L >-;•

* <yv*» • • '

••

;-
7C

Figure 7. Ultrastructural localization of ovoperoxidase. The pointed T-form microvillar projections on
normal Na*-FE (15 min after activation) and the rounded I-form microvillar projections on ChCl-FE are
demonstrated (A and B, respectively). After DAB localization of ovoperoxidase the FEs (C, normal Na+; D,
ChCl) stained more intensely than those of the controls (A and B) in both normal Na* and ChCl-substituted-
SW. Furthermore, the staining of the normal Na* was darker than that of the ChCl. This suggests that less
ovoperoxidase was incorporated into the FE of eggs activated in ChCl-suhstituted SW. D is a montage
showing the FE and cortex of the same egg. Scale bar = 0.5 //m.

FERTILIZATION ENVELOPE ASSEMBLY

353

within the perivitelline space. This conclusion is strength-
ened by the fact that we observed a greater (approx. eight-
fold) increase in total proteins secreted into the ambient
seawater in Tris and ChCl seawaters. Glucanase and ovo-
peroxidase secretions also increased significantly. Because
ovoperoxidase is normally incorporated into the FE
(Somers and Shapiro, 1989). the increase in soluble ovo-
peroxidase activity is probably related to both increased
permeability and decreased incorporation of this enzyme
into the nascent FE. The substitution of K+ for Na+ did
not significantly interfere with hardening (structuraliza-
tion), nor did it change protein or ovoperoxidase secretion
significantly. This observation is consistent with the nor-
mal FE formation in K+- or Li '-substituted seawaters
(Schudetal.. 1982).

The results reported herein are similar to those obtained
inCl -deficient seawaters (Lynn el al.. 1988; Green el a/..
1990). Moreover, our results are strikingly similar to those
of Battaglia and Shapiro (1988). who reported similar
findings when they inhibited egg surface transglutaminase
activity with primary amines. This similarity suggests that
both Cl and Na+ may be important for the transgluta-
minase-catalyzed early cross-linking that occurs before the
ovoperoxidase-catalyzed cross-linking. However, we can
not ignore the possibility that the observed effects may be
the result of ChCl or Tris addition, rather then exclusion
of Na+. This is a drawback to any substitution experiment.
The size of the substituted ionic species may be important
because K+ and Li+ are closer in size to Na+ than are
either Tris or ChCl. This question, too. remains to be
resolved.

Another interesting observation of these experiments
was the apparent paucity of hyalin in the perivitelline
space of eggs activated in ChCl-substituted-SW (e.g.. Figs.
3A, D and 7A, B). This observation and the increased
glucanase activity in the ambient sodium-deficient sea-
water may be related to the possibility that glucanase per-
forms its major function on the hyaline layer of the egg
(Wessel el al.. 1987) in a Na+-dependent manner. This
possibility awaits further experimentation.

Our results demonstrate that the ionic composition of
the seawater significantly influences the formation of the
FE in the sea urchin 51. purpnrutus. These results are con-
sistent with other published reports. Moreover, the phe-
nomenon of regulated extracellular assembly remains an
intriguing field of study, to which the study of sea urchin
FE formation can contribute.

Acknowledgments

The authors wish to thank Drs. Frank N. Low and
Joseph B. Delcarpio for their SEM technical assistance,
and Drs. William J. Swartz and John W. Lynn for their
suggestions regarding this paper. This work was supported

by an Edward G. Schlieder Educational Foundation Grant
toJ.D.G.

Literature Cited

Battaglia, D. E., and B. M. Shapiro. 1988. Hierarchies of protein cross-
linking in the extracellular matrix: involvement of an egg surface
transglutaminase in early stages of fertilization envelope assembly.
J. Cell Biol. 107: 2447-2454.

Carroll, E. J., and A. G. Endress. 1982. Sea urchin fertilization en-
velope: uncoupling of cortical granule exocytosis from envelope as-
sembly and isolation of an envelope intermediate from Strongy/o-
centrotus purpuratus embryos. Dev. Biol. 94: 252-258.

Cavanaugh, G. M., ed. 1956. Pp. 67-69 in Formulae and Methods of
the Marine Biological Lahoralory Chemical Room. Marine Biological
Laboratory, Woods Hole. MA.

Chambers, E. L., B. C. Pressman, and B. Rose. 1974. The activation
of sea urchin eggs by the divalent ionophores A23187 and X-537A.
Kioehem Btoi'hys. Res Coinni. 60: 126-132.

Cheng, S.-D., P. S. Glas, and J. D. Green. 1989. Enzymatic and mor-
phological analysis of fertilization envelope (FE) assembly in low
Na+ sea water. J Cell Biol 109: 128a.

Deits, T., M. Farrance, E. S. Kay, L. Medill, E. E. Turner, P. J. Weidman,
and B. M. Shapiro. 1984. Purification and properties of ovoperox-
idase. the enzyme responsible for hardening the fertilization mem-
brane of the sea urchin egg. J. Biol Client. 21: 13,525-13.533.

Foerder, C. A., and B. M. Shapiro. 1977. Release of ovoperoxidase
from sea urchin eggs hardens the fertilization membrane with tyrosine
crosslinks. Proc. Nail. Acaci Sci. USA 14: 4214-4218.

Green, J. D., and R. G. Summers. 1980. Formation of the cortical
concavity at fertilization in the sea urchin egg. DOT Growth Differ
22(6): 821-829.

Green, J. D., P. S. Glas, S.-D. Cheng, and J. VV. Lynn.
1990. Fertilization envelope assembly in sea urchin eggs inseminated
in chloride-deficient sea water: II. Biochemical effects. Mol. Reprod.
DOT 25: 177-185.

Hall, II. G. 1978. Hardening of the sea urchin fertilization envelope
by peroxidase-catalyzed phenolic coupling of tyrosines. Cell 15: 343-
355.

Jaffe, L. A. 1980. Electrical polyspermy block in sea urchin eggs: nic-
otine and low sodium experiments. DOT'. Growth Differ. 22: 503-
507.

Kay, E. S., and B. M. Shapiro. 1985. The formation of the fertilization
membrane of the sea urchin egg. Pp. 45-80 in Biology of Fertilization,
\'ol 3. The Fertilisation Response of the Egg. C. B. Metz and A.
Monroy, eds. Academic Press. Orlando.

Klebanoff, S. J., C. A. Foerder, M. Eddy, and B. M. Shapiro.
1979. Metabolic similarities between fertilization and phagocytosis.
J. Exp. Med. 149: 938-953.

Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.
1951. Protein measurement with the folin phenol reagent. J. Biol.
Chem. 193: 265-275.

Lynn, J. VV., R. L. Goddard, P. Glas, and J. D. Green.
1988. Fertilization envelope assembly in sea urchin eggs inseminated
in Cr-deficient sea water: I. Morphological effects. Gamete Res. 21:
135-149.

Nishioka, D., and N. Cross. 1978. The role of external sodium in sea
urchin fertilization. Pp. 403-413 in Cell Reproduction: In Honor of
D Mazia. E. R. Dirksen, D. M. Prescott, and C. F. Fox, eds. Academic
Press. New York.

Reynolds, E. S. 1963. The use of lead citrate at high pH as an electron
opaque stain in electron microscopy. J. Cell Biol. 17: 208-212.

Schuel, H., W. L. Wilson, R. S. Bressler, J. W. Kelly, and J. R. Wilson.
1972. Purification of cortical granules from unfertilized sea urchin
egg homogenates by zonal centnfugation. Dev. Biol. 29: 307-320.

354

S.-D. CHENG ET AL.

Schuel, H., J. \V. Kelly, E. R. Berger, and W. L. Wilson. 1974. Sulfated
acid mucopolysacchandes in the cortical granules of eggs. Effects of qua-
ternary ammonium salts on fertilization. Exp. Cell Re* 88: 24-30.

Schuel, H., and R. Schuel. 1981. A rapid sodium-dependent block to
polyspermy in sea urchin eggs. Dev. Bid/ 87: 249-258.

Schuel, H., R. Schuel, P. Dandekar, J. Boldt, and R. G. Summers.
1982. Sodium requirements in hardening of the fertilization en-
velope and embryonic development in sea urchins. Bio/. Bull 162:
202-213.

Shapiro, B. M., C. E. Somers, and P. J. \\eidman. 1989. Extracellular
remodeling during fertilization. Pp. 25 1-276 in The Cell Biology of
Fertilization, H. Schatten and G. Schatten. eds. Academic Press. San
Diego.

Somers, C. E., D. E. Battaglia, and B. M. Shapiro. 1989. Localization
and developmental fate of ovoperoxidase and proteoliaisin, two pro-

teins involved in fertilization envelope assembly. Dev. Biol. 131: 226-

235.
Somers, C. E., and B. M. Shapiro. 1989. Insights into the molecular

mechanisms involved in sea urchin fertilization envelope assembly.

Dev. Growth Difj. 31(1): 1-7.
Steinhardt, R. A., and D. Epel. 1974. Activation of sea urchin eggs

by calcium lonophore. Proc. Nail. Acad. Sci. I'SA 71: 1915-

1919.
Weidman, P. J., E. S. Kay, and B. M. Shapiro. 1985. Assembly of the

sea urchin fertilization membrane: isolation of proteoliaisin. a cal-
cium-dependent ovoperoxidase binding protein. J. Cell Biol. 100:

938-946.
Wessel, G. M., M. R. Truschel, S. A. Chambers, and D. R. McClay.

1987. A cortical granule-specific enzyme, /i-l,3-glucanase, in sea

urchin eggs. Gamete Res 18: 339-348.

Reference: Biol Bull. 180: 355-371. (June. 1991)

Embryonic Development of the American Lobster

(Homams americanus): Quantitative Staging and

Characterization of an Embryonic Molt Cycle

S. M. HELLUY AND B. S. BELTZ
Department of Biological Sciences. Wellesley College, Wellesley, Massachusetts 02181

Abstract. The growth of a single brood of lobsters
(Homarus americanus Milne-Edwards 1837) maintained
at constant temperature is studied from the naupliar stage
to hatching, and the sequence of appearance of morpho-
logical, anatomical, and behavioral characteristics ob-
served. A percent-staging system based upon Perkins' eye
index (1972) is presented, and ten equally spaced embry-
onic stages are illustrated and characterized at different
levels of resolution: whole eggs, dissected embryos, an-
tennulae and telsons. The tegumentary and setal changes
in the telson show that a complete molt cycle takes place
in the egg starting at about 12% embryonic development
(El 2%) with the molt of the nauplius into the metanau-
plius and ending just after hatching when the metanau-
plius molts into a first stage larva (LI, first zoea). At E30%,
the cuticle begins to separate from the setae in the telson;
this signals the start of Orach's (1939) stage D0 of the
metanaupliar embryonic molt cycle. At that time, the first
sign of organogenesis of the LI, the formation of the en-
dopod of the antennulae, becomes visible; presumed sen-
sory neurons and their axons are observed at the tip of
the exopod of the antennulae where a giant sensillum is
differentiating. During D0 the setae of the first larval stage
are forming proximally and medially in the bilobed telson
under the metanaupliar cuticle. At E90%, these setae are
retracting, and the embryo has entered stage D,. After
hatching (£100%), the telson of the free metanauplius
(prelarva) shows the characteristics of stage D:_3 and ec-
dysis soon follows. The arrested development observed at
constant temperature in the experimental brood occurred
at stage D0 of the metanaupliar molt cycle, whereas de-
velopment was resumed as the embryos entered stage D, .
These changes in developmental pace from D0 to D! in
the embryonic molt cycle are parallel to those occurring

Received 3 December 1990; accepted 8 March 1991.

in older lobsters (Aiken, 1973). The quantitative staging
of lobster development from extrusion to hatching, and
the description of the embryonic molt cycle will facilitate
future investigations on particular aspects of the embryo-
genesis of Homarus such as neural differentiation.

Introduction

Studies on lobsters and other crustaceans have made a
significant contribution to our understanding of neural
organization and the control of behavior (see Wiese el ai.
1990). There is increasing interest in examining the on-
togenesis of particular behaviors and the cellular archi-
tecture that is the basis for those behaviors (Kravitz, 1988;
Govind. 1989; Sandeman and Sandeman, 1990). Re-
search on neural development at the embryonic level in
Homarus is flourishing (Cole and Lang. 1980; Beltz and
Kravitz, 1987; Beltz el a/.. 1990; Helluy and Beltz, 1990;
Meier and Reichert, 1990), but progress has been limited
by the lack of adequate documentation on the general
development of this organism in the egg, as well as by the
absence of a staging system for the total embryonic period.
These two problems are addressed in this paper.

Recent developmental studies in Homarus have dealt
primarily with the perihatching period (Davis, 1964; En-
nis, 1975; Charmantier and Aiken, 1987), and larval and
postlarval life (Phillips and Sastry, 1980; Charmantier,
1987), whereas most of the literature concerned with the
prehatching period dates back to the nineteenth century
(Bumpus, 1891; Herrick, 1895). The latter studies are a
remarkable achievement of patient and detailed obser-
vation and are illustrated by elegant drawings (Herrick,
1895), but the modern microscopic and photographic
methods used in this study are necessary to provide added
resolution. The nineteenth century studies also tend to
focus on early embryogenesis while providing little or no
information about middle and late development in the

355

356

S. M. HELLUY AND B. S. BELTZ

egg, and the embryonic molt cycle. A deeper knowledge
of lobster embryology could also provide more insight
and understanding of studies that examine particular as-
pects of development, such as the influence of temperature
on growth rate (Templeman. 1940; Perkins. 1972). pop-
ulation dynamics (Schuur et al. 1976; Hepper and Gough,
1978), the chemical composition and calorific content of
the eggs (Pandian 1970a, b; Sasaki, 1984; Sasaki et al.
1986), or the differentiation of particular organs or sys-
tems, such as heart and gut (Burrage, 1978; Burrage and
Sherman, 1979), and, again, nervous system.

The principal features involved in the reproduction and
early development of the lobster Homarus americanus
are well known. After copulation, spermatozoa are stored
by the female for several months until oviposition and
fertilization occur (Aiken and Waddy, 1980). In New
England waters, egg development spans about 10 months,
from egg extrusion in July or August to hatching the fol-
lowing May or June (Bumpus, 1891; Herrick, 1895). Fol-
lowing extrusion, the eggs are carried on the abdomen of
the mother, attached to the pleopods. Homarus has rel-
atively large, telolecithal eggs. Superficial cleavage leads
to the formation of a blastoderm, and the central mass
of yolk remains undivided (Bumpus. 1891). After only a
few days, the naupliar organization is apparent. The nau-
plius, which is a developmental hallmark of crustaceans,
is characterized by the presence of a median eye and three
pairs of appendages: the antennulae, antennae, and man-
dibles (Shiino, 1988). The metanauplius arises from the
differentiation and growth of the postmandibular ap-
pendages. Homarus hatches as a mature metanauplius
(prelarva. prezoea) that molts rapidly into the first larval
stage (Davis, 1964). There are three pelagic larval stages
swimming with the feathery exopodites of six pairs of tho-
racic limbs. A metamorphosis leads to the formation of
a postlarva (fourth stage) with most of the adult charac-
teristics (Charmantier, 1987). The postlarva, which swims
in a fully extended posture using its pleopods. later settles
on the substrate. The duration of larval life, in the order
of a few weeks, depends largely on temperature.

For the present study, behavioral, morphological, an-
atomical, and morphometric data were gathered from
whole eggs and dissected embryos. A percent-staging
scheme using the size of the pigmented area in the lateral
eyes [the eye index (Perkins, 1972)] was adopted. Sub-
sequently, ten equally spaced developmental stages were
documented in detail with the eggs of different females.
Particular attention was given to the growth and differ-
entiation of the antennulae and telson. The antennulae,
which are lined with aesthetascs (olfactory sensilla) in
postembryonic animals from second larval stage on, were
examined to gain insight into the ontogeny of the olfactory
sensory apparatus. The telson was studied to elucidate
how the round bilobed telson of the embryo is transformed
into the triangular telson of the first larval stage.

Materials and Methods

Lobster and egg maintenance

Egg-bearing female lobsters Homarus americanus
(Crustacea, Malacostraca, Decapoda, Reptantia, Asta-
cidea, Nephropidae) were obtained from the Massachu-
setts State Lobster Hatchery on Martha's Vineyard, Mas-
sachusetts, and kept in recirculating artificial seawater. In
addition, eggs detached from the mother's abdomen were
provided by the New England Aquarium in Boston, Mas-
sachusetts, where lobsters were reared in filtered, temper-
ature-controlled seawater. These detached eggs were
maintained in our laboratory in free-floating net enclo-
sures in artificial seawater. We found that hanging the
clumps of eggs with surgical thread, and allowing them
to float, led to good survival rates. Three tanks were
maintained at temperatures of 10 ± 2°C, 18 ± 2°C. and
20 ± 2°C, to slow or accelerate the rate of development
of the eggs, at a salinity between 27 and 32 ppt in a 12:
12 lightdark cycle.

The experimental brood

We have not had any success promoting egg extrusion
in females held in recirculating tanks, probably because
of the variety of complex environmental factors necessary
for this event (Waddy and Aiken, 1984). Therefore, in
mid-October, the egg-bearing female containing the
youngest eggs was chosen from a collection of approxi-
mately 200 gravid females collected by fishermen for the
State Lobster Hatchery. The earliest stage observed in the
experimental brood was a cleavage stage. The approximate
date of extrusion was calculated as follows: in Temple-
man's (1940) experiments, the period from the late nau-
plius to the first appearance of pigment in the lateral eyes
(26 days) lasted about 45% of the time required for the
development from extrusion to appearance of eye pigment
(58 days) at 12-13°C, and 41% (11/27) in Herrick's ex-
periments (1895, p. 56) at 21°C. In the lobster (Temple-
man, 1940; Perkins, 1972) and in insects (Bentley et al.,
1979), developmental events are more condensed or ex-
panded in time depending on temperature, but the pro-
portion of the total duration of embryogenesis devoted to
each developmental event does not change with temper-
ature. In the present study at 18°C, the development from
late nauplius to the first appearance of eye pigment took
9 days; therefore, by extrapolation from the data of Tem-
pleman (1940) and Herrick (1895), the period from ex-
trusion to first appearance of eye pigment would be pre-
dicted to last 20-22 days. Thus, the estimated date of
extrusion was calculated to be 21 days prior to the ap-
pearance of eye pigment. Note that extrusion did occur
in the wild in water at seasonal temperatures.

Observations were made on the experimental brood
kept at 18 ± 2°C for five months (mid-October to mid-

EMBRYONIC DEVELOPMENT OF THE LOBSTER

357

March, see Table I). The female died in mid-January,
when the eggs were at 66% development: she was stripped
of eggs and the spawn was suspended in nets in the tank.
The eggs were agitated daily to try to replace the vigorous
beating of the pleopods of the mother. In the experimental
brood, the majority of the eggs attained the hatching stage
but very few actually hatched into free metanaupliae; still
fewer molted into first larval stages. Those larvae that did
emerge were perfectly normal animals. The smoothness
of growth curves of the experimental brood (see Results)
and numerous observations on the progeny of other fe-
males confirmed that the free eggs of the experimental
brood followed a normal course of development after the
death of the mother.

Five live eggs from the experimental brood were ex-
amined every two or three days for the first two months,
then once a week until hatching. As soon as the heart was
formed, the heart beat was confirmed in each embryo to
ensure that the observed eggs were alive. During each ob-
servation period, the width and length of the pigmented
area in the lateral eyes (Fig. 1 ) and the greatest axis of the
egg were measured in intact eggs: following dissection, the
length of the cephalothorax was measured ventrally from
the median eye to the anterior margin of the abdomen
(Fig. 2). Behavioral observations, such as antennal
twitching or tail flipping during dissection, were also
noted. Photographs of whole eggs and dissected embryos
were taken with a Zeiss stereomicroscope.

Developmental staging system

A developmental scale was designed that used the eye
index (Perkins, 1972) as a marker of developmental pro-
gress. The eye index is defined as the average of the length
and the width of the brown screening pigment spot (in
micrometers) in the lateral eyes. The first measurable eye
pigment spot had an eye index of 70 ^m (Perkins, 1972;
present study). Therefore, development prior to the ap-
pearance of eye pigment was characterized using time
rather than the eye index. The estimated duration of de-
velopment of the experimental brood was 159 days (Table
I). Eye pigment first appears at 13.2% of the total time
from extrusion to hatching, while the eye index at first
appearance of pigment (70 jum) is 12.2% of the eye index
of the experimental brood at hatching (578 /^m) (Table
I). These values indicate that there is little difference during
early embryogenesis between staging based on time and
that based on the eye index; time-staging was used prior
to, and eye index-staging after 1 5% development (Table
I). Later in embryogenesis, because of the period of de-
velopmental arrest (see Results), staging based upon time
is no longer valid; the morphometric marker (eye index)
must then be used.

Table I

Dales of observation ot the experimental brood ol Homarus
amencanus maintained at 1S°C. age. eye index, and
percent- staging svstem. The dotted line signals the transition between
percent of total lime from extrusion to hatching and percent ol eye
index at hatching

Date
8-89

Embryonic
age (days)

Eye index
(Mm)

Stage
(%)

1
1

1
1
1
]

0-08
0-16
0-18
0-20
0-23
0-25
0-27
0-29

(I
8
10
12
15
17
19
21

70.6

0
5.3
6.3
7.6
9.4
10.7
12.0
13.2

0-31

72.5

14.5

1-02

98.0

17.0

1-04

139.2

24.1

1-06

145.0

25.1

1-08

156.8

27.1

1-1 1

160.7

27.8

1-18

213.6

37.0

1-24

248.9

43.1

2-01

282.2

48.8

2-08

307.7

53.2

2-15

329.3

57.0

2-23

352.8

61.0

2-29

366.5

63.4

(

M-05

386.1

66.8

(

H-12

382.2

66.1

(

)1-19

103

425.3

73.6

H-26

110

447.0

77.3

32-02

117

441.0

76.3

J2-09

124

458.6

79.3

32-16

131

466.5

80.7

32-23

138

474.3

82.1

33-02

145

474.3

82.1

33-09

152

542.9

93.9

33-16

159

578.2

100.0

Characterization often embryonic stages and oj the
embryonic molt cycle

Following the adoption of the percent-staging system,
eggs from different broods were studied in more detail at
every 10% increment in development. The eye index at
hatching was estimated at 570 ± 20 Mm (see Discussion),
and therefore each 10% increment in developmental ma-
turity was characterized by an increase of 57 /*m in the
eye index. The stage described as 10% was reached on
day 16 in the experimental brood: the late egg-nauplius.
Eggs were also examined when their eye indices measured
1 14 ± 6 ^m (E20%), 171 ± 4 Mm (E30%), 228 ± 7 urn
(E40%), 285 Mm ± 14 (E50%), 342 ± 1 1 M™ (E60%), 399
± 17 ^m (E70%), 456 ± 5 Mm (E80%), 513 ± 20 Mm
(E90%), 570 ± 20 Mm (£100%). The varying range for
each stage reflected embryo availability and limitations

358

S. M. HELLUY AND B. S. BELTZ

imposed by the precision of the ocular micrometer.
Twenty micrometers represents 3.5% of the total devel-
opment scale. To characterize each of the ten stages and
the early postembryonic stages, the same protocol de-
scribed earlier for the experimental brood was used. Pho-
tographs of whole eggs (Figs. 3, 4) and dissected embryos
(Fig. 5) were taken with a Zeiss stereomicroscope. In ad-
dition, antennulae (Fig. 6) and telsons (Fig. 7) were also
severed, examined fresh, and photographed using a Zeiss
IM35 photoinvertoscope equipped with modulation con-
trast optics (Hoffman, 1977).

Drach (1939) and Drach and Tchernigovtzeff (1967)
designated the phases of ecdysis in crustaceans by letters
from A to E: A and B are the postmolt periods, C the
intermolt, D the premolt period, and E the ecdysis proper.
In the present study, this system was used to characterize
the molt cycle of Homarus that occurs within the egg
envelopes. The period of the embryonic molt cycle was
determined by matching setal changes in the telson of
embryos (Figs. 7 and 8) with the setal changes in the telson
previously documented in larvae of Homarus americanm
(Rao el al., 1973; Sasaki, 1984) and in the pleopods in
juveniles (Aiken, 1973; 1980) during molt cycles. The
subdivisions of stage D (D0, D,, D:_,) and their distin-
guishing features are those described by Sasaki (1984).

Terminology

More than 70 terms have been used to refer to the
various embryonic and larval stages of decapods (Gore,
1985). The form that arises from the differentiation and
growth of the postmandibular appendages in the Amer-

ican lobster egg has been called a "post-nauplius" by Her-
nck ( 1 895) or "postnauplius" by Helluy and Beltz ( 1 990).
In the present study "metanauplius" is used, a term com-
monly assigned to the form developing just past the nau-
pliar stage (Wear, 1974; Williamson. 1982;Shiino, 1988).
The form that is released from the egg envelopes and rap-
idly molts into the first larval stage is usually called a
"prelarva" or "prezoea"; however, the term "mature me-
tanauplius" seems more biologically relevant (see Dis-
cussion). The first larval stage of Homarus is sometimes
referred to as a "mysis" (Shiino, 1988) based on the num-
ber of its appendages, or a first "zoea" because it locomotes
with its thoracic appendages (Anderson, 1982; William-
son, 1982). Finally, "embryogenesis," "prehatch," and
"egg development" are used interchangeably, although
the latter part of egg development in Homarus is devoted
to larval organogenesis rather than to embryogenesis
strictly defined.

Results

/. Timing of development, sequence of events,
and characterization of embryonic
and early postembryonic stages

In the following account of the embryogenesis of Hom-
arus americanus, the sequence of developmental events
and morphometric data on eye index (El) and cephalo-
thoracic length (Figs. 1, 2) were obtained by studying the
experimental brood, whereas the illustration and char-
acterization of equally spaced embryonic and early post-
embryonic stages was achieved by studying many clutches

Eye

index (um) % developmen

600

TIOO

•A""

500

: $ -

T I a £ so

400

gnC.'

gS * eo

,/*

300

I1*1 i'

1°

'''' /''',,

9 40 .

200

//''/

•''''/' '''''''

100

: f '-

1;^

i i i i § i i i

'"•

Age (days)

20 40 60 80 100 120 140 160 180

Metaiiaupliar Molt

Stage D2 -3 and hatching

Haemolymph blue

Setae invaginating in telson

Stage Di

(15+15) setae on telson

Yolk in 1/2 of egg volume

Tailflips

Eyes oval

Stage Do

Red pigment

Intestinal granules

Heart beats

Eye pigment

Naupliar molt

(6+6) setae on telson

Twitches

Fertilization

5
o. c

03 <U
~ tfl
O, *

Figure I . Eye index versus age of the experimental brood of Homarus americanus. maintained at 1 8°C.
Developmental landmarks are indicated along a percent-scale based on the eye index. Perkins' eye index
(1972) is the mean of the length and the width of the screening pigment spot in the lateral eyes. Each data
point represents the mean of measurements on five individuals ot the experimental brood ± one standard
deviation.

EMBRYONIC DEVELOPMENT OF THE LOBSTER

359

2.4
2.2

- Q Egg
i Cephalothorax

2.0

: B i|I

1.8
1.6
1.4
1.2
1.0

;e^°D° ftf11
; ^ ^*r

0.8

i*1

0.6

: I1

0.4

~ *

0.2
00

- EI (|im)

100 200 300 400 500 600

(

) 10 20 30 40 50 60 70 80 90 100

% Development

Figure 2. Greatest axis of egg and cephalothoracic length versus eye
index and percent-development scale, in Homarits americanus. Greatest
axis of egg: each data point represents the mean of five measurements
± one standard deviation (data points from different broods). Cephalo-
thoracic length: the length of each bar represents one standard deviation
on each side of the mean of five measurements (all individuals from
experimental brood).

of eggs at different levels of resolution [whole eggs (Figs.
3, 4), dissected embryos (Fig. 5), antennulae (Fig. 6), and
telsons (Figs. 7, 8)]. The stage of appearance of develop-
mental events is expressed in percent-development of total
embryogenesis. The percent-staging scheme is explained
in "Materials and Methods." Dates of observation of the
experimental brood, age, eye index, and percent-staging
system are related in Table I and in Figure 1 . The metan-
aupliar molt cycle is described in the second part of "Re-
sults."

Sequence of events prior to 10% development. The first
organized structure to appear at the surface of the green
yolk at 5% development (E5%, estimated day 8 of the
experimental brood) is a typical crustacean nauplius with
three pairs of appendages: the antennulae, the antennae,
and the mandibles. The mandibles are first visible as two
dots medial to the endopods of the antennae. This is
equivalent to stage "M" of Bumpus with the eye lobes
and the thoracoabdominal process still undefined. At E6%
(day 10), the optic lobes appear as a white cloud of cells,
and the thoracoabdominal process is clearly outlined. At
E8% (day 12, stage "N" of Bumpus) the optic lobes are
also delineated, and the embryo is easily separated from
the yolk. The dorsal side of the embryo is apposed to the
yolk, and the abdomen grows folded on the ventral side
of the thorax. The tip of the abdomen reaches the level
of the mandibles and is beginning to part medially at E9%

(day 15, stage "O" of Bumpus); the buds of four pairs of
post mandibular appendages line the trunk.

70% development (no eye pigment present: Figs. 3.4,
5A, 6A, 7 A). At E10%, the yolk occupies approximately
95% of the volume of the egg (Fig. 3A) whose greatest
axis measures about 1.6 mm. The antennulae are unira-
mous and end with five setae whereas the antennae are
biramous with five setae at the tip of the exopods and
three setae at the tip of the endopods (Fig. 6A). The ex-
tremity of the abdomen nearly reaches the level of the
labrum; the telson is beginning to part but setae are not
yet present at its tip. This stage is equivalent to stage "O"
of Bumpus (1891).

Sequence of events from 10% to 20% development. At
El 1% (day 17) at least 8 appendages are formed past the
mandibles and at about this time, the first twitches in the
two pairs of antennae occur upon dissection. Pigment is
then visible in the median eye under the compound mi-
croscope. At about E 1 2%, when pigment is already visible
in the median eye but not yet in the lateral eyes, an em-
bryonic molt occurs (see II, below). At least two envelopes
surround the antennulae (Fig. 6B). and one envelope is
stretched at the tip of the six setae (6 + 6) on each side
of the telson indicating the occurrence of a molt (Figs.
7B, 8A). At about El 3% (day 21), pigment is seen in the
lateral eyes as a small dark crescent lining the posterior
part of the lobes; the eye index at that stage is approxi-
mately 70 jum. At El 4% (day 23), the first heartbeats are
seen in approximately 10% of the embryos examined. By
El 7% (EI 98) heartbeats are seen in half of the eggs, and
small refringent granules are present in the intestine.

20% development (EI = 114 fim; Figs. 3B, 5B, 6C. 7C).
Muscular twitches are readily observed in the antennae,
around the mouth, and in the abdomen, and the heart is
beating. Between 10 and 20% of embryonic development,
the nauplius has molted into a metanauplius (see II, be-
low). This stage is equivalent to stage "P" of Bumpus
( 1 89 1 ), defined as the stage when the tips of the third pair
of maxillipeds reach the point of insertion of the anten-
nulae and the telson reaches the level between the mouth
and the median eye. The telson is provided with 6-7 setae
on each of the two lobes (Fig. 7C).

Sequence of events from 20% to 30% development. At
E24% (EI 139), red pigment spots (chromatophores) ap-
pear on each side of the brain. At E25% (EI 145) the
telson reaches the anterior edge of the optic lobe (stage
"Q" of Bumpus). The full complement of appendages
present in the metanauplius is formed by E27% (EI 157),
and by E28% (EI 161 ) the telson reaches beyond the optic
lobes.

30% development (EI = 171 ^m; Figs. 3C. 5C. 6D,
ID). The cephalothorax of the embryo is nearly 1 mm
long. Red chromatophores are present on each side of the
brain. A cluster of presumed sensory neurons has formed
in the exopod of each antennula and their axons follow

360

S. M. HELLUY AND B. S. BELTZ

EMBRYONIC DEVELOPMENT OF THE LOBSTER

361

the anterior edge of these appendages in a bundle of a few
micrometers (Fig. 6D). Serial plastic sections have shown
that the bundle of axons projects to the olfactory lobes
(in the deutocerebrum), which measure about 40 nm in
diameter at that stage (unpub. results). The cluster of neu-
rons is very similar to the cluster of bipolar sensory neu-
rons that innervate each aesthetasc (olfactory sensillum)
in the antennulae of spiny lobsters (Griinert and Ache,
1988). At E30% also, the endopod of each antennula
tipped with a pointed seta, is visible under the cuticle (Fig.
6D). The endopods are freed after hatching when the me-
tanauplius molts into a first larval stage (LI). The ap-
pearance of the endopod of the antennulae is the first
visible sign of the formation of the LI under the cuticle
of the metanauplius. All postmandibular appendages are
present and the trunk is lined with six pairs of prominent
appendages: a pair of third maxillipeds and five pairs of
walking legs. During the metanaupliar phase, the trunk
appendages, which are uniramous, cannot be separated
from each other. The tips of the third maxillipeds reach
a level between the point of insertion of the antennulae
and the anterior edge of the optic lobes whereas the telson
is at the level of the anterior edge of the optic lobes [stage
"Q" of Bumpus (1891)]. In the telson, the metanaupliar
cuticle begins to separate from the side of the setae but
the tip of these setae is still attached to the cuticle (Fig.
7D); this signals the start of the premolt stage Dn of the
metanaupliar molt cycle (see II, below).

Sequence of events from 30% to 40% development. Stage
"R" of Bumpus is reached between E30% and E37% when
the tip of the third maxillipeds is at the level of the an-
tennae and the telson grows beyond the optic lobes. By
E37%, the eye pigment spots have become oval rather
than crescent-shaped, and red pigment granules line the
sides of the nerve cord.

40% development (El = 228 urn; Figs. 3D. 5D, 6E, IE).
A giant sensillum (260 /urn) is visible as a long straight
rod inverted at the tip of the exopod of each antennula.
Setae are present at the extremities of trunk appendages.
Red chromatophores are seen on the sides of the nerve
cord, on the anterior edge of the optic lobes, and on the
growing carapace. The third maxillipeds reach the anterior
edge of the optic lobes, and the telson reaches anteriorly
to the optic lobes. This stage is more advanced than stage
"R." the most advanced stage described by Bumpus
(1891).

Sequence of events from 40% to 50% development. The
red chromatophores have invaded the appendages and
the growing carapace by E43% and the abdomen by E49%.

50% development (El = 285 urn; Figs. 3E, 5E, IF). By
E50%, some embryos perform very clear tailflips after re-
moval of egg envelopes; also, the first caeca of the paired
digestive glands (hepatopancreas) are seen, with the
stereomicroscope, at the anterior end of the midgut where
it comes in contact with the mass of yolk. The rostrum
of the differentiating LI is folded ventrally between the
optic lobes and is visible upon dissection. The gap between
the cuticle and the six or seven most distal and lateral
setae on each side of the telson has widened, but the tips
of these setae are still in contact with the cuticle (Fig. 7F).
Other setae are growing more medially and more proxi-
mally beneath the cuticle of the telson. By now, the distal
ends of the third maxillipeds, as well as the telson, reach
anteriorly to the level of the optic lobes.

Sequence of events from 50% until hatching. There are
no obvious changes in the general external morphology
of the embryo from E50% until hatching (£100%). How-
ever, the embryo grows dramatically and the structures
typical of the LI are forming progressively beneath the
cuticle of the metanauplius.

60% development (El = 342 urn: Figs. 3F. 5F, 6F, 7G).
At this stage the yolk occupies about half the volume of
the egg. At least 1 0 setae are formed on each side of the
telson (Fig. 7G).

70% development (El = 399 n>n: Figs. 3G, 5G, 7H.
8B). The full complement of setae ( 14 or 1 5 on each side)
of the first larval stage is present on the telson under the
metanaupliar cuticle; the median spine begins to differ-
entiate (Figs. 7H, 8B).

50% development (El = 456 urn: Figs. 3H, 5H, 6G,
71). In the telson, only the most medial setae are in contact
with the metanaupliar cuticle. These setae have not yet
assumed the shape of spines, and the embryos are still in
stage DO of the metanaupliar molt cycle (Fig. 71).

90% development (El = 513 \im; Figs. 4A, 51, 6H, 7J.
8C). The egg is now enlarging rapidly, and its largest axis
measures about 2.0 mm (Fig. 2). The yolk is turning yellow
(Fig. 4A). The telson manifests a number of dramatic
changes (Figs. 7J, 8C). The cuticle has lifted entirely from
the setae and also from the epidermis on the lateral sides
of the telson. The two most lateral setae are now pointed
and sharp like spines, and they begin to retract. About a
third of each seta is visible beneath the tegument. The

Figure 3. Unfixed, intact eggs of Homarus americanus at (A) 10, (B) 20, (C) 30, (D) 40. (E) 50, (F) 60,
(G) 70, and (H) 80% embryonic development. The figures in the lower left corners refer to the percentage
of development. In all photographs, the dorsal side is at the top, and the head and telson of the embryo are
on the right. At 10% development (E10%), the embryo is seen as a small halo at the bottom part of the egg.
The eye pigment is visible in the lateral eyes (le) by E20%. The red chromatophores (ch) already present by
E40% are labeled at E60%. The intestinal granules (ig) are particularly clear at E70%. Scale bar: 500 /jm.

362

S. M. HELLUY AND B. S. BELTZ

Figure 4. Perihatching development of Homarus americanus. In all these photographs of unfixed spec-
imens, dorsal side is at the top, and anterior is right. (A) 90% embryonic development. (B) Embryo just
prior to hatching (100%, blue embryo): note the blue tinge of the hemolymph, the blue stomach (st) and
the red chromatophores (ch) in which the pigment is still concentrated. (C) Hatchling: the outer (oe) egg
envelope has burst, and the telson (te) is piercing the inner egg envelope; the red pigment has spread in the
star-shaped chromatophores (ch). (D) The metanauplius (prelarva, prezoea) is now free of both outer (oe)
and inner (ie) egg envelopes. (E) Early first larval stage (first zoea): the exuvia (ex) of the metanauplius has
been sloughed. (F) Mature first larval stage: rostrum, abdominal spines, and other acuminate structures are
now erect. Scale bars: 500 nm.

EMBRYONIC DEVELOPMENT OF THE LOBSTER

363

epidermis forms papillae around each seta, and appears
scalloped. Retraction of setae and scalloped epidermis are
characteristic of stage D, (Sasaki, 1984).

100% development (El = 570 urn; Figs. 4B. 7K). At
this stage, just prior to hatching, the egg (2.2 mm) is
brightly colored (Fig. 4B). The stomach is deep blue and
the hemolymph pale blue. The yolk, which has been nearly
entirely absorbed, is yellow or pale green. The two pairs
of yolk caeca that were filling the egg earlier are attached
to the digestive tube dorsally by this time, between the
pyloric stomach and the numerous tubular digestive
glands. The bilateral spines of the telson are entirely re-
tracted, whereas the setae are only partially so.

Eclosion of the metanauplius (fiatcliling) (Figs. 4C, 61,
7L, 8D). The outer egg envelope has burst. The red pig-
ment disperses in star-shaped chromatophores (Fig. 4C).
The giant sensillum is everted and projects from the exo-
pods of the antennulae, but is still confined within the
cuticle of the metanauplius (Fig. 61). The spines and setae
of the telson begin to expand (Figs. 7L and 8D). The epi-
dermis becomes very distinct and forms a pronounced
bulging around the invaginated setae; these are two char-
acteristics of stage D:_3 of Sasaki (1984).

Free metanauplius (prelarva, prezoea; Fig. 4D). The
metanauplius is freed of the two external egg envelopes;
it is mostly still, but occasionally performs strong tail-
flips. These movements presumably facilitate the molting
process. Within hours after the egg membranes are shed,
the metanauplius molts into a first larval stage.

Molt of the metanauplius and emergence of first larval
stage (Figs. 4E and F, 6J. 7M). The exuvia peels away
from the metanauplius. Slowly the rostrum, the abdom-
inal spines, and all the other acuminate structures become
erect. The telson opens like a fan into a triangular structure
(Fig. 7M). The setae evaginate. The endopod of each an-
tennula is released (Fig. 6J), as well as the feathery exo-
podites of the six pairs of thoracic appendages. Each an-
tennula at hatching has one giant sensillum (550 /*m) and
three setae at the tip of the exopod, and one seta at the
tip of the endopod ( Fig. 6J) as reported by Herrick (1895).
In brief, the curvaceous metanauplius molts into an
angular larva ready to assume a pelagic life.

//. Metanaupliar embryonic molt cycle, growth curves
and developmental plateau

Metanaupliar embryonic molt cycle. At about El 2%,
an envelope is seen enshrouding the telson (Figs. 7B and
8A), stretched at the tips of the 12 (6 + 6) bilaterally paired
setae on the telson of the nauplius. This envelope is
thought to be the exuvia of the naupliar stage; it is flat
and was formed in the nauplius when the tip of the ab-
domen had not yet acquired any setae. The metanauplius
that emerges at the naupliar molt has been forming during
the naupliar stage. From this molt until the emergence of

the first larval stage after hatching (metanaupliar molt),
a complete molt cycle is observed in the setal changes of
the telson. The cuticle begins to lift away from the telson
at about E30% (Fig. 7D) when the metanauplius enters
stage DO . The shape of the 6 + 6 setae on the telson of
the metanauplius is well defined on this cuticle. Setae form
then medially and proximally, and by E70% (Fig. 7H,
8B), the full complement of setae of the first larval stage
(15 + 15) is formed. Between E80% (Fig. 71) and E90%
(Fig. 7J), dramatic setal and tegumentary changes occur
as the metanauplius enters stage D, . Particularly striking
is the transformation of the most lateral setae into straight
and sharp spines (compare Figs. 71 and J, 8B and C).
These spines are invaginating as well as all the setae. In
addition, the epidermis becomes scalloped, and the cuticle
lifts from the sides of the telson. Just prior to hatching
(E100%), retraction of spines and setae is maximal. When
the outer egg envelope ruptures, the bulging of the epi-
dermis around the setae is pronounced, and the metanau-
plius has entered stage D2_3. After hatching, the metanau-
plius molts into the first stage larva and the cuticle which
is discarded still has the typical metanaupliar shape with
the imprint of the (6 + 6) metanaupliar setae.

Growth cun'es and developmental plateau. The greatest
axis of the egg increases gradually from 1.6 mm at E10%
to about 1.8 mm at E80% and more rapidly to 2.2 mm
at hatching (Fig. 2). In the experimental brood raised at
1 8°C, both the eye index (Fig. 1 ) and the cephalothoracic
length showed a logarithmic growth from the first time
these variables could be measured to approximately day
110. In Figure 2, cephalothoracic length appears linear
because it is expressed as a function of the eye index. The
eggs of the experimental brood reached a developmental
plateau at an eye index of about 474 (E82%). Until this
stage, development of the eggs was synchronous with little
interindividual variability within the brood. This was
shown by the low standard deviation of the eye index and
of the cephalothoraxic length (Figs. 1, 2). Until 82%, all
eggs were "green" and taken at random. However, after
E82%, the population was no longer homogeneous, and
the naked eye could distinguish by size and color three
categories of eggs: "green," "yellowish" (Fig. 4A), and
"blue" (Fig. 4B). In addition, the eggs hatched over a pe-
riod of about a month, again indicating significant vari-
ability between eggs in a single brood. After E82% it was
no longer possible to choose eggs randomly for observa-
tion; to assign an approximate age to each of these stages,
"yellowish eggs" were examined on day 152 when the
majority of eggs had reached this stage, and "blue eggs"
(the hatching stage) were examined a week later. Indi-
vidual eggs took about two weeks to change from "green"
to "blue" eggs at 18°C.

Discussion

In the present paper, different aspects of the embryonic
development ofHomams americanus are examined from

EMBRYONIC DEVELOPMENT OF THE LOBSTER

365

the formation of the naupliar stage until the emergence
of the first larval stage. In the discussion that follows, the
percent-staging scheme presented is compared to that used
in other invertebrate systems, and anatomical and mor-
phological observations of earlier authors are related to
that staging system. The developmental plateau in the
growth curve of the eggs is discussed in the context of the
embryonic molt cycle. The occurrence of embryonic molt
cycles in other crustaceans is reviewed and the significance
of the prelarva debated.

Staging system

Embryonic studies on invertebrates have used staging
systems based upon particular developmental events, and
arbitrary notations such as letters or figures (Bumpus,
1891;FigueiredoandBarraca, 1963; Fernandez. 1980) to
demarcate stages. Nevertheless, when intermediate stages
are likely to be needed, a continuous rather than incre-
mental staging system, and in particular a percent-staging
system, is more flexible and communicable (Bentley et
a/.. 1979). In addition, a percent-staging method takes
into account the entire embryonic life of the organism
without ignoring periods when no particular biological
events seem noticeable. For example, Bumpus (1891),
Herrick (1895), and Templeman (1940) observed Horn-
arm eggs only until the lateral eye pigment spots became
oval, about 40% embryonic development in the present
study.

Age in warm-blooded animals is generally a good in-
dicator of the stage of development, but in invertebrates
the rate of development is strongly dependent on tem-
perature. To circumvent this problem, some methods have
relied on a percent-staging scale of total embryonic time
(Schistocerca: Bentley et ai. 1979; Helisoma: McKenney
and Goldberg, 1989; Cherax: Sandeman and Sandeman,
in press). In these studies, the staging scale is "calibrated"
at a given constant temperature. The time from fertiliza-
tion to hatching is then transformed into a percent-staging
scale. This scale is applicable to animals raised at other
temperatures because all developmental events are com-
pressed or expanded proportionally, depending upon the
temperature.

However, as pointed out by Bentley et ai (1979), a
staging system based on percent of total time of embryo-
genesis cannot be applied in species with a period of de-
velopmental arrest. Homarus embryos manifest a period

of arrested development in natural conditions (Perkins,
1972) and at constant temperature (present study) and
these results indicate that factors other than temperature
have a strong influence on the rate of embryonic devel-
opment in lobsters. Therefore, we have used a morpho-
metric index, the size of the screening pigment spot in
the lateral eyes (the eye index of Perkins, 1972). as the
basis for a percent-staging scheme. The eye index has been
used as an indicator of developmental stage in a number
of embryonic studies (Schuur et ai, 1976; Hepper and
Gough, 1978; Cole and Lang, 1980; Sasaki, 1984; Sasaki
et ai, 1986; Beltz and Kravitz, 1987; Beltz et ai, 1990;
Helluy and Beltz, 1990; Meier and Reichert, 1990).

There are two obvious limitations of a staging system
based upon the eye index. First, the eye pigment does not
appear until approximately three weeks after egg extru-
sion, and at the first possible measurement is about 70
nm. To calibrate the relatively brief period from extrusion
to appearance of eye pigment, a time-staging analysis has
been used (see Materials and Methods). Thus, a percent-
staging system has been established that covers the entire
embryonic period, from extrusion until hatching. The
second drawback of the proposed method is related to the
variation of the eye index at hatching. Perkins (1972) re-
ported that the eye index at hatching is 560 urn. Just prior
to hatching in four broods that we have examined, it
ranged from 570 urn to 586 /^m and, taking into account
Perkins' figure of 560 /urn, 570 ± 20 ^m was chosen as
the eye index at hatching. This figure was used as the end
point in establishing the percent-staging scale. The 20 ^m
variation represents a small proportion (3.5%) of the total
percentage scale. The variation of the eye index at hatching
could be due to a combination of factors such as genetic
variability or perturbations due to environmental con-
ditions. For instance, it has been reported that molting
and development of morphological characteristics proceed
somewhat independently in decapods (Gore, 1985), and
it is conceivable that the physiological changes that reg-
ulate hatching and molting could be advanced or delayed
with respect to morphogenesis and growth.

Growth ciiwes and the developmental plateau

Lobster egg masses kept at seasonal water temperature
show a developmental plateau when the temperature is
low during the winter months (Perkins, 1972). More sur-
prising is the fact that our experimental brood, which was

Figure 5. Ventral view of embryos of Homarus americanus dissected from the yolk and unfixed at (A)
10. (B) 20, (C) 30, (D) 40, (E) 50, (F) 60, (G) 70, (H) 80, and (I) 90% development. In all photographs, the
head is at the top and the abdomen is folded ventrally onto the thorax. The figures in the lower left corners
refer to the percentage of development. The line drawing (J) represents a schematic metanauplius. Abbre-
viations, ab: abdomen, anl: antennula, an2: antenna, ch: chromatophores, he: heart, ig: intestinal granules,
le: lateral eye, me: median eye, mxp3: third maxilliped, te: telson. Scale bar in (F), valid from (A) to (I):
500 Mm.

366

S. M. HELLUY AND B. S. BELTZ

«^^»

^ an2

an1

Figure 6. Unfixed antennulae of embryos of Hatnants americamis at (A) 10, (B) 12, (C) 20, (D) 30. (E)
40, (F) 60, (G) 80, and (H) 90% development, in (I) a hatchling (HA) and in (J) a first larval stage (LI). In all
photographs, the figures in the lower left corners refer to the percentage of development. Thick arrows point
at the anterior end of the animals. The antenna (an2) is also shown in (A) and (C) in addition to the antennula
(an 1 ). By 30% development (E30%), a cluster of presumed sensory neurons (sn) and their axons (ax) are present
in the antennula and are particularly visible at E40%. At E30%, the endopod (edp) of the antennula and the
seta (se) at its tip are growing: the organogenesis of the first larval stage has already started. The endopod

EMBRYONIC DEVELOPMENT OF THE LOBSTER

367

raised at constant temperature, also showed a develop-
mental plateau. The arrest in development characterized
by a lack of growth in either the eye index or the cephalo-
thoracic length, occurred at E82% development (El 474).
At E80%, the eggs are green and the metanauplii are still
at stage D0, whereas by E90% eggs are yellowish and have
entered stage D, . The heterogeneity of the egg population
and the changes in pace of development observed in the
experimental brood prior to hatching were therefore re-
lated to the transition from D0 to D, of the metanaupliar
molt cycle.

Developmental plateaus also occur during stage D0 of
the molt cycle in juvenile lobsters (Aiken, 1973). The ar-
rest in development takes place at different times during
stage D0, from the first indication of epidermal retraction
to maximal epidermal retraction. Lobsters pause in their
development during the cold winter months, and the
transition to the irreversible stage D, does not proceed
until the water warms up in the spring. Aiken (1973) shows
that when a lobster has passed beyond pleopod stage 2.5 —
and therefore entered stage D, — development then pro-
ceeds at a rate regulated by temperature. It is possible that
the embryonic metanauplius goes through the same cycle.
Indeed, developmental plateaus have been observed at
different eye indices during stage D0 (from 350 ^m to 450
nm) in different broods of eggs (Thomann, Beltz, and
Helluy, unpub. results). Additionally, Perkins (1972) notes
that during the winter months development is arrested in
older eggs (extruded early in the summer) and still con-
tinues in younger eggs extruded later. It appears, therefore,
that in the wild the eggs may spend a variable amount of
time in stage D0 (shorter in younger eggs); in the spring,
internal and external cues could trigger the transition from
DO to D, . This could explain why extrusion of eggs is a
prolonged event in a population of females in the wild,
whereas hatching occurs during a more limited period
(Herrick, 1895; Perkins, 1972).

Embryonic molt cycles

In the present study, evidence is presented for two molts
occurring in Homarus prior to the first larval stage and
associated with the beginning and the end of the embry-
onic metanaupliar stage. Other crustaceans are also known
to pass through molt cycles within the egg envelopes
(Wear, 1974; Goudeau, 1976; Goudeau and Lachaise,
1983). Graf (1972) characterizes the embryonic molt cycle
of an amphipod on the basis of changes in the epidermis,

setae, and calcium storage; these changes parallel those
occurring during juvenile and adult molt cycles. In Horn-
ants both Herrick (1895) and Bumpus (1891) mention
the existence of embryonic molts. Based on the number
of membranes enclosing the embryo, Herrick (1895, p.
183) presumes that at least three embryonic molts have
occurred by the time the pigment appears in the lateral
eyes and predicts that many more may take place during
the long embryonic life. Bumpus ( 1 89 1 ) notes that the
cuticle lifts from the embryo in the region of the com-
pound eye between stages N and O, and that a true ecdysis
follows; this molt is probably the same as that observed
in the present study around El 2%, which is slightly after
stage O of Bumpus. Goudeau et al. (1990), using electron
microscopy, detect five envelopes originating from the
embryo ofHomams gam mams secreted beneath the inner
and outer egg envelopes and show that the secretion of
the embryonic envelopes is associated with high liters of
ecdysteroids; however, the embryos are not staged and
the timing of secretion is not studied. The fact that the
metanaupliar cuticle that begins to lift from the telson at
30% development possesses the imprint of the 6 + 6 setae
that were present at the naupliar molt, and the fact that
this same metanaupliar cuticle with the imprint of the
6 + 6 setae is discarded at the metanaupliar molt, dem-
onstrate that there is only one instar during that period,
not the many that were predicted by Herrick (1895). The
progressive setal changes observed in the metanaupliar
telson also support this conclusion. The several envelopes
seen at the level of the antennulae (Fig. 6B) and antennae
at stage El 2% may indicate that additional molts occur
prior to El 2%, during the naupliar phase.

Whereas the setal changes occurring in the telson of
the metanauplius seem very similar to those occurring
during the molt cycle of larval and juvenile lobsters
(Aiken, 1973; Rao et al., 1973; Aiken, 1980; Sasaki,
1984), the cellular and biochemical changes in the epi-
dermis and cuticle must be somewhat different. In the
growing metanauplius there is no fixed postecdysial
volume as there is in postembryonic animals. Indeed,
the cephalothorax of the embryo grows by a factor of
about 4 from the early 12% molt to the hatch molt (Fig.
2). Therefore, we presume that there is no mineraliza-
tion of the metanaupliar cuticle. In that respect, it has
also been noted before (see review in Gore, 1985) that
the prezoeal cuticle is different from the exuvia of older
lobsters.

of the antennula is also seen under the cuticle of the metanauplius at E80%, at E90%, and in the hatchling
but is out of focus in the photographs of other stages. The giant sensillum (gs) at the tip of the exopod of
the antennula is clearly seen forming inverted at E40% development, everted in the hatchling under the
cuticle of the metanauplius, and free and erect with three other setae at the tip of the exopod in the antennula
of the first larval stage. The red pigment in the chromatophores (ch) is seen concentrated at E60% and E80%
and dispersed at E90%. Scale bars. A: 100 jim, B and C: 50 ftm, D to J: 100 Mm.

368

S. M. HELLUY AND B. S. BELTZ

7Q -i —

EMBRYONIC DEVELOPMENT OF THE LOBSTER
BCD

369

Figure 8. Line drawings of telsons of Hiimuru\ unn'ricaiuix at (A) 12, (B) 70, (C) 90% development,
and (D) in a hatchling showing the naupliar ecdysis(Ec) and representative stages D0. D, , D2_3. For photographs
of those stages and legend, see Figure 7.

Lobster embryonic development in perspective

In the present study, the metanaupliar molt cycle and
the organogenesis of the first larval stage of Homarus are
examined. This aspect of development is generally ignored
in the literature. For example. Bumpus ( 1 89 1 ) studies the
early embryology of Homarus only until stage R (between
E30% and E40% development). Herrick (1895, p. 209)
implies that the organogenesis of the L 1 is extremely brief
and takes place just prior to eclosion when he observes
that the antennulae "remain single until just before the
time of hatching when the inner branch of the flagellum
begins to grow." In the present study the endopod (inner
branch of the flagellum) of the antennulae is first seen at
about 30% development. Therefore, our examination of

both the antennulae and the telson indicate that the or-
ganogenesis of L 1 begins early and continues throughout
the embryonic molt cycle.

Overlooking the embryonic metanaupliar molt cycle
has led to some confusion as to the status of the prezoea
(prelarval form). The existence of a prezoea has been noted
in many families of decapods, but its significance has been
largely debated (see review in Gore, 1985). We agree with
Wear ( 1974), who observes that "in decapods which hatch
at a zoea stage, the prezoea] cuticle is associated with the
metanauplius stage relegated to embryonic life, rather than
to the preceding nauplius." This is clearly the case in
Homarus: the ephemeral prelarva (prezoea) is the mature
metanauplius between the moment it is freed of the two
external egg envelopes and the time it molts (Fig. 4D).

Figure 7. Telsons of embryos of Homarus americanus (unfixed) at (A) 10. (B) 12, (C) 20, (D) 30, (E)
40, (F) 50. (G) 60. (H) 70, (I) 80, (J) 90, (K) 100% development, in (L) a hatchling (HA), and (M) a first
larva] stage (LI ). Panel M is a montage. In all photographs, distal is at the top. The figures in the lower left
corners refer to the percentage of development. The tegumentary and setal changes typical of different stages
of the molt cycle as described by Aiken (1973 and 1980). Rao el al. (1973). and Sasaki (1984) in larval,
juvenile, and adult lobsters are indicated with an asterisk in the following text. At about 12% development
(El 2%). an embryonic exuvia is lifting from the telson of the nauplius (arrows). The telson of the metanauplius
forming under that exuvia is provided with 6 + 6 setae. At £30%. the metanaupliar cuticle begins to separate
from the side of the setae* but the tips of these setae are still attached to the cuticle (arrows); this stage is
equivalent to the premolt stage D0 of the molting cycle of older lobsters. During the metanaupliar molt
cycle, the setae present on the triangular telson of the first larval stage (first zoea) are forming gradually,
proximally and medially in the telson of the metanauplius. By E60% at least 10 + 10 setae (arrows) are
visible under the metanaupliar cuticle. At E70%, the full complement of setae of the first larval stage ( 1 5
+ 15) is formed. Between E80% and E90%, dramatic setal and tegumentary changes occur. At E90% the
epidermis is scalloped* (black line): setae and lateral spines are invaginating* (arrows) and the cuticle lifts
from the sides of the telson (arrowheads); this stage is equivalent to D,. Just prior to hatching (E100%)
retraction of spines and setae is maximum. Note the crumpled tissue at the base of the lateral spines (arrow).
In the hatchling (HA), this tissue forms a dark ring (arrow), the lateral spines are half extended, the epidermis
is very distinct* and the bulging of the epidermis around the setae is pronounced* (circle) which is characteristic
of stage D2_3. Ecdysis takes place thereafter, and the metanaupliar cuticle bearing the shape of the 6 + 6
metanaupliar setae (arrows) is shed. After ecdysis the triangular telson of the first larval stage (LI ) unfolds.
Note that two individuals (J and L) had two lateral spines on one side. Scale bars, A to D: 50 ion, E to M:
100 /im.

370

S. M. HELLUY AND B. S. BELTZ

One interpretation of the coupling of hatching and molting
is that hatching is actually a by-product of the molting
process. For instance, the extension of the lateral spines
of the telson (Fig. 7L) could provoke the breaking of the
inner and outer egg envelopes (Fig. 4C, D). Indeed, the
lateral setae of the telson are smooth and extended until
80% development (Fig. 71), begin to invaginate as soon
as they become sharp (Figs. 7J, 8C), are entirely invagi-
nated in the blue embryo (El 00%) just prior to hatching
(Fig. 7K), and are seen half evaginated in the hatchling
(Figs. 7L, 8D) whose telson has just pierced the egg en-
velopes (Fig. 4C). No other part of the mature meta-
nauplius is quite as hard and sharp as the lateral spines
of the telson and it is therefore possible that the extension
of these spines triggers the rupture of the egg envelopes.
By this means, the metanauplius at the end of its molt
cycle would precipitate hatching, and the beating of the
pleopods of the mother would help the larva to slip out
of its swaddling envelopes.

Molt cycles in postembryonic crustaceans are under
hormonal control, and it is likely that embryonic molt
cycles are regulated in a similar way. The circulating
molting hormone is an ecdysteroid whose liters increase
in DO and peak in D2-Di in Homarus (Snyder and Chang,
1991) and in D, in Penaeus (Chan et a/.. 1988). Because
steroids influence neuronal development and survival in
other systems (Weeks and Truman, 1986), the awareness
of embryonic molts and the prediction of the timing of
potential changes in steroid levels could be critical for
future developmental neurobiological studies.

The nauplius is a form common to all crustaceans, and
in some taxa (e.g., Cirripedia, Anostraca) eggs hatch as
nauplii. In other species, the naupliar stage is followed
within the egg envelopes by the organogenesis of a more
complex body form characterized by the morphogenesis
and growth of the postmandibular region (Anderson,
1979, 1982; Weygoldt, 1979; Williamson, 1982; Gore,
1985; Schram, 1986; Shiino, 1988). It has been shown in
several taxa [amphipods: Graf (1972); isopods: Goudeau
(1976); decapods: Wear (1974), and the present study]
that envelopes equated to embryonic exuvia are found
during embryonic life as the postmandibular region dif-
ferentiates. In amphipods (Graf, 1972) and decapods
(present study), embryonic molt cycles are demonstrated
with the progressive setal and tegumentary changes oc-
curring in the telson. The existence of embryonic molt
cycles in different taxa suggests that the relegation oflarval
stages to life in the egg, or, rather, the delay of hatching
with regard to molting, is a widespread and distinctive
evolutionary strategy in crustaceans. Besides leading to
evolutionary considerations, the characterization of the
metanaupliar molt cycle and the percent-staging scheme
for lobster eggs should lend added insight in future in-
vestigations of neural, physiological, and ecological aspects
of Homarus embryonic life.

Acknowledgments

We wish to thank Maureen Ruchhoeft for her kind and
skillful help, Joe Gagliardi and Kay Leland for printing
photographic plates, Michael Syslo and Kevin Johnson
from the Massachusetts State Lobster Hatchery who pro-
vided the egg-bearing female lobsters, as well as Colleen
Boggs and Tom Coffee who maintained them at the New
England Aquarium. (Supported by NSF-BNS-87 18938,
NIH-NS 25915, and NSF- Presidential Young Investigator
Award BNS- 8958169 to B.S.B.)

Literature Cited

Aiken, D. E. 1973. Proecdysis, setal development, and molt prediction
in the American lobster (Homarus americanus). J. Fish. Res. Board
Can. 30: 1337-1344.

Aiken, D. E. 1980. Molting and growth. Pp. 91-163 in The Biology
anil Management of Lobsters. Vol. 1. J. S. Cobb and B. F. Phillips,
eds.. Academic Press, New York.

Aiken, D. E., and S. L. \\addy. 1980. Reproductive biology. Pp. 2 1 5-
276 in The Biology and Management of Lobsters, Vol. 1. J. S. Cobb
and B. F. Phillips, eds.. Academic Press, New York

Anderson, D. T. 1979. Embryos, fate maps, and the phytogeny of ar-
thropods. Pp. 59-105 in Arthropod Phytogeny. A. P. Gupta, ed., Van
Nostrand-Remhold, Princeton. NJ.

Anderson, D. T. 1982. Embryology. Pp. 1-41 in The Biology of Crus-
tacea, vol. 2, Embryology. Morphology, and Genetics. L. G. Abele.
ed. Academic Press. New York.

Beltz, B. S., and E. A. Kravitz. 1987. Physiological identification, mor-
phological analysis, and development of identified serotonin-proctolin
containing neurons in the lobster ventral nerve cord. J. Neurosci. 7:
533-546.

Beltz, B. S., M. S. Pontes, S. M. Helluy, and E. A. Kravitz.
1990. Patterns of appearance of serotonin and proctolin immu-
noreactivities in the developing nervous system of the American lob-
ster. J. Neurohiol. 21: 521-542.

Bentley, D., H. Keshishian, M. Shankland, and A. Toroian-Raymond.
1979. Quantitative staging of embryonic development of the grass-
hopper, Schistocerca nil ens. J Embryol. E\p. Morphol. 54: 47-74.

Bumpus, H.C. 1891. The embryology of the American lobster. / Mor-
phol. 5: 215-262.

Burrage, T. G. 1978. Fine structural development and activity in the
heart and midgut of the embryonic lobster. Homarus americanus
(Milne-Edwards). Ph. D. thesis, Clark University, Worcester. MA.

Burrage, T. G., and R. G. Sherman. 1979. Formation of sarcomeres
in the embryonic heart of the lobster. Cell Tissue Res 198: 477-486.

Chan, S.-M., S. M. Rankin, and L. L. Keeley. 1988. Characterization
of the molt stages in Penaeus vannamei: setogenesis and hemolymph
levels of total protein, ecdysteroids, and glucose. Biol. Bull. 175: 185-
192.

Charmantier, G. 1987. Le developpement larvaire et la metamorphose
chez les Homards (Crustacea, Decapoda). Oceanis 13: 137-165.

Charmantier, G., and D. E. Aiken. 1987. Osmotic regulation in late
embryos and prelarvae of the American lobster Homarus americanus
H. Milne-Edwards, 1837 (Crustacea, Decapoda). / Exp. Mar. Biol.
Ecol 109: 101-108.

Cole, J. J., and F. Lang. 1980. Spontaneous and evoked postsynaptic
potentials in an embryonic neuromuscular system of the lobster.
Homunis americanus. J. Neurobiol. 11:459-470.

Davis, C. C. 1964. A study of the hatching process in aquatic inver-
tebrates. XIII. Events of eclosion in the American lobster, Homarus
americanus Milne-Edwards (Astacura, Homaridae). Am. Midi. Nat.
72:203-210.

EMBRYONIC DEVELOPMENT OF THE LOBSTER

371

Drach, P. 1939. Mue et cycle d'intermue chez les Crustaces Decapodes.
Ann Insl Oceanogr. 19: 103-392.

Drach, P., and C. TchernigovtzefT. 1967. Sur la methode de determi-
nation des stades d'intermue et son application generale aux crustaces.
Vie Milieu A 18: 595-610.

Ennis, G. P. 1975. Observations on hatching and larval release in the
lobster Homanis americanus. J Fish. Res. Board Can 32: 2210-
2213.

Fernandez, J. 1980. Embryonic development of the glossiphoniid leech
Theromyian rude: characterization of developmental stages. Dev. Bio/
76: 245-262.

Kigueiredo, M. J., and I. F. Barraca. 1963. Contnbuicao para o con-
pecimento da pesca e da biologia do Lagostim (Nephrops norvegicus
L.) na Costa Portuguesa. Noias Eslnd. Insl. Bin/. Maril. (Lisb.) 28:
1-28.

Gore, R. H. 1985. Molting and growth in decapod larvae. Crust. Issues
2: 1-65.

Goudeau. M. 1976. Secretion of embryonic envelopes and embryonic
molting cycles in Henuoniscus baluni Buchholtz, Isopodu, Epicaridea.
/ Morphol. 148:427-451.

Goudeau, M., and F. Lachaise. 1983. Structure of the egg funiculus
and deposition of embryonic envelopes in a crab. Tissue and Cell
15: 47-62.

Goudeau, M., F. Lachaise, G. Carpcnlier, and B. Goxe. 1990. High
liters of ecdysteroids are associated with the secretory process of em-
bryonic envelopes in the European lobster. Tissue and Cell 22: 269-
281.

Govind, C. K. 1989. Asymmetry in lobster claws. Am. Sci. 77: 468-
474.

Graf, F. 1972. Stockage de calcium et formation des soies chez 1'em-
bryon d'Orehestia (Crustace. Amphipode, Talitride). Notion d'in-
termue embryonnaire. C. R Acad. Sc. Sene D 275: 1669-1672.

Griinert. I'., and B. \V. Ache. 1988. infrastructure of the aesthetasc
(olfactory) sensilla of the spiny lobster. Panulinis argus. Cell Tissue
Res. 251:95-103.

llelluy, S., and B. S. Beltz. 1990. Stages in the embryonic development
of the American lobster Homanis americanus with special emphasis
on its nervous system. Pp. 530-536 in Frontiers in Crustacean Neu-
robiologv. K. Wiese, W. D. Krenz. J. Tautz. H. Reichert and B.
Mulloney. eds. Birkhaiiser, Basel, Boston.

Hepper, B. T., and C. J. Gough. 1978. Fecundity and rate of embryonic
development of the lobster, Homanis gammanis (L), off the coast
of North Wales. / Cons. In! Explor. Mer 38: 54-57.

Herrick, F. 11. 1895. The American lobster: a study of its habits and
development. Bull U.S. Fish. Commission 15: 1-252.

Hoffman, R. 1977. The modulation contrast microscope: principles
and performance. / Microsc. 110: 205-222.

Kravitz, E. A. 1988. Hormonal control of behavior: amines and the
biasing of behavioral output in lobsters. Science 241: 1775-1781.

McKenney, K., and J. I. Goldberg. 1989. Helisoma embryogenesis:
morphological, behavioral and neural development. Soc. Neurosci.
Abslr. 15: 1016.

Meier, I., and H. Reichert 1990. Neuronal development in the crus-
tacean nervous system studied by neuron-specific antibody labelling.
Pp. 523-529 in Frontiers in Crustacean Neurobio/ogy. K. Wiese,
W. D. Krenz, J. Tautz, H. Reichert, and B. Mulloney, eds. Birkhaiiser,
Basel, Boston.

Pandian, T. J. 1970a. Ecophysiological studies on the developing eggs
and embryos of the European lobster Homanis gammarus. Mar.
Bio/. 5: 154-167.

Pandian, T. J. 1970b. Yolk utilization and hatching in the Canadian
lobster Homanis americanus. Mar Bio/. 7: 249-254.

Perkins, H. C. 1972. Developmental rates at various temperatures of
embryos of the northern lobster (Homanis americanus Milne-Ed-
wards). Fish. Bull 70: 95-99.

Phillips, B. F., and A. N. Sastry. 1980. Larval Ecology. Pp. 1 1-57 in
The Biology and Management of Lobsters, Vol. 2, J. S. Cobb and
B. F. Phillips, eds. Academic Press, New York.

Rao, K. R., S. W. Fingerman, and M. Fingerman. 1973. Effects of
exogenous ecdysones on the molt cycles of fourth and fifth stage
American lobsters, Homanis americanus. Comp. Biochem Physio/.
A 44: 1105-1120.

Sandeman, R., and D. C. Sandeman. 1990. Development and identified
neural systems in the crayfish brain. Pp. 498-508 in Frontiers in
Crustacean Newohiology, K. Wiese, W. D. Krenz, J. Tautz, H.
Reichert. and B. Mulloney, eds. Birkhaiiser, Basel, Boston.

Sandeman, R., and D. C. Sandeman. 1991. Stages in the development
of the embryo of the freshwater crayfish Cherax deslrucli>r. Roux's
Arch. Dev Bio/ (in press).

Sasaki, G. C. 1984. Biochemical changes associated with embryonic
and larval development in the american lobster Homanis americanus
Milne-Edwards. Ph. D. thesis. Massachusetts Institute of Technology/
Woods Hole Oceanographic Institution, WHOI-84-8.

Sasaki, G. C., J. M. Capuzzo, and P. Biesiot. 1986. Nutritional and
bioenergetic considerations in the development of the American lob-
ster Homanis americanus. Can. J Fish. Aaual Sci. 43: 231 1-2319.

Schram, F. R. 1986. Crustacea. Oxford University Press. New York.

Schuur, A., W. S. Fisher, J. C. Van Olst, J. Carlberg, J. T. Hughes,
R. A. Shleser, and R. F. Ford. 1976. Hatchery methods for the
production of juvenile lobsters (Homanis americanus). Insl. Mar.
Res. California 48: 1-20.

Shiino, S. M. 1988. Crustacea. Pp. 333-388 in Invertebrate Embryol-
ogy. M. Kume, and K. Dan, eds. Garland Publishing, New York.

Snyder, M. J., and E. S. Chang. 1991. Ecdysteroids in relation to the
molt cycle of the American lobster, Homanis americanus. I. He-
molymph liters and metabolites. Gen. Comp. Endocnnoi 81: 133-
145.

Templeman, \V. 1940. Embryonic developmental rates and egg laying
of Canadian lobsters./. Fish. Res. Board Can 5: 71-83.

Waddy, S. L., and D. E. Aiken. 1984. Broodstock management for
year-round production of larvae for culture of the american lobster.
Can. Tech. Rep Fish, and Aqua. Sc. No 1272.

Wear, R. G. 1974. Incubation in British decapod Crustacea, and the
effects of temperature on the rate and success of embryonic devel-
opment. J. Mar Biol. Assoc. t/ A 54: 745-762.

Weeks, J. C., and J. VV. Truman. 1986. Steroid control of neuron and
muscle development during the metamorphosis of an insect. J Neu-
robiol 17: 249-267.

Weygoldt, P. 1979. Significance of later embryonic stages and head
development in arthropod phylogeny. P. 107 in Arthropod Phytogeny.
A. P. Gupta, ed. Van Nostrand-Reinhold. Princeton, NJ.

Wiese K., W. D. Krenz, J. Tautz, H. Reichert, and B. Mulloney.
1990. Frontiers in Crustacean Neurobiology. Birkhaiiser, Basel.
Boston.

Williamson, D. I. 1982. Larval morphology and diversity. Pp. 43-1 10
in The Biology oj Crustacea. Vol. 2, Embryology. Morphology, and
Genetics. L. G. Abele, ed. Academic Press, New York.

Reference: Biol. Hull 180: 372-386. (June. 1991)

How Do Temperature and Salinity Affect Relative

Rates of Growth, Morphological Differentiation, and

Time to Metamorphic Competence in Larvae

of the Marine Gastropod Crepidula planal

KERRY M. ZIMMERMAN AND JAN A. PECHENIK1
Biology Department. Tufts University. Mcdford. Massachusetts 02155

Abstract. The influence of environmental conditions on
rates of larval growth has been documented many times
for various marine mollusks. But the factors that influence
rates of morphological and physiological differentiation,
particularly the rate at which larvae within a population
become competent to metamorphose, remain obscure. In
four experiments, we reared larvae of the gastropod Cre-
pidula p/ana at 29°C. 25°C, and 20°C at 30 ppt salinity,
and in two other experiments, in salinities between 4-30
ppt at 25 °C. Rates of shell growth and morphological
differentiation, and rates of becoming competent within
populations were recorded. Larvae were considered to be
competent to metamorphose if they could be stimulated
to metamorphose by exposure to a high concentration of
KG (20 mM above ambient). Larvae consistently became
competent faster at higher temperatures, but in only one
of four experiments did temperature also consistently in-
crease the rates of growth and morphological differentia-
tion. Larvae took longer to become competent when
reared at lower salinities, but the effects were poorly pre-
dicted by the influence of salinity on rates of growth and
morphological differentiation. Competent larvae could
also not be recognized by shell length; many individuals
were competent at shell lengths of 600-800 ^m, while
many other individuals were still not competent at sizes
exceeding 1000 urn. At 29°C, many individuals became
competent at smaller sizes than those reared at lower tem-
peratures. Presence of gill filaments or shell brims also
did not correlate with individual metamorphic compe-

Received 26 February 1990; accepted 9 January 1991.
' Please address reprint requests to J. A. Pechenik.

tence. The data suggest that growth rate, rate of morpho-
logical differentiation, and time required for larvae of C.
plana to become competent can be uncoupled markedly
by shifts in rearing conditions.

Introduction

Competence is a differentiated state in which larvae of
benthic marine invertebrates first become capable of me-
tamorphosing in response to environmental cues (Crisp,
1974;Scheltema, 1974; Chia, 1978; Hadfield, 1978; Miller
and Hadfield, 1986; Coon et al.. 1990; Fitt el al. 1990).
Metamorphosis of gastropod larvae is most easily defined
by the loss of the larval velum, an organ responsible for
larval feeding, swimming, and gas exchange. This trans-
formation marks the transition from a swimming plank-
tonic stage to a largely sedentary benthic stage. The time
required for a larva to become competent thus determines
the obligate planktonic dispersal period (Scheltema, 1978;
Jackson and Strathmann, 1981).

Larvae are often designated as competent based on their
size, age, or the presence of particular morphological
characteristics (Bayne, 1964; Bayne, 1965; Bayne, 1971;
Hickman and Gruffydd, 1 97 1 ; Switzer-Dunlap and Had-
field. 1977; Hadfield. 1978; Pechenik, 1984; Lima and
Pechenik, 1985; Butman el al.. 1988). In at least some
molluscan species, however, such criteria may be poor
indicators of an individual's competence to metamor-
phose. In the bivalves Mytilus edulis and Crassostrea
gigas. for example, neither shell size, age, nor the presence
of eye spots guarantee that larvae will metamorphose in
response to apparently appropriate cues (Eyster and
Pechenik, 1987; Coon et al.. 1990). Similarly, size is an

372

COMPETENCE TO METAMORPHOSE

373

inadequate indicator of metamorphic competence for the
gastropod Crepidulafornicata; larvae from a single larval
culture became competent to metamorphose at shell
lengths ranging between 700 and 1000 ^m (Peehenik and
Heyman. 1987. in response to elevated KC1 concentra-
tions). Neither did behavioral changes successfully signal
the time at which larvae of the opisthobranch Phestilla
sibogae became metamorphically competent in the ex-
periments of Miller and Hadfield (1986). There is growing
reason to doubt, then, that the time required for a larva
to become metamorphically competent is directly coupled
to the rate at which the larva grows or develops most
other conspicuous traits.

To date, few workers have rigorously documented
the rate at which larvae in a population become compe-
tent to metamorphose, or have considered the influ-
ence of environmental factors on that rate. In addition,
the correspondence between the rates of larval growth
and of attaining metamorphic competence have been
poorly explored. Under what conditions do larvae become
competent more quickly, and to what extent can this ac-
celerated attainment of competence be predicted from
the influence of those conditions on rates of growth or
morphological differentiation? Because larval metamor-
phosis can, for a number of species, be triggered by ele-
vating KC1 ambient concentration (Yool el ul.. 1986;
Peehenik and Heyman, 1987), the rate and sizes at
which larvae of those species become competent can
be determined experimentally. The larvae of Crepidula
fornicata can be induced to metamorphose by elevated
K.C1 concentrations at about the same age and size
that larvae become responsive to adult-conditioned sea-
water and surfaces bearing microbial films (Peehenik,
1980; Peehenik and Heyman, 1987). The latter probably
serve as metamorphic cues in the field (McGee and
Target, 1989), but the active constituents have not been
isolated.

In this paper, we report the effects of temperature and
salinity on the rate of larval growth, the rate of morpho-
logical differentiation, and the time required for larvae of
the prosobranch gastropod Crepidula plana to become
metamorphically competent (as indicated by their re-
sponse to elevated potassium concentration). In Crepidula
plana, virtually all larvae eventually metamorphose
"spontaneously" — no cue is deliberately provided — in
glassware that is cleaned and acid-rinsed daily (Lima and
Peehenik, 1985). Thus, the maximum dispersal potential
for these larvae depends on how long metamorphosis can
be delayed after they first become competent. We therefore
also monitored the timing of "spontaneous" metamor-
phosis in relationship to the onset of metamorphic com-
petence. We have thus been able to directly determine
the influence of temperature on the length of time that

metamorphosis can be delayed under laboratory condi-
tions.

Materials and Methods

Maintenance of adults and larvae <>/ Crepidula plana

Adult Crepidula plana were collected near Woods Hole,
Massachusetts. We maintained adults at room tempera-
ture (2 1-25°C) in 1 /urn filtered seawater (collected at Na-
hant, Massachusetts), changing the seawater daily. We fed
adults the green unicellular alga Dunaliella tertiolecta
(clone DUN) daily, until larval release. After their release,
the larvae were isolated on a 1 50 ^m sieve and transferred
to 0.45 ^m filtered seawater (29-30 ppt salinity). In each
of the six experiments conducted, the larvae were all re-
leased on the same day, but not necessarily from one fe-
male.

Larvae were fed the naked flagellate Isochrysis sp.
(Tahitian strain, clone T-ISO) daily; seawater was changed
every other day. At the start of an experiment (2-9 days
after hatching), known numbers of larvae were randomly
assigned to either a 20°C, 25°C, or 29°C temperature
incubator (Percival Manufacturing) stable to 0.1 °C. Lar-
vae of C. plana grow very slowly at temperatures below
20°C, and 29°C seems to be near the upper lethal tem-
perature limit for this species (Lima and Peehenik, 1985).
All larvae were cultured on a 1 1 L: 1 3D light cycle. Larval
concentrations were maintained below one larva • ml ' in
all experiments (I- VI); the aim was to maximize growth
rates and minimize competition for food. Larvae were fed
1.8 X 105 cells- ml ' of T-ISO every other day in Exper-
iments I and II, and daily in all subsequent experiments.
A hemacytometer was used to determine algal cell con-
centrations. To monitor survival, we removed dead or
moribund larvae from the cultures at each water change.
Glassware was cleaned with Bon Ami and rinsed with
deionized water at each water change.

Determining the influence of temperature on rates of
growth and morphological differentiation

In four experiments, we examined how temperature
affects the relationship between rates of larval growth, rates
of morphological differentiation, and rates of becoming
competent to metamorphose. In Experiments I and II,
1 100-1600 larvae were reared at each tested temperature
(20°C, 25°C, and 29°C) in batch culture. Thirty actively
swimming larvae were collected daily (25°C and 29°C),
or every other day (20°C) from the batch cultures. Sea-
water volumes were adjusted after larval collection to
maintain larval densities. In Experiments III and IV, we
determined the growth rates of larvae reared in individual
glass bowls, at densities also below 1 larva- ml" '.

Larval shell lengths were measured at SOX using a dis-
secting microscope equipped with an ocular micrometer;

374

K. M. ZIMMERMAN AND J. A. PECHENIK.
Table I

Inlltieiue nl temperature and salinilv on rale\ ot larval shell i>nmth. morphological differentiation and heeoming competent
lor larvae o/ Crepidula plana

Experiment
number

Temperature

(°C)

Salinity
(ppt)

Growth rate
l/im-day"1)

Days to 50% of Days to 50% of Days to 50% of
the population the population the population
competent gilled brimmed

Mortality
(n)

28.4
(r = 0.96)

12.2

4.0(1620)

40.0
(r = 0.96)

19.0

7.0(1120)

33.8
(r = 0.96)

23.4

13.0(1620)

27.9
(r = 0.88)

12.3

2.5(1300)

22.5
(r = 0.93)

17.0

8.0(1300)

19.2
(r = 0.91)

19.0

15.0(1300)

III

59.1
(r = 0.99)

9.7 9.7 10.4

1.0(1000)

111

52.0
(r2 = 0.89)

13.4 10.3 13.7

2.0(1000)

40.5
(r = 0.79)

11.0 9.6 11.3

1.0(720)

43.0
(r = 0.91)

12.6 11.6 12.0

1.0(720)

29.4
(r = 0.89)

18.6 15.4 18.2

4.0 (720)

39.1
(r = 0.96)

— — —

18.0(48)

27.1
(r = 0.97)

_ _ _

16.0(49)

15.4
(r = 0.91)

— — —

17.5(52)

43.6
(r = 0.96)

14.3 12.5 14.4

2.0 (620)

35.1
(r = 0.94)

17.6 13.3 14.6

2.0 (620)

38.9
(r2 = 0.94)

>22.0 14.0 15.2

1.0(620)

In Experiments I and II. larvae were fed every other day, in all other experiments larvae were fed every day. Dashes indicate sampling trom batch
culture (Expts. I and II) or data not available (Expt. V).

the maximum shell length was measured with the larva
lying on its left side. The presence of gill filaments and
the lateral shell brims characterizing advanced larvae of
this species (Pechenik and Lima, 1984) were also noted.
Growth rates (^m shell growth -day ') were determined
by linear regression analysis of changes in shell length

through time (SPSS Inc., 1988). The percentage of the
larval population that was gilled or brimmed was plotted
against time. From these plots we estimated the number
of days necessary after larvae were released from their egg
masses for 50% of the larvae in a population to become
gilled or brimmed.

COMPETENCE TO METAMORPHOSE

375

Table II

Influence <>j salinity on lan-al sun'ival and rule ol becoming competent
in Experiment I '

%' population competent

after 7 days in each

Salinity

salinity treatment

(PPT)

%. Mortality

X ±SD(n)

18%

37% ±4.0(3)

16%

10.3% ± 9.3 (3)

17.5%

3. 3% ±2.9 (3)

14.5

31%

20% ± 7.2 (3)

85%

0% ± 0.0 (3)

100% in 3 days

—

At the time of K.C1 exposure, larvae were 16 days old. Larvae were
introduced to the reduced salinities after 9 days of culture at full-strength
salinity (29 ppt).

Larvae collected from batch culture were preserved in
10% formalin buffered with sodium borate (BORAX) (pH
~ 8.0), for later determination of larval organic weight;
larvae reared at different temperatures were stored sepa-
rately. Larval organic weights were determined as follows:
one or more larvae of known shell lengths were placed
into pre-weighed aluminum pans; the preserved animals
were first rinsed three times with distilled water to remove
preservatives and salts. The animals were dried overnight
at 60°C in a drying oven, then weighed to determine initial
total (inorganic and organic) dry weights. The animals

100

J, 4 4

•

-f

i i . i i i // i

EXPOSURE TIME (HOURS)

Figure 1. Influence of time exposed to elevated K.CI concentration
on metamorphosis of competent Crepidula plana larvae. KC1 concen-
trations were elevated by 20 mAJ at 22-24°C. Larvae were examined
for loss of velar lobes hourly for 8 h, then at 10 h and 24 h. Each point
represents the mean of five replicates, with 20-21 larvae per replicate.
Vertical bars represent one standard deviation. Average larval shell length
(±SD) was 777 Mm ± 97.8 (n = 100).

were weighed to the nearest microgram (^g) with a Cahn
microbalance with desiccant present in the weighing
chamber to prevent rehydration. The pans were reweighed
after sample combustion in a muffle furnace at 550°C for
6 h; combustion did not change the weight of the alu-
minum pans. The weight lost in combustion is equivalent
to the larval organic weight. Individual body weights were

Table III

Results of ANACO\'As for shell si:e. % of the population competent 10 metamorphose, % fully gilk'd. or fully brimmed
for each experiment by temperature and salinity with age as a covanale

Experiment I:

Experiment II:

Experiment III:

Experiment IV:

Experiment VI:

Influence of temperature (at 30 ppt) on growth rate
Rate at which the population became competent
Rate at which the population became gilled
Rate at which the population became brimmed

Influence ot temperature (at 30 ppt) on growth rate
Rate at which the population became competent
Rate at which the population became gilled
Rate at which the population became brimmed

Influence of salinity (at 25°C) on growth rate
Rate at which the population became competent
Rate at which the population became gilled
Rate at which the population became brimmed

25°C > 20°C > 29°C
29°C> 25°C> 20°C
29°C = 25°C = 20°C
29°C = 25°C = 20°C

29°C > (25°C = 20°C)
29°C > 25°C > 20°C
29°C = 25°C = 20°C
29°C = 25°C = 20°C

29°C > 25°C
29°C> 25°C
29°C > 25°C
29°C > 25°C

25°C > 29°C > 20°C
29°C > 25°C > 20°C
29°C > 25°C > 20°C
29°C > 25°C > 20°C

30 ppt > 20 ppt > 25 ppt
30 ppt > 25 ppt > 20 ppt
30 ppt > (25 ppt = 20 ppt)
30 ppt > 25 ppt > 20 ppt

All differences are significant at P < 0.05, and most were significant at P < 0.001.

376

K. M. ZIMMERMAN AND J. A. PECHENIK.

14 IB 18 20

LARVAL AGE (DAYS FROM RELEASE)

Figure 2. Influence of rearing temperature on the rate at which larvae became competent to metamorphose
in Experiment I. Each point represents the mean percentage metamorphosing in three bowls, with 34-40
larvae per bowl. Vertical bars represent one SD about the mean. Different letters represent larval populations
with different mean growth rates (A < B).

determined for larvae longer than 700 ^m; larvae less than
700 nm were pooled for weight determinations.

Determining the effect of temperature on the rate of
becoming competent to metamorphose

Pechenik and Heyman (1987) found that elevating the
KC1 levels in natural seawater by 20 mA/ induced com-
petent larvae of C. fornicata to metamorphose within 7
h. To determine whether the larvae of C. plana would
respond similarly, we exposed advanced larvae of this
species (22-day-old, 770 ± 98 fim shell length, n = 100)
to a 20 mA/ increase in KC1 concentration. We checked
hourly for larval metamorphosis for the first 8 h, then at
10 h and 24 h; newly metamorphosed larvae were re-
moved at each observation. The experiment was con-
ducted at 22°C, with 5 replicates (2 1 larvae per replicate).

To determine the effect of temperature on the rate at
which larvae in a given population became competent to
metamorphose, we monitored larvae from a temperature
treatment until some individuals reached shell lengths of
about 600 ^m. At 1-3 day intervals, we then transferred
all larvae from three randomly chosen bowls into 3 bowls
of seawater with elevated KC1 concentrations; 30 to 45
glass bowls of larvae (20-40 larvae per bowl, depending
on the experiment) were used for each temperature treat-
ment during the course of an experiment. After exposing
larvae to the elevated KC1 for 6 h, we determined the
number of individuals that had metamorphosed in each
bowl, and measured the shell lengths of those that had
metamorphosed and of those that had not. We also de-
termined whether individuals had gills or shell brims. We
conducted Mests to determine whether there were differ-

ences in the mean shell lengths of competent and pre-
competent larvae in each temperature treatment. The rate
at which larvae in each population became competent
was determined by linear regression analysis. Significant
regression coefficients (r) were obtained in all experiments.
For regressions with correlation coefficients (r) greater
than 0.80, the number of days for 50% of the larval pop-
ulation to become competent was determined from the
regression. For data with r2 values less than 0.80, the
number of days for the populations to become 50% com-
petent was estimated by eye.

Determining the influence of temperature on maximum
length oflamil life

Larvae of C. plana eventually undergo "spontaneous"
metamorphosis in the laboratory, even when maintained
in frequently cleaned glassware (Lima and Pechenik,
1985). Three bowls (20-40 larvae per bowl, depending
on experiment) at each temperature were washed and acid-
rinsed daily, at each change of algal suspension. Larvae
were examined daily; we counted, removed, and measured
newly metamorphosed snails. These data were compared
with observations on the mean age and size, at meta-
morphosis, of individuals cultured in bowls cleaned only
every 48 h ("filmed bowls"). The aim was to determine
whether biological films building up over the 48-h period
would induce a greater number of larvae to metamor-
phose. Such biological surface films have been implicated
as metamorphic inducers in many marine invertebrates
(Meadows and Campbell, 1972; Scheltema, 1974; Kirch-
man et al.. 1982; Lima, 1983; Coon el a/.. 1985; Weiner
etai, 1989).

COMPETENCE TO METAMORPHOSE

377

20°C

Figure 3. Influence of rearing temperature on the number of days for 50% of the larvae in each treatment
population to become competent to metamorphose in Experiments I-IV.

The time required for 50% of the population to me-
tamorphose in the bowls cleaned daily, minus the time
required for 50% of the larvae to become competent in
parallel experiments, was used as an index of capacity for
delaying metamorphosis. This cannot be used to predict
dispersal potential in the field, but should enable us to
assess the influence of temperature and salinity on the
physiological capacity for prolonging larval life, and will
permit future interspecific comparisons of the physiolog-
ical capacity for delaying metamorphosis.

Determining the effects of salinity on rates of growth,
morphological differentiation, and rates of becoming
competent

In two experiments, we examined how salinity affected
the relationship between rates of growth, morphological
differentiation, and becoming competent. In the first ex-
periment, six salinities [29, 25, 19, 14.5. 8, and 4 parts
per thousand (ppt)] were used to determine the salinity
tolerance of larval C. plana; these salinities are equivalent
to osmotic concentrations of 821, 708, 557,403, 223, and
1 16 mOsm, respectively. The five lowest salinities were
made by mixing 0.45 jum filtered seawater with deionized
water; the 29 ppt seawater was composed solely of un-
diluted 0.45 urn filtered seawater. Osmotic concentrations
were measured with a freezing point depression osmom-
eter (Advanced Instruments, Inc.). This experiment was
conducted at 25°C, with three replicate bowls of 20 larvae
per bowl in each salinity treatment. Water and food were
replaced daily. All larvae were reared in full-strength sea-
water for 9 days, and then acclimated to lower salinities
in stages during 1 h. Shell-less, moribund, or dead larvae
were counted and removed daily. Shell lengths were mea-
sured non-destructively (Pechenik, 1984) each day for

growth rate determinations. All larvae were exposed to
an increase of 20 mAl KC1 on the seventh day of the
experiment (the 16th day of larval life) to determine the
percentage of larvae competent to metamorphose in each
salinity.

Based on the results of the first experiment, a second
experiment (Experiment VI) was conducted at 30, 25, 20
ppt (again at 25°C) to examine more fully the effect of
salinity on rates of growth and differentiation. We reared
25 larvae per bowl with 3 1 bowls per treatment. To min-
imize the effects of food supply on salinity — algae are cul-
tured at about 30 ppt — the algae were concentrated by
centrifugation at 3000 X g for 12 min and then resus-
pended in seawater of the appropriate test salinity (Pech-
enik and Fisher, 1979). Algal cells remained alive and
motile in all salinities. Every day, larval shell lengths were
measured non-destructively from randomly selected bowls
at each salinity, presence or absence of gill filaments and
shell brims were simultaneously noted.

Periodically, three bowls of larvae from each salinity
treatment were randomly selected and all individuals (20-
30 larvae per bowl) were exposed to elevated KC1 con-
centrations in seawater to assess metamorphic compe-
tence. Larvae reared at 30 or 25 ppt were exposed to an
increase of 20 mAf K.C1 while those reared in 20 ppt sea-
water were exposed to either a 20 or a 23 mM KC1 in-
crease, to compensate for the lower baseline KC1 concen-
tration at the reduced salinity. All individuals exposed to
KC1 were measured, whether or not they metamorphosed,
and were examined for the presence of gill filaments and
shell brims.

Statistical analyses

Analyses of covariance ( ANACOVA) were conducted
for each experiment. Either temperature or salinity were

378

K. M. ZIMMERMAN AND J. A. PECHENIK

20°C

PRE -
COMPETENT

COMPETENT
25°C

PRE -
COMPETENT

COMPETENT

29UC

PRE -

COMPETENT

400

n n n am a nco(zinii a

4 A •*••*••***• AA

1)3

ana nan 13 nnrjonca

D p on a cnihn an

a n rzn

600

800 1000

SHELL LENGTH ( UM )

1200

1400

Figure 4. A comparison of the shell lengths of competent (D) and pre-competent (A) larvae ofCrcpidula
plana from Experiment IV. The points within each treatment represent the response of larvae from three
bowls (~60 larvae per bowl). Data were taken when larvae at 29°C were 1 1 days old (x = 50.0% larvae
competent; SD = 4.3); larvae at 25°C were 13 days old (x = 54.6% competent; SD = 6.4); larvae at 20°C
were 19 days old (x = 51.3% competent; SD = 14.1).

used as independent variables; age (days from hatch) was
the covariate; and one of the following was taken as the
dependent variable: percent of the larval population com-
petent to metamorphose, percent of the larval population
gilled, percent of the larval population with a complete
shell brim, or shell length (Table I) (Kleinbaum et a/.,
1988; SPSS, Inc. 1988). In Experiments I and II, the gill,
shell brim, and shell length data were obtained from larvae
in batch culture, whereas the rate at which larvae became
competent to metamorphose was determined with larvae
reared in glass bowls. In Experiments III-VI, all data were
obtained from the larvae reared in glass bowls. Percentage
data were arcsine transformed prior to subsequent anal-
ysis, using the formula for proportions with unequal sam-
ple sizes (Draper and Smith, 198 1 ).

Results

Effects oj temperature and salinity on survival

Larval survivorship was high at all temperatures in
Experiments I-IV, with the best survival, greater than
96%, occurring at the highest temperature tested (29°C)
(Table I).

However, larvae were intolerant of very low salinities
(Table II). Within the first two hours at 4 and 8 ppt, larvae
were found clumped together with mucus, mainly on the

bottoms of the rearing bowls, with their velar lobes ex-
tended and velar cilia moving; all treatment bowls at
higher salinities (14. 5, 19, 25, 29 ppt) contained swimming
larvae. On the second day, at 4 and 8 ppt, velar lobes
appeared smaller and velar cilia were less visible. By the
third day, all larvae in the 4 ppt seawater had died and
only two larvae out of the initial 65 survived at 8 ppt.
Larval survivorship was good at salinities of 19 ppt and
above, particularly in the second salinity experiment (Ta-
ble I, Experiment VI).

Effects of temperature on rates of growth ana"
morphological differentiation

Temperature had no significant effect on size-specific
organic weight at 20 and 25°C and at 20 and 29°C (/-
tests between slopes. P > 0.10, t = 0.69, d.f. = 30 and /
= 0.26, d.f. = 42, respectively). Thus, a given change in
shell length reflected comparable growth (in organic
weight) for larvae at 20 and 25°C, and at 20 and 29°C.
However, a given change in shell length reflected greater
growth (in organic weight) for larvae at 25°C as compared
to larvae reared at 29°C (/-tests, P < 0.05, t = 2.05,
d.f. = 50).

The effect of temperature on larval growth rate varied
markedly among experiments (Experiments I-IV, Tables
I and III). There were differences both in the average

COMPETENCE TO METAMORPHOSE

379

Table IV

Influence of temperature on age and size at spontaneous
metamorphosis in glassware cleaned daily (clean bowls) and the delay
period liuinilvr o! days between when >0c"f of the population was
competent and the mean age at metamorphosis in clean howls)

Mean age (days) at

spontaneous Delay

Experiment Temperature metamorphosis (clean) X ± SD period

number (°C) (n) (days)

I 29

19.32 ± 4.9
(75) A

7.16

1 25

24.66 ± 3.8
(98) B

5.67

1 20

28.66 ± 3.8
(100)C

5.26

11 29

18.09 ± 5.4
(99) A

5.79

11 25

26.86 ± 4.9
(44) B

9.86

11 20

30.96 ±3.2
(73) C

12.00

111 29

14.49 ± 2.2
(306) A

4.79

III 25

16.81 ± 1.6
(214) B

3.40

IV 29

16.79 ± 1.7
(24) A

5.79

IV 25

18.54 ± 1.4
(24) B

5.90

IV 20

24.85 ± 2.6
(39) C

6.21

Within each column, letters following sample sizes signify significantly
(P < 0.05) different means within experiments.

amount of daily growth at a temperature and in how tem-
perature affected relative growth rates. For example, larvae
grew the slowest (28 ^m-day"1) at 29°C in Experiment
I, but grew the fastest at 29°C (28 ^m • day"1) in Exper-
iment II (Table I). Over all experiments, average growth
rates ranged between 19 ^m • day ' (Experiment II, 20°C)
and 59 Mm-day ' (Experiment III, 29°C).

Larvae generally developed gill filaments and shell
brims more rapidly at higher rearing temperatures, al-
though rates of gill and brim formation were independent
of temperature in the first two experiments (Table III).
Note that in Experiment IV the effects of temperature on
growth rates did not parallel those on morphological dif-
ferentiation rates. These data indicate that gill formation,
brim formation, and growth rate were affected similarly
by rearing temperature in only one of the four experiments

(Experiment III); only two temperature treatments were
tested in that experiment.

Larvae typically became gilled between about 620-820
^m and brimmed between about 710-850 nm, with no
consistent influence of rearing temperature or feeding fre-
quency. Some larvae within the populations were fully
gilled and brimmed before other larvae in the same pop-
ulation became gilled, indicating much individual vari-
ation in rates of morphological development within each
temperature treatment.

All but seven metamorphosed individuals — out of
thousands of metamorphosed snails examined in these
experiments — had conspicuous gills, suggesting that most
larvae developed gills before they became competent to
metamorphose. The seven gill-less juveniles were all found
at 29°C (Experiments I and II).

Elevated KCl concentration stimulates metamorphosis

Response to elevated KCl was rapid. Of those 22-day-
old individuals (22°C) that eventually responded, in-
creasing KCl concentrations by 20 iruY/ induced at least
90% to metamorphose within 6 h (Fig. 1). Thus, in all
subsequent experiments, larvae were exposed to elevated
KCl concentrations for 6 h to assess metamorphic com-
petence, defined here by the response to elevated potas-
sium.

Effect of temperature on rates of becoming competent to
metamorphose

Despite the unpredictable effects of rearing temperature
on rates of growth and morphological differentiation, in-
creasing larval rearing temperature significantly increased
(P < 0.001) the rates at which larvae became competent
to metamorphose in all experiments (Tables I, III;
Figs. 2, 3).

Larval shell length was a poor indicator of whether a
larva was competent to metamorphose. Although com-
petent larvae were, on average, significantly larger (P
< 0.000 1 ) than pre-competent larvae of the same age and
rearing history, shell lengths of competent and pre-com-
petent larvae overlapped in all experiments, as exemplified
by Experiment IV (Fig. 4).

Effect of temperature on the maximum length oflan'al
life and period of delayed metamorphosis

At higher temperatures, larvae consistently exhibited
"spontaneous" metamorphosis sooner than at lower tem-
peratures (Table IV and Fig. 5). However, average growth
rates failed to predict rates of spontaneous metamorphosis
within a population. In Experiment I, for example, larvae
reared at 20°C or 25°C grew at equivalent rates but meta-
morphosed faster at the higher temperature (Fig. 5). Even

380

R. M. ZIMMERMAN AND J. A. PECHENIR

14 16

18 20 22 24 26 28 30

LARVAL AGE (DAYS FROM RELEASE)

32 34 36 38

Figure 5. Maximum length of larval life for Crepidula plana maintained in glass bowls, acid-washed
daily (Experiment I). Each point represents the mean of three replicates (110 larvae per treatment). Different
letters signify larval populations differing significantly in mean growth rates (A < B < C).

so, individuals exhibiting faster growth within a temper-
ature treatment tended to metamorphose sooner than
slower growing larvae reared at the same temperature,
confirming previous results (Lima and Pechenik, 1985)
(Fig. 6; regression analysis of log growth rate). Within
each temperature, faster growing individuals also tended
to metamorphose at larger shell lengths, although the data
do show considerable scatter (Fig. 7; P < 0.05 at each
temperature). Individual growth rates were estimated us-
ing age and size at metamorphosis (Lima and Pechenik,
1985).

Generally, larvae maintained in bowls cleaned only ev-
ery 48 h metamorphosed significantly sooner (P < 0.05;
/-test), by about 5-10 days, than larvae maintained in
bowls cleaned every 24 h, and at smaller shell lengths
[smaller by about 1 00-300 ^m (Zimmerman, 1989)]. This
indicates that microbial films formed over 48 h could trig-
ger larvae of C. plana to metamorphose, supporting pre-
vious reports (Lima, 1983).

The average delay period, defined here by the difference
(in days) between (a) mean age at "spontaneous" meta-
morphosis in bowls cleaned daily and (b) when 50% of a
larval population was competent to metamorphose, varied
between experiments, and was markedly altered by tem-
perature only in Experiment II (Table IV).

Effect of salinity on rates of growth and morphological
differentiation

The effects of salinity on growth rate differed in the
two experiments. In Experiment V (Table I), larvae grew
more quickly at higher salinities (by about 12 ^m • day"1
for each salinity increase above 1 9 ppt). In the three lowest
salinities (4, 8, and 14.5 ppt), larvae suffered high mortality

(85-100% at 4 and 8 ppt) and exhibited no detectable
growth. In Experiment VI, salinity significantly affected
mean growth rates, but not as dramatically as in Exper-
iment V, and not in direct proportion to salinity. Larvae
reared at 20 ppt grew significantly faster than larvae at 25
ppt in Experiment VI (Tables I. III). In both salinity ex-
periments, larvae reared in full strength seawater (either
29 or 30 ppt) grew at rates comparable to those of larvae
reared under comparable conditions (25°C, full strength
seawater) in Experiments I-IV (Table I).

Salinity over the range of 20-30 ppt had negligible ef-
fects on rates of gill formation (Tables I, V) and on the
shell sizes at which larvae became either gilled or
brimmed. Larvae became gilled and brimmed at shell sizes
between 628-728 /urn and 699-790 ^m, respectively, re-
gardless of rearing salinity. However, every increase in
rearing salinity increased rates of shell brim formation
(Table III). The pattern of significant salinity effects on
rates of growth and on rates of gill and brim formation
(Table III) indicates that rates of growth and morpholog-
ical differentiation were not affected similarly by changes
in salinity.

The relative effect of salinity on rates of becoming
competent to metamorphose and rates of growth

Despite the erratic influence of salinity on rates of
growth, gill, and shell brim formation, larvae reared at
higher salinities typically became competent to meta-
morphose sooner than those reared at lower salinities in
both Experiments V and VI (Tables I-III). These results
suggest that changes in salinity may uncouple rates of
growth, rates of morphological differentiation, and rates
of becoming competent to metamorphose. In Experiment

COMPETENCE TO METAMORPHOSE

381

41.1

•i

INDIVIDUAL GROWTH RATE CJJM-DAY )

29°C

25°C

20°C

Figure 6. Maximum length of larval life as a function of estimated individual growth rate (^rn-day ')
in Experiment IV (r = 0.74, y = - 16.4(ln x) + 88.7). Individual growth rates were estimated from the size
and age at which each individual underwent spontaneous metamorphosis in glass bowls that were cleaned
daily. Larvae were cultured at three temperatures, as indicated (n = 64, 62. and 63 larvae per treatment at
29°C, 25°C and 20°C, respectively).

VI, for example, larvae grew more rapidly at 20 ppt than
at 25 ppt, but took longer to become competent at the
lower salinity (Table III and Fig. 8). Experiment VI was
terminated before all larvae were allowed to metamor-
phose, so calculation of age and size at metamorphosis
was not possible.

There was no significant difference (P > 0.05) in the
percentage of larvae induced to metamorphose when KC1
concentrations were elevated by 20 versus 23 mAl at 20
ppt. Thus, the dilution of full strength seawater to make
20 ppt and 25 ppt seawater did not significantly affect the
ability of KC1 to induce larval metamorphosis.

Discussion

The primary goal of these experiments was to deter-
mine, for Crepidula planu. whether changes in tempera-
ture and salinity alter rates of growth, morphological dif-
ferentiation, and the onset of competence equally. We
must first consider the effects of temperature and salinity
on each of these three components of development in-
dividually.

Larvae grew significantly faster at progressively higher
temperatures (Table I) in only one experiment (Experi-
ment III). Lima and Pechenik (1985) also found an in-

1600
1500
1400
1300
1200

1100
1000

o *

INDIVIDUAL GROWTH RATE (UM DAY )

29"C

25°C

20°C
A

Figure 7. Size at metamorphosis as a function of individual larval growth rate (^m - day ' ) (Experiment
IV). Faster growing larvae tended to spontaneously metamorphose at larger shell sizes (P < 0.05: r = 0.549
at 29°C, n = 64; r = 0.512 at 25°C. n = 62; r = 0.51 1 at 20°C n = 63; combined r = 0.249, n = 189).
Growth rates were estimated from size and age at spontaneous metamorphosis. Larvae were reared at three
temperatures, as indicated.

382

K. M. ZIMMERMAN AND J. A. PECHENIK

Table V

hv nl temperature and salinity on percent changes in rates of shell growth, morphological differentiation, becoming competent to
metamorphose, and spontaneous metamorphosis lor nil experiments

Experiment
number

Temperature, salinity
(°C) (ppt)

Growth rate
(^m -day"')

1 • (Time to 50%
competent) '

1 • (Time to 50%

gilled) '

1 -(Time to 50%
bnmmedr1

1 -(Time to 50%
spontaneously
metamorphosed)"1

20°C. 30 ppt

25°C, 30 ppt

+ 18%

+ 19%

—

+21%

29°C. 30 ppt

-16%

+48%

—

+48%

20°C. 30 ppt

—

25°C. 30 ppt

+ 17%

+ 10%

—

+ 11%

29 °C, 30 ppt

+45%

+35%

—

+48%

III

25 °C. 30 ppt

—

29°C, 30 ppt

+ 14%

+28%

+6%

+24%

+ 18%

20°C, 30 ppt

—

25°C. 30 ppt

+46%

+32%

+25%

+34%

+22%

29°C, 30 ppt

+38%

+41%

+38%

+29%

25°C. 8 ppt

—

25°C. 14.5 ppt

+ 12%

+20%

—

25°C, 19 ppt

+233%

+3.3%

—

25°C, 25 ppt

+ 334%

+ 10%

—

25°C. 29 ppt

+438%

+37%

—

25°C, 20 ppt

—

25°C, 25 ppt

-9%

+20%

+5%

+4%

—

25°C, 30 ppt

+ 12%

+35%

+11%

+6%

—

Each percent change was calculated relative to the lowest temperature, salinity treatment in a particular experiment.

consistent effect of temperature on larval growth rate for
larvae of C. plana fed T-ISO, and the larval growth rates
reported here for C. plana are generally comparable to
those previously reported by Lima and Pechenik (1985)
for larvae reared at identical food concentrations and
temperatures. In our Experiment III, however, larvae
reared at 29°C and 25°C grew 1.5-2.0 times faster than
those reared by Lima and Pechenik (1985) under the same
conditions. Lima and Pechenik (1985) reported compar-
ably high larval growth rates (exceeding 50 /im-day"')
for C. plana reared at 25 °C and 29 °C on a different naked
flagellate, Isochrysis galbana (clone ISO). Larvae of the
congener C. fomicata also grow at rates exceeding 50
nm • day ', at temperatures above 24°C (Lucas and Cost-
low, 1979; Pechenik, 1984; Pechenik and Lima, 1984).

Salinity also influenced larval growth rates, although
the effects were often inconsistent between the two ex-
periments (Tables I, III). Larvae grew faster at progres-
sively higher salinities in Experiment V, but not in Ex-
periment VI (Tables I, III). As with other molluscan larvae,
including the congener C. fomicata (Davis, 1958; Davis
and Ansell, 1962; Davis and Calabrese, 1964; Scheltema.
1965; Calabrese and Rhodes, 1974; Robert el ai. 1988;
Hisi'/fl/., 1989), those of C. plana grew poorly at salinities
below about 20 ppt.

The influence of temperature on rates of morphological
differentiation also varied from one experiment to the
next (Table III). This contrasts with results reported for
C. fomicata by Pechenik and Lima (1984) and Pechenik
(1984), who found that larvae always tended to develop
gills and shell brims more rapidly at higher temperatures.
We have no way of knowing whether the inter-experiment
variation we report for C. plana reflects genetic differences
in the larval populations used, subtle differences in rearing
conditions among experiments, or differences in the
physiological history of the adults that released the larvae
used in these experiments (Bayne ct ai. 1975). Increases
in salinity did not predictably alter rates of gill formation
in Experiment VI (Table III), but shell brims formed more
rapidly at higher salinities.

As reported previously for larvae of C. plana and C.
fornicala (Pechenik and Lima, 1984; Lima and Pechenik,
1985), and for larvae of the blue mussel AI. edidis (Pech-
enik ct al., 1990), temperature apparently altered rates of
growth and morphological development to different de-
grees. In our studies, this is suggested by the fact that
larvae tended to develop shell brims and visible gill fila-
ments at different sizes when reared at different temper-
atures. For example, in Experiment I, larvae formed vis-
ible gill filaments on average between 681 and 721 ^m.

COMPETENCE TO METAMORPHOSE

383

30 PPT

10 11 12 13 14 15 16 17 19 19 20 21 22

LARVAL AGE (DAYS FPOM RELEASE)

Figure 8. Influence of salinity on the rate at which larvae became competent to metamorphose in
Experiment VI. Each point represents the mean percentage of larvae competent in three howls, with 20-23
larvae tested per bowl. Vertical bars represent one SD about the mean. Different letters represent larval
populations with different mean growth rates (A < B < C).

between 675 and 817 ^m, and between 742 and 786 ^m,
at 29°C, 25°C. and 20°C, respectively. Rates of shell
growth would have to be altered by temperature in exact
proportion to any changes in rates of morphological de-
velopment if larvae are to form gills and shell brims at
comparable average sizes in all rearing conditions (Pech-
enik and Lima, 1984; Pechenik el ai. 1990).

Rates of shell growth and morphological differentiation
were also affected to different degrees by salinity. For ex-
ample, in Experiment VI, larvae grew significantly faster
at 20 ppt than at 25 ppt, but the salinity decrease did not
affect rate of gill formation. Indeed, rate of gill formation
was not affected by salinity over the range tested. In con-
trast, shell brims formed faster at the higher salinity.

Despite the generally unpredictable effects of temper-
ature on rates of larval growth and morphological differ-
entiation both among and, often, within experiments, the
influence of temperature on the rates at which larvae be-
came competent to metamorphose was remarkably con-
sistent among all four experiments; larvae always became
competent to metamorphose faster when reared at higher
temperatures (Table III and Fig. 3). Rates of becoming
competent to metamorphose were clearly uncoupled from
rates of morphological differentiation and shell growth.
In Experiment I, for example, larvae reared at 29 °C be-
came competent significantly sooner (and often at smaller
sizes) than larvae reared at 20°C or 25°C, despite signif-
icantly slower average growth for larvae reared at the
higher temperature (Figs. 2, 4). In addition, larvae reared
at 25 °C became competent significantly sooner than lar-
vae at 20°C, even though these larvae did not grow at
significantly different rates at the two temperatures.

The same was true of the experiments (V and VI) ex-

amining the influence of salinity. Here again, larvae reared
at higher salinities generally became competent faster,
while rates of growth and morphological differentiation
were not so predictably affected. For example, in Exper-
iment VI, larvae reared at 20 ppt grew significantly faster
than larvae at 25 ppt, but those larvae reared at 20 ppt
became competent at slower rates (Table III and Fig. 8).
The influence of temperature or salinity on the amount
of time required for larvae in a population to become
competent clearly cannot be predicted from the effects of
environmental change on rates of growth (Table V). In-
dividual competence also cannot be predicted on the basis
of shell length (Fig. 4) or the presence of a shell brim or
visible gill filaments; at least some gill-less larvae were
induced to metamorphose by elevating KG concentration
(at 29°C, Experiments I and II). Also, in every experiment,
at every temperature, some larvae without shell brims
could be induced to metamorphose. Similarly, neither
shell size nor morphological indicators were adequate
predictors of whether individual blue mussel larvae would
or would not attach to filamentous substrates in the lab-
oratory (Eyster and Pechenik, 1987), or when oyster larvae
(Crassostrea gigas) would exhibit settlement behavior in
response to L-DOPA (Coon el al, 1990).

Variation in the rates at which individuals became
competent to metamorphose within treatments (as in Figs.
2 and 8) may be a natural phenomenon that encourages
larvae released from an individual female to metamor-
phose at different times, likely increasing the spread of
siblings among different populations (Strathmann, 1974;
Hadfield, 1977) and minimizing their competition for
food and space as juveniles.

In our experiments, larvae consistently underwent

384

K. M. ZIMMERMAN AND J. A. PECHENIK

32
30

28
26
24

20
18

16
14

DAYS TO 50 J LARVAL POPULATION COMPETENT TO METAMORPHOSE

Figure 9. Influence of temperature on the relationship between the maximum length of larval lite and
the time required for 50% of a population to become competent. Larvae ofCrepidulu plana were reared at
three temperatures, as indicated. For each temperature, different bars represent data from different experiments.

-V

„

I \ \

19 21 23

"spontaneous" metamorphosis sooner at higher temper-
atures (Fig. 5 and Table IV). This phenomenon of mol-
luscan larvae metamorphosing sooner in warmer tem-
peratures has been noted previously (Loosanoff, 1959:
Davis and Ansell, 1962; Davis and Calabrese, 1964;
Bayne. 1965; Pechenik, 1984; Pechenik and Lima, 1984;
Lima and Pechenik, 1985). This relationship is consistent
with the hypothesis that the timing of spontaneous meta-
morphosis is determined by the rate at which larvae pro-
gress through a developmental program with a fixed end-
point (Pechenik, 1980, 1984; Pechenik and Lima, 1984);
the endpoint could be determined by some endogenous
controlling factor or, as suggested recently by Coon et al.
( 1990), could reflect a gradually increasing sensitivity of
receptors for an external chemical cue present naturally
in extremely low concentrations. Although mean growth
rates were not adequate indicators of the rates at which
larvae within a population would undergo spontaneous
metamorphosis (Fig. 5), faster growing individuals did
tend to exhibit spontaneous metamorphosis sooner than
slower growing individuals (Fig. 6). These results are sim-
ilar to those reported previously for this species (Lima
and Pechenik, 1985), for the congener C.fornicata (Pech-
enik, 1984; Pechenik and Lima, 1984), and for the bivalves
Mercenaria merccnaria (Loosanoff, 1959) and M. cctulis
(Beaumont and Budd, 1982).

Despite the pronounced influence of temperature on
rates of becoming competent and rates of spontaneous
metamorphosis (Tables III, IV), temperature had a minor
influence on the length of time that larvae of C. plana

delayed metamorphosis in frequently cleaned glass bowls
in all but one experiment (Table IV and Fig. 9). Only in
Experiment II did temperature affect delay period by more
than one or two days (Table IV). Thus, although larvae
of C. plana will have a longer pre-competent period at
lower temperatures, the capacity for delaying metamor-
phosis in the absence of suitable substrate may be affected
to a much lesser degree.

In our experiments, larvae metamorphosed "sponta-
neously" about 3.5-12 days after becoming competent,
with most delay periods lying between about 5 and 7 days
(Table IV). These data are comparable to earlier laboratory
estimates of delay potential for larvae of this species reared
at comparable temperatures (Lima and Pechenik, 1985:
their Table II), and seem to confirm the reduced capacity
of C. plana for postponing metamorphosis relative to that
exhibited by larvae of C. fornicata; the maximum delay
period of about 20-30 days suggested for C. fornicata
(Pechenik, 1984) is only an estimate, however, and has
not yet been confirmed. The estimates of delay potential
for C. plana reared at different temperatures given by Lima
and Pechenik (1985) were based on the assumption that
competence is attained at a particular shell length. The
good agreement between their estimates and our more
direct determinations suggest that although individual
larvae clearly do not become competent to metamorphose
at a particular size, the simplifying assumption of length-
related competence may permit adequate predictions at
the population level.

Clearly, the various aspects of morphological and

COMPETENCE TO METAMORPHOSE

385

physiological development are affected to different degrees
in C. plana by temperature and salinity changes. In only
one experiment (Experiment III) did increased rearing
temperature significantly increase all components of de-
velopmental rate that were monitored: shell and tissue
growth, timing of gill differentiation and shell brim de-
velopment, and onset of metamorphic competence. The
likely impact of environmental factors on larval dispersal
periods therefore cannot be estimated from data on rates
of growth or morphological development, but clearly must
be determined directly. Our data suggest that changes in
temperature and salinity will have a more consistent in-
fluence on duration of pre-competent and competent pe-
riods of development than on either rates of shell growth
or rates of morphological differentiation.

Acknowledgments

This research was completed in partial fulfillment of
the requirements for the degree of Master of Science to
K. M. Zimmerman. Summer support for K. M. Zim-
merman was provided by NSF Grant OCE-8500857 to
J. A. Pechenik. We thank Durwood Marshall for advising
on analysis of covariance and Carol Valente and Valerie
Ricciardone for typing the manuscript. The manuscript
has benefited from the suggestions of two anonymous re-
viewers.

Literature Cited

Bayne, B. L. 1964. The responses of the larvae ofMylilus edulis L. to
light and to gravity. Oikos 15: 162-174.

Bayne, B. L. 1%5. Growth and delay of metamorphosis of the larvae
ofMytilw; edulis L. Ophelia 2(1): 1-47.

Bayne, B. L. 1971. Some morphological changes that occur at the
metamorphosis of the larvae of Mylilus edulis. Pp. 259-280 in Fourth
European Marine Biology Symposium. D. J. Crisp, ed. Cambridge
University Press. Cambridge.

Bayne, B. L., P. A. Gabbott, and J. Widdovts. 1975. Some effects of
stress in the adult on the eggs and larvae of Mytilus edulis L. J Mar.
Biol. Assoc. V.K. 55: 675-689.

Beaumont, A. R., and M. D. Budd. 1982. Delayed growth of mussel
(Mytilus edulis) and scallop (Pecten maximus) veligers at low tem-
peratures. Mar. Biol. 71: 97-100.

Butman, C. A., J. P. Grassle, and C. M. Webb. 1988. Substrate choices
made by marine larvae settling in still water and in a flume flow.
Nature 333: 771-773.

Calabrese, A., and E. \V. Rhodes. 1974. Culture of Mu/inia laleralis
and Crepidula fornicata embryos and larvae for studies of pollution
effects. Thalassia Jugoslav. 10(1/2): 89-102.

Chia. F. S. 1978. Perspectives: settlement and metamorphosis of marine
invertebrate larvae. Pp. 283-285 in Settlement ami Metamorphosis
of Marine Invertebrate Larvae. F. S. Chia and M. E. Rice, eds. Elsevier,
New York.

Coon, S. L., D. B. Bonar, and R. M. Weiner. 1985. Induction of set-
tlement and metamorphosis of the pacific oyster. Crassostrea gigas
(Thunberg), by L-DOPA and catecholamines. J. Exp. Mar. Biol
Ecol. 94:211-221.

Coon, S. L., W. K. Fitt, and D. B. Bonar. 1990. Competency and delay

of metamorphosis in the Pacific oyster Crassostrea gigas. Mar. Biol.

106: 379-387.

Crisp, D. J. 1974. Factors influencing the settlement of marine inver-
tebrate larvae. Pp. 177-265 in Chemoreception in Marine Organisms.

P. T. Grant and A. M. Mackie, eds. Academic Press, New York.
Davis, H. C. 1958. Survival and growth of clam and oyster larvae at

different salinities. Biol Bull 114(3): 296-307.
Davis, H. C., and A. D. Ansell. 1962. Survival and growth of larvae

of the european oyster, O edulis. at lowered salinities. Biol. Bull.

122: 33-39.
Davis, H. C., and A. Calabrese. 1964. Combined effects of temperature

and salinity on development of eggs and growth of larvae of M. mer-

cenaria and C. virninica. U S Fish Wild. Fish. Bull. 63(3): 643-

655.
Draper, N. R., and H. Smith. 1981. Applied Regression Analysis. John

Wiley and Sons. Inc.. New York. 567 pp.
Eyster, L. S., and J. A. Pechenik. 1987. Attachment ofMylilus edulis

L. larvae on algal and hyssal filaments is enhanced by water agitation.

/ Exp. Mar Biol Kail. 114: 99-1 10.
Fitt, W . K., S. L. Coon, M. Walch, R. M. Weiner, R. R. Colwell, and

D. B. Bonar. 1990. Settlement behavior and metamorphosis of

oyster larvae (Crassostrea gigas) in response to bacterial supernatants.

Mar. Biol 106: 389-394.
Hadfield, M. G. 1977. Chemical interactions in larval settling of a

marine gastropod. Pp 403-4 1 3 in Marine Natural Products Chemistry.

D. J. Faulkner & W. H. Fenical. eds. Plenum Publishing Corp., New

York.
Hadfield, M. G. 1978. Metamorphosis in marine molluscan larvae: an

analysis of stimulis and response. Pp. 165-175 in Settlement and

Metamorphosis of Marine Invertebrate Larvae. F. S. Chia and M. E.

Rice, eds. Elsevier, New York.
Hickman, R. W., and LL. D. Gruffydd. 1971. The histology of the

larva ofOstrea edit/is during metamorphosis. Pp. 28 1-294 in Fourth

European Marine Biology Symposium. D. J. Crisp, ed. Cambridge

University Press, Cambridge.
His, E., R. Robert, and A. Dinct. 1989. Combined effects of temperature

and salinity on fed and starved larvae of the Mediterranean mussel

Mytilus galloprovincialis and the Japanese oyster Crassostrea gigas.

Mar. Biol 100: 455-463.
Jackson, G. A., and R. R. Strathmann. 1981. Larval mortality from

offshore mixing as a link between precompetent and competent pe-
riods of development. .!w. Nat. 118(1): 16-26.
Kirchman, D., S. Graham, D. Reish, and R. Mitchell. 1982. Bacteria

induce settlement and metamorphosis of Janua (Dexiospira) brasil-

iensisGrube (Polychaeta:Spirorbidae). J. Exp Mar. Biol. Ecol. 56:

153-163.
Kleinbaum, D. G., L. L. Kupper, and K. E. Muller. 1988. Applied

Regression Analysis and Other Multivariate Methods. PWS-Kent.

Boston. 718 pp.
Lima, G. M. 1983. The relationship of temperature on growth rates

and the length of larval life of the gastropod. Crepidula plana Say.

Master's Thesis, Tufts University. Medford. MA. 1 15 pp.
Lima, G. M., and J. A. Pechenik. 1985. The influence of temperature

on growth rate and length of larval life of the gastropod. Crepidula

plana (Say). J. Exp. Mar. Biol. Eeol. 90: 55-71.
Loosanoff, V. L. 1959. The size and shape of metamorphosing larvae

of I 'emu (Mereenaria) mercenarm grown at different temperatures.

Biol. Bull 117: 308-318.
Lucas, J. S., and J. D. Costlow, Jr. 1979. Effects of various temperature

cycles on the larval development of the gastropod mollusc Crepidula

fornicata. Mar. Biol 51: 111-117.
McGee, B. L., and N. M. Target!. 1989. Larval habitat selection of

Crepidula (L.) and its effect on adult distribution patterns. J. Exp.

Mar. Biol. Ecol. 131: 195-214.
Meadows, P. S., and J. I. Campbell. 1972. Habitat selection by aquatic

386

K. M. ZIMMERMAN AND J. A. PECHENIK

invertebrates. Pp. 27 1 -36 1 in Advances in Marine Biology, F. S. Rus-
sell and M. Yonge, eds. Academic Press, New York.

Miller. S. E. and Hadfield, M. G. 1986. Ontogeny of phototaxis and
metamorphic competence in larvae of the nudihranch Phestilla si-
bogae Bergh (Gastropoda:Opisthobranchia). J. Exp Mar. Biol. Ecol.
97:95-112.

Pechenik, J. A. 1980. Growth and energy balance during the larval
lives of three prosobranch gastropods. J. Exp. Mar. Biol. Ecol. 44:
1-28.

Pechenik, J. A. 1984. The relationship between temperature, growth
rate, and duration of planktonic life for larvae of the gastropod Cre-
pidula Jormcala (L.). J Exp. Mar. Biol. Ecol. 74: 241-257.

Pechenik, J. A., and N. S. Fisher. 1979. Feeding, assimilation and
growth of mud snail larvae, Nassarius obsoletus (Say), on three dif-
ferent algal diets. J. Exp. Mar. Biol. Ecol. 38: 57-80.

Pechenik, J. A., and G. M. Lima. 1984. Relationship between growth,
differentiation, and length of larval life for individually reared larvae
of the marine gastropod, Crepidula fomicata. Biol. Bull. 166: 537-
549.

Pechenik, J. A., and \V. D. Heyman. 1987. Using K.CI to determine
size at competence for larvae of the marine gastropod Crepidula for-
mcaia (L.). J. Exp. Mar. Biol. Ecol. 112: 27-38.

Pechenik, J. A., L. S. Eyster, J. Widdows, and B. L. Bayne. 1990. The
influence of food concentration and temperature on growth and
morphological differentiation of blue mussel Mytilux edulis L. larvae.
J Exp. Mar. Biol. Ecol. 136: 47-63.

Robert. R., E. His, and A. Dinet. 1988. Combined effects of temperature
and salinity on fed and starved larvae of the European flat oyster
Oslrea edulis Mar. Biol. 97: 95-100.

Scheltema, R. S. 1965. The relationship of salinity to larval survival

and development in Nassarius obsok'lus (Gastropoda). Biol. Bull.
65: 340-354.

Scheltema, R. S. 1974. Biological interactions determining larval set-
tlement of marine invertebrates. Thalassia Jugslav. 10(1/2): 263-
296.

SPSS, Inc. 1988. SPSS-X User's Guide. Third Edition. SPSS. Inc..
Chicago. 1072 pp.

Strathmann. R. 1974. The spread of sibling larvae of sedentary marine
invertebrates. Am. Nat. 108(959): 117-123.

Switzer-Dunlap, M., and M. G. Hadfield. 1977. Observations on de-
velopment, larval growth and metamorphosis of four species of
Aplysiidae (Gastropoda: Opisthobranchia) in laboratory culture. J.
Exp. Mar. Biol. Ecol. 29: 245-261.

Weiner, R. M., M. Walch, M. P. Labare, D. B. Bonar, and R. R. Colwell.
1989. Effect of biofilms of the marine bacterium Alteromonas col-
welliana (LST) on set of the oysters Crassostrea gigas (Thunberg, 1793)
and C. \-irxinica (Gmelm, 1 79 1 ). / Shellfish Res. 8( 1 ): 117-1 23.

YooL, A. J., S. M. Grau, M. G. Hadfield, R. A. Jensen, D. A. Markell.
and D. E. Morse. 1986. Excess potassium induces larval metamor-
phosis in four marine invertebrate species. Biol. Bull. 170: 255-266.

Zimmerman. K. M. 1989. Differential effects of temperature and salinity
on the growth and differentiation of larvae of the marine gastropod
Crepidula plana. Master's Thesis, Tufts University. Medford. MA.
89pp.

Reference: Biol. Bull 180: 387-393. (June, 1991)

Predation Risk and Avoidance Behavior
in Two Freshwater Snails

JAMES E. ALEXANDER, JR.1 AND ALAN P. COVICH

Department of Zoology. University of Oklahoma, Norman, Oklahoma 73019

Abstract. We examined the predator avoidance behav-
iors of two common freshwater snails, Physella virgata
and Planorbella trivolvis, to the crayfish Procambams
simulans. In response to crayfish predation, the snails
crawled above the waterline for several hours, then re-
turned to the water. A significant size-dependent rela-
tionship existed between crawlout (vertical migration
above the waterline) and vulnerability to predation. All
observed size classes of P. virgata, and small P. trivolvis,
were vulnerable and crawled out in response to crayfish
predation. Large, invulnerable P. trivolvis did not display
any overt avoidance behavior, but relied instead on strong
shell architecture for defense. We suggest that, in these
species, crawling above the waterline reduces the proba-
bility of an encounter between vulnerable thin-shelled
snails and crayfish. This behavior is an adaptive response
to predation.

Introduction

Predation is an important cause of evolutionary change
in many prey taxa (Vermeij and Covich, 1978; Vermeij,
1982a, b). Predators influence their prey populations in
various ways; one aspect of predation in freshwater sys-
tems that is receiving increasing attention is the behavioral
interactions that occur between predator and prey (Pec-
karsky, 1984; Sih, 1984). The relative impact of inverte-
brate predators on freshwater snails, and the responses of
the snails to their predators have frequently been studied
(Townsend and McCarthy, 1980; Covich, 1981; Brown
and DeVries, 1985; Lodge et at.. 1987; Brown and Strouse,

Received 2 November 1990; accepted 19 March 1991.

' To whom communications should be sent. Present address: De-
partment of Biology. Box 19498, The University of Texas at Arlington,
Arlington, TX 76019.

1988; Crowl and Covich, 1990; Crowl, 1990; Hanson et
a/.. 1 990; Kesler and Munns, 1 990; Alexander and Covich,
1991). Freshwater snails exhibit predator avoidance
mechanisms, such as burying into substrata, and crawling
into vegetation or above the waterline (Snyder, 1967;
Townsend and McCarthy, 1980; Alexander and Covich,
1991).

Comparative studies on a variety of animals have shown
that closely related or co-occurring species may respond
differently to a predator. In other situations, juveniles or
smaller individuals that are vulnerable to predators show
stronger antipredator responses than larger, older, or other,
relatively less vulnerable prey (Stein, 1977; Schmitt, 1982;
Sih. 1982, 1986; Werner and Hall, 1988). In these studies,
prey appear to assess the tradeoffs between predation risk
and foraging for food; i.e.. the vulnerable species or size
classes forage in different habitats, or at different times,
than the invulnerable prey. Comparative studies, by re-
vealing the variety and relative effectiveness of antipreda-
tor responses, help to elucidate the adaptive nature of a
response. In this paper, we describe the predator avoidance
response of two common, co-occurring freshwater snail
species, Physella virgata (Pulmonata, Physidae, Fig. 1A)
and Planorbella trivolvis (Pulmonata, Planorbidae, Fig.
IB, C), to their predator, the crayfish Procambams simu-
lans (Decapoda, Astacidae). In another paper (Alexander
and Covich, 1 99 1 ), we demonstrated that Physella virgata
performs a chemically mediated predator avoidance be-
havior (crawling above the waterline for a minimum of
2 h) in response to an actively foraging crayfish predator.
Physella virgata appears to react to chemicals emanating
from crayfish and from injured conspecifics. In this study,
we demonstrate a size-dependent avoidance response that
corresponds to the relative vulnerability of a snail to cray-
fish predation.

387

388

J. E. ALEXANDER, JR. AND A. P. COVICH

2mm

Figure 1. The shell morphology of Plmclla viruuta (A) and Planor-
bella tri\-olvis (B and C). The size bar is 2 mm.

Materials and Methods

Study site and general methods

The snails and crayfish used in this study were collected
from Oliver Wildlife Preserve (Norman, Oklahoma).
Oliver Wildlife Preserve is a forested area on the South
Canadian River floodplain that is inundated periodically
by runoff and heavy spring rains. The middle third of the
preserve typically remains under water throughout the
late winter to early summer months (December to June)
and supports large populations of P. virgata. P. trivolvis,
and P. simulans (Alexander. 1987). Woody debris in
Oliver Wildlife Preserve provide abundant substrata onto
which the snails migrate to avoid predators; snails were
observed above the waterline throughout the year at Oliver
Wildlife Preserve and at other sites (pers. obs.).

Laboratory experiments were conducted at night, in
darkness, simulating the natural conditions under which
crayfish are most active. No substratum was included in
these experiments. For the handling time and ingestion
probability experiments (Experiment 1). where crayfish
and snails were under continuous observation, low inten-
sity red light was used to facilitate observations. In the
second experiment, low intensity white light was used
briefly to record observations. When not used in experi-
ments, snails were maintained in 40-80-1 aquaria and fed
commercial fish food (TetraMin) and lettuce ad libitum.
Crayfish were housed individually in 4-1 plastic containers
and fed fish food pellets and lettuce ad libitum. Crayfish
were starved for at least 24 h prior to the start of the
experiments.

Experiment 1: differential vulnerability of
Physella and Planorbella

This experiment was aimed at examining the ability of
P. simulans to handle and ingest different size classes of

P. virgata and P. trivolvis. A 10-1 aquarium was placed so
that the actions of the crayfish and snails could be observed
under low intensity red light illumination, regardless of
their position in the aquarium. The snails were sorted
according to shell length (SL). in 1-mm increments (±0.5
mm), ranging from 5 to 12 mm. For each observation,
50 snails of one size class and species were placed in the
aquarium in 2 1 of previously aerated tap water. One adult
P. simulans [carapace length (CL) = 28-36 mm] was then
added to the aquarium. Two variables were recorded dur-
ing the observation period: (a) handling times (time spent
consuming a prey), and (b) ingestion probabilities (if a
snail was eaten, rejected, or had escaped from the predator
once captured). The crayfish (n = 6) were tested with all
size classes of both species, randomly, during 15-min ob-
servation times, over a 2-week period. Crayfish were ob-
served feeding on one size class of one snail species in all
observation periods. Handling time was defined as the
period including the capture of the snail, the consumption
of the snail, the crayfish cleaning its mouthparts, and the
movement forward by the crayfish to continue foraging.
Each snail capture was noted, as well as the number of
snails that were either consumed or rejected. The ratio oi
number of snails eaten to the number of snails captured
was defined as the ingestion probability.

Experiment 2: size-mediated predator avoidance

To examine the relationship between snail size, pre-
dation vulnerability, and avoidance behavior in both snail
species, P. virgata and P. trivolvis were sorted into five
size categories (4.1-6.0, 6.1-8.0, 8.1-10.0. 10.1-12.0, and
12.1-16.0 mm SL). A total of 100 snails of one species
was added to each 40-1 aquarium (25 X 50 X 30 cm) with
5 1 of previously aerated tap water. Due to unequal num-
bers available from the field in each size class, the size
class categories contained unequal numbers of snails. With
P. virgata, the numbers of snails per size class added were:
10, 30, 30, 25, and 5 snails in each of the increasing size
classes, respectively. With P. trivolvis. the numbers of
snails per size class were: 30. 30, 20, 10, and 10 snails in
each of the increasing size classes, respectively.

To half of the eight replicates per snail species, one
adult (CL = 30-40 mm) P. simulans was added at 2200
h. The other four replicates served as predator- free con-
trols. The crayfish were allowed to feed without interrup-
tion for 2 h in total darkness, then the number of snails
out above the waterline. as well as the number of snails
eaten, were determined for each size class and species.
Because all five snail class sizes were included in each
aquarium, a split-plot ANOVA examined the effects of
the two independent variables (presence or absence of
cravfish and snail size) on the number of snails killed in

PREDATOR AVOIDANCE IN FRESHWATER SNAILS

389

each size class (dependent variable). A second ANOVA
separately analyzed differences in the number of surviving
snails in each size class found above the waterline as the
dependent variable. Each snail species was analyzed sep-
arately. Because the data were expressed as proportions
(proportion of the snails killed and the proportion of the
surviving snails above waterline), the data were arc-sine
transformed prior to analysis (Sokal and Rohlf, 1981 ).

Results

Experiment I: differential vulnerability of
Physella and Planorbella

Handling times increased exponentially with increasing
snail size for both species (Fig. 2A). For P. trivolvis, han-
dling times increased more rapidly with increasing shell
size than did the handling times for P. virgata. For each
snail prey, an exponential equation was fitted by least
squares non-linear regression to the handling time data
of each snail species. The resultant best-fit non-linear
regression between shell length (SL) and handling times
(HT) for P. virgata was HT = 0.095 e°28(SLl (n = 279, r
= 0.75). and for P. trivolvis: HT = 0. 1 1 8 e"4:(SLI (n = 113,
r = 0.74). For both species, the best-fit exponential equa-
tions fit the data well, explaining 74-75% of the observed
variance in the samples.

The ingestion probabilities decreased more rapidly with
increasing shell size for P. trivolvis than for P. virgata (Fig.
2). Approximately 60% of the smallest P. trivolvis (5-7
mm SL) were not eaten once captured, and few of the
larger P. trivolvis (>8 mm SL) were picked up by the
crayfish. In contrast, all small P. virgata (<8 mm SL)
were eaten, once captured. The difference in vulnerability
between the two snail species was significant; P. virgata
were more likely to be eaten, once captured, at all size
classes (Wilcoxin signed-ranks test, T := 0, n = 5, P

< 0.05, Siegel, 1956). The snail size at which 50% of prey
captured were rejected (called R50) was calculated from
linear regression analyses run for each individual crayfish,
using the rejection data (log 10 transformed). The mean
R50 for P. trivolvis was 6.5 mm, and the mean R50 for P.
virgata was 10.7 mm. The R5" for P. trivolvis was larger
than the R50 for P. virgata for each crayfish used in the
experiment (Wilcoxin signed-ranks test, T = 0, n = 6, P

< 0.05).

For both prey species, handling times decreased at the
largest size class tested. The apparent decrease occurred
because the three smaller crayfish used in the study could
not consume snails greater than 8 mm SL (in P. trivolvis)
and 12 mm SL (in P. virgata).

Experiment 2: size-mediated predator avoidance

Two-way analysis of variance results demonstrated that
in P. virgata, only the presence of a predator had a sig-

7 8 9 10
Snoil Size (mm)

10CM

7 8 9 10
Snail Size (mm)

Figure 2. The influence of snail size (shell length) on handling times
(A) and ingestion probabilities (B) in Physella virguta and Planorhella
trivolvia, fed upon by Procambarus xiiniilaii.i. The error bars are standard
errors of the mean.

nificant effect on both dependent variables (the numbers
of surviving snails above the waterline and the number
of snails killed) (Fig. 3A. B, Table I). No significant effect
of snail size was observed in P. virgata: all sizes of P.
virgata were equally vulnerable and were equally likely
to crawl above the waterline. In contrast, for P. trivolvis,
both independent variables (predator presence, snail size)
and the interaction between predator presence and snail
size all were very significant (Fig. 4A, B, Table I). The
significant size effect was due to the inverse relationship
between size and both snail mortality and the number of
surviving snails above the waterline. Smaller P. trivolvis
were more likely than larger individuals to be eaten. In
addition to being more vulnerable to P. simulans pre-
dation, small (4-6 mm SL) P. trivolvis displayed the most
prominent crawlout response, with most of the surviving
snails above the waterline. Medium-sized specimens (6-
12 mm SL) of P. trivolvis were intermediate in vulnera-
bility and were less likely than smaller animals to display
the crawlout response. Larger (12-16 mm SL) specimens
of P. trivolvis were least vulnerable and did not display
an increase in crawlout response over that seen in pred-
ator-free control aquaria. No significant level of mortality

390

J. E. ALEXANDER, JR. AND A. P. COVICH

Physello

<u
Q

5O
40
30
20
10
0

100
?l 80

<T! 5 60

o §

£ .Q 40

o ^2

£ '5 20
(/}

• Predator
O No Predator

=O=

4-6 6-8 8-10 10-12 12-16
Snail Size (mm)

Figure 3. Size-mediated predator avoidance and death in Phyxella
virgata. The upper figure (A) is the percentage of dead snails in each size
class (shell length), the lower figure (B) represents the percentage of sur-
viving snails in each size class above the waterline, in both predator
(darkened circles) and predator-free control (open circles) treatments (n
= 4 in both treatments). The error bars are standard errors of the mean.

or crawlout was observed in the predator-free control
aquaria in either species.

Of the 315 surviving P. trivolvis, 23 (7.3%) had some
shell damage due to crayfish. The damaged shells were
not randomly distributed among the size classes. In the
two largest size classes, 8.3% and 5.1% of the surviving
10-12 mm and 12-16 mm SL size classes were damaged,
respectively. The two smallest (4-6 mm and 6-8 mm SL)
size classes had fewer damaged shells than expected (1.3%
and 6.3%, respectively), based on the number of snails
originally available in each size class, while the interme-
diate (8-10 mm SL) sized class had more damaged shells
than expected, 15.7% (X2 goodness-of-fit test, X2 = 10.9,
d.f. = 4, P < 0.05). In marked contrast, only one out of
the 314 surviving P. virgala (in the 8-10 mm SL size
class) showed shell damage due to crayfish manipulation.

There was no difference in the predation intensity in
aquaria housing P. trivolvis or P. virgata; the crayfish con-
sumed equal numbers of P. virgata (86) and P. trivolvis

Table I

The influence <>l snail size on miwloiii hehavtur in Physella virgata
mill Planorbella tnvolvis

Variable: % dead

Variable: % crawlout

P. trivolvix P. virgata P trirohis P. virgala
Factor (d.f.) F F F F

Predator presence

(1.6)

119.3"*

18.8**

160.5***

146.5***

Snail size

(4, 24)

3.0*

0.4

15.5***

0.3

Predator X Size

(4,24)

3.8*

1.0

12.7***

0.7

The table describes the summary of the ANOVA analyses. Each of
the dependent variables (percent dead, percent surviving snails above
the waterline) were analyzed separately, for each species. (Significance
levels are as follows: *P < 0.05: **/> < 0.01; and *"P < 0.001.)

(85) among the four replicates in each treatment, sug-
gesting that there was no difference in hunger motivation
:- the predators used. On average, each crayfish consumed

Plonorbello

• Predator
O No Predator

if
a>
Q-

100

4-6 6-8 8-10 10-12 12-16

Snail Size (mm)

Figure 4. Size-mediated predator avoidance and death in Planorbella
trivolvis. The treatments and symbols are the same as in Figure 3.

PREDATOR AVOIDANCE IN FRESHWATER SNAILS

391

slightly more than 21 snails of either prey species during
the 2-h observation period.

Discussion

In marine systems, gastropod anti-predator structures
and behaviors are common. Marine snails rely either on
strong shell architecture (Palmer, 1979; Bertness et ai.
1981; Schmitt, 1982; Blundon and Vermeij, 1983; Lowell,
1986) or on escape and avoidance behavior. Many marine
snails crawl towards or above the waterline to temporarily
escape from or avoid their predators, such as crabs, sea
stars, and predatory gastropods (Feder, 1963;Ansell, 1969;
Phillips, 1976; Vaughn and Fisher, 1988). Like many ma-
rine gastropod species, the crawlout responses in P. virgata
and P. trivolvis represent the active use of a potential refuge
(the terrestrial environment) that temporarily protects
these freshwater snails from crayfish predation.

In this study, handling times (time spent consuming
prey) and the ingestion probabilities (probability of con-
suming a prey) were expected to differ between the two
prey, because differences in vulnerability existed due to
differences in relative shell thickness and shell shape be-
tween the two snail species. From these results, it was
clear that P. virgata were much more vulnerable to cray-
fish than similar-sized P. trivolvis. Crayfish could not con-
sume large P. trivolvis. because they could not either crush
the thicker planispiral shell or manipulate the shell to a
position where the mouthparts could crush it or chip the
thickened aperture lip (pers. obs.). Crayfish often dropped
large P. trivolvis (SL > 6 mm) after lengthy handling pe-
riods, and subsequently ignored large P. trivolvis after
several unsuccessful predation attempts. In contrast, in
all size classes, the thinner, elongated spiral shell of P.
virgata could be manipulated and crushed by the same
crayfish, strongly suggesting that specimens of P. virgata
were more vulnerable to crayfish predation than P. tri-
volvis. Crayfish either crushed the shell at the body whorl,
chipped away at the aperture lip, or had broken off the
shell spire (pers. obs.). Because their shells provided little
structural defense, specimens of all size classes of P. virgata
were equally vulnerable to crayfish predation and thus
were equally likely to crawl above the waterline (Fig. 3).
In examining the surviving snails from the second ex-
periment, only one living specimen of P. virgata with a
damaged shell was observed in the experiment, strong
indirect evidence that, once a specimen of P. virgata was
captured, the snail was usually eaten. In addition, when
foraging on P. virgata, crayfish almost always were able
to effectively handle and consume P. virgata encountered,
as shown by the high ingestion probabilities (Fig. 2). For
P. trivolvis, 23 (7.3%) of the surviving animals recovered
in the second experiment were observed with some dam-

age, suggesting that they survived a predatory encounter
with the crayfish.

At any given size, specimens of P. trivolvis required 2-
4 times the handling time by crayfish for successful pre-
dation than did similar-sized P. virgata (Fig. 2A), indi-
cating the much greater difficulty in crushing the P. tri-
volvis shells. Stein et al. (1984), comparing the prey value
of a physid (Physa sp.) and a planorbid (Helisoitui sp.) to
redear sunfish (Lcponiis microlophus), noted that redear
sunfish weakly selected the physid over the planorbid, but
that size selection did not occur within either genus. The
force required to crush the physid (3 Newtons for 10 mm
SL Physa lying aperture down) was less than that required
to crush the planorbid (4 Newtons for 10 mm SL Heli-
soma lying on its side), but the difference in force was not
dramatically different. The shells were crushed in this way
because sunfish were observed orienting snails between
their pharyngeal gill plates (their crushing surfaces) so as
to crush the minimal dimension of the shell. Crayfish in
our study crushed snail shells primarily by chipping with
their mandibles at the shell aperture, holding the snail
with their maxillipeds. Sometimes, crayfish appeared to
use their chelae to balance and press the shell against their
mandibles. Although we did not measure the force re-
quired for crayfish to crush P. virgata and P. trivolvis shells,
nor did we measure the shell thicknesses, the data suggest
that crayfish could more easily crush P. virgata shells than
P. trivolvis shells, because crayfish primarily attempt to
break the aperture lip, particularly in P. trivolvis. and not
the entire shell.

Antipredator mechanisms may be quite dissimilar in
closely related gastropod species. Two congeneric species
of marine snails, Tegula eiseni and T. aureotincta, differ
in their predator defenses; T. aureotincta performed
avoidance behaviors to gastropod and asteroid predators,
while T. eiseni depended more on shell morphology for
defense (Schmitt, 1982). Physella virgata relies on behav-
ioral avoidance much more exclusively than does P. tri-
volvis, which appears to rely more on predator avoidance
when young, and on shell strength as larger adults.

The correspondence between the reactivity of snails of
a given size class and their vulnerability was expected, if
predator avoidance behavior (crawlout) has some costs
associated with reacting inappropriately to the potential
threat of predation. Crawling to or above the waterline
could expose the snail to other predators, including birds,
and certain insects, such as belostomatids (Crowl and Al-
exander, 1989; Kesler and Munns, 1990). Further costs
to crawlout behavior include decreased foraging time (if
the animals cannot forage on food above the waterline),
decreased opportunities for reproduction, and desiccation
(Alexander and Covich, 1991).

392

J. E. ALEXANDER. JR. AND A. P. COVICH

The differences in the antipredator responses between
the two species may be influenced by differences in selec-
tive pressures caused by the distinct physiological adap-
tations used by the two snails in their respective micro-
habitats (McMahon, 1983). Planorbella trivolvis. with its
well-developed neomorphic gill, and its more efficient re-
spiratory pigment (hemoglobin), is much more aquatic
than P. virgata, which retains an air-filled mantle cavity
(lung) as the major organ of gas exchange. Physella virgata
makes periodic excursions to the surface to renew its ox-
ygen store, and subsequently is limited to shallow water
near-shore habitats or those habitats with structure (i.e.,
aquatic macrophytes or woody debris) extending above
the waterline. Planorbella trivolvis. with a much greater
capacity for aquatic gas exchange, makes excursions into
much deeper water, where crawlout sites are likely to be
unavailable. In P. virgata, physiologically restricted to
shallow, near-shore waters, selective pressures may have
caused a retention of a strong crawlout response to avoid
predators and reduced pressure for the development of a
structurally predator-resistant shell. In contrast, P. trivol-
vis, whose range (particularly in adults) extends into
deeper water and consequently has little access to terres-
trial refugia, selection pressures may have been towards
development of a structurally predator-resistant shell and
a reduced dependence on a crawlout response.

Because they can be the dominant primary consumers
in some habitats, mollusks and decapod crustaceans play
important roles in many aquatic communities. Many are
herbivorous, detritivorous, or omnivorous, and are im-
portant for cycling nutrients and providing energy in the
form of variously sized food items for higher trophic level
consumers (Ansell, 1969; Momot el ai, 1978; Grimm,
1988). The study of the behavioral interplay between
freshwater snails and crayfish is essential in understanding
how these behavioral processes influence predator-prey
dynamics and community composition. Rapid snail es-
cape and avoidance behavior, and the subsequent decrease
in encounter probabilities, suggest that in some structur-
ally complex habitats, such as macrophyte-dominated lit-
toral zones or forested wetland areas, vertical migration
above the waterline is an adaptive response to crayfish
predation.

Acknowledgments

The first author thanks his Ph.D. advisory committee:
R. Mellgren, D. Mock, F. Sonleitner, and T. Yoshino, for
their suggestions. Various versions of the manuscript and
statistical analyses were improved by the efforts of T.
Crowl, K. Brown, and P. Rutledge. We thank R. Mc-
Mahon for his help with snail identification, figure draw-
ings, for providing insight into the different physiological

constraints on the two snails, and for critically evaluating
the final manuscript. Two anonymous reviewers helped
to evaluate the final manuscript. The research was sup-
ported by research and teaching assistantships from the
Department of Zoology, University of Oklahoma, and by
NSF grant BSR 8500773.

Literature Cited

Alexander, J. E., Jr. 1987. Predator-prey interactions between fresh-
water snails and crayfish. Ph.D. dissertation. University of Oklahoma.
Alexander, J. E., Jr., and A. P. Covich. 1991. Predator avoidance in

the freshwater snail Physella virgala to the crayfish Procambarus

simulant. Oecologia (in press).
Ansell, A. D. 1969. Defensive adaptations to predation in the mollusca.

Proc. Mar. Biol. Assoc. India Symp. Ser. 3: 487-512.
Bertness, M. D., S. D. Garrity, and S. C. Levings. 1981. Predation

pressure and gastropod foraging: a tropical-temperate comparison.

Evolution 35: 995-1007.
Blundon, J. A., and G. J. Vermeij. 1983. Effect of shell repair on shell

strength in the gastropod Liltorina irrorata. Mar Bio/. 76: 41-45.
Brown, K. M., and D. R. DeYries. 1985. Predation and the distribution

and abundance of a pulmonate pond snail. Oecologia 66: 93-99.
Brown, K. M., and B. H. Strouse. 1988. Relative vulnerability of six

freshwater gastropods to the leech Nephelopxix obscura (Verrill).

Freshwater Bin!. 19: 157-165.
Covich, A. P. 1981. Chemical refugia from predation for thin-shelled

gastropods in a sulfide-ennched stream. Yerh. Int. 1'ereinigitng

Theorel. Augewandte Limnol. 21: 1632-1636.
Crowl, T. A. 1990. Life-history strategies of a freshwater snail in response

to stream permanence and predation: balancing conflicting demands.

Oecologia 84: 238-243.
Crowl, T. A., and J. E. Alexander, Jr. 1989. Parental care and foraging

ability in male waterbugs (Belostoma flumineum). Can J Zoo/. 67:

513-515.
Crowl, T. A., and A. P. Covich. 1990. Predator-induced life-history

shifts in a freshwater snail. Science 247: 949-95 1 .
Feder, H. M. 1963. Gastropod defensive responses and their effective-
ness in reducing predation by starfishes. Ecology 44: 505-512.
Grimm, N. B. 1988. Role of macroinvertebrates in nitrogen dynamics

of a desert stream. Ecology 69: 1884-1893.
Hanson, J. M., P. A. Chambers, and E. E. Prepas. 1990. Selective

foraging by the crayfish Orconectes virilis and its impact on macro-
invertebrates, freshwater Biol. 24: 69-80.
Kesler, D. K., and \V. R. Munns, Jr. 1990. Predation by Belostoma

flumineum (Hemiptera): an important cause of mortality in freshwater

snails. J. N. Am. Ben/hoi. Soc. 8: 342-350.
Lodge, D. M., K. M. Brown, S. P. Klosiewski, R. A. Stein, A. P. Covich,

B. K. Leathers, and C. Bronmark. 1987. Distribution of freshwater

snails: spatial scale and the relative importance of physicochemical

and biotic factors. Am. Malacol. Union 5: 73-84.
Lowell, R. B. 1986. Crab predation on limpets: predator behavior and

defensive features of the shell morphology of the prey. Biol Bull

171: 577-596.
McMahon, R. F. 1983. Physiological ecology of freshwater pulmonates.

Pp. 359-430 in The Mollusca. Vol. 6, Ecology. W. D. Russell-Hunter,

ed. Academic Press, Inc.. Orlando. FL.
Momot, \V. T., H. Gowing, and P. D. Jones. 1978. The dynamics of

crayfish and their role in ecosystems. Am. Midi. Nat. 99: 10-35.
Palmer, A. R. 1979. Fish predation and the evolution of gastropod

shell sculpture: experimental and geographic evidence. Evolution 33:

697-7 1 3.

PREDATOR AVOIDANCE IN FRESHWATER SNAILS

393

Peckarsky, B. L. 1984. Predator-prey interactions among aquatic in-
sects. Pp. l%-254 in The Ecology of Aqital ic //;vr/v. V. H. Resh
and D. M. Rosenberg, eds. Praeger Scientific, New York.

Phillips, D. \V. 1976. The effect of a species-specific avoidance response
to predatory starfish on the intertidal distribution of two gastropods.
Oeco/ogia 23: 83-94.

Schmitt. R. J. 1982. Consequences of dissimilar defenses against pre-
dation in a subtidal marine community. Ecology 63: 1588-1601.

Siegel, S. 1956. \onparametric Statistics For Tilt Behavioral Science*
McGraw-Hill. Inc.. New York.

Sih, A. 1982. Foraging strategies and the avoidance of predation by
an aquatic insect, h'olonecta hoffmanni. Ecology 63: 786-796.

Sih, A. 198-4. The behavioral response race between predator and prey.
Am. Nat 123: 143-150.

Sih, A. 1986. Antipredator responses and the perception of danger by
mosquito larvae. Ecology 67: 434-441.

Snydcr, N. F. R. 1967. An alarm reaction of aquatic gastropods to
intraspecific extract. Cornell University Agricultural Experimental
Station Memoir 403.

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

Stein. R. A. 1977. Selective predation, optimal foraging, and the pred-
ator-prey interaction between fish and crayfish. Ecology 58: 1237-
1253.

Stein, R. A., C. G. Goodman, and F,. A. Marschall. 1984. Using time
and energetic measures of cost in estimating prey value for fish pred-
ators. Ecology 65: 702-715.

Townsend. C. R.. and T. K. McCarthy. 1980. On the defence strategy
of Physa lonlinalis (L.). a freshwater pulmonate snail. Oecologia 46:
75-79.

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

Vermeij, G. J. I982a. Phenotypic evolution in a poorly dispersing snail
after arrival of a predator. Nature 299: 349-350.

Vermeij, G. J. 1982b. Unsuccessful predation and evolution. Am. Nat.
120: 701-720.

Vermeij, G. J., and A. P. Covich. 1978. Coevolution of freshwater
gastropods and their predators. Am. Nat. 112: 833-843.

Werner, E. E., and D. J. Hall. 1988. Ontogenetic habitat shifts in blue-
gill: the foraging rate-predation risk trade-off. Ecology 69: 1352-1366.

Reference: Rial Hull 180: 394-405. (June. 1991)

Shape Variation in Hydractiniid Hydroids

NEIL W. BLACKSTONE1 AND LEO W. BUSS1 2

Department of Biology, and ^Department of Geology and Geophysics,
Yale Universitv, New Haven, Connecticut 065 II

Abstract. Colonies of hydractiniid hydroids consist of
feeding polyps connected by a common gastrovascular
system. The gastrovascular system consists of stolons,
which enclose gastrovascular canals. Stolons may be fused
into a stolonal mat or extend from the periphery of the
colony. Hydractinia forms a stolonal mat early in colony
development; Podocoryne. on the other hand, does not.
To facilitate comparisons of these taxa. we propose a sim-
ple shape metric, perimeter/^ area, and show that this
measure: ( 1 ) correlates closely with relative amounts of
peripheral stolon and stolonal mat structures in Hydrac-
tinia, (2) permits analyses of within- and between-species
variation of growth morphology in Podocoryne and Hy-
dractinia, and (3) allows quantitative analysis of breeding
studies of Hydractinia, both before and after stolonal mat
formation in the progeny.

Introduction

Hydractiniid hydroids encrust hard substrata in the sea.
Hydractinia echinata and related species are commonly
found on the shells of hermit crabs and often exhibit a
species-specific correlation with host hermit crabs (Buss
and Yund, 1989; Cunningham et al, in press). Podocoryne
carnea also encrust hermit crab shells, but commonly in-
habit other substrata as well (Edwards, 1972; Mills, 1976).
Colony development in both taxa begins with the meta-
morphosis of the planula larvae into a primary polyp.
Runner-like stolons extend from the primary polyp. Sto-
lons encase fluid-filled, gastrovascular canals that are con-
tinuous with the gastrovascular cavity of the polyp. Po-
docoryne continues to develop in this way, i.e., by lineal
extension of the stolons, initiation of new stolon tips, and
iteration of feeding polyps on the stolons (Braverman,
1963; McFadden, 1986). Stolons in Hydractinia, however.

Received 7 August 1990: accepted 19 February 1991.

quickly fuse to form a continuous stolonal mat, which
shows sheet-like growth, and from which extend varying
amounts of peripheral stolons (McFadden et al., 1984;
Blackstone and Yund, 1989; Buss and Grosberg, 1990).
Figure 1 provides rough schemata of the differences in
form between these taxa.

While morphological variation within each taxon has
been compared and related to ecological characteristics
(e.g., competitive ability; see McFadden et al.. 1984;
McFadden, 1986; Yund, 1987; Buss and Grosberg, 1990),
quantitative comparisons of between-taxa variation have
been hampered by the differences in growth form, i.e.,
the presence of a stolonal mat in Hydractinia and its ab-
sence in Podocoryne. For instance, competitive ability
among strains of Hydractinia has been shown to correlate
with relative amounts of peripheral stolon and stolonal
mat structures (measured using several methods, see
McFadden et al.. 1984; Yund, 1987; Buss and Grosberg,
1990), but such measures cannot be applied to Podoco-
ryne.

To facilitate comparisons of biological traits between
Podocoryne and Hydractinia. we propose a simple mea-
sure of morphology that can be used in both taxa. We
show that this measure correlates with ratios of peripheral
stolon and stolonal mat structures in Hydractinia. and
we use this measure to examine morphological variation
and its genetic basis both between and within Podocoryne
and Hydractinia. Finally, we relate this variation to eco-
logical, evolutionary, and developmental aspects of these
species and discuss the relevance to other clonal taxa as
well.

Materials and Methods

Growth morphology and shape

We suggest treating hydractiniid hydroids as geometric
shapes for purposes of comparison. We prefer Bookstein's

394

HYDRACTIN1ID HYDROID SHAPE

395

(1978: p. 8) definition: "... a shape is an outline-with-
landmarks from which all information about position,
scale, and orientation has been drained," with the qual-
ification that hydroid colonies have no reliable morpho-
logical landmarks. Further, the aspects of hydractiniid
growth morphology of particular interest are essentially
two-dimensional, comprising those portions of the colony
that adhere to the substratum. Although there are so-
phisticated techniques available for the analysis of two-
dimensional shapes-without-landmarks (e.g.. Lohman,
1983; Person et a/.. 1985), we will take a simpler approach.
The terms previously used to categorize hydractiniid
growth morphology (many peripheral stolons = "net
type," few peripheral stolons = "mat type," see Hauen-
schild, 1954) point out an intuitively obvious correlation
between two-dimensional growth morphology and shape.
Colonies with few peripheral stolons often show approx-
imately circular growth forms, while colonies with many
peripheral stolons exhibit more irregular shapes (Fig. 1).

An appropriate "size-free" metric to quantify these dif-
ferences in shape is perimeter f\ area (cj.. Gould, 1973;
Patton. 1975). We point out several properties of this
measure, by way of introducing it to morphological studies
of encrusting clonal organisms. First, regardless of scale,
this measure is constant for a given geometric shape. For
instance, this measure will equal 2\v for a circle, 4 for a
square, =4.5 for an equilateral triangle, = 5.4 for a "first-
aid" sign, = 5.7 for a cross (length of the long arm is twice
that of the three short arms), and so on. In each case,
these values are constant regardless of the actual size of
the object as long as the same units are used to measure
both perimeter and area. Second, while the same geo-
metric shapes will have similar peri meter/]/ area values,
shapes with the same perimeter/} 'area need not be the
same. In fact, for encrusting clonal organisms, no two
shapes are likely to be the same, yet many may have sim-
ilar peri meter/]/ area values. This shape metric thus only
assesses the degree of circularity of a shape. Shapes with
values close to 2^ approach perfect circularity, while
highly non-circular shapes have much larger values.
Third, perimeter/^ area has a minimum at 2 VTT, possible
values thus have a lower bound, and their distributions
may be skewed. Note that this is not unusual; most mor-
phometric measurements have a lower bound at zero and
thus may form skewed distributions. Regardless of the
lower bound (0 or 2 Vir), a log-transformation usually
provides distributions suitable for parametric analysis (see
Sokal and Rohlf, 1981).

The utility of this measure may be visualized by com-
paring a plot of perimeter and area for two sibling Hy-
dractinia colonies (Fig. 2) grown from primary polyps
under standard conditions (see McFadden et at.. 1984).
While the perimeter versus area trajectories fluctuate as

stolons branch and fuse, it is clear that the colony with a
greater amount of stolons projecting from the center has
a larger perimeter for a given area and larger
perimeter/]/ area values. To assess quantitatively the ca-
pacity of this shape metric to assay the amount of pe-
ripheral stolons, we measured the correlation between
perimeter/]/ area and peripheral stolon development for
the 242 Hvdractinia colonies used in a breeding study
(see protocol below). At age 50 days, each colony was
measured using a digital image analysis system. Briefly,
an Eyecom II camera attached to a Wild Makroskop was
used to project each colony onto a black-and-white mon-
itor (640 X 480 pixels; note that the pixels are orthogonal
and that the length of a pixel is the same in either direc-
tion). Points on the video image of each colony were re-
corded with a digitizing tablet interfaced with a DEC PDP-
1 1 minicomputer. The outline of each colony was traced
with points at 5 pixel intervals, and the perimeter and
area were computed. The outline of the stolonal mat, i.e.,
the fused stolons (see Fig. 1), was also traced, and the
perimeter and area were measured. Scales ranged from
150 pixels/mm for the smallest colonies to 25 pixels/mm
for the largest colonies. Over this range of observation,
these colonies are not fractal, i.e., they do possess a char-
acteristic scale and do not show self-similarity over the
different scales of observation employed here (although
self-similarity may be apparent using other scales of ob-
servation). Thus, while smaller colonies were measured
with slightly greater resolution, this did not bias the results
in a systematic fashion. Data were transferred to an IBM-
PC and uploaded to an IBM 3083 mainframe where anal-
ysis was done using SAS software.

Comparing peripheral stolon development to colony
shape entails methodologic problems. Logical measures
of peripheral stolon development involve a measure of
the total size of the colony divided by the size of the sto-
lonal mat (e.g., total colony perimeter/stolonal mat pe-
rimeter and total colony area/stolonal mat area). Given
the nature of colony growth (i.e., peripheral stolons pro-
jecting from a central area of stolonal mat. Fig. 1 ), the
extent to which these ratios are greater than 1 will measure
the amount of peripheral stolons. A straightforward pro-
cedure would be to correlate these ratios to the total pe-
rimeter divided by the square root of the total area (i.e.,
the shape metric). However, this could result in autocor-
relation, because both ratios necessarily contain measures
of the overall size of the colony (either total perimeter or
total area). We measured these correlations and then as-
sessed the effects of autocorrelation by adjusting the cor-
related ratios to remove similar variables from each. For
instance, the correlation of the ratio, colony perimeter/
stolonal mat perimeter, to the ratio, peri meter/]/ area, can

396

N. W. BLACKSTONE AND L. W. BUSS

be considered equivalent to the correlation of stolonal
mat perimeter to ^larea if there are no effects of autocor-
relation. Further, we considered the biological meaning
of correlations between stolonal mat size and total colony
size and whether these correlations support our interpre-
tation of the shape metric. Spearman's coefficient of rank
correlation (rv) was used; this coefficient is less sensitive
to the statistical pecularities of ratios than parametric cor-
relation coefficients (see Sokal and Rohlf, 1981), although
here both coefficients were similar for all correlations.

Shape variation in Podocoryne and Hydractinia

The perimeter/^ area measure was used to compare
morphological variation within and between field-col-
lected colonies of Podocoryne and Hydractinia using the
technique of clonal repeatibility, i.e.. comparing clonal
replicates of the same colony to gauge broad-sense her-
itability (Falconer, 1981). Colonies were collected from
an intertidal site near Guilford, Connecticut, where Po-
docoryne carnea and Hydractinia symbiolongicarpus, a
sibling species of H. echinaia. commonly co-occur (Buss
and Yund, 1989). although Podocoryne is much less
abundant than Hydractinia. When reproductive polyps
are present, these species can be easily distinguished: Po-
docoryne produces free-swimming medusae, while Hy-
dractinia lacks a medusoid stage and produces fixed gon-
ophores (Mills, 1976). Using a dissecting microscope, Po-
docoryne colonies were identified from large collections
of all hydroid-bearing hermit crab shells. Relatively few
colonies contained reproductive polyps; hence, tentative
identifications were made on the basis of general patterns
of colony appearance (in this area, Podocoryne has few
spines and usually co-occurs with algal epibionts) and
feeding polyp morphology (in this area, Podocoryne tends
to have smaller polyps, a more pronounced hypostome,
and shorter, more tapered tentacles). In this way, 60 col-
onies were tentatively identified as Podocoryne and were
labelled with numbered bee tags attached with cyanoac-
rylate adhesive. Colonies were maintained in 40-liter
aquaria with undergravel filters (20 colonies per tank) at
16°C. Colonies were fed 3-day-old brine shrimp nauplii
(also grown at 16°C) every other day, and 25% of the
water was changed twice a week. In 1-2 weeks all colonies
were reproductive (tentative identifications were correct
in all cases). Medusae from each colony were isolated and
raised in finger bowls at 16°C. Each day. medusae were
examined under a dissecting microscope, fed brine shrimp,
and transferred to fresh seawater. Medusae were raised to
sexual maturity (7-14 days), and the sex of the parent
colony was determined by the morphology of the gonads
(Rees, 1941; Edwards, 1972; identity as P. carnea was
also verified by examining the medusae, see Edwards,

1972; Mills, 1976). From the original 60 colonies. 10 male
and 5 female colonies were selected using a pseudo-ran-
dom number generator. Sixty Hydractinia colonies were
haphazardly collected from the same site and maintained
in the same fashion. Compared to Podocoryne. Hydrac-
tinia requires more time to mature (cf. Hauenschild, 1956;
Braverman. 1963), but within two months all colonies
were fully reproductive, whereupon they were sexed, la-
belled, and 10 male and 5 female colonies were selected
with a pseudo-random number generator. Previous in-
vestigations (McFadden et a/.. 1984; Buss and Grosberg,
1 990) have shown that colony morphology does not differ
on the basis of sex; nevertheless, equal numbers of each
sex from each species were included in this study.

For morphological comparisons, colonies were surgi-
cally explanted onto 22 mm2 glass cover slips and held
in place with loops of thread until attachment whereupon
the threads were removed (see McFadden et al.. 1984;
explants of 3-5 feeding polyps were used). Because of the
work involved, comparisons were made using five field-
collected colonies of each species at a time. Five explants
(hereafter "replicates") for each of the five field-collected
colonies (hereafter "strains") for both Podocoryne and
Hydractinia (hereafter "species") were grown in a floating
rack at 16°C. Three "racks" were used over a two-month
period; rack is thus a proxy for time effects. Each rack
consisted of two side-by-side rows of slots; cover slips were
arranged so that the five replicates for each strain occupied
consecutive slots; strains of each species were randomly
paired, alternating right and left sides. The formal analysis
thus consists of a four-level nested analysis of variance
(see Sokal and Rohlf, 1981 ). Replicates are nested within
strains, which are nested within species, which are nested
within racks. Such an analysis accounts for all sources of
variation except position within racks. Position effects can
be assessed by designating five positions within each rack;
each position then contains the replicates from a pair of
Podocoryne and Hydractinia strains. The analysis then
becomes replicates within species within positions within
racks. Outcome variables were analyzed in both ways.

Using the above protocols, perimeter/} 'area measures
were taken, and counts of polyps and total area measures
were also recorded. Variables were measured at 7 and 14
days after explanting; specific growth rates (see Blackstone,
1987; Blackstone and Yund, 1989) for polyp (polyp/polyp-
dav) and area (mnr /mnr /-day) were also calculated for
this interval. Each rate was calculated by increment in
number or area (for polyp number and total area respec-
tively) per time increment per initial number or area.
While technically "specific" refers to "divided by mass,"
any measure of size can be used, provided the same units
are used in the numerator and denominator, since a spe-
cific growth rate has units of I/time.

HYDRACTINIID HYDROID SHAPE

397

Breeding studies

While studies of clonal repeatibility can establish broad-
sense heritabilities for a trait, breeding studies can provide
further insight into the nature of the genetic variation
underlying a trait (Falconer, 1981). With the same 5 fe-
male and 10 male Hydractinia colonies used above, 10
crosses (2 males per female) were designated using a
pseudo-random number generator, and additional mating
experiments were done to insure that all individuals be-
longed to the same species (see Buss and Yund, 1989).
Matings were carried out every several days for a month.
Pairs of male and female colonies were isolated in the
dark overnight; morning light triggered gamete release (see
Yund el al, 1987, and references therein). Embryos were
transferred to fresh seawater and kept for 3-4 days with
a daily water change. By this time, embryos had developed
into planulae competent to metamorphose (Plickert et
ai, 1988). Metamorphosis was induced by ionic imbal-
ance (Spindler and Muller, 1972; Weis and Buss, 1987).
Competent planulae were transferred to a 53 mA/ CsCl
solution in seawater. After approximately 4 h, planulae
were placed on glass cover slips in seawater-filled six-well
plates ( 1 planula per well). Attachment and metamor-
phosis occurred within 2 days. Six plates per cross (36
planulae total) were metamorphosed. Colonies were fed
3-day-old brine shrimp nauplii, followed by a complete
water change each day.

Colonies were maintained in an incubator at 12.5°C
for 50 days (to a mean size of 1 1 feeding polyps). The
temperature conditions were chosen to reflect the ambient
temperatures in Long Island Sound during the spring and
early summer (Yund et al.. 1987). At this point in the
seasonal cycle, sexual reproduction, recruitment, and in-
traspecific competition occur at high frequencies in this
area (Buss and Yund, 1988). The duration of the exper-
imental period was chosen for the purpose of assessing
colony shape at small colony sizes. Hydractinia planulae
display site-specific settlement on shells, hence the vast
majority of intraspecific competitive encounters occur at
small colony sizes (Yund et al., 1987; Buss and Yund,
1988; Yund and Parker, 1989; see discussion below).

Using the protocols described above, colonies were
measured for area and perimeter as soon as primary polyps
and stolons developed after metamorphosis (<5 days).
Each colony was measured at weekly intervals up to an
age of 50 days (25-50% of the colonies of each cross failed
to survive to this age). We analyzed the data using quan-
titative genetic techniques (Falconer. 1981). Because of
the small size of the laboratory population, we suggest
only very limited interpretation of our results with regard
to the natural population of Hydractinia. Rather, we in-
tended to gain further insight into the results suggested

by the clonal repeatibility experiments; is shape largely
genetically determined, i.e.. does shape variation have a
large broad-sense heritability, and further, is there any
evidence that the broad-sense heritability of shape vari-
ation in this laboratory population is due to narrow-sense
heritability? Analyses were done on initial perimeter/
iarea (age <5 days), on mean perimeter/} 'area (for each
colony, all shape measures up to age 50 days were aver-
aged, and this mean value was used as the outcome), and
on final perimeter/} 'area (age = 50 days). These three
comparisons correspond to before, during, and after sto-
lonal mat formation.

Although our goals were somewhat different from typ-
ical quantitative genetic studies (cf.. Falconer, 1981), we
used standard methods to examine the covariance of full
sibs and the covariance of half sibs. Specifically, the be-
tween-female parent component of variance (i.e., ak.mai^2-
the variance between the means of the half-sib families)
estimates COVHS and measures additive genetic variance
(i.e.. narrow-sense heritability, provided maternal effects
are slight). The between-male parent component of vari-
ance, <rmi,/t.,2. estimates COVFS - COVHS and measures a
combination of additive and non-additive genetic variance
(i.e.. broad-sense heritability, provided environmental ef-
fects are slight). Insight into additive and non-additive
genetic variance can thus be obtained from a nested anal-
ysis of variance. The F-ratio of the male-parent mean
square to the within-brood mean square will measure ad-
ditive and non-additive genetic variance, while the F-ratio
of the female-parent mean square to the male-parent mean
square will measure additive genetic variance (see results
below). We focus on qualitative interpretations of the
analysis of variance rather than exact calculations of her-
itabilities because of the small size of the laboratory pop-
ulation and the limited goals of our breeding study (see
Mitchell-Olds. 1986; Via, 1988).

To properly gauge the inheritance of shape, we at-
tempted to reduce environmental effects in several ways.
First, because we expected a priori that non-additive ge-
netic variance would be large relative to additive genetic
variance (i.e.. COVFS > COVHS, see discussion below),
each female parent was mated to two male parents. Thus,
any maternal or cytoplasmic effects (see discussion in
Mazer. 1987) will inflate the covariance of the half sibs
and inflate our estimate of additive genetic variance. Sec-
ond, because matings were initiated at slightly different
times and because between-mating environmental vari-
ation could inflate the covariance of the full sibs, envi-
ronmental conditions were closely controlled. In addition
to incubation at a constant temperature, seawater chem-
istry was monitored weekly, and nitrates and nitrites were
maintained at low levels (<9.0 ppm and <0.01 ppm, re-
spectively). Salinity was maintained at =26 ppt. Any

398

N. W. BLACKSTONE AND L. W. BUSS

variation in environmental conditions was slight and
showed no systematic trend over the time course of the
experiment. Finally, colony position effects were assessed.
Stacks of culture plates (6 per mating) were kept on a
single shelf in an incubator and positions were varied daily
in a random manner. Individual plates, however, were
kept in descending order (1-6), and culture wells were
also in fixed positions. Because there was only one colony
per well, wells were pooled into left wells, center wells,
and right wells based on their positions in the six-well
plate. The complete analysis was thus well position nested
within plate, plate nested within male parent, and male
parent nested within female parent. This analysis was car-
ried out for intial and average shape measures. By the age
of 50 days, the 25-50% mortality for each cross rendered
the analysis of well position effects and plate effects un-
reliable because of missing values, and the pooled within-
broods mean square was used as the error variance.

Further insight into environmental effects was gained
by two additional experiments. First, for one of the ma-
ternal half-sib families, three 50-day-old offspring from
each paternal cross were explanted onto snail shells oc-
cupied by hermit crabs and cultured in the 40-liter aquaria
until each colony covered its shell and was fully mature.
The 6 colonies were then compared using the method of
clonal repeatibility described above, i.e., 5 explants from
each colony were grown on cover slips in a floating rack
at 16°C and perimeter/} 'area was measured at 10 days
after explanting. These shape measures were then com-
pared to the measures made on the colonies in their first
50 days of growth. Second, two colonies from each of
three crosses were grown in the six-well plates as described
above until they grew to the edge of the coverslip (60-
120 days). Measures of perimeter/^ area were made at
roughly weekly intervals.

Results

Growth morphology and shape

For the 242 50-day-old Hydractinia colonies measured,
indices of peripheral stolon development (total colony
perimeter/stolonal mat perimeter and total colony area/
stolonal mat area) correlate highly with total colony pe-
rimeter divided by the square root of total colony area (rs
= 0.95 and 0.91, respectively). Because the correlated
variables contain similar measures of total colony size
(total perimeter, total area, or the square root of total
area), the possibility of autocorrelation exists. For two
reasons, however, the underlying structure of the data
suggests that autocorrelation has negligible effects.

First, adjusting the correlated ratios to remove similar
variables from each does not alter the correlations. Sto-
lonal mat perimeter is highly correlated with the square

root of total colony area (r> = 0.94; note that r, is insen-
sitive to transformations of the correlated variables so that
the correlation of stolonal mat perimeter and total colony
area is also 0.94). Additionally, total colony perimeter is
highly correlated with the ratio (total colony area)3/:/sto-
lonal mat area (r, = 0.92).

Second, either measure of stolonal mat size (perimeter
or area) shows a high correlation with total colony area
(r, = 0.94 and 0.92, respectively) but much weaker cor-
relations with total colony perimeter (rs = 0.63 and 0.53,
respectively). Stolonal mat size is thus indicative of total
colony area, but less so of total colony perimeter. These
results are consistent with the stolonal mat showing cir-
cular growth in these small colonies, and deviations from
circular growth being caused by peripheral stolons.

Shape variation in Podocoryne and Hydractinia

Measures of perimeter/^ 'area for both 7 and 14 days
after explanting show that Hydractinia has more circular
shapes than Podocoryne (Table I). To analyze these data,
a natural logarithmic transformation was done to better
meet the assumptions of the analysis of variance. The log-
transformed data were first analyzed to assess the effects
of the positions of the colonies within the racks, i.e., rep-
licates nested within species nested within positions nested
within racks. Because of the different numbers of replicates

Table I

Shape variation in 15 strains of Podocoryne and Hydractinia3

Podocoryne

Hydractinia

Strain" n

Age 7

Age 14

Age 7

Age 14

17.87

1.03

21.44

0.99

9.03

0.58

17.46

0.70

13.67

3.96

17.06

3.62

4.17

0.10

5.40

0.43

7.20

2.08

15.70

3.52

3.83

0.08

3.94 0.08

18.54

1.87

25.38

1.94

5.12

0.68

5.44

1.06

15.24

1.67

26.26 0.86

4.330.10

4.05

0.13

23.78

1.92

23.97

1.89

7.23

0.89

15.46

2.70

20.35

1.40

33.43

2.09

4.44

0.33

5.18

0.67

19.06

3.46

30.62

1.10

9.92

2.48

15.12

2.33

20.72

1.79

20.16

0.96

4.52

0.23

6.55

0.87

22.04

2.49

26.50

1.85

6.56

0.52

11.72

0.47

22.59

1.25

23.22

2.55

8.14

3.47

10.53

5.24

13.23

2.08

21.39

2.81

5.63

0.52

10.46

1.48

21.54

0.83

22.63

1.33

7.45

0.74

10.72

1.22

17.87

1.69

21.72

3.30

10.92

1.71

15.75

1.03

10.63

2.43

19.15

0.73

11.07

1.31

19.81

1.06

' Shape measures are perimeter/]/ area: means and standard errors are
shown for n replicates of each strain, 7 and 14 days after explanting.

b For Podocoryne, strains 3, 7, 9, 1 3. and 1 5 are females, and for strain
3 at age 14 n = 2. For Hydractinia. strains 3, 5, 12, 14, and 15 are
females, and for strain 14 at age 14 n = 3.

HYDRACT1NIID HYDROID SHAPE

399

(some replicates were lost due to mortality, see Table I),
the nested ANOVAs were unbalanced, although exami-
nation of the coefficients of the variance components in-
dicated a high reliability of the F-tests carried out (see
discussion in Sokal and Rohlf, 1981). At day 7, there is
a strong effect of species (F = 26.95. d.f. = 15, 98, P
« 0.001). but no effect of position (F = 0.06, d.f. = 12,
15, P> 0.99) or rack (F = 1.05. d.f. = 2, 12, P> 0.35).
Similarly, at day 14, species shows a strong effect (F
= 26.96, d.f. == 15, 96, P <s 0.001), while position (F
= 0.19, d.f. = 12, 15, P> 0.99) and rack (F = 0.70. d.f.
= 2, 1 2, P > 0.50) do not. Based on this analysis, position
effects were dropped from the model: this allowed in-
cluding the strain effects (i.e.. replicates within strains
within species within racks, see Table II). Again, because
of the different numbers of replicates, this ANOVA is also
unbalanced. Examination of the coefficients of the vari-
ance components (Table II) suggests that F-ratios should
be reliable; in particular, the F-ratio assessing the effects
of racks (i.e., MSmiA7MSwat,,) is highly reliable, while the
F-ratio assessing the effects of species (i.e.. MS,,,,,,,.,/
MSS,,,,,,,S) is slightly conservative. The results are similar
to the first analysis; there is no effect of racks (F = 0.02,
d.f. = 2, 3, P > 0.95) and a strong effect of species (F
= 20.45, d.f. = 3, 24, P << 0.001 ). Further, strains within
species show significant variation (F = 4.99, d.f. = 24, 98,
P <$ 0.001 ). Results at 14 days are similar; racks show no
effect (F = 0.03, d.f. = 2. 3, P > 0.95), while species (F
= 1 2.94, d.f. = 3, 24, P « 0.00 1 ) and strains (F = 8. 1 1 ,
d.f. = 24, 96. P <t 0.001) show strong effects.

In addition to shape differences, Podocoryne exhibits
faster growth rates than Hydractinia (Table III). Specific
growth rates of polyps show no effect of racks (F = 0.21,
d.f. = 2, 3, P > 0.80), but significant effects of species (F
= 11. 75. d.f. = 3, 24, P<s 0.001) and of strains (F = 3.79,
d.f. = 24, 96, P <£ 0.001). Similarly, specific growth rates
of colony areas show no effect of racks (F = 0.35, d.f. = 2,
3, P > 0.70), a moderate effect of species (F = 6.70, d.f.

Table II

AnulvMs i>l variance table for log-transformed shape measures at day 7
ot the i'lonul repeatibility experiment

Table III

Speeifie growth rales fur 15 strains ot Podocoryne and Hydractinia3

Source

Mean
d.f. square Composition of mean square

Between racks 2 0.104 a em,,2 + 4.00(7 „,„,„- + 20.00<7J(,m,

+ 40.00(7 ,Mc

Between species within 3 10.897 afrmr2 + 4.02(7 ,,ra,,,: -I- 20.08(7 ,,,,,„,

racks

Between strains within 24 0.497 at,rm2 + 4.22a,,ra,,:2

species

Between replicates 98 0.099 o-,,rror2

Podocoryne

Hvdraciinta

Strain

Area

Polyp

Area

Polyp

0.21 1 0.01

0.1970.01

0.181 0.01

0.1840.01

0.181 0.05

0.1690.02

0.0340.01

0.00 0.00

0.1100.09

0.07 1 0.07

0.059 0.06

0.013 0.04

0.2260.01

0.1660.02

0.0380.01

0.035 0.02

0.1300.06

0.069 0.04

0.1020.01

0.1130.02

0.195 0.01

0. 1 38 0.02

0.092 0.03

0.074 0.03

0.2030.01

0.1700.03

0.1240.01

0.1230.01

0.2200.01

0.1970.01

0.1760.02

0.101 0.02

0.1840.03

0. 1 7 1 0.02

0.0630.01

0.07 1 0.03

0. 1 74 0.02

0.1660.03

0.113 0.03

0.1000.02

1 1

0.191 0.0 1

0.2150.01

0.050 0.03

0.0190.08

0.101 0.06

0.220 0.03

0.1000.01

0.079 0.01

0.1920.02

0.2070.01

0.1020.01

0.1480.02

0.1120.03

0.181 0.02

0.1440.01

0.042 0.03

0.1580.03

0.1370.02

0.1920.01

0.079 0.02

a Specific growth rates ( I/day) for total colony area and polyp number
for 7 to 14 days after explanting; strains are designated by the same
numbers as in Table I. and means and standard errors are shown for n
replicates of each strain.

= 3, 24, P< 0.01) and a strong effect of strains (F = 2.90,
d.f. = 24, 96, P<Z 0.001).

Breeding studies

For the breeding experiments conducted with Hydrac-
tiniu. the shapes of the colonies initially and at 50 days
(Table IV) show little correlation (rs = -0.06, P > 0.30,
for all 242 50-day-old colonies). This likely results from
growth changes associated with stolonal mat formation
(Fig. 1 ). Despite such variation, analyses of initial, average,
and final colony shape all suggest a highly significant effect
of the male parent and a non-significant effect of the fe-
male parent. For initial colony shape, there is no effect
of the female parent (F = 0.98, d.f. = 4, 5, P > 0.45), a
strong effect of the male parent (F = 7.39, d.f. = 5, 50, P
<§ 0.00 1 ), and no effect of either plate (F = 0.85, d.f. = 50,
119, P > 0.70), or well position (F = 0.95, d.f. = 1 19.
173, P > 0.60). Similarly, for average colony shape there
is no effect of the female parent (F = 0.44, d.f. = 4, 5, P
> 0.75) and a strong effect of the male parent (F = 15.21,
d.f. = 5, 50, P « 0.001). Again, there was no effect of
either plate (F = 0.88, d.f. = 50, 1 19, P > 0.65) or well
position (F = 1.04, d.f. = 119, 173, P> 0.40). For both
initial and average shape analyses, the coefficients of the
variance components (Table V) indicate a high reliability
of the F-ratios. For final shape measures, missing values
(because of mortality) rendered the analysis of position

400

N. W. BLACKSTONE AND L. W. BUSS

Table IV

/ ' ve valislicsfor the 10 crosses used in the breeding st

Initial

Average

Final

Parents*

Shape

Polyp

15 X 11

5.86 0.25

8.36 0.47

1 1.44 0.91

19.6 3.1

15 x 13

6.61 0.39

6.71 0.40

6.75 0.54

8.3 1.2

12X7

7.26 0.30

6.170.20

4.99 0.38

11.1 0.9

12 X 2

7.08 0.36

7.85 0.28

8.98 0.70

5.4 1.0

5X8

7.770.42

7.98 0.29

6.78 0.73

12.2 1.4

5X9

7.90 0.35

6.87 0.30

6.76 0.68

1 1.9 1.4

14 x 1

8.56 0.46

8.74 0.34

6.590.61

19.7 1.1

14 x 10

5.55 0.23

5.63 0.22

6.74 0.76

19.52.4

3x4

7.1 1 0.37

6.28 0.27

4.13 0.14

8.4 0.9

3X6

6.09 0.34

6.180.34

5.64 0.56

8.0 1.2

"Sample sizes (n). means and standard errors for shape measures
(perimeter/lfarea) from initial colonies (primary polyps < 5 days old),
average colonies (for each colony, all shape measures up to 50 days were
averaged; this mean value was used for the descriptive statistics), and
final colonies (50 days old). Data on polyp number (mean and standard
error) is also presented for the final colonies.

b Numbers designating female parent and male parent respectively:
the numbers correspond to the strains of Hydraeiinia from Tables I and
III

effects unreliable; nevertheless, using the pooled within-
broods mean square as the error variance, the data suggest
a non-significant effect of the female parent (F := 1.23,
d.f. = 4, 5, P> 0.40) and a large effect of the male parent
(F == 1 1.3, d.f. = 5, 232, P « 0.001). Overall, the slight
effect of the female parent (i.e.. a non-significant covari-
ance of the half sibs) indicates that ff(em.d\fi is relatively
small and that both maternal effects and additive genetic
variance are correspondingly small. On the other hand,
the large effect of the male parent suggests a large co-
variance of the full sibs. a relatively large (7maies2. and likely
a large non-additive genetic variance, given the closely
controlled environmental conditions. The interpretation
of these results should be limited to the small laboratory
population on which the breeding studies were based (see
Discussion).

While environmental effects could not be tested directly
with this experimental design, the six offspring raised on
hermit crab shells and then compared using clonal re-
peatibility allow an assessment of the sensitivity of Hy-
dractinia colony morphology to environmental circum-
stances. These colonies were from crosses 15x11 and
15X13 (see Table IV); Figure 3 shows the shape measures
for the 50-day ontogenies of the young colonies. After
these young colonies were grown to maturity on hermit
crab shells, explants were made; Figure 3 also shows the
mean shape measures for five 10-day-old replicates from
each of the mature colonies (means and standard errors

primary
polyp

stolonal
mat

polyp

Figure I. Rough schemata of (a) a primary polyp of a hydractiniid
hydroid. (b) a small Podocoryne colony, and small Hydraeiinia colonies
of the (c) "mat type." with few peripheral stolons, and the (d) "net type,"
with more peripheral stolons. Colonies are drawn as if encrusting the
surface of the page; polyps would project up out of the plane of the paper.
In the stolonal mat (the central portion of the Hydraeiinia colonies rep-
resented by the stipled pattern), the spaces between the stolons are filled
with tissue; thus these stolons are fused together, while the peripheral
stolons outside the stipled area (and those in Podocoryne) are unfused.
Colonies are drawn to roughly the same scale; stolon width is approxi-
mately 70 microns.

for the 5 replicates of each colony are adjacent to the
symbol for the 50-day shape measure for that colony).
Comparing these shape data generated by different meth-
ods suggests that, despite different culture conditions, both
methods generate roughly similar data for the same col-

Table v

Analysis ot \-ananee table lor the analysis of I he natural logarithms
ol average shape measures for each colour

Mean
Source d.f. square Composition of mean square

Between female parent 4 0.395 n ,.„„,- + 1.93d ,,(.,,: + 5.11 a pia,,2

+ 34.60(7 ma,es- + 6<).2\afemalfs2
Between male parent 5 0.890 cr,.rrar: + 1.94cr,,,,.,,: + 5.78trp/u,,,2

Between plate 50 0.059 afm,2 + 1.95<fHrf/2 + 5.82ff,,to,:

Between well position 119 0.066 amf- + 1.96(i,,,.;/:
Within well position 173 0.064 aern,,2

HYDRACTINI1D HVDROID SHAPE

401

24 0-

200-

~ 16-0-

120

UJ
Q-

80-

4 0

186

93 •

• mm

9 .5

-JV

-? f 5-4

075

1-5

2 25

AREAImm2)

Figure 2. Perimeter versus area plots for two sibling Hydractinia
colonies measured every other day for the first 5 weeks of ontogeny.
Camera lucida tracings (not to scale) and shape measures (perim-
eterffarea) are shown for some of the data points. While shapes fluctuate
as stolons branch and fuse, the two colonies exhibit distinct trajectories
in perimeter versus area space.

ony, and, in particular, either method shows the differ-
ences between the crosses. At 50 days, cross 15X11 pro-
duced significantly more irregular shapes than cross 15
X 13 (Table IV); analysis of the clonal repeatibility data
shows the same pattern. If the log-transformed shape data
are analyzed as replicates nested within crosses nested
within positions, there is an effect of cross (F = 7.9, d.f.
= 3, 24, P < 0.001), but no effect of position (F = 0.05,
d.f. = 2, 3, P > 0.90). Analyzing the data as replicates
nested within strains nested within crosses provides a
similar result (no significant effect of strains F = 0.92, d.f.
= 4, 24, P > 0.45. but a significant effect of crosses F
= 22. 7, d.f. = 1.4, P< 0.01). In either case, cross 15 X 11
exhibits significantly more irregular shapes than cross 1 5
X 1 3. This supports the findings of the breeding experi-
ment and suggests that the differences between crosses are
not the result of some undetected environmental factor
varying over time.

The 6 colonies from 3 different crosses (12 X 7, 5 X 8,
and 3 X 4 in Table IV) which were grown beyond 50 days
(Fig. 4) show some variation in colony shape, but also
suggest that differences among crosses are maintained at

larger sizes. For instance, at 1 10 days, the 4 colonies which
had not yet reached the edges of the cover slips suggest
the same differences in shape, which were apparent for
the complete crosses at 50 days (Table IV; for perim-
eter/^ area, 5 X 8 > 1 2 X 7 > 3 X 4).

Discussion

These results have implications with regard to mor-
phological variation in hydractiniid hydroidsand in other
clonal taxa as well. We discuss ( 1 ) the biological basis of
shape in hydractiniid hydroids, (2) the implications of the
Hydractinia breeding studies, (3) the general phenomenon
of heterochrony in hydractiniid hydroids, and (4) the rel-
evance of these results to other clonal taxa.

172(09)

15-

10-

84(1 8)

20 30

AGE (DAYS)

Figure 3. Shapes (pernnelcr/^area) of 6 colonies from one of the
maternal half-sib families (3 from 15 x 11, shown by circles, 3 from 15
• 13. shown by squares: the crosses are designated as in Table IV) for
the first 50 days of growth (lines connect points for each individual).
These colonies were grown to maturity on hermit crab shells and then
explanted and measured for shape again. Numbers adjacent to the symbol
for the 50-day shape value show the means, with standard errors in
parentheses, for 5 replicates of each colony 10 days after explanting.
Despite differences in culture conditions, the clonal repeatibility shape
measures and the shape measures for the first 50 days both suggest that
colonies from 15- 11 have more irregular shapes than those from 1 5
x 13.

402

N. W. BLACKSTONE AND L. W. BUSS

40 80

AGE (DAYS)

120

Figure 4. Two colonies from each of 3 crosses (5x8, circles; 12
x 7, squares; and 3x4, triangles; the crosses are designated as in Table
IV) were grown beyond 50 days on glass cover slips and measured for
shape (perimeter /y area). Lines connect values for each colony: 2 colonies
reached the edges of the cover slips in <80 days; the remaining 4 were
grown for over 100 days. While shapes fluctuate, at 1 10 days these 4
colonies suggest shape differences which were apparent for all colonies
of each cross at 50 days (Table IV; 5 x 8 > 12 X 7 > 3 X 4).

The biological basis of shape

The results presented show a strong correlation between
measures of peripheral stolon development and pcrim-
eter/^area in Hydractinia. Previous work (McFadden et
al.. 1984; Yund, 1987; Buss and Grosberg, 1990) suggests
that peripheral stolon development is correlated
with competitive ability in Hydractinia. Thus, shape as mea-
sured by perimeter/} 'area will show a similar correlation.
Further, Podocoryne shows more irregular shapes than
Hydractinia and thus greater peripheral stolon develop-
ment; this result agrees with its competitive dominance
over Hydractinia in laboratory studies (McFadden, 1986).
The correlations of shape with competitive ability can
make measures of shape useful to biologists, but clearly
shape differences are not causally related to competitive
ability (see discussion below). Rather, shape, competitive
ability, and peripheral stolon development are likely cor-
related consequences of the underlying dynamics of
growth in these hydroids.

Shape measures bear a clearly interpretable relation-
ship to these growth dynamics. Examining Figure 1 suggests
that perimeter/M area will measure the degree to which

stolons extend from a central ring stolon or network of
ring stolons. Stolons encase the gastrovascular canals; the
combined actions of stolons and, in particular, muscular
polyps drive the gastrovascular fluid through the canals
and nourish the colony (see Schierwater et al.. in press).
Since the gastrovascular system is closed, peripheral sto-
lons are essentially dead-end channels, and a considerable
pressure is likely necessary to supply these stolons with
fluid. Contrast this to the situation in a ring stolon or
network of ring stolons well-supplied by polyps: here, flow
can proceed from polyp to polyp with relatively little ex-
ertion of pressure. Hence, the amount of peripheral stolons
and the degree of non-circularity of colony shape are likely
to be measures of the ability of the colony to maintain
the energetic and physiological costs of supplying these
peripheral elements with gastrovascular flow.

Hydractinia breeding studies

Generally, the clonal repeatibility experiments and the
breeding experiments produced compatible results for the
15 strains of Hydractinia, despite differences in culture
conditions. For instance, the fastest and slowest growing
strains ( 1 and 2, measured by polyp specific growth rates)
produce the fastest and slowest growing offspring (14X1
and 12X2, measured by polyp number at 50 days), the
strain with the most irregular shape ( 1 5) in one cross pro-
duced offspring with the most irregular shapes ( 15 X 11),
and strains with nearly circular shapes ( 3 and 4) produced
nearly circular offspring (3 X 4). The somewhat circular
shape of colony 2 seems at variance with the irregular
shapes of 12 X 2; however, this likely indicates the limi-
tations of comparing developing colonies to clonal ex-
plants. Colony 2 shows extremely slow growth as do its
offspring ( 12 X 2). It is likely that this slow growth reflects
equally slow development of adult colony form and or-
ganization. The irregular shapes of young 12X2 colonies
may indicate a slow transition from early colony devel-
opment to adult morphology.

Despite the agreement of the results of the clonal re-
peatibility experiments and the breeding experiments, the
latter can provide classes of information which are not
available from the former. While only a small breeding
study was carried out here, it is, to our knowledge, the
first example of carefully controlled crosses for a clonal
organism. To stimulate further such work, we will discuss
the general value of such data. Clonal repeatibility studies
demonstrate a significant broad-sense heritability of the
shape variation (the strain-within-species effect in the
ANOVAs). The breeding studies not only demonstrate a
significant broad-sense heritability (the effect of the male
parent in the ANOVAs), but also provide information on
the sorts of genetic variation that constitute this broad-

HYDRACTINIID HYDROID SHAPE

403

sense heritability. For the three analyses (initial, average,
and final shape measures), MS/l.,mj/t.v/MS,,,,,/(., is roughly 1
(0.98, 0.44, and 1 .23 respectively, see Results) and in each
case is non-significant. This suggests that the covariance
of the half-sibs is small, that is, progeny from the same
maternal half-sib family are not appreciably more similar
to each other than to unrelated progeny. On the other
hand, the covariance of the full-sibs is high (hence the
large effect of the male parent). If environmental effects
are slight, these results suggest that non-additive genetic
variance constitutes the bulk of the broad-sense heritability
of shape in this small laboratory population. This result
is bolstered by the additional study of one of the maternal
half-sib families (progeny of female 15, i.e., crosses 15
X 1 1 and 15 X 13). Using clonal repeatibility, this study
shows that the difference between the full-sib families does
not depend on some undetected environmental factor.

Thus, it is possible that within this small laboratory
population of Hydractinia. the genes controlling shape
variation show high levels of dominance and epistasis.
This result is supported by examining the data qualita-
tively. In Table IV, paternal full-sib families are very sim-
ilar (e.g., note the low standard errors). Nevertheless,
within a particular maternal half-sib family, full-sib fam-
ilies are often very different (e.g., progeny of females 3,
12, and 15). Thus, the expression of the shape phenotype
seems to depend on the interactions between the maternal
and paternal genes (i.e., dominance and epistasis). This
result is intriguing in view of what is known about the
ecology of this species. An increasing body of evidence
suggests that competition for space has resulted in selec-
tion on growth morphology in Hydractinia species.
Briefly, when two or more colonies of the same size encrust
the same substratum, the colony with the greater periph-
eral stolon development will predominate (Buss et til.,
1984; Yund et ai, 1987; Buss and Grosberg, 1990). be-
cause peripheral stolons are capable of differentiating into
a specialized aggressive organ, the hyperplastic stolon
(Buss et ai, 1984; Lange et a!.. 1989). Further, such com-
petition is common in nature (Buss and Yund, 1988;
Yund and Parker, 1989), and geographic variation in
growth morphology correlates with the frequency of com-
petition (Yund, 1987). Nevertheless, while this evidence
suggests that natural selection favors colonies with exten-
sive peripheral stolon development, colonies with little
peripheral stolon development are present in the popu-
lation of Hydractinia symbiolongicarpus sampled (see
shape measures in Table I, and see McFadden et ai, 1984)
and in other Hydractinia populations (Yund, 1987).

The results of the breeding experiment indicate a pos-
sible explanation for the maintenance of morphological
variation in Hydractinia populations. While limits to the
effects of natural selection are usually caused by counter-

vailing selection, rather than exhaustion of additive genetic
variance (see Lande, 1988), the latter has been implicated
in a number of studies (e.g., see Falconer, 1981; Lynch
and Sulzbach, 1984; Hilbish and Koehn, 1985; Berven,
1987; Travis et ai. 1987; Emerson et ai, 1988; Gibbs,
1988). Possibly, this has occurred in Hydractinia popu-
lations, i.e., natural selection has removed much of the
additive genetic variation controlling peripheral stolon
development, and what remains may be largely non-ad-
ditive (i.e., subject to epistatic and dominance effects) and
thus masked from selection. We suggest this only as a
possibility for directing future work; the small size of the
laboratory population of Hydractinia precludes any firm
generalization to natural populations.

Heterochrony in hydractiniid hydroids

Using morphological criteria, Podocoryne and Hy-
dractinia have been grouped in the same family (e.g.,
Mills. 1976), and mtDNA sequence data strongly support
this interpretation (C. Cunningham, pers. comm.). In this
phylogenetic context, variation between Podocoryne and
Hydractinia can be described in terms of general patterns
ofheterochrony (Gould, 1977). As Gould points out, cer-
tain morphological traits often correlate with suites of life
history characteristics. This seems to be the case with Po-
docoryne and Hydractinia. Relative to the latter, Podo-
coryne grow and mature rapidly (see Hauenschild, 1956;
Braverman, 1963), produce energetically inexpensive
medusae (cf. Schierwater, 1989; Schierwater and Hauen-
schild, 1990), and disperse widely to new and varied sub-
strata (Podocoryne have swimming medusae and larvae,
while Hydractinia lack medusae and have crawling lar-
vae). Further, the morphology of Podocoryne can be re-
garded as juvenilized relative to Hydractinia. When either
hydroid fully covers the substratum, stolons form densely
packed structures (a fused stolonal mat in Hydractinia,
and a structure resembling a stolonal mat in Podocoryne).
Such a structure is thus the final developmental stage in
both taxa. Hydractinia, however, always forms some sto-
lonal mat early in its colony development, and in many
cases much of the young colony consists of stolonal mat
(hence the nearly circular shapes of some Hydractinia
strains). Very young (and sexually immature) Hydractinia
thus attain a developmental stage (i.e.. fused stolonal mat
and nearly circular shape) that is only approached by fully
mature Podocoryne. The latter can thus be regarded as
paedomorphic or the former peramorphic (see Alberch
etal, 1979).

Shape variation in clonal organisms

Many clonal plants, fungi, and invertebrate animals
are composed of clonally iterated food-gathering units (i.e..

404

N. W. BLACKSTONE AND L. W. BUSS

ramets) connected by vascular canals ( £'.#.. stolons or rhi-
zomes) that adhere to the substratum (Boardman el ai,
1973: Larwood and Rosen, 1979; Jackson cl at., 1985;
Harper etai, 1986). Clonal morphologies of this sort vary
markedly in the development of peripheral stolons or rhi-
zomes (Buss, 1979; Jackson, 1979; Lovett-Doust, 1981;
Harper, 1985). The approach used here to measure vari-
ation in peripheral stolon development in hydractiniid
hydroids may prove useful in other analyses of clonal
form. Simple shape measures such as perimete r/\area can
easily be acquired from properly lighted specimens with
a simple pixel gradient detector (provided by most com-
mercially available image analysis software). Use of such
characters may have considerable technical advantages
for the analysis of ontogenetic and phylogenetic changes
in colony form.

Acknowledgments

The Peabody Museum Morphometrics Laboratory, the
Social Sciences Statistics Laboratory, and the Yale Com-
puter Center were used for image analysis and data anal-
ysis. Comments were provided by M. Dick, R. Lange, B.
Schierwater, and two reviewers. The National Science
Foundation (BSR-88-05961) provided support.

Literature Cited

Albcrch P., S. J. Gould, G. F. Osier, and D. B. Wake. 1979. Size and
shape in ontogeny and phylogeny. Paleobiology 5: 296-317.

Berven, K. A. 1987. The heritable basis of variation in larval devel-
opmental patterns within populations of the wood frog (Rana syl-
valica). Evolutional: 1088-1097.

Blackstone, N. W. 1987. Specific growth rates of parts in a hermit
crab: a reductionist approach to the study of allometry. J. Zoo/. Land.
211:531-545.

Blackstone, N. W., and P. O. Vund. 1989. Morphological variation in
a colonial marine hydroid: a comparison of size-based and age-based
heterochrony. Paleobiology 15: 1-10.

Boardman, R. S., A. H. Cheetam, and W. A. Oliver. 1973. Animal
Colonies, Development, ami Function through Time. Dowden,
Hutchinson. and Ross. Stroundberg. PA.

Bookstein, F. L. 1978. The Measurement of Biological Shape and Shape
Change. Lecture Notes in Biomathematics. No. 24. Springer- Verlag.
New York.

Braverman, M. 1963. Studies of hydroid differentiation II. Colony
growth and the initiation of sexuality. J. Embryol. Exp. Morphol.
11: 239-253.

Buss, L. W. 1979. Habitat selection, directional growth, and spatial
refuges: why colonial animals have more hiding places. Pp. 459-497
in Biology and Systematic! of Colonial Organisms, G. Larwood and
B. Rosen, eds. Academic Press, London.

Buss, L. W., and P. O. Vund. 1988. A comparison of recent and his-
torical populations of the colonial hydroid Hydractinia. Ecology 69:
646-654.

Buss, L. W., and P. O. Yund. 1989. A sibling species group of Hy-
dractinia in the northeastern United States. J. Mar. Biol. Assoc.
U. K. 69: 857-874.

Buss, L. W., and R. K. Grosberg. 1990. Morphogenetic basis for phe-
notypic differences in hydroid competitive behavior. Nature 343: 63-
66.

Buss, L. W., C. S. McFadden, and D. R. Keene. 1984. Biology of hy-
dractiniid hydroids. 2. Histocompatibility effector system/competitive
mechanism mediated by nematocyst discharge. Biol. Bull. 167: 139-
158.

Cunningham, C. W'., L. W. Buss, and C. Anderson, (in press). Molecular
and geologic evidence of shared history between hermit crabs and
the symbiotic genus Hydractinia. Evolution.

Edwards, C. 1972. The hydroids and the medusae Podocoryne areolala.
P horcalis. and l> earned .1 Mar Biol Assoc. i' K 52: 97-144.

Emerson. S. B., J. Travis, and M. Blouin. 1988. Evaluating a hypothesis
about heterochrony: larval life-history traits and juvenile hind-limb
morphology in Hyla crucifer. Evolution 42: 68-78.

Falconer. D. S. 1981. Introduction to Quantitative Genetics. 2nd ed.
Longman, London.

Person, S., V. J. Rohlf, and R. K. koehn. 1985. Measuring shape vari-
ation of two-dimensional outlines. Sysl. Zoo/. 34: 59-68.

Gibbs, II. L. 1988. Hentability and selection on clutch size in Darwin's
medium ground finches (Geospiza fortis). Evolution 42: 750-762.

Gould, S. J. 1973. The shape of things to come. Syst. Zoo/ 22: 401-
404.

Gould, S. J. 1977. Ontogeny and Phytogeny Harvard University Press,
Cambridge, MA.

Harper, J. L. 1985. Modules, branches, and capture of resources. Pp.
1-33 in Biology and Systematic! of Colonial Organisms, G. Larwood
and B. Rosen, eds. Academic Press. London.

Harper, J. L., B. R. Rosen, and J. White (eds.). 1986. The Growth
and Form of Modular Organisms. Royal Society Press, London.

Hauenschild, C. 1954. Genetische entwicklungphysiologische Unter-
suchungen uber Interse-xualitat und Gewebevertraglichkeit bei Hy-
dractinia echinata. Roux's Arehiv. fur Enlwicklungsmech. 147: 1-
41.

Hauenschild, C. 1956. Uber die VererbungeinerGewebevertraglichkeit-
Eigenschaft bei den Hydroidpolypen Hydractinia echinata. Z. Na-
liirforscli. lib: 132-138.

Hilbish, T. J., and R. K. Koehn. 1985. Dominance in physiological
phenotypes and fitness at an enzyme locus. Science 229: 52-54.

Jackson, J. B. C. 1979. Morphological strategies of sessile animals.
Pp. 499-555 in Biology and Systematic! of Colonial Organisms, G.
Larwood and B. Rosen, eds. Academic Press, London.

Jackson, J. B. C, L. W. Buss, and R. E. Cook (eds.). 1985. Population
Biology and Evolution of Clonal Organisms. Yale University Press,
New Haven. CT.

Lande, R. 1988. Quantitative genetics and evolutionary theory. Pp.
7 1 -84 in Quantitative Genetics. B. S. Weir, E. J. Eisen. M. M. Good-
man, and G. Namkoong, eds. Sinauer, Sunderland. MA.

Lange, R., G. Pliekert, and W. A. Muller. 1989. Histoincompatibility
in a low invertebrate. Hydractinia echinata: analysis of a mechanism
of rejection. J. E.\y>. Zoo/. 249: 284-292.

Larwood, G., and B. Rosen (eds.). 1979. Biology and Systematic! of
Colonial Organisms. Academic Press. London.

Lohman, G. P. 1983. Eigenshape analysis of microfossils: a general
morphometnc procedure for describing changes in shape. Math. Geol.
15:659-672.

Lovett-Doust, L. 1981. Population dynamics and local specialization
in a clonal perennial (Ranunculus repens). I. The dynamics of ramets
in contrasting habitats. ./. Ecol. 69: 743-755.

Lynch, C. B., and D. S. Sulzbach. 1984. Quantitative genetic analysis
of temperature regulation in Atits musculus. II. Diallel analysis of
individual traits. Evolution 38: 527-540.

Mazer, S. J. 1987. Parental effects on seed development and seed yield

HYDRACTINIID HYDROID SHAPE

405

in Raphanux raphanistrwn: implications for natural and sexual se-
lection. Evolution 41: 355-371.

McFadden, C. S. 1986. Laboratory evidence for a size refuge in com-
petitive interactions between the hydroids Hydractinia echinata
(Flemming) and Podocoryne ainiea(Sars). Bid Bui/. 171: 161-174.

McFadden, C. S., M. J. McFarland, and L. \V. Buss. 1984. Biology
of hydractiniid hydroids. I. Colony ontogeny in Hydractinia cchinaia
(Hemming). Bio/. Bull. 166: 54-67.

Mills, C. E. 1976. Podocoryne se/ena. a new species of hydroid from
the Gulf of Mexico, and a comparison with Hydractinia cchinaia
Bioi Bull. 151: 214-224.

Mitchell-Olds, I. 1986. Quantitative genetics of survival and growth
in Impatiem capensis. Evolution 40: 107-116.

Patton, D. R. 1975. A diversity index for quantifying habitat "edge."
l\'ildl. Soc. Bull 3: 171-173.

Plickert, G., M. Kroiher, and H. Munk. 1988. Cell proliferation and
early differentiation during embryonic development and metamor-
phosis of Hvdraciinia echinata. Development 103: 795-803.

Rees, W. J. 1941. On life history and developmental stages of the
medusa Padocoryne borealis. J Mar. Biol. Assnc. Plymouth 25: 309-
316.

Schierwater, B. 1989. Experimented Studien zu den energetischen
Kosten der Fortptlanzung bei sexuellem versus vegetativem Modus
an Eleitthmu dicholoma (Athecata, Hydrozoa) und Sly/aria laiiislris
(Naididae. Oligochaeta). Ph.D. Thesis. University of Braunschweig,
Braunschweig, FRO.

Schierwater. B., and C. Hauenschild. 1990. The position and conse-
quences of a vegetative mode of reproduction in the life cycle of a

hydromedusa and an oligochaete worm. Pp. 37-42 in Advances in

Invertebrate Reproduction 5. M. Hoshi and O. Yamashita, eds. El-

sevier. Amsterdam.
Schierwater, B., B. Piekos, and L. \V. Buss, (in press). Hydroid stolon

contractions mediated by contractile vacuoles. J. E.\p. Biol
Sokal, R. R., and F. J. Rohlf. 1981. Biometry. 2nded. W. H. Freeman,

San Francisco.
Spindler, A. N., and \V. A. Muller. 1972. Induction of metamorphosis

by bacteria and by lithium pulse in the larvae of Hydractinia echinata

(Hydrozoa). Wilhelm Roux' Arch. Entwicklungsmech, Org 169:271-

280.
Travis, J., S. B. Emerson, and M. Bluuin. 1987. A quantitative genetic

analysis of larval life history traits in Ilyla crueller. Evolution 41:

145-156.

Via, S. 1988. Estimating variance components: reply to Groeters. Evo-
lution 42: 633-634.
Weis, V. M., and L. W. Buss. 1987. infrastructure of metamorphosis

in Hvdraciinia cchinaia. Poxlilla 199.
Yund, P. O. 1987. Intraspecific competition and the maintenance of

morphological variation in the colonial marine hydroid Hydractinia

symbioacadianus. Ph.D. Thesis. Yale LIniversity, New Haven. CT.
Yund, P. O., C. W. Cunningham, and L. \V. Buss. 1987. Recruitment

and postrecruitment interactions in a colonial hydroid. Ecology 68:

971-982.
Yund, P. O., and H. M. Parker. 1989. Population structure of the

colonial hydroid Hydractinia sp. nov. C in the Gulf of Maine. /

Exp. Mar. Biol. Ecol 125: 63-82.

Reference: Binl. Bull 180: 406-415. (June, 1991)

Inducible Agonistic Structures in the Tropical
Corallimorpharian, Discosoma sanctithomae

J. S. MILES

Marine Science Center, Northeastern University, Nahant, Massachusetts 01908

Abstract. The Corallimorpharia are a group of soft-
bodied anthozoans closely related to the scleractinian
corals. Although numerous reports have documented the
agonistic behaviors of actiniarians and hard corals, only
Chadwick (1987) has shown such behaviors in a coralli-
morph (Corynactis California). The following investigation
confirms the use of inducible aggressive structures in space
competition in the laboratory and in the field by Disco-
soma sanctithomae. This tropical corallimorph used both
modified marginal tentacles and mesenterial filaments to
damage adjacent scleractinians. All colonies ofAgaricia
agaricites transplanted near D. sanctithomae were dam-
aged. Initially, D. sanctithomae adjacent to Meandrina
meandrites were severely wounded. However, 67% re-
covered and retaliated within a one to six month period,
causing damage to M. meandrina that persisted for at
least twelve months.

Introduction

Many benthic cnidarians that reproduce asexually ex-
pand their colonies proximally and radially. New indi-
viduals require space on the substrate to become estab-
lished and to grow. In a coral reef environment, however,
space is limiting, and as an individual, or clone, expands,
competitive interactions are common. These competitive
encounters may have provided important selective pres-
sures for the evolution of agonistic behaviors and struc-
tures to deal with these competition events. Abel (1954)
first described acrorhagi, the inflatable sacks around the
collar of certain Actiniaria. Francis ( 1 973a) noticed a par-
ticular spatial pattern among conspecific anemone clones
ofAnthopleura elegantissima: individuals of a clone were
closely aggregated, but groups of different clones were al-
ways separated by an "anemone-free zone." This led

Received 17 September 1990; accepted 24 January 1991.

Francis to describe a series of behaviors in which A. ele-
gantissima used acrorhagi during agonistic interactions
with non-clonemates. Other researchers have investigated
various aspects of aggressive behavior in members of the
class Anthozoa (Bonnin, 1964; Williams, 1975; den Har-
tog, 1977; Purcell, 1977; Bigger, 1980; Kaplan, 1983;
Sammarco et ai, 1983; Bak and Borsboom, 1984; Hidaka
and Yamazato, 1984; Sebens, 1984; Chadwick. 1987).
Knowledge has evolved from initial descriptions of
straightforward, predictable results of spatial competition
events (Lang. 1971, 1973; Francis, 1973b; Chornesky.
1983; Chornesky and Williams, 1983) to descriptions of
more complex, dynamic interactions. The importance of
temporal scale was recognized, and many competitive
outcomes were discovered to be reversible (Bak et ai,
1982; Logan, 1984; Chornesky, 1985). The initial victor
was not always the ultimate winner. Other factors such
as size, attack angle, and previous aggressive history af-
fected the outcome of competitions (Brace and Pavey,
1978; Brace, 1981; Bak et al. 1982). Also, significant work
has been done elucidating the systems of recognition re-
quired for these agonistic behaviors (Theodor, 1970; Hil-
demann, 1974; Bigger, 1980; Sauer et al.. 1986). The
number of species known to exhibit specialized structures
used in aggressive behaviors has also increased, including
members from four different orders within the class An-
thozoa (reviewed by Lang and Chornesky, 1988).

In addition to acrorhagi, certain species in the order
Actiniaria employ a modified feeding tentacle as a fighting
tentacle (= catch tentacles, Purcell, 1977). Functionally
similar to the acrorhagi, this elongated tentacle can adhere
to neighboring non-clonemate conspecifics, causing tissue
necrosis and ultimately, if successful, retreat of the op-
position. As with A. elegantissimma. these behaviors have
been reported to produce single-clone aggregates separated
by anemone-free zones (Purcell, 1977; Purcell and Kitting,
1982). One report indicated the mechanism also worked

406

CORALLIMORPHARIAN AGGRESSION

407

on an intrasexual level, yielding anemone-free zones be-
tween clones of the same sex (Kaplan, 1983). Some scler-
actmians possess a structure similar to the actiniarians'
fighting tentacle. Sweeper tentacles, so termed because
they sweep the adjacent area, develop on polyps of certain
reef corals (Richardson et at., 1979; Chornesky, 1983;
Chornesky and Williams, 1983; Hidaka and Yamazato.
1984). The development of sweeper tentacles is induced
by the presence of, or aggression by, another coral (Chor-
nesky, 1983). These interactions are primarily interspecific
and are often used in conjunction with a second mech-
anism. Lang(1971, 1973)and Logan (1984) described the
process of extracoelenteric digestion used by reef corals
to avoid being overgrown and to acquire new space. The
extrusion of mesenterial filaments through the mouth and
body wall onto another coral results in partial mortality
of the opposing colony.

Although the Octocorallia use allelochemicals in com-
petitive interactions (Sammarco et a/., 1983, 1985;
LaBarre, 1986; Pawlik et ai. 1987), until recently no
member of this subclass was reported to have specialized
structures used for aggression. Several reports described
sweeper-like tentacles on species of Alcyonacea and Gor-
gonacea, but these tentacles are probably feeding apparati
(Abel, 1970; Muzik, 1983). However, Erythropodium
caribaeorum (Gorgonacea), develops sweeper tentacles
and uses them for aggression (Sebens and Miles, 1988).
These structures function in the same way as the sweeper
tentacles of the scleractinians, but instead of only one or
two tentacles per polyp becoming sweepers, all eight of
the tentacles on many polyps elongate and are able to
damage neighboring corals. It is of interest to note that
E. caribaeorum is the only obligate encrusting gorgonian
in the Caribbean. This growth form inevitably leads to
interactions with a variety of other species requiring space
on the primary substratum.

The Corallimorpharia are another order with members
exhibiting agonistic behaviors. These soft-bodied members
of the Hexacorallia resemble anemones, but are related
more closely, morphologically and phylogenetically. to
the scleractinians. Chadwick ( 1987) reported that the cor-
allimorph Corynactis califarnica used mesenterial fila-
ments against species of anemones and corals in agonistic
interactions in the laboratory. Earlier, den Hartog (1977)
described two types of tentacles along the rim of the oral
disk of the corallimorph Discosoma sanctithomae (Du-
chassaing and Michelotti). Some of these marginal ten-
tacles are thin and hair-like, whereas others are finger-
like and bulbous. He found these bulbous tentacles to
have larger and more dense holotrichous nematocysts than
the thin counterparts and suggested that these might rep-
resent a morphological variant used in agonistic encoun-
ters, although no experimental work was done. Sebens

(1976) examined this species in field and laboratory studies
in Panama but found no evidence of agonistic behavior
in short-term experiments. Here, I report the first field
results demonstrating agonistic behavior in competitions
between a corallimorph and several species of scleractinian
corals. D. sanctithomae used both mesenterial filaments
and enlarged marginal tentacles to damage the scleractin-
ian corals Agaricia agaricites and Meandrina meandrites.

Materials and Methods

Site location and description

Field and laboratory experiments were completed at
the Discovery Bay Marine Laboratory in Discovery Bay.
Jamaica (18°30'N: 77°20'W). The reef crest along the
north coast of Jamaica runs predominantly east to west,
with spur and groove formations jutting out to the north.
At Discovery Bay, the fore reef is separated from a well-
developed lagoon by a conspicuous reef crest that has
mounds of exposed coral rubble accumulated from Hur-
ricane Allen in 1980. Spur and groove formations begin
at 10m on the fore reef and continue to the fore reef
slope, which occurs at 2 1 m at some locations. The east
back reef is predominantly a sandy bottom with Thalassia
beds interspersed with patch reefs. Columbus Park is an
area of the back reef with high concentrations of silt and
of paniculate matter that reduces visibility. Shallow areas
are dominated by benthic soft-bodied zoantharians within
a dead Acropora cervicornis framework. The deeper re-
gions possess a rich sponge and mollusk community. For
a more detailed description, see Goreau (1959), Goreau
and Goreau (1973), and Liddell et ai. (1984). Survey data
were collected from the west back reef and Columbus
Park, and from Long Term Study (LTS), Kinzie's Reef,
and Lynton's Mine on the fore reef. Transplant experi-
ments were located at all the fore reef survey sites between
depths of 10 m and 20 m (Fig. 1 ).

Animal descriptions ami collection

Discosoma sanctithomae is a corallimorpharian com-
mon throughout the Caribbean and Bermuda between
depths of 1-20 m (den Hartog, 1980). Both solitary in-
dividuals and asexually produced clonal aggregates can
be found living within the coral reef framework. The an-
imal is orally-aborally flattened, with the oral disk aver-
aging approximately 4 cm in diameter (Fig. 2A). A margin
at the edge of the oral disk lacks tentacles and is often
tucked up under the disk, but at other times is expanded
well beyond the basal attachment area. The oral disk ten-
tacles of D. sanctithomae are very short, stubby, and are
often ramous. A second group of tentacles extends radially
from the margin of the disk and are thus termed marginal
tentacles.

40S

J. S. MILES

Figure 1. Map of the fore reef at Discovery Bay. Jamaica. Areas marked with stars indicate sites of
transplant experiments and tore reef survey sites.

Specimens ofDiscosoma sanctithomae used in the field
experiments were never removed from the reef or dis-
turbed in any way. Individuals were identified by tags
placed on nearby coral rubble. D. sanctithomae used in
laboratory experiments were collected along with pieces
of the substrate, usually dead Acropora cervicornis, because
attempts to scrape off individuals were always unsuccess-
ful. The collected animals were placed in a running sea-
water table and allowed to acclimate for three to seven
days before being used in experiments.

The sderactinian corals Agaricia agaricites (Pallas) and
Meandrina meandrites (L) were used in the field trans-
plants. Pieces of coral (approximately 6X8 cm) were
collected using a rock hammer and chisel to release them
at their base or at an area of dead coral skeleton. The
corals were collected from the same reefs onto which they
were to be transplanted. They were not brought to the
surface, but were left for two to seven days before being
transplanted. Corals were not used if they showed signs
of tissue damage from the collection methods within this
period. A. agaricites and M. meandrites used in the lab-

oratory experiments were collected in a similar manner.
However, these corals were transferred to running sea-
water tables and allowed to acclimate for three to seven
days.

Field surveys

Field surveys of Discosoma sanctithomae were con-
ducted to determine which organisms lived adjacent to
the corallimorph and what interactions were occurring.
The surveys were done by swimming parallel transects
across depth contours throughout a designated area and
recording every D. sanctithomae observed. Each of the D.
sanctithomae' s neighbors were noted, and any damage on
either D. sanctithomae or any neighbor was recorded.
Percent-cover data were gathered from the same area as
the D. sanctithomae survey. Survey procedures involved
assigning random numbers to a chain-link transect that
was haphazardly dropped within the study area. Species
or substrate type that fell under each of the marked chain
links was recorded (Rogers et a/., 1983). Information from

CORAl.LIMORPHARIAN AGGRESSION

409

Figure 2. (A) Agarkia agarkiic.i (a) transplanted next to a Discnsoma sanclithomae with thin marginal
tentacles (arrows). Scale = 1 cm. (B) D sanctithomae adjacent to Meandrina meandrites (M) developed
large swollen acrospheres (a) at the tips of the marginal tentacles. Areas of the rim have become enlarged
also. Scale ~ 2 mm. (C) D sancnihnmac with acrospheres. Mesenterial filaments (mf) can be seen in the
area of the coelenteron leading to the marginal tentacles. Scale = 2 mm

the D. sanctithomae survey and the percent cover transects
was compared to determine whether D. sanclithomae" s
contact with neighbors was random or reflected some sort
of selection for, or by, neighboring species.

Transplant experiments

Field. Manipulative experiments were done in the field
to study the interactions ofDiscosoma sanctithomae and
scleractinian corals. Pieces of coral were epoxied adjacent
to, but not touching, individual D. sanctithomae, which
were partially retracted. However, transplants were located
so that fully expanded D. sanctithomae would touch the
coral's tissue. The Pettit Underwater Patching Compound
used to fix the corals in place was not toxic when applied
only to the dead base of the coral. Corals showing a general
tissue necrosis (possibly from handling) soon after the

transplant ( 1-2 days) were removed from the study; this
accounts for most of the discrepencies between initial and
final sample sizes. Specific sets of transplants were de-
signed to address the following questions: (1) Can D.
sanctithomae damage scleractinian corals adjacent to
them? (2) Are bulbous marginal tentacles with acrospheres
associated with the damage to corals? (3) Do D. sanctitho-
mae react differently depending on the species of coral
that is next to them?

Three sets of transplants (Series I) were begun in Jan-
uary 1987. In the first set of transplants (Tl) pieces of
Agaricia agari cites (n = 19) were placed next to D. sanc-
tithomaev/ith filiform marginal tentacles (Fig. 2A). A sec-
ond group of transplants (T2) paired A. agaricites (n = 16)
with D. sanctithomae that had bulbous marginal tentacles.
A. agaricites was chosen because it was found frequently
next to D. sanctithomae in the field surveys. Both sets of

410

J. S. MILES

transplants were designed to examine D. sanctithomae's
tendency to damage corals. Also, if the bulbous tentacles
were responsible for damage to the scleractinians, the cor-
als in T2 would be expected to incur damage more quickly
than the corals in Tl. Meandrina meandrites (n = 18)
was used in a third transplant experiment (T3) to test for
any variation in response by D. sanctithomae. Unlike A.
agaricites, this coral is known to use mesenterial filaments
readily in aggressive encounters (Lang, 1973; Logan,
1 984). All experimental pairs were monitored for damage
to D. sanctithomae or to A. agaricites, and for any changes
in the morphology of the marginal tentacles on D. sanc-
tithomae once a week for five weeks, again after five
months, then after one year. Several night dives were done
to confirm damage to coral polyps and to check for de-
velopment of sweeper tentacles (Chornesky, 1983). Pho-
tographs of experimental pairs were taken weekly with a
Nikonos camera with a 2: 1 extension tube, and a Minolta
XL40 1 Super-8 movie camera was left on the reef for four
days at a time, taking photographs at 1.5-min intervals.
A second series of transplants (Series II) was started in
February 1988. These experiments were identical to the
1987 Tl and T2 transplants except that they were mon-
itored once a day for two and a half weeks to examine
the interactions over a shorter time period.

Two types of controls were used to test for the effects
of the transplantation process. Pieces of coral transplanted
near Discosoma sanctithomae were always large enough
so that at least half of their tissue area was out of reach
of the D. sanctithomae. even when fully expanded (op-
posite-side controls). Additional pieces of coral were
transplanted among the experimental pairs, but not within
reach of any D. sanctithomae. These corals were regularly
examined for any signs of damage. A control for D. sanc-
tithomae acrosphere formation was done by observing
the marginal tentacles of two sets of D. sanctithomae that
were surrounded only by algae. One group of D. sanc-
tithomae was monitored once every ten days for two
months. The second group was monitored every day for
up to twenty days. These individuals were studied to de-
termine whether sporadic changes in the marginal ten-
tacles occurred without contact with cnidarian neighbors.

Laboratory experiments. Transplant experiments sim-
ilar to those in the field were done in the laboratory in
running seawater tables. Discosoma sanctithomae indi-
viduals were paired with pieces ofAgaricia agaricites (n
= 5) and Meandrina meandrites (n = 5). None of the D.
sanctithomae had enlarged marginal tentacles, nor did
the corals have sweeper tentacles at the beginning of the
experiment. When both members of an experimental pair
were contracted, neither touched the other. Three pieces
of A. agaricites and two of M. meandrites. as well as three
individuals of D. sanctithomae. were out of reach of any
other anthozoan and acted as the controls. These pairs

were inspected for damage every hour for the first seven
hours. Throughout the experiment a Super-8 movie cam-
era with an intervalometer photographed individual pairs
every 1-5 min. The experiment continued for eight days.

Results

Survcv data

The neighboring species of more than 155 Discosoma
sanctithomae were recorded (Fig. 3A, B). Approximately
37% (n = 238) of them were foliose or turf complex algae,
the largest group total. The second most common group
found adjacent to D. sanctithomae were crustose coralline

300

200

100

r_] Neighbors without damage
B Neighbors with damage

Percent Cover

Percent ot Neighboring Species

Figure 3. (Top) The area adjacent to individual Discosoma sanc-
lillinmac were surveyed at all transplant sites on the tore reef and in
Columbus Park. Neighboring species and substrate type were counted
and classified as damaged or not damaged; exact counts are reported
above bars. Each organism and substrate type counted as " 1 " interaction
regardless of size: this may underestimate the impact of larger organisms
and overestimate those of smaller ones. (Bottom) Surveys of species per-
cent-cover were done at all the neighbor survey sites. Sixteen transects
were completed: 713 chain-link transect points were classified into the
same eight catagories used for the neighbor survey. Numbers above each
bar represent percentages for each group. Percent-cover results (black)
are compared to percentage data of neighboring species (white: n = 64 1 ).

CORALLIMORPHARIAN AGGRESSION

algae (20.4%), followed by the scleractinian corals (18%).
The corals found most frequently adjacent to the coral-
limorphs were Alontastrea annularis. Sidentstreu siderea,
and Agarida agaricites. In more than 75% of the cases
when D. sanctithomae was adjacent to a scleractinian,
there were areas of dead coral associated with the area of
contact. Damage was not readily apparent in any other
group (Fig. 3A).

Although algae were also the most abundant organisms
in the surveys of percent cover (n = 557, 78%), sclerac-
tinian corals (9%-) and sand (8%) were the second and
third most commonly occurring items, respectively (Fig.
3B). The complement of species and groups neighboring
D. sanctithomae proved to be significantly different than
the proportion of species expected from the percent cover
survey using a G-test for independence (G = 21.03, P
<0.05). Algae have been dominant space occupiers in
the fore reef community since the die-off of Diadcnui an-
tillamm in 1983 (Liddell and Ohlhorst, 1983).

Field transplant '.v

Series I. Sixteen of seventeen Discosoma sanctithomae
originally with filiform tentacles had developed bulbous
tentacles with acrospheres in the presence of Aguricia
agaricites within six weeks. The mean time for acrosphere
development was 17.3 ± 2.0 days (mean ± S.E.). During
this time, all 17 of the A. agaricites colonies had been
damaged; the mean time to damage from each colony
was \1.9 ± 1 .8 days (mean ± S.E.) (Fig. 4A). In compar-
ison, only one opposite-side control was damaged. For-
mation of acrospheres and the occurrence of damage was
significantly greater than that which might occur by
chance (G-test with William's correction factor: G = 15.5;
G = 23.0 resp.; P < 0.05). The time to acrosphere devel-
opment was not significantly different from the average
time for damage to occur to the corals (Mann- Whitney;
U'= 170.5, P< 0.05).

All 12 Agaricia agaricites colonies placed next to the
Discosoma sanctithomae that had acrospheres at the start
of the experiment (T2) were damaged. The average time
to damage ( 10.0 ± 1 .7 days, mean ± S.E.) was significantly
faster than the time to damage for the transplants that
later formed acrospheres (Tl ) (Mann- Whitney: U = 204,
P < 0.05) (Fig. 4 A). Again, only one opposite-side coral
control was damaged. Sweeper tentacles on A. agaricites
occurred on only one colony. They formed after the in-
teractions had progressed for about one month and after
the coral had been initially damaged by the corallimorph.
The corresponding D. sanctithomae did not reveal any
damage.

The results of the Discosoma sanctithomae transplants
with Meandhna meandrites were strikingly different.
Within one week, 38% of the D. sanctithomae (n = 16)

.e
=

T1 : A (n=l7) animals w.' acrospherea

Tl n in-17) corals w damage
TZ: D (n=1 2) corals w/ damags

20 30

Time (Days)

ioo r

XII

C 60

Damage lo Discosoma
Damage lo Meandhna

2')

ISO

Time (Days)

Figure 4. (Top) Results of two field transplant experiments (Tl &
T2) are shown. In the first transplant (Tl ) 17 corals were placed next to
Disctmima sancnthonuic with thin marginal tentacles. T1:A (A) charts
the progression of acrosphere development in D. sanctithomae. T1:D
(O) tracks the development of damage to the corals. In T2, 12 corals
were next to D. saiMithomae that possessed acrospheres. T2:D (•) records
the damage incurred to the adjacent corals. (Bottom) Results of field
transplant 3 (T3). Eighteen Meandrina meandrites were placed near D
sanctithomae with acrospheres. Initially 38% of the D. sanctithomae were
severely injured: two died within the first month. Although none of the
corals suffered damage before the end of the first month, 67% of the
remaining transplants were damaged over the subsequent six-month pe-
riod and remained damaged for at least twelve months.

had suffered severe body lesions from the mesenterial fil-
aments of M. meandrites (Fig. 4B). This increased to 63%
within two weeks, culminating in the death of two D.
sanctithomae individuals within the first month. The first
incidence of damage to M. meandrites did not occur until
almost one month had passed. However, of those D. sanc-
tithomae that survived the first two months (n = 12), 67%
went on to damage the M. meandrites over the next four
months. Damage inflicted by these D. sanctithomae was
still visible twelve months later (Fig. 5). None of the op-
posite-side coral controls were damaged.

Series II. Results of the Series II transplants were similar
to Series I results. Ten of eleven A. agaricites corals placed

412

J. S. MILES

Figure 5. DiM-uwniu sanctithomae vs. Mcandnna meandrites: in-
teraction after twelve months. Note the large acrospheres (a) and the
algae-covered dead coral skeleton (d). Live coral polyps (p) can be seen
out of reach of the D. sanctithomae's tentacles. Scale = 2.7 mm.

next to Discosoma sanctithomae with acrospheres were
damaged in 12.0 ± 1.5 days (mean ± S.E.). Only two of
the eleven corals adjacent to D. sanctithomae without ac-
rospheres were clearly damaged (6.0 ± 5.0 days, mean
± S.E.). Only three of eleven had developed acrospheres
within the trial period of 10 to 20 days. This short period
before damage occurred reflects one coral that was exten-
sively damaged after the second day by mesenterial fila-
ments that were released through the marginal tentacles
of the corallimorph. Mesenterial filaments were observed
being extruded by D. sanctithomae through the tentacles
on numerous other occasions, and were extruded fre-
quently from the mouth and through the body wall as
well.

Controls

None of the isolated coral controls experienced any
tissue damage. The lack of incidences of damage to op-
posite-side coral controls were reported with the results
for the particular transplant. The D. sanctithomae indi-
viduals acting as controls for random acrosphere devel-
opment showed little change; no acrospheres were formed.
There were, however, frequent influxes and effluxes of
mesenterial filaments to and from the marginal tentacles.
At times the filaments remained between the mesenteries
in the coelenteron, and at other times they traveled into
the tips of the marginal tentacles.

Laboratory experiments

The results of the laboratory experiments were a brief
accelerated version of the field experiments. All the corals,
and the Discosoma sanctithomae individuals, retracted

their polyps when the experimental pairs were initially
established. Although eight of ten corals partially ex-
panded their polyps within 20 min, and all the D. sanc-
tithomae adjacent to A. agaricites had relaxed within the
first hour, there was no direct contact. The D. sanctitho-
mae next to the M. meandrites remained contracted, with
four of them extruding mesenterial filaments within the
first seven hours.

Meandrina meandrites transplants were equally active.
All five corals released mesenterial filaments onto the
Discosoma sanctithomae within the first 7 h. D. sancti-
thomae near A agaricites also extruded filaments but, in
general, the severity of such attacks was greatly reduced
compared to those with M. meandrites. Within 24 h, 4
D. sanctithomae had mucus layers covering body wounds
inflicted by M. meandrites' mesenterial filaments. Body
postures were strongly evasive, especially when compared
to D. sanctithomae near A agaricites: the latter often laid
their marginal disks over the coral surfaces. By the end
of the third day, four of the five corallimorphs near M.
meandrites were dead. The fifth had severe body lesions
and had partially released its hold on the substrate. None
of the M. meandrites individuals were damaged.

None of the Agaricia agaricites individuals were ob-
served releasing mesenterial filaments, nor using sweeper
tentacles. A general pattern evolved for the D. sanctitho-
mae-A. agaricites interactions of gradual expansion of the
D. sanctithomae onto the coral's surface followed by re-
traction, and intermittent extrusion of mesenterial fila-
ments by D. sanctithomae. Discosoma sanctithomae was
able to damage A. agaricites in two separate cases, al-
though the coral recovered its damaged area in one of
these events. Three D. sanctithomae adjacent to A. agar-
icites died within eight days, but two of these deaths must
be qualified. Two of the fatalities resulted from the D.
sanctithomae releasing its hold near the A. agaricites and
wandering into a colony of M. meandrites. None of the
D. sanctithomae individuals formed marginal tentacles.
All the control corals survived without damage, and one
of the three control D. sanctithomae individuals died.

Discussion

Although lacking a hard skeleton, the soft-bodied rel-
atives of the scleractinians, such as the Actiniaria, have
proven to be able competitors for space (Francis, 1973a;
Purcell, 1 977; Purcell and Kitting, 1982;Chadwick. 1987).
More recently, investigators have discovered that the
Corallimorpharia possess aggressive abilities as well. Dur-
ing prolonged interspecific exposure, Corynactis califor-
nica killed polyps of the actiniarians Anthopleura elegan-
tissima and Metridium senile, as well as the scleractinians
Astrangia lajo/laensis and Balanophyllia elegans by ex-
truding mesenterial filaments (Chadwick, 1987). The

CORAI LIMORPHARIAN AGGRESSION

413

present study supplies evidence that another corallimorph,
Discosoma sanctithomae, can compete successfully with
scleractinian corals for primary space. Every colony of
Agaricia agaricites transplanted next to Discosoma sanc-
tithomae was damaged, whereas none of the associated
D. sanctithomae were damaged. Most of the damage oc-
curred within the first month of a 14-month experimental
period. D. sanctithomae was able to cause severe necrosis
of tissue on the coral Meandrina meandhtcs, which is
considered to be near the top of the Caribbean coral com-
petitive hierarchy due to its effective use of mesenterial
filaments in damaging other scleractinians (Lang, 1973).
Although many of the D. sanctithomae were initially in-
flicted with extensive body lesions by M. meandrites,
many recovered and retaliated successfully, causing dam-
age that persisted at least twelve months. This represents
a clear and dramatic example of a competitive reversal
and places D. sanctithomae near the top of a zoantharian
competitive hierarchy.

Most of the agonistic behaviors of soft-bodied antho-
zoans (and some scleractinians) involve morphological
modifications that provide the capability to inflict damage.
Anemones in the family Actiniidae inflate acrorhagi (Abel.
1954; Francis, 1973a; Sebens, 1984); acontiate anemones
in several families develop "fighting tentacles" from feed-
ing tentacles (Purcell, 1977; Purcell and Kitting, 1982;
Kaplan, 1983). D. sanctithomae uses marginal tentacles
frequently filled with mesenterial filaments and ectoderm
engorged with specialized nematocysts (den Hartog, 1977,
1980). The marginal tentacles changed from thin, filiform
appendages to bulbous acrospheres in the presence of A.
agaricites and M. meandrites. The initial increase in vol-
ume seems to be due to the influx of mesenterial filaments,
which is later compounded by the ectoderm thickening
with nematocysts (as seen by den Hartog, 1977). Unlike
the acrorhagi (Bonnin, 1964; Bigger, 1980), which become
inflated with each aggressive interaction, the marginal
tentacles of D. sanctithomae remain bulbous once en-
larged. In this study, the most extensive acrospheres were
found closest to the site of interaction with the scleractin-
ians. Acrospheres never developed in D. sanctithomae
surrounded only by algae, nor were they found in D.
sanctithomae adjacent to sponges, tunicates, or other non-
cnidarian neighbors in the field surveys.

Every incidence of damage to the experimental corals
was associated with the presence of acrospheres except in
one case. The association between acrospheres and dam-
age is further supported by a decrease in the amount of
time before damage appeared on the corals next to D.
sanctithomae with acrospheres compared to those that
developed acrospheres during the experiment. As the in-
teractions progressed, algae may have acted as a buffer to
contact with the acrospheres. After algae began to settle

on the bare coral skeleton, the acrospheres did not inten-
sify further.

Discosoma sanctithomae responded to the corals ad-
jacent to them by developing acrospheres and by inflicting
damage. However, the response was extremely graded. D.
sanctithomae reacted to Agaricia agaricites much differ-
ently than it did to Meandrina meandrites. In general,
the interactions with A. agaricites appeared to be much
more gradual, progressing slowly, but ultimately resulting
in the development of acrospheres on D. sanctithomae
and damage to the coral. Conversely, the behavior of D.
sanctithomae next to M. meandrites was much more dra-
matic, responding to the aggressive actions of M. mean-
drites. Within less than twenty-four hours, D. sanctitho-
mae individuals had been damaged extensively and were
withdrawn, some for several days. Those that later recov-
ered and attacked M. meandrites did so with well-devel-
oped acrospheres. Often the marginal tentacles and even
the rim of the oral disk were thickened and swollen (pers.
obs.), presumably filled with potent nematocysts (den
Hartog, 1977) (Fig. 2B).

The laboratory experiments and the Series II transplants
served to elucidate the differences in Discosoma sancti-
thomae's behaviors. D. sanctithomae'?: response to adja-
cent Meandrina meandrites was immediate and severe.
M. meandrites' quickness in extruding mesenterial fila-
ments and inflicting damage deterred D. sanctithomae
from approaching M. meandrites. In contrast, D. sane-
til homae placed next to Agaricia agaricites repeatedly re-
laxed and expanded its disk directly on top of A. agari-
cites'living tissue. D. sanctithomae did not appear to be
adversely affected by A. agaricites, although it did peri-
odically retract away from contact with the coral. D. sanc-
tithomae could cause small amounts of necrosis to A.
agaricites' tissue which, at least in the early stages, was
often recovered by the coral. However, sometime after
prolonged exposure and repeated attacks, the A. agaricites
was no longer able to regain lost tissue. Not long after this
stage, algae (usually a green alga) began to settle on the
bare coral skeleton. These dead areas persisted for the
remainder of the study period.

The use of mesenterial filaments in competitive inter-
actions is well documented for scleractinians. Some of the
most aggressive corals use exclusively mesenterial fila-
ments for defense (Lang. 1971, 1973; Logan, 1984). The
corallimorph Corynactis californica extruded mesenterial
filaments primarily out the mouth but also through the
body wall and once through the tentacles during agonistic
encounters (Chadwick, 1987). Discosoma sanctithomae
invoked two mechanisms for inflicting damage that reflect
its phylogenetic relationship with the scleractinians and
its particular tentacle morphology. Like many hard corals,
the corallimorph readily emitted mesenterial filaments out
of the mouth and through the body wall when disturbed.

414

J. S. MILES

In addition. D. sanclithomae continually transferred
mesenterial filaments into the discal and marginal ten-
tacles, sometimes passing them out through the tips. It is
not yet known whether there are permanent holes at the
tips of these tentacles through which the mesenteries can
pass. A similar movement of mesenterial filaments occurs
in the discal tentacles of Rhodactis howesii (Corallimor-
pharia) during feeding behaviors (Hamner and Dunn,
1980).

This regular fluctuation of mesenterial filaments dispose
Discosoma sanctithomae to be capable of quickly sending
mesenterial filaments into and out of the tips of the mar-
ginal tentacles when it is involved in agonistic encounters
with neighboring species. D. sanclithomae was observed
to damage adjacent corals with mesenterial filaments from
its marginal tentacles. Although most incidences of dam-
age occurred after acrospheres had formed, some corals
showed damage before this time that could have been
caused by mesenterial filaments. By extruding the fila-
ments out the tips of the marginal tentacles, D. sanctitho-
mae increases the probability of the filaments landing on
the tissue of the opposing organism. It is a behavior that
the corallimorph can invoke quickly at the time of inter-
action because mesenterial filaments regularly fluctuate
in and out of the marginal tentacles. Hence, there is less
time between the recognition of a competitor and the
commencement of an aggressive response than would be
required to form sweeper or fighting tentacles.

Den Hartog ( 1977) determined that the size and density
of holotrich nematocysts was greater in bulbous marginal
tentacles than in the filiform type. This study confirms
that interactions with scleractinians can induce the for-
mation of bulbous tentacles. After a period of less than a
week, thin transparent marginal tentacles of Discosoma
sanctithomae became thickened and more opaque in the
presence of the coral colonies. Some tentacles doubled in
thickness and became opaque, while others more than
tripled their girth and were associated with a greatly thick-
ened oral rim. A few tentacles elongated, forming distinct
tips with acrospheres (Fig. 2). All forms of these thickened
marginal tentacles were able to cause necrosis of the coral
tissue.

The combination of mesenterial filaments and acro-
spheres enabled D. sanctithomae to respond immediately,
warding orTimminent damage, and to develop an alternate
form of defense that required more time to initiate. D.
sanctithomae may use mesenterial filaments in response
to adverse interactions of short temporal scale, and re-
serves acrosphere formation, involving the costly con-
struction of new tissue and many nematocysts, for pro-
longed interactions. Its soft body allows it to avoid some
acts of aggression from opponents by bending away and
perhaps even by moving the base laterally. All such char-
acteristics make D. sanctithomae an effective competitor.

holding its own on the substratum of the reef against some
of the most aggressive corals.

Acknowledgments

This work was supported partially by the Lerner-Gray
Fund for Marine Research and by Sigma Xi Grants-in-
Aid of Research. My research was aided by the field as-
sistance of Lars Kula, Pete Edmunds, Ruth Gates, and
many members of Northeastern University's East/West
Program. P. Schultze provided insightful discussion on
the statistical analysis. K. P. Sebens, J. Witman, M. P.
Morse, and D. O'Brien, along with two anonymous re-
viewers critically reviewed the manuscript. Thank you
all. This is contribution number 182, Marine Science
Center, Northeastern University, Nahant, MA, and num-
ber 495, Discovery Bay Marine Laboratory, Discovery
Bay, Jamaica.

Literature Cited

Abel, E. F. 1954. Ein Beilrag zur Giftwirkung der Aktmien und Funk-

tion der Randsackchen. Zool. An:. 153: 19-268.
Abel, E. F. 1970. Uber den Tentakelapparat der Edelkoralle (Corallhim

nihriim L.) und seine Funktion beim Beutefangverhalten. Oecologia

4: 133-142.
Bak, R. P. M., and J. L. A. Borsboom. 1984. Allelopathic interaction

between a reef coelenterate and benthic algae. Oecologia 63: 194-

198.
Bak, R. P. M., R. M. Termaat, and R. Dekker. 1982. Complexity of

coral interactions: influence of time, location of interaction and epi-

fauna. Mar. Biol 69: 215-222.
Bigger, C. H. 1980. Interspecific and intraspecific acrorhagial aggressive

behavior among sea anemones: a recognition of self and not-self.

Biol. Bull. 159: 117-134.
Hi HIM 1 1 1. J. P. 1964. Recherches sur la "reaction d'aggression" et sur le

fonctionnement des aerorrhages d' 'Actinia equina L. Bull. Biol. Fr.

Belg. 98: 225-250.
Brace, R. C. 1981. Intraspecific aggression in the color morphs of the

anemone Phymactis clematis from Chile. Mar. Biol. 64: 85-93.
Brace, R. C., and J. Pavey. 1978. Size dependent dominance hierarchy

in the anemone Actinia equina. Nature 273: 752-753.
Chadwick, N. 1987. Interspecific aggressive behavior of the coralli-

morpharian Corynactis califonuca (Cnidaria: Anthozoa): Effects on

sympatnc corals and sea anemones. Biol. Bull. 173: 1 10-125.
Chornesky, E. A. 1985. Repeated reversals of competitive dominance

during spatial competition between reef corals. Ecology 70(4): 843-

855.
Chornesky, E. A. 1983. Induced development ol sweeper tentacles on

the reef coral Agaricia agariciles: a response to direct competition.

Biol. Bull. 165:569-581.
Chornesky, E. A., and S. L. Williams. 1983. Distribution of sweeper

tentacles on Monlaslrea cavernosa. Pp. 61-67 in The Ecology of

Deep and Shallow Reefs, M. L. Reaka, ed. Symp. Ser. Undersea Res..

N.O.A.A. Nat. Undersea Res. Prog.. 1.
den Hartog, J. C. 1980. Caribbean shallow water Corallimorphana.

Zool, Verli 176: 1-83.

den Hartog, J. C. 1977. The marginal tentacles of Rhodactis sanctitho-
mae (Corallimorphana) sweeper tentacles of Monlaslrea cavernosa

(Scleractinia). their cnidom and possible function. The Second Int.

Coral Reef Symposium Univ. Miami Press, Miami. FL. 1: 463-470.

CORALL1MORPHARIAN AGGRESSION

415

Francis, I,. I973a. Clone specific segregation in the sea anemone An-
iliopleura elegantissima. Bio/. Bid I 1-44: 64-72.

Francis, I,. I973b. Intraspedfic aggression and its effects on the dis-
tribution of Anthopleura elegantissima and some related sea ane-
mones. Biol. Bull 1-44: 73-92.

Goreau, T. F. 1959. The ecology of Jamaican coral reefs I. Species
composition and zonation. Ecology 40: 67-90.

Goreau, I . F., and N. I. Goreau. 1973. The ecology of Jamaican coral
reefs II. Geomorphology, zonation. and sedimentary phases. Bull.
Mm Sa. 23: 399-464.

Hamner, VV. M., and D. F. Dunn. 1980. Tropical Corallimorphana
(Coelenterata: Anthozoal: feeding by envelopment. Micronesica, vol
16, June.

Ilidaka, M., and K. Yamazato. 1984. Intraspecific interactions in a
scleractinian coral. Galaxeafascicidaris induced formation of sweeper
tentacles. Coral Reels 3: 77-85.

I lildi in.iiiii. W. H. 1974. Some new concepts in immunological phy-
logeny. Nature 250: 116-120.

Kaplan, 8. A. 1983. Intrasexual aggression in Mclridnim senile. Biol.
Bull. 165: 416-418.

LaBarrc, S. 1986. Competitive strategies of soft corals (Coelenterata:
Octocorallia): III. Spacing and aggressive interactions between al-
cyonaceans. Mar Ecol. Prog Set: 28: 147-156.

Lang, J. C. 1971. Interspecific aggression by scleractinian corals I. The
rediscovery of Scolvmia cubensis (Milne Edwards & Haime). Bull
Mar Sa 21: 952-959.

Lang, J. C. 1973. Interspecific aggression by scleractinian reef corals
II. Why the race is not only to the swift. Bull. Mar. Sci 23: 260-
279.

Lang, J. C., and E. A. Chorneskj. 1988. Competition between scler-
actinian reef corals: a review of mechanisms and effects. In Ecosystems
of I/if World. Coral Reefs. Z. Dubinsky. ed. Elsevier Press, Amster-
dam.

Liddell, W. D.. and S. L. Ohlhorst. 1986. Changes in the benthic com-
munity composition following the mass mortality of Diadenia at
Jamaica. J. E.\p Mar Biol. Ecol. 95: 271-278.

Liddell, VV. D., S. L. Ohlhorst, and A. G. Choates. 1984. Modern and
Ancient Carbonate Environments of Jamaica Sedementa X. Miami
Beach. Univ. of Florida Press. 99 pp.

Logan, A. 1984. Interspecific aggression in herrnatypic corals from
Bermuda. Coral Reefs 3: 131-138.

Mn/ik. K. 1983. Zoom and focus: octocorals. Newton Graphic Science

Magazine I Japan) 3: 30-35.
Pawlik, S. R., M. I . Burch, and \V. Kenical. 1987. Patterns of chemical

defense among Caribbean gorgonian corals: a preliminary survey. J.

E.\p. Mar. Biol Ecol. 108: 55-66.
Purcell, J. E. 1977. Aggressive function and induced development of

catch tentacles in the sea anemone Metrulnini senile Coelenterata:

Actimaria. Biol. Bull 153: 355-368.
Purcell, J. E., and C. L. Killing. 1982. Intraspecific aggression and

population distributions of the sea anemone Metridinm senile. Biol

Bull 162: 345-359.
Richardson, C. A., P. Dustan, and J. C. Lang. 1979. Maintenance of

living space by sweeper tentacles ofMontastrea cavernosa, a Caribbean

reef coral. Mar. Biol 55: 181-186.
Rogers, C. S., M. Gilnack, and H. C. Fitz III. 1983. Monitoring of

coral reefs with linear transects: a study of storm damage. J. E.\p.

.Mar. Biol. Ecol 66: 285-300.

Sammarco, P. W., J. C. Coll, and S. LaBarre. 1985. Competitive strat-
egies of soft corals (Coelenterata: Octocorallia). Coral Reefs 1: 173-

178.
Sammarco, P. W., J. C. Coll, S. LaBarre, and B. Willis.

1983. Competitive strategies of soft corals (Coelenterata: Octocor-
allia): allelopathic effects on selected scleractinian corals. J. E.\p. Mar.

Biol Ecol 91: 199-215.
Sauer, K. P.. M. Muller, and M. Weber. 1986. Alloimmune memory

for glycoproteid recognition molecules in sea anemones competing

for space. Mar Biol 92: 73-79.
Sebens, K. P. 1976. The ecology of Caribbean sea anemones in Panama:

utilization of space on a coral reef. Pp. 67-77 in Coelenterate Ecology

and Behavior. G. O. Mackie. ed. Plenum Publ., NY.
Sebens, K. P. 1984. Agonistic behavior in the intertidal sea anemone

Anthopleura xanthogrammica. Biol Bull. 166: 457-472.
Sebens, K. P., and J. S. Miles. 1988. Sweeper tentacles in a gorgonian

octocoral: morphological modifications for interference competition.

Biol. Bull. 175: 378-387.
Theodor, J. L. 1970. Distinction between "self" and "not self in lower

invertebrates. Nature 221: 690-692.
Williams, R. B. 1975. Catch tentacles in sea anemones: occurrence in

Haliplanella luciae (Verrill) and a review of current knowledge. /

Nat. Hist. 9: 241-248.

Reference: Biol. Bull. 180: 416-431. (June, 1991)

Autotomy in Blue Crab (Callinectes sapidus Rathbun)

Populations: Geographic, Temporal,

and Ontogenetic Variation

L. DAVID SMITH12 AND ANSON H. MINES'

^Smithsonian Environmental Research Center, P.O. Box 28, Edgewater. Maryland 21037 and
"-Department of Zoology, University of Maryland, College Park, Maryland, 20742

Abstract. Blue crab (Callinectes sapidus Rathbun) pop-
ulations were examined at four sites in Chesapeake Bay
and three additional sites along the southeastern Atlantic
coast and Gulf of Mexico; the aims were to assess the
incidence of limb autotomy and to determine whether
injury patterns varied temporally, geographically, and
ontogenetically. These data, which include four years of
information from one site (Rhode River, Maryland, a su-
bestuary of central Chesapeake Bay), make this study the
most extensive and intensive survey of limb autotomy yet
conducted in arthropods. A substantial percentage ( 1 7-
39%) of the blue crab populations were either missing or
regenerating one or more limbs, suggesting that autotomy
is an important mechanism for their survival. The fre-
quency of limb autotomy varied, both within and between
years, and over broad geographical scales. Injury levels
were generally correlated positively with crab size. Limb
autotomy was independent of sex and molt stage, and
frequencies varied little among sites in the Rhode River.
Patterns of limb injury in C. sapidus were remarkably
consistent among all sites. The most frequent injury in-
volved loss of a single cheliped. Swimming legs suffered
the least damage. Severe multiple limb loss was rare. Right
and left limbs were lost with equal frequency in most
populations. This consistency of autotomy pattern sug-
gests differential vulnerability of limbs and standard be-
havioral response by blue crabs to various injury-causing
agents. The frequency of autotomy was density-dependent
in the Rhode River, indicating that intraspecinc interac-
tions (e.g., cannibalism) may be a major cause of limb

Received 2 October 1990: accepted 27 December 1990.

loss in populations in the Rhode River subestuary and
elsewhere.

Introduction

Many invertebrate and vertebrate species respond to
injury or its threat by autotomizing (i.e.. severing) a body
part along a breakage plane (Wood and Wood, 1932;
Needham, 1953; Robinson et a!.. 1970; Vitt et al. 1977;
McVean, 1982; McCallum et al.. 1989). While such be-
havior has immediate survival benefits (Dial and Fitzpa-
trick, 1983; Medel et al.. 1988; Smith, 1990a), autotomy
may handicap individuals when foraging (Slater and
Lawrence, 1980; Smith, 1990a), overwintering (Willis et
al.. 1982); escaping predators (Vitt et al.. 1977; Dial and
Fitzpatrick, 1984; Smith, 1990a), or competing for mates
(Sekkelsten, 1988; Smith, 1 990a) or shelter (Conover and
Miller, 1978;BerzinsandCaldwell, 1983). Energetic costs
of regenerating body parts can reduce reproductive output
(Maiorana, 1977) and growth (Kuris and Mager, 1975;
Smith, 1990b). Theoretical models (Harris, 1989) have
suggested that nonlethal injury could regulate population
abundance, if injury rates were density-dependent and
significantly reduced long-term survival or reproduction.
Detailed knowledge of autotomy patterns and frequencies,
for a single species, over both narrow and broad temporal
and geographic scales, are needed to make more reliable
inferences concerning the fitness benefits and conse-
quences of autotomy.

Quantitative surveys of limb loss in decapod crusta-
ceans exist for only a few species (Cancer magister, Durkin
et al.. 1984; Shirley and Shirley, 1988; Cancer pagurus.
Bennett. 1973; Carcinus maenas, Needham, 1953;
McVean, 1976; McVean and Findlay, 1979; Sekkelsten,

416

AUTOTOMY IN BLUE CRAB POPULATIONS

417

1988, Menippe mercenaria, Sullivan, 1979;Simonson and
Steele, 1981: Simonson, 1985; Paralithodes camtschatica
and Chionoceles bairdi, Edwards, 1972). The percentage
of injury in these species ranged from 13-66%. Inferences
from these data regarding the fitness consequences of au-
totomy have been limited, however, because field data
have not been collected for more than one complete
growing season; smaller individuals in commercial species
frequently have not been sampled; chelipeds have often
been the only limbs assessed; and collections have been
geographically restricted. To understand how the inci-
dence of autotomy varies within and among populations,
multiple-year and -site data on injury are needed for a
range of body sizes for both sexes.

Nonlethal injury often results from unsuccessful attacks
by predators (Vermeij. 1982). Variation in injury levels
among populations and species has been thought to reflect
differences in predation intensity and efficiency over al-
titudinal gradients (Ballinger, 1979; Shaffer, 1978). eco-
logical habitats (Schoener and Schoener. 1980); biogeo-
graphic regions (Vermeij, 1976); geologic time (Vermeij,
1977, 1983), life histories (Vittrffl/.. 1977), and behaviors
(Jaksic and Fuentes, 1980; Schall and Pianka, 1980). Al-
though specific agents responsible for autotomy in nature
are rarely identified (cf., Robinson ct a/.. 1970; Jaeger,
1981: Smith, 1990a), such information is needed to un-
derstand the patterns and impact of injury in populations.
Intraspecific predation is common in the animal kingdom
(Fox, 1975;Polis, 1 98 1; Stevens etai. 1982;Reaka, 1987;
Kurihara and Okamoto, 1987), and may be an important
cause of autotomy in some taxa (e.g., salamanders; Jaeger,
1981). Large Callinectes sapidns are known to prey on
smaller conspecifics (Laughlin, 1982; Hines ct at.. 1990;
Peery, 1989; Smith, 1990a). If intraspecific interactions
are chiefly responsible for autotomy in blue crabs, then
injury levels should correlate positively with population
densities over temporal and spatial scales.

Costs of nonlethal injury to individuals will depend on
the type and number of missing limbs. The relative im-
portance of different limbs to survival, in turn, may be
indicated by the frequency of their repair in the popula-
tion. Limb regeneration in arthropods occurs upon molt-
ing, and crabs may require a number of molts (e.g.. 1-3
in Callinectes sapidux. Smith, 1990b; >4 in Paralithodes
camtschatica. Edwards, 1972) before full limb length is
restored. For most limbs, evidence of past injury disap-
pears once symmetry has been restored. Following the
loss of a major (crusher) claw, however, normal cheliped
dimorphism is often not reestablished (Smith, 1990b);
thus, the absence of such dimorphism can serve as a mea-
sure of survival of past injury.

To assess the impact of autotomy in a population, it is
necessary to: ( 1 ) document spatial, temporal, and onto-

genetic variation in patterns and levels of injury; (2) iden-
tify causal agents; and (3) determine the various costs of
injury to individuals. The present study examines inci-
dences of autotomy in blue crabs (Callinectes sapidns
Rathbun) at four sites in Chesapeake Bay and three ad-
ditional sites along the southeastern LJnited States Atlantic
coast and the Gulf of Mexico. These data, which include
four years of information from one site in central Ches-
apeake Bay, make this the most detailed survey yet con-
ducted on autotomy in arthropods.

Materials and Methods

Sampling procedures

Callinectes sapidns individuals were collected from
1986 to 1989 in the Rhode River, Maryland; at three
additional sites in the Chesapeake Bay in fall 1989; and
at three sites along the southeastern Atlantic coast and
the Gulf of Mexico of the United States (Figs. 1, 2) in
spring 1989. At all locations, crabs were measured or ex-
amined for: ( 1 ) carapace width between tips of lateral
spines. (2) sex, (3) sexual maturity in females (1986-89)
and males (1988-89, only), (4) molt stage, (5) type and
side of any missing or regenerating limbs, (6) lengths of
limb buds, regenerating limbs, and contralateral intact
limbs, and (7) side of the crusher claw.

Sexual maturity in female blue crabs was determined
by examining differences in abdominal allometry (Van
Engel, 1958). For males, sexual maturity was indicated
by the ease with which the abdomen could be pulled away
from the ventral surface of the cephalothorax (Van Engel,
1958; 1990). Molt stages were determined by assessing
carapace hardness and by examining the propodus of the
fifth pereopod for evidence of epidermal retraction (Van
Engel, 1958; Johnson, 1980). A limb stump that was either
scarred, or possessed a papilla or limb bud, was classified
as a missing limb. A regenerating limb was considered to
be a functional appendage that had undergone at least
one molt since autotomy, but was shorter than the intact,
contralateral limb. Crabs that possessed an unscarred
stump wound, indicating possible injury caused during
collection, were not measured. Limb length was measured
as the distance from the autotomy plane in the basi-ischial
segment to the dactyl tip of a fully extended limb.

Site descriptions and collection methods

Rhode River. Alary/and. Callinectes sapidns individuals
were collected from the Rhode River near Edgewater,
Maryland (38°51'N, 76°32'W), between July and No-
vember in 1986, and from May to November each year
from 1987 to 1989 (Figs. 1, 2). The Rhode River is a

418

L. D. SMITH AND A. H. MINES

Chesapeake Bay Region

Rhode River, Md

and
Upper-rrtd bay, Md

Patuxent River, Md

Lower-rnkJ bay, Va

Atlantic Ocean

North Wet, SC

Irxian Rtver, R

Figure 1. Map of the United States Atlantic coast and the Gulf of Mexico showing locations of blue
crab sampling sites from 1986 to 1989.

shallow (maximum depth = 4 m), 485 ha mesohaline
subestuary that empties into the western side of the upper-
central Chesapeake Bay (Hines el al, 1987a,b). Water
temperatures ranged from 8°C to 34°C during the sam-
pling period, with July temperatures averaging 28°C
(±2.4). Salinities typically ranged from 4 to 14%o; but
unusually low salinities (0-10%o) were recorded in
1989.

Several methods were used to sample blue crabs in the
Rhode River as well as at other sites. Potential biases re-
lated to these different collection techniques were exam-
ined and are discussed below. In all four years, blue crabs
were sampled monthly by otter trawl (3 m wide mouth;
5 mm mesh net body; 7 mm mesh cod end; with tickler
chain; Hines el al., 1987a) pulled for 900 m on two con-

secutive days at each of three stations in the Rhode River.
Two stations were located at the river mouth, one over
sandy substrate, and the other over muddy sediment; a
third station was located at the river head over muddy
sediment (Fig. 2).

From 1986 to 1988, blue crabs were also collected bi-
weekly at a fish weir spanning the principal freshwater
tributary (Muddy Creek) of the Rhode River. Crabs mov-
ing up- and downstream were captured separately in single
hoop nets (7 mm mesh). No crabs were sampled at the
weir in 1989 because of storm-related damage. Conse-
quently, in 1989, crabs were collected biweekly at the river
head (0.5-2 m depth); larger individuals were caught in
baited commercial crab pots (57 mm mesh), and smaller
crabs in specially designed crab pots (7 mm mesh). Blue

AUTOTOMY IN BLUE CRAB POPULATIONS

419

. — ' Rjver

Weir • . Head

Trawl

Figure 2. Map of the Rhode River subestuary, Maryland, showing
sampling sites from 1986 to 1989. These include mouth sand and mouth
mud trawl stations ( 1986-89) = River Mouth; nver head trawl station
( 1986-89) and crab pot/seine sites (1989, only) = River Head; and Muddy
Creek up- and downstream weir nets (1986-88) = Creek.

crabs were also sampled periodically in nearshore waters
(depth 0.3-1.2 m) with a 10 m beach seine (1 mm mesh).
Non-Rhode River sites. Locations, dates, physical con-
ditions (e.g.. depth, salinity), and sampling techniques for

additional sites in Chesapeake Bay and for sites in South
Carolina, Florida, and Alabama are summarized in Table
I. Note that the upper-mid Chesapeake Bay site was only
1 km east of the mouth of the Rhode River, Maryland.
Using a dipnet, blue crabs were collected from the sides
of a commercial pound net at this site.

Statistical analyses

Data were treated as categorical, and frequencies were
analyzed by logistic regressions (Cox, 1970; PROC CAT-
MOD with maximum likelihood estimation, 0.5 added
to all cells; SAS Institute, 1985) or two-way contingency
tables. In the Rhode River, data were analyzed for only
those months when 25 or more crabs were obtained. Lo-
cations in the Rhode River were combined into river
mouth, river head, or creek sites, because differences in
autotomy frequency were not detected between sample
stations within each subregion (G-tests, P > 0.05). Crabs
were divided into small (<61 mm carapace width), me-
dium (61 < CW < 1 10 mm), and large (>1 10 mm CW)
size classes. The division between medium and large size
classes corresponded approximately with the onset of sex-
ual maturity. Molt stages were classified as postmolt (stages
A and B), intermolt (stage C), and premolt (stage D)
(Johnson, 1980). Crabs in the act of molting (stage E)
were very rare and were included as premolt animals.

The primary null hypothesis tested whether the fre-
quency of injured crabs (i.e., those animals missing or
regenerating at least one limb) in a population was in-
dependent of one or more of the following independent
variables: ( 1 ) year (Rhode River, only), (2) month (Rhode

Table 1

Sampling sites, dales, physical conditions, and sampling methods used to collect blue crabs in 1989

Location

Sampling

Depth

Salinity

Temp.

Sampling

Site

(Lat., Long.)

dates 1989

(m)

(%»)

(°C)

method

Upper- Mid

38°50'N, 76°31'W

Aug.-Sept.

6-11

24-28

dip net

Chesapeake Bay,

Maryland

Patuxent River,

38°23'N, 76°36'W

Oct.

9-21

12-14

otter trawl

Maryland

Lower-Mid Chesapeake
Bay, Virginia

North Inlet.
South Carolina

Indian River, Florida
Mobile Bay, Alabama

37°25'N-37039'N
75°56'W-76017'W

33°2nM, 79°11'W

27°50'N, 80°29'W
SO'MS'N, 88°00'W

Oct.
May

May

4-18

.2-3

.5-5
.5-5

19
21

23-26
24

19
31

29-32

otter trawl

crab pots, dip net

crab pots, dip net
crab pots, seine

Latitudinal and longitudinal range of sampling transects are given for the lower-mid Chesapeake Bay site. See text for description of 1986-1989
Rhode River surveys.

420

L. D. SMITH AND A. H. MINES

Table II
's and percentages of crabs missing, regenerating, ami both missing and regenerating lunhx in the Rhode River from 1986 lo 19S9

Rhode River, Maryland

1986"

1987"

1988"

Category'

1 Missing = crabs with one or more scarred stumps, papillae, or limb buds.

2 Regenerating = crabs possessing one or more functional but shortened appendages.

3 Miss. + Regen. = crabs possessing both missing and regenerating limbs.

4 Size ratio: (S < 61 mm carapace width, M = 61-1 10 mm CW. L > 1 10 mm CW) for all crabs (injured and intact).
Years with the same superscripted letter did not differ significantly in total autotomy frequency.

1989"

Total intact

1050

75.0

505

81.2

536

82.5

569

82.2

Total injured:

350

25.0

117

18.8

113

17.5

123

17.8

Missing'

211

15.1

9.0

8.2

9.8

Regenerating2

123

8.8

6.7

Miss. + Regen.3

1.1

1.0

0.5

1.3

Total caught

1400

100.0

622

100.0

649

100.0

692

100.0

Sex ratio M:F

67:33

81:19

83:17

76:24

Size ratio S:M:L4

12:48:40

20:26:54

21:38:41

32:18:50

River, only), (3) subestuarine location (Rhode River,
only). (4) body size, (5) sex, (6) sexual maturity, (7) molt
stage, and (8) geographic location. All relevant two factor
combinations of these independent variables were tested
by logistic regression for their relationship to the binary
response variable (i.e.. frequency of injured versus unin-
jured crabs). Expected cell frequencies of injured animals
were often low (<1) and prevented more than two inde-
pendent variables from being tested reliably in a single
model. Significant two-way interactions were not recorded
between independent variables in most instances; hence,
these results, except when specified, are not discussed. If
a test revealed nonindependence. unplanned multiple
comparisons controlling for experimentwise type I error
were used to distinguish differences among frequencies
(simultaneous test procedures, STP tests; Sokal and Rohlf,
198 1, pp. 728). Two-way contingency tables were used to
examine frequencies of injury as a function of limb type
and number, right versus left side, and missing versus re-
generating limbs.

Median carapace widths of injured and uninjured an-
imals were compared within sites by nonparametric pro-
cedures (Mann-Whitney U-test; Sokal and Rohlf, 1981),
because variances for carapace widths were heteroscedastic
(F-max test; Sokal and Rohlf, 1981) even after attempts
at data transformation.

Results

Population structure

In the Rhode River, sex ratios were consistently male-
dominated, but relative frequencies of males and females

differed among all years except between 1987 and 1988
(STP test, 3 df; Table II). Annual size-frequency distri-
butions differed among all years in the Rhode River and
among all other sites in 1 989 (Komolgorov-Smirnov two-
sample tests, P < 0.05; Tables II, III). Outside the Rhode
River, sex ratios were skewed towards females at all sites
except South Carolina and the Patuxent River, Maryland
(G-test, 5 df, P < 0.05; Table III). Collections from the
upper-mid Chesapeake Bay were designed to capture fe-
males and larger individuals; therefore, these sex and size
ratios should not be compared to those from other sites,
which were sampled randomly.

Sampling methods

The frequency of autotomy in crabs collected from
baited crab pots and seines at the Rhode River head (19%)
did not differ from injury levels in otter trawls (22%) at
that site in 1989 (G-test, 1 df, P > 0.05). No significant
differences in injury were observed between otter trawl
and fish weir collections from 1986 to 1988 (G-tests, 1 df,
P > 0.05). At non-Rhode River sites, autotomy frequen-
cies did not differ among crabs collected by otter trawl
(Patuxent River, lower-mid Chesapeake Bay) and crab
pots and seines (South Carolina, Florida, Alabama) (G-
tests, P> 0.05).

A utotomy frequencies

Yearly and geographic variation. Frequencies of blue
crabs missing or regenerating one or more limbs differed
significantly among sites and years sampled (G-tests, P

AUTOTOMY IN BLUE CRAB POPULATIONS

421

Table III

Frequencies and percentages of crabs missing, regenerating, and both missing and regenerating timbs at sites
in the Chesapeake Bay and along the southeastern United Stales in 1 989

Chesapeake Bay

Upper-Mida

Patuxent R."

Lower-Mid h

Inlet SCb

Indian R. FL"

Mobile B.
AL'b

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