Volume 192

THE

Number 1

BIOLOGICAL
BULLETIN

FEBRUARY, 1997

Published by the Marine Biological Laboratory

Charles Baker Metz

As this issue of The Biological Bulletin went to press, we learned that Editorial Board Member and former
Editor Charles B. Metz had passed away on 14 January 1997, in Homestead, Florida. He was 80 years old.

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ERRATUM

The Biological Bulletin, Volume 191, Number 3, page 409

The following correction should be made in the article by Fraser M. Shilling, Ove Hoegh-Guldberg, and
Donal T. Manahan, titled "Sources of energy for increased metabolic demand during metamorphosis of
the abalone Haliolis rufescens (Mollusca)" ( Bio/. Bull. 191 : 402 -4 1 2 ).

In Table II on page 409, the value for "Energy balance: Ratio of available to required energy" should be
a percentage; that is, it should read 38.7%, not 38.7.

Reference: Bid. Bull 192: 1-16. (February, 1997)

Coelenterate Cnidae Capsules: Bisulfide Linkages

Revealed by Silver Cytochemistry and Their

Differential Responses to Thiol Reagents

WALTER M. GOLDBERG AND GEORGE T. TAYLOR

Electron Microscopy Laboratory, Department of Biological Sciences. Florida International
University. University Park. Miami. Florida 33199

Abstract. The sulfur cytochemistry of cnidae from the
Portuguese man-of-war Physalia physalis. the scypho-
zoan Cassiopeia xamachana. and the black coral Cirrhi-
pathes luetkeni was evaluated on the basis of electron mi-
croscopy. X-ray microanalysis, amino acid analysis, and
response to disulfide reducing agents. The cnidae exam-
ined included large and small holotrichous isorhizas in
P. physalis, another small isorhiza in C. xamachana. and
both spirocysts and microbasic mastigophore nemato-
cysts in C. leutkeni. A strong reaction with methena-
mine-silver reagent was characteristic of all cnidae cap-
sules, but the pattern and extent of that argentophilia was
dependent upon the type of cnida and its state of matu-
rity. The large isorhizas of P. physalis reacted primarily
in the outermost capsule layers, but in C. xamachana
isorhizas, silver stained the entire capsule with the excep-
tion of the outermost region. The small isorhizas of P.
physalis and the mastigophore capsules of C. leutkeni
stained throughout, whereas the spirocyst capsules were
outlined by silver, clearly delineating the inner and outer
layers. All of these reactions were abolished with alkyl-
ation, but only after treatment with disulfide reducing
agents; alkylation alone diminished silver staining only
slightly, indicating that the argentophilic response was
due primarily to disulfide linkages. The cystine content
of these cnidae varied from 4. 1 to 4.7 mole percent for a
given species, but amino acid analyses did not separate
components of the cnidom.

Cnidae, both within and among species, exhibited
differential responses to the disulfide reducing agent di-
thiothreitol (DTT). Isolated, unfixed, large isorhizas of

Received IVJuly 1 996; accepted 16October 1996.

P. physalis discharged and appeared to dissolve rapidly
in the presence of this reagent, whereas small isorhizas
from both P. physalis and C. xamachana discharged, but
dissolved slowly if at all. The discharge and solution re-
sponses of the capsule coincided with the complete de-
velopment of the tubule. Cnidae containing an undevel-
oped or partially developed tubule were resistant to
DTT. displayed a weak capsular argentophilia, and con-
tained background levels of sulfur; these results suggest
that formation of disulfide linkages is one of the final
steps in capsular maturation. In contrast, mature nema-
tocyst and spirocyst capsules in C. leutkeni tentacles were
resistant to DTT among other reagents, despite the pres-
ence of disulfides. This suggests that other types of cova-
lent, intermolecular linkages could play a prominent role
in the development of capsular stability in this species.

Introduction

Coelenterate cnidae are among the most complex in-
tracellular secretion products known (Gupta and Hall,
1 984). Each is composed of a double-walled capsule con-
taining an inverted tubule. During eversion the tubule
discharges explosively, completing the process within 3
ms, one of the fastest mechanical events known in the
biological sciences (Holstein and Tardent, 1984). More
than 30 types of cnidae have been described, and they
are classified into three groups: nematocysts. spirocysts,
and ptychocysts (e.g., Mariscal, 1974, 1984). Hydrozo-
ans have the greatest variety of nematocysts, and scypho-
zoans have the least, but only anthozoans produce all
three types. Ptychocysts occur only in cerianthid anem-
ones and are the most phylogenetically restricted of the
cnidae. Spirocysts are also limited in their distribution.

W. M. GOLDBERG AND G. T. TAYLOR

occurring only in the tentacles of various anthozoans;
but spirocysts are also quite common, often outnum-
bering tentacular nematocysts (Mariscal and McLean,
1976; Goldberg and Taylor' 1989).

Nematocysts in particular have been examined closely,
yielding details of structure and function relationships.
For example, upon discharge, the capsule must withstand
internal pressures of up to 140 bar during eversion (Lub-
bock and Amos, 1981; Tardent, 1988). a feat requiring
enormous tensile strength. Recent work has shown that
disulride-linked, woven mini-collagens composing the in-
ternal wall of the nematocyst capsule (Kurz el at., 1991)
may be the key structural element in the resistance of
these cnidae to such pressures. The tensile strength de-
rived from this capsule structure is estimated to be nearly
as high as that of steel (Holstein et a/., 1994).

The occurrence of disulfide-linked collagens in nema-
tocysts was first established by Blanquet and Lenhoff
(1966) after the suggestion by Brown (1950), and subse-
quently by Yanagita and Wada (1954), that since coelen-
terate cnidae are soluble in disulfide reducing agents, they
might be composed of keratins. Hamon ( 195 5 (confirmed
the earlier observations of disulfide linkages by demon-
strating the presence of cystine histochemically. However,
Blanquet and LenhofY clearly showed that cystine is re-
sponsible for stabilizing collagenous proteins, the domi-
nant components of the nematocyst capsule. They also
showed that the cnidae at their disposal dissolved in a
number of disulfide reducing agents including dithiothre-
itol. sodium thioglycolate. and mercaptoethanol.

Disulfide linkages have since been shown to be wide-
spread in nematocyst capsules judging from amino acid
composition (Fishman and Levy, 1967; Blanquet, 1988;
Brand el at.. 1993) and X-ray microanalysis revealing the
presence of sulfur (Mariscal. 1980, 1984. 1988). However,
although the nematocysts from a variety of cnidarians dis-
solve quickly in disulfide reducing agents as might be pre-
dicted, some nematocysts and spirocysts appear to be re-
sistant to such treatment (Mariscal and Lenhoff, 1969;
Mariscal. 1971). Despite a number of subsequent chemi-
cal studies (see review by Blanquet. 1988; Brand el a/.,
1993). little progress has been made in answering the
questions raised by Mariscal and LenhofTs original obser-
vations. To this end, we have examined the chemistry of
cnidae capsules in three coelenterates. each representing a
class within the phylum. The sulfur cytochemistry of each
type of cnida is confirmed by X-ray microanalysis and
correlated with amino acid composition, degree of matu-
rity, and response to disulfide reducing agents.

Materials and Methods

Microscopy

Three species were collected for this study, including
Physalia physalis Lamarck (Hydrozoa: Siphonophora)

from various localities in southeast Florida; Cassiopeia
\iinuichuna Bigelow (Scyphozoa: Rhizostomae) from
several nearshore locations in the Florida Keys, and Cir-
rhipathes luetkeni Brook (Anthozoa: Antipatharia) from
a depth of 25 m off Hollywood, Florida.

Nematocyst batteries separated from fixed tentacles of
P. physalis, individual tentacles isolated from C. luetkeni
polyps, and oral vesicles — the baglike, oval structures as-
sociated with the oral arms (see Bigelow, 1900) — from
C. xanuic/iana were dissected using iridectomy scissors
under low-power microscopy. All of these cnidae-con-
taining structures, referred to as tentacles or tentacular
tissues, were prepared for transmission electron micros-
copy (TEM) by fixation at room temperature for 2-4 h
in an artificial seawater solution containing 2.5% glutar-
aldehyde and 1.0% paraformaldehyde in 0.1 v\/cacodyl-
ate buffer at pH 8.0. The tissue was then stored in 0. 1 M
cacodylate buffer at 4°C. Post-fixation with osmium is
incompatible with silver staining (e.g.. Hayat, 1993) and
was omitted. Tentacular tissues were dehydrated
through ethanols and embedded in Spurr resin. Thick
sections examined in the light microscope were con-
trasted with 0.1%. toluidine blue in 1% borax. For TEM
we used a Philips EM 300 electron microscope operated
at 60 kv. No contrast agents other than silver were em-
ployed in transmission microscopy.

Cnidae fixed as above were also examined by scanning
electron microscopy (SEM). Aldehyde fixation was fol-
lowed by osmication using 1%. OsO4 in 0. 1 M cacodylate
buffer, pH 8.0, for 1 h at room temperature. Dehydra-
tion in ethanols was followed by critical point drying
(CPD) with CO: as the transitional fluid, or by cryofrac-
ture from 100°; ethanol in liquid nitrogen prior to CPD.
Tentacles were then sputter-coated with Au-Pd and ex-
amined in an ISI Super 3A scanning electron microscope
operated at 10 or 15 kv.

Silver staining for disulfide groups

The methenamine-silver stain (Rambourg 1967;
Locke and Krishnan, 1971) was employed for general
electron contrast and sulfur cytochemistry. All reagents
were made fresh daily using double-distilled, deionized
(ddd) water, and all steps employed constant agitation
on an orbital rotator. Fixed tentacular tissues were rinsed
several times in ddd-water, then treated with 5% sodium
metabisulfite for 10 min to block pre-existing aldehyde
groups or those introduced by the fixative. After washing
in three additional changes of ddd-water, tissues were
immersed in methenamine-silver reagent and placed in
closed 1.5-ml polypropylene microcentrifuge tubes in a
70°C oven for 30 min. After a brief ddd-water rinse, all
tissues were treated with 5% sodium thiosulfate to re-
move unreduced silver deposits, then rinsed with ddd-
water again before dehydration and embedment.

DISULFIDES IN CNIDAE CAPSULES

Cytochemical control pr

Before silver staining, additional aldehyde-fixed tenta-
cles were treated with the disulfide reducing agent dithio-
threitol ( = DTT, Cleland's reagent) 0.2 Al in 0.05 M
phosphate buffer pH 8.0 at 37°C for 90 min. Some sam-
ples were stained with methenamine-silver and exam-
ined without further treatment; others were treated with
iodoacetic acid to alkylate naturally occurring sulfhydryl
groups as well as those produced by disulfide reduction
(alkylation blockade). Iodoacetic acid (0.1 Af) was pre-
pared with 0.2 M boric acid in 50% jV-propanol accord-
ing to the method of Swift ( 1 968). Because we used tissue
rather than sections, the reaction time for this blockade
was extended from 4 h at room temperature to 18h.
Blockaded tissues were rinsed first with 20% 7V-propanol,
then several times with ddd-water, and treated with me-
thenamine-silver as above. Samples treated with io-
doacetic acid alone prepared as above, or with the boric
acid-propanol solvent alone, were also examined both
with and without silver treatment, as were tissues treated
with DTT alone. Parallel experiments employed alkaline
0.3 M thioglycolic acid prepared and used under the
same conditions as DTT for disulfide reduction, and
0.1 MTV-ethyl maleimide prepared according to Kiernan
(1990) to block sulfhydryl groups. These sequential con-
trol treatments (disulfide reduction followed by alkyl
bockade) will be referred to as post-reduction alkylation.

Elemental composition

Sections about 100-nm thick (dark gold interference
color) from tissues treated with methenamine-silver were
mounted on carbon-coated copper grids and examined
in a Philips EM 300 transmission electron microscope
operating at 100 kv. X-ray microanalysis employed a
Link Analytical EDS system. The goniometer was tilted
at 36° and counts were obtained using a spot size of
250 nm over an interval of 600 s real time. Count rates
were 1 800-2500 cps; dead time varied from 20% to 25%.
No fewer than four undischarged cnidae of each type (de-
scribed below) were scanned, including two types of ne-
matocysts from P. physalis tentacle, one from C. xama-
c/iana, and both a nematocyst and a spirocyst from C.
luctkeni. In all but C. luetkeni. the spot was moved every
50-60 s to another location on the same nematocyst cap-
sule. In the latter species, the spot was moved every 30-
50 s and changed to a new cnida every 1 50 s due to the
small size of the capsules. Thus each 600-s count for each
type of cnida in C. luctkeni was a composite of four ne-
matocysts and four spirocysts; four such 600-s counts
were taken from a total of 16 cnidae in this species. X-
ray spectra of Spurr resin (blank sections) served as a
means of determining background. A composite peak

showing maximum, minimum, and mean net counts
was constructed for each type of cnida

Isolation ol cnidae and amino acid analysis

Freshly collected P. physalis tentacles, C. xamachana
oral vesicles, and whole segments of C luetkeni colonies
were allowed to autolyze in seawater at 4°C over a period
of 2-7 days. Debris was removed by filtration through a
coarse nylon mesh, and the filtrate was treated briefly
with a sonicator probe to disperse the remaining ma-
terial. Recovery of undischarged cnidae from P.
physalis required an initial low-speed centrifugation
(200 X g) for 4 h over concentrated sucrose (approxi-
mately 2.5 Af). The sucrose layer was diluted and cnidae
were concentrated by centrifugation, then resuspended
in a small volume of 0.1 M cacodylate buffer, pH 8.0.
The crude, buffered autolysate was layered over 2.0 M
sucrose and centrifuged at 10 X g for an additional
60 min, or allowed to settle at 4° overnight. A clean ne-
matocyst preparation was obtained after repeating this
process. The filtered C. xamachana autolysate was sepa-
rated from debris by centrifigation into 1.25 AI sucrose
for 1 h at 1000 X g. Cnidae in the sucrose layer were con-
centrated, resuspended in cacodylate buffer, and briefly
sonicated after addition of 1 yul ml'1 Triton X-100. The
crude cnidae were filtered through a 10-/um nylon mesh,
layered onto a discontinuous gradient of 1 .75, 2.0, 2.25,
and 2.5 Af sucrose, and spun for 1 h with a swinging
bucket rotor at 2000 X g. Cnidae were in the 2.25 and
2.5 M layers. This process was repeated 2-3 times. The
C. luetkeni autolysate contained a considerable amount
of mucus and was treated with 0.5%- cetylpyridinium
chloride in addition to Triton X-100. The treatment and
procedure was otherwise the same as for C. xamachana.
Clean, isolated cnidae from each species were taken
from buffer to distilled water and disrupted with a soni-
cator probe. The fragments were recovered by brief cen-
trifugation, rinsed with distilled water, then dehydrated
in ethanols and oven-dried. Cnidae fragments yielding
1 -2 n& of protein were oxidized in 95% performic acid
for 30 min to convert cystine into cysteic acid and were
subsequently hydrolyzed by microwave digestion in 6 TV
HC1 for 18 min under an atmosphere of nitrogen.
Amino acids were derivitized with Edman's reagent
(phenylisothiocyanate — see Heinrickson and Meredith,
1984) and quantitated by reverse-phase high-perfor-
mance liquid chromatography at Florida State Univer-
sity Analytical Laboratories, using a detection wave-
length of 254 nm. Analyses of each species were per-
formed in triplicate, with the cnidae of each replicate
representing a single individual or colony.

Effects of DTT. thioglycolic acid, and collagenase

Isolated cnidae stored in 0. 1 Af cacodylate buffer were
taken to distilled water. The effects of disulfide reduction

W. M. GOLDBERG AND G. T. TAYLOR

Q'm&

, -. • «

,- • • --f>^^<: , ^^i.'^:m^^^w^^

' •£--••- T^m^ii'^

Figures 1-7. Cnidae from Physalia physalis.

D1SULFIDES IN CN1DAE CAPSULES

were examined for 1 5 min after placing a 1 0-/ul drop con-
taining several hundred cnidae on a slide, followed by
25 ftl of 0.2 A/ DTT or 0.3 M thioglycolic acid in 0.2 M
bicarbonate buffer, pH 10.3 (Brand etui.. 1993). Isolated
cnidae were also tested with collagenase (Type 1A:
320 units mg"1 and 1.7 units mixed protease, Sigma
Chemical Co., St. Louis, MO) by exposing them to 1500
enzyme units in TES buffer, pH 7.5 (10:1 enzxnidae
vol), for 5 h at 37°C. Cnidae were recovered by centrifu-
gation. washed in distilled water, and again exposed to
DTT or thioglycolic acid as above.

Results

Isolated cnidae and electron microscopy

Physalia physalis. Clean, largely undischarged nema-
tocyst preparations were obtained from P. physalis (Fig.
1 ). Cnidae isolated in sucrose were remarkably difficult
to discharge and, in contrast to results reported by Lane
(1960), were often unresponsive to centrifugation or to
immersion in distilled water, 50 mM sodium citrate, N-
HC1 or A'-NaOH. despite capsular permeability to tolu-
idine blue. Two size classes (diameters 8- 1 2 /urn and 20-
35 //m) were obtained from tentacles (Fig. 2). These
ranges are in general agreement with the measurements
obtained by Lane and Dodge (1958), Hulet et at. (1974),
and Cormier and Hessinger ( 1 980). The larger of the two
was characterized by a uniform tubule diameter of about
2.5Mm. with spines of about equal size (0.9-1. 2 ^m),
uniformly distributed along its length in three rows (Fig.
3). The smaller cnidae contained similar tubules and spi-
nation. but these were not examined in the discharged

condition. The designation of these nematocysts as holo-
trichous isorhizas seems appropriate and is consistent
with identifications made previously (Mariscal, 1974;
Brand et ai. 1993).

Cnidae from P. physalis tissues treated with methena-
mine-silver demonstrated a clear and consistent pattern
of argentophilia. The large isorhizas were always strongly
silver-positive, but only in the outermost portion of the
capsule (Fig. 4). Deeper regions, delineated by what ap-
peared to be fibrous annuli, were only lightly stained.
These electron-opaque annuli subdivided the capsule
into as many as five distinct layers. In contrast, the small
isorhiza capsules were most often completely blackened
with metallic silver (Figs. 4, 5). In less intensely stained
capsules, one or two electron-opaque annuli could be
distinguished, but in small isorhizas these layers did not
react differentially with silver. The annuli were present
in isorhizas that were untreated except for aldehyde fix-
ation, and were therefore unrelated to the silver stain.

In P. physalis and all other species examined, the
background stain varied in intensity, probably due in
part to variations in temperature during the 30-min in-
terval at 70°C. Figure 4 shows a low-background prepa-
ration in which some generalized staining of membranes
is evident. However, nothing in the tissue compares to
the intensity of the capsule stain. Figure 5 shows a small
isorhiza with typically strong capsular argentophilia but
with higher background staining of membranes and
small vesicles. Nuclei and mucus cell secretory inclu-
sions were also stained. The nematocyst tubule was quite
variably electron-opaque (cf. Figs. 4 and 5), and silver
deposits were often observed on the tubule periphery. Al-

Figure 1. Large and small isorhizas isolated from tentacular tissue in sucrose. Darkheld microscopy.
Scale bar = 100 ^m.

Figure 2. Freeze-fracture SEM of nematocyst battery cross-section showing large isorhiza and surround-
ing cnidocyte (ct) flanked by two small isorhizas. The opercula (arrows) are visible at the top of each; battery
surface is at the upper left. Scale bar = 5 ^m.

Figure 3. Everted tubule of large isorhiza showing arrangement of three spiral rows of spines. Scale bar
= 5 Mm.

Figure 4. Low-magnification overview of methenamine-silver preparation showing two small isorhizas
(top and right) with surrounding tissue, and a section of a large isorhiza (left). Note that small cnidae capsules
are completely invested with electron-opaque silver deposits, whereas the large isorhiza is primarily reactive
in the outer capsule: annuli marking inner capsule layers are noted by arrows. The tubule (t) of small
isorhizas displays little reactivity toward silver. Scale bar = 2 ^m.

Figure 5. Detail of methenamine-silver reaction on small isorhiza and surrounding cnidocyte. The cap-
sule is clearly delineated from surrounding cellular material by the degree of argentophilia. A tangential
section through the cnidocil complex (cc) is shown at the upper left. Extracellular vesicles (upper right) are
silver-positive, as is mucus secretory material (clear arrow). Tissue was treated with iodoacetic acid, but not
with disulfide reducing agent. Scale bar = 1 ^m.

Figure 6. Low-magnification overview of tentacular tissue after DTT-iodoacetic acid blockade of me-
thcnamine silver reaction. Note that nucleus (n), numerous cnidocyte vesicles (arrows), and membranes are
reactive, but the capsular (c) response is all but completely blocked by alkylation. Scale bar = 2 ^m.

Figure 7. Detail of blockade on small isorhiza. Note that the operculum (op), the periphery of the tubule
(solid, curved arrows), and the cnidocyte cytoplasm are reactive, but the capsule is not. Electron-opaque
spines visible in the tubule center (clear, curved arrows) are not argentophilic. Scale bar = 1 /jm.

W. M. GOLDBERG AND G. T. TAYLOR

though the opacity of the spines in the center of the tu-
bule could be confused with silver stain, they were pres-
ent in unstained controls (see also micrographs in Hulet
el a/., 1974, and Hessinger and Ford, 1988) and were
therefore unrelated to argentophilia.

Control procedures using DTT or thioglycolic acid fol-
lowed by iodoacetate or ethyl maleimide alkylation es-
sentially eliminated capsular argentophilia, while back-
ground staining remained on nuclei, cnidocyte mem-
branes, and many extracapsular vesicles (Fig. 6). At
higher magnification some nonspecific silver was still vis-
ible around the tubule periphery, and the operculum was
also stained (Fig. 7), suggesting that these structures may
have a different chemical composition from that of the
capsule. Tissues treated with DTT or thioglycolic acid
alone exhibited a somewhat less intense capsular argen-
tophilia and a higher background, but were otherwise not
significantly different from untreated tissues. Treatment
with disulfide reducing agents alone, however, removed
the electron-opaque annuli that subdivided the capsule,
resulting in the uniformly electron-lucent appearance of
capsules in Figures 6 and 7. Control tissues treated with
propanol and boric acid, or iodoacetic acid (as in Fig.
4), or ethyl maleimide alone were indistinguishable from
experimental groups.

Cassiopeia xamachana. The nematocysts in the tenta-
cles of this scyphozoan were divided into two distinct re-
gions. The free edges of the oral arms were white and
digitate (called "digitella" by Bigelow, 1900; "tentacles"
by Smith, 1937) and contained primarily ovoid nemato-
cysts 12-15 Mm long, tentatively identified as euryteles.
The oral vesicles, on the other hand, contained primarily
small (6-9 ^m). round cnidae. Clean nematocysts ob-
tained from C. xamachana oral vesicles are shown in
Figure 8. The smaller cnidae contained a tubule about
1 Mm in diameter narrowing gradually (Fig. 9). Spines
could not be measured in our SEM material, but ap-
peared uniform in TEM preparations. These cnidae were
identified by Mariscal and Bigger ( 1976) as holotrichous
isorhizas, and this diagnosis is consistent with our obser-
vations. A smaller number of the eurytele nematocysts
were also present in these preparations and were not sep-
arated by our isolation procedure. In addition, the oral
vesicles contained developmental stages of these cnidae,
some of which were included in our samples.

The capsules of mature isorhizas from C. xamachana
tentacular tissue were 0.4-0.5 j/m thick and were
strongly argentophilic throughout, except for the
~0. 1 Mm thick outermost region (Fig. 10). As in the
small isorhizas of P. physalis, one or two electron-
opaque annuli could be distinguished, but these were
obscured unless the silver stain was omitted. The tubule
of mature isorhizas was electron-opaque in part due to
nonspecific silver deposits and in part due to naturally

occurring electron-opaque material in the tubule
center.

Mature isorhizas were often closely accompanied by
various stages of their development. The most common
of these were capsules containing an electron-opaque
matrix in which the tubule had not yet developed, or in
which an incipient tubule could be observed (Fig. 10).
We refer to the former as immature cnidae and the latter
as submature. In both cases, the capsule was substan-
tially less argentophilic than the mature cnidae. The im-
mature stages reacted variably with silver; reactions
ranged from diffuse and nonlocalized deposits to no re-
action at all. The submature cnidae also exhibited varia-
tion in capsular argentophilia, which ranged from diffuse
deposits to more concentrated metallic silver, but was
always less than in the mature cnidae. In addition to ac-
quiring argentophilia during the transition from subma-
ture to the mature condition, the capsule exhibited a
20%-50% decrease in thickness. Moreover, the electron-
lucent tubule of submature cnidae and their contrasting
electron-opaque matrix (Figs. 10, 11 inset) changed to an
electron-opaque tubule in an electron-gray matrix dur-
ing maturation. The latter change was the most consis-
tent indicator of capsules most likely to exhibit a strong
argentophilic reaction. The background reactivity of tis-
sue from the oral vesicles was similar to that of P. phy-
salis tentacle and included generalized membrane, nu-
cleus, and vesicle staining.

Post-reduction alkylation of mature and submature ne-
matocyst capsules resulted in a virtually complete silver
blockade, except in the outermost capsule layer at the cni-
docyte membrane interface (Fig. 1 1 ) and in the opercu-
lum. Immature nematocysts in blockaded tissues contin-
ued to exhibit a weak response to methenamine-silver.
This response could not be distinguished from the same
weak reactivity in unblocked tissues, suggesting that it
may not be due to sulfur. The response of submature cni-
dae to capsular silver was variable. In some cases, argen-
tophilia was like that of the immature cnidae; in others, a
greater amount of silver was deposited. In the latter case,
silver was effectively blocked by post-reduction alkylation
(Fig. 1 1 inset), although the silver at the capsule periphery
could not be blocked. Mature nematocyst capsules treated
with disulfide reducing agents alone or with iodoacetate
or ethyl maleimide alone were still strongly argentophilic.
but they were less intensely blackened and were accompa-
nied by greater amounts of background silver.

Cirrhipathes hteikcni. Clean cnidae preparations from
whole colonies (Fig. 12) yielded spindle-shaped nemato-
cysts and cylindrical spirocysts, both of which were 15-
1 8 MID long and about 3-4 Mm at the widest diameter. A
small number of nematocysts measuring about 22 X
5 Mm were also present. Nematocysts outnumbered spir-
ocysts about 2: 1 because whole colonies were used to ob-

DISULFIDES IN CNIDAE CAPSULES

a-

,0

Figures 8-11. Cnidae from Cassiopeia .\amacliana.

Figure 8. Cnidae isolated from oral vesicles are primarily small isorhizas with a small percentage of
eur\ teles (arrows). Scale bar = 25 ^m.

Figure 9. SEM preparation of small isorhiza capsule (c) and everted tubule (t). Spines are appressed to
the tubule and are not visible. Scale bar = 5 ^m.

Figure 10. Small isorhizas in various stages of development. Immature isorhizas (ii) at top. mature
isorhizas (mi) in center, and submature isorhiza (si) at bottom. Note diffuse silver deposits on capsules of
immature isorhizas and the electron-opaque tubule matrix (TM) in their centers. Central electron-opaque
matrix of (si) surrounds electron-lucent tubule, whereas in (mi) the tubule is electron-opaque and the
matrix (TM) is electron-gray. Note differences in capsular argentophilia: ii = diffuse, si = slightly greater.
mi = intense. Scale bar = 1 urn.

Figure 11. DTT-iodoacetic acid blockade of mature isorhizas blocks capsular argentophilia; cytoplasm
is still reactive. Inset: submature capsular silver is also occasionally blocked except for the outer capsule
layer. Scale bar = 1 /im.

W. M. GOLDBERG AND G. T. TAYLOR

sn

sn

--.. ^ , -' ,

"

^. 17 ^>^

( r -y«?> >-=&.. TI-2*
Figures 1 2-17. Cnidae from Cirrhipathes luetkeni.

DISULF1DES IN CNIDAE CAPSULES

tain cnide; tentacles alone would have yielded a greater
proportion of spirocysts. Nematocysts and spirocysts
could not be separated and were included in the final
product employed for observation and chemical analy-
sis. Many but not all spirocysts were recovered without
capsules. Isolated nematocysts were resistant to dis-
charge, and unless they were disintegrated by probe son-
ication, most were left intact by the isolation procedure.
Those found in the discharged state (Fig. 13) were con-
sistent with the description of microbasic b-mastigo-
phores(Mariscal, 1974).

The capsules of both nematocysts and spirocysts in
fixed material were strongly argentophilic. Tentacular
cross-sections at low magnification were quite striking
when the silver-blackened outlines of the cnidae were
compared to the rest of the tissue (Fig. 14). The inner and
outer surfaces of the spirocyst capsule were separately
outlined by silver deposits (Fig. 15 and inset). The tubule
wall in C. luetkeni spirocysts was thin, folded at intervals
into pleats, and strongly argentophilic (Fig. 1 5 and inset).
The tubule interior was essentially solid, and thus typical
of the antipatharians examined by Goldberg and Taylor
( 1996). The unstained C. luetkeni tubule contained four
helically arranged bundles of electron-gray material. In
cross-section, the bundles were separated by electron-lu-
cent, cruciform partitions, but in contrast to the tubule
wall and its pleats, none of the material within the tubule
(bundles or partitions) was argentophilic (Fig. 1 5 inset).

Unlike spirocysts, the nematocyst capsules were
strongly silver-positive across the entire capsule cross-
section (Fig. 14). Nematocyst tubules, on the other hand,
displayed an inconsistent pattern of silver deposition.
Often in the same section, the entire tubule was outlined

with silver in one area, but in other areas only the shaft
was argentophilic, or the tubule failed to stain at all.

The silver reactivity of the cnidae capsules in C luet-
keni is persistent. We have tested this species (as well as
four other antipatharian species of the genus Anti-
pathes — see Goldberg and Taylor, 1996) using speci-
mens that had been fixed and stored in ethanol for years,
and they display essentially the same reactivity as freshly
collected material.

Post-reduction alkylation treatment of tissue stored in
cacodylate buffer eliminated all silver reactivity from the
nematocyst capsule, and most but not all reactivity from
the spirocysts. Unfortunately, the background staining
after blockade treatment increased considerably, partic-
ularly on the cell membranes. However, when we em-
ployed fixed material stored in ethanol rather than in
cacodylate, the membrane background was substantially
reduced. No capsular silver was deposited in the spiro-
cyst controls (Fig. 16). but electron-opaque deposits
(Ag?) formed within the tubules. Since tubule precipi-
tates were absent in all other preparations, we suspect
that they are artifacts in this case. The nematocysts were
completely free of silver deposits after blockade treat-
ment (Fig. 17). In C Inetkeni cnidae as in those of C.
.\amachana, treatment with disulfide reducing agents
(DTT or thioglycolic acid) or sulfhydryl blocking agents
(iodoacetate or ethyl maleimide) alone did not signifi-
cantly reduce argentophilia cnidae, but increased back-
ground silver deposits.

Argentophilia of immature cnidae

The relationship between argentophilia and maturity
was evident in C. xamachanu nematocysts because of the

Figure 12. Isolated cnidae from whole colonies are a mixture of nematocysts and spirocysts. with many
more of the former. Scale bar = 100 ^m.

Figure 13. Discharged microbasic b-mastigophore nematocyst on tentacular surface showing shaft (s)
about the same length of capsule (c) with gradual transition to tubule at lower left. Tentacular cilia are
bulbous with pointed tips, possibly artifacts. Scale bars = 5 ^m.

Figure 14. Methenamme-sil ver response of spirocyst capsules (sp) and mature microbasic mastigophore
nematocyst capsules (mn) in tentacular cross-section. Note that capsules in submature nematocysts (sn) are
weakly argentophilic and the tubules within are surrounded by an electron-gray matrix. Mature nematocyst
tubules are matrix-free; their capsules are strongly argentophilic. Scale bar = 2 ^m.

Figure 15. Tangential view of spirocysts treated as above showing uniform silver reactivity of capsule
wall. The tubule and pleats (curved arrows) are also silver-positive; internal portions of the tubule are not.
Note submature spirocyst (ss). lower left, with diffuse silver over electron-opaque matrix; capsule wall is
indistinct and is not argentophilic. Mature nematocyst (mn) is shown at lower right. Scale bar = 2 fim. Inset:
Detail of argentophilia in tubule wall and pleats from area of bar-connected arrowheads in Fig. 15. Note
distinct staining of inner and outer layers of the capsule (straight arrows). The tubule wall is continuous with
the pleats (p). and both are silver-positive. The tubule interior is not argentophilic and contains four bundles
(b) of electron-gray material partitioned in an electron-lucent, cruciform pattern. Scale = 3.0 x Fig. 15 scale
bar.

Figure 16. DTT-iodoacetic acid blockade of mature spirocysts blocks capsular argentophilia (arrows);
pleats are also blocked; electron-opaque deposits in tubule are most likely artifacts. Scale bar = I ^m.

Figure 17. Treatment as above showing complete silver blockade of mature nematocysts (mn) and
submature nematocysts (sn). Scale bar = 1 /im.

10

W. M. GOLDBERG AND G. T. TAYLOR

9

-. m

20

Figures 18-20. Immature nematocysts in Physalia physalis.

Figure 18. SEM cross-section of freeze-fractured nematocyst battery showing mature cnidae at the
outer face (clear arrows). Immature cnidae are formed in the deeper layers of the battery. Mesoglea (m)
supports the inner battery: gastroderm (g) lines the center ol the tentacle (t) and extends into each battery.
Scale bar = 25 j/m.

Figure 19. Toluidine blue-stained thick section taken in plane of trapezoid (Fig. 1 8) with corresponding
locations of mesoglea (m) and gastroderm (g). Mature cnidae at the periphery of the battery contain a
tubule with no matrix. These occur along with capsules containing a basophilic matrix (arrows), possibly
representing submature isorhizas. Immature cnidae (boxed area) are only weakly basophilic. Scale bar =
25 Mm.

Figure 20. Large, immature or submature isorhizas treated with 0.2 M DTT. pH 10.3. are resistant to
depolymerization in contrast to mature stages. Scale bar = 25

proximity of various stages of development. As noted
above, silver deposition in the capsule corresponded with
the extent of tubule development. In P. physalis almost
all of the cnidae on the outer face of the battery were
mature (Fig. 18). Immature isorhizas, located deeper
within the battery, were only weakly reactive toward to-
luidine blue and were virtually unresponsive to methe-
namine-silver reagent. However, with the development
of the submature stage, the tubule matrix and capsule
became strongly basophilic (Fig. 19). Because the tubule
matrix apparently was not rendered electron-opaque
with aldehyde fixation alone, we were unable to deter-
mine whether the basophilia coincided with silver depo-
sition. Thus, in the electron microscope, the submature
cnidae could not be distinguished with certainty from
mature isorhizas.

Developing spirocysts in C. luetkeni occurred just be-
low the outermost, mature cysts. Submature spirocysts
were filled with a granular, naturally electron-opaque
matrix surrounding (he nascent tubule. The matrix also
contained a diffuse si I ,r-reaction product. Thespirocyst
capsule was not clear!;, developed as a double-walled
structure at this stage (Fig. i 5), and it exhibited affinity
for silver only when the ele> \ on-opaque tubule matrix
had almost completely disappeared.

Submature nematocyst capsules were most often lo-
cated within clusters of mature mastigophores and were

readily distinguished from mature nematocysts by their
weak argentophilia. In every case, the weak staining of
the capsules was associated with the presence of some
remaining electron-gray matrix surrounding the tubule
(Fig. 14). This matrix did not occur in mature nemato-
cysts, suggesting that strong argentophilia in the capsule
of these cnidae occurs coincidentally with the complete
maturation of the tubule.

Response of unfixed cnidae to DTT

The large isorhizas of P. physalis discharged within
about 10-15 s of exposure to DTT. After discharge the
capsule dissolved rapidly, followed by the tubule. The
time from discharge to complete solution was generally
30-60 s. Brand el al. ( 1993) also observed rapid solution
in DTT, and found that P. physalis capsules and tubules
dissolved almost simultaneously. It should be noted,
however, that these authors employed discharged cnidae
in their study. Our results contrast with those of Mariscal
and Lenhoff (1969). who noted that disulfide reducing
agents solubilize only fully discharged nematocysts. Sol-
ubility in DTT was typical of the mature isorhizas only.
Those with clearly developed cysts but lacking a devel-
oped tubule failed to dissolve in this reagent. Most of
these immature, DTT-resistant cysts in our P. physalis
samples were the larger isorhizas (Fig. 20), but our isola-

DISULFIDES IN CNIDAE CAPSULES

11

tion procedure favored the larger cnidae. We also found
that some of the large, mature isorhizas with an appar-
ently well-developed tubule were resistant to DTT. How-
ever, these constituted <0.5% of that population, and we
cannot be certain from light microscopic observations
that they were completely mature.

The smaller isorhizas of both P. physalis and C. xama-
chana required 30-60 s for discharge after DTT treat-
ment. Upon discharge, the tubules dissolved slowly over
a period of several minutes, and in some cases failed to
dissolve at all. Many of the small isorhiza capsules were
only partially dissolved and could still be recognized af-
ter 15 min. In contrast, the euryteles in C. xamachana
tentacles discharged and began to dissolve immediately
on exposure to DTT; the tubule dissolved within 30 s.
Small, immature isorhizas in both species were as resis-
tant to DTT as the larger, immature ones in P. physalis.

Unlike the other cnidae in this study, isolated, undis-
charged mastigophores and spirocysts in C. luetkcni
failed to respond to DTT: they did not discharge and the
capsule did not dissolve. Efforts to dissolve these cnidae
in collagenase were unsuccessful, although it should be
noted that all cnidae in this study were collagenase-resis-
tant, consistent with observations made by Blanquet and
Lenhofff 1966). We followed our collagenase treatment
with DTT, again without effect. These cnidae also failed
to discharge or disintegrate after 30 min at room temper-
ature in the presence of 0.5 A/ thioglycolic acid, 3 A/
HC1, 1 A/NaOH,or0.2A/borohydride.

X-ray microanalysis

The spectra presented in Figure 2 1 range from 1 .2 to
5.0 keV. The zinc peak at 1.0 keV and the copper and
zinc peaks above 5.0 keV originated from the grids and
were eliminated from the analysis, as were traces of chro-
mium and iron that originated from the microscope
pole-pieces. We found no elements in these energy
ranges that were natural constituents of nematocysts or
spirocysts. Elements that were consistently present are
depicted in the figure as a solid peak followed by a dashed
line. Mean counts are indicated by a bar at the apex of
the solid peak. The highest and lowest counts are indi-
cated by the highest and lowest bars. Elements not con-
sistently present are shown as peaks without bars or
dashes.

Small amounts of silicon ( 1 .7 keV) were found in all
cnidae except C. xamachana nematocysts, and small
amounts of chlorine (2.8 keV) were found in all but C.
luctkeni nematocysts. In addition, trace amounts of alu-
minum (1.5 keV) were found both in spirocysts and in
nematocysts of C. luetkcni. Sulfur (2. 3 keV), on the other
hand, was present in all cnidae, and was the dominant
naturally occurring element. Nonetheless, since sulfur

counts were often 1 per s or less, we employed samples
that had been treated with methenamine-silver (3. 1 and
3.3 keV) in case a consistent multiple of silver to sulfur
could be demonstrated. However, silver-to-sulfur ratios
were often inconsistent among individual cnidae. The
ratios were generally in the range of 3: 1 for all but spiro-
cysts, which were closer to 2: 1 . The large isorhizas from
P. physalis and the small isorhizas of C. xamachana had
the highest sulfur and silver counts. The immediately ad-
jacent submature cnidae (those without completely
differentiated tubules) exhibited only background levels
of sulfur, corresponding to the cytochemical results. The
second-highest levels of sulfur were found in mature,
large P. physalis isorhizas. Submature capsules could not
be distinguished for separate X-ray examination. Sur-
prisingly, the lowest sulfur counts were found in mature,
small P. physalis isorhizas, followed closely by the mi-
crobasic mastigophores of C Inetkeni. Spirocyst sulfur
counts were unexpectedly higher than those of the small
nematocysts. However, since the spot size was wider
than the diameter of the spirocyst capsule, sulfur counts
were unavoidably collected from both the capsule and
the tubule. This was also the case with the mastigo-
phores, but the tubule alone appeared to contribute little
sulfur to the spectrum. Immature cnidae of both types
from C. luetkcni displayed only background sulfur
counts.

Amino acid analysis

The amino acid analysis included a mixture of cnidae
types, along with various stages of maturity that were un-
avoidable with the procedure employed. These results
(Table I), while not ideal, provide a general impression
of capsule chemistry and are instructive where they cor-
roborate information obtained by cytochemistry and X-
ray microanalysis.

In P. physalis nematocysts, levels of glycine, proline,
and hydroxyproline were consistently high among the
three colonies examined. Cysteic acid was the dominant
sulfur amino acid. In C. xamachana nematocysts, pro-
line and hydroxyproline were substantial constituents,
although levels of these amino acids were not as high as
those of P. physalis. The high levels of sulfur found in
X-ray microanalysis were reflected in the total S-amino
acids found in this species (6.84% ± 1.30%) but about
45% of these occurred as metsulfone. When cysteic acid
and the small amount of residual cystine alone were con-
sidered, the total was not significantly different from that
of C. xamachana cnidae.

Levels of proline and hydroxyproline in C. Inetkeni
cnidae were similar to those found in C. .xamachana, and
the total S-amino acid was similar to totals found in P.
physalis. Cysteic acid residues were highest (3.00 ±

12

W. M. GOLDBERG AND G. T. TAYLOR

Physalia Large Isorhiza

Physalia Small Isorhiza

3000

2500

2000

oo

; 1500

3
O ,.

U '

1000
750
500
250

C

3000
2500
2000

1500
1250
1000

750
500
250

2.3 2.8 3 1 33

keV

Cassiopeia Isorhiza

Ag

23 2 S 3 I 3 3

keV

1500

1250

1000

en

§ 750

u

500
250

17 2.3 28 .VI 3.3

keV

Cirrhipathes Mastigophore

Ag

1.51.7 2.8 31 3.3

keV

Cirrhipathes Spirocyst

Ag

15 1.7 2.3 28 3.1 3.3

keV

Figure 21. Composite TEM X-ray spectra of cnidae capsules. Methenamine-silver preparations show
relative amounts of silver compared to natural constituents of the capsule. Mean counts for each element

DISULFIDES IN CNIDAE CAPSULES

13

Table I

Glycine. proline/hydroxyproline and S-anww acid cuineni <>/ cnidae <;.v
mole.% (pmolex aminoacidas a percent of total pmoles ± xiddevofS
samples)

Amino

Physalia

Cassiopeia

Cirrliipullie\

acid

physalis

xarnachana

luetkeni

Glv

20. 56 ±0.1 5%

18.4 ±3.9%

19.49+ 1.97%

Pro

23.94 ± 0.06%

I2.79± 3.55%

11. 28 ±2.6%

Hypro

10. 72 ±0.04%

1.81 ±0.81%

1.70 + 0.45%

Cvs*

4.36 ±0.17%

4.72 ± 1.13%

4. 14 ±0.61%

Met**

0.25+0.01%

2.12 + 0.29%

0.44 + 0.06%

* Cys is the combined result of cysteic acid and small amounts of
residual cystine. unconverted by performic acid.

** Met is methionine converted b> performic acid to metsullbne.

0.48 mole.%). whereas metsulfone never constituted
more than 0.49 mole.%. However, in contrast to the
other types of cnidae examined, in C. luetkeni cnidae a
substantial amount of cystine (1.14% ± 0.23% of the
amino acids or 27.5% of the S-amino acids) was left un-
hydrolyzed by performic acid.

Discussion

Alkaline silver solutions have long been used to dem-
onstrate neuronbrils. reticulum. Golgi, and other com-
ponents of tissues and cells. The nature of the reaction is
complex, and the degree of specificity often depends
upon the type of fixation employed, the counter ion of
the silver (nitrate, hydroxide, carbonate, methenamine,
etc.) and the post-silver treatment of the tissue (e.g., Hu-
mason. 1979). Specificity is further complicated by the
action of silver as both an oxidizing stain and a redox
reagent (Hayat, 1993). Methenamine-silver, introduced
by Gomori ( 1946), was initially adapted for electron mi-
croscopy as a periodic acid-Schiff reagent. Despite non-
selective staining of nuclear chromatin. melanin gran-
ules and ribosomes, neutral carbohydrate could be spe-
cifically demonstrated by periodic acid oxidation along
with aldehyde blockade reactions (see reviews by Kier-
nan, 1990; Hayat, 1993). Methenamine-silver has also
been employed to demonstrate disulfides in keratin and
in other cystine-containing tissues. The mechanism
probably involves alkaline hydrolysis of the disulfide and
subsequent reduction of an undescribed methenamine-
silver complex to metallic silver (Swift, 1968, 1973;
Thompson and Colvin. 1970). The specificity of the rea-
gent is dependent upon site-specific silver reduction in

tissues that have been aldehyde-blocked and extracted
with thiosulfate to eliminate unreduced silver. In addi-
tion, disulfide reduction and subsequent alkylation must
prevent deposition of reduced silver at those previously
reactive sites. Finally, the method should be employed in
conjunction with parallel chemical techniques to con-
firm the cytochemical results (Thompson and Colvin,
1970). The silver cytochemistry of cnidae capsules is
consistent with the results of post-reduction alkylation
controls. X-ray microanalysis, and amino acid analysis.
All of these data suggest that the argentophilia in the cni-
dae we have studied is due to the presence of capsular
disulfides.

We know of only one other study in which electron
cytochemistry has been used to detect disulfides in cni-
dae. Watson and Mariscal (1984) used performic acid
oxidation coupled with alcian blue staining to study ne-
matocyst development in the anemone Ha/iplanei/a luc-
iae. They found that nematocyst maturation in that spe-
cies is characterized by a 50% reduction in the thickness
of the capsule wall, coincident with the formation of di-
sulfide linkages. Both of these conclusions are in accor-
dance with our observations of Cassiopeia xamachana.
Capsule thinning also occurs with maturity in P/ivxalia
physalis, judging from the differences between immature
and mature isorhizas. We did not attempt to study mat-
urational changes in Chrhipathes luetkeni cnidae.

If the alcian blue technique has an advantage over me-
thenamine-silver, it is in the capacity to distinguish sulf-
hydryl from disulfide groups. We were not able to di-
rectly determine the contribution of either group spe-
cifically because both are argentophilic. The application
of iodoacetic acid as a blockade prevented pre-existing
sulfhydryl groups from reacting with silver, but increased
the background in all but P. physalis. No differences
were noted in this species between mature cnidae treated
with iodoacetic acid and those left unblocked, suggesting
that their argentophilia may be due entirely to disulfide
groups. In C. xamachana and C luetkeni, on the other
hand, there was a small decrease in capsular argentophi-
lia with iodoacetate controls, suggesting that some reac-
tivity was contributed by sulfhydryl groups, although sig-
nificantly less than that contributed by disulfides. In ei-
ther case, the weak reactivity of developing cnidae in our
study strongly suggests that neither sulfhydryl nor disul-
fide groups are present until very late in the process of
capsular maturation.

The amino acid composition of coelenterate cnidae
has been examined by a number of authors (Phillips,

Figure 21. (Continued) are represented by bar atop solid peaks; dashed lines indicate highest counts;
lowermost bar represents lowest counts. Peaks without bars or dashes represent elements that were not
consistently present.

14

W M. GOLDBERG AND G. T. TAYLOR

1956; Lenhoff et a/.. 1957; Lenhoff and Kline, 1958;
Lane and Dodge, 1958; Fishman and Levy. 1967; Lane,
1968, 1974;PhelanandBlanquet, 1985; Blanquet, 1988)
or the patterns of their constituent proteins (Kurz et ai.
1991; Brand et ai. 1993: Hoh in et ai. 1994); but the
universe of species examined is surprisingly small, repre-
senting only four genera with nematocysts (Hydra spp.,
Physalia physalis. Aipu :va pallida, Aletridium spp.) and
Pachycerianthus torrcyi, a species with ptychocysts. Ele-
vated levels of glycine, proline, and hydroxyproline are
characteristic of collagen, and all cnidae analyzed thus
far, including those in the present study, appear to con-
tain these structural proteins. Glycine in nematocyst
protein varies from 31.2-19.2 mole percent (Fishman
and Levy, 1967; Stone et ai, 1970; Lane. 1974; Phelan
and Blanquet, 1985). Glycine in our study is within this
range, albeit at the lower limits. In P. physalis the proline
content we reported is consistent with levels given by
Lane (1968; 1974). Proline levels in C. xamachana and
C. luetkeni cnidae were not unlike those of P. physalis.
Hydroxyproline in P. p/iysa/is. at 10.7 mole percent, is
slightly higher than the range of 6.9-9.2 mole percent re-
ported by Fishman and Levy (1967) and Phelan and
Blanquet (1985). However, hydroxyproline levels have
also been reported as 20%, 8.5%, and 5.4%. of the nema-
tocyst protein (Kline. 1961; Blanquet and Lenhoff, 1966;
Stone et ai. 1970, respectively). Conversion of our data
to this form yields 12.5% hydroxyproline for P. physalis.
but only 1.9% for C. luelkeni and 1.0% for C. xama-
chana. Thus a considerable range of this imino acid ap-
pears to occur in the proteins of the capsule and the tu-
bule. However, these figures have not taken into account
the ratio of collagen to the total nematocyst protein, and
we cannot state with certainty that all of the cnidae
pooled in our analyses (e.g.. spirocysts in C. luetkeni)
contain collagen.

The amount of cystine in cnidocysts is only occasion-
ally reported, and the range is considerable. Phelan and
Blanquet (1985) found 7.24 and 2.39 mole percent cys-
teine in Aiptasia pallida and Pachycerianthus torreyi re-
spectively, and Fishman and Levy (1967) reported
25.7 mole percent in Metridium marginatum. The most
frequent and readily obtainable observation of cnidocyst
chemistry is by X-ray microanalysis. In every case in
which such analyses of capsule composition have been
performed, sulfur has been the dominant element
(Mariscal, 1980; Lubbock et ai. 1981, 1988; Gupta and
Hall, 1984;Tardentt>/rt/., 1990; Zierold el ai. 1991; this
study). In addition, each author has suggested that disul-
fide linkage of collagen is the most likely role of that ele-
ment. Several studies have shown that certain nemato-
cysts dissolve in thiol reagents (e.g.. Yanagita, 1959;
Blanquet and Lenhoff, 1966; Fishman and Levy, 1967;
Mariscal, 1980; Phelan and Blanquet, 1985; Brand et ai.

1993). Yet it is not clear from this database whether the
presence of sulfur alone in nematocysts, or even the pres-
ence of cystine, means that the capsule will be dissolved
by disulfide reducing agents. This uncertainty was first
indicated by Mariscal and Lenhoff ( 1969) who tested the
solubility in dithiothreitol of 16 nematocysts from
squash preparations of 10 coelenterate species. Five of
the nematocysts from four species failed to depolymerize
in this reagent, as did spirocyst preparations from two
additional species. Phelan and Blanquet (1985) found
that ptychocysts from the cerianthid P. torreyi were sim-
ilarly resistant to the effects of disulfide reducing agents,
although cysteic acid residues were present in their hy-
drolysates.

In light of these results, reasonable questions have
been raised as to whether this insolubility might be due
to (a) the absence of disulfide bonds in some cnidae, (b)
the interference of reducing agent activity by mucus in
the fresh preparations used by Mariscal and Lenhoff
(1969), or (c) the inaccessibility of disulfides to reducing
agent activity resulting from the tertiary structure of the
capsule proteins (Blanquet, 1988). As in Phelan and
Blanquet's observations of ptychocysts, our study has
shown that cnidae from C. luetkeni contain cysteic acid
residues. In addition, electron cytochemistry and X-ray
microanalysis strongly suggest that in spite of their resis-
tance to DTT and thioglycolate, nematocysts and spiro-
cysts of this species both possess disulfide linkages. Our
use of clean cnidae preparations rules out chemical in-
terference with disulfide reduction in this study. With re-
spect to the last suggestion (c) above, disulfide linkages
located deep within hydrophobic regions of the protein
could account for the relatively large amount of cystine
remaining after performic acid hydrolysis in this species.
However, since disulfide reducing agents fail to cause
capsular depolymerization in C. luetkeni. it is possible
that disulfides occur within rather than between peptide
chains, or that disulfides are secondary to other types of
covalent, intermolecular linkages as they are in bivalve
byssus (e.g., Benedict and Waite, 1986; Van Ness et ai,
1988) and dogfish egg capsule (e.g., Rusaouen et til..
1976). A lesser degree of dependence on disulfides as a
means of capsule protein stabilization may also account
for the partial depolymerization responses noted in the
small isorhizas of P. physalis and C. xamachana (this
study) and in nematocysts of the sea nettle (Goldner et
ai. 1969; Stone et ai. 1970).

Spirocysts have long been considered a separate class
of cnidae (e.g.. Hyman. 1940), a perspective reinforced
by their distinctive tubule structure (e.g.. Mariscal el ai.
1977; Rifkin, 1991; Goldberg and Taylor. 1996). The
chemistry of the spirocyst capsule is essentially un-
known, except that, like some nematocysts, those of two
actiniarians are resistant to disulfide reducing agents

DISULF1DES IN CNIDAE CAPSULES

15

(Mariscal and Lenhoff, 1969). Despite this property,
Mariscal (1984) found that sulfur was the most strongly
represented element in the spirocyst capsule of the anem-
one Haliplanella lucuic. as it was in the nematocyst cap-
sules of that species. Our study of C. luclkcni not only
shows that sulfur is the principal element of the capsule
(as well as the tubule wall and pleats), but electron cyto-
chemistry strongly suggests that capsular sulfur occurs
primarily in the form of disulnde linkages, as it does in
nematocysts.

Surprisingly, the presence of disultide linkages in spir-
ocyst capsules is not entirely new information. Hamon
( 1955) observed that spirocysts from Anemonia sitlcala
reacted more strongly to nitroprusside after disulfide re-
duction than did the basitrichs of the same species. Un-
fortunately, the effects of disulfide reducing agents on
unfixed cnidae were not investigated. Further study will
be necessary to determine the distribution of resistance
to disulnde reduction among coelenterate cnidae gener-
ally, and spirocysts specifically. Additional work should
also focus on the characterization of spirocyst capsule
proteins and the alternative forms of capsular stabiliza-
tion reflected in their resistance to thiol reagents.

Acknowledgments

We thank P. Blackwelder of E.M. Analytical, Inc., for
assistance with and use of X-ray analytical equipment,
and C. Bigger and B. Fry for assistance in collecting spec-
imens. This work benefited from discussions with K.
Downum and D. Kuhn. We thank R. N. Mariscal (Flor-
ida State University) for helpful critique of the manu-
script. Support from Florida International University is
gratefully acknowledged.

Literature Cited

Benedict, C. V., and J. H. \Vaite. 1986. Location and analysis of hy-
ssal structural proteins of Myii/us edit/is. J. Morphol. 189: 171-181.

BigeloH, R. P. 1990. The anatomy and development of Cassiopeia
.\amachana Mem. Boston Soc. Nat. Hist. 5: 191-236.

Blanquet, R. S. 1988. The chemistry of cnidae. Pp. 407-425 The Bi-
ology of Nematocysts. D. A. Hessingerand H. M. LenhofT. eds. Ac-
ademic Press, New York.

Blanquet, R. S., and H. M. Lenhoff. 1966. A disulfide-linked collage-
nous protein of nematocyst capsules. Science 154: 152-153.

Brand, D. D., R. S. Blanquet, and M. A. Phelan. 1993. Collagenous.
thiol-containing proteins of cnidarian nematocysts: a comparison
of the chemistry and protein distribution patterns in two types of
cnidae. Comp . Bioelwm Physiol 1068:115-124.

Brown, C. H. 1950. Keratins in invertebrates. Nature 166: 439.

Cormier, S. M., and D. A. Hessinger. 1980. Cellular basis for tentacle
adherence in the Portuguese Man-of-War (Physalia pin -M///S). 7h-
sue Cell 12: 7 \3-12l.

Fishman. I... and M. Levy. 1967. Studies on the nematocyst capsule
protein from the sea anemone Metriiiiwn marginalum. Bin/ Bull.
133:464-465.

Goldberg, \V. M., and G. I. Taylor. 1989. Cellular structure and or-

ganization of the black coral Antipathes aperta: I. Organization of
the tentacular epidermis and nervous system./ Morphol 202:239-
253.

Goldberg. \V. M., and G. T. Taylor. 1996. The structure of the spiro-
cyst tubule in black corals (Anthozoa: Antipatharia) and its taxo-
nomic significance. Mar Bin/. 125:655-662.

Goldner, R., J. \V. Burnett, J.S. Stone, and M.S. Dilaimy. 1969.
The chemical composition of sea nettle nematocysts. Proe. Soc.
K.\p. Bio! Med 131: 1386-1388.

Gomori, G. 19-46. The study of enzymes in tissue sections. Am J
Clin. Pat hoi 16: 347-352.

Gupta, B. L., and T. A. Hall. 198-4. Role of high concentrations of Ca,
Cu. and Zn in the maturation and discharge in situ of sea anemone
nematocysts as shown by X-ray microanalysis of cryosections. Pp.
77-95 in To\in.i. Drugs, ami Pollutants in Marine Animals. L. Boils
el <//.. eds. Springer- Verlag, Berlin.

Hamon, M. 1955. Cytochemical research on coelenterate nemato-
cysts. Nature 176: 357.

Hayat. M. A. 1993. Stains and Cyloc/iemical Method*. Plenum
Press. New York. 455 pp.

Heinrickson, R. L., and S. C. Meredith. 1984. Amino acid analysis
by reverse-phase high-performance liquid chromatography: precol-
umn derivitization with phenylisothiocyanate. Anal. Biochem 136:
65-74.

Hessinger, D. A., and M. T. Ford. 1988. infrastructure of the small
cnidocyte of the Portuguese Man-O-War (Physalia p/iysa/is) tenta-
cle. Pp. 75-94 in The Biology of Nematocysts, D. A. Hessingerand
H. M. Lenhoft". eds. Academic Press, New York.

Holstein, T. W., and P. Tardent. 1984. An ultrahigh-speed analytical
exocytosis: nematocyst discharge. Science 223: 830-833.

Holstein, T. V\ '., M. Benoit, G. v. Herder, G. Wanner, C. N. David, and
II. E. Gaub. 1994. Fibrous mini-collagens in Hydra nematocysts.
Science 265: 402-404.

Hulet, \V. H., J. L. Bellemc, G. Musil, and C. E. Lane. 1974. Ultra-
structure of Physalia nematocysts. Pp. 99-1 14 in Bioactive Com-
pounds Irom the Sea. H. J. Humm and C. E. Lane, eds. Marcel De-
kker. New York.

Humason, G. L. 1979. Pp. 162-207 in Animal Tissue Techniques.
W. H. Freeman. San Francisco.

Hyman, L. H. 1940. Pp. 365-661 in The Imenehrales: Protozoa
through Clenophora. McGraw-Hill, New York.

Kiernan. J. A. 1990. Hislological and Histochemical Methods: The-
ory and Practice. Pergammon Press, New York. 433 pp.

Kline, E. S. 1961. Chemistry of nematocyst capsule and toxin of Hy-
dra litlorali.s Pp. 153-168 in The Biology of Hydra and of Some
Other Coelenterates. H. M. Lenhoff and W. F. Loomis, eds. Uni-
versity of Miami Press, Coral Gables, FL.

Kurz, E. M., T. W. Holstein, B. M. Petri, J. Engel. and C. N. David.
1991. Mmi-collagens in Hydra nematocysts. / Cell Biol. 115:
1159-1169.

Lane, C. E. I960. The Portuguese Man-of-War. Sei. Am 13: 371-
393.

Lane, C. E. 1968. Coelenterata: chemical aspects of ecology: pharma-
cology and toxicology. Pp. 263-284 in Chemical Zoology v. 2. M.
Florkin and B. T. Scheer, eds. Academic Press, New York.

Lane, C. E. 1974. Nematocyst toxins of Coelenterates. Pp. 123-137
m Bioactive Compounds from the Sea. H. J. Humm and C. E. Lane.
eds. Marcel Dekker, New York.

Lane, C. E., and E. Dodge. 1958. The toxicity of Physalia nemato-
cysts./W Bull 115:219-226.

Lenhoff, 11. M., E. S. Kline, and R. Hurley. 1957. A hydroxyproline-
rich. intracellular, collagen-like protein of Hydra nematocysts. Bio-
chim. Biophys. Ada 26: 204-205.

16

W. M GOLDBERG AND G. T. TAYLOR

Lenhoff, H. M., and E. S. Kline. 1958. High imino acid content of
the capsule from Hydra nematocysts. Anal Re< 130:425.

Locke, M., and N. Krishnan. 1971. The distribution of phenoloxi-
dasesand polyphenolsduringcuticle formation. Tissue Cell 3: 103-
126.

Lubbock, R., and VV. B. Amos. 1<»K Removal of bound calcium
from nematocyst causi < unite 290: 500-501.

Lubbock, R., B. L. Gupta. 1 I. A. Hall. 1981. Novel role of
calcium in exocyto" : mechanism of nematocyst discharge as
shown by X-ray m.uiunalysis. Proc. Nail Acad Sci USA 78:
3624-3628.

Mariscal, R. N. 1971. The effect of a disulfide reducing agent on the
nematocyst capsules from some coelenterates with an illustrated
key to nematocyst classification. Pp. 1 57- 1 68 in Experimental Coe-
lenterate Biology, H. M. Lenhoff. L. Muscatine. and V. Davis, eds.
Univ. Hawaii Press. Honolulu.

Mariscal, R. N. 1974. Nematocysts. Pp. 1 29- 1 78 in Coelenlerate Bi-
ology: Reviews and New Perspectives, L. Muscatine and H. M. Len-
hoff, eds. Academic Press, New York.

Mariscal, R. N. 1980. The elemental composition of nematocysts as
determined by X-ray microanalysis. Pp. 337-342 in Developmental
and Cellular Biology of Coelenterates, P. Tardent and R. Tardent.
eds. Elsevier/North-Holand. Amsterdam.

Mariscal, R. N. 1984. Cnidaria. Pp. 57-67 in Biology oj the Inlcgu-
incnl vol. 1 Invertebrates. J. Bereiter-Hahn. A. G. Matolsky, and
K. S. Richards, eds. Springer-Verlag. Berlin.

Mariscal, R. N. 1988. X-ray microanalysis and perspectives on the
role of calcium and other elements in cnidae. Pp. 95-1 14 in The
Biology oj Nematocysls. D. A. Hessinger and H. M. Lenhotf, eds.
Academic Press, New York.

Mariscal, R. N., and H. M. Lenhoff. 1969. Effect of a disulfide reduc-
ing agent on coelenterate nematocyst capsules. Expenemia 25:
330-331.

Mariscal, R. N., and C. H. Bigger. 1976. A comparison of putative
sensory receptors associated with nematocysts in an anthozoan and
a scyphozoan. Pp. 559-567 in Coelenlerale Ecology and Behavior,
G. O. Mackie. ed. Plenum, New York.

Mariscal, R. N., and R. B. McLean. 1976. The form and function of
cnidarian spirocysts 2. Ultrastructure of the capsule tip and wall and
mechanism of discharge. Cell Tissue Res 169: 3 1 3-32 I .

Mariscal. R. N., R. B. McLean, and C. Hand. 1977. The form and
function of cnidarian spirocysts 3. Ultrastructure of the thread and
function of spirocysts. Cell Tissue Res 178: 427-432.

Phelan, M. A., and R. S. Blanquet. 1985. Characterization of nema-
tocyst proteins from the sea anemones. liptasia pallula and Pachvc-
enanthns lorreyi (Cnidaria: Anthozoa). Comp Biocheni I'/n'siol
818:661-666.

Phillips, J. H. 1956. Isolation of active nematocysts of Melndiumse-
nile&nd their chemical composition. Nature 178: 932.

Rambourg, A. 1967. An improved silver-methenamine technique for
the detection of periodic acid reactive complex carbohydrates with
the electron microscope. J. Histochem. Cytochem. 15: 409-412.

Rusaouen, M., J-P. Pujol, L. Bocquet, A. Veillard, and J-P. Borel.
1976. Evidence of collagen in the egg capsule of the dogfish. Scvl-
iorhinus canicula. Comp. Biochem. Physiol. 53B: 539-543.

Rifkin, .J. K. 1991. A study of the spirocytes from the Ceriantharia
and Actiniaria (Cnidaria: Anthozoa). Cell Tissue Res 266: 365-
393.

Smith, H. G. 1937. Contribution to the anatomy and physiology of
Cassiopeia frondosa. Carnegie Inst. Wash. Pub. #475, Pap. Drv
Torttigax Lah. 31: 17-52.

Stone, J. H., J. W. Burnett, and R. Goldner. 1970. The amino acid
content of sea nettle (Chrysaora qiiinaitecirra) nematocysts. Comp
Biochem. Physiol. 33: 707-170.

Swift, J. A. 1968. The electron histochemistry of cystine-contaming
proteins in thin transverse sections of human hair. ./ R Microsc.
Site. 88: 449-460.

Swift, J. A. 1973. The electron cytochemical demonstration of cys-
tine disulfide bonds using the silver-methenamine reagent. Histoeh-
inue 35: 307-3 10.

Tardent, P. 1988. History and current state of knowledge concerning
discharge of cnidae. Pp. 309-332 in The Biology of Nematocysts,
D. A. Hessinger and H. M. Lenhoff. eds. Academic Press, New
York.

Tardent, P., K. /Jerold, M. Klug, and J. Weber. 1990. X-ray microa-
nalysis of elements present in the matrix of cnidarian nematocysts.
Tissue Cell 22: 629-643.

Thompson, K., and J. R. Colvin. 1970. Electron cytochemical local-
ization ofcystine in plant cell walls. J Electron Microsc. 91: 87-98.

Van Ness, K. P., T. J. Koob, and D. R. F.yre. 1988. Collagen cross-
linking: distribution of hydroxypyridinium cross-links among in-
vertebrate phyla and tissues. Comp. Biochem. Phvsiol. 91 B: 531-
534.

Watson, G. M., and R. N. Mariscal. 1984. Ultrastructure and sulfur
cytochemistry of nematocyst development in catch tentacles of the
sea anemone llaliplanella luciae (Cnidaria: Anthozoa). / Ultras-
Intel Res. 87: 159-171.

Yanagita, T. M. 1959. Physiological mechanism of nematocyst re-
sponses in sea-anemone 1 . Effects of trypsin and thioglycolate upon
the isolated nematocysts. Jpn. J. /.ool. 12: 361-375.

Yanagita, T. M., andT. Wada. 1954. Effects of trypsin and thioglyco-
late upon the nematocysts of the sea anemone. Nature 173: 171.

Zierold, K., P. Tardent, and S. V. Buravakov. 1991. Elemental map-
ping of cryosections from cnidarian nematocytes. Scanning Mi-
crosc. 5:439-444.

Reference: Biol Bull. 192: 17-26. (February. 1997)

Ovarian Development in the Class Holothuroidea:
a Reassessment of the "Tubule Recruitment Model"

M. A. SEWELL1. P. A. TYLER:, C. M. YOUNG1, AND C. CONAND'

'Department of Larval Ecology. Harbor Branch Oceanographic Institution, 5600 U.S. 1 North. Fort

Pierce, Florida 34946: ^Department of Oceanography, University of Southampton, Southampton

SOI 7 IBJ. United Kingdom: and ^Laboratoire de Biologie Marine, Universite de la Reunion,

97715 Saint-Denis Cedex. La Reunion. France

Abstract. The "tubule recruitment model" for the de-
velopment of the holothurian gonad was proposed (a) to
connect the stages of oogenesis with ovarian morphology
in holothurians throughout the reproductive season and
(b) to emphasize the potential for the holothurian ovary
as a model system for cytological and biochemical study
of echinoderm oogenesis. To reassess the evidence for
this model, we have examined published accounts and
unpublished observations on gonad development in ho-
lothurians from both temperate and tropical habitats, in
shallow water and in the deep sea. A very limited number
of species were found to conform to the predictions of
the tubule recruitment model. The patterns of gonad de-
velopment vary substantially in holothurians, even at the
individual level, and with taxonomic position, geograph-
ical location, and habitat. The tubule recruitment model
can be applied to only a small subset of holothurians.
specifically those in the families Stichopodidae and Ho-
lothuriidae that have gonad morphology similar to that
of Parastichopus californicus. However, the tubule re-
cruitment model is invalid for many other aspidochi-
rotes, and does not have wider applicability within the
class Holothuroidea.

Introduction

Fundamental concepts of reproduction can often be
addressed and tested most easily in marine invertebrates,
where the diversity of reproductive modes is greater than
among terrestrial animals (Giese el al.. 1987). Compara-
tive studies within the marine invertebrates can be used

Received 22 December 1995; accepted 6 November 1996.

to recognize unifying patterns of reproduction and to as-
sist in the development of robust theory (Giese et ai,
1987). An example is the "tubule recruitment model"
proposed by Smiley (1988) to describe gonad develop-
ment in the class Holothuroidea (phylum Echino-
dermata). This conceptual model, based on a careful and
impressive study of ovarian development in the as-
pidochirote sea cucumber Parastichopus californicus.
was proposed to connect the stages of oogenesis with the
ovarian morphology of holothurians throughout the re-
productive season (Smiley, 1988, 1994; Smiley et al.,
1991), and to accentuate the usefulness of the holothu-
rian ovary as a model system for cytological and bio-
chemical study of echinoderm oogenesis (Smiley, 1988,
1990, 1994; Smiley et al.. 1991).

Since the tubule recruitment model was first published,
several studies have documented apparent exceptions to
the model. Moreover, our own work with a variety of ho-
lothurians from throughout the world, and from depths
ranging from the intertidal zone to the deep sea, casts addi-
tional doubt on the broad applicability of the model. Here
we reexamine both the published literature and our own
unpublished data to test the applicability of the tubule re-
cruitment model to the class Holothuroidea in general, and
particularly to the aspidochirote holothurians.

Background

Holothurians differ from other extant echinoderms in
having a single gonad located dorsally in interambula-
crum CD (Hyman, 1955). The gonad consists of many
blind-ending tubules that are united at the anterior end
in a fleshy, saddle-shaped gonad basis in the dorsal sus-
pensor mesentery of the gut. The gonoduct exits anterio-

17

18

M. A. SEWELL ET AL

dorsally from the gonad basis and leads to single or
multiple external gonopores located between or near the
tentacles in interradius CD.

In contrast to the highly con - vative gonad morphol-
ogy found in other echn . lasses, there is consider-
able variability among t onads of different holothuri-
ans (Hyman. 195" ..ugh most species are gono-
choric, three of six holothurian orders have
hermaphroditic representatives (Smiley el a/., 1991). It
is overall gonad morphology, however, that exhibits the
most extreme degree of variability. The tubules attached
to the gonad basis vary in shape (e.g.. tubules, nodules,
globose sacs), length, degree of branching, and thick-
ness— even between species in the same family (e.g.. the
Stichopodidae: Conand, 1993a — fig. 2). For illustrations
of tubule variability, see the following figures: figs. 64,
67, 68, 72-76 (Hyman, 1955); figs. 4, 6 (Conand, 1981);
fig. 1 (Tyler and Gage, 1983); fig. 1 (Tyler et al.. 1985b);
fig. 1 (Tyler and Billett, 1987); fig. 2 (Tyler et al.. 1992);
fig. 2 (Conand, 1993a); fig. 6 (Conand, 1993b);fig. 1 (Ha-
mel eld., 1993); fig. 3 (Sewell, 1994).

In holothurians with an annual cycle, the reproductive
season can generally be divided into five "stages of matu-
rity" as first described for Stichopus japonicus by Tanaka
(1958). After spawning, the spent tubules are usually re-
sorbed and the gonads are defined as being in a post-spawn-
ing, or resting, stage (Stage I). The later stages of gonad ma-
turity are defined based on the stage of development of ga-
metes within the gonad tubules (Recovery, Growth,
Mature, Shedding; Stages II to V). There is not an equal
division of time in each maturity stage; in some species a
considerable portion of the reproductive cycle may be
spent in the resting stage, with little or no gonad material
present. Observations of the presence, size, and appearance
of tubules in the resting-stage gonad are crucial for assess-
ment of the tubule recruitment model in holothurians.

The Tubule Recruitment Model

Overview

The tubule recruitment model of gonad development
was based on histological and ultrastructural observa-
tions of oogenesis in Parastichopus californicus (as Sti-
chopus californicus ). These observations are described in
Smiley and Cloney (1985); Smiley (1988, 1990, 1994);
and Smiley el al. ( 1991 ). In P. californicus the ovarian
tubules are present as three distinct cohorts on the gonad
basis: the smallest or primary tubules most anterior; in-
termediate-sized secondary tubules in a medial position;
and the largest fecund tubules at the posterior end (Fig.
1 ). Central to the model is the progressive recruitment of
primary tubules, which originate in the anterior gonad
basis in Year N, to become the secondary tubules of Year
N+ 1 and the fecund tubules of Year N+2 (Fig. 2).

In simple terms, the entire process of ovary develop-
ment and oogenesis in Parastichopus californicus can be
viewed as a slowly moving conveyor belt carrying con-
tainers of maturing oocytes to the posterior end of the
gonad basis (Fig. 2). After spawning, the spent fecund
tubules are resorbed until only a pigmented plaque re-
mains on the posterior of the gonad basis. Some of the
nutrients obtained from phagocytosis of the spent tu-
bules are transferred to the primary and secondary tu-
bules, where they may provide energy for gamete prolif-
eration and vitellogenesis respectively. For a more com-
plete description of the processes that occur within each
tubule cohort, see the detailed discussions of the model
in Smiley and Cloney (1985); Smiley ( 1988, 1990); Smi-
ley et al. ( 1991 ); and Smiley (1994).

A number of assumptions were made in the formula-
tion of the tubule recruitment model: ( 1 ) that other ho-
lothurians would, like Parastichopus californicus, have a
gonad consisting of distinct tubule cohorts; (2) that re-
sorption of the spent tubules, as suggested by Hyman
( 1955). is a general feature of holothurian reproduction;
(3) that only the recently fecund tubules are completely
resorbed, and (4) that more than one year is required to
produce mature oocytes.

Support for the tubule recruitment model (Smiley,
1988) was drawn from descriptions of gonad morphol-
ogy in the aspidochirotes Stichopus japonicus (Mitsu-
kuri. 1903), Holothuriaparvula(KMe, 1942), Neosticfio-
pus grammalus (Deichmann, 1948), and the dendrochi-
rote Sclerodactyla (Thvone) briareits ( Kille. 1939). The
presence of several size classes of tubules on the gonad
basis in the aspidochirote Mcsothuria intcstinalis (Theel,
1901), the dendrochirote Cucuinana laevigata (Acker-
mann. 1902), and other unspecified species (Delage and
Herouard, 1903; Deichmann, 1930, 1948) were used in
a subsequent review to provide support for the proposed
model (Smiley et al.. 1991 ). Gonad index studies on the
aspidochirotes Stichopus japonicus (Tanaka, 1958),
Thelenota ananas and Holothwia nohilis (Conand,
1981) were also considered to provide data consistent
with the tubule recruitment described in Parastichopus
californicus (Smiley, 1988).

Evidence for the tubule recruitment model was lim-
ited to holothurians of the orders Aspidochirotida and
Dendrochirotida. Comparative information on oogen-
esis in other holothurian orders was considered too lim-
ited to assess the model's general applicability (Smiley.
1988). However, in subsequent reviews it was suggested
that the model had broad-scale applicability to ovarian
development throughout the class Holothuroidea (Smi-
ley. 1990; Smiley el al.. 1991; Smiley, 1994). Several
groups of holothurians were considered to be problem-
atic to the tubule recruitment model; these included
male holothurians of any order, small hermaphroditic or

primary tubule

secondary tubule

oocyles

peritoneum

RFPRODUCTION IN HOLOTHURIANS

ANTERIOR
DORSAL VIEW

19

gonad basis

•dorsal mesentery

inspawned oocyles

WHOLE OVARY

Figure 1. The tubule recruitment model as proposed b> Smiley ( 1988) in Parastichopus californicus.
The whole ovary of P. calij'ornicus is shown. The left side of the figure represents the prespawning condition
with mature oocytes in the fecund tubule and earlier oocyte development in the primary and secondary
tubules. The right side shows the post-spawning, or resting-phase. gonad. The fecund tubules are in the
process of resorption. and the primary and secondary tubules are larger preparatory to posterior migration
on the gonad basis. In this model, three cohorts of tubules are present on the gonad basis at any one time.
Diagram reprinted from Smiley and Cloney (1985) with permission.

short-lived holothurians, and species with more than one
spawning per year or with continuous reproduction (e.g.,
polar and deep-sea species: Smiley, 1988; Smiley et a!.,
1991;Smilev. 1994).

Testing

For a species to conform to the tubule recruitment
model it must have gonad tubules that develop in distinct
primary, secondary, and fecund cohorts, with each cohort
of tubules having synchronous development of oocytes.
After spawning, the relict fecund gonad tubules are re-
sorbed. but the primary and secondary tubules persist on
the gonad basis and migrate posteriorly as new primary
tubules are formed. Consequently, the gonad in the rest-
ing stage following spawning must contain primary tu-
bules as well as secondary tubules that will develop into
the fecund tubules for the next reproductive season.

The tubule recruitment model would not apply to sit-
uations in which a single tubule or a single cohort of sim-
ilar-sized tubules is attached to the gonad basis and re-

sorbed partially or totally after the reproductive period.
It would also not apply where immature previtellogenic
and mature vitellogenic oocytes are found in the same
ovarian tubule (overlapping generations of oocytes), be-
cause this would be evidence that the same "container,"
or cohort of tubules, is being used for multiple reproduc-
tive seasons.

Our survey of the published literature is confined to
those papers on holothurian reproduction that describe
in detail the gross morphology of the gonad during the
reproductive period. We assess the validity of the tubule
recruitment model in two ways. First, we consider stud-
ies in which sufficient information is provided to test for
the distinct gonad morphology required by the model
(i.e.. primary, secondary, and fecund tubules). Second,
we describe the reproductive studies published after 1 988
that explicitly test the tubule recruitment model. Our ex-
amination of those studies, in combination with our own
unpublished observations, leads us to suggest that the ap-
plicability of the tubule recruitment model is relatively
limited in holothurians, even within the family Sticho-
podidae in which it was initially proposed.

20

M. A. SEWELL ET AL

YGM

N

YEAR
N+1

N+2

Germ eel!
in basis

Primary
tubules

Secondary
tubules

Fecund
tubules

Resorbed
tubules

Figure 2. Schematic diagram to show progressive recruitment of tubules in the ovary of Paraslichapus
californium. The gonad morphology is shown for three consecutive years: N, N+l, N+2. In each year the
gonad has three cohorts of tubules ( YGM = year-round gonad morphology), as well as primary germ cells
at the very anterior and the remains of resorhed tubules at the posterior end of the gonad basis. Tubule
cohorts derived from the same primary germ cells are shown with the same pattern (open, hatched, filled,
etc. ). The germ cells of year N migrate on the gonad basis ( M ) to become the primary tubules of year N + 1 .
Oogomal proliferation (P) in this year results in secondary' tubules in Year N+2. Vitellogenesis (V) in the
secondary tubules results in fecund tubules whose gametes are spawned (S) and the empty tubules resorbed
in Year N + 3. Consequently, the gametogenic process in P. californicus from primary germ cells to spawn-
ing takes 4 years.

Evidence from Gonad Morphology
Order Aspidochirotida

A priori we might expect that the strongest support for
the tubule recruitment model would be found in as-
pidochirote holothurians. considering that the model
was based on Parastichopus californicus (Family Sticho-
podidae). The model can be tested in many aspidochi-
rotes because of reproductive studies prompted by their
importance as beche-de-mer fisheries (Conand and
Byrne, 1993).

In holothurians of the family Stichopodidae (30 spe-
cies; Smiley, 1994) the gonad is present as two tufts of
tubules, one on each side of the dorsal mesentery. Al-
though tubule morphology varies considerably in species
of this family, none of the other species examined are
described as having distinct size classes of tubules on the
gonad basis (Stichopus variegatits — Conand, 1993a; S.
mollis — Sewell, 1992; Thelenota ananas — Conand,

1993a). Variability in the pattern of gonad development
is, however, apparent in species with broad geographical
ranges (S. japonicus — Tanaka, 1958; Choe, 1963; S.
mollis— Sewell, 1992). In these species there are differ-
ences in the amount of gonad material found during the
resting stage.

Sewell ( 1992) in a study of gonad development of the
temperate Stichopus mollis in New Zealand showed that
the tubule recruitment model may be applicable in only
some populations. Detailed study of a population of 5.
mollis in northern New Zealand found complete resorp-
tion of the gonad after spawning, with no gonad material
present during the winter months (Sewell and Bergquist,
1990; Sewell, 1992). In contrast, 51. mollis from southern
New Zealand retained a large volume of tubules in the
resting period, and the tubules contained developing oo-
cytes (Sewell, 1992). The evidence for progressive re-
cruitment of tubules is, however, equivocal because the
southern population examined was from a greater depth

REPRODUCTION IN HOLOTHURIANS

21

than the northern one (Sewell, 1992) and the specimens
were preserved and eviscerated, with unattached gonads
(M. A. Sewell, unpub. obs.).

A similar pattern of gonad development with latitude
is observed in the temperate Japanese sea cucumber Sti-
chopus japonicus. A resting-phase gonad with shrunken
tubules observed in S. japonicus at Hokkaido (Tanaka,
1958) was not seen in this species in southern Japan by
Choe (1963). In Atsumi Bay the gonads in S. japonicus
completely disappear after spawning, and a condition
corresponding to the resting stage of Tanaka ( 1958) was
not found (Choe, 1963).

Differences in the resting-phase gonad in distantly sep-
arated populations of 5. japonicus and S. mollis are also
reflected on a smaller scale in two other stichopodids. In
the tropical Stichopus variegatus and Thelenota ananas.
a high variability has been observed in the resting stage
maintained in individual sea cucumbers. After spawn-
ing, the gonad tubules were entirely resorbed, and only
the gonad basis remained in some individuals (C. Con-
and, unpub. obs.). A similar pattern was recently re-
ported in the South African stichopodid Neostichopus
grammatus (Foster and Hodgson, 1995). Although Smi-
ley (1988) considered that to be a species showing pro-
gressive tubule recruitment, Foster and Hodgson ( 1995)
reported that the tubules are either resorbed completely
after spawning or are reduced to a few very short fine
threads.

Almost 50% of the aspidochirote holothurians are
within the Family Holothuriidae (Smiley, 1994). In this
family, gonad tubules are present only on the right side
of the dorsal mesentery. Two species of this family were
considered by Smiley ( 1988) to provide support for the
tubule recruitment model — Holothuria parvula (Kille,
1942) and H. nobilis (Conand, 1981). In H. parvula the
ovary has distinct tubule cohorts, with small immature
tubules at the anterior, a distinct cohort of mature tu-
bules, and the resorption of the most posterior tubules
after spawning (Kille, 1942). Evidence for tubule recruit-
ment in //. nobilis is more limited, however, under the
criteria defined earlier. The gonad in this species has one
tuft of tubules; the length and diameter of these tubules
vary during the reproductive cycle, but no tubule cohorts
are described (Conand, 1981).

Cohorts of tubules with differing stages of oocyte de-
velopment have been observed in individuals of the trop-
ical species Holothuria atra (Pearse, 1968); H. leuco-
spilota (Viet Nam and Britaev, 1992); H.floridana and
H. mexicana (Engstrom, 1980); and the temperate H.
forskali (Tuwo and Conand, 1992). However, the num-
ber of tubule cohorts varies between species (H. atra — 3;
H. leucospilota — 2; H.floridana and H. mexicana— 2 or
3; H. forskali — 5). More importantly for the assessment
of the tubule recruitment model, tubule cohorts are not

consistently seen in all individuals (Pearse, 1968; Eng-
strom, 1980), and in H. leucospilota they are seen in the
summer spawning period but not the spring one (Viet
Nam and Britaev, 1992). Furthermore, in studies of Ho-
lothuria species at other locations, many resting-stage in-
dividuals have no gonads (H. lubulosa — Coulon, 1994;
H. leucospilota— Ong Che, 1990; H. atra—Chao et ai,
\ 994; C. Conand. unpub. obs. ) or only one tubule cohort
(H. mexicana — Hyman. 1955, fig. 64). We, therefore,
conclude that in the genus Holothuria, evidence for the
tubule recruitment model is provided by some individu-
als of selected species, but only at some geographical lo-
cations and some times.

The tropical holothurid Actinopyga shows a pattern
similar to that of the genus Holothuria. The ovary in A.
echinites consists of a single tuft of tubules (Conand,
1982). However, after spawning in A. echinites and A.
mauritiana only a few resting individuals possessed gonad
tubules (Conand, 1993a; C. Conand, unpub. obs.), sug-
gesting that tubule recruitment does not generally occur.

The family Synallactidae comprises 143 species (42%
of the order Aspidochirotida; Smiley, 1994), which are
found at bathyal depths. The hermaphroditic Mesothu-
ria intestinalis. which shows distinct variation in size and
arrangement of male and female gonad tubules (Theel,
1901; Hyman, 1955 fig. 77C), was considered to provide
evidence for the tubule recruitment model (Smiley et at.,
1991). The gonad of a full-grown individual consists of
tufts of sexually differentiated tubules, with male and fe-
male gametes reaching their maturation in different, suc-
cessive tufts (Theel, 1901 ). There is evidence in this spe-
cies for the progressive recruitment of tubules on the go-
nad basis: the youngest tubules at the anterior are
followed posteriorly by tufts of alternating male and fe-
male tubules, and the marks of resorbed tubules from
previous reproductive periods are visible at the most pos-
terior position (Theel, 1901). The female tuft does not,
however, contain cohorts of tubules with oocytes in
different stages of development (Theel, 1901). In terms
of the tubule recruitment model, there is evidence for
progressive recruitment of tubule tufts, but the arrange-
ment of primary, secondary, and fecund female tubules
seen in Parastichopus californicus is lacking.

The gonad structure of the hermaphroditic Paroriza
pallens and P. prouhoi differs from that of other synallac-
tids: the gonad is composed of nodules along a central
tube that connects to the gonoduct (Tyler et ai. 1992).
Each nodule contains oocytes in various stages of devel-
opment; there are no distinct areas with the same oogenic
stage (Tyler et ai. 1992). Among gonochoric synallactids,
the gonad tubules of female Benthothuria funebris and
Palaepatides grisea show evidence of size variation (P. A.
Tyler, unpub. obs.), but the very distinctive gonad tubules
in Bathyplotes natans do not (Tyler et ai, 1994). In the

22

M. A. SEWELL ET AL

ovaries of Afesothuria verrilli and M. laclea the size of the
tubules is uniform, but in the testis of the latter, the tubule
size varies (P. A. Tyler, unpub. obs.)-

Order Dendrochirotida

The order Dendrochirotida is the most speciose in the
class Holothuroidea, with 553 of the 1427 species (Smi-
ley, 1994). At this point, however, sufficient information
to test the tubule recruitment model is available for a
relatively small number of temperate dendrochirotes.

The only species that shows cohorts of gonad tubules
as predicted by the tubule recruitment model is Sclem-
dactyla (Thyone) briareus (Kille, 1939); although Coe
(1912) did not describe an obvious size differentiation in
the gonad tubules of this species (see Hyman, 1955, fig.
67). There is no evidence for variation in tubule size in
the ovaries of Cucumaria pi and (Herouard. 1889; Hy-
man, 1955, fig. 72B), Cucumaria glacialis (Mortensen,
1894; Hyman, 1955, fig. 76H), or Aslia lefevrei (Cos-
telloe. 1985;TuwoandConand, 1994).

In dendrochirotes for which there have been seasonal
studies of reproduction, there is no evidence for progres-
sive tubule recruitment. In A. lefevrei and Pawsonia sax-
icola, relict gametes are resorbed after spawning, but the
tubules themselves are not (Tuwo and Conand, 1 994). A
similar pattern is observed in Psolns fabridi. which has
new oocytes present in its tubules after spawning (Hamel
el a/., 1993). Although the ovary of P. fabridi has two
sizes of gonad tubules, the large and small tubules are
intermixed within the gonads (Hamel el cil.. 1993). Be-
cause the tubules are not resorbed after spawning, large
tubules become small tubules due to oocyte release (Ha-
mel el ai, 1993). In these species the same tubules, or
"containers," are being used for more than one repro-
ductive season.

Examination of reproduction in the temperate Cucii-
maria frondosa along the Atlantic coast of Canada and
the northern United States showed differing gonad mor-
phology with latitude (Hamel and Mercier. 1996). In
northern latitudes the gonad is divided into two distinct
classes of tubules; south of New Brunswick the tubules
are of a uniform size (Hamel and Mercier, 1996). In the
St. Lawrence estuary, where large and small tubules are
present, the gonad tubules are not resorbed after spawn-
ing. As in Psolus fabridi, the small tubules contain ear-
lier stages of gamete development; mature oocytes are
found in the large tubules (Hamel and Mercier, 1996). In
contrast, at more southern, latitudes the gonad tubules
are in the same stage of development and attain maturity
after a single year (Hamel and Mercier, 1996). Thus, in
Cucumaria frondosa the gonad morphology and pattern
of gametogenesis might be related to differing environ-
mental conditions associated with latitude, rather than
being characteristic of the species throughout its range.

In the hermaphroditic species Cucumaria laevigata,
the developing tubules at the gonad basis are sexually in-
different but become female and release eggs as they
lengthen (Ackermann, 1902; Hyman, 1955). With con-
tinuing growth the remaining female tissue is phagocy-
tosed and the same tubules produce sperm (Hyman,
1955). Thus, the gonad of C. laevigata consists of small
basal undifferentiated tubules, larger female tubules, and
very long male tubules (see Hyman, 1955, fig. 76G).
Again, contrary to the tubule recruitment model, the
same tubules are used for more than one reproductive
period.

Order Dactylochirotida

Although there are 31 species of dactylochirote holo-
thurian (Smiley, 1994), reproduction has been described
in only one species, the lower bathyal Ypsilothuria biten-
laculala (as }'. talismani ). In this species Tyler and Gage
(1983) showed that a cluster of small similar-sized tu-
bules are present throughout the year.

Order Molpadula

The few reproductive studies that have been con-
ducted in molpadid holothurians reveal no evidence of
size cohorts within the gonad tubules. The ovaries ofPar-
acaudina diilensis (Kawamoto, 1927; see Hyman, 1955.
fig. 75) and Molpadia rorelzii (Hatanaka, 1939) consist
of elongated tubules of the same size. In the only dedi-
cated study of gonad morphology, Tyler el at. (1987)
showed that Cherbonniera uiriculus has a gonad with
fewer than 10 small, even-sized tubules.

Order Apodida

The few reproductive studies in the apodids are re-
stricted to species within the family Synaptidae, the ma-
jority being in hermaphroditic species. In the simulta-
neous hermaphrodite Leptosynapta tennis, oocytes and
developing sperm are found in the two branching tu-
bules. In rcproductively active animals the tubules are
extensively branched; after spawning the tubules are
shrunken, with little branching (Green. 1978). Oocytes
are present in these tubules throughout the year (Green,
1978). In the protandric hermaphrodite Leptosynapta
clai'ki. after spawning the relict gametes are resorbed
from the posterior ends of the two gonad tubules (Sewell
and Chia, 1994). New gametes for the next reproductive
season form in the anterior end of each gonad tubule,
adjacent to the gonad basis (Sewell and Chia, 1 994). The
type of gametes (oocytes or sperm) formed at the gonad
basis is dependent on the previous sex, animal size, and
other undetermined factors (Sewell, 1994).

The simultaneous hermaphrodite Synaptula hydri-

REPRODUCTION IN HOLOTHURIANS

23

fonnis similarly shows continued development of ga-
metes within the same gonad tubule (Frick el a/.. 1996).
Gametes of many different stages are found simulta-
neously within the two tubules of the ovotestis, and the
entire tubule is not resorbed after reproduction. Progres-
sive tubule recruitment, therefore, does not occur in this
species (Frick et al., 1996).

In the only study ofagonochoricapodan. Rhahdomol-
gus nihcr. the ovary is an unpaired tubule (Menker,
1970). Tubule growth occurs from the gonad basis, the
youngest eggs being most anterior and the older oocytes
occupying positions at the posterior end ( Menker, 1 970).
Unspawned eggs are resorbed in the posterior part of the
gonad in the autumn and winter (Menker, 1970), sug-
gesting that the same tubule is retained year-round.

Order Elasipoda

There has been considerable research on the gonad
morphology of the deep-sea elasipodid holothurians.
Early studies of the gonads were made from the collec-
tions of the Challenger CTheel 1882, plate XLVI;see Hy-
man, 1955, fig. 76D-F) and Galathea expeditions (Han-
sen, 1975). These data, together with more recent studies
of the families Laetmogonidae (Tyler et al.. 1985b), Dei-
matidae (Tyler and Billett, 1987), Psychropotidae (Tyler
and Billett, 1987), and Elpidiidae (P. A. Tyler, unpub.
obs.), provide no evidence for the progressive recruit-
ment of tubules. In most species the ovarian tubules con-
tain one or more large vitellogenic oocytes, together with
a large number of smaller previtellogenic oocytes. In ad-
dition, because most species probably show continuous
reproduction, the seasonal, synchronous development of
tubules as proposed in the tubule recruitment model is
considered unlikely. The one exception to this pattern is
in species of Peniagone in which there is a progressive
increase in the size of the gonadal tubules from anterior
to posterior (Tyler el al., 1985a).

Conclusions from gonad morphology

Review of the gonad morphology of more than 45 ho-
lothurians suggests that size division and progressive re-
cruitment of gonad tubules in Parastichopus californicus
is the exception, rather than the rule, in holothurian re-
production. Holothurian species may show different pat-
terns of gonad development between individuals and lo-
cations, and the use of the same tubule for oocyte growth
in more than one reproductive period is seen in species
of the orders Dendrochirotida, Apodida, and Elasipoda.

Our conclusions many be challenged on the grounds
that the authors of some of the studies we analyzed may
not have specifically looked for the small primary and
secondary tubules on the gonad basis. We, however,
maintain that when reports of primary or secondary tu-

bules are lacking (either because of true absence or an
incomplete examination of gonad morphology), the ob-
servation of the various stages of oogenesis in the same
tubule "container" is, in itself, sufficient to cast doubt
on the tubule recruitment model. Consequently, from
morphological evidence alone, we question the general
applicability of the tubule recruitment model to describe
reproduction in the class Holothuroidea.

Evidence From Explicit Tests

The tubule recruitment model has been explicitly ad-
dressed in nine holothurian species from three of the six
orders (Table I). These include species from different re-
gimes of depth (shallow water or deep sea) and tempera-
ture (temperate or tropical), and in both gonochoric and
hermaphroditic forms.

In the order Aspidochirotida, support for the tubule
recruitment model is found in only one species (Holo-
thitna forskali), and perhaps in southern New Zealand
populations of Stichopus mollis (Table I). The other
three aspidochirote species studied do not show cohorts
of tubules or the progressive recruitment of tubules dur-
ing ovarian development (Table I).

In Holothuria forskali, the tubules attached to the go-
nad basis can be subdivided into five classes (T,-T5 ), and
the tubules show progressive recruitment (Tuwo and
Conand, 1992). Although there are some minor differ-
ences in tubule recruitment between H. forskali and Par-
astichopus californicus (e.g., resorption in the T5 tubules
takes several months, and it can also occur in some T.i
and T4 tubules: Tuwo and Conand, 1992), this species
conforms well to the predictions of the tubule recruit-
ment model.

A more problematic species is Stichopus mollis. which
exhibits different patterns of gonad development in pop-
ulations from the North and South Island of New
Zealand (Sewell, 1992). Detailed visual and microscopi-
cal examination of individuals showed the complete re-
sorption of the gonad tubules after spawning in northern
New Zealand; i.e. no tubule recruitment (Sewell. 1992).
Preserved samples sent from the South Island in June
and August had a large mass of resting-phase gonad tu-
bules (Gonad Index for June: 0.00% in North Island.
0. 1 1% in South Island: Sewell, 1992), with the largest tu-
bules containing previtellogenic oocytes (Sewell, 1992).
Examination of the gonad basis for primary and second-
ary tubules was hindered by poor preservation and evis-
ceration of the gonads in most females (M. A. Sewell,
unpub. obs.). Consequently, although we can be certain
that a resting phase gonad is present in southern New
Zealand populations of Stichopus mollis, an explicit test
of the tubule recruitment model for these populations
awaits further research.

24 M. A. SEWELL ET AL.

Table I

Slitdia nf hit/ill hurian reproduclii >n puNisl. cd after 1 988 in winch the tubule recruitment model is explicitly tested

Order

Family

Tubule

Seasonality of recruitment

Sex1 reproduction2 Habitat1 Study location model4 Reference

Aspidochirotida

Stichopodidae .'• • a\ mollis

Sliclwpus variegatus G A

Thelenoia ananas G A

Holothuriidae Holothuriaforskali G A

Synallactidae Balhyploles naians G C
Dendrochirotida

Psolidae Psolusfabricii G A

Cucumariidae Cuciimana frondosa

Apodida

Synaptidae Leptosynapta clarki PH A

Synaptula hydriformis SH C

S, T, Sub northern New No

Zealand
southern New ?

Zealand
S. Tr, Sub New Caledonia No

S, Tr. Sub New Caledonia No

S, T, Sub Brittany, France Yes

D. T, Sub Bahamas No

S, T, Sub St. Lawrence No

Estuary,

Canada
S, T, Sub St. Lawrence No

Estuary,

Canada

S, T, Int Bamfield, Canada No

S. Tr. Sub Florida Kevs No

Sewell(1992)
Sewell(1992)

Conand(1993a);C.

Conand (unpub. obs.)
Conand (1993b);C.

Conand (unpub. obs.)
Tuwo and Conand ( 1 992 )
Tyler rt a/. (1994)

Hamele/a/. (1993)

Hamel and Mercier ( 1 996)

Sewell and Chia( 1994)
Frickrto/. (1996)

1 G = gonochoric; PH = protandric hermaphrodite: SH = simultaneous hermaphrodite.

2 A = 1 reproductive period per year; C = continuous reproduction.

3 S = Shallow; D = deep sea; T = temperate; Tr = tropical; Sub = subtidal; Int = intertidal.

4 No = tubule recruitment model not supported; Yes = tubule recruitment model supported; ? = further research required.

The three other aspidochirotes (Stichopus variegatus,
Thelenota ananas, Bathyplotes natans) do not conform
to the predictions of the tubule recruitment model (Ta-
ble I). The discrepancy may be partly a result of the
difference in gonad morphology from Parastichopus ca-
lifornicus (Fig. 1 ). These species do not have distinct pri-
mary, secondary, and fecund tubule tufts, but instead
have a number of similar-length tubules attached to the
gonad basis. Each tubule is surrounded with clusters of
gonadal saccules, reminiscent of a bunch of grapes (see
Conand, 1993a, fig. 2; Tyler el al.. 1994, fig. 2). In Sti-
chopus variegatus and Thelenota ananas, many individ-
uals have no tubules at all in the resting phase (C. Con-
and, unpub. obs), suggesting that in these individuals all
gonad material is resorbed after spawning. In Ba-
thyplotes natans, individual tubules produce and resorb
oocytes on a nonseasonal basis; the entire tubule is not
resorbed (Tyler?/ al., 1994).

Sea cucumbers in the orders Dendrochirotida and
Apodida also fail to support the tubule recruitment
model (Table I). Although both dendrochirotes have
large and small tubules, the same "container" is used to
make oocytes for the next reproductive season (Hamel el
al., 1993; Hamel and Mercier, 1996). The apodids.

which have only two gonad tubules, similarly use these
containers for more than one reproductive season (Sew-
ell and Chia, 1994; Frick el al., 1996). In both species
different developmental stages of gamete (oocyte in Lep-
tosynapta clarki and oocytes and sperm in Synaptula
hydriformis) are found within each tubule (Sewell and
Chia, 1 994; Frick i</<;/., 1996).

The tubule recruitment model is, therefore, not gener-
ally upheld in those holothurians where gonad morphol-
ogy and development have been critically examined. Of
the nine species annotated in Table I, only one (llololhu-
ria forskali) provides direct support for the tubule re-
cruitment model.

Conclusions and Recommendations for Future Research

The aim of this review has been to assess the applica-
bility of the tubule recruitment model to species from all
orders of the class Holothuroidea, and the validity of this
conceptual model as a general paradigm for ovarian de-
velopment in holothurians. The reproductive studies
considered here show that gonad development varies in
holothurians as a function of taxonomic position, geo-
graphical location, and habitat, and even within individ-

REPRODUCTION IN HOLOTHUR1ANS

25

uals at the same location. We conclude that the tubule
recruitment model may be appropriate for a number of
species in the aspidochirote families Stichopodidae and
Holothuriidae. However, it is not valid for many other
aspidochirotes and does not have wider applicability
within the holothurians.

The tubule recruitment model may prove to be appli-
cable only to those species that, like Parastichopus calif-
ornicus, have tufted tubular-type gonads. In recent re-
search on species with saccular gonads (e.g., Bathyploles
natans) or without distinct tufts of tubules (e.g., Synap-
titla hydriformis), the authors have questioned the gen-
erality of the tubule recruitment model (Tyler et a/.,
1994; Frick el ai, 1996). The pattern of ovarian develop-
ment in Parastichopus californicus, with synchronous
development of all gametes in discrete subsets of the go-
nad tubules, is apparently not the rule for holothuroid
oogenesisf Frick et al, 1996).

It is apparent from our review that the tubule recruit-
ment model can be applied only to a small subset of ho-
lothurians, specifically those in the families Stichopodi-
dae and Holothuriidae that resemble Parastichopus ca-
lifornicus in gonad morphology. The tubule recruitment
model is useful for such animals and can serve to focus
attention on details of gametogenesis such as ultrastruc-
ture and physiological control. Nevertheless, it is also im-
portant to acknowledge and carefully study the gonads
of species that do not fit the predictions of the tubule
recruitment model. This will lead to a belter understand-
ing of the various mechanisms underlying the wide di-
versity of gametogenic processes that are found in the
class Holothuroidea.

Acknowledgments

The germ cells of this review were conceived during a
dinner at the 8th International Echinoderm Conference
in Dijon in September 1993. We thank our colleagues
and the conference organizers for providing a forum for
this discussion. We also thank J. Pearse for later discus-
sion, J. Frick for comments on an earlier version of the
manuscript, and J-F. Hamel for providing pre-published
data. Harbor Branch Oceanographic Institution Contri-
bution No. 1 174.

Literature Cited

Ackermann, A. 1902. Uber die anatomic und zwittngkeit der Cucu-
maria laevigata. /. H'ixx. 7.ool. 72: 721-749.

Chao, S-M., C-P. Chen, and P. S. Alexander. 1994. Reproduction
and growth of Holothuria aim (Echinodermata: Holothuroidea) at
two contrasting sites in southern Taiwan. Alar. Biol. 119: 565-570.

Choe, S. 1963. h'umuko No Kenkyu (Biology ol l/ic Japanese Com-

mon Sea Cucumber Stichopusjaponicus Selenka). Pusan National
University. Japan. 226 pp.

Coe, XV. R. 1912. Echmoderms of Connecticut. Conn. Slate Geol
Nat. Hist. Sun: 19: 1-147.

Conand, C. 1981. Sexual cycle of three commercially important ho-
lothurian species (Echinodermata) from the lagoon of New Caledo-
nia. Bull. Mar. Sci. 31: 523-543.

Conand, C. 1982. Reproductive cycle and biometric relations in a
population of Actinopyga echinites (Echinodermata: Holothuroi-
dea) from the lagoon of New Caledonia, western tropical Pacific.
Pp. 437-442 in Proceedings <>/ ihe International Echinoderm Con-
ference. Tampa Bay. J. M. Lawrence, ed. A. A. Balkema. Rotter-
dam.

Conand. C. 1993a. Reproductive biology of holothurians from the
major communities of the New Caledonia Lagoon. Mar. Bio/. 1 16:
439_450.

Conand, C. 1993b. Ecology and reproductive biology ofStiehopiis va-
riegalux an Indo-Pacific coral reef sea cucumber (Echinodermata:
Holothuroidea). Bull. Mar Sci. 52:970-981.

Conand, C., and M. Byrne. 1993. A review of recent developments in
the world sea cucumber fisheries. Mar. Fish. Rev. 55: 1-13.

Costelloe, J. 1985. The annual reproductive cycle of the holothurian
Aslia le/evrei (Dendrochirota: Echinodermata). Mar. Biol 88: 1 55-
165.

Coulon, P. 1994. Role du macrobenthos detritivore dans la ecosys-
temes littoraux: etude de 1'holothurie Holothuria titbulosa, espece
commune des herbiers de posidonie en Mediterranee. Ph.D. thesis.
Universite Libre de Bruxelles, Brussels.

Deichmann, E. 1930. The holothurians of the western part of the At-
lantic Ocean. Bull. Mux. Comp /<><)/ 71:41-226.

Deichmann, E. 1948. The holothurian fauna of South Africa. .-Inn
NaialMux. 11:325-376.

Delage, \ '., and E. Herouard. 1903. Lex Echinodermes. Traite de Zo-
ologie Concrete. Yome III. Librarie C. Reinwald. Schleicher Freres
et Cie. editeurs. Paris.

Engstrom, N. A. 1980. Reproductive cycles of Holothuria (Halo-
deima) floridana, H (//. ) mexicana and their hybrids (Echino-
dermata: Holothuroidea) in southern Florida, U.S.A. //)/ J. Inver-
tebr. Reprod. 2: 237-244.

Foster, G. G.. and A. N. Hodgson. 1995. Annual reproductive cycles
of three sympatric species of intertidal holothurians (Echino-
dermata) from the coast of the Eastern Cape Province of South Af-
rica. Invertebr. Reprod. Dev. 27: 49-59.

Frick, J. E., E. E. Ruppert, and J. P. XXourms. 1996. Morphology of
the ovotestis ofSynaplitla Itytlrilimnix (Holothuroidea: Apoda): an
evolutionary model of oogenesis and the origin of egg polarity in
echinoderms. Imencbr. Bio/. 115: 46-66.

Giese, A. C., J. S. Pearse, and X'. B. Pearse, eds. 1987. Reproduction
in Marine Invertebrates, I 'ol. IX. General Aspects: Seeking unity in
diversity. Blackwell Scientific Publications. Palo Alto, CA.

Green, J. D. 1978. The annual reproductive cycle of an apodous ho-
lothunan. Leptosynapta tcnuix: a bimodal breeding season. Biol.
Bull 154:68-78.

Hamel, J-F., and A. Mercier. 1996. Gonad morphology and gameto-
genesis of the sea cucumber Ciicuinaria frondosa. Pp. 22-33 in
Beche-de-mer Information Bulletin. Number 8 (April 1996) South
Pacific Commission, New Caledonia.

Hamel, J-F., J. H. Himmelman, and L. Dufresne. 1993. Gametogene-
sis and spawning of the sea cucumber Pxolus labncii (Duben and
Koren). Biol. Bull. 184: 125-143.

Hansen, B. 1975. Systematic^ and biology of the deep-sea holothuri-
ans. (Jalalhea Report 13: 1-262.

Hatanaka, M. 1939. A study of the caudate holothurian. Molpudia

26

M. A. SEWELL ET AL

rorel-ii (V. Marenzeller). Sci. Rep Tohiiku L'niv. Fourth Ser 14:

155-190.
Herouard, E. 1889. Recherclv- sur les holothuries des cotes de

France. Arch. Zool E\p '' 27:535-704.

Hyman, L. H. 1955. Tin' In* rlebrales: Echinodermaia, The Coelo-

nnnc Bilalcrui Mc(ir\ :;iii Book Company, New York. 763 pp.
Kawamoto, N. 1927. Thi naaimy of Caudinachilensis with especial

reference to the peri\ ; ;ceral cavity, the blood and the water vascular

systems in their relation to the blood circulation. Sci Rep. Toliiiku

Univ. Fourth Scr 2: 239-264.
Kille, F. R. 1939. Regeneration of the tubules following extirpation

in the sea cucumber Thyone briareus. Bin/. Bull 76: 70-79.
Kille, F. R. 1942. Regeneration of the reproductive system following

binary fission in the sea cucumber llolothurui parvula (Selenka).

Biol. Bull. 83: 55-66.
Menker, D. 1970. Lebenszyklus. jugendentwicklung und geschlech-

tsorgane von Rhabdonwlgus ruber (Holothuroidea: Apoda). Mar.

Biol. 6: 167-186.
Mitsukuri, K. 1903. Notes on the habits and life-history of Sticlwpus

japonicus Selenka. Annol. Zool. Jpn. 5: 1-21.
Mortensen. T. 1894. Zur Anatomic und Entwicklungder Citciimana

j?/«tv7/.v(Ljungman). Z HV.v.v. Zool. 57: 704-732.
Ong Che, R. G. 1990. Reproductive cycle of Hololhuria leiicospiloia

Brandt (Echinodermata: Holothuroidea) in Hong Kong and the

role of body tissues in reproduction. Asian Mar Biol 1: 115-132.
Pearse, J. S. 1968. Patterns of reproductive periodicities in four spe-
cies of Indo-Pacificechinoderms. Proc Indian .lead. Sei. Seel. 568:

247-279.
Sewell, M. A. 1992. Reproduction of the temperate aspidochirote

Slichopns inol/is (Echinodermata: Holothuroidea) in New Zealand.

Ophelia 35: 103-121.
Sewell, M. A. 1994. Small size, broodingand protandry in theapodid

sea cucumber Leplosynapla clarki Biol. Bull 187: I 12-123.
Sewell, M. A., and P. R. Bergquist. 1990. Variability in the reproduc-
tive cycle o( Stichopus mollis (Echinodermata: Holothuroidea). In-

vcrtebr. Reprod. Dc\-. 17: 1-7.
Sewell, M. A., and F-S. Chia. 1994. Reproduction of the intraovarian

brooding apodid sea cucumber Leptosynapta clarki (Echino-
dermata: Holothuroidea) in British Columbia. Mar Biol 121:285-

300.
Smiley, S. 1988. The dynamics of oogenesis and the annual ovarian

cycle of Stichopus californicus (Echinodermata: Holothuroidea).

Biol. Bull 175:79-93.
Smiley, S. 1990. A review of Echinoderm oogenesis. ,/ Electron Mi-

crosc. Tech. 16:93-1 14.
Smiley, S. 1994. Holothuroidea. Pp. 401-471 in Microscopic Anal-

omy of Invertebrates, \'ol. 14. Echinodermata, F. W. Harrison and

F-S. Chia. eds. Wiley-Liss. New York.
Smiley, S., and R. A. Cloney. 1985. Ovulation and the fine structure

of the Stichopus californicui (Echinodermata: Holothuroidea) fe-
cund ovarian tubules. Biol. Bull. 169: 342-364.

Smiley, S., F. S. McEuen, C. Chaffee, and S. Krishnan. 1991. Echino-
dermata: Holothuroidea. Pp. 663-750 in Reproduction of Marine
Invertebrates, lolunie 17. Echinoderms and Lophophorates, A. C.
Giese, J. S. Pearse, and V. B. Pearse, eds. The Boxwood Press, Pa-
cific Grove, CA.

Tanaka, Y. 1958. Seasonal changes occurring in the gonad ofSl/cho-
pus laponicus. Bull. Fac. Fish. Hokkaido Univ. 9: 29-36.

Theel, H. 1882. Holothuroidea. Challenger Reports (Zoology) IV (8).
176pp.

Theel, H. 1901. On a singular case of hermaphrodism in holothurids.
Bihang till Kungl. Svensk. I'etensk-acad. Handl 27. Aid 4 No. 6:
1-38.

Tuwo, A., andC. Conand. 1992. Reproductive biology of the holothu-
rian Hi ilot/iuria lorskali( Echinodermata). J. Mar. Biol. Assoc. L'.K.
72: 745-758.

Tuwo, A., and C. Conand. 1994. Fecondite de trois holothuries temp-
erees a developpement pelagique. Pp. 561-568 in Echinodenm
Through Time. Proceedings of the St/i International Echinoderm
Conference. Dijon. France. B. David, A. Guille, J-P. Feral, and M.
Roux, eds. A. A. Balkema. Rotterdam.

Tyler, P. A., and D. S. M. Billet!. 1987. The reproductive ecology of
elasipodid holothurians from the N. E. Atlantic. Biol. Oceanogr. 5:
273-296.

Tyler, P. A., and J. D. Gage. 1983. The reproductive biology of Ypsi-
loilinria talixntani '(Holothuroidea: Dcndrochirota) from the N. E.
Atlantic. J. Mar Biol Assoc t'.A'. 63:609-616.

Tyler, P. A., J. D. Gage, and D. S. M. Billett. 1985a. Life-history bi-
ology of Peniagone azorica and P diaphana (Echinodermata: Ho-
lothurioidea) from the north-east Atlantic Ocean. Mar Biol 89:
71-81.

Tyler, P. A., A.Muirhead.D. S. M.Billett.and J. D.Gage. 1985b. Re-
productive biology of the deep-sea holothunans Laetmogone viola-
ceu and BenlhoKone ri>aea (Elasipoda: Holothurioidea). Mar. Ecol.
Prog Ser 23: 269-277.

Tyler. P. A., D. S. M. Billett, and J. D. Gage. 1987. The ecology and
reproductive biology of Cherhonniera ulnculus and Molpadia bla-
kei from the N. E. Atlantic. J. Mar Biol. Assoc. U.K. 67: 385-397.

Tyler, P. A.,C. M.Young.D.S. M.Billett.andL. A.Giles.1992. Pair-
ing behaviour, reproduction and diet in the deep-sea holothurian
genus Paroriia (Holothurioidea: Synallactidae). J. Mar. Biol.
Assoc U.K 72:447-462.

Tyler, P. A., K. Eckelbarger, and D. S. M. Billett. 1994. Reproduc-
tion in the holothurian Bathyploles nalans (Holothurioidea: Synal-
lactidae) from bathyal depths in the northeast and eastern Atlantic
Ocean. J Mar. Biol Assoc UK 74: 383-402.

Viet Nam, N., and T. A. Britaev. 1992. Reproductive cycle of the sea
cucumber Holothuria leucospilota in Nha Trang Bay (Southern
Viet Nam). RUSH. J. Mar Biol. 18: 185-191.

Reference: Biol. Hull 192: 27-40. (February. 1997)

Energy Use During the Development of a
Lecithotrophic and a Planktotrophic Echinoid

O. HOEGH-GULDBERG' AND R. B. EMLET2

1 School oj Biological Sciences. Building AOS. University of Sydney, 2006 NSW, Australia; and
2 Institute of Marine Biology and Department of Biology, University of Oregon, Charleston, OR 97420

Abstract. The energy required for development was
measured in two closely related echinoids with differing
modes of development. Heliocidaris tubercitlata hatches
from a 95-jum egg (~0. 1 Mg dry organic mass) and devel-
ops via a planktotrophic larva over 2 1-30 days into a ju-
venile (5.3-7.5 jug). H. erythrogramma hatches from a
~400/jm egg (1 1.6-19.0 jug) and develops over 3.5-
4 days via a lecithotrophic larva into a juvenile with a
mass not detectably different from that of the egg. Oxygen
consumption increased exponentially in H. tuberculata
and peaked at about 200-500 pmol indiv ' h ', whereas
the oxygen consumption of//, erythrogramma increased
rapidly, reaching a plateau at about 800 pmol indiv" ' h"1
on the second day. Metabolic energy expenditure for de-
velopment to metamorphosis was twofold higher for //.
tuberculata (52-60 mJ indiv"1) than for //. erythro-
gramma (26-35 mJ indiv"1). The interspecific compari-
son suggests that about half the metabolic expenditure for
planktotrophic development goes toward building and
operating the larval feeding apparatus and that the return
on this investment is 400%-600% over the larval period.
When the energy equivalents of the organic masses of the
juveniles are included, the energy for constructing a juve-
nile on a per mass basis is essentially the same for both
species (cf. H. tuberculata: 37-42 mJ /ug~'; //. erythro-
gramma: 34-36 mJ /zg~') and implies the absence of de-
velopmentally based energetic barriers or benefits to
changes in modes of development. Substantial amounts
of metabolically inactive material may be present in em-
bryos with nonfeeding development and should be con-
sidered in physiological measurements and comparisons.

Introduction

Most marine invertebrates have life cycles that include
a pelagic larva (Thorson, 1950) which may feed (plank-
Received 26 April 1996; accepted 14 November 1996.

totrophic) or not feed (lecithotrophic) on food particles.
Nonfeeding larval development has evolved repeatedly
in echinoderms and many other marine invertebrate
phyla (e.g.. Strathmann, 1978a, 1993; Emlet, 1990,
1994; Wray, 1995) and is associated with increased size
of eggs. With sufficient materials and energy in the larger
eggs, lecithotrophic development is relatively rapid, and
morphogenesis can be quite modified from that of re-
lated taxa with feeding larval development (e.g.. aster-
oids: Byrne, 1991, 1995; McEdward, 1992; Janies and
McEdward, 1993; echinoids: Raff, 1987; Wray and Raff.
1989, 1990; nemerteans: Martindale and Henry, 1995).
Among echinoids, studies on species with feeding and
nonfeeding larval development have demonstrated re-
markable changes in early embryology and larval form.
Patterns of cell cleavage (Raff, 1987), cell lineages (Wray
and Raff, 1989, 1990), and mechanisms of blastulation
and gastrulation (Henry et a!.. 1991; Wray and Raff.
1991; Schatt and Feral, 1996) have apparently changed.
The timing of expression of larval and juvenile traits has
been rearranged (e.g.. Raff, 1987; Wray and Bely, 1994;
Emlet, 1995); larval shape and degree of retention of the
ancestral larval structures varies (e.g.. Amemiya and
Emlet, 1992; Olson et al. 1993; Emlet, 1995; Morris,
1995). The length of the larval period and the size at
metamorphosis can change (Lawrence el al.. 1984;
McClintock and Pearse, 1986; Emlet et al.. 1987; Emlet
and Hoegh-Guldberg, 1997).

Our understanding of how development evolves is
particularly enhanced by comparisons between closely
related taxa with differing modes of development. One
area in which we lack such comparative data is the en-
ergy required for development. Energy required for de-
velopment includes (a) the maternal energy contributed
in the egg, (b) the total metabolic expenditure during de-
velopment, and (c) the energy in the mass change be-
tween fertilization and metamorphosis. Larger eggs do

27

28

O. HOEGH-GULDBERG AND R. B. EMLET

contain more energy, but few studies have been able to
provide precise estimates of the energy required for
different developmental modes (Jaeckle, 1995). We
know little about whet! ; holism changes when de-

velopment changes. Be use lecithotrophic species have
their own energv and usually develop relatively

rapidly, how does iheir metabolic activity change after
fertilization? If rates are adjusted for mass-specific me-
tabolism, how do planktotrophs and lecithotrophs com-
pare? The large yolky eggs of many lecithotrophs may
contain metabolically inert materials: how might this
confound interpretations of mass-specific metabolism?
Do lecithotrophic larvae, which have a finite maternal
energy supply, require more or less energy to produce a
juvenile than is required by planktotrophic larvae that
must gather materials and energy through feeding? Al-
though the evidence suggests that nonfeeding develop-
ment has evolved repeatedly and a considerable effort
has been made to understand this in terms of life-history
evolution (e.g., Vance, 1973; Strathmann, 1985; Rough-
garden, 1989; Havenhand, 1993, 1995). we know of no
studies (or theories) that have searched for developmen-
tally based energetic differences associated with particu-
lar modes of development.

The present study compares the energy use by two
closely related species with differing modes of develop-
ment. By providing several genera that include both
planktotrophic and lecithotrophic species, the echi no-
derm fauna of southeast Australia presents an unusual
opportunity to minimize evolutionary distance and en-
vironmental differences. The echinoid genus Helioci-
daris includes two species that co-exist in the Sydney re-
gion. The adult distributions partially overlap in shallow,
subtidal, rocky habitats, and in general both species ex-
perience similar thermal and other environmental regi-
mens during their life cycles. Laegdsgaard et al. ( 1991 )
found different seasons of reproduction for the two spe-
cies, but viable gametes of both species were readily ob-
tained for this study during Austral summers. // luher-
culala (Lamarck) hatches from a small egg (diameter
— 95 A<m) and completes development via a feeding larva
over 21-30 days at 22°C (this study). //. erythrogramma
(Valenciennes) hatches from a relatively large egg
(~400 ^m) and completes development in 3.5 to 4 days
at 22°C (Williams and Anderson, 1975: Raft", 1987; Em-
let, 1995). The divergence time for the two species has
been estimated as 5-8 million years, on the basis of
differences in mitochondrial DNA (McMillan el al..
1992), and as 10-13 million years, on the basis of DNA
hybridization methods (Smith et al.. 1990). The small
genetic distance (ca. 10 million years vs. 10's to 100's of
millions of years), and the similarity of adult habitats and
physical regimens of the life cycles between sibling He-
liocidaris species minimizes the influence of factors un-
related to the comparison of developmental mode.

This study utilizes the close relationship yet contrast-
ing developmental modes of sibling species of Helioci-
daristo compare biomass changes and metabolism from
fertilization through metamorphosis. With this informa-
tion, differences in the energy budgets of a lecithotroph
and a planktotroph are analyzed. We also take advantage
of the fact that the blastocoelic lipid reserves of H. eryth-
rogramma can be largely removed (Emlet and Hoegh-
Guldberg, 1997) and use this manipulation to provide a
perspective on the partitioning of energy use in the two
types of development. Comparison between the two spe-
cies of the total energy required for development yields
a quantitative measure of the maternal contribution of
energy required to produce a juvenile. This interspecific
comparison also permits us to estimate of the cost of
building and maintaining a larval feeding apparatus and
its efficiency for accumulating the energy required to
construct a juvenile echinoid.

Materials and Methods

Collection, maintenance, and culture of embryos and
larvae

The experiments described here were conducted over
three reproductive seasons (primarily December 1992-
January 1993 and January-February 1994, but also Jan-
uary-February 1996). Heliocidaris erythrogramma and
H. tuberculata were collected, under permit, from Shelly
Beach. Fairy Bower, and Clovelly near Sydney, Aus-
tralia. Adults were maintained at the University of Syd-
ney in recirculating aquaria for up to a week. Animals
were spawned by coelomic injection with 0.5A/KC1.
Eggs were washed with several changes of 1-^m filtered
seawater and fertilized with dilute concentrations of
sperm. Fertilized eggs were placed in dishes without stir-
ring for the first day, and gentle stirring of cultures began
on day 2.

Embryos and larvae were cultured at 22°C and culture
water was changed every 2-3 days. Cultures of//, eryth-
rogramma were checked for juveniles beginning on the
third day. Cultures of//, tuberculata were fed algal cells
(Chaetoceros gracilis or Rhodomonas lens, CSIRO. Ho-
bart) at final concentrations of 20-50 X 103 cells ml~',
and the first individuals completed development in 21-
30 days. Larvae of both species either metamorphosed
spontaneously or were induced to metamorphose with
scrapings from algal-encrusted rocks.

Removal of blastocoelic lipid from embryos of
Heliocidaris erythrogramma

Lipid-rich vesicles are extruded into the blastocoel of
//. erythrogramma during the first 8-1 2 h of develop-
ment and prior to hatching (Henry el al.. 1991 ). These
blastocoelic vesicles were removed from newly hatched

ENERGY USE BY SEA URCHIN LARVAE

29

blastulae ( 12-14 h post-fertilization) by centrifuging
blastulae for two 15-s intervals in a microcentrifuge
(Beckman Microfuge E). Blastocoelic lipid extrudes
through tiny rips in the blastoderm of the blastulae dur-
ing eentrifugation. Embryos at this stage do not have
mesenchyme cells and hence the loss of cells from the
blastula is minimal. This manipulation removes almost
all the blastocoelic lipid, which occupies about 40% of
the volume of the blastula (Emlet and Hoegh-Guldberg,
1 997), and embryos treated in this manner develop nor-
mally to the juvenile stage in the same time as untreated
larvae (Emlet and Hoegh-Guldberg, 1997). We refer to
this as the "reduced-lipid" treatment.

Dry organic mass

Dry organic mass was measured for eggs, embryos,
and larvae of the two Heliocidaris species at various de-
velopmental stages. These measurements were used to
follow changes in biomass during development and to
make calculations on energy use. Five or six replicate de-
terminations of biomass based on 15-200 (//. erythro-
gramma) or 300-2800 (//. tubercidata) individuals per
determination were made for eggs, embryos, and larvae.
Individuals for organic-mass determination were con-
centrated by removing seawater around the embryos or
larvae. Initial samples were frozen in small amounts of
seawater, but later (and most) samples were rinsed for a
few seconds in distilled water (McEdward, 1984) prior to
being frozen (— 20°C). Frozen larvae were added to small
aluminum dishes (pre-ashed at 458°C for 6 h) and were
dried at 80°C to constant mass (which occurred between
5 and 10 d). Samples were then weighed (total dry mass)
to the nearest microgram on a mass-calibrated electroba-
lance (Cahn 25. Cahn/Ventron), and subsequently ashed
at 458°C for 6 h. Ashed samples were weighed (ash mass)
on the same balance, and dry organic mass was calcu-
lated as the difference between the total dry mass and the
ash mass, and expressed as dry organic mass per larva.

Polarographic respirometry of growing and
differentiating larvae

Polarographic respirometry was used to measure the
metabolic rate of embryos and larvae at various stages
during development and for embryos in which lipid re-
serves had been removed. Microrespirometry chambers
(100-^1 and 500-/ul) and polarographic oxygen electrodes
(Strathkelvin Instruments, Glasgow. U.K., OM780 and
SI 1 302) connected to a data acquisition and analysis sys-
tem (DATACAN IV, Sable Systems, Los Angeles, CA)
were used to measure the metabolic rate of the embryos
and larvae. Chamber temperature was 22°C and was
controlled to within ±0. 1 C° by a temperature-controlled
water bath (HAAKE 6) connected to the water jacket of
each microrespirometer cell. Respirometry chambers

contained known numbers of individuals (range 5-175,
usually 10-30 for //. erythrogramma, and range 20-
1 1 79, usually 50- 1 50 for H. tuherculata) for each mea-
sure of oxygen consumption. The highest numbers were
for eggs and cleavage stages, which had low rates of oxy-
gen consumption, and the lowest numbers were for lar-
val stages. Metabolic rate was calculated as oxygen con-
sumption in pmol O: individual ' h '. Specific meta-
bolic rates (SMR == metabolic rates standardized to
mass) were calculated by dividing respiratory rates by the
mean dry organic mass of samples of embryos or larvae
taken within ± 1 day of each reported SMR.

Calculation of energy budgets and the maternal
investment of each species in its eggs

Energy budgets for the entire development of each spe-
cies were constructed to compare energy use between the
two types of development. Energy values for biomass
and oxygen consumption were calculated with data col-
lected from two to four cultures. Biomass of eggs and
changes in biomass during development were converted
into units of energy, joules ( J ), by using the energy equiv-
alents for combustion enthalpy for each fraction (lipid =
39.5 kJ g ', protein = 24.0 kJ g~', and carbohydrate =
17.5 kJ g"'; Gnaiger, 1983). For changes in biomass, the
material laid down was assumed to have the same com-
position as that of eggs. The composition of eggs (percent
lipid, protein, and carbohydrate) of the two Heliocidaris
species was estimated from data collected for a wide
range of planktotrophic and lecithotrophic echinoderm
species (table 6 in Jaeckle, 1995). These data indicate
that the eggs of lecithotrophic and planktotrophic species
have characteristically distinct percentages of constitu-
ents. Assuming that the remainder fraction of the egg re-
sembles the composition of the measured portion, the
mean composition of the eggs of lecithotrophic species is
57.0% lipid, 40.0% protein, and 3.0%- carbohydrate and
of planktotrophic species is 28.6%> lipid, 65.6% protein
and 5.8% carbohydrate. Our use of these estimates is sup-
ported by preliminary biochemical analyses of the com-
position of eggs of //. erythrogramma: the results indi-
cate that larvae with a normal complement of blastocoe-
lic lipid consist of 58% lipid, 39%. protein, and 3%
carbohydrate (A. Moran. unpubl. data). The composi-
tion of gonadal tissue from H. tiibereidata and H. eryth-
rogramma (Lawrence and Byrne, 1994) also supports
these values estimated from Jaeckle ( 1995). The respec-
tive percentages of lipid, protein, and carbohydrate in tis-
sues of the ripe ovaries were as follows: H. erythro-
gramma— 50%, 38%., and 8%; //. tnherculata—21%,
54%., and 12%. (table 3 in Lawrence and Byrne, 1994).

For each species, total metabolic expenditure for de-
velopment was calculated from the total amount of oxy-
gen taken up over the entire course of development. To-

30

O. HOEGH-GULDBERG AND R. B. EMLET

tal oxygen consumption was determined by integrating
3rd or 4th order polynomial equations fitted to respirom-
etry data (instantaneous metabolic rates) with a curve-
fitting program (Solver. Microsoft, USA r > 0.95). The
total oxygen was converted into energy units. J. by as-
suming that the substrate being combusted was a mix-
ture oflipid, protein, and carbohydrate and by using the
oxyenthalpic equivalent for this mixture (480 kJ/
mol O2) determined from Gnaiger (1983).

The maternal investment of energy into the eggs of the
two species was compared using calculations of the en-
ergy contained within the eggs and the energy required
for complete larval development (see above). Some cal-
culations of the energy content of the eggs of H. erythro-
gramma also took into consideration the fact that about
half the egg (Table I) consists of a blastocoelic lipid-rich
component that is used primarily for juvenile develop-
ment (Emlet and Hoegh-Guldberg, 1997). In this case,
egg energy content was calculated by excluding the pro-
portion of the egg mass removed by centrifugation of
blastulae and assuming that the rest of the egg has the
same lipid. protein, and carbohydrate fractions as the
original egg. This last assumption was made because the
biochemical composition of the reduced-lipid embryos
was not measured and will cause an overestimate of en-
ergy content of reduced-lipid embryos by as much as
15% if centrifugation produced embryos with a compo-
sition similar to that of planktotrophic eggs.

Results

Changes in dry organic mass

He/iocidaris tuberculata. Three cohorts of larvae were
raised through metamorphosis with the first individuals
metamorphosing on days 21.29, and 30 for the separate
cultures. Dry organic mass was measured for these co-
horts and for others not raised through metamorphosis.
The eggs from two cohorts of Heliocidaris tuberculata
had estimated dry organic masses of 0. 1 2 ± 0.0 1 /ug egg" '
and 0.22 ± 0.02 /ug egg ' (mean ± 1 SEM), respectively.
The egg masses of these cohorts are significantly different
(Mest: T = 4.82, df = 1 0, P = 0.00 1 ), but the higher value
for one cohort may be in error because it was based on
samples frozen in small amounts of seawater and these
may have absorbed moisture upon initial weighing. The
value of 0. 1 2 /ug egg' ' falls on the regression line of egg
volume and dry organic weight for eggs of echinoderm
species with lecithotrophic and planktotrophic develop-
ment (fig. 1 in Jaeckle, 1995) and is used for calculations
throughout this study.

Larval masses increased exponentially during feeding
larval development. Competent larvae, recognized by well-
developed juvenile rudiments including definitive spines
and shortened larval arms, had masses of 4. 18 ±
0.93 /ug larva"1 to 5.97 ± 0.34 ^g larva"1 (mean ± 1 SEM),

representing a 35- to 50-fold increase from a 0. 1 2 /ug egg '
over the 2 1-30-day larval period (Fig. 1 A). The calculated
change in dry organic weight ranged between 4.06 and
5.86 ug larva"1. Two-day-old juveniles of two cohorts had
dry organic masses of 5.26 ± 0.24 /ug juvenile"' (mean ±
1 SEM) and 7.49 ± 0.39 ,ug juvenile ', respectively.

Heliocidaris erythrogramma. The dry organic mass of
the eggs of Heliocidaris erythrogramma differed signifi-
cantly among seven cultures (ANOVA, F = 8.83, df = 6,
P < 0.001) and ranged from 11.59 ± 0.93 Mg egg"' to
18.97 ± 1.03 /ugegg"' (mean ± 1 SEM). Ovoid eggs had
equivalent, mean spherical diameters of 369 to 418 /um
for six cultures. The dry organic mass of embryos of H.
erythrogramma from which the blastocoelic contents
had been removed by centrifugation was measured to es-
timate the percentage of the egg mass that was contrib-
uted to the blastocoelic contents (Table I A). The mean
percentage that was blastocoelic contents was 52.2 ±
5.1% (mean ± 1 SEM) and ranged from 39.8 to 64.7% ( =
mean ±95% CD.

The mean dry organic masses of H. erythrogramma
remained unchanged in one culture, increased in an-
other, and decreased in two others after 4 days of larval
development (Fig. IB). Three of these cohorts showed
nonsignificant changes in mass, and one showed a sig-
nificant drop over this interval (/-tests. Table IB). Larvae
of//, erythrogramma from which the blastocoelic lipid
had been removed developed normally to the juvenile
stage in the same time (3.5-4 d) as control echinoids (see
also Emlet and Hoegh-Guldberg, 1997).

At metamorphosis, control juveniles of //. erythro-
gramnia had substantially greater organic mass than re-
duced-lipid juveniles. One-day-old control juveniles
from four cohorts had mean dry organic masses of 1 2.4,
16.5, 18.9, and 19.4 /ug individual"' (n = 3 to 6 replicate
samples per cohort). Sibling reduced-lipid juveniles from
the same cohorts had mean dry organic masses of 4.4,
7.2, 10.2. and 13.8 /ug individual"1, respectively (/; = 2 to
5 replicate samples per cohort). These values for re-
duced-lipid juveniles range from 0.8 to 1 .8 times the dry
organic mass of juvenile H. tuberculata. Because meta-
bolic studies were conducted on cohorts of H. erythro-
gramma from the upper part of the egg-mass distribu-
tion, even reduced-lipid juveniles from these cohorts had
masses greater than those of//, tuberculata. These values
indicate that juveniles of the two species differ in mass
and energy content even after blastocoelic lipid materials
are taken into account. See Emlet and Hoegh-Guldberg
(1997) for comparisons of juvenile size and growth be-
tween the control and reduced-lipid treatments of H.
erythrogramma and between the congeners.

Metabolic rates during development

Heliocidaris tubcrculala. The metabolic rate of//. 111-
herculata increased from near zero (1.99 to

ENERGY USE BY SEA URCHIN LARVAE

31

A. Heliocidaris tuberculata

7 --

VI

et ~ 6 --

1 S 4

55 ••« -t

OJD "O

i«3

^52
Q

1

0

U

5 10 15 20 25 30
Age (d)

B. //. erythrogramma

8 10

Age (d)

Figure 1 . Changes in dry organic mass as a function of time for the embryos and larvae of the plankto-
trophic echinoid. Hc/inciilur^ luhcmilala (A), and the lecithotrophic echinoid, // erylhmgramma (B).
Shown are means ± 1 SEM (n = 5-6 replicate weight determinations at a given age), with different symbol
types representing cultures derived from separate parental pairs. Drawings indicate the morphology of
larvae at various stages during development (adapted from Emlet, 1995). C. competent larval stages; J.
juveniles. The arrow indicates the approximate age at which H. erythrogramma reaches metamorphic
competence.

4.75 pmol O: larva ' h ') to metabolic rates that ranged
between 200 and 500 pmol O: larva"1 h"1 (Fig. 2A).
Metabolic rates generally followed changes in biomass.
However, specific metabolic rates (SMR) indicated that
this was not strictly so. Three distinct phases could be
identified (Fig. 3A): (1) An initial increase in specific
metabolic rate over the first 2 days; (2) a relatively stable
phase from days 2 to 10 in which the SMR ranged be-
tween 150 and 250 pmol O2 jtg~' h '; and (3) a final
phase (days 10-22) in which the SMR dropped to be-
tween 50 and 1 50 pmol O: /ug~ ' h~'.

Heliocidaris erythrogramma. The metabolic rate of//.
erythrogramma showed some dramatic changes during
development. Metabolic rates increased steadily from 1 7
to 27 pmol O: larva"' h"1 just after fertilization to ap-
proximately 600-800 pmol O2 larva" ' h" ' at 36 h (22°C,
Fig. 2B). A transient spike in the metabolic rate was seen
in some cultures between 25 and 31 h after fertilization
(corresponding to late gastrulation, early vestibule for-
mation). This spike was not seen in all cultures, however.
Metabolic rates ranged from 600 to 1000 pmol O2
larva"1 h"1 from 1.5 days after fertilization until meta-
morphosis and then declined with time after metamor-
phosis (Fig. 2B). Two days after metamorphosis, juve-
niles (two cohorts), for example, had metabolic rates that
ranged between 200 and 400 pmol O: larva ' h '
(Fig. 2B).

Specific metabolic rates of H. erythrogramma larvae
were lower than those of//, tuberculata for most of their
development (cf. SMR ranges, //. erythrogramma: 0-
100 pmol O2Mg~' h"'. Fig. 4; and H. tuberculata: 0-
300 pmol O2Mg~' h"1. Fig. 3A). However, if SMRs are
calculated using dry organic masses from which the blas-
tocoelic lipid fraction has been removed, SMR values
showed a greater resemblance to those of//, tuberculata
(Figs. 3A and B, 5). The metabolic rates of embryos with
and without blastocoelic lipid were measured in order
to investigate the metabolic activity of the blastocoelic
fraction of H. erythrogramma embryos. Removal of
lipid rich contents from embryos in five separate cultures
revealed that larval metabolic rates were not different be-
tween uncentrifuged (control) and reduced-lipid em-
bryos (Fig. 6, Nested ANOVA. testing for effects of re-
moving lipid contents F^2x = 0.25, P = 0.94).

Energy budgets, maternal investment, and the energy
required to produce a juvenile

Energy budgets were constructed to provide precise
measurement of the energy required for development in
both species. Calculated totals were then used together
with the energetic contents of eggs to provide estimates
of the proportion of energy for development that was
provided as maternal investment in the egg.

32

O. HOEGH-GULDBERG AND R. B. EMLET

Table I

Drv organic mass (micrograms per individual) lor Heliocidaris
erythrogramma

A. Embryos (24-30 h) with (Control) and without (Reduced IJpid)
blastocoelic lipid from 7 independent cultures

Control Reduced lipid

embryos embryos

Blastocoelic
Culture ID (//*) Mean 1 SEM Mean I SEM Contents (%)t

HE013(5.6)

17.7

1.4

10.5

1.3

40.8

HEOI6(6,6)

18.6

1.4

11.9

I.I

35.9

HE017(6,6)

18.9

0.8

10.2

0.8

46.1

HE94/3(4.5)

12.4

0.5

4.4

0.5

64.3

H £94/4(5. 5)

14.4

2.3

4.4

0.8

69.1

HE94/5(6,6)

12.7

0.7

4.4

0.3

65.0

H £94/6(5, 5)

18.0

2.5

10.0

1.6

44.4

Mean

16.1

8.0

52.2

SEM

I.I

1.3

5.1

Min(95%CI)

13.4

4.9

39.8

Max(95%CI)

18.7

1 1.1

64.7

B. Eggs and competent larvae from cultures shown in Figure I B

Competent
Eggs larvae Mests(dflO)

Culture ID («*) Mean I SEM Mean 1 SEM

T. prob.

HEOIO(6,6)

15.10

0.52

14.61

0.47

0.70, P = 0.50

HEOI3(6.6)

16.35

0.42

18.96

1.21

2.03, P= 0.07

HEOI7(6,6)

18.97

1.03

18.62

0.80

0.82, />= 0.94

HE94/4(6,6)

12.59

0.64

10.85

0.25

2.53. P = 0.03

* The number of replicate samples tor each treatment.
t The blastocoelic contents (%) is the percent of the original mass
removed by centrifugation of blastulae.

Heliocidaris tuberculata. During the course of devel-
opment H. tuberculata accumulated biomass that repre-
sented between 114 and 164 mJ individual" ' (Table
IIA). Between 1 08.9 and 1 24.6 nmol of oxygen or 52 and
60 mJ individual ' was utilized in routine metabolism
during this same period (Table IIB). Excluding maternal
investment in the egg, the energy for development is
approximately equal to the sum of these two compo-
nents and ranged between 174 mJ individual ' and
216 mJ individual"1 ii one includes the energy invested
into the egg, the energy tor development is slightly higher
and is equal to 17 20 mJ individual" '. On a per
weight basis, the energy for producing a juvenile of H.
liibercitlata ranges from 3 2 mJ ^g~'.

The proportion of energy for producing a juvenile that
comes from maternal investment in the egg can be cal-
culated by dividing the energy invested into an egg by the
total energy for development. Values calculated in this
way yielded estimates of maternal investment by //. lu-
berculata of less than 2% of the total energy required to
produce a juvenile ( 1 .3% and 1 .6% for two cohorts).

Heliocidaris erythrogramma. For three of four cul-
tures, changes in the biomass were not significant over
the 3.5-4 days it took //. erythrogramma to develop into
a juvenile (Table IB). Based on the integrated metabolic
rate of//, erythrogramma over the time course of devel-
opment, the expected decline in biomass (total energy
burned in respiration divided by 27 kJ g~', the energy re-
leased per gram of a mixture of protein, carbohydrate,
and lipid aerobically combusted; Gnaiger, 1983) was be-
tween 0.9 and 1.3 ^g individual"1. This is below the
precision of the method used to measure dry organic
weight.

//. erythrogramma utilized 55.2 to 74. 1 nmol O: indi-
vidual ' (range of means from four cultures) to develop
into a juvenile. In energetic equivalents this is 26 to
35 mJ individual ' (Table IIIB). The energy required for
H. erythrogramma to develop into a juvenile (excluding
maternal investment in the egg) was therefore 29 ±
6 mJ individual ' (mean ± 95% CI; Table IIIC). If the
maternal investment in the egg was added (including
that used for juvenile development), the total energy for
//. erythrogramma to develop into a juvenile was 571 ±
87 mJ individual"1 (mean ± 95% CI; Table HID). If one
takes into account that about 52.2% of the //. erythro-
gramma egg is used for juvenile development and is not
an investment in embryonic and larval development
(Emlet and Hoegh-Guldberg, 1997). the energy for de-
velopment is 325 ± 68 mJ individual" ' (mean ± 95% CI;
Table HID).

The energy for producing a juvenile was estimated by
dividing the total energy for development by the mass of
juvenile produced. The estimated energy for develop-
ment per microgram of juvenile body produced ranged
between 34.1 and 34.8 mJ /ug^1 across the four cohorts
examined. The mass-specific energy for producing a ju-
venile of//, erythrogramma was similar even when the
blastocoelic contents were excluded from the calculation
of both the total energy required for development and
the total energy supplied to the egg (range 35.4-
36.3 mJMg~', Table HIE).

Maternal investment including the lipid-rich blasto-
coelic component was 94.8% ± 1.3% (mean ± 95% con-
fidence interval, n = 4) of the total energy required to
make a juvenile. Maternal investment by //. erythro-
gramma excluding this blastocoelic component was
90.8% ± 1 .6%. In either case this was many times greater
than the maternal investment by H. tiibercidata (<2%).

Discussion

Despite the active discussion for more than 50 years
on larval life-history patterns of marine invertebrates
(e.g.. Thorson, 1950; Crisp, 1976; Strathmann, 1985:
Havenhand, 1995), precise estimates of the energetics of
development are lacking for invertebrates with differing

ENERGY USE BY SEA URCHIN LARVAE

33

A. Heliocidaris tuberculata

B. H. erythrogramma

a -
o ' *

£ 5

3 .

O •-
y ^

B =

600 r

500

400

300

M "5 200
*^> g

O 3
100

Competence

•:|

mjo •?
!f T

0

10 15 20 25
Age (d)

01

Age (d)

Figure 2. The oxygen consumption of embryos and larvae of the planktotrophic echinoid. Heliocidaris
tiihcrailata (A), and the lecithotrophic echinoid, H erythrogramma (B). Symbols represent individual
respirometry measurements (a chamber of larvae at a given stage) with different symbol types representing
individual cultures derived from separate parental pairs. Arrows indicate the approximate ages at which
metamorphic competence occurred in each species.

modes of development. This study addresses this issue
for two species of congeneric urchins, estimating the en-
ergy required for development, the energy required to
build and operate a feeding larva and that required to
transform an egg into a juvenile, and the percentage of
the total energy required for development that is invested
into the two egg types by the mother. The key observa-
tion of the present study is that although the two species
differ greatly in terms of developmental mode, metabolic
expenditures, and maternal investment, the energy re-
quired to make a juvenile is essentially the same when
scaled to juvenile mass.

Changes in metabolic rates as a function of
developmental stage

A rapid increase in the metabolic rate of both species
of sea urchins characterizes the first 2 days of develop-
ment. The egg rapidly differentiates into the cellular
components required for further development during
this period. For Heliocidaris tuberculata, this involves
the formation of the feeding apparatus required to accu-
mulate further resources over the 3-4-week period of de-
velopment. H. erythrogramma develops directly into a

juvenile sea urchin over 3.5-4 days at 22°C. In this case,
the differentiation presumably results in a minimal
swimming apparatus and the juvenile rudiment. Al-
though comparisons are complicated by the differences
in the duration of development between the two species,
some interesting trends are revealed.

The rapid rise in metabolic activity slows at the end of
the second day in both species and leads to a period in
which the metabolic activity per gram of tissue (specific
metabolic rate, SMR) remains relatively constant (Figs.
3, 4). In the case of H. tuberculata. the SMR during this
second phase ranges between 150 and 250 pmol O:
ftg~' h~'. H. erythrogramma had SMR values ranging
between 30 and 70 pmol O: ^g~' h~". Part of the differ-
ence between the SMR of the two species is due to the
presence of a large amount of blastocoelic lipid in the
larvae of//, erythrogramma. This lipid-rich store, repre-
senting about 50% of the dry organic mass of the larva,
does not appear to influence the success or timing of lar-
val development (Emlet and Hoegh-Guldberg, 1997),
and its removal does not affect the metabolic activity of
larvae (Fig. 6). The SMR of H. erythrogramma was cor-
rected for the presence of non-metabolically active lipid
by standardizing rates to masses excluding blastocoelic

34

O. HOEGH-GULDBERG AND R. B. EMLET

A. Ueliocidaris tuberculata

300 -

•

Kv fl K\ f) no

^— .

250 -

•<> o \Hf W

'js

200 - o « ^ "^

• *

7M

150- . .. ,

=L

o"

100 »- o •

*: '21

"o

0^

r i i i i i i i i i i i

s.

0 2 4 6 8 10 12 14 16 18 20 22

i-
g

B. H. erythrogramma

"o
£

150 -

. " • l^i 1 fe)

4>

• ^ v^/

1 M •

S

100 -

• • • • _
• » ^

c

• n DB«va v "v.

• is °° o' « i ••

B •*

w

50 -

a

t/3

|,l'

oJ

r

J D * , i , i , i , i

1734

Days after fertilization

Figure 3. The specific metabolic rate of Helioadurn tithmiilata (A) and //- erythrogramma (B) as a
function of age. The SMRs for // erylhrugramma were calculated by dividing larval metabolic rates by the
dry organic mass of the reduced-lipid embryos for a given culture. This "corrected" SMR assumes that not
all embryo or larval mass is metabolically active and is corroborated by data presented in Fig. 6. Symbols
represent individual respirometry measurements (a chamber of larvae at a given stage), and different sym-
bol types represent cultures derived from separate parental pairs. Drawings indicate the morphology of
larvae at various stages during development (adapted from Emlet.

contents (cf. Figs. 3B, 4). SMR values of H. erythro-
gramma larvae corrected to active metabolic tissue were
similar to the SMR of H. tuberculata over the last
lOdays of its development (cf. Ht: 50-150 pmol O:
jug'1 h"1; He: 50-100 pmol O; Mg~ ' rT1).

The slower anc nore stable growth of the feeding larva
during the last pa he development of//, tuberculata
suggests that the 1 SMR earlier in the develop-

ment of this species 250 pmol O2 ^g~' h"1 as op-

posed to 50-150 pnu ' h~'; Fig. 3) is associated

with the greater amount o >olic activity required to

form larval arms, skeleton. sociated feeding struc-

tures. Coeloms are also giv luring this period of in-

creased metabolism, but the vestibule or amniotic inva-
gination has not yet formed (Emlei. pers. obs.). In this
case, the difference between the larval structures of the
early larval stages of//, tuberculala and those seen later
in this species and in //. erythrogramma is that //. tuber-

culata does not have a significant amount of rudiment
tissue early in development. This difference implies that
metabolic rates of young //. tuberculala larvae are being
standardized to smaller masses of relatively more active
tissue and that rudiment tissues are relatively less active
metabolically. If the SMR of rudiment tissues is lower
than that of functioning larval tissues, then (in the pres-
ence of ample food or energy reserves) energetic savings
might be gained by forming rudiment tissues early in de-
velopment rather than investing in more expensive lar-
val tissues.

The SMR values reported here are at the lower end of
a large range of values published for feeding and growing
invertebrate larvae (268-893 pmol O: jug" ' h ' dry or-
ganic mass), which were reviewed by Hoegh-Guldberg
and Manahan (1995). based on numbers presented by
Crisp ( 1 976). The viability of larvae during respirometry
measurements was also investigated during experiments

ENERGY USE BY SEA URCHIN LARVA1

35

« 100 -,

BHP

//. erythrogramma

• i

a 1>a 75 -
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1,1,1 , 1

0

Days after fertilization

Figure 4. The "uncorrected" specific metabolic rate of the echinoid Heliocidaris erythrogramma stan-
dardized to the mass of the egg (including blastocoelic lipid). These uncorrected SMRs are roughly half
those reported in Fig. 3B. However, the relative positions of some measurements have shifted between
Figs. 3B and 4 because the % blastocoelic lipid varies among larvae from different cultures. Metamorphic
competence occurred at 3.5-4 days.

reported here. Larvae were always intact and swimming
normally after respirometry measurements. Ongoing
work by Moreno and Hoegh-Guldberg (unpublished)
has revealed that the problems reported by Hoegh-Guld-
berg and Manahan (1995) are probably the result of the
small chambers used in the latter experiments rather
than an inherent problem with the polarographic oxygen
technique (leaking of KC1, effect of electric fields, etc.).

Energy budgets, maternal investment, and the energy for
producing a juvenile

H tuberculata increases in size at an average rate of
between 0.20 and 0.27 ^g d~ ' over its developmental pe-
riod of 3+ weeks. During the same time, between 108.9
and 124.6 nmol of oxygen were consumed. By compari-
son, H. erythrogramma did not increase in size. In this
case, the decrease in mass was probably in the range of
1-1.5 Mg individual' ' (based on its metabolic rate) over
its short developmental period and hence was too small
to detect with the methods used here. The embryos and
larvae of//, erythrogramma also consumed about half as
much (45%-68%) oxygen as H. tiiberculata (between
55.2 and 74.1 nmol individual^ ', range of means from
four cultures of//, erythrogramma).

Although the oxygen consumption of //. erythro-
gramma was lower, its maternal investment was higher
than that of//, tiiberculata. In echinoids, the energy re-
serves of the egg are used by embryos and larvae as
sources of both nutrients and energy. H. tuberculata pro-
duces eggs that are 95 /jm in diameter and contain about
3.3 mJ of energy (assuming a mixture of lipid, protein,
and carbohydrate that is typical of planktotrophic eggs;
Jaeckle, 1995). By comparison, H. erythrogramma pro-

duces eggs that are 370 to 420 n in diameter and contain
between 490 and 6 1 9 mJ of energy (assuming typical lec-
ithotrophic egg composition; Jaeckle, 1995) or 241 and
334 mJ of energy when blastocoelic lipid is excluded.

The difference in the size of egg energy reserves be-
tween H. tuberculata and //. erythrogramma contrib-
uted to the difference in energy required to make a juve-
nile in the two species. Results from two separate cul-
tures revealed that it takes between 177 and 220 mJ of
energy to make a juvenile of//, tuberculata. These values
are both below the lower 959! confidence interval
(257 mJ) of the energy required for making a juvenile
of H. erythrogramma (mean = 325 mJ, see Table HID).
When the lipid-rich component is removed from em-
bryos of//, erylhrogramma. the resulting juveniles have
test and overall dimensions at settlement that are similar
to those of//, tuberculata (Emlet and Hoegh-Guldberg,
1997), but in some instances the dry organic masses of
these juveniles were still greater than those for juveniles
of//, tuberculata. Thus part of the absolute difference in
energy for development is reflected in different masses of
the resulting juveniles, even when blastocoelic lipids are
excluded.

If the total energy to make a juvenile is standardized
to the mass of juvenile produced, the difference between
the two species largely disappears (cf. values for H. tuber-
culata: 37, 42 mJ Mg ' and //. erythrogramma: 34, 35,
35, 34 mJ jug"1 when blastocoelic contents are included
and 35. 36, 36, 36 mJ ^g ' when they are excluded). This
result means that the energy to make a juvenile via feed-
ing larval development is essentially the same as the en-
ergy to make one directly from reserves added to the egg.
This suggests that larval acquisition of materials from the
environment to produce a juvenile may be nearly as

36

O. HOEGH-GULDBERG AND R. B. EMLET

0 6 12 18 24 30

Time after fertilization (h)

Figure 5. The specific metabolic rate of Heliocidaris tuberculata
(open symbols) and H erythrogramma (closed symbols) during early
development. The SMRs of//, erythrogramma were calculated using
masses excluding blastocoelic lipids. Symbols represent individual res-
pirometry measurements. The arrow indicates the approximate point
at which hatching occurred.

efficient as the "pre-packaging" of materials provided in
the lecithotrophic egg. Comparable calculations of en-
ergy per microgram for planktotrophic larval develop-
ment of the crown-ot-thorns seastar, Acanthaster planci
(from table 1 in Hoegh-Guldberg, 1994; measurements
done at 27°C), and for the lecithotrophic larvae of the
red abalone, Haliolis niti'M'cn\ (from tables 2 and 3 of
Jaeckle and Manahan, 1989. at 16°-17°C), yield values
of 37 mJ Mg ' and 36-38 mJ ^g ' respectively, suggest-
ing that the energy for development corrected for organic
mass is strikingly similar among echinoids, asteroids,
and gastropods. Further studies that include both plank-
totrophic and lecithotrophic comparisons are required
and are in progress to substantiate this generalization
and these studies (Moreno and Hoegh-Guldberg, unpub-
lished).

The interspecific comparisons of metabolism and en-
ergy requirements allow some measure of the effective-
ness with which feeding larvae acquire energy. Metabolic
expenditures (measured as oxygen consumed) by larvae
of//, tuberculata were roughly twice those measured for
// erythrogramma over their respective developmental
intervals. Because eggs of//, erythrogramma contain all
the necessary reserves foi -nile construction, the me-
tabolism measured can Iv msidered to be the metabolic
cost to turn these reserves into a juvenile and maintain it
(range for four cohorts = 26 to '. 5 mJ). If one assumes
that half of the metabolic expenditure of//, tuberculata
goes (ultimately) toward the same purpose, then the re-
maining half of the metabolic expenditure is used to
build and power the larval feeding apparatus and diges-
tive system to acquire these reserves. In return for this

investment in larval feeding (half of the metabolic ex-
penditure was 26 to 30 mJ individual '), the offspring
increases 35- to 50-fold in organic matter, representing
an increase in energy content of 1 14 to 164 mJ individu-
al"'. These numbers suggest that for every millijoule ex-
pended to power the feeding systems there is roughly a
return of 5 to 7 mJ ( 1 mJ used to power the feeding sys-
tems and 4 to 6 mJ that supply materials for juvenile
construction). In other words, return on the investment
is 400% to 600% over the 21- to 30-day interval! These
numbers may be high for several reasons. If our measure-
ments of metabolic rates are low, then the actual invest-
ment in feeding is higher and the relative return lower.
Our studies were conducted at high food concentrations
under laboratory conditions. High food concentrations
reduce the time for development and minimize the en-
ergy required by restricting the period over which meta-
bolic expenditures occur. The developmental period for
pluteus larvae can be extended by weeks to months on
limited food (e.g.. Paulay et ai. 1985; Fenaux el cil..
1988, 1994; Pedrotti and Fenaux. 1993). in which time
larvae are metabolically active but not gaining biomass
at maximal rates (see Strathmann, 1978b, 1987, for ex-
amples of the varying planktonic durations). Under situ-
ations of food limitation with prolonged development,
the return on the investment in the feeding apparatus
would be expected to drop.

The values for the effectiveness of the larval feeding
were based on a congeneric, interspecific study of energy
for development, and we know of no similar studies for
comparison. Similar measurements on other congeners
with differing modes of development should permit
comparisons of effectiveness of different kinds of feeding
larvae. In a recent comparison of feeding rates among 1 1

1000

Control
Reduced-lipid

013 017 94-3 94-4 94-6

Figure 6. Metabolic rates of larvae of Heliocidaris erythrogramma
with (control) and without blastocoelic lipid reserves (reduced-hpid).
Each bar represents the mean ± 1 SEM and is based on three or four
chambers oflarvae. Each pair of bars is identified by a culture ID, rep-
resenting separate parental pairs.

ENERGY USE BY SEA URCHIN LARVAE

37

Table II

Mass and energy summaries for the entire development "I
Helicocidaris luherculala constructed from ilaui collectedfrom /i
independent culiitrcs

Culture ID

HKI04

HT-CKI

A. Changes in biomass during development.

(l)Biomassofegg(Mgegg ') 0.12 0.12

(2) Biomassatend of development

(^g larva ') 5.97 4.18

Biomass change (jjglarva^1) +5.86 +4.06

(3) Energy equivalents of biomass

change (J larva ') 0.164 0.1 14

B. Metabolic expenditure

Total oxygen consumption

(pmol individual ') 108,908

(4) Energ> expenditure (J individual ') 0.052

124.571
0.060

C. Knergy for development excluding maternal investment (.) larva ')

(5 = 3 + 4)J larva ' 0.216 0.174

D. Knergy for development including maternal investment (J larva ')

(6) Egg investment (J egg ') 0.0033 0.0033

(7 = 5 + 6)Jlarva"') 0.220 0.177

(8 = 7/2)

E. Energy for development per mass of juvenile (J ng ')

0.037 0.042

See Materials and Methods for the basis by which biomass and oxy-
gen consumption were converted into units of energy.

species of echinoderm larvae, representing four classes
(and two body forms), Hart (1996) suggested that the
pluteus body plan might be "an energetically inexpen-
sive adaptation for suspension feeding in the plankton"
(Hart. 1996, p. 42). He based his suggestion on the obser-
vations that pluteus larvae (both echinoid and ophiuroid
larvae) developed and grew faster than non-pluteus lar-
vae (asteroids and holothuroids) under similar feeding
conditions, and that even though the maximal clearance
rates are lower overall for the pluteus than for non-plu-
teus forms, the pluteus larvae also had a higher maximal
clearance rate for a given number of cells in the ciliary
feeding apparatus. Measurements of the energy for de-
velopment of planktotrophic larvae of Acanthaster
planet allow a partial and heuristic comparison with the
values for larvae of the Hcliocidaris species. With the
same assumptions for A. planci as were used for plankto-
trophic // niherculata, the mean egg energy = 28.6 mJ,
the range of energy of the added biomass == 39.3-
44.3 mJ, and the range of energy expended during respi-
ration 16. 3-29. 2 mJ (calculated from table 1 of
Hoegh-Guldberg, 1994). Like the values for H. tubereii-
lata (Table II), the energy expended in respiration was
roughly half the value of the energy equivalents calcu-
lated for the biomass added during the larval period. In

the absence of a comparison with nonfeeding develop-
ment in a related seastar, it is not clear how much of the
respiratory costs to associate with juvenile formation
versus building and running the larval feeding apparatus
of the seastar larva. If we assume that these costs parti-
tion as they did for Hcliocidaris (50% to juvenile forma-
tion and 50% to operation of the feeding system), then
larvae of Acanihaster may experience about 270% to
540%. return on investment in the feeding apparatus.
This range extends lower than but overlaps the return
estimated for pluteus larvae and hence is consistent with
Hart's (1996) suggestion of difference in effectiveness of
larval body plans. Obviously the data are limited and
many assumptions are untested within this comparison.
Its greatest use. however, is demonstrating the potential
for inferences if appropriate comparisons are made.

Comparison of the amount of energy invested into the
egg with the total energy required to produce a juvenile
permits the calculation of the maternal investment in
each species. //. luherculala invests 3.3 mJ of organic en-
ergy into each egg; this amounts to less than 2% of the
total energy needs of embryonic and larval development
(177 and 220 mJ individual ', Table IIC). In compari-
son, //. erythrogramma invests between 241 and 334 mJ
into each egg; this contributes 90.8% ± 1.6% (mean ±
95% CI) of the total energy required to make a juvenile
H. erythrogramma (325 ± 68 mJ individual ', Table
HID). This study verified that a planktotrophic egg con-
tains only a small contribution to the total energy re-
quired by a developing larva, whereas the opposite is true
with a lecithotrophic egg, which makes a huge contribu-
tion to the energy for development.

Implications Jor the evolution of development and
metabolic studies

The values for energy required to produce a juvenile
imply that there are no developmentally based energetic
barriers or benefits to changes in modes of development.
The total energy for development (per individual) in the
species with planktotrophic larvae is essentially the same
as that in the species with pelagic, lecithotrophic larvae,
once juvenile body size is taken into account. Selection
for the increase in egg size, the loss of feeding function,
and the reorganization of morphogenesis do not require
increases or decreases in the overall energy required to
produce a juvenile beyond those that are considered in
life-hisi .r> theory. The additional comparison that we
offer is for the energy required for development of //.
er\'tlv> -gramma with and without the blastocoelic lipid
components. This comparison not only demonstrates
that this species invests materials that are not used in lar-
val development (see also Emlet and Hoegh-Guldberg,
1 997), but also corroborates the observation that similar
energy for development is involved when juvenile size is

38

O. HOEGH-GULDBERG AND R. B. EMLET
Table III

Muss and energy summaries lor the enlii i - 'A '/'""'"' "/ Heliocidaris erythrogramma constructed /nun data collected from four independent
cultures

Culture ID

HE007

HEOIO

HE013

(5 = 3 + 4)J larva"

C. Energy for development excluding maternal investment

0.026 0.029

0.035

See Materials and Methods for the basis by which biomass and oxygen consumption were converted into units of energy.
* Changes in biomass were not significant, see Table IB.
— , no data.

HE017

A. Changes in biomass during development

( 1 ) Egg biomass ( jjg egg ' )

16.09

15.10

16.35

18.97

(!') Est. biomass. excl. blastocoelic cont. (/igegg ')

7.37

9.0

9.68

10.22

(2) Biomass of at end of development (/ig larva ')

—

14.61

18.96

18.86

Biomass change (j<g larva ')

—

-0.49

+ 2.61

- 0. 1 1

(3) Energy equivalents of biomass change (J larva ')*

0

0

0

0

B. Metabolic expenditure

Total Oxygen consumption (pmol individual ')

55,156

62,643

74,107

59,627

(4) Energy expenditure (J individual ')

0.026

0.029

0.035

0.028

0.028

Mean

95r; CI

Lower

Upper

0.029

0.006

0.023

0.036

D. Knerg) for development including maternal investment (J larva ')

(6) Egg investment (complete egg) 0.525

0.490

0.534

0.619

(6') Egg investment (without blastocoelic contents) 0.241

0.292

0.316

0.334

(7 = 5 + 6) Energy for development (complete egg) 0.55 1

0.519

0.569

0.647

Mean

95% CI

Lower

Upper

0.571

0.087

0.485

0.658

(7' = 5 + 6') Energy tor development 0.267

0.32 1

0.351

0.362

(excluding blastocoelic contents)

Mean

95% CI

Lower

Upper

0.325

0.068

0.257

0.393

E. Energy for development per mass of juvenile (J ^g ')

(8 = 7/1 (Juvenile with blastocoelic contents 0.0343

0.0346

0.0348

0.0341

(8' = 7'/l') Juvenile without blastocoelic contents 0.0362

0.0359

0.0363

0.0354

taken into account. A bigger juvenile can be made with
increased maternal investment, but the energy for devel-
opment per individual does not change from that of a
juvenile developing from planktotrophic larvae when
corrected for juvenile size.

Though the overall energy for development per mass
of juvenile appears unchanged, there are substantial
differences in the magnitude and patterns of metabolism.
Having sufficient enerj y reserves to fuel development
appears to permit the 1 hotrophic species to rapidly
increase metabolic activit .! sustain it at a high rate
throughout development. In i imparison, the metabolic
rate of//, luberciilata, after us initial increase during
embryogenesis, only increases further as larval mass and
tissues are acquired. As stated before, this can take time
and depends on local conditions (phytoplankton con-
centrations, etc.). Although these differences are in part

attributable to a greater number of cells present earlier in
development for //. erythrogramma relative to H. tuber-
culata, these patterns indicate that greater numbers of
cells are metabolically active in H. erythrogramma soon
after gastrulation.

That lecithotrophs may contain a substantial amount
of metabolically inactive biomass is an important finding
of this study. If this material were not excluded from our
estimates of specific metabolic rate, the differences be-
tween species would be far greater. For example, the spe-
cific metabolic rates of the planktotrophic and lecitho-
trophic echinoid show roughly similar patterns and ab-
solute values when inert materials are excluded from the
calculations of specific metabolic rate. If we had ne-
glected to exclude the metabolically inert blastocoelic
components present in H. erythrogramma, however, we
would have come to quite different conclusions. In that

ENERGY USE BY SEA URCHIN LARVAE

39

case, the lower SMR of//, erythrogramma compared to
that of//, tubcrculata would have led us to conclude that
specific metabolic rates are inversely related to larval
size, as appears to be the case for a large proportion of
the kingdom Animalia(Zeuthen, 1947). The presence of
metabolically inert materials in the egg, embryos, and
larvae of invertebrates is likely to be a problem in any
comparison of species with differing egg size, especially
in cases where the measurement and removal of these
energy reserves is not as easy as for //. erythrogramma.
Two other sea urchins, Asthenosoma i/iinai (Amemiya
and Emlet, 1992) and Holopnenstes ptirpwescens (V.
Morris, pers. comm; Emlet, pers. obs.), are known to ex-
trude at least some lipid-rich material into their blasto-
coels, but this has not been reported for most other echi-
noderms with large, "yolky" eggs (e.g., Patiriella exigua,
Cerra and Byrne. 1995).

Acknowledgments

Supported by an A.R.C. small grant and a University
(of Sydney) Research Grant to O.H.-G and by NSF
grants INT-91 14655 and IBN-9396004 to R.B.E. We
thank G. Moreno, C. King, and S. Dove, and especially
P. Selvakumaraswamy for help throughout this research
effort. M. Byrne and V. Morris provided many helpful
comments during discussion. Comments by B.
Hentschel, A. Moran, R. Strathmann, and two anony-
mous reviews helped us improve the manuscript. This is
contribution number 97-01 from the Oregon Institute of
Marine Biology.

Literature Cited

Amemiya, S., and R. B. Emlet. 1992. The development and larval
form of the echinQlhurioidechmoid, Asthenosoma ijimai. revisited.
Bio/ Still 182: 15-30.

Byrne, M. 1991. Developmental diversity in the starfish genus Pati-
riella (Asteroidea: Asterinidae). Pp. 499-508 in Biology of Eeltino-
Llcnnala. T. Yanagisawa, I. Yasumasu, C. Ogura. N. Suzuki, and T.
Motokawa. eds. A. A. Balkema. Rotterdam.

Byrne, M. 1995. Changes in larval morphology in the evolution of
benthic development by Patiriella exigua (Asteroidea: Astennidae).
a comparison with the larvae of Pa Uric/ In species with planktonic
development. Biol Hull. 188:293-305

Cerra, A., and M. Byrne. 1995. Cellular events of wrinkled blastula
formation and the influence of the fertilization envelope on wrin-
kling in the seastar Patiriella exigua. Ada Zool 76: 1 55- 1 65.

Crisp, D. J. 1976. The role of the pelagic larva. Pp. 145-155 in Per-
spectives in Experimental Biology. P. Spencer Davies. ed. Perga-
mon Press. Oxford.

F.mlet. R. B. 1990. World patterns of developmental mode in echi-
noid echinoderms. Pp. 329-334 in Advances in Invertebrate Repro-
duction. \'o!5. M. Hoshi and O. Yamashita. eds. Elsevier, Amster-
dam.

Emlet, R. B. 199-1. Body form and patterns of ciliation in nonfeeding
larvae of echinoderms: functional solutions to swimming in plank-
ton? Am. Zoot. 34: 570-585.

Emlet, R. B. 1995. Larval spicules. cilia, and symmetry as remnants

of indirect development in the direct developing sea urchin Heho-
cidaris erythrogramma. Dev. Biol. 167:405-415.

Emlet, R. B., and O. Hoegh-Guldberg. 1997. Effects of egg size on
post-larval performance: experimental evidence from a sea urchin.
Evolution 51 (in press).

Emlet, R. B., I.. R. McEdward, and R. R. Strathmann. 1987. Echino-
derm larval ecology viewed from the egg. Pp. 55-136 in Eckino-
derm Studies. I'ol. 2. M. Jangoux and J. M. Lawrence, eds. A. A.
Balkema, Rotterdam.

Fenaux, I.., C. Cellario, and F. Rassoulzadegan. 1988. Sensitivity of
different morphological stages ol the larva of Paracenlrolus livulus
(Lamarck) to quantity and quality of food. Pp. 259-266 in Eckino-
derm Biology. Proc fiih Inln'l Echmoderm Con/.. I'iclor/ti. R. D.
Burke. P. V. Mladenov. P. Lambert, and R. L. Parsley, eds. Bal-
kema, Rotterdam.

Fenaux, L., M. F. Strathmann, and R. R. Strathmann. 1994. Five
tests of food-limited growth of larvae in coastal waters by compari-
sons of rates of development and form of echinoplutei. Limnol
Oceanogr. 39: 84-98.

Gnaiger, E. 1983. Calculation of energetic and biochemical equiva-
lents of respiratory oxygen consumption. Pp. 337-345 in Polaro-
graphic Oxygen Sensors. Aquatic and Physiological Applications, E.
Gnaiger and H. Forstner, eds. Springer-Verlag. Berlin.

Hart, M. F. 1996. Variation in suspension feeding rates among larvae
of some temperate, eastern Pacific echinoderms. Invertebr. Biol.
115:30-45.

Havenhand, J. N. 1993. Egg to juvenile period, generation time, and
the evolution of larval type in marine invertebrates. Mar. Ecol.
Prog SIT. 97: 247-260.

Havenhand, J. N. 1995. Evolutionary ecology of larval types. Pp. 79-
122 in Marine Invertebrate Larvae. L. McEdward, ed. CRC Press.
Boca Raton. FL.

Henry, J. J., G. A. Wray, and R. A. Raff. 1991. Mechanism of an
alternate type of echinoderm blastula formation: the wrinkled blas-
tula of the sea urchin Heliocidaris erylhrogramma. Dev (Jrowih
Differ. 33:317-328.

Hoegh-Guldberg, O. 1994. Uptake of dissolved organic matter by lar-
val stage of the crown-of-thorns starfish Acanthaster planci. Mar.
Biol. 120:55-63.

Hoegh-Guldberg, O., and D. T. Manahan. 1995. Coulometric mea-
surement of oxygen consumption during development of marine
invertebrate embryos and larvae. J Exp. Biol. 198: 19-30.

Jaeckle, \V. B. 1995. Variation in the size, energy content, and com-
position of invertebrate eggs: correlates to the mode of larval devel-
opment. Pp. 49-77 in Marine Invertebrate Larvae. L. McEdward.
ed. CRC Press, Boca Raton. FL.

Jaeckle, \V. B., and I). I. Manahan. 1989. Growth and energy im-
balance during the development of a lecithotrophic molluscan larva
(Halioli\ rii/excen^i Biol. Bull. 177:237-246.

Janies, D. A., and I . R. McEdward. 1993. Highly derived coelomic
and water-vascular morphogenesis in a starfish with pelagic direct
development Biol. Bull 185: 56-76.

Laegdsgaard, 1'., M. Byrne, and D. T.Anderson. 1991. Reproduction
of sympatric populations of Heliocidaris erythrogramma and //
lithen ill /(lichinoidea) in New South Wales. Mar. Biol. 110:359-
374.

Lawrence. J. M., and M. Byrne. 1994. Allocation to body compo-
nents in Heliocidaris erythrogramma and Heliocidaris tiiherculaia
(Echinodermata: Echinoidea). Zoo/. Sci. 11: 133-137.

Lawrence, J. M., J. B. McClintock, and A. Guille. 1984. Organic
level and caloric content of eggs of brooding asteroids and an echi-
noid (Echinodermata) from Kerguelen (South Indian Ocean). In!
./ Inverlehr. Reprod. Dev. 7: 249-257.

Martindale, M. Q., and J. Q. Henry. 1995. Modifications of cell fate
specification in equa-cleaving nemertean embryos: alternate pat-
terns of spiralian development. Development 121: 3175-3185.

40

O. HOEGH-GULDBERG AND R. B. EMLET

McClinlock, J. B., and J.S. Pearse. 1986. Organic and energetic
content of eggs and juveniles of" Antarctic echinoids and asteroids
with lecithotrophic development. < I'/n/'- Bioehem. Physiol. 85A:
341-345.

McEdward, L. R. 1984. Morphomctnc and metabolic analysis of the
growth and form of an echinoplmeus. ./ £.Y/>. Mar. Bioi. EcoL 82:
359-287.

McEdward, L. R. 1992. Morphology and development of a unique
type of pelagic larva in the starfish Pleraster tesselatus (Echino-
dermata: Asteroidea). Bid. Bull. 182: 177-187.

McMillan, \V. O., R. A. Raff, and S. R. Palumbi. 1992. Population
genetic consequences of developmental evolution in sea urchins
(Genus Heliocidaris). Evolution 46: 1 299- 1312.

Morris, V. B. 1995. Apluleal development of the sea urchin Holop-
neiixtc.i purpui rscms Agassiz (Echinodermata: Echinoidea:Euechi-
noidea). Zoo/. J. Linn. Sue. 1 14: 349-364.

Olson, R. R., J. L. Cameron, and C. M. Young. 1993. Larval devel-
opment (with observations on spawning) of the pencil urchin Phyl-
laeanlhux imperialis: a new intermediate larval form? Bioi Bull.
185: 77-85.

Paulay, G., L. Boring, and R. R. Strathmann. 1985. Food limited
growth and development of larvae: experiments with natural sea
water./ Exp Mar. Bioi. Ken/. 93: 1-10.

Pedrotti, M. L., and L. Fenaux. 1993. Effects of food diet on the sur-
vival, development and growth rates of two cultured echinoplutei
(Paracentrotus livulux and Arhaeia lixuhi). Invcnchr. Reproil. Dcv
24: 59-70.

Raff. R. A. 1987. Constraint, flexibilitv. and phylogenetic history in
the evolution of direct development in sea urchins. Dev. Bioi. 1 19:
6-19.

Roughgarden, J. 1989. The evolution of marine life cycles. Pp. 270-
300 in Mathematical Evolutionary Theory. M. W. Feldman, ed.
Princeton Univ. Press.

Schatt, P., and J. P. Feral. 1996. Completely direct development of
Ahalu.i cordatus, a brooding schizasterid (Echinodermata: Echi-
noidea) from Kerguelen, with a description of perigastrulation, a
hypothetical new mode ofgastrulation. Binl. Bull. 190: 24-44.

Smith, M. J., J. D. G. Boom, and R. A. Raff. 1990. Single-copy DNA
distance between two congeneric sea urchin species exhibiting radi-
cally different modes of development. Mo/ Bioi. Evol 7: 315-326.

Strathmann, M. F. 1987. Reproduction and Development ol Marine
Invertebrates of the Northern Coast. Univ. Washington Press. Seat-
tle.

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 in echinoderms
with feeding larvae from the northeast Pacific. J. E.\p. Mar. Bioi.
Eeol 34: 23-27.

Strathmann, R. R. 1985. Feeding and nonfeeding larval development
and life-history evolution in marine invertebrates. Ann. Rev Eeol.
Syxl. 16: 339-361.

Strathmann. R. R. 1993. Hypotheses on the origins of marine larvae.
Ann. Re\: Eeol. Sy.\t. 24: 89-1 17.

Thorson, G. 1950. Reproductive and larval ecology of marine bottom
invertebrates. Bioi. Rev 25: 1-45.

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

Williams, D. II. C., and D. T. Anderson. 1975. The reproductive sys-
tem, embryonic development, larval development and metamor-
phosis of the sea urchin Heliocidaris erylhro.vramma (Val.) (Echi-
noidea: Echinometridae). Aust. J. Zoo/. 23: 37 1-403.

Wray, G. A. 1995. Evolution of larvae and developmental modes.
Pp. 413-447 in Marine Invertebrate Larvae. L. McEdward. ed.
CRC Press. Boca Raton. FL.

Wray, G. A., and A. E. Bel). 1994. The evolution of echinoderm de-
velopment is driven by several distinct factors. Dcv Suppl. 1994:
97-106.

Wray, G. A., and R. A. Raff. 1989. Evolutionary modification of cell
lineage in the direct-developing sea urchin. Ilcliociiluri* ervlhro-
xrummu. Dcv. Bioi 132:458-470.

Wray, G. A., and R. A. Raff. 1990. Novel origins of lineage founder
cells in the direct-developing sea urchin Heliocidaris ervthro-
grammu. Dcv Bioi 141:41-54.

Wray, G. A., and R. A. Raff. 1991. Rapid evolution ofgastrulation
mechanisms in a sea urchin with lecithotrophic larvae. Evolution
45: 1741-1750.

/euthen, E. 1947. Body size and metabolic rate in the animal king-
dom with special regard to the marine microfauna. C R Trciv.
Carkbcrg. Ser. Chun 26: 17-161.

Reference: liiol Hull 192: 4 1-52. (February, 1447)

Stages of Larval Development and Stem Cell

Population Changes During Metamorphosis

of a Hydrozoan Planula

VICKI J. MARTIN AND WILLIAM E. ARCHER
Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556

Abstract. Scanning electron microscopy and light his-
tology were used to reveal the changes in overall mor-
phology and in stem cell differentiation and distribution
that occur as a free-swimming, solid hydrozoan planula
larva is transformed into a sessile, hollow adult polyp.
Eight stages of development are described: young 10-
hour planula, mature 48-hour planula, attaching plan-
ula. disc, pawn, crown, immature polyp, and primary
adult polyp. The larval interstitial stem cell population
(interstitial cells, nematocytes, ganglion cells) undergoes
dramatic changes during metamorphosis: ( 1 ) distribu-
tion patterns change, (2) certain larval derivatives disap-
pear, (3) new types of derivatives differentiate, and (4)
migration patterns become more complex. This study is
the first to examine how a stem cell system develops in
an organism that goes from embryo to larva to adult.

Introduction

The stem cell, found in metazoans from sponges to
vertebrates, is an intriguing but little understood cell
type. Stem cells, by definition, are not terminally differ-
entiated: they have the ability to divide, not only gener-
ating more stem cells (self renewal) but also yielding a
variety of differentiated cell types. The mechanisms by
which stem cells differentiate into different phenotypes
and arrive at the appropriate location are some of the
most important questions in developmental biology and
metazoan evolution. Three major migratory stem cell
systems have been extensively studied: vertebrate neural
crest cells, vertebrate hematopoietic cells, and inverte-
brate cnidarian interstitial cells (Bode and David, 1978;
LeDouarin, 1979; Heimfeld and Bode, 1986; Martin and

Received 1 3 October 1 995; accepted 15 November 1996.

Archer, 1986; Lumsden, 1988; Bronner- Eraser and Era-
ser. 1988;PottenandLoeffler, 1990;Weston, 1991: Mar-
tin, 1991;Medvinskyf/rt/.. 1993).

The study of stem cells in evolutionarily primitive
metazoans may reveal some of the earliest mechanisms
used for setting up patterns of cell distribution and over-
all morphology. Certain marine cnidarians have charac-
teristics that make them ideal for such a study. Embryo-
genesis and metamorphosis are relatively rapid and,
more importantly, both embryos and adults contain a
population of migratory stem cells, making it possible to
compare stem cell behavior during embryogenesis with
behavior in the adult.

To date, studies on cnidarian metamorphosis are few
and virtually nothing is known about the behavior of the
interstitial cell system during metamorphosis (Martin et
a/., 1983;Berking, 1984; Thomas et a/., 1987; Weis and
Buss, 1987; Plickert el a/., 1988; Schwoerer-Bohning et
ai. 1990; Sommer, 1990). Thus it is not known how the
interstitial stem cell lineage develops in an organism that
goes from an embryo to a larva to an adult.

The embryonic interstitial cell system of the marine
hydrozoan Pennana tiarella has been characterized by
Martin and associates (Martin and Thomas, 1977, 1980,
1981a, b; Martin and Archer, 1986; Martin, 1988a,
1990, 1991 ). Embryos possess a well-defined population
of migratory interstitial stem cells that either divide to
replenish the population or differentiate into ganglion
cells or nematocytes. The adult stem cell system of this
hydro/oan has also been examined, though in less detail
(Martin, 1988b). Because embryos and adults of Penna-
! : tiarella are easy to obtain and manipulate, develop
quickly, and are small and transparent, the interstitial
stem cell system can be examined continuously starting
from the moment it arises in the embryo, progressing

41

42

V. J. MARTIN AND W. E. ARCHER

through embryogenesis and formation and metamor-
phosis of the planula larva, and continuing in the adult.
In this study we used both scanning electron micro-
scopy and light histology to examine the changes in over-
all morphology and in stem cell differentiation and dis-
tribution that occur as the free-swimming, solid planula
larva of Pennaria tiarella is transformed into the sessile,
hollow adult polyp. Our results show that the larval in-
terstitial cell system is extensively modified during meta-
morphosis to produce the adult pattern.

Materials and Methods

Culture o /'Pennaria adults and embryos

Mature colonies of Pennaria tiarella were collected
from pier pilings in Wilmington and Morehead City,
North Carolina. Fronds from male and female colonies
were mixed together in large finger bowls of filtered sea-
water; these bowls were placed in the dark at 1 800 hours,
and returned to the light at 2 100 hours. Within 1 h after
exposure to light, early cleavage stages were observed in
the bottoms of the dishes. These embryos were trans-
ferred to small dishes of filtered seawater and reared at
23°C to the desired planular stage.

Forty-eight-hour planulae were incubated in cesium
chloride to initiate metamorphosis (Archer and Thomas,
1983). A stock solution containing 9.76 g CsCl/100 ml
distilled water was mixed with Millipore-filtered seawa-
ter at a ratio of 1 (stock): 10 (seawater). Planulae were
placed in 4 ml of this mixture for 3 h; some of these ani-
mals were fixed for scanning electron microscopy (SEM)
immediately after the cesium treatment. Planulae placed
in cesium for 3 h were also returned to dishes of Milli-
pore-filtered seawater and allowed to continue their de-
velopment. Treated planulae were fixed for SEM after a
recovery period of 2 (disc stage), 6 (pawn stage), 12
(crown stage), 14 (immature polyp), or 18 (primary
polyp) h.

Scanning electron microscopy (SEM)

For SEM, 10- and 48-h control planulae. cesium-
treated planulae immediately after treatment, and ce-
sium-treated planulae allowed to recover for various
times were fixed for 1 h in 2.5% glutaraldehyde in 0.2 M
Millonig's phosphate buffer, pH 7.4. Samples were
rinsed three times in the phosphate buffer, postfixed for
1 h in 2% osmium tetroxide in 1.25% sodium bicarbon-
ate buffer, pH 7.2. then rinsed three times in the sodium
bicarbonate buffer. Animals were dehydrated through a
graded series of ethanols to 100%', critical-point dried us-
ing CO;, mounted on metal stubs, and sputter coated
with gold palladium for 1 min in a Denton sputter
coater. Samples were viewed and photographed with a

JEOL JSM T-300 scanning electron microscope oper-
ated at 25 kV.

Light microscopy

Control planulae (various ages) and stages of meta-
morphosis comparable to those processed for SEM were
fixed for 1 h in 10% formalin in seawater. Samples were
dehydrated for 15 min each through an ascending alco-
hol series (25%-100% ethanol), followed by a 20-min
rinse in 100% ethanol: 100% tertiary butyl alcohol (1:
1), and an overnight incubation in 100% tertiary butyl
alcohol. After being infiltrated and embedded in Para-
plast Plus paraffin, animals were serially sectioned at
8 ^m. The sections were mounted on glass slides and
stained with azure B, which specifically stains the inter-
stitial cells and their derivatives (Martin, 1991). Slides
were viewed and photographed using a Zeiss standard
research microscope.

Results

Planular morphology and stages of metamorphosis:
General observations

Between 8 and 10 h postfertilization the gastrula of
Pennaria tiarella elongates in an anterior-posterior di-
rection to produce a fat, ciliated, free-swimming planula
larva (Fig. 1 ). This 10-h larva has a distinct enlarged an-
terior apical pole and a narrower posterior basal pole.
The surface cells are numerous, small, and uniform in
size. By 24 h postfertilization 10-h planulae narrow and
elongate to form mature, metamorphosis-competent
planulae. Although planula larvae are competent to
metamorphose at 24 h, as shown by induction with ce-
sium chloride (Archer and Thomas, 1983), many planu-
lae swim in the water column for 2 to 3 days before at-
taching and metamorphosing. During this swimming pe-
riod the larvae continue to elongate. The 48-h hydrozoan
planula is solid, elliptical, and moves in the water col-
umn with its enlarged apical end directed forward
(Fig. 2).

During metamorphosis the solid, nonfeeding. motile
planula is transformed into a hollow, sessile, feeding
adult polyp (Figs. 3-9); this process takes about 1 8-20 h.
The apical end of the planula forms the base of the polyp,
the middle region forms the stalk, and the basal end
forms the hypostome and tentacles. As planulae meta-
morphose their morphology changes dramatically, in
distinct stages known as shortening planula (Fig. 3), disc
(Fig. 4), pawn (Fig. 5), crown (Fig. 6). immature polyp
(Fig. 7), and primary polyp (Figs. 8 and 9).

Mature planulae of Pennaria attach to substrates, usu-
ally pier pilings in the wild, via their anterior, apical
poles; shortly thereafter metamorphosis begins.

STEM CELL DEVELOPMENT

43

Figure I. Ten-hour planula. The young larva is 350 urn long and 1 70 fim wide. A, apical end; B, basal
end. Bar = 50/jm.

Figure 2. Forty-eight-hour pre-metamorphic planula. The ciliated larva moves with its enlarged apical
end (A) directed forward. It is 900 pm long, 1 .10 ^m wide in the apical region, 70 ^m wide in the mid area,
and 50 /xm wide at the basal end. B. basal. Bar = 100 ^m.

Figure 3. Attached metamorphosing planula. The original anterior, apical end (A) of the larva attaches
to the substrate and flattens over it while the original basal end (B) contracts towards the attached end.
Thus the animal becomes short and fat. measuring 320 /im long, 160 fim wide in the apex, 93 ^m wide in
the middle, and 45 urn wide in the basal region. Bar = 50 jim.

Figure 4. Disc stage. The disc is a flattened ball, 180 tim in diameter, on the substrate. Bar = 50 ^m.

Figure 5. Pawn stage. The base (B) of the pawn, now considered the posterior end of the animal, arises
from the apical end of the planula. The anterior, apical region (A) of the pawn, derived from the basal
region of the planula. forms the head and tentacles of the primary polyp. Small amounts of perisarc mate-
rial (arrow) are deposited at the base. The pawn is 350 ^m tall. 160 ^m wide in the anterior head, 73 ^m
wide in the mid-stalk, and 106 ^m wide at the posterior base. Bar = 50 ^m.

44

V. J. MARTIN AND W. E. ARCHER

Figure 6. Crown stage. A distinct head region (HE), stalk (Si. and base (Blare evident. Perisarc material
(arrows) covers the surface. The crown is 427 ^m tall, 1 25 pm wide in the apical crown region, SO ^m wide
in the stalk region, and 166 pm wide at the base. Bar = 50 ^m

Figure 7. Immature polyp. The head region consists of a conical mound, the hypostome(H), a mouth
(arrow), and a ring of forming filiform tentacles (F). A stalk (S) connects the head to the base (B). The polyp
is 340 /jm tall, 120/jni wide in the anterior hypostome region, 73 jim wide in the mid-stalk area, and
206 nm wide in the posterior base. Bar = 50 ^m.

Figures. Primary polyp. The head is composed of the hypostorne(H), short capitate tentacles (C), and
the longer filiform tentacles (F). A narrow stalk (S), covered by perisarc (arrow), extends from the head to
the base (B) of the adult. Stolons (T) emerge from the base. The primary polyp is 50(1 ^m tall. 200 ^m wide
in the crow n area, 70 fitn wide in the mid-stalk area, and 200 ^m wide in the basal area. Bar = 50 /urn.

Figure 9. Enlarged head region of a primary polyp showing hypostome (H) with capitate (C) and fili-
form (F)u-i v These tentacles are armed with nematocxtes (arrows). Bar = 50 ^m.

Shortening planulu (Fig. 3). Once attached the apical
planula end flattens and expands o\ er the substrate while
the basal end contracts down towards the expanded api-
cal pole. Thus the attached larva becomes short and fat.

Disc (Fig. 4). Within 2 h of attachment the basal end
of the larva has completely contracted and a round disc
shape is formed. The animal appears as a small, flattened
ball on the surface of the substrate: apical and basal ends

STEM CELL DEVELOPMENT

45

are not discernible. Cilia are absent and the surface is
relatively smooth.

Pawn (Fig. 5). Six h after attachment a tiny bleb ap-
pears in the center of the disc and begins to elongate in
an upright direction to form a shape that resembles the
pawn of a chess set. The formation of the pawn from
the disc stage requires 4 h. The original apical end of the
planula forms the base of the pawn and the original basal
end of the planula forms its head. The surface of the
pawn is smooth and the first beginnings of a perisarc, an
outer noncellular protective coating, are seen at the base.

Cmwn (Fig. 6). During the next 6 h the pawn elon-
gates; the anterior head widens, forming a crown; and a
sharp demarcation appears between the head and the
stalk. This crown stage is formed 12 h after attachment,
and it is during this stage that general features of the adult
polyp begin to take shape: crown (future hypostome).
stalk, base. The surface at this stage is smooth and cov-
ered with perisarc material.

Immature polyp (Fig. 7). After 2 h, 14 h after attach-
ment, an immature polyp is formed. Its head region con-
sists of a hypostome, a conical mound bearing the mouth
at its tip, and a ring of forming filiform tentacles. These
tentacles arise as tiny evaginations of the body wall at
the base of the crown and lengthen to achieve the adult
tentacle morphology. A clear division between the polyp
head and the narrowing stalk is evident. The stalk con-
nects the head to an enlarging base. The surface of the
immature polyp below the region of the head is covered
with perisarc.

Primary polyp (Figs. 8.9). Within 4 h. 18 h after at-
tachment, a primary polyp is formed. A row of long, fi-
liform tentacles and a new row of short, evaginating cap-
itate tentacles, just above the filiform tentacles, charac-
terize the crown region, constituting the fully formed
adult hypostome (Fig. 9). A mouth is present at the very
tip of the hypostome just above the whorl of capitate ten-
tacles, a perisarc covers the stalk and basal region of the
polyp, and stolon formation has begun in the basal re-
gion of the polyp (Fig. 8). These stolons produce addi-
tional polyps that remain attached to the original pri-
mary polyp, thus creating a colony.

Interstitial stem cell system

Planulae of Pcnnaria tiarel/a contain a population of
migratory stem cells, interstitial cells, that either divide
to replenish the population or differentiate into two
classes of somatic products: nematocytes (stinging cells)
or ganglion cells (neurons). Early differentiating interme-
diates of the nematocyte lineage are called nematoblasts,
and intermediates of the neural lineage are referred to
as neuroblasts. Interstitial cells and their derivatives are
easily identified in larval and adult tissue at the light mi-

«t

-• "-"" * 1 *' ''••'•' "•

<"*£•• -V<

/& -^H ^^M

•i' A*i»>" *'s

iSt;«^ali«i^A« ;

TV* w

A&, . IS^^*
".-* . -v ^ _ «

Figure 10. Interstitial cells (arrows) in the central endoderm of a
mature planula. Each cell has a lightly stained cytoplasm plus a nucleus
with a darkly stained nucleolus. M. mesoglea. Bar = 10 urn.

Figure 11. Nematoblasts with large dark capsules (D). small dark
capsules (arrows), or large clear capsules (ST) in the endoderm of a
mature planula. Bar = 10 jim.

Figure 12. Nematoblast with a bullet-shaped capsule (arrow) in the
endoderm of a mature planula. Developing desmonemes (large dark
capsules) and microbasic heterotrichous b-mastigophores (small dark
capsules) are also seen. E. ectoderm; EN. endoderm; M. mesoglea. Bar
= 10 Mm.

Figure 13. Bipolar ganglion cell (arrow) in the ectoderm of a ma-
ture planula. The endoderm lacks neurons. EN. endoderm; M, meso-
glea. Bar = 10 /jm .

croscopic level (Figs. 10-13). In the following sections we
describe the behaviors of the interstitial cells, the nema-
toblasts and nematocytes, and the neuroblasts and gan-
glion cells during embryogenesis, in the metamorphosis-
competent planula. during metamorphosis, and in the
adult polyp.

Interstitial cells

Interstitial cells are small round cells measuring 7.5
urn in diameter (Fig. 10). They contain a centrally lo-
cated nucleus with one or more darkly stained nucleoli.

46

V. J. MARTIN AND W. E. ARCHER

These cells arise during gastrulation (8-10 h postfertil-
ization), in the central core of the endoderm along the
entire length of the young planula (Table I). They divide
in the endoderm and by 1 3-14 h postfertilization begin
to emigrate to the ectoderm, migrating as single cells
through the interstitial spaces of the endoderm and
through the mesoglea. By 15 h postfertilization, the in-
terstitial cells reach the base of the ectoderm. Migration
occurs along the entire apical-basal axis of the young
planula. As planulae age, the numbers of interstitial cells
in both the ectoderm and endoderm increase, and mi-
gration from the endoderm to the ectoderm continues
along the entire planular axis. Thus the metamorphosis-
competent planula has many interstitial cells at the base
of its ectoderm and in the endoderm along its entire body
axis (Table I).

During metamorphosis, as the apical pole of the plan-
ula attaches to a substrate and the basal end contracts
towards the attached end, the interstitial cells located in
the mid to basal regions of the larva move into the ecto-
derm and endoderm of the attachment region (Figs. 14-
16; Table I). Their mechanism of movement is unknown
but probably involves active cellular migration. The ec-
toderm and the endoderm of the remaining, still con-
tracting basal portion of the attached larva become de-
void of interstitial cells (Fig. 15: Table I). Once the disc
stage is formed, interstitial cells fill both the ectoderm
and the endoderm (Table I). As the center of the disc
elongates in an upright direction, a pawn is produced
(Fig. 1 7). The growing, apical upright tissue of the pawn,
destined to form the head and stalk of the adult polyp, is
devoid of interstitial cells (Fig. 17; Table I). These cells
remain in the attached, now basal, end of the metamor-
phosing animal (Fig. 18); where the upright portion of
the pawn connects to the basal disc is a sharp demarca-
tion between presence of interstitial cells in the base and
absence of these cells above the base. The distribution
pattern of interstitial cells in the base of the pawn re-
mains unchanged from that of the disc.

By the time the crown stage has formed, the interstitial
cells have migrated out from the basal attachment site to
populate the entire body axis of the animal (Fig. 19; Ta-
ble I). In the attachment area (basal disc) a few interstitial
cells are found in both the ectoderm and the endoderm.
Along the body stalk, the region of the animal that con-
nects the basal disc to the head, are scattered ectodermal
interstitial cells and a few endodermal interstitial cells
(Table I). The head of the crown stage has interstitial cells
in both the ectoderm and the endoderm (Fig. 19; Table
I). This same distribution pattern of interstitial cells is
maintained in the immature polyp and in the adult pri-
mary polyp (Table I). In the primary polyp many inter-
stitial cells are seen at the base of each filiform tentacle,
but none are seen within the tentacles.

Nematoblasts and ncmatocytcs

Nematoblasts, immature nematocytes, range from 10
to 12.5 /um in diameter and form distinctive dark-stain-
ing or light-staining capsules (Figs. 10-12); each capsule
contains a nematocyst thread that may possess barbs and
spines. Nematoblasts are found in both the ectoderm
and the endoderm throughout embryogenesis, meta-
morphosis, and in the adult polyp. Once nematoblasts
move to the outer surface of the ectoderm or project into
a forming gastric cavity, they complete their differentia-
tion and are considered functional nematocytes. A few
nematoblasts with dark capsules are first detected in the
apical endoderm of the young 10-h planula (Table I). Mi-
gration of the nematoblasts begins by 13 h postfertiliza-
tion, and these cells are the first of the interstitial cell sys-
tem to appear in the ectoderm (by 14 h postfertilization)
of the planula. Nematoblasts in the apical endoderm mi-
grate as single cells into the apical ectoderm; they do not
divide and syncytial clusters of nematoblasts are not ob-
served at any stage of the life cycle. As planulae mature
the nematoblasts increase in number in both the ecto-
derm and the endoderm and are largely confined to the
apical two-thirds of the planular axis (Table I). At least
four types of capsules have been observed in the mature
planula (Figs. 10-12): a large clear capsule (stenoteles), a
large dark capsule (desmonemes), a small dark capsule
(microbasic heterotrichous b-mastigophores), and a met-
achromatic bullet-shaped capsule (microbasic heterotri-
chous b-mastigophores with inclusions). Stenoteles and
desmonemes predominate; only a few microbasic heter-
otrichous b-mastigophores with inclusions are seen.

Fully differentiated nematocytes are found only at the
surface of the mature planula, the majority in an area
extending from the apical end of the planula to the mid
planula (Table I); only a few nematocytes are found at
the surface in the basal (posterior) region. Fully differ-
entiated nematocytes of planulae contain either a large
clear capsule (stenoteles), a large dark capsule (desmo-
nemes) or a bullet-shaped capsule (microbasic heterotri-
chous b-mastigophores with inclusions); no fully differ-
entiated nematocytes housing the small dark capsules
(microbasic heterotrichous b-mastigophores) are found
in the planula.

As planulae attach to substrates, all nematoblasts move
into the ectoderm and endoderm of the apical attachment
region (Figs. 14 and 15). Nematocytes are confined to the
outer surface of the ectoderm of the attachment area.
Hence, the contracting basal portion of the attached larva
is devoid of nematoblasts and nematocytes in both the
ectoderm and the endoderm (Table I). All four types of
nematoblast capsules are detected in the ectoderm and en-
doderm of the attachment area. The bulk of these cells are
differentiating stenoteles, desmonemes, and microbasic

STEM CELL DEVELOPMENT 47

Table I
Distribution «/ the interstitial cell system during development ol Pennaria tiarella

Stage Interstitial cells Nematoblasts Nematocytes Ganglion cells

10-Hour Planula

Apical Ectoderm

Mid Ectoderm

Basal Ectoderm

Apical Endoderm + +

Mid Endoderm +

Basal Endoderm +
48-Hour Planula

Apical Ectoderm ++ ++ ++ ++

Mid Ectoderm ++ ++ ++ ++

Basal Ectoderm ++ + + + +

Apical Endoderm ++ + +

Mid Endoderm ++ + +

Basal Endoderm ++ +

Attaching Planula

Apical Ectoderm (Attachment site) ++ ++ +

Mid Ectoderm

Basal Ectoderm

Apical Endoderm ++ + +

Mid Endoderm

Basal Endoderm
Disc Stage

Ectoderm ++ ++ +

Endoderm ++ + +

Pawn Stage

Apical Ectoderm (Head and Stalk)

Basal Ectoderm (Foot) ++ ++ + +

Apical Endoderm (Head and Stalk)

Basal Endoderm (Foot) ++ + +

Crown Stage

Apical Ectoderm (Head) + + +

Mid Ectoderm (Stalk) + + + +

Basal Ectoderm (Foot) + ++ + + +

Apical Endoderm (Head) + +

Mid Endoderm (Stalk) + +

Basal Endoderm (Foot) + +

Immature Polyp

Apical Ectoderm (Head) + + + ++

Apical Ectoderm (Tentacle) + ++ + +

Mid Ectoderm (Stalk) + + + +

Basal Ectoderm (Foot) + ++ + + +

Apical Endoderm (Head) + +

Apical Endoderm (Tentacle)

Mid Endoderm (Stalk) + +

Basal Endoderm (Foot) + +

Primary Polyp

Apical Ectoderm (Head) + + + + +

Apical Ectoderm (Tentacle) + ++ + +

Mid Ectoderm (Stalk) + + + +

Basal Ectoderm (Foot) + ++ + + +

Apical Endoderm (Head) + +

Apical Endoderm (Tentacle)

Mid Endoderm (Stalk) + +

Basal Endoderm (Foot) + +

Table Key: ++ = Abundant to moderate in number. + = A few present. — = Absent.

48

V. J. MARTIN AND W. E. ARCHER

M

EN

M

14

•

_i ir

15

time the crown stage has formed, the interstitial cell sys-
tem has migrated from the basal attachment site to popu-
late the entire body axis of the animal. Nematoblasts are
the first of the line to appear apically (Figs. 22-24), and
distinct patterns of nematoblast and nematocyte distribu-
tion are observed. In the ectoderm of the attachment area
are all four types of nematoblasts and two types of nema-
tocytes (stenoteles and desmonemes). On either side of the
basal disc just above the substrate attachment zone, the
perisarc is connected to the basal disc and lower body col-
umn. In these regions of perisarc attachment to the ecto-
derm, the ectoderm has an abundance of nematoblasts
(desmonemes and stenoteles) (Fig. 22). Along the body
stalk of the crown in the ectoderm are the four types of

HE

EN

B

tit-

EN

* A.

.ta

16

>•* '' ' •. -'»•'

Figure 14. Apical region of an attaching planula. The interstitial
cell system has moved into the attachment area; note the large number
of dark nematoblast capsules in this region. E. ectoderm: EN, cndo-
derm; M, mesoglea. Bar = 50 urn.

Figure 15. Mid to basal region of an attaching planula. Note the
absence of the interstitial cell system in the basal portion (B) of the
animal. E. ectoderm, EN, endoderm: M, mesoglea. Bar = 50 Mm.

Figure 16. Interstitial cells (arrows) in the attachment region of a
metamorphosing planula. E, ectoderm; EN, endoderm. Bar = 10 Mm.

Figure 17. Pawn. The apical head region (HE) is devoid of the in-
terstitial cell system. These cells remain in the basal region (B) of the
pawn. Dark nematoblast capsules are abundant in the base. Bar =
50 Mm.

M

EN

heterotrichous b-mastigophores: only a few developing
microbasic heterotrichous b-mastigophores with inclu-
sions are found. Once the disc stage is reached, the four
types of nematoblasts fill the ectoderm and endoderm
(Fig. 20); a few nematocytes (stenoteles and desmonemes)
are observed around the ectodermal surface of the disc
(Table I). As the center of the disc elongates to form the
pawn, the nematoblasts and nematocytes remain in the
attachment disc (Figs. 1 7 and 2 1 ). Thus, the upright grow-
ing tissue is devoid of nematoblasts and nematocytes.
These cells are confined to the substrate-attached basal
disc region of the metamorphosing animal and resemble
the pattern described for the disc stage (Table I). By the

Figure 18. Interstitial cells (arrows) in the endoderm at the base of
the pawn. These cells are migrating as indicated by the presence of a
single hlopodial-like extension. Bar = 10 Mm.

Figure 19. Head region of the crown stage. Interstitial cells (arrows)
are detected; however, ganglion cells are absent in this area. E, ecto-
derm; EN, endoderm; M, mesoglea. Bar = 10 ^m.

Figure 20. Disc stage. Nematoblasts (arrows) are abundant in the
endoderm; ganglion cells are absent. E. ectoderm: EN, endoderm. Bar
= 10 nm.

Figure 21. Nematoblasts (arrows) in the endoderm at the base of
the pawn. Bar = 10 Mm.

STEM CELL DEVELOPMENT

49

PE

EN

24

Figure 22. Crown stage. The perisarc (PE) is attached to the ecto-
derm (E) just above the foot region of the forming polyp. This region is
rich in ganglion cells (arrows) and nematoblasts. Bar = 10 jim.

Figure 23. Stalk region of the crown stage showing multipolar gan-
glion cells (arrows) and nematoblasts. EN. endoderm. Bar = 10 ^m.

Figure 24. Stenoteles (arrows) and a nematoblast with a dark bul-
let-shaped capsule in the head region of the crown stage. E. ectoderm;
EN. endoderm. Bar = 10 ^m.

Figure 25. Head region of a primary pohp showing nematoblasts
(arrows) in a capitate tentacle (C) and a filiform tentacle (F). Bar =
10 Mm.

nematoblasts and at the surface a few nematocytes (sten-
oteles and desmonemes). The endoderm of the stalk has a
few nematoblasts (desmonemes and Stenoteles) (Fig. 23).
In the head of the crown in both the ectoderm and the
endoderm are three types of nematoblasts: desmonemes,
Stenoteles, and microbasic heterotrichous b-mastigo-
phores with inclusions (Fig. 24). Three kinds of nemato-
cytes, the same varieties as the head nematoblasts, project
from the ectodermal surface of the head. Prior to the
crown stage, the nematoblasts with bullet-shaped capsules
(microbasic heterotrichous b-mastigophores with inclu-
sions) are seen sparingly along the whole body axis of met-
amorphosing animals; however, by the crown stage many
nematoblasts of this type have accumulated in the head.

As the crown stage transforms into the immature
polyp, the ectoderm of the forming filiform tentacles be-
comes filled with three types of nematoblasts and nema-
tocytes: Stenoteles, desmonemes, and microbasic hetero-
trichous b-mastigophores with inclusions. Other than
this change, the distribution pattern of the nematoblasts
and nematocytes is the same as seen in the crown stage.

As immature polyps form primary adult polyps, a sec-
ond group of short tentacles, the capitate tentacles, ap-
pears just above the whorl of filiform tentacles (Fig. 25).
These capitate tentacles are populated with the same
three types of nematoblasts and nematocytes that oc-
cupy the filiform tentacles (Fig. 25). Along the body col-
umn mature nematocytes with small dark capsules (mi-
crobasic heterotrichous b-mastigophores) are visible.
Other than these changes, the nematoblast and nemato-
cyte system of the primary polyp resembles that of the
immature polyp. Thus in the primary polyp the concen-
tration of nematoblasts and nematocytes is high in the
head and in the foot and scattered in the stalk. The dis-
tribution pattern of nematocytes is specific: in the head
are Stenoteles, desmonemes, and microbasic heterotri-
chous b-mastigophores with inclusions; along the body
column are Stenoteles, desmonemes, and microbasic
heterotrichous b-mastigophores; and in the foot are des-
monemes and Stenoteles.

Neuroblasts and ganglion cells

Differentiating neuroblasts are detected as early as 16-
20 h postfertilization in both the ectoderm and endo-
derm of the planula(Brumwell and Martin, 1996). These
first neuroblasts arise in the apical region of the larva;
shortly thereafter they are found along the entire length
of the planula. Neuroblasts are small round cells, similar
in size to interstitial cells, that contain cytoplasm rich
in neurosecretory vesicles (Brumwell and Martin, 1996).
These differentiating intermediates migrate as single cells
from the endoderm to the base of the ectoderm; neuro-
blasts are positioned closer to the mesoglea than are the
interstitial cells of the ectoderm. Neuroblasts seemingly
emigrate in a straight path from the endoderm to the ec-
toderm; there is no evidence that they migrate in an api-
cal or basal direction in the larva. Once neuroblasts reach
the basal ectoderm they stop moving and complete their
differentiation by extending neural processes. These pro-
cesses are filled with neural vesicles and form an exten-
sive neural plexus of transversely and longitudinally ori-
ented processes throughout the length of the planula. As
the planula ages additional ganglion cells differentiate
and incorporate into the larval network. Fully differen-
tiated larval ganglion cells are 5 ^m in diameter, bipolar,
spindle-shaped, and positioned in the ectoderm just
above the mesoglea along the entire apical, basal axis of
the planula (Fig. 13; Table I).

50

V. J. MARTIN AND W. E. ARCHER

* T

Figure 26. Neurons (arrows) and ncmatohlasls with dark capsules
in the hasal disc of the crown stage. E, ectoderm; EN, endoderm; M.
mesoglea. Bar = 10 ^m.

Figure 27. Ganglion cells (arrows) in the head region of a primary
polyp. Bar = 10 jim.

Figure 28. Filiform tentacle of a primary polyp. Note the abun-
dance of neurons (arrows) at the base of the tentacle and along its
length. Bar = 10 ^m.

Figure 29. Stalk of a primary polyp. Note the ganglion cells (ar-
rows) in the ectoderm. PE, perisarc. Bar = 10 ^m.

During attachment and the early stages of metamor-
phosis, the larval ganglion cells disappear, by the disc
stage they are gone (Table I). In the pawn a few ganglion
cells differentiate in the basal disc (Table I). These neu-
rons are found in the ectoderm just above the mesoglea:
they are triangular or star-shaped and are multipolar.
These neurons have smaller cell bodies and thinner pro-
cesses than did the planular ganglion cells. By the crown
stage ganglion cells are found in the ectoderm of the basal
disc and the body stalk (Table I). The basal disc contains
a large number of ectodermal bipolar and multipolar
ganglion cells (Fig. 26), and multipolar ganglion cells are
abundant in the region of perisarc attachment to the
basal disc and lower body column (Fig. 22). Along the
body stalk in the ectoderm are at least three types of gan-

glion cells: triangular-, star- or spindle-shaped (Fig. 23).
Ganglion cells are not detected in the head of the crown
stage. As the crown stage develops into the immature
polyp, many ganglion cells (bipolar, multipolar) appear
in the head ectoderm and tentacles (Table I). By the pri-
mary polyp stage, the head of the animal is enriched in
ganglion cells, especially around the mouth (Fig. 27).
Ganglion cells are found at the base of each filiform ten-
tacle and in the ectoderm along the lengths of the tenta-
cles (Fig. 28). In the ectoderm of the body stalk are scat-
tered ganglion cells (Fig. 29), and the distribution pattern
in the basal disc mimics that of the immature polyp (Ta-
ble I). Thus in the primary polyp there is a high concen-
tration of ganglion cells in the ectoderm of the head and
basal disc and some scattered ganglion cells in the ecto-
derm of the body column (Table I). The ganglion cells of
the polyp are three types: spindle-shaped bipolar, star-
shaped multipolar, and triangular-shaped multipolar.

Discussion

The transformation of the cnidarian planula larva into
the adult phenotype is rapid, taking only 18-20 h in the
hydrozoan Pcnnuria tiarella, and is characterized by
general body reorganization and modification of the
stem cell system. During metamorphosis the hydrozoan
planula ceases swimming, loses its cilia, and attaches to
the substrate by its apical (aboral) pole. Both glandular
secretions and nematocytes may be used for securing
planulae to a substrate (Martin el ai, 1983). Shortly after
attachment the basal (oral) end of the larva contracts
down towards the apical pole until it disappears into the
attached pole. A tiny circular disc is formed. Next, a tiny
bleb appears in the center of the disc and begins to elon-
gate in an upright direction forming a pawn shape. Three
distinct regions of the pawn are evident: an apical head,
a mid-stalk region, and a basal disc. The pawn grows and
reshapes to produce a crown stage, in which general fea-
tures of the adult polyp begin to take shape: head, stalk,
and base, with a clear separation between the head and
the stalk. Tentacles evaginate from the head region and
a mouth breaks through at the tip of the head, producing
an immature polyp. This stage becomes a primary polyp
when a row of long filiform tentacles and a row of short
capitate tentacles adorn the head. A mouth is present at
the very tip of the head just above the whorl of capitate
tentacles, and a perisarc covers the stalk and basal region
of the polyp. To form a colony, the polyp extends stolons
from the base and asexually buds additional polyps, all
of which remain connected together.

Four major changes occur in the interstitial cell system
during metamorphosis: ( 1 ) the distribution pattern of the
cells along the body axis changes, (2) certain larval deriv-
atives disappear, (3) new types of derivatives different!-

STEM CELL DEVELOPMENT

51

ate, and (4) migration patterns become more complex.
Comparisons of the distribution patterns of the intersti-
tial cell system in the planula and in the adult clearly
show that these cells undergo dramatic reorganization
during metamorphosis. This is not surprising given the
fact that the entire polarity of the animal changes as a
planula forms an adult: the apical end of the planula be-
comes the basal end of the polyp, and the mid-to-basal
end of the planula forms the stalk and hypostome of the
adult. In the planula, interstitial cells and ganglion cells
are found along the entire apical-basal axis, whereas the
majority of the nematoblasts and nematocytes are con-
fined to the apical two-thirds of the planula. During
metamorphosis this whole larval pattern is lost; from the
disc stage onward a new adult pattern of the interstitial
cell system is established. By the time the primary polyp
has formed interstitial cells, nematoblasts and ganglion
cells are found along the entire body axis; the concentra-
tion of ganglion cells and nematoblasts is high in the foot;
the concentration of ganglion cells is high in the hy-
postome; the concentration of ganglion cells and nema-
tocytes is high in the tentacles; and specific types of nem-
atocytes are confined to distinct regions of the polyp.

In the mature planula two major kinds of derivatives
differentiate from interstitial cells: a single type of bipolar
ganglion cell and four types of nematoblasts. Of the latter
only three types (desmonemes, stenoteles, and microbasic
heterotrichous b-mastigophores with inclusions) com-
plete differentiation in the planula; only a few nemato-
cytes with bullet-shaped capsules (microbasic heterotri-
chous b-mastigophores with inclusions) were ever found
in the planula. The other variety (microbasic heterotri-
chous b-mastigophores) begins development in the plan-
ula but completes differentiation only during metamor-
phosis. The larval bipolar ganglion cells disappear during
planular attachment, suggesting that these neurons play a
role in the attachment of the planula to the substrate. A
subpopulation of these larval ganglion cells contain a
RFamide-like peptide that may be used to initiate meta-
morphosis, to propagate it, or both (Brumwell and Mar-
tin. 1996). In fact, several investigators have previously
proposed that the larval neurons are involved in propa-
gating the metamorphic signal to other cells in the planula
(Martin and Thomas, 1981b; Thomas el al, 1987; Plick-
ert, 1988:Leitz6V«/.. 1 994; Leitz and Lay, 1995;Gajewski
etai. 1996; Brumwell and Martin, 1996).

In the adult polyp two types of somatic derivatives
arise from interstitial cells: ganglion cells, of which there
are at least three varieties, and nematocytes, of which
there are four kinds. These new neurons begin to appear
at the pawn stage and are found throughout the remain-
ing phases of metamorphosis. Three types of nemato-
cytes (desmonemes, stenoteles, and microbasic hetero-
trichous b-mastigophores with inclusions) first appear in

the planula and are found throughout metamorphosis,
whereas microbasic heterotrichous b-mastigophores,
which begin but never complete differentiation in the
planula. appear at the ectodermal surface between the
crown stage and the polyp stage. Furthermore, by the
polyp stage, fully differentiated microbasic heterotri-
chous b-mastigophores with inclusions have increased in
number and are abundant in the head. The appearance
of new somatic stem cell derivatives during metamor-
phosis, notably the three kinds of ganglion cells, indi-
cates that the interstitial cells have a greater difterentia-
tive potential than demonstrated in the planula.

The migration patterns of the interstitial cell system
are more complex in the adult than in the planula. In the
planula the interstitial cells, nematoblasts, and neuro-
blasts emigrate from the endoderm to the ectoderm.
There is no evidence that once in the ectoderm, these
cells migrate in an apical-basal direction (Martin and
Archer, 1986) — they appear to stay close to the site at
which they entered the ectoderm. During metamorpho-
sis this changes: interstitial cells, nematoblasts. and pos-
sibly neuroblasts migrate apically in large numbers. As
the pawn stage arises from the disc, the emerging stalk
and head of the pawn form and initially are completely
devoid of a stem cell system. Between the pawn stage and
the crown stage, the stem cells migrate apically in large
numbers and populate the stalk and head of the crown.
The first cells of the stem cell line to appear anteriorly are
nematoblasts, followed by interstitial cells and lastly by
ganglion cells. Interestingly, this is the order of appear-
ance of interstitial cell types in the ectoderm of the plan-
ula shortly after larval interstitial cell migration begins
(Martin and Archer. 1986). The introduction of the stem
cell system into the forming apical region of the crown
may be essential for shaping of the hypostome and for
tentacle formation. Coincidentally, it is during the crown
stage that general features of the adult hypostome begin
to take shape and the stem cell system first appears in
the apical end of the metamorphosing animal. Because
interstitial cells and nematoblasts are found in the endo-
derm of adult polyps, it is possible that these cells can
move from the endoderm to the ectoderm. Thus in the
adult, multiple directions of migration are probable. Fur-
thermore, since specific types of cells accumulate in spe-
cific regions of the polyp — for example, neurons are
abundant in the head and the foot — some sort of di-
rected migration must occur during metamorphosis.

Acknowledgments

This research was supported by National Science
Foundation Grants DCB-8702212, DCB-8942149,
DCB-9046094, DUE-9552116 and Career Advance-
ment Award DCB-87 1 1 245 and a John Yarborough Me-

52

V. J. MARTIN AND W. E. ARCHER

morial Undergraduate Research Grant from the North
Carolina Academy of Science, Inc.

Literature Cited

Archer, \V., and M. Thomas. 1983. An SEM study of morphological
changes during metamorphosis in Pennaria liarella Pi'/ic. Soutli-
eaxl Electron Microc. Sue. 7: 2 1 .

Hri kini:. S. 1984. Metamorphosis in Hydractinia eehinala Insights
into pattern formation in hydroids. Wttlielm Rou.\'.\ .ircli Dev
Biol 193: 370-378.

Bode, II., and C. David. 1978. Regulation of a multipotent stem cell,
the interstitial cell of hydra. Prog. Biophys. Mul liiol. 33: 189-206.

Bronner-Fraser, M., and S. Fraser. 1988. Cell lineage analysis reveals
multipotency of some avian neural crest cells. Nature 335: 161-
164.

Brumnell, G., and V. Martin. 1996. Llltrastructural localization of
RFamide-like peptides in neuronal dense-cored vesicles of a cnid-
arian plan u la larva. Invertehr. Bin/. 115: 13-19.

Gaje»ski, M., I. Leitz, J. SchloBherr, and G. Plickert. 1996. LWam-
ides from cnidaria constitute a novel family of neuropeptides with
morphogenetic activity. Dev Bin/ 205:232-242.

Heimfeld, S., and II. Bode. 1986. Growth regulation of the interstitial
cell population in llnlra. Dev liiol 1 16: 5 1-58.

LeDouarin, N. 1979. Cell Lineage, Stem (V/A ami Cell Determina-
tion. Elsevier/North Holland, Amsterdam.

Leitz, T., and M. Lay. 1995. Metamorphosin A is a neuropeptide.
II 'il/ielin Koit.\'xAreh Dev Biol 204:276-279.

Leitz, T., K. Morand, and M. Mann. 1994. Metamorphosin A: a
novel peptide controlling development of the lower metazoan //r-
draclinia echinata (Coelenterata, Hydrozoa). Dev. Bid 163: 440-
446.

Lumsden, A., 1988. Multipotent cells in the avian neural crest.
Trend* Ncurosci. 12:81-83.

Martin, \ . 1988a. Development of nerve cellsin hydrozoan planulae.
I. Differentiation ofganglionic cells. Biol Bull 174: 319-329.

Martin, V. 1988b. Development of the interstitial cell system in a ma-
rine hydrozoan polyp. Am. /.ool. 28: 95A.

Martin, V. 1990. Development of nerve cells in hydrozoan planuiae.
111. Some interstitial cells traverse the ganglionic pathway in the
endoderm. Biol Bull 178: 10-20.

Martin, V. 1991. Differentiation of the interstitial cell line in hydro-
zoan planulae. I. Repopulation of epithelial planulae. llvdrohio-
IOKM 216/217: 75-82.

Martin, V., and \V. Archer. 1986. Migration of interstitial cells and
their derivatives in a hydrozoan planula. Dev. Biol. 116:486-496.

Martin, V., and M. Thomas. 1977. A fine-structural study of embry-
onic and larval development in the gymnoblastic hydroid Pennaria
liarella Biol Bull. 153: 198-218.

Martin, V., and M. Thomas. 1980. Ultrastructure of the nervous sys-
tem in the planula larva of Pennaria liarella ./ .\forphol. 166:27-
36.

Martin, V., and M. Thomas. 1981a. The origin of the nervous system
in Pennaria liarella as revealed by treatment with colchicine. Biol.
Bull 160:303-310.

Martin, \ ., and M. Thomas. I981b. Elimination of the interstitial
cells in the planula larva of the marine hydrozoan Pennaria liarella.
J Exp tool 217:303-323.

Martin, \ ., F. Chia, and R. Koss. 1983. A fine-structural study of
metamorphosis of the hydrozoan Mitrocomella polrdiademaht .1
Morphol 176:261-287.

Medvinsky, A., N. Samoylina, A. Muller, and E. Dzierzak. 1993. An
early preliver intraembryonic source of CFU-S in the developing
mouse. Nan/re 364: 64-67.

Plickert, G., 1988. Proportion altering factor (PAF) stimulates nerve
cell formation in llydraelinia eeliinala Cell Di/ler Dev. 26: 19-28.

Plickert, G., M. Kroiher. and A. Mum k 1988. Cell proliferation and
early differentiation during embryonic development and metamor-
phosis of Hydraetinui eehinala Development 103: 795-803.

Potten, C., and M. I.oerller. 1990. Stem cells: attributes, spirals, pit-
falls and uncertainties. Lessons for and from the crypt. Development
110: 1001-1020.

Schwoerer-Bohning, B., M. Kroiher, and W. Muller. 1990. Signal
transmission and covert prepattern in the metamorphosis of //>•-
draetimu eehimtta (Hydrozoa). ll'iltielm Roux'.i Arch Dev. Biol.
198:245-251.

Sommer, C '". 1990. Post-embryonic larval development and metamor-
phosis of the hydroid Eudendrium raccmosum (Cavolini) (Hy-
drozoa, Cnidaria). Helgol. .\feere.\itnler.\. 44: 425-444.

I homas, M., G. Freeman, and V. Martin. 1987. The embryonic ori-
gin ot neurosensory cells and the role of nerve cells in metamorpho-
sis of Pliialidiiim grcgarimn (Cnidaria. Hydrozoa). Inl ./ Inverlehr.
Reproa Dev 11:265-287.

\\eis, \'., and L. Buss. 1987. infrastructure of metamorphosis in 7/r-
dractinia echinata. I'osiilla 199: 1-20.

\\ eston, J. 1991. Sequential segregation and fate of developmentally
restricted intermediate cell populations in the neural crest lineage.
Curr. lop Dev Biol. 25: 133-153.

Reference: B/o/ Bull 192: 53-6 [.(February, 1997)

Subcuticular Rejection: an Advanced Mode of the

Allogeneic Rejection in the Compound Ascidians

Botrylloides simodensis and B. fuscus

EUICHI HIROSE1, YASUNORI SAITO:, AND HIROSHI WATANABE3

'Biological Laboratory. College of Bioresource Sciences, Ni/ion University. Fitjisawa, Kanagawa 252,

Japan: 2Shimoda Marine Research Center. University of Tsukuba. Shiinoda. Sluzuoka 415, Japan:

and3 Tokyo KaseiGakuin Tsukuba College, Tsukuba, Ibaraki 305, Japan

Abstract. Allogeneic rejection between colonies (col-
ony specificity) was described by electron microscopy in
two compound ascidians. Botrylloides simodensis and
B. fuscus. When two incompatible colonies are brought
into contact at their growing edges, the tunic cuticle dis-
solves and the tunics of the colonies partially fuse. Allo-
reactive, humoral factors may diffuse to the opposite col-
ony through the partially fusing tunic, and the tunic cells
(free cells distributed in the tunic) possibly recognize
these factors and induce a rejection reaction. Then,
blood cells — mainly morula cells — infiltrate into the tu-
nic, while tunic cells are disintegrating near where the
partial fusion of the tunic is occurring. The infiltrating
blood cells aggregate, disintegrate, and discharge elec-
tron-dense materials in the tunic at the subcuticular re-
gions where the tunics have partially fused. Since the re-
jection lesion is restricted to the subcuticular area, some
regulatory systems may be involved in this restriction.
At the end, new walls are formed in the tunic matrix to
separate the rejection lesion from the contacting colo-
nies. The new wall is a continuous layer composed of
electron-dense fibers and is structurally identical to the
regenerating tunic cuticle. The mode of occurrence of
colony specificity (Hirose et a/.. 1994) and the present
results indicate that tunic cells are the only allorecogni-
tion sites in B. fuscus.

Introduction

Self or non-self recognition is one of the most funda-
mental interactions among individuals, and its occur-

Received 26 January 1996; accepted 23 October 1996.

rence has been observed in many animal phyla. For in-
stance, rejection or acceptance of grafted tissues among
allogeneic individuals (histocompatibility) is one of the
immediate examples of its occurrence. Whereas a tissue
graft is an artificial treatment, a kind of tissue transplan-
tation occurs naturally in many colonial forms of sessile
animals, from sponges to ascidians. This phenomenon is
known as colony specificity; when a sessile colony grows
on a substratum and comes in contact with another con-
specific colony, the two colonies either fuse to form a
single colony, or they do not fuse. The outcome depends
on allogeneic recognition between the two colonies. In
some ascidians, the genetic control of this allorecogni-
tion has been demonstrated and is thought to be an an-
cestor of the major histocompatibility complex in verte-
brates (Oka and Watanabe, 1957, I960; Sabbadin, 1962;
Scofieldrttf/., 1982; Weissmanf/tf/.. 1990).

Studies on colony specificity in colonial ascidians have
been focused on species of the family Botryllidae (botryl-
lid ascidians), and every botryllid ascidian studied has
exhibited colony specificity (reviewed in Saito et a/..
1994). These species all form sheetlike colonies in which
the zooids are connected with a vascular network, and
the whole of the colony is embedded in a gelatinous tu-
nic, an integumentary matrix that covers the epidermis.
Free cells (tunic cells) of a few different kinds are distrib-
uted throughout the tunic (Hirose et a/., 1991). and a
cuticular layer overlaying the tunic matrix is the outer-
most structure of the colony (Hirose et ai, 1990a).

When two compatible colonies are brought into con-
tact at their growing edges, they fuse into a single colony.
The course of the fusion reaction is fundamentally the
same in all botryllids, as follows: ( 1 ) contact of the tunic

53

54

E. HIROSE ET AL.

surfaces (= cuticle); (2) dissolution of the tunic cuticle at
the contact area; (3) penetration of ampullae (termini of
the blood vessels at colony periphery) into the opposite
colony; (4) contact between the penetrating ampullae
and those of the opposite colony; (5) ampullar fusion and
establishment of vascular connection between the two
colonies. In contrast, the rejection reaction in each spe-
cies is initiated at a specific stage of the fusion process. In
Botryllus primigenus, for example, rejection is initiated
after the penetration of ampullae (Taneda, 1 985).

Some Botrylloides species show a subcuticular rejec-
tion (SCR) that begins at the earliest step in the fusion
process — immediately after the partial fusion of the tu-
nic. The rejection reaction in SCR involves blood cell
infiltration and occurs in only a small area of the tunic
contacting an allogeneic colony. The loss of tissue is min-
imized in this rejection, which seems, therefore, to be the
most advanced mode of rejection found thus far among
botryllid ascidians. In other ascidians, such as Botryllus
primigenus, all of the ampullae interacting with their al-
logeneic counterparts disintegrate and are cut offthe col-
ony (Taneda el a!., 1985, for review).

Among species of Botrylloides that show SCR, two
different reactions are elicited when incompatible colo-
nies are brought into contact at artificial cut surfaces.
Whereas intensive rejection occurs at the contacting
boundaries in B. simodensis (Hirose el a/.. 1990b), vas-
cular connections are established between incompatible
conspecifics (surgical fusion) in B. fuscus and B. viola-
ceus( Hirose el al.. 1988, 1994). Surgical fusion indicates
that the effector system inducing the rejection in the lat-
ter two species is not distributed in the vascular system.
During SCR, therefore, allorecognition is probably car-
ried out in the tunic. We have used light and electron
microscopy to examine the process of SCR in B. simo-
densis and B. fuscus. This study should provide a better
understanding of the mechanisms of allogeneic rejection
and bring essential insights to the questions: when is self
or non-self recognized; and what is the agent of recogni-
tion?

Materials and Methods

Animals

Colonies of Botrylloides simodensis and B. fuscus were
collected in the vicinity of Shimoda (Shizuoka Prefec-
ture, Japan). The colonies were attached on glass plates
and reared in culture boxes immersed in Nabeta Bay
near the Shimoda Marine Research Center.

The fusibility of colonies was tested with fusion exper-
iments, as follows: Two pieces of colony of the same size
were brought into contact at their growing edges on a
glass slide. After 1-2 h in a moisture chamber, the colo-
nies attached to the glass slide and were then reared in

running seawater in an aquarium in the laboratory; they
were observed every day under a binocular stereomicro-
scope. Within several days, either a fusion or a rejection
reaction occurred between the paired colonies. Here 'fu-
sion1 means establishment of a common vascular system
between the two colonies, and 'rejection' means its inter-
ruption or prevention.

Light microscopy

The specimens were fixed with 2.5% glutaraldehyde
solution containing 0.45 At sucrose buffered with 0.1 M
cacodylate at pH 7.4. The fixed specimens were then de-
hydrated through a butanol series, embedded in Para-
plast, sectioned at 5 ^m, and stained with Congo red, De-
lafield's hematoxylin, and eosin-orange G.

Scanning electron microscopy

To observe the structures of internal tissues, the
method of Armstrong (1971) was applied, as follows:
Specimens embedded in Paraplast were cut with a razor
blade or a microtome blade to expose the inner struc-
tures. The specimens were washed in xylene ( 1 h, three
changes) to remove the Paraplast and then cleared with
absolute ethanol. These specimens were dried in a criti-
cal point dryer and sputter-coated with gold-palladium.
They were examined with a Hitachi S-570 scanning elec-
tron microscope at 20 kV.

Transmission electron microscopr

The specimens were fixed at room temperature in a
2.5% solution of glutaraldehyde containing 0.45 Af su-
crose and buffered with 0. 1 Af cacodylate at pH 7.4. An
alternative fixation, on ice for 2 h. was in a 2.5% solution
of glutaraldehyde containing 2% NaCl buffered with
0.1 M Millonig's phosphate buffer at pH 7.4. The latter
medium is essentially the same as that of Sugino et al.
(1987). These prefixed specimens were rinsed in the
same buffer, and then postfixed with 1% osmium tetrox-
ide in the same buffer for 1 to 2 h. After dehydration
through an ethanol series, the specimens were cleared
with «-butyl glycidyl ether and embedded in epoxy resin.
Thin sections were double-stained with uranyl acetate
and lead citrate. They were examined with a Hitachi HS-
9 transmission electron microscope at 75 kV.

Results

General features oj siihcuticii/ar rejection

In both Botrylloides simodensis and B. fuscus. when
incompatible colonies are brought firmly into contact at
their growing edges, they never fuse (Fig. 1 ). Yet the signs
of inflammatory rejection are barely observable under a

ALLOGENEIC REJECTION IN COMPOUND ASC1DIANS

55

R-

' «:-.. -•-• .••

^ v • ^

-* ..

s---

^*

4A

"

4B

Figure 1 . Subcuticular rejection in Botrylloides sinwilcnsis. Two incompatible colonies ( right and left)
are contacted at their growing edges. Rejection lesions are barely seen under the stereomicroscope. Bar =
0.5 mm.

Figure 2. Histological sections of subcuticular rejection in Bolrvlloiiles ximodenxis (A) and B.fuscus
(B). Arrows, rejection lesions; arrowheads, tunic cuticle; t, tunic matrix; v, vascular vessel. Bar = 200 ^m
(A)and lOO/irnlB).

Figure 3. Fusion of'the tunic between incompatible colonies of Botrylloides fuscus (A) and an enlarge-
ment (B). Arrowheads, tunic cuticle of each colony; e, epidermis of vascular ampulla; t, tunic matrix. Bar
= 5 jim (A). I jim(B).

Figure 4. Vacuo-granular tunic cells of Botrylloides fuscus. Intact cell with vacuoles and granules (A).
and a disintegrating one discharging electron-dense materials that produce filamentous structures (ar-
rowhead. B). Bar = 5 jim.

56

E. HIROSE ET AL.

binocular stereomicroscope. In histological sections, re-
jection lesions are restricted to a limited part of the sub-
cuticular region in the contact area (Fig. 2). In this rejec-
tion, infiltrating blood cells — mainly eosinophilic mor-
ula cells — aggregate and disintegrate in the tunic at the
subcuticular region; thus the lesion is also well stained by
eosin. The morphological process of SCR is fundamen-
tally the same in B. siiuodensis and B.fuscns.

In this report, SCR is divided into the following three
stages: (1) partial fusion of the tunic at the contact area;
(2) blood cell infiltration, migration, and disintegration
in the tunic; (3) separation of rejection lesion from the
colonies. Since we cannot observe these processes in liv-
ing specimens, the time course is difficult to define with
precision. When actively growing colonies were brought
into contact and reared at about 20°C, the time course
could be estimated on the basis of histological data, as
follows: ( 1 ) partial fusion of the tunic cuticle occurs 1 to
2 days after contact; (2) blood cell infiltration and disin-
tegration begins shortly after partial fusion (2 to 3 days
after contact) and continues until the rejection lesion is
separated from the colony; (3) separation of the lesion is
usually completed within 4 to 7 days after contact. The
time course depends on the growth rate of the colonies
as well as on temperature.

Partial fusion oj the tunic

The first stage of SCR between the two colonies is the
partial fusion of the contacting tunics; in other words,
the dissolution of the tunic cuticles (Fig. 3). Partial fusion
occurs in 1 to 2 days after the contact of the colonies. The
tunic cuticle is a continuous, electron-dense sheet with
minute protruberances (Hirose el a/., 1990a), and it is a
boundary between the two contacting colonies. At the
contact area, the tunic cuticles of both colonies adhere
closely to each other. Thereafter, the cuticles dissolve
here and there, and the tunic matrices of both colonies
become continuous. In these areas, the edges of the ad-
hering tunic cuticles become continuous and have a hair-
pin form (Fig. 3B). At this stage, the reaction between
incompatible colonies shows no sign of a rejection reac-
tion.

Blood cell infiltration, migration, and disintegration

The first signs of a rejection reaction are the infiltration
of blood cells and the disintegration of tunic cells (vacuo-
granular tunic cells; see Hirose el ai. 1991) in the sub-
cuticular regions where the partial tunic fusion is occur-
ring (Figs. 4-6); we are not sure which of these events
occurs earlier. Many of the infiltrating blood cells are
morula cells that have several vacuoles filled with elec-
tron-dense material. The disintegrating tunic cells dis-
charge electron-dense materials that probably originate

from their granules. The discharged materials seem to
bind to the filamentous components of the tunic matrix
and form electron-dense filaments around the cells (ar-
rows in Fig. 4). Afterward, the infiltrating blood cells mi-
grate around the regions of partial tunic fusion (Fig. 7),
and then they disintegrate (Fig. 8). The disintegrating
morula cells discharge electron-dense materials, as seen
around disintegrating tunic cells, and electron-dense fil-
amentous structures are also formed in the tunic (Fig. 8).
The infiltrating blood cells increase in number, and
they disintegrate and discharge their contents in the tu-
nic (Figs. 9 and 10). Electron-dense fibers appear around
the disintegrating cells (arrows in Fig. 10). This inflam-
matory-like reaction occurs in a limited area, because the
infiltrating cells usually disintegrate after migrating to a
subcuticular location where the tunics have partially
fused. The cell infiltration, migration, and disintegration
proceed further, and eventually the migrating cells form
an aggregate in those subcuticular regions where the tu-
nics of the allogeneic colonies have partially fused (Figs.
1 1 and 12). Within the cell aggregates, disintegrating cells
discharge their contents, i.e.. electron-dense, eosino-
philic materials. Around them is some electron-dense
substance that may be derived from the discharged ma-
terials.

Separation of the rejection lesion

When the rejection is complete, a dense continuous
layer appears in the tunic matrix, and it separates both
colonies from the rejection lesion (Figs. 13-16). This
new wall consists of aggregates of electron-dense fibers
(arrows in Figs. 14 and 16).

Discussion

When colonies are brought into contact at their grow-
ing edges, the first interaction is a dissolution of the cuti-
cles of the contacting tunics. Due to the cuticle dissolu-
tion, the tunics of the two colonies undergo partial fu-
sion. Even when rejection is induced by contact between
cut surfaces, the tunic matrixes of the two colonies ap-
pear to fuse shortly after the contact (Hirose el al..
1990b). The tunic matrices facing each other probably
fuse without allorecognition. At this stage, morphologi-
cal differences are not observed between compatible and
incompatible combinations, but the signs of rejection
soon appear. Allorecognition probably occurs shortly af-
ter the tunics partially fuse. Blood plasma induces the
allospecific reaction in both Botryllus primigenus
(Taneda and Watanabe, 1982) and Botrylloides simo-
densix (Saito and Watanabe, 1984). Probably, alloreac-
tive factors diffuse into the opposite colony through sites
of partial fusion, and the alloreactive cells respond to
these factors. Furthermore, the allorecognition sites are

ALLOGENEIC REJECTION IN COMPOUND ASCID1ANS

57

*<T\'.» -'••— •> .'•'-'
' '

Figure 5. Blood cell infiltration (Botrvlloides simodensis). Arrow, infiltrating hlood cell (morula cell):
e. epidermis of blood vessel: lu. vascular lumen: t. tunic matrix. Bar = 5 ^m.
Figure 6. Blood cells infiltrating in the tunic (Botrylloidesfuscus). Bar = 5 Mm.

Figure 7. Blood cells migrate in an area of partial tunic fusion (Botrylloides simodensis). Arrowheads
indicate tunic cuticle. Bar = 2 Mil-
Figure 8. Disintegration ot infiltrating blood cells (morula cells) in the tunic (Botrylloidesfuscus). Elec-
tron-dense materials are discharged in the tunic. Bar = 5 urn.

probably restricted to the tunic, particularly in species in of blood cells may also be alloreactive in B. simodensis.

which surgical fusion occurs, i.e.. B. fuscus; moreover, because an intensive rejection occurs between incompat-

the tunic cells may well be the sole allorecognition sites. ible colonies that make contact at artificial cut surfaces.

On the other hand, the ampullar epithelia or some kinds The first signs of SCR are the disintegration of vacuo-

58

E. HIROSE ET AL

;$»•;£ -v

•//'/'•*';'"'?• I
-'- -*~*

^KT^:

M '

, TMTjrB — ' M \-*J**-. •

Figures 9 and 10. Disintegration of blood cells in the vicinity of a partial fusion of tunic cuticles (Fig.
9, Bo/nV/c/i/i's .s7jn<K/mv/.v,- Fig. 10. B lusais). Electron-dense fibers are formed in the tunic (arrows in Fig.
10). Arrowheads, tunic cuticle. Bar = 2 fitn. Fig. 9: 5 ^m. Fig. 10.

granular tunic cells and the infiltration of blood cells,
mainly morula cells. The disintegrating tunic cells may
release chemotactic factors that induce blood cell infil-
tration. Whether the tunic cell disintegration is a primary
response to the allorecognition or an induced one re-
mains uncertain. The infiltrating blood cells migrate and

disintegrate at limited sites in the subcuticular region
during SCR; the discharged contents from blood cells
might also contain chemotactic factors that induce blood
cell infiltration and disintegration.

The disintegrating tunic cells and morula cells dis-
charge electron-dense materials, and these materials ap-

ALLOGENEIC REJECTION IN COMPOUND ASCIDIANS

59

.-

Figures II and 12. Cell aggregates at the rejection lesion (Fig. 1 1. Botrylloides simodensis; Fig. 12.
B /iiM'its). Arrowheads, tunic cuticle: t, tunic matrix. Bar = 5 ^m.

pear to bind to the tunic matrix to form filamentous formation of filaments in the tunic around the disinte-

structures around the disintegrating cells. The dis- grating cells was reported in the allogeneic rejection of

charged contents probably rearrange the tunic matrix Botryllm primigenu.s (Tanaka, 1973; Tanaka and Wata-

and induce the disintegration of the tunic structures. The nabe, 1 973). In some solitary ascidians, morula cells (or

60

E. HIROSE ET AL

Figures 13-16. Now wall separating the colony from the rejection lesion (Figs. 1 3 and 14, Bdlrylloiclex
Miiinilcnsix. Figs. I5and 16, K /;/sf».\). Figures 1 3 and 15 are scanning and Figures 14 and 16are trans-
mission electron micrographs. Lower left parts are lesion areas in these figures. Arrows, new wall; ar-
rowheads, tunic cuticle. Bar = 5 j/m. Figs. 1 3 and 14; 10/im, Fig. 15;2nm,Fig. l(i.

vacuolated cells) contain phenoloxidase, which has cyto-
toxic activity (Jackson el ul.. 1993), and they release the
enzyme in response to allogeneic blood cells (Akita and
Hoshi, 1995). In Boiryllus schlosncri, a botryllid ascid-
ian, Ballarin ci ul. ( 1993) showed that the morula cells
contain phenoloxidase, as well as peroxidase and arylsul-
fatase: and these authors also showed that phenoloxidase
is involved in the allogeneic rejection reaction (Ballarin
etui., 1994, 1995). In the SCR of Botrylloides species, the
discharged substances from morula cells also probably
contain phenoloxidase.

In the SCR, the infiltrating blood cells form aggregates
in the subcuticular region where partial fusion occurs.
There may be a regulatory system to restrict the rejection

reaction to the limited areas. In contrast, when rejection
is induced by contact between cut surfaces in B. sinio-
densis, the infiltrating cells disintegrate at random
around the cut surface facing the incompatible colony,
and the rejection lesion is large enough to be observed as
a clear, black line (Hirose el ul., 1990b). In this case, the
regulatory system proposed above would not work.

After the rejection reaction, the new wall appears, sep-
arating the rejection lesion from the colonies. The syn-
thesis of the new wall was first described in the allogeneic
rejection in B. phmigenns (Tanaka, 1973; Tanaka and
Watanabe, 1973), and the same structures were also re-
ported in the rejection induced by the contact of alloge-
neic colonies of B. siniodensis at cut surfaces (Hirose el

ALLOGENEIC REJECTION IN COMPOUND ASCID1ANS

61

ai. 1 990b). Since the fine structure of the new wall is the
same as that of regenerating tunic cuticle (Hirose el a/..
1995), the new wall should eventually become a tunic
cuticle. During cuticle regeneration, the electron-dense
fibers are probably produced from the aggregates of tunic
matrix, and proteolysis is probably involved. In SCR, the
disintegrating cells may release factors that induce the
aggregation of tunic matrix and lead to the production of
the electron-dense fibers. The new wall is supposed to act
as ( 1 ) a barrier that prevents the migration of cells to the
disintegrating tissue. (2) a barrier preventing the diffu-
sion of factors into the colony from the allogeneic colony
and disintegrating cells. (3) an outer covering of the tunic
once the rejection lesion has been cleared.

What cell types in a colony discriminate self from non-
self? The most likely candidates are the tunic cells, par-
ticularly in B. fitscns where surgical fusion occurs be-
tween incompatible colonies at the cut surface (Hirose el
ai. 1994). In this species, surgical fusion indicates that
the distribution of allorecognition sites is restricted to the
tunic, and thus tunic cells may be the sole allorecogni-
tion sites. The morphological changes that occur during
the fusion and rejection reactions also suggest that the
allorecognition is carried out only in the subcuticular re-
gion of the tunic that is partially fused with the contact-
ing colony.

Acknowledgments

The present study was partly supported by Grants-in-
Aid to EH and YS from the Ministry of Education, Sci-
ence and Culture of Japan. Most of this study was per-
formed at the Shimoda Marine Research Center
(SMRC). University of Tsukuba, and we thank the staff
members. We wish to express our gratitude to Dr. Y.
Sugino (Tosa Women's High School) for providing ad-
vice on electron microscopy and to anonymous referees
for their valuable comments and suggestions. This study
includes the contribution no. 599 from SMRC.

Literature Cited

Akita, N., and M. Hoshi. 1995. Hemocytes release phenoloxidase

upon contact reaction, an allogeneic interaction, in theascidian Hu-

locynthm rorelzi. Cell Struct. Func. 20: 81-87.
Armstrong, P. B. 1971. A scanning electron microscope technique

for study of the internal microanatom> of embryos. Microscope 19:

281-284.
Ballarin. I,., F. Cima, and A. Sabbadin. 1993. Histoenzymatic stain-

ingand characterization of the colonial ascidian Boiryllux Wi/m.vcn

hemocytes. Bui. /<«</. 60: 19-24.
Ballarin, L., F. Cima, and A. Sabbadin. 1994. Phenoloxidase in the

colonial ascidian Botryllux schlosseri (Urochordara: Ascidiacea).

Anun Bin/ 3:41-48.

Ballarin, L., F. Cima, and A. Sabbadin. 1995. Morula cell and histo-
compatihihty in the colonial ascidian Butryllus xchlussi-ri. '/.mil. Sci.
(Tokyo) 12:757-764.

Hirose, E., Y. Saito, and II. Watanabe. 1988. A new type of manifes-
tation of colony specificity in the compound ascidian. Botrvlloidex
violaceus Oka. Biol. Bull. 175: 240-245.

Hirose. E., Y. Saito, K. Hashimoto, and II. Watanabe. I990a. Minute
protrusions of the cuticle: fine surface structures of the tunic in as-
cidians. ./. Morphol 204: 67-73.

Hirose, E., Y. Saito, and II. \\atanabc. 1990b. Allogeneic rejection
induced by cut surface contact in the compound ascidian. Botryl-
loides simodensis Invenehr Reproil Dev. 17: 159-164.

Hirose, E., Y. Saito. and II. \\alanabe. 1991. Tunic cell morphology
and classification in botryllid ascidians. Zoo/. Sci. (Tokyo) 8: 951-
958.

Hirose, E., Y. Saito, and II. Watanabe. 1994. Surgical fusion between
incompatible colonies of the compound ascidian. Bolrylloulex /»s-
cus. Dev. Coin/) Imminnit. 18: 287-294.

Hirose, E., Y. Saito, and II. Watanabe. 1995. Regeneration of the
tunic cuticle in the compound ascidian. Botrylloides simodensis.
Dev Comp Immunol 19: 143-151.

Jackson. A. D., V. J. Smith, and C. M. Peddle. 1993. In vuro phenol-
oxidase activity in the blood of dona intestinalis and other ascidi-
ans. Dev Comp Immumil. 17:97-108.

Oka, II.. and II. Watanabe. 1957. Colony-specificity in compound
ascidians as tested by fusion experiments (a preliminary report).
Proc. Jpn. Acad 33: 657-659.

Oka. H., and H. Watanabe. 1960. Problems of colony-specificity in
compound ascidians. Bull Mar. Biol. Sin. Asamushi 10: 153-155.

Sabbadin, A. 1962. Le basi genetiche della capacita di fusione fra co-
lonie in Boiryllux schlossen (Ascidiacea). Rend. Accad. A'ar. Lincci
32(Ser.8): 1031-1035.

Saito, Y., and H. Watanabe. 1984. Partial biochemical characteriza-
tion of humoral factors involved in the nonfusion reaction of a bo-
tryllid ascidian, Botrylloides simodensis. Zool. Sci. (Tokyo) 1: 229-
235.

Saito, Y., E. Hirose, and II. Watanabe. 1994. Allorecognition in
compound ascidians. Int. J. Dev. Biol. 38: 237-247.

Scoheld, V. I,., J. M. Schlumpberger, L. A. West, and I. I,. Weissman.
1 982. Protochordate allo-recognition is controlled by a MHC-like
gene system. Suture 295: 499-502.

Sugino, Y. M., A. Tominaga. and Y. Takashima. 1987. Differentia-
tion of the accessory cells and structural regionalization of the oo-
cyte in the ascidian dona savignyi during early oogenesis. / Exp.
Zool. 242:205-214.

Tanaka, K. 1973. Allogeneic inhibition in a compound ascidian, Bo-
tryllusprimigenusOka. II. Cellular and humoral responses in "non-
fusion" reaction. Cell. Immunol. 7: 427-443.

Tanaka, K., and H. Watanabe. 1973. Allogeneic inhibition in a com-
pound ascidian, BotryllusprimigenusOka. I. Processes and features
of "nonfusion" reaction. Cell Immunol 7:410-426.

Taneda, Y. 1985. Simultaneous occurrence of fusion and nonfusion
reaction in two colonies in contact of the compound ascidian, Bo-
tryllus primigenus. Dev. Comp Im/niinol.9: 371-375.

Taneda, Y., Y. Saito, and II. Watanabe. 1985. Self or non-self dis-
crimination in ascidians. Zool. Sci. (Tokyo) 2: 433-442.

Taneda, Y., and II. W atanabe. 1982. Studies on colony specificity in
the compound ascidian. Botryllux pnmigenus Oka. II. In vivo bio-
assay for analyzing the mechanisms of "nonfusion" reaction. Dev.
Comp. Immunol 6: 243-252.

Weissman, I. L., Y. Saito, and B. Rinkevich. 1990. Allorecognition
histocompatibility in a protochordate species: Is the relationship to
MHC semantic or structural'.' Inuniinol. Rev 113:227-241.

Reference: Bio! Bull 192: 62-72. (February. 1997)

Effects of Common Estuarine Pollutants on the Immune

Reactions of Tunicates

DAVID RAFTOS AND AIMEE HUTCHINSON
School of Biological Sciences, Macquarie University, North Ryde. NSW, 2109, Australia

Abstract. Tunicates are filter-feeding estuarine and
marine animals that are frequently exposed to chronic
environmental pollution. This study demonstrates that
exposure to low-level (i.e., below the threshold of acute
lethality) contamination with tributyltin, creosote, and
copper can have substantial effects on natural immune
reactions in tunicates. Sublethal doses of toxicants ad-
ministered either in vitro or in vivo profoundly affected
phagocytosis, cellular cytotoxicity, and hematopoietic
cell proliferation. Effects were not always inhibitory, and
responses often varied depending on the route of toxi-
cant administration. The data suggest that pollutants can
activate cascades of cellular processes and compensatory
mechanisms, as well as directly inhibiting some of the
responses tested. Some evidence indicates that toxicants
exert their effects by altering the relative frequencies of
circulatory hemocytes.

Introduction

Marine invertebrates can be profoundly affected by
aquatic pollution. Detrimental effects have been identi-
fied using tests for acute lethality (e.g., 96-h LD5()), toxi-
cant bioaccumulation, anatomical and biochemical ab-
erration, or altered biodiversity and abundance (Giam
and Ray, 1987; Landis and Yu, 1995; Peakall, 1992).
However, there is relatively little information regarding
the effects of environmental contamination on natural
immune reactions in invertebrates, even though modu-
lation of the immune system may dramatically alter pop-
ulations by affecting their resistance to infection ( Ander-

Received 17 May 1995; accepted 24 October 1996.

Abbreviations: FSW, filtered seawater; MAC. marine anticoagulant;
MS. marine saline; PAH. polycyclic aromatic hydrocarbon; RRBC.
rabbit red blood cells; TBT. tributyltin; TBS. Tris-buffered saline.

son el ai, 1990; McCarthy and Shugart, 1990; Roales
and Perlmutter, 1977; Sarot and Perlmutter, 1976; Steb-
bing. 1985). Some evidence suggests that the effects of
decreased disease resistance resulting from low-level,
chronic pollution may not be reflected accurately in as-
says that test acute lethality. For instance, heavy metal or
polychlorinated biphenyl contamination in worms can
significantly inhibit lysozyme activity, wound healing,
phagocytosis, rosette formation, and tissue transplanta-
tion rejection at concentrations that are not acutely le-
thal (Cooper and Roch, 1992; Fitzpatrick et ai. 1992;
Rodrigues-Grau et ai, 1989).

The aim of the current study is to demonstrate that
subacute contamination with three common estuarine
pollutants — the antifouling agents tributyltin (TBT),
copper, and creosote — can significantly affect the immu-
nological defenses of tunicates. Tunicates (Urochordata,
Ascidiacea) are aquatic filter-feeding invertebrates that
are ubiquitous components of estuarine and coastal ma-
rine systems (Berrill, 1950; Goodbody, 1974). They oc-
cur in large populations on marinas, moorings, and
wharves that are often subjected to chronic pollution,
particularly from antifouling treatments. The effects of
pollution are likely to be compounded in tunicates by
their filter-feeding lifestyle. Large volumes of potentially
polluted water pass over the sensitive endothelial sur-
faces of the pharynx (up to 1 00 1 of water per day), greatly
increasing the propensity of tunicates to absorb toxicants
(Goodbody, 1974).

Here, we test the effects of toxicants on a number of
well-characterized assays of immunological reactivity in
tunicates. Those assays quantify hemopoietic cell prolif-
eration (Raftos and Cooper, 1991; Raftos et ai. 1991),
phagocytosis (Beck et ai, 1993: Kelly et ai, 1993), and
cellular cytotoxicity (Parrinello et ai, 1993; Peddie and
Smith, 1993, 1994). Tunicates lack adaptive antipatho-

62

TUNICATES AND MARINE POLLUTANTS

63

genie defense mechanisms that are analogous to the
mammalian adaptive immune system (Ratios, 1994).
Hence, innate (or natural) immunological reactions,
such as phagocytosis and cellular cytotoxicity, are prob-
ably the major protective responses of these animals.

Materials and Methods

Tunicates

Specimens of Styela plicata were collected from two
sites on Sydney Harbor. Australia (Balmoral Beach and
Birkenhead Point). After collection, tunicates were
maintained in 40-1 aquaria filled with seawater. Aquaria
were held at 14°C under constant aeration. These condi-
tions can maintain S. plicata for up to 2 months with
limited mortality. In vivo exposures were conducted in
partitioned aquaria containing 40 1 of seawater per com-
partment.

Toxicants

Tributyltin oxide and copper sulfate were purchased
from ICN Chemicals (Sydney, NSW, Australia) and cre-
osote was obtained from BBC Pty Ltd (Chatswood,
NSW, Australia). Tributyltin and copper were diluted di-
rectly in filtered seawater (FSW; 0.45-^m filtration). Cre-
osote was prepared as a saturated stock solution in FSW
by vigorously agitating 1% v/v creosote in toxicant-free,
filtered seawater overnight and then filtering the solution
(0.45-Mm) to remove insoluble material. Concentrations
of creosote are cited as percentages (v/v) of filtered, satu-
rated creosote solutions. Chemical analysis (Australian
Analytical Laboratories, Asquith, NSW, Australia) re-
vealed that 5% v/v of a saturated solution of creosote
contained 1 mg/1 total polycyclic aromatic hydrocar-
bons (PAH) (napthalene, 280 /ig/1: anthracene, 250 ^g/1;
phenanthrene. 150/wg/l; acenapthene, 93 /ig/1; remain-
ing PAHs < 80 Mg/')- No PAHs (practical quantitation
limit = 1 Mg/0 we detected in the normal seawater used
in aquaria.

Treatment protocols

Two forms of toxicant treatment were applied. Tuni-
cate cells were exposed to toxicants in vitro to assess
effects on isolated tissues, and live tunicates were ex-
posed to toxicants in aquaria to identify effects that were
derived from interactions between organ or physiologi-
cal systems. Where possible, the effects of toxicants were
tested over a range of doses and at a number of time
points for exposure periods of up to 9 days. For brevity,
only representative data reflecting trends in both dose re-
sponse and kinetic analysis are presented here.

Dosages

The ranges of doses used for the three toxicants were
selected to incorporate concentrations that yielded sig-
nificant differences from nontreated controls in at least
one of the assays tested. Copper was the only compound
that proved to be acutely toxic at high doses. All tuni-
cates (n = 8) died within 8 days of exposure to >5 Mg/ml
copper. Mortality was assessed by the sensitivity of tuni-
cates to touch (failure to retract siphons) and by an anal-
ysis of hemocyte (blood cell) viability. None of the other
doses tested, including <5 Mg/ml copper, caused mortal-
ity within 20 days (n = 4 per dose).

Hemocyte harvesting and manipulation

Hemocytes were harvested from incisions in the buc-
cal siphons of S. plicata. The exuded hemolymph
(blood) was collected in equal volumes of ice-cold ma-
rine anticoagulant buffer (MAC; 0. 1 A/ glucose. 1 5 mA/
trisodium citrate, 13mA/ citric acid, 10mA/ EDTA.
0.45 A/NaCl, pH 7.0; Peddie and Smith, 1994) or FSW.
Debris and cell aggregates were removed from the hem-
olymph by sedimentation for 5 min (1 X g). As required,
hemocytes were washed by centrifugation (400 X g,
5 min, 4°C) through either MAC or FSW.

Cell viability and morphology

Hemocyte viabilities and the relative frequencies of
distinct hemocyte subpopulations were determined us-
ing a FACScan flow cytometer with an argon-ion laser
tuned to 488 nm (Becton Dickenson, Mountain View,
CA). Hemocytes for flow cytometry were obtained either
by bleeding tunicates that had been exposed to toxicants
in aquaria or by harvesting cells that had migrated from
cultured pharyngeal explants during in vitro exposures
("emigrant hemocytes"; see Proliferative activity ofio.\i-
cant-treated tunicate cells section). In viability studies,
hemocytes (1 X 106/ml) were stained with ethidium bro-
mide (0.1% v/v) immediately prior to analysis. Dead
cells were detected by their increased red (800 nm) flu-
orescence reflecting the intercalation of ethidium bro-
mide into cellular DNA. The relative frequencies of dis-
tinct hemocyte subpopulations were determined by ana-
lyzing forward angle versus 90° light-scatter plots.

Proliferative activity of toxicant-treated tunicate cells

To quantify hemopoietic cell proliferation, tunicate
tissue cultures were established by excising small por-
tions (2x2 mm) of the pharynx for explant culture in
tunicate tissue culture medium (T-RPMI; Raftos and
Cooper, 1990). Each 100 ml of T-RPMI contained 10 ml
RPMI-1640 tissue culture medium (with sodium bicar-

64

D. RAFTOS AND A. HUTCHINSON

bonate, without L-glutamine; Sigma Chemicals, St.
Louis, MO), 1 ml 20% w/v NaCl, 1 ml antibiotic stock
solution (4 mg/ml streptomycin sulfate, 103 lU/ml peni-
cillin sulfate), 100/il 1 M L-glutamine, and 88 ml FSW.
Cultures were maintained at 1 5°C without CO2 supple-
mentation. Under these conditions explant cultures nor-
mally maintain cell viability and function for up to
70 days (Raftos and Cooper, 1990).

For in vitro exposure trials, explants were cultured for
up to 8 days in 96-well flat-bottomed tissue culture plates
containing 200 jul/well T-RPMI and various concentra-
tions of toxicants. Explants were moved to fresh medium
every 2-4 days. After appropriate exposure periods of up
to 8 days, explants were incubated (overnight, 1 5°C) with
18.5MBq/ml 3H-thymidine (Amersham, NSW, Aus-
tralia, 740GBq/mmol). Non-incorporated 3H-thymi-
dine was removed after incubation by extensive washing
in FSW. Explants were then digested (37°C, overnight) in
2.0% w/v trypsin (Sigma Chemicals) to facilitate liquid
scintillation counting in Ecolite scintillation cocktail
(ICN, Seven Hills, NSW, Australia) (Raftos et a/., 1991).

Explants from tunicates exposed to toxicants in
aquaria were excised, incubated immediately in T-RPMI
containing 18.5 MBq/ml 3H-thymidine (overnight, 15°C),
and then washed and digested as described above.

Phagocvtic activity of toxicant-exposed hemocytes

To test the phagocytic activity of tunicates that had
been exposed to toxicants in aquaria, hemocytes from
treated tunicates were harvested into FSW and their den-
sities adjusted to 3 X 106 cells/ml without washing. For
in vitro exposures, toxicants were added to hemocyte sus-
pensions harvested from nonexposed tunicates (3 X
106 cells/ml in T-RPMI). Aliquots (200 n\) of hemocyte
suspensions were cultured (15°C) on autoclaved glass
coverslips (22 X 22 mm) for either 2 h (aquarium
exposures) or overnight (in vitro exposures). Adherent
hemocytes were washed with 400 ^\ FSW before being
overlaid with 50 n\ yeast (Baker's yeast type II, Sigma
Chemicals; 5 X 106 yeast/ml) that had been prepared and
opsonized with S. plicata plasma according to the
method of Beck et al. (1993). Noningested yeast were re-
moved by extensive washing with FSW after a 30-min
incubation (15°C), and phagocytic activity was quanti-
fied microscopically (Beck et al.. 1993; Kelly et til.,
1993).

Cytotoxic activity oj treated cells

Hemocytes from tunicates that had been exposed to
toxicants in aquaria were harvested in MAC and tested
immediately in cytotoxicity assays. For /;; vitro
exposures, hemocytes were harvested from nontreated

tunicates and cultured overnight ( 1 5°C) in T-RPMI con-
taining various concentrations of toxicants before being
tested for cytotoxic activity.

Cytotoxic activities of hemocytes were tested in two
assays that used either K.562 human chronic myeloge-
nous leukemia cells or rabbit red blood cells (RRBC) as
targets. The capacity of hemocytes to kill K-562 cells was
assessed by a modification of the method of Peddie and
Smith (1993). Hemocyte suspensions were adjusted to
4 X 107 cells/ml in MAC; 50-100 n\ of these hemocyte
suspensions were then mixed in round-bottomed 5-ml
flow cytometry tubes with equal volumes of K-562 cells
(4 X 10h cells/ml) suspended in marine saline (MS;
12mA/ CaCl2-6H:O, 11mA/ KC1, 26mA/ MgCl2-
6H2O, 45 mM Tris, 38 mA/ HC1, 400 mA/ NaCl, pH
7.4). The cell mixtures were incubated at 15°C for
90 min, stained with ethidium bromide and then imme-
diately tested, using a FACScan flow cytometer, for the
uptake of red fluorescence (800 nm). Specific cytotoxic
activities were calculated as the percentage of dead K562
cells in a particular sample minus the percentage of dead
cells in controls that contained K562 cells but no hemo-
cytes. K562 cells were obtained from the American Type
Tissue Culture Collection (Rockville, MD) and were
grown in RPMI-1640 tissue culture medium. Immedi-
ately prior to their use in cytotoxicity assays, K562 cells
were conditioned to the high tonicity of MS by incuba-
tion (30 min, 20°C) in an intermediate saline solution
(12mA/ CaCl2.6H2O, 11mA/ KC1, 26 mM MgCl2-
6H20, 45 mA/Tris, 38 mA/HCl, 300 mA/NaCl, pH 7.4;
Peddie and Smith, 1993).

The ability of hemocytes to lyse RRBC was quantified
by the method of Parrinello et al. (1993). Hemocytes
were washed once through Tris-buffered saline (TBS;
10 mA/Tris, 150 mA/NaCl, pH 7.4)andresuspendedin
TBS supplemented with 10 mMCaC!2 (TBS-Ca) to yield
2 X 106 cells/ml. One-hundred microliters per well of the
suspensions were then incubated (60 min, 37°C) with an
equal volume of RRBC (4 X 107 cells/ml in TBS-Ca) in
96-well round-bottomed tissue culture plates. After in-
cubation the plates were centrifuged and 100^1 of the
resulting supernatant was transferred to flat-bottomed
plates so that the absorbance (405 nm) of hemoglobin
released from lysed cells could be quantified on a mi-
croplate spectrophotometer. Specific cytotoxic activities
were calculated as percentages relative to maximum re-
lease (4 X 10" RRBC/well in H2O) and spontaneous re-
lease (4 X 10" RRBC/well in TBS-Ca. no hemocytes)
values.

Statistical analysis

Nontreated controls were included in all experiments.
A minimum of four tunicates were tested for each dose

TUNICATES AND MARINE POLLUTANTS

65

Table I

Percentage viahililien ofhemocyles after N Jays n/in vitro or aquarium
c.v/'r >\ure in a variety oj toxicants

copper (ug/ml)
0.01 0.1 1 10 100

Treatment

viable cells ±SEM(n> 4)

Aquarium

Control (no treatment)

87.3 ± 1.6

95.0 ±0.6

TBT(IOMg/D

89.3 ± 3.4

94.3 ± 1.2"

Cu(0.5Mg/ml)

75.1 ±2.1''

93. 1 ± 2.3

Creosote (1%)

72.6 ±4.8''

96.6 + 0.7

" Tested after 6-day exposure.
" P< 0.05 v.v. nontreated control.

and time point in aquarium trials. More than three rep-
licates were tested for each dose and time point during in
vitro exposures. The statistical significance of differences
between treatments was determined by Student's two-
tailed /-test using the Statview SE+ (Abacus Concepts
Inc. Berkeley. CA) and Statworks (Cricket Software Inc.,
Philadelphia. PA) statistical packages.

Results

Effects of toxicants on hemocyte viability

The viability of hemocytes emigrating from in vitro
pharyngeal cultures was decreased by exposure to copper
or creosote (Table I). Decreases in viability were dose-
dependent, with concentrations greater than 0.001%
creosote or 0.05 ^g/ml copper significantly lowering
hemocyte survival after 8 days of exposure (P < 0.05 vs.
nontreated controls; Fig. 1). Ten percent creosote or
50 Aig/ml copper decreased viability by 37% and 48% rel-
ative to nontreated controls.

In contrast, TBT did not significantly alter hemocyte
viability at any of the doses or times tested during in vitro
exposures (P> 0.05). Moreover, the viabilities of hemo-
cytes harvested from tunicates that had been exposed to
creosote, copper, or TBT for up to 9 days in aquaria did
not differ significantly from those of nontreated controls
(P> 0.05; Table I).

Alteration of proliferative activity

Exposure to toxicants profoundly affected the uptake
of 3H-thymidine by pharyngeal explants (Table II).
Exposure to TBT in vitro was the only treatment that did
not significantly alter proliferation at any of the doses or
times tested (P > 0.05; Fig. 2A). TBT treatment in
aquaria reduced proliferative activity by as much as 62%
after 6 days of exposure ( 10 ^g/1; Fig. 2A)and copper in-
hibited 3H-thymidine incorporation by up to 82%

100

90-

1 80H
o

25 70-\

CO

60-
50-

40

—a — creosote
---*--- copper

0.01
creosote

100

Figure 1. The percentage of viable cells (±SEM. n > 4) in the pop-
ulation of hemocytes that had emigrated from tunicate pharyngeal ex-
plants after 8 days of ~ in vitro exposure to various doses of creosote and
copper.

(0.5 fig/ml, day 8; Table II) when compared to non-
treated controls.

The effects of creosote on proliferation were both
time- and dose-dependent. After 4 days of exposure to
high doses of creosote in aquaria (> 1%), proliferative ac-
tivity was significantly increased relative to nontreated
controls (P < 0.05; Fig. 2B). In contrast, treatment with
0.5% creosote had no effect after 4 days of exposure but
significantly enhanced 3H-thymidine uptake (252% vs.
controls) after 8 days. At this latter time point, prolifera-
tive activity in tunicates exposed to high doses (> 1% cre-
osote) had subsided. Significant effects of TBT on prolif-
eration were evident after 4 days of exposure and did not
subside over time (Fig. 2A).

Table II

Effect on hematopoict/c cell proliferation oj exposure for 8 days to
various toxicants either i n vitro or in aquaria: data are represented as
percentages of1 II- ihymidinc uptake detected in nontreated controls

Treatment

% of nontreated control1 ± SEM (n > 6)

Aquarium

TBT(10Mg/D
Cu(0.5Mg/ml)
Creosote (0.5%)

124.1 ±23.2"
31.6±4.2
71.3 ±8.0

38.8±7.lrt
1 7. 7 ±4.8
252.1 ±2.1

" P> 0.05 r.v. nontreated controls.
'' 6-day exposure.

1 Mean 'H-thymidine uptake by nontreated controls = 987 ± 23
cpm.

66

D. RAFTOS AND A. HUTCH1NSON

A. TBT

^

180 -i

8

o

160-

T

o

"o

140-

T ^*--

°^

120-

l^ "^^^-^

f

100-

*.,.

0

~ ^

CC

CD

80-

\

03

60-

,---

2

40-

— a — in vitro \ .-'

8

Q.

---*--- aquarium "

0.1 1 10 100

TBT(ug/l)

B. Creosote

snn

150

100-

50-

0

ing/i

10ug/l
1 00 ng/l

0 2 4 6 8 10
period of exposure (days, aquarium)

aoo

mnn

-^— ^ (J\JU

8

T

— a — in vitro *

I UUU "

— • — 0.5%

0

/

HDD-

T ---*--- 1%

B 60°-

^. aquarium <

ouu

• _._^._ CO/

/ s 3/o

jO

'

/ '\

'

600-

1 \

j \

•f" 400-

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> \

t3

CC

/'I

400-

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! \

CD

/

! ,*«

~ 200-

/

' ' *•» \

2

/

200-

//' ^^

^

"** — X

m — 4- -^ N\5

o

B a

J

X 0

CL U

i i i i

•

1 1 1 1 1 1

0.5 1

creosote (%)

0 2 4 6 8 10
period of exposure (days, aquarium)

Figure 2. Proliferative activity (percentage of nontreated control values ± SEM, n > 4) in pharyngeal
explants that were treated with various concentrations of (A) TBT or (B) creosote by either in vitro or
aquarium exposure and harvested from tunicates after various periods of exposure. Uptake by controls:
Day 2 = 322 ± 47 cpm; Day 4 = 874 ± 7 I cpm; Day 8 = 944 ± 79 cpm.

f/^kts 0/Vax/caw/s on phagocytosis

Incubation of hemocytes with copper in vitro did not
significantly (P > 0.05) alter phagocytic activity (Table
III). However, copper substantially decreased phagocyto-
sis (62% vs. control, 8 days, 0.5 Mg/ml) during aquarium
exposures. Exposure to 10 ^g/1 TBT in vitro significantly
(P < 0.05) stimulated phagocytosis, whereas hemocytes
taken from tunicates that had been treated with TBT in
aquaria had lower phagocytic activities than those of non-
treated controls (Table III). Creosote exposure signifi-
cantly reduced phagocytosis after in vitro treatment, but
greatly enhanced the phagocytic activity of hemocytes
from tunicates exposed in aquaria (Table III).

The effects of in vitro TBT treatment on phagocytosis
were dose-dependent (Fig. 3 A). //; vitro doses of TBT
ranging from 1 to 10 /ug/1 enhanced phagocytosis by up
to 220% relative to nontreated controls (P < 0.05),
whereas a higher dose ( 100 Mg/1) reduced phagocytic ac-
tivity to 51%> of its normal level (P < 0.05 v.v. control).
Other treatments consistently enhanced (creosote in
aquaria) or inhibited (creosote in vitro, TBT in aquaria)
phagocytosis over the range of doses tested (Fig. 3 A).

Modulation of phagocytic activity by toxicants was
also time-dependent (Fig. 3B). Alterations of phagocyto-
sis resulting from treatment with both TBT and creosote
in aquaria were most pronounced after short periods of
exposure (creosote = 314% of control value, day 2; TBT

TUNICATES AND MARINE POLLUTANTS

67

Table III

hagocytic activities, relative to nontreaied controls, "I ninicute
<:mocvle\ thai had been exposed to various toxicants for either 24 h in

Phag(

hem

vitro or S days in aquaria

% of nontreated control1 ± SEM (n > 4)

Treatment

In vitro

Aquarium

TBT(10Mg/D
Cu(0.5ng/ml)
Creosote (1%)

220.1 ±3.2
89.2 ± 8.4"
56.8+ 12.5

75.6 ±3.1"
37.4 ± 15.7
196.5 ±2 1.1

" 6-day exposure.
a P> 0.05 v.v. nontreated controls.

1 Mean phagocytic activity in nontreated controls = 0.43 ± 0.11
yeast ingested per hemocyte.

= 69.4% of control value, day 3) and returned to levels
similar to control values after longer treatment times
(creosote = 148% of control, 8 days; TBT = 105%. of con-
trols, 9 days).

Cvtotoxic activities after toxicant treatment

Creosote was the only toxicant that significantly
affected the cytotoxic activity of hemocytes toward either

K562 or RRBC targets (Table IV). Exposure to creosote
in aquaria inhibited cytotoxic activity toward K562 cells
in a dose-dependent fashion (Fig. 4A). Treatment with
5% creosote for 8 days reduced anti-K.562 cytotoxicity
to levels that did not differ significantly from those of
negative controls in which hemocytes and K562 cells
were mixed at 4°C (P > 0.05; Fig. 4A). Significant (P <
0.05) inhibition of cytotoxic responses toward K.562 cells
was evident at all of the creosote doses tested (Fig. 4 A).
A different pattern emerged in assays that used RRBC as
targets. Exposure to creosote doses greater than 0.5% for
8 days significantly enhanced the cytotoxic activity of he-
mocytes toward RRBC (Fig. 4A).

Figure 4B shows that the inhibitory effect of creosote
on cytotoxicity toward K.562 cells was reversible. The cy-
totoxic activity of hemocytes from tunicates that had
been exposed to 5% creosote for 4 days and then trans-
ferred to toxicant-free seawater for a further 4 days
(13.9% specific cytotoxicity) did not differ significantly
from those of nontreated controls (15.3% specific cyto-
toxicity; P > 0.05). However, it was significantly greater
than the cytotoxic activities evident among hemocytes
taken from tunicates that had been exposed to 5% creo-
sote continuously for 8 days (5.4% specific cytotoxicity;

£ 400-

T3
0)

'5

" 300-
o

o

£

>,

o

CO

200-

100-

o

en
CO

— -&-—

cresosote 24 h, in vitro
creosote 2 day, aquarium
TBT 24 h, in vitro
TBT 3 day, aquarium

350

B

300-
250-
200-

150-

100

50

0

I 0.1 1 10 100 1000
creosote dose (%) / TBT (ug/l)

creosote, 5%
TBT, 10ug/L

T
»-

1

2 4 6 8 10
period of exposure (days)

Figure 3. Phagocytic activities (percentage of nontreated control values ± SEM. n > 4) of tunicate
hemocytes that were (A) exposed to various doses of creosote or TBT overnight in vitro or harvested from
tunicates that had been treated with creosote or TBT for 2 or 3 days in aquaria, or (B) harvested from
tunicates after various periods of exposure to 5% creosote or 10 Aig/l TBT in aquaria.

68

D. RAFTOS AND A. HUTCHINSON

Table IV

Effect iij r treatment with various toxicants either overnight fin vitro
exposure) or for S clays (aquarium exposures) on cytoloxic activity of
tunicate hemocyles toward k-562 or rabbit red blood cells (RRBC)

% of nontreated control1 ± SEM (n > 4)

Treatment

//; vitro/
K.562

Aquarium/
RRBC

Aquarium/
K.562

TBT(10Mg/D
Cu(0.5Mg/ml)
Creosote (1%)

nt"
85.8 ± 5.2
nt

121.1 ± 14.3

96.8 ±6.4
138.9 + 6.9*

119.0 + 26.9"
108.1 ±21.3
53.6 ± 15.4*

" Not tested.
" 6-day exposure.
* P < 0.05 vs. nontreated control.

v Mean cytotoxic activities for nontreated controls = 43.6 ± 6.1 for
K562and21.2±3.1 for RRBC.

Effect of creosote on the frequency of hemocyte
subpopulations

Creosote was the only toxicant that affected the fre-
quencies of hemocyte subpopulations in tunicates ex-
posed to toxicants in aquaria (Fig. 5). Three distinct sub-
populations of hemocyte were identified by analysis of
forward angle v.v. 90° light-scatter plots. They were desig-
nated small (low forward scatter), large (high forward
scatter, low 90° scatter) and large granular (high forward
and high 90° scatter) hemocytes. Treatment with creo-

sote significantly decreased the frequency of large granu-
lar hemocytes relative to the other hemocyte subpopula-
tions. The frequency of large granular hemocytes was re-
duced by two-thirds relative to nontreated controls after
4 days of exposure (P < 0.05; Fig 5A). This effect was
apparent within 2 days of exposure to high doses of cre-
osote (5%) and was evident for all of the doses tested
within 4 days (Fig 5A). There was a corresponding in-
crease in the frequency of small hemocytes: after 4 days
about two times more small hemocytes were present
within treated tunicates than in nontreated controls (Fig
5B). The frequency of large hemocytes did not vary from
control levels at any of the doses or times tested (Fig 5B).

Discussion

This study has demonstrated that sublethal contami-
nation by common estuarine pollutants can significantly
alter innate immunological reactions in tunicates. The
three compounds tested here (TBT, copper, and creo-
sote) are particularly relevant. All three are major com-
ponents of antifouling films that are used to prevent the
growth of sessile invertebrates. TBT can leach from ma-
rine paints and accumulate to hazardous levels in har-
bors and marinas (Bryan et ai. 1986; Huggett el ai,
1986). It is known to inhibit chemiluminescence re-
sponses, chemotactic activity, phagocytic oxidative
bursts and phagocytosis by fish leukocytes (Rice and
Weeks. 1991; Warinner et al., 1988; Weeks et ai. 1986).

B

D K562

•B 40-

'x

0

^~

D RRBC

T

1,30-

o

0

T

T

8 20-

Q.

_z^

T

55 10-
n-

T

T

0.5 1

creosote

%)

20

15-

10-

5-

4°C

not
exposed

5%
creosote

4 day
recovery

Figure -4. (A) Cytotoxic activities (percentage of nontreated control values ± SEM, /; = 4 )ol hemocytes
taken from tunicates that were exposed to various doses of creosote in aquaria for 8 days: 4°C represents
hemocytes from nontreated controls that were mixed with targets at 4°C to suppress cytotoxic activity. ( B)
Cytotoxic activities (percent specific cytotoxicity toward K562 cells + SEM. n = 4) of hemocytes harvested
from tunicates that had either been held in aquaria without creosote (not exposed), exposed to 5% creosote
in aquaria for 8 days (5% creosote), or exposed to 5% creosote in aquaria for 4 days and then transferred to
fresh seawater without creosote for a further 4 days (4-day recovery).

TUNICATES AND MARINE POLLUTANTS

69

B

^a-

5%

40-

creosote (%)

1%

— •-•-

0.5%

c

0

H 30-

T ..I. -~

*~' "->.

0%

1

0

CL
0)

\

0

o

™, t

t on

\ >

CD ^U

\ ,

.c

\ *(
\«
\\

T

2

\J ,.-.-

o

.^

5? 10-

~f—^^;

-a

1 1

n

2 4 6 8 10
exposure period (days)

60

50-

40-

30-

20-

10-

•
1

0.1

1
creosote (%)

10

FigureS. Frequencies (percentages of the total hemocyte population ± SEM. n = 4) of (A) large granu-
lar hemocytes in hemolymph harvested from tunicates after various periods of exposure to a range of
creosote concentrations in aquaria, or (B) small, large, and large granular hemocytes harvested from tuni-
cates that been exposed to various doses of creosote for 4 days in aquaria.

Although the use of TBT on pleasure craft is now pro-
hibited widely, it is still applied to larger vessels and re-
mains a common harbor contaminant (NSW Environ-
ment Protection Authority, pers. comm.; Stebbing,
1985). For use on small craft, TBT has been superseded
by copper-based antifouling products. Like TBT. copper
is known to have substantial immunological effects on
aquatic organisms at subacute doses (Roales and Perl-
mutter. 1977). Creosote is a hydrocarbon-based protec-
tive coating that is frequently used on pylons, wharves,
and netting. It is composed of about 85% PAH. 10% phe-
nolic compounds, and 5% heterocyclic compounds (Fai-
sal et ai, 1991). PAHsin particular have been associated
with a variety of physiological effects including carcino-
genesis and the alteration of immune reactivity in hu-
mans and other animals (Faisal et ai, 1991: National Re-
search Council, USA, 1972). In fish, PAHs are responsi-
ble for the development of eye lens cataracts, gill
necrosis, degeneration of renal epithelia, and neoplasia.
They can also inhibit macrophage function, cellular cy-
totoxicity, and cellular proliferation (Faisal el ai. 1991 ).
The range of doses that were tested in this study include
levels that can frequently be found in the environment.
TBT concentrations >2 ^g/1 have been detected in ma-
rine areas (Waldcock and Miller. 1983). PAH levels

greater than 10 times the maximum dose used here are
reportedly common for harbor waters, and levels of cop-
per up to 2.5 pg/ml have been detected in heavily utilized
environments (Hyland and Scheider. 1976; Pluarg In-
ternational Joint Commission. 1978; Waldhauer et at,
1978).

Only high doses of copper (>5 Mg/ml) were acutely le-
thal to tunicates. However, all of the toxicants tested here
exerted powerful effects on at least some immunological
reactions (summarized in Table V). The ability of toxi-
cants, particularly creosote, to simultaneously affect a
variety of apparently unrelated parameters, such as
phagocytosis, proliferation, and cellular cytotoxicity,
does not simply reflect general morbidity or metabolic
downturn resulting from toxicant poisoning. The obser-
vation that toxicants enhanced some hemocyte-medi-
ated responses but inhibited or had no effect on others
confirms that tunicate cells were not generally inacti-
vated by toxicant treatment. There is also no evidence
that any of the toxicants affected the viability of cells
from tunicates that were exposed in aquaria (except after
death at lethal doses of copper), even though both creo-
sote and copper decreased hemocyte viability in vitro.
This discrepancy has three plausible explanations. First.
the observed differences in hemocyte viabilities may

70

D. RAFTOS AND A. HUTCHINSON

have been due to the rapid clearance of dead hemocytes
//; vivo. Those dead cells may not have appeared in the
circulating hemolymph, and so may not have been de-
tected in viability assays. Second, tunicates could possess
mechanisms to detoxify, sequester, or prevent the pene-
tration of copper and creosote in vivo. This possibility is
not, however, supported by the observation that some
immunological reactions were similarly affected by iden-
tical doses of toxicants applied in vitro and in aquaria
(e.g.. copper's inhibitory effect on cell proliferation).
Third, differences between in vitro and aquarium trials
might have been due to the existence of compensatory or
interactive mechanisms that cannot operate in isolated
/// vitro systems.

The latter explanation is supported by differences that
were evident between the effects of in vitro and aquarium
exposures on immunological parameters such as phago-
cytosis, cell proliferation, and cytotoxicity. For instance,
in ritm creosote treatment inhibited phagocytic activity
and cell proliferation, whereas tunicates treated with cre-
osote in aquaria had an enhanced capacity for phagocy-
tosis and a transient increase in proliferative activity.
Such contrasting results indicate that some effects in vivo
may result from interactive mechanisms rather than
from direct toxicity toward the response being examined.
Creosote poisoning, for instance, may have stimulated
regulatory activity that specifically enhanced phagocyto-
sis and proliferation. Mechanisms that are capable of
such cellular regulation are well characterized in tuni-
cates. Regulatory molecules in the hemolymph can en-
hance phagocytosis and cell proliferation in a manner
analogous to the activities of vertebrate cytokines (Beck
etai. 1993;Raftos, 1994; Raftostf ai. 1991).

The data also suggest that tunicates have mechanisms

Table V

Summary of the effects of different toxicants on a ninety ol responses
thai were tested either in vitro (vit) or hy aquarium exposure

Toxicant/treatment

TBT

Creosote

Copper

Responses tested

vit aqu

vit

aqu

vit

aqu

Cell viability

0 0

1

0

J

0

Phagocytosis

4_t

I

t

0

1

Proliferation

0 J

1

«-»

J

I

Cytotoxicity (K562)

0

—

I

0

0

Cytotoxicity (RRBC)

0

—

t

—

0

t = enhanced by toxicant treatment; , = inhibited by toxicant treat-
ment; «-» = enhanced or suppressed depending upon dose or time of
exposure; 0 = no significant alteration relative to nontreated con-
trols; — = not tested.

that can confer some degree of adaptive protection, and
allow recuperation, from the effects of toxicants. The ex-
istence of mechanisms for adaptive protection is indi-
cated by the kinetics of toxicant poisoning. Alterations in
the levels of phagocytosis among tunicates treated with
creosote or TBT in aquaria were short lived. Phagocytic
activity returned to more-or-less normal levels after
8 days of continuous exposure. This amelioration can be
explained in two ways. First, creosote and TBT may have
become detoxified over the 8-day exposure period. Sec-
ond, adaptive mechanisms that reduced the effect of tox-
icants on phagocytosis may have been activated. The lat-
ter possibility is supported by evidence indicating that
both creosote and TBT retain their toxicity toward other
immunological reactions for at least 8 days. The exis-
tence of processes that allow recuperation from the
effects of toxicants is indicated by the rapid recovery of
cytotoxic activity when tunicates are transferred from
contaminated to fresh seawater. There are many possible
mechanisms by which toxicants could gain their effects,
or by which tunicates might adaptively ameliorate those
effects. For instance, the observed recuperation of some
reactions from poisoning may reflect the existence of in-
ducible detoxication mechanisms that directly affect tox-
icant bioavailability. Inducible low molecular weight
metal-binding proteins (metallothioneins) that can bind
and detoxify a variety of metals such as copper and tin
have been identified in many species (George, 1990).

An alternative explanation for the varied effects of tox-
icants is that alterations in the frequencies of specific
effector cells are responsible both for altering immune
functions and for the later recovery of those immune re-
actions from poisoning. In fish, it has been reported that
the frequencies of specialized, ion-transporting chloride
cells vary significantly during copper poisoning as those
cells migrate to and from fixed tissues (Pelgrom et ai.
1995). Similarly, the frequency of large granular hemo-
cytes in 5. plicata decreased rapidly upon exposure to
creosote. The large granular hemocyte population in-
cludes vesiculated cell types that have been implicated in
wound repair and that become localized in fixed tissues
during inflammation-like responses (Goodbody, 1974;
Wright, 1981). These cells may have been sequestered in
fixed tissues damaged during creosote treatment, and so
may have altered the relative mix of hemocyte types in
the hemolymph. The loss of large granular hemocytes
from the circulation may also have activated compensa-
tory hematopoietic cell proliferation. Such hematopoi-
etic proliferation was detected directly by increased 3H-
thymidine uptake and is also indicated by the specific
increase observed in the frequency of small hemocytes, a
population that includes immature hemoblasts derived
from hematopoiesis (Ermak, 1982; Rinkevich and Rab-

TUNICATES AND MARINE POLLUTANTS

71

inowitz, 1993; Wright, 1981). The regulatory activity
that may have activated cell proliferation might also
have affected phagocytic activity. Tunicate cytokine-like
molecules have pleiotropic effects that include the simul-
taneous activation of phagocytosis and proliferation
(Beck el a/.. 1993; Raftos, 1994; Raftos et at.. 1991). This
role of altered hemocyte frequencies on immune func-
tions remains speculative, but is currently being tested.

In conclusion, it is clear that environmental contami-
nants have profound effects on immunological reactions
in tunicates. Those effects are unlikely to be the result of
general morbidity, and they are not reflected by acute
lethality. However, because of the implicit relationship
between innate immune reactions and antipathogenic
defenses, it is likely that the effects demonstrated here
alter the capacity of tunicates to defend themselves
against infection. The relevance of these effects to the vi-
ability of tunicate populations remains unclear. Little is
known about the level of surveillance that is required by
tunicates for survival. We are investigating that relation-
ship between innate or natural immunological compet-
ance and long-term population health by testing the
effects of environmental toxicants on the ability of tuni-
cates to deal with artificial and natural infections.

Acknowledgments

This study was funded in part by a grant from the Aus-
tralian Research Council and an Internal Research
Grant from the University of Technology, Sydney. We
thank Monika Burandt for help with field work and for
other technical assistance.

Literature Cited

Anderson, D. P., G. \V. Bryan, P. E. Gibbs, L. C. Hummerstone, and
G. R. Burl. 1990. Immunological indicators: effects of environ-
mental stress on immune protection and disease outbreaks. Am
f-'txli. Soi: Symp. 8: 38-50.

Beck, G., R. F. O'Brien, G. S. Habicht. D. L. Stillman, E. L. Cooper,
and D. A. Raftos. 1993. Invertebrate cytokines III: Interleukin-1-
like molecules stimulate phagocytosis by tunicate and echinoderm
cells. Cell Immunol 146:284-299.

Berrill, N. J. 1950. The Tunicata. The Ray Society. London.

Bryan, G. \V.. P. E. Gibbs, L. C. Hummerstone, and G. R. Burl. 1986.
The decline of the gastropod, Nucella lapu/iis. around South-West-
ern England: evidence for the effect of tributyltin from antilouling
paints. / Mar. Biol Assoc t'.A'. 66: 61 1-640.

Cooper, E. I.., and P. Roch. 1992. The capacities of earthworms to
heal wounds and to destroy allografts are modified by polychlori-
nated biphenyls(PCBs). J Inverlehr Pallwl. 60: 55-63.

Ermak, T. H. 1982. The renewing cell populations of ascidians. Am.
Zool. 22:795-805.

Eaisal, M., M. S. M. Marzouk, C. L. Smith, and R. J. Iluggett. 1991.
Mitogen induced proliferative responses from spot exposed to poly-

cyclic aromatic hydrocarbon contaminated environments. Immii-
nopharmacol. Immunotoxicol, 13: 31 1-327.

Fit/patrick, L. C., R. Sassani, B. J. Venables, and A. J. Goven. 1992.
Comparative toxicity of polychlorinated biphenyls to the earth-
worms Eisenia foenda and Lumhriciis lerrestns. Environ. Pollut.
77: 65-79.

George, S. G. 1990. Biochemical and cytological assessments of
metal toxicity in marine animals. Pp. 123-142 in Heavy Aletalx in
llic Environment. R. W. Furness and P. S. Rainbow, eds. CRC
Press. Boca Raton, FL.

Giam, G. S., and L. E. Ray. 1987. Pollution Studies in Marine Ani-
mals. CRC Press. Boca Raton, FL.

Goodbody, I. 1974. The physiology of ascidians. Pp. 1-149 in Ad-
vances in Marine Biology. F. S. Russel and M. Yonge, eds. Aca-
demic Press. London.

Hyland, J. L., and E. D. Schneider. 1976. Petroleum hydrocarbons
and their effects on marine organisms, populations, communities
and ecosystems. Pp. 61-76 in Sources. Effects, and Sinks of llv-
drocarbons in I he Aquatic Environment. Proc. Symp. AIBS, Wash-
ington. DC.

Huggett, R. J., M. E. Bender, and D. J. VVestbrook. 1986. Organotin
concentrations in the Southern Chesapeake Bay. Pp. 12-62 in
Oceans 86 Proceedings. Organotin Symposium. IEEE Publishing.
New York.

Kelly, K., E. L. Cooper, and D. A. Raflos. 1993. A humoral opsonin
from the solitary urochordate. Stye/a clam. Dev. Comp. Immunol.
17:29-39.

Landis, W. G., and M.-H. Yu. 1995. Introduction In Environmental
Toxicology Lewis. Boca Raton. FL.

McCarthy, J. F., and L. R. Shugart. 1990. Biomarkers oj ' Environ-
mental Contamination. CRC Press. Boca Raton. FL.

National Research Council, U.S.A. 1972. Paniculate Polrcyclic Or-
ganic Mailer. U.S. National Academy of Sciences. Washington.
DC.

Parrinello, N., V. Arizza, M. Cammarata, and D. M. Parrinello. 1993.
Cytotoxic activity of dona intestinalis hemocytes: properties of the
in vitro reaction against erythrocyte targets. Dev. Comp. Immunol.
17: 19-27.

Peakall, D. 1992. Animal Biomarkers as Pollution Indicators. Chap-
man and Hall, New York.

Peddie, C. M., and V. J. Smith. 1993. //; vitro spontaneous cytotoxic
activity against mammalian target cells by hemocytes of the solitary
ascidian, Ciona intestinalis. J. E.\p. Zool. 269: 616-623.

Peddie, C. M., and V. J. Smith. 1994. Blood cell mediated cytotoxic
activity in the solitary ascidian, C. intestinalis. Ann. A'. }'. Acad. Sci.
712:332-340.

Pelgrom, S. M. G. J., R. A. C. Lock, P. H. M. Balm, and S. E. \\ende-
laar Bonga. 1995. Integrated physiological response of tilapia, Or-
eochromis mossambicus. to sublethal copper exposure. Aaual. To.\-
icol. 32: 303-320.

Pluarg International Joint Commission. 1978. Environmental man-
agement strategy for the Great Lakes. International Reference
Group on Great Lakes Pollution from Land Use Activities. Wind-
sor, Ontario, Canada.

Raftos, D. A. 1994. Allorecognition and humoral immunity in tuni-
cates. Ann. A'. Y Acad Set. 712: 227-243.

Raftos, D. A., and E. L. Cooper. 1990. //; vitro culture of tissues from
the solitary tunicate, Slyela clava In I 'itro Cell. Dev Biol. 26: 962-
970.

Raftos, D. A., and E. L. Cooper. 1991. Proliferation of lymphocyte-
like cells from the solitary tunicate, Slyela clava. in response to al-
logeneic stimuli. 7. E\p. ~/.ool. 260: 391-400.

72

D. RAFTOS AND A. HUTCHINSON

Raftos, D. A., E. L. Cooper, G. S. Habicht, and G. Beck. 1991. Tuni-
cate cytokines: tunicate cell proliferation stimulated by an endoge-
nous hemolymph factor. Pmc. Null. Arad. Sri. U.S.A. 88: 9518-
9522.

Rice, C. D., and B. A. Weeks. 1991. Tributyltin stimulates reactive
oxygen formation in toadtish macrophages. Dev Co/up. Imtnunol.
15:431-436.

Rinkevich, B., and C. Rabinowitz. 1993. //; vilro culture of blood cells
from the colonial protochordate Bolry/lus .sc/;/n.v.vc/v //; I 'itro Cell.
Dc\- Biol. 29A: 79-85.

Roales, R. R., and A. Perlmutter. 1977. The effects of sub-lethal doses
of methyl mercury and copper, applied singly and jointly on the
immune response of blue gourami to viral and bacterial antigens.
Arch. Environ. Contain. To.\k'ol. 5: 325-331.

Rodrigues-Grau, J., B. J. Venables, L. C. Kitzpatrick, and A. J. Goven.
1989. Suppression of secretory rosette formation by PCBs in
Lumhncits terrestris: an earthworm assay for humoral immunotox-
icity of xenobiotics. Environ. Toxicol. Chan. 8: 1201-1207.

Sarot, D. A., and A. Perlmutter. 1976. The toxicity of zinc to the im-

mune response of the zebra fish (Brachydanio rano) injected with
viral and bacterial antigens. Trans. Am. Fish. Soc. 105: 456-459.

Stebbing, A. R. D. 1985. Organotins and water quality — some les-
sons to be learned. Mar. Pollul. Bull. 10: 383-390.

W aldcock, M. J., and D. Miller. 1983. The determination of total
and tributyltin in seawater and oysters in areas of high pleasure craft
activity. ICES Papers CM 1983/E:I2 (mimeograph). International
Council for the Exploration of the Sea. Copenhagen.

\\ aldhauer, R. A., A. Matte, and R. E. Tucker. 1978. Lead and cop-
per in the waters of Raritan and Lower New York Bays. Mar. Pollut.
Bull. 9: 38-42.

\\arinner, J. E., E. S. Mathews, and B. A. Weeks. 1988. Preliminary
investigations of the chemiluminescent response in normal and pol-
lutant-exposed fish. Mar. Environ. Res. 24: 281-284.

Weeks, B. A., P. L. Warinner, J. Mason, and D. S. McGinnis. 1986.
Influence of toxic chemicals on the chemotactic responses of fish
macrophages. J. l-'ish Biol. 28: 653-658.

Wright, R. K. 1981. Urochordates. Pp. 565-627 in Invcrlehrale
Blunt! Cell* N. A. Ratcliffe and A. F. Rowley, eds. Academic Press,
London.

Reference: fl/o/. Bull. 192: 73-86. (February, 1997)

Prey Capture by the Sea Anemone Metridium senile (L.):

Effects of Body Size, Flow Regime,

and Upstream Neighbors

KENNETH R. N. ANTHONY*

The Roval Swedish Academy of Sciences. Kristineberg Marine Research Station, Kristineberg 2130,

S-450 34 Fiskebackskil, Sweden

Abstract. The sea anemone Metridium senile is a
quantitatively important passive suspension feeder in
hard-bottom communities on the west coast of Sweden
and occurs in aggregations with different size distribu-
tions. This study tests the hypothesis that different polyp
sizes have different optimal flow regimes maximizing
prey capture. Results showed that prey capture by M.
senile is a function of both flow regime and polyp size,
and different optimal flow regimes exist for different size
classes. Large anemones had a maximum feeding effi-
ciency at the slowest flow, medium-sized anemones at
moderate flow, and small anemones at moderate- to
high-flow regimes. Small anemones showed consistently
higher feeding rates (per unit of biomass and area of ten-
tacle crown) at all velocities above 10 cm s~ ' and exhib-
ited less flow-induced deformation of the tentacle crown,
suggesting that small anemones are better at feeding in
moderate- to high-flow habitats. Different vertical pro-
jections of large and small anemones in the boundary
layer could only partly account for differences in feeding
success among size classes. Feeding rate was also a func-
tion of upstream conspecifics, declining asymptotically
to 30% of the maximum rate. The negative effects of
neighbors on feeding in aggregations with clonal rather
than polyp growth appear to be compensated for by the
generally higher feeding efficiency of small polyps.

Introduction

Flow habitat and body size are both important factors in
the feeding biology of benthic, passive suspension feeders.

Received 1 8 October 1995: accepted 25 October 1996.
* Current address: James Cook University of North Queensland, De-
partment of Marine Biology, Townsville Qld. 4811. Australia.

Due to the nature of near-bottom hydrodynamics, how-
ever, flow exposure and body size are inseparable factors
(Koehl, 1977a; Vogel, 1981). As suspension feeders grow
taller they become exposed to faster currents within and
beyond the substratum-associated boundary layer, and
consequently to both greater drag and higher flux of poten-
tial prey (Wainwright and Koehl, 1976; Vogel, 1981). The
height of a passive suspension feeder in a given flow habitat
is therefore likely to involve a trade-off between maximiz-
ing food availability and minimizing flow forces and phys-
ical stress. In soft-bodied passive suspension feeders such as
sea anemones, limitations as to how tall an individual can
grow in a high-flow environment may, in part, be governed
by the extent to which flow forces cause deformation of
the feeding apparatus (Koehl, 1976, 1977a), reducing prey
capture and thus energy for growth and reproduction (Seb-
ens, 1979, 1981, 1982; Lesser el al, 1994). Vertically ori-
ented passive suspension feeders of different sizes are there-
fore likely to have different ranges of optimal flow velocities
at which the physical stress is minimized and potential for
prey capture is maximized. Many sessile cnidarians are,
however, capable of polyp degrowth and indeterminate
growth of the genet, in part through variable extents of
asexual proliferation such as pedal laceration (Bucklin,
1987; Anthony and Svane, 1995), polyp fission (Francis,
1979;Sebens, 1980), and colony fission (McFadden, 1986).
These capabilities result in size plasticity of the polyp (or
colony) as well as of the clone. Small clones of large polyps
(or colonies) in low-flow habitats and large clones of small
polyps (or colonies) in high-flow habitats constitute alterna-
tive solutions for maximizing prey capture and the positive
energy balance of the genet. In the latter case, however, the
extent to which the relationship between polyp size, flow,
and prey capture is modified by the presence of neighbor-

73

74

K. R. N. ANTHONY

ing conspecifics (or clone members produced by asexual
reproduction) has been demonstrated in only a few passive
(octocorals: McFadden, 1986) and active (phoronids:
Johnson, 1990; bryozoans: Okamura, 1992) suspension
feeders.

In this paper, the sea anemone Metridium senile is
used for testing the effects of flow, polyp size, and up-
stream neighbors on the feeding success of soft-bodied,
passive suspension feeders. Five features render M senile
a good model for such studies: ( 1 ) The projected area of
the tentacle crown increases more-or-less isometrically
with body size (Sebens, 198 1 ): (2) new tentacles of a de-
termined size are added to the tentacle crown as the
anemone grows (Sebens, 1979), so that the spacing and
size of filtering elements are the same for large and small
anemones; (3) the size of prey (zooplankton) taken does
not increase significantly with anemone size (Purcell,
1977; Sebens, 1981; Sebens and Koehl, 1984); (4) it is
capable of polyp degrowth (in part through asexual re-
production by pedal laceration) and thus has an intrinsic
potential for adjusting its body size to the flow environ-
ment; and finally (5) it often forms dense, clonal aggre-
gations (Anthony and Svane. 1994) in which prey cap-
ture per polyp is likely to be influenced by the presence
of adjacent clonemates.

Metridium senile is a ubiquitous member of the hard-
bottom community of most northern waters (Hoffmann,
1976; Fautin el ai, 1989) and occurs in different size dis-
tributions in different flow environments (Hoffmann,
1976, 1986; Shick et ai. 1979; Shick and Hoffmann,
1980; Anthony and Svane, 1994). For example, in high-
flow channels on the west coast of Sweden where the cur-
rent velocity frequently exceeds 100 cms"1, the walls
and bottoms are carpeted by dense populations of small
(2-3-cm-high) M. senile. Conversely, in low-flow habi-
tats where the current rarely exceeds 5 cm s~', large (20-
30-cm-high) M. senile predominate (Anthony and
Svane, 1994, 1995). In moderate-flow habitats, popula-
tions of M. senile are generally composed of intermedi-
ate-sized anemones (Shick, 1991; and pers. obs.). Previ-
ous studies have suggested that differences in size distri-
butions of M. senile can be attributed to the physical
effects of flow on the anemone body plan ( Koehl, 1 977a;
Shick and Hoffmann, 1 980); genetic differences between
local populations or clones (Shick el til., 1979; Shick and
Dowse, 1985; Hoffmann, 1986; Anthony and Svane,
1994); and effects of substratum instability, which influ-
ence the rate of laceration and thereby inversely affect
polyp and clone size (Anthony and Svane, 1995). Trade-
offs between polyp size and prey capture in different flow
regimes are likely to structure populations of this ecolog-
ically and morphologically plastic species (Shick, 1991).
Nevertheless, no studies have experimentally tested the
hypothesis that different polyp sizes of M. senile have

different optimal flow regimes that reflect the size distri-
butions in natural populations.

The objectives of this study are to ( 1 ) quantify prey cap-
ture in M. senile at a range of polyp sizes and flow veloci-
ties, (2) test whether combinations of polyp size and flow
regime that maximize prey capture reflect size-frequency
distributions found in different flow habitats in situ, and
(3) test whether prey capture in M. senile is, in part, a
function of the number of upstream conspecifics.

Materials and Methods

All experiments were conducted in a laboratory flume
(300-cm long, 47-cm wide, 18-20-cm water depth), de-
signed as described by Vogel ( 1 98 1 ). A constant flow was
ensured by controlling revolutions of the propeller by a
Panasonic 501 inverter. Flow straighteners were
mounted at the entrance of the flume channel to reduce
turbulence to a level resembling near-bed field condi-
tions. Prey used in all experiments were newly hatched
nauplii of Anemia salinn. which measure about 600 ^m
in length and are within the mid-range of prey sizes re-
ported to be taken by M, senile in the field (Sebens and
Koehl, 1984). Anemia nauplii do not show the escape
responses characteristic of some natural prey (Trager et
nl.. 1994), so their use may produce overestimates of
prey-capture rates in situ. That potential drawback was
outweighed by the advantages that the non-evasive Ar-
temia nauplii are less likely to confound flow effects on
capture rates and can be reared in replicable batches.

Flow measurements

Feeding experiments were carried out at six preset flow
regimes: 4, 10. 17, 20, 28, and 44 cm s~", measured at
10cm above the flume floor by visually tracking and
timing particles over a 25-cm trajectory within the work-
ing section of the flume. To determine the local flow ve-
locity experienced by each anemone size class in a given
flow regime, the vertical flow profile between 0.2 and 15
cm above the flume floor (1-cm intervals) was deter-
mined from a matrix of 16 X 6 point samples taken
within a plane normal to flow. These local flow velocities
were measured with a thermistor probe (LaBarbera and
Vogel, 1976) calibrated according to the method de-
scribed by Vogel (1981, p. 316). The development of a
turbulent boundary layer was evident at all flow regimes,
and the local flow velocities measured at 0.2- 1 cm above
the flume floor were generally 50% of those measured at
a height of 1 0 cm ( Uw). The flow profiles obtained in the
flume at the six experimental flow regimes are depicted
in Figure 1 .

Distribution of nauplii in the flume

A comparison of the prey-capture success of different
size classes of passive suspension feeders requires the as-

PREY CAPTURE BY SEA ANEMONES

75

O

o

GJ)

15.0

10.0

5.0

1.0

0.2

0

10

15

20

25

30

35

40

45

50

-I,

Flow velocity (cm s )

Figure 1. Flow profile as a function of flow regime in the working section of the flume. Flow regime is
referred to as the velocity measured at 10cm above the flume floor ((.',„). Error bars represent ±1 SE of 6
isobath measurements.

sumption of a uniform and constant distribution of prey
items in the water column. Therefore, deviations from
an even, vertical distribution of Anemia due to nauplii
swimming or hydrodynamic sorting were determined by
simultaneous sampling from 10 different heights above
the bottom in the working section of the flume. Samples
were collected with a comb of 4-mm acrylic pipes spaced
15 mm apart and pointing into the current, each pipe
connected to a silicone tube (see Muschenheim, 1987).
One-liter samples were taken at each height, and sam-
pling was replicated three times ( 1-h intervals) for every
flow regime. The numbers of nauplii were standardized
to a percentage distribution and tested using a replicated
G-test for goodness-of-fit (Sokal and Rohlf, 1981). Sig-
nificantly higher concentrations of nauplii were found
close to the bottom of the flume channel at Uto =
4 cms"1 (Gn = 55.4. P < 0.0001) and Vw = 10 cms'1
(G,, = 20. 1, P < 0.01), but the vertical distribution was
uniform at L'1(1 = 1 7, 20, 28, and 44 cm s' ' (P > 0.05).

Vertical feeding zone and tentacle-crown deformation

The height range of the tentacle crown above the flume
floor (measured at the uppermost (r( ) and lowermost

(r/) tentacle tips) was determined for each group of
anemones during experiments. These values were used
to compare vertical feeding zones and local flow veloci-
ties experienced by different size classes and to calculate
feeding efficiencies. Furthermore, the ratio of tentacle-
crown surface area (S) to ash-free dry weight of individ-
ual polyps (AFDW) was used to indicate and compare
the degree of tentacle-crown deformation caused by flow
forces. Ash-free dry weight was used as denominator
rather than a linear dimension because it provides the
least variable measure of size. During each feeding ex-
periment, tentacle-crown diameter (TCD, mean of two
diameters taken at right angles to each other and de-
scribed by the outermost tentacle tips) for each anemone
was measured with callipers and used in calculating ten-
tacle-crown area (S = jr(TCD/2)2).

Feeding experiments: sampling and experimental
design

Three size classes of anemones within the full size
range of M. senile were sampled in the Gullmarsfjord on
the west coast of Sweden, from three sites that experience
different prevailing current regimes. Large anemones

76

K R. N. ANTHONY

(6.0-6. 5-cm pedal disc diameter, pdd) were collected in
the central Gullmarsfjord where the current is generally
low (<5 cm s~'), medium-sized anemones (2.5-3.0-cm
pdd) were collected in a sound experiencing an interme-
diate current regime (10-15 cm s"1), and small anemo-
nes (1-2 cm pdd) were sampled in a narrow channel
where the current frequently exceeds 100 cm s~' (see also
Anthony and Svane, 1994). In the laboratory, the anem-
ones were allowed to attach to panels (terracotta tiles
measuring 10 by 20 cm, 3 mm thick) submerged in sea-
water tables. Excess anemones were removed from the
panels so that a single row of 2-4 contiguous anemones
was established on each panel.

Prior to each experiment the flume was filled with fil-
tered (<20 /urn) seawater freshly collected at 30-m depth
in the fjord (31-33%o salinity and 8-1 PC). The volume
of the system when full (20-cm depth) was 400 ±21. Two
panels with anemones attached were placed aligned on
the bottom of the flume in the working section, so that a
single row of 3-8 anemones was oriented perpendicular
to flow. The anemones were allowed 12-24 h to accli-
mate to the selected flow regime and to fully expand be-
fore the feeding experiment was run. Immediately before
an experiment, a standardized initial concentration (C\,)
of 150± 20 Anemia nauplii 1~' was obtained by carrying
out dilution series in 2000-ml beakers and calculating
batch concentrations and C0. This concentration was as-
sumed to be below that causing saturation of the anemo-
nes, since only a small percentage (<5%) of the tentacles
were occupied in prey capture at any given time. The
overall prey capture obtained in the experiments was
therefore assumed to be insignificantly affected by the C0
chosen.

So that concentration of prey could be plotted as a
function of time, five 140-ml water samples were taken
simultaneously from the upstream section of the tank at
t = 0 and after every 10 min during the following 70-
90 min. Because the total sampling volume was less than
3% of the system volume, sampling volume was not re-
placed or corrected for in the analysis. Samples were
taken using a rack of fixed pipettes (2 mm thick and
20 cm long) arranged along a horizontal transect normal
to the direction of flow and connected by silicone tubes
to a rack of syringes on the outside of the tank. Sampling
was done manually and isokinetically during 30 ± 3 s to
minimize variation due to prey patchiness. The samples
were immediately filtered through a plankton mesh (60-
Mm) to give 50-ml concentrated suspensions, and the
nauplii in each sample were counted directly using a dis-
secting microscope. The proportion of nauplii damaged
by handling and by the flume propeller was less than 5%.
To determine feeding relative to anemone biomass, the
anemones were dislodged from the panels 24 h after each
experiment, any food boluses were removed with a sy-

ringe, and anemones were frozen and freeze-dried for
48 h. After weighing, the dried anemones were com-
busted for 1 2 h at 500°C and the ash-free dry weight
(AFDW) was obtained by subtracting ash weight from
dry weight.

Analysis of feeding experiments

The natural logarithm of prey concentration was plot-
ted with time, and the expected relationship

Ln(Q = -Ft + LnC0

(1)

was fitted to the data using linear regression analysis and
tested using Pearson's product-moment correlation (So-
kal and Rohlf, 1981 ). The relationship is a linearization
of the function

C, =

(2)

describing the exponential decrease in prey concentra-
tion with time in a closed system (Leversee, 1976) where
C, and C0 are the concentrations of prey (nauplii 1 ') at
the times / and /,,. respectively, and F is the clearance
rate (min~') of the suspension feeder. F depends on the
features of the feeding structures such as the size and
density of tentacles per square centimeter of tentacle-
crown area (Sebens, 1981), orientation relative to flow
(Leversee, 1976: Johnson and Sebens. 1993), height
above the substratum (Muschenheim, 1987), and prey
availability. The total clearance rate of the group of
anemones (F,ol) was the slope of the regression line ±95%
CL, and the maximum feeding rate was determined as
Fto,C0K (nauplii min '), where I' is the water volume (1)
of the system. To account for settlement and accumula-
tion of prey that could not be explained by anemone
feeding, control experiments were conducted using
anemone mimics. To be able to compare the retention of
prey among anemone size classes in a given flow regime,
feeding rates were expressed based on ash-free dry weight
(AFDW) and square centimeter of tentacle-crown area
(S) at /,,. The empirical, maximum feeding rate per
square centimeter of tentacle-crown area was thus deter-
mined as

ino

ing

" (•'lot ~ ^ control)

.,,

(3)

and the maximum feeding rate per gram of ash-free dry
weight was calculated as

Feeding rateMax.AFDw-' =

(4)

Only the control experiment run at the lowest flow ve-
locity (4 cms"1) produced a significant depletion rate
(^"controiC()r ± CL (nauplii min"1) = 0.36 ± 0.18, P =
0.02), and this was accordingly subtracted from feeding

PREY CAPTURE BY SEA ANEMONES

77

experiments run at this flow speed. Feeding rates of
different size classes at a given flow regime were com-
pared using an unplanned comparison (the Tukey-
Kramer method; Sokal and Rohlf, 1981).

Feeding efficiencies (E) are useful in comparing feed-
ing performance among size classes as well as in compar-
ing feeding performance of a size class among flow re-
gimes, and were calculated as the number of prey items
captured relative to the number that would pass through
the space occupied by the feeding appendages if the latter
were not there (see Patterson, 1991). The maximum
number of prey items passing the projected feeding sur-
face perpendicular to flow (S,.pr, cm2) per unit time is a
product of the concentration of prey at /0(C0, nauplii 1 ' )
and the flow velocity (t/,, cm s"1) at the level of the feed-
ing structures (.r), hence

1 60s

1000cm3 min

(5)

S, pr was calculated as an ellipsoid described by the height
and width ranges of the tentacle crown (perpendicular to
the flow direction), from which the projected, transverse
sectional area of the uppermost part of the column (the
parapet; see Manuel, 1988) was subtracted. The feeding
efficiency (E) was then expressed as

E =

aplured _

FV

16.7

cm- 1

* passing

where

pr(7, s mm
= C0 VF (nauplii min"1).

(6)

Effect of upstream neighbors

To determine the effect of upstream neighbors on the
feeding rate per square centimeter of tentacle crown area,
a series of experiments analogous to the above were con-
ducted using six patch sizes of anemones. Patch size was
expressed as the number of parallel rows of anemones
aligned perpendicular to flow on the bottom of the
flume. To exclude variation caused by flow and anem-
one size, all experiments were conducted using anemo-
nes within an intermediate size range (3-4-cm pdd), and
run at the flow speed at which this size class had its max-
imum feeding efficiency (see Results). Effect of upstream
neighbors on prey capture per square centimeter of ten-
tacle crown was tested using the Tukey-K.ramer method
(Sokal and Rohlf, 1981).

Results

Vertical feeding "one and tentacle-crown deformation

The height of tentacle crowns above the flume floor
(r, from lowermost to uppermost tentacle tips), and thus
vertical feeding zone, of large anemones was 2-3 times
that of medium and small anemones, and showed a con-

sistent decline with increasing flow regime (Fig. 2). The
feeding zone of large anemones was especially affected
by flow — their mean height at the highest flow ( yMean ±
SE = 4.2 ± 0.2 cm) was only 50% of that recorded at
the lowest flow regime (vMcan ± SE = 8.2 ± 0.9 cm). A
pronounced downstream bending of anemone columns
with increasing flow occurred (Fig. 2). The three size
classes experienced different local flow velocities ( t/,, es-
timated from flow profiles in Fig. 1 ) in a given flow re-
gime because of their different positions in the boundary
layer. The uppermost tentacles of large anemones expe-
rienced about twice the flow velocity of medium and
small anemones, which were positioned deeper in the
turbulent boundary layer (Figs. 1 and 2). Differences in
local flow speeds were less noticeable among medium
and small anemones.

Tentacle-crown deformation, indicated by a decreas-
ing ratio of projected tentacle-crown area to ash-free dry
weight (S/AFDW) with increasing flow, was most pro-
nounced for large anemones (Table I). The projected
prey-capture surface of large anemones at U10 =
44 cm s"1 was only one-third of that recorded at U\Q =
4 cm s~'. Tentacle-crown deformation of small and me-
dium-sized anemones was noticeable only at flows be-
tween 4 and 1 7 cm s '. In that range they showed an ini-
tial increase in S/AFDW, presumably indicating incom-
plete tentacle-crown extension at the lowest flow
velocity. Moreover, the generally lower S/AFDW ratios
of large relative to small anemones corroborates earlier
findings (Sebens, 1981) that larger M. senile have a
smaller prey-capture surface per unit of biomass.

Feeding rates

Feeding rates of the three size classes of M. senile
showed drastic changes with increasing flow, regardless
of unit (per polyp, square centimeter of tentacle-crown
area, or gram of ash-free dry weight. Fig. 3). Feeding rates
tended to increase linearly and monotonically with flow
over the range 4- 1 7 cm s~ ' for all size classes, indicating
that the availability of prey is a function of flow in low to
moderate flow regimes. On a per-polyp basis, large
anemones captured 3-10 times more nauplii than small
and medium-sized anemones within the flow range 4-
17 cm s"1 (Fig. 3 A). Above 20 cm s'1, however, the feed-
ing rate of large anemones declined rapidly with flow and
converged with those of small and medium anemones,
which remained constant above 20 cm s^1. On a per-bio-
mass basis, the pattern was partly reversed: small anem-
ones showed 2-3 times higher feeding rates than me-
dium-sized anemones, which in turn showed higher
feeding rates than large anemones, over the flow range
10-44 cms"1 (Fig. 3B). At the slowest flow, medium-
sized anemones showed significantly lower feeding rates

78

K, R. N. ANTHONY

I

e
§

o

15

10

0

D Small, A Medium, * Large

10

20

30

-i

Flow regime (U]0, cm s" )

40

Figure 2. Vertical feeding zone (range of tentacle-crown heights) of the three size classes ofMetridium
senile as a function of flow regime. Error bars are ±1 SE of 3 to 9 anemones. The inset shows the typical
posture of a medium-sized anemone at moderate flow; the arrow indicates flow direction. Small anemones:
1 .0-2.0 cm in pedal disc diameter (pdd). medium: 2.5-3.0 cm pdd. and large: 6.0-6.5 cm pdd.

than did both small and large anemones. Interestingly,
feeding rates based on feeding-surface area (S, Fig. 3C)
and on anemone biomass displayed the same general
patterns, perhaps reflecting isometric growth (Sebens,
1981). One noticeable difference among the sets of data
is, however, that large anemones showed a significant de-

crease in prey capture per unit biomass at Uw >
17 cms"1.

Feeding efficiencies

The three size classes of M. senile demonstrated three
distinct flow optima, inversely related to size class, at

Table I

Flow- induced tentacle-crown deformation and anemone biomass in three size classes ot Metndium senile

Small (1.0-2.0 cm pdd)

Medium (2. 5-3. Ocm pdd)

Large (5.5-6.0 cm pdd)

C/,o

(cm s ')

11

5/AFDW ±
SE(cnrg~')

AFDW ± SE

(g)

n

S/AFDW ±

SE(cm:g ')

AFDW ± SE

(g)

n

S/AFDW ±

SEfcnrg ')

AFDW* + SE

(g)

4

9

64.99 ±6. 24

0.156 + 0.019

7

43.86 ± 2.44

0.635 + 0.048

3

56.31 ±7.14

2. 827 ±0.403

10

6

84.09 ±3. 18

0.1 48 ±0.009

5

49.52± 3.31

0.682 + 0.052

3

53.67 ± 7.48

2.827 ± 0.403

17

8

52.48 ± 3.99

0.1 59 ±0.022

5

40.86 ±3. 31

0.663 ±0.058

3

43.39 ± 3.92

2.827 ± 0.403

20

8

47. 80 ±2. 83

0.1 60 ±0.020

5

36.48+ 1.92

0.650 + 0.061

3

29.61 ±3.17

2.827 ±0.403

28

7

47.80± 3.61

0.101 ±0.015

—

—

—

—

44

6

44.74 ± 3.96

0.148 + 0.009

4

36.37 ± 1.09

0.541 ±0.016

3

20.56 ±4. 16

2.827 ± 0.403

Size classes were based on measurement of pedal disc diameter (pdd). The degree of tentacle-crown deformation is quantified as the decrease in
ratio of tentacle crown area(S) to ash-free dry weight (AFDW) with increasing flow (t',0). SE denotes standard error of n = 3 to 9 anemones.
* The same three large anemones were exposed to all flow regimes.

PREY CAPTURE BY SEA ANEMONES

79

800

600

a

00

c

•5

400

200

1500

'g 1200

'oo

1. 900

3

n

£

% 60°

00

=5 300

Small

Medium

Large

tain up to 25.7 ± 2.0% of the nauplii passing the pro-
jected area of the tentacle crown at their optimal flow
speed ({7]0 = 20 cm s~'). The feeding efficiency of all size
classes decreased monotonically beyond their optimum.
At all flow velocities except the lowest one, small anem-
ones were significantly (2-3 times) more efficient than
medium-sized anemones, which in turn were about
twice as efficient as large anemones; these results are
analogous to the pattern of feeding rates based on bio-
mass and area of feeding surface.

Effect of upstream neighbors

The feeding rate per unit area of tentacle crown of me-
dium-sized M. senile declined significantly with increas-
ing numbers of conspecifics upstream (6.45 ± 0.45 to
2.25 ± 0.30 nauplii min~' cm"2, both ±95% CL) (Fig. 5).
The effect of upstream neighbors was most pronounced
within small aggregations of anemones (2-4 rows). In-
creasing the number of aligned rows of anemones from
7 to 16 did not reduce the feeding rate per unit area of
tentacle crown, and thus indicated a threshold at which
the feeding rate per polyp was unaffected by the addition
of upstream neighbors.

30

J 24

U

1. 18

i

if 12

•8

u

u.

10 20 30

Flow regime (U,0, cm s" )

40

Figured. Feeding rates (nauplii min~ ') per (A) polyp. (B) gram of
ash-free dry weight, and (C) unit area of tentacle crown of Meindium
senile as a function of body size and flow regime. Error bars are ±95%
confidence limits. All comparisons among size classes are significant at
any given flow regime except at l'M) = 4 cm s~'. Groups with overlap-
ping error bars are not significantly different by the Tukey-Kramer test.
Regressions for the determination of F were highly significant in all
experiments (P < 0.001 . R > 0.88).

which the feeding efficiency (E) was maximized (Fig. 4).
Large anemones showed a maximum feeding efficiency
at the slowest flow (L'\(> = 4 cms"1), medium-sized
anemones at low to moderate flows ( 1 0- 1 7 cm s~ ' ), and
small anemones at moderate to high flows (10-
20 cm s" ' ). Small anemones were able to capture and re-

Discussion

Feeding rates and efficiencies

The combinations of polyp size and flow regime at
which prey capture ofMetridium senile is maximized are
in good agreement with the pattern of population struc-
tures and flow habitats observed in the field, suggesting
that size distributions of M. senile are, in part, based on
the ability to utilize seston flux. Interestingly, small
anemones were generally more efficient at retaining prey
than both medium and large anemones over the full flow
range. Large anemones were, however, more than twice
as efficient as medium-sized anemones at the slowest
flow, but this relationship was reversed at moderate to
high flow. The generally higher feeding rate per unit area
and per unit biomass of small anemones may allow rapid
growth to a size at which they can better compete for
space, avoid size-selective predation (Harris, 1986), and
reproduce sexually (Anthony and Svane, 1994), pro-
vided that the energy input is used in polyp growth rather
than in clonal growth by pedal laceration (see below).
The maximized feeding at a high flow speed of small AI.
senile is inconsistent with other studies on prey capture
(e.g., Okamura, 1984, 1985; McFadden, 1986; Best,
1988; Leonard et a/.. 1988, Dai and Lin, 1993) and
growth (Okamura. 1992; Eckman and Duggins, 1993) of
tentaculate suspension feeders. In these studies maxi-
mum rates of feeding, growth, or both were generally as-
sociated with low flow speeds. A direct comparison

80

K. R. N. ANTHONY

25

g 20

1 „

o

£

u

01)

1)

PH

10

Small

Medium

Large

10

20

30

40

Flow regime (Uv, cm s )

Figure 4. Feeding efficiency (number of nauplii captured per number of nauplii passing, see Eq. 5 and
6) as a function of flow regime in three size classes of Meiridiuni senile. Error bars are ±95% confidence
limits. All comparisons among size classes are significant at any given flow regime except between small
and large anemones at t'R, = 4 cm s"'. Groups with overlapping error bars are not significantly different by
the Tukey-Kramer test.

among studies of flow, body size, and prey capture is
difficult, however, because different ranges of size and
flow regime are used in different studies. Also, compari-

sons of size per se among different taxa of suspension
feeders are complicated by differences in their structural
organization.

o,
cd

€ 3

.5 2

T3

<U

0>
r T , i

N = 6

= 37

N= 102

5 10

Rows of anemones perpendicular to flow

15

Figure 5. Effects of upstream neighbors in Metridium senile. Feeding rate (nauplii min ') per square
centimeter of tentacle crown as a function of the number of upstream conspecilics. Error bars are ±95%
confidence limits. Groups underlined at the same level are not significantly different by the Tukey-Kramer
test.

PREY CAPTURE BY SEA ANEMONES

81

The maximum prey-capture efficiency of M senile
from the Gullmarsfjord (25.6 ± 2.0%, small anemones)
is only half of that reported by Lesser et at. ( 1 994) for M.
senile from the Gulf of Maine (40% for offshore and 52%
for coastal anemones). Data on prey capture were, how-
ever, not given in relation to flow speed although a sig-
nificant tentacle-crown deformation (50%>) at increasing
flow was recorded, as also found in this study. The feed-
ing efficiencies determined for At. .senile in this study are
also generally lower than those of the sea pen Ptilosarcus
gurneyi (Best, 1988) which were up to 42%, decreasing
with flow to 30% within a narrow low-flow range ( 1.5-
5.0 cm s~ '), but are comparable with whole-colony effi-
ciencies of the octocoral Alcyoninm siderium (Patterson.
1991) in moderate flow regimes. However, the feeding
efficiency of A. siderium is inversely related to both col-
ony size and flow speed (Patterson, 1991). whereas the
relationship between feeding, size, and flow shows a
more complex pattern in M. senile. These different pat-
terns may, in part, be due to the different filtering struc-
tures and tentacular organizations between solitary and
colonial anthozoans, and to differences in structural
flexibility.

The increasing feeding rates as a function of ambient
flow within the low to moderate flow range corroborates
earlier assumptions that moderate-flow habitats provide
higher seston availability than low-flow habitats (Shick
and Hoffmann, 1980; Sebens, 1984; Shick, 1991). The
flow range at which food intake per polyp or per biomass
is maximized, however, is different between size classes.
Large anemones showed a relatively distinct maximum
at 10-17 cm s~', whereas small and medium anemones
maintained a constant food intake above 10 cms"1.
These different responses to flow are likely to explain, in
part, the differences in size distribution of M. senile
across flow habitats. In high-flow habitats M. senile typi-
cally forms dense aggregations of small anemones mo-
nopolizing vast areas of rock wall and consisting of only
a few clones, whereas populations in low-flow habitats
often comprise large and usually more scattered individ-
uals from numerous clones (Anthony and Svane, 1 994).

Only at the slowest flow were large anemones able to
achieve feeding rates and feeding efficiencies comparable
to those of small and medium-sized anemones. Large
anemones had a significant feeding disadvantage relative
to small and medium anemones at flows greater than
10 cm s~", and displayed a per-biomass food intake only
one-eighth that of the small anemones in very high flow
regimes. One explanation for this pattern is based on
differences in the geometry of the tentacle crown of large
and small anemones. Although the tentacles of large and
small M. senile have comparable geometry and size (Seb-
ens, 1981), the relative size of the oral disc increases with
body size. Therefore, the proportion of tentacle crown

effective in upstream capture (the circumference of ten-
tacles facing upstream or perpendicular to flow) when
the anemone is bent downstream relative to the total ten-
tacle crown area is likely to be greater for small anemo-
nes. Subtracting the projected area of the upper part of
the column from the surface area of the tentacle crown
accounted to some extent for such geometric differences.
However, size-related differences in the spacing of open-
ings in the capitulum at the base of the tentacles were not
accounted for. If such differences exist, mainly as a result
of differential convolutions of the tentacle crown, they
are likely to affect the size-specific feeding efficiencies (J.
M. Shick, pers. com.).

In moderate to high flow velocities, a great proportion
of the tentacle crown of large anemones is hidden from
the upstream flux and is thus engaged in wake feeding,
which is likely to be less effective than upstream feeding
(Shimeta and Jumars, 1991 ). Small anemones may ben-
efit from both upstream and wake feeding at comparable
flow rates, whereas wake feeding is likely to be the pre-
dominant mode of feeding for large anemones in mod-
erate to high flow. However, due to the higher Reynolds
number associated with large animals (Shimeta and Ju-
mars, 1991 ), downstream vortex formation is more pro-
nounced for large anemones. This effect may, in part,
be beneficial in providing a leeward region of enhanced
retention efficiency through reduced flow velocities (Pat-
terson, 1984).

Although the probability of prey encounter increases
with flow, so does the risk of dislodgement of captured
prey, and an optimum local velocity must be within the
range over which retention significantly exceeds dis-
lodgement of encountered prey. Both the capacity for re-
tention by and the risk of dislodgement from the tenta-
cles of sea anemones and of passive suspension feeders
in general may vary among size classes and among geno-
types. The small M. senile from high-flow habitats may
be better adapted to feeding in a high-flow environment
by being equipped with a more efficient cnidom for both
subduing and retaining intercepted prey. Effects of flow
forces on dislodgement of intercepted prey in cnidarian
suspension feeders are, however, poorly understood
(Patterson, 1984: Shimeta and Jumars, 1991).

I 'cnical feeding zone and tentacle-crown deformation

The prey availability can for some passive suspension
feeders be adjusted to a given flow regime by means of
behavior or change of posture, for example by regulating
the height and orientation of the feeding appendages
(ophiuroids: Warner and Woodley (1975);crinoids: Hol-
land et al. (1987), Leonard (1989); sea anemones: Rob-
bins and Shick ( 1 980), this study; polychaetes: Muschen-
heim (1987), Shimeta and Jumars. ( 1 99 1 )) or bv vertical.

82

K. R. N. ANTHONY

oriented locomotion of the whole animal (Anthony and
Svane, 1995). Differences in prey capture among small,
medium, and large A/, senile in this study could not be
explained fully by different local flow velocities at differ-
ent heights in the boundary layer. In fact, the local flow
velocities experienced at the mean level of the tentacle
crown of the three size classes in low to moderate flow
regimes did not differ by more than 1-2 cm s~'. Con-
trasting local flow velocities due to differences in vertical
position were found only between large and small or me-
dium-sized anemones at the highest flow regime (6-
8cms~'). A higher degree of downstream bending of
medium-sized and especially large anemones due to pro-
portionately greater drag (Koehl, 1977a) explains the
overlapping, vertical feeding zones of different size
classes. Flow forces thus act in moving the feeding appa-
ratus of large anemones into the boundary layer and
away from the maximum exposure, enabling some,
though reduced, prey capture at even the highest ambi-
ent flow.

Tentacle-crown deformation, and thereby a reduced
prey-capture area normal to flow, could to some extent
account for the significantly lower feeding success of
large anemones in regimes of moderate to high flow.
Prey-capture surface per unit biomass decreased by more
than 50% when altering the flow regime from 4 to
44 cm s~', affecting the volume of water filtered and the
rate of potential prey encounter accordingly, and proba-
bly also the spacing between tentacles (see also Lesser el
al. 1994). Best (1988) also found that the deformability
of the sea pen Ptilosarcus gurneyi strongly affected fil-
tration efficiency and volume of water filtered. The
effectiveness of the feeding apparatus of a large, soft-bod-
ied suspension feeder is intrinsically apt to be more con-
strained by flow forces than is that of a small suspension
feeder, because large animals are subject to proportion-
ately greater drag (Koehl. 1977a) and shear stresses
(Koehl, 1977b) than are small animals in similar flow
conditions. Since the potential prey capture of, for exam-
ple, a sea anemone is a function of the surface area of the
feeding apparatus (Sebens, 198 1 ), the S/AFDW ratio is
likely to be a useful indicator of flow-habitat suitability.

Feeding rates vs. metabolic cost

To determine flow optima of passive suspension feed-
ers, information on both energy intake and metabolic
cost is necessary for every flow regime (Sebens, 1979,
1982). Although the pattern of feeding rates per unit of
biomass suggests that small anemones generally have a
greater energy surplus than large and medium-sized
anemones regardless of flow regime, energy demand is
likely to vary among size classes as well as among flow
regimes. Gas exchange, and thus metabolic cost, in cnid-

arians is largely governed by the thickness and character-
istics of the boundary layer surrounding the organism,
which in turn are functions of organism size and flow
regime (Patterson, 1992a). For example, Patterson and
Sebens ( 1989) found that the rate of respiration in large
specimens ofMetridium doubled over the flow range 7-
15 cm s~' due to the effect of water motion. A useful
method for comparing metabolic cost across flow re-
gimes as well as size classes is the relationship between
Sherwood number and Reynolds number ( Patterson and
Sebens, 1989; Patterson, 1992a, b). Sherwood number
(Sh) is a dimensionless index of metabolism, defined as
the ratio of mass flux per unit surface area assisted by
convection (Fcon%, /* cm"2 IT ' ) to that which would occur
if diffusion through a stagnant layer of water was the only
mechanism of gas exchange. Reynolds number (Re) is
the ratio of inertia! to viscous forces acting on the flowing
water, and is an index of the strength of flow experienced
by the organism. For Metridium. the functional relation-
ship between Sh and Re is Sh = 0.28 Re1 "4 as found by
Patterson and Sebens ( 1989). Since Re is a function of
body size and flow [Re = {/,. X TCD/v, where {/,. is local
flow speed (cms"'), TCD is tentacle-crown diameter
(cm), and v is the kinematic viscosity of seawater ( 1 .04 X
10~2 cm2 s~')], Sh can be readily obtained for all combi-
nations of flow regime and size class in this study. Ex-
pected (flow-assisted) metabolic cost can be derived from
Sh as the convective mass flux (-Fconv, the numerator of
Sh), normalized to biomass by multiplying with the ratio
of surface area to biomass (SA/AFDW, cm2 g~'), where
SA is the sum of tentacle surface area [see regressions in
Sebens ( 198 1 )] and surface area of the column. Accord-
ing to Patterson ( 1 992b) convective mass flux can be cal-
culated as Fcorn = Sh X D(CC - Q/TCD, where D is the
diffusion coefficient for oxygen (7.2 X 10~2 cm: h~'), Cc
is the oxygen concentration in the surrounding water
( = 7 mgP1). and C, is the oxygen concentration at the
site of metabolism. For azooxanthellate cnidarians, C,
can be regarded as negligible (pers. com.. Mark Patter-
son). Expected metabolic rate per unit biomass (Rexp =
ShXDX Ct,XSA/[TCDX AFDW],Mg(gdrywt)~' h~')
as a function of flow regime for the three size classes of
M. senile is shown in Figure 6.

Expected metabolic rate for all size classes increases as
a linear function of flow regime, with a 10-fold increase
in metabolic rate between lowest and highest flow speeds.
In accordance with allometric studies (e.g.. Sebens,
1981), metabolic rate is greater for small and medium-
sized compared to large anemones, by a factor of about
3/i. Interestingly, expected metabolic rate is higher for
medium-sized compared to small anemones at flow re-
gimes greater than 1 0 cm s~ ' , probably due to the higher
local flow speeds (and hence Sh) experienced by me-
dium-sized anemones (Figs. 1 and 2). Although energy

PREY CAPTURE BY SEA ANEMONES

83

30

u o

td 2 25

o _*

o

J3

w

-a
o

20

15

-a
oo

Small

Medium

Large

10

20

30

40

Flow regime (U10, cm s )

Figure 6. Expected metabolic rate as a function of flow regime for the three size classes of Melridium
senile, based on mean Reynolds numbers lor the study anemones and empirical mass-transfer relations
from Patterson and Sebensl 1 989). See text for calculations.

balance also depends on local food availability, food
quality, and absorption efficiency (e.g., Zamer. 1 986),
the patterns of potential food intake (feeding rate) and
expected metabolic cost provide a basis for relative com-
parisons of potential flow optima among size classes. The
flow speed at which energetic cost counterbalances food
intake will be highest for the small anemones, intermedi-
ate for medium-sized anemones, and lowest for large
anemones. Due to very high feeding rates, small anemo-
nes are able to maintain a more positive energy balance
than both medium and large anemones at all flow speeds
> 10 cm s~'. The almost 10-fold increase in feeding rate
of small anemones in the flow range 4-10 cm s~' amply
exceeds the concomitant increase in metabolic cost over
this flow interval, and is likely to allow rapid growth of
juvenile anemones and lacerates in low-to-moderate
flow regimes. At higher flow speeds, however, feeding
rate remains constant, whereas metabolic rate increases
linearly with flow. If the feeding rate of small anemones
at 4 cm s~' is sufficient to meet basic energy demands, so
is the feeding rate at 44 cm s"1 because both feeding rate
and metabolic cost increase 10-fold between lowest and
highest flow. At the lowest flow speed, however, large
anemones are likely to have a more positive energy bal-
ance than the smaller size classes, given the comparable
feeding rates of small and large anemones but the lower
mass-specific metabolic cost of large anemones. Since
large anemones only showed low weight-specific feeding
rates without a convincing maximum, any energetic op-
timum of this size class must be confined to low-flow re-
gimes because of the rapid increase in metabolic rate

with flow. The flow optimum of medium-sized anemo-
nes, on the other hand, is likely to be located in the range
10-20 cm s"1. as their feeding rate is minimal at 4 cm s~'
('/3 of large and small anemones) and their expected met-
abolic cost increases with flow at a relatively higher rate
than for the two other size classes.

Use of mass-transfer theory to compare metabolic cost
between individual M. senile in different flow regimes
does not, however, take into account the differences in
spatial distributions between large and small anemones.
Large anemones are often widely separated from one an-
other, whereas small anemones (due to their pronounced
clonality) form dense patches structurally resembling
coral colonies (Anthony and Svane, 1994). Flow around
individual polyps in such patches is likely to be further
reduced, resulting in a thicker diffusive boundary layer
and a lower gas exchange than for small, spatially iso-
lated individuals in similar flow habitats (see also Patter-
son and Sebens, 1989).

Effects of flow and feeding on reproductive patterns

One way in which M. senile might maintain an ener-
getically optimal polyp size in the prevailing current re-
gime is by altering the rate of pedal laceration. Lacera-
tion is a loss of tissue to the individual polyp (facilitating
degrowth), but also an effective means of clonal growth,
so differential rates of laceration between local popula-
tions may have consequences for their size distributions.
The generally higher rates of laceration in populations of
small M. senile from high-flow habitats relative to popu-

84

K. R. N. ANTHONY

lations of large anemones from low-flow habitats support
this hypothesis (Shick and Hoffmann, 1980; Anthony
and Svane, 1994). Since large anemones cannot main-
tain a positive energy balance in high-flow habitats,
whereas small anemones can, any excess energy acquired
by high-flow anemones is likely to be allocated to clonal
growth (through pedal laceration) rather than to polyp
growth. Laceration is also stimulated by feeding
(Bucklin, 1987), suggesting that asexual reproduction is
one pathway by which energy surplus is translated into a
greater genet biomass. Effects of feeding and ambient
flow on laceration are, however, apt to be closely linked
due to the direct proportionality between flow and seston
flux, and hence potential particle encounter. By obtain-
ing an optimal polyp size in a given flow habitat through
growth or degrowth (via laceration), the energy available
for clonal growth, sexual reproduction, and hence genet
fitness can be further enhanced. Clonal growth as op-
posed to polyp growth in high-flow habitats creates the
energetic potential for the genet to grow indefinitely
without size-energy constraints, because the addition of
new polyps to the clone increases feeding surface and en-
ergetic cost linearly (Sebens, 1979). Conversely, growth
to a maximum polyp size in low-flow habitats enables
maximum feeding and thus energy input (this study),
and also a maximum per-biomass reproductive output
(Anthony and Svane, 1994).

Effect of upstream neighbors

Considering the clonal nature and high polyp densities
of most populations of AI. senile, the presence of up-
stream neighbors is likely to be an important factor
affecting the energy input of both the polyp and the
clone. Furthermore, the effect of upstream neighbors on
near-bed prey availability downstream may select for
large size in some flow habitats, despite a relatively
greater food intake in small anemones, and thereby con-
tribute to structuring local populations. The asymptotic
relationship between feeding rate per unit area of tenta-
cle crown and number of upstream conspecifics, on the
other hand, indicated that the potential feeding capacity
was not reduced to below about 30% of the situation
without neighbors upstream. A likely explanation for
this threshold is that individual anemones function as
roughness elements, increasing turbulence and eddy
diffusivity downstream (Denny, 1988), thereby reducing
local particle depletion around tentacle crowns by mix-
ing mainstream water into the turbulent boundary layer.
In high-flow habitats, the generally higher density of
anemones may also provide shelter for individual anem-
ones by moving the turbulent boundary layer outward.
As suggested by Patterson (1984), particle lift in the
boundary layer over the tentacle-crown canopy may be

responsible for reduced downstream concentrations, and
thereby, in part, account for the asymptotic rather than
linear reduction in retention with increasing number of
upstream neighbors (see also Frechette el a/.. 1989). Fur-
thermore, gravitational deposition is likely to be more
important in dense clones than in populations of scat-
tered individuals on horizontal substrata, providing a
"rain" of seston that is independent of the number of
upstream neighbors. Overall, these results suggest that
increased clonal growth of AI. senile in high-flow habitats
at the expense of polyp growth (see review by Shick,
1991) may negatively affect net prey capture by the genet
through feeding interaction between clonemates. Con-
versely, predominant polyp growth and reduced clonal
growth in low-flow habitats is likely to enhance prey cap-
ture by excluding the effect of neighbors. The generally
higher feeding efficiency of small polyps of AI. senile
may, however, compensate for the negative effects of
neighbors in dense, clonal aggregations if the prevailing
flow conditions provide a correspondingly higher flux of
prey.

Acknowledgments

I thank the administration and staff of Kristineberg
Marine Research Station for providing laboratory facili-
ties and technical assistance. Thanks are especially due
to Ken Sebens for reviewing the penultimate draft, and
to Terry Hughes, Bette Willis, The Coral Discussion
Group at James Cook University, Henrik Glenner, Pia
Anthony, and Robert George for valuable discussions
and comments on earlier versions of the manuscript. I
am grateful to Mark Patterson for his advice on calcula-
tions of expected metabolic cost. Malcolm Shick, Wil-
liam Zamer, and Barbara Best improved the final manu-
script. Thanks are also due to Walt Doyle for assistance
in the laboratory. This work was supported by the Nor-
dic Council for Marine Biology, Nordic Academy for
Advanced Studies, the Axel Hemmingsen Grant, and the
Hierta-Retzius Foundation (Royal Swedish Academy of
Sciences).

Literature Cited

Anthony, K. R. N., and I. Svane. 1994. Effects of flow-habitat on
body size and reproductive patterns in the sea anemone Melridiwn
viv;//i'(L.) in the Gullmarsfjord, Sweden. Mar. Ecol, Prog. Scr. 1 13:
257-269.

Anthony, K. R. N., and I. Svane. 1995. Effects of substratum instabil-
ity on locomotion and pedal laceration in Metridium senile L. (An-
thozoa: Actiniaria). Mar. Ecu/. Prog. Ser. 124: 1 7 I - 1 80.

Best, B. A. 1988. Passive suspension feeding in a sea pen: effects of
ambient flow on volume flow rate and filtering efficiency. Bio/. Bull.
175: 332-342.

Bucklin, A. 1987. Growth and asexual reproduction of the sea anem-
one Mclndiiiin: comparative laboratory studies of three species. J.
Exp. Mar. Biol. Ecol. 110:41-52.

PREY CAPTURE BY SEA ANEMONES

85

Dai, C.-K., and M.-C. Lin. 1993. The effects of flow on feeding of
three gorgonians from southern Taiwan. ./ Exp. Mar. Bitil. Ecol
173:57-69.

Denny, M. 1988. Biology and the mechanisms ol the wave-swept en-
vironment Princeton University Press, Princeton.

F.ckman, J. E., and D. O. Duggins. 1993. Effects of flow speed on
growth of henthic suspension feeders. BioL Bull 185: 28-4 1

I .nniii. D. G., A. Bucklin, and C. Hand. 1989. Systematics of the sea
anemones belonging to genus Metridium (Coelenterata: Actim-
aria). with a description of M. giganteum new species. (I 'asmann .1
Biol. 47(1 -2): 77-85.

Francis, L. 1979. Contrasts between solitary and clonal lifestyles in
the sea anemone Anthopleura elegantissima. Am /on/ 19: 669-
681.

Frechette. M., C. A. Butman, and \V. R. Geyer. 1989. The impor-
tance of boundary-layer flows in supplying phytoplankton to the
benthic suspension feeder Mytilus edulis L. Limnol. Oceanogr 34:
19-36.

Harris, L. G. 1986. Size-selective predation in a sea anemone, nudi-
branch. and fish food chain. 1 'cliger 29: 38-47.

Hoffmann. R. J. 1976. Genetics and asexual reproduction of the sea
anemone Metridium senile Biol. Bull 151: 478-488.

Hoffmann, R. J. 1986. Variation in contributions of asexual repro-
duction to the genetic structure of populations of the sea anemone
Meiridmm senile. Evolution 40(2): 357-365.

Holland. IS. D., A. B. Leonard, and J. R. Strickler, 1987. Upstream
and downstream capture during suspension feeding by Oligimetra
serripinna (Echinodermata: Cnnoidea) under surge conditions.
Biol. Bull. 173: 552-556.

Johnson, A. S. 1990. Flow around phoronids: consequences of a
neighbor to suspension feeders. Limnol Oceanogr. 35: 1395-1401.

Johnson, A. S., and K. P. Sebens, 1993. Consequences of a flattened
morphology: effects of flow on feeding rates of the scleractiman
coral Meandrina meandrites. Mar. Ecol. Prog. Ser. 99: 99- 1 14.

Koehl, M. A. R. 1976. Mechanical design in sea anemones. Pp. 23-
31 in Coelenlerate Ecology and Behavior. G. O. Mackie. ed. Ple-
num Press. New York.

Koehl, M. A. R. 1977a. Effects of sea anemones on the flow forces
they encounter. / E\p. Biol. 69: 87-105.

Koehl, M. A. R. 1977b. Mechanical organisation of cantilever-like
sessile organisms: sea anemones. J. Exp. Biol. 69: 127-142.

LaBarbera, M. J., and S. Yogel. 1976. An inexpensive thermistor
flowmeter for aquatic biology. Limnol. Oceanogr 21: 750-756.

Leonard, A. B. 1989. Functional response in Anicilon medilerranea
(Lamarck) (Echinodermata: Cnnoidea): the interaction of prey
concentration and current velocity on a passive suspension feeder.
J Exp. Mar. Biol. Ecol 127: 81-103.

Leonard, A. B., J. R. Strickler, and N. D. Holland. 1988. Effects of
current speed on nitration during suspension feeding in Oligometra
serripinna ( Echinodermata: Crinoidea). Afar. Biol. 97: I 1 1-125.

Lesser, M. P., J. D. Witman. and K. P. Sebens. 1994. Effects of flow
and seston availability on scope for growth of benthic suspension-
feeding invertebrates from the Gulf of Maine. Biol. Bull 187: 319-
335.

Leiersee. G. J. 1976. Flow and feeding in the fan-shaped colonies of
thegorgonian coral. Leptogorgia. Biol Bull. 151: 344-356.

Manuel. R. L. 1988. Synopsis ol the British h'aumi. ,\Vu Series 18.
British Anthozoa. Academic Press. London.

McFadden, C. S. 1986. Colony fission increases particle capture rates
of a soft coral: advantages of being a small colony. ./. Exp Mar
Biol Ecol. 103: 1-20.

Muschcnheim, D. K. 1987. Thedynamicsof near-bed seston flux and
suspension-feeding benthos. J Mar Res 45:473-496.

Okamura, B. 1984. The effects of ambient flow velocity, colony size.

and upstream colonies on the feeding success ot Bryozoa. I. Bugulii
slolomlera Ryland, an arborescent species. J Exp Mar Biol Ecol.
83: 179-143.

Okamura. B. 1985. The effects of ambient flow velocity, colony size,
and upstream colonies on the feeding success of Bryozoa. II. Cono-
peum reticulum L.. an encrusting species. J Exp. Mar. Biol Ecol.
89: 69-80.

Okamura. B. 1992. Microhabitat variation and patterns of colony
growth and feeding in a marine bryozoan. Ecology 73(3): 1502-
1513.

Patterson. M. R. 1984. Patterns of whole colony prey capture in the
octocoral Ah yoniuin sidenum. Biol Bull. 167:613-629.

Patterson, M. R. 1991. Passive suspension feeding by an octocoral in
plankton patches: empirical test of a mathematical model. Biol.
Bull. 180:81-92.

Patterson, M. R. I992a. A mass transfer explanation of metabolic
scaling relations in some aquatic invertebrates and algae. Science
255: 1421-1423.

Patterson, M. R. 1992b. A chemical engineering view of cnidarian
symbioses. Am. /.ool. 32: 566-582.

Patterson, M. R., and K. P. Sebens. 1989. Forced convection modu-
lates gas exchange in cnidanans. Proc. Nail. Acad. Sci. USA 86:
8833-8836.

Purcell, J. E. 1977. The diet of large and small individuals of the sea
anemone Metridium se/n/e. Bull. S C Acad. Sci. 76: 168-172.

Robbins, R. E., and J. M. Shick. 1980. Expansion-contraction be-
havior in the sea anemone Metridium senile: environmental cues
and energetic consequences. Pp. 1 0 1 - 1 1 6 in Nutrition in the Lower
,\Ieta:oa. D. C. Smith and Y. Tiftbn, eds. Pergamon Press, Oxford.

Sebens, K. P. 1979. The energetics of asexual reproduction and col-
ony formation in benthic marine invertebrates. Am. Zool. 19: 683-
697.

Sebens, K. P. 1980. The regulation of asexual reproduction and inde-
terminate body size of the sea anemone Anthopleura elegantissima
(Brandt). Biol Bull 158:370-382.

Sebens, K. P. 1981. The allometry of feeding, energetics, and body
size in three sea anemone species. Biol Bull. 161: 1 52- 171.

Sebens, K. P. 1982. The limits to indeterminate growth: an optimal
size model applied to passive suspension feeders. Ecology 63(1):
209-222.

Sebens, K. P. 1984. Water flow and coral colony size: interhabitat
comparisons of the octocoral Alcyonium siderium. Proc. Nail.
Acad. Sa. USA 81: 5473-5477.

Sebens, K. P., and M. A. R. Koehl. 1984. Predation on zooplankton
by the benthic anthozoans Alcyonium siderium (Alcyonacea) and
Metridium senile (Actiniaria) in the New England subtidal. Mar.
Biol. 81:255-271.

Shick, J. M. 1991. .1 Functional Biology oj Sea Anemones. Chapman
& Hall. London.

Shick, J. M., R. J. Hoffmann, and A. N. Lamb. 1979. Asexual repro-
duction, population structure, and genotype-environment interac-
tions in sea anemones. Am. Zool 19:699-713.

Shick, J. M., and R. J. Hoffmann. 1980. Effects of the trophic and
physical environments on asexual reproduction and body size in
the sea anemone Metridium senile Pp. 21 1-216 in Developmental
and Cellular Biology of Coeletlterates, P. Tardent and R. Tardent,
eds. Elsevier/North Holland Biomedical Press, Amsterdam.

Shick, J. M., and II. B. Dowse. 1985. Genetic basis of physiological
variation in natural populations of sea anemones: intra- and in-
terclonal analyses of variance. Proc. I ^th European Marine Biology
Symposium. Cambridge. P. E. Gibbs. ed. Cambridge University
Press, UK.

Shimeta, J., and P. A.Jumars. 1991. Phvsical mechanisms and rates

86

K. R. N. ANTHONY

of particle capture by suspension feeders. Occunogr Mar, BioL Ann.
Rev. 29: 191-257.

Sokal, R. R., and F. J. Rohlf. 1981 . Biometry 2 ed. W. H. Freeman
& Co, San Francisco.

Trager, G., V. Achituv, and A. Genin. 1994. Effects of prey escape
ability, flow speed, and predator feeding mode on zooplankton cap-
ture by barnacles. Mar. Biol. 120:251-259.

Vogel, S. 1981 . Life in Moving l-'liiul\: the Physical Biology of Flow.
Willard Grant Press. Boston, Massachusetts.

\Vainw right, S. A., and M. A. R. Koehl. 1976. The nature of flow and
the reaction of benthic cnidaria to it. Pp. 5-2 1 in Coelenlerate Ecol-
ogy anil Behavior. G. O. Mackie, ed. Plenum Press, New York.

Warner, G. F. and J. D. Woodley. 1975. Suspension feeding in the
brittle star Ophiotrixfragilis. J Mar. Biol. Assoc. L'. A.' 55: 1 99-2 10.

Zamer, W. E. 1986. Physiological energetics of the intertidal sea
anemone Anthopleura elegantissima. I. Prey capture, absorption
efficiency and growth. Mar. Biol. 92: 299-3 14.

Reference: Biol. Hull 192: 87-97. (February, 1997)

Patterns and Consequences of Whole Colony Growth in
the Compound Ascidian Polyclinum planum

ALAN R. HOLYOAK
Department of Biology, University of California, Santa Cruz, California 95064

Abstract. The size and shape of colony-forming mod-
ular animals can convey ecological advantages, but
many patterns and consequences of colony-level growth
are not well understood. I carried out a longitudinal
study on an intertidal population of the pedunculate as-
cidian Polyclinum planum to determine the patterns and
consequences of its colony growth. I found that each P.
planum colony is a nonfragmenting genet, and that col-
ony size is limited by water-flow forces and reproductive
state. P. planum mitigates the effects of water-flow forces
by having an attenuating pattern of growth and by pro-
ducing a laterally flattened, zooid-bearing lobe atop its
tough flexible peduncle. Growth slows as the colony
nears a size limit set by the environment and as it be-
comes reproductively active. The laterally flattened lobe
allows colonies to increase their surface-to-volume ratio,
to house increased numbers of zooids (thereby increas-
ing reproductive potential), and to minimize the effects
of the acceleration reaction of water. P. planum's growth
pattern fits predictions for colonies living in wave- or
surge-impacted environments. The growth of P. planum
provides insight into how indeterminate modular growth
conveys ecological and reproductive advantage, even
amidst a physically stressful environment.

Introduction

Theories describing the indeterminate growth of col-
ony-forming modular organisms suggest that the colo-
nies have the potential to grow linearly or exponentially
throughout their postlarval lives (Jackson, 1977; Sebens.
1 987). Sebens (1987) defines three patterns of indetermi-
nate growth, two of which (Indeterminate Growth Types

Received 24 July 1995; accepted 24 October 1996.
Current Address: Department of Biology. Manchester College,
North Manchester, Indiana 46962.

II and III from his paper) are applicable to colony-form-
ing modular organisms. In Sebens' ( 1987) Indeterminate
Growth Type II (Plastic Exponential Growth), energy in-
take and expenditures occur at the level of each member
(module) of a colony, thus allowing each module to con-
tribute independently to the colony and producing con-
tinuous linear or exponential growth — a growth pattern
reported by Hughes and Jackson (1985) for stony corals,
by Karlson ( 1 988) for a zoanthid, by Patzold el al. (1987)
for a bryozoan, and by Bak el al. ( 1 98 1 ) for a compound
ascidian. In Sebens' ( 1987) Indeterminate Growth Type

III (Plastic Attenuating Growth), colony growth rates de-
crease as colony size increases, growth being limited
largely by environmental factors before internal ener-
getic constraints take effect — a growth pattern reported
by Hughes and Connell ( 1987) for stony corals, by Karl-
son (1988) for a zoanthid, and by Denny el al. (1985) for
a hydrocoral.

External factors therefore constrain growth in some
situations, but internal constraints are also known to be
limiting in some taxa. For example, Millar ( 1952, 197 1 )
and Bak et al. ( 198 1 ) report that ascidian colonies grow
rapidly when they are not reproductive, but that growth
slows or ceases during reproduction. For at least some
modular species, colony growth and reproduction are
physiologically incompatible processes.

To evaluate the applicability of growth predictions for
a colony-forming modular organism, one must un-
derstand how environmental effects (external factors)
and life-history constraints (internal factors) affect col-
ony growth. It is, unfortunately, difficult to collect those
kinds of life-history data for many colony-forming mod-
ular species. The difficulty arises because there is often
no way of knowing whether a colony encountered in the
field developed directly from a larva or was one of many
ramets produced by fragmentation (for examples, see
Hughes, 1984; Hughes and Jackson, 1985; Karlson,

87

A. R. HOLYOAK

1986, 1988; Rosen, 1986; Lasker, 1990; McFadden,
1991; Stocker, 1991). That uncertainty eliminates the
possibility of measuring age-related effects at the level of
genets. To further complicate matters, ramets of some
modular species can fuse with other ramets of the same
or different genotype (Hughes and Jackson, 1985; Rin-
kevich and Weissman, I987a, b; Stocker, 1991; Pancer
el al., 1995). One way to be sure of the origins, ages, and
fates of colonies of modular organisms is to conduct lon-
gitudinal site surveys, as suggested by Berrill ( 1 950) when
he stated that "the age of (compound) ascidians is prac-
tically impossible to estimate, unless a certain inhabited
area is followed closely through seasons and years." That
is sound advice if one hopes to understand the life histo-
ries of modular species, because age, size, and shape of
modular colonies may not be as tightly linked as those
parameters are to body size among unitary organisms
(Hughes and Jackson, 1980; Jackson and Coates, 1986;
Hughes and Connell, 1987).

In the present study I use monthly site surveys con-
ducted over 2.5 years to describe the pattern of whole
colony growth and the consequences of that growth for
intertidal Polyclimnn planum growing in situ. In so doing
I try to determine whether the growth pattern of P. pla-
num colonies more closely approximates Sebens' (1987)
Type II or III pattern of growth, or some other pattern,
and to discern what factor or factors constrain whole col-
ony growth in this species.

I hypothesize that the growth pattern of P. planum will
be more similar to Sebens' (1987) Growth Type III (Plas-
tic attenuating growth) than to his Growth Type II (Plas-
tic exponential growth). 1 chose Type III rather than
Type II because the colonies at my study sites live in a
wave- and surge-impacted environment, and the associ-
ated water-flow forces may impose limits to colony size.
Water-flow forces present an obvious ecological risk
to erect, though flexible, intertidal P. planum colonies.
Denny et al. (1985) demonstrated how the water-flow
forces of drag, lift, and, most importantly, acceleration
can dislodge or limit the size of intertidal organisms. Be-
cause the P. planum colonies at my sites are constantly
subjected to water-flow forces, they should have adapta-
tions for dealing with those forces that will be evidenced
as the colonies increase in size.

I also hypothesize that the onset of reproduction in P.
planum colonies will limit growth, and that large colo-
nies will show more evidence of reproductive activity
than small colonies. A P. planum colony grows by stro-
bilation of its zooids. Strobilation, however, precludes
gonadogenesis and larval brooding by the zooid because
gonads develop only in its post-abdomen and larvae are
brooded only in its atrial chamber — structures that be-
come disorganized during Strobilation ( Holyoak, 1 992).
P. planum, an aplousobranch ascidian, is well suited

; V- -o • .'.•' ;P ."• •„••£• JQ-":V '• : ' =%al"

Figure 1 . (a) Small "recruit-sized" Ptilyclimini plunwn colony with
a spheroidal zooid-hearing lobe atop its zooid-free peduncle, (b) Large
/' planum colony with a laterally flattened lobe. Note: Silhouette
shapes of larger colonies are highly variable.

to a longitudinal investigation of colonial growth. It has
a distinctive colony form — a fleshy, zooid-bearing lobe
supported by a tough and flexible peduncle — that makes
it readily identifiable in the field even when colonies are
small (Fig. 1). P. planum colonies are not believed to
fragment, so a single colony represents an entire genet
(Pearse et al.. 1989). The nonfragmenting growth of
these colonies eliminates the confounding effects of not
knowing whether a colony is a ramet or an unfragmented
genet. The loss of a P. planum colony is consequently
a greater loss to the population, from an evolutionary
perspective, than the loss of one ramet from a population
of fragmenting genets. Since a genet of P. planum does
not spread its genes across several physiologically iso-
lated ramets, one would expect this species to display
strategies that allow individual colonies to minimize eco-
logical risks and to maximize reproductive productivity.

Materials and Methods

Between December 1989 and May 1992, I monitored
the whole colony growth of intertidal Polyclinum pla-
num in situ at the Hopkins Marine Station (HMS), Pa-
cific Grove, California. The colonies there live attached

COLONY GROWTH OF POLYCLIM \l

89

6000 -i

" 5000

S

S 4000

<

| 3000

o

| 2000

•I 1000

u

o -

0

I I I

12 18 24

Colony Age (months)

Figure 2. Mean growth trajectory ofPolyclinum planiim. A quadratic regression provides a significant
fit to the data ( )' = -377.06 + 283.09.V - 9.38.Y2; r = 0.734; / = -3.59; df = 1 7; P < 0.005). Numbers
indicate the number of observations included in each age class, error bars are standard deviations, and the
horizontal dashed line indicates the maximum size (colony-silhouette area in mnr) of the growth trajec-
tory. The single outlier at month 23 was not included in the curve-fitting analysis. Discrepancies between
numbers of colonies indicated for each age class (month) on this figure compared to Figure 7 are due to the
fact that I was typically able to map all colonies at my sites each month, even with marginal tide conditions,
but I was not able to photograph all colonies in months with marginal tides.

to granitic substrates below about -0.3 m MLLW(Mean
Lower Low Water tide) and are most abundant in areas
where granitic outcrops covered by surfgrass (Phyllos-
padix sp.) are protected from direct wave action. The
highest density of P. plamim I encountered during pre-
liminary surveys prior to establishing my study sites was
95 colonies in a single 0.25-m: quadrat, though the mean
of 7.8 colonies per 0.25-nr quadrat (SD = 14.95 colo-
nies; n = 45 0.25-nr quadrats surveyed in the intertidal
zone at HMS) was more representative of P. plamim den-
sities at HMS.

I established three study sites, each about 1 m:, at
-0.3 m MLLW; all had P. plamim colonies present at
the beginning of the study. The sites were within 10 m of
each other and all of them had a dense canopy of surf-
grass covering a mosaic of encrusting organisms, foliose
algae, and bare rock surfaces. The three sites were on the
inshore side of massive granitic outcroppings along the
outermost reaches of the HMS intertidal zone. Most of
the force produced by waves was typically expended on
those rocks. P. plamim colonies were nevertheless sub-
jected to surge conditions as water from breaking waves
flowed over and around the outcroppings during all but
the lowest tides. Because P. plamim lives low in the in-

tertidal zone, my study sites were accessible for only
hours at a time and only in months with sufficiently low
tides. This somewhat limited access occasionally ham-
pered data collection. For this paper describing patterns
of colony growth, data from the three sites were pooled.

Three lag bolts were anchored at each site and used to
locate the sites and to aid in mapping the locations of
all P. plamim colonies. A colony's position was mapped
monthly by measuring the distance between its pedun-
cular point of attachment and the three lag bolts. A
mapped colony was then manipulated gently so that the
full silhouette view of its zooid-bearing lobe (along with
a 15-cm ruler, for scale) could be photographed. Colo-
nies thus censused could thereafter be identified by their
map coordinates and photorecords. Colonies that ap-
peared on the study sites for the first time and were large
enough to be positively identified as P. plamim (with a
lobe-silhouette area typically between 50 and 200 mm2)
were referred to as "recruits." A colony's post-recruit-
ment life span ended when it was dislodged from its
mapped location.

Photographs of colonies were digitized, and the silhou-
ette areas of zooid-bearing lobes were measured by
means of video imagery analysis (Image version 1.43).

90

A R HOLYOAK

s

M

5J
g
O.
(L>

4250 -

May - Oct

4000

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1750

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1500

1250 -

a

1000 -
750 -

d

°^

500 -

po

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250 -

Mo

OO o

^°0

0 0 C0°
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o o §

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2000 4000 6000

Initial Colony Silhouette
Area mm"

0 2000 4000 6000
Initial Colony Silhouette
Area mm"

Figure 3. Relative percent growth ofPolyclinum planum colonies by size and season. The graph on the
left shows growth of 55 colonies during calm, warm-water months; the graph on the right shows growth of
53 colonies during months with colder, rough sea-state conditions. Vertical dashed lines delineate colony
size classes (small = <200 mm" lobe-silhouette area; medium = 200-1800 mnv; large = >1800 mm2).

Lobe silhouette areas were used to generate a mean
growth trajectory for P. planwn. I used stepwise polyno-
mial regression (Zar, 1984) as a descriptive tool to fit a
curve to the mean growth data. That curve should not be
used to make statistical comparisons, however, because
the monthly data included individual colonies that were
sampled multiple times (i.e.. in consecutive months),
and therefore violate the assumptions of independence
required for regression analysis. Consequently, compar-
isons of P. planning mean growth trajectory to theoreti-
cal growth curves are unavoidably qualitative.

To determine where new material (zooids and tunic)
is added to zooid-bearing lobes of growing colonies, I
manipulated eight of the colonies on my sites. I tied loops
of cotton-wrapped ny! ad through three points on

their lobes — one loop through the lobe near the pedun-
cle, one loop through the «; writer of the lobe, and one loop
through the lobe near the 1 In successive months

I measured distances between those marks, and between
the marks and lobe edges.

In April 1991 I began measuring the thickness of lobes
and calculating a lobe flatness value for each colony by
dividing a lobe's maximum width by its maximum thick-

ness. Spheroidal lobes had flatness values of 1 .0, and col-
onies with lobes wider than they were thick had flatness
values greater than 1 .0. Those values were used to gener-
ate a mean trajectory of lobe flattening for P. planum. I
again used stepwise polynomial regression to fit a curve
to the trajectory of lobe flattening.

At the end of my study, in May 1992, I collected 77
P. planum colonies from the study sites to test for rela-
tionships between colony age, size, and reproductive
state. Those colonies ranged from 66 mm: to 566 1 mm2
in size (colony-silhouette area) and from 1 to 24 months
in age.

I determined the reproductive state of those colonies
by cutting three wedge-shaped pieces from the lobe of
each colony — one from the lobe tip and one from each
lateral edge of the colony. I counted the total number of
exposed zooids on one side of each wedge, the number
of those zooids undergoing strobilation, and the total
number of larvae brooded by the nonstrobilating zo-
oids. I also dissected the post-abdomen from four non-
strobilating zooids per wedge and measured the largest
oocyte from each one. I used chi-square analyses to test
for effects of colony age and colony size on reproductive

COLONY GROWTH OF POLYCLINUM

91

condition of P. planum. I also used linear regression to
test for the effect of colony age on colony size.

Results

Potycliniim p/anum colonies were present on the re-
search sites throughout the study. Recruitment and dis-
lodgement of colonies occurred year-round, as did col-
ony growth. The colonies did not fragment: each recruit
produced only one pedunculate colony. During the
study 2 1 1 P. planum colonies appeared at the study sites
as recruits and became dislodged between 1 and 23
months later. The mean value for maximum lobe-sil-
houette area attained by those colonies was 803.0 mm:
(SD = 1224.4 mm:; n = 211 colonies). The largest of
those colonies had a lobe-silhouette area of 85 1 1 mm2 at
17 months of age.

The mean post-recruitment life span of P. planum was
5.52 months (SD = 5.49 months: n = 2 1 1 colonies). The
longest-lived colony on my sites persisted an exceptional
24 months. That colony did not become dislodged dur-
ing the course of this study, so it was not used in analyses
that included data only from colonies that did become
dislodged.

Mean growth trajectory

Mean colony size was plotted against age in months to
generate a growth trajectory for P. p/ainim (Fig. 2). The
mean growth trajectory included a period of rapid
growth throughout the first 10-12 months, followed by a
period of attenuating growth to a mean maximum size
of 1759 mm2 at month 15. The growth trajectory also
indicated a decline in colony size among older colonies.
Most colonies aged 1 4 months or older (2 1 of 27 colonies
= 77.8%) decreased in lobe-silhouette area before being
dislodged. The 21 colonies that experienced a size de-
crease lost an average of 42.4% (SD = 25.5%) of their
maximum lobe-silhouette area prior to dislodgement.
Some colonies decreased in size gradually, by abrasion;
others lost larger pieces of their lobes, presumably in a
single event, the explanation of which remains problem-
atic.

The growth data also revealed increasing size variabil-
ity within age classes throughout the first 9 months. After
the 9th month standard deviations were so wide in some
age classes that they overlapped the mean values of vir-
tually all other age classes (for example, see Fig. 2,
months 9. 12. 13. and 18). This trend suggests that col-
ony age and size were not as tightly linked among larger
colonies as among smaller ones, thus demonstrating the
indeterminate nature of P. planum colony growth.

Relative percent growth of P. planum colonies, com-
paring colony-silhouette area at the beginning and end of
6-month intervals — May through October (months with

12/91

1/92

Figure 4. Patterns of lobe expansion for four colonies with marked
lobes. An "X" indicates the location where a loop of thread was tied
through the zooid-bearing lobe.

calmer sea-state conditions) or November through April
(months with rougher sea-state conditions) — confirmed
the general trends of size-specific growth indicated by the
mean growth trajectory. Smaller colonies grew more rap-
idly than larger colonies (Fig. 3). Those growth data also
suggest a seasonal effect on growth rates: the smallest size
class of colonies exhibited much greater increases in rel-
ative percent growth during periods of calmer and
warmer sea-state conditions (between May and October)
than did colonies of similar initial size during rougher
and cooler sea-state conditions (between November and
April).

To test for seasonal effects on size-specific relative per-
cent growth, I assigned colonies to three size classes: (1)
small (<200mm2 colony-silhouette area: the size of
most recruits): (2) medium (200-1800 mm2: colonies
larger than recruits but smaller than the maximum size
indicated by the growth trajectory); and (3) large
(> 1 800 mm2; colonies larger than the maximum colony
area indicated by the growth trajectory). 7-tests showed
a significant seasonal effect on growth of medium-sized
colonies (t = 3.309; df = 65; P = 0.0014). There was no
seasonal effect on growth of large colonies (t = 1.874; df

92

A R HOLYOAK

8 -i

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2 6 -
a

•t-t

tL,

4H

0

0

12

18

Colony Age (months)

24

Figure 5. Mean trajectory of lobe flattening for Palyclinum plainim. A linear regression provides a
significant fit to the data (}' = 1.528 + 0.106.Y, r = 0.568; / = 4.73; df = 17; P < 0.001). Error bars are
standard deviations, and numbers indicate the number ot observations included in each age class. The
single outlier at month 23 was not included in the curve-fitting analysis. The numbers of colonies indicated
per month on this figure differ from those of Figure 2 because I began measuring lobe thicknesses in April
1991. and 1 began measuring lobe silhouette areas (shown in Fig. 2) in December 1989.

= 22; P = 0.074) or small colonies (/ = 1 .246; df = 1 5; P
= 0.232). The lack of a seasonal effect on small colonies
is probably due to the extremely wide variability of
growth displayed by small colonies in the May-October
group (Fig. 3).

Intra-colony growth

Four of eight colonies with marked lobes persisted
long enough to show where P. planum can add new ma-
terial to the zooid-bearing lobe (Fig. 4). Colony A added
new material in a diffuse manner throughout the length
and width of its lobe. More material was added between
the middle mark and the mark furthest from the pedun-
cle than between the middle and proximal marks. After
July 1991, colony A also added more material along its
right side than along its left side. Colony B decreased
slightly in size in January 1 99 1 , after which material was
added to the left side of the colony. Colony C added more
material to the lobe proximal to the peduncle than dis-
tally, causing the two marked spots on this colony to
move away from the peduncle but not much farther
from each other. Colony C also added material along the
left and right sides of the lobe. Colony D added material
along the right and left sides of its lobe, producing a
spade-shaped silhouette. The peduncle of that colony
also expanded upward into the lobe, causing the marked
spots to appear to move closer to the peduncle.

Mean trajectory of lobe flattening

Zooid-bearing lobes of small colonies changed grad-
ually from a spheroidal shape to a laterally flattened dis-
coidal shape as they grew (Fig. 5), becoming what were

essentially bilayered sheets of zooids. Lobes of recruits
had a mean cross-sectional flatness value of 1.1 (SD =
0.3; /; = 121 recruits appearing between April 1991 and
May 1992). The flattest colony measured was 20 months
old; it had a lobe width of 1 1 3.5 mm and a flatness value
of 12.6. Stepwise polynomial regression showed that a
linear function best described changes in colony flatness.
The mean flatness value of the regression was 2.53. The
linear function of the regression shows that ratios of lobe
width to thickness typically increase throughout a colo-
ny's life span. Increases in lobe-flatness values are largely
the result of the expansion of lobe width, because lobe
thickness typically did not decrease during a colony's life
span.

Recruitment and survivorship

Simple (least squares) linear regression showed that
the number of P. planum recruits appearing at the study
sites each month was not significantly dependent upon
surface water temperatures (Fig. 6: r = 1.46X 10~6;df =
1,21; P > 0.75) (seawater temperatures were measured
daily by HMS staff). Likewise, the number of recruits
appearing monthly at the study sites was not dependent
upon the number of days of strong surge conditions per
month(r = 0.022; df= 1,2 \;P= 0.5). Seawater temper-
ature and surge conditions were measured by HMS staff.
Linear regression of the number of colonies dislodged
per month on water temperature did, however, indicate
a significant relationship (r = 0.345; df = 1.21; P <
0.005), as did the regression of the number of colonies
dislodged per month on the number of days of strong
surge per month (r = 0.256: df= 1,21; P< 0.025).

CXH.ONY (iROWTH OF POLYCUNL'M

93

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(a)
18 -]

15 -

12 :

Cold : Warm ; Cold ; Warm ; Cold

12/89

6/90

12/90 6/91

12/91

Calm

Rough

Calm ; Rough

12/89

6/90

12/90 6/91

12/91

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T3

30 -
20 -

10 H

o

12/89
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20 -
10 -

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.•ill.ll ..•lllhlll

12/90

6/91

12/91

12/89

6/90

12/90

6/91

12/91

Month/Year

Figure 6. (a) Mean monthly surface seawater temperature at Hop-
kins Marine Station (HMS): (b) number of days of strong surge condi-
tions per month at HMS; (c) number of recruits appearing at the re-
search sites per month: (d) and number of colonies dislodged from re-
search sites per month. Vertical dashed lines indicate break points
between warm months with calm sea-state conditions and colder water
months with rougher sea-state conditions. Asterisks on the two lower
graphs indicate months during which data could not be collected. Data
on seawater temperature and sea-state condition were collected by and
provided courtesy of HMS staff.

Significantly more colonies were dislodged from the
study sites during months with rough (as denned by
HMS staff) sea-state conditions (Nov-Apr; .v = 7.2 days
of strong surge per month; SD = 3.7 days) than during
months with calmer sea-state conditions (May-Oct; .v =
1.0 day of strong surge per month; SD = 1.3 days), as
indicated in Table I.

For both small and large colonies, the proportion of
colonies dislodged was significantly larger during rough
months than during calm months (Table I). There was,
however, no statistical difference between the proportion

Table 1

Tin.1 number uf Polyclinum planum colonies dislodged per .virc
cla.i.\ />!' sca.ion

Colony size class
(colony-silhouette area in mnv)

0-200

200-1800

>1800

Strong surge (Nov-Apr)

75(62.5)

74(69.9)

44(31.4)

Calm (May-Oct)

21(33.5)

39(43.1)

13(25.6)

Column x2 values

7.16*

0.63 NS

11.26***

Contingency table \2

value = 19.05***

The results of a chi-square analysis of this 2x3 contingency table
and the results of chi-square tests for each column are indicated. Num-
bers in parentheses are the expected values for each cell.

* = P <0.01; *** = P<0.001;NS = not significant.

of medium-sized colonies dislodged in rough m-mscalm
months. The medium-sized colonies had survived early
post-recruitment mortality but had not grown large
enough to exceed the maximum colony size of the mean
growth trajectory.

Age- and size-dependent survivorship data (Fig. 7)
showed that the greatest mortality occurred among the
youngest colonies (39.3% — 83 of 211 colonies) and
among the smallest colonies (47.9% — 101 of 21 1 colo-

OB

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'E

3

on

8 l(H

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6 12 18

Colony Age (months)

24

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C/}

c/:
OJ

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10 -

2000

4000

6000

8000

Colony Silhouette Area mm

Figure 7. Age-dependent and size-dependent survivorship curves
for Polvclinum planum.

94

A. R. HOLYOAK

Table II

Comparisons of number of colonies in each sue class with looids
undergoing strobilalion. bearing oocyles. and brooding larvae

Colony size classes
(colony silhouette area in mm2)

0-200 200-1800 >1800

Strobilation

100%zooidstrobilation 11(5.45) 23(23.64) 1(5.91)

<1 00% zooid Strobilation 1(6.55) 29(28.36) 12(7.09)
Column x2 values 10.35** 0.18NS 7.48*

Contingency table x2

value = 18.01***
Oocytes

Oocytes present 0(2.03) 7(8.78) 6(2.19)

Oocytes not present 12(9.97) 45(43.2) 7(10.81)

Column x- values 2.44 NS 0.47 NS 7.97**

Contingency table x2

value = 10.88**
Brooded Larvae

Brooded larvae present 0(0.94) 2(4.05) 4(1.01)

Larva not present 12(11.06) 50(47.95) 9(11.99)

Column x2 values 1.02NS 1.12NS 9.60**

Contingency table x2
value = 11.74**

The results of a chi-square analysis of this 2x3 contingency table
and the results of chi-square tests (breach column are indicated. Num-
bers in parentheses indicate the expected values for each cell. * = P <
0.01;** = P < 0.005: *** = /><O.OOl;NS = not significant.

age class (colonies 1 month old), but the other age classes
did not differ significantly from expected values for Stro-
bilation. Age-class comparisons of the frequency of col-
onies bearing developing oocytes or brooded larvae did
not differ from expected values (Table III). There was,
however, a significant relationship between colony age
and colony size (Fig. 8). Colony size appears to be a bet-
ter predictor of reproductive state that colony age, but
colony size is affected by colony age, because the smallest
colonies tend to be young, and larger colonies tend to be
older.

I used multiple regression analysis to ascertain the rel-
ative effects of colony size and colony age on oocyte size.
That analysis was significant, explaining 36.87% of ob-
served variability (F = 2 1 .96; df = 2,75; P < 0.00 1 ). Col-
ony size accounted for 99.9% of the variability explained
by the regression, whereas colony age contributed only
an additional 0.01%- to the explanation (F = 0.035 NS;
df = 1 ,75; P > 0.75). Colony size may thus be invoked as
a primary determinant of oocyte size; colony age has a
secondary effect on oocyte size because it affects colony
size.

Discussion

Polyclinuni plununi colonies do not fragment into
physiologically isolated ramets as they grow. This obser-
vation bears out the assumption made by Pearse el al

nies with silhouette areas smaller than 200 mm:). After
those initial losses, both age- and size-specific survivor-
ship curves indicated a nearly constant rate of mortality,
with possible increased mortality among the largest col-
onies.

Reproductive state

Results of chi-square analyses of the effects of colony
size and colony age on reproductive state of colonies
collected in May 1992 are shown in Tables II and III,
respectively. For colonies of the smallest size class
(<200-mm2 colony-silhouette area), the frequencies of
colonies bearing strobilating zooids were higher than ex-
pected; in medium-sized colonies (200-1800 mm2) the
frequencies did not differ from expected values; and in
large colonies (> 1800 mm2) the frequencies were lower
than expected. The large size class also had higher-than-
expected frequencies of colonies with developing oocytes
and of brooding larvae, whereas the other size classes did
not differ significantly from expected values for those pa-
rameters (Table II).

Results of chi-square analyses on colony age class and
reproductive state showed a significant age effect on the
frequency of strobilating colonies among the youngest

Table III

Comparisons of number of colonies in each age class with zooids
undergoing slrobilalion. bearing oocyles. and brooding larvae

Colony age classes (months)

2-15

Strobilation

100"! zooid Strobilation 10(4.85) 23(27.38) 1(1.76)

< 100% zooid Strobilation 1(6.15) 39(34.62) 3(2.24)

Column x: values 9.78** 1.25NS 0.59 NS

Contingency table x2

value = 11.62**
Oocytes

Oocytes present 0(1.85) 12(10.47) 1(0.68)

Oocytes not present 11(9.15) 50(51.53) 3(3.32)

Contingency table x2

value = 3.20 NS
Brooded larvae

Larvae present 0(0.86) 6(4.83) 0(0.31)

Larvae not present 11(10.14) 56(57.17) 4(3.69)

Contingency table x2

value = I.57NS

The results of a chi-square analysis of this 2x3 contingency table,
and, when the contingency test is significant, the results of chi-square
tests for each column are indicated. Numbers in parentheses indicate
expected values for each cell. ** = P< 0.005; NS = nonsignificant.

COLONY GROWTH OF POLYCLINUM

95

6000 -i

I

S 4000 -

o

B
_O

O

U

2000 -

0 -

= 0.449

12

18

24

Colony Age (months)

Figure 8. The relationship between colony age and colony size for
Polvcliinim planum collected in May 1992 (n = 77 colonies). The solid
line indicates the linear regression, which was significant at « = 0.05.

(1989) that this is a nonfragmenting species and means
that each P. planum colony represents an entire genet.
As such, the loss of a P. planum colony is a greater loss, in
an evolutionary sense, than if it were only one of several
physiologically isolated ramets. P. planum has conse-
quently developed growth-related adaptations that in-
crease the chances of colony survivorship even in physi-
cally stressful intertidal environments.

The most obvious ecological risk to erect, though flex-
ible, intertidal colonies of P. planum is dislodgement by
water-flow forces. Denny el al. (1985) demonstrated how
such forces can dislodge or limit the size of intertidal or-
ganisms living in wave-impacted environments. Since
intertidal P. planum colonies are subjected to those kinds
of water flow forces, it is not surprising that this species
exhibits growth-related adaptations for dealing with
stresses associated with water flow. The pattern of P. pla-
num colony growth reveals two strategies for mitigating
the effects of water flow: ( 1 ) an attenuating pattern of
growth; and (2) allometric expansion of the zooid-bear-
ing lobe into a laterally flattened structure.

Attenuating growth, like that demonstrated by P. pla-
num (see Fig. 2), is common among colony-forming
modular organisms such as corals (Hughes and Connell,
1987; Karlson, 1988;Lasker, 1 990 ),bryozoans( Hughes,
1990; Kauffman. 1981). and ascidians (Ryland el al,
1984; Stoner, 1989; Stocker, 1 99 1 ). In the case of P. pla-
num, it appears that environmental stress limits colony
size. The general pattern of P. planum's attenuating
growth is several months of rapid growth followed by

slowing and eventual cessation of growth. A few colonies
did, however, grow continuously until they were dis-
lodged. The typical pattern of P. planum growth does not
differ markedly from Sebens' ( 1987) predictions for In-
determinate Growth Type III (Plastic Attenuating
Growth), but the growth pattern of a few colonies ap-
proximated Sebens' Indeterminate Growth Type II
(Plastic exponential growth).

The maximum size for a P. planum colony is set, at
least in part, by water-flow forces. This is evidenced by
the fact that some colonies, probably in protected micro-
habitats, produced lobes much larger than 1800 mm2
(colony-silhouette area — the maximum colony size indi-
cated by P. planning mean growth trajectory — see Fig.
2), even though susceptibility to dislodgement increased
for other colonies when their silhouette areas exceeded
1 800 mm2 (see Table I). Similar water-flow-induced size
restrictions have been suggested or demonstrated for col-
ony-forming modular organisms such as sea fans (Birke-
land, 1974) and coral (Denny el al. 1985), and for uni-
tary organisms including sea urchins, limpets, mussels,
and snails (Denny el al., 1985).

Life-history constraints also limit colony growth. As P.
planum colonies increase in size, they tend to become
reproductively active (see Table II). The onset of repro-
duction is a critical event in the life history of any organ-
ism (Lloyd, 1980;Sebens, 1982; Kozlowski and Weigert,
1986). A shift from growth to reproductive activity in P.
planum appears to occur as the rate of colony growth
slows. This relationship between growth and reproduc-
tion apparently occurs not only in colonies of P. planum
but among colonies of some other ascidians (Bak el al.,
1981; Brunetti el al.. 1988). For some clonal taxa, how-
ever, the event triggering the onset of reproductive activ-
ity can vary greatly and may include colony age, size, or
environmental stimuli; in other cases neither colony age
nor size is a good predictor of the onset of reproduction
(HarvellandGrosberg, 1988).

Slowed growth at the onset of reproductive activity in
P. planum is a consequence of life-history constraints on
individual zooids. P. planum zooids are hermaphroditic,
producing sperm and oocytes in gonads housed in the
post-abdomen (Ritter and Forsyth, 1917). Iterative pro-
duction of new zooids (i.e.. colony growth) involves stro-
bilation of the post-abdomen, where gonads develop,
and disorganization of the atrial chamber (Holyoak,
1992), where larvae are brooded. Asa result, a P. planum
zooid cannot simultaneously undergo strobilation and
produce gametes or brood larvae.

The second growth-related strategy that P. planum
uses to minimize the effects of water flow involves mor-
phogenesis of the zooid-bearing lobe from a small sphe-
roidal structure into a large laterally flattened one.
Changes in the silhouette shape of marked colonies sug-

96

A. R. HOLYOAK

gest that P. planum is able to add material to just about
any part of a growing lobe, th r, bj producing a wide
range of colony shapes (see Fi;

The ecological advantage lobe flattening is evident
when the morphology .mum colonies is consid-

ered in light of the ace in of water, which, accord-

ing to Denny et al. • >5), is the water-flow force with
the greatest ability to dislodge sessile organisms. The im-
pact of the acceleration reaction of water is directly pro-
portional to an object's volume, not to its exposed sur-
face area (Denny, 1993). If the spheroidal lobe of a P.
planum recruit were to increase in size isometrically, pro-
ducing a large spherical lobe, the stress from the acceler-
ation reaction and the chance of dislodgement would hy-
pothetically be greater for that colony than for one with
a flattened lobe having the same frontal silhouette area.

Increased reproductive potential is a direct benefit of
colony growth and lobe flattening. A flattened lobe has a
greater surface-to-volume ratio than a spheroidal lobe.
Because zooids are located only at the surface of P. pla-
num colonies (Abbott, 1987). a flat lobe can house more
zooids than a spheroidal lobe with the same volume. And
since each zooid has the potential to produce gametes
and brood larvae, the reproductive capacity of a colony
is directly proportional to the number of zooids it con-
tains. P. planum is therefore able to increase its surface
area, maximize the number of zooids it can bear, and
simultaneously minimize the effects of the water acceler-
ation reaction by producing a flattened lobe.

One final consequence of growth is its impact on the
mortality rate of recruits (Table I). Though growth be-
yond an environmentally imposed size limit puts the
largest colonies at risk, the greatest observed mortality
occurred among the smallest and youngest colonies ( Fig.
7). Birkeland (1974) reported a similar trend of high
mortality among smaller members of a population of sea
fans growing in heavy surf. High mortality among the
smallest size class of P. planum colonies is almost cer-
tainly a function of site selection by larvae at settlement.
The long-term suitability of a site is not tested until col-
onies grow large enough to extend beyond the boundary
layer. Only colonies with the best settlement sites and the
firmest peduncular attachments will survive to grow into
the larger size classes. Mortality among small colonies
is almost certainly a consequence of growth because P.
planum has no ; n predators, and I saw no evidence
of predation o :olonies during this study.

In conclusion, a level growth and morphogene-

sis of modular organisi ..re large-scale effects of self-
assembly processes. Thos processes include the tempo-
ral and spatial iteration nd arrangement of modules
(Rosen, 1986; Ryland and Warner. 1986). The result of
those processes is colony morphology that can convey
ecological advantage (e.g., Ryland and Warner. 1986,

and references therein). Though we are learning more
about the complexities and significance of modular
growth, we are still largely ignorant of the mechanisms
that regulate it. My data suggest that growth in P. planum
may be regulated by a combination of external and in-
ternal factors. As we unravel the rules of colony-level
modular growth, we may gain insights into the processes
that regulate and drive development at other levels of bi-
ological organization.

Acknowledgments

I thank A. T. Newberry, J. S. Pearse, C. M. Young, B.
Rinkevich, and two anonymous reviewers for their help-
ful comments on drafts of the manuscript. Support for
this study was provided by the Department of Biology,
University of California at Santa Cruz: Friends of the
Long Marine Laboratory; the American Museum of
Natural History (Lerner-Gray Fund for Marine Re-
search); the Earl and Ethel Myers Oceanographic and
Marine Biology Trust: and Manchester College.

Literature Cited

Abbott, D. P. 1987. Observing Marine Invertebrates. Stanford Univ.
Press. Stanford.

Bak, R. P. M., J. Sybesma, and F. C. vanDuyl. 1981. The ecology of
the tropical ascidian Trididemnvm solidum. II. Abundance, growth
and survival. Mar Ecol, Prog Ser. 6: 43-52.

Berrill, N. J. 1950. The Tunicaia. The Ray Society, London.

Birkeland. C. 1974. The effect of wave action on the population dy-
namics of Gorgoniu ventalina Linnaeus. Stud. Trop. Oceanogr. 12:
115-126.

Brunetti, R., M. Bressan, M. Marin, and M. Libralato. 1988. On the
ecology and biology of Diplosoma listenanum (Milne Edwards.
1 84 1 ) ( Ascidiacea, Didemnidae). I 'if Milieu 38: 128-131.

Denny, M. \V. 1993. Airand Water: The Biology and Physics of Life's
Media. Princeton Univ. Press, Princeton.

Denny, M. \V., T. L. Daniel, and M. A. R. Koehl. 1985. Mechanical
limits to size in wave-swept organisms. Ecol. Monogr. 55: 69-102.

Harvell, C. D., and R. K. Grosberg. 1988. The timing of sexual matu-
rity in clonal animals. Ecology69: 1855-1864.

Holyoak, A. R. 1992. Morphogenetic movements and assembly of
strobilae into zooidal systems in early colony development of the
compound ascidian Polyclinum planum. Bio/ Bull. 183: 432-439.

Hughes, T. P. 1984. Population dynamics based on individual size
rather than age: a general model with a reef coral example. Am. Nal.
123:778-795.

Hughes, T. P. 1990. Recruitment limitation, mortality, and popula-
tion regulation in open systems: a case study. Ecologv1\: 12-20.

Hughes, T. P., and J. B. C. Jackson. 1980. Do corals lie about their
age? Some demographic consequences of partial mortality, fission,
and fusion. Science 209: 7 1 3-7 1 5.

Hughes,!. P.,andJ. B.C.Jackson. 1985. Population dynamics and
life histories of foliaceous corals. Ecol. Monogr 55: 141-166.

Hughes, T. P., and J. II. Connell. 1987. Population dynamics based
on size or age? A reef coral analysis. Am. Nal. 129: 818-829.

Jackson, J. B.C. 1977. Competition on marine hard substrata: the
adaptive significance of solitary and colonial strategies. Am. Nal.
111:743-767.

Jackson, J. B. C., and A. G. Coates. 1986. Life cvcles and evolution

COLONY GROWTH OF I'OLYCLINL'M

97

ofclonal (modular) animals. Phil. Trans R Site Lond. B 313: 7-
22.

karlson, R. H. 1986. Disturbance, colonial fragmentation, and size-
dependent lite history variation in two coral reef cnidarians. Mar.
Ecol. Prog Sir. 28: 245-249.

Karlson. R. H. 1988. Size-dependent growth in two zoanthid species:
a contrast in clonal strategies. Ecology 69: 1219-1 232.

Kauffman, K. \V. 1981. Fitting and using growth curves. Oecologia
49: 293-299.

Kozlonski, J., and R. G. Weigert. 1986. Optimal allocation of energy
to growth and reproduction. Tlwor. Pofiiil. Bin/. 29: 16-37.

Lasker, H. R. 1990. Clonal propagation and population dynamics of
gorgonian coral. Ecologyll: 1578-1589.

Lloyd, D. G. 1980. Benefits and handicaps of sexual reproduction.
Evol. Bwl. 13:69-111.

McFaddcn, C. S. 1991. A comparative demographic analysis of
clonal reproduction in a temperate soft coral. Ecology 72(5): 1 849-
1866.

Millar, R. H. 1952. The annual growth and reproductive cycle in four
ascidians. J. Mar. Biol. Assoc. {.'. A. 31 : 4 1 -6 1 .

Millar, R. H. 1971. The biology of ascidians. Adv. Mar Bio/. 9: I-
100.

Panccr, 7... H. Gershon, and B. Rinkevich. 1995. Coexistence and
possible parasitism of somatic and germ cell lines in chimeras of the
colonial urochordate Botryllux.ichlox.wri. Biol. Bull 189: 106-1 12.

Patzold, J., H. RistedC, and G. \\efer. 1987. Rate of growth and lon-
gevity of a large colony ofPentaforafoliacea (Bryozoa) recorded in
their oxygen isotope profiles. Afar. Biol. 96: 535-538.

Pearse, J. S., V. B. Pearse, and A. T. Newberry. 1989. Telling sex
from growth: dissolving Maynard Smith's paradox. Bull. Mar. Sci.
45: 433-446.

Rinkevich, B., and I. L. VVeissman. 1987a. Chimeras in colonial in-
vertebrates: a synergistic symbiosis or somatic- and germ-cell para-
sitism? Symbiosis 4: 117-1 34.

Rinkevich, B., and I. L. Weissman. I987b. A long-term study on
fused subclones in the ascidian Botrylliix xchlosseri: the resorption
phenomenon (Protochordata: Tunicata). J. Zoo/. (Lond.) 213: 7 1 7-
733.

Ritter, W. E., and R. A. Forsyth. 1917. Ascidians of the littoral zone
of southern California. Univ. Calif. Pub/. Zoo/. 16:439-512.

Rosen, B. R. 1986. Modular growth and form of corals: a matter of
metamers? Phil. Trans. R Soc. Lond. B313: 1 15-142.

Ryland, J. S., and G. F. Warner. 1986. Growth and form in modular
animals: ideas on the size and arrangement of zooids. Phil. Trans.
R. Soc. Lond. B 313: 53-76.

Ryland, J. S., R. A. VVigley, and A. Muirhead. 1984. Ecology and co-
lonial dynamics of some Pacific reef flat Didemnidae (Ascidiacea).
Zoo/. J. Linn. Soc. 80: 261-282.

Sebens, K. P. 1982. The limits of indeterminate growth: an optimal
size model applied to passive suspension feeders. Ecology 63: 209-
222.

Sebens, K. P. 1987. The ecology of indeterminate growth in animals.
Ann. Rev Ecol. Sy.xi. 18: 371-407.

Stoner, D. S. 1989. Fragmentation: a mechanism for the stimulation
of genet growth rates in an encrusting colonial ascidian. Bull. Mar.
Sci. 45:277-287.

Stocker, L. J. 1991. Effects of size and shape of colony on rates of
fission, fusion, growth and mortality in a subtidal invertebrate. J.
E.\p. Mar. Biol. Ecol 149: 161-175.

Zar, J. H. 1984. Bioxtutixtical Analyxix. 2nd cd. Prentice-Hall, Inc.,
Englewood Cliffs, N.I.

Reference: Biol. Bull 192: 98-1 10. (February. 1997)

Conflicting Morphological and Reproductive Species
Boundaries in the Coral Genus Platygym

KAREN MILLER1 * AND RUSSELL BABCOCK:t

1 Marine Biology Depl. James Cook University of North Queensland, Townsville, QLD 481 1.
Australia: and 2 Australian Institute of Marine Science, PMB 3, Townsville, QLD 4810, Australia

Abstract. In mass-spawning corals, potential exists for
gametes of a number of species to mix in the water col-
umn. The existence of morphologically distinct sympat-
ric coral populations despite such an event implies the
presence of isolating mechanisms to prevent hybridiza-
tion and maintain species boundaries. Over 380 fertiliza-
tion trials were conducted to determine the extent of re-
productive isolation among the seven morphologically
defined species (morphospecies) of the scleractinian
coral genus Platygyra. on the Great Barrier Reef. Results
from these experiments demonstrate that fertilization
between-morphospecies can occur at rates equivalent to
within-morphospecies fertilizations, indicating that no
gametic-level barriers to fertilization exist among these
morphological species. Observations of spawning times
both in the field and in the laboratory have shown that
all seven morphospecies spawn on the same night and
that there is considerable overlap in the hour of spawning
among them. These data indicate that few, if any, tem-
poral barriers to fertilization exist among morphospecies
of Platygyra on the Great Barrier Reef. In addition, lar-
vae resulting from between-morphospecies crosses are
capable of settlement and subsequent growth equivalent
to that of within-morphospecies larvae. Our results re-
veal a discontinuity between reproductive and morpho-
logical species boundaries within the scleractinian genus
Platygyra and challenge species definitions within the
Scleractinia. It is not yet clear what mechanisms might
maintain morphu!. /i^al boundaries in Platygyra in the
face of the clear pt raial for gamete mixing. The dis-

Received ISJuly I996;an , November 1996.

* Present address: National 1 1 ' Water and Atmospheric Re-

search, PO Box 14-901. Kilbirnie, Wellington. New Zealand.

t Present address: University of Auckland. Leigh Marine Labora-
tory. P.O. Box 349, Warkworth. New Zealand.

junct distributions of certain morphospecies along lati-
tudinal and habitat boundaries, and the small levels of
variation in reproduction may be two such mechanisms.

Introduction

Species boundaries in scleractinian corals are primar-
ily based on skeletal morphology (e.g.. Ellis and So-
lander, 1786; Vaughan and Wells, 1943; Veron el a/..
1977; but see Lang, 1984). It is generally assumed that
morphological differences between coral species are
highly correlated with reproductive incompatibility (i.e.,
biological species boundaries) (Willis, 1 990) and that tra-
ditional theories of hierarchical evolution and speciation
(e.g., Darwin, 1 859) will apply to corals.

Recognition that most of the corals on the Great Bar-
rier Reef reproduce annually during a mass spawning
event (Harrison et a/., 1984; Willis et al., 1985; Babcock
el u/.. 1986) has prompted an examination of assump-
tions that morphological species boundaries reflect bio-
logical species boundaries within the Scleractinia (Wal-
lace and Willis, 1994). The persistence of morphologi-
cally distinct species groups (e.g.. Veron et al., 1977)
coupled with mass spawning events, whereby gametes of
many species become mixed within the water column,
implies the presence of some mechanism to prevent hy-
bridization and hence maintain species boundaries (Wil-
lis, 1990). The benefit of such mechanisms was sup-
ported by results from experimental crosses in the genus
Montipora. in which hybridization was documented but
considered to be maladaptive due to arrested develop-
ment in hybrid embryos (Hodgson. 1988). More re-
cently, however, fertilization experiments have indicated
that successful fertilization in vitro is possible among
some morphological coral species (Willis el al.. 1992),
although how experimental fertilization relates to field
fertilization is unknown.

98

CONFLICTING SPECIES BOUNDARIES IN PLATYdYRI

99

The coral genus Platygyra includes morphological
species (morphospecies) from the Great Barrier Reef;
Platygyra daedalea, P. sinensis, P. pini. P. lanu-llina, P.
ryukyuensis. Platygyra 'B', and Platygyra 'H' (see Miller,
1994a, b). These morphological species have been recog-
nized by traditional taxonomy (e.g.. Chevalier, 1975;
Veron ct ai. 1977; Veron, 1993) and numerical taxon-
omy (Miller, 1 994a), although both approaches have rec-
ognized a continuum of separate characters among the
species. Based on the occurrence of morphologically dis-
tinct species of Platygyra, it seems highly likely that
mechanisms exist that promote reproductive incompat-
ibility and subsequently maintain species differences
within the genus.

Contrary to expectations, genetic comparisons show
no differentiation between the morphological species of
Platygyra (Miller and Benzie. in press). The high genetic
similarity of Platygyra morphospecies may reflect recent
speciation events following which little genetic diver-
gence has taken place (Miller and Benzie, in press). Al-
ternatively, gene exchange may occur naturally between
the morphological species groups, and hence the mor-
phological boundaries in this genus may not necessarily
represent reproductive isolation between species. The re-
lationship between reproductive and morphological spe-
cies boundaries within Platygyra and the presence or ab-
sence of reproductive isolation between the morphologi-
cal species therefore need to be established.

A number of mechanisms, including pre- and post-zy-
gotic barriers, could mitigate reproductive isolation be-
tween coral species. In Platygyra, there is some indica-
tion of temporal differences (in the order of hours) in
spawning times among some of the morphospecies (Bab-
cock el al., 1986), although these observations involved
only a few colonies, and variability in spawning times
within morphospecies has not been documented. Coral
gametes remain viable for at least 5 h in the water col-
umn (Oliver and Babcock, 1992), and it is not known
whether these short temporal differences in spawning act
as a complete isolating mechanism.

Sperm chemotaxis is used by a variety of marine in-
vertebrates to attract conspecific sperm (e.g.. Miller,
1979, 1985), and may well be important in reducing po-
tential for hybridization, for example, in pelagic commu-
nities of hydromedusae (Miller, 1979). Chemical sperm
attractants have also been implicated as a mechanism to
reduce hybridization in the coral genus Monripora (Coll
et al, 1994). However, even in Montipora, where sperm
attractants have actually been identified, chemotaxis is
not completely specific, and heterospecific attractants
may still elicit a degree of chemotactic behavior between
species (Coll et ai, 1994; Richard Miller, pers. comm.).
At this point, the role of sperm chemotaxis as an effective

isolating mechanism during mass spawning is still spec-
ulative.

Species-specific gamete-binding proteins have been
described in some marine invertebrates, including aba-
lone (Vacquier ct al.. 1990) and sea urchins (Palumbi,
1992; Metz and Palumbi, 1996). Similar proteins may
play an important role in the reproductive isolation of
many free-spawning invertebrates, including scleractin-
ian corals.

The high predictability of spawning in Platygyra spe-
cies on the Great Barrier Reef (Babcock et al., 1986)
makes it possible to explore reproductive relationships
and potential isolating mechanisms between these corals
at the gamete level. In this paper, we document results
from more than 380 trials that show potential for fertil-
ization between the morphological species of Platygyra.
Parallel field observations of spawning times suggest that
few if any temporal reproductive barriers exist between
the morphological species. This evidence suggests there
are no pre-zygotic isolating mechanisms between the
morphological species of Platygyra. Furthermore, we
found no differences in the ability of larvae from within-
morphospecies and between-morphospecies crosses to
settle and grow. We also examined the effects of gamete
aging, sperm dilution, and parental genotype on fertiliza-
tion success.

The discovery that successful fertilization can occur
between morphological species of Platygyra calls into
question the previous assumption that morphological
species boundaries equate with reproductive isolation
and subsequently with evolutionary units. Our results
prompt a reexamination of species definitions within the
Scleractinia and indicate that species groupings based
solely on morphology are clearly not always "biological"
species ( Mayr, 1 942 ). Our data, combined with other re-
cent findings in this field (Knowlton et al., 1992; Van
Veghel and Bak, 1993; Van Veghel, 1994; Wallace and
Willis, 1 994 ), suggest that species definitions in the Scler-
actinia should perhaps be based on a range of criteria,
rather than solely on reproductive or morphological evi-
dence. In addition, we suggest that in Platygyra, even
with genetic exchange, discrete morphological groups
will be maintained through a combination of spatial and
temporal variability in reproductive processes and fitness
in a variety of habitats.

Materials and Methods

We conducted fertilization experiments and observed
spawnings over a 3 '/2-year study period during seven con-
secutive spawnings at three reefs in the central Great Bar-
rier Reef: Magnetic Island— 19° 10' S, 146°51'E (1990,
1991, 1992, 1993); Davies Reef— 1 8°50' S, 147°39'E
(1990, 1 991); and Orpheus Island— 18°35'S, 146°29'E

100

K. MILLER AND R. BABCOCK

(1992). On the afternoon of the predicted mass spawn-
ing, gravid Platygyra colonies wj> osferred from the
reef into separate containers; pi under natural light

to simulate normal condiiio ; olonies generally com-
menced spawning from k onwards. Once a colony
had spawned, gamete 's were collected from the

surface of the water and sperm and eggs were separated
by gentle agitation and use of a 105-/um sieve. Sperm
were stored at room temperature (about 27°C) in a con-
centrated form to reduce aging (see Chia and Bickell.
1983; Oliver and Babcock, 1992) until required for fer-
tilization. Eggs were taken through a series of 8 to 10
washes of 'sperm-free water' (collected half a kilometer
from the edge of the reef on the afternoon before the
commencement of spawning) to ensure the absence of
any trace sperm. Eggs were stored in glass jars with gentle
agitation until needed for experiments.

Eggs and sperm from different colonies were com-
bined experimentally in 20-ml glass scintillation vials to
examine fertilization success between colonies of differ-
ent morphologies. Morphological species identification
of all colonies used in fertilization trials was based on
Miller (1994a). Concentrations of the stored sperm were
estimated by five replicate hemacytometer counts and,
just before fertilization, diluted to a concentration of ap-
proximately 2.5 X 106 sperm/ml. This sperm concentra-
tion has been shown to give optimum rates for fertiliza-
tion in trials with Platygyra sinensis and other coral spe-
cies (Oliver and Babcock, 1992). About 100 eggs were
added to 20-ml aliquots of diluted sperm in a vial. Each
cross was replicated either two or three times. Sperm
concentrations within experimental vials were checked
randomly and found to be within 10% of estimated con-
centrations.

Experiments were designed in a matrix system in
which all colonies were crossed reciprocally with all other
colonies (eggs A X sperm B and eggs B x sperm A, etc.),
although trials between some morphological species
have not yet been conducted due to the time constraints
associated with coral spawning. All colonies were tested
for their ability to self-fertilize (eggs A x sperm A, eggs B
X sperm B. etc.). Replicate controls were prepared by
placing eggs from each colony in 20 ml of sperm-free wa-
ter to determine whether there had been any sperm con-
tamination of washed eggs. Glass vials containing eggs
and sperm were kept at ambient sea temperature with
gentle agitation to a w fertilization to take place.

Fertilization in all icate vials was recorded after 2-
3 hours and again after -J hours by censusing the first
100 eggs sampled fron, one vial. Fertilization was
scored as the initiation of cleavage. 2-3 h after fertiliza-
tion (Babcock and Heyward. 19 86). After about 7 h most
of the embryos were blastulae. The condition of eggs and
embryos in all crosses was recorded as either 'regular'—

with even, symmetrical cleavages and apparently normal
development (see Babcock and Heyward, 1986); or 'ir-
regular'— with uneven, asymmetrical cleavages and ab-
normal development.

Analysis offertili:ation data

Percent fertilization in crosses after 2-3 h and 6-8 h
was based on the total number of eggs censused in each
vial. Percent fertilization after 6-8 h was calculated to
detect any increase in fertilization over egg-sperm con-
tact time. The percentage of fertilized eggs developing to
blastulae between the two counts for each vial was calcu-
lated as the number fertilized after 6-8 h (including
cleavages and embryos) expressed as a percentage of the
number of fertilized eggs after 2-3 h. Mean percent fer-
tilization of the three replicate vials in each cross (colony
pairing) was used for the analyses. Any crosses for which
the controls showed sperm contamination of eggs during
the washing process were not used in the analyses. Re-
sults from the seven spawnings at three reefs were com-
bined at the level of morphological species for analysis.

A two-way analysis of variance (ANOVA) based on
the percent fertilization data after 2-3 h was carried out
to determine whether fertilization rates differed either
between between-morphospecies and within-morpho-
species fertilizations or between the different morpho-
species (based on maternal colony type). The main
effects were fertilization type (2 treatments; within and
between morphospecies) and egg species (7 treatments;
Platygyra B, P. daedalea. Platygyra H, P. lamellina, P.
pini. P. rynkyiiensis, P. sinensis). The analysis was re-
peated using the percent fertilization data after 6-8 h. A
further two-way ANOVA was carried out to compare the
development of cleaved eggs through to blastulae in
within- and between-morphospecies crosses. All of these
analyses were done using both arcsine-transformed and
raw data. Both methods produced similar results and
only results from analyses of raw data are presented here.

A one-way ANOVA comparing fertilization rates after
2-3 h between morphospecies combinations was also
done to determine whether there were significant differ-
ences in fertilization between morphological species-pair
crosses. Replication was too low to compare all morpho-
species-pair crosses: consequently this analysis was car-
ried out only on crosses in which P. daedalea was the
maternal colony, and for the sperm of P. daedalea, P.
lamellina. P. pini. P. ryiikyuensis. P. sinensis and selfs.
The incidence of irregular and regular embryos was com-
pared between crosses to determine whether irregular
embryos were more common in between-morphospecies
crosses.

Effect oj sperm concentration on fertilization rates

To determine whether fertilization success was an ar-
tifact of using optimal sperm concentrations in expert-

CONFLICTING SPECIES BOUNDARIES IN PL.-lTYtiYRA 101

Table I

Mean fertilization rates fr<»nfenili:ation trials between murplwloxical species <>/ Platygyra based on all crosses tried over the 3 '/r.mir study period

Sperm

PD

PH

PL

PP

PR

PS

PB

Sells

PD

45.2

18.3

66.8

65.4

36.3

52.6 1.3

3.1

(0-100)

(0-58.9)

(41-90)

(0-100)

(0-84.1)

(0-99.1) (0-6.3)

(0-6)

n = 49

n = 5

n = 1 3

n = 1 1

n = S

H = 1 8 n = 2

« = 28

PH

47.5

51.5

—

24.6

—

77.1

1.4

(0-94J)

(9-95.8)

(0-62.5)

(0-100)

(0-4.1)

>7 = 5

n = 2

n = 4

n = 2

n = 2

PL

68.2

—

58.1

—

63.8

72.3

2.1

(1-100)

(41-84)

(40-77)

(41-91)

(0-6.2)

n = 1 3

n = 6

n = 2

11 = 2

« = 4

PP

66.6

17.4

—

58.6

—

77.4

8.9

(6.5-99)

(0-41.8)

(1-96.7)

(26-98)

(0-15)

n= 10

n = 4

H = 4

n = 2

n = 5

PR

66.1

—

90.5

—

57.7

70.6

6.2

(7-100)

(90-92)

(31-71)

(27-92)

(0-14.3)

n = 8

n = 2

n = 2

n = 4

/i = 2

PS

71.7

26.6

45.4

7.3

65.3

50.1

3.7

(0-100)

(13-44)

(0-90.8)

(0-27.7)

(7-100)

(0-100)

(0-11)

n = 50

n = 2

n = 2

n = 2

« = 4

n = 8

n = 6

PB

43.5

—

—

—

—

— —

—

(31-57)

n = 1

n = total number of crosses between morphospecies (i.e.. colony pairs). All crosses between colony pairs were replicated three times and the range
of % fertilization over all replicates of all crosses is presented in parentheses. Conspecific crosses are in bold and crosses between morphospecies in
normal type. ' — ' denotes cross not yet tried. 'Sells' are crosses using eggs and sperm from the same colony. PD — P. daedalea, PL — P. lamellina.
PP—P pmi. PR— P. mtkvuensis. PS— P. sinensia. PB— Platygyra B. PH—Plutygyra II

mental crosses, a sperm dilution series was carried out
using two colonies each of Platygyra daedalea and P. ry-
iikyuensis from Orpheus Island. Sperm was diluted to
105, 104, and 103 per ml and used in reciprocal crosses
between all four colonies (within-morphospecies, be-
tween-morphospecies, selfs. and controls). Percent fertil-
ization in all crosses was calculated after 2-3 h as de-
scribed above.

Effect of egg age on fertilization rates

Due to the complex washing procedures and the na-
ture of the experiments described above, it can be up to
6 h after spawning before eggs and sperm are combined
in fertilization trials. Sperm kept in a concentrated form
has been shown to age slowly (Chia and Bickell, 1983;
Oliver and Babcock, 1992), although nothing is known
about the effect of aging on the egg — particularly with
regard to its ability to be fertilized with foreign sperm.

To test whether egg aging was influencing the results
of fertilization trials, an experiment was carried out to
look at fertilization rates in eggs of varying ages. Three
colonies each of P. daedalea and P. sinensis from Mag-
netic Island were used. Once two colonies had spawned.

gametes were washed as described above and immedi-
ately crossed with each other. As each successive colony
spawned, gamete bundles were washed and immediately
crossed with al! other colonies that had already spawned.
This was repeated until all six colonies had spawned, af-
ter which all crosses were repeated at 2-h intervals until
eggs were 8-h old. All crosses were reciprocal and each
cross was replicated twice at each time interval. Controls
with sperm-free water were done at all stages of the ex-
periment. Percent fertilization of within- and between-
morphospecies crosses at various egg ages was compared
for both P. daedalea and P. sinensis. and regression anal-
ysis was used to detect general trends associated with the
effect of egg age on fertilization success for all cross types
(within-morphospecies, between-morphospecies, self,
and control).

Fertilization success as a function of genotype

Tissue samples from all colonies used in the fertiliza-
tion trials in 1991 and 1992 were collected for genetic
analysis to determine whether any genetic structuring,
independent of morphology, was associated with breed-
ing groups within the genus and whether incompatible

102

K. MILLER AND R. BABCOCK

colonies were clonemates. Colonies were screened using
allozyme electrophoresis at nine polymorphic loci
(PGM*, MPI*. CK*. LP*, LT-1*, LT-2*. LG-1*. LG-2*.
and GPI*; see Miller, 1 994b; Miller and Benzie, in press,
for further details of electrophoretic techniques). The ge-
netic difference between mated colonies, based on the
nine-loci genotype, was calculated as a percentage based
on the number of different alleles (i.e.. alleles not shared
by the two colonies). The fertilization success between
any two colonies and the genetic difference between
them was then compared.

Larval rearing

In addition to the fertilization experiments described
above, large numbers (>1000) of larvae from both
within- and between-morphospecies crosses were reared
and allowed to settle so that their subsequent develop-
ment could be monitored through planulae to polyp.
Larvae were raised using the methods described by Bab-
cock and Heyward ( 1986). After 4-5 days, larvae were
transferred from plastic jars (2-4 1) to closed aquaria in
which settlement substrata (either coral rubble or terra-
cotta paving tiles) were provided. Once all larvae had set-
tled, settlement plates were transferred to a flow-through
aquarium system where they were maintained to obtain
specimens for examination of colony morphology.

Results

Fertilisation trials

The fertilization trials among colonies of Platygyra
showed that fertilization took place between all the mor-
phological species tested (Table I). The gametes of the
seven morphological species crossed were highly com-
patible, and mean fertilization rates among different
morphospecies were similar (between 50% and 70%, Ta-
ble 1). In total, 389 crosses were conducted including
1 140 separate vials. Although variability within replicate
vials was sometimes high (Table II), the controls indi-
cated no contamination of trials by foreign sperm. The
high levels of fertilization (>50%) measured in crosses
demonstrates that colonies were reproductively compat-
ible. Interestingly, self-fertilization rates were low in all
morphospecies (Table I).

Fertilization success was highly variable both among
and within morphological species, with most differences
occurring at the level of individual crosses. For example,
fertilization success between any two P. daedalea colo-
nies ranged from 9.8% to 67. 1% in crosses carried out on
17 October 1992 (Table II) and between 0% and 81%
between P. sine nsis colonies on the same night (Table II).
Similarly, the success of between-morphospecies crosses
seems largely dependent on colony compatibility (Table

Table II

Mean fertilization rales (± standard deviation) between three colonies
nt Platygyra daedalea and three colonies of P. sinensis from
fertilization trials carried out at Magnetic Island on 17 October 1992

Eggs

Sperm PDI

PD2

PD3

PS1

PS2

PS3

PDI 31.6 24.8 3.4 7.1 0.0

(0.0) (14.07) (1.84) (3.82) (0.00)

PD2 9.8 67.1 68.9 40.1 7.7

(0.21) (3.96) (0.0) (3.18) (1.98)

PD3 27.5 47.5 70.6 43.1 16.7

(6.51) (15.63) (1.13) (15.91) (23.55)

PS1 72.0 67.7 91.6 45.1 7.15

(5.66) (1.34) (4.74) (3.04) (10.11)

PS2 73.2 56.1 95.4 73.3 0.0

(3.25) (6.79) (0.0) (2.69) (0.00)
PS3 23.1 18.2 74.9 81.2 10.7

(11.24) (4.31) (0.71) (2.05) (7.00)

Self-fertilization rates (eggs and sperm from the same colony, de-
noted by "- -") were <1% in all colonies except PDI which was <3%.
All controls (eggs in sperm-free water) had 0% fertilization.

II). No colony had consistently low fertilization with all
other colonies (e.g.. Table II), so variability was not due
simply to low overall viability of the gametes in any one
colony.

In crosses for which P. daedalea was the maternal col-
ony, ANOVA indicated significant variability in fertil-
ization rates with different sperm types (df = 5, MS =
1 9043.3 1 , F = 25.65, P < 0.00 1 ). However, the source of
most of this variability lay in differences between rates of
self fertilization rather than in crosses within and be-
tween morphological species. Tukey's HSD test showed
no differences between fertilization rates for within-mor-
phospecies crosses [PD X PD (x = 45.2, n = 49)] and
between-morphospecies crosses [PD X PS (.Y = 7 1 .7, n =
50), PD X PL (x_= 68.2, n = 1 3), PD X PP (x = 66.6, n =
10), PD X PR (.Y = 66. 1 , n = 8)]. These fertilization rates
were, however, significantly different from rates of self
fertilization in P. daedalea (x = 3. 1, /; = 28).

Fertilization success between some morphological
species was low. Crosses between Platygyra H eggs and
P. daedalea sperm (n = 5 pairs), Platygyra H eggs and P.
pini sperm (/? = 3 pairs), P. pini eggs and P. sinensis
sperm (n = 2 pairs) and Platygyra B eggs and P. daedalea
sperm (/; = 1 pair) had fertilization rates below 20% (Ta-
ble I). However, the low fertilization rates between these
morphospecies are within the range observed between
colonies of the same morphospecies (Table I) and may
not reflect species incompatibility. Larvae produced by
crosses with low fertilization rates were regular in appear-
ance and seemed to develop normally.

Reciprocal crosses between different morphological

CONFLICTING SPECIES BOUNDARIES IN PLATYdYRA

103

Table I II

Results Irom multiple two- \vay analyses oj variance to compare
fertilization success (in both within- and between- morphospecies
crosses) for the seven different morphological species of Platygyra

Experimental
data analyzed

Source of variation

df

F

PT>F

2-3 h

fertilization type

I

0.08

0.77 (ns)

egg species

6

1.64

0.13(ns)

fert-type • egg species

5

1.75

O.I2(ns)

6-8 h

fertilization type

1

0.53

0.46 (ns)

egg species

6

2.20

0.04*

fert-type v egg species

5

0.42

0.83 (ns)

Development

fertilization type

1

1.03

0.31 (ns)

egg species

6

4.60

0.00***

fert-type x egg species

5

0.59

0.71 (ns)

'Fertilization type' is within/between morphospecies crosses; 'egg
species' is the morphological species from which eggs in crosses origi-
nated. Analyses were performed on data from both counts at 2-3 h
and 6-8 h following fertilization, and to compare the development of
embrvos from both within- and between-morphospecies crosses, (ns)-
not significant. * P < 0.05. *** P < 0.00 1 .

species generally produced similar fertilization rates (i.e.,
eggs and sperm from two different morphological species
readily fertilized each other) (Table I). In some cases,
however, compatibility in reciprocal crosses was asym-
metrical. For example, P. sinensis eggs were readily fer-
tilized by P. pini sperm (77.4% fertilization), whereas P.
pini eggs were poorly fertilized by P. sinensis sperm
(7.3% fertilization; Table I). A similar pattern existed be-
tween Platygyra B and P. daedaleu (Table I), but replica-
tion was too low to determine if these differences were
significant.

The overall rates of between-morphospecies crosses
(all morphospecies pooled) were not significantly differ-
ent from the rates of within-morphospecies fertilization
('fertilization type' — Table III; Fig. la, b), nor was there
any difference in within- or between-species fertilizations
among the different morphospecies ('fert-type X egg spe-
cies'— Table III). These results indicate that fertilization
can occur at the same rate both within and between mor-
phological species of Platygyra.

There were no differences in mean fertilization rates in
either within- or between-morphospecies crosses be-
tween 2-3 h and 6-8 h (Fig. la, b); therefore percent fer-
tilization did not increase with egg-sperm contact time.
About 80% of all eggs fertilized after 2-3 h had developed
into blastulae after 6-8 h, and survival rates were the
same in both within- and between-morphospecies
crosses (Fig. Ic).

A comparison of regular versus irregular cleavages and
embryos, at both 2-3 h and 6-8 h following fertilization,
showed that the number of irregular cleavages was sim-

ilar in both within- and between-morphospecies crosses
and between 2-3 and 6-8 h (Fig. 2). Irregular embryos
made up about 10%-1 5% of the 100 eggs counted (fertil-
ized and unfertilized) after both 2-3 h and 6-8 h,
whereas regular cleavages were, on average, 40%-50% of
the 100 counted. The occurrence of irregular embryos
does not appear to be related to differing morphology of

100
80

40

T:

20

100

Between Within

80 -

| eo

I 40

£

20

$ 100

B

— r~
Between Within

a BU

:

| 6°

-§ 40

in
o
£•
•° 20

d>

o
^ n

Between Within

Figure 1. Fertilization success. Mean within- and between-mor-
phospecies fertilization rates in fertilization trials between morpholog-
ical species of Plalygyra. Bars denote standard errors. (A) 2-3 h follow-
ing gamete mixing; (B) 6-8 h following gamete mixing; (C) develop-
ment of embrvos after 6-8 h.

104

K. MILLER AND R. BABCOCK.

parent colonies but may be a consequence of the experi-
mental method (see Oliver and Babcock, 1992).

Effects of sperm concentration on fertilisation rates

There were no effects of sperm concentration on fertil-
ization rates in either P. daedalea or P. ryukyuensis
crosses. Fertilization rates for P. daedalea colonies fertil-
ized with conspecinc sperm did not differ significantly at
sperm concentrations ranging from 101 to 105 per ml
(Fig. 3). Similarly, sperm concentration did not affect
fertilization rates in crosses of P. daedalea eggs with P.
rvtikvuensis sperm (Fig. 3) or in crosses of P. ryukyuensis
eggs with conspecific sperm and with P. daedalea sperm
(Fig. 3).

Effects of egg age on fertilization rates

The ability of an egg to be fertilized by sperm from a
different morphological species does not appear to be a
function of egg age. There was no significant change in
within-morphospecies or between-morphospecies fertil-
ization rates with egg age in either P. daedalea or P. si-
nensis. Regression analysis of percent fertilization with
egg age was not significant for either P. daedalea or P.
sinensis for any fertilization type (Fig. 4): within-mor-
phospecies (r = 0.005, 0.05 respectively), between-mor-
phospecies (r = 0.04, 0.1 respectively), self (r = 0.07.
0.1 respectively), or controls (r = 0.0004. 0.0 respec-
tively). Although r was not significant (possibly due to
high variation in fertilization rates; see Fig. 4), fertiliza-
tion (both within- and between-morphospecies) showed
an overall decrease with egg age in P. daedalea and a
general increase with egg age in P. sinensis (Fig. 4). Be-
tween-morphospecies fertilization rates were generally
higher than within-morphospecies fertilization rates in
P. daedalea, whereas between-morphospecies fertiliza-
tion rates were lower than within-species fertilizations in
P. sinensis (Fig. 4); however, neither difference was sig-
nificant.

Fertilization success as a junction of genotype

Fertilization success in colonies of Platygyra was not
related to the genetic difference between parent colonies.
No two colonies used in fertilization trials had identical
nine-locus genotypes (i.e.. none were clonemates). Colo-
nies that were between 40% and 60% different genetically
had fertilization rates varying between 0% and 100%'
(Fig. 5). No evidence of what might be considered a self-
recognition allele (Grosberg. 1988) was seen on the basis
of the nine-locus genotypes of incompatible colonies.
Colony pairs exhibiting low levels of fertilization did not
possess consistent allelic or genotypic characteristics.

Temporal separation in .spawning times

There was some evidence of temporal separation of
spawning times between some morphological species
of Platygyra (Fig. 6). However, this separation was
not clearly defined and an overlap in spawning time ex-
isted between the different morphospecies. Although
Platvgyra daedalea colonies have been observed spawn-
ing throughout the night, P. lamel/ina and P. pini tend to
spawn earlier in the evening (spawning 50 to 200 min-
utes after sunset), while P. sinensis. P. ryukyuensis. and
Platygyra H generally spawn later (more than 200 min-
utes after sunset) (Fig. 3). The ranges of spawning times
observed in the field and laboratory show that there was
considerable overlap in spawning, and that the gametes
of different morphological species were often released si-
multaneously.

Larval rearing

The embryos of within- and between-morphospecies
crosses developed into ciliated planulae larvae within 2
days of fertilization and were competent to settle 4-
5 days after spawning. Metamorphosed larvae from both
fertilization types gained zooxanthellae within 2 days af-
ter settlement and subsequently began skeletal construc-
tion. After 5 months, recruits ranged in diameter from 2
to 10 mm. In March 1993 the oldest colony from a be-
tween-morphospecies cross had reached 3'/2 years of age,
and colony diameters ranged between 2 and 5 cm. Un-
fortunately, all colonies died in 1993 because of a prob-

in
o>

O)

•5

60

40 -

20

T I

Between Within Between Within

2-3hrs

6-8hre

Figure 2. Embryo condition. Occurrence of regular and irregular
embryos in both within- and between-morphospecies fertilization trials
expressed as a percentage of the first 100 eggs counted per vial (all mor-
phospecies pooled). Bars represent 95% confidence limits. • — regular
embryos; • — irregular embryos.

CONFLICTING SPECIES BOUNDARIES IN PLATYdVRA

105

P. daedalea eggs

100 -i

T r-

-? 80 ~

I

"TO
,y 40 -

I

-

'•c

CD

20 -

y
/

\

>
>

>

n _

In

IE

]

1 d\\ 2 d\\ 3 control

P. ryukyuensis eggs

100 -n

_ 80 -

^p
o^

c 60 -

I

I

I I within morphospecies

t;.".'/-;;j between morphospecies

o

'-*— '

05

^ 40 -

CD

20 -
n

L
I

dil 1 dil 2 dil 3 control
sperm concentration

Figure 3. Effects of varying sperm concentration. Results from crosses between two colonies each of
Plalvgvra daedalea and P mikyucnxis in which sperm concentrations were varied 100-fold. Sperm con-
centrations: Dil 1 = 1 < \Q- ml ', Dil 2 = 1 x 104 ml ', Dil 3 = 1 x 10' ml '. Error bars are standard
deviations. Controls are with sperm-free water.

lem with the seawater system. Development of colonies
from between-morphospecies crosses appeared to be
normal up to that point, although their skeletal charac-
teristics were not well enough developed to reveal
whether they resembled any of the predefined morpho-
logical species or perhaps were intermediate in morphol-
ogy between the parent colonies.

Discussion

Fertilization is clearly possible between the different
morphological species of Platygyra in vitro. There ap-
peared to be no relationship between morphology and

reproductive compatibility since fertilization was re-
corded in all crosses. Fertilization occurred between
most morphological Platygyra species at rates equiva-
lent to within-species fertilizations (Table I, Fig. la, b).
Nonsignificant differences in fertilization rate for com-
parisons among crosses in which P. daedalea was the
maternal colony probably reflect widespread gametic
compatibility within the genus. The low fertilization
rates in some crosses between morphological species
may reflect small sample sizes and differences in com-
patibility between individual colonies (Table III, Fig.
la, b) rather than overall morphospecies incompatibil-
ity. Embryos resulting from between-morphospecies

106

K. MILLER AND R. BABCOCK.

100

60

40

20

P daedalea eggs

•

100

80

is eo

40

20

0-22-44-66-8

P smensis eggs

o

0-2 24 4-6 6-8
Egg age at fertilization (hours)

Figure 4. Effects of egg aging. Mean fertilization rates with increas-
ing egg age in crosses between three colonies each ofPlotygyra daedalna
and f .v/Hivj.y/.y. Bars represent 95"i confidence limits. • — within-mor-
phospecies crosses. • — between-morphospecies crosses.

crosses apparently did not have reduced survivorship
(Fig. Ic).

Percent fertilization was high after 2 h (the minimum
time after fertilization in which cleavage becomes evi-
dent), and fertilization rates did not increase with egg-
sperm contact time. This suggests that fertilization be-
tween gametes occurred promptly after the eggs were in-
troduced into the vials, irrespective of the morphotype of
the parent colonies. Variations in egg age, sperm dilu-
tion, and parental genotype failed to affect patterns of
fertilization in crosses within or among morphospecies.

No evidence of pre-zygotic barriers to fertilization was
detected among the morphological species ofPlatygyra.
The seven morphological species have overlapping
spawning times (Fig. 6), and high levels of fertilization in
trials indicate that no species-specific gamete recognition
systems (e.g., Uehara el a/., 1990; Vacquier el ai. 1990;
Palumbi, 1992) are operating in Platygyra. Preliminary
investigations of sperm chemotaxis in Platygyra showed
limited evidence of sperm attraction, but no evidence of
species-specificity of sperm attraction in Platygyra (K.
Miller and R. Miller, unpubl. data). Even if present, spe-
cies-specific sperm chemotaxis could not be completely
effective as a barrier to fertilization without membrane-
level recognition or selectivity.

Mixed parentage does not appear to affect the devel-

opment of Platygyra larvae or the competence of larvae
to settle, metamorphose, and grow. Sterility of these off-
spring may be a post-zygotic isolating mechanism be-
tween morphological species of Platygyra, although costs
associated with sterile progeny (e.g., gamete wastage, loss
of resources) may be high. However, in histological stud-
ies all mature-sized colonies of P. sinensis had gonads
( Babcock, 1986) and in the 389 fertilization trials carried
out, no colony was ever found that did not fertilize with
at least one other colony, suggesting that sterile Platygyra
are either rare or nonexistent.

Results from this study, combined with genetic studies
(Miller and Benzie, in press), lead us to believe that fer-
tilization and gene flow will occur between morphologi-
cal "species" of Platygyra. This raises a number of per-
plexing questions. First, should these morphological
groups within the genus Platygyra (Miller, 1994a) be
considered true species, or is there simply one highly
polymorphic species (e.g., Skulason and Smith, 1995)
within the genus? And second, if gene exchange occurs
freely between all morphological types, how can the mor-
phologically distinct groups be maintained in this genus?

Morphological species boundaries within the genus
Platygyra are clear (Miller 1994a), and morphological
differences appear to be far greater between Platygyra
spp. than between other controversial species groups: for
example, the three morphological variants of Montas-
traea annularis that are now considered sibling species
(Knowlton et ai. 1992; Weil and Knowlton, 1994); the
two morphs ofAfontipora digitata that have been identi-
fied as reproductively isolated species (Stobart and Ben-
zie, 1994); and Acropora cuneata and A. palifera, which
are difficult to distinguish by morphology but are geneti-
cally distinct (Ay re el a/., 1991 ). Hence it is not surpris-
ing that the seven morphological variants of Platygyra
have been considered separate species in the past.

However, genetic and reproductive data indicate that
Platygyra morphospecies are much more closely related
than the Montaslrea spp., the Montipora spp., or the two
Acropora species. Furthermore, many groups of corals
show discrepancies between species characters including
morphology, genetics, and reproduction (Potts and
Garthwaite, 1986; McMillan et at., 1991; Willis el ai.
1992; Wallace and Willis, 1994; Romano and Palumbi,
1996). Defining species boundaries in corals is clearly a
controversial issue. Nonetheless, our results show that in
the case of Platygyra, it is inappropriate to consider the
taxonomic unit as the minimal evolutionary unit and
that evolutionary processes in different morphological
species of Platygyra will be linked.

The maintenance of seven morphologically distinct
entities in Platygyra in spite of genetic interchange poses
a challenging question. Progeny from between-morpho-
species crosses may be expected to have morphological

CONFLICTING SPECIES BOUNDARIES IN I'l.ATYdYRA

107

100
80
60
40
20
0

• . • • '

• i : i . . •

20 40 60

100
80
60
40
20
0

B

20

40

60

80

100
80
60
40
20
0

: i

i -

20 40 60 80

% difference (genetic)

100
30
60
40
20
0

D

• « •

20 40 60 80
% difference (genetic)

Figure 5. Fertilization success as a function of genotype. Comparison of percent fertilization (based on
the mean of three replicates) and allelic differences between colonies used in fertilization trials. (A) Mag-
netic Island, 1 99 1 :(B) Magnetic Island, 1 992; (C) Davies Reef, 1991; (D) Orpheus Island, 1 992.

characters intermediate between those of the parent col-
onies and, with continued interbreeding, distinctions be-
tween the morphological species would be likely to break
down. However, nothing is known of the mechanisms
that produce skeletal differences in corals; if morphology
is linked to dominance alleles, then between-morphos-
pecies progeny may display the skeletal characteristics of
only one parent (e.g., Strathmann, 1981; Byrne and An-
derson, 1994). Alternatively, processes such as introgres-
sion may play a role in the maintenance of morphologi-
cal differences in Platygyra (Miller and Benzie, in press).

Reproductive and distributional variation may also
provide some level of segregation between the morpho-
species, thus helping to maintain morphological differ-
ences. There is some separation among spawning times
of Platygyra morphospecies on the Great Barrier Reef,
but these do not constitute complete temporal barriers
to fertilization (Fig. 6). Nevertheless, most egg-sperm in-
teractions take place in the first few seconds after mature
eggs are in contact with a sperm suspension (Fig. la;
Denny and Shibata. 1989; Mundy et a/., 1994), and col-
onies are more likely to be fertilized by close neighbors
(Oliver and Babcock, 1992). These factors in combina-
tion could result in a degree of temporal reproductive
segregation, if not total isolation.

Sophisticated egg-sperm recognition systems are
clearly operational at several levels in Platygyra. Colo-
nies of Platygyra do not frequently self-fertilize (Table I),

unlike Goniastrea favulus, which commonly self-fertil-
izes (Heyward and Babcock, 1986;Stoddarte/tf/., 1988).
In addition, the high variability in fertilization rates both
within and between morphological species also indicates
some level of incompatibility or individual recognition
between certain pairs of individuals. This degree of re-
productive separation in combination with small differ-
ences in the timing of spawning is probably insufficient
to produce fixed allelic differences in the populations,
but it may produce the indications of nonrandom mat-
ing present in the genotypic structure of Platygyra popu-
lations (Miller and Benzie, in press).

No clear-cut habitat separation is apparent in
Platygyra, where all morphospecies can be found in var-
ious habitats on a single reef (Miller, 1994a). At large
scales, however, trends in the distribution of morpho-
species are apparent. For example, P. lamellina is rare on
mid-shelf and shelf-edge reefs in the central Great Barrier
Reef; P. pint is rare on coastal reefs; P. ryukyuensis is
most common on coastal fringing reef flats; and P. la-
mellina, P. ryukyuensis. and Platygyra H are rare or ab-
sent on the reefs from the southern Great Barrier Reef
(Miller, 1994b). Across even larger scales, further discon-
tinuities in species distributions are evident (Veron,
1993), and observations of spawning times of Platygyra
in other regions (e.g., Okinawa: Heyward et ai, 1987;
Hayashibara et ai. 1993) indicate that on geographical
scales segregation of spawning times among Platygyra

108

K. MILLER AND R. BABCOCK.

<D
0)

cr

in

0)

>

CO
Q

0)

_c

Q_

6

P. sinensis

P ryukyuensis

P. pini

P. lamellina

Platygyra H

Platygyra F.

P daeda/ea <X> O

O

O

O O

P. sinensis X X

P. ryukyuensis

P pini X X X X

P. lamellina X X

Platygyra H
Platygyra B
P. daedalea XX X XX

X X

X X

X X
X XXXM^CX

XX

X

T3

P sinensis
P. ryukyuensis

A

A

1

P pini

-

O

P lamellina

_

A

H)

O>
(0

5

Platygyra H
Platygyra B

-

^E

P daedalea

4& A AA A £& AA A AA A

AAAA A AAAAAAA

A A A AA A

0 100 200 300

Spawning time (minutes following sunset)

Figure 6. Temporal separation in spawning. Spawning times of Pltilygyru colonies at three reefs on the
Great Barrier Reef. Spawning times include observations made by scuba divers in situ, spawning times of
experimental colonies in aquaria (these have been shown not to differ from in siiu spawning times, see
Miller. I994b). and spawning times recorded by Babcock el al ( 1 986).

400

morphospecies could result in reproductive isolation.
The distribution of the morphological species of
Platygyra clearly exhibits some habitat-level differences
that may contribute to the continued coexistence of the
morphospecies, specifically through differential fitness in
various habitats and the subsequent spatial and temporal
reproductive isi ilation in certain habitats.

It has recently 1 •• ro posed that reticulate evolution
occurs within the £ ':ia (Veron, 1995). Platygyra

morphospecies are wi( -oughout the Indo-Pa-

cific (Veron, 1993) ami. ' ive discussed, there are

varied levels of differ I'onnectedness be-

tween the morphological omic units. Surface-

circulation vicariance mechanic & (Veron, 1995) and
reticulate evolution may well be the basis for the mor-
phological and genetic variation in Plarygyra popula-
tions across both local and geographic scales, and the

likelihood of this linkage among morphological species
ofPlatygyra should be accepted.

Acknowledgments

We thank Craig Mundy, Annabel Miles, and Dick
Miller for invaluable assistance during coral spawning
trips; we are also grateful to the many other people who
helped out during fertilization experiments. Also thanks
to Maria Byrne, Alina Szmant, and Charlie Veron, who
provided many useful comments on this manuscript.
This work was financed by grants to K. Miller from The
Australian Coral Reef Society and The Australian Mu-
seum, ARC grants to T. Hughes and to B. Willis/K. Mil-
ler/B. Stobart, and a James Cook University Post-gradu-
ate research award. Support was also provided by the
Australian Institute of Marine Science. This is contribu-
tion 149 from the James Cook University Coral Group.

CONFLICTING SPECIES BOUNDARIES IN PLATYGYRA

109

Literature Cited

Ayre, D. J., J. E. N. Veron, and S. L. Dufty. 1991. The corals Acro-
pora palifera and Acropora cuneala are genetically and ecologically
distinct. Coral Reels 10: 13-18.

Babcock, R. C. 1986. The population ecology of reef flat corals of the
family Faviidae (Goniaslrea. Plaiygyra). Doctor of Philosophy
Thesis. James Cook University. Townsville. 163 pp.

Babcock, R. C., and A. J. He>ward. 1986. Larval development of cer-
tain gamete spawning scleractinian corals. Coral Reds 5: 111-116.

Babcock, R. C., G. D. Bull, P. L. Harrison, A. J. Heyward, J. K. Oli-
ver, C. C. Wallace, and B. L. Willis. 1986. Synchronous spawn-
ings of 105 scleractinian coral species on the Great Barrier Reef.
Mar Biol 90: 379-394.

Byrne, M.. and M.J. Anderson. 1994. Hybridization of sympatric
Palinella species (Echinodermata: Asteroidea) in New South
Wales. Evolution 48: 549-577.

Chevalier. J. P. 1975. Les scleractinaires de la Melanesie Francaise
(Nouvelle Caledonie, lies Chesterfield, lies Layaute. Nouvelles Heb-
rides). 2eme Panic. Exped. Francaise recifs corallens Nouvelle
Caledonie. Edn. Fond. Singer-Polignac. Paris. 507 pp.

Chia, F. S., and t.. R. Bickell. 1983. Echinodermata. Pp. 545-620 in
Reproductive Biology of Invertebrates, Vol. 2: Sperm iogenesis and
sperm function. John Wiley & Sons. New York.

Coll, J. C., B. F. Bowden, G. V. Meehan, G. M. Konig, A. R. Carroll,
D. M. Tapiolas, P. M. Alino, A. Heaton, R. De Nys, P. A. Leone,
M. Maida. T. L. Acerel, R. H. Willis, R. C. Babcock, B. L. Willis,
Z. Florian, M. N. Clayton, and R. L. Miller. 1994. Chemical as-
pects of mass spawning in corals. I . Sperm attractant molecules in
the eggs of the scleractinian coral Monlipora digitala. Mar. Biol.
118: 177-182.

Darwin, C. 1859. On !/ic Origin of Species. John Murray, London.
513pp.

Denny, M. W., and M. F. Shibata. 1989. Consequences of surf zone
turbulence for settlement and external fertilization. Am Kal. 134:
859-889.

Ellis, J., and D. Solander. 1 786. The Natural History ofManv Curi-
ous and Uncommon Zoophytes. Benjamin White and Peter Elmsly,
London. 208 pp.

Grosberg, R. K. 1988. The evolution of allorecognition specificity in
clonal invertebrates. Q. Rev BioL 63: 377-412.

Harrison, P. L., R. C. Babcock, G. D. Bull, J. K. Oliver, C. C. Wallace,
and B. L. Willis. 1984. Mass spawning in tropical reef corals. Sci-
ence 223: I 186-1189.

Hayashibara, T., K. Shimoike, T. Kimura, S. Hosaka, A. Heyward, P.
Harrison. K. Kudo, and M. Omori. 1993. Patterns of coral spawn-
ing at Akajima Island, Okinawa. Japan. Mar Ecol. Prog Ser 101:
253-262.

Heyward. A. J., and R. C. Babcock. 1986. Self- and cross-fertilization
in scleractinian corals. Mar. Biol. 90: 191-195.

Heyward, A., K. Yamazato, T. Yeemin, and M. Minei. 1987. Sexual
reproduction of corals in Okinawa. Gala.\eat>: 331-343.

Hodgson, G. 1988. Potential gamete wastage in synchronously
spawning corals due to hybrid inviability. Proc. bill Int. Coral Reel
Symp. 2:707-714.

Knowlton, N., E. Weil, L. A. Weight, and H. M. Guzman. 1992. Sib-
ling species in Montastn>a annularis, coral bleaching, and the coral
climate record. Science 255: 330-333.

Lang, J. C. 1984. Whatever works: the variable importance of skele-
tal and of non-skeletal characters in scleractinian taxonomy.
Pa/aeonlogr. Am. 54: 18-44.

Mayr, E. 1942. Systematics and the Origin of Species. Dover publi-
cations Inc., New York. 334 pp.

McMillan, J., T. Mahoney, J. E. N. Veron, and D. J. Miller. 1991.

Nucleotide sequencing of highly repetitive DN A from seven species
in the coral genus Acropora (Cnidaria: Scleractinia) implies a divi-
sion contrary to morphological criteria. Mar. Biol 1 10: 323-327.
Metz, E. C., andS. R. Palumbi. 1996. Positive selection and sequence
rearrangements generate extensive polymorphisms in the gamete
recognition protein bindin. Mol Biol. Evot. 13: 397-406.
Miller, K. J. 1994a. Morphological species boundaries in the coral
genus Plaiygyra: environmental variation and taxonomic implica-
tions. Mar. Ecol. Prog. Ser 1 10: 19-28.

Miller, K. J. I994b. The Plaiygyra species complex: implications for
coral taxonomy and evolution. Ph.D. Dissertation. James Cook
University of North Queensland. 164 pp.

Miller, K. J.. and J. A. H. Benzie. In press. No clear genetic distinc-
tion between morphological species within the coral genus
Plaiygyra Bull Mar Sa. 60: ???

Miller, R. 1979. Sperm chemotaxis in the hydromedusae. I. Species
specificity and sperm behaviour. Mar. Biol 53: 99-1 14.

Miller, R. 1985. Demonstration of sperm chemotaxis in the Echino-
dermata: Holothuroidea. Ophiuroidea./ E.\p. Zool. 234: 383-414.

Mundy, C., R. Babcock, I. Ashworth, and J. Small. 1994. A portable,
discrete-sampling submersible plankton pump and its use in sam-
pling starfish eggs. Biol. Bull. 186: 168- 171.

Oliver, J., and R. Babcock. 1992. Aspects of the fertilization ecology
of broadcast spawning corals: sperm dilution effects and in situ mea-
surements of fertilization. Biol. Bull 183:409-417.

Palumbi, S. R. 1992. Marine speciation on a small planet. TREE 7:
114-118.

Potts, D. C., and R. L. Garthwaite. 1986. Population genetics of the
genus Porites. Abstr. Annu. Meet. Int. Soc Reef Stud. p. 38.

Romano, S. L., and S. R. Palumbi. 1996. Evolution of Scleractinian
corals inferred from molecular systematics. Science 271 : 640-642.

Skulason, S., and T. B. Smith. 1 995. Resource polymorphisms in ver-
tebrates. TREE 10: 366-370.

Stobart, B., and J. A. H. Benzie. 1994. Allozyme electrophoresis
demonstrates that the scleractinian coral Montipora digitata is two
species. Mar. Biol 118(2): 183-190.

Stoddart, J. A., R. C. Babcock, and A. J. Heyward. 1988. Self-fertil-
ization and maternal enzymes in the planulae of the coral Gonias-
lrea Javiilus. Mar Biol 99: 489-494.

Strathmann, R. R. 1981. On barriers to hybridization between Stron-
gylocentrotus drobachiensis (O. F. Muller) and S pallidus (G. O.
Sars). / E\p. Mar. Biol. Ecol. 55: 39-47.

Uehara, T., H. Asakura, and Y. Arakaki. 1990. Fertilisation blockage
and hybridisation among species of sea urchins. Pp. 305-310 in Ad-
vances in Invertebrate Reproduction 5. Elsevier Science, Amsterdam.

Vacquier, V. D., K. R. Carner, and C. D. Stout. 1990. Species-specific
sequences of abalone lysin, the sperm protein that creates a hole in
the egg envelope. Proc. Natl. Acad. Sci. USA 87: 5792-5796.

Van Veghel, M. L. J. 1994. Reproductive characteristics of the poly-
morphic Caribbean reel building coral Monlaslrea annularis. 1.
Gametogenesis and spawning behavior. Mar. Ecol. Prog. Ser. 109:
209-219.

Van Veghel, M. L. J.,andR. P. M. Bak. 1993. Intraspecific variation
of a dominant Caribbean reef building coral. Monlaslrea annularis:
genetic behavioural and morphometric aspects. Mar. Ecol. Prog.
Ser 92: 255-265.

Vaughan, T. W., and J. W. Wells. 1943. Revision of the sub-orders,
families and genera of the Scleractinia. Geol. Soc. Am. Spec. Pap
44: 1-363.

Veron, J. E. N. 1993. Biogeography of hermatypic corals. Australian
Institute oj Marine Science Monograph Series I 'olume 10. Towns-
ville. 433 pp.

Veron, J. E. N. 1995. Corals in Space and Tune. University of New
South Wales Press, Sydney. 321 pp.

110

K. MILLER AND R. BABCOCK

Veron, J. E. N., M. Pichon, and M. Wijsman-Best. 1977. Scleractinia
of eastern Australia, Part 2. Families Faviidae, Trachyphyllidae.
Australian Institute of Marine Science Monograph Series I 'olume
3. Townsville. 233 pp.

Wallace, C. C., and B. L. Willis. 1994. The systematics of the coral
genus Acroptiru: implications of the new biological findings for spe-
cies concepts. Annu. Rev. Eeol. Sy.il. 25: 237-262.

Weil, E., and N. know lion. 1994. A multi-character analysis of the
Caribbean coral Monlaslaea annularis (Ellis and Solander, 1786)
and its two sibling species. M. foveolata (Ellis and Solander, 1 786)
and M. franksi (Gregory, 1895). Bull. Mar. Sci. 55: 151-175.

Willis, B. L. 1990. Species concepts in extant scleractinian corals:
considerations based on reproductive biology and genotypic popu-
lation structures. Syst. Bol 15: 136-149.

Willis, B. L., R. C. Babcock, P. L. Harrison, J. K. Oliver, and C. C.
Wallace. 1985. Patterns in the mass spawning of corals on the
Great Barrier Reef from 1981 to 1984. Proc 5th 1m. Coral Reef
Symri. 4: 343-348.

W illis, B. L., R. C. Babcock, P. L. Harrison, and C. C. Wallace. 1992.
Experimental evidence of hybridisation in reef corals involved in
mass spawning events. Proc. 7l/i Int. Coral Reef Svinp. Abstracts:
109.

The Future

of Aquatic Research in Space:

Neurobiology,
Cellular and Molecular Biology

Proceedings

of a workshop

sponsored by

THE CENTER FOR ADVANCED STUDIES
IN THE SPACE LIFE SCIENCES

AT THE MBL

13- 15 May 1996

Marine Biological Laboratory,
Woods Hole, Massachusetts

Funded by

THE NATIONAL AERONAUTICS
AND SPACE ADMINISTRATION
under Cooperative Agreement
NCC 2-896

CONTENTS

Tin' Future <ij Aquatic Research in Sjitice:
Nturobiology, (Cellular and Molecular Biology

Dawidowicz, E. A.

Introduction . .

15

Discussion

144

MECHANOSENSITIVITY

Loewenstein, Werner R.

Mechanosensitive channels: introduction

Morris, Catherine E., Howard Lesiuk, and Linda R.
Mills

How do neurons monitor their mechanical status?
1 1 .mi ill. Owen P., and Don W. McBride Jr.

Mechanogated channels in Xenoput oocytes: differ-
ent gating modes enable u channel to switch from a
phasic to a tonic mechanotransducer

118

Discussion

NEUROBIOLOGY/SENSORY BIOLOGY

Eaton, Robert C., Audrey L. Guzik, and Janet L.

Casagrand

Mauthner system discrimination of stimulus direc-
tion from the acceleration and pressure components
at sound onset

Fetcho, Joseph R., Kingsley J. A. Cox, and Donald

M. O'Malley

Imaging neural activity with single cell resolution in
an intact, behaving vertebrate

DlSCUSSIon

Chalfie, Martin

A molecular model for mechanosensation in Ceieno-
rhabditis elegans

Blount, Paul, Sergei I. Sukharev, Paul Moe, and

Ching Rung

125

Kawasaki, Masashi

Complex signal processing by weakly electric fishes
Bass, Andrew H., Deana A. Bodnar, and Jessica R.
McKibben

From neurons to behavior: vocal-acoustic communi-

146

1 50
154

157

Mechanosensitive channels of E. coir, a genetic and

158

126

161

128

PLANT BIOLOGY
Hader, Donat-Peter

Fischer, Thomas M., and Thomas J. Carew

Activity-dependent regulation of neural networks:
the role of inhibitory synaptic plasticity in adaptive
gain control in the siphon withdrawal reflex of

164

131

Kiss, John Z.

Gravitropism in the rhizoids of the alga Chura:
a model system for microgravitv research

134

Baxter, Douglas A., and John H. Byrne

Complex oscillations in simple neural systems ....

167

170

Discussion .

137

Edwards, Erin Swint, and Stanley J. Roux

The influence of gravity and light on develop-
mental polarity of single cells of Ceratopteris richardii

gametophytes

Nick, Peter, Rea Godbole, and Qi Yan Wang

Probing rice gravitropism with cytoskeletal drugs
and cytoskeletal mutants

DEVELOPMENTAL BIOLOGY

Elinson, Richard P.

139 Getting a head in frog development 172

Angerer, Robert C., and Lynne M. Angerer

Fate specification along the sea urchin embryo an-
141 imal-vegetal axis 175

113

114

FUTURE OF AQUATIC RESEARCH IN SPACE

Maxson, Rob, Hongying Tan, Sonia L. Dobias, Hai-
lin Wu, Jeffery R. Bell, and Liang Ma

Expression and regulation of a sea urchin .Wu class
homeobox gene: insights into the evolution and
function of a gene family that participates in the pat-
terning of the early embryo 178

Discussion

179

CYTOSKELETON/CELL MOTILITY

Burnside, Beth, and Christina King-Smith

Actin-dependent pigment granule transport in reti-
nal pigment epithelial cells

181

Lin, C. H., E. M. Espreafico, M. S. Mooseker, and
P. Forscher

Myosin drives retrograde F-actin flow in neuronal

growth cones 183

Gillespie, Peter G.

Multiple mvosin motors and mechanoelectrical
transduction by hair cells 186

Ditcussion

191

Chairs and Speakers 197

Participants 200

Reference: Bin/. Bull. 192: 1 15-1 16. (February,

Introduction

The Center for Advanced Studies in the Space Life Sci-
ences at the Marine Biological Laboratory was estab-
lished through a cooperative agreement between the
MBL and the National Aeronautics and Space Admin-
istration. This NASA-sponsored Center serves as an in-
terface between NASA and the basic life science commu-
nity, addressing issues of mutual interest. A series of
symposia, workshops, and seminars will be held at the
MBL to advise NASA on a wide variety of topics in the
life sciences, including cell biology, developmental biol-
ogy, molecular biology, neurobiology. plant biology, and
systems biology. Special attention will be directed at ex-
amining how gravity affects biological processes, and
how variation in gravity can be utilized as a probe to bet-
ter understand such processes. This setting provides a fo-
rum for scientists to think about and discuss, often for
the first time, the role that gravity may play in funda-
mental cellular and physiological processes.

"The Future of Aquatic Research in Space: Neurobi-
ology, Cellular and Molecular Biology" held at the MBL
from May 12 to 15, 1996, is the first workshop sponsored
by the new Center. This meeting was chaired by George
Langford, who participated in the overall planning of the
sessions on mechanosensation, developmental biology,
neurobiology, and cell biology. Fred Sack was responsi-
ble for organizing the session on plant biology.

Although the gravitropic and gravitactic responses of
plants to changes in gravity were discussed at this meet-
ing, the responses of animal cells to gravity are not well
documented. Yet mechanical force can affect both eu-
karyotic and prokaryotic cells. At this workshop, Gilles-
pie described how a mechanically sensitive hair bundle
serves the hair cell in mechano-electrical transduction.
Furthermore, specific ion channels are activated by
stretch. Exciting developments in this area, described at
this meeting, include the genetic analysis of mechano-

This paper was originally presented at a workshop titled I he l-'uiurc
nl .li/iiulK. Research in Spine: \eiiri>hinlof>\: Cellular iintl Molecular
Biology. The workshop, which was held at the Marine Biologieal Lab-
oratory, Woods Hole. Massachusetts, from 13 to 15 May 1996, was
sponsored by the Center for Advanced Studies in the Space Life Sci-
ences at MBL and funded by the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

sensation in C. elegans and the identification, purifica-
tion, and reconstitution of a mechanosensitive channel
from E. coli. Studies on related channels in neurons and
Xenopus oocytes were also discussed. Mechanical force
can also be generated within the cell by the action of mo-
lecular motors; the subject of the final session at this
workshop.

The potential effects of microgravity on sensory sys-
tems were considered in the discussion and remain to be
explored. The notion that the microgravity environment
might influence the development of Xenopus eggs by al-
tering the rotation of the entire egg cortex after fertiliza-
tion appears to have been dispelled by direct experimen-
tation. Xenopus eggs develop normally during space-
flight. In his presentation, Elinson suggested that this
finding might reflect the control of cortical movement
by a microtubule-based mechanism that is insensitive to
changes in gravity. He further suggested that larger eggs,
such as those from the Puerto Rican terrestrial-breeding
frog coqui. might lack this microtubule-based mecha-
nism and could therefore exhibit altered development in
microgravity.

Several theoretical studies indicate that the effects of
microgravity would be minuscule at the subcellular level,
but Baxter, in his abstract, cites empirical evidence that
gravity does affect cellular processes such as signal trans-
duction. To reconcile this apparent contradiction, Bax-
ter presents the intriguing hypothesis that nonlinear mo-
lecular systems within the cell provide the amplification
required for cells to sense gravity. This controversy un-
derscores the complexities that must be considered in
predicting potential responses of cells to changes in grav-
ity; it must also be a central consideration in the planning
of future studies on the effects of microgravity.

Scott Brady provided the following remarks at the con-
clusion of the workshop. He pointed out the unique ex-
perimental environments afforded by spaceflight. which
will lead to new approaches for addressing fundamental
questions in biology: "The emphasis in this workshop
and in much of the life sciences research supported by
NASA has been on the effects of microgravity and the
ways in which an organism responds to microgravity.
This is appropriate, and such questions will continue to

115

116

FUTURE OF AQUATIC RESEARCH IN SPACE

be important. However, we should not forget that the
conditions of spaceflight include other parameters that
may affect biological processes, and we may be able to
use the unique environment to address a wider range of
biological questions.

For example, the unique behavior of fluids under con-
ditions of microgravity suggests that the conditions of
spaceflight may help us understand how fluid mechanics
are important for biological processes. Activities that
might be altered include membrane currents and second
messenger signaling, among others. Differences in fluid
mechanics have already led to at least one practical ap-
plication, as seen in the current interest in production of
protein crystals that cannot be grown on earth and lead
to new insights into the structure of biologically impor-
tant molecules.

Several people have remarked about the effects of ra-
diation encountered during spaceflight. An understand-
ing of the biological hazards associated with this radia-
tion is clearly important for people, animals, and plants
during the extended periods of spaceflight expected for
the space station or a trip to Mars. There are surely other
environmental factors that will also be important to eval-
uate during extended exposure to spaceflight.

Finally, a critical challenge for any organism is achiev-
ing homeostasis in response to a changing environment.
Much remains to be understood about the homeostatic
mechanisms that living organisms employ to assure their
continued survival as an individual entity and as a spe-
cies. The conditions of spaceflight, including micrograv-
ity, represent a profound perturbation of the normal en-
vironment. By observing the responses of an organism to
this perturbation and denning the molecular mecha-
nisms that underlie these responses, we have a rare occa-
sion to explore biological homeostasis. As opportunities
to conduct long term studies of different organisms un-
der conditions of spaceflight increase, we must be more
creative in identifying important questions and devising
experimental models."

E. A. DAW1DOWICZ

Director

Center for Advanced Studies

in the Space Life Sciences

Woods Hole

Julv 1996

Reference: Biol. Hull 192: I 17. (February, 1997)

Mechanosensitive Channels: Introduction

WERNER R. LOEWENSTEIN

Laboratory oJ'Cell Communication, Marine Biological Laboratory,
Woods Hole. Massachusetts 02543

Our subject in this session is mechanoreception, a
function that is at the bottom of much of what goes on in
an organism — from the basic sensing of cell movements,
osmotic changes, and touch, to the elaborate sensing of
gravity, balance, hearing, and so on. The hub of this
function is a class of tubular membrane proteins that lets
ions in or out of cells. Very little is known yet about the
molecular makeup of this mechanosensitive class of pro-
teins. Their better-known cousins, the membrane chan-
nels that transduce chemical or electrical stimuli, fall
into three basic building plans: arrangements of four,
five, or six subunits with a central axis of symmetry form-
ing an aqueous passageway through the lipid bilayer of
the cell membrane — the channel pore. The channel I
have been playing with — the cell-to-cell channel — be-
longs to the latter class; here polar a-helical stretches
from the neighboring subunits line a 1 6- 1 8 A pore. This
channel is quite large, and we are just beginning to un-
derstand what conngurational changes in the subunits
are involved in the closing and opening of the pore. The
narrower and more selective ion channels have fewer
subunits, and the polar space between the helices is
smaller.

This paper was originally presented at a workshop titled The Future
oj Aquatic Research in Space: Neiirobiology, Cellular and Molecular
Biology. The workshop, which was held at the Marine Biological Lab-
oratory. Woods Hole. Massachusetts, from 1 3 to 15 May 1996, was
sponsored by the Center for Advanced Studies in the Space Life Sci-
ences at MBL and funded by the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

We should keep our eyes peeled on this polar space.
Here is where much of the action in the channel takes
place — the opening and closing of the pore, or "channel
gating," as it is called. So, the overarching question is
how the mechanical stimulus, namely strain on the cell
surface, is coupled to gating — how that strain is con-
verted into an electrical current. The mechanosensory
channels are probably among the oldest channel entities,
and so we may hope that the conversion is relatively sim-
ple. The prospect of that simplicity — perhaps, a rather
direct transducer mechanism — has attracted many of us
here to try our hand at this channel modality. But what
nature actually holds in store for us remains to be seen.
We will get a firsthand feel for the problem; the speakers
in this morning session will offer us a broad range of
mechanosensitive proteins, from unicellular organisms
to vertebrates.

Cathy Morris will start things off with a picture of how
neurons sense their mechanical state — a low-rate sensing
that comes into play during the vagaries of their long
lives. Owen Hamill will deal with the transduction and
gating mechanisms of mechanosensitive channels in
Xenopus oocytes. Martin Chalfie will tell us about his ge-
netic dissections of a channel in nematodes, and center
on the role of the extracellular- and intracellular matrix
in the coupling between stimulus and gating. And Ching
Rung ends the session with a look at a mechanosensory
channel in E. coli. which he has managed to put into
liposomes and to analyze both genetically and chemi-
callv.

117

Reference: Bin/. Bu/l. 192: 1 18-120. (February, 1997)

How Do Neurons Monitor Their Mechanical Status?

CATHERINE E. MORRIS1, HOWARD LESIUK1, AND LINDA R. MILLS2

1 Loeb Institute, Ottawa Civic Hospital, Ottawa Kl Y 4E9, Canada, and2 Playfair Institute, Toronto

Western Hospital, Toronto M5T 2S8, Canada

Neurons lead mechanically active lives. In conse-
quence, the neuronal plasma membrane must mechani-
cally adjust itself as it is subjected to a multitude of in-
ternally and externally generated forces. Neuronal arbo-
rizations are not fixed structures; they are plastic and can
be refashioned and repaired. Neuronal cytoplasm churns
with motor-driven axonal transport and with the to-and-
fro of membrane trafficking. Neurites continually gener-
ate tension while they are growing (see Heidemann and
Buxton, 1 994); nonadherent neurites cannot sustain ten-
sion and are retracted. Neural processes cope with
stretching and compressive forces from surrounding tis-
sues, and they cope with osmotically induced swelling
and shrinking. Arborizations of a given neuron extend in
many directions and may traverse many environments.
While some processes are only micrometers long, others
can be meters long. In spite of the potentially hazardous
nature of their arborized geometry and the abundance
of mechanical perturbations they experience, individual
neurons evidently survive for 100 years or more! And
to survive, they must ensure that the continuity of their
plasma membrane is never breached.

How is it that, always and at all locations in a neuron,
there is sufficient membrane to prevent rupture? We can
rule out two possibilities a priori. First, "wrinkles," or
microvilli. cannot be maintained everywhere; the excess
capacitance would appreciably compromise fast neuro-
nal signaling. Second, central directives from the soma

This paper was originally presented at a workshop titled The Future
nl .-It/italic Re.wtirc/i in Space: Neurobiology, Cellular ami Molecular
Biology. The workshop, which was held at the Marine Biological Lab-
oratory. Woods Hole. Massachusetts, from Ijt to 15 Max 1996. was
sponsored by the Center for Advanced Studies in the Space Life Sci-
ences at MBL and funded by the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

to add or subtract plasma membrane globally would be
grossly inappropriate for a highly arborized cell.

For effective regulation of cell surface area, the neuro-
nal plasma membrane must detect and respond to ten-
sion in a local fashion. We hypothesize that membrane
area is regulated by a sensor-effector system ofmechano-
.scm/Vnv membrane disposition — i.e., a system which as-
sures that cytoplasmic stores of membrane are rapidly
recruited to the plasma membrane when tension rises,
then restored to the cytoplasm when membrane tension
falls. If exocytosis and endocytosis are tension-sensitive,
this should make the disposition of membrane mecha-
nosensitive, but this example does not exhaust the possi-
bilities. Although it is possible to imagine how mechano-
sensitive ion channels might contribute tension-sensitive
regulation of plasma membrane area, the most stream-
lined system would respond to tension with no interme-
diary chemical or voltage signals. The system would be
in greatest demand during neural development (which
involves both outgrowth and retraction of processes) and
subsequently could provide an emergency response sys-
tem to prevent rupture, membrane excess, or both when-
ever local stresses changed.

In molluscan and mammalian neurons, we have been
examining membrane dynamics that seem to reflect the
exaggerated workings of such a sensor-effector system,
i.e., a system of mechanosensitive membrane disposition
(Want-/«/., 1995: Reuzeauf/rt/., 1995). Osmomechani-
cal perturbations of cultured neurons induce Vacuole
Like Dilations ( VLDs). VLDs, which can grow to about
10 //m across, form when neurons shrink (Fig. la, stages
B to C, D, or Fig. 1 b) and disappear as they swell (Fig. la,
stages D to B). It seems counterintuitive for a membra-
nous cytoplasmic structure to swell as the cell itself
shrinks. The explanation is that, initially, VLDs are not
true vacuoles. VLDs start as invaginations of membrane

118

MECHANOSENSITIVITY

119

swell

VLD formation
shrink ^.

|T7^* '
Ort $°-°J

— r~^ r< swell

VLD reversal

Recovery sensitive to
Cytochalasm
NEW

Brefeldin A
— Noeedagole

RECOVERY

cycle of VLD formation and reversal
can be repeated

VLDs form as invaginations

Figure 1. (a) Schematic showing that VLDs form (BtoC) when neurons shrink and that VLDs reverse
(D to B) upon subsequent reswelling. Repeated cycles of VLD formation and reversal (B-C-D-B . . . etc.)
can be effected repeatedly with about 2 min between solution exchanges. If neurons with VLDs are left in
isosmotic medium (D to E), the VLDs disappear by a drug-sensitive process termed recovery (D to F). The
schematic depicts experiments in which neurons first swell (A to B), so that return to an isosmotic medium
(B to C) constitutes a shrinking stimulus. Between C and D, the VLDs enlarge to full size, then recovery
(post-D) begins. For VLD formation, the critical stimulus is shrinkage; for VLD reversal, the critical stim-
ulus is swelling. Accordingly (though not depicted), VLDs also form when neurons shrink by going from
isosmotic to hyperosmotic medium: these VLDs reverse when the neurons are returned to isosmotic me-
dium, (b) Vertical section schematic to show how VLDs form: when the neuron shrinks, VLDs are initiated
as invaginations from the adherent surface; then, as recovery proceeds, they pinch off near the adherent
surface, yielding true vacuoles. A confocal slice (in cross section) made through a cell shortly after VLDs
form is depicted, as in the image in Figure 2, bottom.

at discrete sites on the substrate-adherent surface. Con-
focal microscopy using membrane dyes (Figs. Ib and 2)
and aqueous-phase dyes (Reuzeau et ai, 1995) reveals
that VLD membrane and VLD contents are initially
contiguous with plasma membrane and with extracellu-
lar fluid, respectively. VLD formation and reversal are
rapid, occurring over tens of seconds. If stimuli that elicit
VLD formation and reversal are given repeatedly (re-
peated cycles of swelling and shrinking, as depicted in
Fig. la), then VLD formation and reversal occurs repeat-
edly at the same site. In cells left in normal saline to re-
cover, VLD membrane is reprocessed and the VLDs dis-
appear on a time scale of tens of minutes (at room tem-
perature); this recovery is blocked by cytochalasin, by N-
ethylmaleimide, and by Brefeldin A, but not by nocada-
zole. By contrast, these drugs do not block the osmo-
mechanically driven formation and reversal of VLDs.
During recovery (i.e.. the drug-sensitive set of events),
VLDs can pinch off and become true vacuoles. This
pinching-off, which involves actin rearrangements, inter-
nalizes membrane so that it is no longer, strictly speak-
ing, plasma membrane.

Although the perturbations we use are osmomechani-
cal, our evidence indicates that the membrane dynamics
of VLD formation and reversal (but not VLD recovery
dynamics) are driven predominantly by the mechanical
rather than the chemical aspects of the perturbations.

The evidence: absolute osmolarity is not important
(VLDs form with shrinking stimuli whether the pertur-
bations are hyposmotic-to-normal or normal-to-hyper-
osmotic), and extracellular calcium and other ions are
not required.

Gastropod neurons are mechanically robust in the
face of extreme swelling stimuli (Wan et a/.. 1995).
Though solute loss and the mechanics of the cortical cy-
toskeleton may reduce the rate of swelling, neurons can
swell enormously and survive; 5-fold volume increases
are tolerated. In effect, compliance, in the form of recruit-
ment of additional membrane, appears to protect the cell
from rupture when swelling is excessive (Wan et ai.
1995; Fejtl et a/.. 1995). The membrane capacitance of
these neurons increases with swelling and subsequently
decreases with shrinking (Wan et ai. 1995). Membrane
tension (see Dai and Sheetz, 1995) in Lymnaea neurons
(J. Dai, M. Sheetz, and C. Morris. 1996. Am. Soc. Cell
Biol. abstract) exposed to 0.5X normal osmolarity in-
creased significantly, but to a level well below lytic ten-
sion, then was redressed upon return to normal saline.
The return from 50% to normal osmolarity also elicited
VLDs; hence, VLD formation occurred when the cell
was reshrinking and membrane tension was falling back
to normal. Taken together, the VLD, capacitance, vol-
ume, and tension observations are consistent with the
possibility that neurons have a sensor-effector system

120

FUTURE OF AQUATIC RESEARCH IN SPACE

Figure 2. Phase contrast (top) and contbcal fluorescence (middle,
bottom) images of a cultured Lymmwa slagnalis neuron before (top,
middle) and 1.5. min after) a solution change that elicited VLDs (bot-
tom). The fluorescence images are confocal slices near the substrate.
Fluorescence is from the membrane dye, dil. Neurons were stained
(and made to swell) by adding distilled water plus dil (yielding a swell-
ing medium osmolarity of about 0.2x normal) for 2 min. Excess dye
was removed by several washes with the isosmotic saline. These washes
shrank the swollen neuron, eliciting VLDs, bottom. (Because only a
single confocal slice of the cell near the substrate is seen, volume

that enables them, in the face of changing mechanical
stresses, to regulate their cell surface area and to keep
membrane tension close to a set point.

So, how do neurons monitor their mechanical status?
Adhesion to a substrate is critical to the VLD events we
observe. Therefore, we suggest that neurons monitor and
adjust their membrane tension and their surface area by
using a sensor-effector system (i.e., mechanosensitive
membrane disposition) that works in concert with a cy-
toskeletal adhesion-based sensor-effector system (see
Wang el a/.. 1993). Although the popular idea of a mon-
itoring role for mechanosensitive neuronal channels is
not completely ruled out, evidence suggests that ion
channel mechanosensitivity is deployed effectively in
specialized receptor neurons (Oliet and Bourque, 1996),
whereas it is suppressed in general-purpose neurons
(Morris and Horn, 1991; Small and Morris, 1994; Pao-
letti and Ascher, 1994).

Acknowledgments

Supported by grants from NSERC, Canada, and by
the Heart and Stroke Foundation of Ontario.

Literature Cited

Dai, J., and M. P. Sheetz. 1995. Mechanical properties of neuronal
growth cone membranes studied by tether formation with laser op-
tical tweezers. Biophys. J 68: 988-996.

Fejll, M., D. H. Szarowski, D. Decker, K. Buttle, D. O. Carpenter, and
J. N.Turner. 1995. Three-dimensional imaging and electrophys-
iology of live Aplysia neurons during volume perturbation: confo-
cal light and high-voltage electron microscopy. JMSA 1: 75-85.

lleidemann, S. R., and R. E. Buxlon. 1994. Mechanical tension as a
regulator of axonal development. Neurotoxicology 15: 95-108.

Morris, C. E., and R. Horn. 1991. Failure to elicit neuronal macro-
scopic mechanosensitive currents anticipated by single channel
studies. Science 251 : 1 246- 1 249.

Oliet, S. H. R., and C. \V. Bourque. 1996. Gadolinium uncouples
mechanical detection and osmoreceptor potential in supraoptic
neurons. Neuron 16: 175-181.

Puoletti, P., and P. Ascher. 1994. Mechanosensitivity of NMDA re-
ceptors in cultured mouse central neurons. Neuron 13: 645-655.

Reuzeau, C, L. R. Mills, J. A. Harris, and C. E. Morris. 1995. Dis
crete and reversible vacuole-like dilations induced by osmomecha-
mcal perturbations of neurons. J. Membr. Biol. 145: 33-47.

Small, D. R., and C. E. Morris. 1994. Delayed activation of single
mechanosensitive channels in Lymnaea neurons. Am. ./. Physiol.
267: C598-606.

Wang, N., J. P. Butler, and D. E. Ingber. 1993. Mechanotransduc-
tion across the cell surface and through the cytoskeleton. Science
260: I 124-1127.

Wan, \., J. A. Harris, and C. E. Morris. 1995. Responses of neurons
to extreme osmomechanical stress. J. Membr. Biol. 145: 22 1 -3 1 .

changes are not evident.) Before eliciting VLDs (middle), dil stains only
the plasma membrane. Its subsequent presence in VLD membrane
(bottom) indicates that VLD membrane is contiguous with plasma
membrane. Scale: phase contrast image is 140 jjm high.

Reference: Biol Bull 192: 121-122. (February. 1997)

Mechanogated Channels in Xenopus Oocytes: Different

Gating Modes Enable a Channel to Switch From a

Phasic to a Tonic Mechanotransducer

OWEN P. HAMILL AND DON W. MCBRIDE JR.

Department of Physiology and Biophysics. The University oj'Texas Medical Branch,

Galveston, Texas 77555

Critical to the survival of any cell is its ability to sense
and respond appropriately to changes in its environ-
ment. In the case of the mechanical environment, there
are both static and dynamic components that the cell
may be required to selectively detect. Such detection
takes place in the presence of a dynamic background of
mechanical stimulation arising from Brownian motion,
gravitational force, and various forces generated within
a cell (e.g., due to molecular motors and cycles of cy-
toskeletal polymerization and depolymerization) that
maintain cell shape and also mediate shape changes dur-
ing growth and adhesion. In addition to such back-
ground forces, a cell may experience other mechanical
perturbations, ranging from steady indentations to high-
frequency vibrations, and from osmotic challenges to
fluid shear stresses. Detection and appropriate responses
to such perturbations may be critical for the function
and, perhaps, survival of the cell. Therefore, cells require
biological mechanotransducers that can extract specific
information regarding relevant mechanical stimuli while
filtering out irrelevant stimuli.

A variety of mechanosensitive processes have been
identified including (i) mechanosensitive enzymes such
as adenylate cyclase (Watson, 1990) and phospholipase
A2(Jukkarta/., 1995), (ii) mechanosensitive transmitter
release (Chen and Grinnell, 1995), (iii) mechanosensi-
tive gene activation (SadoshimatYa/.. 1992), and (iv) the

This paper was originally presented at a workshop titled The Future
o/ Auiialic Research in Space: Ncurohiology. Cellular and Molecular
Biology. The workshop, which was held at the Marine Biological Lab-
oratory, Woods Hole. Massachusetts, from 13 to 15 May 1996, was
sponsored by the Center for Advanced Studies in the Space Life Sci-
ences at MBL and funded by the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

widely expressed class of mechanogated (MG) mem-
brane ion channels (Martinac, 1992). Of these different
processes, the MG channels have proven the most ame-
nable to detailed biophysical study. This has been due, in
part, to the development of high-resolution patch-clamp
recording (Hamill el al, 1981) and fast pressure-clamp
stimulating techniques (McBride and Hamill, 1992,
1993, 1995; Hamill and McBride, 1995a). We have used
these techniques to study the dynamic properties of sin-
gle MG channels, mainly focusing on the cation-selec-
tive channel endogenously expressed in Xenopus oocytes
(Hamill and McBride, 1992, 1994, 1995b, 1996a; Zhang
el al. 1996). This channel is blocked by amiloride and
its analogs, aminoglycoside antibiotics and gadolinium
(for review see Hamill and McBride, 1996b). One of the
most interesting kinetic features of the channel is that its
gating mode can be shifted from a highly nonstationary,
phasic or "high pass" mode to a stationary, tonic or "low
pass" mode (i.e., the input-filter characteristics change).
When in the phasic mode the MG channel activity ex-
hibits rapid and complete adaptation (i.e., the channels
close) despite the presence of maintained mechanical
stimulation. This adaptation is highly voltage dependent
and is similar to that seen in audiovestibular hair cells
(Crawford el al., 1991). For example, at -100 mV the
decay time constant of adaptation is about 100 ms, while
at + 1 00 m V it is more than 2 s. However, unlike the hair
cell, this adaptation does not depend on either extracel-
lular or intracellular Ca++ nor on the polarity of stimula-
tion (i.e., suction or pressure). We find that the adapta-
tion in the oocyte MG channel is due to a shift of the
stimulus-response relation (Boltzmann) to the right (i.e..
towards higher pressures) with no change in shape of the
relation. This adaptation reduces response saturation

121

122

FUTURE OF AQUATIC RESEARCH IN SPACE

while preserving the differential sensitivity to transient
changes in mechanical stimulation.

When the MG channel is in the tonic mode, the open
channel probability becomes time independent but in-
creases with increasing suction or pressure stimulation.
However, the sensitivity of the MG channel to mechani-
cal activation is decreased. Although voltage-dependent
adaptation is clearly absent in this mode, the voltage de-
pendence of MG channel lifetime is preserved and single
channel conductance and ion selectivity remain unal-
tered. The switching between gating modes can be me-
chanically induced in the patch-clamp configuration and
is most likely due to a physical decoupling of the mem-
brane from the underlying cytoskeleton, which presum-
ably contains the required viscoelastic elements. This
idea of decoupling is supported by the observed develop-
ment of a clear space within the patch pipette between
the membrane and the underlying cytoplasm; develop-
ment of this space accompanies the mechanically in-
duced channel mode switching.

The channel mode switching from phasic to tonic, as
studied here, probably reflects a pathological situation
associated with patch-clamp recording (Hamill and
McBride, 1997). Nevertheless, it does indicate a mecha-
nism by which a single molecular mechanotransducer
may alter its input filter characteristics (i.e., go from a
transient to a steady-state detector) of mechanical sig-
nals. It remains to be determined whether the remodel-
ing of the cortical cytoskeleton that occurs during cell
development and differentiation (t'.#.. see Vale. 1991;
Sardet el al., 1994) may also modulate membrane mech-
anosensitivity by such a mechanism.

Acknowledgments

Our research is supported by grants from the National
Institute of Arthritis and Musculoskeletal and Skin Dis-
eases, Grant RO 1 -AR42782, the National Science Foun-
dation, and the Muscular Dystrophy Association.

Literature Cited

Chen, B.-M., and A. D. Grinned. 1995. Inlegrins and modulation of
transmitter release from motor terminals by stretch. Science 269:
1578-1580.

Crawford, A. C, M. G. Evans, and R. Kettiplace. 1991. The action of

calcium on the mechanoelectrical transducer current of turtle hair
cells. J. Physiol. 434: 369-398.

Hamill, O. P., A. Marty, E. Neher, B. Sakmann, and F. Sigworth. 1981.
Improved patch clamp techniques for high current resolution from
cells and cell-free membrane patches. PJIugers Arch. Eur J. Physiol.
391:85-100.

Hamill, O. P., and D. \V. McBride, Jr. 1992. Rapid adaptation of the
mechanosensitive channel in Xenopus oocytes. Proc. Nail. Acad.
Sci. USA 89: 7462-7466.

Hamill, O. P., and D. W. McBride, Jr. 1994. Molecular mechanisms
of mechanoreceptor adaptation. News in Physiological Sciences 9:
53-59.

Hamill, O. P., and D. W. McBride, Jr. 1995a. Pressure/patch-clamp
methods. Pp. 75-87 in Patch Clamp Techniques and Protocols,
A. A. Boulton, G. B. Baker, and W. Walz, eds. Humana Press, New
Jersey.

Hamill, O. P., and D. W. McBride, Jr. 1995b. Mechanoreceptive
membrane ion channels. Am Sci. 83: 30-37.

Hamill, O. P., and D. VV. McBride, Jr. 1996a. Voltage and tension
sensitivity of a mechanogated channel. Binphys. J 70: A348.

Hamill, O. P., and D. W. McBride, Jr. 1996b. The pharmacology of
mechanogated channels. PharmtKol. Rev. 48: 231-252.

Hamill, O. P., and D. W. McBride, Jr. 1997. Induced membrane
hypo/hyper-mechanosensitivity: a limitation of patch-clamp re-
cording. Annu. Rev. Physio! . 59: 62 1 -63 1 .

Jukka, Y., A. Lehtonen, and P. K. J. Kinnunen. 1995. Phospholipase
A: as a mechanosensor. Biophys J 68: 1888-1 894.

Martinac, B. 1992. Mechanosensitive ion channels: biophysics and
physiology. Pp. 327-352 in Thermodynamics of Cell Surface Re-
ceptors. M. B. Jackson, ed. CRC Press, Boca Raton. FL.

McBride, D. W., Jr., and O. P. Hamill. 1992. Pressure clamp: a
method for rapid step perturbation of mechanosensitive channels.
PJIugers Arch. Eur. J Physiol 421: 606-612.

McBride, D. W. Jr., and O. P. Hamill. 1993. Pressure-clamp tech-
nique for measurement of the relaxation kinetics of mechanosensi-
tive channels. Trends Neurosci. 16:341-345.

McBride, D. W. Jr., and O. P. Hamill. 1995. A fast pressure-clamp
technique for studying mechanogated channels. Pp. 329-340 in
Single Channel Recording. 2nd Edition, B. Sakmann, and E. Neher.
eds. Plenum Press. New York.

Sadoshima, J-I., T. Takahashi, L. Jahn, and S. Izumo. 1992. Roles of
mechano-sensitive ion channels, cytoskeleton, and contractile ac-
tivity in stretch-induced immediate-early gene expression and hy-
pertrophy of cardiac myocytes. Proc. Nail. Acad. Sci. USA 89:
9905-9909.

Sardet, C., A. McDougall, and E. Houliston. 1994. Cytoplasmic do-
mains in eggs. Trends Cell Biol. 4: 1 66- 1 72.

Vale, R. D. 1991. Severing of stable microtubulesby a mitotically ac-
tivated protein in .Xenopus egg extracts. Cell 64: 827-839.

Watson, P. A. 1990. Direct stimulation of adenylate cyclase by me-
chanical forces in S49 mouse lymphoma cells during hyposmotic
swelling. J. Biol. Chem. 265: 6569-6575.

/.hang, \ '., F. Gao, D. W. McBride, and O. P. Hamill. 1996. On the
nature of mechano-gated channel activity in cytoskeleton deficient
vesicles shed from Xenopus oocytes. Biophys. J 70(2): A349, 1996.

Reference: Biol. Bull 192: 123-124. (February. 1997)

Discussion

MORRIS: I would like to ask Owen
Hainill a question. Both my labora-
tory (in collaboration with Rick
Elinson) and now your laboratory
have shown that, if you use pharma-
cological agents to block these chan-
nels, embryogenesis of the oocyte is
not impeded. Is this a fair statement?
Could you comment on what it
means?

HAMILL: Frogs eggs have been
taken up on the space shuttle, where
they can be happily fertilized, and
embryogenesis occurs normally in
the microgravity environment. Once
the oocytes are out of the animal,
they don't seem to be especially de-
pendent on mechanical stimuli, at
least as reflected by mechanogated
channels. For example, you can
block the mechanosensitive channel
and still watch the whole process,
from fertilization to a developed
embryo. Apparently the tadpoles
also develop normally in micrograv-
ity. But of course all of the mechani-
cally interesting events of oocyte de-
velopment occur before fertilization
and are associated with the growth of
the oocyte from 10~9 grams to 1 mg.
This involves enormous expansion
of the oocyte with enormous cy-
toskeletal rearrangement, and it's
our bet that that's dependent on the
mechanogated channel.

LOEWENSTEIN: Dr. Morris, what
you call the adhesion portion seems
to start out with an invagination pro-
cess. What structures do you actually
see or suspect might be initiating
this?

MORRIS: We haven't actually any
clue about that as yet. We know that
once the VLDs form, actin accumu-
lates around them. However, if you
treat the cells with cytochalasin, you
can still make these VLDs. It's clear
that the cytochalasin has worked be-
cause the phalloidin staining is
wildly different. We need to look at
spectrin staining and things like that.
I don't want to do this with the mol-
luscan cells; so we are going to try it
in skeletal muscle fibers, because
they make very big cells in culture
and we can use the antibodies with
them. A recent paper from Brad
Amos and Jack Lucy's lab in Cam-
bridge (J. Muscle Res. Cell Motil.
1995. 16: 401-11) has shown basi-
cally the same thing: reversible vacu-
olation in skeletal muscle. In their
case, it's the entotubular system
that's involved, which originates at
the Z-line. They don't know either
whether any other structures are as-
sociated with this. The only struc-
tural detail that they have is that it's
associated with the Z-line. We don't
have any molecular information.

LOEWENSTEIN: How do you de-
fine adhesion?

MORRIS: It's the attached surface:
and we know the cells are adherent.
I don't mean that there are specific
adhesion molecules associated with
this. I don't know that. It's not hap-
pening on the upper surface of these
cultured cells, which is a very low-
level definition of adhesion.

LOEWENSTEIN: What do you sug-
gest for the mechanism of reversibil-
ity here?

MORRIS: I suspect that the mem-
brane is flowing back out and up
over the surface when swelling is oc-
curring. That's the reversibility. I am
amazed that membrane would then
flow back into the same site, but it
does. I presume that some structure
there is acting as a scaffold to support
it, but we have no idea yet about
what it is.

QUESTION: Rick Steinhart has re-
cently been describing how cells rup-
ture and reseal very quickly with
membrane vesicles. I wonder
whether you are looking at rupture
that occurs upon swelling at the sites
of attachment, where the maximal
mechanical force would be experi-
enced. Recruitment of membrane
vesicles to seal that rupture could be
what makes your internal vacuoles.

MORRIS: If so, we are not picking
up any of the extracellular dyes into
the cell.

QUESTION: How big are these
dyes?

MORRIS: We used Rhodamine
3000. The structures that we see are
not tiny vesicles; they grow to be an
average of lO^m across once they
are fully formed.

QUESTION: What happens to the
membrane voltage during the swell-
ing?

MORRIS: I don't know what hap-
pens to the membrane voltage. We

123

124

FUTURE OF AQUATIC RESEARCH IN SPACE

know that we can activate the potas-
sium channels with slow swelling,
and we think that these channels
may actually be the stretch-activated
channels. We don't know \vhether
they are being stretch-activated. We
could be getting additional conduc-
tance because we've added more
membrane carrying the channels, or
because they are getting second mes-
senger signals. Since slow swelling is
activating these potassium currents,
I would imagine that, like Aplysia
neurons, ours hyperpolarize with
swelling. We haven't actually mea-
sured them.

CHALFIE: You showed what hap-
pens after 2-min changes. What hap-
pens when the cells reach osmotic
equilibrium after the change? Do the
vacuoles go away?

MORRIS: Yes, that's in recovery.

ELINSON: Owen (Hamill), those
oocytes have an animal and vegetal
difference. Do you see any disparity
in the presence of those channels on
the animal or vegetal pole? Or if you
look at oocytes early in oogenesis,
when things are moving, do you see
any differences in these mechanical
channels?

HAMILL: No, we don't.

ELINSON: So we still have no role
for these channels in the oocyte?

HAMILL: We don't know the role
yet.

QUESTION: Dr. Morris, is contact
with a cover slip required for forma-
tion of VLDs?

MORRIS: Not necessarily. We
have images of hippocampal cells
growing on glial cells and they make
VLDs, but these are hard to image.
So I don't think that contact with a
cover slip is critical, but mechanical
contact of some sort is.

HAMILL: Do you see these VLDs
with mild osmotic changes, or do
you really have to stress the cells?

MORRIS: You see them if you step
them down from normal to 70% nor-
mal osmotic concentration.

LOEWENSTEIN: Dr. Hamill, in
your interesting model there seem to
be two components: one is nonme-
chanical and the other, superim-
posed on that, is mechanical and act-
ing somewhere in the coupling pro-
cess. Could you elaborate on the
latter? I could easily see an adapta-
tion process being promoted me-
chanically, but I can't quite visualize
a process in which adaptation is
abolished mechanically, as in your
case.

HAMILL: If the viscoelastic ele-
ment is in the cytoskeleton and that's

decoupled, adaptation would be
abolished. You can think of this in
terms of a gating spring and a dash-
pot in series. Normally, when you
pull on the dashpot the spring
stretches. Then the dashpot relaxes
the spring, and you get adaptation.
What we imagine in the oocyte is
that the adaptation occurs because
the underlying cytoskeleton relaxes.
Once the cytoskeleton is decoupled,
or one element (the viscous element)
is decoupled, you can still have gat-
ing, but not the relaxation. We have
tried to dissect out the viscous from
the elastic elements. At one point we
thought that one might be the micro-
tubule and the other actin, but they
don't seem to be very sensitive to ei-
ther colchicine or cytochalasin.

CHALFIE: What is the effect of fer-
tilization in activating these chan-
nels?

HAMILL: There certainly are po-
tential changes associated with the
block to polyspermy. We have tried
very hard to see effects of all these
mechanosensitive channels on poly-
spermy, fertilization, and even mat-
uration. Up to maturation there is
no sensitivity blocking of the mecha-
nogated channel. But that's only half
the story because many events pre-
cede maturation, and we have not
yet examined them.

Reference: Biol. Bull 192: 125. (February, 1997)

A Molecular Model for Mechanosensation in
Caenorhabditis elegans

MARTIN CHALFIE

Department of Biological Sciences, 1012 Fairchild, Columbia University, Mail Code 2446, 1212

Amsterdam Avenue, New York, NY 10027

Sensory signaling by chemicals and light are fairly well
understood in molecular terms, but the molecules
needed for mechanical signaling, which underlies the
senses of touch, hearing, and balance, are not known. By
analyzing the genes needed for mechanosensation in the
nematode Caenorhabditis elegans, we hope to gain this
molecular understanding. Six touch receptor neurons re-
spond to gentle touch in C. elegans. Mutations produc-
ing touch insensitivity have identified 12 met. (mechano-
sensory abnormal) genes needed for touch cell function
(Chalfie and Sulston, 1981; Chalfie and Au, 1989). Eight
of these genes have been cloned by ourselves and others.
Two genes (mec-4 and mec-10) encode similar subunits
(degenerins) of a channel needed for mechanosensation
(Driscoll and Chalfie. 1991; Huang and Chalfie, 1994).
Gene interaction studies suggest that many of the re-
maining mec genes interact with this channel (Huang
and Chalfie. 1994; Gu et al.. 1996).

Predicted protein sequences, gene interactions, and ul-
trastructural studies of the touch receptor neurons sug-
gest the following model for mechanosensation in these
cells (Gu et al., 1996). Mechanosensation requires the
degenerin channel formed from MEC-4, MEC-10, and

This paper was originally presented at a workshop titled The l-'iilurc
ot Aquatic Research in Space: Neurobiology, Cellular ami .Molecular
Biology. The workshop, which was held at the Marine Biological Lab-
oratory. Woods Hole. Massachusetts, from 13 to 15 May 1996. was
sponsored by the Center for Advanced Studies in the Space Life Sci-
ences at MBL and funded by the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

probably MEC-6. This channel is attached to the extra-
cellular matrix, perhaps through interactions with MEC-
5 (a collagen) and possibly MEC-1 and MEC-9 (Du et
al., 1996). Intracellularly, the channel is attached to the
microtubules formed by MEC- 12 (a-tubulin)and MEC-
7 (|tf-tubulin), through an interaction with the stomatin-
like protein MEC-2 (Huang et al., 1995). The other mec
genes may be modifying the conductance of this channel.
In this model, physical manipulation of the channel ei-
ther by displacement of the cuticle or the microtubules
opens the channel.

Literature Cited

Chalfie, M., and M. Au. 1989. Genetic control of differentiation of
the C elegans touch receptor neurons. Science 243: 1027-1033.

Chalfie, M., and J. Sulston. 1981. Developmental genetics of the
mechanosensory neurons of Caenorhabditis elegans. Dev. Biol 82:
358-370.

Driscoll, M., and M. Chalfie. 1991. The mec-4 gene is a member of a
family of Caenorhabditis elegans genes that can mutate to induce
neuronal degeneration. Nature 349: 588-593.

Du, H., G. Gu, C. William, and M. Chalfie. 1996. Extracellular pro-
teins needed for C' cU't;an\ mechanosensation. Neuron 16: 183-
194.

Gu, G., G. A. Caldwell, and M. Chalfie. 1996. Genetic interactions
affecting touch sensitivity in Caenorhabditis elegans. Proc. Nail.
ACM/. Sci L'SA 93: 6577-6582.

Huang, M.. and M. Chalfie. 1994. Gene interactions affecting mech-
anosensory transduction in Caenorhabditis elegans. Nature 367:
467-470.

Huang, M.,G.Gu,E. L.Ferguson, and M. Chalfie. 1995. Astomatin-
like protein necessary for mechanosensation in C. elegant Nature
378:292-295.

125

Reference: Biol. Bull 192: 126-127. (February. 1997)

Mechanosensitive Channels of E. coli: A Genetic and

Molecular Dissection

PAUL BLOUNT1, SERGEI I. SUKHAREV1, PAUL MOE' \ AND CHING RUNG1 -

'Laboratory of Molecular Biology and 2 Departments of Bacteriology and ^Genetics, University of

H 'isconsin, Madison. \\ Isconsin 53 706

Our understanding of mechanosensation is poor, es-
pecially at the level of molecule. There is no shortage of
phenomena, from the sensation of gravity, touch, bal-
ance, and hearing in animals, gravitropism and thigmo-
morphogenesis in plants, to the detection of osmotic
forces in all cells including microbes. Yet to name a pro-
tein or gene that is responsible for these sensations is
difficult. This knowledge vacuum is all the more striking
given that we know, in exquisite detail, the structure and
function of a myriad of ligand receptors, and even the
receptors of light, rhodopsins.

One class of "receptors" are ion channels. These are
integral membrane proteins that line gated pores. By
"gated," physiologists mean that the pore is usually
closed until the channel protein "senses" a stimulus.
There are ion channels gated by external ligands (e.g..
neurotransmitters), second messengers (Ca:+, cyclic nu-
cleotides), or membrane potentials (i.e., transmembrane
voltage drop) (Hille, 1992). Channel pores, when open,
allow passive fluxes of permeant ions. The advent of the
patch clamp has greatly enhanced our capability to mon-
itor these ion fluxes: even the ion current through a single
channel protein can clearly be registered and analyzed
(Sakmann and Neher, 1995). Activities of channels that
open when the membrane patch in a patch-clamp pi-
pette is mechanically stretched were first observed in
muscle cells (Guharay and Sachs, 1984; Brehm et ill..
1984). This kind of activity has now been reported from
studies of neurons, oocytes, heart cells, lens cells, blood
cells, and plant cells (Sackin, 1995). Moreover, mecha-

This paper was originally presented at a workshop titled The f-'unire
i>/ Ac/uatie Research in Spuec: Neurobiology, Cellular ami Molecular
Biology. The workshop, which was held at the Marine Biological Lab-
oratory. Woods Hole. Massachusetts, from 13 to 15 May 1996, was
sponsored by the Center for Advanced Studies in the Space Life Sci-
ences at MBL and funded by the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

nosensitive (MS) channel activities have also been ob-
served in microbes: budding yeast, fission yeast, bean-
rust fungus, and the bacteria Escherichia coli and Bacil-
lus sitbtilis.

When E. coli is cultured in the presence of cephalexin,
the cells do not remain septate but continue to grow into
filaments, some reaching 100 ^/m in length. They can be
digested with lysozyme and EDTA and then collapsed
into spheres 3 to 10 /urn in diameter. Patch-clamp exper-
iments on such giant spheroplasts revealed two types of
MS-channel activities, one with a very large conductance
(2.5 nS) and one with a smaller conductance (0.8 nS).
These activities are observed as the appearance and dis-
appearance of unitary currents indicative of channel
opening and closing. The open probability increases with
the suction applied to the patch-clamp pipette (Martinac
el ai. 1987; Sukharev et ai. 1993). The MS channels of
E. coli can be reconstituted. In other words, bacterial
membrane vesicles, even after solubilization in a mild
detergent, can be placed in liposomes made of foreign
lipids and the MS channels remain functional (Delcour
et ai. 1989). We followed the activity of the 2.5-nS MS
conductance through different series of column chro-
matographic enrichments by reconstituting the column
fractions into soybean liposomes and examining patches
excised from them with the patch clamp. By tracing the
activities in the more and more enriched fractions, a pro-
tein of about 17,000 molecular weight was identified.
From the N-terminal sequence of this protein, the corre-
sponding gene, mscL. was cloned (Sukharev et ai.
1 994a). Disrupting mscL by a marker insertion removed
the 17-kD membrane protein and the 2.5-nS conduc-
tance. Replenishing this null strain with mscL on a plas-
mid restored both. The mscL gene, subcloned into ap-
propriate plasmids, has been functionally expressed in
two heterologous systems: rabbit reticulocyte lysate and
yeast. Therefore mscL alone is necessary and sufficient

126

MECHANOSENSITIVITY

127

for the 2.5-nS MS-channel activities (Sukharev et al..
1994a, h).

Conceptual translation ofmscL yields a protein of 1 36
amino-acid residues. The first three-quarters of the MscL
protein is highly hydrophobic according to its hydropho-
bicity plot. Four lines of recent evidence have indicated
that each MscL peptide traverses the membrane twice,
with both the N- and the C-terminus in the cytoplasm.
Cross-linking and other experiments showed that MscL
monomers assemble into a hexamer that probably en-
closes the pore. A number of deletions and point substi-
tutions have been made and tested for MS-channel prop-
erties. These results indicate the importance of both the
transmembrane domains and the linking loop between
these domains. Substitutions of certain hydrophilic resi-
dues changed the channel kinetics, the mechanosensiti-
vity, or both (Blount el al., 1996a. b). Well-conserved
mscL homologs have recently been found in several bac-
teria, including gram-positive bacteria (Moe el al., un-
pub. data).

Our current effort is directed toward ( 1 ) understanding
the molecular basis of mechanosensitivity by applying
forward and reverse genetics on E. coli mscL, (2) eluci-
dating the function of these MS channels in bacterial
physiology, and (3) searching for mscL homologs in
other species, including plants and animals.

Acknowledgments

Work in our laboratory was supported by NIH

GM47856.

Literature Cited

Blount, P., S. 1. Sukharev, P. C. Moe, M. J. Schroeder, H. R. Guy, and
C. Rung. I9%a. Membrane topology and multimeric structure of
a mechanosensitive channel protein of Eschenchia coli. EMBO
(Eur. Mi>l. Binl. Organ) J 15:4798-4805.

Blount, P., S. I. Sukharev, M. J. Schroeder, S. Nagle, and C. Kung.
1996b. Single residue substitutions that change the gating proper-
ties of a mechanosensitive channel in Escherichia coli. Proc. Null.
Acad. Set. ISA 93: 1 1652-1 1657.

Brehm, P., R. Kullberg. and K. Moody-Corbett. 1984. Properties of
non-junctional acetylcholine receptor channels on innervated mus-
cleof.\Vmy>H.\/<«T/\ ./ Physiol (Lund.) 350: 631-648.

Delcour, A. H., B. Martinac, J. Adler, and C. Kung. 1989. Modified
reconstitution method used in patch-clamp studies of Eschericia
coli ion channels. Biophys. ./. 56: 63 1-636.

Guharay. F., and F. Sachs. 1984. Stretch-activated single ion channel
currents in tissue-cultured embryonic chick skeletal muscle. J.
Physiol (Lonct ) 352: 685-70 1 .

Hille, B. 1992. Ionic Channels of Excitable Membrane's. Sinauer As-
sociates Inc., Sunderland. MA.

Martinac, B., M. Buechner, A. II. Delcour. J. Adler, and C. Kung.
1987. Pressure-sensitive ion channel in Eschericia coli Proc Null.
Acad. So. i'SA 84: 2297-2 301.

Sackin, H. 1995. Mechanosensitive channels. Annu. Rev Physiol. 57:
333-353.

Sakmann, B., and E. Neher. 1995. Single-Channel Recording. 2nd
Edition. Plenum Press, New York.

Sukharev, S. I., B. Martinac, V. V. Arshavsky, and C. Kung. 1993.
Two types of mechanosensitive channels in Eschericia coli cell en-
velope: solubilization and functional reconstitution. Biophys J 65:
177-183.

Sukharev, S. I., P. Blount, B. Martinac, F. R. Blattner, and C. Kung.
1994a. A large-conductance mechanosensitive channel in F. coli
encoded by mscL alone. Nature 368: 265-268.

Sukharev, S. I., B. Martinac, P. Blount. and C. Kung. I994b. Meth-
ods: A Companion to Methods in Enzymology. 6: 5 1-59.

Reference: Bin/. Bull 192: 128-130. (February. 1997)

Discussion

CHALFIE (in response to an inau-
dible question): We have been able
to identify 1 7 genes in the screen for
touch-insensitive genes. If you mu-
tate them, you get a selectively defec-
tive animal that just doesn't respond
to touch. Of the five genes required
for cell differentiation that we have
identified, three are transcription
factors that are needed to make ei-
ther precursors or the cell itself,
another appears to be a transcrip-
tion factor needed to maintain the
differentiated state, and the fifth is a
splicing factor needed to process one
or more of the function genes. This
last gene is the work of Lundquist el
at. (Development 1996. 122: 1601-
1610).

We also know that there are genes
whose products act negatively in the
touch system, but we would not have
been able to screen for mutations in
these genes, because we were not
looking for touch supersensitive ani-
mals. We are trying to develop ways
to look at such mutants. For exam-
ple, the touch circuit is one that re-
quires gap junctions between the in-
terneurons and the touch receptor
cells. At the same time as the poste-
rior touch cells are making gap junc-
tions to one set of imerneurons, they
are also making < ical synapses
to another set c irons to

which the anterior c e making

gap junctions. Getting of the
chemical synapses wi
touch insensitivity, but o.iiers have
identified genes needed for chemical
transmission.

QUESTION: Twelve seems to be a
small number for what seems to be a
saturation screen.

CHALFIE: As I tried to indicate, we
have over 500 mutations in 1 7 genes,
quite a number of alleles for every
single one of those genes that we
study. So we have probably saturated
for genes that can be mutated to the
touch-insensitive phenotype. In ad-
dition, we have done an experiment
to get dominantly enhancing muta-
tions with two different function
genes, and we have not been able to
find any new players. That says that
no dosage-dependent gene is in-
volved. We have not been able to
eliminate genes that may be impor-
tant for the mechanosensory appara-
tus (for example microtubule-associ-
ated protein) and that also produce
lethality or are perhaps redundant.

HAMILL: By analogy to the hair
cell, your model envisages gating be-
ing mediated by the cytoskeleton. In
the hair cell, the tip links are directly
gated. What do you see as a role for
MEC-5 in your system?

CHALFIE: The three products that
we think are important in the extra-
cellular matrix are MEC-1, MEC-5,
and MEC-9. mec-1 is not cloned, but
when you get rid of it genetically
there is no extracellular matrix for
those cells, and the animals are touch
insensitive. Both mec-5 and mec-9
have been cloned: mec-5 encodes a
collagen that's made by the cells that
surround the touch cells, and MEC-

9 is a secreted protein made by the
touch cells. We have very strong ge-
netic interactions between the two of
them, so we think they are coupled.
We have somewhat weaker evidence
for an interaction between the mec-
5 collagen and the mcc-4 and mec-6
genes whose products we think con-
tribute to the channel. So a collagen
is associated with the channel.
There's also another one of this fam-
ily of channel proteins, the product
of the unc-105 gene, that's being
worked on by J. D. Liu in Bob Wa-
terston's lab in St. Louis (Liu el at.
1996. Science 273: 361-364). What
they have found is that a suppressor
of line-] 05 activity is also a collagen.
So this family of channel proteins
seems to interact with collagens.

Let me address one other thing
that you said. I don't think that the
models are very different. It's like
the distinction between pulling and
holding on, and to what. We're say-
ing that whether you're holding on
with the tip link or the extracellular
matrix, you are also tethered to the
actin cytoskeleton or the microtu-
bule cytoskeleton in the two systems.
In fact, the question is. Does the tip
link move to open that channel, or
does the hair cell move and pull the
channel away from the tip link? I
don't think that there is any evidence
bearing on which element is actually
the moving component. Peter (Gil-
lespie) may have more to say about
that. It seems to me that it would be
easier for the microtubules to float
within the cell than for the extracel-

MECHANOSENSITIVITY

129

lular matrix to be moved. In fact, if
you hit the matrix hard enough, that
will jar the microtubules. It's hard to
know who is pulling on whom; I just
think that the microtubules are a lit-
tle more liable to be moved relative
to the matrix.

LOEWENSTEIN: When you men-
tioned the inside sensor, did you re-
ally mean sensor or coup/ing'? The
actual sensor is the transducer. I
think you probably meant coupling,
and that is what makes your system
different from Rung's.

CHALFIE: We don't know how
these things are interacting. One of
the things that we have tried to ex-
plain in the model is the morphology
of the cell with the microtubules sit-
uated within the processes. In fact,
the microtubules are not needed for
the outgrowth of the cells or any
other function of the cell that we can
find; but the microtubules are abso-
lutely essential for those cells func-
tioning as touch receptors. In addi-
tion, the association with the MEC-
2 protein and genetic interactions all
suggest that the microtubules are ac-
tually part of the apparatus. This
model is very difficult to test by try-
ing to reconstitute all of these com-
ponents in some sort of heterologous
system.

KAWASAKI: You find these touch
receptors in E. coli and hair cells.
How ubiquitous are they?

CHALFIE: Owen Hamill probably
has a better handle on this in terms
of the pharmacology of channels ex-
amined with cells in culture. Me-
chanically activated and mechani-
cally inactivated channels have been
found in a wide variety of cell types.
If you are asking whether channel
proteins similar to the E. coli or C
eli'gans proteins can be found in ver-
tebrates, the answer is that Ching
Rung and I both hope so. Our chan-
nel proteins are certainly found in

nematodes, and there are similar
members of what could be a super-
family of proteins in vertebrates.
Whether these proteins act as mech-
anosensory channel components in
vertebrates is not known.

RUNG: If I may add to the matter
of ubiquity: By the criterion of the
patch clamp these channels are all
over the place. You can take just
about any cell membrane, attach a
patch pipette, apply suction and you
will see something open. If you do
not use the patch clamp as a crite-
rion, but look for the genes, then we
are looking for mec-4, mec-10, or
mscL homologues. That's in prog-
ress. When you say homologue, you
must be careful because you do not
know how much of the region is
crucial to mechanosensation. One
doesn't know how to search the
database at this point, because our
knowledge of these molecules is
fairly primitive.

LOEWENSTEIN: Dr. Rung, a com-
ment on your model propsing a hex-
amer. A hexameric channel implies
a mechanistic possibility that I think
is quite attractive in a mechanosen-
sing device. If your six subunits are
slanted, as in the channel I have been
working with, you might get a direct
coupling in a rather simple way. A
hexamer with slanted units can
switch between an open and closed
configuration by a slight change in
the tilt of the subunits. A very small
movement, due to a pressure change
in the lipid bilayer of the cell mem-
brane, would change the angle of tilt,
making the channel go into the open
configuration — a coupling as direct
as you are proposing. With this
model, one could easily imagine that
a minute strain perpendicular to the
membrane could be amplified by the
lever action of the slanted subunits
and be transduced into a current — a
channel opening or closing produced
in the most directly coupled way.

RUNG: I have to point out that I
don't propose, I prove. That's not a
proposal, that's actually an observa-
tion. You ask me whether this is a
possible model. I certainly think that
this is a possible model. My position
is that I believe in experiments.

BARLOW, R.: I have a question re-
garding degenerins. You said that
three mutations cause cell death, cell
lysis. As I understood it, your inter-
pretation is that faulty operation of
the channel leads to cell death. 1 am
curious about an analogous situa-
tion, in vision, where multiple muta-
tions of rhodopsin lead to retinal
degeneration. Retinal degeneration
may not necessarily be caused by
rhodopsins not working well, but
rather that the mutated proteins trig-
ger natural cell death, apoptosis.
Could something similar be happen-
ing with these mutations?

CHALFIE: That's a great question.
In C. elegans there is some beautiful
work, primarily by people in Bob
Horvitz's lab, on genes that are
needed for programmed cell death in
the animal. A cell that's dying by
programmed cell death in C. elegans
becomes very condensed and very
refractile in the light microscope.
Mutations in the genes ced-3 and
ceil-4 abolish these cell deaths, so the
cells persist. The deaths that we see
in the mec-4 and mec-10 animals,
and in mutants defective in one
other gene, deg-1 (all three genes en-
code proteins we call degenerins), are
very different. The cells don't be-
come refractile; they swell up to
many cell diameters and vacuolate.
Genetically, mutations in ced-3 and
ced-4 genes have absolutely no effect
that we have been able to discern on
the degenerations caused by muta-
tions in mec-4 or deg- 1, and soon. In
addition, any of the mutations that
prevent the cell deaths from the pre-

130 FUTURE OF AQUATIC RESEARCH IN SPACE

sumed channel mutations have no in hypocalemic periodic paralysis changes, such as membrane infold-

effect on programmed cell death, so in humans and other mammals in ing and multilamellar bodies. We see

they seem to be two independent sys- which a defect in the dihydropyri- these detects in mec-4 and deg-1 ani-

tems. Rather than apoptotic cell dine calcium channel leads to de- mals. Because of the similarities in

death, a better analogy might be to fects in muscle with a fair amount morphology, we think that the chan-

some of the defects that are seen of vacuolization and ultrastructural nel defect causes the degeneration.

Reference: Kiol Bull 192: 131-133. (February. 1997)

Gravitaxis in Flagellates

DONAT-PETER HADER
Institutefor Botany and Pharmaceutical Biology, Staucltsir. 5. D-91058 EHangen, Germany

The green, unicellular flagellate Euglena gracilis ori-
ents itself in the water column by responding to external
stimuli such as light and gravity (Ha'der, 1987; Ha'der ct
al.. 1995). While the photoreceptor has been character-
ized spectroscopically and biochemically in this organ-
ism, our knowledge of gravity-dependent orientation
mechanisms has been rather limited. Earlier hypotheses
assumed that the cell has an asymmetric mass distribu-
tion and is passively oriented in the water column like
a buoy. However, we found that the cells change their
orientation with age: young cells (up to 4 days after inoc-
ulation) show positive gravitaxis, but older cells show a
negative one. Another indication that the cells use an ac-
tive receptor and a physiological sensory transduction
chain for graviperception is the finding that positive
gravitaxis (in young cells) can be reversed by the addition
of micromolar concentrations of certain heavy metal
ions (cadmium, mercury, lead).

During a recent space flight on the American shuttle
Columbia the cells could be subjected to increasing ac-
celerations on a centrifuge microscope (NIZEMI)
(Ha'der ct ul.. 1995). These experiments indicated that
the threshold for gravitaxis in Euglena is at about 0. 1 6 X
g; above 0.64 X g the precision of orientation saturates.
The dose response curve has a typical sigmoidal shape,
again indicating the involvement of a physiological
gravireceptor in contrast to a passive orientation in the
water column.

One option for an active gravireceptor is an intracellu-
lar statolith exerting pressure on a sensor, as has been

This paper was originally presented at a workshop titled The Future
oj Aquatic Research in Space: Neurobiolugy. Cellular ami Molecular
Biology. The workshop, which was held at the Marine Biological Lab-
oratory. Woods Hole, Massachusetts, from 13 to 15 May 1996, was
sponsored by the Center for Advanced Studies in the Space Life Sci-
ences at MBL and funded by the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

proposed for gravitropism in higher plants. This assump-
tion could be ruled out by the following experiment. The
density of the outer medium was increased with Ficoll.
At low Ficoll concentrations (2.5%), negative gravitaxis
was only slightly impaired compared to the control (Fig.
la, b). With increasing concentrations, the density of the
medium approached the density of the Euglena cell body
(1.04 g mr1), and negative gravitactic orientation was
strongly affected (Fig. Ic, d). At higher concentrations,
the cells showed positive gravitaxis (Fig. le) because the
direction of the pressure of the cell body on its outer
membrane is reversed, and so the direction of orienta-
tion also reverses. Changing the density of the medium
cannot affect an intracellular gravisensor. Therefore it
was assumed that the whole cell — being heavier than the
surrounding medium — exerts pressure on the lower cell
membrane which has also been suggested for the ciliate
Paramecium (Machemer and Bra'ucker, 1992). This
could be sensed by stretch-sensitive ion channels which
have been demonstrated in a number of different biolog-
ical systems.

Stretch-sensitive ion channels can be inhibited by gad-
olinium ions (Franco el al., 1991; Lacampagne el al..
1994; Sukharev el al.. 1993). At low concentrations,
Gd3+ (100 nAf) strongly affected graviorientation, which
was totally inhibited after 90 s. This result indicates
strongly that Ca2+-conducting, stretch-sensitive ion
channels in the cell membrane of Euglena gracilis are
involved in gravitaxis; and a further indication is that a
modulation of the electrical membrane potential is one
element in the signal transduction chain. TPMP+, a lipo-
philic cation, passes through membranes driven by the
existing membrane potential and thus neutralizes the po-
tential. TPMP+ strongly impaired graviorientation even
at low concentrations ( 10 juA/), indicating that the mem-
brane potential plays a crucial role in gravitactic orienta-
tion. In addition, sudden changes of the ionic composi-
tion of the outer medium had drastic effects on gravitac-

131

132

FUTURE OF AQUATIC RESEARCH IN SPACE

270°

90°

2700

80°

Q0°

1800

270°

00°

2700

1800

000

180°

270°

180°

ii 'grams of gravitactic orientation of Eiigli'iui graci/is cells in different Ficoll con-

centraii,M 1 y; (h). ?% (c), 1.5% (d) and 10% (e). 0° indicates upwards. The swimming

directions ul th have been binned in 64 sectors of 5.6° each: the length of each sector indicates the

normalized mm;!" i of cells swimming in the respective direction.

PLANT BIOLOGY

133

tic orientation. Another interpretation could be based on
localized ion fluxes and Ca:+ gradients.

Based on these results, the following model was pro-
posed for gravitaxis in Eiiglena: The cell has a higher
density than the surrounding medium, and depending
on the position of the cell, the resulting pressure deforms
different parts of the cell membrane. These deformations
activate stretch-sensitive ion channels; the resulting
change in the ion flux alters the membrane potential,
which is the primary event of gravity sensing. Another
assumption of the hypothesis is that stretch-sensitive ion
channels are not evenly distributed over the cell. Most
likely, channels are concentrated at the front pole of the
cell. Modulation of the membrane potential would be
minimal only during vertical upward swimming.

In preparation for future experiments on the space sta-
tion, a terrestrial bioreactor that allows the long-term,
stable culture of unicellular algae (CEBAS) has been de-
veloped and tested. A fully automatic monitoring system
determines all important physical and chemical param-
eters, such as temperature, light, oxygen, pH, nitrogen,
and ammonia. Furthermore, absorption spectra are re-
corded demonstrating changes in pigmentation. Cell

density, motility, velocity, and graviorientation were
measured frequently with a real-time, computer-con-
trolled image analysis system developed for this purpose.
This bioreactor has been running for more than 500
days.

Literature Cited

Franco, A., Jr., B. D. \\inegar, and J. B. Lansman. 1991. Open chan-
nel block by gadolinium ion of the stretch-inactivated ion channel
in mdx myotubes. Biochem. J 59: 1 164-1 170.

1 lacier, D.-P. 1987. Polarotaxis, gravitaxis and vertical phototaxis in
the green flagellate, Eiiglena gracilix. Arch Microhm/ 147: 179-

183.

Hader,D.-P.,A.Rosum,J.Schafer,andR.Hemmersbach.l995. Grav-
itaxis in the flagellate Euglcmi f-nicilix is controlled by an active
gravireceptor. / Plant PhyxioL 146:474-480.

Lacampagne, A., F. Cannier, J. Argibay, D. Gamier, and J.-V. Le
Guennec. 1994. The stretch-activated ion channel blocker gado-
linium also blocks L-type calcium channels in isolated ventricular
myocytes of the guinea-pig. Biochnn. Bwphys. Ada 1191:205-208.

Machemer, H., and R. Braucker. 1992. Gravireception and gravires-
ponsesin ciliates. Actu Protozool. 31: 185-214.

Sukharev, S. I., B. Martinac, V. V. Arshavsky, and C. Kung. 1993.
Two types of mechanosensitive channels in the Exchcncia coll cell
envelope: solubilization and functional reconstitution. Biophys. J.
65: 177-183.

Reference: Biol. Bull- 192: 134-136. (February. 1997)

Gravitropism in the Rhizoids of the Alga Chara:
A Model System for Microgravity Research

JOHN Z. KISS

Department of Botany, Miami University, Oxford, Ohio 45056

Gravitropism in plants can be divided into the follow-
ing stages: perception, transduction, and response (Ev-
ans el ai, 1986). In higher plants, there is a cellular and
spatial separation between the perception of and re-
sponse to gravity, and a signal must be transmitted over a
relatively large distance. However, the rhizoid (a rootlike
extension) of the alga Chara is a model system in which
all stages of gravitropism can be analyzed in a single cell
(Braun and Sievers, 1994; Kiss, 1994; Sievers el ai.
1 996). The cell biology of gravitropism is therefore much
more readily analyzed in such single-celled systems than
in multicellular organisms.

Chara rhizoids have a distinctive structural polarity,
with a group of 50-60 vesicles near the apex (Fig. 1 ). The
bulk of the cell consists of a large vacuole, and the nu-
cleus is located immediately adjacent to this zone (Kiss
and Staehelin, 1993). Like pollen tubes and root hairs,
these rhizoids can be characterized as tip growing cells
since all extension occurs at the extreme apex (Sievers
and Volkmann, 1 979). The Chara rhizoid is an ideal sin-
gle-celled subject for space biology studies because ( 1 ) a
great deal of background information is available about
its structure and gravitropic kinetics; (2) rhizoid forma-
tion can easily be induced under controlled conditions;
and (3) these cells have already been successfully flown
aboard the U.S. Space Shuttle and in sounding rockets
(e.g., Volkmann et ai. 1991).

Gravity perception in plants is hypothesized to be me-
diated by the interaction between dense organelles
(termed statoliths) and other cytoplasmic structures

This paper was originally presented at a workshop titled The Future
of Aquatic Research in Space Neurobiology, Cellular ami Molecular
Biology. The workshop, which was held at the Marine Biological Lab-
oratory. Woods Hole, Massachusetts, from 13 to 15 May 1996, was
sponsored by the Center for Advanced Studies in the Space Life Sci-
ences at MBL and funded by the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

(Sack, 1991; Salisbury. 1993). In higher plants, much ev-
idence suggests that amyloplasts function as statoliths
(Kiss et ai. 1989, 1996). But, in Chara rhizoids, the
membrane-bound vesicles that are located close to the
rhizoid apex appear to serve this function (Fig. 1 ). These
vesicles sediment to the lower wall within minutes of
horizontal reorientation (Figs. 2, 3), and this sedimenta-
tion precedes gravitropic curvature (Sievers and Volk-
mann, 1979).

Research in our laboratory has focused on three major
areas. First, we have been characterizing the statolith
compartment and its membrane to determine their po-
tential roles in mechanisms of gravitropism (Wang-Ca-
hill and Kiss, 1995). Statoliths in Chara rhizoids are very
distinctive in that they contain barium sulfate (Sievers
and Volkmann. 1 979) in an organic matrix consisting of
protein and carbohydrate (possibly 3-linked polysaccha-
rides) moieties. Second, we have been manipulating the
number of statolith vesicles in Chara rhizoids (by alter-
ing the growth medium) and have found that the re-
sponse to gravity is correlated with the number of stato-
liths ( Kiss, 1 994). These results are comparable to studies
with higher plants in which it was found that starch-de-
ficient mutants (i.e., less total mass of statoliths per cell)
were not as sensitive to gravity as their respective wild-
types (Kiss et ai. 1989, 1996; Kiss and Sack, 1990).

We also have been investigating the potential role of
integrin-like proteins in gravity perception and subse-
quent transduction mechanisms in both Chara and in
higher plants. Integrins are a large family of integral
plasma membrane proteins that link the extracellular
matrix to the cytoskeleton in animal cells ( Hynes, 1 992).
They exist as heterodimers of a and /3 subunits and have
been shown to activate intracellular signaling cascades
involved in both "inside-to-out" and "outside-to-in" sig-
naling. While several studies have shown that integrin-
like proteins are present in plant and fungal cells (Mar-

134

PLANT BIOLOGY

135

N

Figures 1-3. Light microscopy of Chum rhizoids. N = nucleus; S = statolith vesicles; V = vacuole. (1)
A rhizoid imaged with differential-interference-contrast optics. This cell has a distinctive structural polarity
in which statolith (S) vesicles are located near the apex, and the nucleus (N) is near the vacuole (V) region.
Scale bar = 40 ^m. (2) A rhizoid viewed with hnghttield optics through agar (that was used in the growth
medium). After reonentation ol'the rhizoid, statolith vesicles (arrowhead) sediment to the lower wall. This
sedimentation is followed by gravitropic curvature in the rhizoid. Scale bar = 80 pm. (3) A rhizoid at a
latter stage of gravitropic curvature. Note the position of the statolith vesicles, which are indicated by an
arrowhead. Scale bar = 80 ^m.

cantonio and Hynes, 1988; Quatrano el a/.. 1991;Kam-
inskyj and Heath, 1995), unequivocal evidence for inte-
grins in plant cells is still lacking. Nevertheless, there is
some evidence that integrin-like proteins may play a role
in gravity perception in characean algae, particularly in
the gravity-regulated cytoplasmic streaming in inter-
nodal cells (Wayne el ai, 1992).

As a first step in determining whether integrin-like
proteins are involved in the gravitropic signal transduc-
tion pathway in plant cells, we have investigated their
distribution in Chara. Western blot analyses and immu-
nofluorescence microscopy were used to gain informa-
tion about the size, abundance, and location of integrin-
like proteins in plants (Katembe et a/., 1997). Several
different polypeptides in the rhizoids and internodal cells
of Cham are recognized by a chicken anti-integrin anti-
body (against the 0, subunit; see Marcantonio and
Hynes, 1988). These cross-reactive polypeptides appear

to be associated with cellular membranes, a feature
which is consistent with the known location of integrins
in animal systems. In immunofluorescence studies of
rhizoids, the antibody stained throughout Chara rhi-
zoids, but the highest density of immunolabel was at the
tip. Furthermore, the anti-integrin antibody cross-re-
acted with the higher plant Arabidopsis in both roots and
shoots and in a specific manner. Based on our data and
given the central role that integrins play in cell-cell in-
teractions and cell signaling in animal cells, they are
strong candidates for a mediating role in gravity signal
transduction in plant cells.

Acknowledgments

The author thanks his colleagues at Miami University
who have supported this project: Chris Makaroff, Fan
Wang-Cahill, Jira Katembe, and Lucinda Swatzell. Fi-
nancial support was provided by grants to JZK from the

136

FUTURE OF AQUATIC RESEARCH IN SPACE

National Aeronautics and Administration
(NAGW-3783 and NAG : ). the Research Chal-
lenge Program (Ohio Bo: egents), and the Com-
mittee for Faculty Res utmi University).

•c Cited

Braun. M., and A. Sifvers. ;'.;tM. Role of the microtubule cytoskele-
ton in gravisen ru rhizoids. Eitr. J. Cell Bin/. 63: 289-298.

Evans, M. L., R. Moore, and K.-H. Hasenstein. 1986. How roots re-
spond tograuu. Si1/ Am 255: 1 12-1 19.

Hynes, R. O. 1992. Integrins: versatility, modulation, and signaling
in cell adhesion. Cell 69: 1 1-25.

Kaminskyj, S. G., and I. B. Heath. 1995. Integrin andspectrin homo-
logues. and cytoplasm-wall adhesion in tip growth. / Cell Set. 108:
849-856.

Katembe, W. J., I.. J. Swatzell, C. A. Makaroff, and J. Z. Kiss. 1997.
Immunolocalization of integrin-like proteins in Arabidopsis and
Chara. Physiol Plain. 99: in press.

Kiss. J. Z. 1994. The response to gravity is correlated with the num-
ber of statoliths in Cluira rhizoids. Plunl Physiol. 105: 937-940.

Kiss, J. Z., J. Hertel, and F. D. Sack. 1989. Amyloplasts are neces-
sary for full gravitropic sensitivity in roots of Arabidopsis lhaliana.
Planta 177: 198-206.

Kiss, J. Z., and F. D. Sack. 1990. Severely reduced gravitropism in
dark-grown hypocotyls of a starch-deficient mutant of fs'ieoliuna
sylveslns. Plant Physiol. 94: 1867-1873.

Kiss, J.Z., and L. A. Staehelin. 1993. Structural polarity in the
Cham rhizoid: a reevaluation. Am .1 Hal 80: 273-282.

Kiss, J. Z., J. B. Wright, and T. Caspar. 1996. Gravitropism in roots
of intermediate-starch mutants of Arabidopsis. Physiol. Plunl. 97:
237-244.

Marcantonio, E. E., and R. O. Hynes. 1988. Antibodies to the con-
served cytoplasmic domain of the integrin (i-\ subunit react with
proteins in vertebrates, invertebrates and fungi. J. Cell. Biol. 106:
1756-1772.

Quatrano, R. S., L. Brian, J. Aldridge, and T. Schultz. 1991. Polar
axis fixation in fueiix zygotes: components of the cytoskeleton and
extracellular matrix. Di'v. Suppl 1: 11-16.

Sack, F. D. 1991. Plant gravity sensing. Int. Rev Cyloi 127: 193-
252.

Salisbury, F. B. 1993. Gravitropism: changing ideas. Home. Rev. 15:
233-278.

Sievers, A., and D. Yolkmann. 1979. Gravitropism in single cells. Pp.
567-572 in Eneyelopeilia ol Plant Physiology. Vol. 7. W. Haupt and
M. Feinleib, eds. Springer-Verlag. Berlin.

Sievers, A., B. Buchen, and D. Hodick. 1996. Gravity sensing in tip-
growing cells. Trends Plant Sci. 1:273-279.

\ oik m:mii. D., B. Buchen, Z. Hejnowicz, M. Tewinkel, and A. Sievers.
1991. Oriented movement of statoliths studied in a reduced grav-
itational field during parabolic (lights of rockets. Planui 185: 153-
161.

Wang-Cahill, F., and J. Z. Kiss. 1995. The statolith compartment in
Churn rhizoids contains carbohydrate and protein. Am ./ Bui 82:
22(1-229.

Wayne, R., M. P. Staves, and A. C. Leopold. 1992. The contribution
of the extracellular matrix to gravisensing in characean cells. J. Cell
Sei. 101:611-623.

Reference: Biol. Bull. 192: 1 37- 1 38. (February, 1997)

Discussion

NICK: Is it possible to purify the a bowing rather than a bulging
statoliths? mechanism.

Kiss: Our group and others have
tried for some while, but it does seem
to be difficult. The problem is that
they are almost too dense and there
are many technical difficulties.

NICK: If they are so very dense,
they should be more easily isolated.

Kiss: Yes and no. The density of
pure barium sulfate is about 4.5, and
it goes through every gradient. We
would be more interested in isolating
the statoliths with the membrane
around them, and that is certainly
very difficult because you have this
solid rock in a membrane. The
membrane is lost somewhere in the
gradient. So although I agree with
you, there may also be a problem of
quantity.

JACOBS: Dr. Kiss, you implied that
the statoliths in Chara are not active
themselves, but just diverting mate-
rial to the upper side of the cell. Do
you think that is the case, and if so,
what's the evidence for this as op-
posed to a more direct effect?

Kiss: The model I presented is pri-
marily from the Sievers group. I
think that other things are involved
besides Golgi vesicles being physi-
cally displaced.

QUESTION: Has anyone measured
the two sides of the curving rhizoid
to see which of them is bowed more
than normal?

KJSS: There have been detailed
studies with beads and particles. It's

NICK: I have a small contribution
to the question of this diversion hy-
pothesis. There's an old paper (un-
fortunately forgotten because it's
written in German) by Friedrich and
Hertel (see Nick abstract). They have
shown that, when you apply differ-
ent strengths of acceleration, the cur-
vature goes with the dose even if the
sedimentation is already saturated.
This speaks against a pure proximity
mechanism.

Kiss: I would agree with you on
that.

Roux: Have either one of you
tried to use RODS to try to inhibit
your gravity responses? [Ed: RODS
is the tetrapeptide Arg-Gly-Asp-Ser
contained in the integrin recognition
site of many protein ligans (Schwartz
etal. 1995. Annu. Rev. Cell. Bio! 11:
549-599.)]

Kiss: Randy Wayne has done that
with the internodal cells (Wayne el
a/. 1992. J. Cell. Sci. 101(3): 611-
623). This indicates that the gravi-
receptor in Chara may be an inte-
grin-like protein. The approach that
we are trying now is to microinject
anti-integrin antibodies into the rhi-
zoid. We have not done the RGDS
experiments with the rhizoids.

BARLOW, R.: Dr. Ha'der, you pre-
sented a model indicating that the di-
rection of motion is dependent on
the location of channels in the mem-
brane. You placed the channels in

one part of the cell so that it would
move in one particular direction. It
isn't clear to me why you impose that
restriction on the model.

HADER: The problem in the
mechanism of reorientation is that,
at a given point in time, the cell
swings out its flagellum, which
brings the front end over. This has
been shown to be the case in both
graviorientation and phototaxis.
When the cell is swimming on a heli-
cal path, it is not only moving coni-
cally, it is really rotating, so the fla-
gellum moves all the way around.
Whenever you have a light source
from one side, and this stimulus hits
the cell in the right direction, the
flagellum will swing out on the other
side and turn the front end stepwise
in the direction of the light. By anal-
ogy, for graviorientation you need to
have the cell in a certain position
when it swings out its flagellum.
When you have the channels all the
way around, some channels would
be activated all the time. This would
be independent of whether the cells
are swimming horizontally, down-
wards, or upwards. So we assume
that the channels are in a certain po-
sition, and the cell aims for minimal
modulation of the signal. This oc-
curs when the channels are at the
top. When the cell is swimming up-
wards, the whole pressure goes
downwards where there are no chan-
nels, or fewer channels. Each time
the cell deviates from that course,
moving either horizontally or down-
wards, it will activate the channels.
This will then lead to reorientation

137

138

FUTURE OF AQUATIC RESEARCH IN SPACE

of the flagellum, which will brin
cell upwards again.

BARLOW, R.: I can cei
derstand why having s uni-

formly located all over the cell leads
to a problem, but it's not at all clear
to me, even with your explanation,
why having the channels on one part
or the other of the cell would lead to
the directionality you observe. It
seems to me that you could develop
a workable model with channels at
either pole of the cell. Am I missing
something important?

HADER: We did that by running a
model. When the channels are at ei-
ther pole, the cells would go up or
down equally well. That might ex-
plain the crossover that is observed
between the young and the old cells.
One thing that we cannot distinguish
in that model is whether the chan-
nels are distributed symmetrically
round the top of the cell, or whether
they are oriented only on the same
side as the flagellum.

QUESTION: How did you rule out
the presence of channels in the fla-
gella?

HADER: The flagellum moves with
a frequency of 50 Hz in this flagel-
late, and it will be in various posi-
tions; so the flagellum is not a good
choice for positioning the channels.
One possibility is that the front end
of the cell has an invagination, re-

ferred to as a reservoir. The flagellum
doesn't sit on the tip of the flagellate,
but rather inside the reservoir. This
reservoir does not have the pellicule,
which consists of proteinaceous
strips going round the cell, and could
be a candidate for the location of the
channels.

QUESTION: Did you look at the
effect of the RODS peptide on your
cells?

HADER: We did not try this pep-
tide with Euglena.

QUESTION: Dr. Kiss, did you try
any buoyancy experiments with the
rhizoid?

Kiss: No, these were done with in-
ternodal cells by Randy Wayne and
his colleagues (in the Chani in-
ternode).

MORRIS: Dr. Hader, I have a ques-
tion about your experiments with Fi-
coll. Presumably this is also going to
have an osmotic effect. Do you know
the general effects of the osmotic per-
turbation? Also, can you just take
the flagella off these Euglena? If so,
what happens to the cells, do they
just sink? I wondered if you could
address the problem of using gado-
linium, because it also blocks
calcium channels, and that is going
to have enormous effects on motility
and so on. Finally, have you tried
any other inhibitors to block the
mechanoresponses?

HADER: In answer to your first
question, the Ficoll we were using
has a molecular weight of 400,000.
We checked osmotic effects with Fi-
colls of lower molecular weight and
could find no effect on graviorienta-
tion. As I mentioned, we did all the
experiments with phototaxis and
gravitaxis in parallel. Another poten-
tial concern to us was that Ficoll was
also changing the viscosity of the me-
dium. As a control for this, we used
methyl cellulose, which has much
higher viscosity. Our concern was
that there could be a disturbance of
the timing in an organism that ro-
tates, where it might take a certain
time between the application of a
stimulus and the response. During
this time, the flagellum could be one
quarter or half way round the cell.
However, when we used higher con-
centrations of methyl cellulose in an
attempt to decrease the velocity of
this movement, we saw no effect. In
answer to the next question, we can
distinguish very easily between
effects on motility or swimming ve-
locity and on orientation. Even
when the cells are swimming very
slowly, we can determine whether
they are moving up or down. Thus
we can distinguish whether we are
affecting graviperception or any-
thing in the cellular metabolism that
could slow the cell down. As long as
the cells are swimming in a specific
direction, we can detect this.

Reference: «/../ Hull 192: 1 39-140. (February. 1997)

The Influence of Gravity and Light on Developmental
Polarity of Single Cells of Ceratopteris richardii

Gametophytes

ERIN SWINT EDWARDS AND STANLEY J. ROUX
Department of Botany, The University of Texas, Austin, Texas 78713

The gametophyte generation of Ceratopteris richardii
is being used in a number of laboratories as a model plant
system for developmental studies (Chasan, 1992). It is an
especially valuable system for the study of the effect of
gravity on plants in that the detection and response to
gravity take place in the single-celled spore during its ger-
mination. The most visible evidence of the control of C.
richardii spore polarity by gravity is that the direction of
growth of the primary rhizoid, when it emerges, is down-
ward, in line with the vector of gravity (Edwards and
Roux, 1994). However, the direction of rhizoid growth is
determined prior to its actual emergence through the
spore coat. The influence of gravity on this growth polar-
ity is, in fact, determined irreversibly during a "polarity-
determination window" — a period that occurs after ger-
mination has been initiated by exposure of spores to
light, but before the first cellular division. Gravity does
not appear to have an independent effect on the later
events of prothallus and secondary rhizoid growth in the
gametophyte. for the growth of prothallial cells follows
the same direction as that of the first prothallial cell, and
the growth of secondary rhizoids follows the same direc-
tion as that of the primary rhizoid, no matter how the
gametophyte is oriented.

The polarity-determination window is specified by a
simple assay in which the spore position is changed by
180° at various times after the initiation of germination
by light, but before rhizoid emergence, and then the sub-
sequent direction of rhizoid emergence and growth is re-
corded (Edwards and Roux, 1994). Turning the spore

This paper was originally presented al a workshop tilled The h'ulurc
ol Aquatic Research in Space.- Neiirobiology, Cellular and Molecular
Biology. The workshop, which was held at the Marine Biological Lab-
oratory. Woods Hole. Massachusetts, from I 3 to 15 May 1996, was
sponsored by the Center for Advanced Studies in the Space Life Sci-
ences at MBL and funded by the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

upside down before the window opens will also change
the direction of rhizoid emergence by 180°: the rhizoid
will grow downward in accord with its second position of
down. But, if the orientation of a spore is changed 180°
after the polarity-determination window closes for that
individual spore, the primary rhizoid will grow up, in ac-
cord with what was down before the orientation change.
If the orientation of a population of spore is changed dur-
ing the polarity-determination window for that popula-
tion, some of the primary rhizoids grow in the first direc-
tion of down, while the others grow in the second direc-
tion of down. Thus, the closing of the polarity-
determination window for a population of spores can be
determined as the time at which the orientation change
has no effect on the orientation of primary rhizoid
growth; i.e., the direction of rhizoid growth has already
been fixed by gravity prior to this time. The exact time of
opening this window for a population of spores and the
duration of the open state vary from spore lot to spore lot
and can be influenced by growth conditions (Edwards,
1996).

The direction of rhizoid growth is not the earliest visi-
ble response of germinating spores to gravity. Before the
first cellular division, but after the polarity-determina-
tion window, the first indication of cell polarity is migra-
tion of the nucleus down (with respect to gravity) along
the proximal face of the spore (Edwards and Roux,
1994). This downward migration of the nucleus sets up
an asymmetric first cell division, determining the devel-
opmental fate of the two daughter cells, one giving rise
to the primary rhizoid, and the other giving rise to the
prothallus. Thus, in dictating the direction of nuclear mi-
gration, gravity also dictates the developmental polarity
of the spore. When spores are germinated on a clinostat,
the direction of nuclear migration is random, as is the
direction of rhizoid growth. However, in each case, the
direction of nuclear migration predicts the direction of
rhizoid emergence and elongation (Edwards, 1996).

139

140

FUTURE OF AQUATIC RESEARCH IN SPACE

Although nuclear migration is the most obvious
nuclear movement, it is no) mclear movement.

During the polarity-determ n window, the nucleus

exhibits random excurs" icred around the middle

of the spore. These ex< can be visualized through

the transparent sr . and followed in reference to

a fixed trilete marking on the spore coat called the laesura
(Edwards, 1996). They can be clearly described in plots
showing change in nuclear position over time (Fig. 1 ).
Since the nucleus is the only organelle clearly visible
through the intact spore coat by light microscopy, the
very likely possibility remains that other organelles move
during the polarity-determination window in response to
gravity as well. However, nuclei possess sufficient mass
to contribute significantly to gravity detection (Sack,
1991). Their centered movements prior to their migra-
tion may play some role in the sensing of the vector of
gravity, for, as described by Mesland (1992), nuclei are
typically tethered to the cell periphery by cytoskeletal el-
ements, and these elements could convey tension and
compression forces as the nucleus moves in various di-
rections. Both the centered movements of the nucleus
and its directional migration exhibit the same periodic
variance in speed (Edwards, 1996). The cause for this is
not clear at this time, but may indicate that the same
kind of molecular motor machinery is driving both
movements.

Figure 1. Path of nuclear m a representative spore ger-

minating on a vertically oriented >li e. The position of the nucleus was
recorded every half hour. The original position of the nucleus is as-
signed the position 0.0. Its initial movement is restricted to a region
near the center of the spore before it starts its migration downward 1 5
h after exposure to light. All number values are in micrometers.

Another environmental gradient used to control the
polarity of fern spore development is unilateral light
(Raghaven, 1 989). Light gradients do affect polarity of C.
richardii spores, but initial experiments using total flu-
ences of about 100 ^mol/m2 of unilateral white light in-
dicate that the influence of light gradients is secondary to
that of gravity.

Tests of the effects of agonists and antagonists on the
gravity and light responses are impeded by the limited
permeability of the thick spore coat. To overcome this
problem, prothallus protoplasts may be used as an al-
ternate experimental system for studying processes re-
lated to development of cell polarity. A protocol has been
developed that consistently yields viable protoplasts that
are capable of regeneration, development into fertile ga-
metophytes, and production of sporophytes. Further-
more, the protoplasts follow a path of regeneration in
which a primary rhizoid is formed with the first cell divi-
sion, thus resembling spore germination. As in spores,
polarity of regenerating protoplasts is influenced by uni-
directional light and gravity (Edwards, 1996).

The gametophyte generation of C. richardii has many
advantages that make it ideal for spacetlight studies. Its
small size and easy culture mean that large numbers of
individuals can be utilized in a single experiment. Since
germination is initiated by light, samples can easily be
prepared ahead of time, and germination initiated only
after launch with its attendant hyper-# and strong vibra-
tional stimuli. In addition, the primary gravity response,
fixing the polarity of nuclear migration and rhizoid
growth, occurs in a relatively synchronized population
of cells relatively soon after light-induced initiation of
germination, and it can be visualized easily through the
clear spore coat of Ceratopteris with video microscopy
equipment already developed and used in previous shut-
tle experiments (STL-B). The simplicity of these single
isolated cells should facilitate discoveries that will be ap-
plicable to more complicated systems and thus lead to
advances in the study of the effects of gravity and light
stimuli on a wide range of organisms.

Literature Cited

( h.is.ni. R. 1992. Cerutopleris, a model plant for the 90s. Plant Cell
4: 113-1 15.

Edwards, E. S., and S. J. Roux. 1994. Limited period of gravirespon-
siveness in germinating spores of Ceratopteris richardii f/anui 195:
150-152.

Edwards, E. S. 1996. The influence of gravity and light on develop-
mental polarity of single cells of Ceraloplcnx richardii gameto-
phyes. Ph.D. dissertation. The University of Texas at Austin.

Mesland, D. 1992. Possible actions of gravity on the cellular ma-
chinery. .((A: Space Res. 12: 15-25.

Raghmen, V. 1989. Developmental Biology of Fern (Jametuphviex.
Cambridge Univ. Press. Cambridge.

Sack, F. D. 1991. Plant gravity sensing. //;/ Rev Cvlnl 127: 193-
251.

Reference: Bioi Bull. 192: 141-143. (February, 1997)

Probing Rice Gravitropism With Cytoskeletal Drugs
and Cytoskeletal Mutants

PETER NICK1, REA GODBOLE:, AND QI VAN WANG1

llnstitutfiir Biologic II, Schan-lestrasse I. 79104 Freiburg, Germany, and
2Institutfiir Biologie III, Schanilestrasse I. 79104 Freiburg, Germanv

In multicellular organs, phototropic sensing is based
upon gradients in stimulus strength across the entire tis-
sue. The differences in the strength of the gravitational
field between the two flanks of a plant are certainly too
small to be sensed. This implies that each cell, individu-
ally, must align its polarity with the direction of gravity.
As the first step in gravity sensing, the gravitational field
must be translated into a mechanical force that can then
be perceived in the second step. It is generally believed
that statoliths of some kind are the targets of the gravita-
tional field. In plants, sedimentable amyloplasts have
been conceived of, since the turn of the century, as po-
tential statoliths (Haberlandt, 1900; Nemec, 1900).

In principle statolith sedimentation might be trans-
duced into a new alignment of cell polarity in two ways.
Displacement of the statolith could interfere with diffus-
ible molecules, causing a concentration gradient that can
then direct the new cell polarity (proximity mechanism).
Alternatively, the statoliths could be pressed into a struc-
ture that is sensitive to mechanical stress, triggering a sig-
nal cascade that would cause a reorientation of cell po-
larity (pressure mechanism). The observation that the
gravitational response is pressure dependent, even for ac-
celerations where amyloplast sedimentation already
seems to be saturated, favors a pressure mechanism at
least for higher plants (Rawitscher, 1932; Johnsson,
1965; Friedrich and Hertel, 1973). The pressure-perceiv-

This paper was originally presented at a workshop titled The Fit/tire
ol Aanalic Research in Space: Neurobiology, Cellular ami Molecular
Biology The workshop, which was held at the Marine Biological Lab-
oratory. Woods Hole. Massachusetts, from 1 3 to 15 May 1996. was
sponsored by the Center for Advanced Studies in the Space Life Sci-
ences at MBL and funded by the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

ing structure must embody some kind of directionality,
if it is to interact with a transducing mechanism and alter
cell axis and polarity. Cytoskeletal elements, actin mi-
crofilaments and microtubules, are good candidates for
the pressure-perceiving system (Table I): they have a dis-
tinct axis and polarity, and microtubules have repeatedly
been found to be highly sensitive to mechanical stress.
Polymerization and depolymerization of tubulin into
microtubules is one of the few biochemical processes that
have been reported to respond directly to the direction of
gravity (Tabony and Job, 1992).

With these considerations as background, we tried to
assess the role of microtubules and microfilaments in the
gravitropic response of rice coleoptiles. The rice coleop-
tile is highly sensitive to gravity, and the abundance of
amyloplasts can be controlled by submerged growth. The
cuticle of rice is very thin, so the cells are more accessible
to drugs than are coleoptiles from other species. The co-
leoptile grows exclusively by cell elongation, and there
is a wealth of old physiological data about this process.
Accessibility of molecular techniques and a very small
genome also make rice an excellent system for molecular
studies.

Several steps of the gravitropic response chain were
probed with Cytoskeletal drugs (Table I). Ethyl- A-phe-
nylcarbamate (EPC) and oryzalin block tubulin poly-
merization and rapidly eliminate microtubules. Taxol
blocks depolymerization of tubulin, yielding an in-
creased number of microtubules, and cytochalasin D
rapidly eliminates actin microfilaments. The drugs were
found to be effective within 15-30 min after application
to the gravity-sensing tissue of the Japanese rice cultivar
Nihonmasari.

Amyloplast sedimentation, the putative first step of
the response chain (Sack, 1991), was investigated in vi-

141

142

FUTURE OF AQUATIC RESEARCH IN SPACE

Table I

Elements of the plant cyloskelelon an,! •:.',• <//•».!."> '"''

in this sliidv

Cytoskeletal element Co

Drug

Microtuhles

•ulin Elminating:

Ethyl-A'-phenylcarbamate

(EPC)
Oryzalin
Colchicin
Stabilizing:

Taxol
Microfilaments Actin Eliminating:

Cvtochalasin D(CD)

bratome sections after starch was stained with Lugol so-
lution. Sedimentation was not affected by cytochalasin
D, only slightly promoted by microtubule elimination,
and strongly inhibited by taxol. In this system, therefore,
microtubules, but not microfilaments. are in physical
contact with the statoliths. The gravity-induced reorien-
tation of microtubules in the gravity-sensing tissue was
analyzed, after paraformaldehyde fixation, in vibratome
sections by immunofluorescence and confocal laser mi-
croscopy. The usual reorientation from transverse to
longitudinal was inhibited by taxol. Cytochalasin D pro-
moted this reorientation, even in the absence of gravi-
tropic stimulation. The gravitropically induced lateral
transport of auxin was followed by measuring the asym-
metry of radioactively labeled auxin fed to the coleoptile
tip. Lateral transport was inhibited by suppression of
both tubulin polymerization and depolymerization.
This inhibition could be separated from effects on longi-
tudinal transport. Cytochalasin D inhibited auxin
transport per se, yet we failed to observe a specific inhi-
bition of lateral transport. Gravitropic bending was de-
layed by the microtubule drugs (irrespective of whether
they inhibit polymerization or depolymerization of tu-
bulin), whereas cytochalasin D caused a precocious on-
set of bending at a slower rate. It should be emphasized
that gravitropism occurs, although at low efficiency, in
the absence of microtubules and microfilaments. Fur-
thermore, the gravitropic response could be inhibited at
concentrations that would permit phototropism (as in-
ternal control ibr differential growth) to proceed.

These data fa\or the idea that microtubules interact
with the gravity-perceiving system in the rice coleoptile.
It is not enough that microtubules are present to fulfill
this task, they must also be dynamic. Reorientation of
microtubules from transverse to longitudinal is a prereq-
uisite for the establishment of a new (radial) cell polarity
resulting in a lateral transport of auxin. This would ex-

plain why cytochalasin D. promoting such a reorienta-
tion, causes a precocious gravitropic response.

To test this hypothesis further, the gravitropic re-
sponse was analyzed in two cytoskeletal rice mutants. In
the mutant ER31. the reorientation response of micro-
tubules to the auxin signal is interrupted, although auxin
per se is perceived normally, and microtubules are able
to respond to other signals (Nick el at., 1994). In this
mutant, gravity can reorient microtubules, and gravi-
tropism functions with about the same bending rate as in
the wild type, but the bending is delayed by several hours.
In the mutant Yin-Yang, the auxin-induced stimulation
of actin polymerization is disturbed, resulting in the par-
tial decay of actin microfilaments and the formation of
nuclear actin baskets in response to auxin (Wang and
Nick, unpubl. data). In this mutant, gravitropism begins
earlier than in the wild type, but develops at a slower rate.
The gravitropic behavior of the mutant ER31 can be
mimicked by treatment with antimicrotubular drugs; the
gravitropic behavior of the mutant Yin-Yang is similar
to that of the wild type after application of cytochal-
asin D.

The data can be discussed in terms of a physical con-
tact between amyloplasts and microtubules during grav-
ity perception. Microtubules serve as amplifiers for the
mechanical stress exerted by the amyloplasts. This stress
is then perceived by mechanosensitive channels that are
probably located in the endoplasmic reticulum and
causes the establishment of a new cell polarity. For this
polarity shift, reorientation of microtubules seems to be
a time-limiting step. The shifted polarity then causes a
shift of auxin transport in the transverse direction and,
eventually, gravitropic bending. Parallel to this pathway,
a second, slower gravitropic pathway that is independent
of microtubules seems to exist. This pathway might func-
tion independently of auxin. The observation that rice
can respond gravitropically under submerged conditions
(when amyloplasts are small) opens up the possibility
that two completely different gravitropic pathways exist.
The extremely high gravitropic sensitivity of the rice co-
leoptile might have evolved in response to a situation in
which amyloplasts, due to submergence, are small. This
high sensitivity might be the result of two mutually en-
hancing signal chains. We can ask whether the two
chains can be separated under conditions of micrograv-
ity, when only the more sensitive of the two chains con-
tributes to the gravitropic response.

Acknowledgments

The authors thank Wolfgang Michalke and Rainer
Hertel for technical advice and valuable discussions. This
work has been supported by a grant of the Graduierten-

PLANT BIOLOGY

143

kolleg to R.G. and by a fellowship of the Deutsche
Forschungsgemeinschaft (Habilitandenprogram) to P.M.

Literature Cited

Friedrich, t'., and R. Hertel. 1973. Abhangigkeit der geotropischen

Kriimrnung der Chant — rhizoide von den Zentrifugalbeschleuni-

gung. /. P/lan:cnphysii>l. 70: 173-184.
Haberlandt, G. 1900. liber die Perception des geotropischen Reizes.

Bcr Dim-In: Bol Gcs. 18:261-272.
Johnsson, A. 1965. Investigation of the reciprocity rule b\ means of

geotropic measurements. Physio/ Plant 18: 945-967.

INemec, B. 1900. Uber die Art der Wahrnehmung des Schwerkraf-
treizesbei den Pflanzen. Ber Disc/it: Bui GV.v. 18: 241-245.

Nick, P., O. Vatou. M. Furuya, and A. M. Lambert. 1994. Auxin de-
pendent microtubule responses and seedling development are
affected in a rice mutant resistant to EPC. Plan/ J. 6: 65 1-663.

Rawilscher, F. 1932. DerGeotropismusder Pflanzen.Gustav Fischer
Verlag. Jena.

Sack, F. D. 1991. Plant gravity sensing. Inl. Rev Cylot. 127: 193-
252.

Tabony, J., and D. Job. 1992. Gravitational symmetry breaking in
microtubular dissipative structures. Proc. Nail. Acad. Sci. USA 89:
6948-6952.

Reference: Bin/ Bull 192: 144-145. (Fehruan. 1997)

Discussion

Kiss: Do you think that detection
by proximity and detection by pres-
sure are mutually exclusive?

NICK: No, of course not. If you
think about pressure perception, you
must consider stretch-activated
channels. Although such channels
seem to exist in plants, we don't
know anything about them, and it's
easier to think about proximity
mechanisms.

LOMAX: Peter (Nick), it seems
that there's something circular going
on here, because you have the micro-
tubules reorienting in response to
auxin. You also have auxin reorient-
ing in response to microtubules. Are
you saying that all of this is going on
in one cell, or in different cell types?

NICK: The cells I have shown here
are really those that perceive gravity.
This means that they are not the cells
of the epidermis, but below. The
story is more complex because, in
the epidermis where gravity is not
perceived, the auxins induce a reori-
entation of microtubules. So we have
two components. I don't know the
exact relationship, in those gravity-
perceiving cells, between the direc-
tion of auxin transport and the direc-
tion of the microtubules. In regener-
ating tissues, the direction of auxin
flow and the direction of the micro-
tubules are perpendicular to each
other. However, it's difficult to sort
out what's the cause and \\ hat's the
effect. At this point, it's a correlation.
It's also a correlation when you reor-
ient the microtubules by some other
means, either dilatation or with cy-

tochalasin B, before you get gravi-
tropism. At the moment, I do not
understand what the causal relation-
ship is.

QUESTION: In dicots at least, those
cells in which the amyloplasts are
sedimenting are also the cells where
the major polar auxin transport is
taking place. Consequently, there
should be high concentrations there
holding the microtubules in an ori-
entation.

NICK: It is probably in those cells
where auxin is actually transported,
but in the end the epidermis is the
response site.

LOMAX: Stan (Roux), what kind
of light does it take to change the ori-
entation?

Roux: We have used only white
light so far.

Li: Have you isolated any genes by
your differential display?

Roux: We have perfected the
method to the point that we have
been able to eliminate some false
positives. We now have 1 7 candi-
dates; the primers are amplified, and
we are using them to probe Northern
blots during that period.

Li: Peter (Nick), did you check
auxin transport in the late phase for
cytochalasin-treated rice?

NICK: In cytochalasin-treated rice
coleoptiles, auxin transport (both
lateral and longitudinal transport) is
reduced to the same degree by this
drug. We checked auxin transport at

2.5 h after the onset of stimulation,
somewhere in the middle of curva-
ture development.

Li: Do you see some reduction in
polar auxin transport?

NICK: We see some reduction in
the rate of bending.

Li: But not completely blocked for
polar auxin transport.

NICK: No, we can't block polar
auxin transport completely with cy-
tochalasin. We tried various concen-
trations, but could never block more
than about 60%.

MANDOLI: Stan (Roux), your first
graph showed that 20% of the popu-
lation of the Ceratopteris spores are
not responding, or they are not in the
population at all. Could you com-
ment on that? Were you talking
about the time to respond to fixed
polarity determination? The data
come down to 20%- and then stop.

Roux: That means that 20% can
still reorient even at that time point.
Some cells, apparently, do not show
complete fixation of the polarity by
gravity within that window. That
may reflect a little bit of asynchroni-
zation within the population that's
not perfectly synchronized. We are
just looking at a window. If you went
out another 7 or 8 h, you might see
that dropdown.

MANDOLI: I am wondering what
information you have about the ex-
treme variability (mentioned in your
abstract) from spore lot to spore lot,
and the exact timing of the window

144

PLANT BIOLOGY

145

for a population of spores that you
say is very variable. In your model,
you have a polarity determination
window just before nuclear migra-
tion. (Roux: It's always just before
nuclear migration. The sequence
never changes.) Do you see that
when you analyze an individual
spore, and is that invariant from
spore lot to spore lot? I'm confused
by such extreme variability from
population to population.

Roux: On an individual spore
base, the sequence is always the
same. So the fixation always occurs
before nuclear migration. However,
that time of fixation will vary from
spore lot to spore lot. It's related to
ripening. After the spores are har-
vested, they have to ripen for some
period. That phenomenon also oc-
curs in seeds. The longer the ripen-
ing, the shorter the window. We may
actually be looking at synchroniza-
tion of the population. That is, as
ripening lengthens, more and more
of the spores are synchronized so
that they are fitting into a sharper
window. We have also found out re-
cently that we can shrink that win-
dow by temperature modulation,
which is very exciting. If we incubate
them at a low temperature (20°C) for
about five h, and then switch to the
high temperature, we shrink the win-
dow by half. Maybe that's another
way of better synchronizing the pop-
ulation.

MANDOLI: That sounds very sim-
ilar to what Lew Feldman has pub-

lished on changing the phytochrome
response of corn seeds. He imbibed
the seeds in cold and could switch
them from a low to a very low flu-
ence response by the temperature of
imbibition. That might suggest a
membrane property, or such.

Peter (Nick), I have a question on
gravitropism where we have stato-
liths settling. What's really known,
in either of these systems, about the
membranes surrounding the parti-
cles? In amyloplasts, people have
talked for decades about the fact that
the starch granule itself is settling.
Maybe we have been looking at the
wrong thing; maybe it's the mem-
brane that is settling, but as I un-
derstand from John (Kiss), it is very
difficult to purify membranes from
Chant. What would happen if you
took amyloplasts out of plants and
put them into Cham, or purified the
membrane from amyloplasts and
started looking there instead?

NICK: I think that some people
have tried to make antibodies
against the amyloplast surface. I
don't know where this is published,
but there have been attempts like
this, and you can stain amyloplasts
with the antibodies. However, the
problem is that amyloplasts are quite
sticky, and when you really look
carefully with all of the negative con-
trols, you find that you can stain it
with any secondary antibody you
want. It's quite difficult to work with
amyloplasts because they have all
this carbohydrate sticking out. I

think that it would be very difficult
to get some specific response.

MANDOLI: So if the response is
dose dependent, does that help you
at all to correlate the numbers of
statoliths or amyloplasts with the
graviresponse?

NICK: I think it's difficult to use a
biochemical approach on amy-
loplasts. But it will be necessary, of
course.

Kiss: I don't think I can answer
the question any better. Our original
goal was to try to isolate the statolith
vesicles with the membrane. The
statoliths are very dense, and we
could not get a membrane fraction
isolated. It would be very interesting
to look at interactions, possibly with
cytoskeleton, that way. I think Fred
Sack had done some work along
these lines.

SACK: We isolated amyloplasts
but were unable to purify the mem-
brane. The question is. What would
your assay be for a functional pro-
tein? How would you reconstitute
this, and how would you identify
something special?

NICK: I would like to throw one re-
mark into this debate. There is some
old work (quoted in 1932 in a book
by Rawitscher on Geotropismus)
[see Nick abstract] pointing out that
you don't necessarily need the action
of sedimentation; something is al-
ready going on even if the moving
particles just drag or pull on some el-
ement.

Reference: Biol. Bull 192: 1 46- 1 49. (February, 1997)

Mauthner System Discrimination of Stimulus Direction
From the Acceleration and Pressure Components at

Sound Onset

ROBERT C. EATON, AUDREY L. GUZIK, AND JANET L. CASAGRAND

Center for Neuro\eienee. Universitv of Colorado, Boulder, Colorado 80309-0334

When an animal turns away from an aversive stimu-
lus, what are the underlying neurophysiological pro-
cesses that extract the correct sensory information and
produce an appropriate motor output? The goal of our
research program has been to use the Mauthner system
as a model neural network to understand such sensori-
motor processes (Eaton el ai, 1991 ). The Mauthner cells
are a bilateral pair of brain stem neurons. They receive
acoustic and other sensory inputs and connect directly to
spinal motoneurons. Together with other reticulospinal
cells, the Mauthner cells trigger the C-start, a character-
istic escape movement of fishes. This behavior occurs
when the animal is presented with a sudden aversive
stimulus, such as is produced by a predator during an
attack. Previously my laboratory focused on the produc-
tion of the escape movement and the role of the
Mauthner neurons in organizing the descending flow of
motor signals that activate escape. Now, however, we
have an excellent idea of other candidate neurons that
participate in the escape response (Foreman and Eaton,
1993; Lee et a/., 1993; Fetcho el at.. 1995). To further
understand the underlying sensorimotor process, we re-
cently began studying how the Mauthner system com-
putes the direction of sounds so that the animal makes
the correct initial decision to turn left or right away from
the stimulus (Eaton et ai, 1995a). In this paper we pres-

This paper was onginalh presented at a workshop titled The Future
oj Aunalic Research in Space: Neurobiology, Cellular and Molecular
Hinli'iH The workshop, which was held at the Marine Biological Lab-
oratory, Woods Hole, Massachusetts, from 13 to 15 May 1996, was
sponsored by the Center for Advanced Studies in the Space Life Sci-
ences at MBL and funded by the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

ent the rationale, the theoretical background, and some
of our preliminary findings from these recent studies.

The end organs of the inner ear are conserved in ver-
tebrate evolution and convey information about transla-
tional acceleration and gravity (Davis et ai. 1995). Tele-
ost fishes, such as the goldfish, use the otolithic end or-
gans to detect sounds ranging from very low frequencies
to more than 1 KHz (Popper and Fay, 1993). These end
organs are experimentally accessible. Moreover, the sac-
cule of some teleosts has about an order of magnitude
more receptors than the human saccule (Corwin, 1981).
Finally, some central neural networks that receive affer-
ent input from these organs are well studied. One of these
is the Mauthner system.

The Mauthner system consists of the two Mauthner
cells, their sensory afferents. controlling interneuronal
networks, and output cells to motor circuits both in the
brain stem and spinal cord. The Mauthner axons cross
the midline and descend into the spinal cord on the side
opposite the Mauthner soma. When one Mauthner cell
fires, the animal turns toward the side opposite the acti-
vated Mauthner neuron. The other Mauthner cell is in-
hibited from firing.

Fishes can detect the direction of underwater sounds,
particularly those that activate Mauthner-initiated es-
cape responses (Eaton et ai. 1995a). But they probably
do not use the same mechanisms as terrestrial animals,
which obtain their major cues of sound direction by an-
alyzing interaural time and intensity differences. Instead,
as proposed by Schuijf ( 1981) on the basis of the physics
of underwater sound propagation, sound direction can
be determined if the fish compares the phase of the par-
ticle acceleration component and the phase of the pres-
sure component of the sound wave. Experimental evi-
dence supports this theory (Buwalda et ai, 1983). We

146

NELIROBIOLOGY/SENSORY BIOLOGY

147

have recently shown that the physical basis of this phase
model is most readily apparent for the sounds that the
Mauthner system is specialized to detect; that is, the on-
set of a sound that originates relatively close to the fish
(Guzik and Eaton, 1994; Eaton et at., 1995a). This is be-
cause the Mauthner-initiated escape response is adapted
to detecting ambush attacks from predators that strike
from within a short distance.

Let us assume that the predator is on the left side of
the fish and lunges forward to bite. This causes particle
acceleration from left to right and positive sound pres-
sure. But the source location cannot be computed from
the acceleration component alone. Particles moving left
to right conk! be caused by a source beginning with com-
pression (positive pressure) on the left, or a source begin-
ning with rarefaction (negative pressure) on the right, as
would be caused by a predator on the right trying to suck
the prey into its mouth. This ambiguity is resolved by
referring to the pressure component.

For instance, consider the phase polarity from the per-
spective of the left Mauthner cell. Left-to-right accelera-
tion will be registered as a positive phase relative to this
cell. If acceleration is from left to right, and the source is
on the left, pressure will be increasing. The left Mauthner
cell will detect the phase of these two components as both
being positive, and thus it should fire. This would cause
the animal to correctly turn to the right.

Alternatively if the acceleration were from left to right,
but the pressure was decreasing, this could be caused
only by a sound source on the right beginning with rar-
efaction (negative pressure). Thus, the acceleration and
pressure components are of opposite phase (or polarity),
and the left Mauthner cell should not fire. Using these
conventions, the components from the perspective of the
right Mauthner cell would both be positive, or in-phase,
and the cell should fire, causing the animal to turn cor-
rectly to the left.

In the most general terms, whenever the initial com-
ponents of the aversive sound stimulus are in-phase with
respect to one Mauthner cell (either both positive or both
negative), that cell should fire; and whenever they are
out-of-phase, the cell should not fire. This is equivalent
to the logical operator known as the equivalence func-
tion, EXCLUSIVE-NOR or XNOR (Guzik and Eaton,
1 994). Whenever its inputs are the same, it is ON; when-
ever they are different it is OFF (Guzik and Eaton. 1 994;
Eaton et a/., 1995a). This is the problem that must be
solved by the two Mauthner cell networks to detect the
direction of impulse sounds created by predators.

How might the Mauthner system perform XNOR and
compute sound direction? The acceleration component
is detected as a translational motion of the body, which
has roughly the same density as the water. The end or-
gans of the inner ear contain otoliths that have high den-

sity and act as inertial devices to cause a shearing motion
on the underlying sensory epithelium whenever the body
accelerates in phase with the water. In the goldfish, ex-
perimental evidence shows that these accelerations are
detected by all three end organs of the inner ear, saccule,
utricle and lagena (Fay, 1984), though it has been argued
that the utricle and lagena are most important for this
detection (Schellart and Popper, 1992). The pressure
component is transduced by the swimbladder, a gas-
filled chamber in the abdomen. The swimbladder
changes in size as a result of changes in the impinging
sound pressure field. These vibrations are conveyed
through a series of ossicles to the fluids of the inner ear
where they vibrate the otoliths, most likely in the saccule,
and activate the sensory epithelium of the inner ear. This
leads to the firing of the acoustic afferents that activate
the Mauthner cells (Canfield and Eaton, 1990).

We have performed neurocomputational simulations
to give insights into how the underlying neurons might
be implementing the XNOR function. If the Mauthner
cell can solve XNOR. it must receive all four combina-
tions of sensory afferents (positive and negative pressure,
left-to-right and right-to-left acceleration) but fire only
to those combinations appropriate for sound from the
"correct" direction. This would require a nonlinear in-
teraction of these inputs, perhaps through an appropriate
spatial arrangement of synapses on the dendritic mem-
brane.

Alternatively, the XNOR solution might emerge from
interactions of the Mauthner network as a whole. Math-
ematically. XNOR can be solved by an indefinite num-
ber of equivalent circuits made of various types of logical
operators that together perform the overall XNOR func-
tion. Thus, each neural element could be performing a
subsidiary function that contributes to the overall
XNOR logic. Our simulation studies show that the PHP
neurons may be playing an essential role in this direc-
tional computation (Eaton el at., 1995a).

The PHP cells were discovered and extensively inves-
tigated by Faberand Korn (Faberand Korn, 1978; Faber
et a/., 1991), who showed that these neurons operate in a
feed-forward manner to regulate the firing threshold of
the Mauthner cell to sound. These cells are strongly acti-
vated by low-level acoustic stimulation and strongly in-
hibit the Mauthner cell. However, at higher levels of ac-
tivation appropriate for eliciting escape, the PHP cells
saturate, whereas the Mauthner neuron becomes pro-
gressively excited and eventually exceeds the PHP cell
inhibition, thus leading to an action potential (Faber et
al.. 1991).

How might the PHP cells be performing part of the
XNOR function? In the simplest simulations, these cells
are divided into two populations, both synapsing on each
Mauthner cell (Eaton el al.. 1995a). Each population of

148

FUTURE OF AQUATIC RESEARCH IN SPACE

PHP cells is turned on by the two, out-of-phase, pressure
and acceleration combinations that should not activate
the associated Mauthner ceil on that side of the brain.
Thus, the left Mauthner cell would be turned off by PHP
cells whenever the sound was originating on the right,
regardless of whether it began with compression or rar-
efaction. This would allow the right Mauthner cell to fire
off its pressure input, while the right PHP cell popula-
tions would not be activated. In these simulations, the
PHP cells act as AND gates. Other more complex simu-
lations more closely duplicate the feed-forward dynam-
ics of Faber and Korn. In these models, the PHP cells
perform an OR function (Guzik et al., 1995).

We are currently doing neurophysiological and behav-
ioral experiments to evaluate the XNOR model. We
have devised an experimental test chamber based on a
design of Fay (Fay, 1 984); ours produces accelerations at
90 degrees to the long axis of the fish. In the center of this
chamber is a sound pressure null; a fish at this location
perceives only the acceleration component. We can arti-
ficially control the polarity of sound pressure at the null
point by producing a sound pulse in the air above the
tank. Thus, we can independently control the initial
phase of the acceleration, and the sound pressure com-
ponents, and thereby artificially influence the apparent
direction of the sound stimulus. In preliminary behav-
ioral trials, the fish escapes in the directions predicted by
the phase model (Eaton et al., 1995b). In our trials, the
ratio of amplitude of acceleration to pressure is higher
than for typical monopole or dipole sound sources.

The test chamber can also be used for fishes restrained
for neurophysiological recording from the Mauthner sys-
tem neurons. So far, we know that the Mauthner cell is
widely tuned to sound pressure frequencies from 100 to
2 KHz (Casagrand et al., 1995). We are currently inves-
tigating Mauthner responsiveness to acceleration alone,
and to combinations of acceleration and pressure, to de-
termine whether the Mauthner cell itself performs the
XNOR function. If the Mauthner cell performs XNOR,
this should be detectable as differences in amplitude or
timing of EPSPs evoked to different acceleration and
pressure combinations of our artificial sounds. If it does
not, we would expect to see differences in the timing or
amplitude of the EHP, the extracellular field potential
generated by the PHP cells. This will indicate that the
PHP cells are solving XNOR.

Although ou; :ients are still in the preliminary

stage, enough is alre known about the Mauthner sys-
tem so that we can use it to understand how a computa-
tional problem like the phase model is being solved in
neurophysiological terms with identified cells. In many
types of experiments involving central auditory or ves-
tibular processing, the relevance of a neuron's response
to a particular component of the stimulus is often diffi-

cult to know. The Mauthner system has important ad-
vantages. We have a good physical theory of the problem
being solved by the system. Also, we can access the out-
put state of the system both neurophysiologically and be-
haviorally. We therefore know that the system has inter-
preted the stimulus as having a particular salience; a
predator attacking from a certain location relative to the
body axis of the fish. This makes possible concrete state-
ments about the real importance of the underlying neu-
rophysiological events. We therefore believe that such
work may provide new insights into understanding basic
principles of central vestibular and auditory functions.

Acknowledgments

Work described in this paper was supported by grants
from NIH and ONR.

Literature Cited

Bimalda, R. J. A., A. Schuijf, and A. D. Hawkins. 1983. Discrimina-
tion by the cod of sounds from opposing directions. J. Camp. Phys-
/<>/. .! 150: 175-1X4.

Canfield, J.G., and R. C. Ealon. 1990. Swimhladder acoustic pres-
sure transduclion initiates Mauthner-mediated escape. Nature347:
760-762.

Casagrand, J. 1,., A. L. Guzik, and R. C. Eaton. 1995. Frequency de-
pendence of auditory PSPs in the goldfish Mauthner cell. Soc. Ncu-
ro.fci Al-nir 21:399.

Corwin, J. T. 1981. Postembryonic production and aging of inner ear
haircellsin shark./ Cinnp. Ncurtil. 201: 541-553.

Davis, J. G., J. C. Oberholtzer, F. R. Burns, and R. I. Greene. 1995.
Molecular cloning and characterization of an inner ear-specific
structural protein. Science 267: 1031-1034.

Ealon, R. C'., R. DiDomenico, and J. Nissanov. 1991. Role of the
Mauthner cell in sensorimotor integration by the brain stem escape
network. Brain Bchav Evol. 37: 272-285.

Eaton, R. C., J. G. Canfield, and A. L. Guzik. 1995a. Left-right dis-
crimination of sound onset by the Mauthner system. Brain Bchav.
Evol 46: 165-179.

Eaton, R. C., A. L. Guzik, and J. L. Casagrand. 19956. The neural
mechanism for directional escape in the goldfish./ .\cous. Soc Am
98: 2940.

Eaber, D. S. and H. Korn. 1978. Electrophysiology of the Mauthner
cell: basic properties, synaptic mechanisms and associated net-
works. Pp. 47-131 in Ncnrobiology of the Maulhncr Cell. D. S.
Faber and H. Korn eds. Raven Press. New York.

Faber, D. S., H. Korn, and J.-\V. Lin. 1991. The role of medullary
networks and presynaptic membrane properties in regulating
Mauthner cell responsiveness to sensory' excitation. Brain Bchav.
Evol 37: 286-297.

Fay, R. R. 1984. The goldfish ear codes the axis of acoustic particle
motion in three dimensions. Sru>/;iic225: 951-954.

Fetcho, J. R., V.-H. Kao, and D. M. O'Malley. 1995. Functional
roles of serially homologous reticulospinal neurons studies by in
I'/voconfocal calcium imaging in the zebrafish. Soc AViirnvtv. Ab.tlr.
21:687.

Foreman, M. B., and R. C. Eaton. 1993. The direction change con-
cept for reticulospinal control of the goldfish escape. / Ncurnxci.
13:4101-41 13.

NEUROBIOLOGY/SENSORY BIOLOGY 149

Guzik, A. L., and R. C. Eaton. 1994. Directional hearing by the Schellart, N. A. M., and A. N. Popper. 1992. Functional aspects of

Mauthner system. Adv \enrallnl ' /Voa-.v.v . Syxl 6: 574-58 I. the evolution of the auditory system of actinopterygian fish. Pp.

Guzik, A. L., R. C. Eaton, and D. \V. Mathis. 1995. A backpropaga- 295-322 in The Kvolutionary Biology <>l Hearing. D. B. Webster.

tion model of the Mauthner system. Sue. Netirosci. Ahslr. 21: 147. R. R. Fay, and A. N. Popper, eds. Springer, New York.

Lee, R. K. K., R. C. Eaton, and S. J. /.ottoli. 1993. Segmental ar- Schuijf, A. 1981. Models of acoustic localization. Pp. 267-310 in

rangement of reticulospmal neurons in the goldfish hindbrain. ./. Hearing and Sound Communication in F/.v/it'.v. W. N. Tavolga.

Comp. N enrol 327: I - 1 8. A. N. Popper, and R. R. Fay, eds. Springer, New York.
Popper, A. N., and R. R. Fay. 1993. Sound detection and processing

by fish: critical reviews and major research questions. Brain lielntv.

Erol. 41: 14-38.

Reference: Bio/. Bull 192: 150-153. (February. 1997)

Imaging Neural Activity With Single Cell Resolution in
an Intact, Behaving Vertebrate

JOSEPH R. FETCHO. KINGSLEY J. A. COX. AND DONALD M. O'MALLEY

Department ofNeurobiology and Behavior, Stale University of New York at Stony Brook,

Stonv Brook, New York 11794-5230

Introduction

Most behaviors are produced by activity in popula-
tions of neurons, but the physiological approaches com-
monly used to study neural circuits allow the activity of
only one or very few neurons to be monitored at a time.
What is needed are approaches that allow the monitoring
of activity in a group of cells — preferably a large group —
while simultaneously permitting the identification and
the recording of activity from each cell. Progress along
these lines has been made with the use of electrode arrays
(Wilson and McNaughton, 1994). An alternative, very
promising approach — i.e.. imaging — offers an easy de-
termination of both the activity and the identity of cells
(Wu et al. 1994; O'Donovan el al.. 1993). In this
method, the neurons are labeled with an indicator dye
that signals their activity, and the dye is then used to
monitor the cells that are active during a particular be-
havior. The ideal situation would be one in which a pop-
ulation of neurons could be labeled and their activities
observed with single-cell resolution in an intact, behav-
ing animal. This ideal is difficult to achieve with verte-
brates because most of them arc opaque, so the neurons
cannot be seen in the intact animal. Notable exceptions
are the larvae of many fishes, which are transparent and
thus especially suitable for imaging neurons. We have
developed approaches in which a fluorescent calcium in-
dicator is used to monitor neural activity in intact fish.
Neurons that are labeled with the indicator increase in

This paper was originally piesented at a workshop titled The Finnic
nl Aquatic Research in ,S/u< «.;M. Cellular and Muk'culai

Binlngy. The workshop, which was IvM at the Marine Biological Lab-
oratory, Woods Hole. Massachusetts, from 13 to 15 May 1996, was
sponsored by the Center for Advanced Studies in the Space Life Sci-
ences at MBL and funded by the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

brightness due to the calcium influx associated with their
activity. Thus, neural activity during behavior can be de-
termined by watching for the increase in brightness in
particular cells (O'Donovan el al.. 1993; Fetcho and
O'Malley, 1995; Fetcho et al.. 1995: Cox and Fetcho,
1 996; O'Malley el al., 1 996). The transparency of the fish
has allowed us to image activity in neuronal populations
with single-cell resolution in an intact vertebrate. Our
work so far has focused on the neuronal populations in-
volved in the escape behavior. This behavior has been
the subject of intensive study with single-cell recording
techniques in the past (Faber and Korn. 1978; Fetcho
and Faber, 1988; Fetcho. 1992), but the ability to moni-
tor groups of neurons is providing a new view of the re-
lationships between neural activity and the escape be-
havior.

Labeling

Central neurons were labeled with a calcium indicator,
calcium green dextran. Neuronal activity causes an in-
flux of calcium through voltage-dependent calcium
channels, so the calcium levels sensed by the indicator
can be used as a measure of activity. The neurons in lar-
val, post-hatching zebrafish (Danio rerio) were labeled
in two ways. In the first, the indicator was injected into
muscle or into the central nervous system, backfilling the
neurons of interest. Calcium green dextran is taken up
by the damaged processes of neurons, resulting in both
retrograde and anterograde labeling (O'Donovan et al.,
1993; Fetcho and O'Malley, 1995). This approach led
to a very robust labeling of populations of neurons. The
neurons could be observed and individually identified in
the spinal cord and in the brain of the living fish either
by confocal microscopy or enhanced video imaging. The
labeling was very similar to that seen in material that had

150

NEUROBIOLOGY/SENSORY BIOLOGY

151

been cleared and stained with horseradish peroxidase
(HRP). We could see many of the individually identifi-
able neurons of zebrafish (Bernhardt el ul.. 1990), includ-
ing the three primary motoneurons (rostral, middle, and
caudal — RoP. MiP, and CaP, respectively) located in
spinal segments, and the Mauthner cell and its two seri-
ally homologous neurons (MiD2cm, MiD3cm) located
in the hindbrain. Other identifiable spinal neuronal types
(e.g., secondary motoneurons, Rohon Beard cells, dorsal
root ganglion cells, circumferential descending (CiD)
cells, commissural posterior ascending (CoPa) cells) and
brain neurons (e.g., vestibulospinal neurons, nucleus of
the medial longitudinal fasciculus) were also evident.
The relatively high fluorescence of calcium green at basal
calcium levels produced well-labeled dendrites and ax-
ons. The quality of the labeling, and the high resolution
and optical sectioning ability of the confocal microscope,
allowed the collection of serial optical sections through
the neurons of interest. Three-dimensional reconstruc-
tions of the cells from live fish were produced from opti-
cal sections using the VolVis program (Sobierajski el ul..
1995). The reconstructions allowed a detailed examina-
tion of the neurons whose physiology was being studied
optically. This allowed the unambiguous identification
of the cells and also provided information about the re-
lationships of the axons and dendrites of the imaged neu-
rons.

Although labeling by injection into the larval fish pro-
duced well-labeled neurons, the injection may in some
instances disrupt portions of the neural circuits of inter-
est. To circumvent this problem, we also labeled neurons
by injecting calcium green dextran into blastomeres of
one-, two-, four-, or eight-celled embryos. In this proce-
dure, the indicator is labeling the progeny of the blasto-
mere, just as lineage tracers are used in developmental
studies. The fish were then raised to post-hatching larval
stages. These injections produced larval fish in which a
large fraction of cells contained the calcium indicator,
from one-eighth to all of the cells should be labeled, de-
pending on the number of blastomeres that had been
formed at the time of injection. The proportion of la-
beled cells, in our experience, was roughly consistent
with this expectation, although the intensity of the label-
ing of individual cells varied. The confocal microscope
effectively removed fluorescence that was out of focus,
and allowed visualization of the labeled cells in the intact
animal. This approach has the advantage of not disrupt-
ing the neural circuitry, at least so far as we can tell. The
lack of damage is evidenced by our ability to raise such
blastomere-labeled fish to adulthood after imaging their
neural activity. The labeling produced by blastomere in-
jections is not, however, as intense as that by injection
into larvae, so the type of each cell is more difficult to
identifv. Nevertheless, we have been able to observe

identifiable classes of spinal neurons (e.g.. Rohon Beard
cells) and some neurons, such as olfactory epithelial cells,
are very brightly labeled (Cox and Fetcho, 1996).

Responses of Neurons

We initially used electrical stimuli to determine
whether the indicator was responsive in the neurons and
to demonstrate that we could resolve differential re-
sponses in adjacent cells (Fetcho and O'Malley, 1995).
Motoneurons in larval fish, typically 3-5-days old, were
backfilled by injections of calcium green dextran into
muscle and then, a day or more later, were embedded
in agar and observed with a Biorad MRC 600 confocal
imaging system usingaZeissIM 35 inverted microscope.
The fish remain healthy in the agar, probably because
they depend on cutaneous respiration at these early
stages. An electrical stimulus applied to the skin overly-
ing the muscle near the site of calcium green injection
could increase the fluorescence of the labeled motoneu-
rons. A single stimulus typically produced increases of
5%-10%; additional stimuli, applied in rapid succession,
further increased the fluorescence (over 100% change for
a train of 40 stimuli in some cells) consistent with the
accumulation of calcium expected upon repeated activa-
tion of the motoneurons. By varying the strength of the
electrical stimulus, we could detect differential responses
in adjacent motoneurons, with one cell showing a large
increase in fluorescence (e.g., 70%) and an immediately
adjacent one showing none. Thus, we could resolve
differences in adjacent cells which were roughly 10 ^m
diameter and only about 1 /jm apart.

Having confirmed that the cells behaved as expected
to electrical stimulation, we examined their responses
during escapes (Fetcho and O'Malley, 1995). Escape be-
haviors were elicited by an abrupt touch applied to the
head or tail with a piezoelectric tapping device. In the
escape behavior, a touch on one side of the head leads to
a massive, rapid C-bend to the opposite side of the body,
which turns the fish away from a threatening stimulus.
When we imaged spinal motoneurons on the side of a C-
bend during the escape, we found a massive activation,
with every cell that we imaged responding during the es-
cape bend. In individual cells, the increases in fluores-
cence ranged from 16% to 151%. The activated moto-
neurons included the larger primary motoneurons, as
well as the smaller secondary ones. Most of our observa-
tions were obtained with low temporal resolution, usu-
ally 400 ms for each image. However, we could achieve
resolutions on the millisecond time scale of synaptic
events by taking advantage of the line-scanning ability of
the confocal microscope. This allowed us to look at one
line through a group of cells every 2 ms, and revealed a
svnchronous activation of the motoneurons. The pri-

152

FUTURE OF AQUATIC RESEARCH IN SPACE

mary motoneurons innervate extensive regions of the ax-
ial muscle and are known to be important in escapes,
based upon evidence that they receive a monosynaptic
input from the reticulospinal Mauthner cell that initiates
the escape (Myers et ai. 1986; Westerfield el a/.. 1986;
Fetcho and Faber, 1988; Liu and Westerfield, 1988). The
secondary motoneurons are more numerous and their
role in escapes has been less clear. Our data indicate that
the motoneurons activated in escapes include both pri-
mary and secondary pools (Fetcho and O'Malley, 1 995 ).
We observed a similar massive activation of cells in ven-
tral cord that had been labeled by blastomere injections
and imaged in the larval fish (Cox and Fetcho, 1996).
The identity of these cells in the blastomere-labeled fish
was not certain because of the less complete filling of cells
from blastomere injections. But, based upon their loca-
tions, they were most likely motoneurons. The observa-
tions from backfills and blastomere injections are consis-
tent with the massive activation of muscle that occurs
during the escape bend. The most powerful C-bends oc-
cur in response to stimulation of the head (Eaton et a/.,
1984; Foreman and Eaton, 1993). These bends are the
most forceful motor behavior produced by the fish, so
such a large recruitment of motoneurons is not sur-
prising.

We began our imaging studies with motoneurons, be-
cause enough was known about their activity patterns
that we could use previous data to evaluate the reliability
of the approach. More recently we have begun to study
other, less well understood systems, including the olfac-
tory system, reticulospinal neurons, and spinal interneu-
rons. Our studies of reticulospinal neurons, for example,
have demonstrated that the brain as well as the spinal
cord, can be examined by in vivo imaging. We have used
the approach to evaluate predictions about the pattern of
activation of the Mauthner cell and its serially homolo-
gous neurons in hindbrain ( Fetcho et nl.. 1995; O'Malley
et ai. 1996). The hindbrain consists of a series of seg-
ments that contain repeated, morphologically similar
neurons in successive segments. One of these sets of seri-
ally repeated cells includes the reticulospinal Mauthner
cell (in hindbrain segment 4), which is known to play a
role in escapes, and two reticulospinal cells (MiD2cm,
MiD3cm in segments 5 and 6 respectively) that are very
like the Mauthner cell in their dendritic structure and
axonal projection. The functional role of the Mauthner-
like cells was unknown, but Foreman and Eaton ( 1993)
predicted that the observed variability in the strength of
the C-bend, in response to sensory stimuli at different
locations relative to the fish, might be determined in part
by variability in the activation neurons in the set (includ-
ing the Mauthner cell, MiD2cm, and MiD3cm). We
have examined and confirmed their predictions by
calcium imaging, which has allowed us to study the func-

tion of a set of cells that has been difficult to study with
more conventional techniques (Fetcho et ai, 1995;
O'Malley et a/.. 1996). Our observations suggest that the
hindbrain consists of serial sets of functionally similar
neurons that may act together in various combinations
to produce behavioral variability.

Conclusions

Although still in its infancy, the use of calcium im-
aging in the larval zebrafish offers the possibility of study-
ing the activity of populations of neurons with single-
cell resolution anywhere in the brain or spinal cord of an
intact, behaving vertebrate. This will provide important
information about the relationships between the activity
of neuronal populations and behavior in the normal an-
imal, but there are other advantages of the zebrafish as
well. Thousands of mutant lines of zebrafish have now
been generated by saturation mutagenesis, including
many with sensory and motor deficits (Mullins et ill.,
1994; Driever et a/.. 1994). The calcium imaging ap-
proach should prove useful in analyzing the functional
deficits of these mutant lines. The transparency of the
fish will also facilitate the optical killing of particular,
fluorescently labeled neurons. The behavior and neuro-
nal activity of the fish can be studied both before and
after particular neurons or sets of neurons have been
killed, allowing a more causal link between neurons and
behavior. It may also be possible to use local optical un-
caging of neuroactive substances to activate or inhibit
neurons. This would allow noninvasive perturbations of
activity. The combination of imaging activity, genetics,
and cell ablation will permit a powerful analysis of the
neural basis of behavior in a vertebrate.

Acknowledgments

Supported by NIH NS26539, NS-09113, and the
Howard Hughes Medical Institute.

Literature Cited

Bcrnhardt, R. R., A. B. Chitnis, L. Lindamer, and J. Y. Kuwada. 1990.

Identification of spinal neurons in the embryonic and larval zebra-
fish. J. Comp. Neiirol. 302: 603-616.

Cox, K. J. A., and J. R. Fetcho. 1996. Labeling blastomeres with a
calcium indicator: a non-invasive method of visualizing neuronal
activity in zebrafish. J. Neitnixei. Mel hods 68: 1 85- 141.

Driever, \V., D. Stemple, A. Schier, and S. Solinica-Krezel. 1994. Ze-
brafish: genetic tools for studying vertebrate development. Trends
(Jem-/. 10: 152-159.

Eaton, R. C., J. Nissanov, and C. M. Wieland. 1984. Differential ac-
tivation of Mauthner and non-Mauthner startle circuits in zebra-
fish: implications tor functional substitution. J. COIH/>. Physiol. 155:
8 1 3-820.

Kaber, D. S., and II. Korn, eds. 1978. Neurobiology oj the Mauthner
Cell. Raven Press. New York.

NEUROBIOLOGY/SENSORY BIOLOGY

153

Fetcho, J. R. 1992. Excitation of motoneurons by the Mauthncr
axon in goldfish: complexities in a "simple" reticulospinal pathway.
J. .\cun>i<iiy.<ii<>/. 67: 1574-1586.

Fetcho, J. R., and D. S. Kaber. 1988. Identification of motoneurons
and interneurons in the spinal network for escapes initiated by the
Mauthner cell in goldfish. J. \ewvxci. 8: 4 1 92-42 1 3.

Fetcho, J. R.. and D. M.O'Mallej. 1995. Visualization of active neu-
ral circuitry in the spinal cord of intact zebralish. J Neurophysiol.
73: 399-406.

Fetcho, J. R., Y.-H. Kao, and D. M. O'IMalley. 1995. Functional
roles of serially homologous reticulospinal neurons studied by //;
nvo confocal calcium imaging in zebrafish. Sin: Neuroxei. Ahslr.
21:687.

Foreman, M. B., and R. C. Eaton. 1993. The direction change con-
cept for reticulospinal control of goldfish escape. / Ncurosci. 13:
4101-4113.

Liu, D. \V., and M. \\esterfield. 1988. Function of identified moto-
neurons and coordination of primary and secondary motor systems
during zebrafish swimming. J Physiol. (Limit.) 403: 73-89.

Mullins, M. C., M. Hammerschmidt, P. MarTter, C. Nusslein-Volhard.
1994. Large-scale mutagenesis in the zebrafish: in search of genes
controlling development in a vertebrate. Curr Biol 4: 189-202.

Myers, P. Z., J. S. Eisen, and M. \\estertield. 1986. Development
and axonal outgrowth of identified motoneurons in the zebrafish. /
,\eim>sei. 6: 2278-2289.

O'Donovan, M. J., S. Ho, G. Sholomenko, and \V. Yee. 1993. Real-
time imaging of neurons retrogradely and anterogradely labeled
with calcium-sensitive dyes. J. ,\ciir»xci. Methods 46: 91-106.

O'Malley. D. M., Y.-H. Kao, and J. R. Fetcho. 1996. Imaging the
functional organization of zebrafish hindbrain segments during es-
cape behaviors. Neuron 17:1 145-1 155.

Sobierajski, L. M., R. S. Avila, D. M. O'Malley, S. Wang, and A. E.
kaufmann. 1995. Visualization of calcium activity in nerve cells.
lEEEComp. (jruph. .I/'/1/ 15: 55-61.

Westerfield, M., J. V. McMurray, and J. S. Eisen. 1986. Identified
motoneurons and their innervation of axial muscles in the zebra-
fish. J. \eumsei. 6: 2267-2277.

Wilson, M. A., and B. L. McNaughton. 1994. Reactivation of hippo-
campla ensemble memories during sleep. Science 265: 676-679.

\Vu, J.-Y., L. B. Cohen, and C. \. Falk. 1994. Neuronal activity dur-
ing different behaviors in Aplysiu: a distributed organization? Sci-
ence 263: 820-823.

Reference: Biul. Bull 192: 154-156. (February, 1997)

Discussion

KAWASAKI: It appeared that the
image you showed lasted for a sec-
ond or so. Does this reflect the time
course of the calcium concentration?

FETCHO: That's a good question.
The recovery time is slow: the rise is
very fast, but the recovery time takes
many seconds. This time course oc-
curs in a number of different cells.
There's quite a bit of debate about
the basis for that time course. Some
people think that the dye might be
affecting the time course of the
calcium increase. We think that the
calcium returns slowly to resting lev-
els; but this is a very difficult issue to
resolve, and one that is not my main
focus. I just want an indication of
which cells are active.

WIEDERHOLD: How many action
potentials have been fired in the
Mauthner cell to get that much of a
rise?

FETCHO: As far as we know, in
these teleost fishes, the Mauthner cell
fires only a single action potential.
That's a big change that we see. It
turns out there's some variability in
the size of the observable changes. In
the course of an experiment, the cell
changes the way it responds. It will
start out with relatively modest re-
sponses and then, with repeated tri-
als, will convert to these larger re-
sponses. We think that there are
some interesting things going on,
perhaps with respect to intracellular
calcium being released, perhaps
from stores.

BAKER: I have a question to either
of you. First, I totally reject the hy-

pothesis that there are iterative, ho-
mologous, reticular spinal neurons
in the hindbrain. For three or four
hundred million years, each hind-
brain segment has had an indepen-
dent evolutionary history. Don't you
think that your evidence demon-
strates that the reticulospinal neu-
rons are not iteratively homologous,
because they each exhibit their own
physiological sensitivity, properties,
and projections? Wouldn't it be
more useful to think about each of
these segments containing reticulo-
spinal neurons having their own in-
dependent role in the escape behav-
ior?

FETCHO: The issue is the distinc-
tion between homology and iterative
homology; what was the origin of
these cells? Surely they arose a long
time ago, and they have been inde-
pendently evolving for a long time.
However, they might at first have
been the result of a duplication event
that (to me) would represent a serial
homologous type of organization. I
would agree that they have indepen-
dently evolved for a long time. This
certainly is the case.

BAKER: Joe (Fetcho), Model T's
can be duplicated, but they will
never become Cadillacs. In other
words, duplication and divergence
occurred so long ago that iterative
homology is not a useful physiologi-
cal tool.

FETCHO: They have both morpho-
logical similarities and reasonable
functional similarities as well, at
least to the extent that thev seem to

be involved in the escape behavior. It
remains to be seen whether the ho-
mologues are involved in other
things as well. We haven't explored
that very much.

EATON: I don't know if either one
of us wants to get caught in a debate
about homology and analogy. From
my point of view, I mean only what
has been stated in the previous pa-
pers that have come before us: these
particular cells have similar genetics,
developmental history, immunohis-
tochemistry, and so on. That doesn't
have to imply that there aren't going
to be functional differences between
the cells as well. In fact, we would ex-
pect that once the cells are deter-
mined, they will go off on different
paths and different features of their
function will be emphasized. That's
as far as I would like to go on that.

BAXTER: I wonder if you have
looked at the ability of the Mauthner
circuit to adapt. For example, if you
turn the animal upside down, must
it now reverse its reactions?

EATON: That's a very good ques-
tion. We don't know anything about
that yet.

BAXTER: Some of the synapses in
your circuit were electrotonic, and
one doesn't typically think of those
as being very plastic.

EATON: The inputs to the
Mauthner cell from the inner ear fi-
bers are mixed chemical-electrotonic
junctions. The important point here
is that the fish is using its vestibular
organs to hear. Or, to put it in the

154

NEUROB1OLOGY/SENSORY BIOLOGY

155

other perspective, humans don't use
their vestibular organs to hear in the
same way. As far as I know, there's
not much known about how the fish
discriminates between those two
components — the gravitational field
and the acoustic particle accelera-
tion— at the same time. What hap-
pens then, when you disturb that
process by putting the fish in zero
gravity or by turning it upside down
in a gravitational field, would be
most interesting to evaluate. I don't
think anyone has started to look at
that, but it's certainly an issue that
could be evaluated.

BASS: Do those PHP cells encode
sound intensity? Joe (Fetcho) are
you able to visualize those cells?

EATON: We don't know that they
encode sound intensity perse. In the
only very controlled experiments,
electrical shocks were applied to the
eighth nerve. As the intensity in-
creased, the nerves fired more ro-
bustly. We don't know how they re-
spond to controlled sound pulses.
None of that has been evaluated.

FETCHO: The short answer is that
we don't know whether we can visu-
alize the PHP cells. We think that we
can look at any neurons anywhere in
the brain: it's just a matter of getting
the dye into them without messing
up the circuits that you're interested
in. We haven't tried to label the PHP
cells.

MORRIS: I want to get back to this
business of up and down in your ex-
periments. I was assuming that the
fish was lying on its side — in the con-
focal experiments anyway — while
you were stimulating and imaging. Is
this correct?

FETCHO: That depends on the ex-
periments. In the reticulospinal ex-
periments, the fish is actually lying
on its back, so that we are looking
through the entire head to see the
cells at the bottom of the brain.
That's why we can look anywhere in

the brain; basically we are looking
through the whole brain to see those
images. When we are looking at spi-
nal neurons, we look from the side
because it's very transparent from
that angle. There's a lot of pigment
at the top of the fish.

BARLOW, R.: Joe (Fetcho), I
would like to get a better feel for
what you were seeing in your
calcium measurements. It appeared
that there is a signal without stimula-
tion. Is there background activity—
that is, free intracellular calcium
without stimulation?

FETCHO: That's right.

BARLOW, R.: And when you stim-
ulate, there's an increase in calcium?

FETCHO: That's right. Calcium
green dextran has fairly good fluo-
rescence at basal calcium levels,
which is very important for us. be-
cause we would like to know what
cells we are looking at. If you can't
see them at rest, then you might have
trouble.

BARLOW, R.: Where is the action
potential fired with regard to the in-
creased free calcium in the cell?
Where's the axon hillock region?
Does your imaging data make sense
as to where the major conductance
changes are taking place?

FETCHO: In goldfish, where it's
best understood, the spikes are initi-
ated right where you might expect—
at the initial segment region. You get
a fairly substantial depolarization of
the soma as well, but it's passively in-
vaded by the spike. We don't know
much about the distribution of
calcium channels in the cell.

In the case of the auditory inputs,
they come in on the lateral dendrite.
The big lateral dendrite that goes out
to the ear is passive, and postsynaptic
potentials are passively conducted to
the initial segment. The spikes origi-
nate at the initial segment right near
where the axon goes off, and then it's

passively propagated back into the
soma.

BARLOW, R.: But you are detect-
ing calcium changes mainly in the
cell body.

FETCHO: Well, that's all we've
looked at. We haven't explored
what's happening out in the den-
drite. We don't see much in the cell
body, unless the cell fires an action
potential. We can do thresholding
experiments where we are stimulat-
ing right near the threshold for es-
capes. Under those conditions, we
would expect a massive subthreshold
synaptic input to the cell. In cases
where the fish doesn't carry out an
escape, you don't see any change.
You only see the change when you
get the escape — when the Mauthner
cell has fired a spike.

COMMENT: One possibility may
be that the channels in the larval
Mauthner cell are different from
those in the adult, in what is being
experienced. In fact, that's the case
in some embryonic amphibian neu-
rons which show calcium spikes that
don't appear later in development.
That's certainly a possibility here.

HIGHSTEIN: I have a comment. I
don't think that fish can tell the
difference between hearing sound
and vibration. It's all vibrational en-
ergy with different frequencies. In
fact, there's good evidence that the
mammalian saccule transduces so-
called auditory stimuli. 1 have heard
anecdotal reports of sound underwa-
ter so intense that it rattles paper on
a clipboard used for writing under-
water. A propus of that, I wonder
what the parties here think they are
stimulating to make the fish escape.
I doubt that he's using his lateral line
to hear.

EATON: Both of us have avoided
saying anything about the lateral
line, because we don't know what
possible role it plays in these re-
sponses. We have some circumstan-

156

FUTURE OF AQUATIC RESEARCH IN SPACE

tial evidence suggesting that bi-lh the
lateral line and the ear an vuolved
in the responses to tail ,nulation.
We don't have any ; ,j direct evi-
dence though. The lateral line could
certainly contribute to the resolution
of acoustic directionality in adult
fish in ways that are very similar to
what the inner ear would be doing. I
should also mention that the acceler-
ations we use are somewhat higher
than you would expect for a standard
classical monopolar or dipole sound

source. But as you say, it's sort of a
continuum of intensities of accelera-
tions that vary from normal acoustic
levels to levels that are higher than
those you might call vibration. But
in the fish, how can you distinguish
which is which?

SACK: I have a question of a tech-
nical nature. Dr. Fetcho, what con-
centration of calcium green did you
inject without affecting the intracel-
lular calcium concentration?

FETCHO: We inject about five na-
noliters of a 5% w/v solution into
blastomeres. If you inject too much,
they die. If you inject too little, the
dye gets diluted. If you get the right
amount in, the cells are visible in the
larvae and their responses can be im-
aged. The fish can be taken out of the
agar after imaging and raised to
adulthood. The dye does not cause
any obvious problems.

Referenc-o: liiol. hull 192: 157. (February. 1997)

Complex Signal Processing by Weakly Electric Fishes

MASASHI KAWASAKI
Department of Biology. University of\'irginia. Gilmer Hall. C/uirlotlexvillc. I 'irginia 22903

How does the nervous system extract meaningful in-
formation from complex sensory stimuli in order to per-
form appropriate behaviors? The jamming avoidance re-
sponse of weakly electric fishes, such as Gymnarchus ni-
lolicus. provides an attractive model system with which
to study the neuronal substrate of pattern recognition.

The African electric fish Gymnarchus generates elec-
tric organ discharges at an individually fixed frequency
for electrolocation. But when two fish with slightly
different discharge frequencies meet, they shift their dis-
charge frequencies away from each other and thus avoid
effects of jamming on their electrolocation capabilities.
To produce the correct jamming avoidance response.
Gymnarchus must extract information about the sign of
the frequency difference between its own discharge and

This paper was originally presented at a workshop titled '1 he l-'iilnre
nt .ti/nalic Research in Space': Neurobiology. Cellular anil Molecular
lti«li>K\- The workshop, which was held at the Marine Biological Lab-
oratory. Woods Hole. Massachusetts, from 13 to 15 May 1996. uus
sponsored by the Center for Advanced Studies in the Space Life Sci-
ences at MBL and funded by the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

its neighbor's. This stimulus feature, the sign of fre-
quency difference, is concealed in the temporal pattern
of amplitude and phase modulation in the sensory stim-
ulus. The amplitude modulation and the phase modula-
tion are sampled by different classes of electroreceptors
and processed in parallel by the hindbrain (Rose el at.,
1988; Kawasaki, 1993, 1996: Kawasaki and Guo, 1996).

Literature Cited

Kawasaki, M., and V.-X. Guo. 1996. Neuronal circuitry for compari-
son of timing in the electrosensory lateral line lobe of an African
wave-type electric fish. Gymnarchus niloticus. J Neurosci. 16: 380-
391.

Kawasaki, M. 1996. Comparative analysis of the jamming avoidance
response in African and South American wave-type fishes. Biol.
Bull- 191: 103-108.

Kawasaki, M. 1993. Independently evolved jamming avoidance re-
sponses employ identical computational algorithms: a behavioral
study of the African electric fish. Gyinnarclius nilalicus. J. Camp.
Physiol. 173:9-22.

Rose, G. J., M. Kawasaki, and \V. llciligenberg. 1988. "Recognition
units" at the top of a neuronal hierarchy? Prepacemaker neurons in
Eigenmanma code the sign of frequency differences unambigu-
ously. J Cuinp l'ln-\n>l. 162:759-772.

157

Reference: Biol. Bull 192: 158-160. (February, 1997)

From Neurons to Behavior: Vocal-Acoustic
Communication in Teleost Fish

ANDREW H. BASS, DEANA A. BODNAR. AND JESSICA R. McKIBBEN
Section of Neurobiology and Behavior, Cornell University, Ithaca, New York 14853

Overview

Sound communication is not unique to humans and
other mammals, but rather, is a trait shared with most
nonmammalian vertebrates. The focus of our studies has
been one species of sound-producing teleost fish, the
plainfin midshipman (Porichthys notatits). Our research
program has taken three principal directions: (1) func-
tional organization of the vocal organ and brain circuitry
establishing the physical attributes of vocalizations; (2)
development of, and hormonal influences on, the ex-
pression of sexually dimorphic vocal traits; and (3) en-
coding of vocal signals by the peripheral and central au-
ditory system.

Midshipman Fish: Spawning and Vocal Behaviors

The plainfin midshipman has a wide distribution
along the western coastline of northern California and on
into southern Canada. There are two male reproductive
morphs, type I and type II. with distinct spawning and
vocal behaviors (Brantley and Bass, 1994). Type I males
build nests under rocks in the intertidal and subtidal
zones where they fertilize and then guard eggs deposited
on the roof of their nest by females. In contrast, type II
males gain access to gravid females and their eggs by es-
sentially parasitizing the type I male's reproductive tac-
tic. Thus, type II males lie perched outside of, or sneak
into, a type I male's nest and shed sperm in an attempt
to compete with the type I male for eggs; they do not
build nests or guard egg-

This paper was originally pi . .-, workshop titled The l-'inure

n/ Ai/ualic Research in Space ,\cin 'ni>lt>f>y. Cellular ami Molecular
Biology. The workshop, which was held at the Marine Biological Lab-
oratory, Woods Hole, Massachusetts, from 13 to 15 May 1996, was
sponsored by the Center for Advanced Studies in the Space Life Sci-
ences at MBL and funded by the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

Nesting type I males generate two classes of vocaliza-
tion. Trains of short duration I50-ms grunts are pro-
duced at intervals of about 400 ms during defense of a
nest against potential intruder males. Type I males also
produce long-duration (minutes to over 1 hour), quasi-
sinusoidal calls known as hums. The hum has a simple
harmonic structure; the fundamental frequency is near
90-100 Hz with one or two prominent higher harmon-
ics. During the breeding season, nesting males often clus-
ter in groups of two or more and produce hums simulta-
neously. Observations of captive populations of nesting
type I males, together with playbacks of natural or com-
puter-synthesized acoustic signals through underwater
loudspeakers, show that hums, but not grunts, can at-
tract females to an artificial nest site (Brantley and Bass,
1994; McKibben etat.. 1995). Type II males and females
have only been observed to produce isolated, low-ampli-
tude grunts in nonspawning contexts.

Vocal Motor Circuitry

Anatomical and neurophysiological studies have iden-
tified a vocal control system in midshipman (Bass and
Baker, 1990; Bass el at., 1994, 1996a). The vocal organ
consists of a pair of sonic muscles attached to the lateral
walls of the swimbladder. Each sonic muscle is inner-
vated by a single nerve formed by branches of the ventral
occipital nerve roots that exit the hindbrain just caudal
to the vagus nerve. The occipital nerves carry motor ax-
ons originating from two clusters (nuclei) of vocal moto-
neurons extending along the midline of the caudal hind-
brain (medulla oblongata) and rostral spinal cord. Each
motoneuron cell body (soma) has a single, unbranched
axon that innervates muscle fibers located on the same
(ipsilateral) side of the body axis, and a dendritic arbor
that extends throughout both motor nuclei. Intracellular

158

NEUROBIOLOGV/SENSORY BIOLOGY

159

recording and staining studies have identified vocal pace-
maker neurons that are ventrolateral to the motoneu-
rons. The firing frequency of the pacemaker neurons is
matched 1:1 with that of the sonic motoneurons and, in
turn, to the fundamental discharge frequency of the vo-
cal motor volley as recorded intracranially from the oc-
cipital nerve roots. A single pacemaker neuron inner-
vates the neurons in both motor nuclei, consistent with
the hypothesis that their role is to synchronize the firing
of motoneurons positioned on both sides of the brain.
The latter, in turn, leads to the simultaneous contraction
of both sonic muscles at a fundamental discharge fre-
quency that establishes the fundamental frequency of
vocalization. Hence, there is a direct relationship be-
tween the rhythmic, patterned output of a pacemaker-
motoneuron circuit and the physical attributes of vocal-
ization. Sex- and morph-specinc vocal behaviors are
reflected in a divergence of neurobiological and endocri-
nological traits, ranging from the size of the sound-pro-
ducing (sonic) muscles to the rhythmic firing properties
of vocal neurons (Bass, 1992, 1996).

Encoding of Species-Specific Acoustic Signals

Among many species of teleost fish, acoustic signals
are primarily encoded by fibers that innervate the saccu-
lus division of the inner ear (Popper and Fay. 1993). The
most extensive studies of auditory coding in teleost fish
have been carried out in the goldfish, Carassiits auratits
(Popper and Fay, 1993). Although goldfish have served
as a useful model for identifying the general coding
mechanisms utilized by the vertebrate auditory system,
their lack of obvious vocal behavior makes it difficult to
examine the coding and processing of behaviorally rele-
vant stimuli, such as communication signals. A practical
way to address questions of vocal signal coding has been
to identify neural mechanisms in nonmammalian spe-
cies that utilize acoustic communication signals in their
social behavior; one such species is the midshipman fish.

A fundamental problem faced by the auditory system
of any vocalizing species is the segregation and recogni-
tion of two concurrent vocal signals from independent
sources. Midshipman are routinely faced with the acous-
tic problem of segregating concurrent signals when nu-
merous males congregate during the breeding season and
vocalize simultaneously. Within a natural population,
the fundamental frequencies (FOs) of individual hums
differ within a range of about 10 Hz. The summation of
two concurrent multiharmonic signals, such as hums
that differ in their FOs, results in a complex sinusoidal
waveform with multiple envelope periodicities at differ-
ence frequencies (dF ) between ( 1 ) the FOs of each signal;
(2) their higher harmonics; and (3) the FOs and higher
harmonics. So far, we have assessed the encoding of sim-

ple beats produced by the summation of two tones near
the FOs of natural hums with dFs less than 10 Hz. Play-
back experiments suggest that midshipman can discrim-
inate and choose between two concurrent hums that
differ in FO (McKibben et a/., 1995). Hence, midship-
man must have a neural mechanism for segregating and
discriminating between two signals on the basis of FO.

Peripheral Coding (McKibben and Bass, 1996;
McKibben et til., 1995): Acoustic stimuli were used to
evoke single-unit responses in primary afferent fibers of
the Vlllth cranial nerve that innervate the sacculus. At
intensity levels comparable to those of natural signals
( 100-130 dB, re: 1 ^Pa). auditory fibers respond to pure
tones over a frequency range of 60-300 Hz by changes in
their average spike rate. Fibers respond best to frequen-
cies below 120 Hz within the range of FOs of midship-
man vocalizations. Phase-locking of spikes begins at
lower intensity levels and seems to better encode signal
frequency. Two tones at slightly different FOs (dFs of 2-
20 Hz) were added to generate stimuli that mimic the
simple acoustic beats that result from the overlap be-
tween the hums of Type I males. In response to simple
beats, auditory afferents synchronize best to one of the
two tones, usually the one with the lower FO. In general,
synchronization to dF was much lower than to either of
the two tones comprising the beat stimulus.

Auditory niidbrain (Bass el a/.. 1996b; Bodnar and
Bass, 1996; Bodnar el a/.. 1996): In midshipman, as in
other teleosts, the midbrain's torus semicircularis in-
cludes a nucleus centralis that receives input from hind-
brain nuclei encoding acoustic information. Multi- and
single-unit recordings in the nucleus centralis show that
the majority of auditory units exhibit changes in their
spike rate with changes in frequency from 70 to 200 Hz.
At least two types of units, tonic and phasic, are present.
Tonic units respond throughout the duration of the stim-
ulus, while phasic units respond only at stimulus onset.
In response to simple beats with combinations of pri-
mary tones in the range of 80-100 Hz, a subset of tonic
units are tuned to low frequency dFs (4-10 Hz); units
exhibit low synchronization to individual tones. About
half of the dF encoding neurons exhibit no significant
change in their dF tuning with different primary tones.
Thus, some units appear to be tuned to a specific dF, while
in others, coding of dF and the FOs may be coupled.

In summary, the results suggest that a peripheral peri-
odicity code of the individual components of a beat
waveform is transformed into a midbrain dF code.

Acknowledgments

Research support from NSF, NIH, NIMH, Cornell
University. U. C. Bodega Marine Laboratory, and New
York State Hatch Grant.

160

FUTURE OF AQUATIC RESEARCH IN SPACE

Literature Cited

Bass, A. H. 1992. Dimorphic ma ;md alternative reproduc-

tive tactics in a vocalizing (H rends Neurosci. 15: 139-145.

Bass, A. H. 1996. Slu. Duality. Am. Sci. 84: 352-363.

Bass, A. H., and R. Baku Sexual dimorphisms in the vocal

control system of'a tek morphology of physiologically iden-

tified neurons./ A >/. 21: 1 155-1 168.

Bass, A. H., M. A. Marchaterre, and R. Baker. 1994. Vocal-acoustic
pathways in a teleovl ii.sh. / ,\eitr<i.\ci. 14: 4025-4039.

Bass, A. H., D. A. Bodnar, and M. A. Marchaterre. 1996b. Auditory
pathways in a vocal fish: inputs to a midbrain nucleus encoding
acoustic beats. Sot: Ncurosci. Abstr. 22: 447.

Bass, A. H., B. J. Horvath, and E. B. Brothers. 1996a. Non-sequen-
tial developmental trajectories lead to dimorphic vocal circuitry for
males with alternative reproductive tactics. / Neumbiol. 30: 493-
504.

Bodnar, D. A., and A. H. Bass. 1996. The coding of concurrent sig-
nals (beats) within the auditory midbrain of a sound producing fish.

the plainfin midshipman. Association for Research in Otolaryngol-
«.?.>•. No. 617.

Bodnar, D. A., J. R. McKibben, and A. H. Bass. 1996. Temporal
computation of the difference frequency of concurrent acoustic sig-
nals in the central auditory system of a vocal fish. SOL: Nc-nrosci.
Abstr 22: 447.

Brantley, R. K., and A. H. Bass. 1994. Alternative male spawning
tactics and acoustic signals in the plainfin midshipman fish. Poricli-
tliya •/i<i/£//H.v(Teleostei, Batrachoididae). /:"//;<>/< J.tr.r 96: 213-232.

McKibben, J. R., and A. H. Bass. 1996. Peripheral encoding of be-
haviorally relevant acoustic signals in a vocal fish. Sot: Ncnmsci.
Abstr 22:447.

McKibben, J. R.. D. A. Bodnar. and A. H. Bass. 1995. Everyone's
humming, but is anyone listening? Acoustic communication in a
marine teleost fish. 4ih Ini \cunvthol. Coiigr Abslr. p. 35 1 .

Popper, A. N., and R. R. Fay. 1993. Sound detection and processing
by fish: critical review and major research questions. Brain Bcliuv
Evol.4l: 14-38.

Reference: Riol Hull 192: 161-163. (February, 1997)

Discussion

QUESTION: Dr. Bass, is one hum
better than the other? Why should a
female care which male's nest she
goes into? What's the value of it?

BASS: Certainly that is a critical
question which we are pursuing. I
don't have the answer to it. In acous-
tic systems that have been studied in
terms of female choice, a fundamen-
tal frequency of the signal can often
be predictive of body size; in this sys-
tem, however, we have found no
such correlate. In fact, this goes back
to work that I did with Bob Baker,
here at the MBL, where we were re-
cording from the CNS. I found no
correlation between the fundamen-
tal frequency of the motor discharge
and an animal's body size. So body
size is not a cue in that context.

QUESTION: Didn't you say that the
population in this range is all within
the same temperature?

BASS: Actually I don't know that.
Unfortunately the temperature was
not monitored in those experiments
for each of the individual nests. This
is a critical issue that we hope to in-
vestigate.

HIGHSTEIN: Obviously, these fish
are fairly deaf because the noise is so
loud. Maybe this is just some varia-
tion in frequency, so they know
whether to go in one direction or an-
other. If all the frequencies were the
same, they might get confused about
the sources. By having slight differ-
ences in frequency the sources might
become clearer and lead them in one
direction or the other.

BASS: So you're suggesting that the
frequency differences could play a
role in localization. This may very
well be the case. As you can imagine,
working in an aquatic environment
is extremely difficult. Furthermore,
doing those field experiments has
proven extremely difficult, and we
are still pursuing them. I wish I had
the answer to your question. All I
know is that you see that variation in
the natural signal. It just so happens
that there are units in the midbrain
that perfectly encode the difference
frequency.

MORRIS: I have a question for Ma-
sashi (Kawasaki). I was really in-
trigued by the fact that in neither of
these different evolutionary lines was
the internal reference information
used. It seems sort of amazing not to
make use of that readily available
piece of information. Do you have
any notions from an evolutionary
engineering point of view? What's
the point?

KAWASAKI: To make it more
amazing, there is a species related to
the African fish that does process the
internal reference system for gating.
This was a double shock to us be-
cause the African species is function-
ally similar to the unrelated fish and
dissimilar from the fish that belongs
to the same family. I think that the
timing comparison involved in the
systems that I described is very accu-
rate. The threshold for the timing
comparison is sub-microsecond, yet
the fish can perform the behavior
when the only available phase is 100

ns. The internal reference mecha-
nisms, the sort of mechanism that
predicts feedback timing and gating,
may not be accurate enough to do
this. For internal reference mecha-
nisms, for this gating, only millisec-
ond accuracy may be enough. How-
ever, when you are interested in mi-
crosecond and sub-microsecond
timing differences, using parallel sys-
tems and subtracting between the
two channels could be a good idea.
In the bat's echolocation system,
there is an internal reference mecha-
nism that is also not used as a refer-
ence for some aspects. In the echo
delay comparison, the system com-
putes a timing difference between
outgoing cry and returning echo, but
without using the internal reference
system. But, for blocking self stimu-
lation, it does use the internal refer-
ence system.

BAKER: Do either of you believe
that the role of electrotonic coupling
has been either overemphasized or
overstressed in your system analysis?
Do either of you have any evidence
that it's necessary for these behav-
iors?

BASS: No. As you know. Bob
(Baker), we did electron microscopy
on sonic motor system early on. Al-
though I did find gap junctions, I re-
member being amazed in the begin-
ning when I didn't see a lot of them.

BAKER: Is this also true in your
system, Masashi (Kawasaki)? Is elec-
tronic coupling absolutely necessary
for the behavior?

161

162

FUTURE OF AQUATIC RESEARCH IN SPACE

KAWASAKI: We have done stain-
ing of calcium-binding proteins (cal-
hindin or calretinin) in this differen-
tial phase coding circuitry. Not all of
the elements are labeled. Although I
haven't done any electron micro-
scopy on this system, we have seen
that the giant cell, which is one of the
components of this system, doesn't
light up with calretinin. This might
indicate that this neuron does not
use gap junctions.

BAKER: In this particular case,
what role does the cerebellum play?
Is it essential for the behavior?

KAWASAKI: Lesion of the cerebel-
lum doesn't abolish the behavior.

QUESTION: Andy (Bass), were the
differential frequency responses re-
corded in males or females? Is there
any difference, and do two males sit-
ting side by side try to separate their
frequencies?

BASS: We have no evidence that
there is anything like a jamming
avoidance response. In answer to
your question, let me first say that fe-
males do not generate signals. They
generate grunts — very isolated
grunts — infrequently, and the fun-
damental frequency of their signal is
on the order of 15%-20% lower,
which is matched by a difference in
the CNS output. Nevertheless, if you
sample a single, hour-long hum from
males at different intervals, the stan-
dard deviation is on the order of 1
Hz. It is a highly stable behavior. In
all the experiments on the CNS that
Bob ( Baker) and I have done over the
years, we've never seen any evidence
that the animals can modulate their
central frequency. Although males
may be trying to jam each other in
this behavior, there is no evidence
that they can actively produce some-
thing like a jamming avoidance re-
sponse as a social response.

QUESTION: Are those differential
frequency responses from the re-
sponding neurons present in both
males and females?

BASS: Everything I've mentioned
so far occurs in both males and fe-
males. We haven't seen anything
that's obviously sexually dimorphic.
Another interesting issue here is that
it is important to understand the
difference between the responses of
the auditory and electrosensory sys-
tems. The fish that Masashi (Kawa-
saki ) is studying are more or less con-
tinuously generating an output. Our
fishes do not continuously generate.
There are two conditions under
which it would be useful for a neuron
to produce a difference frequency.
One is when the animal is actively
vocalizing, trying to detect a neigh-
bor whose pulse is essentially super-
imposed on its own. This is distinct
from the case in which an individual
is silent, but is listening to two other
animals out there. It would seem that
both males and females would want
to do that. We've only studied the sit-
uation in which an animal is essen-
tially listening to two other pulses.

QUESTION: Dr. Kawasaki, do the
fish turn to maximize their signal?

KAWASAKI: This system cannot
use propagation time. There is no
propagation time associated with an
electric field. Therefore, no distances
could be encoded into time per sc.
The whole system is concerned more
with dynamics than with stationary
steady-state levels. So the fish will al-
ways operate in a moving situation.
The majority of neurons respond to
modulation of amplitude or phase.

ATEMA: Andy (Bass), you have
eliminated so many possibilities for
the real value of the signal. Wouldn't
it be helpful to consider the cost to
the sender of producing the signal?
Perhaps the female is assessing the
male's ability to maintain a constant
and powerful sound for a long time.
The female may be going around
and comparing constancy between
different males. Is there any evi-
dence, on either the part of the
sender or the receiver, that sound

constancy and power are the param-
eters that constitute signal value? All
your data seem to point in that direc-
tion, including temperature com-
pensation.

BASS: That's an excellent point.
We haven't really assessed our data
carefully to determine whether the
intensity coding in the CNS involves
signal value and power output.
There is a lot of power output from
an individual male. We just don't
have enough data yet — it's very hard
to collect — but we are looking at in-
tensity at an individual nest and how
that might vary with male size. One
would predict that the larger males
might be able to produce a signal for
a longer time. But it also might be a
louder signal, and part of the goal in
the field work this summer is to col-
lect more data on that particular is-
sue. The neurobiology is ahead of the
behavior, as is the case in so many
systems. What's beautiful about this
system is that we can directly corre-
late male vocal signals with repro-
ductive success. We can count the
number of eggs in a male's nest, and
we can in fact assess the number of
clutches the individual male has.
That's where the field work is going.
Your question is excellent. Clearly
the issue is identifying the value of
the signal to an individual female.

BARLOW, R.: Andy (Bass), one of
your slides indicates that the play-
back experiment was performed at
night. What happens during the day?

BASS: We were really surprised.
Basically this is a nocturnal behav-
ior— that's when you pick up the
humming. To get a decent video of
the behavior, we decided to see what
happens at 4 o'clock in the af-
ternoon, instead of 8 at night. Al-
though they do show the response, it
is not as robust and consistent as at
earlier hours. It's remarkably locked
into some sort of circadian rhythm.

BARLOW, R.: Did you do experi-

NEUROBIOLOGV/SENSORV BIOLOGY

163

ments in constant dark throughout
the day and night?

BASS: These experiments are per-
formed outdoors in those circular
tanks. We have no control over the
lighting.

BASS: Another issue we need to as-
sess here is a female's initial ap-
proach to the signal. The way a fe-
male makes that decision may not be
the same as when she makes her de-
cision when she is close to the signal.
This vocal signal could function
merely as a beacon to let females
know where males are breeding. If
the female can assess on the basis of
the beat frequency, she might be able
to extract the number of males that
are in a cluster, and that's why she
goes to that cluster. But when she
gets close to the cluster, how she
makes the decision between different
males could involve an entirely
different set of rules. This could in-
volve olfactory stimuli. Maybe at
that point, the lateral line plays an
important role in terms of intensity
coding of that signal, which as you
say is a very intense vibrational sig-
nal. I think we have such a long way

to go in teasing apart those two
things. These fish differ from the
toadfish here at Woods Hole. If you
listen to the calls of the toadfish,
there is not much overlap in their sig-
nals. What's going on in these fish is
different. We have done some re-
cordings in toadfish and have not
found any units that appear to be en-
coding DF. So the toadfish here at
Woods Hole and our midshipman
fish may have very different strate-
gies.

COMMENT: The toadfish at Woods
Hole undergo a huge temperature
range. Presumably, as the muscle
gets warmer, it's able to operate at a
higher frequency, and the call fre-
quency goes up with temperature. If
we went to the west coast and there
was also a large temperature range
there (which is probably not the
case), the call frequency would also
increase with increasing tempera-
ture. One might imagine the case in
which a cold female cannot hear a
warm male, because the warm male
is sounding out frequencies at 200
Hz and the female is, let's say, at 15
degrees, where her neurons only op-

erate at 100-1 10 Hz. The suggestion
is that the difference in temperature
sensitivity, or the selection of differ-
ent frequencies by the female, may
just be a temperature effect on their
neurons. That would be a necessity
if they were to operate over a large
temperature range.

BASS: That reminds me of some
experiments that I did here at the
MBL with the motor system, where
the temperature and the firing fre-
quency of the motor neurons were
tightly coupled. The fundamental
frequency of the hum varies directly
with temperature. Your point is well
taken that there is temperature cou-
pling between the receiver and the
sender. (Comment: There has to be
or else it would not be heard!) Well,
the caveat to that is, if you look at
tuning curves in these animals, they
are pretty broad, even if you are
shifting temperature. Within the
range of variation of the animal's ex-
perience, rather than our own artifi-
cial manipulations, things are still
falling pretty much within their tun-
ing curve, given the intensity of the
signal.

Reference: Bin/. Bull 192: 164-166. (February, 1997)

Activity-Dependent Regulation of Neural Networks:

The Role of Inhibitory Synaptic Plasticity in Adaptive

Gain Control in the Siphon Withdrawal Reflex

ofAplysia

THOMAS M. FISCHER AND THOMAS J. CAREW

Department of Psychology. Yale University. New Haven. Connecticut 06520

Neural networks can be dynamic systems that enable
organisms to adapt to, and learn about, complex and
variable environments. The broad objective of our re-
search program is to understand the nature of this type
of adaptive process by examining the functional signifi-
cance of neural plasticity observed at both cellular and
network levels during adaptive behavioral modifica-
tions. Our primary goal is to characterize the role of in-
hibitory modulation in network function and plasticity,
by focusing upon a recurrent synaptic circuit formed by
the L29 excitatory interneurons and the L30 inhibitory
interneurons within the siphon withdrawal reflex (SWR)
network ofAplysia californica. Recurrent inhibitory cir-
cuits such as this are a common feature of motor net-
works, providing a mechanism for rapid control of be-
havioral output. Moreover, intrinsic and extrinsic mod-
ulation of recurrent inhibitory circuitry can endow
motor networks with a high degree of flexibility and an
enhanced capability for adaptive modification. By focus-
ing upon identified inhibitory elements within a well-de-
fined circuit with direct behavioral relevance, and per-
forming experiments ranging from cellular to behavioral
levels of analysis, we are beginning to define a functional
role for inhibitory processing in mediating adaptive gain
control of the SWR in response to changing environ-
mental conditions.

This paper was originally presented at a workshop titled The Finnic
i>l Aquatic Research in Space Neurobiology, Cel/ii/ur and Molecular
Biology. The workshop, which was held at the Marine Biological Lab-
oratory, Woods Hole. Massachusetts, from 13 to 15 May 1996, was
sponsored by the Center for Advanced Studies in the Space Life Sci-
ences at MBL and funded by the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

Our cellular experiments have demonstrated that dy-
namic interactions between the L29s and L30s can sig-
nificantly regulate reflex input to the LFS-type siphon
motor neurons (MNs). The L29s (5 total) and L30s
(3 total) are reciprocally interconnected by both chemi-
cal and electrical synapses: the L29s directly excite the
L30s which, in turn, directly inhibit the L29s. The L29
excitatory interneurons, which are known to act as facil-
itatory neuromodulatory neurons (Hawkins el al..
1981), also provide substantial excitatory input to LFS
MNs; a single L29 can account for as much as 50% of
reflex input to these MNs evoked by siphon stimulation.
Yet surprisingly, direct intracellular activation of the
L29s causes a significant, transient decrement of reflex
input to the MNs. The mechanism of this inhibition is
through the recruitment and enhancement of recurrent
L30 inhibitory synaptic transmission. Activation of the
L30s, either directly through current injection or indi-
rectly by means of activation of the L29s, results in a
significant potentiation of the L30 synapse. This activity-
dependent potentiation ( ADP) of the L30s occurs at low,
behaviorally relevant activation frequencies (2 to 10 Hz),
and has a time course of expression matching that of
the reflex inhibition produced by activation of the L29s
(Fischer and Carew, 1993). The recurrent interactions
between L29 and L30 interneurons can thus provide a
mechanism for activity-dependent regulation of excit-
atory input to the siphon motor neurons.

Our identification of the dynamic network interac-
tions described above raised the important issue of the
functional significance of the L29/L30 circuit. To ad-
dress this question, we performed a series of experiments
in which we first examined the kinds of behaviorally rel-

164

NEUROB1OLOGY/SENSORY BIOLOGY

165

evant stimuli that activate the L29/L30 circuit, and then
how this activation modulates reflex responding. These
experiments were performed in a reduced preparation
consisting of the tail, mantle (siphon and gill), and cen-
tral nervous system; this preparation provides a useful
interface between behavioral experiments in intact ani-
mals and analysis of cellular events. We found that weak
(non-noxious) tactile input readily activates the L30s,
and that this activation is sufficient to potentiate the L30
inhibitory synapse onto L29 neurons. This same stimu-
lus also produces significant inhibition of siphon-evoked
reflex responses in both L29 interneurons and siphon
MNs, with a time course corresponding closely to the
time course of L30 ADP( Fischer and Carew. 1995). Sim-
ilar results were obtained when intact, freely moving an-
imals were used (Blazis et a/.. 1994). Finally, we directly
tested the role of the L30s in mediating this inhibitory
process by reversibly inactivating (hyperpolarizing) them
during tactile stimulation; this significantly attenuates
the inhibition of siphon-evoked responses (Fischer and
Carew, 1995).

These results led us to propose an adaptive role for
L30-mediated inhibition in regulating SWR responsive-
ness under conditions of different levels of ambient (tac-
tile) environmental stimulation. Specifically, we hypoth-
esize that activation of the L30s by weak tactile input
from the environment transiently increases the strength
of L30-mediated inhibition in the SWR network. To
produce a siphon response in the face of this increased
inhibition, a stronger input from the siphon would be
required. In this fashion, the increase in L30-mediated
inhibition caused by weak tactile stimulation can elevate
the effective threshold for evoking a motor neuron re-
sponse based upon the recent tactile experience of the
animal (Fischer and Carew, 1995). Recent behavioral ex-
periments, which are discussed below, provide support
for this model ( Yuan et ai, 1996).

In contrast to the effects of weak tactile stimulation on
L30 inhibitory processing, we found that a more intense
stimulus, tail shock, selectively attenuates specific forms
of L30 ADP. At least two temporally separate forms of
ADP can be distinguished: ( 1 ) frequency facilitation
(FF), which is a short-term enhancement that is ex-
pressed during L30 activation; and (2) augmentation
(AUG), which is a longer-term form of enhancement
that persists for up to 60 s following activation. We found
that a single tail shock selectively attenuates AUG with
no effect on FF. This suppression of AUG lasts for hours,
and is mimicked by bath application of the monoamine
serotonin, a neuromodulator implicated in behavioral
sensitization. The mechanism of this suppression is due
to a reduction in calcium influx during L30 activation,
to a level at which FF is fully expressed, but below the
level required for AUG (Fischer and Carew, 1994).

These findings were extended in a series of cellular ex-
periments examining the calcium dependence of the
different forms of synaptic potentiation exhibited by the
L30s. Using the photo-activated calcium chelator diazo-
4, we demonstrated that all forms of L30 ADP require
presynaptic elevation of free calcium for their expression
(Fischer et til., 1996). The selective suppression of
different components of L30 ADP by tail shock suggests
that L30-mediated effects on reflex responsiveness
should be differentially expressed following shock de-
pending upon the recent activation history of the L30s.
Under conditions in which L30 is active, L30 mediated
inhibition should be normal, since FF is relatively un-
affected. Conversely, at time points tens of seconds fol-
lowing L30 activity, inhibition should be attenuated,
since the prominent form of ADP at these time points
(AUG) is suppressed. We are currently carrying out both
behavioral and computational analysis to examine the
implications of these cellular predictions.

The findings described above provided us with an op-
portunity to examine the behavioral implications of the
functional suppression of a specific form of synaptic
plasticity. In separate experiments utilizing both reduced
preparations and intact animals, we found that a single
tail shock of the same intensity used in our cellular ex-
periments abolished the inhibition normally produced
following weak tactile input. Further, we found that the
level of shock used to abolish the inhibition was insuffi-
cient to produce sensitization of the reflex, suggesting
that low levels of tail shock may induce a lower threshold
component of sensitization by reducing L30 inhibitory
modulation within the SWR circuitry (Blazis et ai,
1994). A corollary of this view is that the low-threshold
component of sensitization may be expressed not by an
increase in reflex amplitude, but rather by a reduction in
the threshold of reflex activation under stimulus condi-
tions that normally induce L30-mediated behavioral in-
hibition. Recent behavioral experiments support this
view (discussed below). Thus, tail-shock-induced sup-
pression of L30 ADP may be viewed as an initial step
in behavioral sensitization; it would also have secondary
effects such as allowing greater levels of activity in facili-
tatory neuromodulator neurons such as the L29s. Previ-
ous work in other laboratories has demonstrated that
sensitization of the SWR may be accompanied by a re-
duction in network inhibition (Frost, 1987; Trudeau and
Castellucci. 1993). Our work is consistent with, and an
extension of, these results.

Our results to date thus show that L30-mediated inhi-
bition can provide for a dynamic regulation of the SWR
that is dependent upon the recent tactile experience of
the animal. To further test our cellular predictions, we
examined the effects of changing environmental condi-
tions on SWR responsiveness in intact animals by indue-

166

FUTURE OF AQUATIC RESEARCH IN SPACE

ing a low-level tactile stimulus, artificial turbulence
(mimicking tidal wave actio into the animal's home
aquarium. We found ' Hulence produced two

main effects on the SV 3 significant decrease in the
duration of the SWK ;onse to a siphon stimulus of

fixed intensity, and .ncant increase in the siphon

stimulus intensit\ : ; •; shold) required to elicit a siphon
response. Both t Liiese effects were manifest within
1 min of the change in conditions, and remained stable
for the 10 min in which the turbulent conditions were
maintained. Rapid recovery dynamics were revealed
when calm conditions were restored; both measures re-
turned to baseline (pre-turbulence) values within 1 min
upon restoration of calm conditions. The overall charac-
teristics of this adaptive response were consistent with
the dynamics of L30 ADP. We next investigated the
effects of tail shock on turbulence-induced adaptation.
Tail shock did not alter the behavioral inhibition pro-
duced during turbulence, which is consistent with obser-
vations that L30 is active during turbulence, and that the
FF component of L30 ADP is intact following shock. We
have yet to systematically examine the recovery dynam-
ics following turbulence, where effects on the AUG com-
ponent of L30 ADP would be manifest. Further, we
found that following shock, the threshold stimulus inten-
sity for evoking a siphon response in calm conditions was
significantly decreased. Turbulent conditions still in-
duced an increase in reflex threshold, but this increase
was significantly smaller than in nonshocked animals.
These results are again consistent with previous cellular
predictions. We plan to perform additional cellular ex-
periments to directly test the role of L30 ADP in mediat-
ing this adaptive process.

Taken collectively, our results indicate that activity-
dependent inhibition in the SWR network promotes a
rapid and dynamic adjustment of reflex behavior in re-
sponse to changing environmental conditions. Future

experiments will be directed at examining how this in-
hibitory system may interact with the cellular and net-
work processes underlying learning, and how modifica-
tions of inhibition through tail shock may modulate this
interaction.

Acknowledgments

This work was supported by NIH grants MH48672 to
TJC and MH 1 0334 to TMF.

Literature Cited

Blazis, D. E. J., N. J. Priver, T. M. Fischer, and T. J. Carew. 199-4.

Modulation of tail-induced inhibition of the siphon withdrawal re-
flex of Ap/ys/u SKI: AVi<m«r Abslr. 20: 814.

Fischer, T. M., and T. J. Carew. 1993. Activity dependent recurrent
inhibition: a mechanism for dynamic gain control in the siphon
withdrawal reflex of Aplysia. J \curn.ici. 13: 1302-1314.

Fischer, T. M., and T. J. Carew. 1994. Tail shock differentially mod-
ulates two forms of synaptic plasticity in inhibitory interneuron L30
ofAplyxui. Sue. Neiimsci. Abs/r. 20: 1072.

Fischer, T. M., and T. J. Carew. 1995. Cutaneous activation ol the
inhibitory L30 interneurons provides a mechanism for regulating
adaptive gain control in the siphon withdrawal reflex ofAplysia. J.
Ni'iimsd. 15: 762-773.

Fischer, T. M., R. S. Zucker, and I . J. Carew. 1996. Activity-depen-
dent potentiation of synaptic transmission from L30 inhibitory in-
terneurons of Aply.\ia depends upon residual calcium. Sue. Neu-
msr/. Ahxtr. 22: 327.

Frost, VV. N. 1987. Mechanisms contributing to short- and long-term
sensitization in Aplysia. Ph.D. thesis. Columbia University.

Hawkins, R. D., V. R. Castellucci, and E. R. Kandel. 1981. Interneu-
rons involved in mediation and modulation of gill-withdrawal re-
flex in Aplytid. I. Identification and characterization. J. Ncumphys-
inl 45:304-314.

Trudeau, L.-E., and V. R. Castellucci. 1993. Functional uncoupling
of inhibitory interneurons plays an important role in short-term
sensitization of Aplyxia gill and siphon withdrawal reflex. J. Neu-
rosci. 13:2126-2135.

Yuan, J. \V., T. M. Fischer, and T. J. Carew. 1996. Dynamic regula-
tion of the siphon withdrawal reflex ofAplysia in response to chang-
ing environmental conditions. Sue. Ni'itrosci. Abstr. 22: 1403.

Reference: Bin! Bull 192: 167-169. (February, 1997)

Complex Oscillations in Simple Neural Systems

DOUGLAS A. BAXTER AND JOHN H. BYRNE

Department ofNeurobiology and Anatomy, The University oj Texas Medical School at Houston,

Houston, Texas 77030

One of the goals of this workshop is to explore how
research on aquatic plants and animals can improve our
understanding of the basic mechanisms underlying the
effects of gravity on biological processes. In general, the
focus of "gravitational biology" has been to investigate
gravitational effects at the organismal level rather than
at the cellular, subcellular, or molecular levels. Indeed,
previous theoretical studies have indicated that the
effects of gravity would be minuscule at the subcellular
level (Albrecht-Buehler, 1991; Nace, 1983; Pollard,
1965, 1971; Todd, 1989). However, recent spaceflight
and ground-based experiments have demonstrated that
a broad range of basic cellular properties can be influ-
enced by gravity, including cytoskeletal structure and
cell morphology, cell-cell interactions, cellular metabo-
lism, exocytosis and endocytosis, concentrations of elec-
trolytes, voltage-gated channels, intracellular levels of
second messengers, and enzymatic activities (Cogoli et
a/., 1990; Cogoli and Gmunder, 1991; Edgerton and
Roy, 1994; Gruener and Hoeger, 1990, 1991; Hughes-
Fulford, 1991; Hy mere/ al., 1994; Krasnov, 1994; Mor-
rison, 1994; Reitstetter et al.. 1991; Rijken et al., 1994;
SaloetaL, 1992;Schatz?/a/., 1994;Sibongac?a/., 1989;
Todd, 1 989). Thus, perturbations in the gravitational en-
vironment can induce modifications in cellular activi-
ties, which in turn may contribute to gravity-dependent
changes at the organismal level.

How is it possible to resolve the theoretical and empir-
ical findings? An intriguing explanation for the influence
of gravity on cells was derived, in part, from nonlinear

This paper was originally presented at a workshop titled Tlw h'ulurc
of Aquatic Research in Space: Neurobiology, Cclliilur and Molecular
Biology. The workshop, which was held at the Marine Biological Lab-
oratory, Woods Hole. Massachusetts, from 13 to 15 May 1996. was
sponsored by the Center for Advanced Studies in the Space Life Sci-
ences at MBL and funded by the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

dynamical systems theory (Kondepudi and Prigogine,
1983; Kondepudi and Strom, 1992; Mesland, 1987,
1990, 1992a, 1992b). Specifically, even if the force of
gravity at the subcellular level is extremely weak by com-
parison with other forces, the nonlinearities of most mo-
lecular systems may provide the amplification required
to allow a weak signal to influence subcellular processes.
Thus, gravity may be a bifurcation parameter capable
of inducing state transitions. Since nonlinear dynamical
systems are the rule rather than the exception in biology,
the application of these principles may help us to design
new experiments that will provide insights into basic cel-
lular physiology and the molecular basis of gravitational
effects on cells.

In the present paper, we describe how some of the con-
cepts and analytical techniques of nonlinear dynamical
systems have been applied to computational and experi-
mental analyses of single-cell and multicellular neuronal
oscillators (Baxter et al., 1996; Butera el al, 1995;
Byrne et al, 1994; Canavier et al, 1991, 1993, 1994;
Lechner et al., 1996). Our analysis has focused on the
R15 neuron, which is located in the abdominal ganglion
of the marine mollusc Aplysia. R15 has intrigued neuro-
biologists for decades, principally because of its intrinsic
ability to produce complex bursting activity. This cell
also exhibits many different modes of oscillatory activity.

Previous experimental studies have demonstrated two
conventional methods of shifting the activity of R 1 5 be-
tween different modes of activity. First, the activity can
be altered by intracellularly injecting a constant bias cur-
rent. Sufficiently large depolarizing bias currents shift the
activity of Rl 5 from bursting to beating (i.e., tonic spik-
ing activity), whereas sufficiently large hyperpolarizing
bias currents shift R15 into a silent mode. Second, the
activity of R15 can be altered by applying modulatory
transmitters, such as serotonin (5-HT). By modulating
biophysical parameters (e.g.. membrane conductances)

167

168

FUTURE OF AQUATIC RESEARCH IN SPACE

various concentrations of 5-HT can shift the electrical
activity of R 1 5 between a variety of bursting modes,
which vary in the intensity of spiking during the burst
and the duration of the interburst interval.

We have used a Hodgkin-Huxley type model of the
R15 neuron and computer simulations to investigate a
novel method for shifting the electrical activity of R15
among different modes of oscillatory activity. This novel
method does not rely on alterations in model parame-
ters, but rather exploits the intrinsic nonlinear properties
of the cell. The simulations indicated that, for a single set
of parameter values, the model cell can generate up to
eight distinct modes of oscillatory activity. These modes
of electrical activity corresponded to multiple stable at-
tractors, whose coexistence in phase space was an emer-
gent property of the nonlinear dynamics of the cell. Brief
perturbations, which were simulated postsynaptic poten-
tials (PSPs), could switch the activity of R15 between
different modes. These mode transitions did not require
any changes in the biochemical or biophysical parame-
ters of the neuron and provided an enduring response to
a transient input. Moreover, the nature of the mode shift,
as well as whether the mode shift occurred at all, was
dependent upon the timing of the PSP (i.e.. perturba-
tion) relative to the ongoing activity, thus providing a
mechanism for phasic sensitivity (i.e., temporal specific-
ity). Finally, the multistability of this neuronal oscillator
was regulated by modulatory transmitters. The actions
of modulatory transmitters were simulated by systemi-
cally altering the conductances of specific membrane
currents within physiological ranges (e.g.. ±20% of con-
trol values). Changing these parameters regulated the
number of coexisting modes of electrical activity, such
that at control values eight modes coexisted, at interme-
diate values two or three modes coexisted, and at the
maximum and minimum values only a single mode ex-
isted. Thus, by modulating membrane conductances,
transmitters could position the neuronal oscillator in
different regions of its parameter space; some regions
supported multistability, whereas others did not.

Recently, we have begun to extend our analyses of the
dynamics of neuronal oscillators to include multicellular
oscillators (Baxter el a/.. 1996, 1997; Canavier el ul..
1995). These analyses have focused on a ring network
composed of identical cellular and synaptic elements.
The individual cells consist of modeled R15 neurons.
The parameter values that were used in these simulations
positioned the cells in a region of their parameter space
that supported only a single attractor, which was a burst-
ing limit cycle. Thus, the individual circuit elements by
themselves did not support multistability. We found,
however, that the ring network exhibited multistability.
Simulations also illustrated that, as the number of cells in
the ring network was increased, the number of coexisting

patterns also increased. Similarly, if the parameter values
for the individual cells were modified such that the cells
themselves became multistable, then the number and di-
versity of coexisting oscillatory patterns increased dra-
matically.

Electrophysiological experiments have tested several
key predictions of the computational studies. First, we
examined whether the oscillatory electrical activity of in-
dividual R15 neurons exhibited multistability. Second,
we examined whether modulatory transmitters regulated
the multistability. Finally, we examined whether net-
works of R15 neurons that were artificially connected
into a ring network could support multistability. The re-
sults of these electrophysiological experiments were con-
sistent with the predictions of the computational studies
and indicated that nonlinear processes may endow indi-
vidual cells with a richer repertoire of cellular state tran-
sitions than has been previously appreciated.

In summary, the data described above indicate that
there are two fundamentally different methods of pro-
ducing an enduring change in the mode of oscillatory
activity in R15. One method is parameter dependent
(e.g.. application of a bias current or a modulatory
transmitter), whereas the second method is parameter in-
dependent. Parameter-independent changes in the mode
of activity emerge from the proliferation of attractors in
the phase space of this nonlinear system. Each of these
attractors correspond to a different pattern of oscillatory
activity, and brief perturbations (e.g.. synaptic inputs)
can switch the electrical activity from one stable pattern
of oscillation to another. These mode transitions do no!
require any changes in the parameters of the model, and
such parameter-independent transitions can provide an
enduring response to a transient input. These studies
have provided novel insights into the ways in which non-
linear dynamics at the cellular level might be exploited
in the generation and control of oscillatory patterns of
electrical activity and to process and store information.

Note, finally, that electrical activity is only one of the
nonlinear dynamical activities in cells. The interactions
among most, if not all. biochemical reactions and second
messenger systems are highly nonlinear. Thus, nonlinear
state transitions (i.e., bifurcations) at the molecular level
might function to amplify relatively weak signals (e.g.,
gravity) above the level of random thermal noise, and the
weak signals could thus be manifest at the cellular level.
R 1 5 may provide a useful model system with which to
investigate these possibilities. For example, R15 can be
maintained in vitro for many days while its electrical ac-
tivity is being monitored by means of a number of non-
invasive techniques (e.g.. Parsons el a/.. 1991). More-
over, the parameter-independent mode transitions that
we have characterized could serve as a very sensitive
measure of the effects of gravity on cellular processes.

NEUROB1OLOGY/SENSORY BIOLOGY

169

Literature Cited

Albrecht-Buehler, G. 1991. Possible mechanisms of indirect gravity
sensing by cells. ASUSB Bull 4: 25-34.

Baxter, D. A.. C. C. Canavier, R. J. Butera, J. \V. Clark, and .1. II.
Byrne. 1996. Complexity in individual neurons determines which
patterns are expressed in a ring circuit model of gait generation. Sin:
Neurosci. Abxlr. 22: 1437.

Baxter, D. A., C. C. Canavier, H. A. Lechner, R. J. Butera, A. A. De-
Franceschi, J. VV. Clark, and J. H. Byrne. 1997. Coexisting stable
oscillatory states in single cell and multicellular neuronal oscilla-
tors. In press, in Oscillations in fseiiral Systems. D. Levine, V.
Brown, and T. Shirey, eds. Lawrence Erlbaum Assoc.. Hillsdale.
NJ.

Butera, R. J., J. VV. Clark, C. C. Canavier, D. A. Baxter, and J. H.
Byrne. 1995. Analysis of the effects of modulatory agents on a
modeled bursting neuron: dynamic interactions between voltage
and calcium dependent systems. J Coin/tin. Nciirosci. 2: 19-44.

Byrne, J. H., C. C. Canavier, H. Lechner, J. VV . Clark, and D. A. Bax-
ter. 1994. Role of nonlinear dynamical properties of a modeled
bursting neuron in information processing and storage. Nelh ./.
Zool. 44: 339-356.

Canavier, C. C., J. VV. Clark, and J. H. Byrne. 1991. Simulation of
the bursting activity of neurons R 1 5 in A/>ly\iu: role of ionic cur-
rents, calcium balance, and modulatory transmitters. / \\-iiropliv.\-
ic/. 66: 2 107-2 124.

Canavier, C. C., D. A. Baxter, J. \V. Clark, and ,1.11. Byrne. 1993.
Nonlinear dynamics in a model neuron provide a novel mechanism
for transient synaptic inputs to produce long-term alterations of
postsynaptic activity. J. Neurophysiol. 69: 2252-2257.

Canavier, C. C., D. A. Baxter, J. VV. Clark, and J. H. Byrne. 1994.
Multiple modes of activity in a model neuron suggest a novel mech-
anism for the effects of neuromodulators. ./. Neurophysiol. 72: 872-
882.

Canavier, C. C. R. J. Butera, D. A. Baxter, J. VV. Clark, and J. H.
Byrne. 1995. Networks of physiologically based neuronal oscilla-
tors may provide improved models of pattern generation. SOL: Ncu-
rosci.Absir. 21: 147.

Cogoli, A., and F. K. Gmunder. 1991. Gravity effects on single cells:
techniques, findings and theory. Adv. Space Bin/. Med. 1: 183-248.

Cogoli, A., B. Bechler, and G. Lorenzi. 1990. Responses of cells to
microgravity. Pp. 97-1 12 in Fundamentals o/' Space Bio/ogv. M.
Asashima and G. M. Malacinski. eds. Japan Scientific Societies
Press. Tokyo.

Edgerton, V. R., and R. R. Roy. 1994. Neuromuscular adaptation to
actual and simulated weightlessness. Adv. Space Biol. Med. 4: 33-
67.

Gruener, F., and G. Hoeger. 1990. Vector-free gravity disrupts syn-
apses formation in cell culture. Am. J. Physiol. 27: C489-C494.

Gruener, F.,andG. Hoeger. 1991. Vector-averaged gravity alters my-
ocyte and neuron properties in cell culture. Aviat. Space Environ
Med. 56: 1159-1 165.

Hughes-Fulford, M. 1991. Altered cell function in microgravity.
Exp. Gemntnl. 26: 247-256.

Hymer, W.C., K. Shcllenberge, and R. Grindeland. 1994. Pituitary
cells in space. Adv Space Res. 14:61-70.

Kondepudi, D. K.. and I. Prigogine. 1983. Sensitivity of nonequilib-
rium chemical systems to gravitational field. Adv. Space Res. 3:
171-176.

kondepudi, D. K., and P. B. Strom. 1992. Gravity detection through
bifurcation. . \dv. Space Res. 1 2: 7- 1 4.

Krasnov, I. B. 1994. Gravitational neuromorphology. Adv. .Space
Biol. Med .4:85-1 10.

Lechner, H. A., D. A. Baxter, J. W. Clark, Jr., and J. H. Byrne. 1996.
Bistability and its regulation by serotonin in the endogenously
bursting neuron R 1 5 in Aplysia. J Neurophysiol. 75: 957-962.

Mesland, D. A. M. 1987. Biorack experiments in Spacelah D-I and
1ML-1: further developments in gravitational biology. Pp. 305-312
in Proceedings ol the Third European Symposium on Life Sciences
Research in Space. J. Hunt, ed. ESA SP-271, Noordwijk, The
Netherlands.

Mesland, D. A. M. 1990. Gravity effects on cells. Pp. 22 1-227 in Pro-
ceedings ol Fourth Symposium on Life Sciences Research in Space.
ESA SP-307, Trieste. France.

Mesland, D. A. M. 1992a. Mechanisms of gravity effects on cells: are
there gravity-sensitive windows?. litv. Space Biol. Med. 2: 21 1-228.

Mesland, D. A. M. I992b. Possible actions of gravity on the cellular
machinery. Adv. Space Res. 12: 15-26.

Morrison. D. R. 1994. Cellular changes in microgravity and the de-
sign of space radiation experiments. Adv. Space Res 14: 1005-
1019.

Nace, G. \V. 1983. Gravity and positional homeostasis in the cell.
Adv. Space Res. 3: 159-168.

Parsons, T. D., B. M. Salzberg, A. L. Obaid, F. Raccuia-Behling, and
D. Kleinfeld. 1991. Long-term optical recordings of patterns of
electrical activity in ensembles of cultured Aplysia neurons. J. Neu-
rophysiol 66:316-333.

Pollard, E. C. 1965. Theoretical considerations on living system in
the absence of mechanical stress. J Theor. Biol. 8: I 1 3- 123.

Pollard, E. C. 1971. Physical determinants of receptor mechanism.
Pp. 25-34 in Gravity and the Organism, A. Gordon and M. Cohen,
eds. The University of Chicago Press. Chicago.

Reitstetter, R., A. Schatz, A. Linde-Hommes, and VV. Briegleb. 1991.
Changes in ion channel properties related to gravity. Physiologist
34,Suppl. 1:S68-S69.

Rijken, P. J., J. Boonstra, A. J. Verkleij, and S. W. de Laat. 1994.
Effects of gravity on the cellular response to epidermal growth fac-
tor. Adv. Space Biol. Med. 4: 159-188.

Sato, A.,T. Nakajima, Y.Kumel.T.Hongo.andK.Ozawa. 1992. Grav-
itational effects on mammalian cells. Physiologist 35, Suppl. 1:
S43-S46.

Schatz, A., R. Reitstetter, A. Linke-Hommes, VV. Briegleb, K. Slenzka,
and H. Rahamann. 1994. Gravity effects on membrane processes.
Adv. Space Res, 14:35-43.

Sibonga, J. D., T. N. Fast, P. X. Callahan, and C. M. Winget, eds.
1989. Cells m Space. NASA SP-10034. Moffett Field, CA.

Todd, P. 1989. Gravity-dependent phenomena at the scale of the sin-
gle cell. ASGSB Bull 2: 95- 113.

Reference: Bioi Bull 192: I 70-17 1. (February. 1997)

Discussion

MORRIS: Dr. Baxter, I wonder
whether a system that has so many
attractors in it is subject to knocking
between one attractor and the next
by noise.

BAXTER: We have addressed that
issue. The EPSP that we typically
used as a perturbation was modeled
from experimental measurements of
the giant EPSP onto R15. That's the
largest EPSP that R15 receives. We
found that these mode transitions
were fairly insensitive to low ampli-
tude noise. If you had large ampli-
tude inputs, activity of R15 could be
bounced between modes. Another
consideration is that the phase-por-
traits I showed were two-dimen-
sional, which implies a nice linear re-
lationship between the location of
these attractors; however, that's not
the case. These attractors have 1 1 di-
mensions to them, and even though
they may look far apart on this two-
dimensional plot, they could be right
next to each other. One attractor
could be very large, whereas another
attractor could be very small. It's a
very complex question, and there is
no simple answer. Noise can be a
problem.

NICK: What happens to your iso-
lated cell when you turn it by 90 de-
grees?

BAXTER: We e not examined
the response of R i : i changes in di-
rection of the gravi'} vector. How-
ever, we can induce shifts between
bursting activity and beating activity
in the isolated cell by perturbing the
cell with brief current pulses.

BAXTER: Tom (Fischer), what's
the effect of serotonin on the L30
synapse?

FISCHER: Serotonin has the same
effect as tail shock; it inhibits a
calcium channel. We don't know
what type of calcium channel at this
point. However, if you look at other
indices of calcium conductance, it
just decreases the amount of calcium
influx. We need to see what kind of
channels are being regulated, using
the appropriate voltage clamp exper-
iment. It seems that there is a direct
interaction with that channel to de-
crease its conductance.

BAXTER: You say directly. Are
there no second messengers in-
volved?

FISCHER: No, I didn't mean that.
It probably acts through cyclic AMP.
This was shown through the work of
Bill Frost at Texas — Houston.

BARLOW, R.: It may be of interest
to point out that in the Linntlns lat-
eral eye there is a possibly analogous
situation to R15 bursting. Theoreti-
cal analyses by Hartline, Ratliff, and
Knight showed that the system of
equations that describe the response
of the eye does not have a unique so-
lution, and for a long time it wasn't
clear what that meant. In our experi-
ments we found that the sensitivity
of the cells to inhibition depended on
the cell's own level of excitation.
This introduced a striking nonlinear-
ity into the formulation, which indi-
cates that the eye will go into oscilla-
tion. All receptors in the eye are re-

sponding in synchrony, oscillating at
about 5/s. This is a network property
rather than the property of an indi-
vidual cell. It's almost exactly as you
describe for R15.

BAXTER: We have extended our
analysis to small networks of RI5,
from both a computational and ex-
perimental standpoint. In the com-
puter simulations, we can take R 1 5s,
hook them together and manually
"position" them in a region of their
parameter space, where the individ-
ual cells do not support multistabil-
ity, yet the network can. If one com-
bines multistability at the cellular
level with the network level, the
combinations and perturbations be-
come quite complex.

ELINSON: Dr. Baxter, I'm quite
unfamiliar with this kind of thing,
but all of those phases seem rather
scary. It seems as though any hy-
pothesis would be possible. Is there
some way of predicting which is the
strongest track and which are less
likely?

BAXTER: I guess what you are get-
ting at is the question. How attrac-
tive is an attractor? How strong is
one attractor as compared to an-
other? That's very difficult to answer
with the full model because it's such
a high-dimensional system, so we
have addressed the question in two
ways. One is to reduce the model.
We have reduced it down to a four-
variable model, which allows us to
do analytical system analysis and
quantitatively evaluate the attrac-
tors. Second, with the full system.

170

NLUROBIOLOGY/SENSORY BIOLOGY

171

one can do a random search of the
state space by just creating, for exam-
ple, 1000 random combinations of
initial conditions, and then running
simulations with each set of values
and determining the relative fre-
quency with which the system stabi-
lizes in a given attractor. Our think-
ing is that, if you have a very big at-
tractor or a very strong attractor,
you'll end up there most often. So
you can get some feeling for how
large an attractor is. I don't remem-
ber, when I showed you the eight or
so attractors. what their relative
strengths are. I think the beating and
the outer bursting were very strong
attractors, but that's going to vary
under conditions of modulation as
well.

BAKER: Dr. Fischer, how do you
envision the tail shock acting to
block that response?

FISCHER: We think that the tail
shock affects calcium channels on
the presynaptic terminals of the L30s
and thus regulates presynaptic facili-
tation. We have carried out a num-
ber of studies to show that the en-
hancement in the L30s is strictly de-
pendent upon the levels of
intracellular calcium. What's impor-
tant is how much calcium you get in
the cell per activity. Tail shock and

serotonin seem to act on a calcium
channel, such that less calcium gets
in per unit activity.

MORRIS: Dr. Baxter, when you re-
duce the system from 1 1 to 4 dimen-
sions to be able to look at it analyti-
cally, you still have very serious deci-
sion making to do. Which seven
parameters are you going to make in-
stantaneous and which are you going
to let run?

BAXTER: We don't do this arbi-
trarily. This was an important part of
Robert Butera's Ph.D. thesis. In gen-
eral terms, what we did was to divide
the system into slow and fast sys-
tems. The slow system comprises the
conductances underlying oscilla-
tions, and the fast system comprises
the conductances underlying the ac-
tion potentials. We then collapsed
the fast system into a single variable
that represents the perturbation of
the fast onto the slow system. Thus,
the reduced model has three vari-
ables describing the slow system, and
one variable represents this lumped
effect of the fast onto the slow. We
then tested the reduced model and
compared its output to that of the
full model. The reduced model re-
sponded to modulatory transmitters
in the same way as the full system
and also manifested multistability.
Thus, the reduced model captured

many of the salient features of the
full system.

COMMENT: I want to say for the
record that Jim Blankenship's lab
has shown that serotonin facilitates
motor activity in feeding behaviors
in Aplysia. Dr. Fischer, has serotonin
been shown to facilitate motor activ-
ity in vertebrates in contrast to inver-
tebrates?

FISCHER: I can't really speak for
the vertebrate work. Serotonin has a
lot of effects within the feeding cir-
cuits, where it can facilitate both
ends of the neuromuscular junction.
It also affects siphon withdrawal re-
flex facilitation, which is probably
the most famous form of plasticity
studied in this animal; but you see
inhibition of inhibitory interneurons
as well as facilitation of sensory neu-
rons as a result of serotonin applica-
tion. Thus, one neuromodulatory
system seems to perform whatever
function it has to do to get the re-
sponse up. It can up regulate sensory
neuron transmission and motorneu-
ron activity in this system, but con-
versely it decreases inhibition. I
don't think it's appropriate to speak
of serotonin as having a net facilitory
or net inhibitory role, it just pro-
duces reflex facilitation and, at least
in our hands, does what it has to do
to accomplish this.

Reference: B'wi Bull. 192: 172-174. (February. 1997)

Getting a Head in Frog Development

RICHARD P. ELINSON
Department of Zoology. University of Toronto, 25 Harbord Street, Toronto. Canada AI5S 3G5

The development of a head in frog embryos depends
on the vegetal cortex of the egg. This at first seems odd
because the head develops on the opposite side of the
egg, close to the animal cortex. Nonetheless, the vegetal
cortex not only is the repository of interesting localized
RNAs, but it also causes the cytoplasmic movement that
initiates a chain of events, culminating in the head.

During oogenesis, RNAs are specifically transported
to the vegetal cortex (Forristall et a/.. 1995; Kloc and Et-
kin, 1995). These RNAs are of two types. One type is
involved in formation of germ cells and the other in for-
mation of the head and other dorsoanterior structures.
Germ cells arise from germ plasm, located in the vegetal
cortex. Xcat-2 RNA is localized to the germ plasm, and
like nanos RNA in Drosophila, with which it has se-
quence similarity, it may be involved in germ cell forma-
tion (Mosquera el ai. 1993). Other RNAs, such as Vgl
and Xwnt-1 1, are spread more evenly along the vegetal
cortex. Processed Vgl protein is an inducer of dorsal
mesoderm, and both it and Xwnt- 1 1 RNA can cause the
formation of a dorsoanterior axis experimentally
(Thomsen and Melton, 1993: Ku and Melton, 1993).
These or other RNAs are the likely source of a dorsaliz-
ing activity found at the vegetal cortex in the mature egg,
ready for fertilization (Fujisue et a!., 1993; Holowacz
andElinson, 1993).

The Cortical Rotation, Gravity, and
Dorsoanterior Development

An hour or so after fertilization, the entire egg cortex
rotates by 30° relative to the cytoplasm. This rotation es-

This paper was originally presented at a workshop titled The i'ltiurc
nl tqnatic Research in Space Neurobiology, Cellular ami Molecular
Biology. The workshop, which was held at the Marine Biological Lab-
oratory. Woods Hole, Massachusetts, from 13 to 15 May 1996. was
sponsored by the Center for Advanced Studies in the Space Life Sci-
ences at MBL and funded by the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

tablishes the dorsoanterior axis and depends on a tran-
sient array of parallel microtubules at the vegetal cortex
(Elinson and Rowning, 1988; Larabell el ai. 1996). A
kinesin-like protein is associated with the microtubules
and is thought to move the cortex along the microtu-
bules. which are anchored in the cytoplasm (Houliston
etai, 1994).

This critical rotation can be influenced by gravity in
several ways. First, extremes of gravity, caused by cen-
trifugation, can overcome the microtubule mechanism
and produce a dorsoanterior axis on the centripetal side
(Black and Gerhart, 1 985). Second, gravity, acting alone,
can produce a dorsoanterior axis in the absence of the
microtubule mechanism (Scharf and Gerhart, 1980).
Third, gravity alone can orient the microtubules prior to
their formation, thereby directing where the dorsoanter-
ior axis will form (Zisckind and Elinson, 1990). Gravity
in these cases acts by moving the heavy yolk-rich cyto-
plasm downwards, producing a cytoplasmic re-
arrangement.

These gravity effects have led to repeated attempts to
place frog eggs in space to see how they develop in zero
gravity. In the most successful of such experiments, there
was little or no perturbation of the dorsoanterior axis
(Souza et ai. 1995). A normal head formed, indicating
that some form of cytoplasmic rearrangement occurred.
This arrangement was probably due to the functioning of
the parallel microtubule mechanism. One would guess,
although it has not been shown, that the various cyto-
plasmic regions of different densities retained their iden-
tity (Smith and Neff, 1986) thanks to the egg cytoskele-
ton.

Dorsalizing Activity and the Dorsoanterior Axis

When cytoplasm is withdrawn from the vegetal cortex
of the fertilized egg and microinjected into the ventral
side of a cleaving embryo, an extra head and other dor-
soanterior structures form. There is some connection be-

172

DEVELOPMENTAL BIOLOGY

173

tween this dorsalizing activity in the fertilized egg and
the components of the vegetal cortex of the oocyte, since
UV irradiation of the oocyte eliminates the later dorsal-
izing activity (Holowacz and Elinson. 1993). An attrac-
tive model is that an RNA, localized to the vegetal cortex
of the oocyte, is translated during oocyte maturation to
produce a protein with dorsalizing activity in the vegetal
cortex of the fertilized egg.

Induction of a dorsoanterior axis, such as occurs with
injected vegetal cortex, can be due to one of two activi-
ties: mesoderm induction or competence modification
(Elinson and Holowacz, 1995). Some molecules, such as
processed Vgl protein or activin, are mesoderm induc-
ers. They induce the formation of dorsal mesoderm. thus
generating a dorsoanterior axis. Other molecules, such as
Xvvnt-8 RNA, are competence modifiers. They convert
ventral mesoderm to dorsal mesoderm, and in so doing,
they can create a complete dorsoanterior axis, including
a head. The teratogen lithium acts in this way. The dor-
salizing activ ity of the vegetal cortex behaves more like a
competence modifier than a mesoderm inducer (Holo-
wacz and Elinson. 1995).

The cortical rotation moves the dorsalizing activity to
an asymmetric equatorial position in the egg (Fujisue et
al.. 1993), where its activity specifies the dorsoanterior
axis. The way that the dorsalizing activity is brought into
position is not important, because microinjection or
gravity-mediated rearrangements can substitute for the
normal microtubule mechanism. In zero gravity, the mi-
crotubule mechanism should still function.

Why Can Gravity Substitute for
the Microtubule Mechanism?

After fertilization, the whole frog egg rotates in its jelly
capsule (note this involves the whole egg, not just the
cortex, and occurs earlier than the cortical rotation). As
a result, the animal-vegetal axis of the fertilized frog egg
is perpendicular to gravity, and there is no asymmetry
of gravitational force on the yolk mass to produce the
cytoplasmic rearrangement required for specifying the
dorsoanterior axis. If gravity normally plays no role in
this specification, why can it have an effect? One possi-
bility is that gravity-induced rearrangement is an evolu-
tionarily primitive mechanism, which has been super-
seded by the microtubule mechanism. If there are any
frogs lacking the microtubule mechanism, their eggs
would be interesting objects to put in space, with zero
gravity.

We have limited knowledge of the mechanism of cor-
tical rotation in different species, but one place to look
would be in frogs with large eggs (Elinson et al.. 1990).
Most terrestrially breeding frogs have eggs greater than
3 mm in diameter, more than 1 0 times the volume of the

1 .3-mm egg ofXenopus laevis, the current model for frog
development. The way that these species specify dor-
soanterior development is unknown, but it is easy to
imagine a role for gravity. First, it seems unlikely that
these huge eggs could quickly set up a large microtubule-
based system powerful enough to cause a massive corti-
cal movement. Second, a slight asymmetry of the huge
yolk mass is all that would be needed to shift the cyto-
plasm and set up a dorsoanterior axis.

Frogs with large eggs usually breed on land, so they are
out of the domain of this conference on aquatic research
in space. The eggs can. however, develop in water (Elin-
son, 1987) and may be intriguing to examine under zero
gravity conditions.

Literature Cited

Black, S. D., and J.C. Gerhart. 1985. Experimental control of the
site of embryonic axis formation in Xenopits laevi.\ eggs centrituged
before first cleavage. Dt-r. Biol. 108: 3 10-324.

I linsun. R. P. 1987. Fertilization and aqueous development of the
Puerto Rican terrestrial-breeding frog. Eleullieroiltictvlus coi/i/i. .1
Morphol. 193:217-224.

Elinson, R. P., and T. Holowacz. 1995. Specifying the dorsoanterior
axis in frogs: 70 years since Spemann and Mangold. Curr. Top. Dev
Biol. 30: 253-285.

Elinson, R. P., and B. Rowning. 1988. A transient array of parallel
microtubules in frog eggs: potential tracks for a cytoplasmic rota-
tion that specifies the dorso-ventral axis. Dev. Biol. 128: 185-197.

Elinson, R. P., E. M. Del Pino, D. S. Townsend, F. C. Cuesta, and P.
Eichhorn. 1990. A practical guide to the developmental biology
of terrestrial-breeding frogs. Bin/ Bull 179: 163-177.

Eorristall, C., M. Pondel, L. Chen, and M. L. King. 1995. Patterns of
localization and cytoskeletal association of two vegetally localized
RNAs. Vgl and Xcat-2. Development 121: 201-208.

Eujisue, M., V. Kobayakawa, and K. Yamana. 1993. Occurrence of
dorsal axis-inducing activity around the vegetal pole of an un-
cleaved Xenopits egg and displacement to the equatorial region by
cortical rotation. Development 118: 163-170.

Holowacz, I., and R. P. Elinson. 1993. Cortical cytoplasm, which in-
duces dorsal axis formation in Xenopu.\, is inactivated by UV irra-
diation of the oocyte. Development 119: 277-285.

Holowacz, T., and R. P. Elinson. 1995. Properties of the dorsal activ-
ity found in the vegetal cortical cytoplasm ofXenopits eggs. Devel-
opment 121:2789-2798.

Houliston, E., R. LeGuellec, M.Kress, M. Philippe, and K. LeGuellec.
1994. The kinesin-related protein Eg5 associates with both in-
terphase and spindle microtubules during \enopii.f early develop-
ment. Dev. Biol. 164: 147-159.

Kloc, M., and L. D. Etkin. 1995. Two distinct pathways for the local-
ization of RNAs at the vegetal cortex in Xenopim oocytes. Develop-
menl 121: 287-297.

Ku, M., and D. A. Melton. 1993. \vvnt-l I: a maternally expressed
Xenopu\ uwgene. Development 119: 1 161-1 173.

Larabell, C., B. A. Rowning, J. Wells. M. \Vu, and J. C. Gerhart. 1996.
Confocal microscopy anal) sis of living .Yi'myw.v eggs and the mech-
anism of cortical rotation. Development 122: 1281-1289.

Mosquera, L., C. Korristall, Y. /hou, and M. L. King. 1993. A
mRNA localized to the vegetal cortex ofXenopus oocytes encodes
a protein with a nanos-like zinc finger domain. Development 117:
377-386.

Scharf, S. R., and J. C. Gerhart. 1980. Determination of the dorsal-

174 FUTURE OF AQUATIC RESEARCH IN SPACE

ventral axis in eggs of Xenoptts A.<T;.V complete rescue of uv-im- development in the \irtual absence of gravity. Proc. Nail. Acatl. Sci.

paired eggs by oblique oru-n!. > m before first cleavage. Dcv. Bml U.S.A. 92: 1975-1978.

79: 181-198. Thomsen, G. H., and D. A. Mellon. 1993. Processed Vgl protein is

Smith, R. O, and A. \V. NeiV. i486. Organisation of Xcnoptu eggcy- an axial mesoderm inducer in Xcnopus. Cell 74: 433-441.

toplasm: response I ---..iluted microgravity. J. E.\p. Zoo/. 239: Zisckind, N., and R. P. Elinson. 1990. Gravity and microtubules in

365-378. dorsoventral polarization of the Xmopus egg. Dev. Growth Dili 32:

Souza, K. A., S. D. Black, and R. J. Wassersug. 1995. Amphibian 575-581.

Reference: Bin/. Bull 192: 175-177. (February, 1997)

Fate Specification Along the Sea Urchin Embryo
Animal- Vegetal Axis

ROBERT C. ANGERER AND LYNNE M. ANGERER
Department of Biology, University of Rochester, Rochester, New York 14627

Introduction

Like those of a large majority of taxa, sea urchin em-
bryos establish a spatial coordinate system for the initial
body plan from one axis, the animal-vegetal (A-V). that
is fixed during oogenesis by asymmetric deposition of
maternal molecules (the embryologists" "determinants")
and a second axis, dorsal-ventral (or, more descriptively,
oral-aboral), that is specified sometime during the first
few cleavage divisions (reviewed by Davidson. 1989).
The ability of sea urchin embryos to establish these axes
while continuously reorienting in culture suggests that
neither axis is sensitive to the earth's gravitational field.
In embryos of many sea urchin species, A-V polarity is
evidenced by the unequal sizes of blastomeres of the 16-
cell embryo, which consists of tiers of eight mesomeres,
four macromeres, and four micromeres. Classical exper-
imental micromanipulations of embryos (reviewed by
Horstadius, 1973). have established that the fates of mi-
cromeres are determined by inheritance of maternal
molecules. In addition, the micromeres provide a vegetal
focus of inductive influence that is critical in the normal
embryo for appropriate specification of fates of overlying
animal blastomeres. and that can induce vegetal differ-
entiation (gut, secondary mesenchyme) in cells of more
animal tiers when micromeres are transplanted to ec-
topic sites (Khaner and Wilt. 1991: Ransick and David-
son, 1993). Thus, specification of fates along the AV axis
utilizes both major mechanisms familiar to developmen-
tal biologists— inheritance of maternally provided posi-

This paper was originally presented at a workshop titled The Future
u/' Aquatic Research in Spiice: Ncurohiolony. Cellular and Molecular
Biology. The workshop, which was held at the Marine Biological Lab-
oratory. Woods Hole. Massachusetts, from 13 to 15 May 1996. was
sponsored by the Center for Advanced Studies in the Space Life Sci-
ences at MBL and funded by the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

tional information, and communication among cells,
presumably by ligand-receptor interactions.

Studies with the Very Early Blastula Gene Set

The Very Early Blastula (VEB) gene set provides an
entree to the mechanism of maternal determination of the
A I " developmental axis. Several years ago, we used sub-
tractive cDNA screening methods to identify a set of
mRNAs encoded by four different genes expressed tran-
siently in the very early blastula ( ~ 1 50 cells), and absent
from both the maternal pool of mRNAs and the message
complement of later differentiated cells (Reynolds el ai.
1 992). Surprisingly, the mRNAs identified by this simple
temporal screen accumulate in the embryo with congru-
ent spatial patterns and very similar temporal patterns.
These genes, which we named the VEB genes, have two
important features. First, the messages they encode ac-
cumulate asymmetrically along the AV axis, being pres-
ent throughout the animal —85% of the embryo but ab-
sent from the region around the vegetal pole. Second, the
messages accumulate in embryonic blastomeres that are
continuously separated beginning at the 2-cell stage. This
latter property strongly suggests that activation of VEB
gene expression, and consequently the asymmetry of ac-
cumulation, is regulated by a corresponding asymmetric
distribution of maternal regulatory activities that corre-
sponds to part of the molecular mechanism that estab-
lishes this axis. Our approach is to investigate the cis-
acting elements in the regulatory apparatus of the VEB
genes, and the /ram-acting factors that drive their expres-
sion, to identify these critical molecular components.

Regulatory regions of the SpHE <///<7SpAN genes iden-
tify multiple positively acting factors that drive nonvege-
tal expression. We have analyzed the regulatory regions
of two of the VEB genes: SpHE. which encodes the
hatching enzyme (Wei el ai. 1995), and SpAN (Kozlow-

175

176

FUTURE OF AQUATIC RESEARCH IN SPACE

ski el a/., 1996), which encodes an astacin-family prote-
ase that is likely to be involved in regulating the activities
of cell signaling molecules (see below). These analyses
include functional assays of expression of VEB pro-
moter-driven transgenes microinjected into sea urchin
eggs that are then allowed to develop into embryos, as
well as standard //; vitro assays of interaction of VEB pro-
moter cis elements with proteins in nuclear extracts.

These studies have demonstrated that about 300 bp of
sequence upstream of the transcription initiation site of
Sp/IE and SpAN are each sufficient to drive expression
of a /j-galactosidase reporter in the correct nonvegetal
pattern — i.e.. expression of the transgene is never ob-
served in primary mesenchyme cells (PMCs) derived
from the vegetal micromeres. Despite the congruence of
expression of SpAN and SpHE. and the similar, compact
structure of their regulatory regions, the repertoire of
positively acting factors regulating these genes is rather
different. On the basis of rate of accumulation of tran-
scripts, the endogenous SpHE promoter is very strong.
Reflecting this characteristic, it carries an apparent ex-
cess of positive regulatory sites (Wei el til., 1995): The
region within 300 bp of the transcription start site con-
tains at least nine sites of protein binding which interact
with six different factors. No one cis element appears to
be critical for expression since replacement or deletion
of individual sites decreases promoter activity no more
than about twofold. Mutation of combinations of cis el-
ements has little effect until three or four are inactivated.
In contrast, the SpAN promoter contains fewer sites of
protein-DNA interaction, two of which are essential for
high transgene transcriptional activity (Kozlowski el ul..
1996).

The multiplicity of positive factors driving VEB gene
transcription argues against the simple hypothesis that
a single spatially restricted, positive factor regulates the
nonvegetal accumulation of VEB mRNAs. At least three
mechanisms could achieve spatial regulation of the VEB
genes: ( 1 ) the presence of a vegetal negative regulator
sufficient to prevent expression: (2) exclusion of the ma-
jority of positive regulators from vegetal pole cytoplasm;
and (3) repression of the VEB genes in vegetal cells by
means of a unique chromatin structure. To test for a neg-
ative regulator operating in the vegetal region, we made
a series of deletions across the -300 SpHE promoter.
None of these led to ectopic expression in PMCs, indi-
cating that if a negative mechanism operates, it must in-
volve multiple, independently sufficient regulatory sites.
A vegetal-specific repressive chromatin structure might
be established in micromeres which, as a result of une-
qual cleavage at 16-cell stage, would inherit a lower
quantity of any uniformly distributed positively acting
factors that were stockpiled in the egg cytoplasm. Con-
sistent with this hypothesis, micromeres have a distinc-

tive bulk chromatin structure that is less accessible to
nuclease digestion than is that of the larger blastomeres
(Cognetti and Shaw. 1981). Furthermore, these small
vegetal cells are believed to contain a special cytoplasmic
domain, as reflected by a loss of the pigment granules
that are tethered in the egg cortical cytoskeleton
(Schroeder, 1980). Experiments are underway to dis-
criminate among these possibilities by examining the
DNA-binding proteins present in separated animal and
vegetal blastomeres, as well as by using ;/; vivo foot-
printing coupled with ligation-mediated PCR to exam-
ine actual occupancy of cis elements in animal and veg-
etal blastomeres.

SpAN may function to convert maternal gene regula-
tory information asymmetrically arrayed along the Al'
a.\is inlo spat in! regulation of cell-cell interactions. Be-
cause the sea urchin embryo cellularizes rapidly, and be-
cause the fates of all but the most vegetal blastomeres
(micromeres) are context-dependent, the maternal AV
asymmetry must be translated into, or supplemented by,
extensive cell-cell interactions. Interestingly, one of the
VEB genes, SpAN. encodes a member of the astacin pro-
tease family. Other members of this family include Dro-
sophila tolloid (Schimell el a/.. 1 99 1 ) and tolloid related
(Nguyen el til.. 1994; Finelli el al.. 1995). These metal-
loendoproteases consist of an astacin protease module
linked to domains expected to be involved in protein-
protein interactions (complement Clr/s; EOF), imply-
ing that they may function as tethered proteases. Tolloid
has been shown to interact genetically with decapen-
lap/egic (Ferguson and Anderson, 1992a, b), a member
of the TGF/i superfamily of signaling molecules, and
both of these genes are essential for regulating the fates of
cells along the dorsal-ventral axis in the early Drosophila
embryo. The only vertebrate astacin protease identified
to date, BMP-1, has recently been shown to be a colla-
gen-processing enzyme (Kessler el al.. 1996). However,
a member of the TFG/j superfamily. BMP4 (which is
functionally homologous to dpp: Padgett el al., 1993),
has been strongly implicated in regulating mesodermal
patterning along the DV axis of Xenopus embryos (re-
viewed by Hogan el ai. 1994). Because BMP4 is synthe-
sized as a pro-protein, like other members of the TGF-/3
family, it presumably requires activation by proteolytic
cleavage, and astacin proteases are potential candidates.
We have therefore explored the effect of over- or misex-
pressing Xenopus BMP4 and sea urchin SpAN in early
sea urchin embryos by injecting the corresponding
mRNAs into unfertilized eggs. Both proteins cause sim-
ilar and dramatic phenotypic effects, interpreted most
simply as suppression of differentiation of vegetal struc-
tures. Misexpression in Xenopus embryos of both BMP4
and SpAN also perturbs the dorsal-ventral axis leading
to ventralized phenotypes. We hypothesize that SpAN

DF.VEIOPMENTAL BIOLOGY

177

operates in sea urchin embryos in a BMP4-like pathway
which functions to limit the range of inductive influence
emanating from the vegetal pole. In this way, the SpAN
gene, through its regulation by maternal factors, could
convert maternal spatial information into regulation of
cell signaling along the AV-axis.

Acknowledgments

This work was supported by NIGMS grant GM25553.

Literature Cited

Cognetti, G., and B. R. Shaw. 1981 . Structural differences in the chro
matin from compartmentalized cells of the sea urchin embryo:
differential nuclease accessibility of micromere chromatin. Nucleic
Acids Res. 9: 5609-5621.

Daiidson, E. II. 1989. Lineage specific gene expression and the regu-
lative capacities of the sea urchin embryo: a proposed mechanism.
Development 105:421-445.

Ferguson, E. 1... and k. V. Anderson. I992a. Decapemaplegk acts as
a morphogen to organize dorsal-ventral pattern in the Drosophila
embryo. O// 71: 45 1-461.

Ferguson, E. 1,., and K. V. Anderson. 1 992b. Localized enhancement
and repression of the activity of the TGF-/J family member, ilcca-
penlaplegic, is necessary for dorsal-ventral pattern formation in the
Drosophila embryo. Development \ 14: 583-547.

Finelli. A. 1,., T. Xie, C. A. Bossie, R. k. Blackman, and R. \V. Padgett.
1995. The tolkin gene is a tolloid-BMP-1 homologue that is essen-
tial for Drosophila development. Genetics 141:271 -28 1 .

llogan, B. 1.., M. Blessing, G. E. \\innier, N. Sizuki, and C'. M.. Jones.
1994. Growth factors in development: the role of TGF-beta re-
lated polypeptide signaling molecules in embryogenesis. AT
Sup/'/: 53-60.

llorstadius, S. 1973. Chapter 6 in Experimental Embryology of Echi-
noilcrms Oxford Llniv. Press (Clarendon), New York.

kessler, K., k. I akahara, I Biniaminov, M. Brusel, and D. S.
Greenspan. 1996. Bone morphogenetic protein- 1: the type I pro-
collagen C-proteinase. Science 271: 360-362.

Khaner, O., and F. Wilt. 1991. Interactions of different vegetal cells
with mesomeres during early stages of sea urchin development. De-
velopment 112:881-890.

kozlonski, I). .)., M. I.. Gagnon, J. Marchant, L. M. Angerer, S. D.
Reynolds, and R. C. Angerer. 1996. Characterization of a SpAN
promoter sufficient to mediate correct spatial regulation along the
animal-vegetal axis of the sea urchin embryo. De\'. Bin/. 176: 95-
107.

Nguyen, T., J. Jamal, M. J. Shimell, k. Arora, and M. B. O'Connor.
1994. Characterization of tolloid-related-1: a BMP-1 -like product
that is required during larval and pupal stages of Dros< iriliilu devel-
opment. AT Biol. 166: 569-5X6.

Padgett, R. \\ ., J. M. Wozney, and \V. M. Gelbart. 1993. Human
BMP sequences can confer normal dorsal-ventral patterning in the
Drosophila embryo. Proc Sail. .had. Sci. I'SA 90: 2905-2909.

Ransick, A., and E. II. Daudson. 1993. A complete second gut in-
duced by transplanted micromeres in the sea urchin embryo. Sci-
ence 259: I 134-1 138.

Reynolds, S. D., L. M. Angerer, J. Palis, A. Nasir, and R. C. Angerer.
1992. Early mRNAs. spatially restricted along the animal-vegetal
axis of sea urchin embryos, include one encoding a protein related
totolloidand BMP- 1. Development 114: 769-786.

Schroeder, T. E. 1980. Expression of the pretertilization polar axis in
sea urchin eggs. AT. Bio/. 79: 428-443.

Shimell, M. J., E. L. Ferguson, S. R. Childs, and M. B. O'Connor.
1991. The Drosophila dorsal-ventral patterning gene In/Inn/ is re-
lated to human bone morphogenetic protein- 1. Cell 67: 469-481.

\Vei,Z.,L. M. Angerer, M. L.Gagnon.and R. C. Angerer. 1995. Char-
acterization of the SpllE promoter that is spatially regulated along
the animal-vegetal axis of the sea urchin embryo. AT, Biol 171:
195-21 1.

Reference: Bin/. Bull 192: 178. (February. IW)

Expression and Regulation of a Sea Urchin Msx Class

Homeobox Gene: Insights Into the Evolution and

Function of a Gene Family That Participates in the

Patterning of the Early Embryo

ROB MAXSON, HONGYING TAN, SONIA L. DOBIAS, HAILIN WU,
JEFFERY R. BELL, AND LIANG MA

Department oj Biochemistry and Molecular Biology, A". R. Norris Cancer Hospital. University of
Southern California School of Medicine, 1441 Eastlake Avenue, Los Angeles, California 90033

Inductive tissue interactions, in which signals from
one tissue alter the fate of another, are an essential fea-
ture of the development of all metazoans. Secreted and
membrane-bound signaling molecules, their receptors,
and a series of intracellular signal transducers are key
molecular components of inductive interactions.
Equally important are systems of transcription factors
that respond to and regulate such intracellular signaling
pathways (Davidson, 1990). Among these transcription
factors are the Msx proteins. Named for the Drosophila
insh. the Msx proteins are homeodomain-containing
DNA-binding proteins that can function as both tran-
scriptional activators and repressers (Davidson, 1995).
Characterized by a distinct and highly conserved homeo-
domain, Msx proteins have been identified in a wide va-
riety of metazoans from vertebrates to coelenterates. Al-
though there is evidence that they participate in induc-
tive tissue interactions that underlie vertebrate
organogenesis, including those that pattern the neural
crest, there is little information about their function in
simple deuterostomes.

To learn more about the ancient function of Msx
genes, to shed light on the evolution of developmental
mechanisms within the lineage that gave rise to verte-
brates, and finally, to provide a molecular baseline for
assessing the effects of microgravity on key regulatory

This paper was originally presented ;it a workshop titled The Future
of Aquatic Research in Spun' Veuroi - -ln.vy. Cellular anil Molecular
Biology. The workshop, which was hcKl at the Marine Biological Lab-
oratory, Woods Hole. Massachusetts, from 13 to 15 May 1996, was
sponsored by the Center lor Advanced Studies in the Space Life Sci-
ences at MBL and funded by the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

events of embryonic patterning, we have isolated and
characterized Msx genes from ascidians and echino-
derms(Mai'/«/., 1995; Dobias el a/., 1996). We demon-
strate that portions of the Msx protein sequence, includ-
ing the homeodomain and flanking amino acids, are
highly conserved across wide phylogenetic distances.
Also conserved, particularly among echinoderms, ascid-
ians, and vertebrates, are aspects of Ats.\ expression pat-
terns during early embryogenesis. In an analysis of Msx
gene function, we show that overexpression of a sea ur-
chin Alsx gene (SpMs.\) causes profound anomalies in
gastrulation and spiculogenesis, but does not alter the
specification of the major embryonic territories. Further,
SpMs.\. like its mammalian congeners, is regulated by
members of the BMP family (TGF-/CJ superfamily) of sig-
naling molecules, and that the downstream targets of
SpAIsx likely includes components of the extracellular
matrix. We conclude that Alsx genes function in signal-
ing processes that underlie patterning of early embryos.
We suggest that Msx gene activity may provide a useful
marker for inductive interactions that may be perturbed
during the stresses of space flight.

Literature Cited

Davidson. D. 1995. The function and evolution of Mx.\ genes:
pointers and paradoxes. Trends Gene/. 1 1 : 405-4 1 I

Davidson. K. II. 1990. How embryos work: a comparative view of
diverse modes of cell fate specification. Dc-celupmenl 108: 365-369.

Dobias, S. L., L. Ma, J. R. Bell, and R. Maxson. 1996. Expression
and regulation of a sea urchin .l/s.v class homeobox gene: insights
into the evolution of A/.v.vgene function. Mceh. Dcv. In press.

Ma, L., B. J. Swalla, J. /hou, J. Chen, R. Maxson, and VV. Jetfery.
1995. Expression of an A/.s.v homeobox gene in ascidians: devel-
opmental insights into the origin and evolution of vertebrates. Dev.
Dm. 205: 308-318.

178

Reference: Biol Bull 192: 179-180. (February. 1997)

Discussion

NICK: Dr. Elinson. you have
shown that the microtubules may
not be necessary for establishing the
dorsal-ventral axis, that gravity
alone can do the job. Maybe the ar-
rangement of the microtubules is re-
sponding to gravity, and by this we
have a sort of self-enhancing loop. It
may not be a cause of the axis, but it
may be the means of perceiving the
effect of gravity in this system.

ELINSON: I showed that when you
irradiate with UV and then tilt the
egg. the microtubules don't re-form.
So that's a microtubule-free system
that's working. I do know that there
is a model by Tabony and Job that
you cited on microtubule patterning
by gravity. The time course of that
model just doesn't fit here. The
whole array forms in a couple of
minutes, and I think it took hours for
Tabony and Job to get some kind of
array generating. I don't think that's
involved here.

ANGERER, R.: I want to ask Rick
(Elinson) about the big-egg-little-egg
experiment. It seemed to me that
one interpretation for the lack of de-
pendence on gravity for Xenopus de-
velopment in space is simply that the
motor lies between the cortex and
deeper layers. It's just ratcheting one
against the other, whereas the exper-
imental embryologist needs gravity
to hold the center in place while he
rotates the outside. This means that
gravity will never be sufficient. Even
the big egg is going to require some
kind of microtubule motor, and
would be likely to work the same
way in space (as in a small egg).

ELINSON: The normal Xenopus
egg is, in effect, suspended in space,
because it's floating in a capsule. I
think that's why you're able to get
this cortex to move relative to the cy-
toplasm. If you have it pinned down,
then yes the cytoplasm will be driven
relative to the cortex. I don't think
that driving can occur in very large
eggs; I would be amazed if it did.
Nonetheless, in the very large egg. it
would just have to be a tiny bit off
center, and you would get a shift of
cytoplasm due to gravity. So why not
take advantage of that opportunity?
That's why I might imagine that such
a huge egg may work with a gravity
mechanism rather than with a mi-
crotubule mechanism.

ANGERER. R.: Don't you think
that you will still need some cyto-
plasmic localization event to make a
shift? The egg pops out and now has
an orientation: nothing is moving.
Now you're saying that something
has to move, so gravity alone
couldn't be sufficient under that cir-
cumstance.

ELINSON: I guess what I'm assum-
ing is that the likelihood of an egg
ending up perfectly vertical — ani-
mal-vegetal— is very small. Just a
tiny little shift off-axis of such a huge
egg would be enough to move some
cytoplasm around and provide the
net effect of an axis. No one knows
anything about these eggs in that re-
gard.

HAMILL: Richard (Elinson), you
said that the cytoplasmic factor you
extracted was onlv from the cortical

layer. Did you get any effect with ma-
terial removed from deeper layers?

ELINSON: The only way we get this
activity is if the pipette is put down
onto the surface of the egg and the
surface is drawn into the pipette,
breaking the membrane. When you
do that, the cortical cytoplasm flows
into the pipette. If you stick your
needle into the egg and remove ma-
terial, you don't get activity.

CASSIDV: Richard (Elinson), has
the embryogenesis and the whole de-
velopmental sequence of the cocjui
been well established already?

ELINSON: Yes, we have a repro-
ductive colony of these frogs in the
laboratory. We are doing embrv olog-
ical and molecular experiments on
them, so I'm suggesting that maybe
we should look at their eggs as well.
The stages have all been worked out:
the egg develops into a little frog
within 3 weeks.

QUESTION: Dr. Maxson, it seems
that when you disrupted the msx
gene in the mice you got several
deficits, but evidently that's not the
case in the humans. Can you address
this point?

MAXSON: In many cases mice
have supernumerary digits. Some
humans with this disease have tri-
phalangeal thumbs. So there does
seem to be a limb defect in humans.
Some of the people in this family
have visual field defects, so it's possi-
ble that there's an eye connection. As
far as anvbodv knows, they have nor-

179

ISO

FUTURE OF AQUATIC RESEARCH IN SPACE

mal hearing and normal \ uilar
function.

ELINSON: Rob (Anuovn. if you
move the micromeres around, do
they inhibit this SpA ! expression?

ANGERER, R.: Christian Gache
has done that experiment, and the
answer is no, they don't. The micro-

meres are not antagonistic to SpAN,
as near as we can tell.

QUESTION: (Dr. Angerer) You get
this mosaic expression with DNA;
do you get it with RNA?

ANGERER, R.: The reason for the
mosaic expression with DNA is this:

The first thing that happens when
you inject the DNA is that 2000 cop-
ies concatenate into a single particle
which integrates sometime in the
first few cell divisions. One person
that I know of, Andy Cameron in
Eric Davidson's lab, injected mRNA
that was labeled and followed it.
That message went uniform rapidly.

Reference: B/W. Bull 192: 1 8 1-1 82. (February, 1997)

Actin-Dependent Pigment Granule Transport in Retinal

Pigment Epithelial Cells

BETH BURNSIDE AND CHRISTINA KING-SMITH

Department of Molecular anil Cell Biology, 335 LSA-3200. L wtrrv/Vr oj California.

Berkeley. California 94720-3200

Introduction

Since diffusion of particles as large as vesicles or organ-
elles is constrained by the high viscosity of cytoplasm,
directed translocation of these particles from one cyto-
plasmic site to another must be achieved by cytoskeletal
intervention. In most cases, this is achieved by attaching
the particle to a molecular motor which then navigates
along a microtubule or an actin filament track (Path
and Burgess. 1994). Although microtubule-dependent
transport in animal cells has been more extensively stud-
ied, important roles for actin-dependent transport have
also recently been recognized (Langford. 1995).

Results and Discussion

Our laboratory has been using the migration of mem-
brane-bound melanin pigment granules in the teleost
retinal pigment epithelium (RPE) as a model system for
studying intracellular particle transport (Burnside el ai.
1983; King-Smith el a/.. 1995. 1996). The RPE is a sim-
ple epithelium that lines the back of the vertebrate eye
and lies between the photoreceptors and the choroidal
blood supply. In the fish RPE, pigment granules undergo
dramatic migrations in response to changes in light con-
dition: they aggregate into the RPE cell body in the dark
and disperse into the cell's long apical projections in the
light, migrating distances as great as 100 ^m. Several
properties of RPE cells make them technically advanta-

This paper was original!) presented at a workshop titled The h'ulure
<>l Aquatic Research in Space: Neitrobiology, Cellular and Molecular
Biology. The workshop, which was held at the Marine Biological Lab-
oratory. Woods Hole. Massachusetts, from 13 to 15 May 1996, was
sponsored by the Center for Advanced Studies in the Space Life Sci-
ences at MBL and funded b\ the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

geous for a study of particle transport. In these cells, pig-
ment granules migrate within fixed apical projections;
thus transport-related cytoskeletal dynamics can be ex-
amined in the absence of change in cell shape. Either pig-
ment granule aggregation or dispersion can be triggered
at will by incubating RPE cells with cAMP or dopamine.
respectively, and pigment granule translocation is slow
enough to permit experimental intervention or biochem-
ical isolation while granules are traveling in either direc-
tion (Burnside and Basinger. 1983; Dearry and Burnside.
1988). Pure preparations of RPE sheets (containing a
single cell type) can be isolated in different functional
states in sufficient amounts to permit both molecular
and biochemical analyses. Finally, normal aggregaton
and dispersion can be induced in single isolated cells in
vitro, thereby permitting analysis of transport kinetics
under different experimental conditions by time-lapse
videomicroscopy (King-Smith et ai. 1995, 1996).

Video analysis has shown that pigment granule move-
ments in RPE cells are slower but otherwise similar to
those previously reported for dermal melanophores(un-
publ. obs.). During dispersion, pigment granule move-
ment is saltatory and bidirectional, with individual pig-
ment granules moving independently; mean centrifugal
and centripetal velocities were 3.2 and 1.5 ^m/min re-
spectively. The net pigment dispersion rate in isolated
cells (LS^m/min) is similar to that observed in vivo
(2.2 ^m/min). After full dispersion is achieved, bidirec-
tional saltations of pigment granules continue within the
apical projections. When aggregation is triggered by
cAMP, all granules undergo a coordinated, smooth, non-
saltatory centripetal movement, with a mean velocity of
3.6 Mm/min, which is comparable to the mean in vivo
velocity of 3.4 //m/min. In the time-lapse movies, other
forms of motility are visible in the apical projections, in-
cluding bidirectional translocations of mitochondria.

181

182

FUTURE OF AQUATIC RESEARCH IN SPACE

formation and migration of cytoplasmic bridges between
projections, and modest e >ns and retractions of

the tips of the project;

To identify the cy'- . ::al mechanisms of RPE pig-
ment migration, v • stigated whether disruption of
microtubules with -i-cadozole would block pigment
granule movemcm in isolated sunfish RPE cells in vitro.
Neither aggregation nor dispersion was inhibited by the
complete disruption of the microtubules of the apical
projections (unpubl. obs.). Microtubule disruption had
no effect on the mean velocities of individual granules,
or on the rate or extent of pigment granule aggregation or
dispersion. Maintenance of the aggregated or dispersed
states was also unaffected by microtubule disruption.
These observations strongly suggest that microtubules
are not required for pigment granule migration in iso-
lated RPE cells.

As a first step toward evaluating the role of actin fil-
aments in RPE pigment granule transport, we also inves-
tigated the effects of cytochalasin D on pigment granule
migration in isolated RPE cells (unpubl. obs.). Net pig-
ment granule aggregation and dispersion were both
strongly and reversibly inhibited by cytochalasin D, the
IC50 for dispersion (0.5 nM] being lower than that for
aggregation (2.5 nM}. Video analysis revealed that cyto-
chalasin has a surprising effect on the movements of in-
dividual pigment granules. Although most granules
stopped moving altogether within minutes of exposure
to cytochalasin. several granules in each projection be-
gan to undergo very rapid (up to 40 ^m/s), bidirectional
excursions. When cytochalasin was applied to isolated
RPE cells in which the microtubules had been previously
disrupted by nocodazole, all pigment granule movement
stopped, suggesting that the very rapid bidrectional ex-
cursions observed in cytochalasin-treated cells are mi-
crotubule-dependent.

These inhibitor studies suggest that actin filaments
play important roles in both aggregation and dispersion,
although the mechanism of actin filament participation
is not clear. Since the effects of cytochalasin D on aggre-
gation and dispersion have different IC50s, the force-
producing mechanisms of the two processes may differ.
The low IC50 for dispersion suggests that interfering with
plus-end assf i.vbK of actin filaments is sufficient to block
centrifugal trans] ort. The higher IC50 for aggregation,
on the other i ests that additional effects of cy-

tochalasin, such as n of actin filament organiza-

tion, may be necessary to block motility in this case. The
implication that both centrifugal and centripetal pig-
ment granule movements are actin-dependent is some-
what surprising, since all known myosin motors move
only toward the plus ends of actin filaments. The roles of
myosin motors and actin filament dynamics in pigment
granule transport are not yet clear.

In parallel with physiological analyses of RPE
transport, we have also identified myosin motor proteins
expressed in fish RPE cells (unpubl. obs.). Using degen-
erate primers based on conserved sequences in the myo-
sin motor domain for RT-PCR. we have identified 1 1
myosin motor proteins that are expressed in teleost RPE.
These include one apparently novel myosin selectively
expressed in RPE and retina, and two selectively ex-
pressed in RPE. We are currently making isotype-spe-
cific antibodies to these motors for isolation of native
proteins and subcellular localization of each myosin iso-
type. Ultimately we plan to analyze the roles of these my-
osin motors in pigment granule transport and other mo-
tile processes of RPE cells.

Acknowledgments

Supported by NIH grant R37-EY03575.

Literature Cited

Burnsidc, B., R. A. Adler, and P. O'Connor. 1983. Rclmomotor pig-
ment migration in the teleost retinal pigment epithelium. I. Roles
for actin and microtubules in pigment granule transport and cone
mo\ement. Invent. Oplnhalmul I 'M. Sei 24: 1-15.

Burnsidc. B., and S. Basingcr. 19X3. Relinomotor pigment migration
in the teleost retinal pigment epithelium. II. Cyclic adenosine 3',
5'-monophosphate induction of dark-adaptive movement in vitro.
///res/ OphllHilnml I'M ,S'c/ 24: 16-23.

Dearry, A., and B. Burnsidv. 1988. Stimulation of distinct D2 dopa-
minergic and alpha2-adrenergic receptors induces light-adaptive
pigment dispersion in teleost retinal pigment epithelium. J Ncuro-
c/iem 51: 1516-1523.

Fath, K. R., and D. R. Burgess. 1994. Membrane motility mediated
by unconventional myosin. C'nrr Opin. Cell Biol 6: 131-135.

king-Smith, C., L. Bost-l'singer, and B. Burnsidc. 1995. Expression
of kinesin heavy chain isoforms in retinal pigment epithelial cells.
Cell . \lnlil. Cylii^e/elii/i 31:66-81.

King-Smith, C., P. Chen, D. Garcia, H. Rey, and B. Burnside. 1996.
Calcium-independent regulation of pigment granule aggregation
and dispersion in teleost retinal pigment epithelial cells. J. Cell Sci.
109: 33-43.

I.angford. G. M. 1995. Actin- and microtubule-dependent organelle
motors: interrelationships between the two motility systems. Curr.
O/'in Cell Bu>l 7:82-88.

Reference: Biol. Bull. 192: 183-185. (February. 1997)

Myosin Drives Retrograde F-Actin Flow in Neuronal

Growth Cones

C. H. LIN*. E. M. ESPREAFICO#, M. S. MOOSEKERt AND P. FORSCHERt

i-Dept. Biology, Yale University, New Haven. Connecticut 0651 l;#Dept. Morphology, Faculdade de
Medicina de Ribeirao Preto-USP, SP, Brazil; *National Yang-Ming University. Taipei, Taiwan

Neuronal growth cones guide axons in the developing
nervous system toward distant target sites. Recent evi-
dence suggests that growth cones decode both diffusible
and substrate-bound molecular signals during the guid-
ance process (Kennedy and Tessier-Lavigne, 1995; Tes-
sier-Lavigne, 1992), which involves pathfinding. branch-
ing, and ultimately target recognition. All of these behav-
iors depend on precise regulation of growth cone
motility. Bray pioneered the investigation of growth
cone motility and was the first to observe that inert par-
ticles placed on the growth cone surface would move in
a retrograde direction, suggestive of an underlying cen-
tripetal membrane or actin filament flux (Bray. 1970).
Early studies also demonstrated that growth cone motil-
ity depends on actin filament assembly (Yamada and
Wessells. 1973), and a critical role for actin assembly in
axonal guidance emerged when axons in the developing
grasshopper nervous system were shown to lose all path-
finding capabilities upon treatment with cytochalasin
(Bentley and Toroion-Raymond. 1986). Later, studies
characterizing actin dynamics in Aplysia growth cones
/// vitro suggested that the retrograde flux mentioned
above is actually due to actin filament assembly at nucle-
ation sites along the leading edge, followed by retrograde
translocation and filament disassembly or recycling by
means of severing at a proximal site. This process was
referred to as "retrograde flow" (Forscher and Lin, 1991:
Forscher and Smith. 1988: Smith, 1988).

The peripheral actin ultrastructure of the growth cone

This paper was originally presented at a workshop titled The Future
o! Ai/uulic Retcarcli in Space: Neitrobiology. Cellular unit Molecular
Biology. The workshop, which was held at the Marine Biological Lab-
oratory. Woods Hole. Massachusetts, from 13 to 15 May 1996, was
sponsored by the Center for Advanced Studies in the Space Life Sci-
ences at MBL and funded by the National Aeronautics and Space Ad-
ministration under Cooperative Agreement NCC 2-896.

has also been well characterized as being composed of
two distinct structural domains: filopodia containing ar-
rays of uniformly polarized (barbed end distal) actin fil-
aments, and intervening lamellipodium domains with
less polarized filament structure (cf. figure 6a; Lewis and
Bridgman, 1992). Both domains appear to exhibit uni-
form retrograde flow relative to an external substrate ref-
erence as judged by actin fluorescence photobleaching
studies (Lin and Forscher. 1995). Assembly of actin fil-
aments, followed by centripetal displacement relative to
the leading cell margin, has been observed in a wide va-
riety of motile cells and appears to be a fundamental
property of directed growth processes and cell migration
(Cramer £>/«/.. 1994; Bray and White. 1988; Fisher*? ai.
1988:Abercrombiei'/<;/.. 1970).

In our initial characterization of actin filament dy-
namics in Aplysia growth cones, we noted that when
cones were treated with 2-5 fiM cytochalasin, retrograde
flow did not appear to be markedly affected. Continued
retrograde flow in the absence of new actin assembly re-
sulted in formation of an F-actin-free gap along the
growth cone margin and eventual clearance (typically in
about 3 min) of F-actin from lamellipodia and filopodia.
This persistent retraction of actin filament networks in
the absence of actin assembly demonstrated that poly-
merization could not be supplying the driving force for
retrograde F-actin flow; therefore, we suggested that a
myosin-like molecular motor might be involved
(Forscher and Smith. 1988). Despite recent molecular
cloning of several brain myosins (Bahler el at.. 1994;
Ruppert el ai. 1993; Cheney el ai. 1993) and localiza-
tion of myosins to growth cones (Rochlin et ai. 1995:
Espreafico et ai, 1992; Cheng et ai. 1992: Miller et ai.
1992), little or no information about the functional role
of myosins in growth cone motility has emerged (cf. Ta-
naka and Sabrv. 1995).

183

184

FUTURE OF AQUATIC RESEARCH IN SPACE

Given the functional implications of our previous
work, we designed experiments aimed at global inhibi-
tion of myosin activity to test whether any myosin was in
fact involved in driving retrograde F-actin flow in growth
cones. All known myosins have an evolutionarily con-
served N-terminal head domain containing the site for
ATP and F-actin binding as well as force generation
(Mooseker and Cheney, 1995). Chymotryptic digestion
of muscle myosin results in cleavage of a head domain
subfragment (SI ) from the rest of the molecule (Margos-
sian and Lowey, 1982). SI exhibits ATP-dependent ac-
tin-filament binding, but lacking the C-terminal tail, is
incapable of generating force unless artificially tethered
(e.g., by an antibody) to a substrate. In addition, further
treatment of SI with the sulfhydryl reagent, A'-ethylma-
leimide (NEM), results in a myosin head species (NEM-
Sl ) that remains tightly bound to actin filaments, even
in the presence of ATP, and thus can serve as a potent

specific inhibitor of actomyosin function (Cande, 1986;
Meeusen and Cande, 1979). Our first experimental ap-
proach then was to competitively inhibit myosin func-
tion by injection of purified SI or NEM-S1 and to look
at effects on growth cone motility. actin dynamics, and
structure.

We compared the results of SI or NEM-S1 injection
with those obtained after treatment with 10-30 m^/2,3-
butanedione-2-monoxime (BDM). a pharmacological
inhibitor of endogenous myosin ATPase activity (Fig.
IB). BDM has previously been shown to affect cross-
bridge kinetics and to inhibit both conventional muscle
and nonmuscle myosin ATPases including myosin V.
platelet myosin II. and adrosophila myosin ATPase frac-
tion without affecting kinesin ATPase activity or actin
assembly (Cramer and Mitchison. 1995; Backx a a/..
1994;McK.illop£V(//., 1994; Schramm el a/.. 1994: Zhao
and Kawai. 1994). We found that both types of myosin

B

120

100

o
01

80

a eo

40 T

20 -

12345
NEM-S1 (mg/ml)

0 10 20 30 40 50
BDM (mM)

'

o

r

. NEM-S1
o BDM

20

40 60

Retrograde flow

100

120

Figure 1. Rales of filopodium growth and retrograde F-actin (low are inverse!) proportional. (A) Silica
beads (200-nm diameter) derivatized with Con-A were applied to the growth cone surface with a single-
beam gradient IR laser trap (red graphic top) and used as a noninvashe tool to quantify F-actin flow rates.
Video sequence shows bead displacement over time ( 1 5-s intervals) under control conditions (green arrow)
and immediately after myosin inhibition with 10 m.U BDM (red arrow). Yellow arrow denotes filopodial
growth. (B) Dose-response curves for NEM-S1 and BDM treatments. Rates are % control retrograde flow.
(C) Rates of lilopodium elongation ycrxii.\ retrograde flow. Data from different experiments (growth cones)
were normalized by control retrograde flow rates.

CYTOSKELETON/CELL MOTII.ITY

185

inhibition produced essentially the same effects: dose-de-
pendent attenuation of retrograde F-actin flow accom-
panied by filopodial and leading edge extension at rates
directly proportional to the degree of flow inhibition
(Fig. 1C). Filopodial growth stimulated by the myosin
antagonists was cytochalasin sensitive, indicating that it
is due to barbed end filament assembly. These experi-
ments demonstrate that retrograde actin flow is indeed
driven by a myosin ATPase. and that actin filament as-
sembly and myosin motors function independently. Our
results suggest that simple superimposition of actin poly-
merization and the action of myosin motors underlie the
process of retrograde F-actin flow.

Literature Cited

Abercrombie. M.,J. K. M. lleaysman, and S. M. Pegrum. 1970. The

locomotion of fihrohlasts in culture. III. Movement of particles on
the dorsal surfaces of leading lamella. E.\i> Cell Rex. 62: 389-398.

Backx, P. H., \V. D. Gao, M. D. Azon-Backx, and E. Marban. 1994.
Mechanisms of force inhibition by 2.3-butanedione rnonoximc in
rat cardiac muscle: role of calcium and crossbridge kinetics. / Pliys-
ml 476: 487-500.

Bahler, M., R. Kroschewski, H. E. Stoffler, and T. Behrmann. 1994.
Rat my r 4 defines a novel subclass of myosin I: identification, dis-
tribution, localization, and mapping of calmodulin-bindmg sites
with differential calcium sensitivity. ./ C 'ell Bml 1 26: 375-389.

Benlley, D.. and A. Toroion-Raymond. 1986. Disordered pathfinding
by pioneer neuron growth cones deprived of nlopodia by cytocha-
lasin treatment, \aiiirc 323: 7 1 2-7 1 5.

Bray, D. 1970. The surface movement during growth of single ex-
planted neurons. Proc. Sail Acini. Sci. L'S.-l 65: 905-910.

Bray. D., and J. G. White. 1988. Cortical flow in animal cells. Science
239: 883-888.

Cande, \\ . /. 1986. Preparation of A-ethylmaleimide-modified
heavy meromyosin and its use as a functional probe of actomyosin-
based motility. Mcl/mi/x Enzymol. 134: 473-477.

Cheney, R. E., M. A. O'Oshea, J. E. Heuser, M. V. Coelho, J. S. \Vo-
Icnski, E. M. Esprcafico, P. Eorscher, R. E. Larson, and M.S.
Mooseker. 1993. Brain myosin-V is a two-headed unconven-
tional myosin with motor activity. CV//75: 13-23.

Cheng, T. P., N. Murakami, and M. Elzinga. 1992. Localization of
myosin MB at the leading edge of growth cones from rat dorsal root
ganglionic cells. h'KBSLell. 311:91-94.

Cramer, I.. P., I. J. Mitchison, and J. A. Theriut. 1994. Actin-depcn-
dent motile forces and cell motility. Curr. O/'in. Cell Bin/. 6: 82-86.

C'ramer, L. P., and I . J. Mitchison. 1995. Myosin is involved in post-
mitotic cell spreading. / Cell Bml. 131: 179-189.

Espreafico, E. M., R. E. Cheney. M. Matteoli, A. A. Nascimento, P. V.
De Camilli, R. E. Larson, and M.S. Mooseker. 1992. Primary
structure and cellular localization of chicken brain myosin-V
(p!90). an unconventional myosin with calmodulm light chains. J.
Cell Bml 119: 1541-1557.

Fisher, G. \\ ., P. A. Conrad, R. L. DeBiasio, and D. L. Taylor. 1988.

Centripetal transport of cytoplasm, actin, and the cell surface in
lamellipodiaoffibroblasts. Cell M»til. Crloxke/e/on 11:235-247.

Eorscher, P., and C. Lin. 1991. Polycationic bead translocation
driven by actin assembly in neuronal growth cones. ./. Cell. Bid
I15:368a.

Korscher, P., and S. J. Smith. 1988. Actions of cytochalasins on the
organization of actin filaments and microtubules in a neuronal
growth cone. J Cell Bml 107: 1505-1516.

Kennedy, T. E., and M. lessier-Lavigne. 1995. Guidance and induc-
tion of branch formation in developing axons by target-derived
diffusible factors. Curr. Opin. Neurobiology 5: 83-90.

Lewis, A. K., and P. C. Bridgman. 1992. Nerve growth cone lamelli-
podia contain two populations of 'actin filaments that differ in orga-
nization and polarity. ./ Cell Bml 119: 1219-1243.

Lin, C.-IL, and P. Korscher. 1995. Growth cone advance is inversely
proportional to retrograde F-actin flow. Neuron 14: 763-77 1 .

Margossian, S. S., and S. Lowey. 1982. Preparation of myosin and
its subfragments from rabbit skeletal muscle. Melh. linivmol 85:
55-71.

Mckillop, K. E., N. S. Eortsne, K. \V. Ranatunga, and M. A. Gecves.
1994. The influence of 2.3-butanedione-2-monoxime on the in-
teractions between actin and myosin in solution and skinned mus-
cle fibers. ./ Mmclc Rex. Cell Molii 15: 309-3 1 8.

Meeusen, R. I.., and \\ . /.Cande. 1979. N-Ethylmaleimide modified
heavy meromyosin: a probe for actomyosin interactions. / Cell
Bml. 82: 57-65.

Miller. M., E. Bower, P. Levitt, D. Li, and P. D. Chantler. 1992. Myo-
sin II distribution in neurons is consistent with a role in growth cone
motility but not synaptic vesicle mobilization. Neuron S: 25-44.

Mooseker, M. S., and R. E. Cheney. 1995. Unconventional myosins.
.•Inmt. Rev Cell l)e\- Bml. 11:633-675.

Rochlin, M. W., K. I. Koh, R. S. Adelstein. and P. C. Bridgman. 1995.
Localization of myosin II A and B isofbrms in cultured neurons. ./
Cell Sci. 108:3661-3670.

Ruppert. C., R. Kroschewski, and M. Bahler. 1993. Identification,
characterization and cloning of myr I. a mammalian myosin-1. ./
Cell Bml. 120: 1393-1403.

Schramm, M., II. G. Klieber, and J. Daut. 1994. The energy expen-
diture of actomyosin ATPase, calcium-ATPase and sodium-potas-
sium-ATPase in guinea pig cardiac ventricular muscle. ./ Physiol.
481:647-662.

Smith, S. J. 1988. Neuronal cytomechanics: the actin-based motility
of growth cones. Science 242: 708-7 1 5.

Tanaka, E., and J. Sabry. 1995. Making the connection: cyloskeletal
rearrangements during growth cone guidance. CV//83: 171-176.

Tessier-Lavigne, M. 1992. Axon guidance b\ molecular gradients.
Curr Onin. Neurobiology 2: 60-65,

Yamada, K. M., and N. K. \\essells. 1973. Cytochalasin B: effects on
membrane ruffling, growth cone and microspike activity, and mi-
crofilament structure not due to altered glucose transport. Dev.
Biol. 31:413-420.

Zhao, Y., and M. Kawai. 1994. BDM affects nucleotide binding and
force generation steps of the crossbridge cycle in rabbit psoas muscle
fibers. Am .1 Plivxiol. 266: C437-C447.

Reference: Biol Bull 192: 186-190. (February. 1997)

Multiple Myosin Motors and Mechanoelectrical
Transduction by Hair Cells

PETER G. GILLESPIE

Departments of Physiology and \cnroscicncc. The Johns Hopkins University.
Baltimore, Maryland 2120?

Hair cells are exquisitely specialized mechanorecep-
tors, responding only to specific frequencies of sound or
to distinct head movements (reviewed in Hudspeth.
1989, 1992). A hair cell carries out mechanoelectrical
transduction with its mechanically sensitive hair bundle,
a beveled collection of stereocilia and one solitary kino-
cilium. Although the kinocilium is a true cilium, with
the familiar 9 + 2 arrangement of microtubule doublets,
stereocilia are actin-based. Stereocilia contain several
hundred cross-linked actin filaments, so they are quite
rigid. A stereocilium pivots at its flexible basal insertion
point, where only a few dozen actin filaments penetrate
the cell. Deflection of the bundle causes adjacent ste-
reocilia to slide along each other, stretching elastic gating
springs that tug open transduction channels located at
the top of the bundle.

Hair cells adapt to sustained stimuli; for example, hair
cells in the bullfrog's sacculus can detect transient verti-
cal accelerations of less than 1CT5 g. despite a constant
1 # stimulus from gravity (Koyama etui., 1982). Gating-
spring tension, and hence channel open probability, is
controlled by adaptation motors, which likely contain
myosin molecules (reviewed in Hudspeth and Gillespie,
1994; Gillespie, 1995). During an excitatory stimulus,
when gating-spring tension is high. Ca:* entering
through open transduction channels triggers the adapta-
tion motors to slip down the cytoskeleton and reduce
tension (Fig. 1). By contrast, during inhibitory bundle

This paper svus originally presented at a workshop titled The l-'u/iirc
»l .U/naln- h in Spiicc \'eurt>hn>la?;y. C'c/lii/iu ami Mok'culur

BioloKY The workshop, which was held at the Marine Biological Lab-
oratory. Woods Hole, Massachusetts, from 13 to 15 May 1996, was
sponsored iy the Center for Advanced Studies in the Space Life Sci-
ences at M I and funded by the National Aeronautic-sand Space Ad-
ministrate i :.T Cooperative Agreement NCC 2-896.

deflections that slacken the gating springs, the motors
climb towards the apical ends of the stereocilia and re-
store tension. The adaptation motor thus acts as a nega-
tive-feedback control mechanism that ensures that gat-
ing-spring tension remains at its optimal level.

To prove that adaptation is carried out by myosin mol-
ecules and to identify the responsible isozyme, we have
taken advantage of years of intensive study of the prop-
erties of skeletal-muscle myosin. We have applied gen-
eral principles derived from muscle actomyosin to de-
sign of experiments for hair cells. The ATPase cycle of
myosin is illustrated in a simplified form in Figure 2. Are
the properties of skeletal-muscle myosin II likely to re-
semble those of a hair-cell myosin, even one of an unu-
sual class? Recent results have suggested that this is so.
Detailed kinetic characterization of two Acanthamoeba
myosin-I isozymes indicates that ATPase hydrolysis uti-
lizes the same mechanism as vertebrate myosin II. Fur-
thermore, the rate and equilibrium constants defining
the amoeba myosin-I cycle are nearly identical to those
of vertebrate myosin II (Ostapand Pollard, 1996). These
results give us confidence that the same will hold true for
the myosin molecules of hair bundle.

To provide evidence that myosin mediates adaptation
in hair cells, we dialyzed hair cells of the bullfrog's saccu-
lus with tight-seal, whole-cell recording electrodes filled
with compounds that should interfere with the ATPase
cycle. In one series of experiments, we introduced into
hair cells adenine nucleoside diphosphates. such as ADP
and the metabolism-resistant analog ADP/^S, expecting
that they would promote the population of the diphos-
phate-bound state of myosin (Gillespie and Hudspeth.
1993). We saw the expected results: adaptation was
blocked, but the transduction currents remained robust,
as if the adaptation motors were arrested along the actin

186

CYTOSKELETON/CELL MOTILITV

187

Displacement

Transduction
current

Bundle
movement

Adaptation motor
Transduction channel
Tip link

Actin cytoskeleton

Rest

(channels
partly
open)

Positive step

(channels all

open)

Adaptation

(channels

mostly

closed)

Overshoot
(channels
all closed)

Recovery

(channels

partially

reopen)

Figure I. Transduction-channel gating and adaptation by hair cells. The response of a hair cell's trans-
duction apparatus to a 100-ms, excitatory displacement is depicted. Transduction channels are gated by
tension in tip links, and tension in tip links is controlled both b\ bundle position and by adaptation-
motor position. When the displacement is applied, transduction channels open and a large inward current
develops. In response to high tip-link tension and the influx of Ca:*, however, the adaptation motor slips
down the actin cytoskeleton. As it does so, tension in the tip link diminishes and channels close. When the
bundle is returned to its rest position, the adaptation motor is caught below its rest position, so tip-link
tension drops precipitously and transduction channels close completely. Responding to the reduced ten-
sion, the adaptation motor restores resting tip-link tension by reascending the actin cytoskeleton. Modified
from Hudspeth and Gillespie ( 1 W4).

filaments, unable to climb or slip. In addition, channel
open probability increased, as the number of myosin
molecules in force-producing states increased. These re-
sults are entirely analogous to the effects of ADP on iso-
metrically contracting muscle fibers, where ADP blocks
both shortening and lengthening, as well as increasing
isometric tension (Cookeand Pate, 1985).

In a second series of experiments, we filled hair cells
with phosphate analogs, reasoning that this should lead
to decreased actin-myosin interaction ( Yamoah and Gil-
lespie. 1996). Phosphate analogs such as vanadate, ber-
yllium fluoride, and sulfate eliminated motor-force pro-
duction, blocked climbing adaptation, and slowed slip-
ping adaptation. The effects of phosphate analogs on
slipping adaptation were quantitatively explained by a
simple model that uses parameter values obtained from
skeletal-muscle myosin.

The remarkable similarity of the effects of adenine nu-
cleoside diphosphates and phosphate analogs on hair-
cell adaptation with their effects on skeletal-muscle be-
havior lends strong support to the hypothesis that myo-
sin molecules mediate adaptation. To date, no other
model for adaptation fits more than a fraction of these
and other data.

The evidence that myosin molecules probably medi-
ate adaptation has triggered a search for isozymes of my-
osin expressed in hair cells. Early evidence that hair bun-
dles contain myosin (Macartney el a/., 1980) was later
disputed (Drenckhahn el al., 1982), and may have been
due to anti-actin-antibody contamination of anti-myo-
sin polyclonal antisera (Gillespie el al., 1993). Because of
the large number of myosin isozymes (Cheney el al..
1993) and lack of antibodies that recognize all myosin
isozymes, we developed a photoaffinity-labeling ap-

188

FUTURE OF AQUATIC RESEARCH IN SPACE

Actin

Myosin

ATP

Strong-
binding
states

ADP

\

Weak-
binding
states

ADP

Power stroke

Figure 2. ATP hydrolysis by myosin. In the absence of bound nucleotide, myosin is bound tightly to
actin. The consequence of ATP binding is a dramatic decrease in the affinity of myosin for actin: the
complex then enters several so-called "weak-binding states." in which myosin only transiently interacts
with actin. ATP is hydrolyzed to ADP and P, in a readily reversible step, then P, is released and myosin
rehinds actin. Following P, release, a kinetically irreversible step is surmounted, which is probably associ-
ated with the power stroke that generates forces and displaces actin and myosin filaments. When ADP is
bound, like the subsequent nucleotide-free state, myosin is bound tightly to actin. The effect of adenine
nucleotide diphosphates like ADP and ADP/i should be to lock the myosin in a tightly bound complex
with actin; adaptation motors would therefore be unable to release from actin or carry out power strokes.
By contrast, phosphate analogs like vanadate. beryllium fluoride, and sulfate should increase the popula-
tion of weakly bound states, decreasing force production and inhibiting climbing b\ the adaptation motors.

proach that relies on the properties of myosirTs ATPase
cycle. In this method, purified hair bundles (Gillespie
and Hudspeth, 1991) are incubated with a radioactive
nucleotide, such as [«-32P]UTP, and a trapping phos-
phate analog, such as vanadate. After the nucleotide is
hydrolyzed and P, is released, vanadate binds to myosin
and traps the nucleotide in a slowly dissociating state.
Thorough washing eliminates nucleotides bound to
other bundle proteins, then radioactive nucleotides are
covalently crosslinked to myosin molecules by UV irra-
diation. We found that three myosin isozymes, of 120,
160, and 230 kD, were most consistently labeled in puri-
fied hair bundles (Gillespie el u/., 1993). Although all
three proteins have properties that suggest they are myo-
sin molecules, the behavior of the 120-kD protein was
most consistent with that of an adaptation motor. Label-
ing of the 120-kD protein was blocked by antagonists of
myosin, including ADP. ADP/iS, and NANTP, and the
rank order of effectiveness of three trapping analogs
(vanadate, beryllium fluoride, and aluminum fluoride)
was identical to the order of stability of the correspond-

ing myosin 11-analog complexes in the presence of actin
(Gillespie el at. 1993: Yamoah and Gillespie. 1996). The
120-kD protein bound calmodulin, which is the Ca2+-
binding mediator of adaptation (Walker and Hudspeth.
1996). The other photolabeled proteins shared some, but
not all, of these characteristics. The data thus argue that
bundles contain three myosin isozymes, and that the
120-kD myosin has the most appropriate behavior for an
adaptation-motor myosin.

We have used selective antibodies to identify and lo-
calize three myosin isozymes in saccular hair bundles.
Two of these isozymes, myosin VI and myosin Vila,
have been shown, by genetic methods, to play essential
roles in hair cells (Avraham ct at. 1995; Gibson et at,
1995). Using an anti-myosin-VI antiserum, we showed
that an unusually high-mass form of this isozyme is
found exclusively in hair bundles (T. Hasson, P. G. Gil-
lespie. and D. P. Corey, unpubl. data). This 160-kD form
probably corresponds to the 160-kD photoaffinity-la-
beled protein. The location of myosin VI in bullfrog hair
cells is complex. A large amount of myosin VI was found

CYTOSKELETON/CELL MOTIL1TY

189

in the cytoplasm, and it can readily diffuse out of perme-
ahilized hair cells. Another fraction was apparently
tightly hound within the cuticular plate. In hair bundles,
myosin VI was associated with basal tapers of a fraction
ofstereocilia (only 10%-25%). In other bundles, usually
small bundles from newly formed hair cells, myosin VI
was found throughout the bundle. We suspect that myo-
sin VI may be playing a structural role in hair bundles.

Myosin Vila was abundant in purified hair bundles,
and it comigrated with the 230-kD photoaffinity-labeled
protein (T. Hasson, P. G. Gillespie, and D. P. Corey, un-
publ. data). In frog bundles, myosin Vila was largely
concentrated in a band 1-2 yum in height just above the
basal tapers. Because one class of interstereociliary link-
ages is found in that location, we suspect that myosin
Vila serves as the intracellular anchor for these basal
linkages. This localization is entirely consistent with its
distributed localization in cochlear hair bundles ( Hasson
et a/., 1995), where this class of linkages appears to be
found along the length of the stereociliary opposition.

Finally, myosin 10 appears to be the 120-kD photo-
affinity-labeled bundle protein. Myosin 1/5 has been de-
finitively localized to hair bundles by using two antibod-
ies. The first antibody, mT2, is selective for this isozyme
overall other known myosin-I isozymes(Metcalf. 1996).
In immunoblots, mT2 identified a 120-kD protein pres-
ent at 100-200 molecules per stereocilium (Gillespie et
a/.. 1993). A polyclonal antiserum raised against the tail
of cloned myosin 1/5 also detected, on immunoblots, a
120-kD protein of similar abundance (T. Hasson, P. G.
Gillespie. and Corey, D. P.. unpubl. data). Both antibod-
ies are selective, but additional confirmation of the re-
sponsible isozyme derives from separation, on SDS-
PAGE, of the 120-kD protein from myosin I« (unpubl.
data), the only other myosin-I isozyme known to be ex-
pressed in hair-cell-containing tissues (Sole et a/.. 1994).

Both antibodies labeled stereociliary tips, as expected
for an adaptation-motor myosin (Gillespie et a/.. 1993;
T. Hasson. P. G. Gillespie, and D. P. Corey, unpubl.
data). But only a fraction of tips were labeled, and a sub-
stantial amount of myosin was found below the stereo-
ciliary tips; this observation is consistent with a recent
demonstration that the transduction apparatus is highly
dynamic and can re-form after the tip links are broken
(Zhao et ai. 1996). In fixed cells observed with anti-my-
osin-I/5 antibodies, myosin molecules may be immobi-
lized while carrying transduction channels and tip links
to their proper locations.

Proof that myosin 1/5 is indeed the adaptation motor
will come from three classes of experiments, all in prog-
ress. Because the adaptation motor is thought to reside
in the insertional plaque found at the upper end of a tip
link (Hudspeth and Gillespie. 1994). immunolocaliza-
tion of myosin 1/5 at the plaque by immunoelectron mi-

croscopy will provide one level of proof. More convinc-
ing would be the inhibition of adaptation by isozyme-
selective inhibitors of myosin 1/5, such as inhibitory anti-
bodies or selective peptides. Most definitive would be in-
troduction of a mutated myosin gene into the genome of
an animal with its myosin-I/5 gene deleted; if the proper-
ties of the reintroduced myosin gene were sufficiently
distinctive, adaptation would be affected in a predictable
manner.

Acknowledgments

This work was supported by the NIH (R01 DC02368
and P60 DC00979). P.G.G. is a Pew Scholar in the Bio-
medical Sciences.

Literature Cited

Avraham, K. B., T. Hasson, K. P. Steel. D. M. Kingsley, L. B. Russell,
M. S. Mooseker, N. G. Copeland, and N. A. Jenkins. 1995. The

mouse Snell's wullzcr deafness gene encodes an unconventional
myosin required for structural integrity of inner ear hair cells. Na-
lure Genetics 11:369-375.

Cheney, R. E., M. A. Rile>, and M. S. Mooseker. 1993. Phylogenetic
analysis of the myosin superfamily. Cell Molil. Cvloskc/elnii 24:
215-223.

Cooke, R., and E. Pate. 1985. The effects of ADP and phosphate on
the contraction of muscle libers. Bitiphv.s: J. 48: 789-798.

Drenckhahn, D., J. Kellner, H. G. Mannherz, U. Grbschel-Stewart, J.
Kendrick-Jones, and J. Scholey. 1982. Absence of myosin-like
immunoreactivity in stereocilia of cochlear hair cells. Nature 300:
531-532.

Gibson. F., J. Walsh, P. Mburu. A. Varela, K. A. Brown, M. Antonio,
K. \V. Beisel, K. P. Steel, and S. D. M. Brown. 1995. A type VII
myosin encoded by the mouse deafness gene shaker- 1. \ulure 37-1:
62-64.

Gillespie, P. G. 1995. Molecular machinery of auditory and vestibu-
lar transduction. Curr Opi/i .\curuhiul. 5:449-455.

Gillespie, P. G., and A. J. Hudspeth. 1991. High-purity isolation of
bullfrog hair bundles and subcellular and topological localization of
constituent proteins./ Cell Biol. 112:625-640.

Gillespie, P. G., and A. J. Hudspeth. 1993. Adenine nucleoside di-
phosphate block adaptation of mechanoelectrical transduction in
hair cells. Proc \ull .Iciul Set. i'SA 90: 2710-2714.

Gillespie, P. G., M. C. \Vagner,and A. J. Hudspeth. 1993. Identifica-
tion of a 120-kD hair-bundle myosin I located near stereociliary
tips. Neuron U: 581-594.

Hasson, T., M. B. Heintzelman. J. Santos-Sacchi, D. P. Corey, and
M. S. Mooseker. 1995. Expression in cochlea and retina of myo-
sin Vila, the gene product defective in Usher syndrome type IB.
Pwc. .\\itl. Acad Sci. L.SA 92: 9815-9819.

Hudspeth, A. J. 1989. How the ear's works work. Xanire 341: 397-
404.

Hudspeth, A. J. 1992. Hair-bundle mechanics and a model for mech-
anoelectrical transduction by hair cells. Pp. 357-370 in Semorv
Transduction, D. P. Corey and S. Roper, eds. Rockefeller Univer-
sity Press. New York.

Hudspeth, A. J., and P. G. Gillespie. 1994. Pulling springs to tune
transduction: adaptation by hair cells. Neuron 12: 1-9.

Koyama, H., E. R. Lewis, E. L. Leverenz, and R. A. Baird. 1982.
Acute seismic sensitivity in the bullfrog ear. Brain Res. 250: 168-
172.

FUTURE OF AQUATIC RESEARCH IN SPACE

Macarlnv), ,1. C., S. D. Comis, and J. O. Pickles. 1980. Is rmosm in
the cochlea a basis for active motilit>? \uiiirc 288: 4l)l-4l>2.

Metcalf, A. B. 1996. Molecular characterization of amphibian imo-
sm Ip. a candidate for the hair bundle's adaptation motor. Ph.D.
thesis. Urmersit> of lexa-. Southwestern Medical Center. Dallas.
TX.

Oslap, E. M.. and I . I). Pollard. 1996. Biochemical kinetic charac-
terization of Ai'iini/ianii '<•/></ myosin-l ATPase. / Cell liiol. 132:
1053-1060

Solc.C. K., B. II.Urrflvr.G. M. l>u>k,and 1). P. Corey. 1994. Molec-

ular cloning of myosins from bullfrog saccular macula: a candidate
for the hair cell adaptation motor. Aiulnorv AViimvi1/. 1: 63-75.

\\alker. R. G.. and A. J. lludspeth. 1996. Calmodulin controls adap-
tation of mechanoelectrical transduction by hair cells of the bull-
frog's sacculus. PrtK- \iitl . Icatl .Sci. L'SA 93: 2203-2207.

Yamoah, K. N., and P. G. Gillespie. 1996. Phosphate analogs block
hair-cell adaptation by inhibiting adaptation-motor force produc-
tion. i\ciiriin 17: 523-533.

Zhao, V.-d., E. N. Yamoah, and P. G. Gillespie. 1996. The hair cell's
tip links rapidly regenerate. Proc \ail. Acatl. Sci L'SA, 93: 15469-
15474.

Reference: Bin/ Bull. 192: 191-196. (February. 1997)

Discussion

LANGFORD: Beth (Burnside). it
was very exciting to see those results
with particle motion on actin fila-
ments. You used the decoration of
the actin filaments with the SI heads
to show directionality. You men-
tioned that, to prepare the cells for
this experiment, you have to extract
a great deal, so you may have lost the
actin.

BURNSIDE: That was done with in-
tact sheets of RPE with attached ret-
ina, not on isolated cells. And the
distribution was that 75% of the ac-
tin filaments in apical projections
were oriented with plus ends towards
the tips of the projections, and 25%
were oriented otherwise. We have
not repeated the procedures with iso-
lated cells, because they are rather
tenuously attached to their cover
slips. With the intact retina/RPE
preparations, we have to permeabil-
ize rather drastically to get the myo-
sin subfragments into the region of
the apical projections, since they are
not exposed. Thus some of the apical
projections may have been mechan-
ically disrupted enough to stir
around their actin filaments. I am
more inclined to believe the 75+%
with plus ends distal; the other ±25%
may have been produced by tissue
disruption.

LANGFORD: Do you ever see evi-
dence of bundles of actin, or do they
always appear as single filaments?

BURNSIDE: There are no bundles
in these fish. In other fish that I've
looked at, there are really tight bun-
dles. Parrot fish and chick RPE cells

have bundles, but they don't move
their granules.

QUESTION: (Dr. Burnside) What is
the source of the dopamine?

BURNSIDE: Dopamine is released
from interplexiform cells located in
the inner retina. This dopamine
would have to diffuse 50-100 ^m to
reach the RPE cells and thus is acting
as a diffusible neuromodulator in
this case. We have shown that the
dopamine receptors located on the
photoreceptors are D4 receptors,
with binding constants for dopamine
in the nanomolar range. We have
not specifically characterized the re-
ceptors on RPE cells, but they have
similar affinities for dopamine.

ELINSON: Paul (Forscher), with-
out the myosin inhibition we see this
tremendous retrograde flux. If you
showed us a time lapse of that same
effect under myosin inhibition,
would we see the same speed of ex-
tension forward due to the actin po-
lymerization?

FORSCHER: That's exactly what
you see. There is an inverse propor-
tional relationship between growth
and flow. If you incrementally slow
the flow, you get incremental growth
by exactly the same amount.

ELINSON: So the myosin is actu-
ally competing against growth?

FORSCHER: I wouldn't say it's
competing; there's a steady state.
There are two processes going on:
Under the conditions of these exper-
iments, where the growth cone isn't

really interacting with anything in
the outside world, there's a futile cy-
cle that is essentially assembling ac-
tin, and the myosins are pulling it
back. The rate of assembly is
matched by the rate of myosin ac-
tion, and that's the retrograde flow
rate. Superimposed on that, you can
see fluctuations of the leading edge
and fillopodia. This means that the
rate of assembly is fluctuating
slightly above and below the rate of
retrograde flow, but they are not cou-
pled. Basically the point of my talk
was that, if you slow retrograde flow
by an increment, you'll get extension
that's perfectly proportional to that.

QUESTION: Does that mean that
retrograde flow is an artifact of not
being able to grasp?

FORSCHER: That's a second semi-
nar. Briefly, we have measured retro-
grade flow and growth cone target in-
teractions with neuronal substrates.
When the growth cone can grab on
to something with its myosin intact,
it grabs on. The microtubules go to-
ward the contact site, and the growth
cone starts moving forward. There's
a perfect correlation between growth
and slowing of retrograde flow. But
in this case, instead of turning the
motor off, the motor is intact, and
the actin inside is interacting
through some adhesion proteins to
grab onto the outside world.

BRADY: Growth cones don't nor-
mally go across glass cover slips, or
for that matter, sheets of cells. They
are often going through three-di-
mensional matrices of extracellular

191

192

FUTURE OF AQUATIC RESEARCH IN SPACE

matrix. That would prohahl
more with the specific interactions
with a substrate.

FORSCHERiOnec >e problems is
that we've put these eeils into an ex-
tremely artificial environment. In
fact, Aplysiu growth cones also look
and behave somewhat differently
when cultured on a more physiolog-
ical substrate.

LANGFORD: Peter (Gillespie), I
have a question about the other my-
osins. You mentioned that myosin
\fi is probably the adaptation motor.
You have identified other myosins;
what do you think they are doing?

GILLESPIE: One of the more strik-
ing localizations of myosin VI is in
the cuticular plate, to which the ster-
ocilia insert. The myosin seems to be
rigidly associated there. In the cutic-
ular plate there's an actin meshwork
that's cross linked together, perhaps
by myosin VI; maybe myosin VI is
involved in forming the cuticular
plate. There's more to it than that. I
have looked at newly formed bun-
dles from the cells most recently
turned into hair cells. These cells
have a lot of myosin VI in the ste-
reocilia — not concentrated down at
the tapers, but actually throughout
the stereocilia. I don't know what it's
doing there, but it's striking. My
guess is that the bundle needs myo-
sin VII for these linkages to hold the
stereocilia together, so that the whole
bundle can move, even when dis-
placement is applied to only one part
of the bundle. The nature of the link-
ages is unknown, but they probably
also neei, intracellular anchor,
and this \osin VII. That's

our hypothec ie time being.

BURNSIDE: (D ,pie) In your

preparations, do » ou have

myosin without actin'.'

GILLESPIE: There's a tremendous
amount of the myosin — both myo-
sin VI and myosin VII — that's not
particularly localized in any way. If

you look at the hair-cell bodies, they
have a ton of both myosin VI and
myosin VII in the cytoplasm. If you
permeabilize fixed cells with strepto-
lysin O, most of the myosin VI
diffuses away. It seems to be just sit-
ting there, presumably not interact-
ing with actin filaments. We don't
know whether this is a reservoir, or
what it's doing.

GILLESPIE: David Corey has
shown that there are multiple splice-
variants of myosin VI. We saw a 160-
kD form in the hair bundle, whereas
in the cell body there is the 150-kD
form. We don't know what the
source of that difference is, but it's
intriguing.

GILLESPIE: We haven't done sys-
tematic studies in too many organ-
isms. Most of the work is in frog and
guinea pig. Unfortunately, labeling
of myosin IfJ hasn't been done yet in
a mammal. Labeling of myosin VI
and myosin VII is consistent in the
frog and the guinea pig, although it's
not identical. With myosin VII you
get labeling up and down the ste-
reocilia— where these linkages are.
Myosin VI is not at all prominent in
stereocilia in guinea pig, and it took
unusual experiments to see this la-
beling associated with tapers in frogs.

QUESTION: Peter (Gillespie),
what's the evidence that the actin in
the stereocilia is dynamic in any way;
or is it always stable?

GILLESPIE: As far as we can tell it's
stable, in the sense that the structure
is not sensitive to cytochalasin. The
actin is heavily crosslinked, and
there's no evidence for any rapid
turnover. On the other hand, it
seems very unlikely that you make
the actin when you build a hair cell,
and that's it. There's got to be some
mechanism for turning over the ac-
tin filaments or actin monomers, but
there have been no systematic ap-
proaches to studying that yet. So we
really don't know. I'm sure it's dy-
namic on some time scale.

BRADY: Beth (Burnside), your re-
sults quite clearly show that actin
and myosins were involved in both
directions of movement for your pig-
ment granules.

BURNSIDE: We don't absolutely
know that it's myosin.

BRADY: It certainly isn't microtu-
bule-based, which is the point of the
question. It's also true that the pig-
ment-granule movement that you
see looks very similar to pigment-
granule movement in a wide variety
of cell types; yet there's a very large
literature arguing that microtubule-
based transport occurs in these other
cells. Would you like to comment on
that?

BURNSIDE: We were shocked
when we did the experiments. I
talked to Leah Haimo who studies a
fish dermal melanophore in which
the microtubules are very clearly in-
volved in the pigment migration.
The literature of pigment migration
in melanophores is extremely vari-
able. So there really just may be a va-
riety of different ways that cells
move. One thing is that the speed of
movement in the dermal melano-
phores is much faster. In our case,
this is very slow and occurs at a rate
that's similar to what happens in frog
dermal melanophores. The evidence
for the involvement of microtubules
in frog dermal melanophores is not
very good at all. I think it may be an
adaptation to a slower movement,
but I don't know why you should see
saltation out, and smooth move-
ment in. That's the same in both the
microtubule-based system and in
our system. I don't understand why
that's the case.

BRADY: The other possibility is
connected with the point you made
about the organization. When you
get rid of the microtubules in these
cells, the processes stay pretty much
the same. That's not necessarily true
in some other cell types in which the

CYTOSKELETON/CELL MOT1LITY

193

disruption of cytoplasm may be
more pronounced.

BURNSIDE: That's a good point.
Experiments in which microtubules
are disrupted //; vivo are much more
difficult to interpret because neigh-
boring cell types are also affected and
indirect effects, for example, on sig-
naling, cannot be ruled out. When
we disrupted microtubules in iso-
lated RPE-retina preparations, pig-
ment aggregation was inhibited. I in-
terpret that to mean that when the
RPE apical projections are interdigi-
tated with the photoreceptors as they
are in situ, then microtubules are
needed, perhaps for structural
purposes such as maintaining a pat-
ent pathway for granule transport.
Once the apical projections are stuck
down to a cover slip, microtubules
are not needed for normal pigment
migration in either direction, sug-
gesting that the structural support
may be provided by the substrate un-
der these conditions. In fact, when
we disrupt microtubules in isolated,
unattached RPE sheets, the apical
projections disappear. We do not
know whether they retract or be-
come too fragile to withstand our
handling procedures, but they are
gone, suggesting that microtubules
are needed for structural support
when the projections are not at-
tached to a substrate.

MANDOLI: Peter (Gillespie), you
said that the sacculus was involved in
orienting the frog. I was wondering
whether you think that you would
see an atrophy of the hairs in micro-
gravity?

GILLESPIE: People have actually
looked, and others could probably
comment better on that than I can.
Most species use the sacculus as a
true vertical-acceleration detector
for vestibular information. The frog
uses it as a specialized detector for
ground-borne vibration. When you
sneak up and try to grab that frog for
dinner, it feels you from 30 yards

away, because it has adapted its sac-
culus to detect ground-borne vibra-
tion. 1 don't know what happens to
the hair cells in space, but I know
that it's been looked at.

MANDOLI: These linkages be-
tween the stereocilia, you said that
they run all the way up and down?

GILLESPIE: You see the crosslinks
between adjacent stereocilia from
top to bottom.

MANDOLI: Is the localization of
myosin VII consistent with that link-
age?

GILLESPIE: The answer is yes, al-
though that hasn't really been looked
at systematically. It will be very in-
formative to look at the chicken,
where those linkages have been
mapped out in different regions of
different auditory and vestibular or-
gans. We know what to expect there,
so that will be a strong test of the hy-
pothesis.

MORRIS: In mammals at least,
hair cells have to last a lifetime. It's
kind of intriguing to think of how
you maintain this really nifty three-
dimensional system and do repairs. I
was wondering if there are any
thoughts on whether a modification
of the mechanism that's used for ad-
aptation is used for getting new tip-
link structures with the channels out
to the tip.

GILLESPIE: That's exactly how I
think of it. We've been recently do-
ing experiments in which we break
tip links with calcium chelators and
watch them regenerate over a num-
ber of hours. As far as we can tell, the
tip links seem to be coming from be-
low the tips; we see evidence of link-
ages from below the tips. Transduc-
tion comes back over the same time
course as tip links. Interestingly,
there's much less adaptation after 24
h of regeneration in the chelator-
treated regenerated cells than in the
controls. I think that the adaptation
motor or another mvosin is involved

in assembling everything together.
This makes sense from a lot of points
of view. We also have a strong suspi-
cion that the stereociliary surface has
much more than just one tip-link
monomer per stereocilia. We have
some indirect evidence that there's a
reserve of that protein. One simple
model is that you have a hemi-tip
link on each stereocilium, and that
these can come together and form an
intact tip link. Each hemi-tip link is
controlled by a motor. I don't actu-
ally believe that hypothesis for a
number of reasons, but at least it's
plausible. I think it's more likely that
the extended part of the tip link is
solubilized when we treat with
calcium chelators, and that it has re-
ceptors on either stereocilia. But we
don't know how it's all put together.

QUESTION: What is the tip link
made of?

GILLESPIE: We have direct and rel-
atively indirect evidence that it's gly-
coprotein. Regeneration is not
blocked by cyclohexamide, so new
protein synthesis is not needed, at
least over 12 h. We don't know
whether the tip link is stored in some
reserve in the cell body and then
brought out. or (as I have suggested
before) whether there's a lot of the
tip-link protein on the stereociliary
surface that's brought together. We
are interested in getting back to the
myosin story. In the case of myosin
1/3, we always see fewer than every
single stereocilia lit up at the tip by
either of the myosin 1/3 antibodies.
That correlates with what is seen
with scanning electron microscopy,
particularly in the frog hair cell,
where there are relatively few tip
links. I think there's evidence that
there is less than a full set of tip links
at any given time in a frog's hair cell,
perhaps because there's much more
dynamic turnover of the transduc-
tion apparatus than we initially
thought. The balance between tip
link, transduction-apparatus inser-
tion, and retrieval is affected.

194

FUTURE OF AQUATIC RESEARCH IN SPACE

BARLOW, R.: You show extreme
changes in the angle of the stereocilia
in your cartoons. How much move-
ment actually takes place?

GILLESPIE: Very little. The ste-
reocilia here are 8000 nm tall, and in
the cochlear hair cell the stereocilia
are 4000 nm tall. At the limits of hu-
man hearing you can detect displace-
ment of the stereocilia of about
0.1 nm. You can show a pretty ro-
bust displacement of transduction
current in response to a 1-nm dis-
placement with mammalian coch-
lear hair cells /// vitro. So the move-
ments that are needed for gating the
transduction channels are very
small.

QUESTION: Is there any evidence
as to exactly what type of glycopro-
tein makes up the tip link?

GILLESPIE: Not at the moment.
There's a protein in muscle called
titin that's involved in connecting
the myosin filaments to the Z-line.
Titin has the right properties to be a
tip-link protein. It's an elastic, long
and elongated (1 nm long), 4 MDa
protein. Actually I don't think that
the tip link is a titin homologue.

BARLOW, R.: The tip link would
not work well if it were elastic.

GILLESPIE: We know that the ele-
ment that gates the transduction
channels is elastic. We need to have
some elasticity in it. We do know
whatever gates the transduction
channel that we measure has a stiff-
ness of about 0.5 mN/m.

BARLOW, R.: Being elastic,
wouldn't the tip link lend itself to ad-
aptation?

GILLESPIE: Not really. If you
move the bundle, you want the dis-
placement to supply a force to the
transduction channel.

BARLOW, R.: But you don't want
the tip link to stretch, rather you
want it to be inelastic.

GILLESPIE: No, you actually want
it to stretch some because you want
the channels gate to respond to the
tension in the gating spring, rather
than to the displacement of the chan-
nel. We know that is how it works.
Whether you want that or not is an-
other matter.

BARLOW, R.: Beth (Burnside), you
mentioned that the movement of
these granules and the movement of
the photoreceptors is to place the
photoreceptors in what appears to be
the preferred position. What's the
evidence that this lends itself to sen-
sitivity changes in the retina?

BURNSIDE: There isn't any evi-
dence. Maureen Powers did a sab-
batical in my lab and we used ERGs
to try to ascertain whether changes in
sensitivity could be correlated with
changes in retinomotor position. In
the whole fish it is extremely difficult
to be sure you have produced a
change in retinomotor position with-
out changing anything else in the ret-
ina. We did show changes in sensitiv-
ity, but we could not convince our-
selves that we were looking at
specific effects of retinomotor posi-
tion. We tried to look at isolated A-
waves by knocking out photorecep-
tor synaptic transmission, but most
of the effect we could detect was on
B-waves, and thus possible contribu-
tions of altered signaling pathways in
the inner retina could not be ruled
out.

BARLOW, R.: Paul (Forscher), in
the first experiment, was concanava-
lin A on the beads?

FORSCHER: In our initial experi-
ments we used polycationic beads
that were derivatized with polyethyl-
eneimine. To our surprise we found
that with highly charged polycatio-
nic beads you'd get triggering of new
actin assembly. A bead that was
moving retrograde would suddenly
start whizzing around on a little jet
of newly assembled actin. Since we
didn't want that, we screened a bat-

tery of lectins and found that ConA
binds to many Aplysia membrane
proteins. If we derivatize a bead with
ConA, we get a flow-coupled bead
every time.

BARLOW, R.: When actin moves
under the membrane, are membrane
proteins moving with it?

FORSCHER: That's a very interest-
ing question, but I didn't say that.
We don't know whether the mem-
brane proteins that bind ConA are
actually moving with the actin be-
fore they bind to the bead. We have
studied the detailed dynamics of the
protein apCAM, which is an N-
CAM analog. When a bead that's de-
rivatized with an antibody against an
extracellular epitope for that protein
initially binds, it diffuses freely in the
plane of the membrane. As the bead
accumulates more and more of that
protein over time, it couples to the
actin flow. If you have enough den-
sity of antibody on the bead, it will
trigger actin assembly by crosslink-
ing apCAM. So it's complicated. We
also know that unligated apCAM is
normally a freely diffusible mem-
brane protein; it doesn't tend to ag-
gregate.

KUNG: Peter (Gillespie). if you cut
the tip link and then stain for I/i, does
this protein still stay on top?

GILLESPIE: I haven't done that sys-
tematically, but I have done it over a
short period of time, then treated
with chelators and looked to see if
there are any changes. There's noth-
ing obvious. On the other hand, now
that we know about the time course
for tip-link regeneration and are also
starting to get an idea about the time
course of tip-link retrieval, we know
that we are going to have to do those
experiments over longer time peri-
ods.

BRADY: One point that didn't
come out quite as much in these
talks as it could have is that a lot of
times different systems of motors do

CYTOSKELETON/CELL MOTILITV

195

interact with each other, and differ-
ent cytoskeletal elements also in-
teract with each other. Paul's
(Forscher) work showed some of
that, as did Beth's (Burnside) to
some extent. There are examples in
which actions or a mutation in a my-
osin has a suppressor that is a
kinesin-like protein. This has bog-
gled the imagination of people work-
ing on microtubule and micronla-
ment motors. There are also exam-
ples in which knocking out one
motor disrupts the mitotic spindle,
and knocking out a second motor
that happens to have a similar local-
ization suppresses the initial muta-
tion and restores function. Finally,
there is an example that may be rele-
vant to the comment about the dy-
namics of the stereocilia. Molecular
dissections of regenerating cilia in
Chlamydomonas have used genetic
approaches to identify a number of
motor proteins, in addition to the
dyneins, that are important for as-
sembly of cilia and apparently, in
some cases, for the delivery of pro-
tein to construct a cilium. There are
particles analogous to what Paul
(Forscher) sees moving along the
outside of cilia on the membrane,
and there are kinesins in the center
portion of the flagella. It's not clear
what all of these different interac-
tions may do. One thing that we
probably want to consider is that
many motors may serve not for
movement, but for generating a dy-
namic tension on a structure neces-
sary for function, cr for mediating
shape changes, or for assembly and
disassembly of structures like the mi-
totic spindle.

SACK: I realize this may not be
trivial. If one were to be able to place
a microscope in a horizontal orienta-
tion and maintain the optics with a
vertical stage, you might actually be
able to test whether gravity affects
the motility processes you described.
This has been described in plant
(Cham) cells — for example, in terms

of the streaming rate. If you put the
cell in a vertical orientation you can
see differences in motility, down-
ward-directed versus upward-di-
rected. We don't know why that's
important to the particular cells in
which it occurs. The cytoskeleton
certainly evolved to prevent stratifi-
cation, but in plant cells motility is
found to be under control. There's
lots of signaling controlling it, and
it's not a simple question of passive
drag. I don't know if anybody has
looked for this in animal systems. It
would take a horizontal microscope,
but it might uncover interactions of
gravity with cytoskeleton.

FORSCHER: There is recent evi-
dence that the localization of adhe-
sion proteins may actually be influ-
enced by tension generated by either
tyrosine kinase dependent phos-
phorylation or myosin. This means
that you can biochemically alter a
protein's state by applying the appro-
priate force.

FORSCHER: (Dr. Burnside) Have
you looked at particle movement on
the surface of your cells?

BURNSIDE: We tried to do that us-
ing your derivatized-bead approach
with positively charged beads, but we
could never get them to stick.

QUESTION: I have one comment
and a question about the sacculus in
the frogs. First of all, it is known that
the sacculus of frogs also has acoustic
sensitivity, just as it serves as a gra-
vistatic organ. There's evidence not
only in mammals, but in frogs, and
of course in fishes, where the saccu-
lus can function as an acoustic or-
gan, a gravistatic organ, or both. My
question to Dr. Gillespie is. How
confident can we be that what you've
found about the sacculus is going to
be true of other otolithic end organs,
or even of what's going on in hair
cells in the cochlea?

GILLESPIE: That's something that
has concerned us for many years.

and Jim Hudspeth in particular who
has developed the molecular ap-
proach to hair cells with the frog sac-
culus. Although that question is con-
tinuously raised, every time that we
have found something interesting
and important in the frog hair cell —
be it adaptation or gating compli-
ance in the transduction channel ap-
paratus, or what have you — it turns
out to have a parallel in every other
system that's been looked at. We've
started looking at myosin 1/3 in utric-
ular hair cells, in collaboration with
Richard Baird. It seems to be there
with a relatively similar distribution.
We are particularly interested in
what's going on in the utricular cells.
Richard has shown that different cell
types in the utriculus, which has a
much less uniform population of cell
types, have different adaptation ki-
netics. We are wondering whether
that will give us any more clues as to
the identity and properties of the ad-
aptation. I think that the sacculus is
a very useful model system in which
many of the basic principles will ap-
ply. There are going to be some
differences, and there are going to be
some specializations for its particu-
lar role, but by and large I think that
it's a useful system to work on.

QUESTION: Dr. Gillespie) what is
your opinion on why the hair cells in
the cochlea of mammals don't have
kinocilium?

GILLESPIE: The hypothesis that
makes the most sense to me is that
cochlear hair cells respond to ex-
tremely high frequencies, and a mi-
crotubule-based cilium may be an
impediment to very fast movement
of a hair bundle. The stereocilia may
be moved very readily and faster, be-
cause they are tapered and pivot
readily at their bases.

HIGHSTEIN: Another hypothesis
that might be more appropriate is
that the inner hair cells are not di-
rectly attached to the tectorial mem-

196 FUTURE OF AQUATIC RESEARCH IN SPACE

brane. The difference in these hair- you can sense angular acceleration, movement itself may be what's devi-

cell systems seems to be the acces- linear acceleration, and so forth. In ating the cilia.

sory apparatus, rather than the hair the cochlea the frequency argument

cells themselves. Depending on how has been put forward. Another pos- GILLESPIE: That's true. Outer hair

the bundle is coupled into the acces- sibility is that there is no direct at- cells do seem to be attached, and

sory apparatus, this energy is cou- tachment to the tectorial membrane they have no kinocilia either. You

pled into the accessory apparatus; in the inner hair cells, and the fluid are absolutely right.

Reference: Biol Bull 192: 197- 149. (February. 1997)

Chairs and Speakers

Workshop Chair

GEORGE M. LANGFORD, Ph.D.

Department of Biological Sciences

6044 Oilman Labs

Dartmouth College

Hanover, NH 03755

603-646-1331

Fax:603-646-1347

E-mail: George. M.Langford@Dartmouth.EDU

ROBERT C. ANGERER, Ph.D.

Department of Biology

University of Rochester

Rochester. NY 14627

716-275-8715

Fax:716-275-2070

E-mail: rangerer@rca.biology.rochester.edu

LYNNE M. ANGERER, Ph.D.

Department of Biology

University of Rochester

Rochester, NY 14627

716-275-3215

Fax:716-275-2070

E-mail: langerer@la.biology.rochester.edu

ANDREW H. BASS, Ph.D.

Department of Neurobiology and Behavior

Seeley G. Mudd Hall

Cornell University

Ithaca. NY 14853-2702

607-254-4340

Fax: 607-254-4308

E-mail: ahb3@cornell.edu

DOUGLAS A. BAXTER, Ph.D.

Department of Neurobiology and Anatomy

University of Texas Medical School at Houston

Houston, TX 77030

713-792-5720

Fax:713-500-0621

E-mail: dbaxter@nbal9.med.uth.tmc.edu

SCOTT T. BRADY, Ph.D.

Department of Cell Biology and Neuroscience

Southwestern Medical Center at Dallas

5323 Harry Hines Blvd.

Dallas, TX 75235

214-648-1830

Fax:214-648-1801

E-mail: SCOTT.BRADY@EMAIL.SWMED.EDU

BETH BURNSIDE, Ph.D.

Department of Molecular and Cell Biology

335 LSA-3200

University of California

Berkeley, CA 94720

510-642-3200

Fax:510-643-6791

E-mail: burnside@violet.berkeley.edu

MARTIN CHALFIE, Ph.D.

Department of Biological Sciences

1012 Sherman Fairchild

Columbia University

New York, NY 10027

212-854-8870

Fax:212-865-8246

E-mail: chalfie@cubsps.bio.columbia.edu

ROBERT C. EATON, Ph.D.

Center for Neuroscience

Department of Biology (EPO Box 334)

University of Colorado

Boulder, CO 80309

197

198

FUTURE OF AQUATIC RESEARCH IN SPACE

303-492-6536

Fax: 303-492-8699

E-mail: mauthner@coloi .-.edu

RICHARD P. ELINSO

Department of Zook

University of Ton

25 Harbord Stree

Toronto, M5S 3G5 Canada

416-978-4445

Fax:416-978-8532

E-mail: elinson@zoo.utoronto.ca

JOSEPH R. FETCHO, Ph.D.

Department of Neurobiology and Behavior

State University of New York at Stony Brook

Stony Brook, NY 1 1794-5230

516-632-8698

Fax:516-632-6661

E-mail: JFetcho@ccmail.sunysb.edu

THOMAS M. FISCHER, Ph.D.

Department of Psychology

P.O. Box 208205

Yale University

New Haven, CT 06520-8205

203-432-4676

Fax: 203-432-4690

E-mail: fischer@compuslug.psych.yale.edu

PAUL FORSCHER, Ph.D.

Department of Biology KBT 338

P.O. Box 208 103

Yale University

New Haven, CT 06520

203-432-6344

Fax:203-432-6161

E-mail: paul.forscher@yale.edu

PETER GILLESPIE, Ph.D.

Department of Physiology

The Johns Hopkins University School of Medicine,

WBSB 205

725 N.Wolfe Street

Baltimore, MD 2 1205-2 185

410-614-308;

Fax: 410-955-046!

E-mail: Peter_j cspie@qmail.bs.jhu.edu

EDWARD M. Gooi is Ph.D.

Space Station Bun search Project

NASA Ames Resean

T20G-2

Moffett Field, CA 94035

415-604-1961

Fax:415-604-1701

E-mail: ed_goolish@qmgate.arc. nasa.gov

DR. DONAT-PETER HADER

Institute for Botany and Pharmaceutical Biology

Staudtstrasse 5, D-91058

Erlangen, Germany

49-9131-858216

Fax:49-9131-858215

E-mail: dphaeder@biologie.uni-erlangen.de

OWEN HAMILL, Ph.D.

Department of Physiology & Biophysics

University of Texas Medical Branch

Galveston, TX 77555-0641

409-772-5464

Fax:409-772-3381

E-mail: ohamill@beach.utmb.edu

MASASHI KAWASAKI, Ph.D.
Department of Biology
Gilmer Hall, Room 277
University of Virginia
Charlottesville, VA 22903
804-982-5763
Fax: 804-982-5626
E-mail: mk3u@virginia.edu

JOHN Z. KJSS, Ph.D.
Department of Botany
Miami University
Oxford, OH 45056
513-529-5428
Fax: 5 1 3-529-4243
E-mail: kissjz@muohio.edu

CHING KUNG, Ph.D.

Laboratory of Molecular Biology

R. M. Bock Laboratories

University of Wisconsin

1525 Linden Drive

Madison, WI 53706- 1596

608-262-2059

Fax: 608-262-4570

E-mail: ckung@macc.wisc.edu

WERNER R. LOEWENSTEIN, Ph.D.

Laboratory of Cell Communication

Marine Biological Laboratory

7 MBL Street

Woods Hole, MA 02543

508-289-7430

Fax: 508-548-2003

E-mail: lhoward@mbl.edu

CHAIRS AND SPEAKERS

199

ROBERT E. MAXSON, Ph.D.

633 Morris Hospital

USC Medical School

1441 Eastlake Avenue

Los Angeles, CA 90033

213-764-0633

Fax:213-342-2764

E-mail: maxson@zygote.hsc.usc.edu

CATHERINE E. MORRIS, Ph.D.

Neurosciences Department — Loeb Institute

Ottawa Civic Hospital

1053 Carling Avenue

Ottawa, Ontario, Kl Y 4E9 Canada

613-761-5073

Fax:613-761-5330

E-mail: morris@civich.ottawa.on.ca

DR. PETER NICK

Institut fur Biologic II/Botanik

Schanzlestrasse 1

D-79 104 Freiburg, Germany

49-761-203-2646

Fax:49-761-203-2612

E-mail: pnick@ruf.uni-freiburg.de

SIMON OSTRACH, Ph.D.

Mechanical and Aerospace Engineering

Case Western Reserve University

Cleveland, OH 44 106

216-368-2940

Fax:216-368-6445

E-mail: sxo3@po.cwru.edu

STANLEY J. Roux, JR., Ph.D.

Department of Botany

University of Texas at Austin

Austin, TX 787 13-7640

512-471-5858

Fax:512-471-3878

E-mail: sroux@uts.cc.utexas.edu

FRED D. SACK, Ph.D.

Department of Plant Biology

Ohio State University

1 82 Botany and Zoology Bldg.

1735 Neil Avenue

Columbus, OH 432 10

614-292-0896

Fax:614-292-6345

E-mail: fsack@magnus.acs.ohio-state.edu

Reference: Bin/ Bull 192: 200-201. (February. 1997)

Participants

JELLE ATEMA, Ph.D.
B.U. Marine Program
Marine Biological Laboratory
Tel: 508-289-7499
E-mail: atema@bio.bu.edu

MEL AVERNER, Ph.D.

NASA Headquarters

E-mail: maverner@gm.olmsa.hq.nasa.gov

ROBERT BAKER, Ph.D.

New York University Medical Center

Tel: 2 12-263-5402

ROBERT BARLOW, Ph.D.
Marine Biological Laboratory
Tel: 508-289-7356

FR. J. D. CASSIDV, O.P., Ph.D.
Providence College

MARY ANNE FREV, Ph.D.

NASA Headquarters

E-mail: mafrey@gm.olmsa.hq.nasa.gov

CHANDLER FULTON, Ph.D.

Brandeis University

Tel: 6 17-736-3 150

E-mail: fulton@binah.cc.brandeis.edu

STEPHEN M. HIGHSTEIN, M.D., Ph.D.

Washington University

St. Louis

E-mail: highstein@WUMS.WUSTL.EDU

WILLIAM P. JACOBS, Ph.D.
Department of Molecular Biology
Princeton University
Tel: 609-924-2

CELESTE JARVIS
Information Dynani
Tel: 202-488-5 126
E-mail: cjarvis@hq.na- <

Yi Li, Ph.D.
Kansas State University
Tel: 9 13-532-6360
E-mail: yili@ksu.ksu.edu

TERRI LOMAX, Ph.D.
Oregon State University
E-mail: lomaxt@bcc.orst.edu

BRIAN LOWE
Boston University
Tel: 508-548-5 169
E-mail: btlowe@bio.bu.edu

DINA MANDOLI, Ph.D.

University of Washington

E-mail: mandoli@u. washington.edu

ALLEN MENSINGER, Ph.D.

Washington University

St. Louis

E-mail: MENSINA@TFNEURON.WUSTL.EDU

ENRICO NASI. Ph.D.

Boston University School of Medicine

Tel: 6 17-638-4347

E-mail: enasi@acs.bu.edu

KENNA PEUSNER, Ph.D.
George Washington University
Tel: 202-994-3489

D. MARSHALL PORTERFIELD
Louisiana State University
Tel: 504-388- 1464
E-mail: MPFIELD@AOL.COM

JOHN D. RLIMMEL, Ph.D.
Marine Biological Laboratory
Tel: 508-289-72 18

TOM SCOTT, Ph.D.

NASA Headquarters

Tel: 202-488-5 145

E-mail: tscott@gm.olmsa.hq.nasa.gov

ROBERT B. SILVER, Ph.D.
Marine Biological Laboratory
Tel: 508-548-3705
E-mail: rsilver@mbl.edu

200

PARTICIPANTS

201

RAQUEL SUSSMAN, Ph.D.
Marine Biological Laboratory
Tel: 508-289-7241

DANIEL TOMSIC, Ph.D.

NIH

Tel: 30 1-496-3868

E-mail: TOMSIC@CODON.NIH.GOV

PEI-LAN Tsou

Department of Botany

North Carolina State University

Tel: 9 19-5 15-3 345

E-mail: ptsou@unity.ncsu.edu

CHARLES E. WADE, Ph.D.
NASA Ames Research Center
Tel: 4 15-604-3943

NORMAN WAINWRIGHT, Ph.D.
Marine Biological Laboratory

Tel: 508-289-7343
E-mail: nwainwri@mbl.edu

RON J. WHITE, Ph.D.

NASA Headquarters

Tel: 202-358-2 194

E-mail: rjwhite@gm.olmsa.hq.nasa.gov

MICHAEL WIEDERHOLD, Ph.D.
University of Texas Health Science Center
San Antonio
E-mail: WIEDERHOLD@UTHSCSA.EDU

SARAH WVATT, Ph.D.
Department of Botany
North Carolina State University
E-mail: Sarah_Wyatt@ncsu.edu

STEVE ZOTTOLI, Ph.D.

Williams College

E-mail: Steven.J.Zottoli@williams.edu

CONTENTS

CELL BIOLOGY

Goldberg, Walter M., and George T. Taylor

Coelenterate cnidae capsules: disulfide linkages re-
vealed bv silver cytochemistry and their differential
responses to thiol reagents 1

DEVELOPMENT AND REPRODUCTION

Sewell, M. A., P. A. Tyler, C. M. Young, and
C. Conand

Ovarian development in the class Holothuroidea:

a reassessment of the "tubule recruitment model" 1 7

Hoegh-Guldberg, O., and R. B. Emlet

Energy use during the development of a lecitho-
trophic and a plankton ophic echinoid 27

Martin, Vicki J., and William E. Archer

Stages of larval development and stem cell popula-
tion changes during metamorphosis of a hydrozoan
planula 41

IMMUNOBIOLOGY

Hirose, Euichi, Yasunori Saito, and Hiroshi Wata-
nabe

Subcuticular rejection: an advanced mode of the al-
logeneic rejection in the compound ascidians, Butnl-

loidfssimodensisandB.fuscus 53

Raftos, David, and Aimee Hutchinson

Effects of common estuarine pollutants on the im-
mune reactions of umicates 62

ECOLOGY AND EVOLUTION

Anthony, Kenneth R. N.

Prey capture by the sea anemone Mrtridium \rinh-
(L.): effects of body size, flow regime, and upstream
neighbors 73

Holyoak, Alan R.

Patterns and consequences of whole colom growth

in the compound ascidian Pohrluntm pl/iinim .... 87

Miller, Karen, and Russell Babcock

Conflicting morphological and reproductive species
boundaries in the coral genus Pltit\gyrti 98

THE FUTURE OF AQUATIC RESEARCH IN SPACE:
NEUROBIOLOGY, CELLULAR AND
MOLECULAR BIOLOGY

Introduction 11")

Mechanosensitivity 117

Plant Biology 131

Neurobiology/ Sensory Biology 145

Developmental Biology 172

Cytoskeleton/Cell Motility 181

Volume 192

THE

Number 2

BIOLOGICAL
BULLETIN

APRIL, 1997

Published by the Marine Biological Laboratory

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MAY 1 3 1997

THE

BIOLOGICAL BULLETIN

PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY

Associate Editors

LOUIS E. BURNETT, Grice Marine Biological Laboratory. College of Charleston
WILLIAM D. COHEN, Hunter College. City University of New York

CHARLES D. DERBY. Georgia State University
DAVID EPEL, Hopkins Marine Station. Stanford University

Editorial Board

PETER B. ARMSTRONG. University of California. Davis
THOMAS H. DIETZ, Louisiana State University

RICHARD B. EMI ET. Oregon Institute of Marine Biology.

University of Oregon

DAPHNE GAIL FALITIN. University of Kansas

WILLIAM F. GILLV, Hopkins Marine Station. Stanford

University

ROGER T. HANLON, Marine Biological Laboratory

MAKOTO KOBAVASHI. Hiroshima Prefectural Uni-
versity

MICHAEL LABARBERA, University of Chicago
DONALT. MANAHAN, University of Southern California

MARGARET MC.FALL-NGM. Kewalo Marine Labora-
tory. University of Hawaii

TATSUO MOTOKAW v Tokyo Institute of Technology
K. RANGA RAO. University of West Florida

BARUCH RINKEVICH. Israel Oceanographic &
Limnological Research Ltd.

RICHARD STRATHMANN. Friday Harbor Laboratories.
University of Washington

STEVEN VOGEL. Duke University

J. HERBERT WAITE. University of Delaware

SARAH ANN WOODIN. University of South Carolina

RICHARD K. ZIMMER-FAUST. University of California.

Los Angeles

Editor: MICHAEL J. GREENBERG. The Whitney Laboratory, University of Florida
Managing Editor: PAMELA L. CLAPP. Marine Biological Laboratory

APRIL, 1997

Printed and Issued by
LANCASTER PRESS, Inc.

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Reference: Biol. Bull 192: 203-207. (April. 1997)

Choreography of the Squid's "Nuptial Dance"

WARWICK H. H. SAUER1, MIKE J. ROBERTS2, MAREK R. LIPINSKI2,
MALCOLM J. SMALE3, ROGER T. HANLON4, DALE M. WEBBER5,

AND RON K. O'DOR5

1 Department of Ichthyology and Fisheries Science, Rhodes University, P.O. Box 94, Grahamstown

6140. South Africa: 2Sea Fisheries Research Institute, Cape Town 8012. South Africa: *Port Elizabeth

Museum, Humewood 6013, Port Elizabeth, South Africa: * Marine Biological Laboratory. Woods

Hole, Massachusetts 02543. USA: and ^Dalhousie University. Halifax, Canada B3H 4JI

A mass spawning of squid resembles, at first glance, a
chaotic "nuptial dance" (1). But for the first time, we have
applied 3-D. radio-linked acoustic positioning (R.AP) to
this confusing process, and our early results now reveal a
choreography that is, in fact, well organized in time and
space. Remote tracking with R.4P of individual Loligo
vulgaris reynaudii off' South Africa has provided insights
into the daily sequence of behaviors that lead these ani-
mals to aggregate for sexual selection. Each dawn, the
squid navigate for several kilometers, toward the shore, to
small, well-defined zones near egg beds on the substrate.
After several hours of circling above these egg beds, a pe-
lagic, 3-D lek-like aggregation of large males forms: fe-
males are drawn in. and the aggregation condenses as
the females and males pair, mate, and lay eggs. Smaller
"sneaker males" remain on the periphery of the mating
arena and, from this station, attempt extra-pair copula-
tions (EPCs). The mating system of squids is thus unex-
pectedly complex, rivaling those of mammals and birds
(2. 3). Commercial squid-jigging fishermen in South Af-
rica have recently been attracted to the spawning
grounds, and they have been successful. Moreover, their
activities may be selective for large males. Thus, atten-
tion should be devoted to ensuring that such targeted
fishing does not alter the characteristics of squid popula-
tion genetics. Remote tracking and video observations,
in combination with genetic analyses, may offer a new
opportunity to monitor mating effort and reproductive
success, and thus to manage the fishery.

Determining the reproductive behavior of highly mo-
bile marine animals is challenging, but the results can be
rewarding, particularly in situations in which the species

Received 23 May 1996; accepted 29 January 1997.

is the target of a fishery that is conducted directly on the
spawning grounds. Collaborative efforts between the
fishermen's cooperatives and the government's Sea Fish-
eries Research Institute led to the present study, which
was conducted in November 1993 and November 1994
in Oyster Bay, South Africa. Underwater visibility was
limited and diving conditions were treacherous, thus the
observations of squid behavior by divers during these pe-
riods were substantially complemented by an analysis of
data collected by remote tracking.

Eight squid were fitted with acoustic transmitters that
produced pulses of 50-80 kHz "pings." The devices were
placed within the mantle cavity and attached to the man-
tle wall by a hypodermic needle that was thrust through
the wall, crimped, and secured with silicone washers (see
4). Four of the eight squid were large males (430 g; mantle
length [ML] about 32 cm), two carrying Vemco V 1 6T-3H
telemetry tags with a thermistor-controlled pulse interval,
one a V 1 6-3H pinger (all, 70 x 16mm diameter), and one
a V8-2L pinger with a constant pulse interval of about 1 s
(35 X 8 mm diameter). These transmitters encoded tem-
peratures in the intervals between pings (as the data on
depth and jet pressure had been telemetered in previous
studies; 4). In addition, two small "sneaker" males (70 g;
about 15 cm ML) and two females (180g; about 21 cm
ML) each carried V8-2L pingers. The data supplied by all
of these transmitters are equivalent to those provided by
long-term, focal sampling of animal behavior (5).

The acoustic transmitters borne by the c luids were
tracked for 14 days by four hydrophone-e«. pped RAP
buoys (Vemco Ltd., Shad Bay, NS, Canada) moored in
Oyster Bay in a 45-m square over two •• iiall egg beds
(each about 1 X 2 m) located 1 km offslv re. The buoys
monitored the arrival times of signals rom the tagged

203

204

W. H. H. SAUER ET AL

squid and transmitted them by radio to a base station,
which used a triangulaiion routine (6) to calculate 3-D
positions. The eight squid were tracked for a total of 54
animal-days (maximum for one individual, 14 d). They
produced 35 megabytes of positional data, and the four
large male squid produced temperature data as well.

Data from transmitters fixed to the substrate were
compared with data from diver-transported units so that
the accuracy of the software in its present state of devel-
opment could be evaluated. These tests showed position
accuracy of less than 1 m inside the array of RAP buoys,
decreasing to 5- 10 m at a 150-m radius from the center
of the array (note Fig. 2). Full resolution of point-by-
point tracks requires a complex post-analysis that is still
in progress, but 10-20 s averaging gave reasonable, real-
time tracks for typical squid speeds of 10-50 cm s"1. At
worst, a squid moving 50cm s"', signaling its position
every 20 s, produces points 10 m apart at 150 m from

the center of the array; smaller movements are usually
recorded at slower speeds.

The eight squid being tracked were consistently pres-
ent near the array during the day. The diel rhythmicity
with which the three types of squid appeared on the egg
beds is illustrated in Figure 1. These behavioral observa-
tions are limited in number, but they are consistent; this
consistency, together with our previous experience (4),
indicates that acoustically tagged squid show the normal
range of movement.

At dawn, the squid arrived in the vicinity of the egg
beds. The large males made circles (50- 150 m radius)
around the area (records of these and subsequent move-
ments are shown in Fig. 2). Females and other males
gradually joined the large males as they coalesced into a
3-D mating arena that was generally situated within
10 m of the substrate (typical depths were 20- 30 m). Ag-
onistic behavior among males was very striking and

Squid Types Present on Egg Bed

0 2 4 6 8 10 12 14 16 18 20 22

Time of Day (h)

Females — Males

Sneakers

Figure 1. Diel patterns of squid visitations to the egg bed, shown as the ratio of hours present to hours
monitored. Three large males (blue, 32 cm mantle length) were monitored over 8 full days, two females
(green, 2 1 cm ML) over 7 days, and two small "sneaker" males (red, 1 5 cm ML) over 2 days. Females and
sneakers from offshore appear at dawn and often remain past dusk. Large males also appear at dawn,
although numbers peak later; they often disappear by noon and make brief return visits before moving
offshore at dusk. A trade-off between detailed tracking of individuals and continuous monitoring of all
frequencies makes statistical analysis of daytime data difficult, but continuous monitoring at night
(1800 h-0400 h)did not indicate the presence of large males or females, so patterns are clearly distinct.

CHOREOGRAPHY OF THE SQUID'S "NUPTIAL DANCE'

205

150

150

-100

-50 0 50

Offshore/SW (m)

100

150

Figure 2. Overhead view of a typical full day of tracks for a large
male (blue), a female (green), and a sneaker male (red): full details of
behaviors can be replayed on computer animations. The black dot
identities an egg mass confirmed by divers; video surveys also showed
numerous squid and scattered egg fingers beneath the area of heavy
tracks on the opposite side of the array. Ten days of similar records
show that males and females begin at dawn making circles of 50- 1 50 m
radius, high in the water column, around the sonobuoy array
(A.B.C.D). Circling decreases by mid-morning, and in the afternoon
females spend more time near bottom at individual egg patches.
Sneaker males remain outside the main arena, low in the water column
and near egg patches: direct observations show that they rush in to in-
tercept pairs. Speeds average 18 cm s"1 for females. 14 cm s ' for large
males, and 1 7 cm s 'for small sneakers.

common as they competed for temporary pairings with
females (7; unpubl. results). The paired large males and
females mated, and the female then moved with her part-
ner to the substrate to deposit her "egg fingers" (Fig. 3).
Although these squid need adjacent bottom for egg-fin-
ger attachment, criteria for such beds are not stringent.
Lone large males often waited near the egg beds to in-
tercept pairs moving to and from the communal egg
beds.

This activity — repeated mating and egg laying with a
series of temporary partners — often continued tor 1-
2 h after sunset (Fig. 1). Meanwhile, the sneaker males
remained all day at the periphery of the arenas (Figs. 1
and 2). In this station, presumably, they are maximizing
their opportunities for extra-pair copulations (EPCs):
that is, watching for females temporarily paired with
larger males; then, after the pair has mated, swimming
rapidly into the arena to mate briefly with those females
(Fig. 4). The mean duration of a sneaker male's mating
was only 6 s; in contrast, large males mated for 16-20 s
(7). Large males occasionally left during the day, and one

Figure 3. Photo of a large male escorting a female squid (Loligo
v»/(,'<;m reviiiiiuln) as she attaches an "egg finger" of about 100 eggs to
an egg bed. Photo by MJS.

was tracked manually by boat to another mating arena
1.5 km alongshore. Tagged large males were apparently
unsuccessful in mating, as evidenced by the short time
they spent near the egg beds. In contrast, paired males
escort female mates to the egg beds and guard them as
their eggs are deposited (Fig. 3 ). This does not necessarily
imply that tags interfered with normal behavior, because
males outnumber females.

Most squid moved offshore at dusk; one large male
was tracked 2 km offshore, where he remained relatively
stationary (perhaps feeding or resting) until his return at
dawn. During this male's onshore migration, his mean
swimming speed was. as expected. 1 body length per sec-
ond (45 cms"1), three times his speed in the mating
arena. In contrast, sneakers were smaller and faster, av-
eraging nearly 1 body length per second for the whole
day; their tactic of avoiding large males and achieving
sneak EPCs requires swift swimming.

Figure^. This enhanced video image of a "sneak-, male (left) in-
tercepting a temporary pair and attaching a sperm package to the fe-
male with no response by the escort (right) is direct e\ idence of sneak-
ing in squid mating systems. Video by RTH.

206

W. H. H. SAUER ET AL.

Males of various terrestrial and aquatic taxa assemble
in large leks or lek-like aggregations to attract females
and then compete for mating access (2, 3, 8, 9). Among
loliginid squids I'vcver, the tactic that appears to be
primary is the . cnsive sexual selection that occurs in
these near^' mating arenas, with many of the requi-
sites for Ickb (2). We suggest that the daily offshore-to-
inshore movements and circling aggregation of male
squid draw in new male and female schools from off-
shore, increasing the size of the aggregation and the num-
ber of females. Our picture of the dynamics of these lek-
like aggregations suggests that high-biomass mating are-
nas ensure that most eggs are deposited in locations with
a high probability of hatching success. Other potential
benefits of larger aggregations are more intensive compe-
tition for mates and more gene mixing in the population.

Overall, the squid mating system first includes a period
of offshore mating that provides sperm to most females
before the onset of the inshore migration. This sperm is
stored and, as an alternative tactic, can be used by indi-
vidual females that never make it to inshore mating are-
nas. Females that do participate in the inshore mating
aggregations probably use the sperm of the large males
with which they pair and mate in these arenas (10, 11).
In the end, then, females that mate with large males and
sneakers, as well as with males while offshore, have at
least three sources of sperm (7, 11, 12). Therefore, the
potential for genetic mixing — already increased by the
spawning aggregation — will be further augmented. This
type of mating system may be typical of loliginid squids,
and assessment of paternity by some molecular tech-
nique will help resolve this possibility (12).

The fishery for the squid Loligo vitlgaris rcynuiulii in
South Africa began when fishermen located squid aggre-
gations by sonar and jigged them intensively. At the out-
set, low-intensity jigging often yielded tons of squid daily
for several months, primarily during what we now recog-
nize as the early morning period of circling and pairing
over mating arenas (13). Previous sporadic diving obser-
vations confirmed that the aggregated squid were spawn-
ing around communal egg beds (14, 15). At present fish-
ing levels, the aggregations seem more transitory and iso-
lated, and offshore spawning may have increased.

By now, the fishery, although relatively new. has be-
come the third most valuable in South Africa (16); this
fits the worldwide trend in which cephalopods have risen
to third in dollar value (behind shrimp and tuna) as fin-
fish stocks have declined due to fishing pressure (17). But
this fishery clearly targets spawning squid. Therefore,
fishery biologists and fisherman's cooperatives are keen
to avert both biological and commercial disaster; thus
they have been supporting the acquisition of baseline
data on population dynamics, especially as it is influ-
enced by reproductive behavior. Field trials combining
molecular genetic and behavioral observations with ex-

perimental fishing are planned to develop management
strategies that will avoid alteration or breakdown of the
critical spawning aggregations. In addition to the poten-
tial for altering the mating dynamics (and thus gene dis-
tribution) on the mating arenas, recruitment could also
be adversely affected by increases in the alternative tactic
of mating and spawning offshore, where development
temperatures and success may be lower ( 1 8).

The novel remote tracking project reported here is part
of a larger program that includes videography of behav-
iors recorded by divers, analysis of the recapture of pre-
viously tagged squid, bottom surveys carried out by
video using the Global Positioning Systems for location,
and biomass assessments from sonar and trawl samples.

In summary, the use of an unorthodox technique (tag-
ging and monitoring with RAP) led to the surprising con-
clusion that the squid's "nuptial dance" seems to be cho-
reographed by the large males each morning, and that
the nature of the interactions is reminiscent of lek aggre-
gations of birds and mammals. Because the annual life
cycle of squid is brief (19), and because the fishery has
only recently developed, the system has great potential
as a model of genetic shifts in populations that are sub-
jected to natural selection from predators, sexual selec-
tion due to squid behavior, and selective fishing pressure
from human behavior. Fishery managers might take ad-
vantage of this useful model system.

Acknowledgments

We acknowledge the advice of Mart Gross and support
from the Foundation for Research and Development
(South Africa), South African Squid Management In-
dustrial Association (South Africa), Sea Fisheries Re-
search Institute (South Africa), National Institutes of
Health grant RR01024 (USA), and National Sciences
and Engineering Research Council (Canada).

Literature Cited

1. Coustcau, J.-Y., and P. Diole. 1973. Last dance on the mating
ground. ,\ut. HIM 82(4): 45-48.

2. Hoglund, J., and R. V. Alatalo. 1995. Leks. Princeton Univ. Press,
Princeton, NJ.

3. Widemo, F., and I. P. F. Owens. 1995. Lek size, male mating
skew and the evolution oflekking. Nature 373: 148- 15 1.

4 O'Dor, R. K., J. A. Hoar, D. M. Webber, F. G. Carey, S. Tanaka,
H. Martins, and F. M. Porteiro. 199-4. Squid (Lolixo forbesi) per-
formance and metabolic rates in nature. Mar. Frexh. Behav. Phys-
/()/. 25: 163-177.

5. Martin, P., and P. Bateson. 1993. Measuring Behaviour: An In-
troductory Guide. 2nd edition. Cambridge University Press, Cam-
bridge. U.K.

6. O'Dor, R. K., D. M. Webber, \V. H. H. Sauer, M. Roberts, and M.
Smale. 1996. High-resolution, 3-D tracking of squid on spawn-
ing grounds. Pp. 193-198 in Prm: 13th Int. Symp. Biotelemetry,
C. Cristalli. C. J. Amlaner, Jr.. and M. R. Neuman. eds. Univ. Ar-
kansas Press. Favetteville.

CHOREOGRAPHY OF THE SQUID'S "NUPTIAL DANCE"

207

7 Hanlon, R. I., M. J. Smale, and \\ . H. H. Sauer. 1944. An etho-
gram of body patterning behavior in the squid Loligo vulgar/* ivyihiit-
iln on spawni ng grounds ofl South Africa. Biol. Hull 1S7: 363-372.

8. McKaye, K. R., S. M. Londa, and J. F. Stautfer. 1940. Bower
size and male reproductive success in a cichlid fish lek. Am. ,\al.
135:597-613.

9. Colin, P. L., and I.. J. Bell. 1991. Aspects of the spawning of la-
brid and scarid fishes (Pisces: Labroidei) at Eniwetok Atoll. Mar-
shall Islandswith notes on other families. Environ. Biol. Fishes3\\
229-260.

10. Hanlon, R. I '.. and J. B. Messenger. 1996. Ccphalonoil Behaviour
Cambridge Um\. Press. Cambridge. U.K.

1 1 . Boyle, P. R., G. J. Pierce, and L. C. Hastie. 1995. Flexible repro-
ductive strategies in the squid Loligo torbcsi. Mar. Bin/. 121: 501-
508.

12. Hanlon, R. T. 1996. Evolutionary games that squids play: fight-
ing, courting, sneaking, and mating behaviors used for sexual se-
lection in Loligo pealei. Biol. Bull. 191: 309-310.

13. Sauer. \V. H. H. 1993. The ecology of spawning squid Loligo
vulguris rcynaudii in the inshore waters of the Eastern Cape. Ph.D.
Thesis. U. of Port Elizabeth. 121 pp.

14. Sauer, \V. H. H., M. J. Smale, and M. R. Lipinski. 1992. The

location of spawning grounds, spawning and schooling behav-
iour of the squid Loligo vulgar ix reynaudii (D'Orbigny) (Cepha-
lopoda: Myopsida) off the eastern Cape coast. Mar Biol 114:
97-107.

15. Sauer, \V. H. II., and M. J. Smale. 1993. Spawning behavior of
Li>l/i!<> vulgarix rcynaudii in shallow coastal waters of the south-
eastern Cape. South Africa. Pp. 489-498 in Recent Advance-* in
Cephalopoil h'i\lwries Biology, T. Okutani. R. K. O'Dor. and T.
kubodera. eds. Tokai University Press. Japan.

16. Stuttaford. M. 1994. Fishing Industry Handbook, South Africa,
\unuhui and Musuinhiuui'. Pub: Marine Information. Stellen-
bosch. S. A. 434 pp.

17. FAO Fisheries Department. 1993. Fisheries and the Law of the
Sea: A Decade of Change. I-'AO l-'ishcncs Circular. No. 853: Rome.
66 pp.

18. Roberts, M. J., and \V. H. H. Sauer. 1994. Environment: the
key to understanding the South African chokka squid (Loligo
vulgans rcviunuln) life cycle and fishery. Anlarct. Sci. 6(2): 249-
258.

19 Augustyn, C. J., M. R. I.ipinski, VV. H. H. Sauer, M. J. Roberts,
and B. A. Mitchell-Innes. 1994. Chokka squid on the Agulhas
Bank: life history and ecology. S. Air. J Sci. 90: 143-1 54.

Reference: Biol. Bull 192: 208-216. (April. 1997)

Uptake and Persistence of Homologous and

Heterologous Zooxanthellae in the Temperate Sea

Anemone Cereus pedunculatus (Pennant)

SIMON K. DAVY*. IAN A. N. LUCAS, AND JOHN R. TURNER

School of Ocean Sciences, University of\\ 'ales, Bangor. Marine Science Laboratories.
Menai Bridge. Anglesey, LL59 5EY, UK

Abstract. The uptake and persistence of symbiotic di-
noflagellates (zooxanthellae) were measured in the tem-
perate sea anemone Cereus pedunculatus (Pennant).
Aposymbiotic specimens of C. pedunculatus were inoc-
ulated with zooxanthellae freshly isolated from a range
of temperate and subtropical Anthozoa. Each inoculate
consisted of zooxanthellae from a single host species and
was either homologous (zooxanthellae from a host of the
same species as the one being inoculated) or heterolo-
gous (from a host of a different species than the one being
inoculated). The densities of zooxanthellae in host tis-
sues were determined at regular intervals. C. peduncula-
tus took up homologous and heterologous zooxanthellae
to similar degrees, except for zooxanthellae from the
temperate Anlhopleura ballii, which were taken up to a
lesser extent. The densities of all zooxanthellae declined
between 4 hours and 4 days after uptake, indicating that
zooxanthellae were expelled, digested, or both during
this period. The densities of all zooxanthellae increased
between 2 and 8 weeks after inoculation, indicating zoo-
xanthella growth. Over the entire 8-week period after up-
take, densities of homologous zooxanthellae were always
greater than those of heterologous zooxanthellae. Be-
tween 8 and 36 weeks after infection, densities of homol-
ogous zooxanthellae declined markedly and densities of
some heterologous zooxanthellae increased further, re-
sulting in homologous and heterologous zooxanthella
densities being the same at 36 weeks. These densities

Received 12 January 1996; accepted 9 January 1997.

* Present address: Department of Symbiosis and Coral Biology. Di-
vision of Marine Science, Harbor Branch Oceanographic Institution,
5600 U.S. 1 North, Fort Pierce, Florida 34946.

were the same as those in naturally infected C. peduncu-
latus of similar size. The results suggest that zooxanthel-
lae from a range of host species and environments can
establish symbioses with C. pedunculatus and that, over
long periods under laboratory conditions, heterologous
zooxanthellae may populate C. pedunculatus to the same
extent as homologous zooxanthellae.

Introduction

Many benthic marine Cnidaria possess endosymbiotic
dinoflagellates (zooxanthellae) of the genus Symbiodin-
iitni. These Cnidaria may lose zooxanthellae entirely
(i.e., become aposymbiotic) when under environmental
stress (Williams and Bunkley-Williams, 1990; Glynn,
1991) or when zooxanthellae are not inherited mater-
nally (Trench, 1987; Shick 1991). Consequently, they
may be susceptible to reinfection and colonization by
zooxanthellae released from a range of host species
(Schoenberg and Trench. 1980c; Rowan and Powers,
1991b; Buddemeier and Fautin, 1993). Despite this sus-
ceptibility and the high diversity of hosts and symbionts
(Trench, 1987; Rowan and Powers, 1991b; Trench,
1993; McNally et a/.. 1994), many species of Cnidaria
have been reported to harbor just one strain or species of
Symbiodinium (Schoenberg and Trench, 1980a, b;
Rowan and Powers, 199 la, b; Trench, 1993). Other
hosts have been found to contain more than one strain
or species of zooxanthella, but even in these cases the
algae are the same across the host's geographical range
(Trench and Winsor. 1987; Rowan and Powers, 1991b;
Rowan and Knowlton, 1995).

The uptake and persistence of zooxanthellae following

208

PERSISTENCE OF ZOOXANTHELLAE IN SEA ANEMONES

209

inoculation of aposymbiotic hosts has been investigated
in a range of tropical marine Cnidaria (Kinzie, 1974;
Kinzie and Chee, 1979; Schoenberg and Trench, 1980c;
Trench, 1981; Trench et ai. 1981; Colley and Trench,
1 983; Fitt and Trench, 1983a,b; Bernerrttf/., 1993). The
zooxanthellae were either homologous (from a host of
the same species of cnidarian as the one being inocu-
lated) or heterologous (from a host of a different species
than the one being inoculated). In contrast, similar work
has been performed using just one species of temperate
marine Cnidaria, the North American sea anemone An-
thopleura elegantissima (Trench, 1969, cited in Trench,
1971;Muller-Parker, 1996; Weis and Levine, 1996).

The temperate sea anemone Cereus pedunculatus
(Pennant) (family Sagartiidae) is locally abundant
around the south and west coasts of Europe, where it is
found partially buried in sand and mud from the mid-
shore to a depth of 25 m (Manuel, 1981). C. peduncula-
tus may reproduce by oviparity, but frequently it is her-
maphroditic or parthenogenetic and viviparous (Rossi,
1975;Schafer, 1981;Shick, 1991). The zooxanthellae of
C. pedunculatus are located within the host's endoder-
mal cells and have been identified as Symbiodinium sp.
(Davy et a/., in press). Oocytes may contain up to 300
zooxanthellae, suggesting that C. pedunculatus acquires
its symbionts through maternal inheritance (Turner,
pers. obs.). Brooded and recently released juvenile C.
pedunculatus always harbor zooxanthellae, but densities
in recently released individuals may be less than 10% of
those in adult anemones (Darmayati, 1993; Davy et ai,
1996). The fact that C pedunculatus produces offspring
that lack a full complement of zooxanthellae means that
they may be susceptible to infection by zooxanthellae re-
leased from other Anthozoa.

The sea anemones Anthopleura ballii (Cocks) (family
Actiniidae) and Anemonia viridis (Forskal) (family Ac-
tiniidae), and the zoanthid Isoioanthus sulcatus (Gosse)
(family Parazoanthidae) are frequently found in the
same localities as C. pedunculatus, and also harbor Sym-
biodinium sp. (Davy et al., in press). A ballii and A. vir-
idis regularly expel zooxanthellae in large boluses, as well
as in mucous strands (Davy, pers. obs.); it is also proba-
ble that /. sulcatus releases zooxanthellae, but this has
not been observed. This expulsion suggests that zooxan-
thellae from a variety of sources are available to infect
new hosts.

This research therefore aimed to determine whether
heterologous zooxanthellae can infect C. pedunculatus.
and to compare the persistence of homologous and het-
erologous zooxanthellae after infection. From this infor-
mation the potential for heterologous zooxanthellae to
establish a lasting symbiosis with C. pedunculatus in the
field could be inferred.

Materials and Methods

Collection and maintenance of symbiotic Anthozoa

The sea anemones Cereus pedunculatus (Pennant),
Anemonia viridis (Forskal), and Anthopleura ballii
(Cocks), and the zoanthid Isozoanthus sulcatus (Gosse)
were collected from Lough Hyne Marine Nature Re-
serve, Eire (5 1 °29' N; 9° 1 8' W). All four species are exclu-
sively subtidal at this location. The anemones were all
collected from a depth of 1 to 3 m, and the zoanthids
from 5 to 9 m. Specimens of A. viridis were also collected
from the intertidal zone at Shell Island, North Wales (52°
47' N; 004° 06' W). For comparative purposes, speci-
mens of the subtropical sea anemone Aiptasia pallicla
(Verrill), cloned from one individual, were obtained
from Dr. C- B. Cook, Bermuda Biological Station. The
A. pallida had originally been collected from a mangrove
root in Walsingham Pond, Bermuda.

Adult Anthozoa were kept in seawater from the Menai
Strait, North Wales. They were maintained at 21°C, illu-
minated at 80 ^mol photons irT2 s~' on a cycle of 12 h
light: 12 h dark, and fed twice weekly with Anemia sp.
(Bonneville Anemia International Inc.). The light and
temperature regimes were comparable to those experi-
enced during a warm summer in Lough Hyne (Turner,
1988, and his pers. obs.).

Preparation oj aposymbiotic Anthozoa

Juvenile specimens of C. pedunculatus were squeezed
from the enterons of brooding adult anemones and
placed in a dark-box to render them aposymbiotic. Sea-
water from the Menai Strait, at the ambient temperature
(10-18°C), flowed through the dark-box continuously. It
is unlikely that zooxanthellae were present in this seawa-
ter because Anthozoa-zooxanthella associations do not
occur locally. In addition, the examination of dark-
treated anemones suggested that infections had not oc-
curred under these conditions (see below). The juveniles
were collected on two occasions, with the first batch (n =
250) remaining in the dark for more than 3 years and
the second batch (n = 120) remaining in the dark for
5 months. After collection, the anemones were fed every
2 months with Anemia sp; the anemones also received
planktonic food via the flow-through seawater system. It
is likely that the juveniles were genetically related to one
another because all adult anemones were collected from
the same site and spawning of gametes has not been ob-
served in this population (Davy and Turner, pers. obs.).

Prior to use, 3-year dark-treated anemones (;; = 180)
with oral disc diameters of about 2 to 3 mm were placed
in 100-ml containers filled with filtered (0.45 nm) seawa-
ter (FSW). Five anemones were placed in each container.

210

S. K. DAVY ET AL.

These anemones were maintained at 21°C, illuminated
at 80 ^mol photons m : s ' on a cycle of 1 2 h light: 1 2 h
dark, and fed weekly with Anemia sp. for 2 weeks. The
anemones were ne\er observed to change from white to
brown over thi- ocriod and so were assumed to be apo-
symbiotic. This assumption was checked by squashing a
subsample of five anemones (anemones sampled from
the same container: n was limited by anemone availabil-
ity) and examining their tissues under bright-field and
epifluorescent illumination using a Leitz Orthoplan mi-
croscope. Neither zooxanthellae nor chlorophyll a were
detected, confirming that the 3-year dark-treated anem-
ones were aposymbiotic. Therefore, immediately after
being maintained in the light for 2 weeks, these anemo-
nes were used for monitoring patterns of long-term in-
fection by zooxanthellae. Non-inoculated control polyps
used in these experiments remained zooxanthella-free
over a period of 36 weeks, reconfirming that the anemo-
nes were aposymbiotic.

In contrast, squashes made of 5-month dark-treated
polyps (n = 5) before they were placed in the light re-
vealed a small number of residual zooxanthellae (less
than 1000 zooxanthellae per polyp). These anemones
were therefore used for short-term monitoring of infec-
tion patterns, where densities of residual zooxanthellae
were negligible in comparison to densities of phagocy-
tosed zooxanthellae. To keep residual zooxanthellae to a
minimum, these anemones were maintained in the dark
until inoculation.

Injection of aposymbiotic Cereus pedunculatus when co-
existing with symbiotic C. pedunculatus

Under nonstressful conditions, C. pedunculatus regu-
larly releases a small percentage (<0.3% per hour) of its
zooxanthellae (Darmayati, 1993). A motile stage has yet
to be observed in the life history of these zooxanthellae,
either following release or when in culture (Davy el a/.,
in press; Davy, pers. obs.). To establish whether zooxan-
thellae released by one anemone could infect another
anemone, 3-year dark-treated C. pedunculatus (n = 5)
were placed in a container with symbiotic C. peduncula-
tus (n = 5). Three-year dark-treated C. pedunculatus (n
= 5) in a second container acted as controls. All anemo-
nes were illuminated at 80 ^mol photons irr: s ' on a
cycle of 1 2 h light: 1 2 h dark, maintained at 2 1 °C, and fed
weekly with Anemia sp. After 8 weeks, the presence or
absence of zooxanthellae in the formerly aposymbiotic
polyps (which were still distinct due to their lighter pig-
mentation) was determined. This involved squashing the
polyps and using bright-field and epifluorescent micros-
copy to search their tissues. Densities of zooxanthellae
were not quantified.

Persistence of homologous and heterologous
zooxanthellae

To investigate the extent to which zooxanthellae from
different host species could establish a symbiosis with C.
pedunculatus, the uptake and persistence of homologous
and heterologous zooxanthellae following inoculation
were measured.

Three oral discs plus attached tentacles of C. peduncu-
latus, 5 tentacles from each of 10 individuals of A. hallii
and A. viridis, 30 polyps of/, sii/catus, and 10 complete
A. pa/lida were collected for isolation of zooxanthellae.
Host tissue for each host species was pooled, homoge-
nized in 10 ml FSW in a glass tissue-grinder, and centri-
fuged for 10 min at 1200 rpm. The supernatant was dis-
carded, and the algal pellet was resuspended in 5 ml FSW
and centrifuged for a further 5 min at 1 200 rpm. The su-
pernatant was again discarded and the algal pellet ad-
justed with FSW to give a concentration of 2 X 107 cells
ml '. Isolates from different host species were kept sepa-
rate.

Dark-treated specimens of C. pedunculatus (n = 150
and n = 90 for anemones maintained in darkness for
3 years and 5 months, respectively) with oral disc diam-
eters of about 2 to 3 mm were maintained in 1 00-ml con-
tainers (five anemones of the same dark-treatment per
container) and inoculated with homologous or heterolo-
gous zooxanthellae. Anemones in each container were
inoculated with zooxanthellae from just one source, thus
preventing cross-infection by zooxanthellae from differ-
ent sources. A 1-ml hypodermic syringe was used to de-
posit 0.2 ml of suspension (i.e.. 4 X 10" zooxanthellae)
onto the oral disc of each aposymbiotic anemone. This
number of zooxanthellae was sufficient to saturate the
uptake sites of these anemones (Davy, 1994). Once the
anemones had expanded again, 0. 1 ml of an Anemia sp.
suspension was pipetted onto each oral disc. This sus-
pension enhanced ingestion of zooxanthellae (Davy,
1 994). To remove any zooxanthellae and Anemia sp. not
captured by the anemones, the FSW was then changed.
Uninoculated 3-year and 5-month dark-treated anemo-
nes (/; = 25 and n = 15, respectively; five anemones per
1 00-ml container) acted as controls for spontaneous in-
fection. All anemones were maintained at 2 PC, illumi-

nated with 80 ^mol photons m - s ' on a cycle of 12 h
light: 12 h dark, and fed weekly with Anemia sp. The
densities of zooxanthellae in 5-month dark-treated
anemones were measured after 4 h. 2 days, and 4 days,
and the densities of zooxanthellae in 3-year dark-treated
anemones were measured after 2, 4, 6, 8, and 36 weeks.

Following the incubation period, polyps (/; = 4 or 5 for
each group; replicate anemones sampled from the same
container) were narcotized in 1.5% magnesium chloride

PERSISTENCE OF ZOOXANTHELLAE IN SEA ANEMONES

211

in FSW. Oral disc diameters were then measured using
an ocular micrometer, and oral disc areas were calcu-
lated. This method (i) facilitated measurement of large
numbers of polyps; (ii) was repeatable; and (iii) gave
units (square millimeters of oral disc) proportional to the
protein content of the polyp (r2 = 0.97 for anemones
with disc diameters of 1.0 to 3.0 mm; Davy. 1994). Pol-
yps examined between 4 hours and 4 days after reinfec-
tion were cut longitudinally, and residual Artemia sp.
and zooxanthellae were washed from their enterons; this
step was not necessary for anemones examined after
2 weeks or more. Each polyp was then homogenized in
I ml FSW. and the number of zooxanthellae in the ho-
mogenate was counted using a Fuchs Rosenthal hema-
cytometer. The density of zooxanthellae in the anemo-
ne's tissues was expressed as zooxanthellae per square
millimeter of oral disc.

The same method was used to determine the densities
of zooxanthellae in five similarly sized, naturally infected
C. pedunculatus. These values were compared with those
from the infection experiments.

Statistical analysis

Significant differences (P < 0.05) were identified using
one-way analysis of variance (ANOVA) followed by
Bonferroni pair-wise comparisons. When variances were

not homogeneous (as determined using Bartlett's statis-
tic), data were logarithmically transformed.

Results

Infection ofaposymbiotic Cereus pedunculatus when
co-e.\ixtinK with xymhioticC. pedunculatus

All aposymbiotic Cereus pedunculatus maintained in
the presence of symbiotic C. pedunculatus turned brown
after 2 months. Light microscopy revealed that this
change in color resulted from the presence of zooxan-
thellae in the endodermal cells. In contrast, control
anemones remained white and free of zooxanthellae af-
ter 2 months.

Short-term persistence of zooxanthellae

Figure 1 summarizes the patterns of infection of C.
pedunculatus by homologous zooxanthellae and by zoo-
xanthellae from Anthopleura ba/lii. Anemonia viridis,
Isoioantlnts sulcatus. and Aiptasia pa/lida over the first
4 days (i.e.. over the short-term).

The densities of homologous zooxanthellae were not
significantly different from the densities of any of the het-
erologous zooxanthellae at 4 h ( Bonferroni post-hoc AN-
OVA, P > 0.05); but note that the homologous zooxan-
thellae were 2.5 times denser than the zooxanthellae

Time (days)

B

1

Time (days)

Figure 1. Short-term persistence patterns of homologous and heterologous zooxanthellae in infected
Cereus pedunculatus- All anemones dark-treated for 5 months prior to infection and unfed over period
shown, n = 5. Points at 4 days offset for clarity. All values are means ± 1 SE. (A) Persistence of homologous
zooxanthellae from C. pedunculanm (• — •). and heterologous zooxanthellae from Anthopleura hallii
(A Aland Isi>:<>antlnt\ •>ulcalu.i(O- • -O). D- - D represents zooxanthellae in non-inoculated dark-
treated controls. (B) Persistence of heterologous zooxanthellae from Anemonia viridis from Lough Hyne
(4 _ . _ *)and Shell Island (V V), and.-liplasia pallidu (•• • ••).

212

S. K DAVY KT AL

Homologous zooxanthellae

4 6 8 36

/

B

H h

468
Time (weeks)

i
36

Figure 2. Long-term persistence patterns of homologous and heterologous zooxanthellae in infected
Cereus pedunculatus. All anemones dark-treated for more than 3 years prior to infection and fed weekly
over period shown, n = 5, except n = 4 for zooxanthellae from Antlwpleura halln and laoioanlhus sulcatus
at 36 weeks. Points at 36 weeks offset for clarity. All values are means ± 1 SE.(A) Persistence of homologous

zooxanthellae from C. pedunculatus (• — •). and heterologous zooxanthellae from A. balln (A A)

and / sulcatus (O- • -O). D - • - D represents zooxanthellae in non-inoculated dark-treated controls, and
white diamond represents /ooxanthellae in naturally infected C pedunculatus (B) Persistence of
heterologous zooxanthellae from Anenwnia viridis from Lough Hyne (*-•-*) and Shell Island
(V V), and Aiptasia pallida (•• • ••).

from A. ballii (Fig. 1A). Subsequently, homologous zoo-
xanthellae persisted at the highest densities. Homolo-
gous zooxanthellae were present at significantly greater
densities than any of the heterologous strains at 2 days
( Bonferroni post-hoc ANOV A, P < 0.0 1 ).

Short-term infection patterns by heterologous zooxan-
thellae were similar to one another, with the only major
difference being the relatively low uptake of zooxanthel-
lae from A. bal/ii. Zooxanthellae from A. ballii were pres-
ent at significantly lower densities than zooxanthellae
from /. sulcatus at 4 h (Bonferroni post-hoc ANOVA, P
<0.05).

Long-term persistence of zooxanthellae

Figure 2 summarizes the densities of zooxanthellae 2,
4, 6, 8, and 36 weeks after reinfection (i.e., over the long-
term), and the density of zooxanthellae in naturally in-
fected C. pedunculatus.

Homologous zooxanthellae persisted at significantly
greater densities than any of the heterologous zooxan-
thellae at 2 and 6 weeks (Bonferroni post-hoc ANOVA,
P < 0.05). Homologous zooxanthellae also persisted at
significantly greater densities than zooxanthellae from A.
bal/ii, A. viridis (Shell Island), and A. pallida at 4 weeks.

and zooxanthellae from A. ballii. A. viridis (Shell Island),
and /. sulcatus at 8 weeks ( Bonferroni post-hoc ANOVA,
P < 0.05). However, at 36 weeks, the densities of homol-
ogous zooxanthellae, heterologous zooxanthellae, and
zooxanthellae in naturally infected C. pedunculatus were
not significantly different (ANOVA, P > 0.05).

Zooxanthellae from A. pallida were present at signifi-
cantly lower densities than zooxanthellae from A. viridis
(Lough Hyne) at 4 weeks (Bonferroni post-hoc ANOVA,
P < 0.05). Zooxanthellae from A. viridis (Shell Island)
were present at significantly lower densities than any
other heterologous zooxanthellae at 2 and 4 weeks, and
zooxanthellae from A. ballii and A. viridis (Lough Hyne)
at 6 weeks (Bonferroni post-hoc ANOVA, P < 0.02). No
other significant differences were evident between the
densities of heterologous zooxanthellae (Bonferroni
post-hoc ANOVA, P> 0.05).

Anemone size

Prior to infection, anemones had oral disc diameters
of about 2 to 3 mm. At the end of the 36-week experi-
mental period, all anemones infected with either homol-
ogous or heterologous zooxanthellae still had oral disc
diameters of 2 to 3 mm and were not significantly differ-

PERSISTENCE OF ZOOXANTHELLAE IN SEA ANEMONES

213

ent in size (average diameter 2.39 ± 0.03 mm; ANOVA.
P> 0.95). As oral disc area is proportional to polyp pro-
tein content (see Materials and Methods), these results
suggest that changes in anemone hiomass were minimal
during the experimental period, presumably reflecting
the limited feeding regime (once per week). Thus the sub-
stantial changes in zooxanthella densities were most
likely due to changes in the number of zooxanthellae
rather than in the normalizing parameter (oral disc area).

Discussion

The results reveal that zooxanthellae from a range of
host species, localities, and environments can establish
symbioses with Cereus pedunculutiis. In the short-term,
homologous zooxanthellae persist to a greater extent
than heterologous zooxanthellae, but over the long-term
the two types of zooxanthellae achieve similar densities.
The possible reasons for these infection patterns and
their implications will be discussed.

Short-term persistence

C peditncidatus took up zooxanthellae from An-
thopleiira hallii at a lower rate than zooxanthellae from
the other host species, perhaps indicating discrimination
based upon the surface characteristics of the zooxanthel-
lae. However, uptake rates of zooxanthellae from the
other host species were similar. This suggests that during
the initial phases of infection C. pediinciilatus'was unable
to discriminate between these zooxanthellae, perhaps be-
cause of the similarity of their surface characteristics or
the host material contaminating their surfaces (Trench
etai. 1981; Colley and Trench, 1983).

Following phagocytosis, all populations of zooxan-
thellae declined, with the homologous populations de-
clining less rapidly than most of the heterologous popu-
lations. Symbiont populations also declined following
infections of Hydra viridissima (Jolley and Smith, 1980)
and the jellyfish Cassiopeia xamachana (Colley and
Trench. 1983), but did not do so following infections of
a number of other species of Cnidaria (Muscatine et al.,
1975; Schoenberg and Trench, 1980c; Berner el al.,
1993). The decline may represent the nonselective elim-
ination of a proportion of the phagocytosed zooxanthel-
lae or the selective elimination of unhealthy and incom-
patible zooxanthellae. Elimination may occur via expul-
sion or digestion. Regular expulsion of zooxanthellae is
a common feature of Anthozoa-zooxanthella symbioses
(Steele, 1975; Heegh-Guldberg et al., 1987; Stimson and
Kinzie, 1991; McCloskey et al.. 1996). and the produc-
tion of zooxanthella-containing boluses was observed
during the course of experiments. In contrast, digestion

is thought not to play a major role in the regulation of
populations of zooxanthellae (Colley and Trench, 1985).
The elimination of some zooxanthellae more quickly
than others has also been observed in C. xamachana
(Colley and Trench, 1983). This trend may indicate
differences in zooxanthella survivorship following isola-
tion or discrimination among genotypically distinct zoo-
xanthellae by means of post-phagocytotic recognition
(Colley and Trench, 1983; Trench, 1988. 1992, 1993;
Markell et al., 1992; Markell and Trench, 1993). If zoo-
xanthellae were being discriminated against by post-
phagocytotic recognition, it is curious that a small num-
ber of zooxanthellae always persisted. Perhaps there was
some variation within the source populations, with the
retained zooxanthellae being the few that possessed cer-
tain appropriate characteristics.

Long-term persistence of zooxanthellae

Homologous zooxanthellae repopulated C. peduncu-
latus much more rapidly and achieved higher densities
than did heterologous zooxanthellae. Similar patterns
have been observed in C xamachana (Colley and
Trench. 1983) and Aiptasia tagetes(= pallida) (Schoen-
berg and Trench, 1980c). In contrast, zooxanthellae
from Anemonia viridis from Shell Island repopulated C.
pednncidatus more slowly than did any of the other zoo-
xanthellae. These repopulation trends may result from
host-algal recognition and greater expulsion or digestion
of some types of zooxanthellae than others. However, the
repopulation patterns could also be determined by
differing growth rates of zooxanthellae; changes in host
biomass (either growth or shrinkage), which could cause
the 'dilution' or 'concentration' of zooxanthella popula-
tions, were probably not responsible for the density
changes. Comparison of growth and expulsion rates of
homologous and heterologous zooxanthellae during re-
population of aposymbiotic anemones would be an in-
teresting topic for future work.

Several factors could influence the growth of zooxan-
thellae during repopulation. Firstly, the light and tem-
perature regimes may favor growth by one type of zoo-
xanthellae over another. However, this possibility is not
supported by the similarity of the specific growth rates
(0.25-0.29 d~') of zooxanthellae from C. pedunculatus,
A. ballii. and A. viridis, when cultured in vitro under the
same light and temperature regimes as those used here
(Davy. 1994). Secondly, the growth of zooxanthellae
may depend upon the ability of a particular strain or spe-
cies to survive in the intracellular environment of the
host (Rahat and Reich, 1988). But this hypothesis is in-
consistent with evidence from analogous plant-micro-
bial symbioses (Trench, 1993). Thirdly, hosts may be

214

S. K. DAVY ET AL

able to control the growth of zooxanthellae directly
(Smith and Muscatine. 1996). It is therefore possible that
C. pedunculatus was able to exercise more control over
some types of / i.xanthellae than others. Finally, the
growth of zo •• nellae may be limited by the intracel-
lular space ,iable within the host, with small zooxan-
thellae achieving greater densities than large zooxanthel-
lae. It seems unlikely that this hypothesis can explain the
densities achieved by zooxanthellae from A. viridis
(Lough Hyne) and A. pallula (Fig. 2B), given that these
zooxanthellae are similar in size to zooxanthellae from
C. pedunculatus when in their original hosts, after resid-
ing in reinfected C. pedunculatus for 36 weeks, and when
cultured in vitro (Davy, 1994; Davy et al., 1996, and in
press). It is unknown, though, whether these zooxanthel-
lae maintained similar sizes throughout the repopulation
process.

In spite of previous differences, at 36 weeks the densi-
ties of homologous and heterologous zooxanthellae, and
zooxanthellae in naturally infected C. pedunculatus,
were similar. The decline in the density of homologous
zooxanthellae to a "normal" level is unique. If the pop-
ulation of zooxanthellae was unialgal, then the decline
may result from the population being "brought under
control." However, why this occurred only several
months after the establishment of the symbiosis is un-
known. One possibility is that control mechanisms be-
come repressed in aposymbiotic anemones and are re-
stored during the repopulation process. Alternatively, if
the homologous population contained a mixture of zoo-
xanthella types, then the decline may result from an ini-
tially successful type becoming unstable and being re-
placed by a type better suited to the culture conditions.

The ultimate success of heterologous zooxanthellae
that were initially slow to establish a symbiosis with C.
pedunculatus can also be interpreted in different ways de-
pending upon the nature of the zooxanthella popula-
tions. Firstly, if the populations were unialgal, then host-
symbiont adjustment over time may have enhanced the
compatibility of the partners (Roughgarden, 1975;
Smith, 1980). It would be interesting to re-isolate heter-
ologous zooxanthellae or make reinfected anemones
aposymbiotic again, and determine whether the repopu-
lation process is faster when repeated. In a comparable
experiment, however, the growth of heterologous zoo-
xanthellae in A. tagetcs was not enhanced by their previ-
ous association with the same host species (Schoenberg
and Trench. 1980c). Alternatively, if the populations
contained a mixture of zooxanthella types, then the
"normal" densities achieved by heterologous zooxan-
thellae at 36 weeks may represent the proliferation of a
small number of zooxanthellae identical to those usually
harbored by C. pedunculatus. But such a mechanism is

unlikely to explain the gradual increase in the density of
zooxanthellae from A. pallida. This anemone is found
at subtropical latitudes in the western Atlantic, and so
probably contains zooxanthellae that are quite different
from those in C. pedunculatus. In fact, the zooxanthellae
of this anemone are believed to be a single species, Sym-
biodinium bermudense (Banaszak et al., 1993). Further-
more, evidence from carbon-flux studies suggests that
the homologous and heterologous zooxanthella popula-
tions were not identical at 36 weeks (Davy, 1994).

Host-symbionl recombination in the field

Molecular genetic techniques must be employed to de-
termine the precise nature of the source populations of
zooxanthellae and whether heterologous zooxanthellae
actually establish symbioses with C. pedunculatm in the
field. The long-term infection patterns (Fig. 2) suggest
that host-symbiont recombination may be possible — if,
that is, the new symbiosis is competitive under the pre-
vailing environmental conditions and the heterologous
zooxanthellae are not overgrown by homologous zoo-
xanthellae in the short-term (Schoenberg and Trench,
1 980c; Colley and Trench, 1983). The ability to establish
symbioses with zooxanthellae from a range of sources
may enable C. pedunculatus to adapt to different envi-
ronmental regimes, as has been suggested for corals
(Buddemeier and Fautin, 1993; Rowan and Knowlton.
1995). Alternatively, it may simply increase the anemo-
ne's chances of survival should it lose all of its zooxan-
thellae and have to acquire new symbionts. There are,
however, no reports of "bleached" C. pedunculatus in
the field. How zooxanthellae behave in mixed homolo-
gous-heterologous inoculations and the ecological ad-
vantages of maintaining heterologous zooxanthella pop-
ulations are important questions for future research,
both in temperate and tropical systems.

Acknowledgments

We thank Drs. R. K. Trench and C. B. Cook, and sev-
eral anonymous reviewers for valuable comments on
earlier drafts of the paper. We also thank the Irish Wild-
life Service for permission to work and collect speci-
mens in Lough Hyne Marine Nature Reserve. This
work was carried out whilst SKD was in receipt of a
SERC award.

Literature Cited

Banaszak, A. T., R. Iglesias-Prielo, and R. K. Trench. 1993. Scripp-
sii'lla n'k'llac sp. nov. (Peridiniales) and Gloeodinium vismm sp.
nov. (Phytodiniales), dinoflagellate symbionts of two hydrozoans
(Cmdaria)./ Phycol. 29:517-528.

PERSISTENCE OF ZOOXANTHELLAE IN SEA ANEMONES

215

Berner, T., G. Baghdasarian, and L. Muscafine. 1993. Repopula-
tion of a sea anemone with symbiotic dinoflagcllates: analysis by
in vivo fluorescence. / E.\p. Mar. Bin/. Ecol. 170: 145-158.

Buddemeier, R. \V., and D. G. Fautin. 1993. Coral bleaching as an
adaptive mechanism: a testable hypothesis. Bioaciemv 43: 320-
326.

Colley, N. J., and R. K. Trench. 1983. Selectivity in phagocytosis
and persistence of symbiotic algae by the scyphistoma stage of the
jellyfish Cassiopeia xamachana. Proc. R Soc. Lond Ser B 219:
61-82.

Colley, N. J., and R. K. Trench. 1985. Cellular events in the re-estab-
lishment of a symbiosis between a marine dinoflagellate and a coe-
lenterate. Cell Tissue Res 239: 93-103.

Darmayati. Y. 1993. The effect of elevated temperature on the sym-
biosis between the sea anemone Cereus pedunculatus Pennant and
Symbiodinium sp. M.Sc. thesis. University of Wales. 54 pp.

Davy, S. K. 1994. The specificity of temperate anthozoan-dinoflag-
ellate symbioses. Ph.D. thesis. University of Wales. 546 pp.

Davy, S. K., I. A. N. Lucas, and J. R. Turner. 1996. Carbon budgets
in temperate anthozoan-dmoflagellate symbioses. Mar. Bin! 126:
773-783.

Fin, \V. K., and R. K. Trench. I983a. Infection of coelenterate hosts
with the symbiotic dinoflagellate Symbiodinium microadriaticum.
Pp. 675-681 in Endocytobiology II, W. Schwemmlerand H. E. A.
Schenk. eds. Walter deGruyter, Berlin.

Fitt. \V. K., and R. K. Trench. 1983b. Endocytosis of the symbiotic
dinoflagellate Symbiodinium microadriaticum Freudenthal by en-
dodermal cells of the scyphistomae of Cassiopeia xamachana and
resistance of the algae to host digestion. J. Cell Sci. 64: 1 95-2 1 2.

Glynn, P. W. 1991. Coral bleaching in the 1980's and possible con-
nections with global warming. Trends Ecol. Evol. 6: 1 75- 1 79.

Heegh-Guldberg, O., L. R. McCloskey, and L. Muscatine. 1987. Ex-
pulsion of zooxanthellae by symbiotic cnidarians from the Red Sea.
Coral Reefs 5: 201-204.

Jolley, E., and D. C. Smith. 1980. The green hydra symbiosis. II. The
biology of the establishment of the association. Proc. R Soc. Lond.
Ser £207:311-333.

Kinzie, R. A. 1974. Experimental infection of aposymbiotic gorgo-
nian polyps with zooxanthellae. / Exp. Mar Bio/. Ecol 15: 335-
345.

Kinzie, R. A., and G. S. Chee. 1979. The effect of different zooxan-
thellae on the growth of experimentally reinfected hosts. Biol Bull
156:315-327.

Manuel, R. L. 1981. British Antho:oa. Academic Press. London. 241
pp.

Markell.D. A..R. K.Trench,andR.Iglesias-Prieto.l992. Macromol-
ecules associated with the cell walls of symbiotic dinoflagellates.
Symbiosis 12: 19-31.

Markell, D. A., and R. K. Trench. 1993. Macromolecules exuded by
symbiotic dinoflagellates in culture: amino acid and sugar compo-
sition. / Phycol. 29: 64-68.

McCloskey, L. R., T. G. Cove, and E. A. Verde. 1996. Symbiont ex-
pulsion from the anemone Antlwplcura clt'gantissiina (Brandt)
(Cnidaria: Anthozoa). J. Exp .Mar Biol Ecol. 195: 173-186.

McNally, K. L., N. S. Govind, P. E. Thome, and R. K. French. 1994.
Small subunit nbosomal DNA sequence analyses and a reconstruc-
tion of the inferred phylogeny among symbiotic dinoflagellates
(Pyrrophyta). J Phycol. 30: 316-329.

Muller-Parker, G. 1996. A comparison of temperate and tropical al-
gal-anemone associations. 8th Int. Coral Reef Symp. Abstracts:
139.

Muscatine, L., C. B. Cook, R. L. Pardy, and R. R. Pool. 1975. Up-

take, recognition and maintenance of symbiotic Chlorella by Hydra
vindis. Symp. Soc. E\p. Biol. 29: 175-203.

Rahat, M., and V. Reich. 1988. The establishment of algal/// ydra
symbioses — a case of recognition or preadaptation? Pp. 297-310
in Cell to Cell Signals in Plant, Animal and Microbial Svmbioses,
NATO ASI Series HI 7, S. Scannerini, D. C. Smith. P. Bonfante-
Fasolo and V. Gianinazzi-Pearson, eds. Springer- Verlag. Berlin.

Rossi, L. 1975. Sexual races in Cereus pedunculatus (Boad.). Pubbl
Sta:. Zoo/. Napoli (Suppl. . I '/// Ettr. Mar. Biol. Symp.) 39: 462-
470.

Roughgarden, J. 1975. Evolution of marine symbiosis — a simple
cost-benefit model. Ecology 56: 1201-1208.

Rowan, R., and N. Knowlton. 1995. I ntraspecific diversity and ecolog-
ical zonation in coral-algal symbiosis. Proc. Nail. Acad. Sci. USA
92:2850-2853.

Rowan, R., and D. A. Powers. 1 99 1 a. Molecular genetic identification
of symbiotic dinoflagellates (zooxanthellae). Mar. Ecol. Prog. Ser.
71:65-73.

Rowan, R., ajid D. A. Powers. 1991 b. A molecular genetic classifica-
tion of zooxanthellae and the evolution of animal-algal symbioses.
Science 25: 1348-1351.

Schafer, \V. 1981. Fortpflanzung und Sexualitat von Cereus pcdun-
culaliis und Actinia euuina (Anthozoa. Actiniaria). Helgol. Meere-
sunters. 34: 45 I -46 1 .

Schoenberg, D. A., and R. K. Trench. 1980a. Genetic variation in
Symbiodinium (= Gymnodinium) microadriaticum Freudenthal.
and specificity in its symbiosis with marine invertebrates. I. Isoen-
zyme and soluble protein patterns of axenic cultures ofS. microa-
drialicum. Proc R Soc. Lond. Scr. B 207: 405-427.

Schoenberg, D. A., and R. K. Trench. 1980b. Genetic variation in
Symbiodinium (= Gymnodinium) microadriaticum Freudenthal.
and specificity in its symbiosis with marine invertebrates. II. Mor-
phological variation in S. microadriaticum. Proc. R. Soc. Lond. Ser.
5207:429-444.

Schoenberg, D. A., and R. K. Trench. 1980c. Genetic variation in
Symbiodinium (= Gymnodinium) microadriaticum Freudenthal.
and specificity in its symbiosis with marine invertebrates. III. Spec-
ificity and infectivity of S. microadriaticum. Proc. R Soc. Lond
Ser B 207: 445-460.

Shick. J. M. 1991. .1 Functional Biology of Sea Anemones. Chapman
and Hall. New York. 395 pp.

Smith, D. C. 1980. Principles of colonisation of cells by symbiontsas
illustrated by symbiotic algae. Pp. 317-332 in Endocytobiology I.
W. Schwemmlerand H. E. A. Schenk, eds. Walter deGruyter. Ber-
lin.

Smith, G. J., and L. Muscatine. 1996. Why don't symbiotic dino-
flagellates overgrow theircnidarian hosts? 8th Int. Coral Reef Symp.
Abstracts: 183.

Steele, R. D. 1975. Stages in the life history of a symbiotic zooxan-
thella in pellets extruded by its host Aiptasia tagetes (Duch. and
Mich.) (Coelenterata. Anthozoa). Biol. Bull 149: 590-600.

Stimson. J., and R. A. kinzie. 1991. The temporal pattern and rate of
release of zooxanthellae from the reef coral Pocillopora danucornis
(Linnaeus) under nitrogen-enrichment and control conditions. J.
Exp. Mar Biol. Ecol. 153: 63-74.

Trench, R. K. 1971. The physiology and biochemistry of zooxanthel-
lae symbiotic with marine coelenterates. III. The effect of homoge-
nates of host tissues on the excretion of photosynthetic products in
vitro by zooxanthellae from two marine coelenterates. Proc. R. Soc.
Lond. Ser. B 177: 25 1-264.

Trench, R. K. 1981. Cellular and molecular interactions in symbioses
between dinoflagellates and marine invertebrates. PureAppl. Chcm.
53:819-835.

216

S. K. DAVY ET AL.

Trench, R. K. 1987. Dinoflagellates in non-parasitic symbioses. Pp.

530-570 in The Biology <>/ Dino/Jage/lates. F.3.R. Taylor, ed.

Blackwell Scientific, Oxford.
Trench, R. K. 1988. Specificity in dinomastigote-marine invertebrate

symbioses: an evaluation of" hypotheses of mechanisms involved in

producing specificity. Pp. 325-346 in Cell W Cell Signals in Plant,

Animal and Microbial Symbioses. NATO ASI Series H 1 7, S. Scan-

nerini, D. C. Smith, P. Bonfante-Fasolo and V. Gianinazzi-Pear-

son. eds. Springer-Verlag, Berlin.
Trench, R. K. 1992. Microalgal-invertebrate symbiosis, current

trends. Pp. 129-142 in Encyclopedia of Microbiology, Vol. 3, J.

Lederberg, ed. Academic Press, New York.
Trench, R. K. 1993. Microalgal-invertebrate symbioses: a review. En-

docytobioxix Cell Res 9: 135-175.
Trench, R. K., N. J. Colley, and XV. K. Fin. 1981. Recognition phe-

nomena in symbioses between marine invertebrates and "zooxan-
thellae"; uptake, sequestration and persistence. Her. Deulscli. Bui
Ges. Bil 94: 529-545.

Trench, R. K., and H. XX insor. 1987. Symbiosis with dinoflagellates
in two pelagic flatworms, .Amphixcolops sp. and Haplodiscus sp.
Symbiosis 3: 1-22.

Turner, J. R. 1988. The ecology of temperate symbiotic Anthozoa.
D. Phil, thesis. University of Oxford, 484 pp.

XX'eis, V. M., and R. P. I.evine. 1996. Differential protein profiles re-
flect the different lifestyles of symbiotic and aposymbiotic An-
thiipleiira elegantissima, a sea anemone from temperate waters. J.
li.\p. Biol. 199: 883-892.

XX illiams, E. H.. and L. Bunkley- XX illiams. 1990. The world-wide
coral reef bleaching cycle and related sources of coral mortality.
Aioll Res. Bull 335: 1-71.

Reference: Bio/ Bull 192: 217-230. (April. 1997)

p58, a Cytoskeletal Protein, Is Associated With Muscle
Cell Determinants in Ascidian Eggs

WILLIAM R. BATES

Bamfield Marine Station, Bam field, British Columbia. Canada I 'OR 1BO. and the Department of
Biology, University of Victoria. Victoria. British Columbia, Canada \'SW 2Y2

Abstract. The theory that p58, a cytoskeletal protein,
has an important role in ascidian muscle cell develop-
ment was tested by altering normal distributions of or-
ange-pigmented myoplasm in Boltcnia villosa embryos
and determining if muscle development is correlated
with the presence of p58. Removal of the animal region
of fertilized Boltenia eggs resulted in the redistribution of
myoplasm into the anterior endoderm cells of the
embryo. Despite alterations in the normal distribution
of myoplasm, these embryos developed into larvae.
However, when four-celled embryos that exhibited al-
tered distributions of pigmented myoplasm were stained
with NN18, an antibody that stains p58, a maximum of
two blastomeres were stained, as in control embryos.
Compression of Boltenia embryos at the four-celled
stage caused the myoplasm to be partitioned into four
blastomeres of an eight-celled embryo, instead of into
two blastomeres. Compressed and cleavage-arrested
eight-celled embryos developed myosin and muscle ac-
tin RNA in a maximum of four blastomeres, compared
to a maximum of two blastomeres in control embryos.
When compressed eight-celled embryos were stained
with NN 1 8, p58 was present in a maximum of four blas-
tomeres. These results support the idea that the cytoskel-
etal protein p58 is associated with muscle cell determi-
nants in ascidian eggs.

Introduction

Egg cytoskeletons are known to have important roles
in the determination of embryonic cells (Jeffery, 1982;
Jeffery, 1989; Elinson. 1990; Hill el at., 1990). Experi-
ments performed by Jeffery and Meier (1983), Jeffery

Received 23 September 1996; accepted 3 January 1997.

and Swalla (1990a), Swalla el a/. (1991), and Marikawa
( 1 995) support the idea that cytoplasmic factors required
for the development of larval muscle cells are associated
with myoplasmic cytoskeletal domain (MCD) of ascid-
ian eggs. The MCD consists of two distinct, yet inte-
grated, cytoskeletal systems: a plasma membrane lamina
(PML) and a deep filamentous lattice (DFL). The PML
is an actin-containing skeleton, sensitive to DNase I
treatment, that contracts towards the vegetal pole during
ooplasmic segregation (Jeffery and Meier, 1983). The
DFL (Jeffery and Meier. 1983; Swalla el ai, 1991) is sit-
uated beneath the PML; contains pigment granules, mi-
tochondria, and other components: and co-segregates
with the PML during ooplasmic segregation. Swalla el al.
(1991) have shown that a protein termed p58, which
binds to a vertebrate intermediate filament antibody, is a
component of the DFL. In ascidian species that produce
tadpoles, the DFL was present in the cortical cytoplasm
of the unfertilized egg and was partitioned exclusively
into embryonic muscle progenitor cells. Eggs produced
by direct-developing ascidians, which do not produce
larvae with muscular tails, lack p58 (Swalla el al.. 1991;
Bates, 1995).

Normal distributions of myoplasm were altered in as-
cidian embryos by using a microcompression technique
(Whittaker, 1980). Whittaker positioned four-celled Sty-
e/a embryos between glass plates during the third cell
cycle and applied pressure that resulted in the reorienta-
tion of mitotic spindles according to Hertwig's Rule. In
compressed eight-celled embryos, all of the blastomeres
were in the same plane, instead of four animal blasto-
meres being positioned over four vegetal blastomeres.
Furthermore, in compressed embryos, rm 'plasm was
present in four blastomeres, instead of in two blasto-
meres. Compressed and non-compress eight-celled
embryos were then treated with cytoch. asm B to pre-

217

218

W. R. BATES

B

Figure 1. A schematic drawing showing the distribution of cyto-
plasmic determinants in unfertilized and fertilized ascidian eggs. (A)
In the unfertilized egg, the myoplasm (bold line) is situated in the egg
periphery. The ectoplasm (white region) is located inside the germinal
vesicle, and the endoplasm (stipled region) fills the inner egg region. (B)
After an egg is fertilized, the myoplasm moves into the vegetal pole
region. The ectoplasm forms a band of cytoplasm above the myoplasm,
and the endoplasm resides above the ectoplasm. (C) During the second
phase of ooplasmic segregation, the myoplasm and ectoplasm shift into
the equatorial region. At the end of the second phase of ooplasmic seg-
regation, the egg cytoplasm is divided into five regions: ( 1 ) ectoplasm:
(2) neuroplasm; (3) notochord plasm; (4) myoplasm: and (5) endo-
plasm (based on Conklin, 1905).

vent subsequent cell divisions. Treatment with cytocha-
lasin facilitated the identification of myoplasm-contain-
ing blastomeres in eight-celled embryos after they were
cultured for the time required for control embryos to de-
velop into hatched larvae. When these embryos were ex-
amined for the activity of acetylcholinesterase (AchE), a
muscle-specific enzyme, a maximum of four blastomeres
comprising a compressed embryo expressed AchE activ-
ity, whereas in non-compressed eight-celled embryos a
maximum of two blastomeres exhibited AchE activity.
These elegant experiments performed by Whittaker pro-
vide rigorous support for Conklin's idea, published in
1905. that ascidian eggs contain muscle cell determi-
nants.

Bissection of fertilized eggs has also been used to alter
the normal distributions of myoplasm in ascidian em-
bryos. When the animal hemispheres of fertilized eggs

were removed, myoplasm was partitioned into more
than the normal number of blastomeres (Bates. 1988).
During the first phase of ooplasmic segregation, fertilized
eggs were bissected into vegetal fragments that contained
all of the egg myoplasm. termed myoplasm-enriched
(ME) fragments, and into animal fragments that lacked
myoplasm. ME fragments composed of 40%-50% of the
total egg volume usually cleaved normally, completed
gastrulation, and in some cases developed into larvae. At
each developmental stage, it was shown that more than
the normal number of blastomeres contained my-
oplasm. In larvae derived from ME fragments, most of
the myoplasm was contained in tail muscle cells, al-
though significant quantities of myoplasm were present
in some of the head endoderm cells.

When the spatial expressions of tissue-specific markers
were examined in ME larvae, each marker was normally
expressed. For example, AchE activity and myosin were
expressed in the cytoplasm of tail muscle cells, and alka-
line phosphatase ( AP) activity was expressed in the cyto-
plasm of endoderm cells situated in the head region. In
another set of experiments, smaller ME fragments com-
posed of 10%-30% of the total egg volume were pro-
duced from the vegetal pole region of fertilized eggs at
the first stage of segregation, and these fragments could
sometimes undergo several rounds of cleavage before cell
division stopped. When cleavage-arrested four-celled
ME embryos composed of 10%-30% of the total egg vol-
ume were tested for AchE activity after the controls de-
veloped into larvae, in some cases AchE activity was de-
tected in all four blastomeres. In cleavage-arrested con-
trol four-celled embryos and ME four-celled embryos
composed of 40%-50% of the total egg volume, AchE

animal

B

animal

vegetal

vegetal

Figure 2. Schematic drawings showing the designation of blasto-
meres composing four-celled and eight-celled ascidian embryos. (A)
The A3 blastomeres of a four-celled embryo lack myoplasm. whereas
the B3 blastomeres contain myoplasm and develop muscle cell features
when isolated from the rest of the embryo. ( B) B4. 1 blastomeres contain
myoplasm and produce most of the tail muscle cells. mesench> me cells,
and some of the endoderm cells and notochord cells. A4. 1 blastomeres
produce spinal cord cells, endoderm, notochord. and distal tail muscle
cells. a4.2 blastomeres produce epidermal and brain cells. b4.2 blasto-
meres produce epidermis, spinal cord cells, and distal tail muscle cells
(Conklin, 1905; Nishida and Satoh. 1983).

p58 AND ASCIDIAN MUSCLE CELL DETERMINANTS

219

B

• i

^f

Figure 3. Light photomicrographs of detergent-extracted larvae.
(A) A control larva showing the pigmented myoplasm within tail mus-
cle cells (t). Endoderm cells situated within the head region (h) do not
contain myoplasm. (B) A myoplasm-enriched (ME) larva showing the
pigmented myoplasm within tail muscle cells (t) and the pigment gran-
ules e\ident within some of the endoderm cells situated in the head
region (h). The dotted white lines shown in (A) and (B) designate the
boundary between the larval head and and tail regions. Scale bars equal
50 Mm.

activity was detected in a maximum of two blastomeres
that corresponded to the posterior B3 muscle progenitor
cells.

The contrasting results obtained from these two kinds
of myoplasm redistribution experiments present an in-
triguing paradox. Why were muscle cell fates altered in
compressed embryos and in small ME embryos pro-
duced from the vegetal pole region, whereas muscle cell
fates were normal in ME embryos composed of 40%-
50% of the total egg volume? The present study resolves
this paradox by examining the distribution of MCD
components in ME and compressed Boltenia villosa em-
bryos and provides experimental support for the idea
that p58 anchors ascidian muscle cell determinants.

Materials and Methods

Adult ascidians and embryo cultures

Boltenia villosa (Stimpson 1864) adults were pur-
chased from Westwind Sealab Supplies, Victoria, British
Columbia. Canada. Adults were maintained under con-
ditions of constant light to prevent spawning (West and
Lambert. 1975). Eggs and sperm were dissected from the
gonads of two or more individuals. The eggs were cross-
fertilized, washed several times with large volumes of
seawater, and cultured at 1 1°C until the desired develop-
mental stages were obtained.

Surgical methods

The spatial patterns of five kinds of cytoplasmic fac-
tors present in unfertilized and fertilized ascidian eggs

are shown in Figure 1. These factors determine epider-
mal, muscle, endoderm, notochord, and nerve cell fates.
The present study is focused on muscle cell factors local-
ized in the myoplasm of ascidian eggs. Prior to fertiliza-
tion, the myoplasm is present in the cortical cytoplasm
that surrounds the egg. Within minutes of fertilization,
the myoplasm is dramatically segregated into the vegetal
hemisphere by a series of precisely controlled cyto-
plasmic movements, termed ooplasmic segregation. A
fixed cleavage pattern subsequently partitions the my-
oplasm into specific blastomeres of the embryo (Fig. 2).
In four-celled embryos, the posterior B3 blastomeres
contain myoplasm, whereas the anterior A3 blastomeres
lack it. At the eight-cell stage, the B4. 1 blastomeres con-
tain myoplasm, and these cells produce most of the tail
muscle cells of the larva (Conklin, 1905; Nishida and Sa-
toh. 1983).'

The normal distribution of myoplasm within embry-
onic progenitor cells was altered using a surgical method
previously described by Bates and Jeffery (1987). Eggs
were fertilized and allowed to undergo the first phase of
ooplasmic segregation, in which the cortical myoplasm
is moved into the vegetal hemisphere and surrounds the
vegetal pole. At a position corresponding to the animal
pole, a tear was made in the follicular envelope (FE) that
surrounds the egg. In some cases, 50%-60% of the total
egg was extruded through the torn FE, leaving the my-

n

•• " .•',.

Figure 4. Light photomicrograph of a thick section through a my-
oplasm-ennched (ME) larva show ing pigment granules ( pg) in tail mus-
cle cells and in the cytoplasm of endoderm cells situated in the head
region (h). The notochord (n) is displaced into a more anterior position
than normal. Scale bar equals 1 00 ^m.

220

W. R. BATES

.: WA

Figure 5. Transmission electron micrographs showing the ultrastructural features of muscle and endo-
derm cells of a myoplasm-enriched (ME) larva. (A) Sacromeres are evident in tail muscle cells near the
notochord (n). Magnification equals 6.300X. (B) A higher magnification of (A) showing well-developed
sacromere structure. Magnification equals 13.500X. Arrow points to the notochord sheath that surrounds
the notochord (n). (C) Fine structure of yolky endodermal cells situated in the head region. Magnification
equals 10.500X. (D) Features of endoderm and epidermal cells in the head region. Magnification equals
10.500X.

oplasm-containing vegetal hemisphere within the FE.
The cytoplasmic bridge connecting the extruded egg re-
gion with the myoplasmic region was cut using a fine

needle. In other cases, 70%-90% of the total egg volume
was extruded through the tear, and the cytoplasmic
bridge was cut leaving the vegetal pole ME fragment

p58 AND ASCIDIAN MUSCLE CELL DETERMINANTS

221

Figure 6. Photomicrographs showing myosin heavy chain expression in a control larva, a cleavage-
arrested eight-celled embryo, and a compressed and cleavage-arrested eight-celled embryo. (A) Section ol
a control lar\a showing the expression of myosin heavy chain protein in tail muscle cells (arrow) situated
along the notochord. ( Bisection of a cleavage-arrested eight-celled embryo showing myosin in the periph-
eral cytoplasm of B4. 1 blastomeres (long arrows). (C) Section of a compressed and cleavage-arrested eight-
celled embryo showing myosin in the peripheral cytoplasm of B4.1 blastomeres (long arrows) and two
additional blastomeres (short arrows). Scale bar in (A) equals 50 ^m/Same magnifications in (B and C) as
in (A).

within the FE. ME fragments of both size classes were
either fixed for electron microscopy or immunocyto-
chemistry, or they were transferred to tissue culture wells
containing filtered seawater and cultured until the de-
sired developmental stages were obtained.

Microcompression of embryos

Whittaker (1980) modified T. H. Morgan's micro-
compression technique (1910) to reorient mitotic spin-
dles of eight-celled Stye/a embryos. Compression re-
sulted in the partitioning of the myoplasm of eight-celled
Slvela embryos into four blastomeres instead of two blas-
tomeres. A compression method similar to Whittaker's
was used to alter the normal distributions of myoplasm
in B. villasa embryos. Microcompression chambers were
prepared by positioning strips of lens paper about 20 mm
apart on a glass microscope slide. A drop of seawater
containing four-celled embryos was positioned between
the paper strips, and a coverslip was placed over the em-
bryos. Seawater was gradually withdrawn from beneath
the coverslip by capillary action to exert a gentle pressure
on the embryos. The extent of compression was moni-
tored under a microscope. Pressure was applied to em-
bryos for about 15 to 20 min. Next, the coverslip was
floated away from the compressed embryos and the em-
bryos were transferred to a well containing 2 jig/ml cyto-
chalasin B (CB; Sigma Chemical Co., St. Louis, MO) dis-
solved in seawater. CB treatment inhibits cell division, as
previously described by Whittaker (1980). Control and
compressed eight-celled CB embryos were cultured until
the control eggs developed into hatched larvae. CB em-
bryos were subsequently processed for in situ hybridiza-
tion or immunocytochemistry.

Identification of myoplasm

Orange pigment granules embedded in the cortex of
B. villosa eggs were used to produce myoplasm-enriched
(ME) egg fragments. Some specimens were treated with
0.5% Triton X-100 detergent to make the myoplasm-
containing cells more visible in photographs (Jeffery and
Meier, 1983; Bates, 1988).

Transmission electron microscopy

Larvae were fixed with 2% glutaraldehyde in 0.1 M
phosphate buffer, pH 7.4, for 30 min at room tempera-
ture, as previously described by Jeffery and Meier ( 1983).
Specimens were rinsed with phosphate buffer and post-
fixed in 1% osmium tetroxide dissolved in 0.1 M phos-
phate buffer, pH 7.4, for 1 h at room temperature. After
the samples were rinsed with the phosphate buffer, they
were dehydrated through a graded series of ethanol. Fol-
lowing gradual infiltration with Spurr's resin, specimens
were embedded. Thick sections (0.5 j/m) and thin sec-
tions were cut. The thin sections were stained with 2%
aqueous uranyl acetate and lead citrate and viewed using
a Phillips 420 electron microscope at 80 kV.

Scanning electron microscopy

Specimens were prepared for scanning electron mi-
croscopy (SEM) using a modification of a method pre-
viously described by Jeffery and Meier (1983). Speci-
mens were extracted with 0.5% Triton X-100 detergent
dissolved in seawater for 45 to 60 min at room tempera-
ture and then washed in seawater. Triton X-100 de-
tergent removes cell membranes to expose the underly-
ing MCD for SEM (Jeffery and Meier. 1983). Specimens

222

W. R. BATES

B

vN|S
•i&JF.

. "vivo.'

(fW •

'fvV' :

^»

\

Figure 7. Light photomicrographs showing the spatial distribution ot imoplusm in control and com-
pressed eight-celled embryos and in silu hybridizations of sectioned control and compressed cleavage-
arrested eight-celled embryos using SpMA3C anti-sense RNA. (A) A detergent-extracted control embryo
showing myoplasm contained in the B4.I blastomeres (long arrows). (B) Bright-field image of a cleavage-
arrested embryo showing the hybridization of SpMA3C probe to the cytoplasm of B4.1 blastomeres (long
arrows). (C) A detergent-extracted, compressed embryo showing myoplasm in the B4. 1 blastomeres (long
arrows) and in two additional blastomeres (short arrows). (D) Bright-field image of a compressed and
cleavage-arrested eight-celled embryo showing the hybridization of SpM A3C probe to the cytoplasm in the
B4.1 blastomeres (long arrows) and in two additional blastomeres (short arrows). Scale bar in (A) equals
50 ^m. Same magnification for (B-D) as (A).

were rinsed with PBS and then fixed in 2% glutaralde-
hyde in 0.1 M sodium phosphate buffer, pH 7.4, for
30 min at room temperature. Samples were washed three
times in 0. 1 M sodium phosphate buffer. Fixed speci-
mens were immersed in 1% osmium in the same buffer
•or 1 h at room temperature, followed by dehydration
rough a graded us of ethanol (10%, 30%, 50%, 70%,
100%) for 10 min at each step,
nmens we ansferred into a specimen holder
is inserted e a Tousimis Autosamdri-814

-loint drying hamber. Dried specimens were
o double- d tape on an aluminum stub
' inside a Sputtering System, Hummer VII
coati hine. A 20-nm gold/palladium metal alloy

coating ipplied to the surface of each specimen.

and the specimens were viewed using a JSM-6400
scanning electron microscope.

In situ hybridization

The in situ hybridization method previously described
by Tomlinson et al. (1987) was used in the present study.
Normal and compressed eight-celled embryos, cultured
in seawater containing cytochalasin B until the controls
developed into larvae, were fixed for 20 min in 3: 1 etha-
nol-glacial acetic acid at -20°C. After the fixed embryos
were dehydrated in a graded series of ethanol, they were
gradually infiltrated with Paraplast and embedded in
BEEM capsules. Specimens were sectioned at 7 ^m and
dried on gelatin-coated slides. SpMA3C DNA cloned

p58 AND ASCIDIAN MUSCLE CELL DETERMINANTS

223

Figure 8. Scanning electron micrographs of detergent-extracted unfertilized and fertilized eggs. (A) An
unfertilized egg showing the myoplasmic cytoskeletal domain (MCD) in the peripheral egg cytoplasm. (B)
A fertilized egg at the first stage of ooplasmic segregation showing the segregation of the MCD in the
cytoplasm of the vegetal hemisphere. AP — animal pole; VP — vegetal pole. Same magnification for (A) and
(B); Scale bar in (A) equals 10 ^m.

into Bluescribe vector was linearized using EcoRl or
Hind III to serve as templates for anti-sense and sense
RNA probes respectively. Linearization was checked by
running some of the cut and uncut DNA on 1% agarose
gels followed by ethidium bromide staining. SpMA3C
DNA cut with EcoRI was transcribed in the presence of
50 MCi of 3H-UTP (Amersham) or 3?S-UTP (Amersham)
and cold ATP, CTP. and GTP (400 »M of each nucleo-
tide) using T3 polymerase. SpMA3C DNA was cut with
Hind III and transcribed with T7 polymerase to produce
sense probes.

The hybridization buffer contained 50% formamide.
10% w/v dextran, 0.01 M Tris, 0.3 M NaCl, 0.001 M
EDTA, 500 jug/ml tRNA, Denhardt's solution ( 1 : 10 di-
lution of 50x stock), and 500 ng/m\ polyadenylic acid.
Slides were probed at low stringency (washed in 1 X SSC
for 30 min at room temperature) or at high stringency
(washed in 1 X SSC for 30 min at 45°C), air dried, dipped
in Kodak NTB-2 nuclear track emulsion, and exposed
for up to 6 weeks.

Immunocytochemistry

Specimens were fixed and embedded in polyester
(Steedman's) wax, as previously described by Mita-
Miyazawa^a/. (1987). Specimens were fixed for 20 min
in absolute methanol and then immersed in cold abso-
lute ethanol for 20 min. Specimens were infiltrated in
50% polyester wax in absolute ethanol for 1 h at 42°C,
then infiltrated in 100% polyester wax for 1 h at 42°C.
Specimens were embedded in BEEM capsules, sectioned

at 7 //m, and mounted on gelatin-coated stripes of cover-
slips. Sections were de-waxed in 100% ethanol, rehy-
drated, and washed in PBS prior to treatment with the
primary antibody.

Myosin was detected using Mu-2 antibody (Mita-
Miyazawae/fl/., 1987) diluted 1:300 with PBS. Sections
were immersed in the primary antibody for 1 h at room
temperature, washed in PBS at room temperature, and
immersed in FITC-conjugated anti-mouse IgG (Sigma
Chemical Company, St. Louis. MO) diluted 1:60 with
PBS. After a 30-min incubation at room temperature,
sections were washed in PBS for 30 min, mounted in
80% glycerol in PBS, and viewed with an Olympus flu-
orescence microscope. Stained sections were photo-
graphed using Tri X film, ASA 400.

Monoclonal anti-neurofilament 160 antibody (clone
NN18; Sigma Chemical Company, St. Louis, MO) was
the primary antibody used to detect p58 in embryos
(Swalla el al., 1991). Sections were immersed in NN18
diluted 1:25 with PBS for 1 h at room temperature. Sec-
tions were washed with PBS and treated for 1 h at room
temperature with anti-mouse IgG-POD (Sigma Chemi-
cal Company, St. Louis, MO) diluted 1 :60 with PBS. Per-
oxidase activity was detected using Sigma FAST DAB
peroxidase substrate tablets. Sections were incubated in
the substrate solution for 10 min, washed in PBS, and
viewed with a Zeiss Axioplan microscope.

Results

The myoplasm of an ascidian egg is normally parti-
tioned into the embryonic progenitor cell that produce

224

W. R. BATES

Figure 9. Higher magnification scanning electron micrographs of
myoplasmic and non-myoplasmic egg regions. (A) The plasma mem-
brane lamina (PML) of the myoplasmic cytoskeletal domain (MCD) is
evident beneath a small patch of plasma membrane that was not dis-
solved by Triton X- 100 treatment. Numerous pigment granules are ev-
ident. Scale bar equals 1 Mm. (B) A distinct boundary is evident between
the MCD and non-MCD regions of a fertilized egg. Scale bar equals
10 ^m.

muscle cells situated in the larval tail region (Fig. 3A).
When the animal hemisphere region was surgically de-
leted from fertilized eggs at the first stage of ooplasmic
segregation, the nucleated vegetal merogons that con-
tained segregated myoplasm developed into myoplasm-
enriched (ME) larvae (Fig. 3B). In striking contrast to
normal larvae, in which the pigmented myoplasm was
present only in the tail muscle cells, in ME larvae myo-
'asrnic pigment granules were present in tail muscle
md many of the endoderm cells situated in the lar-
>ad region (I -B). To determine more precisely
cell types o '. larvae contained pigmented my-
thick sections of ME larvae were examined, as
Fig. 4. Pigmented myoplasm was evident in the
of many endoderm cells situated in the head
•tioned ME larvae, whereas myoplasmic pig-
i: ?s were not evident in the cytoplasm of epi-

dt Hochord cells. Thick sections of ME larvae

also revealed that their notochords were displaced into
more anterior positions, as compared to normal larvae.

Can endoderm cells of ME embryos that contain pig-
mented myoplasm develop muscle cell features? This
important question was addressed by using transmission
electron microscopy to examine the cytoplasm of head-
region endoderm cells of ME larvae for myofilaments.
Twelve ME larvae were sectioned and thick sections
were produced to identify head and tail regions prior to
cutting thin sections. The ultrastructural features of ME
tail muscle cells and head endoderm cells are shown in
Figure 5. Sacromeres were evident in the peripheral cy-
toplasm of tail muscle cells (Fig. 5 A, B); however, there
was no evidence for the development of myofilaments in
the head region of ME larvae in the more than 300 sec-
tions that were examined (Fig. 5C, D).

Next, the expressions of two muscle-specific markers,
myosin and muscle actin RNA, were examined in cleav-
age-arrested control (non-compressed) and compressed
eight-celled embryos. Figure 6 shows the expression pat-
terns of myosin heavy chain protein in a control larva
(Fig. 6A), in a cleavage-arrested eight-celled embryo (Fig.
6B), and in a compressed and cleavage-arrested eight-
celled embryo (Fig. 6C). Myosin development was re-
stricted to the tail muscle cytoplasm in normal larvae (in
all 40 larvae tested). In cleavage-arrested eight-celled em-
bryos, a maximum of two blastomeres expressed myosin
(40 larvae tested). In contrast, compressed and cleavage-
arrested eight-celled embryos developed myosin in a
maximum of four blastomeres (40 embryos tested).

In another set of experiments, the development of
muscle-specific actin RNA was studied in control and
compressed cleavage-arrested eight-celled embryos (Fig.
7). A maximum of two blastomeres in control embryos
showed hybridization signals (50 embryos examined:
Fig. 7B). whereas a maximum of four blastomeres exhib-
ited hybridization signals in the compressed embryos (40
embryos examined; Fig. 7D). The redistribution of pig-
mented myoplasm into more than the normal number
of blastomeres by compression (compare A and C in Fig.
7) promoted the ectopic development of these muscle
cell markers.

Why were muscle cell fates altered in compressed Bol-
tenici embryos, whereas cell fates were normal in ME
Bo/lenia embryos? This question was examined by
studying the distribution of MCD components in nor-
mal embryos compared to ME embryos and compressed
embryos.

Spalial distributions o/'t/ie myoplasmic cytoskeletal
domain and p58

The structure of the pigmented MCD of an unfertil-
ized Boltenia egg treated with Triton X-100. as revealed

p58 AND ASCIDIAN MUSCLE CELL DETERMINANTS

225

Figure 10. Scanning electron micrographs (SEM) and transmission electron micrographs (TEM) of
myoplasm-deficient and myoplasm-enriched egg fragments. (A) SEM of a detergent-extracted myoplasm-
dencient egg fragment. The pigmented myoplasmic cytoskeletal domain (MCD) is absent. Scale bar equals
10 Mm. (B) SEM of a detergent-extracted myoplasm-ennched egg fragment. The pigmented MCD covers
about 60%-70% of the surface. Scale bar equals 10 /jm. (C) TEM of cytoplasm of a myoplasm-deficient egg
fragment. Yolk granules and a few scattered mitochondria are evident. Magnification equals 6,300x. (D)
TEM of cytoplasm of a myoplasm-enriched egg fragment. Pigment granules surrounded by many mito-
chondria. Magnification equals 6.300X.

by scanning electron microscopy, is shown in Figure 8A.
After fertilization, the MCD segregated into the vegetal
hemisphere and covered about 40%-50% of the total egg
perimeter (Fig. 8B). The animal hemisphere region
lacked the MCD and contained only a few scattered pig-
ment granules. Higher magnification SEM images of the
animal and vegetal hemisphere regions of a fertilized egg
are shown in Figure 9. A small patch of plasma mem-
brane that was not dissolved by Triton X-100 is evident
in Fig. 9A. The distinct edge of the MCD in a fertilized
egg is shown in Fig. 9B.

Animal fragments produced from fertilized eggs dur-
ing the first phase of ooplasmic segregation lacked the

MCD (Fig. 10A) and contained fewer mitochondria than
found in the ME fragments (compare C and D in Fig.
10). The MCD surrounded about 60%-70% of the pe-
rimeter of ME fragments (Fig. 10B), and the MCD con-
tained more mitochondria than did the egg fragments
produced from the animal hemisphere (Fig. 10D). Re-
moval of the animal hemisphere resulted in a 10%-20%
increase in the surface area of an ME fragment composed
of MCD (compare Figs. 8B and 10B). This increase in
the surface area that contained the pigment- J MCD re-
sulted in the development of orange-h< ided larvae
(Fig. 3B).

Seasonal variations in the distribution /f myoplasmic

226

W. R. BATES

Figure 1 1 . Scanning electron micrographs ol a four-celled embryo
showing the pigmented mvoplasmic cytoskeletal domain (MCD) pres-
ent in all four blastomeres. (A) The MCD is present in the cytoplasm of
posterior B3 blastomeres and in the cytoplasm of anterior A3 blasto-
meres. Scale bar equals 10 ^m. (B) Higher magnification showing the
structure of the MCD in B3 cytoplasm. The MCD consists of a network
of filaments with underlying pigment granules. Scale bar equals 10 ^m.
(C) Higher magnification of the MCD in the posterior region of an A3
blastomere. Actin filaments (AF) composing the plasma membrane
component of the MCD cover the underlying pigment granules (PC)
that are embedded in the deep filamentous lattice ( PG). Scale bar equals
1 ^m.

Table I

Distribution oj'ihe myoplasmic cytoskeletal domain (MCD) in the
blastomeres of Jour-celled embryos produced in the autumn thai
showed pigment granules in all blastomeres

Number of blastomeres
containing MCD

Number of
embrvos

0
0
0

1
29

pigment granules in Bollenia villosa embryos were ob-
served. In late autumn, about one-quarter of the B. vil-
losa adults produced clutches of eggs in which orange
pigment granules were present in the cortical cytoplasm
of all four blastomeres in four-celled embryos, as com-
pared to clutches of eggs produced at other times of the
year in which pigment granules were restricted to the cor-
tical cytoplasm of B3 blastomeres of four-celled em-
bryos. Most of the embryos that exhibited altered pig-
mentation patterns developed into normal larvae. The
spatial distributions of the MCD were mapped in four-
celled embryos; SEM was used to determine if the MCD
was restricted to the B3 blastomeres or was also found in
A3 cytoplasm. These results are shown in Figure 1 1 and
Table I. Whereas most of the pigmented MCD was con-

Figure 12. Light photomicrograph of a four-celled embryo stained
with NN18 antibody in which the myoplasmic cytoskeletal domain is
present in all four blastomeres. Two blastomeres are stained with NN 1 8
antibody. Scale bar equals 100/jm.

p58 AND ASC1DIAN MUSCLE CELL DETERMINANTS

227

Table II

Distribution of p58 in unoperated and myoplasm-enriched (ME)
four-celled embryos

Maximum number of blastomeres
stained with NN18 antibody

Specimens

0

1

3

'Autumn' embryos

ME embryos composed of

40%-50% of total egg

volume
ME embryos composed of

20% -30% of total egg

volume

25

15

10

* Three blastomere per embryo were in the plane of section.

tained in the B3 blastomeres, A3 blastomeres contained
significant quantities of the pigmented MCD.

The spatial distributions of p58. a protein associated
with the DFL component of the MCD, were examined
using NN18 antibody in four-celled embryos that con-
tained pigmented MCD in all blastomeres. A maximum
of two blastomeres per embryo were stained with NN 1 8
antibody (Fig. 12 and Table II). Therefore, although A3
blastomeres contained the anteriormost region of the
MCD, this region of the MCD lacked p58.

In another set of experiments, four-celled ME em-
bryos composed of 40%-50% and 20%-30% of the total
egg volume were stained with NN18 antibody. In em-
bryos made up of 40%-50% of the total egg volume, a
maximum of two blastomeres were stained with NN 1 8
as in control four-celled embryos (Fig. 13A and Table
II). In contrast, when four-celled embryos derived from
smaller ME fragments produced from the vegetal pole
region were stained with NN18, in most cases (95%; /; =

19) more than two blastomeres reacted with the antibody
(Fig. 13B and Table II). When compressed eight-celled
embryos were stained with NN18, a maximum of four
blastomeres reacted with this antibody (Fig. 1 3C and Ta-
ble III). In control eight-celled embryos, a maximum of
two blastomeres were stained with NN 1 8.

Discussion

The results of the present study demonstrate that ( 1 )
compression of four-celled Boltcnia villosa embryos in-
creased the number of blastomeres of eight-celled em-
bryos that could develop muscle cell features; (2) the de-
velopment of muscle cell features in compressed em-
bryos was correlated with the presence of the cytoskeletal
protein p58; (3) p58 was restricted to the cytoplasm of
posterior B3 blastomeres of four-celled ME embryos
composed of 40%-50% of the total egg volume; (4) p58
was present in the cytoplasm of anterior A3 and posterior
B3 blastomeres of four-celled embryos that were derived
from small ME fragments produced from the vegetal
pole region of fertilized eggs at the first stage of ooplasmic
segregation; and (5) p58 was concentrated in the vegetal
pole region of the MCD at the first stage of ooplasmic
segregation.

The present results provide rigorous experimental sup-
port for the idea, first suggested by Swalla el al. (1991),
that the cytoskeletal protein p58 is associated with mus-
cle cell determinants in ascidian eggs. Another cytoskel-
etal protein. myoplasmin-C 1 , is localized in ascidian my-
oplasm (Nishikata ct al.. 1987), and it has been suggested
that myoplasmin-C 1 may interact with p58 through a-
helical rod regions formed by hydrophobic heptad re-
peats present in both of these proteins (Nishikata and
Wada, 1996; B. J. Swalla, pers. comm.). Therefore, «-
helix coiled-coil complexes composed of p58 and my-

Figure 13. Light photomicrographs of NN18 staining patterns of myoplasm-enriched (ME) and com-
pressed embryos. (A) A section of a four-celled ME embryo composed of 40^ of the total egg volume that
shows two blastomeres stained with NN18. (B) A section of a four-celled ME embryo composed of 30% of
the total egg volume in which four blastomeres reacted with NN 1 8. (C) A section of a compressed embryo
showing four blastomeres stained with NN 1 8. Scale bars equal 50 ^m.

228

W. R. BATES

Table HI
Distribution ofp58 in i compressed eight-celled embryos

Maximum number of blastomeres per
embryo stained with NN 1 8 antibody

Specimens

0 1

5678

Normal embryos 22240 0 0000

Compressed embryos 31 I 0 22* 0 0 0 0

* In five embryos, five or six blastomeres were in the plane of section.

oplasmin-Cl may be the cytoskeletal scaffold that an-
chors muscle cell determinants.

The expression of a muscle actin promoter-/ac'Z re-
porter gene construct, MocuM A 1 /lacZ, was recently ex-
amined in the tailless larvae of Molgula occult a. In these
larvae, which lack p58 and do not develop functional
muscle cells, low levels of /i-galactosidase activity were
detected in a few posterior cells (Kusakabe el ai. 1996).
This result and the presence of insertions, deletions, and
codon substitutions in the coding regions of orthologous
larval muscle actin genes isolated from the urodele spe-
cies Molgula oculata suggest that mutations in muscle
genes rather than changes in trans-acting regulatory fac-
tors are responsible for the regression of muscle cells.
These investigators also reported that MocuMAl//acZ
constructs are expressed in mesenchyme and other non-
muscle cell types in urodele larvae and that the M. oc-
culta cells expressing /3-galactosidase activity did not cor-
respond to the cells expressing low levels of vestigial
AchE activity. Therefore, these results suggest that the
activity of the MocuM A 1 promoter is somewhat "leaky"
in some cell types, as is the transcription of AchE genes
in vestigial muscle cells of several anural ascidian species
(Whittaker, 1979; Jeffery and Swalla, 1990b; and Bates
and Mallett, 1991). The results obtained by Jeffery and
Meier (1983), Swalla el ai (1991), Nishikata and Wada
(1996) and those reported in the present study suggest
that determinants associated with the myoplasmic cy-
toskeleton are required for the normal promoter activi-
ties of muscle genes.

Mapping the distributions of p58 in ME and com-
pressed embryos has resolved the paradox of why cell
fates were normal in embryos derived from ME frag-
ments composed of 40%-50% of the total egg volume
(Bates, 1988), but muscle cell fates were altered in com-
pressed embryos (Whittaker, 1980; and the present
study) and small ME embryos produced from the vegetal
pole region of fertilized eggs (Bates, 1988). Removal of
the animal hemisphere of a fertilized egg during the first
stage of ooplasmic segregation resulted in a 10%-20% in-
crease in the cortical cytoplasm of an ME fragment that

contained the MCD. Increasing the surface area of MCD
would produce larger than normal myoplasmic crescents
after the second phase of ooplasmic segregation. These
enlarged crescents would position the anteriormost
MCD into the endodermal determinant domain of the
egg(Conklin, 1905; Nishida, 1994). The normal cleavage
pattern of an ME embryo (Bates, 1988) would then par-
tition the anterior MCD into endoderm cells, resulting
in the development of larvae in which some of the head
endoderm cells contain pigmented cytoplasm.

When the regional expressions of three muscle-specific
markers, myosin heavy chain (Bates, 1988), AchE activ-
ity (Bates, 1988), and myofilaments (present study) were
examined in ME larvae, only tail muscle cells were able
to develop these markers. The endoderm cells present in

or

Figure 14. A summary of the present results. (A; top) The myoplas-
mic cytoskeletal domain (MCD) resides in the more posterior cyto-
plasm of a fertilized egg at the second stage of ooplasmic segregation
(anterior — top; MCD is outlined). The MCD contains pigment gran-
ules (dots), proteins, RNA. and mitochondria. The posterior region of
the MCD contains p58 (short lines). (A: bottom) In late autumn, some
adults produce eggs in which some of the MCD was extended into the
anterior cytoplasm, yet p58 was restricted to the more posterior MCD.
(B) In myoplasm-enriched (ME) fragments composed of 40%-50% of
the total egg volume, there was an increase in cortical cytoplasm that
contained the MCD. As in autumn eggs that exhibited altered pigmen-
tation patterns (Fig. I4A; bottom), some of the pigmented MCD was
extended into the more anterior cytoplasm. This pigmented MCD was
partitioned into some of the head endoderm cells of the larva; however,
endoderm cell fates remained normal because p58 was localized in the
more posterior region of the MCD. (C) In smaller ME fragments pro-
duced from fertilized eggs, MCD that contained p58 was present in the
anterior cytoplasm as well as in the posterior cytoplasm. After two cell
divisions, the resulting four blastomeres contained p58, and all four
blastomeres of cleavage-arrested smaller ME embryos expressed AchE
activity (Bates, 1988). (D) Compression of four-celled embryos resulted
in the partitioning of MCD that contained p58 into four blastomeres of
eight-celled embryos, in contrast to normal eight-celled embryos that
have p58 in the two B4. 1 blastomeres. The presence of p58 in four
blastomeres of a compressed and cleavage-arrested eight-celled embryo
promoted the development of myosin and muscle actin RNA in four
blastomeres.

p58 AND ASCID1AN MUSCLE CELL DETERMINANTS

229

Table IV

Summary of results

Type of embryo'

Maximum number of
cells/embryo expressing a Distribution
muscle cell marker ofp58

Cleavage-arrested four-
celled control
embryos

Cleavage-arrested four-
celled ME embryos
40%-50%TEV

Cleavage-arrested four-
celled ME embryos
10%-3(KTEV~

Cleavage-arrested eight-
celled embryos

Cleavage-arrested and
compressed eight-
celled embrvos

two cells expressed AchE two cells

activity :

two cells expressed AchE two cells

activity2

four cells expressed AchE four cells

activity2

two cells expressed myosin two cells

and muscle actin RNA
four cells expressed myosin four cells

and muscle actin RNA

1 ME = myoplasm-enriched; TEV = total embryo volume.
: From Bates (1988).

the head region of ME larvae developed an endoderm-
specific marker, alkaline phosphatase activity, as did the
control larvae (Bates, 1988). The detection of p58 in the
cytoplasm of B3 blastomeres of four-celled ME embryos
but not in A3 blastomeres explains why muscle cell fates
in these embryos were normal.

Two observations described in the present study indi-
cate the probable distribution of p58 in the MCD in fer-
tilized eggs just prior to first cleavage. During most of
the year, Boltenia adults produce clutches of eggs that
develop into four-celled embryos in which the MCD re-
sides exclusively in the cytoplasm of the B3 blastomeres
(Jeffery and Meier, 1983). However, in late autumn
some animals produce four-celled embryos in which the
MCD is present in A3 blastomeres and in B3 blasto-
meres. When these embryos were stained with NN18,
only two blastomeres contained p58. These results sug-
gest that p58 may be more concentrated in the posterior
region of the MCD prior to first cleavage than in the
more anterior region of the MCD. This idea is further
supported by the detection of p58 in the cytoplasm of all
four blastomeres of four-celled ME embryos produced
from the vegetal pole region of fertilized eggs (see
Fig. 14).

The present results indicate that the distribution of the
cytoskeletal protein p58 is correlated with the ectopic de-
velopment of muscle cell features in compressed ascid-
ian embryos. Whittaker ( 1980) observed that the maxi-
mum number of blastomeres that can develop AchE ac-
tivity is four in compressed and cleavage-arrested eight-
celled Styela embryos, but only two in non-compressed

and cleavage-arrested embryos. The present results have
confirmed and extended Whittaker's findings by showing
that compression can alter muscle cell fates in Boltenia
villosa embryos and that p58 is present in four, instead of
two, blastomeres of compressed eight-celled embryos.

The results of the present study are summarized in Fig-
ure 14 and in Table IV. During the first phase of ooplas-
mic segregation following fertilization, the MCD is
moved towards the vegetal pole. The MCD is then sec-
ondarily shitted into the equatorial-vegetal region of the
egg prior to first cleavage (Fig. 14 A, top), and this region
designates the future posterior region of the larva (Con-
klin, 1905). Embedded within the MCD are pigment
granules, mitochondria, RNA, and proteins. The cy-
toskeletal protein p58 is concentrated in the deep fila-
mentous lattice of the MCD (Swalla el a!.. 1991). In
some clutches of eggs produced in late autumn, the sur-
face area of an egg containing pigmented MCD was
greater than in eggs produced at other times of the year
(Fig. 14A, bottom). This increase resulted in the devel-
opment of four-celled embryos in which the MCD was
present in the cytoplasm of all four blastomeres. How-
ever, staining with NN18 showed that only the primary
muscle progenitor B3 cells contained p58. These obser-
vations suggest that p58 is associated with the posterior
MCD contained within the cytoplasm of B3 blastomeres,
but absent from the anterior region of the MCD con-
tained in A3 blastomeres.

In ME fragments composed of 40%-50% of the total
egg volume, there was an increase in the surface area that
contained the pigmented MCD (Fig. 14B). This increase
resulted in the redistribution of some of the pigmented
MCD into the anterior blastomeres. When four-celled
ME embryos derived from this size class of egg fragments
were immersed in NN 1 8, a maximum of two blastomeres
were stained. These results demonstrate that p58 distribu-
tions were not altered in the ME embryos and explains
why the fates of embryonic ME cells were normal. In con-
trast, nearly the entire cortical cytoplasm of smaller ME
fragments produced from the vegetal pole region of fertil-
ized eggs contained the MCD (Fig. 14C). When four-
celled ME embryos derived from these fragments were
stained with NN18, p58 was detected in all four blasto-
meres. This observation explains why AchE activity was
sometimes detected in all four blastomeres of small cleav-
age-arrested ME embryos, as compared to a maximum
of two blastomeres in the control cleavage-arrested four-
celled embryos (Bates, 1988). Compression of four-celled
embryos caused p58 to be partitioned into four, rather
than two, blastomeres of an eight-celled embryo (compare
Figs. 2B and 14D). The presence of p58 in four blasto-
meres was correlated with the development of myosin and
muscle actin RNA in four blastomeres of o inpressed and
cleavage-arrested eight-celled embryos.

230

W. R. BATES

In conclusion, mapping the distribution of p58 has re-
solved the paradox of why cell fates were normal in ME
embryos composed of 40%-50% of the total egg volume,
but muscle cell fates were altered in small ME embryos
and in compressed embryos. Immunoprecipitation ex-
periments, now in progress, will coprecipitate muscle cell
determinants with p58 for microinjection into non-mus-
cle lineage blastomeres of urodele ascidian embryos and
injection into anural ascidian eggs.

Acknowledgments

Monica Lovis and Mike Swallow are thanked for their
technical assistance. SpMA3C DNA was kindly pro-
vided by Dr. William Jeffery, and Mu-2 antibody was
kindly provided by Dr. N. Satoh. The comments pro-
vided by the reviewers were most helpful in preparing
this manuscript. The author is most thankful for the
award of an operating grant by the Natural Sciences and
Engineering Research Council (NSERC) of Canada.
This paper is dedicated to the memory of Dr. Rick Bro-
deur.

Literature Cited

Bates, \V. R. 1995. Direct development in the ascidian Molgula re-

wrliformis (\eu\\\. 1871). fi;u/. Bull 188: 16-22.
Bales, \V. R. 1988. Development of myoplasm-enriched ascidian

embryos. Dev Biol 129: 241-252.

Bates, \V. R., and \V. R. Jeffery. 1987. Localization of axial determi-
nants in the vegetal pole region of ascidian eggs. Dev. Biol 124: 65-

76.
Bates, W. R., and J. E. Mallett. 1991. Ultrastructural and histo-

chemical study of anural development in the ascidian Molgula pa-

c-//(c-« (Huntsman). Roux'sArch. Dcv Bin/ 200: 143-201.
Conklin, E. G. 1905. The organization and cell lineage of the ascidian

egg. J. Acail. Nail, Sci. (Philadelphia) 13: 1-114.
Elinson, R. P. 1990. Cytoskeleton and embryo polarity. Curr. Opin.

O//fl/<>/. 2:43-48.
Hill, D. P., S. Strome, and G. P. Radice. 1990. The cytoskeleton in

development. Pp. 177-200 In Cytop/asmie Organisation System.

G. M. Malacinski. ed. McGraw-Hill. New York.
Jeffery, VV. R. 1982. Messenger RNA in the cytoskeletal framework:

analysis by in situ hybridization. J Cell Bml. 95: 1 -7.
Jeffery, \V. R. 1989. Localized mRNA and the egg cytoskeleton. Int.

Rev Cylol. 119: 151-195.
Jeffen, \V. R., and S. Meier. 1983. A yellow crescent cytoskeletal

domain in ascidian eggs and its role in early development. Dev. Biol.
96: 125-143.

Jeffery, VV. R., and B. J. Swalla. 1990a. The myoplasm of ascidian
eggs: a localized cytoskeletal domain with multiple roles in embry-
onic development. Semin. Cell Biol. 1: 373-38 1 .

Jeffery, VV. R., and B. J. Swalla. 1990b. Anural development in as-
cidians: evolutionary modification and elimination of the tadpole
larva. Scmin. Dev Biol. 1: 253-261.

Kusakabe, I., B.J. Swalla, N. Satoh, and \V. R. Jeffery. 1996. Mecha-
nism of an evolutionary change in muscle cell differentiation in as-
cidians with different modes of development. Dev Biol. 174: 379-
342.

Marikawa, Y. 1995. Distribution of myoplasmic cytoskeletal do-
mains among egg fragments of the ascidian Ciona savignyi: the con-
centration of deep filamentous lattice in the fragment enriched with
muscle determinants. J. Exp. Zoo/. 271: 348-355.

Mita-Miyazawa, I., T. Nishikata, and N. Satoh. 1987. Cell- and tis-
sue-specific monoclonal antibodies in eggs and embryos of the as-
cidian Haloeynlhia rorelii. Development 99: 155-162.

Morgan, T. H. 1910. The effects of altering the position of the cleav-
age planes in eggs with precocious specification. \\'ilhelm Rmtx'
Arch Entwickliinxsmechanik Org. 29: 205-224.

Nishida, H. 1994. Localization of egg cytoplasm that promotes
differentiation to epidermis in embryos of the ascidian Halocynthia
roret-i. Development 120: 235-243.

Nishida, H., and N. Satoh. 1983. Cell lineage analysis in ascidian em-
bryos by intracellular injection of tracer enzyme. I. Up to the eight-
cell stage. Dev. Biol. 99: 382-344.

Nishikata, T., and M. \Vada. 1996. Molecular characterization of
myoplasmin-CI: a cytoskeletal component localized in the my-
oplasm of the ascidian egg. Dev Genes Evol. 206: 72-76.

Nishikata, T., I. Mita-Miyazawa, T.Deno, and N. Satoh. 1987. Mono-
clonal antibodies against components of the myoplasm of eggs of
the ascidian Ciona intesiinalis partially block the development of
muscle-specific acetylcholinesterase. Development 100: 577-586.

Swalla, B. J., M. R. Badgett, and \V. R. Jeffery. 1991. Identification
of a cytoskeletal protein localized in the myoplasm of ascidian eggs:
localization is modified during anural development. Development
111:425-436.

Tomlinson. C. R., R. L. Beach, and \V. R. Jeffery. 1987. Differential
expression of a muscle actin gene in muscle cell lineages of ascidian
embryos. Development 101: 751-765.

West, A. B., and C. C. Lambert. 1975. Control of spawning in the
tunicate Slvela pheaia by variations in a natural light regime. J
Exp. Zool. 195:265-270.

\\hittaker, J. R. 1979. Development of vestigial tail muscle acetyl-
cholinesterase in embryos of an anural ascidian species. Biol. Bull.
156:343-407.

VVhittaker, J. R. 1980. Acetylcholinesterase development in extra
cells caused by changing the distribution of myoplasm in ascidian
embryos. J Emhryol. Exp Murphol. 55: 343-354.

Reference: Biol. Bull 192: 231-242. (April, 1997)

Embryonic Coat of the Grass Shrimp
Palaemonetes pugio

PATRICIA S. GLAS1, LEE A. COURTNEY2*, JAMES R. RAYBURN1.
AND WILLIAM S. FISHER:

' National Research Council Associates and 2 U.S. Environmental Protection Agency, National Health

and Environmental Effects Research Laboratory, Gulf Ecology Division.

1 Sabine Island Dr., Gulf Breeze, Florida 32561

Abstract. The embryo of the grass shrimp, Palaemo-
netes pugio, is surrounded during development by a
protective extracellular coat designated as the embry-
onic coat (EC). At hatching, this EC is composed of four
embryonic envelopes (EE), each of which is composed
of multiple layers. The outermost layer of the EC, the
outer investment coat (QIC), is derived primarily, if not
completely, from pleopods of the female. The first en-
velope (EE1) forms as a bilayered envelope, EEla and
EElh, immediately after oviposition. The OIC becomes
closely associated with EE1 and remains in close con-
tact with EE1 until hatching occurs. An additional
layer, EE1C, is added to the inner side of EE1 between 3
and 5 d after oviposition. Three more embryonic enve-
lopes, EE2, EE3, and EE4. are formed between the
embryo and EE1 by 7 d after oviposition. Formation of
embryonic envelopes continues until 10 d after ovipo-
sition; by this time each envelope is morphologically
distinct in composition, with "outer" and "inner" sides
clearly identifiable. All but the innermost embryonic
envelope (EE4) are shed by the embryo about 6 h before
hatching. Permeability of the EC during the 12-d incu-
bation period is found to decrease between 0 and 5 d
after oviposition, and then increase until hatching. Flu-
orescently labeled lectins react positively with the OIC,

Received 1 9 August 1996; accepted 20 December 1996.

Abbreviations: embryonic coat (EC): embryonic envelope (EE):
outer investment coat (OIC): scanning electron microscopy (SEM);
transmission electron microscopy (TEM); fluorescein isothiocyanate
(FITC); Tris[hydroxymethyl]aminomethane (Tris).

Mention of commercial products does not constitute endorsement
by the U.S. Environmental Protection Agency.

* Author to whom correspondence should be addressed.

indicating the presence of glucose and jV-acetylglucos-
amine residues. Thus, the palaemonid EC is a dynamic
structure throughout embryonic development.

Introduction

Grass shrimp, Palaemonetes pugio, inhabit estuaries
in the coastal regions of the eastern United States. Dur-
ing mating, the male places a spermatophore on the tho-
rax of a mature female, near the opening of the gono-
pores. As eggs are extruded from the oviduct, they pass
across the spermatophore and are fertilized externally.
Eggs are deposited, or oviposited, on setae of the pleo-
pods of the female. After about 2 weeks of incubation,
the embryos hatch as zoeal larvae (Broad, 1957). During
this time, the embryonic coat (EC) must protect the em-
bryos from microbial, physical, and possibly chemical
conditions of the ambient water while allowing passage
of gases and other metabolites.

Various nomenclature has been used to describe the
embryonic coat in marine decapods. Lobsters have a fer-
tilization envelope or chorion (Talbot. 1981; Talbot and
Goudeau, 1988); crabs have an extra-cellular capsule or
fertilization envelope (Goudeau and LaChaise, 1980a, b;
Goudeau and Becker, 1982), penaeid shrimp have a
hatching envelope (Clark and Lynn, 1977; Pillai and
Clark, 1987), and Palaemon has an extracellular capsule
(Goudeau et a/.. 1991). Investigations have revealed at
least one additional "coat" besides the fertilization or
hatching envelope as part of the protective covering of
the developing embryo. Lynn et al. (1993) reported the
presence of three or more "envelopes" duri r g later stages
of development in the penaeid shrimp Sit mia ingentis.
Goudeau and LaChaise (1983) and < oudeau et al.

231

232

P. S. GLAS ET AL.

EE1 3

200.pm

Figures 1-9. Adult and embryonic stages of the grass shrimp, Palaemonetes pugio.

Figure 1. Photograph of P. pugio adults. Top. mature female: middle, ovigerous female; bottom, ma-
ture male.

Figure 2. Embryos 8 d after oviposition are shown attached to the ventral abdomen of a female. The
embryos were attached to setae on the female pleopod by a cement that also forms the outer investment
coat (OIC) of the embryonic coat.

Phase contrast micrograph of fertilized eggs on Day 0 within 4 h of ovipositioning. The
embno had progressed through karyokinetic divisions without cytokinesis.

Figure 4. Cytokinesis began after the embryos had completed three karyokinetic divisions.

The tissue cap stage was seen 3 d after oviposition. The tissue cap (clear area at the animal
pole) is the developing embryo. The attachment of the egg by the funiculus. Fu. may provide spatial ori-
entation for embryo development.

GRASS SHRIMP EMBRYONIC COAT

233

Table I

Development <>/ Palaemonetespugioa/27°Com/.?0%o salinity

Day of Diameter Standard
Embryonic stage development (mm) deviation

Oviposition/karyokinesis/

cytokinesis

0

0.59

0.026

Late cleavage/early

gastrulation

1

0.62

0.021

Gastrulation

2

0.67

0.028

Tissue cap

3

0.66

0.027

Cephalothorax delineation

4

0.66

0.021

Heartbeat initiation

5

0.69

0.020

Eye pigmentation

6

0.72

0.016

Embryonic eye

7

0.76

0.021

Early compound eye

8

0.80

0.019

Completed compound eye

9

0.84

0.048

Protozoeal embryo

10

0.84

0.030

Protozoeal embryo

11

0.82

0.047

Protozoeal embryo/pre-hatch

12

0.98

0.030

Hatch — 1 st zoeal larva

12

2.28

0.340

For each stage, the diameter is the mean value for 24 embryos mea-
sured across the longer axis of the oval-shaped egg. The hatch length is
the "unfolded" length of the zoeal stage larva. Days of development
represent 24-h periods at 27°C and 20%o salinity.

(1990) reported five "coats" in the brachyuran crab Car-
cinus maenas and six "coatings" in the lobster Homants
gammarus. Morphological studies of the EC in palae-
monid shrimp have been published (Sandifer and Lynn,
1980; Fisher and Clark, 1983; Lynn and Clark, 1983;
Goudeau el al, 1991), but none of these studies have
examined embryos beyond the first few hours after ovi-
position.

On the basis of observations of development in Palae-
monetes pugio, we have adapted terminologies from Tal-
bot and Goudeau (1988) for the lobster and from Lynn
et al. (1993) for penaeid shrimp. Crustacean decapods,
in general, have an embryonic coat (EC) surrounding the
developing embryo. This coat comprises one or more
embryonic envelopes (EE) plus, in animals that brood
their young, an outer investment coat (OIC) or cement
that attaches the embryos to the female. Each EE, in
turn, may be formed of several different layers. Thus,

EEs are defined as layers that generally appear together
as a single unit. The layers are morphologically distinct
regions of the envelope. The origin of the layers that form
the EE is usually embryonic, whereas the OIC origin may
be maternal, embryonic, or both. In this study, we show
that the EC is morphologically and functionally dynamic
throughout embryogenesis.

Materials and Methods

Grass shrimp, Palaemonetes pugio, were collected
from waters in east Escambia Bay, Pensacola, Florida,
during the summer and fall of 1995. Adults were main-
tained at 24°C temperature and 20%o salinity in flow-
through aquaria. Animals were held in these conditions
for 2 weeks before ovigerous females were removed. This
period ensured that eggs removed from the females were
oviposited under laboratory conditions. Embryos were
gently removed from females and their ages were deter-
mined by using developmental stages as indications of
approximate times postextrusion, i.e., after oviposition
(Tyler-Schroeder, 1978). A method modified from
Fisher and Foss (1993) was used to culture the embryos.
Each embryo was placed in a separate well of a 24-well
plastic tissue culture plate. The plates were incubated at
27°C and 20%o salinity with continual agitation at
60 rpm on a rotary shaker. Under these conditions,
hatching was consistently 1 1 - 1 3 d after oviposition, with
most embryos hatching after 12 d (Fisher and Foss,
1993).

Embryo development

Embryos at different developmental stages were re-
moved from culture plates, placed on glass slides in sea-
water in a ring of petroleum jelly, covered with glass cov-
erslips, and photographed using phase microscopy on an
inverted microscope. Embryo lengths were obtained us-
ing a Microcomp particle analysis system attached to a
video capture system mounted on a dissecting micro-
scope. Twenty-four embryos were measured daily
throughout the course of development. Mean embryo
lengths and standard deviations were calculated for each
day of development.

Figure 6. At 7 d after oviposition, eye development (arrow) was well advanced. The cephalothorax and
abdominal regions were well denned. Fu, funiculus.

Figure 7. By 10 d after oviposition, eye formation appeared complete. The telson (arrowhead) was
wrapped around the cephalothorax of the embryo. Lipid droplets (Dr) were seen in the region of the devel-
oping hepatopancreas. Fu, funiculus.

Figure 8. At 12 d after oviposition, about 6 h before hatch, EE1-EE3 were shed (EE1-3), remaining
attached only at the point of the funiculus. Only EE4 remained around the embryo. Arrowhead indicates
the telson still within EE4. Figures 3-8 are presented at the same magnification to demonstrate the increase
in embryo size during development. Bar equals lOO^m.

Figure 9. The first zoeal larvae hatched 1 2 d after oviposition. Bar equals 200 Mm.

234

P. S. GLAS ET AL.

12

Figures 10-19. Electron micrographs ofPalaemonetespugio embryos showing the development of the
embryonic coat.

Figure 10. A scanning electron micrograph of an embryo at 3 d after oviposition shows the smooth
outer surface of the outer investment coat (QIC) with the t'uniculus, Fu. The funiculus was the point of
attachment of the embryo to a seta of the female pleopod.

Figure 11. At 3 d after oviposition. the QIC and first embryonic envelope (EE1 ) were the only parts of
the EC seen with TEM. EE1 had two layers, a thin, electron-dense layer, "a." and a thicker, less electron-
dense layer, "b."

Figure 12. By 5 d after oviposition. EE1 had 3 layers, "a" and "b" as defined above, and layer "c," a
flocculent, loosely defined layer. A second envelope, EE2, had formed and, in some areas, elevated to just
below EE1.

Figure 13. In a section of another embryo 5 d after oviposition, EE2 (arrow) remained close to the
embryo. The third envelope. EE3. was seen forming internally to EE2 and had a thin, electron-dense outer
layer (d) and a looser, inner tibrillar layer (f).

Embryonic coat development

Morphological changes in the embryonic coat (EC)
were examined using scanning electron microscopy
(SEM) and transmission electron microscopy (TEM).
Embryos for SEM and TEM were fixed for 24 h in 1 .6%
formaldehyde and 0.8% glutaraldehyde in seawater. Al-
ternatively, embryos were fixed for 4 h in 3% glutaral-
dehyde, then 24 h in Bouin's fixative. Samples were
washed three times with 0.2 M phosphate buffer and
post-fixed in 1% osmium tetroxide in phosphate buffer.

then washed in distilled water. SEM samples were then
frozen in liquid nitrogen, loaded into the Zeiss DSM
962 scanning electron microscope, sublimated, then
sputter-coated with palladium-gold. For freeze-fracture
samples, the embryos were fractured before sublima-
tion. Samples for TEM analysis were dehydrated with
acetone, infiltrated with Spurr's resin, and then em-
bedded in fresh resin. Thin sections were cut, dou-
ble stained with lead citrate (Venable and Cogges-
hall, 1965) and aqueous uranyl acetate, and observed
with TEM.

GRASS SHRIMP EMBRYONIC COAT

235

Figure 14. Between 5 and 7 d after oviposition. multiple envelopes of the EC had formed and are visible
in thisSEM of a freeze-fractured 7-d embryo. Three of the envelopes. EE1. EE2. and EE3. were seen, with
EE3 having an elaborate system of ridges (arrowhead).

Figure 15. A cross-section of a ridge (arrow) in EE3 was seen in TEM from a 7-d embryo. The dense
layer (d land fibrillar layer (f) of EE3 were better organized than in earlier micrographs.

Figure 16. By 7 d after oviposition. a fourth envelope had formed interiorly to EE3. This envelope.
EE4, remained close to the embryo and developed a thin, electron-dense outer bilayer (d) and a fibnllar
inner layer (f).

Figure 17. By 10 d after oviposition. four EE surrounded the embryo, with EE1-EE3 found juxtaposed
and relatively distant from EE4 and the embryo. This micrograph of the EC at 10 d shows all the layers of
EE 1 (a. b. and c). as well as EE2 and the two layers of EE3.

Figure 18. At 10 d after oviposition, EE4 was still found close to the embryo. The dense outer layer (d)
had a bilaminar. railroad-track appearance, while the inner fibrillar layer (0 had become more organized.
EE4 formed large folds with a wavelike appearance.

Figure 19. Before hatching from EE4, the embryo formed the cuticle (Cu) of the exoskeleton. This
cuticle had a tnlaminar appearance in contrast to the bilaminar appearance of EE4. The characteristic
layering of the cuticle had already begun.

236 P. S. GLAS ET AL

Embryonic Coat of Palaemonetes pugio

ve?

QIC

EE1c

EE2

EE3

Figure 20. A schematic presentation of the sequence of formation of the embryonic coat of Pulacmn-
nclc.s pitg/o. including the embryonic envelopes (EE) and layers, ve, vitelline envelope; OIC. outer invest-
ment coat; EEla, EEIh, and EE1C, the three layers of the first embryonic envelope; EE2, EE3, EE4. the
second, third and fourth embryonic envelopes, respectively. The layers and distance from the embryo are
not to scale.

Embryonic coat permeability

Permeability assays were performed as reported by
Glas et al. (1995) and Legge (1995). Embryos at various
stages of development were incubated with fluorescein
isothiocyanate (FITC)-labeled dextrans of five molecular
weights | Sigma Chemical Co.) by adding 10 ^1 of fluo-
rescently ;x-!ed dextran (stock = 50 mg/ml in distilled
water) in idual wells containing embryos and sea-

water at 2 nity. After 30 min incubation in the

dark at 27" os were rinsed three times with

filtered seawa; xamined for dextran penetration

1e the EC filters on an inverted micro-

At least i as were observed for each

of dextran. ition of the EC by the dextran

rmined b\ ig through the embryo. For il-

embryos w examined and photographed
>cal microscopy. The embryos were scanned
usini; -qe settings for laser intensity (488 nm blue

lasei ' gain.

Lectin-binding affinity

Embryos with intact ECs at 8 d and 1 2 d after oviposi-
tion were incubated with FITC-labeled lectins. The lee-
tins (and their affinities for oligosaccharides) used in this
study were Concanavalin A agglutinin (Con A), a-D-glu-
cosyl residues, especially glucose and 7V-acetylglucos-
amine; Limuliis polyphemus bacterial agglutinin (LPA),
jV-acetylated D-hexosamines, especially D-glucuronic
acid and /V-acetylneuraminic acid; and Triticwn viilgaris
agglutinin (wheat germ agglutinin, WGA), Ar-acetyl-/3-D-
glucosaminyl residues, especially yV-acetyl-/3-D-glucos-
amine oligomers. Lectin stock solutions, I mg/ml for
LPA and WGA and 2 mg/ml for Con A, in 0.05 A/ Tris
(pH 7.2), with 0.0 1 A/ calcium, magnesium, and manga-
nese chlorides as trace metals, were made according to
the procedures of Kiernan (1 990); 10 ^1 of stock solution
was added to individual wells containing embryos in
1 .0 ml seawater at 20%o salinity. The embryos were incu-
bated in the dark for I h at 27°C, then rinsed three times

GRASS SHRIMP EMBRYONIC COAT

237

Figures 21-24. Permeability of the embryonic coat (EC) of Palaemonetes pi/f;i<> was determined using
FITC-labeled dextrans. Embryos were viewed using confocal microscopy.

Figure 21. At 3 d after oviposition, the embryo EC was permeable to FITC-labeled dextrans weighing
9 kDa. The dextran inside the EC showed the entire embryo as a bright image.

Figure 22. An example of an embryo 3 d after oviposition incubated with dextrans weighing 38 kDa.
The dextran does not remain in the inside of the EC so the outline of the embryo is visible.

Figure 23. Embryo EC was permeable to the FITC-labeled dextran weighing 72 kDa at 10 d after ovi-
position, so the embryo appears as a bright oval.

Figure 24. Embryo EC was impermeable to the dextran weighing 148 kDa at 10 d after oviposition.

with seawater. The embryos were then observed for flu-
orescence on the outside of the QIC. To block for non-
specific binding, embryos were pretreated for 30 min
with 1 mg/ml bovine serum albumin in the Tris buffer,
then rinsed with seawater. As sugar controls, lectins were
incubated with 1 M solutions (0.3 M for 7V-acetylneur-
aminic acid) of specific oligosaccharides before being

Table II

Permeability of the embryonic coal <>/ Palaemonetes pugio
to FITC-lahcled dextrans

Day of
development

FITC-labeled dextran molecular weight (kDa)

4.3 9.3 19.6 38.9 71.2 148.3

3
4
5
7
10
12

Permeability is recorded as + (permeable) or - (nonpermeable) on
the basis of the presence or absence of fluorescent dye in the perivitel-
line space; ± indicates that fluorescence was found in the perivitelline
space of 50'"; of the eggs. Each symbol represents the observation of at
least four embryos.

added to the wells (Kiernan, 1990). Sugars used were su-
crose, glucose, and 7V-acetylglucosamine (for Con A); D-
glucuronic acid and jV-acetylneuraminic acid (for LPA);
and jV-acetylglucosamine and A'-acetylneuraminic acid
(forWGA).

Results

Embryo development

Development and hatching of P. pugio closely fol-
lowed the sequence of events described by Broad ( 1 957),
Davis (1965), and Thomas (1970). Ova of P. pugio were
released from the female oviduct and passed over the
male spermatophore, fertilized, and attached to the fe-
male pleopods during oviposition (Figs. 1 and 2). At the
time of attachment, the zygotes were single celled, carry-
ing both male and female pronuclei. After pronuclear fu-
sion, the zygote proceeded through three karyokinetic di-
visions (Fig. 3) before cytokinesis (Fig. 4). The orienta-
tion of the embryo within the EC remained the same,
with the funiculus. or attachment stalk, at the distal edge
of the presumptive cephalothorax.

By 3 d, or 72 h after oviposition, embryos reached the
tissue cap stage as described by Tyler-Shroeder (1978):
the clear region was at the animal end of the embryo, and
yolk filled much of the remainder of the i.-gg (Fig. 5). Af-

238

P. S. GLAS ET AL.

Embryos at 8 d and 12 d after oviposition were exposed to FITC-labeled lectins.
: luiirol embryos. Con, at 8 d after oviposition had some autotluorescence, especially in the
eyes, yolk, and olher pigmented areas. Arrowheads indicate the outline of the embryo.

Figure 2< After the outer EE I -EE3 were shed (arrow), the remaining inner coat (arrowheads) also did
not show an\ fluorescence.

GRASS SHRIMP EMBRYONIC COAT

239

ter 5 d of development, the heart had begun beating, and
segmentation was apparent as the embryos were elongat-
ing. Eye pigmentation was evident by 7 d after oviposi-
tion (Fig. 6), with development continuing until 9 d,
when the compound eye was fully formed. At this time,
the embryos appeared to be completely formed exter-
nally. At 10 d (Fig. 7), lipid "droplets" were visible in the
region destined to become the hepatopancreas, and the
amount of dark yolk had greatly diminished.

The size of P. piigio embryos cultured at 27°C and
20%o salinity increased throughout development (Table
I). After 12 d of development and about 6 h before hatch-
ing, the embryos shed the outer portion of the EC (Fig. 8)
and rapidly increased in size from 0.82 mm to 0.98 mm
within 24 h (Table 1). The embryos finally broke through
the remaining envelope to hatch as first zoeal larvae (Fig.
9). Development was consistent with that described by
Fisher and Foss (1993) so that hatching consistently oc-
curred after 1 1-13 d of incubation.

Embryonic coat formation

During oviposition. the female released a "cement"
that coated the newly fertilized eggs in a sticky layer for
attachment to setae on the pleopods (Figs. 2 and 10). By
4 h after oviposition, the cement had formed an outer
investment coat (QIC) that surrounded the embryo (Fig.
10) and also comprised the attachment strands, or funi-
culi, which connected the embryos to the pleopods. The
embryos were always attached in the same relative posi-
tion; i.e.. the funiculus was always at the position that
became the posterior end of the carapace on the dorsal
side (see Davis, 1965). The funiculus and attachment
points to other embryos were continuous with the QIC
surface. Externally, the QIC had no distinguishing fea-
tures other than the funiculus attachments (Fig. 10). The
smooth exterior surface of the embryos sometimes had
bacterial growth during later stages of embryonic devel-
opment, a common occurrence for externally brooded

decapod embryos (Fisher, 1983). Otherwise, no dramatic
external changes occurred until the outer envelopes were
shed. The stickiness of the cement was no longer notice-
able by 3 d after oviposition as the eggs no longer
clumped together on contact.

Four hours after oviposition, the embryo was sur-
rounded by the QIC and a bilayered embryonic enve-
lope, EE1. Transmission electron micrographs revealed
that the QIC comprised the outermost layer of the coat
surrounding the embryo and was about 0.4 /urn thick
(Fig. 1 1). The first EE initially consisted of a thin, outer
layer, EEla,andathick, inner layer, EElb. The thickness
of the EElh had increased to 0.38-0.4 /urn by 3 d after
oviposition (Fig. 1 1 ), after which no further increase was
seen. Within 3-5 d after oviposition, a third layer, EE1C,
was added to the previous two layers. This layer was
composed of an electron-dense, filamentous material
with a loosely organized structure (Fig. 12). This layer
did not always stain well, appearing as a sparsely filled
space between EE1 and EE2 after its formation. Usually
EE1 was closely associated with the QIC.

The second envelope, EE2, began to appear late in the
4th d after oviposition. At 5 d after oviposition, EE2 ele-
vated to remain closely associated with EE1 (Fig. 12). In
a section of another 5-d embryo, EE2, which had not yet
elevated could be seen as a dense envelope outside the
newly forming EE3 (Fig. 13). When stained for electron
microscopy, EE2 measured about 0. 12-0.2^111, with a
thin, dense outer layer and a less dense inner layer.

A third envelope, EE3, appeared by 5 d after oviposi-
tion. This envelope consisted of an electron-dense, thin
outer layer and a wide band of heterogeneous electron-
dense and lucent regions (Fig. 1 3). By 7 d, this envelope
had condensed and formed ridges in some parts of the
embryo (Fig. 14). The ridges did not circumscribe the
embryo. In section, a ridge was seen as an area where
EE3 had folded back on itself (Fig. 15). The condensed
EE3 had an outer electron-dense layer about 0.13 ^m
thick and a fibrillar, less dense inner layer about 0.26 ^m

Figure 27. The lectin Concanavalin A (Con Al had a high affinity for the outer investment coat (OIC)
of the 8-d embryo.

Figure 28. After the outer EEs were shed immediately prior to hatching, the Con A lectin had only a
slight affinity for the remaining envelope. EE4. The discarded EEI-EE3 and OIC (arrow) still reacted
strongly with the lectin.

Figure 29. The wheat germ agglutinin lectin. WGA. had a very strong affinity for the OIC of the 8-d
embryo.

Figure 30. At 1 2 d after oviposition, after the outer EE 1 -EE3 and OIC were shed, the WGA lectin had
no affinity for EE4. which remained around the embryo (arrowheads). The discarded EE1-EE3 and OIC
(arrow) still reacted to the lectin.

Figure 31. Limiilus polyphemus agglutinin showed only low nonspecific affinity for the OIC of the 8-d
embryo (arrowhead).

Figure 32. At 12 d after oviposition, after EE1-EE3 and the OIC were shed immediately before hatch-
ing, the LPA lectin had no affinity for EE4 (arrowheads), which remained around the embryo. Bar equals
100 Mm.

240

P. S. GLAS ET AL.

thick (Fig. 15). The fibrils tended to lay parallel to the
embryo surface. By 10 d after oviposition, this envelope
was about 0.5 ^m wide and, in regions where EE3 had
not formed ridges, was found subjacent to EE2 (Fig. 17).

The final envelope, EE4, was first seen at 7 d after ovi-
position. The morphology of EE4 was initially similar to
EE3 during formation in that the outer layer was elec-
tron-dense and the inner layer was fibrillar and irregular
(Fig. 16). The layers were near the embryo surface and
measured about 0.5 nm thick. By this time, EE3 had
moved away from the embryo toward the outer enve-
lopes that were layered beneath the QIC (Fig. 17). At 10 d
after oviposition, EE4 had large wave-shaped folds (Fig.
18) and measured about 1.2 ^m thick. The outer layer of
EE4 had formed a bilaminar "railroad-track" appear-
ance, and the inner fibrillar layer was oriented parallel
to the embryo surface. The developing embryo was seen
immediately below the waves of EE4 (Fig. 18). EE4 re-
mained near the embryo surface throughout the rest of
embryonic development.

Six hours before hatching, the QIC and the outer en-
velopes, EE 1 -EE3, were shed, remaining attached to the
embryo only at a point on the posterior dorsal edge of the
cephalothorax (Davis, 1965). The innermost envelope,
EE4, no longer had folds in it, but was found close to
the embryo (Fig. 19). The thickness of the envelope was
greatly reduced, as though stretched. The fibrils were
scattered throughout the space between the dense outer
layer and the embryo. At this time, the cuticular layer
of the embryonic exoskeleton had formed (Fig. 19). The
cuticular layer could be distinguished from EE4 by the
morphology of the dense outer layer. In EE4, this layer
was bilaminar, or "railroad track," in appearance,
whereas the dense layer of the forming cuticle had a tri-
laminar appearance.

The sequence of events for the formation of the extra-
embryonic coat is summarized in Figure 20. The relative
widths of the embryo, envelopes, layers, and perivitelline
space are not drawn to scale. The origin of EE1 from a
precursor layer or "vitelline envelope" is speculative at
this time.

FITC-labeled dextrans indicate permeability change
•vith time

'.'hen embryos were incubated with FITC-labeled

ans ." 1 after oviposition, only those dextrans

ng less than 9 kDa were able to penetrate the EC

' and 22). 7 Tmeability became more restrictive

-r oviposu i, when only the dextrans weighing

less . 9 kDa co sld penetrate the EC. This restriction

conti; >ntil 7 i after oviposition, when larger size

dextrin • able to penetrate the EC. As the embryos

aged 1'u.i: the permeability to dextrans increased.

with only the largest dextran ( 148 kDa) being excluded
from the EC by 10 d after oviposition (Figs. 23 and 24).
Immediately prior to hatching, 12 d after oviposition, all
dextrans tested penetrated the inner envelope, EE4 (see
Table II).

Lectin specificity indicates p-glucoside saccharides

The OICs of embryos were exposed to three lectins —
Concanavalin A (Con A), Limulus polyphemus aggluti-
nin (LPA), and wheat germ agglutinin (WGA) — at 8 d
and 12 d after oviposition to test for the presence of spe-
cific terminal oligosaccharides (Figs. 25-32). Embryos
without any lectins added showed slight autofluores-
cence in pigmented regions (see Thomas, 1970, for de-
scription) but no fluorescence in the EC (Figs. 25 and
26). The OICs of embryos incubated with Con A 8 d after
oviposition gave a positive fluorescent reaction (Fig. 27),
indicating terminal sucrose, D-glucosyl and A'-acetyl-D-
glucosamine residues. After the EEs were shed at 12 d
after oviposition, there was little fluorescent reaction
with EE4 (Fig. 28), but the discarded EEs and OICs still
reacted strongly. At 8 d after oviposition, the OIC had a
strong fluorescent reaction with WGA (Fig. 29), indicat-
ing terminal A'-acetyl-D-glucosaminyl residues as well as
/3-jV-acetylglucosamine oligomers. Embryos 12 d after
oviposition had little fluorescent reaction with EE4 when
treated with WGA (Fig. 30), although the discarded EEs
and OICs still reacted. At 8 d after oviposition, LPA
showed only nonspecific binding with the OIC (Fig. 3 1 ).
At 12 d after oviposition, when the outer EE and OIC
were shed, EE4 showed no affinity for the LPA lectin
(Fig. 32). When blocked for nonspecific binding using
bovine serum albumin, the Con A and WGA lectins con-
tinued to react strongly with the OIC, but LPA lectin
showed no reaction with the OIC. When each lectin was
preincubated with the corresponding sugars, fluores-
cence was blocked in all treatments (data not shown).

Discussion

The formation of the EC within the first four hours
after oviposition has been documented in the prawn Pa-
laemon serratus by Goudeau el al. ( 1 99 1 ) and in Macro-
brachium by Sandifer and Lynn ( 1 980) and by Lynn and
Clark (1983). The formation of additional coats after the
EC has formed has also been reported in other species
such as penaeid shrimp (Lynn et al., 1993) and lobster
(Talbot and Goudeau. 1988). What has not been de-
scribed in previous studies is that formation of the EC
may continue, as in P. pugio. for up to 10 d or so after
oviposition. In P. pugio during this time, at least three
additional EEs are formed around the developing
embryo that are not present at 3 d after oviposition.
These envelopes show distinct morphological character-

GRASS SHRIMP EMBRYONIC COAT

241

istics that allow each to be identified during develop-
ment. The EEs form and mature morphologically for
10 d after oviposition of the embryos. Together, the QIC
and all the EEs form the protective EC of the palaemonid
shrimp. At 12 d after oviposition, the outer envelopes,
EE1-EE3, are shed; only the innermost envelope, EE4,
remains around the embryo. Hatching occurs when the
embryo emerges from EE4. The hatching of P. pugio is
described by Davis (1965). However, the role of the fu-
niculus in the development of the embryo has not been
closely examined.

Broad (1957) states that a prezoeal molt takes place
before hatching. We saw no evidence of a molt that, as
Helluy and Beltz (1991) have seen in lobster, conforms
to the surface of the embryo before hatching. The cuticle
layer seen at 1 2 d conformed to the surface of the
embryo, but was clearly separate from EE4. Although
the innermost envelope, EE4, was morphologically sim-
ilar to the adult crustacean exoskeleton, there were dis-
tinct morphological differences when compared to the
embryonic exoskeleton (compare Fig. 18 and Fig. 19).

Changes in permeability of the EC to labeled dextrans
coincide with the formation of the envelopes. As EEs
were added, the permeability of the EC decreased. After
all the envelopes had formed and the embryo had devel-
oped further, the permeability increased. The permeabil-
ity was greatest as the embryo approached hatching; i.e..
after the QIC and EE 1 -EE3 had been shed and only EE4
remained around the embryo. Some of these results may
explain in part the difficulty in embryo fixation reported
by Thomas (1970) and also seen in this laboratory.

Lectin affinity studies lend some insight into the com-
position of the EC. The lectins are too large in molecular
weight to penetrate the EC. Only Con A may be small
enough to pass in and out of the EC; however, it appears
to bind to the EC, as indicated by the strong fluorescent
image. The strong Con A and WGA lectin affinity indi-
cates the presence of a terminal glucosyl and yV-acetyl-
glucosamine residues. Contrast the binding of lectin to
the O1C with the lack of binding to EE4 after the outer
coat has been shed.

The results of this study are important to those using
P. pugio for bioassays to understand the effects of toxi-
cants and microorganisms on the developing embryo.
The changing morphological and functional aspects of
the EC throughout the development of the embryo pro-
foundly affect the stage-specific sensitivity and suscepti-
bility of the embryo. Further research is needed to ad-
dress the importance of the QIC and funiculus in suc-
cessful development, the functional roles of the different
layers of each EE, and the effects of physical and chemi-
cal stressors on the progression of EC formation.

In this paper, we propose terminology to describe a
complex protective structure. We use terms that are de-

scriptive yet familiar, avoiding commonly used terms
such as "extracellular coat" because we feel they are mis-
leading. Because we are studying the envelopes around
an embryo with multicellular organization, not just the
newly fertilized zygote, the word "extracellular" could be
misleading to those unfamiliar with the historical usage.
We use "coat" to encompass the entire protective struc-
ture, since normally we think of only one coat being used
at a time. We have continued to use "envelope" since it is
now widely accepted in several species. Ideally, this usage
will further promote the standardization of terminology
among developmental biologists.

Acknowledgments

The authors give special thanks to Steve Foss, EPA,
for assistance with the embryo cultures. John Lynn and
Prudence Talbot were helpful with pertinent discussions
on Macrobrachium reproduction and lobster develop-
ment, respectively. Jeff Green and Gerry Cripe provided
valuable criticism of the manuscript. This work was per-
formed while PSG and JRR held National Research
Council-EPA, Gulf Ecology Division Research Asso-
ciateships. This is EPA, Gulf Ecology Division contribu-
tion No. 979.

Literature Cited

Broad, A. C. 1957. Larval development of Palaemonetes pugio Hol-
thius. Biol. Bull. 1 1 2: 1 44- 1 6 1 .

Clark, \V . H., Jr., and J. \V. Lynn. 1977. A Mg** dependent cortical
reaction in the eggs of penaeid shrimp. J. Exp. Zoo/. 200: 177-183.

Davis, C. C. 1965. A study of the hatching process in aquatic inverte-
brates. XIV. An examination of hatching in Palaemonetes vulgaris
(Say). CnislaceanaS: 233-238.

Fisher, W. S. 1983. Eggs of Palaemon macrodactylus: II. Association
with aquatic bacteria. Biol. Bull 164:201-213.

Fisher, W. S., and W. H. Clark, Jr. 1983. Eggs of Palaemon macro-
dactvlits: I. Attachment to the pleopods and formation of the outer
investment coat. Biol. Bull 164: 189-200.

Fisher, W. S., and S. S. Foss. 1993. A simple test for toxicity of num-
ber 2 fuel oil and oil dispersants to embryos of grass shrimp, Palae-
monetes pugio. Mar. Pol/iil. Butt. 26: 385-391.

Glas, P. S., J. D. Green, and J. W. Lynn. 1995. Oxidase activity asso-
ciated with the elevation of the penaeoid shrimp hatching envelope.
Biol. Bull. 189: 13-21.

Goudeau, M., and J. Becker. 1982. Fertilization in a crab. II. Cyto-
logical aspects of the cortical reaction and fertilization envelope
elaboration. Tissue Cell 14: 273-282.

Goudeau, M., H. Goudeau, and D. Guillaumin. 1991. Extracellular
Mg2+ induces a loss of microvilli. membrane retrieval, and the sub-
sequent cortical reaction, in the oocyte of the prawn Palaemon ser-
ratus.Dev Biol. 148:31-50.

Goudeau, M., and F. Lachaise. 1980a. 'Endogenous yolk' as the pre-
cursor of a possible fertilization envelop in a crab (Carcinus mae-
nax). Tissue Cell 1 2: 503-5 1 2.

Goudeau, M., and F. Lachaise. 1980b. Fine structure and secretion of
the capsule enclosing the embryo in a crab (Carcinus maenas (L)).
Tissue Cell 12: 287-308.

242

P. S. GLAS ET AL.

Goudeau, M., and F. Lachaise. 1983. Structure of the egg funiculus
and deposition of embryonic envelopes in a crab. Tissue Cell 15:
47-62.

Goudeau, M., F. Lachaise, G. Carpentier, and B. Goxe. 1990. High
tilers of ecdysteroids are associated with the secretory process of
embryonic envelopes in the European lobster. Tissue Cell 22: 269-
281.

Helluy, S. M., and B. S. Beltz. 1991. Embryonic development of the
American lobster (Homants americanus): Quantitative staging and
characterization of an embryonic molt cycle. Biol. Bull 180: 355-
371.

Kiernan, J. A. 1990. Histological and Histochemical Methods: The-
ory and Practice. Permagon Press, Oxford.

Legge, M. 1995. Oocyte and zygote zona pellucida permeability to
macromolecules. J. Exp. Zoo/- 27: 145-150.

Lynn, J. \V., and W. H. Clark, Jr. 1983. A morphological examina-
tion of sperm-egg interaction in the freshwater prawn, Macrobra-
chium rosenbergii. Biol. Bull 164: 446-458.

Lynn, J. W., P. S. Glas, Q. Lin, and J. D. Green. 1993. Assembly of
extracellular envelopes around the eggs and embryos of the marine
shrimp, Sicyonia ingenlis. J Reprod Dev. 39: 90-91.

Pillai, M. C., and VV. H. Clark, Jr. 1987. Oocyte activation in the
marine shrimp, Sicyonia ingenlis. J. E.\p. Zoo/. 24: 325-329.

Sandifer, P. A., and J. W. Lynn. 1980. Artificial insemination of car-
idean shrimp. Pp. 271-288 in Advances in Invertebrate Reproduc-
tion, Vol. II. W. H. Clark, Jr. and T. S. Adams, eds. Elsevier/North
Holland, New York.

Talbot, P. 1981. The ovary of the lobster, Homants americanus. II.
Structure of the mature follicle and origin of the chorion. J. Ultras-
Intel. Res 76: 249-262.

Talbot, P., and M. Goudeau. 1988. A complex cortical reaction leads
to formation of the fertilization envelope in the lobster, Homants.
Gamete Res. 19: 1-18.

Thomas, M. B. 1970. A descriptive morphological and electropho-
retic study of the embryonic development of Palaemonetes pugio
(Crustacea: Decapoda). M. S. Thesis. Department of Zoology, Uni-
versity of North Carolina, Chapel Hill.

Tyler-Shroeder, D. B. 1978. Culture of the grass shrimp (Palaemo-
netes pugio) in the laboratory. Pp. 69-72 in Bioassay Procedures
for the Ocean Disposal Permit Program; EPA/600/9-78-010, U.S.
EPA. Environmental Research Laboratory, Gulf Breeze. FL.

Venable, J. H., and R. Coggeshall. 1965. A simplified lead citrate
stain for use in electron microscopy. J. Cell Biol. 25: 407-408.

Reference: Biol Buli 192: 243-252. (April. 1997)

Morphology and Development of Odostomia

columbiana Ball and Bartsch (Pyramidellidae):

Implications for the Evolution

of Gastropod Development

RACHEL COLLIN1 * AND JOHN B. WISE2

1 Department of 'Zoology, University of Washington, Box351800, Seattle, Washington 98 195; and
2 Houston Museum of Natural Science, 1 Herman Circle Drive. Houston, Texas 77030

Abstract. Although pyramidellid gastropods are a phy-
logenetically important group of diverse and abundant
ectoparasites, little is known about their life histories.
Herein, we describe the adult morphology and develop-
ment of the pyramidellid Odostomia columbiana, which
parasitizes the scallops Chlamys hastata and C. rubida
in the Northeast Pacific. Anatomically, adult O. colum-
biana resemble other known pyramidellids although
they lack the tentacular pads typical of other Odostomia
species. Embryonic development is similar to that de-
scribed for other pyramidellids: cleavage is unequal, gas-
trulation is partially by invagination, and considerable
growth occurs before hatching. However, embryonic and
larval development are much slower than for other de-
scribed species. The planktotrophic larvae hatch after
19 days of intracapsular development and metamor-
phose about 2 months later. O. columbiana veligers have
a large black pigmented mantle organ to the right of the
midline, a distinct metapodial tentacle, and three or four
long bristles that project over the operculum from be-
hind the foot. Observations of newly metamorphosed ju-
veniles suggest that previous disagreements regarding the
development of heterostrophy are due to variation in the
degree of heterostrophy among species. Our observa-
tions also generally corroborate certain scenarios ex-

Received 16 August 1996; accepted 17 December 1996.

* Address for correspondence: Committee on Evolutionary Biology.
Culver Hall. 1025 E. 57th Street, Chicago. IL 60637; e-mail: rcollin®-
midway.uchicago.edu

Abbreviations: PMO = pigmented mantle organ; hpl = anterior huc-
cal pump; bp2 = posterior buccal pump; VCSGL = ventral ciliated
strip gland.

plaining the evolution of gastropod cleavage type and
larval heterochrony. Unequal cleavage and larvae that
hatch without well-developed eyes and tentacles may be
characteristic of the common ancestor of pyramidellids
and opisthobranchs; however, late development of the
larval heart is probably a derived condition of opistho-
branchs.

Introduction

Pyramidellids are common ectoparasites in many ma-
rine communities, but little is known about their biology
and life histories (Haszprunar, 1988; Wise, 1996). In par-
ticular, data concerning their development and larval bi-
ology are lacking. As in many benthic marine inverte-
brates with limited adult dispersal, the duration of a
planktonic larval stage may be a key factor influencing
pyramidellid distribution and population dynamics.
Long-lived planktonic stages may increase colonization
of patchily distributed hosts, whereas species that hatch
as crawling juveniles may have lower probabilities of lo-
cal extinction (Thorson, 1950: White et al., 1984; Gum-
ming, 1993). Therefore, knowledge of pyramidellid de-
velopment and larval biology is key to understanding
their life histories and host-parasite interactions, and to
planning effective pest-control strategies for aquaculture.

Pyramidellid development is also particularly infor-
mative when viewed in a phylogenetic context. Gener-
ally, pyramidellids are placed, with other families in the
order Heterostropha, at the base of the heterobranch
clade (Haszprunar, 1988). Although the relationships
among these families are poorly resolved, pyramidellids
clearly represent basal members of the clade that in-

243

244

R. COLLIN AND J. B. WISE

eludes opisthobranchs and pulmonates, and which is the
sister group of the Caenogastropoda (Haszprunar, 1988;
Bieler, 1992; Mikkelsen, 1996). Knowledge of such basal
groups can be useful in tracing evolutionary transitions,
determining ancestral character states, and testing evolu-
tionary scenarios. This knowledge may be particularly
useful in gastropod development, where evidence for ev-
olutionary scenarios is often available from derived rep-
resentatives of only a few clades. For example, van den
Biggelaar ( 1996) found a trend in cleavage type and D-
quadrant specification from equal cleavage and late spec-
ification in "archaeogastropods" to unequal cleavage
and early specification in opisthobranchs and pulmo-
nates. Additionally, Page (1994) suggested that hetero-
chronic shifts in gastropod development decelerated the
formation of larval and adult structures in opistho-
branch larvae relative to prosobranchs. These scenarios
were generated primarily from observations of derived
caenogastropods, opisthobranchs, and pulmonates.
Moreover, detailed descriptions of pyramidellid devel-
opment could be used to further support or refute these
types of scenarios.

To date, accounts of pyramidellid reproduction and
embryology are brief and limited to a small number of
European (Lebour, 1932, 1936; Rasmussen, 1944, 1951;
Fretter and Graham, 1949), eastern North American
(Robertson, 1978; White el at.. 1985), and Indo-Pacific
(Amio, 1963;Nishinorttf/., 1983; Gumming, 1993) spe-
cies (Table I). Although more than 50 species have been
reported from western North America (Dall and Bartsch.
1909), there exists only a single account of pyramidellid
development from the region (LaFollette, 1979). No de-
tailed embryological studies of pyramidellids have been
published, and larvae that do not metamorphose imme-
diately after hatching are seldom reared through settle-
ment (see Robertson, 1967, for an exception). Conse-
quently, the details of early development, the duration of
the planktonic period, and the events at metamorphosis
have been the subject of much speculation (Thorson,
1946; Fretter eta/., 1982).

Published accounts of pyramidellid reproduction and
development are often obscured by taxonomic confu-
sion within the group. Criteria used to identify species
are often not explicitly stated in published reports, or the
taxonomy is so conjectural that species cannot be identi-
fied with much certainty. For example, Clark (1971) and
'Vnann (1979) did not state how they identified their
as Odostomia columbiana. and Cumming ( 1993)
able to definitely identify "'Turbonilla sp." to ge-
;pecies. This taxonomic uncertainty makes it
^ interpret comparative reproductive and de-
'1 data.

'poecilogony, a rare condition in gastropods
(Ho 'd Robertson, 1988; Bouchet. 1989) in

which two or more developmental modes occur in one
species, may be an example of such taxonomic confu-
sion. Boonea impressa has been reported to have plank-
totrophic development in North Carolina and lecitho-
trophic development in Texas. However, these popula-
tions may not represent the same species (Bouchet,
1989). Both small eggs that develop into planktotrophic
larvae and large eggs with direct development have also
been reported for Brachystomia rissoides in areas of
different salinities in Europe (Pelseneer, 1914; Rasmus-
sen, 1944, 1951; Thorson, 1946).

This paper adds to the data on pyramidellid develop-
ment while avoiding the taxonomic confusion of previ-
ous studies by describing both the adult morphology and
the development of the pyramidellid Odostomia colum-
biana Dall and Bartsch, 1907. This new information is
discussed in the context of the general characteristics of
pyramidellid development and its implications for the
evolution of development within the gastropods.

Materials and Methods

Mature Odostomia columbiana were found on the
scallops Chlamys hastata and C. rubida dredged
from 90- 130m in San Juan Channel, Washington
(48°34'10"N, 123°2'0"W) during March 1995. Both the
snails and their hosts were kept at ambient sea tempera-
ture (8°-12°C) in flow-through sea-tables at Friday Har-
bor Laboratories, Washington. Snails ranged from 5 to
8 mm in length and were identified as O. columbiana on
the basis of the original description of the shell provided
by Dall and Bartsch (1907) and by comparison with the
holotype and five syntypes on deposit at the National
Museum of Natural History (lot #126658). Voucher
specimens have been deposited at the Field Museum of
Natural History (FMNH 293334: dry shells; FMNH
293334: formalin-fixed animals).

Adull morphology

Adult snails were extracted for dissection by first
cracking their shells with a vise. Snails were dissected
whole, and structures of the gut and reproductive tract
were routinely stained with toluidine blue (Wise, 1993,
1996).

Development

Egg masses were present on the scallops when they
were collected, and adult snails continued to produce egg
masses in the laboratory until May 1996. Scallop shells
were checked for new egg masses several times a month
to determine whether reproduction was seasonal. Indi-
vidual egg masses were removed from the scallop shells
and kept in small glass dishes while their development

TRENDS IN GASTROPOD DEVELOPMENT

245

was observed. Although we did not observe egg laying,
several egg masses were collected before the beginning of
first cleavage. Observations of these egg masses were used
to produce a developmental timetable. Eggs and egg cap-
sules were measured prior to first cleavage, and measure-
ments were taken in the plane of the chalazae. After
hatching, larvae were transferred to filtered (0.45 ^m)
seawater in small glass dishes. They were fed Isochrvsis
galbana and Rhodomonas sp. ad libitum, and the water
was changed every 2 to 3 days. Because the larval shells
are hydrophobic. flakes of cetyl alcohol were added to the
surface of the cultures to prevent larvae from becoming
trapped in the surface tension. When the veligers began
to spend time crawling, juvenile scallops were added to
the cultures to induce metamorphosis and as food for
newly metamorphosed juveniles. All observations re-
ported are of animals reared or maintained in the labo-
ratory.

Larval shells were prepared for scanning electron mi-
croscopy by rinsing them in dilute bleach and distilled
water following the procedure of Hickman ( 1 995). They
were mounted on stubs with double-sided tape, coated
with gold and palladium, and examined with a JEOL
JM35 scanning electron microscope.

Results

Adult morphology

The following is given in the concise style of a species
description.

Shell: Relatively thin, elongate conical, chalky white,
5-8 mm in length, composed of 5 to 6 adult whorls.
Adult whorls with numerous fine spiral growth lines.
Whorl shoulders rounded, with moderately deep sutures.
Shell etched and pitted, particularly above body whorl.
Lenticular aperture, with flared lower portion. Single,
short, acute columellar fold on upper half of columella
perpendicular to columellar axis. Smooth, sinistrally het-
erostrophic protoconch oriented 130 degrees to teleo-
conch axis, with 40% submerged within first adult whorl.
Operculum brown, lenticular, with subcentric nucleus.

Head-Foot: White to yellow, with numerous scattered
white cells. Propodium with slight medial indentation
and rounded lateral edges. Foot narrows posterior to pro-
podium, then widening to gradually taper to a blunt
apex. Pedal gland opens in middle of groove extending
along posterior half of ventral surface of foot. Attach-
ment thread present. Tentacles subtriangular, connate,
ventrolaterally folded; tentacular pads apparently ab-
sent. Black round eyes beneath epithelium on median
side of tentacles. Mentum unnotched, not bifurcate.

Alimentary Tract: Retracted introvert-proboscis ex-
tending posteriorly from its aperture on ventral side of
head, dorsal to mentum base and entering cephalic

Figure 1. Diagram of alimentary tract of Odostmnia atlumhuina.
aes = anterior esophagus, bp I = buccal pump 1 , bp2 = buccal pump 2,
bs = buccal sac. p = proboscis, pes = posterior esophagus, sb = stylet
bulb, sd = salivary gland duct, sgl = salivary gland, su = sucker. Scale
bar is 500 ^m.

hemocoel (Fig. I ). Introvert joining buccal sac (contain-
ing buccal sucker, stylet, and stylet bulb), which is con-
nected to buccal pump. Buccal pump divided into an-
terior (bpl) and posterior (bp2) sections, with bp2
one-third longer than bpl; bpl narrow, round in cross-
section; bp2 wider, laterally flattened, distally rounded.
Short, straight anterior esophagus originating on ventral
surface of alimentary tract at bpl-bp2 juncture, extend-
ing posteriorly to join posterior esophagus and paired
salivary glands, forming a four-way junction. Long,
highly coiled posterior esophagus extending posteriorly
to enter visceral mass and join stomach. Long, narrow
salivary glands equal to bp2 in length. Salivary gland
ducts highly folded and attached to anterior esophagus.
Ducts penetrating alimentary tract just anterior to bpl -
bp2 juncture, extending parallel to one another within
walls of bpl and entering stylet bulb without exiting gut.
Salivary glands not attached distally to esophagus.

Pallia! Cavity: Mantle and mantle organs typical of
members of the Odostominae, with exception of position
of ventral ciliated strip gland ( = VCSGL). Small, spheri-
cal, yellow VCSGL underlying 20% of ventral ciliated
strip about halfway from anterior edge of mantle floor.
Ventral and dorsal ciliated strips joining on mantle roof
posterior end of mantle cavity. Small, round-to-oblong
pigmented mantle organ ( = PMO), composed primarily
of cells containing a bright yellow exudate, framed by
numerous brown cells. A thick, bright yellow exudate is
released when snail is disturbed.

Reproductive System: Opaque-to-transparent repro-
ductive organs located on columellar side. Common pal-
lial gonoduct extends anteriorly from visceral mass to
open on right side of head just anterior and beneath the
right tentacle.

Reproduction and development

Our observations of reproduction and larval develop-
ment of O columbiana generally agree \v: i previous ac-
counts of pyramidellid development (I Iseneer. 1914;

246

Review of pyramidellid

R. COLLIN AND J. B. WISE
Table I

Specie -

Egg size

(^m)

Capsule size
(Mm)

Days to
hatching

Larval duration
(days)

Temp.
(°C)

Reference

Boonea inr

130*

180-280*

3-5

7

-25

White etui. 1985

Brad: > issoides

120

500

25

0

7

Rasmussen, 1951

Brui •/: L! rissoides

60

200

7

7

?

Thorson, 1 946

Braclii <i<imia rissoides

70

200

6

?

-20

Rasmussen, 1944

Brachystomia rissoides

100

380

25

0

7

Pelseneer, 1914

Chrysallidu cincla

9

320

22-27

0

20

LaFollette. 1979

Chrysallida deaissata

90- 1 20t

240-350

7

7

7

Lebour, 1936

Menestho diaphana

80'

200

16

?>8

11

Kristensen, 1970

Odoslomia ucma

-80*

160x200

a few

7

?

Haisaeter, 1989

Odostomia columbiana

72-74

153-178

19

-70

12

this study

Odoslomia. desimana

80

1 30 x 1 60

14

9

7

Amio. 1963

Odoslomia eulimoides

7

9

10-12

3-4

18

Fretter and Graham. 1949

Odoslomia eulimoides

90*

160

7

9

7

Lebour, 1932

Odoslomia fujilaii

7

?

15-16

8-12

15

Minichev, 1971

Odostomia sp.

-100*

-200*

7

3(?)

22

Nishinorta/.. 1983

Odoslomia omaensis

60

1 20 X 1 50

8

7

7

Amio, 1963

Turbonilla sp.

130

300 x 400

10-11

3-5 or 0

23

Cumming, 1993

* Measured from figures,
t 4-8 eggs per capsule.

Lebour, 1932, 1936: Rasmussen, 1944, 1951; Fretter and
Graham, 1949; White et ai. 1985). However, both em-
bryonic (used here to refer to development prior to
hatching) and larval development are much slower than
reported for other species (Tables I and II). Although we
searched for spermatophores like those described by
Robertson (1978), at no time did we find any attached
to the shells or bodies of these snails. Egg masses each
containing up to several hundred eggs were deposited on

Table II
Developmental time table for O. columbiana ai III-I2°C

Age

Stage

—6 h First cleavage

1 8 h 4 cells
24 h 8 cells

48 h Round blastula

2-3 days Gastrulation: blastula flattens and then forms a

horseshoe-shaped gastrula
3-7 days Gastrula

8 days Foot and head rudiments begin to differentiate, some

trochal ciliary motion; the shell is not yet present
10 days Shell, cilia, and foot with operculum and statocysts

present, light red PMOjust visible
1 7 days Heartbeat and ciliary motion in gut visible

1 9 days Veligers hatch, PMO is black
72-80 days Veligers begin to crawl

— 90 days Settlement and metamorphosis

the hosts' shells continually from March 1995 until May
1 996 when the last of the snails died. Irregular egg masses
are often deposited in groups, making it difficult to dis-
tinguish individual masses. Each snail generally pro-
duced several egg masses per week.

Individual eggs are white and range from 7 1 to 77 ^m
in diameter (mean = 74.5; SD = 1.61; n = 23). Egg size
did not differ between summer (July) and winter (Janu-
ary). Each round egg, surrounded by an oval layer of
opaque albumin, is enclosed in a thick transparent ge-
latinous capsule (Fig. 2A-F). The length of the space
enclosing the albumin is 154-170 /urn (mean = 162.5;
SD = 4.6; n = 23) and the outer length of the capsule is
193-222 Mm (mean = 21 1; SD = 6.9; n = 23). In some
previous accounts of pyramidellid development (e.g.,
White et ai, 1985), capsular length has been reported as
egg size, whereas other studies measure the egg cell. In
this paper "egg" is used to denote the actual egg cell,
and "capsule" refers to the egg and the albumin con-
tained in one gelatinous covering. The capsules are con-
nected to one another by thin strands called chalazae. In
O. columbiana. chalazae are composed of a thin extra-
embryonic layer that surrounds the albumin layer be-
neath the capsule, traverses the capsule, and continues
between capsules as a thin tubular strand (Fig. 2C). The
egg, albumin, and capsular covering are embedded in a
transparent gelatinous matrix that is often covered with
diatoms.

All eggs within a single egg mass develop synchro-

TRENDS IN GASTROPOD DEVELOPMENT

247

r ' f

Figure 2. Stages in OcloMoniia columbiana development. (A) Two-cell stage; (B) four-cell stage; (C)
blastula; (D) begining of differentiation of the foot, velum, and shell; (E) hatchling veliger; (F) empty egg
capsule. In A and B the capsule appears round because it is viewed end on. Chalazae are clearly visible in
the upper right of C and the upper left of F. All figures are to the same scale, and scale bar is A is

nously, and the initial cleavages are simultaneous. First
cleavage is clearly unequal (Fig. 2A), but the second
cleavage appears to be more-or-less equal. This results in

a four-cell stage with two relatively large cells and two
small cells (Fig. 2B). Third cleavage is also unequal. Be-
cause the polar bodies are initially obscured by the

248

R. COLL1N AND J. B. WISE

Figure 3. Scanning electron micrograph of Odostomia columhiana lar\al shell at hatching. Apical,
apertural. and umbilical \ieus. from left to right. Shell length is 150-160/jm for these three shells.

opaque albumin, the polarity and direction of this divi-
sion could not be determined.

After 2 days the initially spherical blastula flattens
(Fig. 2C) and in the next few days develops into a heart-
shaped or cup-shaped gastrula. Thusgastrulation occurs
at least partially by imagination, as is typical of many
opisthobranchs. After 8 days the foot, head, and velum
anlangen appear (Fig. 2D), and short cilia present on the
velar lobes beat weakly. Even after the cilia are fully
formed the embryos rotate very slowly, if at all, within
the capsules. After 10 days the shell and statocysts are
clearly visible, and the pigmented mantle organ begins to
develop. As the PMO grows it darkens and changes from
dark red to black before the larvae hatch. At 1 7 days the
beating heart is visible, and the cilia of the gut are active.
The embryo continues to absorb the surrounding albu-
min and grows until it eventually fills the capsule. At no
time during development did we observe paired larval
kidneys similar to those described by Pelseneer (1914).
Additionally, methods used to demonstrate protein up-
take in morphologically similar caenogastropod larval
kidneys (Collin. unpubl. data) did not detect protein up-
take in decapsulated embryos. Inspection of capsules, af-
ter hatching, shows that larvae hatch through an opening
in the capsule along one of the chalazal strands (Fig. 2F).

At hatching the smooth, sinistral protoconch com-
prises nearly an entire whorl (Fig. 3) and is 150-160 ^m
in length (n = 7; Fig. 3). Newly hatched larvae have a
black PMO just to the right of the midline, a small bi-
lobed velum, and no eyes, tentacles, or obvious apical
tuft (Fig. 2E). Except for the PMO and the stomach and
digestive gland, whose color reflects larval diet, the veli-
ger is entirely transparent. Although larvae retracted into
their shells when placed under the microscope, the foot

points slightly to the animal's right when they swim. As
the foot grows, the metapodial tentacle, the posterior
protuberance of the foot (distinct from adult metapodial
tentacles) and its terminal cilia, which extend over the
operculum, become more obvious. On each side of the
foot, three or four bristles, which seem to originate be-
hind the foot, project radially over the edge of the oper-
culum. Veligers develop eyes within 18-23 days of
hatching. After 2 months, the velum shrinks and the lar-
vae, which are about 290 urn in shell length, begin to
crawl. Larvae at this stage settle and metamorphose
within days of the addition of juvenile scallops to the cul-
ture. Larvae that were the same age but had not begun to
crawl did not settle in response to scallops. Water in
which scallops had been kept overnight did not induce
metamorphosis in crawling larvae. The PMO remained
dark for at least 1 month after metamorphosis (Fig. 4),
but had turned yellow after 6 months.

Discussion

TiiMvioniu nule

The snails used in this study were identified as O. co-
lumhiana on the basis of the original description of the
shell (Dall and Bartsch. 1907. 1909) and by comparison
with the type specimens. All animals were collected at-
tached to Chlainys spp. However, previous papers have
described O. colunihiunu found on Tricholropix can-
ccllala in Puget Sound, but did not state how the snails
were identified (Clark, 1971; Hoffmann, 1979). Pyrami-
dellid snails on T. canccllata are much smaller (2-4 mm.
pers. obs., RC) than the snails used in this study and
those described by Dall and Bartsch (up to 8 mm). The
differences in size and host suggest that these may be two

TRENDS IN GASTROPOD DEVELOPMENT

249

Figure 4. Juvenile Odostomia culumhiana at the edge of a scallop
shell about I month after settlement. The black PMO is visible through
the shell in the middle of the body whorl. Shell length is about 700 Mm.

different species. Nevertheless, the adult shell shape,
sculpture, protoconch morphology, and adult anatomy
are quite similar.

Previous studies have reported that some pyramidel-
lids may not be host specific, or their preference may
change as they grow (Boss and Merrill, 1965: Powell el
at., 1987), suggesting that the association of small ani-
mals with T. cancc/lata and large animals with Chlamys
spp. may represent an ontogenetic host shift. This is un-
likely because juvenile O. columbiana raised in this study
did not move onto T. cancc/lala when given the choice
between juveniles of Chlamys spp. or T. cancellaia. No
egg masses were discovered on T. cancellata. so it is un-
clear whether the pyramidellids from these hosts were
sexually mature. Our preliminary results suggest that
these are closely related species.

Adult morphology

The morphology of O. columbiana is very similar to
that of other members of the family. However, it differs
in several ways from the morphology of known species
of the genus Odostomia. Odostomia columbiana lacks
tentacular pads, and its gut arrangement is similar to that
found in members of the genus Boonea except that the
salivary gland ducts are attached to the anterior esopha-
gus, which with the posterior esophagus and paired sali-

vary glands form a four-way junction (Fig. 1). Odo-
stomia columbiana. as well as other members of the sub-
family Odostominae, will ultimately be relegated to
other genera (Wise, in prep.).

Pyramidellid development

The reproduction and development of 0. columbiana
is similar to that previously described for other pyrami-
dellids (Lebour, 1932. 1936: Rasmussen. 1944. 1951;
Fretter and Graham, 1949: White el at.. 1985), although
most of these descriptions are brief and small differences
may have been overlooked. Eggs of O. columbiana are
relatively small, but not unusually so (Table I). Because
embryos grow to fill the capsule before hatching, capsule
size is a good predictor of larval size in pyramidellids. O.
columbiana deposits a single egg per capsule, as do other
pyramidellids except Chrysallida decussata (Table I).
Development of O. columbiana is slower than in other
species with comparable egg size: only species with large
eggs and direct development have a similarly long em-
bryonic period (Table I). Because water temperatures
were either not reported or varied considerably in several
previous studies, it is difficult to determine whether these
differences in development rate were due to temperature.

In O columbiana the long embryonic period is fol-
lowed by an unusually long planktonic larval stage. Al-
though times to metamorphosis may be different in the
laboratory than in nature, veligers in two non-overlapping
cultures settled at the same age. Furthermore, recent im-
provements in larval culture technique and larval diet
have reduced the time to metamorphosis in cultures of
other invertebrate larvae (Strathmann, 1987). This sug-
gests that the longer larval period of O. columbiana in
comparison to other laboratory-reared pyramidellid spe-
cies from previous studies is real (Table I). Unfortunately
these studies did not indicate if the larvae studied were
planktotrophic or if food was added to the larval cultures.
A distinctive characteristic of the O columbiana veligers
is the presence of bristles extending over the operculum
from the side of the foot. These are not figured or men-
tioned in any other description of pyramidellid larvae.

Our observations increase the data on the distribution
of several developmental characters that are used in het-
erobranch systematics. Robertson (1985) suggested that
chalazae may be a useful synapomorphy shared by pyra-
midellids, architectonicids, and opisthobranchs. Chala-
zae are clearly present in egg masses of O. columbiana
and many other pyramidellids. Haisaster (1989), how-
ever, suggests that some pyramidellids lack chalazae and
that their presence may be a useful systematic character
within the pyramidellids. He did not find chalazae in O.
acuta, and they were not mentioned in descriptions of
egg masses from Brachystomia eulim ndes (Lebour,

250

R. COLLIN AND J B. WISE

1932) and Odostomia sp. (Nishino et al., 1983). Addi-
tionally, the shape and thickness of the chalazae of O.
fujitanii figured by Minichev (1971) are clearly different
from those in O. columbiana and other pyramidellids.
Spermatophores have also been used as a systematic
character within pyramidellids to define the genus
Boonea (Robertson, 1978). O. columbiana apparently
lacks spermatophores; however, this character has been
observed in some other species occurring in the north-
eastern Pacific (A. J. Kohn, pers. comm.).

Our observations of pyramidellid development from
hatching to metamorphosis illuminate several points
that have been the subject of much conjecture. At hatch-
ing, veligers of O. columbiana are morphologically sim-
ilar to veligers of other planktotrophic pyramidellid spe-
cies, all of which seem to have small, nonpigmented ve-
lums. Previous studies that failed to rear larvae to
metamorphosis often concluded that the larval period is
short because the velum is small (e.g., Thorson, 1946).
This is not the case for O. columbiana. which has a long
planktonic period during which its velum remains small.
Small velar lobes are also typical of planktotrophic opis-
thobranch larvae (pers. obs., RC), although caenogastro-
pods with long-lived feeding larvae often have large com-
plex velar lobes (Thorson, 1 946).

Confusion exists as to how differential shell growth
around the aperture results in heterostrophy (the change
of coiling direction from a left-handed protoconch to a
right-handed teleoconch). Thorson (1946) illustrated
several pyramidellid shells near metamorphosis and
stated that the outer apertural margin of the larval shell
thickens to become a new columella at metamorphosis.
Fretter et al. ( 1 982 ) disagreed and suggested that the pro-
toconch's outer lip is continuous with the outer lip of the
teleoconch. In O columbiana, the position of the colu-
mella shifts clockwise around the aperture as the coiling
direction changes, but not to the extent described by
Thorson (1946). These conflicting observations of
changes in shell shape at metamorphosis may be due to
varia'tK in degree of heterostrophy among pyramidellid
specie 'though the coiling direction reverses in all
cases, : :onsiderable variation in the displacement

of the CL is of the teleoconch relative to that of the

protocont ist of the species figured by Thorson

( 1 946), the , h is offset roughly perpendicular to

the teleoco: -ever, in O. columbiana and

hystomia ei one species studied by Fretter

^raham, 19' angle between the coiling axes

• trotoconch and onch often appears to be

clos 1 40 degrees! ..-neretal., 1982).

Evolia >f gastropod development

Detail. udies of embryology and larvae of "archae-
ogastropot caenogastropods, and opisthobranchs re-

veal several trends in gastropod development. Among
gastropods there is a trend in both caenogastropods and
opisthobranchs toward unequal cleavage and early D-
quadrant specification (Freeman and Lundelius, 1992;
van den Biggelaar, 1996; van den Biggelaar and Hasz-
prunar, 1996). Equal cleavage with 4d cell formation at
the 64-cell stage as in "archaeogastropods" is assumed to
be the ancestral condition. In opisthobranchs, the first
one or two cleavages are unequal, and the 4d cell is pro-
duced by the 24-cell stage. Intermediate conditions occur
in the Architaenioglossa and the Valvatoidea, and there
is considerable variation in basal opisthobranchs (van
den Biggelaar, 1996). Although Pelseneer(1914)and van
den Biggelaar and Haszprunar (1996, citing Pelseneer)
report equal cleavage in pyramidellids, first cleavage in
O. columbiana is clearly not equal. In addition. White et
al. (1985) figured an unequal two-cell stage of Boonea
impressa in the process of division. This suggests that ei-
ther cleavage varies among pyramidellids (an unusual
occurrence in gastropod groups) or Pelseneer's account
is inaccurate. Regardless, it is interesting that early cleav-
age divisions in pyramidellids are similar to those in an-
aspideans and some other opisthobranchs.

Page ( 1994) suggested that heterochronic shifts in the
timing of development of larval and adult organs ac-
count for some of the variation in gastropod larval devel-
opment. In "archaeogastropods" the adult organs de-
velop directly from the embryo, whereas in caenogastro-
pods the larva hatches with well-formed larval organs
and subsequently develops the definitive adult struc-
tures. Development is retarded in opisthobranchs, and
they hatch at a stage of morphogenesis similar to that of
unhatched caenogastropods: some larval organs develop
after hatching, and the definitive adult organs develop
during late larval life or at metamorphosis (Page, 1994).
Page ( 1994) paid particular attention to the stage of for-
mation of the eyes, tentacles, and larval heart. Caenogas-
tropod veligers hatch with well-formed eyes and tenta-
cles, and the larval heart develops long before hatching
(Page, 1994, and pers. obs). In opisthobranchs, the larvae
hatch before the eyes or tentacles have formed, and the
larval heart usually forms during the last half of the
planktonic stage (Page, 1994). The observations of O. co-
lumbiana reported here suggest that pyramidellid larval
development may combine characters of opisthobranch
and caenogastropod development. As in opisthobranchs,
the eyes and tentacles did not appear until well into larval
development; as in caenogastropods, the heart began to
beat well before hatching.

These observations support recognized trends in the
evolution of gastropod development and suggest ances-
tral character states. Unequal cleavage is characteristic of
pyramidellids and some basal opisthobranchs, whereas
caenogastropods have a polar lobe; thus unequal cleav-

TRENDS IN GASTROPOD DEVELOPMENT

251

age may be a heterobranch synapomorphy. However,
additional groups at the base of this clade must be exam-
ined to rule out parallel evolution of unequal cleavage
from an equally cleaving ancestor, because many other
opisthobranchs and pulmonates have equal cleavage.
Additionally, larvae that hatch before the development
of eyes and tentacles were probably ancestral for pyrami-
dellids and opisthobranchs. On the other hand, early ap-
pearance of the larval heart relative to hatching in pyra-
midellids and caenogastropods suggests that late devel-
opment of the heart is a derived feature of the
opisthobranchs. It is clearly necessary to have detailed
descriptions of more phylogenetically intermediate
groups and to formulate robust phylogenetic hypotheses
before any firm conclusions regarding the evolution of
these characters can be drawn.

Acknowledgments

We thank A. Kabat (US National Museum of Natural
History) for lending us the type specimens of O. colnm-
biana, R. Bieler (Field Museum of Natural History, Chi-
cago) for accepting the voucher specimens, E. Kozlofffor
translating Minichev (1971), and D. Willows and the
staff of Friday Harbor Laboratories for their support. B.
Fernet and R. Strathmann made useful comments on
the manuscript. This research was supported by a Na-
tional Science Foundation Graduate Fellowship and a
Pacific Northwest Shell Club Scholarship to RC, and
grant OCE 9301665 to R. Strathmann.

Literature Cited

Amio, M. 1963. A comparative embryology of marine gastropods,
with ecological considerations. Shimonoskei L'niv h'ish J. 12: 15-
144.

Bieler, R. 1992. Gastropod phylogeny and systematics. Aimu. Rev.
Eail.Sym .23:311-338.

Biggelaar.J. A. M. van den. 1996. The significance of the early cleav-
age pattern for the reconstruction of gastropod phylogeny. Pp. 155-
160 in Origin and Evolutionary Radiation oj lite Mollusca. J. D.
Taylor, ed. Oxford University Press. Oxford. UK.

Biggelaar, J. A. M. van den, and G. Haszprunar. 1996. Cleavage and
mesentoblast formation in the Gastropoda: an evolutionary per-
spective. Evolution SO: 1520-1540.

Boss, K. J., and A. S. Merrill. 1965. Degree of host specificity in two
species of 0</fu7f>;;i/u( Pyramidellidae: Gastropoda), Proc. Malacol.
Sin: London 36: 349-355.

Hum In i. P. 1989. A review of poecilogony in gastropods. ./ Mull
Stud 55: 67-78.

Clark, K. 1971. Host texture preference of an ectoparasitic opistho-
branch, Odostomia columbtuna Dall and Bartsch 1909. I 'eliger 14:
54-56.

Cumming, R. L. 1993. Reproduction and variable larval develop-
ment of an ectoparasitic snail. Turbonilla sp. (Pyramidellidae. Opis-
thobranchia) on cultured giant clams. Bui/. .Men: Sci. 52: 760-77 1 .

Dall, \V. H., and P. Bartsch. 1907. The pyramidellid mollusks of the

Oregonian faunal area. Proc. V S Nail. Mus 33: 49 1-534.

Uall, \V. H., and P. Bartsch. 1909. A monograph of west American
pyramidellid mollusks. Bull. I' S Nail. Mus 68: 1-258.

Freeman, G., and J. \\ . Lundelius. 1992. Evolutionary implications
of the mode of D quadrant specification in coelomates with spiral
cleavage. J. Evol. Biol 5: 205-247.

Fretter, V. M., and A. Graham. 1949. The structure and mode of life
of the Pyramidellidae. parasitic opisthobranchs. J. Mar Biol. Assoc.
UK. 28:493-532.

Fretter, V. M., A. Graham, and E. B. Andews. 1982. The proso-
branch molluscs of Britain and Denmark. Part 9 — Pyramidellidae.
J. Mo/1. Si uii. Suppl. 1 1: 363-434.

Haszprunar, G. 1988. On the origin and evolution of major gastropod
groups, with special reference to the Streptoneura. J Moll. Stud. 54:
367-441.

Hickman.C. S. 1995. Asynchronous construction of the protoconch/
teleoconch boundary: evidence for staged metamorphosis in a ma-
rine gastropod larva. Invertebr. Biol 1 14: 295-306.

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

Hoffmann, D. L. 1979. An attachment structure in an epiparasitic
gastropod. I 'eliger 22: 75-77.

Haisaeter, I. 1989. Biological notes on some Pyramidellidae (Gastro-
poda: Opisthobranchia) from Norway. Sarsia 74: 283-297.

Kristensen, J. H. 1970. Fauna associated with the sipunculid Phas-
colion strombi (Montagu), especially the parasitic gastropod Men-
estho diaphana (Jeffreys). Ophelia 1: 257-276.

LaFollette, P. 1979. Observation on the larval development and be-
havior of Chrysallida Carpenter. 1857 (Gastropoda: Pyramidelli-
dae). West. Sot: Malacol. Annii Rep 10: 18-23.

Lebour, M. 1932. The eggs and early larvae of two commensal gas-
tropods. Stililer stylifer and Odosiomia eu/imoides. J. Mar. Biol.
Assoc. U.K. 18: 117-1 19.

Lebour, M. 1936. Notes on the eggs and larvae of some Plymouth
prosobranchs. J Mar Biol Assoc. I' A. 20: 547-566.

Mikkelsen, P. M. 1996. The evolutionary relationships of Cephalas-
pidea s.l. (Gastropoda: Opisthobranchia): a phylogenetic analysis.
Malacologia31: 375-442.

Minichev, Y. S. 1971. On the biology of some Pyramidellidae of the
Possjet Bay of the Sea of Japan. Acad. Sci. USSR Zool. Insl. Explor-
ations oj the Fauna of the Seas%: 221-229 [in Russian].

Nishino, T., S. Nojima, and I. kikuchi. 1983. Quantitative studies of
life history and interspecific relationship of two gastropod species,
Odostomia sp. (ectoparasite) and U mbonium (Suchnim) monilif-
crum (Lamarck) (host). I. Life history and population dynamics of
Odostomia sp. Publ. Amakusa Mar. Biol. Lab. Kyushu Univ. 7: 61-
67.

Page, L. R. 1994. The ancestral gastropod larval form is best approx-
imated by hatching-stage opisthobranch larvae: evidence from
comparative developmental studies. Pp. 206-223 in Reproduction
and Development ol Marine Invertebrates. W. H. Wilson. S. A.
Strieker, and G. L. Shinn, eds. Johns Hopkins University Press. Bal-
timore, MD.

Pelseneer, P. 1914. Ethologie de quelques Odostomia et d'un Mons-
trillide parasite de 1'un d'eux. Bull Sci. France Be/g 7: 1-14.

Powell, E.N., M. E. White, E. A. Wilson, and S. M. Ray. 1987.
Change in host preference with age in the ectoparasite pyramidellid
snail Boonea impressa (Say). J Moll. Stud. 53: 285-IS6.

Rasmussen, E. 1944. Faunistic and biological note' marine inver-
tebrates. I. The eggs and larvae of Brachystomia isoides (Hani.),
Eulimella natidissima (Mont.). Rettisa iruncai, Brug.), and Em-
blelonia pallida (Alder and Hancock). (Gastr >oda marina). \'i-

252

R. COLLIN AND J. B. WISE

demk. Medd. Dan. Naturhist. f'oren. 107: 207-233.

Rasmussen, E. 1951. Faumsticand biological notes on marine inver-
tebrates II. The eggs and larvae of some Danish marine gastropods.
Vidensk. Medd. Dan. Nalurlii.it. Foren. 113: 201-249.

Robertson, R. 1967. The life history of Odostmnia bi.iutitralis. and
Odostomm spermatophores (Gastropoda: Pyramidellidae). Year
Book Am. Philos. SOL: 1966: 368-370.

Robertson, R. 1978. Spermatophores of six eastern North American
pyramidellid gastropods and their systematic significance (with the
new genus Boonea). Biol. Bull 155: 360-382.

Robertson, R. 1985. Four characters and the higher category system-
atics of gastropods. Am. Malacol Bull. Spec. Ed. 1: 1-22.

Strathmann, M. ¥. 1987. Reproduction and Development oj Marine
Invertebrates of the Northern Pacific Coast. University of Washing-
ton Press, Seattle. WA. 670 pp.

Thorson, G. 1946. Reproduction and larval development of Danish
marine bottom invertebrates with special reference to the plank-

tonic larvae in the Sound (Oresund). Medd. Dan. Fisk-Ha.vund.ers,
Ser. Plane/on 4: 1-523.

Thorson, G. 1950. Reproductive and larval ecology of marine bottom
invertebrates. Biol Rev 25: 1-45.

White, M. E., E. N. Powell, and C. L. Kitting. 1984. The ectopara-
sitic gastropod Boonea { = Odoslomia) impressa: population ecology
and the influence of parasitism on oyster growth rates. Mar. Ecol.
5: 283-299.

White, M. E., C. L. Kitting, and E. N. Powell. 1985. Aspects of re-
production, larval development, and morphometrics in the pyra-
midellid Boonea impressa (=Odostomia impressa) (Gastropoda:
Opisthobranchia). I 'eliger 28: 37-5 1 .

Wise, J. B. 1993. Anatomy and functional morphology of the feed-
ing structures of the ectoparasitic gastropod Boonea impressa (Pyr-
amidellidae). :\fuluci>lt>xiu35: 1 19-134.

Wise, J. B. 1996. Morphology and phylogenetic relationships of cer-
tain pyramidellid taxa ( Heterobranchia). Malacologia 37: 443-5 I 1 .

Reference: Biol. Bull. 192: 253-261. (April.

Bacterial Endosymbionts in the Gills of the Deep-Sea

Wood-Boring Bivalves Xylophaga atlantica and

Xylophaga washingtona

DANIEL L. DISTEL1 AND SUSAN J. ROBERTS2

1 Department of Biochemistry, Molecular Biology and Microbiology. University of Maine, Orono,
Maine 04469-5 7 35; and2 MB, DCS, NCI. NIH, 9000 Rockville Pike. Bethesda. Maryland 20892

Abstract. Bacterial endosymbionts found in gill tissues
in several bivalve families convert otherwise unavailable
energy sources (sulfide, methane, or cellulose) to forms
readily metabolized by their hosts. We investigated the
existence of such a symbiosis in two species of Xylophaga
(family Pholadidae). The genus Xylophaga includes op-
portunistic species that are the predominant colonizers
of wood at depths greater than 150 m. It has been hy-
pothesized that, like their shallow-water counterparts the
shipworms (family Teredinidae), species of Xylophaga
utilize wood for nutrition. Results from transmission
and scanning electron microscopy of A", atlantica and A.
washingtona clearly demonstrate the presence of endo-
symbionts that resemble the shipworm endosymbionts
both morphologically and in their anatomical location
within the gills. Xylophaga and the teredinids both have
a caecum packed with wood chips but lack the dense
populations of microorganisms associated with cellulose
digestion in termites or ruminants. These observations
suggest that Xylophaga has evolved a symbiotic solution
to wood digestion similar to that seen in shipworms.
Hence, the Xylophaga symbiosis suggests a mechanism
for the conversion of terrestrially derived cellulosic car-
bon from wood into animal biomass in the deep sea.

Introduction

In 1973 Popham and Dixon (Popham and Dickson.
1973) demonstrated the presence of endosymbiotic bac-
teria in the gills of wood-boring bivalves of the family
Teredinidae, commonly known as shipworms. The role
of these svmbionts, however, was not elucidated until a

Received 2 October 1996; accepted 3 January 1997.

decade later when it was shown that the symbiotic bacte-
ria, when cultivated /'/; vitro, both digest cellulose and fix
atmospheric nitrogen (Waterbury et ai, 1983). This
novel combination of symbiont metabolic activities is
now thought to be essential for the survival of teredinids
on a diet composed primarily of wood, a food source
that, although rich in carbon and energy, is deficient in
nitrogen and cannot be digested by most animals.

Whereas teredinids are the predominant wood-boring
bivalves in shallow waters (intertidal to 100 m), bivalves
of the subfamily Xylophagainae (family Pholadidae) fill
the same niche in the deep oceans, occurring primarily
at depths from 150 m to greater than 7000 m (Turner,
1972). Like teredinids, xylophagainids bore into woody
substrates, ingest the excavated wood particles, and store
them in a specialized caecum formed by an outpocketing
of the stomach (Purchon. 1941; Turner, 1973). The role
of wood in the diet of Xylophaga has not, however, been
determined experimentally. On the basis of morphologi-
cal and ecological arguments, it has been proposed that
Xylophagainids derive (1) significant (Purchon, 1941;
Turner, 1973), (2) little (Potts, 1923), or (3) no (Yonge,
1937, 1938; Yonge and Thompson, 1976) nutrition
from the ingestion of wood.

The utilization of wood by xylophagainids is a subject
of considerable ecological interest since wood is abun-
dant in many areas of the deep sea (Wolff, 1979) and the
opportunistic Xylophagainae are among the most com-
mon colonizers of wood at these depths (Turner, 1973).
Hence the deep-sea Xylophagainae could perform a role
analogous to that of termites in terrestrial habitats and
shipworms in shallow marine habitats by .inverting the
refractory cellulosic carbon deposited in \ ood and other
plant remains to a more readily availab: form (Turner,

253

254

D. L. DISTEL AND S.I. ROBERTS

1973). Both termites (Kane, 1997) and shipworms (Wa-
terbury el a/.. 1983) utilize cellulolytic and nitrogen-fix-
ing symbionts to survive on wood as the sole food source.
In this report we present evidence that bacterial endo-
symbionts are present in the gill tissue of two species of
Xvloplmga, and that these bacteria appear morphologi-
cally similar to the gill endosymbionts previously identi-
fied in shipworms.

Materials and Methods

Specimens of A', washingtona were collected in pine
boards submerged for 2-3 months in Monterey Bay
(depth 61 m, coordinates 36°39.8'N. 121°52.88'W;
courtesy of Dr. E. C. Haderlie, Naval Postgraduate
School) or in Scripps canyon off the San Diego coast
(depth 274 m, coordinates 32°3 1' N, 1 1 7° 16.5' W). Spec-
imens of A', atlantica were collected from oak boards ( 1"
X 2" X 2' lobster-trap skids) submerged for about 1 year
at 80-100 m depth 12 miles off the coast of SW Harbor,
Maine. Animals were kept alive in chilled seawater tanks
until they were removed from the wood and prepared for
microscopy (less than 2 weeks). Animals with an average
valve diameter of 3-4 mm were dissected and fixed for
1 - 1 .5 h in 3% glutaraldehyde buffered with 0. 1 M caco-
dylate/HCl (pH 7.3) and 0.4 M NaCl or 3% glutaralde-
hyde buffered with 0.1 M sodium phosphate (pH 7.4),
3% NaCl. and 4.5% sucrose as in Eckelbarger et al.
(1990). After fixation the specimens were post-fixed in

1% osmium tetroxide and dehydrated through a graded
ethanol series. Specimens for transmission electron mi-
croscopy (TEM) were then transferred to propylene ox-
ide and embedded in Epon/Araldite for sectioning. Spec-
imens for scanning electron microscopy (SEM) were
fractured under liquid nitrogen after ethanol dehydra-
tion and then were critical-point dried from CO: and
sputter coated with gold. A Phillips CM 10 transmission
electron microscope and an AMR 1000 scanning elec-
tron microscope were used to examine samples.

Both Xylophaga atlantica and Xylophaga washing-
tona were used for TEM of the gills and light microscopy
of the digestive tract. Similar results were observed with
both species. A", atlantica was used for SEM of the gills,
and A. washingtona was used for TEM of the digestive
tract.

Results
Gross morphology of the ctenidium

The gross morphology of the gills of Xylophaga has
been described in detail by Purchon (1941) and will be
briefly summarized here. The ctenidia (gills) of Xylopha-
gainae, like those of the Teredinidae, are formed by a
single (outer) demibranch on either side of the visceral
mass (Fig. 1 ). The marginal groove is absent, there is no
ciliary sorting mechanism, and the labial palps are
greatly reduced. These features are shared by most tere-

fr

eb

m

ab

1C

Figure 1 . Diagram of \yluphaxa in sagittal section shown in the burrow (left) and in transverse section
(right), aa. anterior adductor muscle; ab. afferent branchial vein; ar, abfrontal region of gill demibranch;
eb. efferent branchial vein; es, exhalent siphon; f, loot; IV. frontal region of gill demibranch; ic, infrabran-
chial chamber of mantle cavity; is. inhalant siphon; Id. left demibranch of gill; m, mantle; pa, posterior
adductor muscle; rd. right demibranch of gill; sc. suprabranchial chamber of mantle cavity; va, ventral
adductor muscle; v, outline of valves. Boxed area is shown in Figure 2C.

GILL ENDOSYMBIONTS OF XYLOI'll HiA

255

dinids but are divergent from the filter-feeding gill mor-
phology of other lamellibranchs (Purchon, 1941). The
ctenidia are connected to the visceral mass dorsally at
the efferent branchial vein and ventrally at the afferent
branchial vein, forming an arc that separates the mantle
cavity into suprabranchial and infrabranchial chambers.
Each filament is composed of two distinct regions: a nar-
row frontal region (fr) and a broad, flattened abfrontal
region (ar). As is typical of lamellibranch gills, the frontal
region is composed of frontal, laterofrontal, and lateral
ciliated cells: a central blood vessel; and supporting con-
nective tissue. The abfrontal region, however, is distinc-
tive, consisting of an extensively developed interlamellar
junction that forms a crescent-like sheet bridging the arc
of the filament (Fig. 1 ). The filaments are joined laterally
by interfilamentar cellular junctions in the frontal re-
gion. Whereas cellular interfilamentar junctions are ab-
sent in the abfrontal region, filaments appear to be con-
nected by lateral ciliary junctions in this region (Fig. 2E).

Identification and localization ofintracellular bacteria

in the ctenidium

In the Xylophagainae, as in other symbiont-bearing
bivalves, the frontal region of each gill filament is devoid
of symbionts. The most conspicuous cytoplasmic com-
ponents of the cells of the frontal region include mito-
chondria. Golgi. endoplasmic reticulum, ciliary struc-
tures, centrioles, and glycogen granules. Bacterial symbi-
onts are, however, abundant in the abfrontal region of
the ctenidium (Fig. 2D-F and Fig. 3). This region forms
a broad, flattened plate composed of two closely ap-
pressed single-layered sheets of epithelial cells surround-
ing a narrow blood sinus.

The epithelium of the abfrontal region consists of two
recognizable cell types here referred to as bacteriocytes
and intercalary cells in keeping with terminology in cur-
rent use for other bivalve symbioses (Fisher, 1 990: Fren-
kiel et a/., 1996; Gros et a/.. 1996). Bacterial endosymbi-
onts are absent from the intercalary cells but dominate
the cytoplasm of the bacteriocytes. The symbionts are
distributed in clusters throughout the bacteriocytes, with
mitochondria infrequently interspersed in the cytoplasm
between clusters. Large membrane-bound inclusions,
possibly lysosomes or lysosomal residual bodies, also oc-
cur in the bacteriocyte cytoplasm (Fig. 3). These inclu-
sions frequently display a biphasic appearance with a
granular low-density region and a slightly denser reticu-
late region that appears to consist of whorled mem-
branes. Some inclusions contain bodies suggestive of
partially degraded bacteria. Bacteriocytes and interca-
lary cells are uniformly distributed in the epithelium of
the abfrontal region with about equal frequency. The
apical surfaces of both cell types are densely covered with

microvilli at points that directly contact the external en-
vironment (Fig. 3C).

The bacteriocytes are typically spherical or cylindrical.
Their broad basal surfaces compose much of the internal
lining of the blood sinus. Intercalary cells, on the other
hand, are typically narrow at their basal ends, expanding
to a broad and irregular apical surface. Thin sheet-like
projections of the apical end of the intercalary cells ex-
tend over the apical surfaces of adjacent bacteriocytes,
apparently shielding most but not all of the surface of
the bacteriocytes from direct contact with the external
environment. The outer surface of the abfrontal epithe-
lium is elaborated into a series of larger rounded hum-
mocks (formed by the spherical outline of the bacterio-
cytes) and smaller papillae (formed by the irregular sur-
faces of intercalary cells) surrounded by many deep folds
and pits (Fig. 2F and Fig. 3A). The inner blood-facing
surface of this epithelium also contains numerous ima-
ginations (Fig. 3). These elaborations greatly increase the
surface areas that are exposed to blood internally and to
seawater externally in the symbiont-containing abfrontal
zone.

The endosymbionts are straight to gently curved rods
that display a cell-wall morphology typical of gram-neg-
ative bacteria and range from 0.4 to 0.7 //m in width and
up to 5.0 nm in length (Fig. 3). No conspicuous internal
structures, such as the sulfur granules seen in thiotrophic
symbionts or the stacked internal membranes seen in
methanotrophic symbionts of other bivalve families
(Fisher, 1990), are observed in the symbionts of A'y/c-
phaga.

The symbionts are not in direct contact with the cyto-
plasm but are contained within membrane-bound vesi-
cles ranging from 10 to 20 ^m in diameter. A single vesi-
cle may contain many bacteria. The orientation of sym-
bionts within vesicles is not random. Most symbionts are
oriented with their long axes perpendicular to the lateral
surface of the filament. Few symbionts are oriented with
their long axes parallel to the lateral surface and of those,
fewer still align with the frontal-abfrontal axis of the fil-
ament. This distribution of orientations is readily ob-
servable in both SEM and TEM images.

Extensive examination of TEM images failed to pro-
vide evidence of connections between the symbiont-con-
taining vesicles and the external environment. In all sec-
tions examined, the vesicular membrane appears dis-
tinct from and unconnected to the plasma membrane.
Similarly, examination of the exterior surfaces by SEM
revealed no evidence of the kind of ducts or openings
to the bacteria-containing vesicles that arc seen in the
symbiont-containing light organs of soim ininous fish
(Haygood. 1993). Although deep pits *d infoldings
were observed on the lateral surfaces ot e filaments in
SEM images. TEM images showed v . these are not

256

D. L. DISTEL AND S. J. ROBERTS

*•' mm

^-^^m ^v * '

"^Wi ^ V<V;

>; iK* ^ £M ^ -

I

,

L_''.^
micro-

^»--j « r tyv^^-rtrsjim*-'-, ^ ' .f^-a^f^

Figure 2. (A\) Xylophagu waxhint'tona with siphon fully extended. (B-F) Scanning electron ,,,,t,w-
graphs of ,V alanlica gill. ( B) Lateral surface of a gill filament near the posterior tip of the right demibranch.
(C) Frontal surface of the right demibranch. (D) Transverse fracture of right demibranch showing three gill

GILL ENDOSYMB1ONTS OF XYLOPHAGA

257

continuous with the symbiont-containing vesicles (Fig.
3C). Hence, at minimum, the bacterial symbionts are
separated from the external environment by the plasma
membrane, cytoplasm, and vesicular membrane of the
host.

H 'ood and flora of the digestive tract

Xylophaga has a digestive system similar to that de-
scribed for shipworms, including a large caecum that is
typically distended with wood shavings (Fig. 4). The di-
mensions of the shavings (width and height) match the
contours of the dentition on the shell, consistent with the
ingestion of wood excavated during burrowing. Wood
was the predominant component of the gut contents
identifiable at the level of light microscopy. The caecum
was the most densely packed space, while the stomach
contained dispersed wood fibers. Fibrous material was
found in proximity with the crystalline style and at low
density in the style sac. Examination of serial thick sec-
tions of the guts of several animals failed to reveal any-
conspicuous community of microorganisms resembling
those found in wood-eating insects. Electron microscopy
confirmed this observation and further indicated that
bacteria are absent from cells of the ciliated epithelium
lining the gut and occur only sparsely in the digestive
spaces (Fig. 4D-E).

Discussion

The discovery of bacterial endosymbiont populations
in a number of marine invertebrate species has been the
key to understanding the remarkable ability of these in-
vertebrates to survive on unusual nutrient sources. For
example, the identification of cellulolytic, nitrogen-fix-
ing bacteria in the gills of the wood-boring teredinid
clams (shipworms) pointed toward an explanation of
how these bivalves are able to thrive with wood as their
primary food source. This capability, which is quite rare
among higher animals, has led to the great success of the
Teredinidae as colonizers of wood in coastal marine en-
vironments. In the deep oceans, the dominant colonizers
of wood and other plant materials are the Xylophagainae
( family Pholadidae). Similarities in the gut anatomy and
wood-boring habits of the Teredinidae and the Xylopha-
gainae led us to examine whether species of Xylophaga

maintain similar symbiotic associations. In this report
we present morphological evidence for the existence of
bacterial endosymbionts in the gill tissue of two of these
deep-sea wood-boring bivalves — Xylophaga atlantica
and Xylophaga washingtona.

In many respects, the gill of Xylophaga closely resem-
bles the symbiont-containing gills of other bivalves in-
cluding the Lucinidae, Vesicomyidae, Modiolinae, and
Solemyidae (Distel and Felbeck, 1987; Fisher 1990;
Frenkiel et at., 1996; Gros et a/.. 1996). The gill of Xylo-
phaga is composed of two regions: a heavily ciliated,
symbiont-free frontal region that is like the typical lamel-
libranch gill structure, and an abfrontal region that ap-
pears to be specifically adapted to harbor symbiotic bac-
teria. The abfrontal region, an extension of the interfil-
amentar junction, is much broader than the frontal
region and composes the bulk of the mass of the gill. The
symbionts are completely surrounded by a vacuolar
membrane of host origin and lack contact with the exter-
nal environment. In addition to harboring symbionts,
the bacteriocytes contain large membranous structures
resembling lysosomes in various stages of development.
In some instances, these lysosome-like compartments
contain bodies that resemble partially degraded b'acteria,
which suggests that lysosomal digestion is a mechanism
for regulation of the bacterial population.

The symbiont-containing tissue in Xylophaga also re-
sembles that of the teredinids. Although teredinid gills
are highly modified, the symbionts are found in an ana-
tomically analogous region (Distel et al.. 1991). This re-
gion is referred to as the gland of Deshayes (Sigerfoos.
1908) in teredinids and corresponds to the interlamellar
junction of the fused right and left demibranchs (Turner,
1966; Distel eta/., 1991). Bacteriocytes in this tissue con-
tain symbionts that resemble the symbionts of Xylo-
phaga; both are gram-negative rods averaging about 0.5
ium in width and up to 5 ^m in length, and both lack
internal membranes or other conspicuous internal or ex-
ternal structures.

It is striking that xylophagainids and teredinids not
only harbor symbionts that are similar in appearance
and location, but that they also share similar adaptations
to their woody habitats. Both are obligatory wood borers
that excavate burrows by using their shells as rasps, ingest
the excavated wood shavings, and store the wood parti-

filaments (plane of fracture indicated by dashed lines in B and C). Abfrontal (ar) and frontal (fr) regions
indicated by side bar. (E) Detail from box 1 in D, showing deep invaginations (arrows), lateral ciliary tufts
(Ic), and numerous papillae decorating the lateral surfaces of the abfrontal region. (F) Detail from box 2 in
D. showing an intercalary cell (it) and portions of two bacteriocytes (be). Rod-shaped cells (arrows) within
bacteriocytes are symbionts. me. lumen of mantle cavity: bs. blood sinus: es, exhalent siphon; is, inhalant
siphon: v. valve. Scale bars: A. 0.5cm: B-C. 100 ^m; D. 50 urn: E. lO^m; F. l.Ojirn.

258

D. L. DISTEL AND S. J. ROBERTS

P:

^"rs ''•••'*?^-^r* --•::-,..
^? -*'•"*%&•&&>}''••'- ' '**'; M ' "

a"-

^wii E

'V'-

'•<*J

?-,j i r»v**^i

* : , I 4 ' '

. i -*%.

t ^Wr- . . • 1

i*

1

x M

, ... , •.- -- :vz~?.*g •'"-_
*<•: ',„.:• ,o-^Wlry^tv i

"v: '•' " - ..

W^

me

M

.* ,

••''/

i-2ai'J^fi^ JSli

. . ^ „

Transmission electron micrographs of a section through the abfrontal region of the gill of
\ i tiiluiiiicu. (A) Low-magnification view of a region comparable to that shown in 2E. Symbionts

.11 M :i on-dense rods. Note deep invaginations of the microvillar surface of gill (arrows). ( B) Detail

GILL ENDOSYMBIONTS OF XYLOPHAGA

259

cles in a large caecum prior to passing them through the
stomach and gut. Purchon ( 1 94 1 ) argued that several fea-
tures common to the xylophagainid and teredinid diges-
tive systems indicate a departure from reliance on filter
feeding and represent specific adaptations to the diges-
tion of wood. These include the lack of a marginal food
groove, limited ciliary sorting mechanisms in the cteni-
dia, reduced labial palps and crystalline style sac, and
presence of a wood-storing caecum with ciliated grooves
capable of providing a steady stream of wood particles to
the stomach.

It is now well established that wood is a primary con-
stituent of the teredinid diet (Gallager et ai, 1981). al-
though nutrients may also be obtained from suspension
feeding (Mann and Scott, 1985). In fact, at least one ship-
worm species has been shown to be capable of sustaining
normal growih and reproduction with wood as the sole
source of paniculate nutrients (Gallager et ai, 1981).
The shipworm symbiont has been cultivated in vitro and
shown to fix atmospheric nitrogen and secrete cellulo-
lytic, xylanolytic, and proteolytic enzymes (Greene and
Freer, 1986; Greene et ai, 1988; Greene, 1989, Greene
and De Wispeleare, pers. comm.). Although these sym-
biont activities are appropriate for a wood-based diet and
are thought to be critical for the shipworm's success in
colonizing woody substrates, no direct evidence exists
for their participation in the nutrition of the shipworm
hosts.

Since the Xylophaga symbiont has not yet been culti-
vated, the question of whether its metabolic capabilities
are similar to those of the shipworm symbiont is unre-
solved. Hence, it is possible that the two bacteria play
different roles in their respective symbioses. For exam-
ple, the bacterial symbionts in Xylophaga may be nitro-
gen fixing but not cellulolytic, or they may contribute
essential nutrients that are absent from wood. On the
other hand, phylogenetic analyses based on 16S rRNA
sequences indicate that the Xylophaga symbiont is the
closest relative of the shipworm isolates identified to date
(Distel and Roberts. 1996). That finding increases the
likelihood that these two symbionts play similar physio-
logical roles.

Although the presence of similar bacteria in the same
regions of the gills of the Teredinidae and the Xylopha-

gainae does not demonstrate a common function for the
two symbioses the additional features they have in
common — their wood-boring habits and parallel ana-
tomical modifications of the digestive tract — lend sup-
port to this hypothesis. To our knowledge, no animal
species has yet been demonstrated to utilize wood as a
primary nutrient source without the aid of symbiotic mi-
croorganisms; thus if wood is the primary dietary con-
stituent of Xylophaga, a nutritional role for its symbionts
is strongly implicated. If wood is not a primary food
source for the xylophagainids (as it is for teredinids), it is
difficult to explain the function of the caecum, account
for the fact that wood is the principal constituent of the
caecal and gut contents, or identify the diet of the xylo-
phagainids. The scarcity of alternative food sources in
the deep sea, as well as the notable paucity of bacteria,
phytoplankton, and other microorganisms in the gut of
Xylophaga, seems to preclude the possibilities that Xylo-
phaga subsists by filter feeding, by grazing on wood-as-
sociated microorganisms, or by utilizing an extracellular
symbiotic gut microfauna as do termites and ruminants.
Nonetheless, the possibility cannot be ruled out that A'y-
lophaga (or the teredinids) produces sufficiently active
endogenous cellulases to facilitate wood digestion, as has
been suggested for some termites (Breznak and Brune,
1994).

To determine whether the Xylophaga symbiosis is
cellulolytic. it is necessary to identify the activity of cel-
lulolytic or nitrogen-fixing enzymes in the symbionts.
demonstrate the depletion of cellulose in fecal material,
and analyze the nutritional utilization of carbon de-
rived from cellulose. These properties have been dem-
onstrated for the shipworm symbiosis, although it is still
not clear how the cellulolytic enzymes produced by bac-
terial endosymbionts in the gill might be transported to
the gut where wood digestion must occur. In the tere-
dinids, it has been proposed that a duct in the afferent
branchial vein connects the gills to the esophagus (Sar-
aswathy, 1971). Such a duct could serve as a conduit for
cellulolytic enzymes; however, the complete vessel was
not observed in all species examined, and its existence
has not been independently confirmed. Our results in-
dicate that the symbionts of Xylophaga (this study) and
those of teredinids (unpubl. obs.; Trytek and Allen

from lower right quadrant of A. showing a bacteriocyte and portions of two neighboring intercalary cells.
(C) Detail from upper left quadrant of A, showing symbionts near the base of a deep lateral invagination.
Note that membranes surrounding symbionts are distinct from the microvillar surface of the bacteriocyte
plasma membrane. Inset in C shows high magnification of a symbiont cell from \ washingtona. Large
arrow indicates host-derived membrane of symbiont-containing vesicle. Small arrows indicate double-lay-
ered gram-negative cell envelope of the symbiont cell. bs. blood sinus; bn, bacteriocyte nucleus; in, interca-
lary cell nucleus; ly. lysosome-like granules; me, lumen of mantle cavity; s, symbionts. Scale bars: A. -'
Mm, B-C. 1.0 firm inset, O.I ^m.

260

D. L. DISTEL AND S. J. ROBERTS

«

\

'.!

cc

1, ' ' „ V' "

> -• *: ; . •

eg

fc <v

.\ .-_• . . _

N.

**^ f ' * • X ^^'

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•

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.' '<

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%

i

/ v*
* V

;, \s

k^>

X

*

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V*.

;• I

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»

f

f

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l\

V

* *

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^

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'• \

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« i

L

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••

cc

-'•

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UT

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i*^r<- •

£••£•"
'"^•.

Figure 4. Gut content of .\ylophiigu washingtona (A) Light micrographs ot caecal contents showing

ciliated groove (eg) that channels wood shavings into the stomach. (B) Detail of caecal contents from A. Note

predominance of wood shaungs and absence ot algae, fungi, and protozoans. (C-E) Transmission electron

micrographs. (C) Ciliated epithelial cells in the region of the caecal wall (ciliated groove) shown in A. The

cilia in the lower left face into the lumen of the caecum. No bacteria were observed within the cells of the gut

epithelium. (D)One of the rare bacteria seen among the caecal contents. The dark, electron-dense pentagons

>od shavings. (E) Cluster of bacteria observed adjacent to the gastric shield of the stomach, b, bacteria;

cc, coelomic cavity: cm, caecum; w, wood. Scale bars: A, 250 ^m; B. 150 ^m;C-E. 1.0 /urn.

(19i truly intracellular. Consequently, the plau- an intracellular bacterial population are ultimately

sibilit\ symbiotic mechanism depends on the transported to the lumen of the gut to participate in cel-

i of a pathway whereby enzymes from lulose degradation.

GILL ENDOSYMBIONTS OF XYl.OPHAGA

261

Acknowledgments

We thank Dr. Ruth D. Turner and Dr. George Somero
for helpful discussion and encouragement; Dr. Kevin
Eckelbarger, Dr. Nick Holland, and Kelly Edwards for
expertise in electron microscopy of marine invertebrates;
Wendy Morrill for technical assistance; and Jack Merrill,
Ron McConnaughey, and Dr. E. C. Haderlie for aid in
collecting specimens. This work was supported by NSF
EPSCoR Grant # EHR 91-08766, awards from the Reg-
ular Faculty Research and Summer Faculty Research
Funds of the University of Maine, Orono, and a research
award from the Scripps Industrial Associates.

Literature Cited

Breznak, J. A., and A. Brune. 1994. Role of microorganisms in the
digestion of lignocellulose by termites. Annii. Rev. Entomol. 39:
453-487.

Distel, D. L., and H. Felbeck. 1987. Endosymbiosis in the lucinid
clams Lueinoma aequi:onala, Lueinoma anniilaia and Liicinu
/toriWana/areexamination of the functional morphology of the gills
as bacteria-bearing organs. Mar. Bid. 96: 79-86.

Distel, D. L., and S. J. Roberts. 1996. Bacteria] endosymbionts in the
gills of the deep-sea, wood-boring pholad clams, V.r/r iphaga allanlica
and Xylophuga washinglona. P. 350 in Abstracts: American Society
for Microbiology 96th General Meeting. New Orleans, LA. Ameri-
can Society for Microbiology, 1325 Massachusetts Ave., N.W.,
Washington, DC.

Distel, D. L., E. F. Delong, and J. B. \\aterbury. 1991. Phylogenetic
characterization and in situ localization of the bacterial symbiont
of shipworms (Teredinidae: Bivalvia) by using 1 6S rRNA sequence
analysis and oligonucleotide probe hybridization. Appl. Environ
Mierobid. 57:2376-2382.

Eckelbarger, K., B. Bieler, and P. Mikkelsen. 1990. Ultrastructure of
sperm development and mature sperm morphology in three species
of commensal bivalves (Mollusca: Galeommatoidea). ./. Morphol.
205:63-75.

Fisher, C. R. 1990. Chemoautotrophic and methanotrophic symbio-
sesin marine invertebrates. Rev Aijiial Sci. 2: 399-436.

Frenkiel, L., O. Gros, and M. Moueza. 1996. Gill structure in Liietmi
peel i mi I a (Bivalvia:Lucinidae) with reference to hemoglobin in bi-
valves with symbiotic sulphur-oxidizing bacteria. Mar. Bid 125:
51 1-524.

Gallager, S. M., R. D. Turner, and C. J. Berg. 1981 . Physiological as-
pects of wood consumption, growth, and reproduction in the ship-
worm Lyrodus pedicellatits Quatrefages. / Exp. Mar. Bin/. Eeol.
52:63-77.

Greene, R. V. 1989. A novel, symbiotic bacterium isolated from ma-
rine shipworm secretes proteolytie activity. Citrr Microbiol. 19:
353-356.

Greene, R. V., and S. N. Freer. 1986. Growth characteristics of a

novel nitrogen-fixing cellulolytic bacterium. Appl. Environ. Miero-
hid. 52(Nov.): 982-986.

Greene, R. V., H. L. Griffin, and S. N. Freer. 1988. Purification and
characterization of an extracellular endoglucanase from the marine
shipworm bacterium. Arch Biochem Biopliys. 1(15): 334-341 .

Gros, O., L. Frenkiel, and M. Moueza. 1996. Gill ultrastructure and
symbiotic bacteria in the tropical lucinid. Linga pensylvainea
(Linne). Symbiosis 20: 259-280.

Haygood, M. G. 1993. Light organ symbioses in fishes. Crit. Rev. Mi-
crobiol. 19(4): 191-216.

Kane, M. D. 1997. Microbial Fermentation in Insect Guts. Chap. 8,
pp. 231-265 in Gastrointestinal I-ermentation and Ecosystems,
Vol. I.R.I. Mackie and B. A. White, eds. Chapman and Hall, New
York.

Mann, R., and M. G. Scott. 1985. Growth, morphometry, and bio-
chemical composition of the wood boring molluscs Teredo navalis
L., Bankia goitlili (Bartsch), and Nototerendo knoxi (Bartsch). J
Exp. Mar. Bid. Ecol. 85: 229-25 I .

Popham, J. D., and M. R. Dickson. 1973. Bacterial associations in
the teredo Bankia uustralis (Lamellibranchia, Mollusca). Mar.
Bio/. 19:338-340.

Potts, F. A. 1923. The structure and function of the liver of Teredo.
the shipworm. Proc. Cainb. Phil Sac \: 1-17.

Purchon, R. D. 1 94 1 . On the biology and relationships of the lamelli-
branch Xylophaga dorsalis (Turton). J. Mar. Biol. Assoc. U.K.
25(1): 1-39.

Saraswathy, M. 1971. Observations on the structure of the ship-
worms, Nausitora hedleyi. Teredo furcijera and Teredora pricesae
(Bivalvia: Teredinidae). Trans. Roy. Sue. Edinh. 68(14): 508-562.

Sigerfoos, C. P. 1908. Natural history, organization and late develop-
ment of the Teredinidae or shipworms. Bull. Bur. fish. 37: 191 -
231.

Trytek. R. E., and W. V. Allen. 1980. Synthesis of essential amino
acids by bacterial symbionts in the gills of the shipworm Bankia
setacea (Tryon). Comp. Biochem. Physiol. 67A: 419-427.

Turner, R. D. 1966. A Survey and Illustrated Catalogue of the Tere-
dinidae (Mollusca: Bivalvia). The Museum of Comparative Zool-
ogy, Harvard University. Cambridge. MA.

Turner, R. D. 1972. A new genus of deep water wood-bonng bivalve
(Mollusca, Pholadidae. Xylophagainae). Basleria 36(2-5): 98-
104.

Turner, R. D. 1973. Wood-boring bivalves, opportunistic species in
the deep sea. Seimce 180: 1 377- 1 379.

Waterbury, J. B., C. B. Calloway, and R. D. Turner. 1983. A cellulo-

lytic-nitrogen fixing bacterium cultured from the Gland of Des-

hayes in shipworms (Bivalvia: Teredinidae). Science 221: 1401-

1403.

Wolff, T. 1979. Macrofaunal utilization of plant remains in the deep

sea. Sar.siu 1(2): 117-136.
Yonge, C. M. 1937. Evolution and adaptation in the digestive system

ofmetazoa. Bid. Rev 12: 87- 1 15.
Yonge, C. M. 1938. Recent work on the digestion of cellulose and

chitin by invertebrates. Sei. Prog 32: 638-47.

Yonge, C. M., and T. E. Thompson. 1976. Borers in rock and timber.
Chap. 16, pp. 205 -219 in Living Marine Molluscs. William Collins
Sons. London.

Reference: Biol. Bull. 192: 262-278. (April, 1997)

Decline in Pelagic Cephalopod Metabolism With

Habitat Depth Reflects Differences

in Locomotory Efficiency

BRAD A. SEIBEL, ERIK V. THUESEN1, JAMES J. CHILDRESS,
AND LAURA A. GORODEZKY2

Oceanic Biology Group, Marine Science Institute, University of California.
Santa Barbara. California V3106

Abstract. The metabolic rates of 33 species of pelagic
cephalopods from California and Hawaii were measured
and correlated with minimum depth of occurrence.
Mean metabolic rates ranged from 0.07 ^mol O: g~' h~'
for the deep-living vampire squid, I 'ampyroteuthis infer-
nalis, to 8.79 /imol O2 g ' h ' for Gonatus onyx, a verti-
cally migrating squid. An individual of I', infernalis,
which lives within the oxygen minimum layer off Cali-
fornia, had the lowest mass-specific metabolic rate ever
measured for a cephalopod (0.02 ^molO:g"' h~', 1050g
wet weight). For species collected in sufficient quantity
and size range, metabolism was related to body size. Crit-
ical partial pressures of oxygen (Pc) were determined for
Hawaiian and Californian cephalopods. Pt values for
Hawaiian animals were considerably higher than for
those taken off California, a trend that corresponds to the
higher levels of environmental oxygen in the Hawaiian
waters. Buffering capacity (ft) of mantle muscle, assayed
in eight cephalopod species, was used to estimate the ca-
pacity for glycolytic energy production. Mean ft ranged
from 1.43 slykes for a bathypelagic octopod, Japetella
heathi, to 77.08 slykes for an epipelagic squid, Stlieno-
teuthis oualaniensis. Significant declines with increasing
depth of occurrence were observed for both metabolism
and ft. The decline in metabolic parameters with depth

Received 31 January 1996; accepted 18 November 1996.

' Present address: The Fvergreen State College. Olympia. WA 98505.

- Present address: Channel Islands National Marine Sanctuary, 1 13
Harbor Way, Santa Barbara, CA 93 109-23 1 .

Abbreviations: ft, buffering capacity; MDO. minimum depth of oc-
currence; MDdO, minimum depth of daytime occurrence: />c>2, partial
pressure of dissolved oxygen; Pf, critical oxygen partial pressure.

is interpreted as a decreased reliance on locomotory abil-
ities for predator/prey interactions in the light-limited
deep sea. The decline in metabolism with depth observed
for pelagic cephalopods was significantly steeper than
that previously observed for either pelagic fishes or crus-
taceans. We suggest that since strong locomotory abili-
ties are not a priority in the deep sea, deeper-living ceph-
alopods may rely more heavily on means of locomotion
that are more efficient than jet propulsion via mantle
contractions — means such as fin swimming or medusoid
swimming utilizing the arms and extensive webbing
present in many deep-living species. The greater effi-
ciency of deeper-living cephalopods may be responsible
for the observation that the decline in metabolic rates
with depth is more pronounced for pelagic cephalopods
than for fishes or crustaceans.

Introduction

Cephalopods are morphologically diverse, visually ori-
enting predatory molluscs. The five groups of extant
cephalopods — squids (Teuthoidea), cuttlefishes (Sepi-
oidea), octopuses (Octopoda), vampire squids (Vampyr-
omorpha), and the chambered Nautilus (Nautiloidea) —
are easily distinguished by morphological characteristics,
among which are locomotory adaptations to their habi-
tat (Roper et al.. 1984). Locomotory differences are also
reflected in an animal's physiological properties. Previ-
ous physiological studies on cephalopods have primarily
focused on the more commercially important squids,
and on the shallow-water octopuses and cuttlefishes
(Grieshaber and Ga'de, 1976; Baldwin. 1982; O'Dor,
1982; O'Dor and Webber, 1986: Portner et al., 1993).

262

METABOLISM OF PELAGIC CEPHALOPODS

263

Some of these animals are among the most metabolically
active poikilotherms known (O'Dor and Shadwick.
1989), which stems in part from the inherent inefficiency
of jet propulsion. Other studies have investigated the me-
tabolism of Nautilus spp. and its ability to withstand low
levels of oxygen (O'Dor el ai. 1990; Wells el al.. 1992;
Boutilier el al.. 1996). Very little is known, however,
about the physiological adaptations of cephalopods to
the deep sea. The current study is a comprehensive com-
parison of the metabolic rates of midwater cephalopods
living at depths down to 2 km off California and Hawaii.

Many studies have demonstrated a decline in the met-
abolic rates of pelagic fishes and crustaceans with in-
creasing habitat depth (Childress, 1975; 1995; Torres el
al.. 1979; Torres and Somero, 1988;Ikeda, 1988;Cowles
el nL 1991). Additional studies found no clear relation-
ship between metabolism and minimum depth of occur-
rence in chaetognaths and medusae (Thuesen and Chil-
dress, 1993a, 1994). Bathypelagic representatives of
these groups have metabolic rates comparable to those of
their epipelagic counterparts when measured at the same
temperature.

Childress and Mickel (1985) put forth the visual in-
teractions hypothesis to explain the decline in metabolic
rates of fish and crustaceans with increasing minimum
depth of occurrence. They observed that reduced meta-
bolic rates reflect decreased locomotory abilities in many
deeper-living groups. They hypothesized that this de-
crease resulted from relaxed selection for strong locomo-
tory abilities for visually cued predator/prey interactions
in the low ambient light levels of the deep sea. Although
deep-living fishes and crustaceans do possess well-devel-
oped eyes, these are apparently used primarily for in-
teractions via bioluminescence (Marshall, 1979). which
is a relatively weak, often transient light source. Under
these conditions, interactions are likely to take place over
short distances that do not require strong locomotory
abilities (Marshall, 1979; Herring et al.. 1994; Fleisher
and Case, 1995). The visual interactions hypothesis is
supported by the presence of a decline in metabolic rates
of visually orienting pelagic groups (fishes and crusta-
ceans), and by the absence of a decline in non-visually
orienting gelatinous organisms (e.g.. chaetognaths and
medusae).

Cephalopods, like fishes and crustaceans, are highly vi-
sual predators that occupy a range of depths and habitats.
As such, they are an obvious choice for further testing of
the visual interactions hypothesis. In the present study,
the oxygen consumption rates of 33 species of pelagic
cephalopods were measured and correlated with mini-
mum depth of occurrence. Because of differences in local
productivity and ambient oxygen levels between the two
regions, comparisons between Hawaiian and Californian
animals are used to determine possible effects of oxygen

and food availability on metabolic rates. Buffering ca-
pacity was assayed to indicate an animal's capacity for
anaerobic work (Castellini and Somero, 1981).

Materials and Methods

Cephalopods were captured on nine cruises aboard the
R/V Point Sur and R/V New Horizon between Septem-
ber 1992 and September 1996. Sampling was done pri-
marily in an area 160 km west of Point Conception, Cal-
ifornia (34° 37'N, 1 22° 42'W to 34° 30'N, 1 23° 20'W) and
off Oahu, Hawaii (21° 20'N, 158° 20'W to 21° 35'N, 158°
35'W). Animals were collected using an opening/closing
Mother Tucker trawl with a 10-rrr mouth. The net was
equipped with a 30-1 thermally protecting cod end that
reduced mechanical damage and heat shock to animals
during recovery (Childress et ai. 1978). Ship speed was
kept very low (0.5-1 kn) to decrease turbulence and
abrasion in the net and to reduce the number of animals
collected in the cod end. Upon reaching the surface,
specimens were immediately transferred to 5°C seawater
and allowed to recover for several hours. Only animals
in the best physical condition were selected for physio-
logical study. Animals were identified to species with the
aid of several sources (Roper et al.. 1984; Sweeney et al.,
1992; Young, 1972; Hochberg, pers. comm.; Young,
pers. comm.). Voucher specimens were preserved for
verification of identifications.

Routine oxygen consumption rates were determined
on board ship by allowing individual specimens to de-
plete the available oxygen in a sealed, water-jacketed
chamber filled with filtered seawater containing
50mgl~' streptomycin (Childress, 197 la). All experi-
ments were carried out at atmospheric pressure because
hydrostatic pressure has been shown to have negligible
effects on the metabolism of a mesopelagic squid, I list i-
oteuthis heteropsis ( Belman, 1978), and on several meso-
and bathypelagic gelatinous zooplankters (Childress and
Thuesen, 1993). Chambers were kept in darkness and, in
most cases, the temperature was maintained at 5°C by
means of a refrigerated water bath. Rates for individuals
of some species were measured at either 2°, 3.5°, 10°, or
1 5°C and corrected to 5°C either using measured Qw val-
ues or according to the methods outlined in Childress et
al. ( 1990). The rate of change of the oxygen partial pres-
sure within the respirometer was monitored using a
Clark-type oxygen electrode (Mickel et al.. 1983) cali-
brated to air- and nitrogen-saturated seawater before and
after each experiment. A magnetic stir pump mixed the
water in the respirometry chamber and maintained wa-
ter flow past the electrode without damage to the ani-
mals. Individual respiration experiments lasted from 2
to 24 h. Oxygen concentrations were averaged over 2-
min intervals and recorded using a coir outer-based data

264

B. A. SEIBEL ET AL

acquisition system. The mean rates of oxygen consump-
tion between partial pressures of 30 and 70 mm Hg were
used for comparisons because this allowed a period for
the animals to calm down after being introduced to the
chamber and <• Jed before oxygen concentrations be-
came lim \ gen consumption rates of the smallest
specimens were measured using glass syringes (3-50 ml)
as miniature respiration chambers (Thuesen and Chil-
dress, 1993a, b) maintained in darkened water baths.
Water samples were withdrawn periodically from the in-
cubation syringe through a three-way valve using a gas-
tight syringe, and the new incubation volume was noted.
The oxygen content of the water sample was then ana-
lyzed using a gas chromatograph.

Oxygen consumption was calculated as micromoles of
oxygen consumed per gram wet weight per hour. At sea,
immediately following respiration experiments, animals
were weighed using a motion-compensated shipboard
precision balance system (Childress and Mickel, 1980).
For additional comparisons, wet-weight-specific rates of
oxygen consumption were corrected by covariance to
10 g by using measured scaling coefficients or an as-
sumed scaling coefficient of -0.20 (Childress el til..
1990). The value of 10 g was chosen because it was the
approximate modal weight of the animals measured.
Both corrected and unsealed values for mean rates of ox-
ygen consumption are presented in Table I.

Mean rates of oxygen consumption were regressed
against habitat depth. Because animals live at a range of
depths, minimum depth of occurrence (MDO) was
taken as the primary description of habitat depth. MDO
is denned as that depth below which 90% of the individ-
uals of a given species are captured in a given region
(Childress, 1975). Ten meters was taken as the MDO for
animals living at that depth or shallower to avoid distor-
tions in regressions of In-transformed data. MDO refers
to the adult distribution except for species of the genus
Leachia, as discussed later. For consistency with previ-
ous studies, animals that undergo a strong diel vertical
migration are considered at their shallowest depth
whether day or night. For comparison, regressions
against minimum depth of daytime occurrence (MDdO)
are also plotted. Both MDO and MDdO are listed in Ta-
ble I. Estimates of cephalopod depth were based on col-
lections from a variety of studies as well as on personal
observations and communications (Pickford, 1946:
Roper and Young, 1975; Young, 1978; Lu and Roper,
1979; Roper el al.. 1984; Sweeney el al., 1992; James
Hunt, MBARI, pers. comm.).

Regulation of oxygen consumption was measured by
plotting specific rates of oxygen consumption versus par-
tial pressures of oxygen. The critical partial pressure (Pc)
was taken as the point at which oxygen consumption was
no longer maintained independent of oxygen concentra-

tion. The P, was determined by calculating regression
lines for the two distinct parts of the relationship between
oxygen consumption and Po2< the regulated (higher Po2)
segment and the highly sloped (low Po2) segment. The Pc
was designated as the intersection of these two lines.

In vitro buffering capacity (0) of mantle muscle, due
to non-bicarbonate buffering compounds, was assayed
following the methods ofCastellini and Somero (1981).
0 is defined as the micromoles of base needed to change
the pH of the homogenate by 1 pH unit per gram wet
weight of muscle tissue. A unit of 0 is termed a "slyke."
Although many cephalopod species use fins for sustained
aerobic swimming, most cephalopods rely on mantle
musculature for jet propulsion during burst escape re-
sponses (Baldwin, 1982). For this reason, and because it
usually represents the largest percentage of body mass,
mantle tissue was chosen for measurements of buffering
capacity. Muscle tissue was homogenized at a dilution
of 1:20 (weight:volume) in normal saline (0.9% NaCl).
Homogenates were equilibrated to 20°C and stirred con-
tinuously during experiments. Homogenates were ti-
trated from pH 6 to 7 using 0.2 A' NaOH (less concen-
trated NaOH was used for animals with extremely low
buffering capacities to get a sufficient number of points
in the titration curve). If the homogenate had an initial
pH greater than 6.0, it was acidified by the addition of
HC1 before the titration. As observed by Castellini and
Somero (1981), more weakly buffered muscles yielded
curves that tailed upward at pH values near 7.0 and
above. In these cases, the linear portion of the curve (usu-
ally between pH 6.0 and 6.5) was used for calculating fl.
An Orion digital research ionalyzer (model 701 A) was
used with a glass pH electrode for monitoring pH
changes. A microburet was used to add the NaOH solu-
tion.

All data analyses were performed with Statview II or
SuperANOVA statistics programs (Abacus Concepts,
Inc., Berkeley, CA). Simple linear regressions, / tests, and
analysis of covariance ( ANCOVA) were used. Mean val-
ues given are followed by the standard error. All regres-
sions were carried out on In-transformed data to linear-
ize the data and maintain consistency with previous
studies. The figures, however, are semilog plots. Confi-
dence intervals for regression exponents are at the 95%<
level, P values for regression coefficients are F tests. AN-
COVA was used to test whether the slopes and elevations
of the various relationships were significantly different
from zero and from each other. Regression slopes were
declared significant when their slopes differed from zero
at the 5% confidence level.

Results

The metabolic rates of 33 species of pelagic cephalo-
pods from four orders are presented in Table I. An indi-

METABOLISM OF PELAGIC CEPH.ALOPODS

265

10-,

o

c
o
O

Q)
O5
>.
X

O

_
o

p
b

0.1,

'260' '460' 'eio'

's6o'

1000

Depth of Occurrence (m)

Figure 1. Mean oxygen consumption rates (y = /tmol O? g 'h ')of
23 species of pelagic cephalopods as a function of minimum depth of
occurrence (A = meters). There is a significant decline in metabolism
with minimum depth of occurrence for California (•) species (r =
707.7.Y ' :5 ' ° •", P = 0.001: R- = 0.68) and Hawaiian (D) species (;• =
264.0.Y"1-05-0-": P = 0.001: R- = 0.87). There is also a significant de-
cline in metabolism with minimum depth of daytime occurrence for
both Calilbrnian ( • • -)species(r= 1.54*10s.v 107±- ": P = 0.01: R2 =
0.43) and Hawaiian (....) species ( v =1.1 2* 10V2 24 = ' 65; P = 0.002:
R: = 0.62). Results of ANCOV.A show that there is not a significant
difference in the slopes or magnitudes of the regressions of Californian
and Hawaiian animals at either MDOor MDdO(P> 0.4).

vidual of Vampyroteuthis infernalis. which lives within
the oxygen minimum layer between 600 and 800 m, had
the lowest mass-specific metabolic rate ever measured
for a cephalopod (0.02 ^mol O: g~' h"1, 1050g wet
weight), r. infernalis had the lowest mean rate as well
(0.07 ± 0.03 jumol O2 g~ ' h~ ' ; « = 1 5, 223.4 g mean wet
weight). Gonatus onyx, a shallow-living vertical migra-
tor, had a mean metabolic rate of 8.79 ^mol O: g ' h '.
the highest rate measured in this study. Rates corrected
to 10 g of wet weight ranged from 0.06 ^mol O: g~' h~'
for I '. infernalis measured off Hawaii, to 6.55 ^mol O:
g"1 h"1 for Gonatus onyx. The mean rate presented here
for Histioleuthis heteropsis, 1 .02 ± 0. 1 5 /umol O: g ' h '
(0.87 /umol O:, corrected to 4.25 g for comparison with
published values), is comparable to the value of
0.83 jumol O: g ' h ', 4.25 g mean wet weight (Belman,
1978), the only other measurement of this kind available
for a midwater cephalopod. Most physiological studies
of cephalopods have been done on fast-swimming epi-
pelagic squids that are capable of avoiding midwater
trawls. DeMont and O'Dor ( 1984) reported a metabolic
rate of 14 ^mol O2 g"1 h ' for Illex illecebrosus at rest
and 56 jimol O;g~' h"1 at 100% activity (100-g animal,
13°C measurement temperature). These rates are similar
to those observed for Loligo forbesi by Boucher-Rodoni
and Mangold (1989).

Even with the exclusion of the faster epipelagic squids,
a significant decline in mean metabolic rate (r) with
MDO (A) was observed for both Californian and Hawai-
ian pelagic cephalopods (Fig. 1). There was no significant
difference in either the slope (ANCOVA; P = 0.47) or the
elevation (ANCOVA; P = 0.98) of the regression lines
between Californian (r = 707.7.V'1 --5±(I-5S; P = 0.001; R:
= 0.68) and Hawaiian (v = 264.0^' 05±055; P = 0.001;
R2 = 0.87) species. A significant decline in metabolic rate
(v) with minimum depth of daytime occurrence
(MDdO) was also observed for both Californian and Ha-
waiian pelagic cephalopods (Fig. 1). Because of the ab-
sence of vertical migration among the deepest living spe-
cies, estimates of daytime depth (x) result in a signifi-
cantly steeper slope (ANCOVA; P = 0.002) for both
Californian (r = 1.54*10V307 ± : "; P = 0.012; R2
0.43) and Hawaiian (.r = 1.12*10V224± ' 65; P = 0.002;
R2 = 0.62) regressions.

Normalization of metabolic rates to 10 g wet weight
did not have a significant effect on the slopes or eleva-
tions of Hawaiian or Californian regressions against
MDO or MDdO. The regression of cephalopod metabo-
lism corrected to 10 g against MDO (Fig. 2) had a sig-
nificantly steeper slope than the slopes for both fishes
(ANCOVA; P = 0.004) and crustaceans (ANCOVA: P =
0.001) (data from Childress, 1975; Torres et al.. 1979).
This relative steepness is driven both by higher metabolic
rates among shallower animals (cephalopods living at
100m have metabolic rates comparable to fishes and
crustaceans living at the surface) and by lower metabolic
rates among some deep-living cephalopods. It is interest-
ing that the squids (order Teuthoidea) living below
300 m had significantly higher metabolic rates than
other cephalopods (orders Octopoda and Vampyromor-
pha) (unpaired t test. P = 0.000 1 ). The metabolic rates of
teuthoids (r = 1 17.9.Y ~"sil±" [S; P = 0.0001; R2 = 0.66)
and octopods with I 'ampyroteuthis infernalis (y =
16.8.V"0-74*"-12; P = 0.0007; R2 = 0.87) alone still decline
with depth with a significantly steeper slope than either
fishes or crustaceans.

Several specimens were excluded from the above anal-
yses and are plotted separately (Fig. 3) because small
numbers of captures and various life-history characteris-
tics (i.e., ontogenetic vertical migration, or pelagic stages
of otherwise benthic animals) make depth distributions
and metabolic data difficult to interpret. Inclusion of
these species into the regression using roughly estimated
MDOs does not significantly alter the slope or elevation
of the regression of cephalopod metabolism with mini-
mum depth of occurrence. OcythoetuberculatasndAm-
p/iitretus pelagicus (both pelagic octopods). planktonic
juveniles of the benthic octopus Octopus nibescens, and
seven species of the family Cranchiidiv: (Teuthoidea)
were among those excluded.

266

B. A. SEIBEL ET AL

Table I

Metabolic rales and weigh/ and depth data for 33 species oj pelagic cephalopodsfrom California and Hawaii

Depth (m)*

Wet weight (g)

MOi (^mol Oi g ' wet wt

h ')t

Species

MDO

MDdO

n

rc

Range

Mean

Range

Mean ± SE

Corrected

California

Order Teuthoidea

GonalM onyx

100

400

1

5

—

2.30

—

8.79

6.55

Gonaiuspyros

100

400

5

5

2.17-31.28

8.58

3.15-7.60

4.38 ±0.84

3.41

Abrahiipxis lei is

50

300

1

5

—

0.99

—

3.44

2.16

1

10

—

0.36

—

6.70

Oclopoleiillns delelron

100

300

1

5

—

8.19

—

1.28

1.22

Histioteuthis heleropsis

150

500

17

5

0.23-36.98

9.99

0.45-2.98

1.02±0.15

0.73

3

10

0.23-2.85

1.38

1.32-2.5

1.74 ±0.38

Histioteuthis lioylei

150

500

1

5

—

8.51

—

1.13

1.09

Chiroteuthis calyx

300

500

II

5

5.87-89.02

38.88

0.15-0.85

0.47 ± 0.07

0.67

I 'albyteuthis oligobessa

900

900

1

2

—

25.40

—

0.55

0.66

Family Cranchiidae

Galiteuthis phyllura

300

500

1

5

—

5.19

—

0.61

0.54

Cranclua scabra

10

10

I

5

—

35.39

—

0.29

0.37

Leachia dislocata

10

300

1

5

—

3.27

—

0.70

0.55

llelicocranclua pjejleri

300

300

6

5

0.15-3.60

0.88

0.4 1 - 1 .94

0.97 ±0.22

0.59

Order Octopoda

Ocythoe tubemilata

10

10

1

5

—

1.21

—

4.17

2.73

Japelella licalhi

600

600

1 1

s

0.84-162.5

35.19

0.03-0.28

0.18 ±0.03

0.16

1

10

—

6.32

—

0.48

Octopus rnbescens^

10

2

5

0.04, 0.08

0.06

7.43-7.53

7.48 ± 0.05

2.78

3

10

0.07-0.12

0.10

10.19-10.90

10.43 ±0.23

Order Vamp\ romorpha

I 'ampyroteuthis infernalis

600

600

15

5

0.41-1050

223.4

0.025-0.41

0.07 ±0.03

0.09

1

10

—

4.74

—

0.31

Hawaii

Order Sepioidea

Heieroteutlus huH-aiiensis

1 10

250

1

_s

—

5.88

—

4.81

4.31

Order Teuthoidea

Abraliopsis pacificus

50

300

5

s

0.40-1.90

1.22

1.86-3.94

2. 39 ±0.39

1.63

2

15

1.89-2.91

2.40

4.96-5.90

5.43

Enoploieuthis higginsi

50

300

1

5

—

6.47

—

5.59

5.12

Plerygioieuthis microlampas

50

300

1

5

—

0. 1 3

—

6.46

2.71

Oclopoteulhis nielseni

100

300

1

2

—

0. 1 3

—

1.20

0.61

Histioteuthis hoylei

150

500

i

5

0.40, 1.27

0.84

1. 66-1. 77

1.71

1 .03

Chiroteuthis imperalor

300

500

1

5

—

14.94

—

0.70

0.76

Joubiniteuthis portieri

500

500

3

5

36.00-48.99

41.85

0.20-0.39

0.31 ±0.06

0.41

Mastigoieuthisfamelica

375

675

1

5

—

4.06

—

0.70

0.59

Clenopleryx siculus

50

625

1

5

—

4.24

—

2.81

2.37

2

15

1.28.2.11

1.70

5.07-5.41

5.24

Bathyteuthis ahyx.ticolu

800

800

2

5

1.53,37.7

19.6

0.56-0.61

0.59

0.59

Family Cranchiidae

Leachia pacifica

50

50

3

3.5

0.90-2.12

1.52

0.24-1.83

0.81 ±0.51

0.56

Liocranchia raldivia

500

500

12

S

0.17-21.28

2.92

0.27-1.61

0.56 + 0.11

0.27

1 1

15

0.02-19.0

2.25

1.47-3.47

2.47 ±0.23

Megalocranchia /i.iheri

10

600

1

5

—

47.9

—

0.39

0.54

Cranchia scabra

10

10

1

5

—

6.39

—

0.40

0.37

Order Octopoda

Japelella diapliana

700

700

12

S

0.02-242

59.49

0.04-0.89

0.25 ±0.07

0. 1 5

4

15

0.02-17.15

5.04

0.53-4.04

1.79 ±0.82

0.52

Eledonella pyt>maea

975

975

5

S

2.03-40.00

15.88

0.05-0.43

0. 1 7 ± 0.07

0.18

6

15

2.00-67.0

24.03

0.08-1.03

0.49 ±0.11

A mphitrelus pelagicus

300

—

1

5

—

30.3

—

0.10

0. 1 3

Order Vampyromorpha

1 ampyroleuthis infernalis

800

800

1

—

0.5

—

O.I 1

0.06

* MO2: mean oxygen consumption rates measured from 70 to 30 mm Hg oxygen partial pressure. Values in the rightmost column are corrected
to 10 g wet weight and 5°C ( using scaling coefficients and (?io values or assuming a scaling coefficient of -0.20 and a Qw of 2.0).
tMDO: minimum depth of occurrence; MDdO: minimum depth of daytime occurrence.
JThe individuals of Octopus rubescens measured here are pelagic juveniles of an otherwise hcnthic species.

METABOLISM OF PELAGIC CEPHALOPODS

267

10-,

Is

O) 1-

t— c\

o O

O _o

c o

<u P

co Ej_

X ^~"

O

0.1-

0 200 400 600 800 1000 1200

Minimum Depth of Occurrence (m)

Figure 2. Oxygen consumption rates of cephalopods (•). fish ( ).
and crustaceans (D) normalized to 10 g wet weight and 5°C as a func-
tion of minimum depth of occurrence. Results of ANCOVA show that
the slope of the regression for cephalopods is significantly steeper than
for either fish (P = 0.004) or crustaceans (P = 0.001 ) (data from Chil-
dress, 1 973: Torres el a!.. 1979).

The one specimen ofOcythoetiiberculata measured in
this study was a male caught in an oblique tow from
2500 m to the surface. The animal's excellent physical
condition upon reaching the surface suggests that it was
caught at a shallow depth near the end of the trawl, but
very little is known about its vertical distribution. It had

a metabolic rate of 4.17 ^mol O2 g ' h '. The specimen
of Amphitretus pelagian measured was captured in a
closing net at night between 700 and 820 m. There is
some evidence that the juveniles of the species are found
shallower than the adults, but adults have been captured
as shallow as 250 m at night (Roper and Young, 1975).
Nothing is known of its daytime distribution. Its meta-
bolic rate (0.10 /^mole O: g ' rT1) is very close to that of
other deep-living pelagic octopods and to I'ainpyro-
teitthis injernalix.

Seven species of the family Cranchiidae were mea-
sured in this study. Limited data and questionable ver-
tical distribution information failed to reveal a signifi-
cant relationship with depth in these species. He/ico-
cranchia pfc/fcri had the highest corrected (to 10 g wet
weight) metabolic rate among the Cranchiidae
(0.59 //mol O: g'1 h~', n = 6), but was not significantly
different from other cranchiid species. Because many
cranchiids undergo an ontogenetic migration (Young.
1975a, b), MDO was difficult to pinpoint. In the case
of Leachia pacifica and Leach ia dislocata, minimum
depth refers to the larval habitat. Individuals of this spe-
cies spend the majority of their lives in epipelagic waters
and then migrate suddenly to great depths where matu-
ration and spawning occur (Young, 1975a). The speci-
mens of L. pacifica measured in this study were cap-
tured in shallow water, so its minimum depth is listed
here as 50 m. Certainly for most of its life, L. pacifica is
subject to the selection regime associated with epipela-
gic waters. Gal it cut his phyllnra, //. pfefferi, and Lio-

c
o

0.

E

1
o
O

c
0

1 ;

o
E

O.IT

200 400 600 800

Minimum Depth of Occurrence (m)

1000

Figure 3. Mean oxygen consumption rates (jimol O: g ' h ') normalized to a wet weight of 10 g (as-
suming a scaling coefficient of -0.20) of seven species from the family Cranchiidae (A) as a function of
habitat depth (m). The slope of the regression line is not significantly different from zero (P = 0.57). The

regressions of Californian ( ) and Hawaiian ( ) cephalopods other than cranchiids are plotted as a

function of minimum depth of occurrence for comparison (from Fig. 1 ). Ocvlhoc tubcmilalu (D) had an
oxygen consumption rate of 4. 17 ^mol O: g ' h '. Paralarval Octopus nihi:\ccn\ ( ) had a mean oxygen
consumption rate of 7.27 fimol O: g ' h '. Amphitretus pelagicus (x) had a metabolic rate of OJ
02g 'h '.

268

B. A. SEIBEL ET AL
Table II

Critical oxygen partial pt " weight-specific oxygen consumption rale* ( MO :. ninol O2x ' »<•'

CM). anilQiu value-, foi agici cfihalopotls with different life-history characteristics

h ') as a function o/ hotly »cii;/ii

Species

Wet weight
Pc 5°C <g)

VO; = aM*

MeanMO;(±SE,

n)

<?io

(mm Hg) range

a: /i(±95%CI, /i)

5V 10°C

1ST

California

Order Teuthoidea

Abraliopsisfelis

0.36-0.99

ns

3.44 ( — .1) 6.70( — . 1)

—

3.79

HIS/K iiciithi.i heteropsis

6.97 0.23-36.98

1.16; -0.20(0.09.17)

1.09* 1.74(0.38.3)

—

2.55

Order Octopoda

Japi'lclla heal hi

6.10 0.84-162.5

ns

0.22( — .2) 0.481 — .1)

—

4.76

Octopus rubescens^

0.04-0.12

ns

7.48( — ,2) 10.43(0.23.3)

—

1.94

Order Vampyromorpha

I 'ampyroteuthis infernalis

3.10 0.41-1050

0.18; -0.30(0.10,15)

0.11* 0.31 (—,1)

—

7.90

Hawaii

Order Teuthoidea

A braliopsis paa fiats

0.40-2.91

ns

2.39(0.39.5)

5.43 (-.2)

2.27

Clenoplvn :\ siculus

1.28-4.24

ns

2.81 ( — . 1)

5.24<-,2)

1.86

Liocranchia valdivia

0.17-21.28

ns

0.56(0.1 1.12)

2.47(0.23. 1 1)

4.41

0.01-0.25f

ns

1.05( — .2)

2.56(0.70.3)

2.43

0.37-21.3}

ns

0.47(0.08.9)

2.43(0.28.8)

5.17

Balhyteulhis abyssicola

18.0

ns

Order Octopoda

Japi'lclla diaphana

18.6 0.02-242

0.24; -0.27(0.05, 12)

0.44(0.1 1,6)

1.79(0.82,4)

4.10

0.56-2.97t

ns

0.26(0.01.3)

0.601 — . 1)

2.31

17.0-36.0J

ns

0.1 1 ( — .2)

0.53 ( — . 1)

4.82

Eledonella pygmaca

2.03-4(1.0

ns

0.17(0.07,5) —

0.57(0.14,5)

3.35

Critical partial pressure of oxygen (PJ is defined as that partial pressure of oxygen at which oxygen consumption is no longer independent of
oxygen concentration. All confidence intervals for scaling coefficients (/>) are / tests at the 95"; level of significance. Q,u values were calculated using
rates from similar-sized animals.

* Rates corrected to appropriate weight for Qm measurements using measured scaling coefficients.

t Weight range undergoing ontogenetic descent (captured between surface and 600 m depth ).

\ Adult weight range captured below 600 m depth.

tj Pelagic juveniles stage of an otherwise benthic species.

cranchia valdivia undergo a gradual ontogenetic de-
scent. The specimens measured here were primarily
small ( 10-30 mm mantle length) and are plotted at in-
termediate depths ranging from 300 to 500 m. Cranchia
scabra has a very wide distribution. It has been captured
from the surface to depths greater than 2000 m. It is
plotted here at an MDO of 10 m.

Temperature effects

Metabolic rates were measured at 5°, and either 10° or
15°C for 10 cephalopod species with various life-history
characteristics. QRI values are presented in Table II. Qw
values were significantly lower for species that undergo
diurnal vertical migrations through large temperature
changes than for species with permanently deep (cold)
habitats (unpaired / test, P = 0.049). The mean Q[0 for
vertical migrators was 2.62 ± 0.42 (n = 4), which is con-
sistent with temperature responses observed previously

for vertically migrating crustaceans (Cowlest'/w/.. 1991).
The mean Qw for non-migrators was 4.90 ± 0.78 (n =
5). Pelagic juveniles of Octopus rubescens were excluded
from this comparison because nothing is known of their
diel movements. The Qw for O, rubescens was 1.94. Of
the non-migrators, two species are known to undergo on-
togenetic migrations from the surface to great depths.
Liocranchia valdivia and Japelella c/iap/uina both mi-
grate from the surface as paralarvae to great depths as
subadults. The Qt() values measured for individuals cap-
tured in the process of migration are similar to those of
diurnally migrating cephalopods (2.31, 2.43), whereas
the Qw values for deep-living adults are considerably
higher (4.82, 5.17). It should be pointed out that the non-
migrators (including the adults of ontogenetically mi-
grating species) live in water of about 5°C or below and
rarely if ever encounter water as warm as 10°C. Their
responses to temperatures of 10°C and above are thus
not expected to be adaptive.

METABOLISM OF PELAGIC CEPHALOPODS

269

1-r

.2

w

O v

c o

0 P

>! §-

X ^

o

0.1-

I t I I > > I t I I I I I I I I 1-4 t I I I I | t t t * I * I I I I

0 10 20 30 40 50 60 Vo

Oxygen Partial Pressure (mm Hg)

Figure 4. Mean oxygen consumption rates (/imol O: g 'h '(aver-
aged o\er 2-min intervals as a function of available oxygen (mm Hg)
at 5°C. Individuals from five species are plotted. Mean critical partial
pressures are 7.2 ± 1.4 mm Hg for Vampyroieuthis infemalis (A), 6.1
mm Hg for Japetella healhi (•). 2 1 .8 ± 3.2 mm Hg for Japetella dui-
pluimi ( + ), 1 1.99 ± 2.28 mm Hg for Histioteuihis heleropsis (A), and
1 8.0 for Bathyieuthis ahyssicola (D).

Regulation

The regulation of oxygen consumption at 5°C was in-
vestigated in five species of pelagic cephalopods (Fig. 4).
Critical partial pressures of oxygen (Pc) are presented in
Table II. Vampyroteuthis infemalis, which lives perma-
nently within the well-developed oxygen minimum layer
ofFCalifornia, had a mean Pc of 7.2 ± 1 .4 mm Hg (n = 6),
which is comparable to that of the mysid Gnathophaitsia
ingens (Childress, 1968; Sanders and Childress, 1990).
One individual of I '. infemalis demonstrated regulatory
abilities down to 3.1 mm Hg, considerably lower than
the minimum A> of 6 mm Hg found within the Califor-
nia oxygen minimum layer. Similarly, Japetella heathi,
a bathypelagic octopod found off the California coast,
was capable of regulating its oxygen consumption to as
low as 6.1 mm Hg (n = 2). Japetella diaphana off Ha-
waii, however, is apparently able to regulate its oxygen
consumption to only 2 1 .8 ± 3.2 mm Hg (/; = 3), despite
a metabolic rate very similar to that observed for J. hea-
thi (Table I). Bathyteuthis abyssicola from Hawaii regu-
lated to 18 mm Hg (/; = 2). The regulatory abilities of/
diaphana and B. abyssicola are consistent with the min-
imum value of 20 mm Hg found for the partial pressure
of oxygen within the Hawaiian oxygen minimum layer.
Regulatory abilities were assessed in four individuals of
the vertically migrating squid Histioteuthis heteropsis
captured off California. The mean critical partial pres-
sure was 1 1 .99 ± 2.28 mm Hg. One individual regulated

to 6.97 mm Hg, consistent with oxygen concentrations
found at its deeper, daytime habitat.

Buffering capacity

The mean buffering capacities (fi) in mantle muscle of
eight species of pelagic cephalopods are listed in Table
III. f3 ranged from 1.43 slykes for the Californian bathy-
pelagic octopod Japetella heathi to 77.08 slykes for the
epipelagic Hawaiian squid Sthenoteuthis oualaniensis.
Significant scaling relationships could not be derived for
any species measured. A significant decline in buffering
capacity (y = slykes) with increasing MDO (x = meters)
was observed (.v = 275.9A-~() "7 ± ° 19; P = 0.004; R2 = 0.85;
Fig. 5). The slope of this regression is significantly lower
than the slope of the regression of oxygen consumption
with depth (-ANCOVA: P = 0.009).

Metabolic scaling

Regressions of weight-specific oxygen consumption
against wet weight were significant in only three individ-
ual species (Fig. 6). I 'ampyroteuthis infemalis had a scal-
ing coefficient, b. of -0.30 ± 0.10 (P = 0.0001; R2
0.75), with weights ranging from 0.41 to 1050.0 g. Hisii-
oteuthis heteropsis had a scaling coefficient of -0.20 ±
0.09 (P = 0.001; R2 = 0.50), with weights ranging from
0.23 to 36.98 g. The scaling coefficient found for Jape-
tella diaphana was -0.27 ± 0.05 (P = 0.000 1 ; R2 = 0.95).
Weights ranged from 0.02 to 242.17 g. The scaling co-
efficients for all three of these species are consistent with
scaling data available for a wide range of animals
(Schmidt-Nielsen, 1983). Sufficient range in size and
numbers of Chiroteuthis calyx (5.97-89.02 g, n = 11)
and Japetella heathi (0.84- 1 62.5 g, /; = 11) failed to yield
a significant scaling relationship. Individuals of/ healhi
larger than 10 g appear to follow a scaling pattern similar
to that of/ diaphana, although the relationship was not
significant.

There is a significant increase in wet weight (In y =
grams) with increasing MDO (In .v = meters) among the
species for which oxygen consumption was measured (y
= 1 .22.v - 4.08; P = 0.000 \;R2 = 0.39). The largest ceph-
alopod measured in this study, an individual of Vam-
pyroteuthis infemalis ( 1050 g), had the lowest metabolic
rate measured (0.02 ^mol O: g ' h"1). We were unable
to measure oxygen consumption for the faster epipelagic
squids, many of which reach sizes considerably larger
than any species reported in the present study. Typical
sizes of commercial species range from 0. 1 to 1 .0 kg
(Roper etui. 1984).

Discussion

A decline in metabolic rate with a species' minimum
depth of occurrence (MDO) was obsi ved for pelagic

270

B. A. SEIBEL ET AL
Table III

Buffering capacit r '"tie muscle of pelagic cephalopodsfrom California and Hawaii measured al 20°C:

wet weight and minimum depth of adult occurrence (MDO) are also given

Species

Wet weight (g)

MDO(m)

Range

Mean ± SE

Range

Mean ± SE

California

Order Teuthoidea

Gonatusonyx

100

1

—

1.06

—

32.61

C 'hiroleitlhis calyx

300

5

3.43-70.08

36.96

7.43-9.86

8.37

Mastigoteuthis pyrodes

375

2

67.50,96.^8

82.25

8.00. 1 1.76

9.88

Cranchia scabra

10

2

16.14,35.38

25.76

14.99,30.77

22.88

Order Octopoda

Japetflla heat hi

600

3

25.50-162.5

86.59

0.94-2.33

1.43 ±0.45

Order Vampyromorpha

I 'ampyroteuthis mjernalis

600

13

5.93-600

224.35 ± 52.39

0.86-6.89

3. 33 ±0.45

Hawaii

Order Teuthoidea

Sthenoteuthis oualaniensis

10

1

—

1 10

77.08

Chiroleuthis imperawr

300

3

27.7-53.56

40.07 ± 7.50

5.98-9.66

8.03 ± 1.08

Order Octopoda

Japetella diaphana

700

7

8.96-325.8

1 19. 10 ±40.06

1.34-2.44

1.67 + 0.15

cephalopods off California and Hawaii. This decline with
increasing MDO is significantly steeper in slope than that
observed for pelagic fishes and crustaceans (Fig. 2) (Chil-
dress, 1975; Torres el al., 1979). The large decline results
from both higher rates among shallower cephalopods
and lower rates among many cephalopods living at
depth. This means that many deep-living cephalopods
have metabolic rates similar to those of midwater jelly-
fishes (Thuesen and Childness, 1994). The presence of

100T

10-.

I

'8

D)

m

Minii n Depth of Occurrence (m)

Buffering en. • Uy (slykes) of mantle muscle as a function
of mini::, nth ofoccuru-nce in meters for nine cephalopods mea-

sured fn M rniaand Hawaii. The regression is y= 275.9.v~067±0lW

(f = 0.()() . 85).

a strong decline in cephalopods, fishes, and crustaceans
suggests, however, that the selective factors that influ-
ence metabolic parameters are similar at any given depth
for all three groups. Buffering capacity has been used in
the past to indicate the capacity of muscle tissue for an-
aerobic work (Castellini and Somero, 1 98 1 ). Like oxygen
consumption, ft declined with increasing MDO. This is
consistent with the results of Castellini and Somero
(1981), who saw lower ft values in deep-sea fishes than in
either actively foraging pelagic ectotherms or in warm-
bodied fishes.

There are manv variables confounded with habitat

o
'o.^

E !c

D

1-

W
O

O

O)

cT

o °-1^

0.01
0.

)1

ol

1 10

Mass (g)

> iit«4 1 t * MUII

ibo 1000

Figure 6. Oxygen consumption rates (/^mol O:g 'h ') as a func-
tion of wet weight (g). The slope of the regression line is r =
\A(>\ "2"±"m(P= O.OOI;R: = 0.50) for Histioteuthis heteropsis (+), y
= 0. 1 8.v~° 30±0 I0 (P = 0.000 1 : R1 = 0.75 ) for I 'ampynneiilhis infernalis
(D), and y = 0.24.v'° 27±" "' ( P = 0.000 1 ; R2 = 0.95 ) for Japctella dia-
phuini (•).

METABOLISM OF PELAGIC CEPHALOPODS

271

depth. These include light, productivity, temperature,
phylogeny. neutral buoyancy, vertical and horizontal
migrations, oxygen concentration, and body size, among
others. However, in the discussion below it will become
apparent that the decline in metabolic parameters with
increasing habitat depth most likely reflects decreased lo-
comotory abilities in deeper-living animals. The steep
slope of the regression of cephalopod metabolism with
depth relative to that of fishes and crustaceans may re-
flect the relative efficiencies of locomotion at the surface,
where high speeds are required for visually cued preda-
tor/prey interactions, and at depth, where sit-and-wait
predation strategies are prevalent.

Light and visual interactions

According to the visual interactions hypothesis, re-
duced reliance on visual predator/prey interactions in
the light-limited deep sea decreases selection for locomo-
tory abilities, thus resulting in the lower metabolic rates
observed in deep-living fishes, crustaceans, and cephalo-
pods (Childress, 1995). This idea is supported by the
presence of a decline in metabolism with depth among
these visually orienting groups and the absence of such a
decline among organisms that lack eyes (medusae and
chaetognaths). That light-limited predator/prey interac-
tions affect animal behavior is not a new idea. Light has
long been recognized as a primary controlling factor in
the daily movements of midwater organisms (Kerfoot,
1970). Limits on concealment from predation through
counterillumination determine daytime depth distribu-
tion in some squids and fishes (Young el ai. 1980), and
young cranchiid squids undergo ontogenetic changes
that minimize their visibility at a given light level
(Young, 1975b). These cranchiids can also control the
orientation of their body and eyes to reduce the size of
their own silhouette against surface illumination (Seapy
and Young, 1986). The extent to which animals are con-
cealed from visual predators and prey is suggested here
to strongly influence the evolution of loccmotory abili-
ties and metabolic rates of midwater organisms. The
metabolic rate of Lolliguncida brevis, which is typically
found in complex and shallow coastal waters where op-
portunities for refuge and crypsis are greater, is half that
of the more powerful squid living in open epipelagic wa-
ters. It was suggested that L. brevis is not required to
swim great distances or at high speeds to capture prey or
to escape predators (Finke el a/.. 1996). The metabolic
rates of fishes and crustaceans in benthic habitats, where
opportunities for refuge and crypsis are greater, do not
decline with increasing habitat depth to nearly the extent
observed in pelagic animals (Childress et ai. 1990). The
apparent lack of a decline in metabolic rates among cran-
chiid squids in the present study is probably a result of
concealment from predation through transparency.

Productivity

The food limitation hypothesis suggests that animals
living in relatively food-poor environments such as the
deep sea have evolved lower metabolic rates as a way of
conserving energy (Childress, 1971b; Smith and Hessler,
1974). If this were true, one might expect metabolic rates
of deeper-living species to vary from region to region in
correlation with surface primary productivity. Contrary
to this prediction, comparisons between tropical and
temperate regions of differing productivity have shown
that among pelagic crustaceans living continuously be-
low 400 m, there is no significant variation in metabolic
rate with depth. In fact, shallow-living crustaceans actu-
ally have higher metabolic rates in Hawaii than in Cali-
fornia where productivity is considerably higher (Cowles
et al., 199 1 ). This may indicate an increased demand for
strong locomotion for predator/prey interactions over
greater distances in the clearer, more strongly illumi-
nated surface waters. No significant differences were
found between the slopes or magnitudes of the regres-
sions of metabolism with depth for Californian and Ha-
waiian cephalopods. Although the magnitudes of the re-
gressions were not significantly different, it may be worth
noting that I 'ampyroteuthis infernalis seems (based on
small numbers of captures) to come as much as 100 m
shallower off California than off Hawaii. Similarly, Ja-
petella heathi comes about 100 m shallower off Califor-
nia than does J. diaphana off Hawaii (Roper and Young,
1975; Young, 1978). This may reflect differing light re-
gimes between the two regions.

Temperature effects

Temperature generally has large effects on metabolic
processes. A 10°C increase in temperature typically causes
a doubling of metabolic rate (Q\0 = 2). Deep-living ani-
mals may be expected to have lower metabolic rates on
the basis of lower temperatures alone. Donnelly and Tor-
res ( 1 988) report that the decline in aerobic metabolism of
midwater fishes and crustaceans with depth in the eastern
Gulf of Mexico is no greater than that due to the decrease
in temperature. However, the observation that metabo-
lism declines with increasing MDO in the isothermal wa-
ters of Antarctica (Torres and Somero, 1988;Ikeda, 1988)
seems to eliminate temperature as the primary factor pro-
ducing lower metabolic rates in the deep sea.

The Qn, values measured here (Table II) fall into two
groups. Vertical migrators, which experience a wider
range of temperatures every day, have typical responses to
temperature, but non-migrators have mu< larger re-
sponses. Non-migrators typically live at or ;:ear 5°C and
rarely, if ever, experience temperatures ne 10°C. The re-
sponses to temperatures outside the non 1 range for the
species are not expected to be adaptive, few of the non-

272

B. A. SEIBEL ET AL.

migrators undergo ontogenetic descents through the wa-
ter column. The > iig stages experience a wider range
of temperatures over a relatively short (though unknown)
period of time. Individuals captured during the process of
ontogenetic descent have more typical responses to tem-
perature than do the adults of the species. A Qw of ap-
proximately 20 would be required to explain the slope of
the decline in metabolic rates with depth observed here.

Phytogeny

Harvey and Pagel (1991) warned that interspecific
comparisons may be invalid because phylogeny, instead
of selection, may have played the greater role in deter-
mining the properties of the species. Although there are
obvious phylogenetic effects, the observed variation with
depth in the metabolism of cephalopods, like that of
fishes and crustaceans, is probably not an artifact of phy-
logeny for two reasons (Childress, 1995). First, the phys-
iological and morphological characteristics of related
species seem to diverge as a function of habitat depth.
Single genera or families of fishes and crustaceans often
occupy the entire depth range studied, and the variation
in a characteristic being studied may approach the entire
range of variation for such a parameter within a data set
(Childress el al.. 1990; Cowles el a!., 1991 ). Second, the
evidence for convergent evolution among genera, fami-
lies, and even phyla is strong. The origin of many of these
groups lies outside the deep sea and thus can hardly pro-
duce a phylogenetic bias. Many deep-living groups share
characteristics of chemical composition, general muscu-
lature, and physiology that all lead to decreased locomo-
tory abilities. Cowles and Childress ( 1 988) demonstrated
directly that bathypelagic mysids have weaker swimming
capabilities than do epipelagic species. Enzymatic activ-
ities provide evidence for reduced locomotory abilities
in deeper-living fishes also (Childress and Somero, 1979;
Sullivan and Somero. 1980).

Divergence of related species as a function of habitat
depth can be seen at the ordinal level among cephalo-
pods. Epipelagic squids (order Teuthoidea) are ex-
tremely active predators, and some are capable of speeds
sufficient to propel their bodies out of the water (Cole
and Gilbert. 1 970). A study on Il/e.\ illecebrosus reported
a respiration rate nearly twice as high as any cephalopod
^easured in this study (DeMont and O'Dor, 1984).
ing the teuthoids measured here, nearly 2 orders of
utude separu Gonatus onyx, a vertical migrator
ves below luOm, and Bathyteuthis ahyssicola,
lives continuously below 800 m. Similarly,
nberculala (order Octopoda), believed to be
e^ had a metabolic rate nearly 2 orders of mag-

nitui! r than that measured for the pelagic octo-

pods . 'ins pe/agicus ( captured at 700 m ) and Ja-

petella diaphana (MDO = 700). Convergence of cepha-
lopods with fishes and crustaceans living at similar
depths is evidenced by both their morphology and me-
tabolism. Voss( 1967 (recognized morphological similar-
ities in cephalopods living at a given habitat depth. He
observed a decrease in mantle musculature and the ap-
pearance of a gelatinous material that formed the bulk of
the mantle in deep-living forms. In general, deeper-living
cephalopods, like fishes and crustaceans, appeared to be
relatively poor swimmers compared to their epipelagic
counterparts.

Phylogeny does, however, appear to play at least some
role in the evolution of cephalopod metabolism. Deep-
living octopods have significantly lower metabolic rates
than squids at similar depths. It appears from observa-
tions of the musculature (Roper, 1969), gill areas (Ma-
dan and Wells, 1996: Seibel and Childress, 1996), and
metabolic rates that perhaps Bathyteuthis abyssicola
leads a more active life than the deep-living octopods and
I 'ampyroteuthis infemalis. However, the metabolic rate
of B. abyssicola is still comparable to that of deep-living
fishes with sit-and-wait predation strategies (Cowles and
Childress, 1996).

The only family with representatives from a wide
range of depths measured in this study is the Cranchii-
dae. Cranchiids comprise a monophyletic group distin-
guished from other cephalopods by many unique fea-
tures (Nixon. 1983; Voss and Voss, 1983) that allow rel-
atively low metabolic rates regardless of habitat depth.
Foremost among these is the presence of a coelom. which
provides neutral buoyancy (Denton and Gilpin-Brown,
1973; Clarke et ai, 1979) and may be contracted to cre-
ate a flow of water over the gills. The mantle musculature
is rarely used and then only for escape responses (Clarke,
1962). All cranchiids are largely transparent (Clarke.
1962; Seapy and Young, 1986). which decreases the se-
lection associated with higher predation risks in higher
levels of ambient light (McFall-Ngai. 1990). When dis-
turbed, Cranchia sctibra is also capable of filling its man-
tle cavity with seawater, which effectively doubles its vol-
ume (shipboard observations). It apparently shares this
ability with at least one other cranchiid (Nixon, 1983).
This sort of defense mechanism may reduce the need for
lengthy escape swimming, and further emphasizes the
trend among cranchiids to reduce metabolic costs. The
limited data on metabolic rate and vertical distribution
suggest that metabolism may be independent of habitat
depth within this family (Fig. 3). Inclusion of the cran-
chiids does not significantly alter the slope or elevation
of the regression in Figure 1.

Buoyancy

Many midwater cephalopods possess some means of
achieving neutral buoyancy (Denton and Gilpin-Brown,

METABOLISM OF PELAGIC CEPHALOPODS

273

1973; Clarke, 1988; Clarke et ai. 1979; Voight el al.,
1994), whereas most epipelagic squids are negatively
buoyant and rely on constant swimming for lift. It is con-
ceivable that the metabolic rates of midwater cephalo-
pods might be reduced simply by achieving neutral
buoyancy, and that the higher metabolic rates of epipel-
agic cephalopods result from the added cost of support in
the water column. Childress and Nygaard (1974) found,
however, that neutral buoyancy in crustaceans does not
appear to be a means of conserving energy per xe. Meta-
bolic rates were not correlated with density at a given
depth. In epipelagic waters, locomotion is apparently a
greater priority and dictates a high content of protein.
Protein, associated with locomotory muscles, provides
the density responsible for the negative buoyancy in such
epipelagic squids as Loligo sp. and Ommaxt replies sp.
(Denton and Gilpin-Brown, 1973). Buoyancy can actu-
ally have a detrimental effect on locomotory abilities.
Buoyancy organs make animals bulkier, which increases
the energy needed for swimming at a given speed (Alex-
ander, 1990; O'Dor and Webber, 1991). Although me-
tabolism and buoyancy are certainly confounding fac-
tors, evidence suggests that the need for strong swim-
ming determines the practicality of buoyancy. The
energy required for support in the water column may be
insignificant compared to an animal's overall activity
level (Childress and Nygaard. 1974). However, when lo-
comotion is not a high priority and activity levels are rel-
atively low, the cost of support in the water column may
be large relative to that of overall activity. In such situa-
tions, neutral buoyancy may be a cost-effective option.
Among neutrally buoyant cephalopods there is still a de-
cline in metabolic rates with increasing habitat depth.

I 'ertical migration

Many organisms undergo daily vertical migrations,
spending the daylight hours at greater depths. Because
vertical migrators are never exposed to surface daylight
conditions, one might expect that light limitation on
predator/prey interactions would result in metabolic
rates more similar to those of deeper-living non-migra-
tors than of epipelagic non-migrators. In previous studies
(Childress and Nygaard, 1974), vertical migrators were
considered at their shallowest depth (MDO) because it
was the location of their primary food source. Evidence
suggests that vertically migrating fishes and crustaceans
are more similar in composition to shallow-living than
to deep-living non-migrators (Childress and Nygaard,
1974). It was suggested that vertical migrators are essen-
tially epipelagic animals that take refuge during the day
at depth. However, Gonatits sp. and Histioteuthis sp.,
both vertically migrating cephalopods, have energy
contents lower than those of epipelagic squids (Clarke et

al, 1985). The lower wet-weight-specific energy contents
in some species are believed to be a result of increased
water content, suggesting decreased locomotory abilities
in those species. As discussed previously, Voss (1967)
characterized organisms from various depths on the ba-
sis of morphological features such as body musculature,
size and shape of fins and mantle, eye size, and skin
color. He considered Histioteuthis to be a resident of the
bathypelagic zone (700-2000 m) along with Bathy-
teitthis. By his criteria, Gonatm would have been placed
in the mesopelagic zone (200-700 m), at a depth consis-
tent with its deeper daytime habitat. The vast majority
of epipelagic cephalopods exhibit some diel movements.
Loliginids and ommastrephids (among others) migrate
away from the surface during the day. A full moon, the
equivalent of daylight at 300 m depth, has been shown
to keep some ommastrephids away from the surface at
night ( Wormuth, 1 976). This suggests that vertically mi-
grating cephalopods may be following an isolume to
which their metabolic rate may be adapted. The day and
night habitats of vertical migrators are distinctly differ-
ent, and both presumably have strong effects on the
physiology of vertically migrating organisms. Both
MDO and MDdO reveal a strong decline in cephalopod
metabolism with increasing habitat depth (Fig. 1 ).

Horizontal migration

In addition to migrating vertically, many epipelagic
squids migrate long distances horizontally. O'Dor ( 1 992)
has suggested that powerful squids have evolved to take
advantage of current systems, and that this requires
strong swimming and perhaps high metabolic rates as
well. The deep sea has relatively weak currents, which
may relieve animals from the selective pressures associ-
ated with current systems. However, this hypothesis
probably applies only to truly large and active predators,
all of which are capable of avoiding midwater trawls. Al-
though nothing is known of the horizontal migration
patterns of midwater cephalopods, none of the species
studied here appear to be powerful swimmers of the cali-
ber discussed by O'Dor (1992). Selection due to strong
currents also cannot explain the lack of a decline in me-
tabolism with depth in some phyla (cnidarians and chae-
tognaths; Thuesen and Childress, 1993a; 1994); patterns
of vertical migration through varying current strengths;
or differences in metabolic rates observed between Ha-
waii and California for shallow-water crustaceans
(Cowles et al., 1991).

Oxygen minimum layer

Another factor possibly selecting for </er metabolic
rates at depth is oxygen concentration. ! lyers of low ox-
ygen are found throughout the worlds' jceans at depths

274

B. A. SEIBEL ET AL

between 200 and 1000 m. The oxygen minimum layer
off California is particularly well developed. Partial pres-
sures of oxygen reach a minimum of about 6 mm Hg be-
tween 600 and 800 m. Fischer and Bottjerf 1995) suggest
that the oxygen minimum layer provides refuge from
large predators and that low oxygen has enforced passiv-
ity in animals that live within it. Certainly some larger
predators are excluded from hypoxic waters (Carey and
Robison, 1980). Reduced predation pressure may allow
passive lifestyles. I 'ampyroteuthis infernalis is "oligoaer-
obic" — found only in low oxygen, with a peak concen-
tration in the oxygen minimum layer (Pickford. 1946,
1952) — and had a metabolic rate of 0.09 /umol O2 g~'
h~'. Species of the genus Japetella also live within the
oxygen minimum layer off both California and Hawaii
and are relatively inactive metabolically. However, the
mean metabolic rate of J. diaphana off Hawaii, where
the oxygen seldom drops below 20 mm Hg, is not sig-
nificantly higher than that of J. hcutlii off California.
Fishes and crustaceans living below the oxygen mini-
mum layer have been shown to have lower metabolic
rates than others living directly within (Childress, 1995).
This suggests that the low metabolic rates are not a spe-
cific adaptation to low oxygen and that passive lifestyles
are selected for at great depths regardless of oxygen
content.

Some animals possess specific adaptations to the oxy-
gen minimum layer. Gnathophausia ingcns. a deep-liv-
ing mysid, has been shown to live aerobically within the
minimum layer by being especially effective at extracting
oxygen from the water. Its ability to regulate oxygen con-
sumption to Pc>2 values as low as 3 mm Hg is due in part
to its ability to maintain a high ventilatory flow and to
remove up to 50%-80% of the oxygen from the inhaled
water (Childress, 1968, 197 la; Belman and Childress,
1976). The latter ability appears to stem from a hemocy-
anin with an unusually high affinity for oxygen and co-
operativity of oxygen binding (Sanders and Childress,
1 990). Regulation of oxygen consumption was measured
in Japetella diaphana, J. heathi, Histioteuthis heteropsis.
and \ 'ampyroteuthis infernalis. Like G. ingens. both J.
heathi and V. infernalis off California can regulate oxy-
gen consumption to at least 6 mm Hg. allowing aerobic
living even at the lowest environmental oxygen levels
fi >und there. Two individuals of V. infernalis regulated
essfully helow 4 mm Hg. J. diaphana, however, reg-
d oxygc onsumption to a mean of 21.8 mm Hg,
• correspo is to the minimum environmental Po2
if 20 mm I Ig off Hawaii. Histioteuthis heteropsis,
igrates into the oxygen minimum layer during
thu as previously reported by Belman ( 1 978) to be

ini. f regulating to oxygen concentrations re-

quired obic living within the minimum layer (Pc

= 20.0 rr.i •). In contrast to those earlier studies, in

our studies H. heteropsis was able to regulate its oxygen
consumption to at least 7 mm Hg. The mean critical par-
tial pressure for this species was 1 1.99 ± 2.28 mm Hg.
The lowered metabolism at low temperatures may allow
aerobic living for some animals, such as H heteropsis,
migrating into the cooler waters of the oxygen minimum
layer during the day. Cowles et al. ( 199 1 ) found a strong
effect of temperature on the critical oxygen partial pres-
sures of vertically migrating crustaceans. This is consis-
tent with findings by Sanders and Childress ( 1990) that
the oxygen affinity of hemocyanin from several of these
species is temperature-dependent and increases greatly
at low temperatures.

Oxygen extraction and jet propulsion are incompati-
ble functions in cephalopods (Wells, 1988). To maxi-
mize oxygen extraction, water must be passed relatively
slowly over the gills and out the jet. Maximization of pro-
pulsion requires the ejection of water at a velocity suffi-
cient to generate the required thrust. Nautilus pompilius,
for example, can extract 20% of the available oxygen
from the ventilatory stream at rest, but only 5% or less
during active propulsion (Wells and O'Dor. 1991). Epi-
pelagic squids have maximized locomotion and are able
to extract oxygen at levels necessary to fuel high speeds
only because water flow over the gills increases as swim-
ming speed increases. Circulatory adaptations, including
enhanced unloading of oxygen at the tissues and a large
positive Bohr shift, allow efficient use of nearly all of the
oxygen taken up by the blood (Zammit. 1978). Benthic
octopods, on the other hand, have optimized oxygen ex-
traction. The respiratory pigments of octopods have a
greater affinity for oxygen than do pigments in the faster
squid and sepioid species (Brix et a/.. 1989). In addition,
their slow benthic lifestyle allows a reduction in mantle
contractions and a slow flow of water over the gills. The
decreased reliance of deep-living cephalopods on loco-
motory abilities, implied by the decline in metabolism
with depth, would, as in benthic octopods, allow a slow-
ing of the ventilatory stream and more efficient extrac-
tion of oxygen — adaptations compatible with life in a re-
duced oxygen environment.

Buffering capacity

Reduced buffering capacities in mantle muscle at
depth are further evidence that locomotory abilities are
not strongly selected for in the deep sea. A high buffering
capacity allows prolonged glycolytic work such as that
required for extended burst escape responses. The sit-
and-wait predation strategies prevalent through much of
the deep sea do not require a high capacity for anaerobic
work. Differences in oxygen levels between California
and Hawaii might be expected to yield higher short-term
anaerobic capacities in animals that live in the lower ox-

METABOLISM OF PELAGIC CEPHALOPODS

275

ygen habitat. A lesser oxygen supply would more quickly
lead to functional anoxia (tissue energy demand out-
strips aerobic capacity). However, no difference in 0 was
seen between Japele/la diaphana and J. heat hi. Both spe-
cies, like Gnathophausia ingem. appear to have very lim-
ited anaerobic abilities. Buffering capacity is believed to
be an adaptable parameter that can be altered to match
metabolic function (Castellini and Somero, 1981) and
has been correlated with tissue capacity for glycolytic en-
ergy production in response to functional anoxia (Eb-
erlee and Storey, 1984). The fact that no difference was
seen between similar animals in different oxygen envi-
ronments is evidence that the reduced locomotory abili-
ties are not a response to ambient oxygen levels.

Metabolic scaling

The body size of the pelagic cephalopods measured in
this study increased with increasing MDO (largely be-
cause the large epipelagic species were not sampled). Be-
cause mass-specific metabolism has. with notable excep-
tions (DeMont and O'Dor, 1984; Thuesen and Chil-
dress, 1994), been repeatedly shown to decrease with
increasing body mass (Schmidt-Nielsen, 1 983), body size
could have significant effects on metabolic estimates
based on depth of occurrence. Scaling coefficients could
be derived for only three species: I 'ampyroteuthis injcr-
nalis. Japetella diaphana, and Histioteuthis heteropsis.
Scaling coefficients for all three (b = -0.30, -0.27. and
-0.20 respectively) fall within the range of most animals
(Schmidt-Nielsen, 1983). Sufficient range in size and
numbers failed to yield a significant scaling relationship
for Chiroteiithis calyx. This is perhaps due to the drastic
change in body proportion from the "doratopsis" para-
larval stage, a characteristic of this genus, to adulthood
( Vecchione etal.. 1992). Significant scaling relationships
could not be derived for the remaining species, owing
either to lack of sufficient numbers of specimens or to
insufficient size ranges. Normalizing metabolic rates to
lOg, either by assuming a scaling coefficient of -0.20
(Schmidt-Nielsen, 1983) or by using measured coeffi-
cients where possible, did not have a significant effect on
the slopes of the regressions with depth. A scaling co-
efficient of approximately - 1 .8, based on the increase in
body size with depth observed in this study, would be
required in order for size to account for the observed
variation with depth. A significant increase in body size
with depth was observed among the animals for which ft
was assayed as well. However, no significant size-scaling
was observed for buffering capacity.

Locomotory efficiency

Packard (1972) compared fish and cephalopod
"modes of life" and distinguished these organisms from

crustaceans and other molluscs in terms of their "great
ability to travel." He observed that functionally, squid
are fish. But many deep-living cephalopods actually have
much lower metabolic rates than do fishes or crustaceans
living at similar depths. The slope of the regression of 1 0-
g-corrected oxygen consumption with MDO is steeper
for cephalopods than for both fishes and crustaceans
(Fig. 2). The metabolic rates of I 'ampyroteuthis infer-
nalis and the deep-living pelagic octopods are more
comparable to those of similar-sized scyphomedusae
(Thuesen and Childress, 1994) than to those of fishes or
crustaceans. Although the rates of deep-living squids are
similar to those offish with sit-and-wait predation strat-
egies (Cowles and Childress, 1996), shallow squids have
higher rates than do fish or crustaceans at similar depths.
The slope of the regression of metabolism with depth is
steeper for teuthoids and octopods, considered sepa-
rately, than for fishes and crustaceans. More efficient lo-
comotion among deeper-living cephalopods may be re-
sponsible for this difference. Shallow-living cephalopods
rely extensively on jet propulsion to gain high speeds.
Because the energy required for thrust increases with the
velocity squared, high-speed jet propulsion is inherently
inefficient (Vogel, 1994). Yet most shallow-living squids
retain jet propulsion as the primary means of locomo-
tion (Baldwin, 1982; Hoar et a/.. 1994). Hoar et al.
(1994) did note, though, that the slower coastal species
such as the loliginids use their fins, which are large, more
actively than do the oceanic ommastrephids. which use
theii s./.a'ler fins as rudders during extremely high-speed
swimming.

Deep-living cirrate octopods have been observed using
the arms and web to swim in a manner similar to the
"bell-swimming" of medusae (Vecchione and Roper,
1991). Cirrothauma nuimiyi relies very heavily on fins
for locomotion ( Aldred el al. , 1 983). It has also been seen
using the arms and web as a "balloon" for defense, elim-
inating costly escape swimming (Boletzky et al.. 1992).
V ampyroteuthis inferna/is, Chiroteiithis calyx, and His-
tioteuthis heteropsis, among others, have been observed
swimming primarily with fins (James Hunt, pers.
comm.). Fin- and bell-swimming are cost efficient com-
pared to jet propulsion by means of mantle contractions,
due to reduced speeds (reduced drag) and to the large
water masses (relative to body mass) being processed
(Vogel. 1994). Although their morphology suggests that
Octopoteuthis spp. (MDO = 100 m) rely heavily on fins
for swimming, limited video footage shows O de/etron
swimming with slow contractions of the m. ntle while
holding the large fins curved over the body ' that water
passes beneath them (James Hunt, pers. i omm.). The
metabolic rates of 0. deletron and ofO n Iseniare low
compared to those of other squids at siniii.tr depths. Per-
haps the curious fin posture provides s .tic lift that re-

276

B. A. SEIBEL ET AL

duces the metabolic costs associated with support in the
water column. This species also attains considerable size,
which would deter predation ( Roper et ai. 1984). Again,
among the cranchiids, where transparency has alleviated
much of the need for high-speed swimming, mantle con-
tractions are rarely used for locomotion (Clarke, 1962).

Morphology and limited shipboard observations indi-
cate that some deep-living cephalopods do utilize jet pro-
pulsion as a primary means of locomotion. At extremely
low speeds, jet propulsion may not be so costly, which
might explain the existence of slow jetters such as jelly-
fish and salps ( Vogel, 1 994). It might also explain the ex-
tremely low metabolic rates of deep-living pelagic octo-
pods. These organisms process large amounts of water
through a relatively large funnel opening, resulting in
efficient, low-speed swimming. It is also possible that
they utilize the arms and web for bell-swimming, much
as the cirrate octopods do. There is tremendous diversity
in the swimming behaviors of pelagic cephalopods but,
perhaps because high speeds are not a priority in the deep
sea, a more efficient means of locomotion is desirable.

Despite differences in their locomotory efficiencies,
buoyancy mechanisms, and structural compositions,
fishes, crustaceans, and cephalopods show similar meta-
bolic trends with depth; these trends probably reflect the
presence of highly evolved eyes in these groups. The re-
sults presented here emphasize the importance of differ-
ential selection for locomotory abilities with habitat
depth in determining the metabolic rates of organisms.
Available evidence implicates vision as a selective factor
strongly affecting the evolution of locomotory abilities.
Although one may intuitively suspect food availability
or low oxygen, the declines in both oxygen consumption
rates and pH buttering capacity of locomotory muscles
with depth in pelagic cephalopods point to a different
conclusion. These factors support the hypothesis that the
extent to which visual predator/prey interactions occur
is largely responsible for the decline in metabolic rates of
visually orienting, midwater organisms with increasing
minimum depth of occurrence. Although cephalopods
have i 'it-n been compared with fishes in discussions of
ecology d locomotion, we conclude that pelagic ceph-
alopods in the deep sea might be more appropri-
ately coni|<,- 1 with medusae in these contexts.

••knowledgments

We thank F.G. ' ichberg and A.L. Alldredge for con-

jctive comments OM the manuscript. We are grateful

•aptains, crews, and scientists aboard the research

•w Horizon and Point Snr for their assistance at

sea. • ' ank R.E. Young and J. Hunt for informative

discuss concerning midwater cephalopods, and C.

Braby. ;ly. T. vanMeeuen, J. Freytag. and B. Rab-

kin for their assistance at sea, in the laboratory, or both.
Research was supported by NSF grants OCE-94 15543
and OCE-9 1 1 555 1 to J.J. Childress.

Literature Cited

Aldrcd, R. G., M. Nixon, and J. /.. Young. 1983. Cirrothaumu miir-
rar; Chun, a finned octopod. Philn\. Trans R Soc. Loml. B30\: 1-
54.

Alexander, R. M. 1990. Size, speed and buoyancy adaptations in
aquatic animals. Am /.not 30: 189-196.

Baldwin, J. 1982. Correlations between enzyme profiles in cephalo-
pod muscle and swimming behavior. Puc Sci. 36: 349-356.

Belman, B. 1978. Respiration and the effects of pressure on the me-
sopelagic vertically migrating squid Hisli<neultus hcieropsis. Lim-
nul Occannnr. 23: 735-739.

Belman, B. \\ ., and J. J. Childress. 1976. Circulatory adaptations to
the oxygen minimum layer in the bathypelagic mysid Gnathophau-
sui ingcua. Hint. Bull 150: 15-37.

Boletzky. S. \ ., M. Rio, and M. Roux. 1992. Octopod 'ballooning'
response. Nature 356: 199.

Boucher-Rodoni, R., and K. Mangold. 1989. Respiration and nitro-
gen excretion by the squid Loligoforbesi. Mar. Bio/. 103: 333-338.

Boutilier, R. G., I. G. West, G. H. Pogson, K. A. Mesa, J. Wells, and
M. J. Wells. 1996. Nautilus and the art of metabolic mainte-
nance. AV»ji;v382: 534-536.

Brix, O., A. Bardgard, A. Cau, A. Colosimo, S. G. Condo, and B. Giar-
dina. 1989. Oxygen-binding properties of cephalopod blood with
special reference to environmental temperatures and ecological dis-
tribution./ E.\p /<»)/ 252: 34-42.

Carey, F. G.. and B. II. Robison. 1980. Daily patterns in the activities
ofswordfish, Xiphias gladius, observed by acoustic telemetry. Fish.
Bull 79: 277-292.

Castellini, M. A., and G. N.Somero. 1981. Buffering capacity of ver-
tebrate muscle: correlations with potentials for anaerobic function.
/ Camp. Phyxinl. 143: 191-198.

C'hildress, J.J. 1968. Oxygen minimum layer: vertical distribution
and respiration of the mysid Gnalhophausia ingcns. Science 160:
1242-1243.

Childress, J. J. 1971a. Respiratory adaptations to the oxygen mini-
mum layer in the bathypelagic mysid (.inullinpliaiixia ingcns. Bin/
Hull 141: 109-121.

C'hildress, J.J. 1971 b. Respiratory rate and depth of occurrence of
midwater animals. Limnol. Oceanogr. 16: 104-106.

Childress, J. J. 1975. The respiratory rates of midwater crustaceans
as a function of depth occurrence and relation to the oxygen mini-
mum layer off Southern California. Coinp. Biochem. Pliysinl 50A:
787-799.

C'hildress, J. J. 1995. Are there physiological and biochemical adap-
tations of metabolism in deep-sea animals? Trends Ecu/, /-.'m/ 10:
30-36.

C'hildress, J.J., and T.J. Mickel. 1980. A motion compensated ship-
board precision balance system. Deep-Sea Res 27A: 965-970.

C'hildress, J. J., and T. J. Mickel. 1985. Metabolic rates of animals
from the hydrothermal vents and other deep-sea habitats. Bin/. Sac.
li'iis/i Bull 6:249-260.

C hildress, J. J., and M. II. Nygaard. 1974. The chemical composi-
tion and relative buoyancy of midwater crustaceans as a function of
depth off Southern California. Mar. Bin/ 21: 225-238.

Childress, J. J., and G. N. Somero. 1979. Depth related enzymic ac-
tivities in muscle, brain and heart of deep-living pelagic marine te-
leosts. Mar. Biol 52: 273-283.

C'hildress. J. J., and E. V. Thuesen. 1993. The effect of hydrostatic

METABOLISM OF PELAGIC CEPHALOPODS

277

pressure on the metabolic rates of six species of gelatinous zoo-
plankton. Limnol. Oceanogr. 38: 665-670.

Childrcss, J. J., A. T. Barnes. L. B. Quetin, and B. II. Robison. 1978.
Thermally protecting cod ends for recover, of living deep-sea ani-
mals. Deer-Sea Res. 25: 419-422.

Childress, J. J., D. L. Cowles, J. A. Favuzzi, and T. J. Mickel. 1990.
Metabolic rates of benthic deep-sea decapod crustaceans decline
with increasing depth primarily due to the decline in temperature.
Deep-Sea Res. 37: 929-949.

Clarke. A., M. R.Clarke, I.. J.Holmes.andT. D. Waters. 1985. Calo-
rific values and elemental analysis of eleven species of oceanic
squids (Mollusca: Cephalopoda). / Mar. Biol. Assoc. I'.k. 65: 983-
986.

Clarke, M. R. 1962. Respiratory and swimming movements in the
cephalopod Cranclua seabra. Nature 196: 35 1-352.

Clarke, M. R. 1988. Evolution of buoyancy and locomotion in recent
cephalopods. Mollusca 12: 203-213.

Clarke. M. R., E. J. Denton. and J. B. Gilpin-Brown. 1979. On the
use of ammonium for buoyancy in squids. J. Mar. Biol. Assoc. I K
59: 259-276.

Cole, S. K.. and D. L. Gilbert. 1970. Jet propulsion of squid. Biol.
Bull 138: 245-246.

Cowles, D. L., and J. J. Childress. 1988. Swimming speed and oxy-
gen consumption in the baths pelagic mysid Gnathophausia ingen.s.
Biol. Bull. 175: 111-121.

Cowles, D. L., and J. J. Childress. 1996. Aerobic metabolism of the
anglerfish Melanocetusjohnsoni, a deep-pelagic marine sit-and-wait
predator. Deep-Sea Res 42: 1 63 1 - 1 638.

Cowles, D.C., J.J. Childress, and M. E. Wells. 1991. Metabolic
rates of midwater crustaceans as a function of depth of occurrence
offthe Hawaiian Islands: food availability as a selective factor? Mar
Bi«l. 110:75-83.

DeMont, M. E., and R. K. O'Dor. 1984. The effects of activity, tem-
perature and mass on the respiratory metabolism of the squid. Illcx
illeeebrosns. J. Mar Biol. Assoc. i'K 64: 535-543.

Denton, E. J., and J. B. Gilpin-Brown. 1973. Floatation mechanisms
in modern and fossil cephalopods. Adv. Mar Biol. 11: 197-268.

Donnelly, J., and J. J. Torres. 1988. Oxygen consumption of mid-
water fishes and crustaceans from the eastern Gulf of Mexico. Mui
Biol. 97: 483-494.

Eberlee. J. C., and K. B. Storey. 1984. Buffering capacities of the tis-
sues of marine molluscs. Physiol. Zool. 57: 567-572.

Finke, E., H. O. Portner, P. G. Lee, and D. M. Webber. 1996. Squid
(Lolliguncula hrevis) life in shallow waters: oxygen limitation of me-
tabolism and swimming performance. J. Exp. Biol 199: 91 1-921.

Fischer, A.G., and D. J. Bottjer. 1995. Oxygen-depleted waters: a
lost biotope and its role in ammonite and bivalve evolution. A'. Jh
Geol. Palaont Ahh. 195: 133-146.

Fleisher, K. J., and J. F. Case. 1995. Cephalopod predation facili-
tated by dinoflagellate luminescence. Biol. Bull. 189: 263-27 1 .

Grieshaber, M.. and G. Gade. 1976. The biological role of octopine
in the squid. Luligo vu/garis (Lamarck). J. Comp. Physiol. 108:
225-232.

Harvey, P. H., and M. D. Pagel. 1991. The Comparative Method in
Evolutionary Biology. Oxford University Press. New York. 239 pp.

Herring, P. J., P. N. Dilly, and C. Cope. 1994. The bioluminescent
organs of the deep-sea cephalopod Vampyroteulhh inlemalis
(Cephalopoda: Vampyromorpha). J. Zool. Land. 223:45-55.

Hoar, J. A., E. Sim, D. M. Webber, and R. K. O'Dor. 1994. The role
of fins in the competition between squid and fish. Pp. 27-43 in Me-
chanics ami Physiology of Animal Swimming. L. Maddock. Q.
Bone, and J. M. V. Rayner. eds. Cambridge University Press, New-
York.
Ikeda. T. 1988. Metabolism and chemical composition of crusta-

ceans from the Antarctic mesopelagic zone. Deep-Sea Res. 35:
1991-2002.

Kerfoot, W. B. 1970. Bioenergetics of vertical migration. Am. Nat.
104: 529-546.

Lu, C. C., and C. F. E. Roper. 1979. Cephalopods from deepwater
dumpsite 106 (western Atlantic): vertical distribution and seasonal
abundance. Smilhson. Contrih. Zool. 288: 1-36.

Madan, J. J., and M. J. Wells. 1996. Why squid breathe easy. Nature
380: 590.

Marshall, N. B. 1979. Developments in Deep-Sea Biology. Blandford
Press. Poole. England. 566 pp.

McFall-Ngai, M. J. 1990. Crypsis in the pelagic environment. Am.
Zool. 30: 175-178.

Mickel, T. J., L. B. Quetin, and J. J. Childress. 1983. Construction
of a polarographic oxygen sensor in the laboratory. Pp. 81-89 in
Polamgruphic Oxygen Sensors. E. Gnaiger and H. Forstner. eds.
Springer-Verlag. Berlin.

Nixon, M. 1983. Teiithonvnia megalops. Pp. 233-247 in Cephalopod
Life Cycles P. R. Boyle, ed. Academic Press, London.

O'Dor, R. K. 1982. The respiratory metabolism and swimming per-
formance of the squid. Loligo opulescens. Can. J. Aauat. Sci. 39:
580-587.

O'Dor. R. K. 1992. Big squid in big currents. 5. Afr. J Mar. Sci. 12:
225-235.

O'Dor, R. K., and R. E. Shadwick. 1989. Squid, the Olympic cepha-
lopods../ Ceph. Biot 1:33-54.

O'Dor, R. K., and D. M. Webber. 1986. The constraints on cephalo-
pods: why squid aren't fish. Can. J Zool. 64: 1591-1605.

O'Dor, R. K., and D. M. Webber. 1991 . Invertebrate athletes: trade-
offs between transport efficiency and power density in cephalopod
evolution. J. Exp. Biol. 160: 93-1 12.

O'Dor, R. K., J. Wells, and M. J. W ells. 1990. Speed, jet pressure,
and oxygen consumption relationships in free-swimming .\aiililtts
J. Exp. Biol. 154: 383-396.

Packard, A. 1972. Cephalopods and fish: the limits of convergence.
Biol. Rev 47:241-307.

Pickford, G. E. 1946. I 'ampyroteiil/us inlemalis Chun, an archaic di-
branchiate cephalopod. 1: Natural history and distribution. Dana-
Rep 29: 1-40.

Pickford, G. E. 1952. The Vampyromorpha of the Discovery expedi-
tions. Discovery Rep 26: 197-210.

Portner, H. O., D. M. Webber, R. K. O'Dor, and R. G. Boutilier. 1993.
Metabolism and energetics in squid (Illex illecehrosns. Loligo
pealei) during muscular fatigue and recovery. Am. J. Physiol 265:
R157-R165.

Roper, C. F". E. 1969. Systematics and zoogeography of the
worldwide bathypelagic squid Balhyteuthis (Cephalopoda: Oegop-
sida). L'. S Nat. Mus. Bull. Washington D.C. 291.

Roper, C. F. E., and R. E. Young. 1975. Vertical distribution of pe-
lagic cephalopods. Smnhson. Contrih. Zool 209: 1-51.

Roper, C. F. E., M. J. Sweeney, and C. E. Nauen. 1984. l-'AO Species
Catalogue. I'ol. 3 Cephalopods of the World. An Annotated and
Illustrated Catalogue o/ Species ol Interest to Fisheries. 277 pp.

Sanders. N. K., and J. J. Childress. 1990. Adaptations to the deep-
sea oxygen minimum layer: oxygen binding by the hem<v\anin of
the bathypelagic mysid. Gnalhophaiisia ingen.s Dohrn liiol. Bull
178:286-294.

Schmidt-Nielsen, K. 1983. Animal Physiology: Adapt. • " and I-'nn-
ronment. Cambridge University Press. Cambridge. < ' ^ pp.

Seapy, R. R., and R. K. Young. 1986. Concealmenl i pipelagicpter-
otracheid heteropods (Gastropoda) and cranchiiu quids (Cephalo-
poda). J. Zool. 210: 137-147.

Seibel, B. A., and .I.J. Childress. 1996. Deer-breathing cephalo-
pods11 Nature 384: 42 1 .

278

B. A. SEIBEL ET AL.

Smith, K. L., and R. R. Hessler. 1974. Respiration of benthopelagic
fishes: in silii measurements at 1 230 m. Science 184: 72-73.

Sullivan. K. M., and G. N. Somero. 1980. Enzyme activities of fish
skeletal muscle and brain as influenced by depth of occurrence and
habits of feeding and locomotion. Mar. Biol 60: 91-99.

Sweeney, M. J., C. F. E. Roper, K. M. Mangold, M. R. Clarke, and
S. V. Boletzky. 1992. "Larval" and juvenile cephalopods: a man-
ual for their identification. Smilhson. Contnh. Zoo! 513: 282.

Thuesen, E. V., and J. J. Childress. I993a. Enzymatic activities and
metabolic rates of pelagic chaetognaths: lack of depth-related de-
clines. Limnol. Oceanogr. 38: 935-948.

Thuesen, E. V., and J. J. Childress. 1993b. Metabolic rates, enzyme
activities, and chemical compositions of some deep-sea pelagic
worms, particularly Nectonemenes mirabilis (Nemertea; Hoplo-
nemertinea) and Poeobius meseres (Annelida; Polychaeta). Deep-
Sea Res. 140:937-951.

Thuesen, E. V., and J. J. Childress. 1994. Respirator, rates and met-
abolic enzyme activities of oceanic California medusae in relation
to body size and habitat depth. Biol Bu/l. 187: 84-98.

Torres, J. J., and G. N. Somero. 1988. Vertical distribution and me-
tabolism in antarctic mesopelagic fishes. Com/' Biochem. Physiol.
908:521-528.

Torres, J. J., B. W. Belman, and J. J. Childress. 1979. Oxygen con-
sumption rates of midwater fishes as a function of depth of occur-
rence. Deer-Sea Rex. 26A: 185-197.

Vecchione, M., and C. F. E. Roper. 1991. Cephalopods observed
from submersibles in the Western North Atlantic. Bull Mar Sci.
49:433-445.

Vecchione, M., B. II. Robison, and C. F. E. Roper. 1992. A tale of two
species: tail morphology in paralarval Chirotcut/iis (Cephalopoda:
Chiroteuthidae). Pr,n Biol Soc llas/i 105(4): 683-692.

Voight, J. R., H. O. Former, and R. K. O'Dor. 1994. A review of am-
monia-mediated buoyancy in squids (Cephalopoda: Teuthoidea).
Mar. Fresh Behav Phyxiol. 25: 193-203.

Vogel, S. 1994. Life in Moving Fluids. Second Edition. Princeton
University Press. Princeton. 467 pp.

Voss, G. L. 1967. The biology and bathymetric distribution of deep-
sea cephalopods. Stud. Trap. Oceanogr. 5: 5 1 1-535.

Voss, N. A., and R. S. Voss. 1983. Phylogenetic relationships in the
cephalopod family Cranchiidae (Oegopsida). Malacologia 23(2):
397-426.

Wells, M. J. 1988. Oxygen extraction and jet propulsion in cephalo-
pods. Can. J. Zoo/. 68: 8 1 5-824.

Wells, M. J.. and R. K. O'Dor. 1991. Jet propulsion and the evolu-
tion of the cephalopods. Bull Mar. Sci. 49:419-432.

Wells, M. J., J. Wells, and R. K. O'Dor. 1992. Life at low oxygen
tensions: the behaviour and physiology of Nautilus pompilius and
the biology of extinct forms. J. Mar. Biol. Assoi: L'.fi. 72: 313-
328.

\\ormuth, J. II. 1976. The biogeography and numerical taxonomy
of the oegopsid squid family Ommastrephidae in the Pacific Ocean.
Bull. Scrip/is Ins/ Oceanogr L'niv. Calif. 23.

Young, R. E. 1972. The systematics and areal distribution of pelagic
cephalopods from the seas off southern California. Smithson. Con-
inh /.i>i>l 97: 1-159.

Young, R. E. I975a. l.eachia pacijica (Cephalopoda, Teuthoidea):
spawning habitat and function of the brachial photophores. Pac.
Sci. 29: 19-25.

Young, R. E. 1975b. Transitory eye shapes and the vertical distribu-
tion of two midwater squids. Pac. Sci. 29: 243-255.

Young, R. E. 1978. Vertical distribution and photosensitive vesicles
of pelagic cephalopods from Hawaiian waters. Fish Bull 76: 583-
615.

Yuung, R. E., E. M. kampa, S. D. Maynard, F. M. Mencher, and
C. F. E. Roper. 1980. Counterillumination and the upper depth
limits of midwater animals. Deep-Sea Res 27A: 67 1 -69 1 .

Annum. V. A. 1978. Possible relationship between energy metabo-
lism of muscle and oxygen binding characteristics of haemocyanin
of cephalopods. J. Mar Biol.Assoc. V K 58:421-424.

Reference: Bio/ Bull 192: 279-289. (April. 1997)

Plasticity in the Sclerites of a Gorgonian Coral: Tests of
Water Motion, Light Level, and Damage Cues

JORDAN M. WEST
Section of Ecology and Systematics, Corson Hall, Cornell University, Ithaca, New York 1 4853-2701

Abstract. The gorgonian coral Briareum asbestinum
contains skeletal elements (sclerites) that vary in length
and density within and among local populations. Data
from previous work suggested that the sclerite composi-
tions of colonies may be altered in response to environ-
mental cues such as predator damage, water motion, and
light level. To test these hypotheses, colonies from shallow
reefs were transplanted to racks at a single location where
the three environmental factors of interest were artificially
manipulated. After 9- 14 weeks of growth, sclerite mor-
phologies and densities had not changed in response to
shading or to water-motion reductions that mimicked
deep-water conditions. However, colonies did respond
significantly to two types of simulated predator damage.
Following tip amputation, sclerites in the regenerated tips
of damaged colonies were shorter and more dense than
in the controls. In contrast, mid-branch scarring caused
colonies to produce longer sclerites at lower densities.
Since long sclerites deter feeding by predatory snails, the
increase in sclerite length in response to scarring of mid-
branch regions may function as an inducible defense.

Introduction

Phenotypic plasticity — or differential phenotypic ex-
pression of a genotype under varying environmental
conditions — has been a subject of increasing interest to
evolutionary ecologists since Bradshaw's (1965) exten-
sive review (e.g.. Bradshaw, 1974; Via and Lande, 1985;
Schlichting, 1986; Stearns, 1989; Scheiner, 1993). Al-
though plasticity can be nonadaptive or maladaptive, re-
searchers have been primarily interested in adaptive
plasticity as a mechanism by which organisms cope with

Received 1 8 September 1996; accepted 6 February 1997.
Present address: University of Washington. Friday Harbor Labora-
tories. 620 University Road, Friday Harbor, Washington 98250.

changing environments. Adaptive plasticity is especially
well documented in plants, for which the literature con-
tains numerous examples of induced responses to physi-
cal factors such as shade (Turesson, 1920), desiccation
(Harlan, 1945), soil fertility (Sorensen, 1954), tempera-
ture (Mooney and West, 1964), and water stress (Roy
and Mooney, 1982). Bradshaw (1965) reasoned that
plasticity should often be favored in plants because they
are sessile organisms whose autotrophic lifestyle requires
that they inhabit relatively open spaces where they may
be exposed to environmental extremes. When the scale
of temporal variability is shorter than the lifespan of the
organism, or where spatial variability is at a scale smaller
than the dispersal range of the organism, adaptive plas-
ticity is a viable strategy.

In marine habitats, sessile colonial invertebrates, such
as scleractinian and gorgonian corals, share many char-
acteristics with plants. Colonies are permanently sessile,
often with upright branched growth forms (Barnes,
1987); and many corals depend on symbiotic algae (zoo-
xanthellae) for a large proportion of their energy (Mus-
catine, 1974; Muscatine et a/., 1975; Svoboda, 1978;
Sebens, 1987) and are thus largely autotrophic. In addi-
tion, populations with wide distributions are routinely
exposed to variation in abiotic factors such as light level
(McCloskey and Muscatine, 1984; Miles. 1991), wave
exposure (de Weerdtr 1981: Sebens. 1984). and sedi-
mentation rate (Foster, 1979). Indeed, variation in col-
ony morphology or physiology in response to tempera-
ture, depth, or water movement has been documented
in sponges (Bavestrello et a/.. 1993), scleractinian corals
(Wijsman-Best, 1974; Foster. 1979; Lesser et <//.. 1994).
and gorgonian corals (Grigg, 1972; West et n 1993).

In both plants and corals, inducible defer- :s represent
a special category of adaptive plasticity tli, involves bi-
otic rather than abiotic cues. Defined as e' • ironmentally
triggered phenotypic responses that defl ,d against spe-

279

280

J. M. WEST

cific biotic selective agents (Adler and Harvell, 1990), in-
ducible defenses have emerged as prominent phenom-
ena only in the past 20 years. A variety of plant responses
to real or simulated herbivore damage include increased
morphological defenses such as spines or resistant
growth forms (Young, 1987; Lewis el ai. 1987) and pro-
duction of physicochemical deterrents such as polyphe-
nolic compounds, alkaloids, or silica (McNaughton and
Tarrants, 1985; Schultz, 1988; Van Alstyne, 1988; Bald-
win and Ohnmeiss, 1993). Among colonial inverte-
brates, various species respond to the presence of com-
petitors by producing agonistic weaponry such as diges-
tive filaments, stolons, or sweeper tentacles (hker, 1972;
Francis, 1973; Lang, 1973; Wellington, 1980; Sebens
and Miles, 1988; Harvell and Padilla, 1990; Miles, 1991).
However, although predator-induced defenses, such as
spines and helmets, have been documented in various
clonal freshwater invertebrates (e.g., Gilbert and Stemb-
erger, 1984; Havel, 1986). examples of induced defenses
against predators in colonial marine invertebrates (Har-
vell, 1984) are surprisingly rare.

In the Caribbean coral Briareum asbestinum (Gor-
gonacea), both biotic and abiotic environmental agents
may contribute to plastic variation in skeletal features.
Unlike the hard corals with their massive calcium car-
bonate skeletons, gorgonian corals consist of a central
axis surrounded by an outer cortex of a soft, polyp-bear-
ing matrix, the structural integrity of which is main-
tained by small (0.2-1.2 mm), calcitic skeletal elements
(sclerites) (Fig. 1 ). Within and among local populations,
B. asbestinum colonies vary significantly in the mean
lengths and densities of their sclerites. Furthermore, a re-
ciprocal transplant of colonies between shallow and deep
sites indicated that sclerite morphology is phenotypically
plastic in response to one or more environmental cues
(West?/ a/.. 199 3; West, 1996).

One such cue may be damage from predators. In addi-
tion to skeletal support, sclerites fulfill a second role as
structural defenses against predators. B. asbestinum con-
tain^ hthyodeterrent, diterpene secondary compounds
(Paw i. ' a/.. 1987), and predation by fishes appears to
be m; 'C. D. Harvell, unpubl. data). However, the

gastropo'- I'oma gibbosum is a major predator of
gorgonian (Kinzie, 1970; Lasker and Coffroth,

1 988; Lask 1 988) and is an important source of

damage to B. a urn (Hazlett and Bach, 1 982). C'r-

phoma gibbosum ains biotransformation enzymes
capable of detoxilv asbestinum allelochemicals

( Vrolijk and Targett, 1 9<>2) and consumes artificial foods
containing B. asbestinum extracts and pure compounds
just as readily as it consumes control foods (C. D. Har-
vell, unpubl. data). Therefore, the sclerites of B. asbesti-
num may be its only defense against this predator, such
that differences in skeletal composition will correlate

0.1 mm

Figure 1 . Briareum asbexlinum. Diagram of typical sclerites, show-
ing the 23'1! difference in mean sclerite length that is inducible and
affects snail feeding (West, 1996). Colonies within the same local pop-
ulation can differ in mean sclerite length by as much as 32% (West,
1996).

with the ability of colonies to deter snail feeding (West el
ai, 1993; West, 1996). As sclerites increase in size, they
render artificial foods less palatable to C. gibbosum (Van
Alstyne and Paul, 1992; West et ai, 1993; West, 1996).
Hence, the inducibility of long, defensive sclerites as a
reaction to damage would be a potentially advantageous
plastic response.

In addition to biotic induction, sclerite plasticity
among B. asbestinum populations may also be cued by
changing abiotic conditions. At two islands in the Carib-
bean, colonies of B. asbestinum are distributed along a
depth gradient of 1-30 m. At the shallow end of the gra-
dient, sclerites are short and of high density, whereas
deep colonies contain sclerites that are long and of low
density. Sclerite length and density within colonies are
negatively correlated, such that colonies with both long
and densely packed sclerites do not occur (West et ai,
1 993; West, 1 996). Although biotic induction by a patch-
ily distributed predator could account for variability
within sites, the pattern of increasing sclerite length with
depth cannot be explained solely by the presence of pred-
ators. Snails are indeed present at all of my study sites,
but their densities and damage actually decrease with
depth, whereas sclerite length increases with depth
(West, 1996;C. D. Harvell, unpubl. data). Hence, larger-
scale patterns of sclerite variability with depth may be
generated by abiotic environmental factors.

Along the depth cline. declining light penetration and

SCLERITE PLASTICITY IN A CORAL

281

water motion both show some degree of correlation with
sclerite variation (West. 1996). Both factors might affect
skeletal composition in different ways and might func-
tion as cues that induce a plastic response in sclerite
length and density. Observed correlations between scle-
rite variation and percent penetration of photosyntheti-
cally active radiation (West, 1996) may be related to
effects of light on colony growth rates (C. D. Harvell, un-
publ. data), because gorgonians depend on their symbi-
otic zooxanthellae for most of their energy (Sebens,
1987). In addition, strong correlations between sclerite
variation and water motion (West. 1996) may relate to
the function of sclerites as skeletal support structures.
The sclerites of B. asbestinum act as rigid reinforcing
points of attachment within the soft matrix, providing
resistance to deformation; both smaller sclerites and
greater densities of sclerites confer greater stiffness
(Wainwright et al. 1976; Koehl, 1982; Palumbi, 1986).
Therefore, colonies may display depth-related shifts in
skeletal composition according to water motion and
light level cues.

In this study, I tested the ability of one biotic agent
(predator damage) and two abiotic factors (water motion
and light) to induce skeletal modifications in the soft
coral B. asbestinum. Because the large collections
needed for this work would have denuded the sparsely
populated deep reefs at my sites, I focused on shallow-
water B. asbestinum. To examine separately the effect of
each type of cue, two large transplant experiments were
conducted in which colonies from a shallow population
were grown on racks at a single site where water motion,
light, and damage were manipulated under controlled
conditions. I hypothesized that shallow-water colonies
subjected to reduced water motion and reduced light
(simulating deep-water conditions) and colonies sub-
jected to mechanical scarring (simulating predator dam-
age) should respond in each case by producing longer
sclerites at lower densities compared to controls.

Materials and Methods

Experiment I: water motion and damage

To assess whether differences in water motion and
simulated predator damage would induce plastic
changes in sclerite composition, I conducted a transplant
experiment at San Salvador, Bahamas, from 1 June to 3
August, 1991. Branches from a shallow ( 1-3 m) popula-
tion of Briareum asbestinum were transplanted to racks
on which they reattached themselves and grew as inde-
pendent new colonies. The racks were arrayed at a single
location where water motion and damage were varied.
The 12 racks were constructed according to a design
modified from West et al. (1993). Each rack consisted of
an acrylic plate, dimensions 30.0 X 23.0 X 2.5 cm, that

snapped into an aluminum angle frame. Each plate ac-
commodated 12(2 rows of 6 (colonies, each inserted into
a recessed well and secured with cushioned cable ties to
an acrylic post.

The racks were rigidly affixed to cement blocks at a
shallow (3m) site that exposed the colonies to high-en-
ergy waves and surge. Water motion was reduced inside
half of the racks by clear, small-mesh (0.20 mm) nylon
screening that was attached to form walls 1 5 cm tall (Fig.
2). Water motion inside control and walled racks was
quantified by recording the percent dissolution of plaster
of Paris (a water-motion index) using methods adapted
from Muus (1968), Doty (1971), Day (1977), Bushek
(1988), and Jokiel and Morrissey (1993). For this inte-
grated relative measure of water movement due to cur-
rent velocities and turbulence (Doty, 1971), I measured
the weight loss of five replicate plaster domes (24 g) for
each rack type over a 24-h period (see West, 1996, for a
more detailed description). When compared with mea-
surements made at various other B. asbestinum habitats,
the reduction in water motion between walled and con-
trol racks was found to be similar to differences in water
motion between colonies growing deep within crevices
and colonies out on the open reef flat. Naturally occur-
ring B. asbestinum colonies from these habitat types
differ significantly in sclerite composition (West, 1996).

All racks were fitted with clear, large-mesh (2.5 cm)
roofs that afforded protection from disturbance by fishes
without affecting light penetration. Furthermore, place-
ment of the racks in a sand patch several meters from
the reef prevented discovery of the colonies by benthic
predators (confirmed through weekly monitoring). Light
measurements with a quantum/radiometer/photometer
(LI-185B with LI-192SB underwater sensor; LI-COR,
Inc., Lincoln. Nebraska) showed that penetration of pho-
tosynthetically active radiation (PAR) was the same in-
side and outside of walled and control racks. During the
experiment, the walls and roofs were scrubbed regularly
with a stiff brush to remove fouling organisms.

The effects of damage due to simulated predation or
breakage were tested by assigning each colony to one of
three damage treatments: ( 1 ) control; (2) scar; and (3) tip
amputation (Fig. 2). The controls were left undamaged.
The scar treatment simulated damage by Cyphoma gib-
bositm (Gastropoda), a predator that is typically found
on the middle region of colony branches (Harvell and
Suchanek, 1987; Gerhart, 1990), where it can rasp the
cortex as deep as the axis (Harvell and Suchanek, 1987;
West, 1996). Hence, starting about 3 cm from the tip and
working downward, I damaged the mid-re;, ins of colo-
nies by gouging them to the axis to create a 1- X 3-cm
scar. Finally, tip amputation involved sr ering the top
1 cm of the colony to simulate predation or breakage.
Briareum asbestinum is a preferred piv species of Her-

282

10 cm

J. M. WEST

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CONTROL

SCAR TIP AMPUTATION

CONTROL

REDUCED
EXPOSURE

CONTROL

REDUCED
LIGHT

EXPERIMENT 1

EXPERIMENT 11

Figure 2. Design of transplant experiments to test sclerite plasticity in Briareum asbestimim. Damage
types: control = undamaged, scar = 1- x 3-cm gouge, tip = 1-cm tip amputation. Experiment I (exposure
and damage): control and reduced exposure racks. Experiment II (light and damage): control and reduced
light racks.

modice canmciilata (Polychaeta), which commonly
feeds by a characteristic removal of branch tips (Vree-
land and Lasker, 1989). Thus, tip removal may be sim-
ilar to the damage inflicted on colonies by worms. Yet,
tip removal also lays bare a cross-sectional area of the
colony, so amputation also mimics the end product of
breakage resulting from heavy wave action. The original
cortex material from each scar or tip amputation was
preserved in 70% ethanol for later analysis.

Because the number of samples was large, the experi-
ment was set up over 2 days. Each day, 72 undamaged
branches, 10 cm long, were collected from a shallow ( 1-
3 m) reef of high wave energy. To maximize the genetic
diversity of the collection, divers swam linearly along the
reef and sampled single branches from colonies that were
separated from any others by at least 3 m. After collec-
tion, the branches were suspended in flow-through mesh
bags from the side of the boat at the nearby transplant
site. They were then assembled onto racks (equal num-
bers of damage types, randomized with respect to rack
position) inside large containers of seawater in the boat,
and each completed rack was immediately conveyed to
the transplant site below. Virtually all branches began
extending their polyps within 2 h of transplantation.
Within a few weeks, the branches had attached them-

selves to the racks and were growing as independent col-
onies. The colonies were allowed to heal and grow for
9 weeks, and survival exceeded 95%. During the experi-
ment, more than 50% of the colonies increased in height
by 0.1-1.5 cm; and all survivors showed positive growth
in the sense that they produced new cortex material that
encrusted their supportive posts and ties, filled in their
scars, and covered over their tip wounds. At the end of
9 weeks, the colonies were collected and preserved in
70% ethanol until they could be processed in the labora-
tory.

Experiment II: light and damage

To test the effects of reduced light and colony damage,
another transplant experiment was conducted from 7
February to 20 April, 1 99 1 . The methods were the same
as those described for experiment I with the following
modifications. Branches were collected from throughout
a shallow, relatively calm bay and transplanted to a 12-
m location nearby. The colonies were subjected to the
same three types of damage and were distributed sim-
ilarly within the racks, but instead of walls, half of the
racks were fitted with roofs made of a double layer of
dark window screening (Fig. 2). This screening (1-mm

SCLERITE PLASTICITY IN A CORAL

283

mesh) reduced the light levels typical of 12-m depth to
light levels typical of 30-m depth (J. Miles, pers. comm.,
calibrated with Li-Cor light meter). The other half of the
racks (controls) were fitted with roofs made of clear ny-
lon netting (2.5-cm mesh) to discourage disturbance by
fishes without affecting light penetration. The roofs were
scrubbed regularly to remove fouling organisms.

To prevent access by benthic predators, the racks were
floated about 2 m above the reef flat with steel cables and
subsurface buoys. Because the racks could move with the
currents, the effects of water motion were moderated.
Positive growth of the transplanted colonies was similar
to that of experiment I, and colony survival again ex-
ceeded 95%. After 14 weeks, the colonies were preserved
in 70% ethanol until they were processed in the labora-
tory.

Sclerite measures

At the end of each experiment, subsets of 1-3 colonies
per damage type per rack were measured. Sclerite lengths
were recorded in the laboratory with the MORPHOSYS
image analysis program (Meacham and Duncan, 1990;
version 1 .26). For colonies subjected to scarring or tip
amputation, both the original cortex material collected
when the colonies were damaged ("before" sample) and
newly regenerated material from the healed wound ("af-
ter" sample) were analyzed. In experiment I, a mid-
branch cortex sample from the side opposite the scar
("opposite" sample) was also analyzed to see whether the
response to scarring was regionwide. Hence, cortex ma-
terial was sampled from the following regions of the col-
onies: ( 1 ) tip region of damaged colonies, before and
after amputation; (2) mid-branch region of damaged col-
onies, before, after, and opposite the scars; and (3) mid-
branch and tip regions of control colonies at the end of
the experiment (after).

For sclerite length measurements, cortex subsamples
were taken from the edges of scars before and after heal-
ing, from original and healed tips at a distance of 1 cm
down from the apex, and from the same locations (by
distance from the apex) in the controls. In each case, two
small (3-4 mm3) cortex samples were excised, and the
organic matter was dissolved away with a solution of
2.6% sodium hypochlorite. The isolated sclerites were
rinsed and distributed in their entirety across six slides
(three slides per tissue sample). Video images of the first
4 intact sclerites encountered per slide were measured,
for a mean of 24 sclerites per region per colony.

From the same regions of the colonies, the proportion
of cortex weight consisting of sclerites (the sclerite weight
fraction, a measure of density) was estimated according
to Harveli and Suchanek's (1987) protocol. From mid-
branch regions, a 1- X 3-cm cortex scraping at the edge

of the original scar (or from the middle region of the con-
trol) was taken, and from tip regions, the distal 1 cm of
cortex material was used in all cases. The samples were
separately dried for 24 h at 60°C and weighed; a drying
test on a subset of samples showed that they did not con-
tinue to lose weight after 24 h. Each sample was then
ashed in a muffle furnace at 450°C for 1 h. This process
burned the organic matter away, but left the sclerites in-
tact (Harveli and Suchanek, 1987). The sclerite weight
fraction was calculated as the proportion of the total cor-
tex weight consisting of sclerites (ash weight/dry weight).

Statistical analyses

All length and weight fraction distributions were nor-
mal and homoscedastic, so groups were compared using
parametric tests. The "after" data from experiments I
and II were subjected to split-unit (or split-plot) analysis
of variance (Neter et al, 1990). Blocks, which tested for
microenvironmental effects within the experimental site,
consisted of pairs of adjacent racks (units), one unit be-
ing a control rack while the other was a reduced exposure
(experiment I) or reduced light (experiment II) rack.
Within racks, individual colonies (subunits) received the
different damage treatments. Because previous work had
indicated that different regions within colonies may
differ in sclerite composition (West, 1996), mid-branch
and tip regions were examined separately. Hence, for
each experiment, four split-unit analyses were per-
formed: (1) mid-branch sclerite length; (2) mid-branch
sclerite weight fraction; (3) tip sclerite length; and (4) tip
sclerite weight fraction. Because the Unit*Damage term
was not significant in any of the analyses (P > 0.29 in all
cases), it was removed from the model and its sum of
squares was pooled with the error sum of squares for final
calculation of F-statistics (Neter et a/.. 1990).

Mid-branch and tip-damage responses detected in the
split-unit ANOV As were confirmed through comparison
of "before" and "after" material from within damaged
colonies. Both before and after measurements were
taken for each mid-damaged or tip-damaged colony, and
my a priori expectation was that after and before samples
would differ in the same direction as would the after and
control samples; hence, I analyzed the before and after
data using paired one-tailed Mests. Data from opposite
the scars (experiment I only) were also compared to be-
fore data using a paired one-tailed /-test.

In both experiments, the different contrasts of interest
involved multiple comparisons and contained variables
that lacked independence from variables in other tests
(e.g.. sclerite length and sclerite weight fraction are from
the same colony; control mid-branch and control tip are
from the same colony). Hence, a sequential Bonferroni
correction for multiple tests was performed (Holm,

284

J. M. WEST

1979; Rice, 1989). The sequential Bonferroni method is
less conservative and more powerful than the standard
Bonferroni method yet still restricts the probability of a
type-I error for the .Miiire test as well as each step of the
testto« = 0 (Holm, 1979; Rice, 1989).

Results

Damage

In both experiments I and II, mean sclerite length in-
creased 23% in regenerated mid-branch scars (see Fig. 1 ),
whereas sclerite weight fraction decreased 2.3% (Fig. 3).
The increase in sclerite length was significant for the after
and control comparisons of the split-unit ANOVAs (Ta-
bles I, II) as well as for the before and after paired com-
parisons (Fig. 3A). Mean sclerite weight fraction de-

MID-BRANCH RESPONSE
Experiment I Experiment II

A

0.5-

-J-

*

4

Sclerite Length (mm

D 0 0 0

j k> UJ f-

Table I

Summary ofsplii-unii analyses of variance on sclerite length and
sclerite weight fraction tor Bnareum ashestinum in experiment I
(June-August 1991)

P value

Comparison

region

Source

Length

Wt. fraction

Tip

Block

0.954

0.040

Exposure

0.465

0.294

Block* Exposure

0.201

0.946

Damage

0.062

0.742

Mid-branch

Block

0.284

0.340

Exposure

0.388

0.240

Block* Exposure

0.663

0.896

Damage

0.005*

0.939

Control Before After Opposite

N=12 N=20 N=21 N=21

Control Before After

N=30 N=20 N=22

Note: Tip region: comparison of protected to exposed colony tips and
comparison of damaged colony tips to undamaged control tips; mid-
branch region: comparison of protected to exposed mid-regions and
comparison of damaged colony mid-regions to undamaged control
mid-regions. Blocks (pairs of adjacent racks) test for microenvironmen-
tal effects. *denotes significance under sequential Bonferroni adjust-
ment.

creased in cortex material after scar damage, but the
effect was statistically significant only for the before and
after comparison of experiment II (Tables I, II; Fig. 3B).
Thus, simulated predator damage did alter the average
lengths and (in one case) the average weight fractions of
B. asbestinum sclerites. Moreover, the mid-branch in-
crease in sclerite length was not restricted to newly
formed cortex material within the healed scars. Sclerites

B

084-|

0.78

T

T

T

T

T

T

Control Before After

N=12 N=20 N=21

Control Before After

N=31 N=31 N=33

Figure 3. Bnareum asbestinum. Response of colony mid-branch
regions to simulated predator damage: (A) mean sclerite lengths (mm);
and (B) mean sclerite weight fractions. Error bars are 1 standard error.
Groups: control = undamaged mid-branch tissue at end ot experiment;
before = original mid-branch tissue gouged at start of experiment; after
= regenerated mid-branch scar tissue at end of experiment; opposite =
tissue from opposite the scar, sampled at the end of the experiment.
The sample sizes for paired Mests are indicated by the /; values of the
before groups. Under sequential Bonferroni adjustment, "denotes sig-
nificance of both the control versus after group comparison and the
before versus after paired /-test; *denotes significance of the paired /-
test only.

Table II

Summary of split-unit analyses <>/ variance on sclerite length and
sclerite weight fraction for Briareum asbestinum in experiment II
(February-April 1991)

Comparison
region

Source

P value

Length

Wt. fraction

Tip

Block

0.870

0.294

Shading

0.977

0.433

Block* Shading

0.286

0.126

Damage

0.005*

0.971

Mid-branch

Block

0.666

0.532

Shading

0.191

0.865

Block*Shading

0.348

0.092

Damage

0.003*

0.559

Note: Tip region: comparison of shaded to unshaded colony tips and
comparison of damaged colony tips to undamaged control tips; mid-
branch region: comparison of shaded to unshaded mid-regions and
comparison of damaged colony mid-regions to undamaged control
mid-regions. Blocks (pairs of adjacent racks) test for microenviron men-
tal effects. *denotes significance under sequential Bonferroni adjust-
ment.

SCLERITE PLASTICITY IN A CORAL

285

from material opposite the scar were also significantly
longer than the sclerites before damage (Fig. 3 A), indi-
cating that this length response was regionwide.

The response to tip removal was opposite to that of
mid-branch scarring. Colonies subjected to tip amputa-
tion produced new tips that contained sclerites that were
up to 16% shorter and made up 2.5% more of the total
cortex weight (Fig. 4). The significant difference between
tips after damage and control tips is reflected in the dam-
age term of the split-unit ANOVA for experiment II (Ta-
ble IIA). The difference between damaged tips and con-
trols was not statistically significant in experiment I (Ta-
ble IA; Fig. 4A). However, within colonies, both
experiments showed a significant decrease in sclerite
length from before to after damage (Fig. 4A). Increases
in mean weight fraction in healed tips were statistically
significant only for the before and after comparison of
experiment I (Tables I, II; Fig. 4B).

H 'ater motion

Dissolution of plaster of Paris domes over 24 h showed
that water motion inside the walled racks was signifi-
cantly reduced compared to water motion within control
racks (P = 0.01, unpaired I test; Fig. 5). The magnitude
of the reduction was similar to water-motion differences
measured for colonies growing within crevices versus on
the open reef flat at a variety of sites around San Salvador
(Fig. 5). Such microhabitat differences have been corre-
lated with significant sclerite variation, with micropro-
tected colonies containing longer sclerites at lower den-
sities than microexposed colonies (West. 1 996).

When colonies from a shallow site of high-energy
waves and surge were shielded from water motion within
the walled racks, their sclerites did not differ from the
sclerites of control colonies after 9 weeks. This result was
consistent for both mid-branch and tip regions of colo-
nies (Table I). In summary, the skeletal composition of
Briareum asbestinum colonies was not modified in re-
sponse to the degree and duration of water-motion re-
ductions tested here.

Light

In experiment II, shallow-water colonies were sub-
jected to greatly reduced light levels by shading them
with dark screens that had been previously determined
to reduce the light levels typical of 12 m to those typical
of 30 m (J. Miles, pers. comm.). After 14 weeks, shaded
colonies did not differ significantly from unshaded con-
trols in either sclerite length or sclerite weight fraction.
This result was consistent for both mid-branch and tip
regions of colonies (Table II). Thus, as with water mo-
tion, there was no indication that the light reductions

TIP RESPONSE
Experiment I Experiment 11

A

0.5 -i

•c
u

~ 03-
tfl

T

Control Before

N=12 N=ll

After

N=12

Control Before After

N=29 N=26 N=27

0.78 H

op
"S

0.75-

T

T

T

Control Before After

N=24 N=ll N=19

Control Before After

N=35 N=29 N=36

Figure 4. Briareum asheslinum. Response of colony tip regions to
simulated predator damage: (A) mean sclerite lengths (mm); and (B)
mean sclerite weight fractions. Error bars are 1 standard error. Groups:
control = undamaged tips at end of experiment; before = original tips
amputated at start of experiment; after = regenerated tips at end of
experiment. The sample sizes for paired /-tests are indicated by the n
values of the before groups. Under sequential Bonferroni adjustment,
"denotes significance of both the control versus after group compari-
son and the before versus after paired /-test: *denotes significance of the
paired /-test only.

tested in this experiment triggered a plastic response in
sclerite composition.

Discussion

Environmental cues

Briareum asbestinum colonies responded to one of the
three types of environmental cues tested in this study.
Simulated predator damage caused clear and significant
changes in sclerite morphology, and these changes actu-
ally reversed patterns of sclerite length that were ob-
served in colonies without mid-branch or tip damage.
Samples collected from naturally growing colonies in the
field display a consistent pattern in which the sclerites are
longer at the tips of colonies than at the basal regions;
similarly, the experimental controls contained longer
sclerites at the tips than at the mid-branch regions (Fig.

286

J. M. WEST

I 55

Nm
Point

controls

Water Motion
Racks

Rice Bay

Site

Figure 5. Comparison of mean water-motion index (percent disso-
lution of plaster of Paris domes over 24 h) for walled and control exper-
imental racks and for other Briamim asbestinwn sites of similar depth
(1-4 m) (n = 4-5 domes in all cases). Error bars are one standard error,
p = protected microhahitat (where colonies are growing within shel-
tered crevices), e = exposed microhabitat (where colonies are growing
on the open reef flat).

6). In contrast, sclerite length decreased in regenerated
tips and increased in mid-branch scars, such that the
usual pattern of variation within undamaged branches
was fully reversed (Fig. 6). Furthermore, sclerite length
increased not only within mid-branch scars, but also in
material located on the opposite side of the colony from
the actual wound. Hence, this is a regionwide response
that likely affects the entire band of cortex material sur-
rounding a scar.

These mid-region increases in mean sclerite length are
more likely due to sclerite turnover than to the simple
addition of many long sclerites. If the average length of
sclerites was shifted by the accumulation of very long
sclerites without the loss of any small sclerites, then we
would expect to see greater variances associated with scar
means than with control means and higher sclerite
weight fractions within scars than in controls. Instead,
scarred colonies have length variances that are compara-
ble to controls, and they have reduced weight fractions
(Fig. 3).

In general, the changes in sclerite weight fraction re-
flected the previously observed negative correlation be-
tween sclerite length and sclerite density. Weight fraction
'ied to decrease as sclerite length increased within
i scars, and the opposite was true for regenerated
nvever, for each region (tip and mid-branch), the
nduced change in weight fraction was statisti-
cal <icant for only one out of six contrasts tested.
s, the significant weight fraction responses
that 'ected, when combined with the dramatic
and o changes in sclerite length ( 16%-23%) ob-
served, pi . strong evidence of plasticity.

In contrast to the damage responses, there was no in-
dication that the sclerites of shallow-water colonies were
altered in response to the abiotic cues of reduced water
motion or reduced light. Depth-related sclerite variation
may therefore include a component of genetic differen-
tiation. With its ability to reproduce asexually through
fragmentation (Brazeau, 1989) and its brooded, nega-
tively buoyant larvae (Brazeau and Lasker, 1990), B.
asbestinwn is probably relatively philopatric. Indeed, an
electrophoretic study performed concurrently with this
work indicates significant genetic differentiation be-
tween shallow and deep populations (Brazeau and Har-
vell, 1 994). Yet there is also some indication that sclerites
are altered in response to wounds that mimic breakage
(see discussion below), and reciprocal transplantation re-
vealed a plastic change in sclerites with very extreme
differences in depth (West el al.. 1993). Here. I tested
the more usual variation in light and water motion that
colonies in highly populated shallower environments en-
counter. As such, the exposure and light treatments ap-
plied to B. asbestimtm over 9-14 weeks did not induce
significant changes in sclerite length or sclerite weight
fraction. Further testing is warranted to determine
whether a longer duration of acclimation could eventu-
ally result in plastic shifts in sclerite composition.

Ecological and evolutionary implications

When colony tips regenerate, the new cortex contains
sclerites of decreased length and increased weight frac-

D Tip

D Middle/Base

040-

0 36 -

v 028-

<" 0 24 -

0.20

Bonefish Bay Control Colonies Damaged Colonies

Source

Figure 6. Briarcum asbestinum Comparison of mean sclerite
length (mm) in different regions of naturally growing colonies IW.VH.V
experimental colonies. Source groups: Bonefish Ba\ = samples from
tip and base regions of colonies (n = 16) growing naturally near the
transplant sites. Control Colonies = samples from tip(» = 41 land mid-
branch!// = 42 (regions of pooled expen mental controls. Damaged Col-
onies = samples from regenerated tips (n = 39) and healed mid-branch
scars (11 = 43) of pooled damage treatments. Error bars are I standard
error.

SCLER1TE PLASTICITY IN A CORAL

287

tion. Since tip amputation is a characteristic feeding
mode of the worm Hermodice carunculata, then perhaps
increased sclerite densities are an induced defense that
makes feeding difficult for worms, or renders the food
less palatable (Vreeland and Lasker, 1989). This hypoth-
esis seems unlikely for the following reasons. First, //.
carunculata does not feed exclusively on colony tips, but
also feeds on middle regions of B. asbestinum branches
(Vreeland and Lasker, 1 989) where the response to dam-
age is a decrease in sclerite density. Second, Vreeland
and Lasker (1989) found no relationship in this predator
between preferences for particular gorgonian species and
the ash content (sclerite density) of those species. Finally,
H. carunculata routinely feeds on milleporid fire corals
(Witman, 1988), and one hungry worm in the laboratory
attempted to engulf the tip of a plastic pen cap (pers.
obs.). Hence, a 2.5% increase in sclerite weight fraction
at colony tips would probably be a poor defense against
this predator.

Instead, the plasticity of sclerites in colony tips after
amputation may be a response that decreases further
breakage. Survival is lower for small fragments than for
larger fragments in other gorgonians( Lasker, 1990), thus
breakage of B. asbestinum colonies may result in partial
mortality as water motion abrades loose branch frag-
ments against the reef. In addition, Gerhart ( 1 990) found
that fouling by epibionts such as algae is a serious prob-
lem for gorgonian colonies that have suffered major
damage such as the baring of large wounded areas at
scars or break points. For these reasons, it may be advan-
tageous for colonies to cover such wounds with new cor-
tex material that is fortified against further breakage
through increased packing of smaller sclerites.

Biomechanical assays using both artificial and real tis-
sues have indicated that small sclerites at high densities
confer greater stiffness than longer and more sparsely
packed sclerites (Koehl, 1982). This may be because
small sclerites at high densities provide more surface area
for tissue attachment and also leave smaller spaces con-
sisting solely of deformable soft matrix. In more recent
work, Koehl (1996) has also shown that the strength and
toughness of sclerite-reinforced materials increases —
and then decreases — with increasing sclerite density.
Biomechanical testing of actual B. asbestinum branches
is needed to determine whether the tip response renders
the surrounding matrix stronger and tougher, or more
brittle and breakable. A finding of increased strength and
toughness in regenerated tips would support the hypoth-
esis that the observed sclerite modifications function in
colony reinforcement. This hypothesis could be further
tested by breaking additional colonies at the mid-branch
region instead of the tip region and observing their re-
sponse. To adopt a fortification response of shorter,
denser sclerites at mid-branch, colonies would have to be

capable of reversing the scar-induced sclerite shifts de-
tected in this study (see below).

At the mid-branch region, B. asbestinum colonies re-
spond dramatically to mechanical scarring, producing
sclerites that are 23% longer. The middle region of colo-
nies is the typical feeding location of the snail Cyplioma
^/Mmf/wf Harvell and Suchanek, 1987; Gerhart, 1990),
and longer sclerites have been shown to reduce snail
feeding significantly (West el ai, 1993; West, 1996). At
St. Croix (U.S. Virgin Islands), B. asbestinum colonies
from two habitats contain sclerites that differ in length
by about 22%) (see Fig. 1 ), and individuals of C. gibbosum
that are given a choice feed at a higher rate and spend
more time on the colonies with shorter sclerites than on
those with longer sclerites (West, 1996). It is unlikely that
this effect is due solely to chemistry because C. gibbosum
has gorgonian-detoxifying enzymes ( Vrolijk and Targett,
1992) and appears indifferent to B. asbestinum extracts
in sclerite-free artificial foods (C. D. Harvell, unpubl.
data). Conversely, artificial food assays that employed
isolated sclerites in the absence of chemistry have indi-
cated that this predator is significantly deterred by long
sclerites (West ct ai. 1993; West, 1996). Hence, the pro-
duction of 23%- longer sclerites in the mid-branch regions
of scarred San Salvador colonies may represent an in-
duced defense.

Harvell (1984) hypothesized that predator-induced
defenses should be favored in clonal taxa that suffer in-
termittent, unpredictable nonfatal encounters with pred-
ators. Cyphoma gibbosum frequently causes significant
damage to colonies of B. asbestinum, but it seldom com-
pletely kills them (Kinzie, 1970; Birkeland and Gregory,
1975). At shallow sites of greatest snail activity, the aver-
age amount of surface area scarred per B. asbestinum
colony can approach 30% (West, 1996). Although the
tenure time of individual C. gibbosum on particular gor-
gonian colonies averages only about 10 days (Harvell
and Suchanek, 1987), snails tend to form aggregations
that have been observed to stay together in a localized
area (and even on a single colony) for up to 4 months
(Kinzie, 1970; Birkeland and Gregory, 1975;Hazlettand
Bach, 1982; Gerhart, 1986). Therefore, colonies within
such a feeding area may be grazed upon multiple times,
by multiple snails, over a period of weeks to months.
Meanwhile, B. asbestinum in areas adjacent to large snail
aggregations may be virtually free of predator damage
(pers. obs.). Experiments on feeding behavior have
shown that when given a choice, C. gibbosum tends to
move off of colonies containing long sclerites and onto
colonies containing short sclerites (West, 1996). There-
fore, production of snail-deterrent sclerites by B. asbesti-
num, occurring over a period of months, would be an
appropriate and advantageous defensive response.

A potential cost to this induced defense may relate

288

J. M. WEST

once again to the effects of changes in skeletal composi-
tion on the biomedr.mical behavior of colonies. Tissues
that contain long sclerites at low densities will be less stiff
and more elastic than tissues with short sclerites at high
densities. In the region of a shift to long, sparse sclerites,
a branch will indeed be in little danger of breaking due to
brittleness; yet too great a propensity for pliability might
itself be disadvantageous. A branch that is too soft may
not be able to support the optimal orientation for light or
prey capture, and excessive bending and swaying of the
colony in waves and surge could lead to damage as tissue
is abraded against adjacent coral heads. If so, this may
explain why long sclerites are an induced rather than a
constitutive defense.

The evolution of adaptive plasticity is favored in many
plants and corals because of their distributions across
changing habitats and their inability to escape extreme
environmental conditions through movement (Brads-
haw, 1965). In plants, inducible chemical and morpho-
logical defenses against predators have been widely re-
ported (Schultz, 1988). In colonial invertebrates such as
bryozoans, gorgonians, and scleractinians, it is morpho-
logical defenses against competitors rather than against
predators that have been most widely documented (Ad-
ler and Harvell, 1990). Examples of predator-induced
defenses have been uncommon in colonial invertebrates
and have been cited only for temperate bryozoans (Har-
vell, 1 984). Now, B. asbestinum may represent a new ex-
ample of a tropical colonial invertebrate with an induc-
ible defense against a predator. Another gorgonian (Ple\-
aurella dichotoma) also responds to snail damage by
increasing both the size and density of its sclerites
(Nowlis, West, and May, unpubl. data). These results for
two different genera of gorgonians suggest that the func-
tioning of sclerites as an inducible defense may be wide-
spread in this order of corals.

Acknowledgments

This project benefited from suggestions and field assis-
tant --,m C. D. Harvell. A.-M. Walls, D. F. Shapiro,
J. Nov. S. Lewis, and S. Nolon. Statistical advice was
provide; Buttel and S. J. Schwager. I thank C. D.

Harvell, Her, N. J. Hairston, Jr., R. Strathmann,

the FHL d i group, and two anonymous review-

ers for consti mments on the manuscript. This

research was si by grants from the Mellon Foun-

dation, the Ameri useum of Natural History, the

Houston Underwater Club (Seaspace), Sigma Xi

Grants-in-Aid of Resuai (national chapter), and the
Cornell Graduate School to J. M. West, and NSF grant
OCE 90-012034 to C. D. Harvell.

Literature Cited

Adler, F. R., and C. D. Harvell. 1990. Inducible defenses, phenotypic
variability and biotic environments. TREE 5(1 2): 407-410.

Baldwin, I.T., and T. E. Ohnmeiss. 1993. Alkaloidal responses to
damage in Nicotiana native to North America. J Chem. Ecol.
19(6): I 143-1153.

Barnes R. D. 1987. Invertebrate Zoology. Saunders, New York.

Bavestrello, G., M. Bonito, and M. Sara. 1993. Influence of depth on
the size of sponge spicules. 5V;. Ma 57(4): 415-420.

Birkeland, C., and B. Gregory. 1975. Foraging behavior and rates of
feeding of the gastropod. Cyphoma gibbosum. Bull. Nat. Hist. Mus.
Los Angeles Co. 20:57-67.

Bradshan, A. D. 1965. Evolutionary significance of phenotypic plas-
ticity in plants. Adv. Genet. 13: 115-155.

Bradshaw, A. D. 1974. Environment and phenotypic plasticity.
Brookhawn Symp. Biol 25: 75-94.

Brazeau, D. A. 1989. A male-biased sex ratio in the Caribbean octo-
coral, Briamim asbestinum: sex ratio evolution in clonal organ-
isms. Doctoral Dissertation, State University of New York at
Buffalo, NY.

Brazeau, D. A., and C. D. Harvell. 1994. Genetic structure of local
populations and divergence between growth forms in a clonal in-
vertebrate, the Caribbean octocoral Briart'iim asbestinum. Mar.
Biol. 104: 465-474.

Brazeau, D. A., and H. R. Lasker. 1990. Sexual reproduction and ex-
ternal brooding by the Caribbean gorgonian Briareitm asbestinum.
Mar Biol. 104:465-474.

Bushek, D. 1988. Settlement as a major determinant of intertidal oys-
ter and barnacle distributions along a horizontal gradient. / Exp.
Mar. Biol Ecol. 122: 1-18.

Day, R. \V. 1977. The ecology of settling organisms of the coral reef
at Heron Island, Queensland. Doctoral Dissertation. University of
Sydney, Australia.

de Weerdt, \V. H. 1981. Transplantation experiments with Carib-
bean Millepora species, including some ecological observations on
growth forms. Bijdr. Dierkcl. 51: 1-9.

Doty, M. S. 1971. Measurement of water movement in reference to
benthic algal growth. Bot Mar. 14: 32-35.

Foster, A. B. 1979. Phenotypic plasticity in the reef corals Alonias-
traea annulari.i (Ellis & Solander) and SiJeraxlrea siderca (Ellis &
Solander). ./. E\p. Mar. Biol. Ecol. 39: 25-54.

Francis, L. 1973. Intraspecific aggression and its effects on the distri-
bution of Anlliofileura eleganlixxima and some related anemones.
Biol. Bull 144: 73-92.

Gerharl, D. J. 1986. Gregariousness in the gorgonian-eating gastro-
pod Cyphoma gibbosum: tests of several possible causes. Mar. Ecol.
PmK.Scr 31:255-263.

Gerhart, D. J. 1990. Fouling and gastropod predation: consequences of
grazing for a tropical octocoral. Mar Ecol. Prog. Ser. 62: 1 03- 1 08.

Gilbert, J. J.. and R. S. Stemberger. 1984. Asp/anchna-mduced poly-
morphism in the rotifer Kcratella xlaeki. Limnol Oceanogr. 29:
1309-1316.

Grigg, R. VV. 1972. Orientation and growth form of sea fans. Limnol.
Oceanogr. 17(2): 185-192.

Marian. J. R. 1945. Cleistogamy and chasmogamy in Broinus cari-
nalus Hook, and Arn. Am. J Bot. 32: 66-72.

Harvell, C. D. 1984. Predator-induced defense in a marine bryozoan.
Science 224: 1357-1359.

Harvell, C. D., and D. Padilla. 1990. Inducible morphology, het-
erochrony and size hierarchies in a colonial invertebrate monocul-
ture. Pme. Nail. Acatl. Set i'SA 87: 508-512.

Harvell, C. D., and T. S. Suchanek. 1987. Partial predation on tropi-
cal gorgonians by Cyphoma gibbosum (Gastropoda). Mar. Ecol.
Prog. Ser. 38: 37-44.

Havel, J. 1986. Predator-induced defenses: a review. Pp. 263-278 in
Prcdalion: Direct and Indirect Impacts on Aquatic Communities.

S( I I HI II PI AS IK 'in IS \ (OR \l

289

W. C. Kertbot and A. Sih, eds. University Press of New England. Han-
over, NH.

Hazlett. B. A., and C. E. Bach. 1982. Distribution pattern of the Fla-
mingo Tongue Shell (Cyplioma nibhostim) on its gorgonian prey
(Briarcuin asbeslnmin). Mar Behav. Physiol. 8: 305-309.

Holm, S. 1979. A simple sequentially rejective multiple test proce-
dure. Seand. J. Sun. 6: 65-70.

Ivker, K. 1972. A hierarchy of histo-incompatibility in Hydractinia
eckmala Bin/. Bull. 143: 162-174.

Ink id. P. L_ and J. I. Morrissey. 1993. Water motion on coral reefs: eval-
uation of the 'clod card' technique. Mar. Ecol. Prog. Ser. 93: 175-181.

Kin/ir. R. A. 1970. The ecology of the gorgonians (Cnidaria: Octo-
corallia) of Discovery Bay, Jamaica. Doctoral Dissertation, Yale
University, New Haven, CT.

Koehl, M. A. R. 1982. Mechanical design of spicule-reinforced tissue:
stiffness. J. Exp Bio/. 98: 239-267.

Koehl, M. A. R. 1996. Mechanical design of sclente-reinforced skele-
tons. Am. ~/.ool. 36(5): 55A.

Lang, J. C. 1973. Interspecific aggression by scleractiman corals. II.
Why the race is not onh to the swift. Bull. Mar Sci. 23: 260-279.

Lasker, H. R. 1990. Clonal propagation and population dynamics of
agorgonian coral. Ecology 1\(4): 1578-1589.

Lasker, H. R., and M. A. Coffroth. 1988. Temporal and spatial variability
among grazers: variability in the distribution of the gastropod Cyphoma
gibboHim on octocorals. Mar Ecol. Prog. Ser 43: 285-295.

Lasker, H. R, M. A. CofTroth, and L. M. Fitzgerald. 1988. Foraging pat-
terns ofCyphomagibbosum on octocorals: the roles of host choice and
feeding preference. Bull. Mar Biol Lah. ll'oods Hole 174: 254-266.

Lesser, M. P., V. M. VVeis, M. R. Patterson, and P. L. Jokiel. 1994.
Effects of morphology and water motion on carbon delivery1 and
productivity in the reef coral, Pocillopora damicomis (Linnaeus):
diffusion barriers, inorganic carbon limitation, and biochemical
plasticity./ Exp. Mar. Biol. Ecol 178: 153-179.

Lewis, S. M., J. N. Norris, and R. B. Searles. 1987. The regulation of
morphological plasticity in tropical reef algae by herbivory . Ecology
68(3): 636-641.

McCloskey, L. R., and L. Muscatine. 1984. Production and respira-
tion in the Red Sea coral Stylophora pislillala as a function of depth.
Proe. R Soc. Lond Ser. B 222: 2 1 5-230.

McNaughton, S. J., and J. L. Tarrants. 1985. Grass leaf silicification:
natural selection for an inducible defence against herbivores. Proc
Nail. Acad Sci. L'SA 80: 790-791.

Meacham, C. A., and T. Duncan. 1990. Morphosys version 1.26. Uni-
versity Herbarium, University of California, Berkeley, CA.

Miles, J. 1991. Inducible agonistic structures in the tropical coralli-
morphanan, Discoma sanclilhomae Biol. Bull 180:406-415.

Mooney, H. A., and M. West. 1964. Photosynthetic acclimation of
plants of diverse origin. Am J. Boi. 51:825-827.

Muscatine, L. 1974. Endosvmbiosis of cnidanans and algae. Pp. 359-
395 in Coelenterate Biology. L. Muscatine and H. M. Lenhoff, eds.
Academic Press, New York.

Muscatine, L., R. R. Pool, and R. K. Trench. 1975. Symbiosis of algae
and invertebrates: aspects of the symbiont surface and the host-
symbiont interface. Trans. Am Microsc. Soc 94(4): 450-469.

Muus, B. J. 1968. A field method for measuring "exposure" by
means of plaster balls. Sar.tia34: 61-68.

Neter, J., \V. \\asserman, and M. H. Kutner. 1990. Applied Linear
Statistical Models. Richard D. Irwin. Inc.. Boston. MA.

Palumbi, S. R. 1986. How body plans limit acclimation: responses of a
demosponge to wave force. Ecology 67(1): 208-2 14.

Pawlik, J. R., M. T. Bun h. and \V. Kenical. 1987. Patterns of chemi-
cal defense among Caribbean gorgonian corals: a preliminary sur-
vey. J. Exp Mar. Biol Ecol 108: 55-66.

Rice, \V. 1989. Analyzing tables of statistical tests. Evolution 43(1):
223-225.

Roy, J., and H. A. Mooney. 1982. Physiological adaptation and plas-
ticity to water stress of coastal and desert populations of Hcliotro-
pium curassavicitm. Oecologia 52: 370-375.

Scheiner, S. M. 1993. Genetics and evolution of phenotypic plastic-
ity, .-(mm. Re\\ Ecol Sysl. 24: 35-68.

Schlichting, C. D. 1986. The evolution of phenotypic plasticity in
plants. Annii. Rev Ecol. Syst. 17: 667-693.

Schullz, J. C. 1988. Plant responses induced by herbivores. TREE
3(2): 45-49.

Sebens, K. P. 1984. Water flow and coral colony size: interhabitat
comparisons of the octocoral Alcyonium siderium. Proc. \ail
Acad. Sci. L'SA 81: 5473-5477.

Sebens, K. P. 1987. Coelenterata. Pp. 55-120 in Animal Energetics.
T. J. Pandian and F. J. Vemberg. eds. Academic Press. San Diego, CA.

Sebens, K. P., and J. C. Miles. 1988. Sweeper tentacles in a gorgo-
nian octocoral: morphological modifications for interference com-
petition. Biol Bull 175:378-387.

Sorensen, T. 1954. Adaptation of small plants to deficient nutation and
a short growing season. Illustrated by cultivation experiments with
Capsella bursapasloris (L). Med. Botan. Tidsskrift 51: 339-36 1 .

Stearns, S. C. 1989. The evolutionary significance of phenotypic
plasticity. Bio.icU'nce39(l): 436-445.

Svoboda, A. 1978. In MIII monitoring of oxygen production and res-
piration in Cnidana with and without zooxanthellae. Pp. 75-82 in
Physiology and Behavior of Marine Organisms, D. S. McLusky and
A. J. Berry, eds. Permagon Press, New York.

Turesson, G. 1920. The cause of plagiotrophy in maritime shore
plants. Litnds Vim- Arsskr. Avd. 2(NF) 16(2): 1-33.

Van Alstyne, K. L. 1988. Herbivore grazing increases polyphenolic
defenses in the intertidal brown alga Fucus distichus. Ecology 69(3):
655-663.

Nan Alstyne, K. L., and V. J. Paul. 1992. Chemical and structural
defenses in the sea fan Gorgonia venlalma: effects against generalist
and specialist predators. Coral Reels 11: 1 55- 1 59.

Via, S., and R. Lande. 1985. Genotype-environment interaction and
the evolution of phenotypic plasticity. Evolution 39(3): 505-522.

Vreeland, H. \ ., and H. R. Lasker. 1989. Selective feeding of the
polychaete Hermodice carunculala Pallas on Caribbean gorgo-
nians. / Exp. Mar. Biol Ecol. 129: 265-277.

Vrolijk, N. H., and N. M. Targett. 1992. Biotransformation enzymes
in Cyphoma gihhosum (Gastropoda: Ovulidae): implications for
detoxification of gorgonian allelochemicals. Mar. Ecol. Prog. Ser.
88: 237-246.

\\ ainwright, S. A., VV. D. Biggs, J. D. Currey, and J. M. Gosline. 1976.
Mechanical Design in Organisms Edward Arnold. London.

Wellington, G. M. 1980. Reversal of digestive interactions between
Pacific reef corals: mediation by sweeper tentacles. Oecologia 47:
340-343.

West, J. M. 1996. Skeletal variation in the Caribbean coral Briamun
asbestinum (Gorgonacea): pattern and process across environmental
gradients. Doctoral Dissertation. Cornell University. Ithaca, NY.

West, J. M., C. D. Harvell, and A.-M. Walls. 1993. Morphological
plasticity in a gorgonian coral (Briareiun asbestinum) over a depth
cline. Mar. Ecol. Prog Ser 94: 61-69.

Wijsman-Best, M. 1974. Habitat-induced modification of reef corals
(Faviidae) and its consequences for taxonomy. Proc. 2nd Int. Coral
ReefSymp. 2:217-228.

Witman, J. D. 1988. Effects of predation by the fire worm Hermotliee
ccininciilalaon millepond hydrocorals. Bull Mar. Sci 42: 446-458.

Young, T. P. 1987. Increased thorn length in Acacia depranolo-
hn/in — an induced response to grazing. Oecologia 71: 436-438.

Reference: Biol. Bull 192: 290-299. (April, 1997)

Life-History Variation in a Colonial Ascidian: Broad-
Sense Heritabilities and Tradeoffs in Allocation to
Asexual Growth and Male and Female Reproduction

PHILIP O. YUND, YVETTE MARCUM1, AND JOHN STEWART-SAVAGE

Department of Biological Sciences, University of New Orleans. New Orleans, Louisiana 70148

Abstract. Intraspecific variation in life-history strategy
provides a valuable opportunity for examining how nat-
ural selection acts on life-history variants to mold repro-
ductive strategies. Evaluating the consequences of selec-
tion requires knowledge of the range of phenotypic vari-
ation in life histories, the extent to which variation is
genetically based, and possible correlations among
different traits that might constrain or promote the effect
of selection on individual traits. We explored life-history
variation in the colonial ascidian Botryllus sc/ilosseri (a
cyclical hermaphrodite) by growing clonal replicates of
1 8 genotypes in a common-garden experiment. Colonies
of this species have previously been shown to vary in egg
production and growth rate. We demonstrate that geno-
types also vary in sperm production, which is manifested
as variation in testis size. We then calculate broad-sense
heritabilities for a suite of life-history traits and demon-
strate correlations among traits that suggest a three-way
tradeoff in resource allocation to asexual growth and sex-
ual reproduction via male and female function. This cor-
relation structure suggests that selection cannot act inde-
pendently on individual life-history traits.

Introduction

Marine habitats tend to subject organisms to unique
selective pressures that are reflected in the life-history
strategies that evolve in this environment (Hendler,
v Strathmann et ai. 1984; McEdward and Coulter,
198 Fmlet et ai. 1987; Strathmann, 1990). For free-
marine invertebrates, fertilization via the re-

>ctober 1996; accepted 3 January 1997.

'Currci: ss: School of Veterinary Medicine, Louisiana State

Universi i Rouge, LA 70803.

lease of sperm into the water column may lead to distinc-
tive selective pressures on life-history strategies (Ghi-
selin, 1987; Strathmann, 1990). In particular, recent field
experiments and assays of fertilization success in nature
have suggested that selection via fertilization processes
may profoundly influence both the quantity of gametes
produced and specific attributes of those gametes (re-
viewed in Levitan, 1995; Levitan and Petersen, 1995).

Because many species exhibit little apparent intraspe-
cific variation in life-history strategy, comparative stud-
ies among taxa have contributed immensely to the study
of the evolution of marine invertebrate life histories (e.g.,
Menge. 1975; Strathmann and Strathmann, 1982; Eck-
elbargerand Watling, 1995). However, selection actually
acts on variation within a species, and some marine
species do exhibit substantial intraspecific variation
in different life-history characters (e.g., Hughes and
Hughes, 1986;Grosberg, 1988; Yund, 1991; Cohen and
Strathmann, 1996). These variable taxa yield particu-
larly valuable opportunities for evaluating the action of
natural selection on life-history variants.

Evaluating the consequences of natural selection on
intraspecific variation in life-history traits requires three
types of information: first, which life history traits exhibit
phenotypic variation, and hence can potentially be sub-
ject to selection; second, for each variable trait, what is
the extent to which phenotypic variation is genetic; third,
since selection ultimately acts on the total phenotype
and not just on independent traits, are there positive and
negative correlations among traits. Correlations among
traits could either constrain or promote the effect of se-
lection acting on each individual trait (Lande and Ar-
nold, 1983).

We present data from a common-garden experiment
with the colonial ascidian Botryllus schlosseri, a cyclical

290

ASCID1AN LIFE HISTORY VARIATION

291

hermaphrodite, that addresses these three points. Colo-
nies of this species are known to vary in growth rate, ter-
minal size, and egg production (Grosberg, 1982, 1988;
Chadwick-Furman and Weissman. 1995). We demon-
strate additional variation among genotypes in sperm
production and reproductive cycle duration, and esti-
mate broad-sense heritabilites for all five of these traits.
Finally, we explore possible correlations among traits to
assess the potential for selection to act independently on
individual traits.

Materials and Methods

Study species

Botryllus schlosseri, a colonial ascidian with a cosmo-
politan distribution, is common on firm substrata in the
shallow subtidal zone of New England waters (Gosner,
1971). Colonies are composed ofasexually produced zo-
oids arranged in clusters, or systems, with all zooids in a
system sharing a common exhalant siphon. All zooids in
the colony synchronously undergo an asexual cycle of
zooid replacement in which a new generation of zooids
(buds) forms between the existing generation of zooids
(Milkman, 1967). Over a period of about 6-12 d, these
buds grow and expand, and then take over the function
of the previous generation of zooids, which are quickly
resorbed. Colonies grow as long as bud production ex-
ceeds the number of zooids in the current generation,
and growth continues until colonies reach a terminal size
(generally associated with the onset of sexual reproduc-
tion), which is then maintained over a number of subse-
quent asexual cycles (Boyd el al, 1986; Grosberg, 1988).

Sexual reproduction begins when colonies exceed a
minimum size (Harvell and Grosberg, 1988). Eggs
brooded by each new generation of zooids are fertilized
at the time of take-over, when the zooids' siphons first
open and admit water to the atrial chambers. Sperm re-
lease does not begin until about 1-2 d after ovulation,
effectively preventing self-fertilization (Milkman, 1967;
Yund and McCartney. 1994). Because sexual reproduc-
tion is linked to the asexual zooid replacement cycle, col-
onies cycle between male and female function.

Relationship between test is cross-sectional area ami
sperm count

When colonies are grown in culture on glass substrata,
variation in sperm production is readily apparent as vari-
ation in testis size (viewed from the underside; pers.
obs.). To facilitate monitoring a number of colonies and
to permit repeated sampling of individuals over time, we
needed an assay of sperm production that was relatively
quick and nondestructive. Consequently, we assayed
sperm production as testis cross-sectional area. To vali-

date this approach, we first tested for a correlation be-
tween testis cross-sectional area and the number of
sperm in individual testes. We used an ocular microme-
ter to measure the length and width of 27 mulberry-
shaped testes in zooids of different colonies and then sur-
gically removed each measured testis from its zooid. All
testes were sampled at the time of maximum size during
the reproductive cycle (day 2-3 by the criteria of Milk-
man, 1967). Each testis was removed by making a longi-
tudinal incision in the wall of the zooid, lifting the testis
with forceps, and cutting the connective tissue attached
to the underside of the testis. Excised testes were pre-
served in 2% glutaraldehyde in buffered seawater and
later macerated in 1 00 n\ of seawater by five gentle grinds
with a glass rod. The number of mature spermatozoa in
each testis was estimated as the mean of four hemocy-
tometer counts of the sperm suspension. We analyzed
these data by examining the correlation between mean
sperm count and testis cross-sectional area (calculated as
the length multiplied by the width of each testis).

Common-garden experiment

We examined life-history variation among 18 B.
schlosseri genotypes by growing clonal replicates in a
flowing seawater system at the University of Maine's
Darling Marine Center. Colonies used in this experiment
were initially collected from widely spaced ( = 10-20 m.
apart) locations within a field population located in 3 to
8 m of water (MLW) on the western shore of Carlisle Is-
land in the Damariscotta River, Maine (adjacent to the
experimental field site of Yund and McCartney, 1994;
Yund, 1995; Atkinson and Yund, 1996). Field-collected
colonies were subdivided and explanted onto glass mi-
croscope slides (2.5 X 7.6 cm), with between 5 and 1 1
replicate colonies established for each genotype.

The 18 genotypes selected for inclusion in the com-
mon-garden experiment were chosen on the basis of ini-
tially possessing less than three eggs per bud. Although
this criterion restricted us to exploring only a subset of
the total life-history variation in this population, this
limitation was necessary in order for us to collect data
on each colony over a series of asexual or sexual cycles.
Genotypes with higher egg production were present (al-
though rare) in this population, but such colonies typi-
cally die after one brooding cycle (Grosberg, 1988).

The experiment began on 20 June 1994, when all col-
onies were trimmed to an average of 33.5 zooids
(±1.9 SE) arranged in two or three systems of zooids.
Colonies were housed in a vertical position in acrylic
racks, with neighboring slides separated b\ 1.3 cm. The
racks were placed in a single large, shallow tank (130 X
100 X 9 cm) in the flowing seawater sy m. Racks had
no fixed position within the tank, but systematic posi-

292

P. O. YUND KT AL.

tional effects are unlikely because racks were moved and
relocated daily throughout the experiment. Similarly,
colonies were shuffled among positions within racks
about once a week (at the time of each data collection).
No supplemental food was added, because past experi-
ence indicated that colonies have reasonably high growth
and fecundity under these culture conditions.

Data on colony size (number of zooids), growth rate
(number of buds), egg production (number of eggs per
bud for a subsample of 10 buds), and sperm production
(testis cross-sectional area for a subsample of 10 testes in
5 zooids) were collected for each surviving colony (some
replicates died before the end of the experiment) once
during each asexual or sexual cycle for seven cycles, be-
ginning the week of 27 June. Colonies were viewed from
either the top (colony size) or bottom (all other variables)
under a dissecting microscope. Testis cross-sectional
area was calculated by measuring the length and width of
testes with an ocular micrometer at 25 X magnification.
Testes measurements were standardized within each
asexual or sexual cycle by sampling within the window of
maximum testis size (day 2-3 by the criteria of Milkman,
1967). Testes measurements and bud counts were per-
formed on 5 and 10 zooids (respectively) widely scattered
throughout the colony to prevent possible biases from
positional effects within colonies. For a subset of eight
genotypes, we also directly assayed variation in sperm
production by excising three testes from each of three
replicate colonies in cycle 6 and estimating the number
of mature sperm in each testis as the mean of four hemo-
cytometer counts of a 100-^1 dilution of the macerated
testis (as described in the preceding section). For each
colony, we also recorded the time (number of days) re-
quired to complete each asexual and sexual cycle. Com-
parison of these values permits an assessment of varia-
tion in cycle duration among genotypes.

Life-history data were analyzed through a series of
ANOVAs performed with a computer software package
(JMP. SAS Institute, Cary, NC). Unequal sample sizes
among genotypes and sample dates due to the death of
some replicate colonies and values missed during data
collection necessitated an unbalanced design. Similar
analyses were conducted for six dependent variables:
eggs per bud, buds per zooid (calculated by dividing the
number of buds by the number of zooids in each colony
) each cycle), testis cross-sectional area, sperm count in
. 6, terminal size (the maximum number of zooids
ii by each colony), and cycle duration. All analy-
ded the random effects 'genotype' and 'replicate
'iin genotype', except for the analysis of termi-
nal SL ich did not include a 'replicate' effect because
there v >|y a single size value for each replicate col-
ony. Ana for the four dependent variables eggs per
bud, buds ooid, testis cross-sectional area, and cycle

duration included 'cycle' as an effect and were performed
as repeated measures ANOVAs by testing 'cycle' with re-
spect to the random effects. The 'genotype' by 'cycle' in-
teraction effect was omitted from these models due to
inadequate sample sizes in some cells. Data collection
for the two dependent variables terminal size and sperm
count during cycle 6 was restricted to a single cycle, and
hence no 'cycle' effect could be included in these models.
Variance components from these analyses were used
to calculate the clonal repeatability of each life-history
trait (a form of broad-sense heritability; Falconer, 1981).
The component of variance attributable to the 'geno-
type' effect was used as an estimate of genetic variance
and was divided by the sum of all variance components,
which provides an estimate of total phenotypic variance
( Falconer, 1981). We also recorded the range of genotype
means and least square means (which adjust for a 'cycle'
effect in four of the six analyses) for each life-history trait,
and calculated the magnitude of variation in each trait
by dividing the highest genotype least square mean by
the lowest. Finally, we explored possible univariate and
multivariate correlations among life-history traits. First,
we calculated Pearson product-moment correlations
among genotype least square means for each possible set
of paired traits. For a subset of three of the traits (eggs per
bud, testis area, and buds per zooid) we also calculated
partial correlation coefficients and employed a principal
components analysis to characterize the dimensionality
of the three-way relationship.

Results

Relationship between testis cross-sectional area ami
sperm count

Testis cross-sectional area, calculated as testis length
multiplied by width, was highly correlated with the ac-
tual number of mature spermatozoa in each testis (Fig.
1 ; r = 0.7 1 , P < 0.00 1 ). Testes are three-dimensional ob-
jects, and so variation in cross-sectional area alone (a
two-dimensional measurement) might be expected to
scale nonlinearly with actual sperm counts. However,
there is no evidence of a nonlinear relationship between
these variables (Fig. 1 ). Nonlinear functions fit to these
data yielded substantially lower correlation coefficients
than the linear function (exponential, r = 0.52; power, r
= 0.48; logarithmic, r = 0.62). Either variation in testis
thickness (the third, unmeasured dimension) is minimal,
or the effect of this aspect of variation in testis volume on
sperm counts is negligible within the size range measured
in this study.

Genotvpic variation and clonal repeatabilities
of life-history traits

All six life history traits assayed in this study varied
greatly among genotypes (Table I). The effect of geno-

ASCIDIAN LIFE HISTORY VARIATION

293

"cb
X

CD
Q.
CO

CD
.0

150-1

100-

50-

r = 0.71

0 0.1 0.2 0.3 0.4 0.5 0.6

Testis Cross-sectional Area (mm2)

Figure 1. The relationship between testis cross-sectional area and
number of mature spermatozoa within 27 testis measured and excised
from different colonies.

type was extremely significant (P < 0.0001) in all six
analyses. In contrast, the effect of replicate nested within
genotype (an indicator of environmental variance) was
not significant in four out of the five analyses in which it
was included (Table I). Testis cross-sectional area was the
only trait that exhibited significant variation among rep-
licate colonies within a genotype, though the magnitude
of the replicate effect was only about half that of the ge-
notype effect (Table I). In all four analyses for which data
were collected in multiple asexual and sexual cycles, the
effect of cycle was also highly significant (Table I). Aver-
aged across all genotypes, the number of eggs per bud
and testis cross-sectional area both initially increased
over the first two reproductive cycles and then declined
in the final two cycles (Table II). The number of buds
per zooid decreased over subsequent cycles as colonies
approached a terminal size (Table II). Cycle duration
fluctuated over subsequent cycles with no apparent pat-
tern (Table II).

The temporal patterns of life-history traits can be bet-
ter appreciated by examining the performance of three
sample genotypes, chosen to represent the extremes of
life-history strategy present among experimental colo-
nies (Fig. 2). All three genotypes exhibited high initial
growth rates and then leveled off at a maximum size (Fig.
2;numberofzooids), but the high-growth genotype (Fig.
2C) grew more rapidly than the other two (Fig. 2A, B)
and never completely ceased growing. Egg production
generally increased over subsequent cycles, but substan-
tial variation was present between adjacent cycles (Fig.
2; eggs per bud). Finally, though sperm production gen-
erally increased over subsequent cycles (Table II), sperm
production in the high-sperm-production genotype ac-
tually decreased over time (Fig. 2 A; testis area), a pattern
that was repeated for the second highest sperm-produc-

tion genotype in this study (unpubl. data). All of the
lower sperm-production genotypes either increased
sperm production over time or remained more-or-less
constant (Fig. 2B, C).

Clonal repeatabilities calculated from variance compo-
nents from these analyses varied widely among traits (Ta-
ble III). The two variables with the highest expected envi-
ronmental component to variance (buds per zooid and
cycle duration) had the lowest clonal repeatabilities (Table
III). The relatively high clonal repeatability for actual
sperm counts may be due to a combination of sampling
in a single cycle, limiting data collection to only eight ge-
notypes, and utilizing a higher resolution sampling tech-
nique (in comparison to testis cross-sectional area), all of
which could have reduced environmental variance.

Although all six life-history traits varied significantly
among genotypes (Table I), the magnitude of that varia-
tion differed greatly among traits (Table III). Cycle dura-
tion exhibited the least variation, with the least square
mean for the slowest genotype only 40% greater than that

Table I

A NO I A rcxulix lor I he six life-history variahlc.t

Source

df

Sum of
squares

A. Dependent: Eggs per bud

Genotype 17 199.1 38.9 0.0001

Replicate [Genotype] 123 34.4 0.7 0.9979

Cycle 6 78.4 30.4 0.0001

B. Dependent: Testis cross-
sectional area

Genotype 17 21.8 11.4 0.0001

Replicate [Genotype] 93 11.3 1.7 0.0005

Cycle 6 8.6 19.9 0.0001

C. Dependent: Sperm count in
cycle six

Genotype 7 183,698 28.7 0.0001

Replicate [Genotype] 16 14,609 1.0 0.4781

D. Dependent: Buds per zooid

Genotype 17 16.7 9.3 0.0001

Replicate [Genotype] 107 10.6 0.7 0.9958

Cycle 6 115.1 150.8 0.0001

E. Dependent: Terminal colony
size (number of zooids)

Genotype 17 39,101 9.0 0.0001
I Dependent: Cycle duration

Genotype 17 116.6 13.0 0.0001

Replicate [Genotype] 106 44.7 0.3 0.9999

Cycle 5 136.1 22.1 0.0001

Note: All analyses contain genotype as a random main effect. Analy-
ses for all dependent variables except for terminal colony size contain
replicate nested within genotype as a second random p-.i:n effect. Anal-
yses for all dependents except for terminal colony si/ nd sperm count

in cycle 6 contain cycle as an additional main efti-n hese four analy-
ses were performed as repeated measures by testin; :e cycle effect with
respect to the random effects.

294

P. O. YUND ET AL

Table II

Cvcle effects tor the lour lite-history variables measured over
subsequent asexual and sexual cycles

Cycle

number

Eggs
per bud

Testis
area

Buds per
zooid

Cycle
duration

1

1.12

0.64

2.25

2

1.63

0.99

1.85

6.74

3

1.61

0.78

1.1 1

8.10

4

2.01

0.79

1.01

7.22

5

2.21

0.99

1.08

7.06

6

1.57

0.80

0.96

7.78

7

1.34

0.58

8.17

Note: Least square means, adjusted tor genotype and replicate nested
within genotype effects, are reported for each cycle. Cycle duration val-
ues are missing for the first cycle because the start of the cycle preceded
the initiation of data collection. Similarly, buds per zooid data are miss-
ing for cycle seven because the number of zooids was not counted in
what would have been the eighth cycle.

of the fastest genotype (Table III). Thus, although we
were able to detect significant variation in cycle duration
among genotypes, the magnitude of variation in this trait
relative to other life-history traits is comparatively mi-
nor. The other five variables exhibited variation in geno-
type least square means ranging from more than a factor
of 2 (buds per zooid) to almost a factor of 5 (sperm count
in cycle six; Table III).

Correlations among life-history variables — genotype
means

Two pairs of correlations among the six life-history
traits that we measured exist because the pairs of vari-

ables measure traits produced by the same or very similar
underlaying processes. First, for the subset of eight geno-
types for which we actually counted sperm in cycle 6, this
variable was highly correlated with genotype least square
means for testis cross-sectional area (Table IVA). Be-
cause the two variables just represent two different ways
of measuring sperm production, this correlation is to be
expected and conveys little biological information (be-
yond confirming the relationship between testis size and
sperm number; see Fig. 1 ). Secondly, genotype least
square means for growth rate (buds per zooid) were sig-
nificantly correlated with genotype means for terminal
size (Table IVA). Again, genotypes with higher growth
rates (adjusted for variation among cycles) should in gen-
eral attain a larger terminal size. These two correlations
are assumed to be biologically relatively trivial, and the
remainder of our presentation considers only one of each
of these two pairs of variables (genotype least square
means for testis cross-sectional area and buds per zooid,
since these two traits are measured at the zooid level and
adjusted for variation among cycles).

Of the remaining four life-history variables, genotype
least square means for cycle duration were not signifi-
cantly correlated with genotype least square means for
any of the other variables (Table IVA). Since the magni-
tude of variation in cycle duration was so much smaller
than for the other three variables (Table III), correlations
might exist that could be detected only with much larger
sample sizes. Alternatively, variation in cycle duration
may be truly independent of other life-history traits.

The genotype least square means of the three remain-
ing variables, which represent allocation to female repro-

A. High Sperm Production Genotype

o
o

N "S

3-

= 1 -

Y \

— • — Eggs/Bud
B. High Egg Production Genotype

- 3-

- 2-

Cycle

*

Cycle

-a — Testis Area

C. High Growth Genotype

- 3-

- 2-

-0.4

-0.3

-o,

Cycle

Figure 2. Growth and sexual reproduction trajectories for three of the more extreme Botryllus schlos-
seri genotypes in this study. Average eggs per bud, colony size, and testis cross-sectional area are plotted
against cycle number. Error bars represent 1 standard error. (A) A high-sperm-production genotype. (B) A
high-egg-production genotype. (C) A high-growth genotype.

ASCIDIAN LIFE HISTORY VARIATION
Table III

295

Clumil repeatabilities, genotype ranges, genotype least .square means ranges (adjusting lor cycle e/leets in lour of the variables), ami lite magnitude
i >/ genotype least square means variation (highest genotype value divided hy lowest genotype value) for the six life-history variables

Genotype means

Genotype least square means

Variable

Clonal
repeatability

Highest

Lowest

Highest

Lowest

Magnitude

Eggs per hud

0.47

2.63

0.68

2.77

0.62

X4.5

Testis area (mm2)

0.42

0.23

0.08

0.25

0.07

X3.5

Sperm count ( x 10')

0.75

196.4

42.0

196.4

42.0

X4.7

Buds per zooid

0.21

1.89

0.99

1.92

0.79

X2.4

Terminal size

0.57

338.3

78.3

338.3

78.3

X4.3

Cycle duration

0.20

8.19

6.33

8.68

6.28

X1.4

duction (eggs per hud), male reproduction (testis cross-
sectional area), and asexual growth (huds per zooid) at
the zooid level, exhibit somewhat more complex corre-
lations. First, there are significant negative univariate
correlations between buds per zooid and both eggs per
bud and testis area (Table IVA). In contrast, the correla-
tion between eggs per bud and testis area is virtually zero
(Table IVA). However, the partial correlation coeffi-
cients between each pair of traits (which in each case ad-
just for the effect of the third variable) suggest a much
stronger relationship in three-dimensional space. All
three partial correlations are much more strongly nega-
tive than the respective univariate correlations, espe-
cially the partial correlation between eggs per bud and
testis area (Table IVB). This pattern suggests a negative.

three-way correlation among these three variables that is
consistent with a tradeoff in resource allocation among
male, female, and asexual reproduction.

These negative correlations can also be examined by
visualizing the relationships among these variables in
three dimensions (Fig. 3). The data points fall primarily
on a plane that intersects the egg per bud, testis cross-
sectional area, and buds per zooid axes at relatively high
values (Fig. 3). A principal components analysis provides
an additional assessment of the dimensionality of this
data cloud (Table V). The first two principal components
have relatively high eigenvalues and in combination ex-
plain more than 92% of the variance in the data set (Ta-
ble V). The third principal component has a low eigen-
value and explains less than 8% of the variance (Table
V). This result indicates that the data points cluster

Table IV

Correlations among genotype least square means

Testis Sperm Buds per Terminal Cycle
area count zooid size duration

A. Univariate (Pearson's product moment) correlation coefficients
between each pair of variables (all pairs)'

Eggsperbud 0.01 NS -0.21 NS -0.59** -0.34 NS -0.06 NS

Testis area -0.87** -0.48* -0.46 NS 0.12NS

Sperm count -0.41 NS -0.30 NS 0.02 NS

Buds per zooid 0.52* 0.19NS

Terminal size -0.09 NS

B. Partial correlation coefficients among the three zooid-level
variables2

Eggs per bud -0.48*
Testis area

-0.67**
-0.59**

Note: *P < 0.05; **P < 0.0 1 ; NS = Not Significant.

' n = 18 genotypes in all comparisons ( 16 df) except those involving
sperm count in cycle 6 for which ;; = 8 (6 df).

- n = 18 in each comparison ( 1 5 df). These values represent the cor-
relations between each pair of variables after adjusting for the effect of
the third variable.

0.5

Figure 3. The three-dimensional relationsh : among genotype
least square means for eggs per bud. testis cross-sr t .mal area, and buds
per zooid. Values for the 18 genotypes fall n a plane that slopes
downward from the upper back to the lower h of the figure.

296

P. O. YUND ET AL.

Table V

Principal component* analysis of the three-way relationship among
eggs per bud. testi* cross-sectional area, and budx per :ooid

Principal components

PC 1

PC 2

PC 3

Eigenvalue

1.77

0.99

0.24

Percent of variance

59.00

33.04

7.96

Cumulative percent of variance

59.00

92.04

100.00

Eigenvector loadings

Eggs per hud

0.55

-0.63

0.55

Testis area

0.45

0.78

0.44

Buds per zooid

-0.71

0.00

0.71

Note. The first two principal components explain most of the vari-
ance in the data and are heavily loaded on the three variables, indicat-
ing that the data points fall largely on a plane that represents a three-
way tradeoff in allocation to male, female, and asexual reproduction
(see test for details). Because there are only three variables in the anal-
ysis, the third principal component is completely determined by the
first two; it has to explain all of the remaining variance. However, the
very low magnitude of the third component is not inevitable, and it is
the relative magnitude of the three principal components that describes
the planar nature of the relationship among the three primary variables.

mainly on and around a plane denned by the first two
principal components. The first principal component is
most heavily loaded (negatively) on the variable buds per
zooid, whereas the second principle component is
heavily loaded on eggs per bud (negatively) and testis
cross-sectional area (positively; Table V). Again, the
principal components analysis demonstrates a three-way
negative correlation among traits associated with male,
female, and asexual reproduction.

Discussion

Genetic and environmental variation

As previously reported for other populations (Gros-
berg, 1988; Chadwick-Furman and Weissman, 1995), B.
schlosseri colonies in the Damariscotta River exhibited
a great deal of variation in egg production and growth
rate (Table III). In addition, we found colonies to be
highly variable in sperm production, with the highest
sperm-production genotype yielding 4.7 times as many
sperm per testis as the lowest sperm-production geno-
(Table III). Variation in sperm production is re-
d in the size of testes, and measurement of testis
•tional area provides a reasonable nondestruc-
of actual sperm production (Fig. 1 ).
ast to a previous study (Grosberg, 1982, 1988),
t significant variation in reproductive cycle
dun, >ng genotypes. Since a colony's growth rate

in rea1 i function of both bud production per zo-

oid and c ration, the existence of genotype-specific

cycle duration suggests that caution must be used in
comparing colony growth rates on the basis of budding
rates alone. However, the magnitude of variation in cycle
duration is relatively small compared to variation in bud
production (Table III), and cycle duration does not ap-
pear to be correlated with other life-history traits. Conse-
quently, for many purposes the inclusion of information
on cycle duration may only increase variance in growth
rates.

To assess temporal patterns in four of the life-history
variables, we were forced to exclude genotypes with very
high egg production from our common-garden experi-
ment. Colonies with higher levels of egg production than
those included here typically exhibit semelparous repro-
duction and then die (Grosberg, 1988), precluding the
collection of data on subsequent asexual and sexual cy-
cles. Consequently, our value for the magnitude of vari-
ation in egg production (Table III) somewhat underesti-
mates the total variation in this natural population.
Qualitatively, however, total variation in egg production
in the Damariscotta River still appears to be quite a bit
lower than in the Eel Pond at Woods Hole (Grosberg,
1988). Although we have collected colonies that pro-
duced up to 6 eggs per bud, colonies with egg production
> 3 eggs per bud appear to be rare at all times of the year
(unpubl. data). In contrast, colonies producing 8 to 12
eggs per bud dominate populations in the Eel Pond in
early summer (Grosberg, 1988). Although we know that
we underestimated total phenotypic variation in egg pro-
duction, we can only speculate on the effect that exclud-
ing higher egg producers may have had on our estimates
of variation in other life-history traits. If the negative cor-
relations among male, female, and asexual reproduction
(Fig. 3, Tables IV and V) are maintained across higher
levels of egg production, then the range of variation in
the other life-history traits may have been affected as
well. Consequently, our range estimates for life-history
traits (Table III), though demonstrating a large degree of
variation in this population, are likely to be conservative.
However, the correlation structure itself may be altered
if more extreme genotypes are included in the analysis
(Grosberg. 1988).

Following colonies through subsequent asexual and
sexual cycles allowed us to evaluate temporal trends in
female (eggs per bud), male (testis cross-sectional area),
and asexual (buds per zooid) reproduction as well as cy-
cle duration (Table II). All colonies were sexually mature
when collected from the field, so the temporal patterns
reported here do not simply reflect the onset of reproduc-
tion. In particular, significant variation in egg and sperm
production among subsequent cycles suggests that esti-
mates of reproductive output based on observations of a
single cycle, even of sexually mature colonies, should be
interpreted with caution. Additionally, the different tern-

ASCIDIAN LIFE HISTORY VARIATION

297

poral patterns exhibited by different genotypes (Fig. 2)
are suggestive of genotype by cycle interaction effects,
which we could not explicitly test because of inadequate
sample sizes in some cells. The temporal component of
variation in life-history strategy in B. schlosseri merits a
more detailed examination.

All six life-history variables that we measured exhib-
ited significant variation among genotypes (Table I),
with clonal repeatabilities (a form of broad-sense herita-
bility) ranging from 0.20 to 0.75 (Table III). These broad-
sense heritability estimates may include some forms of
environmental variance, and so set an upper limit for the
proportion of additive genetic variance (Falconer, 1 98 1 ).
Although the relatively large magnitude of most of our
broad-sense heritability estimates suggests a large genetic
component to phenotypic variation, environmental
effects are also likely to be substantial. Grosberg ( 1988)
has previously demonstrated that food levels can signifi-
cantly alter both asexual growth rates and egg production
levels in B. schlosseri. The temporal patterns that we ob-
served in egg and sperm production among reproductive
cycles (Table II) may reflect temporal variation in plank-
tonic food availability. In particular, the decrease in re-
productive output during the last two cycles coincided
with a previously reported seasonal decrease in phyto-
plankton in the Damariscotta River (Incze el ai, 1980).
Likewise, temporal patterns in cycle duration (Table II)
may be due to variation in water temperature (Grosberg,
1982). Clearly, the results reported here do not constitute
a definitive statement about the genetic basis of these life-
history traits. Breeding experiments will ultimately be
necessary to estimate narrow-sense heritabilities.

The terminal sizes of colonies in our study were sub-
stantially smaller than those reported in other studies
that did not employ clonal replication (Grosberg, 1988;
Chadwick-Furman and Weissman, 1995; etc.). Either
colonies in the Damariscotta River cease growth at a
smaller size than colonies in some other populations
(Monterey Bay and the Eel Pond), or our estimates of
terminal size were affected by subdividing colonies to
produce clonal replicates. Field colonies in the Damaris-
cotta River appear to frequently attain a larger size than
those in our study (pers. obs.), lending credence to the
latter interpretation. Although subdivision may have
affected our absolute values for terminal size, all geno-
types were subdivided, and hence the terminal size of
subdivided colonies should nevertheless yield a valid es-
timate of the relative performance of each genotype.

Correlations and selection

The six life-history variables that we measured were
not independent of one another. In addition to a couple
of correlations between variables that measure the same

or similar traits (testis cross-sectional area and sperm
counts, growth rate and terminal size), we found nega-
tive partial correlations among genotype least square
means for male (testis cross-sectional area), female (eggs
per bud), and asexual (buds per zooid) reproduction (Ta-
ble IV). The three-dimensional relationship of these vari-
ables (Fig. 3) and the results of a principal component
analysis (Table V) both suggest a three-way tradeoff
among these variables. Although this three-dimensional
relationship is greatly strengthened by the inclusion of
the three most extreme genotypes, the remaining 15 ge-
notypes still cluster around a plane (Fig. 3) and display
the same basic relationship.

One element of this multivariate correlation appears
to differ slightly from the result previously reported by
Grosberg ( 1982), who found no correlation within itero-
parous colonies between asexual growth (buds per zooid)
and female reproduction assayed as eggs per bud (male
reproduction was not assayed). However, Grosberg
(1982) did detect a negative correlation between asexual
growth and lifetime egg production, which is another as-
say of female reproduction. Strikingly, the correlation
between asexual growth and female reproduction (as-
sayed as eggs per bud) became strongly positive when
semelparous genotypes were also included in the analysis
(Grosberg, 1982, 1988).

The existence of negative correlations alone indicates
little about the proximate causes of life-history tradeoffs.
Surgical manipulation of gamete production and growth
patterns (Grosberg. 1988) could be employed to explore
possible physiological mechanisms underlaying these
negative correlations. However, the correlation structure
that we detected is consistent with a simple energetic
tradeoff in allocation to male, female, and asexual repro-
duction. Although a tradeoff in allocation between male
and female reproduction is a common assumption of
sex-allocation models for hermaphrodites (Charnov,
1 979, 1 982), this assumption has rarely been empirically
evaluated. Although the likely consequence of a shift in
resource allocation between male and female reproduc-
tion is relatively straightforward, variation in allocation
to asexual reproduction has potentially more complex
ramifications. Asexual reproduction increases the num-
ber of zooids in a colony and hence is likely to funda-
mentally alter the future energy budget of a colony by
determining total food intake. Could increased alloca-
tion to asexual growth at the expense of current sexual
reproduction be associated with increased sexual repro-
duction effort at some later point in colony ontogeny?
Again, temporal allocation patterns of different geno-
types (genotype by cycle interactions) mer i further con-
sideration.

The negative correlations among rr e, female, and
asexual reproduction that we detects aave important

298

P. O. YUND ET AL

implications for evaluating the possible consequences of
selection on these life <n story traits. These negative cor-
relations are based on genotype least square means, and
so represent bnxui-sense genetic correlations roughly
comparable to estimates based on family mean values
in breedir .lies (Via, 1986). To the extent that these
broad-sense genetic correlations reflect narrow-sense ge-
netic correlations, they indicate that selection cannot act
independently on these three traits. A change in fitness
via increased allocation to one form of reproduction will
be balanced by reduced allocation, and hence presum-
ably reduced fitness, via the other two modes of repro-
duction. In this scenario, selection would result in evolu-
tionary change if fitness advantages due to increased al-
location to one mode of reproduction more than offset
fitness costs due to decreased allocation to the other two
modes.

The selective pressures acting on life-history traits in
B. schlosseri are not completely understood. Overgrowth
by colonies of the con-familial Botrylloides diegense has
been suggested to select against semelparous, high-egg-
production genotypes of B. schlosseri in the Eel Pond at
Woods Hole (Grosberg, 1982, 1988), and this introduced
spatial competitor has also been present in the Damaris-
cotta River since the late 1970s (Yund and Feldgarden,
1992). Selection via fertilization processes may also be
important, especially in favoring different production
levels of male and female gametes (Levitan, 1995; Levi-
tan and Petersen, 1995). For example, ecological situa-
tions in which males compete for fertilizations (Yund
and McCartney, 1994) or levels of egg fertilization are
sperm limited (Levitan, 1995) could both result in selec-
tion for increased production of sperm (Yund, 1997).
The relaxation of those conditions would in turn favor
increased allocation to egg production due to tradeoffs in
allocation.

Acknowledgments

T. Miller and the other staff members of the University
of Maine's Darling Marine Center made this work possi-
ble, and R. Grosberg provided valuable comments on
correlations among life-history traits in B. schlosseri. Fi-
nancial support was provided by the National Science
Foundation (OCE-92-02805 and OCE-94- 16548).

Literature Cited

Atkinson, O. S., and P. O. Yund. 1996. The effect of variation in pop-
ulation density on male fertilization success in a colonial ascidian.
J. Exp. Mar Biol. Ecol. 195: 1 1 1-123.

Boyd, H.C., S. K. Brown, J. A. Harp, and I. L. Weissman. 1986.

Growth and sexual maturation of laboratory-cultured Monterey
Botrvllus schlosseri. Biol Bull 170: 91-109.

Chadwick-Furman, N. E., and I. L. Weissman. 1995. Life histories
and senescence of Botryllus schlosseri (Chordata. Ascidiacea) in
Monterey Bay. Biol. Bull. 189: 36-41.

Charnov, E. L. 1979. Simultaneous hermaphroditism and sexual se-
lection. Proc. Nail. Actul. Sci. USA 76: 2480-2484.

Charnov, E. L. 1982. The Theory of Sex Allocation. Princeton Uni-
versity Press, Princeton, New Jersey.

Cohen, C. S., and R. R. Strathmann. 1996. Embryos at the edge of
tolerance: effects of environment and structure of egg masses on
supply of oxygen to embryos. Biol. Bull. 190:8-15.

Eckelbarger, K. J., and L. Watling. 1995. Role of phylogenetic con-
straints in determining reproductive patterns in deep-sea inverte-
brates. Im-eriehr. Biol 114: 256-269.

Emlet, R. B., L. R. McEdward, and R. R. Strathmann. 1987. Echino-
derm larval ecology viewed from the egg. Pp. 55-1 36 in M. Jangoux
and J. M. Lawrence, eds. Eclunoderm Studies. Vol. 2. Balkema,
Rotterdam.

Falconer, D. S. 1981 . Introduction lo Quantitative Genetics (2nd ed.).
Longman. Essex. England.

Ghiselin, M. T. 1987. Evolutionary aspects of marine invertebrate re-
production. Pp. 609-665 in A. C. Giese. J. S. Pearse, and V. B.
Pearse. eds. Reproduction of Marine Invertebrates: Seeking Unity in
Diversity. Boxwood Press. Pacific Grove, CA.

Gosner, K. L. 1971 . Guide to the Identification of Marine and Eslua-
nnc Invertebrates: Cape Halleras lo the Bay of Fundy. John Wiley,
New York.

Grosberg, R. K. 1982. Ecological, genetical, and developmental fac-
tors regulating life history variation within a population of the colo-
nial ascidian Botrylliis schlosseri ( Pallas) Savigny. Thesis. Yale Uni-
versity. New Haven, CT.

Grosberg, R. K. 1988. Life-history variation within a population of
the colonial ascidian Botryllus schlosseri. I. The genetic and envi-
ronmental control of seasonal variation. Evolution 42: 900-920.

Harvell, C. D., and R. K. Grosberg. 1988. The timing of sexual matu-
rity in clonal animals. Ecology 69: 1855-1864.

Hendler, G. 1975. Adaptational significance of the patterns of ophi-
uroid development. Am Zoo/. 15:691-715.

Hughes, D. J., and R. N. Hughes. 1986. Life history variation in Cel-
leporella hyalina (Bryozoa). Proc. Royal Soc. London Ser. B228:
127-132.

Incze, L. S., R. A. Lutz, and L. Watling. 1980. Relationship between
effects of environmental temperature and seston on growth and
mortality ofMytilus cdulis in a temperate northern estuary. Mar.
Biol. 57: 147-156.

Lande, R., and S. J. Arnold. 1983. The measurement of selection on
correlated characters. Evolution 37: 12 10- 1226.

Levitan. D. R. 1995. The ecology of fertilization in free-spawning in-
vertebrates. Pp. 123-156 in L. McEdward. ed. Ecology of Marine
Invertebrate Larvae. CRC Press, Boca Raton, FL.

Levitan, D. R., and C. Petersen. 1995. Sperm limitation in the sea.
Trends Ecol. Evol. 10: 228-23 1 .

McEdward, L. R., and L. K. Coulter. 1987. Egg volume and ener-
getic content are not correlated among sibling offspring of star-
fish: implications for life history theory. Evolution 4\: 914-917.

Menge, B. 1975. Brood or broadcast? The adaptive significance of
different reproductive strategies in the two intertidal sea stars
Leptaslerias he.\actis and Pisaster ochraceus. Mar Biol 31: 87-
100.

Milkman, R. 1967. Genetic and developmental studies on Bolryllus
schlosseri Biol. Bull. 132:229-243.

Strathmann, R. R. 1990. Why life histories evolve differently in the
sea. Am. Zool. 30: 197-207.

Stralhmann, R. R., and M. F. Strathmann. 1982. The relationship be-

ASCID1AN LIFE HISTORY VARIATION

299

tween adult size and brooding in marine invertebrates Am. Nal.

119:41-101.
StraChmann, R. R., M. F. Strathmann, and R. II. Emson. 1984. Does

limited brood capacity link adult size, brooding, and simultaneous

hermaphroditism? A test with the starfish Axlcrina p/iyliifiicn lin

Nat. 123:796-818.
Via, S. 1986. Genetic covariance between oviposition preference and

larval performance in an insect herbivore. Evolution 4/0: 778-785.
Yund, P. (). 1991. Natural selection on hydroid colony morphology

by intraspecinc competition. Evolution 45: 1 564- 1573.
Yund, P.O. 1995. Gene flow via the dispersal of fertilizing sperm in a

colonial ascidian (Botryllus .\chlos.wri): the effect of male density.
Mur Bu>l 122:649-654.

Yund, P. O. 1997. The effect of sperm competition on the relation-
ship between sperm production and reproductive success in a ma-
rine invertebrate. Ideology In press.

Yund, P. ()., and M. Kcldgardcn. 1992. Rapid proliferation of histor-
ecognition alleles in populations of a colonial ascidian. J. Exp. Zool.
263: 442-452.

Yund, P. O., and M. A. McCartney. 1994. Male reproductive success
in colonial invertebrates: competition for fertilizations. Ecology 75:
2151-2167.

Reference: Biol. Bull. 192: 300-308. (April, 1997)

Compound Eye Fine Structure in Paralomis multispina

Benedict, an Anomuran Half-Crab From 1 200 m Depth

(Crustacea; Decapoda; Anomura)

EISUKE EGUCHI1, MARI DEZAWA2, AND V. BENNO MEYER-ROCHOW-

1 Department of 'Biology, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 2 36,

Japan, from Japan Marine Science and Technology Center (JAMSTEC), Natsushima-cho,

Yokosuka, 237, Japan; ^-Department of Anatomy, School of Medicine, Chiha University, Inohana,

Chuo-ku, Chiba 260, Japan: and ^Department of Biology (Section Animal Physiology),

University ofOitlu, SF-90570 Oulit, Finland

Abstract. Fully grown, unsexed specimens of the ano-
muran half-crab Paralomis multispina Benedict were
obtained from a depth of 1200 m, and the eyes of three
individuals were prepared for light and electron micros-
copy. In their outer appearance the compound eyes of
Paralomis resemble those of common shallow-water
half-crabs (e.g., Petrolistlies), but facets in Paralomis
were about 3 times larger in diameter (i.e., 60 ^m)and at
least twice as long. Interommatidial angles ranged from
3° to 5°. The proximal width of the crystalline cone in
Paralomis was 10 times that of its equivalent in the Pet-
rolistlies eye, and the rhabdom — although only twice as
long — had a radius that was 7 times greater distally and
4 times greater proximally. A clear-zone between cones
and rhabdom was not developed, and cross sections of
crystalline cones revealed rounded rather than square
profiles. A distal retinula cell (R8) was absent, and all
regular retinula cells (R1-R7) protruded microvilli of
about 0. 1 1 nm diameter in many (and not only two) di-
rections. A maximum rhabdom occupation ratio of 85%
was found in the Paralomis retinula, whereas in the shal-
low-water half-crabs the comparable figure was 35%.
Paralomis featured a wide, rhabdomless space between
basement membrane and proximal rhabdom ends; the
space was occupied by reflecting cells. Primary screening
pigment cells and their dark granules were present; sec-
ondary screening pigment cells, however, were replaced

Received 19 June 1996; accepted 18 November 1996.

by reflecting cells. The anatomical modifications in the
Paralomis eye are consistent with habitat-related adap-
tations seen in the eyes of other benthic and slow-moving
deep-water crustaceans, but not with those of euphausi-
ids. We conclude that the eye of Paralomis functions as
an apposition eye, designed to maximize photon cap-
ture, especially from point sources (i.e.. biolumines-
cence) rather than extended sources. We estimate that
the Paralomis eye is at least 1 50 times more sensitive to
light than the eye of shallow-water Petrolisthes.

Introduction

Animals that live in or colonize greater oceanic depths
face three major physical challenges (Marshall, 1957;
Thorson, 1972): (a) atmospheric pressure increases by 1
with every 10 m of water; (b) ambient light levels become
progressively reduced, and the spectral composition of
the downwelling light changes as depth increases: and (c)
the temperature of the water falls as the distance to the
surface increases, except in the polar oceans (where bot-
tom temperatures may actually lie a few degrees above
those of the surface) and near hydrothermal vents.

The eyes of animals are usually attuned to the photic
conditions under which they operate (Forward el ai,
1988), but environmental temperature and pressure also
influence certain structural and functional parameters of
photoreception through their effects on membrane fatty
acid content and composition (Cossins and Macdonald,
1989; Sebert el ai. 1992; Kashiwagi et a/., 1996). Based

300

COMPOUND EYE ULTRASTRUCTURE IN PARALOMIS MILT1SPINA

301

on a number of light microscopical (Beddard, 1890;
Welsh and Chace, 1937, 1938; Zharkova, 1970, 1975;
Bursey, 1975) and electron microscopical studies of
deep-water crustacean eyes (Elofsson and Hallberg,
1977; Ball, 1977; Hallberg, 1977; Meyer-Rochow and
Walsh. 1977, 1978; Hallberg et at.. 1980; Gaten el al.,
1992;Gaten, 1994; Nuckley et al.. 1996), certain general
trends concerning their anatomy and performance in re-
lation to depth have become apparent.

Bath pelagic and benthic species of depths exceeding
1000 m usually exhibit small and degenerate eyes (sim-
ilar to those of species known from marine caves (Meyer-
Rochow and Juberthie-Jupeau, 1987). Crustaceans in-
habiting zones above 1000m, on the other hand, fre-
quently possess adaptations such as enlarged ommatidia,
more voluminous rhabdoms, presence of retinal reflec-
tors, etc.. to improve the efficiency of photon capture.
Often such adaptations enhance overall sensitivity at the
expense of acuity, but in cases where acuity apparently
suffers little degradation, regional eye modifications and
special optical designs may be employed as, for instance,
in the Euphausiaceae (Land et al.. 1979; Hiller-Adams
and Case, 1984). Most euphausiids, however, are lumi-
nescent and thus not necessarily representative of other
groups of crustaceans. For that reason and the fact that
few species of deep-sea crustaceans have had their pho-
toreceptors studied, we decided to examine the eyes of
the anomuran decapod half-crab Paralomis multispina
from a depth of 1 200 m and compare them with those of
shallow-water anomurans investigated earlier (Eguchi et
al.. 1982; Meyer-Rochow et al., 1990; Gaten, 1994).

Materials and Methods

Several unsexed specimens of Paralomis multispina
Benedict (Decapoda, Anomura, Galatheoidea) with ca-
rapace widths ranging from 5 to 1 1 cm and maximum
body lengths (from head to tail) of 1 1.5 cm (Fig. la) were
obtained in March 1992. Collections were made from
the "Hatsushima seep" (Ohta et al.. 1987) at a depth of
1200 m about 5 km off Hatsushima Island in Sagami
Bay (Shizuoka Prefecture, Japan) during a cruise of the
manned research submersible Shinkai 2000 (JAMS-
TEC). The half-crabs themselves are not considered to
be thermophilic, although they occurred in association
with the giant clam Calyptogena soyae, vestimentiferan
tube worms, gastropods, and polychaetes (Hashimoto et
a/.. 1987) in an area that was characterized by an ex-
tremely high methane content. The half-crabs were
picked up from the seafloor with remotely controlled ar-
tificial arms and put in a basket attached to the outside
of the submersible. It took about 1 h for the submersible,
with the collected animals, to reach the surface at about
1700h.

Figure I. Photographs of Paralomis multispina. (a) Dorsal view of
specimen with carapace width of 1 1 cm and legs 30 cm long, (b) Close-
up of head region with pair of eyestalks and black shiny eyes, each mea-
suring about 4 mm in length in this specimen.

During capture, the animals were exposed to 10,000-
20,000 lux bright sunlight for about 20 min, but imme-
diately after they had been hauled on board the moth-
ership Natsushima, the compound eyes of three individ-
uals were fixed for 12 h at 4°C in 2% glutaraldehyde and
2% paraformaldehyde solution, buffered to a pH of 7.3
with 0. 1 M cacodylate buffer. The dissections were car-
ried out under dim red light to minimize further
exposure to light and structural damage (cf. Meyer-Ro-
chow, 1994). After a brief wash in buffer, the specimens
were postfixed for 2 h in 2% OsO4 solution, using the
same buffer as before, and dehydrated in a graded series
of acetone before being embedded in Epon 812. Ultra-
thin sections, cut with a diamond knife, were picked up
on uncoated 200-mesh copper grids and stained with
uranyl acetate and lead citrate for a few minutes. Obser-
vations were carried out under a JEM 1200EX transmis-
sion electron microscope, operated at 80 kV.

Results

In their external appearance the compound eyes of the
deep-sea anomuran Paralomis multispina (Fig. Ib) re-
semble those of other common anomuran shore-crabs
(e.g., genus Petrolisthes: Eguchi et al.. 1982; Meyer-Ro-

302

E. EGUCHI ET AL.

chow et al.. 1990), but overall the eyes are considerably
larger. They -re oval in outline and measure 3.5 X
2.5 mm in an individual of about 10 cm carapace width.
Each eye -.its at the tip of an eyestalk that is 4-5 mm thick
and 1 2 mm long; thus inter-eye distances and the precise
location of the eyes in space are to a certain extent vari-
able. Ommatidial numbers increase with age: whereas a
specimen with a carapace length of 5 cm has about 1 500
facets, some 2400 were counted in a specimen with a ca-
rapace width of 10 cm.

Interommatidial angles apparently do not change sig-
nificantly with age and measure about 3°-5°. Figure 2
provides a comparison between the ommatidia. shown
in identical scale, of a shallow-water anomuran and the
deep-water species Paralomis multispina. Biometrical

Figure 2. i f comparable ommatidia of Petmlislhcs sp.

( A-E), found in s, -r, and Paralomis mnllisi>ina(F,G), found

in the deep-sea at ;:; \escale. A8 = axon of distal retinula

R8; Bm = basemc.; Cc = crystalline cone; Cg = cor-

L-nous cell; Cr = cor = distal retinula cell; Re = retinula

= rhabdom; Rp pigment cell; Sp = screening pig-

1'. (A) longitudinal section; (B, C) cross sections at the level of

i;t bands in the distal layers of the rhabdom; (D. E) cross

> adjacent bands in the proximal rhabdom layers. (F)

11111 ion; (C) cross section al distal rhabdom layers.

data of the constituent parts of one representative central
ommatidium of the compound eye of the two crusta-
ceans are given in Table I. From Figure 2 and Table I it
is evident that the ommatidium of the deep-sea half-crab
is much larger than that of the shallow- water species,
even if differences in body size are taken into consider-
ation.

Dioptric apparatus

A single facet of the eye of Paralomis is about 3 times
larger in diameter and has a corneal lens that is 1 .8 times
thicker than that of a comparable shallow-water Pet-
rolisthes. No significant difference between the two types
could be detected, however, in the 200 ^m thick periodic
layers, revealed in longitudinal sections of the cornea
along the optic axis. Two corneagenous cells, not notice-
ably different from those of Petrolisthes or any other
decapod crustacean, occupied the space between cornea
and cone.

The crystalline cone of Paralomis tapered only very
gently and retained a much wider proximal diameter
(Fig. 3a) than that of Petrolisthes. Whereas in Pct-
rolisthcs. cross sections through distal and central re-
gions of the cone displayed square profiles and a content
of electron-dense material, sections through the cone of
Paralomis at corresponding levels exhibited rather circu-
lar outlines and a content of much looser consistency
(Fig. 3b). When related to overall ommatidial length, the
dioptric apparatus in the eye of Paralomis (though
greatly enlarged in diameter) occupied significantly less
space than the equivalent structure in the eye of the shal-
low-water Petrolisthes.

Retinula and rhabdom

In the eyes of other anomuran species — for example,
Petrolisthes spp. (Eguchi et al., 1982; Meyer-Rochow et
al.. 1990) and Municla spp. (Bursey, 1975; Gaten.
1994) — a distal retinula cell (R8) with four cytoplasmic
lobes occupies the tier between the crystalline cone and
the seven regular retinular cells, but in Paralomis an om-
matidial retinula is composed of only seven regular cells
(1-7) and lacks the distal eighth cell. The distal end of
the rhabdom is thus made up of seven regular retinula
cells, which are in contact with the proximal end of the
crystalline cone. It is in this region that the mottled retin-
ula cell nuclei, with a maximum diameter of 7.5 /urn, can
be found.

The rhabdoms in Paralomis are extraordinarily well
developed and occupy up to 85% of the available cyto-
plasmic space in the distal and central regions of the re-
tinula (Fig. 4). The estimated membrane surface of an
ommatidial rhabdom of Paralomis (231 X 104), calcu-

COMPOUND EYE ULTRASTRUCTURE IN PAR.4LOMIS Mi'LTISPINA 303

Table I
Comparison of biometrical data (in urn) ofommatidia in the shallow-water Petrolisthes sp. and the deep-sea Paralomismultispina

Petrolisthes

Paralomis

Para/Pelro

Remarks

Cornea

diameter

21

60

2.9

thickness

16

28

1.8

Crystalline cone

distal diameter

20

56

2.8 length of a square side

prox. diameter

4

40

10.0

length

110

1 10

1.0

Ommatidial retinula

length

60

390

2.6

diameter

20

40

2.0

Rhabdom

diameter at nuclear layer

3.5

25

7.1

diameter at proximal layer

3x8*

18

3.7 'rectangular

length

50

210

1.9

thickness of one band

7

4-7

0.6-1.0

diameter of one microvillus

0.08

0.11

1.4

area of rhabdom membrane

8.5 x 104

231 X 104

27.2 total surface of microvilli/ommatidium

Rhabdom occupation ratio fr)

distal and central region

12

85

6.3

proximal region

35

45

1.3

Distance from rhabdom end to BM

12

180

15.0

Interommatidial angle

4-6°

3-5°

ca. 0.9

Sensitivity

0.23

34.7

1 5 1 relat. sensitivities (after Land, 1981)

lated from the data in Table I. is about 27 times larger
than that of Petrolisthes (8.5 X 104). Another compari-
son could be made with Limulus, which — even though it
is not a crustacean — has a compound eye (Fahrenbach,
1969) superficially similarto that of Paralomis. but with

rhabdom occupation ratios generally lower than 10%.
On the other hand, the hydrothermal vent shrimp Rimi-
caris exociilata occurs in a habitat similar to that of Para-
lomis, but its eye is highly aberrant, with volume densi-
ties of rhabdoms reaching 70%-80% (O'Neill el al,

Figure 3. The deep-sea half-crah Pura/om/*, mulnspina. (a) Longitudinal section through the proximal
region of the crystalline cone and the distal tip. C = crystalline cone: R = rhabdom. Scale bar = 5 ^m. (b)
Cross section through proximal part of crystalline cone with its four components. Numerous screening
pigment granules surround the crystalline cone. Scale bar = 5 ^m.

304

E. EGUCHI ET AL

ff' ;?»-x.

ir

\ •

"

•

• •

- ••

'

f '

«

'-

Figure 4. Cross section of rhabdom at the retinula cell nuclear layer. Almost the entire cytoplasmic
space of the retinula cells isoccupied by the rhabdomeres. N = nucleus of retinula cell; R = rhabdom. Scale
bar = 1 ^m.

1995). The retinula cells do not form proper rhabdoms
in the proximal region; instead they gradually turn into
slender axonal processes ( Fig. 5).

Longitudinal sections reveal that the regular "bands,"
so typical for the rhabdoms of other decapods (including
those of the shallow-water anomuran species), are almost
lost in Paralomis and are replaced by microvilli running
in many directions. This gives the rhabdom a somewhat
irregular, disorderly appearance. Individual microvilli in
Paralomis (Fig. 6) were thicker (0.1 1 ^m) than those of
fully grown shallow-water Petrolisthes (0.08 nm: Eguchi
etal, 1 982; Meyer-Rochow and Reid, 1996). This differ-
ence has to be interpreted with caution, since it is known
from other crustacean eyes (e.g., Orchomene sp.: Meyer-
Rochow, 1981; Mysisrelicta:Lmdslr6m etal., 1988) that
'xlom microvilli have a tendency to swell and increase
i meter when suddenly exposed to very bright light,
•(•-filament, usually identifiable in the lumen of a
ibdom microvillus of the crustacean eye, was
1 or fragmented into smaller pieces (Fig. 7).
Sonu ; rhabdom microvilli exhibited flattened or

swollei tures in the place where core-filaments with

their as -d side-arms should have been. Since core-

filaments and their associated side-arms in compound
eyes are fragile and easily destroyed by irradiation with
bright light ( Blest etal.. 1982; Tsukita el a/.. 1988), their
disruption in our material could stem from the brief
exposure to sunlight during capture.

Screening pigment

The eye of Paralomis lacks secondary pigment cells;
two primary pigment cells are found around the crystal-
line cones and contain spherical electron-opaque pig-
ment grains of about 0.4 ^m in diameter (Fig. 3a). The
density of these granules seems not to differ from that of
granules in the shallow-water half-crabs, but screening
pigment granules in the retinula cells are far less numer-
ous in Paralomis. In place of secondary pigment cells are
an unknown number of cells presumed to contain re-
flecting granules.

Reflecting pigment

The distance between the proximal end of the rhab-
dom and the basement membrane of an ommatidium is
relatively short in Petrolisthes and other shallow-water

COMPOUND EYE ULTRASTRUCTURE IN PAR.ILOMIS MI'LTISPINA

305

c

-
1

^- .' '

Figure 5. Cross section through proximal retinal layer. A small rhabdom (R) is seen at the center of a
cluster of seven retinula cells. A few scattered screening pigment granules are visible in some of the retinula
cells. Well-developed reflecting pigment cells fill the interommatidial spaces. Scale bar = I nm.

Figure 6. Cross section through a segment of a retinula cell demonstrating the multidirectional orien-
tation of microvilli in the rhabdom. Scale bar = 1 ^m.

Figure 7. Higher magnification of transversely cut microvilli of the rhabdom. Note that core-filaments
are lost in some of the microvilli. Scale bar = 0. 1 ^m.

decapods. In Paralomis, however, this same distance is
strikingly long (ca. ISO^m). In the proximal layer, the
retinula cells become slender as shown in Figure 5. The
space thus made available is filled with enormously de-
veloped cells containing large amounts of reflecting pig-
ments. The extensions of the reflecting pigment cells,
which are easily identifiable by their innumerable 0.3-
^m-wide vesicles, penetrate between the retinula cells of
individual ommatidial units, thus apparently increasing
their effectiveness in reflecting light towards the more
distally placed rhabdom.

Discussion

Eguchi el al. (1982) suggested that anomuran half-
crabs of the superfamily Galatheoidea possess reflecting
superposition eyes. Research by Meyer-Rochow el al.
( 1 990) on the galatheid Petrolisthes elongatm and by Ga-
ten (1994) on Mimida rugosa lent further support to this
notion, but specified that this was true only for the dark-
adapted eye: in the light-adapted state apposition optics

were used. It is generally assumed that superposition eyes
are more useful than apposition eyes in dim light, for the
former are typical of many nocturnal crustaceans and
deep-sea forms.

It is, therefore, a little surprising to find that the eye of
the deep-sea anomuran galatheid Paralomis nndtispina
(a) lacks a wide clear-zone, which is normally considered
a prerequisite for any form of superposition vision
(Land, 1981), and (b) possesses roundish rather than reg-
ular, square cones, which are an essential requirement
for reflecting superposition (Land, 1976; Vogt, 1980).
The species does, however, exhibit other kinds of modi-
fications that are more in keeping with adaptations to an
extremely dim environment: compared with the shal-
low-water half-crabs of the genus Petrolisthes (Eguchi el
al. 1982: Meyer-Rochow el al.. 1990), in Paralomis the
corneal diameter is three times greater, and cone as well
as rhabdom diameters are even more enlarged (Table I).
The reflecting tapetum on the proximal sk'e of the retin-
ula is massively developed, and it is evki . nt that the eye
is designed to maximize photon capture The fine-struc-

306

E. EGUCHI KT AL.

tural disruptions and larger diameters of the rhabdom
microvilli seen in Paralomis are almost identical to those
reported from the eyes of deep-water amphipods from
the Antarctic ( f ••' <- Rochow, 1981) and are most likely
caused by tru- •-•.•sure to bright light during capture. In-
directly the disruptions thus point to a high absolute sen-
sitivity to light, but at the same time they obscure signs
for or against membrane shedding (<;/.' Chamberlain and
Barlow, 1984).

On the basis of the definition that Land (1981) pro-
vided for "absolute sensitivity," we calculated sensitivi-
ties of light-adapted eyes of Paralomis and those of shal-
low-water Petrolisthes: the eye of Paralomis was 150
times more sensitive. The comparison is based on the
assumptions that the extinction coefficient (k) is the
same for the two species and that the types and densities
of pigments found in the rhabdoms are identical (cf. dis-
cussion in Ziedins and Meyer-Rochow, 1990). If one as-
sumes a superior photopigment content in the dark-
adapted Paralomis eye and considers thermal noise re-
duction at low environmental temperatures (Aho el al,
1988), the overall sensitivity advantage of Paralomis
over Petrolisthes to extended light sources may be even
higher.

If the lack of a clear-zone is real and not artifactual
(clear-zones in the superposition eyes of deep-sea deca-
pods can easily collapse and, on account of their fragility
and delicateness, may remain undetected as shown by
Nilsson, 1990), the closer approximation of the mas-
sively developed rhabdom to the much wider dioptric el-
ements, in combination with the backing of a tapetum
from behind, could be interpreted as an adaptation to
improve sensitivity, especially to point sources. The
shortening of both cornea and cone, relative to the over-
all length of one ommatidium, and the loss of the orderly
arrangement of microvilli in the rhabdom also point to-
ward an adaptation to minimize photon loss and maxi-
mize photon capture (Laughlin et al., 1975). The consid-
erably greater rhabdom-occupation ratio in the eye of
Paralomis as compared with the shallow-water species
not only allows more photopigment molecules to be
packed into the visual membranes, but also indicates low
energy demand and slow cellular metabolism, both ad-
aptations that are extremely useful in the deep-sea envi-
ronment (Elofsson and Hallberg, 1977).

In the shallow-water Petrolisthes elongatus the eye en-
larges as the half-crab grows; ommatidia are added and
sensitivity to both extended and point sources increases.
P. elongatus uses vision to detect and approach hiding
places (Meyer-Rochow and Meha, 1994). Since signs of
eye regression in adult Paralomis are missing, we must
assume that the general growth pattern resembles that of
Petrolisthes. This, however, raises the question of what

Paralomis could possibly see at a depth of 1 200 m, the
"limit" beyond which sunlight can no longer be detected
(Clarke and Kelly, 1964). Biological light sources, how-
ever, abound at this depth (Omori, 1974), and it may
well be in the interest of a benthic, sedentary detritus and
filter feeder to notice them. Any visual signal adult Para-
lomis could possibly be interested in would almost never
come from below, and this could explain the lack of re-
gional eye specializations seen in so many mesopelagic
shrimps (Galen £>/«/., 1992).

We know nothing about the spectral sensitivity of
Paralomis, but the visual pigments of eight other ano-
muran species all exhibit a single absorption peak in the
bluegreen region of the spectrum (Cronin and Forward,
1988). Ziedins and Meyer-Rochow ( 1990) electrophysi-
ologically measured spectral sensitivity peaks of dark-
and light-adapted eyes of P. elongatus and also found
them to lie in the bluegreen part of the spectrum. Since
even the eyes of the hydrothermal vent species Rimicaris
exoculata. which are strongly modified morphologically
(O'Neill et al., 1995), possess a sensitivity peak in the
bluegreen (Johnson et al.. 1995), we do nol expecl the
eyes of Paralomis to differ in this respect. However, the
lack of secondary screening pigments and retinula cell
8 in Paralomis suggests that the eye of Al. rugosa, for
example, is less well adapted to the greatest depth of its
range (shallow water down to 1250m:Gaten, 1994) than
that of Paralomis. M. rugosa appears to be a relative
"newcomer" to the deep-sea, while Paralomis has been
exploiting that habitat for a longer evolutionary period.
How much longer is hard to say, but Nuckley et al.
(1996) speculate that a hydrothermal vent shrimp with
modified eyes may have "migrated from the surface pos-
sibly in the last 5,000-10,000 years" and over thai period
evolved its present eye morphology.

In conclusion, Ihe hypertrophied rhabdoms in Ihe eye
of Paralomis, Ihe loss of Ihe orderly microvillus arrange-
ment Ihe reduction of the cytoplasmic componenl of the
retinula cells, the massively developed layer of reflecting
vesicles in Ihe proximal half of Ihe relinula, and (consid-
ering overall ommalidial length) the relative shortening
of the dioptric elements coincident with greally enlarged
diamelers in Ihe eye of Paralomis are all consistent with
deplh-relaled adaplalions seen also in Ihe eyes of deep-
sea mysids (Elofsson and Hallberg. 1977), amphipods
( Hallberg et al.. 1980; Meyer-Rochow et al., 1991), and
to some extenl olher benlhic decapods (Hiller-Adams
and Case, 1985) and mesopelagic shrimps (Galen et al.,
1992). However, Ihe eyes of deep-waler euphausiids
(Hiller-Adams and Case, 1988) are least similar to Ihose
of Paralomis and Ihis, we believe, has lo do wilh (a) Ihe
widespread abilily of euphausiids lo produce light, (b)
the greater mobility and pelagic lifeslyles of euphausiids.

COMPOUND EYE ULTRASTRUCTURE IN PAR.-ILOMIS MVLTISP1NA

307

and (c) the longer evolutionary period euphausiids have
had to adapt their photoreceptors to the deep-sea envi-
ronment.

Acknowledgments

We thank the Shinkai 2000 operation team and the
Japan Marine Science and Technology Center (JAMS-
TEC) (Natsushimacho, Yokosuka, Japan) for their kind
offer to assist in the procurement of the material. We also
wish to acknowledge that through the constructive criti-
cism of two anonymous referees we were able to improve
the paper.

Literature Cited

Aho, A. C., K. O. Donner, C. Hyden, L. O. Larsen, and T. Reuter. 1988.

Low retinal noise in animals with low body temperatures allows
high visual sensitivity. Nature 334: 348-350.

Ball, E. E. 1977. Fine structure of the compound eyes of the mid-
water amphipod Ptmmima in relation to behaviour and habitat.
TissueCell9:251-536.

Beddard, F. E. 1980. On the minute structure of the eye in some shal-
low-water and deep-sea species of the isopod genus Acturus. Proc.
Zool SOL: Lond. 26: 365-375.

Blest, D., S. Stowe, and \V. Eddy. 1 982. Cytoskeleton i n rhabdomeral
microvilli ofblowflies. Cell Tissue Res. 223: 553-573.

Bursey, C. R. 1975. The microanatomy of the compound eye of
Munida irrasa (Decapoda: Galatheidae). Cell Tissue Res. 160: 505-
514.

Chamberlain, S. C., and R. B. Barlow, Jr. 1984. Transient membrane
shedding in Limulus photoreceptors: control mechanisms under
natural lighting./ Neurosci. 4: 2792-2810.

Clarke, G. I,., and M. G. Kelly. 1964. Variation in transparency and
in bioluminescence on longitudinal transects in the western Indian
Ocean. Bull lust Oceanogr. (Monaco) 64: 1 -20.

Cossins, A. R., and A. G. Macdonald. 1989. The adaptation of bio-
logical membranes to temperature and pressure: fish from the deep
and cold. J. Bioenerg. Biomembr. 21 : 115-135.

Cronin, T. \V., and R. B. Forward. 1988. The visual pigments of
crabs. I. spectral characteristics. J. Comp. Physiol A 162: 463-478.

Eguchi, E., T. Goto, and T. H. Waterman. 1982. Unorthodox pattern
of microvilli and intercellular junctions in regular retinular cells of
the porcellanid crab Pelrolisllies. Cell Tissue Res. 222: 493-5 1 3.

Elofsson, R., and E. llallberg. 1977. Compound eyes of some deep-
sea and fiord mysid crustaceans. Ada Zool. (Stockh.) 58: 1 69- 1 77.

Fahrenbach, \V. H. 1969. The morphology of the eyes of Limulus —
II. Ommatidia of the compound eye. Z. Zellforxch. 93:451-483.

Forward, R. B., T. W. Cronin, and J. K. Douglass. 1988. The visual
pigments of crabs. II. environmental adaptations. J. Comp. Pliysiol.
A 162:479-490.

Gaten, E. 1994. Geometrical optics of a galatheid compound eye. /
Comp. Physiol. A 175: 749-759.

Gaten, E., P. M. J. Shelton, and P. J. Herring. 1992. Regional mor-
phological variations in the compound eyes of certain mesopelagic
shrimps in relation to their habitat. / Mar Bio/ Assoc tA' 72:61-
75.

Hallberg.E. 1977. The fine structure of the compound eyes of mysids
(Crustacea: Mysidacea). Cell Tissue Res 184:45-65.

Hallberg, E., H. Nilsson, and R. Elofsson. 1980. Classification of am-

phipod compound eyes — the fine structure of the ommatidial units
(Crustacea. Amphipoda). Zoomorphology94: 279-306.

Hashimoto, J., T. Tanaka, S. Matsuzana. and II. Hotta. 1987. Sur-
veys of the deep-sea communities dominated by the giant clam Ca-
lyptogena soyac along the slope foot of Hatsushima Island. Sagami
Bay. JAMSTECTR Divpsea Res 3: 37-5 1 .

Hiller-Adams, P., and J. F. Case. 1984. Optical parameters of eu-
phausiid eyes as a function of habitat depth. J. Comp. Phvsiol. 154:
307-318.

Hiller-Adams, P., and J. F. Case. 1985. Optical parameters of the
eyes of some benthic decapods as a function of habitat depth. Zoo-
morphology 105: 108-1 13.

Hiller-Adams, P., and J. F. Case. 1988. Eye size of pelagic crusta-
ceans as a function of habitat depth and possession ofphotophores.
I 'ision Res. 28: 667-680.

Johnson, M. L., P. M. J. Shelton, P. J. Herring, and S. Gardner. 1995.
Spectral responses from the dorsal organ of a juvenile Rimicaris
exoculata from the TAG-hydrothermal vent site. Bridge Newslett.
8:38-42.

Kashinagi, T., V. B. Meyer-Rochow, K. Nishimura, and E. Eguchi.
1996. Fatty acid composition and ultrastructure of photorecep-
tive membranes in the crayfish Procamharus clarkii under condi-
tions of thermal and photic stress. J Comp. Physiol. B: In press.

Land, M. F. 1976. Superposition images are formed by reflection in
the eyes of some oceanic decapod Crustacea. Nalure 263: 764-765.

Land, M. F. 1 98 1 . Optics and vision in invertebrates. Pp. 47 1 -492 in
( 'ision in Invertebrates (Handbook of Sensory Physiology. Vol. V1I/
6B), H. Autrum. ed. Springer, New York.

Land, M. F., F. A. Burton, and V. B. Meyer-Rochow. 1979. The op-
tical geometry of euphausiid eyes. J. Comp. Physiol. 130: 49-62.

Laughlin, S. B., R. Menzel, and A. W. Snyder. 1975. Membranes, di-
chroism and receptor sensitivity. Pp. 237-262 in Photoreceptor Op-
tics, A. W. Snyder and R. Menzel, eds. Springer, New York.

I indstrom, M., H. Nilsson, and V. B. Meyer-Rochow. 1988. Recov-
ery from light-induced sensitivity loss in the eye of the crustacean
Mysis relicta in relation to temperature: a study of ERG-deter-
mined V/log I relationships and morphology at 4°C and 14°C. Zool.
Sa. 5: 743-757.

Marshall, N. B. 1957. Tiefseebiologie. VEB, Jena. Germany.

Meyer-Rochow, V. B. 1981. JheeyeofOrchomenesp. cf. O rossi.an
amphipod living under the Ross Ice Shelf (Antarctica). Proc. R. Soc.
Lond. B Biol. Sci. 21 2: 93- 1 I 1 .

Meyer-Rochow, V. B. 1994. Light-induced damage to photorecep-
tors of spiny lobsters and other crustaceans. Cruslaceana 67: 97-
111.

Meyer-Rochow, V. B., and J. Juberthie-Jupeau. 1987. An electron
microscope study of the eye of the cave mysid Heteramysoides cotli
from the Island of Lanzarote (Canary Isl.). Slvgologia 3: 24-34.

Meyer-Rochow, V. B., and W. P. Meha. 1994. Tidal rhythm and the
role of vision in shelter-seeking behaviour of the half-crab Pet-
rolisthes elongatus (Crustacea; Anomura). J. R. Soc. NZ 24: 423-
428.

Meyer-Rochow, V. B., and \\ . A. Reid. 1996. Does age matter in
studying the crustacean eye? / Comp. Pliysiol. B: In press.

Meyer-Rochow, V. B., and S. Walsh. 1977. The eyes of mesopelagic
crustaceans I. Gennadas sp. (Penaeidae). Cell Tissue Res 186: 87-
101.

Meyer-Rochow, V. B., and S. Walsh. 1978. The eyes of mesopelagic
crustaceans III. Thysanopoda iricuspidata (Euphausiaceae). Cell
Tissue Res. 195:59-79.

Meyer-Rochow, V. B., D. Towers, and I. Ziedins. 1990. Growth pat-
terns in the eye ofPelrolisihcs e/onxuii/.s (Crustacea; Decapoda; An-
omura). E.\p. Biol. 48: 329-340.

308

E. EGUCHI ET AL

Meyer-Rochow, V. B.,H.Stephan,andS. D.Moro.1991. Morpholog-
ical and anatomical observations on the hairy eyes of males and
females of the manneamphipod Dulic/na porrecia( Crustacea, Am-
phipoda. Podoceridae). Boll. ZooL 58: 59-69.

Nilsson, D.-F. 1990. Three unexpected cases of refracting superposit-
ion eyes in crustaceans. J. Comp. Pkysiol. A 167: 7 1-78.

Nuckley, D. J., R. N. Jinks, B.-A. Battelle, E. D. Herzog, L. Kass,
G. H. Renninger, and S. C. Chamberlain. 1996. Retinal anatomy
of a new species of bresiliid shrimp from a hydrothermal vent field
on the mid-atlantic ridge. Biol. Bull. 190: 98-1 10.

Ohta, S., H. Sakai, A. Taira, K. Otmada, T. Ishii, M. Maeda, K. Fuji-
oka, T. Saino, K. kogure, T. Camo, V. Shirayama, T. Furuta, T.
Ishizuka, K. Endow, T. Sunn. H. Hotta, J. Hashimoto, N. Handa,
T. Masuzawa, and M. Horikoshi. 1987. Report on multi-disci-
plinary investigations of the Calyptogena communities at the Hat-
sushima site. JAMSTECTR Deepsea Res. 3: 52-65.

Omori, M. 1974. The biology of pelagic shrimps. Pp. 233-324 in Ad-
vances in Marine Biology. F. S. Russel and M. Yonge. eds. Aca-
demic Press, New York.

O'Neill, P. J., R. N. Jinks, E. D. Herzog, B.-A. Battelle, L. Kass, G. H.
Renninger, and S. C. Chamberlain. 1995. The morphology of the
dorsal eye of the hydrothermal vent shrimp, Rimicans
I'isual Ncumsci. 12: 861-875.

Sebert, P., B. Simon, and L. Barthelemy. 1992. Fluidite membranaire
et allometrie: interet ecophysiologique. Bull. Soc. Ecophvsiol. 17:
115-120.

Thorson, G. 1972. Erforschung des Meeres — eine Bestandsauf-
nahme. Kindler, Munich.

Tsukita, S., S. Tsukita, and G. Matsumoto. 1988. Light induced
structural changes of cytoskeleton in squid photoreceptor microvilli
detected by rapid-freeze method. J. Cell Biol 106: 1 1 5 1 - 1 1 60.

Vogt, K. 1980. Die Spiegeloptik des Flusskrebsauges. J. Comp. Plivs-
iol. 135: 1-19.

Welsh, J. H., and F. A. Chace. 1937. Eyes of deep-sea crustaceans I.
Acanthephyridae. Biol. Bull 72: 57-74.

Welsh, J. H., and F. A. Chace. 1938. Eyes of deep-sea crustaceans II.
Sergestidae. Biol. Bull 74: 364-375.

/.harkova, 1. S. 1970. Reduction of the organs of vision in deep-sea
mysids. Zoo/. Zh. 49: 685-693.

/.harkova, I. S. 1975. Reduction of organs of sight in deep-water Iso-
poda, Amphipoda, and Decapoda. ZooL Zh. 54: 200-208.

Ziedins, I., and \ . B. Meyer-Rochow. 1990. ERG-determined spec-
tral and absolute sensitivities in relation to age and size in the half-
crab Petrolimhes elongatus (Crustacea; Decapoda; Anomura). Exp.
Biol 48:319-328.

Reference: Biol. Bull 192: 309-320. (April, 1997)

Heat-Shock Protein Expression in Mytilus

californianus: Acclimatization (Seasonal

and Tidal-Height Comparisons) and

Acclimation Effects

DEIRDRE A. ROBERTS, GRETCHEN E. HOFMANN1, AND GEORGE N. SOMERO2
Department of Zoology, Oregon State University, Corvallis. Oregon 97331-2914

Abstract. Heat-shock protein (hsp) expression was ex-
amined in gill of field-acclimatized and laboratory-accli-
mated mussels (Mytilus californianus) from the Oregon
coast. Endogenous levels of heat-shock proteins in the
70-kDa class (hsp70 isoforms) and profiles of induction
temperature for newly synthesized hsp70 were measured
in freshly field-collected specimens as functions of loca-
tion height in the intertidal and season, and in mussels
after 7 weeks of laboratory thermal acclimation. There
were significant differences in endogenous levels of
hsp70 as functions of season and collection height.
Strong induction of new hsp70 synthesis occurred at
body temperatures within the range measured in field
specimens. Profiles of hsp70 thermal induction varied
significantly with season, but not with height of collec-
tion. In contrast to the large differences in hsp70 expres-
sion between winter- and summer-acclimatized mussels,
no differences related to temperature occurred in the
differently acclimated mussels. The differences found be-
tween the effects of field acclimatization and laboratory
thermal acclimation suggest that the stress response is
modulated by environmental factors in addition to body
temperature. Thus, caution is required in extrapolating
from laboratory acclimation studies to acclimatization
effects in field populations. The seasonal and tidal-height
variations in the heat-shock response are discussed in the
context of energy costs of protein turnover.

Received 4 June 1996; accepted 30 January 1997.

1 Direct correspondence to Dr. Hofmann at the Department of Biol-
ogy, University of New Mexico, Albuquerque, NM 87131-1091.

2 Current address: Hopkins Marine Station, Stanford University, Pa-
cific Grove, CA 93950-3094.

Introduction

All but one species so examined have been found to
synthesize heat-shock proteins (hsps) in response to
exposure to temperatures of a few to several degrees Cel-
sius above those normally experienced by the organism
(Craig, 1 985; Bosch <?/«/.. 1988; Welch, 1993; Parsell and
Lindquist, 1993; Becker and Craig, 1994). At tempera-
tures near the upper limit of thermal tolerance, hsps may
be the major, and in some cases the only, proteins syn-
thesized (Morimoto el a/., 1990). The presence of the
heat-shock response across taxa suggests an ancient ori-
gin for the response (Gupta and Singh, 1992). In addi-
tion, many studies suggest that hsps play a critical role
in thermal tolerance at the cellular level (for review see
Parsell and Lindquist. 1994). The recognition that some
hsps are members of a broad class of proteins termed mo-
lecular chaperones — proteins that prevent improper ag-
gregation of structurally non-native proteins and assist
in correct protein folding and compartmentalization—
has helped to clarify the potential roles of hsps in re-
sponse to heat stress. Thus, hsps prevent aggregation of
heat-damaged proteins in the cell, and therefore may in-
directly assist in restoring the native structure of proteins
that are reversibly damaged by high temperatures
(Wiech el a/.. 1992; Jakob et a/., 1993; Parsell and Lind-
quist, 1993; Becker and Craig, 1994; Haiti, 1996).

Although major advances have been made using iso-
lated cell lines and unicellular organisms to resolve the
mechanisms by which molecular diaper »nes function
and by which their synthesis is regulate relatively few
studies have examined the heat-shock r >onse in organ-
isms in their natural habitats, in whl i thermal stress
may greatly vary in time and space. '• adies of the heat-

309

310

D. A. ROBERTS ET AL.

shock response in natural populations are important for
several reasons, including (1) determining what temper-
atures are in fact sufficiently high to induce the response
under natural hab tai conditions; (2) characterizing the
plasticity of the response (e.g., changes in induction tem-
peratures and endogenous levels of hsps) in concert with
variation in seasonal thermal regimes; and (3) establish-
ing the relative magnitude of the heat-shock response in
conspecifics exposed to different thermal regimes in their
distinct microhabitats. In view of the high fraction of me-
tabolism directed to protein synthesis and protein turn-
over (Hawkins and Bayne, 1992), all of this information
could be useful in developing models of ecological ener-
getics— for example, in understanding the energy cost of
existence in the face of fluctuating temperatures. Further
understanding of the heat-shock response could be espe-
cially important in developing and refining conceptual
models in community ecology. These would include
models of environmental stress, distribution and zona-
tion, and diversity gradients, which invoke the impor-
tance of sublethal abiotic stress in determining organ-
isms' distribution limits, competitive relationships, and
life-history strategies (Menge and Olson, 1990; Bertness
andCallaway, 1994).

As part of a broad study of the physiological ecology
of temperate, rocky intertidal invertebrates, we have ex-
amined several attributes of the heat-shock response in
the California mussel, Mytilus calijornianus. The rocky
intertidal zone exposes its inhabitants, especially sessile
species like M. calijornianus, to wide ranges of tempera-
ture, as well as to desiccating conditions, wave-exposure
stress, variations in access to oxygen and nutrients, and
high levels of UV radiation (Newell, 1979). Variation in
physical condition occurs both seasonally and as a func-
tion of height in the intertidal zone. In general, the high-
intertidal zone is characterized by more extreme abiotic
conditions than the low-intertidal zone, due to wider
fluctuations in temperature and greater exposure to ae-
rial conditions during tidal cycles.

Mytilus californianus is an appropriate species for
studies of natural variation in the heat-shock response
with season and microhabitat location of conspecifics.
Mytilus spp. are a prominent component of many tem-
perate, wave-swept rocky shores (Seed and Suchanek.
1992). Mytilus californianus occurs along the Pacific
coast of North America from Alaska to Baja California
( Morris el at., 1980), and the upper and lower limits of its
beds typically define the boundaries of the mid-intertidal
zone (Suchanek, 1978). The lower limit of the M. califor-
nianus zone is primarily determined by biotic interac-
tions, predation and competition (Paine. 1966. 1974),
whereas the upper limit is generally considered to be
most influenced by physical constraints involving tem-
perature and desiccation (Seed and Suchanek, 1992).
Harger ( 1 970) found that M. californianus at higher tidal

heights had decreased growth rates and attained smaller
maximal sizes than conspecifics found lower in the inter-
tidal zone.

We examined the heat-shock response in Mytilus cali-
fornianus to determine how it varies with season and
with microhabitat location. Two characteristics of the re-
sponse were studied: the temperatures at which hsp in-
duction occurs and the endogenous levels of hsps. We
focused on hsps of the 70-kDa size class (i.e., hsp70 iso-
forms) and found that both attributes of the heat-shock
response change significantly with season, and that the
concentration of hsp70 in high- and low-intertidal indi-
viduals of M. californianus also differs significantly. We
performed a companion laboratory study of thermal ac-
climation to determine whether temperature per se, in-
dependent of other environmental factors like aerial
exposure, influenced variations in the heat-shock re-
sponse. We discuss these results in the context of the ex-
pression of heat-shock proteins in organisms under eco-
logically relevant habitat conditions and in relation to
how laboratory acclimation studies may obscure the
complete range of expression patterns found in field-ac-
climatized organisms.

Materials and Methods

Acclimatization studies: field collections and body-
temperature measurements

Mytilus californianus (shell length 60-80 mm) was
collected at Strawberry Hill on the central Oregon coast
(44° 15TM. 127° 07'W) in July of 1993, and in February,
March, May, June, and August of 1 994. Collections were
performed on a single day each month. In February,
mussels were collected from a mid-intertidal site. In all
other months, mussels were collected from low- and
high-intertidal sites corresponding to the lower and up-
per limits of the M. californianus zone at Strawberry Hill.
For the heat-shock induction experiments, freshly col-
lected mussels were transported to the laboratory in am-
bient-temperature seawater (8° to 12°C) within 4-6 h of
collection. In the laboratory, mussels were kept at 10°C
in tanks of recirculating seawater and used in hsp induc-
tion experiments within 1-3 days of collection.

Samples of gill tissue for solid-phase immunochemical
quantification of hsp70 levels (western blotting) were
collected in July 1993 and February 1994. In the field,
gill lamellae were dissected from mussels gathered at the
high- and low-intertidal sites at the beginning and end of
the emersion period, during the more extreme low tide
on the collection day. The tissue samples were immedi-
ately frozen on aluminum blocks chilled on dry ice,
transported to the laboratory on dry ice, and stored at
-70°C until processed for protein electrophoresis and
western blotting.

The body temperatures of mussels at the field collec-

HEAT-SHOCK PROTEINS IN MYTILL'S

311

tion sites were measured with a thermocouple connected
to a hand-held digital thermometer (Omega Inc.). The
probe was inserted into a small hole drilled through a
valve, and temperatures were recorded at intervals of
about 20 min throughout the emersion period. Between
measurements, the holes were plugged with modeling
clay to prevent evaporative water loss.

Acclimation studies: field collections ami acclimation
procedure

Specimens were collected at Strawberry Hill on 15
May 1994, during low tide. Gill-tissue samples for west-
ern analysis were collected from mussels dissected in the
field, as described above. Mussels for acclimation exper-
iments (/; = 125) were haphazardly selected from a mid-
intertidal rock bench, within an area of about 4 nr.
Mussels were transported to the laboratory in ambient-
temperature seawater ( 12°C) within several hours of col-
lection and immediately placed in a tank with recirculat-
ing seawater at 1 3°C. The next day, the mussels were di-
vided into four tanks, each containing 30 mussels. One
tank was maintained at 13°C and the other three tanks
were adjusted to one of three temperatures, 10°, 17°, or
20°C, at a rate of 2°C per day. All tanks were maintained
for 7 weeks after acclimation temperatures (10°, 1 3°, 1 7°,
and 20°C) were reached. Mussels were fed an algal con-
centrate mixture (Algae Preserve Diet 'B': Coast Sea-
foods Co., Bellevue, WA) every 4 days.

Heat-shock-protein induction experiments

For the field-acclimatization studies, hsp induction ex-
periments were conducted on freshly collected mussels
in February, March. May, June, and August of 1994. For
the laboratory -acclimation studies, hsp induction exper-
iments were conducted in May 1994 on freshly field-col-
lected mussels and in July 1994, at the end of the 7-week
acclimation period, on mussels acclimated to tempera-
tures of 1 0°C. 1 3°C, 1 7°C and 20°C. All induction exper-
iments were conducted with gill tissue because of the ease
with which several similar fragments of this tissue could
be obtained from each individual, and because of the
ability of gill to take up dissolved amino acids from the
medium. Whole gill lamellae were dissected from mus-
sels held in chilled seawater. The lamellae were cut into
small fragments weighing about 200 mg. These frag-
ments, one for each temperature tested, were immedi-
ately placed into 500 /il of incubation medium (Hepes-
buffered artificial seawater [20 mA/ Hepes, 7.57 mM
(NH4):SO4, 375 mA/ NaCl, 9.35 mM KC1, 2.7 mM
NaHCO,, 17.95 mA/ Na;SO4, 37.7 mA/ MgCl:-6H:O,
8 mA/ CaCl:-2H:O, 10 mA/ glucose]) in 1.5-ml micro-
centrifuge tubes that had been pre-equilibrated at the
heat-exposure temperatures. Immediately prior to the
addition of gill, 5 ^1 of ^S-labeled methionine and cys-

teine (Trans-35, ICN Radiochemicals) was added to the
incubation medium. Gill fragments were radiolabeled
for 2 h at the following temperatures: 10°, 13°, 17°, 20°,
23°, 25°, and 28°C. During the incubation period, gill
fragments were aerated frequently by blowing a stream
of air onto the surface of the incubation medium with
sufficient force to stir the medium and oxygenate the
buffer. After the incubation period, gill fragments were
rinsed in 2 vol of nonradioactive incubation medium
and then either frozen immediately on dry ice or pro-
cessed for protein electrophoresis.

Protein electrophoresis and Jluorography

Gill fragments from hsp induction experiments were
placed in mierocentrifuge tubes containing 300 ^1 of ly-
sis buffer (32 mA/Tris-HCl, pH 6.8; 2% sodium dodecyl
sulfate (SDS) with 1 mA/ phenylmethylsulfonylfluoride
(PMSF), a protease inhibitor, added immediately before
use), and boiled for 2 min. Samples were then homoge-
nized with a Teflon pellet pestle, boiled again for 5 min,
and homogenized a second time. The homogenates were
centrifuged at 16,000 X g in a mierocentrifuge for
15 min. The supernatants were removed and the radio-
activity, in counts per minute (CPM), of a 10-^1 fraction
of each sample was determined with a liquid scintillation
counter; the remainder of the sample (about 200 ^1) was
stored at -20°C.

Proteins in the supernatants were separated by electro-
phoresis on 1 2% polyacrylamide gels in an SOS-buffer
system (Laemmli, 1970). For ease of comparison and
consistency, all of the samples (one gill fragment at each
of the heat-exposure temperatures) from an individual
mussel were analyzed on a single gel. Each sample
was loaded in equivalent counts, approximately
600,000 CPM per lane. Gels were electrophoresed with
20 mA current for about 3.5 h, fixed for 1 h in an aque-
ous solution of 10% acetic acid and 30% methanol, and
then incubated in EN3HANCE (NEN) according to the
manufacturer's protocol. Dried gels were exposed to film
(Kodak X-OMAT) at -70°C for 1 5 h. After the film was
developed, protein bands were analyzed with a densi-
tometer (Molecular Dynamics), and the relative inten-
sity and size of bands were quantified using ImageQuant
software (Molecular Dynamics).

We measured the amount of newly synthesized pro-
tein (radioactivity) in a given band with a volume quan-
tification procedure in ImageQuant, which calculated
the intensity and the area of the band. The hsp bands
were normalized relative to a non-heat-induced, strongly
labeled 46-kDa protein band within the same sample
lane to eliminate the variation in background labeling
between samples. All comparisons of amounts of newly
synthesized hsp70 were made relative to the amounts of
newly synthesized 46-kDa protein ("relative amounts of

312

D. A. ROBERTS ET AL

hsp70 synthesis"), for reasons given below. Normaliza-
tion to the 46-kDa protein is appropriate because our ob-
jective was to estimate the temperatures at which en-
hanced synthesis of hsp70 relative to other size classes of
proteins occurred, regardless of whether the change in
relative synthesis rates was due to enhanced synthesis of
hsp70 or to reduced synthesis of proteins, such as the 46-
kDa protein, not induced at high temperatures. At the
highest incubation temperatures, synthesis of proteins
other than hsps was reduced. This observation was con-
sistent with a denning characteristic of the heat-shock re-
sponse: preferential synthesis of hsps over normal cellu-
lar proteins at abnormally high temperatures. Thus, the
ratio of hsp70: 46-kDa protein could potentially increase
as a result of an increase in hsp70 synthesis, a decrease in
synthesis of the 46-kDa protein, or both. Our analysis of
newly synthesized proteins therefore is appropriate for
determining when hsp70 synthesis is induced by heat
stress, even though this autoradiographic procedure can-
not provide an estimate of the absolute amount of new
hsp70 that is synthesized. Another reason for normaliz-
ing to the 46-kDa protein is the need to control for tem-
perature effects on rate processes (Qw effects). It could be
argued that increases in hsp synthesis with rising temper-
ature could simply be a consequence of Qw effects on
the rate of protein synthesis, and not a result of thermal
induction of the heat stress response per se. By normal-
izing synthesis of hsp70 to synthesis of a non-heat-in-
duced protein, we eliminated potential artifacts due to
Qw effects on synthesis rates.

For the graphical representations, but not for the sta-
tistical analyses, the relative amounts of hsp70 synthe-
sized were normalized to the corresponding value at
10°C within each gel (i.e.. within each mussel), so that
among the autoradiographs(/>.. among the mussels) the
relative intensities of hsp70 at 10°C were equivalent.
This temperature is within the range of ambient sea water
temperatures recorded at Strawberry Hill, and from pre-
liminary experiments was shown not to induce synthesis
of hsps in this population of M. califomianus. By stan-
dardizing the relative intensity values for hsp70 to this
common temperature, we could make graphical com-
parisons of the heat-shock response between sites and on
a seasonal basis.

7sp70 western blotting

dogenous hsp70 levels in gill samples were quanti-
ith immunoblotting techniques. For the field stud-
dissected in the field in July 1993 and February
used; for the acclimation experiments in the
labo gills dissected in the field in May 1994 and

from n s acclimated to 10°C and 20°C for 7 weeks
were use II fragments dissected from mussels in the
field ami i i immediately were homogenized in lysis

buffer, following the same protocol used on samples for
the induction experiment. Equivalent amounts of pro-
tein (5 jug) were electrophoresed on 7% or 7.5% SDS-
polyacrylamide gels. This amount of protein was deter-
mined to be within the linear range of detection for the
final immunochemical detection step (data not shown;
see Hofmann and Somero, 1995). After electrophoresis,
separated proteins were transferred from the gel to a ni-
trocellulose membrane via a semi-dry transfer blot appa-
ratus (Owl Scientific). Transfers were conducted for 1 .5 h
at 1 1 5 mA with a transfer buffer containing 25 mM Tris
base, 192 mMglycine, and 20% methanol. The nitrocel-
lulose membrane was hydrated for 3 h prior to transfer,
and the filter paper used to sandwich the membrane and
gel was saturated in transfer buffer. After the transfer, the
membrane was blocked overnight in blocking solution
(5% nonfat dry milk, 0.02% thimerosal, in phosphate-
buffered saline (PBS: 10 mM sodium phosphate,
150 mJl/NaCl, pH 7.4)) and then rinsed three times for
10 min in PBS containing 0.1%- Tween-20. The mem-
brane was then sequentially incubated in solutions of rat
monoclonal anti-hsp70 antibody (hybridoma 7.10; pro-
vided by Dr. Susan Lindquist of the University of Chi-
cago), bridging antibody (rabbit anti-rat IgG; Vector
Laboratories) and Protein A-horseradish peroxidase
(HRP) conjugate (Bio-Rad). These methods are more
fully described in Hofmann and Somero (1995). The en-
hanced chemiluminescence detection method (ECL de-
tection reagents; Amersham) was used to visualize pro-
teins that cross-reacted with the anti-hsp70 antibody. In
order to compare multiple western blots, a biotinylated
ECL molecular weight marker was run on each gel and
visualized by adding a streptavidin-HRP incubation af-
ter the Protein A-HRP incubation step (see Hofmann
and Somero, 1995). For individual western blots, the in-
tensity of the ECL marker was used to standardize the
intensities of the hsp70 bands. The chemiluminescent
signal was detected according to the manufacturer's in-
structions, using pre-flashed Hyperfilm-ECL X-ray film
(Amersham). Exposure times of the blot to the film were
typically 5-15 s. Relative amounts of hsp70 were then
quantified with densitometry and ImageQuant software.

Statistical analysis

SAS (SAS Institute Inc.) and SYSTAT (Systat Inc.)
software programs were used for the statistical analyses.
In induction experiments with field-acclimatized mus-
sels, we compared the relative level of newly synthesized
hsp70 in relation to incubation temperature, tidal height,
and time of collection (i.e.. month). Using ANOVA tech-
niques, incubation temperature (TEMP), collection time
( MONTH ), and tidal height of collection site (HEIGHT)
were designated as factors, or main effects (i.e.. indepen-
dent variables), and the relative level of newly synthe-

HEAT-SHOCK PROTEINS IN MYTILUS

313

sized hsp70 was designated as the response variable (i.e.,
dependent variable). Because individual mussels were di-
vided into separate gill fragments, suhiinils. in the induc-
tion experiments, a split-plot ANOVA was used (Sokal
and Rohlf. 1981). MONTH and HEIGHT were unit
(whole mussel) level effects, and TEMP was a subnnit
(gill fragment) level effect. A variable to designate
"among whole mussel variation" (MUSS) was also in-
cluded in the analysis. In this case, the null hypothesis
was that the variation in hspVO synthesis was random,
and not due to TEMP, MONTH, or HEIGHT. Because
the relative level of newly synthesized hsp70 was calcu-
lated as a ratio, and because exploratory data analyses
revealed unequal variances, these data were log-
transformed prior to running the analysis. A similar
model was used to analyze the results of the hsp induc-
tion experiments with the laboratory-acclimated mus-
sels. We were interested in comparing the relative level
of newly synthesized hsp70 in relation to incubation
temperature and acclimation treatment (pre-acclima-
tion (field), 10°, 13°, 17°, and 20°C acclimation). Incuba-
tion temperature (TEMP) and acclimation treatment
(ACCLTRT) were the independent variables, and the
relative level of newly synthesized hsp70 was the depen-
dent variable.

ANOVA was also used to analyze the results of the
hsp70 western blotting. For the field studies, the relative
level of endogenous hsp70 was the dependent variable,
and collection season, tidal height, and collection time
were the independent variables. For the laboratory accli-
mation studies, the relative level of endogenous hsp70
was the dependent variable, and acclimation treatment
was the independent variable (Sokal and Rohlf, 1981).
The Bonferroni correction procedure (Systat) was used
for post hoc multiple comparisons (Schlotzhauer and
Littell, 1987).

Results

Tissue temperatures of mussels in the field

Internal tissue temperatures of attached mussels dur-
ing emersion at low tide were recorded seasonally at
Strawberry Hill, using mussels from both high- and low-
intertidal sites. Tissue temperatures in May and July
ranged from about 9° to 28°C. Tissue temperatures on
a day in July 1993 are shown in Figure 1 (top). Tissue
temperatures were above 1 8°C for 2.5 h during emersion
at the high site and for less than 30 min at the low site.
Temperature data from a day in May 1993 (Fig 1. bot-
tom) show that tissue temperatures were above 1 8°C for
about 3.5 h at both sites. The temperatures of the low-
site animals reached slightly higher peak values than con-
specifics at the high site.

These data indicate the potential range and variability
in tissue temperatures, but because measurements were

28

20

E 16

12

• low
O high

JULY

700

800

900

1000

1100

1200

28 -

24 -

= 20

O

0)
CL

E 16

V
h-

12

MAY

re-immersion

800 900 1000 1100 1200

Time of Day (Hours)

1300

1400

Figure 1. //; situ tissue temperatures ofAIytilns californianus in re-
lation to time of day over a single low-tide emersion period in July (top)
and May (bottom) 1993, at high- and low-intertidal sites at Strawberry
Hill, Oregon. Each point represents the mean temperature of 5 mussels.
Error bars are ± 1 standard error of the mean.

only made over discrete time intervals around low tide,
and because weather conditions (e.g., wind, cloud cover)
can fluctuate immensely on a daily basis, the data should
not be interpreted to represent general seasonal patterns.
In addition, these data show the unpredictable nature of
the temperature ranges experienced by mussels on any
given day. The time of day of the lowest low tide each
day during spring tides varies seasonally; in the fall and
winter the extreme low tides are in the early evening and
night, and in the spring and summer the extreme low
tides are in the early to mid morning (7995 Tide Tables
for the Pacific Coast of North and South America.
NOAA, US Dept. of Commerce).

Heat-shock-protein induction profiles

General patterns of newly synthesized proteins in field-
acclimatized mussels. Experimentson induction of heat-
shock proteins were conducted on freshh tield-collected
mussels in February, March, May, June, and August of
1994. In most cases, direct visual analysis of labeled pro-
tein bands revealed a clear profile of isp induction: as

314

D. A. ROBERTS ET AL.

incubation temperature increased, the relative intensi-
ties of bands representing newly synthesized proteins
changed markedly, with bands of molecular mass corre-
sponding to major classes of heat shock proteins showing
the strongest increase in intensity (Fig. 2). The most
prominent bands of heat-inducible protein were of ap-
parent A/r 68-74 kDa and 31.5 kDa — bands that we as-
sume to represent heat shock proteins of the 70-kDa and
30-kDa classes. These bands were faint or absent at tem-
peratures below 20°C, and exhibited a marked increase
in intensity and width at temperatures of 23°C and
higher (Fig. 2). The appearance of two strong bands with
A/r values near 70 kDa indicated de novo synthesis of
more than one hsp70 isoform (Fig. 2). Other protein
bands, whose synthesis was not induced by heat, were
consistently present in all samples and at all tempera-
tures tested. Most notable were two bands of 39 and
46 kDa (Fig. 2). However, synthesis of these non-heat-
induced proteins decreased at 25° and 28°C, tempera-
tures at which hsp synthesis remained strong.

Seasonal and tidal-height patterns in hsplO induction
profiles. In February, in mussels collected from a mid-
intertidal site, there was a significant increase in the rela-

tive amount of hsp70 synthesis at 23°, 25°, and 28°C as
compared to synthesis at lower temperatures (Fig. 3; AN-
OVA, P < 0.00 1 in all cases). Between 23° and 28°C there
was a twofold increase in the relative amount of hsp70
synthesized, and synthesis levels at 28°C were about four
times higher than at 10°, 17°, and 20°C (Fig. 3). The im-
portance of normalizing hsp70 synthesis to the 46-kDa
protein to determine induction temperatures is illus-
trated by comparing data in Figures 2 and 3. Although
Figure 2 appears to show an increase (induction) in
hsp70 synthesis at 20°C, when the 70-kDa band is nor-
malized to the 46-kDa band (Fig. 3) no change in the
relative amount of hsp70 synthesis is found until 23°C.

Beginning in March, hsp induction experiments were
performed on mussels collected at high- and low-inter-
tidal sites, to examine site location and seasonal effects
on patterns of hsp synthesis. The overall trends in the
March and February specimens were similar: a strong
increase in relative synthesis of hsps of both general size
classes occurred at 23°C, and hsp70 levels were about
fourfold higher at 28°C than at temperatures of 20° and
lower (Fig. 4). In March, the relative magnitude of the
response was somewhat elevated in the low-intertidal

Incubation Temperature (°C)

10 13 17 20 23 25 28

: hsp70

: 46 kDa

•=hsp30

:

3 4

6 7 8 9 10 11 12 13 14

Figure 2. Autoradiograph from an experiment on the induction of heat shock proteins in Mytilus
calif ornianus. Fragments of gill tissue from a single individual (collected in February 1994) were radiola-
beled with a mixture of '5S-methionine and 35S-cysteine for 2 h. Samples were loaded on a 12% polyacryl-
amide gel in duplicate; approximately 600,000 CPM per lane were loaded. Lanes 1-2, 10°C; lanes 3-4.
1 3°C; lanes 5-6, 1 7°C: lanes 7-8, 20°C; lanes 9- 1 0, 23°C: lanes 11-12, 25°C; lanes 13-14. 28°C.

HEAT-SHOCK PROTEINS IN MYTILUS

315

FEBRUARY

10

15

20
Temperature (°C)

25

30

Figure 3. Hsp induction in gill of Mytilus califomianus in relation
to incubation temperature. Mussels were collected in February 1994
from a mid-intertidal site at Strawberry Hill. Each point represents the
mean relative level of newly synthesized hsp70 of 6 mussels; error bars
are±l standard error of the mean.

mussels as compared to the high-intertidal mussels at all
temperatures, although this difference was not signifi-
cant (Fig. 4). The induction profiles in the May samples
are similar to those in February and March, although
there was an overall reduction in the magnitude of the
response. The June samples showed a further dampening
of the induction response (note that 13° and 17°C were
not tested in the June collection; Fig. 4), and in the Au-
gust samples there was no longer a sharp induction pro-
file in either the high or low samples (note that 13°C was
not tested; Fig. 4).

A split-plot ANOVA indicated that incubation
temperature (TEMP, P < 0.0001), collection month
(MONTH, P < 0.0007), and the interaction term be-
tween temperature and collection month (MONTH*
TEMP, P < 0.0001) were statistically significant in ex-
plaining variation in relative amounts of new synthesis
of hsp70. There was significant individual variability in
hsp70 expression among individual mussels (MUSS
(MONTH * HEIGHT), P< 0.0001), but the tidal height
of the collection site was not significant in the model
(HEIGHT, P< 0.7470).

Hsp70 synthesis patterns in laboratory-acclimated
mussels. Freshly collected (= pre-acclimation) speci-
mens of mid-intertidal M. califomianus collected in May
1994 exhibited an hsp70 expression pattern (data not
shown) similar to that observed with high- and low-in-

MARCH

JUNE

AUGUST

10 15 20 25

Temperature (°C)

10 15 20 25

Temperature (°C)

Figure 4. Hsp70 induction in gill of Mytilus califomianus in relation to incubation temperature in
field-collected mussels in March. May, June, and August 1 994, from high- and low-intertidal sites at Straw-
berry Hill. Points represent means: March low, n = 5; March high. n = 4; May low. n = 8: May high, n = 8.
June low, n = 5: June high, n = 4; August low, /; = 3; August high. /; = 3. Error bars are ± I standard erro
ot the mean.

316

- 3

i -

10
5 ~

^ 3

D. A. ROBERTS ET AL.

5

10°C

15

20

25 30

17°C

10 15 20 25 30

Temperature (°C)

10

10

13«C

15

20

25

30

20°C

15

20 25

Temperature (°C)

30

Figure 5. HspVO induction in gill of laboratory-acclimated Mylilm californianus in relation to temper-
ature. Mussels were collected in May 1 994 from a mid-intertidal site at Strawberry Hill and acclimated in
the laboratory tor 7 weeks. Each point represents the mean relative level of newly synthesized hspVO; error
bars are ±1 standard error of the mean. Sample sizes were IO°C-acclimation. n = 5; !3°C-acclimation,
n = 6; 17"C-acclimation. n = 7; 20°C-acclimation, n = 1.

tertidal mussels from the same month (Fig. 4): amounts
of newly synthesized hsp70 were relatively constant and
low between 10°C and 20°C, but between 20°C and 25°C
there was a sharp increase in synthesis of hsp70. The
amounts of newly synthesized hspTO at 25°C and 28°C
were more than three times the levels at 10°C and 1 3°C.

A comparison of these data with results from the tem-
perature-acclimated mussels (Fig. 5) revealed several
differences. Overall, the relative magnitude of the
amount of newly synthesized hsp70 was reduced in the
mussels acclimated to 10°, 13°, and 17°C, when com-
pared to the pre-acclimation mussels (compare May data
in Fig. 4 with dula in Fig. 5). In all but the 20°C-accli-
mated specimens, the relative amount of new hsp70 syn-
thesis was maximal at 28°C and decreased at the highest
incubation temperature 32°C. Protein synthesis in the
20°C-acclimated mussels was more resistant to high tem-
perature, and no decrease in hsp70 synthesis was noted
at 32°C. Acclimation had no consistent effects on the
temperature at which a significant increase in hsp70 syn-
thesis first occurred.

The split-plot ANOVA model showed that acclima-
tion treatment ( ACCLTRT, P < 0.000 1 ), temperature

(TEMP, P < 0.0001 ). and the interaction between these
two factors (ACCLTRT * TEMP, P < 0.0001 ) explained
a significant portion of the variation in relative levels of
newly synthesized hsp70. This model also shows that the
variation in response among individual mussels was sig-
nificant (P < 0.0052).

Endogenous hsp70 levels: constitutive and heat-induced
isoforms

Western blotting with a rat monoclonal anti-hsp70 an-
tibody was used to measure the relative amounts of
hsp70 present in field-acclimatized M. californianus over
the low-tide emersion period, in high- and low-intertidal
populations, and in summer- and in winter-acclimatized
specimens (Fig. 6). For quantification and comparison,
the hsp70 bands were divided into two groups of iso-
forms; a lower molecular mass group (LMM-hsp70, ap-
parent A/r: 66-68 kDa) and a higher molecular mass
group (HMM-hsp70, apparent A/r: 69-73 kDa). These
two molecular-mass classes may include both constitu-
tively synthesized isoforms of 70-kDa chaperones (hsp70
cognates = hsc70) and heat-induced isoforms (hsp70s)

HEAT-SHOCK PROTEINS IN MYTILUS
1 234 56 7 8 9 10 11 12 13 14 15 16

317

HI

LI

H2

L2

SUMMER

1 234 56 7 8 9 10 11 12 13 14 15 16

HI

LI

H2

L2

WINTER

Figure 6. Western blot analysis of hsp70 isoforms in gill tissue of summer- and winter-acclimatized
Mylilus calijornianus. Mussels were collected at high- and low-intertidal sites just as the water was receding
(HI and L 1 . respectively) and just prior to re-immersion ( H2 and L2. respectively). Lanes were loaded with
equal amounts of protein (5 ^g). and immunodetection was performed using ECL reagents. Two samples
from each site were loaded in duplicate (e.g.. lanes 1 and 2 are the same sample). It should be noted that
one individual (H2 in Winter) displayed significantly lower levels of endogenous hspVO: the reason for this
difference is unknown.

because this anti-hsp 70 antibody reacts with both types
of 70-kDa isoforms.

As shown in Figure 7, there were no significant differ-
ences in hsp70 levels over a single low-tide emersion pe-
riod in either the high- (HI, H2) or low- (LI, L2) inter-
tidal sample, in either the summer or winter collections.
However, distinct seasonal and tidal height differences,
both in the quantity and banding pattern of hsp70 pro-
teins, were observed (Fig. 7). The relative amount of
LMM-hsp70 isoforms was significantly greater in the
high-intertidal mussels than in the low-intertidal mussels
in the summer collection (P < 0.0 1 ). In the winter, there
was no significant difference in the amount of LMM-
hsp70 in the gill of high- and low-intertidal mussels (Fig.
7a). Within the high-intertidal site, there were signifi-
cantly higher levels of LMM-hsp70 isoforms in the sum-
mer than in the winter (P < 0.01). Within the low site,
there were no significant seasonal differences.

Consistent with the patterns shown by LMM-hsp70,
the amount of HMM-hsp70s in summer was signifi-
cantly higher in the high-intertidal mussels than in the
low-intertidal mussels (Fig. 7b; P < 0.01). However,
within the high-intertidal site, there were no significant
differences seasonally, and within the low intertidal site,
the amount of HMM-hsp70 was higher in winter than in
summer.

Using western analysis, we also compared hsp70 levels
in the pre-acclimation group of field-collected mussels
(May 1994), and in the laboratory-acclimated mussels

after 7 weeks of acclimation to either 10° or 20°C (data
not shown). There were no significant differences among
any of these three treatment groups for either the LMM
or HMM forms of hsp70.

Discussion

In situ tissue temperatures and expression of heat-shock
proteins

The tissue temperatures recorded in situ during emer-
sion at all seasons frequently reached levels at which in-
creased synthesis of hsp70 was observed in the in vitro
labeling studies (Figs. 1-4). These observations, in con-
cert with the seasonal and tidal-height variations ob-
served in endogenous levels of hsp70 (Figs. 6 and 7),
show that the heat-shock response is likely to be induced
in mussels at all seasons, but particularly during summer
when low tides typically occur during midday. Because
the intensity of thermal stress is due to the product of
temperature multiplied by duration of exposure, mussels
in the higher regions of the intertidal zone experience
greater heat stress, even though the absolute tissue tem-
peratures reached during emersion on hot days may not
differ between high- and low-intertidal individuals (Fig.
1 ). Other data support our conclusion thai tissue temper-
atures of mussels frequently become hi;;h enough to in-
duce the heat-shock response. Elvin and Conor (1979)
recorded tissue temperatures ranging >m 0° to 34°C in
M. calijornianus on the Oregon a .t. Hofmann and

318

D. A. ROBERTS ET AL.

0.4 -

0.0

Figure 7. Mean relative level of low molecular mass (66-68 kDa)
hsp70 isoforms (a) and high molecular mass (69-73 kDa) hsp70 iso-
forms (b) in Mviilus californianus g\\\ in relation to intertidal collection
site, over a low-tide emersion period in summer and in winter. H and
L designate samples from high and low sites, respectively; 1 and 2 des-
ignate samples collected at the beginning and end, respectively, of the
period of emersion. Error bars are ±1 standard error of the mean (n
= 5).

Somero (1995) reported tissue temperatures as high as
32°C during emersion of M. trossulits during midday low
tides in coastal intertidal sites on San Juan Island, Wash-
ington. These data on body temperature, taken in con-
junction with the hsp induction profiles presented here
and in other studies of intertidal invertebrates (Hofmann
and Somero, 1995, 1996), highlight how important
stress-protein expression may be to eurythermal ecto-
therms, such as intertidal invertebrates, for tolerance of
fluctuations in environmental temperature and the de-
velopment of seasonal thermotolerance (Coleman et a/.,
1995).

Seasonal patterns: induction profiles and endogenous
levels of heat-shock proteins

The results of the in vitro labeling experiments showed
that patterns of hsp expression varied significantly over

the 7-month period of the field experiment (Figs. 3, 4; P
< 0.0007). Two observations illustrate the plasticity of
the stress response in field organisms. First, there was sig-
nificant seasonal variation in the induction profiles with
respect to induction temperatures and the magnitude of
new synthesis of hsp70 isoforms (Figs. 3 and 4). In gill
from mussels sampled in February and March, hsp70
synthesis was strongly induced at temperatures greater
than 20°C. By May, the induction response was damp-
ened, and this trend continued in June and August.
Thus, in what may appear to be a paradoxical trend, the
amount of new synthesis of hsp70 was lowest in the
months when average temperatures during emersion
were likely to be highest.

Few other studies have examined seasonal changes in
the heat-shock response. In an estuarine goby fish, Gil-
Ikiithys niirahi/is, the induction temperature for synthe-
sis of hsp90 increased from 28°C in winter-acclimatized
fish to 32°C in summer-acclimatized fish (Dietz and
Somero, 1992). The plasticity observed in invertebrates
(this study) and fishes (Dietz and Somero, 1992) in the
induction of hsp synthesis indicates that the regulation
of hsp expression is not a genetically fixed characteristic
of an organism, but is instead a trait subject to acclimati-
zation.

The second type of plasticity noted in the field-acclima-
tization experiment was the strong effect of season on en-
dogenous levels of hsp70 isoforms (Figs. 6 and 7). During
summer, when the highest body temperatures during
emersion are likely, the levels of the LMM isoforms of
hsp70 were significantly elevated over levels measured in
winter mussels in the high-intertidal location, but not at
the lower site (Fig. 7; see below). For the HMM isoforms
of hsp70, summer levels were significantly elevated in
mussels from the high-intertidal site as compared to levels
in mussels from the low-intertidal site (Fig. 7). The sea-
sonal changes noted in endogenous levels of hsp70 iso-
forms may provide a partial explanation (but see below)
for the seemingly paradoxical result found in the induc-
tion experiments — namely, the dampened induction re-
sponse noted in summer specimens. The higher levels of
hsp70 isoforms maintained in summer may be a type of
"anticipatory" adaptation to the likelihood of increased
thermal stress in these months. Higher endogenous levels
of hsps may preclude the need for induction of new hsp
synthesis in concert with emersion during the tidal cycle.

Tidal height patterns: induction profiles and endogenous
levels

No significant differences were found in the induction
profiles of mussels from high- and low-intertidal sites
(Fig. 4), even though the endogenous levels of hsp70 iso-
forms in the two groups were significantly different (Fig.
7). In summer, the levels of LMM and HMM hsp70 were

HEAT-SHOCK PROTEINS IN HfYTlLUS

319

significantly higher in mussels from the high site than in
individuals from the low site, an observation consistent
with greater amounts of thermal stress in the high inter-
tidal individuals. In winter, for both isoforms of hsp70,
high- and low-site mussels had the same hspTO levels.
These data show that the temperatures at which new syn-
thesis of hsp70 isoforms is induced are not established
entirely by the endogenous levels of hsp70. One reason
for the observed lack of a consistent correlation between
endogenous levels of hsp70 isoforms and induction tem-
perature may be that the data from the western analysis
includes both constitutively expressed and heat-induced
isoforms of hsp70. whereas the data from in vitro labeling
primarily represents new synthesis of the heat-inducible
isoforms. A clearer understanding of the correlation be-
tween endogenous levels of an hsp and the induction
temperature for new hsp synthesis thus requires the abil-
ity to to distinguish between constitutively expressed and
heat-induced isoforms, something our antibody analysis
did not do.

Studies with other intertidal invertebrates have also
observed differential expression of stress proteins as a
function of intertidal location. The limpet Collisella sea-
bra, which occurs relatively high in the intertidal zone,
expressed more isoforms of hsp70 and hsp60 than a con-
gener, C. pelta. which occurs lower in the intertidal in
shaded surge channels (Sanders et ai, 1991). In the sea
anemone Anemonia virldis. intertidally occurring indi-
viduals have higher constitutive levels of a low molecular
weight hsp than do anemones from subtidal collections
(Sharp cl ul.. 1994). In M. trossuhis, intertidal popula-
tions had higher endogenous levels of hsp70 than subti-
dal populations (Hofmann and Somero, 1995).

Acclimation patterns: induction profiles and endogenous
levels of heat-shock proteins

The acclimation experiments were conducted to de-
termine whether the seasonal acclimatizational effects
noted in induction profiles and endogenous levels of
hsp70 could be mimicked through cold- and warm-accli-
mation in the laboratory. That is, the acclimation exper-
iments were designed to test the hypothesis that changes
in temperature alone were responsible for seasonal accli-
matization of the heat-shock response. Our data refute
this hypothesis. The induction profile data (Fig. 5) show
that the warm- and cold-acclimated mussels had indis-
tinguishable heat-shock responses. Both acclimation
groups exhibited a relatively attenuated response, with
maximal increases in hsp70 synthesis of no more than
about 2.5-fold, in contrast with the greater than 4-fold
increases seen in winter-acclimatized mussels (Figs. 3
and 4). Similarly, no significant differences were found
between pre-acclimation, 10°-, and 20°C-acclimated
groups in the endogenous levels of LMM and HMM
hsp70 isoforms.

The differences that distinguish the results of the accli-
mation and acclimatization experiments with M. califor-
nianus raise caveats about the design and interpretation
of heat-shock experiments. Particularly for sessile inter-
tidal species like M. californiamis, which encounter a va-
riety of emersion-related stresses, including those due to
desiccation and anaerobiosis, it may be unrealistic to as-
sume that temperature is the only, or even the primary,
stress that necessitates the heat-shock (stress) response. A
common signal for hsp induction is the appearance in
the cell of unfolded proteins (Ananthan et al.. 1986;
Parsell and Lindquist, 1993). In addition to thermal
stress, changes in intracellular ionic strength and pH dur-
ing emersion could affect protein structure and, thereby,
induce the synthesis of heat-shock proteins. Because the
mussels in the acclimation experiment were continu-
ously submerged, stresses due to desiccation and anaero-
biosis were not present. Therefore, acclimation to a sin-
gle environmental variable, for example, temperature,
may not lead to a heat-shock response that mimics the
response occurring under in situ conditions.

General conclusions

Synthesis of stress proteins, like all protein synthesis,
is expensive in terms of the energy budget of an animal
(Creighton, 1993). Hawkins (1991) has estimated the
costs of protein synthesis to constitute 20%-25% of the
energy budget of the bay mussel, Mytilus edulis. This
cost represents an additional energy burden because
stress proteins do not directly contribute to increases in
growth or reproduction, and because under stress condi-
tions they may be synthesized preferentially, such that
other proteins critical for the normal functioning of the
organism are either synthesized at reduced rates or not
synthesized at all. Furthermore, the function of stress
proteins may require considerable ATP turnover; refold-
ing of a protein may consume in excess of 100 ATP mol-
ecules (Creighton, 1991; Martin et al.. 1991; Parsell and
Lindquist, 1993). The extent to which different intensi-
ties of thermal stress found in high- and low-intertidal
sites impact protein synthesis and growth is not known.
However, significant differences in growth rates (Menge
et al., 1994) and in capacity for protein synthesis as indi-
cated by RNA:DNA ratios (Dahlhoff and Menge, 1996)
have been found between mussels in different tidal loca-
tions at Strawberry Hill. Although these height-related
differences may be due in large measure to variation in
food availability from site to site (Dahlhoff and Menge,
1996), other explanations are possible. The data also are
consistent with the conjectures that the heat shock re-
sponse can exact a measurable toll on the energy budgets
of organisms (Krebs and Loeschcke, 1994; Coleman et
al.. 1995), and that thermal damage to proteins could
play a key role in defining the habitat ranges of marine
species (Somero, 1995).

320

D. A. ROBERTS ET AL.

Acknowledgments

We thank Dr. Susa; 1 mdquist for providing the anti-
bodies used in this s'udy. Team Mussel was supported
by an OSU Zoological Research Fund Award to DAR,
an NSF Man no Biotechnology Postdoctoral Fellowship
to GEH, and NSF grant IBN 9206660 to GNS.

Literature Cited

Ananthan, J., A. L. Goldberg, and R. Voellmy. 1986. Abnormal pro-
teins serve as eukaryotic stress signals and trigger the activation of
heat shock genes. Science 232: 522-524.

Becker, J., and E. A. Craig. 1994. Heat-shock proteins as molecular
chaperones. Eur. J Biochem 219: 1 1-23.

Bertness, M. D., and R. Galloway. 1994. Positive interactions in com-
munities. Trends Eco/. Ewl. 9: 191-193.

Bosch, T. C. G., S. M. Krylow, H. R. Bode, and R. E. Steele. 1988.
Thermotolerance and synthesis of heat shock proteins: these re-
sponses are present in Hydra allenuata but absent in Hydra oli-
gaclis. Proc. Nail. Acad. Sci. USA 85: 7927-793 1 .

Goleman, J. S., S. A. Heckathorn, and R. L. Hallberg. 1995. Heat-
shock proteins and thermotolerance: linking molecular and ecolog-
ical perspectives. Trends Ecol. Evol. 10: 305-306.

Craig, E. A. 1985. The heat shock response. CRCCrit. Rev. Biochem
18: 239-280.

Creighton,T. E. 1991. Unfolding protein folding. Nature^!: 17-18.

Creighton, T. E. 1993. Proteins: Structure and Molecular Properties.
2nd. ed. W. H. Freeman, San Francisco.

Dahlhoff, E. P., and B. A. Menge. 1996. Influence of phytoplankton
concentration and wave exposure on the ecophysiology of the Cali-
fornia mussel Mytilus ca/ijornianus. Mar Ecol. Prog. Ser. 144: 97-
107.

Dietz, T. J., and G. N. Somero. 1992. The threshold induction tem-
perature of the 90-kDa heat shock protein is subject to acclimatiza-
tion in eurythermal goby fishes (genus Gillichthys). Proc. Nail.
Acad. Sci. USA 89: 3389-3393.

Elvin, D. W., and J. J. Conor. 1979. The thermal regime of an inter-
tidal Mytilus californianus Conrad population on the central Ore-
gon coast. J. Exp. Mar. Biol. Ecol. 39: 265-279.

Gupta, R. S., and B. Singh. 1992. Cloning of the HSP70 gene from
Halobacterntm marismornii: relatedness of archaebactenal HSP70
to its 24 eubactenal homologs and a model for the evolution of the
HSP70gene. J Baclenol. 174:4594-4605.

Harger, J. R. E. 1970. The effect of wave impact on some aspects of
the biology of sea mussels. Veliger 1 2: 40 1 -4 1 4.

Hartl, F. II. 1996. Molecular chaperones in cellular protein folding.
/Vamrt'381:57l-580.

Hawkins, A.J.S. 1991. Protein turnover: a functional appraisal.

Fund Ecol 5: 222-233.

Hawkins, A. J. S., and B. L. Bayne. 1992. Physiological interre-
lations and the regulation of production. Pp. 171-222 in The Mus-
sel Mytilus: Ecology. Physiology, Genetics and Culture. E. Gosling,
ed. Dev. Aquae. Fish. Sci. 25, Elsevier, Amsterdam.
Hofmann, G. E., and G. N. Somero. 1995. Evidence for protein dam-
age at environmental temperatures: seasonal changes in levels of
ubiquitin conjugates and hsp70 in the intertidal mussel Mytilus
tro.ssu/us. J. Exp. Biol 198: 1509-1518.

Hofmann, G. E., andG. N. Somero. 1996. Protein ubiquitination and
stress protein synthesis in Afytilus trossulus occurs during recovery
from tidal emersion. Molec. Mar. Biol Biotech. 5: 175-184.

Jakob, U., M. Gaestel, K. Engel, and J. Buchner. 1993. Small heat
shock proteins are molecular chaperones. J Biol. Chem. 268: 1517-
1520.

Krebs, R. A., and V. Loeschcke. 1994. Costs and benefits of activa-
tion of the heat shock response in Drosophila melanogaster. Fund.
Ecol. 8:730-737.

Laemmli, E. K. 1970. Cleavage of structural proteins during the as-
sembly of the head of bacteriophage T4. Nature356: 683-689.

Martin, J., T. Langer, R. B. Boteva, A. Schramel, A. L. Horwich, and
F.-U. Hartl. 1991. Chaperonin-mediated protein folding at the
surface of groEL through a 'molten globule'-like intermediate. iVa-
lure 352: 36-42.

Menge, B. A., E. L. Berlow, C. A. Blanchette, S. A. Navarrete, and
S. B. Vamada. 1994. The keystone species concept: variation in
interaction strength in a rocky intertidal habitat. Ecol. Monogr. 64:
249-286.

Menge, B. A., and A. M. Olson. 1990. Role of scale and environmen-
tal factors in regulation of community structure. Trends Ecol. Evol.
5:52-57.

Morimoto, R. I., A. Tissieres, and C. Georgopoulos. 1990. Stress Pro-
teins in Biology and Medicine. Cold Spring Harbor Laboratory
Press. New York.

Morris, R. H., D. P. Abbott, and E. C. Haderlie. 1980. Intertidal In-
vertebrates of California. Stanford University Press, Stanford, CA.

Newell, R. C. 1979. Biology oj Intertidal Animals. 3rd ed. Marine
Ecological Surveys, Faversham, UK.

Paine, R. T. 1966. Food web complexity and species diversity. Am.
Nat. 118:65-75.

Paine, R. T. 1974. Intertidal community structure: experimental
studies on the relationship between a dominant competitor and its
principal predator. Oecologia 15: 93-120.

Parsell, D. A., and S. Lindquist. 1993. The function of heat-shock
proteins in stress tolerance: degradation and reactivation of dam-
aged proteins. Annit. Rev. Genet. 27: 437-496.

Parsell, D. A., and S. Lindquist. 1994. Heat shock proteins and stress
tolerance. Pp. 457-494 in The Biology of Heat Shock Proteins and
Molecular Chaperones. R. I. Morimoto, A. Tissieres, C. Georgo-
poulos, eds. Cold Spring Harbor Laboratory Press, New York.

Sanders,B. M.,C.Hope,V. M.Pascoe,andL. S.Martin. 1991. Char-
acterization of the stress protein response in two species ofCollisella
limpets with different temperature tolerances. Physio/. Zool. 64:
1471-1489.

Schlotzhauer, S. D., and R. C. Littell. 1987. SAS System for Elemen-
tary Statistical Analysis. SAS Institute. Inc., Cary, NC.

Seed, R., and T. H. Suchanek. 1992. Population and community
ecology of A f vi Hits. Pp. 87-169 in The Mussel Mytilus: Ecology.
Physiology. Genetics and Culture. E. Gosling, ed. Dev. Aquae. Fish.
Sci. 25., Elsevier, Amsterdam.

Sharp, V. A., D. Miller, J. C. Bythell, and B. E. Brown. 1994. Expres-
sion of low molecular weight HSP70 related polypeptides from the
symbiotic sea anemone Anemonia virdis Forskall in response to
heat shock. J Exp. Mar Biol .Ecol. 179: 179-193.

Sokal, R. R., and F. J. Rohlf. 1981. Biometry. 2nd ed. W. H. Free-
man, San Francisco.

Somero, G. N. 1995. Proteins and temperature. Annu. Rev Physiol.
57: 43-68.

Suchanek, T. H. 1978. The ecology of Afylilus edulis L. in exposed
rocky intertidal communities. J. Exp. Mar Biol Ecol. 31: 105-120.

\Viech, H., J. Buchner. R. Zimmerman, and U. Jakob. 1992. Hsp90
chaperone protein folding ;/; vitro. Nature 358: 169-170.

Welch, \V. J. 1993. How cells respond to stress. Sci. Am. (May): 56-
64.

Reference: Biol Bull 192: 321-331. (April, 1997)

The Route of Ion and Water Movements Across the Gill

Epithelium of the Freshwater Shrimp Macrobrachium

olfersii (Decapoda, Palaemonidae): Evidence From

Ultrastructural Changes Induced by Acclimation

to Saline Media

JOHN C. McNAMARA1 AND ALICE GONCALVES LIMA2

{DepartamentodeBiologia, FFCLRP. Universidade de Sao Paulo, Ribeirao Preto

14040-901 SP, Brasil; and 2Departamento de Fisiologia. Institute de Biociencias,

Universidade de Sao Paulo. Sao Paulo. Brasil

Abstract. The ultrastructure of the pillar cells in the gill
lamellae of the freshwater shrimp Macrobrachium olfer-
sii was examined to evaluate the routes of salt and water
movement across the gill epithelium and into the hemo-
lymph. Alterations were morphometrically quantified in
shrimp maintained in fresh water (FW, <0.5%« salinity)
and after acclimation to saline media (21%o or 28%» sa-
linity). The tissue interface between the hemolymph and
the external medium consists exclusively of the thin api-
cal flange regions of the pillar cells, the upper membrane
of which is highly amplified by dense microvilli and over-
lain by a thin cuticle. The lower flange membrane,
bathed by the hemolymph, is smooth and not invagi-
nated. Contiguous flanges are strongly bound by junc-
tional structures including desmosomes and septate
junctions. The basal surface of the pillar cell perikaryon
is linked to the adjacent septal cells through many baso-
lateral junctions. The septal cell plasmalemma is abun-
dantly and deeply invagmated, each infolding enclosing
numerous mitochondria; these characteristics are typical
of salt-transporting machinery. After shrimps were accli-
mated to saline media for 10 days, the thickness of the
pillar cell flanges was significantly reduced (from 1.3 to
<^QA Mm), as was the height (from 0.8 to 0.3 ^m) and
density (from 4.0 to = 1.8 microvilli/^m) of the apical
microvilli. This reduction in the apical surface area of
the pillar cells appears to lead to decreased ionic perme-

Received 6 May 1996; accepted 20 December 1996.

ability, concomitant with a reduction in Na+/K+-ATP-
ase activity, thus limiting Na+ uptake. In contrast to the
brachyurans, in which the respiratory and ion-transport-
ing mechanisms are differentially located in the anterior
and posterior gills, in palaemonid shrimps the pillar cells
apparently play a dual role: ions move preferentially
through ion transporters in the microvilli above the pillar
cell perikaryon, while respiratory gases are exchanged
through the fine flange regions in contact with the hem-
olymph.

Introduction

Salt uptake in hyperosmoregulating, freshwater deca-
pods and in hyper-regulating, euryhaline brachyurans
experimentally exposed to dilute media takes place pri-
marily through the gill tissues (see Mantel and Farmer,
1983; Gilles and Pequeux, 1986; Freire and McNamara,
1995, for discussion and references).

In brachyurans, specific ion-transporting regions of
the posterior gills account for much of this salt uptake,
while the anterior gills appear to be responsible mainly
for respiratory gas exchange (Pequeux, 1995). Such salt-
transporting gills exhibit a typical microanatomy: the
highly flattened, cuticle-bounded gill lamellae essentially
consist of a continuous layer of epithelial cells enclosing
a narrow hemolymph space; linked pairs of sustaining
pillar cells extend across this space between the opposing
epithelial sheets, their apical flanges often forming part
of the epithelium (Mantel and Farmer, 1983; Taylor and

321

322

J. C. McNAMARA AND A. G. LIMA

Taylor, 1986; Maina, 1990). The apical surfaces of the
epithelial cells are characteristically amplified by mem-
brane infoldings in the form of leaflets, whereas the ba-
solateral membranes are deeply invaginated and associ-
ated with mitochondria (Copeland, 1968; Copeland and
Fitzjarrell. 1968; Gilles and Pequeux, 1985; Maina,
1990). A fenestrated, partial intralamellar septum, which
bisects the gill lamella into two symmetrical portions,
may be present (Barra et al., 1983; see Taylor and Taylor,
1992, for review).

The apical infolding system of the gill epithelial cells
of decapod crustaceans undergoes structural reorganiza-
tion in response to short-term exposure or acclimation to
salinities different from that predominant in the habitat
(Bubel, 1976; Compere et a/., 1989; see Taylor and Tay-
lor, 1992; Freire and McNamara, 1995, for review and
references). After exposure to dilute media, the apical
leaflets and microvilli become more numerous and elab-
orate, and an extracellular, subcuticular compartment
enlarges due to osmotic swelling (Gilles and Pequeux,
1985). During short- and medium-term exposure (hours
to days) to concentrated media, the apical infolding sys-
tem is disrupted and regresses while the subcuticular
space disappears (see Shires etai, 1994; Pequeux, 1995).
The apical region of the epithelial cells thus appears to
represent a labile, primary barrier to osmotic alteration
deeper within the gill tissue.

The routes by which salt uptake is effected in the gills
ofhyperosmoregulating, freshwater palaemonid shrimps
have received very little attention. Although phyllobran-
chiate palaemonid gill is comparable to the brachyuran
gill in gross morphology, very little is known about its
fine structure (cf., Nakao, 1974; Doughtie and Rao,
1978; see Taylor and Taylor, 1992); ultrastructural stud-
ies of the effects of acclimation to saline media on the
apical infolding system are entirely lacking. Freire and
McNamara (1995) analyzed the gill structure of Macro-
brachium olfersii, a representative freshwater palae-
monid shrimp. Each flattened hemilamella constitutes a
narrow, 'molymph-filled space delimited by the flanges
of two o ing layers of pillar cells, the bases of which
adjoin in u <1-region of the lamella. A layer of septal
cells, com. ith the pillar cell bases, divides the in-

tralamellar i two parallel compartments, form-

ing a lattice-; ; of lacunae through which the

hemolymph flow nithelium forming the hemo-

lymph-water interi (insists exclusively of the ex-

panded apical flanges <: ic pi liar cell perikarya. The up-
per membranes of these fl ; cells are modified to form
an extensive system of microvilli, although the lower
flange surfaces and perikarya show no membrane-ampli-
i :ealures that would suggest salt transport. This role
h; n assumed by the cells of the intralamellar sep-
tum. ia membranes of which are extensively in-

vaginated and rich in associated mitochondria. Other
cell types are rare within the septum.

This relocation of the salt-transporting machinery —
from the basolateral membranes of epithelial cells whose
apical surfaces are in direct contact with the cuticle adja-
cent to the external medium, in the brachyurans, to the
intralamellar septum, surrounded on both surfaces by
flowing, Na+-rich hemolymph, in the freshwater palae-
monids — poses several engaging questions in terms of
the physical routes by which salt might be transported
across the gill tissue to the hemolymph. The present
study thus examines the ultrastructure of the pillar cells,
particularly their apical microvilli and junctions, in the
gills of Macrobrachium olfersii, a strongly hyperosmore-
gulating, freshwater palaemonid shrimp (McNamara,
1987). Gills were examined both in animals maintained
in fresh water (<0.5%o salinity) and in those acclimated
to media considered to be strongly saline (2 1 %o and 28%o
salinity) for this species.

Materials and Methods

Adult female specimens of Macrobrachium olfersii,
measuring 4 to 6 cm in total length, were collected some
2 km from the mouth of the Paiiba Stream on the south-
ern coast of Sao Paulo State, Brazil. In the laboratory, the
shrimps were maintained in 250-1 aquaria in water from
the collection site (fresh water [FW], <0.5%« salinity) and
were fed 3 times a week with fish, beef, and carrot.

To examine the effects of exposure to saline media on
gill ultrastructure, groups of shrimps in stage C-D0 of the
molt cycle were acclimated to salinities of <0.5%o, 2 1 %o,
or 28%o (20, 630, and 840 mOsm/kg H2O; 5, 287, and
383 mEq Na+/l; 5, 335, and 447 mEq CP/l, respectively)
over a 10-day period. Salinities, verified using an optical
refractometer, were prepared by diluting seawater with
FW from the collection site.

After the acclimation period, the ventral nerve cord
was severed, the eyestalks and rostrum were removed,
and the shrimps were perfused through the ventral ab-
dominal sinus with primary fixative at the rate of 1 ml/
min for lOmin. The sixth gill was then dissected, the
middle third was removed and bisected, and the two por-
tions, each comprising about 1 5 lamellae, were placed in
primary fixative on ice for 2 h.

The primary fixatives for the different groups of accli-
mated shrimps were adjusted according to the hemo-
lymph osmolality measured for each group. For shrimps
maintained in FW, the fixative consisted of (in milli-
moles) paraformaldehyde (200). glutaraldehyde (250),
sodium cacodylate (100); and Na+ (28), K+ (8), Ca+ +
(25), and Mg++ (4) as chlorides (effective osmolality
360 mOsm/kg H:O, pH 7.3). The effective osmolalities
(565 and 725 mOsm/kg H:O) of the fixatives for shrimps

SALT MOVEMENT ACROSS FRESHWATER SHRIMP GILLS

323

dors

post

vent

ev

Figure 1. A summary and location diagram showing the general anatomy of the sixth, right posterior
gill (A) and of a constituent gill lamella (B) in the freshwater shrimp Macrobrachium nlfersii. Hemolymph
flows from the lateral efferent vessels (ev), through the outer marginal canals (me), across the hemilamella
(hi) by way of the gill capillaries (gc) to the inner marginal canals, and back through the central afferent
vessel (av). (C) A cross section of the hemilamella (between arrows in B) reveals the lattice-like organization
of the gill tissue, resulting from the semi-regular arrangement of opposing pillar cells (pc). The perikarya
of the pillar cells are surrounded by hemolymph spaces (hs) and abut the lateral regions of the median,
intralamellar, septal cells (sc). The fine pillar cell flanges (pcf). in contact with the thin cuticle (c), form the
primary epithelial interface between the hemolymph and the external medium.

acclimated to saline media of 2 1 %> and 28%o were ad-
justed by using final NaCl concentrations of 140 and
225 mA/, respectively.

The gill fragments were then rinsed (3X5 min) in the
respective buffer solutions alone on ice (composition as
above, less the aldehydes) and post-fixed in 1% osmium
tetroxide in the same buffer systems for 1 .5 h on ice.

The fragments were dehydrated in a graded ethanol
series (65 min total), transferred into propylene oxide (2
X 15 min), infiltrated overnight, and embedded in Aral-
dite 6005 resin. Thick sections were cut with glass knives
at 0.5 Mm thickness on a Porter-Blum Sorvall MT2 ultra-
microtome, stained with 1 % methylene blue and 1 % azur
II in 1% aqueous borax, and photographed using Kodak
T-Max 100 ASA film on a Nikon AFX II photomicro-
scope. Thin sections of 50-80 nm thickness prepared
similarly were stained with aqueous uranyl acetate and

Reynolds' (1963) lead citrate and examined at an accel-
erating voltage of 80 kV in a Jeol 100-CX electron mi-
croscope.

The effects of acclimation to the experimental media
(<0.5%o, 21%o, or 28%o) were evaluated morphometri-
cally using as criteria the alterations induced in the thick-
ness (in micrometers) of the intralamellar septal cells and
the pillar cell flanges; and the height (in micrometers)
and numerical density (as microvilli/micrometer of api-
cal membrane) of the apical microvilli on the pillar cells.
Measurements were made on between 10 and 15 micro-
graphs of sections taken at random in a plam transverse
to the long axis of the gill lamellae (see Fig. and Freire
and McNamara, 1995) from 3 to 5 shrim for each sa-
line medium. The material was photogra ed at 8000 to
1 0,000x and the negatives were printed a final magni-
fication of 25,000x.

324

J. C. McNAMARA AND A. G. LIMA

r

,
*

, - ; .• I', -

',;

s*
23S

*

H

, ' ' t^

J/n.

*• \/..: '"

Figure 2. Thick (0.5 ^m), epoxy section, taken transversely to the long axis of a gill lamella from Ufac-
robrachium olfer.iii acclimated to fresh water (FW. < 0.5%o), showing the pillar cell perikarya (p) and
thick flanges (f) below the subcuticular space. The intralamellar septum (s) adjoins the pillar cell perikarya
(arrowhead). A hemocyte (*) is present in the hemolymph space (h). Scale bar = 10 ^m.

Figure 3. Thick, epoxy section taken transversely to the long axis of a gill lamella from M. olfersii
acclimated to seawater (SW) 2%%a for 10 days. The marked reduction in thickness of the pillar cell flanges
(f (and septal cells (s) results in extensive hemolymph spaces (*). There is a distinct difference in cytoplasmic
density (arrowheads) between the dark pillar (p) and light septal cells (s). Scale bar = 10 ^m.

Figure 4. Transmission electron micrograph of a thin section taken transversely to the long axis of a gill
lamella from M. olfersii in FW. showing the apical cytoplasm directly above the perikarya of two adjacent
pillar cells, separated by a long, junctional complex (arrowheads). Apical microvilli (m). numerous vesicles
(v), mitochondria, cisternae of rough endoplasmic reticulum (er), and microtubule bundles (t) are present. A
basal lamina (b) separates the pillar cell membrane from the hemolymph space. Scale bar = 0.7 ^m.

Figure 5. Electron micrograph of the apical region of a pillar cell perikaryon (p) and the bases of its
lateral flanges (f) in a section taken transversely to the long axis of a gill lamella from M. olfersii acclimated
to SW 2 1 %« for 1 0 days. The reduction in the height and numerical density of the apical microvilli (m) and
in the thickness of the flanges is evident. The subcuticular space (*) between and below the microvilli is still
present. Scale bar = 0.75 /jm.

SALT MOVEMENT ACROSS FRESHWATER SHRIMP GILLS

325

Morphometric measurements were made using a
transparent test overlay consisting of parallel line seg-
ments each 1 cm in length, equally spaced at 1-cm in-
tervals (Weibel el al, 1966; Freire and McNamara,
1995). To measure the thickness of the septal cells, the
test system was placed perpendicularly to the plane of
the gill cuticle; valid transects were represented by single
planes in which the line segments intersected both cell
margins. To estimate the thickness of the pillar cell
flanges, valid transects were single planes in which a line
segment intersected at least one cell margin.

The heights of the apical microvilli were sampled both
in the region above the pillar cell body and in the mid-
flange region. The test system was placed parallel to the
plane of the microvilli, and the heights of all microvilli
intersecting one of the extremities of each line segment
were measured. A height of 1 mm on the micrograph was
considered to be the minimum length criterion repre-
senting a single microvillus.

The numerical density of the apical microvilli was also
sampled in the same regions of the pillar cells. A second
test system comprising a straight line 125 mm in length
(equivalent to 5.0 ^m at 25.000X) was placed over the
micrograph at random, although parallel to the plane of
the cuticle. All microvilli lying directly above the plane
of the test line were counted.

All data were tested for normality of distribution using
the Kolmogorov-Smirnov test. Non-normal data were
normalized by transformation using the inverse func-
tion. Single- or two-factor analyses of variance were then
performed to detect the effect of acclimation salinity on
the various response variables. This was followed by
multiple means testing, using the Student-Newman-
Keuls test, when an effect was found. The data on micro-
villus height could not be normalized by transformation
and were analyzed nonparametrically with the Friedman
two-factor and Kruskal-Wallis one-factor analyses of
variance. Differences between groups were located using
the Wilcoxon-Mann-Whitney U test. All tests were per-
formed using a minimum significance level of P = 0.05.
The data are presented in the text as the mean ± 1 SEM
(n) unless otherwise indicated.

Results

Figure 1 , a diagram of the localization and general
morphology of the gill lamella in M. olfersii, illustrates
the organization of the gill tissue into a semiregular lat-

ticework of pillar and septal cells within the lamella. The
present study focuses particularly on the ultrastructure
of the flange and perikaryon regions of the pillar cells,
which form the principal epithelial barrier between the
hemolymph and the external medium.

Fine structure of I lie gill tissue in shrimps acclimated to
fresh water

Pillar cells. The electron-dense pillar cells are highly
differentiated epithelial cells constituted by two distinct
regions: the pillar cell perikaryon, 7.8 ± 1 .4 ^m (n = 10)
in height by 9. 1 ± 5.8 ^m (n = 14) in width (Fig. 2); and
the pillar cell flange, a fine, roughly elliptical, radial, api-
cal expansion of the perikaryon (Fig. 2), 56 ± 10 ^m («
= 3) in diameter and 2. 86 ±0.16 nm (n = 6) in thickness
near the perikaryon.

The apical membrane of the pillar cells, overlain by
the fine gill cuticle [249.0 ± 4.4 nm (/; = 10) thickness],
is folded into an extensive system of microvilli (Fig. 4)
that are organized into small tufts of from 4 to 8 villi (Fig.
7). These 10-nm diameter, cylindrical projections of the
apical cytoplasm (Fig. 8) are frequently arranged into
regularly spaced, often hexagonal, units and measure
roughly 700 nm in height. The distribution and height of
the microvilli are not uniform over the pillar cell surface:
they are longer (Table I) and numerically more dense
(Fig. 13) above the perikaryon (Fig. 4) than in the outer
flange region (Fig. 9). A distinct subcuticular space, con-
sisting mainly of the deeper invaginations between the
tufts of microvilli (Figs. 2 and 7) and of large vesicles
lying near the apical membrane (Fig. 9), is apparently
continuous with these invaginations.

The electron-dense subapical cytoplasm immediately
above the perikaryon (Fig. 4) contains numerous small
vesicles; polyribosomes; mitochondria, frequently dis-
posed with their long axes parallel to that of the perikar-
yon; cisternae of rough endoplasmic reticulum; and bun-
dles of 23-nm diameter microtubules that insert into the
base of each tuft of microvilli (Fig. 4). The nucleus occu-
pies the basal region of the pillar cell perikaryon (Fig. 2),
which is coupled to the adjacent cells of the intralamellar
septum by regions of thick, basolateral junctions (Fig.
12). These junctions consist of a wide (109. 3 ± 35.5 nm)
intercellular space of variable form and length. The
space, which contains granular material, leads to often
complex, although limited, interdigitations between the
plasmalemmae of the two cell types (Fig. 12, insert).

Figure 6. Electron micrograph of the apical region of a pillar cell perikaryon (p) and bases of its flanges
(f) from M. olfersii acclimated to SW 28%o for 10 days. The apical microvilli and subcuticular space have
disappeared completely, being replaced by a few invaginations of the plasma membrane (arrowheads).
Numerous vesicles (v) and mitochondria are present in the subapical cytoplasm. Scale bar = 0.4 ^m.

326

J. C. McNAMARA AND A. G. LIMA

'^' %'• ''7( -f[-. ~Mty('-- -: ";- '•'• ' 'f\ •'• ";i~r:';;=-l ^^'iVA^/^/J./l^' • 'Vv-^-?-.^i'-.'i''

,;»

•»-

' ' _

•

•M& "..^. ••;•«*.: a^*^-

' '•, • V V'a^rf^" - ' :" T

• • '• - m . 7

-*» • - • • :'«•?«

- -: - i* ---*:• •• >

•^^^^i^^t^^-i W'&k&M

i-sf^-'', --^.\:'-'^r:^^'\^' .-, -^"7 - •• " •

^':V. iH* :. '"; * ",V ^fy&Ji* • r •*.•'•'*' •', -t ' v*i

^.'4;i.';^ v- ,.^x fv-V-:j?-^ ^'•^-- •'-,:•• ^m

'"D*?=^sfe^a^r ,i;-rr-~ I r^;-.

o

V .. J <t Jj

Figure 7. Transmission electron micrograph of a section taken transversely to the long axis of a gill
lamella from Macrobrachium oltcr.tii in FW, showing a region of the pillar cell flange, near the perikaryon.
in junctional contact (arrowheads) with a flange from an adjacent pillar cell. Well developed, apical micro-
villi (m) are evident below the thin gill cuticle, as are numerous apical microvesicles (v). mitochondria,
and the basal lamina (b) separating the uninvaginated. lower plasma membrane from the hemolymph.
Subcuticular spaces (*) are present among and below the microvilli. Scale bar = 0.7 ^m.

Figure 8. Thin section taken slightly obliquely to and just below the base of the gill cuticle (c) from M.
•'//irv/i in FW. demonstrating that the apical infolding system is constituted by microvilli (arrowheads)

(her than leaflets, seen here in transverse section. Scale bar = 0.7 ^m.

;gure9. Extreme, lateral region of a line, pillar cell flange (f), distant from the perikaryon. underlain
basal lamina (b) from M nl/crMi in FW. The apical microvilli (arrowheads) have virtually disap-
: elow the cuticle (c), and only a few vesicles of the subcuticular space (*) and microtubules are

pi h • granular cytoplasm. Scale bar = 0.5 ^m.

Figure Extreme, lateral region of an apical, pillar cell flange, distant from the perikaryon, in the gill

epithelium rom M. t>lfcrxn acclimated to SW 21%o for 10 days. No apical microvilli are distinguishable
below the cuticle (c), and only a very thin layer of cytoplasm containing a few microtubules is visible above
the basal lamina (b) and underlying hemolymph space. Scale bar = 0.3 Mm.

SALT MOVEMENT ACROSS FRESHWATER SHRIMP GILLS

327

Table I

Effect of acclimation u> saline media for 10 davs on various
morphometric characteristics analyzed in the gill lamellae of the
freshwater \/irnnp Macrobraehium olfersii

Acclimation salinity (%o)

Characteristic

<0.5

28

Thickness of pillar 1.30 ±0.20 '0.34 ± 0.04 a0.40 ± 0.05

cell flange (/jm) (II) (10) (II)

Density of h4.00 ± 0.50 "2. 70 ±0.40 bcd1.00± 0.40

microvilli in (11) (12) (12)

pillar cell flange

Density of 6.20 ± 0.40 5.00±0.20 C4.00 ± 0.60

microvilli above (12) (12) (12)

pillar cell

penkaryon

Thickness of 7.80 ±0.50 7. 10 ±0.50 6.50 + 0.60

septal cell (jim) (10) (10) (13)

Data are given as mean values ± SEM (n). Numerical densities are
expressed as the number of microvilli per micrometer of linear apical
membrane.

' P < 0.05 compared to control thickness in 0 %»; *P < 0.05 compared
to values for perikaryon; CP < 0.05 compared to value for flange in 0 %»;
<Y><0.05 compared to value for flange in 21 %0','P < 0.05 compared to
value for perikaryon in 0 %».

The apical flange region of the pillar cells becomes at-
tenuated and thinner as the distance from the perikaryon
increases, attaining only 1 .38 ± 0.30 /*m (n = 6) in thick-
ness at the extreme margins (Fig. 9). The number and
height of the microvilli and organelles likewise decrease
markedly; the mitochondria exhibit no specific orienta-
tion. Like the pillar cell perikaryon (Fig. 4), the entire
lower surface of the pillar cell flange is underlain by a
fibrous basal lamina (Figs. 7 and 9), thickness 92.8 ±
1 1.0 nm (n = 8), that separates it from the hemolymph
space. The plasma membrane of this lower surface is not
folded or amplified in any way.

The regions of contact between adjacent pillar cell
flanges constitute highly structured junctional com-
plexes (Figs. 7 and 1 1 ). These typically comprise a short.
dense desmosome of 287.4 ± 30.2 nm (n = 6) length,
followed by an extensive septate junction of about
1.5 nm in length, and a long region of simple apposition
of the two cell membranes, separated by a constant nar-
row distance of 17.8 ± 1.7 nm (n = 10).

Intralamellar septal cells. The electron-lucent septal
cells, 12.0±0.7^m(/7 = 10) in thickness (Figs. 2 and 3),
make contact with the hemolymph over most of their
surface, which is greatly amplified and is characterized
by many invaginations of the plasmalemma; these pene-
trate deeply into and extensively throughout the septal
cell cytoplasm and appear to individually envelop each
of the numerous ovoid mitochondria present within the

cytoplasm (Fig. 12). The membrane infoldings define an
extracellular space of constant width (22.0 ± 0.7 nm, n
= 10) and maintain contact with the hemolymph. Typi-
cally, a single septal cell connects the bases of two adja-
cent pillar cells, its lateral ends interdigitating in a re-
stricted manner with their basolateral membranes (Fig.
12, insert). Golgi bodies and glycogen granules are fre-
quently found in the septal cells.

Ultrastructural and morphometric alterations induced
in the gill tissue of shrimps acclimated to saline media

Various ultrastructural alterations appear in the pillar
cells of the gill lamellae of M. olfersii after acclimation to
the two saline media (2 l%o and 28%o) for 10 days. Quali-
tatively similar, these modifications comprise substantial
reductions in the thickness of the pillar cell flanges ( Figs.
3 and 10) and in the height and density of the apical mi-
crovilli, both in the region above the pillar cell perikarya
(Figs. 5 and 6) and in the attenuated flange regions (Fig.
1 0). In 2 1 %o salinity, the microvilli lose their characteris-
tic tuft-like arrangement (Fig. 5), and the associated mi-
crotubule bundles are less evident. In 28%o, the microvilli
are virtually absent, and only a few invaginations of the
apical membrane are evident (Fig. 6).

There is a more subtle reduction in the thickness of the
intralamellar septal cells (Fig. 3, cf. Fig. 2), the structural
organization of which is maintained. With the reduction
in thickness of the pillar cell flanges, and to a lesser extent
of the intralamellar septal cells, there is a corresponding,
marked increase in the volume of the hemolymph lacu-
nae (Fig. 3, cf. Fig. 2).

These qualitative ultrastructural alterations were
quantified through morphometrical evaluation; the prin-
cipal findings for normally distributed data are presented
in Table I. Figure 13 presents the data on the height of
the apical microvilli for which a normal distribution
could not be obtained.

Discussion

In the gill epithelium of Macrobraehium olfersii, the
apical surface of the pillar cells is highly amplified by an
extensive system of microvilli (type 2, see Cioffi, 1984).
This system is found principally above the perikaryon,
becoming attenuated in the extreme lateral regions. The
lower surface of the pillar cell flange is not invaginated or
associated with mitochondria, and it does not appear to
be involved in active transport and salt movement. This
is in strong contrast to the epithelial cells ; the gill in
brachyurans (Gilles and Pequeux, 1° •), penaeids
(Couch, 1977; Foster and Howse, 1978). damphipods
(Kikuchi et ai. 1993: Kikuchi and V ,umasa, 1995:
Shires et a/., 1994). In these crustacei- . Na+/K+-ATP-
ases are typically associated with the asolateral infold-

328

J. C. McNAMARA AND A. G. LIMA

• v*^r ' '::- .'. ^^ " -.--•'&

»«JF ' •*, • ''*' ': '--V-§ - <

^^-i »„«% r*| vir:.**-' *sSP»-8$'

Figure 11. Detail of a characteristic junctional complex between the extreme, lateral tlange regions of
i adjacent pillar cells from Macrobrachium olfersn in FW. The sequence of a small desmosome (d).
hy septate junction (s)and region of close membrane apposition (a) is typical. Small groups of micro-
arrowheads) are loosely associated with the apical region of the junction. Scale bar = 100 nm.
"". Transverse section of a gill lamella from M. o/lersii in FW, showing a complex region of
een the bases of two electron dense, pillar cell penkarya (p) and an electron lucent cell in
tlu septum (s), characterized by extensive, deep invaginations (i) of the plasma membrane

associ. ;':tnchondria. Areas of dense, basolateral junctional complexes (arrowheads) link the two

cell types cell nucleus (n), hemolymph space (h). Scale bar = LO^m. Insert: Detail of a thick,

basolateral ji' . ion (arrowheads) between an interlamellar septal cell (s) and an abutting pillar cell (p),
showing abuiu mt, finely granular material in the wide extracellular space. Scale bar = 0.? ^m.

SALT MOVEMENT ACROSS FRESHWATER SHRIMP GILLS

329

08

o

06

o
y
'a.

0

o>

X

04

02

[v] Penkoryon O Flange

b,c

b,c

28

Acclimation salinity, %,

Figure 13. The effect of acclimation salinity (<0.5%o, 21%o, and 28%o) on the height of the apical mi-
crovilli in the region immediately above the pillar cell perikarya and in the extreme flange regions. The
non-normally distributed data are given as the median values (arrows); the upper and lower box boundaries
indicate the interquartile range; and the whiskers depict the minimum and maximum values. "P < 0.05
compared to value for perikaryon in fresh water (< 0.5%o), bP < 0.05 compared to value for flange in fresh
water, CP < 0.05 compared to respective values for perikarya in same salinities.

ings of the epithelial cells (see Towle, 1984; Towle and
Kays, 1986; Taylor and Taylor, 1992) and actively drive
salt uptake from the external medium directly into the
hemolymph up a Na+ gradient across the epithelium (see
Pequeux, 1995, for review).

The differential distribution of the apical microvilli in
M. olfersii attests to a dual role for the pillar cells in the
regulatory physiology of the gill. The principal ion move-
ments across the apical membrane occur through ion ex-
changers like the Na+/NH4 (Armstrongs al.. 1981) and
Cr/HCOj (Pequeux, 1995) counter transporters most
likely present in the membranes of the long, dense mi-
crovilli located immediately above the pillar cell perikar-
yon, the region closest to the basolateral junctions with
the septal cells. The exchange of respiratory gases, how-
ever, would be preferentially effected through the far less
amplified surface of the shorter, less dense microvilli in
the very thin, extreme flange regions that form a cyto-
plasmic barrier only 1 .4 ^m in thickness, directly above
and in contact with the hemolymph space. This situation
contrasts sharply with that known for brachyurans, in
which respiratory gas-exchange functions predominate

in the anterior gills, while ion transport mechanisms are
more restricted to the posterior gills ( Pequeux, 1995).

The notable reductions in the height and numerical
density of the microvilli on the apical surface of the pillar
cells, and in the thickness of the flanges, which occur as
a result of acclimation to saline media in M. olfersii, are
qualitatively similar to the modifications seen in the gill
epithelia of a variety of osmoregulating crustaceans in
response to exposure to either hyper- or hypo-osmotic
media. In the brachyuran crabs Eriochier sinensis and
Carcinus maenas acclimated to fresh water and 30% sea-
water, respectively, the apical infolding system becomes
deeper and more pronounced, as does the subcuticular
space (Gilles and Pequeux, 1985, 1986). In the latter
crab, the depth of the basolateral invaginations and the
number of mitochondria also appear to increase, with a
closer degree of apposition between them (Compere et
al., 1989).

In the marine shrimp Penaeus aztecus exposed to a
dilute medium of 0.9%o, the apical membranes of the epi-
thelial cells become highly and deep!} infolded com-
pared to those of shrimps kept in seawater (Foster and

330

J. C. McNAMARA AND A. G. LIMA

Howse, 1978). In this penaeid and P. diiorarum (Couch,
1977) and P. vannamei (Taylor and Taylor, 1992, Fig.
25A), and in the palaemonid Palaemonetes pugio
(Doughtie and Rao, 1978), the pillar cell perikarya ap-
pear to constitute part of the intralamellar septum. How-
ever, no changes seem evident in the septal cells of P.
aztecus after low-salinity exposure (Foster and Howse,
1978). In M. olfersii, subtle changes in septal cell mor-
phometry do occur after high-salinity acclimation: the
number of membrane invaginations interposed between
adjacent mitochondria increases, as does the surface
density (square micrometers of membrane surface per
cubic micrometer of cytoplasm) of the septal cells and
the mitochondria! volume fraction; the mitochondria!
profiles also become more elongate (Freire and McNa-
mara, 1995).

In one of the few studies directly comparable to the
present report (Shires el a!.. 1994), a marked reduction
occurred in the height and organization of the apical la-
mellae in the gill epithelial cells of the amphipod Gam-
mants duebeni within 1 h of exposure to seawater; the
mitochondria relocated to the cell center and appeared
to lose their intimate contact with both the apical and
basolateral membrane systems. These alterations were
transient, however, as the lamellae appeared to reorga-
nize after 10-16 h.

These various data demonstrate the labile nature of
the apical infoldings of the epithelial and pillar cells in
the gill lamellae of osmoregulating crustaceans. The
membrane surface area of this primary interface between
the external medium and the hemolymph increases
markedly during the acclimation of marine decapods to
dilute media, and is notably decreased in freshwater
Crustacea acclimated to saline media. This membrane
system may thus serve primarily to modulate apical per-
meability by regulating the density of constituent ion-
exchange molecules. In contrast, the system of basolat-
eral infoldings and mitochondria in the epithelial cells of
marine decapods appears largely unresponsive to de-
creased salinity; in freshwater Crustacea, exposure to sa-
line media produces subtle alterations in membrane sur-
face area and stacking, and in the location of the mito-
chondria.

The data on M. olfersii, both from shrimps main-
tained in fresh v (FW) and in those acclimated to
saline media, alsc - ide pertinent information about
the routes of ion and \ ter movements into the hemo-
lymph through the s. : tissue. In FW-acclimated
shrimps, the gill epithelium appears to be a tight epithe-
lium, well protected from paracellular water and ion
movements by extensive and characteristic junctions —
constituted by a small desmosome and a lengthy septate
junction — between adjacent pillar cell flanges. These
junctions are similar to those found in the gill epithelia

of the freshwater amphipods Sternomoera yezoensis (Ki-
kuchi el ai. \ 993) and Gammarus duebeni (Shires e! ai,
1994). The absence of mitochondria and infoldings of
the basolateral membrane strongly suggests that the
lower flange regions are not involved in active salt move-
ment in M. olfersii.

However, the ultrastructure of the intralamellar septal
cells, with their deep and numerous invaginations inti-
mately associated with mitochondria, is typical of an ac-
tive, salt-transporting epithelium in crustaceans and in a
variety of invertebrate tissues (seeCioffi, 1984; Pequeux,
1995). Sites of Na+/K+-ATPase activity, demonstrated
ultracytochemically, are present on the cytoplasmic sur-
face of the leaflets of this extensive membrane system in
M. olfersii (Torres and McNamara, 1996); their activity
would be fueled by ATP furnished directly by the abun-
dant adjacent mitochondria. In FW-acclimated shrimps,
the activity of the Na+/K+-ATPases in the membrane in-
vaginations of the septal cells creates the driving force for
net Na+ uptake from the freshwater medium across the
apical membranes of the pillar cells into the perikarya.
Na+ would then pass through the extensive intercellular
junctions between the base of the pillar cell perikarya
and the lateral ends of the intralamellar septal cells into
the septal cell cytoplasm, and from there directly into the
hemolymph via the Na+/K+-ATPases in the membrane
invaginations. Corroborating this proposed route of ion
movement. Lima et a/. (1997) have shown a significant
reduction in Na+/K+-ATPase activity in preparations of
gill membrane vesicles from AI. olfersii after acclimation
to saline media (21 and 28%o salinity) for 20 days. This
observation suggests a reduction in the number of ATP-
ase molecules in these membranes available for active
Na+ transport.

The alterations occurring at the apical pillar cell in-
terface, and other changes in the characteristics of the
intralamellar septal cells, including the reduction in
Na+/K+-ATPase activity, thus appear to reflect the struc-
tural transformations underlying the molecular mecha-
nisms of long-term adaptation to hyperionic media in
freshwater palaemonids, particularly those that restrict
the uptake of Na"*.

Acknowledgments

This study represents part of an MSc thesis submitted
by AGL to the Departamento de Fisiologia/IBUSP; it
was financed by a research grant to JCM (FAPESP 9 1/
2467-2) and a post-graduate scholarship (FAPESP 90/
3807-9). The authors thank Drs. Joao Lunetta (CEBI-
M AR), Sergio Oliveira (DHE/ICB), and Valder de Melo
(DM/FMRP) for access to essential facilities; Jose Au-
gusto Maulin (DM/FMRP) for photographic work; and
Marcos Ribeiro de Souza (FFCLRP) for the line
drawing.

SALT MOVEMENT ACROSS FRESHWATER SHRIMP GILLS

331

Literature Cited

Armstrong, D. A., K. Strange, J. Crowe, A. Knight, and M. Simmons.
1981. High salinity acclimation by the prawn Macrobrachium ro-
senbergii: uptake of exogenous ammonia and changes in endoge-
nous nitrogen compounds. Biol. Bull. 160: 349-365.

Barra, J.-A., A. Pequeux, and \V. Humbert. 1983. A morphological
study on gills of a crab acclimated to fresh water. Tissue Cell 15:
583-596.

Bubel, A. 1976. Histological and electron microscopical observations
on the effects of different salinities and heavy metal ions on the gills
ofJaera nordmanni (Rathke) (Crustacea, Isopoda). Cell Tis.s. Res.
167:65-95.

Cioffi, M. 1984. Comparative ultrastructure of arthropod transport-
ing epithelia. Am. Zool. 24: 139-156.

Compere, Ph., S. \Vanson, A. Pequeux, R. Gilles, and G. Goffinet.
1989. Ultrastructural changes in the gill epithelium of the green
crab Carcinus madias in relation to external salinity. Tissue Cell
21:299-318.

Copeland. D. E. 1968. Fine structure of salt and water uptake in the
land crab Gecarcmus lutcralis. Am. Zool. 8: 4 1 7-432.

Copeland, D. E., and A. T. Fitzjarrell. 1968. The salt absorbing cells
in the gills of the blue crab(Callineeles sapuhis Rathbun) with notes
on modified mitochondria. Z. Zellforsch. 92: 1-22.

Couch, J. A. 1977. Ultrastructural study of lesions in gills of a marine
shrimp exposed to cadmium. / Invcnebr. Palhol. 29: 267-288.

Doughtie. D. G., and K. R. Rao. 1978. Ultrastructural changes in-
duced by sodium pentachlorophenate in the grass shrimp, falemo-
neiespugio. in relation to the molt cycle. Pp. 2 1 3-250 in Pentachlo-
rophenol: Chemistry. Pharmacology, and Environmental Toxieol-
ogy. Vol. 12, K. R. Rao, ed. Plenum Press, New York.

Foster, C. A., and H. D. Howse. 1978. A morphological study on gills
of the brown shrimp, Penaeus a:lecus. Tissue Cell 10: 77-92.

Freire, C. A., and J. C. McNamara. 1995. Fine structure of the gills
of the fresh-water shrimp Macrobrachium olfersii (Decapoda):
effect of acclimation to high salinity medium and evidence for in-
volvement of the intralamellar septum in ion uptake. J Cnislac.
Biol. 15: 103-116.

Gilles, R., and A. Pequeux. 1985. Ion transport in crustacean gills:
physiological and Ultrastructural approaches. Pp. 136-158 in
Transport Processes. lono- and Osmoregulation. R. Gilles and M.
Gilles-Biallien.eds. Springer-Verlag. Berlin.

Gilles, R., and A. Pequeux. 1986. Physiological and Ultrastructural
studies of NaCl transport in crustacean gills. Bo/. Zool. 53: 173-
182.

kikuchi, S., M. Matsumasa, and V. Yashima. 1993. The ultrastruc-
ture of the sternal gills forming a striking contrast with the coxal
gills in a fresh-water amphipod (Crustacea). Tissue Cell 25: 915-
928.

Kikuchi, S., and M. Matsumasa. 1995. Pereopodal disk: a new type

of extrabranchial ion-transporting organ in an estuarine amphipod.
Melila selif/age/ii (Crustacea). Tissue Cell 27: 635-643.

Lima, A. G., J. C. McNamara, and VV. R. Terra. 1997. Regulation of
hemolymph osmolytes and gill Naf/K*-ATPase activities during
acclimation to saline media in the freshwater shrimp Macrobra-
cliium olfersii (Wiegmann. 1836) (Decapoda. Palaemonidae). J.
E\p. Mar. Biol. Ecol. In press.

Maina, J. N. 1990. The morphology of the gills of the freshwater Af-
rican crab Potamon ni/olicus (Crustacea: Brachyura: Potamoni-
dae): a scanning and transmission electron microscopic study. J.
Zool. 221:499-515.

Mantel, L. II., and L. L. Farmer. 1983. Osmotic and ionic regulation.
Pp. 53-161 in The Biology of Crustacea. Vol. 5, Internal Anatomy
and Physiological Regulation, L. H. Mantel, ed. Academic Press,
New York.

McNamara, J. C. 1987. The time course of osmotic regulation in the
freshwater shrimp Macrobrachium olfersii (Widgmann) (Deca-
poda. Palaemonidae). J. Exp. Mar. Biol. Ecol. 107: 245-25 1 .

Nakao, T. 1974. Electron microscopic study of the open circulatory
system of the shnmp, Caridina japonica. I. Gill capillaries. J Mor-
pliol. 144:361-380.

Pequeux, A. 1995. Osmotic regulation in crustaceans. J. Cnislac.
Biol. 15: 1-60.

Reynolds, E. S. 1963. The use of lead citrate at high pH as an elec-
tron-opaque stain in electron microscopy. / Cell Biol. 17: 208-212.

Shires, R., N. J. Lane, C. B. Inman, and A. P. Lockwood. 1994. Struc-
tural changes in the gill cells ofGammarus duebeni (Crustacea. Am-
phipoda) under osmotic stress; with notes on microtubules in asso-
ciation with the septate junctions. Tissue Cell 26: 767-778.

Taylor, H. H., and E. W. Taylor. 1986. Observations of valve-like
structures and evidence for rectification of flow within the gill la-
mellae of the crab Carcinus maenas (Crustacea. Decapoda). Zoo-
morphology 106: 1-11.

Taylor, H. H., and E. W. Taylor. 1992. Gills and lungs: the exchange
of gases and ions. Pp. 203-293 in Microscopic Anatomy of Inverte-
brates, Vol. 10. Decapod Crustacea. F. W. Harrison and A. G.
Humes, eds. Wiley-Liss, Inc., New York.

Torres, A. tL.andJ. C.McNamara, 1996. Localizacaoultracitoquim-
ica da ATPase Na+/K+-dependente em celulas de branquia e
glandula antenal do camarao de agua doce Macrobrachium olfersii.
IV Simposio de Iniciacao Cientifica da Universidade de Sao Paulo.
Abstracts, p. 132.

Towle, D. \V. 1984. Membrane-bound ATPases in arthropod ion-
transporting tissues. Am. Zool 24: 177-185.

Towle, D. VV., and \V. T. Kays. 1986. Basolateral localization of Na+
+ K*-ATPase in gill epithelium of two osmoregulating crabs. Calli-
necles sapidus and Carcinus maenas. J Exp. Zool. 239: 311-318.

VVeibel, E. R., G. S. Kistler, and \V. F. Scherle. 1966. Practical ste-
reological methods for morphometric cytology. J. Cell Biol. 30: 23-
28.

Reference: Biol. Bull 192: 332-339. (April, 1997)

Effect of Salinity on Ionic Shifts in Mesohaline
Scyphomedusae, Chrysaora quinquecirrha

DAVID A. WRIGHT1 AND JENNIFER E. PURCELL2

University of Maryland System, Center for Environmental and Estuarine Studies, ' Chesapeake

Biological Laboratory, Solomons, Maryland 20688-0038, and2 Horn Point Environmental

Laboratory, Cambridge, Maryland 21613

Abstract. Mesohaline populations of the scyphomedu-
sae Chrysaora quinquecirrha are found in salinities rang-
ing from 5%o to 25%o. Osmotic and ionic adjustments
within this salinity range were investigated using C. quin-
quecirrha ephyrae budded from polyps in the laboratory
and young medusae collected from the mid-salinity re-
gion of the Patuxent River, Maryland. When medusae
were transferred from 20%» salinity to lower salinities
(8%o, 12%o), concentrations of sodium and magnesium
in tissue and mesogleal fluid fell rapidly and approached
those of dilute seawater within 6 hours. There was some
recovery of these levels relative to the 8%o medium, and
they were significantly higher than the dilute seawater
concentration after 1 week. Tissue concentrations of
calcium showed no evidence of being regulated, whereas
potassium was strongly regulated such that levels did not
fall significantly following transfer of medusae to lower
salinities. However, after 1 week, the concentration of
potassium in mesogleal fluid approached that of the di-
lute medium. Extracellular space measured by direct
blottn.a and weighing or using 35S was about 40%. As a
result, i fi mates for intracellular potassium were revised
to 17 m.'- I '. The concentration of potassium in tissue
remained Ne following transfer to lower salinity, de-
spite a subs, ial osmotic influx of water. This influx
was measureu ->20% gain in body weight over 24 h
following transk nedusae from 16%o to 8%o. Meso-
gleal fluid was slit ^/po-osmolar to the medium at
15% and 20%o and M. vperosmolar to the medium

at 5%o and 12%o. Sulla oncentrations in mesogleal
fluid were 66%-70% those ol the external medium. Me-
dusae died or were unable to achieve positive buoyancy

Recc' • i:8 May 1 996; accepted 1 3 January 1 997.

at 5%o, which is probably very close to a lower salinity
limit for C. quinquecirrha in the mesohaline Chesapeake
Bay.

Introduction

Most members of the phylum Cnidaria are marine.
Only a few hydrozoans (e.g., Craspedacusta) live in fresh
water. Some cnidarians occur in low-salinity waters, but
species diversity decreases sharply with decreased salin-
ity (Dumont, 1994). In Chesapeake Bay, medusae of the
scyphozoan Chrysaora quinquecirrha are unusual in tol-
erating salinities as low as 5%o; the scyphistomae (polyps)
are not found where salinities are less than 7%o and thrive
at 10%o to 25%o (Cargo and Schultz, 1966, 1967). Purcell
(unpubl. data) has determined that asexual reproduction
in C. quinquecirrha in the mesohaline Chesapeake Bay is
limited by both low (<5%o) and high (>25%o) salinities.
Differences in reproduction at different salinities may re-
sult in part from the physiological cost of salinity adjust-
ment; i.e., energy required for volume regulation may
detract from that available for reproductive effort.

Mills ( 1 984) found that a variety of marine hydrome-
dusae and ctenophores adjusted osmotically within a few
hours to abrupt changes in salinity between 23%o and
38%o. Most species tested were able to osmoconform to
this salinity range. However, salinity of 19%o was usually
lethal to animals normally found at a salinity of 30%. In
experiments using Percoll to change the density but not
the tonicity of seawater, medusae made no buoyancy ad-
justments. Therefore, Mills (1984) concluded that the
process was passive osmotic accommodation to the sa-
linity changes, not active density regulation. Studies such
as those of Schick ( 1 973) have suggested that amino acids
play an important role in maintaining cellular volume

332

SALINITY AND IONIC SHIFTS IN MEDUSAE

333

in osmotically stressed cnidarians. In freshwater forms,
however, sodium and potassium extrusion followed by
water loss from cells (Benos and Prusch, 1972) seems to
provide the principal means of regulating cellular vol-
ume. Webb el ai (1972) examined free amino acids
(FAA) in scyphistomae of three species of scyphomedu-
sae, including C. quinquecirrha. They found that al-
though FAA concentrations increased linearly with in-
creasing salinity, they were only 0.9% to 4.6% of the total
osmotically active substances in the scyphistoma and
could not account for its osmotic balance. In summary,
there is evidence for both active and passive osmotic pro-
cesses in pelagic cnidarians.

Little work has been done on ionic regulation in pe-
lagic cnidarians. Robertson (1949) summarized some
earlier work on A urelia aurita medusae, showing that the
ionic composition of the medusae was not the same as
that of the surrounding water and thus suggesting that
the medusae can actively control their ionic composi-
tion. The finding that sulfate ions were particularly low
stimulated interest in the exclusion of heavy ions, spe-
cifically sulfate, as a mechanism of buoyancy control in
gelatinous zooplankton (Denton and Shaw, 1962;
Mackay, 1969; Bidigare and Biggs, 1980; Mills and Vogt,
1984). Mackay (1969) described a saturable, probably
active, sulfate transport mechanism with Km values of 1 7
to 22 mA/r1 in two species of hydromedusae. Bidigare
and Biggs ( 1980) determined that active sulfate loss from
the ctenophore Beroe cuciimis could be compensated by
isosmotic chloride exchange. The current research inves-
tigated changes in tissue and mesoglea osmolality and
cation levels in C. quinquecirrha medusae at different sa-
linities. Results were considered in light of salinity-re-
lated changes in body size and extracellular space deter-
mined either directly or using sulfate.

Materials and Methods

Medusae between 1 and 2 g wet weight (25-30 mm
bell diameter) were obtained from the Patuxent River,
Maryland, and acclimated in 40-1 aquaria containing
16%o Patuxent River water for 1 wk before experiments.
Medusae were fed Anemia salina nauplii during the ac-
climation periods, but they were not fed during experi-
mental treatments unless otherwise stated. Ephyrae were
budded from scyphistomae at 20%« in the laboratory.

Cation measurements

Medusae were netted out of holding tanks, blotted dry
in a standardized manner using laboratory wipes, and
weighed by introducing them to tared individual poly-
ethylene beakers containing 400 ml of experimental me-
dium. In the two experiments reported here, medusae
were transferred from 20%o to 8%o and 12%o at time =

0 h. Six animals were prepared for immediate analysis,
and six animals from each treatment were sampled at
6 h, 24 h, and 7 d. Animals were lifted from beakers with
large plastic forceps, blotted dry on laboratory wipes, and
weighed in plastic weighing dishes. The bell and tentacles
were then separated, and the mesoglea was allowed to
drain into the weighing dish. A 50-^/1 sample of mesogleal
fluid was pulled into a hematocrit tube and then expelled
into a small Nalgene vial containing 200 n\ of Ultrapure
concentrated nitric acid. A weighed amount of tentacu-
lar tissue (50-100 mg) was stored in Nalgene vials with
500 j/1 of nitric acid. Water samples from each beaker
were collected at the end of each experiment and stored
in Nalgene vials with 100 n\ of nitric acid. When diges-
tion was complete, all samples were diluted with nano-
pure water and refrigerated until analysis.

Sodium, potassium, calcium, and magnesium were
analyzed using flame atomic absorption spectrophotom-
etry. Lanthanum chloride was used to offset anionic in-
terference with calcium and magnesium analyses.

Effect of salinity on osmolality of mesogleal fluid

Medusae were acclimated for 1 wk at salinities of 5%«,
12%o, 15%o,and20%0. Mesogleal fluid from both bell and
tentacles was drawn into calibrated hemocrit tubes and
osmotic pressure determined using a Wescor 5500 vapor
pressure osmometer. Osmotic pressure was also mea-
sured in the corresponding samples of external media.
Osmolality in media and mesogleal fluid was expressed
per kilogram, based on weight determinations made of
both samples.

Size and weight changes in ephyrae and medusae

Ephyrae used for experiments were 2-3 mm in diam-
eter; others, fed on Anemia nauplii, were grown to
greater sizes for determinations of diameter and weight.
A binocular microscope equipped with a micrometer
eyepiece was used to measure diameters; a Cahn micro-
balance was used for weights. Weights of ephyrae used
in experiments were estimated from a regression curve
relating empirical measurements of diameter and wet
weight over a range of animal sizes.

Medusa weights were determined directly by remov-
ing animals from the media, rinsing and blotting them
lightly to remove surface water and gastrovascular fluid,
and transferring them to a plastic weighing tray on a top-
pan balance. To estimate the relative contributions of
mesogleal fluid and tissue to total medusa '.'.eight, eight
medusae were weighed as above. Then the bell and ten-
tacles were separated and weighed. The beli and tentacles
were next sliced into small pieces, the' mghly blotted
with laboratory wipes, and reweighed liis weight was
subtracted from the total wet weigh 10 estimate the

334

D. A. WRIGHT AND J. E. PURCELL

mesogleal fluid weight. No correction was made for the
fibrous componeri' ae mesoglea, which was included
in the tissue we: it. Some contamination of the meso-
gleal fluid c unent by gastrovascular fluid may have
occurred. ' . most of the latter was removed by the ini-
tial bio ,,g. Finally the residue was dried thoroughly
and rev. eighed to obtain a percentage solid material.

During salinity transfer experiments, we observed
weight losses that were probably caused by starvation. To
better understand the role of starvation in weight loss, a
single controlled feeding experiment was conducted us-
ing ephyrae fed cultured rotifers (Brachionis). The diam-
eters of 24 ephyrae acclimated to a salinity of 20%o were
measured. The animals were placed into individual
multi-wells containing 2 ml of 20%o water. The ephyrae
were divided into three feeding groups: unfed. 5 rotifers
d~', and 10 rotifers d~'. At 24 h, the ephyrae were mea-
sured, their water was changed, and the appropriate
number of rotifers was added to each well. Measure-
ments were repeated at 48 h. and growth was recorded as
the mean daily increase in ephyra diameter.

Sulfate in medusa tissue

Weighed tissue samples were dissolved in Ultrapure
concentrated nitric acid and diluted with nanopure wa-
ter. Total sulfate was determined with a Dionex ion chro-
matograph. Sulfate exchange was measured using
Nai35SO.4. Small medusae were exposed to radioisotope
for periods up to 60 h. The exposure medium had a sa-
linity of 16%o, with Nay^SQj added to a specific activity
of 250 nC\/mAl SO4. After isotope exposure the medusae
were netted, then triple rinsed, blotted, and weighed. The
specific activity of sulfate in the animals was recorded as
a percentage of the specific activity of the external me-
dium. "S was measured with a Packard Tricarb 4000 se-
ries liquid scintillation counter.

Statistics

Statistical differences between the mean ionic concen-
trations of tissue and seawater were tested using /-tests
with significance determined at the 5% level. Paired !-
tests wert used to evaluate osmolality differences be-
tween me^. :a and medium in individual replicates (P
< 0.05). A lir. f best bit fit through 35S exchange data
was estimated p.fj an iterative (NLIN) hyperbolic re-
gression progran, S).

'esults

Cation concentrations in tissue and mesogleal fluid

Ionic concentrations in tissue and mesogleal fluid gen-
erally underwent similar changes when medusae were
transferred from a salinity of 20%o to either 8%o (experi-

ment 1. Fig. 1 ) or 12%o (experiment 2, Fig. 2). Following
the transfer of animals from 20%o to either 8%o or 12%o,
sodium concentrations in tissue and mesogleal fluid were
characterized by a rapid drop over the initial 6 h to levels
approaching those seen in the external media (Figs. 1A,
2A). Mesogleal sodium also fell, but much slower in the
12%o medium; at 8%o, the tissue Na concentration made
an apparent "recovery" after 1 wk, to a level higher than
that of the external medium.

Tissue potassium concentration in animals held at
20%» was at least twice the corresponding seawater con-
centration and was generally maintained at levels be-
tween 1 1 and 14 myl/kg"' over a period of a week in
both experiments. However, in experiment 2, tissue po-
tassium in control (20%o) animals rose to a mean level of
17m;Ukg '. Tissue potassium levels represented a
differential of at least 12raA/kg~' compared with the
most dilute medium (2 m;U kg P1). In experiment 1 the
initial concentration of potassium in mesogleal fluid was
significantly higher than at 20%o (P < 0.05). When ani-
mals were transferred from 20%o to 8%o, mesogleal potas-
sium fell to a concentration similar to that of the initial
medium (20%o) but significantly higher than the 8%o me-
dium (Fig. IB). After a subsequent rise at 24 h it fell
again, but remained significantly higher than the 8%o me-
dium after 1 wk. On transfer from 20%o to 12%o (experi-
ment 2), mesogleal potassium concentration showed no
significant dilution and remained consistently higher
than that of the 12%« medium over 1 wk. Tissue potas-
sium levels did not change significantly following the
transfer of medusae to either 8%o (Fig. IB) or 12%o
(Fig. 2B).

In animals transferred from 20%o to 8%o, concentra-
tions of calcium in both tissue and mesogleal fluid fell
after 6 h (Fig. 1C). Thereafter, mesogleal calcium con-
centration remained significantly higher than concentra-
tions in tissue or in the external medium. Overlap of
standard deviations associated with calcium levels of the
medium at 20%» and 12%o (Fig. 2C) rendered calcium
data from experiment 2 equivocal.

Magnesium levels in both salinity transfer experi-
ments showed trends similar to those of calcium. Follow-
ing transfer of animals from 20%o to either 8%» (Fig. 1 D)
or 12%o (Fig. 2D), magnesium levels in tissue remained
consistently higher than in mesogleal fluid. Both tissue
and mesogleal magnesium concentrations remained
higher than those of dilute external media for a period of
a week.

Transference of medusae from 20%o to 5%o caused all
animals to fall to the bottom of the experimental beakers.
Three out of six medusae began to disintegrate within
36 h; one was unhealthy but alive after 3 days, and two
were swimming weakly near the bottom of the beakers.
No ionic data were recorded from these animals.

SALINITY AND IONIC SHIFTS IN MEDUSAE

335

ID

CO
CO

o
"co"

"5>
o

0)

T3

C
CO

0)
CO

168 0 10 20 30
30

10 20 30

168 o 10 20 30

168

TIME(h)

TIME(h)

Figure 1. Ion concentrations in mesoglcal fluid and tissue ofChrysaora quinquecirrha medusae trans-
ferred from 20%o salinity to 8%». (A) sodium, (B) potassium, (C) calcium. iD) magnesium. Each symbol
represents the mean ± I SD of six animals. Solid symbols represent ion concentrations in animals trans-
ferred from higher to lower salinity at I = 0: squares = mesogleal fluid, triangles = tissue. Open squares and
triangles represent concentrations in mesogleal fluid and tissue respectively, in animals maintained at 20%o.
Dotted horizontal lines represent the mean ionic concentrations of the media in experimental containers
(upper = control, lower = treatment) at the end of the experiments: shaded areas represent ± 1 SD.

Effect oj salinity change on body and tissue weight

All of the foregoing measurements of ionic shifts
should be considered against changes in tissue hydration
resulting from osmosis. Several experiments were carried
out to quantify this phenomenon, although it proved
difficult to obtain consistent results. Typical results indi-
cate a wide variation in weight change, even over 24 h
(Fig. 3). Changes in salinity from 16%o to 8%o resulted in
body weight increases between 10% and 15% in whole
medusae over 24 h, at which time their body weight was
>30% higher than control animals. The time course of
the subsequent weight loss was reflected by a concomi-
tant loss in weight of medusae maintained in 16%o for
1 wk.

Some of this weight loss could have been due to lack
of food during the experiments. The diameter of unfed
ephyrae in a 48-h controlled feeding experiment was re-

duced at a daily rate of 2.3% ± 20.5% (SD) (Fig. 4).
Ephyra size was positively related to food; diameters in-
creased by 10.6% ± 13.8% d~' in those fed 5 rotifers d^1
and by 29.2% ± 25.4% d"1 in those fed 10 rotifers d~'.

Osmolality of mesogleal jluid

The osmolality of mesogleal fluid and corresponding
seawater was measured in medusae acclimated for 1 wk
in 5%o, 12%o, 15%o, and 20%0 (Fig. 5). All animals at
12%o-20%o exhibited overlap between seawater and
mesogleal osmolality, although paired /-tests indicated
that the osmolality of mesogleal fluid was significantly
lower than that of the corresponding exk-r <al medium at
15%o and 20%o (P < 0.05). Conversely, mimals at 5%o
and 12%» had significantly higher mes( gleal osmolality
than that of seawater.

336

D. A. WRIGHT AND J. E. PURCELL

10 20

168

0

10 20 30

TIME (h)

168

Figure 2.

(erred from
Figure I.

30
TIME(h)

Ion concentrations in mesogleal fluid and tissue ofChrysuura c/iiimiiurirr/ni medusae trans-
20%o salinity to I2%«. (A) sodium. (B) potassium. (C) calcium. (D) magnesium. Symbols as in

Sulfale

35S exchange at a salinity of 16%o was complete in
about 40 h (Fig. 6) and indicated a freely exchanging
component representing about 40r< of the total weight.
This o "responded closely to the "mesogleal space" of
43<£ is estimated from direct measurement of

whole me!.' hell and tentacular tissue weight (Fig. 7).
Thesuli , ions in mesogleal fluid of medusae

acclimai. 16%o, and 12%o are shown

in Table l iiMeal sulfate levels were sig-

nificantly lovvei the external medium.

Mills ( 1984) found i >glea of Aequorea vic-

toria hydromedusae maint. \ h at equilibrium in

salinities between 23%o and 3 is generally hypoos-

molar to the corresponding extei nal medium, although
''OS between seawater and mesoglea were very
.it the lower end of the salinity range. A similar
palt^ -.\ as seen here, although between 15%o and 12%o
u/iiecur/ui mesoglea switched from being

hypo-osmolar to hyperosmolar relative to the external
medium. Although there is some overlap in the osmol-
alities of seawater and mesogleal fluid at salinities be-
tween 12%o and 20%o, paired /-tests involving individual
replicates reveal significant differences in each case. Nev-
ertheless, differences between mesogleal fluid and seawa-
ter remained very small, and C. quinquecirrha appeared
to be an osmoconformer throughout this range.

Of the ions measured, the most clearly regulated was
potassium. Total tissue potassium concentration was ap-
proximately 1 3 m.M kg" ' and remained stable following
transference of medusae from a salinity of 20%o to 8%o or
12%o. The apparent rise in tissue potassium in control
animals (maintained at 20%o) in experiment 2 (Fig. 2B)
was unexplained but did not differ significantly from the
corresponding tissue levels in 12%o. The potassium con-
centration in mesogleal fluid appeared more variable,
and values of 8.8mAfkg~' and 6mA/kg ' were re-
corded at / = 0 in experiments 1 and 2. Transference of
medusae from 20%o to 8%o resulted in fluctuating meso-
gleal concentrations, including an apparent rise in meso-
gleal potassium 24 h after the salinity change (Fig. IB).

SALINITY AND IONIC SHIFTS IN MEDUSAE

337

<u

01

C

O

2

O)

I

24

Time (h)

Figure 3. Weight changes in Chrysaora quinquecirrha medusae fol-
lowing transfer from 16%o salinity to 8%o over 1 week. Means ± 1 SD
for eight animals.

This elevation in mesogleal potassium concentration
was seen only after the larger salinity change (20%o to
8%o), and it is possible that osmotic shock may have re-
sulted in a transient increase in cellular potassium per-
meability or. perhaps, in some cell damage leading to po-
tassium contamination of the mesogleal fluid. A further
possibility is that potassium may be actively extruded
from cells to mitigate cellular hydration, as has been
demonstrated in the freshwater hydromedusa Craspeda-
custa sowerbyi (Fleming and Hazelwood, 1967; Hazel-
wood et a/., 1970). However, in Craspedacusta, sodium
was also involved (Hazelwood et a/.. 1970). There is no
significant evidence of sodium involvement in volume
regulation in C. quinquecirrha, and amino acid regula-
tion is probably more important in this regard (Wright,
unpubl. data). In the euryhaline sea anemone Diadu-
mene leucolena, Pierce and Minasian (1974) demon-

40

$ 30

0)
TO

b

_C

0)
O)

TO

-C

O

20

10

-10

0

10

Number of Rotifers Fed per Day

Figure 4. Effect of food ration on size of Chrysaora quinquecirrha
ephyrae over a 48-h period as determined by measurement of diameter.

600

~ 500

E
en

O

O
CO

O

400

300

ro 200

D)
O
CO
0>

100

0

0 100 200 300 400 500 600
Seawater Osmolality (m Osm I'1 )

Figure 5. Osmolality of mesogleal fluid and external medium in
Chrysaora quinquecirrha medusae acclimated for I week in salinities
of 5%o, 12%«. 15%o. and 20%o. Six individuals were measured at each
salinity except for 5%». where n = 5. Some symbols overlay others.

strated that volume regulation was achieved through the
regulation of the intracellular amino acid pool.

The coincidence of measured sulfate space and our
physical determination of extracellular fluid indicated an
extracellular space of about 40% in C. quinquecirrha. Us-
ing this figure and a concentration of 7.4 mA/kg~' for
mesoglea potassium, one can determine a corrected
value for intracellular potassium. The corrected esti-

Figure 6. Sulfate exchange in Chrysaora quinquecirrha medusae.
Exchange was determined as the percentage internal "S specific activity
versus external "S specific activity over 63 h.

338

D. A. WRIGHT AND J. E. PURCELL

100

c

CD

O

O

<u
2
o>
Q.

whole

bell tentacle

Type of Tissue

l:::::-| extracellular
L^J water

Iintracellular
water

Bother

U substances

Figure 7. Mesogleal fluid space in bell, tentacles, and whole medu-
sae of Chrysaora quinquecirrha. The space, expressed as a percentage
of all constituents, was measured directly by weighing the components.
"Other substances" refers to dry residue following final drying and re-
weighing.

mate of 1 7 mAf kg ' calculated in this way is intermedi-
ate between the mean values of 10.6mA/kg~' and
29.5 m/V/kg~' determined by Steinbach ( 1963) for intra-
cellular potassium in the hydroids Chlorohydra viridis-
sima and Hydra littoralis in freshwater media. Sulfate
space shows considerable variation both within and be-
tween species of hydromedusae. Mills and Vogt (1984)
reported the sulfate space to be between 10% and 80%
in six species of marine hydromedusae. They found that
equilibration times varied between <4 h for Aglantha
digitale and about 85 h for Mitrocoma cellularia. The
current estimate of 40%> sulfate space for C. quinquecir-
rha, with an apparent equilibration time of about 40 h.
falls more or less in the middle of this range. At salinities
between 20%» and 12%o, sulfate in the mesogleal fluid of
C. quinquecirrha is maintained at concentrations lower
than those of the external media. Similar results were ob-
tained by Newton and Potts ( 1 993), who reported meso-
gleal sulfate levels of 40% and 52% of seawater levels in
Cyanea capillata and Rhizostoma pulmo respectively.

In both experiments 1 and 2, there was evidence that
the magnesium concentrations in tissue and mesogleal
fluid were maintained at levels above that of the external
medium. Following transfer of medusae from 20%o to
12%o, magnesium was substantially diluted in tissue and
mesogleal fluid, yet even 7 d after the salinity change, re-
mained significantly higher than the concentration of the
dilute medium (Fig. 2D). A similar phenomenon was
seen following transfer from 20%o to 8%o (Fig. ID). The
concentration of sodium also remained significantly (P
< 0.05) higher in tissue than in the 8%» medium.

Dilution resulting from water influx obviously con-
tributed to the fall in tissue ions in most cases. Weight
changes in medusae transferred to lower salinities indi-
cated that water influx reached 10%-20% of total body
weight within the first 24 h following transfer. But tissue
concentrations of sodium, magnesium, and calcium in
animals transferred from 20%o to 8%o fell by more than
40% in the first 6 h, indicating that diffusional losses of
these ions were at least as important as increased tissue
hydration in explaining these losses. The apparent sub-
sequent "recovery" of tissue sodium and magnesium to
levels above those of the dilute medium suggests that,
although these ions may not be actively regulated, the
tissue is not freely permeable to them. It is also possible
that tissue permeability to these ions changes after the
initial osmotic shock. The relative stability of tissue po-
tassium, despite the osmotic swelling, emphasizes the
efficiency of the mechanism that regulates cellular potas-
sium in C. quinquecirrha.

The degree to which medusae of C. quinquecirrha be-
have like osmometers with changing salinity is difficult
to exactly quantify in view of the likely influence of food
on weight changes. The controlled feeding experiment
illustrated that lack of feeding may account for a weight
loss greater than 20% over a 24-h period. This supports
the data shown in Fig. 3B. If results from 8%o animals are
corrected for controls ( 1 6%o), the 24-h weight change due
to osmotic stress may exceed 30%-. Medusae of Aurelia
unriui and C. quinquecirrha also decreased in diameter
when unfed (Hamner and Jenssen, 1974; Rosen and
Purcell, unpubl. data).

In studies conducted here, medusae were able to
achieve at least neutral buoyancy in all salinities except
one — at 5%« they remained on the bottom of experimen-
tal containers. Bidigare and Biggs ( 1 980) suggested a role
for sulfate in adjusting buoyancy in jellyfish. They
showed that, by active elimination of more than half of
its body SO4 relative to seawater, the ctenophore Berne
ci/cumis could neutralize its protein mass and achieve
neutral buoyancy in dilute seawater. It was postulated
that sulfate elimination was offset by isosmotic replace-
ment by chloride. Our data show sulfate concentrations

Table I

Sulfate concentrations in mesogleal Jluid of Qaysaora quinquecirrha
medusae adapted to different salinities for 48 h

Seawater sulfate

Mesogleal sulfate

Mesogleal sulfate

Salinity

concentration

concentration

as percentage

%0

(m.U/l)

(mM/l)

seawater

20

13.2

9.1

69

16

9.7

6.8

70

12

7.9

5.2

66

SALINITY AND IONIC SHIFTS IN MEDUSAE

339

to be consistently lower in tissue of C. quinquecirrha
than in the external media, but there was no indication
of a change in tissue:medium sulfate ratio on transfer of
medusae from higher to lower salinity. Therefore, we
conclude that C. quinquecirrha does not regulate buoy-
ancy by excluding sulfate ions. These medusae are strong
swimmers and perhaps would not benefit substantially
from ionic buoyancy compensation.

Our results suggest the possibility that medusae of
Chrysaora quinquecirrha are unable to regulate volume
or buoyance at salinities <5%o. This agrees with the dis-
tribution of polyps and medusae in situ (Cargo and
Schultz, 1966, 1967) and with laboratory experiments on
asexual reproduction (Purcell et a/., unpubl. data).

Acknowledgments

This research was funded by NSF grant OCE-
9019404. We thank H. Lemke, G. Aldridge, and T.
Lewis for assistance with experiments. University of
Maryland, Center for Environmental and Estuarine
Studies Contribution No. 2793.

Literature Cited

Hi-mis, D., and R. Prusch. 1972. Osmoregulation in fresh-water Hy-
dra. Comp. Biochem. Physiol. 43A: 165-171.

Bidigare, R. R., and D. C. Biggs. 1980. The role of sulfate exclusion
in buoyancy maintenance by siphonophores and other oceanic ge-
latinous zooplankton. Comp. Biochem. Physiol. 66A: 467-47 I .

Cargo, D. G., and L. 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 L. P. Schultz. 1967. Further observations on the

biology of the sea nettle and jellyfishes in the Chesapeake Bay. Ches-
apeake Sd. 8: 2W-220.

Denton, E. J., and 1 . 1. Shaw. 1962. The buoyancy of gelatinous ma-
rine animals. J . Physiot 161: 14-15.

DiiiMi.ni. H. J. 1994. The distribution and ecology of the fresh- and
brackish-water medusae of the world. Hydrobiologia 272: 1-12.

Fleming, \V. R., and D. H. Hazelwood. 1967. Ionic and osmoregula-
tion in the freshwater medusa Craspedacusta sowerbyi. Comp. Bio-
chem. Physiol. 23:911-915.

Hamner, W. M., and R. M. Jenssen. 1974. Growth, degrowth, and
irreversible cell differentiation in Aurelia aurila. Am. Zoo/. 14: 833-
849.

Hazelwood, D. H., W. T. W. Potts, and W. R. Fleming. 1970. Fur-
ther studies on the sodium and water metabolism of the freshwater
medusa, Craspedacusta sowerbyi. Z I'gl. Physiol. 67: 186-191.

Mackay, W. C. 1969. Sulfate regulation in jellyfish. Comp. Biochem.
Physiol. 30:481-488.

Mills, C. E. 1984. Density is altered in hydromedusae and cteno-
phores in response to changes in salinity. Biol. Bull 166: 206-2 1 5.

Mills, C. E., and R. G. Vogt. 1984. Evidence that ion regulation in
hydromedusae and ctenophores does not facilitate vertical migra-
tion. Biol. Bull. 166:216-227.

Newton, C., and W. T. VV. Potts. 1993. Ionic regulation and buoy-
ancy in some planktonic organisms. / Afar. Biol. Assoc. (7.A 73:
15-23.

Pierce, S. K., and L. L. Minasian, Jr. 1974. Water balance of a eury-
haline sea anemone, Diadumene leucolena. Comp. Biochem. Phys-
iol. 49 A: 159-167.

Robertson, J. D. 1949. Ionic regulation in some marine inverte-
brates. / E\p. Biol. 26: 1 82-200.

Snick, J. M. 1973. Effects of salinity and starvation on the uptake
and utilization of dissolved glycine by Aurelia aurila polyps. Biol.
Bull 144: 172-179.

Steinbach, II. B. 1963. Sodium, potassium and chloride in selected
hydroids. Biol. Bull 124: 322-336.

Webb, K. L., A. L. Schimpf, and J. Olmon. 1972. Free amino acid
composition of scyphozoan polyps of Aurelia aurila. Chrysaora
quinquecirrha and Cyanea capillata at various salinities. Comp.
Biochem. Phvsiol. 43B: 653-663.

CONTENTS

RESEARCH NOTE

Sauer, Warwick H. H ., Mike J. Roberts, Marek R.
Lipinski, Malcolm J. Smale, Roger T. Hanlon, Dale
M. Webber, and Ron K. O'Dor

Choreography of the squid's "nuptial dance" .... 203

CELL BIOLOGY

Davy, Simon K., Ian A. N. Lucas, and John R.
Turner

Uptake and persistence of homologous and heterol-
ogous zooxanthellae in the temperate sea anemone
Cereus pedunculatus (Pennant) 208

DEVELOPMENT AND REPRODUCTION

Bates, William R.

p58, a cytoskeletal protein, is associated with muscle

cell determinants in ascidian eggs 217

Glas, Patricia S., Lee A. Courtney, James R. Ray-
burn, and William S. Fisher

Embryonic coat of the grass shrimp Palaemonetes
Pug>o 231

ECOLOGY AND EVOLUTION

Co 1 1 in, Rachel, and John B. Wise

Morphology and development of Odostomia colum-
biana Dall and Bartsch (Pyramidellidae): implica-
tions for the evolution of gastropod development 243
Distel, Daniel L., and Susan J. Roberts

Bacterial endosymbionts in the gills of the deep-sea
wood-boring bivalves Xylophaga atlantica and Xv/o-
phaga washinglona 253

Seibel, Brad A., Erik V. Thuesen, James J. Childress,
and Laura A. Gorodezky

Decline in pelagic cephalopod metabolism with hab-
itat depth reflects differences in locomotory effi-
ciency 262

West, Jordan M.

Plasticity in the sclerites of a gorgonian coral: tests
of water motion, light level, and damage cues .... 279
Yund, Philip O., Yvette Marcum, and John Stewart-
Savage

Life-history variation in a colonial ascidian: broad-
sense heritabilities and tradeoffs in allocation to
asexual growth and male and female reproduction 290

NEUROBIOLOGY AND BEHAVIOR

Eguchi, Eisuke, Mari Dezawa, and V. Benno Meyer-
Rochow

Compound eye fine structure in Paralomis multispina
Benedict, an anomuran half-crab from 1200 m
depth (Crustacea; Decapoda; Anomura) 300

PHYSIOLOGY

Roberts, Deirdre A., Gretchen E. Hofmann, and
George N. Somero

Heat-shock protein expression in Mytilus califor-
tiianus: acclimatization (seasonal and tidal-height
comparisons) and acclimation effects 309

McNamara, John C., and Alice Goncalves Lima
The route of ion and water movements across the
gill epithelium of the freshwater shrimp Macrobra-
chium olfersii (Decapoda, Palaemonidae): evidence
from ultrastructural changes induced by acclima-
tion to saline media 321

Wright, David A., and Jennifer E. Purcell

Effect of salinity on ionic shifts in mesohaline scy-
phomedusae, Chiysaora quinquecirrha 332

Volume 192

E

Number 3

BIOLOGICAL
BULLETIN

JUNE, 1997

Published by the Marine Biological Laboratory

1997

THE

BIOLOGICAL BULLETIN

PUBLISHED BY

THE MARINE BIOLOGICAL LABORATORY
Associate Editors

Louis E. BURNETT, Grice Marine Biological Laboratory. College of Charleston
WILLIAM D. COHEN, Hunter College, City University of New York

CHARLES D. DERBY, Georgia State University
DAVID EPEL, Hopkins Marine Station. Stanford University

Editorial Board

PETER B. ARMSTRONG, University ofCalifornia. Davis
THOMAS H. DIETZ. Louisiana State University

RICHARD B. EMLET. Oregon Institute of Marine Biology,

University of Oregon

DAPHNE GAIL FALITIN, University of Kansas

WILLIAM F. G\LL^ , Hopkins Marine Station. Stanford

University

ROGER T. HANLON, Marine Biological Laboratory

MAKOTO KOBAYASHI, Hiroshima Prefectural Uni-

versity

MICHAEL LABARBERA, University of Chicago
DONAL T. MA\ AHAN, University of Southern California

MARGARET McFALL-NGAi, Kewalo Marine Labora-
tory, University of Hawaii

TATSLIO MOTOKAWA. Tokyo Institute of Technology
K. RANGA RAO, University of West Florida

BARUCH RINK.EVICH, Israel Oceanographic &
Limnological Research Ltd.

RICHARD STRATHMANN, Friday Harbor Laboratories,
University of Washington

STEVEN VOGEL. Duke University

J. HERBERT WAITE. University of Delaware

SARAH ANN WOODIN, University of South Carolina

RICH ARD K. ZIMMER-FAUST. University of California.

Los Angeles

. MICHAEL J. GREENBERG. The Whitney Laboratory. University of Florida
HK Eihlor: PAMELA L. CLAPP. Marine Biological Laboratory

JUNE, 1997

Printed and Issued by
LANCASTER PRESS, Inc.

3575 HEMPLAND ROAD

LANCASTER, PA

Cover

Embryo of Hydra vitlgaris encased in a multilay-
ered cuticle adorned with ornate spines. The
embryo remains in the cuticle for varying lengths
of time. At hatching, the cuticle breaks open and
a hatchling emerges head-end first. See Martin el
a/., this issue.

CONTENTS

RESEARCH NOTE

Anderson, Erik J., Patrick S. MacGillivray, and
M. Edwin DeMont

Scallop shells exhibit optimi/.ition of i iblet climen-
sions for drag reduction 341

DEVELOPMENT AND REPRODUCTION

Martin, Vicki J., C. Lynne Littlefield, William E.
Archer, and Hans R. Bode

Embryogenesis in India 345

Hanlon, Roger T., Michael F. Claes, Susan E. Ash-
craft, and Paul V. Dunlap

Laboratory culture of the sepiolid squid Eiipn'iinin
Mil/apt'.^: a model system for bacterial -animal symbi-
osis 364

NEUROBIOLOGY AND BEHAVIOR

Preuss, Thomas, Zora Lebaric, and William F. Gilly

Post-hatching development of circular mantle mus-

cles in the squid L/iligu <ijxih^n-ti\ 375

Marois, Rene, and Thomas J. Carew

Fine structure of the apical ganglion and its seroto-
nergic cells in the larva of Apl\si« iiili/nniiin 3KN

Pires, Anthony, and Robert M. Woollacott

Serotonin and dopamine have opposite effects on
phototaxis in larvae ol the bryozoan Bugula in'ittnin 399

Moore, Paul A., and Deborah M. E. Lepper

Role of chemical signals in the orientation behavior

of the sea star A.\t<-i-i<i\ /i»-ln:\i 410

Schivell, Amanda E., Samuel S.-H. Wang, and Stuart

H. Thompson

Behavioral modes arise from a random process in

the nuclibranch Melihf . 418

PHYSIOLOGY

McManus, Michael G., Allen R. Place, and William
E. Zamer

Physiological variation among clonal genotypes in
the sea anemone Haliplanella liin'riln: growth and

biochemical content 426

Nii, Calvin M., and Leonard Muscatine

Oxidative stress in the symbiotic sea anemone A//I-
III\KI jiulihi'/l/i: (Calgren, 1943): contribution of tin-
animal to superoxide production at elevated tem-
perature 444

Index for Volume 192 457

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VOLUME CONTENTS

CELL BIOLOGY

No. 1, FniKi ARV 1997

MECHANOSENSITIVITY

Goldberg, Walter M., and George T. Taylor

Coelenterate cnidae capsules: disulhde linkages re-
vealed by silver cytochemistry and their differential
responses to thiol reagents ]

DEVELOPMENT AND REPRODUCTION

Sewell, M. A., P. A. Tyler, C. M. Young, and
C. Conand

Ovarian development in the class Holothuroidea:

a reassessment of the "tubule recruitment model" 1 7

Hoegh-Guldberg, O., and R. B. Emlet

Energy use during the development of a lecitho-
trophic and a planktotrophic echinoid 27

Martin, Vicki J., and William E. Archer

Stages of larval development and stem cell popula-
tion changes during metamorphosis of a hydrozoan
planula 41

IMMUNOBIOLOGY

Hirose, Euichi, Yasunori Saito, and Hiroshi Wata-
nabe

Subcuticular rejection: an advanced mode of the al-
logeneic rejection in the compound ascidians, B<>1>\/-

loides simodensisz.i\AB.fuscus 53

Raftos, David, and Aimee Hutchinson

Effects of common estuarine pollutants on the im-
mune reactions of tunicates 62

ECOLOGY AND EVOLUTION

Anthony. Kenneth R. N.

Prey capu v the sea anemone M,'lnt/iini/ wmlr

(L.): effect si/e. flow regime, and upstream

neighbor* 73

Holyoak, Alan

Patterns and i s whole colony growth

in thecompoiuu 'mm /;/tiiniiii .... 87

Miller, Karen, and 1

( :<)nflicting morphi , ,1 , .ductive species
boundaries in the KM „ 98

HE FUTURE OF AQUATI- ^SEARCH IN SPACE:
NEUROBIOLOGY ..LULAR AND
MOLECULAR i OLOGY

Loewenstein, Werner R.

Mechanosensitive channels: introduction 117

Morris, Catherine E., Howard Lesiuk, and Linda R.
Mills

How do neurons monitor then mechanical status? 1 18
I lamill. Owen P., and Don W. McBride Jr.

Mechanogated channels in AVm//)i;\ oocytes: differ-
ent gating modes enable a channel to switch from a
phasic to a tonic mechanotransducer 121

DI

123

Chalfie, Martin

A molecular model for mechanosensation in C.urnii-
rhabditis elegam 125

Blount, Paul, Sergei I. Sukharev, Paul Moe. and

Ching Kung

Mechanosensitive channels of E. culi: a genetic and
molecular dissection 126

Discussion

128

PLANT BIOLOGY

Hader, Donat-Peter

Gravitaxis in flagellates 131

Kiss, John Z.

Gravitropism in the ihizoids of the alga Chtirti:

a model system for microgravity research 134

137

. A.

I !

115

Edwards, Erin Swint, and Stanley J. Roux

The influence of gravity and light on develop-
mental polarity of single cells of Ceratopteris in /innli/

gametophytes 1 39

Nick, Peter, Rea Godbole, and Qi Yan Wang

Probing rice gravitropism with cytoskeletal drugs

and cytoskeletal mutants 141

Z)/.(c».s.w'»» . 144

NEUROBIOLOGY/SENSORY BIOLOGY

Eaton, Robert C., Audrey L. Guzik, and Janet L.
Casagrand

Mauthner system discrimination of stimulus direc-
tion from the acceleration and pressure components
at sound onset 1 46

CONTENTS

Fetcho, Joseph R., Kingsley J. A. Cox, and Donald
M. O'Malley

Imaging neural activity with single cell resolution in
an intact, behaving vertebrate .............

Discussion ...........................

Kawasaki, Masashi

Complex signal processing by weakly electric fishes
Bass, Andrew H., Deana A. Bodnar, and Jessica R.
McKibben

From neurons to behavior: vocal-acoustic communi-
cation in teleost fish ...................

Fischer, Thomas M., and Thomas J. Carew

Activity-dependent regulation of neural networks:
the role of inhibitory s\ naptic plasticity in adaptive
gain control in the siphon withdrawal reflex ol
Apl\v(i ...........................

Baxter, Douglas A., and John H. Byrne

Complex oscillations in simple neural systems ....

150
154

157

158
llil

DI

164
167

170

Angerer, Robert C., and Lynne M. Angerer

Fate specification along the sea urchin embryo an-
imal-vegetal axis 175

Maxson, Rob, Hongying Tan, Sonia L. Dobias, Hai-

lin Wu, Jeffery R. Bell, and Liang Ma

Expression and regulation of a sea urchin A/.\\ class
homeobox gene: insights into the evolution and
function of a gene family that participates in the pat-
terning of the early embryo 178

179

CYTOSKELETON/CELL MOT1LITY

Burnside, Beth, and Christina King-Smith

Actin-dependent pigment granule transport in reti-
nal pigment epithelial cells 181

Lin, C. H., E. M. Espreafico. M. S. Mooseker, and

P. Forscher

Myosin drives retrograde E-actin How in neuronal
growth cones 183

Gillespie, Peter G.

Multiple myosin motors and mechanoelectrical
transduction by hair cells . 186

191

DEVELOPMENTAL BIOLOGY

Elinson. Richard P.

Getting a head in frog development

Chairs and Speakers 197

72 Participants .

200

No. 2, APRIL 1997

RESEARCH NOTE

Sauer, Warwick H. H., Mike J. Roberts, Marek R.
Lipinski, Malcolm J. Smale, Roger T. Hanlon,
Dale M. Webber, and Ron K. O'Dor
Choreography of the squid's "nuptial dance" .... 203

CELL BIOLOGY

Davy, Simon K., Ian A. N. Lucas, and John R.
Turner

Uptake and persistence of homologous and heterol-
ogous zooxanthellae in the temperate sea anemone
Cereus pedunriilatus (Pennant) 208

DEVELOPMENT AND REPRODUCTION

Bates, William R.

p58, a cvtoskeletal protein, is associated with muscle

cell determinants in ascidian eggs 217

Glas, Patricia S., Lee A. Courtney. James R. Ray-
burn, and William S. Fisher

Embryonic coat of the grass shrimp Palarmonftrs

piigin 231

ECOLOGY AND EVOLUTION

Collin, Rachel, and John B. Wise

Morphology and development of O(lu\l<i»/i<i m/iim-
/IKIIKI Dall and Bartsch (Pyramidellidae): implica-
tions for the evolution of gastropod development 243

Distel, Daniel L.. and Susan J. Roberts

Bacterial endosymbionts in the gills of the deep-sea
wood-boring bivalves Xylophaga titltnitirti and \\l<>-
/ihtiffa washingtona

Seibel, Brad A., Erik V. Thuesen, James J. Childress,

and Laura A. Gorodezky

Decline in pelagii cephalopod metabolism with hab-
itat depth reflects differences in loi cinotoiv effi-
ciency

West, Jordan M.

Plasticity in the sclerites of a goigonian coral: tests

of water motion, light level, and damage cues .... 279

262

CONTENTS

Yund, Philip O., Yvette Marc n. and John Stewart-
Savage

Life-historv \;n iat ; ,n.il ascidian: broad-

sense herital'iln' , adeoffs in allocation to

asexual growth le and female reproduction

NEUROBIOLOGY AND BEHAVIOR

Eguchi, Eisuke, Mari Dezawa, and V. Benno Meyer-
Rochow

Compound eye fine structure in Paralomu multispinu
Benedict, an anonuiran hall-crab from 1200 in
depth (Crustacea; Decapoda; Anomura)

290

PHYSIOLOGY

Roberts, Deirdre A., Gretchen E. Hofmann, and
George N. Somero

Heat-shock protein expression in M\li/u\ mlifor-
iiniiiiis: acclimatization (seasonal and tidal-height
comparisons) and acclimation effects 309

McNamara, John C., and Alice Goncalves Lima
The route of ion and water movements across the
gill epithelium of the freshwater shrimp Macrobra-
ili/iun iil/rniii (Decapoda, Palaemonidae): evidence
from ultrastructural changes induced by acclima-
tion to saline media 321

Wright, David A., and Jennifer E. Purcell

Effect of salinity on ionic shifts in mesohaline scy-
phomedusae, Chn'saurn t/nhx/in-in rhu 332

No. 3, Jt \i- 1997

RESEARCH NOTE

Anderson, Erik J., Patrick S. MacGillivray, and
M. Edwin DeMont

Scallop shells exhibit optimization of riblet dimen-
sions for drag reduction 341

DEVELOPMENT AND REPRODUCTION

Martin, Vicki J., C. Lynne Littlefield, William E.
Archer, and Hans R. Bode

Embryogenesis in hydra 345

Hanlon, Roger T., Michael F. Claes, Susan E. Ash-
craft, and Paul V. Dunlap

Laboratory culture of the sepiolid squid Kuf»-\iintn
\iul<i/if\: a model system for bacterial-animal symbi-
osis 364

NEUROBIOLOGY AND BEHAVIOR

Preuss, Thomas, Zora Lebaric, and William F. Gilly

Post-hatching development of cin ular mantle mus-

cles in the squid Lo/igo o/ui/Mrriij 375

Marois, Rene, and Thomas J. Carew

Fine structure of tlu api< al ganglion and its serolo-

ii --it cells in ili. larva of » i uili/nnmn 388

Pires, Anthony, and Robert M. Woollacott

Serotonin and dopamine have opposite effects on
phototaxis in larvae of the bryozoan Bugulfi iii'iilnni 399

Moore, Paul A., and Deborah M. E. Lepper

Role of chemical signals in the orientation behavior

of the sea star .-Wr/vrM /iii/x'w 410

Schivell, Amanda E., Samuel S.-H. Wang, and Stuart

H. Thompson

Behavioral modes arise from a random process in

the nudibranch Mrlihr . 418

PHYSIOLOGY

McManus, Michael G., Allen R. Place, and William
E. Zamer

Physiological variation among clonal genotypes in
the sea anemone H<iiijilniii'lln inn-nln: growth and

biochemical content 426

Nii, Calvin M., and Leonard Muscatine

Oxidative stress in the symbiotic sea anemone Ai/i-
ttmri pulclifllti: (Calgren, 1943): contribution of the
animal to superoxide production at elevated tem-
perature 444

Index for Volume 192 457

Reference: B/o/. Bull. 192: 341-344. (June. 1997)

Scallop Shells Exhibit Optimization of Riblet
Dimensions for Drag Reduction

ERIK J. ANDERSON, PATRICK S. MACGILLIVRAY, AND M. EDWIN DEMONT*

Biology Department, St. Francis Xavier University. Antigonish. Nova Scotia, Canada B2G JM'5

Drag reduction by streamwise surface grooves, or rib-
lets, has been observed by engineers and has been sug-
gested to apply to certain biological systems. Drag reduc-
tions as high as 8% have been observed (1), leading to
practical nautical and aeronautical applications (2, 3. 4).
The shells of several species of scallop, including Placo-
pecten magellanicus, display rib/els arranged radially.
and therefore roughly parallel to the How during swim-
ming (Fig. la). The dimensions of these riblets on partic-
ular scallops fall within the region necessary for drag re-
duction at experimentally measured swimming speeds.
Moreover, the actual spacing of the riblets gradually mi-
grates into the theoretically optimal spacing region as
shell length increases beyond 40 mm (Figs. 2. 3). Speci-
mens of P. magellanicus 40 to 80 mm in length demon-
strate the greatest swimming ability (5): our data strongly
suggest that streamwise riblets may be a contributing fac-
tor to the swimming success in scallops of this siie range.

The giant scallop, Placopecten magellanicus, was
the subject of a recent comprehensive work on the hy-
drodynamics and energetics of locomotion (6, 7, 8, 9).
Scallops utilize a jet-propulsion system in swimming:
water is taken in when the shells open, and the valve-
like velum traps a volume of water between the shells
which is expelled in two jets adjacent to the hinge as
the shells clap shut. The scallop is therefore propelled
through the water gape-leading, hinge-trailing, at
speeds up to about 0.55 m/s, the swimming speed
common to scallops 65 mm in ventral-dorsal length.
LI (5). Their swimming ability enables scallops to es-
cape from predators, such as starfish, and, as some sug-
gest, to migrate with the seasons (5). Certainly if the

Received 2 December 1 996; accepted 1 4 April 1997.
* Author for correspondence.

latter behavior were a significant part of the scallop life
cycle, development of drag-reducing structural fea-
tures would not be surprising.

Recent years have seen the application of streamwise
grooves, or riblets, to aircraft and the hulls of racing boats
as a means of drag reduction and, therefore, enhanced
speed capability and decreased energy requirements.
The mechanism involves a reduction in the surface shear
stress arising from a turbulent boundary layer. Similar
grooves in aquatic organisms may offer the same advan-
tage (3, 4, 10, 1 1). We examined about 440 species of
bivalves from 60 families representing over 160 genera
and found that, with the exception of 7 genera, radial
riblets of dimensions similar to those in P. magellanicus
occur almost exclusively in swimming bivalves (ob-
served in 16 genera). The Limidae. which swim hinge
first, in the direction opposite to that of the scallop, also
exhibit radial riblets.

Theoretical and experimental work by fluid engineers
has elucidated the function and efficacy of riblets ( 12.
13) with drag reductions of 3%-8% observed in various
systems, from flat plates, to the hulls of racing boats (2),
the surfaces of aircraft ( 3 ), and the skin of sharks (3,4, 10,
1 1 ). Though riblet function is still not fully understood,
researchers have put forth a theory, based on the organi-
zation of surface vortices, to explain the drag-reducing
mechanism. According to the theory, there is a reduction
in the component of drag arising from a particular high
shear occurrence generated by the action of so-called
hairpin vortex tubes. In turbulent boundary layers, hair-
pin-shaped vortex tubes occur near the surface, oriented
so the bend of the hairpin is downstream and the two
prongs of the hairpin are pointed upstream and aligned
roughly parallel to the flow. As the bend of the hairpin is
swept further downstream, the remaining vortex tubes
move toward each other in a spanwise direction, which

341

342

E. J. ANDERSON ET AL

Figure I. Photographs of rihlets on the scallop Placopecien magellanicm (a) Whole shell. Shell length,
L, = 63 mm. (h) Magnified image of rihlets. Note the two newly added riblets marked with circles. For
scale, the distance between the two circles is 1 mm.

ultimately results in a downwash of fluid onto the sur-
face. This phenomenon is known as near-wall burst. Rih-
lets may inhibit this spanwise movement by organizing

adjacent surface vortex tubes into streamwise corridors
and thus restricting the vortex tubes from interacting to
cause near-wall burst.

1.8

1.6

I 1'4
^ 1.2

g> 1.0
ro 0.8

Q.

0.6
<D

S 0.4
S 0.2-

0.0

= 20

= 10

shell length = 32 mm

15 20 25 30 35
•om leading edge, x (mm)

E
JE

V)

at

c

o

P3
OL

.Q

a:

1.0

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0

* =20

+ =10

shell length = 48 mm

) 5 10 15 20 25 30 35 40 45 50

Distance from leading edge, x (mm)

; ;Het spacings (squares), at positions A = 0. 0.25/-,, 0.5L,. and 0.75L,. plotted against

uiing edge of the shell, from two representative scallops: (a) L, = 32 mm: (b) L,

present the boundaries of the optimal riblet spacing region for rihlet-based drag

reduction v.hiehr to each scallop on the basis of shell length and swimming speed (Eqs. Iand2).

A series (// -.HH^IH shells of L, from 10 mm to 90 mm was used in this study. Arcs were

established at posi = 0. 0.25/.,, 0.5LV, and 0.75L, over the central 30 degree sector of each shell, and

the number of riblt s on each arc was counted: Bioquanl OS/2 image analysis software was used for posi-
tioning and counting. Real riblet spacings were calculated by dividing the arc length. /„„ = w(L, - .v)/6. at
the four positions by the number of riblets on the arc. Swimming velocities, needed to calculate
,ir stresses (t.q. 2}. vary significantly with shell length and were taken from published data obtained
from film of I' inaxc/liinicia swimming ui •iini (5). Note that despite the radial pattern of rihlets. spacing
remains relatively constant due to riblet addition. Note also that the spacing measurements move into the
optimal region between A' = Klandi' = 20 as shell length increases from 32 mm to 48 mm.

SCALLOP RIBLET-BASED DRAG REDUCTION

343

Rihlet spacing for optimal drag reduction can be de-
termined from the following expression.

(Eq.

s = vs \ —

where s is actual rihlet spacing, v is the kinematic viscos-
ity of the fluid, s+ is a dimensionless expression of riblet
spacing for which the range 10-20 has been determined
to be optimal (11, 14). r,, is the wall shear stress due to
the flow, and p is the fluid density. The equation is simply
a rearrangement of the standard expression for a dimen-
sionless length parameter, in terms of v, ra and p. Such
dimensionless parameters are used in fluid dynamics for
the reason that, in general, any two systems with the
same s^ value will behave similarly, though values of ac-
tual spacing, .v, may differ. Wall shear stress for the swim-
ming scallop was determined from the equation for flow
over a flat plate with a turbulent boundary; the equation
was obtained through the standard practice of applying
the momentum integral equation to a '/7 power velocity
profile.

1 . 0.0594

•, = — (Eq. 2)

where U is the flow velocity over the scallop. Re* is the
length (or local) Reynolds number, and .v is the distance
measured along the line bisecting the valve, starting from
the most ventral edge of the valve (i.e., the leading edge
of the scallop). Due to the shell curvature, the shear
would be slightly higher than the equation predicts for
the leading section of the scallop. A turbulent boundary
layer is assumed on the basis of Reynolds numbers close
to the transition between laminar and turbulent flow; an
early breakdown of the laminar boundary layer is proba-
bly triggered by the combined effects of leading edge
roughness and the rapid clapping of the leading edge of
the scallop during swimming.

The average riblet spacing as a function of distance
from the leading edge was determined for each scallop
observed, then plotted together with the theoretically
predicted optimal riblet spacing for s* = 10 and s+ = 20
(Fig. 2). At smaller shell lengths, the actual riblet spacing
data fall below the optimal region (Fig. 2a). Since scal-
lops of lengths up to about 30 mm are attached to the
ocean bottom by byssal threads (5), there would be no
advantage for optimized riblet spacing in these smaller
scallops. At a shell length of about 50 mm — the size at
which P. inagellanicus exhibits the greatest swimming
speed (in body lengths per second) — the actual riblet
spacing data fall near the center of the optimal region
for riblet-based drag reduction (Fig. 2b). When the riblet
spacing at A = 0.5LX for each scallop observed is plotted
against shell length, the riblet spacing on scallop shells
larger than 40 mm remains in the optimal region (Fig.

1.6

1.4

12

1.0
I 0.8

03

g- 0.6
| 0.4
01 0.2

0.0

s* = 10

20 40 60 80

Shell length, Ls (mm)

100

Figure 3. Real riblet spaeing at position .v = 0.5L, plotted against
shell length. The plot compares the riblet spacings of the entire series (n
= 20) of shells observed at the same relative position on each shell.
Thus, each data point (circle) is produced from the spacing data of a
different shell. The solid lines are the predicted optimal rihlet spacing
for .f4 = 10 and .v* = 20. at A = 0.5L, using swimming speeds corre-
sponding to the given shell lengths (5). Notice that as shell length in-
creases past 40 mm, the data points enter the optimal region.

3). Moreover, the higher Reynolds numbers. Re = 2.0-
4.0 X 104, at these larger scallop sizes, increase the likeli-
hood of transition from laminar to turbulent boundary
flow. Thus, scallops of the size reported to swim most
vigorously are highly favored for riblet-based drag reduc-
tion, suggesting an optimization of shell design. Re-
search on P. magellanicus reveals that scallops larger
than 80 mm are generally less active swimmers due to
inferior hydrodynamic characteristics and heavy bodies
(5, 7). Scallops of this size are also commonly covered
with embionts which reduce swimming ability.

An equally impressive aspect of riblet design in these
scallops is best introduced by examining the equations
for determining optimal riblet spacing, s. Combining
equations 1 and 2 reveals that optimal riblet spacing is
inversely proportional to distance from the leading edge
to the '/!„ power. Therefore, as the optimal curves show
(Fig. 2), the best arrangement would be to have, at the
leading edge of an object (.v = 0), narrow spacing which
gets wider as you move toward the trailing edge (.v = LJ.
The radial pattern of the scallop riblets. which has its ver-
tex at the trailing edge, would be the opposite of this op-
timal arrangement if it were not for the fact that riblets
are added to the pattern, by intercalation, as the scallop
grows (Fig. Ib); i.e., there are more riblets at the leading
edge than near the vertex. Shells showed an average of 3
to 4 times as many riblets at the leading edge as at A
= 0.75L,.. For example, in a shell 54 mm in length, riblet

344

E. J. ANDERSON KT AL

spacing decreases, from 0 i \ = 0.75L,, to a sur-

prising 0.66 mm at the i.dge (A = 0). In contrast,

if the scallop did : , the geometrically calcu-

lated spacing v m at the leading edge. If the

riblet spacing v maintained by intercalation, the

riblet spacinc scallops could not remain within

the optimal spacing region for riblet-based drag reduc-
tion. Of the 37 species of nonswimming bivalves ob-
served to have shells with radial riblets, none added rib-
lets except 12 species of ribbed mussels and 7 species of
Arcidae. Glycymerididae, and Psammobiidae. Although
there are surely more bivalves with this trait, the occur-
rence and addition of riblets does not appear to be wide-
spread; yet this characteristic is fairly common in the
scallops and limids.

The combination of the correlation of optimal riblet
spacing with previously observed swimming capability
and the maintenance of riblet spacing by intercalation
implies that the apparent fine-tuning of riblet spacing on
P. magellanicus is functionally significant.

Acknowledgments

This research was funded by an NSERC (Canada) re-
search grant to M.E.D. P.S.M. was supported by an
NSERC Summer Research Scholarship. We thank R.
Antonia (The University of Newcastle) for his valuable
comments and suggestions throughout this study, A. An-
derson (St. F. X.) for the use of his image analysis equip-
ment, the Nova Scotia Museum of Natural History for
opening their bivalve collection to us, E. Kechington at
the Department of Fisheries and Oceans for sending
specimens, and collector M. Le Quement for his contri-

bution of bivalves from Brittany. We thank also J. Flynn
(Dalhousie), W. Quinn (St. F.X.) and S. Vogel (Duke)
for reading the manuscript.

Literature Cited

1 . Walsh, M. J., and I,. M. VVeinstcin. 1978. Drag and heat transfer
on surfaces with longitudinal fins. AIAA Paper 78-1 161.

2. Bushnell, D. M., and K. J. Moore. 1991. Drag reduction in na-
ture. Annu Rev l-liiid Mccli 23: 65-74.

3. Moin, P., and J. Kim. 1997. Tackling turbulence with supercom-
puters. Sci. Am. 276(1): 62-68.

4. Mullins,J. 1 997. Secrets of a perfect skin. NewSci. 153(2065): 28-
31.

5. Dadswell, M. J., and D. VVeihs. 1990. Size-related hydrodynamic
characteristics of the giant scallop Placopecten magcllaniais (Bi-
valvia: Pectinidae). Can J Zool. 68: 778-785.

6. Cheng, J.-V., and M. E. DeMont. 1996. Hydrodynamics of scallop
locomotion: unsteady fluid forces on clapping shells. / Fluid
Mciii. 317: 73-90.

7. Cheng, J.-Y., and M. E. DeMont. 1996. Jet-propelled swimming
in scallops: swimming mechanics and ontogenic scaling. Can J
Zoo/. 74: 1734-1748.

8. Cheng, J.-Y., I. G. Davison, and M. E. DeMont. 1996. Dynamics
and energetics of scallop locomotion. J. Exp. Bial 199: 1931-
1946.

9. Vogel, S. 1997. Squirt smugly, scallop. Nature 385: 2 1 -22.

10. Reif, \\.-E. 1982. Morphology and hydrodynamic effects of the
scales of fast swimming sharks. Nenes. Jahrb. Geol. Paleontol 164:
184-187.

I 1. Bechert, D. W ., G. IIoppc, and \\.-E. Reif. 1985. On the drag re-
duction of shark skin. AIAA Paper 85-0546.

12 Kline, S. J., VV. C. Reynolds, F. A. Schraub, and P. W. Runstadler.
1967. The structure of turbulent boundary layers. / Fluid Mech.
30:741-773.

I 3. Djenidi, L., and R. A. Antonia. 1993. Riblet flow calculation with a
low Reynolds number k-t model. Appl. Sci. Res. 50: 267-282.

14. Walsh, M. J. 1982. Turbulent boundary layer drag reduction us-
ing riblets. . 1 1. 1 A Paper 82-0 1 69.

Reference: Biol. Bull 192: 345-363. (June. 1997)

Embryogenesis in Hydra

VICKI J. MARTIN1, C. LYNNE LITTLEFIELD234, WILLIAM E. ARCHER1,

AND HANS R. BODE: '

1 Department of Biological Sciences, University of Notre Dame. Notre Dame. Indiana 46556;

^-Developmental Biology Center and Department of Developmental and Cell Biology. University of

California at Irvine. Irvine. California 92 71 7; ami * Department of Cell and Molecular Biology.

Tidane University. New Orleans. Louisiana 701 18

Abstract. Embryogenesis in hydra includes a variable
period of dormancy; and this period, as well as subse-
quent stages through hatching, takes place within a
thick cuticle that hinders observation. Thus, although
the early stages of development have been well-charac-
terized qualitatively, the middle and later stages are
only poorly understood. Here, we provide a detailed de-
scription of the stages of embryogenesis, including the
time required to traverse each of the stages, and the
changes that occur in the type and number of cells
throughout the stages. The events of cleavage and gas-
trulation occur within the first 48 h. Cleavage is holo-
blastic and unipolar and leads to a single-layered coe-
loblastula. Gastrulation occurs by ingression and is fol-
lowed by the deposition of the thick cuticle. Thereafter,
during the variable period of dormancy ranging from
2-24 weeks, little occurs: the important events are the
conversion of the outer layer into an ectoderm and the
appearance of the interstitial cell lineage. During the
last 2 days before hatching, the endoderm and gastric
cavity form, while stem cells of the interstitial cell lin-
eage proliferate and differentiate into neurons, nemato-
cytes. and secretory cells. Finally, the cuticle cracks, and
the hatchling enlarges and emerges from the cuticle as a
functional animal. The formation of the gastric cavity
and the hatching of the embryo are both explicable in
terms of the osmotic behavior of the animal and the hy-
drostatic forces generated by this behavior. Characteris-
tics of development that are common to hydra and trip-
loblastic phyla are presented.

Received 1 5 November 1996: accepted 26 March 1997.

Introduction

Hydra, like all cnidarians, arose very early in evolu-
tion. Its diploblastic body plan is therefore simple, as is
its embryogenesis. A comparison of this simple embryo-
genesis with those of the more complex triploblastic
metazoans would reveal those features that are common
to all animals. And a further investigation of those com-
mon features should then uncover basic genetic mecha-
nisms underlying development. The Hox genes, for ex-
ample, play an important role in specifying morphologi-
cal regions in insects and vertebrates — and these genes
also occur in cnidarians (Schierwater et a/.. 1991: Mur-
thaetal., 1991;SchummertV«/., 1992); C'/;av-2 is a Hox
gene that is involved in maintaining the adult pattern of
hydra (Shenk et ai. 1993a, b). Identifying these regula-
tory genes and understanding their roles in development
will depend on a detailed knowledge and understanding
of hydra embryogenesis.

The epithelial cells in the adult hydra are constantly
proliferating, changing their relative position along the
body axis, and differentiating. Thus the patterning pro-
cesses that maintain the form of the animal are continu-
ously active in the adult as well as the embryo (Campbell,
1967a, b; Otto and Campbell. 1977). The effects of the
patterning processes on cells and tissues are well un-
derstood, and current efforts are focused on understand-
ing their molecular bases. The processes that set up the
pattern in the embryo could be the same, overlap with,
or differ from those maintaining pattern in the adult. Re-
solving this uncertainty in hydra will require a precise
description of embryogenesis.

After more than a century of study, the early stages
of hydra embryogenesis — cleavage through cuticle

345

346

V. J. MARTIN KT AL

formation — have been well described qualitatively
(Kleinenberg, 1872: Brauer. 1891; Tannreuther. 1908;
Kanev, 1952; Tardent, 1966). But the stages have not
been timed, and neither the rates of cell division nor the
number of cells in each stage have been measured. In
contrast to the early stages of embryogenesis, the later
ones are poorly understood; a single report (Brauer.
1 89 1 ) provides a rough outline of the events, but detail is
lacking. This lack of information is due to four factors.
First, many strains of hydra species do not produce ga-
metes under culture conditions in the laboratory. Sec-
ond, most cultured strains and species also have low
hatching rates. Third, the early and late stages of embry-
ogenesis are separated by a period of dormancy that is
extremely variable (2-52 weeks). Finally, a thick cuticle
covers the embryo through most of embryogenesis: thus
development is difficult to examine during the dormant
period or during the final stages leading to hatching.

We have taken advantage of two strains, one female
and one male, that produce gametes continuously in the
laboratory. Although the hatching time remains quite
variable, the number of embryos produced is high and
the hatching rate is close to 100%. We could therefore
describe the stages of development in detail; we could
also time them and quantify the cell populations during
embryogenesis.

Materials and Methods

Culture of adult hydra and embryos

Most analyses were carried out with two strains. One
was PA 1 , a female strain of Hydra vulgaris, isolated from
a pond on the Haverford College campus near Philadel-
phia, Pennsylvania, by Dr. Carolyn Teragawa. The
other, CA7, a male strain of Hydra vulgaris, was isolated
by Drs. Lynne Littlefield and Carolyn Teragawa at Boul-
der Creek, near Susanville, California. Both strains were
maintained in the laboratory for 3 years before the cur-
rent studies were undertaken. Some analyses were car-
ried out with the male E2 and female A5 strains, which
were derived from crosses of PA 1 and CA7. All strains
were cultured in hydra medium, which consisted of a
commercial spring water (Arrowhead) supplemented
with 1 mM CaCN. Animals were maintained at 15°C
with a light/dark regime of 12 hours light and 12 hours
dark; they were fed once a week with the nauplii of the
brine shrimp. Anemia salinas (San Francisco Brand),
and the medium was changed three times per week. Un-
der these culture conditions, the males produced sperm
most of the time. Individual females produced one-to-
several eggs at roughly monthly intervals, and often a
culture of females produced eggs simultaneously.

To obtain fertilized eggs, six male hydra bearing testes
and 50 female hydra bearing eggs were cultured together

in 100 ml of hydra medium. The males were removed
weekly and replaced with new males bearing testes; 50-
100 fertilized eggs, identified by the initiation of cleav-
age, were harvested. In most cases, the strands attaching
the embryo to the parent were cut with a surgical knife
and the detached egg was transferred to a petri dish
(60 mm X 15 mm) containing hydra medium. In other
cases, females carrying fertilized eggs were transferred to
a fresh dish containing hydra medium, and the embryos
were allowed to develop while still attached. Cultures of
20 embryos or 20 females bearing embryos were main-
tained at 15°C, and the medium was replaced daily. Em-
bryos were cultured until they hatched, unless used for
experiments at some point during embryogenesis.

Analysis of embryonic stages

} 'ideo microscopy. Progress through the stages of
embryogenesis was monitored on live embryos in two
ways. For the early stages of development, embryos from
the fertilized egg to the early cuticle stages were continu-
ously monitored with a time-lapse video recorder (JVC
BR-9000U) mounted on a dissecting microscope (Olym-
pus SZH) and attached to a Sony Trinitron color moni-
tor (model KX-2501). One frame per second was re-
corded over 3 days. Second, the entire process of embry-
ogenesis, from fertilized egg to hatching, was monitored
with an optical disc recorder (Panasonic) attached to a
Wild dissecting microscope and a Sony color monitor.
One frame per 1 5 seconds was recorded over 30 days.
The resultant time-lapse films were viewed and analyzed
at playback speeds 10-100 times normal speed.

Histology. At various stages from early cleavage to
hatchlings. embryos or young animals were fixed and
sectioned to examine their histological structure. The
embryos were fixed in Lavdowsky's fixative (ethanol: for-
malin: acetic acid: water in the ratios of 50:10:4:40), ei-
ther for 60 min at room temperature or, for embryos
with a cuticle, overnight at 4°C. Embryos at the last
stage — bilayer formation, which occurs 2 days before
hatching — and young hatchlings were relaxed in 2% ure-
thane in hydra medium for 30 s prior to fixation. Fixed
embryos and hatchlings were dehydrated through an as-
cending alcohol series (25%- 100% ethanol), rinsed for
20 min in a 1:1 mixture of 100% ethanol and tertiary bu-
tyl alcohol, incubated overnight in tertiary butyl alcohol,
and then infiltrated and embedded in Paraplast Plus par-
affin (Fisher Scientific). Serial sections (lO^m) of em-
bryos and hatchlings were prepared, mounted on glass
slides, and stained with Scruffs reagent, toluidine blue,
or hematoxylin and eosin. The stained sections were ob-
served and photographed with a Zeiss compound micro-
scope.

Scanning electron microscopy. Relaxed hatchlings and

HYDRA t MBRYOGENESIS

347

embryos at various stages were fixed for 90 min with glu-
taraldehyde (2.5fV in 10mA/ Millonig's phosphate
butter, pH 7.2). and then rinsed three times for 15 min
each in 10 mA/ Millonig's butter. While in butter, some
samples were cut transversely with a straightedged razor
blade. Samples were postfixed for 60 min in 2% osmium
tetroxide in 10 mM Millonig's butter, and then rinsed
three times in 10mA/ Millonig's butter. Subsequently,
they were dehydrated through a graded series of ethanols
to 100%. critical-point dried with CO:, mounted on
metal stubs, and sputter-coated with gold-palladium for
1 min in a Denton sputter-coaler. Samples were exam-
ined and photographed with a scanning electron micro-
scope (JEOL JSM T-300) operated at 25 kV.

Quantification ot cell numbers and types. Individual
embryos were submerged in 50 n\ of maceration fluid
(acetic acid:glycerol:water in the ratios of 1 : 1 :26: David,
1973) and maintained at 5°C for 2-3 days. For eggs and
embryos up to the cuticle stage, the cell suspension was
gently agitated with a stream of air blown through a mi-
crocapillary pipette. The cells dispersed in this way were
postfixed with 8% formaldehyde and transferred to a gel-
atin-coated slide. A piece of fishline coated with 1%
Tween 80 was used to spread the cells evenly over a 1 X
1 -2 crrr area of the slide. For later stages (post-cuticle
formation), individual embryos were placed on a gelatin-
coated slide in a drop of maceration fluid, and the cuticle
was removed with sharpened forceps. This often caused
some of the larger embryonic cells to be sheared as they
were extruded through the opening in the cuticle layer.
Nevertheless, the cells were postfixed with formaldehyde
and spread on a gelatin-coated slide as described above.
The cells from early and late stages were dried on a slide
warmer (40°C) overnight.

To aid in distinguishing embryonic cells (or the nuclei
released when cells were sheared) from the nurse cells
that are present in eggs and embryos at all stages through
to hatching, macerated cells were stained with a 2.5 /*g/
ml solution of DAPI (4,6 diamidino-2-phenylindole-
2HCI: Accurate Scientific and Chemical Corp.) in
10mA/ MgCl2/McIlvaine buffer (18mA/ citric acid,
164 mM Na:PO4, pH 7.0) for 30 min and rinsed in
10 mM MgCU/McIlvaine buffer. The cells were exam-
ined microscopically at 375X and classified using termi-
nology established for cell types of adult hydra (David,
1973; Campbell and Bode, 1983). Classification criteria
were based on nuclear morphology (examined with epi-
fluorescence) for cells at early stages of embryogenesis
and on both nuclear and cell morphology (viewed with
phase optics) for cells at later stages.

Hydroxyurea treatment

To block cell division during gastrulation. blastulae
were exposed to 10 mA/hydroxyurea(HU) in hydra me-

dium for 6- 1 2 h at 1 5°C. Thereafter, the embryos were
washed three times in hydra medium and allowed to de-
velop in hydra medium until hatching. Some embryos
(controls and treated) were fixed and processed for DAPI
staining to determine if the treatment blocked cell divi-
sion during the time of treatment. In detail, embryos
were fixed in Lavdowsky's fixative for 60 min, rinsed in
hydra medium, incubated for 5 min in DAPI, washed
three times for 10 min each in phosphate buffered saline
(PBS), mounted in glycerin:PBS (3:1), and examined
with epifluorescence. The number of nuclei per embryo
was scored. Some HU-treated embryos were processed
for histology immediately after gastrulation as described
above.

Immunocytochemical detection ofmesoglea and
interstitial cells

Hydra embryos (all stages of development) and
hatchlings were examined for the presence of interstitial
cells and mesoglea. The monoclonal antibody MG52
(kindly provided by Michael Sarras), which recognizes
laminin (Sarras el al. , 1 99 1 ), was used to detect the pres-
ence ofmesoglea. CP4, a monoclonal antibody that rec-
ognizes cells of the interstitial cell lineage (Javois and
Bode, unpubl. data), was used to identify cell types of
this lineage. Indirect immunofluorescence was used for
visualization.

The procedure used for immunocytochemistry was a
modification of the one described by Dunne el al. ( 1985).
Animals were fixed for 60 min in Lavdowsky's fixative,
washed three times for 15 min each in PBT (PBS con-
taining 0.25% Triton-X), and incubated overnight at 4°C
in blocking serum (PBS containing 10% neonatal calf se-
rum [Irvine Scientific] and 0. 1% sodium azide). Thereaf-
ter, antibody diluted in PBS ( 1 : 100 dilution of CP4 asci-
tes fluid or a 1 :20 dilution of MG52 tissue culture super-
natant) was added to the samples, and they were
incubated overnight at 4°C. Subsequently, samples were
washed two times for 10 min each in PBT followed by a
30-min rinse in PBT. Then, samples were incubated in a
1:50 dilution of FITC-conjugated goat anti-mouse Ig's
(Boehringer-Mannheim) in blocking serum for 30 min
in the dark at 22°C. Samples were washed three times for
10 min each in PBT and counterstained in 0.01% Evans
blue in PBT for 10 min. Finally, animals were washed
ten times for 2 min each in PBT, rinsed twice in PBS.
and mounted on slides in 3:1 glycerin:PBS containing
0.5% 77-propyl gallate. and examined with epifluores-
cence.

Results
Aspects ofoogenesis

In hydra, an egg forms in the following manner (Hon-
egger, 1981; Honegger et al.. 1989; Littlefield. 1994).

348

V. J. MARTIN ET AL.

Stem cells located in the ectoderm, whose only differen-
tiation products are oocytes and nurse cells, continu-
ously enter the gamete differentiation pathway. Under
the appropriate environmental conditions they complete
traversal of the pathway, and the products, which have
the morphology oflarge interstitial cells, accumulate in
the ectoderm. One of these interstitial cells forms an oo-
cyte that increases in mass by engulfing or fusing with
large numbers of the remaining interstitial cells, referred
to as nurse cells. As the oocyte grows in mass, it distends
and eventually ruptures the ectoderm (Fig. 1 ). Thereaf-
ter, the ectoderm recedes around the edge of the egg to
form the egg cup. a raised ring of tissue at the base of the
egg (Fig. 2).

Once an egg is exposed to the medium, meiosis and
fertilization occur. The egg nucleus, which is located at
the distal end of the cell, the point furthest from the body
column of the parent, undergoes meiosis I and II, result-
ing in the formation of three polar bodies (Honegger,
198 1 ). Thereafter, in the female strain used here, the egg
has to be fertilized within 2 h for normal embryogenesis
to occur. After 2 h the addition of fresh sperm did not
result in the initiation of cleavage divisions. Instead the
egg began to swell and eventually disintegrated.

The nurse cells engulfed by the developing oocyte dur-
ing oogenesis remain in the egg throughout embryogen-
esis as spherical, refractile cells with pycnotic nuclei
(Zihler, 1972; Honegger el a/.. 1989) (see Fig. 11). They
are located within the cytoplasm of all cells until cuticle
formation; thereafter, they persist in many of the epithe-
lial cells until hatching. They occur in large numbers,
ranging from 2000-9000 per embryo, with an average of
4500.

Stages of embryogenesis

1 Cleavage. Fertilization occurs at the distal end of
the egg, which is the end farthest from the adult body
column. The distal end is also the future head of the
animal. This was established by marking the distal end
with a vital dye and finding that the head was stained
in the resulting hatchling. Cleavage in hydra embryos
is holoblastic and unipolar; that is, the cleavage furrow
progresses inward from one side of each cell. The first
meridional division is initiated at the distal end of the
embryo and moves in a distal-proximal direction per-
pendicular to the axis of the parent (Figs. 3 and 4).
The second cleavage division, which begins shortly
fore the first is complete, also occurs in a distal-prox-
rection and is perpendicular to the first one.
i visions result in four equal-sized blasto-
As the first cleavage furrow bisects the
egg, small n ': rovilli, or filopodia, form in the region
imn;c i i *ehind the leading edge of the furrow

(Fig. 4). These microvilli, which also occur in the next
cleavage divisions, appear to hold the forming blasto-
meres together. Occasionally the blastomeres separate
at the two-cell stage, or less frequently at the four-cell
stage, resulting in the development of two or four em-
bryos in a single egg cup.

The third division is equatorial, starting at the side of
the embryo closest to the head of the adult. The furrow
occurs somewhat closer to the distal end of the embryo,
resulting in two tiers of unequal-sized blastomeres (Fig.
6). Because the furrow starts at one end, the division of
the four blastomeres is asynchronous. Thereafter, the
cleavage divisions are very irregular. They occur asyn-
chronously and the division planes are often oblique,
yielding unequal-sized blastomeres. Cells near the distal
end are generally smaller than those at the proximal end
(Fig. 7).

By the fourth to fifth cleavage division, a cavity begins
to form in the interior of the cell mass (Fig. 1 1 ), while the
surface remains uneven (Fig. 8). Over the next 2 h the
cavity enlarges to its final size. The shape of the embryo
smooths out into a spherical shell (Fig. 9), which is orga-
nized in a single cell layer surrounding the blastocoel
(Fig. 10). At this point, the embryo has reached the
blastula stage and consists of 64- 128 cells (ave. = 76
± 47 cells. Table I). Each cell is filled with about 40 nurse
cells (Fig. 1 1 ). Since each cell division lasts about 60 min,
the blastula stage is reached within 6 to 8 h of fertiliza-
tion.

2. Gastrulation. Gastrulation occurs by ingression.
Shortly after the blastula is formed, individual cells begin
elongating and extending their basal surfaces into the
blastocoel (Figs. 15 and 16). Simultaneously, the lateral
contacts with neighboring cells are reduced and eventu-
ally severed, resulting in these blastomeres moving into
the blastocoel. The process of ingression occurs in about
2 to 4 h; when it is completed, the embryo consists of
an outer layer of columnar cells and a central mass of
unorganized spherical cells (Figs. 12, 17, 18). There are
nurse cells in all blastomeres, although many more in the
cells of the inner cell mass (Fig. 12). No cavity remains,
and the embryo is somewhat compacted.

Ingression begins at the distal end and proceeds in a
wave traveling across the embryo in a proximal direc-
tion. It is accompanied by a transient inward distortion
of the surface of the blastula as cells ingress. This distor-
tion apparently involves considerable force — an embryo
detached from its parent will actually roll over the sur-
face of the culture dish as ingression takes place.

During gastrulation, the number of cells increases
roughly fourfold, from 76 ± 47 to 315 ± 200 (Table I).
The fact that cell division occurs while cells are accumu-
lating in the blastocoel suggests delamination as an al-
ternative explanation for generating cells in the cavity. If

HYDRA I MBRYOGENESIS

349

Figure 1. Hydra forming an egg. As the egg increases in size, it distends the overlying ectoderm of the
parent. The ectoderm has not yet ruptured. Bar = 100 /im.

Figure 2. Hydra egg that has broken through the ectoderm. This developing egg is loosely attached to
the parent at the egg cup (EC|. Bar = 100 ^m.

Figure 3. Hydra egg undergoing first cleavage. Note the distinctive heart shape created by unilateral
furrowing. EC. egg cup. Bar = 100 ^m.

Figure 4. Two-cell stage. Microvilli (arrow) form in the region of the cleavage furrow and may help
hold the blastomeres together. Bar = 100 ^m.

the plane of cell division were parallel to the surface of
the embryo, cell division would result in one daughter
remaining at the surface while the other would be in the
interior. To distinguish between delamination and in-
gression, embryos were treated with hydroxyurea (HU)

for 6 h covering the period of gastrulation. HU blocks
cell division in hydra (Bode. 1983). In HU-treated em-
bryos, gastrulation, the subsequent development of the
embryo, and the resulting hatch I ings were all normal.
The only differences were two. One was that the cells of

350

V. J MARTIN ET AL.

FigurtS. Four-cell embryo. Bar = 1 00 urn.

Figure 6. Forming eighl-cell embryo of hydra showing two unequal-sized tiers of cells. Bar = 100 ^m.

Figure 7. Mid-cleavage embryo. Note the unequal size of the blastomeres. Cells at the distal end are
smaller than those at the proximal end. Bar = 100 jim.

FigureS. Late cleavage stage. The cells are more uniform in size and resemble a morula. Bar = lOO^m.

Figure 9. Coeloblastula. Note the microvilli (arrows) that delineate the margins of the cells. Bar =
100 urn.

Figure 10. Cross-section of the coeloblastula. Note the single layer of cells surrounding the blastocoel.
These cells contain many nurse cells (arrows). Bar = 100 ^m.

treated animals were somewhat larger
i 'compare Figs. 12 and 13), as would be

expec ision had been interrupted. The other

was that the i imbei of cells did not increase during gas-

trulation in the treated animals. Thus, ingression of cells,
not delamination, is the most likely explanation for the
origin of the interior cells.

Gastrulation is complete by 8 to 12 h postfertilization.

HYDRA F.MBRYOGENESIS

351

13

Figure 11. Histological section of the morula. Note the forming
hlastocoel (B) and the darkly stained nurse cells (arrows) within the
hlastomeres. Bar = 100 ^m.

Figure 1 2. Gastrula of control embryo. A central mass of cells filled
with darkly stained nurse cells (arrows) is surrounded b\ an outer epi-
thelial layer(E). Bar = 100 i/m.

Figure 13. Gastrula of hydroxyurea-treated embryo. Note the large
cells (arrows) filled with nurse cells. Bar = 100 i^m.

Figure 14. Cuticle deposition in a control embryo. Cells of the
outer layer exhibit filopodia (arrows) extended into the outside me-
dium. Cuticle material is deposited around the filopodia. producing
spines. Bar = 100 ^m.

or about 2 to 4 hours after the blastula is formed. There-
after, little activity is visible for the next 10- 12 h.

3. Cuticle format ion. The next stage involves the for-
mation of the cuticle, a thick protective outer layer that
is also commonly referred to as the embryotheca. About
20 h after fertilization, the cells of the outer layer begin
to extend filopodia into the surrounding medium (Fig.
14). Cuticular material is deposited in layers around the
filopodia and on the apical surfaces of the cells (Fig. 19).
Three hours later the material has built up to the point
that spines are visible. Over the next 18-24 h, material
is continually deposited, producing a multilayered struc-

ture over the surface of the embryo (Figs. 20 and 21 ) with
ornate spines 25-50 ^m long (Fig. 22) forming where fi-
lopodia were located. Towards the end of this period, the
filopodia are retracted and the resulting channels filled
with cuticular material. The process is complete 40-48 h
postfertilization.

Cuticle deposition commonly starts on the proximal
side in the egg cup and slowly progresses around the
embryo in a distal direction. In general, the final distri-
bution of material is somewhat asymmetric, with the cu-
ticle being thickest (~60 ^m) at the proximal end and
thinnest (~45 ^m) at the distal end. Once this process
is complete, a second, very thin, membranous layer is
deposited by the outer cell layer beneath the cuticle (Fig.
24). Thereafter, the embryo detaches from the parent,
although detachment occurs occasionally at any point
from the two-cell stage onward. The time of detachment
has no bearing on the progress of embryogenesis.

4. Cuticle stage. Unlike the early and late stages of
embryogenesis. the middle stage is of undefined length,
ranging in the laboratory from 2-24 weeks. It is thought
to correspond to an overwintering period of the embryo
in nature. Two important events occur during this stage:
the cells of the outer layer acquire characteristics of the
epithelial cells of the adult ectoderm: and the cells of the
interstitial cell lineage first appear.

Shortly after cuticle formation is complete, the outer
layer is observed to consist primarily of smaller squa-
mous epithelial cells, most of which are devoid of nurse
cells (Figs. 23 and 24). In contrast, the interior cells are
irregular in shape and all contain nurse cells (Fig. 25).
A change in nuclear morphology is correlated with the
appearance of these smaller epithelial cells. In macerates.

Table I

Increase in number »l hlu.\i«mcn'x and epithelial ccllx iltrtntgli
embryogenesis

Number of

Time after

epithelial cells'

Stage

fertilization

(± 1 SD)

Blastula

6-8 h

76 ±47

Gastrula

8-12h

315 + 200

Cuticle formation

20 h

400 ± 200

Cuticle

40 h

370+ 120

Cuticle

6d

440 ± 1 60

Cuticle

1 1 d

420 ± 200

Bilayer

3000+ 100

Hatchling

2830 ± 1080

1 Through 144 h. the cells are blastomeres and all are included.
Thereafter, epithelial cells are counted, but cells of the interstitial cell
lineage are not. Sample size = 5-13.

: Bilayer embryos are analyzed 2 days before hatching. Bilayer and
hatchling data are from embryos with a diameter of 400 ^m.

352

V. J. MARTIN KT .11.

Figure 15. Cross-section ot'a hydra hlastula showing cells (arrows) ingressing into the hlastocoel from
all sides. Bar = 100 ^m.

Figure 16. Cells ingressing into the hlastocoel during gastrulation (enlargement ofFig. 15). Individual cells
extending filopodia (arrows) detach from their neighbors, move into the blaslocoel. and fill it. Bar = 100 ^m.

Figure 17. Cross-section of a control hydra gastrula. The outer layer of cells (E) will give rise to the
epidermis of the adult polyp, while some of the cells of the inner mass (EN) will contribute to the gastro-
dermis of the adult. Nurse cells (arrows) are abundant in the inner layer of cells. Bar = 100 jim.

Figure 18. Cross-section of a control gastrula. Note the outer layer of columnar-shaped cells (E) that
surround the central spherical cells (EN). A mesoglea is not detected between the outer and inner cells. Bar
= 10 jim.

of blastomeres of the gastrula and early cuticle

rge and amorphous. By 6 days postfertiliza-

tion i i. after cuticle formation is complete, some

of the cells are much smaller and have nuclei that resem-
ble those of the epithelial cells of adults. They have a
prominent nucleolus and a clear cytoplasm. Thus, by

HYDRA KMBRYOGENESIS

353

Figure 19. Early cuticle stage. Cuticular material (arrows) is deposited on the apical surfaces of the cells
and around filopodia that project from the outer cells. Bar = 10 ^m.

Figure 20. Hydra embryo with fully developed cuticle. The cuticle is adorned with thick spines (ar-
rows). Bar = 1 00 jim.

Figure 21. Cross-section of a cuticulated embryo. A thick cuticle is tightly attached to an outer thin
layer of squamous cells (not visible here). The interior of the embryo contains spherical cells rich in nurse
cells (arrows). Bar = 100 ^m.

Figure 22. Enlargement of cuticle spines (S) and nurse cells (arrows) of a cuticulated embryo. Bar =

this stage the outer layer becomes morphologically sim-
ilar to the epithelium of the adult ectoderm.

The second event is the appearance of interstitial cells.
All nonepithelial cells in hydra are part of the interstitial

cell lineage. It consists of a population of interstitial cells,
some of which are multipotent stem cells, and four
classes of differentiation products: neurons, nemato-
cytes, secretory cells, and gametes (Bode, 1996). During

354

V. J. MARTIN ET AL.

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Figure 23. Early cuticle stage. The outer layer of lightly staining
cells (E) is flattened against the cuticle (C). Nurse cells (darkly staining)
fill the central mass of cells. Bar = 50 /im.

Figure 24. Mid-cuticle stage. Note the flattened squamous-like cells
of the outer layer (E) and the inner membranous layer (arrow) of the
cuticle. Bar = 10 ^m.

Figure 25. Central mass of cells, containing nurse cells, of a cuticle
stage embryo. Bar = 10 jim.

Figure 26. Young interstitial cells (arrows) among cells of the outer
layer. Bar = 10 jim.

the first 6 days of embryogenesis, the morphology of the
cells is either large with amorphous nuclei or epithelial in
character. By 1 1 days postfertilization, however, another
distinct change has occurred in a subpopulation of the
cells. Much smaller cells with a morphology of the large
and small interstitial cells of the adult have appeared
(Fig. 26). Most of these cells are found among the epithe-
lial cells of the thin outer layer (Fig. 26). and some are
also observed in the inner mass of cells. As shown in Ta-
ble II, most of these cells are large interstitial cells.

ver formation. During this last stage of embry-
ogt- 1 AO epithelial layers separated by a basement

membrane are formed; these closely resemble the cell
layers of the adult animal. Because the cuticle stage is of

variable length, the timing of the onset of bilayer forma-
tion is not easy to predict. However, the fact that an
embryo is in the final stage is signaled by a clear morpho-
logical change that occurs 2 days before hatching. In gen-
eral, the embryo is opaque due to the refractility of the
several thousand nurse cells in the cytoplasm of the em-
bryonic cells. Two days before hatching the outer edge of
the embryo becomes translucent. The thin outer layer
of squamous cells devoid of nurse cells becomes thicker,
consisting of columnar epithelial cells and large numbers
of interstitial cells (Figs. 27 and 28). Between the 1 1-day
postfertilization stage and the bilayer stage, the number
of interstitial cells increases markedly (Table III). When
sections are treated with the antibody CP4, which spe-
cifically recognizes large interstitial cells, the majority of
stained cells appear in the outer layer (Fig. 29). In addi-
tion, the small interstitial cells (cell types derived from
the large interstitial cells) appear in substantial numbers
(Tables II and III). Small numbers of neurons and nests
of nematoblasts are also observed. At this point, the layer
closely resembles the ectoderm of the adult.

The formation of the ectoderm must precede the for-
mation of the endoderm because the outer layer is clearly
defined 2 days before hatching, while the cells in the in-
terior remain in an unorganized mass (Fig. 27). Once the
ectoderm has formed, some of the cells of the interior
mass line up along the ectoderm and change in shape
from spherical to columnar (Fig. 30). This alignment oc-
curs simultaneously in different parts of the embryo, and
the enlarging aligned regions of the developing endo-
derm fuse into a complete spherical layer. As this occurs,
spaces appear among the cells within the interior cell
mass (Figs. 28 and 30). These spaces enlarge and coalesce
with time, thereby forming the gastric cavity. When
these processes are complete, two layers have formed
(Figs. 31 and 32). Most of the cells of the inner mass have
become part of the endoderm, although some remain in
the cavity and later appear to degenerate. The cells of the
inner layer have the typical appearance of endodermal
epithelial cells of the adult in that one or more cilia pro-
trude from their surfaces into the gastric cavity (Fig. 32).

The last step is the formation of the mesoglea, the base-
ment membrane that separates the two layers in the
adult. Like the basement membranes found in higher
metazoans, this structure is mainly composed of collagen
IV, fibronectin, laminin, and heparan sulfate proteogly-
can (Sarras el al., 1991). Whole embryos at different
stages of development were stained with the monoclonal
antibody MG52, which recognizes the laminin compo-
nent of mesoglea. Embryos at the blastula, gastrula, cuti-
cle deposition, and early presumptive ectoderm stages
did not stain with the antibody, indicating that the meso-
glea had not yet been synthesized (data not shown). Em-
bryos in which both layers had formed showed staining

HYDRA EMBRYOGENESIS 355

Table 1 1
Relative increase in the population wro nt ihcci-ll iypc\ of the interstitial cell lineage during embryogenesis

Number of cells of a cell type/epithelial cell

Stage'

1 a i ye
interstitial cells

Small
interstitial cells

Nematohlasts
+ Nematocytes

Neurons

Secretory
cells

Cuticle (6 d pt")

—

—

—

—

Cuticle (lid pf )

0.33

0.06

—

—

—

Bilayer

0.61

0.40

0.023

—

0.013

Hatchling (0-5 ID

0.52

0.58

0. 1 3

0.025

0.058

Hatchlmg (2-2l)li)

0.44

0.61

0.70

0.1 1

0. 1 2

Adult:

0.64

0.70

1.32

0.25

0.27

' pf = postfertilization.

: Data from Bode i-t ai. 1973.

between the layers where the mesoglea normally forms
(Fig. 33). The endodermal layer need not be complete
for the appearance of the mesoglea. which begins to form
shortly after the endodermal cells align on the ectoderm.
With the formation of the mesoglea, the overall structure
of the embryo is complete.

6. Hatching. Once the bilayer has formed, the
embryo begins to pulsate rhythmically and continues to
do so until hatching is complete. As the embryo pul-
sates, perforations and channels appear in the cuticle,
forming a honeycomb pattern and suggesting that the
structure is beginning to break down (Fig. 34). Within
24 h of the formation of the two epithelial layers, the
cuticle cracks open on its thinnest side, the original dis-
tal side of the embryo where the head forms. To confirm
that the crack was always on the distal side, embryos
that had formed a cuticle were detached from the par-
ent, embedded in soft agar with known orientation, and
incubated in hydra medium. These embryos hatched in
the agar, and in each case the cuticle opened at the distal
end. Furthermore, the head end of the hatchling always
came out first.

Once the cuticle opens, the spherical embryo contin-
ues its pulsatile contractile activity and begins to elon-
gate and emerge from the cuticle (Fig. 35). For the next
2.5 h the periodic elongations and contractions con-
tinue, and the shape changes from a compact sphere to
an elongate cylinder 550-700 ^m in length. During the
early stages of hatching, the embryo is still enveloped in
the membrane that was laid down at the beginning of the
cuticle stage, but as the embryo enlarges, the membrane
ruptures. This results in increased activity of the hatch-
ling as it frees itself of membrane and cuticle.

The apical end of the hatchling undergoes dramatic
morphological changes during the final stages of hatch-
ing. Before the membrane ruptures, the apical end has
the shape of a smooth dome (Fig. 35). Within as short a

period as 15 min after rupture, the apical end narrows
into a conical shape, and one to five tentacle primordia
evaginate in a ring below the apical tip (Fig. 36). At this
point, the head of the hatchling is morphologically com-
plete and resembles that of a mature bud. The foot is
also completely functional. Once free of the cuticle, the
hatchling is able to stick to the surface of the petri dish.
This ability is also found in adult hydra, and indicates
the presence of functional mucous-producing cells in the
foot region.

Other signs that the hatchling is essentially fully func-
tional are apparent. The body column elongates and
contracts, indicating that the muscle processes of the ep-
ithelial cells in each of the layers are fully developed. Fur-
ther, the nerve net that coordinates the contractile activ-
ity must have formed. The hypostome, the dome above
the ring of tentacles in the head, contains the mouth. In
adults, treatment with glutathione results in opening of
the mouth, mimicking a feeding response (Lenhoff.
1983). A freshly hatched hydra treated with glutathione
opens its mouth, indicating that the mouth is fully
formed and functional. When the hypostome is touched
with a brine shrimp larva the mouth opens and the larva
is ingested, and then digested. Because the tentacles are
short upon hatching, capture of brine shrimp larvae is
difficult. But within 2 days of hatching, after the tentacles
have grown in length, the hatchling is fully capable of
capturing shrimp larvae and feeding itself. Hence, the
nematocytes necessary for the capture of the shrimp, the
mucous cells of the head, which are inferred to be in-
volved in ingestion. and the gland cells necessary for di-
gestion have all formed. All of these activities indicate
that the hatchling has a full complement of the somatic
cell types found in an adult. As shown in Table II. the cell
composition of a hatchling less than a day old is rapidly
approaching that of an adult, which confirms the infer-
ence from the behavior of the young animal.

356

V. J. MARTIN /;'/ .11

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Figure 27. Early bilayer stage. An outer layer ( E, future ectoderm )
of columnar cells and interstitial cells (arrow (surround an unorganized
mass of interior cells (EN, future endoderm). Bar = 100 ^m.

Figure 28. Forming endoderm (EN) of a bilayer embryo. As the
endodermal epithelial cells line up along the ectoderm (E). spaces (S)
appear among the interior mass of cells. Note the interstitial cells (ar-
rows) in the outer layer. Bar = 50 /im.

Figure 29. Bilayer stage stained with CP4 monoclonal antibody. A
large number of labeled interstitial cells (arrows) are seen in the outer
epithelial layer. Bar = 100 ^m.

Figure 30. Bilayer stage. An outer ectoderm (E) and inner endo-
derm (EN) are complete. Interstitial cells (arrows) are found in both
layers. Bar = 50 ^m.

Discussion

Timing ofembryological events

Because the body plan of hydra is simple, the events of
embryogenesis are few when compared to the develop-
ment of the body plans of more complex metazoans. The
early events can be summarized as shown in Figures 37-
\flcr fertilization, the cleavage divisions lead to the
n of a single-layered blastula. Gastrulation is
due to tl mgression of cells from the blastula layer into
the caviiv ,til it is filled. Thereafter, a thick protective
outer la\vi ' .uticle and a thin inner membranous layer

are laid down around the embryo (Fig. 40). During the
prolonged cuticle stage, the major events are the conver-
sion of the outer layer of blastomeres into a layer closely
resembling the adult ectoderm and the appearance of the
interstitial cell lineage (Figs. 41 -43). In the final days be-
fore hatching, the endoderm, the mesoglea separating
the two layers, and the gastric cavity form (Figs. 43
and 44).

When kept in the laboratory at a constant temperature
of 15°C, embryos of the species used here complete de-
velopment at any time from 2 weeks to 6 months. Yet,
this is not an accurate reflection of the time required to
undergo the events of embryogenesis. From fertilization
through the completion of cuticle formation requires 40
to 48 h. The time from the appearance of the translucent
stage, indicating a fairly complete ectoderm at the onset
of the bilayer stage, until hatching is also about 48 h. The
very large variable period occurs during the cuticle stage.
In its normal freshwater habitats, the embryo is inactive
during this period of embryogenesis.

There are indications that the actual time required for
embryogenesis maybe much shorter. Other authors (Ka-
naev, 1952) have noted that fertilized eggs can hatch in
13- 14 days. We found that 5% -10% of the embryos
hatched in about 2 weeks, but the percentage increased
to 40% -50% if the embryos were subjected to electro-
poration at 5 days postfertilization (P. Bode, pers. obs.).
These results suggest that, in addition to the 2 days at the
beginning and 2 at the end of embryogenesis, the cuticle
stage can be traversed in about 9 to 10 days. Because rel-
atively few embryonic events occur during this stage
compared to the beginning and end of embryogenesis, it
is unclear whether this amount of time is obligatory.

The two epithelial cell lineages form seiinentiully

The development of both epithelial lineages involves
relatively few steps. A hatchling derived from a large
embryo (~400 ^m in diameter) has about 3000 epithe-
lial cells (see Table III). Most of the cell divisions giving
rise to this population occur at the very beginning of
embryogenesis. In the approximately 10 h from the fer-
tilized egg through gastrulation, the cell number in-
creases to about 300 cells, with each of the eight to nine
cell divisions lasting about an hour. Thereafter, the rate
of cell division slows down dramatically. In the next 10 h
before cuticle formation begins, some of the cells un-
dergo another division and raise the number to about
400 cells. Since the interstitial cells most likely arise by
asymmetric cell divisions of blastomeres in the cuticle
stage, one can consider the 400 blastomeres at the begin-
ning of cuticle formation to be the direct precursors of
the 3000 epithelial cells of the hatchling. This implies
that these blastomeres, or later as epithelial cells, un-

HYDRA EMBRYOGENESIS
Table III

357

iHi during embryogenesis

Stage

Epithelial
cells

Large
interstitial cells

Small
interstitial cells

Nematoblasts
+ Nematocytes

Secretory
Neurons cells

Blastula

76

Gastrula

315

—

—

—

— —

Cuticle formation

400

—

—

—

— —

Cuticle (40 h)

370

—

—

—

— —

Cuticle (fid)

440

—

—

—

— —

Cuticle (1 1 d)

420

140

25

—

— —

Bilayer

3000

1830

1230

70

40

Hatchling(0-5 ID

2830

1470

1640

370

70 160

Hatchlmg(2-20h)

2830

1390

1730

1480

310 340

Data for epithelial cells are from Table I. The number of cells of the interstitial cell lineages are calculated from the numbers of epithelial cells
and the ratios of interstitial cell lineage cell type to epithelial cells from Table 11.

dergo only another two to three divisions. And. if the
minimum hatching time is about 13-14 days, about ten
cell divisions occurred in the first day, and the remaining
two to three occurred in the last 12-13 days.

The outer layer of blastomeres formed at gastrulation
will give rise to the ectoderm. After ingression is com-
plete, these cells still contain nurse cells, although they
are fewer and mostly located near the basal portions of
the cells (Fig. 39). In animals whose hatching time was
not artificially reduced, a distinct change occurs by
6 days postfertilization. The outer layer next to the cuti-

cle consists of a layer of squamous cells devoid of nurse
cells (Fig. 41 ). In addition, the morphology of the nuclei
of these cells changes from the amorphous nucleus of a
blastomere to the clear nucleus, characterized by a prom-
inent nucleolus, of an adult epithelial cell. This layer
most likely arose from blastomeres of the outer layer un-
dergoing a division parallel to the surface and giving rise
to the squamous cells next to the outer cuticle and
deeper, somewhat larger, cells that contain nurse cells.

The next distinct change in this layer occurs 2 days
before hatching when the ectodermal layer becomes

Figure 31. Cross-section of a bilayer stage embryo. Two distinct layers (E. EN) surrounding a gastric
cavity (G) are visible. C, cuticle. Bar = 100 ^m.

Figure 32. Bilayer stage embryo. A distinct ectoderm (E) and endoderm (EN) are separated by a meso-
glea (arrow). Nurse cells (N) are found in the cells ot the endoderm. G. gastric cavity. Bar = 10 jim.

358

V. J. MARTIN KT AL

Figure 33. Mesoglea (arrow) of the hilayer stage stained with an
anti-laminin monoclonal antibody, MG52. E, ectoderm; EN, endo-
derm. Bar = 50 ^m.

translucent. The layer becomes two to three times
thicker, with the ectodermal cells changing from squa-
mous to columnar in shape. The increased width and the
absence of the opaque nurse cells render the layer
translucent. This change may occur primarily because
of the rapid accumulation of cells of the interstitial cell
lineage among the cells of the outer layer during the bi-
layer stage. Since the surface area of this layer is constant,
the increasing mass of the interstitial cells may force the
epithelial cells to undergo the observed shape change. At
this point the outer layer is very similar to the adult ecto-
derm.

At the bilayer stage when the ectoderm is mostly com-
plete, the definitive endoderm is nonexistent. The cells
that will form the endoderm are still a disorganized mass
in the interior of the embryo. Between the bilayer stage
and hatching 2 days later, many of these cells begin to
align themselves on the overlying ectoderm, changing in
shape from roughly spherical to columnar (Figs. 43 and
44). This alignment occurs independently in several
places, with the enlarging patches merging and fusing
into a complete endodermal layer. These cells still con-
tain large numbers of nurse cells and will do so for several
days after hatching. The role of the nurse cells is unclear,
although the most probable explanation is that they pro-
vide a source of nutrients.

The last step in the development of the two epithelial
layers is the formation of the mesoglea between them
(Figs. 43 and 44). This occurs after the alignment of the
endodermal cells with the overlying ectodermal cells. Ev-
idence in adult animals indicates that both layers are in-
volved in the formation of the mesoglea ( Epp ct a/. , 1986;
Sarras ct al., 1993). Most likely the same process occurs
here.

These events lead to the formation of two layers sepa-
rated by a basement membrane. An unanswered ques-
tion concerns the changes in these epithelia that will lead
to formation of the head and foot. Shortly after hatching,
tentacles emerge from the apical dome, and upon stimu-

Figurc 3-4. Degenerating cuticle just prior to hatching. Perforations and channels (arrows) appear in
the cuticle, producing a honeycomb pattern. Bar = 10 ^m.

Figure 35. Hydra hatchlmg emerging from the cuticle. Arrow indicates a dome-shaped anterior head.
Bar = 100 jim.

Figure 36. Hydra hatch ling with four tentacle bumps. Bar = 100 jim.

HYDRA EMBRYOGENES1S

359

lation with glutathione, the mouth opens. In addition,
once free of the cuticle, the hatchling sticks to the surface,
indicating that it has a fully formed foot. When do the
epithelia undergo the changes that set up the tissue for
these morphogenetic and differentiation events?

Development of the interstitial cell lineage

All nonepithelial cells in hydra are part of the intersti-
tial cell lineage. The formation of this lineage occurs in
parallel with the development of the two epithelial layers.
Large interstitial cells are rare or absent at 6 days postfer-
tilization, but are present by 1 1 days (Tables II and III).
Since large interstitial cells are considerably smaller than
blastomeres. the simplest explanation is that some of the

%r

37 •••*•»•'

Figure 37. Coeloblastula. A single layer of cells filled with darkly
colored n urse cells (P) surround a fluid-filled blastocoel(B). N. nucleus.

Figure 38. Cells ingressing (arrow ) into the blastocoel during early
gastrulation. As cells move to the interior they elongate, detach from
their neighbors, and round up once in the filling blastocoel. N. nucleus.

Figure 39. Embryo at the completion of gastrulation. An outer
layer of columnar cells has formed; within these cells the nurse cells are
found at the basal tips. Spherical cells filled with nurse cells occupy the
center of the embryo. A blastocoel is obliterated.

Figure -40. Early cuticle stage of hydra. The cells of the outer layer
extend tiny filopodia (arrows) upon which cuticle material is deposited.
Note that the darkly colored nurse cells have accumulated in the central
cells. C. cuticle.

Figure 41 . Embryo a tew days after cuticle deposition is completed.
Note that the outer layer of cells has flattened against the cuticle (C) to
assume a squamous-like morphology. Nurse cells are virtually absent
from the outer layer. Spherical cells containing numerous nurse cells
fill the center of the embryos. The cuticle is thick and bears beautiful
geometric spines. Filopodia of the outer cells have been retracted.

Figure 42. Mid-cuticle stage of hydra. Interstitial cells (arrows) arise
in the outer layer. Cells of the outer layer have a flattened, squamous
morphology. The inner cells are spherical and filled with nurse cells: no
nurse cells remain in the ectoderm. C. cuticle.

Figure 43. Bilayer stage. The two layers, the ectoderm (E) and the
endoderm (EN), are distinct and are separated by a mesoglea (double
arrows). A forming gastric cavity (G) is present. Interstitial cells (single
arrows) are found in both germ layers. C, cuticle.

blastomeres undergo asymmetric cell divisions to gener-
ate the interstitial cells. Though such divisions were not
observed directly here, Noda and Kanai (1980) reported
them in another species of hydra, Pelmatohydra robiista.
and FennhofT(1980) described similar divisions in Tit-
bit/aria crocea, a marine relative of hydra.

Where do these asymmetric cell divisions take place?
Noda and Kanai ( 1980) placed them at the base of the
outer layer, and Fennhoff ( 1980) localized them in the
central mass of cells. In our study there is clear evidence
of large interstitial cells in the thin outer layer where the
epithelial cells are devoid of nurse cells. However, some
cells in the interior mass of cells also have the morphol-
ogy of interstitial cells. Results of the CP4 staining in bi-
layer stage embryos (Fig. 29) indicate that most of the
interstitial cells are in the ectoderm, and some are in the

360

V J. MARTIN ET AL.

Figure 44. Emerging hatchling. As the cuticle (C) breaks open, the
hatchling exits head-end first. The cells of the ectoderm (E) and endo-
derm (EN (expand, and the gastric cavity (G) increases in size. Intersti-
tial cells (single arrows) are abundant in both layers, and clusters of
nematoblasts (NB) (differentiating nematocytes) are found in the ecto-
derm.

endoderm. Since interstitial cells can migrate (Martin
and Archer, 1986; Teragawa and Bode, 1990), it is quite
plausible that they arise in both areas and that most even-
tually migrate into the ectoderm.

The development of the population of the interstitial
cell lineage can be conveniently examined in three
stages: the cuticle and bilayer stages and shortly after
hatching. The large interstitial cells, which contain the
multipotent stem cells (David and Murphy, 1977), arise
during the cuticle stage, appearing between 6 and
1 1 days postfertilization. At 1 1 days the ratio of large in-
terstitial cells to epithelial cells is 0.3: 1.0, and rises to 0.5:
1.0 by the time of hatching (Table II). In the adult, this
ratio of these two ever-growing populations is main-
tained atO. 65: 1.0(Bodet'/rt/., 1973). These results imply
that the population of large interstitial cells arises, grows,
and approximates the adult ratio of large interstitial cells
to epithelial cells during the cuticle stage. Whether the
population of large interstitial cells reaches this size pri-
marily by the division of a few interstitial cells that arise
by asymmetric divisions, or whether most arise from
asymmetric divisions is not known.

During the cuticle stage, most of the large interstitial

cells are involved in proliferation. By the bilayer stage,
however, a substantial fraction are differentiating, as
measured in terms of the small interstitial cells, which
are the dividing intermediates of the neuron and nema-
tocyte pathways (David and Gierer, 1974). Shortly after
hatching, fully differentiated neurons, nematocytes, and
secretory cells appear; within a day there are substantial
numbers of all three classes of these differentiation prod-
ucts. Since the time required for the three classes of
differentiation products to traverse the differentiation
pathways is equal to, or considerably longer than, the
length of the bilayer stage, large interstitial cells must
have begun entering the differentiation pathways late in
the cuticle stage. Within a day after hatching, when the
first wave of differentiation is complete, the ratios of the
populations of these differentiation products to epithelial
cells have reached half those found in adult animals (Ta-
ble II).

In summary, the interstitial cell lineage develops in a
straightforward manner. Large interstitial cells, perhaps
all stem cells, appear during the cuticle stage and prolif-
erate. Starting one-to-several days before the bilayer
stage, these cells enter the three differentiation pathways
in large numbers, resulting in the formation of numbers
of neurons, nematocytes, and secretory cells within a day
after hatching. Thus, this initial round of differentiation
results in a cell composition that is close to that of the
normal adult.

A mechanism for the formation of the gastric cavity and
hatching

Unexplained so far is how the gastric cavity forms, and
how an embryo emerges from the cuticle during hatch-
ing. Both events are probably part of a single process that
is related to the osmotic behavior of the animal and the
hydrostatic forces generated by this behavior.

There is a considerable difference in osmolarity be-
tween the external environment and the cytosol of the
cells of an adult hydra (Lilly, 1955). Since the two envi-
ronments are separated only by a plasma membrane, wa-
ter is continually transported from the surrounding me-
dium into, and through, the cells of the two epithelial
layers into the gastric cavity. The increasing volume of
the gastric cavity is periodically reduced as the animal
contracts, thereby expelling fluid through the mouth.

Observations of developing aggregates indicate that
both layers are necessary for this transport. When viable
cells obtained by dissociating whole hydra are centri-
fuged into a pellet, or aggregate, the aggregate will de-
velop into a normal animal (Gierer el a/.. 1 972). Initially,
ectodermal epithelial cells migrate to the surface, form-
ing an epithelium. Thereafter, endodermal epithelial
cells align themselves on the ectoderm and form patches

HYDRA L-:MBRYOGENESIS

361

of epithelium, which eventually coalesce into a complete
endoderm. Internal spaces of fluid first form directly be-
hind the patches of endodermal epithelium (Sawada.
1994), which suggests that the transport of water from
the surrounding medium into the interior requires both
the ectodermal and endodermal layers.

A similar process occurs during embryogenesis. Dur-
ing the bilayer stage, as the endodermal layer forms,
fluid-filled spaces appear among the internal cells: these
eventually coalesce into the gastric cavity. Because the
size of the cavity becomes substantially larger than the
volume of the space occupied by the interior cells at the
beginning of the bilayer stage, it is unlikely that the cavity
arises simply by the ordering of these cells into a layer.
Instead, it is more likely that the formation of the gastric
cavity and the subsequent hatching are due to the
transport of water into the internal space. This could
happen in the following manner.

By the bilayer stage, the cuticle has developed cracks.
Because the cuticle is thinnest at the distal end where the
head will form, it is likely that water seeps through the
cuticle in significant quantities in this location first, and
is transported into the interior of the embryo. That the
gastric cavity is first visible as an elipse-shaped space near
the distal end is consistent with this view. As the cuticle
weakens, increasing amounts of water are transported
into the gastric cavity from all over the surface of the
embryo. This increases the hydrostatic pressure of the
cavity, which in turn increases the pressure on the weak-
ening cuticle. Eventually the cuticle cracks at the thin-
nest, and thus, weakest, point, which is at the distal end
where the head is forming. With the embryonic surface
now in direct contact with the surrounding medium,
even more water is transported across the epithelial lay-
ers. Without the restraint of an intact cuticle at the distal
end, the ever-increasing hydrostatic pressure in the gas-
tric cavity distends the epithelial layers. Hence, the
embryo slowly emerges through the crack in the cuticle
like an inflating balloon, increasing in size as it emerges.
Once liberated, the hatchling is at least twice the size it
was just before hatching.

Another effect of water transport is taking place at the
same time. Adult ectodermal epithelial cells have vacu-
oles that make up 90% -95% of the cell volume (Camp-
bell, 1985). The size of these vacuoles is inversely corre-
lated with the ion concentration of the surrounding me-
dium (Trenkner cl ul.. 1973). Shortly before hatching,
the ectodermal epithelial cells have no, or small, vacu-
oles. As water enters the late stage embryo during pre-
hatching and hatching, the ectodermal cells begin to
form vacuoles and increase in size. The resulting increase
in the size of the ectoderm as a whole acts synergistically
with the increasing volume of the gastric cavity to force
the hatchling out of the cuticle.

Embryogenesis in hydra anddeuterostom.es shares
several characteristics

The evolutionary distance is considerable between the
diploblastic. radially symmetrical cnidaria and the trip-
loblastic bilateral phyla of both deuterostomes and pro-
tostomes. Yet, in examining the evolutionary conserva-
tion of developmental processes and mechanisms, it is
useful to compare the features of embryogenesis in hy-
dra, or cnidaria in general, with those of other phyla. A
tentative conclusion is that embryogenesis in hydra has
a number of features that are characteristic of deuteros-
tomes.

The cleavage pattern of hydra is radial for the first
three divisions, becoming irregular thereafter. Both are
characteristic of some vertebrates, such as amphibians.
The resulting hydra blastula is a spherical shell consisting
of a single layer of cells surrounding a blastocoel, as is
found in sea urchins. And, as with many deuterostomes,
hydra embryos are regulative during early cleavage
stages. The two cells resulting from the initial cleavage
sometimes separate, with each blastomere developing in-
dividually into a normal animal. Each of these embryos
is half the size of the original egg. Occasionally, the cells
resulting from the first two cleavage divisions separate to
form four embryos with one-fourth the volume of the
original egg. They too undergo normal embryogenesis
and yield normal hatchlings.

In many deuterostomes, gastrulation occurs by invo-
lution or invagination of parts of the blastula. The same
is true for some cnidarians, as many scyphozoans and
anthozoans gastrulate by invagination (Hyman, 1940;
Berrill, 1949; Chia and Spaulding. 1972). Other cnid-
arians use ingression for gastrulation; hydra clearly uses
this process (Hyman. 1940). The endoderm and meso-
derm of the chick embryo are also formed by ingression.
as are the primary mesenchyme cells of the sea urchin
embryo.

Another interesting feature is that, like vertebrates, hy-
dra has several stem cell systems that arise during embry-
ogenesis. Both the ectoderm and the endoderm of hydra
are epithelial stem cell systems; they are similar to the
epidermis and intestinal epithelium of vertebrates in that
all consist of stem cells and differentiation products.
Even more striking is the hydra interstitial cell system,
which has many similarities to the hemopoietic system
of the mouse. In both cases the cells of the tissue are mi-
gratory, and a multipotent stem cell gives rise to a num-
ber of differentiation products.

Finally, the cnidaria are usually considered to be dip-
loblastic animals with an ectoderm and an endoderm.
but no mesoderm. They may, in fact, have a primitive
mesoderm. The interstitial cell lineage is a separate lin-
eage even though it is physically intermingled with the

362

V. J. MARTIN ET AL

cells of the other two layers. Like many mesodermal tis-
sues, this interstitial c lineage is mesenchymal rather
than epithelial in oler. Moreover, in some cnid-

arians there an: interstitial cell lineage in the

mesoglea, SUR; DC beginnings of a physical sep-

aration of a " . -derm "precursor" from the other two
layers.

Conclusion

This study provides a clearer understanding of the se-
quence and timing of embryonic events in hydra, the
changes in cell population sizes, and the development
of the three cell lineages that constitute the adult ani-
mal. We are now in a better position to examine the
expression pattern of a variety of genes that have been
isolated in hydra, and thereby investigate the relation-
ship among the patterning processes that govern embry-
ogenesis, budding, and maintenance of the form of the
adult body.

Acknowledgments

The authors thank Lydia Gee for her technical assis-
tance, Jason VanLieshout for the line drawings, and Pat
Bode, Ann Grens, Andy Shenk, Robert Steele, and Hir-
oshi Shimizu for providing help and suggestions
throughout the course of these studies. We thank Mi-
chael Sarras, University of Kansas Medical Center, for
supplying the monoclonal antibody to hydra mesoglea,
and we are grateful to Beverly Marcum, California State
University at Chico, for the use of her time-lapse video
system. This work was supported in part by NIH grants
HD24511 (HRB) and HD27173 (HRB), NSF grants
DCB-8702212 (VJM), DCB-8711245 (VJM). DUE-
95521 16 (VJM), DCB-8819247 (CLL), and funds from
the Jesse Jones Foundation (VJM).

Literature Cited

Itci i ill. N. 1949. Developmental analysis of scyphomedusae. Biol

Rev 24: 343-410.
Bode, H. 1983. Reducing populations of interstitial cells and nema-

toblasts with hydroxyurea. Pp. 29 1-294 in Hydra: Research Meth-
ods. H. tendon", ed. Plenum Press, New York.
Bode, H. 1996. The interstitial cell lineage of hydra: a stem cell system

that arose early in evolution./ CcllSci. 109: 1 155-1 164.
Bode, H., S. Berking, C. David, A. Giercr, H. Schaller, and E. Trenk-

ner. 1973. Quantitative analysis of cell types during growth and

morphogenesis in hydra, ll'il/ielm Rou.\' Arch 171: 269-285.
Brauer, A. 1891. Uberdie entwicklung von hydra. Z H'iss. Zool 52:

167-216.
Campbell, R. I967a. Tissue dynamics of steady state growth in Hydra

liiiora/is: 1. Pattern of cell divisions. Dev. Bin/. 15:487-502.
Campbel; R. I967b. Tissue dynamics of steady state growth in

Hydra ',. ra/;,\ II. Patterns of tissue movement. ./ Morphol

121: 19-:,-
C'ampbell, R. r .v Tissue architecture and hydroid morphogenesis:

the role of locomotory traction in shaping the tissue. Pp. 221 -238
in The Cellular and Molecular Biology of Invertebrate Develop-
nienl. R. Sawyer and R. Showman, eds. Univ. of South Carolina
Press, Columbia.

Campbell, R., and II. Bode. 1983. Terminology for morphology and
cell types. Pp. 5- 14 in Hydra: Research Methods: H. Lenhoff. ed.
Plenum Press, New York.

Chia, F-S., and J. Spaulding. 1972. Development and juvenile
growth of the sea anemone. Tcalia crassicornis. Bio/. Bull. 142:
206-218.

David, C. 1973. A quantitative method for maceration of hydra tis-
sue. I \'ilhcl»i Rou.s 'Anh 171: 159-268.

David, C., and A. Gicrer. 1974. Cell cycle kinetics and development
of Hydra atteuuala III. Nerve and nematocyte differentiation, j.
Ce/lSci. 16:359-375.

David, C., and S. Murphy. 1977. Characterization of the interstitial
stem cells in hydra by cloning. Dev. Biol. 58: 372-383.

Dunne, J., L. Javois, L. Huang, and II. Bode. 1985. A subset of
cells in the nerve net of Hydra oligaclis defined by a monoclonal
antibody: its arrangement and development. Dev Biol 1(19:
41-53.

Epp, L., I. Smid, and P. Tardent. 1986. Synthesis of the mesoglea by
ectoderm and endoderm in reassembled hydra. J. Morphol 189:
271-279.

FennhofT, F. 1980. Embryonic development of Tiihularia crocca
Agassiz with special reference to the formation of the interstitial
cells. Pp. 127-131 in Developmental and Cellular Biology of Coe-
lenieraies. P. Tardent and R. Tardent. eds. Elsevier North-Holland.
Amsterdam.

Gierer, A., S. Berking, II. Bode, C. David, K. Flick, G. Ilansmann,
H. Schaller, and E. Trenkner. 1972. Regeneration of Hydra from
reaggregated cells. Nat. Ne» Biol. 239: 98-101.

Ilonegger, T. 1981. Light and scanning electron microscopic investi-
gation of sexual reproduction in Hydra carnea Ini .1 Inverlehr.
Rcprod Dev 3:245-255.

Ilonegger, I., D. /.urrer, and P. Tardent. 1989. Oogenesis in Hydra
carnea: a new model based on light and electron microscopic anal-
ysis of oocyte and nurse cell differentiation. Tissue Cell 21: 38 I -
393.

llyman, I.. 1940. The Invcrtchrales Protozoa through Clcnophora.
McGraw-Hill Book Company, New York.

Kanaev, I. 1952. Hydradn Russian). Izd. Akad. NaukSSR, Moscow-
Leningrad.

Kleinenberg, N. 1872. Hydra. Eine Anatomisch-entwicklungsg-
eschichtliche Untersuchung. Leipiz, pp. I -90.

I.enhofT, II. 1983. Labeling with gaseous I4CO: or by feeding on ra-
dioactive tissues. Pp. 193-196 in Hydra. Research Methods. H.
Lenhoff, ed. Plenum Press, New York.

Lilly, S. 1955. Osmoregulation and ionic regulation in hydra. J. Exp.
Biol 32:423-429.

I n i Kin Id C'. 1994. Cell-cell interactions and the control of sex deter-
mination in hydra. Scnun Dev Biol 5: 13-20.

Martin, \ '., and \\ . Archer. 1986. Migration of interstitial cells and
their derivatives in a hydrozoan planula. Dev. Biol 1 16:486-496.

Murtha, M., J. Leekman, and F. Ruddle. 1991. Detection of homeo-
box genes in evolution and development. Pnic. Nail. Acad Sci.
t'5,-188: 1071 I- 10715.

Noda, K., and C. Kanai. 1980. An ultrastructural observation on the
embryogenesis of Pelmatohydra rohusia. with special reference to
"germinal dense bodies." Pp. 127- 131 in Developmental and Cel-
lular Biology of Coelenterales, P. Tardent and R. Tardent, eds. El-
sevier North-Holland. Amsterdam.

Otto, .}., and R. Campbell. 1977. Tissue economics of hydra: regula-

HYDRA F.MBRYOGENESIS

363

lion of cell cycle, animal size and development by controlled feed-
ing rates. J Cell Sci. 28: 1 1 7 - 1 32.

Sarras, M.. M. Madden, \. /hang. S. Gunnar. .). Huff, and B. Hudson.
1991. Extracellular matrix (mesoglea) of H\'dra ri/fec/nv. I. Isola-
tion and characterization. Dcv. Bid/. 148: 48 I -444.

Sarras, M.. X. /hang. J. Huff. M. Accavilli, P. St. John, and D. Abra-
li. mis. in 1993. Extracellular matrix (mesoglea) of Hydra r;//i,'<//7\
III. Formation and function during morphogenesis of hydra cell
aggregates. De\: Biol. 157: 383-398.

Sawada, V. 1994. Regeneration of hydra from dissociated cell aggre-
gates. Proceedings of the 33rd NIBB Conference: approaches to the
cellular and molecular mechanisms of regeneration. Okazaki. Ja-
pan, p. 28.

Schierwatcr, B., M. Murtha, M. Dick, K. Ruddle, and L. Buss. 1991.
Homeoboxes in cnidarians. J. E\p /-<><>/. 260:413-416.

Schummer, M., I. Scheurlen, C. Schaller, and B. Galliot. 1992.
HOM/HOX homeoho.x genes are present in hydra (Chlorokydra

r;//<//wm<;| and are differentially expressed during regeneration.
i:\lfiOJ 11: 1815- 182V

Shenk, M., H. Bode, and R. Steele. I993a. Expression of Cnox-2. a
HOM/HOX homeohox gene in hydra, is correlated with axial pat-
tern formation. Development 1 17: 657-667.

Shenk, M., L. Gee, R. Steele, and U. Bode. 1993b. Expression of
Cno.x-2, a HOM/HOX gene, is suppressed during head formation
in hydra. Dcv Biol. 160: 108- I 18.

Tannreuther.G. 1908. The development of hydra. Biol Bull 14:261.

Tardent, P. 1966. Zur sexualhiologie von Hydra ullemuila (Pall.)
Rc\: Snixxc'/.iHil. 73(2): 357-380.

Teragawa, C., and II. Bode. 1990. Spatial and temporal patterns of
interstitial cell migration in Hydra vulgaris. Dc\'. Bid. 138: 63-81.

Irenkner, E., K. Flick, G. llansmann, 11. Bode, and P. Bode. 1973.
Studies on hydra cells in vitro. ./. li\p /<W 185:317-326.

Zihler, J. 1972. Zur gametogenese und befructungsbiologie von Hy-
dra. Wilhelm Roux" Arch. 169: 239-267.

Reference: Bin/ Bull 192: 364-374. (June, 1997)

Laboratory Culture of the Sepiolid Squid

Euprymna scolopes: A Model System

for Bacteria-Animal Symbiosis

ROGER T. HANLON1, MICHAEL F. CLAES:*. SUSAN E. ASHCRAFT1,
AND PAUL V. DUNLAP3*

1 Marine Resources Center, Marine Biological Laboratory, Woods Hole, Massachusetts 02543:

2 Marine Science Center, Northeastern University, Nahant, Massachusetts 01908; and 3 Department

of Biology. H 'oods Hole Oceanographic Institution. H 'oods Hole. Massachusetts 02543

Abstract. The small Hawaiian sepiolid Euprymna sco-
lopes, with its symbiotic luminous bacterium I'ihrio
fischeri. was cultured through one complete life cycle in
4 months. Paralarval squid hatchlings were actively
planktonic for the first 20-30 days, after which they set-
tled and assumed the typical adult mode of nocturnal
activity and diurnal quiescence. Squids were aggressive
predators that preferred actively swimming prey up to 2-
4 times their size; the only diet that yielded good survival
and rapid growth for paralarvae was large adult mysids.
Survival to settlement was 73% on this diet, whereas it
was 0%-17% on controls and three other diets. Paralar-
vae initially lacked both detectable luminescence and I '.
fischeri cells in their incipient light organs: all remaining
stages produced luminescence, and their light organs
were colonized by apparently pure cultures of >105 I'.
tischcri typical of E. scolopes symbiont strains. Survival
from settlement to sexual maturity was 76%. Mating and
egg laying commenced at 2 months, yet attempts to cul-
ture the next laboratory generation of hatchlings were
not as successful. The results indicate that the host or-
ganism of this symbiosis can soon be cultured with con-
sistency thn •'.! "h its brief life cycle, thus opening new av-
enues of rest, .: intodevelopmental aspects of this sym-
biosis.

Introduction

The symbiotic association between the Hawaiian sepi-
olid Euprymna scolopes and the bioluminescent bacte-

Received 14 November 1446; accepted 4 March 1997.
*Present address: Center for Marine Biotechnology, University of
Maryland Biotechnology Institute. Baltimore, Maryland 2 I 2(12.

rium Vibrio fischeri has been developed into an experi-
mental marine model in cell and molecular biology (Wei
and Young, 1989; McFall-Ngai and Ruby, 1991; Ruby
and McFall-Ngai. 1992). Various strains of I'ihrio
ftscheri (both natural and mutant) have been cultured
so that components of the symbiotic association can be
manipulated experimentally (e.g., Boettcher and Ruby.
1990; Ruby and Asato. 1993: Graf tv ill.. 1993; Dunlap
el til.. 1995). However, full development of this marine
model system has been hampered by the inability to cul-
ture the host organism — the squid — completely through
its life cycle with some degree of consistency and stan-
dard methodology.

Euprymna scolopes is a very small species that is en-
demic to the Hawaiian Islands, where it spends much of
its life buried in the sand. There have been no field stud-
ies of its behavior and life cycle, so most of what is known
comes from laboratory studies (Singley, 1983). Of partic-
ular interest is how these small squids might use their
relatively huge light organ in their daily lives.

Since the mid- 1 980s. several teams of researchers have
studied details of the symbiosis by bringing wild-caught
adults to the laboratory, allowing them to mate and lay
eggs, and then using the hatchling squids (i.e.. the para-
larvae) to explore the infection process as well as the ini-
tial development of the light organ. However, thus far
investigators have been unable to examine events be-
yond the first week posthatching because the squid para-
larvae deplete their internal yolk supplies by that time
and perish; several unreported rearing attempts in the
1 990s have failed. Nevertheless, before this model sys-
tem was developed, Euprymna had been reared from

364

SQUID CULTURE FOR SYMBIOSIS RESEARCH

365

eggs through a portion of its life cycle. Euprynina hcrryi
was reared to 2 months in Korea by Choe and Oshima
( 1963) and Choe ( 1966). Arnold ct til. (1972) reared 10
E. scolopes (from a starting number of 26) to 28 days,
and two survived to 202 days without reproducing. Sin-
gley (1983) reared 13 of 16 E. scolopes to 28 days and
one to 120 days, although its reported length was very
small (<4 mm mantle length). The encouraging results
of Arnold et al. ( 1 972) and Singley ( 1 983) on E. scolopes,
coupled with the rapid development of this symbiosis
model system in the last 5 years, led us to initiate the
trials reported here. The overall goal was to develop stan-
dard methods for culturing Euprynina and to learn as-
pects of its biology that would enhance its successful cul-
ture in captivity. We hypothesized that behavioral fea-
tures related to feeding would be most important to the
successful culture of Euprynina hatchlings through the
paralarval stage, as they have proved to be in many other
squid species (e.g.. Boletzky and Hanlon. 1983; Yang et
al.. 1986: Hanlon et al.. 1989; Hanlon, 1990; Lee el al..
1994).

Although we hope to eventually culture successive
generations of this small, fast-growing cephalopod, a
near-term goal is to develop a method for rearing apo-
symbiotic hatchlings (i.e.. those deprived of the bacte-
rium) through most of the life cycle, so that cellular and
molecular aspects of the complex development of the
symbiosis can be studied in even greater detail. We re-
port here our initial findings that Euprynina scolopes can
be cultured through its brief life cycle under controlled
laboratory conditions, and we highlight some of the
more important needs of this species in captivity. We
also describe our observations on some critical features
of the behavior of this species, including multiple effects
of light and some aspects of reproductive biology that
require future experimentation.

Materials and Methods

The term "paralarvae" is used to describe young
squids from hatching to about 3 weeks of age; it accu-
rately distinguishes the behavior of hatchlings from that
of juveniles and adults (see details in Young and Har-
man, 1988). Euprynina scolopes is a member of the fam-
ily Sepiolidae in the order Sepioidea (according to the
nomenclature of Voss, 1977), which includes cuttlefish.
The term "squid" is commonly applied tosomesepioids,
although true squids are members of the order Teu-
thoidea.

Brood stock acquisition and egg care

Adults were collected on Oahu, Hawaii, and shipped
via airfreight to the Marine Resources Center of the Ma-
rine Biological Laboratory in Woods Hole, Massachu-

setts. Squids were shipped individually in 3-1 plastic bags
containing 1.5 1 of natural seawater. The seawater was
filtered to 10 ^m, heavily aerated, and spiked with 1 g/1
tris buffer: the remaining 1 .5 1 of each bag was filled with
oxygen and the bags were fitted in insulated shipping
boxes. The total time spent in these bags by each squid
was typically 2 1 hours. When the bags arrived at the lab-
oratory, the temperature was slightly depressed (down to
1 8°- 1 9°C), the salinity was 31-32 ppt, the concentration
of dissolved oxygen was 7-1 1 mg/1, the pH was 8.29-
8.41, and the levels of ammonia and nitrite were low
(however, colorimetric reading were often impossible
due to interference from ink in the bags). Ammonia was
measured with LaMotte kits that are based on the salicy-
late method and colorimetric analysis. Nitrite and ni-
trate were measured with Hach kits that also used color-
imetric methods; the precision of the ammonia and ni-
trite methods is ±0.01 mg/1, and the lowest sensitivity is
0.05 mg/1.

Adult males were housed individually, and females
individually or in pairs, in small chambers (25 cm
X 33 cm; water depth 18 cm) and were fed ad libitum
with live shrimp (Palaemonetes sp. or Crangon sp.).
Each chamber had 10 cm of crushed coral sand for the
squids to bury in.

Mating was induced once per week by transferring one
male into a female's chamber overnight. To avoid dam-
aging the squid they were transported in small clear glass
vials of water rather than in nets. Transferred males al-
ways mated, and eggs were generally laid the night after
the male was removed. In the trials reported here, five
females and three males produced 13 clutches of eggs,
but we used only five clutches for the culture trials.

It was essential that eggs be handled as little as possible
so that they would develop fully and not hatch prema-
turely. The key was to keep temperature, salinity, pH,
nitrogen levels, and light cycles as steady as possible, and
to keep disturbance at a minimum (not bumping tanks,
changing light levels, inspecting eggs, etc.). Adults were
kept in closed, recirculating seawater systems to ensure
that water quality was consistent. Eggs laid on coral fin-
gers were left on the coral and moved to aerated mesh
containers. Eggs laid on the flat tank wall were gently
scraped from the wall with a glass slide; they generally
came off as a single unit, which was then transferred to a
mesh container. Eggs were never exposed to air, and an
airstone was placed into each mesh container adjacent to
the eggs so that water was constantly circulating near
them. The eggs were kept in the same water that adults
had laid them in.

Rearing chambers and seawater systems

The actual rearing chamber for each paralarval rearing
trial was a circular black container, 25 cm in diameter.

366

R. T. HANLON ET AL

Figure 1. The culture tanks for Eupryinnti .icolitpe.t. (A) Each para-
larval rearing chamber ( 1 ) held 30 squids and had a viewing port (2).
Water flowed in through two tubes (3) in each chamber and exited ver-
tically through the sand-covered sieve bottom, then flowed horizontally
through mesh screens (4) to the drain (5). The white ruler is the stan-
dard 12 inches. (B) The complete closed system and A-frame, showing
the filter apparali i he location of the biological filter substrate (2)

and the chambe: nning adults and for mating (3).

with a 200-Mm-mesh screen bottom (Fig. 1). The cham-
ber was immersed in a seawater tray tank to achieve shal-
low water depths of only 3-6 cm. Water flowed into the
top through rubber tubing and flowed out through the
mesh bottom. The two inflow tubes were arranged along
the edge to create a gentle circular water flow, which

helped keep the hatchlings and prey items away from the
sides. A thin layer of sand coated the mesh bottom.

Two small seawater systems were used in the Marine
Resources Center: one was a completely recirculating
system of 340 1, and the other was an open system of
600 1. Local Woods Hole water was the original source,
and this water was passed through a 1-^m filter and
heated to about 23°C. The closed system consisted of an
A-frame with a shallow tray tank for the rearing experi-
ments above (70 cm X 78 cm X 9 cm deep) and a deeper
tank below to house the biological filter. The biological
filter was composed of crushed oyster shell with an un-
dergravel filter; the oyster shell was 1 5 cm deep and
spread over an area of 5250 cnr. The water was then
pumped through a canister with a particulate filter and
then through activated carbon and a UV filter. The flow
rate was about 22 1/min, and about 30% of the water was
replaced weekly. The open system was constructed sim-
ilarly, with a top tray size of 76 cm X 130 cm X 9 cm
deep and a tank below with 9956 cnr crushed oyster
shell as a substitute substrate. The open system was only
used for some squids after Day 49 to help reduce crowd-
ing. Immersion heaters in each system helped maintain
temperature. Water quality was checked two or three
times per week and ammonia, nitrite, and nitrate were
determined according to the methods listed above.

The tanks were situated 3 m from a window that
provided indirect natural light. Overhead fluorescent
fixtures provided indirect light on a 12:12 light cycle.
The typical light level falling on the tray tanks was 2-
8 H'm :MA.

One small trial (/; = 8 squids) was performed in a semi-
closed seawater system at the Marine Science Center of
Northeastern University. Natural seawater was used in
an A-frame that held a total of 350 1. Crushed coral was
the biological filter, and no other UV or charcoal system
was used. Lighting was by direct overhead incandescent
bulbs (50cm away) on a 12:12 cycle. The paralarvae
were reared in a black circular PVC chamber (20 cm in
diameter, 20 cm in height) with 1 50-^m-mesh sides and
a bare plastic bottom.

For behavioral observations, the top trays were fitted
with glass panels for horizontal viewing into portions of
the round rearing chambers. Observations were made
from the side and also from the top of the tanks, and
night observations were accomplished with a night-vi-
sion device that amplified existing dim light. A very
weak red light was aimed at the ceiling to produce a soft
glow of reflected light in the room (approximately 0.3 w
m'1 nA.). A video camera (Sony HiBand 8 mm) was
used to record behavior; the night-vision device could
be fit to the front of the camcorder to record nocturnal
behavior.

SQUID CULTURE FOR SYMBIOSIS RESEARCH

367

Hatchlings. stocking densities, and foods

Most embryos hatched in the first 2 h of the dark cycle,
and individuals were transferred with a turkey baster to
the culture chamber. For individual 30-day feeding tri-
als, 30 hatchlings were stocked in each round rearing
chamber. The exceptions were one trial in which 50
hatchlings were stocked and one trial in which only 8
hatchlings were taken to the Nahant laboratory. By day
50, squids were removed from the small round chambers
and the juveniles were reared in the divided tray tanks,
each of which had dimensions of 35 by 39 cm.

Food items included live zooplankton, crustaceans,
and larval fishes. Mysid shrimps of the genera Mysi-
dopsis and Neomysis. and larval fishes, Menidia berry-
Una, were commonly used. The term "postlarval mys-
ids" was applied to those that were newly hatched from
the brood sacks of females (Lussier et a/.. 1988). They
were of body lengths 0.5-1.5 mm, whereas adult mysids
were typically 4-10 mm long. Hatchlings were fed 1-
5 times per day between 0700 and 2300 to ensure ade-
quate food availability. Progressively larger shrimp
(Crangon and Palaemonetes) were provided to juvenile
and adult squids as they grew.

Luminescence measurement and quantification of
symbiotic bacteria

Light produced by hatchlings, juveniles, and subadults
was detected qualitatively with a Turner 20e lumino-
meter (Sunnyvale. CA). Individual live animals were
placed in 5 ml (50 ml for subadult animal) of filter-ster-
ilized (0.2-^m pore size) natural seawater in 30-ml glass
scintillation vials (300-ml glass beaker for subadult ani-
mal, placed in a foil-lined funnel to channel light into
the luminometer detector), and the light produced was
recorded for 30 s. Three measurements were taken. The
data reported (as arbitrary light units, LU, per animal)
are the highest of the light levels detected for each ani-
mal.

I '. fischeri cells colonizing the animal light organ were
quantified by plate counts using a seawater complete
agar (SWC; Nealson. 1978). To minimize the presence
of surface-associated bacteria, hatchlings and juveniles
were removed from culture tanks in a minimal volume
of seawater and rinsed by three passages in 5 ml of filter-
sterilized seawater in autoclave-sterilized scintillation
vials. The animals were then homogenized in a 15-ml
Ten Broeck tissue homogenizer with 1 ml of filter-steril-
ized seawater. The homogenate was serially diluted and
plated in quadruplicate on SWC agar plates, which were
incubated at room temperature for 24 h before colonies
were counted. For subadults, the light organ was re-
moved aseptically and homogenized and handled as
above. Representative colonies of bacteria arising on the

spread-plates, which were uniform in appearance for
those animals that produced light, were confirmed to be
I '. fischeri strains that characteristically colonize light or-
gans of E. scolopes (Boettcher and Ruby, 1990; Dunlap
el at.. 1995) by (i) their production of luminescence, al-
though at a very low level in culture, (ii) their lumines-
cence response to the I '. fischeri autoinducer-producing,
nonluminous strain MJ-203, and (iii) their effect on the
luminescence of the autoinducer nonproducing strain
MJ-21 5 (Kuo <•/<//.. 1994; Kuoelal.. 1996).

Results

Hatching success, water iiuulity, and system design

An essential improvement that led to successful cul-
ture was providing a very stable environment so that em-
bryos developed to full term and did not hatch prema-
turely. Failure to do this affected early trials adversely.
Temperature, light, and pH were nearly constant, and
the nitrogen levels were kept within standard limits (i.e.,
0. 10mg/l for ammonia and nitrite, 20 mg/1 nitrate;
Spotte, 1973; Hanlon, 1990). Hatching occurred after
about 18-22 days at 21-23°C. Particular care was taken
not to bump the tank or handle the eggs during the last
days of development. A sample check under a dissection
microscope indicated that hatchlings had no external
yolk amidst the arms, and normal amounts of internal
yolk along the midgut (Arnold et ai, 1972).

Concentrations of ammonia (NH3) and nitrite (NO:)
remained near the recommended levels of 0. 10 mg/1 ex-
cept for two brief spikes of ammonia to 0.40 mg/1 on day
22 and 0.20 mg/1 on day 71, and one brief spike of nitrite
to 0.20 mg/1 on day 46. No mortalities or unusual behav-
ior could be correlated with these spikes. Nitrate (NO3)
levels were very low throughout the trials, ranging from
1 to 4 mg/1, well below the recommended level of 20 mg/
1. The pH always stayed above 8.0, and salinity ranged
from 30 to 37 ppt although the mean was near 35 ppt.
Temperature ranged from 21 to 25°C, but on one occa-
sion the temperature in the Northeastern University
tank decreased slowly to 4°C during the night. The tem-
peratures were returned to 21°C in the course of 5 h. All
of these tropical Hawaiian squids survived!

The utility of a shallow rearing chamber was evident;
in particular it helped concentrate prey organisms for the
very young squids. The system also allowed easy visual
inspection of the tank, including the behavior of the
squids and their food sources.

Behavior of paralarvae and juveniles

The paralarvae in this study showed two notable be-
havioral features: (i) they were strong swimmers, espe-
cially compared to other cephalopod hatchlings of com-

368

R. T. HANLON ET AL

parable size; and (ii) they were alternately active and
planktonic as well hie for the first month post-

hatching. The first ! , ;;s validated in field observa-

tions when RTH o a the hatchlings and juveniles

in their nat .n Hawaii in August 1995. Very

small squi' *• -art towards prey 20 cm away in only

a second o a large distance for a paralarval ceph-
alopod.

Diurnal paralarval swimming activity is illustrated in
Figure 2. There were many squids swimming during the
day in both the unsuccessful and successful feeding trials.
Nevertheless, even the youngest hatchlings spent a large
proportion of their day buried in the sand substrate. By
day 30 in our trials, all of the squids remained benthic
during the day like the adults. At all phases of the life
cycle, the squids were active throughout the night (only
occasional observations were made from midnight to
0600 h). The behavior of juveniles was seemingly identi-
cal to that of adults: they remained buried in the sand
during the day and hunted for prey at night.

At all life stages. Euprymnu tolerated high densities.
By the time the squids had settled (ca. 30 days) and
grown for a total of 40-50 days, their density was quite
high considering that they were still in the small round
rearing chambers. At this time some were transferred to
an adjacent tank system, and in both systems the squids
were distributed into rectangular sections measuring 35
X 39 cm with a sand substrate of about 1-2 cm. There
were no cases of cannibalism, which is common in many
cephalopods.

Unlike the squid in most of the trials, the group of
eight taken to Northeastern University were given no
sand substrate. These squids never sat on the bottom but
swam all the time. After 2-3 weeks, they settled on the
bare PVC bottom; the five squids that survived in this

* • J.

B

Figure 3. Predation by paralarval squids. (A) A 15-day-old para-
larva subduing a very large mysid. which it ate successfully. (B) A 7-
day-old paralarva eating a mysid and holding it in a typical position at
mid-carapace.

system were brought to Woods Hole and reared with oth-
ers to adulthood.

100-

f 8°H

to

•o

O)

C

60-

40-

Successful G1 trial with
diet of adult mysids
Unsuccessful G1 trials
of various diets

B

3D°0

oo
-oo

QB D D 0 • 00

10

20

Age (days)

30

40

Figurv j . rval swimming behavior, indicating the percentage

of squids thai vas actively swimming during daylight hours. After
30 days all vjuid-, lemained benthic during the day.

Feeding

Substantial effort was devoted to studying the feeding
behavior of squids, the swimming behavior of various
prey, and the interactions of predator and prey. The most
important finding was that hatchlings of Eitprynuui ,vr«-
lopes are voracious predators that prefer very large prey
relative to their own size. As indicated in Figure 3, it was
common for hatchlings to successfully attack and ingest
prey that were much larger than themselves. Conversely,
they were capable of eating very small prey, on the order
of 0.5 mm. Hatchlings were already strong swimmers
and could easily make vigorous forward attacks of 12
body lengths in 1 s (as measured from videotape). Field
observations at night in Hawaii also confirmed this
strong swimming ability.

To assess different preferences, six diets were tried for

SQUID CULTURE FOR SYMBIOSIS RESEARCH

369

the critical paralarval period of 1-30 days post-hatching.
Figure 4 indicates the clear results: large adult mysids
were preferred and also produced the best survival; 22
of 30 squids survived to settlement. These mysids were
usually stocked at levels of about 2-4 per squid, but the
numbers fluctuated a great deal. Control animals re-
ceived no food and all died by day 8. as did all 30 squids
provided with a smorgasbord of live zooplankton. Post-
larval mysids, which were expected to be the best food,
produced poor results too; only 5 of 50 squids survived
to settlement. When postlarval mysids and larval fishes
were provided together, the mortality was initially high
but stabilized within a week; 5 of 30 squids survived to
settlement. Similar results were obtained with irregular
mixed diets that consisted of postlarval mysids and
fishes, as well as zooplankton and adult mysids; only 4 of
30 survived to settlement. It is worth emphasizing that
food was presented in abundance in every case, and for
all but a few of the 30 days in each trial.

The behavior of squids during active periods (i.e.,
when in the water column) seemed to be related solely to
foraging. Very rarely was a prey item attacked near the
substrate. Generally the squids preferred to strike up-
ward, which often required that they descend under the
prey, then move rapidly towards it at about a 45 degree
vertical angle. Because their two tentacles were not evi-
dent until about 25 days post-hatching, squids used their
eight arms to grasp the prey. Feeding was clearly stimu-
lated by the addition of prey items to the tank; possibly
the increased motion and swimming activity of prey was
enough of a stimulus to provoke more attack attempts
by squids. For this reason it is advisable to feed squids
many times per day rather than once or a few times. Mys-
ids were clearly preferred, even when other prey such as
fish larvae were present in the tank in far greater quanti-
ties. Squids were commonly observed to make 2-8
strikes at mysids before a successful capture. Usually a

•v.-.

V)

— « — Control

— v — Zooplankton

— x — Postlarval mysids

— * — Irregular, mixed tood

Postlarval mysids & larval fish

^ Y ( Hosoarval mysids & I

\A \ ">-«\»^ — • — Adult mysids

\ \ \ ^^

»-Ji *-,^ \ %»-»-»-»-«-t-«-»-»-»-»-«-»

15 20

Age (days)

25

30

Figure 4. Survival on different diets during the I -month paralarval
period.

large mysid was held at mid-carapace (Fig. 3B), and it is
possible that the squids were biting through the dorsal
nerve cord to immobilize the mysid. We occasionally
saw two squids eating the same large mysid and, more
rarely, one squid attacking a mysid while in the process
of eating another; this occurred only with the smaller
postlarval mysids. Extremely large mysids (e.g.. more
than 4 times the size of squids) were not eaten and prob-
ably disrupted the squids in small chambers.

Paralarval squids showed considerable interest in fish
larvae (Menidia) on some days. But the Menidia larvae
seemed to present problems for capture, and survival of
squids on this diet was poor. For example, on day 27,
squids were presented with fishes that were nearly
12 mm long. The squids strongly pursued the fish larvae,
yet often made 4-6 unsuccessful strikes and sometimes
as many as 20; recall that squids of this age and size were
well developed and strong swimmers. In a typical feed-
ing, only 1-3 squids would have a fish 15 min after 30-
40 fishes were added and 30 attacks were observed. By
contrast, if 30 shrimp (Crangon) were placed in the same
tank, commonly 8 squids would have successfully cap-
tured one within 5 min. It was often observed that squids
could not hold onto a fish even when they made contact,
suggesting that the suckers were not able to grasp the fish
well. Once captured, fish were harder to subdue than
shrimp; it took on the order of 2-3 min, as compared
with 10-30 s for shrimp. When a fish was being eaten,
the stomach of the squid was black and highly visible
compared to a much less distinct dark color when shrimp
were ingested.

Food densities varied greatly due to variable food sup-
ply. However, in the early weeks, about 2-4 large adult
mysids were supplied to each squid in trial 1 . These mys-
ids, which were about 1-3 times the average body length
of the very young squids, were generally eaten in the
course of 24 h. By days 35-40, these squids were being
fed a combination of adult mysids and Crangon shrimp
( 1 .0- 1 .5 cm length) at a level of 5-6 times as many prey
as predator; the tanks appeared very crowded when the
food was first put in. By day 52, 15 juvenile squids were
being fed about 20 Crangon (1.5cm length) and 100
large mysids.

Paralarval squids foraged throughout day and night,
but this was not quantified by counting the number of
attacks on prey per unit time. After settling to the adult
behavior mode by day 30, all squids foraged at night, and
food was generally added to tanks late in the day and
cleaned out the following morning. Juveniles and adults
continued to feed vigorously on prey that were generally
their own size or larger.

Lighting levels appeared to be important to successful
feeding: lower light enhanced feeding, whereas bright
light seemed to retard it. For example, when tops were

370

R. T. HANLON ET AL

placed over the rearing di uiber during the day, the re-
duced light level stir^ d feeding. Very dark overcast
days also resulted quids spending more time

foraging and f<= iight in the rearing chambers

was arrange adividual squids could view prey

objects in mig light against a black background,

and this r uiy increased the contrast of the prey or-
ganism.

Growth, age, maturity, and mortality

Growth was rapid and adult sizes were reached in
about 2 months. Figure 5 illustrates growth in mantle
length and wet weight over the culture period. Hatch-
lings ranged from 1.6 to 1.9 mm in mantle length and
from 4.2 to 5.8 mg in wet weight. Despite the limited
data set (i.e.. few points between days 10 and 70) and
the highly conservative growth measurements (nearly all
taken from freshly dead squids), rapid growth in wet

35
30

I25

.c

|> 20 -

o>

0 15

1 10

5

-y = 0.10215 + 021722X R= 091424

First mating & egg laying

i

20 40 60 80 100 120 140
Age (days)

10 •

S

£
g>

I
-01-

ID

001

0001

-y = 0.0032968 ' eA(008373x) R= 094481

First mating & egg laying

40 60 80 100

Age (days)

120

140

Figure 5. Growth of cultured Eiipryitinu si Wo/K'.v. TOP: Linear plot
of mamle length increases versus age. BOTTOM: Wet weight growth
wasexpi .. '.-ntial through day 83 (R = 0.95). Data ("or males and females
are lumped - RISC identification was not always possible.

weight through day 83 was still best fit by the exponential
equation y = 0.0033e(""84vl. From this function, the in-
stantaneous relative growth rate from hatching to day 83
was an 8.4% increase in weight per day. After day 83, the
data were not amenable to curve fitting because growth
was very slow and the data were highly variable. The ex-
tremely high growth rate of paralarvae is mostly due to
the high feeding rate; hatchlings that were feeding on 2-
3 very large mysids per day were probably ingesting more
than their body weight per day. The length/weight rela-
tionship, based upon 4 1 measurements from hatching to
day 1 33, was expressed by the equation

Wet weight in grams

= 0.0015 X (mantle length in millimeters)2674

The complete life cycle (i.e.. from egg to egg) was com-
pleted in about 80 days, and the longest-lived squid
reached an age of 1 39 days. As noted in the next section,
many squids were sexually mature by 60 days post-
hatching. During the paralarval stage, mortality was
27%, then the population stabilized and the remaining
mortalities occurred over a protracted period that was
marked by reproductive activity. From settling to sexual
maturity, mortality was only 24%; thereafter, there was a
slow attrition. Early mortalities were probably related to
nutrition, but later ones were inexplicable and may have
been associated with maturity and "old age" or with
some unknown pathogen.

Reproduction

Sexual dimorphism is only slightly evident in this spe-
cies. Males have slightly enlarged suckers on some arms
and tend to have slimmer posterior mantles, especially
compared to fully mature females whose posterior man-
tles become broader as they fill with eggs. The testis of
the male can sometimes be seen dorsally when all the
chromatophores are retracted.

The first eggs were laid on day 58 and the first mating
was observed on day 61, when 24 squids were still alive.
Mating occurred at night, often just at the onset of dark-
ness. Overall, 1 6 matings were observed between days 6 1
and 1 16; the last squid died on day 139, so that mating
occurred over the last one-third to one-half of the brief
life cycle. Matings were not controlled in any way and
possibly more matings occurred throughout the nights
than we observed. Mating (Fig. 6A) seemed to be initi-
ated by the male (bottom), who grasped the female and
placed a spermatophore somewhere in her mantle. Mat-
ing lasted about 30-50 min in the few cases in which the
whole mating was observed. Competition for mates was
observed only once: two males grabbed a female, some
wrestling followed, then they all moved to corners of the
tray tank. One male was removed, but the remaining

SOUID CULTURE FOR SYMBIOSIS RESEARCH

371

B

Figure 6. Mating (A) and egg laying (B) by cultured Kiipryiuna .«•<>-
AI/VY The male grasped the female from underneath and they mated
for 30-50 min. Later, the female affixed one egg at a time to a PVC pipe
and coated the eggs with sand to camouflage them.

male and female did not mate that night. In this particu-
lar case, the female was larger than the males.

Egg laying (Fig. 6B) was observed four times and, cu-
riously, took place in the mornings and often lasted until
midday. In total, 1 3 egg clutches were laid by this gener-
ation of adults. Attachment of each egg took 10 s on av-
erage, and about 40 s elapsed between the deposition of
each egg, so that it took about 25-30 min to lay a clutch
of 30 eggs. As usual, the eggs were soon coated with sand
that was somehow placed there by the females.

To minimize handling, individual squid were not
marked; thus fecundity could be estimated only from the
number of eggs laid by individual females. All tray tanks
contained males and females in high densities, even
when the squids were separated on day 49; i.e., there
were 2-3 squids per 35 X 39 cm chamber.

Reestablishment of the bacterial .symbiosis in reared
squids

To determine whether symbiosis with I', fischeri was
reestablished in the light organ of the reared squids.

hatchlings, juveniles, and subadult animals were exam-
ined for the production of light and for the presence of
r. fischeri cells. The hatchling squids initially lacked lu-
minescence, and no colony-forming units of I', fischeri
were detected in their nascent light organs. By day 5,
however, the animals were luminous, and their light or-
gans contained 105 or more I', fischeri 'cells (Table I). Fig-
ure 7 illustrates bacterial cells in the light organ of a
reared squid.

Discussion

We have identified three essential keys for successful
laboratory culture of Enprymna scolopes: (i) the eggs
must be provided with conditions that lead to complete
embryonic development, thus rendering fully competent
hatchlings; (ii) water quality must be good, and the tank
configuration and lighting must be tailored to the specific
needs and behavior of the species; and (iii) the proper
type and quantity of prey organism must be provided.
Observations in the sea as well as in the laboratory indi-
cated that E. scolopes was a voracious predator for its
size, and that relatively large prey were preferred.

Because our main interest in culturing E. scolopes is to
advance the use of this model of symbiosis, it was also
important to demonstrate that laboratory animals were
competent to receive bacteria in a manner similar to that
of wild-caught Enprymna. Our results are consistent with
previous demonstrations that hatchlings initially are apo-
symbiotic and that they acquire symbiotic strains of I '.
fischeri, and consequently produce light, within about a
day of hatching (Wei and Young, 1989; McFall-Ngai and
Ruby, 1991). Furthermore, squids that had reached the
juvenile benthic and subadult stages also produced lumi-
nescence, and their light organs contained increasingly
larger populations of I '.fischeri (Table I; Fig. 7).

Table I

Re-establishment of hacicruil symbiosis in culnircil Euprymna
scolopes

Animal stage

Approximate
size (mm)

Animal light
production'

Symbiont
CFU2

Hatchling (Day 0- 1)

2x2

not detected

not detected

(Planktonic)

(') = 5)

(n = 5)

ParalarvaKDay 5)

2.5x2

44

6.0 x 105

(Planktonic)

3.2

5.0 x I05

Juvenile (Day 15)

4x3

220

8.0 X 10"

(Benthic)

Juvenile (Day 30)

7 x 4.5

16

2.0 x I07

(Benthic)

Subadult (Day 130)

35 • 20

640

8.0 x 107

1 LU (arbitrary light units) per animal.

: Symbiont CFU = colony-forming units of I 'ihnu /

372

R. T. HANLON ET AL.

Figure 7. (A) Scanning electron micrograph of half the light organ
of a mature, cultured squid. Letter "p" indicates the pore. Symbol in-
dicates the approximate region of interest shown in panel B. (B) High
magnification of I'ihriu lischcri cells within the internal cavity of the
distal end of the light organ. Bar is I ^m.

Behavior, life cycle, and laboratory culture comparisons

It has been found repeatedly that teuthoid and sepioid
squids are visual predators that generally prefer actively
swimming prey (e.g., Boletzky ct a/., 197 1 ; Boletzky and
Boletzky, 1973: Boletzky, 1974; Boletzky and Hanlon,
1983; Yang et at.. 1986; Hanlon ct a/.. 1989; Hanlon,
1990; Lee el ai. 1994). The peculiar decapod arrange-
ment of eight arms and two tentacles allows capture and
ingestion of both very small and unusually large prey.
Nevertheless, as observed in many of the studies just
cited, many seemingly good prey items are not preferred
by paralarval squids, thus diets must be determined ex-
perimentally. It was predictable that E. scolopes would
ingest mysids given the enormous success of this diet for
other squids (studies cited above) and cuttlefish (For-
sythe el ai, 1994) and the results of rearing work on E.
herryi (Choe and Oshima, 1963; Choe, 1966) and E. sco-
lopes (Arnold et ai, 1972; Singley, 1983). It was not pre-
dictable that E. scolopes paralarvae would prefer such
large prey and only survive well on them as compared to
mysids of other sizes. However, field observations made
with a night-vision device by RTH in Hawaii indicated
thai \ >' young E. scolopes were exceptionally strong
swimnK s that could jet forward at great lengths. Labo-
ratory \ > in this experiment documented forward at-

tacks of at least 12 body lengths in about a second, a feat
that small squids such as Loligo cannot perform.

How might the paralarvae live in nature? Our study indi-
cates that their activity patterns are flexible compared with
those of adults (Fig. 2). They can bury in the sand like adults,
yet they are surprisingly strong swimmers that can capture
prey with a very rapid forward attack and avoid predators by
combining a swift backwards jet escape with inking. We did
not see any evidence that the light organ was used by para-
larvae, juveniles, or adults in our study, despite many obser-
vations at night. It seems unlikely that paralarvae commonly
use the light organ while seeking prey because we probably
would have observed it through the viewing ports in the side
of the tray tanks (Fig. 1 ). A more plausible use for the biolu-
minescence would be in defense against predators from be-
low, when Eupryinna of any life stage are higher in the water
column. Since the organ is directed downward, it probably
is used to eliminate or interrupt the squid's shadow against
the downwelling light.

Eupryinna scolopes has one of the shortest life cycles of
any cephalopod (Boyle, 1983), mainly as a result of its
rapid exponential growth, the warm temperatures it lives
in, and its small adult size. The fast exponential growth
through day 83 is typical for many squids: since first mat-
ing and egg laying were seen at about day 60, we expected
that growth would slow substantially soon thereafter,
which it did. The shortest life cycle reported for a cepha-
lopod is in the small tropical species Idiosepius pygmaeus:
statolith ring analysis indicates that this species matures
in 1 .5-2.0 months and lives only about 79 days (Jackson,
1989); this species was not cultured in the laboratory. E.
scolopes is relatively easy to rear because of the large
strong hatchlings and their propensity for mysid shrimps.
Close relatives of E. sco/opes share rapid growth, small
size, and preference for mysids. Eupryinna berryi was
reared to 2 months by Choe and Oshima (1963) and Choe
(1966); four species of Sepiola and two of Sepiella were
reared by similar techniques by Boletzky el ai (1971);
Sepietla oweniana was cultured by Summers and Berg-
strom (1981); and Russia macrosoma was reared to
8 months by Boletzky and Boletzky (1973). Only E. sco-
lopes, however, lives in warm water, which accelerates
growth (Forsythe and Van Heukelem. 1987) and pro-
motes a short life cycle. Could these techniques be appli-
cable to Eupryinna inorsei from Japan and to similar sep-
ioids with light organs?

Behavioral and physiological factors that require future
attention

Now that progress has been achieved with the paralar-
val stages, the next logical stage is to focus on reproduc-
tive behavior. Practically nothing is known about the
mating system of any member of the subfamily Sepioli-

SQUID CULTURE FOR SYMBIOSIS RESEARCH

373

naeU'.A'. Boletzky el al.. 1971; Boletzky, 1975; Moyni-
han, 1983; also reviewed by Hanlon and Messenger.
1996), and optimal conditions for brood stock manage-
ment will have to be determined before "normal" repro-
ductive behavior and high fecundity can be expected.
The density of adults, the diet, the light cycle (especially
gradual changes that imitate natural changes), the com-
binations of females and males and the nature of their
pairings, agonistic behavior, courtship, and any form of
sperm competition will all influence mating, egg laying,
and the quality of the progeny. The matings observed in
this study averaged 35 min; Moynihan (1983) and Sin-
gley (1983) reported matings of 25-80 min. These long
mating times combined with the presence of a seminal
receptacle (called the pharetra. located internally near
the opening of the oviduct) strongly suggest the possibil-
ity of sperm competition behavior among males. A prac-
tical problem is to determine how many adult squids
must be maintained as brood stock under optimal con-
ditions (physical and social) to ensure sufficient genetic
diversity in subsequent generations.

A misbalance in the mating system can put stress on
the females, resulting in poor egg production, low fertil-
ization rates, and thus poor hatching rates or lack of
vigor in the progeny. Crowded laboratory conditions in
cuttlefish and loliginid squids can lead to forced copula-
tions of females (J.G. Boal. pers. comm.. 1996) and a
disruption of the mating system. Lack of attention to
these key issues in reproduction is one reason why most
cephalopods cultured in captivity have not been cultured
through multiple generations. Another sepioid. the cut-
tlefish Sepia ojlicinalis. is the one cephalopod that has
successfully been cultured through many generations
(Forsythe el at.. 1994), and is currently in the 14th labo-
ratory generation at the University of Texas Medical
Branch in Galveston.

The second-generation hatchlings in this culture trial
did not do well, but no obvious cause was detected. Be-
cause we had no access to a control group of hatchlings
from wild-caught adults, we were unable to determine
whether the problem lay with our techniques or with a
lack of vigor in the first filial generation. We also had no
certain clues to the cause of the mortalities that occurred
throughout the trials, although several of the adults had
whitish patches of ulceration that are common in many
laboratory-reared cephalopods (Hanlon and Forsythe.
1990). Certainly other factors should be analyzed more
closely — for example, different light cycles and types of
substrates (cf. Shears. 1988) — to see how they affect the
health and well being of squid in captivity.

Future possibilities lor this marine model <>l symbiosis

The immediate application of our culture techniques
is in exploring new questions about the developmental

biology of this symbiosis. The light organ requires bacte-
ria before it can develop (Montgomery and McFall-Ngai.
1994), and the bacteria need the light organ to become
luminescent (Boettcher and Ruby. 1990). On one hand,
the ability to culture the host organism — the squid-
opens the prospect of studying late development of the
light organ, although it first requires that the paralarvae
be reared in the absence of the bacteria. On the other
hand, one can now address issues of involvement of bac-
teria in developmental programs at the level of tissue,
organ, and whole animal.

The longer-term application of Etiprymna scolopes
culture is to develop this species into a genetic model.
This is not a trivial task, but E. scolopes has characteris-
tics that favor success. Unlike other cephalopods. this
species is small and short-lived, and each female usually
lays its clutch of eggs in a single night and in a discrete
clutch, so that parentage and reproductive success can be
assessed.

Acknowledgments

We are grateful for advice from John Arnold, Alan
Kuzirian. Bill Mebane. Richard Young, Ned Ruby, and
Margaret McFall-Ngai. Alan Kuzirian helped produce
the fine SEMs in Figure 7, and John Forsythe helped
with the growth analysis. Kurt Fiedler collected adults
for us in Hawaii. Bill Mebane. Janice Hanley, Louis
Kerr, and David Remsen of the MBL provided essential
logistical support. We thank the Aquatic Resources Di-
vision of the MBL, Springborn Laboratories (MA),
Aquatic Research Organisms (NH), the Marine Biomed-
ical Institute (TX), Dave Bengston (RI), and especially
Ray Lewis of Aquatic Indicators (FL) for help in provid-
ing food organisms. Roxanna Smolowitz performed nec-
ropsies of dead squids. This work was partially funded by
NSF Grant MCB 94-08266 to PVD.

Literature Cited

Arnold. J. M., C. I. Singley, and I.. D. \\ illiams- Arnold. 1972.

Embryonic development and post-hatching survival of the sepiolid
squid Eupn'inna uo/c/'o under laboratory conditions. I eliger 14:
361-364.

Boettcher, K. J.. and E. G. Ruby. 1990. Depressed light emission by
symbiotic I 'ihrio lisclicri of the sepiolid squid Eiiprymna scolopes.
J.Baaeriol. 172:3701-3706.

Boletzky, S. v. 1974. Elevage de Cephalopodes en aquarium. I 'ic Mi-
//«<24(2-A): 309-340.

Bolelzky, S. v. 1975. The reproductive cycle of Sepiolidae(Mollusca.
Cephalopoda). /V>W filu: /.out. \iipoli 39. Suppl.: 84-95.

Boletzky, S. v., M. V. \ . Boletzky, D. Frosch, and V. Gatzi. 1971 . Lab-
oratory rearing of Sepiolinae (Mollusca. Cephalopoda). Mar. Binl
8(1): 82-87.

Boletzky, S. v., and V. v. Boletzky. 1973. Observations on the embry-
onic and early post-embryonic development of Rossia macn>\«nhi
(Mollusca. Cephalopoda). Helgol. ll'i\\. Mccreminiers.S: 135-161.

Boletzky, S. v., and R. I. Hanlon. 1983. A review of the laboratory

374

R. T. HANLON ET AL.

maintenance, rearing and culture- of cephalopod molluscs. Mem.
Neil I. Mus. (Vc. 4-1: 14?

Boyle, P. R. 1983. ('<•/'/;. Cycles, Vol. 1 : Species Account

Academic Press. !

Choe, S. 1966. ():• rearing, habits of the try. and growth of

somecepl- i Mar.Sei. 16:330-347.

Choe, S.. ;i: ::>'a. 1963. Rearing of cuttlefishes and squids.

Nanirel97:

Dunlap, P. \ ., K. Kita-Tsukamoto, J. Waterbury, and S. M. Callahan.
1995. IMVUIUII and characterization of a visibly luminous variant
of I'ibrin lischeri strain ESI 14 from the sepiolid squid Euprymna
seoloi'cs Anil. Microhiol. 164: 194-202.

Forsythe, J. W., and VV. F. Van Ileukelem. 1987. Pp. 135-155 and
203-204 in Cephalopod Li/e Cycle*. I 'ol. II Comparative Reviews,
P. R. Boyle, ed. Academic Press, New York.

Forsythe, J. W., R. H. DeRusha, and R. T. Hanlon. 1994. Growth,
reproduction and life span of Sepia officinalis (Cephalopoda: Mol-
lusca) cultured through seven consecutive generations. J. Zoo/.
(L»ml.)23: 175-192.

Graft, J., P.V. Dunlap, and E. G. Ruby. 1993. Effect of transposon-
induced motility mutations on colonization of the host light organ
by \'ihno1i\chcri.J. Bacterial. 176:6986-6991.

Hanlon, R. T. 1990. Maintenance, rearing and culture of teuthoid
and sepioid squids. Pp. 35-62 in Squid as Experimental Animals,
D. L. Gilbert, W. J. Adelman. Jr., and J. M. Arnold, eds. Plenum
Press, New York.

Hanlon, R. T., and J. W. Forsythe. 1990. Diseases of Mollusca: ceph-
alopoda: I.I. Diseases caused by microorganisms; 1.3 Structural ab-
normalities and neoplasia. Pp. 23-46 in Diseases ol Marine Ani-
mals I 'ol. Ill: Cephalopoda to Urachordala, O. Kinne, ed. Biolog-
ische Anstall Helgoland. Hamburg.

Hanlon, R. T., and J. B. Messenger. 1996. Cephalopod Behuvioin
Cambridge University Press, Cambridge, U.K.

Hanlon, R. T., W. T. Yang, P. E. Turk, P. G. Lee, and R. F. Hi\on.
1989. Laboratory culture and estimated life span of the eastern
Atlantic squid, Luligo forbesi Steenslrup, 1856, (Mollusca: Cepha-
lopoda). Ai/iun nil. Eish. Manage 20: 15-34.

Jackson, G. D. 1989. The use of statolith microstructures to analyze
life-history events in the small tropical cephalopod Idiosepius pyg-
maeus Fish Hull 87:265-272.

Kuo, A., N. V. Blough, and P. V. Dunlap. 1994. Multiple A-acyl-ho-
moserine lactone autoinducers of luminescence in the marine sym-
biotic bacterium Vibriofischeri J Bacterial. 176:7558-7565.

Kuo, A., S. M. Callahan, and P. V. Dunlap. 1996. Modulation jof lu-
minescence operon expression by A-octanoyl-homoserine lactone
in ainS mutants of I 'ihno lischeri. J Bacterial. 178: 97 1-976.

Lee, P. G., P. E. Turk, W. T. Yang, and R. T. Hanlon. 1994. Biologi-
cal characteristics and biomedical applications of the squid Sepio-
lenlliis lessoniana cultured through multiple generations. Biol.
Hull 186:328-341.

Lussier, S. M., A. Kuhn, M. J.Chammas, and J. Sewall. 1988. Tech-
niques for the laboratory culture of Mysidop.sis species (Crustacea:
Mysidacea). Envir Toxicol Chem. 7: 969-977.

McFall-Ngai, M.J., and E.G. Ruby. 1991. Symbiom recognition
and subsequent morphogenesis as early events in an animal-bacte-
rial mutualism. Science 254(5037): 1491-1494.

Montgomery, M. K., and M. McFall-Ngai. 1994. Bacteria] symbi-
onts induce host organ morphogenesis during early postembryonic
development ol the squid Euprymna sco/opes. Development 120:
1719-1729.

Moynihan, M. 1983. Notes on the behaviour of Euprymna .scolopes
(Cephalopoda: Sepiolidae). Behaviour 85: 25-41.

Nealson, K. II. 1978. Isolation, identification, and manipulation of
luminous bacteria. Methods Enivmol. 57: 153-166.

Ruby, E. G., and L. M. Asato. 1993. Growth and flagellation of I '//>-
nolischcn during initiation of the sepiolid squid light organ symbi-
osis. .-1*7). Microhinl. 159: 160-167.

Ruby, E. G., and M. J. McFall-Ngai. 1992. A squid that glows in the
night, development of an animal-bacterial mutualism. J Bacterial.
174:4865-4870.

Shears, J. 1988. The use of a sand-coat in relation to feeding and diel
activity in the sepiolid squid Euprymna scolopes Malacologia 29:
121-133.

Singlcy, C. T. 1983. Euprymna scolopes Pp. 69-74 in Cephalopod
Lite Cycles. Vol. I, P. R. Boyle, ed. Academic Press. New York.

Spotte, S. II. 1973. Marine Aquarium Keeping. Wiley, New York.

Summers, \V. C., and B. Bergstrom. 1981 . Cultivation of the sepiolid
squid, Septetla oHeniana, and its ecological significance. Am. /.out.
20:927.

Voss, G. L. 1977. Present status and new trends in cephalopod sys-
tematics. Pp. 49-60 in The Biology o/ Cephalopods. Symposia ol
the Zoological Society o/ London 3H M. Nixon and J. B. Messen-
ger, eds. Academic Press. London.

Wei, S. L., and R. E. Young. 1989. Development of symbiotic bacte-
rial bioluminescence in a nearshore cephalopod, Euprymna sco-
lopes Mar. Biol. 103:541-546.

Yang. \V. T., R. F. Hixon, P. E. Turk, M. E. Krejci, \V. H. Hulet, and
R. T. Hanlon. 1986. Growth, behavior, and sexual maturation of
the market squid. Loliga opalescens, cultured through the life cycle.
l-'ish Bull 84: 771-798.

Young, R. E., and R. E. Harman. 1988. "Larva", "paralarva" and 'sub-
adult' in cephalopod terminology. Malacologia 29: 201-207.

Reference: Bio/, Bull. 192: 375-387. (June, 1997)

Post-Hatching Development of Circular Mantle
Muscles in the Squid Loligo opalescens

THOMAS PREUSS, ZORA N. LEBARIC, AND WILLIAM F. GILLY

Hopkins Marine Station. Department of Biological Sciences, Stanford Universitv,
Pacific Grove. California 93950

Abstract. Post-hatching development of the circular
muscles in the mantle of squid was studied morphomet-
rically to identify structural changes and to quantify hy-
perplasia and hypertrophy of the muscle fibers. Superfi-
cial, mitochondria-rich (SMR) fibers and central, mito-
chondria-poor (CMP) fibers are present at hatching.
Although both fiber types increase in size and, even more
so, in number during post-hatching development, CMP
fibers increase at a much higher rate than do SMR fibers.
As a result, the relative proportion of SMR to CM P fibers
shifts from about 1:1 in a hatchling to about 1 :6 in an 8-
week-old animal; it then apparently remains constant to
adulthood. These structural changes are consistent with
developmental changes in muscular activity. During
slow, jet-propelled swimming, 1 -week-old animals show
mantle contractions that have twice the relative ampli-
tude and frequency of those in adults. The presence of
Na-channel protein in mantle muscle was detected bio-
chemically by using site-directed antibodies: the protein
was found to be preferentially expressed in CMP fibers.
These results suggest that SMR fibers are an important
source of locomotory power at hatching, but become
progressively less important during the first 8 weeks of
development as CMP fibers assume the dominant role in
jet locomotion.

Introduction

Jet propulsion in squids is caused by the antagonistic
action of circular and radial mantle muscles in connec-

Received 2 December 1996: accepted 3 April 1997.

Abbreviations: CMP = central mitochondria-poor muscle fibers;
DML = dorsal mantle length; GFL = giant fiber lobe; SMR = superfi-
cial mitochondria-rich muscle fibers; SR = sarcoplasmic reticulum.

tion with two layers of stiff, collagenous tunics and a net-
work of intermuscular connective tissue fibers (Ward
and Wainwright, 1972: Bone el at.. 1981; Gosline et a/.,
1983; Kier, 1988; Wells, 1988). The circular muscle
mass, responsible for producing thrust during jet propul-
sion, is divided into three layers, largely on the basis of
the relative mitochondrial content of individual muscle
fiber types. Two thin layers of superficial mitochondria-
rich, oxidative fibers on the outer and inner mantle sur-
face enclose a much thicker layer of mitochondria-poor,
glycolytic fibers in the central zone (Bone el a/.. 1981;
Mommsen et a/.. 1981 ). In light of these structural and
metabolic differences, it has been proposed that the su-
perficial layers are active during respiration and slow jet-
propelled swimming, whereas the central circular fibers,
presumed to be innervated by the giant axon system,
power the jet-escape and rapid locomotion (Bone et a/..
1995). In general agreement with these ideas, recent elec-
trophysiological studies have revealed that at least two
classes of circular muscle fibers can be distinguished on
the basis of their electrical properties and that some of
the circular fibers, presumably those innervated by giant
axons, display Na-channel-based excitability (Gilly et
at.. 1996).

Jet propulsion, mantle mechanics, and mantle struc-
ture have been well studied in adult squid, but relevant
work on hatchlings and developing juvenile squid is
comparatively limited (Zuev, 1966; v. Boletzky, 1982,
1987;O'Dorrta/., 1986; Moltschaniwskyj. 1994, 1995;
Matsuno, 1987). Although squid begin actively swim-
ming by jet propulsion as soon as they hatch (Packard.
1969), the full flexibility and fine coordination of the lo-
comotor system is lacking (Gilly el ai, 1991; Chen et ai.
1996). The aim of the present study was to analyze the
maturation of the mantle musculature during post-

375

376

T. PREUSS ET AL.

hatching development and to identify structural and
functional characteristics of the different types of muscle
fiber associated v -propelled locomotion. In pursuit

of that goal, v ned three approaches. First, ana-

tomical an.i .^metric methods at light- and elec-

tron-mien K ievels were used to reveal structural

changes as squu mature from hatchlings to adults. Sec-
ond, the kinematics of mantle contractions in free-swim-
ming animals were investigated to shed light on func-
tional characteristics of locomotion at different maturity
stages. Third, biochemical analysis of the presence of Na-
channel protein in superficial and central fibers provided
a tool to characterize the location and abundance of pu-
tative fast-twitch fibers in the developing mantle muscu-
lature.

Our results indicate that superficial, mitochondria-
rich (SMR) fibers are an important source of locomotory
power at hatching but become progressively less impor-
tant during the first 8 weeks after hatching. Over this
same period, central, mitochondria-poor (CMP) fibers
increase in number until they contribute most of the
overall mantle muscle mass and play the dominant role
in jet locomotion.

Materials and Methods

Experimental animals

L»/;#0 o/w//rotwi.v was collected in Monterey Bay, Cal-
ifornia, between August and November of 1995. Ani-
mals were maintained at Hopkins Marine Station in cir-
cular tanks (2.5 m diameter: 1 m deep) plumbed with
flow-through natural seawater. Spawning typically oc-
curred in these tanks within 3-5 days of collecting the
squid. Clusters of egg cases were removed and cultured
at the Monterey Bay Aquarium, Monterey, California,
in flow-through circular 320-1 tanks (temperature range
13-16°C) until natural hatching occurred. During the
first 10 weeks, squid received a diet, ad libitum, of brine
shrimp nauplii (Anemia salina) enriched with algae and
Super Selco (a nutrient medium rich in lipids, fatty acids,
and vitamins; produced by Artemia Systems N. V., Bel-
gium). Copepods (Acartia sp.) and mysids (Acanmo-
mysis sp.) were added to the diet when available (about
twice per week).

Mantle anatomy

The anatomy of the mantle musculature was exam-
ined by light microscopy (LM) and transmission electron
microscopy (TEM). Prior to dissection, all animals were
anesthetized for 20 min in 7.5% MgCl; diluted 1 : 1 in ox-
ygenated seawater and then killed by decapitation. Tis-
sue for fixation was removed from adult squid within 1

day after collection. Blocks of mantle muscle were cut
parallel to the main body axis and taken from an antero-
dorsal region close to the stellate ganglion of five adults
with dorsal mantle lengths (DML) between 80 and
140 mm. For studies of hatchling and juvenile squid, six
healthy looking animals that displayed vigorous swim-
ming capability were collected for fixation weekly over
a 10-week period. These animals were fixed whole, and
smaller tissue samples for ultrastructural analysis of the
mantle muscles were taken after fixation. In all animals
(except hatchlings), the skin was carefully removed be-
fore fixation.

For LM, the tissue was fixed in 4% paraformaldehyde
in filtered seawater for 3-5 days at 4°C, dehydrated in
graded ethanol, and either infiltrated with paraffin under
vacuum or embedded in plastic (JB4; Polysciences, Inc.).
Thick sections (6-10 ^m) and semithick sections (2-
4 ^m) were cut and stained with conventional histologi-
cal stains and viewed with an Olympus BH-2 micro-
scope.

For TEM, the tissue samples were fixed in 0.065 A/
sodium phosphate buffer (pH 7.4) with 3% glutaralde-
hyde, 0.5% tannic acid, and 6% sucrose for 8 h at 4°C
(first 1 5 min at room temperature), rinsed in 0.065 M so-
dium phosphate buffer without sucrose, and postfixed in
a 1:1 mix of 0.13 Mcacodylate buffer (pH 7.2) with 2%
potassium ferrocyanide and 2% osmium tetroxide for
40 min at 4°C. Thereafter, the tissue was rinsed in
0.065 M cacodylate buffer, dehydrated in graded etha-
nol, and infiltrated overnight (10-12 h) in resin (LR
White: Sigma). For sectioning, the blocks were oriented
to obtain transverse, sagittal, and tangential sections of
the circular mantle muscles. Thin (0.8-1 fim) and ultra-
thin (gray-silver) sections were obtained in alternating
section series, thereby collecting ultrathin sections every
4-6 /jin in hatchlings and juveniles and every 50-60 ^m
in adults. Up to five consecutive section series were col-
lected in this manner. Thin sections were stained with
toluidine blue and examined with an Olympus BH-2 mi-
croscope. Ultrathin sections were collected onto Form-
var-coated mesh and slot grids (Electron Microscopy Sci-
ences), stained either with a saturated aqueous uranyl ac-
etate solution and Reynolds' lead citrate or with 2%
phosphotungstic acid, and examined with Phillips EM
201 or EM 401 electron microscopes. The magnification
stops were calibrated with a diffraction grating replica
(Ted Pella. Inc.).

For morphometric analysis of the muscle fibers, a
computer-aided image analysis program (NIH-Image
1.60) was used on a Power Macintosh 7100/80 com-
puter. TEM micrographs of muscle fibers were digitized
at 600-dpi resolution using a scanner (Microtek Scan-
Maker HHR), and stored on a magneto-optical drive (Pin-

SQUID MANTLE MUSCLE DEVELOPMENT

377

nacle Tahoe 230 MB). In addition. LM video-images
were obtained using an Olympus BH-2 microscope
equipped with a CCD B/W camera (Sony SSC-M374).
and selected frames were digitized with a high-resolution
video capture card (Scion LR-3).

Measurements were taken on longitudinal mantle sec-
tions (i.e.. on a cross section of the circular muscle fi-
bers). Two measurements of each muscle fiber visible in
a given electron micrograph were taken: (i) the total
cross-sectional area of a single muscle fiber and (ii) the
area occupied by its mitochondria! core. The difference
between these values approximates the remaining myo-
filament area; this value also includes cytoplasm and sar-
coplasmic reticulum (SR). Additional measurements
were taken in selected micrographs to determine the
cross-sectional area (and the calculated diameter) of syn-
aptic vesicles in nerve processes.

Mantle kinematics

A dorsal view of freely swimming juvenile (1 -week-
old) and adult squid in their respective holding and rear-
ing tanks was filmed with a high-resolution CCD B/W
video camera (Sony SSC-M374) mounted above the
tanks on a remote controlled panning motor (Prinz
Power-Fanner 430-62), and recorded on a Sony Hi-8 re-
corder (EVO-9700). Single video-frame analysis of digi-
tized video sequences was carried out using the image
analysis system described above. The mantle diameter
was measured at its widest point (the anterior mantle end
in juveniles and about '/3 from the anterior mantle end in
adults) in successive, enlarged video images (calibrated
by dorsal mantle length). To compare mantle diameter
dimensions in animals of different size, the fractional
mantle diameter was calculated by defining the largest
diameter in a given measurement sequence as 100%.

Biochemistry

Production oj antibodies. Two antibodies (one poly-
clonal and one monoclonal) directed against distinct re-
gions of a putative squid sodium channel encoded by the
cDNA GFLN1 (Rosenthal and Gilly, 1993) were used.
mRNA corresponding to this gene is expressed widely
throughout the squid nervous system (Liu and Gilly,
1995).

Polyclonal antisera (produced in collaboration with
Dr. S.R. Levinson. University of Colorado) were raised
against a bacterial fusion protein that contained amino
acids (aa) 483-576 of the predicted GFLN1 sequence.
These residues compose the C-terminal half of the cyto-
plasmic linker between domains I and II. Construction
of the fusion protein is described in detail elsewhere
(Rosenthal. 1996). Polyclonal antisera (Ab4K3-57<,) were

affinity-purified using the soluble fraction of the fusion
protein coupled to an affinity column (AminoLink,
Pierce) according to manufacturer's protocol.

A monoclonal antibody (mAb|i0?.:i) was produced in
collaboration with J. Burkhard and S. L. Feng. Univer-
sity of California, San Francisco. This antibody was di-
rected against a synthetic peptide (synthesized by the
Protein and Nucleic Acid Facility, Stanford University.
Stanford, CA) corresponding to aa 1305-1321 of the
GFLN1 sequence. These residues make up the part of
the cytoplasmic linker between domains III and IV that
is well conserved in most sodium channels (Gordon el
a/.. 1988).

Preparation of protein samples. Specificity of the anti-
bodies was tested with tissue samples of cleaned giant ax-
ons and giant fiber lobes (GFL) taken from adult squid.
Animals were rapidly decapitated before tissues were dis-
sected in cold, Ca-free artificial seawater (480 mAI NaCl,
10 mM MgCl:, 5 mM EGTA, 10 mM HEPES, pH 7.4)
for biochemical analysis. Segments of stellar nerves were
ligated and cleaned by manually stripping off the small
nerve fibers under microscopic observation until only
the giant axon and its Schwann-cell sheath remained.
Cleaned axons were cut at both ends and immediately
placed in ice-cold lysis buffer containing proteinase in-
hibitors (Knudson et ai. 1989). Several axons were
pooled, homogenized in the same buffer, and centrifuged
at 1 500 X g for 1 0 min at 4°C. The supernatant was then
used for protein-concentration analysis (bicinchoninic
acid assay. Pierce, Rockford, 1L) and for immunoblot-
ting. Giant fiber lobe, brain, and cornea tissues were dis-
sected and homogenized in the lysis buffer as described
above. The resulting supernatant was centrifuged at
1 00.000 X g for 30 min at 4°C to form a crude membrane
pellet. This pellet was resuspended in lysis buffer for de-
termination of protein concentration and for immu-
noblotting.

Muscle samples were obtained from animals anesthe-
tized in 0.5% urethane in artificial seawater and dissected
in cold, Ca-free artificial seawater. Thereafter, all muscle
tissue was processed as described for giant fiber lobes (see
above). To test for Na-channel protein within individual
layers of circular mantle muscle, tissue samples from su-
perficial and central layers were dissected and processed
separately (outer and inner superficial layers were
pooled). To test for the presence of Na-channel protein
in the mantle muscle mass during post-hatching devel-
opment, samples were taken from skinned mantles of
hatchling and juvenile squid at four maturity stages (3
days, 2 weeks, 3 weeks, and 14 weeks) and from adults.

Immunoblotling. Protein samples ( 10 ^g of total pro-
tein per lane) were separated by standard SDS-PAGE
electrophoresis using 5% gels and transferred to nitrocel-

378

T. PREUSS KT AL.

lulose. Nonspecific binding of the antibodies was mini-
mized by pretreating the nitrocellulose with 10% nonfat
dry milk in PBS ( / Na:HPO4, 0.02 M NaH:PO4,

0.1 A/NaCl) . UTinity-purified antibodies were

diluted ;)v n the figure legends. Undiluted hy-

bridoma1 ; . ni was used as a source of monoclonal

antibody. Goal anti-rabbit or goat anti-mouse secondary
antibodies conjugated to horseradish peroxidase (Sigma)
were used at 1:5000 dilution in conjunction with an en-
hanced chemiluminescence detection system (Renais-
sance. Du Pont NEN Research Products). For control
experiments with blocked Ab^.svh. purified antiserum
was incubated overnight at 4°C with the fusion protein
antigen at a concentration of 0. 1 mg/ml.

Results

Structural aspects of muscle fiber maturation

Squid hatchlings (1-2 days old) already possess the
overall organization of radial and circular muscle fibers
shown by adults (Bone el at., 1995). Profiles of two dis-
tinct types of circular muscle fibers are visible in a longi-
tudinal mantle section (Fig. 1A). Layers of superficial,
large-diameter fibers on the outer and inner mantle sur-
face enclose a central layer composed of fibers of smaller
diameter. The superficial fibers and the central fibers differ
in their mitochondrial content. In any given cross section,
superficial mitochondria-rich (SMR) fibers often contain
several large mitochondria, whereas central mitochon-
dria-poor (CMP) fibers rarely display more than a single
small one (Fig. 1 ). In both fiber types, the mitochondria
are surrounded by loosely packed myofilaments (Fig. 1 B,
C). Myofilaments are rather poorly organized at this time,
and sizable areas of cytoplasm without myofilaments are
common. Similarly, the SR network is poorly developed
and organized at this stage, with only a few SR tubules
scattered among the myofilaments (Fig. 1 B, C). Fibers of
both types display very large nuclei (Fig. 1 A).

By 8 weeks after hatching, several of the above charac-
teristics show signs of substantial maturation. In addi-
tion to their difference in size and mitochondrial content
(see also below), SMR and CMP fibers now clearly dis-
play differences in the organization of their myofila-
ments and SR (Fig. 2). The myofilament density in-
creases considerably in both fiber types, but CMP fibers
show a much thicker myofilament zone, both in absolute
size and in relation to the mitochondrial core (Figs. 2B,
2C, and 3). Moreover, myofilaments in each fiber type
are now organized in a characteristic manner. In cross
sections of CMP fibers, myofilaments are divided by the
SR and the Z-bodies into radially oriented, trapezoidal
bloci »Fig. 2C). In the same sections, SMR fibers lack
such u ,MIIS, and the myofilaments form a continuous

ring around the central mitochondrial core (Fig. 2B).
This difference between CMP and SMR fibers was found
in cross sections examined at all maturity stages from
week 1 on.

Longitudinal sections confirm the difference in width
of the myofilament zone in relation to the mitochondrial
core of the two circular fiber types (Fig. 3). These sections
also reveal differences in myofilament staggering in the
two fiber types. In CMP fibers, the myofilaments and Z-
bodies form an oblique pattern across the fiber, which
characterizes them as regular, obliquely striated muscles
similar to those in other cephalopods (Gonzalez-San-
tander and Garcia-Blanco, 1 972; Amsellem and Nicaise,
1980, Kier. 1985). In contrast, the myofilaments and Z-
bodies of SMR fibers are lined up nearly in register across
the fiber (Fig. 3). Although the appearance of the SMR
fibers in cross section and longitudinal section is sugges-
tive of cross striation rather than oblique striation, fur-
ther morphological studies are necessary to clarify this
point. The degree of myofilament staggering in a given
section depends on the sectioning angle and on the de-
gree of contraction of the muscle fiber (Rosenbluth,
1972; Kier 1985).

In all maturity stages, numerous profiles of neuronal
processes containing round, clear vesicles were found
(Fig. 4). These processes contact CMP and SMR muscle
fibers at presumptive chemical synapses with clefts about
20 nm wide (Fig. 4C). A rich, ramifying network of these
processes exists within the zone of CMP fibers, and indi-
vidual processes appear to contact multiple fibers (Fig.
4A, B). Within the SMR fiber zone, nerve processes are
less prominent and appear to run in grooves of the mus-
cle fibers (Fig. 4D). The cross-sectional areas of synaptic
vesicles were measured at a magnification of 240.000 on
scanned micrographs, and vesicle diameters were calcu-
lated by assuming a spherical shape. Vesicles from pro-
cesses associated with SMR fibers (mean diameter of
39 nm ± 0.7 SEM) are significantly smaller than those
from processes associated with CMP fibers (mean diam-
eter of 45.8 nm ± 0.6 SEM). These values are signifi-
cantly different by Student's t test (df = 225, P< 0.005).

Quantitative aspects of muscle fiber maturation

In longitudinal sections, the circular mantle muscle
mass is divided by bands of radial fibers into rectangular
muscle segments (Figs. 1 A and 2A). The dimensions of
such a muscle segment, in a given section, can be de-
scribed by (i) its thickness (i.e., the distance between the
outer and inner tunics) and (ii) its width (i.e.. the distance
between two successive radial-fiber bands). Horizontal
sections through the entire body of a juvenile showed
that the thickness and the width of the muscle segments

SQUID MANTLE MUSCLE DEVELOPMENT

379

Figure 1. Transmission electron micrographs of superficial (outer) and central layers of mantle muscle
from a squid hatchling ( 1-2 days old). (A) Cross section of circular muscle fibers showing the outer layer
composed of 1-2 superficial mitochondria-rich fibers (SMR) and half of the central zone composed of
mitochondria-poor fibers (CMP). Radial fibers (RF; cut longitudinally) divide mantle muscle into rectan-
gular segments. N = nucleus; oT = outer tunics. Scale bar = 2 ^m. (B) Cross section of a single SMR fiber.
Many large mitochondria (Mil) are located in the central core. Mf = myofilaments. Scale bar = I nm. (C)
Cross section of several CMP fibers. Usually one mitochondrion is located in the central core of an indi-
vidual CMP fiber. Note the irregular shape of CMP fibers and the lack of any myofilament organization.
Scale bar = 2 ^m.

vary considerably along the mantle. The thickest and
widest segments are found about one-third of the way
from the anterior mantle end, whereas more anterior and
posterior segments become progressively thinner and
narrower. Transverse mantle sections, on the other
hand, show almost no variation in the thickness of the
muscle segments in a given section, although segments
become very narrow dorsally at the location of the pen.
In light of these results, the mantle region that contains
the thickest muscle segments was chosen for analyzing
the growth of the mantle muscle, and two animals of
each age group with similar dorsal mantle length (DML)
were studied.

In adults, muscle segments from this same area of the
mantle were analyzed. Although adult animals were part
of a spawning population, only very robust males that
showed no sign of skin damage or senescence were stud-
ied. We therefore consider it unlikely that any selective
"deconstruction" of the mantle muscle mass took place
prior to fixation (Giese, 1969; O'Dor and Wells, 1978).

A muscle segment in a hatchling (2 mm DML) en-
closes the profiles of about 20-22 circular muscle fibers,
of which 12-14 are CMP fibers (i.e., « 60%). During
growth, the number of fibers enclosed in a single muscle
segment increases considerably, but SMR and CMP fi-
bers increase at different rates (Fig. 5). By 8 weeks
(12 mm DML). the number of CMP fibers has increased
about 10-fold, whereas the number of SMR fibers in-
creases only 2.5-fold (Fig. 5 A). Thus, the relative propor-
tion of CMP fibers increases duringgrowth (Fig. 5B), and
by week 8, about 84% of the fibers in a muscle segment
are CMP fibers. This proportion is comparable to that
derived from analysis of a single muscle segment in a ma-
ture animal (110mm DML; muscle segment 2.8 X
0.12 mm), which revealed a total of about 12.000 circu-
lar fibers, 86% of which were CMP fibers (Fig. 5A, B).

Morphometric measurements on the ultrastructural
level for developing CMP fibers are summarized in Fig-
ure 6A. Mean cross-sectional area of CMP fibers doubles
between 1 week and 8 weeks of age and increases by a

380

T. PREUSS £7 AL.

Figure 2. Transmission electron micrographs of superficial (outer) and central layers of mantle muscles
from a juvenile squid (8 weeks old). (A) Cross section of circular muscle fibers showing the outer layer
composed of several SMR fibers and part of the central zone composed of CMP fibers. Radial fibers (RF)
are cut longitudinally. CMP fibers show a well-developed sarcoplasmic reticulum (SR). oT = outer tunics.
Scale bar = 2 ^m. (B) Cross section of a single SMR fiber. A continuous ring of myofilaments (Mf) sur-
rounds a massive core of large mitochondria (Mit). Scale bar = 1 ^m. (C) Cross section of a single CMP
fiber. The myofilament area is divided into trapezoidal blocks by SR and Z-bodies. Only a small proportion
of the fiber isoccupied by mitochondria (Mit). Scale bar = I ^m.

total of 3- to 4-fold in mature animals. This increase in
fiber cross-sectional area is almost exclusively due to an
increase in the filament and SR area. The mitochondrial
core contributes relatively little to the absolute fiber size
and, moreover, does not grow in proportion to the rest
of the fiber. As a consequence, the relative area occupied
by the mitochondrial core in CMP fibers decreases dur-
ing growth from 16.3% ± 0.6% SEM in 1-week-old ani-
mals to 6% ± 0.3% SEM in adults.

In Figure 6B, results of the same ultrastructural analy-
sis are presented for SMR fibers. The mean cross-sec-
tional area of SMR fibers shows a pattern of increase sim-
ilar to that described above for CMP fibers, although the
absolute fiber area is much larger in SMR fibers (Fig. 6 A,
B left). Growth in SMR fibers, however, reflects a sub-
stantial increase in both the filament and SR area and
the mitochondrial core. As a result, the proportional area
occupied by the mitochondrial core in SMR fibers re-
mains more-or-less constant at about 40%.

Functional maturation ol circular muscle fibers in
locomotion

The anatomical data described above indicate that the
circular muscle of the mantle changes in composition
during post-hatching maturation. At hatching, SMR and
CMP fibers contribute almost equally to the circular
mantle mass. Subsequent growth is mostly due to an in-
crease in the contribution of CMP fibers, and the relative
contribution of SMR fibers to the mantle mass in an
adult is only about 1 4%. These structural changes suggest
that the mantles of hatchlings and adults may have
different contractile and endurance properties. For ex-
ample, the endurance capabilities of SMR fibers should
be manifested in respiration-related slow swimming
(Bone el ai. 1995) or hovering (Zuev. 1966), and their
relative contribution to jet-propelled locomotion should
be most apparent at the earliest stages of maturation. To
test this idea, we compared the kinematics of mantle

SQUID MANTLE MUSCLE DEVELOPMENT

381

Figure 3. Transmission electron micrograph of mantle muscle fi-
bers from a juvenile squid (8 weeks old). Longitudinal section of circu-
lar muscle fibers showing portions of two SMR fibers and three CMP
fibers. Note the differences in mitochondrial content and in myofila-
ment (MF) staggering angle between SMR and CMP fibers. Mil = mi-
tochondria; iT = inner tunics. Scale bar = 2 /JPI

contractions in freely swimming 1 -week-old and adult
squid during hovering behavior.

Adults and juveniles show quite different locomotor
behavior during hovering. Adults move slowly back and
forth, either in a slightly head-down or head-up position,
due to gentle jets and undulatory fin movements. Juve-
niles, on the other hand, continuously bob up and down
in a definite head-down position. Although juveniles
beat their very small fins rapidly (up to 16 Hz), locomo-
tion at this stage is apparently driven primarily by jetting.
Comparison of the mantle kinematics in Figure 7 shows
that hovering juveniles (2.5 mm DML) produce jets
about twice as frequently (2.7 Hz) as do adults ( 1 .3 Hz).
In addition, the fractional change in mantle diameter in
juveniles ( = 32%) is about three times that in adults
(=12%; Fig. 7).

Identification of Na-channel protein in developing
mantle muscle

Both Na-channel antibodies described in this study
were produced against predicted sequence for the puta-

tive squid Na channel encoded by the cDNA GFLN1
(Rosenthal and Gilly, 1993). mRNA corresponding to
GFLN1 is expressed throughout the squid nervous sys-
tem, particularly in neurons, including those of the GFL,
whose axons are large, long, or both (Liu and Gilly,
1995). These tissues were therefore used to test the spec-
ificity of the Na-channel antibodies.

Results of the monoclonal antibody mAb^i^, with
GFL and cleaned axon samples are shown in Figure 8A.
A prominent band with an apparent molecular weight of
about 250 kD is present in both lanes. Control experi-
ments with secondary antibody alone gave no signal ( not
illustrated). Similar results were obtained using the poly-
clonal antibody Ab4X.i-576 with GFL samples (Fig. 8B). In
this case, a control experiment employing blocked-
Ab483-57(, (see Methods) demonstrates specificity. The
specificity of these antibodies for Na-channel protein is
also supported by the fact that both antibodies give sim-
ilar results, even though they are directed against two dis-
tinct portions of the protein encoded by GFLN 1 .

As detected by the polyclonal antibody, a specific (i.e.,
blockable) Na-channel band centered around 210-
220 kD is prominent in protein samples derived from
mantle muscle (Fig. 8C). The monoclonal antibody also
recognizes a comparable band in muscle tissue (not illus-
trated). Thus, it appears that Na-channel protein is rela-
tively abundant in mantle muscle tissue.

Results obtained with a control tissue expected to
show minimal Na-channel protein are also shown in Fig-
ure 8C. The cornea of the eye is a simple arrangement of
a layer of epithelial cells supported by a transparent layer
of muscle fibers (unpubl. obs.). Whole-cell patch clamp
recordings made, using established methods (Gilly et til..
1990, 1996), from enzymatically dissociated cells from
both layers failed to reveal the presence of any voltage-
gated Na currents (unpubl. results); the immunoblot re-
sults also fail to reveal a strong Na-channel band (Fig.
8C). The weak bands in the cornea lane may arise from
axonal membrane, because the muscle fiber layer of the
cornea presumably is innervated. These bands are
blocked in the control experiment (Fig. 8C).

Electrophysiological recordings of Na currents in cir-
cular muscle fibers of squid mantle have been reported,
and it was proposed that the muscle fibers with Na cur-
rents were small-diameter, CMP fibers (Gilly el ai,
1 996). To test this idea, mantle tissue was dissected from
an adult squid (see Methods) to provide samples of pure
CMP circular fibers (plus radial fibers) and samples con-
taining SMR fibers (plus contaminating CMP fibers as
well as radial fibers). The results of an immunoblot with
these samples are shown in Figure 9. The Na-channel
band is most prominent in the central-zone sample.

382

T. PREUSS KT AL

•j"-v

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L^

^;^i|if^,

Figure 4. Transmission electron micrographs of synaptic profiles contacting circular mantle muscle
fibers (juvenile: 8 weeks old). (A and B) Longitudinal mantle section (circular fibers in cross section). A
nerve process (NP) runs within the central muscle layer and forms putative synaptic contacts (arrows) onto
several CMP fibers. Note the relatively large dimensions of the synaptic profiles in relation to the size of the
muscle fiber. (C) Neuromuscular junction onto a CMP fiber. The synaptic profile is filled with round, clear
vesicles. (D) Synaptic profile running within a groove of an SMR fiber. Scale bars = 0.5 Aim for A. and
0.2 Aim for B-D.

much weaker in the superficial (inner/outer) sample, and
quite strong in the whole-mantle sample.

If Na channels are preferentially expressed in CMP fi-
bers, the relative abundance of Na-channel protein in
whole-mantle samples should increase during the post-
hatching period of maturation described in this study.
Mantle samples were therefore collected from squid dur-
ing tl: id and processed for immunoblotting. Re-

sults in Figure 10 confirm the predicted pattern. The
muscle-type Na-channel band of low apparent molecu-
lar weight (relative to the neuronal form detected in
brain; see also Fig. 8A) increases steadily in intensity be-
tween days 3 and 100 post-hatching. At this latter time,
the band is comparable to that in the adult squid. Be-
cause each lane in Figure 10 was loaded with the same
amount of protein, these results strongly suggest that the

SQUID MANTLE MUSCLE DEVELOPMENT

383

relative abundance of Na-channel protein in mantle
muscle is increasing during maturation.

Discussion

Comparison of the histology of the circular muscle of
squid mantle during development from hatchling to
adult reveals large changes in the size and number of the
muscle fibers. Moreover, clear changes in the relative
proportions of the different types of fiber are associated
with maturation; these, in turn, affect mantle kinematics
and jet-propelled locomotion.

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SMR fibers

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Figure 5. Recruitment of CMP and SMR fibers during mantle
growth. (A) Absolute fiber counts are from individual muscle segments
from hatchling, l-week-old. 8-week-old. and adult squid. (B| Histo-
gram of relative proportion of SMR and CMP fibers at different matu-
rity stages.

total fiber filament mitochondria

Figure 6. Characteristics of muscle fiber growth in CMP (A) and
SMR (B| fibers. Individual histograms are given for the total cross-sec-
tional fiber area, the filament and SR area, and the area occupied by
the mitochondrial core for three maturity stages (means ± SEM; N =
number ofanimals; n = number of measurements).

Mantle growth in squid is due to an increase in the size
of existing muscle fibers (hypertrophy) and to recruit-
ment of new fibers (hyperplasia). The extensive increase
in fiber number in a single muscle segment, however,
suggests that the latter mechanism is dominant in overall
growth, and may also be responsible for the rapid so-
matic growth rates reported in squid (Forsythe and Van
Heukelem. 1987). Similar growth mechanisms have
been found in other squid (Moltschaniwskyj, 1994) and
in teleost fish (Weatherley et ai. 1988).

On an ultrastructural level, hypertrophy in both SMR
and CMP fibers is based on a steady increase in the num-
ber of myonlaments and, in the case of SMR fibers, the

384

T. PREUSS ET AL.

1 week old

adult

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video frames [30/s]

Figure 7. Manlle kinematics analyzed from video recordings of
hovering or slow-swimming adult and juvenile squid. Mantle diameter
was measured at the widest point (dorsal view), and the fractional man-
tle diameter was calculated by defining the largest diameter in a given
measurement sequence as 100''! . Time resolution is 30 frames/s.

size of the mitochondrial core. CMP fibers, however,
show a selective hypertrophy of the myofilament and SR
area and thus add a disproportional amount of force-
generating capability at the individual fiber level. Never-
theless, hypertrophy of individual muscle fibers is lim-
ited by physiological constraints. For example, fibers re-

quiring rapid excitation-contraction coupling in the ab-
sence of a transverse tubular system need to be small in
diameter (Bone and Ryan, 1973; Bone el at., 1995). This
constraint would especially affect the presumptive fast-
twitch CMP fibers with their high myofilament volume-
to-surface ratio (Fig. 2). Such limitations on fiber diame-
ter would indeed necessitate extensive recruitment of
new CMP fibers (Fig. 5) to maintain rapid muscular re-
sponses as an animal grows throughout development.

Two biochemical results described in this paper sup-
port the idea that at least some CMP fibers display Na-
channel-based excitability. Such excitability is consistent
with the hypothesis that these are fast-twitch fibers re-
sponsible for all-or-none mantle contractions (Young,
1938; Gilly et a/., 1996). The first supporting evidence is
that Na-channel protein is primarily expressed in the
CMP fiber layer. Second, the increase of Na-channel pro-
tein seen in developing mantle is comparable to the se-
lective hypertrophy and hyperplasia of CMP muscle fi-
bers as shown by histology. The Na-channel band in
muscle tissue is of lower apparent molecular weight than
that detected in neuronal tissue (Figs. 8-10), and this
suggests that a distinct Na-channel isoform exists in mus-
cle. The exact relationship of this Na channel to the
GFLNl-derived channels, which have a predicted core-
polypeptide mass of 203 kD, is currently unknown.

Nerve terminals have been previously described in the
mantle of other squid (Bone eta/., 1981, 1982, 1995)and

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KigureS. Specificity of Na-channel antibodies determined with immunoblots of samples of giant fiber
lobe (GFL), cleaned giant axon. cornea, and mantle muscle. (A) mAbm,5.2i was used as undiluted hybrid-
oma supernatant. Secondary goat anti-mouse antibody dilution was 1:5000. (B) AbJKM76 was used at 1:
1000 dilution, while dilution of secondary goat anti-rabbit antibody was 1:10.000. (C) Ab48v!7t, used at I:
2000. dilution of secondary goat anti-rabbit antibody was 1 :5000. Samples were prepared as described and
separated on 5't SDS-PAGE gels. Each lane was loaded with lO^g of total protein. Following transfer and
antibody incubation, bands were detected by chemiluminescence reagent. A 260-280 kD band is detected
in GFL and clean axon by monoclonal (A) and polyclonal (B) antibodies. A specific 210-220 kD band is
present in mantle muscle, but not in cornea tissue (C). Blocking control was carried out as described in
Methods.

SQUID MANTLE MUSCLE DEVELOPMENT

385

d>

0)

200 -

Ab 483-576

Blocked

Figure 9. Identification of Na-channel protein in different layers of
mantle muscle. Mantle muscle blocks were dissected to represent CMP
fibers. SMR fibers, and whole mantle samples. Ab4K,.57^ was used at I:
1 000 dilution : secondary goat anti-rabbit antibody dilution was 1:5000.
Blocking control was prepared as described in Methods. Each lane was
loaded with 10 Mg of total protein. A 2 10-220 kD blockable band is
detected in all samples. The band is most prominent in the CMP fiber
sample, weaker in the SMR fiber sample, and strong in the whole-man-
tle sample. Although the CMP sample contains no SMR fibers, the
SMR sample does contain some CMP fibers.

more negatively buoyant than adults (Zuev. 1966), and
that, in contrast to adults, fin beating plays only a minor
role during hovering (and for locomotion in general;
v. Boletzky, 1982; Hoar et al., 1994).

The disproportional increase of CMP fibers, on the
other hand, suggests a higher demand for acceleration
power in adults than in juveniles. It is clear that CMP
fibers are necessary to produce the fast and powerful
mantle contractions required for the high accelerations
seen during escape- and attack-jets in all maturity stages
(Gilly et ai. 1991 ). Indeed, Packard (1969) showed that
acceleration power increases during development in Lol-
igo (i.e., an increase of power per unit weight of mantle
muscle during escape-jets), which would be consistent
with the disproportional increase of CMP fibers de-
scribed in the present study.

The disproportional increase of CMP fibers may also
reflect metabolic constraints due to the restricted capac-
ity of hemocyanin to deliver oxygen (Wells, 1983; O'Dor
et al., 1990). This might effectively limit the proportion
of aerobic SMR fibers in the mantle that can be supplied
with oxygen (O'Dor, 1988), despite the heavy vasculari-
zation of this layer (Bone el al., 1981). Even considering
the fact that SMR fibers can be supplied with oxygen di-
rectly by diffusion through the skin (Wells and Wells,
1 983; Portner, 1 994), the size of the SMR fiber layer that

in other muscles of Sepia and 0i7o/w.v(Graziadei, 1966).
Although there is no direct evidence, the likely func-
tional relationship between giant axons and CMP fibers
(see above) and the large size of the neuronal processes
contacting multiple CMP fibers (Fig. 4A, B) suggest that
these processes may be the terminal branches of the giant
axons. The origin of the terminal branches found within
the SMR fibers remains unknown. Nevertheless, the de-
tection of distinct size populations of vesicles in nerve
profiles associated either with CMP or SM R fibers is con-
sistent with the idea that different types of motor axons
innervate the two fiber types (Bone et al., 198 1 ).

Muscle fiber recruitment takes place at different rates
in CMP and SMR fibers during maturation; i.e.. the rel-
ative proportions of SMR to CMP fibers change progres-
sively from 1 : 1 in hatchlings to 1 :6 in adults. SMR fibers
possess a high relative abundance of mitochondria (Figs
1-3) and a high content of oxidative enzymes (Momm-
sen et al.. 1 98 1 ). which suggests that hatchlings and juve-
niles may have a higher demand for aerobic, fatigue-re-
sistant muscles than do adults. This is consistent with
our findings that the frequency and extent of mantle con-
tractions during hovering (slow swimming) are substan-
tially higher in juveniles than in adults (Fig. 7). These
differences undoubtedly reflect the fact that juveniles are

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Figure 10. Apparent increase in abundance of Na-channel protein
in de\ eloping mantle muscle. Mantle samples were collected Irom ma-
turing squid and processed as described. In addition, a brain sample
was included. Ab4n3.57B was used in 1 : 1000 dilution, secondary antibody
dilution was 1:5000. Each lane was loaded with 10 Mg of total protein.
The muscle samples show a 210-220 kD band of increasing intensity,
whereas the brain sample shows a 260-280 kD band.

386

T. PREUSS ET AL

can thus be ventilated v e restricted. These con-
straints would again favor Uu recruitment of anaerobic
CMP fibers during p.

A final faclcv • uie different ratios of SMR

and CMP fibei :i hatchlings and adults pertains

to growth-relat hunges in the forces acting on the
body of a squid during jet-propelled swimming such as
drag, thrust, and acceleration (Johnson el a/., 1972). In-
deed, hatchlings and adults live at regimes of low and
high Reynolds number respectively (Hoar el ui, 1994:
Moltschaniwskyj, 1995); which, in turn, determines the
relative importance of viscous and inertial effects on the
hydrodynamic resistance to motion (Blake. 1983). For
example, the movement of hatchlings ceases almost im-
mediately when jet-propelled thrust stops, whereas
adults coast over a considerable distance with a single jet
(unpubl. data). These observations are consistent with
the idea that viscous forces dominate over inertial forces
in hatchlings and vice versa in adults. Thus, to cover any
distance efficiently, hatchlings have to jet continuously
because they cannot coast and therefore have a higher
demand for aerobic, fatigue-resistant muscles than do
adults.

Increasing size, on the other hand, places an additional
constraint on the developing mantle muscle if rapid ac-
celeration is to be maintained during growth. Inertial re-
sistance is proportional to body mass, which increases as
the third power of DML. Driving muscle force, on the
other hand, is proportional to myofilament cross-sec-
tional area, which increases with the square of DML
(Daniel and Webb, 1987). Consequently, a dispropor-
tionate increase in fast-twitch CMP fibers would lead to
a greater driving force for a given body size and, by tend-
ing to compensate for the increased inertial resistance as
an animal grows, would help to maintain proper acceler-
ations during escape- and attack-jets.

Acknowledgments

This material is based upon work supported by the Na-
tional Institutes of Health under Grant No. NS-17510-
1 4 and by the National Science Foundation under Grant
No IBN-963151 1. We are also grateful to Gilbert Van
Dykhuizen and Reginald C. Gary (Monterey Bay Aquar-
ium, California) for providing juvenile squid and to Dr.
M. W. Denny for helpful comments on the manuscript.

Literature Cited

Anisellcm, J., and G. Nicaisc. 1980. I Jltrastructural study of muscle
: >and their connections in the digestive tract of Sepia qfficinalis.

!/< rose Cylol 12(2): 214-231.

Blake. . 1983. Fish Locomoiion Cambridge University Press,
Cai

Bolelzky, S. v. 1982. Developmental aspects of the mantle complex
in coleoid cephalopods. Malacologia 23( 1 ): 1 65- 1 75.

Bolelzky, S. v. 1987. Juvenile behaviour. Pp. 45-60 in Ceplialopod
Lite Cycles — 1'nlitme II — Comparative Renews. P. R. Boyle, ed.
Academic Press. London.

Bone, Q., and K. P. Ryan. 1973. The structure and innervation of lo-
comotor muscles of salps (Tumcata: Thalicea). J Mar Biol. I s\oc
I'. K 53:873-883

Bone, Q., A. Pulsford, and A. D. Chubb. 1981. Squid mantle muscle.
J.Mur Biol . Issoc T A .61:327-342.

Bone, 0- A. Packard, and A. L. Pulsford. 1982. Cholinergic innerva-
tion of muscle fibres in squid. J Mar. Biol Assoc. U. A' 62: 193-
199.

Bone, Q., E. R. Brown, and M. Usher. 1995. The structure and phys-
iology of cephalopod muscle fibers. Pp. 301-329 in Cep/ialopo</
Neurobiology, N. J. Abbott, R. Williamson, and L. Maddock. eds.
Oxford University Press, New York.

Chen, D. S., G. Van Dykhuizen, J. I lodge, and W . F. Gilly. 1996. On-
togeny ot copepod predation in juvenile squid (Lolit>o opalescens).
Hml Hull 190:69-81.

Daniel, I. L., and P. W. Webb. 1987. Physical determinants of loco-
motion. Pp. 343-369 in Comparative Physiology: Life in Water anil
On Land. P. Dejours. L. Bolis, C. R. Taylor, and E. R. Weibel. eds.
IX-Liviana Press, Padova.

Forsythe, J. W., and W . F. Van Heukelem. 1987. Growth. Pp. I 35-
156 in Ceplialopod 1. lie Cycles — I'oliinic II — Comparative Re-
views. P. R. Boyle, ed. Academic Press. London.

Giese, A. C. 1969. A new approach to the biochemical composition
of the mollusc body. Oceunogr. Mar. Biol. Annu Rev 7: 1 75-229

Gilly, W. F., M. T. Lucero, and F. T. Horrigan. 1 990. Control of the
spatial distribution of sodium channels in giant fiber lobe neurons
of the squid. Neuron 5: 663-674

Gilly, W . F., B. Hopkins, and G. O. Mackie. 1991 . Development of
giant motor a.xons and neural control of escape responses in squid
embryos and hatchlings. Biol. Hull 180: 209-220.

Gilly, W. F., T. Preuss, and M. B. McFarlane. 1996. All-or-none
contraction and sodium channels in a subset of circular muscle fi-
bers of squid mantle. Biol. Bull 191: 337-340.

Gonzalez-Santander, R.. and E. S. Garcia-Blanco. 1972. Ultrastruc-
ture of the obliquely striated or pseudostnated muscle fibers of the
cephalopods: Sepia, Octopus and Eledone. J Suhmierose. Cylol. 4:
233-245.

Gordon, D..D.Merrick, I). A. \\ollner.and\V. A.Catterall. 1988. Bio-
chemical properties of sodium channels in a wide range of excitable
tissues studied with site-directed antibodies. Biochemistry 27:
7032-7038.

Gosline.,1. M..J. D. Sleeves, A. D. Ilarman.and M. E. DeMonl. 1983.
Patterns of circular and radial muscle activity in respiration and
jetting of the squid I.oligo opalescens. J E\p. Biol. 104: 97-109.

Graziadei, P. 1966. The ultrastructure of the motor nerve endings in
the muscles of cephalopods. J. iltiastnicl Res 15: 1-13.

Hoar, J. A., E. Sim, D. M. Webber, and R. K. O'Dor. 1994. The role
of fins in the competition between squid and fish. Pp. 27-43 in Me-
chanics and Physiology ol Animal Swimming. L. Maddock, Q.
Bone, and J. M. V. Rayner, eds. Cambridge University Press. New
York.

Johnson, W., P. D. Soden, and E. R. Trueman. 1972. A study in jet
propulsion: an analysis of the motion of squid. Loligo vulgaris. .1
E\p. Biol 56: 155-165.

Kier, W. M. 1985. The musculature of squid arms and tentacles: ul-
trastructural evidence for functional differences. J Morphol 185:
223-239.

Kier, W. M. 1988. The arrangement and function of molluscan mus-

SQUID MANTLE MUSCLE DEVELOPMENT

387

cle. Pp.21 I -252 in The Mollusea. I'ol II Form and Function, E. R.
Trueman and M. R. Clarke, eds. Academic Press, San Diego.

Knudson.C. M., N.Chandhari, A. H. Sharp, J. A. Powell, K. G. Beam,
and K. P. Campbell. 1989. Specific absence of the « suhunit ot'the
dihydropyridine receptor in mice with muscular dysgenesis. / Binl.
Client 264: 1345-1348.

Liu, T. I., and \V. K. Gilly. 1995. Tissue distribution and subcellular
localization of Na+ channel mRNA in the nervous system of the
squid Loligo opalescens. Reeepi. Channels^: 243-254.

Matsuno, A. 1987. Ultrastructural studieson developing oblique-stri-
ated muscle cells in the cuttlefish. Se/iiel/u iuponieu Sasaki, '/.not.
Set. 4. 53-59.

Moltsehaninskyj, N. A. 1994. Muscle tissue growth and muscle fibre
dynamics in the tropical loliginid squid Plintnloligo up. (Cephalo-
poda: Loliginidae). Can. J Fish. .U/tiul. Sei. 51: 830-835.

Moltschaniwskyj, N. A. 1995. Changes in shape associated with
growth in the loliginid squid Pholo/olii;o V morphometric ap-
proach. Can J /<><)/. 73: 1335-1343.

Mommsen, T. P., J. Ballanlyne. D. MacDonald, J. Gosline, and P. \V.
Hochachka. 1981. Analogues of red and white muscle in squid
mantle. Proe. \all. .laid Sei. USA 78: 3274-3278.

O'Dor, R. K. 1988. Limitations on locomotor performance in squid.
J.AppI Phvsiol. 64(1): 128-134.

O'Dor, R. K., and M. J. Wells. 1978. Reproduction versus somatic
growth: hormonal control in Oelopus vnlguns. .1 K\p Binl. 77: 1 5-
31.

O'Dor, R. K., E. A. Foy, P. L. Helm, and N. Balch. 1986. The loco-
motion and energetics of hatchling squid. llle.\ illecebrosus. .Inter.
Muliicol. Bull. 4(1): 55-60.

O'Dor, R. K.. H.O. Portner, and R. E. Shadwick. 1990. Squid as elite
athletes: locomotory. respiratory, and circulatory integration. Pp.
481-503 in Stiuiil As Experimental Animals, D. L. Gilbert. W. J.
Adelman, and J. M. Arnold, eds. Plenum Press, New York.

Packard, A. 1969. Jet propulsion and the giant fibre response of Lnl-
it><>. \ainre 221: 875-877.

Portner, II. O. 1994. Coordination of metabolism, acid-base regula-
tion and hemocyanin function in cephalopods. Mar Fresh. Bchav
Physiol. 25: 131-148.

Rosenbluth, J. 1972. Obliquely striated muscle. Pp. 389-419 in The
Strtieltire and Function i>l Muscle — I'ol. I. G. H. Bourne, ed. Aca-
demic Press. New York.

Rosenthal, J. J. C. 1996. Molecular identification of the ion channels
underlying the action potential in the squid giant axon. Ph. D. The-
sis. Department of Biological Sciences, Stanford University.

Rosenthal, J., I. C., and \V. K. Gilly. 1993. Amino acid sequence of a
putative sodium channel expressed in the giant axon of the squid
Loligo opule.Miis. Proe. Nail. .lead. Sei. US. I 90: 10026-10030.

Ward, D. V ., and S. A. \Yainwright. 1972. Locomotory aspects of
squid mantle structure. ./. /.ool. (Loiui i 167: 437-449.

\\eatherley, A.M., M.S. Gill, and A. F. Lobo. 1988. Recruitment
and maximal diameter of axial muscle fibers in teleosts and their
relationship to somatic growth and ultimate size. J. Fish Binl. 33:
851-859.

Wells, M. J. 1983. Circulation in cephalopods. Pp. 239-290 in The
Mollusea. I'ol 5, K.. M. Wilbur, ed. Academic Press, London.

Wells, M. J. 1988. Mantle muscle and mantle cavity in cephalopods.
Pp. 287-300 in Tlw.MoHiiseii — Form uiul Fnnelion. E. R. Trueman
and M. R. Clarke, eds. Academic Press. San Diego.

Wells, M. J., and J. Wells. 1983. The circulatory response to acute
hypoxia in Oetopns. J K.\p. Biol 104: 59-7 I .

Young, J. Z. 1938. The functioning of the squid giant nerve fibres of
the squid./ E.\p. Biol. 15: 170-185.

/uev, G. V. 1966. Characteristic features of the structure of cephalo-
pod molluscs associated with controlled movements. Fisheries Re-
search Board ol Canada Translation Series No. 101 1 1968.

Reference: Bin/ Bull 192: 388-398. (June. 1997)

Fine Structure of the Apical Ganglion and Its
Serotonergic Cells in the Larva of Aplysia californica

RENE MAROIS1 * AND THOMAS J. CAREW2

1 Inicrdeparlmental Neuroscience Program and ~ Departments of Biology and Psychology.
Yale Universitr. New Haven. Connecticut 06520

Abstract. The apical ganglion is a highly conserved
structure present in various marine invertebrate larvae.
Although one of the hallmarks of this ganglion is the
presence of serotonergic cells, little is known about the
structure and function of these cells. We have examined
this ganglion in larvae of the marine mollusc Aplysia
with light- and electron-microscopic immunocytochem-
istry. The results indicate that the cellular composition
of the apical ganglion of Aplysia is very similar to that of
other opisthobranchs. It consists of three classes of sen-
sory cells (ampullary, para-ampullary, and ciliary tuft
cells) and of other nerve cell types. Almost a third of the
cells in the apical ganglion of Aplysia are serotonergic,
and these can be divided into two classes: three para-am-
pullary and two interneuronal cells. All of the serotoner-
gic cells extend an axon into the central nervous system.
The variety of sensory and serotonergic cell types sug-
gests that each type processes distinct attributes of the
sensory environment. We argue that the apical ganglion,
by virtue of its serotonergic cells, is well-suited to play
important roles in the integration of sensory information
to achieve proper motor adaptation to variable seawater
conditions.

Introduction

One of the most highly conserved neuronal structures
across phyla is the apical ganglion (AG) (Nielsen, 1994).
Also referred to as the apical sensory organ, the apical
organ, or the cephalic sensory organ, the AG has so far
been extensively described in the embryos, larvae, or
both of cnidarians (Chia and Koss, 1979; Fukui, 1991).

1 7 October 1 996; accepted 12 April 1997.

:u1dress: Department of Diagnostic Radiology. Yale Uni-
versity Si h i.i Medicine. New Haven, CT 06520-8042.

turbellarians(Lacalli, 1982; 1983), polychaetes (Lacalli.
1981; 1984), molluscs (Bonar, 1978; Chia and Koss,
1984; Page, 1 992, Tardy and Dongard, 1993; Kempf and
Page, 1995); brachiopods (Hay-Schmidt. 1992); phoro-
nids (Hay-Schmidt, 1989; Lacalli, 1990), echinoderms
(Bisgrove and Burke. 1986; Chia el a/.. 1986; Nakajima.
1988; Nakajima et a/.. 1993), and hemichordates (Dau-
tov and Nezlin, 1 992). A homologous structure may also
occur in cephalochordates (Lacalli, 1994; Lacalli et a/..
1994). Although the precise function of this organ re-
mains to be determined, its subcellular and cellular
structures, as well as its superficial anterior position just
above the mouth, have led to the suggestion that the AG
is likely to be involved in sensing ambient water condi-
tions during locomotion, feeding, and metamorphosis
(Bonar. 1978; Chia and Koss, 1984).

Despite considerable variations in their fine structure,
the apical ganglia of all species examined to date appear
to share two characteristics: first, the presence of modi-
fied epithelial or subepithelial cells that give rise to an
external tuft of nonmotile cilia; and second, the presence
of serotonergic cells (Bisgrove and Burke, 1986. 1987;
Nakajima, 1988; Hay-Schmidt, 1990, 1992, 1995;
Kempf el al.. 1991; Nakajima et a/., 1993; Moss et a/..
1994; Lacalli, 1994; Kempf and Page, 1995). Despite
their pervasive nature, very little is known of the struc-
ture, identity, and functions of the serotonergic neurons
in the apical ganglion.

The present study describes the fine structure of the
AG and its serotonergic cells in the larva of the marine
mollusc Aplysia californica. In addition to shedding
some light on the biology of the serotonergic (5HT) cells,
this detailed study of the serotonergic constituents of the
AG also aims at achieving a better understanding of the
functions of this anatomical structure. This work is also
the first to describe the presence of an apical ganglion in

388

APICAL GANGLION OF LARVAL ,1/V >.S7 I

389

Aplysiu. Surprisingly, despite the fact that this animal
has been a favorite preparation of neurobiologists and
the focus of a number of neurodevelopmental studies
(Saunders and Poole, 1910; Kriegstein, 1977a, b;
Schacher el at.. 1979a, b; Jacob, 1984). the apical gan-
glion has hitherto gone unnoticed in Aplysia.

Some of the results presented in this paper have been
previously reported in abstract form (Marois et at.. 1992.
1993).

Materials and Methods

Mariculture

Animals were collected and maintained as described
in Marois and Carew (1997a). Seven-day-old embryos
(7 days after oviposition), hatchlings (9 days after ovipo-
sition). and 2-day-oId larvae (1 1 days after oviposition)
were used in this study. In addition, older (Stage 2 and
3) larvae were used for histological characterization and
localization of the apical ganglion. The animals were
staged according to the criteria of Kriegstein ( 1 977 a).

Immunocytochemistry (ICC)

All the immunocytochemical and ultrastructural tech-
niques were performed as described in Marois and Ca-
rew (1997a). The specificity of the serotonin antibody
used has been previously demonstrated (Marois and Ca-
rew, 1997a). The results are based on semi-thin and ul-
tra-thin sectioning of the apical ganglion of at least 10
animals, and on whole-mount processing of at least 50
animals.

\\hole-tnoimt immunocytochemistry. Animals were
first anesthetized in a MgCl; solution isotonic to seawa-
ter for 5 min at room temperature (rt), followed by 8 min
on ice. Animals were then immersed in three changes of
ice-cold fixative solution (4% paraformaldehyde in Mil-
lonig's phosphate buffer saline [PBS]) for 30 min, and
then left in the fixative solution at 4°C for an additional
2.5 h (total fixation time: 3 h). After five 4-min washes in
PBS. Stage 1 -6 larvae were exposed to trypsin (Type 1,
Sigma, St. Louis, MO, 0.1% in PBS for 5 to 15 min at rt)
and specimens were then immersed in 4% Triton X-100
(TX-100) in PBS for 1 h, rinsed in PBS, exposed to 10%
EDTA in PBS for 45 min at rt to decalcify the shell, and
rinsed in PBS. This was followed by pre-incubation in
2% goat serum (GS), 0.5%. TX-100 in PBS for 1 h at 4°C,
and by a primary ( 1°) Ab incubation (rabbit anti-seroto-
nin, Incstar, Stillwater, MM; 1:650 in pre-incubation se-
rum) for 2.5 days at 4°C on a shaker. The animals were
then rinsed in PBS, pre-incubated in 2% GS in PBS for
1 h at 4°C, and immersed in secondary (2°) Ab solution
(fluorescein isothiocyanate [FITC]-linked goat anti-rab-
bit IgG. Sigma, St. Louis, MO, 1:50 in PBS with 2% GS,

0.5%. TX-100) for 2.5 h at 4°C, and rinsed in PBS. The
specimens were mounted in a 3:1 glycerine:PBS solu-
tion, viewed under FITC optics (excitation filter,
480 nm; barrier filter, 520 nm) on a Nikon Optiphot-2
microscope, and photographed with Ilford XP2 400 or
Kodak Ektachrome 400 film.

Sectioned tissue immunocytochemistry. Embryos and
larvae were prepared as above, with the following modi-
fications. 2° Ab (goat anti-rabbit IgG. Cappel; 1 :50 in 2%
GS, 0.5% TX-100 in PBS for 2 h at rt), and 3° Ab (rabbit
peroxidase anti-peroxidase (PAP), Cappel; 1:50 in 2%.
GS, 0.5% TX-100 in PBS for 2 h at rt). Following the
PBS rinses after the 3° Ab solution, the animals were pro-
cessed for horseradish peroxidase (HRP) reaction
( 1 5 min in 0.05%. DAB in PBS at rt; followed by 45 min
in 0.005% H2O: , 0.05%. DAB in PBS), rinsed in PBS, and
dehydrated in an alcohol series (50. 70, 80, 95, and 3x
100%. ethanol), and infiltrated in Epon (3X propylene
oxide (PO): 2:1 PO:Epon; 1:2 PO:Epon, and pure Epon).
A few animals were not processed for ICC and instead
were stained with the Richardson's solution ( Richardson
et a/.. 1960). All animals were sectioned on aSorvall MT-

2 ultramicrotome. Sections were viewed under a Nikon
Optiphot-2 microscope and photographed with Kodak
T-MAX 100 film.

Immune-electron microscopy

Embryos or larvae were anesthetized as described
above. They were fixed for 30 min on ice and then for

3 h at 4°C on a shaker in 4% paraformaldehyde, 0.12%
glutaraldehyde, 20% sucrose in Millonig's phosphate
buffer (PB). After PBS rinses, the animals were decalci-
fied in 10%. EDTA in PBS (PB with 0.9%. NaCl) for
45 min at rt, exposed to 1% NaH:B4 in PBS for I h at rt,
rinsed in PBS, exposed to 0.05% trypsin for 15 min, and
rinsed in PBS. This was followed by freeze-thawing: the
animals were first immersed at 4°C for 2 h in cryoprotec-
tant (25%. sucrose, 10% glycerol in 0.1 M PB), then sub-
sequently dipped in liquid Ni-cooled iso-pentane and in
liquid N:, and rinsed in PBS. The 1°. 2°, and tertiary (3°)
Ab incubations were performed as for sectioned tissue
ICC except that the pre-incubations lasted 2 h and no
TX-100 was present in the pre-incubation and incuba-
tion solutions. The HRP reaction was performed as for
sectioned tissue ICC, except that a metal-enhanced DAB
substrate was used (Pierce, Rockford, IL; 45-60 min in-
cubation followed by PBS rinses). The animals were sub-
sequently osmicated in 2% OsO4 in PBS for 1 h at rt on a
shaker, and dehydrated and infiltrated in Epon as de-
scribed for sectioned tissue ICC. Serial silver and gold
sections were cut on a Sorvall MT-2 microtome ^d col-
lected serially on either Formvar-coated slot cr <" grids
or Thin-200 copper grids (EMS, Fort Washington, PA).

390

R. MAROIS AND T. J. CARHW

The sections were viewed ;• ider a Philips 300 or Zeiss
EM-10 transmission el on microscope at 80 kV.

Ultrastructun

Anr u/ed as described above and

fixed in '^ <•• aldehyde, 20% sucrose in 0.1 M PB

for 30 min on i z followed by 2.5 h at 4°C on a shaker.
After iiS rinses, animals were osmicated, rinsed

in PBS, decalcified in 10% EDTA in PBS, rinsed in PBS,
and dehydrated and infiltrated as described above. Silver
and gold sections were cut and collected as described
above. The grids were then stained for 1 2 - 1 5 min in 3%>
uranyl acetate and for 5 min in 0.3% lead citrate, and
viewed as described above.

Results

General observations

The veliger of Aplysia califomica possesses an apical
ganglion (AG), located above and between the cerebral
ganglia (Figs. 1. 2). It sits atop the cerebral commissure
and is composed of 15 to 20 cells, many of which are
heavily ciliated (Fig. 2). Since this structure is strikingly
similar to the AG in the nudibranch Rostanga pulchra
(Chia and Koss, 1984), we have adopted the same no-
menclature to describe the components of this ganglion
in Aplysia. As in Roslunga. three major types of cells
were observed in the AG of Aplysia: ( 1 ) four ampullary
cells with large, heavily ciliated lumina; (2) three para-
ampullary cells that extend one or two cilia from their
apical surface; and (3) two ciliary tuft cells that project

numerous long cilia from their apical surface. All of these
cells send anterior apical projections that follow one of
three tracts (left, right, and medial tracts) to reach the
epithelial surface (Fig. 3). In addition to these cell types,
the AG contains a few posterior cells that do not have
any apical projections, and a dense neuropilar region lo-
cated posterior and medial to all of these cells (Fig. 2B).
This neuropil is in contact with the underlying cerebral
commissure (Fig. 2A). Immunocytochemical staining
for serotonin reveals that five cells of the AG are seroto-
nergic (the three para-ampullary cells and two posterior
cells; Fig. 4A). These are the only serotonergic cells in the
entire CNS of the newly hatched Aplysia veliger (Marois
andCarew, 1997a).

Fine structure of the major cell types in t/ieAG

Ampullary cells. These are four centrally positioned
cells, each containing a large lumen densely populated
with cilia (Figs. 2, 3A, 4B). Posteriorly the cells border
the neuropilar region, while anteriorly their cytoplasm
funnels to a constricted neck to expand again as a swell-
ing at the epithelial surface (Figs. 3A, 4B). Numerous mi-
crovili and one or two cilia protrude externally from
these swellings (Figs. 3 A, 4B). These apical cilia are dis-
tinct from the cilia in the lumen. The latter are entirely
contained inside the lumen and do not pierce through
the epithelial surface (Figs. 3A. 4B). The bases of these
internal cilia are anchored into the cytoplasm of the am-
pullary cells (Fig. 3B). None of the ampullary cells are
serotonergic.

Para-ampullary cells. This set of three cells surround-

Kigure 1. Position of the apical ganglion (AG) in Aplyxia veligers. (A) Cross-section of a Stage 2 larva
showing the AG (arrow) between the cerebral ganglia (CG) and above the oesophagus (O). (B) Horizontal
section through the AG (asterisk). E, eye; PC. pedal ganglion. Scale bar: A, 20 /jm; B. 15 yum.

APICAL GANGLION OF LARVAL Al'l 1 SI 1

391

^tflETSSW

^^ • . ^•••-•\ m , H.

;\M* : •'. . v.Ww >

;«-l- .' 1 Jf..---^

B

Figure 2. Llltraslructure of the apical ganglion. (A) Cross-section shows the AC above the cerebral
commissure (cc) and between the cerebral ganglia (CG). Note the cilia (arrow) in the ampullary cells. (B)
Horizontal section through the AG. Anterior is up. Note ciliary bundles (white arrow) inside the ampullary
cells and a neuropil (N) posterior to these cells. O, oesophagus. Scale bar: A and B, 2 nm.

ing the ampullary cells is immunoreactive for serotonin:
two of the cells are laterally positioned, and the third is
centrally located between the two pairs of ampullary cells
(Fig. 4A). These cells correspond to the para-ampullary
cells of Rostanga (Chia and Koss, 1984). Each para-am-
pullary cell sends an anterior projection to the epithelial
surface of the apical ganglion (Figs. 4, 5. 6). The projec-
tions of the lateral pair follow the lateral tracts (Figs. 4,
6), whereas the projection of the median cell emerges
from the ventral side of the cell and bends anteriorly to
reach the epidermal surface beneath the central tract

(Fig. 5B). These three processes enlarge at the epithelial
surface. From the swellings, one or two short, curly cilia
extend into the external environment (Figs. 3A, 4B, 5A).
The swellings also contain numerous mitochondria
(Figs. 3A, 4B) and bear microvili (Fig. 3A). These expan-
sions are linked together and to the adjacent epithelial
cells by zonula adherens (Fig. 3A). In addition to their
anterior projections, each of these three cells also sends
a projection into the central neuropil (Fig. 4A), which
appears to be heavily populated with serotonergic fibers
(Fig. 5A). The distant target tissues of these central neu-

392

R MAROIS AND T. J. CAREW

Figure 3. Cilia in the apical ganglion. Horizontal sections, anterior is up. (A) The apical projections of
the AG cells follow tracts to the epithelial surface. The left lateral tract consists of two ampullary cell
processes (A) and one para-ampullary cell process (P); the median tract has a single ampullary cell projec-
tion (A). The apical swellings of these projections extend cilia (arrow) and microvili (open arrow). The
swellings are linked to each other and to the adjacent epithelial cells with zonula adherens (arrowheads).
An ampullary cell contains an internal ciliary bundle (ci). (B) A ciliary bundle (ci) is attached to the cyto-
plasm of an ampullary cell, m, mitochondria: nu, nucleus. Scale bar: A. 0.5 Mm; B. 0.5 ^m.

ropilar projections have been described elsewhere (Mar-
ois and Carew, 1 997b). Under conventional electron mi-
croscopy, the cytoplasm of the para-ampullary cells ap-
pears very granular and contains lipid yolk droplets,
mitochondria, and small (40-60 nm) clear and dense-
core vesicles (Fig. 4C). Unlike the ampullary cells, the
para-ampullary cells do not contain lumina densely
packed with cilia.

Ciliary tuft cells. A bilateral pair of rectangular cells is
located at the anterior and ventral edge of the AG (Fig.
6). Each cell sends a cytoplasmic projection anteriorly
into the lateral tracts, underneath the projections of the
ampullary and para-ampullary cells. After narrowing in
the lateral tracts, the projections expand considerably at
the apical surface of the AG (Fig. 7). At least five long
cilia emerge from each of these two large apical swellings
(Figs. 6, 7). These cilia are anchored to the swellings by
long ciliary rootlets and dense basal bodies (Fig. 7). Mi-
tochondria are found at the bases of the cilia. The swell-
ings are linked by zonula adherens to epidermal cells, to
the serotonergic swelling of the unpaired median para-
ampullary cell, and to each other.

Oilier serotonergic and non-serotonergic cells. There

are six to eight other cells in the AG at hatching. Two

of them are immunoreactive for serotonin (Figs. 4A, 6).

e 5HT cells are located immediately posterior and

tiy medial to the lateral pair of para-ampullary sero-

tonergi cells (Figs. 4A, 6). Each of these cells extends a

single : -ess into the neuropil of the AG (Fig. 4A).

Since these cells have not been identified in Rostanga
(Chia and Koss, 1984) and since they do not extend any
apical projections, they are referred to as serotonergic in-
terneurons. Little is known about the remaining cells of
the AG except that they do not appear to have any apical
processes and are not immunoreactive for serotonin.
The structure of the apical ganglion and of its principal
cellular constituents in Aplysia is summarized in Fig-
ure 8.

Discussion

General struct we of the AG o/'Aplysia and other
gastropods

The AG of Aplysia is strikingly similar to the apical
ganglion of the opisthobranch Rostanga pulchra (Chia
and Koss, 1984). They both have the same number and
major types of cells: four ampullary cells, three para-am-
pullary cells, and two ciliary tuft cells. The only notable
difference is that the cell bodies and apical projections of
the ciliary tuft cells are ventral to the other cells in the
AG of Aplysia. but they seem to occupy a dorsal position
in Rostanga (Chia and Koss. 1984).

It is intriguing that the structure of the AG in both
Aplysia and the nudibranch Rostanga (Chia and Koss,
1 984) differs markedly from that of another nudibranch.
Phestilki sihtigae (Bonar. 1978): Ciliary tuft cells, char-
acterized by apical projections having many long, deeply
rooted cilia, appear to be absent in Phestilla. Neverthe-

APICAL GANGLION OF LARVAL I/'/ T.SYI

393

~6 4? k

Figure 4. Serotonergic cells of the apical ganglion. Horizontal sections, anterior is up. (A) The lateral
(P) and unpaired median (LI) para-ampullary cells are serotonergic Two interneurons (I) posterior to the
lateral para-ampullary cells are also serotonergic. Note the apical projection (small black arrows) of a para-
ampullary cell, the apical swelling of another (black arrowhead), and the central projections (small white
arrows) of a para-ampullary cell and an interneuron. (B) Apical swelling (arrow) of a serotonergic para-
ampullary cell. A cilium (arrowhead) extends from the external surface of the swelling. Note the cilia (ci)
and apical process of the adjacent ampullary cell (A). (C) Fine structure of a para-ampullary cell processed
for conventional electron microscopy. The cytoplasm contains lipid yolk droplets ( L). and clear and dense-
core vesicles (arrowheads). A. ampullary cell: ci, ciliary bundle; O. oesophagus; nu, nucleus; LI, unpaired
median para-ampullary cell. Scale bar: A. 2 ^m: B 0.5 urn; C. 0.5

less, other cell types appear structurally similar to those
ofAplysia. Thus, the ampullary cells ofAply.sia and Ros-
tanga are very similar to the flask-shaped cells of Phcs-

lilla (Bonar, 1978). with the notable difference that the
ciliary bundles in PlH-stilhi arc not restricted to the lu-
men but extend to the surface of the animal. Likewise.

394

R. MAROIS AND T. J. CAREW

Figure 5. Serotonergic cells of the apical ganglion. (A) Dorsal view, anterior is up. Whole-mount im-
munocytochemistry in a late-stage embryo shows central projections in the neuropil (arrowhead) and api-
cal projections with terminal cilia (small arrows) of the para-ampullary cells. (Bl Frontal whole-mount ICC
shows the apical projection terminating as a swelling (arrow). P, para-ampullary cell. Scale bar: A, 10 ^im;
B, 5/jin

;

Figure 6. Oblique horizontal section through the apical ganglion showing two serotoncrgic para-am-
pullary ( P) cells and interneurons ( I ) and a ciliary tuft cell (asterisk). Note the apical projection (arrowheads)
of a para-ampullary cell, and the long cilia (arrow) of a ciliary tuft cell. O. oesophagus. Scale bar: 2 ^m.

the cells referred to as support cells in Phe.villa (Bonar,
1978) strongly resemble the para-ampullary cells of Rox-
langa and Aply.siu. They surround the flask-shaped cells.

and each has a narrow process extending to the surface
and giving rise to microvilli and one or two cilia. Al-
though the striking similarities between the AG cells of

APICAL GANGLION OF LARVAL AI'LYSI I

395

Figure 7. Apical projections of the ciliary tuft cells. Horizontal sec-
tion, anterior is up. b, basal bodies of cilia: r. rootlet of cilia: z. zonula
adherens. Scale bar, 0.5 ^m.

Rostanga. P/iestilla, and Aplysia suggest that these struc-
tures are truly homologous, more detailed phyletic stud-
ies of the AG in various prosobranchs and opistho-
branchs are required before the homology is demon-
strated conclusively.

Functions of the ciliated cells of the apical ganglion

The morphology of the three ciliated cell types of the
AG is very similar to the known morphology of epithelial
chemoreceptorand mechanoreceptor cells in sensory or-
gans of adult Aplysia and other invertebrates and verte-
brates (Laverack, 1974; Wright. 1974; Emery and Aude-
sirk. 1978; Altner and Prillinger. 1980; Dorsett. 1986).
In addition, ultrastructural examinations of the cilia of
apical ganglion cells have consistently indicated that, un-
like the motile cilia of the velar cells, these cilia are non-
motile: They have a 9 + 2 microtubular arrangement
lacking the dynein arms that confers motility to the cilia
(Bonar. 1978; Dorsett, 1986; Nakajima, 1988). Taken
together, these findings strongly suggest that the ciliated
cells of the AG are sensory cells. The additional finding
that these ciliated cells can be classified into three mor-
phological groups in Aplysia and Rostanga implies that
each class may serve a distinct sensory function. Chia
and Koss (1984) have proposed that in Rostanga the in-
ternal ciliary bundles of the ampullary cells have a role
in vibration or pressure detection, the long numerous
cilia of the ciliary tuft cells act as distance chemorecep-
tors, and the short curly cilia of the para-ampullary cells
act as contact chemoreceptors. Although only the para-
ampullary cells were examined for central axonal projec-
tions in the present study, in Rostanga the three cell
types extend axons into the AG neuropil (Chia and Koss.

1984). These results suggest that information about the
veliger's aquatic surroundings is first gathered by the pri-
mary sensory ciliated cells of the AG and subsequently
conveyed to the CNS of the animal.

Serotonergic cells in the apical gang/ ion of Aplysia and
other gastropods

Despite the notable differences in the structure of the
AG across phyla, when serotonergic neurons have been
looked for in this organ, they have invariably been
found. Serotonergic neurons have been observed in the
AG or apical region of the nudibranch Berghia (Kempf
el al.. 1991; Kempf and Page, 1995), the prosobranch
Haliotis (Barlow and Truman, 1992), phoronids (Hay-
Schmidt, 1990). polychaetes (Hay-Schmidt, 1995),
brachiopods (Hay-Schmidt, 1992) and various echino-
derms (Moss et cil.. 1994; Bisgrove and Burke, 1986;
1987; Nakajima, 1988; Nakajima et al., 1993). In addi-
tion, serotonergic neurons are associated with the ex-
treme anterior end of the amphioxus nerve cord (Hol-
land and Holland, 1993), a region postulated to be de-
rived from the apical organ of invertebrate ancestors
(Lacalli, 1994; Lacalli et al.. 1994). However, it is diffi-
cult to compare the fine structure of the serotonergic cells
of Aplysia with those of other species because these other
studies have predominantly been limited to a whole-
mount, light microscopic level of analysis. Nevertheless,
serotonergic cells with the gross morphology of the para-
ampullary cells of.lp/ysia (a short apical projection and
a basal axonal process) have been observed in the apical
organs of some echinoderms (Bisgrove and Burke, 1986;
1987; Nakajima, 1988). and serotonergic cells extending
only basal processes, similar to the serotonergic interneu-
rons of Aplysia. have been observed in polychaete and
brachiopod larvae (Hay-Schmidt. 1992; 1995). Although
these findings could be interpreted as indicating that the
two classes of serotonergic cells observed in Aplysia may
be differentially represented in other phyla, an examina-
tion of the fine structure of the 5HT cells in these other
animal groups may reveal instead that they do not corre-
spond to either of the two serotonergic classes in Aplysia.

Functions of the sero/onergic cells in the AG

The serotonergic cells make up about a third of the
entire cellular population of the AG. This sheer number
suggests that serotonergic cells are important compo-
nents of the AG. Furthermore, the distinct morphology
of the para-ampullary and interneurons suggests that
these two types of serotonergic cells serve different func-
tions. As mentioned above, the three para-ampullary se-
rotonergic cells are probably sensory (Chia and Koss,
1984). Because the serotonergic interneurons do not pos-
sess an apical process or any cilia, it is unlikely that they

396

R MAROIS AND T. J. CARI-W

Figure 8. Schematic diagram of the apical ganglion of larval .lply.ui The diagram illustrates both
horizontal and frontal plane views. The three para-ampullary (P| and the two interneuron (1) cells are
serotonergic. All 5HT cells send projections into the neuropil (N). The ampullary (A) and the ciliary tuft
(C) cells are also illustrated. Except for the 5HT interneurons, all the labeled cell types extend apical pro-
cesses to the surface of the animal. See text for details, ci. cilia; b. ciliary bundle; O. oesophagus.

act as sensory receptors. Instead, they may act as inter-
neurons along the information pathway that links the
AG to the rest of the CNS and to effector tissues. These
cells, as well as the para-ampullary cells, extend an axo-
nal process into the AG neuropil and the cerebral com-
missure; these processes subsequently course in various
directions to reach and innervate muscles, nerve cells,
and ciliated cells of the velum (Marois and Carew,
1997b). There is circumstantial evidence that the para-
ampullary cells provide the serotonergic input to the ve-
lum and the interneurons innervate the CNS of Aplysia
(Marois and Carew, 1997b). There is also biochemical
evidence from bath-application experiments that seroto-
nin exerts a potent modulatory effect on ciliary activity
and locomotion of various molluscs (Koshtoyants ct ui.
1961; Diefenbach et a/., 1991; Marois and Hofstadter,
unpubl. obs.). Given that these velar cells are directly
contacted by serotonergic varicosities (Marois and Ca-
rew, 1997b), it is very likely that the modulatory effects
I IT on ciliary beating are mediated by these seroto-
ner synapses. Thus, the serotonergic cells of the AG
cann- e regarded as strictly sensory or interneuronal
sini y appear to have direct effector functions on cil-

iary activity, and probably on muscular and neuronal ac-
tivity as well.

These findings suggest that the serotonergic cells in lar-
val Aplysia are multimodal neurons involved in the
modulation of ongoing physiological and behavioral ac-
tivity (see Marois and Carew, 1997b). Although there is
evidence from studies on the gastropod Ilyanassa that
serotonin may also be involved in the induction of meta-
morphosis (Levantine and Bonar. 1986; Leise, 1996;
Couper and Leise, 1996), no comparable effect has been
observed in Aplysia and other gastropods (Morse et al,
1979;Hadtield, 1984; Coon ci ul.. 1985; Marois and Hof-
stadter, unpubl. obs.).

In conclusion, this study, together with other recent
work (Marois and Carew, 1997a. b), has begun to reveal
the anatomical and functional properties of the seroto-
nergic system in the apical ganglion of the gastropod
Aplysia. However complex the functions of the seroto-
nergic cells may turn out to be, they probably represent
only a fraction of the roles played by the entire apical
ganglion. The AG may therefore be best regarded as a
complex nerve center for the regulation of larval-specific
behaviors, integrating sensory information to achieve

APICAL GANGLION OF LARVAL APLYSIA

397

proper motor adaptation to variable environmental con-
ditions. Thus, more than simply a sensory structure, the
apical organ is a bonafide neuronal ganglion.

Acknowledgments

We thank Isabel Gauthier for assistance with the draw-
ing. This work was financially supported by an NSERC
(Canada) pre-doctoral fellowship to R.M. and by NSF
grant IBN9221 1 17 and NIMH Merit Award R01-MH-
14-1083 to T.J.C.

Literature Cited

Altner, H., and L. Prillinger. 1980. Ultrastructure of invertebrate
chemoreceptors. thermoreceptors and hydroreceptors and its func-
tional significance. Int. Rev Cylol. 67: 69- 140.

Barlow, L. A., and J. \V. Truman. 1992. Patterns of serotonin and
SCP immunoreactivity during metamorphosis of the nervous sys-
tem of the red abalone. Haliolis ru/esccns. J Ncwobiol. 23: 829-
844.

Bisgrove, B. \\ '., and R. D. Burke. 1986. Development of serotoner-
gic neurons in embryos of the sea urchin, Slrongylocenlrolus purpu-
ralus Dcv. (Jrowlh Differ. 28: 569-574.

Bisgrove, B. \\ ., and R. D. Burke. 1987. Development of the nervous
system of the pluteus larva of Slrongylocentrolus droebaclnensis.
Cell Tissue Res. 248: 335-343.

Bonar. D. B. 1978. Ultrastructure of a cephalic sensory organ in lar-
vae of the gastropod Pheslilla sihogue ( Aeolidacea. Nudibranchia).
Tissue Cell 10: 153-165.

Chia, F. S., and R. Koss. 1979. Fine structural studies of the nervous
system and the apical organ in the planula larva of the sea anemone
Anthopleura eleganlissima. J Morphol. 160: 275-298.
Chia, F. S., and R. Koss. 198-4. Fine structure of the cephalic sensory
organ in the larva of the nudibranch Rostanga pulchra (Mollusca.
Opisthobranchia. Nudibranchia). Zoomorpho/ogy 104: 131 - 139.
Chia, F. S., R. D. Burke, R. Koss, P. V. Mladenov, and S. S. Rumrill.
1986. Fine structure of the doliolaria larva of the feather star /•'/<>/-
ometra serratissima (Echinodermata: Crinoidea), with special em-
phasis on the nervous system. / Morphul. 189: 99- 1 20.
Coon, S. L., D. B. Bonar, and R. M. \\einer. 1985. Induction of set-
tlement and metamorphosis of the Pacific oyster. Crassostreagigas
(Thunberg) b> L-DOPA and catecholamines. J. E\p. Mar Biol.
Ea>l. 94:21 1-221.

Couper, J. M., and E. M. Leise. 1996. Serotonin injections induce
metamorphosis in larvae of the gastropod mollusc llyanassa obso-
leta. Biol. Bull 191: 178- 186.

Dautov, S. Sh., and L. P. Nezlin. 1992. Nervous system of the Tor-
naria larva (Hemichordata: Enteropneusta). A histochemical and
ultrastructural study. Biol. Bull 183: 463-475.

Diefenbach, T. J., N. K. Koehncke, and J. I.Goldberg. 1991. Charac-
terization and development of rotational behavior in Helisoma em-
bryos: role of endogenous serotonin. / \eitrohiol. 22:922-934.
Dorset!, D. A. 1986. Brain to cells: the neuroanatomy of selected gas-
tropod species. Pp. 101 - 187 in The Mollusca (Vol. 9): Neurobiol-
ogy ami Behavior Part 2. A. O. D. Willows, ed. Academic Press.
New York.
Emery, D. G., and T. E. Audesirk. 1978. Sensory cells in Aplysia. J

Neurobiol.9:33-46.

Fukui, Y. 1991. Embryonic and larval development of the sea anem-
one Haliplanellahneala from Japan. Hvdrohiologia 216/21 7- 137-
142.

Hadfield, M. G. 1984. Settlement requirements of molluscan larvae:

new data on chemical and genetic roles. Ac/iiaculliirc 39: 283-298.

Hay-Schmidt, A. 1989. The larval nervous system ofPhoroius muel-

leri. Zoomorphology 108: 333-351.

Hay-Schmidt, A. 1990. Catecholamine-containing, serotonin-like
and FMRFamide-like immunoreactive neurons and processes in
the nervous system of the early actinotroch larva of Plwroms van-
couvcrcnsis (Phoronida): distribution and development. Can. J
Zoo/. 68: 1525-1536.

Hay-Schmidt, A. 1992. Ultrastructure and immunocytochemistry of
the nervous system of the larvae of Lingula analina and (jlotiidia
sp. (Brachiopoda). /.oomnrphology 112: 189-205.
Hay-Schmidt, A. 1995. The larval nervous system of Polvgonlius lac-
tens Scheinder. 1868 (Polygordiidae. Polychaeta): immunocyto-
chemical data. Ada Zool. 76: 121-140.

Holland, N. D., and L. /.. Holland. 1993. Serotonin-containing cells
in the nervous system and other tissues during ontogeny of a lance-
let, Branchiostomajloridae. Ada Zoo/. 74: 195-204.
Jacob, M. H. 1984. Neurogenesis in Aplysia calitoriuca resembles
nervous system formation in vertebrates. J. Neurosci. 4: 1225-
1239.

Ki m|il, S. C., and L. Page. 1995. Development of the apical seroto-
nergic complex in opisthobranch larvae. Larval Biol Meet 4bs-/r
2:20.

Kempf, S. C., A. Saini, and A. Jones. 1991 . The ontogeny of neuronal
systems expressing SCP-like and serotonin-like antigens in Berghia
verrucicornis. Soc. Neurosci. Abstr. 17: 1356.

Koshtoyants. K. H. S., A. G. Buznikov, and B. N. Manukhin. 1961.
The possible role of 5-hydroxytryptamine in the motor activity of
some marine gastropods. Comp Bun-hem. Physio/. 3: 20-26.
Kriegstein, A. R. I977a. Stages in the post-hatching development of

Aplysia californica. J. L'xp. Zool. 199:275-288.
Kriegstein, A. R. 1977b. Development of the nervous system of

Aplysia californica. Proc. Nail. AcacJ. Sci. USA 74: 375-378.
Lacalli, T. C. 1981. Structure and development of the apical organ in
trochophores ofSpirobranchuspolycerus, PhylloJoce maciilala and
Phyllodocemucosa(Polychaeata). Proc. R Soc. Loud. B 212: 381 -
402.
Lacalli, T. C. 1982. The nervous system and ciliary band of Muller's

larva. Proc. R Sue Loiul. #217: 37-58.
Lacalli, T. C. 1983. The brain and central nervous system of Muller's

larva. Can. J Zool. 61: 39-51.

Lacalli, T. C. 1984. Structure and organization of the nervous system
in the trochophore \arvaofSpirobranchus. Philos Trans R Soc.
Lorn/. B Biol Set 306: 79- 1 35.

Lacalli, T. C. 1990. Structure and organization of the nervous system
in the actinotroch larva ofPhoronis vancoin-erensis. Philos. Trans.
R Soc l.oml. B Biol. Sci 327: 655-685.
Lacalli, T. C. 1994. Apical organs, epithelial domains, and the origin

of the chordate central nervous system. Am. Zool. 34:533-541.
Lacalli, T. C., N. D. Holland, and J. E. West. 1994. Landmarks in
the anterior central nervous system of amphioxus larvae. Philos
Trans. R Soc. Loud B Biol Sci. 344: 165- 185.

Laverack, M. S. 1974. The structure and function of ohemoreceptor
cells. Pp. I -48 in Chenmreeeplion in Marine Organisms. P. T.
Grant and A. M. Mackie. eds. Academic Press, New York.
Leise, E. M. 1996. Selective retention of the fluorescent dye DASPEI
in a larval gastropod mollusc after paraformaldehyde fixation.
Micros. Res Tech. 33:496-500.
Levantine, P. L.,and I). B. Bonar. 1986. Metamorphosis of llyanassa

ohsolelu: natural and artificial inducers. Am. Zool. 26: 14A.
Marois, R., and T. J. Carew. 1997a. Ontogeny of serotonergic neu-

rons in Ap/ysia californica J Comp \eurol In press.
Marois, R., and T. J. Carcvt. I997b. Projection patterns and develop-

398

R. MAROIS AND T. J. CAREW

mental functions of serotonergic cells in larval Aplysia californica.
J Comp. N enrol. In i-

Marois, R., P. Hofstadto. i' .! T. J. Carew. 1993. Birthdate and iden-
tification of seroton.-r ; cells in embryonic and larval Aplysia. Soc:
Nenrosci Absli f9: 1287.

Marois, R., G. M. Kelly, S. Hockfield, and T. J. Carew. 1992. An
ultrastruciural study of serotonergic cells in the CNS of embryonic
and lar\ al Aplysia. Soc. Nenrosci. Ahstr. 18: 1 47 1 .

Morse, D. E.. N. Hooker, H. Duncan, and L. Jensen. 1979. Gamma-
aminobutyric acid, a neurotransmitter. induces planktonic abalone
larvae to settle and begin metamorphosis. Science 204: 407-410.

Moss, C, R. D. Burke, and M.C. Thorndyke. 1994. Immunocyto-
chemical localization of the neuropeptide SI and serotonin in lar-
vae of the starfish Pi.iasier ochraceus and Aslerias ruhens. J Mar.
Biol.Assoc. UK. 74:61-71.

Nakajima, Y. 1988. Serotonergic nerve cells of starfish larvae. Pp.
235-239 in Echinoderm Biology. Proceedings of the Sixth Interna-
tional Echinoderm Conference. R. D. Burke. P. V. Mladenov, P.
Lambert, and R. L. Parsley, eds. Balkema. Rotterdam.

Nakajima, V., R. D. Burke, and Y. Noda. 1993. The structure and
development of the apical ganglion in the sea urchin pluteus larvae
of Strongylocentrotiis droebachiensis and Mespilta gluhulHs. Dev.
Growl h Differ. 35: 531 -538.

Nielsen, C. 1994. Larval and adult characters in animal phylogeny.
Am. Zoo/. 34:492-501.

Page, L. R. 1992. New interpretation of a nudibranch central ner-
vous system based on ultrastructural analysis of neurodevelopment
in Me/ibe leonina. I. Cerebral and visceral loop ganglia. Biol Bull.
182:348-365.

Richardson, K. C., L. Jarrett, and E. II. Einke. 1960. Embedding in
epoxy resins for ultrathm sectioning in electron microscopy. Slain
Techno!. 35:313-323.

Saunders, A. M.C., and M.Poole. 1910. The development of Aplysia
punckiUi Q J Microsc. Sci 55: 497-539.

Schacher, S., E. R. Kandel, and R. Woolley. 1979a. Development of
neurons in the abdominal ganglion of Aplysia californica. I. Axoso-
maticsynaptic contacts. Dev. Biol. 71: 163- 175.

Schacher, S., E. R. kandel, and R. Woolley. 1979b. Development of
neurons in the abdominal ganglion of Aplysia californica. II. Non-
neural support cells. Dev Biol 71: 176-190.

Tardy, J., and S. Dongard. 1993. Le complexe apical de la veligere de
Riiditapi'.\ philippinarum (Adams et Reeve, 1850) Mollusque Bi-
valve Veneride. Biol. Mar. 316: 177-184.

Wright, B. R. 1974. Sensory structure of the tentacles of the slug. Ar-
ton <;/cT(Pulmonata, Mollusca) 2. Ultrastructure of the free nerve
endings in the distal epithelium. Cell Tissue Res. 151: 245-257.

Reference: Biol Hull. 192: 399-409. (June, 1997)

Serotonin and Dopamine Have Opposite Effects on
Phototaxis in Larvae of the Bryozoan Bugula neritina

ANTHONY FIRES' AND ROBERT M. WOOLLACOTT2

1 Department of Biology. Dickinson College, Carlisle, Pennsylvania 1 7013; and 2 Department of
Organising and Evolutionary Biology, Harvard University, Cambridge, Massachusetts 02138

Abstract. Adult colonies of the bryozoan Bugula neri-
tina release short-term anenteric larvae that initially are
strongly photopositive. Over the course of several hours
larvae lose their initial photopositivity and either be-
come photonegative or alternate between positive and
negative phototaxis. We report that newly released pho-
topositive larvae rapidly become photonegative upon
exposure to ICT^-IO 5 M serotonin or its metabolic pre-
cursor. 5-hydroxytryptophan. This behavior was not ob-
served in two congeners of B. neritina, nor in larvae of
three other species of bryozoans and seven species from
four additional phyla. Antibodies to serotonin label cells
in the region of the equatorial nerve-muscle ring and in
two tracts extending from the apical disc to this ring. In
a separate series of experiments, larvae treated with do-
pamine( 10 7-10 5 Af) significantly prolonged their pho-
topositive period. This effect was also obtained with
the D2 dopamine receptor agonist, quinpirole (10~6-
1(T5 A/). HPLC analysis determined that newly released
photopositive larvae contained 0. 120pmol dopamine/
Mg protein. These findings implicate serotonin and dopa-
mine as important neurochemical regulators of photo-
taxis in larvae of B. neritina.

Introduction

Regulation of the vertical distributions of pelagic lar-
vae of marine invertebrates is most likely restricted to the

Received 1 I February I 997; accepted 7 April 1997.

Ahhri'vialionx: 5HT. serotonin: 5HTP, 5-hydroxytryptophan; DA,
dopamine: DHBA. 3.4-dihydroxybenzylamine; DDW, deionized dis-
tilled water: HPLC. high-pressure liquid chromatography; MBL-ASW.
MBL artificial seawater; MPB. Millonig's phosphate buffer. PBS + .
phosphate-buffered saline containing 0.1% Triton X-IOO and
O.I'; NaN,.

vectoral factors of current velocity, light direction, and
gravity (Crisp, 1984). Of these, most is known about the
influence of light. Studies of the role of phototaxis in lar-
val behavior were pioneered by Thorson (1964), who
documented the photic responses exhibited by larvae of
141 species of shallow-water benthic marine inverte-
brates from 1 1 phyla. In Thorson's survey, the most fre-
quently observed response to light was for larvae to be
initially positively phototactic (82%). Only 6% re-
sponded negatively to light throughout larval life, and
only 12% were indifferent to light. In 76% of the cases
examined, larvae that were initially photopositive be-
came photonegative before the end of the larval period.
Apparently, an ontogenetically regulated switch govern-
ing the sign of phototaxis is found in larvae of many spe-
cies. Positive phototaxis early in the larval period may
bring larvae up into the water column and facilitate dis-
persal; later negative phototaxis may bring them down
to the substratum where settlement occurs. In addition
to changes in phototaxis during development, environ-
mental signals may influence phototactic behavior of lar-
vae (see Crisp, 1984; Young and Chia, 1987, for reviews).
Factors such as temperature, salinity, light intensity,
ionic shock, pH, exposure to chemical cues from settle-
ment-specific substrates, and pollutants all have been
implicated in environmentally induced changes in pho-
totaxis.

Despite the wide phylogenetic distribution and ecolog-
ical significance of larval phototaxis, very little is known
about the internal mechanisms responsible for generat-
ing and modulating this behavior. This lack of knowl-
edge stands in contrast to current understanding of the
neural bases of stereotyped behaviors in many adult in-
vertebrates. Although that literature is far too extensive

399

400

A. PIRES AND R. M. WOOLLACOTT

to review here, it is important to point out a common
theme that has emcrp d i: the last two decades: Many
behaviors are initia maintained, altered, or termi-
nated by the nonoamine or peptide modula-
tors on neural P i see reviews by Harris- Warrick
and Marder, >•; Katz, 1995). Examples involving
monoamines include initiation of swimming in an anne-
lid (Willard, 1981; Mangan et ai, 1994); modulation of
phototaxis, swimming, respiration, and feeding in gas-
tropods (Crow and Forrester, 1986; McClellan el ai,
\994;Syedetal., 1990; Wieland and Gelperin, 1983;Ky-
riakides and McCrohan, 1989); starting and stopping
flight in an insect (Claassen and Rammer, 1985); and
switching of body postures and modulation of pyloric
motor output in decapod crustaceans (Livingstone et ai,
1980; Flamm and Harris- Warrick, 1986).

Although evidence has been presented for the exis-
tence of neuroactive monoamines in the larvae of
many marine invertebrates — including hydrozoans
(McCauley, 1995; Walther et ai, 1996), a nemertean
(Hay-Schmidt, 1990a), a polychaete (Hay-Schmidt,
1995). bivalve and gastropod molluscs (Coon and Bonar,
1986; Goldberg and Kater, 1989; Marois and Carew,
1990; Barlow and Truman, 1992; Pires et ai. 1992), sev-
eral echinoderms (Toneby, 1980; Burke, 1983; Burke et
ai, 1986; Bisgrove and Burke, 1986, 1987; Nakajima,
1987, 1988;Thorndyket'/fl/., 1992), brachiopods (Hay-
Schmidt, 1992), phoronids (Hay-Schmidt, 1990b,c), a
hemichordate (Dautov and Nezlin, 1992), and a cepha-
lochordate (Holland and Holland, 1993) — less is known
of the roles of these compounds in the mediation or
modulation of larval behaviors. Monoamines have been
implicated in the control of metamorphosis in a variety
of taxa including hydrozoans (McCauley. 1995; Walther
L'tai, 1996), polychaetesf Biggers and Laufer, 1992;Oka-
moto et ai, 1995), gastropods (Couper and Leise, 1996;
Pires el ai, 1995), bivalves (Coon and Bonar, 1987; Bo-
nar el ai. 1990; Chevolot el ai, 1991; Kingzett et ai,
1990), a barnacle (Yamamoto et ai, 1996), and an echi-
noid (Burke, 1983). Effects of monoamines on ciliary lo-
comotion have also been reported. Bath-applied dopa-
mine (DA) induces ciliary reversal and backward swim-
ming in some echinoid plutei (Lacalli and Gilmour,
1990; Mogami et ai, 1992), while serotonin (5HT) in-
creases the speed of forward swimming (Mogami et ai,
1992). Serotonin also accelerates ciliary beat frequency
in encapsulated embryos of the gastropod Helisoma tri-
volvis. while DA has no effect (Diefenbach el ai. 1991;
Goldberg et al., 1994). However, no data have been pub-
1 ',cd on the regulation of larval phototaxis by mono-
•s or any other neuroactive substances.

oans are excellent material for experimental
stiu larval phototaxis because most species retain

their early developmental stages and, on stimulus of
light, release larvae that generally settle within a few
hours of eclosion. Cohorts of larvae all released within a
few minutes of each other can thus be obtained. Ryland
(1976, 1977) reviewed in detail the early studies of the
phototactic behavior of bryozoan larvae, and several im-
portant conclusions come from these studies. First, a
range of responses to light are possible, depending on the
species. During the course of its larval existence an indi-
vidual could remain neutral to light throughout; react
positively throughout; change from positive to negative
or positive to partial negative; and change from positive
to alternation between positive and negative (Ryland,
1960). Second, in larvae of Bugula flabellata and B. tur-
rita, it is possible to artificially force a switch in photic
response with agents such as elevated pH, hypotonic sea-
water, or CuCl (Lynch, 1947). Third, in larvae of Cryp-
tosula pal/assiana, a rise in temperature decreases the
photopositive phase, but the amount of illumination has
no effect on the rate of change in sign of the response to
light (Ryland, 1962). Furthermore, Ryland observed
that pipetting larvae also results in an immediate change
from positive to negative phototaxis.

Larvae of many species with phototactic responses
possess pigmented epidermal structures that, on the basis
of anatomical criteria, are assumed to be photoreceptors
( Ryland, 1976, 1977). Not all species with larvae that ori-
ent to light have pigmented "eyespots," however (Ry-
land, 1 960); in these species, other specialized epidermal
cell types are hypothesized to be photoreceptors (Zim-
merand Woollacott, 1989). To date, all pigmented ocelli
of bryozoan larvae examined at the ultrastructural level
have been based on a sensory cell with an elaboration of
multiple cilia that are thought to be the receptoral organ-
elles (Woollacott and Zimmer, 1972; Hughes and Wool-
lacott, 1978, 1980; Reed ««/.. 1988).

We selected Bugula neritina for detailed investigation
of the monoaminergic control of phototaxis in the lar-
vae of marine bryozoans. B. neritina is cosmopolitan in
temperate to tropical waters and is often abundant in
specific locales at certain times of the year. Larvae of B.
neritina are barrel-shaped and about 300 /urn in diame-
ter. General features of larval anatomy and events in
metamorphosis are well documented for this species
(Woollacott and Zimmer, 1971, 1972, 1978; Reed and
Woollacott, 1982, 1983). The motive force for swim-
ming is provided by the coordinated beating of the cilia
of some 300 elongate coronal cells that form the exten-
sive lateral surface of the larva. Two pigmented "eye-
spots" are situated on the posterior surface, and these
connect with a neural plate in the apical disc by way of
an underlying equatorial nerve-muscle ring. Larvae are
strongly photopositive on release and with time either

MONOAMINES MODULATE PHOTOTAXIS

401

change to photoneutral or alternate between photopos-
itive and photonegative.

We report that 5HT and its immediate metabolic pre-
cursor. 5-hydroxytryptophan (5HTP), rapidly caused
photopositive larvae of B. neritina to become photonega-
tive. Antibodies to 5HT label cells with processes that are
associated with the apical disc and extend orally to the
equatorial nerve-muscle ring. In contrast, we show that
DA and the D: DA receptor agonist quinpirole prolonged
photopositivity in newly released larvae, and we present
chromatographic data to confirm the presence of endoge-
nous DA. We also report qualitative observations on the
effects of 5HT and DA on the photic behavior of larvae
from five other species of bryozoans and from seven spe-
cies in four additional phyla. These results demonstrate
that the responses observed in B. neritina to 5HT and DA
are generalizable neither to larvae of congeners nor to a
broad range of marine invertebrate larvae.

Materials and Methods

Behavior oj larvae of Bugula neritina

Sexually mature colonies of Bugula neritina (Linnaeus)
1 758 were collected in 1 993, during February and March,
from the sides of floating docks and other submerged ob-
jects in the Indian River near the Smithsonian Marine
Station at Link Port, Fort Pierce, Florida. Colonies were
maintained in 2-gal aquaria provided with aeration. Re-
lease of larvae was initiated by exposing colonies to sun-
light following an overnight period of dark adaptation.

Phototaxis experiments were conducted in MBL arti-
ficial seawater (MBL-ASW, Cavanaugh, 1956) with the
salinity adjusted to habitat level (20%o). Phototactic
behavior of larvae was assessed in the presence of
5-hydroxytryptamine hydrochloride (serotonin, 5HT,
Sigma); 5-hydroxy-L-tryptophan (5HTP, Sigma); 3-hy-
droxytyramine hydrochloride (dopamine, DA, Sigma);
(±)-SKF-38393 (a D, DA receptor agonist. Research
Biochemicals Inc.); and ( — )-quinpirole hydrochloride (a
selective D: DA receptor agonist. Research Biochemicals
Inc.). Twenty milliliters of MBL-ASW served as the con-
trol in all experiments. Drugs were dissolved and diluted
directly into MBL-ASW. Final working solutions were
in 20-ml volumes and contained 1CT5, lO"6, 10~7, or
10~8 M concentrations of the drug.

Experiments were conducted in glass Slender dishes
with a capacity of 30 ml and an inside diameter of
4.5 cm. Larvae were transferred into dishes by pipetting,
usually carrying over about 0.2-0.3 ml of Indian River
water. It was not feasible to accurately determine the
number of larvae being transferred; consequently, dishes
contained unequal numbers of larvae. The number of
larvae varied from 20 to 78 per dish, but 25 to 35 was the

most common range of values. Pipetting did not affect
the responses of larvae of B. neritina to light; this result
is contrary to the observations of Ryland ( 1 962) with lar-
vae of another bryozoan, Cryptosula pallasiana. A 4-ft
GE F400CW cool white fluorescent lamp was used as
the light source. Dishes containing larvae were placed on
white paper 3 in. from the light. Photon flux was mea-
sured with a LI-COR model LI- 1000 light meter
equipped with a detector calibrated for air and measur-
ing between 400 and 700 nm. Irradiance measurements
were made in air and ranged between 4.76 X 1015 and
5.78 X 1015 photons/s/cnr along the length of the tube.
The gradient across the dishes was 6.69 X 1015 to 4.88
X 1013 photons/s/cm2. Temperature remained within
PC over the course of individual experiments and
ranged from 20°C to 23°C over the course of all experi-
ments.

The number of larvae in the half of the dish that was
closest to the light was counted at designated time in-
tervals throughout an experiment. Total numbers of lar-
vae were then counted and the data converted to per-
centages. A repeated-measures ANOVA was used on
arcsin-transformed data to determine whether signifi-
cant differences existed among treatments, and a Fisher
probability least significant difference test was used to lo-
calize where these differences, if any, resided (Statview
4.0, Abacus Concepts).

Behavior of other marine invertebrate larvae

The phototactic responses of larvae of 12 additional
species of marine invertebrates were evaluated qualita-
tively (Table I). Some of these studies were conducted
at the Museum of Comparative Zoology in Cambridge,
Massachusetts, and others at the Kewalo Marine Labo-
ratory in Honolulu, Hawaii. In all cases, 5HT and DA
were applied at a concentration of 10" ? M. As above, 20-
ml volumes in 30-ml Slender dishes were used. A com-
bination of fluorescent and natural light was employed,
but irradiance was not quantified. Responses of larvae
were gauged qualitatively.

Scanning electron microscopy

Larvae of Bugula neritina were fixed in 1% OsO4 in sea-
water for 30 min, rinsed in distilled water, and dehydrated
in a graded series of acetone. Specimens were critical-
point dried in CO2, gold-palladium coated, and examined
with an AMR 1000 scanning electron microscope.

Immunohistochemistry

The immunohistochemical protocol was adapted from
that of Kempf el a/. (1987). Larvae were fixed for 1 h at

402

A. PIRES AND R M. WOOLLACOTT

room temperature in 4% paruformaldehyde containing
0.1 4 M NaCl and 0.2 M Millonig's phosphate buffer
(MPB). After fi.vni. vere washed twice in MPB

(10 mineach/. ' >. ashed in deionized dis-

tilled w;it Jrated through an ethanol

series to x , 80%, 95%, 100%, 100%, xy-

lene. 10 n rehydrated through the same se-

ries back to This procedure increases tissue perme-

ability to a > iodies. Larvae were then washed twice
(lOmin each) in 20mA/ phosphate-buffered saline in
0.15 M NaCl, pH 7.3, containing 0.1%. Triton X-100 and
0. 1% NaNj (PBS+). Subsequent steps were carried out on
a shaker table at 4°C. Larvae were incubated for 4 h in a
blocking medium consisting of 5% heat-inactivated goat
serum in PBS+, then incubated overnight in primary an-
tibody solution. This was a 1:325 dilution (in blocking
medium) of polyclonal rabbit anti-5HT (Incstar #20080).
A batch of larvae was treated overnight with blocking me-
dium instead of primary antibody solution, as a control
for specificity of immunofluorescent labeling. All larvae
were then washed four times with PBS+ over a 1 2-h pe-
riod, and incubated overnight in the secondary antibody
solution. Secondary antibodies were goat anti-rabbit im-
munoglobulin G conjugated to fluorescein isothiocyanate
(Organon-Teknica-Cappel #12121671), diluted 1:300 in
blocking medium. Following treatment with secondary
antibody, larvae were again washed four times with PBS+
over a 12-h period, then mounted in a Tris-buffered (pH
9.5) glycerol medium containing 4% //-propyl gallate (Gi-
loh and Sedat. 1982). Larvae were examined and photo-
graphed under UV epi-illumination.

Chromatographic analysis of dopamine

DA content of larvae was analyzed by high-pressure
liquid chromatography (HPLC) with electrochemical
detection. Homogenization of larvae and extraction of
catecholamines differed only in a few details from the
methods of Coon and Bonar (1986). Approximately
75 p.\ of packed larvae was concentrated by gentle cen-
trifugation and homogenized in a glass tissue grinder on
ice in 1 .0 ml of 0.4 A' perchloric acid with 4 mM reduced
glutathione, 4.7 mAl EGTA. and 100 nAl 3,4-dihydrox-
ybenzylamine (DHBA, Sigma) as an internal standard.
The homogenate was centrifuged at 15,000 X g for
5 min. Two 400-/ul aliquots were transferred to 10
X 75 mm glass tubes for extraction of catecholamines
over alumina (Anton and Sayre, 1962). To each tube was
added 600 n\ DDW. 50 mg alumina, and 1 ml extraction
buffer (1.5 M Tris plus 50mA/ Na: EDTA. pH 8.6).
> were agitated 10 min by inversion. Alumina was
wash twice with DDW, transferred to microcentrifuge
till semblies (Rainin #39-402), and catechols were

eluted with 200 ^10. 1 N perchloric acid. This extract was
injected directly into the HPLC system (standard injec-
tion volume 80 n\). Each extraction therefore provided
enough material for two full sample injections; the re-
maining material from each extraction was used in
smaller injections to help confirm the DA peak while
varying the organic content of the mobile phase and
"spiking" the chromatographic standards mix (see be-
low). The recovery efficiency of the DHBA internal stan-
dard (64%-68%) was calculated for each extraction and
used to correct the endogenous catecholamine data from
that extraction.

Separation of catecholamines was accomplished by
HPLC with an Alltech #28922 Adsorbosphere catechol-
amine column ( 100 x 4.6 mm, 3 nm C-18 reverse phase
packing). The aqueous portion of the mobile phase con-
tained 100mA/ monochloroacetic acid. 1.3mA/ Na2
EDTA, and 1 .3 mAl sodium octyl sulfate adjusted to pH
3.00-3.05 with NaOH (85 mA/ final concentration). To
this was added l%-5% (v/v) acetonitrile. Flow rate was
set at 1.0 ml/min.

Catecholamines were detected with an EG & G/
Princeton Applied Research #400 amperometric electro-
chemical detector with glassy carbon working electrode
set at an oxidizing potential of 700 mV against a Ag/AgCl
reference electrode (Riggin and Kissinger, 1977; Krstu-
lovic, 1982). Oxidation current range was set at 10 or
20 nA full-scale for the smaller peaks in the first part of
the chromatogram, and at 50 nA for DA, which eluted
later and was present at higher concentration. Detector
output was low-pass filtered ( 1 .0-s time constant) and sent
to a Beckman #427 integrator which printed chromato-
grams and calculated peak areas and retention times.

The DA chromatogram peak was identified by com-
paring its retention time to that of authentic DA at vari-
ous concentrations of acetonitrile (l%-5%). A standard
curve of DA peak area as a function of amount injected
was used to quantify DA concentration in larval homog-
enates. Reported values of larval DA are derived from
the mean of two 80-^1 injections, one from each extrac-
tion, run at the acetonitrile concentration (5%) that
yielded best resolution of the DA peak. Dopamine con-
centration was expressed as picomoles of free base per
microgram of protein. Protein content of the homoge-
nate was assayed by the Coomassie blue dye-binding
method (Bradford, 1976) with bovine serum albumin as
the standard (Bio-Rad kit, #500-0002).

Results

Monoamine modulation ofphototactic behavior of
larvae of Bugula neritina

Preliminary experiments conducted during June 1992
in Honolulu, Hawaii, established in a qualitative fashion

MONOAMINES MODULATE PHOTOTAX1S

403

that the addition of exogenous 5HT at 10 5 M concen-
tration brought about an immediate transformation
from positive to negative phototaxis in newly released
larvae of B. neritina. At that time, stocks of adult colo-
nies were insufficient to provide the quantities of larvae
needed for a full experimental analysis, but the response
was reconfirmed in Hawaiian B. neritina during May
1993.

The effects of 5HT and, subsequently, DA on photo-
taxis were evaluated in detail in February and March
1993 in Fort Pierce, Florida, where a large supply of sex-
ually mature colonies was available.

The effect of 5HT on the percentage of positively pho-
totactic larvae was evaluated at concentrations of 1(T5,
10~6, 10~7, and l(TKMovera 140-m in period (Fig. 1A).
In controls without applied 5HT, the percentage of lar-
vae remaining photopositive decreased from a mean of
88% to 70% over the 140-min period. There was no sig-
nificant difference from the control in experimental
treatments with 10~8 M exogenous 5HT. However, the
decrease in photopositivity seen after 140 min in larvae
treated with 10"7, 10~6, and 10~5M 5HT was much
greater than that seen in controls (P < 0.05 for 10~7 M;
P < 0.02 for 10~6 and 10~5 M 5HT). The change from
photopositive was especially rapid at 10~6 and 10~5M
concentrations. At these concentrations, most larvae
switched to photonegative within about 1 min after the
addition of 5HT; in every trial at 10~5 M all larvae were
photonegative after 1 10 min. Parallel studies of change
in sign of phototaxis were conducted using 5HTP, the
immediate metabolic precursor of 5HT (Fig. IB). Re-
sults of these studies documented a shift in phototaxis
even more dramatic than that observed with 5HT. The
decrease in percentage of positively phototactic larvae af-
ter 140 min was significantly greater at 10~5, 10~6, and
10"7 M 5HTP than in control trials (P < 0.02). The re-
sponse to 5HTP was also more rapid at the higher con-
centrations than was the response to 5HT at those same
concentrations.

The effect of DA on the percentage of positively pho-
totactic larvae was assessed at 10~5, 10~6, 10~7, and
10~8 M concentrations over a 240-min period (Fig. 2A).
In controls lacking exogenous DA, the percentage of lar-
vae remaining photopositive decreased from a mean of
83% to 41% over 240 min. The addition of DA at 10~7,
10~6, and 10~5 M levels caused more larvae to remain
photopositive than in the untreated controls (P < 0.02).
Larvae treated with 10~5 M DA did not show any appre-
ciable loss of positive phototaxis up to 240 min after ap-
plication of DA. The response of larvae of B. neritina to
DA was qualitatively reconfirmed using Hawaiian mate-
rial in May 1993. Application of the D, DA receptor ag-
onist SKF-38393 had no significant effect on phototactic

A

SEROTONIN

50 100

TIME (MIN)

150

B 5-HYDROXYTRYPTOPHAN

50 100

TIME (MIN)

150

Figure 1. Percentage of larvae of Biigula nerilina that were photo-
positive after timed exposure to varying concentrations of (A) serotonin
and (B) 5-hydroxytryptophan. Each point represents the mean (±1
SEM) of 5 (A) or 4 (B) replicate trials. Open circles, 10~5 M. open trian-
gles. 10~6A/. filled triangles, 1CT7.W, filled circles, 1CT8A/. squares,
control. Single and double asterisks indicate significant differences
from control at P < 0.05 and P < 0.02.

404

A PIRES AND R. M. WOOLLACOTT

DOPAMINE

B

100

50 100 150
TIME(MIN)

200 250

QUINPIROLE

100 150
TIME(MIN)

200 250

Figure 2. Percentage of larvae ofBugitla ncrilina that were pholo-
• after timed exposure to varying concentrations of (A) dopa-
and (B) quinpirole. Each point represents the mean (±1 SEM) of
ic trials. I • , i .nation of symbols as in Figure 1 .

•of larvae of B. neritina at concentrations of 10 5
to ;. but was clearly toxic to the larvae. The D:

DA >r agonist quinpirole, however, mimicked the

effect o )\ in prolonging positive phototaxis. The per-

centage of photopositive larvae was significantly greater
after 240 min in quinpirole treatments of 10~5 and
10^6 M than in the untreated controls (P< 0.02, Fig. 2B).
Application of quinpirole at 10~7 and 10~s M produced
an initial decrease in the percentage of photopositive lar-
vae compared with the controls, but the difference was
no longer significant after 240 min.

Localization of serotonin

Larvae of B. neritina have two pigmented eyespots sit-
uated in two depressions on the posterior lateral surface
of the larva (Fig. 3). The lateral surfaces of the larva are
formed by heavily ciliated cells of the corona, the larval
locomotory organ. An apical disc complex with a central
neural plate is situated at the aboral pole of the larva.

Immunolocalization of 5HT was achieved in larvae of
B neritina, but the procedure resulted in distortion of
larvae and made precise determination of sites of the re-
action difficult (Fig. 4). Strong 5HT-like immunoreactiv-
ity is evident, however, in the region of the equatorial
nerve-muscle ring and in two tracts extending from the
apical disc to this ring. These tracts are in a position oc-
cupied by a nerve-muscle tract extending from the roof
of the metasomal sac and equatorial nerve-muscle ring
to the neural plate in the center of the apical disc. The
equatorial nerve-muscle ring underlies the two posterior
lateral pigmented eyespots. No fluorescence above back-
ground was observed in control larvae in which the anti-
5HT primary antibody was omitted.

Chromatographic analysis of dopamine

Reversed-phase HPLC of two alumina extracts of a
homogenate of newly released larvae of B. neritina
yielded chromatograms that included several peaks, in-
cluding one that was identified as DA on the basis of its
co-elution with authentic DA over a range of mobile-
phase acetonitrile concentrations (Fig. 5). The two ex-
tractions resulted in DA values of 516 and 481 pmol/
homogenate; these were averaged and divided by the to-
tal soluble protein content of the homogenate to yield
a final estimate of larval DA content of 0.120 pmol/^g
protein. Dihydroxyphenylalanine and norepinephrine
may also be present in larvae of B. neritina (Fig. 5): all
chromatograms showed at least a partially resolved peak
corresponding to each of these monoamines, but we did
not have enough material to justify reporting quantita-
tive values.

Comparative analysis of larval phototaxis

We examined the effects of 5 HT at 10"5 ^/concentra-
tion, and in some cases 1 0 ~ ' M DA as well, on phototaxis

MONOAMINES MODULATE PHOTOTAXIS

405

Figure 3. Scanning electron micrograph ofBugula nmiimi larva taken from lateral view. The meta-
chronal waves of cilia mark the locations of the strap-like elongated coronal cells that together form the
larval locomotory organ. Depressions in which the two pigmented eyespots (E) are situated are visible on
the posterior lateral surface. The opening of the metasomal sac marks the oral pole (O) of the larva, and the
ciliated apical disc (AD) is located at theaboral pole (A). -200

Figure 4. Light photomicrograph of 5HT-like immunoreactivity in larva ofBugula ncritina, with ori-
entation oflarva positioned to match that in Figure 3. Fluorescence is associated with position of equatorial
nerve-muscle tract that underlies eyespots and is continuous with two fluorescent tracts that extend into
apical disc. x200

of larvae from a number of species (Table I). Although
the analysis was based exclusively on a qualitative evalu-
ation of responses to the addition of these monoamines,
we were unable to document a pattern similar to that
observed in B. nerilina in larvae from two congeners and
three additional species of bryozoans. In a broader sur-
vey we did not detect any effects of monoamines on pho-
totaxis of larvae of one copepod crustacean, one gastro-
pod, two demosponges, and two ascidians. The larvae
of the gastropod Phestilla sibogac, however, did present
increased motility and greater positive phototaxis when
exposed to 1(T53/5HT.

Discussion

Bath-application of 10 - M or 10 "M5HT rapidly in-
duced negative phototaxis in newly released photoposi-
tive larvae of B. ncri/ina (Fig. 1A). A similar response
was obtained with the same concentrations of 5HTP. the
immediate metabolic precursor of 5HT, although the
onset of negative phototaxis took about 10-15min
longer with 5HTP than with 5HT (Fig. 1 B). This result is
consistent with the notion that serotonergic cells may
take up exogenous 5HTP and convert it to 5HT, thus
augmenting releasable endogenous stores of that neuro-

modulator. In the gastropod Lymnaea stagnalis, injec-
tion of 5HT activates rhythmic shell movements; the
same motor program can be obtained by injection of
5HTP. which also increases levels of 5HT and firing of
serotonergic neurons (Kabotyanski el a/., 1992).

Dopamine. when bath-applied in concentrations from
10~7 AIlo 10~5 M, prolonged the initial period of photo-
positivity in larvae of B. ncritina (Fig. 2A). This effect
was mimicked by the mammalian D: DA receptor ago-
nist quinpirole (Fig. 2B) at 10^ M and 10~5M. The
sharp initial decrease in the percentage of photopositive
larvae seen after treatment with 10~7 M quinpirole, con-
trasted with enhancement of phototaxis at higher con-
centrations, may reflect interactions with more than one
class of DA receptor; no pharmacological profiles of
bryozoan DA receptors are available.

Under normal laboratory conditions, larvae of B. ner-
ilina remain photopositive for 2-3 h (see controls in Fig.
2), then make a transition to a state in which they al-
ternate between positive and negative phototaxis (Ry-
land, 1960), and eventually may become photonegative
(Mawatari, 1951 ). Lynch ( 1947) never observed negative
phototaxis in this species under normal laboratory con-
ditions of lighting, temperature, and salinity, but he was
able to induce larvae to become photonegative by

406

A P1RES AND R M. WOOLLACOTT

n

2nA

5nA

—I 1 1 —

468
RETENTION TIME (MIN)

10

12

Figure 5. Separation of catecholamines by HPLC with electro-
chemical detection. Upper trace: chromatogram of catecholamine
standards mixture (3.2 pmol each of [I] dihydroxyphenylalanine, [2]
norepinephrine, [3] epinephrme; 6.4 pmol [4] DHBA; 64 pmol [5]
DA). Lower trace: chromatogram of alumina extract of homogenateof
newly released larvae of Bugula ncnlina. Current scale changes at time
marked by vertical arrows.

exposing them to intense light. He inferred from distri-
butions of adult colonies in the field that most larvae in
nature are probably photonegative at the time of settle-
ment and metamorphosis.

t is possible that 5HT and DA are neurochemical
'an ontogenetic switch in the sign of photo-
taxi, nsing ••• ripheral cilioexcitatory and both pe-
riphei central cilioinhibitory roles, respectively of
nd doparnine, have been described in the gill
epithe! .he bivalve Mytilus edulis (Catapane et al,
1978; o ei al.. 1986). However, such mechanisms

are probably not adequate to explain the aminergic con-
trol of cilia-driven phototaxis documented in this report.
Because 5HT and DA exert opposite effects on the sign
of phototaxis when applied homogeneously to the entire
animal, it seems more likely that they modulate the in-
teraction between photoreceptor organs and the ciliated
coronal cells that are the effectors of phototaxis (Woolla-
cott and Zimmer, 1972). Larvae of B neritina always
swim with the aboral pole directed forward, while rotat-
ing clockwise about the oral-aboral axis. In both positive
and negative phototaxis the sensory feedback to the lar-
va's locomotion should operate to equalize the light in-
put to the two laterally situated photoreceptor organs;
this would keep the oral-aboral axis parallel to the direc-
tion of the light stimulus. The crucial difference between
positive and negative phototaxis is expected to be in the
larva's course-correction mechanism. In positive photo-
taxis, asymmetric illumination of the two photoreceptor
organs should result in inhibition of cilia on the more-
illuminated side, or excitation of cilia on the less-illumi-
nated side (Woollacott and Zimmer, 1972). To achieve
negative phototaxis, the opposite course-correction strat-
egy would be required: inhibition of cilia on the less-illu-
minated side, or excitation of cilia on the more-illumi-
nated side.

If phototaxis is synaptically mediated by cells of the
equatorial nerve-muscle ring, as has been suggested on
anatomical grounds (Woollacott and Zimmer, 1972),
one can propose that a functional "rewiring" of the pho-
totaxis control system might be accomplished by mono-
amine neuromodulators. That is, whether a light stimu-
lus to a photoreceptor organ excites or inhibits a given
population of cilia might depend on the neuromodula-
tory milieu of the synapses in the phototaxis control sys-
tem. In the pyloric network of the stomatogastric gan-
glion of the lobster Panulirus interntptus. the qualitative
pattern of functional connections between identified
neurons has been shown to depend critically upon the
modulatory neurohormonal environment of the circuit
(Johnson el al., 1995). In two instances DA actually re-
verses the sign of an identified mixed chemical/electrical
synaptic connection, enhancing chemical inhibition and
reducing electrical coupling so that the net synaptic in-
teraction changes from excitatory to inhibitory (Johnson
eta/.. 1993).

Although we have demonstrated clear and dramatic
effects of bath-applied 5HT and DA on the photic behav-
ior of larvae of B. neritina, participation of endogenous
5HT and DA in the control of phototaxis remains to be
established. However, such roles for these amines seem
quite likely. Processes of 5HT-immunoreactive cells are
localized in the equatorial nerve-muscle ring (Fig. 4),
where they are situated to modulate the interactions be-

MONOAM1NES MODULATE PHOTOTAXIS

407

Table I

Change in phcloiaelie swimming behavior <>} 'initially photopositive larvae In exugi'ntni.ily applied nwnoamines (I0~5 M)

Phylum

Species

Larval type

Photota.xis
examined

Monoamine

Response

Source

Date

Arthropoda

Acartia IOHMI

Nauplius

Positive

5HT

None

North Atlantic

11/93

Bryozoa

Amalhia dislans

Coronate

Positive

5HT

None

Honolulu. HI

5/93

DA

None

Bitgii/ii nerilina

Coronate

Positive

5HT

Abrupt switch to negative

Honolulu, HI

6/92

Abrupt switch to negative

Fort Pierce, FL

2/93

Abrupt switch to negative

Honolulu. HI

5/93

DA

Slows switch to negative

Fort Pierce, FL

2/93

Slows switch to negative

Honolulu. HI

5/93

Bugula slolonijera

Coronate

Positive

5HT

None

Woods Hole. MA

9/93

Bii/gK/a inrriia

Coronate

Positive

5HT

None

Woods Hole. MA

9/93

Hippopodinafeegeensis

Coronate

Positive

5HT

None

Honolulu. HI

5/93

DA

None

Schizoporella sp.

Coronate

Positive

5HT

None

Honolulu. HI

5/93

Mollusca

Crepidula liirnieaia

Veliger

Neutral

5HT

None

Woods Hole. MA

11/93

DA

None

Phestilla xihogae

Veliger

Positive

5HT

>motility, > positive taxis

Honolulu. HI

5/93

DA

None

Porifera

Aplysilla sp.

Parenchymella

Positive

5HT

None

Honolulu. HI

8/94

Halichondria coemlea

Parenchymella

Neutral

5HT

None

Honolulu, HI

8/94

Llrochordata

Aseidia eeraiiidex

Tadpole

Positive

5HT

None

Monterey, CA

11/93

DA

None

Ciniui intestinalis

Tadpole

Positive

5HT

None

Boston, MA

11/93

DA

None

tween the putative photoreceptoral organs and the coro-
nal effectors of locomotion (Woollacott and Zimmer,
1972). Dopamine was not localized in the present study,
but amounts of DA per microgram of total protein in
newly released larvae of B. neritina are within the range
reported for molluscan larvae, in which DA has been im-
plicated in the control of settlement (Coon and Bonar,
1986; Bonar el al, 1990) and metamorphosis (Pires el
al.. 1992: Pires el al, 1995).

It is indeed puzzling that newly released photopositive
larvae of B. slo/onifera and B. turrita were unresponsive
to 10~5 M bath-applied 5HT. It may be that in these spe-
cies the conditions that would permit 5HT to influence
phototaxis do not apply early in larval life, or at all. Res-
olution of this issue will require detailed neurochemical
and behavioral investigations across the duration of the
larval period.

Acknowledgments

This study was made possible through the kindness of
M. Hadneld (Kewalo Marine Laboratory, University of
Hawaii, Honolulu, Hawaii) and M. Rice (Smithsonian
Marine Station at Link Port. Fort Pierce, Florida), who
sponsored our work at their respective facilities. SEM
analysis was conducted by E. Seling at the Museum of

Comparative Zoology, Harvard University. During this
project AP was a postdoctoral fellow supported by NSF
Grant # DCB89-03800 to M. Hadneld. Support pro-
vided RMW by a Short-Term Visitor Award from the
Smithsonian Institution and a Putnam Expedition
Grant from the Museum of Comparative Zoology, Har-
vard University, is gratefully acknowledged. This is con-
tribution number 428 from the Smithsonian Marine Sta-
tion at Link Port.

Literature Cited

Aiello, E., K. Hager, C. Akiwumi, and G. B. Stefano. 1986. An opioid
mechanism modulates central and not peripheral dopaminergic
control of ciliary activity in the marine mussel Mytilux ediilis. Cell.
Mol. .\ennihiol 6: 17-30.

Anton, A. II.. and D. F. Sayre. 1962. A study of the factors affecting
the aluminum oxide-trihydroxyindole procedure for the analysis of
catecholamines. J Phannacol. Exp. Ther 138: 360-375.

Barlow, L. A., and J. \V. Truman. 1992. Patterns of serotonin and
SCP immunoreactivity during metamorphosis of the nervous sys-
tem of the red abalone Halioti.i rufescens. J. Ncurobiol. 23: 829-
844.

Biggers, \V. J., and II. I.aufer. 1992. Chemical induction of settle-
ment and metamorphosis of Capilella ci/pila/ii Sp. I (Polychaeta)
larvae by juvenile hormone-active compounds. Invenehr. Reproil
Dev. 22: 39-46.

Bisgrove, B. \\ ., and R. D. Burke. 1986. Development of serotoner-

408

A PIRES AND R. M. WOOLLACOTT

gic neurons in embryos of i' lin Strongylocentrotus purpu-

ralus. Dev Gnm-lli Dtfii" '. 74.

Bisgrove, B. W.,andR ;

1987. Development of the nervous

system of the pit; >igylocentrotiis droebachiensis.

Cell Tissue R.
Bonar, D. B.. S. . ii, K. M. \Veiner, and VV. Fitt. 1990.

Control . Jnd metamorphosis hy endogenous

ande\. :• cues. Bull. Mar Si 7. 46: 484-498.

Bradford. M A rapid and sensitive method for the quanti-

tati. yam quantities of protein utilizing the principle of

proien nding. Anal. Biochem. 72: 248-254.

Burke, R. D. 1983. Neural control of metamorphosis in Dcndraslcr
exceniricus. Biol. Bull 164: 176-188.

Burke, R. D., D. G. Brand, and B. \V. Bisgrove. 1986. Structure of the
nervous system of the auricularia larva of Parastichopus californi-
eits. Biol. Bull. 170:450-460.

Catapane, E. J., G. B. Stefano, and E. Aiello. 1978. Pharmacological
study of the reciprocal dual innervation of the lateral ciliated gill
epithelium by the CNS ofMytilusedulis(BlvaMa). J Exp. Biol. 74:
101-1 13.

Cavanaugh, G. M., ed. 1956. Formulae and Methods 1 of the Marine
Biological Laboratory Chemical Room. Marine Biological Labora-
tory, Woods Hole, Massachusetts.

Chevolot, L., J.-C. Cochard, and J.-C. \\in. 1991. Chemical induc-
tion of larval metamorphosis ofPcetcn maximitx with a note on the
nature of naturally occurring triggering substances. Mar Ecol
Prog. Ser 14: 83-89.

Claassen, D. E., and A. E. Kammer. 1985. Effects of octopamine, do-
pamine, and serotonin on production of flight motor output by tho-
racic ganglia of Maiuluca sexla. J Neurobiol. 17: 1-14.

Coon, S. L., and D. B. Bonar. 1986. Norepmephrine and dopamine
content of larvae and spat of the Pacific oyster, Crussosirea #WM.
Biol Bull. 171:632-639.

Coon, S. L, and D. B. Bonar. 1987. Pharmacological evidence that
alpha- 1 adrenoceptors mediate metamorphosis of the Pacific oys-
ter. Crassoslrea gigas. Neuroseienee 23: 1 169-1 174.

Couper, J. M., and E. M. Leise. 1996. Serotonin injections induce
metamorphosis in larvae of the gastropod mollusc Ilyanassa ohso-
Iciu Biol Bull 191: 178-186.

Crisp, D. J. 1984. Overview of research on marine invertebrate lar-
vae, 1940-1980. Pp. 103-126 in MarineBiodeterioration: An Inter-
disciplinary Study. J.D. Costlow and R.C. Tipper, eds. Naval Insti-
tute Press, Annapolis. Maryland.

Crow, T., and J. Forrester. 1986. Light paired with serotonin mimics
the effect of conditioning on phototactic behavior of Hermissenda.
Proc. Nail. Aeud. Set. USA 83: 7975-7978.

Dautov, S. S. H, and L. P. Nezlin. 1992. Nervous system of the tor-
naria larva (Hemichordata: Enteropneusta). A histochemical and
ultrastructural study. Biol Bull. 183:463-475.

Diefenbach.T. J., N. K. Koehncke.and J. I.Goldberg. 1991. Charac-
terization and development of rotational behavior in flelixomaem-
bryos: role of endogenous serotonin. J. Neurobiol. 22: 922-934.

Flamm, R. E., and R. M. Harris- Warrick. 1986. Ammergic modula-
tion in lobster stomatogastric ganglion. I. The effects on motor pat-
tern and activity of neurons within the pyloric circuit. J Neuroptiys-
- S47-865.

i. W. Sedat. 1982. Fluorescence microscopy: reduced
ig of rhodamine and fluorescein protein conjugates by
- • s1, ,'rmv217: 1252-1255.

5. B. Kater. 1989. Expression and function of the
milter serotonin during development of the Helisoina
e\ Biol 131:483-495.

Gold!); .1. 1., N. K. Koehncke, K. J. Christopher, C. Neumann, and

T. J. Diefenbach. 1994. Pharmacological characterization of a se-
rotonin receptor involved in early embryonic behavior ofHi'lisoma
trivolvis. J Neurobiol. 25: 1545-1557.

Harris- VVarrick, R. M., and E. Marder. 1991. Modulation of neural
networks for behavior. Annu. Rev \eurosei. 14: 39-57.

Hay-Schmidt, A. 1990a. Catecholamine-containing. serotonin-like,
and neuropeptide FMRFamide-like immunoreactive cells and pro-
cesses of the nervous system of the pilidium larva (Nemertini). Zoo-
morphology 109: 23 1-244.

Hay-Schmidt, A. 1990b. Distribution of catecholamine-containing,
serotonin-like and neuropeptide FMRFamide-like immunoreac-
tive neurons and processes in the nervous system of the actinotroch
larva of Pliorimi\ muelleri (Phoronida). Cell Tissue' Res 259: 105-
118.

Hay-Schmidt, A. I990c. Catecholamine-containing, serotonin-like,
and FMRFamide-like immunoreactive neurons and processes in
the nervous system of the early actinotroch larva of Phoronis van-
eowerenms (Phoronida): distribution and development. Can. J.
/on/. 68: 1525-1536.

Hay-Schmidt, A. 1992. Ultrastructure and immunocytochemistry of
the nervous system of the larvae of Lingula unatina and Gltillidia
sp. (Brachiopoda). Zoomorphology 1 12: 1 89-205.

Hay-Schmidt, A. 1995. The larval nervous system ofPolygordius lae-
leus Scheinder. 1868 (Polygordiidae. Polychaeta): immunocyto-
chemical data..4c/a Zool. 76: 121-140.

Holland, N. D., and L. 7.. Holland. 1993. Serotonin-containing cells
in the nervous system and other tissues during ontogeny of a lance-
let. Brain luosloma Jloridae. Acta Zool. 74: 195-204.

Hughes, R. L., Jr., and R. M. \\oollacott. 1978. Ultrastructure of po-
tential photoreceptoral organs in the larva ofScrupocellaria benho-
/<•///( Bryozoa). Zoomorphologie91: 225-234.

Hughes, R. L., Jr., and R. M. Woollacott. 1980. Photoreceptors of
bryozoan larvae (Cheilostomata. Cellularioidea). Zool. Ser. 9: 129-
138.

Johnson, B. R., J. H. Peck, and R. M. Harris- Warrick. 1993. Dopa-
mine induces sign reversal at mixed chemical-electrical synapses.
Brain Re-, 625: 159-164.

Johnson, B. R., J. H. Peck.and R. M. Harris- VVarrick. 1995. Distrib-
uted amine modulation ol graded chemical transmission in the py-
loric network of the lobster stomatogastric ganglion. J Neurophys-
10! 74:437-452.

Kabotyanski, E. A., VV. Winlow, D. A. Sakharov, L. Bauce, and K. Lu-
kowiak. 1992. 5-h>droxytryptophan elicits sustained CPG activ-
ity for rhythmic shell movements in Lvmnaea slagnalis. Soe. Neu-
rosa.Abstr 18:531.

Katz, P. 1995. Intrinsic and extrinsic neuromodulation of motor cir-
cuits. Citrr. Opin \eurobiol. 5: 799-808.

Kempf, S. C., B. Masinovsky, and A. O. D. Willows. 1987. A simple
neuronal system characterized by a monoclonal antibody to SCP
neuropeptides in embryos and larvae of Trilonia diomedeu (Gas-
tropoda. Nudibranchia). / \eurobiol. 18: 217-236.

Kingzett, B.C., N. Bourne, and K. Leask. 1990. Induction of meta-
morphosis of the Japanese scallop Palmopeelen vessoensis Jay. J.
Shellfish Res 9: 119-124.

Krstulovic, A. M. 1982. Investigations of catecholamme metabolism
using high-performance liquid chromatography: analytical meth-
odology and clinical applications. J Chromatogr, 229: 1-34.

kyriakides, M. A., andC. R. McCrohan. 1989. Effect of putative neu-
romodulators on rhythmic buccal motor output in Lymnaea slag-
nalis J \eurobiol. 20:635-650.

Lacalli, I. C., and II. J. Gilmour. 1990. Ciliary reversal and locomo-
tory control in the pluteus larva of Lytechinus pictus. Phil. Trans.
R Soe. Land. B 330: 391-396.

MONOAMINES MODULATE PHOTOTAX1S

409

Livingstone, M.S., R. M. Harris- VVarrick, and E. A. Kravitz. 1980.

Serotonin and octopamme produce opposite postures in lobsters.
Science 208: 76-79.

Lynch, \\ . R. 1947. The behavior and metamorphosis ofthe larva of
Bugula ncrinna (Linnaeus): experimental modification of the
length ofthe free-swimming period and the responses ofthe larvae
to light and gravity. But/ Bull. 92: 115-1 50.

Mangan, P. S., G. A. Curran, C. A. Hurney, and W. O. Friesen. 199-4.
Modulation of swimming behavior in the medicinal leech. III. Con-
trol of cellular properties in motor neurons by serotonin. / Com/7.
Physiol.A 175:709-722.

Marois, R., and T. J. Carew. 1990. The gastropod nervous system in
metamorphosis./ Neiirobiol. 21: 1053-1071.

Mawatari, S. 1951. The natural history of a common fouling bryo-
zoan. Bugula neritina (Linnaeus). Mine. Rep Res Inst. Nat Re-
sour. (Tokyo) 20: 47-54.

McCauley, D. \V. 1995. Serotonin mediates metamorphosis in plan-
ulae ofthe hydrozoan Phialidilim gregariwn. Am Zool. 35: 1 1 2A.

McClellan, A. D., G. D. Brown, and P. A. Getting. 1994. Modulation
of swimming in Tritonia: excitatory and inhibitory effects of sero-
tonin. ./ Comp. P/tysiol. A 174: 257-266.

Mogami. N . k. YVatanabe, C. Ooshima, A. kawano, and S. A. Baba.
1992. Regulation of ciliary movement in sea urchin embryos: do-
pamine and 5-HT change the swimming behaviour. Comp. Bio-
ehem Phystol C' 101: 251-254.

INakajima, Y. 1987. Localization of catecholaminergic nerves in lar-
val echinoderms. Zool. Sei. 4: 293-299.

Nakajima, \. 1988. Serotonergic nerve cells of starfish larvae. Pp.
235-239 in Eehinoderm Biology. R.D. Burke, P.V. Mladenov, P.
Lambert, and R.L. Parsley, eds. Balkema. Rotterdam.

Okamoto, K., N. \\atanabe, A. VVatanabe, and k. Sakata. 1 995. I nduc-
tion of larval metamorphosis in serpulid polychaetes by L-DOPA
and catecholamines. Fish. Sei. 61: 69-74.

Pires, A., S. L. Coon, and M. G. Hadfield. 1992. Analysis of catechol-
amines and DOPA in a gastropod larva. Am. Zool. 32: 1 19A.

Pires, A., M. G. Hadfield, and J. A. Skiendzielewski. 1995. Deple-
tion of dopamine is accompanied by inhibition of metamorphosis
in two gastropods. Am Zool. 35: 10A.

Reed. C. G., and R. M. VVoollacott. 1982. Mechanisms of rapid mor-
phogenetic movements in the metamorphosis ofthe bryozoan Bu-
gula neritina (Cheilostomata. Cellularioidea). I. Attachment to the
substrate. J. Morphol 172: 335-348.

Reed, C. G., and R. M. VVoollacott. 1983. Mechanisms of rapid mor-
phogenetic movements in the metamorphosis ofthe bryozoan Bu-
gula nenlina (Cheilostomata, Cellularioidea). II The role of dy-
namic assemblages of microfilaments in the pallia! epithelium. J.
Morplwl. 177: 127-143.

Reed, C. G., J. M. Ninos, and R. M. Woollacott. 1988. Bryozoan lar-
vae as mosaics of multifunctional ciliary fields: ultrastructure ofthe
sensory organs of Bugula stolonifera (Cheilostomata: Cellulari-
oidea)./ Morplwl. 197: 127-145.

Riggin, R. M., and P. T. kissinger. 1977. Determination of catechol-

amines in urine by reverse-phase liquid chromatography with elec-
trochemical detection. Anal. Chern 49: 2 109-2 111.

Ryland, J. S. 1960. Experiments on the influence of light on the be-
havior of polyzoan larvae. / Exp. Biol. 37: 783-800.

Ryland, J. S. 1962. The effect of temperature on the photic responses
of polyzoan larvae. Sarsia 6: 4 1 -48.

Ryland, J. S. 1976. Physiology and ecology of marine bryozoans.
Adv. Mar. Biol. 14: 285-443.

Ryland, J. S. 1977. Taxes and tropisms of bryozoans. Pp. 41 1-436
in Biology of Bryoioans, R.M. Woollacott and R.L. Zimmer, eds.
Academic Press, New York.

Syed, N. I., A. G. M. Bulloch, and k. Lukowiak. 1990. In vitro recon-
struction ofthe respiratory central pattern generator ofthe mollusk
Lymnaea. Seienee 250: 282-285.

Thorndyke, M. C., B. D. Crawford, and R. D. Burke. 1992. Localiza-
tion of a SALMFamide neuropeptide in the larval nervous system
ofthe sand dollar Dcndra.tter excen/rictis. Ada Zool. 73: 207-2 12.

Thorson, G. 1964. Light as an ecological factor in the dispersal and
settlement of larvae of marine bottom invertebrates. Ophelia 1:
167-208.

Toneby, M. 1980. Dopamine in developing larvae ofthe sea urchin
Psammechinns milians Gmelin. Comp. Bioeheni. Phvsiol. C 65:
139-142.

Walther, M., L). Ulrich, M. kroiher, and S. Berking. 1996. Metamor-
phosis and pattern formation in Hydractinia eehinaia. a colonial
hydroid. Int. / Dev Biol. 40: 313-322.

Wieland, S. J., and A. Gelperin. 1983. Dopamine elicits feeding mo-
tor program in Limax maximiis. J. Neumsci. 3: 1735-1745.

Willard, A. 1981. Effects of serotonin on the generation of the motor
program for swimming by the medicinal leech. / Neurosei. 1: 936-
944.

Woollacott, R. M., and R. L. Zimmer. 1971. Attachment and meta-
morphosis of the cheilo-ctenostome bryozoan Bugula neritina
(Linne). /. Morphol. 134: 35 1-382.

Woollacott, R. M., and R. L. Zimmer. 1972. Fine structure of a po-
tential photoreceptoral organ in the larva of Bugula neritina (Bryo-
zoa). Z. Zellforseh. Mikrosk. Anal. 123: 458-169.

VVoollacott, R. M.. and R. L. Zimmer. 1978. Metamorphosis of cel-
lularioid bryozoans. Pp. 49-63 in Settlement and Metamorphosis of
Marine Invertebrate Larvae, F.-S. Chia and M.E. Rice, eds. Elsevier
North-Holland, New York.

Yamamoto, II. A. Tachibana, S. kawaii, k. Matsumura, and N. Fuset-
ani. 1996. Serotonin involvement in larval settlement ofthe bar-
nacle, Balanus amphitrite. J. Exp. Zool. 275: 339-345.

Young, C. M., and F.-S. Chia. 1987. Abundance and distribution of
pelagic larvae as influenced by predation, behavior, and hydro-
graphic factors. Pp. 385-463 in Reproduetion of Marine Inverte-
brates. Volume IX. General Aspects: Seeking Unity in Diversity.
A.C. Giese, J.S. Pearse, and J.B. Pearse, eds. Blackwell Scientific
Publ, Palo Alto, CA and The Boxwood Press, Pacific Grove, CA.

Zimmer, R. L., and R. M. Woollacott. 1989. Intercoronal cell com-
plex of larvae of the bryozoan H'atersipora arcuata (Cheilostomata:
Ascophora)./ Morphol 199: 151-164.

Reference: Biol. Bull 192: 410-417. (.June, 1997)

Role of Chemical Signals in the Orientation Behavior
of the Sea Star Asterias forbesi

PAUL A. MOORE AND DEBORAH M. E. LEPPER

Laboratory for Sensory Ecology, Department of Biological Sciences, Bowling Green State University.

Bowling Green. Ohio 43403

Abstract. The importance of chemical signals as forag-
ing and orientation cues has been demonstrated for
many marine organisms. It is still unclear whether sea
stars use chemical signals during orientation and
whether chemoreception occurs in the absence of mac-
roscale flow. To determine whether the sea star Asterias
forbesi can perceive chemical signals in the absence of
flow and what role such signals play in orientation and
foraging behavior, we tested the orientation behavior of
sea stars to prey and nonprey items under conditions of
nondirectional flow. Prey items were whole and broken
clams (Mercenaria mercenaria) and mussels (Mytilus
ethtlis); the nonprey item was squid flesh. Asterias forbesi
showed the ability to successfully locate odor sources ir-
respective of the type of odor. Only in trials with the bro-
ken clam did the animals reveal an initial directional
choice towards the odor source. There were significant
changes in the movement rates and heading angles dur-
ing orientation for all three stimuli. In addition, orienta-
tion paths were different for each of the chemical stimuli
tested. From these results, we conclude that sea stars can
detect and respond to chemicals in the absence of mac-
roscale flow. Orientation paths appear to be more of a
taxis, in which heading is directly guided by the stimulus
field.

Introduction

Asteroids are marine benthic invertebrates found in

abundance in the littoral and subtidal zones of most

lines. Most asteroids are carnivores and can play

1 leant role as keystone predators in these habitats

(1 . Hid Christensen, 1966; Jangoux. 1982). Prey

Receiv : l

1996; accepted 3 March 1997.

items consist of large epifaunal species including gastro-
pods, various bivalves, and some crustaceans, typically
barnacles (Feder and Christensen, 1966; Jangoux, 1982).
Some species show food preferences that are not due ex-
clusively to prey availability (Christensen, 1957; Jan-
goux, 1982). The sensory mechanisms that asteroids use
to forage for prey have been debated for a number of
years.

Most of the previous research, focusing on the chemo-
sensory or mechanosensory abilities of asteroids, has re-
sulted in mixed conclusions. Sloan and Campbell (1982)
have reported evidence for olfaction in some species,
while concluding that others have no such abilities.
Other researchers have shown that olfaction is important
for foraging (McClintock and Lawrence. 1981; McClin-
tocki'/tf/., 1984; Valentincic, 1985; Valentincic and Ota,
1985). In addition, chemical orientation also has been
seen in both field (Christensen, 1957) and laboratory set-
tings (Castilla, 1972a;Castilla and Crisp, 1970; Rochette
el al.. 1994). Conversely, it was noted that A. ntbens
showed an avoidance response to damaged or spawning
prey and living predators in the static-flow Y-maze and
the flow tank (Castilla and Crisp, 1970). Crossaster pap-
posus was observed to be attracted to prey extracts by
choosing the arm in a Y-maze in which the extract was
added. They also avoided the extract of carnivorous con-
specifics by moving downstream (Sloan and Northway,
1982). However, orientation to live oysters, mussels, and
nudibranchs could not be demonstrated in other in-
stances (Sloan and Campbell. 1982).

Many of these studies were done in flow tanks (Cas-
tilla. 1972b; Castilla and Crisp. 1970; Sloan and North-
way, 1982;Zafiriou, 1972;Zafiriou etai. 1972), to which
sea stars show a positive rheotaxis (Valentincic. 1983).
Observed movement toward the odor source may or may
not have been mediated bv chemical stimulation but

410

CHEMORECEPTION AND BEHAVIOR IN ASTEROIDS

41

may have been strictly a response to water flow. Avoid-
ance responses in these situations may be negative rheo-
taxis, initiated by the presence of the chemical signal. Be-
havioral trials with no macroscale flows can help elimi-
nate the possibility of rheotaxis but may have some
confounding factors due to small-scale water dynamics
in the tank. Sea stars, when excited by a food item in the
aquarium, often pass directly by it or turn away just as
they near it (Federand Christensen, 1966). These behav-
ioral results could be interpreted as being a result of
small-scale circulation patterns within the aquarium.

The spatial distribution of chemical signals in marine
environments is determined by the hydrodynamics of
the environment. In most marine habitats, turbulent
diffusion is the major force that disperses chemicals.
Whether in air or water, turbulent odor concentrations
in plumes are heterogeneous when measured at fast tem-
poral and small spatial scales (air: Murlis and Jones,
1981; Murlis el al, 1991; water: Atema, 1985; Moore
andAtema, 1988, 1991;Zimmer-Faust ct al., 1988). The
exact nature of concentration fluctuations within any
habitat depends on the interaction between the size of
the turbulent eddies and the size of the odor plume. As a
result of these interactions, animals using chemical sig-
nals to locate potential prey items will experience differ-
ent stimulus patterns in different flow regimes (Moore el
al., 1994). Thus, if changes in behavior occur simulta-
neously with changes in flow, it is often difficult to deter-
mine whether those changes are due to the differences in
the chemical signal or the mechanical signal. In previous
work, mechanical and chemical signals were presented
simultaneously (Castilla, 1972b; Castilla and Crisp,
1970; Rochette et al., 1994; Sloan and Northway, 1982;
Zafiriou, 1972; Zafiriou el al., 1972). To determine
which source of information is important in orientation
behavior, it is important to separate these two sources of
information during orientation trials.

Marine animals use many different behavioral mech-
anisms to orient to a chemical source. Categories for ori-
entation behaviors have been based on a variety of cri-
teria, including locomotor output, distribution and
number of sensory receptors, and information available
to and used by the animal (Bell, 1984; Dusenbery. 1992;
Kennedy, 1986; Preface for Bell and Carde, 1984;
Schone, 1984). In abroad sense, it appears that chemical
signals either play a direct guidance role (Johnson and
Teeter, 1980; Moore et al. 1991; Reeder and Ache,
1980; Rochette el al., 1994), serve to initiate maneuvers
that depend on nonchemical stimuli (Mafra-Neto and
Carde, 1994), or can be used in conjunction with me-
chanical signals ( Weissburg and Zimmer-Faust, 1993).

From a sensory perspective, it is important to differ-
entiate between potential sources of directional informa-
tion. Y-mazes and other choice experiments demon-

strate that chemoreception can play a role in directional
decisions, but under these conditions, reliable direc-
tional information is still provided by the unidirectional
flow. Although there are problems of small-scale circula-
tion, the reason for performing experiments in still water
is the removal of any directional information provided
by macroscale currents. Although it is highly unlikely
that flow is completely absent in still-water trials, it is
equally unlikely that reliable directional information on
the odor source is provided by any small-scale circula-
tion present within the tank.

Chemical cues are apparently important in orienta-
tion byA.farbexi. but it is still not clear whether chemical
signals or mechanical and chemical signals mediate for-
aging and orientation. The purpose of this study was to
demonstrate chemoreception by A. forbesi in the ab-
sence of directional information provided by macroscale
flow and to investigate the role of chemical signals in ori-
entation behavior. We hypothesize that this species of
sea star has the ability to use chemical signals in the ab-
sence of macroscale flow to successfully orient to poten-
tial prey. Thus, still-water trials become crucial for deter-
mining the source of information controlling or guiding
the orientation of the organism. A better understanding
of the chemoreceptive abilities of this sea star and
sources of sensory information used in foraging may lead
to a broader understanding of intertidal foraging mecha-
nisms in sea stars.

Materials and Methods

Animals

Sea stars, Asterias forbesi, were obtained from the
Aquatic Resources Division of the Marine Biological
Laboratory in Woods Hole, Massachusetts. The animals
were wild caught off the coast of Woods Hole and re-
mained in flow-through seawater tanks before shipping
to our laboratory. The animals measured 7.5-15.25 cm
in diameter and were kept in 35-gallon aquaria, main-
tained at a salinity of 25%o-30%o, a temperature of 15°-
20°C, and a cycle of 12 h light and 12 h dark. An under-
gravel filter was used for maintaining water quality in
the tank, and filtered air was pumped into the system for
aeration and circulation. Each sea star was isolated from
the others in the aquarium by plastic crating, which al-
lowed water movement between the chambers but sepa-
rated animals for individual identification. No more
than four individuals were housed in a single aquarium.
The sea stars were fed once a week at a maintenance
level, about 2 g of thawed squid flesh. Any uneaten food
was removed from the aquarium after 2 h.

Hard-shelled clams (Mercenaria mercenaria) and blue
mussels (Mylilus edulis) were obtained from the Aquatic
Resources Division of the Marine Biological Laboratory

412

P. A. MOORE AND D. M. E. LEPPER

in Woods Hole. Both of these molluscs are common prey
items of A. /r >/•/>< v in ?h<s area. The clams (5-10 cm in
width) and muv- cm in width) were placed in a

35-gallon ai under the same conditions of salin-

ity, temper .u- nd light as the sea stars. They were fed
suspended algae twice a week for 4 h each time by plac-
ing them in beakers with the algae.

Experimental procedure

The testing arena was an acrylic plastic aquarium, di-
mensions 57.5 cm X 1 17.5 cm, with a water depth of
5 cm. It was located within the same room as the housing
aquaria and under the same lighting conditions. The sa-
linity of the tank was matched to the salinity of the hous-
ing aquarium. The bottom of the tank was marked off in
a 5-cm grid with the origin (0, 0) at the center of the tank.
Between each trial, the tank was rinsed with cold tap wa-
ter to avoid any chemical contamination from the previ-
ous trial. After filling, the tank was allow to sit for at least
1 h before orientation trials were started.

For every trial, the animal was placed at the origin,
with its madreporite as a fixed point of reference. Previ-
ous observations indicated that handling did not ad-
versely affect the behavior. In trials with a chemical stim-
ulus, the source was placed in a position (.\ = 0 cm, r
= -45 cm) that allowed the sea star to move both toward
and away from the stimulus by several body lengths and
prevented accidental contact with the source if the ani-
mal was only following the contours of the tank walls.

During each 25-min trial, the position of the sea star
(determined using the madreporite as a reference) was
recorded by eye every 30 s. Animals that did not move
for a period of 6 min were rejected. For experimental tri-
als, the chemical stimulus (squid, mussel, or clam) was
placed in the tank before the sea star was added. If the
animal walked within 5 cm of the stimulus source, the
run was considered successful. If the madreporite of the
animal was within 5 cm of the odor source, one of the
arms would be in contact with the source. By accepting a
distance of 5 cm for the madreporite, we had a criterion
that was consistent between trials and independent of the
arm size of the animals. Control studies were conducted
in the same way as experimental trials except that no
chemical stimulus was placed in the tank. After each
trial, the sea star was returned to its chambered aquar-
ium and any stimulus source discarded.

Stimulus preparation and distribution

In chemical stimulus trials, five stimulus sources were
used: squid flesh (a broad-based amino acid source),
whole live clam and mussel (potential prey items), and
cracked clam and mussel (wounded potential prey
items). For the squid stimulus, a 2-g piece of flesh was

cut from the body of the squid with a sharp blade. The
frozen flesh had been obtained in bulk and was allowed
to thaw before use. For whole prey trials, the bivalve was
placed in the tank 5 min prior to an orientation run to
allow it to begin filtering. For broken prey trials, the bi-
valve was crushed and left in the tank for 10 min prior to
an orientation run. Mussels were prepared by holding
the animal directly over the source point and crushing
the shell with ph'ers; clams were placed on plastic wrap,
hit with a hammer, and then transferred to the source
point. Additional control trials used cleaned and dried
empty shells from the clam or mussel. For these trials,
an intact shell of each species was recovered from dead
individuals in the holding aquarium. The shells were vig-
orously scrubbed with biodegradable soap and a nylon
brush. The shells were then rinsed under tap water for
1 min, soaked in tap water for 1 h, and soaked in dilute
bleach ( 1:10) for 10 min. After this the shells were rinsed
under tap water for 1 min, soaked three times for 30 min
each in distilled water, and rinsed between each period.
Just prior to the trials, the shells were soaked for 30 min
in 30%o salt water.

To qualitatively analyze the spatial movement of
chemicals within the testing arena, we used fluorescein
dye in gelatin blocks to mimic the spatial distribution
of chemicals emanating from the nonliving prey items.
Gelatin blocks with 2 g/1 of fluorescein dye were placed
within the testing arena using the methods outlined
above for broken prey items. After 10 min, the spatial
distribution of the dye was mapped visually using four
broad categories of dye concentration. This process was
repeated six times. This qualitative analysis showed that
each of the six replicates resulted in a different spatial
distribution and that the lowest visible concentration of
dye was 35 ± 0.2 cm (SEM) away from the odor source.
For each replicate, the distance that 10 individual fila-
ments of dye moved during a 1-min period was recorded
as an estimate of the maximum water velocity within the
tank. The estimate of the maximum water velocity was
3 ± 0.5 cm/min (SEM) or 0.05 ± 0.008 cm/s for all 60
samples.

Data analysis

The orientation paths from each run were recorded as
a single point (A. r) every 30 s as previously described.
From these paths, several orientation parameters were
analyzed. These included walking speed, net-to-gross ra-
tio (NGR), turning angles, and headings. NCR values
were transformed using an arcsine method (specifically,
// = arcsine p</2, Zar, 1984). Standard ANOVA tech-
niques were used to compare the average value for each
parameter between each of the trials. If the ANOVA was
significant, it was followed by a post-hoc comparison us-

CHEMORECEPTION AND BEHAVIOR IN ASTEROIDS

413

ing a Neuman-Keuls analysis. A linear regression analy-
sis was used to correlate changes in parameter values
with distance from source. Distance from the source for
regression analysis was the Euclidean distance between
the source point (0, 0) and the animal's position within
the tank (described above). NGR was calculated by di-
viding the Euclidean distance from start to finish (net) by
the total path length (gross). These values can range from
0 to 1, with 0 being a very circuitous path, and 1 being a
linear path. Turn angle was denned as the angle between
the path connecting the previous (/ = -!) position to the
present (/ = 0) position and the path connecting the pres-
ent to the next (/ = +!) position (Moore el ai, 1991).
A sea star's heading was defined as the angle between a
straight line towards the source and the direction from
the present position to the next position on the track,
with an angle of zero pointing directly at the source
(Moore el ai. 1991). All angles were calculated in a
clockwise fashion.

The initial directional choice for each sea star was re-
corded as the heading angle for initial movement from
the starting point. A modification of the Rayleigh's test
(The I 'test) was used to determine whether the heading
angles were uniformly distributed around the circle or if
there was a significant directionality to the movement
(Zar, 1984). For this test, we assumed a priori that an

angle of zero degrees towards the odor source was ex-
pected.

Results

General orientation results

Our criteria for successful orientation resulted in rejec-
tion of 52% of the total number of trials. Successful trials
resulted in different paths that depended upon the odor
source (Fig. 1 ). Most rejections were due to the animal's
inability to locate the odor source; very few of the trials
were rejected due to lack of movement by the animal.
This high rate of rejection may be due to the unnatural
experimental condition of no macroscale flow. Subse-
quent testing under flowing conditions may produce a
higher success rate, but would not allow us to pursue the
hypothesis proposed in the introduction.

Orientation to squid flesh

The odor of squid flesh in the test arena caused the
animal to modify its walking behavior. This was shown
by a significantly slower walking speed in the experimen-
tal animals compared with controls (/ test, P < 0.0 1 , Fig.
2). Sea stars in the presence of odor walked at 0.06 ± 0.0 1
(SEM) cm/s and control animals at 0.18 ± 0.02 cm/s. In

20

10

-10

-20 -

20

10

-20

A. Broken Clam

20

-10

-20 •

B. Whole Clam

•10 0 10 20 30 40 50 60 70 80 90 100 -10 0 10 20 30 40 50 60 70 80 90 100
30 i i • i • I 30 r

C. Broken Mussel

20

10

-10

D. Whole Mussel

-10 0 10 20 30 40 50 60 70 80 90 100

-10 0 10 20 30 40 50 60 70 80 90 100

Figure 1. Randomly selected trials for four different sea stars for tour different stimuli. Odor sources:
(A) broken clam, (B) whole clam. (C) broken mussel. (D) whole mussel. Stimulus source is located at 0,0
(asterisk) and starting point of orientation path is at 45,0 (solid square).

414

P. A. MOORE AND D. M. E LEPPER

Ifl

(A

s

o

Control
Whole Prey
Broken Prey
Prey Shell

Figure 2. Average (±SEM) net-to-gross ratio and walking speed of sea stars under no odor stimulation
(control, blank bar), whole prey (gray bar), broken prey (black bar), and empty prey shell (hatched bar).
Prey items: (A) clam, (B) mussel. (C) squid flesh, n = 14 for all trials for clam and mussel; n = 22 for squid.
Asterisk indicates a value that is significantly different from all other values (ANOVA, then Newman-
Keuls test, P< 0.05).

addition, the paths of sea stars walking during chemical
stimulation were more linear than those of control ani-
mals. This was shown by a significantly higher NCR in
the experimental animals than in the controls (/ test, P
<0.01, Fig. 2). Experimental animals had an average
net-to-gross ratio of 0.58 ± 0.04 (SEM), whereas control
animals had ratios of 0.14 ± 0.02. Further analysis of
orientation paths showed a significant relationship be-
tween heading angles and distance from the odor source
(Table I). Sea stars had lower heading angles as they ap-
proached the odor source, but this same relationship was
not present in the turning angles. Analysis of the initial
directionality of sea star orientation to squid flesh
showed no significant initial directional choice for either
the control or the experimental groups.

Orientation to natural prey items

Clam. Sea stars walked significantly faster when a bro-
ken clam was in the test arenas than they did in the other
test situations (Fig. 2). There was no difference in walk-
ing speeds among the control, whole clam, or empty
clam shell trials. Sea stars showed a more circuitous path
in the presence of a broken clam than in the other test
situations (Fig. 2). There was no difference in NGR
among the control, whole clam, or empty clam shell tri-
als. Regression analysis of orientation paths showed a

significant relationship between heading angles and dis-
tance from the odor source during whole clam stimula-
tion (Table I). As with the squid odor, heading angles
during orientation significantly decreased as sea stars ap-
proached the odor source. This relationship was absent
from heading angles for the control, empty shell, and

Table I

Regression summary of changes in heading and turning angles
with distance from odor source

Odor source

Heading angles

Turning angles

Control

N.S.

N.S.

Squid

]•= 37. 7 + 0.59 .v*

N.S.

Whole mussel

v = 46.0 + 0.52 .VB

N.S.

Broken mussel

v= 31.8 + 0.86.vc

N.S.

Mussel shell

N.S.

N.S.

Whole clam

r = 26.2 + 0.82 .v"

N.S.

Broken clam

N.S.

N.S.

Clam shell

N.S.

N.S.

All regressions were run on the population of sea stars within each
trial.

AF( 1 ,393) = 20. 1 , P < 0.05, r = 0.22.
"/=•(!, 348)= 12.5, /><0.05, r = 0.18.
CF( 1 , 1 94) = 1 6.4, P < 0.05, r = 0.28.
°F( 1 . 1 20) = \\A.P< 0.05, r = 0.30.
All nonsignificant results had P> 0.10.

CHEMORECEPTION AND BEHAVIOR IN ASTEROIDS

415

broken clam situations and was absent from all turning
angle analysis. Analysis of the initial directional choices
of sea stars during clam stimulation showed a significant
directional choice towards the odor source for the broken
clam only (The I ' test, M<> = 0°, u = 2.0. P < 0.05, Fig.
3). All other heading angles were uniformly distributed
around the circle, indicating no significant initial head-
ing angle.

Ahissel. Sea stars did not alter either their walking
speed or the NCR in any of the trials involving either
mussels, broken mussels or empty mussel shells when
compared to control values (Fig. 2). There was a signifi-
cant change in heading angles for both the whole and
broken mussel trials. Sea stars significantly decreased
their heading angles as they approached the odor source
(Table I). This relationship was absent from heading an-
gles for the control and empty mussel shell and was ab-
sent from all turning angle analysis. Analysis of the ini-
tial directional choices of sea stars during mussel stimu-
lation showed that there was not a significant directional
choice towards the odor source for any of the odor stim-
ulations (Fig. 4).

Discussion

In previous orientation studies using sea stars, it has
been difficult to differentiate between a true chemically
mediated response, in which the animal is responding
only to the odors, and a combination of chemically and
mechanically mediated responses. In fact. Rochette el al.
( 1994) showed conclusively that Leptasterias polaris has
a strong orientation response to current in both the pres-
ence and absence of chemical stimulation. The orienta-
tion behavior that results from multiple inputs of che-
mosensory and mechanosensory information could be
due to a range of orientation strategies from a chemically
triggered rheotaxis to a flow-triggered chemotaxis (for a

review of different orientation strategies, see Shone,
1 984). This study demonstrates three results concerning
the chemosensory abilities ofA.forbesi. First, this species
perceives and responds to chemical signals in the absence
of macroscale flow. Many previous studies on both the
foraging and orientation behavior of other sea stars have
been in flow tanks (Castilla, 1972b: Castilla and Crisp,
1970; Sloan and Northway, 1982; Zafiriou, 1972; Zafi-
riou el al., 1972). In many of these situations, the sea
stars show a positive rheotaxis (Valentincic, 1983), and
it is not clear whether they are responding to the mac-
roscale flow, to the chemical, or to both.

Second, we demonstrate that A. forbesi is capable of
locating odor sources without the aid of external direc-
tional cues provided by macroscale flow. The analysis of
the orientation paths shows that orientations are proba-
bly a response to information from the chemical signal
within the experimental arena. Several features of the
orientation paths reveal an orientation strategy that may
be a taxis (as defined in both Dusenbery, 1992, and
Schone, 1984). Using these definitions, a taxis is an ori-
entation that is a result of biased or directed turns with
respect to some aspect of the stimulus field. This would
result in decreased heading angles as the organism ap-
proached the odor source. As has been demonstrated for
other sea stars (Rochette ct al.. 1994), and suggested for
A. forbesi under different flow conditions (Dale, 1996),
these animals use concentration differences as measured
at the tips of different arms to guide orientation to food
sources. This is a tropotactic response, in which the ori-
entation path is guided by simultaneous information ob-
tained by spatially separate sensory cells. If A. forbesi is
performing a tropotaxis by comparing different concen-
trations at the tips of its arms, we would expect to find
some initial directional decisions towards the odor
source and decreased heading angles as the animal ap-
proached the source.

Control

Broken Clam

Clam Shell

Whole Clam

27O(-w

.90 270

135

135

315

180

Figure 3. Initial directional choices for sea stars during different odor stimulations for clam sources.
Solid dots represent heading angle in relation to odor source for the first movement of the animal. Sig-
nificant mean angles are indicated by a solid black arrow. I' test. All other groups were uniformly distrib-
uted around the circle: >i = 1 4 for all groups.

416

Control

P. A. MOORE AND D. M. E. LEPPER

Broken Mussel Mussel Shell

Whole Mussel

315

315

270

135

315

45

90 270

225

13S

31 3

225

135

180

180

Figure 4. Initial directional choices for sea stars during different odor stimulations for mussel sources.
Solid dots represent heading angle in relation to odor source for the first movement of the animal. There
were no significant mean angles in any of the groups, indicating that the initial headings were uniformly
distributed around the circle; n = 14 for all groups.

We found changes in the orientation paths to be a
function of distance from the odor source. For all of our
stimulus conditions except one, we found that the head-
ing angle relative to the odor source decreased as an ani-
mal approached the odor source. In other words, as it
approached the odor source, the animal began to walk in
a straighter line towards the source. Since we did not find
similar results with the control or shell studies, we con-
clude that the locomotory output of the animal is influ-
enced by the chemical stimulus whether it is perceived
spatially or temporally. In addition, walking speed and
turning angles did not change as a function of distance.
Although these results are consistent with either orienta-
tion mechanism, we feel that the behavioral patterns of
our animals in the absence of macroscale flow are more
compatible with a taxis-based orientation strategy then
with a kinesis strategy (as denned by Dusenbery, 1 992).
This finding is similar to that of Rochette el al. ( 1 994),
who convincingly demonstrated that other species of sea
stars were capable of showing directed responses, but
that these directional choices were dependent upon their
physiological state (starved vs. fed) and the ambient cur-
rent flow. Their finding may be due to decisions based
on dual sensory information (that is, from both the odor
signal and ambient coalitions of macroscale flow),
whereas we provided main, -hemosensory cues.

Third, A. forbesi respond^ ;fferently to the various
stimulus sources by having dis ' walking patterns for
each of the stimulus conditions. L :ip, stimulation with
squid flesh, the sea stars walked sk r. in a straighter
line, with a characteristic decrease in heading angle as
they approached the odor source. The sea stars walked
faster and had a more circuitous path only for the broken
clam and showed only the characteristic changes in head-
ing angles for the mussel stimulus. Different locomotory
outputs may indicate cither that these animals can per-
ceive and identify different prey items or that the outputs
are responses to concentration differences between stim-

ulus sources. Since we neither quantified the stimulus
patterns nor analyzed the chemical composition under
the different treatments, it is difficult to make any defi-
nite conclusions based on the differences in locomotion.

Many researchers have studied chemical orientation
in marine animals. Animal models include fish (Kleere-
koper, 1967; Kleerekoperefa/., 1969) and decapod crus-
taceans (Moore el al.. 1991; Reeder and Ache, 1980;
WeissburgandZimmer-Faust, 1993). These studies have
shown that the spatial and temporal distribution of
chemical signals plays an important part in the orienta-
tion ability of these animals. It has also been shown that
animals can have different responses to different odors.
Certainly, for sea stars, some odors trigger an attraction
and other odors an avoidance (Castilla and Crisp, 1970;
Sloan and Northway, 1982). Similar findings have been
recorded for the mud snail (Atema and Burd, 1975). All
of these results taken together show that marine organ-
isms can have different behavioral responses for different
chemical sources, but it has yet to be shown whether a
single animal has distinct orientation strategies for
different odors.

In summary, A. forbesi has chemosensory responses in
the absence of macroscale flow and can locate the source
of odors using only chemosensory information. It is still
unclear whether these animals strictly use a chemotaxis
or a kinesis strategy and whether they use both spatial
and temporal sampling of concentrations to guide orien-
tation to the odor source. In addition, the sea stars show
different orientation paths for different odors. Further re-
search is needed to determine whether these animals
have different orientation strategies that are prey depen-
dent.

Acknowledgments

The authors thank Dr. Steve Vessey for reading an ear-
lier version of this manuscript and three anonymous re-

CHEMORECEPTION AND BEHAVIOR IN ASTEROIDS

417

viewers for insightful comments. This research is sup-
ported by NSF grant OCE-9596270.

Literature Cited

\u m.i. J. 1985. Chemoreception in the sea: adaptation of chemore-
ceptors and behavior to aquatic stimulus conditions. Stic. Exp. Bioi
Symp. 39: 387-423.

Atema, ,)., and G. D. Burd. 1975. A lield study of chemotactic re-
sponses of the marine mud snail Nassaritis obsoletus. J Chem
Ecol 1:243-251.

Bell, \\.J. 1984. Chemo-orientation in walking insects. Pp. 93-106
in Chemical Ecology ol Insects, W. J. Bell and R. T. Carde, eds.
Sinauer Associates. Sunderland. MA.

Bell, \V. J., and R. T. Carde. 1984. Chemical Ecology of Insects. Si-
nauer Associates. Sunderland, MA.
Castilla. J. C. 1972a. Responses ofAsterias ruhens to bivalve prey in

a Y-maze. Mar. Biol 12: 222-228.

Castilla, J.C. 1972b. Avoidance behavior of Aslerias rithens to ex-
tracts of Mviilits ediilis. solutions of bacteriological peptone, and
selected amino acids. .Mar. Biol. 15: 236-245.
Castilla, J. C., and D. J. Crisp. 1970. Responses ofAsterias rubens to

olfactory stimuli./ Mar. Bio/. Assoc. L: A 50: 829-847.
Christensen, A. M. 1957. The feeding behavior of the seastar Eva-

steriaslroschelii Stimpson. Limnol. Oceanogr. 2: 180-197.
Dale, J. 11. 1996. Co-ordination of chemosensory orientation in the

starfish. Aslerias jorhesi. Chem Senses Abslr. (in press).
Dusenbery, D. B. 1992. Sensory Ecology: How Organisms Acquire

and Respond to Information. W.H. Freeman. New York.
Keder H., and A. M. Christensen. 1966. Aspects of asteroid biology.
Pp. 87-127 in Physiology of Echinodermata, R. A. Boolootian, ed.
Interscience. New York.

Jangoux, M. 1982. Food and feeding mechanisms: Asteroidea. Pp.
117-159 in Echiiwderm Nutrition. M. Jangoux and J. M. Law-
rence, eds. Balkema, Rotterdamm.
Johnsen, P. B., and J. H. Teeter. 1980. Spatial gradient detection of

chemical cues by catfish. J. Comp. Physio/. A 140: 95-99.
Kennedy, J. S. 1986. Some current issues in orientation to odour
sources. Pp. I 1-25 in Mechanisms in Insect Ol/aclion, T. L. Payne,
M. C. Birch, and C. E. J. Kennedy, eds. Claredon Press, Oxford.
Kleerekoper, H. 1967. Some aspects of olfaction in fish, with special

reference to orientation. Am. 7.ooi 7: 385-295.

Kleerekoper, H., A. M. Timms, G. F. Westlake, F. B. Davy, T. Malar,
and V. M. Anderson. 1969. Inertial guidance system in the orien-
tation of the goldfish (Carassitisaiiralits). Nature 223: 501-502.
Mafra-Neto, A., and R. T. Carde. 1994. Fine scale structure of pher-
omone plumes modulates upwind orientation of flying moths. Na-
ture 369: 142-144.

McClintok,J.B.,T. S. Klinger.and J. M.Lawrence. 1984. Chemore-
ception in Liicidia elathraia (Echinodermata: Asteroidea): qualita-
tive and quantitative aspects of chemotactic responses to low mo-
lecular weight compounds. Mar. Biol 84: 47-52.
McClintok, J. B., and J. M. Lawrence. 1981. An optimization study
on the feeding behavior of Liicidia clalhrala Say (Echinodermata:
Asteroidea). Mar. Behav. Physio/ 10: 167-181.

Moore, P. A., and J. Atema. 1988. A model of a temporal filter in
Chemoreception to extract directional information from a turbulent
odor plume. Biol. Bull 174: 355-363.

Moore, P. A., and J. Atema. 1991. Spatial information in the three-
dimensional fine structure of an aquatic odor plume. Biol. Bull
181:408-418.

Moore, P. A., N. Scholz, and J. Atema. 1991. Chemical orientation of
lobsters, Homarus americanus, in turbulent odor plumes. ./. Chem
Ecol. 17: 1293-1307.

Moore, P. A., M. J. Weissburg, J. M. Parrish, R. K. Zimmer-Faust,
and G. A. Gerhardt. 1994. Spatial distribution of odors in simu-
lated benthic boundary layer flows. / Chem. Ecol 20: 255-279.

Murlis J., and C. D. Jones. 1981. Fine-scale structure of odour
plumes in relation to insect orientation to distant pheromone and
other attractant sources. Phys. Ent. 6: 7 1-86.

Murlis, J., M. A. Willis, and R. T. Carde. 1991. Odour signals: pat-
terns in space and time. Pp. 6-17 in Proceedings ol the Tenth In-
ternational Symposium on Ol/aclion and Taste. K. Doving, ed.
Graphic Communication System, Oslo.

Reeder, P. B., and B. W. Ache. 1980. Chemotaxis in the Florida spiny
lobster. Pamilints argils. Anim Behuv 28: 831-839.

Rochette, R., J.-F. Hamcl, and J. H. Himmelman. 1994. Foraging
strategy of the asteroid Leptasterias polaris: role of prey odors, cur-
rent and feeding status. Mar Ecol. Prog Ser. 106: 93-100.

Shone, II. 1984. Spatial Orientation. Princeton University Press,
Princeton, NJ.

Sloan, N. A., and A. C. Campbell. 1982. Perception of food. Pp. 3-23
Echinoderm Nutrition. M. Jangoux and J. M. Lawrence, eds. Bal-
kema. Rotterdam.

Sloan N. A., and S. M. Northway. 1982. Chemoreception by the as-
teroid Crossaster papposus (L). J. Exp. Mar. Biol. Ecol. 61: 85-98.

Valentincic, T. 1983. Innate and learned responses to external stimuli
in asteroids. Pp. 1 1 1-137 Echinoderm Nutrition. M. Jangoux and
J. M. Lawrence, eds. Balkema, Rotterdam.

Valentincic, T. 1985. Behavioral study of Chemoreception in the sea
slarMarlhastcriusglacia/is: structure-activity relationships of lactic
acid, amino acids, and acetylcholine. J. Comp. Physio/. A 157: 537-
545.

Valentincic, T., and D. Ola. 1985. Comparison of chemical senses in
the sea star Marthasterias g/acialis with chemical senses in some
fishes. Pp. 563-569 in Echinodermata: Proceedings oj the Filth In-
ternational Echinoderm Conference. B. F. Keegan and B. D. S.
O'Connor, eds. Balkema, Rotterdam.

Weissburg, M. J., and R. K. Zimmer-Faust. 1993. Life and death in
moving fluids: hydrodynamic effects on chemosensory-mediated
predation. Ecology 14: 1428-1443.

/.iln urn. O. 1972. Response of Aslerias vulgaris to chemical stimuli.
Mar. Biol. 17: 100-107.

Zafiriou, O., K. J. Whittle, and M. Blumer. 1972. Response of Aste-
ncis vulgaris to bivalves and bivalve tissue extracts. Mar. Biol. 13:
137-145.

Zar, J. H. 1984. Biostali.slical Analysis, second edition. Prentice
Hall, Englewood Cliffs. NJ.

Zimmer-Faust, R. K., J. M. Stanfill, and S. B. Collard, III. 1988. A
fast, multichannel fluorometer for investigating aquatic Chemore-
ception and odor trails. Limnol Oceanogr. 33: 1 586- 1595.

Reference: Biol. Bull 192: 418-425. (June. 1997)

Behavioral Modes Arise From a Random Process
in the Nudibranch Melibe

AMANDA E. SCHIVELL1, SAMUEL S.-H. WANG2, AND STUART H. THOMPSON3

Department of Biological Sciences and the Hopkins Marine Station, Stanford University.

Pacific Grove. California 93950

Abstract. Stochastic analysis was applied to observa-
tions of spontaneous behavior in the carnivorous mol-
lusc Melibe leonina. Six behaviors were defined that
could be easily recognized on inspection and it was
found that transitions between each of these behaviors
could be fully described by a first-order random process
without memory of past behavioral choices. The behav-
iors are organized by frequency of transition into two
modes, a feeding mode and a resting mode. Transitions
within modes are more likely than transitions between
modes, and the feeding and resting modes are linked by
a preferred pair of behavioral transitions. The amount of
time spent in the feeding mode is positively correlated
with body size, but the average length of a feeding epi-
sode is independent of size. This suggests that body size
regulates the probability of entry into feeding behavior
but does not influence the basic pattern of feeding. In the
presence of food the animals express nearly continuous
feeding behavior, suggesting that food reduces the prob-
ability of exiting the feeding mode. This model of spon-
taneous behavior in Melibe is used to form hypotheses
amenable to further exploration through neurophysio-
logical experiments.

Introduction

Analysis of animal behavior provides the background
necessary for studies of the underlying neural circuitry
and circuit function. Such analysis sometimes permits

Received 2 December 1996; accepted 28 February 1997.

'resent address: Dept. of Zoology. University of Washington. Seat-
tle, W'V

i address: Dept. of Neurobiology. Bryan Research Bldg.,
Duke i n rsity Medical Center, Durham, NC 277 10.
3 Auti horn correspondence should be addressed.

useful predictions to be made about specific properties of
the underlying neural networks — for example, the extent
to which stochastic processes in the nervous system in-
fluence the ordering of behaviors in a sequence (Heili-
genberg, 1973). Molluscs have been used to advantage
by neuroethologists because the relatively limited behav-
ioral repertoires and accessible nervous systems of these
animals permit investigation of synaptic connections be-
tween identified neurons (Davis et a!., 1974: Kandel.
1976; Gelperin, 1983; Willows, 1985: Getting, 1989).
Most studies have concentrated on reflex or conditioned
responses evoked by stimuli presented in defined pat-
terns, an approach that simplifies the design of electro-
physiological experiments. Important generalizations
about the organization of small neural networks have
emerged from such studies, including the idea that ani-
mal behaviors are organized in hierarchies of succes-
sively more dominant or more strongly commanded re-
sponses (Tinbergen, 1951: Davis el al.. 1977; Getting,
1989).

Concentration on evoked and conditioned behavior
ignores, however, the phenomenon of unstimulated be-
havioral choice (Lorenz. 1 98 1 ). We recorded the tempo-
ral sequence of behaviors in the nudibranch Melibe leo-
nina as it evolved spontaneously in the absence of overt
stimulation, then used standard techniques of random-
process analysis to model this behavioral sequence
(Chatfield and Lemon, 1970). Our results lead to several
conclusions. (1) In the absence of overt stimuli, the
choice of behavior is well described by a first-order ran-
dom process. (2) Consideration of the transition proba-
bilities of this process indicates that spontaneous behav-
iors in Melibe fall into two behavioral modes. (3) There
is a preferred transition that links the behavioral modes.
(4) The presence of strong stimuli such as food organisms
influences the individual transition probabilities. Fi-

41S

BEHAVIORAL MODES IN MELIBE LEON1NA

419

nally. (5) physiological parameters, such as body size,
also influence behavioral transition probabilities.

Mclibe is well suited to this study. We were able to
define a set of six canonical behaviors that are easily rec-
ognized on inspection. Transitions between these behav-
iors were infrequent and could be adequately scored by
making observations at 15-min intervals. This permitted
us to examine behavioral transitions over an extended
time period and to perform replicate sets of observations.
Furthermore, the Mclibe central nervous system is suit-
able for microelectrode and optical recording methods
for monitoring cellular activity in identified neurons in
restrained whole animal preparations (Willows, 1973;
Cohen et a/.. 1991). This provides the opportunity to
study mechanisms of spontaneous behavioral expression
at the level of neural networks.

Materials and Methods

Melibe leonina, a carnivorous nudibranch of the fam-
ily Tethyidae, is distinguished by an extended oral veil
used in prey capture (Agersborg, 1923). Two groups of
10 animals were studied between 1 and 29 May. Adult
specimens weighing 40-250 g were collected from the
Macrocystis kelp forest in Monterey Bay, California. The
animals were held in 45-liter tanks supplied with contin-
uously flowing sand-filtered seawater for 2-28 days be-
fore observations were begun. Changes in motivation
were minimized by allowing the animals to acclimate to
the experimental tanks and by simulating a natural envi-
ronment. No correlation was found between the number
of days in captivity and the behavioral pattern. For ob-
servation, animals were placed in five identical 17-liter
acrylic plastic tanks provided with flowing sand-filtered
seawater and a frond of kelp. The tanks rested on sea
tables in a roofed aquarium building where they were
protected from direct sun and rain but exposed to natu-
ral cycles of light and temperature. Methylene blue dye
was injected into the first right ceratum of one animal of
the two animals in each tank to facilitate identification.
The animals were fed every other day at the same time
with the same amount of their natural food, live mysid
shrimp collected from the wild. The animals were other-
wise left undisturbed for eight days of observation, which
included four nonfeeding days from each group.

We define six behaviors that occur frequently but
without overlap in the absence of overt stimulation (Fig.
1 ). Together they account for greater than 90% of the
activity in the observation tanks. The behaviors are dis-
tinguished by characteristic body postures involving the
oral hood, the rhinophore processes, and the cerata, as
well as by the presence or absence of pedal locomotion.
The six behaviors are as follows: ( 1 ) Feeding (F): rhyth-
mic extension and contraction of the oral hood in a cast-

ing motion through the water (Hurst 1968; Watson and
Trimarchi, 1992). (2) Open hood (OH): full extension of
the oral hood with the cerata and rhinophore processes
extended. (3) "Alert'Vprocesses extended posture (AL):
oral hood partially closed with the oral tentacles tucked
inside, cerata and rhinophore processes extended. (4)
Roaming/open hood (R/OH): OH behavior combined
with pedal locomotion. (5) Roaming/processes extended
(R/AL): AL behavior combined with pedal locomotion.
(6) Resting (RST): locomotion absent, hood closed and
held against the substrate, cerata contracted against the
sides of the body, rhinophore processes folded down.

Several other behaviors occurred too infrequently for
analysis. These included (1) pedal locomotion in the
RST posture; (2) the "crumple" reflex, an alarm re-
sponse elicited by tactile or vibratory stimuli and charac-
terized by forceful contraction of most of the body wall
musculature (Scott, 1990); (3) swimming; (4) egg-laying.
Copulation was observed occasionally but was not
scored independently because the six behaviors we fol-
lowed occurred at the same frequency independent of
copulation.

Observations were made at 1 5-min intervals for 6
hours a day beginning between 0800 and 1 200 h. During
preliminary studies, we observed behavior continuously
for periods of several hours. By parsing the data into time
periods of varying duration, we determined that a 15-
min sample interval is adequate to capture behavioral
transitions. Observations from one nonfeeding day for
the 10 animals in group 1 are tabulated in Figure 2. It
is apparent that behavior changes infrequently and that
individuals tend to perform the same behavior over sev-
eral time points. On feeding days the animals spent
about 80% of their time performing feeding behavior (F),
indicating that the presence of prey organisms has a pow-
erful influence on behavioral choice (Watson and Tri-
marchi, 1992; Watson and Chester, 1993). Analysis was
limited to nonfeeding days in order to include a more
diverse range of behaviors and to obtain observations un-
der relatively constant physiological conditions.

Results

The frequency of occurrence of each of the 36 possible
behavioral transitions, including consecutive occur-
rences of the same behavior, was tallied by observing be-
havior at 1 5-min intervals. The data are expressed in Ta-
ble I as transition probabilities for each of two replicate,
4-day experiments. The sum of the transition probabili-
ties from a given starting behavior is normalized to 1.0.
The predominance of certain one-step transition proba-
bilities over others suggests that the six behaviors can be
grouped into two distinct behavior modes (Fig. 3). Be-
haviors F, OH, and R/OH form the feeding mode, while

420

A. E. SCHIVELL ET AL

Figure I. Body postures during six behaviors: (A) Open hood (OH) and roaming/open hood (R/OH)
behaviors. (B) Resting (RST) behavior. (C) Feeding (F) behavior. (D) "AlerT/processes extended ( AL) and
roaming/alert (R/AL) behaviors.

behaviors RST, AL, and R/AL form the resting mode.
This grouping of behaviors is based on the observation
that transitions among mode elements occur much more
frequently than transitions to behaviors outside of the
mode. The frequencies of intermode transitions are
listed in Table II. This qualitative observation is borne
out by calculating between-mode transition frequencies
for all possible ways of grouping the six behaviors into
two modes. The correct mode structure will give fewer
between-mode transitions than any other grouping.

There are 25 ways to structure two modes: 10 in which
each mode contains three behaviors, and 1 5 in which one

mode contains two behaviors and the other contains
four. For data set 1, the number of between-mode tran-
sitions for the mode structure proposed in Figure 3 was
145, whereas between-mode transitions for the other
possible groupings ranged from 267 to 389. This sup-
ports the behavioral relationships diagrammed in Figure
3. According to this analysis, animals stay in one mode
for an average of 1 hour and 1 1 minutes (5. 2 observation
intervals) before switching to the other.

Transitions between modes occur most frequently be-
tween behaviors OH and AL in either direction. The OH
to AL transition accounts for 35% of all transitions from

THURSDAY, MAY 9, 1991. 0900-1500 hrs

Interval «

. 2

3

4

5

6

7

B

9

10

11

12

11

U

IS

16

17

IB] w

20

21

22

21

24j 25,

1A R/OH f

F

F

F

F

F

F

F

F

F

F

F

F

F

F

F

F F

F

F

F

F

F F

41

OH

OH

f

F

OH

R/OH

F

OH

OH

AL

OH

OH

F

F

OH

OH

OH

OH ! OH

OH

AL

OH

OH

OH i F

u

R/OH

F

AL

R/AL

RST

AL

R/AL

RST

RST

R/OH

AL

OH

AL

OH

OH

OH

R/OH RST

OH

R/ALj RST

RST

RST

RST | R/OH

2B

RST

R/OH OH

R/AL

AL

AL

R/AL

AL

CR ! RST

RSt R/AL

RST

RST

RST

R/AL

CR AL

RST

RST i AL

RST

RST

RST R/AL

M

RST

OH

R/ALl OH

OH

OH

OH

OH

OH

OH ( OH OH

OH

OH

OH

OH

RST

OH R/OHl OH OH R/OHI F

OH F

M

RST

RST

RST

RST

AL

AL

R/AL

AL

AL

AL AL AL

R/AL

R/AL

AL

R/AL R/AL

AL

R/AL AL

AL

R/AL R/AL

R/OH R/AL

4A

R/OH

RST

OH

RST

R/OH

OH

OH

OH

OH

OH OH OH

AL

OH

OH i AL

AL AL OH

AL

AL S

AL

OH OH

11

RST

RST

RST

RST

RST

RST

RST

RST

RST

RST

RST RST

RST

RST

RST RST RST

RST RST

AL ' RST RST

RST

RST RST

SA

F

F IR/OH

R/OH

RST

R/AL

RST

R/AL

OH

OH

R/OH RST

R/OH

AL R/OH| R/AL

OH

R/OH

R/AL

AL ! RST RST

OH

R/OH RST

SB

RST

RST

RST

AL

AL

AL

AL

AL

AL

AL

AL AL

AL

AL

AL

AL

R/AL

AL

R/AL

RST

AL AL

R/AL

R/AL RST

Figure 2. Sample data set from one full day of observations. Observations of each of 10 animals were
made at 15-min intervals for 6 h. The duration of a behavior in continuous intervals is referred to as its
dwell time. An example of a seven-interval dwell time in the open hood (OH) behavior can be seen in
animal 4A from intervals six to twelve. Abbreviations: Feeding (F), open hood (OH), "Alert"/processes
extended posture (AL), roaming/open hood (R/OH), roaming/processes extended (R/AL). resting (RST).
crumple (CR), and swimming(S).

BEHAVIORAL MODES IN MELIBE LEON1NA

Table 1

Transition probabilities between all behaviors for both sets of data

421

Table II

Transition probabilities within and between modes

SET1

Ending behavior

Starting

behavior

F

OH

R/OH

AL

R/AL

RST

F

0.597

0.223

0.095

0.038

0.019

0.028

OH

O.I S3

0.486

O.I 15

0.101

0.014

0.101

R/OH

0.299

0.253

0.218

0.080

0. 1 5 1

0.226

AL

0.041

0.142

0.027

0.473

0.122

0.196

R/AL

0.085

0.149

0.085

0.298

0.149

0.234

RST

0.040

0.081

0.054

0.117

0.054

0.664

SET 2

Ending behavior

Starting

behavior

F

OH

R/OH

AL

R/AL

RST

F

0.459

0.208

0.138

0.101

0.075

0.0 1 9

OH

0.188

0.370

0.087

0.268

0.043

0.043

R/OH

0.293

0.200

0.227

0.093

0.120

0.067

AL

0.065

0.094

0.039

0.526

0.101

0.175

R/AL

0.135

0.122

0.135

0.446

0.122

0.041

RST

0.005

0.020

0.010

0.256

0.054

0.655

Each row of probabilities totals 1. 00. Probabilities for transitions oc-
curring within a behavioral mode are given in boldface.

the feeding to the resting mode, and the AL to OH tran-
sition accounts for 30%' of transitions from the resting to
the feeding mode. None of the 16 other possible be-
tween-mode transitions accounted for more than 1 5% of
the transitions in either direction.

Individual variability

The time spent in each behavior varied a great deal
between individual animals. As a measure of this vari-

FEEDING MODE

OH

it

AL ^

R/OH

> R/AL

RESTING MODE

RST

Figure 3. Feeding and resting modes. Arrows indicate the 10 most
frequent transitions between different states. The mode structure min-
imizes intermode transitions: all other possible mode arrangements
were considered (see text).

Probability that
the next transition is to:

Feeding mode

Resting mode

Starting from:

Feeding mode

0.79

0.21

Resting mode

0.18

0.82

ability, we calculated coefficients of variation (CV) for
the number of transitions from a behavior back to itself
(i.e.. F -* F) using the second set of 10 animals. The CV
ranged from 64% to 1 57%. Some of the variability can be
linked to a physiological correlate. For example, we
found that body weight and time spent in the feeding
mode are positively correlated (r = 0.87; Fig. 4). An 80-g
individual spent 10% of the time in the feeding mode as
compared to 80% for a 250-g animal. A similar correla-
tion exists between size and time spent in the feeding (F)
behavior within the feeding mode. The 250-g animal
spent 58% of the total time in F, but the 80-g animal did
not exhibit this behavior at all. This bias on both inter-
and intramodal transition probabilities may reflect a
difference in the nutritional needs of small vs. large ani-
mals and is worthy of further study. We did not find a
correlation between body size and average dwell time in
the F behavior in the absence of food organisms. This
suggests that large animals enter feeding behavior more
often than small ones, but that the duration of feeding
episodes is about the same once feeding begins. Although
body size has little effect on feeding dwell time, this pa-

o 100

o

03

C

^

o

<D

E

80
60
40
20
0

100

200

300

volume (ml)

Figure 4. Body size and total time spent in the feeding mode are
correlated. Data taken from set 2. Total time in the feeding mode from
Figure 3 was calculated by counting the total number of intervals spent
in feeding mode behaviors. Bod) volume was determined by water dis-
placement. The correlation coefficient is ;• = 0.87.

422

A. E. SCHIVELL ET AL.

rameter is influence-- e presence of food; Watson

and Trimarchi ( 1 Q< e shown that dwell time varies

inversely with the . . ntration of available prey.

Two-step r ''• probabilities

A sequence : ,t behavioral acts is stochastic if the tran-
sition from one behavior to the next is independent of
preceding behavioral changes. If this is so, the frequency
of two-step transitions observed experimentally should
match the transition frequencies predicted from a two-
step stochastic model. Expected probabilities for two-
step transitions were calculated from the measured one-
step probabilities following the equation P(a -» b -* c)
= />(a -* b) * P(b -+ c), where a, b, and c represent any
of the six canonical behaviors. Behavioral observations
from the 10 individuals in each data set were pooled and
used to tabulate the probability of occurrence of each of
the 2 16 possible two-step behavioral transitions. Transi-
tions from four of the six possible starting behaviors oc-
curred frequently enough to permit this extensive analy-
sis (F, OH, AL, and RST). The expected and observed
probabilities were placed in 6 X 6 matrices for compari-
son. The matrices for starting behavior a = AL from data
set 2 are tabulated and plotted in Figure 5. The expected
and observed three-dimensional plots of behavioral fre-
quency show peaks and valleys at the same positions and
with approximately the same amplitudes.

We attempted to test the significance of differences be-
tween the expected and observed 6x6 matrices by cal-
culating chi-square values. When the two data sets were
treated separately, chi-square scores ranged from 42.5,
for set 2, starting behavior AL, to 62.9 for set 1, starting
behavior F. When both sets of data were pooled, chi-
square scores ranged from 66.5 to 81.5. All the chi-
square values were significant by standard tests using 25
degrees of freedom (P < 0.001 to P < 0.01). The chi-
square statistic is known to be unreliable, however, in the
case of behavior-sequence matrices with cells containing
low values (Chatfield and Lemon, 1970), and the two-
step transition matrices we tabubted have a number ot
cells with values of 0. Also, vana. in transition rates
was high among individuals (CV tor F — F as high as
1 57%), which adds to the chi-square score. The presence
of this type of added uncertainty is demonstrated by the
fact that higher scores result when data from two periods
of observation are pooled. Instead of a chi-square test,
Chatfield and Lemon (1970) suggest selecting the most
frequent sequences and comparing expected and ob-
served values by inspection. The plots of Figure 5 follow
that principle and show that the two-step transition pre-
dictions match the observations fairly well.

Behavioral dwell-t'une distributions

We defined behavioral dwell time as the duration of
a continuous single bout of the same behavior. This is

equivalent to the 'interval distribution' (Heiligenberg,
1973) or the 'hazard function' (Cox and Lewis, 1966) but
differs in that exits into any other behavioral state are
allowed. Dwell-time histograms for four of the six behav-
iors are plotted in Figure 6. If behavioral choice is deter-
mined stochastically, the distribution of dwell times will
be exponential. R/OH and R/AL behaviors did not oc-
cur frequently enough to permit dwell-time analysis. OH
and AL dwell times were exponentially distributed. F
dwell times were also exponentially distributed, with the
exception of a few lengthy episodes. The presence of
longer episodes has implications for the organization of
the feeding neural network and suggests that the network
may be modulated by stimuli such as the presence of
stray food organisms or by a long-lasting effect of food
presented on the previous day (Watson and Chester,
1993). The RST dwell-time histogram was best fit by the
sum of two exponentials. This suggests two possibilities:
first, there may be two resting states which have indepen-
dent transition probabilities but are indistinguishable
by observation; alternatively, the transition probabilities
for exiting RST may vary with time spent in the RST
behavior.

Discussion

These observations demonstrate that spontaneously
evolving sequences of behavioral transitions in Melibe
leonina are stochastic such that the probability of a given
transition is independent of preceding ones. At the same
time, behavior is organized by the fact that transitions
between behaviors within a mode are more likely than
transitions between behaviors in different modes. This
creates a cul-de-sac within the transition-probability ma-
trix so that one behavior pattern can only be reached by
way of another. As a result, the organization is not in-
consistent with hierarchical models of behavioral orga-
nization like that proposed by Tinbergen (1951) since
specific stimuli might move the animal into a mode in
which certain behaviors are more likely than others even
though the individual transition probabilities are con-
stant and independent. This view is supported by evi-
dence that the neural circuits underlying spontaneous
behavioral choice are influenced by external cues such as
the presence of prey and by physiological signals corre-
lated with body size.

Spontaneous behavior in Melibe demonstrates two
features characteristic of a stochastic system. First, the
two-step transition probabilities predicted from consid-
eration of measured single-step transition frequencies
closely match the two-step frequencies observed experi-
mentally. This shows that behavioral transitions are in-
fluenced by the starting behavior but not by previous be-
haviors. Second, for three of the four behaviors for which

d-Al.

F

OH
AL

R/OH

R/AL

RST

BEHAVIORAL MODES IN MEL1BE LEON1NA

EXPECTED

c
F OH AL R/OH R/AL RST

423

OBSERVED

c

8.8 4.1 2.1 2.6 1.5 03

5.3 10.3 7.3 2.3 1.2 1.2

10.0 14.4 81.2 6.2 15.5 27

3.2 2.3 1.2 2.6 1.5 0.9

4.1 3.5 13.2 4.1 3.5 1.2

0.3 1.2 13.2 0.6 2.6 33.7

d-AL

F

OH

AL

R/OH

R/AL

RST

F

5

4

6

0

4

1

OH

5

7

9

5

0

2

AL

10

12

96

3

12

27

R/OH

1

2

2

4

2

1

R/AL

3

2

14

3

5

1

RST

0

0

11

2

5

27

Figure 5. Top. Expected and observed two-step transitions with data from starling behavior AL (set 2).
Expected numbers were calculated from single-step transition frequencies using the equation P(a -» b -»
c) = P(a)P(a -» b)/J(h -» c) (see text). Bottom. 3-D plots corresponding to the expected and observed two-
step transition matrices.

there was sufficient data to evaluate behavioral dwell-
time distributions, dwell times were distributed expo-
nentially. This supports a model based on independent
and unvarying transition probabilities. The dwell-time
distribution for the exception, the RST behavior, was
best fit by the sum of two exponentials. This result might
be explained by the existence of two resting states with
different average duration. We were unable to identify
multiple resting states but, like sleep and quiescent be-
havior in other animals, the two states may have a
different neurophysiological basis while appearing iden-
tical to the observer. Another possible interpretation is
that the exit probability from RST decreases with time
spent in the RST state, perhaps reflecting a decrease in
arousal. The data are not sufficient to evaluate these pos-
sibilities, but they raise questions of a general nature that
might be accessible to neurophysiological investigation
in Mel i be.

A property that emerges from a consideration of the
transition-probability matrix is that spontaneous behav-
ior can be interpreted as a series of jumps between two

modes named the feeding mode and the resting mode.
This grouping of behaviors follows directly from the one-
step transition probabilities and the observation that
transits within modes are far more probable than transits
between modes. It is a robust conclusion based on over
1900 observations of transitions, far more than the 360
(10 n2) required to analyze a repertoire of /; behaviors
(Fagen and Young, 1978). This organization represents
a way in which higher order structure can be generated
by a coupled system of first-order random processes.

The probability of transitions between modes did not
fluctuate during 6 h observations, but this period is short
relative to the life span of the animal. Preliminary data
suggest that transition frequencies do change with matu-
ration and that juveniles demonstrate both a different
repertoire of behaviors and different frequencies of tran-
sitions between the behaviors they share with adults (S.E.
Gelber, pers. comm.). In addition, Watson and Trimar-
chi (1992) have shown that the presence of prey organ-
isms, sensed in response to chemical and tactile cues
(Watson and Chester. 1993), influences feeding dwell

424

A. E. SCHIVELL ET AL

o> 200 | 1 1 1— —I— — r— — i

— I

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c

1 I I 1

| 150

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| 150

1 RST

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L.

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\

J2 100

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\

o

I

o

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I

E

V

W

rt

\

5 50

v

o 50

A

»*—
o

=jt n

MQlSfemriEinit^ o-u

=*= n
0 60 120 180 240 300 360 0 60 1 20 1 80 240 3C

time (minutes)

time (minutes)

0> ^3VJ

C

I 200
E
* 150

I OH

\

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time (minutes)

180

250

o 200
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100
50
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60 120

time (minutes)

Figure 6. Dwell-time histograms for feeding (F), open hood (OH), resting (RST). and alert-processes
extended (AL) behaviors. The OH and A L distributions are lit by exponentials (solid curve), consistent with
the exit from these behaviors being random. The feeding (F) dwell-time distribution is also approximately
exponential, except for outlying points that account for 1% of the data. The outlying points with long dwell
times may reflect behavior driven by external stimulation. Two fits are plotted for the resting (RST) data, a
single exponential (solid line) and the sum of two exponentials (dashed line). All distributions were fitted
using a nonlinear least-squares method.

180

time in a fashion that is graded with the concentration of
prey.

A particular pair of behavioral transitions, OH<-»AL,
acts as a gateway between the feeding and resting behav-
ior modes. The OH and AL behaviors share features of
body posture such as the positions of the rhinophores
and cerata, but they different in oral hood posture. The
change between behavior modes, therefore, is bridged by
a relatively small postural adjustment, but it signals a
long-term change in the animal's behavior pattern. A
neurophysiological interpretation is that slight but mea-
surable shifts in the activities of neurons involved in OH
and AL behaviors car. result in a stable, lasting shift in
motoi - • v.tem function.

Incn a ' body size is correlated with an increase in

time spent feeding, but the average dwell time of feeding
episodes is independent of size. This suggests that the
probability of entering feeding behavior increases with
body size. Conversely, it is apparent that the presence
of food organisms decreases the probability of exit from
feeding but does not have a clear effect on entry into feed-
ing behavior. If we assume that separate entry and exit
circuits govern transitions into and out of the feeding
state, it should be possible to measure changes in excit-
ability in those circuits that correspond to the behavioral
observations. One simple and testable prediction is that
body size modulates the entry circuit, while the presence
of food modulates the exit circuit. Modulation of the exit
circuit by food could confer competitive advantage by
maximizing foraging efficiency in the face of a variable

BEHAVIORAL MODES IN MEL1BE LEON1NA

425

food supply. Such shifts in behavioral transition proba-
bilities might be mediated by neuromodulatory or hor-
monal effects that cause a dynamic restructuring of neu-
ral networks by influencing either postsynaptic excitabil-
ity or the amplitudes of synaptic potentials (Getting,
1989;Katzrtfl/.. 1994).

Neuronal circuits that mediate behavior in Melibe can
be studied with intracellular recording in a whole-animal
preparation using techniques pioneered by Willows
(1973) and Getting (1981). Using this preparation, we
have made progress in elucidating the networks involved
in swimming locomotion and feeding movements of the
oral hood; Cohen et al. (1991) have succeeded in using
optical imaging techniques to monitor activity simulta-
neously in dozens of neurons in the Melibe buccal gan-
glion. Neurophysiological methods are available, there-
fore, to study changes in neuronal function that are cor-
related with behavioral choice. The behavioral model
developed here provides a framework for the interpreta-
tion of physiological experiments.

Acknowledgments

We thank Russ Fernald for comments and R.C. Rao
for original artwork. We also thank the staff of the Hop-
kins Marine Station. Supported by NSF grant 9021217
(S.H.T) and the Lerner-Gray Fund for Marine Research
(S.S.-H.W.).

Literature Cited

Agersborg, H. P. K. 1923. The morphology of the nudibranchiate
mollusc Melibe (syn. Chioraera leonina (Gould.)). Q J Microsc.
Sa. 67: 508-592.

Chatfield, C., and R. E. Lemon. 1970. Analysing sequences of behav-
ioral events. J. Thtvr. Biol. 29: 427-445.

Cohen, L. B., W. Watson, J. Trimarchi, C. X. Falk, and J.-Y. Wu.
1991 . Optical measurement of activity in the Melibe leonina buc-
cal ganglion. Sue. Nenrosci. Abslr. 17: 1593.

Cox, D. R., and P. A. Lewis. 1996. The Statistical Analysis of Series
oj Events, p. 276. Methuen. London.

Davis, W. J., G. J. Mpitsos, M. V. S. Siegler, J. M. Pinneo, and K. B.

Davis. 1974. Neuronal substrates of behavioral hierarchies and
associative learning in Pleurobranchaea. Am Zool. 14: 1037-1050.

Davis, W. J., G. J. Mpitsos, J. Pinneo, and J. L. Ram. 1977. Modifi-
cation of the behavioral hierarchy of P/eiirobranchacu. J Comp
Phvsiol A 117:99-125.

Fagen, R. M., and D. V. Young. 1978. Temporal patterns of behav-
iors: durations, intervals, latencies, and sequences. Pp. 79-1 14 in:
Quantitative Ethology. P.W. Colgan, ed. John Wiley & Sons, New
York.

Gelperin, A. 1983. Neuroethological studies of associative learning in
feeding control systems. Pp. 189-205 in: Neuroethology and Behav-
ioral Physiology — Roots and Growing Points. F. Huber and H.
Markl. eds. Springer- Verlag. New York.

Getting, P. A. 1981. Mechanisms of pattern generation underlying
swimming in Tritonia. I. Neuronal network formed by monosyn-
aptic connections. / Neiirophysiol. 46: 65-79.

Getting, P. A. 1989. Emerging principles governing the operation of
neural networks. Annu. Rev. Nenrosci. 12: 184-204.

Heiligenberg, \V. 1973. Random processes describing the occurrence
of behavioral patterns in a cichlid fish. Anim. Behav. 21: 169-182.

Hurst, A. 1968. The feeding mechanism and behavior of the opistho-
branch Melibe leonina. Symp. Zool. Soc. Loud. 22: 151-166.

Katz, P. S., P. A. Getting, and \V. N. Frost. 1994. Dynamic neuro-
modulation of synaptic strength intrinsic to a central pattern gener-
ator circuit. Nature 3ftl: 729-731.

Kandel, E. R. 1976. Cellular Basis of Behavior p. 727. W.H. Free-
man and Company. San Francisco.

Lorenz, K. Z. 1981. The Foundations o/ Ethology, p. 380. Springer-
Verlag. New York.

Scott, D. L. 1990. The shrug response in Melibe leonina: behavioral
and neurophysiological observations. Unpublished ms. on file at
Hopkins Marine Station library. Pacific Grove. CA.

Tinbergen, N., 1951. The Study of Instinct, p. 228. Clarendon Press.
Oxford.

Watson, W. H., Ill, and J. Trimarchi. 1992. A quantitative descrip-
tion ofMelihe feeding behavior and its modulation by prey density.
Mar. Behav Phys/ol 19: 183-194.

Watson, W. H., Ill, and C. M. Chester. 1993. The influence of olfac-
tory and tactile stimuli on the feeding behavior of Melibe leonina
(Gould, 1852) (Opisthobranchia: Dendronotacea). I'cliger 36(4):
311-316.

Willows, A. O. D. 1973. Interactions between brain cells controlling
swimming in a mollusc. Pp. 233-247 in: Neurobiology of Inverte-
brates. J. Salanki. ed. Plenum, New York.

Willows, A. O. D., ed. 1985. The Mollusca: Neurobiology and Behav-
ior, Pan I. Academic Press. Inc. Orlando.

Reference: Biol. Bull 192: 426-443. (June. 1997)

Physiological Variation Among Clonal Genotypes

in the Sea Anemone Haliplanella lineata:

Growth and Biochemical Content

MICHAEL G. McMANUS1, ALLEN R. PLACE2, AND WILLIAM E. ZAMER1

^Department of Biology, Lake Forest College, 555 N. Sheridan Rci, Lake Forest, Illinois 60045:

and ^Center of Marine Biotechnology. University of Mary/and Biotechnology Institute,

Suite 236 Columbus Center, 701 E. Pratt St., Baltimore. Man-land 21202

Abstract. We have explored physiological variability
among clonal genotypes from a single population of the
sea anemone Haliplanella lineata located at Indian Field
Creek, Virginia. Information about the correlation be-
tween physiological variability and genetic differences
may provide a foundation for a mechanistic understand-
ing of the breadth of adaptation of individual genotypes
(i.e., the nature of "general purpose genotypes") and of
the concept of localized adaptation in clonal anemones.
Anemones from three clones (A, B, C) were fed mea-
sured rations of adult Anemia, after which growth, ab-
sorption efficiency, and net growth efficiency were deter-
mined. Biochemical constituents were measured in the
tissue of this group of anemones as well as in the tissue of
anemones from the same clones that had fed ad libitum
on Anemia nauplii. Anemones from the different clones
did not differ significantly in growth, or gravimetric ab-
sorption or growth efficiencies, but significant differences
were found in biochemical composition. Regardless of
feeding regime and diet composition, clone B anemones
consistently had lower tissue averages of triacylglycerols,
fatty acids, sterol and wax esters, glycerol ethers, and car-
bohydrates than did clone A and clone C anemoi.es. As
a result of differences in the carbohydrate and lipid con-
stituents, the energetic content of tissues from clone B
anemones that had been fed rations was significantly
lower than the energetic content of tissues of anemones
from clone C. This clonal pattern in biochemical com-
position and energetic content may be due to differences

Recc . ! ?9 August 1996: accepted 12 April 1997.

in substrate absorption among anemones from the
different clones, to differences in metabolic rate, or to a
combination of both. Because anemones from this pop-
ulation may encyst in mucus and stop feeding when wa-
ter temperatures are less than 10°C, the genotypic differ-
ences in storage lipids and carbohydrate may have im-
plications for the winter survivorship of clone B
anemones in this population.

Introduction

Many species of sea anemones reproduce asexually,
resulting in the production of genetically identical indi-
viduals in natural populations. Local populations may
consist of a single clone (e.g., the actinian Haliplanella
lineata: Shick, 1976; Shick and Lamb. 1977), or they
may be composed of many clones and approach the ge-
netic diversity expected for a sexually outcrossing pop-
ulation (see Shick, 1 99 1 . pp. 270-277). Such differences
in population genetic structure may have multiple
causes. For example, monoclonal populations may re-
sult from genetic founder effects (i.e.. settlement of a
single adult or planula larva, followed by asexual prolif-
eration), followed by competitive exclusion of other
clonal genotypes (Ayre, 1982, 1983, 1995; Hoffmann,
1986). Alternatively, monoclonal populations could re-
sult from the settlement of multiple genotypes, followed
by differential selection leading to the elimination of all
but one highly locally adapted genotype (see Ayre,
1985, 1995). Multiclonal populations could result from
the asexual proliferation of multiple genotypes that
have the same relative fitness, although formal tests of

426

CLONAL VARIATION IN SEA ANEMONES

427

fitness differences among clonal genotypes are rare
(Ayre, 1995).

On theoretical grounds, asexual reproduction may be
viewed as a means of preserving and increasing the size
of locally adapted multiple-locus (i.e.. clonal) genotypes
(Williams, 1975), and a number of researchers have pro-
vided evidence consistent with this theoretical prediction
of local adaptation (see especially, Ayre, 1985, 1995).
However, the functional basis for such local adaptation
has not been explored. Indeed, Shick (1991) points out
that studies yielding evidence of local adaptation have
not tested whether locally adapted clones exist in any
other localities, i.e.. the extent to which any single clone
is a broadly adapted, "general purpose genotype" (Shick.
1976; Shick and Lamb, 1977).

Although the ecological physiology of sea anemones
is rather well-studied (cf. Shick, 1991), there have been
surprisingly few investigations of physiological variation
among clonal genotypes present in individual popula-
tions or individual habitats within populations of asexu-
ally reproducing anemones (e.g., Shick and Dowse,
1985). Such information could provide the initial basis
fora mechanistic understanding of localized adaptation,
and for determining whether natural selection or genetic
founder effect is more important to the genetic structure
of these populations. Of particular note in this context is
a study by Shick and Dowse (1985), who concluded, on
the basis of an examination of literature data on sea
anemones, that intraclonal variance in a variety of phys-
iological measurements is smaller than interclonal vari-
ance in these traits, and that the variance for some traits
could be explained largely by clonal identity. Thus they
provided strong evidence of genetically correlated per-
formance variability. However, much of their work fo-
cused on comparisons of variances between geographi-
cally separated monoclonal and multiclonal populations
of the same species, or between genetically diverse popu-
lations of one species and monoclonal populations of an-
other species. Although many studies have persuasively
shown that localized adaptation of clones occurs (e.g.,
Ayre 1985, 1995;Sebens. 1981; Zamer and Shick, 1989),
such comparisons reflect the fact that few studies have
directly examined physiological variation among clonal
genotypes collected from a single population or habitat,
and maintained under the same environmental condi-
tions so as to remove acclimation effects on perfor-
mance.

Some field-based studies have provided limited evi-
dence for physiological variation among clonal geno-
types within and between populations. For example,
Jennison (1979) interpreted differences in tissue lipid
among clones of the anemone Anthopleura elegant is-
sima to be the result of either genetically encoded
differences in lipid metabolism or micro-environmen-

tal differences in food availability between clones at a
single field site. Differences in reproductive characteris-
tics between high- and low-intertidal clones of A. ele-
gantissima were attributed to higher temperatures in
the upper intertidal (Sebens, 1981), the likeliest expla-
nation, but the possibility remains that these clones
were genetically adapted to the different habitats in this
single population.

More persuasive evidence of localized genetic adapta-
tion among anemone clones comes from controlled ex-
perimental studies. When acclimated to common condi-
tions, high-intertidal clones of A. elegantissima exhibit
different physiological energetic characteristics (e.g., ab-
sorption efficiency, net growth efficiency) than low-inter-
tidal clones of this anemone; these results were inter-
preted to mean that adaptive, genetic divergence of the
clones had occurred in response to low food availability
in the upper intertidal zone (Zamer, 1986; Zamer and
Shick, 1987, 1989). Ayre (1985) showed that local clones
of the anemone Actinia tenebrosa had greater capacities
for asexual reproduction than did transplanted foreign
clones; he concluded that highly localized adaptation of
the clones had occurred in colonies of the anemone that
were only 2-4 km apart. And Shick el al. (1979) trans-
planted clones of the anemone H. lineata from a Rhode
Island population to a Maine population site and ob-
served 100% mortality of the transplants. These investi-
gators also interpreted their results to mean that the sep-
arate populations were genetically distinct and contained
locally adapted clones. However, little information
about functional diversity among clones within any sin-
gle habitat or population is available from the aforemen-
tioned studies. More recently, variation in performance
traits among clones has been explored in polyps of the
jellyfish Aiirelici anrita (Keen and Gong, 1989), in the
corallimorpharian Corynactis califomica (Chadwick and
Adams, 1991), and in the anemone A. elegantissima
(Tsuchida and Potts, 1994).

Genetic variation in growth among individual organ-
isms has been examined generally by two approaches.
The first, quantitative genetics, statistically partitions
phenotypic variance among relatives (e.g., parents, off-
spring, half-siblings) into genetic and environmental
components (Falconer, 1989). This approach has suc-
cessfully documented significant genetic variation and
genotype-environment interactions in growth in a num-
ber of shellfish species (Jones el al., 1996; Rawson and
Hilbish, 1991). Such studies cannot, however, address
the physiological mechanisms underlying genetic varia-
tion in growth (cf. Clark, 1990; Koehn, 199 1 ), and there-
fore cannot provide relevant information about the
mechanisms of localized adaptation among clones of sea
anemones.

The second approach is physiological energetics.

428

M. G. McMANUS ET AL

which focuses on performance traits that constitute the
energy and materials budgets of an organism (e.g., Hil-
bish and Koehn, 1985; Hawkins et ai. 1986; Zamer and
Shick, 1987; 1989; Koehn and Bayne, 1989; Present and
Conover. 1992). We have taken this approach to exam-
ine physiological variation among clonal genotypes in
the eurytolerant sea anemone //. lineala collected from
a single population. The present paper focuses on growth
and on the biochemical content of the tissue. The bal-
anced energy equation ( Winberg, 1 956) has been used as
a conceptual framework in examining variation in or-
ganismal performance. Bayne and Newell (1983) ex-
pressed the equation as

Pg+Pr= C-AE-(R,,, + R,)

where Pg is somatic production (growth), /Vis reproduc-
tive production (gametes), C is energy consumed, AE is
the efficiency with which consumed energy is absorbed,
R,,, is the metabolic cost of body maintenance, and R,
includes all other metabolic costs (Koehn and Bayne,
1989). Variation in growth among individuals can origi-
nate from differences in the components of the balanced
energy equation, such as metabolic costs, consumption,
and absorption efficiency (Koehn and Bayne, 1989; Pres-
ent and Conover, 1992).

Ha/ip/anel/a lineata is a widely distributed, colonizing
species, which in natural populations rapidly increases
in numbers by asexual reproduction and may disappear
from an area suddenly (Shick, 1976). Although this spe-
cies is extremely euryhaline and eurythermal (Shick.
1976), sudden disappearances have been attributed to
the existence of one or only a few clonal genotypes in
populations and to environmental factors that exceed ge-
netically based tolerance limits (Shick, 1976; Shick etai,
1979). Although sexually reproducing populations occur
in Japan (Fukui, 1991), North American and laboratory
populations of H. lineala reproduce only asexually,
mainly by longitudinal fission (Johnson and Shick, 1977;
Minasian, 1979), and the species can be classified as an
agametic, cloning anemone (Hughes, 1989; Carvalho,
1994).

A physiological advantage of studying anemones that
reproduce exclusively by asexual fission is that sexual re-
production (Pr) is eliminated from consideration of en-
ergy balance, simplifying the analysis. The animal is eas-
ily cultured in the laboratory, and the rate of longitudinal
fission may be increased by maintaining the anemones at
relatively high temperatures (Minasian, 1<O>; Minasian
and Mariscal, 1979; Zamer and Mangum, 1979), so that
large numbers of genetically identical clonemates are ob-
tained easily. It is this latter feature, and the eurytolerant
nature of the animal, that affords an ecophysiological
and gene ; advantage, in that clonemates may be stud-
ied at a variety of relevant environmental conditions.

Using this approach we can replicate our physiological
measurements on genetically identical individuals.
Eventually, to better understand localized adaptation in
this species, we can study the physiological characteris-
tics of clonal genotypes from separate populations. We
will also be able to partition physiological variation in
H lineata into genetic (comparisons among anemones
from different clones) and nongenetic (comparisons
within each clone) components (Shick and Dowse, 1985;
Vrijenhoek, 1994).

In this study genetic variation in growth was examined
in anemones from different clones, all of which were fed
similar, measured food rations, thereby eliminating con-
sumption differences in the energy balance equation as a
source of variation in growth. In terms of physiological
energetics we asked: Do growth, absorption efficiency,
and growth efficiency differ among anemones from
different clones? Because genetic variation in physiolog-
ical energetics has been associated with differences in
lipid and protein metabolism in some organisms (Me-
drano and Gall, 1976a, b; Hawkins et ai, 1986), we ex-
amined the biochemical composition of the ration-fed
anemones from the different clones. Finally, we mea-
sured the biochemical composition of tissues from
anemones that were fed Anemia nauplii to test whether
consumption differences associated with the capture of
suspended prey could affect any of the biochemical pat-
terns.

Materials and Methods

Collection and maintenance

In October 1990, individuals of H. lineata were col-
lected from Indian Field Creek, a tributary of the York
River in Virginia (37°16'N, 76°33' W), and shipped by
air to our laboratory at Lake Forest College. Anemones
were collected from tens of square meters at the site (C.
P. Mangum, pers. comm.), so that this original sample
was likely to have included a representative sampling of
clones in this population. At the time of collection, water
temperature was about 20°C (C.P. Mangum. pers.
comm.). Salinity ranges from 15-18 parts per thousand
(ppt) at this site (W.E.Z., pers. obs., and C.P. Mangum,
pers. comm.), and surface water temperature ranges an-
nually from about 5°C to 27°C (Coast and Geodetic Sur-
vey, 1 960. as cited in Sassaman and Mangum, 1 970). Air
temperatures to which the anemones are exposed at low
tide may reach 30°-32°C during the summer, and may
be near freezing during the winter (C. P. Mangum, pers.
comm.).

Each anemone was placed in its own beaker (30 ml)
containing 16 ppt seawater (Instant Ocean), held at
room temperature ( 15°-25°C), and fed Anemia nauplii
(San Francisco Bay brand) ad libitum every other day.

CLONAL VARIATION IN SEA ANEMONES

429

Under these conditions, most anemones underwent re-
peated longitudinal fission. Fission products within indi-
vidual beakers constitute a separate genetic lineage, and
were eventually transferred to individual "stock"
aquaria (9.5 1), where the separate lineages are currently
maintained under continuous immersion in recirculat-
ing filtered seawater. Other maintenance conditions are
the same as stated above.

Anemones from each of the aquaria were genotyped
by using starch gel electrophoresis at five polymorphic
loci [superoxide dismutase (SOD), isocitrate dehydroge-
nase 1 (IDH 1), glucose-6-phosphate isomerase (GPI),
octopine dehydrogenase (ODH), and 6-phosphogluco-
nate dehydrogenase (6Pgdh)] with standard gel electro-
phoresis techniques that will be described elsewhere
(Zamer. unpubl. data). Based on the five polymorphic
loci, five unique, multiple-locus genotypes, designated as
A. B, C. D. E, and referred to hereafter as clones, were
detected among the different lineages of anemones. For
most of the work described below, three clones (A-C)
were used.

Anemones from these different clones were accli-
mated in monoclonal aquaria (9.5 1) in an incubator at
1 5°C for at least 5 weeks prior to the experiments. At this
temperature these anemones do not readily undergo lon-
gitudinal fission, but they do grow. All acclimation and
experimental conditions described in this paper included
continuous immersion of the anemones.

Growth experiment

In 1994 we initiated a controlled growth experiment
using anemones from our stock cultures. Sixty-five
anemones from clones A, B, and C were selected from
the stock cultures so as to minimize any size differences
among the clones (Table I). Sample sizes were » = 13,
26, and 26. in clones A, B, and C, respectively (unequal
sample size was due to the slow rate of fission by clone A
anemones in the stock aquaria). Before weighing each
anemone, adhering debris was removed, and the gastro-
vascular water was removed to absorbent paper by ap-

plying gentle pressure to the body wall with a small spat-
ula. Anemones were allowed to attach to individual 15-
ml plastic beakers, which were then floated on the sur-
face of monoclonal aquaria at 15°C. Anemones were fed
Anemia nauplii in the aquaria by submerging the bea-
kers.

From the 65 anemones, an initial group of 25 was cho-
sen for calculation of dry-to-wet-mass regressions. Each
anemone in this initial group (« = 5, 10, and 10 respec-
tively for clones A, B, and C) was removed from its bea-
ker, sliced longitudinally to release all gastrovascular wa-
ter, rinsed briefly with deionized water to remove salts,
and blotted. The anemones were dried at 50°C for 24 h,
cooled to room temperature in a desiccator, and weighed
to the nearest 0.01 mg. For each of the clones, the dry
mass of the anemones in the initial group was regressed
on their wet mass (previous paragraph). Clonal regres-
sion equations were used to estimate the starting dry
mass from the wet mass of the remaining individual
anemones in each clone. These dry mass estimates were
used in the growth experiment, and the size range of ini-
tial anemones was selected to ensure that the estimates
were interpolations and not extrapolations of the regres-
sions (Weisburg, 1985).

The 40 remaining anemones used in the growth ex-
periment (hereafter called the experimental anemones)
were attached to individual glass beakers that were ran-
domly assigned to one of two 38-1 aquaria maintained
at 15°C and filled with 24 1 of 16-ppt salt water. Anem-
ones were randomly assigned to a position on the floor
of each aquarium. The box filter in each aquarium was
moved once a day to a different corner of the aquarium
to prevent any bias in airflow and filtration. These
aquaria served as experimental blocks, which mini-
mized any effects of temperature heterogeneity within
the incubator.

Each experimental anemone received a daily ration of
frozen adult Artemia (San Francisco Bay brand) for the
next 10 days. For the first 2 days, the size of the ration
was 6% of the estimated starting dry mass of each anem-

Table I

Anemone si:e (in milligrams)

Clone

Initial Wet Mass

Initial Dry Mass
(Regression-Estimated)

Final Drv Mass

A

13

66.1 (50.

5,

86.7)

8

10.96(7

.67,

15.67)

8

14.30(10.46

. 19.53)

B

26

62.7(48,

5,

81.2)

16

8.96(6

54.

12.28)

16

11.98(9.00.

15.94)

C

26

63.5(52,

1,

77.4)

16

10.46(8

,45.

12.99)

16

14.45(1 1.51

. 18.16)

The values for the size variables are the hack-transformed means with their 95"r confidence limits in parentheses. Based on analysis of variance
for each \ariable. clonal identity did not have a significant effect on anemone size (initial wet mass P = 0.96. initial dry mass P = 0.56, final dry
mass P= 0.46).

430

M. G. McMANUS ET AL.

one. To make the we; ! it • >f the Anemia easier, the ra-
tion size was ir.r j 8.5% for the remaining 8 days.
Each ration w;< vJ on the oral disk of each anemone
to ensure that i was ingested. On the following day,
egesta produced from each anemone was removed a few
hours prior to that day's feeding. The daily egesta for
each anemone was rinsed with deionized water to re-
move salts and stored on a weigh boat in a desiccator.
Egesta obtained from individual anemones was pooled
over the 10-day experiment, dried at 50°C, and then
weighed. The pooled egesta mass was used in the calcu-
lation of gravimetric absorption efficiency for each
anemone (see below). On the day following collection of
the last egesta, "blotted" wet mass (described below) and
dry mass (described earlier) were determined for each of
the 40 experimental anemones.

Physiological energetics

The relative growth (RGR) of the experimental anem-
ones was calculated as: RGR = [(final dry mass — esti-
mated starting dry mass) -(estimated starting dry
mass)"1] X 100%. Gravimetric absorption efficiency [Ag,
(g ingested - g egested)-(g ingested)"' X 100%] and net
growth efficiency [A":, g growth -(g absorbed ration)"1
X 100%] were calculated as in Zamer (1986).

Biochemical analyses and energetic content

Tissue hydration, and protein, carbohydrate, and lipid
content of tissues were measured. Tissue hydration was
calculated for the initial and experimental groups of
anemones as: ( blotted wet mass - dry mass) • ( blotted wet
mass)'1 X 100%. Blotted wet mass (mean ± SE = 83.1
±4.55 mg) was obtained on anemones after cutting their
body walls to express gastrovascular cavity water, rinsing
to remove salts, and blotting as described previously for
the initial group of anemones.

After the dry mass of an anemone was measured, the
material was placed in a shell vial, ground with a glass
rod, and stored at -70°C. Total protein and carbohy-
drate was measured in samples (3. 1-8.4 mg) of oven-
dried tissue sonicated in 500 /ul of deionized water. An
800-jil volume of cold 10% trichloroacetic acid (TCA)
was added to a 200-^1 aliquot of the homogenate, the
sample was kept on ice for lOmin and swirled every
5 min, then centrifuged at 4500 X g at 2°C for 30 min.
The supernatant was discarded, and the tube containing
the precipitated protein was inverted and drained for 1 h
at room temperature. The protein was then dissolved in
1.0 ml of 10% sodium hydroxide (NaOH). Two 200-^1
aliquots of this protein solution were removed and each
ixed with 400 n\ of 10% NaOH and 1.4 ml of de-
ioni/:ecl water. Microbiuret (Itzhaki and Gill, 1964) de-
terminations of protein were made on this solution. Bo-

vine serum albumin is an inappropriate standard for es-
timating protein content in sea anemones (Zamer et at.,
1989). Protein was isolated from anemones (kept in the
stock aquaria) representing all five clones, using the pro-
cedures of Zamer et a/. (1989). This Haliplanella protein
was dissolved in 10% NaOH and used as the standard.

Total carbohydrate was isolated from the remaining
300 n\ of homogenate as described in Zamer etal. (1989)
and quantified spectrophotometrically using the method
of Dubois et al. (1956). The relatively mild wet biochem-
ical methods that we used to isolate carbohydrate do not
cleave the carbohydrate residues from glycoproteins as-
sociated with collagen in anemones (Zamer el al.. 1989).
Consequently, this source of structural carbohydrate was
not included in our measurements of carbohydrate
content of the tissues.

Total lipid was extracted from 1.4-6.0 mg of oven-
dried tissue by using a modified Bligh and Dyer ( 1959)
technique with methylene chloride and methanol as the
solvents (Carlson, 1985). Lipid class composition was
measured by thin-layer chromatography/flame ioniza-
tion detection (TLC/FID) on an latroscan TH- 1 0 TLC/
FID Analyzer (latron Laboratories, Tokyo, Japan).
About 20 Mg of lipid from each extracted lipid sample
was spotted onto activated S-III chromarods in dupli-
cate in 1 -2 n\ of methylene chloride:methanol ( 1 : 1 ) for
TLC/FID lipid class analysis. As controls for both lipid
class retention time and FID efficiency, we included
with each sample analysis two chromarods, which were
spotted with a standard mixture of phosphatidyl cho-
line, cholesterol, triolein, l-O-hexadecyl-2-3-dipalmi-
toyl-rac-glycerol (glycerol ethers) and cholesteryl oleate
in proportions similar to those found in anemone sam-
ples, as determined by a preliminary analysis of the
samples. Rods spotted with lipids were prefocused twice
in chloroform:methanol (1:1), then developed in hex-
ane:diethyl etherformic acid (85:15:0.1) for 45 min.
Racks containing the chromarods were then dried at
100°C for 5 min before being scanned. Developed
chromarods were scanned at 30cm-min~'. Gas flow
rates for hydrogen and air were 190ml-min ' and
20 1-min"1, respectively. Peak areas for each lipid com-
ponent were quantified by using a Hewlett Packard
3390A integrator.

Standard curves from 0 to 20 /ug were created for phos-
pholipids, sterols, fatty acids, triacylglycerols. glycerol
ethers, sterol esters, and wax esters. The specific lipid
used to represent each lipid class and the r value for each
standard curve are as follows: phospholipids (phosphati-
dyl choline), r = 0.992; sterols (cholesterol), r2 = 0.996;
fatty alcohols (hexadecanol), r2 = 0.998; fatty acids (oleic
acid), r2 = 0.985; triacylglycerols (triolein), r: = 0.984;
glycerol ethers. ( l-O-hexadecyl-2-3-dipalmitoyl-rac-gly-
cerol), ;-: = 0.992; sterol esters (cholesteryl oleate), r2

CLONAL VARIATION IN SEA ANEMONES

431

= 0.987 and wax esters (cetyl oleate), r = 0.914. Sterol
and wax esters are not resolved in the solvent system
used. Periodic checks of area responses for standards in-
dicated that standard errors of replicates were less than
3% of mean values in all cases. Total lipid was estimated
by summing peak areas for each of the lipid classes.

Anemones in the experimental group were of suffi-
cient size so that enough tissue was available for protein,
carbohydrate, and lipid measurements, including the six
lipid classes. The masses (in milligrams) of protein, lipid,
and carbohydrate in each experimental anemone were
multiplied by their corresponding specific enthalpy of
combustion (A,/;, kJ-g~'; Gnaiger, 1983), and the prod-
ucts were summed to estimate tissue energetic content
(in kilojoules) of each anemone.

Anemones in the initial group, which were fed nauplii
and used for estimating starting dry mass, had only
enough tissue for determination of lipid class content.
Therefore, in two other experimental groups (X and Y),
we measured biochemical composition of tissues of
anemones. These two groups of anemones were main-
tained simultaneously (in this case for over 5 weeks) in
a single set of five monoclonal aquaria under the same
conditions of feeding, temperature, and salinity as the
initial group of anemones. Anemones in groups X and Y
consisted of individuals from all five clones (A, B, C, D,
and E), and were attached to the surfaces of the aquaria;
unlike the anemones in the initial and experimental
groups, they were not confined to beakers. The initial
group of anemones and anemones in groups X and Y
allowed us to test for the effects of clonal identity on bio-
chemical composition when H. lineata consumed sus-
pended nauplii as opposed to rations of frozen adult Ar-
temia. Protein, carbohydrate, and tissue hydration were
measured on the group X anemones; ash content and
tissue hydration were measured on group Y anemones.
Ash content was determined after the dried anemone tis-
sue was combusted at 500°C for 6 h in a muffle furnace.

Statistical analysis

Relative growth, gravimetric absorption efficiency,
and gravimetric net growth efficiency of the experimen-
tal anemones were analyzed with a randomized block
analysis of variance (ANOVA) (Steel and Torrie, 1980).
The categorical factors were block, represented by the
two aquaria, and clone. Relative growth was arcsine
transformed, whereas gravimetric absorption efficiency
required a logit transformation (Cox and Snell, 1992) to
meet the assumptions of the analysis. Gravimetric net
growth efficiency did not require any transformation.
Based on regression analysis, these three physiological
energetic traits did not vary with body size.

Tissue hydration (percent) was measured on anemo-

nes from all four data sets: initial, experimental, and
groups X and Y. The analysis of tissue hydration for the
experimental anemones followed the randomized block
design. Tissue hydration was examined by using an anal-
ysis of covariance ( ANCOVA) for anemones in the other
three data sets because significant regression slopes
showed size-dependence, which was not the case for the
experimental group. Tissue hydration in all cases was
arcsine transformed, and the covariate, dry mass of the
anemones, was transformed using natural logarithms.

All the biochemical variables (protein, carbohydrate,
total lipid, and the six lipid classes) were initially ex-
pressed as proportions of the dry mass of tissue samples.
These biochemical variables were "scaled up" (and ex-
pressed in milligrams) by multiplying the proportions by
the dry mass of each anemone. The estimate of ash was
made from an entire anemone, not from a tissue sample,
so "scaling up" of that variable (milligrams of ash) was
not needed. All the biochemical response variables, ash
and anemone dry mass were transformed using natural
logs.

Next, to acknowledge Lie metabolic relationships
and potential covariance among similar dependent
variables (e.g., protein, carbohydrate, and lipid in tis-
sues of experimental anemones), all of which had dry
mass as a covariate, we grouped such variables and ini-
tially analyzed them with a multivariate analysis of Co-
variance (MANCOVA; Huitema, 1980). This set of
analyses also guards against over-interpretation of only
a series of univariate analyses for these same variables.
We used MANCOVA to analyze the effect of clone on
protein, carbohydrate, and total lipid in experimental
anemone tissue; the effect of clone on the six lipid
classes in tissues of initial and experimental anemones;
and the effect of clone on protein and carbohydrate
content in tissues of group X anemones. When a MAN-
COVA resulted in a significant effect of clone on the
group of dependent variables being analyzed, we pro-
ceeded with a series of ANCOVA, in which the effect of
clone was examined for individual dependent variables
having dry mass as covariate. Block and clone were the
categorical variables for the experimental group of
anemones for all these analyses. Clone was the only cat-
egorical variable for the other data sets (initial group,
and groups X and Y) in all of these analyses.

The energetic contents of the experimental anemones
were size-dependent, so they were analyzed with an AN-
COVA. Block and clone were the categorical variables,
and the natural log of dry mass was the covariate. No
transformation was necessary for energetic content.

When ANOVA or ANCOVA resulted in a significant
effect of clone on a dependent variable, we employed un-
planned multiple comparison procedures (Day and
Quinn, 1989). For the ANOVAs, all pairwise compari-

432

M. G. McMANUS ET AL.

sons of means were made with the Tukey-Kramer pro-
cedure, which adjusts for unequal sample sizes (Day and
Quinn, 1989). In the case of significant clonal effects
from ANCOVAs, the Bryant-Paulson-Tukey (BPT) pro-
cedure was used to compare the size-adjusted means
(Huitema. 1980). The BPT procedure takes into account
that these size-adjusted means are not statistically inde-
pendent due to the use of a pooled regression slope (Day
and Quinn, 1989).

The relationship between the amount of water in the
anemones (measured as the difference between blotted
wet mass and dry mass; in milligrams) and the amount of
total lipid (in milligrams) in the experimental and initial
anemones was examined by partial correlation analysis,
which measures the correlation between this pair of vari-
ables, keeping anemone body size constant (Sokal and
Rohlf, 1981).

In the tables, the clonal means are presented with their
95% confidence limits (CL), and in the graphs, the clonal
means are presented with their 95% confidence intervals
(CI). For the size-independent variables (physiological
energetic traits and tissue hydration of experimental
anemones), their transformed means and standard er-
rors were used to calculate the 95%. CL, the means and
95% CL were back-transformed, and these values are re-
ported here (Sokal and Rohlf, 1981, p. 419). The un-
transformed means for gravimetric absorption efficiency
are presented. For the size-dependent variables (tissue
hydration for the initial, X, and Y groups of anemones,
and all the biochemical data) transformed, size-adjusted
means, and their size-adjusted standard errors (Sokal
and Rohlf, 1981, p. 525) were used to calculate 95% CL,
and both means and confidence limits were then back-
transformed. We present these back-transformed values
of means and confidence intervals. The significant
differences from the unplanned multiple comparison
procedures, Tukey-Kramer or BPT, are indicated on the
graphs, where different lowercase letters indicate signifi-
cantly different clonal means.

Results

Physiological energetics

Our comparison of the physiological energetics of the
anemones from the different clones revealed substantial
intraclonal variation. Average relative growth for the ra-
tion-fed anemones was 35% (Fig. 1), and the effect of
clone was not significant (F; ih = 1 .3, P = 0.29). The ini-
tial dry mass and final dry mass averages did not differ
significantly among the clones (F2 37 = 0.60, P = 0.56,
F2 ,:„, = 0.80, P = 0.46, respectively; Table I). Nor was
there a significant effect of clone on gravimetric absorp-
tion efficiency (F2.26 = 0.36, P = 0.70), for which clonal
averages ranged between 92.7% and 93.5%. The clonal

45 r

40

| 35

o
O
o>
•| 30

2
01

25

20

Clone

Figure I. Relative growth, expressed as a percentage of the esti-
mated initial dry mass, of the experimental anemones fed frozen adult
Anemia rations. The filled squares are the clonal. back-transformed
means, and the vertical bars are 95% confidence intervals (CI). The
clonal sample sizes were nA = 8, >IB = 16. and «c = 16. Growth did not
vary among the clones (P = 0.29).

pattern in net growth efficiency (A"2) was similar to that
for relative growth. Clonal averages for A": were 40.4%,
43.8%, and 51.3% for clones A, B, and C, respectively.
There was no significant effect of clone on A"2 (F2 2(, = 1 .9,
F-=0.16).

Tissue hydration and lipid content

At the biochemical level of organization, interclonal
variation was frequently greater than intraclonal varia-
tion. Clonal genotype significantly affected biochemical
content in the experimental anemones, as revealed by a
MANCOVA in which carbohydrate, protein, and lipid
values were analyzed collectively (Wilks' A = 0.33, F6..,2
= 7.6, P< 0.001).

Tissue hydration differed significantly among clones
of the experimental anemones (F2 v, = 15.2. P < 0.001),
with those from clone B having the highest average (Fig.
2 A). The effect of clone accounted for 43% of the varia-
tion in tissue hydration. For the initial group, anemones
from the three clones also differed significantly in tissue
hydration (F2.2| =; 14.8, P < 0.001); anemones from
clone B had the highest average at 84.5% (Fig. 2B). Tissue
hydration was not significantly different among the
anemones from the five clones of group X (Fj u = 2.4, P
= 0.07; Table II), but it was in group Y anemones (F424

A. Experimental

85 r

s

-

•o

84

o

a 83

o

82

80

CLONAL VARIATION IN SEA ANEMONES

Table II
Tissue hydration for group X and Y ancmoni'\

433

a

B
Clone

B. Initial

88 r

86

O

2 84

82

80

78

a

a

ABC
Clone

Figure 2. Tissue hydration. expressed as a percentage of the blotted
wet mass, of the experimental ( A ) and initial ( B) anemones. Clonal aver-
ages that do not share the same lowercase letter are significantly different
from one another (P < 0.05). For the experimental anemones (A), the
Tukey-Kramer procedure was used, and the symbols and sample sizes
are as described in Figure 1. For the initial anemones (B), the Bryant-
Paulson-Tukey (BPT) procedure was used, and the filled squares are the
size-adjusted, back-transformed means, and the vertical bars are the 95%
CI. For the initial anemones, tissue hydration was calculated for an
anemone with an average dry mass of 8.40 mg. The clonal sample sizes
for the initial anemones are «A = 5. nB = 10. and /)c = 10.

Clone

Group X

Tissue Hydration (%)

Group Y
Tissue Hydration (%)

A

82.3(81.6,83.0)

81.8(81.0.82.6)

B

82.3(81.6,83.0)

82.2(81.4.83.0)

C

81.0(80.3,81.8)

81.6(80.8,82.5)

D

81. 6 (80.8, 82.4)

81.5(80.4.82.6)

E

82.1 (81.3,82.8)

83.1 (82.2,84.1)

For each of the five clones, the sample sizes are/; = 8 in group X and
n = 6 in group Y. All values are the size-adjusted, back-transformed
means and their 95pr confidence limits. Tissue hydration was calcu-
lated (bran anemone with an average dry mass of 8. 73 mg for group X
and I 7.80 mg for group Y.

= 2.8, P = 0.048; Table II). However, in group Y none
of the pairwise comparisons among clonal means were
significant.

The amount of water and lipid (both in milligrams) in
tissues of the experimental anemones showed an inverse
relationship that was significant according to the partial
correlation analysis (/• = -0.34; ro.o5.i5dt = -0.32). The
effect of clone was significant on the amount of total lipid
(F2.,j = 3.3. P = 0.05: Fig. 3A). which composed 15.5%
of the dry mass, on average, of the experimental anemo-
nes. On the basis of the Tukey-Kramer procedure, none
of the pairwise comparisons were significant. For the ini-
tial group of anemones, amounts of tissue water and total
lipid were not significantly correlated (partial correlation
coefficient = -0.30, r,,.,,,. 22dl- = -0.40). Also, initial
anemones from the three clones were not significantly
different in the amount of total lipid (F2,2, = 2.8, P =
0.08: Fig. 3B). Total lipid constituted 20.4% of the dry
mass, on average, of the initial anemones.

The analysis of the individual lipid classes proved
more informative than the examination of total lipid.
Overall, lipid class content differed significantly among
clones of both the experimental anemones (Wilks' A =
0.245. Fl2,h,, = 5.10, P < 0.001) and the initial group of
anemones (Wilks' X = 0.107. FI2.,2 = 5.48, P < 0.001).
In general, anemones from clone B had less lipid than
anemones from clones A and C. The three clones of ex-
perimental anemones differed significantly in the
amounts of triacylglycerols (F2 .35 = 4.3, P = 0.02; Fig.
4A), sterol esters and wax esters (F2J5 = 7.3, P = 0.002;
Fig. 5A), glycerol ethers (F2.35 = 15.1, P < 0.001; Fig.
6A), and free fatty acids (F2J5 = 5.5, P = 0.009; Fig. 7A).
In the initial anemones, clonal identity significantly
affected levels of triacylglycerols (F2.2| = 3.9, P = 0.04;
Fig. 4B), glycerol ethers (F2.2I = 8.3, P = 0.002; Fig. 6B).
and free fatty acids (F2.2l = 4.3. P = 0.03; Fig. 7B).

434

A. Experiment?

2.6

2.4

J 2.2

•o
a

5 2.0

o

1.8

1.6

M. G. McMANUS ET AL.

A. Experimental

0.8

0.7

B
Clone

B. Initial

2.5

-5 2-°

5.

1.5

1.0

B

Clone

Figure 3. (A) Total lipid (mg)in the experimental anemones. Filled
squares are size-adjusted, back-transformed means, and the vertical
bars are the 95% Cl. Sample size is as described in Figure I. Total lipid
was calculated for an anemone with an average dry mass of 13.38 mg.
Clonal identity significantly affected total hpid (/' = 0.05). (B) Total
111 ig) in the initial anemones. Symbols and sample size are as de-
scnl i Figure 2B. Total lipid was calculated for an anemone with an
averag. .!i\ mass of 8.40 mg. Clone did not significantly affect total
lipid i; 8).

0>

i

o

«

0.5

0.4

0.3

ab

a

B
Clone

B. Initial

0.9 r

0.8

g 0.6

«
o

_>.

™ 0.5

o

S.

H 0.4

0.3

0.2

ABC

Clone

Figure -4. Triacylglycerol (mg) in the experimental (A) and initial
(B) anemones. Clonal averages that do not share the same lowercase
letter are significantly different from one another (P < 0.05) based on
the BPT procedure. (A) Symbols for the experimental anemones are
as described in Figure 3A, and sample size as described in Figure 1.
Triacylglycerol was calculated for an anemone with an average dry
mass of 1 3.38 mg. ( B) Symbols and sample size for the initial anemones
are as described in Figure 2B. Triacylglycerol was calculated for an
anemone with an average dry mass of 8.40 mg. Clone significantly
affected tnacylglycerol content in experimental (P = 0.002) and initial
anemones!/3 = 0.004).

CLONAL VARIATION IN SEA ANEMONES

435

A. Experimental

0.25

0.22

"5

9 0.19

s

o

W

0.16

0.13

0.10

a

J_

B
Clone

B. Initial

0.25

0.21

"5

I 0.17

UJ

X

to

2
5)

0.13

0.09

o.c

B

Clone

Figure 5. Sterol esters and wax esters (mg) in the experimental (A)
and initial (B) anemones. Symbols and sample sizes of experimental
and initial anemones are as described in Figure 4. Clone significantly
affected sterol and wax ester content in experimental anemones (P =
0.002). but not in initial anemones (P = 0.25).

clones B and C in the initial group yielded a generalized
Studentized range statistic (Qnom.\aa\) = 3.47) from the
BPT procedure (Huitema, 1 980) that was not significant
(Quoit) = 3.67).

Among the experimental anemones, those in clone B

A. Experimental

0.15 r

0.13

« 0.11

UJ

1 0.09
u

o.o?

0.05

'

B
Clone

B. Initial

0.15 r

0.13

a

E 0.11

2 0.09

"5
0

5

0.07

0.05 -

0 O3

a

a

On average, experimental anemones from clone B had
35.6% less triacylglycerol than anemones in clone C (Fig.
4A), and clone B anemones in the initial group had the
lowest levels of triacylglycerol (Fig. 4B). However, com-
parison of the average amount of triacylglycerol between

Clone

Figure 6. Glycerol ethers (mg) in the experimental (A) and initial (B)
anemones. Symbols and sample sizes of experimental and initial anem-
ones are as described in Figure 4. Clone significantly affected glycerol
ether content in experimental (P < 0.001) and initial anemones (P =
0.002).

436

M. G. McMANUS ET AL

had the lowest amount of sterol esters and wax esters,
averaging 36.4% less than anemones in clone A and
22.5% less than anemones in clone C (Fig. 5A). Although
the effect of clone was significant on the amount of sterol
esters and wax esters in the initial group of anemones
(f"2.2t = 3.8, P = 0.04; Fig. 5B), an outlier, the maximum
value for sterol esters and wax esters, was identified, and
dropping this value resulted in no detectable effect of
clone (/V,,,= 1.5, P = 0.25).

Similar patterns were found for glycerol ethers and
free fatty acids in both experimental and initial anem-
ones. In the experimental group of anemones, clone
B anemones had at least 41% less glycerol ethers than
anemones in clones A and C (Fig. 6A), and they had
36%. less free fatty acids than anemones in clone C (Fig.
7A). Among initial anemones, clone B anemones had
64% less glycerol ethers than anemones in clone A, and
45% less than anemones in clone C (Fig. 6B). And free
fatty acids in initial clone B anemones were 62% less
than the value in anemones in clone C (Fig. 7B).

Sterol content did not vary among the clones of exper-
imental anemones (F2..,5 = 0.20, P = 0.82; Fig. 8A), or
among clones of the initial anemones (F22] = 2.6, P
= 0.10; Fig. 8B). Similarly, there was no effect of clone
on the amount of phospholipids for the experimental
anemones (/•":. 35 = 0.45, P = 0.64; Fig. 9A) or the initial
anemones (F2.2\ = 0.92, P = 0.4 1 ; Fig. 9B).

Carbohydrate, protein, and ash contents

Clones of experimental anemones differed signifi-
cantly in tissue carbohydrate content (F2 33 = 27.0. P
< 0.001), which constituted 6.7%. of anemone dry
mass, on average (Fig. 10A). Carbohydrate, like lipid.
was lowest in anemones from clone B — on average,
24% less than that of anemones in clones A and C (Fig.
10A). In group X anemones, a MANCOVA revealed
significant clonal effects on protein and carbohydrate
contents (Wilks' A = 0.43, F8.52 = 3.4, P < 0.001 ), and
univariate analysis of carbohydrate content showed
that clonal identity affected levels of this biochemical
class (F4.27 = 5.0, P = 0.004; Fig. 10B). Anemones in
clones A and C had higher amounts of carbohydrate
than anemones in clones B, D, and E, and the latter
clones had similar levels (Fig. 10B). Carbohydrate con-
stituted, on average, 8.3%. of the dry massamonggroup
X anemones.

The clonal pattern for the amount of protein in the
experimental anemones resembled that seen for relative
growth, with anemones in clone A having the lowest
amount and those in clone C the highest amount (Fig.
1 1 A). Like relative growth, the amount of protein in the
experimental anemones showed no significant effect of
clone a •',,) = 1.7..P = 0.19). Protein constituted the bulk

of the anemone's dry mass, averaging 69.2%. For group
X anemones, the ranking of lowest to highest amount of
protein was the same as for the experimental anemones.
The effect of clone was not significant on amount of pro-

A. Experimental

0.19 r

0.17

•S 0.15

0

0.13

0.11

0.09

ab

a

B
Clone

B. Initial

0.2O r

~ 0.16

O>

u.

0.12

0.08

0.04

ab

Clone

Figure 7. Free fatty acids (mg) in experimental (A) and initial (B)
anemones. Symbols and sample sizes of experimental and initial anem-
ones are as described in Figure 4. Clone significantly affected free fatty
acid content in experimental (P = 0.009) and initial (P = 0.03)
anemones.

CLONAL VARIATION IN SEA ANEMONES

437

A. Experimental

On average, ash constituted 8.6% of the dry mass of an
anemone (group Y; Table III). Clonal genotype did not

U.15

affect ash content (F4.:, = 1.5, P = 0.24; Table III). The

0.14

_

A. Experimental

1.2 r

"" 0.13

-

1

I

00

1

1

1

~ 0.12

-

1.1

-

1

0.11

-

•8

1

i

2 1.0

-

o

a

i

i

0.10

iii a>

i

o

ABC £

Clone °-9

-

B. Initial

0.24

-

00

i i i

ABC

0.21

-

Clone

B. Initial

1 0.18

0.9

CO

2

I

i

& °'15

-

i

0.8

~

1

i

i

0.12

\

1 3 0.7

~

a
o

1

i i

i

0.09

1 1 1 "n. n n

_

S- 0.6

ABC 5

^™

(^

Clone

0.5

-

Figure 8. Sterol (mg) in experimental (A) and initial (B)

anemones. Symbols and sample sizes of experimental and initial

anemones are as described in Figure 4. Clone had no significant

effect on sterol content of experiments

ar initial (P = 0.4

i |

1 (P - 0.82)

O.IO) anemones. ABC

tein in group X anemones (F4.2S = 1.2. P = 0.34; Fig.
1 IB), and protein composed, on average. 77.8% of the
anemone dry mass.

Clone

Figure 9. Phospholipids (mg) in experimental (A) and initial (B)
anemones. Symbols and sample sizes ol experimental and initial anem-
ones are as described in Figure 4. Clone had no significant effect on
phospholipid content of experimental (P = 0.64) or initial (P = 0.4I)
anemones.

438
A. Experimental

M. G. McMANUS ET AL.

A. Experimental

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A B C ABC

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Figure 10. Carbohydrate (mg) in experimental (A) and group X
anemones (B). Clonal averages that do not share the same lowercase
letter are significantly different from one another based on the BPT
method (P < 0.05). (A) Symbols for the experimental anemones are as
described in Figure 3 A, and clonal sample sizes are HA = 8. «„ = 14, and
lie = 16. Carbohydrate was calculated for an anemone with an average
dry mass of 14.37 mg. (B) The tilled squares for group X anemones are
the size-adjusted, back-transformed means, and vertical bars are 95%
confidence intervals. Clonal sample sizes are «A = 6, /;„ = 6, nc = 6. nD

S. and iif_ = 1. Carbohydrate was calculated for an anemone with an
average dry mass of 10.47 mg. Carbohydrate content was significantly
difi M : among clones of experimental anemones (/> < 0.001) and
among d". •• of the group X anemones (P = 0.004).

A B C D E

Clone

Figure II. Protein (mg) in the experimental ( Aland group X anem-
ones (B). (A) Symbols for the experimental anemones are as described
in Figure 3A. Clonal sample sizes are «A = 8. «B = 15, and nc = 16.
Protein was calculated for an anemone with an average dry mass of
13.96 mg. (B) Symbols for the group X anemones are as in Figure 10B.
Clonal sample sizes are nA = 6. nB = 6, nc = 7, nD = 8, and >i£ = 7.
Protein was calculated for an anemone with an average dry mass of
10.45 mg. Protein was not significantly different among clones of either
experimental (P = 0. 19) or group X (P = 0.34) anemones.

CLONAL VARIATION IN SEA ANEMONES

439

Table III

Axil ciwlcnl o] gnnip )' ancnwnc\

Clone

Ash(mg)

A

1.44(1.33, 1.55)

B

1.51 (1.41, 1.61)

C

1.46(1.36, 1.57)

D

1.57(1.44, 1.72)

E

1.61 (1.49, 1.75)

The sample size is /; = 6 for all of the clones except clone A, where /(A
= 5. All values are the size-adjusted, back-transformed means and their
95% confidence limits. Ash content was calculated for an anemone with
an average dry mass of 1 7.83 mg.

amount of ash in H. lineata is comparable to the 9.1%
ash content in the sea anemone A. elegantissima fed Ar-
temia nauplii in the laboratory (Zamer, 1986).

Energetic content

Tissue energetic content (in kilojoules), as calcu-
lated from biochemical composition, differed signifi-
cantly among the three clones of experimental anemo-
nes (F: u = 5.6, P = 0.008). The average energetic
content of anemones from clone B was 17% less than
that of anemones from clone C (Fig. 12).

Discussion

We observed a consistent, significant pattern of ge-
netic variation in tissue hydration, carbohydrate
content, and the content of several lipid classes among
clones of H. lineata. The same clonal differences oc-
curred in anemones from the two feeding regimes. Com-
pared to anemones from clones A and C, clone B anem-
ones consistently had lower averages for the lipid and
carbohydrate contents of their tissues, and higher values
for tissue hydration. Similar to clone B, anemones from
clones D and E had less carbohydrate in their tissues than
anemones from clone A (Fig. 10B). The net result of
these differences in tissue constituents was lower ener-
getic content in tissues of clone B anemones.

Although the similarity in clonal pattern for the tissue
constituents between nauplii-fed and ration-fed anemo-
nes may reflect the relatively short duration of the growth
experiment (10 days of ration feedings) compared with
the extended acclimation of the nauplii-fed anemones
(groups X and Y; more than 5 weeks), the experimental
anemones (ration fed) tended to contain less protein
(69.2%) and carbohydrate (6.7%) than the anemones in
group X (average protein and carbohydrate contents:
77.8% and 8.3%, respectively), which were fed ad libitum
on suspensions of Anemia nauplii. Thus biochemical
composition responded to the change in feeding regime

during the 10-day experimental period. Changes in bio-
chemical constituents in tissues of A. elegantissima also
were evident after just 6 days of feeding on even smaller
rations (1% of dry body mass) of adult Anemia (Zamer,
1986; Zamer and Shick, 1989), and the same trend in
protein and carbohydrate content was found between
groups of A. elegantissima that were fed adults and
nauplii of Anemia (Zamer, 1986; Zamer and Shick,
1989). Clearly, cnidarian biochemical composition can
be altered by differences in diet composition and feeding
frequency (Szmant-Froelich and Pilson, 1980; Fitt and
Pardy, 1981; Zamer and Shick, 1989).

Yet despite this trend in biochemical content associ-
ated with the different feeding regimes in our study, we
observed overriding and repeatable clonal patterns in
biochemical composition of the tissues of//, lineata. We
cannot convincingly argue that the 10-day period of
feeding on adult Anemia was sufficient time to remove
a clonal pattern in biochemical content differences that
could have been established by clonal differences in ten-
tacular capture of suspended prey. However, the similar
and rapid changes in protein and carbohydrate that oc-
cur when both H lineata and A. elegantissima are
switched from nauplii to ration feeding suggest that the
persistent pattern of clonal differences observed under
both feeding regimes is not the result of prey capture
differences, but rather the result of differences in meta-

-0.45 r

-0.42

£ -0.39

o
o

u

••g -0.36
a

u

-0.33

-0.30

a

ab

Clone

Figure 12. Energetic content, in kilojoules, of the experimental
anemones, with symbols as described in Figure 3A. Clonal sample sizes
are nA = 8, nB = 14, HC =16. Energetic content, calculated for an anem-
one with an average dry mass of 14.37 mg, was significantly different
among clones of the experimental anemones (P = 0.008).

440

M. G. McMANUS ET AL

bolic rates or food conversion efficiencies among the sep-
arate clones of H lineal a (see below).

The high amounts of storage lipids and carbohydrate
in anemones in clones A and C compared to anemones
in clone B is the same pattern of genetic covariation in
these biochemical classes that has been found in different
genetic lines of Drosophila melanogaster (Clark and
Keith, 1988; Clark, 1 990) and in different species of Dro-
sophila (Clark and Wang, 1994). Mechanisms that may
produce this covariation include genetic modulation of
the activities of enzymes associated with lipid and carbo-
hydrate pathways of metabolism (Clark and Keith.
1988), and such genetic modulation of metabolic path-
way performance has been associated with genotypes at
the glucose-phosphate isomerase locus in the anemone
Metric/him senile (Zamer and Hoffmann, 1989). Alter-
natively, clone B anemones may have been unable to ab-
sorb energy from the digested rations as well as anemo-
nes from the other two clones. Differential substrate-spe-
cific absorption has been demonstrated in anemones
(Zamer and Shick, 1989), and could produce differences
in the biochemical and energetic content of their tissues.
Low amounts of carbohydrate and lipid in tissues of
clone B anemones resulted in low values for the calcu-
lated energetic content of these tissues, even though
anemones in clone B received the same rations as anem-
ones in clones A and C.

The lower energetic content of clone B anemones de-
rives primarily from the lower levels of what may be con-
sidered energy-storage forms of lipid: fatty acids, triacyl-
glycerols, and sterol esters and wax esters. All of these
lipid classes have been associated with energy storage in
anemones (e.g., Pollero, 1983; Hill-Manning and Blan-
quet, 1979). We know of no data concerning the turn-
over of glycerol ethers in tissues of sea anemones, but
given the low levels of this class of lipids in clone B anem-
ones, we infer that it may also be a storage form.

In contrast, sterol (with cholesterol being the principal
sterol in the anemone Actinostola callosa; Bergmann el
a/.. 1956) and phospholipids (often considered to be im-
portant as structural or membrane classes) are not vari-
able among the three anemone clones examined here.
These results indicate that storage of energy in the form
of lipid is somehow impaired in clone B anemones.

In this context, we also note the inverse relationship
between tissue water and total lipid in the experimental
anemones. Small differences in tissue hydration have
been reported between high- and low-intertidal individ-
uals of A. elegantissima and between continuously im-
mersed individuals of H. lineata and those maintained
ui fluctuating immersion conditions (Shick. 1991;
.In i and Shick, 1977). In both cases, anemones pe-
riodic exposed to air have slightly higher tissue hy-
dration. ; .' elegantissima freshly collected from upper

and lower intertidal areas, a significant difference in lipid
content was not observed, although high-shore speci-
mens tended to have lower lipid than low-shore ones,
consistent with the inverse relationship between tissue
hydration and lipid content found in this study for //.
lineata. Shick ( 1 99 1 ) also points out that tissue hydration
may be genetically correlated, given that variance in tis-
sue hydration is significantly greater in multiclonal pop-
ulations of//, lineata than in monoclonal ones (Shick
and Dowse, 1985). Our findings of significant differences
in tissue hydration among clones of//, lineata are con-
sistent with this observation.

No genetic variation in tissue protein was detected
among the anemones. Likewise, none was found for the
flour beetle Triholium custaneuin. either in genetic lines
selected for 2 1-day pupa weight or in control, unselected,
genetic lines (Medrano and Gall, 1 976a). Protein content
in //. lineata is less variable than carbohydrate or lipid.
For the experimental anemones (n = 38), the coefficient
of variation of protein was 8.3%, whereas it was 15.4%
for carbohydrate and 19.1% for lipid. Although our static
measurements of protein were not different among our
anemone clones, genetic variation has been associated
with nitrogen metabolism, protein turnover rate, and
physiological energetics of the blue mussel Mytihts edulis
(HilbishandKoehn, 1985; Hawkins el at.. 1986).

The physiological energetic values reported here for //.
lineata are within typical ranges for sea anemones
(Shick, 1991), and the experimental anemones from
clones A. B, and C did not differ in the traits of relative
growth, absorption efficiency, and net growth efficiency,
which are all expressed gravimetrically. Recently, Tsuch-
ida and Potts (1994) also reported that, in two separate
feeding experiments, clonal identity had no effect on
weight change in A. elegantissima. Differences in absorp-
tion efficiencies and net growth efficiencies have been de-
tected among clones of A. elegantissima from different
shore levels (Zamer. 1986), and those differences could
not be erased by acclimation to common conditions.
However, variation among clones within each tidal re-
gime was not examined in that study. A. elegantissima
had an average gravimetric absorption efficiency of
69.6% (Zamer. 1986), compared to 93% measured for//.
lineata in this study. The net gravimetric growth effi-
ciencies for these two species are about 45% (cf. Zamer.
1986). The relative growth reported here for //. lineata is
about 15% greater than that measured in A. elegantis-
sima (Zamer, 1986). but the difference is probably due
to the larger rations received by H. lineata in this study
(6%-8.5% of dry body mass compared to 4%-5.6% for A.
elegantissima).

The lack of clonal differences in these components of
the energy budget does not necessarily mean that the ge-
notypes of//, lineata are equivalent in their physiologi-

(l()\\l \\KI\I10N IN SI A \NIMONIS

441

cal energetics. First, an examination of energetically ex-
pressed absorption and net growth efficiencies, in which
the energy content (rather than mass) of tissue growth,
rations, and egesta are used in the calculations of these
quantities, may reveal features of the physiological ener-
getics of these clones that are not apparent from the pres-
ent analysis of gravimetric values (cf. Zamer, 1986). The
similar average body masses of anemones in all three
clones examined (Table I), in combination with both the
low energetic content of the tissues of clone B anemones
and the similar growth rates in anemones among these
clones, is circumstantial support for the hypothesis that
energetic values for net growth efficiency may be more
informative than the present gravimetric ones.

Moreover, estimates of the biochemical content of
food and egesta, which are values needed for determin-
ing energetic contents of these substances, may also be
used to test one hypothesis concerning the differences in
biochemical and energetic content of the tissues from
anemones in the three clones. If we find the biochemical
composition of the egesta to be the same, regardless of
clonal genotype, then we can eliminate the differential-
substrate-absorption hypothesis as an explanation for
differences in the biochemical content of tissue.

Second, metabolic rate was not measured in the pres-
ent study. If clone B anemones have a higher metabolic
rate, on average, than anemones in clones A and C, then
a greater proportion of the absorbed ration would be
catabolized for maintenance, and consequently less en-
ergy would be stored in the tissues. In comparing clone
B anemones acclimated to 25°C with anemones in
clone C at the same temperature, we have observed con-
sistently higher rates of longitudinal fission and smaller
average body mass in clone B animals (Zamer, unpubl.
data). The metabolic rate in clone B anemones may be
elevated owing to greater costs associated with higher
fission rate at 25°C. But we do not know whether eleva-
tion of metabolic rate underlies the low energetic
content of clone B anemone tissues at 15°C. Genetic
variation in metabolic rate, measured as oxygen con-
sumption, has been detected among selected and con-
trol lines of T. castanenm (Medrano and Gall. 1976b),
among D. melanogaster lines selected for desiccation
resistance and control lines (Hoffmann and Parsons,
1989ai 1989b), in bivalves differing in multiple-locus
heterozygosity (Bayne, 1987), in common garter snake
(Thamnophis sirtalis) offspring from different families
(Garland, 1 994), and among strains and populations of
the deer mouse Pemmvscits manicitlalits that differed
in «-chain hemoglobin genotypes (Chappell and Sny-
der, 1984). Such variation in metabolic rate can be
manifested as variation in maintenance efficiency, spe-
cifically in the rate of protein turnover (Hawkins el a/.,
1986). Our ongoing experiments are aimed at determin-

ing energetically expressed values for absorption effi-
ciency and growth and measuring oxygen uptake rates
among anemones from our different clones. These stud-
ies will yield information about the mechanisms un-
derlying the present clonal differences in physiological
energetic traits as well as additional data on genetically
correlated physiological variation.

In addition to providing these two mechanistic
hypotheses, our study of the physiological variation that
is associated with clonal genotype potentially has im-
plications for the relative fitness of anemones from the
different clones. At 1 0°C in the laboratory, individuals of
//. lineata commonly encyst in mucous secretions, and
in Indian Field Creek encysted and nonencysted individ-
uals occur in the winter when the surface water tempera-
ture is below 10°C (Sassaman and Mangum. 1970). Al-
though metabolic rate is correspondingly low at this tem-
perature, and is likely to be even less in encysted
compared to nonencysted individuals, survivorship of
encysted anemones probably depends on reserves of
storage lipid and carbohydrate. During extended encyst-
ment, anemones from clone B, which have less of these
reserves at 15°C, may have lower survivorship than
anemones in clones A and C, which have more of these
reserves. The relationship between individual variation
in the physiological characteristics of organisms and the
variation among organisms in fitness is an essential com-
ponent oforganismal performance (Rough, 1989).

Acknowledgments

Thanks to Mike Lynch for assistance in the lipid anal-
ysis, to M. Amsler for doing the electrophoresis, and to
C.P. Mangum for collecting the anemones at Indian
Field Creek. This research was supported by NSF grant
DCB-9057315 to W. E. Z. This is contribution No 294
from the Center of Marine Biotechnology, University of
Maryland Biotechnology Institute. Versions of this
manuscript benefitted from comments by C. O. Deetz,
A. T. Weglinski, and two anonymous reviewers.

Literature Cited

Ayre, D. J. 1982. Inter-genotype aggression in the solitary sea anem-
one Actinia tcwhrosa. Mar Bin/ 68: 199-205.

Ayre, D. J. 1983. The effects of asexual reproduction and inter-geno-
typic aggression on the genotypic structure of populations of the sea
anemone Actinia tenebrosa. Omilogia (Bcr/jSl: 158-165.

Ayre, D. J. 1985. Localized adaptation of clones ol the sea anemone
Actinia tenebrosa. Evolution 39: 1250-1260.

Ayre, D. J. 1995. Localized adaptation of sea anemone clones: evi-
dence from transplantation over two spatial scales. J. An/in, EcoL
M: 186-196.

Bayne, B. L. 1987. Genetic aspects of physiological adaptation in bi-
valve molluscs. Pp. 169- 1 89 in Evolutionary Physiological Ecology,
P. Calow. ed. Cambridge University Press, New York.

Bayne, B. L., and R. C. Newell. 1983. Physiological energetics of ma-

442

M. G. McMANUS ET AL

rine molluscs. Pp. 407-5 1 5 in The Mollnsca, Volume 4, Physiology,
Part I, A. S. M. Saleuddin and K. M. Wilbur, eds. Academic Press,
New York.

Bergmann, \V.,S. M.,Creighton,and VV. M. Stokes. 1956. Contribu-
tions to the study of marine products. XL. waxes and triglycerides
of sea anemones. / Org. Chem. 21: 72 1-728.

Bligh, K. G., and \V. T. Dyer. 1959. A rapid method of total lipid ex-
traction and purification, din, J Biochcm. Physiol. 37: 91 1-917.

Carlson, L. A. 1985. Extraction of lipids from human whole serum
and lipoproteins and from rat liver tissue with methylene chloride-
methanol: a comparison with extraction with chloroform-metha-
nol. Clin. Chim. Ada 149: 89-93.

Carvalho, G. R. 1994. Genetics of aquatic clonal organisms. Pp. 29 1-
323 in Genetics and Evolution oj Aquatic Organisms, A. R. Beau-
mont, ed. Chapman and Hall, London.

Chadwick, N. E., and C. Adams. 1991. Locomotion, asexual repro-
duction, and killing of corals by the corallimorpharian Corynactis
calilornica Hyarobiologia 216/217:263-269.

Chappell, M. A., and L. R. G. Snyder. 1984. Biochemical and physi-
ological correlates of deer mouse a-chain hemoglobin polymor-
phisms. Proi: Nail. .lead. Sci, USA 81: 5484-5488.

Clark, A. G. 1990. Genetic componentsof variation in energy storage
in Drosophila melcinogasler. Evolution 44: 637-650.

Clark, A. G., and L. E. Keith. 1988. Variation among extracted lines
of Drosophila melanogaster in triacylglycerol and carbohydrate
storage. Genetics 1 19: 595-607.

Clark, A. G., and L. Wang. 1994. Comparative evolutionary analysis
of metabolism in nine Drosophila species. Evolution 48: 1230-
1243.

Coast and Geodetic Survey. 1960. Surface water temperature and sa-
linity Atlantic Coast North and South America. C. & G. S. Publica-
tion 31-1, 76 pp. Government Printing Office, Washington, DC.

Cox, D. R., and E. J. Snell. 1992. Analysis <>/ Binary Data Chapman
and Hall, New York.

Day, R. \\ '., and G. P. Quinn, 1989. Comparisons of treatments after
an analysis of variance in ecology. Ecol. Monogr. 59: 433-463.

Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith.
1956. Colonmetnc method for determination of sugars and re-
lated substances. A mil Chem. 28: 350-356.

Falconer, D. S. 1989. Introduction to Quantitative Genetics. Wiley,
New York.

Fill, \V. K., and R. I.. Pardy. 1981. Effects of starvation, light and
dark on the energy metabolism of symbiotic and aposymbiotic sea
anemones, Anlhopleura eleganlissima. Mar. Biol. 61: 199-205.

Fukui, Y. 1991 . Embryonic and larval development of the sea anem-
one Haliplanella lineatafrom Japan. Hydrohiologia 216/217: 137-
142.

Garland, T., Jr. 1994. Quantitative genetics of locomotor behavior
and physiology in a garter snake. Pp. 25 1-277 in Quantitative tic-
nclic Studies of Behavioral Evolution. C. R. B. Boake, ed. The Uni-
versity of Chicago Press, Chicago.

Gnaiger, E. 1983. Appendix C. Calculation of energetic and bio-
chemical equivalents of respiratory oxygen consumption. Pp. 337-
345 in Polarograpflic Oxygen Sensors: Aquatic ami Physiological
Applications. E. Gnaiger and H. Forstner, eds. Springer, New York.

Hawkins, A. J. S., B. L. Bayne, and A. J. Day. 1986. Protein turn-
over, physiological energetics and heterozygosity in the Blue Mus-
sel. Mytilus edulis: the basis of variable age-specific growth. Proc R.
Sue. Lond. B Bio/ Sci. 229: 161-176.

Hilbisli, T. J., and R. K. koehn. 1985. The physiological basis of nat-
u ' -.flection at the lap locus. Evolution 39: 1 302- 1317.

Hill-lN H. D.N., and R.S. Blanquet. 1979. Seasonal changes in

the hpu the sea anemone, Melridium senile (L.) / Exp. Mar,
Biol /:, 249-257.

Hoffmann, A. A., and P. A. Parsons. 1989a. An integrated approach
to environmental stress tolerance and life-history variation: desic-
cation tolerance in Drosophila Biol. J. Linn. Soc. 37: 117-136.

Hoffmann, A. A., and P. A. Parsons. 1989b. Selection for increased
desiccation resistance in Drosophila melanogaster: additive genetic
control and correlated responses. Genetics 122: 837-845.

Hoffmann, R. J. 1986. Variation in contributions of asexual repro-
duction to the genetic structure of populations of the sea anemone
Metridium senile. Evolution 40: 357-365.

Hughes, R. N. 1989. .-1 Functional Biology of Clonal Animals Chap-
man and Hall, New York.

Huitema, B. E. 1980. The Analysis oj Covariance and Alternatives.
Wiley. New York.

Itzhaki, R. F.. and D. M. Gill. 1964. A micro-biuret method for esti-
mating proteins. Anal. Bine/win 9:401-410.

Jennison, B. L. 1979. Annual fluctuations of lipid levels in the sea
anemone Anlhopleura elegantissima (Brandt, 1835). J. Exp. Mar.
Biol Ecol 39:211-221.

Johnson, L. L., and J. M. Shick. 1977. Effects of fluctuating temper-
ature and immersion on asexual reproduction in the intertidal sea
anemone Haliplanella luciae( Verrill) in laboratory culture. J Exp.
Mar. Biol. Ecol 28: 141-149.

Jones, R.,J. A. Bates, D. .1. 1 lines, and R. J.Thompson. 1996. Quanti-
tative genetic analysis of growth in larval scallops (Placopeclen ma-
Kellamcus). Mar Biol 124:671-677.

Keen, S. L., and A. J. Gong. 1989. Genotype and feeding frequency
affect clone formation in a marine cnidanan (Aureha aunia La-
marck 1816). h'unct. Ecol 3: 735-745.

Koehn, R. K. 1991. The cost of enzyme synthesis in the genetics of
energy balance and physiological performance. Biol J Linn. Soc.
44:231-247.

Koehn, R. K., and B. L. Bayne. 1989. Towards a physiological and
genetical understanding of the energetics of the stress response. Biol.
J. Linn. Soi. 37: 157-171.

Medrano, J. F., and G. A. E. Gall. 1976a. Growth rate, body compo-
sition, cellular growth, and enzyme activities in lines of Tribolium
custom-urn selected for 2 1 -day pupa weight. Genetics 83: 379-39 1 .

Medrano, J. F., and G. A. E. Gall. 1976b. Food consumption, feed
efficiency, metabolic rate, and utilization of glucose in lines of
Tribolium caslaneum selected for 2 1 -day pupa weight. Genetics 83:
393-407.

Minasian, L. L., Jr. 1979. The effect of exogenous factors on mor-
phology and asexual reproduction in laboratory cultures of the in-
tertidal sea anemone, Haliplanella luciae(Veni\\) (Anthozoa:Acti-
naria) from Delaware. J. Exp. Mar. Biol. Ecol 40: 235-246.

Minasian, L. L., and R. N. Mariscal. 1979. Characteristics and regu-
lation of fission activity in clonal cultures of the cosmopolitan sea
anemone, Haliplanella luciac (Verrill). Biol Bull. 157: 478-493.

Pollero, R. J. 1983. Lipid and fatty acid characterization and metab-
olism in the sea anemone Phymactis clematis (Dana). Lipids 18:
12-17.

Pough, F. H. 1989. Orgamsmal performance and Darwinian fitness:
approaches and interpretations. Physiol Zoo/. 62: 199-236.

Present, T. M. C, and D. O. Conover. 1992. Physiological basis of
latitudinal growth differences in Menidia menidia: variation in con-
sumption or efficiency, Fund. Ecol. 6: 23-3 1 .

Rawson, P. D., and T. J. Hilbish. 1991. Genotype-environment in-
teraction for juvenile growth in the hard clam Mercenaria mercen-
ana ( L. ). Evolution 45: 1 924- 1935.

Sassaman, C., and C. P. Mangum. 1970. Patterns of temperature ad-
aptation in North American Atlantic coastal actinians. Mar. Biol.
7: 123-130.

Sebens, K. P. 1981. Reproductive ecology of the intertidal sea anem-
ones Anlhopleura xanthogrammica (Brandt) and A. elegantissima

CLONAL VARIATION IN SEA ANEMONES

443

(Brandt): body size, habitat, and sexual reproduction. ./ /-.'A/I. Mar.
Bin/ Ecol. 54: 225-250.

Shick, .1. M. 1976. Ecological physiology and genetics of the coloniz-
ing actinian Haliplanella luciae. Pp. 137-146 in Coelenterate Ecol-
ogy and Behavior. G. O. Mackie. ed. Plenum Publishing, New
York.

Shick, J. M. 1991. A Functional Biology of Sea Anemones. Chapman
and Hall. New York.

Shick, ,J. M., and II. B. Dowse. 1985. Genetic basis of physiological
variation in natural populations of sea anemones: intra- and in-
terclonal analyses of variance. Pp. 465-479 in Proceedings of the
Nineteenth European Marine Biology Symposium, P. E. Gibbs, ed.
Cambridge University Press, U. K.

Shick, J. M., R. J. Hoffmann, and A. IN. Lamb. 1979. Asexual repro-
duction, population structure, and genotype-environment interac-
tions in sea anemones. Am Zoot 19:699-713.

Shick, .1. M., and A. N. Lamb. 1977. Asexual reproduction and ge-
netic population structure in the colonizing sea anemone llalipla-
iwllu luciae. Bio/. Bull. 53: 604-6 1 7.

Sokal, R. R., and F. J. Rohlf. 1981. Biometry. W. H. Freeman and
Co.. San Francisco.

Steel, R. G. D., and J. H. Torrie. 1980. Principles and Procedures of
Statistics. McGraw-Hill, New York.

Szmant-Froelich. A., and M. E. Q. Pilson. 1980. The effects of feed-
ing frequency and symbiosis with zooxanthellaeon the biochemical
composition ofAstrangia danae Milne Edwards and Haime 1849.
./ E\p. Mar. Bio/ Ecol. 48: 85-97.

Tsuchida, C. B., and D. C. Potts. 1994. The effects of illumination,
food and symbionts on growth of the sea anemone Anlhopleura cl-
egantissima (Brandt, 1835). I. Ramet growth. J Exp. Mar. Biol.
Ecol. 183:227-242.

Yrijenhoek, R.C. 1994. Unisexual fish: model systems for studying
ecology and evolution. Annu. Rev. Ecol. Syst. 25: 71-96.

\Veisberg, S. 1985. Applied Linear Regression. John Wiley and Sons,
New York.

\\ illiams.G. C. 1975. Sex and Evolution. Princeton University Press,
Princeton. NJ.

\\inberg, G. C. 1956. Rate of metabolism and food requirements of
fishes, l-'ish Res. Board Can Trans. Ser. 194: 1-202.

Zamer, VV. E. 1986. Physiological energetics of the intertidal sea
anemone Anthoplcura cIcKanns.sima. I. Prey capture, absorption
efficiency and growth. Mar. liiol. 92: 299-3 14.

Earner, W. F... and R. .1. Hoffmann. 1989. Allozymes of glucose-6-
phosphate isomerase differentially modulate pentose-shunt metab-
olism in the sea anemone Mctridium senile. Proc. Null. ACM/. Sci.
i'SA 86: 2737-274 1.

Zamer, \V. F,., and C. P. Mam>um. 1979. Irreversible nongenetic tem-
perature adaptation of oxygen uptake in clones of the sea anemone
Haliplanella luciae (\em\\). Biol. Bull. 157: 536-547.

Zamer, \V. E.. and J. M. Shick. 1987. Physiological energetics of the
intertidal sea anemone Anlhopleura I'leganli.ssinia. II. Energy bal-
ance. Mar. Biol 93: 48 1 -49 1 .

/amer, W. E., and J. M. Shick. 1989. Physiological energetics of the
intertidal sea anemone Anlhopleura elegantissima. III. Biochemical
composition of body tissues, substrate-specific absorption, and car-
bon and nitrogen budgets. Oveologia 79: 1 17-127.

Zamer, \V. E., J. M. Shick, and D. VV. Tapley. 1989. Protein mea-
surement and energetic considerations: comparisons of biochemi-
cal and stoichiometric methods using bovine serum albumin and
protein isolated from sea anemones. Limnol. Oceanogr. 34: 256-
263.

Reference: Biol Bull 192:444-456. (June. 1997)

Oxidative Stress in the Symbiotic Sea Anemone

Aiptasia pulchella (Carlgren, 1943): Contribution

of the Animal to Superoxide Ion Production

at Elevated Temperature

CALVIN M. Nil AND LEONARD MUSCATINE
Department of Biology, University of California, Los Angeles, California 90095-1606

Abstract. Production of superoxide ions within tissues
of the symbiotic sea anemone Aiptasia pulchella was de-
tected using SOD-inhibitable cytochrome c reduction
and quantified by SOD-inhibitable reduction of nitro
blue tetrazolium (NBT). Intact aposymbiotic and sym-
biotic specimens of A. pulchella produced superoxide in
response to acute, sublethal thermal stress. Neither light
nor inhibition of symbiont photosynthesis by (3,4-di-
chlorophenyl)-l,l-dimethylurea (DCMU) affected su-
peroxide production. The time course of superoxide ion
production strongly resembled the time course of in-
creased dark respiration by intact anemones, suggesting
that the effect of elevated temperature on host mitochon-
dria may account for increased superoxide production.
Interestingly, freshly isolated algae (FIZ) did not release
superoxide ions in response to elevated temperature, and
net oxygen production decreased greatly in both intact
symbiotic anemones and in FIZ within 20 minutes after
temperature elevation. These results show that oxidative
stress in A. pulchella is primarily an animal response, and
suggest that the presence of symbiotic algae, although
sufficient to cause hyperoxia, is not necessary for the ap-
pearance of oxidative stress in these anemones at ele-
vated temperature.

Introduction

Elevated temperature can adversely affect the stability
of symbioses between cnidarians and symbiotic dino-
flagellates and result in bleaching (Glynn, 1990). Cni-
darian bleaching may be manifest as a decline in the pop-

Receiveu '.July 1996; accepted 27 February 1997.

ulation density of the symbiotic algae (Fisk and Done,
1985; Hoegh-Guldbergand Smith, 1989), a reduction in
the amount of chlorophyll a (Coles and Jokiel, 1977;
Kleppel et a/., 1989; Porter et ai, 1989; Szmant and
Gassman, 1990) and accessory pigments (Kleppel et al.,
1989) per alga, or both (Glynn and D'Croz, 1990; Lesser
et at., 1990). While the loss of symbionts/?w se has been
described at the organismic level (Gates et al., 1992;
Brown el at., 1995), there are few experimental studies of
the cellular or molecular mechanism of cnidarian
bleaching.

Oxidative stress is thought to play a role in bleaching
(Lesser and Shick, 1989; Lesser et al, 1990; Shick et al..
1991. Shick et al., 1995; Lesser, 1996). Active oxygen
species may be produced within the algae and within the
host in response to a combination of elevated seawater
temperature and high levels of photosynthetically active
radiation (PAR) or ultraviolet radiation (Lesser and
Shick. 1989; Lesser et al.. 1990; Dykens et ai. 1992).
Symbiotic dinoflagellates may respond by exhibiting in-
creased activities of the protective enzymes superoxide
dismutase (SOD) and catalase (CAT; Lesser and Shick,
1989; Lesser et al., 1990; Shick et ai. 1991; Malta and
Trench, 1991; Shick et ai, 1995). Production of hy-
droxyl radicals in the light has been detected in homoge-
nates of both symbiotic and aposymbiotic anemones and
in isolated symbiotic algae (Dykens el ai, 1992). Yet, the
explicit source of active oxygen species in the intact sym-
bioses under stress conditions is still unknown.

Active oxygen species could originate from the in-
teraction of molecular oxygen from symbiont photosyn-
thesis and electrons from animal tissue sources (Dykens
and Shick, 1982, 1984; Dykens, 1984; Dykens et ai.

444

OX1DATIVF. STRESS IN SEA ANEMONES

445

1992), or from o.\idants(O: and H:O:) produced within
the algae and released to host tissues (Lesser and Shick,
1989; Lesser el a/., 1990). Lesser et al. (1990) also hy-
pothesized that active oxygen species from algae may sig-
nal the bleaching response in symbiotic cnidarians.

Whereas the foregoing observations emphasize oxy-
gen production and univalent reduction in algae, oxygen
derived from the ambient medium may also undergo
univalent reduction in the animal. About 4% of the total
oxygen that is consumed by animal mitochondria during
aerobic respiration undergoes a further univalent reduc-
tion to form superoxide ion (Boveris and Cardenas,
1982), and exposure to elevated temperature increases
superoxide ion production in animal mitochondria
( Burden?/ al.. 1990).

In this study, we demonstrate that oxidative stress is
concomitant with acute, sub-lethal thermal stress in the
tropical sea anemone Aiptasia pulchella. and that at ele-
vated temperature, increased superoxide production
represents primarily an animal response.

Materials and Methods

Animal collection and maintenance

The symbiotic sea anemone Aiptasia pulchella (Carl-
gren, 1943) was collected from reef flats near the Hawaii
Institute of Marine Biology, Kaneohe, Hawaii, and
transported to the University of California, Los Angeles.
These anemones were maintained in 1.5-liter glass bowls
containing natural seawater (Redondo Beach, CA; 33%o,
pH 8.3) and kept in a temperature (26° ± 1°C) and light-
controlled (70 ^E/rrr/s: 12 h light/12 h dark) incubator.
Anemones were fed three times weekly with freshly
hatched Anemia nauplii. Unless stated otherwise, artifi-
cial seawater ("FSW"; 33%o, pH 8.3, Tropic Marin) fil-
tered through glass fiber niters (Whatman GF/C) was
used in all experiments. All chemicals were obtained
from Sigma (St. Louis. MO).

Aposymbiotic anemones were generated by chilling
symbiotic A. pulchella in 4°C seawater for 4 h and then
maintaining them in darkness at 26° ± 1°C (Steen and
Muscatine, 1987). The anemones lost about 99% of their
algae within 1 week after "cold shock," but were not used
in experiments until they had been maintained in dark-
ness for at least 10 weeks. Aposymbiotic anemones were
fed three times weekly with Anemia nauplii, but those
used in the hyperoxia experiments were fed twice weekly
with adult Anemia.

Algae-free tissue homogenates from individual ani-
mals were prepared by homogenizing a single specimen
of A. pulchella in 1.0 ml FSW on ice and removing the
algae by centrifugation (IEC; 500 X g) for 2 or 3 min.
The supernatant containing the algae-free animal tissue
homogenate was decanted, brought to a final volume of

2.0 ml with FSW, and kept on ice briefly until needed. A
0.2-ml aliquot of the homogenate was removed for mea-
surement of soluble protein.

Freshly isolated symbiotic dinoflagellates (FIZ; Sym-
biodinium pulchronim. Banaszak et al., 1993) were pre-
pared by homogenizing an anemone in 2 ml FSW and
then centrifuging this homogenate at 500 X g for 2-
3 min. The supernatant was discarded, and the algal pel-
let was washed by suspension and centrifugation up to
five times until it was free of nearly all animal contami-
nation. Samples were then taken for algal cell counts.

Assay for superoxide ions in intact host tissues by NBT
reduction

Superoxide ions were measured by the reduction of
nitro blue tetrazolium (NBT) to its diformazan deriva-
tive (formazan; Seidler. 1991: Thorn et al.. 1993). For-
mazan production was quantified by a modification of
the methods used by Chacon and Acosta (1991). NBT
formazan was first isolated by homogenization of an in-
tact anemone in 1.0 ml FSW in a glass tissue grinder.
The homogenate was then centrifuged (IEC; 500 X g),
and the supernatant was removed and saved for mea-
surement of soluble protein. NBT formazan crystals re-
mained as a thin bluish layer above the algal pellet. Pellet
and formazan were then transferred to a 1.5-ml plastic
microfuge tube and then extracted in 1 .0 ml of 80% or
95% ethanol at 4°C for at least 60 min to remove algal
pigments that interfere with the spectrophotometric
measurement of the ethanol-insoluble NBT formazan.
Complete removal of algal pigments was verified by spec-
trophotometric examination (between 400 and 800 nm)
of the ethanolic extract.

The extracted suspension was then centrifuged (Fisher
microcentrifuge; 5000 X g) to pellet the ethanol-insolu-
ble formazan crystals. The ethanol supernatant and re-
sidual algae were then discarded and replaced by 1 .0 ml
of dimethylformamide (DMF) to dissolve the formazan.
Dissolution was accelerated by sonication ( Branson: four
15-s pulses at 35 W) and the absorbance of the resulting
bluish solution was then measured spectrophotometri-
cally at 550 nm. The concentration of formazan was
then calculated from the measured absorbance and the
molar extinction coefficient of the NBT formazan («
= 30,000). Results are expressed as nmol NBT formazan
produced per milligram of soluble animal protein.

Specificity of the NBT assay for superoxide ions

Separate volumes (2.0 ml) of animal homogenate were
prepared as described above. After removing 0.2 ml for
soluble protein analysis, four 0.3-ml samples were used
in NBT reduction experiments. These were performed
in microfuge tubes ( 1.5 ml). A series of four treatments

446

C. M. Nil AND L. MUSCAT1NE

was performed: samples with animal homogenate only
(control); samples with Cu-Zn SOD only (from bovine
erythrocytes; 50-500 U); samples with both SOD and
1 mA/diethyldiihiocarbamate (DDC), a copper chelator
that inhibits SOD activity in vivo (Enger and Kensler,
1985); and samples with DDC only. DDC did not reduce
NBT in control experiments.

The reaction mixtures consisted of animal homoge-
nate (0.3 ml), NBT dissolved in FSW (0.3 ml. to a final
concentration of 10~4 A/), 1 mM DDC, and 100 U Cu-
Zn SOD to a final reaction volume of 1 ml. The residual
volume in treatments that omitted SOD, DDC, or both
was made up with FSW (0.4 ml). Control treatments
contained only animal homogenate and NBT; FSW was
added to achieve the final reaction volume.

The NBT was added last to the sample mixture to ini-
tiate the reaction. Reaction mixtures were incubated in
darkness for 60 min, including a 5-min centrifugation
(5000 X g) to isolate any NBT formazan produced. For-
mazan was quantified using the methods described
above. The results are expressed as the percent stimula-
tion or inhibition of NBT reduction (per milligram of
soluble animal protein) by SOD, DDC, or both com-
pared with control samples (no SOD/DDC).

The effect of SOD concentration on NBT reduction in
animal homogenates was investigated by adding 50 to
500 U Cu-Zn SOD to a mixture (final volume = 1 .0 ml)
containing animal homogenate (0.3 ml) and NBT
dissolved in FSW (0.3 ml; final NBT concentration
= 10 4 A/).

Cytochrome c reduction assay for superoxide production

Cytochrome c reduction was used as an independent
assay for superoxide production in algae-free tissue ho-
mogenates of symbiotic A pnlctiella. The tissue homog-
enates were prepared by homogenizing three to five sym-
biotic anemones in a reaction buffer (0.5 mA/ potassium
phosphate, 0. 1 mA/ EDTA. pH 7.8) and then separating
the algae from the homogenate by centrifugation as de-
scribed above. The soluble protein conten! of the ho-
mogenate was adjusted to 5 mg/ml by dilution with re-
action buffer. The homogenate was maintained briefly at
room temperature until needed. The Cytochrome c re-
duction assay used was modified from the method of
Flohe and Otting (1984). Briefly, the reaction mixture
(total volume 1.5ml) contained 0.5mA/ potassium
phosphate buffer (pH 7.8). 0.1 mA/ EDTA and 20 fiA-f
acetylated Cytochrome c. The latter is refractory to re-
duction by mitochondria! Cytochrome c oxidases (Azzi
etal., 1975). To verify the efficacy of this modified assay,
we used xanthine oxidase (0.2 U/ml) and 50 nAI xan-
thine to generate superoxide in the reaction medium,
causing red tion of Cytochrome c. Cytochrome c reduc-

tion was determined by measuring the change in absor-
bance at 550 nm using a UV-VIS spectrophotometer
(Uvikon) fitted with temperature-controlled cuvette
holders. The cuvette holders were connected to a recir-
culating water bath maintained at 26° ± 1°C. The addi-
tion of 1. 10. or 100 U Cu-Zn SOD to the reaction mix-
ture inhibited Cytochrome reduction by 55%, 95%, and
98% respectively. Superoxide production by tissue ho-
mogenates was determined by substituting 100 n\ of tis-
sue homogenate for the xanthine and xanthine oxidase.
To verify the assay specificity for superoxide ions, Cu-Zn
SOD (10, 20, 50, or 100 U) was added to inhibit cyto-
chrome c reduction in reaction mixtures containing ho-
mogenates.

NBT reduction hy intact symbiotic and aposymbiotic
anemones

Symbiotic and aposymbiotic anemones of similar oral
disc diameter (~1 cm) and column height (~1.5 cm)
were placed in 15-ml glass test tubes containing 2.5 ml
FSW and allowed to attach to the bottom of the tube.
Groups of 12 anemones were pre-incubated in light
(300 juE/rrr/s) or dark at ambient temperature (26°C) for
at least 1 h before the start of an experiment. Another
group was pre-incubated in FSW containing 0.5 mA/
(nominal concentration) 3-(3,4-dichlorophenyl)-l,l-di-
methylurea (DCMU) at ambient temperature. Prelimi-
nary experiments (n = 3) showed that DCMU did not
affect dark respiration in intact symbiotic and aposym-
biotic animals or in FIZ. but did inhibit photosynthetic
oxygen production in the light (300 /uE/irr/s) in intact
symbiotic animals and FIZ.

The FSW used for pre-incubations was discarded from
each tube and replaced with 2.5 ml FSW containing 1.2
X 10~4 A/ NBT pre-warmed to the desired temperature
(either 26° or 32°C). All anemones were incubated in the
presence of NBT at the desired temperature for an addi-
tional 60 min. DCMU-treated anemones were exposed
to 0.5 mA/ DCMU during the 60 min incubation with
NBT at the desired temperature. The elevated tempera-
ture condition was set at 32°C because this is a sublethal
(and environmentally relevant) temperature that evokes
bleaching in A. pit/c/iella (C. B. Mahnke and L. Musca-
tine, unpubl.).

Incubations in light were carried out by placing the
test tubes containing the anemones in the reservoir of a
circulating water bath (Neslab) illuminated by six 40 W
fluorescent tubes (Sylvania "Cool-White"; 300 ^E/rrr/s)
and maintained at the desired incubation temperature.
Dark incubations were conducted in a photographic
darkroom illuminated only by a safelight (Kodak). After
the incubations were completed, the seawater containing
the NBT in each test tube was replaced by an equal vol-

OXIDATIVE STRESS IN SEA ANEMONES

447

ume of fresh seawater and the anemones were allowed to
expand in the dark or in the light for about 30-45 min
before formazan production and soluble protein content
were measured.

The effect ofpO? on NBT redact ion in intact anemones

Hyperoxic conditions were established by bubbling
50 ml of FSW(33%»;pH 8.3) with a gas mixture contain-
ing 67% O:, 32.97% N2, and 0.03% CO: at 26°C for 60
minutes (Malta and Trench, 1991). The oxygen content
of the FSW bubbled with this gas mixture was deter-
mined using a microcathode oxygen electrode (Strath-
kelvin Instruments). The oxygen content of the hy-
peroxic medium was 13.88 ml O:/l (466 mmHg). This
value is comparable to the pO2 measured in tissues of
symbiotic anemones in the light (Snick and Brown,
1977; Dykensand Shick, 1982). To control for the effect
of bubbling, normoxia (4.75 ml O;/l, 160 mmHg) was
achieved by bubbling 50 ml of FSW with ambient air for
60 min.

Nine aposymbiotic A. pnlchella were allowed to accli-
mate to either hyperoxic or normoxic conditions for 1 h
in darkness with continuous bubbling in a 50-ml Erlen-
meyer flask. The FSW containing 10~4 A/NBT was pre-
bubbled with the special gas mixture or with air for
60 min to establish either hyperoxic or normoxic condi-
tions. A 1-h NET reduction experiment as described ear-
lier was then conducted with these animals in darkness.
The flasks were bubbled continuously with the special
gas mixture or with ambient air to maintain hyperoxic
or normoxic conditions during the experiment.

NBT reduction by freshly isolated algae

A sample of FIZ containing 106 cells was transferred
to a 10-ml test tube and centrifuged to pellet the cells.
The algae were then resuspended with 2.5 ml of FSW
containing 10~4 M NBT, preheated to the desired tem-
perature. Six replicate tubes were incubated under the
experimental conditions at either ambient or elevated
temperature either in darkness or in the light.

Enzyme analyses

The activities of superoxide dismutase (Cu-Zn SOD
and Mn SOD) and catalase (CAT) in symbiotic and apo-
symbiotic host tissues were determined using the meth-
ods of Lesser et al. (1990). The single modification was
that host tissue homogenates were prepared in ice-cold
0.0 1 A/ phosphate buffer containing 400 mA/ NaCl (pH
8.3) and 0.1 mA/ phenylmethyl sulfonyl fluoride
(PMSF).

OxYgen flux in intact anemones at ambient and elevated
temperature

Dark respiration rates of intact symbiotic anemones
were measured at time = 1 , 20, and 60 min at both 26°C
and 32°C using the method described by Hoegh-
Guldberg and Smith (1989) with two modifications.
First, the incubation chamber ( 1 5 ml) used for these
measurements was connected to a circulating water bath
set at either 26°C or 32°C. Second, between each oxygen
measurement the electrode was briefly removed from the
chamber and then replaced to prevent hypoxia within
the chamber.

The effect of acute temperature elevation on anemone
dark respiration was studied in an individual anemone
after a 1-h incubation period at 26°C. The FSW in the
chamber was immediately replaced with FSW preheated
to 32°C, and host dark respiration was then measured
over the next 60 min as described above. The FSW in the
incubation chamber was changed every 15 min with a
fresh volume of FSW preheated to 32°C. The anemone
was then sacrificed by homogenization in 2 ml of FSW.
The algae were separated from the animal tissue by cen-
trifugation (500 X g) and the supernatant containing al-
gal-free animal homogenate was then decanted and
saved for analysis of soluble protein. Dark respiration
rates for intact anemones are reported as microliters of
O: consumed per hour per milligram of soluble protein
(jul O:/h/mg soluble animal protein). The soluble protein
content of animal homogenates was determined using
the method of Bradford (1976).

To determine the effect of acute temperature elevation
on algal photosynthesis in hospitc, oxygen flux in intact
symbiotic anemones was measured in the light (300 /*E/
rrr/s) at ambient (26°C) and elevated temperature
(32°C). First, rates of dark respiration photosynthesis for
intact animals were measured at 26°C. The FSW used for
these initial measurements was then replaced by FSW
preheated to 32°C, and changes in the O: concentration
were monitored for another 60 min in the light after 1,
20, and 60 min. After 60 min, the light was turned off
and a final measurement of dark respiration was made.

Oxygen flux in freshly isolated algae at ambient and
elevated temperature

Algae (106 cells) from a single anemone were resus-
pended in 3 ml of FSW and placed in the incubation
chamber. Oxygen flux in freshly isolated algae was mea-
sured at both 26°C and 32°C over a 60-min incubation
period as described for intact animals. After dark respi-
ration and photosynthesis were measured at 26°C, 3 ml
of algal suspension in the incubation chamber was re-
moved and the algae were isolated by centrifugation (500
X g). The supernatant (FSW) was discarded and the algal

448

C. M. Nil AND L. MUSCATINE

pellet was resuspended in 3 ml of FSW preheated to
32°C. This suspension was then returned to the incuba-
tion chamber for oxygen flux measurements in the light
( 300 ME/m2/s) for another 60 min at 32°C. Algal dark
respiration was measured at the beginning and end of the
60-min period. Oxygen flux for freshly isolated algae is
expressed as n\ O2/h/ 106 cells. The concentration of F1Z
was determined using a hemacytometer (Spencer Bright-
line) and the mean of eight separate cell counts.

Statistical analyses

Nonparametric statistical methods were used for all
analyses because some data subsets within the same ex-
periment were heteroscedastic (Fma, test, (Sokal and
Rohlf. 1981) and Kolmogorov-Smirnoff analysis (Zar,
1984)). Homoscedastic data sets were also analyzed us-
ing parametric methods; in these cases, parametric and
nonparametric analyses led to identical conclusions.

Formazan production by anemones was evaluated for
the effects of temperature, light, and "condition" (sym-
biotic versus aposymbiotic) by a nonparametric three-
way analysis of variance (Zar, 1984). The effect of
DCMU on NBT reduction in intact symbiotic and apo-
symbiotic anemones was evaluated separately because
DCMU effects were investigated only in the light and not
in darkness. Including DCMU results would cause an
unbalanced analysis of the results obtained for the other
treatments (light/temperature/condition). Treatment
effects were evaluated at a significance level of 0.05.

Where significant treatment effects occurred, a non-
parametric multiple comparison test based on a modifi-
cation of the Kruskal-Wallis method (Zar, 1 984) was ap-
plied at the 0.05 significance level to identify individual
differences among data sets. The Mann-Whitney (/-test
was used to determine differences in antioxidant enzyme
activities of symbiotic and aposymbiotic animal tissue
homogenates and in NBT reduction by aposymbiotic
anemones subjected to normoxia or hvperoxia.

Results

Superoxide ion specifically reduces NBT

To determine if superoxide ions specifically duced
NBT, we measured the effect of SOD on NBT redu, tion
in algae-free tissue homogenates of A. pulchella. The
addition of 100, 250, or 500 U Cu-Zn SOD to reaction
mixtures inhibited NBT reduction by more than 95%
(Fig. 1).

Conversely, the addition of DDC, an SOD inhibitor,
to the reaction mixture increased NBT reduction by
nearly 100% in mixtures that contained no SOD and in
mixture-, that contained 100 U SOD (Fig. 2). Neither
DDC nor SOD caused NBT reduction in controls. These

results strongly suggest that superoxide ion is the primary
rcductant of NBT in these homogenates.

We also used the SOD-inhibitable reduction of acety-
lated cytochrome c as an independent method to verify
superoxide ion production in algae-free A. pulchella tis-
sue homogenates. The homogenates reduced acetylated
cytochrome c at rates comparable to positive control ex-
periments in which superoxide ions were generated en-
zymatically using purified xanthine oxidase and xan-
thine. Xanthine oxidase reduced cytochrome c at a rate
of 0.040 ± 0.004 absorbance units/min. A. pulchella tis-
sue homogenates reduced cytochrome c at a rate of 0.035
± 0.008 absorbance units/min. The addition of 10 U
SOD to homogenates decreased the rate of cytochrome c
reduction by 35%. (0.023 ± 0.006 absorbance units/min).
SOD added in excess of 1 0 U ( 20, 50, 1 00, or 200 U ) did
not cause any further inhibition of cytochrome c' reduc-
tion (0.025 ± 0.005 absorbance units/min). These results
show that superoxide ions are produced in algae-free A.
pulchella tissue homogenates, despite the presence of na-
tive SOD, which interferes with the assay. These results
also indicate that cytochrome c reduction by homoge-
nates may still occur by alternate mechanisms.

Some studies have reported that elevated />O: may
affect NBT reduction /// wm>(Auclairand Voisin, 1985,
Seidler and Van Noorden. 1 994). To determine the effect
of elevated />O: on NBT reduction by anemones in vivo.
we imposed hyperoxia on aposymbiotic A. pulchella in
darkness and then conducted an NBT reduction experi-
ment at ambient temperature (26°C). As there was no
significant difference in NBT reduction between apo-

100-

80-

60-

<u

H

pa

40-

20 -

control 50 100 250 500
SOD (U)

Figure 1. The effect of SOD concentration on NBT reduction in
algae-tree AifiUiMa /nilclu'lla tissue homogenates. Six anemones were
homogenized individually. From each homogenate a sample was taken
lor N BT reduction at each SOD concentration. Each bar represents the
mean ± SD of the six samples.

OXIDATIVE STRESS IN SEA ANEMONES

449

I

o
o

E

60

B

JS

o

-100

+SOD/-DDC +SOD/+DDC -SOD/+DDC

treatment

Figure 2. The effect of I m.U DDC on NBT reduction by algae-free
Aiplaxia pulchella tissue homogenates. Six anemones were homoge-
nized individually. From each homogenate a sample was taken for
NBT reduction under the following conditions: +SOD/— DDC;
+SOD/ + DCC; and -SOD/+DDC. Controls contained neither DDC
nor SOD. Each bar represents the mean ± SD of the percent increase
or decrease in NBT reduction compared to controls determined for the
six samples.

o
o.
u

30

E

~5
c

50"

Q 26C

40-

• 32C

I

30-

•r

i

20-

I

io-

A 1

I 1

1

; i

'

20 40

time (minutes)

60

Figure*!. Timecourseof NBT reduction by symbiotic Aiplaaia pul-
chclla at 26° and 32°C. Two groups of 1 2 anemones were selected for a
60-min NBT reduction experiment at either ambient (26°C) or elevated
(32°C) temperature. Three anemones at each temperature were sacri-
ficed 1, 20, 40 and 60 min after starting the experiment, and the
amount of NBT formazan produced by each anemone was measured.
Each point represents the mean ± SD for three anemones per time
point.

symbiotic anemones under hyperoxia or normoxia
(Mann- Whitney U. P> 0.10: Fig. 3), it appears that ele-
vated /O: does not influence NBT reduction by these
organisms.

Acuic thermal stress causes oxidative stress in intact
symbiotic anemones

To determine if acute thermal stress causes oxidative
stress in intact symbiotic anemones, we measured the

c

•5

e

o.

JU

.0

oo
E

o

16

14-
12-

10-

8-
6-

4-

1

I

normoxia hyperoxia

Figure 3. The effect of hyperoxia on NBT reduction by aposymbi-
otic Aiplama pulchella. Groups of three anemones were acclimated to
either normoxia or hyperoxia for 60 mm in darkness at 26°C. A 60-min
NBT reduction experiment using these anemones was then conducted
under normoxia or hyperoxia. The amount of NBT formazan pro-
duced by each anemone was then measured. Each bar represents the
mean ± SD for nine anemones per treatment.

production of NBT formazan in the animal tissues of
anemones exposed to ambient (26°C) and elevated tem-
perature (32°C). A 60-min incubation was sufficient to
resolve differences in NBT reduction between control
anemones and those exposed to acute thermal stress
(Fig. 4).

Temperature had a significant effect on formazan pro-
duction by symbiotic anemones (ANOVA, P < 0.001).
Symbiotic A. pulchella produced significantly more for-
mazan at 32°C in both darkness and light, and in the
presence of DCMU than at 26°C (Fig. 5). Neither light
(ANOVA, P > 0.25) nor DCMU (ANOVA, P > 0.25)
had a significant effect on formazan production in intact
anemones at either ambient or elevated temperature. Ex-
cept for the significant interaction between light and
temperature (ANOVA, P < 0.05), there were no signifi-
cant interactions between temperature, light, and "con-
dition" (ANOVA, all P> 0.10) in either intact symbiotic
or aposymbiotic anemones. These results show that
acute thermal stress increases superoxide ion production
and that the increase may be independent of the presence
of algae.

Is oxidative stress an "animal" rather than an "algal"
phenomenon?

To further investigate the observation that symbiotic
algae may be sufficient but not necessary for oxidative

450

C. M. Nil AND L. MUSCATINE

00

I symbiotic. 0 uc
D symbiotic. 300 nE

symbiotic, 300iiE. DCMU

26 32

temperature (°C)

Figure 5. Superoxide production by symbiotic Aiptaxia pulchella
at ambient (26"C) and elevated (32°C) temperature in darkness, in light,
and in light in the presence of 0.5 mM DCMU. Groups of six anemones
were used in each treatment. A 60-min NET reduction experiment was
then conducted under each defined condition, after which the amount
of formazan produced by each anemone was measured. Each bar rep-
resents the mean ± SD for six anemones per treatment.

stress to occur in these anemones, aposymbiotic A. pul-
chella were exposed to FSW containing 1CT4 M NBT in
both darkness and light at both ambient and elevated
temperature. Figure 6 shows that temperature had a sig-
nificant effect on formazan production by intact apo-
symbiotic anemones (ANOVA, P < 0.001 ): in darkness
and light these anemones produced significantly more
formazan at elevated temperature (Kruskal-Wallis, P
< 0.001). Neither light nor DCMU had a significant
effect in aposymbiotic anemones (ANOVA, P > 0.50
and P > 0.25, respectively). Interestingly, at ambient
temperature, formazan production in darkness was sig-
nificantly higher by aposymbiotic anemones than by
symbiotic anemones in darkness (Kruskal-Wallis, P <
0.01).

Do symbiotic algae release superoxide ions'.'

Because NBT does not penetrate intact algae and re-
mains in the external medium (Nii, unpubl. obs.), it can
be used to measure the possible release of superoxide ion
by FIZ.

To determine if symbiotic algae release superoxide
ions, FIZ from A. pulchella were incubated in FSW con-
taining 10~4A/ NBT under the following incubation
conditions: ambient and elevated temperature; darkness
or light; and with or without DCMU. If superoxide ions
are released by the algae, these radicals should then re-
duce NBT present in the external medium. NBT can de-
tect the presence of superoxide ions in nanomolar con-
centration (>5 nmol; Auclair and Voisin, 1985). How-
ever, torn: >7.an was not detected in the external medium
under any >i he incubation conditions (data not shown).

If these algae released superoxide ions under these exper-
imental conditions, the amount was less than 5 nmol.

SOD and CAT activity in symbiotic and aposymbiotic
A. pulchella

To determine if differences in NBT reduction were re-
lated to the ability of the host to detoxify potential oxi-
dants, the specific activities of Cu-Zn SOD, Mn SOD,
and CAT were measured in animal homogenates pre-
pared from symbiotic and aposymbiotic A. pulchella.
The specific activities of Cu-Zn SOD (Mann-Whitney U;
P < 0.05, Fig. 7), Mn SOD (Mann-Whitney U. P < 0.05,
Fig. 7), and CAT (Mann-Whitney U, P < 0.05, Fig. 8)
were significantly higher in aposymbiotic anemones
than in symbiotic ones. The specific activity of CAT was
an order of magnitude higher in aposymbiotic A. pul-
chella. These results suggest that aposymbiotic anemo-
nes may be subject to chronic oxidative stress.

The effect of acute thermal stress on oxygen flux by
intact anemones and FIZ

In a 60-min incubation, acute thermal stress immedi-
ately increased the dark respiration rate of intact symbi-
otic anemones more than twofold (Fig. 9). Further, the
time course of the rate increase (Qw = 14.1) resembled
the apparent time course of NBT reduction (Qw = 34.2)
by anemones at elevated temperature (Fig. 4).

Net oxygen flux in intact anemones was relatively con-
stant in the light at ambient temperature (26°C; Table I).
However, within 20 min of the initiation of acute ther-

300

Xi

3

I

op

temperature (°C)

Figure 6. Superoxide production by aposymbiotic Aiptaxia />»/-
chi'lla at ambient (26°C) and elevated (32°C) temperature in darkness,
in light, and in light in the presence of 0.5 mA/ DCMU. Groups of
six anemones were used in each treatment. A 60-min NBT reduction
experiment was then conducted under each defined condition, after
which the amount of formazan produced by each anemone was mea-
sured. Each bar represents the mean ± SD for six anemones per treat-
ment.

• aposymbiotic. OnE

D aposymbiotic, 300(iE

Q aposymbiotic. 300>iE. DCMU

OX1DAT1VE STRESS IN SEA ANEMONES

451

.£ 350

aposymbiotic

symbiotic

Figure 7. Specific activities (U/mg soluble protein) ofCu-Zn SOD
and Mn SOD in symbiotic and aposymbiotic Aifiiasia pulchella. Six
anemones were homogenized individually. From each homogenate,
samples were taken for SOD measurement. Each bar represents the
mean ± SD of the six homogenates.

mal stress, net oxygen production by intact anemones
ceased (32°C; Table I). After 60 min, intact anemones
began to consume oxygen in the light at rates compara-
ble to their dark respiration rates at elevated temperature
(Table I).

Net oxygen flux in FIZ also decreased dramatically
within 20 min after exposure to elevated temperature
(Table I). This decrease was accompanied by an increase
in dark respiration. There was considerable variability in
the mean response of FIZ to elevated temperature. In

c 6000 -

1

a- 5000 -
u

12
•f 4000 -

tn

T

00

- 3000 -

•f ' 2000 '

^ 1000 -

1

r=_1

GO

aposymbiotic symbiotic

Figure 8. Specific activity (U/mg soluble protein) of CAT in sym-
biotic and aposymbiotic Aipla\ia pulchella. Six anemones were homog-
enized individually. From each homogenate. samples were taken for
CAT measurement. Each bar represents the mean ± SD of the six ho-
mogenates.

o
S.

300-

-I 250

ao

<N

O

200-
150-

100-

50-
0

32°C

0

20 40

time (mm)

60

Figure 9. Time course of the dark respiration response of intact
anemones to acute thermal stress. Dark respiration by an individual
anemone was measured first at 26°C over 60 min (at 1.20. and 60 min).
The 26°C seawater was then immediately replaced with seawater pre-
heated to 32°C. Dark respiration was then measured for another 60 min
at 32°C (at 1 , 20. and 60 min). Each point represents the mean ± SEM
for three anemones.

three of the five experiments performed, net oxygen con-
sumption in the light began within 20 min of exposing
the FIZ to elevated temperature. Although net oxygen
production was observed in the two remaining experi-
ments, the rates were <20% of the net flux at ambient
temperature. Further, in two of the five experiments,
dark respiration rates increased more than fivefold at el-
evated temperature. These results suggest that the mo-
lecular oxygen produced by algae during acute thermal
stress is mostly consumed by the algae themselves.

Discussion

Acute thermal stress causes oxidative stress in intact
anemones

Our results show that superoxide ions are produced
within symbiotic and aposymbiotic specimens of Aip-
tasia pulchella in response to acute thermal stress. Inter-
estingly, production of superoxide ions at elevated tem-
perature was not affected by exposing anemones to light
and DCMU. Moreover, net oxygen production in the
light ceases when either intact anemones or FIZ are sub-
jected to acute thermal stress. These results strongly sug-
gest that, in response to acute thermal stress, oxidative
stress in A pulchella is largely an animal rather than an
algal phenomenon. Symbiotic algae are sufficient but not
necessary for the manifestation of oxidative stress in host
tissues at elevated temperature.

452

C. M. Nil AND L. MUSCATINE
Table I

Ncl <>vr?t'H lhi\ fur intact Aiptasia pulchella ami freshly isolated Symbiodinium pulchrorum. in light (300 iiE/rrf/s) and darkness, exposed lit
ambient (26°C) and aeinely elevated <32°C) iciniwrulure

Net oxygen flux* (mean ± 1 SEM)

Temperature

Time after

Intact anemones'

Freshly isolated algae:

Process (°C)

exposure

(tt\ O2/h/mg soluble protein )

(MlO2/h/106 cells)

Photosynthesis 26°

58.80 ± 13.95

8.49 ±0.59

32°

0 min

12.75 + 2.10

9.I4±5.30

20min

-10.95 ±4.50

2.63 ±3.66

60 mm

-38.85 + 9.60

2.02 ± 2.70

Dark respiration 26°

-14.70 ±4. 80

-5.55 + 0.96

32°

-39.30 ±8.40

-8.68 + 2.50

*A negative value indicates net oxygen consumption.
'A' =3.
:A' = 5.

Superoxide production in animal tissues

Despite the caveats regarding tissue homogenates (Dy-
kens el ai. 1992; Verde and McCloskey, 1996), we mea-
sured superoxide production in A. pulchella tissue ho-
mogenates by using two independent methods (SOD-in-
hibitable NBT and cytochrome t reduction), and we find
evidence to support the interpretation that superoxide
ion is the primary reductant of NBT in these anemones.
The inhibition of NBT reduction by SOD and increase
in NBT reduction in the presence of the inhibitor, DDC,
is consistent with this interpretation. Further, because A.
pulchella tissue itself exhibits SOD activity, both meth-
ods (NBT or cytochrome c reduction) may actually un-
derestimate the amount of superoxide ion produced ei-
ther in homogenates or in intact animals due to SOD
interference with each assay. We also observed that the
addition of the SOD did not completely inhibit cyto-
chrome c reduction. This result is not entirely surprising
considering the complex, dynamic nature of tissue ho-
mogenates. Although acetylated cytochrome c has been
shown to resist reduction specifically by mitochondrial
cytochrome c reductases (Azzi el at., 1975), other reduc-
tases released during homogenization may still reduce
cytochrome c under the assay conditions.

The effect of molecular oxygen on NBT reduction in
intact anemones

The reduction in photosynthetic oxygen production
by intact symbiotic anemones and by FIZ at elevated
temperature argues against the possible confounding
el.' >s of molecular oxygen on NBT reduction. Molecu-
lar o, --en is thought to compete with NBT for reduc-
tants (i.e., electrons; see Halliwell and Gutteridge, 1987,
for a discussion of this hypothetical phenomenon).

which may result in a decreased and therefore inaccurate
estimation of NBT reduction by superoxide ions. How-
ever, the evidence for the existence of such competition
is equivocal (Seidler, 1991). In this study, we demon-
strate empirically that elevated pO2 does not affect NBT
reduction in aposymbiotic A. pulchella during imposed
hyperoxia (Fig. 3). We attribute the lack of hyperoxic en-
hancement in these aposymbiotic anemones primarily
to their increased antioxidant enzyme activity (com-
pared to symbiotic anemones).

We noticed that the magnitude of NBT reduction in
the aposymbiotic anemones used in the hyperoxia exper-
iment was lower than that in other experiments (Figs. 3
and 6). This is probably due in part to differences in the
antioxidant enzyme activities present in aposymbiotic A.
pulchella, as well as to differences in the soluble protein
content of the aposymbiotic specimens used in the
different experiments. The animals used in the hyperoxia
experiment contained similar amounts of protein, but
this amount was up to 10 times higher than that in ani-
mals used in other experiments. This difference may be
due in part to changes in the maintenance regime for
aposymbiotic anemones (i.e., feeding on Anemia adults
rather than nauplius larvae), as well as to poorly un-
derstood variation in these artificially generated aposym-
biotic animals. Nevertheless, under these conditions, tis-
sue-specific NBT reduction by aposymbiotic anemones
was three times that of symbiotic anemones at ambient
temperature.

Symbiotic algae do not release superoxide

The lack of detectable NBT reduction by FIZ incu-
bated in FSW containing NBT preheated to the identical
temperatures used in whole-animal experiments (26°

OXIDATIVE STRESS IN SEA ANEMONES

453

and 32°C) indicates that, at least in vitro. little(<5 nmol),
if any. superoxide ion is released by the algae to the me-
dium during acute thermal stress. Although it is possible
that other oxidant species such as hydrogen peroxide
(H;O;) may be released from the algae during environ-
mental stress (Lesser et al.. 1990. Lesser, 1996a), three
observations argue against this conjecture: ( 1 ) algae ex-
hibit robust antioxidant activity ( Lesser and Shick, 1989;
Malta and Trench. 1991); (2) transport of superoxide
ions is relatively slow (Fisher. 1987); and (3) oxidant spe-
cies are highly reactive (Dykens et a/.. 1992). Tytler and
Trench (1988) did not detect the release of H:O: from
cultured symbionts in vitro, although H2O2 can cross bi-
ological membranes more readily than superoxide ions
(cf. Lesser. 1996). Our short-term, acute stress experi-
ments with S. piilchrorwn do not exclude the possibility
that exposure to chronic stress may cause symbiotic al-
gae to release oxidants directly (Lesser et al., 1990). How-
ever, there is yet no published evidence that oxidants are
released directly by symbiotic algae under any denned
condition (Lesser, 1996).

Does hyperoxia contribute to oxidative stress in intact
anemones during thermal stress?

Hoegh-Guldberg and Smith (1989) observed greatly
reduced net oxygen production in intact corals exposed
to chronic temperature stress, and Iglesias-Prieto et al.
(1992) showed that in cultured Symbiodinium, net oxy-
gen flux decreases precipitously in vitro at nonlethal tem-
peratures exceeding 3 PC. In the present study, at ele-
vated temperature, net oxygen production in anemones
and FIZ ceases, probably because of impairment of pho-
tosynthetic electron transport at or near the reaction cen-
ter of PSII (Warner el al.. 1996). As photosynthetic oxy-
gen production is greatly reduced, any oxidative stress
attributed directly to hyperoxia in symbiotic anemone
tissues (Dykens and Shick, 1982) is also greatly reduced.
Our results show that, whereas symbiotic algae may con-
tribute to hyperoxia. they are not required for the evoca-
tion of oxidative stress in these anemones at elevated
temperature.

On the other hand, it remains unclear from our data
whether photosynthesis has actually ceased at elevated
temperature as it is not possible to measure oxygen flux
due to photosynthesis and respiration in the light simul-
taneously (Table I). Net oxygen flux could be reduced, in
part, by the increase in dark respiration of the host (Fig.
9). That is, the oxygen produced within the algae could
be consumed by the algae before it diffuses into host tis-
sues. However, a very low, but positive, net oxygen flux
was sometimes observed in FIZ during exposure to ele-
vated temperature (Table I). This observation suggests
that oxygen production may still occur within the algae

at elevated temperature, but may be largely masked by
the increase in algal respiration.

At ambient temperature, the slightly elevated produc-
tion of superoxide ions by symbiotic anemones in the
light compared with those in darkness is probably due to
hyperoxia generated in the host cytoplasm as a result of
algal photosynthesis (Shick and Brown, 1977; Dykens
and Shick. 1982). However, hyperoxia cannot account
for oxidative stress in aposymbiotic anemones during
acute thermal stress in darkness or light. These results
suggest that superoxide ion production may be limited
by the supply of electrons or other excitatory inputs such
as photodynamic effects (Valenzano and Pooler, 1987;
Dykens et al.. 1992; Lesser, 1996) that can participate in
univalent reduction of available molecular oxygen,
rather than by the supply of molecular oxygen itself.

Do aposymbiotic anemones experience chronic
oxidative stress?

We observed that superoxide ion production by apo-
symbiotic A. pulchella in darkness at ambient tempera-
ture was significantly higher than superoxide ion produc-
tion by symbiotic anemones under the same conditions
(Figs. 5 and 6). To account for this observation, we as-
sessed the activities of the antioxidant enzymes superox-
ide dismutase (SOD) and catalase (CAT) in aposymbio-
tic versus symbiotic A. pulchella. We found significant
differences in the specific activities of Cu-Zn SOD, Mn
SOD. and CAT between the two forms of the anemone.
The specific activity of Mn SOD was fivefold higher in
aposymbiotic anemones. The MnSOD activity in apo-
symbiotic A. pulchella is much higher than MnSOD ac-
tivity reported for other eukaryotic organisms (Asada et
al., 1980), including symbiotic dinoflagellates (Lesser
and Shick, 1989; Matta and Trench, 1991). The CAT
activities measured using a spectrophotometric method
were very similar to CAT activities measured previously
using a polarigraphic method (Tytler and Trench, 1 988).
Like Tytler and Trench (1988), we also found that the
specific activity of CAT was an order of magnitude
higher in aposymbiotic specimens.

At present, we find it difficult to explain the increased
MnSOD activity observed in aposymbiotic A. pulchella.
The increase may represent one of potentially many, and
as yet uncharacterized, physiological changes which oc-
cur in A. pulchella when it is rendered aposymbiotic due
to cold shock. Overexpression of MnSOD in aposymbi-
otic individuals may be a response to increased hetero-
trophy or may reflect an increase in mitochondrial den-
sity. The former explanation is difficult to reconcile as
Asada et al. (1977) found that Euglena graci/is raised
phototrophically has almost three times the SOD activity
of E. gracilis raised heterotrophically. The latter expla-

454

C. M. Nil AND L. MUSCATINE

nation also seems unlikely because the protein-specific
dark respiration rates for symbiotic and aposymbiotic A.
pulchella are very similar (Nii, unpubl.). We infer from
the increased MnSOD and CAT activities in aposymbi-
otic A. pulchella that these anemones may manifest
chronic (oxidative) stress, although the origin of this con-
dition is still unknown.

The origin of increased superoxide production at
elevated temperature

The dark respiration rate of symbiotic A. pulchella tri-
ples under acute thermal stress (Fig. 9). The time course
of host dark respiration over 60 min (Fig. 9) resembles
the time course of formazan production by anemones at
elevated temperature (Fig. 4). The Qw values calculated
for the respective processes are very different, but both
reflect the acute sensitivity of each process to elevated
temperature. These observations suggest that increased
respiration by the animal host may contribute to an in-
crease in the rate of electron leakage from mitochondrial
electron transport and may account, in part, for the in-
creased production of superoxide ions (Burdon el ai,
1990; Richterf /<//.. 1995;Garcia-Ruiz etal.. 1995; Van-
Fleteren and De Veese, 1996).

Our results also suggest that in these anemones, super-
oxide is produced primarily within the host. Animal cells
have many sites of superoxide generation (Bendich,
1989). Production of superoxide ion has been localized
between complexes I and III of the mitochondrial elec-
tron respiratory chain at the sites of NADH dehydroge-
nase (Krishnamoorthy and Hinkle. 1988; Turrens and
Boveris, 1980) and ubiquinone(Nohl and Jordan, 1984).
Other potential sites for active oxygen production in the
host cell include the respiratory chain enzymes NADH-
ubiquinone reductase and NADH-cytochrome b~, reduc-
tase, xanthine oxidase, and the lipid metabolism en-
zymes NADPH-cytochrome P45<> reductase and 6-9-de-
saturase( Bendich, 1989).

How are oxidative stress ami cnidarian bleaching
related?

Direct comparison between our results using NET and
other studies of oxidative stress in symbiotic cnidarians
is difficult, largely because of differences in the species;
contrast between intact animals, FIZ and cultured algae;
and dissimilarities in the methods used in recent studies
attempted in vivo. On the basis of enhanced antioxidant
enzyme activities in symbiont and host. Lesser et ai
(1990) concluded that elevated temperature caused oxi-
dative stress and bleaching in Palythoa carihaeorum. Dy-
kens ' ( 1992), using a methane sulfinic acid bioassay
for h\ >xyl radicals in vivo, found greater oxidant pro-
duction within the symbionts (5. calijomiiim) of An-

thopk'itra elegantissima than in host tissues. They also
observed enhanced hydroxyl radical production in S. ca-
lifornium due to chronic UV radiation exposure, but did
not investigate the effects of elevated temperature in ei-
ther the symbiont or the host. More recently. Lesser
( 1996) measured increased production of superoxide ion
and hydrogen peroxide in cultured algae (S. bermudense
from Aiptasia pallida) due to chronic elevated tempera-
ture and UV radiation; specific fluorochromes were used
for each oxidant species. Using these observations as a
point of departure, we now advance the hypothesis that
oxidative stress may also represent an animal response
to acute thermal stress, and we suggest that the symbiont
may not necessarily be required for oxidative stress to
occur under these conditions.

The link between oxidative stress and cnidarian
bleaching is still unknown. The production of active ox-
ygen species can lead to a number of damaging effects,
including the peroxidation of membrane lipids (Gut-
teridge, 1987) or the direct disruption of cell adhesion
molecules by oxygen radicals. Such damage may then
lead to release of host cells (Gates et ai. 1992). As the
host animal alone may manifest oxidative stress — that
is, without the contribution of photosynthetic oxygen by
symbiotic algae — oxidative stress originating in host
cells may explain the correlation observed between high
rates of colony respiration and thermal bleaching in reef
corals (Jokiel and Coles. 1990; P. Edmunds, in prep.).
The inherent susceptibility of host cells themselves to
oxidative stress may be an important mechanistic link
between temperature stress and cnidarian bleaching.

Acknowledgments

We thank Drs. J. Dykens, P. Edmunds, R. Gates, M.
Lesser. J. M. Shick, R. K. Trench, and three anonymous
reviewers for critically evaluating earlier versions of the
manuscript, and D. Nii for editorial assistance. This
work was supported by NSF grant OCE # 9 1 1 5834.

Literature Cited

Asada, K., S. Kancmatsu, and K. I'chida. 1977. Superoxide dismu-
tases in photosynthetic organisms: absence of the cuprozinc enzyme
in eukaryotic algae. Arch Biochem Biophys. 179:243-256.

Asada, K., S. Kanematsu, S. Oka, and T. Hayakawa. 1980. Phylo-
genic distribution of superoxide dismutase in organisms and in cell
organelles. Pp. 136-153 in Chemical and Biochemical Aspects oj
Superoxide and Superoxide Dismuiase. J.V. Bannister and H.A.O.
Hill. eds. Elsevier/North-Holland, Amsterdam.

Auclair, C'., and E. Voisin. 1985. Nitroblue tetrazolium reduction.
Pp. 123-132 in CRC Handbook of Methods for Oxygen Radical
Research. R.A. Greenwald, ed. CRC Press, Boca Raton. FL.

Azzi, A., C. Montecucco, and C. Richter. 1975. The use of acetylated
ferricytochrome c for the detection of superoxide radicals produced
in biological membranes. Biochem. Biophys. Res. Comnuin 65:
597-603.

OXIDATIVE STRESS IN SEA ANEMONES

455

Banaszak, A. I ., R. Iglesias-Prieto. and R. K. Trench. 1993. Seripp-
sii'llu vele/laesp. nov.. Peridiniales and Gloedinium viscum sp. nov.,
Phytodiniales. dinoflagellate symhionts of two hydrozoans (Cnida-
ria).//)yj.vro/.29:517-528.

Bendich. A. 1989. Antioxidant nutrients and immune functions: an
introduction. Pp. 1-12 in Anlioxidani Nutrients and Immune Func-
tions, A. Bendich, M. Phillips, and R. Tengerdy. eds. Pergamon
Press. New York.

Boveris, A., and E. Cardenas. 1982. Production of superoxide radi-
cals and hydrogen peroxide in mitochondria. Pp. 15-30 in Super-
oxide Disnuiiasc. L.W. Oberly, ed. CRC Press. Boca Raton, FL.

Bradford, M. M. 1976. A rapid and sensitive method for the quanti-
tation of microgram quantities of protein utilising the principle of
protein-dye binding. Anal. Biochcm. 72: 248-254.

Brown, B. E., M. D. A. Le Tissier, and J. C. Bythell. 1995. Mecha-
nisms of bleaching deduced from histological studies of reef corals
sampled during a natural bleaching event. Mar Bin/. 122:655-66.

Burdon, R. H., V. Gill, and C. Rice Evans. 1990. Active oxygen and
heat shock protein induction. Pp. 19-25 in Stress Proteins. M.I.
Schlesinger. M.G. Santoro. and E. Garaci, eds. Springer-Verlag.
Heidelberg.

Chacon, E., and D. Acosta. 1991. Mitochondria! regulation of super-
oxide by Ca:*: an alternate mechanism for the cardiotoxicity of
doxorubicin. Toxicol. Appl. Pluirmacol 107: I 17-128.

Coles, S. L., and P. L. Jokiel. 1977. Effects of temperature on photo-
synthesis and respiration in hermatypic corals. Mar. Bin/. 43: 209-
216.

Dykens, J. A. 1984. Enzymic defenses against oxygen toxicity in ma-
rine cnidarians containing endosymbiotic algae. Mar, Biol. Lell. 5:
291-301.

Dykens, J. A., and J. M. Shick. 1982. Oxygen production by endo-
symbionts control superoxide dismutase activity in their animal
host, \aniiv297: 579-580.

Dykens, J. A., and J. M. Shick. 1984. Photobiology of the symbiotic
sea anemone. Anlliopleura eleganlissima. defenses against photo-
dynamic effects, and seasonal photoacclimitization. Bin/ Bull 167:
693-697.

Dykens. J. A., J. M. Shick, C. Benoit, G. R. Buettner, and G. \V. Win-
ston. 1992. Oxygen radical production in the sea anemone .4/1-
ihnpk'iira elegantissima and its endosymbiotic algae. J Exp Bio/.
168:219-241.

Enger, P. A., and I . XV. Kensler. 1985. Effects of a biomimetic super-
oxide dismutase on complete and multistage carcinoma in mouse
skin. Careinngenesis 6: I 167-1 172.

Fisher, A. B. 1987. Intracellular production of oxygen-derived free
radicals. Pp. 34-39 in Oxygen Radicals and Tissue Inniry. B. Halli-
well. ed. Federation of American Studies for Experimental Biology.
Bethesda. MD.

Fisk, T. A., and T. J. Done. 1985. Taxonomic and bathymetric pat-
terns of bleaching in corals. Myrmidon Reef (Queensland). Proc.
Filth Intl Coral Reel Congr. 6: 149-154.

Flohe. I,., and F. Otting. 1984. Superoxide dismutase assays. Pp. 93-
104 in Mel hod), in Enzymology. L. Packer, ed. Academic Press.
New York.

Garcia-Ruiz, C., A. Colell, A. Morales, N. Kaplowitz, and J.C. Fernan-
dez-Checa. 1995. Role of oxidative stress generated from the mi-
tochondrial electron transport chain and molecular glutathione sta-
tus in loss of mitochondria! function and activation of transcription
factor nuclear factor-kappa B: studies with isolated mitochondria
and rat hepatocytes. Mol Pharmacol. 48: 825-834.

Gates, R. D., G. Baghdasarian. and L. Muscatine. 1992. Tempera-
ture stress causes host cell detachment in symbiotic cnidarians: im-
plications for coral bleaching. Biol. Bull. 182: 324-332.

Glynn, P. \V. 1990. Coral mortalitv and disturbances to coral reefs in

the tropical eastern Pacific. Pp. 55-126 in Global Ecological Conse-
quences nl ihc 1982-83 El Nino-Southern Oscillation. P.W. Glynn.
ed. Elsevier. Amsterdam.

Glynn, P. XV., and L. D'Croz. 1990. Experimental evidence for high
temperature stress as the cause of El-Nino coincident coral mortal-
ity. Coral Reels 8: 181-192.

Gutteridge, J. M. C. 1987. Lipid peroxidation: some problems and
concepts. Pp. 9- 1 9 in Oxygen Radicals and Tissue Injury, B. Halli-
well, ed. Federation of American Societies tor Experimental Biol-
ogy. Bethesda. MD.

Halliwell, B., and J. M. C. Gutteridge. 1987. Free Radicals in liiol-
ogy and Medicine, 2ndEdition. Blackwell, London, p. 75.

Hoegh-Guldberg, O., and G.J. Smith. 1989. The effect of sudden
changes in temperature, light and salinity on population density
and export of zooxanthellae from the reef corals Serialopora hysinx
and Slylophorapistillaia. J Exp. Mar Biol. Ecol. 129: 279-303.

Iglesias-Prieto. R., J. L. Matta, XV. A. Robins, and R. K. Trench. 1992.
Photosynthetic response to elevated temperature in the symbiotic
dinoflagellate Symbiodinium microadriaticum in culture. Proc.
Nail. Acad. Sci. i'SA 89: 10302-10305.

Kleppel, G. S., R. E. Dodge, and C. J. Reese. 1989. Changes in pig-
mentation associated with the bleaching of stony corals. Limnol.
Oceangr.34: 1331-1335.

Krishnamoorthy, G., and P. C. 1 1 ink U 1988. Studies on the electron
transfer pathway, topography of iron sulfur centers and sites of cou-
pling in NADH-Q oxido-reductase. J. Biol. Client. 263: 566-575.

Lesser, M. P. 1996. Elevated temperature and ultraviolet radiation
cause oxidative stress and inhibit photosynthesis in symbiotic dino-
flagellates. Limnol Oceangr. 41: 27 1-283.

Lesser, M.P., and J.M. Shick. 1989. Effects of irradiance and ultra-
violet radiation on photoadaptation in the zooxanthellae of Aip-
lasia pallida: primary production, photomhibition and enzymatic
defenses against oxygen toxicity. Mar Biol. 102:243-255.

Lesser, M. P., XV. R. Stochaj, D. XV. Tapley, and J. M. Shick. 1990.
Bleaching in coral reef anthozoans: effects ot irradiance. ultraviolet
radiation and temperature on the activities of protective enzymes
against active oxygen. Coral Reels 8: 225-232.

Matta, J. L., and R. K. Trench. 1991. The enzymatic response of the
symbiotic dinoflagellate Symbiodinium microadriaticum (Freun-
denthal) to growth under varied oxygen tensions. Symbiosis 11:31-
45.

Nohl, H., and XV. Jordan. 1984. The biochemical role ot ubiquinone
and ubiqumone-derivatives in the generation of hydroxyl radicals
from hydrogen peroxide. Pp. 155-160 in Oxygen Radicals in
Chemistry and Biology. W. Bors. M. Saran. and D. Tail, eds. Walter
deGruyter. Berlin.

Porter, J. XV., XV. K. Fitt, H. J. Spero, C. S. Rogers, and M. XV. XX hilc.
1989. Bleaching in reef corals: physiological and stable isotopic
responses. Proc. .\a/l Acad Sci. L'SA 86: 9342-9346.

Richter, C., V. Gogvadze, R. l.affranchi, R. Schalpbach, M. Schweizer,
M. Suter, P. XX alter, and M. Yaffee. 1995. Oxidants in mitochon-
dria: from physiology to diseases. Bioehim. Biop/iys. Ada 1271:67-
74.

Seidler, E. 1991. The tetrazolium-formazan system: design and his-
tochemistry. Progr. Hi.slochem Cvtochem. 24: 1-86.

Seidler, K., and C. J. F. Van Noorden. 1994. On the mechanism of
tetrazolium salts with special reference to the involvement of tetra-
zolium radicals. Ada Ilistoclicm 96: 43-49.

Shick, J. M., and XV. I. Brown. 1977. Zooxanthellae-produced O:
promotes sea anemone expansion and eliminates oxygen debt un-
der environmental hypoxia./ E\p. Zool. 201: 149-155.

Shick, J. M., M. P. Lesser, and VV. R. Stochaj. 1991. Ultraviolet ra-
diation and photooxidative stress in zooxanthellate anthozoa: the

456

C. M. Nil AND L. MUSCATINE

sea anemone Phyllodisciu \emoni and the octocoral Clavularia sp.
Symhiosis 10: 145-173.

Shick, J.M., M.P. Lesser, W. C. Dunlap, \V. R. Stochaj, B. E.
Chalker, and J. Wu Won. 1995. Depth-dependent responses to
solar ultraviolet radiation and oxidative stress in the zooxanthellate
cora\ Acropora microphthalma Mar Bin! 122:41-51.

Sokal, R. R., and K. J. Rohlf. 1981. Biometry. 2ml edition W.H.
Freeman. New York. 859 pp.

Steen, R. G., and L. Muscatine. 1987. Low temperature evokes rapid
exocytosis of symbiotic algae by a sea anemone. Bin/. Bull. 172:
246-263.

Szmant, A. M., and N. J. Gassman. 1990. The effects of prolonged
bleaching on the tissue biomass and reproduction of the reef coral
Mnnlastrea annularis. Coral Reeh 8: 2 1 7-224.

Thorn, S. M., R. W. Horobin, E. Seidler, and M. R. Barer. 1993. Fac-
tors affecting the selection and use of tetrazolium salts as cytochem-
ical indicators of microbial viability and activity. J. Appl Buclenol
74: 433-443.

Turrens. J. F.. and A. Bo»eris. 1980. Generation of superoxide anion

by the NADH dehydrogenase of bovine heart mitochondria. Bio-
clieiii.J. 191:501-507.

Tytler, E. M., and R. K. Trench. 1988. Catalase activities in cell-
free preparations of various invertebrate hosts. Symhinsis 5: 247-
254.

Valenzano, D. P., and J. P. Pooler. 1987. Pholodynamic action. Bio-
SciciK r 37: 270-276.

\ anKleteren, J. R., and A. De\'reese. 1996. Rate of aerobic metabo-
lism and superoxide rate production in the nematode Cacnorliiih-
iltn-, ek'saHx. J. E.\r tool 274: 93-100.

Verde, E. A., and L. R. McCloskey. 1996. Carbon budget studies of
symbiotic cnidarian anemones — evidence in support of some as-
sumptions. J. £.v/>. Mar Bail. Ecol. 195: 161-171.

\\arner, M. E., V\ . K. Fitt, and G. W. Schmidt. 1996. The effects of
elevated temperature on the photosynthetic efficiency of zooxan-
thellae ;/; ln>.\i'itt' from four different species of reel coral: a novel
approach. 1'Uinl Cell ami linvirnn. 19: 291-299.

/ar, J. H. 1984. Biostatistical Analysis, 2mt edition Prentice-Hall.
Englewood Cliffs, N.J. 718 pp.

INDEX

A molecular model for mechanosensation in Caenorhabdilh clc^nn\.
125

Acclimation, .109

Acclimitization. 309

Acoustic telemetry, 203

Actin. 181. 183

Actin-dependent pigment granule transport in retinal pigment epithe-
lial cells, 181

Activity-dependent regulation of neural networks: the role of inhibitory
synaptic plasticity in adaptive gain control in the siphon with-
drawal reflex of. -l/>/rwi<. 164

Allograft. 53

Allorecognition, 53

ANDFRSON. ERIK J., PATRICK S. MAC-GILLIVR.\> , and M. EDWIN DE-
MONT, Scallop shells exhibit optimization of rihlet dimensions for
drag reduction. 341

ANGERER. LYNNE M., see Robert C. Angerer. 1 75

ANOERER, ROBERT C., and LYNNE M. ANGERER, Fate specification
along the sea urchin embryo animal-vegetal axis. 1 75

Animal behavior, 410

Animal-vegetal axis. 175

ANTHONY. KENNETH R. N.. Prey capture by the sea anemone Mclrtd-
inin senile (L.): effects of body size, flow regime, and upstream
neighbors. 73

Anthozoa, 73

Ap/ysia cali/oniiai. 164. 167,388

ARCHER, Wn.i i \M E.. see Vicki J. Martin. 41, 345

Ascidian. 62. 87. 217

ASHCR-XFT. SUSAN E.. see Roger T. Hanlon. 364

Axis, dorsoanterior. 172

Axon. 183

B

BABCOCK. RUSSELL, see Karen Miller. 98

Bacteria. 126

Bacterial endosymbionts in the gills of the deep-sea wood-boring bi-
valves Xyltiphuga ailantica and Xylophaga washinglona, 253

BASS, ANDREW H., DEANA A. BODNAR. and JESSICA R. Me KIBBFN,
From neurons to behavior: vocal-acoustic communication in tele-
ost fish, 158

BATES. WILLIAM R.,p58,acytoskeletal protein, is associated with mus-
cle cell determinants in ascidian eggs. 2 1 7

BAXTER. DOUGLAS A., and JOHN H. BYRNE, Complex oscillations in
simple neural systems, 167

Behavior. 146, 164,388.410

Behavioral modes arise from a random process in the nudibranch Mcl-
ihe. 4 1 X

BELL, JF.FFERY R.. see Rob Maxson. 1 78

Blood cell. 53

BLOUNT, PAUL, SERGEI I. SUKHAREV, PAUL MOE, and CHING KUNG,
Mechanosensitive channels of/;', colt: a genetic and molecular dis-
section. 126

BODE. HANS R., see Vicki J. Martin. 345

BODNAR. DEANA A., see Andrew H. Bass. 1 58

Body size, 73

Bone morphogenetic protein. 1 75

Botrylloides. 53
Briareum asbeslinum, 279
Bryozoan. 399

BURNSIDE, BETH, and CHRISTINA KING-SMITH, Actin-dependent pig-
ment granule transport in retinal pigment epithelial cells. 1 8 1
BYRNE, JOHN H., see Douglas A. Baxter, 1 67

Caenorhabditis, 1 25

Calcium. 150

CAREW, THOMAS J., see Thomas M. Fischer, 164: Rene Marois. 388

CASAGRAND, JANET L.. see Robert C. Eaton, 146

Cassiopeia. I

Cell

area of, I 1 8
blood, 53
stem. 41
volume of, I 18

Center for Advanced Studies in the Space Life Sciences, 1 1 I

Cephalopod. 203, 262, 364. 375

Ceratopteris, 139

CHALFIE. MARTIN. A molecular model for mechanosensation in
Caenorhabditi'i elegans. 125

Cham. 134

Chemical ecology. 410

CHILDRESS. JAMES J., see Brad A. Seibel. 262

Choreography of the squid's "nuptial dance," 203

Clir\:sa«ra quinquecirrha. 332

Cilia. 388

Cirrhipathes, 1

CLAES, MICHAEL F., see Roger T. Hanlon, 364

Clonal repeatability, 290

Cnidae. 1.41

Cnidaria, 345

Coelenteratecnidae capsules: disulfide linkages revealed by silver cyto-
chemistry and their differential responses to thiol reagents. I

COLLIN, RACHEL, and JOHN B. WISE, Morphology and development
ofOdoxlomiu caliiinhiiina Dall and Bartsch (Pyramidellidae): im-
plications for the evolution of gastropod development, 243

Colony, 87
specificity of, 53

Common garden experiment, 290

Complex oscillations in simple neural systems. 167

Complex signal processing by weakly electric fishes, 1 57

Compound eye fine structure in Paralomis imiltispina Benedict, an an-
omuran half-crab from 1200 m depth (Crustacea; Decapoda; An-
omura). 300

Computational neuroscience. 167

CONAND, C, see M. A. Sewell, I 7

Conflicting morphological and reproductive species boundaries in the
coral genus Platygyra, 98

Copper, effects of. 62

Coral

plasticity in. 279
reproduction in. 98

COURTNEY, LEE A., see Patricia S. Glas, 23 1

Cox, KINGSLEY J. A., see Joseph R. Fetcho. 1 50

457

458

INDEX TO VOLUME 192

Creosote, effects of. 62
Crustacean, 300
Cues, abiotic and biotic, 279
Cyphiniw glbbosum, 279
Cytoplasmic arrangement, 172
Cvtoskeleton. 141, 181, 183. 217

D

DAV> , SIMON K... IAN A. N. LUCAS, and JOHN R. TURNER, Uptake

and persistence of homologous and heterologous zooxanthellae

in the temperate sea anemone Ccirn.\ pcdunculalits (Pennant),

208
DAWIDOWICZ. E. A.. Introduction — the future of aquatic research in

space: neurobiology, cellular and molecular biology. 1 15
Decline in pelagic cephalopod metabolism with habitat depth reflects

differences in locomotory efficiency. 262
Deep-sea, 262, 300
Degenenns, 125

DtMoNT, EDWIN M., see Erik J. Anderson. 34 1
Developmental. 172, 178,345
DEZAWA, MARI, see Eisuke Eguchi, 300
DISTEL, DANIEL L., and SLISAN J. ROBERTS, Bacterial endosymbionts

in the gills of the deep-sea wood-boring bivalves Xylophaga allan-

licit and \r//iplui^u washingtona, 253
Disultides, 1

DOBIAS. SONIA L., see Rob Maxson, 1 78
Dopamme. 399
Drag. 34 1
DUNLAP, PAUL V., see Roger T. Hanlon, 364

E

E. coll. 1 26

EATON, ROBERT C., AUDREY L. GUZIK., and JANET L. CASAGRAND,
Mauthner system discrimination of stimulus direction from the
acceleration and pressure components at sound onset, 146

Echinoderm, 27

EDWARDS, ERIN SWINT, and STANLEY J. Roux, The influence of grav-
ity and light on developmental polarity of single cells ofCmi/op-
Icris richiirtlii gametophytes, I 39

Effect of salinity on ionic shifts in mesohaline scyphomedusae, Chry-
\ti/ii-a quinquecirrha, 332

Effects of common estuanne pollutants on the immune reactions of
tunicates, 62

EGUCHI, EISUKF, MARI DEZAWA, and V. BENNO MEYER-ROCHOW.
Compound eye fine structure in Paralomis multispina Benedict,
an anomuran half-crab from 1 200 m depth (Crustacea: Decapoda;
Anomura), 300

Electric fish. 157

Electron microscopy. 388

ELINSON, RICHARD P.. Getting a head in frog development, 1 72

Embryo development, 175.231

Embryogenesis in hydra, 345

Embryonic coat of the grass shrimp Palaemonetes pugio, 23 1

EMLET, R. B., see O. Hoegh-Guldberg, 27

Energy for development, 27

Energy use during the development of a lecithotrophic and a plankto-
trophic echmoid, 27

Escape behavior, 146. 150

Escherichia coh. 1 26

ESI-REAFICO, E. M., seeC. H. Lin, 183

/ , 'unit. 364

I . >n of development, 243

1 M '"i and regulation of a sea urchin ,U.v.v class homeobox gene:
n its into the evolution and function of a gene family that par-
ti x in the patterning of the early embryo. 178

Eye. cm-:, ;;nd, ~i '()

Fate specification along the sea urchin embryo animal-vegetal axis. 1 75

Feeding. 364

Fern. 1 39

FETCHO, JOSEPH R., K.INGSLEY J. A. Cox, and DONALD M. O'MAL-
LEV, Imaging neural activity with single cell resolution in an intact,
behaving vertebrate. 1 50

Fine structure of the apical ganglion and its serotonergic cells in the
larva ofAplysiu catifornica, 388

FISCHER, THOMAS M., and THOMAS J. CAREW, Activity-dependent
regulation of neural networks: the role of inhibitory synaptic plas-
ticity in adaptive gain control in the siphon withdrawal reflex of
Aplysiu, 1 64

FISHER, WILLIAM S.. see Patricia S. Glas, 23 1

Flagellates, 13 1

Flow, 73

FORSCHER, P.,seeC. H. Lin. 183

Frog. 1 72

From neurons to behavior: vocal-acoustic communication in teleost
fish, 158

Gametogenesis, 17

Gastropod development, 243

Gene expression, 175

Genetic constraint, 290

Germination, 139

Getting a head in frog development, 1 72

Gill tissue, 321

GlLLESPlE, PETER G.. Multiple myosin motors and mechanoelectrical

transduction by hair cells, 186
GILLY, WILLIAM F.. see Thomas Preuss, 375
GLAS, PATRICIA S., LEE A. COURTNEY, JAMES R. RAYBURN. and Wu

LI AM S. FISHER. Embryonic coat of the grass shrimp Palaemoneles

pugio. 23 1

GODBOLE, REA, see Peter Nick, 141
GOLDBERG, WALTER M., and GEORGE T. TAYLOR, Coelenterate cni-

dae capsules: disulfide linkages revealed by silver cytochemistry

and their differential responses to thiol reagents, I
Goldfish, 146
Gorgonacea, 279

GORODEZKY. LAURA A., see Brad A. Seibel, 262
Gravitaxis in flagellates. 131
Gravitropism, 134, 141
Gravitropism in the rhizoids of the alga C/nira a model system for

microgravity research, 134
Gravity, 111, 134, 139, 172
Growth, 87, 375

cone, 183
GUZIK, AUDREY L., see Robert C. Eaton. 146

H

HADER. DON AT-PETER, Gravitaxis in flagellates. 1 3 1

Hair cells. 186

HAMILL, OWEN P., and DON W. McBRiDE, JR., Mechanogated chan-
nels in Xerwpus oocytes: different gating modes enable a channel
to switch from a phasic to a tonic mechanotransducer, 1 2 1

HANLON, ROGER T.. MICHAEL F. CLAES, SUSAN E. ASHCRAFT, and
PALIL V. DLINLAP, Laboratory culture of the sepiolid squid Eu-
prnnnu scolopes: a model system for bacterial-animal symbioses,
364

HANLON, ROGER T., see Warwick H. H. Sauer, 203

Hearing, 158

Heat-shock protein expression in Mylihis californianus acclimitiza-
tion (seasonal and tidal-height comparisons) and acclimation
effects, 309

Hemocytes. 62

Heritability. 290

INDEX TO VOLUME 192

459

Hmdhrain. 158

HIROSE. EUICHI, YASUNORI SAITO, and HIROSHI WATANABE, Subcu-
ticular rejection: an advanced mode of the allogeneic rejection in
the compound ascidians, Botrvlluulex .xnnaJi'nxis and B liixcux.
53

HOEGH-GULDBERG, O., and R. B. EMLET, Energy use during the de-
velopment of a lecithotrophic and a planktotrophic echinoid. 27

HOFMANN, GRETCHEN E.. see Deirdre A. Roberts, 309

Holothuroidea. 17

HOL^OAK, ALAN R.. Patterns and consequences of whole colony
growth in the compound ascidian Polyclinum planum, 87

Homeobox, I 78

How do neuronal processes monitor their mechanical status?. I I 8

HUTCHINSON, AIMEE, see David Ratios. 62

Hydra. 345

Hvdrozoan. 41

I

Imaging. 150

Imaging neural activity with single cell resolution in an intact, behaving

vertebrate, 150
Immunocytochemistry. 388
Immunology. 62
Inducible defense, 279
Inner ear. 146

Input filter characteristics, 121
Integrins. 134

Interspecific variation in life-history strategy, 290
Intracellular transport. 181
Introduction — the future of aquatic research in space: neurobiology.

cellular and molecular biology, 1 15
Invertebrate immunology, 62
Ion channel, 126

Jamming avoidance response, 157
Jet propulsion, 375

K

KAWASAKI, MASASHI. Complex signal processing by weakly electric

fishes, 157

KING-SMITH, CHRISTINA, see Beth Burnside. 1 8 1
KISS, JOHN Z., Gravitropism in the rhizoids of the alga Chuia: a model

system for microgravity research, 134
KUNG, CHING. see Paul Blount, 1 26

Laboratory culture of the sepiolid squid Knpryinnu .vro/o/w a model

system for bacterial-animal symbioses, 364
Larva. 27, 41,399

LEBARIC. ZORA. see Thomas Preuss, 375
Lecithotrophic. 27
Lek-like. 203

LEPPER, DEBORAH M. E.. see Paul A. Moore. 410
LESIUK, HOWARD, see Catherine E. Morris. 1 1 8
Life history. 87, 290
Life-history variation in a colonial ascidian: broad-sense heritabihties

and tradeoffs in allocation to asexual growth and male and female

reproduction. 290
Light. 139

LIMA, ALICE GONCALVES, see John C. McNamara. 32 1
LIN. C. H., E. M. ESPREAFICO, M. S. MOOSEKER, and P. FORSCHER,

Myosin drives retrograde F-actin flow in neuronal growth cones.

183

Lipid. 27
LIPINSKI. MAREK R.. see Warwick H. H. Sauer. 203

LITTLEFIELD, C. LVNNE, see Vicki J. Martin. 345
Locomotion, 262. 341. 375

LOEWENSTEIN, WERNER R., Mechanosensitive channels: an introduc-
tion, I 17
LUCAS, IAN A. N., see Simon K. Daw, 208

M

MA, LIANG, see Rob Maxson. 178

MACGiLLivRAY, PATRICKS., see Erik J. Anderson, 341

Machrobrachium, 32 1

MARCLIM, YVETTE, see Philip O. Yund, 290

Mariculture, 364

MAROIS, RENE, and THOMAS J. CAREW, Fine structure of the apical
ganglion and its serotonergic cells in the larva of Aplvsia califor-
nica. 388

MARTIN. VICKI J.. and WILLIAM E. ARCHER, Stages of larval develop-
ment and stem cell population changes during metamorphosis of
a hydrozoan planula. 4 1

MARTIN, VICKI J., C. LYNNE LITTLEFIELD, WILLIAM E. ARCHER, and
HANS R. BODE, Embryogenesis in hydra, 345

Maternal determinants, 175

Mating systems. 203

Mauthner. 150
neuron. 146

Mauthner system discrimination of stimulus direction from the accel-
eration and pressure components at sound onset, 146

MAXSON, ROB, HONGYING TAN, SONIA L. DOBIAS, HAILIN Wu, JEFF-
ERY R. BELL, and LIANG MA, Expression and regulation of a sea
urchin A/.V.V class homeobox gene: insights into the evolution and
function of a gene family that participates in the patterning of the
early embryo, I 78

Me BRIDE. DON W., JR., see Owen P. Hamill, 121

McKlBBEN, JESSICA R.. see Andrew H. Bass, 158

MCMANUS, MICHAEL G.. ALLEN R. PLACE, and WILLIAM E. ZAMER.
Physiological variation among clonal genotypes in the sea anem-
one /laliplanella lincala: growth and biochemical content, 426

MCNAMARA, JOHN C.. and ALICE GONCALVES LIMA, The route of ion
and water movements across the gill epithelium of the freshwater
shrimp Macrobrachium olfcrxii (Decapoda, Palaemonidae): evi-
dence from ultrastructural changes induced by acclimation to sa-
line media. 32 1

Mechanical transduction. 186

Mechanogated channels in \vnitpm oocytes: different gating modes en-
able a channel to switch from a phasic to a tonic mechanotrans-
ducer. 121

Mechanoreceptors, 1 17

Mechanosensitive channels: an introduction, 1 1 7

Mechanosensitive channels of E coli: a genetic and molecular dissec-
tion, 126

Mechanosensitivity, I 17. 1 18. 12 I, 125. 126

Mechanotransduction. 117, 121

Membrane dynamics, 118

Metabolism. 27. 262

Metamorphosis. 41

Methenamine-silver, 1

Mclndium senile, 73

MEYER-ROCHOW, V. BENNO, see Eisuke Eguchi, 300

Microgravity. I 1 1

Microtubules. 141

Midbrain, 158

MILLER, KAREN, and RLISSELL BABCOCK, Conflicting morphological
and reproductive species boundaries in the coral genus Plutmvra,
98

MILLS. LINDA R.. see Catherine E. Morris, 1 1 8

Modular organism. 87

MOE, PAUL, see Paul Blount. 1 26

Mollusc, 388

MOORE, PAUL A., and DEBORAH M. E. LEPPER, Role of chemical sig-
nalsin the orientation behavior of the sea star Axleriasforbcsi, 410

MOOSEKER. M.S., see C. H.Lin. 183

460

INDEX TO VOLUME 192

Morphology and development of Odoslomia columbiana Dall and
Bartsch (Pyramidellidae!: implications for the evolution of gastro-
pod development, 24?

MORRIS, CATHERINE E., HOWARD LESIUK, and LINDA R. MILLS, How
do neuronal processes monitor their mechanical status?. I 1 8

MscL. 126

Msx. 178

Multiple myosin motors and mechanoelectrical transduction by hair
cells, 186

MUSCATINE. LEONARD, see Calvin M. Nil. 444

Muscle cell determinants. 2 1 7

Myosin, 181, 183, 186

Myosin drives retrograde F-actin flow in neuronal growth cones, 183

Mytilus califurnianiis. 309

N

National Aeronautics and Space Administration. I I 1

Nematocysts, I

Neural circuits. 4 1 8

Neurite, 183

Neurocomputation, 146

Neuron network. 157

Neuronal populations, 150

NICK, PETER, REA GODBOLE, and Qi YAN WANG, Probing rice gravi-
tropism with cytoskeletal drugs and cytoskeletal mutants, 141

Nil, CALVIN M., and LEONARD MUSCATINE, Oxidative stress in the
symbiotic sea anemone Aipiasiapuli'hellalCa\gren, 1943): contri-
bution of the animal to superoxide production at elevated temper-
ature, 444

Nonlinear dynamics. 167

Nuclear migration. 139

Nudibranch.418

o

O'DOR, RON K... see Warwick H. H. Sauer. 203
O'MALLEY, DONALD M.. see Joseph R. Fetcho. 1 50
Orientation, 410
Osmomechanical, 118

Osmoregulation. 32 I

Ovarian development, 17

Ovarian development in the class Holothuroidea: a reassessment of the

"tubule recruitment model." 17
Oxidative stress. 444
Oxidative stress in the symbiotic sea anemone Aiplasia pulchella (Cal-

gren, 1943): contribution of the animal to superoxide production

at elevated temperature, 444
Oxygen minimum layer. 262

p58, a cytoskeletal protein, is associated with muscle cell determinants

in ascidian eggs, 2 1 7
Pacemaker. 158
Palacmtmeie*, 231
Patch clamp. 126
Patterns and consequences of whole colony growth in the compound

ascidian Polyclinum planum, 87
Phase model, 146
Phenotypic plasticity, 279
Phototaxis, 399
PHP cell. 146
f'lmalia. 1
I 'logical variation among clonal genotypes in the sea anemone

' liiliplani'lla lineala: growth and biochemical content, 426
Pii', 181

PIKI rHONV, and ROBERT M. WOOLLACOTT, Serotonin and do-

le have opposite effects on phototaxis in larvae of the bryo-

zoan •'iliinerinna. 399
PLACE, .' . R.. see Michael G. McManus, 426

Planktotrophic, 27

Plant gravitropism, 134

Planula larvae, 41

Plasticity in the sclerites of a gorgonian coral: tests of water motion,
light level, and damage cues, 279

Polarity, 139

Pollution, 62

Polyclinum. 87

Post-hatching development of circular mantle muscles in the squid Lo-
ligo opalescens, 375

Predation, 73. 146

Predator. 146

PREUSS, THOMAS, ZORA LEBARIC, and WILLIAM F. GILLY, Post-
hatching development of circular mantle muscles in the squid Lit-
ligo opalescens, 375

Prey. 146

Prey capture by the sea anemone Metridium senile (L.): effects of body
size, flow regime, and upstream neighbors, 73

Probing rice gravitropism with cytoskeletal drugs and cytoskeletal mu-
tants, 141

Protease. 175

PURCELL, JENNIFER E., see David A. Wright. 332

Pyramidellids. 243

RAFTOS, DAVID, and AIMEE HLITCHINSON, Effects of common estua-
rine pollutants on the immune reactions of tunicates, 62

RAVBURN, JAMES R., see Patricia S. Glas, 23 1

Recurrent inhibition, 164

Reproduction, 87

Respiration, 27

Rhabdom, 300

Rhizoid, 134, 139

Riblet, 341

ROBERTS, DEIRDRE A., GRETCHEN E. HOFMANN, and GEORGE N.
SOMERO, Heat-shock protein expression in Mytilus californiamis:
acclimitization (seasonal and tidal-height comparisons) and accli-
mation effects, 309

ROBERTS, MIKE J., see Warwick H. H. Sauer. 203

ROBERTS, SUSAN J., see Daniel L. Distel, 253

Role of chemical signals in the orientation behavior of the sea star As-
icriasforhesi. 410

Roux, STANLEY J.,see Erin Swint Edwards, 139

SAITO, YASUNORI. see Euichi Hirose, 53

Salinity

acclimation to, 32 1
effects of, 332

Salt movement, 32 I

SAUER, WARWICK H. H.. MIKE J. ROBERTS, MAREK R. LIPINSKI.
MALCOLM J. SMALE, ROGER T. HANLON, DALE M. WEBBER, and
RON K. O'DOR, Choreography of the squid's "nuptial dance," 203

Scaling, 262

Scallop shells exhibit optimization of riblet dimensions for drag reduc-
tion. 341

Scallops. 341

SCHIVELL, AMANDA E., SAMUEL S.-H. WANG, and STUART H.
THOMPSON, Behavioral modes arise from a random process in the
nudibranch Metibe, 418

Sclerite, 279

Sea anemone, 73, 208

Sea cucumber, 17

Sea urchin, 175. 178

SEIBEL, BRAD A.. ERIK V. THUESEN, JAMES J. CHILDRESS, and LAURA
A. GORODEZKY, Decline in pelagic cephalopod metabolism with
habitat depth reflects differences in locomotory efficiency, 262

Selection on life history variants, 290

Sensory cell, 388

INDEX TO VOLUME 192

461

Serotonin. 399

Serotonin and dopamme have opposite effects on phototaxis in larvae

of the bryozoan Biit>u/u ncriiina. 399

SEWELL, M. A.. P. A. TYLER, C. M. YOUNG, and C. CONAND, Ovarian
development in the class Holothuroidea: a reassessment of the "tu-
bule recruitment model." 17

Sex allocation. 290

Sexual selection, 203

Signal processing. 157

SMALE, MALCOLM J.. see Warwick H. H. Sauer. 203

Sodium channel. 375

SOMERO. GEORGE N., see Deirdre A. Roberts. 309

Sound, 146

Space, biological research in. I I 1

Spatial regularity, 175

Species boundaries, 98

Spirocysts, I

Spontaneous behavior. 4 1 8

Squid. 203.262. 364

Stages of larval development and stem cell population changes during
metamorphosis of a hydrozoan planula, 41

Starfish. 410

Stem cell. 41

Stereocilin. 186

STEWART-SAVAGE, JOHN, see Philip O. Yund. 290

Stochastic processes. 4 1 8

Stomatin. 125

Stretch-activated channels. 1 3 1

Subcuticular rejection: an advanced mode of the allogeneic rejection in
the compound ascidians Butryl/nides sirnodensis and B. fiiscus, 53

SUKHAREV, SERGEI I., see Paul Blount, 1 26

Superoxide anion. 444

Suspension feeding, 73

Swimming, 341

Symbiosis, 208. 253, 364
re-establishment of, 208

Synaptic plasticity, 164

Tributyltin, see TBT

Tubule recruitment method. 17

Tunic cell, 53

Tunicate, 53,62

TURNER, JOHN R., see Simon K. Davy, 208

TYLER, P. A.,seeM. A. Sewell, 17

U

Ultrastructural modifications, 321

Uptake and persistence of homologous and heterologous zooxanthellae
in the temperate sea anemone Cereuspedunciilalux (Pennant), 208
Urochordates, 62

Vacuole, 1 18
Vibrio. 364
Vocalization. 158

w

WANG. Qi YAN. see Peter Nick, 1 4 1

WANG, SAMUEL S.-H., see Amanda E. Schivell, 41 8

WATANABE, HIROSHI, see Euichi Hirose, 53

WEBBER. DALE M.. see Warwick H. H. Sauer, 203

WEST, JORDAN M., Plasticity in the sclerites of a gorgonian coral: tests

of water motion, light level, and damage cues, 279
WISE, JOHN B., see Rachel Collin, 243
WOOLLACOTT, ROBERT M., see Anthony Pires, 399
WRIGHT, DAVID A., and JENNIFER E. PURCELL, Effect of salinity on

ionic shifts in mesohaline scyphomedusae. Chrvsaora yitinqiiecir-

rha. 332
Wu. HAlLlN.seeRobMaxson, 178

TAN, HONGYING, see Rob Maxson, 178

TAYLOR, GEORGE T., see Walter M. Goldberg. 1

TBT. effects of. 62

The future of aquatic research in space: neurobiology, cellular and mo-
lecular biology. I 1 1

The influence of gravity and light on developmental polarity of single
cells of Ceratopteris nclmrdn gametophytes, 139

The route of ion and water movements across the gill epithelium of the
freshwater shrimp Macrobrachium olfersii (Decapoda, Palaemon-
idae): evidence from ultrastructural changes induced by acclima-
tion to saline media, 32 1

THOMPSON. STUART H., see Amanda E. Schivell. 4 1 8

THUESEN, ERIK V.. see Brad A. Seibel. 262

Touch. 125

Tracking, 3-D, 203

Transcription, 175

Xenopus, 121

oocytesof. 121
XNOR. 146
Xylophaga. 253

Y

YOUNG, C. M., see M. A. Sewell, 1 7

YUND, PHILIP O., YVETTE MARCUM, and JOHN STEWART-SAVAGE,
Life-history variation in a colonial ascidian: broad-sense heritabil-
ities and tradeoffs in allocation to asexual growth and male and
female reproduction. 290

ZAMER, WILLIAM E., see Michael G. McManus, 426
Zebransh. 150
Zooxanthellae. 208

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