(navigation image)
Home American Libraries | Canadian Libraries | Universal Library | Community Texts | Project Gutenberg | Children's Library | Biodiversity Heritage Library | Additional Collections
Search: Advanced Search
Anonymous User (login or join us)
Upload
See other formats

Full text of "The Biological bulletin"

Volume 184 



THE 



Number 1 



BIOLOGICAL 
BULLETIN 




LIBRARY 
FEB191993 



WuOv'S 'I-' 1 ''!. Vi.TSS. 



FEBRUARY, 1993 



Published by the Marine Biological Laboratory 



al Laooratoiy 



THE 



1 9 1993 



BIOLOGICAL BULLETIN 



PUBLISHED BY 
THE MARINE BIOLOGICAL LABORATORY 



Associate Editors 

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

DAVID EPEL. Hopkins Marine Station, Stanford University 

J. MALCOLM SHICK, University of Maine, Orono 



Editorial Board 



WILLIAM D. COHEN, Hunter College 
DAPHNE GAIL FAUTIN, University of Kansas 

WILLIAM F. GILLY, Hopkins Marine Station, 
Stanford University 

ROGER T. HANLON, Marine Biomedical 

Institute, 
University of Texas Medical Branch 



CHARLES B. METZ, Llniversity of Miami 
K. RANGA RAO, University of West Florida 

RICHARD STRATHMANN, Friday Harbor Laboratories, 
University of Washington 

STEVEN VOGEL, Duke University 

SARAH ANN WOODIN, University of South Carolina 



Editor: MICHAEL J. GREENBERG, The Whitney Laboratory, University of Florida 
Managing Editor PAMELA L. CLAPP, Marine Biological Laboratory 



FEBRUARY, 1993 



Printed and Issued by 
LANCASTER PRESS, Inc. 

3575 HEMPLAND ROAD 
LANCASTER, PA 



THE BIOLOGICAL BULLETIN 



THE BIOLOGICAL BULLETIN is published six times a year by the Marine Biological Laboratory, MBL 
Street, Woods Hole, Massachusetts 02543. 

Subscriptions and similar matter should be addressed to Subscription Manager. THE BIOLOGICAL BUL- 
LETIN, Marine Biological Laboratory, Woods Hole. Massachusetts 02543. Single numbers, $35.00. Sub- 
scription per volume (three issues), $87.50 ($175.00 per year for six issues). 

Communications relative to manuscripts should be sent to Michael J. Greenberg. Editor-in-Chief, or 
Pamela L. Clapp, Managing Editor, at the Marine Biological Laboratory. Woods Hole, Massachusetts 02543. 
Telephone: (508) 548-3705, ext. 428. FAX: 508-540-6902. E-mail: pamclig'hoh. mbl.edu. 



POSTMASTER: Send address changes to THE BIOLOGICAL BULLETIN. Marine Biological Laboratory. 

Woods Hole, MA 02543. 

Copyright 1993, by the Marine Biological Laboratory 

Second-class postage paid at Woods Hole, MA, and additional mailing offices. 

ISSN 0006-3 185 



INSTRUCTIONS TO AUTHORS 



The Biological Bulletin accepts outstanding original research 
reports of general interest to biologists throughout the world. 
Papers are usually of intermediate length (10-40 manuscript 
pages). A limited number of solicited review papers may be ac- 
cepted after formal review. A paper will usually appear within 
four months after its acceptance. 

Very short, especially topical papers (less than 9 manuscript 
pages including tables, figures, and bibliography) will be pub- 
lished in a separate section entitled "Research Notes." A Re- 
search Note in The Biological Bulletin follows the format of 
similar notes in Nature. It should open with a summary para- 
graph of 150 to 200 words comprising the introduction and the 
conclusions. The rest of the text should continue on without 
subheadings, and there should be no more than 30 references. 
References should be referred to in the text by number, and 
listed in the Literature Cited section in the order that they appear 
in the text. Unlike references in Nature, references in the Re- 
search Notes section should conform in punctuation and ar- 
rangement to the style of recent issues of The Biological Bulletin 
Materials and Methods should be incorporated into appropriate 
figure legends. See the article by Lohmann et al. (October 1990, 
Vol. 179: 2 1 4-2 1 8) for sample style. A Research Note will usually 
appear within two months after its acceptance. 

The Editorial Board requests that regular manuscripts con- 
form to the requirements set below: those manuscripts that do 
not conform will be returned to authors for correction before 



1. Manuscripts. Manuscripts, including figures, should 
be submitted in triplicate. (Xerox copies of photographs are not 
acceptable for review purposes.) The original manuscript must 
be typed in no smaller than 12 pitch, using double spacing (in- 
cluding figure legends, footnotes, bibliography, etc.) on one side 
of 16- or 20-lb. bond paper, 8'/2 by 1 1 inches. Please, no right 
justification. Manuscripts should be proofread carefully and er- 
rors corrected legibly in black ink. Pages should be numbered 
consecutively. Margins on all sides should be at least 1 inch (2.5 
cm). Manuscripts should conform to the Council of Biology Ed- 
itors Style Manual. 5th Edition (Council of Biology Editors, 
1983) and to American spelling. Unusual abbreviations should 



be kept to a minimum and should be spelled out on first reference 
as well as defined in a footnote on the title page. Manuscripts 
should be divided into the following components: Title page. 
Abstract (of no more than 200 words). Introduction, Materials 
and Methods, Results, Discussion. Acknowledgments. Literature 
Cited, Tables, and Figure Legends. In addition, authors should 
supply a list of words and phrases under which the article should 
be indexed. 

2. Title page. The title page consists of: a condensed title 
or running head of no more than 35 letters and spaces, the 
manuscript title, authors' names and appropriate addresses, and 
footnotes listing present addresses, acknowledgments or contri- 
bution numbers, and explanation of unusual abbreviations. 

3. Figures. The dimensions of the printed page. 7 by 9 
inches, should be kept in mind in preparing figures for publi- 
cation. We recommend that figures be about 1 '/: times the linear 
dimensions of the final printing desired, and that the ratio of 
the largest to the smallest letter or number and of the thickest 
to the thinnest line not exceed 1:1.5. Explanatory matter gen- 
erally should be included in legends, although axes should always 
be identified on the illustration itself. Figures should be prepared 
for reproduction as either line cuts or halftones. Figures to be 
reproduced as line cuts should be unmounted glossy photo- 
graphic reproductions or drawn in black ink on white paper, 
good-quality tracing cloth or plastic, or blue-lined coordinate 
paper. Those to be reproduced as halftones should be mounted 
on board, with both designating numbers or letters and scale 
bars affixed directly to the figures. All figures should be numbered 
in consecutive order, with no distinction between text and plate 
figures. The author's name and an arrow indicating orientation 
should appear on the reverse side of all figures. 

4. Tables, footnotes, figure legends, etc. Authors should 
follow the style in a recent issue of The Biological Bulletin in 
preparing table headings, figure legends, and the like. Because 
of the high cost of setting tabular material in type, authors are 
asked to limit such material as much as possible. Tables, with 
their headings and footnotes, should be typed on separate sheets, 
numbered with consecutive Roman numerals, and placed after 



CONTENTS 



No. I, FEBRTARY 1993 



CELL BIOLOGY 



Costas, Eduardo, Angeles Aguilera, Sonsoles Gon- 
zalez-Gil, and Victoria Lopez-Rodas 

Contact inhibition: also a control for cell prolifer- 
ation in unicellular algae? 



DEVELOPMENT AND REPRODUCTION 

Fenteany. Gabriel, and Daniel E. Morse 

Specific inhibitors of protein synthesis do not block 
RNA synthesis or settlement in larvae of a marine 

gastropod mollusk (Haliati* n//o//v) 

Freeman, Gary 

Metamorphosis in the brachiopod Ti >< 'Innlulni: ev- 
idence for a role of calcium channel function and 
the dissociation of shell formation from settlement 



ECOLOGY AND EVOLUTION 

Curtis, Lawrence A., and Karen M. K. Hubbard 

Species relationships in a marine gastropod-tre- 

ntatode ecological system 

Douillet, Philippe, and Christopher J. Langdon 
Effects of marine bacteria on the culture of axenic 
oyster Crassostrea gigas (Thunberg) larvae 



15 



25 



36 



Okamura, Beth, and Lita Ann Doolan 

Patterns of suspension feeding in the freshwater 

bi vo/oan Plumatella irpens 52 

Scheltema, Amelie H. 

Aplacophora as progenetic aculiferans and the coe- 
lomate origin of mollusks as the sister taxon of Si- 
puncula 57 

IMMUNOLOGY 

Rinkevich, B., Y. Saito, and I. L. Weissman 

A colonial invertebrate species that displays a hi- 
erarchy of allorecognition responses 79 

S.i u .id.*, Tomoo, Jeffrey Zhang, and Edwin L. Cooper 
Classification and characterization of hemocytes in 

Sl\t-/a f/ava . 87 



PHYSIOLOGY 

Hidaka. Michio, and Kiwamu Afuso 

Effects of cations on the volume and elemental 
composition of nematocysts isolated from acontia 
of the sea anemone ('.ulliacti* f>nl\j)ii.\ 97 

Mangum, Charlotte P. 

Hemocyanin subunit composition and oxygen 
binding in two species of the lobster genus Homiinn, 
and their hybrids 105 



No. 2, APRIL 1993 



DEVELOPMENT AND REPRODUCTION 

Abraham, Vivek C., Sunita Gupta, and Richard A. 
Fluck 

Ooplasmic segregation in the medaka (On~ias ta- 
lipes) egg 115 

Hamel, Jean-Francois, John H. Himmelman, and 

Louise Dufresne 

Gametogenesis and spawning of the sea cucumber 
Psolus fabririi (Duben and Koren) 125 



ECOLOGY, EVOLUTION AND BEHAVIOR 



Bridges, Todd S. 

Reproductive investment in four developmental 
morphs of Streblospw (Polychaeta: Spionidae) .... 



144 



Emschermann, Peter 

On Antarctic Entoprocta: nematocyst-like organs 
in a loxosomatid, adaptive developmental strategies, 
host specificity, and bipolar occurrence of species 153 

Saigusa, Masayuki 

Control of hatching in an estuarine terrestrial crab. 
II. Exchange of a cluster of embryos between two 
females 186 

Takeda, Satoshi, and Minoru Murai 

Asymmetry in male fiddler crabs is related to the 
basic pattern of claw-waving display 203 

PHYSIOLOGY 

Ellington, W. Ross 

Studies of intracellular pH regulation in cardiac 
myocytes from the marine bivalve mollusk, Merce- 
naria campechiensu 209 



CONTENTS 



Matsushima, O., T. Takahashi, F. Morishita, M. 
Fujimoto, T. Ikeda, I. Kubota, T. Nose, and W. Miki 

Two S-Iamidf peptides, AKSGEYRIamide and 
VSSEYRIamide, isolated from an annelid. 



McFarland, F. K., and G. Muller-Parker 

Photosynthesis and retention of zooxanthellae 
within the aeolid nudibranch Amluhti papillosa . . 



216 



223 



Rees, Bernard B., and Steven C. Hand 

Biochemical correlates of estivation tolerance in the 
moiiniainsnailOwi//i-//\(Piilmonata:Oreohelicidae) 230 
Wright, Jonathan C., and John Machin 

Atmospheric water absorption and the water budget 

of terrestrial isopods (Crustacea, Isopoda. Onisci- 

dea) 243 



No. 3, JUNE 1993 



REVIEW 

McEdward, Larry R., and Daniel A. Janies 

Life cycle evolution in asteroids: what is a larva.' 



PHYSIOLOGY 



255 



DEVELOPMENT AND REPRODUCTION 

Buckland-Nicks, John 

Hull cupules of chiton eggs: parachute si rue lures 

and sperm focusing devices? 269 

Bollner, Tomas, and I. A. Meinertzhagen 

The patterns of bromodeoxyuridine incorporation 
in the nervous system of a larval ascidian, Ciinin ni- 
testinalis 277 

Harvell, C. Drew, and Richard Helling 

Experimental induction of localized reproduction 

in a marine bryozoan 286 

Montgomery, Mary K., and Margaret McFall-Ngai 
Embryonic development of the light organ of the 
sepiolid squid Euprymna sculopes Berry 296 



BIOCHEMISTRY 

Weis, Virginia M., Mary K. Montgomery, and Mar- 
garet J. McFall-Ngai 

Enhanced production of ALDH-like protein in the 
bacterial light organ of the sepiolid squid Eiijji-yiiniii 
seolupes 309 



Gaus, Gabriele, Karen E. Doble, David A. Price, Mi- 
chael J. Greenberg, Terry D. Lee, and Barbara-Anne 
Battelle 

The sequences of five neuropeptides isolated from 
Liinulii-, using antisera to FMRFamide 322 

Tamura, Shouhei, Takahiko Shimizu, and Susumu 

Ikegami 

Endocytosis in adult eel intestine: immunological 
detection of phagocytic cells in the surface epithe- 
lium . 330 



RESEARCH NOTE 

Smith, Andrew M., William M. Kier, and Sonke 
Johnsen 

The effect of depth on the attachment force of lim- 
pets 338 

VIEWS AND DISCUSSION 

Rinkevich, Baruch 

Immunological resorption in Bo/n7/in schlosseri 
(Tunicata) chimeras is characterized by multilevel 
hierarchial organization of histocompatibility alleles. 
A speculative endeavor 342 

Index to Volume 184 . 346 



the Literature Cited. Figure legends should contain enough in- 
formation to make the figure intelligible separate from the text. 
Legends should be typed double spaced, with consecutive Arabic 
numbers, on a separate sheet at the end of the paper. Footnotes 
should be limited to authors' current addresses, acknowledg- 
ments or contribution numbers, and explanation of unusual 
abbreviations. All such footnotes should appear on the title page. 
Footnotes are not normally permitted in the body of the text. 

5. Literature cited. In the text, literature should be cited 
by the Harvard system, with papers by more than two authors 
cited as Jones el al.. 1980. Personal communications and ma- 
terial in preparation or in press should be cited in the text only, 
with author's initials and institutions, unless the material has 
been formally accepted and a volume number can be supplied. 
The list of references following the text should be headed Lit- 
erature Cited, and must be typed double spaced on separate 
pages, conforming in punctuation and arrangement to the style 
of recent issues of The Biological Bulletin. Citations should in- 
clude complete titles and inclusive pagination. Journal abbre- 
viations should normally follow those of the U. S. A. Standards 
Institute (USASI). as adopted by BIOLOGICAL ABSTRACTS and 
CHEMICAL ABSTRACTS, with the minor differences set out below. 
The most generally useful list of biological journal titles is that 
published each year by BIOLOGICAL ABSTRACTS (BIOSIS List of 
Serials; the most recent issue). Foreign authors, and others who 
are accustomed to using THE WORLD LIST OF SCIENTIFIC PE- 
RIODICALS, may find a booklet published by the Biological 
Council of the LI.R. (obtainable from the Institute of Biology, 
41 Queen's Gate, London, S.W.7, England, U.K.) useful, since 
it sets out the WORLD LIST abbreviations for most biological 
journals with notes of the USASI abbreviations where these differ. 
CHEMICAL ABSTRACTS publishes quarterly supplements of ad- 
ditional abbreviations. The following points of reference style 
for THE BIOLOGICAL BULLETIN differ from USASI (or modified 
WORLD LIST) usage: 



A. Journal abbreviations, and book titles, all underlined 
(for italics) 

B. All components of abbreviations with initial capitals 
(not as European usage in WORLD LIST e.g.. J. Cell. Comp. 
Physiol. NOT/ cell. comp. Physiol.) 

C. All abbreviated components must be followed by a pe- 
riod, whole word components must not (i.e.. J. Cancer Res.) 

D. Space between all components (e.g., J Cell. Comp. 
Physiol., not J. Cell. Comp. Physiol.) 

E. Unusual words in journal titles should be spelled out 
in full, rather than employing new abbreviations invented by 
the author. For example, use Rii I 'fsindafjelags Islendinga with- 
out abbreviation. 

F. All single word journal titles in full (e.g.. I 'eliger. Ecol- 
ogy. Brain). 

G. The order of abbreviated components should be the 
same as the word order of the complete title (i.e.. Proc. and 
Trans, placed where they appear, not transposed as in some 
BIOLOGICAL ABSTRACTS listings). 

H. A few well-known international journals in their pre- 
ferred forms rather than WORLD LIST or USASI usage (e.g.. 
Nature. Science, Evolution NOT Nature, Land.. Science, N.Y.; 
Evolution. Lancaster. Pa.) 

6. Reprints, page proofs, and charges. Authors receive their 
first 100 reprints (without covers) free of charge. Additional re- 
prints may be ordered at time of publication and normally will 
be delivered about two to three months after the issue date. 
Authors (or delegates for foreign authors) will receive page proofs 
of articles shortly before publication. They will be charged the 
current cost of printers' time for corrections to these (other than 
corrections of printers' or editors' errors). Other than these 
charges for authors' alterations. The Biological Bulletin does not 
have page charges. 



Reference: Biol Bull 184: 1-5. (February. 



Contact Inhibition: Also a Control for Cell 
Proliferation in Unicellular Algae? 



EDUARDO COSTAS, ANGELES AGUILERA, SONSOLES GONZALEZ-GIL, 
AND VICTORIA LOPEZ-RODAS 

Genetica Production Animal, Facnltad de 1'eterinaria. Universidad Complutense, Madrid, Spain 



Abstract. According to traditional views, the prolifera- 
tion of unicellular algae is controlled primarily by envi- 
ronmental conditions. But as in mammalian cells, other 
biological mechanisms, such as growth factors, cellular 
aging, and contact inhibition, might also control algal 
proliferation. Here we ask whether contact inhibition reg- 
ulates growth in several species of unicellular algae as it 
does in mammalian cells. Laboratory cultures of the di- 
noflagellate Prowcentrum lima (Ehrenberg) Dodge show 
contact inhibition at low cell density, so this would be an 
autocontrol mechanism of cell proliferation that could 
also act in natural populations of P. limn. But, Synecho- 
cystis spp., Phaeodactylum tricornntnm (Bohlin), Skele- 
tonema costatmn (Greville), and Tetraselmis spp. do not 
exhibit contact inhibition in laboratory cultures because 
they are able to grow at high cellular density. Apparently 
their growth is limited by nutrient depletion or catabolite 
accumulation instead of contact inhibition. Spirogyra in- 
signis (Hassall) Kutz, Prorocentrum triestinum Schiller, 
andAlexandrium tamarense (Hsfiaa) Balech show a com- 
plex response, as they are able to grow in both low and 
high cell density medium. These results suggest that con- 
tact inhibition is more adaptative in benthic unicellular 
algae. 

Introduction 

Environmental conditions (light, nutrients, tempera- 
ture, and turbulence) are thought to be the main controls 
of proliferation in unicellular algae. Thus, axenic cultures 
of algae progressively increase in cell number until division 
slows due to nutrient depletion, the shadowing of some 
cells by others, or metabolite accumulation. But other 
mechanisms could play an important role in autocontrol 

Received 26 March 1992; accepted 23 October 1992. 



of algal proliferation. In this respect, endogenous rhythms 
have been proposed as pacemakers of algal proliferation 
(reviewed by Edmunds, 1988). Also, mucilage production 
has been considered a mechanism of biological autocon- 
trol in unicellular algae (Margalef, 1989). Recently, Wyatt 
and Reguera (1989) proposed that the onset of phyto- 
plankton blooms and red tides are due to a mechanism 
of ecological autocontrol acting at the Gaian level. 

Several biological mechanisms that control the cell di- 
vision cycle in mammalian cells have recently been elu- 
cidated. They are based on growth factors, genes, and gene 
products that respond to growth factors (Baserga ft ai. 
1986; Goustin ft ai, 1986; Cantley et ai. 1991; North, 
1991 ). Although these mechanisms have been interpreted 
as adaptations for regulating cellular proliferation in mul- 
ticellular organisms, they are common to all eukaryotic 
cells, even regulating the cleavage of zygotes (Murray and 
Kirschner, 1989). Recently, we have proven that the cell 
division cycle in unicellular algae from different phyla 
(Cyanophyceae, Dinophyceae, Bacillarophyceae, and 
Chlorophyceae) are regulated by growth factors just as are 
mammalian cells (Costas and Lopez-Rodas, 199 la; L6- 
pez-Rodas et ai, 1991). 

In addition to regulation by growth factors, other 
mechanisms control the cell proliferation of mammalian 
cells. For example, some cells are genetically programmed 
to degenerate and die of old age after a determined number 
of generations. Also, the unicellular algae Spirogyra in- 
signis (Conjugatophyceae) undergoes cellular aging as do 
mammalian cells (Costas and Lopez-Rodas, 1991b). 

In mammals, another important regulator of cellular 
proliferation is contact inhibition. Mammalian cells grow 
in monolayers, colonizing the bottom of culture flasks, 
but they only increase until their growth is inhibited by 
contact with neighboring cells. Various mechanisms seem 
to be involved in this complex phenomenon, from growth 



E. COSTAS ET AL 



Table 

Characteristics ofllie species used 



Species 


Phyla 


Charactenstic 


Synechocystis spp. 


Cyanobacteria 


unicellular. 






planktonic 


Prorocenlrum lima 


Dmophyceae 


unicellular. 


(Ehrenberg) Dodge 




benthic 


Prorocenlrum Iriestinum 


Dinophyceae 


unicellular. 


Schiller 




swimming 


Alexandrium tamarense 


Dinophyceae 


unicellular. 


(Halim) Balech 




swimming 


Tetraselmis spp. 


Praxinophyceae 


unicellular. 






swimming 


Skek'lonema costatuin 


Bacillarophyceae 


cenobial 


(Greville) 




filamentous. 






planktonic 


Pliaeodaclylum tricormitum 


Bacillarophyceae 


unicellular. 


(Bohlin) 




benthic 


Spirogyra insignis* 


Conjugatophyceae 


cenobial 


(Hasak) Kutz 




filamentous. 






benthic 



* Spirogyra insignis grows in cenobial filaments anchored to the bottom 
of the flask by the distal cell. Every cell of the filament can divide (more 
detail in Costas and Lopez-Rodas, 1991b). 



factor competence to cell shape changes related to intercell 
contacts (review Alberts el a!., 1983). 

This paper attempts to determine whether contact in- 
hibition can limit the growth of unicellular algae, as is the 
case in mammalian cells. Several species of unicellular 
algae from different phyla are analyzed in a combined 
ecological and evolutionary approach. 



Materials and Methods 



Cultures 



Isolation and culture procedures for the species used 
were previously described in detail (Costas, 1990; Costas 
and Lopez-Rodas, 199 la, b, c), so only a brief description 
is provided here. 

The characteristics of the eight species employed are 
summarized in Table I. Freshwater and marine species 
were grown, respectively, in Petri dishes with 20 ml of 
WC medium or f/2 medium (Guillard, 1975), at 22.5 
0.5C and 80 Mmol nT 2 s~', 12:12 h light-dark cycle. 

Cultures were treated with 150 mg 1 ' penicillin and 
100 mg 1~' streptomycin and were, therefore, axenic. Be- 
fore the experiments were performed, the cultures were 
tested for the presence of bacteria using epifluorescence 
procedures as previously described (Costas, 1990). The 
possible effects of antibiotics on algal proliferation were 
obviated, because the antibiotic treatment was applied 
two months before the experiments took place, so the 
cultures were grown under axenic conditions. 



Cultures were maintained by serial transfers of a 500 
30 cell inoculum to fresh medium once every day. The 
cells grew exponentially for 20-30 days, and then the cul- 
tures showed density-dependent inhibition of growth. We 
determined that a culture reached saturation when its 
growth rate approached zero and its cell density reached 
the maximum. Saturation was easily detected because, 
growth rates and cell densities were determined daily. The 
experiments took place three days after the cultures were 
saturated. 

Experimental Design 

Many factors act in the cell density-dependent inhibi- 
tion of growth. In this investigation, we attempted to an- 
alyze whether contact inhibition also takes part in this 
process. Clonal cultures of each species were grown until 
saturation density was reached, and then the following 
two experiments were performed. 

Experiment 1: Cells at saturation density growing in 
fresh medium. All the cells of each saturated culture were 
collected (by centrifugation at 1000 rpm for 20 min), and 
resuspended in the same quantity of fresh medium. In 
this way we obtained a culture in fresh medium with sat- 
urated density of cells. Growth rates and cellular densities 
were measured during the five following days. Five rep- 
licates were performed for each species. 

Experiment 2: Cells at low density growing in saturated 
medium. In the second experiment, the saturated medium, 
after centrifugation, was filtered through a 0.22 ^m pore 
filter to produce a completely axenic, saturated medium 
that was free of cells. In this saturated, cell-free medium, 
a centrifuged inoculum of the same species growing ex- 
ponentially, was cultured. Growth rates and cellular den- 
sities were measured during the five following days. Five 
replicates were performed for each species. 

If inhibition of growth by contact inhibition and other 
factors are mutually exclusive, then contact inhibition of 
growth can be detected by this system, according to the 
following logic. If a species exhibits contact inhibition, 
then it will probably be able to grow in Experiment 2. but 
it won't be able to proliferate in Experiment 1. On the 
contrary, if the growth inhibition is due to other factors 
(nutrient depletion or catabolite accumulation), then it 
will probably be able to grow in Experiment 1 but not in 
Experiment 2. But, if other factors (i.e., soluble factors), 
as well as contact inhibition affect growth, then the simple 
two possibility choice won't happen. 

To determine whether contact inhibition is a factor in 
growth inhibition of those algae that grow in monolayers, 
the following experiment was performed; i.e.. the same 
method used to detect contact inhibition in mammalian 
cells was applied to algae. The cells from half a Petri dish 
were removed mechanically from each saturated culture 



CONTACT INHIBITION IN MICROALGAE 

Table II 

Growth rates and percentage increase oj cell density in fresh medium at saturation density and in saturated medium at low density 



Cells at saturation density 
in fresh medium 



Cells at low density 
in saturated medium 



Exponential 
growth-rates 



Saturated 
growth-rates 



Growth rates 



% increase 
cell density 



Growth rates 



cell density 



Syiicchocystis spp. 


0.79 0.01 


-0.07 0.006 


0.49 0.06" 


64 5% 


-0.04 0.01** 


-4 i"; 


Prorocentrum lima 


0.38 0.03 


0.01 0.01 


0.03 0.02** 


2 1% 


0.39 0.07** 


47 2% 


Prorocentmm triestinitm 


0.91 0.05 


0.01 0.01 


0.29 0.02** 


33 1% 


0.07 0.02** 


7 2% 


Alexandnum tamarense 


0.43 0.03 


-0.03 0.02 


0.12 + 0.05* 


12 5% 


0.07 0.02* 


7 1% 


Telraselmis spp. 


0.96 0.05 


-0.04 0.01 


0.87 0.07** 


1 38 6% 


0.01 0.01** 


1 1% 


Ske/etonema costaturn 


1.01 0.07 


-0.02 0.03 


0.58 0.03** 


78 3% 


-0.07 0.01" 


-6 r; 


Phaeodactilum tricomiaum 


0.88 0.04 


0.02 0.01 


0.52 0.01** 


68 2% 


-0.03 0.02** 


-2 3% 


Spirnt>yra insignis 


0.94 0.05 


0.03 0.02 


0.18 0.05** 


19 4% 


0.68 0.05** 


97 6% 



* Statistically no significant differences were found (P > 0.05). 

** Statistically significant differences (P < 0.01) were found between growth rates of Exp. 1 and Exp. 2. 



sample of monolayer species. If contact inhibition exists, 
the cells on the full side will continue growing into the 
cell-free half of the dish. Five replicates were performed 
in each case. A continuous recording by video microscopy 
helped us to evaluate this experiment. 

Control of hand/ ing effects 

Because some dinoflagellates are very sensitive to shear 
stress, we performed the following two preliminary ex- 
periments to determine whether manipulation would have 
detectable effects on the analyzed species. 

(a) Exponentially growing cells of each species were 
collected by centrifugation at 1000 rpm for 20 min and 
resuspended in the same quantity of fresh medium. Their 
growth rates (5 replicates of each species) were measured 
during the following five days and compared with the 
growth rates of uncentrifuged exponentially growing con- 
trols (5 replicates of each species). ANOVA analysis 
showed no significant differences (P > 0.05) between 
growth rates of centrifuged and uncentrifuged cells. Fur- 
thermore, the number of dead cells was estimated by the 
yellow cosine exclusion procedure (more details in Costas, 
1986; Gonzalez-Chavarri, 1991), and ANOVA showed 



Table III 

Growth rates of Prorocentrum lima and Spirogyra insignis after cells 
were mechanically removed from half a Petri dish 

Border where Zone where cells 
cells had been had not been 

removed removed F 



no significant differences (P > 0.05) in the rate of cell 
death between centrifuged and uncentrifuged cells. 

(b) A similar procedure was employed with saturated 
cells, and the same results were obtained; (i.e.. there were 
no significant differences (P > 0.05) between centrifuged 
and uncentrifuged cells). More details about the proce- 
dures used to control the effects of handling are set out 
in Costas ( 1 986) and Gonzalez-Chavarri (1991). 

Experimental evaluation 

Once an experiment was initiated for each of the five 
replicates, both the mean growth rates (during the sub- 
sequent five days) and the percentage of cell density in- 
crease (during the subsequent 24 h) were determined. Cell 
density was estimated as the number of cells per square 
or cubic centimeter in monolayer or suspension cultures, 
respectively. The number of cells in each culture was de- 
termined by counting samples in a hemocylometer. The 
number of samples counted was determined according to 
the mean progressive technique (Williams, 1977) to obtain 
95% accuracy. 

Growth rates were calculated as doublings per day: 



Prorocentrum lima 
Spirogyra insignis 



0.31 0.07 
0.47 0.03 



0.01 0.01 
0.13 0.02 



dd' 1 = l/Ln2 Ln(Nt/No)/t, 

Where Nt = cells at time t; No = cells at time 0; and t 
= number of days between times t and (more detail in 
Costas, 1990). 

Results and Discussion 

Growth inhibition of saturated cultures of unicellular 
algae is a complex process, influenced by various factors, 
such as nutrient depletion, catabolite accumulation, 
shading effects, and possibly by contact inhibition. Be- 
cause these factors do not act independently, their inter- 



E. COSTAS ET AL 




Figure 1 . Growth of Prorocenlntm lima and Spirogyra insignia when cells were mechanically removed 
from half a Petn dish. The arrows represent the border produced in the experiment. Only the cells bordering 
the cell-free zone were able to grow, (a) Saturated P. lima culture at the time of removal, (b) P. lima culture 
72 h after the removal. New cells have only proliferated into the open half of the plate, (c) Saturated 5. 
inaignis culture at the time of removal, (d) 5. insignia culture 72 h after the removal. New cells have only 
proliferated into Ihe free half of the plate. 



actions complicate a precise evaluation of the relative im- 
portance of each. Thus, our experimental design was 
aimed only at detecting whether contact inhibition takes 
part in cell dependent inhibition of growth. 

Table II summarizes the growth rates and the percent- 
age of cell density increases in both fresh and saturated 
culture media. Apparently, the dinoflagellate P. lima 
showed contact inhibition of growth. Both the growth rates 
and the cell densities of Experiments 1 and 2 were sig- 
nificantly different (P < 0.0 1 ). P. lima cells were not able 
to grow at saturation density in fresh medium (Experiment 
1 ), but their growth started again in saturated medium 
when their cell density decreased (Experiment 2). 



In contrast, Synechocystis spp., Phaeodactilum iricor- 
nutitm, Skeletonema costatum and Tetraselmis spp. did 
not exhibit contact inhibition. In all the cases, statistically 
significant differences (P < 0.01) were detected between 
both the growth rates and the cell densities of Experiments 
1 and 2. Apparently, their growth was limited by nutrient 
depletion or catabolite accumulation; thus they could 
proliferate at high cellular density in fresh medium (Ex- 
periment 1 ), but were not able to grow in saturated me- 
dium at low cell density (Experiment 2). 

Contact inhibition of growth may be an important 
mechanism in Spirogyra insignis. Although this species 
grew slowly at saturation density in fresh medium (Ex- 



CONTACT INHIBITION IN MICROALGAE 



periment 1), its growth was significantly increased (P 
> 0.01) at low density in saturated medium (Experiment 
2). So, in S. insignis. the contact inhibition component 
seems to prevail because proliferation is faster in a satu- 
rated medium with low cell density than in fresh medium 
with high cell density. 

In Pmwcentrum tricstimun. however, a nutrient de- 
pendent inhibition or catabolite accumulation seemed to 
be more important than contact inhibition. P. tricstinum 
was able to grow in both experiments, although its growth 
in fresh medium at high cellular density was significantly 
(P > 0.01) faster than that in saturated medium at low 
cell density. In Ak'xandnwn tamarcnsc, all of the factors 
seemed to slow down proliferation. A. tamarcnsc cells 
were scarcely able to grow in either experiment. 

The cells of P. lima and 5. insignis were mechanically 
removed from half a Petri dish, and the resulting growth 
rates are summarized in Table III. In agreement with pre- 
vious experiments, the growth of P. lima and S. insignis 
seemed to be inhibited by a contact inhibition mechanism. 
In particular, only the cells bordering the cell-free zone 
were able to grow (Fig. 1 ). This experiment, which employs 
the traditional method of detecting contact inhibition in 
mammalian cells (Alberts et a/.. 1983), supports the hy- 
pothesis that contact inhibition takes place in the growth 
inhibition of P. lima and S. insignis saturated cultures. 

Only two of the three benthic species analyzed seemed 
to exhibit contact inhibition. These results suggest that 
contact inhibition is a more adaptative mechanism in 
benthic unicellular algae. 

Contact inhibition is usually thought of as a mechanism 
developed by animal cells to limit cell division. The results 
obtained in these experiments suggest an alternative in- 
terpretation. The dinoflagellates, which could be consid- 
ered the earliest group of protist, but which are also far 
removed from actual eukaryotes (Dodge. 1955; Herzog 
et ai, 1984; Costas and Goyanes, 1988), have developed 
contact inhibition, thereby suggesting that such a mech- 
anism had already been developed by unicellular organ- 
isms in an early era, probably as an autocontrol mecha- 
nism regulating natural populations. Nevertheless, contact 
inhibition has also evolved in the Conjugatophyceae (a 
recent group of higher algae that are phylogenetically far 
removed from dinoflagellates), suggesting that such 
mechanisms may have been developed independently in 
phylogenetically different groups of unicellular organisms. 

Acknowledgments 

Supported by DGICYT grants IN89-0163 and PS89- 
0014. 



Literature Cited 

Alberts, B., D. Bray, J. Lewis, M. Raff, K. Roberts, and J. D. Watson. 
1983. Molecular Biology <>/ I lie Cell. Garland Publishing, New 
York. 

Baserga, R., L. Kaczmarek, B. Calabrelta, R. Battini, and S. Ferrari. 
1986. Cell cycle genes as potential oncogenes. Pp. 3-12 in Cell 
Crc/e ami Oncogenes. W. Tanner ami D. Gallwitz. eds. Springer- 
Verlag. New York. 

Cantley, L. C., K. R. Auger, C. Carpenter, B. Duckworth, A. Graciani, 
R. Kapeller, and S. Snlloff. 1 99 1 . Oncogenes and signal transduc- 
tion. CV//64: 281-302. 

Costas, E. 1986. Vltraesinictura cromosomica en dinoflagelados. Con- 
sidcntcioncs fvo/n/m/.v. Ph.D. Thesis. Univ. Santiago de Compostela. 
240 pp. 

Costas, E. 1990. Genetic variability in growth rates of marine dinofla- 
gellates. Genenea 83: 99-102. 
Costas, E., and V. J. Goyanes. 1988. Comparative analysis of dinofla- 

gellate chromosomes and nuclei. Genet. (Lije Sci. Adv.) 7: 15-18. 
Costas, E., and V. Lopez-Rodas. 1 99 la. On growth factors, cell division 
cycle and the eukaryotic origin. Endocytobiosis & Cell Res 8: 89- 
92. 

Costas, E., and V. Lopez-Rodas. 1991 b. Persistence of cell division 
synchrony in S/'/n^vra mvn,vi/s (Gamophyceae): membrane pro- 
teoglycans transmitting synchronizing information throughout gen- 
erations. Chronohiol Int 8(2): 85-92. 

Costas, E., and V. Lopez-Rodas. I99lc. Evidence for an annual rhythm 
in cell aging in Spin>f>yra m.v/#m.v (Chlorophyceae). Phmilngia 30(6): 
S97-S99. 

Dodge, J. D. 1955. Chromosome structure in the dinoflagellates and 
the problem of the mesokaryotic cell. 2ml. Internal. Con] on Pro- 
lo-ool. Exc Med Inter Cont-r Ser. No. 91: 39. 
Edmunds, L. N. 1988. Cellular and molecular hasis <>/ 'biological docks 

Spnnger-Verlag, New York. 497 pp. 

Gonzalez-Chavarri, E. 1991. I'roduccion de biomasa a base de 1111- 
eroalgas v sus a/ilicacioncs en la prutliiecion animal. Ph.D. Thesis. 
Universidad Complutense. 142 pp. 
Goustin, A. S., E. B. Leof, G. D. Shipley, and H. L. Moses. 

1986. Growth factors and cancer. Cancer Res 46: 1015-1029. 
Guillard. R. 1975. Culture of phytoplankton for feeding marine in- 
vertebrates. Pp: 26-60 in Culture of Marine Invertebrate Animals, 
W. Smith and M. Chanley. eds. Plenum Publ. Co., New York. 
Herzog, M., S. Boletzky, and M. O. Soyer. 1984. Ultrastructural and 
biochemical nuclear aspects of eukaryote classification: independent 
evolution of the dinoflagellates as a sister group of the actual eu- 
karyotes. Origins of Lite 13: 205-215. 

Lopez-Rodas, V., M. Navarro, L. De La Campa, E. Gonzalez De Cha- 
varri, S. Gonzalez-Gil, A. Aguilera, R. Segura, and E. Costas. 
1991. Tras las pistas de los primeros mecanismosde control de la 
division celular: Una aproximacion evolutiva. Pp. 94-108 in Crimo- 
canccrolngia. F. Chavarria, ed. Fundacion Cientifica A.E.C.C. Ma- 
drid. 

Margalef, R. 1989. Condiciones de aparicion de la purga de mar y 
presiones de seleccion sobre sus componentes. Cuademos da Area 
de Ciencias Marinas 4: 1 3-20. 
Murray, A. W., and M. W. Kirschner. 1989. Dominions and clocks: 

The union of two views of the cell cycle. Science 246: 614-621. 
North, G. 1991. Starting and stopping. Nature 351: 604-605. 
Williams, M. 1977. Stereological techniques. Pp. 226 in Practical 
methods in Electron Microscopy !'<>/ 17 M. Hayat. ed. Elsevier 
Sci. Publ. Co., New York. 

Wyatt, T., and B. Reguera. 1 989. ( , Ha alcanzado el cultivo de mejillon 
en Galicia su masa critica? Cuademos da Area de Ciencias Marinas 
4:63-71. 



Reference: Bio/, Bull. 184: 6-14. (February, 1993) 



Specific Inhibitors of Protein Synthesis Do Not Block 

RNA Synthesis or Settlement in Larvae of a Marine 

Gastropod Mollusk (Haliotis mfescens) 

GABRIEL FENTEANY 1 AND DANIEL E. MORSE 2 

Department of Biological Sciences and the Marine Biotechnology Center, Marine Science Institute. 
University of California, Santa Barbara, California 93106 



Abstract. Antibiotic inhibitors of protein synthesis were 
tested for their effectiveness in larvae of the red abalone, 
Haliotis mfescens (gastropod mollusk). Emetine and an- 
isomycin proved highly effective in this system, while cy- 
cloheximide, fusidic acid, puromycin. and tetracycline 
were less effective. Emetine and anisomycin specifically 
inhibited protein synthesis but not RNA synthesis. The 
contribution to protein synthesis by chloramphenicol- 
sensitive prokaryotic contaminants was found to be un- 
detectable, except following the onset of symptoms of 
toxicity resulting from prolonged exposure to emetine or 
anisomycin. The induction of larval settlement and plan- 
tigrade attachment by 7-aminobutyric acid (GABA), a 
functional analog of the natural inducer of settlement, 
occurred even under conditions in which most protein 
synthesis was inhibited, as expected for a chemosensory 
system response, whereas subsequent developmental 
metamorphosis was completely blocked. Because emetine 
and anisomycin block protein synthesis including the 
synthesis of new transcription factors but do not block 
early transcription, treatment of marine invertebrate em- 
bryos and larvae with these inhibitors can be used to ob- 
tain a selective enrichment in the mRNA population of 
"early gene" transcripts induced directly by GABA and 
other morphogenetic signals, without dilution by new 
mRNAs, the appearance of which is dependent on the 
synthesis of new protein transcription factors. 



Received 5 August 1992; accepted 18 November 1992. 

Abbreviations: GABA, -j-aminobutync acid; TCA, tnchloroacetic acid; 
SSC, standard sodium chlonde-sodium citrate buffer. 

Present address: ' Program in Cell and Developmental Biology, Har- 
vard Medical School. Boston, Massachusetts 021 15. 

2 Author to whom correspondence should be addressed. 



Introduction 

Developmentally competent larvae (0.2 mm in diam- 
eter) of the marine gastropod mollusk Haliotis mfescens 
(red abalone) are induced to settle from the plankton and 
begin metamorphosis by oligopeptides and proteins as- 
sociated with the surfaces of crustose red algae (Morse et 
al., 1979a, b, 1984; Morse and Morse, 1984), and by 
functional analogs of these natural inducers, such as 
7-aminobutyric acid (GABA), muscimol, and baclofen 
(Morse et al.. 1979a.b, 1980a. Morse, 1992). These com- 
pounds apparently bind to chemosensory receptors, with 
subsequent transduction of the signal mediated by the 
second messengers cyclic AMP and Ca ++ (Trapido- 
Rosenthal and Morse, 1986a; Morse, 1992). This trans- 
duction pathway culminates in an excitatory depolariza- 
tion that is apparently triggered by the regulated opening 
of chloride ion channels (Morse et al.. 1980; Baloun and 
Morse, 1984; Morse, 1990, 1992). The morphogenetic 
response can be facilitated or amplified by the presence 
of lysine or lysine analogs (Trapido-Rosenthal and Morse, 
1985, 1986b), acting through a separate lysine receptor 
that, in turn, stimulates a G protein-diacylglycerol signal 
transduction cascade (Baxter and Morse, 1987, 1992; 
Wodicka and Morse, 1991; Morse, 1990, 1992). Before 
we can understand the molecular mechanisms by which 
these convergent chemosensory pathways regulate larval 
settlement behavior and the subsequent induction of gene 
expression controlling cellular differentiation and prolif- 
eration (cf. Cariolou and Morse, 1988; Groppe and Morse, 
1989; Spaulding and Morse, 1991; Degnan and Morse, 
1993), we must first determine the requirements for de 
novo protein synthesis in these processes. 

Specific inhibitors of protein synthesis, such as cyclo- 
heximide and puromycin, have proved invaluable for such 



INHIBITORS OF PROTEIN SYNTHESIS 



studies in many other systems. Yet these well-known in- 
hibitors were ineffective with larvae of the red abalone 
(Haliotis mfesL'cns: gastropod mollusk) developing in sea- 
water. This finding prompted our search for inhibitors of 
mRNA translation that would specifically block protein 
synthesis in abalone larvae in seawater media, while not 
inhibiting RNA synthesis. 

The antibiotic protein synthesis inhibitors emetine and 
anisomycin exhibit the necessary effectiveness and spec- 
ificity. These compounds do not inhibit RNA synthesis 
or the induction of settlement and plantigrade attachment 
of the planktonic abalone larvae, as would be expected if 
these processes are mediated by a chemosensory system, 
but they completely block the subsequent metamorphosis 
which, as expected, is apparently dependent on de novo 
protein synthesis. These inhibitors should therefore help 
investigators identify the primary response genes (the 
transcription of which does not depend on de novo protein 
synthesis) and messenger RNAs responsible for the in- 
duction of metamorphosis. 



Materials and Methods 

Haliotis rufescens broodstock was collected off the coast 
of Santa Barbara, California, and production and culti- 
vation of larvae conducted as previously described (Morse 
et ai. 1977. 1978, 1979b). Spawning was induced by ex- 
posing gravid adults to 10 mA/ hydrogen peroxide. Male 
and female gametes were collected and washed separately, 
and then mixed to allow fertilization. Embryos and larvae 
were maintained in 5 ^m-filtered, U.V. -sterilized flowing 
seawater at 15 1C. 

Antibiotic inhibitors of protein synthesis were pur- 
chased from Sigma Chemical Company (St. Louis, Mis- 
souri), dissolved to make concentrated stock solutions and 
used fresh on the day of preparation. Tetracycline was 
purchased as the hydrochloride, fusidic acid as the sodium 
salt, and puromycin and emetine as the dihydrochlorides. 
Stock solutions were prepared in 0.22 /urn-filtered distilled 
water, either alone, or containing the minimum amount 
of ethanol required to completely solubilize the antibiotic. 
After addition of antibiotics to experimental samples, no 
more than 0.2% (v/v) ethanol was present in any seawater 
sample. Control experiments showed that the presence of 
0.3% ethanol had no effect on larval behavior and settle- 
ment or on the level of [ 3 H]leucine incorporation into 
TCA-insoluble material in the larvae; higher concentra- 
tions of ethanol (above ca. 0.75%) and other organic sol- 
vents induced settlement of the larvae, with a rapidity 
corresponding to the concentration of solvent (data not 
shown). Similar results were reported earlier by Penning- 
ton and Hadfield (1989) for larvae of the nudibranch 
mollusk Phestilla sibogae. 



Synthesis of protein and RNA was measured by incor- 
poration of radioactive amino acid or nucleoside into acid- 
precipitable macromolecules. For each assay ca. 2000 lar- 
vae were placed in 10 ml of 5 /urn-filtered, U.V. -sterilized 
seawater in 40 ml Oakridge tubes. Rifampicin, a specific 
inhibitor of bacterial RNA synthesis, was added to a final 
concentration of 2.4 jtA/ in all samples to limit bacterial 
growth, except where otherwise noted. After incubations 
in the presence or absence of inhibitors, either L-[4,5- 
3 H]leucine (150 Ci/mmol; Amersham Corporation, Ar- 
lington Heights, Illinois) or [5,6- 3 H]uridine (42 Ci/mmol; 
Amersham) was added to 0.1 or 0.2 jiCi/ml, as noted in 
the figure legends. For each treatment at each time point, 
three larval samples were used. Larvae were kept at 1 5 
1 C, except as noted. To end the labeling, nonradioac- 
tive L-leucine or uridine was added to a final concentra- 
tion of 0.8 mA/ or 0.4 mA/, respectively. The Oakridge 
tubes then were centrifuged (16,000 rpm; 4C) for 5 min 
(Sorvall RC5 or RC5C Superspeed Centrifuges, Clare- 
mont, California); Sorvall SA-600 or SS-34 fixed angle 
rotors were used to pellet the larvae. The tubes were placed 
on ice, the water was drained off, 2 ml of cold 1 X SSC 
was added, and the larvae were re-suspended and ho- 
mogenized completely in an ice-cold Dounce homoge- 
nizer (7 ml Pyrex tissue grinder). One aliquot of 0.5 ml 
for each sample was removed, placed in a 1.5ml microfuge 
tube, and frozen for future quantitation of protein. For 
each sample, 1.5 ml of the homogenate was then placed 
into another microfuge tube on ice, and 100% (w/v) tri- 
chloroacetic acid (TCA) was added to yield a final con- 
centration of 10% (w/v). After acid precipitation for 30 
min at 4C, each sample was poured through a 2.4 cm 
glass microfiber filter (GF/C; Whatman International Ltd., 
Maidstone, UK), washed three times with 5% TCA, then 
washed twice with 100% ethanol. The filters were com- 
pletely dried in an oven at 55C and then placed in liquid 
scintillation vials; 1 ml of scintillation cocktail (Bio-Safe 
II; Research Products International, Mount Prospect, IL) 
was added and radioactivity determined by liquid scin- 
tillation. Protein was quantitated by the method of Brad- 
ford ( 1976) according to the protocol of the reagent man- 
ufacturer (Bio-Rad Protein Assay; Bio-Rad Laboratories, 
Richmond, California); assays were conducted in triplicate 
and evaluated relative to a bovine serum albumin standard 
measured in parallel. The incorporation data presented 
are the means of triplicate determinations, with error bars 
representing one standard deviation. 

Assays of settlement and metamorphosis were con- 
ducted with larvae in glass scintillation vials (ca. 200 larvae 
in 10 ml of rifampicin-containing 5 ^m-filtered, U.V.- 
sterilized seawater) maintained under low illumination 
and observed with a dissecting microscope. Each treat- 
ment was conducted in triplicate. Larvae also were placed 
at a density comparable to that used in the incorporation 



G. FENTEANY AND D. E. MORSE 



150 



^ 100 



O 
O 



2 
o 

Q. 

o 
o 

_c 





50 




100 



200 



300 



400 



200 - 



g 150 - 



m 
CD 
CC 




100 200 300 400 

[Antibiotic] (\iM) 



500 



Figure I. Incorporation of ['H]leucine as a function of the concen- 
tration of antibiotic. After incubating 8-l()-day-old larvae (ca. 2.000 lar- 
vae/10 ml rifampicin-containing seawater) for 2 h in the presence or 
absence of blocker at the concentrations indicated, [ 3 H]leucine was added 
to 0.1 /iCi/ml, and the pulse allowed to proceed for 2 h. Mean control 
values (representing incorporation in the absence of antibiotic) are dis- 
placed on the abscissa for clarity. (A) Incorporation in the presence of 
emetine (diamonds) or anisomycin (rectangles). (B) Incorporation in the 
presence of cycloheximide (diamonds), puromycin (rectangles), tetra- 
cycline (triangles), or fusidic acid (circles). Details as described in Materials 
and Methods. 



assays (ca. 2000 larvae/ 10 ml seawater), and observed for 
mortality and other responses to the protein synthesis in- 
hibitors. 

Results 

Inhibition of protein synthesis 

Larvae of H. rufescens take up exogenous amino acids 
from seawater, as demonstrated by these and other in- 
vestigations (Jaeckle and Manahan, 1989), although these 
larvae are lecithotrophic. Several commonly used inhib- 
itors of protein synthesis, including cycloheximide, fusidic 
acid, puromycin, and tetracycline, had little or no inhib- 
itory effect on the overall incorporation of [ 3 H]leucine 
into TCA-insoluble material at concentrations that were 
not toxic to the larvae (Fig. 1). In marked contrast, both 



emetine and anisomycin proved strongly inhibitory in a 
concentration-dependent manner. 

Emetine (9 n.\f) efficiently blocked the incorporation 
of [ 3 H]leucine into Haliotis larvae under conditions in 
which 100 nM chloramphenicol (an inhibitor of protein 
synthesis only in prokaryotes) had no significant effect 
(Fig. 2A). Identical results were obtained for a range of 
chloramphenicol concentrations (50-600 n.\f), both in 
the presence or absence of 2.4 pM rifampicin (an inhibitor 
of bacterial RNA polymerase). Thus, in the absence of 
emetine, prokaryotic incorporation of [-'H]leucine was not 
detectable. Inhibition by emetine was quite rapid; incu- 
bation for 10 min with 9 fiM emetine prior to addition 
of radiolabel was sufficient to block incorporation to a 
level comparable to that produced by an incubation for 
2 h (data not shown). The inhibitory effect of a single 



200 



150 



c 100 

' 



50 



o 



"I 200 

2 
o 
9- 

8 15 

c 



100 



50 









Figure 2. Incorporation of [ 3 H]leucine in 7-day-old larvae in the 
presence or absence of emetine (9 nM) or anisomycin (200 //A/) and 
chloramphenicol (150 nKf). 7-day-old larvae were used. Pulse-labeling 
at 24 h following the time of initial addition of emetine was for 2 h (0.1 
nCi/ml). (A) Treatments: ( 1) No emetine or chloramphenicol. (2) Chlor- 
amphenicol added at 2 1 h. (3) Emetine added at h. (4) Emetine added 
at h and chloramphenicol added at 21 h. (5) Emetine added to a con- 
centration of 9 ^M at h and the same amount added again at 12 h. 
(B) Treatments: (1) No anisomycin or chloramphenicol. (2) Chloram- 
phenicol added at 21 h. (3) Anisomycin added at h. (4) Anisomycin 
added at h and chloramphenicol added at 2 1 h. (5) Anisomycin added 
to a concentration of 200 /iA/ at h and the same amount added again 
at 12 h. 



INHIBITORS OF PROTEIN SYNTHESIS 



addition of emetine relaxed with time (Fig. 3A), and a 
second addition of the same amount of emetine at 12 h 
reduced incorporation slightly further (Fig. 2A). However, 
following prolonged incubation in the presence of eme- 
tine, the addition of chloramphenicol 3 h before labeling 
led to significantly lower levels of incorporation, partic- 
ularly at 24 h and 48 h following the addition of emetine 
(Figs. 2A, 3A). Therefore, some of the apparent relaxation 
of inhibition by emetine may be due to an increase in the 
proportion of protein synthesis attributable to contami- 
nating chloramphenicol-sensitive prokaryotes. This is 
likely to be the result of bacterial growth on the emetine- 
treated larvae themselves, as these larvae become weaker, 
although rifampicin (2.4 (iAf) was present throughout. 

The inhibitory effect of a single addition of anisomycin 
(200 pM) persisted longer than that caused by 9 /J.M eme- 
tine (Figs. 2B, 3B), and a second addition 12 h after the 
first did not reduce the incorporation of [ 3 H]leucine fur- 
ther (Fig. 2B). In the presence of anisomycin, the addition 
of chloramphenicol 3 h before pulse-labeling did not lead 
to significantly lower levels of incorporation up to 24 h 
(Figs. 2B, 3B). Much of the inhibition of protein synthesis 
by a single addition of anisomycin was reversed between 
24 and 48 h (Fig. 3B). This late apparent relaxation was 
blocked by chloramphenicol (Fig. 3B), suggesting that it 
was due to an increase in prokaryotic incorporation. 

Effects of emetine and anisomycin on RNA synthesis 

To test whether emetine affects RNA synthesis, larvae 
were pulsed with [ 3 H]uridine both in the presence and 
absence of 10 6 M GABA (added 30 min following the 
addition of emetine). No significant inhibition of RNA 
synthesis was observed except at 6 h in the presence of 
both GABA and 9 nM emetine (Fig. 4A). The presence 
of emetine (9 n.M) may have a stimulatory effect on the 
incorporation of ['H]uridine in Haliolis larvae after 
12.5 h. A similar experiment showed that the incorpo- 
ration of [ 3 H]uridine also was not inhibited by the addition 
of 200 pM anisomycin (added 60 min before addition of 
GABA; Fig. 4B). 

Effects of emetine and anisomycin on settlement, 
metamorphosis, and survival 

At concentrations sufficient to inhibit most protein 
synthesis, emetine and anisomycin did not block the initial 
induction of larval settlement and plantigrade attachment 
by GABA, although subsequent metamorphosis was 
completely blocked. Toxicity of these inhibitors was both 
time- and concentration-dependent. Initial settlement and 
plantigrade attachment of larvae induced by GABA ( 10~ 6 
M and 10~ 3 M) occurred normally in the presence of 9 
nM emetine (Fig. 5 A, B). Both in the presence and absence 
of emetine, larvae ceased their swimming behavior after 



c 
'CD 

2 

Q. 
O) 



300 



200 



100 



. A 




12 18 24 30 36 42 48 



= 200 

2 
o 

Q. 



150 



100 



50 



B 




12 



18 24 30 
Time (h) 



36 42 



48 



Figure 3. Incorporation of ['HJleucme in the presence or absence 
of emetine (9 pM) or anisomycin (200 n\I) and chioramphenicol (150 
nM) as a function of time following addition of emetine. Larvae were 
pulsed at the times after addition of emetine or anisomycin indicated 
for 2 h (O.I jiCi/ml). In the chloramphemcol-treated samples, chlor- 
amphenicol was added 3 h prior to pulse-labeling. Mean values are dis- 
placed slightly on the abscissa for clarity. (A) 4-day-old larvae were used 
(6 days old by the end of the experiment in the last 3 sets of samples). 
No antibiotic (diamonds); emetine (rectangles); emetine plus chioram- 
phenicol (triangles). (B) 5-day-old larvae were used (7 days old by the 
end of the experiment in the last 3 sets of samples). No antibiotic (dia- 
monds); anisomycin (rectangles); anisomycin plus chloramphenicol (tri- 
angles). 



addition of GABA, and plantigrade attachment followed. 
Attached larvae exhibited normal pedal locomotion in 
the presence of emetine. Abscission of the velum was also 
observed in the presence of emetine and occurred whether 
GABA (10~ 6 A/) was present or not, although at 9 /j.M 
emetine abscission occurred at lower levels when GABA 
was not present. Abscission was often premature or in- 
complete, particularly at higher concentrations of emetine. 
By 6 h after the addition of 10~ 6 M GABA, most of the 
larvae had settled both in the presence and absence of 9 
juM emetine (Fig. 5 A). New shell growth was not observed 
in the presence of emetine when larvae were induced to 
settle with 10~ 6 M GABA, although it was observed nor- 
mally in settled larvae in the absence of the inhibitor by 
48 h. Attachment proceeded more rapidly at the higher 



G. FENTEANY AND D. E. MORSE 



c 

'0) 

"o 

Q. 
O5 



40 



30 



20 



10 



I -o 








12 15 18 21 



24 



-.5 40 

2 
o 

Q. 



O 

o 



30 



20 



10 



B 




9 12 15 18 

Time (h) 



21 24 



Figure 4. Incorporation of [ 3 H]uridine in the presence or absence 
of emetine or anisomycin as a function of time after addition of GABA 
( ICT 6 M). The larvae were pulsed for 20 min with radiolabeled nucleoside 
(0.2 ^Ci/ml) at the times indicated. Mean values slightly displaced on 
the abscissa for clarity. (A) I0-day-old larvae were used. Where indicated, 
emetine (9 p.\l) was added 30 min prior to the addition of GABA. No 
emetine or GABA (diamonds); emetine with no GABA (rectangles): no 
emetine, plus GABA (triangles); emetine plus GABA (circles). (B) 9-day- 
old larvae were used. Where indicated, anisomycin (200 nl\f) was added 
60 min prior to the addition of GABA. No anisomycin or GABA (dia- 
monds); anisomycin with no GABA (rectangles): no anisomycin, plus 
GABA (triangles): anisomycin plus GABA, (circles). 

concentration of GABA; virtually all of the larvae were 
attached within 20 min, with no inhibition by emetine 
(Fig. 5B). Although emetine did not inhibit the initial rate 
of attachment of the larvae induced by GABA. the larvae 
failed to maintain their plantigrade attachment (Fig. 5A. 
B), and progressively more were found on their sides, ap- 
parently due to the toxic effect of prolonged exposure to 
emetine. There also was some attachment in the presence 
of emetine when GABA was absent (Fig. 5A. B). 

Prolonged exposure of larvae to emetine proved lethal. 
Even before any mortality was observed, larvae treated 
with 9 fiM emetine appeared to spend more time on the 
bottom of the test vial than larvae in control vials. By 
36 h after the addition of emetine (9 nAf) both in the 
presence and absence of GABA (ca. 20 larvae/ml), few 
larvae were swimming and many appeared dead, while 



in the control vials lacking GABA, many of the larvae 
remained swimming and virtually all remained alive. All 
the larvae were dead by 54 h in the presence of 9 ^M 
emetine at ca. 20 larvae/ml, and by 72 h at ca. 200 larvae/ 
ml. Toxicity was progressively accelerated by higher con- 
centrations, although the initial rate of GABA-induced 
attachment remained unimpaired below 80 ^/emetine; 
in the presence of 18 nM and 40 nM emetine, virtually 
all of the larvae were attached within 20 min following 
the addition of 1(T 3 M GABA (data not shown). Exposure 
to 80 pM or 160 fiM emetine produced marked symptoms 
of toxicity; GABA-induced settlement was reduced, pre- 
mature abscission of the velum occurred in the presence 
and absence of GABA. and all of the larvae died within 
6-12 h at the low density. 

Anisomycin appeared to exert a stimulatory effect on 
the activity of the larvae, particularly on the movement 
of the cilia. The level of swimming activity of the larvae 
was markedly greater in the presence of 200 nAI aniso- 
mycin than in control or emetine-treated vials, even after 
only 20 min following addition. This effect appeared to 
partially antagonize the initial GABA-induced attach- 
ment, with the attached larvae abnormally continuing 
sustained beating of their swimming cilia and displaying 
little pedal locomotion; plantigrade larvae often were dis- 
placed by collision with other swimming larvae, and 
sometimes began swimming again. The initial rates of 
settlement and attachment induced by 10 3 M GABA 
were relatively unaffected by anisomycin, although long- 
term attachment was reduced (Fig. 5C, D). [The weak 
settlement-inducing activity of high concentrations of an- 
isomycin itself (cf. Fig. 5C, D) may explain the biphasic 
settlement observed in the presence of GABA.] Aniso- 
mycin also produced concentration-dependent and time- 
dependent symptoms of toxicity, with complete mortality 
resulting from prolonged exposure (96 h) of larvae, at 
high or low density, to 200 ^M concentration. 

Discussion 

Inhibition of protein synthesis 

Emetine and anisomycin were found to be highly ef- 
fective inhibitors of protein synthesis in Haliotis larvae, 
whereas cycloheximide, fusidic acid, puromycin, and tet- 
racycline proved far less effective. Possible reasons for the 
limited effectiveness of these widely used inhibitors may 
include their instability or low solubility in seawater, or 
their inefficient diffusion into the deeper layers of larval 
tissue. There is some structural evidence supporting the 
suggestion that membrane permeability may be an im- 
portant determinant of effectiveness in the marine larval 
system. Emetine contains four methoxy groups, while an- 
isomycin contains one methoxy and one acetoxy group, 
all carbon-linked to cyclic nuclei in both of these anti- 



INHIBITORS OF PROTEIN SYNTHESIS 



11 




40 60 

Time (h) 



20 



40 60 

Time (h) 



80 



100 



Figure 5. Attachment of larvae in the presence or absence of emetine or anisomycin as a function of 
time following addition of GABA. Cu 200 were placed in 10 ml rifampicin-treated seawater in triplicate, 
as described in Materials and Methods. Following a 30-min incubation with or without emetine (A and B) 
or a 2-h incubation with or without anisomycin (C and D), GABA was added to the samples indicated, and 
the mean percentage of the larvae showing attachment was scored at the times indicated. Standard deviations 
for all points, assayed in triplicate, were <4<V (A) 8-day-old larvae treated with or without 10 * M GABA 
and 9 juA/ emetine. No emetine or GABA (diamonds); emetine with no GABA (rectangles); no emetine, 
plus GABA (triangles); emetine plus GABA (circles). (B) 8-day-old larvae treated with or without 10~ 3 A/ 
GABA and 9 nM emetine. No emetine or GABA (diamonds); emetine with no GABA (rectangles); no 
emetine, plus GABA (triangles); emetine plus GABA (circles), (C) 10-day-old larvae treated with or without 
10" A/ GABA and 200 pM anisomycin. No anisomycin or GABA (diamonds); anisomycin, with no GABA 
(rectangles); no anisomycin, plus GABA (triangles); anisomycin plus GABA (circles). (D) 10-day-old larvae 
treated with or without 10 ' A/ GABA and 200 ^M anisomycin. No anisomycin or GABA (diamonds); 
anisomycin with no GABA (rectangles); no anisomycin, plus GABA (mangles); anisomycin plus GABA 
(circles). 



biotics. These lipophilic groups may facilitate entry into 
cells and diffusion through tissues. This lipid solvent-like 
effect also might account for the weak settlement-inducing 
activity of these antibiotics, analogous to that reported by 
Pennington and Hadneld (1989) for some organic sol- 
vents. Puromycin contains only one methoxy group, and 
fusidic acid contains one acetoxy group. 

Puromycin, which was not an effective inhibitor of 
protein synthesis in Haliotis larvae, has been successfully 
used in sea urchin embryos. Gong and Brandhorst (1988) 
found puromycin to effectively inhibit most protein syn- 
thesis in gastrulae of the sea urchin Lytechhnts pictus in 
artificial seawater. On a molar basis, however, it was less 



effective than emetine, anisomycin, or the less readily 
available pactamycin. Cycloheximide dissolved in sea- 
water-based media has limited use as a protein synthesis 
inhibitor in sea urchin eggs (K. Foltz, pers. comm.) and 
is a poor inhibitor of protein synthesis in sea urchin em- 
bryos (Hogan and Gross, 1971). This could be due in part 
to the fact that cycloheximide is rapidly inactivated in 
dilute alkali at room temperature, and thus may be un- 
stable at the pH of seawater (ca. pH 8). This could also 
help to explain its lack of effectiveness in Haliotis larvae. 
Emetine and anisomycin are specific inhibitors of pro- 
tein synthesis in eukaryotes; they bind to specific sites on 
eukaryotic ribosomes, and are ineffective in prokaryotes 



12 



G. FENTEANY AND D. E. MORSE 



(Grollman. 1967; Barbacid el ai. 1975; Carrasco et ai, 
1976; Jimenez et ai. 1977; Sanchez et ai. 1977). Inhi- 
bition of protein synthesis by emetine is irreversible 
(Grollman, 1968). whereas the inhibition by anisomycin 
is reversible (Barbacid and Vazquez, 1975). Both are ef- 
fective inhibitors of protein synthesis in the eggs and em- 
bryos of several species of sea urchins (Hogan and Gross, 
1971; Epel, 1972: Wagenaar and Mazia, 1978; Hille et 
ai. 1981: Wagenaar, 1983; Dube. 1988; Gong and 
Brandhorst, 1988: Sluder et ai. 1990). Our extension of 
these findings to the anatomically more complex larvae 
of the mollusk, H allot is nifescens, suggests that these 
compounds may be generally useful for inhibiting protein 
synthesis in marine invertebrate embryos and larvae. 

Settlement, metamorphosis, and toxicity 

The density ofHaliotis larvae used in the incorporation 
assays was about ten-fold higher than in the samples used 
to investigate the effects on settlement, metamorphosis 
and survival. This reflected the practical requirements for 
a large number of larvae needed to obtain reliable values 
in the isotope incorporation studies, and a low density of 
larvae needed to make accurate observations of behavior 
and metamorphosis. Therefore, one would expect that 
the actual level of inhibition of protein synthesis that oc- 
curred in the samples in which settlement and metamor- 
phosis were investigated would be at least equal to and 
probably greater than the inhibition estimated in the in- 
corporation assays. The survival and behavior of the larvae 
at the higher density in the presence of the inhibitors nev- 
ertheless were found to parallel those at the lower density, 
although mortality generally was delayed at higher density. 

The fact that the larvae initially settle and attach nor- 
mally in response to GABA in the presence of emetine 
or anisomycin at concentrations sufficient to block nearly 
all protein synthesis suggests that the induction of settle- 
ment and plantigrade attachment does not require de novo 
protein synthesis, consistent with the notion that these 
behavioral responses are controlled by a chemosensory 
mechanism mediated by the preformed larval nervous 
system. It remains possible, however, that the inhibition 
of protein synthesis may not have been sufficient to block 
the synthesis of some new proteins required for this re- 
sponse. While the highest concentrations of emetine tested 
(80 fiM and 160 pM) did interfere with initial settlement, 
these concentrations rapidly caused acute symptoms of 
toxicity, and all the larvae died within 6-12 h at ca. 20 
larvae/ml. The initial high level of induced attachment 
observed in response to 10 ? M GABA in the presence of 
anisomycin was transitory, and appeared to be antago- 
nized by the stimulatory effect of this compound on 
swimming activity. Emetine had no such stimulatory ef- 
fect on the larvae. Anisomycin may directly or indirectly 



stimulate the movement of the cilia, independently of its 
effects as an inhibitor of protein synthesis. 

Both emetine and anisomycin completely blocked the 
induction of metamorphosis (in conjunction with their 
effect on protein synthesis) at concentrations that did not 
inhibit initial settlement and plantigrade attachment. 
There was no shell growth, nor were any other landmarks 
of developmental metamorphosis observed (beyond the 
abnormal abscission of the velum in the presence of eme- 
tine). This may be the direct result of the inhibition of 
protein synthesis, suggesting that the biosynthesis of new 
proteins is required even for the early processes of meta- 
morphosis to follow settlement. New proteins made fol- 
lowing the induction of metamorphosis in H. nifescens 
have been shown to include a sulfatase (Spaulding and 
Morse, 1 99 1 ), a chymotrypsin-like protease (Groppe and 
Morse. 1989) and a new conchiolin shell matrix protein 
(Cariolou and Morse. 1988). It also is possible that the 
failure to progress through metamorphosis may have re- 
sulted from other toxic effects of emetine and anisomycin 
beyond their inhibition of protein synthesis. Emetine has 
well-documented cardiotoxic effects in vertebrates (for re- 
views, see Wenzel, 1967; Yang and Dubrick, 1980). The 
abscission of the velum observed in the presence of eme- 
tine, in the presence or absence of GABA, apparently is 
a result of toxicity of this compound. This suggestion is 
supported by the facts that this abscission also occurs in 
the absence of GABA and that similar responses of the 
larvae to unrelated toxic compounds have been observed 
previously (Morse et ai, 1980). Moreover, the fact that 
anisomycin, an equally potent but longer-lasting inhibitor 
of protein synthesis in the larvae, does not induce this 
abscission and causes less rapid mortality than does eme- 
tine at the concentrations used, supports the interpretation 
that this abscission is the result of toxicity rather than 
normal morphogenesis. However, whether the inhibition 
of metamorphosis is a direct result of an inhibition of 
protein synthesis, or the consequence of other toxic effects 
of the inhibitors, does not alter the principal conclusion 
of this study: both the initial induction of larval settlement 
and attachment and the overall rate of RNA synthesis are 
not significantly inhibited under conditions in which 
emetine and anisomycin block nearly all protein synthesis. 

Conclusions and prospects 

The embryos and larvae of marine invertebrates such 
as abalones and sea urchins provide highly tractable model 
systems for analyses of the molecular mechanisms of gene 
expression and gene regulation controlling behavior, cell 
function, cellular differentiation, and proliferation. Ad- 
vantages of these systems include: egg-to-egg cultivation; 
the large numbers of gametes, embryos, and larvae that 
are readily obtainable (in some species numbering in the 



INHIBITORS OF PROTEIN SYNTHESIS 



13 



millions): the ability to trigger development and other 
responses with high synchrony: and the accessibility of 
the genes, messenger RNAs and proteins for experimental 
analysis and manipulation. 

The conditions reported here can be used to selectively 
obtain mRNAs induced in marine invertebrate embryos 
and larvae by GABA and other morphogenetic signals in 
the absence of de now protein synthesis. The isolation 
and characterization of such primary response (or "im- 
mediate-early") transcripts will be essential to understand 
the cascade of gene expression and regulatory events 
that transduce signals such as those induced by GABA 
into morphogenetic development in the larvae of //. 
rufescens. 

Acknowledgments 

This research was supported by grants R01-RR06640 
and R01-CA53105 from the National Institutes of Health 
to D. E. M., and a National Defense Science and Engi- 
neering Graduate Fellowship to G. F. 

Literature Cited 

Balnun, A. J., and D. E. Morse. 1984. Ionic control of settlement and 
metamorphosis in larval Haliolis rufescens (gastropoda). Bid. Bull. 
167: 124-138. 

Barbacid, M., and D. Vazquez. 1975. Ribosome changes during trans- 
lation. J A/o/. Biol. 93: 449-463. 

Barbacid. M., M. Fresno, and D. Vazquez. 1975. Inhibitors of poly- 
peptide elongation on yeast polysomes. J. Antibiotics 28: 453-462. 

Baxter, G., and D. E. Morse. 1987. G protein and diacylglycerol regulate 
metamorphosis of planktonic molluscan larvae. Proe. Natl. Acad. 
Sci. L'SA 84: 1867-1870. 

Baxter, G., and D. E. Morse. 1992. Cilia from abalone larvae contain 
a receptor-dependent G protein transduction system similar to that 
in mammals. Biol. Bull. 183: 147-154. 

Bradford, M. A. 1976. A rapid and sensitive method for the quantitation 
of microgram quantities of protein utilizing the principle of protein- 
dye binding. Anal. Biochem. 72: 248-254. 

Cariolou, M. A., and D. E. Morse. 1988. Purification and character- 
ization of conchiolin shell peptides from the marine mollusc. Haliolis 
rufescens. as a function of development. / Cmp. Biochem. Physiol. 
B 157: 717-729. 

Carrasco, L., A. Jimenez, and D. Vazquez. 1976. Specific inhibitors of 
translocation by tubulosme in eukaryotic polysomes. Etirnp. J 
Biochem. 64: 1-5. 

Degnan, B. M., and D. E. Morse. 1993. Identification of eight homeo- 
box-contaming transcripts expressed during larval development and 
at metamorphosis in the gastropod mollusc Haliotis rufescens. Molcc. 
Mar Biol. Biotechnoi (in press) 

Dube, F. 1988. Effect of reduced protein synthesis on the cell cycle in 
sea urchin embryos. J. Cell. Physiol. 137: 545-552. 

Epel, D. 1972. Activation of Na + -dependent amino acid transport system 
upon fertilization of sea urchin eggs. Exp. Cell Res 72: 74-89. 

Gong, Z., and B. P. Brandhorst. 1988. Stabilization of tubulin mRNA 
by inhibition of protein synthesis in sea urchin embryos. A/o/. Cell. 
Biol. 8:3518-3525. 

Grollman, A. P. 1967. Inhibitors of protein synthesis. II. Mode of action 
of anisomycin. J. Biol. Chem. 242: 3226-3233. 



Grollman, A. P. 1968. Inhibitors of protein synthesis. V. Effects of 
emetine on protein and nucleic acid biosynthesis in HeLa cells. / 
Biol. Chem. 243: 4089-4094. 

Groppe, J., and D. E. Morse. 1989. Cloning and sequence analysis of 
novel serine protease cDNAs from the abalone. Haliotis rufescens. 
Pp. 285-288. in Current Topics in Marine Biotechnology, S. Miyachi, 
I Karuhe, and Y. Ishida, eds. Fuji Technology Press. Tokyo. 

Hille, M. B., D. C. Hall, Z. Yablonka-Reuveni, M. V. Danilchik, and 
R. T. Moon. 1981. Translational control in sea urchin eggs and 
embryos: initiation is rate limiting in blastula stage embryos. De\: 
Biol. 86:241-249. 

Hogan, B., and P. R. Gross. 1971. The effect of protein synthesis in- 
hibition on the entry of messenger RNA into the cytoplasm of sea 
urchin embryos. ./ Cell Biol 49: 692-701. 

Jaeckle, \V. B., and D. I. Manahan. 1989. Feeding by a "nonfeeding" 
larva: uptake of dissolved amino acids from seawater by lecithotrophic 
larvae of the gastropod Haliolis ni/esccns. Mar Biol. 103: 87-94. 

Jimenez, A., L. Carrasco, and D. Vazquez. 1977. Enzymic and non- 
enzymic translocation by yeast polysomes. Site of action of a number 
of inhibitors. Biochemistry 16: 4727-4730. 

Morse, A. N. C., and D. E. Morse. 1984. Recruitment and metamor- 
phosis of Haliolis larvae induced by molecules uniquely available at 
the surfaces of crustose red algae. ./ Exp. Mar. Biol. Ecol. 75: 191- 
215. 

Morse, A. N. C., C. Froyd, and D. E. Morse. 1984. Molecules from 
cyanobacteria and red algae that induce larval settlement and meta- 
morphosis in the mollusc Haliolis rufescens. Mar. Biol. 81: 293-298. 

Morse, D. E. 1985. Neurotransmitter-mimetic inducers of larval set- 
tlement and metamorphosis. Bull. Mar Sci. 37: 697-706. 

Morse, D. E. 1990. Recent progress in larval settlement and meta- 
morphosis: closing the gaps between molecular biology and ecology. 
Bull. Mar. Sci. 46: 465-483. 

Morse, D. E. 1992. Molecular mechanisms controlling larval meta- 
morphosis and recruitment in abalone larvae. Pp. 107- 1 19 in Abalone 
of the World. S. A. Shepherd. M. J. Tegner, and S. Guzman del Proo. 
eds. Blackwell. Oxford. 

Morse, D. E., H. Duncan, N. Hooker, and A. Morse. 1977. Hydrogen 
peroxide induces spawning in mollusks. with activation of prosta- 
glandm endoperoxide synthetase. Science 196: 298-300. 

Morse, D. E., N. Hooker, and A. Morse. 1978. Chemical control of 
reproduction in bivalve and gastropod molluscs. III: An inexpensive 
technique for mariculture of many species. Proc. World Maricull. 
Soc. 9: 543-547. 

Morse, D. E., N. Hooker, H. Duncan, and L. Jensen. 1979a. -y-ami- 
nobutyric acid, a neurotransmitter, induces planktonic abalone larvae 
to settle and begin metamorphosis. Science 204: 407-410. 
Morse, D. E., N. Hooker, L. Jensen, and H. Duncan. 1979b. Induction 
of larval abalone settling and metamorphosis by -y-aminobutyric acid 
and its congeners from crustose red algae, II: Applications to culti- 
vation, seed-production and bioassays; principal causes of mortality 
and interference. Proc. WorldMariw.lt. Soc. 10: 81-91. 
Morse, D. E., H. Duncan, N. Hooker, A. Baloun, and G. Young. 
1980. GABA induces behavioral and developmental metamorphosis 
in planktonic molluscan larvae. Fed. Proc. 39: 3237-3241. 
Pennington. J. T., and M. G. Hadh'eld. 1989. Larvae of a nudibranch 
mollusc (Phestilla silwgae) metamorphose when exposed to common 
organic solvents. Biol. Bull 177: 350-355. 

Sanchez, L., D. Vazquez, and A. Jimenez. 1977. Genetics and bio- 
chemistry of cryptopleurine resistance in the yeast Saccharomyces 
cerevisiae. A/o/. Gen. Genetics 156: 319-326. 

Sluder, G., J. M. Miller, R. Cole, and C. L. Rieder. 1990. Protein 
synthesis and the cell cycle: Centrosome reproduction in sea urchin 
eggs is not under translational control. / Cell Biol. 1 10: 2025-2032. 



14 



G. FENTEANY AND D. E. MORSE 



Spaulding, D. C.. and D. E. Morse. 1991. Puntication and character- 
ization of sulfatases from Halioti\ ri</t'.va'/i.v Evidence for changes 
in synthesis and heterogeneity during development. J. Comp. Phyvul 
B 161:498-515. 

Trapido-Rosenthal, II. G., and D. E. Morse. 1985. L-a.a!-Diamino 
acids facilitate GABA induction of larval metamorphosis in a gas- 
tropod mollusc (Hulmtis ni/i'.vavfv). J Cunip Pliyuol. B 155:403- 
414. 

Trapido-Rosenthal, H. G., and D. E. Morse. 1986a. Availability of 
chemosensory receptors is down-regulated by habituation of larvae 
to a morphogenetic signal. Prnc. Null. .icad. Set USA 83: 7658- 
7662. 

Trapido-Rosenthal, H. G., and D. E. Morse. I986b. Regulation of re- 
ceptor-mediated settlement and metamorphosis in larvae of a gas- 
tropod mollusc (Haliotis m/exivnx). Bull. Mar. Set. 39: 383-392. 



\\agenaar, E. B. 1983. The timing of synthesis of proteins for mitosis 

in the cell cycle of the sea urchin embryo. E.\p. Cell Ri", 1-44: 393- 

403. 
\\agenaar, K. B., and D. Mazia. 1978. The effect of emetine on hrst 

cleavage division in the sea urchin. Strongylocentrotus purpiiratiis. 

Pp. 539-545 in Cell Reproduction. E. R. Dirksen. D. M. Prescott. 

and C. F. Fox, eds. Academic Press. New York. 
\\enzel, D. G. 1967. Drug-induced cardiomyopathies. J. Pharm. Sci 

56: 1209-1224. 

\\odicka, L. M., and D. E. Morse. 1991. cDNA sequences reveal 
mRNAs for two G signal transducing proteins from larval cilia. 
linil Hull 180: 318-327. 

Yang. \V. C. I., and M. Dubrick. 1980. Mechanism of emetine car- 
diotoxicitv. Pharm. Ther. 10: 15-26. 



Referenc 



Bull 18-: 15-24. (February, 1993) 



Metamorphosis in the Brachiopod Terebratalia: 
Evidence for a Role of Calcium Channel Function and 
the Dissociation of Shell Formation from Settlement 



GARY FREEMAN 

Friday Harbor Laboratories of the L'niversity of Washington ami Center for Developmental Biology, 
Department of Zoology, University of Texas, Austin, Texas 78712 



Abstract. Larvae of Terebratalia will not undergo 
metamorphosis when maintained in a sterile environment 
unless they are 9-10 days old; under these conditions the 
frequency of normal metamorphosis is low. Four-day lar- 
vae are normally induced to metamorphose when they 
contact a suitable substrate. They will also undergo meta- 
morphosis when they are treated with high K ' seawater 
in the presence of Ca 2+ . Additional experiments indicate 
that both substrate-induced and high K f sea water-induced 
metamorphosis may involve the function of voltage-de- 
pendent calcium channels. 

Metamorphosis involves settlement of the larva fol- 
lowed by formation of the protegulum, the initial shell. 
In larvae that have been aged in a sterile environment 
and in larvae treated with high K* in seawater with low 
Ca :+ . partial metamorphosis takes place. Under these 
conditions the larva does not settle, however a protegulum 
forms. Substrate-induced metamorphosis does not occur 
in the absence of the distal end of the pedicle lobe of the 
larva which normally makes contact with the substrate, 
however, treatment with high K + seawater containing 
Ca 2+ induces partial metamorphosis in these larvae. These 
experiments suggest that there are at least two centers in 
the larva that control metamorphosis. 

Introduction 

Adult articulate brachiopods are sessile organisms. The 
larvae of these animals do not feed; they disperse the spe- 
cies as a consequence of their behavior and their ability 
to settle and metamorphose at appropriate sites. The pro- 
cess of settlement and metamorphosis has been described 

Received 14 May 1992; accepted 25 October 1992. 



for several species of articulate brachiopods (See Long 
and Strieker, 1 99 1 . for a review). These events are similar 
in all of the species examined (Fig. 1). The larva swims 
close to the substrate for a variable period of time. Settle- 
ment begins when the larva orients itself perpendicular 
to the substrate and becomes attached to it via a secretory 
product produced by the distal tip of its pedicle lobe. After 
attachment of the larva to the substrate, the mantle lobe 
flips so that instead of partially covering the pedicle lobe 
it now partially covers the apical lobe. In Terebratalia 
transversa the mantle lobe secretes a periostracum prior 
to flipping (Strieker and Reed, 1985a). Within one day 
after the mantle lobe moves to its new position, a pro- 
tegulum (initial shell) containing calcium carbonate is de- 
posited on the periostracum, which lies over the new 
outer surface of the mantle lobe and part of the pedicle 
lobe (Strieker and Reed, 1 985b). By four days, the post 
settlement part of the pedicle lobe has secreted a cuticle 
(Strieker and Reed, 1985c). At an unknown time after 
settlement the endoderm makes contact with a region 
of the apical lobe ectoderm, and a mouth is formed 
giving the metamorphosed individual the capacity to 
feed. 

Virtually nothing is known about the factors that are 
responsible for the induction of settlement and meta- 
morphosis in brachiopods. In order to induce metamor- 
phosis in Terebratalia transversa. investigators have in- 
troduced fragments of brachiopod shell, various pelecypod 
shells or Sahellaria tubes into dishes with the larvae. The 
larvae frequently settle and metamorphose on these sub- 
strates. Long and Strieker (1991) state that larvae of Ter- 
ebratalia transversa will not settle on clean glass surfaces 
or on shell fragments from which diatoms and bacteria 
have been removed. Long (1964) is cited for this 



15 



16 



G. FREEMAN 




C) 




E) 




Figure 1. Diagrammatic view of swimming larva and larvae at various 
stages of substrate induced metamorphosis. (A) Side view of swimming 
larva. The larva is composed of three lobes. The apical lobe (AL) is at 
the anterior end of the larva. Behind the apical lobe there is a mantle 
lobe (ML) that partially covers the pedicle lobe (PL). The pedicle lobe 
comprises the posterior end of the larva. The apical lobe has a ciliary 
field that is responsible for larval locomotion. This lobe also has pigmented 
eye spots at its anterior end and a population of vesicular cells at its 
distal end bordering the cleft between the apical and mantle lobes. Setae 
extend from the distal end of the mantle lobe. An internal endodermal 
mass (E) is present. (B) Larva that has attached to the substrate by its 
pedicle lobe. (C) Longitudinal section showing the pedicle and part of 
the mantle lobe of a larva that has attached to a substrate. A periostracum 
(P) has formed externally between the upper part of the pedicle lobe and 
the inner surface of the mantle lobe. The inside of the larva has a pair 
of muscles (M) that insert in the mantle and pedicle lobes. The endo- 
dermal mass is not shown. (D) Side view of a larva where the mantle 
lobe has reversed and partially covers the apical lobe. Note the new 
position of the setae. A protegulum (S) has been laid down on the penos- 
tracum secreted by the mantle and pedicle lobes. The distal part of the 
pedicle lobe is forming a cuticle (C). (E) Side view of an individual that 
has completed metamorphosis. The endodermal region has formed a 
gut which is connected to the mantle cavity by a mouth. Shell has been 
added to the protegulum by the mantle. 



information, however his dissertation does not make these 
statements. 

In some animals the timing of metamorphosis appears 
to depend on internal signals that are only indirectly in- 
fluenced by the external environment (e.g., amphibia. 
White and Nicoll, 1981). In other organisms specific cues 
from the external environment play a necessary role in 
inducing metamorphosis (Burke, 1983a). Metamorphosis 
of larvae in several groups of marine invertebrate animals 
appears to fit this latter category. One way to distinguish 
between these two possibilities is to rear larvae in an en- 
vironment that is devoid of the cues that are thought to 
induce metamorphosis and to see if metamorphosis will 
occur spontaneously. Larvae from several groups of an- 
imals that are competent to metamorphose will not un- 
dergo metamorphosis under these conditions (e.g., Free- 
man, 1981, for hydrozoans). One aim of this study is to 
find out if this is the case for brachiopod larvae. 



In those cases where specific chemical cues induce 
metamorphosis, the cues appear to activate receptor cells 
on the surface of the larva, and the receptor cells then 
activate a neuro-endocrine pathway that mediates meta- 
morphosis (Morse, 1990). In some cases where meta- 
morphosis is activated by an external chemical cue, this 
process can also be activated by depolarizing cells that 
are presumably part of the receptor or neuro-endocrine 
system that mediates metamorphosis by treating intact 
larvae with seawater containing excess K + (Yool el ai, 
1986; Cameron el al.. 1989). Presumably the K + treatment 
activates membrane potential dependent ion channels 
such as Ca 2+ or Na + channels. Another aim of this study 
is to provide evidence that settlement and metamorphosis 
in Terebratalia transversa occurs as a consequence of the 
opening of voltage-dependent calcium channels. 

Metamorphosis involves a number of changes in the 
larva that occur at specific times after settlement. These 
changes appear to be coordinated. During the course of 
this work, larvae regularly have been observed that have 
not settled and have not reversed their mantle lobe but 
have secreted a protegulum. This observation indicates 
that different components of the metamorphic response 
can be dissociated from each other. 

Materials and Methods 

The biological material 

Terebratalia tramversa were dredged or collected by 
SCUBA at various subtidal localities near San Juan Island. 
Washington. The animals were maintained in aquaria 
with running seawater. Artificially inseminated cultures 
were prepared using the methods outlined in Strathman 
(1987). Because most T. transversa oocytes from a given 
female do not fertilize, cleavage stage embryos were picked 
out of the mass culture and washed in several changes of 
pasteurized seawater (PSW) with 100 units of Strepto- 
mycin per ml to dilute out the bacteria and inhibit their 
growth. Pasteurized seawater was prepared by filtering 
seawater through a 0.45 fiM filter and heating it to 80- 
90C. for 15 min followed by cooling and aeration. The 
embryos were reared in PSW with 100 units of strepto- 
mycin per ml in Falcon 1008 35 X 10 mm petri dishes 
until they had formed larvae. Streptomycin was always 
added to PSW immediately before use. The PSW with 
streptomycin and the petri dishes used were changed every 
other day. All experiments were carried out at 12-13C. 

For some experiments, four-day larvae were produced 
that had the distal tip of their pedicle removed. This op- 
eration was done by placing a larva in a Falcon plastic 
dish and using an electrolytically sharpened tungsten nee- 
dle to cut through the pedicle lobe. 



IONIC CONTROL OF METAMORPHOSIS 



17 



The induction of metamorphosis 

Two methods were used for eliciting metamorphosis. 
Larvae were either exposed to natural substrates that in- 
duce metamorphosis or they were treated with seawater 
containing an excess of K* for a short time period to 
depolarize their cells. Natural substrate experiments were 
carried out in sterile flat bottom 10 X 15 mm Linbro 
sterile plastic dishes. One ml of cloth-filtered natural sea- 
water was placed in the dishes and about 40 fragments of 
shell with external surface from a freshly smashed T. 
transversa or sand grains from the outside of a Sabellaria 
tube were added to the dish. About 20 four-day-old larvae 
were placed in the dish and the dish was incubated in the 
dark for 1 day. If 50% or more of the larvae had attached 
and reversed their mantle lobe, all of the animals were 
removed from the dish and the dish was set aside for sub- 
sequent experiments. Between 25 and 50% of the dishes 
were suitable for experimental purposes. When high K + 
seawater was used to induce metamorphosis, larvae were 
incubated in the high K f seawater for a defined period of 
time (30 min in most experiments), then washed in several 
changes of PSW to dilute out the K + and set aside in 
sterile Linbro dishes. They were assayed for metamor- 
phosis at 24 h. In those experiments where larvae were 
treated with high K + in a modified seawater that lacked, 
or had a lower or a higher than normal amount of a given 
ion, the larvae were washed several times in the modified 
seawater before treatment with the high K/ modified sea- 
water. Table I gives the ionic composition of the different 
seawaters used. 

Histological work 

Larvae were fixed in 1% osmium in PSW in the cold 
for one hour, stored in 70% ethanol, dehydrated through 
an alcohol series, transferred to propylene oxide and 



embedded in an Epon equivalent. Sections were cut at 
and stained with methylene blue. 



Results 

Can metamorphosis occur autonomously? 

The first aim of this study was to find out if articulate 
brachiopod larvae would autonomously settle and un- 
dergo metamorphosis when reared in an environment 
with no external cues. Cohorts of 20 four-day-old larvae 
were set up in Linbro dishes in PSW with streptomycin. 
Each day of the experiment all of the larvae were examined 
for settlement and reversal of the mantle lobe and a cohort 
of larvae was examined with a compound microscope 
equipped with polarizing filters for shell mediated bire- 
fringence. Every other day. the larvae that were not used 
for birefringence studies were transferred to new sterile 
Linbro dishes with PSW containing streptomycin. The 
results of one of these experiments is presented in Ta- 
ble II. 

Normal metamorphosis, settlement, reversal of the 
mantle lobe and formation of the protegulum was initiated 
only after larvae had been cultured in a sterile environ- 
ment for at least 9 days; the percentage of larvae showing 
normal metamorphosis was low (29% at 10 days). Partial 
metamorphosis also took place in this experiment. Partial 
metamorphosis occurs when a larva does not settle and 
the mantle lobe is not reversed, but a birefringent mass 
forms in association with the mantle lobe (compare Fig. 
2A and B). In a swimming larva, the eye spots in the 
apical lobe and the retractor muscles inside the pedicle 
lobe are birefringent. These can easily be distinguished 
from mantle lobe birefringence which is much stronger. 
The birefringent mass associated with the mantle lobe 
can be isolated by placing living larvae in 10%. sodium 
hypochlorite in a 0.1 M phosphate buffer at pH 7 and 



Table I 



Ionic composition of artificial seawaters (inM) 



Salt 


Artificial 
SW 


High K + 
SW 


Na + -free 
SW 


High K* 
Na+-free SW 


Ca 2+ -free 
SW 


High K + 
Ca 2+ -free SW 


Mg 2+ -free 
SW 


High 1C 
Mg 2+ -free SW 


NaCl 


425.0 


262.5 


0.0 


0.0 


425.0 


262.5 


425.0 


262.5 


KC1 


9.4 


279.5 


9.4 


280.8 


9.4 


279.7 


9.4 


279.5 


CaCl, 2H 2 O 


9.0 


9.0 


9.0 


9.0 


0.0 


0.0 


9.0 


9.0 


MgCl : H 2 O 


22.1 


111 


22.1 


11.05 


22.1 


11.1 


0.0 


0.0 


MgS0 4 


25.6 


12.8 


25.6 


12.8 


25.6 


12.8 


0.0 


0.0 


NaHCO, 


2.1 


1.1 


0.0 


0.0 


2.1 


1.1 


2.1 


1.1 


TES buffer 


10.0 


5.0 


10.0 


5.0 


10.0 


5.0 


10.0 


5.0 


Choline Cl 






425.0 


262.5 










ICHCOj 






2.1 


1.1 










Na,S0 4 














25.0 


12.5 



All seawaters had their pH adjusted to 7.9; IM NaOH was used to adjust the pH except in Na :+ -free seawater where l.U KOH was used. 



18 G. FREEMAN 

Table II 

Settlement, normal metamorphosis, and partial metamorphosis in larvae reared under sterile conditions 



Experiment 
number 


Days in 
culture 


Number 
settled 


Mantle lobe 
reflected 


Sample of 
swimming 
Birefnngent larvae 


Percent 

birefringent 


1 


5 


0/126 




19 







6 


0/102 




18 







7 


0/81 




19 







8 


0/59 




17 







9 


1/40 


1 


1 IX 


39 




1(1 


6/21 


6 


3 15 


87 


2A 


7 


0/78 




15 







8 


0/61 




14 







9 


1/47 


1 


13 







Id 


0/3 1 




15 


40 




11 


0/14 




14 


50 


2B 


7 


0/76 




14 







8 


0/60 




13 







9 


0/44 




13 


(1 




III 


2/29 


2 


14 


36 




11 


1/14 


1 


1 13 


54 



changing the solution at frequent intervals until the or- 
ganic material that makes up the larva is dissolved. One 
is left with a birefringent mass (Fig. 2C). When the bire- 
fringent mass was transferred to weak acid (0.1 M HC1), 
it effervesced as it dissolved indicating that it was com- 
posed of calcium carbonate. Sections through partially 
metamorphosed larvae showed that the calcium carbonate 
mass was located on the side of the mantle lobe that cov- 
ered the pedicle lobe (Fig. 2D). This is the side of the 
mantle lobe on which the protegulum would have formed. 
The onset of partial metamorphosis was variable; In Ex- 
periment 1 in Table 2 it was observed as early as day nine. 
In a replica of this experiment with another batch of larvae 
(data not shown) partial metamorphosis was not observed 
until day 12 of culture. By the second day after the initial 
appearance of partial metamorphosis it was seen in 50- 
75% of the larvae. 

A group of larvae may produce enough of a metabolite 
that induces metamorphosis during a two-day culture pe- 
riod to potentiate group metamorphosis. This possibility 
was examined in Experiment 2 (Table II). The onset of 
metamorphosis was compared for larvae from the same 
batch reared in groups of 16-20 in one ml dishes (Exper- 
iment 2A) or individually in one ml dishes (Experiment 
2B). There was no difference in the onset of metamor- 
phosis under these two culture conditions. 

The ionic induction of metamorphosis: evidence for the 
involvement of voltage-dependent calcium channels 

Four-day-old larvae were treated with high K + seawater 
(Table 1) for 30 min and set aside to see if they would 



metamorphose. The high K* seawater presumably de- 
polarizes the cells of the larvae. In the high K + seawater 
the larvae stop swimming and show signs of sticking to 
the container. This treatment induces normal metamor- 
phosis and partial metamorphosis in from 25-90% of the 
larvae, depending on the batch, when metamorphosis is 
assayed 24 h later (Fig. 3). Among the larvae that respond 
to high K + seawater, 40-90% undergo normal metamor- 
phosis while the remainder undergo partial metamorpho- 
sis. Experiments where cohorts of larvae from the same 
batch were treated with high K + seawater for varying pe- 
riods of time showed that a 5-min incubation in high K + 
seawater will not induce normal or partial metamorphosis 
while treatment with high K + seawater for 15, 30, or 45 
min induces normal or partial metamorphosis with the 
same frequency. Treatment with high K + seawater for 
one or two hours reduces the percentage of larvae under- 
going normal or partial metamorphosis (data not shown). 
While these experiments were done with a seawater with 
280 mM K + , artificial seawater with 1 14 mM K + , 9 mM 
Ca 2+ and slightly higher concentration of the other salts 
are equally effective (data not shown). These experiments 
suggest that the opening of voltage-dependent ion channels 
in appropriate cells may play a role in inducing meta- 
morphosis. 

The most common voltage-dependent ion channels are 
the calcium, sodium and potassium channels (Hille, 
1984). The function of these different ion channels fol- 
lowing membrane depolarization can be distinguished 
using the following criteria. ( 1 ) The movement of ions 
across cell membranes via these channels depends on their 
concentration in the external medium and the cytosol of 



IONIC CONTROL OF METAMORPHOSIS 



19 







Figure 2. Photographs of larvae that have undergone normal or partial metamorphosis and the protegulum 
from a larva that has undergone partial metamorphosis. (A,) Larva undergoing normal metamorphosis. 
The mantle lobe partly covers the apical lobe. Note the position of the setae and the eye spots of the apical 
lobe. (A 2 ) The same larva viewed with polarized light showing its birefnngent protegulum and eye spots. 
(B,) Partially metamorphosed larva. The mantle lobe partially covers the pedicle lobe and the setae have a 
position typical of a swimming larva. (B : ) Same larva viewed with polarized light showing the birefnngent 
protegulum and eye spots. (C,) Isolated protegulum from a partially metamorphosed larva. Note the setae 
embedded in the protegulum. (C 2 ) The same protegulum viewed with polarized light showing its birefringence. 
A-C are at the same magnification; the bar indicates 50 tiM (D) Longitudinal section through a partially 
metamorphosed larva. Note the periostracum (P) between the mantle (ML) and the pedicle lobes (PL) and 
the space where the calcium carbonate (S) had been deposited. The bar indicates 100 tiM 



the cell. For example, in Ca 2+ -free seawater the depolar- 
ization of cells should have no effect if internal Ca 2 ' levels 
must rise via calcium channels to initiate metamorphosis, 
(2) The function of specific ion channels is inhibited by 
channel blockers. For example, the ions Mg 2f and Co 2+ 
and the drug nifedipine block calcium channel functions 
at concentrations that have no effect on sodium or po- 
tassium channel function. 

Treatment of four-day-old larvae for 30 min with high 
K + in Na + -free seawater induces normal and partial 
metamorphosis with the same frequency that normal and 
partial metamorphosis are induced in high K + seawater 
(Fig. 3A). Treatment of four-day-old larvae for 30 min 
with Na'-free seawater, had no effect on metamorpnosis 
(data not shown). Treatments of four-day-old larvae with 



high K + in Ca 2+ -free seawater inhibited both normal and 
partial metamorphosis (Fig. 3B). These larvae were not 
damaged by the treatment in Ca 2+ -free seawater because 
when some of these treated larvae were subsequently 
treated with high K + seawater, they were induced to un- 
dergo normal and partial metamorphosis. 

The effects of the calcium channel blockers Co 2+ and 
nifedipine on high K 4 seawater induced metamorphosis 
were tested (Fig. 3C, D). The Co 2+ was prepared as a 360 
mM CoCl 2 stock solution that was diluted appropriately 
with high K + seawater. The nifedipine was prepared as a 
10 mAl stock solution in ethanol and diluted appropriately 
in high K + seawater. When four-day-old larvae were in- 
incubated for 30 min in high K + seawater with either 
50 mM Co 2+ or 0.2 mM nifedipine, normal and partial 



20 



G. FREEMAN 



High K* SW 

High K* in Na free SW 

High K* in Mg 2 * free SW 

High K* SW 
High K*m Ca free SW 

High K* SW 

High K* SW *10 mM Co 2 ' 
High K* SW + 50 mM Co 2 * 

High K*SW 

High K* SW + 1 mM Niledipme 
High K* SW + 2 mM Ntfedipme 

High K* SW + 2 25 mM Ca 2 * 

High K* SW + 4 5 mM Ca 2 * 

High K* SW * 9 mM Ca 21 

HighK'SW* lemMCa 2 * 

High K* SW + 27 mM Ca 2 ' 



] 




139 






* 1 




1 




35 












a 








'//A 


1 3fi 




46 

;;::;;;: 

ISO 
33 


3- 










9 


35 






,..'A 
20 

134 

"j 


H37 

]4T 












. ] 




Z]33 










Z]37 








'.: 1 


|3J 



20 40 60 80 100 

Percenl Metamorphosed 

Figure 3. Histograms showing the effect of treatment with high K* 
in seawaters that lack Na*, Ca 2 *, or Mg 2 *, in high K* seawater with 
different concentrations of Ca 2+ and in high K* seawater with calcium 
channel blockers on metamorphosis. The hatched segment of each bar 
indicates the proportion of cases that underwent normal metamorphosis. 
The clear segment of the bar indicates the percentage of cases that un- 
derwent partial metamorphosis. The number of cases is indicated at the 
top of the bar. Experiments A-E were each done with a batch of larvae 
from a separate female. 



metamorphosis was completely inhibited. When four-day- 
old larvae were incubated for 30 min in high K + seawater 
with the concentration of ethanol used to make the 0.2 
mM nifedipine solution, metamorphosis was not inhibited 
(data not shown). The larvae that were treated with 50 
roA/ Co 2+ or 0.2 mM nifedipine were not damaged by 
these treatments because when some of these larvae were 
subsequently treated with high K + seawater in the absence 
of these calcium blockers, many of them were induced to 
undergo normal or partial metamorphosis. The calcium 
channel blocker Mg 2+ is a normal constituent of seawater. 
Treatment of four-day-old larvae with high K + in Mg 2+ - 
free saltwater induces both normal and partial metamor- 
phosis in a higher percentage of cases than in high K + 
seawater (Fig. 3A). Treatment of four-day-old larvae for 
30 min with Mg 2+ -free seawater had no effect on meta- 
morphosis (data not shown). When four-day-old larvae 
are treated with high K f seawater in the presence of the 
sodium channel blocker tetrodotoxin at a concentration 
of 20 nA/, metamorphosis was not inhibited. 

In an additional experiment, the effect of varying the 
external concentration of Ca 2+ on high K + seawater me- 
diated metamorphosis was tested by incubating four-day- 
old larvae for 30 min in an appropriate high K + seawater. 
The normal Ca 2+ concentration in seawater is 9 mM. 
Concentrations of Ca 2+ were used that were lower or 
higher than the normal concentration (Fig. 3E). At con- 
centrations of 2.25 mMCa 2+ high K + seawater had almost 



no effect on metamorphosis. At concentrations between 
4.5 and 9 mA/Ca :+ the percentage of larvae undergoing 
metamorphosis increased; between 4.5 and 18 mMCa 2+ 
the proportion of larvae showing normal metamorphosis 
increased. Incubating four-day-old larvae for 30 min in 
artificial seawater with 18 or 27 mM Ca 2+ had no effect 
on metamorphosis. The response to high K. 4 seawater in 
the presence of elevated levels of Ca 2+ was comparable to 
the responses to high K + seawater in the absence of the 
calcium channel blocker Mg : + . In both cases there was a 
significant increase in the percentage of larvae that un- 
derwent normal as opposed to partial metamorphosis. 

Does substrate induced metamorphosis involve putative 
voltage-dependent calcium channel function'.' 

Substrate induced metamorphosis presumably involves 
a random walk on the part of the larva until it makes 
contact with a substrate bound inducer that elicits meta- 
morphosis. These experiments were done to find out if 
inhibitors of calcium channel function, Co 2+ . nifedipine, 
and Ca 2+ -free seawater, also inhibit substrate induced 
metamorphosis and if Mg 2 + -free seawater or high Ca 2+ 
seawater, which facilitate calcium channel function, also 
facilitate substrate induced metamorphosis. Some of these 
experiments were not feasible. Incubation of larvae in Ca 2+ 
or Mg :+ -free seawater for 24 h leads to a marked decrease 
in the adhesive bonds between cells, causing some cell 
dissociation. It was possible to do experiments in low Ca 2+ 
seawater (2.25 mM). When larvae were incubated in sea- 
water with 50 mM Co 2+ or 0.2 mM nifedipine, both agents 
caused the larvae to stop swimming after a few hours ren- 
dering them unable to sample the substrate. After about 
12 h the nifedipine began to come out of solution and 
form crystals at the air-water interface; as this happened, 
the larvae began to locomote again. It was possible to 
incubate larvae in seawater with 10 mMCo 2+ ; under these 
conditions larval locomotion slowed down but did not 
stop. 

Four-day-old larvae were used for these experiments. 
Substrate induced metamorphosis differs from high K + 
induced metamorphosis in that the larvae either undergo 
natural metamorphosis, which was assayed 24 h after the 
experiment had been set up, or they retain their larval 
character. Very few cases of partial metamorphosis were 
observed. Over 100 larvae that were swimming after 
24 h in the experiments without the channel blocker Co 2+ , 
or low or high Ca :+ were examined; only three of these 
cases had a birefringent mantle lobe. Both 10 mM Co 2+ 
and low Ca 2+ seawater inhibited substrate induced meta- 
morphosis (Fig. 4A, B). Treatment with Co 2+ or low Ca 2+ 
seawater for 24 h did not damage these larvae because 
when they were introduced into a dish with a substrate 
that would induce metamorphosis or treated with high 



IONIC CONTROL OF METAMORPHOSIS 



21 



Nalural SW 
Natural SW. 10 mM Co 2 * 

Artifical SW 
Artificial SW + 2 25 mM Ca 2 * 
Artificial SW. IBmMCa 


'WZfZM: 


. ...' ' 1 


1 69 


|w 








<%%%%%%& 


'%%%%%%%, 


|S9 


79 






;...;_:/:_ 


'MfM 77 


-I 1 1 1 1 1 
20 40 60 8 

Percent Metamorphosed 



Figure 4. Histograms showing 
with lower than normal or higher 
or the calcium channel blocker Co 2+ 
The hatched segment of each bar 
underwent normal metamorphosis 
the top of the bar. Experiments A 
separate females. 



the effect of treatment with seawater 
than normal concentrations of Ca 2 * 
on substrate induced metamorphosis, 
ndicates the proportion of cases that 
The number of cases is indicated at 
and B were done with larvae from 



K + seawater for 30 min, over half of these larvae under- 
went metamorphosis. The Co 2+ and low Ca 2+ seawater 
treatments did not alter the ability of the substrate in the 
Linbro dishes to induce metamorphosis. When the sea- 
water with the Co 2+ and the low Ca 2+ seawater was re- 
moved from the wells and replaced by natural seawater 
and a batch of four-day larvae were added to the dish, 
metamorphosis was induced in more than 50% of the 
cases at 24 h. When larvae were placed in dishes with a 
substrate that would induce metamorphosis under con- 
ditions where the Ca 2+ was higher than normal ( 1 8 mA/), 
a higher proportion of larvae underwent metamorphosis 
than in dishes of seawater with the normal amount of 
Ca 2+ (9 mA/) (Fig. 4B). These experiments suggest that 
calcium channel function may also play a role in substrate 
induced metamorphosis. 

The role oj the pedicle lobe in metamorphosis 

Prior to substrate induced metamorphosis the larva ap- 
proaches the site where it will settle with its pedicle lobe, 
makes contact and adheres to the substrate with the distal 
end of its pedicle lobe. The possibility exists that there 
are substrate receptor cells in the pedicle that have to be 
activated to initiate metamorphosis. These may be the 
same cells that contain putative voltage-dependent cal- 
cium channels whose activation is necessary for meta- 
morphosis. 

In order to test this hypothesis four-day-old larvae were 
operated on to remove the distal portion of their pedicle 
lobe (Fig. 5), and two hours after the operations were 
completed they were tested to see whether or not they 
could undergo substrate induced or high K + seawater in- 
duced metamorphosis (Fig. 6). None of these larvae settled 
or reversed their mantle lobe. Settling should not be ex- 
pected because the distal part of the pedicle lobe is missing, 
and reversal of the mantle lobe is also not expected because 




Figure 5. Diagram of a swimming larva showing the operation to 
remove the distal part of the pedicle lobe. 



the retractor muscles that presumably play a role in mov- 
ing the mantle lobe have been cut (see Discussion). All 
of the larvae with the distal end of the pedicle lobe missing 
swam around the Linbro dish among the substrates in a 
normal manner; at 24 h none of these larvae exhibited 
mantle birefringence indicating that substrate induced 
metamorphosis had not occurred. Many of the larvae with 
the distal end of the pedicle lobe missing that were treated 
with high K + seawater underwent partial metamorphosis 
when assayed at 24 h; however, the percentage of cases 
undergoing partial metamorphosis was not as high as it 
was for intact four-day-old larvae from the same batch. 
These results suggest that substrate receptors needed for 
metamorphosis are located in the pedicle lobe and that 
the removal of the distal region of the pedicle makes the 
larva unresponsive to substrate mediated cues, however, 
other cells with putative voltage-dependent calcium 
channels whose activation is necessary for metamorphosis 
are located in another region of the larva. 

Can larvae that have undergone partial metamorphosis 
subsequently undergo normal metamorphosis? 

It is not clear whether partial metamorphosis is the 
result of the activation of a metamorphic pathway or an 
epiphenomenon that is not related to metamorphosis. The 
strongest evidence that partial metamorphosis is related 
to normal metamorphosis is that both can be induced 
with high K + seawater and that both can occur sponta- 
neously at the same time in aging larvae reared under 



Substrate - Intact larvae 

Substrate No peddle 

H.gh K* SW - Inlacl Larvae 

High K + SW No pedicle 



20 40 60 

Percent Metamorphosed 

Figure 6. Histograms showing the effect of pedicle removal on sub- 
strate or high K + seawater induced metamorphosis. Metamorphosis was 
measured as larvae with birefringent mantle lobes. The number of cases 
is indicated at the top of the bar. The histograms combine data from 
two experiments on larvae from separate females. 



22 



G. FREEMAN 



sterile conditions. To better understand partial meta- 
morphosis, experiments were done to find out if larvae 
that had undergone partial metamorphosis could respond 
to substrates that induce metamorphosis or high K + sea- 
water by undergoing normal metamorphosis. 

A large batch of four-day-old larvae were induced to 
undergo metamorphosis using high K/ seawater. Under 
these conditions some of the larvae underwent normal 
metamorphosis, some underwent partial metamorphosis 
and some larvae had not metamorphosed when they were 
assayed one day later. The five-day-old larvae that had 
undergone partial metamorphosis and those that had not 
metamorphosed as a consequence of the high K + seawater 
treatment and five-day-old larvae from the same batch 
that had not previously been treated with high K* seawater 
were exposed to a substrate that would induce metamor- 
phosis or high K + seawater with 18 mM Ca :+ which in- 
duces normal metamorphosis in a high percentage of 
cases. The results of these experiments (Fig. 7) show that 
larvae which have already undergone partial metamor- 
phosis will not respond to a natural substrate that induces 
metamorphosis or to high K* seawater with 18 mA/Ca 2+ 
by settling or reversing their mantle. Reversal of the man- 
tle may not be possible for these larvae because of the 
calcification of their mantle lobe; some movement of the 
lobe in these partially metamorphosed larvae is possible 
because when they are stimulated by prodding with a 
tungsten needle, the setae of the larvae take on a transient 
position perpendicular to the body. This same kind of 
movement takes place in normal larvae. There is no ob- 
vious reason why partially metamorphosed larvae should 
not be able to attach to the substrate. When larvae that 
had been treated with high K. + seawater that did not un- 
dergo normal or partial metamorphosis were treated with 
a natural substrate that induces metamorphosis or high 
K + seawater with 18 mMCa 2+ , many of these larvae un- 
derwent natural or partial metamorphosis. The percentage 
of pretreated larvae that underwent metamorphosis was 
lower than the percentage of larvae metamorphosing that 
had not been pretreated with high K + seawater. This sug- 
gests that larvae which have been exposed to a metamor- 
phic stimulus and do not respond, may either be less 
competent to respond to a metamorphic inducer and have 
been selected for via the pretreatment, or prior exposure 
to a metamorphic inducer may render these larvae less 
competent to respond to a subsequent metamorphic cue 
even though they have not undergone metamorphosis. 

As part of this experiment some larvae that settled, but 
had not yet undergone mantle reversal, were gently re- 
moved from their substrate with a tungsten needle so that 
their pedicle lobe was not damaged and transferred to 
PSW in a sterile Linbro dish, while other larvae that had 
not undergone mantle reversal were left in place. All of 
the larvae that had not yet undergone mantle reversal and 



Melamorphic substrate - Partial melamorphosis 
High K* SW - Partial metamorphosis 
Melamorphic subslrale - No metamorphosis 
High K* SW - No metamorphosis 

Metamofphic substrate 
High K* SW 


18 
19 

W////////S 


W&42 








.'"1 \43 






,: r : >%/. 


80 






.. -. - \ \ss 




1 1 -nr | i [ 







20 40 60 80 

Percent Metamorphosed 

Figure 7. (A) Histograms showing the effect of pretreatment at four 
days of larvae with high K + seawater that either induced partial meta- 
morphosis or no metamorphosis on the ability of these two categories 
of larvae to undergo metamorphosis after treatment with a metamor- 
phosis substrate or high K + seawater at five days. (B) Histograms showing 
effect of treatment with a metamorphosis substrate or high K* seawater 
at five days on metamorphosis of larvae that had not been pretreated 
with high K + seawater at four days. The hatched segment of each bar 
indicates the proportion of cases that underwent normal metamorphosis. 
The clear segment ol the bar indicates the percentage of cases that un- 
derwent partial metamorphosis. The number of cases is at the top of the 
bar. 



were not disturbed underwent normal metamorphosis by 
the next day (six cases). The larvae that were removed 
from their settlement site did not resettle but underwent 
partial metamorphosis (four out of six cases). This ex- 
periment suggests that settlement is necessary for normal 
metamorphosis. 

Discussion 

The role of voltage-dependent Ca 2+ channels in 
metamorphosis 

The following lines of evidence indicate that voltage- 
dependent calcium channels may play a role in meta- 
morphosis: ( 1 ) Treatment of larvae with high K + seawater 
which presumably depolarizes the cells of the larva induces 
metamorphosis and treatment of larvae with high K + in 
Na + -free seawater is just as effective in inducing meta- 
morphosis, (2) Treatment of larvae with high K 1 in Ca 2+ - 
free seawater inhibits metamorphosis, (3) Treatment of 
larvae with high K + in seawater with elevated Ca 2+ levels 
or Mg 2+ -free seawater increases the percentage of cases 
metamorphosing, (4) Treatment of larvae with high K + 
seawater in the presence of the calcium channel blockers 
Co 2+ and Nifedipine inhibits metamorphosis. In order to 
make this work more convincing one would have to dem- 
onstrate electrophysiologically that target cells are not only 
depolarized but give an action potential which is typical 
of voltage-dependent Ca 2+ channels and that Ca 2+ moves 
into the target cells from the external environment during 
depolarization. 

The identities of the target cells where voltage-depen- 
dent calcium channels function to mediate the meta- 
morphic stimulus is not known. One possible target cell 



IONIC CONTROL OF METAMORPHOSIS 



23 



candidate is a subset of cells in the larval nervous system. 
There is evidence that the nervous system receives and 
mediates the metamorphic stimulus in echinoid larvae 
(Burke, 1983b). Unfortunately, virtually nothing is known 
about the organization of the nervous system in articulate 
brachiopod larvae; however, nerve cell processes have 
been noted in ultrastructural studies done on these larvae 
for other purposes (Strieker and Reed, 1985a). Another 
possible set of target cells could be some of the cells that 
make up the surface epithelium of the larva (e.g., the cells 
of the distal part of the pedicle lobe). After these cells 
receive a metamorphic stimulus, it could be transferred 
to other epithelial cells of the larva by epithelial conduc- 
tion. There is evidence that epithelial conduction mediates 
the metamorphic stimulus in hydrozoans (Freeman and 
Ridgway, 1990). 

Both substrate and high K + seawater induced meta- 
morphosis appear to depend on calcium channel function. 
Substrate induced metamorphosis also depends on the 
pedicle lobe while high K + seawater induced metamor- 
phosis does not. The simplest model that accounts for 
these results is that there is a substrate-induced meta- 
morphosis receptor at the distal end of the pedicle lobe. 
When this is activated a metamorphic signal is sent from 
this site to cells outside of the distal region of the pedicle 
lobe that must have their putative voltage-dependent cal- 
cium channels activated in order to spread the metamor- 
phic stimulus (Fig. 8). When the cells outside the distal 
region of the pedicle lobe are activated, they also send an 
inhibitory signal to the cells in the distal region of the 
pedicle lobe preventing them from responding to substrate 
mediated metamorphic cues (Fig. 7). 

The signijii'iiiur <>/ '"partially metamorphosed" larvae 

The partially metamorphosed larva is most probably 
the result of an abnormal metamorphic response. This 
larva is characterized as a larva that forms a protegulum 
in the absence of mantle reversal and settlement. Because 
the formation of a protegulum under these conditions 
probably renders the mantle lobe incapable of reversal 
and because the mantle lobe does not spread out to occupy 
a larger area as it does after reversal, this metamorphic 
response is probably maladaptive. I have made only lim- 
ited attempts to look for later manifestations of normal 
metamorphosis in partially metamorphosed larvae. Two 
partially metamorphosed larvae were fixed and sectioned 
four days after the initiation of high K + seawater induced 
metamorphosis. Both of these larvae showed suggestions 
of cuticle deposition by the pedicle. In order to make this 
point with certainty, it would be necessary to do a study 
of these larvae at an electron microscope level of resolu- 
tion. I did not observe any indication of mouth formation 
in these partial larvae; however, they may not have been 
cultured long enough. 





N 






PI 








/ 




!/ 


i 


>* 




i \ 








\ 


\ 











Figure 8. Diagrammatic view of a swimming larva with an apical 
lobe (AL). mantle lobe (ML), and pedicle lobe (PL). At the distal end of 
the pedicle lobe there is a postulated center composed of cells ( 1 ) which 
may use voltage-dependent calcium channels to transduce a substrate 
mediated metamorphic signal. This center sends a stimulatory meta- 
morphic signal to other cells in the larva including center (2) which 
functions via voltage-dependent calcium channels that acts as a secondary 
metamorphic center. Here this center is shown in the mantle lobe but 
it could be any place outside of the distal end of the pedicle lobe. The 
cells of this secondary metamorphic center send a stimulatory meta- 
morphic signal to other cells of the larva and an inhibitory signal to the 
cells that transduce the substrate mediated metamorphic signal turning 
off the metamorphic stimulus from these cells. This model accounts for 
the experiments described in this paper. 



A variety of factors probably play a role in generating 
the partial metamorphosis phenotype. In larvae that have 
been reared for a number of days in a sterile environment 
intrinsic maturational changes may occur so that various 
parts of the metamorphosis signaling pathway or cells that 
respond to the signaling pathway may be activated. If the 
postulated distal pedicle lobe substrate receptor cells were 
activated, an aged larvae may undergo normal metamor- 
phosis. This happened in a small percentage of cases (Ta- 
ble II). If cells that are part of the metamorphic pathway 
that reside outside of the distal region of the pedicle lobe 
are activated or if cells that will form the protegulum are 
activated, a larva that shows the partial metamorphosis 
phenotype would be generated. There is evidence that in 
some species with a bathy-pelagic life cycle that larvae 
which do not see an appropriate metamorphic cue in na- 
ture will metamorphose or partially metamorphose and 
still continue a pelagic existence (Thorson, 1946; Paine, 
1963). 

The mechanics of mantle lobe reversal during meta- 
morphosis are not understood. There is a pair of muscles 
that insert in the mantle lobe and the pedicle lobe that 
are thought to contract during metamorphosis causing 
the mantle lobe to flip (Franzen, 1969; Long, 1964). Sub- 
strate adhesion by the pedicle lobe may be necessary for 
these muscles to contract or to cause the pedicle lobe to 
be compressed in an appropriate way as the muscles con- 
tract so that the mantle lobe is reversed. The production 
of larvae that show partial metamorphosis following sub- 
strate detachment could occur because protegulum for- 
mation is activated even though mantle reversal is inhib- 
ited. The small number of cases where partial metamor- 



24 



G. FREEMAN 



phosis occurs following the culture of larvae in the 
presence of substrates that induce metamorphosis can be 
explained in this way. Partially metamorphosed larvae 
and the conditions where they are formed provide an in- 
sight into the normal metamorphosis process. 

Acknowledgments 

I am grateful to Dr. A. O. D. Willows and the staff of 
the Friday Harbor Laboratories for their hospitality. I want 
to thank Sarah Cohen and her diving companions for 
collecting animals using SCUBA, Dr. Craig Staude for 
saving animals for me that were collected on dredging 
trips, and Drs. Alan Kohn and Patricia Morse for letting 
me use animals that were dredged for class use. I want to 
thank Judith Lundelius, Bob Goldstein, and Hyla Sweet 
for their comments on this manuscript. This work was 
supported by NSF grant DCB-8904333 and a URI re- 
search leave from The University of Texas. 

Literature Cited 

Burke, R. D. 1983a. The induction of metamorphosis of marine in- 
vertebrate larvae: stimulus and response. Can. J Zoo/. 61: 1701- 
1719. 

Burke, R. D. I983b. Neural control of metamorphosis in Dendraster 
excentricus. Biol. Bull. 164: 176-188. 

Cameron, A., T. Tosteson, and V. llensley. 1989. The control of sea 
urchin metamorphosis: ionic effects. Develop. Growth Differ. 31: 589- 
594. 

Franzen, A. 1969. On larval development and metamorphosis of Ter- 
cbralulina Brachiopoda. /<><>/, Bid. Uppsala 38: 155-174. 

Freeman, G. 1981. The role of polarity in the development of the hy- 
drozoan planula larva. Rou.\'s Arch. Dev Biol. 190: 168-184. 



Freeman, G.. and K. B. RidgHa\. 1990. Cellular and intracellular path- 
ways mediating the metamorphic stimulus in hydrozoan planulae. 
Rou\'sArch. Dcv. Biol. 199: 63-79. 

Hille, B. 198-1. Ionic Channels and Excitable Membranes Sinauer As- 
soc., Sunderland, MA. 

Long, J. A. 1964. The embryology of three species representing three 
superfamilies of articulate brachiopoda. Ph.D. Dissertation. University 
of Washington. 

Long, J. A., and S. A. Strieker. 1991. Brachiopoda. Pp. 47-84 In Re- 
production in Marine Invertebrates, I 'ol. 6 Echinoderms ami Lopho- 
phorales. A. C. Giese, J. S. Pearse. and V. B. Pearse, eds. Boxwood 
Press. Palo Alto. CA. 

Morse, D. K. 1990. Recent progress in larval settlement and meta- 
morphosis: closing the gaps between molecular biology and ecology. 
Bull. Mar Sci. 46: 465-483. 

Paine, R. 1963. Ecology of the brachiopod Glottidia pyramidata. Ecol. 
Monogr. 33: 187-213. 

Strathman, M. 1987. Reproduction ami Development of Marine I/t- 
vertehrates oj the Northern Pacific Coast. University of Washington 
Press, Seattle. WA. 

Strieker, S. A., and C. G. Reed. 1985a. The ontogeny of shell secretion 
in Terehratalia transversa (Brachiopoda, Articulata) I. Development 
of the mantle. ./ Morphol. 183: 233-250. 

Strieker, S. A., and C. G. Reed. 1985b. The protegulum and juvenile 
shell of a recent articulate brachiopod: patterns of growth and chemical 
composition. Lclhaia 18: 295-303. 

Strieker, S. A., and C. G. Reed. 1985c. Development of the pedicle in 
the articulate brachiopod Terebratalia transversa ( Brachiopoda, Ter- 
ebratulida) '/.oomorphology 105: 253-264. 

Thorson, G. 1946. Reproduction and larval development of Danish 
marine bottom invertebrates. Medd. Komm. Dan Fisk: Havundersog 
Ser. Plankton 4: 1-523. 

White, B. II., and C. S. Nicoll. 1981. Hormonal control of amphibian 
metamorphosis. Pp. 363-396 in Metamorphosis: A Problem in De- 
velopmental Biology. L. I. Gilbert and E. Frieden, eds. Plenum, New 
York. 

Yool, A. J., S. M. Green, M. Hadfield, R. Jensen, D. Markell, and I). 
Morse. 1986. Excess potassium induces larval metamorphosis in 
four marine invertebrate species. Biol. Bull. 170: 255-266. 



Reference: Biol. Bull 184: 25-35. (February, 1993) 



Species Relationships in a Marine 
Gastropod-Trematode Ecological System 

LAWRENCE A. CURTIS' AND KAREN M. K. HUBBARD* 

University Parallel, * School of Life Sciences, and College of Marine Studies. 
University of Delaware. Newark, Delaware 19716 



Abstract. Individual snails (Ilyanaxsa obsoleta) on Cape 
Henlopen, Delaware, frequently are host to one or more 
trematode species. When different species occupy the same 
host, interactions might be expected. We investigated five 
species of parasites to determine whether their existence 
in different combinations would lead to altered within- 
host distributions or changed numbers of shed cercariae. 
Snails (32 samples, total = 379) were collected from June 
to August, in 1989, and microscopically examined. Par- 
asite species and stages present in five sections through 
each snail were recorded. Before examination, 206 of these 
snails were held in individual chambers in the field. 
After two high tides (ca. 24 h), the chambers were 
checked for species and the numbers of cercariae shed. 
Overall, 22 trematode combinations in single hosts were 
observed. Analysis revealed that co-occurrence with 
other species had no significant effects on any trema- 
tode. Further, analyses of species richness of infecting 
assemblages over two distinct intervals failed to show 
that competition is important in determining assem- 
blage richness. One pair of trematodes (Himasthla 
quissetensis and Lepocreadium setiferoides) has been 
reported not to co-occur. We observed co-occurrences, 
but so few that the apparent conflict between them could 
not be statistically demonstrated. We suggest that, in 
this system, parasites are adapted to the host only, they 
may interact, but they are not adapted to each other. 
Chances for a parasite to live free from other parasites 
seem too great for evolved (adapted) relationships to 
develop. The host, for similar reasons, is probably not 
adapted to the parasites. 



Received 14 February 1992: accepted 6 October 1992. 
1 Mailing address: Cape Henlopen Laboratory, College of Marine 
Studies, University of Delaware, Lewes, DE 19958. 



Introduction 

For one species to be adapted to another, they must 
interact in such a manner that one consistently exerts a 
selective pressure on the other. Species interactions may 
be thought of as a continuum from local to global. A local 
interaction (as used here) results in genetic changes in 
restricted parts of a gene pool (and may result in local 
ecotypes). On the other hand, if an interaction is global, 
one species can be a source of biotic selective pressure 
over the whole operating gene pool of another. Reciprocal 
genetic changes between species amount to coevolution 
(Futuyma and Slatkin, 1983). This paper considers the 
species interactions in a marine gastropod-trematode 
system. Because the host gastropod has a planktonic larva 
and the trematodes are dispersed by highly mobile defin- 
itive hosts, both local and global phenomena must be 
considered. 

The interactions between hosts and parasites have been 
much discussed (see Moore, 1987 for an extensive review), 
and the levels at which such discussions may be focussed 
should be distinguished. In this work, two levels are nec- 
essary. The component community includes all parasite 
species using a particular host species (population). The 
infracommunity includes all the parasites in a single host 
(Esch el ai, 1990). An individual host, harboring a mul- 
tispecies parasite assemblage, is a biological unit where 
parasite-parasite as well as host-parasite interactions can 
occur. 

There are four basic patterns of evolutionary relation- 
ships that may be found in any host-parasite system (Fig. 
la-d). In scheme a, the parasites are adapted to the host 
(the minimal condition), whereas in scheme b, the host 
is also adapted to the parasites. Scheme c illustrates the 
case where the parasites are adapted to the host and to 



25 



26 



L. A. CURTIS AND K. M. K. HUBBARD 



PARASITE 
A 



PARASITE 
B 



HOST 



PARASITE 
A 




HOST 




PARASITE 
B 



PARASITE 
A 




PARASITE 
B 



PARASITE 
A 




HOST 




PARASITE 
B 



Figure 1. Four models of possible adaptive relationships among spe- 
cies in a snail-trematode system. Parasites A and B may coexist in a 
single host. An arrow from one participant to another indicates that the 
participant at the origin of the arrow has evolved adaptations to selection 
pressures coming from the other (i.e.. "PARASITE A - PARASITE 
B" means A is adapted to B). One-way interactions between parasites 
(/.('., A adapts to B but not the reverse) are possible, but not figured. 



each other. Scheme d shows the case where parasites are 
coevolved with the host and with each other. There should 
be evidence of an adaptive relationship between species 
before it is assumed to exist (Williams, 1966). In this work, 
we have tested for species interactions among trematodes 
inhabiting the same gastropod host. The goal is to gather 
evidence to support the elimination of one or more of the 
above schemes and thereby improve our understanding 
of host-parasite systems. 

Studies of trematodes infecting gastropod populations 
have often revealed patterns of species co-occurrence that 
suggest interactions (see Rohde, 1981 for references). 
However, few workers have examined trematode assem- 
blages in individual gastropods taken from their natural 
habitat, to determine whether fitness of certain members 
is consistently affected by co-occurrence with other 
members (see DeCoursey and Vernberg, 1974). This is 
largely because multiply-infected hosts are difficult to ob- 
tain in numbers for study. The prevalence of trematodes 
in the population of Ilyanassa obsoleta (Prosobranchia, 



Neogastropoda) on Cape Henlopen, Delaware Bay is high. 
and a diversity of multiply-infected snails may be ob- 
tained (Curtis, 1985, 1987, 1990; Curtis and Hubbard, 
1990). This allowed us to test for species interactions in 
a variety of trematode ensembles. 

Of the nine trematode species in Ilyanassa obsoleta ob- 
served in Delaware, five are commonly observed in the 
Cape Henlopen population and figure in this study: ///- 
masihla quissetensis, Lepocreadium setiferoides, Zoo- 
gonus rubellus. Aiistmbilharziu variglandis, and Gvnae- 
cotyla adunca. The snail is the first intermediate host. A 
variety of second intermediate hosts is used by these spe- 
cies. Various shorebirds serve as definitive hosts for H. 
quissetensis. A. variglandis and G. adunca, whereas fish 
species are used by L. setiferoides and Z. rubellus (see 
Stunkard, 1983 for life-cycles and taxonomic matters). 
Any direct species interactions among these parasites must 
occur in the snail, the only host they all have in common. 

There is no indication that Ilyanassa obsolete! lose in- 
fections (Curtis and Hurd, 1983). so the ensembles ob- 
served in snails probably represent relatively longstanding 
(period unknown) assemblages. Enduring species assem- 
blages, proximity in a natural habitat unit, and utilization 
of similar resources (Smyth and Halton. 1983). suggest 
that strong interspecific interactions might occur. 

If competitive interactions are frequent within individ- 
ual hosts whereby dominant species come to monopolize 
the host population through time, a pattern should emerge 
at the component community level. Early on, most snails 
should have single species infections; as time progresses 
species accumulate and there should be a preponderance 
of double and triple infections; and eventually there should 
be mostly single infections again, as the dominant species 
evict subordinates (Sousa, 1990). We searched for such a 
component community pattern among our snails at two 
time scales, through the summer and over several years. 

To examine within-snail parasite interactions, we tested 
individual species to see whether existence in different 
assemblages had consequences in terms of ( 1 ) alterations 
of within-snail spatial distributions, (2) complete suppres- 
sion of cercarial production, and (3) changes in numbers 
of cercariae released from hosts. 

Materials and Methods 

One sandbar (Fig. 2), located near the mouth of the 
Delaware Bay on Cape Henlopen (75 06'W, 38 471^), 
was chosen as the source for snails. Certain species of 
trematodes affect the behavior, distribution and temporal 
occurrence of Ilyanassa obsoleta on sandbars (Curtis, 
1987, 1990). To avoid over-representing snails harboring 
particular parasite ensembles, we randomly chose collec- 
tion sites according to the angle and distance from a ref- 
erence point at the peak of the sandbar (Fig. 2). Samples 



GASTROPOD-TREMATODE INTERACTIONS 



27 




"0 10 20 30 40 

METERS (ALONG BEACH) NE 

Figure 2. An elevational contour map of the 1989 sandbar on Cape 
Henlopen. Delaware where samples ofllyanassa ohsolctu were collected 
for this study. The 32 randomly selected sample sites are indicated by 
filled diamonds. The highest point on the map (the sandbar peak at 
center) is 56 cm above the lowest. 



were taken between 16 June and 17 August 1989 on both 
day and night low tides. We wanted many multiply-in- 
fected snails in the samples, and the 379 snails obtained 
(Table I) were purposely biased to include them. The snails 
came from an area where many multiples were likely to 
be found (e.g., Curtis, 1987), and large snails that were 
likely to be infected were chosen (Curtis and Hurd, 1983). 
Usually, two collections of 10 to 13 snails were collected 
and processed at a time. All the snails were dissected and 
206 were also tested for cercarial release. 

We were interested in revealing gross within-snail dis- 
placements of individual parasite species by other species 
or combinations of species. Such displacements would be 
required if dominant species gradually evicted subordi- 
nates from the snail. During dissection each snail was 
removed from its shell and examined in sections to de- 
termine how individual parasite species, and stages 
thereof, were distributed within. Heavily parasitized snails 
are virtual bags of trematodes; they retain no consistent 
morphological landmarks that are useable as standard 
points of reference. Consequently, each snail was pinned 
to a board and cut crosswise into five equal lengths with 
a razor blade. Section 1 was the most dorsal portion of 
the snail (the spire), and section 5 was the most ventral 
(head and foot). The razor was cleaned between cuts and 
scrupulous care was taken to prevent contamination of 
one section with material from another. 

Sections were placed separately in small vials containing 
5 ml filtered baywater. Each vial was vigorously shaken 



50 times to release the contained trematode stages into 
the water. A small amount of the water was placed on a 
slide and examined with the aid of dissecting (32X) and 
compound (100X) microscopes. We took two samples 
from each vial. The species and stages of the trematodes 
were recorded for each section of each snail as follows: 
parental stages (rediae or sporocysts) and cercariae (PC); 
cercariae only (C); parental stage without mature cercariae 
(P); or absent (A). Observed cercariae may have been lib- 
erated from parental stages during the procedure, but this 
does not matter as we were only interested in whether 
formed (mature) cercariae were present. Trematodes were 
never found in section 5, and after the hundredth snail 
we stopped examining this section. 



Table I 

Trematode infections in Ilyanassa obsoleta collected for this study 
from a sandbar area (Fig. 2) on Cape Henlopen. Delaware 



Infecting 

species 


n 


Mean shell 
height (mm) 


Range shell 
height (mm) 


uninfected 


18 


21 


17-25 


singles 








Hq 


74 


24 


20-27 


Ls 


25 


24 


20-27 


Zr 


29 


22 


20-26 


Av 


5 


23 


22-24 


Ga 


29 


22 


18-25 


doubles 








HL 


4 


23 


22-23 


HZ 


42 


24 


20-27 


HG 


10 


23 


21-25 


LZ 


8 


24 


20-28 


LA 


1 


25 





LG 


32 


23 


20-26 


ZA 


1 


24 





ZG 


24 


23 


20-27 


AG 


1C) 


22 


19-24 


AD 


1 


23 





GD 


1 


21 





triples 








HZG 


33 


24 


18-26 


LZA 


1 


28 





LZG 


22 


23 


17-26 


LAG 


4 


21 


20-24 


ZAG 


5 


23 


23-25 


Total = 


379 


Overall = 23 


17-28 



For each infection, number collected (n), and mean and range of shell 
heights are given. Snails infected by a single species (singles) are repre- 
sented by the genus and species initials of the trematode (Hq = Himasllila 
qitissetensis, Ls = Lepocreadium setiferoides, Zr = Zoogonus rubellits. 
Av = Austrobilhariia variglandix. Ga = Gynaecotyla adunca). Double 
and triple infections are represented with the generic initials of the species 
involved (e.g., a snail infected with H. quissetensis, Z. rubellus. and G 
adunca goes in the HZG category). Diplostomum nassa (D) occurred 
only in double infections. Shell height = siphonal canal to apex of shell 
(e.g., 21 = 20.5 to 21.4 mm). 



L. A. CURTIS AND R. M. K. HUBBARD 



The frequencies of parasite presence or absence in the 
snails were crosstabulated according to the following cri- 
teria: parasite assemblage (those species infecting the 
snail); snail section (1 - 4); and the stage of the parasite 
(sporocysts, rediae, cercariae). Contingency table analyses 
were employed to test for significant displacements of 
parasite stages within snails. For each parasite, we used 
log linear models (Sokal and Rohlf, 1981 ) to calculate the 
expected frequencies of occurrence of the stages (PC, P, 
C, or A) in various sections of hosts harboring various 
trematode ensembles. A saturated log linear model for 
this kind of analysis includes seven terms: Assemblage; 
Section; Stage; Assemblage x Section; Assemblage 
X Stage; Section X Stage; and Assemblage X Section 
X Stage. The purpose of this analysis is to learn which of 
these terms are necessary to calculate a set of expected 
frequencies that do not deviate significantly from the ob- 
served frequencies. After unnecessary terms are elimi- 
nated, we are left with the accepted model. The accepted 
model is expressed in hierarchical form. For example, an 
Assemblage X Section X Stage hierarchical model would 
nest all seven terms of the full model; and an Assemblage, 
Section X Stage model would nest all three one-way terms 
and the Section X Stage two-way term. 

If species interactions lead to spatial rearrangements 
within snails, a 3-way interaction term (i.e.. Assemblage 
X Section X Stage) would be necessary in the accepted 
model for any displaced species. For example, suppose 
species "a" were usually distributed throughout the snail 
from spire to mantle when it occurred alone, but in the 
presence of species "b" (i.e., assemblage "ab"), "a" were 
consistently absent from the spire section. The three-way 
interaction term would be necessary in the accepted model 
because absence (A) of species "a" from Section 1 would 
be a consequence of Assemblage composition. That is, 
the presence of species "b" in Section 1 would change 
Stage entries for "a" in Section I to absent (A) from one 
of the present categories (PC, P, or C). Therefore, a table 
of expected frequencies that matched observed frequencies 
could not be calculated without the Assemblage X Section 
X Stage term in the accepted model. 

We used cercarial release as a measure of fitness to 
learn whether parasites were affected by within-host in- 
teractions with other infecting species. We evaluated cer- 
carial output from assemblages during one short period, 
and tested similar assemblages throughout the summer. 
(An alternative, more manipulation laden, approach 
would be to follow cercarial output from individual as- 
semblages over a longer period of time.) Individual snails 
were confined in chambers in the natural environment 
for two high tides (ca. 24 h), and the water in which they 
had been immersed was examined for numbers and spe- 
cies of cercariae. We used 24-h periods to encompass any 
daily shedding patterns. The procedure is described in 



more detail in Curtis and Hubbard (1990). We used a 
Kruskal-Wallis test (Hollander and Wolfe, 1973) to de- 
termine, for each species, whether the number of cercariae 
shed was significantly different when in various co-oc- 
curring assemblages of parasites. All statistical calculations 
were done with the software package. Number Cruncher 
Statistical System, 5X Series. 

Results 

A competition model (Sousa, 1990) suggests that trem- 
atodes might invade a snail population, accumulate in 
snails, compete, and eventually complete the process by 
having dominant trematodes evict subordinates. If true, 
then over the relevant time we should see infecting species 
richness start low (mostly single infections), increase 
(mostly doubles and triples), and then decrease again. We 
looked for such a pattern within two distinct intervals, 
over the summer (Table II A), and over several years (Table 
IIB). We divided the sampling period into four two-week 
intervals; the fourth interval was extended to encompass 
the 25 snails collected on August 1 7. There were significant 
changes in richness from one period to the next, but the 
expected pattern was not seen. In particular, triple infec- 
tions were quite abundant early in summer and were most 
abundant in the last sampling period. This would not have 
been observed if dominant trematodes had defeated sub- 
ordinates in this period of time. 

Using size-classes of snails (Table IIB), the interval can 
be extended from months to years. At the beginning of 
its third summer, a snail on Cape Henlopen is about 
14-15 mm; by the end of that summer, it has grown to 
about 17-18 mm (Curtis and Hurd, 1983). This means 
that the smallest snails we collected (17 mm. Table I) 
were probably in their fourth summer. If 3 mm/summer 
is used as an estimate of growth for parasitized snails, 
then the < =22 group in Table IIB is 4-5 yr old; the 23- 
25 group is 5-6 yr old; and the 26-28 group is 6-7 yr old. 
The interval encompassed by Table IIB is about three 
years using this estimate. Parasitized snails may not grow 
this rapidly, and the interval is possibly longer. In this 
years-long interval (size-class range), there were signifi- 
cant changes in infecting species richness. Note (Table 
IIB) that single infections were more abundant than triple 
infections in the youngest snails, but that the proportion 
of triples increased among older snails. This is not the 
pattern predicted by the competition model. 

Occurrence of stages of five trematodes in sections of 
Ilyanassa obsoleta harboring different assemblages is 
shown in Table III. Recall that section 1 was dorsal (spire) 
and section 4 ventral (mantle). In Table IV. models for 
all five species (except Austrobilliarzia vanglandis) require 
the one-way Assemblage term because of the widely dif- 
ferent numbers of snails infected with each assemblage 



GASTROPOD-TREMATODE INTERACTIONS 



29 



Table II 

Tremalode infections in Ilyanassa obsoleta examined during tins work crosstabulated by number of inled IIIR Iremalode species (richness), time of 
collection in summer 19S9 (A), and si:e (age) of snail (B) 

Infecting Species Richness 



(n = 18) 



% Singles 
(n = 162) 



% Doubles 
(n = 134) 



% Triples 
(n = 65) 



A. Time Collected 
16 Jun-29 Jun 
30Jun-13Jul 

14 Jul-27 Jul 
28 Jul-17 Aug 

B. Size Class (mm) 
< = 22 

23-25 

26-28 

2-way contingency analyses: 
Time X richness, Xf,, = 43.69, P < 0.001 
Size X richness, X, 2 6) = 25.16, P < 0.001 



0.9 
15.0 

1.1 
4.1 

10.5 
1.0 
0.0 



39.8 

38.7 
50.0 
42.9 

43.8 
43.6 
32.3 



44.3 
40.0 
30.7 

25.5 

33.3 
36.9 
35.5 



15.0 
6.3 
18.2 

27.5 

12.4 
18.5 
32.3 



113 
80 

X8 
48 

153 

195 

31 



Size class ranges are in terms of shell height as in Table I. 



(see Table III). The frequencies for Lepocreadium setifer- 
oides can be modeled by taking into account, beyond the 
Assemblage term, only the one-way Stage term because 
most of the stage entries are in the PC category. The rest 
of the models require the Section X Stage term because 
there was some specificity as to what sections were likely 
to harbor which stages. This is clearest for Zoogonus ru- 
bcllus and Gynaecotyla adunca. Stages were often (clearly 
not always) absent (A) from sections 1 and 4. However, 
this was not significantly correlated with the assemblage 
of species infecting the snail. For none of the five species 
tabulated is an Assemblage X Section X Stage (three- 
way) interaction term necessary in its accepted hierarchical 
log linear model (Table IV). That is, co-occurring trem- 
atodes did not significantly affect the distribution of any 
of the five species tested. In most snails, parasite stages of 
all species present occurred throughout. 

Parasite species interactions could lead to cercarial 
suppression in a section rather than species eviction. For 
example, if the presence of species "a" suppressed cercarial 
production by species "b", the accepted log linear model 
for species "b" would have to include the Assemblage 
X Stage term. This would be necessary because, for species 
"b" in the presence of species "a", the frequency of the 
PC category would decrease, while the frequency of the 
P category would increase as compared to other assem- 
blages involving species "b". Expected values that 
matched this shift in observed frequencies could not be 
predicted (modeled) without incorporating the influence 
of Assemblage on Stage. No species' cercarial production 
was completely suppressed in this manner (Table I V, lack 
of Assemblage X Stage terms). 



The question now becomes: given that cercariae were 
being produced, was the number released from snails 
changed as a function of assemblage composition? To 
answer this, we used data from cercarial release chambers. 
Prepatent infections (those with no cercariae present) were 
eliminated from this analysis because their prepatency 
was not caused by assemblage composition (Table IV, no 
Assemblage X Stage terms in the accepted models). In- 
cluding prepatents would add meaningless variability. 
Absent cercariae are not germane to this analysis if they 
are not caused by the presence of other species. Table V 
describes statistically the cercarial output of each of the 
five species in various assemblages. The magnitude of 
variability should be noted. 

Table VI presents the results of Kruskal-Wallis tests 
that were used to determine whether the assemblage com- 
position significantly affected the numbers of cercariae 
released by particular (patent) assemblage members. The 
results show that although cercarial output (mean rank) 
did decrease for all species when additional species were 
present, there was not a significant depression of cercarial 
output for any one species. 

Finally, because Himasthla quissetensis and Lepo- 
creadium setiferoides have not previously been observed 
together, note that in Table V such a co-occurrence is 
listed, and that both species shed cercariae concurrently. 
Four snails contained both H. quissetensis and L. setifer- 
oides (Table I). Based on observations of a few mature 
(often moribund) H. quissetensis rediae and cercariae 
among many L. setiferoides rediae and cercariae, it ap- 
peared that L. setiferoides was evicting H. quissetensis 
from the snails. There were not enough of these snails to 



30 



L. A. CURTIS AND K. M. K.. HUBBARD 
Table III 



Spatial distributions of five trematode species (see Table I for parasite abbreviations) within sint>l\- and multiply-infected Ilyanassa obsoleta. 
Observed frequencies of parasite occurrence thy stage*), in snail sections 1-4 (see text), are given for each species 



Section 


Infecting 
trematodes 


Species 
tabulated 


1 
Stage 


2 

Stage 


3 
Stage 


4 
Stage 


PC 


C 


p 


A 


PC 


C 


p 


A 


PC 


C 


p 


A 


PC 


C 


p 


A 


Hq(n = 74) 


Hq 


72 


1 


1 





73 





1 





73 


1 








69 


2 


1 


2 


HZ(n = 42) 


Hq 


38 





2 


2 


40 








2 


40 


1 





1 


33 


6 


2 


1 


HG(n = 10) 


Hq 


6 





3 


1 


10 











9 








1 


8 





1 


1 


HZG (n = 33) 


Hq 


30 





2 


1 


33 











32 





1 





27 


2 





4 


Ls(n = 25) 


Ls 


25 











25 











24 


1 








22 


3 








LZ (n = 8) 


Ls 


8 











8 











8 











7 


1 








LG (n = 32) 


Ls 


26 


4 


1 





32 








(I 


30 





2 





23 


2 


3 


4 


LZG (n = 22) 


Ls 


19 


2 


1 





21 


1 





(] 


21 


1 








17 


5 








Zr(n = 29) 


Zr 


27 








1 


27 








2 


27 








2 


17 








12 


HZ 


Zr 


25 








17 


37 








5 


38 








4 


26 


2 





14 


LZ 


Zr 


4 








4 


8 











7 


1 








2 








6 


ZG (n = 24) 


Zr 


22 








2 


24 











23 








1 


10 








14 


HZG 


Zr 


20 








13 


30 








3 


32 








1 


17 








16 


LZG 


Zr 


12 








10 


21 








1 


21 








1 


8 





1 


13 


ZAG (n = 5) 


Zr 


3 








2 


4 








1 


5 




















5 


Av (n = 5) 


Av 


4 


1 








5 











4 








1 


2 








3 


AG(n = 10) 


Av 


7 








3 


10 











10 











2 








8 


ZAG (n = 5) 


Av 


3 








2 


5 











1 








4 











5 


Ga(n = 24) 


Ga 


25 








4 


28 








1 


26 








3 


16 


1 





12 


HG 


Ga 


6 








4 


9 








1 


10 











4 


1 





5 


LG 


Ga 


23 


1 


1 


7 


29 





1 


2 


29 


1 





2 


16 


2 





14 


ZG 


Ga 


20 








4 


22 








2 


24 











15 








9 


AG 


Ga 


9 








1 


9 








1 


9 








1 


8 








2 


HZG 


Ga 


21 








12 


28 








5 


32 








1 


10 








23 


ZG 


Ga 


14 








8 


17 








5 


21 








1 


13 








9 


AG 


Ga 


4 


1 








5 











5 











4 








1 



* Stage abbreviations: PC = parental stage (i.e., sporocysts or rediae) plus cercariae: P = parental stage only; C = cercariae only; and A = all stages 
absent. 

Individual species occurred in the context of several different combinations of infecting species (e.g.. Hg occurred alone, in HZ and HG doubles, 
and in HZG triples). For each species, frequencies are tabulated for each context. The number (n) of snails infected by particular trematode assemblages 
is indicated. Assemblages found in fewer than four snails are not tabulated. 



be included in the above log linear or Kruskal-Wallis 
analyses. 

Discussion 

Ilyanassa obsoleta is the only shared host in the life- 
cycles of these trematode species and is, therefore, the 
only place they might directly interact. They are tightly 
packed together in the snail, gather resources in similar 
ways, and are abundant on Cape Henlopen. Antagonistic 
interactions between trematode assemblage members have 
been noted by several investigators (e.g.. Lie el a!.. 1965: 
Basch el al, 1969; DeCoursey and Vernberg, 1974; Kuris, 
1990; Sousa, 1990). On such grounds we anticipated that 
trematodes co-occurring in /. obsoleta would interact and 
most likely compete. A between-snail (component com- 



munity) analysis indicated that competition within snails 
was not an important determinant of the number of trem- 
atodes infecting individual snails. Further, regarding 
within-snail phenomena, no effect of assemblage com- 
position on any individual species could be discerned sta- 
tistically. However. Himasthla quissetensis and Lepo- 
creadiwn setiferoides were seen to co-occur in this study 
for the first time (Tables I, V), and this observation de- 
serves special comment. By virtue of their rare co-occur- 
rence, which eliminated the pair from our statistical anal- 
yses, these species apparently do interact negatively when 
they occur in the same snail. 

Our sample of trematode assemblages from the Cape 
Henlopen sandflat naturally included only those species 
combinations that can coexist long enough to be observed 
by the methods used. These included most of the possible 



GASTROPOD-TREMATODE INTERACTIONS 



31 



Table IV 

Results l log! i near analyses testing the influence of three factors 
ttrenuitode Assemblage, snail Section, and parasite Stage) on the 
frequencies of within snail occurrence reported in Table I' 



Species 
analyzed 


Hierarchical log-linear 
model accepted 


x : 


d.o.f. 


P =* 


Hq 


Assemblage, Section 


54.21 


45 


0.163 




x Stage 








Ls 


Assemblage, Stage 


67.33 


57 


0.165 


Zr 


Assemblage. Section 


63.25 


90 


0.985 




X Stage 








Av 


Section x Stage 


25.79 


32 


0.773 


Ga 


Assemblage, Section 


60.99 


105 


0.999 




x Stage 









* An insignificant X : (P > 0.05) without the three-way interaction 
term means that it is unnecessary; no significant displacement occurred. 

If a trematode's stages (/.<.. sporocysts. rediae. cercariae) were displaced 
from one snail section to another by the presence of a co-occurring 
species or combination of species, the accepted model for that trematode 
would require the three-way interaction term (i.e.. Assemblage X Section 
> Stage) to calculate expected frequencies without significant deviation 
from the observed. 



assemblages and virtually all that might have been ex- 
pected to occur. Twenty, of the 32 possible for five species 
analyzed, were actually observed (Table I). Missing as- 
semblages were the quintuple, the five quadruples, mul- 
tiples involving the scarce Amtrobilhania variglandis, and 
two triples involving Himasthla quissetensis and Lepo- 
creadiwn seiiferoides. 

A major concern is whether interparasite competitions 
occur that require considerable time for completion. In 
the early to middle phases of competition there may be 
no noticeable effect on any one species. We may have 
examined most assemblages at a time when coexistence 
is possible, and erroneously concluded that species do not 
interact. If such a time-course for competition is involved, 
how much time is necessary, and was our collection of 
parasite assemblages (in snails) biased by this? Two pos- 
sibilities present themselves: competitions could play 
themselves out over the summer; or over several summers. 
There was no indication that trematodes assemble in 
snails, compete, and ultimately evict subordinate species 
in either the short or the long interval (Table II). To the 
contrary, species appear to collect in snails as a function 
of time. Note that older snails, and not either younger 
group, have the largest proportion of triple infections (Ta- 
ble IIB). Sousa (1990) looked for a hyperbolic relationship 
between snail size and infecting species richness and sim- 
ilarly did not find one. 

Direct measurements of within-snail species dynamics 
also indicate no interactions among assemblage members. 
The occurrence of parasitic stages in different sections of 
variously infected snails is shown in Table III. No species 



was excluded from sections of snails because of co-oc- 
curring species (lack of Assemblage X Section x Stage 
terms in Table IV). If one species (or combinations of 
species) leads to gradual eviction of another species from 
snails, this phenomenon should have been quite common. 
Neither was cercarial production (from existing parental 
stages) of any species shut down by co-occurring species 
(lack of Assemblage X Stage terms in Table IV). Also, 
there was no indication that cercarial output from hosts 
(an estimate of fitness) was influenced by co-occurring 
species. There was no statistically significant reduction of 
cercarial output of any species as a function of assemblage 



Table V 

Descriptive statistics associated with numbers oftremalode cercariae 
released per hosl (Ilyanassa obsoleta) in 24 h in the field. Information 
is grouped bv species of cercariae being tabulated (.see Table I for 
species abbreviations) 



Infecting Cercariae 
trematodes tabulated 


Mean* 


S.D. 


Max. 


Med. 


Min. 


Hq(n = 42) 


Hq 


527 


709 


2739 


225 





HL(n = 1) 


Hq 


18 





18 


18 


18 


HZ(n = 23) 


Hq 


211 


344 


1428 


90 





HG (n = 4) 


Hq 


696 


1135 


2388 


177 


42 


HZG(n = 22) 


Hq 


155 


274 


1233 


60 





Ls(n = 18) 


Ls 


319 


590 


2394 


129 





HL(n = 1) 


Ls 


567 





567 


567 


567 


LZ(n = 2) 


Ls 


130 


185 


261 


131 





LG(n = 10) 


Ls 


42 


66 


165 


9 





LZG(n = 15) 


Ls 


121 


153 


483 


45 





LAG(n = 2) 


Ls 


18 


25 


36 


18 





Zr(n = 12) 


Zr 


249 


378 


1095 


15 





HZ(n = 20) 


Zr 


68 


199 


882 


1 





LZ(n = 2) 


Zr 

















ZA(n = 1) 


Zr 


1065 





1065 


1065 


1065 


ZG (n = 8) 


Zr 


61 


67 


189 


42 





HZGln = 19) 


Zr 


47 


73 


210 


6 





LZG(n = 13) 


Zr 


79 


138 


474 


21 





ZAG (n = 2) 


Zr 


12 


8 


18 


12 


6 


Av (n = 3) 


Av 

















ZA (n = 1) 


Av 

















AC (n = 5) 


Av 


9 


16 


36 








LAG(n = 3) 


Av 


21 


16 


33 


27 


3 


ZAG (n = 1 ) 


Av 


6 





6 


6 


6 


Gain = 14) 


Ga 


222 


454 


1398 








HG(n = 3) 


Ga 


6 


10 


18 








LG(n = 8) 


Ga 


74 


168 


483 








ZG (n = 6) 


Ga 

















AG(n = 5) 


Ga 


3 


5 


12 








HZG(n = 17) 


Ga 


2 


9 


36 








LZG(n = 13) 


Ga 


27 


96 


345 








LAG (n = 4) 


Ga 


6 


12 


24 








ZAG(n = 1) 


Ga 


18 





18 


18 


18 



Only infections that were patent for the species being tabulated are 
considered (n). For example, there were 33 HZG-infected snails (from 
Table I): 22 of these were patent for Hq; 19 for Zr; and 17 for Ga. A 
total of 206 snails were tested for cercarial release. 



32 



L. A. CURTIS AND K. M. K. HUBBARD 



Table VI 



Ri'Htlts ofKruskal-H 'ulli\ tests I'vuliuitmx the null hypothesis for each 
trematode species (see Table I for species abbreviations), thai the 
inimher of cercariat! shed was unaffected hv coeMSlhig species 



Effect of 
coexisting 
species on Infecting Mean rank 
fitness of species (# cercariae) 


Kruskal- 
d.o.f. Wallis H P = 


Hq 


Hq(n = 42) 


52.583 






HZ(n = 23) 


41.957 






HG (n = 4) 


53.500 






HZG (n = 22) 


36.295 


3 6.454 0.09 1 


Ls 


Ls(n = 18) 


23.972 






LG (n = 10) 


15.850 






LZGIn = 15) 


23.733 


2 3.186 0.203 


Zr 


Zr(n= 12) 


42.417 






HZ(n = 20) 


29.575 






ZG (n = 8) 


42.563 






HZG(n = 19) 


33.763 






LZG(n = 13) 


41.962 


4 5.254 0.262 


Av 


No test 






Ga 


Ga(n = 14) 


39.536 






LG(n = 8) 


39.688 






ZG (n = 6) 


26.500 






AG (n = 5) 


38.100 






HZG(n = 17) 


28.471 






LZG(n = 13) 


33.537 






LAG(n = 4) 


34.375 


6 8.075 0.233 



Prepatent infections and trematode assemblages observed fewer than 
four times were excluded. 



composition (Table VI). There was much variation, even 
in single infections (Table V), suggesting that sources of 
variability other than co-occurring species control cercarial 
output. 

DeCoursey and Vernberg (1974) studied assemblages 
of trematodes infecting Ilyanassa obsoleta in North and 
South Carolina. At the level of the component commu- 
nity, they noted that some species co-occur in multiple 
infections more or less often than would be expected based 
on the abundance of each in the system. They proposed 
that such patterns are produced by antagonisms or affin- 
ities among assemblage members. About 80 snails were 
dissected, with 30 of these being serially sectioned. The 
number of snails examined in each assemblage category 
is not reported. The authors noted "marked overlap in 
territory and habitat preferences," as we did in this study. 
Contrary to our conclusion (based on arbitrary snail sec- 
tions) that the parasites are not displaced, they concluded 
that some species are displaced from preferred sites (spe- 
cific snail organs) by other species. Even if small scale 
displacements (i.e., from organ to organ within our ar- 
bitrary sections) do occur, they would have to result in 
reductions in cercarial output (fitness) to have evolution- 
ary consequences. Cercarial output was not significantly 



reduced (Table VI). We also note that, if the interest is in 
adaptation of one parasite to others (Fig. 1 ), then section- 
ing snails along snail organ boundaries confounds adap- 
tation to other parasites with adaptation to the host. 

In the laboratory, DeCoursey and Vernberg ( 1974) also 
counted the cercariae released from 10 infected snails. 
Three were infected with Zoogonus lasius ( = rubellus) and 
five with Lepocreadium setiferoides. The remaining two 
were doubly infected with these same species. The num- 
bers of cercariae released in the laboratory by each species 
of trematode were averaged and compared. When Z. ru- 
be/Ins and L. setiferoides occurred alone, they each re- 
leased approximately 3500 cercariae in 24 h. When the 
species co-occurred, they released 901 and 1477, respec- 
tively. The authors concluded that L. setiferoides sup- 
pressed cercarial release by Z rubellus. Data show that 
cercarial production of both species was lower when they 
co-occurred. In any case, the number of observations pre- 
cludes meaningful statistical inference. 

We are interested in eliminating inoperative models 
from the four presented in Figure 1. Williams' (1966) dis- 
tinction between "functions" and "effects" seems useful 
here. Functions are biological characteristics that are direct 
products of natural selection (adaptations), whereas effects 
are characteristics that are a consequence of functions 
("side" effects, not directly selected). Holmes (1986) points 
out that parasitic ". . . interactions should be important 
[in structuring helminth communities] only when species 
regularly co-occur at substantial population densities" 
(p. 203, brackets ours). We note, more specifically, that 
interactions based on adaptive responses (functions) of 
one parasite species to another cannot arise unless there 
is frequent co-occurrence over global gene pools. 

We cannot imagine how the parasites under study here 
could have adapted to one another. Definitive hosts (fish 
and birds) are highly mobile and scatter parasite eggs 
widely and unevenly. Consequently, spatial distribution 
of these trematodes within and among host snail popu- 
lations is patchy (Curtis and Hurd, 1 983; Curtis and Hub- 
bard, 1990), and there are abundant opportunities for 
trematode species to exist in isolation. The probability of 
co-occurrence generation after generation, particularly for 
specific parasites, is very low. Therefore, evolved parasite- 
parasite relationships are unlikely in this system. If inter- 
actions occur, they most likely result from effects, not 
functions. Our data indicating the lack of interactions 
among the majority of co-occurring trematodes. and the 
above considerations, justify eliminating models "c" and 
"d" (Fig. 1 ). Any evolved features of this system probably 
stem ultimately from the evolution of parasites to host, 
or possibly of host to parasites (models "a" and "b". 
Fig. 1 ). 

In deciding between models "a" and "b", many of the 
same arguments apply. Gooch et al. (1972) found that 



GASTROPOD-TREMATODE INTERACTIONS 



33 



Ilvanassa obsolete! were electrophoretically homogeneous 
all along the eastern seaboard, pointing to extensive dis- 
persal of larvae as the main cause. The planktonic larvae 
of/, obsoleta would then function analogously to parasite 
definitive hosts in the dispersal of progeny. Given the het- 
erogeneity of trematode prevalence in /. obsoleta popu- 
lations, many snail larvae would settle where parasites are 
not a frequent environmental challenge. If a snail were 
to obtain, by mutation, resistance to infection by one or 
more trematode species, its fitness probably would be en- 
hanced in parasite-ridden environments such as parts of 
Cape Henlopen. Yet its progeny would very possibly settle 
where parasites are infrequent. The mutation, there, would 
be at best neutral. These considerations suggest that model 
"a" (Fig. 1) is the operative one the only adaptive re- 
sponses between species in the /. obsoleta system are most 
likely those of the parasites to the host. 

The negative interaction between Hiniasthla quisse- 
lensis and Lepocreadium setiferoides in the Ilyanassa ob- 
soleta system deserves comment because it seems to 
counter the proposition that these parasites are not 
adapted to each other. A lack of co-occurrence of these 
abundant species in / obsoleta has been reported (Vern- 
bergetal., 1969; Curtis. 1985), but the detailed dissection 
methods used in this study revealed four co-occurrences 
(Table I). Obviously, miracidia of both species reach the 
same host, and there is a subsequent eviction (apparently 
of H. quissetensis). This eviction is important in terms of 
determining composition of the infra- and component 
assemblages observed, but is it based on adaptation of one 
parasite to another? In keeping with the above reasoning 
that parasite co-occurrence is not globally predictable 
enough to result in adaptations to other parasites we 
interpret this negative co-occurrence as based on an effect 
rather than a function [an exaptation (Gould and Vbra, 
1982)] because it results from the way these species have 
evolved to the host, not to each other. In ecological terms, 
such a phenomenon is a competitive exclusion. However, 
in our hypothesis, the exclusion occurs between two spe- 
cies that are adaptively unaware of each other. If species 
interactions are an evolutionary force driving the struc- 
turing of interactive, co-adapted species assemblages, then 
we should distinguish between function- and effect-based 
relationships among species. A deeper appreciation of 
causal relationships in ecological systems will require un- 
derstanding these relationships. 

Factors structuring the assemblage of larval trematodes 
in populations of the California estuarine snail Cerithidea 
californica have been examined by Sousa (1990) and Kuris 
(1990). Two direct lines of evidence convinced these au- 
thors that competitive exclusions were occurring among 
C. californica trematodes. Sousa (1990) cites personal 
laboratory observations in which dominant species preyed 
upon stages of subordinates. Both authors reported trem- 



atode species replacements in individual snails periodically 
reexamined for infection by cercarial release. Kuris (1990) 
constructed a competitive hierarchy among trematode 
species in infracommunities, which he concluded would 
produce component community structure. In the Ily- 
anassa obsoleta system, such cercarial release data would 
have to be used judiciously because cercariae, even if 
present, often are not shed (Curtis and Hubbard, 1990). 
Data are not presented that assess this source of error for 
the C. californica system. In any event, there is consid- 
erable heterogeneity in prevalence of trematodes among 
C. californica populations (Kuris, 1990; Sousa, 1990). 
Parasite progeny are dispersed by definitive hosts similar 
to those in the I. obsoleta system, giving species the same 
opportunities to exist in isolation. This may mean that, 
whether they interact or not, parasites are not co-adapted 
in the C. californica system either. Because C. californica 
has direct development (Sousa, 1990) making popula- 
tions more insular the host may have the ability to 
evolve to its parasites. 

Several authors have examined snail-trematode sys- 
tems for interactions among parasites infecting the same 
host individual. Some have emphasized direct micro- 
scopical observations of antagonisms occurring in fresh- 
water snails (e.g.. Lie el al, 1965; Basch el al. 1969; 
Mouahid and Mone, 1990). Based upon such observa- 
tions, there can be no doubt that antagonisms between 
trematodes can and do occur, but their frequencies in 
natural snail populations are less certain. Other authors 
have emphasized observations of multiple infections in 
marine (e.g., Kuris, 1990; Sousa, 1990) and freshwater 
(e.g.. Fernandez and Esch, 199 la, b; Williams and Esch, 
1991 (gastropods. In no case are multispecies assemblages 
reported to be particularly frequent. Such species-rich as- 
semblages are more frequent and various in the Ilyanassa 
obsoleta system on Cape Henlopen (Curtis, 1985, 1987, 
1990; present study) than in any studied so far (see Cort 
el al. ( 1937)). The most frequent assemblage observed on 
Cape Henlopen is Lepocreadium setiferoides with Gy- 
naecotyla adiinca. and it occurred in only 4.4% of snails 
(n = 4870) examined by dissection (Curtis, unpub. data). 
Individual occurrence of each was 16.9 and 20.3%, re- 
spectively. Thus, even when species can and do co-occur, 
the probability of co-occurrence is slight. The opportunity 
to evolve adaptive responses to other particular trematodes 
seems minimal or nonexistent, which suggests that models 
"c" and "d" (Fig. 1 ) may be generally inoperative. The 
best opportunity for trematode-trematode adaptive re- 
sponses would be in a situation where all the necessary 
hosts are confined to one habitat, such as in a freshwater 
pond, as described by Williams and Esch ( 1 99 1 ) and Fer- 
nandez and Esch (199 la). However, Williams and Esch 
(1991) and Fernandez and Esch (1991b) conclude that 
within-snail trematode interactions in their system are in- 



34 



L. A. CURTIS AND R. M. K HUBBARD 



frequent and not the factor structuring the infra- and 
component communities. 

Can the host be adapted to its parasites? The evolution 
of a host to several parasites is a problem of "overwhelm- 
ing complexity" (McLennan and Brooks, 1991). and the 
issue is not resolvable with the data at hand. Dobson and 
Merenlender ( 199 1 ) suggest, as we content here, that the 
probability of such evolutionary responses would depend 
on host and parasite dispersal abilities, llyanassa obsoleta. 
because of its widespread dispersal, is unlikely to evolve 
to its parasites (model "b"), but it is a possibility with a 
snail in a more insular system, such as a pond. 

How can the coexistence, in a small habitat unit, of 
several species with similar resource requirements be ex- 
plained? This study has provided considerable compar- 
ative data on the fitness of parasites when they occur in 
different assemblages. The extensive variation in cercarial 
output (Table V) is not explainable by looking to presence 
or absence of other species. Perhaps resources for trem- 
atodes living in llyanassa obsoleta are somehow not lim- 
iting. We have suggested that the only adaptations (func- 
tions) in the system are those of the parasites enabling 
them to live in the snail (model "a". Fig. 1 ). We offer the 
following possible explanation. Each of these five trem- 
todes has evolved to castrate the host snail. Castration of 
the host stems from a parasite adaptation to channel en- 
ergy to the parasite that would otherwise go to the support 
of host gonadal tissue (Baudoin, 1975). llyanassa obsoleta 
is a long-lived host (7 years or more); the largest (oldest) 
snails are nearly all parasitized where trematodes are 
prevalent; and they appear not to lose infections (Curtis 
and Hurd, 1983). The host must survive the rigors of suc- 
ceeding winters. A trematode adapted to such a host may 
have been selected to exact intermediate to minimal 
damage (besides castration) because it could then "farm" 
the host for many years (see Minchella et al. (1985) and 
Gill and Mock (1985) for similar interpretations of host- 
parasite systems). We propose that, if the trematodes of 
/. obsoleta operate this way, then they should not singly, 
or in multiples, drain resources to the extent that they 
become limiting. In brief, they can coexist if they are all 
adapted to live well below the level at which the host is 
stressed. 

Acknowledgments 

We would like to thank the Undergraduate Research 
Office and the School of Life Sciences, University of Del- 
aware for a Science and Engineering Scholar grant, and 
two Peter White Fellowships awarded to support K. H.'s 
undergraduate thesis, from which this paper is adapted. 
We thank J. Moore for helpful comments on an earlier 
version. We are also grateful for the efforts and comments 
of two anonymous reviewers. This is contribution number 
173 from the Program in Ecology, School of Life Sciences. 



Literature Cited 

Basch, P. K., K. J. Lie, and D. Heyneman. 1969. Antagonistic inter- 
action between strigeid and schistosome sporocysts within a snail 
host. J. Punisiiol. 55: 753-758. 

Baudoin, M. 1975. Host castration as a parasitic strategy. Evolution 

29: 335-352. 

Curtis, L. A. 1985. The influence of sex and trematode parasites on 
carrion response of the estuarine snail llyanassa obsoleta. Biol. Bull. 
169: 377-390. 

Curtis, L. A. 1987. Vertical distribution of an estuarine snail altered 
by a parasite. Science 235: 1 509- 1511. 

Curtis, L. A. 1990. Parasitism and the movements of intertidal gastro- 
pod individuals. Biol. Bull 179: 105-1 12. 

Curtis, L. A., and K. M. Hubbard. 1990. Trematode infections in a 
gastropod host misrepresented by observing shed cercanae. ./. IC.\[t 
Mar Biol Ecu/ 1-43: 131-137. 

Curtis, L. A., and L. E. Hurd. 1983. Age. sex and parasites: spatial 
heterogeneity in a sandflat population of llyanassa obsoleta. Ecology 
64: 819-828. 

Cort, VV. W., D. B. McMullen, and S. Bracken. 1937. Ecological studies 
on the cercariae in Stugnicolu cmar^inala angulata (Sowerby) in the 
Douglas Lake region. Michigan. ./ Parusilol. 23: 504-532. 

DeCoursey, P. J., and \V. B. Vernberg. 1974. Double infections of 
larval trematodes: competitive interactions. Pp. 93-109 in Symbiosis 
;/; l/ie Sea. W. B. Vernberg. ed. University of South Carolina Press. 
Columbia. SC. 

Dobson, A. P., and A. Merenlender. 1991. Coevolution of macropar- 
asites and their hosts. Pp. 83-101 in Parasite-Host Associations Co- 
existence or Conflict? C. A. Toft, A. Aeschlimann, and L. Bolis. eds. 
Oxford University Press. New York. 

Esch, G. W., A. W. Shostak, D. J. Marcogliese, and T. M. Goater. 
1990. Patterns and processes in helminth parasite communities: an 
overview. Pp. 1-19 in Parasite Communities: Patterns and Processes, 
C. Esch. A. Bush, and J. Aho, eds. Chapman and Hall. London. 

Fernandez, J., and G. \V. Esch. 1991a. Guild structure of larval trem- 
atodes in the snail Helisoma anceps: patterns and processes at the 
individual host level. / Parasitol. 77: 528-539. 

Fernandez, J., and G. \V. Esch. 1991 b. The component community 
structure of larval trematodes in the pulmonate snail Helisoma anccps 
J Panisiiol 11: 540-550. 

Futuyma, D. J., and M. Slatkin. 1983. Introduction. Pp. 1-13 in Co- 
solution. D. J. Futuyma and M. Slatkin. eds. Sinauer Associates. 
Sundcrland, MA. 

Gill, D. E., and B. A. Mock. 1985. Ecological and evolutionary dy- 
namics of parasites: the case of Trypanosnma diemyctyli in the red- 
spotted newt Nolophthalmus viridescens. Pp. 157-183 in Ecology 
and Genetics of Host-Parasite Interactions, D. Rollinson and R. M. 
Anderson, eds. Academic Press, New York. 

Gooch, J. L., B. S. Smith, and D. knupp. 1972. Regional survey of 
gene frequencies in the mud snail Nassarius obsoletus. Biol. Bull. 
142: 36-48. 

Gould, S. J., and E. Vbra. 1982. Exaptation a missing term in the 
science of form. PuleohiologyS: 4-15. 

Hollander, M., and D. A. Wolfe. 1973. Nonparametric Statistical 
Methods. John Wiiey and Sons. New York. 

Holmes, J. C. 1986. The structure of helminth communities. Pp. 203- 
208 in PurasilologyQuo vadil^. M. J. Howell, ed. Proceedings 6th 
International Congress of Parasitology, Australian Academy of Sci- 
ences, Canberra. 

Kuris, A. 1990. Guild structure of larval trematodes in molluscan hosts: 
prevalence, dominance and significance in competition. Pp. 69-100 
in Parasite Communities: Patterns and Processes, G. Esch, A. Bush, 
and J. Aho, eds. Chapman and Hall, London. 



GASTROPOD-TREMATODE INTERACTIONS 



35 



Lie, K. J., P. F. Basch, and T. Umathevy. 1965. Antagonism between two 
species of larval trematodes in the same snail. Nature 206: 422-423. 

McLennan, D. A., and D. R. Brooks. 1991. Parasites and sexual 
selection: a macroevolutionary perspective. Q Rev Biol 66: 
255-286. 

Minchella, D. J., B. K. Leathers, K. M. Brown, J. M. McNair. 
1985. Host and parasite counteradaptations: an example from a 
freshwater snail. Am Nat. 126: 843-854. 

Moore, J. 1987. Some roles of parasitic helminths in trophic interac- 
tions. A view from North America. Revixta Chilena de Hisloria Nat- 
ural 60: 159- 179. 

Mouahid, A., and H. Mone. 1990. Interference of Echinoparyphium 
elegans with the host-parasite system Bulmus truncatus-Schistosoma 
bovis in natural conditions. Ann. Trop. Med. Parasitol. 84: 341-348. 

Rohde, K. 1981. Population dynamics of two snail species, Planaxis 
sulcatus and Cerithium moniliferum, and their trematode species at 
Heron Island, Great Barrier Reef. Oecologia 49: 344-352. 

Smyth, J. D., and D. W. Halton. 1983. The Physiology of Trematodes. 
2nd ed. Cambridge University Press, Cambridge. 



Sokal, R. R., and F. J. Rohlf. 1981. Biometry, 2nd ed. Freeman and 
Company, New York. 

Sousa, \V. P. 1990. Spatial scale and the processes structuring a guild 
of larval trematode parasites. Pp. 4 1 -67 in Parasite Communities 
Patterns and Processes. G. Esch, A. Bush, and J. Aho. eds. Chapman 
and Hall, London. 

Stunkard, H. \V. 1983. The marine cercariae of the Woods Hole, 
Massachusetts region, a review and a revision. Biol. Bull. 164: 
143-162. 

Vernberg, \V. B., F. J. Vernberg, and F. W. Beckerdite. 1969. Larval 
trematodes: double infections in the common mud-flat snail. Seienee 
167: 1287. 

Williams, G. C. 1966. Adaptation and Natural Selection. Princeton 
University Press, Princeton, NJ. 

Williams, J. A., and G. \V. Esch. 1991. Infra- and component com- 
munity dynamics in the pulmonate snail Helisoma anceps. with spe- 
cial emphasis on the hemmnd trematode Ha/ipegus occidualis. J 
Parasitol. 77: 246-253. 



Reference: Biol. Bull 184: 36-51. (February, 1993) 



Effects of Marine Bacteria on the Culture of Axenic 
Oyster Crassostrea gigas (Thunberg) Larvae 

PHILIPPE DOUILLET 1 AND CHRISTOPHER J. LANGDON 

Oregon State University, Department of Fisheries, Hatfield Marine 
Science Center, Newport, Oregon 97365 



Abstract. Bacteria-free oyster larvae ( Crassostrea gigas) 
were cultured under aseptic conditions; they were fed 
axenic algae (Isochrysis ga/bana), and the medium was 
inoculated with isolated strains of marine bacteria. 
Twenty-one bacterial strains were tested, and most were 
detrimental to larval survival and growth. However, ad- 
ditions of strain CA2 consistently enhanced larval survival 
(21-22%) and growth (16-21%) in comparison with con- 
trol cultures that were fed only algae. Size-frequency dis- 
tributions of populations of larvae cultured for 10 days 
on axenic algae were skewed due to the poor growth of 
many individuals; whereas size-frequencies from popu- 
lations of larvae fed axenic algae supplemented with CA2 
bacteria were distributed normally. Strain CA2 may 
therefore make a nutritional contribution to the growth 
of oyster larvae. /. galbana did not grow under the light 
intensities used for larval culture; thus the improvement 
in larval growth cannot be attributed to bacterial en- 
hancement of algal growth and, consequently, food avail- 
ability. Naturally occurring microflora from Yaquina Bay, 
Oregon, depressed survival or growth of larvae-fed live 
algae. 

Introduction 

Bivalve larvae in culture vary substantially in survival 
and growth (Davis, 1953;Loosanoff, 1954;Walne, 1956a). 
Between 25% and 50% of the variability in the growth of 
a single population of mussel larvae (Innes and Haley, 
1977), or different populations of larval Crassostrea vir- 
ginica (Newkirk el al, 1977), are due to genetic factors. 
A significant proportion of the variability in the survival 
of C. gigas larvae was similarly attributed to genetic factors 



Received 3 June 1992; accepted 10 November 1992. 
' Present address: The University of Texas at Austin, Marine Science 
Institute, P.O. Box 1267, Port Aransas. Texas 78373. 



(Lannan, 1980). Exogenous factors, such as temperature 
(Loosanoff, 1959), salinity (Bayne, 1965), pH (Calabrese 
and Davis, 1970), food quantity (Walne, 1965), food 
quality (Davis, 1953), age of the algal food (Dupuy, 1975), 
larval concentration (Loosanoff et al., 1953), size of con- 
tainer (Dupuy, 1975), silt (Davis and Hidu, 1969), exu- 
dates of unfavorable algal species (Bayne, 1965), water 
quality (Millar and Scott, 1967) and toxicants (Walne, 
1970) have been found to contribute significantly to vari- 
ability in larval growth. Nonetheless, even different cul- 
tures of larvae obtained from the same parents and grown 
under identical conditions of temperature, salinity and 
ration have been commonly reported to vary in their 
growth (Bayne, 1983). 

The role of bacteria as beneficial or harmful agents in 
the culture of bivalve larvae has been the subject of many 
investigations, but this role has not been fully evaluated. 
Thirteen different isolates of marine bacteria did not sup- 
port the growth of oyster larvae when provided as the sole 
source of paniculate food (Davis, 1950, 1953). High bac- 
terial densities in cultures of bivalve larvae are generally 
considered to be deleterious to the larvae (Walne, 1956a, 
1956b, 1958), and even innocuous bacteria in large num- 
bers have been reported to depress the rate of algal inges- 
tion (Ukeles and Sweeney, 1969). Some bacterial strains 
are reportedly able to invade larvae, to produce toxins, 
or both (Guillard, 1959; Tubiash et al.. 1965; Tubiash et 
al., 1970; Brown, 1973; Di Salvo, 1978; Nottage and 
Birkbeck, 1986). In contrast, bacteria have also been im- 
plicated as a food source for bivalve larvae (Carriker, 1956; 
Hidu and Tubiash, 1963) or as improving the growth of 
larvae fed on algae (Martin and Mengus, 1977; Beese, in 
Prieur et al., 1990). 

The elimination of microbial contaminants is prereq- 
uisite to a study of the effects of a bacterial strain on an 
organism in culture. This approach has been used to study 
the effects of several bacterial strains on cultures of the 



36 



BACTERIAL EFFECTS ON OYSTER LARVAE 



37 



protozoan Amoeba nitrophila (Frosch, 1897 in Luck et 
al., 1931); the cladoceran Moina macrocopa (Stuart et ai, 
1931); and larvae of the clam Mercenaria mercenaria 
(Guillard, 1959). 

In the present study, axenic larval Crassostrea gigas, 
obtained without the use of antibiotics, were used in a 
series of experiments meant to reveal whether selected 
strains of marine bacteria can consistently improve the 
survival and growth of algal-fed oyster larvae. 

Materials and Methods 

Maintenance of larvae, bacteria and algae 

Bacteria-free oyster larvae were obtained according to 
the method of Langdon ( 1983). Adult oysters Crassostrea 
gigas were held at 1 8C in a recirculating seawater system 
for a period of 4 to 6 weeks, depending on the initial 
reproductive condition of the broodstock. After this con- 
ditioning period, the oysters were opened and shucked. 
Using aseptic techniques in a laminar-flow hood, we dis- 
infected the external surface of the gonads of each oyster 
with a 1% solution of sodium hypochlorite. A small in- 
cision was made through the surface of the gonads with 
a heat-sterilized scalpel, and gametes from each oyster 
were removed with sterile Pasteur pipettes and transferred 
to separate sterile flasks containing 0.2 ^m-filtered, au- 
toclaved seawater (FSSW). Eggs were fertilized by the ad- 
dition of a few drops of sperm suspension and then were 
transferred to Erlenmeyer flasks containing FSSW at a 
density of 1 00 eggs ml ' . Eggs were incubated on an orbital 
shaker at 25 C for 48 h. When the trocophore larvae had 
developed into veligers (straight-hinged larvae), subsam- 
ples of larvae were aseptically withdrawn for axenicity 
tests, and the remaining larvae were then held at 5C for 
5 days. Axenicity of larvae was determined by epifluo- 
rescence microscopy using 4'6-diamidino-2-phenylindole 
(DAPI) staining techniques (Porter and Feig, 1980). Sam- 
ples of larvae were also added to 1/10 recommended con- 
centration of Difco marine broth 2216 (3.74gr', salinity 
30 ppt) and incubated at 25C under aerobic or anaerobic 
conditions (BBL GasPak Pouch). Larvae from cultures 
that showed no evidence of microbial contamination from 
either the epifluorescence test or the 5 day broth incu- 
bations were considered adequate for experimentation. 
To confirm that the larvae were axenic, broth incubations 
were continued for 30 days. Axenic straight-hinged larvae 
were transferred to 250 ml Erlenmeyer flasks, each con- 
taining 1 50 ml of FSSW, closed with cotton plugs and 
capped with aluminum foil. Final larval density was 
5 ml" 1 . Growth experiments were then initiated by the 
addition to the culture flasks of axenic algae and the dif- 
ferent bacterial strains. Shell lengths of 100 randomly se- 
lected larvae were measured, either with an optical mi- 
crometer fitted to a compound microscope, or with an 
image analysis system (Zeiss Videoplan 2). 



Strains of marine bacteria were isolated from cultures 
of algae or oyster larvae at the Whiskey Creek Hatchery 
in Netarts Bay, Oregon. Other bacteria were isolated, ei- 
ther from the guts of adult oysters, or from incubations 
of protein capsules (Langdon, 1989) suspended in unfil- 
tered seawater. Pure bacterial strains were obtained by the 
dilution method of Rodina (1972). Strains were grown, 
at 25C, on marine agar 22 16 or brain heart infusion agar 
(Difco). Bacteria grown on such solid media for 3 to 5 
days were resuspended for 24 h in FSSW; they were then 
washed by centrifugation at 20,000 X g for 10 min and 
resuspended in FSSW. 

Strains were added to larval cultures at concentrations 
of 10 5 -10 6 cells ml" 1 . Cell concentrations were derived 
from equations relating spectrophotometric absorbance 
(600 nm) and bacterial concentration; the latter value was 
determined by direct count after staining with DAPI 
(Porter and Feig, 1980). Such equations were developed 
and used for each strain tested. 

Axenic Isochrysis galbana Parke (clone ISO) was ob- 
tained from the Culture Collection of Marine Phyto- 
plankton (Maine). Algal cultures were grown at 20C in 
200 ml f/2 medium (Guillard and Ryther, 1962) illumi- 
nated by 1000-1500 lux of cool white fluorescent light 
under a 12 h light/ 12 h dark photoperiod. Algal axenicity 
was determined as described above for larvae. 

All glassware was washed in 10% nitric acid, rinsed 
seven times with distilled water, and baked overnight at 
450C. Disodium ethylenediamine-tetraacetate (EDTA) 
was added at a final concentration of 1 ppm to all seawater 
to reduce the load of dissolved organic matter (Utting and 
Helm, 1985). Salinity of seawater after sterilization varied 
between 28 and 3 1 ppt. Heat sterilization was carried out 
for 15 min at 121C and 1.06 kg cm 2 pressure. 

Larvae fed on live algae and bacteria 

Twenty-one marine bacterial isolates were tested in 
three culture experiments for their effects on the survival 
and growth of larvae fed axenic Isochrysis galbana. Ex- 
periment I included seven microbial isolates from the 
Whiskey Creek Hatchery (H1-H7) and five isolates from 
the guts of adult oysters (G1-G5). Control treatments were 
either larvae fed only algae or starved larvae. 

In Experiment II, two strains (H6, H7) that improved 
larval growth in Experiment I were tested along with five 
strains isolated from the Whiskey Creek Hatchery (H8- 
H12), one strain isolated from the gut of an adult oyster 
(G6), and three strains isolated from protein capsules in- 
cubated in seawater (CA1-CA3). Control treatments in- 
cluded starved larvae and larvae fed only algae. In third 
control (SW), cultures of larvae were inoculated at the 
beginning of the experiment with naturally occurring 
bacteria present in 5 ml samples of 1 ^m-filtered seawater 
collected from Yaquina Bay, Oregon. The larvae in the 



38 



P. DOUILLET AND C. J. LANGDON 



third control treatment were fed axenic algae every other 
day. Experiments I and II were carried out with four rep- 
licates per treatment. 

Experiment III was designed to retest strains that had 
enhanced larval survival and growth in Experiment II (H7, 
CA2). Control treatments similar to those described for 
Experiment II were included. Experiment III was carried 
out with eight replicates per treatment. 

Cultures of bacteria-free oyster larvae (75.5-82 ^m shell 
length) were inoculated once at the beginning of each ex- 
periment with bacterial strains. Bacteria-free algal cells, 
harvested from cultures in exponential growth phase, were 
added to the larval cultures every two days. The seawater 
of the larval cultures was not renewed during the culture 
period. The concentration of algal cells in each larval cul- 
ture flask was estimated, as follows, before each feeding. 
A 2-ml sample of the larval culture medium was asepti- 
cally removed from each flask with a pipet; to prevent 
removal of larvae, the end of the pipet was covered with 
a 64 j/m Nitex screen. Algal cells were preserved with 
formalin, concentrated by centrifugation, and re-sus- 
pended in 100 jul of 0.2 ^m-nltered seawater. Algal con- 
centrations in the samples were then determined with a 
hemocytometer. Fresh algae were then added to larval 
culture flasks to provide cell concentrations at pre-deter- 
mined levels. Algal cell concentrations were increased by 
15,000 cells ml ', from 40,000 to 100,000 cells ml" 1 over 
a 10 day culture period. To provide uniform food quality 
during the experiments, algae from a single culture were 
added at each feeding period, to all larval cultures receiving 
an algal diet. 

Larval culture flasks were placed randomly on orbital 
shakers in a temperature-controlled room at 25C. The 
cultures were exposed to a light intensity of 50-70 lux for 
12 h each day. No algal growth occurred at this low light 
intensity. After 10 days of culture, samples of water were 
aseptically withdrawn from flasks containing starved lar- 
vae or larvae fed only axenic algae; these samples were 
analyzed for microbial contamination as described above. 
The experimental data were analyzed only if these control 
treatments were bacteria-free at the end of the 10 day 
culture period. 

Effects ofCAl bacteria on the growth of algae in tan-al 
cultures 

Cells of axenic /. galhana were initially suspended at a 
concentration of 40,000 ml~' in f/2 medium and then 
subdivided in sixteen 250 ml Erlenmeyer flasks. CA2 cells 
were added at 10 5 cells ml ' (final concentration) to eight 
flasks, while FSSW was added to the other eight flasks to 
maintain similar initial algal concentrations in all flasks. 
The final volume of each algal culture was 200 ml. Four 
algal cultures inoculated with bacteria and four cultures 
that had received only FSSW were placed in conditions 



conducive to the growth of I. galbana (1000-1500 lux 
and 20C); the remaining algal cultures were exposed to 
the conditions used for larval culture (50-70 lux and 
25 C). The algal cultures were incubated on orbital shak- 
ers for three weeks. Every second day, 10 ml samples were 
removed aseptically from each algal culture, and algal 
concentrations determined with a Coulter counter (Mo- 
del ZB1). 

Larvae fed on dead algae and bacteria 

Interactions between strain CA2 and living Isochrvsis 
galbana that could modify algal food quality were not 
addressed in the previous experiments. To determine 
whether bacteria could enhance cultures of larvae fed on 
non-living diets, live /. galbana were replaced with dead 
algae. 

In Experiment IV, known concentrations of axenic /. 
galbana were frozen at -5C. Freezing and thawing broke 
the cell walls and membranes of the algal cells. Larvae 
were fed dead freeze-killed algae (FA) every two days ac- 
cording to the same protocol used with live algae. One 
group of larval cultures fed FA was maintained bacteria- 
free, and two groups were inoculated at the beginning of 
the experiment with either strain H6 at 10 5 cells ml" 1 
(final concentration), or with an inoculum of naturally 
occurring bacteria (SW). The wild strains were added in 
5 ml samples of 1 /jm-nltered seawater collected from 
Yaquina Bay, Oregon, at a concentration of 10 5 -10 6 cells 
ml" 1 . Other larval cultures received on alternate days, ei- 
ther additions of strain H6 (at a final concentration of 10 ? 
cells ml ') alone, or naturally occurring bacteria (SW) (5 
ml of 1 ^m-nltered seawater) alone. Control treatments 
included starved larvae and larvae fed every second day 
on live axenic /. galbana. Culture conditions and sample 
treatments were similar to those of experiments carried 
out with live algae. Four replicates were tested per treat- 
ment. 

Algal cells were also killed by w 'Co-irradiation (5 me- 
garads) at the Radiation Center at Oregon State Univer- 
sity. Non-viability of irradiated algae (IA) was evident by 
the lack of growth of cells in f/2 medium at 20C under 
1000-1500 lux of fluorescent light emitted 12 h a day. 
The irradiation process also destroyed contaminants, as 
demonstrated by incubations, at 25 C, of irradiated algae 
in 1/10 diluted marine broth 2216 (3.74 g 1~', salinity of 
30 ppt) under either aerobic or anaerobic conditions (BBL 
GasPak Pouch). The integrity of the irradiated algal cells 
was verified by microscopic examination. Cell volumes 
of irradiated and non-irradiated algae from seven different 
cultures were determined with a Coulter counter (Model 
ZB1) equipped with a calibrated Coulter channelyser 
(Model 256). 

To ensure that IA were acceptable to larvae as a food 
source, the ingestion rates of larvae fed on either IA or 



BACTERIAL EFFECTS ON OYSTER LARVAE 



39 



live /.SW///T.V/.V galbana were compared. Ingestion rates 
were calculated according to the methods described by 
Checkley (1980). Larval ingestion rates for live and ''"Co- 
irradiated algae were compared with a 2 sample t-test, 
after verifying homocedasticity by Cochran's test for ho- 
mogeneity of variances at the 0.05 level of probability 
(Douillet, 1991). 

In Experiment V, oyster larvae were fed IA every second 
day according to the methods employed with live algae 
in Experiments I to III. Three groups of larval cultures 
were fed IA. One group was maintained bacteria-free, 
while the two others were inoculated at the beginning of 
the experiment with strains H7 or CA2. Control treat- 
ments included starved larvae or larvae fed every two days 
on live axenic Isocfuysis galbana. Eight replicates were 
tested per treatment. Larval survival and growth were de- 
termined as described below. 

Data collection and analysis 

At the end of each experiment, the larvae were carefully 
transferred to scintillation vials containing buffered form- 
aldehyde (2% final concentration, pH = 8). The larval 
tissues were stained with rose of Bengal, so that the larvae 
that were alive could be distinguished from empty shells. 
The whole larval population in each flask was counted 
with a dissecting microscope, and the shell lengths of 100 
randomly selected larvae were measured, either with an 
optical micrometer fitted to a compound microscope, or 
with an image analysis system (Zeiss Videoplan 2). Sur- 
vival and growth data were transformed to satisfy as- 
sumptions of ANOVA. Survival data were transformed 
as: 

arcsin (square root (percent survival 100 ')) 
Growth data were transformed as: 

arcsin (square root ((In L, - In L,,)t ')) 



where L, is the final mean shell length (^m); L u is the 
initial mean shell length (^m); and t is the culture period 
(10 days). 

These transformations were successful in reducing the 
heterocedasticity of the survival data but not of the growth 
data (Cochran's test for heterogeneity of variances, at the 
0.05 level of probability). Treatment effects on larval sur- 
vival were tested with one-way ANOVA. Where significant 
differences were indicated, Tukey's honestly significant 
difference test (T-HSD) was applied to determine the sta- 
tistical significance of differences among individual treat- 
ments at the 0.05 level of probability. Treatment effects 
on larval growth were analyzed with the Kruskal-Wallis 
test (KW). Differences among individual treatments were 
determined by means of the Games and Howell test 
(G&H) of equality of means with heterogeneous variances 
(Sokal and Rohlf, 1981), at the 0.05 level of probability. 



All tests were performed with the computer program Sta- 
tistix (NH Analytical Software), except the Games and 
Howell test which was carried out with the program Biom 
(Rohlf, 1982). 

The size-frequency distributions of populations of algae- 
fed larvae that were bacteria-free were compared with 
those fed algae supplemented with CA2 bacteria in Ex- 
periments II and III. Skewness coefficients (gl : Sokal and 
Rohlf, 1981) of larval populations from each replicate 
flask were calculated and used to compare larval size fre- 
quency distributions. A normal size distribution would 
have a gl coefficient equal to 0. A skewness coefficient 
higher than indicates that the size distribution is posi- 
tively skewed (higher proportion of small-sized individ- 
uals), while a coefficient smaller than indicates negative 
skewness. After confirmation of homocedasticity of gl 
values by Cochran's test at the 0.05 probability level, data 
were analyzed by two-way ANOVA with treatment (algae, 
algae + CA2) and experiment as factors. As dictated by 
the results of ANOVA, appropriate multiple comparisons 
of means were conducted at the 0.05 level of probability 
using the Student-Newman-Keuls procedure (SNK), 
controlling for experiment-wide error (Underwood, 198 1 ). 

Cryopreservation ot bacteria 

Bacteria have been described as adaptable chimaeras, 
the metabolic plasticity of which results from widespread 
transfer of genetic information though plasmids or pro- 
phages (Sonea. 1988). This strategy for adaptation to 
changing environments may result, during evolution, in 
the loss of beneficial characteristics of selected bacterial 
strains. In order to reduce the possibility of changes in 
bacterial characteristics between successive experiments, 
selected strains were cryopreserved at -70C in 10% 
(V/V) glycerol in sterile 1/10 diluted marine broth 2216. 

Identification of strain CA2 

The identification of bacterial strain CA2 was based on 
Bergey 's Manual of Systematic Bacteriology (Holt, 1 984). 
The methodology used for different procedures followed 
the Manual of Methods of General Bacteriology (Gerhardt 
et ai, 1981). Exponentially growing cells cultured on ma- 
rine agar 22 1 6 were used for the following tests performed 
at the Hatfield Marine Science Center, Newport, Oregon, 
(a) Cells were Gram stained, (b) Motility was determined 
by observations of wet mounts with light microscopy, (c) 
Oxidase activity was determined by spreading CA2 cells 
with sterile cotton swabs over Pathotec cytochrome oxi- 
dase test strips (General Diagnostics), which contained a 
derivative of dimethyl-p-phenylenediamine and -naph- 
thol. (d) Cultures of CA2 cells were flooded with 3% hy- 
drogen peroxide for catalase testing, (e) Oxidation and 
fermentation of glucose was assayed with the modified 
O-F medium of Leifson (1963). (0 Utilization of inorganic 



ORIGIN OF BACTERIA ADDED TO LARVAL CULTURES 
m H rcn c 



Q 

on 



I 
> 

cc 

Z) 





A 






100- 


r\ 










80- 










/ 




/ 




/ 




1 


60- 








/ 




f 




/ 

/ 




/ 






1 

X 










7 




y 


1 


' 




/ 
/ 




/ 






X 












/ 




. 




/ 




. 




/ 
/ 




' 






X 






40- 






J, 




/ 




', 




^ 




; 




/ 
/ 
/ 




; 


A 


X 
X 

X 




X 

X 




20- 






/ 




/ 




' 




/ 




^ 




/ 




/ 


X 




X 


1 


X 










/ 




y 




/ 




/ 




^ 




/ 




/ 


a.. X 




X 


M 


X 




- 


r 1 , 




y 




/ 




/ 




/ 




/ 




/ 

/ 




/ 


M v 




X 


H 


X 

V 




STARVED ALGAE H1 H2 " 2 H4 H5 H6 H7 G1 G2 


G3 G4 G5 


ALGAE + BACTERIA 





155 



in 



145- 



E 135- 



O 



125- 



1 15- 



105- 



T 



JL 



I 

/ 





STARVED ALGAE 



HI H2 H3 H4 H5 H6 H7 Gl G2 G3 G4 G5 



ALGAE + BACTERIA 



Figure I. Effects of different bacterial strains on oyster larvae cultured on a diet of axenic Isochrrsis 
galhana for 10 days. (A) Survival in Experiment I. (B) Survival in Experiment II. (C) Growth in Experiment 
I. (D) Growth in Experiment II. Bacteria were isolated from the Whiskey Creek Hatchery. Oregon (H). from 
the guts of adult oysters (G), from incubations of protein capsules in seawater (CA), or were naturally- 
occurring in 1 nm-nltered seawater (SW). Larval control treatments were starved or fed axenic /. galbana. 
Results of Tukey's HSD pairwise comparisons and Games and Howell's tests are displayed below the his- 
tograms of survival and growth, respectively. Squares that occur together on any one of the horizontal lines 
indicate mean values that are not different at the 0.05% level of significance. 

40 



BACTERIAL EFFECTS ON OYSTER LARVAE 



41 



ORIGIN OF BACTERIA ADDED TO LARVAL CULTURES 
KS Sw I~TI H GZ) C E\S CA 



100- 


B 




1 1 I 1 1 


oo 80- 




/ 




/ 


1 


/ 


f 


X 




A 




;' 6 - 






> 




/ 




1 




/ 




1 




^ 




1 




X 
X 












| 










/ 









/ 




/ 








/ 




. 




X 




v 




^ 








> 






y 




/ 




' 








/ 




./ 




' 




~x 




V 




X 




s^ 




40- 
co 






i 




^ 




/ 




/ 




/ 




^ 




', 




/ 




x 
x 
x 




1 












20- 






X 




/ 




/ 




'. 




V, 




/ 




/ 




/ 




~x 




^ 




^ 




N 
v \ 




- 


i 




1 




^ 




^ 




/ 




/ 




/ 




/ 




/ 




>(. 
X 




1 




1 




1 




cTiOv/irn !r.r SW H6 H7 H8 H9 HlO H1 1 HI 2 G6 CA1 CA2 CA3 



ALGAE + BACTERIA 



o 

CO 



E 

X 
I 

o 



I 

CO 



75 



150- 



125- 



iOO- 



75 



I 



I 



I 



STARVED ALGAE 



SW H6 H7 H8 H9 HlO H11 H12 G6 CA1 CA2 CA3 



ALGAE 



BACTERIA 



Figure 1. (CiiHtinued) 



42 



P. DOUILLET AND C. J. LANGDON 



sources of nitrogen was evaluated by culturing CA2 cells 
on media prepared with NH 4 C1 or NaNO, (0.5 g 1 '), 
glucose (0.1 g 1 '), Na 2 HPO 4 (0.1 g 1 '), FePO 4 (0.004 g 
1 '(and 1 ml 1 ' off/2 vitamin mix (Guillard and Ryther, 
1962). The culture media used as controls were prepared 
by replacing NaNO, or NH 4 C1 with peptone or tryptone 
(Difco) at 0.5 g 1 '. (g) Anaerobic growth was determined 
by transferring CA2 cells either into solid media in Petri 
dishes, or into 25 ml 1/10 diluted marine broth 2216 (3.74 
gT 1 ; salinity 30 ppt) contained in 50 ml Erlenmeyer flasks, 
placing these cultures in anaerobic GasPak pouches (BBL), 
and incubating the cells at 20C for up to one month. 

The following tests were carried out by Dr. Ronald 
Weiner (University of Maryland at College Park). Meth- 
odology followed the Manual of Methods for General 
Bacteriology (Gerhardt et ai, 1 98 1 ). (a) Salt requirements 
were evaluated by culturing CA2 cells in tryptic soy agar 
(TSA) prepared at different salt concentrations; NaCl was 
added at 1% increments up to 10% of the control level. 

(b) As evidence of anaerobic growth and motility, CA2 
cells on a straight needle were used to inoculate a tube 
containing semisolid tryptic soy broth enriched with 0.8% 
agar and 1%. NaCl, and the pattern of growth observed. 

(c) Flagellar staining was carried out by the Leifson 
method (Gerhardt et ai, 1981). (d) Synthesis of exopoly- 
saccharides was evaluated by the phenol-sulfuric acid re- 
action (Gerhardt et ai, 198 1 ). (e) The mole percent gua- 
nine plus cytosine (mol% G + C) in extracted deoxyri- 
bonucleic acid (DNA) was determined by the thermal 
melting (denaturation) methods of Marmur and Doty 
( 1962) with a Gilford UV programmable spectrophotom- 
eter. (f) Antibodies of 20 different bacteria strains be- 
longing to the Alteromonas/Shewanella group were tested 
for reaction with exopolysaccharides of CA2 cells, (g) Fatty 
acid analyses of strain CA2 were carried out for compar- 
ison with profiles of other marine bacteria by Dr. Fred 
Singleton (Center for Marine Biotechnology, University 
of Maryland) and by Dr. Warren L. Landry (Food and 
Drug Administration, Dallas, Texas). 

Results 

Larvae fed on live algae and bacteria 

Single additions of marine bacterial isolates to oyster 
larvae cultures significantly affected larval survival (AN- 
OVA, P< 0.01) and growth (KW, P < 0.01 ) after 10 days 
of culture in all experiments (Figs. 1, 2). The microbes 
tested can be divided into categories depending on their 
effects upon oyster larvae: adverse, neutral, or beneficial. 
Bacteria belonging to the last category were tested further, 
and their effects upon oyster larvae were designated as 
either variable or consistently beneficial. 

Adverse strains. Strains Gl. G2 and G4 adversely af- 
fected larval survival (T-HSD, P < 0.05), whereas strains 
Gl, G2, G4, G5, H8, and H10 adversely affected larval 



growth (G&H, P < 0.05). Bacteria present in 5 ml aliquots 
of 1 ^m-filtered seawater depressed larval survival (T- 
HSD. P < 0.05 ) in Experiment II and larval growth (G&H. 
P < 0.05) in Experiment III. 

Neutral strains. A large proportion of the strains (HI, 
H2. H3. H4. H5. H9. Hll, HI 2, G3, CA1, and CA3) 
added to cultures of oyster larvae had no significant effect 
on larval survival (T-HSD, P > 0.05) or growth (G&H, 
P > 0.05) compared with cultures fed algae alone. 

( 'ariable strains. Addition of strains H6 and H7 to larval 
cultures caused inconsistent improvements of larval 
growth. For example, larval growth was enhanced (G&H, 
P < 0.05) in cultures inoculated with strains H6 and H7 
in Experiment I. but the enhancement with strain H7 was 
statistically insignificant in Experiments II and III (G&H, 
P> 0.05). Moreover, larval growth was depressed (G&H, 
P < 0.05) when strain H6 was added to larval cultures in 
Experiment II. 

Beneficial strains. In both Experiments II and III. larvae 
grown in cultures inoculated with strain CA2 had a sig- 
nificantly greater shell length than control larvae fed only 
axenic algae (G&H, P < 0.05). Larval survival was en- 
hanced in cultures inoculated with strain H7 and CA2, 
but this enhancement was statistically significant only in 
Experiment III (T-HSD, P < 0.05). 

Size frequency distributions of populations of larvae 
fed axenic algae were skewed compared to those from 
cultures fed algae supplemented with CA2 bacteria (Fig. 
3; Table 1 ). Analysis of variance indicates a significant 
interaction between treatment and experimental factors 
(Table 2). In both Experiments II and III, skewness coef- 
ficients for populations of larvae fed axenic algae alone 
were significantly larger (SNK, P < 0.05) that those for 
populations of larvae fed algae and inoculated with CA2 
bacteria. The difference between the skewness coefficients 
of treatments in Experiment II is larger than that in Ex- 
periment III, explaining the significant interaction deter- 
mined by the two-way ANOVA test. 

Effects ofCA2 bacteria on the growth of algae in larval 
cultures 

Cells of Isochrysis galbana, with or without inoculations 
of CA2 bacteria, did not grow under the conditions used 
to culture larvae (Fig. 4). The occurrence of CA2 cells in 
the culture medium had no effect on algal growth under 
favorable light intensity (1000-1500 lux) and tempera- 
ture (20C). 

Larvae fed on dead algae and bacteria 

Significant differences among treatments in Experi- 
ments IV and V were determined for larval survival (AN- 
OVA, P < 0.0 1 ) and growth (KW. P < 0.0 1 ). The survival 
of larvae cultured on axenic FA or IA alone was signifi- 
cantly lower (T-HSD, P < 0.05) than that of larvae 



BACTERIAL EFFECTS ON OYSTER LARVAE 




20 



STARVED ALGAE 



ALGAE + BACTERIA 



O 

uo 



+ 1 



I 
\ 
O 



UJ 

I 
in 




STARVED ALGAE 



ALGAE + BACTERIA 



Figure 2. Survival and growth of oyster larvae after 10 days of culture on axenic Isochrysis galbana 
supplemented with different bacterial strains (Experiment III). Bacteria were isolated from the Whiskey 
Creek Hatchery, Oregon (H) or from incubations of protein capsules in seawater (CA). Naturally-occurring 
bacteria present in 1 Aim-filtered seawater (SW) were added in a control treatment. Other control treatments 
included larvae fed axenic / ga/hana or starved. Results of Tukey's HSD pairwise comparisons and Games 
and Howell's tests are displayed below the histograms of survival and growth, respectively. Squares that 
occur together on any one of the horizontal lines indicate mean values that are not different at the 0.05% 
level of significance. 



44 



P. DOUILLET AND C. J. LANGDON 



CO 

i 

rr 



z 

CO 



o 

UJ 
M 

LO 



IT 



DIETS 
O O AXENIC ALGAE 

AI GAF 4- CA2 



DIETS 

O O AXENIC ALGAE 

ALGAE + CA2 




105 



140 



175 



210 



245 



280 



LARVAL SHELL LENGTH 

Figure 3. Size frequency distributions of larvae cultured for 10 days on a diet oflsuchrysis galhana with 
or without addition of CA2 cells. Points represent percent larvae for each shell length interval of 30 ^m. 
Lines used for illustrative purposes only. Data from Experiments II (n = 400) and III (n = 800) for each 
treatment. 



cultured on live axenic algae alone (Figs. 5, 6). However, 
the survival of larvae fed FA or IA was higher (T-HSD, 
P < 0.05) than that of starved larvae. In contrast, no sig- 
nificant differences in larval survival were detected be- 
tween cultures fed live algae and cultures fed FA or IA 
inoculated with strains H6 and H7, respectively (T-HSD, 
P> 0.05). Survival of larvae fed every two days on bacteria 
H6 alone was not significantly different (T-HSD, P > 0.05) 
from that of larvae fed live algae, and was significantly 
higher (T-HSD, P < 0.05) than that of starved larvae (Fig. 
5). Larvae from cultures inoculated every two days with 
5 ml of 1 /jm-filtered seawater (SW) also showed higher 
survival (T-HSD, P < 0.05) than that of starved larvae. 
Larvae fed on FA or IA were significantly smaller than 
larvae fed on live axenic algae (G&H, P < 0.05), and were 



not different from the size of starved larvae (G&H, P 
> 0.05) at the end of the experiment (Figs. 5, 6). Additions 
of single bacterial strains to cultures of larvae fed FA or 
IA did not improve larval growth compared to larvae fed 
FA or I A alone (G&H, P > 0.05). In contrast, growth of 
larvae fed FA inoculated with 5 ml of 1 ^in-filtered sea- 
water was significantly enhanced (G&H, P < 0.05) com- 
pared to that of larvae fed FA alone or starved larvae (Fig. 
5). Similarly, additions every two days of 5 ml of 1 //m- 
filtered seawater or strain H6 alone to larval cultures sig- 
nificantly enhanced the growth of larvae (G&H, P< 0.05) 
compared to that of starved larvae. 

The poor growth of larvae fed FA may have been due 
to the rupture of the freeze-killed algal cells. 60 Co-irradia- 
tion did not affect the integrity of the algal cells but re- 



BACTERIAL EFFECTS ON OYSTER LARVAE 



45 



Table I 

Skewness coefficients (gl) jrom si:efm/neney ilistrihulianx 
of populations oj larvae cultured in Experiments II and III 



Experiment 



Diet 



Average skewness of 
populations 1 S.D. 



11 
II 

II! 
Ill 


ISO 
ISO + CA2 
ISO 
ISO + CA2 


0.7906 0.21 34 (n = 4) 
-0.0605 0.2235 (n = 4) 
0.3801 0.1720 (n = 8) 
-0.0466 0.29 10 (n = 8) 



Larvae were cultured with either axenic Isoclin'sis galbana (ISO) alone 
or /. galbana plus CA2 bacteria. 



duced their volume from 44.4 1 .92 ^m 3 to 26.3 0.59 
/urn 3 (x 1 SD; n = 7). A high proportion of irradiated 
cells remained intact while in suspension in seawater, as 
demonstrated by the small decrease in cell concentration 
in control flasks, from 59,043 1,1 19 cells ml~ ', to 58,539 
1,505 cells mr' (x 1 SD; n = 4) in 105 min. IA cells 
were ingested by oyster larvae at rates significantly (2 
sample t-test, P < 0.01 ) greater than that for live cells. 

Identification of strain CA2 

Strain CA2 was presumptively identified as Altero- 
nwnas sp. on the basis of the following characteristics: 
Gram negative rod; aerobic; oxidase positive; requires 250 
nA/ salt; motile with polar flagella; exopolysaccharide 
synthesis; and guanine plus cytosine 43 mol% (T m ). 

The exopolysaccharides of CA2 bacteria did not react 
with antibodies to 20 species of A/temmonas. Further- 
more, both analyses of fatty acids revealed a very unusual 
fatty acid profile with a high proportion of C-14, C-15 
fatty acids (Table 3); this is not characteristic of the genus 
Alteromonas. However, the fatty acid profile was not sim- 
ilar to any of the species profiles listed in Dr. Landry's 
marine library. Therefore, strain CA2 may be an Altero- 
monas species not typical of the genus. 

Further characteristics of strain CA2 include yellow 
pigment production, oxidation and fermentation of glu- 
cose, but no gas production, and inability to utilize in- 
organic sources of nitrogen, such as NH 4 C1 or NaNO 3 for 
growth. Catalase was weakly positive. 

Discussion 

Axenic larval Crassostrea gigas were used to determine 
the effects of additions of single bacterial strains on the 
survival and growth of larvae cultured with algae. Bacteria 
can be categorized as adverse, neutral or beneficial, de- 
pending on their effects upon oyster larvae. Furthermore, 
bacteria found beneficial in one experiment were reiested 
in subsequent experiments and could be further catego- 
rized as either variable or consistently beneficial strains. 



Additions of strain CA2 to larval cultures consistently 
enhanced larval survival (21-22%) and growth (16-21%) 
compared with that of larvae fed on algae alone. 

The specificity of bacterial strains as food for grazers 
has frequently been reported (Frosch, 1897 in Luck a ai. 
1931; Stuart el al., 1931; Curds and Vandyke, 1966). Fur- 
thermore, Curds and VanDyke (1966) found that one 
bacterial strain was either slightly toxic, unfavorable, or 
favorable depending on the ciliate species tested. In con- 
trast, a single bacterial strain (PM-4) was found to promote 
the growth of both shrimp (Penaeus monodon) and crab 
(Port units tridentatus) larvae (Maeda, 1988; Maeda and 
Nogami, 1989). Consequently, no generalization about 
the beneficial effects of specific bacterial strains can be 
made; i.e., each strain must be tested again with each new 
target species. 

Bacteria may be used directly as a food item by oyster 
larvae (Douillet, 1991). Starved axenic oyster larvae 
showed poor survival and did not grow after 10 days of 
culture. In contrast, larvae in cultures inoculated with 
single bacterial strains or mixtures of naturally-occurring 
marine bacteria had higher survival rates than starved lar- 
vae, but lower growth rates than larvae fed on algal diets. 
Consequently, the bacterial strains tested did not provide 
all the nutritional requirements for larvae, but appeared, 
at least, to partially satisfy larval metabolic requirements, 
as demonstrated by the beneficial effects of bacteria on 
larval survival and growth. Straight-hinged oyster larvae, 
fed for 10 min on l4 C-labeled CA2 cells at 1.5 X 10 7 cells 
ml ' and purged of undigested l4 C-material, retained 
enough bacterial carbon to meet over 140% of their active 
carbon metabolic requirements during a 10 min period 
(Douillet, 1991). Beese(in Prieureta/.. 1990) determined 
that xenic, starved larval Crassostrea gigas grew 60% in 
size after seven days of culture, whereas starved axenic 
larvae did not grow. The ability of starved xenic bivalve 
larvae to grow has been determined to be greater for larvae 



Table II 

Two-way analysis of variance of skewness coefficients (gl)for size 
frequency distributions of populations of larvae cultured 
in Experiments II and III 



Source of 
variation 


d.f. 


Sum of 
squares 


Mean 
squares 


F-ratio 


Sig. 
level 


Experiment (A) 


1 


0.31468 


0.31468 


5.79 


0.0259 


CA2 addition (B) 


1 


3.2656 


3.2656 


60.12 


0.0000 


Interactions (A*B) 


1 


0.36039 


0.36039 


6.63 


0.0180 


Replicates (C) 












Residual (A*B*C) 


20 


1.0864 


0.05432 






Total 


23 


5.0271 









Larvae were cultured with Isochrysis galbana alone or /. galbana plus 
CA2 bacteria. 



46 



P. DOUILLET AND C. J. LANGDON 



= 3 



V 

<J 



o 



u 

z 
o 
u 



o 

_1 

< 



O O AXENIC 
- ALGAE 
V V AXENIC 

A A ALGAE 

/ 

Rt 9. 


ALGAE \ 
+ CA2 CELLS )'000-,500LUX.2C*: 

^ \ 50-70 LUX. 25t T i 
+ CA2 CELLS / y '"^y 

* s' ^ 

^6/ 

/ 

X^^ vi^ ^i^ ^? ^17 rr* 


D 4 


i A i*i lAiJLim.jti i 
6 8 10 13 15 17 19 21 



Figure 4. 

(50-70 lux: 



TIME (days) 

Effects of CA2 bacteria on growth of Isochr r.v/.v giilhani.1 under conditions used to raise larvae 
25C) or under conditions found optimal for algal growth ( 1000-1500 lux; 20C). 



of the mussel Mytilus edit/is than for larval C. gigas (His 
ct til.. 1989). But bacteria lack long-chain polyunsaturated 
fatty acids (PUFA) (Kates. 1964; Perry el at., 1979) and 
sterols (Lehninger, 1975), both of which may be essential 
for the growth of marine bivalves (Trider and Castell. 
1980; Langdon and Waldock, 1981). This lack of es- 
sential nutrients could explain why larvae grew more 
poorly on a diet of bacteria alone than on a diet of algae 
alone. 

Size-frequency distributions of bacteria-free oyster lar- 
vae cultured for 10 days on axenic live algae were always 
positively skewed due to a high proportion of larvae that 
exhibited poor growth. Algae were always present in cul- 
tures at satisfactory concentrations for larval growth 
(Breese and Malouf, 1975); therefore, the poor growth of 
some larvae in populations fed axenic algae could not be 
due to insufficient algal food. In contrast, additions of 
CA2 bacteria to cultures of algae-fed larvae consistently 
normalized larval size-frequency distributions. Larval 
survival was equal (Experiment II; Fig. IB, D) or higher 
(Experiment III; Fig. 2) in cultures inoculated with strain 
CA2 than in cultures fed algae alone; therefore, changes 
in size-frequency distributions were not due to the selective 
death of slow growing larvae in bacterized cultures. In- 
stead, additions of strain CA2 to larvae fed on algae ap- 
parently shifted larval size-frequency distributions by 
promoting the growth of larvae that would grow poorly 
on an algal diet alone. This result suggests that some oyster 
larvae in cultured populations require supplements of 
bacteria in order to grow, and that an algal diet of Iso- 
c/irysis galhana alone is not sufficient to meet their nu- 
tritional requirements. 



The inability of a single algal food species to support 
larval growth rates comparable to those obtained on mix- 
tures of algal species suggests that diets of single algal spe- 
cies can be nutritionally inadequate for maximum larval 
growth (Davis and Guillard. 1958; Walne, 1970). Mi- 
crobes could provide dietary micronutrients, such as vi- 
tamins (Kutsky, 1981) or other growth factors, that could 
be deficient in algal diets. Vitamin deficiencies in the me- 
dia used to culture axenic Anemia have arrested growth 
and caused the early mortality of this crustacean (Provasoli 
and D'Agostino, 1962). Vitamin supplements increased 
the growth rate of larval Crassostrea virginica, when given 
alone or in combination with Chlorella (Davis and Chan- 
ley, 1956). The high nutritional value of bacteria is in- 
dicated by the success of bacterial supplements in im- 
proving the quality of algae (Provasoli el a/., 1959), or of 
dried diets of different chemical composition (Douillet, 
1987). 

Bacterial enhancement of larval cultures may also have 
been due to other mechanisms apart from bacterivory. 
Bacteria could have acted as a symbiont for larvae, con- 
tributing to the larva's protein nutrition through nitrogen 
fixation (Benemann, 1973; Carpenter and Culliney, 1975; 
Guerinot and Patriquin, 1 98 1 ), or by aiding in the diges- 
tion and assimilation of ingested algae. The bacterial flora 
of bivalve larvae consists of a high proportion of strains 
that produce extracellular enzymes, such as proteases and 
lipases (Prieur, 1982). 

Oyster larvae were grown for 10 days with no change 
in the culture medium; thus metabolites excreted by bi- 
valves (Cockcroft, 1990) and algae (Hellebust, 1974) 
would accumulate in the larval cultures. Strain CA2 may 



BACTERIAL EFFECTS ON OYSTER LARVAE 



47 



o 

I/I 






o: 



a? 



100 
90 
80- 
70- 
60- 
50- 
40- 
30- 
20- 
10- 
0- 



rb. 




STARVED ALGAE FA 



SW 



H6 



SW 



H6 



FA + BACTERIA 



BACTERIA 



Q 
00 



+ 1 



o 

z 



LJ 

X 

oo 



< 

LJ 




STARVED ALGAE 



SW 



H6 



SW 



H6 



FA + BACTERIA 



BACTERIA 



Figure 5. Survival and growth of oyster larvae after 10 days of culture when fed on a diet of either 
bacteria alone (strain H6, naturally-occurring bacteria present in 1 ^m-tiltered seawater (SW)) or freeze- 
killed Isoi-hrysisgalbana (FA) with or without supplements of bacteria (H6 or SW) (Experiment IV). Control 
treatments were starved or fed axenic / galbana. Results of Tukey's HSD pairwise comparisons and Games 
and Howell's tests are displayed below survival and growth histograms, respectively. Squares that occur 
together on any one of the horizontal lines indicate mean values that are not different at the 0.05% level of 
significance. 



48 



P. DOUILLET AND C. J. LANGDON 



Q 
GO 



4-1 



CL 

ID 
CO 



60- 



50- 



40- 



30- 



20- 



10- 







STARVED ALGAE 



IA 



H7 



CA2 



IA + BACTERIA 



O 

oo 



+ 1 
If 

I 

I 

o 

z 

(jj 



Ld 

I 
00 

z 
< 

LJ 



160 
150 
140- 
130- 

120- 

1 10- 

100- 

90- 

80- 

70 



STARVED ALGAE 



IA 



H7 



CA2 



IA + BACTERIA 



Figure 6. Survival and growth of oyster larvae after 10 days of culture on 60 Co-irradiated 
galhana (IA) with or without supplements of H7 and CA2 bacteria (Experiment V). Control treatments 
were starved or fed axenic / galhana. Results of Tukey's HSD pairwise comparisons and Games and Howell's 
tests are displayed below survival and growth histograms, respectively. Squares that occur together on any 
one of the horizontal lines indicate mean values that are not different at the 0.05% level of significance. 



have enhanced larval cultures by removing toxic metab- 
olites. This may have stimulated larval growth and may 
have normalized the size-frequency distribution by pro- 
moting the growth of larvae that were more sensitive than 



others to the adverse growth effects of metabolites. How- 
ever, the growth of xenic larvae was also enhanced by the 
addition of CA2 bacteria in cultures where the water was 
replaced every second day (Douillet, 1991); therefore. 



BACTERIAL EFFECTS ON OYSTER LARVAE 



49 



Table III 

acnl composition oj CA2 bacteria 



Fatty acid 



composition 



Unknown 1 1.541 


1.73 


14:0 ISO 


4.17 


14:0 


0.76 


15.1 ISOG 


4.99 


15:0 ISO 


18.99 


15:0 ANTEISO 


8.07 


15:1 B 


6.62 


15:0 


4.66 


16:1 ISOH 


5.71 


16:0 ISO 


2.02 


16:1 CIS 9 


4.08 


16:0 


0.99 


15:0 ISO3OH 


12.44 


15:0 3OH 


1.89 


17:1 C 


2.24 


16:0 ISO3OH 


15.98 


16:0 3OH 


1.00 


17:0 ISO 3OH 


1.78 


17:0 2OH 


1.21 



larvae of the American oyster Crassostrea virginiui. Lar- 
vae of M. mercenaria grew when fed on a diet of lyoph- 
ilized /. galbana (Hidu and Ukeles, 1962) or frozen / 
galbana (Chanley and Normandin. 1967), whereas larval 
C. virginica did not grow when fed either of these non- 
living diets. The failure of larval Crassostrea gigas to grow 
when fed on dead algae impeded the evaluation of the 
direct nutritional contribution by bacteria under condi- 
tions where potential bacteria-algal interactions were 
eliminated by the use of killed, rather than living, algal 
cells. 

In summary, bacteria added as single strains or as 
natural communities were found to be major sources 
of variation in cultures of Crassostrea gigas larvae. Se- 
lection of a consistently beneficial bacterium (strain 
CA2) for bivalve larval culture offers a valuable tool for 
research on the role of bacteria in the nutrition and 
culture of marine invertebrates. In addition, the use 
of beneficial microbes in aquaculture may contribute 
to the reduction of undesirable variation in culture 
success. 



bacterial removal of toxic metabolites from culture waters 
is less likely the mechanism of enhancement of larval 
growth. 

Strain CA2 did not indirectly affect larvae by increasing 
algal growth and food availability in larval cultures, be- 
cause no enhanced algal growth occurred in the presence 
of CA2 bacteria. 

Larvae did not grow when fed on freeze-killed or w 'Co- 
irradiated Isochryxis galbana, and additions of bacteria 
did not significantly improve the growth of larvae fed on 
either of the two killed algal diets. 60 Co-irradiated algal 
cells were grazed by larvae at rates that were significantly 
higher than those for live algal cells (Douillet, 1 99 1 ). This 
suggests that the poor growth of larvae fed on killed algal 
diets was not due to a lack of available paniculate matter, 
but more likely to the destruction or loss of essential nu- 
trients from killed algal cells. Supplements of bacterial 
strains or mixtures of naturally occurring bacteria did not 
overcome these possible nutritional deficiencies of the 
killed algal diets. 

The ability of the larvae of some bivalve species to uti- 
lize dead algae as food under xenic conditions has been 
well documented. Larvae of the mussel Mytilns gallo- 
provincialis grew at similar rates whether fed on live or 
frozen Monochrysis lutherii(Masson, 1977). Chanley and 
Normandin (1967) reported that larvae of the clam Mer- 
cenaria mercenaria grew and survived equally well when 
fed on either live or frozen cells of Isoclim'ix galbana. 
However, different species of bivalves appear to have dif- 
ferent nutritional requirements, as indicated by the find- 
ings of Loosanoff (1954) on the ability of M. mercenaria 
larvae to utilize a greater variety of natural foods than the 



Acknowledgments 

Support for this research was provided by a Markham 
Award as well as by the Oregon Sea Grant, NOAA grant 
#NA 85AA-D-SG095 (to C. J. L.). We are grateful to Drs. 
A. Robinson, R. Y. Morita, R. Griffiths and C. Dungan 
for helpful discussions; to Drs. R. Weiner, F. Singleton 
and W. Landry, for help in the taxonomic identification 
of bacteria; and to L. Hanson (Whiskey Creek Hatchery, 
Netarts Bay, Oregon), for encouragement and collabo- 
ration throughout the years. This research is part of a 
doctoral thesis submitted by P. D. to Oregon State Uni- 
versity. 

Literature Cited 

Bayne, B. L. 1965. Growth and the delay of metamorphosis of the 

larvae of Mytilns eitnlix ( \ ). Ophelia 2: 1-47. 
Bayne, B. L. 1983. Physiological ecology of marine molluscan larvae. 

Pp. 299-343 in The M.illiixca. Vol. 3, K. M. Wilbur, ed. Academic 

Press, New York. 
Benemann, J. R. 1973. Nitrogen fixation in termites. Science 181: 164- 

165. 
Breese, \V. P., and R. E. Malouf. 1975. Hatchery manual for the Pacific 

oyster. Oregon Agricultural Experiment Station Special Report No. 

443, and Oregon State Llniversity Sea Grant College Program Pub- 
lication No. ORESU-H-75-002. 22 pp. 
Brown, C. 1973. The effects of some selected bacteria on embryos and 

larvae of the American oyster, Cruxxoxtrea virginica. J. Invert Puthitl. 

21:215-223. 
Calabrese, A., and H. C. Davis. 1970. Tolerances and requirements of 

embryos and larvae ot'bivalve molluscs. Hclgol. Wiss. MeervMintcrs 

20: 553-564. 
Carpenter, E. J., and J. L. Culliney. 1975. Nitrogen fixation in marine 

shipworms. Science 187: 551-552. 
Carriker, M. R. 1956. Biology and propagation of young hard clam, 

Mercenaria mercenaria. J Klixha Mitchell Sd. Soc. 12: 57-60. 



50 



P. DOUILLET AND C. J. LANGDON 



Chanley, P., and R. F. Nurmandin. 1967. Use of artificial foods for 
larvae of the hard clam, Mercenana mercenana (L.). Proc. Null. 
Shellfish Ass. 57: 31-37. 

Checkley, D. M. 1980. The egg production of a marine planktonic 
copepod in relation to its food supply: laboratory studies. Limnol. 
Oceanogr 25(3): 430-446. 

Cockcroft, A. C. 1991). Nitrogen excretion by the surf zone bivalves 
Dona.\ si'm: and /) sordulus. Mar. Ecol. Prog. Ser. 60: 57-65. 

Curds, C. R., and J. M. Vandyke. 1966. The feeding habits and growth 
rates of some fresh-water ciliates found in activated-sludge plants. J 
Appl. Ecol. 3: 127-137. 

Davis, II. C. 1950. On food requirements of larvae ofOstrea virginica. 
Anal. Rec 108: 132-133. 

Davis, H. C. 1953. On food and feeding of larvae of the American 
oyster, C. virginica. Biol. Bull 104: 334-350. 

Davis, H. C., and P. E. Chanley. 1956. Effects of some dissolved sub- 
stances on bivalve larvae. Proc. Nail. Shellfish. Assoc. 46: 59-74. 

Davis, II. C., and R. R. Guillard. 1958. Relative value often genera 
of micro-organisms as foods for oyster and clam larvae. Fish. Bull. 
58: 293-304. 

Davis, H. C., and H. Hidu. 1969. Effects of turbidity-producing sub- 
stances in sea water on eggs and larvae of three genera of bivalve 
molluscs. I'eliger 11: 316-323. 

Di Salvo, L. H. 1978. I'ihrio anguillarum and larval mortality in a 
California coastal shellfish hatchery. Appl. Environ. M Microbiol. 
35(1): 219-221. 

Douillct, P. 1987. Effect of bacteria on the nutrition of the brine shrimp 
Anemia fed on dried diets. Pp. 295-308 in Anemia Research and 
Its Applications. Vol. 3, P. Sorgeloos, D. A. Bengtson, W. Decleir. 
and E. Jaspers, eds. Universa Press, Wetteren. 

Douillct. P. 1991. Beneficial effects of bacteria on the culture of larvae 
of the Pacific oyster Crassostrea gigas (Thunberg). Ph.D Thesis, Or- 
egon State University, 185 pp. 

Dupuy, J. L. 1975. Some physical and nutritional factors which affect 
the growth and setting of the larvae of the oyster, Crassostrea virginica. 
in the laboratory. Pp. 319-331 in Physiological Ecology of Estuarine 
Organisms. F. J. Vernberg, ed. University of South Carolina Press. 
Columbia. 

Gerhardt, P., R. G. E. Murray, R. N. Costilow, E. \V. Nester, W. A. 
Wood, N. R. Krieg, and G. B. Phillips. 1981. Manual of methods 
for general haclenology. Am. Soc. Microbiol., Washington DC. 524 
pp. 

Guerinot, M. L., and D. G. Patriquin. 1981. N : -fixing vibrios isolated 
from the gastrointestinal tract of sea urchins. Can. J. Microbiol. 27: 
311-317. 

Guillard, R. R. L. 1959. Further evidence of the destruction of bivalves 
larvae by bacteria. Biol. Bull. 55: 260-282. 

Guillard, R. R. I.., and J. H. Ryther. 1962. Studies on marine planktonic 
diatoms. I. Cyclolela nana Hustedt and Detonula conjervacea Cleve. 
Can. J. Microbiol. 8: 229-239. 

llellebust, J. A. 1974. Extracellular products. Pp. 838-963 in Algal 
Physiology and Biochemistry. W. D. Stewart, ed. University of Cal- 
ifornia Press. Berkeley. 

Hidu, H. J., and R. Ukeles. 1962. Dried unicellular algae as food for 
the larvae of the hard shell clam, Mcrccnana mercenana. Proc. Nail. 
Shellfish Assoc. 53: 85-101. 

Hidu, H., and H. S. Tubiash. 1963. A bacterial basis for the growth of 
antibiotic treated bivalve larvae. Proc Nati Shellfish Assoc. 54: 25- 
39. 

His, E., R. Robert, and A. Dinet. 1989. Combined effects of temperature 
and salinity on fed and starved larvae of the Mediterranean mussel 
Mylilus galloprovincialis and the Japanese oyster Crassostrea gigas. 
Mar Biol 100(4): 5-463. 

Holt, J.G. 1984. Bergey's Manual of Determinative Bacteriology. Wil- 
liams and Wilkins, Baltimore. 964 pp. 



Innes, D. J., and L. E. Haley. 1977. Genetic aspects of larval growth 
under reduced salinity in Mylilus edu/is. Biol. Bull. 153: 312-321. 

Kates. M. 1964. Bacterial lipids. Adv. Lipid Res 2: 17-90. 

Kutsky, R. J. 1981. Handbook ofl'itaminx. Minerals, and Hormones 
Van Nostrand Reinhold, New York. 492 pp. 

Langdon, C. J. 1983. Growth studies with bacteria-free oyster (Cras- 
sostrea gigas) larvae fed on semi-defined artificial diets. Biol. Bull 
164: 227-235. 

Langdon. C. J. 1989. Preparation and evaluation of protein microcap- 
sules for a marine suspension-feeder, the Pacific oyster Crassostrea 
gigas Mar Biol 102: 217-224. 

I.angdon, C. J., and M. J. \\aldock. 1981. The effect of algal and 
artificial diets on the growth and fatty acid composition of Crassostrea 
gigas spat. J Mar. Biol 61: 431-448. 

I.annan, .]. E. 1980. Broodstock management of Crassostrea gigns I. 
Genetic and environmental variation in survival in the larval rearing 
system. Aquacullure 21: 323-336. 

Lehninger, A. L. 1975. Biochemistry. Worth Publishers. New York. 
1104 pp. 

Leifson, E. 1963. Determination of carbohydrate metabolism of marine 
bacteria. ./ Baclenol. 85: 1 183-1 184. 

Loosanoff, V. L. 1954. New advances in the study of bivalve larvae. 
Am. Sci 42: 607-624. 

Loosanoff, V. L. 1959. The size and shape of metamorphosing larvae 
of I'enus /Mercenarial mercenana grown at different temperatures. 
Biol Bull 117: 308-318. 

Loosanoff, V. L., H. C. Davis, and P. E. Chanley. 1953. Effect of ov- 
ercrowding on rate of growth of clam larvae. Anal. Rcc. 117(3): 645- 
646. 

Luck, J. M., G. Sheets, and J. O. Thomas. 1931. The role of bacteria 
in the nutrition of protozoa. Quart. Rev Biol. 6: 46-58. 

Maeda, M. 1988. Microorganisms and protozoa as feed in manculture. 
Prog. Oceanog 21: 201-206. 

Maeda, M., and K. Nogami. 1989. Some aspects of the biocontrolling 
method in aquaculture. Pp. 395-398 in Current Topics in Marine 
Biotechnology. S. Miyachi, I. Karube, and Y. Ishida, eds. Jap. Soc. 
Mar. Biotechnol, Tokyo. 

Marmur, J.. and P. Doty. 1962. Determination of the base composition 
of deoxyribonucleic acid from its thermal denaturation temperature. 
J. Mul Biol 5: 109-118. 

Martin, Y. P., and B. M. Mengus. 1977. Utilisation de souches bac- 
teriennes selectionees dans ('alimentation des larves de Mylilus gal- 
loprovincialis (LMK) (Mollusque bivalve) en elevages experimentaux. 
Aquacullure 10: 253-262. 

Masson, M. 1977. Observations sur la nutrition des larves de Mylilus 
galloprovincialis avec des aliments inertes. Marine Biologv 40: 1 57- 
164. 

Millar, R. H., and J. M. Scott. 1967. Bactena-free culture of oyster 
larvae. Nature 216: 1 139-1 140. 

Newkirk, G. F., L. E. Haley, D. L. Waugh, and R. Doyle. 1977. Genetics 
of larvae and spat growth rate with the oyster Crassoslrea virginica. 
Mm Biol 41:49-52. 

Nottage, A. S., and T. H. Birkbeck. 1986. Toxicity to marine bivalves 
of culture supernatant fluids of the bivalve-pathogenic }'ibrio strain 
MCNB 1338 and other marine vibrios. J. Fish. Dis. 9: 249-256. 

Perry, G. J., J. M. Volkman, R. B. Johns, and H. J. Bavor, Jr. 
1979. Fatty acids of bacterial origin in contemporary marine sed- 
iments. Geochim. Cosmochim. Ac/a. 43: 1715-1725. 

Porter, K. G., and Y. S. Feig. 1980. The use of DAPI for identifying 
and counting aquatic microflora. Limnol. Oceanogr. 25: 943-948. 

Prieur, D. 1982. Les bacteries heterotrophes dans les elevages experi- 
mentaux et industriels de larves de bivalves marins. Oceania 8(8): 
437-457. 

Prieur, D., G. Mevel, J.-L. Nicolas, A. Plusquellec, and M. Vigneulle. 
1990. Interactions between bivalve molluscs and bacteria in the 
marine environment. Oceanogr. Mar. Biol. Annu. Rev 28: 277-352. 



BACTERIAL EFFECTS ON OYSTER LARVAE 



51 



Provasoli, L., K. Shiraishi, and .1. R. l.ance. 1959. Nutritional idio- 
syncrasies of Artemia and Tignopu.i in monoxenic culture. Ann. New 
YorkAcad. Sci. 77: 250-261. 

Provasoli, L., and A. D'Agostino. 1962. Vitamin requirements of Ar- 
temia salina in aseptic culture. Amur. Zooi 2: 439. 

Rodina, A. G. 1972. Methods in Auuattc Microbiology. R. R. Colwell 
and M. S. Zambruski. eds. University Park Press, Baltimore. 
461 pp. 

Rohlf, F. J. 1982. liinni. . I Package of Statistical Programs lo Accom- 
panvlhe Text Biometry. State University of New York at Stony Brook, 
New York. 859 pp. 

Sokal, R. R., and F. J. Rohlf. 1981. Biometry. W. H. Freeman and 
Co.. San Francisco, CA. 859 pp. 

Sonea, S. 1988. A bacterial way of life. Nature 331 : 2 1 6. 

Stuart, C. A., M. McPherson, and H. J. Cooper. 1931. Studies on 
bacteriologically sterile Moina macrocopa and their food require- 
ments. Physiol. Zoo/. 4: 87-100. 

Trider, D. J., and J. D. Castell. 1980. Effect of dietary lipids on growth, 
tissue composition and metabolism of the oyster (Crassostrea vir- 
ginica).J. ofNitlr. 110(7): 1303-1309. 

Tubiash, II. S., P. E. Chanley, and E. Leifson. 1965. Bacillany necrosis, 
a disease of larval and juvenile bivalve mollusks. I. Etiology and 
Epizoology. J. Bad 90(4): 1036-1044. 

Tubiash, H. S., R. R. Colwell, and R. Sakazaki. 1970. Marine vibrios 



associated with bacilliary necrosis, a disease of larval and juvenile 

bivalve mollusks. ./ Bad 103(0:272-273. 
Ukeles, R., and B. M. Sweeney. 1969. Influence of dinoflagellate 

trichocysts and other factors on the feeding of Ora.v.vo.v/m; virginica 

[arvse on Monochrysis lutheri. Limnoi Oceunogr. 14(31:403-410. 
Underwood, A. J. 1981. Techniques of analysis of variance in exper- 
imental marine biology and ecology. Oceanogr. Mar. Biol. A Rev. 

19: 513-605. 
Utting, S. D., and M.M. Helm. 1985. Improvement of sea water quality 

by physical and chemical pre-treatmenl in a bivalve hatchery. Aaua- 

cii/litrc44: 133-144. 
\\alne, P. R. 1956a. Experimental rearing of the larvae ofO.itren ciluhs 

(L.) in the laboratory. Ministry of Agriculture, Fisheries and Food. 

Fishery lim-M Sene\ 11 20(9): 22 pp. 
VYalne, P. R. 1956h. Bacteria in experiments on rearing oyster larvae. 

Nature 178: 91. 

\\alne, P. R. 1958. The importance of bacteria in laboratory experi- 
ments on rearing the larvae of Oslrea edulis (L.). J. Mar. Biol 37: 

415-425. 
\Yalne, P. R. 1965. Observations on the influence of food supply and 

temperature on the feeding and growth of the larvae ofOxtrea edulis 

L. Fish. Im-esl . London, Ser. 2 24(1): 1-45. 
VYalne, P. R. 1970. Present problems in the culture of the larvae of 

Oxtrea eiliilix. Helgol. Wiss. Meeresunters. 20: 514-525. 



Reference: Biol Bull 184: 52-56. (February, 1993) 



Patterns of Suspension Feeding in the Freshwater 
Bryozoan Plumatella repens 

BETH OKAMURA 1 AND LITA ANN DOOLAN : 

^Department of Zoology, South Parks Road, Oxford, U. K. OX1 3PS and ~27 Hawthorn Avenue, 

Headington, Oxford, U. K. OX3 9QJ 



Abstract. Feeding of large and small colonies of Plu- 
matella repens was assessed under two flow conditions. 
Large colonies ingested greater numbers of particles than 
small colonies and feeding of colonies of both sizes in- 
creased with flow. However, the rate of increase depended 
on colony size. Small colonies increased feeding to a 
greater degree than large colonies. Mechanisms that may 
explain these patterns are discussed. These results contrast 
with an earlier study of feeding in a freshwater bryozoan. 
The conflicting results may reflect experimental condi- 
tions. In the previous study a small volume of still water 
likely entailed greater food depletion by large colonies. In 
our study food depletion did not occur and ambient flow 
carried away filtered water. We discuss how the relatively 
large, U-shaped lophophores of freshwater bryozoans 
function to produce powerful feeding currents that are 
suited to feeding in lotic and lentic habitats. 

Introduction 

Assemblages of colonial suspension feeders are com- 
mon to both marine and freshwater habitats. In general, 
marine assemblages are characterized by high levels of 
competition for space and food as asexual growth results 
in numerous interactions (e.g., Stebbing, 1973; Jackson, 
1979; Buss, 1980; Kay and Keough, 1981; Rubin, 1982; 
Okamura, 1988; Lopez Gappa, 1989). By contrast, very 
little is known about the dynamics of freshwater systems 
where assemblages of colonial invertebrates such as bry- 
ozoans and sponges are common (Bushnell, 1966; Wood, 
1973; Frost, 1991; Karlson, 1991; Ricciardi and Lewis, 
199 1 ). However, competition for food and space has been 
shown to influence patterns of distribution and abundance 
of freshwater insects (e.g., Hildrew and Townsend, 1980; 

Received 26 March 1992; accepted 23 October 1992. 



McAuliffe, 1984; Hart, 1985; Chance and Craig, 1986; 
Lamberti et at., 1987; Ciborowski and Craig, 1989). 

Investigations of marine bryozoans have revealed that 
colony size, neighbors, and ambient flow conditions can 
influence feeding success (Buss, 1980; Okamura, 1984, 
1985, 1988) and subsequent colony growth (Okamura, 
1992). Thus patterns of suspension feeding play an im- 
portant role in the dynamics of these assemblages that 
are typically limited by space. Unlike their marine coun- 
terparts who feed with a circular lophophore (an apical 
tentacular crown), freshwater bryozoans (Cl: Phylactolae- 
mata) possess a relatively larger and U-shaped lophophore. 
However, little is known about comparative patterns of 
feeding in freshwater bryozoans and how these may in- 
fluence patterns of distribution and abundance of fresh- 
water populations. For these reasons we undertook to 
characterize how colony size and ambient flow conditions 
influence suspension feeding in the freshwater bryozoan 
Plumatella repens. 

Materials and Methods 

Plumatella repens is probably the most common fresh- 
water bryozoan and shows a cosmopolitan distribution 
(Wood, 1989). As its name implies, colonies are repent, 
adhering to the substratum as a series of branching zooe- 
cial tubes that can spread to cover large areas. Lopho- 
phores typically are deployed some distance apart as a 
result of elongation of zooecial tubes. Colonies are found 
in lakes, ponds, and streams on a variety of substrata in- 
cluding the undersurfaces of aquatic vegetation (especially 
lily pads: pers. obs.), submerged branches and roots, and 
rocks and stones (Wood, 1989). 

Colonies for feeding studies were collected from a 47- 
year-old gravel pit located at Cassington Nurseries in Cas- 
sington, Oxfordshire. Pieces of lily pads containing large 



52 



SUSPENSION FEEDING IN PLVMATELLA 



53 



(5-7 cm in diameter) and small (2-3 cm in diameter) 
colonies were brought to the laboratory where feeding 
studies were conducted. Lily pads were cut to approxi- 
mately equal sizes for each treatment (7 cm for large col- 
onies and 5 cm for small colonies; colonies were located 
centrally). 

During feeding trials, cut lily pads containing colonies 
were floated on the surface of the water in the working 
area of a recirculating flow tank that contained a suspen- 
sion of polystyrene particles. Lily pad segments were re- 
tained in position with thin lengths of wire. Colonies on 
the undersurfaces of the lily pads were thus immersed just 
below the surface of the water as they are in the field. 

Polystyrene particles were suspended in distilled water 
at a concentration of 200 particles -ml ' (SD = 3.95, 
n = 10). The concentration of particles was estimated by 
counting 10 haphazardly chosen fields of 10 samples of 
suspension. Polystyrene particles have been used to study 
feeding in marine bryozoans (e.g., Okamura, 1984, 1985), 
and preliminary investigation revealed Plumatella would 
ingest them in large numbers. The diameter of the particles 
was 19. 1 jjm (SD = 0.6 ^m) (Duke Scientific Corporation, 
Palo Alto, California) and lies within the size range of 
normal food items ingested by Plumatella (Kaminski, 
1984). The concentration of particles was well within the 
range experienced by suspension feeders in their natural 
environment (DeMott. 1986). 

Two standard flow conditions were created by effecting 
two known velocities in the mid-channel section of the 
flow tank (approx. 3-10 cm below the air-water interface, 
2 cm in from the sides of the flow tank, and 20 cm long) 
(total cross section of water in the flow tank was 15X17 
cm) during feeding trials. In the slower flow condition, 
velocity in the mid-channel section was 2.5 cm s~' (SD 
= 0.40 cm -s ', n = 15), and in the faster flow condition 
it was 5.3 cm-s" 1 (SD = 0.80 cm-s~', n = 15). These 
velocities were determined by timing the passage of par- 
ticles (hydrated Anemia eggs) over a known distance (15 
cm) of the mid-channel section. Since lily pads with col- 
onies were floated on the surface of the water, interaction 
of flow with the air-water interface and with the lily pad 
substrata would have created a velocity gradient (a 
boundary layer) during feeding trials (Vogel, 1981). Thus 
colonies experienced slower ambient flow velocities than 
those measured at the mid-channel section. Although we 
did not have the means to characterize flow at the level 
of lophophores, casual observations indicated that colo- 
nies experienced qualitatively different flow conditions 
during the feeding trials. 

Colonies were starved for 24 h and then allowed to feed 
on particles for 10 min. Feeding trials of longer than 15 
min were found to result in the ingestion of too many 
particles to assure accurate counting. A feeding trial time 
of 10 min is shorter than the gut passage time of various 



marine bryozoans (Winston, 1977). As the polystyrene 
particles were slightly denser than freshwater, a poultry 
baster was used at two minute intervals to resuspend par- 
ticles. Immediately after each feeding run. colonies were 
placed facing down in individual petri dishes containing 
distilled water. Feces obtained after 12 h were collected 
and preserved in 80% ethanol until sampling. 

Feeding rates were estimated by determining the mean 
number of particles per fecal pellet. Individual fecal pellets 
were gently squashed under a cover slip on a microscope 
slide, and the number of particles was counted using a 
compound microscope. Forty fecal pellets were sampled 
per colony when colonies produced large numbers of fecal 
pellets, otherwise all fecal pellets were sampled. As the 
relative proportions of actively feeding zooids varied in 
colonies during feeding trials, the total number of pellets 
produced per colony is not particularly informative. 
However, to check that fecal pellets did not vary with 
colony size, the maximum lengths and widths of 3-5 fecal 
pellets of large (n = 17) and small (n = 8) colonies were 
determined (colonies were not used in feeding trials). 

Data on mean number of particles per fecal pellet per 
colony in the different treatments and on fecal pellet size 
were checked for normality and heterogeneity of variances 
prior to analysis. F-max tests revealed heterogeneous 
variances in the number of particles per fecal pellet per 
colony so data were log-transformed for ANOVA. 

Results 

Zooids of phylactolaemates produce mucus-encased 
fecal pellets that appear to be of fairly fixed sizes and reg- 
ular shapes. Observations suggest that pellet dimensions 
reflect the dimensions of the packaging region of the 
hindgut (Okamura, pers. obs.). Data on fecal pellet size 
collected for small and large colonies support these ob- 
servations. The mean volume of fecal pellets did not vary 
with colony size (mean volume of fecal pellets for large 
colonies was 3.24 X 10" 3 mm 3 [SD = 1.8 X 10~ 3 ] and for 
small colonies was 4.38 X 10~ 3 mm 3 [SD = 2.67 X 10~ 3 ]; 
t = 1.264, df = 23, P = 0.219). Furthermore, although 
the total number of fecal pellets produced by colonies was 
not determined (since the number of feeding zooids per 
colony was variable and very difficult to count during 
feeding trials), fecal pellet production during experiments 
seemed generally to parallel feeding estimated by the 
number of particles per fecal pellet per colony. We there- 
fore are confident that the data collected are good esti- 
mates of feeding rates. 

Two-way ANOVA indicated significant size and flow 
effects on feeding (as measured by mean number of par- 
ticles per fecal pellet per colony) (see Table I). The inter- 
action of size and flow was also significant. These effects 
are shown in Figure 1 . Large colonies had greater feeding 



54 



B. OK.AMURA AND L. A. DOOLAN 



Table I 

Two-way ANOl'A of the tffeas of flow and colony size on the mean 
number particles per fecal pellet per colony. Analysis 
an log-transformed data 







Sum of 


Mean 






Source 


df 


squares 


square 


F-value 


f-value 


Flow 


1 


1.169 


1.169 


42.690 


0.0001 


Size 


1 


1.820 


1.820 


66.457 


0.0001 


Row x Size 


1 


0.400 


0.400 


14.592 


0.0006 


Residual 


32 


0.876 


0.027 







rates, and feeding increased with flow in colonies of both 
sizes, however, when feeding from faster flow small col- 
onies increased feeding rates approximately five times, 
while feeding rates of large colonies increased by a factor 
of only 1.8. 

Discussion 

Flow conditions and feeding 

Increased feeding of Phimatella in conditions of greater 
flow contrasts with feeding patterns of marine bryozoans. 
In general, marine bryozoans experience reduced feeding 
success with increased ambient flow (e.g.. Okamura, 1984, 
1985, 1988, 1990). The explanation for this difference 
may relate to the relative sizes and shapes of their loph- 
ophores. Phylactolaemate lophophores are large and U- 
shaped and have numerous ciliated tentacles. For instance, 
in Phimatella repens tentacle number ranges from 40-60 
(Lacourt, 1968), while the number of tentacles in various 
marine bryozoan groups ranges from 8-34 (Winston, 
1977). Best and Thorpe (1986) found that the strength of 
feeding currents related positively to lophophore size in 
marine bryozoans. This suggests that phylactolaemates 
should produce relatively more powerful feeding currents. 
Furthermore, deflection of lophophoral arms in phylac- 
tolaemates places cilia lining the arms in relatively closer 
proximity than the cilia that line the arms of the circular 
lophophores of marine bryozoans. This closer ciliary 
proximity should also contribute to creation of stronger 
feeding currents. 

Bishop and Bahr (1973) report feeding currents ex- 
tending 5 mm from lophophores of the phylactolaemate 
Lophopodella carleri (tentacle number = 50-95; Lacourt, 
1968). McKinney el al. (1986) observed feeding currents 
to be effective at a distance of 3 mm in the marine chei- 
lostome Biigula neritina (tentacle number = 23; Winston. 
1978). (Both observations of feeding currents were in still 
water.) Greater pumping capacity of phylactolaemates 
may thus explain why, over the velocity range tested in 
this study, feeding by large lophophores of P/imiatella was 
not constrained. Ultimately, of course, phylactolaemate 



feeding will be constrained by flow, when feeding currents 
can no longer overcome friction drag imposed by flow. 

Greater feeding rates in the faster flow condition by 
Phimatella may reflect higher concentrations of particles 
in volumes of water diverted by feeding currents and. 
consequently, a greater flux of particles through the 
lophophore. Profiles of particle availability near surfaces 
can change: as flow increases higher particle concentra- 
tions and fluxes can occur closer to surfaces in boundary 
layer flows (Muschenheim, 1987; Frechette et al., 1989). 
Whether this is true for particle profiles near the air-water 
interface is not known to us (colonies floating on lily seg- 
ments were situated just below this interface). Alterna- 
tively, there may be a greater propensity to feed at in- 
creased flow. 

These results suggest that phylactolaemates possess 
powerful lophophores that allow them to feed from lotic 
and lentic environments (many, including Plumatella, 
occur in both). In diverting fluid from great distances, 
powerful lophophores may be significant for feeding in 
still conditions where food-depletion close to surfaces may 
be common and where lack of flow precludes resource 
renewal. Powerful lophophores will also be less over- 
whelmed by friction drag imposed by water moving 
downstream in lotic environments. 

Colony size and feeding 

The relatively high feeding rates of large Phimatella 
colonies may be explained by several mechanisms. The 
many lophophores of large colonies may conceitedly 
pump greater volumes of water under varying flow con- 
ditions than can the fewer lophophores of small colonies 
(i.e.. the many lophophores of large colonies may con- 
ceitedly produce a stronger pump). Alternatively, large 



o> 

-*-- 

CO 
DC 



c 
o 



60 
50 
40 
30 
20- 



0) 

0) 

- 10-1 





1 2 




I 




Slow Fast 

Flow 

Figure 1. Ingestion rates (mean number of particles per fecal pellet 
per colony) of small (S) and large (L) colonies in slow and fast ambient 
flow (numbers above columns indicate number of colonies sampled). 
Bars represent two standard errors. 



SUSPENSION FEEDING IN PLUMATELLA 



55 



colonies may have relatively greater metabolic demands 
(possibly they invest more in statoblast or larval produc- 
tion per unit mass) than small colonies and therefore have 
a higher propensity to feed. However, the greater increase 
in the rate of feeding in small colonies with increased flow 
(by a factor of five) relative to large colonies (in which 
feeding increased by a factor of 1.8) (see Fig. 1 ) suggests 
that small colonies respond more strongly to increases in 
particle flux (or flow). Why small colonies should show 
such a marked response is not apparent. Perhaps small 
colonies create stronger ciliary currents in response to an 
abundance of food (particle flux serving as a cue) as has 
been observed in marine bryozoans (Best and Thorpe, 
1983). Concerted pumping in large colonies may preclude 
the necessity to create individually stronger feeding cur- 
rents and may provide for a more constant food supply. 
Our results contrast with those obtained by Bishop and 
Bahr (1973) who found that clearance rates of the phy- 
lactolaemate Lophopodella carteri decreased with colony 
size. This discrepancy may relate, in part, to differences 
in colony morphology and growth in the two species, but 
it also is complicated by comparing feeding studies con- 
ducted under static and dynamic conditions and in dis- 
similar volumes of suspension. 

Lophopodella is a higher phylactolaemate, producing 
gelatinous, globular colonies with no branching (Wood. 
1991 ). Colonies of Lophopodella do not grow indefinitely 
but undergo fission, the resulting colonies slowly creeping 
apart. Fission in Lophopodella may result in avoidance 
of lophophoral feeding interference that occurs as colonies 
get bigger, hence maximizing filtering efficiency (Bishop 
and Bahr. 1973; Hughes, 1989). To some extent, our re- 
sults for feeding in Plumalella support this contention. 
Plumatella does not undergo fission and its feeding does 
not decrease with increased colony size. The lack of in- 
terference in feeding in Plumatella may partly reflect its 
morphology. Plumatella colonies are tubular and branch- 
ing and their lophophores are spaced much further apart 
than those of Lophopodella. However, we also believe it 
is crucial to consider differing patterns of excurrent flow 
and food depletion in our experiments and in those of 
Bishop and Bahr (1973). 

In Bishop and Bahr's study (1973), Lophopodella col- 
onies were placed in small vials (diameter = 22 mm) that 
contained 10 ml of an algal suspension. Thus colonies 
will have had ample opportunity to resample previously 
filtered water because the total volume of water was small 
and because, under conditions of still water, previously 
filtered water was not carried away. Thus, it is not sur- 
prising that clearance rates were lower for large colonies. 
The volume of suspension in our study was large (25 1), 
and food depletion was not significant. Furthermore, in- 
corporation of ambient flow meant food-depleted water 
was carried away from colony surfaces. 



Conclusion 



This study indicates that feeding by freshwater bry- 
ozoans is less constrained by increased flow than it is in 
marine forms. As suggested above, the relatively large 
lophophores of phylactolaemates create powerful feeding 
currents that may be beneficial in both lotic and lentic 
environments. The complex hydrodynamics characteristic 
of marine habitats (see Denny, 1988) may ensure delivery 
of food to the level of small, circular lophophores of ma- 
rine bryozoans. Furthermore, small, circular lophophores 
maximize the collective surface area for feeding, and col- 
onies can benefit from the larger energy surplus associated 
with small size (Sebens, 1979, 1982; Ryland and Warner, 
1986; Hughes, 1989). Thus lophophore size and shape in 
marine and freshwater bryozoans may reflect different so- 
lutions to different kinds of problems faced by small, co- 
lonial suspension feeders in the two sorts of environments. 
However, the role of phylogenetic constraint in deter- 
mining lophophore morphology cannot be ruled out (tra- 
ditional views hold U-shaped lophophores to be primi- 
tive). Although the majority of freshwater bryozoans 
possess large, U-shaped lophophores, small, circular lo- 
phophores are found in the phylactolaemate Fredericella 
and in the few gymnolaemates that have invaded fresh- 
water habitats. These exceptions to the rule indicate that 
the significance of lophophore size and shape in freshwater 
habitats merits further investigation. 

Acknowledgments 

We thank Pauline and David Whittington for their 
friendly interest and kind permission to collect Plumatella 
from their pond at Cassington Nurseries and Mark Brown 
for technical help. This work was submitted in partial 
fulfillment for the Zoology Honours Degree in the De- 
partment of Zoology, University of Oxford by L. Doolan. 
The manuscript has been improved by comments from 
two reviewers. 

Literature Cited 

Best, M. A., and J. P. Thorpe. 1983. Effects of particle concentration 
on clearance rate and feeding current velocity in the marine bryozoan 
Flustrellidra Impida. Mar. Biol. 77: 85-92. 

Best, M. A., and J. P. Thorpe. 1986. Effects of food particle concen- 
tration on feeding current velocity in six species of marine Bryozoa 
Mar. Bid/ 93: 255-262. 

Bishop, J. VV., and L. M. Bahr. 1973. Effects of colony size on feeding 
by Loplwpiiciella carteri. Pp. 433-437 in Animal Colonies: Devel- 
opment and Function through Time. R. S. Boardman. A. H. Chee- 
tham, and W. A. Oliver, eds. Dowden. Hutchinson and Ross, 
Stroudsberg, PA. 

Bushnell, J. H. 1966. Environmental relations of Michigan Ectoprocta. 
and dynamics of natural populations of Plumalella repens. Ecol. 
Monogr. 36: 95-123. 

Buss, L. W. 1980. Bryozoan overgrowth interactions-the interdepen- 
dence of competition for space and food. Nature 281: 475-477. 



56 



B. OKAMURA AND L. A. DOOLAN 



Chance, M. M., and D. A. Craig. 1986. Hydrodynamics and behaviour 

of Simuliidae larvae (Diptera). Can. J Zoo/. 64: 1295-1304. 
Ciborowski, J. J. H., and I). A. ( rain. 1989. Factors influencing dis- 
persion of larval black (lies (Diptera: Simuliidae): effects of current 

velocity and food concentration. Can. J. Fi\h. Aqnat. Sci. 46: 1329- 
1341. ' 
DcMott, \V. R. 1986. The role of taste in food selection by freshwater 

zooplankton. (Ic^'logia 69: 334-340. 
Denny, M. W. 1988. Biology and the Mechanics of the Wave-Swept 

Environment. Pnnceton University Press, Princeton. N. J. 329 pp. 
Frechette, M., C. A. Butman, and W. R. Geyer. 1989. The importance 

of boundary -layer flows in supplying phytoplankton to the benthic 

suspension feeder, Mytilus edulis L. Limmil. Oceanogr. 34: 19-36. 
Frost, T. M. 1991. Porifera. Pp. 95-124 in Ecology and Classification 

nt \nrili American Freshwater Invertebrates J. H. Thorp and A. P. 

Covich, eds. Academic Press, Inc.. San Diego. CA. 
Hart, D. D. 1985. Causes and consequences of territonality in a grazing 

stream insect. Ecology 66: 404-414. 
Hildrew, A. G., and C. R. Townsend. 1980. Aggregation, interference, 

and foraging by larvae of Pleetroenemia conspersa (Trichoptera: 

Polycentropodidae). Amm. Behav. 28: 553-560. 
Hughes, R. N. 1989. .-I Functional Biology of Clonal Animals. Chapman 

and Hall, London. 331 pp. 
Jackson, J. B. C. 1979. Overgrowth competition between encrusting 

cheilostome ectoprocts in a Jamaican cryptic reef environment. / 

Anim. Kcul 48: 805-823. 
kaminski, M. 1984. Food composition of three bryozoan species (Bry- 

ozoa, Phylactolaemata) in a mesotrophic lake. Pol Arch. Hviirobtol. 

31:45-53. 
karlson, R. H. 1991. Recruitment and local persistence of a freshwater 

bryozoan in stream riffles. Hydrobiologia 226: 91-101. 
Kay, A. M, and M. J. Keough. 1981. Occupation of patches in the 

epifaunal communities on pier pilings and the bivalve Pinna hicolor 

at Edithburgh, South Australia. Oecohgia 48: 123-130. 
Lacourt, A. W. 1968. A monograph of the freshwater Bryozoa-Phy- 

lactolaemata. /.oologische Verhandelingen No. 93. 
Lamberti, G. A., J. W. Feminella, and V. H. Resh. 1987. Herbivory 

and intraspeciftc competition in a stream caddisfly population. 

Oeeologia 73: 75-81. 
Lopez Gappa, J. J. 1989. Overgrowth competition in an assemblage 

of encrusting bryozoans settled on artificial substrata. Mar. Ecol. Prog. 

Ser. 51: 121-130. 
McAuliffe, J. R. 1984. Competition for space, disturbance, and the 

structure of a benthic stream community. Ecology 65: 894-908. 
McKinney, F. K., M. R. A. Listokin, and C. D. Phifer. 1986. Row and 

polypide distribution in the cheilostome bryozoan Bitgula and their 

inference in Archimedes. Letliaia 19: 81-93. 
Muschenheim, D. K. 1987. The dynamics of 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 of Bryozoa. I. Bitgula 
stolotujera Ryland, an arborescent species. J. E\p. Mar. Biol Ecol. 
83: 179-193. 

Okamura, B. 1985. The effects of ambient flow velocity, colony size, 
and upstream colonies on the feeding success of Bryozoa. II. Cono- 
pcuin rcticiiluin (Linnaeus), an encrusting species. J. E.\p. Mar Biol. 
Ecol 89: 69-80. 

Okamura, B. 1988. The influence of neighbors on the feeding of an 
epifaunal bryozoan. / Exp. Mar. Biol Ecol. 120: 105-123. 

Okamura, B. 1990. Particle size, flow velocity, and suspension-feeding 
by the erect bryozoans Bugula neruina and B stolonifera. Mar. Biol. 
105: 33-3X. 

Okamura, B. 1992. Microhabitat variation and patterns of colony 
growth and feeding in a marine bryozoan. Ecology 73: 1502-1513. 

Ricciardi. A., and D. J. Lewis. 1991. Occurrence and ecology of Lo- 
phupodella carleri (Hyatt) and other freshwater Bryozoa in the lower 
Ottawa River near Montreal, Quebec. Can J Zoo/. 69: 1401-1404. 

Rubin. J. S. 1982. The degree of intransitivity and its measurement 
in an assemblage of encrusting cheilostome Bryozoa. J. Exp. Mar. 
Biol Ecol. 60: 119-128. 

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 Loud. B313: 53-76. 

Sebens, K. P. 1979. The energetics of asexual reproduction and colony 
formation in benthic marine invertebrates. Am. Zool. 19: 683-697. 

Sebens. K. P. 1982. The limits to indeterminate growth: an optimal 
size model applied to passive suspension feeders. Ecology 63: 209- 
222 

Stebbing, A. R. D. 1973. Competition for space between the epiphytes 
of Fiicits semilu.s L. / Mar. Biol. Assoc. (/.A". 53: 247-261. 

Vogel, S. 1981. Life in Moving Fluids. Princeton University Press, 
Pnnceton. N. J. 352 pp. 

Winston, J. K. 1977. Feeding in marine bryozoans. Pp. 233-271 in 
Biology of Bryozoans. R. M. Woollacott and R. L. Zimmer, eds. 
Academic Press, New York. 

Winston, .J. E. 1978. Polypide morphology and feeding behavior in 
marine ectoprocts. Bull. Mar. Sci. 28: 1-31. 

Wood, T. S. 1973. Colony development in species of Plumalella and 
Fredericella (Ectoprocta: Phylactolaemata). Pp. 395-432 in Devel- 
opment and Function of Animal Colonies through Time. R. S. Board- 
man, A. Cheetham, and J. Oliver, eds. Dowden. Hutchinson & Ross. 
Stroudsburg. PA. 

Wood, T. S. 1989. Ectoproct bryozoans of Ohio. Ohio Biol. Surv Bull 
\ew Series 8(2): X + 70 pp. 

Wood, T. S. 1991. Bryozoans. Pp. 481-499 in Ecology and Classifi- 
cation />/ \orih American Freshwater Invertebrates. J. H. Thorp and 
A. P. Covich. eds. Academic Press. Inc.. San Diego. CA. 



Reference: Bin/. Bull 184: 57-78. (February, 1993) 



Aplacophora as Progenetic Aculiferans 

and the Coelomate Origin of Mollusks 

as the Sister Taxon of Sipuncula 1 

AMELIE H. SCHELTEMA 
\Vootls Hole Oceanographic Institution, Woods Hole, Massachusetts 02543 



Abstract. Evidence is presented in support of the fol- 
lowing phylogenetic hypotheses: ( 1 ) Sipuncula are the sis- 
ter taxon of Mollusca; (2) the two aplacophoran taxa, 
Neomeniomorpha (= neomenioids) and Chaetodermo- 
morpha (= chaetoderms), are monophyletic with a com- 
mon neomenioid-like ancestor, and of the two taxa, 
Chaetodermomorpha are more derived; (3) Aplacophora 
and Polyplacophora are sister taxa and form a clade, Acu- 
lifera; (4) Aculifera are the sister group of the remaining 
extant mollusks, Conchifera; and (5) Aplacophora are 
progenetic Aculifera. 

The evidence is based on homologies of early and late 
emhryological development, adult morphologies, and 
molecular analyses. Embryological development in si- 
punculans and mollusks shows a close relationship be- 
tween them, and embryological development of the shell 
separates Aculifera and Conchifera. Adult morphologies 
indicate: ( 1 ) monophyly of Aplacophora; (2) sister-group 
relationship between Aplacophora and Polyplacophora; 
(3) a molluscan plesiomorphy of nonsegmented serial 
replication of organs; and (4) progenesis in Aplacophora. 
Molecular evidence supports the embryological and mor- 
phological relationships between Sipuncula and Mollusca. 

Mollusca are thus hypothesized to be coelomate Eu- 
trochozoa, which share an ancestor that probably had se- 
rial replication of organs. Differences in size and structure 
of the coelom among Eutrochozoa are hypothesized to 
have been brought about by changes in the timing and 
the process of cavitation of the mesodermal bands that 
arise from cell 4d. Through the process of progenesis 
Aplacophora retained an ovoid embryological shape and 



Received 19 August 1992; accepted 25 November 1992. 
' Contribution Nos. 8205 from the Woods Hole Oceanographic In- 
stitution, and 314 from the Smithsonian Marine Station at Link Port. 



several internal structures that, although they appear to 
be in a primitive state, are actually secondarily derived as 
is quadrant D specification during early cleavage. 

Introduction 

The uniqueness of Aplacophora among Mollusca lies 
in their derived vermiform body in combination with an 
internal organization that appears to reflect a primitive 
molluscan state, especially the simple ladderlike nervous 
system, serial musculature, distichous radula (two teeth 
per row) in its plesiomorphic aplacophoran state, simple 
digestive system, and epidermis that produces an aculif- 
erous cuticle. Their evolutionary significance to the phy- 
lum has long been a matter for conjecture. First came the 
question of whether Aplacophora were even mollusks, as 
they lack a number of "typical" characters such as a shell, 
mantle, and kidneys (e.g., Thiele, 1902; H. Hoffmann, 
1929-30), but they have more usually been considered to 
belong within the phylum because of similarities to chitons 
in their nervous system ( Amphineura) and spicules (Acu- 
lifera) (e.g., Spengel. 1881; Heath, 1911). Further discus- 
sions were concerned with whether aplacophorans were 
"degraded" or truly "primitive" mollusks (see Hyman, 
1967, pp. 68-70 for a historical account). 

There have been no current arguments which separate 
Aplacophora from Mollusca since evidence for a close 
relationship between Aplacophora and Polyplacophora 
was published by S. Hoffman (1949), but under present 
discussion is their origin and position within the phylum 
(Salvini-Plawen, 1972. 1981a, 1985; Scheltema, 1978, 
1988), as well as the origin of the phylum Mollusca itself. 
Mollusca have been argued either to have a noncoelomate 
origin and to be the sister taxon of the eucoelomate An- 
nelia-Echiura-Sipuncula (Salvini-Plawen, 1972, 1985 fig. 
42), or to be eucoelomates with an ancestor in common 



57 



58 



A. H. SCHELTEMA 



with other coelomates (Wingstrand, 1985; Scheltema. 
1988). In either argument, Aplacophora have been con- 
sidered stem forms and therefore preceded the Monopla- 
cophora with serial replication of organs. 

Hypotheses for a noncoelomate origin rest on the ar- 
gument that the worm-like Aplacophora with replicated 
lateroventral musculature evolved from a turbellario- 
morph ancestor, and that consequently the molluscan 
coelom is not homologous to that in the Eutrochozoa. A 
coelomate origin has been hypothesized from annelid- 
mollusk relationships, including the presence of a cell 4d 
that gives rise to mesoblasts and consequently a homol- 
ogous coelom, the presence of a trochophore larva, and 
serial repetition of body parts. Because the molluscan 
coelom is small and unsegmented, the idea that annelids 
and mollusks form a clade with a common segmented 
ancestor is poorly accepted. The dichotomous choice be- 
tween either a turbellariomorph or an annelid-like ances- 
tor for mollusks has dominated recent thinking about 
molluscan evolution (e.g., Hyman, 1967; Haszprunar, 
1992), and the relationship of mollusks to other Eutro- 
chozoa has not been examined. However, recent molec- 
ular data discussed below urge reconsideration of mol- 
luscan relationships to other phyla. 

Evidence is presented here to support the hypotheses 
that ( 1 ) Mollusca are eucoelomates with their closest living 
relatives in Sipuncula, their sister group; (2) Aplacophora 
and Polyplacophora are sister groups in the subphylum 
Aculifera (contradicting Scheltema, 1978, 1988); (3) Acu- 
lifera are the sister group of the remaining living mollusks, 
Conchifera; (4) the aplacophoran taxa Chaetodermo- 
morpha (= Caudofoveata, here also called chaetoderms) 
and Neomeniomorpha (= Solenogastres sensu nomine 
Salvini-Plawen, here also called neomenioids) are mono- 
phyletic, sharing a neomenioid-like ancestor; and (5) 
aplacophorans are progenetic Aculifera. Considered in the 
discussion is the homology of the eutrochozoan coelom 
and the evolutionary difference between metamerism, or 
segmentation as it occurs in the annelids, and serial rep- 
lication of organs, as found in Neopilina and Vema 
(Wingstrand, 1985). The term "metamerism" is used here 
only to denote a segmented coelom; "serial replication" 
is used to denote the more general case of serial repetition 
of organs, whether or not by metameres. 

Evidence that Mollusca are Descended 
from Coelomates 

Mollusca have a coelom consisting of gonadal lumina, 
pericardium, and kidneys, as well as part of the gameto- 
ducts in Aplacophora. A noncoelomate ancestry calls for 
the widening of a pericardial space lined by mesoderm as 
protection for a heart (Salvini-Plawen, 1968a, 1972; not 
discussed 1985, 1990) and for gonads separate from the 
pericardium. This development of coelomic spaces would 



be a molluscan apomorphy. not homologous with annelid 
or sipunculan coelom. Alternatively, the molluscan peri- 
cardium can be considered as reduced from a large coe- 
lomic space homologous to that in other eutrochozoa. 
The involvement of the pericardial coelom in excretion 
is unique to mollusks. Ultranltration of blood occurs 
through podocytes that are present in most molluscan 
classes including Aplacophora (Andrews, 1988; Reynolds 
and Morse, 1991). 

Five independent lines of evidence indicate that re- 
duction of coelom is the case, and that Mollusca are eu- 
trochozoan coelomates: ( 1 ) presence of the molluscan 
cross in mollusks and sipunculans and (2) homology of 
certain characters in larvae of mollusks and sipunculans 
indicate that mollusks and sipunculans are sister taxa; (3) 
a large pericardium among "primitive" mollusks indicates 
that it is a molluscan plesiomorphy; (4) the embryological 
development of mesoderm in annelids, mollusks, sipun- 
culans, and nemertines is similar, and the coelom in the 
four groups is homologous; and (5) molecular data groups 
mollusks with other eutrochozoans. 

Sipunculans as sister taxon of the mollusks 

An evolutionary relationship between sipunculans and 
mollusks lies in their early embryological development 
and in morphological features of sipunculan pelagosphera 
and molluscan larvae. 

Molluscan cross. The molluscan cross is found in the 
embryological development of Gastropoda, Polyplaco- 
phora, Scaphopoda, and Aplacophora by the end of the 
64-cell stage (Verdonk and van den Biggelaar, 1983; 
Heath, 1899; van Dongen and Geilenkirchen. 1974; Baba, 
1951). It is formed by la' 2 -ld 12 cells and their descen- 
dents, with cells la" 2 -ld" 2 , called peripheral rosette cells, 
forming the angle between the arms of the cross (Fig. 1 A, 
B, D, peripheral cells solid black). In Annelida, however, 
it is cells la" 2 - Id" 2 that form the cross (Fig. IE, cross 
cells solid black) (Wilson, 1892). In the 64-cell stage of 
the neomenioid aplacophoran Epimenia vermcosa figured 
by Baba (1951), a molluscan cross seems apparent from 
Baba's shading (Fig. ID), although Salvini-Plawen (1985) 
found "no definite cross formation" in the same source. 
Manuscript drawings by G. Gustafson of developing 
Chaetoderma nitidulum eggs likewise show a molluscan 
cross. In contrast to most mollusks, early cleavage in Pe- 
lecypoda is asynchronous and bilateral, and no cross is 
formed; its absence would seem to be an apomorphy. 
Likewise, development in Cephalopoda seems an apo- 
morphy of that group, which has telolecithal eggs, early 
bilateral cleavage, and no molluscan cross. 

In Sipuncula, a molluscan not an annelid cross is 
formed, as Rice ( 1975, 1985) has emphasized and refig- 
ured from Gerould ( 1906), who first described its presence 



APLACOPHORA: PROGENETIC COELOMATES 



59 




Figure 1. (A-D) The molluscan cross. (A) Gastropoda (Lyniiiucu 
siagnalis. after Verdonk and van den Biggelaar. 1983, p. 1 1 1 fig. 3b); 
(B) Polyplacophora (Stenoplax heathiana. after Heath, 1899. pi. 32, fig. 
23); (C) Sipuncula (Golfingia vulgaris. after Gerould, 1 906. p. 99, fig. D, 
as published in Rice, 1975, p. 99. fig. 17); (D) Aplacophora (Epimenia 
verrucosa. after Baba, 1951. p. 46, fig. 18). The apical rosette la'"-ld'" 
is shown in fine, close stippling; arms of the cross la' : - Id' 2 and daughter 
cells are shown in fine, open stippling; tip cells of cross 2a"-2d" are 
shown in coarse stippling; peripheral rosette cells la" 2 -ld" : are solid; 
and trochoblast cells I a 2 - Id 2 are clear. In Epimenia (D), the cleavage 
stage appears to be earlier than shown in A-C, as the tip cells have not 
yet separated from 2a' and 2c' (indicated by question marks), and the 
arms of the cross are not quite straight, similar to an earlier stage in 
Polyplacophora (Heath. 1899. pi. 32, fig. 17). In B, only one tip cell was 
discernible in Heath's illustration, and in C tip cells were not indicated 
in Gerould's original figure. (E) Annelid cross, Polychaeta (Nereis) (after 
Wilson, 1892, p. 396. diagram II). The apical rosette la 1 "-Id"' is shown 
in fine, close stippling; peripheral cells la' 2 -ld 12 are shown in fine, open 
stippling; and the arms of the cross from la" 2 -ld" 2 are solid. 



in sipunculan development (Fig. 1C). The presence of a 
molluscan cross during embryological development is 
understood here to be of phylogenetic importance, and 
sipunculans and mollusks share a character not found in 
either annelids or flatworms (Freeman and Lundelius, 
1992). Its presence can be considered apomorphic to the 
embryonic morphology of turbellarians, which lack a 
cross. 

Similarities between sipunculan and molluscan lan'ae. 
Gerould (1906) noticed certain other resemblances to 
mollusks besides the molluscan cross in the development 
of sipunculans. In particular, he found similarity between 
sipunculan pelagosphera and molluscan larvae. The pela- 
gosphera is unique to sipunculans. It is a swimming larva 



that metamorphoses from a trochophore stage (Rice, 
1975, 1985). Gerould noted the resemblance of the pela- 
gosphera lip glands to chiton larval pedal glands, and of 
the pelagosphera buccal organ to the radula sac in chiton 
larvae (Figs. 2, 3, 4). Pelagosphera larvae can either swim 
upright with the large metatroch or creep, head-down, 
along a solid surface. These activities are lost along with 
the larval head at metamorphosis. Jagersten (1963) first 
described creeping in living pelagosphera, and he related 
it to a creeping gastropod. He also noted that the buccal 
organ (= pharyngeal bulb, Schlundkopf) was used in 
feeding. Later Jagersten (1972) proposed a possible, but 
not certain, homology of the pelagosphera lip, which is 
the creeping surface posterior to the mouth, and the 
creeping lobe, or foot, between mouth and anus of mol- 
luscan larvae. 

Rice (1975, pp. 120-121) described the creeping lo- 
comotion of pelagosphera as follows: "The larva is able 
to ... glide along with . . . [the] head flattened against 
the bottom. Frequently the larvae . . . may crawl in the 
manner of an inchworm. presumably scraping material 
from the bottom. The continual eversion of the buccal 
organ during feeding probably aids in the removal of food 
from the substratum. This tough muscular organ [covered 
by cuticle. Rice, 1973] is believed to function in breaking 
up material into small panicles for feeding. . ." A mucus- 
like substance from the lip glands is secreted as the animal 
moves along a natural substratum (Rice, 1981 and pers. 
comm.). My own recent observations on living pela- 
gosphera corroborate many of Rice's. 

Precise descriptions of the protrusible buccal organ and 
lip gland have been given by Rice (1973). The buccal 
organ is a muscular sac, ventral and posterior to a cuticle- 
lined invagination called the buccal groove that lies below 
the esophagus. The epithelium of the buccal organ is 
overlain by the cuticle of the ventral side of the buccal 
groove and is the area first protruded (Fig. 2). Although 
the precise innervation of the buccal organ was not dem- 
onstrated, the circumesophageal connectives, which arise 
from the dorsal cerebral ganglion, are closely associated 
with the organ. Both the topography and function of the 
buccal organ and groove are remarkably similar to those 
of the radular apparatus in mollusks: ventral odontophore 
= buccal organ; ventral radula sac = buccal groove; and 
ventral cuticular radula = ventral portion of the cuticular 
buccal groove. Furthermore, the odontophore and prob- 
ably the buccal organ are innervated through connectives 
united with the cerebral ganglion. The homology would 
be more certain if it were known whether the buccal organ 
musculature is formed from mesoderm, as is the odon- 
tophore of mollusks (Raven, 1966), or whether it is myo- 
epithelial as in archiannelids (Jagersten, 1947; Rice, 1973). 

The lip gland takes several forms in various pelago- 
sphera, from a bilobed to a paired or four-lobed body 




A. H. SCHELTEMA 



Figure 2. Peiagosphera larvae of Sipuncula. (A) Frontal view of head. 
Siinmailu* sp. (from Rice, 1981, fig. 4). (B) Entire larva. Aspidosiphon 
sp. (from Rice, 1981, fig. 6). Numbers as in Figure 3: I huccal gland, 
3 pore of lip gland, 4 mouth, 5 lip. 



which opens either directly, or by way of a ciliated duct 
or ducts, into the lip pore. In comparison, the anterior 
pedal gland in larval chitons and in Aplacophora is duct- 
less (cf.. Figs. 2A, 4B, 6C). Aplacophora, but not chitons, 
have a central ciliated pit. 

Similarities in form and function in these three struc- 
tures lip and foot, lip glands and pedal glands, and buc- 
cal organ and radula with its sac are striking. Their 
morphologies are particularly clear in sagittal sections of 
a pelagosphera and a chiton larva (Figs. 3, 4). There are 
also similarities in their development, as they all arise 
from posttrochal ectoderm, with these differences: in si- 
punculans, the origin of all three structures is stomodeal, 
whereas in mollusks, the ventral somatic plate, usually 
from cell 2d, gives rise to the foot and its glands, and only 
the radula sac is stomodeal (Raven, 1966). In Sipuncula 
as well, cell 2d gives rise to the somatic plate, which forms 
the ectoderm of the trunk (Rice, 1976). In mollusks, how- 
ever, the proximity and functional interdependence of 
the somatic and stomodeal structures are indicated by the 
pedal contribution to feeding in veliger larvae. An anterior, 
medial ciliary tract is formed on the foot by which particles 
unsuitable for ingestion are rejected (Moor, 1983). 

Only the head region of the pelagosphera, which is rad- 
ically altered during metamorphosis to a juvenile sipun- 
culan. can be compared to the Mollusca. The posterior 
part of the body with its large coelomic sac, nephridia, 
mid-dorsal anus, and ventral nerve cord, are already de- 
finitive adult structures. 

Evidence from the presence of the molluscan cross and 
from locomotary and feeding structures that are similar 
in mollusks and larval sipunculans is sufficiently strong 
that the two phyla can be considered as sister groups, and 
mollusks, therefore, as eucoelomates. Of course, if the 
primitive mode of sipunculan development should prove 
to be by way of a nonfeeding, lecithotrophic larva, then 
the similarities between planktotrophic pelagosphera and 
molluscan larvae would be convergent. However, Rice 
(1985) most recently considered evolutionary questions 
of sipunculan larval development and concluded that a 
yolky egg and short-lived planktotrophic pelagosphera was 
the primitive mode of development. 

Other considerations. Two further observations can be 
made to support arguments for a sipunculan-molluscan 
sister relationship, one embryological, the other paleon- 
tological. The first is the embryological development of 
Echiura (Newby, 1940) compared to that of the sipun- 
culans. Echiurans have traditionally been linked with si- 
punculans, both having worm- or sac-like, unsegmented 
coelomate bodies, but echiurans afford a contrast to si- 
punculans in their closer relationship to annelids. They 
have an annelid cross rather than a molluscan cross during 
early cleavage, and as in annelids, the major ciliary band 



API ACOPHORA: PROGENETIC COELOMATES 



61 




Figure 3. Midsagittal section of the pelagosphera larva, Phascolosoma 
agassi:ii (from Rice, 1973, pi. 5). 1 buccal organ, 2 lip gland, 3 pore of 
lip gland, 4 mouth, 5 lip, 6 stomach, 7 coelom, 8 esophagus. 



of older echiuran larvae is the prototroch anterior to the 
mouth. In sipunculan pelagosphera. the metatroch below 
the mouth, not the prototroch, is the major swimming 
organ. Indeed, the region in pelagosphera that forms the 
head with its locomotory lip, lip gland, and buccal organ, 
is represented in echiuran larvae by only a few rows of 
cells between the prototroch and metatroch, and no larval 
organs are present. 

If sipunculans are sister taxon of the Mollusca, they 
must have arisen, like mollusks, early in the evolution of 



metazoans. One piece of evidence for an early sipunculan 
history is the mid-Cambrian genus Ottoia from the Bur- 
gess Shale. Considered priapulids by Conway Morris 
( Whittington, 1985) and close to priapulids by Banta and 
Rice (1976), the genus indicates great diversity of spe- 
cialized sacciform, coelomate or pseudocoelomate, worm- 
like animals already in the early Paleozoic. Sipunculans 
therefore could have a very long, but unobservable and 
unverified, geologic history. A second piece of evidence 
is that sipunculans contain hemerythrins, found also only 
in priapulids, lingulid brachiopods, and some annelids 
(Curry and Runnegar, 1 990). Because lingulids and prob- 
ably priapulids and annelids are known from the early 
Cambrian, the presence of hemerythrins indicates a very 
long history for all forms having these oxygen transport 
molecules. 

Si:e of the pericardium in "primitive" mollusks 

The pericardium is larger relative to the heart in Apla- 
cophora, Monoplacophora, and Polyplacophora than it 
is in Gastropoda, Pelecypoda, and Cephalopoda (Schel- 
tema, 1973, 1988; Scheltema and Kuzirian, 1991) (Figs. 
5, 6A). Ontogenetically, the pericardium is already large 
before the heart develops from pericardia! epithelium in 
Aplacophora (Baba, 1938), and in Polyplacophora de- 
velopment of the pericardium precedes development of 
the gonad (Hammersten and Runnstrom, 1925). Thus 
the polarity of pericardia! size is from large to small in 
Mollusca, and the continued reduction within the phylum 
is considered to be a derived condition of the Mollusca. 




B 



Figure 4. Newly settled larvae of Polyplacophora. (A) Midsagittal 
section of Acanthochiton discrepans (after Hammarsten and Runnstrom, 
1925, fig. E, figure reversed). (B) Ventral view of Stetwplax hcalhiana 
just after metamorphosis (after Heath. 1899, fig. 59). The opening of the 
pedal gland (3) lies posterior to the mouth (4); the gland opens through 
"a series of. . . intercellular channels" rather than a duct (Heath, 1899, 
p. 631); compare with Figure 6C. 1 radula sac, 2 anterior pedal gland, 
3 opening of pedal gland, 4 mouth, 5 foot, 6 larval eye. Structures num- 
bered 1-5 are homologous to structures with the same numbers in Figs. 
2 3. 



62 




A 9 



9 9 





Figure 5. Large pericardia! space and heart in primitive molluscs. (A) Aplacophoran, Chaetaderma 
nitidulum, sagittal section. Gametes pass from the gonads through the pericardium with its large, paired, 
lateral extensions ("horns") and thence into gametoducts leading to the mantle cavity (from Scheltema. 
1973, fig. 2, and Scheltema, 1988, fig. 1 3). (B) Polyplacophoran, Chiton sine/am, cross section (after Wissel, 
1904, pi. 24, fig. 49). (C) Monoplacophoran. Neopilina galathea. dorsal view, with paired pericardia! sacs, 
paired ventricles, and two pairs of auricles (after Lemche and Wingstrand, 1959, from Scheltema, 1988, fig. 
1 3). (D) Polyplacophoran, Acanlhopleiira echinata, dorsal view, with two pairs of openings between auricles 
and ventricle (after Plate, 1898. from Scheltema, 1988. fig. 13). 1 pericardium, 2 ventricle, 3 auricle, 
4 opening between auricle and ventricle, 5 auriculoventricular valve, 6 aortal bulb, 7 gonopericardial duct, 
8 lateral extension of pericardium, 9 gametoduct. 



Development ofmesoderm 

The interpretation that the coelom is reduced in Mol- 
lusca assumes that the molluscan pericardium is homol- 
ogous to the coelom in other spiralian coelomates, namely 
Annelida and Sipuncula. In all three, the coelom is formed 



from mesoderm that originates from embryonic cell 4d. 
This cell gives rise to a pair of mesodermal teloblasts, 
which migrate inward to a ventrolateral position, one on 
each side of the midline ( Verdonk and van den Biggelaar, 
1983; Anderson, 1973; Rice, 1975) and proliferate forward 
into two lateral mesodermal bands. Mesodermal bands 



APLACOPHORA: PROGENETIC COELOMATES 



63 




Figure 6. (A) Cross-section through the pericardium of a neomenioid aplacophoran, Helicoradomenia 
juani (from Scheltema and Kuzirian. 1991, fig. 5C). (B) Cross-section through the pedal gland and pedal 
pit of a neomenioid aplacophoran, Ocheyoherpia sp. The voluminous pedal gland occupies most of the 
head region; the lobes of the gland are in varying stages of secretion. (C) Ciliated pedal pit of Helicoradomenia 
juani. The pedal gland discharges into the pedal pit, not through distinct ducts, but through numerous 
channels as described for chitons (Fig. 4B). (D) Secretory epidermal papillae of the neomenioid aplacophoran, 
Helicoradomenia juani (from Scheltema and Kuzirian, 1991, fig. 2C). (E) Secretory epidermal papillae of 
the polyplacophoran. Acanthochiton fascicularis (from Fischer el ai, 1980, fig. 3). 1 pedal gland, 2 ciliated 
pedal pit, 3 dorsal blood sinus, 4 dorsal cecum of midgut, 5 cerebral ganglion, 6 oral cavity, 7 pericardium, 
8 auricle, 9 ventricle, 10 ovum. 11 U-shaped gametoduct, 12 copulatory spicule pocket, 13 foot. Asterisks 
in D and E, cavities of dissolved spicules. 



64 



A. H. SCHELTEMA 



are present as well in Nemertini (Turbeville, 1986). In 
annelids, sipunculans, and nemertines, the coelom is 
formed by cavitation (schizocoely) of the bands. The coe- 
lom constitutes the major body cavity in annelids and 
sipunculans, but in nemertines it forms only vessels for 
blood circulation (Turbeville, 1986). In mollusks, the me- 
sodermal bands break up into masses of coelenchyme, 
from which is formed a solid anlage or pair of anlagen 
that cavitate to form the pericardium, heart and kidneys 
(Raven, 1966; Moor, 1983). In some mollusks with paired 
anlagen, the pericardium begins as paired cavities before 
becoming united (Raven, 1966). In Neopilina the peri- 
cardium is still paired (Fig. 5C), and the large pericardia! 
"horns" in some Aplacophora (Fig. 5A) may reflect an 
ancestral paired condition. 

The coelom among the spiralian protostomes described 
here is interpreted as being homologous because of sim- 
ilarities in early embryological development. Differences 
in coelom formation among the four phyla apparently 
arise from variations in the timing of cavitation after the 
mesodermal bands have formed; but the differences in 
process are not considered sufficient to deny homology 
of the coelom. A single pericardium formed by fusion in 
mollusks other than Neopilina is thus an apomorphy. 

Molecular evidence 

Recent sequencing of 18S ribosomal RNA among 22 
classes (not including Aplacophora), in 10 animal phyla, 
split off acoelomate Platyhelminthes as sister group of the 
remaining bilaterian taxa, the eucoelomates, which fall 
into four closely rooted groups (Field ct ai, 1988). The 
group termed Eutrochozoa (Ghiselin, 1988) includes five 
analyzed phyla: Mollusca, Annelida. Brachiopoda, Po- 
gonophora, and Sipuncula. More recently Turbeville el 
al. (1992) have added Nemertini to the Eutrochozoa, bas- 
ing their results on 18S rRNA and analyzing two Platy- 
helminthes, in addition to the single flatworm analyzed 
by Field el al. (1988). A re-analysis by Lake (1990) of the 
1988 data positioned Sipuncula closest to Mollusca and 
Brachiopoda, with Annelida and Pogonophora as sister 
groups. The presence of hemerythrins in Brachiopoda, 
Sipuncula, and some Annelida affords independent sup- 
port from molecular data for some of the results of Field 
t't al. (Curry and Runnegar, 1990). 

The relationships among Sipuncula, Mollusca, and 
Brachiopoda, however, remain unresolved, and possible 
synapomorphies of sipunculan and molluscan larval 
characters were not taken into account by Lake. Although 
the molecular evidence is still incomplete, it suggests that 
mollusks have descended from a coelomate ancestor, and 
that sipunculans are their closest sister group. In proposing 
that the last common ancestor of the Annelida-Mollusca 
lineage was hemocoelic and segmented. Lake did not dis- 



cuss the presence or absence of a coelom. Ghiselin ( 1988) 
considered the evolution of Mollusca in light of the mo- 
lecular evidence given in Field et al. (1988), amplifying 
the data with an analysis of specific nucleotides and a 
useful history of molluscan phylogenetic hypotheses. 
Ghiselin favored a segmented, coelomate eutrochozoan 
ancestor, with loss or reduction of segmentation in the 
Mollusca. Salvini-Plawen (1990), however, retained a 
preference for a turbellariomorph molluscan ancestry and 
refuted the validity of the sequencing by Field et al. (1988) 
and Ghiselin (1988), because "for some selected, tradi- 
tionally monophyletic groups [including mollusks] eu- 
phemistic premises are made" by eliminating some data 
as convergences. Willmer and Holland (1991) also con- 
sidered that mollusks had a flatworm origin and suggested 
that RNA analysis of several Platyhelminthes might show 
them to be poly- or paraphyletic, but the work of Turbe- 
ville et al. (1992) indicates that they are monophyletic. 

Monophyly of Aplacophora 

A proposed homology of the chaetoderm oral shield 
with the creeping sole of the archimollusk was the basis 
for separating the two aplacophoran taxa into two classes 
(Fig. 7B, C; Fig. 8A) (Salvini-Plawen, 1972, 1985, 1990). 
This homology was based on the innervation of the oral 
shield (Salvini-Plawen, 1972), the character of the epi- 
dermis, and the presumed homology of cuticular struc- 
tures (Fig. 8C, arrowhead) (S. Hoffman, 1949), but it is 
not upheld either by light or transmission electron mi- 
croscopy (Scheltema et al., in press, fig. 9; Tscherkassky. 
1989). The oral shield cuticle is continuous with that of 
the pharynx and is a lip. and the innervation of the shield 
is cerebral, lying anterior to that part of the anterior ner- 
vous system considered "tentacular," and thus part of the 
head region, by Ivanov ( 1 99 1 ). Accordingly the two apla- 
cophoran taxa cannot be separated on the basis of the 
chaetoderm oral shield, although Salvini-Plawen (1990) 
recently argued that the homology holds because the fore- 
gut and oral-shield epithelia are different, and the presence 
of the cuticle is secondary. In a schematic drawing through 
an oral shield, Salvini-Plawen (1990, fig. 7) showed a sep- 
aration, the "mantle rim," between the oral shield cuticle 
and body cuticle, but this separation does not exist in my 
experience (Scheltema et al., in press, fig. 9B). The ar- 
gument would be clarified if it were known whether the 
oral shield is stomadeal in origin. 

Several synapomorphies suggest that the two aplacoph- 
oran taxa are monophyletic. The outgroup for comparison 
is Polyplacophora. 

The tetraneural nervous system, including the cerebral 
commissure, lateral and ventral nerve cords, and supra- 
rectal commissure, is more heavily ganglionated in both 
neomenioids and chaetoderms than in chitons. The radula 



APLACOPHORA: PROGENETIC COELOMATES 



65 




Figure 7. (A-C) Chaetodermomorpha. (A. B) (.'kern 'derma lurnerae. entire animal (antenor to left) and 
divided oral shield (I'rom Scheltema, 1985, fig. 3L, O. P). (C) Oral shield of SculOfiim megaradulalu.i (cf., 
Fig. 8A) (from Scheltema, 1988, fig. 6). (D. E) Neomeniomorpha. (D) Dorymenia sp. (E) A new neomenioid 
genus and species in the family Simrothicllidae. D and E are drawn to the same scale, anterior to left; the 
midgut and gonad lie between X-X and Y-Y. 



in its plesiomorphic state in Aplacophora is distichous, 
that is, only two teeth per row (Scheltema, 1988; Schel- 
tema el al, 1989), a reduction in number from the doco- 
glossate chiton radula. Both neomenioids and chaeto- 
derms have a dorsoterminal sense organ (= dorsocaudal 
sensory pit), or sometimes several, in the epidermis. It is 
of unknown function, although homology to the osphra- 
dium has been conjectured (Spengel, 1881; Haszprunar, 
1987). Whether or not this homology is correct, the po- 
sition of the dorsoterminal sense organ is an autapomor- 
phy of the Aplacophora, for there is no compelling evi- 
dence that this position, postulated to be primitive for the 
molluscan osphradium (Salvini-Plawen, 1985), is other 
than an apomorphy shared only by neomenioids and 
chaetoderms. 

The two aplacophoran taxa share a similar reproductive 
system unique among mollusks. Paired gonads, some- 
times fused, open directly into the pericardium, and paired 



U-shaped gametoducts lead from the posterior end of the 
pericardium, first anteriorly and then posteriorly, to the 
mantle cavity (Figs. 5 A, 6 A, 9D). Separate gonaduct 
openings in species of Phyllomenia (Salvini-Plawen, 
1978) are interpreted here as a derived condition of that 
genus. 

The mantle cavity in both neomenioids and chaeto- 
derms is small and posterior, acting as little more than a 
cloaca. In neomenioids, the groove on either side of the 
foot-fold can also be considered as reduced mantle grooves 
(Figs. 6A, 8C). The paired ctenidia in chaetoderms, which 
fill most of the mantle cavity, is probably a plesiomorphy. 
with loss in the neomenioids resulting from the space re- 
quirements of a secondarily more complicated reproduc- 
tive system, including sometimes very large copulatory 
spicules. 

Finally, the worm shape itself is here considered a syn- 
apomorphy of the Aplacophora. and not separate. 




Figure 8. (A) Cross-section through the oral shield of a chaetoderm, Scutopus megaradulatus, showing 
continuity between pharyngeal and oral-shield cuticle. Arrow indicates transition between homogeneous 
pharyngeal cuticle and more specialized fibrillar oral-shield cuticle with a thickened outer layer (from Schel- 
tema, 1988, fig. 5). (B) Sagittal section through a neomenioid, liymnomenia sp., showing serial lateroventral 
musculature. (C) Cross-section through the nonmuscular, heavily ciliated foot of a neomenioid, Helicora- 
domeniajuani. The arrowhead indicates the nonspiculose cuticle of the mantle cavity extending along each 
side of the foot groove, which was considered homologous to the chaetoderm oral shield by S. Hoffman 
(1949). (D) Cross-section through the radula. radula bolsters, and paired, hollow radula vesicles in Helicor- 
adomenia /lunu (from Scheltema and Kuzinan, 1991. fig. 4D). 1 oral-shield cuticle, 2 pharyngeal cuticle, 3 
cuticle of body wall, 4 nerve fibers from precerebral ganglion, 5 ovarian region of hermaphroditic gonad, 6 
digestive cells of undifferentiated midgut. 7 copulatory spicule pocket, 8 foot. 9 radula vesicle, 10 radula, 
1 1 dorsal cecum of stomach/digestive gland. 

66 



APLACOPHORA: PROGENETIC COELOMATES 



67 




Figure 9. Nervous system and reproductive system in a neomenioid, Strophomenia scandens (A, D) 
and nervous system in a chaetoderm, Limifossor lalpoideus (B, C). (A) Lateral (= pleural, visceral) cord 
with its ongm in the cerebral ganglion separate from the origin of the ventral (= pedal) cord. Lateral and 
ventral cords remain separate posteriorly (after Heath, 1904, pi. 27, fig. 2). (B) Anterior end; the lateral and 
ventral cord have a single origin in the cerebral ganglion (after Heath, 191 1, pi. 10, fig. 8). (C) Posterior end: 
the ventral cord runs close to the lateral cord and fuses with it. The suprarectal commissure is ganglionated 
(after Heath, 1905. pi. 43, fig. 18). (D) Posterior end; the lateral and ventral cords are well separated, with 
the separation maintained throughout. The gonad empties into the pericardium, which is shown in fine 
stippling. The U-shaped gametoduct, with a many-lobed seminal receptacle, is shown in coarse stippling; it 
runs from the posterior end of the pericardium to the mantle cavity, indicated by dashed lines (after Heath, 
1904, pi. 27, fig. 6). 1 cerebral ganglion, 2 lateral cord, 3 pedal cord, 4 suprarectal ganglion/commissure, 
5 buccal ganglion, 6 gonad, 7 pericardium, 8 seminal receptacle, 9 mantle cavity. 



68 



A. H. SCHELTEMA 



convergent apomorphies in the two taxa. When this char- 
acter and those mentioned above are considered together, 
the Aplacophora clearly emerge as a monophyletic taxon. 

Chaetodermomorpha, derived Aplacophorans 

Neomeniomorpha are more similar than Chaetoder- 
momorpha to the outgroup, the Polyplacophora, in ner- 
vous system (Fig. 9A, D), form of epidermal papillae (Fig. 
6D, E), presence of anterior pedal glands (present only in 
the larvae of chitons) (Figs. 4, 6B), presence of paired 
pharyngeal glands, serial lateroventral musculature (Fig. 
8B), and inequality of height and width dimensions. Sev- 
eral autapomorphies indicate that the burrowing Chae- 
todermomorpha have been derived from a creeping neo- 
menioid-like ancestor. Criteria for considering a structure 
to be apomorphic are fusion or elaboration. 

Changes in the nervous system are pronounced. In 
chaetoderms the lateral and ventral cord on each side have 
a single origin from the cerebral ganglion, whereas in 
nearly all neomenioids lateral and ventral cords have sep- 
arate origins (Fig. 9A, B). In chaetoderms, the lateral and 
ventral cords on each side soon run close to each other, 
finally fusing into a single cord anterior to the suprarectal 
ganglion (Fig. 9C). In neomenioids, the cords remain apart 
and are well separated from each other (Fig. 9 A, D). There 
are few commissures between the ventral cords in chae- 
toderms, and many in neomenioids. In chaetoderms, the 
suprarectal commissure and precerebral ganglia are larger 
and more swollen than in neomenioids. 

Related to the loss of the ventral cord commissures, 
chaetoderms have entirely lost the foot and anterior pedal 
glands. The homology of mucous glands of the oral shield 
with pedal glands, proposed by S. Hoffman (1949), does 
not hold in TEM studies (Scheltema el ai. in press. Fig. 
9A). In some species of Scut opus and Psilodens, ventral 
fusion of the mantle is marked by a longitudinal furrow 
between the spicules (Salvini-Plawen, 1968b; author's 
unpub. data). 

The gut of chaetoderms is modified from the simple 
combined stomach-digestive gland midgut of neomenioids 
to a separate stomach and blind digestive gland. In its 
most derived state in Chaetodermatidae, there is a gastric 
shield and style sac with a mucoid rod (Scheltema, 1978; 
Salvini-Plawen, 1981b). 

The serial lateroventral musculature of neomenioids 
(Fig. 8B) is lost in chaetoderms, although a few vestigial 
anterior bundles have been reported in a species of Scu- 
topus (Salvini-Plawen, 1985). Body form in chaetoderms 
is circular in cross-section; in neomenioids there is usually 
a small but measurable difference between height and 
width. The circulatory system is somewhat better denned 
in chaetoderms than in neomenioids, with anterior and 
posterior vertical septa defining hemocoelic sinuses, and 



with an often thick-walled aorta and aortal bulb (Schel- 
tema, 1973) (Fig. 5A). Finally, the chaetoderm oral shield 
represents a specialized cuticular structure. 

The autapomorphies of Chaetodermomorpha all seem 
to be related to their form of locomotion burrowing in 
muds and silts and feeding habits, either as carnivores 
on small benthic organisms, or as detritivores. Autapo- 
morphies also exist in the Neomeniomorpha, particularly 
the sensory vestibule and rather complicated reproductive 
system with accompanying loss of mantle cavity ctenidia, 
but specializations of the Chaetodermomorpha mark 
them as the more derived of the two taxa. 

Relationship of Aplacophora and Polyplacophora 

Aplacophora and Polyplacophora are here considered 
to be sister taxa, the Aculifera, on the basis of shared char- 
acters of nervous system, spicules, and epidermal papillae. 
An attempt is made to determine the polarities of these 
characters, and other anatomical similarities are noted. 

Nervous system 

Aplacophora, Polyplacophora, and the monoplacoph- 
oran Tryblidiacea Neopilina and Vema all have a fully 
developed tetraneury, with paired lateral and pedal nerve 
cords arising from a cerebral commissure or ganglia and 
a circumoral or circumesophageal nerve ring. In the 
monoplacophorans, both cords are joined posteriorly 
ventral to the rectum, whereas in Aplacophora (Fig. 9) 
and Polyplacophora, only the lateral cords are joined, and 
they unite above the rectum in a commissure (chitons) 
or ganglion (aplacophorans). There is only a single cross- 
pedal commissure in the monoplacophorans and numer- 
ous ones in chitons and neomenioid aplacophorans. 

What is the polarity of these two plans, both of which 
are plesiomorphic to more specialized nervous systems in 
other mollusks? Obvious outgroups for comparison, An- 
nelida, Echiura, Nemertini, and Sipuncula, appear to have 
a reduced nervous system and offer no clues. In Annelida 
there is only a paired ventral cord, except for a secondarily 
derived tetraneury in Amphinomidae (Gustafson. 1930); 
in Nemertini there is a pair of lateral cords joined either 
above or below the rectum; in Echiura, there is a single 
ventral cord; and in sipunculans there is also a single ven- 
tral cord which is paired in the larval pelagosphera of 
Phascolosoma agassizii (Rice, 1973). One might surmise 
that Neopilina, a deep-sea deposit or xenophyophore 
feeder (Tendal, 1985), is less mobile than either aplacoph- 
orans or polyplacophorans and has retained a simpler 
nervous system, and the aplacophoran-polyplacophoran 
system is more specialized (derived) owing to habitat (chi- 
tons) or to carnivory (aplacophorans). Of course, a sec- 
ondary loss and shifting of nerve elements in the mono- 
placophorans might also be considered, and Wingstrand 



APLACOPHORA: PROGENETIC COELOMATES 



69 



(1985) and Salvini-Plawen (1972) suggest that the sub- 
rectal commissure is an apomorphy. Whichever interpre- 
tation is correct, one can say that monoplacophoran and 
aplacophoran-polyplacophoran nervous systems are each 
apomorphic to some unknown ancestral state, and the 
suprarectal ganglion or commissure of the Aplacophora- 
Polyplacophora serves to relate them phylogenetically and 
set them apart from the Monoplacophora. 

Spicule formation 

Spicule formation in aplacophorans and polyplacoph- 
orans has most recently been investigated by Haas (1981) 
(Fig. 10). Spicules in both taxa are aragonite and formed 
extracellularly within an invagination of a single basal 
cell, which secretes calcium carbonate within a crystalli- 
zation chamber sealed by neighboring cells (Scheltema a 
al., in press, fig. 6D). In chitons, megaspines are formed 
from a proliferation of the basal cell and do not occur in 
aplacophorans. 

Spicules of the Aculifera are usually considered to be 
a plesiomorphic state of calcium carbonate formation 
within Mollusca, since both spines and shell occur in chi- 
tons and only spines occur in Aplacophora, both being 
"primitive" groups in the general sense. However, Mono- 
placophora, likewise considered primitive, have no spines. 

The dorsal, calcium-carbonate-secreting epidermis of 
Mollusca, in combination with a ventral locomotary sur- 
face, is probably an apomorphy. However, the shell-bear- 
ing Brachiopoda are rooted with the Mollusca-Annelida 
group by RNA sequencing (Field el al.. 1988). and some 
boring Sipuncula have calcium carbonate deposits at the 
dorsal anterior end of the trunk (Rice, 1969). Further 
comparative work needs to be done to compare calcium 
carbonate secretion among the Eutrochozoa before ho- 
mology can be assumed. 

It cannot be concluded from outgroup comparison that 
spicules and shell are homologous structures (and the ar- 
gument will be made further on that they are not), or that 
either is the plesiomorphic state. It can be concluded, 
however, that because of the way in which they are formed, 
spicules of Aplacophora and Polyplacophora are homol- 
ogous and can be construed as a synapomorphy. 

Epidermal papillae 

The epidermis of both chitons and aplacophorans are 
liberally supplied with secretory papillae (Fig. 6D, E). In 
chitons, papillae are homologous with aesthetes (Fischer 
c/ al.. 1980). Although homology with other conchiferan 
shell-penetrating structures has been suggested (Salvini- 
Plawen, 1985), the homology was considered spurious by 
Wingstrand, who reviewed the literature on the subject 
(1985, pp. 58-59). The presence of these papillae is COn- 




Kigure 10. Spicule formation in (A) Aplacophora and (B) Polypla- 
cophora (after Haas. 1981. figs. 6, 12). The spicule is formed within an 
invagination of a basal cell which secretes CaCOj. The crystallization 
chamber is sealed by a nng of neighboring cells, which in Polyplacophora 
produce a pellicle around the spicule. 1 spicule, 2 neighboring cell, 
3 CaCO r secreting basal cell. 



sidered here to be an apomorphy of the Aplacophora- 
Polyplacophora. 

Rciluccd aerial replication 

Compared to Monoplacophora, there is less serial rep- 
lication in both Polyplacophora and Aplacophora, but 
both have greater serial replication than other mollusks. 
Serial replication appears as regular, lateroventral mus- 
culature in Neomeniomorpha (Fig. 8B) and as 8-fold rep- 
etition of muscles and shell plates in chitons. 

Other anatomical homologies 

Aplacophorans and polyplacophorans share certain 
other anatomical structures that are probably homolo- 
gous, but they may be plesiomorphies of the Mollusca. 
Dorsal paired gonads, becoming fused during ontogeny 
in chitons and most Chaetodermomorpha, lie like sacs 
more or less free above the gut and digestive gland in the 
dorsal hemocoel. In Neopilina, the gonad is ventral to the 
digestive system (Lemche and Wingstrand, 1959), and in 
many other Mollusca the gonad is intermingled closely 
with lobes of the digestive gland. The circulatory system 
in both groups is extremely open with posterior paired 
auricles and a ventricle, a dorsal aorta leading to the head 
(lacking in many Neomeniomorpha), and open sinuses, 
the latter more profuse in chitons. 

Taken together, the above reasons are sufficient for 
concluding that Aplacophora and Polyplacophora belong 
together in a single taxon, the Aculifera, which is therefore 
a clade, and not a grade. 



70 



A. H. SCHELTEMA 



Aculifera as the Sister Taxon of the Conchifera 

Chitons provide evidence that Aculifera are separate 
from their sister group, the Conchifera. The evidence is 
based on shell ontogeny, shell structure, and perhaps mo- 
lecular data. 

She/I ontogeny 

In Conchifera, the shell originates within an ectodermal 
invagination. the shell-field invagination. which is covered 
by an organic pellicle (Eyster and Morse, 1984) (Fig. 1 1 ). 
In Aeolidia papillosa, long cytoplasmic processes overlie 
the pellicle. In chitons, there is no shell field invagination, 
and shell plate anlagen are deposited within transverse 
depressions which are sealed, not by a pellicle, but by 
long, overlapping microvilli that lie beneath a gelatinous 
mucoid substance (Kniprath, 1980; Haas cl a/., 1980; 
Haas, 1981; see Scheltema, 1988, for a more complete 
discussion). Furthermore, in healthy larvae, shell is not 
deposited as separate granules, as illustrated by Kowalev- 
sky (1883), but as uninterrupted rods (Kniprath, 1980). 
This fact conflicts with the hypothesis that chiton shell 
arose from fused spicules (Salvini-Plawen, 1985. 1990). 

Shell structure 

The crystallography of chiton shell has been said to 
indicate an autapomorphy of chitons by Haas ( 1976), who 
found that "The . . . c-axis of [the] hypostracum lies in 
the bisectrix of the crystalline fibers. The whole complex 
acts crystallographically as a single crystal" (p. 392). If 
this crystallographic orientation is correct, then no ho- 
mology exists between polyplacophoran and conchiferan 
shell. Further differences are a lack of true periostracum 
in chitons (although Haas [ 1 98 1 ] has demonstrated a thin 
cuticle overlying the shell plates) and a lack of a nacreous 
layer (for further discussion see Wingstrand, 1985; and 
Scheltema, 1988). On the other hand, the shell of the try- 
blidiacean Monoplacophora does not differ from other 
primitive conchiferan shells (Lemche and Wingstrand, 
1959). 

Molecular evidence 

The evolutionary tree of 1 8S rRN A has three branches 
for three classes of mollusks a nudibranch, two species 
of clam, and a chiton. This trifurcation of mollusks also 
appears in Lake's (1990) re-analysis of the data. Further 
molecular data for all molluscan classes should resolve 
the branching, but there is a hint of molecular distance 
between chitons and the two other classes analyzed. 

Evidence for Progenesis in Aplacophora 

A vermiform body is a character that could have been 
added rapidly by a small change in a regulatory gene or 




Figure 11. Shell deposition in larvae of Conchifera and Aculifera. 
(A, B) Gastropod, Aolidia papillosa- An organic pellicle (arrowheads) 
covers the lumen of the shell held invagination; a cytoplasmic extension 
shown in B seals the edge of the pellicle (after Eyster and Morse, 1984, 
figs. 1,2; from Scheltema, 1988, fig. 4). (C, D) Polyplacophoran, Ischno- 
chiton rissoi. The shell plate is first secreted beneath microvilli (stragulum) 
which are covered by a layer of mucus (C); later (D) the microvillar 
processes have pulled apart and a cuticle begins to form (after Kniprath. 
1980, fig. 5, from Scheltema. 1988, fig. 4). Haas (1981) illustrated a 
similar process except for showing that cuticle covered the stragulum 
before CaCO, deposition. 1 shell field invagination, 2 cytoplasmic ex- 
tension, 3 microvillar process (stragulum), 4 calcium carbonate of shell 
plate, 5 mucous layer, 6 ?mucous cell, 7 cuticle. 



in timing of cell assembly early in the ontogeny of an 
aculiferan mollusk (for mechanisms and examples see Raff 
and Kaufman, 1983; McKinney and McNamara, 1991). 
In the embryological development of the chiton Lepido- 
pleums asellus, swimming larvae are first oval and then 
become secondarily flattened and sink to the bottom 
(Christiansen. 1954). Even with development of the foot, 
chiton larvae remain ovoid for a time (Heath, 1899, Fig. 
52; Eernisse, 1988, Fig. 7). One can imagine that larvae 
of some aculiferan, not necessarily a chiton, might not 



APLACOPHORA: PROGENETIC COELOMATES 



71 



have become dorsoventrally flattened through a small 
change in gene regulation and the worm-like shape arose. 

The change to a vermiform shape could have occurred 
either early in the evolution of Mollusca or late. Recent 
phylogenies presume that a vermiform shape evolved as 
an early offshoot of the Mollusca, placing Aplacophora 
closest to the stem form, either as a monophyletic clade 
(Scheltema, 1988; Wingstrand, 1985), or as two separate 
clades, with the Chaetodermomorpha evolving first as the 
sister-group to all extant Mollusca (Salvini-Plawen, 1972, 
1985). Serial replication thus was seen to he an apomor- 
phy. If Aplacophora are closest to the molluscan ancestor, 
then the imperatives following from that phylogenetic 
construct fit poorly with the arguments given above, that 
is: ( 1 ) Aplacophora and Polyplacophora are a clade; (2) 
shell is not formed by fusion of spicules; and (3) chiton 
shell is not homologous to conchiferan shell. The question 
of when serial replication evolved in mollusks becomes 
critical, for it is either a plesiomorphy of mollusks. or not. 

Polyplacophora, belonging to Aculifera, have some 
structures homologous with Monoplacophora, belonging 
to Conchifera, that are not shared with the Aplacophora 
(Wingstrand, 1985); radula dentition and radular appa- 
ratus including musculature; 8-serial pedal retractors; pre- 
oral unpaired fold, or velum; perhaps the heart with two 
pairs of atria; and coiled intestine. Wingstrand noted that 
some of these structures "could be plesiomorphic, i.e., 
could have been present already in some Aplacophoran 
ancestors" (1985, p. 74), but considered that the radula 
and radular apparatus, in particular the paired, hollow 
radula vesicles, are synapomorphies. It was not then 
known that paired radular vesicles are also present in some 
neomenioids (Fig. 8D). Here, structures argued to be apo- 
morphic by Wingstrand are considered plesiomorphic 
with exception of the coiled gut, a character widely con- 
vergent among mollusks. Thus, serial replication is here 
considered a plesiomorphy of Mollusca. 

The possibility that a worm shape was acquired by 
aplacophorans late in aculiferan evolution leads to a 
wholly different concept of molluscan phylogeny. It calls 
for progenesis in Aplacophora, wherein nonserial but ple- 
siomorphic-appearing anatomical characters are retained. 
The following evidence supports the hypothesis that 
Aplacophora are progenetic; i.e., that they have retained 
ancestral juvenile characters in adult form through ac- 
celeration of sexual maturation (Gould, 1977). 

( 1 ) If narrowing of the body by acquisition of a worm 
shape arose early in aculiferan evolution without proge- 
nesis, then this process should be reflected somehow in 
the internal anatomy, and the more elongate (that is, nar- 
rower) the shape, the more pronounced should the internal 
changes become. Within the Neomeniomorpha, the least 
derived aplacophoran taxon, there is little organizational 
difference between short and elongate species in anterior 



and posterior ends or in musculature. Elongation of ex- 
ternal form is accompanied internally by a simple length- 
ening of the gonad and midgut (Fig. 7D, E). The situation 
in the more derived chaetoderms differs and does not serve 
the argument. 

A comparison can be made to Cryptoplax. a genus of 
chiton with a derived worm-like shape. In Cryptoplax 
there are at least four specializations of adult characters: 
(a) the mantle is very thick relative to internal body di- 
ameter; (b) there is loss of circulatory pathways; (c) there 
is loss of shell and shell musculature; and (d) the intestinal 
tract is remarkably long and complicated, turning back 
on itself in numerous spirals (Wettstein, 1904; H. Hoff- 
mann, 1929-30). Furthermore, an analysis of the allo- 
metric equation defining shape in 408 chiton species in 
39 genera indicated great uniformity in allometry, except 
in the carnivorous Placiphorella and in genera of Cryp- 
toplacidae (Watters, 1991). Species ofCryptoplacidae, ex- 
cept those in the most primitive genus, are allometrically 
similar to each other but have shifted markedly from the 
allometry of other chitons. Although no allometric studies 
have been made of neomenioids, the extremes in ver- 
miformity (Fig. 7D, E) do not predict uniformity. Thus 
there may be an ontogenetic difference in the evolutionary 
pathway to a worm-like shape taken by the two aculiferan 
taxa. Progenesis, an intrinsic process, is hypothesized for 
Aplacophora, and selection working on structural genes, 
an extrinsic process, for Cryptoplax. 

(2) Progenesis results in early reproduction (Gould, 
1977). One abundant northwestern Atlantic aplacophoran 
species living at 2000 m, Prochaetoderma yongei, is known 
to mature within one year, a remarkably rapid rate, given 
the ambient temperature (~3C) and in comparison with 
other cold-water mollusks. P. yongei is interpreted as being 
an opportunistic species (Scheltema, 1987), but since it 
is the only aplacophoran for which even part of the life 
history is known, one cannot be sure that early repro- 
duction is the usual case in Aplacophora. 

(3) Progenesis results in a reduced body size (Gould. 
1977), but the size of the nearest ancestor to Aplacophora 
is, of course, unknown. Most neomenioids are usually 
less than 5 mm long, and one can only infer from the 
generally larger size of chitons that the first ancestral apla- 
cophoran was already small. Like some other deep-sea 
taxa, such as protobranch bivalves (Sanders and Allen. 
1973) and isopods (Hessler et ai, 1979), aplacophorans 
have evolved primarily in the deep sea, where they reach 
their greatest diversity (Scheltema, 1990). Food is limiting 
there, and small body size of macrobenthic organisms is 
the norm (Monniot and Monniot, 1978; Allen, 1983; 
Soetaert and Heip, 1989). Large neomenioids do exist in 
the deep sea, but they are usually either specialized (giant 
Neomenia species: Baba, 1975; Kaiser, 1976) or live in 
environments where there is high productivity (e.g., high 



72 



A. H. SCHELTEMA 



latitudes: Proncomenia sluitcri, Derjugin. 1915, 1928). 
Large body size in Aplacophora is probably an apo- 
morphic character because it is found scattered amongst 
unrelated families, some of which have derived characters 
such as loss of radula or a thick dermis. 

(4) Certain structures in Aplacophora are less devel- 
oped than homologous structures in Polyplacophora or 
other mollusks. (a) The organic compos : *ion of the cuticle 
is simpler than in chitons (Beedham ano Trueman, 1968). 
(b) The radula in its plesiomorphic state in neomenioids 
has only two teeth per row (distichous), a condition found 
in the early ontogeny of several gastropods (Kerth, 1983; 
Scheltema, 1988; Scheltema ct a!., 1989). (c) The apla- 
cophoran mantle cavity, located ventroposterior to pos- 
terior, is small, serving as little more than a cloaca (Fig. 
7A, D). (d) Both neomenioids and chaetoderms lack kid- 
neys, (e) The foot is developed only as a ciliated ridge 
without musculature in neomenioids (Fig. 6A). (f ) Gonads 
and pericardium are united in aplacophorans, reflecting 
the early ontogenic state in chitons, where the gonad orig- 
inates as an anlage of the pericardium (Hammarsten and 
Runnstrom, 1925) (Figs. 5 A, 6A, 9D). (g) The gut in neo- 
menioids is simple, with a united stomach and digestive 
gland; the digestive gland is separate from the stomach in 
other mollusks. 

(5) Aplacophora have retained a structure found in 
chitons only as larvae. The anterior pedal glands are large 
and specialized in neomenioids (Fig. 6B, C), but are lost 
soon after metamorphosis in chitons, where they serve 
only for early postmetamorphic attachment (Heath, 1899) 
(Fig. 4B). 

Although progenesis results in primitive-appearing 
structures, they are actually derived. Therefore, some 
process within the Aculifera should be primitive in the 
Polyplacophora but derived in the progenetic Aplacoph- 
ora. Such seems to be the case in early embryological 
development. Freeman and Lundelius (1992) have pro- 
posed that, among the spiralian coelomates Mollusca, 
Annelida, Sipuncula and Echiura, two mechanisms de- 
termine which blastomere is specified as the D quadrant. 
They hypothesized that the primitive mechanism for D 
quadrant specification is by induction after the fifth cleav- 
age, when one of the four macromeres has maximum 
contact with the micromeres. The derived mechanism is 
by segregation of the cytoplasm into one macromere, 
which is then specified as the D quadrant; it occurs by 
the second cleavage. In Polyplacophora, macromeres 
cleave equally and the D quadrant is specified by induc- 
tion, the primitive mechanism. But in the cleaving egg of 
the neomenioid Epimenia, a polar body is formed and 
therefore macromeres of unequal size; thus the D quadrant 
is specified by cytoplasmic determinants, the derived 
mechanism (Baba, 1951; Freeman and Lundelius, 1992). 



The evidence for progenesis presented here argues for 
heterochrony in the Aplacophora, but this idea cannot be 
tested either against fossils, which are unknown, or against 
a more complete phyletic lineage, as has been done for 
progenetic meiofaunal forms (Westheide, 1987) and deep- 
sea tunicates (Monniot and Monniot, 1978). When the 
early embryological development of aplacophorans is 
better known, and with further intrataxon comparative 
studies, the validity of the hypothesis may be clarified. 

Phytogeny of the Mollusca 

The phylogeny represented in Figure 12 proposes a 
coelomate molluscan ancestor with serial replication; two 
separate evolutionary molluscan lineages, the Conchifera 
and the Aculifera, based on synapomorphies of differences 
in CaCO, deposits; and morphologies arising from pro- 
genesis in the Aplacophora. 

The molluscan ancestor is considered to have had the 
following plesiomorphies: (1) extracellular CaCO 3 depo- 
sition by the dorsal epidermis (Mollusca generally); (2) 
serial replication, probably originally 8-fold (Monopla- 
cophora, Polyplacophora, Nautilus, neomenioids, some 
bivalves); (3) coelom from the 4d cell, paired pericardial 
cavities (in Monoplacophora, and fused but large in Apla- 
cophora and Polyplacophora); (4) radula, radular appa- 
ratus with hollow radula vesicles (Polyplacophora, 
Monoplacophora. Aplacophora, Fig. 8D); (5) nervous 
system poorly ganglionated, with cerebral ganglia and 
commissure, circumenteric ring, and paired lateral and 
pedal cords with cross-commissures and posterior con- 
nection (Monoplacophora in part, Aplacophora and 
Polyplacophora); (6) dorsoventrally flattened, small size 
(Cambrian Mollusca: Runnegar and Pojeta, 1985; Hasz- 
prunar, 1992; but note that the Cambrian fossil halkierids 
and Wiwaxia, perhaps near relatives of mollusks, are cen- 
timeters in length [Conway Morris, 1985; Conway Morris 
and Peel, 1990]); (7) dorsal cuticle (Aplacophora, Poly- 
placophora); (8) ventral ciliated locomotory sole (Mollusca 
generally); (9) head separate from the locomotory sole 
and with cerebral ganglia (Mollusca generally); (10) a 
groove between the dorsal and ventral surfaces, the future 
mantle cavity (Mollusca generally): (11) pre-oral fold; (12) 
the presence of podocytes in pericardial tissue (mollusks 
generally); (13) ductless anterior pedal mucous glands (as 
a glandular epithelium in Monoplacophora; Lemche and 
Wingstrand, 1959); (14) a one-way gut with mouth, anus, 
large digestive gland poorly differentiated from stomach 
(Neomeniomorpha, Monoplacophora); (15) paired pha- 
ryngeal diverticula; (16) poorly defined circulatory system; 
and (17) gonad and pericardium joined at least during 
ontogeny (Mollusca generally). 

The phylogeny presented in Figure 12 requires that the 
original calcium carbonate deposition in mollusks was 



APLACOPHORA: PROGENETIC COELOMATES 



73 



ACULIFERA 



APLACOPHORA 



CONCHIFERA 




0. 

O 



tr oc 


65 
ili 

< o 



39' 
23 



B 



-- 22' 



39' 
18 



6-17 [c?| 

- 5 

4 [d?| 

- 3 
2 
1 

Figure 12. Proposed phylogeny of extant "primitive" Mollusca. (A) 
Apomorphies of Mollusca: I extracellular CaCO, deposition by dorsal 
epidermis; 1 eight-fold serial replication; 3 paired coelom, including 
pericardium; 4 radula; 5 poorly ganglionated tetraneury; 6 small size, 
dorsoventrally flattened; 7 dorsal cuticle; 8 ventral locomotory sole; 9 
head separate from sole; 10 groove between dorsal and ventral surfaces; 
1 1 pre-oral fold; 12 nonsegmented pericardium, pencardial tissue with 
podocytes; 13 ductless anterior pedal gland; 14 poorly differentiated 
stomach/digestive gland (model: Neopilina); 15 paired pharyngeal di- 
verticula; 16 poorly denned circulatory system; 17 joined gonad/pen- 
cardium during early ontogeny. (B) Separation of Conchifera and Acu- 
lifera: 18 calcareous shell; 19 spicules; 20 epidermal papillae; 21 supra- 
rectal ganglion/commissure; 22 reduced serial replication and fused 
pericardium. (C) Separation of Polyplacophora and Aplacophora 
(24-31 the result of progenesis): 23 eight shell plates; 24 worm shape; 
25 reduced foot; 26 reduced mantle cavity; 27 joined gonad/pericardium; 
28 kidneys absent; 29 chemically simple cuticle; 30 serial lateroventral 
musculature; 31 distichous radula; 32 U-shaped gametoducts; 33 gan- 
glionated nervous system; 34 dorsoterminal sense organ. (D) Separation 
of Chaetodermomorpha and Neomeniomorpha: 35 ventrally fused cu- 
ticle, foot lacking; 36 oral shield; 37 fused, reduced nervous system; 38 
serial replication absent; 39 stomach separate from digestive gland; 40 
large anterior pedal gland; 41 elaborated reproductive system; 42 ctenidia 
absent. * = convergent morphologies; c? = presence of ctenidia ques- 
tionable; d? = radula questionably docoglossate. 



neither as spicules nor as shell. CaCO, was first deposited, 
perhaps, as granules within a dorsal cuticle, which was 
thereby stiffened. Such a reinforced cuticle could act as 
the antagonist to the dorsoventral pedal musculature. 
During chiton ontogeny, the pedal musculature develops 
earlier than the shell plates (Hammarsten and Runnstrom, 
1925). One can speculate from this fact that, perhaps, the 
various forms of shell and spicules among mollusks have 
resulted from selection for different modes of locomotion 
in various habitats, rather than selection just for protec- 
tion. 

In terms of CaCO 3 secretion among phyla, the impor- 
tant synapomorphy for mollusks, which sets them off from 
other spiralian coelomates, is the locomotary sole in com- 
bination with a cuticle- and CaCO r secreting dorsal epi- 
dermis. Certain rock-boring sipunculans also secrete 
CaCOj dorsally, forming a plug for their tubes (Rice, 
1969), and Brachiopoda, which fall in with spiralian coe- 
lomates in molecular analysis, also have calcium carbon- 
ate shells. However, animals in neither of these phyla have 
the combination of dorsally produced CaCO, and a ven- 
tral locomotary surface unique to mollusks. 

It is hypothesized that after, or as, Conchifera diverged 
from the stem line, the mantle deepened and gills devel- 
oped. Serial replication was retained in Monoplacophora 
but lost in the rest of the Conchifera, except for serial 
pedal musculature in some taxa and the renal system in 
cephalopods. Aculifera may have evolved either at the 
same time as Conchifera or later. By the Upper Cambrian 
or Lower Ordovician, the serial shell plates of Polypla- 
cophora had evolved (Runnegar and Pojeta, 1985). This 
event was preceded by the loss of serial replication other 
than lateroventral muscles and perhaps by an increase in 
size. In a separate evolutionary event of progenesis, the 
Aplacophora evolved with probable reduction in size, 
further loss of serial replication, loss of nephridia, retention 
of gonad-pericardial connection, and acquisition of a 
worm shape with concomitant reduction of the foot. 
Chaetodermomorpha were derived from the neomenioid- 
like stem with complete loss of foot, reduction and fusion 
of the nervous system, and specializations of the gut. 

This hypothesized phylogeny does not call for an evo- 
lutionary process in which CaCO, deposits, or the cells 
that produce them, become fused. Furthermore, it should 
allow some of the Early Cambrian sclerite-bearing forms 
now coming to light, such as the shell-bearing, articulated 
halkieriid described recently from the Lower Cambrian 
of Greenland (Conway Morris and Peel, 1990), to find 
their place in relation to the extant Mollusca. 

In this phylogeny, the Monoplacophora with clear serial 
replication are not evolved after Aplacophora, and mol- 
luscan serial replication is considered to be a plesiomor- 
phy. As Wingstrand (1985) pointed out, it is difficult to 
imagine that serial replication evolved after the shell. The 



74 



A. H. SCHELTEMA 



careful and original anatomical analysis of Wingstrand 
showing close affinities of the monoplacophoran Trybli- 
diacea and Polyplacophora are upheld here as retained 
plesiomorphies of the common ancestor. Whether Pru- 
vot's neomenioid larva with its supposed seven rows of 
spicules actually exists does not change the argument (see 
Salvini-Plawen, 1972, 1981a, 1985; Scheltema, 1988 for 
discussions and figures of the larva). Manuscript drawings 
of Chaetoderma nitidithtm larvae made by G. Gustafson 
show eight rows of spicules for this taxon as well. If further 
observations on aplacophoran development prove that 
serial rows of spicules do exist, the larva still would not 
necessarily reflect progressive evolution from spicules to 
fused shell plate formation, but more likely would indicate 
a breakdown of plate formation similar to the breakdown 
of larval chiton shell plates caused experimentally by 
Kniprath (1980) (See also Scheltema, 1988). 

Age of the Aplacophora 

If known fossils reflect the actual time of evolutionary 
events, then the evolution of Polyplacophora late in the 
Cambrian (Runnegar and Pojeta, 1985) from a continuing 
line of aculiferous creatures was probable, with increased 
size and muscles being the determinants of shell plates 
rather than vice versa (see Hammarsten and Runnstrom, 
1925, p. 276, for ontogenetic development of muscle be- 
fore shell). Aplacophora, with their highly derived shape 
and paedomorphic internal organization, give information 
about the primitive conditions of mollusks without being 
themselves primitive. A Late Cambrian-Early Ordovician 
origin from an aculiferan form with a developed mantle 
groove and posterior mantle cavity is postulated for Apla- 
cophora, with the 8-fold dorsoventral muscles rearranged 
in neomenioids into a series of indeterminate number. 

Cautionary Notes on Convergences 

Digestive system 

The molluscan gut appears to have evolved similar 
morphologies more than once (Fig. 12, no. 39). Evidence 
for convergence lies in presence of the style sac and gastric 
shield, found in a number of molluscan classes. In the 
aplacophoran family Chaetodermatidae, one of the most 
derived of the chaetoderm groups based on radula mor- 
phology (Scheltema, 1972, 1981), the gut is the most 
complicated among chaetoderms. with a gastric shield and 
a mucoid rod in a style sac (Scheltema, 1978; Salvini- 
Plawen, 1981b). The polarity of a less to a more compli- 
cated gut configuration within the chaetoderms is clear 
(Scheltema, 1981). Thus, the presence of a style sac and 
gastric shield is convergent among Mollusca. 

Metamerism 

Reduction of serial replication (Fig. 12, no. 22) is hy- 
pothesized for several molluscan classes Cephalopoda, 



Bivalvia, Polyplacophora. and Aplacophora. The evidence 
from morphology, ontogeny, and molecular analysis 
seems not to favor the hypothesis that replication origi- 
nated in annelids. If the altogether unsegmented Sipuncula 
are sister taxon of the mollusks, then arguments that the 
molluscan coelom is the result of a reduced annelid-like 
segmented coelom are not convincing. 

Evidence presented here could be interpreted in three 
ways (Fig. 13, s'-s 4 ). (1) A nonsegmented ancestor that 
had serial replication of organs and a coelom lies at the 
base of the lineage giving rise to Eutrochozoa (s 1 ). (2) The 
eutrochozoan ancestor had no serial replication, which 



O 

z 



O 
0) 



Q 

_l 
UJ 



9* 



6-8 

o 2. 



10 



11 

9* 

S3 



1-5 



Figure 13. Phylogenetic relationship among Sipuncula, Mollusca. 
and Annelida. 1 spiral cleavage; 2 paired coelom originating from two 
teloblasts derived from 4d; 3 trochophore larva (?); 4 tetraneury; 5 ciliated 
creeping sole; 6 molluscan cross; 7 ventral, cuticular, pharyngeal (sto- 
madeal), protrusihle invagmation and attendant musculature; 8 anterior 
pedal gland; 9 fused nerve cords; 10 reduced coelom; 1 1 loss of creeping 
sole, s = serial replication: s 1 symplesiomorphic for all three taxa, but 
lost in Sipuncula; s 2 symplesiomorphic for Sipuncula and Mollusca, but 
lost in Sipuncula, and convergent with s 1 as metamerism in annelids; s 3 
metamerism plesiomorphic for Annelida, convergent with either s 2 or 
s 4 ; s 4 plesiomorphic for Mollusca, convergent with s 1 . * = convergent 
morphologies. 



APLACOPHORA: PROGENET1C COELOMATES 



75 



later arose de novo twice: once in the stem form leading 
to mollusks and sipunculans (s 2 ). which was lost in the 
latter, and secondly in the ancestral annelid as metamer- 
ism (s 3 ). (3) Molluscan 8-fold serial replication (s 4 ) evolved 
after the stem form that gave rise to Sipuncula and Mol- 
lusca; and annelidan metamery (s 3 ) [as in (2)], arose as 
an unrelated evolutionary event. The first interpretation 
is perhaps closest to what may actually have occurred and 
seems the most parsimonious explanation. 

Differences in the coelom among eutrochozoan groups 
can be related to locomotion, a theme emphasized cor- 
rectly, I believe by Salvini-Plawen (e.g., 1972, 1985). 
Locomotion among Eutrochozoa is most rapid in annelids 
and mollusks. Serial pedal musculature is related to a 
creeping locomotion and is the most conservative serial 
structure in mollusks, present as a plesiomorphy in 
Monoplacophora. Polyplacophora, Aplacophora, and 
(much reduced) Pelecypoda and perhaps the neritid Gas- 
tropoda. In Annelida, coelom and muscle have combined 
in the perfection of a hydraulic locomotion (Clark, 1964). 
Perhaps, then, a re-examination of the relationship of 
muscles and coelom during ontogeny would be a useful 
exercise in providing insights into understanding the de- 
velopment of metamerism in Eutrochozoa. For instance, 
in at least some Annelida, ectodermal segmentation of 
the three anterior segments precedes segmentation of me- 
soderm (Anderson, 1973, pp. 36-37). 

Radula 

Wingstrand (1985) gave a detailed description of the 
radular apparatus in Polyplacophora and Monoplacoph- 
ora, demonstrating their great similarity, especially the 
docoglossate radula and radula vesicles. There are three 
possibilities: such a radula is a molluscan plesiomorphy; 
it is an apomorphy of Polyplacophora and Monopla- 
cophora; or it is convergent. 

Evidence from Aplacophora and ontogeny of some 
Gastropoda suggests that the plesiomorphic radula in 
mollusks was distichous (Keith, 1983; Scheltema el ai, 
1989). An outgroup for comparison is the Cambrian 
sclerite-bearing Wiwaxia (Conway Morris, 1985) with two 
or three rows of teeth which appear much like the ple- 
siomorphic radula in Aplacophora. The phylogenetic po- 
sition of Wiwaxia. however, remains enigmatic, consid- 
ered either to be close to mollusks (Conway Morris, 1985) 
or to be an annelid (Butterfield, 1990). If the plesiomor- 
phic radula is distichous, then the docoglossate radula is 
convergent in Polyplacophora, Monoplacophora, and 
patellacean Gastropoda. If the docoglossate radula is a 
molluscan plesiomorphy, it is difficult to imagine how it 
functioned in a small Cambrian mollusk and what evo- 
lutionary steps would be necessary to account for all other 
molluscan radulae. 



The strongest evidence given by Wingstrand (1985) for 
monophyly of polyplacophorans and conchiferans is 
presence of a pair of hollow, presumably liquid-filled rad- 
ula vesicles found at that time only in Polyplacophora 
and Monoplacophora. None had been reported in Apla- 
cophora. However, a re-examination of the neomenioid 
Helicoradomenia juani and other species in the genus, 
which have a plesiomorphic aplacophoran radula, has led 
me to conjecture that paired, elongate, hollow vesicles 
present in this genus are a homolog to the radula vesicles 
in Polyplacophora and Monoplacophora (Fig. 8D). 
Therefore these vesicles are a molluscan plesiomorphy. 
However, further study of the aplacophoran radula and 
its apparatus is needed. 

Larval forms 

The phylogenetic significance of larval forms in Spiralia 
is not addressed here. There is still no agreement on 
whether a pelagic organism gave rise to benthic forms 
U'.t,'.. Nielsen and Norrevang, 1985), or vice versa, and 
whether the trochophore larva arose once or several times 
(Ivanova-Kazas, 1985a, b, for careful discussions). Within 
Mollusca. Salvini-Plawen ( 1972, 1985) regarded the peri- 
calymma larva, which lacks purely larval organs except 
the swimming test and is found only in aplacophorans 
and protobranch bivalves, as the ancestral type. The 
questions are left here as unresolved and not affecting the 
arguments for homology of early cell fate among Eutro- 
chozoa, although my preference is indicated by use of the 
latter term. 

Classification of Extant Molluscan Classes 

With shell and spicules considered as synapomorphies 
for Conchifera and Aculifera, respectively, the following 
classification of extant Mollusca emerges: 

Phylum Mollusca 

Subphylum Conchifera 
Class Monoplacophora 
Class Bivalvia 
Class Gastropoda 
Class Scaphopoda 
Class Cephalopoda 
Subphylum Aculifera 
Class Polyplacophora 
Class Aplacophora 

Subclass Neomeniomorpha 
Subclass Chaetodermomorpha 

This arrangement is similar to that already proposed 
in the last century with little knowledge of the soft anat- 
omy of Monoplacophora. Garstang (1896) considered the 



76 



A. H. SCHELTEMA 



Aplacophora as "degraded" from an ancestral chiton-like 
form, but although he later stressed the importance of 
paedomorphosis in evolution, he did not see it as per- 
taining to Aplacophora. It is curious that a classification 
based on what are here inferred to be synapomorphies 
and on progenesis should be much the same as classifi- 
cations of a hundred years ago. 

Conclusions 

The hypotheses, arguments, and pieces of evidence 
presented here lead to the conclusions that Mollusca ( 1 ) 
are eucolomates with an ancestry in common with spiral- 
ian trochozoans; (2) are related to Annelida, but not as 
closely as they are to Sipuncula: (3) have a reduced coelom 
which was never segmented; (4) are not directly descended 
from an aplacophoran-like or turbellariomorph prede- 
cessor; and (5) are descended from an ancestor with serial 
replication. 

Acknowledgments 

The idea that aplacophorans may have evolved through 
progenesis originally came from David R. Lindberg. I have 
benefitted from critical discussions with Dave and with 
Carole S. Hickman, Bruce Runnegar, Claus Nielsen, Tom 
Waller, Douglas Eernisse, and Bertil Akesson, all of whom 
also steered me towards relevant literature. Doug Eernisse 
and Bruce Runnegar read an earlier version of this paper 
as well. Thanks are also due to Gerhard Haszprunar, who 
read the manuscript in its present form. Two reviewers 
most helpfully suggested literature that I had overlooked. 
I have tried to keep the phylogeny presented here as 
straightforward as criticisms and discussions suggested, 
and hopefully there is not too much "story telling." My 
gratitude goes to each of my critics. 

Mary E. Rice opened the way for an understanding of 
the Sipuncula-Mollusca relationships presented here, and 
the visit with her at the Smithsonian Marine Station at 
Link Port, Ft. Pierce, Florida, afforded the opportunity 
to work with both living pelagosphera larvae and apla- 
cophorans. I thank her deeply, and for prints of the splen- 
did photographs of pelagosphera. 

I thank Franz P. Fischer for providing me with a copy 
of his photograph of polyplacophoran epidermis, and 
Claus Nielsen for a copy of G. Gustafson's original draw- 
ings of Chaetoderma nitidulum larvae. 

As always. I gratefully acknowledge helpful discussions 
with Rudolf Scheltema, who has provided me space and 
who has always taken an energetic interest in my work. 

The following credits for previously published illustra- 
tions are acknowledged: figures 5A, C, D, 7C, 8A, and 1 1 
from American Malacological Bulletin 6 (1988): 57-68, 
figs. 4, 5, 6, 13; figure 2A, B from American Zoologist 21 



(1981): 605-619, figs. 4, 6; figure 3 from Smithsonian 
Contributions to Zoology 132 ( 1973): pi. 5; figures 6A, D, 
and 8D from Ve liger 34 (1991): 195-203, figs. 2C, 4D, 
and 5C; figure 6E from Zoomorphologie 94 (1980): 
1 2 1 - 1 3 1 , fig. 3; figure 7 A, B from Biological Bulletin 169 
(1985): 484-529, fig. 3L, O, P. 

Note added in proof: Two papers have just been published that have 
direct bearing on the ideas presented here. (1) Bengston S. 1992. The 
cap-shaped Cambrian fossil Maikhanella and the relationship between 
coelosclentophorants and molluscs. Lethaia 25: 401-420. Maikhanella 
is a genus of Lower Cambrian halkienids with a cap-shaped shell formed 
of rows of embedded spicules, which were added by marginal accretion. 
Bengston discusses the possible homology with spicules of extant mollusks 
and with polyplacophoran shells. (2) Eernissee. D. J.. J. S. Albert, and 
F. E. Anderson. 1992. Annelida and Anthropoda are not sister taxa: a 
phylogenetic analysis of spiralian and metazoan morphology. Sysl. Biol. 
41: 331-344. Analysis by maximum parsimony among 141 morpholog- 
ical and embryological characters supports the concept of Eutrochoza. 
including the Mollusca. 



Literature Cited 

Allen, J. A. 1983. The ecology of deep-sea molluscs. Pp. 29-75 in 
The Mollusca. Vol. 6- Ecology. W. D. Russell-Hunter, ed. Academic 
Press, Orlando, FL. 

Anderson, D. T. 1973. Embryology and Phytogeny in Annelids and 
Arthropods. Pergamon Press, Oxford and New York, xiv + 495 pp. 

Andrews, E. B. 1988. Excretory systems of molluscs. Pp. 381-448 in 
The Mollusca. Vol. 11. Form and Function, E. R. Trueman and 
M. R. Clarke, eds. Academic Press, San Diego, CA. 

Baba, K. 1938. The later development of a Solenogastre, Epimenia 
verrucosa (Nierstrasz). / Dept Agric. Kyitsyu Imp. Univ. 6: 21-40. 

Baba, K. 1951. General sketch of the development in a Solenogastre. 
Epimenia verrucosa (Nierstrasz). Misc. Repts. Res. Insl. Nat. Res. 
19-21: 38-46. [In Japanese with English summary'-] 

Baba, K. 1975. Neomenia yamamoloi spec, nov., a gigantic Solenogaster 
(Mollusca: Class Solenogastres. Family Neomeniidae). occurring in 
the north-eastern part of Japan. Publ. Seto Mar. Biol. Lab. 22(5): 
277-284. 

Banta, W. C., and M. E. Rice. 1976. A restudy of the Middle Cambrian 
Burgess Shale fossil worm, Oltoia prolifica. Pp. 79-90 in Proceedings 
of the International Symposium on the Biology oj the Sipuncula and 
Echiura, M. E. Rice and M. Todorovic, eds. Institute for Biological 
Research "SmisStankovic," Belgrade, and Smithsonian Institution, 
Washington, DC. 

Beedham, G. E., and E. R. Trueman. 1968. The cuticle of the Apla- 
cophora and its evolutionary significance in the Mollusca. ,/ Zoo/. 
154: 443-451. 

Butterfield, N. J. 1990. A reassessment of the enigmatic Burgess Shale 

fossil Wiwaxia corrugata (Matthew) and its relationship to the poly- 

chaete Canadia spinosa Walcott. Paleobiology 16: 287-303. 
Christiansen, M. E. 1954. The life-history of Lepidopleurus asellus 

(Spengler) (Placophora). Nytt Mag. Zool. 2: 52-72. 
Clark, R. B. 1964. Dynamics in Metazoan Evolution: The Origin of 

the Coelom and Segments Clarendon Press, Oxford. 313 pp. 
Conway Morris, S. 1985. The Middle Cambrian metazoan \fiwaxia 

corrugata (Matthew) from the Burgess Shale and Ogygopsis Shale. 

British Columbia. Canada. Phil Trans. Roy. Soc. London. B 307: 

507-586. 
Conway Morris, S., and J. S. Peel. 1990. Articulated halkienids from 

the Lower Cambrian of north Greenland. Nature 345: 802-805. 



APLACOPHORA: PROGENETIC COELOMATES 



77 



Curry, G. B., and B. Runnegar. 1990. Partial amino acid sequences of 

hemerythrins from Lmgula and a pnapulid worm, and the evolution 

of oxygen transport in early metazoans. No. 4314, Geol. Soc. Am 

Abstr Dallas, TX. 
Derjugin, K. M. 1915. [Fauna of the Kola gulf and its life conditions.]. 

.Mem Acad Imp Sci. Petrograd (ser. 8)34(1): 1-929, t. 1-14 [Pp. 

499-501 Solenogastres]. [In Russian] 
Derjugin, K. M. 1928. Fauna des Weissen Meeres und ihre Existenz- 

bedingungen. Exp. Mcrs V.R.S.S. fasc. 7-8, 1-511 [Pp. 294-295, 

Solenogastres]. 
Eernisse. D. J. 1988. Reproductive patterns in six species of Lepi- 

dochitona (Mollusca: Polyplacophora) from the Pacific Coast of 

North America. Biol Bull 174: 287-302. 
Eyster, L. S., and M. P. Morse. 1984. Early shell formation during 

molluscan embryogenesis, with new studies on the surf clam, Spisula 

so/idissima. Am. Zoo/ 24: 871-882. 
Field, K. G., G. J. Olsen, D. J. Lane, S. J. Giovannoni, M. T. Ghiselin, 

E. C. Raff, N. R. Pace, and R. A. Raff. 1988. Molecular phylogeny 

of the animal kingdom. Science 239: 748-753. 
Fischer, F. P., W. Maile, and M. Renner. 1980. Die Mantelpapillen 

und Stacheln von Acanthochitonfascicularis L. (Mollusca, Polypla- 
cophora). Zoomorphologie 94: 121-131. 
Freeman, G., and J. \V. Lundelius. 1992. Evolutionary implications 

of the mode of D quadrant specification in coelomates with spiral 

cleavage. ./ Evol Biol 5: 205-247. 
Garstang, VV. 1896. On the aplacophorous Amphineura of the Bntish 

Seas. Proc. Malacol. Soc. London 2: 123-125. 
Gerould, J. H. 1906. The development of Phascolosoma. Studies on 

the embryology of the Sipunculidae I. Zoo/ Jalirh.. Anal. 23: 

77-162. 
Ghiselin. M. T. 1988. The origin of molluscs in the light of molecular 

evidence. Pp. 66-95 in Oxford Surveys in Evolutionary Biology Vol. 

>. P. H. Harvey and L. Partridge, eds. Oxford University Press. Ox- 
ford. 
Gould. S. J. 1977. Ontogeny and Phylogeny. Harvard University Press, 

Cambridge. MA, and London. 
Gustafson, G. 1930. Anatomische Studien uber die Polychaten-familien 

Amphinomidae und Euphrosynidae. Zoo/. Bidr. Uppsala 12: 

305-471. 
Haas, W. 1976. Observations of the shell and mantle of the Placophora. 

Pp. 389-402 in The Mechanisms of Mineralisation in Invertebrates 

and Plants, N. Watabe and K. M. Wilbur, eds. Belle W. Baruch 

Library in Marine Science 5. 
Haas, VV. 1 98 1 . Evolution of calcareous hardparts in primitive molluscs. 

Malacologia 21: 403-418. 
Haas, W ., K. Kriesten, and N. Watabe. 1980. Preliminary note on the 

calcification of the shell plates in chiton larvae (Placophora). Pp. 

67-72 in The Mechanisms of Biomineralization in Animals and 

Plants. M. Omori and N. Watabe, eds. Tokai University Press, 

Tokyo. 
Hammarsten, O. D., and J. Runnstrom. 1925. Zur Embryologie von 

Acanthochiton discrepans Brown. Zoo/. Jahrb. Anal. 47: 261-318. 
Haszprunar, G. 1987. The fine morphology of the osphradial sense 

organs of the Mollusca. IV. Caudofoveata and Solenogastres. Phil 

Trans Roy Soc. London 5315: 63-73. 
Haszprunar, G. 1992. The first molluscs small animals. Boll. Zoo/. 

59: 1-16. 
HeaCh, H. 1899. The development of Ischnochiton. Zoo/. Jahrb.. Anal. 

12: 567-656. 
Heath, H. 1904. The nervous system and subradular organ in two 

genera of Solenogastres. Zoo/. Jahrb . Anal. 20: 399-408. 
Heath, H. 1905. The morphology of a Solenogastre. Zoo/. Jahrb. Anal. 

21: 703-734. 



Heath, H. 1911. The Solenogastres. Reports on the scientific results 
of the expedition to the tropical Pacific . . . by the "Albatross" . . . 
Mem. Mus. Comp Zoo/. (Harvard University) 45(1): 1-179. 

Hessler, R. R., G. D. Wilson, and D. Thistle. 1979. The deep-sea 
isopods: a biogeographic and phylogenetic overview. Sarsia 64: 67- 
75. 

Hoffman, S. 1949. Studien uber das Integument der Solenogastren nebst 
Bemerkungen uber die Verwandtschaft zwischen den Solenogastren 
und Placophoren. Zoo/ Bidr. Uppsala 27: 293-427. 

Hoffmann, H. 1929-30. Aplacophora. Pp. 1-134, 383-453 in Bronns 
Klasscn und Ordnungen des Tier-Reichs. Band III, Abt. I. 

Hyman, L. H. 1967. The Invertebrates. Vol. VI. Mollusca I McGraw- 
Hill, Inc., New York, vii + 792 pp. 

Ivanov, D. L. 1991. [Tentacular region of the central nervous system 
of the Mollusca and the homology of the organs of locomotion.] 
Rulhenica 1: 81-89. [In Russian] 

Ivanova-Kazas, O. M. 1985a. Origin and phylogenetic significance of 
trochophore larvae. Zoo/. Zluir 64(4): 485-497. [In Russian. English 
translation no. 1112119, National Museum of Canada, in manu- 
script.] 

Ivanova-Kazas, O. M. 1985b. Origin and phylogenetic significance of 
trochophore larvae. Zoo/. Zhur. 64(5): 650-660. [In Russian. English 
translation no. 1112120, National Museum of Canada, in manu- 
script.] 

Jagersten, G. 1947. On the structure of the pharynx of the Archian- 
nelida with special reference to there occurring muscle cells of aber- 
rant type. Zoo/ Bull. Uppsala 25: 551-570. 

Jagersten, G. 1963. On the morphology and behaviour of Pelagosphaera 
larvae (Sipunculoidea). Zoo/ Bidr. Uppsala 36: 27-35. 

Jagersten, G. 1972. Evolution of the Metazoan Life Cycle Academic 
Press. London and New York. 

Kaiser, P. 1976. Neomenia herwigi. sp. n.. ein bemerkenswerter Ver- 
treter der Solenogastren (Mollusca, Aculifera) aus Argentimschen 
Schelfgewassern. Mitt llamb Zoo/ Mus Insi 73: 57-63. 

Kerth, K. 1983. Radulaapparat und Radulabildung der Mollusken. II. 
Zahnbildung. Abbau und Radulawachstum. Zoo/. Jahrb.. Anal 110: 
239-269. 

Kniprath. E. 1980. Ontogenetic plate and plate field development in 
two chitons, Middendorffia [= Lepidochitona] and Ischnochiton. 
IVilhelm Roux's Arch. Devel. Biol. 189: 97-106. 

Kowalevsky, M. A. 1883. Embryogeme du Chiton polii Phil., avec 
quelques remarques sur le developpement des autres Chitons. Ann. 
Mus. Hist. Nat. Marseille 1: 1-46. 

Lake, J. A. 1990. Origin of the Metazoa. Proc. Nat. Acad. Sci. USA 
87: 763-766. 

Lemche, H., and K. G. Wingstrand. 1959. The anatomy of Neopilina 
galatheae Lemche, 1957 (Mollusca Tryblidiacea). Galathea Kept. 
3:9-71. 

McKinney, M.L., and K. L. McNamara. 1991. Heterochrony: the Evo- 
lution of Ontogeny. Plenum Press, New York and London, xi.x 
+ 437 pp. 

Monniot, C., and F. Monniot. 1978. Recent work on the deep-sea tu- 
nicates. Oceanogr Mar. Biol. Ann. Rev 16: 181-228. 

Moor, B. 1983. Organogenesis. Pp. 123-177 in The Mollusca. Vol. 3. 
Development. N. H. Verdonk, J. A. M. van den Biggelaar, and A. 
S. Tompa, eds. Academic Press, New York. 

Newby, W. W. 1940. The embryology of the echiuroid worm Vreehis 
caupo. Mem. Am. Philos. Soc. 16: 1-213. 

Nielsen, C., and A. Narrevang. 1985. The trochaea theory: an example 
of life cycle phylogeny. Pp. 28-41 in The Origins and Relationships 
of Lower Invertebrates. Syst. Assoc. Spec. Vol. No. 28. S. Conway 
Morris, J. D. George, R. Gibson, and H. M. Platt. eds. Clarendon 
Press, Oxford. 



78 



A. H. SCHELTEMA 



Plate, L. H. 1898. Die Anatomic und Phylogenie der Chitonen. Zool. 
Jahrb.. Suppl IV (Fauna Cln/ensis I): 1-243. 

Raff, R. A., and T. C. Kaufman. 1983. Embryos, Genes, and Evolution. 
Macmillan Publishing Co., Inc., New York & Collier Macmillan 
Publishers, London. 

Raven, C. P. 1966. Morphogenesis: The Analysis of Mottuscan Devel- 
opment. 2nd ed. Pergamon Press, Oxford etc. xiii + 365 pp. 

Reynolds, P. I)., and M. P. Morse. 1991. Morphological evidence for 
ultrafiltration of blood in the Aplacophora. Amer Zool 31: 137A. 

Rice, M. K. 1969. Possible bonng structures of Sipunculids. Amer 
/.ool 9: 803-812. 

Rice, M. E. 1973. Morphology, behavior, and histogenesis of the pe- 
lagosphera larva of Phascolosoma agassizii (Sipuncula). Smithsonian 
Contr. Zool. 132: 51 pp. 

Rice, M. E. 1975. Sipuncula. Pp. 67-127 in Reproduction of Marine 
Inver/ehrali's. \'ol. 2. Enloprocls and lesser coeloma/es, A. Giese, 
and J. Pearse, eds. Academic Press, New York. 

Rice, M. E. 1981. Larvae adrift: Patterns and problems in life histories 
of sipunculans. Am. Zool. 21: 605-619. 

Rice, M. E. 1985. Sipuncula: developmental evidence for phylogenetic 
inference. Pp. 274-296 in The Origins and Relationships of Lower 
Invertebrates, S. Conway Morns, J. D. George, R. Gibson, and 
H. M. Platt, eds. Oxford University Press, Oxford. 

Runnegar, B., and J. Pojeta, Jr. 1985. Origin and diversification of the 
Mollusca. Pp. 1-57 in The Mollusca. I '<>!. 10, Evolution. E. R. True- 
man, and M. R. Clarke, eds. Academic Press, Orlando, FL. 

Salvini-Plawen, L. v. 1968a. Die 'Funktions-Coelom Theorie' in der 
Evolution der Mollusken. Sysl. Zool. 17: 192-208. 

Salvini-Plawen, L. v. 1968b. Uber Lebendbeobachtungen an Caudo- 
foveata (Mollusca. Aculifera), nebst Bemerkungen zum System der 
Klasse. Sarsia 31: 105-126. 

Salvini-Plawen, L. v. 1972. Zur Morphologic und Phylogenie der Mol- 
lusken: Die Beziehungen der Caudofoveata und der Solcnogastres 
als Aculifera, als Mollusca und als Spiralia. Z. wiss. Zool. 184: 
205-394. 

Salvini-Plavven, L. v. 1978. Antarktische und subantarktische Solen- 
ogastres (eine Monographic: 1898-1974). Zoohgica 44: 1-315. 

Salvini-Plawen, L. v. I981a. On the origin and evolution of the Mol- 
lusca. Alt Convegni Lincei 49: 235-293. 

Salvini-Plawen, L. v. 1981 b. The molluscan digestive system in evo- 
lution. Malacologia 21: 371-401. 

Salvini-Plawen, L. v. 1985. Early evolution and the primitive groups. 
Pp. 59-150 in The Mollusca. Vol. 10. Evolution. E. R. Trueman, 
and M. R. Clarke, eds. Academic Press, Orlando, FL. 

Salvini-Plawen, L. v. 1990. Origin, phylogeny and classification of the 
phylum Mollusca. Ibents9: 1-33. 

Sanders, H. L., and J. A. Allen. 1973. Studies on deep-sea Protobran- 
chia (Bivalvia); Prologue and the Pristiglomidae. Bull. Mus. Comp. 
Zool. 145: 237-262. 

Scheltema, A. H. 1972. The radula of the Chaetodermatidae (Mollusca. 
Aplacophora). Z Morph. Tiere72: 361-370. 

Scheltema, A. H. 1973. Heart, pericardium, coelomoduct openings, 
and juvenile gonad in Chaetoderma nitidulum and Falcidens cau- 
dalus (Mollusca, Aplacophora). Z. Morph. Ticreld: 97-107. 

Scheltema, A. H. 1978. Position of the class Aplacophora in the phylum 
Mollusca. Malacologia 17: 99-109. 

Scheltema, A. II. 1981. Comparative morphology of the radula and 
alimentary tracts in the Aplacophora. Malacologia 20: 361-383. 

Scheltema, A. H. 198?. The aplacophoran family Prochaetodermatidac 
in the North American Basin, including Chevroderma n.g. and Spa- 
ihoderma n.g. (Mollusca; Chaetodermomorpha). Biol. Bull 169: 
484-529. 

Scheltema, A. H. 1987. Reproduction and rapid growth in a deep-sea 
aplacophoran mollusc, Prochaetoderma rongei. Mar. Eeol. Progr. 
Set: 37: 171-180. 



Scheltema, A. H. 1988. Ancestors and descendents: relationships of 
the Aplacophora and Polyplacophora. Amer. Malacol. Bull 6: 
57-68. 

Scheltema, A. H. 1990. Aplacophora as a Tethyan slope taxon: evidence 
from the Pacific. Bull. Mai: Sci. 47: 50-61. 

Scheltema, A. H., K. Kerth, and A. M. Kn/irian. 1989. The primitive 
molluscan radula. P. 220 in L'nitas Malacologicu Iff 1 ' International 
Congress, Tubingen. [Abstract] 

Scheltema, A. H., and A. M. ku/irian. 1991. Helieoradomema juani 
n.g. n. sp.. a Pacific hydrothermal vent Aplacophora (Mollusca, 
Neomeniomorpha). I'eliger34: 195-203. 

Scheltema, A. H., M. Tscherkassky, and A. M. Kuzirian. (In 
press). Aplacophora. In Microscopic Anatomy 01 'Invertebrates. \'ol. 
5. Mollusca: Monoplacophora, Aplacophora. Polyplacophora, and 
Gastropoda. F. W. Harrison and A. J. Kohn. eds. Wiley-Liss, New- 
York. 

Soetaert, K., and C. Heip. 1989. The size structure of nematode as- 
semblages along a Mediterranean deep-sea transect. Deep-Sea Res 
36:93-102. 

Spengel, J. \V. 1881. Die Geruchsorgane und das Nervensystem der 
Mollusken. Z wiss. Zool. 35: 333-383. 

Tendal, O. S. 1985. Xenophyophores (Protozoa, Sarcodina) in the diet 
of Neopilma galatheae (Mollusca. Monoplacophora). Galalhea Repi 
16: 95-98. 

Thiele, J. 1902. Die systematische Stellung der Solenogastren und die 
Phylogenie der Mollusken. Z wiss. Zool. 12: 249-466. 

Tscherkassky, M. 1989. Pedal shield or oral shield in Caudofoveata 
(Mollusca. Aculifera)? P. 254 in Unilas Malacologica, Abstracts of 
l(f h International Malacological Congress. Tubingen. [Abstract] 

Turbeville, J. M. 1986. An ultrastructural analysis of coelomogenesis 
in the hoplonemertine Prosorhochmus americanus and the poly- 
chaete Magelona sp. / Morphol. 187: 51-60. 

Turbeville, J. M., K. G. Field, and R. A. Raff. 1992. Phylogenetic po- 
sition of phylum Nemertini, inferred from 18S rRNA sequences: 
molecular data as a test of morphological character homology. Mol. 
Biol. Evol. 9: 235-249. 

van Dongen, C. A. M., and W. L. M. Geilenkirchen. 1974. The de- 
velopment of Denlalium with special reference to the significance 
of the polar lobe. I. II and III. Division chronology and development 
of the cell pattern in Dentalium </tvtfa/c(Scaphopoda). Proc. A'. Ned- 
erl. Akad. Helens., ser. C. 77: 57-100. 

Verdonk, N. H., and J. A. M. van den Biggelaar. 1983. Early devel- 
opment and the formation of the germ layers. Pp. 91-122 in The 
Mollusca. \'ol. 3. Development. N. H. Verdonk. J. A. M. van den 
Biggelaar, and A. S. Tompa, eds. Academic Press, New York. 

Walters, G. T. 1991. LUilization of a simple morphospace by polypla- 
cophorans and its evolutionary implications. Malacologia 33: 
221-240. 

\Vestheide, VV. 1987. Progenesis as a principle in meiofauna evolution. 
/ Nat. Ilia. 21: 843-854. 

\\ettstein, E. 1904. Anatomic von Cryptoplax. Jenaische Z. Naturwiss. 
38: 473-504. 

\\hittington, H. B. 1985. The Burgess Shale. Yale University Press, 
New Haven and London, xv + 151 pp. 

Willmer, P. G., and P. \V. H. Holland. 1991. Modern approaches to 
metazoan relationships. J. Zoo/., London. 224: 689-694. 

Wilson, E. B. 1892. The cell-lineage of Nereis. / Morphol 6: 
361-480. 

Wingstrand, K. G. 1985. On the anatomy and relationships of Recent 
Monoplacophora. Galalhea Rep/. 16: 7-94. 

Wissel, C. v. 1904. Pacifische Chitonen. Zool. Jahrb. Syst. 20: 
591-676. 



Reference: Biol. Bull 184: 74-86. (February. 1993) 



A Colonial Invertebrate Species that Displays a 
Hierarchy of Allorecognition Responses 

B. RINKEVICH 1 . Y. SAITO : . AND I. L. WE1SSMAN' 

^Marine Biology Department, Israel Oeeanographic & Limnological Research. Tel-Shikmona, 
P.O.B. 8030, Haifa 31080, Israel, 2 Shimoda Marine Research Center, University of Tsukuba, 

Shimoda 5-10-1. S/ii:iioka 415, Japan, and ^Howard Hughes Medical Institute. 
Stanford University Medical Center. B-257. Beckman Center. Stanford, California 94305 



Abstract. When two colonies of the compound ascidian 
Botryllus schlosseri come into contact with each other, 
they either fuse or reject. This allorecognition is governed 
genetically by multiple, codominantly expressed alleles at 
a single, highly polymorphic haplotype called the fusibil- 
ity/histocompatibility (Fu/HC) locus. Two colonies shar- 
ing one or both alleles at this locus can fuse via their 
extracorporeal tunic blood vessels. Thereafter, in labo- 
ratory studies, one partner in the chimera is usually re- 
sorbed. The direction of resorption appears to be inherited, 
as multiple subclones of asexually-derived individuals 
from colony A always resorb paired subclones from colony 
B, independent of laboratory conditions or colony age. 

We established 121 pairs of chimeric partners by fusions 
of relatives from four generations within a pedigree, all 
homozygotes (AA line) at their Fu/HC haplotype. This 
was carried out by self- and defined-crosses done in the 
laboratory on two outbred founder colonies (each AB at 
the fusibility locus) which were taken from the field. We 
found that the resorption phenomenon is characterized 
by a linear hierarchy within each generation of colonies, 
which is expressed by the existence of at least 5 inter- 
mediate groups. However, the time for resorption did not 
correlate with the position in the hierarchy. Analysis of 
resorption hierarchies between different generations re- 
vealed that mother colonies always resorbed their self 
crossed offspring. More interesting, colonies low in the 
hierarchy within a specific generation reproducibly re- 
sorbed the self crossed offspring of a superior kin. Chi- 
meras between defined-crossed offspring of different gen- 
erations revealed nontransitive types of hierarchies which 
were correlated with the relative position of each colony 



Received 24 April 1992; accepted 14 September 1992. 



in the linear hierarchy established for the colonies within 
each generation. We propose that colony resorption in 
colonial botryllid ascidians is controlled by several allo- 
recognition elements that determine a resorption hi- 
erarchy. 

Introduction 

The compound ascidian Botryllus schlosseri is a cos- 
mopolitan metazoan of the subfamily Botryllinae, inhab- 
iting shallow waters abundantly throughout the world, 
especially in harbors. Adults are made of several to 
hundreds of genetically identical units (each one is called 
a zooid), which are grouped in typical star-shape structures 
(systems), and are embedded within a translucent, gelat- 
inous matrix, the tunic. All systems, as well as zooids 
within a single system, are interconnected to each other 
by a network of blood vessels, which bear spherical to 
elongate termini (called ampullae) near the surface of the 
tunic, between the systems and around the borders of the 
colony. 

Colonies originate from a sexually produced tadpole 
larva. After a short free-swimming phase, the larva at- 
taches to the substrate, resorbs its tail components, and 
undergoes metamorphosis to a founder individual, the 
oozooid. Oozooids grow by a typical asexual budding 
(blastogenesis), a cyclic phenomenon: i.e., every six to 
seven days all parental zooids in a colony are synchro- 
nously resorbed and a new generation of buds matures 
to the zooid stage. Each adult zooid can give rise to one 
to four buds per generation (Boyd et a/., 1986). At the 
end of each blastogenic cycle, all of the zooids of one 
generation are resorbed. This event, called "takeover", is 
characterized by a massive phagocytosis, and is completed 
within 24 h (Harp et at., 1988). During takeover, some 



79 



80 



B. RINKEV1CH ET AL 



buds can be resorbed together with their parent, and thus 
the total number of zooids in a colony can either increase, 
remain constant, or decrease. 

Allorecognition by complex metazoans can result in a 
variety of manifestations of histoincompatibility. The 
most detailed information concerning the genes that en- 
code histocompatibility determinants, and the cells and 
receptors that recognize these determinants, comes from 
studies of mouse and man. In these species, as in all ver- 
tebrates tested, a single, highly polymorphic haplotype of 
linked histocompatibility genes called the major histo- 
compatibility complex (MHC) is the primary determi- 
nant of rapid graft rejection mediated by allospecific T 
lymphocytes (Klein, 1986). However, when grafts are ex- 
changed between individuals that share both MHC alleles. 
but are otherwise genetically distinct, a T cell-mediated 
graft rejection occurs, albeit at a slower pace than when 
MHC mismatched grafts are applied (Bevan. 1975; Love- 
land and Simpson, 1986). The genes encoding these de- 
terminants almost certainly proteolytically derived 
peptides that are embedded in an MHC protein cleft 
(Bjorkman et ill.. 1987) and thereby presented to MHC- 
restricted, allospecific T cells are called minor histo- 
compatibility (H) antigens. Genetic studies indicate that, 
with any two distinct mouse strains, the combinatorial 
association of minor H antigenic peptides with highly 
polymorphic MHC genes results in the elaboration of tens 
of alloantigens, encoded by genes residing on all (or nearly 
all) chromosomes (Bailey, 1978: Johnson, 1981;Zaleski 
eta/.. 1983; Klein, 1986). 

Allorecognition is also genetically denned in botryllid 
ascidians, as manifested by several distinct phenomena. 
Colonies that meet naturally in the wild or under labo- 
ratory conditions, very soon after initial contact, either 
fuse their adjacent extracorporeal tunic blood vessels to 
form a natural vascular parabiont (cytomictical chimera), 
or undergo a rapid series of inflammatory phenomena 
culminating in rejection, and the formation of a fibrous 
barrier between them (Scofield el a/.. 1982; Taneda et al.. 
1985; and literature therein). This colony specificity phe- 
nomenon is determined by a single, highly polymorphic 
fusibility/histocompatibility (Fu/HC) locus (or haplotype). 
In laboratory experiments, if two individuals sharing a 
single allele (or both alleles) at this locus fuse at some later 
time, the genetic colonial descendants (zooids) from one 
partner in the chimera are all resorbed by massive phago- 
cytosis, leaving the genetic descendants of the other colony 
intact (Rinkevich and Weissman, 1987a, b, 1989, 1992; 
Weissman et al.. 1990). This phenomenon, called colony 
resorption, typically occurs at the end of a blastogenic 
cycle, when the new generation of zooids fails to develop 
to the mature phase, or does not develop at all (Rinkevich 
and Weissman, 1987a). Moreover, colony resorption also 
appears to be controlled genetically, insofar as all sub- 
clones from colony A will resorb all subclones from colony 



B, whatever the laboratory microenvironment in which 
the subclones are reared. The microenvironmental vari- 
ables tested include different temperature regimens, types 
of food, running versus standing seawater systems, and 
the number of asexual generations separating the founder 
of the colony and the particular subclone tested (Taneda 
el al.. 1985; Rinkevich and Weissman, 1987a, b; 1989, 
1992). 

Here we provide evidence that the resorption of non- 
identical partners in a chimera of the colonial tunicate 
Botryllus schlosseri, from Monterey, California, is at least 
partly controlled by additional recognition elements un- 
linked to the Fu/HC haplotype of this species. These col- 
ony resorption responses have a hierarchial property in 
that dominant, intermediate, and inferior responses are 
maintained through many asexual cycles, independent of 
environment. 



Materials and Methods 



Animals 



Botryllus schlosseri colonies were kept in 1 7 1 glass tanks 
supplied with 50-70 ml/min of filtered seawater that had 
been preconditioned in a large plastic holding tank con- 
taining 235 1. The water in each glass tank was aerated 
with an airstone and maintained at 18C (with a 50 W 
aquarium heater). The animals were fed daily with 0.55 
gr/tank of powdered Similac (a milk substitute) and sub- 
jected to a 14: 10 hour light:dark regimen. Colonies were 
grown on 5 X 7.5 cm glass slides, one colony per slide, 
and kept vertically in slots of glass staining racks, within 
the glass tanks. 

Colony allorecognition assays (CAAs) 

Only large, healthy colonies were used. Small pieces of 
growing edges (subclones. ramets; containing one to three 
systems each) were isolated by careful dissection from each 
colony without injuring their surrounding ampullae. 
Subclones from two genetically distinct colonies were 
paired on glass slides, so that they contacted one another 
with their extended ampullae. They were fastened to the 
slides by placing them in a moisture chamber for 30-45 
min before transferring them into the 17 1 running sea- 
water tanks. All of the paired subclones were in the range 
of size differences which does not affect directionality in 
the resorption phenomenon (Rinkevich and Weissman, 
1987a) and were observed under the binocular stereo- 
microscope every day until they formed a well-organized 
chimera (Rinkevich and Weissman, 1987a, 1988, 1989). 
Thereafter they were observed 2 to 3 times a week. During 
the observations, colonies were cleaned with soft, small 
brushes to remove debris, fouling organisms, and trapped 
food particles. The substrate around the colonies was 
carefully cleaned with small pieces of razor blade. 



ALLORECOGNITION RESPONSES IN COLONIAL INVERTEBRATE 



SI 



Experimental procedures 

To analyze further the possible role of heritable ele- 
ments in colony resorption other than the Fu/HC locus, 
we carried out self and defined crosses from one of the 
Fu/HC homozygotic strains that are raised in our labo- 
ratory (Boyd el ai. 1986), the Monterey A A haplotype. 
The genealogical tree relevant to the present study is il- 
lustrated in Figure 1, which shows the pedigree of four 
successive generations. 

Two outbred colonies, each AB at the fusibility locus, 
were taken from the Monterey marina and served as the 
founders of this strain. The fusibility of each offspring was 
determined through CAAs by removing subclones from 
the main colony and placing them with subclones from 
other colonies (Rinkevich and Weissman, 1988). The 
present study focuses on the AA strain. Five healthy col- 
onies of the AA strain served as parent colonies for the 
next generation offspring by self-crosses and defined- 
crosses. Self-crosses result from the fertilization of the eggs 
of one subclone (ramet) from a specific colony with the 
sperm from another ramet from the same colony, in the 
absence of competing sperm. The fastest growing and 
healthiest offspring colonies were either used in the ex- 
periments, with subclones being taken for fusibility assays 
(Rinkevich and Weissman, 1988), or they were used to 
produce the next generation of the AA line. Subclones 
from the indicated colonies were isolated, placed side-by- 
side on colony fusion plates, as described previously, and 
observed closely to record colony resorption (Rinkevich 
and Weissman, 1987a). 



Results 

Colonv resorption hierarchies within different 
generations 

Thirteen chimeras were derived by fusion between the 
four surviving AA colonies of generation II (Fig. 2a). In 
five cases (38.5%), the partners within the chimeras dis- 
connected before resorption was complete (average time 
for disconnection 64 21 days). Colony PI 1 1R was in- 
volved in four of these cases. 

The resorption between this group of colonies (average 
time for resorption 48 23 days) is characterized by a 
linear hierarchy. In this hierarchy, colony P21R is the 
"superior" partner, in that in the absence of dissociation, 
it resorbs all other colonies of this generation. In contrast, 
colony P94R is the "inferior" partner, as it is resorbed by 
the other three members of this generation (Fig. 2a). The 
time for resorption does not correlate with position in the 
hierarchy; subclones from the inferior P94R colony were 
resorbed by subclones from the superior P2 1 R colony at 
the same, or even a slower pace than between the sub- 
clones of the two intermediate members (P32R and 
PI 1 1R; Fig. 2a). During the phase of chimerism, from 
the day of fusion up to the day of complete resorption, 
the superior partners increased in zooid numbers by asex- 
ual budding by 35-300%. 

Eleven chimeras were derived by fusion between the 
self-crossed offspring of generation III themselves, the off- 
spring of PI 1 1R and P21R (Fig. 1 and the two diagrams 
on the left of Fig. 2b). Out of 1 1 cases, one chimera died 
and one disconnected. Two hierarchies emerged when the 
resoiption patterns were observed. Another hierarchy was 



Type of 
colony 

outbred 



inbred 



mbred 



inbred 



Generation 
no 



II 



m 



TV 



Fusibility 
locus 

AB *AB 



all AA 



oil AA 



all AA 




Figure 1. The pedigree of four successive generations of Monterey Bmryllits xcliloxxeri used in this study. 
Two independent outbred colonies, typed as Fu/HC AB, were designated generation I and were mated to 
give rise to generation II of Fu/HC AA colonies. Colonies of generations III and IV (all AA on the Fu/HC 
haplotype) are designated in running numbers with a preface letter which denotes the type of crossing: d 
= denned crossed colony, s = self-crossed colony. Heavy lines represent the pedigree of self-crossed colonies. 



82 



B RINK.EV1CH ET AL 



established by analyzing the outcome of 20 CAAs carried 
out between the defined-cross offspring of generation IV 
(right diagram. Fig. 2b). In this set. three chimeras dis- 
connected and one died. The average time for resorption 
between self-crossed offspring (20 18 days) was signif- 
icantly shorter than the average time for resorption be- 
tween defined-cross offspring (69 43 days; P < 0.001, t 
test). As before (Fig. 2a). a linear hierarchy emerged from 
the analyses of the interactions within generations III and 
IV (Fig. 2b), and there was no correlation between the 
time to resorption and level in the hierarchy. The results 
illustrated in Fig. 2b also indicate the existence of at least 
five intermediate levels in the resorption hierarchy. 

Colony resorption hierarchies between different 
generations 

Fifty chimeras were generated between colonies of gen- 
eration II and their self-crossed offspring of generation III. 
by assaying pairs of similar-sized ramets between parents 
vs. its own offspring, and pairs of generation II colonies 
vs. offspring of a kin colony (Fig. 2c). Death of the chimera 
or a disconnection was recorded in eight (16%) of the 
cases. In the other 42 cases, no matter how large the col- 
onies were at the time of fusion, generation II ramets re- 
sorb generation III ramets (average time 42.4 25.9 days). 
This result was obtained either when parent-offspring chi- 
meras or chimeras of a generation II colony vs. self-crossed 
offspring from a kin were done. Most interestingly, gen- 
eration II inferior colonies in the resorption hierarchy re- 
producibly resorbed the self-crossed offspring of a superior 
kin, such as the cases of P94R, PI 1 1R and P32R vs. off- 
spring of P2 1 R (Fig. 2c). In addition, similar to the cases 
shown in Fig. 2b, a resorption hierarchy is also found be- 
tween the self-crossed offspring of colony P94R (Fig. 2c). 

Twenty-seven chimeras were established between col- 
onies of generation II and the defined-cross offspring of 
generation IV (Fig. 2d); 12 of them (44.4%) died or dis- 
connected. The average time to a complete resorption in 
the other 15 chimeras was 56.1 26.8 days. In this set of 
experiments, five of the IVth generation colonies resorbed 
and two (marked by dashed arrows with arrowheads; Fig. 
2d) started to resorb colonies of the Ilnd generation, while 
in 10 cases, generation II ramets resorbed generation IV 
ramets (Fig. 2d). A closer examination reveals that colony 
d20 of generation IV is superior in the hierarchy of re- 
sorption to all four generation II colonies (Fig. 2d). This 
colony was found to be the superior colony within gen- 
eration IV offspring as well (Fig. 2b). Colony d!5 is the 
most inferior colony in generation IV colonies (Fig. 2b) 
and is resorbed by generation II colonies as well (Fig. 2d). 
Colony P94R (the inferior colony of generation II. Fig. 
2a) was resorbed in all cases where a successful chimera 
was followed with generation IV colonies (Fig. 2d), 
whereas colony P21R (the superior colony of generation 



II, Fig. 2a) was inferior in the resorption hierarchy only 
to colony d20 of generation IV colonies (Fig. 2d). 

Discussion 

The colony resorption phenomenon is limited to in- 
dividuals that are not genetically identical, since two ge- 
netically identical isolates from a single parent colony will 
meet, fuse, and give rise by asexual budding to growing 
colonies (Rinkevich and Weissman, 1987a). The studies 
of tunicate colony resorption reported here and previously 
(Rinkevich and Weissman, 1987a, b. 1989. 1990. 1992; 
Weissman el ai, 1990) reveal a unique hierarchical or- 
ganization in Bolrylhis schlosseri chimeras. Fusion in the 
laboratory between two colonies that are Fu/HC homo- 
zygotes (i.e.. A A vs. A A), but that are not genetically iden- 
tical or Fu/HC heterozygotes (i.e., any combination of 
AX vs. AY), leads to colony resorption. All ramets from 
a superior colony will resorb fused ramets of an inferior 
colony, implying that other resorption elements, most 
likely encoded at other genetic loci, are responsible. Be- 
cause the mother colony ramets usually resorb ramets 
from their more inbred progeny ramets, a simple hierarchy 
is difficult to explain. Perhaps heterozygotes at these loci 
are more likely to resorb homozygotes; or perhaps the 
general "fitness" of progeny of a self-cross allows a weaker 
resorption locus to emerge superior in colony resorption. 
The second suggestion is much less plausible, since the 
"performance" of the studied self-crossed homozygotes 
(either in survivorship, reproductive outputs, or growth 
rates) in our laboratory conditions (Boyd el a/., 1986) was 
as good if not better than that of the control, more het- 
erozygotic colonies (Ishizuka and Rinkevich, in prep.). 

If the above view is correct, then there may be positive 
selection for tunicates heterozygous for several allorecog- 
nition loci. Allorecognition in colonial tunicates therefore 
represents a histocompatibility system of considerable ge- 
netic sophistication and diversity, rivalling the MHC and 
minor histocompatibility loci in vertebrates, such as the 
mouse (Eichwald el a/.. 1958: Eichwald and Weissman, 
1966; Graff et ai. 1966; Lappe el ai. 1969; Graff, 1978; 
Klein, 1986; Townsend et ai. 1986; Weissman, 1988). 
These genes provide means by which each individual is 
likely to be unique in terms of histocompatibility. Whether 
this elaborate system of histocompatibility and allorecog- 
nition in colonial tunicates and vertebrate histocompat- 
ibility was derived from the same ancestral genes, or 
whether the similarities are merely semantic, remains to 
be determined. 

The strength of the chimerism-resorption system, as 
defined by the period needed for a complete resorption, 
is extremely variable, from one week to five months (Fig. 
2). At least part of the variability in the time for resorption 
may have been caused by experimental manipulations 
(such as the length and the structure of the fusion areas. 



ALLORECOGNITION RESPONSES IN COLONIAL INVERTEBRATE 



83 



the numbers of anastomizing blood vessels, etc.; Rink- 
evich and Weissman, 1989). rather than by genetic factors. 
A similar characteristic of variability is also found in the 
murine minor histocompatibility loci (Klein. 1986). where 
there appear to be at least 50-100 distinct histocompat- 
ibility loci, with histocompatibility genes scattered 
throughout virtually every chromosome. 

That the diversity of genetic types in the MHC of the 
vertebrates is the result of past selection for resistance to 
different diseases is a persistent speculation (Black and 
Salzano, 1981; Robertson, 1982; Hedrick and Thomson, 
1983). This suggests that an animal heterozygous for the 
MHC antigens may respond much more efficiently to a 
wider range of pathogens than a homozygote, and that 
polymorphism may be maintained by heterozygous ad- 
vantage or heterosis. In contrast. Flaherty ( 1 988) has pro- 
posed that the high degree of mammalian MHC poly- 
morphism has been established and maintained because 
of a constant, but promiscuous, heterozygote advantage. 
That is, no particular MHC allele has selective advantage; 
rather, all heterozygotes are favored over all homozygotes. 
This promiscuous heterozygote advantage would lead to 
a large allelic pool, because rare alleles would be favored 
and lead to more heterozygotes in the population. Other 
authors have proposed previously that heterozygote ad- 
vantage might be involved in mammalian MHC evolution 
(Gallon, 1967; Robertson, 1982; Hughes and Nei, 1988). 
In general, heterosis refers to allelic combinations in 
which a heterozygote (i.e.. AB) has greater fitness than 
either of its homozygotes (AA or BB) (reviewed in Gros- 
berg, 1988). We therefore postulate that the phenomena 
effusion and chimeric resorption of Fu/HC compatible 
botryllid ascidians may provide substantial fitness benefits. 
If the dominant resorption of offspring settling near, and 
fusing with, maternal colonies (Rinkevich and Weissman, 
1987b) is due to heterozygote advantage, then chimeric 
resorption linked to chromosomally dispersed "resorp- 
tion" loci may serve to promote chromosomal hetero- 
geneity, and therefore may provide substantial fitness 
benefits. Indeed, Grosberg and Quinn (1986) have shown 
in field experiments that sibling larvae of B. schlosseri 
settle non-randomly in aggregations; siblings that cosettle 
in these clusters share at least one Fu/HC allele, which 
should lead to the formation of more chimeras, and the 
resorption of a significant part of the Botryllus population. 
If colony resorption occurs in nature, the survivors would 
not only be at the top of the resorption hierarchy, but 
also are more likely to be heterozygotic at the resorption 
loci; thus colony resorption could contribute to heterosis 
benefits. 

Four classes of benefits: genetic variability, develop- 
mental synergism, mate location, and size-specific eco- 
logical processes, have been attributed to the (.himeric 
state (Buss, 1982). Despite these proposed benefits and 
those that heterosis might engender, however, there are 



potential costs to fusion, as well as mechanisms that could 
prevent this advantage from being passed on. The result 
of mixing genetically distant cell lines could result in germ- 
cell or somatic-cell parasitism when one member of the 
chimera could parasitize the other (Buss, 1982; Rinkevich 
and Weissman. 1987c). It has been reported (Sabbadin 
andZaniolo. 1979; Rinkevich and Weissman, 1987c)that 
short-term chimeras of Botryllus colonies result in free 
exchange of germ cells, and that one individual in the 
chimera may gain a disproportionate share of gametic 
output even after the separation between both members 
in the chimera (Sabbadin and Zaniolo, 1979). 

Thus, while the "heterosis" concept favors fusion as a 
pathway for selection against the less vigorous partner 
(including its soma and the germ line), the "somatic cell 
parasitism" concept, if reproducible and functional in na- 
ture, could lead to the survival of blood cells (especially 
the totipotent stem cells) from the resorbed partner, in 
effect cancelling the advantages gained by heterozygote- 
dominated resorption. In chimeras, therefore, several 
contradicting processes might play a role in colony sur- 
vival until complete resorption occurs. However, suc- 
cessful domination of a feeding surface by chimeras should 
effectively prevent colonization of that surface by other 
competitor species. Whether the resorption "winner" or 
"loser" gives rise to the germ line that will successfully 
give rise to offspring is a critical evolutionary issue. Clearly, 
much more effort will be needed to elucidate the processes 
occurring within Botryllus chimeras in the field. 

The present and previous studies on tunicate resorption 
(Rinkevich and Weissman. 1987a, b, 1989, 1990, 1992) 
used non-inbred Botryllus colonies. In order to study the 
individual histocompatibility gene and proteins of the 
mouse MHC, it was necessary to deal with the problems 
of multiple histocompatibility loci, extensive H-2 poly- 
morphism and heterozygosity of the H-2 genes. These 
obstacles were circumvented by the development of three 
special types of mouse strains: inbred, congenic, and re- 
combinant congenic, which is also the reason why we 
know the murine histocompatibility system better than 
in any other vertebrate. Studies on the colonial tunicate 
Botryllus schlosseri have revealed the Fu/HC system 
which resembles in some ways the vertebrate MHC (Sco- 
field et a!.. 1982), and a multilevel hierarchial organization 
of histocompatibility alleles which lead to the resorption 
of partners within chimeras (this study). The genetic 
structure of the protochordate histocompatibility system 
is only now being slowly revealed. Therefore, the analysis 
of histocompatibility pathways in Botryllus inbred lines 
may elucidate the sophisticated immunological systems 
of both protochordates and vertebrates, and their evolu- 
tion. 

Acknowledgments 

We thank R. Lauzon for critically reading the manu- 
script, K. Ishizuka and K. Palmeri for their endless care 



84 



B RINK.EV1CH ET AL 



58 (062) '223% 



13 (1.23)- 77% 






^! p 1 14(0 ~>3)'3C> /o i _ ] 45(138)*- 3uu% JTTT 






1 ' 62 (081)* 63% " ' i D47( 17 VS 10) " - 
065(14 VS 8) 

53(061)* 194% 
041 (38VS 27) 






073 16 VS 13) 
68 (073)*233/ 







093 ( 16 VS 13) 







L-l 


2O 




r- 






CM 






fi 






9 






CM 






3 








f 

8 




=) d 100 






_*_ 


j 









CO 


c 


f 






~ 


_i 


ie 









K-) 
CO 











O 


ID 












-| d 51 










g 


2 


f 








T 


i/i 

> 











S 


OJ 











QD 


31 











CO, 

_J 


r 

1 


~ 










^ d 16 


l 






j5 






CD 


i* 









6 


i 


P 






in 
o 


O 


Q 









c g 


J-T 3 


49~1 






o 


5?^ 








o 


-i si 








5 


Co 9 










CD 










^ d 


5] 



Figure 2. a. A hierarchy in the resorption between tour eolonies of generation II (refer to Fig. 1). The 
arrowheads point to the inferior partner. Numbers printed along the arrows refer to, respectively: days for 
complete resorption, zooid ratio (in parentheses, calculated as: the number of zooids in the inferior/superior 
partners on the day of fusion), percent increase or decrease of zooids in the superior partner from the day 
of fusion until a complete resorption of the inferior partner. The letter D refers to a case where a disconnection 
between the partners in a specific chimera occurs. In that case, the numbers along the arrow indicate: days 
from fusion to disconnection, number of zooids of the left colony on the day effusion vs. the number of 
zooids of the right colony (in parentheses). Disconnection between the partners within a Botryllus chimera 
is one of the variations in the outcome to chimera formation, resulting from unsuccessful fusion (Rinkevich 
and Weissman, 1989), reciprocal resorption (Rinkevich and Weissman, 1987a, 1989), or from a retreat 
growth phenomenon (Rinkevich and Weissman, 1988). These physiological-genetic-morphological parameters 
may lead to early separation between the partners before a complete resorption of the inferior partners in a 
chimera is obtained (Rinkevich and Weissman, 1988, 1989). The hierarchial tendency in the resorption 
phenomenon is, in most of the cases, already observed before separation between the candidates cancels 
this reaction. However, we did not count disconnection even when figuring hierarchy. In each such case, at 
least one additional chimera, where full resorption was accomplished, is assayed. It should be noted, however, 
that the incompleled results of disconnections are always in agreement with the results where resorption is 
completed. Subclone sizes may alter the direction of chimera resorption. However, this occurs only when 



ALLORECOGNITION RESPONSES IN COLONIAL INVERTEBRATE 



85 










the subordinate partner is much larger than the winner. All subclones used in the present study were matched 
to pairs with zooid ratios, below that may reverse the direction of resorption. b. Hierarchy in the resorption 
within the self-crossed offspring of generation III (the two left schemes) and within the detined-cross offspring 
of generation IV (refer to Fig. 1). The letter M refers to a case where the chimera dies. In that case, the 
numbers along the arrow indicate: days from fusion until the death of the chimera, number of zooids of the 
left and the right partners, respectively, on the day of fusion (in parentheses). A dashed arrow with an 
arrowhead points to a case where the direction of resorption is evident; however, the chimera either died or 
the partners disconnected before the resorption was completed. Additional subclones for doing new chimeras 
were absent; therefore, the hierarchy in resorption was not fully determined, c. Hierarchy in the resorption 
between generation II colonies and the self-crossed offspring of generation III. A dashed arrow without an 
arrowhead indicates a case where hierarchy is not evident before interactions of the partners in a specific 
chimera were interrupted by chimera mortality or disconnection. In those cases, no more chimeras were 
done because of the lack of additional subclones. d. Hierarchy in the resorption between generation II 
colonies and the defined-cross offspring of generation IV. Dashed arrow with arrowhead indicates two cases 
where hierarchy became evident before the interactions in the CAAs were interrupted by disconnection. 



86 



B. RINKEVICH ET AL 



in maintaining the Botryllus colonies, and M. Greenberg 
for valuable remarks on an earlier version. This study was 
supported by NCI Grant CA 42551, partly by a grant 
from the United States-Israel Binational Science Foun- 
dation, by a grant from the Basic Research Foundation 
administered by the Israel Academy of Sciences and Hu- 
manities, and by a Career Development Award from the 
Israel Cancer Research Fund, USA. 

Literature Cited 

Bailey. D. VV. 1978. Sources of subline divergence and their relative 
importance forsublines of six major inhred strains of mice. Pp. 197- 
215 in Origins of Inbred Mice. H. C. Morse, ed. Academic Press. 
New York. 

Bevan, M. J. 1975. Interaction antigens detected by cytotoxic T cells 
with the major histocompatibility complex as modifier. Nature 256: 
419-421. 

Bjorkman, P., M. A. Sapir, B Samraoui. W. S. Bennett, J. L. Strominger, 
and D. C. Wiley. 1987. The foreign antigen binding site and T cell 
recognition regions of class I histocompatibility cell recognition an- 
tigens. Nature 329: 512-518. 

Black, F. L., and F. M. Salzano. 1981. Evidence for heterosis in the 
HLA system. Am. J. Hum. Genet. 33: 894-899. 

Boyd, H. C, S. K. Brown, J. A. Harp, and I. L. Weissman. 
1986. Growth and sexual maturation of laboratory-cultured Mon- 
terey Bolryllm schlosseri Biol Bull- 170: 91-109. 

Buss, L. W. 1982. Somatic cell parasitism and the evolution of somatic 
tissue compatibility. Proc Nail. Acad Sci. i'SA 79: 5337-5341. 

Eichwald, E. J., and I. L. Weissman. 1966. Weak histocompatibility 
loci. Annals N.Y. Acad. Sci. 129: 94-101. 

Eichwald, E. J., C. R. Silmser, and I. L. Weissman. 1958. Sex linked 
rejection of normal and neoplastic tissue. 1. Distribution and speci- 
ficity. J Nail Cancer 1ml 20: 563-575. 

Flaherty, L. 1988. Major histocompatibility complex polymorphism: 
A non immune theory for selection. Human Immiin. 21: 3-13. 

Galton, M. 1967. Factors involved in the rejection of skin transplanted 
across a weak histocompatibility barrier: gene dosage, sex of recipient, 
and nature of expression of histocompatibility genes. Transplantation 
5: 154-168. 

Graff, R. J. 1978. Minor histocompatibility genes and their antigens. 
Transplant Proc 10: 701-705. 

Graff, R. J., W. K. Silvers, R. E. Billingham, W. H. Hildemann, and 
G. D. Snell. 1966. The cumulative effect ol histocompatibility an- 
tigens. Transplantation 4: 605-617. 

Grosberg, R. K. 1988. The evolution of allorecognition specificity in 
clonal invertebrates. Q Rev Biol. 63: 377-412. 

Grosberg, R. K., and J. F. Quinn. 1986. The genetic control and con- 
sequences of kin recognition by the larvae of a colonial marine in- 
vertebrate. Nature 322: 456-459. 

Harp, J. A., C. B. Tsuchida, I. L. Weissman, and V. L. Scofield. 
1988. Autoreactive blood cells and programmed cell death in growth 
and development of protochordates. J. E.\p. Zooi 247: 257-262. 

Hedrick, P. W., and G. Thomson. 1983. Evidence for balancing selec- 
tion at HLA. Genetics 104: 449-456. 

Hughes, A. L., and M. Nei. 1988. Pattern of nucleotide substitution 
at major histocompatibility complex class I loci reveals overdominant 
selection. Nature 335: 167-170. 



Johnson, L. L. 1981. At how many histocompatibility loci do congenic 
mouse strains differ? Probability estimates and some implications. 
/ llered 72: 27-31. 

Klein, J . 1 986. Natural History of the Major Histocompatihility (. '< im- 
plex. John Wiley & Sons, New York. 

Lappe, M. A., R. G. Graff, and G. D. Snell. 1969. The importance of 
target size in the destruction of skin grafts with non H-2 incompat- 
ibility. Transplantation 1: 372-377. 

Loveland, B., and E. Simpson. 1986. The non-MHC transplantation 
antigens: neither weak or minor. Immunol. Today 1: 223-224. 

Rinkevich, B., and I. L. Weissman. I987a. A long-term study on fused 
subclones in the ascidian Botryllus schlosseri: The resorption phe- 
nomenon (Protochordata: Tunicata). J. Zoo/. (Land.) 213: 717-733. 

Rinkevich, B., and I. L. Weissman. 1987b. The fate of Botryllus (As- 
cidiacea) larvae cosettled with parental colonies: beneficial or dele- 
terious consequences? Biol Bull. 173: 474-488. 

Rinkevich, B., and I. L. Weissman. 1987c. 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. 1988. Retreat growth in the ascidian 
Botryllus schlosseri: A consequence of nonself recognition. Pp. 93- 
109 in Invertebrate Historecognition, R. K. Grosberg, D. Hedgecock. 
and K. Nelson, eds. Plenum. New York. 

Rinkevich, B., and I. L. Weissman. 1989. Variation in the outcomes 
following chimera formation in the colonial tunicate Botryllus 
schlosseri. Bull Mar. Sci. 45: 213-222. 

Rinkevich, B., and I. L. Weissman. 1990. Bolryllus schlosseri (Tunicata) 
whole colony irradiation: do senescent zooid resorption and im- 
munological resorption involve similar recognition events? J Exp 
7,ool 253: 189-201. 

Rinkevich, B., and I. L. Weissman. 1992. Allogeneic resorption in co- 
lonial protochordates: consequences of nonself recognition. Dc\. 
Camp. Immun 16: 275-286. 

Robertson, M. 1982. The evolutionary past of the major histocom- 
patibility complex and the future of cellular immunology. Nature 
297: 629-632. 

Sabbadin, A., and G. Zaniolo. 1979. Sexual differentiation and germ 
cell transfer in the colonial ascidian Botryllus schlosseri. J. Exp. Zoo/. 
207: 289-304. 

Scofield, V. L., J. M. Schlumpberger, L. A. West, and I. L. \\eissman. 

1982. Protochordate allorecognition is controlled by an MHC-like 
gene system. Nature 295: 499-502. 

Taneda. Y., Y. Saito, and H. \\atanabe. 1985. Self or nonself discrim- 
ination in ascidians. Zoo/. Sci. 2: 433-442. 

Townsend, A. R. M., J. Bastin, K. Gould, and G. G. Brownlee. 
1986. Cytotoxic T lymphocytes recognize influenza haemagglutinin 
that lacks a signal sequence. Nature 324: 575-577. 

Weissman, I. L. 1988. Was the MHC made for the immune system, 
or did immunity take advantage of an ancient polymorphic gene 
family encoding cell surface interaction molecules? A speculative es- 
say. Int. Rev Immiin. 3: 393-416. 

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

Zaleski, M. B., S. Dubiski, E. G. Niles, and R. K. Cunningham. 

1983. Invnunogenelics. Pitman. Boston. 



Reference: Biol Bull 184: 87-96. (February, 1993) 



Classification and Characterization of 
Hemocytes in Styela clava 

TOMOO SAWADA 1 , JEFFREY ZHANG : , AND EDWIN L. COOPER 

Department of Anatomy and Cell Biology. School of Medicine, 
University of California, Los Angeles. California 90024 



Abstract. Viable hemocytes of the common tunicate 
Styela clava are classified into four groups designated as 
eosinophilic granulocytes, basophilic granulocytes, hyaline 
cells and lymphocyte-like cells. Eosinophilic granulocytes, 
actively amoeboid, have large refractive granules that stain 
with neutral red. Basophilic granulocytes do not stain with 
neutral red and formed couplets or triplets. Hyaline cells, 
which often contain phagosomes. have electron-dense 
small vesicles recognizable only by electron microscopy. 
Hemoblasts have a characteristic large nucleolus which is 
visible by light microscopy. Eosinophilic granulocytes and 
hyaline cells actively ingest yeast particles in vitro. This 
classification simplifies former ones by correlating electron 
microscopy, with light microscopy, and viable with fixed 
hemocytes. Clearly viable tunicate hemocytes can be 
identified by simple methods. We have provided clear 
and more accurate descriptions which will lessen the con- 
troversy often associated with assigning hemocyte func- 
tions in immunodefense responses both in vivo and in 
vitro. 

Introduction 

The classification of tunicate hemocytes remains con- 
fused, not withstanding Wright's attempt ( 1 98 1 ) to devise 
useful categories. Recent progress in tunicate biology, 
however, requires a precise correlation between various 
cellular functions and particular types of hemocytes. 
Styela clava. especially, has been used in investigations 
of immunological responses including those associated 
with hemocytes: allogeneic reactions (Raftos and Cooper. 
1991); cytotoxic reactions (Kelly et ai. 1992a); humoral 



Received 20 May 1992; accepted 9 November 1992. 
Present address: ' Department of Anatomy, Yamaguchi University 
School of Medicine, Ube-city, 755 Japan. 

2 Undergraduate under the SRP program at UCLA. 



opsonin (Kelly et ai. 1992b, 1993a, b, in press); and the 
production of cytokines (Beck et ai. 1989; Raftos et ai. 
1991). Humoral lectins (Yokozawa el ai. 1986; Harada- 
Azumi et ai. 1987). antibacterial substances (Azumi et 
ai. 1990), and a metallo-protease (Azumi el ai. 1991) 
were studied in another species, Halocynthia roretzi. 

Although the classification of Styela clava hemocytes 
began early (Ohue, 1936) and the site of hemopoiesis is 
described (Ermak. 1975. 1976), the literature includes de- 
scriptive morphologies with a plethora of terms, but rel- 
atively little experimental information uniting structure 
with function. Previous analyses of hemocytes failed to 
correlate age, season, and cell behavior in a systematic 
way, and these variables were not related to the various 
techniques used for examining them (e.g.. staining and 
fixation versus observation of live cells). Recent molecular 
and cytological studies focusing on the hemocytes and 
immune system of Stye/a will reveal a more precise picture 
of the functional contribution of individual effector cells. 
But this development depends on a thorough and con- 
sistent classification of the hemocytes. 

To establish an acceptable and predictable classification 
scheme, we examined hemocytes from Stye/a clava and 
correlated the morphological and behavioral character- 
istics of living hemocytes, and compared appearance of 
viable cells with those analyzed by light and electron mi- 
croscopy. Our work offers a strategy for classifying hem- 
ocytes in any invertebrate, especially tunicates which are 
becoming increasingly more important as we decipher 
the nature of effector cell activity during immune re- 
sponses. 



Materials and Methods 



Hcmocvtes 



Hemocytes were harvested by severing the stolons of 
Styela clava after rinsing the outside with 70% ethanol. 



87 



T. SAWADA ET AL 



Exuding hemolymph was collected into 0.5 M NaCl 
(NaCl-solution, pH 7.0 by 0.01 N NaOH) in polystyrene 
tubes; this prevented the nonspecific coagulation of he- 
mocytes and allowed individual hemocytes to be observed. 
Hemolymph was mixed with the NaCl solution one to 
one in final volume. 

Staining 

Hemolymph or hemocyte suspensions in NaCl-solution 
were loaded onto glass slides. After 10 min, adhering 
hemocytes were fixed for 15 min and stained with he- 
matoxylin and eosin (H&E). Cold ethanol, cold methanol 
or 4% paraformaldehyde (0. 1 M sodium cacodylate buffer, 
pH 7.0) were used as fixatives, and the morphological 
preservation was compared. For vital staining, neutral red 
(NR, 0.01% in final concentration) was added to hemocyte 
suspensions: 1 5-30 min later, the hemocytes were loaded 
onto glass slides and observed. 

Correlation of NR-staining with H&E-staining 

We photographed NR-stained hemocytes adhering on 
glass slides, then fixed them for regular light microscopy 
without moving the slides, and photographed them again 
under phase-contrast microscopy. After H&E-staining, we 
found exactly the same cells as in the former two pho- 
tographs (NR-staining and phase-contrast) to compare 
their appearance. 

Transmission electron microscopy (TEM) 

Hemocytes in the hemolymph and inside pharyngeal 
tissue were examined by TEM. Hemolymph collected into 
polystyrene tubes was centrifuged (400 X g for 5 min) 
and the pellet fixed. Pieces of pharynx (about 1.5 mm 
square) were dissected and fixed. Specimens were pre- 
fixed in a mixture of 2% glutaraldehyde and 2% parafor- 
maldehyde (0.75 M sucrose, 0.2 M sodium cacodylate 
buffer, pH 7.0), then post-fixed with 1% osmium tetroxide 
in the same buffer. The specimens were dehydrated in 
ethanol series and embedded in Medcast (Ted Pella, Red- 
ding, CA). Propylene oxide was used to infiltrate the resin. 

Autonomous fluorescence of viable hemocytes 

Hemocytes suspended in NaCl-solution were loaded 
on glass slides and observed with a Nikon EFD2 fluores- 
cence microscope with blue (420-490 nm)-and ultraviolet 
(330-380 nm)-illumination. 

Composition of hemocytes 

Different hemocyte types were counted by light mi- 
croscopy after H&E or NR-staining. and also by TEM. 
A sample of hemocytes was taken from 6 animals, and 
five to ten different viewing fields (1 10-130 cells in total) 



from each sample were examined in light microscopy with 
a 100X objective lens. Five pharyngeal pieces, one each 
from 5 animals (one TEM-section for each piece), and a 
hemocyte-pellet from one animal were examined by TEM. 
About one hundred cells were examined on each section. 

Phagocytic activity against yeast particles 

Saccharomyces cerevisiae (baker's yeast, type II; Sigma 
Chemicals, St. Louis, MO) was stained with Congo red 
and suspended in artificial seawater (approximately 
1 X 10 8 particles/ml), according to Kelly et ai (1993a). 
Hemocyte suspensions in NaCl-solution (100 p\) were 
loaded on cover slips, and yeast particle suspension (100 
^1) was added 5 min later. The hemocytes were incubated 
for 30 min. After the cover slips were gently rinsed to 
remove excess yeast particles, 0.01% neutral-red solution 
was added. Hemocyte types were identified by NR-stain- 
ing. Types of hemocytes which phagocytized yeast par- 
ticles were identified. 

Results 

Light microscopy of hemocytes 

Most hemocytes adhered to glass slides, and some of 
them exhibited amoeboid movement within 5 min. How- 
ever, many small, transparent cells did not adhere well 
enough to resist water movement caused by pressure on 
the cover slip. By phase contrast microscopy, four different 
types were observed (Table !):(!) hyaline cells, which ex- 
hibited significant extensions ( 1 5-20 j/m in diameter); (2) 
round cells (basophilic granulocytes, 6-10 ^m in diam- 
eter), which contained highly refractive small granules and 
often formed couplets or triplets; (3) amoeboid cells (eo- 
sinophilic granulocytes; 8-15 /jm in diameter), which 
contained large granules and exhibited more active 
amoeboid movement than the other types; and (4) small 
spherical cells that did not spread (hemoblasts; 4-6 ^m 
in diameter), which contained a small amount of cyto- 
plasm and had a nucleolus clearly visible by light mi- 
croscopy. The nuclei of hemocytes other than hemoblasts 
were not visible unless they were spread flat on a glass 
slide. 

Phase contrast microscopy was not sufficient to distin- 
guish all eosinophilic and basophilic granulocytes with 
certainty. The eosinophilic granulocytes often contained 
granules as small as those of basophilic granulocytes, and 
when they were not moving they were just as round as 
basophilic granulocytes. 

We observed large cells that had a hyaline cytoplasm 
lacking visible granules, but they did contain pigmented 
or non-pigmented large vacuoles. These cells were also 
identifiable as hyaline cells because they spread wide and 
flat. The spreading of hyaline cells was rapid once it began, 
and these cells did not exhibit active amoeboid movement 
after they spread. 



HEMOCYTES OF TUNICATE 
Table I 



Classification and sonic characteristics o/'Styela clava licmocvies 



89 



Size 


8 ! 5 ^m 


610 f*m 


15-20 JOT 


4-6 j/m 


H&E-staining 


Orange 


Purple 


Very weak purple or 


Purple 


NR-staining 


Orange or red-violet 





pink 
Negative or orange at 










vacuoles 




Granules in LM 


Many retractile 


Many small G refractile 






Granules in TEM 


Not uniform heterogeneous 


Uniform spherical 


Small vesicles 


_ 








electron dense 




Adhesion to glass 


+ + 


ii 






Phagocytosis 


+ + 


+ 


+ + + 




Other characteristics 


Active amoeboid movement 


Forming couplets or 


Widely spread (cell 


Nucleolus visible in LM 




* Blue fluorecence in red-violet cells 


aggregates 


fusion?) 




Previous classifications 


Compartment signet-ring vesiculated 


Finely granular 


Hyaline signet-ring? 


Hyaline lymphocyte-like 




morula coarsely granular 


amoebocyte 




hemoblasts 



Under UV-illumination (330-380 nm). 



NR-staining and some characteristics of viable cells 

NR mainly stained the cytoplasmic granules of amoe- 
boid cells (Fig. 1 ). Hyaline cells were usually not stained 
except for some cases in which small or large cytoplasmic 
vacuoles were stained (Fig. IE, F). Two groups of amoe- 
boid cells were positively stained by NR (eosinophilic 
granulocytes), but the intensity differed. One was stained 
a dense red- violet, whereas the other was orange (Fig. 1 ). 
Both types of cells contained 5-20 large cytoplasmic 
granules, and they were morula-shaped before starting 
amoeboid movement. Other granular cells were usually 
round, unstained by NR, and contained highly refractive 
small granules (Fig. 1; basophilic granulocytes). Most of 
the hemoblasts were not stained (Fig. 1 ). but a few some- 
times stained faintly orange. 

Following staining with NR. hemocytes were easily 
distinguished and their characteristic behaviors examined. 
Both types of NR-positive granulocytes (eosinophilic 
granulocytes) were active in amoeboid movement, ex- 
tending many spine-like pseudopodia. NR-negative gran- 
ular cells (basophilic granulocytes) were less active in 
amoeboid movement, but extended long pseudopodia. 
These cells were often observed as couplets (Fig. ID), 
triplets or small aggregates composed only of this type of 
cell, and they did not separate once they came in contact. 
Occasionally, these cells spread flat after 30-60 min in- 
cubation. 

H&E-staining 

We used three different methods to observe hemocytes 
with H&E-staining after fixation. Ethanol and methanol 
significantly modified hemocyte morphology, so only 
paraformaldehyde fixation was utilized. We observed the 
same cells with NR-staining and H&E-staining. 



Two NR-positive amoeboid cells (red-violet and orange 
cells) were stained intensely red or pink. Both cells con- 
tained various sizes of cytoplasmic granules that stained 
with eosin, so both cells were classified as eosinophilic 
granulocytes. The appearance of the cytoplasmic granules 
was altered by fixation, especially by ethanol and meth- 
anol. Cells that were fixed with these agents appeared as 
vacuolated cells, granular cells, compartment cells or sig- 
net-ring cells. 

NR-negative amoeboid cells that contain small refrac- 
tive granules were stained purple with H&E and were des- 
ignated as basophilic granulocytes. Their cytoplasmic 
granules were no longer evident after fixation, and cyto- 
plasmic staining was relatively weak after they spread on 
slides. However, the nuclei of basophilic granulocytes were 
smaller and more dense than those of hyaline cells. 

The cytoplasm of hyaline cells was very thin after 
spreading so H&E stained them only weakly purple with 
some orange-stained cytoplasmic vesicles. Phagocytic 
vacuoles in some of them were stained red. Some hyaline 
cells contained large cytoplasmic vacuoles and thus ap- 
peared as signet-rings. Also their nuclei became larger as 
they spread. 

A few encapsulations of several small cells were ob- 
served (small encapsulation; Fig. 2). In addition, small 
numbers of multinuclear cells were observed in H&E- 
staining (Fig. 2). These cells spread flat and contained 
several large nuclei and small vesicles stained with eosin. 
Finally, hemoblasts stained purple, and their nucleoli be- 
came unclear after fixation. 

TEM oj hemocytes 

In centrifuged pellets of hemolymph, we observed five 
different hemocyte types (Figs. 3, 4): (1) large cells 



90 



T. SAWADA ET AL 








bg 




hy 



Figure 1. Living hemocytes on glass slides after NR-staining. (A) Eosinophilic granulocytes included 
two groups of granulocytes that stained in different colors (o = orange and r = red-violet). The sizes of the 
cytoplasmic granules are variable in each hemocyte. (B) Neither hemoblasts (hb) and basophilic granulocytes 
(bg) were stained. Nucleoli were evident in hemoblasts. (C) Basophilic granulocytes (bg) contained many 
refractive granules which were smaller than those of eosinophilic granulocytes (o = orange cells). (D) A 
couplet of basophilic granulocytes (bg): these were frequently observed. (E) Hyaline cells (hy) spread wide 
and flat on the glass slide to form a thin cytoplasmic sheet. (F)Some hyaline cells contained granules (arrow) 
that stained with NR. x 1250 



(10-12 ^m in diameter: hyaline cells) containing signifi- 
cant amounts of endoplasmic reticulum (ER); (2) small 
spherical cells (5-6 /urn in diameter: hemoblasts) with little 
cytoplasm and large nuclei; (3-5) three different granu- 
locytes (6-12 nm in diameter: basophilic and eosinophilic 



granulocytes) containing abundant cytoplasmic granules. 
These five types also constituted the entire hemocyte pop- 
ulation within the pharyngeal tissue. 

The large cells contained numerous rough-surfaced and 
smooth-surfaced ER and also small vesicles (0.1 ^m in 



HEMOCYTES OF TUNICATE 



91 




A 

, 






Figure 2. Fixed hemocytes on glass slides with H&E-staining. (A) 
Multinuclear cells with seven nuclei spread wide and flat. Vesicular 
structures in the cytoplasm were slightly stained. (B) Small encapsulation 
(arrow) containing 3-7 small cells; these were sometimes observed in 
the hemolymph. xl 180 



diameter) of high electron density (Fig. 3). A few of these 
cells contained large vacuoles or phagosomes. Their nuclei 
often had nucleoli and coarse and uniform euchromatin, 
although heterochromatin was sometimes observed. These 
cells corresponded to hyaline cells on the basis of size, 
phagosomes, and the absence of large cytoplasmic gran- 
ules. 

The small cells with little cytoplasm contained mito- 
chondria and small amounts of ER (Fig. 4C). Their nuclei, 
with characteristic large nucleoli, were usually larger than 
those of other hemocytes. Chromatin was uniformly dis- 
tributed and slightly more dense in comparison with hya- 
line cells. These cells corresponded to hemoblasts in cell 
size, i.e., little cytoplasm and characteristically large nu- 
cleoli. 

The three different granulocytes (temporarily designated 
as type 1, 2 and 3 granulocytes according to TEM) had 
the same nuclear pattern (usually with dense heterochro- 
matin at the periphery and sometimes small nucleoli) but 
differed in their cytoplasmic granules. Type 1 granulocytes 



(6-10 yum in diameter) contained electron-dense and 
spherical granules with a diameter range of 0.2-0.5 ^m 
(Fig. 4A). These cells correspond to basophilic granulo- 
cytes on the basis of size, the sizes of their cytoplasmic 
granules (they had smallest granules among granulocytes), 
and their frequency in the hemolymph. Type 2 gmnit/o- 
cytes (8-10 /urn in diameter) contained irregular-shaped 
granules that varied in size (0.1 -1.3 /urn in diameter). The 
granules contained homogeneous material of intermediate 
electron-density (Fig. 4B). Type 3 granulocytes ( 8- 1 2 ^m 
in diameter) had cytoplasmic granules that were also ir- 
regularly-shaped and remarkably varied in size (0.1-1.5 
nm in diameter). These granules were composed of het- 
erogeneous materials central spheres with high electron 
density and surrounding material of intermediate electron 
density (Fig. 4B). Type 2 and 3 granulocytes corresponded 
to eosinophilic granulocytes on the basis of size, the ir- 
regular shape of their cytoplasmic granules, and their fre- 
quency of occurrence. 

Hemocyte composition 

We examined percentages of the various hemocytes in 
hemolymph by counting each type after NR- and H&E- 
staining and TEM (Table II). The order of dominance for 
each type was the same in all cases, but the exact values 
were somewhat different. The most abundant cells were 
eosinophilic granulocytes (46.3% in NR-staining); second 
were the basophilic granulocytes (21.0%); hyaline cells 
were third ( 18.5%-): and the smallest population was that 
of the hemoblasts (14.1%). 

The percentage of eosinophilic cells in H&E-staining 
(68.5%) was about the same as the sum of type 2 and 3 
granulocytes in the hemocyte pellets observed by TEM 
(67.8%), but it was larger than the sum of orange- and 
red-violet cells in NR-staining (46.3%). Many fewer he- 
moblasts were found in both H&E-staining (2.3%.) and 
TEM (2.0%) than in NR-staining (14.1%). The proportion 
of hyaline cells ranged from 5.5 to 18.5%, even after the 
percentages of multinuclear cells and phagocytosis were 
added. Multinuclear cells (1.2% in H&E-staining) were 
not found in NR-staining or TEM of pharyngeal tissue. 

A utonomous fluorescence oj hemocytes 

Blue fluorescence was observed in certain granulocytes 
under ultraviolet-illumination. NR-staining of those flu- 
orescent hemocytes, in the same field of view, revealed 
that the autonomous fluorescence was from eosinophilic 
granulocytes which stained in red-violet (Table I). Under 
blue-illumination, no hemocytes exhibited autonomous 
fluorescence. 

Phagocytosis 

Four hemocyte types i.e., hyaline cells, eosinophilic 
granulocytes (including red-violet and orange cells in 



92 



T. SAWADA ET AL 



m 








t!+^& .. 






'JQratfSH 
* ' 

: * *. '?& . 




Figure 3. Transmission electron microscopy of hyaline cells and a hinuclear cell. (A) Hyaline cell (h) in 
the centrifuged pellet, with heterochromatin at the nuclear periphery. (B) A binuclear cell in the centrifuged 
pellet. (C) Hyaline cell (h) in pharyngeal tissue; the nucleus has a large nucleolus and uniform euchromatin. 
All cells (A, B, C) contained electron-dense small vesicles, numerous vesicular structures, and endoplasmic 
reticulum. Bar = 1 jim. 



NR-staining). and basophilic cells ingested yeast parti- 
cles. Among them, hyaline cells and eosinophilic granu- 
locytes had significantly higher activity than basophilic 
granulocytes (Table III). In the cell population that had 
ingested yeast particles, hyaline cells (36-42%) were fewer 
than eosinophilic granulocytes (5 1-68%), as shown in Ta- 
ble IA. However, phagocytic activity was higher in hyaline 
cells, because the phagocytic ratios were higher in hyaline 
cells (32-78%) than in eosinophilic granulocytes (13- 
35%), as shown in Table IB. Many hyaline cells engulfed 



2-5 yeast particles, whereas most eosinophilic granulo- 
cytes incorporated only one particle. 

Discussion 

Classification of hemocytes 

Hemocytes from many species of tunicates have been 
classified by both light and electron microscopy (Ohue, 
1936; George, 1939;Endean, 1960; Andrew, 1961, 1962; 
Overton, 1966; Smith, 1970; Botte and Scippa, 1977; 




Figure 4. Transmission electron microscopy of hemocytes in the centnt'uge pellet of hemolymph. (A) 
Type 1 granular cells ( 1 = basophilic granulocytes) containing relatively uniform and spherical granules. 
(B) Both type 2 (2) and 3 (3) granular cells (eosinophilic granulocytes) containing irregularly shaped granules. 
The granules of type 3 cells contain electron dense cores. (C) Hemoblast (hb) with little cytoplasm and 
without cytoplasmic granules, except for mitochondria and vesicles. The relatively large nucleus contains 
characteristic large nucleolus. Bar = 1 ^m. 

93 



94 



lli'inncvtc composition examined under different conditions 



T. SAWADA ET AL 
Table II 



Hemocyte types 


Viable cells 
(NR-staining) 


Fixed cells 
(H&E-staining) 


EM 
(pellet) 


EM 
(pharynx) 


Eosinophilic 


Orange 


68.5 


5.6% 


38.9% 


15.8 


7.5% 


granulocytes 


16.5 5.0% 














Red-violet 






28.9 


32.8 


11.0 




29.8 8.5 












Basophilic 


21.0 5.1 


21.1 


4.4 


2 1 .0 


21.3 


7.0 


granulocytes 














Hyaline cells 


18.5 7.9 


5.5 


2.9 


8.0 


16.8 


3.8 


Hemoblasts 


14.1 3.2 


2.3 


2.2 


2.0 


8.5 


6.0 


Multinuclear cells 


0.0 


1.2 


1.2 


0.2 




0.0 


Cells of 


0.1 0.2 


1.4 


1.2 


1.1 


2.3 


1.6 


phagocytosis 














No. of individuals 


6 




6 


1 




5 


examined 















average S.D. 



Milanesi and Bunghel, 1978; Fuke. 1979, 1980: Rowley, 
1981, 1982; Mukai et ai, 1990), and in morphological 
terms, such as vacuolated or granular cells, hyaline cells, 
hemoblasts or lymphocytes (Wright, 198 1 ). or by functions 
(Freeman, 1964; Fuke, 1980; Fujimoto and Watanabe, 
1976; Burighel et ai, 1976; Rowley, 1983; Azumi et al, 
1 990, 1 99 1 ; Raftos et al. . 1 990; Raftos and Cooper, 1 99 1 ). 
However, the inapplicability of these classifications from 
one species to the next, and the lack of correspondence 
between different methods (e.g., light versus electron mi- 
croscopy) have produced confusion. 

Hemocytes of S. clava have been classified into morula 
cells, compartment cells, signet-ring cells, granular amoe- 
bocytes, hyaline cells and lymphocyte-like cells (Ohue, 
1936; Wright, 1981). But, among fresh and living hemo- 
cytes, we observed no signet-ring cells nor any cells with 
a stable, morula shape. Instead, there were granulocytes 
that frequently changed their appearance during amoe- 
boid movement. They appeared morula-like when they 
rounded up, and could be compartment cells or granular 
amoebocytes after they had become extended and flat- 
tened. Fixation, especially with ethanol or methanol, 
modified hemocyte morphology significantly, and some 
of the eosinophilic granulocytes and hyaline cells became 
signet-ring in shape. Therefore, we adopted two cautious 
guidelines. First, we avoided using such terms as morula, 
compartment, or signet-ring. Second, we employed no 
Wright- or Giemsa staining because they require methanol 
as the fixative. Instead, we preferred to use formaldehyde 
fixation and H&E-staining. 

We identified five different hemocyte types by vital NR- 
staining and TEM, and four types by H&E-staining of 
fixed cells. We estimated that the granules of the orange 
cells in NR-staining contain less dense material, and so 
correspond to type 2 granulocytes in TEM; similarly red- 



violet cells in NR-staining correspond to type 3 granu- 
locytes in TEM. The difference between type 2 and 3 cells, 
or between orange and red-violet cells, is not significant 
enough to separate them into two cell types. Moreover, 
both the orange and red-violet cells evidently correspond 
to eosinophilic granulocytes in H&E-staining. These two 
granulocytes appear to be similar in amoeboid movement 
and phagocytic activity. Therefore, we classified both of 
them into the same group as eosinophilic granulocytes. 
We suggest that type 2 granulocytes (orange cells) are an 
earlier stage in cell differentiation than type 3 granulocytes 
(red-violet cells). 

The correspondences between the light microscopical 
and TEM images of basophilic granulocytes (type 1 gran- 
ulocytes in TEM), hyaline cells, and hemoblasts were clear 
on the basis of their morphological characteristics and 
their frequencies of appearance. 

Multinuclear cells were classified as hyaline cells for 
the following reasons: (1) morphologically multinuclear 
cells are in all other respects similar to hyaline cells; (2) 
they sometimes contain large, eosinophilic vacuoles that 
we assume to be phagosomes; (3) the morphology and 
behavior of hyaline cells are quite similar to phagocytes 
type 1 (pl-ceQs)ofHalocynthiaroretzi(H. rorelii), which 
evidently fuse together and form multinuclear cell sheets 
(Sawada el al., 1991). But, we have no strong evidence 
for cell fusion between the hyaline cells of S. clava. More- 
over, the frequency of multinuclear cells in fresh hemo- 
lymph is not clear, because they could be identified only 
after spreading on glass. Both of these points require fur- 
ther investigation. 

In this study, therefore, we have identified four hemo- 
cyte types in S clava. ( 1 ) Eosinophilic granulocytes con- 
tain several refractive vacuoles that appeared red in neutral 
red vital stain, red by H&E. and exhibit active amoeboid 



HEMOCVTES OF TUNICATE 



95 



Table III 
Phagocytosis / yeast particles by hemocytes from three Jil/erent individuals 



(A) Composition of hemocytes which ingested yeast particles 




Eosinophilic granulocytes 








Total 






Basophilic 


Hyaline 




cells 


Animals 


(red-violet)* 1 (orange)* 1 


granulocytes 


cells 


Hemoblasts 


examined 


a* 2 


29.2% 22.1% 


6.2% 


42.5% 


0.0% 


113 


b 


41.0 11.0 


10.0 


38.0 


0.0 


100 


c 


41.6 16.8 


5.0 


36.6 


0.0 


nil 



(B) Phagocytosis against yeast particles within each kcinocyte type 

Hemocyte types Animals Ingesting cells 



Non-ingesting cells 



Total cells examined 



eosinophilic granulocytes 


a* 2 


19.4% 


80.6% 


108 


(red-violet) 


b 


30.8 


69.2 


52 




c 


12.9 


87.1 


101 


(orange) 


a 


16.4 


83.6 


110 




b 


ND* 3 


ND 


ND 




c 


1.6 


98.4 


61 


basophilic granulocytes 


a 


7.3 


92.7 


124 




b 


3.8 


96.2 


53 




c 


0.0 


100 


57 


hyaline cells 


a 


78.0 


22.0 


100 




b 


63.6 


36.4 


22 




c 


32.8 


67.2 


61 



*' Two sub-populations of eosinophilic granulocytes different in colors of NR-staimng are indicated in parenthesis. 
* : Animals (a. b. c) in Table A correspond to the animals in Table B. 
* 3 No data. 



movement and phagocytosis. (2) Basophilic granulocytes 
contain numerous small granules that do not stain with 
neutral red, are purple in H&E, and form specific aggre- 
gations with the same cell type. (3) Hyaline cells contain 
fine electron-dense granules in TEM, occasionally contain 
phagosomes that stain red with neutral red and H&E, and 
extend into thin circular sheets on glass. (4) Hemoblasts 
possessed little cytoplasm, large nucleoli visible by light 
microscopy, but adhere only weakly to glass. Possible cor- 
respondence between former classifications are shown in 
Table I. 

Functions and characteristic behavior of each hetnocyte 
type 

Phagocytosis, as is well known, is a ubiquitous and im- 
portant immuno-defense response found throughout the 
animal kingdom. Hyaline cells exhibited the highest 
phagocytic activity, and some of them engulfed more than 
five yeast particles. Eosinophilic granulocytes were less 
active than hyaline cells, but they accounted for the largest 
population because of their abundance and active motility. 

Hyaline cells were the most likely candidates for ef- 
fecting encapsulation of larger particles by their ability to 
spread and form flat sheets and to fuse together into larger 
multinuclear sheets. Hemoblasts have been referred to as 



lymphocyte-like cells (Wright, 198 1 ) and as proliferative 
stem cells (Ermak, 1976). We also observed the charac- 
teristically large nucleolus also in viable cells and con- 
firmed their equivalents by light (Wright, 1 98 1 ) and elec- 
tron microscopy (Ermak, 1976). 

Motility was also an important and definitive, behav- 
ioral characteristic. Only eosinophilic granulocytes ex- 
hibited active movement. In contrast, the basophilic 
granulocytes did not separate after once contacting others, 
which resulted in the formation of couplets or triplets. 
This behavior continues when augmented, resulting in 
small aggregates. Similar behavior was also observed on 
gl -cells of//, roretii (Sawada et ai, 1991), and we suggest 
the presence of common granulocytes that can form spe- 
cific aggregates within the same cell type. 

Correspondence to the hemocytes in other tunicate 
species 

Hemocyte types found in many species have been cat- 
egorized into several groups by Wright (1981). However, 
the hemocytes of a single category often include several 
different types. In addition, certain hemocytes of one spe- 
cies are apparently absent in other species. It would not be 
instructive to compare only morphological aspects of hemo- 
cytes, and only under a single condition, such as in paraffin 



96 



T. SAW A DA ET AL 



sections. Observations of living hemocytes, under different 
conditions and stained with simple dye, coupled with func- 
tional analysis, e.g.. of phagocytosis, would be more useful. 
In such a manner, we compared the hemocytes of St vela 
clava and Halocvntlria rorctzi which have also been clas- 
sified in the living state (Sawada et ai, 1991 ), and found 
interesting correspondences between types. Hyaline cells 
and basophilic granulocytes were similar to the pi -cells 
and gl -cells of Halocynthia roretzi. respectively, in mor- 
phological and behavioral aspects. Hemoblasts, as the 
candidate for hematopoietic stem cells, may correspond 
to the ly-cells of Halocynthia roretzi, but their function as 
the stem cells has not been established in either species. 
Eosinophilic granulocytes seemed to be similar to the v3- 
and v4-cells of Halocynthia roretzi in that refractive vacuoles 
occupy most of the cell volume, and active amoeboid 
movement and addphilic staining occur. But eosinophilic 
granulocytes ofStyela clava were evidently more phagocytic. 
The correspondence between these species of at least two to 
three cell types may be consistent with their phylogeny. 

Acknowledgments 

We thank Sharon Sampogna and Monica Eiserling for 
technical help in light and electron microscopy. This study 
was supported by the National Science Foundation (Grant 
#DCB 90 05061). 

Literature Cited 

Andrew. VV. 1961. Phase microscope studies of living blood-cells of 
the tunicates under normal and experimental conditions, with a 
description of a new type of motile cell appendage. Q. .1. Micr. Sci. 
102: 89-105. 

Andrew, VV. 1962. Cells of the blood and coelomic fluids of tunicates 
and echinoderms. Am. Zoo/. 2: 285-297. 

Azumi, K., H. Yokozawa, and S. Ishii. 1990. Halocyammes: novel anti- 
microbial tetrapeptide-like substances isolated from the hemocytes of 
the solitary ascidian. Halocynthia roretzi Biochemistry 29: 159-165. 

Azumi, K., H. Yokozawa, and S. Ishii. 1991. Lipopolysacchande in- 
duces release of a metallo-protease from hemocytes of the ascidian, 
Halocynthia roretzi. Dev. Comp. Immune/ 15: 1-7. 

Beck, G., G. R. Vasta, J. J. Marchalonis, and G. S. Habicht. 
1989. Characterization of mterleukin-1 activity in tunicates. Comp. 
Bioclwm. Physiol. 92B: 93-98. 

Botte, I,., and S. Scippa. 1977. Ultrastructural study of vanadocytes 
in .-I iciilia malaca. Experienlia 33: 80-8 1 . 

Burighel, P., R. Brunelti, and G. Zaniolo. 1976. Hibernation of the 
colonial ascidian Botrylloides leachi (Savigny): histological obser- 
vations. Boll. Zoo/. 43: 293-301. 

F.ndean, R. 1960. The blood-cells of the ascidian. Phalhisia mammilala. 
Q J Micr. Sii. 101: 177-197. 

Ermak, T. H. 1975. An autoradiographic demonstration of blood cell 
renewal in Styela clava (Urochordata: Ascidiacea). Experientia 31: 
837-839. 

Ermak, T. II. 1976. The hematogenic tissue of tunicates. Pp. 45-56 
in Phylogeny of Thymus and Bone Marrow-bursa Cells, R. K. Wright 
and E. L. Cooper, eds. Elsevier/North-Holland, Amsterdam. 

Freeman, G. 1964. The role of blood cells in the process of asexual 
reproduction in the tunicate Perophora viridis. J Exp. Zool. 156: 
157-183. 



Fujimoto, H., and II. VVatanabe. 1976. The characterization of granular 
amoebocytes and their possible roles in the asexual reproduction of the 
polystyelid ascidian, Poly:oa vesiciiliphora. J. Morphol 150: 623-638. 

Fuke, M. T. 1979. Studies on the coelorruc cells of some Japanese ascidians. 
Bull. Mar. Biol. Sin. Asamushi, Tohoku University 16: 143-159. 

Fuke, M. T. 1980. "Contact reactions" between xenogeneic or allo- 
geneic coelomic cells of solitary ascidians. Biol. Bull. 158: 304-315. 

George, VV. C. 1939. A comparative study of the blood of the tunicates. 
Quart. J Micr Sci. 81: 391-431. 

Harada-Azumi, K., II. Yokozawa, and S. Ishii. 1987. N-acetyl-galac- 
tosamine-specific lectin, a novel lectin in the hemolymph of the 
ascidian Halocynthia roretzi: Isolation, characterization and com- 
parison with galactose-specific lectin. Com. Bioclwm. Physiol 88B: 
375-381. 

Kelly, K. L., E. L. Cooper, and D. A. Raftos. 1992a. In vitro allogeneic 
cytotoxicity in the solitary urochordates. J. Exp. Zool. 262: 202-208. 

Kelly, K. L., E. L. Cooper, and D. A. Raftos. 1993a. A humoral opsonin 
from the solitary urochordate Styela clava. Dev Comp. Imnumol. 
17: (in press). 

Kelly, K. L., E. L. Cooper, and D. A. Raftos. 1992b. Purification and 
characterization of a humoral opsonin from the Styela clava. Comp. 
Biochem. Physiol 103B: 749-753. 

Kelly, K. L, E. L. Cooper, and D. A. Raftos. 1993b. Cytokine-like activities 
of a humoral opsonin from Styela clava. Zool. Sci. (in press). 

Milanesi, C., and P. Burighel. 1978. Blood cell ultrastructure of the 
ascidian Bolryllux schlosseri. I. Hemoblast, granulocytes macrophage. 
morula cell and nephrocyte. Acta Zool 59: 135-147. 

Mukai, H., K. Hashimoto, and H. VV atanabe. 1990. Tunic cords, glom- 
erulocytes, and eosinophilic bodies in a styelid ascidian. Polyandro- 
carpa misakiensis. J. Morphol. 206: 197-210. 

Ohue, T. 1936. On the coelomic corpuscles in the body fluid of some 
invertebrates. III. The histology of the blood of some Japanese as- 
cidians. Sci. Rep Tohoku L'niv. 11: 191-206. 

Overton, J. 1966. The fine structure of blood cells in the ascidian Per- 
ophora viriilix. J. Morph. 119: 305-326. 

Raftos, D. A., D. L. Stillman, and E. L. Cooper. 1990. //; vitro culture 
of tissue from the tunicate Styela clava. In Vitro Cell Dev Biol 26: 
962-970. 

Raftos, D. A., and E. L. Cooper. 1991. Proliferation of lymphocyte- 
like cells from the solitary tunicate. Styela clava. in response to al- 
logeneic stimuli. J. Exp. Zool. 260: 391-400. 

Raftos, D. A, E. L. Cooper, G. S. Habicht, and G. Beck. 1991 . Invertebrate 
cytokines Tunicate cell proliferation stimulated by an interleukin-1- 
like molecule. Proc. Nat. Acad. Sci. USA 88: 9518-9522. 

Rowley, A. F. 1981. The blood cells of the sea squirt, dona intestinalis: 
morphology, differential counts and in vitro phagocytic activity. J. 
Invertebr. Palhol. 37:91-200. 

Rowley, A. F. 1982. Ultrastructural and cytochemical studies on the 
blood cells of the sea squirt, dona intestinalis. I. Stem cells and 
amoebocytes. Cell Tissue Res. 223: 403-414. 

Rowley, A. F. 1983. Preliminary investigations on the possible anti- 
microbial properties of tunicate blood cell vanadium. J. Exp. Zool. 
27: 319-323. 

Sawada, T., Y. Fujikura, S. Tomonaga, and T. Fukumoto. 
1991. Classification and characterization often types of hemocytes 
in tunicate Halocynthia roretzi. Zool. Sci. 8: 939-950. 

Smith, M. J. 1970. The blood cells and tunic of the ascidian Halo- 
cynthia aurantium (Pallas). I. Hematology. tunic morphology and 
partition of cells between blood and tunic. Biol. Bull 138: 354-378. 

Wright, R. K. 1981. Urochordates. Pp. 565-626 in Invertebrate Blood 
Cells 2. N. A. Ratcliffe and A. F. Rowley, eds. Academic Press, 
London. 

Yokozawa, H., K. Harada, K. Igarashi, Y. Abe, K. Takahashi, and S. 
Ishii. 1986. Galactose-specific lectin in the hemolymph of solitary 
ascidian. Halocynthia roretzi. Molecular, binding and functional 
properties. Biochim. Biophys. Acta, 870: 242-247. 



Reference: Bin! Bull 184: 97-104. (February, 1993) 



Effects of Cations on the Volume and Elemental 

Composition of Nematocysts Isolated from Acontia 

of the Sea Anemone Calliactis polypus 

MICHIO HIDAKA AND KIWAMU AFUSO 

Department of Biology, University of the Ryiikyus, Nishihara Okinawa, 903-01 Japan 



Abstract. The hypothesis that exchange of intracapsular 
divalent cations with Na + in seawater increases the inter- 
nal osmotic pressure during discharge of nematocysts of 
marine cnidarians was tested by examining effects of ex- 
ternally applied cations on the volume and elemental 
composition of nematocysts isolated from acontia of the 
sea anemone Calliactis polypus. The volume of isolated 
nematocysts increased with increasing concentrations of 
cations if the cation was monovalent but appeared to de- 
crease if the cation was divalent. Ca 2+ reduced the internal 
osmotic pressure of the nematocysts more efficiently than 
Mg 2+ . X-ray microanalysis of nematocysts incubated in 
1 M solutions of various salts showed that Ca 2 + in isolated 
nematocysts was only partially replaced, if at all, by ex- 
ternally applied Na + and Mg 2+ while most Mg :+ was re- 
placed by Na + and Ca 2+ . The present results suggest that 
exchange of intracapsular divalent cations with external 
monovalent cations increases the internal osmotic pres- 
sure, and that selective binding of Ca 2+ to polyanions in 
the capsule decreases it. Whether the increase in the in- 
ternal osmotic pressure caused by the cation exchange is 
large enough to trigger discharge remains to be investi- 
gated. 

Introduction 

Lubbock and his colleagues proposed that loss of Ca 2+ 
from a nematocyst increases the osmotic pressure of the 
intracapsular fluid and thus causes discharge of the ne- 
matocyst (Lubbock and Amos, 1981; Lubbock el ai, 
1981; Gupta and Hall, 1984). They proposed that poly- 
peptides in undischarged nematocysts are crosslinked by 
Ca 2+ to form polypeptide chains and that the release of 

Received 14 May 1992; accepted 20 October 1992. 



calcium from the nematocyst dissociates the polypeptide 
chains, thereby increasing the number of osmotically ac- 
tive molecules. Because of this report, Ca 2i has been con- 
sidered to play a major role in nematocyst discharge. 

Recently Weber (1989) demonstrated that naturally 
occurring cations of Hydra nematocysts can be replaced 
by externally applied cations. Nematocysts loaded with 
other cations generally retain discharge capabilities. Gerke 
d t/l. ( 199 1 ) found that in situ nematocysts of Hydra con- 
tained high concentrations of potassium (K) instead of 
calcium (Ca). These observations suggest that Ca 2+ is not 
indispensable for the discharge of certain kinds of ne- 
matocysts. 

Weber (1989) proposed that Hydra nematocysts can 
be considered as Donnan-equilibrium dominated osmotic 
systems and that cations associated with polyanions in 
the capsule, rather than polyanions themselves, contribute 
to high intracapsular osmotic pressure. Because Hydra 
nematocysts contain high concentrations of K (Gerke et 
a/., 1991 ) and are surrounded by a membrane that might 
serve as a diffusion barrier against ions of low molecular 
weight (Lubbock et a/., 1981), nematocysts of Hydra 
might be in equilibrium with high concentrations of K + . 
If such nematocysts are exposed to freshwater as a result 
of exocytosis, the osmotic pressure difference across the 
capsule wall would increase, leading to the discharge of 
the nematocysts. Indeed, isolated Hydra nematocysts im- 
mersed in concentrated NaCl or KC1 solutions swell up 
to 1 1 5% of the original volume and tend to discharge 
when the external concentration of the salts is lowered 
(Weber, 1989). 

The above process, however, may not account for the 
discharge of nematocysts of marine cnidarians, because 
nematocysts of marine cnidarians must discharge in sea- 
water, which contains high concentrations of salts. X-ray 



97 



98 



M. HIDAKA AND K.. AFUSO 



microanalysis of frozen sections of various marine cni- 
darians show that the predominant cation of nematocysts 
/// situ is either Ca :+ , Mg : + , or K* (Tardent el a/.. 1990). 
If nematocysts of marine cnidarians also behave as Don- 
nan-equilibrium dominated systems, exchange of intra- 
capsular cations with cations in seawater will occur when 
nematocysts are exposed to seawater as a result of exo- 
cytosis. In Ca- or Mg-containing nematocysts, the ex- 
change of divalent cations in the capsule with monovalent 
cations such as Na + in seawater might increase the internal 
osmotic pressure, since one divalent cation is replaced by 
two monovalent cations to maintain electroneutrality. If 
the increase in the internal osmotic pressure is large 
enough, the nematocysts would discharge. 

The purpose of the present study is to examine the 
hypothesis of divalent-monovalent cation exchange. Un- 
discharged nematocysts isolated from various cnidarians 
contain high concentrations of Ca and Mg (Weber et a!., 
1987; Mariscal, 1988; Hidaka, 1993). These isolated ne- 
matocysts provide a useful model for studying the re- 
sponses of Ca- and/or Mg-containing nematocysts to var- 
ious cations. We determined the effects of mono- and 
divalent cations on the volume of nematocysts isolated 
from acontia of the sea anemone Calliactis polypus. We 
also studied whether Ca 2+ and Mg 2+ found in the isolated 
nematocysts could be replaced by externally applied cat- 
ions as in Hydra nematocysts (Weber, 1989). 

Materials and Methods 

Specimens of Calliactis polypus on the shells of hermit 
crabs belonging to the genus Dardanus. were collected 
from the reef around the Okinawa island, and maintained 
in an aquarium supplied with a subgravel filter. The her- 
mit crabs were fed with chopped Tapes every 2-4 days. 
The anemones were used as a source of acontial nema- 
tocysts 1-3 days after feeding. Acontial filaments were 
obtained by prodding the sea anemone with blunt-tipped 
forceps. 

Undischarged basitrichous isorhiza nematocysts were 
isolated either in artificial seawater (ASW) or in distilled 
water (DW), since nematocysts isolated in ASW and those 
isolated in DW display different discharge capabilities 
(Hidaka and Mariscal, 1988). A piece of acontium was 
placed in a drop of ASW or DW on a glass slide. The glass 
slide was treated with a drop of a 0.1% solution of poly- 
1-lysine (Sigma; approx. mol. wt. 90,000) in distilled water 
for 10 min in a wet chamber prior to use (Mazia et ai, 
1975). Two strips of thin adhesive tape (Scotch 3M) were 
placed on both sides of the drop to make a narrow space 
between the glass slide and a cover slip, and to make it 
easy to replace the solution in this space. When nema- 
tocysts were isolated in ASW, the acontium was squashed 
under a cover slip. Only a small percentage (about 5%) 



of nematocysts discharged during this procedure, and most 
of them only partially discharged eversion of the tubule 
stopped halfway. Cellular debris and partially discharged 
nematocysts were removed by washing the squashed 
acontium with a few drops of ASW. Most of the nema- 
tocysts isolated in this manner from acontia ofCalliaclis 
tricolor discharged when immersed in 5 mAf EGTA (Hi- 
daka and Mariscal, 1988), suggesting that the isolated ne- 
matocysts are functional. When nematocysts were isolated 
in DW, the acontium was immersed in a drop of DW for 
5 min and then the remaining acontium was removed. 
The extruded undischarged nematocysts were allowed to 
settle onto the glass slide for 10 min. Then, the isolated 
nematocysts were washed with more than five drops of 
ASW or DW to remove unattached nematocysts. 

Photomicrographs of nematocysts were taken in ASW 
or DW using a plan objective lens (X 100). Then, test so- 
lutions were applied by perfusing the nematocysts with 
at least eight drops of each test solution (Hidaka and Mar- 
iscal, 1988). Nematocysts isolated in ASW were treated 
with decreasing concentrations of salt solutions, that is, 
1000, 100, 10, 1, and mAf solutions. Nematocysts iso- 
lated in DW were treated with increasing concentrations 
of salt solutions. Nematocysts were immersed in each test 
solution for 10 min, because changes in the volume of 
isolated nematocysts in solutions with or without Ca 2+ 
were completed within 10 min (Hidaka, 1992). After 10 
min a pair of photomicrographs of the nematocysts was 
taken for each test solution. The length and diameter of 
nematocysts capsules were measured to the nearest 0.1 
mm (corresponding to 0.05 /*m) on two photomicrograph 
prints using calipers. The average value was used for each 
capsule. The volume was calculated assuming that the 
capsule was an ellipsoid. The volume of nematocysts im- 
mersed in test solutions was normalized by the original 
volume of the nematocysts in ASW or DW, and expressed 
as relative volume. The original volume (mean SD) was 
1 17.7 18.8 Mm 1 (n = 37) in ASW-isolated nematocysts 
and 100.7 10.1 M' (n = 37) in DW-isolated nema- 
tocysts. For each salt solution, three or four experiments 
were performed and at least seven nematocysts were mea- 
sured. The significance of regression of the relative volume 
of nematocysts on log (salt concentration) was tested in 
each salt solution. When the regression was not significant, 
the relative volume at 1 M salt concentration was com- 
pared with that at 1 mAf using Duncan's multiple range 
test. The difference in the relative volume of nematocysts 
was tested among pairs of cations at 1 M salt concentration 
using the multiple range test. 

For substitution experiments, nematocysts were isolated 
by immersing 20 acontia in 5 ml of DW for 5-10 min. 
Remaining acontial tissues were removed by filtering the 
nematocyst suspension through 60 ^m nylon mesh. Ali- 
quots (0.8 ml) of the filtrate were placed in each of six 



EFFECTS OF CATIONS ON NEMATOCYSTS 



99 



microtubes and centrifuged at 1940 X g for 5 min. One 
ml of each test solution was added to the pellet. Nema- 
tocysts were resuspended in the test solutions and allowed 
to stand for 10 min. Test solutions were ASW and 1 M 
solutions of NaCl, KG, CaCl : , MgCl 2 , and SrCl 2 . Next, 
the nematocysts were washed in DW by centrifuging at 
1940 X g for 5 min and by resuspending the nematocysts 
in 1 ml of DW. The nematocysts were washed in DW 
and collected by centrifugation three times. Finally, the 
nematocysts were resuspended in 1 50 n\ of DW. Aliquots 
(20 n\) of the nematocyst suspension were placed on 
meshes with formval or collodion membranes that had 
been coated with carbon and treated with poly-1-lysine. 
The nematocysts were allowed to settle on the membrane 
for 1 h, then air-dried after the remaining solution was 
soaked up with a piece of filter paper. Specimens were 
observed under a scanning transmission electron micro- 
scope (JEOL JEM-2000EX) equipped with an energy dis- 
persive spectrometer (TN 42 U). X-ray spectra were ac- 
quired at an acceleration voltage of 100 kV. Semiquan- 
titative elemental analyses were performed using an 
application software (Noran Instruments Inc. SMTP) on 
4-6 nematocysts for each test solution. The software, 
which was designed for standardless semiquantitative 
analysis of metallurgical thin films, removes background 
and integrates peak areas. The peak intensities were con- 
verted to ratios of element concentrations by multiplying 
by calculated K-factors. Correction for absorption was 
not made. 

The ASW contained (in mAI): NaCl, 480; KC1, 10; 
CaCl 2 , 10; MgCl 2 , 26; MgSO 4 , 29; and was adjusted to 
pH 8.0 with 10 mM HEPES. All the salt solutions and 
DW were buffered to pH 8.0 with 10 mM HEPES, and 
all the experiments were done at room temperature 
(23-26C). 

Results 

Nematocysts isolated from acontia of Calliaclis polypus 
in ASW swelled in concentrated solutions of monovalent 
cations (Fig. 1 ). There was a significant positive regression 
between the volume of nematocysts and the concentration 
of Na + and K + (regression analysis, P < 0.05). Though 
there was no significant regression between the volume 
of nematocysts and the concentration of divalent cations, 
the volume of nematocysts was significantly smaller in 1 
M MgCl 2 and SrCl 2 than in 1 mM solutions (Duncan's 
multiple range test, P < 0.01). At 1 M concentration, 
nematocysts immersed in divalent cations were signifi- 
cantly smaller than those immersed in monovalent cations 
(Duncan's multiple range test, P < 0.01). When nema- 
tocysts that had been immersed in various salt solutions 
were immersed in a buffer solution without added salts, 
there was no significant difference in the mean volume 



120 



110 



g 100 



JS 90 
0> 

oc 



80 




10 



100 



1000 



Concentration of salts (mM) 

Figure 1. Effects of cations of various concentrations on the volume 
of nematocysts isolated from acontia of Calliaclis polypus in ASW. Ne- 
matocysts isolated in ASW were immersed successively in salt solutions 
of decreasing concentrations. The salt solutions examined were NaCl 
(). KCI (), CaCN (O), SrCl : (A), and MgCl 2 (O). The volume of ne- 
matocysts in each solution is expressed as a percentage of the original 
volume of nematocysts in ASW. Vertical bars represent standard devia- 
tions; some SD bars are omitted for clarity. 



(one-way ANOVA, P > 0.25). Thus the volume of the 
nematocysts increased with increasing concentration of 
cations if the cation was monovalent, but decreased if the 
cation was Mg :+ or Sr + . The volume of the nematocysts 
was smaller in 1 A/CaCl 2 than in 1 A/MgQ 2 (P < 0.01). 

The volumetric behavior of nematocysts isolated in DW 
was almost the same as that of nematocysts isolated in 
ASW (Fig. 2). When the concentration of external K + was 
increased, the volume of isolated nematocysts increased 
(regression analysis, P < 0.05). Though regression between 
the volume of nematocysts and concentration of Na + was 
not significant, nematocysts immersed in 1 M NaCl were 
larger than those immersed in 1 mAf NaCl (Duncan's 
multiple range test, P < 0.05). Nematocysts immersed in 
1 M Cad: and SrCl 2 were smaller than those immersed 
in 1 mM solutions (P < 0.01). Nematocysts immersed in 
1 M CaCl 2 or SrCl 2 were smaller than those immersed in 
1 A/MgCl 2 (/ > <0.01). 

A scanning electron micrograph of a nematocyst sample 
prepared for X-ray microanalysis is shown in Figure 3. 
X-ray spectra of nematocysts were different depending on 
the incubation solutions (Fig. 4). The major elements of 
nematocysts incubated in ASW were Ca and Mg in ad- 
dition to sulfur (S), though a small Na-peak was present 
(Fig. 4A). When nematocysts were incubated in 1 M NaCl, 
the Na-peak increased, the Ca-peak remained high but 
the Mg-peak disappeared (Fig. 4B). When nematocysts 
were immersed in 1 M KCI, a small K-peak appeared, 
but the peaks of the other elements were not affected (Fig. 



100 



M. HIDAKA AND K. AFUSO 



120 r 



- 110 
0) 



100 



o 

> 



0) 



2 90 

4) 

DC 



80 




10 



100 



1000 



Concentration of salts (mM) 



Figure 2. Effects of cations of various concentrations on the volume 
of nematocysts isolated from acontia of Calliactis polypus in DW. Ne- 
matocysts isolated in DW were immersed successively in salt solutions 
of increasing concentrations. The symbols are the same as in Figure 1. 
The volume of nematocysts in each solution is expressed as a percentage 
of the original volume of the nematocysts in DW. The verlical bars 
represent standard deviations; some SD bars are omitted for clarity. 



4C). Nematocysts incubated in 1 A/ CaCl 2 showed large 
Ca- and small Na-peaks in addition to the S-peak (Fig. 
4D). When nematocysts were incubated in 1 M MgCl 2 , 
the Mg-peak increased (Fig. 4E). Nematocysts incubated 
in 1 A/ SrG 2 showed large Sr- and small Ca-peaks in ad- 
dition to the S-peak (Fig. 4F). When discharged nema- 
tocysts were analyzed, peaks of metals were absent re- 
gardless of the incubation solutions. 

Table I shows the relative abundance of metal cations 
in undischarged nematocysts that were isolated in DW 
and then incubated in various salt solutions. Ca accounted 
for about 50% of the metals in nematocysts immersed in 
ASW or 1 A/ MgCl 2 and more than 50% in nematocysts 
immersed in 1 A/ NaCl or KC1. Ca was replaced substan- 
tially only by strontium (Sr). Most of the Mg disappeared 
when nematocysts were immersed in 1 A/ NaCl, CaCl 2 , 
and SrCl : . Only a small amount of K was present in ne- 
matocysts incubated in 1 A/ KC1. 

Discussion 

Weber (1989) studied the volumetric behavior of iso- 
lated stenoteles of Hydra under different ionic conditions. 
He showed that nematocysts immersed in 1 A/ solutions 
of various salts swell when the concentration of salts is 
lowered, regardless of whether the cations are monovalent 
or divalent. The volumetric behavior of isolated nema- 
tocysts of the sea anemone Calliactis polypus was different 
from that of Hydra nematocysts. Calliactis nematocysts 
appeared to shrink in concentrated solutions of divalent 
cations as in Hydra nematocysts, but swelled in concen- 



trated solutions of monovalent cations. Thus the volu- 
metric behaviors of the marine anemone nematocysts and 
the freshwater Hydra nematocysts are different in solu- 
tions of monovalent cations. 

Weber (1989) showed that the volumetric behavior of 
Hydra nematocysts immersed in salt solutions of various 
concentrations can be accounted for by a Donnan-equi- 
librium model. The Donnan potential generates an asym- 
metrical distribution of ions across the capsule wall. Ac- 
cording to Weber's simulation studies, the difference in 
total ion concentration between the inside and outside of 
the capsule increases as the external salt concentration is 
lowered from 3 M to 0. 1-0.01 A/. When the external salt 
concentration is further lowered, the osmolarity difference 
drops due to protonation of polyanions, unless the exter- 
nal pH is high. The volume of Calliactis nematocysts, 
however, decreased as the external concentration of 
monovalent cations was lowered from 1 to A/. Thus the 
volumetric response of the sea anemone nematocysts to 
monovalent cations cannot be accounted for by the simple 
Donnan-equilibrium model. 

Weber ( 1989) showed that naturally occurring cations 
in Hydra nematocysts can be replaced by externally ap- 
plied cations. If this is true for nematocysts of marine 
cnidarians, cations contained in the isolated capsule might 
be replaced by external cations when nematocysts are im- 
mersed in various salt solutions. Isolated Calliactis ne- 
matocysts contained predominantly Ca 2+ and Mg :+ (Hi- 
daka, 1993). IfCa 2+ and Mg 2+ in the isolated nematocysts 
are replaced by monovalent cations, the internal osmotic 
pressure would increase as one divalent cation is replaced 
by the two monovalent cations required to maintain elec- 
troneutrality. The swelling of the sea anemone nemato- 




Figure 3. Scanning electron micrograph of isolated nematocysts used 
for X-ray microanalysis. The white spot represents the site irradiated 
with an electron beam during the acquisition of spectra. These nema- 
tocysts were isolated in DW and incubated in 1 A/ MgCl : for 10 mm. 



I I I I ( IS in (A [ IONS ON NEMATOCYSTS 



101 



flSM- 1 o.d.d 



C- 1 od<J undl ch 






K-l_OflDED UNDISCHRRGED hCMRTOCVST 9O3 



Figure 4. X-ray spectra of nematocysts incubated in various salt solutions. Nematocysts isolated in DW 
were incubated in ASW or 1 A/ solutions of various salts for 10 min. A, ASW. B, NaCl. C, KC1. D, CaCl,. 
E, MgCl,. F, SrCl 2 . 



cysts in 1 M NaCl or KC1 might be partly due to an ex- 
change of intracapsular divalent cations with monovalent 
cations in the bathing solution. 

X-ray microanalysis of Calliactis nematocysts incu- 
bated in 1 M solutions of various salts for 10 min showed 
that the predominant cation in the nematocysts was not 
necessarily the cation in the incubation medium. The 
predominant cation in nematocysts incubated in 1 M 
NaCl, KC1, MgCl : and CaCl 2 was Ca 2+ . Beside Ca 2+ , Sr + 
is the only cation that replaced most of the cations in 
isolated nematocysts and accounted for about 9C% of the 
cations contained in the nematocysts. This implies that 



Ca :+ in isolated nematocysts was replaced only partially, 
if at all, by externally applied cations. This contrasts with 
the observation that Ca 2 ' and Mg 2+ found in isolated Hy- 
dra nematocysts can be replaced almost completely by 
externally applied cations (Weber, 1989). On the other 
hand, most of the Mg 2 * in isolated nematocysts is replaced 
by externally applied Na 2+ , since Mg 2+ almost disappeared 
after incubation in 1 M NaCl. 

However, it was difficult in this experiment to estimate 
what percentage of Ca 2+ and Mg 2+ in isolated nematocysts 
was actually replaced by externally applied cations. The 
present semiquantitative analyses do not provide a mea- 



102 



M. HIDAKA AND K. AFUSO 



Table I 

Relative abundance of metal '.oneum in isolated Calliactis polypus nematocysts incubated in various sail solutions^ 



Solutions 


Na 




K 


Ca 


Mg 


Sr 


N ; 


ASW 


10.9 


5.8 


1.4 


0.4 


52.5 


4.7 


35.2 





2.4 





5 


1 M NaCl 


26.6 


8.3 


1.5 


0.2 


71.7 


8.4 


0.3 


+ 


0.4 





4 


1 M K.C1 


23.2 


7.1 


2.6 


1.8 


61.3 


7.1 


13.0 


+ 


1.9 





4 


1 M CaCI, 


6.5 


6.1 


1.6 


0.3 


91.7 


6.3 


0.2 


+ 


0.3 





6 


1 M MgCl, 


5.9 


5.9 


0.8 


0.4 


48.7 


5.7 


44.5 


+ 


3.2 





5 


1 M SrCl, 










8.4 


0.3 


0.7 


+ 


0.5 


90.1 0.3 


4 



' The relative abundance of each metal element is shown as a percentage of total metal elements listed. Means SD. 
; Number of nematocysts analyzed. 



sure of the absolute concentration of each metal but only 
their relative abundances. Some of the cations that have 
lower affinity for polyanions in the capsule might be lost 
during washing in DW. If this is the case, the actual con- 
centration of Na f and K 1 in the nematocysts immersed 
in 1 M solutions of these monovalent cations must have 
been much higher than estimated. It is likely that most 
of the Mg 2+ and some of the Ca 2+ in isolated nematocysts 
are replaced by Na + or K + when the nematocysts are im- 
mersed in 1 M NaCl or KC1. Such divalent cation-mono- 
valent cation exchange might at least partly account for 
the swelling of nematocysts in solutions containing high 
concentrations of monovalent cations. 

Nematocysts isolated in ASW and those isolated in DW 
responded similarly to changes in external salt concen- 
tration. Nematocysts isolated in DW swelled as the ex- 
ternal concentration of monovalent cations was increased. 
This is probably because a greater percentage of the di- 
valent cations was replaced by monovalent cations as the 
concentration of external monovalent cations was raised. 
The volume of nematocysts isolated in ASW decreased 
as the concentration of external monovalent cations was 
lowered. It is unlikely that the decrease in the nematocyst 
volume was due to an exchange of intracapsular mono- 
valent cations with external divalent cations, since the 
external solutions did not contain divalent cations. The 
affinity of divalent cations for polyanions might have be- 
come higher, and negative charges on the polyanions 
might have become neutralized, by tightly bound divalent 
cations as the ionic strength decreased. This might account 
for the decrease in the volume of the nematocysts at low 
monovalent cation concentrations. 

Since Ca 2+ reduced the volume of isolated nematocysts 
to a greater extent than Mg 2+ , Ca :+ might have a higher 
affinity for polyanions than Mg 2 + . The substitution ex- 
periments also show that Ca :+ had a higher affinity for 
polyanions than Mg :+ . Binding of Ca 2+ to polyanions may 
mask some of the negative charges on the polyanions and 
thus reduce the number of osmotically active cations 



within the capsule. It is also possible that Ca 2f cross-links 
polyanions to reduce the number of osmotically active 
particles, as suggested by Lubbock et al. ( 198 1 ). Thus, the 
osmotic pressure of the intracapsular fluid is determined 
not only by the Donnan-equilibrium, but also by the se- 
lective binding of Ca 2+ to polyanions in the capsule. The 
present observations suggest that not only divalent- 
monovalent cation exchange but also exchange of intra- 
capsular Ca :+ with Mg :+ in seawater will increase the in- 
ternal osmotic pressure when Ca-containing nematocysts 
come into contact with seawater. 

Lubbock el al. (1981) found high concentrations ofCa 
in undischarged holotrichous isorhiza nematocysts by X- 
ray microanalysis of frozen sections of mesenterial fila- 
ments of the sea anemone Rhodactis rhodosloma and ac- 
rorhagi ofAnihopleura elegantissima. They observed that 
an influx of Na accompanies the efflux of Ca during dis- 
charge. Their observation is consistent with the above hy- 
pothesis that the osmotic pressure of the sea anemone 
nematocysts increases due to the exchange of intracapsular 
divalent cations with Na + in seawater. Lubbock et al. 
(1981) suggested that nematocysts become exposed to 
seawater at the time of discharge after exocytotic fusion 
of the nematocyst membrane with the cell membrane. 
Robson (1973) observed that nematocysts of Rhodactis 
rhodostoma swell up to 150% of their original size when 
they are exposed to seawater immediately before dis- 
charge. This observation suggests that the osmotic pressure 
of sea anemone nematocysts increases when nematocysts 
are exposed to seawater. 

Weber (1990, 1991) found that polyanions contained 
in nematocysts of Hydra and various marine cnidarians 
are poly(7-glutamic acid)s with various degrees of poly- 
merization. As for the cations associated with the poly- 
anions, there are differences between species and between 
types of nematocysts. Tardent et al. ( \ 990) reported that 
the predominant cation of marine cnidarian nematocysts 
is either Ca 2+ , Mg 2+ or K + . The predominant cation of 
the tentacular and acontial nematocysts of Calliactis par- 



EFFECTS OF CATIONS ON NEMATOCYSTS 



103 



is K + as in Hydra nematocysts. The tentacular 
nematocysts of Anthopleura elegantissima and Actinia 
et/uina contain Mg 2+ while the acrorhagial nematocysts 
of these species contain predominantly Ca :+ (Tardent et 
ul. 1990). The differences in the dominant cation among 
nematocysts of marine cnidarians suggest that the hy- 
pothesis of divalent-monovalent cation exchange is not 
applicable to all nematocysts of marine cnidarians 
hut only to those that contain predominantly Ca :+ 
and/or Mg : + . 

Potassium was almost absent even in nematocysts in- 
cubated in 1 M KC1, indicating that K 1 had the lowest 
affinity for polyanions. This means that K + is the ideal 
cation to generate a high internal osmotic pressure if ionic 
distribution across the capsule wall is determined by a 
Donnan-equilibrium. K-containing nematocysts may en- 
counter an osmotic pressure difference large enough to 
trigger discharge when they come into contact with sea- 
water. If they fail to discharge, the internal osmotic pres- 
sure would decrease due to the exchange of intracapsular 
K + with Ca :+ and Mg :+ in seawater. 

The problem with the hypothesis of divalent-mono- 
valent cation exchange, is that exposure of nematocysts 
to seawater alone does not elicit discharge of the nema- 
tocysts, as shown by the fact that undischarged nemato- 
cysts can be isolated from marine cnidarians in ASW (e.g., 
Hidaka and Mariscal, 1988). Yanagita (1959), however, 
found that nematocysts isolated from acontia of the sea 
anemone flaliplanella luciae in 1 M glycerol, discharged 
when immersed in concentrated salt solutions such as 
seawater and isotonic NaCl. He also reported that when 
nematocysts isolated in 1 M glycerol are immersed in di- 
luted salt solutions such as 0.03 A/Cadi, the nematocysts 
become unresponsive to concentrated salt solutions that 
would otherwise elicit discharge of the nematocysts. This 
suggests that the isolated nematocysts can remain undis- 
charged if the salt concentration of the surrounding me- 
dium is increased gradually. During artificial isolation of 
nematocysts in ASW, changes in the ionic composition 
of the surrounding medium might be too slow to cause 
nematocyst discharge. Yanagita (1959) also noted that 
nematocysts liberated into a salt solution through cytolytic 
disintegration of acontia often remain undischarged. 

Furthermore, nematocysts isolated in ASW did not 
discharge in 1 M NaCl or KC1. This suggests that the 
increase in the internal osmotic pressure caused by cation 
exchange may not be large enough to trigger discharge. 
However, the possibility remains that the rate of change 
in the ionic composition of the surrounding medium was 
too slow to trigger discharge, because solution exchange 
was performed by perfusing the test solution drop by drop. 
If the "stopper" (a sealing structure of nematocysts) is 
made of viscoelastic material, whether it fractures or not 
depends on the rate of deformation. Nematocysts would 



discharge only when the rate of increase in the internal 
osmotic pressure exceeds a certain limit. When nemato- 
cysts incubated in 1 A/ CaCl : were treated with 1 M NaCl 
using the present perfusion method, the volume of the 
nematocysts increased by 18% and 3-5% of them dis- 
charged (Hidaka and Afuso, unpub. ob.). It is likely that 
more nematocysts would discharge if the surrounding 
medium is changed more rapidly. The observation that 
isolated nematocysts did not discharge in 1 A/ NaCl or 
KC1 does not necessarily contradict the hypothesis of di- 
valent-monovalent cation exchange. 

Nematocysts can be induced to discharge in media that 
contain no or only small amounts of monovalent cations 
by reagents that rupture disulnde bonds or chelate calcium 
(Hidaka, 1993). These reagents seem to induce discharge 
of nematocysts by weakening the nematocyst "stopper." 
The question of whether such weakening of the "stopper" 
is involved in the in situ mechanism of nematocyst dis- 
charge remains to be investigated. 

The present results show that the volumetric behavior 
of isolated Calliactis nematocysts immersed in various 
salt solutions can be explained by the exchange of intra- 
capsular divalent cations with cations in the external me- 
dium and by the selective binding of Ca 2+ to polyanions 
in the capsule. It remains unknown whether the increase 
in the internal osmotic pressure caused by the cation ex- 
change is large enough to trigger discharge, or whether 
nematocyst discharge involves biochemical modification 
of structural components such as the nematocyst 
"stopper." 

Acknowledgments 

The authors would like to thank Drs. R. A. Kinzie, III 
and M. J. Grygier for critically reading this manuscript. 

Literature Cited 

Gerke, I., K. Zierold, J. Weber, and P. Tardent. 1991. The spatial 

distribution of cations in nematocytes of Hydra vulgaris. Hydro- 

biologia 216/217: 661-669. 
Gupta, B. L., and T. A. Hall. 1984. Role of high concentration 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 Toxins. Drugs, ami Pollutants in Marine Animals. L. Bolis, 

J. Zadunaisky, and R. Gilles. eds. Springer-Verlag, Berlin, Heidelberg. 
Hidaka, M. 1992. Effects of Ca 2f on the volume of nematocysts isolated 

from acontia of the sea anemone Calliactis tricolor. Comp. Biochem. 

Physiol. 101 A: 737-741. 
Hidaka, M. 1993. Mechanism of nematocyst discharge and its cellular 

control. Adv. Comp Envir. Physiol. (in press) 
Hidaka, M., and R. N. Mariscal. 1988. Effects of ions on nematocysts 

isolated from acontia of the sea anemone Calliactis tricolor by different 

methods. J. Exp. Biol. 136: 23-34. 
Lubbock, R., and W. B. Amos. 1981. Removal of bound calcium from 

nematocyst contents causes discharge. Nature 290: 500-501. 
Lubbock, R., B. L. Gupta, and T. A. Hall. 1981. Novel role of calcium 

in exocytosis: mechanism of nematocyst discharge as shown by X- 

ray microanalysis. Proc. Natl. Acad. Sci. USA 78: 3624-3628. 



104 



M. HIDAK.A AND K.. AFUSO 



Mariscal, R. N. 1988. X-ray microanalysis and perspectives on the 
role of calcium and other elements in cnidae. Pp. 95-1 13 in The 
Biology of Nemaivcyv U. A. Hessinger and H. M. Lenhoff, eds. 
Academic Press. San Diego, New York. 

Mazia, D., G. Schatu-r and VV. Sale. 1975. Adhesion of cells to surfaces 
coated with poK ;. sine. Application to electron microscopy. J. Cell 
Binl. 66: I u s 00 

Robson, E. A. i973. The discharge of nematocysts in relation to prop- 
erties of the capsule. Puhl. Setn Mar Binl. Lab. 20: 653-665. 

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

\Vebcr, J. 1989. Nematocysts (stinging capsules of Cnidaria) as Don- 



nan-potential-dominated osmotic systems. Eur J. Bioclwin. 184: 465- 
476. 

Weber, J. 1990. Poly(->-glutamic acid)s are the major constituents of 
nematocysts in Hydra (Hydrozoa, Cnidana). ./. Binl. Chcm 265: 
9664-9669. 

Weber, J. 1991. A novel kind of polyanions as principal components 
of cnidarian nematocysts. Cnnip. Biochem. Physiol. 98A: 285-291. 

Weber, J.. M. Klug, and P. lardent. 1987. Detection of high concen- 
trations of Mg and Ca in the nematocysts of various cnidanans. E.\- 
pcricntia43: 1022-1025. 

Vanagita, T. M. 1959. Physiological mechanisms of nematocyst re- 
sponses in sea-anemone II. Effects of electrolyte ions upon the isolated 
cnidae. / Fac. Sci. Univ. Tokyo Sect. II' 8: 381-400. 



Reference: Biol. Bull 184: 105-113. (February. 1993) 



Hemocyanin Subunit Composition and Oxygen Binding 
in Two Species of the Lobster Genus Homarus and 

Their Hybrids 

CHARLOTTE P. MANGUM 

Bodega Marine Laboratory. University of California. P. O. Box 247. Bodega Bay, California 94923 [ 



Abstract. The monomeric subunit composition and O : 
binding properties of the hemocyanins (Hcs) of Homarus 
americamts. H. gammarus and their hybrids are very 
similar, though not identical. //. americanus He has six 
major electrophoretically separable polypeptide chains; 
H. gammarus He has four major and two minor chains; 
and the hybrid He has four major and one minor chain. 
Four chains co-migrate in all three groups, and the fifth 
chain in the hybrid co-migrates with a fifth chain in H. 
gammarus. Thus, qualitatively, the hybrid He is more 
like that of//, gammarus than H. americanus. a similarity 
reflected in respiratory properties. Although the O : affinity 
of the hybrid hemocyanin appears to lie intermediate be- 
tween that of the two parent hemocyanins at 25C, in 
fact it is significantly different from that of//, americanus 
but not H. gammarus. The cooperativity of the hybrid 
He also differs significantly from that of //. americanus 
but not H. gammarus He. The distinctive properties of 
H. americanus hemocyanin at 25C are believed to be 
due to either or both of two chains: a unique and also 
invariant chain in H. americanus. and one that is present 
in H. gammarus and the hybrids but not in H. ameri- 
canus. H. americanus He also appears to be slightly less 
sensitive to the allosteric modulator L-lactate. No differ- 
ence in CaCl 2 sensitivity was found. At lower temperatures 
respiratory properties are indistinguishable. In adult H. 
americanus that had been held under identical conditions 
for long periods, variation in subunit pattern was not en- 
tirely absent, but it was smaller than that found in natural 
populations of other species. No differences in O 2 binding 
at 25C were found in morphs differing qualitatively in 



Received 3 June 1992; accepted 8 October 1992. 
Permanent address: 'Department of Biology, College of William & 
Mary, Williamsburg, VA 23185-8795. 



one chain and quantitatively in two others. No effect of 
a combination of rearing temperature and diet was found 
on the He subunit composition of juveniles. 

Introduction 

The arthropod hemocyanins (Hcs) are multiples of 
hexamers built of 70-80 kDa polypeptide chains. Often 
the 2 X 6-mers predominate in the bloods of adult decapod 
crustaceans, including the lobster Homarus americanus 
(Olson et ul.. 1988). The number of different monomers 
is usually large, with a dozen or more found in several 
species of Uca (Sullivan et ai. 1983: Callicott and Man- 
gum, 1992; Mangum, 1992 and unpub. data). The mono- 
mers have been grouped into one of four categories on 
the basis of their electrophoretic mobilities, immunolog- 
ical reactions and roles in oligomer assembly (Markl, 
1986). Within a species, the monomeric heterogeneity also 
plays a functional role in respiratory adaptation during 
the adult stage (Mason et ai, 1983; Mangum and Rainer, 
1988; deFur et ai. 1990; Mangum et ai. 1991). By com- 
paring morphs, the functional differences have been at- 
tributed to particular electrophoretic bands (Mangum and 
Rainer, 1988; Mangum et ai, 1991; Mangum, 1992). 

The role of He subunit composition in bringing about 
functional differences between species is less clear. A sur- 
vey of forty-two species of various degrees of taxonomic 
relatedness suggests a high degree of specificity (Reese and 
Mangum, 1992). More intensive investigation of the Hcs 
of seven species of the genus Uca. which are extremely 
polymorphic as well as heterogeneous, supports the in- 
ference of species specificity (Callicott and Mangum, 1992; 
Mangum, 1992; C. P. Mangum, unpub. data). In every 
case, including sibling species such as U. panacea and 
pugilator, even low frequency He morphs of a species can 
be readily distinguished from those of another. Functional 



105 



106 



C. P. MANGUM 



properties can also differ in sibling congeners with different 
latitudinal ranges. In comparisons of congeners that are 
less closely related, however, functional properties are 
more clearly related to environmental factors than to 
phylogenetic allinity or subunit composition (Reese and 
Mangum, 1992). 

In the only two species in which the effect of laboratory 
acclimation has been examined, the subunit phenotype 
of an adult individual is not fixed (Mason ct a/.. 1983; 
deFur el a!.. 1990; Callicott and Mangum, 1992). In Cal- 
lincctes sapidus. moreover, the variation both in the lab- 
oratory and in nature can be related to environmental 
factors such as salinity and hypoxia (Mangum, 1990; Pihl 
ct ai. 199 1;C. P. Mangum and S. P. Baden, unpub. obs.). 
Thus the members of the highly polymorphic samples of 
natural populations may have been acclimated to different 
environmental (or nutritional) conditions. 

Here I report data for the monomeric subunit com- 
position and oxygen binding of the Hcs of the adults of 
two species of lobsters in the genus Homarus and the 
hybrid progeny of their spontaneous matings. The two 
parent species had been brought from their native Atlantic 
habitats to the Bodega Marine Laboratory, where they 
were held under identical conditions for periods far longer 
than the species investigated previously. They are known 
to be highly homozygous at 41 loci and, at most loci, the 
allozymic phenotypes of the two are either extremely sim- 
ilar or identical. It is believed that the two speciated al- 
lopatrically when isolated for the first time during the 
Pleistocene (Hedgecockrt al.. 1977). In one species, I also 
examined the He subunit composition of juveniles which 
had been reared on either of two diets, and at different 
temperatures. 



Materials and Methods 



The sample 



All available adults, a total of 36. were examined; they 
were large (28-42 cm from rostrum to tail), intermolt 
individuals. Two (one of each sex) belong to Homarus 
gammarus (Linnaeus), formerly known as H. vulgaris: 
they were collected near lona, Scotland in 1975. They are 
the sole survivors of the larger sample characterized by 
Hedgecock et al. in 1977. Twenty-five adults ( 16 females, 
9 males) are members of Homarus americanus H. Milne 
Edwards. All but one were caught on various dates in 
1988-92 in waters surrounding Martha's Vineyard, Mas- 
sachusetts, and had been held in the mariculture facility 
at the Bodega Marine Laboratory for periods ranging from 
three months ( 1 individual) to more than three years (3 
individuals). One individual of H. americanus (age > 6 
years) was born in the Bodega Marine Laboratory. Nine 
hybrid adults (7 fertile females, 2 infertile males) were 
progeny of spontaneous matings between //. gammarus 



and americanus. Most were produced in 1983 by a female 
H. gammarus and a male H. americanus: one, of un- 
known parentage, was born in 1978. 

All adults had been fed the same diet of surf fish and 
shrimp, and had been held under identical photoperiods 
in the running seawater system of the Bodega Marine 
Laboratory. A seven year compilation of data ( 1 985-9 1 ) 
indicates that the water temperature ranges from 10 to 
15C, and usually varies within about two degrees; over 
the four month period of sampling the salinity varied from 
32.5-33.5%o, which is typical. 

The He subunit composition of 14 juvenile //. amer- 
icanus (8-10 cm length, both sexes), which had hatched 
in the Bodega Marine Laboratory 20-22 months earlier, 
was also examined. Half of these animals, which were 
their natural color, had been fed since stage IV a diet of 
brine shrimp, fish and crabs, and had been held at the 
seawater system temperature. The other half, phenotypic 
albinos, had been fed a diet based on casein; for the past 
year they had been held at room temperature (ca., 23C). 
The diets, rearing conditions and molt history of animals 
such as these were described in detail by Baum ( 1990). 

Preparation oj material and electrophoresis 

Blood was taken from the base of the last leg and serum 
expressed from the clot in a tissue grinder. After centrif- 
ugation an aliquot of the material was dissociated to its 
monomers by dilution with 0.01 mol I" 1 EDTA + 0.05 
mol r 1 Tris (pH 8.9), to reduce light scattering; He con- 
centration was estimated from the absorbance of disso- 
ciated material at 338 nm (Bausch & Lomb Spectronic 
2000 spectrophotometer), using the extinction coefficient 
reported by Nickerson and van Holde (1971). An addi- 
tional aliquot was diluted (1:10 or 1:30, depending on 
concentration) with the dissociating buffer for electro- 
phoresis, and the remainder frozen for future use. Ab- 
sorbance of the material from several individuals, detailed 
below, was compared at 280 and 338 nm. 

PAGE electrophoresis of native monomers was carried 
out at constant current according to Hames and Rickwood 
(1985). Following determination of the He phenotype in 
each individual, the variants among H. americanus were 
examined several times in adjacent lanes on the same 
gels. Representatives of each of the three groups were also 
compared many times on the same gels. Finally, gels were 
overloaded with six times the usual amount of material, 
the presence of Cu was determined according to Bruyn- 
inckx et al. (1978), and then the gels were stained as usual 
with Coomassie Blue. 

Oxygen binding 

On the basis of the PAGE, particular individuals were 
selected for a second bleeding, performed within a week 



HEMOCVANIN-O, BINDING AND SUBUNIT COMPOSITION IN LOBSTERS 



107 



of the final phenotype determination. Serum was dialyzed 
overnight, against seawater for most of the measurements 
or against a Tris maleate buffered salt for the experiments 
on inorganic ion sensitivity. Oxygen binding was deter- 
mined within a few days, using the cell respiration method 
(Mangum and Lykkeboe, 1979). 

Data analysis 

Bohr plots of the values for P 50 (oxygen affinity) were 
described by regression lines and their 95% confidence 
intervals compared. Mean values for n 50 (cooperativity) 
and He concentration were compared by Student's /-test. 
The data for CK binding as a function of [Cad:] were 
analyzed similarly. However, the nonlinearity of the re- 
sponse of Psn to [NaCl] and [Na^SOj] precluded statistical 
analysis. 

Results 

Hemocyanin concentration 

Adults of Homarus americanus had significantly 
(P = .02) higher levels of He [6. 1 1 (0.41 S.E.) g 100ml '] 
than the hybrids [4.18 (0.70 S.E.) g 100 ml" 1 ]. The values 
for the two members of//, gammarus (2.75 and 4.84 g 
100 mr 1 ) also fall below the 95% confidence interval 
around the mean of the H. americanus sample. In the // 
americanus data there is no clear trend with length of 
time in the laboratory, suggesting that the nutritional state 
of the animals was good. The juveniles of this species had 
considerably lower He concentrations [1.02 (0.15) g 
100 mr 1 ]. which were unrelated to diet. 

Monomeric subunit composition 

The two adult Homarus gammarus had identical He 
phenotypes, which were also the same as that of one of 
the two individuals examined two years earlier (C. P. 
Mangum, unpub. obs.). Four high density (or major) and 
two intermediate density (or minor) electrophoretic bands 
separated by charge (Fig. 1 ). All six were positive for Cu. 

The 25 adults of//, americanus exhibited very similar 
but not identical He phenotypes (Fig. 1 ). As many as eight 
bands separated on the lower third of the gels, four of 
which had co-migrants in H. gammarus (Fig. 1 ). The two 
most anodic bands ( 1 and 2) were always present in trace 
quantities, if at all. Material at their position appeared to 
quench the fluorescence of bathocuproine sulfonate, in- 
dicating the presence of Cu. However, it was not possible 
to ascertain the site of the quenching more precisely, only 
one of the two may contain Cu. These bands had no co- 
migrants in H. gammarus or the hybrids. 

In H. americanus bands 3-8 could reach high concen- 
trations. The gels on which the best separation was ob- 
tained exhibited less density in the middle of the material 



designated as bands 3 and 4, suggesting the presence of 
two chains that are similar in charge and extremely dif- 
ficult to resolve. Moreover, the leading edge of this ma- 
terial clearly co-migrated with bands 2 in the hybrid and 
3 in H. gammarus. whereas the trailing edge clearly lagged 
behind. Thus I assigned two numbers (3 and 4) to this 
position of the H. americanus material, even though the 
separation was not great enough to photograph. In ad- 
dition, I was not able to decide whether the trailing ma- 
terial was present in all 25 individuals. The quantities of 
chains 6 and 7 in //. americanus are similar to the cor- 
responding ones in //. gammarus, but chains 4 and 8 
always occurred in higher levels in H. americanus than 
H. gammarus (Fig. 1 ). 

In the early PAGE, band 5 in H. americanus did not 
appear to be sharply delineated at its leading and trailing 
edges. It was the only high density band that clearly varied 
qualitatively as well as quantitatively, ranging from absent 
(3 adults) to low concentration (5) to high concentration 
(17). Since I suspected that this band might not be a He 
chain, I examined the ratio of the absorbance at the protein 
peak (280 nm) and the active site (338 nm). According 
to this index, however, the total Cu content of H. amer- 
icanus samples containing maximal levels of band 5 did 
not clearly differ from those that lacked it; nor did it differ 
from the samples from //. gammarus and the hybrids. 
none of which contained co-migratory material. In ad- 
dition, on subsequent gels band 5 was as sharply delineated 
as the rest (Fig. 1). 

Bands 6 and 8 varied quantitatively, though the mag- 
nitude was not great. They decreased concomitantly to 
intermediate levels in two of the 25 animals; band 8 was 
intermediate in two additional individuals in which band 
6 remained maximal. Band 7 appeared to be absent in a 
single individual in which 6 and 8 were maximal. This 
female, from which larvae had hatched two months ear- 
lier, had been in the laboratory for only three months. 
All other individuals had maximal levels of these three 
chains. 

Although I did not investigate material held at tem- 
peratures above freezing, there was no correlation between 
phenotype and age of frozen preparations; this has been 
true in my experience with Hcs from all species examined 
thus far. In the present case the same banding pattern was 
observed before and after four months. Finally, material 
prepared on a second occasion from four of the same 
individuals three months after the first bleeding showed 
no change in phenotype. 

The hybrids, all adults, exhibited a single phenotype 
which did not vary quantitatively or qualitatively. With 
the exception of the higher levels of band 2, it resembles 
the phenotype of H. gammarus more than that of H. 
americanus (Fig. 1 ). The hybrid He has four major chains 
and one minor chain, all of which correspond in mobility 



108 



C. P. MANGUM 




1 
2 











H. americanus 



hybrid H. gammarus 



1 

i a 

3 



4 
m 5 
6 

Figure 1. Banding patterns of the dissociated hemocyanins of the two parent species of Homarus and 
their hybrids, and a diagram illustrating the correspondence (arrows) of band positions. The anode is at the 
top. In the gel for // americanus, each pair of lanes shows a sample from a different individual in higher 
(left) and lower (right) concentrations. The lanes on the far right were overloaded to show the anodic material 
(numbered I and 2) that occurs in trace quantities. The cathodic triplet of bands in this species is more 
clearly shown in lanes that were not overloaded. The middle panel shows // gammarus ( 1 individual) and 
the hybrid He ( 1 individual) in alternating lanes. 



to one of the six chains of H. gammarus. The hybrids 
differ from both parents, but from H. gammarus only in 
the absence of the most anodic band (H. gammarus band 
1 ) and the higher levels of hybrid band 2. They differ from 
H. americanus in the absence of its three most anodic 
bands (H. americanus bands 1-3), in the absence of H. 
americanus band 5, in the consistently lower quantity of 



their most cathodic chain (hybrid band 5) and in the pres- 
ence of a distinctive band 1. 

As in all other species I have examined (e.g., 
Mangum et a!., 1985; Mangum, 1992), the phenotypes 
of males and females in each of the three groups 
were indistinguishable no sex specific material was 
present. 



HEMOCYANIN-O, BINDING AND SUBUNIT COMPOSITION IN LOBSTERS 



109 



The juvenile H. americanus were indistinguishable 
from the adults. Six of the seven members of each dietary- 
thermal group had the maximum number of bands. One 
in each group lacked chain 5. Chains 6-8 were invariably 
present in maximal concentrations. 

Oxygen binding 

First, the intraspecific variation in //. americanus was 
examined. One adult lacked band 5 and also had minimal 
(= intermediate) levels of chains 6 and 8; at 25C, however, 
the oxygen binding properties of its He (stripped of organic 
co-factors) were indistinguishable from those of another 
individual containing maximal amounts of all eight bands. 
Therefore the data have been combined for presentation. 
The coefficient of determination (r) for the regression 
line describing P 50 in Figure 2 is 0.962, further affirming 
the absence of a perceptible effect of phenotype. The single 
individual with low quantities of band 7 had been sacri- 
ficed at the time the O 2 binding measurements were per- 
formed. 

Second, the Hcs of the two parent species were com- 
pared. At all but the lowest pH investigated. He O : affinity 
at 25 C is significantly lower in H. gammarus than H. 
americanus, though the difference is fairly small (Fig. 2). 
Third, the hybrid He was compared with each of the par- 
ent Hcs. Whereas the data for the hybrid He appear to be 
intermediate between those for the two parent species, the 
difference from //. gammarus is not significant even in 
the middle of the pH range investigated. In contrast, the 
difference between the hybrid and //. americanus is 



significant throughout most of the pH range exam- 
ined (>7. 2). 

The mean value for cooperativity is somewhat smaller 
(P = .001) in H. americanus (3.24 .11 S.E.) than //. 
gammarus and the hybrids (3.95 .13), which do not 
differ from one another (P = .15). Thus the respiratory 
properties of the hybrid He are also more like those of//. 
gammarus than H. americanus. 

At lower temperatures, the significant differences dis- 
appear completely. Ninety-five percent confidence inter- 
vals around regression lines fit to the O : affinity data in 
Figure 3 overlap fully throughout the pH range investi- 
gated. Often this is true because the numerical values di- 
minish and are thus more difficult to distinguish, but in 
this example there is not even an apparent trend. Mean 
values for cooperativity do not differ (P = .2-. 8). As a 
result, //. americanus He is less temperature sensitive than 
the other two Hcs, though only in the 15-25C range. 
For that range, the apparent heat of oxygenation (AH) is 
only 2.4 kcal mor' for H. americanus He (pH 7.6), 
whereas the value for the hybrid He is 5.6, and the value 
for H. gammarus He is -6.6. For the range 5-15C the 
value of AH for all three Hcs is -9.4 kcal mol~' 
(same pH). 

//. americanus He is slightly less sensitive to the allo- 
steric effector L-lactate (Fig. 4) than H. gammarus He; 
once again, the sensitivity of the hybrid He appears to be 
intermediate. At pH 7.6 the addition of 10 mmol 1~' lac- 
tate changes log P 50 of H. americanus He by 0.166, H. 
gammarus He by 0.232, and the hybrid He by 0.203. 
However, O ; affinity in H. gammarus and the hybrids is 



so 



40 



20 



10 
8 



o . 




5 - 



A A 

c 

' o * 



7.2 



7.6 



8.0 



7.2 



7.6 



8.0 



Figure 2. Oxygen binding at 25C of Homarus americanus (closed circles, solid lines), H. gammarus 
(open circles, dotted lines) and their hybrid (triangles, dashed lines) hemocyanins. The curves are fitted 
regression lines 95% confidence intervals. 0.05 mol T' Tris maleate buffered seawater. Material obtained 
from two individuals of H americanus was used (see text), whereas H gammarus and the hybrids are 
represented by a single individual. 



110 



C. P. MANGUM 



S 4 



15 C 



5 C 




;-.** 

, i , i 


A 

00 



o 
in 

Q_ 



70 
SO 

30 



10 

7 
5 




o, 



7.0 7.5 8.0 7.0 7.5 

PH 



8.0 



Figure 3. Oxygen binding al 1 5 and 5C of//, americanus (closed circles), H gammarus (open circles) 
and their hybrid (triangles) hemocyanins. The regression lines and confidence intervals were omitted for 
clarity. 0.05 mol 1~' Tns maleate buffered seawater. Origin of material as in Figure 2. 



not quite significantly different in the presence of lactate, 
even in the middle of the pH range. In the presence of 
lactate, O : affinity of //. gammarits He remains signifi- 
cantly lower than that of H. americanus throughout the 
pH range investigated. In contrast, the hybrid He has a 
significantly lower O 2 affinity than that of H. americanus 
He only at high pH. In all three groups cooperativity is 
significantly diminished in the presence of lactate. The 
mean value drops from 3.2 to 2.86 .05 S.E. (P = .05) 
for H americanus He, from almost 4 to 3.06 .24 (P 
= .05) for H. gammarus He, and from almost 4 to 2.95 
.29 (P = .002) for the hybrid He. 



The sensitivity of the three Hcs to CaCh is indistin- 
guishable (Fig. 5). Regression lines and their 95% confi- 
dence intervals overlap fully throughout the concentration 
range investigated. Mean values for cooperativity do not 
differ (P = .50-.75). 

NaCl clearly raises He O ; affinity and lowers cooper- 
ativity of//, americanus He (Fig. 5). Once again, however, 
the different morphs were indistinguishable, and the data 
were combined for presentation. In contrast to the allo- 
steric effect of Ca 2+ . the relationship between P 50 and NaCl 
is nonlinear on logarithmic coordinates. I used high con- 
centrations of Na2SO 4 , prepared from the decahydrate. 



too 

70 
50 

30 



o 

n 10 

L 7 

5 




7.2 



7.6 



8.0 



7.2 



7.6 



8.0 



7.2 



7.6 



B.O 



Figure 4. Lactate sensitivity of//, americanus (left panel: closed circles, solid regression lines 95% 
confidence intervals), H. gammarus (right panel: open circles, dotted lines) and their hybrid (middle panel: 
triangles, dashed lines) hemocyanins. Origin of material as in Figure 2. Control curves reproduced in each 
panel from Figure 2. 25C, 0.05 mol I" 1 Tris maleate buttered seawater. 



HEMOCYANIN-O, BINDING AND SUBUNIT COMPOSITION IN LOBSTERS 



111 



o 

in 



5.0 
4.5 
4.0 


O* * * 


3.5 j 

3.0 
7 S 


f 8 o * A 
* 

i i i 



o 

7 i 


| 


O 


g 






5 


- 


o. 

e 


4 













3 


~ 


o 


2 


i 


l l I l i 



1.8 - 




0.0 



0.5 



1 .0 



1.5 



log free [CaCI ] 



2.0 



1.5 



1 .0 



o o 



o 



8 



0.0 0.5 1.0 1.5 2.0 2.5 3.0 

log free [NaCI] or [Na 2 SOj 



Figure 5. Inorganic ion sensitivities of lobster hemocyanins. The units of free ion concentrations (antilogs) 
are mmol l~'. Left panels: H amcricanus (closed circles, solid lines). //. gammarus (open circles, dashes) 
and their hybrids (triangles, dots). 0.05 mol 1~' Tns maleate buffer (pH 7.7) + O.I mol T 1 NaCI. Right 
panels: the response of H americanus He to NaCI (closed circles) and Na : SO 4 (open circles). 0.05 mol 1' 
Tris maleate + 0.01 mol 1~' CaCl : . 25C. Ongin of matenal as in Figure 2. 



to examine specificity. The response differed very little 
from that of NaCI (Fig. 5), and the apparent difference 
may lie within the error of preparing an accurate solution 
of a highly hydrated salt (especially at a marine labora- 
tory). 

Discussion 

The essentially non-specific sensitivity of Homarus 
americanus He to NaCI is further evidence that the in- 
organic ion responses of the crustacean Hcs are not all 
alike. The response of this He differs from that of portunid 
crab Hcs (Truchot. 1975; Mason el al, 1983), which are 
insensitive to NaCI, but resembles that of penaid shrimp 
Hcs (Brouwer et al.. 1978; Mangum and Burnett. 1985). 
From a physiological point of view, however, NaCI sen- 
sitivity is unlikely to be important in H. americanus, a 
stenohaline species. 

According to Hedgecock et ill. ( 1977 and pers. comm.), 
the genetic distance between the two parent species of 
Homarus. though significant, is so small that the numer- 
ical value is closer to expectation for subspecies than spe- 
cies. Thus it is of particular interest that the present find- 
ings support the inference of species specificity of He sub- 



unit composition (Reese and Mangum, 1992). Although 
H. gammarus is monomorphic for the common H. amer- 
icanus allele at 30 allozymic loci, neither of the two parent 
Hcs in the present sample could be confused with the 
other. As in the sibling species of Uca (Mangum, 1992 
and unpub. obs.), this inference is true in spite of intra- 
specific variation. In H. americanus. band 3 is both di- 
agnostic of the species and, at least in the present sample, 
invariant. Material that co-migrates with chains 1 and 2 
of H. gammarus is clearly absent from H. americanus. 
Furthermore, the hybrid He is structurally distinct from 
either parent. 

The present findings also support the inference of little 
interspecific genetic distance. Even though they are not 
identical, the two parent Hcs are more similar than any 
of the ca. 50 Hcs we have examined thus far, with the 
exception of Menippe adina and M. mercenaria Hcs 
(Reese 1989). Like the lobsters, these two sibling species 
of stone crabs are also believed to have speciated recently, 
and they also hybridize spontaneously (Bert, 1986). 

In both structural and functional properties the hybrid 
He resembles that of one parent more than the other. It 
has only one less chain than H. gammarus He but several 
fewer than H. americanus He. The electrophoretic be- 



112 



C. P. MANGUM 



havior of each of the five hybrid chains is identical to that 
of some one of the // gammanis chains, whereas hybrid 
chain 1 has no co-migrant in H. americanus. These re- 
lationships are reflected in the O : binding of the Hcs in 
a complete saline, though only at high temperature. 

In stage IV through adult H. americanus, SDS PAGE 
separates three He chains (Olson el ai. 1988; Olson and 
McDowell, 1989). As is often the case (e.g., Sullivan et 
al., 1983), additional bands are revealed when the sepa- 
ration is made by charge. 

In neither juveniles nor adults of this species can the 
He be categorized as strictly monomorphic at the level of 
quaternary structure, despite prolonged acclimation of the 
donors. Moreover, in juveniles the variation is distinctive 
of neither the stage nor the thermal-nutritional history. 
In both stages, however, the variation is much smaller 
than in samples of natural populations of several species 
of brachyuran crabs (Mangum, 1990, 1992; Callicott and 
Mangum, 1992). This generalization is true of respiratory 
properties of the adults as well. Although the sample size 
is much smaller in the present investigation, the inference 
remains unchanged when the comparison is made with, 
for example, the 14-20 individuals of Callinectes sapidus 
investigated by Mangum el al. in 1991. 

I emphasize that the small amount of He variation 
found here may not accurately represent natural popu- 
lations of H. americanus (much less H. gammanis). Nor 
is it clear that the lack of variation results from prolonged 
acclimation rather than limited genetic diversity, as found 
at other loci (Tracey et al., 1975; Hedgecock el al., 1977). 
However, I note that the inference of allozymic similarity 
in the species was made from samples of populations on 
either side of Cape Cod but not Cape Hatteras, the greater 
geographic barrier (e.g., Friedrich, 1973; National Geo- 
graphic Society, 1985). 

The present findings suggest that, given the common 
acclimation, the differences observed both within H. 
americanus, and between this species and the other two 
groups, represent a fixed condition in an adult individual. 
Although only intermolt animals were investigated here, 
the finding of no change with molt stage in Callinectes 
sapidus (Mangum et al.. 1985) has recently been con- 
firmed in H. americanus (N. B. Terwilliger, pers. comm.). 
More important in the present context, the virtual identity 
of most respiratory properties of the three Hcs appears to 
reflect the notable similarity of the electrophoretic phe- 
notypes. Conversely, it is reasonable to suggest that the 
slightly higher O 2 affinity and lower cooperativity of H. 
americanus He at high temperature are due to the chains 
that are unique to one of the three groups. Perhaps the 
most likely candidate is chain 2 in //. gammanis (= 1 in 
the hybrids), which is absent in H. americanus. However, 
the possibility that band 3 is invariant as well as unique 
to //. americanus cannot be excluded. Bands 1 and 2 are 



never present in H. americanus in more than trace quan- 
tities, and morphs containing or lacking chain 5 did not 
differ in O 2 binding. The latter inference would be un- 
warranted only if the effect of chain 5 was exactly com- 
pensated by an equal and opposite effect of chains 6 and 
8, which were also variables in the comparison. 

Acknowledgments 

Supported by NSF DCB 88-16172 (Physiological Pro- 
cesses). I am extremely grateful to the University of Cal- 
ifornia for a Research Fellowship and to the Bodega Ma- 
rine Laboratory for its unfailing hospitality. 

Literature Cited 

|{:iiiin. N. A. 1990. Studies on the role of dietary protein and lecithin 
in molting and cholesterol transport in juvenile lobsters, Homanis 
sp. M. A. Thesis. Sonoma State University, Rohnert Park. Cal.. 65 
pp. 

Bert, T. M. 1986. Speciation in western Atlantic stone crabs (genus 
MenippeY the role of geologic processes and climatic events in the 
formation and distribution of species. Mar. Biol. 93: 157-170. 

Brouwer, M., C. Bonaventura, and J. Bonaventura. 1978. Analysis of 
the effect of three different allosteric ligands on oxygen binding by 
hemocyanin of the shrimp Pcnaeus setiferus. Biochem. 17: 2148- 
2154. 

Bruyninckx, W. J., S. Gutteridge, and H. S. Mason. 1978. Detection 
of copper on polyacrylamide gels. Analyt. Biochem. 89: 174-177. 

Callicott, K. A., and C. P. Mangum. 1992. Phenotypic variation and 
lability of the subunit composition of the hemocyanin of I'ca pugi- 
lulor. J Exp. Afar. Biol. Ecol. (in press). 

deFur, P. L., C. P. Mangum. and J. E. Reese. 1990. Respiratory re- 
sponses of the blue crab Callinectes sapidus to longterm hypoxia. 
Biol. Bull 178:46-54. 

Friedrich, H. 1973. Marine Biology. University of Washington Press. 
United Kingdom. 474 pp. 

Hames, B. D., and D. Rickwood. 1985. Gel Electrophoresis of Proteins. 
1RL Press, Oxford. 290 pp. 

Hedgecock, D., K. Nelson, J. Simons, and R. Shleser. 1977. Genie 
similarity of American and European species of the lobster genus 
Homanis. Biol. Bull. 152:41-50. 

Mangum, C. P. 1990. Inducible O, carriers in the crustaceans. Pp. 92- 
103 in Animal Nutrition and Transport Processes. 2. Transport, Res- 
piration and Excretion. Comparative and Environmental Aspects. 
J.-P. Truchot and B. Lalou, eds. Karger, Basel, Switzerland. 

Mangum, C. P. 1992. Structural and functional polymorphism of the 
hemocyanin O 2 transport system of the sand fiddler crab. L'ca pug- 
ilalor. J. Exp. Mar. Biol. Ecoi (in press). 

Mangum, C. P., and L. E. Burnett. 1986. The CO 2 sensitivity of the 
hemocyanins and its relationship to Cl sensitivity. Biol. Bull. 171: 
248-263. 

Mangum, C. P., and G. Lykkeboe. 1979. The influence of inorganic 
ions and pH on the oxygenation properties of the blood in the gas- 
tropod mollusc Busycon canaliculalum. J Exp. Zoo/. 207: 417-430. 

Mangum, C. P., and J. S. Rainer. 1988. The relationship between sub- 
unit composition and oxygen binding of blue crab hemocyanin. Biol. 
Bull 174: 77-82. 

Mangum, C. P., J. Greaves, and J. S. Rainer. 1991. Oligomer com- 
position and oxygen binding of the hemocyanin of the blue crab 
Callinectes sapidus. Biol Bull 181:453-458. 

Mangum, C. P., B. A. McMahon, P. L. deFur, and M. I. VVheatly. 
1985. Gas exchange, acid-base balance and the oxygen supply to 



HEMOCYANIN-O: BINDING AND SUBUNIT COMPOSITION IN LOBSTERS 



1 13 



tissues during a molt of the blue crab. Callincctcx sapidiis Rathbun. 
J. Crn.it. Bioi 5:207-215. 

Mason, R. P., C. P. Mangum, and G. Godette. 1983. The inllucnce of 
inorganic ions and acclimation salinity on hemocyanin-oxygen binding 
in the blue crab Callmeaes sapidiis. Biol. Bull. 164: 104-123. 

Markl. J. 1986. Evolution and function of structurally diverse subunits 
in the respiratory protein hemocyanin from arthropods. Biol. Bull. 
171: 90-115. 

National Geographic Society. 1985. .-I/An <>/ An///; America Wash- 
ington, DC. Pp. 66-67. 

Nickerson. K. \\ ., and K. E. van ilolde. 1971. A comparison of mol- 
luscan and arthropod hemocyanin. I. Circular dichroism and ab- 
sorption spectra. Comp Biochcin P/IVMO! 39B: 855-872. 

Olson, K., N. B. I erwilliger, and J. McDowell Capuz/.o. 1988. Structure 
of hemocyanin in larval and adult lobsters. Am /.mil 28: 47A. 

Olson, K. S., and J. McDowell. 1989. Structure and function of he- 
mocvamn in American lobsters. Am /.mil. 29: 20A. 



Pihl, I.., S. P. Baden, and R. J. Dia/. 1991. Effects of periodic hypoxia 
on distribution of demersal fish and crustaceans. Mar Binl 108: 
349-360. 

Reese, J. E. 1989. Structure and function of crustacean hemocy- 
anins. MA Thesis. College of William and Mary, Williamsburg. 
VA. 75 pp. 

Sullinin, B., L. Pennell, B. Hutchison, and R. Mulchings. 1983. Genetics 
and evolution of the hemocyanin multigene-I. Genetic variability in 
L'ca pugilator from Beaufort, NC. Comp. Biochem. Physiol 76B: 
615-618. 

Tracey, M. L., K. Nelson, D. lledgecock, R. A. Shleser, and M. I,. 
Pressick. 1975. Biochemical genetics of lobsters: Genetic variation 
and the structure of American lobsters (Homanis americanus) pop- 
ulations. ./ Fish. Res Board Can 32: 2091-2101. 

Truchot. J.-P. 1975. Factors controlling the in vitro and in vivo oxygen 
affinity of the hemocyanin of the crab, Carcinus meanas. Resp. 
Phvswl 24: 173-189. 



CONTENTS 



CELL BIOLOGY 

Costas, Eduardo, Angeles Aguilera, Sonsoles Gon- 
zalez-Gil, and Victoria Lopez-Rodas 

Contact inhibition: also a control for cell prolifer- 
ation in unicellular algae? 



DEVELOPMENT AND REPRODUCTION 

Fenteany, Gabriel, and Daniel E. Morse 

Specific inhibitors of protein synthesis do not block 
RNA synthesis or settlement in larvae of a marine 

gastropod mollusk (Haliotis rujescens) 6 

Freeman, Gary 

Metamorphosis in the brachiopod Terebratalia: ev- 
idence for a role of calcium channel function and 
the dissociation of shell formation from settlement 15 



ECOLOGY AND EVOLUTION 

Curtis, Lawrence A., and Karen M. K. Hubbard 

Species relationships in a marine gastropod-tre- 

matode ecological system 25 

Douillet, Philippe, and Christopher J. Langdon 

Effects of marine bacteria on the culture of axenic 
oyster Crassostrea gigas (Thunberg) larvae 36 



Okamura, Beth, and Lita Ann Doolan 

Patterns of suspension feeding in the freshwater 

bryozoan Pltimatella repens 52 

Scheltema, Amelie H. 

Aplacophora as progenetic aculiferans and the coe- 
lomate origin of mollusks as the sister taxon of Si- 
puncula 57 

IMMUNOLOGY 

Rinkevich, H., Y. Saito, and I. L. Weissman 

A colonial invertebrate species that displays a hi- 
erarchy of allorecognition responses 79 

Sawada, Tomoo, Jeffrey Zhang, and Edwin L. Cooper 

Classification and characterization of hemocytes in 

Stvela dava . 87 



PHYSIOLOGY 

Hidaka, Michio, and Kiwamu Afuso 

Effects of cations on the volume and elemental 
composition of nematocysts isolated from acontia 

of the sea anemone Calliactis polypus 97 

Mangum, Charlotte P. 

Hemocyanin subunit composition and oxygen 
binding in two species of the lobster genus Homarus 
and their hybrids 105 



Volume 184 



THE 



Number 2 



BIOLOGICAL 
BULLETIN 







APRIL, 1993 



Published by the Marine Biological Laboratory 




1993 LATE SUMMER 
COURSES AT THE MBL 

History of Biology: Human Genetics in the Twentieth Century 

(AUGUST 1- AUGUST 11, 1993) 
APPLICATION DEADLINE: MAY 21, 1993 

Open to students from a wide variety of backgrounds and ranks who share an interest in 
the history and philosophy of human genetics and eugenics. This course will focus on 
the history of human genetics in the United Slates, Great Britain, France, Germany and 
Russia in the twentieth century. Themes will include clinical and eugenic aspects of 
human genetic studies, the history of efforts to control human evolution, ethical 
questions arising from present as well as past attempts at such control, and the social 
construction of scientific knowledge. Directors: Garland Allen, Washington Univer- 
sity; John Beany, University of Minnesota; and Jane Maienschein, Arizona Stale 
University. 

Methods in Computational Neurosdence (AUGUST 3- AUGUST 31, 1993) 
APPLICATION DEADLINE: MAY 21, 1993 

This intensive, four week computer laboratory and lecture course for 23 advanced 
graduate students, postdoctoral fellows and faculty, will examine how the biophysical 
and biochemical properties of neurons and synapses, together with the architecture of 
neural circuits, produce observed animal behavior. Lecture material will include the 
dynamics of individual neurons and synapses; the use of exact models of single cells 
versus reduced neural models in analysis of networks; the coding and processing of 
external stimuli within nervous systems; development of the nervous system; and 
applied mathematics. In the laboratory, specific aspects of nervous systems will be 
modeled using a UNIX graphic-color workstation and software designed forthe analysis 
of both single-cell dynamics and large network properties. Directors: David Kleinfeld 
andDavidW. Tank, Biological Computation Research Department, Bell Laboratories, 
Murray Hill, NJ. 

Molecular Evolution (AUGUST 8- AUGUST 20, 1993) 
APPLICATION DEADLINE: JUNE 1, 1993 

A series of lectures and discussions exploring multiple approaches to molecular 
evolution, and a computer laboratory for phylogenetic and sequence analysis. Designed 
for a class of 60 established investigators, postdoctoral fellows, and advanced graduate 
students, this two week program will provide a forum for exchange of information 
among organismic and molecular biologists and ecologists. Director: Mitchell L. Sogin, 
Marine Biological Laboratory. 

Cellular and Molecular Neurobiology and Development of the Leech 

(AUGUST 8-Aucusr28. 1993) 
APPLICATION DEADLINE: JUNE 1, 1993 

This course is for 12 graduate and postdoctoral students and independent investigators 
interested in applying diverse experimental approaches (electrophysiology, biophysics, 
cellular and molecular biology) to the study of the nervous system and development of 
a single organism. Students will leam techniques and concepts of modern experimental 
embryology, including lineage tracing, cell cycle analysis and the analysis of gene 
expression by in situ hybridization and immunohistochemical procedures. Students will 
also use patch clamp techniques to study whole cell and single channel transmembrane 
currents in situ and in isolated neurons in culture . Directors: Pierre Drapeau, McGill 
University; and David Weisblat, University of California, Berkeley. 

Pathogenesis of Neuroimmunologic Diseases (AUGUST 15- AUGUST 27, 1 993) 
APPLICATION DEADLINE: MAY 4, 1993 

This course for 30 advanced graduate students, postdoctoral fellows, and junior faculty 
in neurosciences and immunology, and for residents in neurology, neurosurgery or 
psychiatry , will consist of lectures and discussions describing the application of genetic, 
molecular, and cell physiologic concepts and techniques in current use in immunology 
and neurophysiology to the analysis of pathogenesis in the belter known neurologic and 
psychiatric diseases thought to have an immunologic basis. Directors: J. Murdoch 
Ritchie, Yale University; and Byron H. Waksman, Harvard and New York Universities. 

Optical Microscopy and Imaging in the Biomedical Sciences 

(OCTOBER 20-OcroBER 28, 1993) 
APPLICATION DEADLINE: JULY 19, 1993 

Designed for 22 research scientists, physicians, postdoctoral trainees, and advanced 
graduate students in animal, plant, medical, and material sciences, as well as non- 
biologists seeking a comprehensive introduction to microscopy and video- imaging. The 
course consists of lectures, laboratory exercises, demonstrations, and discussions that 
will enable the participant to obtain and interpret microscope images of high quality, to 
perform quantitative optical measurements, and to produce photographic and video 
records for documentation and analysis. Instruction on state-of-the-art equipment will 
be provided by experienced staff from universities and industry. Director: Colin S. 
Izzard, State University of New York at Albany. 

FOR FURTHER INFORMATION AND APPLICATION FORMS contact: 

Ms. Donanne Chrysler, Admissions Coordinator, Marine Biological Laboratory, 

Woods Hole, MA 02543, USA; (508) 548-3705, ext 401 . 



THE 



BIOLOGICAL BULLETIN 



PUBLISHED BY 
THE MARINE BIOLOGICAL LABORATORY 



Associate Editors 









WAV 1 






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

DAVID EPEL. Hopkins Marine Station. Stanford LIniversity 

J. MALCOLM SHICK. University of Maine. Orono 



I 



Editorial Board 



WILLIAM D. COHEN, Hunter College 
DAPHNE GAIL FAUTIN. University of Kansas 

WILLIAM F. GILLY. Hopkins Marine Station. 
Stanford LIniversity 

ROGER T. HANLON, Marine Biomedical 

Institute. 
LIniversity of Texas Medical Branch 



CHARLES B. METZ. LIniversity of Miami 
K. RANGA RAO. University of West Florida 

RICHARD STRATHMANN, Friday Harbor Laboratories, 
University of Washington 

STEVEN VOGEL, Duke University 

SARAH ANN WOODIN. University of South Carolina 



Editor MICHAEL J. GREENBERG, The Whitney Laboratory, University of Florida 
Managing Editor: PAMELA L. CLAPP. Marine Biological Laboratory 



APRIL, 1993 

Printed and Issued by 
LANCASTER PRESS, Inc. 

3575 HEMPLAND ROAD 
LANCASTER, PA 



THE BIOLOGICAL BULLETIN 



THE BIOLOGICAL BULLETIN is published six times a year by the Marine Biological Laboratory. MBL 
Street, Woods Hole. Massachusetts 02543. 

Subscriptions and similar matter should be addressed to Subscription Manager. THE BIOLOGICAL BUL- 
LETIN. Marine Biological Laboratory, Woods Hole, Massachusetts 02543. Single numbers. $35.00. Sub- 
scription per volume (three issues), $87.50 ($175.00 per year for six issues). 

Communications relative to manuscripts should be sent to Michael J. Greenberg. Editor-in-Chief, or 
Pamela L. Clapp. Managing Editor, at the Marine Biological Laboratory. Woods Hole. Massachusetts 02543. 
Telephone: (508) 548-3705, ext. 428. FAX: 508-540-6902. E-mail: pamclirt'hoh. mbl.edu. 



POSTMASTER: Send address changes to THE BIOLOGICAL BULLETIN, Marine Biological Laboratory, 

Woods Hole, MA 02543. 

Copyright ( Q> 1993. by the Marine Biological Laboratory 

Second-class postage paid at Woods Hole. MA. and additional mailing offices. 

ISSN 0006-3185 



INSTRUCTIONS TO AUTHORS 



The Biological Bulletin accepts outstanding original research 
reports of general interest to biologists throughout the world. 
Papers are usually of intermediate length (10-40 manuscript 
pages). A limited number of solicited review papers may be ac- 
cepted after formal review. A paper will usually appear within 
four months after its acceptance. 

Very short, especially topical papers (less than 9 manuscript 
pages including tables, figures, and bibliography) will be pub- 
lished in a separate section entitled "Research Notes." A Re- 
search Note in The Biological Bulletin follows the format of 
similar notes in Nature. It should open with a summary para- 
graph of 150 to 200 words comprising the introduction and the 
conclusions. The rest of the text should continue on without 
subheadings, and there should be no more than 30 references. 
References should be referred to in the text by number, and 
listed in the Literature Cited section in the order that they appear 
in the text. Unlike references in Nature, references in the Re- 
search Notes section should conform in punctuation and ar- 
rangement to the style of recent issues of The Biological Bulletin. 
Materials and Methods should be incorporated into appropriate 
figure legends. See the article by Lohmann el at. (October 1990, 
Vol. 179: 2 1 4-2 1 8) for sample style. A Research Note will usually 
appear within two months after its acceptance. 

The Editorial Board requests that regular manuscripts con- 
form to the requirements set below, those manuscripts that do 
not conform will be returned to authors for correction before 
review. 

1. Manuscripts. Manuscripts, including figures, should 
be submitted in triplicate. (Xerox copies of photographs are not 
acceptable for review purposes.) The original manuscript must 
be typed in no smaller than 12 pitch, using double spacing (in- 
cluding figure legends, footnotes, bibliography, etc.) on one side 
of 16- or 20-lb. bond paper. 8'/2 by 1 1 inches. Please, no right 
justification. Manuscripts should be proofread carefully and er- 
rors corrected legibly in black ink. Pages should be numbered 
consecutively. Margins on all sides should be at least 1 inch (2.5 
cm). Manuscripts should conform to the Council of Biology Ed- 
itors Style Manual, 5th Edition (Council of Biology Editors. 
1983) and to American spelling. Unusual abbreviations should 



be kept to a minimum and should be spelled out on first reference 
as well as defined in a footnote on the title page. Manuscripts 
should be divided into the following components: Title page. 
Abstract (of no more than 200 words). Introduction. Materials 
and Methods. Results. Discussion. Acknowledgments. Literature 
Cited. Tables, and Figure Legends. In addition, authors should 
supply a list of words and phrases under which the article should 
be indexed. 

2. Title page. The title page consists of: a condensed title 
or running head of no more than 35 letters and spaces, the 
manuscript title, authors' names and appropriate addresses, and 
footnotes listing present addresses, acknowledgments or contri- 
bution numbers, and explanation of unusual abbreviations. 

3. Figures. The dimensions of the printed page, 7 by 9 
inches, should be kept in mind in preparing figures for publi- 
cation. We recommend that figures be about 1 '/: times the linear 
dimensions of the final printing desired, and that the ratio of 
the largest to the smallest letter or number and of the thickest 
to the thinnest line not exceed 1:1.5. Explanatory matter gen- 
erally should be included in legends, although axes should always 
be identified on the illustration itself. Figures should be prepared 
for reproduction as either line cuts or halftones. Figures to be 
reproduced as line cuts should be unmounted glossy photo- 
graphic reproductions or drawn in black ink on white paper, 
good-quality tracing cloth or plastic, or blue-lined coordinate 
paper. Those to be reproduced as halftones should be mounted 
on board, with both designating numbers or letters and scale 
bars affixed directly to the figures. All figures should be numbered 
in consecutive order, with no distinction between text and plate 
figures. The author's name and an arrow indicating orientation 
should appear on the reverse side of all figures. 

4. Tables, footnotes, figure legends, etc. Authors should 
follow the style in a recent issue of The Biological Bulletin in 
preparing table headings, figure legends, and the like. Because 
of the high cost of setting tabular material in type, authors are 
asked to limit such material as much as possible. Tables, with 
their headings and footnotes, should be typed on separate sheets, 
numbered with consecutive Roman numerals, and placed after 



the Literature Cited. Figure legends should contain enough in- 
formation to make the figure intelligible separate from the text. 
Legends should be typed double spaced, with consecutive Arabic 
numbers, on a separate sheet at the end of the paper. Footnotes 
should be limited to authors' current addresses, acknowledg- 
ments or contribution numbers, and explanation of unusual 
abbreviations. All such footnotes should appear on the title page. 
Footnotes are not normally permitted in the body of the text. 

5. Literature cited. In the text, literature should be cited 
by the Harvard system, with papers by more than two authors 
cited as Jones el ai, 1980. Personal communications and ma- 
terial in preparation or in press should be cited in the text only, 
with author's initials and institutions, unless the material has 
been formally accepted and a volume number can be supplied. 
The list of references following the text should be headed Lit- 
erature Cited, and must be typed double spaced on separate 
pages, conforming in punctuation and arrangement to the style 
of recent issues of The Biological Bulletin. Citations should in- 
clude complete titles and inclusive pagination. Journal abbre- 
viations should normally follow those of the U. S. A. Standards 
Institute (USASI), as adopted by BIOLOGICAL ABSTRACTS and 
CHEMICAL ABSTRACTS, with the minor differences set out below. 
The most generally useful list of biological journal titles is that 
published each year by BIOLOGICAL ABSTRACTS (BIOSIS List of 
Serials; the most recent issue). Foreign authors, and others who 
are accustomed to using THE WORLD LIST OF SCIENTIFIC PE- 
RIODICALS, may find a booklet published by the Biological 
Council of the U.K. (obtainable from the Institute of Biology, 
41 Queen's Gate, London, S.W.7, England, U.K..) useful, since 
it sets out the WORLD LIST abbreviations for most biological 
journals with notes of the USASI abbreviations where these differ. 
CHEMICAL ABSTRACTS publishes quarterly supplements of ad- 
ditional abbreviations. The following points of reference style 
for THE BIOLOGICAL BULLETIN differ from USASI (or modified 
WORLD LIST) usage: 



A. Journal abbreviations, and book titles, all underlined 
(for italics) 

B. All components of abbreviations with initial capitals 
(not as European usage in WORLD LIST e.g.. J. Cell. Comp. 
Physiol. NOT/ cell. comp. Physiol.) 

C. All abbreviated components must be followed by a pe- 
riod, whole word components must nut (i.e., J Cancer Res.) 

D. Space between all components (e.g., J Cell. Comp. 
Physiol.. not J.Ccll. Comp. Physiol.) 

E. Unusual words in journal titles should be spelled out 
in full, rather than employing new abbreviations invented by 
the author. For example, use Rit I 'isindajjelags Islendinga with- 
out abbreviation. 

F. All single word journal titles in full (e.g.. Veliger, Ecol- 
ogy. Brain). 

G. The order of abbreviated components should be the 
same as the word order of the complete title (i.e.. Proc and 
Trans, placed where they appear, not transposed as in some 
BIOLOGICAL ABSTRACTS listings). 

H. A few well-known international journals in their pre- 
ferred forms rather than WORLD LIST or USASI usage (e.g.. 
Nature. Science. Evolution NOT Nature. Land.. Science. N.Y.; 
Evolution. Lancaster. Pa.) 

b. Reprints, page proofs, and charges. Authors receive their 
first 100 reprints (without covers) free of charge. Additional re- 
prints may be ordered at time of publication and normally will 
be delivered about two to three months after the issue date. 
Authors (or delegates for foreign authors) will receive page proofs 
of articles shortly before publication. They will be charged the 
current cost of printers' time for corrections to these (other than 
corrections of printers' or editors' errors). Other than these 
charges for authors' alterations. The Biological Bulletin does not 
have page charges. 



Reference: Bioi Bull 184: I 15-124. (April, 1993) 



Ooplasmic Segregation in the Medaka 
(Oryzias latipes) Egg 

VIVEK C. ABRAHAM, SUNITA GUPTA. AND RICHARD A. FLUCK 

Department of Biology, Franklin and Marshall College, P.O. Box 3003, 
Lancaster, Pennsylvania 1 7604-3003 



Abstract. Using time-lapse video microscopy, we found 
that ooplasmic inclusions in the fertilized medaka egg dis- 
played two types of movement during ooplasmic segre- 
gation. The first manifested itself as the movement of 
many inclusions (diameter = 1.5-1 1 ^m) toward the an- 
imal pole at about 2.2 pm min~'; this type of movement 
appeared to be streaming. The second type of movement 
was faster (about 44 //m min ') and saltatory; inclusions 
displaying this type of movement were smaller (diameter 
< 1.0 /urn) and moved toward the vegetal pole. The move- 
ment of oil droplets toward the vegetal pole of the egg 
may represent a third type of motion. All these movements 
began only after a strong contraction of the ooplasm to- 
ward the animal pole, which at 25C began 10-12 min 
after fertilization and <3 min after formation of the second 
polar body. 

In eggs treated with microtubule poisons colchicine, 
colcemid, or nocodazole oil droplets did not move to- 
ward the vegetal pole, saltatory motion toward the vegetal 
pole was absent, and the growth of the blastodisc was 
slowed. Eggs treated with 0-lumicolchicine, an inactive 
derivative of colchicine, showed normal movements. 
Colchicine, while not inhibiting formation of the second 
polar body, did inhibit pronuclear migration. These results 
suggest that microtubules are involved in the movement 
of some ooplasmic inclusions, including oil droplets, to- 
ward the vegetal pole; the movement of ooplasmic inclu- 
sions toward the animal pole; and pronuclear migration. 

Introduction 

Eggs of many animal species display a remarkable va- 
riety of movements soon after they are fertilized. Many 
of these movements, known collectively as ooplasmic 

Received 18 May 1992; accepted 25 January 1993. 



segregation, are important for the rearrangement of egg 
cytoplasm during the minutes and hours following fertil- 
ization. In some animals, amphibians and ascidians for 
example, these movements lead to cytoplasmic localiza- 
tion of morphogenetic determinants, which are subse- 
quently segregated to specific cells during cleavage and 
ultimately affect gene expression in the cells that incor- 
porate them (Reverberi, 1971; Davidson, 1976; Illmensee 
et al.. 1976; Jeffery, 1984; Speksnijder el ai, 1990a). 

In contrast to a relatively detailed understanding of oo- 
plasmic segregation in eggs of ascidians, annelids, and 
amphibians (Vacquier, 1981), relatively little is known 
about it in fish eggs. Except for Roosen-Runge's (1938) 
classic study in which time-lapse cinemicrography was 
used to monitor ooplasmic segregation in the zebrafish 
egg (Brachydanio rerio), there have been no published 
reports of the use of time-lapse microscopy to monitor 
segregation in a fish egg. Given the increasing use of fish 
embryos as model systems in the study of development 
(Kimmel, 1989; Powers, 1989; Kimmel et al.. 1990; 
Schindler, 1991), it is important to examine segregation 
in this group of organisms more closely. 

Microtubules are required for ooplasmic movements 
in several taxa of animals, including amphibians (Wak- 
ahara, 1989; Houliston and Elinson, 1991; Peter et al., 
1991), ascidians (Zalokar, 1974; Sawada, 1988; Sawada 
and Schatten, 1989), and annelids (Eckberg, 1981; Shi- 
mizu, 1982; Astrow et al., 1989). Microtubule poisons 
drugs that block either assembly or disassembly of mi- 
crotubules have been useful tools in these studies (Za- 
lokar, 1974; Eckberg, 1981; Shimizu, 1982; Astrow et al., 
1989; Sawada and Schatten, 1989), in which a role for 
microtubules is presumed when a particular movement 
is inhibited by one or more of these poisons. Because 
these poisons can have cytotoxic effects unrelated to their 
effects on microtubules, many studies have compared the 



115 



116 



V. C. ABRAHAM ET AL 



effects of more than one such class of these poisons and. 
also, have used as controls chemically similar derivatives 
that have low affinity for tubulin, the protein subunit of 
microtubules. For example, /i-lumicolchicine, a derivative 
of colchicine (Wilson and Friedkin, 1967), can be used 
as a control for colchicine (Sabnis, 1981; Achler et al., 
1989; Peter el al.. 1991). 

We have studied ooplasmic segregation in the egg of 
the medaka (Oryzias talipes). This large (diameter = 1.2 
mm) clear egg, with its thin peripheral layer of ooplasm 
surrounding a central yolk vacuole, permits microscopic 
study of both the gross movements of ooplasm as well as 
the movement of ooplasmic inclusions. The objectives of 
the present study were ( 1 ) to describe the movements of 
ooplasmic inclusions, and (2) to monitor the effects on 
these movements of three drugs that block microtubule 
assembly (Wilson et al.. 1974; Dustin, 1984, ch. 5; Bray. 
1992, p. 207). 

A preliminary account of these findings has been pub- 
lished (Abraham and Fluck, 1991). 



Materials and Methods 

We removed gonads from breeding medaka (Yama- 
moto, 1967; Kirchen and West, 1976; Fluck, 1978) and 
placed them in a balanced saline solution (BSS; 1 1 1 mM 
Nad; 5.37 mM KC1; 1.0 mM CaCl 2 ; 0.6 mM MgSO 4 ; 
HEPES, pH 7.3). Eggs were removed from the ovary, and 
the long chorionic fibers at the vegetal pole were removed 
with scissors. Eggs were fertilized in BSS (Yamamoto, 
1967) and transferred to a microscope slide on which a 
cover glass was supported by four pillars of petroleum 
jelly. The cover glass was then pressed gently against the 
chorion to flatten a small region of the egg near its equator. 
Such flattening facilitated optical studies and also enabled 
us to roll the egg to achieve the desired orientation. All 
procedures were performed at room temperature (23- 
26C in most experiments); in this temperature range, 
the first cell division begins after about 70 min. Because 
the rate of development varies inversely with temperature, 
we have reported the timing of events not only as "minutes 
after fertilization" but also as "normalized time" (t n ), 
where t n = 1.0 is the time at which cytokinesis begins. 

We monitored the movements of ooplasmic inclusions 
(or parcels) with time-lapse video microscopy, using a Ni- 
kon Optiphot or Diaphot microscope equipped with 
phase-contrast optics and connected through a Dage/MTI 
camera to a Panasonic NV-8050 time-lapse video cassette 
recorder. Using a 40X phase-contrast objective lens, we 
usually focused on a patch of ooplasm near the equator 
of the just-fertilized egg; with the Optiphot, the field of 
view was approximately 140 ^m X 200 /urn (total mag- 
nification = 882X) and with the Diaphot it was approx- 



imately 225 ^m X 325 nm (total magnification = 542X). 
We measured the diameters of inclusions on the screen 
of the video monitor and corrected for scale. To measure 
the speed and direction of movement of the inclusions, 
we placed a transparent plastic sheet over the video mon- 
itor during playback and mapped the paths of randomly 
chosen inclusions at regular time intervals; the length of 
the time interval chosen, usually either 20 s or 40 s, de- 
pended on the average speed of the inclusions at the time. 

To measure the thickness of the blastodisc, we viewed 
it in profile from the side, measured its thickness along 
the animal-vegetal axis, and corrected for scale. To mea- 
sure the volume of the blastodisc, we viewed it in profile 
from the side and used an image analysis program (Mi- 
crocomp Planar Morphometry, Southern Micro Instru- 
ments, Atlanta, Georgia) to measure three parameters 
(area, centroid x and projected .\) of one-half of the blas- 
todisc, after drawing a line that bisected the image along 
the animal-vegetal axis. We then calculated its volume, 
using the following equation: volume = (2?r) (area) (cen- 
troid .v-projected .\). The validity of this method was es- 
tablished by measuring standard objects. To measure the 
thickness of ooplasm elsewhere on the egg, we measured 
its thickness en face in the Z axis of the objective lens, 
using the presence of inclusions as a marker for ooplasm. 

We used two methods to monitor the timing of the 
second meiotic division. In the first, we fixed eggs in 3.7% 
formaldehyde in BSS at regular intervals after fertilization. 
After rinsing away the fixative and staining the nuclei 
with Hoechst 33258 (10 ^g ml 1 in BSS containing 1% 
Triton X-100), we examined the eggs with epifluorescence 
optics. In the second method, we microinjected Hoechst 
33258 (100 Mg ml' 1 , dissolved in 50 mM K 2 SO 4 and 10 
mM HEPES, pH 7.2) into unfertilized eggs, injecting ap- 
proximately 1.5 nl into the ooplasm at about 45 latitude 
from the animal pole. The method for microinjection was 
similar to that used by Ruck et al. (1991), except we used 
a high pressure microinjection system (Narashige IM-200). 
The injection process parthenogenetically activated the 
eggs, while Hoechst 33258 stained the maternal nuclear 
DNA. After placing these eggs between a coverglass and 
slide, we recorded movements of the ooplasmic inclusions, 
using a SIT camera coupled to the VCR, and monitored 
the second meiotic division by examining the eggs at reg- 
ular intervals with epifluorescence optics. Room temper- 
ature was 19.5C in this latter series of experiments. 

Microtubule poisons 

Stock solutions of colchicine (1 mM in BSS), /8-lumi- 
colchicine ( 1 mM in BSS), colcemid (0.35 mM in BSS), 
and nocodazole (2 mg ml' 1 ) in DMSO were diluted into 
BSS to make working solutions. Working solutions of no- 
codazole also contained 1% DMSO, which had no ap- 



OOPLASMIC SEGREGATION IN MEDAK.A 



17 



parent effect on the eggs. In preliminary experiments, we 
monitored the effects of several concentrations of each 
drug on the movement of oil droplets during ooplasmic 
segregation and found the minimum effective concentra- 
tions that disrupted their normal movement to be 100 
nAf colchicine, 0.35 pAI colcemid, and 0.17 nM nocod- 
azole; we used these concentrations in subsequent exper- 
iments. Eggs were generally incubated with the drugs for 
1 h before fertilization and then fertilized in the same 
medium; however, in some experiments, eggs were in- 
cubated with the drugs for 1.5 h or 2 h before they were 
fertilized. In each experiment, we monitored one egg with 
time-lapse video microscopy and monitored an additional 
15-20 eggs with a stereomicroscope. 

To monitor the effect of colchicine on formation of the 
second polar body and migration of the pronuclei, control 
eggs (nine eggs from two females) and eggs treated with 
100 fiM colchicine (eight eggs from two females) were 
fixed in 3.7% formaldehyde in BSS at t n = 0.45. After 
washing away the fixative, the eggs were stained with 
Hoechst 33258 and examined with epifluorescence optics. 

Chemicals 

Colcemid. colchicine, Hoechst 33258, /3-lumicolchi- 
cine, and nocodazole were obtained from Sigma (St. 
Louis, Missouri) and formaldehyde from Electron Mi- 
croscopy Sciences (Fort Washington, Pennsylvania). 

Results 

An early sign of egg activation was the cortical granule 
reaction, which spread as a wave from the animal pole to 
the vegetal pole in about 90 sec at 26C. a result consistent 
with the time reported by Gilkey et al. (1978). After the 
cortical granule reaction, the ooplasm became relatively 
transparent (Fig. 1 A), and several types of inclusions could 
be seen in it (Fig. 2). One class of inclusions were oil 
droplets, which with phase-contrast optics appeared as 
white spheres with diameters from < 1.0- 100 nm. That 
these spheres were oil droplets was confirmed by staining 
them with a lipophilic fluorescent dye, nile red (data not 
shown). About 1 min after the beginning of the cortical 
granule reaction, there was a strong contraction of the 
ooplasm. marked by the movement of all ooplasmic in- 
clusions toward the animal pole; this fertilization con- 
traction lasted about 1.5 min and thus was over within 
2.5 min after fertilization, times also consistent with 
Gilkey el al. (1978). Our detailed study of the movement 
of ooplasmic inclusions began after the fertilization con- 
traction. 

At 10- 12 min after fertilization (at t n = 0.16at25C), 
a second contraction occurred (Fig. 3), in which all oo- 
plasmic inclusions, including oil droplets, again moved 
toward the animal pole. After this second contraction. 



most inclusions continued to move toward the animal 
pole; however, oil droplets (Fig. 1C-F) and some smaller 
inclusions began to move toward the vegetal pole. Ac- 
cumulation of ooplasm at the animal pole and the move- 
ment of oil droplets and other inclusions toward the veg- 
etal pole proceeded simultaneously for = 70 min, at which 
time the blastodisc underwent its first division (Fig. IF). 
By this time, there were fewer, larger oil droplets, a result 
of their fusion with each other during their movement 
toward the vegetal pole. 

The timing of the second meiotic division was approx- 
imated by examining fixed eggs and was confirmed by 
injecting Hoechst 33258 into live eggs. At 1 9. 5 C the sec- 
ond polar body formed by 13.8 3.3 min (X S.D.. n 
= 4; t n = 0. 1 2) after activation, and the second contraction 
began about 3 min later at 16.7 1.2 min (X S.D., n 
= 6; t n = 0. 15; Fig. 2). In all cases, polar body formation 
preceded the second contraction. 

In the following three sections, we describe (1) the 
streaming movement of inclusions toward the animal 
pole, (2) the saltatory movement of inclusions toward the 
vegetal pole, and (3) the movement of oil droplets toward 
the vegetal pole. The data presented in Figures 4 and 5 
were collected from a single egg at t n = 0.43. The move- 
ments seen in this egg were confirmed in 41 other eggs 
studied between the second contraction and the first cell 
division, and an additional 15 eggs were used to obtain 
the data summarized in Figure 2. 

Streaming 

Essentially all the inclusions in Figure 2 appeared to 
be streaming toward the animal pole. The diameters of 
these inclusions were in the range 1.5-1 1 /urn, and they 
appeared to be distributed throughout the depth of the 
ooplasm. By "streaming" we mean that the movements 
of the individual inclusions did not appear to be inde- 
pendent of each other; in other words, all inclusions 
moved at nearly the same speed and in the same direction. 
The motion of three such inclusions, moving at about 1 .5 
^m min ', is summarized in Fig. 4A. Though this speed 
was typical of streaming motion (2.2 0.8 ^m min~', X 
S.D., n = 31 inclusions from 5 eggs), the speed some- 
times increased to as high as 8.2 ^m min" 1 for periods 
lasting up to 10 min and sometimes decreased to near 
zero for periods lasting up to 12 min. 

Saltatory movement 

The circled inclusion in Figure 2 is one that by its size, 
shape, and appearance (phase-dark) would be expected 
to exhibit saltatory movement. The number of inclusions 
showing such movement was usually not more than three 
per microscopic field. These inclusions were in the same 
optical section as those showing streaming movement 



118 



V. C. ABRAHAM ET AL. 




Figure I. Ooplasmic segregation in the medaka egg. (A) t n = 0.07. The just-fertilized egg consists of a 
chorion covered with hairs, a large yolk vacuole, and a thin peripheral layer of ooplasm between the yolk 
membrane and plasma membrane. Oil droplets are present throughout the ooplasm. and a thin blastodisc 
is visible at the animal pole (AP). (B) t n = 0.25. The thickness of the blastodisc has increased, but oil droplet 
movement toward the vegetal pole has not yet begun. (C) t n = 0.53. The thickness of the blastodisc has 
increased even more, and oil droplets have begun to move toward the vegetal pole. (D) t n = 0.69. A biconvex 
blastodisc has formed, and many oil droplets have left the animal hemisphere. (E) t n = 0.81. The blastodisc 
has become plano-convex, and oil droplet movement continues. (F) t n = 1.00 (about 70 min at 23C). The 
blastodisc has begun to undergo cytokinesis, and most of the oil droplets have formed a crude cap over the 
vegetal hemisphere. Scale bar. 500 ^m. 



toward the animal pole. The movement of such inclu- 
sions (Fig. 4B) differed from those that streamed in the 
following ways: ( 1 ) Their motion was intermittent, hence 
the designation "saltatory." An inclusion showing sal- 
tatory motion typically moved at a constant rate for 1 5- 
120 sec, paused for 5-20 sec and then began moving 
again. (2) Their velocity (44.4 13.8 pm min ', X 
S.D., n = 17 inclusions from 9 eggs) was about 20-fold 
higher than that of streaming inclusions. (3) They moved 
toward the vegetal pole, not the animal pole. (4) Whereas 
streaming inclusions appeared to move directly toward 



the animal pole, the paths of these inclusions, though 
generally directed toward the vegetal pole, were more 
zig-zagged. 

Movement of oil droplets 

Immediately after the second contraction, oil droplets, 
like saltatory inclusions, began to move toward the vegetal 
pole (Fig. 1B-E). Unlike saltatory inclusions, however, 
oil droplets appeared to move directly toward the vegetal 
pole. Moreover, oil droplets moved more slowly than sal- 



OOPLASMIC SEGREGATION IN MEDAKA 



19 




Figure 2. Phase-contrast image of a typical microscopic field near 
the equator of a fertilized egg at t n = 0.43. Ooplasmic inclusions include 
some that streamed toward the animal pole (arrowheads), oil droplets 
of various sizes (arrows), and inclusions that moved saltatorily toward 
the vegetal pole (encircled parcel). The out-of-focus image of chorionic 
hairs distorts the image in places (outlined by dashed lines). Scale bar, 
50 /jm. 



Ef/ccls of microtubule poisons 

Colchicine, colcemid, and nocodazole had the same 
effects on the eggs, while eggs treated with /i-lumicolchi- 
cine behaved as untreated (control) eggs. No effect of these 
poisons was apparent until after the second contraction, 
even in eggs that were incubated in the microtubule poi- 
sons for 1 .5 h or 2 h before fertilization: The cortical gran- 
ule reaction (in 96% of the drug-treated eggs vs. 97% of 
the controls), the fertilization contraction, elevation of 
the fertilization membrane, and the second contraction 
occurred normally in these eggs. 

However, these drugs had dramatic effects on the sub- 
sequent movement of ooplasmic inclusions toward the 
poles of the egg. The most obvious effect was on the oil 
droplets, which floated to the top of the egg instead of 
moving toward the vegetal pole (Fig. 6B). Moreover, sal- 
tatory motion toward the vegetal pole was absent from 
drug-treated eggs. All three poisons also slowed the rate 
of growth of the blastodisc (Figs. 6B; 7). The volume of 
the blastodisc of control eggs at t n 0.85-1.0 was 21.3 
+ 4.0 nl (X S.D.. n = 7), while that in eggs treated with 
microtubule poisons was 1 1 .6 2.6 nl (n = 12). Moreover, 
in poisoned eggs the blastodisc did not undergo the 
changes in shape seen in control eggs from meniscus to 
biconvex to planoconvex (Fig. 1); instead the blastodisc 
appeared only to enlarge while maintaining its meniscus 
form. The microtubule poisons also caused a decrease in 
the velocity of streaming inclusions [2.2 0.2 |/m min ' 
(X S.E.M.. n = 3 eggs) versus 2.9 0.5 (n = 3 eggs) in 
control eggs]. When we looked at the direction of move- 
ment of inclusions during a 10-min period, we found that 



tatory inclusions (17.0 5.7 /urn min ', X S.D., n 
= 1 5 droplets in 3 eggs), and their speed varied more than 
that of saltatory inclusions during a given stretch in which 
they were moving continuously (Fig. 4C). 

Second contraction complex 

We monitored this contraction by observing either oil 
droplet movement at low magnification (at which the en- 
tire egg could be seen simultaneously) or the movements 
of inclusions at higher magnification. In all 18 eggs (from 
8 females) in which we analyzed the second contraction, 
it was composed of at least two components: ( 1 ) a move- 
ment of ooplasmic inclusions, including oil droplets, to- 
ward the vegetal pole, and (2) a subsequent pronounced 
movement of the inclusions toward the animal pole (Fig. 
5A, B). In 5 of the 18 eggs we observed, the movement 
toward the vegetal pole was preceded by a weaker move- 
ment toward the animal pole (data not shown). 



200 



! 150 

D 
O 



m 
o 



100 



50 




U.O 0.2 0-4 0.6 0.8 1.0 1.2 
Proportion of Time to First Cleavage 

Figure 3. Change in thickness of the blastodisc during ooplasmic 
segregation. Data from 1 5 eggs, grown at 14C-24C, were used to con- 
struct this figure; shown are X S.D. Arrows mark the times of occurrence 
of the second meiotic division (M) and the beginning and end of the 
second contraction (C). The thickness of the blastodisc increased from 
= 40 Mm in the just-fertilized egg (t n = 0.05) to =160 ^m at t n = 0.7, 
by which time the blastodisc was biconvex (Fig. ID). The decrease in 
the thickness at t n > 0.7 was caused by a change in the shape of the 
blastodisc from biconvex to plano-convex before the first mitotic division 
(Fig. ID, E). 



120 



V. C. ABRAHAM ET AL 




100 



200 400 600 

Elapsed Time (sec) 



800 



80- 
60- 
40- 
20- 




100 200 

Elapsed Time (sec) 



300 



40- 



100 200 300 
Elapsed Time (sec) 



400 



Figure 4. Graphic summary of the movements of ooplasmic inclusions. The movements of five inclusions 
in a single egg are shown, beginning at t n = 0.4. (A) Streaming of inclusions toward the animal pole. 
Throughout most of this 1 1+ minute period, the speed of the inclusions was & 1.5 Mm min~'. (B) Saltatory 
motion of an inclusion toward the vegetal pole. Movement was intermittent, i.e.. the inclusion sometimes 
moved rapidly (a -* b) and sometimes paused (c). Occasionally such inclusions reversed their direction (d). 
The velocity of the inclusion between t ~ 28 s and t = 200 s was ^25.7 ^m mirT 1 . (C) Movement of an 
oil droplet toward the vegetal pole. The motion summarized here is that of a small oil droplet (diam. =6 
^m). The speed of the droplet was about 30 Mm min~' at 50-100 s and about 9 Mm min~' at 125- 
225s. 



whereas inclusions in control eggs moved in essentially 
the same direction (3.7 10.6 departure from the an- 
imal-vegetal axis, X S.D., n = 30 inclusions; note the 
small standard deviation), inclusions in poisoned eggs 
varied substantially in their direction of movement (10.9 
55.7, X S.D., n = 52 inclusions; note the large stan- 
dard deviation). 

Hoechst 33258 stained three bodies in control eggs fixed 
at t n = 0.45. One was inferred to be the second polar body 



(Fig. 8A) on the basis of its protrusion from the surface 
of the egg, its size, and the presence of a halo of membrane 
ruffles around it (Brummett et a/.. 1985). The other two, 
the male and female pronuclei, were about 5 jim from 
each other and 51.8 10.2 ^m (X S.D., n = 9) from 
the polar body (Fig. 8B, C). As in control eggs, Hoechst 
33258 stained three bodies in eggs treated with 100 pM 
colchicine and fixed at t n = 0.45, one of which was the 
second polar body (Fig. 8D). However, in contrast to the 




30 




Is 20 

ss 



3 0) 

E 2? 

3 0) 



o- 



-10 




4 6 

Elapsed Time (min) 



Figure 5. The second contraction. (A) The "tracks" of five inclusions during the second contraction in 
an egg growing at 2lC. The circles represent the positions of the inclusions at 20 sec intervals, beginning 
at "B" (6 mm after fertilization; t n = 0.06) and ending about 9.5 min lateral "E" (at t n = 0.15). The parcels 
first moved toward the vegetal pole ("down" in this figure: ), and then reversed their direction at "R" 
and began to move toward the animal pole (o-o). Note that the movement toward the animal pole was rapid 
at first and then slowed. Scale bar, 5.65 Mm. (B) Graphic summary of the movement of parcels #1-3 in 
Figure 5A. The parcels moved hardly at all for 2 min, began to move toward the vegetal pole (down in this 
figure) after about 3 min (at t n = 0.09), and then reversed their direction after 5 min (> t n = 0.1 1) and began 
to move toward the animal pole (up in this figure). 



10 



OOPLASMIC SEGREGATION IN MEDAKA 



121 




Figure 6. Effects of nocodazole on ooplasmic segregation. (A) Control egg, t n = 0.84. A large blastodisc 
has formed at the animal pole, and oil droplets have formed a cap over the vegetal hemisphere. (B) Egg 
treated with 0.17 nM nocodazole. t n = 0.80. The blastodisc is smaller than in the control egg, and most of 
the oil droplets, instead of moving toward the vegetal pole, have floated to the top of the egg (i.e.. toward 
the viewer, whose perspective is from above the egg) and coalesced into one large droplet there. Scale bar, 
250 Mm. 



situation in control eggs, in which the male and female 
pronuclei were within 5 ^m of each other, the male and 
female pronuclei in colchicine-treated eggs were far apart 
(132.2 54.8 urn; X S.D., n = 8; Fig. 8E). 

Discussion 

All three microtubule poisons used in this study col- 
chicine, colcemid. and nocodazole affected the move- 




o 

m 
^ 

a> 

c 

JC 

u 



175 

150 

125 

100 

75 

50 

25 




00 0.2 0.4 06 0.8 1.0 1.2 

Proportion of Time to First Cleavage 

Figure 7. Effect of microtubule poisons on the growth of the blas- 
todisc. The thickness of the blastodisc along the animal-vegetal axis was 
measured in untreated eggs ( . four eggs) and in eggs treated with 
100 iiM fj-lumicolchicine ( D , three eggs), 100 pM colchicine ( 
, four eggs), 0.35 nM colcemid ( O . three eggs), or 0.17 tiM 
nocodazole ( A . five eggs). Shown are the mean values; over all the 
treatments and times, the standard deviation averaged 12% of the mean. 
Colcemid, colchicine. and nocodazole all slowed the growth of the blas- 
todisc. while eggs treated with J-lulmicolchicine behaved as untreated 
eggs. The volume of the blastodisc in control eggs_and poisoned eggs at 
t n = 0.85- 1 .0 was 2 1 .3 4.0 nl and 1 1 .6 2.6 nl (X SD), respectively. 



ment of ooplasmic inclusions during segregation in the 
medaka egg. That the poisons acted specifically as micro- 
tubule poisons is a reasonable inference because ( 1 ) their 
effective concentrations were similar to those used in pre- 
vious studies (Zalokar, 1974; Eckberg, 1981; Shimizu, 
1982; Astrow el ai. 1989; Sawada and Schatten, 1989); 
(2) poisons from two classes of microtubule poisons (Bray, 
1992, p. 207) had similar effects on the eggs; and (3) 100 
nM /3-lumicolchicine had no apparent effect on the eggs. 
The results of the present study differ from those of Katow 
(1983), who reported that the blastodisc formed normally 
in zebrafish eggs treated with colchicine. This difference 
could be due to the lower concentration of colchicine used 
in the earlier study (2.5 nAl vs. 100 nM in the present 
study) or to a lower permeability of the zebrafish egg to 
colchicine. It is possible (but unlikely, we believe) that 
microtubules are not required for ooplasmic segregation 
in the zebrafish egg. 

Saltatory movement similar to that observed in the 
present study is often associated with microtubules (Hay- 
den el ai. 1983; Brady and Pfister, 1991). Similarities 
include the intermittent nature of the movement, its in- 
hibition by microtubule poisons, and the speed of moving 
particles (Hamaguchi et ai. 1986; Shimizu el ai, 1991). 
These similarities suggest that in the medaka egg some 
ooplasmic inclusions move toward the vegetal pole via 
microtubules oriented approximately along the animal- 
vegetal axis. 

The normal movement of oil droplets was also affected 
by the poisons, suggesting that microtubules are also in- 
volved in the movement of these droplets. Such an in- 



122 



V. C. ABRAHAM ET AL 




Figure 8. Formation of second polar body and pronuclear migration. 
Untreated eggs (A-C) and eggs treated with 100 n.M colchicine (D-E) 
were fixed at t n = 0.45, subsequently stained with Hoechst 33258, and 
viewed with either phase contrast (A. D) or epifluorescence (B, C, E) 
optics. The second polar body (arrowhead) could be seen near the animal 
pole in both untreated eggs (A) and in eggs treated with 100 ^M colchicine 
(D). Of the three fluorescent bodies present in this region of the egg, one 
corresponded to the polar body (arrowhead in B, E), and the other two 
were the male and female pronuclei (C, E). In the untreated egg shown 
here, the polar body and pronuclei were recorded in separate photographs 
(B, C) because their focal planes were separated by about 25 jim. The 
two pronuclei were close to each other and were about 37 ^m from the 
polar body. In eggs treated with colchicine, the pronuclei were much 
farther apart (E). Scale bars. A, D. 10 ^m; B, C, E, 50 urn. 



volvement would seem to require the presence of a unit 
membrane at the surface of the oil droplets to provide a 
site of attachment for a kinesin-like molecule. Whether 
such a membrane is present around these droplets is not 
known. In other types of cells, unit membranes are present 
around some lipid droplets but not others (Wake, 1974; 
Nedergard and Lindberg, 1982). An alternative expla- 
nation for the effect of these poisons on oil droplet move- 
ment is that in control eggs a dynamic network of micro- 
tubules holds the oil droplets in place; in the presence of 
these microtubule poisons, such a dynamic network would 
eventually disappear as disassembly continues in the ab- 
sence of assembly. This question will require further study. 
The movement of ooplasm toward the animal pole in 
fish embryos has been previously described as streaming 
(Roosen-Runge, 1938; Beams et ai. 1985) or bulk How 
(Gilkey, 1981); our results confirm these reports. Micro- 



tubule poisons slowed both the movement of inclusions 
toward the animal pole and the growth of the blastodisc, 
but they did not inhibit either process entirely, suggesting 
that more than one mechanism is responsible for these 
phenomena. In ascidians (Sawada and Osanai, 1981, 
1984, 1985; Jetfery, 1984; Bates and Jeffery, 1988) and 
an oligochaete (Shimizu, 1982, 1984), actin microfila- 
ments form a cortical network that contracts toward one 
pole of the egg, pulling with it both cortical and subcortical 
components of the ooplasm. F-actin is present in the cor- 
tex and subcortex (Beams et a/.. 1985; Wolenski and Hart. 
1987; Chang, 1991) of the zebrafish egg, and an acto- 
myosin-like ATPase has been identified in cortical prep- 
arations offish eggs (Jorgensen, 1972). Moreover, cyto- 
chalasins (Katow, 1983; Ivanenkov cl ai. 1987; Fluck, 
unpub.) and DNase I (Ivanenkov et ai. 1987) inhibit for- 
mation of the blastodisc in fish embryos. Thus, both mi- 
crotubules and microfilaments may be involved in the 
movement of ooplasm and its inclusions toward the an- 
imal pole in the medaka egg. 

Calcium ion may both trigger and organize such a con- 
traction in the medaka egg just as it does in ascidian egg 
(Jeffery, 1982;Sardetrfa/.. 1 986; Speksnijder <?//.. 1990a, 
b; see also Cheer et ai. 1987). Cytosolic [Ca :+ ] is elevated 
at the animal and vegetal poles of the medaka egg during 
ooplasmic segregation (Fluck et ai. 1992b), and injection 
of the weak calcium buffer. dibromo-BAPTA (Speksnijder 
et ai. 1989), into the medaka egg inhibits formation of 
the blastodisc (Fluck et ai. 1992a). 

In eggs treated with microtubule poisons, the ooplasm 
appeared to be solated compared to that in control eggs 
because oil droplets floated to the top of the egg instead 
of moving toward the vegetal pole. Moreover, the move- 
ment of inclusions toward the animal pole of the egg was 
more disorganized. Both of these effects could be the result 
of the disruption of a dynamic network of microtubules 
in the ooplasm by the microtubule poisons. 

All three of the movements described in this report 
streaming toward the animal pole, saltatory movement 
toward the vegetal pole, and movement of oil droplets 
toward the vegetal pole-began only after formation of the 
second polar body and the second contraction; this was 
true for both control eggs and for eggs treated with mi- 
crotubule poisons. These events thus heralded a radical 
change in the structure and/or activity of the cytoskeleton 
of the egg. Though the present study is the first to describe 
this phenomenon as a contraction (or series of contrac- 
tions), both Sakai (1965) and Iwamatsu (1973) reported 
that oil droplets in the medaka egg oscillate along the 
animal-vegetal axis at this time. 

In addition to its effect on ooplasmic segregation, col- 
chicine inhibited the migration of the pronuclei after for- 
mation of the second polar body, which apparently formed 
normally. In its insensitivity to a microtubule poison, the 



OOPI.ASMIC SEGREGATION IN MEDAKA 



123 



medaka egg is like that of the ascidian Pluillusiu nuuu- 
millata (Zalokar, 1974) and the polychaete Chaetoptcnts 
pergamentaceus (Eckberg, 1981), but unlike that of the 
leech Hclobdella triserialis (Astrow ct ai. 1989) and the 
oligochaete Tuhifex hattai (Shimizu, 1982). In contrast 
to their variable effects on polar body formation, micro- 
tubule poisons consistently inhibit pronuclear migration 
(Zalokar, 1974; Hiramoto ct ai. 1984: Hamaguchi and 
Hiramoto, 1986; Sawada and Schatten, 1989). The results 
of the present study are consistent with these earlier 
studies. 

These results suggest that in the medaka egg microtu- 
bules are necessary for the movement of some components 
of the ooplasm to the vegetal pole and of others to the 
animal pole. Such movement in an animal egg is generally 
considered to constitute "ooplasmic segregation," but 
whether it also constitutes "cytoplasmic localization" 
(Davidson, 1976) remains to be seen. These movements 
in the medaka egg, especially the saltatory ones, may sim- 
ply reflect the need of this large, polarized cell to sustain 
its polarity in the same way that epithelial cells (Rindler 
ct ai. 1987; Achler ct ai. 1989; Breitfield et ai. 1990) 
and other eggs (Peter et ai. 1991 ) do. 

Acknowledgments 

Doug Antonioli, Jesse Fluck, and Justine Fluck assisted 
in the studies summarized in Figure 3; Justine Fluck in 
the analysis of that data and the preparation of Figure 3; 
and Minerva Medina in the studies of the timing of the 
second meiotic division. We thank Dr. Lionel Jaffe and 
Dr. Andrew Miller for helpful discussions of these phe- 
nomena: Dr. Jaffe for helping us improve the text of the 
manuscript; and Dr. George Rosenstein and Dr. Gene 
Johnson for their help in measuring the volume of the 
blastodisc. Supported by NSF DCB-90 1 72 1 and Franklin 
and Marshall College's Hackman Scholar Program and 
STEP program. 

Literature Cited 

Abraham, V., and R. A. Fluck. 1991. Ooplasmic segregation in the 
fertilized egg of Ory:ias lalipex (medaka). J. Cell Bio/. 115: 56a. 

Achler, C., D. Filmer, C. Merle, and D. Drenckhahn. 1989. Role of 
microtubules in polarized delivery of apical membrane proteins to 
the brush border of the intestinal epithelium. J Cell Biol 109: 1 79- 
189. 

Astrow, S. H., B. Holton, and D. A. Donovan. 1989. Teloplasm for- 
mation in a leech. HelobJclla iriserialis. is a microtubule-dependent 
process. Dcv. Biol. 135: 306-319. 

Bates, W. R., and W. R. Jeffery. 1988. Polarization of ooplasmic seg- 
regation and dorsal-ventral axis determination in ascidian embryos. 
Dev. Biol. 130:98-107. 

Beams, H. \V., R. K. Kessel, C. Y. Shin, and H. N. Tung. 
1985. Scanning electron microscope studies on blastodisc for- 
mation in the zebrafish, Brachyclanio rerio. J. Morphol. 184: 4 1 -49. 



Brady, S. I., and K. K. Ptister. 1991. Kinesin interactions with mem- 
brane bounded organelles ;/; vmiand in vitro. J. Cell Sei. Snppl 14: 
103-108. 

Bray, D. 1992. Cell Movement*. Garland Publishing. Inc., New York. 
Breitfield, I'. P., W. C. McKinnon, and K. E. Mostov. 1990. Effect of 
nocodazole on vesicular traffic to the apical and basolateral surfaces 
of polarized MDCK cells. ./. Cell Biol. Ill: 2365-2373. 
Brummett, A. R., J. N. Dumont, and C. S. Richter. 1985. Later stages 
of sperm penetration and second polar body and blastodisc formation 
in the egg of Fiintluhis heteroclilux. J. E\p. Zool. 234: 423-439. 
Chang, D. C. 1991. Structures of actin in zebra fish embryos studied 

by confocal microscopy. Biophyx. ./. 59: 55a. 

Cheer, A., J.-P. Vincent, R. Nuccilelli, and G. Oster. 1987. Cortical 
activity in vertebrate eggs. I. The activation waves. ./. Theor. Biol. 
124: 377-404. 

Davidson, E. H. 1976. Pp. 245-248 in Gene Aclivilv in Early Devel- 
opment. 2nd ed.. Academic Press. New York. 
Duslin, P. 1984. Mierotithiiles. 2nd ed. Springer- Verlag, Berlin. 
Eckberg, \V. R. 1981. The effects of cytoskeleton inhibitors on cyto- 
plasmic localization in Chaetopterus pergamentaceus. Differentiation 
19: 55-58. 

Fluck, R. A. 1978. Acetylcholine and acetylcholinesterase activity in 
early embryos of the medaka Ory:iux lalipex. a teleost. Dev. Growth 
Differ. 20: 17-25. 

Fluck, R. A., V. C. Abraham, A. L. Miller, and L. F. Jaffe. 
1992a. Calcium buffer injections block ooplasmic segregation in 
Orynax talipes (medaka) eggs. Biol Bull 183: 371-372. 
Fluck, R. A., A. L. Miller, and L. F. Jaffe. 1991. Slow calcium waves 
accompany cytokinesis in medaka fish eggs. ./ Cell Biol. 115: 1259- 
1265. 
Fluck, R. A., A. L. Miller, and L. F. Jaffe. 1992b. High calcium zones 

at the poles of developing medaka eggs. Biol. Bull 183: 70-77. 
Gilkey, J. C. 1981. Mechanisms of fertilization in fishes. Am. Zool. 

21: 359-375. 

Gilkey, J. C., L. F. Jaffe, E. B. Ridgway, and G. T. Reynolds. 1978. A 
free calcium wave traverses the activating egg of the medaka, Oryzias 
latipex J Cell Biol 76: 448-466. 

Hamaguchi. M. S., Y. llamaguchi, and Y. Hiramoto. 1986. 
Microinjected polystyrene beads move along astral rays in sand dollar 
eggs. Dev Growth Differ 28:461-470. 

Hamaguchi, M. S., and Y. Hiramoto. 1986. Analysis of the role of 
astral rays in pronuclear migration in sand dollar eggs by the col- 
cemid-UV method. Dev. Growth Differ. 28: 143-156. 
Hayden, J. H., R. D. Allen, and R. D. Goldman. 1983. Cytoplasmic 
transport in keratocytes: direct visualization of particle translocation 
along microtuhules. Cell Motil. 3: 1-19. 

Hiramoto, Y., M. S. Hamaguchi, Y. Nakano, and Y. Shoji. 
1984. Colcemid uv-irradiation method for analyzing the role of 
microtubules in pronuclear migration and chromosome movement 
in sand-dollar eggs. Zool. Sei. 1: 29-34. 

I liui I isi mi. E., and R. P. Elinson. 1991. Patterns of microtubule po- 
lymerization relating to cortical rotation in Xenopiis laevis eggs. De- 
velopment 112: 107-1 17. 

lllmensee, K., A. P. Mahowald, and M. R. Loomis. 1976. The ontogeny 
of germ plasm during oogenesis in Drosophila. Dev Biol 49: 40- 
65. 

Ivanenkov, V. V., A. A. Minin, V. N. Meshcheryakov, and L. E. Mar- 
tynova. 1987. The effect of local microfilament disorganization on 
ooplasmic segregation in the loach (Misgumusfossilis) egg. Cell Dif- 
fer. 22: 19-28. 
Iwamatsu, T. 1973. On the mechanism of ooplasmic segregation upon 

fertilization in Ory:iax talipes. Jpn. J Ichthyol. 20: 273-278. 
Jeffery, \V. R. 1982. Calcium ionophore polarizes ooplasmic segre- 
gation in ascidian eggs. Science 216: 545-547. 



124 



V. C. ABRAHAM ET AL. 



Jeffery, VV. R. 1984. Pattern formation by ooplasmic segregation in 
ascidian eggs. Biol. Bull 166: 277-298. 

Jorgensen, N.-C. 1972. Actomyosin-like ATPase activity at the surface 
offish eggs. Exp. Cell Res 71: 460-464. 

Katow, H. 1983. Obstruction of blastodisc formation by cytochalasin 
B in the zebrafish. Brachydanio rerio. Dev. Growth Differ- 25: 477- 
484. 

kimmel, C. B. 1989. Genetics and early development of zebrafish. 
Trends Genet. 5: 283-288. 

Kimmel, C. B., R. M. Warga, and T. F. Schilling. 1990. Origin and 
organization of the zebrafish fate map. Development 108: 581-594. 

Kirchen, R. V., and W. R. West. 1 976. The Japanese Medaka: Its Care 
and Development. Carolina Biological Supply Company, Burlington, 
North Carolina. 

Nedergard, J., and O. Lindberg. 1982. The brown fat cell. Int. Rev. 
Cytol. 74: 187-286. 

Peter, A. B., J. C. Schittny, V. Niggli, H. Reuter, and E. Sigel. 1991 . The 
polarized distribution of poly(A*)-mRNA-induced functional ion 
channels in Xenopus oocyte plasma membrane is prevented by an- 
ticytoskeletal drugs. J. Cell Biol. 114: 455-464. 

Powers, D. A. 1989. Fish as model systems. Science 246: 352-358. 

Reverberi, G. 1971. Ascidians. Pp. 529-532 in Experimental Em- 
bryology of Marine and Freshwater Invertebrates, G. Reverberi, ed. 
North Holland, Amsterdam. 

Rindler, M. J., I. E. Ivanov, and D. D. Sabatini. 1987. Microtubule- 
acting drugs lead to the nonpolarized delivery of the influenza hem- 
agglutinin to the cell surface of polarized Madin-Darby canine kidney 
cells. / Cell Biol 104: 231-241. 

Roosen-Runge, E. C. 1938. On the early development bipolar dif- 
ferentiation and cleavage of the zebra fish. Brachydanio reno. Biol. 
Bull 75: 119-133. 

Sabnis, D. D. 1981. Lumicolchicine as a tool in the study of plant 
microtubules: some biological effects of sequential products formed 
during phototransformation of colchicine. J. Exp Bot 32: 27 1-278. 

Sakai, V. T. 1965. Studies on the ooplasmic segregation in the egg of 
the fish Orv:ias talipes. III. Analysis of the movement of oil droplets 
during the process of ooplasmic segregation. Bin! Bull 129: 189- 
198. 

Sardet, C., S. Inoue, L. F. Jaffe, and J. E. Speksnijder. 1986. Surface 
and internal movements in fertilizing Phalhisia eggs. Biol. Bull 171: 
488. 

Sawada, T. 1988. The mechanism of ooplasmic segregation in the as- 
cidian egg. Zoo/. Sci. 5: 667-675. 

Sawada. T., and K. Osanai. 1981. The cortical contraction related to 
the ooplasmic segregation in dona iniestmahs eggs, \\llhelm Rout's 
Arch. Dev Biol 190: 208-214. 

Sawada, T., and K. Osanai. 1984. Cortical contraction and ooplasmic 
movement in centnfuged or artificially constricted eggs of dona 
iniexiinalis eggs. Wilhelm Roux's Arch. Dev. Biol. 193: 127-132. 



Sawada, T., and K. Osanai. 1985. Distribution of actin filaments in 
fertilized eggs of the ascidian dona inleslinalis. Dev Biol. Ill: 260- 
265. 

Sawada, T., and G. Schatten. 1989. Effects of cytoskeletal inhibitors 
on ooplasmic segregation and microtubule organization during fer- 
tilization and early development in the ascidian Molgtila occidentals. 
Dev Biol. 132: 331-342. 

Schindler, J. M. 1991. Zebrafish: Drosoplula with a spine (but can 
they fly?). New Biol. 3: 47-49. 

Shimizu, T. 1982. Ooplasrmc segregation in the Tubifex egg: mode of 
pole plasma accumulation and possible involvement of microfila- 
ments. ll'ilhelm Roiix's Arch. Dev. Biol. 191: 246-256. 

Shimizu, T. 1984. Dynamics of the actin microfilament system in the 
Tiihilcx egg during ooplasmic segregation. Dev. Biol. 106: 414-426. 

Shimizu, T., K. Furusawa, S. Ohashi, Y. Y. Toyoshima, M. Okuno, F. 
Malik, and R. D. Vale. 1991. Nucleotide specificity of the enzy- 
matic and motile activities of dynein, kinesin. and heavy mero- 
myosm. / Cell Biol. 112: 1 189-1 197. 

Speksnijder, J. E., A. L. Miller, M. H. \\eisenseel, T.-H. Chen, 
and L. F. Jaffe. 1989. Calcium buffer injections block fucoid egg 
development by facilitating calcium diffusion. Proc. Nail. Acad. Sci. 
{75.486:6607-6611. 

Speksnijder, J. E., C. Sardet, and L. F. Jaffe. 1990a. The activation 
wave of calcium in the ascidian egg and its role in ooplasmic seg- 
regation. / Cell Biol. 110: 1589-1598. 

Speksnijder, J. E., C. Sardet, and L. F. Jaffe. 1990b. Periodic calcium 
waves cross ascidian eggs after fertilization. Dev. Biol. 142: 246- 
249. 

Vacquier, V. D. 1981. Dynamic changes of the egg cortex. Dev Biol. 
84: 1-26. 

VVakahara, M. 1989. Specification and establishment of dorsal-ventral 
polarity in eggs and embryos of Xenopus laevis. Dev Growth Differ. 
31: 197-207. 

Wake, K. 1974. Development of vitamin A-rich lipid droplets in mul- 
tivesicular bodies of rat liver stellate cells. J. Cell Biol 63: 683-69 1 . 

Wilson, L., J. R. Hamburg, S. B. Mizel, L. M. Grisham, and K. M. 
Creswell. 1974. Interaction of drugs with microtubule proteins. 
Fed. Proc 33: 158-166. 

Wilson, L., and M. Friedkin. 1967. The biochemical events of mitosis. 
II. The in vivo and /;; vitro binding of colchicine in grasshopper 
embryos and its possible relation to inhibition of mitosis. Biochem- 
istry 6\ 3126-3135. 

Wolenski, J. S., and N. H. Hart. 1987. Visualization of actin with 
rhodamine phalloidin in the zebrafish egg. Biol. Bull 173: 573a. 

Yamamoto, T. 1967. Medaka. Pp. 101-111 in Methods in Develop- 
mental Biology. F. M. Wilt and N. K. Wessells, eds. Thomas Y. 
Crowell Company, New York. 

Xalokar, M. 1974. Effect of colchicine and cytochalasin B on ooplasmic 
segregation of ascidian eggs. Wilhelm Roux's Archiv. Dev. Biol 175: 
243-248. 



Reference: Bml Bull 184: 125-143. (April. 1993) 



Gametogenesis and Spawning of the Sea Cucumber 
Psolus fabricii (Duben and Koren) 



JEAN-FRANCOIS HAMEL 1 . JOHN H. HIMMELMAN 1 , AND LOUISE DUFRESNE 2 

^Departement de Biologic and GIROQ (Groupe Interuniversitaire de Recherches Oceanographiques 

chi Quebec), Universite Laval, Quebec, Canada G1K 7P4 and 2 Departement d'Oceanograplue, 

Universite du Quebec a Rimoitski and Centre Oceanographique de Rimouski. 

Runouski. Canada G5L 3A1 



Abstract. The reproductive cycle of the sea cucumber 
Psolus fabricii was studied in a population from the St. 
Lawrence Estuary in eastern Canada from May 1988 
through August 1989. The gonad consists of numerous 
germinal tubules which vary greatly in size. The mean 
diameter of the tubules and gonadal mass follow annual 
cycles, increasing from early winter through spring, and 
dropping abruptly during spawning in the summer. Ga- 
metogenesis is generally a prolonged process and begins 
in small tubules in January. By summer the ovarian tu- 
bules contain oocytes with a modal diameter of 400-600 
l/m, and the testicular tubules contain an abundance of 
early spermatogenic stages, but rarely spermatozoa. These 
small tubules of the gonad do not spawn until the follow- 
ing year, and there is little gametogenic activity within 
them until January, when oocyte growth and the pro- 
duction of later spermatogenic stages resumes. The latter 
production continues until summer and results in a 
marked increase in the diameter of the tubules. Then, 
during spawning, these now large fecund tubules are 
transformed into small tubules. Following spawning, the 
predominant activity within the spent tubules is phago- 
cytosis of the residual gametes. The active phase of ga- 
metogenesis (January to summer) coincides with an in- 
creasing photoperiod regime, and an accelerated game- 
togenesis occurs in March when temperature and food 
availability begin to increase. Spawning was one month 
later in 1989 than in 1988 and did not show a consistent 
relationship with either temperature or light conditions. 
However, in both years, spawning coincided with a de- 
crease in the freshwater run-off into the Estuary and with 
the predicted annual increase in phytoplankton. 

Received 2 April 1992; accepted 25 January 1993. 



Introduction 

If we want to describe the reproductive cycle of an in- 
vertebrate, we need information about the gonad (struc- 
ture and development) and gametogenesis (with respect 
to biometry) (Smiley el ai. 1991), and about environ- 
mental factors that control these events (Giese and Pearse, 
1974; Himmelman, 1981). We are interested in repro- 
duction in echinoderms and particularly in holothurians. 
The gonad of many holothurians is unusual in that it 
consists of numerous germinal tubules (Theel, 1882; Tyler 
and Gage, 1983; Smiley and Cloney, 1985; Smiley, 1988) 
that can vary markedly in size and state of gametogenic 
development (Theel, 1901; Kille, 1939, 1942; Smiley and 
Cloney, 1985; Smiley, 1988; Smiley el ai, 1991). Smiley 
( 1988) shows that, in female Stichopus californicus. fecund 
tubules are attached to the posterior part of the gonad. 
He suggests that this arrangement may be a general char- 
acteristic of holothurians. Because of the complex gonadal 
morphology, seasonal changes in gametogenesis are more 
difficult to quantify in holothurians than in invertebrates 
that have a globular gonad. Another problem in studying 
holothurian reproductive cycles is that body size can vary 
drastically due to water uptake and loss (Edwards, 1910) 
so that body component indices are not as constant and 
reliable as they are for many other invertebrates. The 
principal studies on holothuroid reproduction are those 
by Tanaka (1958), Krishnaswamy and Krishnan (1967), 
Rutherford (1973), Green ( 1 978), Engstrom ( 1 980), Con- 
and (198 1,1982), Tyler and Gage ( 1983), Costelloef 1985), 
Smiley and Cloney (1985), Cameron and Fankboner 
(1986), Tyler and Billett (1988), Smiley (1988), Bulteel et 
al. (1992), and Sewell ( 1992). 

The role of environmental factors in controlling ga- 
metogenesis and spawning in marine invertebrates has 



125 



126 



J.-F. HAMEL ET AL 



been well investigated. Temperature, food availability, and 
photoperiod have often been suggested as environmental 
cues based on correlative evidence (Giese and Pearse, 
1974; Himmelman, 1981; Todd and Doyle, 1981; Giese 
and Kanatani, 1987). Laboratory experiments in con- 
junction with field observations have demonstrated that 
specific environmental changes coordinate certain repro- 
ductive events in echinoderms. For example, photoperiod 
has been shown to be the primary factor controlling ga- 
metogenesis in the urchin Strongylocentrotiis purpiiratus 
and the seastar Pisaster ochraceits (Pearse and Eernisse, 
1982; Pearse et ai, 1986). In addition, spawning in several 
echinoderms as well as molluscs, seems to be triggered by 
the spring phytoplankton increase (Himmelman, 1975, 
1981; Starr. 1 990; Starr et al . 1990, 1992). Temperature 
(Tanaka, 1958), light intensity (Conand, 1982; Cameron 
and Fankboner, 1986), water turbulence (Engstrom, 
1980), salinity ( Krishnaswamy and Krisnan, 1967), a 
combination of temperature and light intensity (Costelloe, 
1985), and phytoplankton blooms (Cameron and Fank- 
boner, 1986) have all been suggested as potential spawning 
cues particularly for holothurians, but experiments dem- 
onstrating such a role have yet to be performed. 

In this study we examined the reproductive cycle of 
the holothuroid Psolits fabricii (Duben and Koren) in re- 
lation to environmental conditions for a period of 16 
months. This species was chosen because it possesses the 
complex system of gonadal tubules characteristic of holo- 
thurians, and because it can be readily collected since it 
is abundant on rocky faces in the subtidal zone in our 
region. First, we developed techniques for quantifying 
changes in the gonads and associated body organs. Because 
of the marked variation in the size and state of develop- 
ment of the germinal tubules, we decided to quantify the 
gametogenic events in large and small tubules separately, 
so as to clarify the extent to which tubule size affects in- 
terpretation. Although previous studies on holothurians 
report differences in gametogenetic development in tu- 
bules of different size, this is the first study quantifying 
the rate of gametogenic development in different-sized 
male and female tubules at frequent intervals throughout 
the year. 



Materials and Methods 



Studv site 



The population studied was at Anse a Robitaille (4832' 
N: 6941' W), 2.5 km from Les Escoumins on the north 
shore of the lower St. Lawrence Estuary. Samples of 35- 
40 individuals were collected at monthly or bimonthly 
intervals between May 1988 and August 1989, from a 
bedrock face (45-60) at a depth of about 10 m below 
the lowest water of spring tides. Because the animals could 
not be dissected immediately, they were preserved in 10% 



neutralized formalin in seawater and dissected about a 
month later. By this time, changes due to the preservation 
should have stabilized (Pitmann and Munroe, 1982). 

Determination oj indices of the gonad, respiratory tree, 
and intestine (including its contents) 

The dry mass of the body wall, including the aqua- 
pharyngeal bulb, longitudinal muscle bands, and cloacal 
muscles (Fig. 1 A), was chosen as a denominator for body 
component indices because calculating these indices as a 
proportion of the wet body wall mass would have mark- 
edly increased the confidence intervals (by 12-25% for 
the gonadal index, 9-18% for the intestinal index, and 
16-22% for the respiratory tree index). Moreover, seasonal 
variations in dry body wall mass were probably small since 
the calcareous plates accounted for =87% of this mass. 
All masses were recorded to the nearest 0.01 g, and dry 
masses were determined after drying at 55C for 96 h. 

The intestine (with contents) was removed from 
the posterior end of the stomach to the beginning 
of the cloaca, the gonad from its point of attachment to 
the gonoduct, and the respiratory tree from its point of 
attachment to the cloaca. Intestinal and gonadal indices 
were calculated as the ratio of their wet mass to dry body 
wall mass (this permitted an examination of the intestinal 
contents and gonadal histology), whereas the respiratory 
tree index was calculated as its dry mass relative to the 
dry body wall mass. For each collection date, the various 
indices were determined for 15 males and 15 females, 
ranging from 25 to 34 g in dry body wall mass (equivalent 
to 5.7-6.1 cm in the distance from the mouth to the 
anus). 

In order to compare them with the evolution of the 
gonadal indices, seasonal changes in the diameter of the 
gonadal tubules were quantified as follows: The gonads 
were spread out in a shallow container, and the tubule 
diameter was measured at random points with a binocular 
scope ( 12X). Fifteen measurements for each of 15 males 
and 1 5 females were made for each sampling date. 

Gametogenesis 

Gonads from preserved individuals were removed and 
transferred to Bouin's fixative for four weeks and then 
processed according to standard embedding technique 
(Junqueira el al.. 1986). To determine the variation due 
to differences in tubule size, separate examinations were 
made of small (<1.9 mm) and large tubules (>1.9 mm). 
To prevent the loss of tubule contents during embedding, 
the tubule sections were cut well beyond the segment se- 
lected for sectioning. For each individual, six 5 ^m-mi- 
crotome sections were cut from both the small and large 
tubules. These sections were first placed on gelatin coated 
slides (the gelatin was heated to 42 C) and then transferred 



REPRODUCTIVE CYCLE OF PSOLUS 1-ABR1C11 



127 




Figure 1. Psolus fabricii. (A) Dissected male showing the respirator, tree (R), intestine (I), body wall 
(W), aquapharyngeal bulb (A), cloacal muscles (C), longitudinal muscles (L). and testis (G). (B) Photograph 
of the mouth region showing the feeding podia, one of which is in the mouth and the others extended, and 
papillae surrounding the gonopore (arrow). Photographs of a testis (C) and an ovary (D) just after spawning 
showing the large tubules (L), small tubules (S). and tubules with swellings containing residual gametes 
(RG). The horizontal bars in photographs C and D represent 50 mm. 



to an oven at 37C for 1 h. This technique usually pre- 
vented the breaking of the fragile tubules and the loss of 
gametes. The slides were stained with cosine and hema- 
toxylin, as described by Galigher and Kozloff( 1971), and 
good resolution of the various cell types was achieved. A 
second series of slides was stained with the periodic acid- 
SchifT(PAS) reaction (Humason, 1981) to identify poly- 
saccharides (glycogen). 

Gonadal development was classified into five stages 
(post-spawning, recovery, growth, advanced growth, and 
mature stage) that were adapted from the earlier studies 
of holothurians (Tanaka, 1958; Costelloe, 1985; Cameron 
and Fankboner, 1986). For each male, we made 15 ran- 
dom measurements of the thickness of the gonadal tubule 
wall from slides of both small and large tubules. Only 
intact areas were used in this assay. For each female and 
for both small and large tubules, we determined the di- 
ameters of 200 relatively unbroken oocytes that showed 
a well-centered germinal vesicle. 



Size of the gonad at sexual maturity 

A sample of 132 individuals was collected on 4 May 
1988. For each of these individuals, we measured the go- 
nadal index and made histological sections to determine 
whether mature gametes were present. We also deter- 
mined the total number of tubules in each individual, as 
well as the length of 1 5 randomly selected tubules, and 
the length of the intestine. 

Environmental factors 

Continuous temperature measurements at the study 
site at Anse a Robitaille were made during most of our 
study using a Peabody Ryan thermograph placed at 10 
m in depth. Data on day length and minimum daily sun- 
shine were obtained from the weather station at the Que- 
bec Airport (Environment Canada, Atmospheric Envi- 
ronment Service). Data on freshwater run-off were pro- 
vided by Environment Canada (Climatologic Services) by 



128 



J.-F. HAMEL ET AL 



using the combined discharges from the Montmorency, 
Bastiscan, Saint-Anne and Chaudiere rivers. 

Phytoplankton cells abundance control the spawning 
of a number of marine invertebrates in the Estuary, but 
regular phytoplankton measurements could not be made 
during our study. As an indirect signal of the spring phy- 
toplankton bloom, we determined, in 1989. the time of 
spawning in the green sea urchin Strongylocentrotus droe- 
bachiensis. This species spawns when the adults detect 
the rapid growth of phytoplankton during the spring 
bloom (Himmelman, 1981; Starr, 1 990; Starr elal, 1990, 
1992). Thus, from March to August, when spawning was 
anticipated, gonadal indices (percentage gonadal mass) 
were determined for 15 adult urchins (4.0-6.5 cm in di- 
ameter) of both sexes at each sampling date. 

We used two approaches to examine seasonal changes 
in the intestinal contents of Psolus fabricii. First, for each 
date, the contents of the first centimeter of the intestine 
of each of the 30 individuals were suspended in 5 ml of 
10% formalin, and the various types of undecomposed 
organisms present in a 1 ml subsample were identified 
and counted with a hemacytometer. Large cells were ex- 
amined under white illumination, and the presence of 
small phytoplankton cells was determined by the fluo- 
rometric method of Yentsch and Menzel ( 1963). Second, 
the contents of the following 30 g portion of the digestive 
tract was emptied into a Petrie dish, examined with a 
binocular scope, and the proportion of living phytoplank- 
ton cells (green in color) to non living materials (decom- 
posed cells and inorganic materials) was estimated. The 
phytoplankton cells in the first centimeter of the intestine 
seemed virtually undigested (green in color and intact). 

Buoyancy ofoocytes 

Forty oocytes were collected from five mature females 
(measuring 25-34 g in dry body wall mass) collected on 
14 July 1990. The oocytes were placed in natural seawater 
to provoke breakdown of the germinal vesicle. 

The oocytes were then placed in a 500 ml graduated 
cylinder (5 cm in diameter) at 7-8C and the rate of up- 
ward vertical movement was recorded (Fig. 8). This 
movement was taken as a measure of buoyancy. 



250n 



200- 

_0j 

2 150- 



.2 1001 



50- 



Male* 




b 

I 101 

* 

JD 

a 

73 5H 

G 



Ol 

O 



Male 




Juvenile 



200 i 



Size range selected 





150- 


Male 


X 






<u 




Jt 


TJ 




9 


.5 




* 


73 


100- 





T3 




O 


s 




o o 
o O o o 


C 




o* 0ooo o* 


O 


50- 


^ " o t a 00 S o o 

v Female o n 






nDQ? O O 






Ao Juvenile 




n . 





10 



20 



30 



40 



50 



Dry body wall mass (g) 



Figure 2. Psolus fabricii. The relation of the number of germinal 
tubules (A) and of germinal tubules length (B) to dry body wall mass for 
juvenile and adult males and females in May 1988 (n = 132). (C) The 
relation of the gonadal index to dry body wall mass (n = 132) in May 
1988. The bracket indicates the size range used for gonadal index de- 
terminations. 



Results 

Gonadal morphology and si:e at sexual maturity 

The gonads of Psolus fabricii consist of a large number 
of rarely branched germinal tubules distributed through- 
out the peri visceral cavity (Fig. 1C, D). The tubules join 
at a single gonoduct which exits through a gonopore lo- 
cated between the feeding podia (Fig. IB). The number 
of tubules is greater for males than for females (Fig. 2A, 
Z test, P < 0.01, Tessier's slope analysis, Tessier, 1948), 



whereas tubule length does not vary significantly between 
the sexes (Fig. 2B, Z test, P > 0.05). In some very large 
males and females, the terminal ends of some tubules 
were necrotic, and at times these ends were found floating 
free in the coelomic fluid. 

The size of the gonad at sexual maturity was determined 
from the 132 individuals collected on 4 May 1988 (Fig. 
2C). Gonadal tubules were present in every individual 
examined that had a dry body wall mass of at least 1.2 g 



REPRODUCTIVE CYCLE OF PSOLL'S FABRICll 



129 




Figure 3. Psolus lahncn Light micrographs of sections of gonads of juveniles. (A) Immature gonad 
(from an individual weighing <5.5 g in dry body wall mass) showing the germinal epithelium (GE) and an 
absence of identifiable precursor cells for two germinal tubules (GT); (B) Immature female (between 5.6- 
10 g) with oogonia (O). primary oocytes (PR), but an absence of more advanced stages; (C) Immature male 
(between 5.6-10 g) showing the proliferation zone (PZ) and the lumen containing only a few spermatozoa 
(SP); (D) Young female ( = 10 g) showing the germinal epithelium (GE), primary oocytes (PO) and a few 
vitellogenic oocytes (V); (E) Young male ( = 10 g) showing two distinct germinal tubules (GT) and numerous 
spermatozoa (SP) in the lumen. The horizontal bar in photograph A represents 800 ^m and applies to all 
of the photographs. 



(equivalent to =0.7 cm in distance mouth-anus). The 
relative size of the gonads increased sharply as the dry 
body wall mass rose from 3 to 10 g (Fig. 2C). The size of 
testis and ovary overlapped greatly up to a body wall mass 
of = 1 5 g, but the testis was generally larger than the ovary 
for larger individuals (Kruskal-Wallis, analysis of variance, 
P < 0.01, followed by a non-parametric multiple-range 
test, P < 0.05; Sokal and Rolph, 1981). The variation in 
gonadal size was relatively small between 20 and 34 g (5.5 



to 6.1 cm); beyond that range the relative gonadal size 
dropped. 

Histological preparations showed that only undiffer- 
entiated precursor cells are present along the germinal 
epithelium of individuals weighing <5.5 g( = 3.2 cm) (Fig. 
3A). The sex of larger individuals could be identified by 
the presence of oogonia and young oocytes in females, 
and spermatogonic stages in males (Fig. 3B, C). Beginning 
at 6.5-7.9 g (4.0-4.6 cm), the sexes were readily recognized 



130 



J.-F. HAMEL ET AL 



from gonadal smears (Fig. 2C), although histological ex- 
amination showed that only individuals weighing >10 g 
(=4.8 cm) contained mature gametes with the same mor- 
phology and reaction to PAS and hematoxylin and cosine 
as for very large individuals (Fig. 3D, E). Immature gonads 
were cream in color, whereas the mature testis was pink, 
and the mature ovary reddish brown (individuals > 
10 g). Thus, Psolus fabricii starts to produce mature ga- 
metes at 10 g, but the gonads only attain a plateau in size 
at 15-20 g (Fig. 2C). Individuals weighing 25 to 34 g 
showed no variation due to body size and were used in 
following the gonadal index cycle. 

No significant departure from a sex ratio of 1 : 1 was 
observed in any of the samples, and the ratio for all of 
the samples together was 595 males to 607 females (df 
= 14, X 2 : = 1.44, P > 0.05). No external differences were 
observed between the sexes, and hermaphrodites were not 
encountered. 

Seasonal changes in body component indices 

Throughout the study, the mean gonadal index of males 
was more than twice that of females (Kruskal-Wallis, P 
< 0.01), and on no date did the maximum value for any 
given female attain the minimum observed for the males 
(Fig. 4). Nevertheless, the two sexes showed parallel sea- 
sonal cycles in gonadal size. The index for males and fe- 
males dropped significantly between 1 4 May and 1 2 June 

1988 (Kruskal-Wallis, P < 0.01 ), suggesting the release of 
gametes. The gonads showed no further significant change 
in size until the end of the following winter (in March 

1989 for males and April 1989 for females), when a sig- 
nificant growth was evident (Kruskall-Wallis, P < 0.01). 
Both testicular and ovarian indices attained a peak in mid 
July 1989 and then dropped abruptly by 5 August 1989, 
suggesting a second spawning. 

The diameter of the germinal tubules showed a similar 
pattern, although the annual cycle was more pronounced 
(Fig. 4). In both years, the mean diameter attained a max- 
imum just before spawning (higher in 1989 than in 1988) 
and dropped precipitously during spawning (Kruskal- 
Wallis, P < 0.01). The decrease was by =24% in 1988 
compared with =80% in 1989. The diameter of any given 
tubule was relatively uniform, except after spawning when 
some tubules had swollen sections containing unspawned 
gametes. The mean diameter of male tubules was consis- 
tently larger than that of females during May through 
August (Kruskal-Wallis, P < 0.0 1 ), but not during autumn 
and winter (Kruskal-Wallis, P > 0.05). In spite of the 
distinct seasonal pattern in mean tubule size, extremes in 
tubule size were always evident, and every gonad con- 
tained tubules ranging from small to large. 

The 1989 spawning was also observed directly by per- 
sons diving in our study site on 22 July (Normand Piche 




Male 



Female 



10 



M J JASOND 
1988 



F M A M J I A 
1989 

Figure 4. Psolus fabricii. Seasonal changes in the mean gonadal, 
intestinal and respiratory tree indices and in the diameter of the germinal 
tubules for males and females from May 1988 to August 1989. Vertical 
lines indicate the 95% confidence intervals. 



and Andrea Cantin, pers. comm.). Numerous females 
were seen releasing eggs. Spawning was probably wide- 
spread and massive in the lower St. Lawrence Estuary 
because on 25 and 26 July 1989 other divers observed an 
abundance of Psolus fahricii oocytes and embryos 
throughout the first 3 m of the water column over a long 
section (>5 km) of the southern side of the Estuary near 
Rimouski (Lucie Bosse, Institut Maurice-Lamontagne, 
pers. comm.). 

Variations in the intestinal index were largely attrib- 
utable to changes in the intestinal contents: the mass of 
the wall of the first centimeter of the intestine varied by 
<2% throughout the study (and no significant seasonal 



REPRODUCTIVE CYCLE OF PSOLUS l-'AHRK'll 



131 



changes were detected. Kruskal-Wallis, P > 0.05), whereas 
the mass of its contents varied by = 37% (Kruskal-Wallis. 
P < 0.0 1 ). The intestinal index showed intermediate values 
during the summer of 1988, a minimum between De- 
cember 1988 to March 1989, and then a sharp increase 
to a maximum in July 1989. The maximum attained in 
1989 was much greater than in 1988 (Fig. 4). The respi- 
ratory tree decreased during the winter of 1 988-89, started 
to growth in April 1989, and reached a peak in mid July 
1989 (Fig. 4). 

Female reproductive cycle 

Oogenesis. The development of gametes in Psolusfa- 
bricii was transversal, starting at the surface of the germinal 
epithelium and progressing towards the lumen of the tu- 
bule. In addition, it proceeded in a relatively uniform 
fashion along all surfaces of any tubule. Along the surface 
of the germinal epithelium, oogonia occurred in groups 
at numerous points, whereas primary oocytes (<100 /urn) 
were dispersed. The small oocytes (<250 /um) were sur- 
rounded by follicular cells which persisted until spawning. 
The germinal vesicle is central and also persisted until 
spawning. The small oocytes have a PAS-negative baso- 
philic cytoplasm which becomes slightly PAS-positive 
upon attaining 300 ^m (indicating the beginning of gly- 
cogen accumulation), and increasingly positive as vitel- 
logenesis progressed. The morphology and histological 
staining indicated that the oocytes were mature at =800 
um, although they could attain up to 1 400 ^m in diameter. 
Nutritive phagocytes were associated with the tubules that 
had released gametes. For both males and females, the 
tubule wall became more and more PAS-positive after 
spawning until January-February, and then progressively 
PAS-negative until the following spawning period. The 
following five stages of oogenic development were used 
to quantify the seasonal oogenic changes (Fig. 5). 

( 1) Post-spawning (Fig. 5A). The gonadal tubule wall 
is thin and extremely convoluted. Although some residual 
or unspawned oocytes. measuring 400-800 ^m, remained 
in the tubules, the majority of oocytes measured <300 
/im and are generally PAS-negative. Striking elongated 
empty areas are seen in the tubules, suggesting the passage 
of oocytes along the length of the tubule during spawning. 
Nutritive phagocytes begin to appear and are always inside 
of the follicular cells that surround the residual oocytes. 
The follicular cells around the residual oocytes were de- 
generated. 

(2) Recovery (Fig. 5B, C). The gonadal tubule wall is 
very thick. The germinal epithelium is convoluted, and 
beds of small oocytes (<200 /urn in diameter) are present 
along the epithelium. Nutritive phagocytes are closely as- 
sociated with nearly all of the residual oocytes and the 
follicular cells are poorly denned. 



(3) Growth (Fig. 5D). The thickness of the tubule wall 
reaches its maximum. Along the surface of the germinal 
epithelium, many small oocytes (<200 /urn, PAS-negative) 
and some previtellogenic oocytes (300-600 um, PAS-pos- 
itive) are present and nutritive phagocytes are virtually 
absent. 

(4) Advanced growth (Fig. 5E). The tubule wall is 
thinner, and the diameter of the tubules is increased. In 
the lumen of the tubules, well-defined follicular cells are 
associated with large PAS-positive previtellogenic (400- 
600 ^m) and vitellogenic (>600 nm, PAS-positive) oo- 
cytes. The vitellogenic oocytes are reddish orange. Nu- 
merous small oocytes (<400 um) are present along the 
germinal epithelium. 

(5) Mature (Fig. 5F). The tubules are highly dilated, 
their walls thin and not convoluted, and they are almost 
completely filled with mature oocytes (>800 /urn). Each 
oocyte contains one to four nucleoli and a well-defined 
germinal vesicle, which occupies 30-50% of the surface 
of the oocyte in the histological preparations. Immature 
oocytes are virtually absent. 

Seasonal changes in the oogenesis. Advanced oogenic 
stages (advanced growth and mature stages) predominate 
in the large tubules, and earlier stages (post-spawning, re- 
covery and growth stages) in the small tubules (Fig. 6). 
Nevertheless, both categories of tubules showed a seasonal 
pattern that is correlated with the gonadal index cycle. In 
the large tubules, the post-spawning and recovery stages 
are almost always absent, and the major evidence of the 
June 1988 and July 1989 spawnings was the decrease in 
the mature stage. In contrast, the mature stage in the small 
tubules is rare before spawning and absent in other pe- 
riods, and the major evidence of spawning was a sharp 
increase in the post-spawning stage (from to 80%-). These 
observations suggest that the release of mature oocytes 
during spawning transforms large fecund tubules into 
small tubules in post-spawning condition. This change 
coincides with a sharp drop in the mean diameter of tu- 
bules (Fig. 4). Following spawning there is a period of 
inactivity until mid January; then oocyte development 
resumes. In small tubules, a progressive increase in the 
frequency of the growth stage occurred between January 
and July 1989 coincident with a decrease, first in the post- 
spawning stage, and then in the recovery stage. Mean- 
while, the large tubules show an increase in the advanced 
growth and mature stages (Fig. 6). 

Si:e of oocytes. The seasonal pattern in the size structure 
of oocytes varies markedly between large and small tubules 
(Fig. 7). In large tubules, a striking change in the oocyte 
population occurred during the 1988 spawning. Prior to 
spawning, most oocytes measured >800 /urn, whereas after 
spawning in June 1988, 500-700 /urn oocytes predomi- 
nated. Following this, the oocyte population in the large 
tubules was stable. Then in January 1989, renewed oocyte 



132 



... 

m 

^gggsli* 



\ 




Figure 5. Psolusfabrk'ii Light micrographs of ovarian sections illustrating the oogenic cycle. (A) Portion 
of a post-spawning ovary showing the germinal epithelium (GE). residual oocytes (RO). and a channel 
created by the expulsion ol eggs during spawning (C); (B) Early recovery stage showing primary oocytes 
(PO), mature oocytes (M), and nutritive phagocytes (P) surrounded by follicular cells. (This section was 
across a swelling containing residual gametes in a spent tubule); (C) Late recovery' stage showing an abundance 
of nutritive phagocytes (P); (D) Growth stage showing sites of oogonial proliferation (OP), primary oocytes 
(PO), and mature oocytes (M); (E) Advanced-growth stage showing an abundance of both vitellogenic oocytes 
(V) and mature oocytes (M) with nucleoli (N); (F) Mature stage showing large mature oocytes (M) containing 
the germinal vesicle (GV) and surrounded by follicular cells (F). The bar in photograph A represents 800 
Mm and applies to photographs B, C. D and E, whereas the bar in photograph F represents 400 urn. 



growth was evident, and the predominant mode of oocytes 
attained a peak of 1 100 to 1300 ^m in mid July 1989. 
These mature oocytes largely disappeared during the July 
1989 spawning, and on 5 August the modal oocyte class 
was again 500-700 /urn. Although the loss of large oocytes 
(>800 /urn) during spawning was expected, the presence 
of a strong cohort of intermediate oogenic stages (500- 



700 /urn) in the large tubules after spawning seemed at 
first surprising. This was because spawning transformed 
the large tubules into small tubules. Thus, the small tu- 
bules in June 1988, which were characterized by a strong 
mode of oocytes measuring <300 j*m, were probably those 
which had just spawned. They contained residual oocytes 
that were being attacked by nutritive phagocytes, whereas 



Large tubules 



100 



REPRODUCTIVE CYCLE OF PSOLl'S FABR1CI1 

Female Small tubules 

T 



133 




Post-spawning 
Small tubules 




Male 



Large tubules 




M J 



A S O N D J 
1988 



F M A M 
1989 



J A 



Figure 6. Pxolus tahncii. Relative frequency of different gametogenic 
stages (as defined in the Materials and Methods section) in small and 
large, female and male, tubules for the period from May 1988 to August 
1989. 



at the same time, the large tubules contained an abun- 
dance of intermediate stages, and nutritive phagocytes 
were rare. Most oocytes in the small tubules in mid May 
1988 measured 400-600 ^m, whereas those in the large 
tubules in June 1988 measured 600-800 /urn. The simi- 



u 

c 

0> 

cr 

0) 



Large tubules 

T 




500 1000 1500 500 1000 1500 

Oocytes diameter (|im) 

Figure 7. Psolus fabncn. Oocyte diameter distributions for small 
and large tubules for the period from April 1988 to August 1989. For 
each distribution the vertical axis is from to 60% and the mean oocyte 
diameter is indicated by an arrow. 



larity in size distributions for these two samples suggested 
the transition from small to large tubules during spawning. 
Following spawning, until mid December 1988, the oocyte 



134 



J.-F. HAMEL KT II 



distributions for the small tubules showed little change. 
However, a marked increase in the total number of cells 
per surface of germinal tubule was noted in January 1989. 
Thereafter, oocyte size and number progressively in- 
creased in the small tubules until 20 July 1989 when the 
size structure was virtually identical to that of the small 
tubules prior to spawning in 1988. The sharp reduction 
in >800 ^m oocytes in the large tubules during the 1989 
spawning closely followed the changes in the large tubules 
during the 1988 spawning. 

Buoyancy o/ Oocyles. Thirty three of the oocytes showed 
a positive floatability which clearly increased with di- 
ameter (Spearman rank correlation coefficient, r = 0.67, 
df: 32, P < 0.01). This indicated that 1.2 mm oocytes 
would move upward at a rate of 20-30 mm min~'. The 
other four oocytes showed a slightly negative floatability, 
and we suspect that they were damaged (possibly the egg 
membrane was not intact) (Fig. 8). These observations 
indicate that spawned eggs will move to the surface of the 
water column. This agrees with the abundance of devel- 
oping Psolus fabricii embryos near the surface, as observed 
by divers during the 1989 spawning. 

Male reproductive cycle 

Spermiogenesis. The following five stages of spermio- 
genesis are used to quantify the seasonal changes in the 
small and large tubules (Fig. 9). 

( 1 ) Post-spawning (Fig. 9A). The thickness of the tubule 
wall is at its minimum. In the sections, we observed elon- 
gated empty areas along the length of the tubules, sug- 
gesting the passage of gametes during spawning. A few 
residual spermatozoa are present, and no proliferating 
zone (containing spermatogonia, spermatocytes and 
spermatids) was present. 

(2) Recovery. The tubule wall is extremely thick and 
highly convoluted. The tubules contain small quantities 
of spermatozoa and scattered nutritive phagocytes. 

(3) Growth (Fig. 9B, C). The gonadal tubule wall is 
beginning to decrease in thickness but is still convoluted. 
Spermatogonia are abundant along the surface of the ger- 
minal epithelium. Progressing towards the lumen, there 
is a layer of spermatocytes, one of spermatids, and finally 
a small number of spermatozoa in the lumen. 

(4) Advanced growth (Fig. 9D, E). The tubule wall is 
thinner and slightly convoluted, and the lumen is filled 
with spermatozoa. 

(5) Mature (Fig. 9F). The tubules are stretched to their 
maximum diameter and completely filled with sperma- 
tozoa. The tubule wall is nearly smooth, and earlier sper- 
matogenetic stages are absent. 

Psolus fabricii spermatozoa are flagellated with a round 
head measuring 5-6 /urn. Microscopic observation of a 
sperm suspension in seawater, just prior to spawning, re- 
vealed a low motility of the spermatozoa. 



.5 






g- 

I 
o 

3 

pa 



35- 

30- 

25- 

20 - 

15- 

10 - 

5 - 

0- 

-5- 



-10 




0.3 



0.6 



0.9 



1.2 



Diameter (mm) 



Figure 8. Pxoltis Jahricn. Relation of buoyancy to diameter for oo- 
cytes dissected for mature females and activated by being placed in sea- 
water. The regression line is based only on oocytes with positive buoyancy. 



Seasonal changes in spermatogenesis. In May and June 
1988 and again in July and August 1989, advanced stages 
(advanced growth and mature stages) were found in >85% 
of the large tubules, whereas earlier stages (post-spawning, 
recovery and growth stages) predominated in the small 
tubules (Fig. 6). The most striking evidence of the spawn- 
ings in June 1988 and August 1989 was the abrupt ap- 
pearance of post-spawning stages in the small tubules. 
The tubules classified as large after spawning were char- 
acterized by an abundance of early spermatogenetic stages 
and few spermatozoa. These observations suggested, first, 
that the release of spermatozoa from the large tubules 
during spawning diminish their size, so that after spawning 
they were considered as small tubules. Moreover, the tu- 
bules that were selected as large were those that had re- 
cently attained a diameter of 1.9 mm (the lower limit for 
large tubules). Thus a pattern parallel to that observed for 
the females was found. The post-spawning stage, found 
only in the small tubules, disappeared by late summer 
and was replaced largely by the recovery and growth stages. 
Subsequently, the advanced growth and mature stages be- 
came more common and attained a peak a few month 
prior to spawning. 

Thickness of the gonadal luhit/e wall. In males, the dis- 
tributions for gonadal tubule wall thickness were virtually 
always skewed rather than being symmetrical (Fig. 10). 
Further, all size classes up to 140-160 ^m were present 
in both small and large tubules throughout the study, ex- 
cept for four dates near the time of spawning, when the 
largest classes were absent. In both the small and large 
tubules, changes in the distributions followed an annual 
pattern. Just before spawning in May 1988, the mean 
thickness was 20-40 nm; after spawning it decreased 



REPRODUCTIVE CYCLE OF PSOLUS 1-'ABR1CI1 



135 




Figure 9. Psolus fabricii. Light micrographs of testicular sections illustrating the spermatogenic cycle. 
(A) Post-spawning testis showing the germinal epithelium (GE) and channels where sperm passed during 
spawning (C); (B) Growth stage showing the highly convoluted germinal epithelium (GE) and the proliferation 
zone (PZ); (C) Growth stage showing the germinal epithelium, spermatogonia (SG), spermatocytes (SC) 
spermatids (ST) and spermatozoa (SP) in successive layers progressing towards the lumen; (D) Early advanced- 
growth stage showing the proliferating zone (PZ) and spermatozoa (SP): (E) Late advanced-growth stage 
showing the thin gonadal tubule wall and an abundance of spermatozoa (SP); (F) Mature stage showing the 
thin tubule wall (TW), absence of the proliferation zone, and great numbers of spermatozoa (SP) in the 
lumen. The bar in photograph A represents 800 ^m and applies to photographs B, D, E and F. whereas the 
bar in photograph C represents 300 urn. 



slightly. Subsequently, the thickness of the tubule wall 
progressively increased, although the pattern varied de- 
pending on tubule size. Thus, the large tubules grew more 
rapidly and attained a peak (=120 ^irn) in November 
1988, whereas the small tubules did not attain a peak 
(=140 nm) until February 1989. Subsequently, the size 
of the modal size class again decreased to 20-40 /um fol- 
lowing the 1989 spawning (Fig. 10). 



Environmental factors 

Temperature. The mid May to mid June spawning, in 
1988, coincided with the spring warming period, and 
temperatures attained about 5C at the time of spawning 
(Fig. 11). However, temperatures fluctuated markedly 
during this period. An increase from 4 to 6C was ob- 
served between 20 and 23 May 1988, and a drop of 6.7 



136 



J.-F. HAMEL ET AL. 



Small tubules Large tubules 




40 80 120 160 40 80 120 160 

Gonadal tubule wall thickness (|im) 

Figure 10. Psolusfabricii Frequency distributions! 10 nm size classes) 
of the thickness of the gonadal tubule wall for small and large tubules 
for males collected from May 1988 to August 1989. The arrows indicate 
the mean thickness for each sampling date. 



to 4.4C between 5 and 6 June. These variations were 
due to the semidurnal tides in the Estuary (Demers el al. 
1986). During 1988. the maximum temperature was 
reached in mid July, and the autumnal decrease began in 
late August. 

In 1989, warming began in March, attained a peak in 
late June, and then varied around 6C until the end of 
the study (2-4C daily fluctuations were frequent). The 
accelerated gonadal growth, which began in March or 
April, occurred at about the time that the vernal warming 
began. There was no spawning, either during the warming 
phase or during marked temperature variations in May 
and June; rather, spawning occurred in late July when 
temperatures were more stable. Thus, although spawning 
occurred at about the same temperature in the two years 
(5-6C), the point in the temperature cycle was quite dif- 
ferent (Fig. 1 1). 

Photoperiod. In our study, the renewal of gametogenesis 
in January coincided with the period when day length 
and daily bright sunshine were beginning to increase, and 
the gonadal peak was attained at the photoperiod maxi- 
mum (Fig. 11). Although Psolusfabricii spawned near 
the photoperiod maximum in both years, the 1988 
spawning occurred at the beginning of this maximum and 
the 1989 spawning one month later, when photoperiod 
was just beginning to decline. 

Freshwater run-off' and the predicted timing of the phy- 
toplankton bloom. The period during which freshwater 
run-off decreased in the Estuary was much later in 1989 
(July) than in 1988 (late May) (Fig. 11). Nevertheless, 
spawning in both years coincided with this event, sug- 
gesting a relationship between the two. 1989 was an ex- 
ceptional year in that the run-off was markedly greater 
and more delayed than in the five previous years. It was 
also unusual in that spawning of the green sea urchin 
Strongylocentrotus droebachiensis, a signal of the phyto- 
plankton increase, was much later than in the previous 
years. The urchin spawned abruptly between 14 July and 
5 August, exactly the same period during which Psolus 
fabricii spawned (Fig. 1 1 ). In contrast, when urchins were 
studied in the Estuary in the previous years, spawning 
occurred prior to mid June (Starr, 1990). 

Intestinal contents. The intestine of adult Psolusfabricii 
contains two types of materials: ( 1 ) non living particles 
and (2) phytoplanktonic cells; the major species of plank- 
ton are the diatoms Thalassiosira sp., Coscinodiscus sp., 
Chaetoceros sp., and Skeletonema sp. (Fig. 1 1). During 
the autumn and winter, only 20-50% of the contents were 
phytoplanktonic cells, and this increased during the spring, 
attaining virtually 100% in the summer (Fig. 1 1 ). 

A marked seasonal pattern was evident for the diatoms 
present in the intestines. Most cells in the first samples in 
May and June 1988 were Coscinodiscus sp., Thalassiosira 
sp., and Skeletonema sp., suggesting a diatom bloom at 



REPRODUCTIVE CYCLE OF PSOLL'S 1-'ABRIC11 



137 



41 

t- 
3 

"% 



u 



5 
3' 

1 



ns 

D 



w- 






<u 


8^ ' 


.5 


15] 


. t 


e 




' ' '. .-'"' 


3 






tfl 


10- 


;-. - ^ ... ._ 



'<fl 

D 



T3 



o 

u 




16- 

14 

12' 

ID- 
S' 
6' 
4" 

7 



Strongy/ocentrotus 
droebachiensis 



Female 




u. 3000i 
"o 

*f 2000-j 



s 



u 
u 



Ol 

Xi 
g 

3 

z 



u 

ra 
U 

C 






13 



1000- 



o 

8000 
6000 
4000 
2000 




Coscinodiscus 
tSkeletonema 




Thalassiosira 




Chaetoceros 



1001 
80 
60 
40 
20 







M J J ' A'S 'O 'N 'D ' J 'F M 'A M ') ' J 'A 

1988 I 1989 



this time. Subsequently, there was a progressive decrease 
to the winter minimum. In 1989, Coscinodiscus sp. 
showed an increase during March and April, and Tluil- 
cissiosim sp. and Skeletonema sp. an increase in June (Fig. 
1 1 ). The latter two species increased further to the highest 
level for the study in late July. This suggested an intensive 
bloom at this time. Whereas the above large diatom species 
typically occur in chains in the water column, they were 
always present as individual cells in the intestines. No 
animal structures were observed in Pso/us fabricii intes- 
tines. 

Discussion 

Morphological comparisons with other holothurians 

Non-branched germinal tubules, such as are found in 
Psolus fabricii, also occur in Ciiciinuiriu litbrica and Yp- 
silolhuria talismani (Atwood and Chia, 1974; Tyler and 
Gage, 1983), but branched tubules have been reported in 
other holothurians (Atwood. 1974; Smiley and Cloney, 
1985; Cameron and Fankboner, 1986; Tyler and Billett, 
1987). The increase in the number of gonadal tubules 
with size in P. fabricii (Fig. 2A) is in contrasts with As/in 
Icfevrei. where tubule number is highly variable and not 
related to size (Costelloe, 1985). 

Throughout the year, the gonad of Pso/us fabricii is 
larger in males than in females, and this is primarily due 
to the number of tubules in the testis rather than to tubule 
length or diameter (Fig. 2A, B). This, together with the 
greater drop in gonadal mass during spawning (Fig. 4), 
suggests that males have a greater reproductive output. A 
larger male gonad is not a general holothurian character- 
istic, since the inverse is the case for Slichopus californicits 
(Cameron and Fankboner, 1986), and equal-sized gonads 
are reported for Cucitniaria pseudocurata (Rutherford, 
1973). A sex ratio of 1:1 has been reported for several 
holothurians in addition to P. fabricii (Cameron and 
Fankboner, 1986; Jespersen and Lutzen, 1971;Conand, 
1982; Engstrom, 1982; Mosher, 1982), but numerous 
others have a ratio favoring males (Lawrence, 1987). 

Necrotic fragments of germinal tubules as found in 
Psolus fabricii have previously been described for Sticfio- 



Figure 1 1 . Seasonal variations in temperature, daylight, daily bright 
sunshine, and freshwater run-off, as well as the relative proportion of 
living and non-living materials in a == 30 g portion of the intestinal mass 
and the absolute abundance of the four major phytopiankton species, 
in the first centimeter of the intestine of Psolus fabricii, during the period 
from May 1988 to August 1989. The gonadal index cycle of the urchin 
Strongylocentrotus droebachiensis was quantified in 1989, and the drop 
in the index between 20 July and 5 August suggests that there was a 
phytopiankton bloom at this time. The vertical lines indicate the 95% 
confidence intervals. The arrows above the temperature cycle indicate 
when P. fabricii spawned. 



138 



J.-F. HAMEL ET AL 



fiux califomicus by Cameron and Fankboner (1986), but 
only for males. In both sexes of P. fabricii. necrotic tubules 
were most common in large individuals and thus may be 
related to the decrease in relative gonadal size in very 
large individuals. Aerobic metabolism furnishes most of 
the energy requirements of echinoderms (Ellington, 1982; 
Lawrence and Lane, 1982; Shick. 1983; Feral and Mag- 
niez. 1985), and Hopcroft el al. (1985) demonstrate that 
75% of oxygen required by P. fabricii( individuals weighing 
80 g in wet mass) is obtained through the respiratory tree. 
Thus, the increase in the size of the respiratory tree as the 
gonads grow may indicate its role in supplying oxygen for 
gametogenesis (Fig. 4). 

The follicular cells associated with developing oocytes 
in echinoderms supply nutriments to the oocytes and also 
control the environment around them (Hirai and Kana- 
tani, 1971). In Psolusfabricii, the follicular cells are closely 
associated with the oocytes as they migrate into the lumen 
during maturation, and they may surround the oocyte 
during spawning as reported for Cucumaria elongata 
(Chia and Buchanan, 1969). This contrasts with Stichopus 
califomicus in which the follicular cells stay attached to 
the epithelium as the oocytes migrate into the lumen 
(Smiley and Cloney, 1985). The clearly stratified sper- 
matogenesis of P. fabricii (Fig. 9) contrasts with that in 
Leptosynapta clarki and C. hibrica, in which spermato- 
gonia and spermatids are erratically distributed through- 
out the lumen (Atwood, 1973, 1974). 

Gametogenesis 

In Psolusfabricii, oogenesis begins with the production 
of precursor cells in the small tubules in January. Pro- 
gressively through the winter and spring these cells are 
transformed into oogonia and primary oocytes, and by 
mid summer 400-600 ^m cells are most abundant (Fig. 
7). These stages in small tubules do not contribute to 
spawning. After spawning, some small tubules attain > 1.9 
mm, a diameter sufficient to be classified as large tubules. 
During the autumn, the oocyte distributions in the tubules 
classified as large, remain virtually static, indicating a pe- 
riod of inactivity; then in January, oocytes growth and 
tubule enlargement resumes. This growth continues until 
the following summer when most oocytes measure >800 
^m and the tubules attain their maximum diameter (Fig. 
4). Finally, the release of these large oocytes during 
spawning results in a drop in the size of the tubules. From 
the time of spawning until the following January, nutritive 
phagocytes are active in destroying the residual oocytes 
(Fig. 5). 

Our study also indicates a prolonged spermatogenesis. 
As with oogenesis, it begins with the production of pre- 
cursor cells in the small tubules in mid winter, although 
a prior accumulation of reserves in these tubules is indi- 



cated by the thickening of the gonadal tubule wall during 
the previous autumn (Fig. 10). In late winter and spring, 
as the thickness of the germinal wall decreases, spermato- 
gonia. spermatocytes, and spermatids progressively ac- 
cumulate in the tubules (Figs. 9, 10). Since these tubules 
contain only small amounts of spermatozoa, they prob- 
ably do not participate in spawning. During and after 
spawning they progressively attain the size of large tubules. 
The major change after spawning in what are now the 
large tubules is the thickening of the gonadal tubule wall 
and this peaks in February (Fig. 10). At about the same 
time, the production of spermatozoa increases, and this 
amplifies until a peak just prior to spawning (Fig. 6). Fi- 
nally, with the release of sperm during spawning, these 
large tubules become small; after spawning nutritive 
phagocytes become abundant. This is the first report of 
the testicular cycle taking longer than a year in holothu- 
rians. 

Although the examination of large and small tubules 
indicates that gametogenesis is prolonged, studies with 
radioactive markers are needed to determine its duration 
precisely. Our observations suggest that the production 
of the majority of gametes begins in winter and terminates 
15-18 months later (two summers later). In some tubules, 
however, this process may be much longer or, at times, 
shorter. 

That gametogenesis in Psolus fabricii generally takes 
more than a year was revealed from the separate histo- 
logical studies of small and large tubules. Smiley and Clo- 
ney (1985) and Smiley (1988) examined the gonads of 
female Sticliopus califomicus collected in different seasons. 
Their observations of three size groups of tubules, with 
the most advanced stages of oogenesis only being present 
in the largest tubules, similarly led them to conclude that 
oogenesis was a long process. In contrast to P. fabricii. 
the various sized ovarian tubules of S. califomicus are not 
intermixed. Rather they are arranged in order, the large 
fecund tubules being located posteriorly. Smiley and Clo- 
ney (1985) report that the large tubules are completely 
reabsorbed once the oocytes are released. Based on these 
observations, they propose that the tubules are produced 
at the anterior of the gonad and migrate posteriorly as 
they increase in size and state of development. Reabsorp- 
tion does not occur in P. fabricii, since a new group of 
oocytes is evident in the fecund tubules at the time of 
spawning and persists in spent tubules during the autumn 
when residual gametes are being phagocytised. Thus, the 
pattern of tubule and gamete production in S. califomicus 
contrasts markedly with that in P. fabricii. Resorption of 
tubules has also been noted in Mesothuria intestinalis 
(Theel, 1 90 1 ) and Ypsilothuria talisman! (Tyler and Gage, 
1983), but probably does not occur in S. japonicus (Ta- 
naka, 1958) and three species of sea cucumber examined 
byConand(1981). 



REPRODUCTIVE CYCLE OF PSOLL'S FABRICII 



139 



Gametogenesis in another holothurian, Aslia lefrevrei. 
in the same family as Psolus fabricii (Dendrochirotida), 
follows still another pattern. The tubules are of uniform 
width, and gametogenesis follows an annual pattern that 
is highly synchronized amongst the tubules (Costelloe, 
1985). For example, during numerous periods in the year, 
all of the tubules are at the same stage of gametogenetic 
development. As in P. fabricii. the tubules are not reab- 
sorbed after spawning, the growth continues for a sub- 
sequent year, oocytes (<200 /^m) appearing prior to 
spawning. The above observations indicate that the pat- 
tern of gametogenesis varies markedly even within closely 
related holothurian species. Future studies should there- 
fore consider the pattern of production of the tubules as 
well as the gametogenesis within the tubules. 

Control of gametogenesis 

The active phase of gametogenesis in Psolus fabricii 
begins in January and continues until spawning. In small 
tubules of both males and females, early gametogenetic 
stages proliferate. And in the large tubules, oocyte growth 
increases in females, and more advanced spermatogenic 
stages are produced in males. This renewed gametogen- 
esis occurs when water temperatures are near freezing 
(= -1C, Therriault, 1973; Ouellet-Larose, 1973), and 
an increase does not occur until several months later 
(March-April). Further, food conditions are minimal as 
evidenced by the near absence of phytoplankton cells in 
the intestines. The first increase in phytoplanktonic cells 
in the intestine is in mid March (Fig. 1 1 ). The only notable 
environmental change during the mid-winter renewal of 
gametogenesis is the return to increasing photoperiod. In 
fact, virtually the entire gametogenetic period coincides 
with the period of increasing day length and daily bright 
sunshine, and peak maturity is attained at the maximal 
photoperiod. These observations suggest that an increasing 
photoperiod controls gametogenesis. Photoperiod has 
been experimentally shown to control gametogenesis in 
numerous taxa including echinoderms (Pearse et al, 1986; 
McClintock et al., 1990). P. fabricii occurs where annual 
changes in temperature and food availability are pro- 
nounced, certainly more so than the habitats of other 
echinoderms that have been used to study the photoperiod 
control of gametogenesis. Nevertheless, the activation of 
gametogenesis well before the increase of temperature and 
food from their annual minima suggests the potential im- 
portance of photoperiod for P. fabricii. 

The increased growth in tubule diameter and gonadal 
mass observed in March or April (Fig. 4) suggests a second 
point that may be controlled by environmental factors. 
At this time, photoperiod has been increasing for several 
months, and the most notable environmental change is 
the first increase of phytoplanktonic cells in the intestines 



(Fig. 11). Phytoplankton are probably not abundant at 
this time, but abundant enough that the feeding mecha- 
nism of Psolus fabricii can filter cells from the water col- 
umn, contributing to gonadal production. An influence 
of food availability on gonad development is also sug- 
gested by numerous studies on other invertebrates (Sastry 
and Blake, 1971; Gimazane, 1972; Bayne, 1975). The 
vernal warming is another potential environmental change 
at this time, and our temperature record, which began in 
mid April in 1989, indicates that warming had occurred 
in April. 

Role of nutritive phagocytes and the gonadal tubule wall 
in supporting gametogenesis 

The appearance of nutritive phagocytes and their role 
in eliminating residual gametes is well documented in 
studies of holothurians (Tanaka. 1958; Costelloe, 1985; 
Smiley and Cloney, 1985) and other echinoderms (Lieb- 
man, 1950; Holland and Giese, 1965; Fenaux, 1972). In 
echinoids, nutritive phagocytes have been shown to 
transform reserves to dissolved compounds which are later 
used for gamete production (Holland and Giese, 1965). 
This may occur in Psolus fabricii, but since the phagocytes 
disappear before the active gametogenic period, these 
substances would have to be stored for their eventual use 
in gametogenesis. 

In Psolus fabricii, the gonadal tubule wall thickens dur- 
ing the period of gametogenic inactivity, from autumn to 
mid winter (Fig. 10). This growth, coincident with the 
autumnal decrease in food availability, falling tempera- 
tures, and short photoperiod, appears to be a priority in 
the use of energetic reserves of the animal at this time. In 
a variety of echinoderms. gametogenesis is similarly pre- 
ceded by a thickening of the tubule wall, and this is 
thought to represent an accumulation of reserves for ga- 
metogenesis (Pearse, 1969; Conor, 1973). Costelloe (1985) 
suggests that certain increases in gonadal size in the sea 
cucumber Aslia lefrevrei are due to the storage of materials 
in the tubule wall, but P. fabricii does not show an increase 
in gonadal mass as the tubule wall thickened. The earlier 
growth of the tubular wall in the large tubules, compared 
with the small tubules, suggests that resources are chan- 
neled preferentially to the large tubules. This could be 
because the later stages of gametogenesis in the large tu- 
bules require more resources than the earlier stages in the 
small tubules (Fig. 10). In the small tubules, the massive 
proliferation of the earlier gametogenetic stages during 
January and February precedes the thinning of the wall 
that begins in March. This suggests that the proliferation 
does not require large amounts of reserves. 

External spawning cues 

A massive loss of gametes over a short period strongly 
suggests that spawning is controlled by external factors 



140 



J.-F. HAMEL ET AL. 



(Himmelman, 1981; Giese and Kanatani, 1987; Starr, 
1990; Starr el al.. 1990, 1992). In both years of our study, 
the gonadal indices, measurements of tubule diameter, 
and histological observations indicated an abrupt spawn- 
ing between two successive sampling dates (a 4-weeks in- 
terval in 1988, and 2 weeks in 1989). Temperature more 
than any factor has been suggested as a spawning signal 
in invertebrates (Orton, 19 14; Brown, 1984; Bricelij et al.. 
1987), but we did not observe a consistent relationship 
between temperature and spawning in Psolus fabricii. 
Similar conclusions have been reported for other holo- 
thurians (Costelloe, 1985; Cameron and Fankboner. 
1986). The only study suggesting that temperature might 
control spawning in holothurians is that of Tanaka (1958). 
That spawning time of P. fabricii varies between years 
suggests that spawning is not controlled by photoperiod. 

Our data suggests that spawning in Psolus fabricii may 
be signaled by the phytoplankton increase. Therriault and 
Levasseur( 1985, 1986) demonstrate that the phytoplank- 
ton bloom in the Estuary is always delayed relative to that 
of the Gulf of St. Lawrence because of freshwater run-off: 
the bloom only develops after the surface layer has sta- 
bilized, which occurs when the spring run-off drops. In 
both 1988 and 1989, spawning in P. fabricii coincided 
with the predicted onset of the bloom, based in turn on 
the decrease in freshwater run-off (Fig. 1 1). Such a syn- 
chrony is further indicated by the coincidence of the 
spawning dates of P. fabricii with those of the green sea 
urchin, whose spawning is triggered by phytoplankton 
(Himmelman, 1975; Starr et al.. 1990, 1992). Cameron 
and Fankboner ( 1986) indicated that phytoplankton may 
also initiate spawning in Stichopus calif ornicus. They 
noted that spawning individuals in the field were almost 
only observed after periods of bright sunshine (>5 h d-1 
for >4 d), and further that phytoplankton was abundant 
during some spawnings. 

The spawning in 1989 resulted in the release of a larger 
amount of gametes than in 1988 (Fig. 4). Possibly more 
gametes attained maturity in 1989 because of the delay 
in spawning. We suggest this because the mean tubule 
diameter attained a higher value in 1989 than in 1988, 
and the difference was largely due to the growth that oc- 
curred during June and July (Fig. 4). The longer period 
before spawning may have permitted the completion of 
gametogenesis in additional tubules, tubules that might 
otherwise not have matured until the following year. In 
addition, the gonadal index and tubule diameter did not 
fall as low in 1988 as in 1989. This again suggests that 
fewer gametes were mature when the spawning cue was 
detected in 1988. This discontinuation of gamete pro- 
duction after the early 1988 spawning suggests that phys- 
iological mechanisms prevent further gamete maturation 
and secondary spawnings once spawning has occurred. 
For the urchin, for which phytoplankton has been shown 



to be the spawning cue, spawning in the laboratory in- 
creases with plankton abundance (Starr el al.. 1990). If 
this is true for Psolus fabricii, a more intense phytoplank- 
ton bloom in 1989 might account for the more massive 
spawning in that year. The greater mass of intestinal con- 
tents of P. fabricii in 1989 suggests there was a more in- 
tense bloom in that year (Fig. 1 1). 

Plankton ic stages of Psolus fabricii 

Holothurians with small eggs usually have a larval stage, 
whereas species with large eggs usually develop directly 
into juveniles (Tanaka, 1958; Rutherford, 1973; Green, 
1978; Tyler and Billett, 1988). Psolus fabricii has excep- 
tionally large eggs, sometimes attaining 1400 ^m in di- 
ameter, and lacks a larval phase (pers. obs.). Nevertheless, 
the juvenile stage is pelagic. Probably, as Tyler and Billett 
( 1987) indicate forelasipodid holothurians, the abundant 
nutritive reserves in the egg account for the high degree 
of floatability of the pelagic stage. Warmer temperatures 
near the surface may enhance the rate of development, 
and in addition the pelagic juveniles may further benefit 
from increased food resources, either in the form of dis- 
solved substances or planktonic cells. The feeding podia 
are well developed around the mouth of the pelagic ju- 
veniles of P. fabricii (pers. obs.). which suggests that they 
are capable of feeding on suspended particles. 

Feeding 

Some holothurians feed on organic material at the wa- 
ter-sediment interphase (Hyman, 1955; Reese, 1966; Fer- 
guson, 1969) whereas others feed on planktonic particles 
(MacGinitie and MacGinitie, 1949; Brumbaugh, 1965). 
Psolus fabricii is a highly selective feeder. For example, 
although numerous phytoplankton and zooplankton spe- 
cies are common in the region where we collected P. fa- 
bricii (Cote, 1972; Cardinal and Lafleur, 1977; Fortier et 
al.. 1978; Maranda and Lacroix, 1983; Therriault and 
Levasseur, 1985, 1986), the intestines contained almost 
exclusively four species of diatoms. The proportion of 
these items decreases in abundance in the intestine as 
productivity drops in late autumn and winter and is re- 
placed by nonliving matter. P. chitonoides (Fish, 1967) 
and Cucumaria elongata (Fankboner, 1978) similarly feed 
primarily on suspended living particles. That dendrochi- 
rotes are most abundant in temperate and subtropical 
waters, and rare in tropical areas and at great depths 
(Pawson. 1966; Hansen, 1975; Lawrence, 1987), suggests 
that they require the abundance of small living particles 
such as found in shallow water northern areas (Lawrence, 
1987). Nonliving matter or detritus has been suggested to 
be an important source of food in the diet of suspension 
feeders (Baier, 1935; Newell. 1965: Kirby-Smith, 1976) 
and could provide nutritional resources for P. fabricii 



REPRODUCTIVE CYCLE OF PSOLUS FABRIC/1 



during the winter. The long intestine of dendrochirotides 
may be an adaptation for digesting vegetal matter (Law- 
rence, 1987). P.fabricii has a remarkably long intestine 
relative to its body size (intestinal length = -1.68 + 4.52 
dry body wall mass; r = 0.95, n = 37). For example, an 
adult measuring 6.1 cm in distance mouth-anus, (34 g) 
has a 1 52 cm intestine. This unusually long intestine may 
be an adaptation to its diet of diatoms which are protected 
by siliceous frustules. 

Acknowledgments 

We are greatly indebted to N. Piche for his help in 
collecting the samples and for the underwater photographs 
ofPsolusfabricii. The aid of S. Paradis, M. Claereboudt, 
A. Duval, E. Bourget, A. Cantin, A. Cardinal, H. Gu- 
derley. L.-P. Hamel, O. Hamel. B. Laganiere, and A. 
Tremblay at various points in the project is also gratefully 
acknowledged. Thanks are also due to A. Pusterla (De- 
partement de Pathologic, Universite Laval) and A. J. Col- 
let (Departement d'Anatomie, Universite Laval) and the 
Departement d'Oceanographie (Universite du Quebec a 
Rimouski) for the histological preparations. The first au- 
thor was supported by a FCAR scholarship and the re- 
search was supported by NSERC funding to J. H. H. and 
L. D. 

Literature Cited 

Atwood, D. G. 197.1. Ultrastructure of the gonad wall of the sea cu- 
cumber Lep/osynapta clarki. Z Zclltorscli 141: 319-330. 

Atwood, D. G. 1974. Fine structure of the spermatogonia. spermatocytes 
and spermatids of the sea-cucumber (Echinodermata: Holothuro- 
idea). Can. J /.ool 52: 1389-1396. 

Atwood, D. G., and F.-S. Chia. 1974. Fine structure of an unusual 
spermatozoan of a brooding sea cucumber. Ciiaiinaria lithrica. Can 
J. Zoo/. 52: 519-523. 

Baier, C. R. 1935. Studicn zur hydro-backteriologie stehenden Bin- 
nengerwasser. Arch. Hydmbiol. 29: 183-264. 

Bayne, B. L. 1975. Reproduction of bivalve molluscs under environ- 
mental stress. Pp. 259-277 in Physiological Ecology of Estuariiic 
Organisms. F. J. Vernberg, ed. University of South Carolina. 

Bricelij, V. M., J. Kpp, and R. E. Malouf. 1987. Intraspecific variation 
in reproductive and somatic growth cycles of bay scallops Argopecien 
irradian.s. Mar. Ecol. Prog. Ser. 36: 123-137. 

Brown, R. A. 1984. Geographical variations in the reproduction of the 
horse mussel Modiolus inodiolus (Mollusca: Bivalvia). / Mar. Biol. 
Asxoc. U. A 64: 751-770. 

Brumbaugh, J. H. 1965. The anatomy, diet, and tentacular feeding 
mechanism of the dendrochirote holothurian Ciictimaria curata 
Cowles 1907. Ph.D. thesis. Stanford University, California. 1 19 pp. 

Bulteel, P., M. Jangoux, and P. Coulon. 1992. Biometry, bathymetric 
distribution and reproductive cycle of the holothuroid Holollmria 
titbiilnia (Echinodermata) from Mediterranean seagrass beds. Mar. 
Ecol. 13: 52-62. 

Cameron, J. L., and P. V. Fankboner. 1986. Reproductive biology of 
the sea cucumber Para.siiclwpus calilifornicus (Stimpson) (Echino- 
dermata: Holothuroidea). I. Reproductive periodicity and spawning 
behavior. Can. J. Zoo/. 64: 168-175. 



Cardinal, A., and P.-K. Laflcur. 1977. Le phytoplancton estival de I'es- 

tuaire du Saint-Laurent. Soc. Pliycol l-'rance Bull. 22: 150-160. 
Chia, F.-S., and J. B. Buchanan. 1969. Larval development of Cucii- 
mariaclimgata( Echinodermata: Holothuroidea)./ Mar. Biol.Assoc. 
U. A. 49: 151-158. 

Conand. C. 1981. Sexual cycle of the three commercially important 
holothurian species (Echinodermata) from the lagoon of New Cale- 
doma. Bull. Mm Set 31: 523-543. 

Conand, C. 1982. Reproductive cycle and biometnc relations in a pop- 
ulation of Aclinopyga ci'/iinilcs (Echinodermata: Holothuroidea) 
from the lagoon of New Caledonia, western tropical Pacific, Pp. 
437-442 in Proceeding ol l/ic International Conference on Ecluno- 
dcnm. Tampa Hay. J. M. Lawrence, ed. A. A. Balkema, Rotterdam. 
Costelloe, J. 1985. The annual reproductive cycle of the holothurian 
Axlia letem'i (Dendrochirota: Echinodermata). Mar. Biol. 88: 155- 
165. 

Cote, R. 1972. Influence d'un melange mtensif de diflerents types d'eau 
sur la distribution spatiale et temporelle du zooplancton de I'estuaire 
du Saint-Laurent. Ph.D. thesis. Universite Laval, Quebec. 251 pp. 
Demers, S.. L. Legendre, and J.-C. Therriault. 1986. Phytoplankton 
response to vertical tidal mixing. Pp. 1-40 in Tidal Mixing and 
Plankton Dynamic*. J. Bowman, C. M. Yentsh, and W. T. Peterson, 
eds. Springer- Verlag, New York. 

Edwards, C. L. 191(1. Revision of the Holothuroidea. I. Ciicnmariu 
frondoda (Gunner) 1767. Zool.Jahrb Ahi Svst Ockol. Geogr. Tiere 
29: 334-357. 

Ellington, \V. R. 1982. Intermediary metabolism. Pp. 395-415 in 
Echinoderm Nutrition. M. Jangoux and J. M. Lawrence, eds. A. A. 
Balkema. Rotterdam. 

Engstrom, N. A. 1980. Reproductive cycles of lloloilmnu tloridana. 
H (II ) mexicana and their hybrids (Echinodermata: Holothuroidea) 
in southern Florida. Inl J Invert. Reprod. 2: 237-244. 
Engstrom, N. A. 1982. Brooding behavior and reproductive biology 
of sub-tidal Puget Sound sea cucumber. Ciicuniana lubricu (Clark, 
1901) (Echinodermata: Holothuroidea). Pp. 447-450 in Proceedings 
of /lie International Conference on Ec/iinoderms. Tampa Bar. 3. M. 
Lawrence, ed. A. A. Balkema. Rotterdam. 

Fankboner, P. V. 1978. Suspension-feeding mechanisms of the ar- 
moured sea cucumber Psolits clutonoides Clark. ./. Exp. Mar. Biol. 
Ecol 31: 11-25. 
Fenaux, L. 1972. Modalites de la ponte chez 1'oursin Sphaerecluniis 

graniilans (Lamarck). Rente (/V.v. Hydmbiol. 57: 551-558. 
Feral, J. P., and P. Magniez. 1985. Level, content and energetic equiv- 
alent of the main biochemical constituents of the subantarctic mo- 
ladid holothurian Eumolpadia violacea at two seasons of the year. 
Comp Bioclicin Physiol. 81: 415-422. 

Ferguson, J. C. 1969. Feeding, digestion, and nutrition in Echino- 
dermata. Pp. 71-100 in Chemical Zoology, \'ol III. M. Florkin and 
B. T. Scheer. eds. Academic Press, New Y'ork. 
Fish, J. D. 1967. The biology ofCucumaria e/ongaia (Echinodermata: 

Holothuroidea). J. Mar. Biol. Assoc. U. A' 47: 129-143. 
Fortier, L., L. Legendre, A. Cardinal, and C. C. Trump. 1978. Vanabilite 
a court terme du phytoplancton de I'estuaire du Saint-Laurent. Mar. 
Biol. 46: 349-354. 
Galigher, A. E., and E. N. KozlofT. 1971. Essentials of Practical Micro- 

lec/inii/ues. Lea and Febiger, Philadelphia, PA. 

Giese, A. C., and H. Kanatani. 1987. Maturation and spawning. Pp. 
251-313 in Reproduction of Marine Invertebrates. I'ol. 9, General 
Aspect: Seeking Unity in Diversity. A. C. Giese and J. S. Pearse, eds. 
Blackwell Scientific Publications. California. 

Giese, A. C., and J. S. Pearse. 1974. Introduction: general principles. 
Pp. 1-49 in Reproduction of Marine Invertebrates: Acoelomate and 
Pseitdocoe/oinate Metaioans. Vol. 1. A. C. Giese and J. S. Pearse, 
eds. Academic Press, New York. 



142 



J.-F. HAMEL ET AL 



Gimazane, J. P. 1972. Etude experimentale de I'action de quelqucs 
facteurs externes sur la reprise de 1'activite genitale de la coque. 
Cerastoderma cdulc L. Mollusque bivalve. C. R Sue. Bin! 166: 
587-589. 

Conor, J. J. 1973. So\ ratio and hemaphroditism in Oregon inlertidal 
populations of the edunoid Strongylocentrotus pitrpiiratus Mar Bio/ 
19: 278-2X0. 

Green, J. D. 197S. The annual reproductive cycle of the apodus holo- 
thurian Leptosynapta tennis: a bimodal breeding season. BioL Bull. 
154:68-78. 

Hansen. B. 1975. Systematics and biology of the deep-sea holothurians. 
Galathca. Reports. 1 3 pp. 

Himmelman, J. II. 1975. Phytoplankton as a stimulus for spawning 
in three marine invertebrates. J. Exp. Mar. BioL Ecol. 20: 199-2 14. 

Iliiiuiiflnian, J. H. 1981. Synchronization ot spawning in marine in- 
vertebrates by phytoplankton. Pp. 3-19 in Advances in Invertebrate 
Reproduction. W. H. Clark, Jr. and T. S. Adams, eds. Elsevier/North- 
Holland. New York. 

Hirai. S., and H. Kanatani. 1971. Site of production of meiosis-inducing 
substance in ovary of starfish. E.\p. Cell Rc\ 67: 224-227. 

Holland, N. D., and A. C. Giese. 1965. An autoradiographic investi- 
gation of the gonads of the purple sea urchin (Strongylocentrotus 
piirpiinilus). Biol Bull. 128: 241-258. 

Hopcroft, R. R., B. D. Ward, and J. C. Roff. 1985. The relative sig- 
nificance of body surface and cloacal respiration in Psolus fabricii 
(Holothuroidea: Echinodermata). Can. J. Zool. 63: 2878-2881. 

Humason, G. L. 1981. Animal Tissue Techniques. W. H. Freeman. 
San Francisco. 

Hyman, L. H. 1955. The Inveriehraies: Echinodermata, I'ol /I. 
McGraw-Hill Book Co., New York. 763 pp. 

Jespersen, A., and J. Lutzen. 1971. On the ecology of the aspidochirote 
sea cucumber Stichopus lermulus (Gunnerus). Nor\\: J Zool. 19: 
117-132. 

Junqueira, L. C., J. Carneiro, and J. A. Long. 1986. Basic Histology, 
Fifth edition. Lange Medical Publications, Los Altos, CA. 529 pp. 

Kille, F. R. 1939. Regeneration of gonad tubules following extripation 
in the sea cucumber Throne hriareus. Biol. Bull. 76: 70-79. 

Kille, F. R. 1942. Regeneration of the reproductive system following 
binary fission in the sea cucumber Hololhuria parcu/a. Biol. Bull. 
83: 55-66. 

Kirby-Smith, \V. \V. 1976. The detritus problem and the feeding and 
digestion of an estuarine organism. Pp. 469-479 in Esluanne Pro- 
cesses, Vol. 1. M. L. Wiley, ed. New York. 

Krishnaswamy, S., and S. Krishnan. 1967. A report of reproductive 
cycle of the holothurian Holotlnina scahra Jaeger. Curr. Set. 36: 
155-156. 

Lawrence, J. M. 1987. .-1 Functional Biology of Echinoderms. Croom 
Helm Ltd., Australia. 339 pp. 

Lawrence, J. M., and J. M. Lane. 1982. The utilization of nutrients 
by post-metamorphic echinoderms. Pp. 331-371 in Echinoderm 
Nutrition. M. Jangoux and J. M. Lawrence, eds. A. A. Balkema, 
Rotterdam. 

Liebman, E. 1950. The leucocytes ofArabacia punctulala. Biol. Bull. 
98: 46-59. 

MacGinitie, G. E., and N. MacGinitie. 1949. \atural History ol Munne 
Animals. McGraw-Hill Co., New York. 473 pp. 

Maranda, Y., and G. Lacroix. 1983. Temporal variability of zooplank- 
ton biomass (ATP content and dry mass) in the St. Lawrence Estuary: 
advective phenomena during neap tide. Mar. Biol. 73: 247-255. 

McClintock, J. B., and A. \V. Stephen. 1990. The effects of photoperiod 
on gametogenesis in the tropical sea urchin Euddans trihuloides 
(Lamarck) (Echinodermata: Echinoidea). J Exp. Mar Biol Ecol. 
139: 175-184. 



Mosher, C. 1982. Spawning behavior of aspidochirote holothunan 
Hololhuria me\icana Ludwig. Pp. 467-469 in Proceedings of the 
International Conference on Echinoderms. Tampa Bay. J. M. Law- 
rence, ed. A. A. Balkema. Rotterdam. 

Newell, R. C. 1965. Biology ol Inlertidal Animals. Am. Elsevier. New 
York. 254 pp. 

Orion, J. H. 1914. On some Plymouth holothurians. J. Mar. Biol 
ASSOC. L'. K 10:211-235. 

Ouellet-Larose, D. 1973. Influence des marees sur les fluctuations a 
court terme des biomasses planctoniques dans I'estuaire. Masters 
thesis, Universite Laval, Quebec. 231 pp. 

Pawson, D. L. 1966. Ecology ol holothurians. Pp. 63-71 in Physiology 
of Echinodermata. R. A. Boolootian. ed. Wiley Interscience. New 
York. 

Pearse, J. S. 1969. Reproductive periodicities of Indo-Pacific inver- 
tebrates in theGult of Suez. II. The echinoid Echinometra mathaei 
(de Blainville). Bull Mar. Sci. 19: 580-613. 

Pearse, J. S., and D. J. Eernisse. 1982. Photoperiodic regulation of 
gametogenesis and gonadal growth in the sea star Pisaster ac/irucciis 
Mar. Biol 67: 212-125. 

Pearse, J. S., V. B. Pearse, and K. K. Davis. 1986. Photoperiodic reg- 
ulation of gametogenesis and gonadal growth in the sea urchin 
Strongylocentrolus purpuratus. J. Exp. Zoo/. 237: 107-118. 

Pittman, B.. and B. Munroe. 1982. Effect of preservation on the mass 
of marine benthic invertebrates. Can. J. fish. Aqual. Sci. 39: 220- 
224. 

Reese, E. S. 1966. The complex behavior of echinoderms. Pp. 157- 
218 in Physiology oj Echinodermata. R. A. Boolootian, ed. Inter- 
science Publishers, New York. 

Rutherford, J. C. 1973. Reproduction, growth and mortality of the 
holothunan Cucumaria pseudocurata. Mar. Biol. 22: 167-176. 

Sastry, A. N., and N. J. Blake. 1971. Regulation of gonadal devel- 
opment in the bay scallop. Aeqiiipecten irradians Lamarck. Biol. 
Bull 140: 274-283. 

Sewell, M. A. 1992. Reproduction of the temperate Aspidochirote Sti- 
chopus mollis (Echinodermata: Holothuroides) in New Zealand. 
Ophelia 35: 103-121. 

Snick, J. M. 1983. Respiratory gas exchange in echinoderms. Pp. 67- 
1 10 in Echinoderm Studies I M. Jangoux and J. M. Lawrence, eds. 
Balkema. Rotterdam. 

Smiley, S. 1988. The dynamics of oogenesis and the annual ovarian 
cycle of Stichopus californicus (Echinodermata: Holothuroidea). Biol. 
Bull. 175: 79-93. 

Smiley, S., and R. A. Clone). 1985. Ovulation and the fine structure 
of the Stichopus californicus (Echinodermata: Holothuroidea) fecund 
ovarian tubules. Biol. Bull 169: 342-364. 

Smiley, S., F. S. McEuen, C. Chaffee, and S. Krishnan. 1991. 
Echinodermata: Holothuroidea. Pp. 663-750 in Reproduction of 
Marine Invertebrates: l-ophophorates. Echinoderms. I'ol. 17. Giese 
and Pearse. eds. Boxwood Press, California. 

Sokal, R. R., and F. J. Rohlf. 1981. Biometry. Freeman. San Francisco. 
776 pp. 

Starr, M. 1990. Mecanismes de coordination entre la ponte de certains 
invertebres marins et la poussee printaniere du phytoplancton. Ph.D. 
thesis, Universite Laval, Quebec, Canada. 133 pp. 

Starr, M., J. H. Himmelman, and .1. C. Therriault. 1990. Direct cou- 
pling of marine invertebrates spawning with phytoplankton blooms. 
Science 247: 1071-1074. 

Starr, M., J. H. Himmelman, and J. C. Therriault. 1992. Isolation and 
properties of a substance from the diatom Phaeodactyliim tricor- 
nutiini which induces spawning in the sea urchin Strongylocentrotus 
droebachiensis. Mar. Ecol. Prog. Ser. 79: 275-287. 

Tanaka, Y. 1958. Seasonal changes in the gonad ofStichoptis iaponicus 
Bull. Fac. Fish. Hokkaido Uni\: 9: 29-36. 



REPRODUCTIVE CYCLE OF PSOLUS FABRIC!] 



143 



Tessier, G. 1948. La relation d'allometrie: sa signification statistique 

et hiologique. Biometrics 4: 14-48. 
Therriault, J.-C. 1973. Variations des propnetes physico-chimiques et 

biologiques d'une zone de melange de I'estuaire du Saint-Laurent. 

Masters thesis. L'niversite Laval. Quebec. 155 pp. 
Therriault, J.-C., and M. Levasscur. 1985. Control of phytoplankton 

production in the lower St. Lawrence Estuary: light and freshwater 

run-off. \al Can 112: 77-96. 
I lin i mull. J.-C., and M. Levasseur. 1986. Freshwater run-off control 

of spatio-temporal distribution of phytoplankton in the lower St. 

Lawrence Estuary (Canada). Pp. 251-260 in Proceedings of the 

NATO Freshwater, Sea Workshop. Bodo. Norway, S. Skresled. ed. 

NATO ASI Series. Vol. G7. Springer-Verlag, New York. 
Theel, 11. 1882. Report on the Hithilhuroulea hy 11 M S Challenger 

During the Years 1873-1876. Part I . Report of the Scientific Results 

of the Voyage of the Challenger (Zoology) 4: 1-176. 



Theel, P. A. 1901. A singular case of hermaphroditism in holothuroids. 

Bihang nil Kungi Svenxka I 'ciensk . teat! llandl 27: Afd. 4. No. 

6. 
I odd. C. D.,and R. \V. Doyle. 1981. Reproductive strategies of marine 

benthic invertebrates: settlement-timing hypothesis. Mar. Ecol. Prof;. 

Ecol 23: 55-69. 
Tyler, H., and J. I). Gage. 1983. The reproductive biology of )/>w/<>- 

llntriii uilisiihini from the northeast Atlantic. J Mar. Bio/. Assoc. 

U.K. 63:609-616. 
Tyler, P. A., and D. S. M. Billett. 1988. The reproductive ecology of 

elasipodid holothurians from the N. E. Atlantic. Biol. Oceanogr. 5: 

273-296. 
Yentsh, C. S., and D. \\. Men/el. 1963. A method for the determination 

of phytoplankton chlorophyll and phaeophytin by fluorescence. Deep 

Sea Res 10:221-231. 



Reference:/;/.-/ Hull 184: 144-1 52. (April. 1993) 



Reproductive Investment in Four Developmental 
Morphs of Streblospio (Polychaeta: Spionidae) 



TODD S. BRIDGES* 

Department of Marine, Eurih. and Atmospheric Sciences, North Carolina State University, 

Raleigh, North Carolina 27695-8208 



Abstract. Per brood and per offspring C and N invest- 
ment were examined in four developmental morphs of 
the spionid polychaete Streblospio: S. shruhsolii (direct 
development, D), 5. benedict i (lecithotrophic, L), S. hene- 
dicti (planktotrophic, P), and Strehlospio n. sp. (plank- 
totrophic, P). Large differences were apparent among these 
morphs in fecundity and embryo size. S. shruhso/ii (D) 
and S. benedicti (L) invested about 10 X more C and N 
in each offspring and 30% more C and N in each brood 
than did the morphs with planktotrophic development. 
C and N concentration dug per unit volume) was signifi- 
cantly greater in S. benedicti (L) than in the other morphs. 
though no general relationship with embryo size was ev- 
ident. The C:N ratio of offspring did not differ among the 
four morphs. Comparisons of estimated lifetime repro- 
ductive investment made by the two developmental types 
of S. benedicti indicated that lecithotrophic development 
involved twice the C and N investment in reproduction. 
Positive, significant regressions were evident between em- 
bryo C and N content and embryo volume at the inter- 
morph level. Significant intra-morph regressions were ev- 
ident in all morphs but Streblospio n. sp. (P). However, 
the large amount of variation unaccounted for by embryo 
volume calls into question the use of embryo size as a 
predictor of parental investment in offspring. 

Introduction 

Three modes of development, strongly correlated with 
egg size and fecundity (Thorson, 1946, 1950), are recog- 
nized among marine invertebrates. Planktotrophic de- 
velopment is descriptive of the production of a relatively 



Received 13 March 1992; accepted 25 January 1993. 
* Current address: USAE Waterways Experiment Station, WES-ES- 
F, 3909 Halls Ferry Road. Vicksburg. Mississippi 39180. 



large number of larvae, developed from small eggs, which 
acquire the necessary energy for growth by feeding on 
particulate matter during planktonic life. Lecithotrophic 
larvae are produced from fewer, but larger, eggs. These 
larvae do not feed on particulate matter but subsist, at 
least in part, on the energy supplied by the mother in the 
form of yolk during oogenesis. During direct development, 
offspring complete their development without a plank- 
tonic phase, usually within the mother or an egg mass; 
the energy for development is supplied by the mother 
(Thorson, 1950; Grahame and Branch, 1985). 

The adaptive significance of development mode has 
received extensive consideration. Selection pressures such 
as predation, starvation, and dispersal have been proposed 
for development mode evolution (Thorson, 1950; Chia, 
1974; Strathmann, 1985). Quantitative modeling ap- 
proaches have been employed in an effort to identify the 
selection pressures and processes of importance in life 
history and development mode evolution in marine in- 
vertebrates (Vance, 1973a, b; Christiansen and Fenchel, 
1979;Caswell, 1981; Grant, 1983). 

One fundamental assumption of the models of Vance 
( 1973a, b) and Christiansen and Fenchel ( 1979) concerns 
the relationship between an egg's size and its energy con- 
tent. It is assumed that a positive correlation exists between 
an egg's measured or estimated size and the investment 
in material or energy that egg represents. This seemingly 
reasonable assumption enables the models to make use 
of the large amount of data on egg size and development 
mode available for marine invertebrates. 

Interspecific comparisons of egg size and organic con- 
tent including a broad range of taxa have shown the ex- 
pected positive relationship to exist (Strathmann and 
Vedder, 1977). However, the use of interspecies compar- 
isons to justify the assumed relationship between egg size 
and organic content appears invalid. McEdward and Car- 



144 



REPRODUCTIVE INVESTMENT IN STREBLOSPIO 



145 



son (1987) and McEdward and Coulter (1987) have 
pointed out that for models describing evolutionary pro- 
cesses, the relevant level of variation to examine occurs 
within a single species. For the species of asteroids studied 
by McEdward, Carson and Coulter, egg size was found 
to be a poor predictor of organic content when examined 
intraspecifically due to the large amount of variation in 
organic content unaccounted for by egg size. Likewise, 
Qian (1991) found that egg size was not correlated with 
egg energy content within three populations of the poly- 
chaete Capitella sp. Variation in egg organic composition 
may also make egg size an unreliable predictor of an egg's 
energetic value (Turner and Lawrence, 1979). 

To date, the relationship between egg size and egg or- 
ganic content has been examined in only a small number 
of taxa (mostly echinoderms). One of the questions ad- 
dressed in this paper concerns the extent to which the 
pattern identified in echinoderms is present in other taxa, 
specifically spionid polychaetes. 

Inter- and intraspecific comparisons of reproductive 
and per offspring investment in organisms with different 
development modes have proved to be useful in identi- 
fying the ecological consequences of development mode 
as well as the potential evolutionary forces shaping these 
patterns (Menge, 1974; Perron, 1986; Levin cl nl.. 1991). 
The spionid polychaete genus Strchlospio. which exhibits 
developmental variation both within and between species 
(Levin, 1984; Cazaux, 1985), offers a highly suitable sys- 
tem for examining questions concerning egg size and egg 
organic content and the consequences of development 
mode. Six reproductive variants are known for Strchlos- 
pio, comprising three or more species (Levin, 1984; Ca- 
zaux, 1985; Rice, 1991; Levin and Eckelbarger, pers. 
comm.). 

To assess the nature of reproductive costs associated 
with development mode patterns in Streblospio, both re- 
productive expenditure per offspring and per brood were 
examined in four reproductive variants. These variants 
are similar in body size and ecology but are distinguishable 
in a number of reproductive characteristics (Dean. 1965; 
Levin, 1984; Cazaux, 1985). Two reproductive morphs 
of 5. benedict i have been identified from the Atlantic coast 
of the United States (Levin, 1984; Levin ct til.. 1991). 
Females with planktotrophic development produce a large 
number (100-600) of small eggs (70-90 urn dia.) which 
develop into planktotrophic larvae. Lecithotrophic 
morphs of this species produce a smaller number (10- 
100) of large eggs (100-200 nm dia.) and lecithotrophic 
larvae (Levin, 1984). Streblospio n. sp. from the Gulf of 
Mexico also has planktotrophic development and pro- 
duces a large number (100-700) of small eggs (60-70 ^m 
dia.) (Levin, 1984; Rice and Levin, in prep.). S. shrubsolii 
with direct development from near-shore habitats in 
France produces a very small number (20-50) of large 



eggs (200-230 ^m dia.) which develop directly into crawl 
away juveniles (Cazaux, 1985). 

Materials and Methods 

Animals used during this study were obtained from 
three sources. S. hcncc/icli with both planktotrophic and 
lecithotrophic development were initially collected from 
intertidal salt marsh habitats in Bogue Sound, North Car- 
olina. Worms with lecithotrophic development repre- 
sented second generation laboratory animals whose par- 
ents were collected in September, 1990 from natural pop- 
ulations at Fivers Island near Duke Marine Lab in 
Beaufort, North Carolina. Individuals with planktotrophic 
development were collected at Tar Landing Bay, North 
Carolina in June 1991. Individuals of Strchlospio n. sp. 
were settled in the lab by S. Rice from plankton samples 
made in the Hillsborough River, Tampa Bay, Florida in 
July 1991. Individuals of 5. shrub.wlii were taken from 
laboratory cultures established by L. Levin in 1986 from 
samples collected by C. Cazaux in Arcachon, France. 

Males and females of each variant were incubated in 
pairs at 20C in culture dishes of sieved marsh sediment 
and 34-36%o seawater. according to the techniques out- 
lined in Levin and Creed (1986). To make observations 
of multiple broods from the same female it was necessary 
to collect recently fertilized embryos rather than eggs. 
Embryos are brooded on the dorsal surface in all repro- 
ductive variants and are more accessible than maturing 
eggs which occur within the body coelom. Embryos were 
always collected within 24 h of fertilization and were 
composed of between one (zygotes) and approximately 
250 cells. The developmental stage of sampled embryos, 
estimated from the number of blastomeres present, was 
used as a covariate in data analyses. 

Once embryos were separated from females they were 
collected by pipet, counted, and placed in a dish of filtered 
(0.45 jum) seawater. Two perpendicular (maximum length 
and width) measures of embryo diameter were made with 
a compound microscope and ocular micrometer for ap- 
proximately 20 embryos within a brood. Embryo volume 
was calculated using the mean radius and the formula 
^Trr 1 since the embryos were more spherical than prolate 
or oblate in form. 

Entire broods, produced by a single female, were col- 
lected for C and N analysis by depositing all the embryos 
onto a small square of previously combusted Whatman 
GF/F (glass-fiber) filter paper. A minimum of approxi- 
mately 1 5 embryos of S. shrubsolii (direct developer) or 
S. benedicti (lecithotrophic) and 200 embryos of S. hene- 
dicti (planktotrophic) or Streblospio n. sp. (plankto- 
trophic) were needed to meet the detection limits of the 
analysis (2-3 ^g of C or N). Broods smaller than these 
minimum sizes were not analyzed. Samples were dried 



146 



T. S. BRIDGHS 



Siimnuirv <>l the reproductive cluu\i< /i 



Table I 
<>t the lour developmental inrph\ <<t Streblospio 



Characters 



SD n 



S. hem-dun (L) 



SD 



S. benedicn (P) 



SD 



Stri'Hii\pin n. sp. (P) 



SD 



df 



Embryos/Blood 




8.15 


24 


50.49' 


20.41 


100 


276.04" 


43.27 


6 


315.20" 


132.11 


20 


3. 103 


168.93 


0.0001 


ng C/Brood 


37 53' 


12.76 


24 


41.15' 


13.26 


100 


30.79" 


13.83 


6 


26.80* 


9.00 


20 


3, 103 


8.81 


0.0001 


>ig N/Brood 


7.77** 


1.63 


14 


8.50' 


2.80 


90 


6.75" 


> ">i 


1 


5.14" 


1.55 


20 


3. 86 


8.99 


0.0001 


Embryo Volume 
































(fil X 111-') 


4.67" 


0.78 


24 


3.08" 


0.44 


100 


0.495' 


0.057 


6 


0.366" 


0.041 


20 


3. 103 


1395. 


0.0001 


Mg C/Embryo 


1.09' 


0.25 


24 


0.85" 


0.14 


100 


O.I08 C 


0.026 


6 


0.091" 


0.031 


20 


3, 103 


725.4 


0.000 1 


Mg N/Embryo 


0.229" 


0.048 


19 


0.174" 


0.026 


90 


0.023 C 


0.0033 


1 


0.017" 


0.0051 


20 


3, 86 


949.0 


0.0001 


C cone. (>ig//jl) 


233.3 s 


34.50 


24 


279.7" 


51.54 


100 


217.2" 


48.02 


6 


248.9' 


79.7 


2(1 


3. 102 


12.60 


0.0001 


N cone. (/ig/MD 


48.67' 


5.57 


19 


57.62" 


10.19 


90 


45.72" 


4.67 


1 


47.47' 


12.62 


20 


3. 85 


1 3.58 


0.0001 


C;N Ratio 


4.83' 


0.54 


19 


4.88" 


0.28 


90 


4.97" 


0.69 


1 


5.17" 


0.48 


20 


3. 85 


2.40 


0.0767 



Data on embryo volume was obtained by calculating volume (4/3nr'l using a mean radius determined from two perpendicular estimates of diameter. C and N data 
were obtained from elemental analysis of entire broods of early embryos. Degrees of freedom (df). F. and P values are listed for the ANOVA results for each character. 
Superscripted letters denote those values within a row which are significant!) different 



in an oven at 50C for approximately 24 h, then stored 
in a vacuum desiccator prior to analysis. Appropriate 
blank samples, without eggs, were prepared to distinguish 
background C and N values associated with the collection 
technique. The amount of C and N in each brood was 
determined by use of a Carlo Erba Elemental Analyzer 
(model E.A. 1 108). 

All statistical analyses of data were performed with SAS 
(version 5.18). All data were log transformed to remove 
heteroscedasticity and normalize distributions. When sig- 
nificant differences (P < 0.05) were found among the four 
reproductive variants, an n-posteriori Least Significant 
Difference (LSD) test was performed on means of the four 
reproductive types (a = 0.05). A multiple linear regression 
model was used to examine the relationship between em- 
bryo volume and C and N content. Given the repeated 
measures structure of the data, two covariates were in- 
cluded in the model to distinguish between among-female 
and within-female variation. Covariate 1 (cov 1 ), repre- 
senting mean values of embryo volume for each female, 
allowed the relationship between either C or N content 
and embryo volume to be examined. Covariate 2 (cov 2) 
was formed by subtracting the mean embryo volume of 
all broods produced by a female during the experiment 
from the mean embryo volume of each individual brood. 
These deviations permitted testing for a relationship be- 
tween embryo C and N content and volume for multiple 
broods from a given female. 

Results 
Reproductive ami offspring investment 

Per brood measures of fecundity in S 1 . shrubsolii (D) 
and S. benedicti (L) were significantly lower than 5". bene- 
dicti (?) and Strehlospio n. sp. (P), where (L), (P), and (D) 



designate lecithotrophic. planktotrophic and direct de- 
velopment, respectively (Table I). The differences in fe- 
cundity among reproductive morphs were accompanied 
by differences in embryo volume. S. xlirubsolii (D) em- 
bryos, which were the largest of the four types (4.67 X 10~ 3 
Ml), were 12.7 X the volume of Streblospio n. sp. (P) em- 
bryos, 9.4 X the volume of S. benedict i (P) embryos, and 
1.5 X the volume of embryos of S. benedicti (L) (Table 
I). Embryo volume increased with developmental stage 
(P = 0.0021). 

Comparisons of the C and N investment made to in- 
dividual offspring produced by each reproductive type re- 
vealed differences similar to those found for embryo vol- 
ume (Table I). 5". shrubsolii (D) made the largest average 
investment in each offspring ( 1 .09 /jg C, 0.229 /^g N) fol- 
lowed by 5. benedicti (L) (0.851 ^g C, 0.174 /ig N), S. 
benedicti (P) (0.108 ^g C, 0.023 ng N), and Streblospio 
n. sp. (0.091 M gC, 0.017 jug N). 

In terms of C and N, the lecithotrophic and direct de- 
veloper made a greater material investment in each brood 
than did the morphs with planktotrophic development 
(Table I). Significant differences were present in ^g C per 
brood between S 1 . shrubsolii (D) and both planktotrophic 
developers, and in /ug C and N per brood between 5. bene- 
dicti (L) and both planktotrophic morphs (Table I). Even 
though the planktotrophic morphs produced much larger 
numbers of embryos, the lecithotrophic and direct de- 
velopers were found to have made a 30% greater C and 
N investment in each brood. 

The C:N ratio of brooded offspring was similar among 
the reproductive variants, ranging from 4.83 in S. shrub- 
solii (D) to 5.17 in Streblospio n. sp. (P) (Table I). The 
C:N ratio of embryos decreased with developmental stage 
(P = 0.01 12). 

S. benedicti (L) exhibited significantly greater C and N 
concentration (^g C and ^g N per ^1) than the other re- 



REPRODUCTIVE INVESTMENT IN STRKBLOSHO 



147 



A. CARBON CONTENT VS. EMBRYO SIZE 



I 

w 
o 

tm 



O.OOO O.O01 0.002 O.O03 O.O04 0.005 O.OO6 
EMBRYO VOLUME ( n 1) 

B . NITROGEN CONTENT VS. EMBRYO SIZE 

0.41 




1 

U 



0.3 




O.OOO 0.001 O.O02 0.003 O.OO4 0.005 O.OO6 
EMBRYO VOLUME ( n 1) 

Figure 1. Scatter plots describing the relationship between jig C/ 
embryo (A) and ng N/embryo (B) and embryo volume for all reproductive 
morphs. Both regressions are highly significant (P < 0.0001 ). 



productive types (Table I). Both C (P = 0.0306) and N 
(P = 0.0794) concentrations decreased with developmen- 
tal stage. 

Embryo C and N content versus eiuhryo volume 

Significant, positive correlations were found between 
jig C per embryo and embryo volume (r = 0.87, P 
< 0.0001) and ^g N per embryo and embryo volume (r 
= 0.89, P < 0.0001 ) across reproductive variants ofStre- 
blospio (Fig. 1). The regression model incorporating cov 
1 and cov 2 used in analyzing the relationship between 
Mg C (or jig N) per embryo and embryo volume explained 
95% of the C variation and 97% of the N variation. A 
strong relationship was evident between C and N content 
and embryo volume across reproductive types as indicated 
by the significance of cov 1 in the models (C ANCOVA: 
F U0 2 = 82.34; P < 0.0001; N ANCOVA: F U85 = 56.38; 
P < 0.0001). However, the relationships between jug C 
arid N per embryo and embryo volume within each re- 



productive type were not identical. The significance of 
reproductive type (C ANCOVA: F 3 ., 2 == 646.84; P 
< 0.0001; N ANCOVA: F X85 = 823.51; P < 0.0001) in 
the models indicated that differences existed among the 
four morphs in the nature of the regressions, specifically 
the y-intercept. No differences could be detected in the 
slope of the lines among the four types as seen by the lack 
of significance in the cov 1 X type interaction in both 
models (C ANCOVA: F, ,,, : = 1.85: P = 0.1478; N AN- 
COVA: F 3 . 85 = 2.29; P = 0.0884). Cov 2 was significant 
only in the case of the N model (C ANCOVA: F, lo: 
= 1.98; P =0.1626; N ANCOVA: F,, 85 == 4.37; P 
= 0.0395). indicating that the relationship between N 
content and embryo volume could be detected with data 
from individual females sampled more than once. 

Differences were evident among the four variants in 
the strength of the relationship between embryo C and N 
content and embryo volume within each morph. Signif- 
icant, positive correlations existed between jig C and ^g 
N per embryo and embryo volume for S. shrubsolii (D), 
S. benedict i (L). and 5. benedict i (P). but not for Stre- 
blospio n. sp. (P) (Table II. Figs. 2. 3). The specific regres- 
sion parameters for each of the relationships are listed in 
Table II. The amount of variation accounted for by the 
regressions, and therefore the strength of the relationship, 
was highest for S. shrubsolii (D) (71%- for N content and 
66% for C content). A much smaller amount of variation 
was accounted for by the regressions for 5. benedicli (L) 



Table II 

Slope and v-intcrcepl estimate* lor the regression equation* of ng C 
und ng N/embrvo ver\u* embrvo volume tor Streblospio dill morphsi 
and each morph separately 



Variants 


y-intercept 


SE 


Slope 


SE 


P 


,ug C/Embryo 


versus Embryo Volume 


Slrcblospio (all 












morphs) 


0.0431 


0.0213 


0.247 


0.00713 


0.0001 


5. shrubsolii (D) 


-0.102 


0.187 


0.256 


0.0395 


0.0001 


S benedicli (L) 


0.489 


0.0950 


0.118 


0.0305 


0.0002 


5. benedicli (P) 


0.00382 


0.0414 


0.210 


0.0831 


0.0187 


S. n sp. (P) 


-0.00732 


0.0627 


0.269 


0.170 


0.1306 



fig N/Embryo versus Embryo Volume 

Sireblospio (all 

morphs) 0.00812 0.00431 0.0513 0.00147 0.0001 

S. shnibsolii (D) -0.0276 0.0406 0.0548 0.00855 0.0001 

S. benedict HL} 0.112 0.0187 0.0203 0.00607 0.0012 

S. benedicli (P) 0.00271 0.00421 0.0402 0.00847 0.0001 



S. n sp. (P) 



-0.000504 0.00995 0.0489 0.027 



0.0869 



Estimates and standard errors (SE) are listed for the slope and y-in- 
tercept of the overall regression for Streblospio. including all morphs. as 
well as the specific regressions for each morph. P denotes the significance 
for each relationship. 



148 



T. S. BRIDGES 
S. shruhsolii (D) 




O.OO2 O.OO3 O.OO4 O.OO5 0.006 

EMBRYO VOLUME ( M 1) 

Streblospio n. sp. (P) 



O.lb- 


t"= .12 




p. 13 


0.14 







00 


0.12 


o 


0.10 


e 




6 


O.O8 


O 


0.06 




o 




00 



O.OO03 O.OOO4 O.OO05 

EMBRYO VOLUME ( n 1) 



S. benedicti (L) 



1.6 

1.4 

I- 

i 10 
P 

O 0.8 

tic 
3. 

0.6 




0.4 
OOOO O.O01 O.O02 O.OO3 O.OO4 0.005 

EMBRYO VOLUME ( M 1) 

S. benedicti (P) 




OOOO4 O.OO05 O.OO06 O.OO07 

EMBRYO VOLUME ( n 1) 



Figure 2. Scatter plots describing the relationship between ng C/embryo and embryo volume for each 
reproductive morph of Strehlospio. 



(11% for C and 1 3% for N) and 5. benedicti ( P) ( 2 1 % for 
Cand 51% for N). 

A more meaningful estimate of the strength of the three 
significant regressions can be made by examining confi- 
dence limits for predicted embryo C and N content. Pre- 
dictions of embryo C and N content using each morph's 
regression parameters and a single value of embryo size 
(each morph's mean) produced the following predicted 
values (95% confidence limits): S. shrubsolii (D), C 
= 1.10/ug0.31,N = 0.228 /ug 0.058; S. benedicti (L). 
C = 0.851 0.268, N = 0.174 0.050; S. benedicti (P), 
C = 0.108 0.050, N = 0.023 0.005. These confidence 
intervals envelop a large portion of the range of actual 
values for embryo C and N content found in each of these 
morphs, between 53%- and 99%. The large amount of 
variation about these regressions, which results in such 
large confidence intervals, makes it difficult, if not im- 
possible, to make significantly different predictions of 
embryo C or N content from embryo volume within each 
morph. 



Discussion 



Offspring invest men! 



The negative relationship between offspring size and 
number described for many marine invertebrate taxa 
(Thorson, 1946, 1950; Emlet et al.. 1987) including poly- 



chaetes (Hermans, 1979; Levin et al., 1991), was also 
found in this study (Table I). This tradeoff can be ex- 
plained by assuming there to be a finite and limited 
amount of energy available for reproduction (Vance, 
1973a; Smith and Fretwell, 1974; Stearns. 1976), an as- 
sumption more easily justified among closely related spe- 
cies which accumulate and apportion nutrients in a similar 
fashion. Levin et al. (1991) observed a negative genetic 
correlation between fecundity and egg size in S. benedicti 
reared in the lab, suggesting that evolutionary forces may 
influence this tradeoff. 

The potential evolutionary forces driving differences in 
per offspring investment and development mode are of 
particular interest. One of the key preadaptations allowing 
for the evolution of direct from indirect development may 
be the evolution of a large yolk-filled egg ( Wray and Raff. 
1991). However, experimental embryology has demon- 
strated that in species developing directly, development 
can proceed normally at half the egg size, in a size range 
similar to forms with indirect development (Okazaki and 
Dan, 1954; Henry and Raff, 1990; Wray and Raff, 1991). 

If direct development or lecithotrophy could be ac- 
complished in Slreblospio with only a five-fold increase 
over planktotrophy in per offspring investment (instead 
of the 10-fold increase in investment reported here), and 
the remaining C and N was allocated to increased fecun- 
dity, the resulting fecundity benefit could make such a 



REPRODUCTIVE INVESTMENT IN STREBLOSPIO 



149 



S. shrubsolii (D) 



S. benedicti (L) 





O.O03 OOO4 OO05 0.006 

EMBRYO VOLUME ( n 1) 

Streblospio n. sp. (P) 



O.OOO 0.001 O.O02 0.003 O.O04 O.O05 
EMBRYO VOLUME ( M 1) 

S. benedicti (P) 



0.03O 
0.025 


, ! =.16 
ps.OQ 


o 


g 0.020 

Id 
g- 0.015 


O 

8 




0.010 


O 







O.OO03 O.OOO4 O.OOO5 

EMBRYO VOLUME ( ji 1) 



OOOO4 OOOO5 OOOO8 O.O007 

EMBRYO VOLUME ( n 1) 



Figure 3. Scatter plots describing the relationship between ^g N/embryo and embryo volume for each 
reproductive morph of Streblospio. 



strategy adaptive (Table I). However, greater per offspring 
investment in S. shrubsolii (D) and S. benedicti (L), in 
addition to developmental changes, also produces a larger 
offspring. S. shrubsolii (D) produces a 1000 /um long crawl 
away juvenile (Cazaux. 1985). Larvae of S. benedicti (L) 
are released and settle at half the size of 5. shrubsolii (D) 
(about 550-650 ^m) (Levin, 1984). Planktotrophic larvae 
ofS. benedicti and Streblospio n. sp. are released at about 
250-350 ^m in length and appear to settle at a size com- 
parable to or smaller than S 1 . benedicti with lecithotrophic 
development (Levin, 1984). 

Selection for increased offspring size may have been an 
important factor in development mode evolution in Stre- 
blospio. A shift toward larger offspring size in Streblospio 
offspring with a planktonic phase may be adaptive in the 
face of size-selective planktonic predation (Kerfoot. 1977; 
Greene, 1985; Rumrill et at., 1985; Pennington el a/.. 
1986). The presumed predator avoidance behavior of 
some planktotrophic spionid polychaete larvae, including 
S. benedicti (P), that increase their effective size by flaring 
long swimming setae, is consistent with the importance 
of size-selective predation in this species. Larger size at 
settlement in 5 1 . benedicti (L) and at release from the fe- 
male in S. shrubsolii (D) may also benefit offspring subject 
to negative interactions with permanent meiofauna or 
macrofauna by accelerating passage through vulnerable 
size ranges (Bell and Coull, 1980; Watzin, 1983, 1986). 
Juvenile 5. benedicti are sensitive to interactions with 



macrofauna (McCann and Levin, 1989). Levin and Hug- 
gett (1990) reported a larval and juvenile survivorship 
advantage in S. benedicti (L) (relative to 5. benedicti (P)) 
during a field study of populations of 5. benedicti with 
lecithotrophic and planktotrophic development. 

Offspring composition 

Even though morphological distinctions are evident in 
yolk granules of S. benedicti with lecithotrophic and 
planktotrophic development, differences in gross measures 
of organic composition were not evident in this study 
(Eckelbarger. 1980, 1986). Offspring C:N ratios of the four 
reproductive types could not be distinguished statistically, 
suggesting that the relative proportion of protein to non- 
nitrogen containing compounds is the same among the 
four morphs (Table I). Turner and Lawrence (1979) also 
found that organic composition did not change with egg 
size in the echinoderms they studied. Lawrence et al. 
( 1984) concluded, due to the compositional similarity of 
eggs of different sizes and development modes, that the 
significance of larger eggs was not to accommodate dif- 
ferences in the energetic demands of development, but to 
create a larger offspring. One would expect to see a higher 
proportion of lipid in larger eggs if the change in devel- 
opment involved a greater energetic demand (Lawrence 
et al., 1984). Increased per offspring investment in Stre- 
blospio may have similar importance, i.e., the production 
of larger offspring. 



150 



T. S. BRIDGES 



C and N concentration (j/g//ul) was similar among the 
embryos of three of the four Strcblospio reproductive 
morphs, and no consistent irend with embryo size was 
noted (Table I). Qian a id Chia (1992) found that egg 
energy concentrali >as similar in lecithotrophic and 
planktotrophic C 'upiiella sp. Strathmann and Vedder 
(1977) reported that organic matter per unit volume de- 
creased with egg size in echinoderms with feeding larvae. 
Such a trend has not been observed in echinoderms with 
larger eggs, including pelagic lecithotrophs (Turner and 
Lawrence, 1979; McEdward and Chia. 1991). Energy 
concentration does appear to be significantly greater in 
eggs of echinoderms with nonfeeding larvae than those 
with feeding larvae (Emlet el a/., 1987; McEdward, pers. 
comm.); this observation is consistent with data presented 
by Needham (1963). Thus, important fundamental dif- 
ferences may exist among the eggs of echinoderms with 
different developmental modes. More data are required 
before such trends can be discerned for polychaetes. 

Reproductive investment 

The lecithotrophic and direct developers made greater 
material investments in each brood than either plankto- 
trophic developer. In addition to investing more C and 
N in each offspring, S. benedicti (L) was also found to 
have invested 33% more C and 26% more N in each brood 
than did 5. benedict i (P). However, these values are min- 
imum estimates of the difference in reproductive invest- 
ment since S. benedict i (P) produced more broods that 
were too small to be analyzed for their C and N content. 
Lifetime investment levels can be estimated by combining 
data on per offspring investment made in this study with 
lifetime fecundity data made by Levin el til. (1987), where 
worms were raised under the same experimental condi- 
tions. Using these data, S. benedicti (P) (1324.32 eggs/ 
lifetime) would have a calculated lifetime reproductive 
investment level of 143.03 jug C and 30.46 /ng N, and S. 
henedicti (L) (336.6 eggs/lifetime) would have invested 
286.45 jug C and 58.57 /jg N. Based on these calculated 
values, 5". benedicti (L) makes a two-fold higher investment 
in reproduction than S. benedict i (P). These estimates do 
not technically represent reproductive effort since repro- 
ductive effort is defined as the proportion of resources 
devoted to reproduction (Havenhand and Todd. 1989). 
However, the similarity of these two morphs in size as 
well as ecology (Levin et ai. 1987; Levin and Huggett. 
1990). would suggest that such estimates may represent 
a first approximation of reproductive effort, though some 
caution is warranted (Grahame, 1982). Efforts at deter- 
mining which reproductive pattern, planktotrophy or lec- 
ithotrophy, is more energetically expensive have yielded 
equivocal results (Grahame and Branch, 1985; Strath- 
mann. 1985). 



Differences in apportionment of energy to growth and 
development in S. benedicti with planktotrophic and leci- 
thotrophic development may partially account for the 
difference in reproductive investment. S. benedicti with 
planktotrophic development reaches sexual maturity (first 
reproduction) earlier and at a larger size than the lecitho- 
trophic morph, indicating that growth and developmental 
rates are accelerated in planktotrophs compared to leci- 
thotrophs (Levin et ai. 1987; Levin et ai. 1991). The 
importance of accelerated growth and development in 
planktotrophic 5. benedicti is further suggested by de- 
mographic analyses of the two developmental morphs. 
Similarity in estimated population growth rates (X) in the 
two morphs were the result of a balance between a larval 
and juvenile survivorship advantage in lecithotrophs and 
increased fecundity in early adult stages in planktotrophs 
(Levin el ai. 1987; Levin and Huggett, 1990). Given the 
effect of age at first reproduction and early fecundity on 
population growth rates (Stearns, 1976). females with 
planktotrophic development may be investing in future 
offspring both through energy committed to eggs directly 
and through enhanced early growth and development. 
The evolutionary shift from planktotrophy to lecithotro- 
phy may involve not only changes in offspring size and 
investment, but also age and size at maturity in S. bene- 
dicti. 

Embryo si:e versus C and N content 

Significant, positive relationships have been found be- 
tween egg size and organic content using data from a 
number of species in this study (Fig. 1) as well as others 
(Strathmann and Vedder, 1977: Turner and Lawrence, 
1979; McEdward and Chia, 199 1 ). In general, the strength 
of this relationship when examined at the interspecific 
level, as reflected by r values, appears to be high (present 
study; McEdward and Chia, 1 99 1 ). However, large errors 
in prediction may result when using regression equations 
formulated with interspecific data to predict values of per 
offspring investment from intraspecific and intra-morph 
data on embryo size (Bridges, 1992). The strength of intra- 
morph relationships between embryo C and N content 
and embryo volume ranged from 5. shrubsolii (D), where 
the regressions accounted for 66% of the variation in C 
and 71% of the variation in N to Streblospio n. sp. (P), 
where significant relationships could not be detected (Figs. 
2, 3). Even in the three morphs where significant regres- 
sions were evident, the size of 95% confidence intervals 
on predicted values of C and N content would preclude 
making significantly different predictions of C and N con- 
tent from embryos of different size within developmental 
morphs. Observations in this study of lecithotrophic and 
planktotrophic polychaetes are similar to those in echi- 
noderms with lecithotrophic development where variation 



REPRODUCTIVE INVESTMENT IN STREBLOSP1O 



151 



among species in the nature and strength of the relation- 
ship between egg size and organic content has been found 
(McEdward and Carson, 1987; McEdward and Coulter. 
1 987; McEdward and Chia, 1 99 1 ). Given that egg or em- 
bryo size accounts for minimal variation in organic con- 
tent within species, considerable caution should be taken 
in presuming egg or embryo size as an accurate measure 
of per offspring investment. 

Acknowledgments 

I would like to thank G. Plaia and N. Blair for providing 
assistance and advice with elemental analysis and C. 
Brownie for help with the statistics. Cultures ofStrehloxpio 
were graciously supplied by L. Levin, S. Rice, and C. 
Cazaux. I would also like to thank F. Gould, J. Garlich, 
D. Checkley, D. Wolcott, and three anonymous reviewers 
for their comments on earlier versions of this manuscript. 
Special thanks must go to L. Levin for her conscientious 
advising and critical examination of earlier versions of 
this paper. This project was supported in part by funds 
from EPA grant R8 1-72-52-0 10 to L. Levin. 

Literature Cited 

Bell, S. S.. and B. C. Coull. 198(1. Experimental evidence for a model 
of juvenile maerofauna-meiofauna interactions. Pp. 179-192 in 
Marine Benlluc Dynamics, K.. R. Tenore and B. C. Coull, eds. Uni- 
versity of South Carolina Press, Columbia. SC. 

Bridges, I. S. 1992. Effects of development mode, contaminated sedi- 
ments, and maternal characteristics on growth and reproduction in 
the polychaetes Slreblospio henedieli (Spionidae) and Capilclla sp. 
I (Capilellidae). Ph.D. thesis. North Carolina State University. 

Caswell, H. 1981. The evolution of "mixed" life histories in marine 
invertebrates and elsewhere. Am. Nat 117: 529-536. 

Cazauv, C. 1985. Reproduction et developpement larvaire de 1'annelide 
polychete saumatre Slreblospio shrubsoln (Buchanan. 1980). Call. 
Biol. Mar. 26: 207-22 i . 

Chia, F.-S. 1974. Classification and adaptive significance of develop- 
mental patterns in marine invertebrates. Thalaxx. Jugoslav. 10: 121- 
130. 

Christiansen, F. B., and T. M. Fenchel. 1979. Evolution of marine in- 
vertebrate reproductive pattern. Theor. Pop Bio/. 16: 267-282. 

Dean, D. 1965. On reproduction and larval development of Strchloxpio 
benedicli Bio/. Bull 128: 67-76. 

Eckelbarger, K. J. 1980. An ultrastructural study of oogenesis in Slre- 
blospio benedicli (Spionidae), with remarks on the diversity of vi- 
tellogenic mechanisms in Polychaeta. Zoomorphologie 94: 241-263. 

Eckelbarger, K. J. 1986. Vitellogenic mechanisms and the allocation of 
energy to offspring in polychaetes. Bull Mar Sci 39: 426-443. 

Emlet, R. B., L. R. McEdward, and R. R. Strathmann. 1987. Echinoderm 
larval ecology viewed from the egg. Pp. 55-1 36 in Ecliinodcnn Stud- 
ies vol. 2, M. Jangoux and J. M. Lawrence, eds. A. A. Balkema. 
Rotterdam. 

Grahame, J. 1982. Energy flow and breeding in two species of Lacuna: 
comparative costs of egg production and maintenance. Inl .1 Invert. 
Repro. 5:91-99. 

Grahame, J., and G. Branch. 1985. Reproductive patterns of marine 
invertebrates. Oceanogr. Mar Bio/. Ann. Rev. 23: 373-398. 

Grant, A. 1983. On the evolution of brood protection in marine benthic 
invertebrates. Am. Nat. 122: 549-555. 



Greene, C. II. 1985. Planktivore functional groups and patterns of prey 
selection in pelagic communities. ./ Plank Res 1: 35-40. 

Havenhand, J. N., and C. D. Todd. 1989. Reproductive effort of the 
nudibranch molluscs Adalana proxima (Adler & Hancock) and 
Onclndons inuncaia (Muller): an evaluation of techniques, l-'tinc 
Ecoi 3: 153-163. 

Henry, J. J., and R. A. Raff. 1990. Evolutionary change in the process 
of dorsovcntral axis determination in the direct developing sea urchin, 
llclioctdans ervlhrogi'amma. Dev Bio/. 141: 55-69. 

Hermans, C. O. 1979. Egg size and energetics: polychaete egg sizes, life 
histories and phylogeny. Pp. 1-9 in Reproductive Ecology of Marine 
liiYcrtehraies. S. E. Stancyk, ed. University of South Carolina Press. 
Columbia, SC. 

Kerfoot. \V. C'. 1977. Implications of copepod predation. I.imnol 
Oceanogr 22: 316-325. 

Lawrence, J. M., J. B. McClintock. and A. Guille. 1984. Organic level 
and caloric content of eggs of brooding asteroids and an echinoid 
(Echinodermata)from Kerguelen (South Indian Ocean). Inl ./ Invert 
Repro. Dev 1: 249-257. 

Levin, L. A. 1984. Multiple patterns of development in Slreb/ospio bene- 
dicli Webster (Spionidae) from three coasts of North America. Biol 
Bull 166: 494-508. 

Levin, L. A., H. Caswell, K. D. DePalra, and E. L. Creed. 1987. De- 
mographic consequences of larval development mode: planktotrophy 
vs. lecithotrophy in Slreblospio henedieli. Ecology 68: 1877-1886. 

Levin, L. A., and E. L. Creed. 1986. Effect of temperature and food 
availability on reproductive responses of Slrchlnxpio henedieli 
(Polychaeta:Spionidae) with planktotrophic or lecithotrophic de- 
velopment. Mar Biol. 92: 103-113. 

Lenn, L. A., and D. V. Huggel. 1990. Implications of alternative repro- 
ductive modes for seasonality and demography in an estuarine poly- 
chaete. Ecology 1\: 2191-2208. 

Levin, L. A., J. Zhu, and E. L. Creed. 1991. The genetic basis of life- 
history characters in a polychaete exhibiting planktotrophy and leci- 
thotrophy. Evolution 45: 380-347. 

McCann, L. D., and L. A. Levin. 1989. Oligochaete influence on settle- 
ment, growth and reproduction in a surface-deposil-feeding poly- 
chaete. ./. Exp. Mar Biol. Ecoi 131: 233-253. 

McEdward, L. R., and S. F. Carson. 1987. Variation in egg organic 
content and its relationship with egg size in the starfish Solasler 
siimpsom Mar Ecoi Prog. Ser 37: 159-164. 

McEdward, L. R., and F.-S. Chia. 1991. Size and energy content of eggs 
from echmoderms with pelagic lecithotrophic development. J. E\p. 
Mar Biol Ecoi 147: 95-102. 

McEdward, L. R., and L. K. Coulter. 1987. Egg volume and energetic 
content are not correlated among sibling offspring of starfish: im- 
plications for life-history theory. Evolution 41: 914-417. 

Menge, B. A. 1974. Effect of wave action and competition on brooding 
and reproductive effort in the seastar. Leplas/erias hexaetis. Ecology 
55: 84-93. 

Needham, ,1. 1963. Chemical Embryology. I. Hafner, New York. 613 

PP. 

Okazaki, K., and K. Dan. 1954. The metamorphosis of partial larvae of 
Pcronella laponica Mortensen. a sand dollar. Biol. Bull. 106: 83- 
99. 

Penningtun, J. T., S. S. Rumrill, and F.-S. Chia. 1986. Stage-specific 
predation upon embryos and larvae of the Pacific sand dollar. Den- 
draslcr cxcentncus. by 1 I species of common zooplanktonic pred- 
ators. Bull. Mar. Sci. 39: 234-240. 

Perron, F. E. 1986. Life history consequences of differences in devel- 
opmental mode among gastropods in the genus Conns Bull Mar 
Sci 39: 485-497. 

Qian, P. Y. 1991. Impact of environmental factors on life history strategies 
of the marine polychaete Capilclla sp. Ph.D. thesis. University of 
Alberta. 



152 



T. S. BRIDGES 



Qian, P. V., and F.-S. Chia. 1992. Effects ol'diet type on the demographics 
ofCapilclla sp. (Annelida: PoKchaela): lecithotrophic development 
vs. planktotrophicdevclopmcni. ./. l-.xp. Mar. Biol. Ecol. 157: 159- 
179. 

Rice, S. A. 1991. Reproductive isolation in the I'olyJora ligni complex 
and Strehlospto benedicti complex (Polychaeta: Spionidae). Bull. 
Mai Sa. 48: 432-447. 

Rumrill, S. S., J. T. Pennington, and F.-S. Chia. 1985. Differential sus- 
ceptibility of marine invertebrate larvae: laboratory predation of sand 
dollar, llcndrastcr e\cenlricm (Eschscholtz). embryos and larvae by 
zoeae of the red crab. Cancer prodiielus Randall. J /-.'A/I Mar Biol 
Ecol. 90: 193-208. 

Smith. C. C.. and S. D. Fretwell. 197-1. The optimal balance between 
size and number of offspring. Am. Nat. 108: 499-506. 

Slearns, S. C. 1976. Life-history tactics: a review of the ideas. Q Rev 
Biol 51: 3-47. 

Strathmann, R. R. 1985. Feeding and nonfeeding larval development 
and life-history evolution in marine invertebrates. Ann. Re\- Ecu/ 
Sysl 16: 339-361. 

Strathmann, R. R., and K. Yedder. 1977. Size and organic content of 
eggs of echmoderms and other invertebrates as related to develop- 
mental strategies and egg eating. Mar. Biol. 39: 305-309. 



Thorson, G. 1946. Reproduction and larval development of Danish ma- 
rine bottom invertebrates with special reference to the planktomc 
larvae of the Sound (Oresund). Medd. Komm. For Damn, h'iskeri- 
OgHavunders. Ser. Plankton 4: 1-523. 

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

Turner, R. I,., and J. M. Lawrence. 1979. Volume and composition of 
echmoderm eggs: implications for the use of egg size in life-history 
models. Pp. 25-40 in Reproductive Ecology of Marine Invertebrates. 
S. E. Stancyk, ed. University of South Carolina Press, Columbia, 
SC. 

Vance, R. R. I973a. On reproductive strategies in marine benthic in- 
vertebrates. Am. Nat. 107: 339-352. 

Vance, R. R. 1973b. More on reproductive strategies in marine benthic 
invertebrates. Am Kat. 107: 353-361. 

\\atzin, M. C. 1983. The effects of meiofauna on settling macrofauna: 
meiofauna may structure macrofauna communities. Oeeologia 59: 
163-166. 

\\ atzin. M. C. 1986. Larval settlement into marine soft-sediment systems: 
interactions with the meiofauna. / Exp. Mar. Biol. Ecol. 98: 65- 
113. 

Wray, G. A., and R. A. Raff. 1991. The evolution of developmental 
strategies in marine invertebrates. Trends Ecol. Evoi 6: 45-50. 



Reference: Bail. Bull 184: 153-1X5. (April. 1993) 



On Antarctic Entoprocta: Nematocyst-like Organs in a 

Loxosomatid, Adaptive Developmental Strategies, 
Host Specificity, and Bipolar Occurrence of Species* 



PETER EMSCHERMANN 

Fakulliit fiir Biologic tier Universitat Freiburg i.Br.. Biologic fiir Mediiiner, 
Schdnilestrafie 1. 7800 Freiburg i.Br. BRD 



Abstract. In the southern Weddell Sea and the Brans- 
field Strait a total of eight species of entoprocts were found: 
four Loxosomatidae. originally known to be common in 
the Northern Polar Sea and the Atlantic sector of the sub- 
arctic region (Loxosomella antedonis Mortensen. 1911, 
L. compressa Nielsen and Ryland, 1961, L. varians Niel- 
sen, 1964, and L. antarctica Franzen, 1973); three new 
species of loxosomatids (L. brochobola spec, nov., L. seir- 
yoini spec, nov., and L. tonsoria spec, nov.); and one single 
colonial entoproct Barentsia discreta (Busk, 1886) which 
is distributed worldwide. Loxosomella brachystipes, de- 
scribed by Franzen in 1973 from South Georgia, is shown 
to be synonymous with L. varians Nielsen, 1964. The 
microscopic investigation of the above species revealed 
several morphological characters, previously unknown, 
that add to our knowledge of the Entoprocta in general, 
and also help in characterizing species. The first of these 
novel characters, observed in L. brochobola spec, nov., 
are extruding organs similar to cnidarian spirocysts. This 
is the first description of such organs in entoprocts. Lox- 
osomella antarctica is capable of calyx regeneration and 
thereby becomes the only solitary entoproct known to 
have such a regeneration capacity. Finally, the formation 
of special resting buds in Barentsia discreta is described. 
The range of morphological variation of these species, the 
question of host specificity in the Loxosomatidae, and the 
bipolar occurrence of some of these species is discussed. 

Introduction 

Reports on Antarctic Entoprocta are scarce. Until 1973 
only five colonial forms had been recorded from the 

Received 31 August 1992; accepted 25 January 1993. 
* These investigations have been supported by the Deutsche Forsch- 
ungsgemeinschaft. 



Southern Ocean, predominantly from the subantarctic 
region: Pedicellina anstralis Ridley, 1881 was reported 
from the Magellan Strait, at the Patagonian coast and the 
Falkland Islands (Islas Malvinas); Barentsia capitata Cal- 
vet, 1904, and Barcnisin variahilix Calvet, 1904 were re- 
ported from South Georgia and the Falkland Islands; and 
Barentsia aggrexakt 1 Johnston and Angel, 1940 from 
Macquarie, Heard, Marion, and the Kerguelen Islands. 
These latter three species are probably synonymous. Fi- 
nally, Barentsia discreta (Busk, 1886), common circum- 
antarctically in subantarctic latitudes (Johnston and An- 
gel. 1940: Rogick, 1956; Waters. 1904) as well as on the 
Antarctic shelf itself, was reported in the Commonwealth 
Bay (Johnston and Angel. 1940) and from the northern- 
most tip of the Antarctic Peninsula (Franzen, 1973). In 
1973, Franzen augmented these reports with observations 
of older samples from the 1901-03 Swedish Antarctic ex- 
pedition. He added Pedicel/ inn ccrmia (Pallas, 1774) and 
four Loxosomatidae to the list of entoprocts from Ant- 
arctic waters: Loxosomella compressa Nielsen and Ry- 
land, 1961 var. antarctica; Loxosomella mwmanica (Ni- 
lus, 1909); Loxosomella antarctica spec, nov.; and 
Loxosomella brachystipes spec. nov. There have been no 
more recent studies of the Antarctic entoproctan fauna. 
During the Antarctic summer 1989-90, in the course 
of a survey of the Antarctic benthos supported by the 
Deutsche Forschungsgemeinschaft, the entoproctan fauna 
of the Weddell Sea and the Bransfield Strait were inves- 
tigated aboard the German research vessels PFS POLAR- 



' A sample of barentsiid colonies from the Californian coast near Santa 
Cruz, sent me by Kerstin Wasson (University of California, Santa Cruz), 
proved to consist not only of colonies ofBiirentsia ramosa. being common 
there, but also of Barentsia aggregata J. and A., which had previously 
been believed to occur only in the subantarctic region. 



153 



154 



P. EMSCHERMANN 



STERN and FFS METEOR. Benthic samples were taken 
in the Weddell Sea at 26 locations along the shelf from 
its northeastern most edge down to the base of the Ant- 
arctic Peninsula. Forty-five hauls were taken with both 
an Agassiz trawl and an epibenthic sledge (Fig. 1. Table 
1 ). Except at three stations in the 600- 1 100 m depth range, 
most samples were taken between 100 and 500 m (see 
Table I and Fig. 1 ). The benthic fauna, especially the as- 
sociations of ciliary-feeders, was generally richer, both in 
abundance and species diversity, in the eastern part of 
the Weddell Sea where the steep slope is more exposed 
to the Weddell Sea current than the fauna of the western 
Weddell Sea where bottoms are less sloped and less ex- 
posed to the current. 

In the Bransneld Strait a total of 22 hauls were evalu- 
ated, nine from a quadrangular dredge and 1 3 from a Van 
Veen Grab, taken at a depth range between 120 and 400 
m at 1 5 locations between Elephant Island to the north 
and Adelaide Island to the south. (Fig. 2, Table II). 

Altogether, eight entoproctan species were found. Four 
of them occurred in both areas and one species was found 
only in the Weddell Sea. Three new species were described: 
two from the Weddell Sea and one from the Bransfield 
Strait. 



As a general trend, the abundance and population den- 
sity of Entoprocta in the Bransneld Strait was much higher 
than in the Weddell Sea presumably because of the 
higher primary production and consequently higher nu- 
trient supply in this area. Of special zoogeographical in- 
terest is that four of the species found to be common in 
the Antarctic region also occur in the North Atlantic and 
the Arctic Polar Sea, but seem to be absent from the mid- 
Atlantic coasts. 

Sampling Methods and Treatment of Samples 

The trawling times of the sampling gear varied between 
about 30 and 90 min, according to the bottom structure 
and ice conditions. To obtain undamaged living samples 
for laboratory observations and culture experiments, the 
hauls were immediately subject to rough presorting. Ap- 
propriate growth substrates for entoprocts, such as bryo- 
zoan and hydroid colonies, bivalve shells, small sponges 
and stones, as well as potential entoproctan hosts such as 
errant and sedentary polychaetes with their tubes, sipun- 
culids. echiurans, priapulids, ophiuroids and. occasionally, 
crinoids, were collected as soon as possible and placed in 
separate plastic tubs with running fresh seawater of outside 




-70- 



SO' 



-40' 



-20' 



10' 



65 



>OLAR CIRCLE 



WEDDELL SEA 



/ 



ff\ 






75 



ANTARCTICA 

< 




PFS POLARSTERN 
CRUISE ANT Vlll-5 
STATIONS OF AQASSIZ TRAWL 
AND EPIBENTHrC SLEDGE 



-50' 



-30' 



-20' 



-10' 



Figure 1. Station map of the POLARSTERN cruise ANT VIII-5 in the Weddell Sea. 



ON ANTARCTIC ENTOPROCTA 



155 



Table I 



Station l.M / Pularxicrn-Cntiae AKT 1 7/7-5 in the II cddcll Sen 



Station 



Position 



Depth (m) 



Date 



Gear 



Bottom 



Predominant fauna 



16-346 


571.08 W 11,77 


360-320 


29/12/89 


A 


St 


Sponges. Brso/oa 




-399 


S 72,86 


W 19.30 


380-390 


30/12/89 


A 


St, Sf 


Sponges. Bryozoa. Ascidia 




-403 


S 76,94 


W 49.81 


220-250 


06/01/90 


A 


Ch. G 


Bryozoa, Holothuna 




-405 


S 76,52 


W 52.63 


380-390 


07/01/90 


A 


S 


Ascidia, Pennatulids, 






S 76.53 


W 52,72 










Gorgonia, Sponges: 






S 76,52 


W 52,78 










Bryozoa 




-407 


S 75.46 


W 27,02 


240-250 


12/01/90 


E, A 


Sf 


Sponges, Holothuna 




-41 1 


S 74,54 


W 25,75 


520-530 


14/01/90 


E, A 


S, St 


Cnnoids. Holothuna, Nemerteans. Prawns 


-421 


575,21 


W 27,80 


430-400 


17/01/90 


E, A 


S. G 


Pennat., Crinoids, Echin., Ophmr.. Holothur.. 
















Prawns, Fishes 




-423 


S 74,84 


W 27,56 


460-470 


17/01/90 


A, E 


S. St 


Sponges. Pennat.. Ophiur., Prawns 




-434 


S 73,69 


W 21,75 


260-270 


20/01/90 


E. A 


Cb. St 


Bryoz., Holoth., Prawns 




-437 


S 72,84 


S 19.40 


390-420 


21/01/90 


E 


Cb, St 


Bryoz., Ophiur.. Amphip. 




-454 


571.08 


W 11.69 


210-280 


26/01/90 


E. A 


Sf 


Sponges. Gorgon.. Fishes 




-456 


S 71.25 


\V 21,01 


200-250 


26/01/90 


A. E 


Sf 


Sponges. Ascid., Amphip., Fishes 




-459 


S 70,69 


W 11.19 


350-390 


28/01/90 


E, A 


G. St, Cb 


Sponges, Pennat., Crinoids, Amphipods 




-468 


S 74,74 


W 26.36 


480-460 


09/02/90 


E. A 


S. G 


Pennal.. Amphip.. Prawns. Fishes 




-470 


S 74.28 


W 34.09 


1050-960 


10/02/90 


E. A 


Cb 


Stylasterids. Ophiur.. Ascid.. Sponges. Polychaetes 


-475 


S 76.85 


W 49.45 


280 


1 3/02/90 


E. A 


S 


Sponges, Echin., Ascid., Cnn. 




-477 


S 76.45 


W 53.15 


430-450 


14/02/90 


A, E 


S 


Pennat.. Echin.. Prawns 




-479 


S 75.68 


W 56.72 


340-360 


14/02/90 


E. A 


S. Sf 


Crin., Ophiur.. Prawns, Pantop. 




-481 


S 74,71 


W61.14 


640-620 


15/02/90 


A. E 


St 


Ophiur.. Amphip.. Prawns 




-484 


S 75,28 


W 55.98 


450-440 


16/02/90 


E, A 


S 


Pennatul., Ascid., Ophiur., Polychaetes. 


Brachiopods 


-486 


S 76,50 


W 52,15 


340-330 


1 7/02/90 


E 


Cb 


Spong., Bryoz., Pennat., Ascid. 




-489 


S 73,68 


W 23.13 


980-990 


21/02/90 


A 


St 


Ophiuroids. Prawns 




-490 


S 73,70 


W 22,66 


630-610 


21/02/90 


E 


Sf 


Cnn.. Hololhur., Echin.. Prawns 




-491 


S 73,69 


W 22,42 


390-370 


21/02/90 


E 


S. G 


Cnnoids, Pantopods, Amphip. 




-492 


S 73,69 


W 21,74 


250 


21/02/90 


A 


Cb 


Cnn., Bryo/.. Spong., Ophiur. 




-496 


S 70,63; W 08.09 


80 


27/02/90 


A. E 


Cb 


Bryozoa. Holothuria 





Benthos stations: Gear, sediments, and predominant fauna 

Gear: A, Agassiz trawl: E. epibenthic sledge; Bottoms: Cb. calcareous bryozoan shells: G. sand and coarse gravel; S. sandy silt and mud: Sf. felt ot 
sponge needles; St. larger stones and rocks. 



temperature (-1 to +0.5C). The presorted substrates 
were subsequently checked for entoprocts under the dis- 
section microscope. About half of the zooids of each spe- 
cies found were kept alive, whereas the rest of them were 
preserved, some after narcotization, some without such 
a pretreatment. 

Narcotization and fixation 

A 4% formaldehyde solution in seawater proved to be 
the best fixation medium, yielding usable results even for 
electron microscopical purposes. For fast narcotization, 
especially when specimens were treated while still on their 
host, the gradual addition of an isosmotic solution of 
MgSO 4 gave acceptable results. But the local anesthetic 
amylocaine-hydrochloride [Stovaine R Rhone Poulenc: 1- 
(dimethylamino)-2-methyl-2-butanol-benzoate hydro- 
chloride] was more effective, particularly for small samples 
or single specimens in a small amount of water. By far 
the best results were obtained with a two-step narcotiza- 
tion: 8-10 crystals of amylocaine-hydrochloride were 



gradually added to a small sample of specimens in about 
2 ml of seawater until the animals were completely ex- 
panded and showed no reaction to mechanical stimulation 
(about 10 min). Subsequently, some crystals of MgSOj 
were added. After 5 min the sample could be fixed by the 
addition of 0.2 ml of 4% formaldehyde. 

Treatment of living samples 

Living samples for later culture experiments and ob- 
servations aboard were kept in 5-10 1 aquaria under run- 
ning seawater at 0-0. 5C on their original substrates or 
hosts. They were fed by the moderate addition of fresh 
nanoplankton samples, chiefly diatoms. Only the colonial 
Barentsia discreta could be cultured successfully and 
brought home to the laboratory still alive. None of the 
solitary loxosomatids could be kept alive and actively 
budding for more than three to four weeks, even when 
left on their original hosts. 

Measurements and sketches 

Measurements of a representative number of living 
specimens (30-50 if possible) from every locality were 



156 



P EMSCHERMANN 



FFS METEOR 

CRUISE XI-4 

STA'RONS OF 

VAN VEEN GRAB AND 

JUAURANGULAR DREDGE 




Figure 2. Station map of the Meteor cruise XI-4 in the Bransfield Strait. 



taken aboard and. later on, compared with those of ran- 
dom samples of preserved specimens. These groups of 
measurements were not significantly different. Freehand 
sketches were made of living specimens. When ship con- 
ditions allowed, micrographs of living specimens were 
taken through the dissection microscope. Higher mag- 
nification micrographs were made from preserved mate- 
rial in the home laboratory. 

Some Remarks on Species Determination and the 
Description of New Species in Entoprocta 

Entoprocta in general, and most Loxosomatidae in 
particular, have a scarcity of reliable species characters. 
The majority of morphological parameters, such as size, 
number of tentacles, body proportions, shape of stomach, 
and even conspicuous structures like cuticular pores, and 
spines, and body appendices, exhibit great intraspecific 
variability, and there is often overlap between species. 
Because of this deficiency of reliable morphological fea- 
tures, attempts have often been made to use the host or 
the locality of an entoproct as an aid for identifying its 
species. But neither the number of true species and their 
variation, nor their geographical distribution and possible 



spectrum of hosts, are sufficiently well-known to be useful 
in species identification. 

A rigorous biological species characterization by 
demonstration of their genetic isolation has not been 
possible for the great majority of entoproct species. 
Therefore, any species determination, especially any 
description of new species, founded on the evaluation 
of a few morphological characteristics, should be based 
on an intimate knowledge of all comparable species and. 
if possible, a comparison of the specimens in question 
with the type material of all similar species or. at least, 
with definitively identified samples of the latter. The 
description of a new species is not of value in itself; the 
demonstration of the real distribution range of a species 
is much more important. 

Any description of specimens new to an area should 
be illustrated with precise drawings in frontal and lateral 
view, and, if possible, in the contracted as well as the 
expanded state. Additional micrographs are often very 
helpful. Proof samples or types should be preserved both 
in contracted and expanded state. Because of insufficient 
description and unsatisfactory preservation of type ma- 
terial, not one Loxosomatidae described by Harmer (1915) 
from the Siboga samples can be reidentified. 



ON ANTARCTIC ENTOPROCTA 

I a Mr II 



Sun inn List 2 Meitw-Cnnac \l-4 in the Bransfield Strait 



157 



Station 



Position 



Depth (m) 



Date 



Gear 



Bottom 



08-90 


561.25 W 55.05 


125 


29/01/90 


VG 


S 


14-90 


562.53 W 54.15 


400 


29/12/90 


VG. D 


St 


21-90 


561.00 


W 56.00 


337-426 


30/12/90 


VG 


S 


27-90 


561.75 


W 57.89 


340 


01/01/90 


VG 


S 


28-90 


S 62.09 


W 57.64 


286-383 


01/01/90 


VG 


S 


31-90 


562.99 


W 56.99 


80 


02/01/90 


VG. D 


St. S 


39-90 


5 63.42 


W 59.86 


155 


03/01/90 


VG, D 


St 


50-90 


S 62.25 


W 60.57 


167-147 


05/01/90 


VG, D 


S 


64-90a 


564.15 


W 63.55 


135-150 


08/01/40 


VG, D 


St 


66-90 


S 64.47 


W64.77 


356 


08/01/40 


VG, D 


St, S 


76-90 


S 65.06 


W 66.98 


220 


10/01/90 


D 


St 


77-90 


S 65.39 


W66.18 


330-370 


10/01/90 


VG 


St 


78-90 


565.91 


W 66.85 


75 


11/01/90 


D 


St 


87-90 


S 66.57 


W 68.57 


450 


12/01/90 


VG, D 


S. St 


96-90 


S 62.77 W 60.90 


150 


16/01/90 


VG. D 


S, St 



Benthos stations: gear and sediments 

Gear: VG. Van Veen grab; D, rectangular dredge; Bottom: Cb, calcareous bryozoan shells; G, sand and coarse gravel, S, silt and mud; St, felt of 
sponge needles; St, larger stones and rocks. 



Because of our limited knowledge of the intraspecific 
variability and geographic distribution of most entoproc- 
tan species, every describer of a new entoproctan species 
should be wary that his new species may turn out to be 
synonymous with a species long known, even if all pre- 
cautions have been taken. The following descriptions of 
new species must be seen in this light. 

Description and Discussion of Species 

Loxosomella brochobola spec. nov. 

Holotype. Collected by the author on 20 January 1990 at the type 
locality, stat. 16-434 ANT VIII-5 (73.69S; 21.75W) at a depth of 
260-270 m from sandy and rocky bottoms with abundant calcareous 
Bryozoa; the entoproctan species was growing exclusively on the inner, 
abfrontal surface of tube-shaped Porcllu malouinensis colonies (Bry- 
ozoa). 

Syntypes. Deposited in the British Museum of Natural History, London 
(No. 1992.12.14.1) and the Zoologisk Museum, Kebenhavn. 
Name. From Greek: fipoXof-snare and I3a\\tiv-dis- 
charge, referring to the sticky threads that can be ejected 
by nematocyst-like extrusive organs a unique character 
of this species. 

Description. This is a tall Loxosomella species, about 
1 300 pm in length, the individuals resemble at first glance 
a Pedicellina zooid (Figs. 3a-c; 4). The bulgy goblet- 
shaped calyx is sharply delineated from the long, slender, 
and highly motile stalk. The large tentacular crown with 
14-20 slim tentacles is oriented straight up. In the ex- 
panded state, the calyx is slightly laterally depressed, but 
is nearly globular when contracted. The rectum bulges 
out between the aboral pair of tentacles, and the anus 
opens immediately anterior to the aboral ends of the 



horseshoe-shaped periatrial ciliary rim (Fig. 4f, i). The 
peduncle is slender, cylindrical and one and a half to twice 
as long as the calyx. About halfway down there is usually 
a slight "waist." The basal attachment area is narrower 
than the average diameter of the peduncle and animals 
are not fixed very strongly to their substratum, but can 
be removed easily without damage. Remnants of the foot 
gland normally persist as a plug of globular cells in the 
base of the stalk. As often observed in loxosomatids, the 
perikarya of the peduncular epithelial cells are arranged 
in longitudinal rows between the muscle strands. 

The stomach is voluminous and globular with wide 
lateral pouches bulging out at either side (Fig. 4k). The 
longitudinal musculature of the oral side consists of a 
dense layer of fibers running downwards from the oral 
and orolateral calyx wall to nearly the base of the peduncle, 
while at the aboral side and laterally in the upper portion 
of the stalk, only 2-3 single strands at either side are de- 
veloped. Basalwards these aboral muscle fibers increase 
in number, thus forming together with the oral fibers 
a closed muscular tube in the lower portion of the pe- 
duncle. 

Living specimens in normal expanded posture have the 
peduncle slightly curved, the aboral side of the calyx in- 
clined downwards, the oral side up (Fig. 40. Seen from 
above in this position, four large whitish blue, opaque 
blister-like structures are conspicuously visible at either 
side between the bases of each of the second and third, 
as well as the third and fourth, oral tentacle. Upon irri- 
tation, or sometimes spontaneously, 300-400 nm long 
delicate, helically twisted threads can be ejected from these 
enigmatic organs (Figs. 3d-h; 4b-d, n-g), which resemble 




158 



ON ANTARCTIC ENTOPROCTA 



159 



cnidarian spiro- or nematocysts. The sticky threads are 
as long, or somewhat longer than the tentacles, and remain 
anchored with their proximal ends in the epithelial cells 
from which they originate, floating with their distal ends, 
outside the tentacular crown. At higher magnification 
these extrusive organs each consist of an enlarged barrel- 
shaped and plurinuclear epithelial capsule, about 80 ^m 
long and 45 /j.m in diameter. In the unexploded state, it 
is filled with an invaginated highly coiled tubule, roughly 
square in cross section. The nuclei, generally four, are 
situated basally in a narrow area of marginal plasma. 
When ejected, the evaginated tubule, about 3 jum in 
diameter, has an X-shaped cross section and is cov- 
ered by a thin mucous coat. Loxosomella brochobola 
is the only entoproct known to have such extrusive 
organs. 

The function of these organs is obscure, but defense 
seems unlikely. Possibly these extrusive threads are con- 
nected with a specialized method of feeding: their ar- 
rangement around the mouth supports such a presump- 
tion. The extrusive threads could act as a kind of "fly 
paper" in a marine biotope poor in suspended matter. 
They would collect small particles attached to the sub- 
stratum and inaccessible to the ciliary feeding apparatus, 
and from time to time would be swallowed together with 
any adhering material. But this kind of activity has, so 
far, not been observed. 

From their genesis, the extruding organs are surely true 
kamptozoan organs, not "kleptocnides" somehow ac- 
quired from hydroids growing in their immediate vicinity, 
such as Halecium. Moreover, in both their overall struc- 
ture and extrusion mechanism, they are distinct from 
similar cnidarian organs. Unlike the tubules of cnidarian 
nematocysts, they do not evaginate by turning inside out, 
like a glove finger, but rather they are ejected simply by 
the unfolding of the curled introverted thread through a 
rupture of the extruding capsule at its proximal tip. (An 
ultrastructural investigation of these peculiar organs is in 
progress and will be published separately.) 

Buds, normally two to three on either side, develop 
orolaterally, level with the basal half of the stomach. They 
are fixed to the parent, not by the aboral tip of the foot, 
but by a junctional zone situated basally at the aboral side 
of the calyx, as is known from Loxosomella kefersteini 
(Figs. 3a, d: 4a-e, 1). The long, sickle-shaped glandular 
foot of the bud points upwards. The main body of the 
foot gland is situated just below the stomach. From there. 



a narrow glandular groove bordered by large secreting 
cells runs all along the foot to its aboral tip. After the bud 
has detached from the parent, the foot does not degenerate 
totally, but develops into the basal portion of the adult 
peduncle. The aboral tip of the foot becomes the attach- 
ment site for settling on the substrate. The upper portion 
of the adult stalk above the "slight waist" consequently 
develops by stretching the zone between the calyx base 
and the proximal part of the foot. 

Gonads in different stages of development were ob- 
served in nearly all specimens examined: Immature and 
mature testes were developed only in undetached buds 
and newly settled specimens, while mature ovaries were 
found exclusively in larger animals, usually with 1 -2 eggs 
on either side. The testes are positioned laterally to the 
stomach; the ovaries lie more distally, in the space between 
stomach, esophagus and the atrial bottom. In a few spec- 
imens unhatched larvae lacking eyespots were observed 

(Fig. 4m). 

Measurements. Total length: 1200 ^m (994-1350 Mm); length of calyx: 
380 Mm (260-493 Mm); length of stalk: 800 Mm (423-978 Mm): width 
of calyx: 335 Mm (239-408 Mm); thickness of calyx: 400 Mm (245-554 
Mm); diameter of stalk: 90 Mm (65-1 14 Mm); number of tentacles: 18 
( 12-20). in buds: 12. 

Hahiuit and distribution. Though its bryozoan host is 
abundant all over the Weddell Sea, Loxosomella brocho- 
bola has been found at only two locations in the eastern 
Weddell Sea (stations ANT-VII1-5 16-396; 16-434; 16- 
491; and 16-492). L. hroc/ioho/a grows exclusively on the 
inner, abfrontal surface of the tube-shaped colonies of 
Porella malouinensis (Bryozoa) which is sometimes as- 
sociated with young colonies of an undetermined species 
of Halecium (Hydroida). At the type locality L. brochobola 
was growing in small groups of 20-30 specimens/cm 2 , 
specially on younger host colonies settled only sparsely 
by other epizoans. 

Discussion of the species. The loxosomatid described 
above is the only entoproct known to possess nematocyst- 
like organs. These may be homologous to and derived 
from pearl-like glandular cells or cell complexes, which 
have been found to be more or less regularly scattered 
around the margin of the tentacular crown in a number 
of loxosomatids. These organs alone are a striking species 
character. To date only four other loxosomatids are known 
which show the budding pattern described above: Loxo- 
somella kefersteini Claparede, 1867, L. pseudocompressa 
Konno, 1977, L. annulata Harmer, 1915, and L. mepse 
du Bois-Raymond-Marcus, 1957. In its general appear- 



Figure 3. Loxosomella brochobola spec. nov. a-c: living specimens in abfrontal (a) and lateral view (b, 
c); d: contracted specimen with large bud and ejected sticky threads: e: ejected sticky thread; f: extrusion 
organ in Nomarski contrast, the coiled ejectable tubule is visible (bar 10 Mm); g: the same organ, two of four 
nuclei are visible at the left side (bar: 10 Mm); h: part of an ejected tubule [bar in all micrographs 100 Mm 
unless otherwise indicated). 



160 



P. EMSCHERMANN 




Figure 4. l.i>M>somclla hrnchohola spec. nov. a: contracted zooid from fixed sample; b-e: two expanded 
zooids (b/c; d/e), in lateral-frontal, and latcral-ablrontal view, respectively, with buds and. partly, ejected 
extrusion organ; f: living expanded zooid in normal posture; g, h: bending movements of a zooid after 
irritation; i: contracted tentacular crown seen from above, the rectum bulging out between the aboral tentacles; 
k: calyx seen from the aboral side; lateral pouches of the stomach are visible; I: newly detached bud. the 
navel being visible at the abtrontal side of the calyx base; m: young larva before hatching; n-q: extrusive 
organs in different stages of ejecting the sticky thread (from preserved specimens). 



ance, only the latter shows some similarities to the Weddell 
Sea specimens; but the smaller size and average number 
of tentacles of L. niepse. the shape of its stomach not 
being trilobed. the shorter foot of its buds not being sickle- 



shaped, and most of all, the lack of the conspicuous ex- 
trusive organs clearly distinguishes this species from the 
Weddell Sea specimens. In conclusion, Loxosomella bro- 
c/ioho/a is a reliable new species. 



ON ANTARCTIC ENTOPROCTA 



161 



Loxosomella seiryoini spec. n<n: 

//o/ii/r/'c Collected by the author on 17 January 1990 at the type 
locality, station 16-421 ANT-VII1-5 (75.2 1 S: 27.56 W) at a depth of 
430-470 m from a silts and muddy bottom; specimens were growing 
in dense populations on the rear end of the body and the anterior part 
of the proboscis of (joltingia margariuia-a (Sipunculida). Four of eight 
specimens of this particular sipunculan species, depending on their 
size, were host to 20 to several hundred loxosomatids. 
.SYmr/x's Deposited in the British Museum of Natural History (no. 
1992.12.14.2) and in the Zoologisk Museum Robenhavn. 
Name. The species name is given in honor and remem- 
brance of a Japanese friend and colleague who passed 
away. 

Description. Loxosomella seiryoini is a small species, 
with club-shaped individuals about 700 /jm long. The ca- 
lyx, almost circular in cross section, gradually transforms 
into a peduncle of half to one times the calyx length, ta- 
pering slightly towards its base and affixed to the substra- 
tum by an enlarged attachment disc (Figs. 5a-e; 6). The 
tentacular crown, with eight short, stoutish tentacles, is 
inclined oralwards in an angle of about 45, both in con- 
tracted and expanded state, and is surrounded by a broad 
peritentacular membrane resembling a Stuart-collar in 
expanded animals. In living specimens, this collar is much 
more conspicuous than in preserved ones, where it is 
manifested merely as a swelling of the lophophoral rim 
(Fig. 6a, c). 

The calyx tapers gradually into the stalk when ex- 
panded, but in strongly contracted specimens a deep fold 
demarcates the transition between calyx and peduncle, 
and the latter becomes bulgy and barrel-shaped. The foot- 
plate is fixed very firmly to the substratum by a thin 
brownish layer of the foot gland secretion, and in some 
cases a small remnant of this gland persists as a small 
globular pit in the middle of the adhesive disc (Fig. 5e). 
Specimens settling on the introvert of the host usually 
are enclosed by a felty and stifTcuirass of adhering detritus 
particles which are permanently agglutinated with the cu- 
ticle, leaving only the lophophoral area and the oral side 
uncovered (Fig. 6i-k). Such individuals have a consid- 
erably reduced capacity for expansion, and they have a 
more sturdy, globular shape with a short bulging peduncle. 
The stomach is globular in outline and lacks lateral 
pouches. The longitudinal musculature consists of about 
12 coarse muscle strands running down from the oral 
calyx wall to the foot plate, as well as some more delicate 
lateral and aboral fibers. In the basal portion of the pe- 
duncle, particularly in contracted specimens, 10 to 12 ad- 
ditional delicate spirally arranged fibers can be observed 
(with polarized light!); these fibers crisscross in opposite 
directions, forming a helical lattice. The buds usually 
one at either side of the calyx develop orolaterally, in 
line with the base of the stomach. When mature enough 
to become detached, they have only a short, boat-shaped 
foot-gland (Figs. 5c; 6d). 



In most specimens, gonads in different maturation 
stages are present: immature testes in undetached or newly 
detached buds, ovaries in older specimens and, sometimes, 
embryos and larvae in the brood pouches (Fig. 6f, g). Oc- 
casionally, ovaries with eggs as well as degenerating testes 
were observed in the same specimen, indicating that this 
species, like other loxosomatids, is protandric. 

Meaxiircmciit.\. Total length: 700 m (560-780 Mm); length of calyx: 
400 Mm (318-415 Mm); length of peduncle: 270 Mm (163-318 Mm); 
width of calyx: 280 Mm (160-350 Mm): thickness of calyx: 290 Mm 
(254-349 Mm); diameter of peduncle: about 124 Mm above, tapcnng 
to 100 /jm at its base; number of tentacles: 8. 

In one single, older, not very well preserved sample (stat. 224, Ant 
VII/4. 7P15'S; 1307'W) on a specimen of Goltmgia margaritacea. 
a number of loxosomatids were found that are similar to L seiryoini 
in most characters (calyx shape, swollen lophophoral rim. the helical 
muscular lattice, and remnants of the foot-gland in the peduncular 
base) except that they have a long, slender peduncle (about 1000- 
1400 Mm) and thus achieve a total length of 1400-2000 Mm. Whether 
L seirvoini really attains such a size, or whether these specimens were 
artificially stretched by rude handling during sorting and preserving, 
is unclear. 

Hahitut and distribution. Loxosomella seiryoini has 
been found exclusively on Golfingia margaritacea in the 
Weddell Sea, never on other sipunculans of the same size 
occuring at the same sites. West of the Antarctic Peninsula 
this loxosomatid seems to be absent, although the host is 
as common in this region as in the Weddell Sea. This 
Loxosomella preferably settles at the rear of its host and 
around the foremost part of the introvert where, in most 
cases, it is difficult to detect. Especially at the latter site, 
it can form crowded aggregations. 

Discussion oj the species. This loxosomatid is one of 
numerous medium-sized species, that lack striking species 
characters. Quite a list of such species with 8 tentacles 
and a more or less club-shaped form exists in the literature. 
Most of these species cannot be reidentified due to the 
poor quality of the original description based in some 
cases on only a single specimen and due to the insuffi- 
cient preservation, or the complete lack of any type ma- 
terial. This applies to: Loxosomella breve and L. loricatum 
(Harmer, 1915) from the Siboga samples; Loxosomella 
mimila Osburn, 1910, described from the Woods Hole 
region as growing on sipunculids; Loxosoma (Loxoso- 
mella'. 7 } cingulata, L. infundibulifonnis, and L. rotunda, 
described by Kluge (1946) from the Arctic Polar Sea; and 
Loxosoma (Loxosomella?) singulare Barrels, 1877 and L. 
singii/areHincks, 1880. However, Harmer mentions small 
lateral sensory papillae present in the Siboga specimens; 
so they seem to be different from our species. 

Among the more recently described species, Loxoso- 
mella fauveli Bobin and Prenant, 1953, L. globosa Bobin 
and Prenant, 1953. and L varians Nielsen, 1964, look 
similar to the Weddell Sea form. But even if one does not 
attach too great significance to their smaller size and di- 
vergent hosts, a number of other characters differ consid- 
erably from L. seiryoini: they all lack the conspicuous 




162 



ON ANTARCTIC ENTOPROCTA 



163 



collar-like peritentacular membrane; all normally have 
more than 8 tentacles, except L. var/ans; and the buds of 
only the latter have a small, partly reduced foot-gland. 
L. seirvoini and L. varians on the other hand differ in the 
shape of the extended adult foot plate, which is bordered 
by conspicuously large cells in L. varians, but which are 
lacking in L. seiryoini. Furthermore, the helically arranged 
muscle strands in the basal portion of the peduncle of the 
latter seem to be absent in L. var/ans. So at present, Lo.\- 
osoinella seiryoini may be regarded as a new species. 

Loxosomella tonsoria spec. nov. 

llololvpc Specimens were collected by Dr. U. Wirth on X January 
1490 at the type locality, station 66/90 Meteor XI-4 (64 "3(>'S; 6445'W) 
at a depth of 320 m from a stony and muddy bottom, growing in 
small numbers ( 10 specimens) dorsally on the anterior segments and 
gills of an ampharetid polychaete (ct Glyphanoslomum \pcc.: Fig. 
7a, b). 

Name. From Latin: tonsorius-shaver, because of the 
gibbous appearance of the calyx, which in lateral profile 
resembles an old Norelco 4 ' electric shaver (e.g.. Fig. 50. 
Description. A medium-sized species, 600-800 ^m in 
length, with a characteristic gibbous calyx, a short and 
thin peduncle of about 0.5-0.7 times the calyx length. 
The comparatively large tentacular crown with 8 short 
and stout tentacles faces towards the oral side (Figs. 5f- 

i; 7c-0. 

The calyx is slightly laterally depressed (width/thickness 
ratio 0.8). Below the stomach, the calyx constricts abruptly 
into the thin peduncle, which tapers somewhat towards 
its base and terminates in a small attachment area. The 
latter consists of a small epithelial invagination repre- 
senting a remnant of the genuine foot-gland (Figs. 5k; 7h, 
i). The animals are not fixed very firmly to their substra- 
tum and can be easily removed without damage; they fall 
off easily after fixation. The stomach is almost globular. 
and as a result of the humpbacked calyx, the rectum is 
an unusually long tube. 

The longitudinal musculature consists of only a few 
muscle strands: frontally and at either side 2 to 3 fibers 
each run from the calyx wall down to the base of the 
peduncle. Buds develop orolaterally in line with the upper 
half of the stomach. Only two of the specimens had de- 
veloped very young buds, however, since these did not 
show any trace of a foot-gland, nothing is known of its 
structure. 

Mature gonads (Fig. 7g, were present in all specimens; 
the majority contained ovaries with 3-4 eggs in different 



maturation stages. In one single case, testes filled with 
sperms were observed and in another specimen, the rem- 
nants of degenerating testes were visible below the ovaries. 
This indicates a protandric hermaphroditism also in this 

species. 

\h-ii\urcnn-ni\ Total length: 600 M (366-795 MHI); length of calyx: 
370 Mm (223-461 M'TI); length of peduncle: 197 Mm (95-350); width 
of calyx: 200 M"! ( I I 1-240 Mm): thickness of calyx: 243 Mm (223-254 
MTU), diameter of peduncle: 95 //m (above) to 64 urn (below); number 
of tentacles: 8. 

Habitat ami distribution. Only 10 specimens of this 
loxosomatid were found on the gills and the dorsal side 
of the first segments of an ampharetid polychaete from 
silty and rocky bottom, west of Anvers Island at a depth 
of 320 m. 

Discussion o/ /he species. Since a small foot-gland is 
present even in adult specimens (Figs. 5k: 7h, i) a rem- 
nant of a presumably larger gland in the buds the above 
specimens belong to the genus Loxosomella. The rudi- 
mentary attachment gland seems to remain active 
throughout life. Any conspicuous circular muscle fibers 
in the peduncle base, which would indicate a sucker-like 
function of the basal glandular pit, as is characteristic for 
the genus Loxosonui. are lacking. The body shape is quite 
distinctive: no other loxosomatid so far described has such 
a gibbous calyx with the expanded tentacular crown facing 
exactly towards the front. The species characterization is 
based on only a few individuals; additional examination 
of the foot-gland structure in older buds, as well as an 
investigation of the variation range of this species from 
more numerous samples is highly desirable. The data 
available at present suggest that the specimens described 
above constitute a new species. 

While nothing is known so far about the distribution 
of the above new species beyond their type localities, the 
species described below seem to be distributed not only 
in the whole Atlantic sector of the Antarctic and subant- 
arctic sea, but also in the arctic and subarctic region of 
the northern hemisphere. 

Loxosomella antarctica Franzen 1973 

Material. Collected by the author in the Weddell Sea at stations ANT 
VIII-5/ 16-396, 16-411, 16-42 Land 16-434, growing on the brittle star 
Ophiuroli'pis gelida as well as on the aphroditid polychaete Laetmonice 
pmducta at stations 16-411 and 16-489. In the Bransneld Strait the 
species has been found by Dr. U. Wirth at the stations Met. XI-4/31- 
90. 39-90. and 64-90 growing on the same hosts with an apparent 
preference for Opluurolcpis xi'liila 

The original description of this species given by Franzen 
( 1973) was based on preserved specimens from samples 



Figure 5. LoxiiMimclla .wirvnini spec. nov. a-e: contracted preserved specimens in frontal view (a), and 
lateral view (b); c: specimen with large bud; d: contracted specimen with a small bud; e: stalk of the latter 
with basally visible remnants of the foot-gland (arrow); f-k: Loxosonu'llu liimmriii spec, nov., preserved 
specimens in lateral and frontal view; i: with a small bud; k: stalk of i with basal remnant of the foot-gland 
(arrow) [bar 100 Mm]. 













Figure 6. l.i>.\osomella seirvonini spec. nov. a-g: different zooids from the rear end of (Jolfingia mar- 
garitaci'a. a: living specimen with the conspicuous peritentacular collar; b and c: preserved specimens; d: 
contracted specimen with large bud; e: specimen in semiexpanded state, in abfrontal view; f and g: specimens 
with larvae in their brood pouches in lateral and frontal view, respectively; h-k: different specimens from 
the introvert of the host, k with a robust "lorica" out of detritus particles covering the abfrontal part of calyx 
and stalk. In the foot plates of all specimens remnants of the foot-gland are visible. 



164 



ON ANTARCTIC ENTOPROCTA 



165 




Figure 7. LoxmmnclUi tansoria spec. nov. a: host polychaete Glyphanostomum xpcc.; h: head region of 
the latter with loxosomatids settling on the cirri and the prostomium: c-e: preserved expanded specimen 
with mature ovaries, in lateral, frontal, and ahfrontal view, respectively; f and g: preserved specimens in 
lateral view with mature ovary (0 and mature testes (g); h-i: basal tip of the stalk with remnants of the foot- 
gland. 



166 



P. EMSCHFRMANN 



of the 1902 Swedish Antarctic Expedition; these samples 
were dredged west of the northernmost tip of the Antarctic 
Peninsula. Franzen's description will be supplemented by 
my recent observations on living specimens. 
Description. Loxosoinclla anlarclica is a tall species, up 
to 2 mm in length, with a high goblet-shaped calyx, almost 
lyriform when seen from the oral side, and nearly circular 
in cross section. Only in a strongly contracted state is it 
at times somewhat flattened (Figs. 8a-f; 9a-g). In the ex- 
panded state, the large tentacular crown, generally with 
12 slender tentacles (only 10 in newly detached buds), is 
inclined to the oral side at an angle of about 45; when 
contracted it faces more or less orally. The peduncle of 
large budding specimens varies in length from -h to 3 
times the calyx length. Basally, below a conspicuous con- 
striction, it terminates in an enlarged foot-plate. 

The stomach is variable in shape, voluminous and 
globular to inversely triangular, but in contracted speci- 
mens, transversely oval. The cuticle is comparatively ro- 
bust; in younger specimens it is smooth, but in older in- 
dividuals, especially in the basal portion of their peduncle, 
broadly wrinkled. The body musculature is well devel- 
oped. Longitudinal fibers run upwards from the pedun- 
cular base, fanning out into the calyx where, at either side 
of the esophagus and intestine, they insert into the frontal 
and aboral body walls. The muscular layer, compact at 
the oral side, thins out towards the aboral side into loose 
bundles of single fibers. 

Depending on the nutritional conditions, 1-3 buds ap- 
pear at either side (Fig. 90, developing anterolaterally in 
line with the middle of the stomach. The glandular foot 
of the bud has a long posterior extension and only a knob- 
like frontal protuberance (Figs. 8e: 9h). This is one of the 
striking differences between this species and the similar- 
looking Loxosomella antcdonis (Fig. 14e), which is diffi- 
cult to distinguish from younger specimens of L. untarc- 
tica. But in the buds of the former, the foot is inversely 
T-shaped, extending to a conspicuous anterior as well as 
a posterior, process. Immature and mature gonads are 
present in most specimens testes exclusively in buds and 
newly detached specimens, and ovaries only in older 
zooids. 

So far the Weddell Sea and Bransfield Strait samples 
agree quite well with Franzen's description and illustra- 
tions. But the variability in the ecological conditions of 
the Weddell Sea, and, consequently, in the size and body 
shape of the Weddell Sea specimens, is much higher than 
in the type samples. While the average size of Weddell 
Sea specimens is about 1000 /urn (600-1750 /urn), the 
Bransfield Strait samples average 1500 ^m (640-2100 
^m), and both are smaller than Franzen's specimens. The 
calyx of the latter is, in most cases, distinctly marked off 
from the peduncle, but in some Weddell Sea populations 
it transforms gradually into the stalk. In these samples, 
the stalk usually tapers towards its base to about half of 



its original diameter (Figs. 9i-l; lOc. d). while in Franzen's 
samples, the stalk was cylindrical throughout its length. 
According to Franzen, Loxosomella cintarctica lacks any 
lateral sensory papillae. But in two Weddell Sea popula- 
tions (stat. 16-422 and 16-439), in a number of specimens 
growing on Ophiurolepis gelicla. very small sensory pa- 
pillae were present on either side of the calyx, in line with 
the second pair of oral tentacles (Figs. 9i-l; lOc, d). Usually 
these delicate "sensory spots" are only visible under higher 
microscopical magnification as pointed cuticular protru- 
sions equipped with 1 to 3 stiff cilia (Fig. lOc, inset) that 
protrude from an intraepithelial cluster of sensory cells. 
I have never found such sensory organs in buds and young 
individuals. Most remarkable is the ability of L. antarctica 
to shed and regenerate a calyx (Figs. 1 1: 12) a regener- 
ative capacity unique to this species amongst loxosoma- 
tids. 

.Mi'iiMtivincnl* \\'cddcll Sea \i>d.-inien.f Total length: 1000 Mm (595- 
1750 Mm); length of calyx: 400 nm (380-636 Mm); length of peduncle: 
680 MHI (240- 1 1 30 /im): width of calyx: 3 1 5 Mm (208-4 14 Mm): thick- 
ness of calyx: 317 ^m ( 178-477 Mm); diameter of peduncle: 133 Mm 
(85-180 Mm); diameter of peduncle in specimens with tapering pe- 
duncle: above 155 Mm 1 127-180 Mm), hasally 104 jim (87-135 Mm); 
number of tentacles: 12 (10-12). Bransfield Strait specimens: Total 
length: 1524 M m (636-2142 Mm): length of calyx: 490 Mm (318-625 
Mm); length of peduncle: 1074 Mm (318-1525 Mm): width of calyx: 
280 Mm (143-357 Mm); thickness of calyx: 290 Mm (159-318 Mm): 
diameter of peduncle: 133 Mm (95-220 Mm); number of tentacles: 12 
(10-12). 

Hahiun anil distribution. In the Weddell Sea. L. ant- 
arctica has been found repeatedly at depths ranging from 
100 to 400 m. growing in moderate numbers on the oral 
disc and the arms of the brittle star Ophiurolepis gelida 
(Fig. 8a), and, occasionally, in small numbers, on the dor- 
salmost fine setae (Fig. lOa. b) of the polychaete Laet- 
monice proditcta ( Aphroditidae). 

In the Bransfield Strait, Loxosoinclla antarctica is the 
most common loxosomatid. and grows on the same hosts; 
but it exhibits a conspicuous preference for the brittle 
star. Usually the ventral body surface and the arms of this 
host, as well as the dorsal side of the disc, are occupied 
by crowded populations of the loxosomatid at a density 
of about 4-6 individuals per mm : . 

Be\ond its Antarctic occurrence, the same species possibly has a second 
area of distribution in the Arctic Polar Sea. In the collections of the 
British Museum, a small sample of a loxosomatide (no. 31.7.3.1.70) 
was deposited which was collected from an Epizoanthus arborescens 
colony near Bear Island (Greenland). Although the preservation state 
of these specimens is not the best, and they are identified by Mortensen 
himself as L.\\mclla anleili>ni.\. it is evident that they lack bilateral 
sensory papillae, one of the striking species characters of the latter 
species (if. p. #). Therefore, these Arctic specimens may also belong 
to Loxosomella anlarclica Franzen. 1973. Especially with respect to 
the bipolar distribution of a number of entoprocts, a critical review 
of museum samples, as well as some new investigations in arctic waters, 
would be desirable. 

Discussion of the species and the possibility of hybrid- 
ization. Specimens from different hosts usually did not 
differ significantly, but samples from the Weddell Sea and 




Figure 8. Loxosomella antarctiai. a: Zooids settling tightly on an Ophiumli'pis arm (scale 1 mm); b and 
c: living specimens in frontal and lateral view, respectively; d and e: young bud and newly detached bud; f: 
specimen from Franzen's type sample (bar 100 ^m). 



167 




Figure 9. Lo.WM'iiwIlii iinlanlica a-c: expanded and contracted specimens from the Bransfield Strait; 
a: expanded specimen from Ophiurolepis (horn life); b-c: preserved contracted specimens from Laetmoiucc: 
d-g: expanded and contracted specimens from the Weddell Sea (from life): d and e: from Ophiurolepis; f 
and g: from Laelmonice; h; newly detached bud; i-1: contracted Weddell Sea specimens from Ophiurolepis 
with tiny sensory spots (m) at either side. 

168 





Figure 10. Loxosomclla anlarclica. a and b: Preserved Weddell Sea specimens from the setae of Laet- 
monice products; c and d: expanded and contracted Weddell Sea specimens from Ophiurolepis gelida with 
minute lateral sensory spots (arrows, inset) [bar 100 urn]. 

169 



170 



P. EMSCHERMANN 



the Bransiield Strait differ markedly in their average sizes. 
This may result from the conspicuous differences in the 
nutritional conditions in these regions; primary produc- 
tion, predominantly consisting of diatoms, is much richer 
in the Bransfield Strait than in the Weddell Sea. 

A discontinuous presence of lateral sensory spots, which 
was observed in some rare cases in Weddell Sea specimens 
of L. antarclica, has been reported likewise for Loxoso- 
mella clavijonnis and L. phascolosomata (Vogt, 1876); 
but the latter observations are not well established. Since 
all other characters of such Weddell Sea specimens with 
small lateral sensory papillae were within the normal range 
of variation of L. antarctiai. and since such specimens 
were always found mixed with a majority of "normal" 
antarctica-zooids, they are considered to belong to the 
same species. Of course, such cases could also be produced 
by hybridization between L. antarctica and another spe- 
cies, such as L. antalonis. that is equipped with lateral 
sensory papillae. 

At the one location in the Weddell Sea (stat. 16-369), 
where both of these species, /.. antarctica and L. antalonis. 
occurred, they settled on different hosts and only in small 
numbers: L. antarctica on Ophiurolepis and L. antcdonis 
on Lac'tmonicc. Where Loxosomella antarctica occurred 
abundantly on both the ophiurid and (in smaller numbers) 
the polychaete, L. antedonis seemed to be generally absent. 
It was exactly under these conditions, amidst a majority 
of "normal" Antarctica-zooids, that zooids with tiny lat- 
eral sensory papillae were detected. 

These findings are strongly suggestive of hybridization 
between these species, especially since, within the abun- 
dant L. antarclica populations of the Bransfield Strait 
where L. antedonis appeared to be generally lacking, no 
specimens with sensory papillae were detected. In fact, if 
hybridization between Loxosomella antarctica and Lox- 
osomella antedonis is possible at all, then small dissemi- 
nated populations of L. antedonis may well be absorbed 
by hybridization with the antarctica populations wherever 
the latter species is dominating. A sporadic appearance 
of antedonis characters in such hybridized populations 
must be expected. Both species can maintain themselves 
unhybridized only in macrobiotopes where they live as 
small populations that are spatially separated, e.g.. on dif- 
ferent hosts. 

Regeneration. Loxosomella antarclica is the only sol- 
itary entoproct. for which the ability to regenerate a calyx 
has been demonstrated (Figs. 11; 12). Usually in Loxo- 
somatidae the regenerative capacity is limited to the repair 
of single injured tentacles. 

In abundantly growing populations of Loxosomella 
antarctica on Ophiurolepis from the Bransfield Strait, 
amidst great numbers of large active zooids, sporadic 
headless (no calyx) stalks were found. These stalks were 
still intact and were actively twisting and bending. At their 
headless apical end, they were sealed with a cuticular cap. 




Figure 1 1 . L.t>xo\omclla anlurclica. stages of calkyx regeneration, a: 
headless stalk in total view; b: apical region of a regenerating stalk with 
adhering remnants of the calyx cuticle; c-g: apical portions of several 
stalks in different regeneration stages: the beginning invagination and 
formation of the primary atrial vesicle (c, d), the initial gut formation 
(e), and differentiation of esophagus, stomach, intestine, and rectum (f). 
and (g) the atrial opening, newly broken through, and formation of the 
hrst oral tentacles (drawn alter preserved samples). 



and sometimes remnants of the cuticle of the shed calyx 
were still present. The basal portion of these peduncles 
is. in general, sharply delineated from the apical part by 
a different structure of the cuticle; basally, it is roughly 
wrinkled and coated by detritus particles, while apically 
it is thinner, smooth, and translucent (Figs. 1 la, b; 12a, 
c). The same was observed in the stalks of many large 
active zooids presumably an indication of successive 
growth periods. 

Under the microscope, headless peduncles were ob- 
served in different stages of calyx regeneration. The ear- 
liest, least differentiated stages, have a thickened body wall 
epithelium throughout. At their distal ends they are con- 
tracted by the fibers of the well-developed longitudinal 
musculature, the remaining wound of the shed calyx being 
sealed off by a plug of epithelial cells and covered by a 
newly secreted cuticular cap (Figs, llb-d; 12a, b). The 
innermost strands of the muscular layer are partly dis- 
integrating, and the body cavity is filled with voluminous 
parenchyma cells containing many granules and vesicles, 
presumably storage proteins from phagocytized muscle 
cells. This picture resembles the muscular joints of bar- 
entsiid stalks when transforming into resting buds. 

It can be inferred from the different stages observed 
that the subsequent regeneration of a calyx proceeds in 
the same way as in colonial entoprocts (Figs. 1 Ib-g; 12b, 
d, e): ( 1 ) A primary atrial vesicle is formed by an apical 
invagination of body wall cells. (2) Gut and atrial floor 



ON ANTARCTIC ENTOPROCTA 



171 




Figure 12. Loxmcimella anlarcnca. calyx regenerating stalks; a and b: heavily cuticularized stalk with 
germinating tip; a primary atrial vesicle has formed (arrowhead); b-d: formation of the gut, theatrial opening 
not yet broken through; c and e: in lateral view; d: in frontal view; A - atrial cavity; I - intestine; R - "anlage" 
of the rectum; S - stomach; arrows - residual storage cells (bar 100 #im). 



172 



P. EMSCHERMANN 



differentiate out of a basal cluster of the invaginated cells. 
(3) After the atrial opening has been broken through, the 
tentacles develop along the atrial rim beginning at the 
oral side. 

The size of the headless peduncles, and their obviously 
successive stages of differentiation, preclude the possibility 
that they could merely be young metamorphosing spec- 
imens or old zooids in the course of degeneration. The 
former would be expected to be considerably smaller and 
in any case devoid of adhering apical cuticular remnants 
(Fig. 1 Ib), whereas the atrium of the latter would never 
be completely closed (Figs, llf: 12c-e). 

Whether this calyx regeneration takes place as a con- 
sequence of external injury to the calyces, or is due to a 
periodic transformation of peduncles into resting buds 
under unfavorable environmental conditions, is presently 
indeterminable. All other loxosomatids examined in this 
respect under normal temperatures have an individual 
life span of hardly more than 6-10 weeks. During this 
time, depending on the nutritive supply, they continuously 
develop and release buds. Simultaneously, they pass 
through a short protandric male phase and a subsequent 
longer female phase. After having released about 10-20 
larvae, the zooids degenerate. The larvae require at least 
about a week for metamorphosis. 

The presence of apparently older basal portions and 
younger distal parts of the peduncles in larger zooids in- 
dicates a life span being extended over several growth pe- 
riods. Such an increased ability to regenerate is obviously 
an adaptation to extremely short growth periods dimin- 
ishing the chance of sexual reproduction. Under such cir- 
cumstances an extension of the life span by an optional 
inactive resting phase is advantageous. 

Loxosomella antedonis Mortensen 1911 

Material. Collected by the author in the Weddell Sea at stations ANT 
VIU-5, 16-396 and 16-405 in depths of 300-400 m growing in moderate 
numbers on the dorsalmost tine setae of Laetmonice producta (Poly- 
chaeta. Aphroditidae). 

The species was originally described by Mortensen 
(1911) from the northeast coast of Greenland, growing 
on the cirri of the feather star Antedon prolixa, and has 
been redescribed by Ryland and Austin (1960) from set- 
tlement panels off Swansea. I found it again in 1964, 
growing abundantly for a brief period on rocks and other 
solid substrates at the rocky shore around Helgoland. The 
species is very similar to young specimens of Loxosomella 
antarctica. but does not attain the length of the latter. 

Description. The Antarctic specimens are 700 to max- 
imally 1200 //m long, the slender, almost cylindrical pe- 
duncle being as long, or 1.5 times as long, as the calyx 
(Figs. 13a-d; 14a-e). The calyx, seen from the oral side, 
is inversely triangular in outline and slightly depressed in 
the oral-anal axis. In the expanded state, the calyx trans- 
forms gradually into the stalk. The large tentacular crown. 



with 12-16 tentacles, when expanded, is conspicuously 
inclined to the oral side. In the contracted state the calyx 
is racket-shaped, flattened, with the lophophore facing 
frontally. The stomach is oval to inversely triangular with 
somewhat projecting lateral lobes. The peduncle, which 
bears longitudinal musculature that is not as strongly de- 
veloped as in /.. aniarclica. terminates in an enlarged ad- 
hesive disc. 

As a striking character, this species possesses at either 
side of the calyx, just beneath the lophophore and level 
with the stomach roof, a prominent, non-retractile sensory 
papilla, about 20-30 ^/m long, with a tuft of stiff bristles 
(Figs. 13b-d; 14b-d). Buds develop orolaterally in line 
with the upper half of the stomach. In contrast to those 
of most other loxosomatids, they have a very distinct T- 
shaped foot with a long anterior and posterior process, 
the peduncle inserting in the middle (Fig. 14e). Sometimes 
several fine sensory bristles are visible at the anterior tip 
of the foot. 

Measurements. Total length: 900 /im (690-1450 ^m): length of calyx: 
42(1 urn (336-548 jum): length of peduncle: 508 ^m (361-651 urn): 
width of calyx: 265 ^m (233-308 Mm); thickness of calyx: 185 /jm 
(169-189 fimY, diameter of peduncle: 107 ^m (93-1 17 /jm): number 
of tentacles: 14-16: length of sensory papillae: 20-30 ^m. 
Habitat anil distribution. In Antarctic waters, the spe- 
cies has been found only in the Weddell Sea. growing 
exclusively on Laetmonice producta. West of the Antarctic 
Peninsula it seems to be absent. The actual distribution 
of Loxosomella antedonis probably consists of the arctic 
and subarctic region, where it settles on various living as 
well as dead substrates, showing no host specificity. 

Additional remarks to the species. The Weddell Sea 
specimens agree quite well with Mortensen's original de- 
scription of Loxosomella antedonis, as well as with Ry- 
land's and my own specimens from the Irish- and the 
North Sea. Though the original type specimens have been 
lost, the conspicuous lateral sensory organs, mentioned 
and figured by Mortensen, present a striking species char- 
acter. A sample of specimens without such papillae, de- 
posited in the British Museum (cf. p. #) and identified by 
the late Mortensen as Loxosmella antedonis. is definitely 
different from this species. 

Loxosomella compressa Nielsen and Ryland 1961 

Synonym. Loxosomella compressa var. antarctica Franzen. 1 973. 
Material. This species was abundant at depths from 100 to 400 m at 
almost all stations in the Weddell Sea. but was absent at depths greater 
than 500 m. In the Branstield Strait it was found at one single location 
(Met. Xl-4/39-90). at a depth of 160 m. generally growing on the 
dorsal setae of a great variety of polynoid polychaetes. 
Loxosomella compressa, first described by Nielsen and 
Ryland from the Norwegian coast, growing on the no- 
topodial setae of several polynoids, turned out to be the 
most common entoproct in the Weddell Sea. In this area, 
it apparently prefers the same hosts as in its northern area 
of distribution. 



h 

TV<ft 

. v < 




Figure 13. a-d: Loxosomella antedonis from the Weddell Sea. a: living expanded specimen; b-c: preserved 
specimens, the large sensory papillae (d) are conspicuously visible: e-h: Li>\wmclla >m/r,v,v<j. preserved 
expanded specimens (e-f) and living specimen (g) in natural posture on polynoidan setae; h: preserved 
specimen with large bud (bar 100 /*m). 

173 



174 



P. EMSCHERMANN 





Figure 14. Loxosomella antedonis from the Weddell Sea. a-b: contracted specimens from the Weddell 
Sea (preserved material); c-d: expanded specimens from life, the large sensory papillae being conspicuous 
in all specimens; e: newly detached bud with its typical T-shaped foot. 



Description. A medium size species, easily recognized 
by its peculiar laterally depressed calyx and by the for- 
mation of its buds almost medially, level with the stomach 
roof, and perched on a console-like protuberance of the 
oral calyx wall. 



The total length of mature, budding Weddell Sea spec- 
imens varied between 500 and 750 ^m. Seen from the 
side the calyx is goblet-shaped, with a kind of "paunch" 
below the budding zone (Figs. 13e-h; 15b-e). With no 
conspicuous demarcation, it transforms into the slender 



ON ANTARCTIC ENTOPROCTA 



175 



stalk, which ends in a small attachment disc. Seen from 
the frontal side, the calyx is slim, and of the same diameter 
as the upper part of the peduncle which tapers downwards. 
The comparably large lophophore with 8 tentacles faces 
nearly straight up, both in the expanded and contracted 
state. 

The stomach is large and globular. The buds, rarely 
more than one at either side, are normally oriented in an 
upright position, rather than hanging downwards (Figs. 
13h; 15c). They have a very short stalk and a well-devel- 
oped, posteriorly extended foot, with a glandular groove 
along its whole length (Fig. 150. The latter is lined with 
large glandular cells. The main portion of the gland is 
situated just below the stomach. Gonads were observed 
in detached animals only. Smaller specimens contained 
both immature testes as well as young ovaries, while larger 
specimens exclusively contained mature ovaries and 
brood pouches with embryos and larvae in different de- 
velopmental stages. 

Mi'tniiri'incHls. Total length: 640 /jm (540-750 ^m) (Nielsen: 500 ^m, 
max. 700 Aim; Franzen: 870 ^m); length of calyx: 290 ^m (240-348 
jim) (Nielsen: 178 jim; Franzen: 457 ^m): length of stalk: 360 nm 
(300-460 ^m) (Nielsen: 267 ^m; Franzen: 41 1 jim); width of calyx: 
183 MHI (180-140 Aim) (Nielsen: 78 urn: Franzen: 195 ^m); thickness 
of calyx: 215 ^m (176-246 ^m) (Nielsen: 120 yum; Franzen: 257 jum); 
diameter of peduncle: above 137 /jm (101-168 fim). below: 60 /jm 
(53-75 jim); number of tentacles: 8 (Nielsen: 8-9; Franzen: 8). 
Habitat and distribution. Loxosomella compressa was 
found exclusively perched on the notopodial setae of a 
broad spectrum of Polynoidae (Fig. 15a). It was never 
found on other hosts, although about a thousand speci- 
mens of a great number of other polychaete species oc- 
curring in the same locations were searched for epizoans. 
Predominantly smaller polynoids (4-10 cm) are chosen 
as hosts, and species with short and thick notopodial setae 
covered by the elytrae are preferred; the loxosomellae are 
attached at the setal bases. On average, 2-4 individuals 
were found per parapodium, but at several locations of- 
fering exceptional nutritional conditions, up to 8 individ- 
uals per parapodium were counted. Under such conditions 
of extreme scarcity of unoccupied substrate, other poly- 
noid species with dense bunches of thinner notopodial 
setae, or with dorsal setae not covered by the elytrae, e.g., 
Hennadion ferox, served as hosts for Loxosomella com- 
pressa. 

In culture experiments aboard ship, L. compressa could 
be kept actively budding for about 3-4 weeks. In aquaria 
containing some polynoid hosts with their epizoans. newly 
detached buds were found to settle and become themselves 
actively budding on diverse solid substrates, such as stones 
and small settlement panels. 

In the Weddell Sea, Loxosomella compressa is the most 
common entoproct occurring at depths from 1 00 and 500 
m all along the Antarctic shelf from its northeastern edge 
to Kap Fiske, at the base of the Antarctic Peninsula. West 
of the peninsula, in contrast, this species seems to be rare, 



having been found there at only a single location north 
of Tower Island (stat. 39-90) and in very small numbers. 
Moreover, this species is also distributed in the Indie sector 
of the Antarctic Ocean. I detected it in several samples 
taken by Russian and Soviet Antarctic expeditions: in 
1903 at the Alasheyev Bight, off today's Soviet station 
Molodeshnaya, in 1956 at the Budd Coast, in 1957 off 
the Lars Kristensen Coast, and in 1965 at the Tokarjev 
Island, growing on Harmothoe mo/liiscum and H. gour- 
iloni. 

Franzen reports this species from the subantarctic re- 
gion as occurring abundantly in samples taken in 1902 
at the northernmost tip of the Antarctic Peninsula (Sey- 
mour Island), as well as from the Falkland Islands (Islas 
Malvinas), and from South Georgia where it reaches a 
size of 800-900 /mi. 

In the northern hemisphere. Loxosomella compressa 
is common in the whole arctic and subarctic regions. 
where it is reported to occur abundantly on several po- 
lynoids (Lagiscu extemtata, Gattyana cirrhosa, Acanllu- 
olepis asperrima, and Harmothoe Ini/iaeli). It also occurs 
along the English and Norwegian coasts, in the Skagerrak 
and Kattegatt (Nielsen and Ryland, 1961; Nielsen, 1964a- 
b;Eggleston, 1965, 1969; Eggleston and Bull, 1966; Jones, 
1963), as well as all along the shelf to the Arctic Polar 
Sea, from the Barents to the Kara Sea, living on the poly- 
noids Harmothoe imhricaia. Antinoella badia, A. sarsi 
and Eiinoe hartmannae (Emschermann, unpub.). In the 
entire mid-Atlantic, as well as in the Pacific, this species 
seems to be absent. 



Loxosomella varians Nielsen 



Svnonvni Loxosomella brachystipes Franzen 1973. 

Material. Collected in the Weddell Sea by the author (stations ANT 

VIII-5. 16-396 and 16-454), at depths from 270 to 400 m on stony 

bottoms and in the Bransh'eld Strait by Dr. U. Wirth (stations Met. XI- 

4. stat. 8-90, 39-90, and 96-90) at depths from 100 to 1 50 m on muddy 

grounds, this species grew in moderate numbers on the gills (Fig. 17a) 

of Aglaophamux fulioxitx (Polychaeta. Nephthyidae). The species was 

originally described by Nielsen as common in the North as well as in 

the Baltic Sea living on the gills of several nephthyid polychaetes. 

Description. The specimens from the Weddell Sea, as 

well as from the Bransfield Strait, are in good agreement 

with Nielsen's description: a small variable species of 

about 300-500 /urn total length, with a bulgy goblet-shaped 

calyx on an almost short, sturdy peduncle, distinctly 

marked off from the calyx, and terminating in a more or 

less extended foot plate of variable form (Figs. 16: 17; 

18a-h). 

The calyx is laterally depressed; the comparatively large 
tentacular crown with its 8 slender tentacles faces upwards, 
slightly inclined to the oral side. The stomach is large and 
globular. Buds are formed at the oral side in two adjacent 
paramedial areas in line with the roof of the stomach. 
Under favorable conditions, they can build up a crowded 



176 



P EMSCHERMANN 




Figure 15. Ltixmoinclla comprcssa. a: polynoid parapodium with loxosomalid specimens on the no- 
topodial setae; b-c: living expanded and contracted specimens in lateral and frontal view; d: preserved 
specimen in expanded state, seen from lateral; f: newly detached bud. 



ON ANTARCTIC ENTOPROCTA 



177 




Figure 16. Loxosomcllu varum*. a-c: two more or less expanded Weddell Sea specimens in frontal, 
lateral, and abfrontal view, respectively: d-e: specimen from the Bransfield Strait with a total of six buds in 
two paramedian clusters, in lateral and frontal view, respectively; f: preserved specimen with larvae in the 
brood pouches at either side; g-h: abnormal newly detached buds. 



178 



P. EMSCHERMANN 




, 



Figure 17. Loxosomella various from the Weddell Sea. specimen with a cluster of 7 buds of different 
age on a parapodial cirrus of its host. Aglaophamus foliosus: b-c: Type specimen of Loxosomella brachystipes 
Franzen (c: foot of b enlarged); d: young bud from Franzen's type specimens with the clearly visible foot- 
gland (bar 100 j<m). 



cluster of eight and more buds which appear to originate 
from a single medial budding area (Figs. 16e, d; 17a). 

The foot-gland in young buds forms a small groove; in 
older ones a slit-like invagination is bordered by densely 
arranged, elongated, club-shaped gland cells (Figs. 17d; 
18i-m). In the adult foot plate, parts of the adhesive gland 
usually persist as a row of marginal large cells around the 



rim of the basal disc. Sometimes an additional plug of 
epithelial cells is formed in the middle of the foot (Fig. 
18e, o-p). 

Most specimens contained immature and mature ova- 
ries, whereas testes were seen only in older undetached 
buds. In animals bearing larger embryos or larvae, the 
aboral calyx wall at either side, just below the tentacular 



ON ANTARCTIC ENTOPROCTA 



179 




Figure 18. Loxosomella varians. Comparison of a Bransfield Strait specimen (a) with type specimens 
of Loxosomella brachystipes Franzen (b-c) and paratypes of L. varians Nielsen from the Kattegat (d-h); i- 
n: shape of the foot-gland in buds (i from Franzen's, k from Nielsen's samples) and in newly detached 
specimens (1-n); o-p: adhesive plates in adult specimens from Nielsen's paratype material. 



180 



P. EMSCHERMANN 



crown, bulged out due to the enlarged brood pouches, 
like a clumsy rucksack almost equal in size to the entire 

normal calyx (Fig. 160- 

A/iwwi/rwiWs Total length: .'TO M m (300-485 Mm) (Nielsen 392 Mm: 
Franzen: 426 urn): Length of calyx: 305 ^m (175-325 Mm) (Nielsen: 
285 Mm; Franzen: 375 ftm): length of stalk: 65 ^m (32-1 1 1 ^m) (Niel- 
sen: 107 ftm: Franzen: 51 Mm): width of calyx: 223 Mm (191-254 Mm) 
(Nielsen: 200 Mm; Franzen: 292 Mm); thickness of calyx: 290 Mm ( 1 75- 
461 Mm) (Nielsen: 205 Mm: Franzen: 332 Mm); number of tentacles: 
8 (Nielsen: 8, Franzen: 8); maximal number of buds: 8 (Nielsen: 15). 
Additional remarks on the variability of this species. 
Although highly variable, especially with respect to the 
length of the stalk and shape of the foot plate, the species 
is well-defined by its general body shape, the crowded 
buds, and the paramedial budding areas, as well as by the 
small groove-like foot-gland in the buds. 

In 1973, Franzen described a new loxosomatid found 
in small numbers on the gills of Aglaophamus virginis 
(Polychaeta. Nephthyidae), from old samples collected 
northeast of South Georgia during the 1902 Swedish Ant- 
arctic expedition; he named it Loxosomella brachystipes 
(Figs. 1 7b. c: 1 8b, c). This species, in most instances, looks 
like Loxosomella various, but according to Franzen, the 
shape of the foot is markedly different in these two forms. 
However, judging from the present samples, both of these 
forms must be considered identical, because they cover 
the whole range from /_. varicins- to L. brachystipes- type 
specimens. So, in agreement with Franzen, they must be 
considered synonymous (see Fig. 18 regarding the vari- 
ability of this species). 

Habitat and distribution. Loxosomella various has been 
found living on the gills of a broad spectrum of nephtydid 
polychaetes, never on hosts belonging to other polychaete 
families. In the Atlantic sector of the subantarctic and 
Antarctic Sea, this species is widespread from South 
Georgia, south to the eastern Weddell Sea, and along the 
western coast of the Antarctic Peninsula. In the northern 
hemisphere, Loxosomella various is reportedly common 
in the North and Baltic Seas, but it seems to be absent 
from the midatlantic region and the Pacific Ocean. 

Barentsia discreta (Busk 1886) 

Sy/wnvmv Ascopodaria discreta Rusk, 1886:Kluge, 1946: Thornely. 
\905; Ascopodaria mtieri>pn\ Ehlers. 1890; Robertson, 1900; Barentsia 
unlarclica Johnston and Angel. 1940: Barentsia discreta Annandale, 
1915; O'Donoghue, 1920: Emschermann. 1985: Franzen, 1973; 
Harmer, 1915; Hutchins. 1945: Johnston and Angel 1940: Kirkpatrick 
1888; Konno 1971; Marcus. 1922. 1937. 1953; Mature. 1957; Mukai 
andMakioka, 1980; Okada and Mawatari. 1938;Osburn. 1912. 1914, 
1932, 1944, 1953;Rogick, l956:Toriumi. 1949. 1951: Vigeland. 1937/ 
38; Waters. 1904; Barcmsia gracilis Norman. 1907/10; Barentsia in- 
termedia Johnston and Angel. 1940: Barent\ia miMikicnxis Oka. 1895; 
liarcntxiii tiniula Verrill, 1900. 

Material. In the Weddell Sea. small colonies were found on diverse 
sol d substrates from three locations (ANT VIII-5/ 16-396 and 16-456. 
ami additionally in older samples collected at 76 36,0'S; 30 33.3'W). 
In the Branstield Strait, samples were found at all stations, except 66- 
90. in a depth range of 80 to 400 m. 



Description. Living colonies of Barentsia discreta (Fig. 
19) can immediately be recognized macroscopically by 
the vivid bending and twisting movements of the tall. 4- 
6 mm-long zooids arising from large, cylindrical, and del- 
icately annulated basal sockets. The slender and predom- 
inantly rigid stalk bears a broad, cup-shaped calyx with 
the circle of 20-24 long tentacles facing straight up. The 
rigid part of the stalk, depending on the growth conditions, 
may be the same diameter over its entire length, or may 
widen slightly distally. Its smooth, yellowish to brownish 
cuticle is usually perforated by more or less numerous, 
minute, pore-like openings of subcuticular epithelial or- 
gans; the latter are presumably ion regulating cells ho- 
mologous to protonephridia (Emschermann, 1972. 1982). 
As is normal for Barentsiidae, the rigid portion of the 
stalk distally. just below the calyx, turns into a short mus- 
cular segment with a wrinkled flexible cuticle. This distal 




a ^ 



Figure 19. Barcnt\ia di\m-tti. a-c: three zooids from the Bransfield 
Strait (a) and the Weddell Sea (b. c): d: basal socket with a disc-shaped 
resting bud below it (part of a colony from the Bransfield Strait); e: calyx 
with the characteristic atnal retractor muscle indicated. 



ON ANTARCTIC ENTOPROCTA 



181 



stalk segment has the capacity for calyx regeneration. Un- 
der favorable growth conditions, after degeneration of a 
calyx, and before its regeneration, the muscular section 
may give rise to a second stalk segment, consisting in turn 
of a proximal stiff and a distal muscular portion. The 
primary muscular swelling in such a case persists as an 
intercalating muscular joint, separated from the next seg- 
ment by a cuticular hemiseptum. A stack of 8 to 10 star- 
shaped transverse muscle cells ("star cells" Emschermann, 
1969) forms a sort of diaphragm between stalk and calyx. 
In older well-fed colonies, a cup-shaped secondary in- 
flation can develop below the bases of the zooid muscular 
sockets (Fig. 19d); the inflation is filled with storage cells 
and is separated by a diaphragm from the zooid base. 
These basal inflations function as resting buds, being re- 
sistant to mechanical damage, as well as temperatures up 
to 25C, and even against being embedded in ice or drying 
for at least a week. They give rise to new zooids after the 
primary ones have been damaged or have died. 

The structure of the calyx musculature, in particular 
the shape of the paired atrial retractor muscles, is a useful 
and reliable species character (Fig. 19e), as in most Bar- 
entsiidae (Emschermann. unpub.). In Barentsia discreta. 
three fine muscular strands on either side originate from 
the atrial floor just behind the mouth. Running down- 
wards, in line with the roof of the stomach, they unite to 
form a short muscular ribbon. This in turn bifurcates again 
into an anterior and a posterior branch, each splitting 
into 2 to 5 fine single fibers, which insert in the lateral 
calyx walls at either side of esophagal entrance into the 
stomach. These atrial retractors can best be visualized in 
contracted calyces with polarized light or Nomarski in- 
terference contrast. 

In the Antarctic samples, sexually mature zooids with 
both ovaries and, more rarely, testes were found. 
Measurements. Total length of zooids: 4-6 mm; length of muscular 
base: 0.8-1.1 mm; diameter of muscular base: 0.3-0.44 mm; length 
of the distal muscular portion of the stalk: 180-330 pm: length of 
calyx: 580-700 Mm: number of tentacles: 20-24. 
Additional remarks about the species. Barentsia dixcreta 
has been found worldwide, the size of the zooids varying 
considerably, not only from location to location, but also 
under different nutritive conditions at the same locality. 
The cylindrical (but never barrel-shaped), annulated 
muscular base, the stalk-rigid over nearly its entire length 
with only a short muscular portion below the cup-shaped 
calyx, and the typical structure of the atrial retractor mus- 
cles are reliable, if only morphological, species characters. 
In colonies of different origin (California, Florida, and the 
Mediterranean Sea) cultured in the laboratory under the 
same conditions, no significant morphological differences 
between the specimens of different origin were found 
(Emschermann, unpub.). Their range of variation falls 
within that of the Antarctic material. Interbreeding be- 
tween different populations can be observed in culture to 



the extent that the experimental populations are able to 
be active and become sexually mature under the same 
environmental conditions. In their physiological tolerance 
to environmental conditions, such as temperature, pop- 
ulations from different parts of the world can differ mark- 
edly. An Antarctic colony in my laboratory cultures did 
not remain active at temperatures above 4-5C, but in 
an inactive state, it tolerated temperatures up to 1 5C for 
several weeks. On the other hand, populations from tem- 
perate climates are able to tolerate low temperatures nearly 
to freezing, but they do not develop gonads under these 
conditions. To date, no long term attempts to gradually 
adapt colonies of different origin to lower or higher tem- 
peratures, have been carried out). 

Therefore the genetic exchange between the Antarctic 
populations and others may be considerably reduced, but 
not interrupted. Their morphological conformity can be 
seen as an indication that they are not genetically isolated, 
and the populations of Barentsia discreta reported world- 
wide may be thought of as belonging to the same species 
(cf. Franzen, 1973. p. 185). 

Habitat and distribution. In the Weddell Sea, especially 
in the eastern part, Barentsia discreta is found regularly, 
but never abundantly, at depths between 200 and 400 m. 
This species grows on every solid substrate, preferably on 
primary or secondary hard bottoms, basally on the stems 
of erect hydrozoan and bryozoan colonies, as well as on 
stones, shells, and even on brittle stars. But in the Brans- 
field Strait, the species occurred abundantly everywhere 
at depths from 80 to 500 m, presumably because of the 
more favorable nutrient conditions throughout the year 
in this region. 

In general, this species is distributed worldwide, missing 
only from the Atlantic-subarctic European coasts. Fur- 
thermore, it is reported circumantarctically, along the 
shelves of Antarctica itself and the subantarctic islands 
(Busk, 1886; Franzen. 1973; Johnston and Angel, 1940; 
Rogick, 1956; Vigeland, 1937/38; Waters, 1904). Along 
the South and North American coasts, its distribution 
extends, on the Atlantic side, from Tierra del Fuego, along 
the Argentinian and Brazilian coasts (Marcus, 1937, 
1953), the Caribbean Sea (Osburn, 1914, 1940; Emscher- 
mann, unpub.), and Florida (Nielsen, pers. comm.), up 
to the Massachusetts Bay in the north (Hutchins, 1945: 
Mature. 1957; Osburn, 1912, 1932, 1944); on the Pacific 
side, it extends from southern Chile and along the coast 
of Central America (Osburn, 1953), to California (Rob- 
ertson, 1900; Emschermann, 1985). 

In the Atlantic region, and along the European coasts, 
the species is reported from the Bermuda Islands (Verrill, 
1900; Mature and Schopf, 1968), from Madeira (Norman, 
1907/10; Emschermann, unpub.) and the Azores (Em- 
schermann, unpub.), and from the Mediterranean Sea 
(Ehlers, 1890; Zirpolo, 1927; Emschermann, unpub.). 



182 



P. EMSCHERMANN 



In the Indo-Pacific region, Barentsia discreta seems to 
be common everywhere, from South Africa (O'Donoghue, 
1920), the Indian Ocean (Annandale. 1915: Harmer, 
1899;Kirkpatrick, 1888; Thornely, 1905) and South-Pa- 
cific (Marcus, 1922). to the Chinese- and Japanese Sea 
(Konno, 1971; Oka. 1895; Okada and Mawatari, 1938; 
Toriumi, 1949, 1951:Yamada, 1956). Finally the species 
was reported by Kluge (1946) from the Laptev Sea (Si- 
berian Polar Sea). 

Some General Concluding Considerations 

Besides representing merely a faunistical survey, four 
particular aspects of the above results are of special in- 
terest: ( 1 ) the detection of nematocyst-like organs in an 
entoproct; (2) the ability of a loxosomatid to regenerate 
its calyx; (3) some additional observations on the nature 
of host preference or host specificity of the Loxosomatidae; 
and (4) the bipolar occurance of several Loxosomella spe- 
cies. 

( 1 ) The detection of extruding organs in an entoproct 
raises questions about their comparative morphological 
importance and their phylogenetic significance. Compa- 
rable, usually unicellular, extrusive glandular organs, 
which produce clearly structured secretions, have been 
described in quite a number of invertebrate phyla, in ad- 
dition to coelenterates: in Platyhelminthes (only in Tur- 
bellaria; Reisinger and Kelbertz. 1964; Smith et al.. 1982), 
Gastrotricha (Rieger cl al., 1974), Nemertini (Jennings 
and Gibson. 1969), Gnathostomulida (Rieger and Mein- 
itz, 1977), and the Archiannelida among the annelids 
(Martin, 1978). Except in the Cnidaria. Ctenophora, and 
Turbellaria, these extrusion organs do not represent typical 
characters of the above animal taxa, but occur in isolation 
in one or another species. Only in Cnidaria and Cteno- 
phora do the extrusive organs eject harpoonlike, poisonous 
or sticky threads. In all of the other above taxa the extru- 
sive gland cells produce rod-like mucous secretions of the 
rhabdiite type. The probably syncytial plurinuclear extru- 
sive capsules of Loxosomella brochobola seem, at present, 
to be unique in the animal kingdom and to differ re- 
markably, in development, structure, and extrusion 
mechanism, from comparable organs in other groups. 
Thus they must be considered as an isolated apomorphic 
character of this particular entoproctan species, rather 
than a character of phylogenetic significance. Probably 
they are derived, in a highly specialized form, from con- 
spicuous uni- or pluricellular mucous glands of unknown 
function, which occur in a number of loxosomatids 
around the margin of the tentacular crown. 

(2) The ability l/> regenerate calyces in Loxosomella 
antarctica is unusual for solitary entoprocts. Distinct from 
Loxokalypus socialis (Emschermann, 1972) another 
entoproct species with an enhanced regenerative capacity, 
and in which the budding zone has shifted from the calyx 



wall down to the stalk as a first evolutionary step towards 
the colonial growth pattern-normal asexual budding in 
Loxosomella antarctica proceeds as usual in two paired 
budding areas on the oral wall of the calyx. Therefore, 
the enhanced regeneration capacity of the distalmost tip 
of the stalk epithelum in L. antarctica (Figs. 11: 12) is an 
isolated secondary adaptation to the conditions of Ant- 
arctic life. This is important to the biology of entoprocts. 
but is without phylogenetic significance. 

(3) A marked host specificity is thought by several au- 
thors to be characteristic of, most of the epizoic Loxo- 
somatidae. For example, Nielsen (1966) describes Loxo- 
soma davenport i as normally settling inside the tubes of 
the maldanid polychaete Clyinenella zonalis. but as com- 
pletely absent from the tubes of the closely related Cly- 
inenella torqiiala. which is found much more frequently 
than Clyinenella zonalis on the same sandy bottoms. 
Consequently, many authors consider the host a sufficient 
species character for the identification of loxosomatids. 
But the host can only be employed as a reliable species 
character if its relationship with the loxosomatid is specific: 
i.e.. determined by a strict physiological dependence. A 
shared preference of the host and its epizoan for the same 
microenvironment, or some structural feature of the host 
that offers the epizoan an ideal complex of life conditions 
(C.K.. a combination of mechanical shelter and a water 
current supplying food and oxygen and removing detritus) 
are situations in which host specificity is not a reliable 
species character. A majority of the guest-host relations 
in the loxosomatids seem to be of this latter type. 

Only three of the loxosomatids discussed above are 
known to show a preference for specific hosts independent 
of the respective localities: Loxosomella varians for neph- 
tyid polychaetes, Loxosomella antarctica for the brittle 
star Opliiurolepis gelida. and Loxosomella compressa for 
errant polychaetes of the family Polynoidae. The latter 
two loxosomatids were very abundant at many locations. 
Loxosomella antarctica is found predominantly on silty 
bottoms, on the oral disk between the arms of Opliiuro- 
lepis gelida. never on other ophiurids abundant in the 
same place. If nutrients are abundant, it also builds up 
crowded aggregations on the aboral side of its host and 
laterally along its arms. At adequate sites, where Opliiuro- 
lepis is lacking or very rare, Loxosomella antarctica does 
not switch to an other ophiurid, but rather to an aphroditid 
polychaete, Laelmonice producta. On this second host, it 
occupies exclusively the tips of the dorsalmost notopodial 
setae in the first segments as well as the posterior dozen 
body segments. 

The large robust zooids of Loxosomella antarclica are 
quite resistant to mechanical lesions (cf. regeneration ca- 
pacity), as well as low oxygen supply. As can be seen from 
their stomach contents, consisting mainly of detritus par- 
ticles and some larger ciliates mixed with fine mineral 
material, they are sediment feeders. Their requirements 



ON ANTARCTIC ENTOPROCTA 



183 



are for a nutrient-rich fine sediment and a solid settling 
substrate offering a certain protection against predators 
and against being buried irreversibly under sediments. So 
this species thrives on hosts like Ophiurolepis and Lact- 
iii* mice which creep on. or dwell in. the upper sediment 
layer. 

Loxosomella compressa on arctic and subarctic shelves 
as well as in antarctic waters was detected exclusively on 
polychaetes of the family Polynoidae. attached basally to 
the notopodial setae of their hosts. A more detailed anal- 
ysis of the microhabitat of L. compressa reveals that the 
only polynoidan species infested by this epizoan guest are 
those with notopodial setae that are thick and short, not 
too densely arranged, and covered by the elytrae. This 
loxosomatid has only exceptionally been found on po- 
lynoids with bushy, thinner notopodial setae or with 
parapodia not covered by the elytrae. Usually only smaller 
species, up to 10 cm in length, or younger specimens of 
larger polynoids are chosen as hosts. In culture experi- 
ments, the newly detached buds also settled on diverse 
non-living substrates (cf. p. #) exposed to the current. 

Loxosomella compressa is smaller and less resistant to 
mechanical injury and low oxygen supply than L. ant- 
(irciica. As can be demonstrated by an examination of its 
stomach contents, its diet consists mainly of small algae; 
predominantly small pennate diatoms. Consequently, its 
delicate zooids can grow only in a microhabitat that offers 
shelter against predators as well as against mechanical 
injury, but which also exposes them to a continuous water 
current and provides enough space for optimal feeding 
positions. Such conditions are preferably offered by 
smaller polynoid polychaetes, not dwelling in the sedi- 
ment, but creeping on the exposed surface of sponges and 
on erect bryozoan and hydroid colonies. Other habitats, 
offering comparable physical conditions, may also be 
chosen as a substrate by L. antarctica and L. compressa 
But the small loxosomatids are not easily detected amidst 
the bulk of possible substrates in dredged material; pre- 
sumably they are usually overlooked during sorting. But 
when the hosts were kept for a while in well aerated 
aquaria, the loxosomatids were also found on various 
other non-living substrates. 

From these observations, one can speculate that the 
choice of settling substrate, at least for these loxosomatid 
species, is determined by the physical structure of the mi- 
crohabitat and the supply of an appropriate diet, rather 
than by specific physiological properties of the host itself. 
Thus, although most loxosomatids have preferred hosts, 
these can only be regarded as weak species characters. 

(4) The observed bipolar occurrence of Loxosomella 
antedonis, L. compressa, and L. varians in coastal waters 
suggests, at first glance, a discontinuous, exclusively bi- 
polar distribution of these species. Their northern distri- 
bution in the litoral and sublitoral of the continental coasts 
stretches from Greenland (L. antedonis) and the Eurasian 



polar shelf (L. compressa and L. varians), along the 
northern European coasts, south to about 54 N in the 
southeastern North Sea. In the South Atlantic and the 
Atlantic sector of the Antarctic Ocean, these three species 
are common from the Weddell Sea. and north to the Islas 
Malvinas and South Georgia (about 54S). To date, none 
of them has been found along the eastern or western mid- 
Atlantic coasts, although the entoproctan fauna of the 
Central European shelf, in particular, as well as of the 
Caribbean, Argentinean and Chilean coasts have been well 
investigated. At present, however, nothing is known about 
the depth range of these species and their possible distri- 
bution along the Atlantic deep sea ridges. 

Comparable examples of a suggested bipolar distribu- 
tion of a single species are extremely rare and still con- 
troversial, the best known being the bipolar occurrence 
ofPriapnlus caudatus (Ekman. 1935; van der Land, 1970). 
A discontinuous distribution of taxa above the species 
level can be explained by the break-up of an originally 
continuous area of distribution by geomorphic events, 
such as continental drift, and long term climatic changes. 
At the species level, on the other hand, it seems unlikely 
that populations separated over geological periods could 
remain uniform in their specific characters unless at least 
a limited amount of genetic exchange were maintained 
between them. 

But how can such an exchange take place in the present 
case? Under the conditions of an exclusively bipolar dis- 
tribution, such a genetic exchange between the North and 
South Atlantic populations of the above loxosomatids 
must be excluded, because the life span of individual lox- 
osomatid zooids does not exceed 4-6 weeks, and the mo- 
bile larval phase lasts scarcely more than 8 days. 

Neither passive drifting with currents, nor transport by 
fast swimming hypothetical hosts such as whales could 
proceed quickly enough to maintain a sufficient exchange 
between populations of the North and South Atlantic. 
Nor can the considerable increase of the shipping traffic 
in the past decades be responsible for this distribution. 
One might postulate that the Antarctic faunal region had 
been colonized only recently by these species. But at least 
for Loxosomalle compressa and L. varians, their distri- 
butions in both the Arctic and Antarctic regions were al- 
ready established in the 19th century, as documented by 
the evaluation of several samples from the turn of the 
century (Franzen 1973; this paper). 

Thus a recent continuous distribution by colonization 
along the Atlantic ridges, and possibly the deep sea basins, 
must be postulated as being responsible for the bipolar 
occurrence of these loxosomatids in shallow coastal waters. 
More deep sea samples should be obtained and evaluated 
so that this hypothesis can be tested. 

As far as can be judged to date, the three loxosomatid 
species mentioned above are distributed in the Atlantic 
sector of the Antarctic Sea only, and they seem to be ab- 



184 



P. EMSCHERMANN 



sent from the Pacific sector. The faunal connection be- 
tween the North and South Atlantic must, therefore, be 
much more intense than the circum-Antarctic faunal mi- 
grations. 

Acknowledgments 

I thank Dr. Claus Nielsen (Kobenhavn) and Professor 
Ake Franzen (Stockholm) for their helpful critical remarks 
regarding my species determinations and the newly de- 
scribed species as well as for having placed at my disposal 
their type specimens and comparison samples. Also, I wish 
to thank Dr. E. Androsova (Leningrad) for giving me the 
chance to check the entoproctan samples from Soviet 
Arctic and Antarctic expeditions. I am grateful as well to 
Kerstin Wasson (Santa Cruz, CA) for critically reading 
and correcting the English in this manuscript. 

Literature Cited 

Annandale, N. 1915. Fauna of the Chilka Lake and of brackish water 

in the Gangetic Delta. Mem I mi Mus Calcutta. V: 1 19-134. 
Barrois, J. 1877. Recherches sur 1'embryologie des Bryozoaires. Trar. 

Sin Zoo/. }\'nnereii.\. 1:1-205 (also: These Fac. Sci Pans 396). 
Bobin, G., and M. Prenant. 1953a. La classification des Loxosomes 

selon Mortensen et le Loxosoma singulars de Keferstein et de Cla- 

parede. Bull Soc. Zoo/, France. 78: 84-96. 
Bobin, G., and M. Prenant. 1953b. Sur trois Loxopsomes mediterra- 

neens. Bull, lust. Oeeanogr. (Monaco) 50: 1-9. 
Busk, G. 1886. Report on the Polyzoa collected by H.M.S. Challenger 

during the years 1873-76. Pt. II: The Cyclostomata, Ctenostomata 

and Pedicellinea. In: Report on the Sci. Res 1 or. H.M.S. Challenger. 

Zoo/. XVII: 1-47. Evre & Spattiswood. London. 
Calvet, L. 1904. Diagnoses de quelques especes de Bryozoaires nouvelles 

ou incompletement decrites de la region subantarctique de 1'Ocean 

atlantique. Bull. Soc Zoo/. France XXIX: 50-59. 
Calvet, L. 1904. Ergebnisse tier Magalhaensischen Sammelreise 1S92/ 

93. Ill: Brvoioen und Wiirmer. L. Friedrichsen & Co, Hamburg. Pp. 

1-45. 
Claparede, E. 1867. Sur le Loxosoma kefersteini. Bryozoaire mou du 

Golfe de Naples. Ann Sci Nat. (Paris), ser. 5. Zoologie. VIII: 28- 

30. 
du Bois-Raymond-Marcus, E. 1957. Neue Entoprocten aus der Gegend 

von Santos. Zoo/. An:. 159: 68-75. 
Kggleston, D. 1965. The Loxosomatidae of the Isle of Man. Proc. Zoo/. 

Soc. London 145: 529-547. 
Eggleston, D. 1969. Marine Fauna of the Isle of Man: Revised lists of 

Phylum Entoprocta (= Kamptozoa) and Phylum Ectoprocta ( = 

Bryozoa). Rep. Mar. Biol Sin. Port Erin 81: 57-80. 
Egglestnn, D., and H. O. Buill, 1966. The Marine Fauna of the Cul- 

lercoats District. 3a: Entoprocta. Rep. Dove Mar. Lab. (3 ser) 15:5- 
10. 
Ehlers, E. 1890. Zur Kenntnis der Pedicellineen. Abhandlungen der 

phys Rlasse der kgi Ges. Wtsscnsch Gottingen. 36: 1-200. 
Ekman, S. 1935. Tierge<igraphie des Meeres. Akad. Verlagsges. Leipzig. 

542 pp. 
Emschermann, P. 1969. Ein Kreislauforgan bei Kamptozoen. Z. Zell- 

Jorscli 97: 576-607. 
Emschermann. P. 1972a. Cuticular pores and spines in the Pedicellin- 

idaeand Barentsiidae ( Entoprocta). their relationship, ultrastructure. 

and suggested function, and their phylogenetic evidence. Sarsia 5: 

7-16. 



Emschermann, P. 1972b. l.oxoka/ypiis socialis gen. and spec. nov. 
(Kamptozoa. Loxokalypodidae fam nov.), ein neuer Kamptozoentyp 
aus dem nordlichen Pazifischen Ozean. Ein Vorschlag zur Neufassung 
der Kamptozoensystematik. Marine Biology 12: 237-254. 

Emschermann. P. 1982. Les Kamptozoaires. Etat actuel de nos con- 
naisssances sur leur anatomie. leur developpement. leur biologie et 
leur position phylogenetique. Bull. Soc. Zoo/. France 107: 317-344. 

Emschermann, P. 1985. Factors inducing sexual maturation and in- 
fluencing the sex determination of Barenlsia discrela Busk (Ento- 
procta Barentsiidae). Pp. 101-108 in Krymoa Ordovician to Recent, 
C. Nielsen and G. P. Larwood. eds. Olsen and Olsen. Fredensborg. 
Denmark. 

Eranzen, A. 1973. Some Antarctic Entoprocta with notes on mor- 
phology and taxonomy in the Entoprocta in General. Zoo/. Sci. 2: 
183-195. 

Harmer, S. 1885. On the structure and development of Loxosoma. Q 
J. Microsc. Set. N.S. 25: 261-337. 

Harmer, S. 1915 The Polyzoa of the Siboga Expedition. Part 1. En- 
toprocta, Ctenostomata and Cyclostomata. Siboga Exped. Rep 28a: 
1-180. 

Hincks, Th. 1880a. On New Hydroida and Polyzoa from Barents Sea. 
Ann. Mag Nat Hist. (5. ser. I 6: 277-286. 

Hincks, Th. 1880b. History of the British Marine Polyioa. 2 Vols. 
John van Voorst. London. 

Hutchins, L. \V. 1886. An annotated check-list of the salt water Bryozoa 
of Long Island Sound. Trans. Connecticut Acad Arts Sci. 3: 533- 
551. 

Jennings, J., and R. Gibson 1969. Observations of the nutrition of 
seven species Rhynchocoelan Worms. Biol. Bull. 136: 405-433. 

Johnston, T. H., and L. M. Angel. 1940. Endoprocta. Pp. 2 1 5-23 1 in 
B.A.N.Z. Antarctic Research Expedition 1929-1931. Report Series 
B (Zoology and Botany). IV, part 7, Adelaide, Australia. 

Jones, N. S. 1963. Phylum Kamptozoa (= Polyzoa). Pp. 224-225 in 
Marine Fauna ol the Isle ol Man. 2nd edition. J. R. Bruce. J. S. 
Coleman, and N. S. Jones eds.. Liverpool University Press, Liverpool. 

Kirkpatrick, R. 1888. Reports on the Zoological Collections Made in 
Torres Straits by Professor A. C. Haddon 1888-1889. Hydroida and 
Polyzoa. Set. Proc. R Dublin Soc. (N.S.) 6: 603-626. 

Kluge, G. A. 1946a. Kamptozoa from the Arctic Ocean. Trudy Droy- 
fuviish. Exped. dluvsev. Na Ledok. Parochode "G. Sedov"God. 1937- 
1940. 3:149-156 (in Russian with Engl. summary). 

Kluge, G. A. 1946b. New and little known species of Bryozoa from 
the Arctic Ocean. Trudy Droyfuyush. Exped. Glavsev. Na Ledok. "G. 
Sedov"God. 1937-1940. 3:194-223 (in Russian with Engl. summary). 

konno, K. 1971. On some entoprocts found at Fukaura. Aomori Pre- 
fecture. Rep. Fukaura Mar Biol. Lab.. 3: 2-9. 

Konno, K. 1977. Studies on Japanese Entoprocta VII. On two new 

species of Loxosomatidae. Sci. Rep Hirosaki L'nivers. 24: 81-84. 
Land, J. van der 1970. Systematics. zoogeography, and ecology of the 

Priapulida. Zoo/ Verhandelingen (Leiden) 112: 1-118. 
Marcus, E. 1922. Papers from Dr. Th. Mortensen's Pacific Expedition 
1914-1916 VI: Bryozoen von den Auckland- and Campbell-Inseln. 
I 'iilcnskabe/ige Meddetser fra Dansk Nan/i/nst. Forening Kabenlumi 
173: 85-121. 
Marcus, E. 1937. Bryozoarios marinhos Brasileiros I. Bol. Fac. Phil. 

Cienc. l.clr I'niv Sao Paulo I, Zoologia. 1: 1-224. 
Marcus, E. 1953. Notas sobre briozoos marinhos brasileiros. Arquivos 

do Mus \acional (Rio de Janairo) 42: 273-342. 
Martin, G. E. 1978. A new finding of Rhabdites: Mucus production 

for ciliary gliding. Zooinorphology 9 1 : 235-248. 
Maturo, F. J. S. 1957. A study of the Bryozoa of Beaufort, North 

Carolina, and vicinity. J Elisha Mitchell Scient. Soc. 73: 1 1-68. 
Maturo, E. J. S., and Th. J. M. Schopf. 1968. Ectoproct and Entoproct 
type material: Reexamination of species from New England and Ber- 



ON ANTARCTIC ENTOPROCTA 



185 



muda named by A. E. Verrill, J. W. Dawson and E. Desor. I'osiilla 

(\c Haven) 120: 1-95. 
Morlensen, Th. 1911. A new species of Entoprocta. Loxosomella an- 

ledonis. from North-east Greenland. Danmark-Ekspeditionen til 

Grmlands .\or<l.sikyv l<Mf>-IW>S 5(8): 344-406. 
Mukai. II.. and T. Makioka. 1980. Some observations on the se\ de- 
termination of an Entoproct, Baienlsui discrcla ( Busk). ./. Exp. Zoo/. 

21:45-59. 

Nielsen, C. 1964a. Studies on Danish Entoprocta. Ophelia I: 1-76. 
Nielsen, C. 1964b. Entoprocta from the Bergen Area. Sarxia 17: 1-6. 
Nielsen, C. 1966. Some Loxosomatidae (Entoprocta) from the Atlantic 

Coast of the United States. Ophelia 3: 244-275. 
Nielsen, C., and J. R\land. 1961. Three new species of Entoprocta 

from West Norway. Sarsia I: 39-45. 
Nilus, G. 1909. Notiz iiber Loxosonm niiiriuanica und I.o\osoma 

Imniipti sp. n. Trav. Soc Imper. Nat- Si. I'elershoiirt; 40( 1 ): 157- 

169. 
Norman, A. M. 1907/10. The Polyzoa of Madeira and the Neighbouring 

Islands. / Linn. Soc. (London) 30: 275-277. 
O'Donoghue, Ch. 1920. The Bryozoa (Polyzioa) collected by the S. S. 

"Pickle". Fish anil Mar. Biol. Survey I Cape Town) repl 1: 1-61. 
Oka. A. 1895. S\u \a Barentsia misakiensis ~/.oo\ Mug 7:1-10. 
Okada, V., and S. Mawalari. 1938. On the collection of Bryozoa along 

the coast of Wakayama-ken. the middle part of Honsyu. Japan. Annot. 

/ool Japan 17:445-448. 
Osburn, R. 1912. The Bryozoa of the Woods Hole Region. Hull Bur 

Fish. Washington) 30: 212-214. 
Osburn, R. 1914. The Bryozoa of the Torlugas Islands. Florida. Papers 

from the Tortugas Lab Carnegie lust <>/ Washington 5: 1X3-222. 
Osburn, R. 1932. Bryozoa from Chesapeake Bay. Ohio ./ Sci. 3: 44 1 - 

446. 
Osburn, R. 1944. A Survey of the Bryozoa of the Chesapeake Bay. 

I'uhl Chesapeake Biol Lab. 63: 1-55. 
Osburn, R. 1940. Bryozoa of Puerto Rico with a resume of the West 

Indian Bryozoan Fauna. Pp. 321-486 in Sci. Survey ol Puerto Rico 

and the 1'irgin Island. \ol 16. New York. 
Osburn, R. 1953. Bryozoa of the Pacific Coast of America. Pt. 3: Cy- 

clostomata. Ctenostomata. Entoprocta and Addenda. Mian hancock 

Pacific Expeditions 14: 613-822. 
Pallas, P. 1774. Spicilegia Zoologica. quihns novae el imprimis oh- 

scitrac aniwnahum species iconibus. desciiplionihns ati/uc coniinen- 

tarns illustianlnr vol. 1. fasc. 10. Gottlieb August Lange. Berlin. 
Reger, J. 1969. Studies on the fine structure of muscle fibers and con- 
tained crystalloids in basal socket muscle of the Entoproct. Btirenlsia 

gracih.s J Cell Sci 4: 305-325. 



Reisinger, E.. and S. Kelbertz. 1964. Feinbau und Entladungsme- 

chanismus der Rhahditen. / uns Miksrok Tech 65: 472-508. 
Ridley. St. O. 1881. Polyzoa. In: Account of the Zoological Collections 

made during the survey of H. M. S. "Alert" in the Straits of Magellan 

and on the Coast of Patagonia. Proc. /.ool Soc I London) 1881: 44- 

61. 
Rieger, R. M., and M. Mainit/. 1977. Comparative fine structure study 

of the body wall in Gnathostomulida and their phylogenetic position 

between Platyhelminthes and Aschelminthes. Zoo/. Syst. Evolu- 

tion.s'/orseh 15: 4-34. 
Rieger, R. M., E. Ruppert, G. E. Rieger, and C. Schoepfer-Sterrer. 

1974. On the tine structure of gastrotnchs with description of 

Chordodasvs anlennatus sp.n. ~/,ool. Scr 3: 219-237. 
Robertson, A. 1900. Studies in Pacific Coast Entoprocta. I'roc Culil 

.lead Sci. 3rd.ser '/.ool 2: 323-349. 
Rogick. M. D. 1956. Bryo/oa of the United States Navy's 1447-1948 

Antarctic Expedition, 1-4. Proc. L : . S. Nail. Mus. 105: 221-317. 
Rogick, M. D. 1965. Bryozoa of the Antarctic. Pp. 401-413 in Bio- 

geographv and Ecology in Antarctica. P. Van Oye and J. van 

Mieghem. eds. W. Junk, den Haag. 
KM. mil. J., and A. P. Austin. I960. Three species of Kamptozoa new 

to Bntain. Proc. Zool. Soc London 133: 423-433. 
Smith, J., S. Thyler, M. B. Thomas, and R. M. Rieger. 1982. The 

morphology of Turhellanan Rhabdites: Phylogenetic implications. 

Trans, liner Soc 101: 104-228. 
Thornely, L. R. 1905. Report on the Polyzoa collected by Professor 

Herdman, at Ceylon in 1402. Ceylon Pearl Oyster Fisheries, pt 4. 

suppl rep 26: 107-130. 
I .n mini. M. 1949. On some Entoprocta from Japan. Sci. Rep To/ioktt 

Univ.. 4th set (Biology) 18: 223-227. 
Toriumi, M. 1951. Some Entoprocts found in Matsushima Bay. Sci. 

Rep To/iokn L'niv 4ih scr (Biology) 19: 17-22. 
\errill, A. E. 1900. Additions to the Tumcata and Molluscoidea of 

the Bermudas. Trans Conned lead Sci. 10:588-598. 
Vigeland. I. 1937/38. Bryozoa of Tristan da Cunha. Res. Norweg. Sei. 

Expcd to I'nsiainlaCnnha 1V37-1V3844: 9-18. W. Nygaard. Oslo. 
Voigt, C. 1876. Le Loxosome des Phascolosomes (Loxosoma phas- 

colosomatuin) /.ool Exp el (icn. (Pans) 5: 305-356. 
\\aters, A. \\'. 1904. Bryozoa. Pp. 1-1 13 in Res I 'or. du S. Y Belgica 

en 1S97-ISW. J. E. Buschmann. Anvers. 
N amada, M. 1956. The Fauna of Akkeshi Bay: 24. Entoprocta. J. Fac. 

Sci. Hokkaido L'/uvers. 12: 237-243. 
/irpolo, G. 1927. Sulla presenza della Barenlsia iliscreta Busk nel Golfo 

di Napoli. Boll Soc Nat. Napoli 39: 413-419. 



Reference: Biol Hull 184: 186-202. (April, 1993) 



Control of Hatching in an Estuarine Terrestrial Crab. 

II. Exchange of a Cluster of Embryos 

Between Two Females 

MASAYUKI SAIGUSA 
Okayama University, College of Liberal Arts and Sciences. Tsushima 2-1-1. Okayama 700. Japan 



Abstract. The eggs of an estuarine terrestrial crab, Se- 
sarma haematocheir (akate-gani), are incubated by the 
female for about one month. In estuarine crabs larval 
hatching is synchronized with the nocturnal high tide. To 
investigate whether the female or the embryo controls the 
actual timing of the hatching, one cluster of embryos was 
detached from each of two ovigerous females and recip- 
rocally transplanted. Hatching of the transplanted em- 
bryos was divided into the following three patterns ac- 
cording to the number of nights until either (or both) of 
the females released their larvae. In Pattern I. the trans- 
planted clusters both hatched on the same night that the 
donor females released their larvae. In Pattern II. the 
hatching of one of the transplanted clusters was not con- 
trolled by the host female, whereas hatching of the other 
transplanted cluster was obviously induced. Finally, in 
Pattern III. not only the induction of hatching, but also 
the time of hatching, was controlled by the female. 
Hatching profiles of transplanted embryos transferred to 
aerated conditions indicated that hatching requires three 
nights, and that each embryo also has an endogenous 
rhythm for hatching. The female seems to play two roles 
in hatching: i.e.. initiation of the hatching process, and 
enhancement of hatching synchrony in each embryo. A 
plausible hypothesis explaining the mechanism of induc- 
tion and the synchronization of hatching is presented. 

Introduction 

Clearly demarcated rhythmicities are often observed in 
the reproductive behaviors of both marine and terrestrial 
animals. A persistent question in reproductive rhythm 
research is whether the female or the embryo controls the 

Received 28 May 1 99 1 ; accepted 25 January 1993. 



actual timing of these behaviors. Rhythms of spawning 
(i.e.. shedding of gametes or fertilized eggs) or oviposition 
following gametogenesis must be controlled by the female 
alone. Examples of this phenomenon are the circadian 
rhythm of oviposition in the pink bollworm Pectinophora 
gossypiella (Pittendrigh and Minis, 1971), egg laying in 
the teleost Oryzias latipes (Egami. 1954; Ueda and Oishi. 
1982), and the daily, tidal and lunar rhythms of spawning 
in many kinds of marine invertebrates (Korringa, 1947; 
Pearse, 1990). 

Embryonic development proceeds within the eggs ovi- 
posited by the female, and hatching occurs after a certain 
period. A circadian rhythm of hatching appears in the 
bollworm P. gossypiella. Eggs of this species, maintained 
at 20C, hatched 10-13 days after they were oviposited; 
the eggs were transferred from constant light (LL) to con- 
stant darkness (DD) every 5.5 h during embryonic de- 
velopment, and hatching was monitored (Minis and Pit- 
tendrigh. 1968). This experiment suggested that a circa- 
dian pacemaker controlling hatching is differentiated at 
least around the midpoint of embryogenesis, i.e., 6-7 days 
after oviposition. 

Obvious rhythmic patterns are also observed in the 
hatching of marine crustaceans (Sastry, 1983; DeCoursey, 
1983). But eggs of most marine and freshwater crustaceans 
are incubated by the female until hatching occurs. This 
phenomenon complicates the control of the larval hatch- 
ing rhythm. Indeed, the timing of larval hatching is syn- 
chronized with day-night, tidal, and lunar cycles, but 
whether it is the female or the embryo that controls the 
actual timing of hatching remains unclear. 

This question has only been investigated with respect 
to eggs already detached from the female (Saigusa, 1992c), 
the role of the female is still unknown. This paper focuses 
on the female control of larval hatching in an estuarine 



186 



LARVAL HATCHING IN AN ESTUARINE CRAB 



187 



terrestrial crab, Sesarma haematocheir, and reports on an 
embryonic exchange method that was used in the inves- 
tigation. Eggs of this species consist of eight clusters. Two 
of such clusters, one from each of two ovigerous females, 
were detached and exchanged by reciprocal transplanta- 
tion. The transplanted eggs survived on the host females, 
and most of them successfully hatched. Hatching in the 
transplanted eggs was clearly divided into three patterns 
depending upon the number of nights intervening between 
the exchange and the occurrence of hatching in both or 
either of the females. 

These results suggest that the female triggers the hatch- 
ing process in each embryo, but that each embryo has 
also an endogenous rhythm of hatching. In response to 
some (unknown) stimuli released from the female, each 
embryo must initiate its hatching process around the time 
of nocturnal high tides, and hatching occurs 48-49.5 h 
later. Since all of the female-attached embryos hatch 
within a very short time, the female should have some 
mechanism for enhancing hatching synchrony just before 
the larval release. 

This paper provides evidence that the control of hatch- 
ing involves cooperation between female and embryo. 
Based on the data reported here, I present a hypothesis 
that explains the mechanism controlling the daily timing 
of larval hatching in Sesarma haemaloeheir. 

Materials and Methods 

Maintenance of crabs and monitoring of larval release 
in experimental rooms 

Experimental animals were ovigerous females of the 
terrestrial red-handed crab (akate-gani) Sesarma hae- 
malocheir, randomly collected on 9, 19, 31 July, 16 Au- 
gust 1990, 19 July, and 8 August 1991 from the thicket 
along a small estuary at Kasaoka, Okayama Prefecture. 
The crabs were immediately brought into the experimental 
rooms in the laboratory, and were kept in plastic con- 
tainers (70 cm long, 40 cm wide, and 25 cm high) with 
shallow water ( 1 cm deep) at the bottom, and with hiding 
spaces above it. Light and temperature in the experimental 
rooms were controlled. A 1 5-h light: 9-h dark photoperiod, 
the same phase as that in the field (light-off at 20:00 and 
light-off at 5:00), was employed for all experiments. The 
intensity of illumination in the light phase was 700-1200 
lux at the floor, and in the dark phase, less than 0.05 lux. 
Temperature was constant at 23 1.5C and the crabs 
were fed every few days. 

A female of S. haematocheir incubates 20,000-50,000 
eggs on her abdomen. When embryonic development is 
complete, all of the larvae hatch simultaneously. Hatching 
is completed within a very short time, within 5-30 min 
in the laboratory (Saigusa, 1992c). As soon as hatching is 
finished, the female releases her larvae into the water. The 



time of day of larval release can easily be monitored by 
the photoelectric-switch method (Saigusa, 1992a). Under 
the above-mentioned light conditions, larval release ac- 
tivity shows a circa-tidal rhythm, the phase of which co- 
incides roughly with nocturnal high waters. 

Exchange of egg clusters between two females 

The females of 5. haematocheir incubate their eggs for 
about one month. During this time, the color of the em- 
bryos changes from dark brown to brownish green, ac- 
cording to the stage of development which can, therefore, 
be estimated by visual inspection. In these experiments, 
females with mature embryos (brownish green color) or 
near mature embryos (light brown color) were used. 

The reciprocal exchange of a cluster of embryos between 
paired females is carried out as follows. Two females with 
similar carapace sizes were taken from containers. The 
walking legs and body, except the portion where the em- 
bryos are incubated, were wrapped in a paper towel, and 
the claws were then secured with a rubber band (Fig. 1 A, 
upper panel). To prevent the crabs from removing the 



A 




1cm 

r 




>t 




i V 



cl 
1cm 






Figure 1. Embryo exchange between two females. (A) Upper photo: 
females with their chelae and walking legs restrained with a rubber band 
(rb). Lower photo: a cluster of embryos (cl) detached from each female. 
The base of the ovigerous seta is tied with a thread (/). (B) Females after 
the embryo exchange: a view from behind. //: paper towel. 



188 



M. SAIGUSA 



exchanged cluster, 2-3 mm of one of the (paired) tips of 
both claws was removed with scissors (see the female at 
the left side of Figure 1A). Bleeding was stopped with a 
small soldering iron. 

Females of S. liuciniiiin heir have four pairs of abdom- 
inal appendages, each of them consisting of plumose and 
non-plumose setae (Fig. 2). The eggs are attached, just 
like grapes (8 clusters in all), to ovigerous hairs that grow 
from the non-plumose setae, and are ventilated by the 
female during development. The number of attached em- 
bryos in a cluster is 2000-6000. The first non-plumose 
seta on the right side was cut with scissors (Fig. 2). because 
it is the most convenient place to bind an exchanged clus- 
ter from another female. The excision of the egg cluster 
caused a small amount of bleeding from the base of the 
non-plumose seta, but hemostasis was induced with a 
sharpened soldering iron. These procedures, including the 
removal of an egg cluster, were applied in rapid succession 
to both females. 

Each cluster of removed eggs was tied at the cut end 
of its seta to the center of a long thread (Fig. 1 A, lower 
panel). Each tied cluster was then put into the space where 
the reciprocal cluster had been detached, and the free ends 
of the thread were passed around to the dorsal side of the 
abdomen and knotted at the articulation between the ab- 
domen and the thorax (Fig. 1 B). This prevented the trans- 
planted cluster from being squeezed out of the egg mass 
being incubated by the host female. There was no ex- 
change of blood between the transplanted non-plumose 
seta and the female. 

Paralleling the exchange of a cluster of embryos, another 
small embryo cluster (200-500 eggs) was removed from 
each female and placed in the glass beaker with aeration 
(Saigusa, 1992b). Under such conditions, embryos that 
were detached less than 48-49.5 h before the larval release, 
all hatch on the same night as the eggs incubated by the 
female; moreover, they develop and are able to swim. In 
contrast, embryos separated earlier than 48-49.5 h before 
the release, do not hatch during the experimental period. 
After more than a week of aeration, these embryos grad- 
ually hatch as larvae with no ability to swim (i.e.. the 
prezoea) (Saigusa, 1992c). To determine whether the 
hatching of transplanted embryos is triggered by the host 
female, hatching of the embryos detached from the female 
was monitored (i.e., control experiment). 

The time required for the removal and exchange of a 
pair of egg clusters was about 15-20 min. In addition, 
preparation of the control experiment i.e., detachment 
of a small egg mass, binding it with thread, and then setting 
it onto the apparatus for aeration took only about 5 
min. To avoid nocturnal light, procedures were carried 
out in experimental rooms with a light phase of 24-h LD 
cycle. 




a*- 



an 



1cm 



Figure 2. Abdominal appendages of Si'xurma haematocheir female. 
The abdomen is opened and drawn from the ventral aspect. (/. thorax, 
a: abdomen, an: anus, px: plumose seta, npx. non-plumose seta, ga: genital 
aperture). Ovigerous hairs growing from the non-plumose seta are omitted 
from the drawing. The unlabeled black arrow shows the place where the 
cluster of embryos is cut off with scissors. 



Inspections of hatching in transplanted embryos 

When the exchange of a cluster of embryos had been 
completed, the females were put into individual plastic 
cages with small holes in their sides. (These cages were 
either 1 1 cm in diameter and 1 0.5 cm in height, or 7 cm 
in diameter and 14 cm in height.) Each cage was then 
placed in a beaker containing 10%o clean seawater. The 
time of larval release was monitored with a photoelectric 
device: details of this apparatus have already been de- 
scribed elsewhere (Saigusa. 1992a). 

One of the most important questions in the present 
study was whether the transplanted cluster of embryos 
(Fig. 3A) would successfully hatch, and if so. whether 
this would occur simultaneously with the 7 clusters of 
female-attached eggs. For this purpose, hatching was also 
monitored, not only with the photoelectric apparatus, but 
also by visual inspections, described in detail below (Fig. 
4A. B). 

In intact females of S. haemal ocheir (i.e.. females with- 
out embryo exchange), hatching occurs synchronously, 
possibly within 5-30 min in the laboratory. Eggs were 
frequently found to be wet from the diluted seawater in 
the beaker due to the female's movements within the cage. 
When the hatching started, several zoea larvae were ob- 
served swimming in the beaker (Fig. 4A, middle). As soon 
as hatching was completed, the female released all the 
larvae into the water within 3-5 s (Fig. 4A, right). This 
quick release is associated with an abdominal tanning be- 
havior, which triggers the photoelectric switch. Thus, if 
the seawater in the beaker is frequently checked, an ob- 
vious sign of hatching (i.e.. several swimming zoeas) will 
be noticed about 30 min before the larval release for most 
specimens. Such visual inspections were also applied to 
females with a transplanted cluster of embryos. 



LARVAL HATCHING IN AN ESTUARINE CRAB 



189 



A 




Figure 3. Eggs o( S. haematocheir and their hatching. (A) a cluster of embryos ((7.) the cut base of 
which is tied with a fine thread (.v). os: ovigerous seta. (B) empty egg-cases dr) remaining after larval release 
by the female. (C) very thin membranes dm} protruding from the egg-case upon the liberation of hatched 
larva. This membrane invests the embryo before hatching [described as the "third membrane" by Saigusa 
(1992b). but probably the cephalic portion of the so-called embryonic cuticle]. (D) embryos (cm) dropped 
from ovigerous hairs without hatching. Diameter of each egg. about 330-350 f/m. 



Observations were made every 15-30 min throughout the 
night, using a hand-held light (or head lamp) covered with 
a few sheets of red cellophane. (These red lights were used 
for all of the observations and manipulations carried out 
in the experimental rooms during the dark phase.) When 
several zoeas were found swimming, the beaker was ex- 
amined more frequently, i.e.. at intervals of 5-10 min. 

As the upper diagram of Figure 4B indicates, when the 
photoelectric switch monitoring one of the paired females 
with a transplanted cluster operated (i.e.. the sign of larval 
release), the female was taken out of the cage. The thread 
was cut, and the transplanted cluster was carefully re- 
moved from the female's abdomen. Since empty egg cases 
remain attached to the ovigerous hairs, as they do after a 
normal larval release, it was easy to determine whether 
all of the transplanted embryos had hatched. (The judg- 
ment as to whether all the eggs hatched at the same time 
as the other embryos carried by the female is described 



in the Results section.) This observation was made under 
normal light, outside of the experimental room, because 
a female that had completed larval release was never used 
for further experiments. If hatching had not yet occurred, 
the cluster was quickly transferred into vigorously aerated 
seawater ( 10%o), and examined for the subsequent occur- 
rence of hatching. Some of the aerated clusters were mon- 
itored for hatching every hour in constant darkness (DD) 
or in 24-h light-dark ( LD) conditions; the hatching of other 
clusters was sought during the light phase of the 24-h LD 
cycle. 

Just after the observations and manipulations men- 
tioned above, the other female was also checked to ex- 
amine whether her transplanted cluster had also hatched. 
This was done by observing the water in the beaker under 
red light. As indicated in the middle diagram of Figure 
4B, when hundreds of zoeas were seen swimming in the 
beaker, the transplanted cluster was removed and its 



190 



M. SAIGUSA 



A Larval release in intact females 




B. Larval release in the females ', ;h transplanted clusters 

One fej 




r naicning 

removal of Ihe examination 

"""" transplanted cluster ""*" of hatching 

L* no hatch-* 



removal of the examination 

transplanted cluster """" of hatching 



' 






removal of the 
' transplanted cluster 



examination 
of hatching 



Figure 4. Experimental procedures used to examine hatching in intact 
females and females with transplanted clusters. The female crabs (not 
depicted) are in perforated cages suspended in beakers containing dilute 
seawater (10%). A photoelectric device monitors the seawater for the 
presence of zoeas (see text). (A) The sign of hatching and larval release 
by intact females. Before larval release, the photoelectric switch is "off." 
Upon larval release, the switch operates ("on"): threshold of response: 
10,000-20.000 zoeas in the beaker. (B) Examination of the transplanted 
clusters removed from host females. [ 'pper diagram: Larval release occurs 
in one of the paired females, and the procedures indicated are followed 
to monitor hatching in the transplanted cluster (described in Materials 
and Methods section). Middle diagram: The cluster transplanted to the 
other female has hatched, indicated by hundreds of zoeas swimming in 
the beaker, (observed visually the switch is "off'). The transplanted 
cluster is removed and examined, and the female is reset in the recording 
apparatus. Lower diUKram: No zoeas are seen in the beaker. The female 
is removed from the cage alter the release of her larvae, and the trans- 
planted cluster is then examined. 



hatching was confirmed; a stereo-microscope was used as 
necessary. The beaker was then replaced with another, 
and the female was reset to the recording apparatus. On 
the other hand, as shown in the lower diagram (Fig. 4B). 
there were many cases where no zoeas were seen swim- 
ming. In these cases, when the female finally released lar- 
vae, the transplanted cluster was removed and its hatching 
was examined. 

Notice that the transplanted clusters were examined 
under normal light to determine whether hatching had 
occurred. This was no problem when all of the embryos 
had already hatched. But a question remained in the other 
cases as to whether this light might have affected the timing 
of hatching. To reduce the effect of light, some of the 
transplanted clusters were removed from the female in 
the experimental room under red light and transferred 
into a vigorously aerated medium. The latter experiments 
were carried out in 1991 as specified in the figures. The 
embryo exchange experiments involving 5 1 pairs of fe- 
males were all done in 1990, although the year is not 



identified in the figures. Animals were never used for more 
than one experiment. 

Results 

Ovigerous females with a transplanted cluster released 
their zoea larvae between the night of embryo exchange 
(e.g.. Fig. 5A-a) and 1 1 days after. The release behavior 
was the same as that of intact animals. The results are 
clearly divided into the following three patterns (Pattern 
I. Pattern II. and Pattern III), which are related to the 
number of nights until larval release occurred. 

Pattern I (Fig. 5 A) 

Both females released their larvae within two nights after 
the embryo exchange. The detached eggs of the control 
experiment and the transplanted embryos, all hatched on 
the same night that the donor females released their larvae. 
The results of this data can be further divided into three 
sub-patterns. 

Sub-pattern 1-1 ( 1 pair). As shown in Figure 5A-a, larval 
release of both females (F-l and F-2) occurred on the 
night following embryo exchange. As soon as the release 
of one of the females (F-l ) was recorded, the transplanted 
eggs (cl:F-2) were removed. Almost all eggs remained un- 
hatched, and were quickly transferred to aerated condi- 
tions and monitored. As shown in Figure 5B-a, all of the 
embryos had hatched by about 6:00 on 18 August. On 
the other hand, the larval release of the paired female (F- 
2) occurred 50 min later than F-l (Fig. 5A-a). When the 
implanted cluster (cl:F-l) was removed from F-2, it had 
already hatched. The eggs detached from both females 
(i.e., ae:F-l and ae:F-2) also hatched during the same 
night. 

Sub-pattern 1-2 (6 pairs). Larval release of these females 
occurred on the first and second nights after embryo ex- 
change, respectively (Fig. 5A-b). The egg mass of the con- 
trol experiment (ae:F-3 and ae:F-4) hatched on the same 
night that the donor females released their larvae. The 
transplanted eggs (cl:F-4) were removed from the female 
(F-3) immediately after larval release. No eggs hatched, 
and this cluster was monitored under constant darkness. 
Hatching occurred on the following night (Fig. 5B-b). 
corresponding to the release of the donor female (F-4). 
On the other hand, in the beaker where the female F-4 
was confined, swimming zoeas emerged around the time 
of larval release of female F-3. These larvae had clearly 
hatched from the implanted cluster (cl:F-3). When this 
cluster was removed from the host female F-4, almost 
every embryo had already hatched. The remaining em- 
bryos hatched within a few hours under aerated condi- 
tions. 

Sub-pattern 1-3 ( 1 pair). In only one instance did both 
females release larvae two nights after the embryo 
exchange (Fig. 5A-c). The embryos in the control exper- 



LARVAL HATCHING IN AN ESTUARINE CRAB 



DayO 



Day 1 



Day 2 



191 
72 h 



i 



















F- 1 - 






A 










r o 


Aug. 


7 '! 


-(A)cl:F-2 - 
t 


aeration in darkness 






a 


z 




i 















































F- ^ - 






A 














* 












p / 


Aug i 








^ 




b 








( O )cl' F 


.3 































u 


























Fr- 










t 
















.( ) c 1 F- 6 






F-fi - 


Aug.1 


9 : r~ 






t 




c 


r o 










C ) cl F 


"-5 





















































Figure 5A. Hatching of transplanted clusters: Pattern I (a) Sub-pattern I- 1 F-l and F-2 designate the 
paired females that were set into recording apparatuses. Excision and exchange of embryos occurred at 16: 
55-17:15 on 17 August. Upward black arrows show the time of day of larval release of these females (at 23: 
00 for F-l. and at 23:50 for F-2). cl:F-2 and cl:F-l are the transplanted clusters. ae:F-l and ae:F-2 are the 
control egg masses kept in vigorous aeration. O: Hatching of the egg-cluster or egg mass. A: Partial hatching 
of the egg cluster. (/)) Siih-puttern /-.? Detachment and exchange of eggs. 15:20-15:40 on 4 August. F-3: 
Larval release at 1:00 on 5 August: F-4: at 1:40 on 6 August. <: No eggs hatched from the transplanted 
cluster when it was removed from the host female, (c) Sub-pattern 1-3. Detachment and exchange of eggs: 
12:55-13:10 on 19 August. F-5: Larval release at 23:50 on 20 August: F-fi: at 0:50 on 21 August. : These 
transplanted egg-clusters hatched at the same time as the female-attached eggs. Other symbols as in Fig. 5A- 
a and 5A-b. 



iment (ae:F-5 and ae:F-6) both hatched on the second 
night after the embryo exchange. A feature of this case 
was that hatching of the transplanted cluster (cl:F-6) 
seemed to be synchronized with that of the host female 
(F-5). On the other hand, no swimming zoeas were ob- 
served in the beaker of the female F-6 around the time 
of the release of F-5, although the beaker was checked 
often. Female F-6 released her larvae before the trans- 
planted cluster (cl:F-5) was removed. When this cluster 
was examined, all the egg cases were already empty (Fig. 
3B). Thus, hatching of this transplanted cluster also 
seemed to be synchronized with that of the female-at- 
tached eggs. 

Pattern II (Figs. 6A and 7 A) 

In this pattern, only one of the paired females released 
her larvae within one or two nights after the embryo ex- 
change. The embryos of the control experiment all 
hatched on the same night as the release of the donor 
female. In contrast, the paired females released their larvae 
more than three nights after the embryo exchange. The 
control egg masses taken from these females did not hatch 



at all. A remarkable feature was that the hatching of the 
transplanted cluster was apparently induced by the donor 
female. The results were further divided into the following 
two sub-patterns. 

Suh-pattern II- 1 (II pairs). The first pattern occurred 
when one of the females released larvae on the first night 
after embryo exchange. Five instances are summarized in 
Figure 6A. For example, in Figure 6A-a, larval release of 
F-7 occurred on the night of embryo exchange. The trans- 
planted cluster of embryos (cl:F-8) was removed from the 
female 10 min after the release of F-7, but no eggs had 
yet hatched. This cluster was quickly transferred to aerated 
conditions, and was monitored every hour (Fig. 6B-a). 

The reciprocal cluster (cl:F-7), which had been trans- 
planted to the female F-8. hatched on the same night that 
the donor female (F-7) released her larvae. When the 
beaker of F-8 was observed with the red light shortly after 
the larval release of F-7, hundreds of zoea larvae were 
swimming. The transplanted cluster (cl:F-7) was quickly 
removed from F-8 under red light, and examined. The 
zoea larvae remaining attached to broken egg cases began 
to swim when the cluster was shaken by hand several 
times in the seawater, and hatching was completed within 



192 



M. SAIGUSA 



Dayl 



Day 2 



A8h 




Figure 5B. Hatching of the transplanted egg-clusters. (<;) Hatching 
of the cluster (cl:F-2) removed from the host female (F-l). Downward 
black arrow: time of larval release of the host female (F-l ). (b) Hatching 
of the cluster (cl:F-4) removed from F-3. Open areas in histogram indicate 
the number of premature zoeas (prezoeas) that could not swim and thus 
sank to the bottom of the beaker. 



a few hours. The beaker of F-8 was replaced with another 
one containing 10%o seawater, and larval release of this 
female was monitored. 

The control egg mass (ae:F-8) never hatched even after 
five days (Fig. 6A-a). In contrast, the cluster of embryos 
(cl:F-8) successfully hatched two nights after the start of 
aeration (Fig. 6B-a), and all eggs hatched by noon on 10 
August. Obviously, hatching of the transplanted embryos 
(cl:F-8) was induced by the host female (F-2). 

As four additional cases show (Fig. 6A-b-e), hatching 
of the transplanted clusters (cl:F-10. cl:F-12. cl:F-14, cl: 
F-l 6) was induced by the host females. Moreover, when 
these embryos were transferred into aerated conditions, 
they always hatched two nights after the removal from 
the host female (compare Fig. 6A and 6B). The pattern 
of hatching was similar, whether the clusters were mon- 
itored in constant darkness (Fig. 6B-a-b). or in LD cycles 
(Fig. 6B-c-e). 

Sub-pattern fI-2 (1 pairs). In these cases (Fig. 7A), the 
first larval rei occurred on the second night after the 
embryo exchange. The control egg mass detached from 
these females hatched on the same night. For example 
(Fig. 7A-a), female F-l 7 released her larvae first. The clus- 
ter transplanted to this female (i.e., cl:F-18) was quickly 
removed and examined with the stereo-microscope. No 
hatching had occurred, so this cluster was transferred into 



aerated conditions and monitored. After about 24 h. it 
had completely hatched (Fig. 7B-a). 

At about the time that F-l 7 was releasing her larvae, 
zoeas were observed swimming in the beaker of female 
F-l 8 (Fig. 7A-a). The cluster transplanted to this female 
(cl:F-17) was removed and examined. Most egg cases were 
already empty and the remaining embryos all hatched 
within a few hours in aerated dilute seawater. Female F- 
18 was replaced in 10%o seawater, and monitored until 
the time of larval release (day 3; Fig. 7A-a). 

Other transplanted clusters put into aerated conditions 
(i.e., cl:F-20, cl:F-22, cl:F-24, cl:F-26) also hatched about 
24 h after the larval release of their host females. In the 
first three experiments (Fig. 7A-a-c), the transplanted 
clusters (i.e., cl:F- 1 8, cl:F-20, and cl:F-22) hatched on the 
same night as the larval release of the donor females (i.e.. 
F-l 8, F-20, F-22). But in the other two instances (d and 
e), the donor females (F-24 and F-26) released their larvae 
two or three nights later than their complementary pairs 
(F-23 and F-25). These results further indicate that hatch- 
ing of the transplanted clusters is induced by the host 
female. 

Pattern III (Fig. 8) 

In these experiments (25 pairs), both females released 
their larvae more than three nights after the embryo ex- 
change. In these cases, none of the control egg masses 
ever hatched during the experiment. Five instances of 
hatching by the transplanted clusters are summarized in 
Figure 8. For example, one of the females (F-29) released 
her larvae five nights after the embryo exchange (Fig. 8- 
b). Within a few minutes after the photoelectric switch 
had operated, the transplanted cluster was removed from 
female F-29 and examined resulting in the discovery that 
all the embryos had already hatched (Fig. 3B). The beaker 
containing the paired female (F-30) was frequently 
checked on the night that female F-29 released, but no 
swimming larvae were seen, and larval release occurred 
on the following night. Female F-30 was removed from 
the cage 5 min after the release, and the transplanted clus- 
ter was examined. Again, it was observed that hatching 
was complete. Similar results were obtained from the other 
experiments shown in Figure 8-a and 8-c-e. 

The following evidence suggests that all of the trans- 
planted clusters in Pattern III hatched simultaneously with 
the attached clusters that had been incubated by the donor 
female. ( 1 ) In intact females, a small number of zoea larvae 
begins to swim in the beaker around 20-30 min before 
the larval release. Animals with transplanted embryos ex- 
hibited the same phenomenon. (2) When the transplanted 
cluster of eggs was examined just after the larval release 
of the host female, hatching had already been complete. 
(3) The date of larval release for the complementary 



Day 
_ 24 



LARVAL HATCHING IN AN ESTUARINE CRAB 

1234 



193 



48 



T 



72 






96 



120 



144 h 



P 7 - 




, 4 


cl:F-8 ae 


ration in 


DO 












a 




C" Q 


A 


i 'T < x > 

ug 7 


r o - 

FQ 




hCO) 
ae:F-7. - O 


cl.F-7 
































cl:F-10 ae 


ration in 


DD 






-4 






b 




y - 
Fin 


A 


) 
ug i 

'i ' 


I(J' 
p.i 1 . 




i 
ae:F-9 


(O) 

-~ o 


cl:F-9 










T 




























A 


:l:F-l2 a, 


'ration ir 


LD15.9 


-~ + 






i "" * 




c 




p.i 9. 


A 


ug17 rp^' 

'4 ' 


r i L 
P n 




i 

ae:F-ll 
ae:F-!2 


r <O) 
O 


:I:F-11 






T 






f) cl: 


r -1A - aerat 


on in 1C 


15:9 






-A. 






d 




r i j 

P 1Z. 


A 


jg 17 !!~~~ 

n 


P-lt;. 




i ^ 
ae:F13 


0) cl: 

O 


r -13 










T 






















' ' \ 


:F-16 aera 


lion in L 


3159 










-A 


e 




r i o 

p ic. 


A 


ug.7 I. ft*' C 




a 
a 


1 

?:F-I5 
- F-16 


(0) C 

o 


:F-15 














T 


_ 























Figure 6A. Induction of hatching in the transplanted cluster of embryos: Suit-pattern II-I (a) Embryo 
exchange between females F-7 and F-8. Detachment and exchange of clusters: 19:20-19:40 on 7 August. 
Larval release: F-7. at 1:00 on 8 August; F-8. at 2:00 on 10 August. (/>) Embryo exchange between F-9 and 
F-10. Detachment and exchange of clusters: 15:50-16:20 on 2 August. Larval release: F-9. at 1:50 on 3 
August: F-10. at 23:45 on 5 August, (c) Embryo exchange between F-l I and F-12 at 17:50-18:10 on 17 
August. Larval release: F- 1 1 , at 1:00 on 18 August; F-12, at 1:25 on 20 August, (d) Embryo exchange between 
F-l 3 and F-l 4 at 16:10-16:30 on 17 August. Larval release: F-l 3. at 21:45 on 17 August; F-l 4. at 23:40 on 
20 August, (c) Embryo exchange between F-15 and F-16 at 11:40-12:00 on 17 August. Larval release: 
F-l 5, at 23:40 on 17 August: F-16. at 22:40 on 21 August. The transplanted clusters (cl:F-7. cl:F-9, cl:F-l 1, 
cl:F-13. cl:F-15) all hatched on the same night as the larval release of the donor females. Symbols are the 
same as in Fig. 5A. 



females was usually different. If these females carried their 
eggs for three nights or more after the embryo exchange, 
no swimming larvae appeared until the host female re- 
leased her larvae. (4) When the transplanted cluster was 
examined with the stereo-microscope, a very thin mem- 
brane that invests the embryo had emerged from the egg 
case (Fig. 3C). Emergence of this membrane always ac- 
companies hatching in this species (Saigusa, 1992b). so 
its appearance signifies that larvae had just emerged. 
Eventually this membrane is lost, and the inside of the 
empty egg cases are contaminated with fine detritus. 

To prove that the transplanted clusters hatched si- 
multaneously with the clusters attached to the host fe- 



male, it was necessary to make direct observations of 
the female releasing larvae. Such observations were only 
meaningful if the females could be sampled within 30 
min after the larval release was monitored in the event 
recorder (44 females). Shortly after larval release, the 
females unfold their abdomens and begin eating the 
empty egg cases that remain attached to the ovigerous 
hairs. Thus, six females that were examined more than 
30 min after release had already eaten most of the empty 
egg cases, or the cases had dropped to the bottom of 
the beaker, so the time of hatching of either the trans- 
planted or attached clusters could not be determined. 
In Pattern III, there were only three instances in which 



194 



M. SAIGUSA 



t OOO-i 



500- 




Figure 6B. Hatching ol'the transplanted egg-clusters in vigorous aer- 
ation. In the upper two panels (a and h), the cluster was monitored in 
constant darkness. In the lower three panels (c. d, and e), hatching was 
recorded in 24-h light-dark cycles. Note that the embryos hatch around 
48 h after the larval release of the host females (downward black arrows). 
The horizontal arrow in panel h indicates that the egg masses dropped 
from cluster cl:F-10 during aeration, so the hatching was not fully mon- 
itored. 



a portion of the transplanted cluster did not hatch, but 
remained attached to ovigerous hairs. These clusters were 
shaken by hand several times in 10%o seawater to remove 
adherent zoeas, and were quickly transferred to contin- 
uously dark (DD) conditions and monitored under aerated 
conditions. These eggs all hatched with peak hatching oc- 
curring about 24 h later (not illustrated). 



Loss of the <',, >n%e hy the incubating female 

Figure 9A incJK m example of egg loss, a curious 
phenomenon not usu seen in intact females, either in 
the laboratory or the nek'. In this experiment, one of the 
paired females (F-37) released her larvae on the night of 
embryo exchange. The egg cluster transplanted to this 



female (cl:F-38) was quickly removed and transferred into 
an aerated medium where it hatched about 48 h later. 
However, the reciprocally transplanted cluster (cl:F-37) 
was not removed quickly, therefore the egg sponge of the 
other female (F-38) dropped from her abdomen during 
the daytime of 2 August. At the time they were dropped, 
the embryos were still alive (Fig. 3D). 

Thus, if a transplanted cluster hatched and was not 
removed soon thereafter, the host female's own attached 
eggs often dropped within a few days; indeed, 7 out of 52 
females dropped all of their eggs without hatching. This 
phenomenon appears to be due to a substance released 
outside of the egg membrane and associated with hatching 
(Saigusa, submitted). Nevertheless, as shown in Figure 
9A, the control egg mass (i.e., ae:F-38) never hatched. So 
we can presume that female F-38 would have released 
larvae more than three nights after the embryo exchange. 
These results clearly belong to Pattern II- 1. Eggs were lost 
only by females of Pattern I and Pattern II, and never in 
females of Pattern III. This is indirect evidence that, in 
Pattern III, transplanted embryos hatch on the same night 
as do the female-attached eggs. 

Influence of the light used in monitoring on the hatching 
of the transplanted clusters 

Hatching of transplanted embryos can be induced, if 
they are incubated by a host female that releases her larvae 
within two nights (Figs. 6B, 7B). Indeed, all of the trans- 
planted clusters were removed from the host female under 
red light in the experimental chamber. But the determi- 
nation of hatching was carried out under the normal light 
outside of the experimental room, although the time re- 
quired for this observation was only 5 min. Hence, we 
must question the influence of this light on the hatching 
of these embryos. 

To address this issue, the hatching profile of egg clusters 
exposed to normal light was compared with the profile of 
those exposed to red light. In this experiment, the trans- 
planted cluster was removed from the host female just 
after larval release, and was kept under the light for 5 min. 
This cluster was then transferred into aerated conditions 
and was monitored every hour. In two other experiments, 
the transplanted clusters were also quickly removed from 
the host females just after larval release, but they were 
not exposed to the light outside of the experimental room. 
Instead they were tied to nylon thread under red light and 
monitored for hatching. 

The hatching profiles of the cluster treated with normal 
light showed that hatching occurred two nights after the 
transfer into aeration (data not shown). The other two 
clusters, which had been treated with red light, also 
hatched two nights after the aeration. Furthermore, a small 
peak of hatching was also observed on the third night for 



LARVAL HATCHING IN AN ESTUARINE CRAB 



195 



DayO 



21* 

~T~ 



48 



72 

r~ 



96 
r~ 



120 

i 



144 h 

I 



F-17 










4 


. 

)cl:F-!8 a 


^ration ir 


00 








a 




p. iQ . 


A 


ug.7 ! 

1 










F-1Q - 




ae;F-17 


/ /-, 


)ct:F-17 










J ^ 


















:l:F-20-*ae 


ration in 


DD 








b 




r iy 

F-7D- 


Ju 


' :,:, 


1.22 ,r^ 

. 








F-71- 


ae 
ae 


1 




cl:F-19 












F "'O 


. . 
























A 


.r- 11 aeratic 


n ^^ 










c 




F-97- 


Jul.20 I,' 










F ' 22 LD15:9 ' 


F-7T- 






:F-21 














1 






















A 


r 

F JL aerat ' c 


>n -j^ 




4 






d 




F-7A- 


J 




j!20 








LD15:9 w 


F.9R- 






;F-23 








ae - F ?? 










] 






















A 

(x)cl:F 


- oc aeratio 


n 










e 




r zo 

F 9C 


J 


( 


ji.2o ; r 






LD15:9 


1 /D 






F-25 





































Figure 7A. Induction of hatching in the transplanted cluster of embryos: Suh-ptitlern 1 1-2 (a) Embryo 
exchange between F-17 and F-18 at 19:45-20:00 on 7 August. Larval release: F-17, at 4:00 on 9 August; 
F-18, at 2:40 on 10 August. (/>) Embryo exchange between F-19 and F-20 at 1 1:30-12:00 on 22 July. Larval 
release: F-19. at 2:30 on 24 July; F-20, at 1:30 on 25 July, (c 1 ) Embryo exchange between F-21 and F-22 at 
18:30-19:00 on 20 July. Larval release: F-21. at 0:10 on 22 July; F-22. at 23:20 on 22 July, (d) Embryo 
exchange between F-23 and F-24 at 16:30-17:00 on 20 July. Larval release: F-23, at 0:20 on 22 July; F-24, 
at 1:10 on 24 July, (e) Embryo exchange between F-25 and F-26 at 17:10-17:40 on 20 July. Larval release; 
F-25, at 22:30 on 2 1 July; F-26, at 23:50 on 24 July. For hatching of egg-clusters (cl:F-18, cl:F-20) after they 
were removed, see Figure 7B-a and 7B-b. The other transplanted clusters (cl:F-22, cl:F-24, cl:F-26) hatched 
during one night later (hourly data not obtained). 



one of these clusters. Such a secondary peak of hatching 
was often observed in other experiments treated with nor- 
mal light (e.g.. Fig. 6B-e), so it cannot be attributed to the 
influence of red light (data not shown). 

Some experiments related to the induction of hatching 
by the female 

In two instances, the implanted egg-cluster did not 
hatch at all. Although egg loss occurred in both experi- 
ments, these results clearly belong to Pattern II- 1 (see Fig. 
9B for the result of one of these experiments). A feature 
common to these two experiments is that the interval be- 
tween the embryo exchange and the larval release of the 



host female was very short. The transplanted clusters were 
incubated by the host females for 4 h and 4.5 h, respec- 
tively. In every case of induced hatching, the minimum 
period was 5.5 h (e.g.. Fig. 6A-d). These results suggest 
that at least 5-6 hours are required to induce hatching of 
the transplanted cluster. 

The possibility that some stimulus of hatching in the 
female-attached eggs induced the hatching of transplanted 
clusters was also examined. For this purpose, the trans- 
planted cluster was removed from the host female some 
hours before the larval release (Fig. 9C). In this experi- 
ment, the clusters were incubated by the host females for 
about 1 7 h, and were then transferred into aeration. The 
cluster (cl:F-4 1 ) hatched on the same night that the donor 



196 



M SAIGUSA 



Day 1 



Day 2 



0,2000- 
ns 



ulOOO- 

'r5 

-C 



0. 
.D 

1 1000 



500- 



cl:F-18 
Aug 8 



cl:F-20 
JuL23 




I- inure 7B. Distribution of hatching in the transplanted embryos in 
vigorous aeration: (a) cl:F-18; (/>) cl:F-20. The release of larvae by the 
host females (F-17, F-19) is shown by a downward arrow. (Egg masses 
dropped from the cluster at the time indicated by the horizontal arrow, 
and hatching could not be perfectly monitored during this period.) The 
other transplanted clusters from Figure 7A (cl:F-22. cl:F-24, cl:F-26) 
hatched one night later (hourly data not obtained). 



female (F-41 ) released their larvae. The cluster (cl:F-42) 
also hatched two nights after the aeration. Since no hatch- 
ing was observed in the control experiment (ae:F-42), 
hatching of cl:F-42 can be regarded as having been induced 
by the host female (F-41). 

The possibility that egg-clusters kept under aerated 
conditions for more than three nights after detachment 
may lose their ability to hatch was then tested. In Figure 
9D, a cluster (cl:F-43) was detached from a female and 
placed in aeration for five days. This cluster was then 
transplanted to another female (F-44). This cluster 
hatched in synchrony with the attached eggs of the host 
female F-44. Similar results were obtained in another ex- 
periment (not illustrated). Thus, eggs that were detached 
and transferred into aeration retained their ability to hatch. 
Hatching was obviously inhibited under aerated condi- 
tions. 

The interaction between the female and transplanted 
embryos was also examined. A few clusters were covered 
with a skirt made of thin cellophane. This skirt was open 
at the bottom, ensuring an interchange of water at the 
surface of the eggs. As seen in Figure 10, the transplanted 
clusters were induced to hatch (upper left in each panel). 
But the hatching profiles of the egg-clusters that were 
transferred into aeration (Fig. 10) showed a somewhat 
different pattern. In these three experiments, the cluster 
hatched in two peaks, about 24 h apart. Almost all of the 
eggs hatched in one experiment (Fig. 10-a), but many eggs 



failed to hatch in the other two (Fig. 10-b-c). These results 
are difficult to interpret; one possible explanation is that 
the stimuli recognized by the embryos are attenuated by 
the cellophane, which caused the occurrence of two peaks 
and the decrease of the number of larvae hatched. In any 
event, such a splitting of the hatching pattern has never 
been observed in intact females. 

Does the hatching of transplanted clusters affect the 
day or the time of hatching of eggs attached to the host 
female? For example, the hatching of the transplanted 
cluster (Figs. 5A, 6A) might release a stimulus that acts 
on the female to disturb the time of hatching or to advance 
the day of hatching of the female-attached eggs. To ex- 
amine the former possibility, the time of day of larval 
release by host females (except the females whose eggs 
had dropped) was monitored with the event recorder. As 
shown in Figure 11, the larval release of these females 
coincided roughly with the time of night high water in 
the field, showing a clear circa-tidal rhythm. This suggests 
that at least the daily timing of egg hatching was not dis- 
turbed, either by the exchange of embryos or by the 
hatching of the transplanted eggs. 

To examine whether the hatching of transplanted egg- 
clusters advances the date of hatching in the host female, 
the number of nights between the larval release of paired 
females was compared with respect to differences between 
Pattern II and Pattern III The range was 0-9 days in 
Pattern III (25 pairs), but only 0-4 days in Pattern II ( 19 
pairs with no egg loss). Since the experimental crabs were 
chosen randomly, this difference might suggest that 
hatching of the transplanted cluster can advance the day 
of hatching in the host female. 

Discussion 

The embryos of most marine crustaceans are incubated 
by the female for a certain period before hatching. An 
endogenous factor has been suggested as operating in the 
hatching rhythms of many kinds of marine animals (Sai- 
gusa, 1992c). But does the endogenous component con- 
trolling rhythmicity occur within the embryo, its mother, 
or both? To answer this question, larval hatching must 
be examined, not only in the embryo, but also in the 
female. Although the embryos of crustaceans are attached 
to non-plumose setae by a funiculus that is possibly com- 
posed of chorion, there is no circulation of blood between 
embryo and mother (Yonge, 1937, 1946; Cheung, 1966; 
Goudeau and Lachaise, 1983). Thus, the embryo exchange 
experiments were aimed at revealing the site of the en- 
dogenous clock. 

An endogenous clock times hatching in each embryo 

The present experiments were primarily aimed at de- 
termining whether the implanted embryos would hatch 



LARVAL HATCHING IN AN ESTUARINE CRAB 



197 



Day 
24 h 



10 



p. 97 






( 
( 


cl:F-< 
cl:F- 


!8 
17 


























a 


r L 1 

p. OQ 


A 


ug.5 , 












r zo 
F-7Q- 


a 
a 


[ 




e F-27* 




















































































. 




s 

() 


1 
cl:F-30 

A 


K,:F. 


25 














b 


r /a 
F-^O- 


A 




jg.3,; | 
















F-T1 - 


as. 
an. 


1 : 






r 70. 
























F-30 . 














































































' ^ 


)cl:F-: 


2 




4 


k 

)cl:F 


31 








c 

> 






c 


r j i 
F-"}?- 


k 




ii.22 :r 
















p.-jo. 


a< 

a 


i- 




































> F-32J 






































































) 


1 








) 


cl:F-3 


3 














d 




A 




jg.ljf 










p.oc. 


a< 
a< 


1 






, 
































































x 














































, . 4 


)cl:F-36 




















X 


e 
:l:F-35 


p.oc. 


Jc 


" t . i /5 


i.23,r 
















r jb 


ae 
at 


i 


























-n^ 






































F-lfi*_ 





















































































Figure 8. Induction of hatching in the transplanted cluster of embryos; Pallcrn III (<;) Embryo exchange 
between females F-27 and F-28. Detachment and exchange of clusters: 19:20-19:40 on 5 August. Larval 
release: F-27, at 0:30 on 8 August; F-28. at 0:40 on 8 August, (b) Embryo exchange between F-29 and F-30 
at 14:45- 1 5: 10 on 3 August. Larval release: F-29. at 0:35 on 8 August; F-30, at 3:05 on 9 August, (c) Embryo 
exchange between F-31 and F-32 at 16:30-17:00 on 22 July. Larval release: F-31. at 2:10 on 26 July; F-32, 
at 3:20 on 28 July. (</) Embryo exchange between F-33 and F-34 at 16:25-16:45 on 1 August. Larval release: 
F-33, at 23:35 on 3 August; F-34, at 23:50 on 6 August, (el Embryo exchange between F-35 and F-36 at 
14:15-14:40 on 23 July. Larval release: F-35, at 3:40 on 27 July; F-36, at 22:40 on I August. Symbols the 
same as in Figure 5A. 



synchronously with the attached embryos of the host fe- 
male. A potential procedural problem remaining is that 
the transplanted embryos were exposed to light, although 
for only 5 min, to determine whether hatching had oc- 
curred. Hatching could have been induced by the direct 
influence of this light, e.g., like the oviposition of the te- 
leost Oryiias (Egami, 1954; Ueda and Oishi, 1982). To 
reduce the effect of light, transplanted clusters were re- 
moved from the female, and subsequent procedures in- 
volving aeration were carried out under red light. The 
hatching pattern under red light was similar to that of an 
egg-cluster exposed to normal light. Furthermore, the peak 



of hatching occurred about 24 or 48 h after the trans- 
planted egg-clusters had been transferred into aeration, 
even in constant darkness (Figs. 5B-b, 6B-a-b, 7B-a-b). 
These peaks must have been induced by an endogenous 
rhythm existed in embryo itself, and not caused by the 
light used during examination of hatching or by 24-h LD 
cycles. 

Like hatching, pupation, and emergence, many aspects 
of development occur only once in the life cycle of an 
animal (Saunders, 1976). Whether the timing of those 
phenomena is controlled by an endogenous pacemaker 
can be examined in a population of mixed age e.g.. the 



198 



F-37 



F-: 

F-40 



F-41 



.' 



48 



M. SAIGUSA 
72 



96 



120 



144 h 



- 1 1 




1 ' 






A 


- ifl 


aeration 


n LD159 


. n 
















Aug.! 




b. n 


SS 

jici 


Of 

ly) 


>gg spong 


n 




ae:F-37 
ae F-38 


-iO)ct: 
-0 


F-37 
not removed c 


















1 






(x)cl:F 


jin 




c 


le rat ion 


n LD15.9 








h. Y 




jut. 20 'r 








1 






> of egg 


sponge 




i * 
ae:F-39 - 


(O)cl:F 
-O 


"39( no ' remov 














































A 


I 

at ion 
















J 


f 




aer 


ul.29 I~ 
1990 ; 


cl:F-4; 


) ; 












* 


on 










cl:F-4 


| aerat 





































B 



24 h 



F-43 



























h 

--43 


















) 




: -:'[ 


) 


Jut. 27 






















cl:F 


(19? 


1} 




aer 


atk 


)n 








i 


ae:F-43 

1 




















til 




























in 


:ubat 


on 





















































Figure 9. Hatching profile of transplanted clusters. (A ) Loss ol the egg sponge by the host female. Embryo 
exchange between females F-37 and F-38 at 16:10-16:25 on 1 August. Larval release: F-37. at 23:35 on I 
August. (B) Failure to induce hatching. Embryo exchange between females F-39 and F-40 at 17:50-18:20 
on 20 July. Larval release: F-39, at 22:30 on 20 July. (O Removal of the transplanted cluster before the 
release of the host female. Embryo exchange between females F-41 and F-42 at 16:05-16:25 on 29 July. 
Larval release: F-41. at 3:45 on 31 July; F-42, at 1:35 on 1 August. (D) Hatching of the egg-cluster kept in 
aeration for five days. Detachment of cl:F-43, at 19:50 on 27 July: binding to F-44, at 16:20 on 1 August. 
Larval release: F-43, at 4:55 on 2 August; F-44, at 23:10 on 6 August. 



circadian rhythm of emergence in the fly Drosophilu (Pit- 
tendrigh and Bruce, 1959). But the validity of using such 
a population to demonstrate a circadian rhythm has been 
questioned (Saunders. 1976, chapter 3). So Pittendrigh 
and Skopik (1970) used populations that were develop- 
mentally synchronous at pupation to study the emergence 
rhythm in the fly Drosophila pseudoobuscura, and sug- 
gested that a circadian pacemaker in each developing fly 
dictates the circadian time of emergence, but not that of 
the intermediate developmental stages, such as head ever- 
sion and eye pigmentation. 

The eggs of St'tumia luicmatochcir are oviposited 
within a short time. So the developmental embryos in- 
cubated by a female clearly do not constitute a mixed- 
age population. Like the data presented by Pittendrigh 



and Skopik (1970), the hatching of the egg clusters re- 
moved from the host females was often split into two 
distinct peaks almost 24 h apart (Figs. 6b-e, lOa-c). To 
explain such a splitting of hatching pattern, we can assume 
an allowed zone: (see Pittendrigh and Skopik, 1970) related 
to hatching in the endogenous pacemaker of each embryo. 
In Figure 12, this zone is expressed by the acrophase 
(shown by a dot) in the embryo's pacemaker. The pre- 
ceding paper (Saigusa, 1 992c) indicated that each embryo 
undergoes a hatching process that continues for 48-49.5 
h prior to egg-membrane breakage. If the embryos were 
detached from the female early enough that this time in- 
terval were exceeded, hatching would not occur. One 
speculation is that, in the larval hatching rhythm in Se- 
sanna. a gated phenomenon occurs at the start of the 



LARVAL HATCHING IN AN ESTUARINE CRAB 



199 



Day 1 



Day 2 



Day 3 



Day 4 



96 h 



600 n 




Figure 10. Hatching of egg-clusters from which the cellophane skirt was removed before transfer into 
aeration, (a) Hatching of cl:F-46. Embryo exchange between F-45 and F-46 at 17:25-17:40 on 9 August. 
Larval release: F-45. at 23:45 on 10 August; F-46. at 23:50 on 12 August. (/) Hatching of cl:F-48. Embryo 
exchange between F-47 and F-48 at 14:25-14:35 on 9 August. Lar\al release: F-47. at 1:30 on 1 1 August: 
F-48. at 1:50 on 15 August, d) Hatching of cl:F-50. Embryo exchange between F-49 and F-50 at 19:10-19: 
20 on 9 August. Larval release: F-49. at 0:20 on 10 August: F-50. at 2:20 on 15 August. Downward arrows 
indicate time of dav of release bv the host females. 



hatching process, and not hatching itself. If the hatching 
process is not initiated at a certain acrophase (Fig. 12). 
the embryos must wait until the next allowed zone to start 
the process. 

Induction of hatching in the transplanted cluster 

While each embryo has an endogenous rhythm of 
hatching, the present study indicates that the hatching of 
exchanged clusters is induced by the host female, provided 
that the incubation was longer than 5-6 h (compare Figs. 
6A, 7A and 9B). Since the start of the hatching process 
may be a gated event, it is reasonable to speculate that 
this process begins 48-49.5 h before the hatching, re- 
sponding to some signal to each embryo. But we do not 
know what stimuli trigger the hatching process in each 
embryo. One possibility is mechanical stimuli generated 
by the female perhaps some special movements of the 
abdomen or ovigerous setae as the embryonic develop- 
ment is completed. Another possibility is a hatch-inducing 



substance, produced by the female and recognized by the 
embryos. We also do not know when hatch-inducing 
stimuli are released from the female. Females could gen- 
erate such stimuli at any time of day, or at a particular 
phase of her circatidal rhythm (see the question mark on 
the female pacemaker in Fig. 12). 

Synchronization of hatching between transplanted 
embryos and female-attached embryos 

For most intertidal and estuarine crustaceans, female- 
attached eggs hatch within a very short period, although 
the exact duration cannot be determined because of the 
mass. In S. haematocheir, hatching is completed in 5-30 
min in each female (Saigusa, 1992b). Because the hatching 
synchrony of the embryos detached from the female is 
perturbed (Saigusa. 1992c), some mechanism must un- 
derlie the highly-synchronous hatching in female-attached 
eggs. This cannot be an endogenous clock in each embryo; 



200 



M. SAIGUSA 



' 



18 



Time of day 
It, 



12 



1990 



Jul.20- 



25- 

e 

30- 
Aug.l - 



5- 



O 



10- 



15 



20 




D a 







t 

55 



t 
SR 



Figure 11. Time of day of larval release by females carrying an exchanged cluster and by females from 
which the cluster had been removed. Records were made in the laboratory under 24-h LD cycle, but under 
no tidal influence. Solid diagonal lines connect the times of high water (//III in the field. D: larval release 
on the first night of embryo exchange. A: larval release on the second night after embryo exchange. : larval 
release occurs more than three nights after the embryo exchange, .v.v and AT connect the times of sunset and 
sunnse, respectively. 



the female must produce some unknown stimulus that 
enhances synchronous hatching. 

Females of S. haematocheir release their larvae with 
vigorous abdominal movements. This same behavior 
is observed in other terrestrial crabs (Saigusa, 198 1 ). In 
species that release their larvae under the water, the 
release is effected by the pumping behavior of the ab- 
domen (DeCoursey, 1979; Forward ct ai. 1982: Saigusa, 
1992a). Forward and Lohmann (1983) suggested that 
this behavior enhances hatching synchrony. In contrast. 



hatching in terrestrial species occurs prior to larval re- 
lease. Clearly, larval release behavior itself does not en- 
hance the hatching synchrony in female-attached em- 
bryos. One possible mechanism is that the female 
kneads the egg-clusters several times around the time 
of night high tide. In addition to such physical stimuli, 
the hatching of a few embryos might release a substance 
like the hatching enzyme suggested by De Vries and 
Forward (1991), and thus stimulate the hatching of the 
remaining embryos. 



LARVAL HATCHING IN AN ESTUARINE CRAB 



201 



Tide 
Female 

Embryo 



Female-attached eggs 



hatching process 
*>() 



Detached eggs 



detachment 



detachment 



-o 

- x 



Figure 12. Proposed mechanism of induction of hatching and syn- 
chronization of hatching with nocturnal high water. Endogenous pace- 
makers related to hatching are shown with by a sine curve (female) and 
a rectangle (embryo). Small circle above the female's pacemaker indicates 
time of nocturnal high water. Stippled area: stimuli that induce the 
hatching process in each embryo are released during this period. The 
heavy downward arrow represents stimuli by the female to enhance 
hatching synchrony among embryos. The success of hatching in detached 
embrvos is shown under the horizontal line. See the text for details. 



Timing mechanism of hatching: a hypothesis (Fig. 12) 

As in most other decapods, the oviposited eggs of S. 
haematocheir are incubated by the female until hatching 
occurs. Most of this period is probably related to embry- 
onic development. But hatching does not immediately 
follow the completion of development; i.e.. embryos wait 
for stimuli that initiate the hatching process. So when the 
eggs are detached from the female during this period, 
hatching should be inhibited. As demonstrated in this 
study, one or more hatch-inducing stimuli are produced 
by the female. But once these signals have been received, 
the start of the hatching process would be determined by 
an endogenous clock within each embryo. 

We can assume that a self-sustained oscillation under- 
lies most endogenous rhythms (Pittendrigh and Bruce, 
1 959). As shown in Figure 1 2, we can express the embryo's 
pacemaker for hatching as a rectangular wave with 24.5- 
h period. Similarly, females would also have 24.5-h pace- 
maker for hatching and larval release. Both pacemakers 
would be synchronized with nocturnal high tide in the 
field. Since eggs that are detached from the female more 
than 48-49.5 h before hatching of the female-attached 
eggs do not hatch, the "allowed zone" related to the start 
of this process should be positioned at the phase corre- 
sponding to the time of nocturnal high tide. i.e.. the ac- 
rophase of the embryo's pacemaker in Figure 12. When 
some (unknown) stimuli (stippled area in Fig. 12) have 
been transmitted to the embryos for several hours (at least 
more than 5-6 h), the hatching process starts at this phase, 
and zoeas hatch 48-49.5 h later; i.e.. around the time of 
nocturnal high tide. If the stimuli from the female are 



insufficient to start the hatching process, embryos must 
wait for the next acrophase. This would have resulted in 
the split hatching peaks seen in vigorously aerated dilute 
seawater (Figs. 6B-e and 10 a-c). 

As shown in the lower diagram of Figure 12. if the 
embryos are detached from a female before the hatching 
process, they would not hatch at all. If they are separated 
from a female while the hatching process is in progress, 
then those embryos will hatch at about the same time as 
the embryos that remain attached to the female. But 
hatching synchrony is perturbed in this condition, so the 
process is extended by several hours (Saigusa, 1992c). 
Since female-attached eggs hatch synchronously, the fe- 
males must have some mechanism (a downward arrow 
in Fig. 12) for enhancing hatching synchrony while they 
are still on the hillside awaiting the time of hatching. 

Acknowledgments 

The zoea larvae found after hatching were counted by 
Mr. A. Shiomi and Miss H. Yunoki, students of Okayama 
University. They also helped with some of the daytime 
procedures, such as exchanging clusters, placing females 
in the recording apparatuses, and collecting ovigerous fe- 
males. Supported by a Grant-in-Aid for Scientific Re- 
search (C) (No. 02640582) from the Ministry of Educa- 
tion, Science and Culture. 

Literature Cited 

Cheung, T. S. 1966. The development of egg-membranes and egg at- 
tachment in the shore crab. Curcinus maenas. and some related 
decapods. ,/ Mar Biol Assoc. t'.A'. 46: 373-400. 

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

DeCoursey, P. J. 1983. Biological timing. Pp. 107-162 in The Biology 
of Crustacea I'll: Behavior and Ecology. F. J. Vernberg and W. B. 
Vernberg, eds. Academic Press. New York. 

De \ries, M. C., and R. B. Forward, .Ir. 1991. Mechanisms of crus- 
tacean egg hatching: evidence for enzyme release by crab embryos. 
Mar. Biol. 110: 281-291. 

F^gami, IV 1954. Effect of artificial photoperiodicity on time of ovi- 
position in the fish. Ory:iax lali/vs Annot. Zoo/. Jpn. 27: 57-62. 

Forward, R. B., Jr.. K. Lohmann, and I. \V. Cronin. 1982. Rhythms 
in larval release by an estuarine crab (Rhithroponopeus harrisii). Biol. 
Bull. 163: 287-300. 

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

Goudeau. M., and F. Lachaise. 1983. Structure of the egg funiculus 
and deposition of embryonic envelopes in a crab. Tissue Cell 15: 
47-62. 

korringa, P. 1947. Relations between the moon and periodicity in the 
breeding of marine animals. Ecol. Monogr 17: 347-381. 

Minis, D. H., and C. S. Pittendrigh. 1968. Circadian oscillation con- 
trolling hatching: its ontogeny during embryogenesis of a moth. Sci- 
ence 159: 534-536. 

Pearse, J. S. 1990. Lunar reproductive rhythms in marine invertebrates: 
maximizing fertilization? Pp. 31 1-316 in Advances in Invertebrate 



202 



M. SAIGUSA 



Reproduction 5. M. Hoshi and O. Yamashita. eds. Elsevier Science 

Publishers B. V. 
Pittendrigh, C. S., and V. G. Bruce. 1959. Daily rhythms as coupled 

oscillator systems and their relation to thermoperiodism and pho- 

toperiodism. Pp. 475-505 in Photoperiodism and Related Phenomena 

in flams ami Animals. R. B. Withrow. ed. American Association for 

the Advancement of Science, Washington. DC. 
Pittendrigh, C. S.. and S. D. Skopik. 1970. Circadian systems, V The 

driving oscillation and the temporal sequence of development. Proc. 

Nali Acad. Sci. U.S.A. 65: 500-507. 
Pittendrigh, C. S., and D. H. Minis. 1971. The photopenodic time 

measurement in Pectinophora gossypiella and its relation to the cir- 

cadian system in that species. Pp. 212-250 in Biochronometry, M. 

Menaker, ed. National Academy of Sciences, Washington. 
Saigusa, M. 1981. Adaptive significance of a semilunar rhythm in the 

terrestrial crab Sesarma Biol. Hull 160: 31 1-321. 
Saigusa, M. 1992a. Phase shift of a tidal rhythm by light-dark cycles 

in the semi-terrestrial crab Sesarma pictum. Biol Bull 182: 257- 

264. 



Saigusa, M. 1992b. Observations on egg hatching in the estuanne crab 

Sesarma haematocheir Pin Sci 46: 484-494. 
Saigusa, M. 1992o. Control of hatching in an estuarine terrestrial crab. 

I. Hatching of embryos detached from the female and emergence of 

mature larvae. Biol. Bull. 183: 401-408. 
Sastry, A. N. 1983. Pelagic larval ecology and development. Pp. 2 1 3- 

282 in The Biology of Crustacea, I'ol. VII: Behavior and Ecology, 

F. J. Vernberg and W. B. Vernberg. eds. Academic Press, New York. 
Saundcrs, C. S. 1976. Insect Clock\. Pergamon Press. Oxford. 279 pp. 
I'eda, M., and T. Oishi. 1982. Circadian oviposition rhythm and lo- 

comotor activity in the medaka. Ory:ias talipes. J Interdiscipl. Cycle 

Re\. 13: 97-104. 
Yonge, C. M. 1937. The nature and significance of the membranes 

surrounding the developing eggs of Homarus vitlgaris and other De- 

capoda. Proc. Zool Soc Lund.. Ser A. 107: 499-517 (plus 1 plate 

page). 
Yonge, C. M. 19-46. Permeability and properties of the membranes 

surrounding the developing egg of Homarus vulgaris. J. Mar. Biol. 

Assoc. U.K. 26: 432-438. 



Reference: Biol. Bull 184: 203-208. (April, 



Asymmetry in Male Fiddler Crabs is Related to the 
Basic Pattern of Claw-waving Display 

SATOSHI TAKEDA 1 AND MINORU MURAI 2 

^Marine Biological Station. To/iokn University. Asamushi, Aomori 039-34. Japan and 2 Department 
of Biology. Faculty of Science, Kyiislni University, Hakoiaki. Fukitoka SI J, Japan 



Abstract. Morphological asymmetry was correlated with 
the pattern of claw-waving display in males from five spe- 
cies of fiddler crabs: three vertical wavers ( Uca unillei. 
U. dussumieri, U. vocans), a lateral waver ( U. annulipes), 
and an intermediate waver (U. tetragonon). On the first, 
second and third ambulatory legs of male lateral waver 
crabs, the distance between the inner edge of the basis 
and the outer edge of the merus was larger on the side 
bearing the major cheliped than it was on the side with 
the minor cheliped. A similar asymmetry was observed 
in male intermediate waver crabs, but only the first am- 
bulatory leg was involved. This morphological asymmetry 
is clearly related to the style of waving adopted by these 
crabs. When lateral wavers display, the weight of the major 
cheliped (which forms about one-third of the total body 
weight) is carried largely by the anterior ambulatory legs 
on the same side of the body, but the imbalance of weight 
during display is less in the intermediate waver. In the 
vertical waver crab horizontal motion of the major che- 
liped occurs relatively rarely; thus there is hardly any ad- 
ditional load on the ambulatory legs, which showed no 
asymmetry. 

However, the total length of the five sterna bearing tho- 
racic legs tended to be larger on vertical waver males than 
on the female crabs. Thus the sterna of male crabs bulge 
outwards more than those of female crabs, and the angle 
between the sternum bearing the cheliped and the ground 
surface is larger in male crabs than in females. This may 
be an adaption enabling the cheliped of the male to be 
raised higher during the waving display. 

Introduction 

A characteristic feature of the genus Uca (Ocypodidae; 
Brachyura) is hypertrophy of one of the male chelipeds. 

Received 5 November 1992; accepted 25 January 1993. 



resulting in a striking asymmetry (Crane, 1975). The 
hypertrophic, or major, cheliped plays a very important 
role in antagonistic and courtship behaviors (Crane, 
1957. 1975). especially as a distinctive indicator of the 
male sex during the breeding season (Salmon and Stout. 
1962). 

The mechanism determining which of the chelipeds 
becomes hypertrophic has been examined in some species 
(Morgan, 1923, 1924; Yamaguchi, 1977; Ahmed, 1978), 
and the development of the asymmetry has been analyzed 
by monitoring the growth rate of the major cheliped rel- 
ative to that of the carapace (Huxley and Callow, 1933; 
Tazelaar, 1933; Miller, 1973). In addition, the first and 
second ambulatory legs are longer on the side bearing the 
major cheliped than they are on the contralateral side in 
male U. pugilator (Yerkes, 1901; Duncker, 1903; Huxley 
and Callow, 1933; Miller, 1973), and in U. pugna.\ 
(Yerkes, 1901; Tazelaar, 1933) in North America. This 
asymmetry of the ambulatory legs was thought to help in 
raising the major cheliped higher, thus displaying it to 
more crabs (Miller, 1973). 

Crane ( 1957, 1975) divided Uca into two groups based 
on the male's claw-waving display; i.e., into vertical and 
lateral waving species. U. pugilator and U. pugnax, with 
asymmetry of the ambulatory legs as well as the chelipeds, 
form the lateral waving group (Crane, 1957, 1975). On 
the other hand, the ambulatory legs of male crabs of the 
vertical waving group have not yet been examined for 
possible asymmetries. 

In this study, the degree of asymmetry of certain 
morphological characters, including the length of 
the ambulatory legs, was determined and compared 
among five species of Ucu with different patterns 
of claw-waving display. Three of the five species were 
vertical wavers, one species was a lateral waver, and 
one exhibited an intermediate type of waving dis- 
play. 



203 



204 



S. TAKEDA AND M. MURAI 



Materials and Methods 

Crabs of five species. ('. urvillei, U. dussumieri, U. vo- 
cans, U. tetragonon and U. annulipes, were collected on 
the seashores in Thailand (Table I). Larger individuals 
were selected for examination, since the degree of asym- 
metry in male fiddler crabs increases with body growth 
(Miller, 1973). Crabs lacking thoracic legs or those with 
degenerate, atypical thoracic legs were excluded. 

U. urvillei, U. dussumieri and U. vocans are vertical 
wavers, U. annulipes is a lateral waver, and U. tetragonon 
is an intermediate (Crane, 1957, 1975). The proportion 
of right-handed and left-handed male crabs was nearly 
equal in I', urvillei. U. dussumieri and U. annulipes. and 
almost all male U. vocans and L'. tetragonon are right- 
handed (Takeda and Yamaguchi, 1973; Frith and Frith. 
1977). 

Two indices of body-size were measured: carapace 
width (Fig. 1 A); and the whole body wet-weight. The wet 
weight of the cheliped cut off between ischium and basis 
was also measured. Several morphological measurements 
were made (Fig. 1 ). These measurements on the right and 
left side of each individual include: three dimensions on 
the minor cheliped or ambulatory leg (Fig. 1 B); body depth 
(Fig. 1 A); carapace depth (Fig. 1 A); and the length of each 
sternum bearing thoracic legs (Fig. 1C). 

These measured values were normalized with respect 
to the wet-weight or carapace width. To determine the 
degree of asymmetry, the ratio of the values on the major 
cheliped side to the values on the opposite side in male 
crabs, and the ratio of the values on the right side to those 
on the left side in female crabs, were calculated for each 
individual. These data were examined for significance with 
Student's Mest. 

Results 

The carapace width and body wet-weight of individuals 
differed among the five species of Uca (Table II). 



The weight of the major cheliped relative to the whole 
body wet-weight increased in the following order: L'. te- 
tragonon ^ U. dussumieri ^ U. iinillei ^ U. annulipes 
^ U. vocans (Table III). The relative weight did not differ 
widely among the species of vertical, intermediate, and 
lateral wavers. The relative weight of the minor cheliped 
did not differ significantly (P > 0.05) among individuals 
of the same sex in four species, but U. tetragonon had a 
larger minor cheliped than the other four species (P 
< 0.05). 

The relative dimensions of the minor chelipeds were 
larger in female crabs than in male crabs in each species 
(P < 0.05). The smaller values in male crabs resulted from 
their larger whole body wet-weight, which included the 
weight of the major cheliped. 

The chelipeds of male crabs had a remarkable asym- 
metry (P < 0.001 ). and the ratio of major cheliped weight 
to minor cheliped weight increased in the order of U. 
tetragonon < L'. annulipes 2= U. dussumieri ^ L'. wvillei 
S= U. vocans. The smaller ratio in male crabs of U. tetra- 
gonon (P < 0.05) was caused by their having heavier minor 
chelipeds than the other four species. However, the in- 
tensity of asymmetry did not differ among the species 
with the different display patterns. In female crabs of all 
five species, the asymmetry ratio was near 1.0, which in- 
dicates the chelipeds were the same size. 

Considering the total length of the ambulatory legs, 
which was calculated by adding the lengths of the I, II 
and Ill-sections of each ambulatory leg (Fig. IB), asym- 
metry was perceptible (P < 0.05) only in first (ratio 
= 1 .04 0.02 (mean the 95% confidence interval)) and 
second ambulatory legs (ratio = 1.02 0.01) of male U. 
annulipes (Fig. 2). and the ambulatory legs of the side 
bearing the major cheliped were longer than those of the 
opposite side. Moreover, for each section of the ambu- 
latory legs, asymmetry was present in the I-section of the 
first, second and third ambulatory legs of male U. 



Table I 



Materials, display form and handedness in Uca xpp. 



Species 



1 750' N; 9824' E 
- 1328'N; IOO55'E 
3 744' N; 9825' E 



Display 



Handedness 



Specimens 



Location 



Date 



L'. urvillei 


vertical 


random 


6 males 


Ao Nam Bor 1 


1990. Sep. 








7 females 






L ' dussumieri 


vertical 


random 


9 males 


Smare Kaow 2 


1987. Oct. 








9 females 






L vocans 


vertical 


right 


8 males 


Ao Nam Bor 1 


1990. Sep. 








6 females 






U. leli'iixonon 


intermediate 


right 


1 1 males 


Ao Tang Khen' 


1990. Sep. 








9 females 






U. annulipes 


lateral 


random 


10 males 


Ao Nam Bor 1 


1990. Sep. 








6 females 


Ao Tang Khen 3 


1991. Jan. 



ASYMMETRY IN MALE FIDDLER CRABS 



205 






Figure 1. Diagrams of frontal view of carapace (A), ventral surface of left ambulatory leg (B), ventral 
view of male crab (C). and dimensions measured in this study. (1) carapace width: the minimum distance 
between both tips of the anterolateral angles. (2) body depth: the minimum distance between the carapace 
and a straight line in contact with the plane of the sterna with the first and second ambulatory legs. (3) 
carapace depth: the minimum distance between the tip of one anterolateral angle and the edge of the buccal 
region on the side of the Milne-Edwards opening. (4) I-section: the minimum distance between the inner 
edge of the basis and the outer edge of the merus of the thoracic legs. (5) II-section: the minimum distance 
between the inner edge of the carpus and the outer tip of the propodus ot the thoracic legs. (6) Ill-section: 
the minimum distance between the inner edge and the tip of the dactylus of the ambulatory legs. (7), (8). 
(9). ( 10). and ( 1 1 ) the length of each sternum with cheliped, first, second, third and fourth ambulator, leg, 
respectively. 



anmdipes (P < 0.001), and in the I-section of the first 
ambulatory legs only of male V. tetrugonon (P < 0.01). 
The asymmetry ratios of U. annulipes male crabs were 
1 .09 0.0 1 , 1 .06 0.02 and 1 .05 0.02, and the degree 
of asymmetry decreased posteriorly. The ratio for male 
U. tetragonon was 1 .02 0.0 1 . 

Total lengths of the minor cheliped and the first am- 
bulatory leg on minor cheliped side tended to be greater 
in males than in females, and the difference was significant 
(P < 0.05) in V. dussumieri, L'. letragonon and U. an- 
nulipes for the minor chelipeds. and in U. dussumieri and 
U. letragonon for the first ambulatory legs (Fig. 2). In 
addition, the same tendency (P > 0.05) was observed for 
the total length of the second ambulatory leg in the four 
species other than U. annulipes. The total length of the 



third ambulatory legs did not differ between male and 
female crabs. The total length of the fourth ambulatory 
leg was greater in female U. letragonon than in male crabs 
(P < 0.05). 

The body depths of male crabs were larger on the major 
cheliped side than on the other side (P < 0.001) (Table 
IV). The asymmetry ratios were between 1.05 and 1.07 
for all species, indicating no difference between the species. 
The body depths of female crabs of all five species were 
symmetrical (P < 0.05). 

The relative body depths on the major cheliped side of 
male crabs were larger than those of female crabs (P 
< 0.05). The relative body depth on the side bearing the 
minor cheliped in male crabs showed a similar tendency, 
especially in U. dussumieri and U. tetragonon (P < 0.05). 



Table II 

Carapace width and wet weight in Uca spp. 



Carapace width (mm) 


Wet weight (g) 


Species 


Male 


Female 


Male 


Female 


C itn'illei 


25.19 1.43 
(23.40-26.75) 


23.07 1.49 
(20.85-25.35) 


5.030 0.709 
(4.115-6.169) 


2.989 0.604 
(2.309-4.136) 


1 '"' """' 


29.80 1.18 
(27.65-32.85) 


23.31 1.03 
(21.70-24.95) 


9.273 1.161 
(7.371-12.553) 


3.490 0.508 
(2.680-4.646) 


(.' >WUn .v 


23.05 0.63 
(22.15-24.55) 


17.17 0.66 
(16.15-18.05) 


5.503 + 0.298 
(4.865-5.930) 


1.487 0.294 
(1.191-1.955) 


I' letragonon 


2 1.00 0.87 
(19.40-23.35) 


21.03 0.93 
(19.10-22.95) 


3.754 0.563 
(2.956-5.465) 


3.090 0.339 
(2.333-3.605) 


U annulipe\ 


15.49 0.79 
(14.35-17.50) 


11.98 + 0.60 
(10.95-12.50) 


1.3 19 0.229 
(0.978-1.910) 


0.437 0.048 
(0.355-0.495) 



Mean the 95% confidence interval. 
Numbers in parentheses indicate the range. 



Table III 

Weight of chelipeds relative to whole body wet-weight in Uca spp. 



Male crab 



Female crab 



Species Major side Minor side Right side Left side 



V. urvillei 0.366 0.047 

(31.48 7.15)' 
U. dussumieri 0.358 0.015 

(31.07 2.12)* 
U. means 0.432 0.034 

(40.18 4.35)* 
T tetragonon 0.340 0.013 

(17.58 1.13)* 
U. annulipes 0:380 0.032 

(28.41 4.41)* 



0.012 0.001 0.016 0.001 0.016 0.001 

(1.02 0.04) 
0.012 0.001 0.015 0.001 0.015 0.001 

(1.01 0.07) 
0.011 0.000 0.015 0.002 0.016 0.001 

(0.98 0.04) 
0.0 1 9 0.00 1 0.022 0.00 1 0.022 0.00 1 

(1.01 0.02) 
0.014 0.001 0.018 0.001 0.018 0.001 

(1.00 0.00) 



* Student's (-test, P < 0.00 1 . 

Mean the 95% confidence interval. 

Numbers in parentheses (mean the 95%' confidence interval) indicate the 
asymmetry ratio based on weight (male crab = major cheliped side/minor cheliped 
side: female crab = right side/left side). 



206 



S. TAKEDA AND M. MURAI 



Uca dussumien 



Chetiped 




Uca tetragonon 



Uca annulipes 



1st ambulatory leg 
majo 
mino 
right 



5 major 



minor 
igh 
left 



2nd ambulatory leg 

5 major 
minor 
? right 
left 



3rd ambulatory leg 

S major 
minor 
? right 
left 

4th ambulatory leg 
* major 
O minor 



nghi 

left 



ILJ 



05 10 15 05 10 1.5 









:-M-;->; , 












:::: 




1 






1 


:-'.-:-:-:.: 
































j 




























:-:-:-: 


















:-:-:-:-: : : 












3 


5 l.< 


) 



05 10 15 











!- 


1 ab 




























L ;-', 


ab 








::.-:-:-:- 












-:-; :-x : 




a 


























::-:-:- 












. 
















i 





5 


15 



Length relative to carapace width 

Figure 2. Length of the three sections of the thoracic leg relative to carapace width in L'ca spp. Each 
bar shows the relative length of the I-, II- and Ill-section (Fig. IB) from the left. (a), (b): asymmetry in the 
length of I-section of the thoracic leg and in the total length of the thoracic leg, respectively. 



The carapace depths of crabs was symmetrical (P 
< 0.05) in both sexes of the four species other than in 
male U. vocans (Table V). 

In the male crabs of all species, the sternum bearing 
the major cheliped was larger than that bearing the minor 
cheliped (P < 0.001 ), and the asymmetry ratios were be- 
tween 1.16 and 1 . 1 9 ( Fig. 3 ). The asymmetry is probably 
due to the hypertrophy of the coxa of the major cheliped, 
as indicated by the remarkable development of the pro- 
podus of the major cheliped (see Fig. 1C). In the male 



crabs of all species, the total length of the five sterna with 
thoracic legs was greater on the major cheliped side than 
it was on the opposite side, showing significant asymmetry 
(P<0.05). 

Male crabs of U. itnil/ei. I', dussitmieri and U. vocans 
had larger major cheliped-bearing sterna than did female 
crabs of the same species (P < 0.05). But, there was no 
significant difference between male and female crabs of 
U. tetragonon and U. annulipes (P > 0.05). 

The total length of the five sterna on the minor cheliped 



Table IV 

Length ot body depth relative to carapace width in Uca spp. 



Male crab 



Female crab 



Species 



Major side Minor side Right side 



U.un'illei 0.53 0.01 

(1.06 0.01)* 
U iliissiiiiiten 0.58 0.01 



0.50 0.01 



('. I 



( tfiragmum 0.56 0.01 
(1.05 0.00)* 

U. annulipes 0.55 0.01 
(1.05 0.01)* 



Left side 



0.48 + 0.01 0.48 0.01 
(1.00 0.01) 

0.55 0.01 0.52 0.01 0.52 0.01 
(1.07 0.01)* (1.00 0.01) 

0.56 0.01 
1 .07 0.01) 



0.53 0.01 0.49 0.02 0.49 0.03 

(1.00 0.01) 
0.54 0.01 0.51 0.01 0.51 0.01 

(1.00 0.00) 
0.52 + 0.02 0.52 + 0.01 

(1.00 0.02) 



* Student's /-test. P< 0.001. 

Mean the 95% confidence interval. 

Numbers in parentheses (mean the 95^ confidence interval) indicate the 
asymmetry ratio (male crab = major cheliped side/minor cheliped side; female 
crab = right side/left side). 



Table V 

Length of carapace depth relative to carapace width in Uca spp. 



Male crab 


Female crab 


Species 


Major side 


Minor side 


Right side 


Left side 


U. itrvillci 


0.34 


+ 


0.01 


0.34 


0.01 


0.32 + 


0.01 


0.33 


+ 0.01 




(1.02 


< 


0.02) 






(1.00 


0.01) 








I' lllUUllfllCI'l 


0.33 


. 


0.01 


0.33 


0.01 


0.32 


0.01 


0. 


32 


0.01 




(1.01 


f 


0.01) 






(1.00 + 


0.01) 








U. vocans 


0.31 





0.01 


0.32 


0.01 


0.31 


0.01 


0. 


31 


0.01 




(0.98 





0.0 1)* 






(0.99 


0.01) 








I' iclraxtinon 


0.3 1 


. 


0.00 


0.32 


0.00 


0.30 


0.00 


0. 


31 


0.00 




( 1 .00 





0.0 1 ) 






(1.00 


0.00) 








L' ttnnulipes 


0.33 


- 


0.00 


0.33 


0.00 


0.32 


0.01 


0. 


32 


0.00 




( 1 .00 


1 


0.01) 






(1.00 + 


0.01 









* Student's /-test, P<0.05. 

Mean the 95% confidence interval. 

Numbers in parentheses (mean the 95% confidence interval) indicate the 
asymmetry ratio (male crab = major cheliped side/minor cheliped side; female 
crab = right side/left side). 



ASYMMETRY IN MALE FIDDLER CRABS 



207 





r i 

Uca urvillei 

a c d 
^^;JA......-..V^:.V^ I ' -. , ,>, i ','[-------------; v-v. .....'..'] 1 


minor 


- :::^ -^ r'-''r' - 1 


right 


4 r . " . : 


* left 


H1M1SS ; ( I '. 




Uca dussumieri 
a c d 


* major 


wmmmm ".'- - ,'\ .----.. v:-:-. ":": .::! i 


O minor 


. . x :-::::-:::; : : : : : )T ,, ; ,.;- j - 1 -- . v . .-... ;, ; 


r '9 hl 


-:-: \ ^ ' ^plx-ftffffi "i 


S major 


b 
Uca vocans 
a d 

mmmmw, 1 ' -. '-",-t K/.\V. -..-..v.-.-i 


minor 
g right 


.if :.-;-* >. ' ^my'i ' 


* left 


rr~v:;'""4 ,i i, ...,...:: 




(yea tetragonon 
a d 


* major 


.1 . :. .:- .1 i 


O minor 


r ~ j .. E ."'.... -.-v.i i 


r '9 nt 
left 


. . .'.....-.-i-.-.-.-.- T -rt-ij i ..'-.i ' >.*. = : '. .**. . ;'.."; *. .' ;'..*; '. .' ; 




Uca annulipes 
a c d 

J.:-.::-.-.-.::-.-.-.-.-J.'.'.'l ' ' T I. ... ... ... II 1 


minor 


, /.^ -'...'-.-'** i^.^.1- 1 J 


r '9 h t 


:.::". ^- -f |--v^v^ | 




i i I 



025 0-50 

Length relative to carapace width 



0.75 



Figure 3. Length of each sternum bearing thoracic legs relative to 
carapace width in L'ca spp. Each bar shows the relative length of the 
sternum bearing (from left to right) the cheliped, first, second, third and 
fourth ambulatory leg (Fig. 1C), (a), (b), (c). and (d) indicate asymmetry 
in the length of the sternum bearing the cheliped. first and second am- 
bulatory leg, and in the total length of five sterna with thoracic legs, 
respectively. 



side of male U. dmsitmieri and U. vocans tended to be 
larger than those of female crabs, and a similar, though 
weaker, tendency was observed in U. un'illei. The differ- 
ences between the male and female crabs of U. dussumieri 
and U. vocans were caused by the male crabs having more 
extensive sterna bearing the cheliped and first ambulatory 
leg. The slight difference in the total length of the sterna 
of male and female U. un'illei was due to the smaller 
degree of enlargement of the sternum with the first am- 
bulatory leg in male U. un'illei, compared with that of 
male U. dussumieri and U. vocans. On the other hand, 
female U. tetragonon and U. annulipes tended to have 
slightly longer sterna than the male crabs. This was be- 
cause the sternum bearing the third ambulatory leg is re- 
markably larger in female crabs than in males, although 
the male crabs tended to have a larger sternum bearing 
the first ambulatory leg than did the female crabs. 

Discussion 

Male crabs of U. annulipes, which display with lateral 
waving, had longer first and second ambulatory legs on 



the major cheliped side than on the other side (Fig. 2), 
corresponding with the asymmetry reported for the lateral 
waver males of U. pitgilator ( Yerkes, 1 90 1 ; Duncker, 1 903; 
Huxley and Callow, 1933; Miller, 1973) and U. put>na.\ 
(Yerkes, 1 90 1 ; Tazelaar, 1933). Moreover, comparing the 
major and minor cheliped sides, the I-sections of the first, 
second and third ambulatory legs were longer on the major 
cheliped side, resulting in asymmetry of total lengths of, 
especially, the first and second ambulatory legs. Male U. 
tetragonon have a form of display intermediate between 
the lateral and vertical waving types, and the I-section of 
their first ambulatory leg showed only asymmetry similar 
to that of the male U. annulipes. Asymmetry of the am- 
bulatory legs was not seen in U. un'illei, U. dussumieri 
and U. vocans males, which display in the vertical form, 
or in females of any of the five species. 

The sequence of the display performed by U. annulipes 
male crabs is as follows: the major cheliped, normally 
flexed in front of the buccal region, is extended laterally 
and subsequently raised, then flexed and brought down 
to the starting position from above the eyes and buccal 
region (Crane. 1957, 1975). The major cheliped consti- 
tutes about one-third of the whole body weight (Table 
III), and during such a display the proportion of its weight 
supported by each pair of ambulatory legs will change 
according to the angular position of the major cheliped. 
That is, while the flexed major cheliped is moving toward 
the side, its weight is borne on the anterior ambulatory 
legs and then on the ambulatory legs on the same side as 
the major cheliped. When the major cheliped is fully ex- 
tended laterally, it achieves its maximum loading weight. 
Subsequently, during the raising of the unflexed major 
cheliped and its return to the starting position, the weight 
carried on the side of the body bearing the major cheliped 
will decrease gradually. The degree of asymmetry in the 
I-section of the ambulatory legs was greatest for the first 
ambulatory leg, and decreased gradually until no asym- 
metry was apparent for the fourth ambulatory legs (Fig. 
2). Thus, the distances between the dactylus tips of each 
pair of ambulatory legs are greater anteriorly, especially 
on the side bearing the major cheliped, since waving males 
hold the I-section horizontally rather than vertically 
(Crane, 1975, Fig. 92). In addition, the distance between 
the dactylus tips of the first and fourth ambulatory legs is 
greater on the major cheliped side than on the other side, 
since the ambulatory legs of both sides were extended in 
the antero-posterior direction during display. The in- 
creased horizontal distance on the major cheliped side 
may be a morphological adaptation to bearing most of 
the weight of the major cheliped as it moves laterally from 
the anterior position during display. 

On the other hand, in the sequence of the display of 
male U. iirvi/lei, U. dussumieri and U. vocans, the major 
cheliped initially remains flexed in front of the buccal 
region, and is moved up and slightly forward, without 



208 



S. TAKEDA AND M. MURAI 



unflexing (Crane, 1957, 1975). In such a display, the hor- 
izontal component of the motion of the major cheliped 
will be very small in both the antero-posterior direction 
and laterally, compared with the display of the lateral 
wavers. Therefore, the crab does not have to bear an ad- 
ditional load on the side of the major cheliped during 
waving. Vertical wavers, therefore, do not have an elon- 
gated I-section of the ambulatory legs on the major che- 
liped side. 

The display performed by male U. tetragonon is inter- 
mediate between the lateral and vertical waving types 
(Crane, 1957, 1975). That is, the major cheliped, flexed 
in front of the buccal region, is incompletely extended 
forward at an acute angle. Subsequently, the semi-flexed 
cheliped is raised so that its tip barely reaches to the eye. 
In such a display, the crab does not have to bear a load 
on the ambulatory legs of the major cheliped side, as the 
lateral wavers do. 

The asymmetry in the total length of the ambulatory 
legs or in the length of each section of the ambulatory leg 
was not seen in U. vocans, in which right-handed males 
are predominant (Takeda and Yamaguchi, 1973), or in 
male U. unillei and U. dussumieri in which the ratio of 
left and right handedness is similar (Fig. 2). These facts 
suggest that the asymmetry of the ambulatory legs cor- 
responds with differences in the form of display, rather 
than with differences in the mechanism inducing hyper- 
trophy of one of the chelipeds, whether innate or random. 

The ratio of body depth to carapace width was greater 
on the side bearing the major cheliped than it was on the 
opposite side, in male crabs of all species (Table IV). 
However, the relative carapace depth, which is considered 
to be related directly to the body depth, was the same on 
the two sides of the body (Table V). On the other hand, 
the length of the sternum bearing cheliped was much 
greater on the major cheliped side than it was on the minor 
cheliped side, resulting in the asymmetry of the body 
depth. Because the body depth was measured as the min- 
imum distance between the carapace and a straight line 
in contact with the planes of the sterna bearing the first 
and second ambulatory legs (Fig. 1A), the excessive in- 
crease in the body depth on the major cheliped side, re- 
sulting from the excessive enlargement of the sternum 
bearing the major cheliped, means that the plane of the 
sternum with the major cheliped was inclined more ver- 
tically than was that of the opposing sternum. This more 
vertical sternum position probably contributes to the 
smoother motion of the major cheliped in a vertical di- 
rection. 

The total length of the five sterna on the side with the 
minor cheliped in male U. urvillei, U. dussumieri and U. 



vocans tended to be greater than that of the female crabs 
(Fig. 3). But, there was no such sexual dimorphism in U. 
tetragonon and U. anmdipes. These results indicate that 
the sterna on the minor cheliped side of males extend 
further laterally, like those on the major cheliped side, 
and that the plane of the sternum bearing the minor che- 
liped becomes more vertical than is the case in females 
of the vertically waving species. 

Acknowledgments 

We thank the Phuket Marine Biological Center and the 
National Research Council of Thailand for providing fa- 
cilities for this research in Thailand. We thank Dr. M. 
Matsumasa, Department of Biology, School of Liberal 
Arts and Sciences, Iwate Medical University and Mr. T. 
Koga and Mr. T. Kosuge, Faculty of Science, Kyushu 
University, for their field assistance. This study was par- 
tially supported by Grants-in-Aid for International Sci- 
entific Research for the Japanese Ministry of Education, 
Science and Culture (Nos. 62042019 and 01041069). 

Literature Cited 

Ahmed, M. 1978. Development of asymmetry in the fiddler crab Vca 

ciumdanla Crane. 1943 (Decapoda, Brachyura). Cntstaceana 34: 294- 

300. 
Crane, J. 1957. Basic patterns of display in fiddler crabs (Ocypodidae. 

Genus Uca). Zoologica 42: 69-82. 
Crane, J. 1975. Fiddler Crabs of the World. Ocypodidae: Genus Uca. 

Princeton University Press, New Zealand. 736 pp. 
Duncker, V. G. 1903. Uber Asymmetric bei Gelasimus pugilator Latr. 

Biiimctnka 2: 307-320. 
Frith, D. W., and C. B. Frith. 1977. Observations on fiddler crabs 

(Ocypodidae: Genus Uca) on Surin Island, western peninsular Thai- 
land, with particular reference to Uca letragonon (Herbst). Phuket 

Mar. Bwl Center Res. Bull, 18: 1-14. 
Huxley, J. S., and F. S. Callow. 1933. A note on the asymmetry of 

male fiddler-crabs (Uca pugilator). W RouxArch. Entwicklungsmech. 

129: 379-392. 
Miller, D. C. 1973. Growth in Uca. I. Ontogeny of asymmetry in Uca 

pugilator ( Bosc ) ( Decapoda, Ocypodidae ). Crusiaceana 24: 119-131. 
Morgan, T. H. 1923. The development of asymmetry in the fiddler 

crab. Am. Nat. 57: 269-273. 
Morgan, T. H. 1924. The artificial induction of symmetncal claws in 

male fiddler crabs. Am. Nat. 58: 289-295. 
Salmon, M., and J. F. Stout. 1962. Sexual discrimination and sound 

production in Uca pugilator Bosc. Zoological!: 15-21. 
Takeda, M., and T. Yamaguchi. 1973. Occurrences of abnormal males 

in a fiddler crab Uca marionis ( Desmarest), with notes on asymmetry 

of chelipeds. Pmc. Jpn. Soc. Syst. Zool. 9: 13-20. 
Tazelaar, M. A. 1933. A study of relative growth in Uca ptignax. W. 

RI>IL\ Arch. Entwicklungsmech. 129:393-401. 
Yamaguchi, T. 1977. Studies on the handedness of the fiddler crab, 

Uca lactea. Biol. Bull. 152: 424-436. 
Yerkes, R. M. 1901. A study of variation in the fiddler crab Gelasimus 

pugilator Latr. Pmc. Am. Acad. Arts Sci. 36: 4 1 7-442. 



Reference: Biol. Bull 184: 209-215. (April. 1993) 



Studies of Intracellular pH Regulation in Cardiac 

Myocytes From the Marine Bivalve Mollusk, 

Mercenaria campechiensis 

W. ROSS ELLINGTON 

Department of Biological Science, B-157, Florida State University. Tallahassee, Florida 32306 



Abstract. Myocytes were isolated from the ventricle of 
the marine clam Mercenaria campechiensis by enzymatic 
dispersion procedures. Intracellular pH (pH,) was mea- 
sured via fluorescence imaging techniques using an in- 
verted microscope interfaced with a high sensitivity tele- 
vision camera. Myocyte pH, was similar to values observed 
in other molluscan muscles measured by weak acid dis- 
tribution and nuclear magnetic resonance (NMR) tech- 
niques. Myocytes displayed a good capacity for defending 
pH, against changes in extracellular pH (pH e ) as the pH, 
remained unchanged in the pH c range of 7.1 to 8.0, but 
gradually declined at lower pH e values. Myocytes had a 
relatively high non-bicarbonate intracellular buffering ca- 
pacity. Further, these cells showed recovery from imposed 
acid loads. This recovery was accelerated by increasing 
HCOj concentrations, was not dependent on external 
Na + and was blocked by a stilbene transport inhibitor, 
suggesting that a HCO 3 ~:Cr transporter plays a central 
role in regulation of pH,. Collectively, these data show 
that ventricular myocytes of M. campechiensis have a rel- 
atively high capacity for dealing with potential metabolic 
proton loads associated with environmental anaerobiosis. 

Introduction 

Environmental hypoxia or anoxia imposes important 
energetic and acid/base stresses on marine invertebrates. 
When anaerobic energy yielding processes prevail, there 
appears to be an uncoupling of proton production and 
consumption. The extent of excess production of protons 
is dependent on the specific pathways operating (Portner 
el ai, 1984a; Portner, 1987a, 1989). The major evolu- 
tionary trajectory in highly anoxia-tolerant marine in- 
Received 13 November 1992; accepted 25 January 1993. 



vertebrates (bivalve/gastropod mollusks and certain worm 
groups) is the development and use of anaerobic metabolic 
pathways with a lower H + /ATP ratio, the ratio of proton 
release to ATP produced (Gnaiger, 1980). 

Because proton production will continue throughout 
anoxia, it is readily apparent that specific mechanisms are 
present in these organisms to minimize reductions in in- 
tracellular pH (pH,). Rates of intracellular acidification 
during anoxia (or air exposure) are generally quite low in 
the muscles of bivalves (Barrow el ai, 1980; Ellington, 
1983a; Walsh el ai. 1984) and gastropods (Ellington. 
1983b; Graham and Ellington, 1985). This is also true of 
the sipunculid Sipunatlm midiis (Portner el ai, 1984b; 
Portner, 1987b). Muscles of many of these species have 
moderately high non-bicarbonate intracellular buffering 
capacities (/C?NB) (Eberlee and Storey, 1984; Morris and 
Baldwin, 1984; Portner el ai. 1984a; Wiseman and El- 
lington, 1989). Furthermore, there is good evidence for 
ion exchange of acid/base equivalents between the intra- 
and extracellular compartments. For instance, in S. nudus 
Portner and coworkers (Portner el ai, 1984b; Portner, 
1987b) have shown that the extracellular compartment 
serves as sink during anoxia for metabolically produced 
protons. This is also true of bivalves where the calcareous 
shell serves as an external buffering agent (Crenshaw and 
Neff, 1969; Booth el ai, 1984). In terms of ion exchange 
processes in marine invertebrates, a sodium-dependent 
Cr-HCO} exchanger appears to be the predominant ef- 
fector of regulation of pH, in two well-studied systems 
squid giant axon (Boron and Russell, 1983) and the giant 
muscle fibers of barnacles (Boron el ai, 1979). These ex- 
changers are blocked by stilbene derivatives and have low 
Kms for HCO 3 ~ (around 2-3 mA/). Recently, it has been 
shown that bivalve anterior byssus retractor muscle (ABRM) 
has a stilbene-sensitive anion exchanger (Zange el ai. 1990). 



209 



210 



VV R ELLINGTON 



In the present study, regulation of pH, has been inves- 
tigated in myocytes isolated from the ventricle of the ma- 
rine bivalve Merccnaria campechiensis. This study uses 
fluorescent ratio imaging technology, which permits the 
observation of the dynamics of change in pH, in individual 
myocytes. Experiments focus on the measurement of J NB 
and observation of defense of pH, after exposure of cells 
to acid/base stress. 

Materials and Methods 

Animals and materials 

Specimens of M. campechiensis were collected via a 
dredge by a commercial fisherman from St. Joseph's Bay, 
Gulf County, Florida, and were transported to the Florida 
State University Marine Laboratory within a few hours 
after collection. Animals were maintained in raw (unfil- 
tered, unsettled), continuously flowing seawater. Prior to 
experiments, animals were transported to the main uni- 
versity campus and maintained in recirculating aquaria 
20 1.5Cundera 12:12 (L:D) photoperiod. 

Dispersion enzymes and buffers were purchased from 
Sigma Chemical Co. (St. Louis, Missouri). Nigericin (free 
acid). 2',7'-bi-(2-carboxyethyl)-5-(and -6)-carboxyfluores- 
cein-acetoxymethyl ester (BCECF/AM) and BCECF (free 
acid) were purchased from Molecular Probes (Eugene, 
Oregon). The anion transport inhibitor, 4-acetamido-4'- 
isothiocyanatostilbene-2, 2'-disulfonic acid (SITS), was 
obtained from Sigma Chemical Co. All other chemicals 
were of reagent grade quality. 

Isolation of myocytes 

Procedures for myocyte dispersion were adapted from 
suggestions made by C. Bruce (Department of Pharma- 
cology, University of British Columbia, Vancouver, BC). 
Ventricles were dissected from 3 to 5 specimens of A/. 
campechiensis. After removal of the intestine, tissue was 
cut into very small pieces (1 mm 3 ), suspended in 45 ml 
myocyte artificial seawater (MASW, 440 mA/ NaCl, 10 
mA/KCl, 7.5 mA/CaC! 2 , 23 mA/MgCl 2 , 25 mA/MgSO., 
and 10 mA/ HEPES adjusted to pH 7.75 with NaOH), 
and gently washed in a rotary shaker for 1 h. The sus- 
pension was placed in a 50 ml conical centrifuge tube and 
the tissue pieces were allowed to settle by gravity for a 
few minutes. After aspirating off the MASW, tissue was 
resuspended in 20 ml 0.1% protease VIII (Sigma) in 
MASW and incubated with gentle agitation for 30 min. 
The tissue was again placed in a 50 ml centrifuge tube 
followed by 30 ml MASW. After settling of the tissue, the 
MASW was aspirated off and 50 ml MASW added. After 
settling and aspiration, the tissue pieces were resuspended 
in 20 ml 0.1% collagenase (type 2, Sigma) and incubated 
with agitation for 90 min. Periodically during incubation, 
tissue was gently sucked in and out of a flame-polished 



Pasteur pipette. After incubation, the cell suspension was 
centrifuged for 45 s at low speed (400 rpm) in a clinical 
centrifuge. The supernatant was carefully decanted with- 
out disturbing the loose pellet and centrifuged for 4 min 
as above. The supernatant was discarded and the pellet 
resuspended in MASW and centrifuged for 4 min. The 
final pellets were resuspended in a small volume of 
MASW. Cells were seeded on circular coverslips (Ni- 
cholson Precision Instruments, Gaithersburg, Maryland) 
which were immersed in 15 ml MASW in 10 cm plastic 
culture dishes. Coverslips had been previously washed in 
acid, rinsed exhaustively and then polished with ethanol 
using lens paper. Dishes were placed in a humidified cul- 
ture chamber. Cells were always prepared during the af- 
ternoon and then used for imaging the following morning. 
All isolation and incubation procedures were conducted 
at 18-21C. 

Fluorescent ratio imaging 

The overall rationale and approach for BCECF imaging 
has been previously described (Rink el a/.. 1982; Bright 
et ai. 1987). Cells were loaded with 5 pM BCECF/AM 
for 45 min. After washing, the coverslip was mounted in 
a Dorvak-Stottler chamber (Nicholson Precision Instru- 
ments) which was attached to a Peltier device (Physitemp. 
Clifton, New Jersey) mounted on the stage of a Zeiss IM- 
35 inverted microscope. Cells were superfused (0. 1 
ml/min) by gravity flow from a manifold device consisting 
of a Hamilton (Reno, Nevada) eight-way valve, microbore 
tubing and eight reservoir chambers. Temperature was 
controlled at 20C. 

A xenon lamp (Optiquip, Highland Mills, New York) 
provided illumination. Fluorescence excitation was con- 
trolled via a dual filter wheel/shutter assembly (Ludl, 
Hawthorne, New York). One wheel contained excitation 
filters (490, 450 nm) while the other had a range of neutral 
density filters. A dichroic (530 nm) was positioned on the 
fluorescence emission side. All filters were from Omega 
Optical (Brattleboro. Vermont). Phase and fluorescence 
images were obtained using an Achrostigmat LD 32X/.4 
PHI objective with light passing onto an iCCD camera 
(QUANTEX, Sunnyvale, California). Video signals were 
digitized and processed by IMAGE 1/FL software from 
Universal Imaging Corp. (Westchester, Pennsylvania) us- 
ing a 486-based computer with output on a color monitor 
(Trinitron, SONY). Software controlled the operation of 
the rilterwheel/shutter system. 

Isolated myocytes did not display any auto-fluores- 
cence. Preliminary experiments showed that BCECF- 
loaded myocytes went into contracture when illuminated 
with intense monochromatic light (490 or 450 nm). Un- 
loaded cells did not respond to light in this way. Fur- 
thermore, the ability to regulate pH, was impaired in 



BIVALVE MVOCYTE INTRACELLULAR pH REGULATION 



211 



loaded cells at high light intensities. Thus, during all ex- 
periments we used high range neutral density niters to 
reduce light intensities compensating for the reduced flu- 
orescence by employing high camera gain and intensity 
settings. Furthermore, the period of irradiation of cells 
with monochromatic light was reduced to a minimum 
consistent with camera lag. Images were acquired using 
shade (shade "mask" obtained using 25 n\ of 25 fiM 
BCECF sandwiched between coverslip and slide) and 
background correction capabilities of the IMAGE 1/FL 
software. Image pairs were acquired at specific time in- 
tervals (usually every 20 s). Individual cells were selected 
and fluorescence ratios (I^o/Uso) for each cell versus time 
were stored in a spread-sheet data base. Numerical data 
were transferred as ASCII files to a Macintosh Ilci and 
processed and analyzed using Sigmaplot ( Jandel, San Ra- 
fael, California). 

In vivo calibration of ratios 

Fluorescent ratios with respect to pH were calibrated 
by the nigericin pH clamp approach of Thomas el al. 
( 1979). The calibration solution was identical to MASW 
except that it contained 290 mA/NaCl, 160 mAl KC1 and 
nigericin (5 fig/ml). The concentration of K.C1 chosen 
brackets values for intracellular K 1 as determined in the 
muscles of marine mollusks (Potts, 1958; Robertson, 
1965; Burton, 1983). Cells were allowed to equilibrate 
with each solution until ratios stabilized (generally <5 
min). In routine experiments, pH, was estimated for in- 
dividual myocytes. Mean values for acid-base measure- 
ments in each physiological treatment represent data from 




3.0 



2.8 



2.4 
2.2 
2.0 
1.8 
1.6 




6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 
pH 

F'igure 2. Relationship between the fluorescence intensity ratio (R 
= IWUso) and pH obtained using the nigericin approach with cardiac 
myocytes from Mcrcciuiria campechiensis. Each value corresponds to a 
mean I SD (n ranges from 8 to 12). 



individual cells from up to two independent cell disper- 
sions. 

Results 



Figure 1. Cardiac myocyte from the ventricle of the clam Aiercenaria 
campechiensis. Photograph was taken with Kodak TMAX 100 film. Bar 
corresponds to 100 Mm. 



Dispersion procedures produced a very high yield of 
myocytes. Cells were generally long and spindly (50-400 
fj. X 10 n) (Fig. 1). Immediately after dispersion, a large 
fraction of cells showed spontaneous contractile activity 
but were generally quiescent after 12 h. Dispersed cells 
excluded trypan blue and were responsive to addition of 
10~ 5 M 5-hydroxytryptamine (5-HT). In fact, many cells 
remained viable and responded to 5-HT up to 7 days after 
isolation. The addition of antibiotics (penicillin G. baci- 
tricin) and 5 mAI D-glucose did not enhance survival in 
the short term nor influence results of pH, determinations. 
Thus, these components were not added to MASW. Ven- 
tricles of the congeneric clam Mercenaria mercenaha have 
extremely high glycogen levels, on the order of 240 
^moles/g wet wgt (Ellington, 1985). Thus, it is clear that 
there is a sufficient endogenous reserve of metabolic fuels 
in these myocytes for the period over which they were 
used (15- 18 h). 

Nigericin pH clamp 

Myocytes were subjected to a nigericin pH clamp pro- 
tocol encompassing the range of pH from 6.6 to 7.6 in 
0.2-unit increments. Typically, most cells responded to 
the clamping medium by contracting to approximately 
60% of their initial length and remained so throughout 
the protocol. Fluorescence ratios varied linearly with pH 
(Fig. 2). A regression equation was calculated (RATIO 
= 1.1 1 1 pH -5.724, R = 0.994) and used to transform 
all ratios from the ratio versus time spreadsheets to pH, . 
The compressed ratio range was due to differential 



212 



W. R. ELLINGTON 



7.5 
74 
7.3 

pHi : 

7.1 
7.0 
6.9 
6.8 




6.0 6.4 



6.8 
pHe 



7.2 7.6 8,0 



Figure 3. The relationship between pH, and pH e in cardiac myocytes 
from Mercenaria campechiensis. The continuous diagonal line corre- 
sponds to the iso-pH line. Each value corresponds to a mean 1 SD 
(n = 11). 



gain/level adjustments of the analog-digital converter at 
the two excitation wavelengths as well as the fact that the 
neutral density filter at 490 nm was higher than the one 
used at 450 nm. Under routine superfusion conditions, 
pH, of myocytes was observed to be 7.22 0.08 (mean 
1 SD, n = 20). 

Relationship between intracellular and extracellular pH 

Cells were superfused with MASW (containing 20 mM 
HEPES) adjusted to various pH values (pH e ). Media were 
equilibrated with air. Ratios were observed for 1 5-20 min 



at each pH. pH, was essentially constant in the pH e range 
from 7.1 to 8.0 (Fig. 3). At lower pH e values, the pH, 
declined linearly but still was considerably above the iso- 
pH line indicating good capacity for defense of pH, against 
pH e (Fig. 3). 

Non-bicarbonate buffering capacity (@NB) 

Non-bicarbonate buffering capacity was estimated by 
the NH 4 C1 prepulse method of Boron (1977). Myocytes 
were superfused with MASW and then subjected to a pulse 
of 15 mM NH 4 C1-MASW (pH 7.75). After peak alkalin- 
ization, myocytes were superfused with MASW resulting 
in a pronounced acidification. /} NB values were calculated 
as described by Boron (1977). Buffering capacity is ex- 
pressed as Slykes (dH 4 /dpH). Since the MASW was 
equilibrated in air, [HCO.r] was low (around 0.7 mAf) 
so the contribution of this species to total buffering ca- 
pacity is negligible. Figure 4 is an example of a typical 
pre-pulse experiment. Following alkalinization, there was 
a gradual decline in pH,. After NH 4 C1 wash-out, there 
was a characteristic alkalinization back towards the initial 
condition (Fig. 4). J NB values for individual myocytes were 
somewhat variable ranging from 22 to 65 Slykes with a 
mean of 39.98 8.86 (1 SD, n = 31). The inherent 
limitation of the NH 4 C1 prepulse approach is that some 
recovery of pH, could occur during the early phase of the 
wash-out therebye producing an overestimate of /3 NB (Bo- 
ron, 1977). In the present study, wash-out was extremely 



pHi 



f . \J 


1 1 1 I 


7.7 


1 


7.6 


- 1 I 


7.5 


***4 


7.4 


o 


7.3 


1 


7.2 


- *- 


7.1 


la^v**^^ 


7.0 


^ 


6.9 


- 


fi ft 


1 1 1 1 


10 20 30 40 51 



time (min) 



Figure 4. Time course of changes in pH, in a single Mercenaria campechiensis cardiac myocyte during 
a typical NH 4 C1 pre-pulse experiment. The first arrow indicates the onset of superfusion with 1 5 mM NH 4 CI- 
MASW. The second arrow indicates the onset of washing with MASW. 



BIVALVE MYOCYTE INTRACELLULAR pH REGULATION 



213 



rapid as the peak acidosis occurred within 3 min (Fig. 4). 
Thus, it is likely that pH, recovery processes would po- 
tentially produce only minor errors in /} NB determination 
in this system. 

Recovery from acid loading 

Isolated myocytes regulate pH, after acid-loading as ev- 
idenced by the slow alkalinization following NH 4 C1 wash- 
out using normal MASW ([HCO, ] approximately 0.7 
mM) (Fig. 5A; Table 1). However, the rate of alkaliniza- 
tion was greatly accelerated during wash-out using 0.3% 
CO : /4 mA/ HCO 3 MASW (Fig. 5B; Table I). Recovery 
from acid loading did not appear to be dependent on ex- 
ternal Na + , as the recovery rate was essentially the same 
for MASW and Na + -free MASW (Table I). SITS com- 
pletely blocked recovery. In fact, there was a gradual re- 
duction in pH, once the plateau acidification after wash- 
out had been attained (Table I). Collectively, these results 
show that it is likely that a SITS-sensitive HCO 3 ":CI~ ex- 
changer plays a major role in recovery from acid loading 
in M. campechiensis myocytes. 

Discussion 

Molluscan myocytes have been used on a number of 
occasions as experimental systems for investigating phys- 
iological phenomena ranging from ion channels (Brezden 
eta/., 1 986) to changes in intracellularCa :+ concentrations 
during contraction (Ishii el a/.. 1989). In this regard, car- 
diac myocytes from M. campechiensis appear to be an 
ideal model system for studies of regulation of pH, in that 
these cells are easily isolated, retain viability for extended 
time periods and, of course, can maintain acid-base bal- 
ance in spite of extracellular and intracellular pH distur- 
bances. The average pH, of 7.22 in these cells as deter- 
mined by BCECF-imaging is comparable to values ob- 
served in various marine gastropod muscles (Ellington, 
1983b; Graham and Ellington, 1985; Wiseman and El- 



pHi 



7.0 
6.9 
6.8 




20 30 
time (mm) 



10 20 
time (mm) 



Table I 

Recovery from acid loading in myocytes from Mercenaria 
campechiensis 



Washing medium 



Acid/base 

dpH/dt transport 

(pH units/min) (Aimoles/min) n 



MASW 


0.0040 0.00 18 


0.157 0.055 


10 


MASW-0.3%CO 2 : 








4 mM HCOr 


0.0 152 0.00 12 


0.576 0.115 


9 


Na + -free MASW 


0.0054 0.0030 


0.236 0.159 


9 


MASW-0.5 mM SITS 


No Recovery 


Not estimated 


10 




(-0.0073 0.0056) 







Figure 5. Typical records of change of pH, in single Mercenaria 
campechiensis myocytes during NH 4 C1 pre-pulse experiments when 
MASW (A) or 0.3% CO 2 :4 mM HOV-MASW (B) were used in washing. 



Myocytes were superfused with MASW followed by 15 mM NH 4 C1- 
MASW. After the plateau alkalinization was acheived, myocytes were 
washed with various media. The rate of recovery (dpH/dt) was calculated 
using a regression (Sigma Plot) of the initial, linear portion of the recovery 
curve after NH 4 CI wash-out. Buffering capacity values (dhT/dpH) were 
calculated according to Boron (1977). Rates of acid/base equivalent 
transport (^moles/min) were calculated for each myocyteby multiplying 
the measured individual f? NB value times the corresponding dpH/dt value 
(^moles/mm = /} NB -dpH/mm). Each value represents a mean 1 SD. 
Sample size (n) is indicated. 

MASW-0.3% CO,: 4 mM HCO, was prepared by gassing MASW 
with 0.3% CO 2 (balance air), addition of solid NaHCO 3 followed by 
adjustment of pH. The reservoir was continuously gassed with hydrated 
0.3% CO 2 in air and the superfusion line was contained within a gas 
jacket. Concentrations were calculated using appropriate apparent dis- 
sociation (Mehrbach cl a/., 1973) and solubility (Riley and Skirrow, 1975) 
constants. Na + -free MASW was prepared by replacing NaCl with three 
times crystallized choline chloride (Sigma Chemical Co.). SITS solutions 
were shielded from light to prevent photodecomposition. 



lington, 1989) and is slightly lower (0.1-0.2 units) than 
what has been observed for squid giant axon (Boron and 
Russell, 1983) and various tissues of the mussel Mytilus 
edulis (Walsh el al, 1984; Zange el a/.. 1990). The above 
data on other species were obtained by a variety of tech- 
niques including NMR, weak acid distribution and micro- 
electrode methods. 

Changes in pH e have minimal effect on the pH, of M. 
campechiensis myocytes over what can be viewed as a 
physiologically realistic range of pH e s (7.1-8.0). A similar 
high capacity for defending pH, against changes in pH e 
has been observed in M. edulis ABRM preparations 
(Zange el al., 1990) as well as in hemocytes from the squid 
Sepiateuthis lessoniana (Hemming el al.. 1990). In the 
present study, we have further seen that clam myocytes 
display recovery from experimentally imposed acid-base 
disturbances. Walsh and Milligan (1989) have pointed 
out that there are three potential avenues of regulation of 
pH, available to cells (a) intracellular physico-chemical 
buffering, (b) ion exchange of acids/bases between intra- 
and extracellular compartments, and (c) metabolic pro- 
duction or consumption of acids and bases. The present 
results with M. campechiensis myocytes provide strong 



214 



W. R. ELLINGTON 



evidence for operation of the first two of these mecha- 
nisms. 

The presence of non-bicarbonate intracellular buffers 
constitutes the first line of defense against acid/base stress 
in cells (see reviews by Burton, 1978, and Roos and Boron, 
1 98 1 ). In vertebrate-muscles, there appears to be a general 
correlation between the magnitude of the /3 NB and the 
potential for anaerobic function (Castellini and Somero, 
1981). This general correlation has been suggested for 
molluscan muscles (Eberlee and Storey, 1 984; Morris and 
Baldwin. 1984), however, the validity of these conclusions 
is somewhat in doubt due to the artifacts imposed by ho- 
mogenate titration methods used for /3 NB determinations 
(Wiseman and Ellington, 1989; Portner, 1990). In the 
present study we used the NH 4 Cl-prepulse approach and 
obtained a value of 40 Slykes, which is in the range of 
values determined by NMR-prepulse for whelk radula 
muscle (33 Slykes; Wiseman and Ellington, 1989) and 
mussel ABRM (26.5 Slykes; Zange et ai, 1990), both of 
which have impressive capacities for anaerobic metabo- 
lism. In contrast to these observations, the average /3 NB 
for squid giant axons was 1 1.2 (Boron and Russell, 1983). 
Thus, it is clear that M. campechiensis myocytes have a 
relatively high /j NB , which is consistent with the natural 
history of this species where exposure to hypoxic stress 
may be a regular phenomenon. 

The pH L . in bivalves is alkaline relative to pH, under 
normal conditions (Booth et al., 1984). Given a slightly 
alkaline pH e , the pHj of 7.22, and the undoubtedly neg- 
ative sign of the membrane potential in Al. campechiensis 
myocytes, it is clear that protons are not at equilibrium. 
These cells must continuously export protons or bring in 
base equivalents to maintain pH, . This problem becomes 
greatly exacerbated when the pH e is reduced (therebye 
decreasing or even reversing the transmembrane proton 
gradient) or when acid or base loads are imposed on the 
cells. The relative constancy of pH, with pH c and recovery 
from experimentally imposed acidosis in clam myocytes 
clearly show that such ion exchange processes are oper- 
ating in these cells. 

Our results show that A/, campechiensis myocytes ap- 
pear to regulate pH, via a SITS-sensitive ion exchanger 
which does not have a requirement for external Na + . Most 
likely, this transporter is a HCO 3 :C1~ exchanger as has 
been seen in M. editlis ABRM (Zange et ai, 1 990). Under 
the routine, normocapnic conditions in the present study 
the concentration of HCO 3 was estimated to be around 
0.7 mA/, which appears to be sufficient to promote re- 
covery of pHj after acidosis. However, recovery was greatly 
accelerated when HCO 3 concentration was increased to 
around 4 mA/. Booth et al. (1984) estimated that [HCO-f ] 
in M. edulis hemolymph was 1.8 mA/ in normoxia and 
rose to nearly 3 mA/ during hypoxic stress. It is likely that 
physiological [HCO 3 ] in A/, campechiensis spans a higher 



range that 0.7 mA/, implying greater overall transport rates 
in vivo. The above results are in contrast to the work of 
Boron and Russell (1983) who found that there was an 
absolute Na + requirement (Km = 77 mA/) for HCO 3 : 
Cr exchange in squid axons. However, Hemming et al. 
(1990) found that acid recovery in squid hemocytes was 
Na + -independent. Zange et al. (1990) found that addition 
of 5-hydroxytryptamine (5-HT) elicited activation of a 
Na + :H + exchanger in A/, edulis ABRM. It was not possible 
to investigate this possibility in A/, campechiensis myo- 
cytes, as addition of 5-HT caused rather violent contrac- 
tions of myocytes, which interfered with imaging exper- 
iments. 

The present results show that myocytes from the clam 
M. campechiensis have a good capacity for regulation of 
pH,. This capacity is based a relatively high /3 NB and the 
presence of a SITS-sensitive anion exchanger. Clam myo- 
cytes also appear to be excellent candidates for long-term 
primary culture. Thus, future studies will focus on poten- 
tial phenotypic plasticity of /S NB and ion exchange capacity 
in cells cultured under conditions which might induce 
such changes (altered pH c , hyper- or hypo-capnia or hyp- 
oxia). 

Acknowledgments 

I wish to heartily thank Ms. Carolyn Bruce (University 
of British Columbia) for advice and encouragement in 
the development of the myocyte isolation protocol. I am 
also very grateful to Leavins Seafood, Inc. (Apalachicola, 
Florida) for providing animals. This research was sup- 
ported by NSF grants DIR-9014510 (Instrument and In- 
strument Development Program) and IBN-9 104548 
(Functional and Physiological Ecology Program). 

Literature Cited 

Barrow, K. D., D. D. Jamil-son, and R. S. Norton. 1980. "P nuclear 
magnetic resonance studies of energy metabolism in tissue from the 
marine mollusc Tapes watlingi. Eur. J. Biochem. 103: 289-297. 

Booth, C. E., D. G. McDonald, and P. J. Walsh. 1984. Acid-base bal- 
ance in the sea mussel Mylilus edulis. I. Effects of hypoxia and air- 
exposure on the hemolymph acid-base status. Mar. Biol. Lett. 5: 
347-358. 

Boron, \V. F. 1977. Intracellular pH transients in giant barnacle muscle 
fibers. Am. J. Physiol. 233: C61-C73. 

Boron, \V. F., and J. M. Russell. 1983. Stoichiometry and ion depen- 
dencies of the intracellular-pH-regulating mechanism in squid giant 
axons. ./. Gen. Physiol. 81: 373-399. 

Boron, \V. F., W. C. McCormick, and A. Roos. 1979. pH regulation 
in barnacle muscle fibers: dependence on intracellular and extra- 
cellular pH. Am. J. Physiol. 237: C185-C193. 

Brezden, B. L., D. R. Gardner, and G. E. Morris. 1986. A potassium- 
selective channel in isolated Lymnaea stagnulis heart muscle cells. 
./. Exp. Biol. 123: 175-189. 

Bright, G. R., G. W. Fisher, J. Rogowska, and D. L. Taylor. 
1987. Fluorescence ratio imaging microscopy: temporal and spatial 
measurements of cytoplasmic pH. J. Cell Biol. 104: 1019-1033. 



BIVALVE MYOCYTE INTRACELLULAR pH REGULATION 



215 



Burton, R. F. 1978. Intracellular buttering. Resp Physiol. 33: 51-58. 

Burton, R. F. 1983. Ionic regulation and water balance. Pp. 291-352 
in The Mollusca. Vol 5. A. S. M. Saleudin and K.. M. Wilbur, eds. 
Academic Press. New York. 

Castellini, M. A., and G. N. Somero. 1981. Buffering capacity of ver- 
tebrate muscle: correlations with potentials for anaerobic function. 
J. Comp. Physiol. 143: 191-198. 

Crenshaw, M. A., and J. M. Neff. 1969. Decalcilication at the mantle- 
shell interface in molluscs. Am. Zool 9: 881-889. 

Eberlee, J. O, and K. B. Storey. 1984. Buttering capacities of the tissues 
of marine molluscs. Physiol. Zoo/. 57: 567-572. 

Ellington, VV. R. 1983a. The extent of intracellular acidification during 
anoxia in catch muscle of two bivalve molluscs. J. Exp. Zool. 221: 
313-317. 

Ellington, \V. R. I983b. Phosphorus nuclear magnetic resonance studies 
of energy metabolism in molluscan tissues: effect of anoxia and isch- 
emia on intracellular pH and high energy phosphates in the ventricle 
ofthevihe\k.,Busyconcontrarium.J Comp Physiol 153: 159-166. 

Ellington, \V. R. 1985. Cardiac energy metabolism in relation to work 
demand and habitat in bivalve and gastropod molluscs. Pp. 356- 
366 in Circulation, Respiration and Metabolism, R. Gilles, ed. 
Springer Verlag, Berlin. 

Gnaiger, E. 1980. Das Kalorische Aquivalent des ATP-Umstazes im 
aeroben und anoxischen metabolismus. Thermochim. Ada 40: 195- 
223. 

Graham, R. A., and W. R. Ellington. 1985. Phosphorus nuclear mag- 
netic resonance studies of energy metabolism in molluscan tissues: 
intracellular pH change and the qualitative nature of anaerobic end 
products. Physiol. Zool. 58: 478-490. 

Hemming, T. A., C. O. Vanoye, S. E. S. Brown, and A. Bidani. 
1990. Cytoplasmic pH recovery in acid-loaded haemocytes of squid 
(Scpiateuihis lessoniana). J. Exp. Bio/. 148: 385-394. 

Ishii, N., A. \V. M. Simpson, and C. C. Ashley. 1989. Free calcium at 
rest during "catch" in single smooth muscle cells. Science 243: 1 367- 
1368. 

Mehrbach, C., C. H. Culberson, J. E. Hawley, and R. M. Pytkowicz. 
1973. Measurement of the apparent dissociation constants of car- 
bonic acid in seawater at atmospheric pressure. Linmol. Oceanogr. 
18: 897-907. 

Morris, G. M., and J. Baldwin. 1984. pH buffering capacity of inver- 
tebrate muscle: correlations with anaerobic muscle work. Molec 
Wi.i-.vw/. 5:61-70. 

Portner, H.-O. 1987a. Contributions of anaerobic metabolism to pH 
regulation in animal tissues-theory. J. Exp. Biol. 131: 69-87. 



Portner, H.-O. 1987b. Anaerobic metabolism and changes in acid- 
base status: quantitative interrelationships and pH regulation in the 
marine worm Sipunculus nitihts. J Exp. Biol. 131: 89-105. 

Portner, H.-O. 1989. The importance of metabolism in acid-base reg- 
ulation and acid-base methodology. Can. J Zool. 67: 3005-3017. 

Portner, H.-O. 1990. Determination of intracellular buffer values after 
metabolic inhibition with fluoride and nitrilotriacetic acid. Rcsp 
Physiol. 81: 275-288. 

Portner, H.-O., N. Heisler, and M. K. Grieshaber. 1984a. Anaerobiosis 
and acid-base status in marine invertebrates: a theoretical analysis 
of proton generation by anaerobic metabolism. J. Comp. Physiol. 
155: 1-12. 

Portner, H.-O., M. K. Grieshaber, and N. Heisler. 1984b. Anaerobiosis 
and acid-base status in marine invertebrates: effect of environmental 
hypoxia on extracellular and intracellular pH in Sipunculus nudiis 
L.J Comp. Physiol 155: 13-20. 

Potts, W. T. W. 1958. The inorganic and amino acid composition of 
some lamellibranch muscles. J. Exp Biol. 35: 749-764. 

Riley, J. P., and G. Skirrow. 1975. Chemical Oceanography. Academic 
Press. New York. 

Rink, T. J., R. Y. Tsien, and T. Pozzan. 1982. Cytoplasmic pH and 
free Mg 2+ in lymphocytes. J Cell Biol. 95: 189-196. 

Robertson, J. D. 1965. Studies on the chemical composition of muscle 
tissue. III. The mantle muscle of cephalopods. J. Exp. Biol 42: 153- 
175. 

Roos, A., and W. F. Boron. 1981. Intracellular pH. Physiol. Rev 61: 
296-434. 

Thomas, J. A., R. N. Buschbaum, A. /.imniak, and E. Raeker. 
1979. Intracellular pH measurements in Ehrlich ascites tumor cells 
utilizing spectroscopic probes generated in situ. Biochemistry 18: 
2210-2218. 

Walsh, P. J., D. G. McDonald, and C. E. Booth. 1984. Acid-base bal- 
ance in the sea mussel Mytilus eiiulis. II. Effects of hypoxia and air 
exposure on intracellular acid-base status. Mar. Biol. Lett. 5: 359- 
369. 

Walsh, P. J., and C. L. Milligan. 1989. Coordination of metabolism 
and intracellular acid-base status: ionic regulation and metabolic 
consequences. Can. J. Zool. 67: 2994-3004. 

Wiseman, R. W ., and W. R. Ellington. 1989. Intracellular buffering in 
molluscan muscle: superfused muscle versus homogenates. Physiol. 
Zool. 62: 541-558. 

Zange, J., M. K. Grieshaber, and A. W. H.Jans. 1990. The regulation 
of intracellular pH estimated by -"P-NMR spectroscopy in the an- 
terior byssus retractor muscle of Mytilus editlis L. J. Exp. Biol. 150: 
95-109. 



Reference: BID/. Bull 184: 216-222. (April. 1993) 



Two S-Iamide Peptides, AKSGFVRIamide and 

VSSFVRIamide, Isolated from an Annelid, 

Perinereis vancaurica 



O. MATSUSHIMA 1 , T. TAKAHASHI 1 , F. MORISHITA 1 , M. FUJIMOTO 1 , T. IKEDA 2 , 
I. KUBOTA 3 , T. NOSE 4 , AND W. M1KI 4 

1 Zoological Institute, Faculty of Science, Hiroshima University, Higashi-hiroshima 724, Japan, 

2 Physiological Laboratory. Faculty of Integrated Arts and Sciences, Hiroshima University. 

Hiroshima 730. Japan. 3 Suntory Bio-Pharma Tech Center. Gunma 370-05. Japan. 

and ^Marine Biotechnology Institute, Shimiiu 424, Japan 



Abstract. Two peptides, H-Ala-Lys-Ser-Gly-Phe-Val- 
Arg-Ile-NH : (AKSGFVRIamide), and H-Val-Ser-Ser- 
Phe-Val-Arg-Ile-NH 2 (VSSFVRIamide) were isolated 
from a polychaete annelid, Perinereis vancaurica. 
Both the peptides evoked rhythmic contractions in the 
esophagus of Perinereis with a threshold as low as 
10~'-10~ 9 M. suggesting that the peptides may be in- 
volved in the regulation of gut motility of the animal. 
The sequences of these peptides are very similar to those 
of other S-Iamide family peptides which have been pre- 
viously isolated from an echiuroid worm and some mol- 
luscs. In particular, the sequence of VSSFVRIamide is 
identical to that of an echiuroid S-Iamide peptide. All 
of the molluscan and echiuroid S-Iamide peptides, as 
well as the annelid peptides. were found to produce con- 
tractions in the esophagus of Perinereis. On the other 
hand, the annelid S-Iamide peptides, as well as the mol- 
luscan and echiuroid peptides, were found to inhibit or 
potentiate contractions elicited by electrical stimulation 
in echiuroid and molluscan muscles. S-Iamide peptides 
may be a typical neuropeptide family distributed inter- 
phyletically in the Protostomia. 

Introduction 

In annelids, pharmacological studies have been exten- 
sively done on the actions of classical transmitters such 
as 5-hydroxytryptamine, epinephrine, norepinephrine and 
dopamine mainly on somatic muscles, and these sub- 
Received 6 October 1992, accepted 25 January 1993. 



stances have been suggested to be present in the central 
and peripheral nervous systems (for review, Tashiro and 
Kuriyama, 1978). In addition, bioactive peptides found 
in vertebrates and other phyla of invertebrates have been 
suggested to be present in annelids (Carraway et a/., 1 982; 
Engelhardt et a/.. 1982: Dhainaut-Courtois et a/.. 1985; 
Diaz-Miranda et a/., 1991, 1992). 

Many peptides are known in vertebrates, especially in 
mammals which control the motility of the gut (Holm- 
gren, 1989). However, few gut motility-controlling pep- 
tides have been reported for invertebrates. Immunohis- 
tochemical or immunochemical studies have suggested 
that some vertebrate neuropeptides, such as enkephalin, 
/3-endorphin (Alumets et al. 1979), substance P (Dhai- 
naut-Courtois et al.. 1985; Kaloustian and Edmands, 
1986), cholecystokinin/gastrin, 0-MSH (Engelhardt et a/.. 
1982; Dhainaut-Courtois et al.. 1985) and neurotensin 
(Carraway et al., 1982) may be present in annelids. Ka- 
loustian and Edmands (1986) reported that substance P 
stimulated the rate of spontaneous contraction of intes- 
tinal tissues of the earthworm Lumbricus terrestris. It has 
also been shown that a tetrapeptide (WMDFamide) re- 
lated to cholecystokinin/gastrin has excitatory effects on 
the anterior intestine of a polychaete, Chaetopterus var- 
iopedatus (Anctil et a/.. 1984). Apart from immunocy- 
tochemical and pharmacological studies, most investi- 
gations of bioactive peptides in annelids have centered 
on those involved in reproductive events (Thorndyke, 
1989). Thus, reports on authentic bioactive peptides in- 
volved in the regulation of gut-motility of annelids are 
very few in number. 



216 



S-IAMIDE PEPTIDES OF AN ANNELID 



217 



Recently, Krajniak and Price ( 1990) showed the pres- 
ence of FMRFamide which was first identified in a mol- 
lusc as a cardioexcitatory neuropeptide in the polychaete 
Nereis virens (Price and Greenberg, 1977). Krajniak 
and Greenberg (1992) showed that immunoreactive 
FMRFamide was present in various tissues including the 
gut in Nereis, and that FMRFamide had a relaxing action 
on both the spontaneously active and electrically stimu- 
lated esophagus, suggesting the involvement of the tet- 
rapeptide in the control of gut-motility. Furthermore, 
FMRFamide and its related peptides have been shown to 
be present in other annelid species such as Nereis diver- 
sicolor (Baratte el a/.. 1991) and Hirudo medicinalis 
(Evans et at.. 1991). 

In the present study, we isolated and sequenced two 
bioactive peptides, AKSGFVRIamide and VSSFVRIam- 
ide, from the polychaete Perinereis vancaiiricii, which in- 
duced contraction of the isolated esophagus of the animal. 
These peptides were found to be members of the S-Iamide 
peptide family ( Ikeda t'/ al.. 1991; Muneokaand Kobaya- 
shi, 1992). The name S-Iamide peptide was given after 
the common structure. -SSFVRIamide. Kuroki el al. 
(1992) first isolated one of the S-Iamide peptides, 
LSSFVRIamide, from the prosobranch mollusc Fitsinus 
ferrugineus, and up to the present, S-Iamide peptides have 
been found not only in molluscs but also in an echiuroid 
worm (Ikeda et al., 1991). We also examined the effects 
of several S-Iamide peptides on some invertebrate muscle 
tissues including the esophagus of Perinereis. 



Materials and Methods 



Purification 



Perinereis vancaurica tetradentata are commercially 
available as fishing bait. Approximately 380 worms (500 
g) were rinsed twice with artificial seawater ( ASW). blotted 
lightly with tissue paper and boiled for 10 min in 4 vol- 
umes of 4% acetic acid (21). The animals were homoge- 
nized in 4% acetic acid by using a Waring blender and a 
Polytron. The homogenate was centrifuged ( 15,000 X g, 
40 min, 4C), and the resulting precipitate was again ho- 
mogenized and centrifuged. The two supernatants were 
pooled and concentrated to a volume of about 100 ml by 
using a rotary evaporator (40C). To the concentrated 
supernatant, 1/10 volume of 1 N HC1 was added, and the 
precipitated material was centrifuged off( 1 5.000 X g, 40 
min, 4C). Next, the supernatant was forced through two 
disposable C-18 cartridges in series (Mega Bond-Elut. 
Varian). The retained material was eluted with 50% 
methanol. The eluate was concentrated, loaded on a C- 
18 reversed phase HPLC column (CAPCELL-PAK, Shi- 
seido; 10 mm X 250 mm), and eluted with a linear gra- 
dient of 0-60% acetonitrile (ACN) in 0. 1% trifluoroacetic 
acid (TFA) for 120 min at a flow rate of 1 ml/min. The 



chromatography was monitored at 220 nm. Aliquots of 
2 mi-fractions were evaporated to dry ness, and the residues 
were dissolved in ASW and bioassayed on an isolated 
esophagus of Perinereis as described below. Two con- 
tractile peaks were detected. The fractions of each active 
peak were concentrated and subjected to HPLC using an- 
other C-18 reversed phase column (ODS-80TM, Tosoh; 
4.6 mm X 150 mm) with a linear gradient of 10-20% 
ACN for one activity and 1 5-25% ACN for the other in 
0.1% TFA (0.5 ml/min). Active fractions obtained from 
each HPLC were then loaded onto a cation-exchange col- 
umn (SP-5PW, Tosoh; 7.5 mm X 75 mm) and eluted in 
a linear gradient of 0-0.7 Al NaCl in 10 mM phosphate 
buffer (pH 7. 1 ) for 70 min at a flow rate of 0.5 ml/min. 
Then, the active substances identified as single peaks on 
the cation-exchange HPLC were chromatographed on the 
ODS-80TM column with a linear gradient of 10-16%. 
ACN and 15-25% ACN, respectively, and finally purified 
on the same column with an isocratic elution of 17% and 
19% ACN. 

The two purified substances were subjected to amino 
acid sequence analysis by automated Edman degradation 
with a gas-phase sequencer (Shimazu PSQ-1 ). The results 
of the chemical analyses suggested that the substances were 
members of the S-Iamide peptide family. Therefore, the 
two peptides having the suggested structures were syn- 
thesized by a manual method followed by an HF-anisol 
cleavage and purified by reversed-phase HPLC. Then, the 
synthetic peptides were compared with native ones in the 
behavior on HPLC and in the bioactivity on the Perinereis 
esophagus. 

Bioassay 

The contractile activities of the native and synthetic 
substances were examined on the isolated esophagus of 
Perinereis. The method for contraction recording was 
essentially the same as that reported by Krajniak and 
Greenberg (1992). Both ends of the isolated esophagus 
were ligated with two cotton threads, one being secured 
to a stationary rod at the bottom of a trough (2 ml) and 
the other connected to a force-displacement transducer 
(NEC San-ei Instruments). 

The saline in the trough was constantly aerated through 
a syringe needle connected with an air pump to ensure 
uniform distribution of applied substances (dissolved in 
0.1 ml saline) in the trough. In the present study, we did 
not apply electrical stimulation but just monitored in- 
ductivity of spontaneous contractions of the esophagus 
by test substances. For examination of the bioactivity of 
the material retained by the C-18 cartridges, two more 
assay systems, the inner circular body-wall muscle of an 
echiuroid worm Urechis unicinctus (Ikeda et al., 1991), 
and the radula retractor muscle of a prosobranch mollusc 



218 



O. MATSUSHIMA ET AL 




B 

"i 



JL 



4 
RM 



1 min 



t 

RM 



10 


" y) 


I 


i 













I 


1 


1 g 



s 

.0 

5 




i! 



RM 



1 min 



Figure 1. Effects of the retained materials (RM) on the three muscle 
systems. (A) the esophagus of Pe rinereix. (B) twitch contractions of the 
inner circular body-wall muscle of L'rechis. The twitch contraction was 
produced by an electrical pulse (20 V, 3 ms). (C) twitch contractions of 
the radula retractor of Rapana. The twitch contractions were produced 
by a train of electrical pulses (15V. 1 ms, 0.2 Hz, 5 pulses). In each case. 
1/1000 of total RM. which corresponded with extracts from 0.4 worm, 
was applied to the assay system. The upward arrows indicate application 
of RM to the tissue. The downward arrows indicate washing-out of the 
RM. 



Rapana thomasiana (Muneoka et al., 1991), were em- 
ployed. In these cases, electrical pulses of stimulation were 
applied to the preparations. 

Pharmacology 

The methods used in the pharmacological experiments 
were basically the same as those in the bioassay experi- 



o 

CM 



4.0 



a 

0) 

ra 

^ 

O 
CO 

.0 



2.0 




60 



CJ" 


A B 


'^lA^ 




I 


1 1 I 1 I 


i i 


i 





40 80 
Time (min) 


120 


160 



o 



Figure 2. HPLC profile of the retained materials (RM) on a reversed 
phase column. The RM loaded onto the column was eluted with a linear 
gradient of ACN concentration (0-60%/120 min) in 0.1% TFA (pH 2.2) 
at a flow rate of 1 ml/min and collected in 60 fractions of 2 ml each. 
Aliquots (10 n\ = 1/200) of each fraction were evaporated to dryness, 
dissolved in ASW and applied to the Perinereis esophagus. The contractile 
peaks were indicated by the horizontal bars (A and B). 





nO.7 



20 40 



60 



20 40 



60 



Time (mm) 



Figure 3. HPLC profiles of active fractions (A and B in Fig. 2) on a 
cation-exchange column. Elution was performed in a 70-min linear gra- 
dient of 0-0.7 A/ NaCl in 10 m.U phosphate buffer (pH 7.1) at a flow 
rate of 0.5 ml/min (collected in 1-ml fractions). The activities (A and B) 
were detected in respective peaks indicated by arrows. 



ments. In these experiments, we used three kinds of mus- 
cles, the esophagus of Perinereis, the anterior byssus re- 
tractor muscle (ABRM) of the bivalve mollusc Mytilus 
edulis and the radula retractor muscle of the prosobranch 
mollusc Fusinus ferrugineus. 

Salines 

The saline used for Perinereis and Urechis muscles was 
ASW of the following composition: 445 mAl NaCl, 55 
mM MgCl : , 10 mA/ CaCl : , 10 mA/ KG, 10 mAl Tris- 
HC1; pH 7.6. For the Rapana and Fusinus muscles, low 
Mg-ASW (20 mA/ MgCl 2 ) was used. The low-Mg ASW 
was prepared by replacing a part of MgCl : in the normal 
ASW with osmotically equivalent NaCl. 

Results 

The retained material (RM) eluted with 50% methanol 
was examined for its biological action on three muscle 
systems, the esophagus of Perinereis, the inner circular 
body-wall muscle of Urechis and the radula retractor 
muscle of Rapana (Fig. 1 ). The RM elevated a basal tone 
with rhythmic small contractions in the esophagus of Per- 
inereis and exerted inhibitory effects on twitch contrac- 
tions evoked by electrical stimulations in the latter two 
muscles. After the test solution was replaced with normal 
ASW, the contractions of Rapana radula retractor became 
greater than the control contractions and then returned 
to the control level. We decided to purify at first the sub- 
stance which elicited contractions of the Perinereis 
esophagus. 

At the first step of HPLC, two contractile peaks (peak 
A and B) were found. They were eluted at approximately 
15% and 20% ACN, respectively (Fig. 2). At the second 
step, active substances of peak A and B were eluted at 
13% ACN and 19% ACN, respectively (data not shown). 
Then, the fractions containing active substance A and B 
were respectively subjected to the cation-exchange HPLC 



S-IAMIDE PEPTIDES OF AN ANNELID 



219 



o 

CM 

"0.5 
ra 



20 



J 



40 
Time 



0.1 













20 4 



(min) 




19 



0.5 9 



2 min 



Figure -4. Final purification by HPLC using a reversed-phase column 
(A. B) and the action of each purified substance on the Perincrcis esoph- 
agus (C, D). Isocratic elution with 17% ACN (A) and 19% ACN (B) in 
0.1% TFA at a flow rate of 0.3 ml/min. Aliquots ( 1/100) of the purified 
substances were dissolved in ASW and applied to the isolated esophagus 
at the time indicated by arrows (C. D). 



(Fig. 3). The active substances appeared to be eluted as 
single peaks around 0.35 M NaCl (A) and 0.25 M NaCl 
(B). The final purification was performed on the C-18 
column with an isocratic elution of 17% ACN (A) and 
19% ACN (B) (Fig. 4). The respective single peaks with 
OD at 220 nm of 0.303 (A) and 0.085 (B) were eluted at 
23 min and 16 min after injection. The purified substances 
(1/100) elicited rhythmic contractions of the esophagus 
(Fig. 4C, D). 

Amino acid sequence analysis of the purified substances 
(A and B) revealed the structure to be the octapeptide Ala 
(216)-Lys (43.3)-Ser (8.7)-Gly (7.0)-Phe (5.9)- Val (1.7)- 



Arg (1.3)-Ile ( + ) and the heptapeptide Val (181.5)-Ser 
(66.0)-Ser (45.8)-Phe (118. 6)- Val (150.2)-Arg (28.2)-Ile 
(0.9), respectively (the figures are expressed in pmoles). 
The peptides of the respective sequences with C-terminus 
amidated were synthesized, and HPLC profiles of the syn- 
thetic peptides were compared with those of native ones. 
The synthetic and native peptides showed identical reten- 
tion times on the C-18 reversed-phase column and the 
cation-exchange column (data not shown). Furthermore, 
the mixture of the synthetic and native peptides was eluted 
as a single peak on each column (Fig. 5). The respective 
synthetic peptides evoked contraction of the Perinereis 
esophagus in the similar manner to the corresponding 
native peptides (Figs. 6, 7). The threshold concentrations 
for the synthetic peptides to evoke contraction were found 
to be between 10~' M and 10~ 9 M for both peptides. 

Thus, the structures of substance A and B were con- 
cluded to be AKSGFVRIamide and VSSFVRIamide, re- 
spectively. Both AKSGFVRIamide and VSSFVRIamide 
were members of S-Iamide peptides which had been pu- 
rified from one echiuroid and four mollusks (Table 1 ). 



10' 9 M 





S*N 




iLJ 



* 



.05 




20 40 20 40 

Time (min) 



Figure 5. HPLC profiles of mixtures of native and synthetic peptides. 
AKSGFVRIamide (A) and VSSFVRIamide (B) on a reversed phase col- 
umn with an isocratic elution of 17% ACN and 19% ACN at a flow rate 
of 0.3 ml/min. AKSGFVRIamide (C) and VSSFVRIamide (D) on a 
cation-exchange column with an isocratic elution of 0.27 At NaCl and 
0. 14 M NaCl in 10 mM phosphate buffer (pH 7. Data flow rate of 0.5 
ml/mm. 




2 min 



Figure 6. Comparison of the bioactivities of the native (N) and syn- 
thetic (S) peptides (AKSGFVRIamide) on the isolated esophagus of Per- 
inereis. The peptide solutions were applied at the time indicated by arrows. 
The concentration of the native peptide was estimated by comparing its 
peak height on HPLC with that of the synthetic peptide. 



220 



O. MATSUSHIMA ET AL 

A Ll B 



f ~^-i^-J^ 



, > 

* 



10- 8 M 





10' 7 F 





N 



2 min 



Figure 7. Comparison of the bioactivities of the native (N) and syn- 
thetic (S) peptides (VSSFVRIamide) on the isolated esophagus of Pcri- 
nereis. The peptide solutions were applied at the time indicated by arrows. 
The concentration of the native peptide was estimated by comparing its 
peak height on HPLC with that of the synthetic peptide. 



These S-Iamide peptides and some fragment peptides were 
examined on the Pcrinereis esophagus (Fig. 8). All of the 
S-Iamide family peptides and the fragment peptides more 
or less elicited rhythmic contractions of the esophagus at 
10~ 7 M. 

The biological activities of the two S-Iamide peptides 
isolated from Pcrinereis were examined on three muscle 
systems, the inner circular body-wall muscle of Urechis 
(Fig. 9). the ABRM of Mytilnx (Fig. 10) and the radula 
retractor muscle of Fusinus (Fig. 1 1 ). In the Urechis mus- 



Table 1 



S-Iamide peptides 


Phyla 


Species 


Structures 


Annelida 


Pcrinereis vancaurica 


AKSGFVRIamide 






VSSFVRIamide 


Echiura 


I'rec/iis WHcvmvin 


ASSFVRIamide 






PSSFVRIamide 






VSSFVRIamide 


Mollusca 


Fiifinin Icirugineus 


LSSFVRIamide 




Helix pomatia 


TSSFVRIamide 




Achatina fulica 


SPSSFVRIamide 






APSNFIRIamide 




.inmlimta cygnea 


SGFVRIamide 






t t 4 

10 ? M AKSGFVRIa 10 7 M APSNFIRIa 10' 7 M SPSSFVRIa 10 7 M ASSFVRIa 







I I 

10 'M PSSFVRIa 10 7 M VSSFVRIa 10 7 M LSSFVRIa 10 'M TSSFVRIa 




10 7 M SGFVRIa 10' 7 M SSFVRIa 10 7 M SFVRIa 



I 
10 



M FVRIa 



Figure 8. The actions of 1CT 7 M of various S-Iamide peptides and 
some fragment peptides on the isolated Pcrinereis esophagus. Each pep- 
tide was applied at the time indicated by arrows. 



cle. the twitch contraction evoked by electrical stimulation 
was inhibited by the S-Iamide peptides; AKSGFVRIamide 
was less potent than VSSFVRIamide (Fig. 9). The phasic 
contraction of the Mytilus ABRM evoked by repetitive 
electrical stimulation was inhibited by the peptides (Fig. 
10). The effects of these two peptides on Fusinus muscle 
were somewhat complicated. AKSGFVRIamide poten- 
tiated twitch contractions at the concentration of 10~ 7 M. 
but inhibited at 10~ 5 M. VSSFVRIamide. on the other 
hand, did not show any augmentation of the twitch con- 
traction, but inhibited at concentrations higher than 
10~ 6 M(Fig. 11). 



10' 7 M 
AKSGFVRIa 




1 min 




4 

10' 7 M 
VSSFVRIa 

Figure 9. Effects of AKSGFVRIamide and VSSFVRIamide on twitch 
contraction of the inner circular muscle of the body wall of Vrechis. The 
upward arrows indicate application of the peptides. The downward arrows 
indicate washing-out of the peptides. The twitch contraction was evoked 
by an electrical pulse (20 V, 3 msec). 



S-IAMIDE PEPTIDES OF AN ANNELID 



221 



t 

10" 7 M 
AKSGFVRIa 



10' 6 M 



* 



I J 



5g 



2 mm 



B 



t JV - * J 
10' 7 M 
VSSFVRIa 



t 



I 
10- 5 M 



* J 



5g 



Figure 10. Effects of AKSGFVRIamide and VSSFVRIamide on 
phasic contraction of the ABRM of .\fytilus. The upward arrows indicate 
application of the peptides. The downward arrows indicate washing-out 
of the peptides. The phasic contraction was evoked by repetitive electrical 
pulses (15V. 3 msec, 10 Hz, 50 pulses). 



Discussion 

The principal aim of this study was to find out authentic 
bioactive peptides in annelids. We isolated two S-Iamide 
family peptides, AKSGFVRIamide and VSSFVRIamide, 
from the polychaete annelid, Perinercis. Both the peptides 
showed a contractile effect on the esophagus of the ani- 
mal. AKSGFVRIamide is the novel peptide, and 
VSSFVRIamide has previously been found in the ventral 
nerve cord of the echiuroid worm, Urechis. S-Iamide pep- 
tides have been found so far in one species of Echiura 
and four species of Mollusca (Ikeda et a/.. 1991; Kuroki 
el al.. 1992; Muneoka and Kobayashi, 1992; in prep, for 
Anodonta S-Iamide peptide), as listed in Table I. Thus, 
S-Iamide peptides have been proven to be distributed 
among at least three invertebrate phyla and may range 
throughout the Protostomia. 

The C-termini of the two S-Iamide peptides identified 
in the current study were concluded to be amidated. 
though the purified substances were not subjected to fast 
atom bombardment mass spectrometry. Since the C-ter- 
mini of all the S-Iamide peptides so far isolated from the 
echiuroid and molluscs have been known to be amidated. 
we synthesized AKSGFVRIamide and VSSFVRIamide 
and compared their behavior on HPLC and contractile 
activity with those of the purified native peptides. As a 
result, the identical properties of the native and synthe- 
sized peptides were confirmed. 

The synthetic tetra- and pentapeptides, FVRIamide and 
SFVRIamide, showed only a slight activity for induction 



of the spontaneous contraction in the esophagus of Per- 
inereis. However, the synthetic hexapeptide, SSFVRIam- 
ide (a common structure for most of the S-Iamide pep- 
tides), was active, suggesting that at least six amino acid 
residues would be important for the expression of the ac- 
tivity of S-Iamide peptides. However, SGFVRIamide 
which has been isolated from Anodonta showed weak 
contractile activity in the esophagus. The substitution of 
the amino acid residue, Ser, with Gly seems to be dele- 
terious for contractile activity, and the N-terminal elon- 
gation of SGFVRIamide by Ala-Lys might cancel the del- 
eteriousness. 

The effect of AKSGFVRIamide on twitch contractions 
of the radula retractor of Fusinus was somewhat compli- 
cated. That is, the peptide potentiated the contractions at 
10~ 7 M, but inhibited at 10 5 M. The well-known mol- 
luscan neuropeptide FMRFamide has been known to po- 
tentiate the contractions of the same muscles (Kuroki et 
al.. 1992). Since the C-terminal tetrapeptide sequence of 
the S-Iamide peptide. -FVRIamide, is closely related to 
that of FMRFamide. the potentiating effect of 10~ 7 M 
AKSGFVRIamide may be attributable to the FMRFam- 
ide-like action of the S-Iamide peptide. The inhibition of 
the twitch contractions by 10~ 5 M of AKSGFVRIamide 
is probably the original action of the S-Iamide peptide. 
In this connection, Kuroki et al. ( 1992) reported that an- 
other S-Iamide peptide, LSSFVRIamide, isolated from 
the ganglia of Fusinus showed the same dose-dependent 
actions on the contractions as did AKSGFVRIamide. 

The physiological role of AKSGFVRIamide and 
VSSFVRIamide in Peri nereis is not elucidated at present. 
The threshold concentrations of these two S-Iamide pep- 
tides to induce contraction of the esophagus were between 




AJI_ 4 

10' 7 M 
AKSGFVRIa 



IL^JIUL, 
10 5 M 



JilL 



19 



1 min 



I 

10' 7 M 
VSSFVRIa 



0.5 g 



10' 6 M 



10' 5 M 



Figure 11. Effects of AKSGFVRIamide and VSSFVRIamide on 

twitch contractions ot the radula retractor muscle of Fusinus. The upward 
arrows indicate application of the peptides. The downward arrows indicate 
washing-out of the peptides. The twitch contractions were evoked by a 
train of electrical pulses (15 V, 1 msec, 0.2 Hz, 5 pulses). 



222 



O. MATSUSHIMA ET AL. 



1(T 10 A/and 10 9 M. It seems to be probable that these 
S-Iamide peptides are neuropeptides which regulate the 
gut-motility in the annelid. It has been demonstrated that 
FMRFamide is present in annelids such as Nereis virens 
(Krajniak and Price, 1990), Nereis diversicolor (Baratte 
el al. 1991) and Himdo mcdicinalis (Evans el al.. 1991) 
and that the tetrapeptide relaxed spontaneous and elec- 
trically-induced contractions of the esophagus (Krajniak 
and Greenberg, 1992). Thus, the action of FMRFamide 
on the esophagus of polychaete annelid is opposite to those 
of the S-Iamide peptides. This was also the case for most 
of the molluscan muscles examined (Muneoka and Ko- 
bayashi, 1992). Therefore, FMRFamide and the S-Iamide 
peptides may regulate the esophagus-motility in an an- 
tagonistic manner, and this regulatory relation may be 
also applied to the molluscan muscles. 

The classical neurotransmitters such as norepinephrine, 
epinephrine, acetylcholine, 5-hydroxytryptamine and 7- 
aminobutyric acid also seem to regulate gut-motility in 
the polychaete annelid, Chaetopterus variopedatus (Anctil 
et al., 1984), and the presence of catecholamines (dopa- 
mine, norepinephrine and epinephrine) has been reported 
in the nervous and intestinal tissues of the same species 
(Anctil et al., 1990). Thus, peptides such as FMRFamide 
and the S-Iamide peptides, and classical transmitters seem 
to regulate the gut-motility harmoniously in annelids. 
Further study is necessary to reveal the relationship be- 
tween the physiological role of classical transmitters and 
peptides. 

Acknowledgments 

The authors wish to express their thanks to Professor 
Yojiro Muneoka (Hiroshima University) for his kind ad- 
vice regarding the present study. 

Literature Cited 

Alumets, J., R. Hakanson, F. Sundler, and T. Thorell. 1979. Neuronal 
localisation of immunoreactive enkephalin and 0-endorphin in the 
earthworm. Nature 279: 805-806. 

Anctil, M., M. Laberge, and N. Martin. 1984. Neuromuscular phar- 
macology of the anterior intestine of Chaetopterus variopedatus, a 
filter-feeding polychaete. Coinp. Biochem. Physiol. 79: 343-35 1 . 

Anctil, M., J.-P. De Waele, M.-J. Miron, and A. K. Pani. 
1990. Monoamines in the nervous system of the tube-worm Chae- 
topterus variopedatus (Polychaeta): Biochemical detection and se- 
rotonin immunoreactivity. Cell Tissue Res 259: 81-92. 

Baratte, B., H. Gras-Masse, G. Ricart, P. Bulet, and N. Dhainaut-Cour- 
tois. 1991. Isolation and characterization of authentic Phe-Met- 
Arg-Phe-NH 2 and the novel Phe-Thr-Arg-Phe-NH, peptide from 
Nereis diversicolor. Ew. J Biochem. 198: 627-633. 

Carraway, R., S. E. Ruane, and H. R. Kim. 1982. Distribution and 
immunochemical character of neurotensin-like material in represen- 
tative vertebrates and invertebrates: Apparent conservation of the 
COOH-terminal region during evolution. Peptides 3: 1 15-123. 



Dhainaut-Courtois, N., M. P. Dubois, G. Tramu, and M. Masson. 
1 985. Occurrence and coexistence in Nereis diversicolor O. F. Muller 
(Annelida Polychaeta) of substances immunologically related to ver- 
tebrate neuropeptides. Cell Tissue Res 242: 97-108. 

Diaz-Miranda, L., G. Escalona de Motta, and J. E. Garcia-Arraras. 

1991. Localization of neuropeptides in the nervous system of the 
marine annelid Sabellastarte magnifica. Cell Tissue Res 266: 209- 
217. 

Diaz-Miranda, L., G. Escalona de Motta, and J. E. Garcia-Arraras. 

1992. Monoamines and neuropeptides as transmitters in the sed- 
entary polychaete Sabellastarte magnifica: Actions on the longitudinal 
muscle of the body wall. J Exp. Zooi 263: 54-67. 

Engelhardt, R. P., N. Dhainaut-Coutois, and G. Tramu. 1982. Im 
munohistochemical demonstration of a CCK-like peptide in the ner- 
vous system of a marine annelid worm. Nereis diversicolor O. F. 
Muller. Cell Tissue Res 227: 401-41 1. 

Evans, B. D.. J. Pohl, N. A. Kartsonis, and R. L. Calabrese. 
1991. Identification of RFamide neuropeptides in the medicinal 
leech. Peptides 12: 897-908. 

Holmgren, S. 1989. Gut motility. Pp. 231-255 in The Comparative 
Physiology oj Regulatory Peptides. S. Holmgren, ed.. Chapman and 
Hall, New York. 

Ikeda, T., I. kubota, V. kitajima, and V. Muneoka. 1991. Structures 
and actions of neuropeptides isolated from an echiuroid worm, L're- 
chis vnicinctus. Pp. 29-41 in Comparative Aspects of Neuropeptide 
Function. E. Florey and G. B. Stefano, eds.. Manchester University 
Press. Manchester. 

Ikeda, T., V. kuroki, I. kubota, H. Minakata, k. Nomoto, \V. Miki, T. 
kiss, L. Hiripi, and V. Muneoka. 1991. SSFVRIamide peptides 
A new family of neuropeptides distributed interphyletically. Pp. 65- 
70 in Peptide Chemistry. A. Suzuki, ed.. Protein Research Foundation. 
Osaka, Japan. 

kaloustian, k. V., and J. A. Edmands. 1986. Immunochemical evidence 
for substance P-like peptide in tissues of the earthworm Lumhnciis 
lerreslris: Action on intestinal contraction. Comp. Biochem. Physio/ 
83C: 329-333. 

krajniak, k.. and D. A. Price. 1990. Authentic FMRFamide is present 
in the polychaete Nereis virens. Peptides 11: 75-77. 

krajniak, k., and M. J. Greenberg. 1992. The localization of 
FMRFamide in the nervous and somatic tissues of Nereis virens and 
its effects upon the isolated esophagus. Comp. Biochem Physiol. 101C: 
93-100. 

kuroki, Y., T. kanda, I. kubota, T. Ikeda, Y. Fujisana, H. Minakata, 
and Y. Muneoka. 1992. FMRFamide-related peptides isolated from 
the prosobranch mollusc Fusinus femtgineus. J. Biol Hung. 43: 491- 
494. 

Muneoka, Y., and M. kobayashi. 1992. Comparative aspects of struc- 
ture and action of molluscan neuropeptides. Experienlia 48: 448- 
456. 

Muneoka, Y., Y. kuroki, H. Minakata, T. Ikeda, Y. Fujisawa, k. Nomoto, 
and I. kubota. 1991 . Structure and pharmacological characterization 
of a molluscan neuropeptide related to the crustacean RPCH. Pp. 
274-279 in Molluscan Neurobiohgy. K. S. Kits, H. H. Boer, and J. 
Joose. eds. North Holland, Amsterdam. 

Price, D. A., and M. J. Greenberg. 1977. Structure of a molluscan 
cardioexcitatory neuropeptide. Science 197: 670-671. 

Tashiro, N., and H. kuriyama. 1978. Neurosecretion and pharmacology 
of the nervous system. Pp. 207-242 in Physiology of Annelids. P. J. 
Mill, ed. Academic Press, London. 

Thorndyke, M. C. 1989. Peptides in invertebrates. Pp. 203-228 in The 
Comparative Physiology of Regulatory Peptides. S. Holmgren, ed. 
Chapman and Hall, New York. 



Reference: Bio/. Bull. 184: 223-229. (April, 1993) 



Photosynthesis and Retention of Zooxanthellae and 

Zoochlorellae Within the Aeolid Nudibranch 

Aeolidia papillosa 

F. K. McFARLAND AND G. MULLER-PARKER 1 

Shannon Point Marine Center, Western Washington University. 1900 Shannon Point Road. 

Anaeortes, Washington 98221 



Abstract. Both zooxanthellae and zoochlorellae are 
found in the cerata of Aeolidia papillosa after it has in- 
gested symbiotic Anthoplewa elegantissima containing 
these algae. High rates of photosynthesis were found in 
algae present in the cerata and in algae isolated from nu- 
dibranch feces. For algal cells present in the cerata of nu- 
dibranchs collected in June 1991, carbon fixation by 
zooxanthellae ( 1 . 18 0.36 pg C/cell/h) was significantly 
greater than carbon fixation by zoochlorellae (0.55 0.32 
pg C/cell/h). Algal densities within the cerata of laboratory 
fed nudibranchs were significantly greater for zoochlo- 
rellae (175 82 cells/Mg protein, light treatment; 131 
106 cells/^g protein, dark treatment) than for zoox- 
anthellae (38 18 cells/^g protein, light; 53 30 cells/ 
^g protein, dark). Ceratal densities of zooxanthellae (16 
8 cells/Mg protein) in the field during January 1992 
were low in comparison to ceratal densities in the labo- 
ratory several of the nudibranchs in the field lacked any 
symbiotic algae, and zoochlorellae were always absent. 
Nudibranch algal densities were not stable and dropped 
rapidly if the nudibranchs were starved. Both zoochlorella 
and zooxanthella densities dropped to cells///g protein 
within 1 1 days of starvation. While these results show 
that the relationship between A. papillosa and the two 
algae is not a stable symbiosis, the photosynthetic activity 
of the algae in the cerata suggests that the nudibranch 
and/or the algae may benefit from the association while 
it lasts. 

Introduction 

Several aeolid nudibranchs, as well as other nudi- 
branchs with cerata, contain zooxanthellae of the genus 

Received 6 July 1992; accepted 25 January 1993. 

1 Author to whom reprint requests should be addressed. 



Symbiodinium (Rudman, 1981a. b, 1982; Kempf, 1984, 
1991). Each ceras contains a diverticulum of the digestive 
gland within which the algal symbionts are both extra- 
and intracellularly located (Rudman, 1982; Kempf, 1984. 
1991). Many of these nudibranchs obtain their algae 
through ingestion of marine cnidarians which are sym- 
biotic with zooxanthellae. Zooxanthellae in the cnidarian 
host fix carbon through photosynthesis, and then trans- 
locate much of this carbon to the animal's tissue (e.g.. 
Trench, 1979). The carbon available for translocation may 
represent as much as 95% of the amount fixed (Muscatine 
et ai. 1 984), and is used by the host for respiration, growth, 
and reproduction (Kevin and Hudson, 1979; Davies, 
1984; Rinkevich, 1989). 

The ability of zooxanthellae to fix and translocate 
carbon in nudibranchs, as well as the benefits of such 
an association to the nudibranchs, have been described 
for several relationships. Grassland and Kempf (1985) 
reported that zooxanthellae in the tropical nudibranch 
Alelibe pilosa fixed large amounts of carbon (5.85 mg 
C/mg chlorophyll a/h), and that fixed carbon was 
translocated to the nudibranch for growth and re- 
production. Kempf (1990) reported that the aeolid 
nudibranch Berghia verrucicornis produced 1.7 times 
more eggs when in a symbiotic relationship with 
zooxanthellae than when algae-free. At high densities, 
zooxanthellae in the temperate nudibranch Pteraeolidia 
ianthina can supply carbon well in excess of the nudi- 
branch's respiratory demand during the spring and 
summer (H0egh-Guldberg and Hinde, 1986; H0egh- 
Guldberg et al. 1986). With the exception of the nu- 
dibranch Pteraeolidia ianthina. only tropical species 
have been studied, and all the studies have focused on 
species with zooxanthellae symbionts. 



223 



224 



F. K. McFARLAND AND G. MULLER-PARKER 



The temperate nudibranch Aeolidia papillosa is found 
within the intertidal zone of the northeastern Pacific 
(Kozloff, 1983) where one of its preferred prey species is 
the symbiotic anemone Anthopleura elegantissima (Wa- 
ters, 1973; Edmunds el ai. 1974; McDonald and Nybak- 
ken. 1978). A. elegant issima forms symbiotic relationships 
with both zooxanthellae and the unicellular green algae 
called zoochlorellae (Muscatine, 1971). Fixation and 
translocation of carbon by zooxanthellae in A. elegant is- 
sima is substantial (Trench 1 97 1 a, b), and the contribution 
of these anemones to intertidal gross primary production 
is equal to that of temperate intertidal seaweed populations 
on an areal basis (Fitt ct ai, 1982). Zoochlorellae found 
in A. elegantissima and A. xanthogrammica are also pho- 
tosynthetically active (Muscatine, 1971; O'Brien, 1980). 
While both zooxanthellae and zoochlorellae fix carbon in 
their host species, zooxanthellae translocate much more 
of their fixed carbon to their host than do zoochlorellae. 
Zooxanthellae translocate on the order of 50% of the car- 
bon fixed while zoochlorellae translocate less than 5% 
(Muscatine, 1971; O'Brien, 1980). 

High densities of zooxanthellae and zoochlorellae are 
found in the cerata of A. papillosa after it has been fed 
symbiotic anemones containing these algae (Kellett and 
Wiederspohn, pers. comm.). The following study consid- 
ers the nature of the symbiotic relationship formed be- 
tween A. papillosa and both zooxanthellae and zoochlo- 
rellae. particularly the photosynthetic activity of these al- 
gae and the stability of their populations within the 
nudibranchs' cerata. 

Materials and Methods 

Collection and maintenance oj nudibranchs and 
anemones 

Specimens of A. papillosa were collected from the San 
Juan Islands, WA in June 199 1 and from the Port Orchard 
side of the Sinclair Inlet, WA in January 1992. All ane- 
mones used to feed the nudibranchs were collected from 
Skyline beach in Burrows Bay at Anacortes, WA. No nu- 
dibranchs were found on this beach. Individual nudi- 
branchs were maintained in separate plastic mesh con- 
tainers submerged in flow-through seawater tables at 
Shannon Point Marine Center (Anacortes, WA). Seawater 
tables were cleaned twice weekly. The temperature of the 
water during June 1991 ranged from 10.7C to 13.0C 
with a mean of 1 1.6C. During January 1992 the tem- 
perature ranged from 7.5C to 9.2C with a mean of 
8.4C, and during February 1992 the temperature ranged 
from 8.2C to 10.9C with a mean of 9. 1 C. The average 
salinity during the study was 29 %o. 

Specimens of A. papillosa collected in June 1991 were 
used to determine the productivity of symbiotic algae. 
Continuous light provided to nudibranchs by a bank of 



two fluorescent lamps averaged 28 yumol photons/nr/s at 
the water's surface (LiCor cosine quantum sensor, 400- 
700 nm PAR). One group of nudibranchs was fed brown 
A. elegantissima containing zooxanthellae, another group 
was fed green A. elegantissima containing zoochlorellae, 
and the control group was fed white (algae-free) A. ele- 
gantissima. Personal experience has shown that brown 
anemones always contain at least 98% zooxanthellae (on 
a cell basis) and green anemones contain at least 98% 
zoochlorellae. This was confirmed during the experiment 
by periodic microscopic examinations of tentacle squashes 
from brown and green anemones. 

Specimens of A. papillosa collected in January were 
separated into two groups of twelve to examine retention 
of zooxanthellae and zoochlorellae in the cerata. The ini- 
tial algal complement of field nudibranchs was determined 
by sampling two cerata from each nudibranch within 24 
h of collection. One group was maintained in continuous 
darkness, and the other group was maintained under 12 
h light/ 12 h dark. During the light cycle, irradiance at the 
water's surface averaged 33 /imol photons/m : /s. Each 
group in the light and the dark treatments was further 
separated into three treatments of 4 nudibranchs each. 
One group was fed brown anemones for 28 days, then 
had its diet switched to green anemones for 1 3 days, and 
was then starved. Another group was fed green anemones 
for 28 days and was then switched to a diet of brown 
anemones. The third group was fed brown anemones for 
28 days and was then starved. Each fed nudibranch was 
given 5 anemones per week, provided individually on 
separate days. Fed nudibranchs had no more than two 
consecutive days without feeding. 

Productivity of symbiotic algae 

Algae within the cerata. To determine whether zooxan- 
thellae and zoochlorellae remain photosynthetically active 
within the cerata of the nudibranchs, 3 cerata (one an- 
terior, one middle, and one posterior) were removed from 
each nudibranch and incubated whole with I4 C in 20 ml 
glass scintillation vials. 2.0 ml of filtered seawater (FSW) 
and 0. 1 fid I4 C bicarbonate were added to each vial. The 
cerata were incubated at room temperature (20-24C) at 
249 jumol photons/nr/s for 50-120 min. Control vials 
for dark carbon fixation were also maintained for each 
I4 C experiment. Replicate vials of each treatment were 
wrapped with black electrical tape and incubated under 
the same conditions as light vials. The dark vials were 
used to correct for dark carbon fixation. To determine 
total activity (TA), 100 fil was subsampled from each vial 
and placed in a 7 ml plastic scintillation vial with 5 ml 
of Ecolume (ICN) scintillation fluid. Incubations were 
terminated by removing the cerata and washing them with 
several rinses of FSW. The cerata were then homogenized 



SYMBIOTIC ALGAE IN A. PAPILLOSA 



225 



in 1.5 ml of FSW using a 5-ml Wheaton tissue grinder. 
Two 500 jul subsamples were taken from each homogenate 
solution and placed in separate plastic scintillation vials. 
All unfixed I4 CO : was evolved from the homogenate sub- 
samples by adding 300 n\ of 6N HC1 and then placing the 
vials under a heat lamp in a fume hood for 1 h. The 
subsamples were neutralized with 300 n\ 6N NaOH prior 
to the addition of 5 ml scintillation fluid. The homogenate 
subsamples were counted along with the TA subsamples 
in a Packard TR 1900 scintillation counter using the au- 
tomatic DPM mode. The remaining homogenate suspen- 
sion was used for algal cell counts and then frozen for 
future protein analysis. 

Algae isolated from nudibranchfeces. Nudibranchs fed 
symbiotic A. elegantissima produced green or brown fecal 
pellets consisting mainly of intact symbiotic algae. Fresh 
fecal pellets were collected from nudibranchs and were 
suspended in FSW. Fecal algae were washed three times 
in FSW by centrifugation and resuspension. The final 
suspension was sequentially filtered through 73 jum and 
20 ,um Nitex screening to remove debris. After initial cell 
counts algal densities were adjusted to 4-6 X 10 5 cells/ 
ml. The productivity of fecal algae was measured using a 
protocol similar to that described for the cerata with the 
following exceptions: 2.0 ml of either the green or the 
brown fecal algae suspension was placed in each 20 ml 
glass vial and the algal cells were allowed to incubate for 
30 min at room temperature (20-24C). Incubations were 
at an average irradiance of 249 ^mol photons/irr/s. 

Algae freshly isolated from anemones. The productivity 
of algae isolated directly from A. elegantissima was also 
determined. The oral disk and tentacles of individual 
anemones were excised and homogenized using a tissue 
grinder. Algal cell suspensions were washed and filtered 
as described above. Final algal densities ranged from 
2.5-6 X 1 5 cells/ml. Incubations were performed as above 
except that cells were allowed to incubate for up to 1 h 
at an average irradiance of 102 ^mol photons/ nr/s. 

Algal densities and replacement within the cerata 

Algal population density within the cerata was mea- 
sured twice each week by removing 2 cerata (one posterior 
and one anterior) from each experimental nudibranch 
during January and February 1992. The cerata were ho- 
mogenized in 1.5 ml of cold FSW using a 2-ml Wheaton 
tissue grinder. Algal cell counts of the homogenate solu- 
tions were determined using a hemocytometer, and the 
remaining homogenate solutions were frozen for future 
protein analysis. Protein analysis was performed using the 
Lowry method (Lowry el al.. 1951) and bovine serum 
albumin (BSA) standards with the modification that the 
homogenates and standards were pre-treated at 30C for 
30 min in 0. 1 N NaOH to solubilize the proteins. Cell 



counts and protein content were used to determine cell 
densities within the cerata of nudibranchs fed green and 
brown A. elegantissima. 

Statistical analyses 

Comparison of photosynthetic rates. Photosynthesis 
data for algae in A. papillosa cerata, algae freshly isolated 
from A. papillosa feces, and freshly isolated from A. ele- 
gantissima were analyzed to determine if there was a sig- 
nificant difference in the rates of carbon fixation for zoo- 
xanthellae and zoochlorellae. Zooxanthellae rates of 
carbon fixation were compared to zoochlorellae carbon 
fixation rates using two-sample t-tests. Algae in cerata. 
algae from feces, and algae from anemones were all com- 
pared separately. Comparisons were also made of the 
photosynthetic rates of zooxanthellae and zoochlorellae 
between the different treatments. 

Comparison of algal densities. Algal densities in A. 
papillosa cerata after 28 days of feeding the nudibranch 
either brown or green anemones were analyzed using two- 
sample t-tests to determine if there was a significant dif- 
ference in the densities of zooxanthellae and zoochlorellae 
found in the nudibranchs both under light and dark con- 
ditions. When zooxanthellar densities were compared to 
zoochlorellar densities, the data were logarithmically 
transformed to correct for differences in variance between 
the algal types. The effect of light versus dark on zooxan- 
thellar and zoochlorellar densities was also analyzed using 
two-sample t-tests. 

Comparison of treatment effect on algal replacement. 
Repeated-measures analysis of variance (Potvin and 
Lechowicz, 1990) was used to analyze the effect of light 
versus dark on the replacement (after switching diets) and 
expulsion (during starvation) of algae in the cerata. Zoo- 
xanthellae data for the replacement of zoochlorellae with 
zooxanthellae and the expulsion of zooxanthellae were 
logarithmically transformed to correct for differences in 
variance between the light and dark treatments. 

Results 

Productivity of symbiotic algae 

Both zooxanthellae and zoochlorellae remain photo- 
synthetically active within the nudibranch cerata (Fig. 1 ). 
where the mean rate of carbon fixation by zooxanthellae 
is significantly greater (P = 0.0216) than that of zoochlo- 
rellae. Cerata used for determining the photosynthetic rate 
of zooxanthellae contained 99.9% zooxanthellae on a cell 
basis. Cerata used for determining photosynthetic rate of 
zoochlorellae contained 99.5% zoochlorellae. 

Figure 1 also shows that algal symbionts isolated from 
nudibranch feces also had high photosynthetic rates. There 
is no significant difference (P = 0.078 1 ) between the pho- 



226 



1 k. VlcFARLAND AND G. MULLER-PARKER 



3.5-, 



3.0- 



= 2.5-1 
<U 

\ 2.0 ^ 
TJ 

0) 

x 1.5- 



CTl 
CL 



1.0- 

0.5- 
0.0 



I I zoochlorelloe 
ESS zooxanthellae 



(ns) 
n = 3 



CERATA 



FECES 



n = 7 



* 



ANEMONE 



Figure 1. Mean rates of photosynthesis tor algae incubated within 
whole cerata (.1. papillosa) and for algae isolated from .-( papillosa feces 
and from symbiotic anemones (A. elegantissima). Photosynthesis was 
determined in July 1991 at an average irradiance of 249 fjmol/m : /s for 
cerata and fecal algae and in May 1991 at an average irradiance of 102 
nmol/nr/s for anemone algae. Vertical lines represent 95'" confidence 
intervals. Numbers above each bar represent treatment size. *. P < 0.05; 
**, P< 0.0001. 



ingested an average of 4.5 anemones each per week re- 
gardless of whether they were fed anemones with zooxan- 
thellae or zoochlorellae. 

Comparisons of algal densities between the light and 
dark treatments showed no effect from light on the den- 
sities of zooxanthellae or zoochlorellae in nudibranchs 
fed brown or green anemones, respectively. Zooxanthellae 
densities in nudibranchs maintained in the light (38 18 
cells/jug protein) were not significantly different (P 
= 0.3063) from those in nudibranchs maintained in the 
dark (53 30 cells//^g protein). Zoochlorellae densities 
in the light treatment (175 82 cells/j/g protein) were 
not significantly different (P = 0.3339) from those in the 
dark treatment (131 + 106 cells/^g protein). 

Nudibranchs fed green anemones contained signifi- 
cantly higher algal densities than nudibranchs fed brown 
anemones (P = 0.0004 for nudibranchs maintained in the 
light: P = 0.0152 for nudibranchs maintained in the dark). 
After 28 days of feeding on one type of anemone, the 
cerata of nudibranchs fed brown anemones contained 
99.9% zooxanthellae and the cerata of nudibranchs fed 
green anemones contained 99.0% zoochlorellae. 



tosynthetic rate of zooxanthellae and that of zoochlorellae 
isolated from the nudibranch feces. But there is a signif- 
icant difference (P < 0.000 1 ) between the photosynthetic 
rate of zooxanthellae and the photosynthetic rate of 
zoochlorellae isolated from A. elegantissima. 

In cross comparisons, photosynthesis by fecal zoochlo- 
rellae is significantly higher than photosynthesis of both 
zoochlorellae in the cerata (P = 0.0369) and zoochlorellae 
from the anemone (P = 0.0033). Photosynthetic rates are 
not significantly different between zoochlorellae in the 
cerata and zoochlorellae isolated from the anemone (P 
= 0.5493). For zooxanthellae. photosynthesis in the cerata 
is significantly lower than photosynthesis by both fecal 
zooxanthellae (P = 0.0375) and anemone zooxanthellae 
(P = 0.0003). The latter two are not significantly different 
from each other (P = 0.6772). 

Algal densities within I/re cerata 

Algal densities in freshly collected nudibranchs (field) 
during January 1992 averaged 16 zooxanthellae/^g pro- 
tein and ranged from to 69 zooxanthellae/^g protein. 
Only zooxanthellae were found within the cerata of the 
24 nudibranchs collected from one beach on the Sinclair 
Inlet during a single low tide. No symbiotic algae were 
found in the cerata of nudibranchs collected in June 1991. 
These nudibranchs were collected from beaches where 
symbiotic anemones were not available as a food source. 
The algal densities in the cerata of field nudibranchs were 
low in comparison to those of nudibranchs regularly fed 
svmbiotic anemones in the laboratory. These nudibranchs 



Replacement of algal populations in the cerata 

Algal densities within the cerata do not remain constant 
over time and depend on the algal complement of the 
food source. When the nudibranchs were switched from 
a diet of anemones containing zoochlorellae to a diet of 
anemones containing zooxanthellae. the zoochlorellae 
within the cerata were completely replaced by zooxan- 
thellae within 24 days (Fig. 2), after approximately 15 
anemones containing zooxanthellae had been ingested per 
nudibranch. There is no significant difference between 
the light and dark treatments in either the loss of zoochlo- 
rellae (P = 0.8338) or the gain of zooxanthellae (P 
= 0.056 1 ). 

The replacement of zooxanthellae with zoochlorellae 
in the cerata required even less time. Upon switching the 
diet of nudibranchs from anemones containing zooxan- 
thellae to anemones containing zoochlorellae, the zoo- 
xanthellae were replaced by zoochlorellae within 13 days 
(Fig. 3), after approximately 8 anemones with zoochlo- 
rellae had been ingested per nudibranch. Again, there is 
no significant difference between light and dark treatments 
in either the loss of zooxanthellae (P = 0.7988) or the 
gain of zoochlorellae (P = 0.6664). 

The time it took for each type of alga to be completely 
expelled from the cerata upon starvation was even less 
than the algal replacement times. All algae disappeared 
from the cerata within 1 1 days when nudibranchs con- 
taining either zooxanthellae or zoochlorellae were starved 
(Figs. 3, 4). There is no significant difference between light 
and dark treatments in either the expulsion of zooxan- 



SYMBIOTIC ALGAE IN A. PAPILLOSA 



227 





KHJ 

300 




Dark Treatment 


1 




c 


250 






A - - A ZOOWWTHELmE 


F 




0} 


















ZOOCHUMEI1AE 






~o 


200 










Q_ 












cn 


150 










=i 









S 


i 


^s, 
0) 


100 








\ 


o 

01 
< 


50 








J\I 4- -I 






4 


. 

! J , 


,, 




c 


300- 




_ight Treatment 


t 




CJ 












Q 


250- 












200- 






. 1 






150- 




c 


\ 








( 


)^ / 


\ 
C 






100- 








\ T 




50- 




^T 




?x4--A-^ i 




0- 


i 


1 


.-4 


' "f ]^l~3, '$ ' t 



5 10 15 20 25 30 35 40 45 50 55 

Day 

Figure 2. Mean algal densities in the cerata of nudibranchs initially 
ted anemones containing zoochlorellae, and then switched to anemones 
containing zooxanlhellae on day 28 of the experiment. Closed symbols 
represent dark treatments and open symbols represent light treatments. 
A = zooxanthellae; O = zoochlorellae. Vertical lines represent 95%- con- 
fidence intervals. The size of each treatment was 4 nudibranchs. # = day 
diet switched. 



thellae (P = 0.6674) or the expulsion of zoochlorellae (P 
= 0. 1 337) during starvation. 

Discussion 

Since both zooxanthellae and zoochlorellae obtained 
by the ingestion of Anlhop/eiira eleganlissima remain 
photosynthetically active within the cerata (Fig. 1), it is 
likely that Aeolidia papillosa derives some benefit from 
these algae. Because of the higher density of zoochlorellae 
than zooxanthellae in the cerata, the actual amount of 
carbon fixed per ceras is not as different as the algal pro- 
ductivities would imply. The lack of any significant dif- 
ference between the photosynthetic rates of fecal zooxan- 
thellae and zoochlorellae may be due simply to the limited 
number of replicates. Although incubations were carried 
out at different irradiances (249 /umol photons/nr/s for 
ceratal and fecal algae, 102 /umol photons/irr/s for ane- 
mone algae), and algae in cerata are likely to receive lower 
light during incubations than the isolated alpae. both ir- 
radiances are well above the I k value determined for zoo- 
xanthellae and zoochlorellae (I k = 50 ,umol photons/nr/s) 



in independent experiments also conducted during the 
summer (Aagaard and Muller-Parker, unpub.). 

Zooxanthellae release much more of their photosyn- 
thate than do zoochlorellae within their primary hosts A. 
elegantissima and A. xanthogrammica(Moseatinz, 197 1 ; 
O'Brien, 1 980). If this is also true within the nudibranch's 
cerata, there may be an energetic advantage to the selec- 
tion of prey anemones containing zooxanthellae. A. pap- 
illosa does not appear to selectively retain one alga over 
the other since the expulsion of algae upon starvation was 
the same for both algae (Figs. 3, 4). The replacement of 
zoochlorellae with zooxanthellae took longer than the re- 
verse. This may have been due in part to the size difference 
of the algae; the larger zooxanthellae may have restricted 
the exit of smaller zoochlorellae from the diverticula 
within the cerata. 

Any benefits to the nudibranch of containing photo- 
synthetically active algae would be most evident under 
light conditions. Therefore, if the nudibranch had the 
ability to control expulsion rates, algae should be retained 
longer under light than under dark conditions, especially 





350 


## 




300 


Dark Treatment 




^ 








'QJ 


250 


A - - A ZOOXANTHELLAE 






o 




ZOOCHLORELLAE 


1 


1 




200 






L 


CL 






/ 


\ 


^ 


150 


4 

/ 


1 


\ 


"g" 


100 


/ 
T/ 




\l 


01 




I/ 


\l 


<^ 


50 

Q 




>. 


300- 


Light Treatment ## 


'(n 






c 








Q) 


250- 








Q 












200 


* 










\. 


) 






150- 


/ 


( 


) 




100- 


# rLl/ 










/I I 






50- 
0- 


f -- - I/I 

'i- "Vi 

rS 1 1 S 1 R-^l A: --A ,A 


l /O . Al , 



10 15 20 25 30 35 40 45 50 55 

Day 

Figure 3. Mean algal densities in the cerata of nudibranchs initially 
fed anemones containing zooxanthellae, then switched to anemones 
containing zoochlorellae on day 28 of the experiment, and then starved 
after day 41 of the experiment. Closed symbols represent dark treatments 
and open symbols represent light treatments. A = zooxanthellae; O 
= zoochlorellae. Vertical lines represent 95% confidence intervals. The 
size of each treatment was 4 nudibranchs. # = day diet switched; ## 
= day began starving. 



228 




F. K 


. McFARLAND 


150-1 




# 


# 


1 125 ' 








"o 








- 100 








1 75 ~ 








^ 50- 


1 


/ 


\ 


CO 

1 25- 
n- 


f 




V 



10 15 20 25 

Day 



30 



35 40 



Figure 4. Mean zooxanthellac densities in the cerata of nudibranchs 
initially fed anemones containing zooxanthellae. and then starved after 
day 28 of the experiment. Closed symbols represent dark treatments and 
open symbols represent light treatments. Vertical lines represent 95% 
confidence intervals. The size of each treatment was 4 nudibranchs. ## 
= day began starving. 



if the nudibranch is starved. The rapid expulsion of algae 
under both light and dark conditions suggests that A. pap- 
///osa has little control over the retention or expulsion of 
algae from its cerata, even when starved. 

The large numbers of healthy algal cells present in fecal 
pellets and the photosynthetic rates of the fecal algae (Fig. 
1 ) indicate that at least a portion of the algae consumed 
by the nudibranch pass unharmed through the digestive 
tract. Kempf (1984) found evidence of algal breakdown 
within the tissues of three tropical nudibranchs, but no 
evidence of active digestion in two additional species 
(Kempf 1984, 1991). Whether A. papillosa digests some 
of the ingested symbiotic algae is unknown, but at least 
a large number of the algae remain unaffected by passage 
through the nudibranch. Thus, the fecal material of A. 
papillosa may be important in the dispersal of algae and 
reinfection of temperate anemones as has been suggested 
for Berghia major, a tropical nudibranch that also feeds 
on symbiotic anemones (Muller Parker, 1984). 

Another possibility is that the algae are heterotrophic 
in the nudibranch and thus represent a liability. Zooxan- 
thellae isolated from the sea anemone Aiptasia pulchella 
are capable of heterotrophic growth under low light levels 
(Steen, 1987). The possibility of zoochlorellae being par- 
asitic in A. elcgantissima has been suggested by Muscatine 
(1971). Because of the generally low light levels in the 
Northeastern Pacific region, especially during winter 
months, algae within the cerata of the nudibranch may 
not be able to meet their carbon requirements photosyn- 
thetically. As such, it is possible that algae in A. papillosa 
are a benefit during the summer and a liability during the 
winter. 



Kempf (1990. 1991) suggested that the nudibranch 
Berghia verntcicornis has a primitive mutualistic sym- 
biosis with zooxanthellae based on the following obser- 
vations. Relatively high concentrations of zooxanthellae 
are found in all B. verntcicornis from the field. The zoo- 
xanthellae ( 1 ) reside in peri-algal vacuoles within the nu- 
dibranch's digestive cells, (2) do not appear to be digested 
along with their primary host Aiptasia pallida, (3) remain 
photosynthetically active within the nudibranch, and (4) 
appear to benefit the nudibranch in its reproductive effort. 
Kempf terms the relationship primitive because the sym- 
biosis is not permanent. The zooxanthellae are eventually 
exocytosed back into the gut and defecated in a healthy 
state when nudibranchs are starved in the laboratory. 

The relationship between zooxanthellae, zoochlorellae, 
and .-f . papillosa may also be a primitive form of symbiosis, 
possibly corresponding to a Type IV association as de- 
scribed by Kempf (1991). Both algae are photosyntheti- 
cally active within the nudibranch's cerata (Fig. 1 ). Rapid 
reduction in the density of each alga when it is no longer 
available in the nudibranch's food shows that frequent 
ingestion of symbiotic anemones is required to maintain 
the association. But A. papillosa does not appear to be 
obligately dependent on either alga at any period of the 
year. Several of the A. papillosa collected in the Sinclair 
Inlet lacked symbiotic algae in their cerata, and all of the 
A. papillosa collected in June 1991 on beaches where 
symbiotic Anthopleura sp. were not available, lacked 
symbiotic algae in their cerata. Ultrastructural investi- 
gations are needed to determine whether the algae are 
intracellular, and whether they reproduce while in the 
cerata. Translocation experiments to determine whether 
fixed carbon is utilized by the nudibranch for growth or 
reproduction will help to explain the nature of this rela- 
tionship. Our work to date suggests that A. papillosa will 
be a good model system for comparing and contrasting 
the symbiotic relationships between zooxanthellae and 
zoochlorellae and their animal host. 

Acknowledgments 

A portion of this study was supported by an NSF Re- 
search Experience for Undergraduates Site grant (OCE- 
9000676) to Shannon Point Marine Center and an NSF 
Instrumentation and Laboratory Improvement Program 
award (USE-905 1 180). The technical assistance of Katie 
McFarland was critical to the successful completion of 
the project. The initial work on the symbiotic relationships 
of.-), papillosa by Michael Kellett and David Wiederspohn 
( WWU undergraduates) provided the inspiration for this 
project. M. Kellett's assistance with collection of nudi- 
branchs is greatly appreciated. Brian Bingham provided 
invaluable assistance with the statistical analysis of the 
data. The suggestions of anonymous reviewers are also 
appreciated. 



SYMBIOTIC ALGAE IN ,1 PAPll.LOSA 



229 



Literature Cited 

Crossland, C. J., and S. C. Kempf. 1985. Carbon fixation and com- 

partmentation in the zooxanthellae containing nudibranchs. .\fclihc 

piloxa and Melihe -./> Proceedings of the Fifth International Coral 

Reel' Congress. Tahiti. Vol. 6: 125-130. 

Davies, P. S. 1984. The role of zooxanthellae in the nutritional energy 

requirements of Pocillopora eydmtxi. Coral Rech 2: 181-186. 
Edmunds, M.. G. \V. Potts, R. C. Swinfen, and V. L. Waters. 1974. The 
feeding preference of Aeolidia papillosa (L.) (Mollusca, Nudibran- 
chia). ./ Mar BID! .l.v.v. U.K. 54: 939-947. 

Fitt, VV. K., R. L. Pardy, and M. M. Littler. 1982. Photosynthesis, 
respiration, and contribution to community productivity of the 
symbiotic sea anemone Anthoplcura cIcKanlisfunni (Brandt. 1835). 
J. Y/>. Mar. Biol. Ecol. 61: 213-232. 

Hoegh-Guldberg, O., and R. Hinde. 1986. Studies on a nudibranch 
that contains zooxanthellae. I. Photosynthesis, respiration and the 
translocation of newly fixed carbon by zooxanthellae in Ptcracolidia 
iamhina Proc R Sue Loud B 228: 493-509. 

Hoegh-Guldberg, O., R. Hinde, and L. Muscatine. 1986. Studies on a 
nudibranch that contains zooxanthellae. II. Contribution of zoo- 
xanthellae to animal respiration (CZAR) in Pieraeolulia iamhina 
with high and low densities of zooxanthellae. Pmc. R Soc. Lond 
B 228: 511-521. 

Kempf, S. C. 1984. Symbiosis between the zooxanthella Synihiodinmm 
(=Gymnodinium) microadriaticum (Freudenthal) and four species 
of nudibranchs. Biol. Bull. 166: 110-126. 

Kempf, S. C. 1990. Is the association between the acolid nudibranch 
Berglua vemiacornis and a zooxanthella a true symbiosis'? Am. Zoo/. 
30: 99 A. 

Kempf, S. C. 1991. A 'primitive' symbiosis between the aeolid nudi- 
branch Berglua verrucicornis (A. Costa, 1987) and zooxanthellae. 
J. Mull. Si ud. 57: 75-85. 

Kevin, K. M., and R. C. L. Hudson. 1979. The role of zooxanthellae 
in the hermatypic coral Plesiastrea iirvillea (Milne-Edwards & 
Haime) from cold waters. J. Exp. Mar. Biol. Ecol. 36: 157-170. 

Kozloff, E. N. 1983. Pp. 186 and 246 in Seashore Lite ol the \oriliern 
Pacific Coast, University of Washington Press, Seattle. WA. 

Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 
1951 . Protein measurement with the Folin phenol reagent. ./. Biol. 
Chem. 193: 265-275. 



McDonald, G. R., and J. W. N> bakken. 1978. Additional notes on the 
food of some California nudibranchs with a summary of known 
food habits of California species. \'eliger 21: 110-119. 

Muller-Parker, G. 1984. Dispersal of zooxanthellae on coral reefs by- 
predators on enidarians. Biol Hull 167: 159-167. 

Muscatine. L. 1971. Experiments on green algae coexistent with zoo- 
xanthellae in sea anemones, Puc Sci. 25: 13-21. 

Muscatine, L., P. G. Kalkcmski, J. W. Porter, and Z. Dubinsky. 
1984. Fate of photosynthetic carbon in light- and shade-adapted 
colonies of the symbiotic coral Slylophora pixtillata. Pine R Soc. 
Lond. 5222: 181-202. 

O'Brien, T. L. 1980. The symbiotic association between intracellular 
zoochlorellae (Chlorophyceae) and the coelenterate Aniliopleiira 
xanthogrammica J E\p. /ool 211: 343-355. 

Potvin, C, and M. J. Lechowicz. 1990. The statistical analysis of eco- 
physiological response curves obtained from experiments involving 
repeated measures. Ecology 1\(4): 1389-1400. 

Rinkevich, B. 1989. The contribution of photosynthetic products to 
coral reproduction. Mar. Biol. 101: 259-263. 

Rudman, W. B. I98la. Further studies on the anatomy and ecology 
of opisthobranch molluscs feeding on the scleractinian coral Forties. 
/.ool. .1 Linn Sue 71: 373-412. 

Rudman, W. B. I981b. The anatomy and biology of alcyonarian-feeding 
aeolid opisthobranch molluscs and their development of symbiosis 
with zooxanthellae. Zoo/. J Linn Soc 72: 219-262. 

Rudman, W. B. 1982. The taxonomy and biology of further aeolidacean 
and arminacean nudibranch molluscs with symbiotic zooxanthellae. 
/.ool. J. Linn. Soc. 74: 147-196. 

Steen, R. G. 1987. Evidence for facultative heterotrophy in cultured 
zooxanthellae. Mar. Biol. 95: 15-23. 

Trench. R. K. 197la. The physiology and biochemistry of zooxanthellae 
symbiotic with marine coelenterates. I. The assimilation of photo- 
synthetic products of zooxanthellae by two marine coelenterates. 
Proc R Soc Lond. B 177: 225-235. 

Trench, R. K. 1971 b. The physiology and biochemistry- of zooxanthellae 
symbiotic with marine coelenterates. II. Liberation of fixed I4C by 
zooxanthellae in \-nro Proc R Soc. Lond. B 111: 237-250. 
Trench, R. K. 1979. The cell biology of plant-animal symbiosis. Ami. 

Rev. PI. Phyxiol. 30: 4X5-531. 

Waters, V. L. 1973. Food-preference of the nudibranch Aeolidia pup- 
illoxa. and the effect of the defenses of the prey on predation. I 'eliger. 
15: 174-192. 



Reference: Biol Bull 184: 2.10-242. (April, 1993) 



Biochemical Correlates of Estivation Tolerance in the 
Mountainsnail Oreohelix (Pulmonata: Oreohelicidae) 

BERNARD B. REES 1 AND STEVEN C. HAND 

Department of Environmental, Population ami Organismic Biology, University of Colorado. 

Boulder. Colorado 80309-0334 



Abstract. Biochemical changes occurring over 7 months 
of estivation were studied in two species of land snail, 
Oreohelix strigosa (Gould) and O. subrudis (Reeve), to 
determine whether differential mortality during estivation 
is related to different energetic strategies. Laboratory- 
maintained snails, which were fed ad libitum prior to es- 
tivation, were compared with snails collected from the 
field and induced to estivate without augmenting their 
energy reserves. In all groups, polysaccharide was catab- 
olized early in estivation, and protein was the primary 
metabolic substrate after polysaccharide reserves were de- 
pleted. Lipid was catabolized at a low rate throughout 
estivation. Rates of catabolism were largely statistically 
equivalent between species. Urea and purine bases ac- 
cumulated during estivation as a result of protein catab- 
olism, with the former being quantitatively more impor- 
tant. In both laboratory-maintained and field-collected 
snails, the rate of urea accumulation was greater in O. 
subrudis. resulting in higher tissue urea contents in this 
species at the end of the 7-month experiment. The tissue 
concentrations of urea at 7 months ranged from about 
150 to 300 mA/ and were positively correlated (r = 0.99, 
P = 0.006) with mortality in these snails. Methylamine 
compounds, a class of compounds that can offset disrup- 
tive effects of elevated urea, were measured in one group 
of O. strigosa at 7 months of estivation and found to be 
low relative to urea levels. We suggest, therefore, that in 
the absence of elevated levels of counteracting com- 
pounds, urea may reach toxic levels and may be one factor 
limiting the duration of estivation that is survived by these 
land snails. 



Received 2 June 1992; accepted 9 December 1992. 
1 Present address: Hopkins Marine Station, Department of Biological 
Sciences, Stanford University. Pacific Grove. CA 93950. 



Introduction 

The success of gastropod mollusks in terrestrial habitats 
has been due to various structural, physiological, and be- 
havioral specializations (Riddle, 1983). One specialization 
that is well developed among the pulmonate land snails 
is the capacity to enter the dormant state of estivation 
during periods of hot and dry environmental conditions. 
By entering estivation, snails are able to endure potentially 
desiccating climatic conditions until the return of more 
favorable conditions. Some species are capable of esti- 
vating for remarkable periods of time, ranging up to sev- 
eral years in duration (Stearns. 1877; Machin, 1967). 

There are limits to the duration of estivation that can 
be tolerated, though, and mortality eventually increases 
as estivation is prolonged. Because there is no intake of 
foodstuffs during estivation, the period of estivation that 
can be survived may be limited by the exhaustion of en- 
dogenous energy reserves (Pomeroy, 1969; Schmidt-Niel- 
sen el til.. 1971 ). Metabolic rate reduction, which would 
serve to prolong the energy stores of the animal, occurs 
during estivation, and desert-dwelling species display 
lower rates than species from more mesic environments 
(Schmidt-Nielsen el a/.. 1971; Herreid, 1977; Rees and 
Hand. 1990). These observations have been taken as sup- 
porting the idea that energy reserves are limiting. But since 
the rates of metabolism and evaporative water loss are 
highly correlated in land snails (Barnhart, 1986), the re- 
duction of metabolic rate may reflect an adaptation to 
conserve water rather than energy. A comparison of sur- 
vivorship in snails with differing levels of energy reserves 
prior to estivation would more clearly address the question 
of energy limitation. 

The duration of estivation may also be limited by the 
accumulation of noxious end-products of protein catab- 
olism. Depending upon the species and activity pattern. 



230 



BIOCHEMICAL CHANGES IN ESTIVATION 



231 



land snails can dispose of nitrogen derived from protein 
catabolism in the form of uric acid and other purines, 
urea or gaseous ammonia (Bishop el <//., 1983). In species 
that produce urea, this compound can reach very high 
levels in the tissues during estivation: levels of 260 jimol 
g ' wet mass (ca. 300 mM) have been measured in the 
tissues of Biilinnihts dealbatits (Home, 1971), and 440 
mAI in the blood of Strophocheilus oblongns (Tramell 
and Campbell, 1972). At these levels, urea can have sig- 
nificant deleterious effects on the function of several bio- 
logical processes (Yancey el til.. 1982: Yancey, 1985; 
Yancey and Berg, 1990). In other organisms displaying 
elevated tissue contents of urea, methylamine compounds, 
which can offset the disruptive effects of urea, are com- 
monly accumulated. It is not known whether methyl- 
amines accumulate during estivation in snails with high 
urea. If not, then urea could reach toxic levels and be a 
factor limiting the duration of estivation. 

In the present study, we have investigated the extent 
to which the exhaustion of energy reserves and the ac- 
cumulation of nitrogenous compounds correlate with 
mortality differences observed during laboratory estivation 
in two species of the mountainsnail Orcohclix. We mea- 
sured the biochemical composition of O slrigosa and O. 
xubrudis over a 7-month period of laboratory estivation. 
From these data we have estimated rates of catabolism of 
protein, polysaccharide, and lipid. We compared snails 
that had been fed ad libitum prior to estivation with snails 
that had been collected from the field and induced to 
estivate without feeding to ascertain the effects of elevated 
energy stores. We also measured the accumulation of ni- 
trogenous end-products of protein catabolism. Estivating 
snails were found to accumulate large quantities of urea, 
and we measured the tissue content of methylamines to 
address the possible counteraction of urea effects by these 
compounds. 

Finally, tolerance to desiccation under laboratory con- 
ditions has been correlated with the distribution of a va- 
riety of land snail species in nature, with the more tolerant 
species occurring in drier habitats (Machin, 1967; Cam- 
eron, 1970; Arad el a/., 1989). The genus Orcohclix is 
widely distributed in western North America, ranging 
from mesic riparian areas to semi-arid habitats (Bequaert 
and Miller, 1973; Rees, 1988). In the present study, we 
have characterized the climatic conditions prevailing at 
three collection sites in western Colorado, and we have 
evaluated the distribution of O. strigosa and O. xubrudix 
at these sites in light of their differing capacities for pro- 
longed laboratory estivation. 

Materials and Methods 

Collection sites 

Oreohelix spp. were collected in western Colorado along 
Mitchell and East Rifle Creeks. The snails from the 



Mitchell Creek drainage were collected along the east bank 
of the creek, approximately 100 m downstream of the 
Mitchell Creek Fish Hatchery (3942', 10722'W; 1850 
m). near Glen wood Springs, Colorado. Along the East 
Rifle Creek, snails were collected from areas located ap- 
proximately 1 km upstream and 3 km downstream of the 
Rifle Falls Fish Hatchery (3942', 10742'W; 2100 m). 
The upstream site was about 25 m west of the creek among 
rock slide rubble in Rifle Gorge, and the downstream site 
was adjacent to the creek at the Rifle Falls campground. 
The three collection locales will be referred to as the 
Mitchell Creek, Rifle Gorge, and Rifle Falls sites. The 
Mitchell Creek site has previously been referred to as the 
Glenwood Springs collection site (Rees, 1988). 

The climatic conditions prevailing during the summer 
months in the Mitchell Creek and East Rifle Creek drain- 
ages are shown in Table I. Further information on the 
conditions at the two Rifle sites was obtained with a hand- 
held temperature-humidity sensor on several days during 
the summers of 1 990 and 1 99 1 . Measurements were made 
2-5 cm above the ground between 06:00 and 08:00, and 
again between 13:00 and 16:00 h. On average, the early 
morning humidity was 4% higher, and the mid-day hu- 
midity was 5% higher, at the Rifle Falls than at the Rifle 
Gorge site. Taken together, these data illustrate that mois- 
ture availability at the three collection sites decreases in 
the order Mitchell Creek > Rifle Falls > Rifle Gorge. 

Animals and species identification 

Snails were collected in June and August of 1987 and 
in November of 1989. They were either sacrificed im- 
mediately for determination of the biochemical compo- 
sition of animals in the field, or brought into the laboratory 
and used for estivation studies (see below). The average 
shell-free tissue mass of snails prior to estivation in the 
laboratory was 0.453 0.014 g (SEM, n = 63) for O. 
strigosa and 0.394 0.013 g for O. siibntdis (n = 41). 
Both species are hermaphroditic and bear live young. Only 
individuals without developing young in their oviducts 
were used in this study. 

After the snails had been sacrificed for biochemical 
analyses (see below), the species was determined by starch 
gel electrophoresis of proteins (Rees, 1988). During the 
present study, additional, faster-migrating alleles were re- 
solved in O. strigosa at the phosphoglucomutase and 
phosphoglucose isomerase loci. This finding does not 
compromise the utility of this technique in species deter- 
mination, however, as the occurrence of the slow alleles 
at these loci remains diagnostic ofO. siibntdis. Individuals 
that were not electrophoretically genotyped (snails col- 
lected in June 1987 and those which died during the es- 
tivation series) were separated into species by their shell 
morphology (Rees, 1988). 



B. B. REES AND S. C. HAND 

Table I 

Climalu- iniul/lu>n<i iliinni; the \iininier <>/ IWtl in llic Mitchell Creek and Kust Ri/lc Creek drainages 



Site 


Month 


Daily low 
temp(C) 


Daily high 
temp (C) 


Daily low 
RH (%) 


Daily high 
RH (%) 


Rainfall 
(mm) 


Normal 
rainfall (mm) 


Mitchell Creek 


June 


8 2 


25 4 


30 8 


70 6 


-n 


31 




July 


11 2 


23 2 


37 8 


75 6 


48 


30 




August 


10 2 


23 3 


36 9 


72 8 


15 


36 




June-Aug 


10 2 


23 3 


35 9 


72 7 


85 


97 


East Rifle Creek 


June 


11 3 


28 5 


26 6 


52 15 


18 


21 




July 


13 2 


28 2 


31 6 


65 15 


31 


19 




August 


122 


27 4 


30 7 


61 17 


7 


32 




June-Aug 


12 2 


28 + 4 


29 7 


60 17 


56 


72 



Temperature and humidity readings were made continuously with hygrothermographs located at the Mitchell Creek and Rifle Falls Fish Hatcheries. 
Hygrothermographs were enclosed in Stevenson-style temperature cabinets approximately 10 cm above the ground and were calibrated against a 
hand-held temperature-humidity sensor that had been certified by the National Bureau of Standards. The data reported for June were recorded 
between June 5 and June 30: data for July and August are from all days in these months. Temperature and humidity are reported as the means and 
one standard deviation of the daily values. All monthly temperature and relative humidity averages are significantly different between field sites, 
except for June daily low relative humidity (t-test, P < 0.05). Monthly rainfall data for 1990 and normal rainfall (averages for the years 1951-1980) 
were recorded in the nearby communities of Glenwood Springs and Ritle (ca.. 5 and 20 km from fish hatcheries, respectively) and are taken from 
Climatological Data. Colorado (U.S. Department of Commerce). 



Estivation series 

Two experiments were carried out to assess the effects 
of estivation on the biochemical composition of these 
snails. One was performed with snails collected in No- 
vember of 1989 and fed ad libitum for 2 months prior to 
estivation. These snails were kept in damp terraria and 
fed lettuce and carrots. Chalkboard chalk was provided 
as a source of calcium. This feeding regime was designed 
to saturate the energy reserves of the snails prior to esti- 
vation and to minimize the variation in nutritional status 
due to differing conditions at the collection sites. After 2 
months, these snails were transferred to dry terraria with- 
out food, which induced estivation. These snails are re- 
ferred to as the laboratory-maintained group. In the other 
experiment, snails collected in August 1987 were brought 
into the lab and induced to estivate immediately by place- 
ment in dry terraria. In this experiment, we wanted to 
determine the effect of estivation on snails that did not 
have their energy reserves augmented by laboratory feed- 
ing. These snails are referred to as the field-collected snails. 
In both series, snails were maintained at room temperature 
(23-28C) and humidity (ca. 20-60%) for the duration 
of the experiment. Under these conditions, snails were 
inactive within 2 days after being transferred to dry con- 
ditions, and there was no indication that any of the ani- 
mals became active again once they had entered quies- 
cence. Photoperiod was not controlled. 

Preparation ot snails for chemical analyses 

Snail extracts were prepared and maintained at 0-4C 
unless otherwise stated. Chemicals and biochemicals were 



of reagent grade, and water was purified with a Milli-Q 
Reagent Water System (Continental Water Systems, Inc.). 

At the start of the experiments and at 1, 2, 4, and 7 
months following entry into estivation, snails were sam- 
pled randomly from the terraria. An additional sampling 
interval at 10 days was included in the experiment with 
the field-collected snails. The shell diameter of each in- 
dividual was measured, and the snails were then dissected 
from their shells, briefly blotted, and frozen in liquid ni- 
trogen. Tissues were kept at -70C until biochemical 
analyses could be performed, at which time a small portion 
(5-15 mg) of the digestive gland was removed for electro- 
phoresis, and the remainder of the tissue was lyophilized 
to a constant dry mass. The difference between fresh tissue 
mass and dry tissue mass was recorded as tissue water. 
Dry tissues were then pulverized with a mortar and pestle 
and divided into two subsamples: one fraction (approxi- 
mately 40 mg) was used for determination of protein, 
DNA, polysaccharide, urea, and for the lab-maintained 
snails, purines; and the other fraction (10-25 mg) was 
kept for lipid analysis. At the later time points in the es- 
tivation series, individuals were commonly less than 50 
mg dry mass. This small amount of dry tissue could not 
be divided, so lipid was not measured in these individuals. 

Extracts for the determination of protein, DNA, poly- 
saccharide, urea and purines were prepared as follows. 
Dry tissues were homogenized in 1.0 ml of ice cold 1 N 
perchloric acid with a glass homogenizer. Two 50 ^1 ali- 
quots of the perchloric acid homogenate were removed: 
one was combined with 0.95 ml 0.5 N NaOH and saved 
at 70C for protein assays; and the other was combined 
with 0.95 ml 0.5% (w/v) lithium carbonate and saved at 



BIOCHEMICAL CHANGES IN ESTIVATION 



233 



70C for purine analysis. The remainder of the per- 
chloric acid extract was centrifuged at 10,000 X g for 15 
min. The pellets were washed once with 0.7-0.8 ml of 1 
N perchloric acid and centrifuged as above. The perchloric 
acid insoluble material was saved for DNA measurement. 
Perchloric acid supernatants for each individual were 
pooled, neutralized with 5 Af K 2 CO 3 , and centrifuged at 
10,000 x g for 10 min to remove perchlorate salts. Two 
hundred to 400 n\ of the neutralized extract was combined 
with two volumes of 95% ethanol and stored at -70C 
for polysaccharide assays, and the remainder was saved 
at 70C for urea measurements. 

Biochemical analyses 

Protein was measured by the method of Lowry el al. 
( 1951 ), as modified by Peterson (1977), with bovine serum 
albumin as the standard. For calculations of nitrogen bal- 
ance, it was necessary to determine the mass of nitrogen 
in snail protein. The protein in a perchloric acid homog- 
enate was recovered by centrifugation after the nucleic 
acids had been digested by heating (see below). Lipid was 
removed by washing the PCA-insoluble material with 
methanol. The amount of nitrogen in the PCA-insoluble 
fraction was determined by a micro-Kjeldahl procedure 
that includes direct nesslerization of ammonia following 
digestion of the proteins (Koch and McMeekin, 1924). 
The Nessler reagent was obtained from Sigma Chemical 
Company. The amount of nitrogen in protein determined 
in this manner was not different in the two species and 
was found to account for 16.8 0.9% (S.D., n = 4) of 
the protein mass measured by the Lowry assay. 

DNA was determined by the diphenylamine assay of 
Burton ( 1956) with modifications suggested by Giles and 
Myers (1965). Briefly, perchloric acid insoluble material 
was suspended in 1 .0 ml 1 .5 A' perchloric acid and heated 
at 70C for 20 min. Following centrifugation at 10,000 
X gfor 20 min, an aliquot (50-100 ^1) of the supernatant 
was brought to 2.5 ml with 1.5 N perchloric acid and 
combined with 1.5 ml 4% (w/v) diphenylamine made in 
glacial acetic acid and 0.1 ml 0.16 mg ml ' acetaldehyde 
made in water. The color was allowed to develop for 20 
h in the dark at room temperature. To correct for non- 
specific color development, an absorbance difference ( A 600 
- A 700 ) was determined for each sample. Calf thymus 
DNA was the standard. 

Polysaccharide (glycogen plus galatogen), which pre- 
cipitated in the ethanolic extract, was collected by cen- 
trifugation at 10,000 X g for 20 min, washed once with 
1.0 ml 95% ethanol and centrifuged again. The pellets 
were air-dried and redissolved in 1.0 ml water by heating 
at 70C. Polysaccharide was measured by the anthrone 
method described by Jermyn (1975), except that the ad- 
ditions of hydrochloric and formic acid to the samples 



were omitted. Polysaccharide content was expressed as 
0.9 x glucose mass. 

For urea analysis, samples were thawed and clarified 
by centrifugation at 10,000 X g for 10 min. Urea was 
measured colorimetrically as ammonia after treatment of 
the samples with urease (Sigma Diagnostic Kit No. 640). 
Blanks without urease were subtracted from each sample. 

Purine bases were analyzed with high performance liq- 
uid chromatography essentially as described by Simmonds 
and Harkness ( 1981 ). A LDC/Milton Roy HPLC system 
was employed in conjunction with a Waters /uBondapak 
C-18 column (30 cm X 3.9 mm i.d.). The lithium car- 
bonate solutions were thawed, diluted, neutralized, and 
filtered through Gelman SuporO.45 ^m membrane filters. 
Twenty n\ were injected onto the column, and purines 
were eluted isocratically with a buffer of 4 mA/ potassium 
phosphate (pH 3.6) containing 1% (v/v) methanol. Ab- 
sorbance was monitored at 265 nm, and uric acid, guanine 
and xanthine were quantified by integration of peak area. 

Total lipid was determined after extraction of the tissues 
in chloroform:methanol (Folch el al.. 1957; Ways and 
Hanahan, 1964). For each snail, lyophilized tissues were 
homogenized in 4 ml chloroform:methanol (2:1) with a 
Virtis micro-ultrashear apparatus for 1 min and filtered 
through a fritted disc funnel. The residue was rehomog- 
enized in 4 ml chlorofornrmethanol and filtered. The res- 
idue was finally washed with another 2 ml of chloroform: 
methanol and the filtrates combined. The filtered chlo- 
roform:methanol homogenate was mixed with 0.25 vol- 
ume 0.88%- (w/v) KC1 in water, and after separation, the 
aqueous phase was aspirated. The remaining organic phase 
was mixed with 0.25 volume methanol:water (1:1), and 
the aqueous phase was aspirated after separation. The or- 
ganic phase was then decanted into a pre-weighed alu- 
minum planchet and evaporated to dryness under a 
stream of nitrogen. The dried lipid was held over Drierite 
a further 24 h and weighed to the nearest 0. 1 mg. 

In one group of estivating snails, methylamine com- 
pounds were measured by reineckate precipitation pro- 
tocol modified from Kermack et al. (1955). Lyophilized 
tissues from a whole snail were homogenized in 30 vol- 
umes of 40%> ethanol and centrifuged at 20,000 X g for 
15 min. The pellet was washed with another 30 volumes 
of 40% ethanol, and the combined supernatants were 
boiled for 10 min to precipitate proteins. The ethanolic 
extract was centrifuged at 10.000 X g for 20 min, lyoph- 
ilized, and redissolved in 1.0 ml 0.1 TV HC1. Saturated 
ammonium reineckate, prepared in water and titrated to 
pH 1 with 5.0 N HC1, was added to the each sample in 
the ratio 3:1 (reineckate:sample). Reineckate salts were 
allowed to precipitate at 4C overnight and were collected 
by filtration on polycarbonate membrane filters (Nucleo- 
pore, 0.2 ^m). After washing the precipitate three times 
with 3 ml diethyl ether, the precipitate and membrane 



234 



B B. REES AND S. C. HAND 
Table II 

i^ilinn at lul'tiivlnry nuiinuiincil Orcohelix 



Compound 


O. strigiixa 




O subrudix 






mgg ' dry mass 


% dry mass 


mg g ' dry mass 


% dry mass 


Protein 


512.6 19.2 


51.3 


509.4 13.7 


50.9 


Polysaccharide 


216.2 11.3 


21.6 


230.1 8.8 


23.0 


Lipid 


70.3 1.4* 


7.0 


78.2 1.9 


7.8 


DNA 


14.9 0.3 


1.5 


16.7 0.5 


1.7 




jjmol g ' dry mass 


% dry mass 


^mol g" 1 dry mass 


% dry mass 


Urea 


0.98 0.29* 


<0.1 


2.20 0.76 


<0.1 


Uric acid 


55.1 3.9 


0.9 


45.1 4.0 


0.8 


Guanine 


17.1 1.9 


0.3 


10.3 1.2 


0.2 


Xanthine 


7.1 0.6 


0.1 


8.2 0.9 


O.I 


Total dry mass accounted for 




82.7 




84.5 



Values are given as the mean and standard error of the mean. The sample sizes were 27 O xtrigosa and 2 1 O xubritdix, except for the lipid analyses, 
where sample sizes were 1 8 and 1 2 for O slrigosa and O. subrudis. respectively. Asterisks indicate that species means for these biochemical constituents 
are significantly different. 



were dissolved in 70% acetone, and the absorbance was 
read at 520 nm. Betaine was the standard. 

Following the above protocols, the recoveries of known 
quantities of protein, DNA, urea, uric acid, guanine. xan- 
thine. and lipid were >88%, and we did not correct the 
results for differences in recovery. In the case of polysac- 
charide, this protocol led to a 77 2.5% (S.D., n = 4) 
recovery of glycogen standards, and the polysaccharide 
content of snails was corrected accordingly. 

Data analysis 

Examination of the total tissue contents of various bio- 
chemical compounds revealed a large degree of variation 
due to size differences among individuals. For snails prior 
to estivation (both laboratory-maintained and field-col- 
lected), biochemical constituents were expressed in terms 
of dry mass in order to standardize for size differences. 
Equality of sample variances was tested with Bartlett's 
Box-F (Zar, 1984), and differences among group means 
were evaluated with parametric or nonparametric analyses 
of variance accordingly (Zar, 1984). A posteriori testing 
was done with Scheffe's or Dunn's multiple comparison 
tests (Zar, 1984). 

During estivation, considerable dry mass was lost, so 
some variable other than dry mass was required as an 
index of snail size for standardization of biochemical 
composition. Data from non-estivating, laboratory- 
maintained snails showed that the relationship between 
shell diameter and snail size was quite good: coefficients 
of determination (r) for regressions of whole tissue and 
dry tissue mass versus shell diameter were 0.763 and 0.784, 
respectively. Furthermore, when all snails were consid- 



ered, there was no effect of duration of estivation on shell 
diameter (analysis of variance, P = 0.969), suggesting that 
shell diameter neither increases nor decreases during es- 
tivation. Therefore, tissue mass, water, and biochemical 
contents of estivating snails were adjusted to a snail of 
average shell diameter ( 15.63 mm) based upon the slopes 
of regression equations describing the relationship between 
each component and shell diameter. For each species, the 
rates of change in these adjusted values during various 
intervals of estivation were then determined by regression 
analysis. Differences between species-specific rates of 
change were evaluated with the test for homogeneity of 
slopes in an analysis of covariance package (Zar, 1984). 
Correlations between various biochemical measure- 
ments and mortality at 7 months of estivation were an- 
alyzed with Pearson's product-moment correlation. All 
statistical analyses were performed with SPSS-X, version 
4 (SPSS, Inc.). and a probability < 0.05 was considered 
as statistically significant. Unless otherwise stated, data 
are presented as means and one standard error of the 
mean (SEM). 

Results 

Biochemical composition of laboratory-maintained 
Oreohelix 

Laboratory-maintained Oreohelix strigosa and O. sub- 
nidiswere composed of approximately 51% protein. 22- 
23% polysaccharide, 7-8% lipid. and about 1 .5% DNA 
(Table II). The levels of urea and purine bases were low 
prior to estivation. Urea averaged 1-2 ^molg~' dry mass, 
comparable to the level reported in Bulimulus dealbatus 
prior to estivation (Home, 1971). The levels of purine 



BIOCHEMICAL CHANGES IN ESTIVATION 



235 



bases totaled to 64-79 ^mol g~ ' dry tissue, similar to the 
tissue contents of other non-estivating snails (Jezewska el 
al.. 1963: Home, 1971). On a molar basis, uric acid ac- 
counted for about 70% of the total purine, with guanine 
and xanthine accounting for approximately 20 and 10% 
of the total purine, respectively, in both O. strigosa and 
O. subrudis. Hypoxanthine was not found in the tissues 
of these snails. Taken together, these compounds account 
for more than 80% of the dry mass of these snails. The 
unaccounted fraction is presumed to be other low mo- 
lecular weight organic compounds (e.g.. amino acids) and 
inorganic ash. 

Biochemical composition of field-collected Oreohelix 

Compared with the values obtained for laboratory- 
maintained snails, both O. strigosa and O. subrudis dis- 
played lower polysaccharide levels in the field-collected 
groups (Fig. 1A). Protein constituted a correspondingly 
larger portion of the dry mass in both species (Fig. IB), 
and lipid was somewhat higher in O. strigosa collected in 
the late summer (Fig. 1C). These differences in biochem- 
ical composition reflect the effects of ad libitum feeding 
in the laboratory-maintained group and suggest that snails 
feed less regularly or on food of differing qualities in the 
field. Of the snails collected in the late summer, O. strigosa 
displayed significantly higher levels of polysaccharide than 
O. subrudis. Differences in polysaccharide content may 
influence the capacity of these snails for long-term esti- 
vation (see Discussion). 



Snails of either species collected late in the summer 
demonstrated much more variable urea contents than 
snails in the laboratory-maintained or early summer 
groups (Fig. ID). Among the laboratory-maintained 
snails, only 17% had urea contents greater than 1 ^mol 
g ' dry mass, and among the snails collected early in the 
summer, this percentage was 22%. In these groups, the 
highest urea content measured was 11.7 /urno! g ' dry 
mass. Among the snails collected later in the summer, 
urea was higher than 1 /^mol g ' dry mass in 33% of the 
snails, and the highest value was 93.0 /xmol g" 1 dry mass. 
Since urea accumulates during estivation (see below), the 
occurrence of elevated urea in snails collected late in the 
summer suggests that many of these animals had been 
estivating in the field. 

Mortality during estivation 

Both species of Oreohelix experienced mortality during 
the later months of estivation. In the group of snails that 
had been maintained in the laboratory prior to estivation. 
1 of the remaining 13 O. strigosa had died at 7 months, 
whereas 9 of 30 O. subrudis had died. For snails that were 
brought in from the field, the mortality at 7 months in 
both species was higher: 10 of 24 O. strigosa had died, 
whereas 28 of 34 O. subrudis had died. Among the field- 
collected snails, the proportion of dead O. subrudis at 7 
months was significantly greater than the proportion in 
O. strigosa (G-test, P < 0.05). These results demonstrate 
that O. strigosa tolerates extended periods of estivation 
in the laboratory better than O. subrudis. 



ti O 

g E 200 



' 



A- (211 

(27) |2 ' 



120 



E 80 

o 

51 -a 60 

en 40 



JL 



500 



0.250 

o* 





25 

S 20 

E 

? 15 



Figure 1. Biochemical composition of laboratory-maintained and field-collected O. strigosa (open bars) 
and O .subrudis (solid bars). A. Polysacchande content. B. Protein content. C. Lipid content. D. Urea 
content. Error bars indicate one standard error of the mean. Asterisks indicate that the content of this 
constituent is significantly different from that measured in laboratory-maintained snails of the same species, 
and the crosses indicate that species means are significantly different for that sampling interval. 



236 

Analysis oj changes during estivation 

We were interested in whether the two species have 
different rates of substrate depletion or end-product ac- 
cumulation during c>; vation. Because variation in the 
size of individuals among the sampling intervals and be- 
tween species would tend to obscure these rates, we have 
normalized the tissue mass, water content, and the content 
of biochemical constituents to an average snail size based 
upon shell diameter (see Materials and Methods). Note 
that, since dry mass, water content, and biochemical 
composition can be determined only once for any indi- 
vidual, the rates of change described below reflect average 
rates of loss or accumulation among groups of individuals 
rather than rates of change within individual snails. Fur- 
thermore, shell diameters were not measured on the field- 
collected snails sacrificed prior to estivation (day 0). and 
consequently the data for this group begin at 10 days of 
estivation. 

Loss of tissue mass and water during estivation 

Fresh tissue mass, dry tissue mass, and water decreased 
significantly in both species oWreo/ie/ix during estivation. 
When tissue mass and water content data were corrected 
for size differences among individuals, rates of loss in the 
two species were not significantly different. The loss of 
tissue was characterized by parallel decreases in both dry 
tissue mass and tissue water. These losses were biphasic, 
occurring more quickly at the onset of estivation as the 
snails entered estivation, and then reaching a steady slower 
rate after the initial drop. By 7 months of estivation, the 
tissue mass and water content of snails were reduced by 
approximately 35% in all groups. 

The loss of tissue water from estivating Oreolielix was 
not reflected in a decrease in the percent tissue water be- 
cause the dry mass decreased proportionately. The per- 
centage of tissue water remained between 78 and 81% tor 
both species in both experimental series. In fact, among 
the laboratory-maintained snails, there was a slight but 
statistically significant increase in the percent tissue water 
over the 7 months of estivation despite the overall loss of 
water. Thus a constant percentage tissue water cannot be 
interpreted as indicating no loss of water, as has been 
assumed previously for other species of estivating snails 
(Schmidt-Nielsen et at.. 1971). 

Catabolism of energy reserves during estivation 

Polysaccharide, protein, and lipid were all catabolized 
during estivation, but the substrates that were utilized 
changed as estivation proceeded (Figs. 2-4, Table III). 
Polysaccharide was the primary metabolic fuel for the 
initial months of estivation (Fig. 2). Snails that had been 
maintained in the laboratory began the estivation period 



B. B. REES AND S. C. HAND 



with large polysaccharide stores, and in these snails, ca- 
tabolism of this substrate continued for the first 4 months 
of estivation (Fig. 2A). During the first month of estiva- 
tion, the rate of polysacchande depletion was significantly 
faster in O. suhntdis (Table III). Between 1 and 4 months, 
carbohydrate catabolism continued at moderate rates that 
were similar in the two species. After 4 months, the poly- 
sacchande content of the snails was much reduced and 
its rate of utilization was correspondingly low. In the field- 
collected snails, the polysaccharide stores were smaller, 
and consequently they were depleted earlier (Fig. 2B). Al- 
though the initial rates of utilization were similar in the 
two species, carbohydrate lasted longer in O. strigosa. 
which had begun estivation with larger stores. As in the 
estivation series begun with laboratory-maintained snails, 
rates of polysaccharide utilization were much reduced 
during the later phases of estivation and statistically 
equivalent between species. 

Upon depletion of the polysaccharide stores, net protein 
catabolism occurred (Fig. 3). In the laboratory -maintained 
snails, the onset of net protein depletion occurred at about 
2 months of estivation (Fig. 3 A). Before this time, no net 



Zb 






(26,21) 




i A. 


id 9n 

Q ^t-v 




< 1 


4\ 


I -15 


fc 


\\ 


co cMO 


\T 


^Jl 


i^\V 2) 


5 


(12.12) ~?5^ 




^\1 (10.14) 




1,1 _ _. 




B. 


i i i o p\ 




cE-- 




< l 




I !=15 




o 







(11.11) 


LO CP 1 


J(14.9) 


^ 




" R 

D_ 5 


"\ \ U - 1 





\ (12.9) (,2.6) 

*=-=^-==^^ : 





01 234567 
DURATION OF ESTIVATION (mo) 

Figure 2. Polysaccharide content during estivation in O. strigosa (O) 
and O .suhnulis (). All values have been adjusted to a snail of average 
size based upon shell diameter. A. Laboratory-maintained snails. B. Field- 
collected snails. Sample sizes are given in parentheses with the value for 
O. strigosa appearing first. Bars indicate one standard error of the mean. 



BIOCHEMICAL CHANGES IN ESTIVATION 



237 



Table III 

<>/ polysaccharide, protein, and lipid catabolism ami urea ami purine accumulation in Oreohehx v/>/> during estivation 



Compound 



Experiment 



Interval 



O suhnulis 



Polysaccharide 


A 


0-1 month 


-7.81 2.16 


-13.45 1.71 


0.05 




A 


1-4 months 


-2.66 0.59 


-2.23 0.41 


0.54 




A 


4-7 months 


-0.23 0.22 NS 


-0.27 0.12 


0.84 




B 


10 days-2 months 


-3.84 1.14 


-2.25 0.45 


0.23 




B 


2-7 months 


-0.23 0.08 


-0.18 0.04 


0.64 


Protein 


A 


0-2 months 


-0.72 1.56 NS 


0.95 0.97 NS 


0.37 




A 


2-7 months 


-1.95 0.63 


-2.73 0.35 


0.26 




B 


10 days-7 months 


-2.14 0.58 


-3.03 0.43 


0.26 


Lipid 


A 


0-7 months 


-0.33 0.06 


-0.36 0.07 


0.78 




B 


10 days-7 months 


-0.11 O.I2 NS 


-0.67 0.21 


0.06 


Urea 


A 


0-2 months 


0.98 + 0.21 


1.25 0.32 


0.47 




A 


2-7 months 


6.20 0.87 


8.66 0.67 


0.03 




B 


10 days-7 months 


5.52 0.59 


8.75 0.53 


<0.01 


Uric acid 


A 


0-7 months 


0.43 0.12 


0.56 0.08 


0.36 


Guanine 


A 


0-7 months 


0.1 1 0.04 


0.15 0.02 


0.40 


Xanthine 


A 


0-7 months 


0.03 0.02 NS 


0.05 0.02 


0.51 



Experiment A was done with snails after laboratory maintenance and experiment B with field-collected snails without prior laboratory maintenance. 
Values for rates of catabolism (negative values) and accumulation (positive values) are slopes and their standard errors from regression equations of 
the adjusted tissue content of each compound versus length of estivation over the intervals indicated (see also Figs. 2-6). Units are mg snail ' mo" 1 
for polysaccharide. protein and lipid and /umol snail ' mo ' for urea, uric acid, guanine and xanthine. All slopes were significantly different from 
zero, except where indicated (NS). /"values are from tests of equality of species-specific slopes. 



protein catabolism occurred in either species, as indicated 
by the slopes of regression lines not significantly different 
from zero (Table III). After the onset of net protein ca- 
tabolism, the rates of utilization were fairly linear 
throughout the remainder of the estivation period. Among 
the field-collected snails, significant protein catabolism 
occurred from the beginning of estivation (Fig. 3B). While 
species-specific rates were not significantly different, there 
was a trend toward lower rates of protein catabolism in 
O. strigosa in both experimental series (Table III) a trend 
that likely influenced the rates of end-product accumu- 
lation (see below). 

Lipid was catabolized at a low rate throughout the du- 
ration of estivation in both experimental series (Fig. 4). 
The rates of lipid utilization in the two species were not 
significantly different during the 7-month estivation ex- 
periments (Table III). 

Accumulation oj nitrogenous end-products and nitrogen 
balance 

With the onset of protein catabolism. the nitrogenous 
end-products, urea and purine bases, accumulated in the 
tissues of estivating snails (Figs. 5-6. Table III). The tissue 
levels of urea increased dramatically in both species of 
Oreoheli.\ (Fig. 5). In the laboratory-maintained snails, 
protein was not catabolized early in estivation, and hence 
urea began to accumulate only after 2 months of estivation 



(Fig. 5 A). Between 2 and 7 months of estivation, the rate 
of accumulation was higher in O. subrudis than in O. 
strigosa (Table III). By 7 months of estivation, urea was 
32.9 4.5 ^mol snail ' in O strigosa (n = 10) and 43.7 
3.1 /umol snair 1 in O subrudis (n = 14). In the field- 
collected snails, urea began to increase almost immediately 
upon the commencement of estivation, reflecting the early 
dependence upon protein catabolism (Fig. 5B). Between 
10 days and 7 months, the rate of urea accumulation in 
O. subrudis was again greater than in O. strigosa (Table 
III). The tissue urea contents of these snails after 7 months 
of laboratory estivation were 36.4 4.3 /xmol snair 1 in 
O. slrigosa (n = 12) and 58.2 6.1 yumol snail ' in O. 
subrudis (n = 6). 

The accumulation of purine bases was only measured 
in snails that had been maintained in the laboratory, and 
their patterns of change are shown in Figure 6. Over 7 
months of estivation, uric acid increased by 3 to 4 j/mol 
snail' 1 (Fig. 6A), guanine increased by approximately 1 
^mol snair 1 (Fig. 6B), and xanthine increased by less 
than 0.5 ^mol snail ' (Fig. 6C). Hence the sum of the 
purines increased by only 5 to 6 j/mol snail~'. Over the 
7-month estivation period, the rates for uric acid, guanine 
and xanthine accumulation were not statistically different 
between the two species (Table III). 

Ammonia production was measured as described by 
Speeg and Campbell ( 1968), except that estivating snails 
were kept in a closed chamber for a period of two days. 



238 



B B. REES AND S. C. HAND 



60 



50 



ct: 

Q. o. 20 

10 




60 

50 



O 

rr 



10 



(26,21 ) (1 2.1 2) 




.0..4) 



(11.11) 

1(14.9) (13.10) 



B. 




(4.9) 



(12.6) 

o 



01234567 
DURATION OF ESTIVATION (mo) 

Figure 3. Protein content during estivation in O sirigosa (O) and 
O xnhnidix (). All values have been adjusted to a snail of average size 
based upon shell diameter. A. Laboratory-maintained snails. B. Field- 
collected snails. Sample sizes are given in parentheses with the value for 
O. strigosa appearing first. Bars indicate one standard error of the mean. 



Over this period, the amount of ammonia produced by 
8 snails of either species was below the limit of detection 
(0.02 



Levels of urea-counteracting solutes 

Methylamine compounds were measured in one group 
of field-collected O. .strigosa after 7 months of estivation 
and found to be 2.68 0.27 ^mol snair ' (n = 5). HPLC 
analyses of selected extracts of both species have shown 
that betaine is the predominant methylamine compound, 
and that polyhydric alcohols, another class of protective 
compounds, do not significantly accumulate in snail tis- 
sues during estivation (data not shown). 

Discussion 

In the present study, we undertook an analysis of the 
biochemical changes that occur in Oreohelix strigosa and 
O. .siibniclis during a period of laboratory estivation. The 
temporal nature of substrate utilization and nitrogenous 
end-product accumulation were described for the first time 
in congeneric species of land snails that are dissimilar in 



their capacity for long-term estivation. Differences in the 
patterns of biochemical changes may account, in part, for 
the observed difference in mortality. Below, we evaluate 
the relationships between mortality and both the exhaus- 
tion of energy stores and the accumulation of nitrogenous 
end-products of protein catabolism. We also discuss the 
distributions of these Oreohelix species in the field in light 
of their different survivorship during desiccation stress. 

Mortality anil exhaustion of energy stores 

If the duration of estivation is limited by the depletion 
of energy storage compounds during estivation, then snails 
with larger stores prior to estivation would be predicted 
to survive estivation proportionately longer. We were able 
to elevate the level of polysaccharide, the primary meta- 
bolic substrate during early estivation, by feeding snails 
ad libitum in the laboratory prior to estivation. Subse- 
quently, when these snails were allowed to estivate, poly- 
saccharide stores lasted longer, and mortality in both spe- 
cies was lower than when snails collected from the field 



o 6 

CO 

en 



Q 
CL 



6 

en 

en 



9 3 

Q. 



(17.12) 





0123456 
DURATION OF ESTIVATION (mo) 

Figure 4. Lipid content during estivation in O. slrigosa (O) and O 
xiihritdix (). All values have been adjusted to a snail of average size 
based upon shell diameter. A. Laboratory-maintained snails. B. Field- 
collected snails. Sample sizes are given in parentheses with the value for 
O. strigosa appearing first. Bars indicate one standard error of the mean. 



BIOCHEMICAL CHANGES IN ESTIVATION 



239 



estivated without prior laboratory feeding. In addition, 
among the field-collected snails. O. strigosa began with 
higher polysaccharide levels than O. suhrudis, and the 
former displayed only half the mortality by 7 months of 
estivation. With data from four groups of snails (2 species 
X 2 experimental series), we tested the correlation between 
pre-estivation polysaccharide stores and percent mortality 
at 7 months of estivation. Since snails with higher poly- 
saccharide stores were predicted to survive estivation bet- 
ter (i.e., show lower mortality), the test was one-tailed. 
The negative correlation between pre-estivation polysac- 
charide stores and mortality was statistically significant 
(r = -0.91, P = 0.045). The observation that p