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THE

Number I

GT/^i A ih
ICAL,

We walk in the footsteps of visionaries,
^nd see with the eyes of disciples.

T^ Galileo up close: astronomer, physicist,
tf mathematician, dethroner of Aristotle, defender
( of Copernicus and, yes, credited with making
Double convex lenses and the first biological
with a microscope. A compound microscope.

Hooke: 43 years later, the compound microscope
eveals living things are composed of cells.

Leeuwenhoek: invents the first practical microscope
and discovers bacteria.

Armed with the knowledge and inspiration of these
jnd other Olympians, secure in the history of our own
iccomplishments and aware that the mind's eye should
ilways focus on challenging the enigmatic as well as
he undeniable, Olympus has created the BX2 series of
:linical and research microscopes, noteworthy for their
nhancements in optics, imaging and ergonomics.

And each so individual, it deserves the nomenclature
Ay Microscope.

From possibility to actuality, it is wonderful to behold.
\ posse ad esse est mirabile visu.

TheBX4l

The new standard
in laboratory
microscopy
performance, ver-
satility, operation.

Uncanny digitized imaging:

enhanced with the DP11 digital
camera, delivering high-resolution
image recording beyond the norm.

Unusually insightful
optics: observe
magnifications from
1.25x to lOOx
without changing
the condenser.
Choice of condensers
includes Abbe, swing-
out, phase-c
and darkllol

Unerringly precise-
motion stage:

selectable for right/

left hand. I)i

speed, ease,
comfort, an.
accuracy.

Uncommonly easy-to-operate
controls: extended fine-focus knob;

ed illumination
jtile X-Y control.

croscope Senes

The BX45

The new standard
in ergonomic
microscopy. A
dramatic metamor-
phosis: innova- &Q"
live, intelligent,
insightful design
improvements
specifically addressing
the needs of those spending hours
looking through a microscope.

The lowest low-position stage:
three inches lower than standard
microscopes, offering the promise of
minimum effort and hand movement
when changing specimens.

The tilting, telescoping observation
tube: a marvel of adjustability, it can
be changed to accommodate any
. 80mm higher
and 70mm

to the

operator than
a convention-
al tilting tube.
And the eye-
i iurward by 45mm
from 0-25 degrees.

The new stan- -m,.^«

dard in research

microscopy. . ,(

The words "new"

and "standard"

really don't do it '

justice. How about

"ingenious" or "original," "paragon"

or "prototypical." Prepare yourself

for a higher power.

The 6-position filter cube turret:

Not 4, not 5, but 6, so single and multi-
band imaging of new fluorochromes
and fluorescent proteins is faster,
simpler and exclusively tailored

The rectangul

ours alone. By cleverly matcnms
the camera's field of view, only the
area requiring fluorescence excita-
tion is exposed. Which means that
the surrounding areas are protected
— - from photo
bleaching.

The fluorescence excitation bal-
ancers: another Olympus exclusive.

A continuously variable excitation
bandwidth. Visualization is enhanced,
differentiation of multi-labels is
immeasurably improved, capturing
of images is at an unheard-of level.

Trinle Labeled Specimen (1 to r):

. with prevalent FITC ;

• with prevalent DAPI and Cy3

• with balanced fluorescence

emission

wider mirror aspherical lens

The aspherical collector lens:

sui generis, in a class by itself.
Specimens appear brighter and more
evenly illuminated; and achromatic
performance now incorporates
wavelengths from UV to IR.

The universal ^^
condenser: with
8 positions, it can

accept optical inserts
for DIC, phase,
brightfield and dark-
field illumination.
And the advanced
Nomarski DIC system has
been expanded and optimized
to encompass prisms for enhanced
contrast and image resolution.

OLYMPUS

FOCUS ON LIFE

Visit us at www.olympusamerica.com
or call 1-800-455-8236

THE

BIOLOGICAL BULLETIN

f *VOOnc w~i . ^ ua 'CSl i ah •

AUGUST 2000

Editor
Associate Editors

Section Editor
Online Editors

Editorial Board

Editorial Office

"-uc,,^;,;.

MICHAEL J. GREENBERG

Louis E. BURNETT
R. ANDREW CAMERON
CHARLES D. DERBY
MICHAEL LABARBERA

SHINYA INDUE, Imaging and Microscopy

JAMES A. BLAKE, Keys to Marine
Invertebrates of the Woods Hole Region
WILLIAM D. COHEN, Marine Models
Electronic Record and Compendia

PETER B. ARMSTRONG
ERNEST S. CHANG
THOMAS H. DIETZ
RICHARD B. EMLET
DAVID EPEL
GREGORY HINKLE
MAKOTO KOBAYASHI
ESTHER M. LEISE
DONAL T. MANAHAN
MARGARET MCFALL-NGAI
MARK W. MILLER
TATSUO MOTOKAWA
YOSHITAKA NAGAHAMA
SHERRY D. PAINTER
J. HERBERT WAITE
RICHARD K. ZIMMER

PAMELA CLAPP HINKLE
VICTORIA R. GIBSON
CAROL SCHACHINGER

CAROL MARRAMA

The Whitney Laboratory, University

Grice Marine Biological Laboratory, College of Charleston
California Institute of Technology
Georgia State University
University of Chicago

Marine Biological Laboratory

ENSR Marine & Coastal Center, Woods Hole

Hunter College, City University of New York

University of California, Davis

Bodega Marine Lab., University of California, Davis

Louisiana State University

Oregon Institute of Marine Biology, Univ. of Oregon

Hopkins Marine Station, Stanford University

Cereon Genomics, Cambridge, Massachusetts

Hiroshima University of Economics, Japan

University of North Carolina Greensboro

University of Southern California

Kewalo Marine Laboratory, University of Hawaii

Institute of Neurobiology, University of Puerto Rico

Tokyo Institute of Technology, Japan

National Institute for Basic Biology, Japan

Marine Biomed. Inst., Univ. of Texas Medical Branch

University of California, Santa Barbara

University of California, Los Angeles

Managing Editor

Staff Editor

Editorial Associate

Subscription & Advertising Secretary

Published by

MARINE BIOLOGICAL LABORATORY
WOODS HOLE, MASSACHUSETTS

Cover

Among the "primitive" chitons of the suborder
Lepidopleurina, the eggs have a smooth jelly coat,
and the sperm — equipped with a typical, prominent
acrosome — probably can enter an egg at any point
on its surface. All other chitons have eggs with
more elaborate spinous or cupulous hulls that focus
sperm to specific regions on the surface. Moreover,
these sperm have evolved a long nuclear filament
tipped by a minute acrosome which interacts with
the egg in specific ways. Differences in the form of
the egg hull and in the mechanism of fertilization
among chitons are providing insights into the evo-
lution of this ancient molluscan taxon.

In this issue (pp. 59-67), John Buckland-Nicks and
Alan Hodgson describe fertilization in Cullocliiton
castaneus from South Africa. This chiton retains a
mixture of primitive and derived characters that
together produce a novel mechanism of fertiliza-
tion, which is represented on the cover.

In the background of the cover is an unfertilized egg
of C. castaneus, from which the jelly coat and part
of the vitelline layer have been stripped to reveal a
honeycomb of egg membrane cups. In the intact
egg, these cups coincide with the bases of regularly
spaced pores in the jelly coat. Fertile sperm seeking
the egg locate one of these external pores and swim
down it to the vitelline layer. The minute acrosome
digests a pore, and the needle-like nuclear filament
bridges the distance to the egg membrane cup.

The micrograph superimposed on the background
shows a fertilizing sperm in the process of injecting
chromatin into the egg cortex. The unusual aspect
of fertilization in these chitons is that the sperm
organelles are apparently abandoned in a membrane
bag on the egg surface. If this is indeed the case,
then inheritance, not only of mitochondria, but also
of centrioles and other cytoplasmic components,
would be maternal.

CONTENTS

VOLUME 199. No. 1: AUGUST 2000

RESEARCH NOTE

Bolton, Toby F., Florence I. M. Thomas, and Celere N.
Leonard

Maternal energy investment in eggs and jelly coats sur-
rounding eggs of the echinoid Aitariti punctulata ....

PHYSIOLOGY

Johnson, M. L., P. M. J. Shelton, E. Gaten, and P. J.
Herring

Relationship of dorsoventral eyeshine distributions
to habitat depth and animal size in mesopelagic de-
capods

Dietz, T. H., A. S. Udoetok, J. S. Cherry, H. Silverman,

and R. A. Byrne

Kidney function and sulfate uptake and loss in the
freshwater bivalve T»xt>ln\nia texasensis

Vollmer, Steven V., and Peter J. Edmunds

Allometric scaling in small colonies of the scleractin-
ian coral Siderastrea sidimi (Ellis and Solander) ....

DEVELOPMENT AND REPRODUCTION

Torrado, Mario, and Alexander T. Mikhailov

Frog Lim-1-like protein is expressed predominantly
in the nervous tissue, gonads, and early embryos of

the bivalve mollusc Afyfr'/f/.s gattopravinciatis

Gibson, Glenys D., and Jennifer M. L. Harvey

Morphogenesis during asexual reproduction in Py-
gospio eh'gmis Claparede (Annelida, Polychaeta) ....

Watson, G. J., F. M. Langford, S. M. Gaudron, and
M. G. Bentley

Factors influencing spawning and pairing in the scale
worm Harmothoe imbricata (Annelida: Polychaeta). . .

Buckland-Nicks, John, and Alan N. Hodgson

Fertilization in C.nll/>< Intuit untnni'in (Mollusca)

Hirose, M., R. A. Kinzie III, and M. Hidaka

Early development of zooxanthella-containing eggs
of the corals Pocillo/xn-a vemimsfi and P. ryilouxi with
special reference to the distribution of zooxanthellae

CELL BIOLOGY

Wakefield, Timothy S., Mark A. Fanner, and Stephen C.
Kempf

Revised description of the fine structure of in \ilii
"Zooxanthellae" genus .S'v'"'"'"/'''"""

ECOLOGY AND EVOLUTION

Barnes, David K. A., and Mathew H. Dick

Overgrowth competition between clades: implica-
tions for interpretation of the fossil record and over-
growth indices 85

4 1 Annual Report of the Marine Biological Laboratory

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Reference: Biol. Bull. 199: 1-5. (August 20(10)

Maternal Energy Investment in Eggs and Jelly Coats
Surrounding Eggs of the Echinoid Arbacia punctulata

TOBY F. BOLTON'-*, FLORENCE I. M. THOMAS1, AND CELERE N. LEONARD2

1 Department of Biology, University of South Florida, Tampa, 4202 East Fowler Avenue,
Tampa, Florida 33620-5150: and ~ Department qf Biology, Tuskeegee University, Tuskeegee, Alabama 36088

In free-spawning marine invertebrates, the amount of
maternal energv that is invested in each egg has profound
implications for all life-history stages of the offspring. The
eggs ofechinoids are freely spawned into the water and are
surrounded by several structurally complex extracellular
layers. These extracellular layers, or jelly coats, do not
contribute energy to embryonic development but must im-
pose an energy cost on the production of each egg. The
investment of maternal energy reserves in the jelly coats of
echinoid eggs may have important implications for the
number of eggs that can be produced (i.e.. fecundity) and
the amount of energy that can be invested in each egg. We
estimated the degree to which maternal energy is invested in
the jelly coats surrounding eggs of the echinoid Arbacia
punctulata. Estimates were derived from measurements of
the amount of energy contained in the combined eggs and
jelly coats, and in the eggs alone. The amount of energy
contained in A. punctulata eggs ranged from 2.70 to 5.53 X
10~4 J egg~l. The amount of energy contained in the jelly
coats ranged from 0.13 to 0.48 X 10~4 J jelly coat ~ '. The
mean concentration of energy in the eggs was 2.15 mm ''
and 0.29 J mm~~ in the jelly coats. These results indicate
that bet\veen 3% and 11% (mean = 7%) of the total energy
invested in each A. punctulata egg is partitioned to the jelly
coat alone. A significant positive relationship was found
between the volumes of the jelly coats and the amount of
energy they contained. Based on this relationship and an
analysis of differences in the size of jelly coats between
echinoid species, we suggest that the degree to which en-
ergy is invested in jelly coats may vary among echinoid
species and is therefore likely to be an important life-history
characteristic of these organisms.

Received 2 February 1999; accepted 18 April 2000.
* To whom correspondence should be addressed.

In free-spawning marine invertebrates, the egg contains
all the maternal energy provisioned for the development of
each offspring. The amount of maternal energy invested in
individual eggs is central to many theories on the evolution
of life-history patterns in marine invertebrates and is widely
considered to have profound implications for all stages of
marine invertebrate life cycles (1-5). The eggs ofechinoids
are freely spawned into the water column where fertilization
and development take place. Several extracellular layers
surround the eggs of echinoids. These extracellular layers
(commonly, and from here on, referred to as "jelly coats")
are structurally complex, consisting of several concentric
layers of polysaccharide fiber networks embedded in a
glycoprotein matrix (6, 7). The jelly coats surrounding the
eggs of echinoids are thought to play important roles in
fertilization processes (8-10) and may also protect eggs
from physical forces that they are exposed to during and
after spawning (11, 12). The jelly coats of some echinoid
species disintegrate soon after contact with seawater ( 13) or
following fertilization, and do not contribute energy to
embryonic development.

Although the jelly coats surrounding the eggs of echi-
noids do not contribute energy to embryonic development,
they must impose an energy cost on the production of each
egg. Assuming that the amount of maternal energy available
for reproduction is finite, the investment of energy in jelly
coats may have important life-history implications. These
potential implications include a reduction in the number of
eggs that can be produced (i.e., fecundity), a reduction in the
amount of energy that can be invested in each egg, or both.
Although previous studies have shown that there is substan-
tial extra-embryonic investment in the gelatinous matrices
of egg masses that are deposited on the benthos by some
marine invertebrates (14, 15), the energy invested in jelly

T. F. BOLTON ET AL

coats surrounding the eggs of a free-spawning species has
not been considered specifically.

We estimated the amount of maternal energy that is
invested in the jelly coats surrounding eggs of the echinoid
Arbacia punctulata. These estimates were derived from wet
oxidation (16. 17) measurements of the amount of energy
contained in the combined eggs and jelly coats, and in the
eggs alone. The wet oxidation method yields an estimate of
the amount of organic carbon contained in a sample, which
can be directly interpreted as a measure of the amount of
energy that it contains. This method has been used in
previous studies of maternal energy investment in marine
invertebrate eggs ( 18-21 ), so our data can be directly com-
pared with earlier results.

The amount of energy contained in the combined egg and
jelly coat (mean ± SD = 3.97 ± 0.79 X 1(T4 J egg~' ) was
significantly higher (paired sample t test: / = 8.33, df = 9,
P < 0.0001 ) than the amount of energy contained in the
egg alone (mean ± SD = 3.69 ± 0.57 X 10 ~4 J egg"';
Table 1 ). The average ( ±SD) amount of energy contained in
the jelly coat was 0.28 ± 0.10 X 10~4 J jelly coat"1, and
constituted 7.4% of the total amount of energy contained in
the combined egg and jelly coat (Table 1 ).

The concentrations of energy (mean joules per cubic
millimeter) in the eggs and jelly coats were calculated from
the amount of energy each contained (Table 1 ) and their
respective volumes (Table 2). The concentration of energy
in eggs was 2.15 J mm'-1 (i.e., 3.69 X 10~4 X 5847 = 2.15
J mm"3, where the combined volumes of 5847 eggs are
equivalent to 1 mm3). The concentration of energy in the
jelly coats was 0.29 J mm"-' (i.e.. 0.28 X 10~4 X 10,416 =
0.29 J mm"3, where the combined volumes of 10.416 jelly
coats are equivalent to 1 mm3). The concentration of energy
in the egg was 7.4 times greater than the concentration of
energy in the jelly coats (i.e., concentration of energy in the
eggs [2.15 J mm 3] divided by the concentration of energy
in the jelly coat [0.29 J mm"3] = 7.4).

Before exposure to seawater, the volume (mean ± SD) of
the combined egg and jelly coat was 2.67 ± 0.30 X 10~4
mm3, and the volume of the egg alone was 1.71 ±0.19 X
1()~4 mm3 (Table 2). The volume of the jelly coat alone was
0.96 ± 0.48 X 1() 4 mm3 (Table 2); thus, the jelly coat
constituted 36% of the volume of the combined egg and
jelly coat prior to exposure to seawater (i.e., 0.96/2.67 X
100 = 35.9%). After exposure to seawater, the volume of
the jelly coats increased substantially to 9.27 ± 2.42 •
10~4 mm3 (Table 2) and constituted 84%- of the combined
volume of the egg and jelly coat.

Linear regression analyses on data contained in Tables 1
and 2 showed no significant relationship between the vol-
umes of the jelly coats and the volumes of the eggs. Simi-
larly, no significant relationship was found between the
amount of energy contained in the jelly coats and the
amount of energy contained in the eggs. A significant pos-

Table 1

The iiiinnini of energy contained (mean ± SD x IQ~4 J, n = 10) in the
combine J egg and jelly coat, and in the egg and jelly coat alone for
each female Arbacia punctulata; the proportion of total energy in the
combined egg and jelly coal that is partitioned to jelly coat is also given

Combined egg
Female and jelly coat

Energy partitioned
Jelly coat to jelly coat (<£)

4.06 (0.20)

3.93(0.18)

0.13

3.3

3.50(0.22)

3.15 (0.13)

0.35

100

5.53(0.17)

5.32(0.31)

0.21

3.X

4.45(0.15)

4.12(0.34)

0.32

7.2

4.77(0.69)

4.29(0.43)

0.48

10.1

3.86(0.30)

3.59(0.25)

0.26

6.7

3.42(0.41)

3.05 (0.44)

0.37

10. N

2.70(01)2)

2.47 (0.46)

0.23

8.5

3.46(0.30)

3.11 (0.07)

0.34

9.8

4.04(0.57)

3.89(0.85)

0.14

3.5

Overall

3.97 (0.79)

3.69(0.57)

0.28(0.10)

7.4(2.8)

Specimens of Arbacia punctulata were collected subtidally between July
and August 1998 from marina walls at Panama City. Florida. Eggs were
obtained from 10 of these specimens by intra-coelomic injection of 0.5-1 ml
0.5 M KC1. The amount of energy contained in the combined egg and jelly coat
and in the egg alone was determined using a modification of the wet oxidation
method given by Parsons ct al. ( 16). Energy determinations were made from
large samples of eggs that were estimated to yield at least 7.8 joules (J).

The jelly coats were removed from half of the eggs obtained from each
female by pouring them through a 100-/u,m Nytex screen. Thus, samples of
eggs with and without jelly coats were obtained from each female for analysis.
The concentration of eggs in each sample was determined by replicate counts
(n = 7-20) of 10-/M1 aliquots of well-suspended eggs from each sample. To
ensure that the eggs were not damaged by the removal of the jelly coats, eggs
were examined microscopically (400x magnification) for any signs of injury
to the egg membrane or leakage of yolk from the egg. The viability of eggs
from rive females was assessed from fertilization assays in which samples of
eggs with and without jelly coats were incubated in dilute sperm suspensions
(dry sperm diluted hy 10 J in seawater). Embryos were allowed to divide to
the four-cell stage before being recorded as viable. The proportion of eggs with
jelly coats that were fertili/.ed was compared to the proportion of eggs without
jelly coats that were fertilised from each female (paired sample / test, a = 5%,
on arcsine transformed proportions).

Three subsamples of eggs with jelly coats and without jelly coats were
taken from samples of eggs from each female and placed in separate
containers. The jelly coat material was eliminated from subsamples hy
removing the supernatant above the eggs and refilling the container with
seawater that had been filtered through a 0.22-jiuii membrane. This process
was repeated several times with all subsamples of eggs. To ensure that all
of the jelly coat material had been removed from the subsamples, a vital
stain (Janus green) was added to the final supernatant solutions, which were
then examined microscopically.

The amount of energy contained in each egg (mean ± SD joules egg" ' )
was calculated from the total amount of energy in each subsample and the
number of eggs that each subsample contained. The concentrations of
energy (joules per cubic millimeter) in the eggs and jelly coats were
calculated from the amount of energy each contained (Table I ) and their
respective volumes (Table 2). A paired sample t test (a = 5%) was used
to determine whether there were differences in the amount of energy
contained in the combined eggs with jelly coats compared to the amount of
energy contained in the egg alone. Relationships between the volumes and
the amounts of energy contained in eggs and jelly coats were examined
using linear regression analyses. The significance of these relationships
were tested by one-way ANOVA (a = 5%).

PARTITIONING OF MATERNAL ENERGY TO EGGS AND JELLY COATS SURROUNDING EGGS

Table 2

Volumes (mean ± SD X 10 "J mm', n = 10) i>f llic cimihiitcd CKK <»'</ /<'//v null, ami of the cfiK ami jelly cutil alone hct/>rc
and after contact with seawater, fur Arbacia punctulata

Before contact with seawater

After contact with seawater

Female

Egg

Egg and jelly coat

Jelly coal

Egg and jelly coat

Jelly

coat

.43(0.22)

3.32(0.77)

1.89

10.61 (3.08)

9.18

L14(0.44)

3.49(0.74)

1.35

11.78(1.63)

9.64

.73 (0.79)

2.61 (0.35)

0.84

7.45(1.761

5.75

.16(0.31)

2.78(0.73)

1.62

9.69(4.04)

8.53

.67(0.23)

2.56(0.56)

0.89

15.23(2.84)

13.56

.73 (0.79)

2.42(0.31)

0.64

12.77(1.92)

1 1.04

.73 (0.79)

2.40(0.17)

0.67

11.65(1.60)

9.92

.74(0.18)

2.29(0.32)

0.55

12.22(1.98)

10.42

.70(0.14)

2.22(0.25)

0.52

11.15(1.81)

9.45

II)

.49 (0.52)

2.56(061 )

0.57

7.20(1.19)

5.21

Overall

.71 (0.14)

2.67 (0.30)

0.96(0.48)

11.02(2.51)

9.27

(2.42)

The volumes of the combined eggs and jelly coats and of the eggs alone were calculated from their respective diameters (D) and the equation for the
volume of a sphere (4/3ir[D/2]3). The volumes of the jelly coats were calculated by subtracting the volumes of the eggs alone from the volumes of the
combined eggs and jelly coats. Before the eggs of Arbacia punctulata contact seawater (i.e., prior to spawning), the jelly coats lie in close proximity to
the eggs. After contact with seawater, the jelly coats hydrate and increase substantially in volume. The volumes of jelly coats before hydration were used
in calculations of the amount of energy they contain. To determine the pre-hydration volume, the thickness of the coat was measured, using an ocular
micrometer in a compound microscope (200X magnification), from the distance between adjacent eggs and added to the mean diameter of the eggs. The
edges of jelly coats after exposure to seawater were visualized by adding india ink to the egg suspension, and diameters were measured in the manner
described above.

itive relationship was apparent between the amount of en-
ergy contained in the jelly coats and their volumes (r~
0.482, F == 7.44. P = 0.025). However, no significant
relationship was found between the amount of energy con-
tained in the eggs and the volumes of the eggs.

Microscopic examination of eggs from which the jelly
coats had been removed did not reveal any damage to the
integrity of the membrane surrounding the eggs. Eggs from
which jelly coats had been removed were fertilized at the
same rate as eggs with jelly coats at a standard sperm
concentration. Thus we assume that the removal of the jelly
coats did not result in any leakage of yolk from the eggs or
any reduction in their viability.

Our results indicate that approximately 7% (range =
3%-l 1%) of the maternal energy invested in the combined
eggs and egg jelly coats of A. punctulata is partitioned to the
jelly coats alone (Table 1 ). The amount of energy contained
in the eggs of the A. punctulata tested in this study was
about half of that reported for this species in a previous
study (18). Similarly, the concentration of energy in the
eggs of the A. punctulata measured here is about half of the
average concentration of energy contained in eggs of free-
spawning marine invertebrates with planktotrophic larval
development (22). Large differences in the amount of en-
ergy contained in eggs from different populations of marine
invertebrate species have been reported previously ( 19. 20).
Differences in the quality of the yolk content of eggs be-
tween populations of the echinoid Arbacia lixula have also

been reported (23, 24). These population differences in the
energy content of the egg and of the quality of the yolk may
be the result of variation in the quality and quantity of food
available to the adult (22) or of differences in the produc-
tivity of the waters in which larvae develop (25).

The degree to which maternal energy is partitioned to the
jelly coats of A. punctulata eggs (mean = 7.490 is small
relative to the amount of extra-embryonic energy parti-
tioned to the gelatinous matrices of benthic egg masses of
some other marine invertebrates. Although these gelatinous
matrices contain less energy per unit weight than the eggs
they encompass, they constitute a large proportion of the
total maternal energy investment in the mass. For example,
in species of the prosobranch gastropod genus Conns, up to
50% of the maternal energy invested in egg masses is
partitioned to the gelatinous matrix (14). Similarly, in spe-
cies of opisthobranch gastropods, up to 589r of the total
energy investment in egg masses is partitioned to the gelat-
inous matrix (15).

While the amount of energy invested in the jelly coats of
,4. punctulata eggs is small relative to that of the gelatinous
matrices of benthic egg masses, it may nonetheless have
important life-history implications. Although the jelly coats
of echinoid eggs do not contribute energy to embryonic or
larval development, they do impose energy costs on the
production of each egg. Within the context of current life-
history theory (1-5), the investment of energy in the pro-
duction of jelly coats may influence the number of eggs

T. F. BOLTON ET AL.

Table 3

ize indices of the jelly coats surrounding the eggs of six echinoid species

Diameter of

Source of

Diameter of egg

combined egg and

Relative size index

Echinoids

data*

(fini)

jelly coat (^m)

(±SD)

Strongylocentrotus purpuraius

120

1 .50

Strongylocentrotus franciscanus

130

196

1.51

Strongylocentrotus droebachiensis

160

260

1.61 (0.16)

A rhticia punctulata

100

126

1.83(0.15)

Lytechinus variegalus

12?

143

298

2.09(0.27)

DenJra.iter excentricns

125

205

1.64

The size indices are the ratio of the diameter of the combined egg and jelly coat (after contact with seawater) to the diameter of the egg alone. A larger
index indicates larger jelly coat relative to the size of the egg. SD = standard deviation; NA = not available.
* 1— Lessios, 1990 (25); 2— Bolton and Thomas, unpubl. data; 3— this study; 4— Timko. 1479 (26).

produced, the degree to which energy is invested in indi-
vidual eggs, or both. Assuming that the maternal energy
available for reproduction is finite, and that the amount of
energy in each egg is constant, the investment of energy in
jelly coats may compromise the number of eggs that could
be produced (i.e., may reduce fecundity). This study indi-
cates that approximately 7% of total energy investment in
the combined egg and jelly coats is partitioned to the jelly
coats alone. Accepting the assumptions given above, the
investment of energy in jelly coats may reduce the potential
fecundity of A. punctulata by about 7%. Alternatively,
assuming that the amount of energy available for reproduc-
tion is constant, and that the number of eggs produced is
also constant, the partitioning of energy to jelly coats may
reduce the amount of energy that could be invested in each
egg. If this is the case, the investment of energy in jelly
coats may compromise offspring growth, survivorship, and
reproductive output.

No significant relationships were apparent between the
volumes of jelly coats and eggs or the amount of energy
contained in jelly coats and eggs. This indicates that the
amount of maternal energy invested in jelly coats is inde-
pendent of the amount of energy invested in eggs. Similarly,
no relationship was found between the amount of energy
invested in eggs and the volume of the eggs. A significant
relationship was apparent, however, between the amount of
energy invested in jelly coats and the volume of jelly coats.
This suggests that it may be possible to infer the relative
degree to which maternal energy is invested in the egg jelly
coats of different species from the volumes of these coats.

The proportion of maternal energy invested in jelly coats
relative to that invested in eggs is likely to vary among
echinoid species. For example, an index of relative size of
the jelly coats surrounding eggs of a particular species can
be obtained by taking the ratio of the diameter of the egg
plus jelly coat to the diameter of the egg alone. Thus, the
relative size of the jelly coats to the size of the egg can be
compared among species independently of actual differ-

ences in egg size. When this index is calculated for the few
echinoid species for which data are available (26. 27).
differences are apparent (Table 3). Since the amount of
energy contained in the jelly coats of A. punctulata is
positively related to the volume of the jelly coats, it is
possible that the proportion of energy invested in the jelly
coat relative to that invested in the egg could be inferred
from this index. If this is the case, ecologically important
differences in the degree to which energy is invested in jelly
coats may exist among echinoid species.

The jelly coats surrounding the eggs of echinoids are not
unique: the eggs of many free-spawning marine inverte-
brates are surrounded by extracellular structures and exhibit
enormous diversity in size, structure, and form (28-30).
Therefore, the investment of energy in the extracellular
structures surrounding their eggs may impose substantial
reproductive costs on many of these species and should be
considered in theories of their life-history evolution. Further
measurements of the degree to which maternal energy is
invested in the extracellular structures surrounding the eggs
of free-spawning marine invertebrates are clearly needed.

Acknowledgments

This research was supported by NSF grants IBN-9723779
and OCE-9701434 awarded to Dr. Florence I. M. Thomas.
We thank Julien Lartigue. Chris Cornelisen. Gregory De-
Lozier. Kristen Edwards, and two anonymous reviewers for
helpful comments on this manuscript.

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Reference: Binl. Bull. 199: 6-13. (AucuM 2000)

Relationship of Dorsoventral Eyeshine Distributions

to Habitat Depth and Animal Size

in Mesopelagic Decapods

M. L. JOHNSON1'*, P. M. J. SHELTON1. E. GATEN4, AND P. J. HERRING:

Department of Biology, University of Leicester, University Road, Leicester LEI 7RH, UK; and
Southampton Oceanography Centre, Empress Dock, Southampton SO14 3ZH, UK

Abstract. Eyeshine distribution patterns recorded from
the eyes of 19 mesopelagic decapod species were examined
and related to the depths at which the species are found. For
most species examined, eyeshine was found to be brighter
ventrally than dorsally. Deep-water decapod species that do
not undergo diel vertical migrations had brighter dorsal
eyeshine than migratory species. Eyeshine intensity in-
creased with body size in five of the species examined and
decreased in two. These changes in eyeshine intensity may
be an adaptation to variations in depth distributions that
occur with increasing body size. It is suggested that the
depth and size-related changes reflect the importance of
remaining camouflaged in the mesopelagic realm and are an
example of ecologically functional development.

Introduction

Many species active at low light levels possess a well-
developed reflective tapetum behind the retina that effec-
tively doubles the path length of light through the photore-
ceptor cells (Lythgoe, 1979). This doubling increases the
photon-capturing efficiency of the eye without requiring an
increase in eye size (Land, 1981 ). In most arthropod species
that have superposition compound eyes, light reflected by
the tapetum and not absorbed by the rhabdoms is visible as
eyeshine (Kunze, 1979). Eyeshine consists of a circular
patch of light that tills about half of the eye and represents
the effective aperture (Land, 1981). Since approximately
80%-90% of the blue-green light entering the eye is ab-

Received 18 February 2000; accepted 12 June 2000.
* Present address: Biology Department. University of Hull, (Scarbor-
ough Campus), Filey Road, Scarborough. YO1 1 3ZZ. UK

t To whom correspondence should he addressed. E-mail: gat@le.ac.uk

sorbed by the rhabdoms (Johnson, 1998), eyeshine is or-
ange-red when the eye is illuminated with white light.

The ecology of the mesopelagic realm is dominated by
the vertical distribution of its inhabitants (Herring and Roe,
1988). Few expeditions have comprehensively studied the
way in which species are distributed in the water column in
a particular area over any significant length of time. As a
result, we have little knowledge of how ecological factors
affect vertical distributions and daily vertical migrations. In
addition, the picture is complicated by community variation
and differences in migratory behavior that may be associ-
ated with factors such as the intensity and angular distribu-
tion of light, hydrography, season, reproduction, ontogeny,
feeding, interspecific interactions, and capture methods
(Foxton, 1970; Jerlov, 1974; Longhurst. 1976; Marshall,
1979; Roe. 1984; Domanski. 1985; Herring and Roe, 1988;
Gonzalez el <//.. 1997). Our knowledge of mesopelagic
ecology is derived for the most part from spot samples taken
at various unrelated locations, seasons, and times of day.
These samples have demonstrated that, although there are
clear underlying patterns of diurnal behavior (Foxton,
1970), the preferred depths for all species are highly vari-
able.

For many deep-sea species, shielding and reflecting pig-
ments in the eye probably do not move in response to
changes in light intensity (Nilsson, 1982; Shelton et <//..
1986). In addition, previous studies have suggested that, as
with some nocturnal insects (Laughlin and Weckstrom.
1993), deep-water decapods may lack the physiological
gain-control mechanisms necessary for light adaptation
(Nilsson and Lindstrom, 1983; Johnson et ai. 2000). Me-
sopelagic species are thus limited by the range of light
intensities in which their eyes can function, and they must
migrate to avoid downwelling light intensities that exceed

EYESHINE DISTRIBUTION IN DECAPODS

their physiological limits. Therefore, given the structured
way in which light varies with depth (Kirk, 1983) and the
fact that the dynamic range of the eyes dictates to some
degree the depths inhabited, the eyes of mesopelagic deca-
pods should show physiological and anatomical associa-
tions with their preferred depth.

Here we suggest that, with respect to sensitivity, the ideal
eye would have a complete tapetum equally reflective in all
regions. Such an eye would have uniform sensitivity in all
regions. However, in an earlier study of selected mesope-
lagic shrimps, it was found that tapeta are often incomplete
or have regional variations in their reflectivity (Shelton et
al., 1992). This is in spite of the fact that such animals live
in a dim environment where bioluminescence plays a major
role in inter- and infra-specific communication (Burken-
road, 1943; Herring, 1990; Morin and Cohen, 1991) and
where maximizing sensitivity must be of considerable im-
portance. It was concluded that any reduction in reflectivity
must be an adaptation to reduce visibility to predators. It
was shown that in many species there is a decreasing
gradient of eyeshine intensity along the anteroposterior axis
of the eye. In other cases there is a large hole in the tapetum.
The anteroposterior gradient seems to be associated with
reducing the visibility of the shrimp to predators during the
escape response. At such times, the eye swings forward so
that the least reflective part of the eye is exposed to the
predator (Shelton el al, 2000).

We set out to investigate the features of eyeshine in
mesopelagic decapods and to suggest how the variations
found may be related to their life history and depth distri-
bution. The current paper investigates differences in reflec-
tivity along the dorsoventral axis of a range of mesopelagic
shrimps. It was found that, as with the anteroposterior axis,
there are considerable differences in reflectivity between
dorsal and ventral regions. We conclude that the design of
the eye in mesopelagic species is constrained by competing
factors — the need to see and the need to avoid being seen.

Materials and Methods

Shrimps were taken from depths of between 0 and
2250 m during RRS Discover cruise 204 (1995) in the
eastern Atlantic north of the Cape Verde Islands (25° W,
20° N). The use of an RMT 1 + 8 net system allowed
sampling at discrete depths, and a closing cod-end main-
tained animals in good condition while they were brought to
the surface (Roe and Shale, 1979; Wild et al., 1985). Light-
induced damage of the eye was prevented, and the general
condition of the animals was maintained by sorting and
storing them in dim red light and placing them in refriger-
ated aquaria until required. All were utilized within 2 h of
capture.

Mesopelagic species can be split broadly into two groups;
those that undergo diel vertical migrations and those that

live in deep water and do not migrate (Fig. 1 ). Species such
as Sergestes comiculum, Oplophorus spinosus, Parapandu-
lus richardi, and Systellaspis debilis are generally found
above 1000 m and migrate close to the surface at night
(Hiller-Adams and Case. 1988; Cartes etui. 1994; Institute
of Oceanographic Sciences (IOS) Database). Other species
such as Acanthephyra pelagica, Gennadas valens, and Ser-
xiu rohiistii.s undergo diel vertical migrations of less mag-
nitude and are rarely caught above 300 m (Domanski, 1985;
Hiller-Adams and Case. 1988; IOS Database). Systellaspis
cristata, Acanthephyra grucilipes, and Bentheogennema in-
termedia are examples of virtually nonmigratory species
that are generally found between 700 and 1000 m (Doman-
ski, 1985; IOS Database).

Eyeshine distribution and intensity was examined using a
variation of the protocol developed by Shelton et al ( 1992).
Shrimps were mounted on a rotatable rod projecting into a
polythene chamber filled with chilled seawater. The cham-
ber was sealed with a glass coverslip to prevent distortion of
the image by water surface movement. Measurements were
carried out only on animals that had eyestalks in the later-
ally extended position, as they are during normal forward
swimming. The preparation was examined through a Zeiss
binocular microscope with a video camera attachment, and
illumination was provided by a Schott KL 1500 halogen
light source. The microscope was focused and the specimen
was manipulated under dim red light. To record eyeshine
intensity, preparations were axially illuminated with green
(520 nm broadband filter; Wratten No. 59) light via a small
mirror oriented at about 45° to the light beam just outside
the field of view. Eyeshine was recorded using a JVC
TK-1085E color video camera (with the automatic gain
control switched off) and a Panasonic VHS video recorder.
The preparation was rotated around the longitudinal axis by
20° increments from the dorsal to ventral points. For com-
parison, and to more easily assess any physical damage, the
eye was then observed using white light (no filter). Video
images were analyzed on a Kontron image analysis system.
Average brightness across the eyeshine patch was measured
in gray-scale units (1-255 GSU) and the patch diameter
measured. Depth distributions were taken from the most
appropriate literature or the IOS database. Where the infor-
mation was available, the mean depth and the 95% confi-
dence interval for range were used. If only range was
available, then the midpoint was taken.

Results

Eyeshine distribution patterns were observed and re-
corded from 136 eyes from 19 species (Table 1 ).

Eyeshine patch diameter

There were noticeable differences in the diameter of the
eyeshine patches under the two different colors of light

M. L. JOHNSON ET AL.

<i -

Day Depth (m)

•

T ;

T* l

• I ;

T T

•

Night

Depth (m)

500-

"T"

T x ;

1000-

o-l-o

X 0

•T

ill

1 1

O 1

1 1

o« 1 -1-

1500-

O«

J- o»

C
C

c
Gennadas brevirostris

Gennadas scutatus

Gennadas valens

Notostomus auricu/atus

Opiophorus spinosus
Parapandalus richardi

Plesionika mart/a
Sergestes corniculum
Sergia grandis
Sergia robustus

Systellaspis cristata
Systellaspis debilis

\canthephyra gracilipes

Acanthephyra pelagica

Ithephyra stylorostratis

ttheogennema intermedia
Gennadas talismani

Hymenodora gracilis
'apasiphaea sulcatifrons

Species ^ *

Figure 1. Estimated depth distributions for the 19 species of mesopelagic decapods examined. Data points
indicate the mean or midpoint of the range which, when data are available, is indicated by the error bars. The
species are separated into two depth classifications depending on their depth distribution and migratory behavior.
Those that are commonly found to occur above 1000 m are classed as migratory. Depth data are from the
following sources: Hiller-Adams and Case (1988) — Gennadas valens, Opiophorus spinosus. Parapasiphaea
sulcatifrons, Sergia graiulis, S. robustus, Systellaspis cristata. S. debilis: Cartes et a/. (1994) — Plesionika
mania: Crosnier and Forest (1973) — Notostomus auriciilatus, Sergestes corniculum: Heffernan and Hopkins
(1981) — Gennadas brevirostris. G. scutatus. G. talisman!: Domanski (1985) — Acanthephyra pelagica, A.
stylorostratis, Bentheogennema intermedia, Parapandalus richardi: Institute of Oceanographic Science data-
base— Acanthephyra gracilipes, Hymenodora gracilis.

used. Generally the eyeshine patch diameter appeared
smaller under green light and larger under white light. Often
when illuminated with white light, the exact edge of the
eyeshine patch was not clear since a band of eyeshine of a
different character (color and intensity) would surround the
main patch. To allow eyeshine patch diameters resulting
from green and white light to be compared, the relative
eyeshine patch diameter (eyeshine patch diameter/eye di-
ameter) was calculated. This allowed a better comparison of
animals of different sizes. The results of a two-tailed t test
of the data from all the species examined suggested that
there was a significant difference resulting from illumina-
tion by green or white light (/ = 10.85, P > 0.001, n =
128). The relative eyeshine patch diameter resulting from
green incident light (0.33 ± 0.09 mm) was 27.5% smaller
than that resulting from white light (0.46 ± 0.10 mm).

Because there was a significant difference in the diameter
of the eyeshine patch under white and green light, this study
used only the measurements of diameter and intensity made
under green illumination, which more closely resembles the

light that mesopelagic species would experience normally
(Kirk, 1983).

Eyeshine distribution along the ilorsoventn/l av/'.v

For the 19 species examined, the general trend is for
eyeshine to be brightest ventrally (Table 1 ). The degree to
which eyeshine intensity varies around the eye differs be-
tween species. In nonmigratory and deeper-living species,
such as Acanthephyra pelagica and Acanthephyra styloro-
stratis, eyeshine is usually of similar intensity dorsally and
ventrally (Fig. 2a). Migratory species that come close to the
surface during the night, such as Parapandalus richardi,
Plesionika mania (Fig. 2b), Opiophorus spinosus, and Sys-
tellaspis debilis (Fig. 2 c, d), have eyeshine that is brighter
ventrally than dorsally. In Sergestes cornicttlum, because it
hangs vertically during normal swimming, the eyeshine
pattern is offset by 90° so that the more reflective posterior
region of the eye is directed downwards (Shelton et <//..
1992).

EYESHINE DISTRIBUTION IN DECAPODS

Table 1

Dorsal (in bold te.\t) and ventral eyeshine intensities with standard errors

Species

Eyeshine intensity by size class

(carapace length.

mm)

0-5 5-10

11-15

16+

Acanthephyra gracilipes

84.0 ± 5.5

85.8 ± 7.3

Acanthephyra pelagica

50.4

68.7 ± 17.5

104.5 ± 7.8

78.5

88.7 ± 9.8

98.3 ± 6.5

Acanthephyra stylorostratis

78.1 ±4.6

70.53 ± 2.3

Bentheogennema intermedia

56.0 ± 3.3

78.4 ± 14.6

Gennadas brevirostris

42.8 ± 11.3

1 10.7 ± 18.4

Gennadas scittalus

32.9 ± 4.1

54.2 ± 22.3

Genniulas taiismani

79.6 ± 9.7

95.5 ± 36.6

Gennadas valens

45.1 ± 11.4

96.1 ± 15.3

Hymenodora gracilis

63.7 ± 4.4

72.8 ±11.4

Notostomus aitriciilatus

34.2 ± 1.6

25.2 ± 3.2

37.2 ± 6.8

42.1 ± 6.9

Oplophorus spinosus

30.7 ± 0.2 31.2 ± 0.7

35.8 ± 1.4

48.9 ± 9.6

48.1 ± 10.1 56.1 ± 3.3

48.5 ± 5.7

48.7 ± 24.0

Pcu'iipandulus richardi

27.9 ± 0.9

45.3 ± 15.7

Parapasiphaea sitlcatifrons

75.8 ± 3.5

42.1 ± 0.4

Plesionika mania

31.9

31.1 ± 1.1

46.7

82.4 ± 22.1

Sergestes corniculum *

33.8 ± 0.8

39.6 ± 18.0

30.8 ± 2.5

45.7 ± 8.4

48.6 ± 5.7

69.9 ± 19.1

Sergio grandis

32.2 ± 0.8

33.1

51.3 ± 3.2

33.5 ± 0.8

48.7

94.8 ± 9.1

Sergia robustus

31.4 ± 0.2

40.8

35.5 ± 0.5

46.K

Syste/laspis cristata

43.6

64.3

67.3

82.3

81.2

74.8

Syste/laspis debilis

33.6 ± 2.2 30.8 ± 2.7

28.7 ± 1.6

46.0 ±4.6 41.2 ± 6.0

34. S ± 1.3

Species have been classified (Cl as nonmigratory (N) and migratory (M) according to the depth and distance of their daily vertical migration cycle (Fig.
1). Eyeshine intensity is measured in gray-scale units (GSU).

* For Sergestes corniculum. anterior and posterior eyeshine intensities were used in place of dorsal and ventral (see text).

Animal size, eveshine intensity, and distribution

Good size ranges (>5 mm range) were obtained for nine
species. For five species (Acanthephyra pelagica, Oplopho-
rus spinosus, Sergia grandis, Sergia robustus, and Systel-
laspis cristata) both dorsal and ventral eyeshine increased
with carapace length (Table 1 ). For two species (Plesionika
martia and Sergestes corniculum) there was no clear trend
with regard to dorsal eyeshine, but ventral eyeshine was

greater in larger specimens. In Notostomus auriculatus and
Systellaspis debilis, both dorsal and ventral eyeshine inten-
sity decreased with increasing carapace length. A typical
example of eyeshine distribution in large and small speci-
mens is shown by O. spinosus (Fig. 2c). S. debilis is unusual
in that eyeshine intensity is markedly greater in small spec-
imens than large (Fig. 2d). For four species where size
ranges and numbers captured permitted, dorsal eyeshine
was plotted against carapace length (Fig. 2 e, f). The two

M. L. JOHNSON ET AL
a) Non-migrants b) Migrants

125-

S 100-
cfl

? 75

c
u

= 50-

~* Acanthephyra pelagica

"* Acanthephyra stvloroslratis

50 100 150 200
Degrees from dorsal

• Parapandalus richardi

Z)
O)

125 -
lOOn

75-

* Plesionika martia

«>

50-

£ TTJ

25 -

*^i^_r*^±J- -L

D -

50 100 150 200
Degrees from dorsal

100-

50-1

c ) Oplophorus spinosits

Cl = 4 mm
Cl = 16mm

0 50 100 150 200

Degrees from dorsal

e )Systellaspis debilis &
S. cristata

• ^'yi/t'//«5/?(5 debilis

° Systellaspis cristata

§ 75 H

5 10 15 20

Carapace length (mm)

D
on

60-

d) Systellaspis debilis

150-

100-

5(1 -

I 50 100 150

Degrees from dorsal

f) Oplophorus spinosus &
Acanthephyra pelagica

• Oplophorus spinosus

° Acanthephyra pelagica

5 1(1 15 20 25
Carapace length (mm)

Figure 2. Size- and depth-related variations in eyeshine distributions, (a. b) Dorsoventral eyeshine intensity
distributions for four species of decapod. In the two migratory species Parapandalus richardi and Plesionika
ihi cveshine intensity is markedly brighter ventrally than dorsally. In the deep migratory (Acanthephyra
] and nonmigratory (A. stylorostratis) species, eyeshine does not vary significantly from dorsal to
ventral (c. d) Dorsoventral eyeshine distributions for two species of migratory decapod showing how eyeshine
intensity differs between large and small specimens. In Oplophorus spinosus. eyeshine is brightest in large
specimens. In S\-\iflla.\r>is debilis, eyeshine is brightest in smaller specimens, (e, f) Changes in dorsal eyeshine
intensity with increasing carapace length for four species of mesopelagic decapod. For two species (Syslellaspis
cristata and Oplophorus \y>m«.v»,v). positive and significant correlations were found (/; = 3, r = 0.896, P < 0.05
and n = 16. /• = 0.459, P < 0.10 respectively). Although a positive trend is also found for Acantheph\ra
pelagica (it = 8, i = 0.614). it is not significant. In the case of Systellaspis Jehilis. dorsal eyeshine intensity
decreases with increasing carapace length (n = 15, r = 0.560. P < 0.05).

EYESHINE DISTRIBUTION IN DECAPODS

deep-water species (Acanthephyra pelagica and Systelluspis
cristata) showed a significant increase in dorsal eyeshine
intensity with increasing carapace length. In Oplophorus
spinosus, the increase in dorsal eyeshine intensity was less
pronounced, and in Systellaspis debilis, as is also demon-
strated by Figure 2b, eyeshine actually decreased with in-
creasing carapace length.

Depth distribution and eyeshine intensity

In the present study it was found that for the largest size
classes of each of the 19 species examined (Table 1), there
were significant correlations between loglo depth and loglo
dorsal eyeshine intensity (Fig. 3 a. b). In the case of the
relationship between eyeshine intensity and daytime depth,
the correlation was markedly improved when Oplophorus
spinosus was excluded from the analysis. This species has
much higher dorsal eyeshine intensity for its daytime depth
distribution than would normally be expected. It is possible
that this anomaly is related to the unusually small amplitude

of its vertical migration pattern (Foxton, 1970), which sug-
gests that this species may be able to light adapt (thereby
reducing eyeshine) to some degree. Ventral eyeshine ap-
pears to vary independently of depth (Fig. 3 c, d). Analysis
of variance showed that migratory species have significantly
lower (F = 3.12, P = 0.095) log,,, dorsal eyeshine intensity
(1.69 ± 0.21, n = 13) than nonmigrants (1.85 ± 0.15, n =
6). A comparison of ventral eyeshine between the two
groups showed that there was no significant difference (F =
2.29, P = 0.15) between migratory (1.81 ± 0.14) and
nonmigratory species (1.92 ± 0.15).

Discussion

Eyeshine intensity varies as a result of the efficiency and
quantity of reflecting and absorbing pigments within the eye
(Gaten et «/., unpubl.). Our examination of mesopelagic
decapods has demonstrated that the distribution patterns of
their dorsoventral eyeshine intensity vary with the species'
estimated habitat depths. With increasing habitat depth.

a) Day depth v. Dorsal eyeshine intensity

2.1 -I

0 l.q-
•5 1.8-

.1 1.7-

1 1.6-

1.4

O.s

b) Night depth v. Dorsal eyeshine intensity

1.9-

1.x -
1.7-
1 .6 -
1.5-
1.4

Depth (m)

1.9-
1.8-

1.7-
1.6-

c) Day depth v. Ventral eyeshine intensity

1.5

2.5 3 3.5

Depth (m)

3
C/5

d ) Night depth v. Ventral eyeshine intensity

I 9-
1.8-

1.7-
1.6-

1.5-

1.5

2.5
Deplh (m)

Figure 3. Eyeshine intensity in relation to depth distributions for adult mesopelagic species (n = I9| with
least-squares lines fitted. Significant positive correlations were found for dorsal eyeshine intensity and day depth
[(a) r = 0.68, P < 0.001] and night depth [(b) r = 0.81. P < 0.001]. When Optophoms spinosus (O.s in Fig.
3a) — which exhibits higher dorsal eyeshine intensity than expected for its estimated daytime depth distribu-
tion— is excluded, r = O.X2. Ventral eyeshine intensity was poorly correlated with both day [(c) ;• = 0.34, n.s.]
and night |(d) r = 0.17, n.s.] depths.

3.5

M. L. JOHNSON ET AL.

dorsal eyeshine was brighter, and the difference in intensity
between dorsal and ventral regions of the eye decreased. In
all species examined except Systellaspis debilis and Noto-
stomus aitriculatus. eyeshine intensity in at least one region
of the eye increased with carapace length. Ventral eyeshine
showed no significant depth-related trend. The variation in
eyeshine distribution suggests that dorsal eyeshine intensity
is related to the degree to which each shrimp species is
exposed to downwelling light. This is supported by theo-
retical evidence which suggests that for any given eye, there
are ideal distributions of reflecting and shielding pigments
that optimize both sensitivity and resolution (Warrant and
Mclntyre, 1991). For most superposition compound eyes,
the tapetum should, ideally, be formed of reflecting pigment
enclosing the proximal third of each rhabdom. This has the
effect of doubling the path length of light (by reflecting
unabsorbed photons back through the target rhabdom) and
restricting the bleed of light between adjacent rhabdoms.
Despite the prevailing low levels of ambient light normally
experienced by mesopelagic species, the distributions of
these pigments generally deviate from the theoretical ideal
(for maximum sensitivity) in the dorsal part of the eye
(Gaten el al, 1992; Johnson, 1998). This suggests that there
is a requirement to remain camouflaged that outweighs the
need for highly sensitive vision dorsally.

Generally we have found that with increasing carapace
length, eyeshine intensity increases, and that the increase is
more pronounced dorsally than ventrally. If the supposition
that dorsal eyeshine intensity is determined by habitat depth
is true, then it follows that where eyeshine increases with
carapace length, juvenile mesopelagic decapods should be
found closer to the surface than adults. Size-related differ-
ences in vertical distribution have been observed for some
mesopelagic decapods, euphausiids, and copepods (Foxton.
1970; Baker, 1970; Hays, 1996). The study of the ontogeny
of eye anatomy of mesopelagic decapods has shown that
juveniles often have apposition eyes, that superposition
optics develop with age, and that the ventral portion devel-
ops first (Gaten and Herring, 1995). Our description of the
way in which eyeshine distribution patterns develop with
size agrees with this finding.

The current results are consistent with the view that
gradients of reflectivity along the dorsoventral axis and
dorsal holes in the tapetum reduce the visibility of the eye to
predators (Shelton et al.. 1992, 2000). The gradients are
necessary because of the characteristic distribution of irra-
diance in the ocean. Here the brightness of upwelling light
is two orders of magnitude lower than that of the down-
welling light, and the light field is symmetrical about the
vertical axis (Kirk, 1983). Low reflectivity in upwardly
looking parts of the eye reduces the contrast between the
light reflected from the tapetum and that arising from the
dim background. In downwardly looking parts of the eye, a

highly reflective ventral tapetum is unlikely to increase
visibility, because the levels of upwelling light are low.

The variations in eyeshine distribution shown here are an
example of how the development of sense organs can be
linked, in a functional manner, to variations in depth distri-
bution. Small decapods can only have small eyes and are
limited in the degree to which they can vertically migrate by
the inverse relationship between body size and energy re-
quirements for swimming (Longhurst, 1976). A small ap-
position eye is sufficient in the relatively well-lit upper
regions of the pelagic realm, where juvenile and adolescent
pelagic decapods and euphausiids are to be found (Baker,
1970; Foxton, 1970; Marshall, 1979). but as shrimps in-
crease in size and daily movement to the ecological refuge
provided by depth (King and Butler, 1985) becomes a viable
strategy, their eyes develop to suit a more oligophotic en-
vironment.

Acknowledgments

We gratefully acknowledge the assistance given by the
officers and crew of RRS Discoveiy and Tony Rice of
Southampton Oceanography Centre for allowing MLJ to
participate in cruise 204. Many thanks also to Jeremy
Brooks for technical assistance with eyeshine measurements
and to Matt Sheehy for his comments on the manuscript.
MLJ was supported by NERC grant GT4/92/5/A. PMJS and
PJH acknowledge support of NERC grants GR9/0019A and
GR3/11212.

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Reference: Biol. Bull. 199: 14-20. (August 2000)

Kidney Function and Sulfate Uptake and Loss in the
Freshwater Bivalve Toxolasma texasensis

T. H. DIETZ1 •*, A. S. UDOETOK1. J. S. CHERRY1, H. SILVERMAN1, AND R. A. BYRNE2

^Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803; and
~ Department of Biology, State University of New York College at Fredonia, Fredonia, New York 14063

Abstract. Toxolasma texasensis acclimated to an artificial
pondwater (PW) maintained a concentration of SO4 in the
blood of about 1-2 mmol I"1. The anion transport inhibitor
DIDS (5, 5'-diisothiocyanatostilbene 2, 2'-disulfonic acid)
reduced the uptake of 35SO4 from the bathing medium by
54%. The clearance of polyethylene glycol (PEG) injected
into the blood of T. texasensis ranged between 0.8 and 1.3
ml g ' dry tissue h~', and provided an estimate of renal
filtration in PW-acclimated animals. The clearance of ra-
dioactive ^SO., simultaneously injected into the same ani-
mal was about 16% of the PEG clearance, suggesting that
sulfate was being reabsorbed by the kidney. Para-aminohip-
puric acid was cleared about 4.6 times faster than PEG.
indicating that this organic acid was subjected to secretion
in addition to filtration. When the normal osmotic gradient
was abolished by acclimating T. texasensis to 10% seawater
(SW), the PEG clearance decreased to 0.17 ml g ' dry
tissue h~ '. Sulfate clearance in animals acclimated to PW or
10% SW was the same. However, in mussels acclimated to
10% SW, the calculated amount of SO4 reabsorbed was
significantly reduced relative to mussels acclimated to PW.
T. texasensis conserved SO4 when acclimated to PW, and
reduced reabsorption when acclimated to the sulfate-rich
10% SW. When mussels acclimated to 10% SW were
returned to PW, there was a transient increase in sulfate
clearance during the first 8 h because filtration exceeded
reabsorption.

Received 2 February 201 .0. accepted 16 May 2000.

* To whom correspondence should he addressed. E-mail:
zothom@lsu.edu.

Abbreviations: DIDS. 5, S'-diisothiocyanatostilbene 2, 2'-disultomc
acid; PAH. para-aminohippuric acid: PEG. polyethylene glycol; PW. arti-
licial pondwater: SW. artificial seawater.

Introduction

The characteristics of solute (K, La, Na, sucrose, manni-
tol) penetration through the epithelia of the unionid Toxo-
lasma texasensis are intermediate to those of other fresh-
water bivalves (Dietz and Byrne, 1990; Dietz el ai, 1995;
Wilcox and Dietz, 1995; Byrne and Dietz, 1997; Zheng and
Dietz, 1998b; Dietz and Byrne, 1999). Previous studies
indicated that the passive movements of solutes and water
across the epithelia of T. texasensis were relatively slower
that those of the dreissenid Dreissena polymorpha (Scheide
and Dietz, 1986; Dietz and Byrne, 1997. 1999). To maintain
ionic homeostasis, an animal must be able to accumulate
and retain solutes; then to preserve water balance, it must
excrete a volume of water equivalent to that taken up
osmotically. Kidney filtration can be estimated by measur-
ing the clearance of marker solutes from the blood, and it is
a useful index from which kidney function and osmotic
water movement can be monitored (Potts, 1954b; Murphy
and Dietz, 1976; Hevert, 1984; Kirschner, 1991; Dietz and
Byrne, 1997, 1999).

Unionid bivalves accumulate sulfate at the rather slow
rate of 0.04 jumol g"1 dry tissue h"1 (Dietz, 1978). Thus,
sulfate is a relatively nonpenetrating anion, and therefore it
has been used for short-term studies of independent ion
transport (Krogh, 1939; Scheide and Dietz, 1982: Byrne and
Dietz, 1997; Zheng and Dietz, 1998a; Dietz and Byrne,
1999). However, sulfate is present in millimolar concentra-
tions in the blood of freshwater mussels (Potts, 1954a; Dietz
and Byrne, 1999) and is a component of various organic
molecules (e.g., amino acids, mucopolysaccharides) found
in molluscs (Eriksson ct ai. 1984; Kornprobst et ai, 1998).

Sulfate balance was studied recently in D. polymorpha.
which has the highest epithelial solute permeability of any
freshwater bivalve tested (Dietz and Byrne, 1999). In the
present study, kidney function was examined in a unionid

RENAL FUNCTION IN A FRESHWATER CLAM

bivalve, Toxolasma texasensis. The unionids have a longer
freshwater ancestry (Triassic) than the dreissenids, which
invaded freshwater in the Pleistocene (Haas, 1969). Sulfate
uptake and the characteristics of the renal clearance and
conservation of SO4 that was injected into the body fluids
were studied.

Materials and Methods

Animal acclimation

Toxolasma (=Carunculina) texasensis was collected
from ponds near Baton Rouge, Louisiana. The animals were
stored, unfed for at least 1 week before use, in aerated
artificial pondwater (PW) at 22° ± 2°C. The pondwater
composition (in millimoles per liter) was 0.5 NaCl. 0.4
CaCl2, 0.2 NaHCO,. 0.2 MgSO4, 0.05 KC1 (Dietz et <//.,
1994). Some mussels were acclimated to an artificial sea-
water (SW) that was diluted with PW to be about 10% SW
( — 106 mosm kg"') for more than 13 d before use. In 10%
SW the mussels become isosmotic. and the osmotic gradient
is minimal. The stock SW composition (in millimoles per
liter) was 449.1 NaCl, 27.5 MgSO4, 24.4 MgCU, 9.9 CaCl2,
6.6 KC1. 2.4 KHCO,, 0.8 KBr, 0.4 H,BO3, "l076 mosm
(salinity = 35%c) (Chambers and De Armendi. 1979).

Solute analyses

Samples of blood (200-300 /j,l) were obtained from
mussels by pericardia! puncture and centrifuged for 3 min at
8000 X g before analysis (Fyhn and Costlow, 1975). The
osmolality of blood or bathing medium was determined on
undiluted samples by freezing point depression. Sodium and
potassium concentrations were determined by emission
flame photometry. Calcium and magnesium concentrations
were determined by atomic absorption spectroscopy from
samples diluted with La2O_,/HCl. Chloride concentration
was determined by electrometric titration.

Sulfate was determined by a turbidimetric method that
formed a precipitate with barium in a total volume of 0.8 ml
(Dietz and Byrne, 1999). A 50- jd sample of blood or
bathing medium was added to 450 /u,l deionized water. To
each sample, 100 /A! of 4.1 mol 1~ ' NaCl in 0.2 mol 1~' HC1
was added and vortexed. A 100-/ul aliquot of glycerol,
ethanol, dibutyl phosphate (2:1:0.15 by volume) was added
and vortexed. The SO4 was precipitated as BaSO4 crystals
by the addition of 100 ju.1 of 1 mol 1~' BaCl2 and immedi-
ately sonicated for 10 s to form crystals of a reproducible
size (Dietz and Byrne, 1999). Turbidity was measured spec-
trophotometrically at 400 nm and was compared to a stan-
dard curve that was linear up to 1 mmol 1~' Na^SO4.

Sulfate uptake

Sulfate uptake was measured by the appearance of the
radiolabeled SO4 in the blood (Dietz and Byrne, 1999).

Each animal was placed in a separate container and used
once. The bathing medium was PW (0.2 mmol 1~' SO4),
and trace amounts of 35SO4 were added to give a specific
activity of 100,000 dpm junior'. After 3 h, bath samples
were collected, and the radioactivity was measured using a
xylene/Triton X-l 14 liquid scintillation cocktail (Wiegman
et a!., 1975) and a Beckman LS6000 counter. After the
samples were collected, each mussel was removed, blotted
dry, and weighed; a blood sample was then taken and
processed as described above. The tissue was removed from
the shell, and the soft tissue was dried overnight (90°C) to
determine dry mass. The amount of 35SO4 that accumulated
in the mussel blood during the exposure to the isotope was
used to calculate the uptake of sulfate (nanomoles per
milliliter) in the blood by dividing the amount of radioac-
tivity in a known volume of blood (dpm per milliliter) by
the specific activity of the bathing medium (dpm per nano-
mole). In some studies, SO4 uptake was measured for 3 h
from PW containing 0.5 mmol 1~' of the anion transport
inhibitor 5, 5'-diisothiocyanatostilbene 2, 2'-disulfonic acid
(DIDS) adjusted to pH 7.3 with Tris. The uptake studies
were performed under indirect illumination to minimize
photodegradation of DIDS.

Solute clearance

Radioactive tracers [3H-polyethylene glycol (PEG, 4
kDa), and Na2 35SO4] dissolved in deionized water were
injected [10 /nl, 1 /xCi of each isotope ( 1 Ci = 37 GBq)] into
the foot muscle of each specimen, and the animal was
returned to the appropriate acclimation medium for 3 h to
allow isotope equilibration. The clearance of the radioiso-
tope from the body fluids of T. texasensis was determined
using the procedures of Dietz and Byrne (1997, 1999). In
brief, after the 3-h equilibration, the animals were rinsed
three times to remove adsorbed isotope and transferred to
separate containers with 30 ml of the appropriate experi-
mental bathing medium. The mussels resumed siphoning
within 10-20 min, bath samples were collected at times 0
and 1 h, and the radioactivity was determined by double-
label counting procedures, as needed, using a programmable
Beckman LS6000 scintillation counter. After the final sam-
ple of bathing medium was taken, each mussel was re-
moved, blotted dry. and weighed; a blood sample was then
collected. The tissue was removed from the shell, and the
soft tissue was dried overnight at 90°C and reweighed.

The clearance of the isotope was calculated from the total
amount of radioactivity that accumulated in the bathing
medium during the 1-h interval (dpm per hour) divided by
the isotope radioactivity in the blood (dpm per milliliter) at
the end of the clearance study (Murphy and Dietz, 1976).
The clearance of solute from the blood was expressed as
milliliters of blood per gram of dry tissue per hour. Because
of the potential damage due to pericardial sampling, blood

T. H. DIETZ ET AL

was collected only at the end of the experiment. Calculated
data assumed that the specific activity of the blood remained
constant during the 1-h clearance measurement. The spe-
cific activity of the blood probably decreased exponentially
during the experiment, and clearance may be underesti-
mated by 20%-24% (Murphy and Dietz. 1976; Dietz and
Byrne, 1997).

Urine samples were not collected from the bivalves; thus
the urine volume and urine:blood ratio of radioactive tracer
were not determined directly. The method used for calcu-
lating clearance would determine the equivalent volume of
blood that would have to be cleared of the tracer by all
routes (kidney, epithelial, digestive tract), but our previous
studies indicated that most of the loss is renal (Dietz and
Byrne, 1997) and that contamination of the blood with
bathing medium was rare and minimal (Dietz ct <//., 1997).

The PEG marker was considered to represent the amount
of material filtered by the kidney in bivalves (Dietz and
Byrne, 1997, 1999). Thus, clearance values of other sol-
utes(x) were compared with PEG clearance for each spec-
imen to distinguish filtration (equal clearance values), reab-
sorption [clearance(x) < PEG], or secretion [clearance(x) >
PEG]. Sulfate clearance was calculated by the method de-
scribed above for PEG. The sulfate concentration in the
blood of each specimen was measured and converted into
the amount of sulfate filtered (micromoles of sulfate per
gram of dry tissue per hour) into the kidney by multiplying
the PEG clearance (milliliters of blood cleared of PEG per
gram of dry tissue per hour) by the blood sulfate concen-
tration (micromoles of sulfate per milliliter of blood).
Knowing the specific activity (dpm jamo!"1) of 35SO4 in the
blood, the quantity of 35SO4 excreted (dpm per gram of dry
tissue per hour) was converted into the total quantity of
sulfate eliminated (micromoles of sulfate per gram of dry
tissue per hour) for each specimen, and this value repre-
sented sulfate excretion. The sulfate reabsorption was cal-
culated as the difference between the filtered and excreted
sulfate values for each animal.

Clearance studies were performed on mussels acclimated
either to PW or to 10% SW: animals in PW were hyperos-
motic to the bathing medium; those in 107r SW were
isosmotic. Animals acclimated to 10% SW were transferred
to PW for 1, 4, 8. 24, 48. or 72 h to observe the changes in
renal clearance when they experienced an increased osmotic
gradient. Clearance was measured for I h, ending at each
time interval specified, and the amounts of sulfate filtered,
excreted, and reabsorbed were calculated.

Para-aminohippuric acid (PAH, 194 Da) was injected
into mussels together with PEG ( 15 jul. 1 jaCi) to compare
clearance values. The method used was similar to that
described above tor the double-label 'H-PEG and 35SO4
studies. Both 'H- and "C-label for both PEG and PAH, and
identical results were obtained. The clearance of PAH was

Table 1

Bl<Hnl *<>h<te concentration in pondwater-acctimated Toxoiasma
texasensis with or without exposure to 0.5 mmol I ' D1DS for 3 h

Solute

Control

Treated

Total solute, mosm kg"1

40 ± 2

39 ± 2

Na, mind 1

19.4 ± 1.3

19.9 ± 1.2

K. mmol 1 '

0.4 ± 0.0

0.5 ± 0.1

Ca, mmol P '

3.5 ± 0.2

3.4 ± 0.2

Mg, mmol 1 '

0.3 ± 0.0

0.4 ± 0.0

CI. mmol 1 '

12.3 ± 0.6

12.5 ± 0.8

SO4. mmol 1 ' '

1.8 ± 0.2

1.7 ± 0.1

^SO4, nmol ml '

L3± I

6 ± 2*

Data are mean ± 1 standard error, n = 5, * P < 0.05.

rapid, thus the equilibration time was shortened from 3 h
to 1 h.

Statistical analyses

All data are expressed as the mean ± 1 standard error
(SE). An animal was used once, and /; indicates the number
of animals in each treatment group. Data were analyzed for
differences between treatment groups by performing a one-
way analysis of variance (ANOVA). When the ANOVA
was significant, the Fisher's protected least significant dif-
ference method was used to determine differences between
specific means (P < 0.05).

Results

Solute balance and SO4 uptake

Toxoiasma texasensis is a hyper-regulator in freshwater,
and the solute concentrations measured in the blood were
maintained at higher levels than those in the PW bathing
medium (Table 1 ). Sulfate had the lowest concentration of
any anion in the blood (1.7-1.8 mmol 1" '), but this concen-
tration was about 8 times higher than in the medium (0.2
mmol r '). Thus, SO4 is concentrated to the same level as
Cl (Table 1).

The SO4 influx is slow in unionids (Dietz, 1978); thus we
did not measure the unidirectional influx of SO4 by the
disappearance of isotope from the bathing medium. Less
than 1000 dpm ml"' of 35SO4 would disappear from the
bath after several hours, and compared to the 20,000 dpm
ml"1 (30 ml bath volume) present in the bathing medium,
this difference could not be distinguished with liquid scin-
tillation counting techniques. The amount of 1SSO4 that
accumulated in the blood over the incubation interval was
small, but significantly greater than zero. The uptake of
'SSO4 was reduced 54% (significant at P < 0.05) by expo-
sure to 0.5 mmol I ' D1DS, but none of the other solutes
measured in the blood were affected (Table 1).

RENAL FUNCTION IN A FRESHWATER CLAM

Table 2

Volume of blood cleared of polyethylene gl\col (PEG] and " SO4, and calculated sulfate processing b\ the kidne\ o/Toxolasma texasensis acclimated
to pondwater (PW), 10% seawater (SW). or when returned to PW for various periods

Treatment

Clearance, ml g ' dry tissue h

Sulfate, jiimol g ' dry tissue h

PEG

Sulfate

Filtered

Reabsorbed

Excreted

10% SW

0.17 ± 0.05a

0.09 ± 0.02a

0.48 ± 0.1 4a

0.23 ± O.OSa

0.25 ± 0.07ab

1 h PW

0.57 ± 0.1 7b

0.21 ± 0.05a

1 .34 ± 0.54b

0.84 ± 0.38ab

0.51 ± 0.1 8b

4 h PW

1.24 ± 0.1 2c

0.76 ± 0.1 3b

2.97 ± 0.37c

1.17 ± 0.32b

1.80±0.31d

8 h PW

1.32 ± O.I8c

0.77 ±0.15b

1.77 ± 0.25b

0.81 ± 0.25ab

0.97 ± O.OSc

24 h PW

1.25 ± 0.1 5c

0.09 ± 0.02a

1.66 ± O.llh

1.54 ± O.lObc

0.12 ± 0.02ab

48 h PW

1.32 ± 0.19c

0.10 ± 0.04a

1.74 ± 0.20b

1.62 ± 0.21 be

0.11 ±0.03ab

72 h PW

0.93 ± 0.1 5bc

0.03 ± O.OOa

2.03 ± 0.39b

1.97 ±0.38c

0.06 ±0.0 la

0.76 ± 0.1 Ib

0.12 ± 0.02a

1.33 ± 0.24b

1.17 ± 0.24b

0.17 ± 0.02ab

Data are expressed as mean ± 1 standard error. Values within a column that have different letters are significantly different using Fisher's protected least
significant difference method (P < 0.05).

Solute clearance

We previously reported values for clearance of radioac-
tive inulin from the blood of Ligumia subrostrata and T.
texasensis (Murphy and Dietz, 1976; Scheide and Dietz,
1986); in this study similar results were obtained using
PEG: 0.77 ± 0.04 ml g~' dry tissue h"1 (n = 6). However,
the clearance of 35SO4 administered simultaneously (0.09 ±
0.03 ml g"1 dry tissue h~') was 12% of the clearance of
3H-PEG, suggesting that filtered SO4 was being reabsorbed
by the renal tissue.

The osmotic uptake of water should be high in PW-
acclimated T. texasensis, but low in 10% SW-acclimated
animals. Thus, filtration measured by the clearance of PEG
should be at a relatively high rate in the former and lower in
the latter acclimation medium. The clearances from animals
that were doubly labeled with 35SO4 and 3H-PEG were
measured from animals acclimated to either PW or 10% SW

(Table 2). The animals in 10% SW were isosmotic with the
medium (Table 3) and would have experienced a lower
osmotic uptake of water and therefore reduced filtration, as
reflected in the clearance of PEG. The sulfate clearance was
about 16% of the PEG clearance in the PW-acclimated
animals. However, the mussels acclimated to 10% SW had
similar clearances (P > 0.1) for both "SO4 and ^H-PEG.
The SO4 clearance in 10% SW-acclimated animals ap-
peared to be unchanged relative to the PW-acclimated mus-
sels. However, because the clearances of SO4 and PEG were
similar, these data suggest that 10% SW-acclimated animals
had reduced their reabsorption of SO4 (Table 2). The SO4
concentration in the blood of T. texasensis acclimated to
10% SW was significantly higher (P < 0.05) than in PW-
acclimated controls (Table 3), but was the same as in the
10% SW bathing medium (-2.7 mmol 1~' SO4).
When T. texasensis was transferred from 10% SW into

Table 3

Concentration of solutes in the blood o/Toxolasma texasensis acclimated to pondwater {PW), 10% seawater (SW), or returned to PW
for various periods

Treatment

mosm kg '

Ion concentration, mmol I '

Total solute

S04

10% SW

110 ± If

45.1 ± 0.7e

1.6

± O.le

2.1

± 0.2a

3.3

± 0.3e

41.1

± 1.7e

2.8 ± 0.2c

1 h PW

102 ± 2e

41.0 ± 0.7d

1.1

± O.Od

2.8

± 0.2b

3.6

± 0.2e

36.2

± I. Id

2.1 ± 0.4ab

4hPW

79 ± Id

29.6 ± 0.7c

0.9

± O.Ocd

2.1

± O.la

2.7

±0.2d

24.9

± 0.8c

2.4 ± 0.1 he

8 h PW

67 ± Ic

27.7 ± 0.5c

0.7

± O.lbc

2.6

± O.lb

1.8

± 0.2c

21.0

± 0.3b

1.4 ± 0.2a

24 h PW

55 ± Ib

20.1 ± 0.7b

0.6

± O.Oab

3.0

± 0.2bc

1.3

± O.lbc

15.5

± 0.6a

1.4 ± 0.2a

48 h PW

45 ± 3a

16.4 ± l.la

0.4

± O.Oa

2.6

± O.lb

1.0

± O.lb

12.0

± 0.8a

1.4 ± 0.2a

72 h PW

46 ± la

16.6 ± 0.2a

0.6

± O.lab

2.9

± 0.2b

0.8

± O.lab

12.3

± 0.5a

2.1 ± O.labc

44 ± 2a

19.1 ±0.9b

0.5

± O.Oa

3.5

±0.2c

0.4

± O.Oa

12.7

±0.7a

1.7 ±0.2a

Data are expressed as mean ± 1 standard error, with 5-1 1 animals for each treatment. Values within a column that have different letters are significantly
different using Fisher's protected least significant difference method (P < 0.05).

T. H. DIETZ ET AL.

PW. PEG and "SO4 clearance increased (Table 2). Al-
though there was an immediate rise in osmotic uptake of
water, only PEG clearance increased significantly during
the first hour relative to 10% SW animals, but clearance of
both solutes was elevated by 4 h. The clearance of PEG was
restored to the same level as that found in PW mussels by
72 h. In contrast, the 35SO4 clearance remained elevated for
8 h and then returned to PW control levels by 24 h. The
elevation in SO4 excretion was due to a significant increase
in filtration. During the first hour after transfer to PW, the
SO4 clearance remained statistically the same as the PEG
clearance. By 4 h, the SO4 clearance was significantly less
(P < 0.05) than the corresponding PEG clearance. The
reduction in SO4 clearance was due to the rapid restoration
of sulfate reabsorption (Table 2). During the first hour of
re-acclimation to PW, the sulfate concentration in the blood
returned to the same level as in the PW-acclimated controls;
recovery was due to dilution caused by the osmotic uptake
of water combined with increased levels of filtration (Table
3). However, 48 h were required for the total solute and
most of the other measured ions to return to PW levels
(Table 3).

To determine whether the renal tissue of T. texasensis
could secrete organic acids, radioactive PEG and PAH were
both injected into PW-acclimated animals. The clearance
was 1.26 ± 0.08 mlg~' dry tissue h"1 for PEG, and 5.75 ±
0.65 ml g~' dry tissue h ' for PAH (/; = 10). PAH is a
smaller molecule than PEG, but the volume of blood cleared
of PAH by filtration was likely to be the same as for PEG.
The additional PAH clearance was due to secretory mech-
anisms and amounted to 4.49 ml g~' dry tissue h"1; this
value was 3.5 times the amount of PAH cleared by filtration.

Discussion

Toxolasma texasensis has a tubular kidney with func-
tional characteristics similar to those found in other inver-
tebrates and vertebrates. The kidney forms urine by ultra-
filtration, for which PEG serves as a useful marker (Hevert,
1984). Some solutes can be added to the urine by the
process of secretion, as well as filtration, and PAH is an
organic acid that is subject to secretory activity. Most of the
PAH eliminated by the kidney of T. texasensis was through
secretory mechanisms. The importance of secretion in the
elimination of PAH has also been documented in the snail
Achatina fulica (Martin et ul.. 1965). At low concentrations
of PAH in the blood, most of this solute is secreted by the
snail kidney rather than filtered. The third major process
responsible for urine formation is solute reabsorption. In
this study, we have focused on the characteristics of sulfate
reabsorption by the kidney of T. texasensis.

Toxolasiua texasensis was able to maintain a sulfate
concentration in the blood of about 1-2 mmol 1" ' while
acclimated to an artificial PW containing 0.2 mmol 1 ' SO4.

Sulfate balance was maintained by transport systems in the
epithelia, including the kidney. Sulfate concentrations in the
blood and pericardial fluid are the same, which suggests that
the anion is freely filtered in molluscs (Potts and Todd,
1965) as it is in vertebrates (Mudge et «/.. 1973). Thus,
filtration in bivalves was assumed to be the same for sulfate
as for PEG, but the renal reabsorption of SO4 reduced its
clearance from the blood by more than 80% relative to PEG
clearance. Renal reabsorption of sulfate was reduced in
animals that were acclimated to 10% SW for almost 2
weeks. During acclimation to 10% SW, the concentration of
SO4 in the blood increased to about 2.8 mmol 1~ ' (equal to
the bathing medium). These data contrast with the some-
what more rapid SO4 transport rates observed in Dreissena
polymorphtt, and with the apparent cessation of SO4 reab-
sorption in that species when acclimated to 10% SW (Dietz
and Byrne. 1999). The low blood SO4 concentration of 0.7
mmol 1~' reported for the unionid Anodonla cygnea (Potts,
1954a) is similar to concentrations we observed in T. tex-
asensis, but is less than half the concentration found in D.
pol\morpha (Dietz and Byrne, 1999).

In previous studies, the clearance values for PEG, inulin.
and high-molecular weight dextran from the blood of D.
pol\morpha were similar, and we concluded that these three
solutes were probably measuring the renal filtration rate
(Dietz and Byrne, 1997, 1999). PEG clearance values in
PW-acclimated T. texasensis were about I ml g~ ' dry tissue
h~', and were similar to inulin clearances reported for the
freshwater snail L\mmiea stagnalis (de With and van der
Schors, 1984). When T. texasensis was acclimated to 10%
SW. the PEG clearance decreased to about 0.2 ml g~' dry
tissue h '. Although pondwater-acclimated D. polymorpha
clear the blood of PEG at about double the rate observed for
T. texasensis, the response by the kidney was the same in
both species when the osmotic gradient was abolished by
acclimation to 10% SW (Dietz and Byrne, 1997. 1999; this
study).

In the sulfate-rich 10% SW environment. T. texasensis
became isosmotic and isoionic for SO4 and reduced its renal
reabsorption. All freshwater bivalves studied become isos-
motic when exposed to dilute seawater (Wilcox and Dietz,
1998; Jordan and Deaton, 1999). They have limited toler-
ance, but may survive in an environment in which total
solutes approach 400 mosm kg"1. Freshwater bivalves can
maintain cellular volume regulation under moderate os-
motic challenges; their ability to mobilize free amino acids
is restricted, however, and this restriction may be responsi-
ble for the limit to their survival (Dietz et al., 1998; Jordan
and Deaton. 1999).

Transferring mussels from 10% SW to PW would in-
crease the osmotic uptake of water and the subsequent
excretion of water by the kidney. After the transfer, PEG
clearance was rapidly elevated to values exceeding those
observed for PW-acclimated T. texasensis. Because of ele-

RENAL FUNCTION IN A FRESHWATER CLAM

vated filtration, it was 24 h before SO4 clearance returned to
PW control levels, even though SO4 reabsorption was im-
mediately reestablished. D. polymorpha also required about
24 h to reestablish SO4 reabsorption to PW control levels
(Dietz and Byrne, 1999). However. D. polymorpha did not
elevate PEG clearance above that found in PW-acclimated
animals. Unlike the unionids, D. pol\nwrpha has maximum
renal filtration (PEG clearance) when acclimated to PW, and
filtration cannot be increased even when the mussel is
subjected to higher osmotic uptake of water (Dietz and
Byrne. 1999).

Recent studies have examined mechanisms of sulfate
transport in a variety of preparations (Larsen and Simonsen,
1988; Cole and Rastogi, 1991; Tenenhouse and Martel.
1993; Grassl, 1996; Dietz and Byrne, 1999). Toad skin is
capable of active sulfate influx from a Ringer's solution
containing 1 mmol 1~' SO4, using an anion exchange mech-
anism (Larsen and Simonsen, 1988). The anion transport
inhibitor. DIDS. significantly decreased the amount of
SO4 label that accumulated in the blood of freshwater
bivalves, suggesting that sulfate uptake was linked to an
anion exchange mechanism (Dietz and Byrne. 1999; this
study).

Sulfate reabsorption is subject to regulatory mechanisms
in freshwater bivalves. When mussels were acclimated to
10<7r SW, the concentration of SO4 in the blood rose, and
renal reabsorption was reduced in both unionids and dreis-
senids; thus, conservation of this anion was minimal (Dietz
and Byrne. 1999; this study). Freshwater bivalves that are
acclimated to PW are in a low SO4 environment, but trans-
port systems and conservation mechanisms both allow them
to maintain a SO4 concentration almost 10 times higher than
that in the medium.

Acknowledgments

We thank Paul Bruce and Chris Thibodaux for technical
assistance. The Louisiana Alliance for Minority Participa-
tion provided support for A.S.U. This study was partially
supported by the Louisiana Sea Grant College grant NOAA
46RG0096 Project R/ZMM-1 and 46RG0096 R/ZMM-5.

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20 T. H. DIETZ ET AL

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Reference: Bio/. Bull. 199: 21-28. (August 2000)

Allometric Scaling in Small Colonies of

the Scleractinian Coral Siderastrea siderea

(Ellis and Solander)

STEVEN V. VOLLMER* AND PETER J. EDMUNDS

Department of Biology, California State University, 18111 Nordhoff Street,

Northridge. California 91330

Abstract. Although most physiological traits scale allo-
metrically in unitary organisms, it has been hypothesized
that modularity allows for isometric scaling in colonial
modular taxa. Isometry would allow increases in size with-
out functional constraints, and is thought to be of central
importance to the success of a modular design. Yet, despite
its potential importance, scaling in these organisms has
received little attention. To determine whether scleractinian
corals are free of allometric constraints, we quantified met-
abolic scaling, measured as aerobic respiration, in small
colonies (S40 mm in diam.) of the scleractinian Siderastrea
siderea. We also quantified the scaling of colony surface
area with biomass, since the proposed isometry is contin-
gent upon maintaining a constant ratio of surface area to
biomass (or volume) with size. Contrary to the predicted
isometry, aerobic respiration scaled allometrically on bio-
mass with a slope (b) of 0.176, and colony surface area
scaled allometrically on biomass with a slope of 0.730.
These findings indicate that small colonies of S. siderea
have disproportionately high metabolic rates and SA:B ra-
tios compared to their larger counterparts. The most prob-
able explanations for the allometric scaling of aerobic res-
piration are ( 1 ) a decline in the SA:B ratio with size such
that more surface area is available per unit of biomass for
mass transfer in the smallest colonies, and (2) the small size,
young age, and disproportionately high growth rates of the
corals examined. This allometric scaling also demonstrates
that modularity, alone, does not allow small colonies of S.

Received 23 September 1999; accepted 30 May 2000.

* To whom correspondence should be addressed. Current address: De-
partment of Organismic and Evolutionary Biology. 16 Divinity Ave.. Box
90. Harvard University. Cambridge. MA 02138. E-mail: svollmer@oeb.
harvard.edu

siderea to overcome allometric constraints. Further studies
are required to determine whether allometric scaling is
characteristic of the full size range of colonies of S. siderea.

Introduction

Body size affects diverse biological variables ranging
from physiological to life-history traits (Schmidt-Nielson,
1984). In unitary organisms, most processes scale allometri-
cally (Schmidt-Nielson, 1974) — that is, they change dispro-
portionately with size — as a result of physical and geomet-
ric constraints on body size, structure, and function (Gould,
1966; Schmidt-Nielson, 1974). Classic examples of these
constraints include the limits that the skeleton places on the
size of terrestrial mammals (Schmidt-Nielson, 1974; Econo-
mos, 1981) and the limits that flight muscles place on the
size of flying birds (Pennycuick, 1972). Constraints often
are inherent to the design of organisms, yet they can be
minimized, in theory, by minor changes in geometry or
shape (Brody, 1945; Gould, 1966). Profound changes in
size, however, require design modifications, including elab-
orate structural changes and the development of complex
internal systems (Gould, 1966; Schmidt-Nielson, 1984).
Such changes probably evolve over relatively long time
scales (Gould, 1966, 1977).

The relationship between surface area and volume is
fundamentally important to scaling arguments, especially
for surface-area-related phenomena such as metabolism and
thermal regulation (Gould, 1966; Schmidt-Nielson, 1984).
because most processes scale allometrically as a result of
decreasing ratios of surface area to volume (SA:V) that are
associated with volumetric increases in body size (Gould.
1966). In cases where geometric similarity is maintained
with increasing size (that is. where there is geometric isom-

S. V. VOLLMER AND P. J. EDMUNDS

etry, or constancy of shape), surface area (v) scales to the
two-third power of volume (.v) according to the allometric
equation: v = a.\J'. This equation describes a case of func-
tional allometry arising from geometric isometry, where a is
a constant and b is the scaling exponent with a predicted
value of .067 (Schmidt-Nielson, 1984; Peters, 1983). Thus,
all things being equal and with geometric isometry, all
surface-area-related processes (heat loss, gas exchange, etc.)
also should scale to the two-thirds power of mass, i.e., the
surface rule (Rubner, 1883). However, most organisms do
not maintain geometric similarity as they grow (McMahon,
1973), and therefore the scaling exponents of many surface-
area-related processes deviate from the predicted value of
0.67 (Kleiber, 1932, 1961). Metabolism, for example, often
scales to the three-quarter power in interspecific analyses
(b = 0.75) (Zeuthen, 1953; Hemmingsen, 1960). whereas
intraspecific exponents vary widely depending on the or-
ganism (Altman and Dittmer, 1968; Peters, 1983). Such
deviations can be explained, in part, by geometric allometry
involving changes in the shape of exchange surfaces; these
changes maintain high SA;V ratios and minimize surface-
area-related constraints (Gould, 1966). In an extreme case,
organisms might overcome geometric constraints entirely
by maintaining a constant SA:V ratio with increasing body
size (Gould, 1966). However, the maintenance of a constant
SA:V ratio is likely only in organisms with relatively un-
usual body plans — for example, the dorsoventral flattening
in flatworms (Gould, 1966), and the incorporation of non-
respiring biomass in corals, bryozoans, hydroids, and other
colonial modular organisms (Gould, 1966; Sebens. 1987a).

It has been hypothesized that colonial modular organisms
overcome the allometric constraints typically associated
with volumetric increases in body size by maintaining a
constant SA:V ratio as colony size increases (Jackson, 1979;
Hughes and Hughes, 1986). Purportedly, this is achieved by
subdividing the biomass of the colony into individual units
(i.e., modules) of similar size (Hughes and Hughes, 1986)
and growing through modular iteration (Jackson, 1979;
Hughes and Cancino, 1985). As a result, physiological
processes should not be functionally constrained by declin-
ing SA:V ratios, but instead should scale proportionally
(i.e., isometrically, b = I ) to both the number of modules
and the total colony biomass (Jackson, 1979; Sebens, 1979,
1987a), thereby allowing indeterminate colony growth (Se-
bens, 1987a). In turn, this proposed isometry is thought to
be critical to the success of colonial modular organisms
(Hughes and Hughes. 1986). because it should provide
access to the beneficial fitness consequences of increased
size (Jackson. 1977; Sebens. 1982; Hughes and Jackson,
1985; Karlson, 1988) without the constraints of allometry.

However, despite the theoretical importance of isometry
in colonial modular organisms, few studies have tested this
prediction, and the available data are contradictory. Aerobic
respiration, for example, scales isometrically with mass in

the bryozoan Elect ra pilnsa (b = 0.97; Hughes and Hughes,
1986) but allometrically in the soft coral Alcyoiiiiini side-
riiiin (h = 0.88; Sebens, 1987b). Moreover, chemical engi-
neering and mass transfer theory predict that many colonial
modular organisms with simple geometries should display
allometric scaling (Patterson. 1992a). In this study, we
revisit scaling in colonial modular organisms to determine
whether their body plans do, indeed, provide a comprehen-
sive escape from allometric constraints. More specifically,
we test the null hypotheses that aerobic respiration (hereaf-
ter referred to as respiration) and the surface-area-to-bio-
mass (SA:B) ratio scale isometrically (i.e., proportionately)
in the scleractinian Siderastreu siderea. Respiration was
selected to examine the scaling of physiological traits be-
cause of its importance in generating ATP for synthetic and
muscular work. The SA:B ratio was selected as a proxy for
the SA:V ratio because biomass (B) can be determined
easily with a gravimetric approach, and it is proportional to
volume with a constant tissue density.

Siderastrea siderea was used as a model system for a
colonial modular taxon because, as a scleractinian, it pro-
vides a consummate example of this structural clade. Ad-
ditionally, S. siderea is ecologically important on Caribbean
reefs (Goreau, 1959) and can be identified readily to species
(Foster, 1979, 1980). The study was restricted to small (<40
mm diam.), juvenile colonies (Soong, 1993) because they
are tractable to investigation within the constraints of lab-
oratory chambers designed to measure metabolism. Juvenile
corals also have a strong effect on the population biology
of reef corals (Bak and Meesters, 1999), and thus studies of
their biology are likely to result in a better understanding of
the processes driving coral demography. The full size range
of S. side rea (to = 1 m diam. and > 100 y old. Foster, 1979)
was not included because large colonies were rare at the
study site (the north coast of Jamaica) and cannot be ac-
commodated easily in laboratory apparatus. Thus, although
the results of this study provide a valid test of scaling in an
important life-history stage of a colonial modular taxon, the
findings cannot be extrapolated beyond the size range of the
colonies investigated.

Materials and Methods

Respiration

Small colonies of Siderastrea siderea were collected
from 8.5 m depth on the forereef at Dairy Bull, about 2 km
east of Discovery Bay. Jamaica, in January 1997. They were
transported to the Discovery Bay Marine Laboratory
(DBML) where they were epoxied (Z-Spar A-788) to tiles
made of acrylic plastic. The epoxy was applied to the
exposed skeleton so that only living coral tissue was left
uncovered. Within 24 h. the tiles were secured to racks and
returned to the collection site to recover. After more than 1
week of recovery, corals were selected haphazardly from

ALLOMETRIC SCALING IN SMALL CORALS

the racks, returned to the laboratory, and placed in a dark-
ened container supplied with flowing seawater. The corals
were kept in darkness overnight, prior to respiration mea-
surements, to avoid the confounding effect of light history
on the respiration of symbiotic corals (Edmunds and Da-
vies. 1988).

Respiration rates were measured as oxygen flux using
polarographic oxygen electrodes that were connected to an
oxygen meter (Cameron OM400) and inserted into the top
of clear acrylic chambers. The chambers were designed to
expose the corals to unidirectional flow while retaining the
minimal volumes necessary for respirometry with small
organisms (Fig. 1). A small chamber was used for corals
roughly 20 mm in diameter, and a large chamber for corals
21 to 40 mm in diameter. Both chambers consisted of a
circular working area with volumes of 332 and 680 ml,
respectively, and were regulated at ambient seawater tem-
perature (26°C) using a water jacket and bath (Haake Dl).
Water flow inside the chambers was created by a stirbar
rotating at a constant rate. Flow rates at the periphery of the
chambers, where the corals were located, were quantified
using brine shrimp cysts (Johnson and Sebens, 1993), and
were not significantly different between chambers (Mann

Water Jacket
\

Working
Volume

Stirbar

Electrode

6 cm '

Figure 1. Plan view of the respiration chamber (drawn to scale). The
chamber consisted of a cylindrical working volume (5 cm high x 14 cm in
diam.. 680 ml in volume) surrounded by a water jacket. A centrally located
stirbar created a unidirectional flow (5.8 ± 0.1 cm s~'( over the coral
colony located on the periphery of the chamber; arrows indicate direction
of flow.

Whitney U test, £/, = 683.5, n]2 = 40, P = 0.26). The
pooled flow rate for both chambers was 5.8 ± 0.1 cm s '
(mean ± SE. ;; = 80).

Two oxygen electrodes were used (Strathkelvin E5046
and YSI Model 5739), and both were calibrated using a zero
solution (0.01 M sodium tetraborate and sodium sultite) and
air-saturated seawater. Salinities were determined using a
refractometer, barometric pressure was recorded, and oxy-
gen solubilities were determined from Weiss (1970). Corals
were placed into the chambers filled with filtered seawater
(0.45 n-m. FSW). and respiration rates were measured in
darkness following 15-min acclimation to the chamber. All
measurements were completed at an oxygen saturation
above 80% (Edmunds and Davies, 1986), and data were
recorded using a data acquisition system (Datacan, Sable
Systems). Controls were run daily in the same manner using
FSW alone. The rates of change in pO-, in the experimental
and control trials were calculated using simple linear regres-
sion (r2 > 0.94). After accounting for controls, the respira-
tion rate per coral (micromoles of oxygen per coral per
hour) was calculated to examine metabolic scaling.

Surface area and biomass

After respiration measurements were completed, surface
areas were estimated using the aluminum foil method
(Marsh, 1970). In this technique, aluminum foil was molded
over the surface of the coral; the foil was then removed,
dried, and weighed; and the surface area was estimated
using a previously derived relationship between area and
weight. Dry tissue biomass was quantified by preserving the
corals in 5% formalin in seawater, decalcifying in 5%
HNO3. and drying the resulting tissue tunic at 60°C for 7
days (Edmunds and Davies, 1986). Preliminary experiments
using tissue from the anemone Anthopleura xanthogram-
niica demonstrated that the formalin and acid treatment
resulted in a loss of 2.7% ± 0.7% (mean ± SE, n = 10) of
the dry tissue. Therefore, the values of dry tissue biomass in
the present study are likely to be slightly conservative.

Statistical analyses

Logarithmic linear regression was used to examine the
scaling relationships. The slope of the regression provides
the scaling exponent (/?), and all analyses were completed
using natural logarithms (In). The scaling of metabolism
was estimated by a regression analysis with the log of
respiration (per coral) as the dependent variable and the log
of dry tissue biomass as the independent variable. Changes
in the ratio of surface area to volume were estimated by
regression analysis with the log of surface area as the
dependent variable and the log of dry tissue biomass as the
independent variable, assuming that biomass and volume
are related linearly. Model II (reduced major axis) regres-
sion analyses were used because the independent variables

S. V. VOLLMER AND P. J EDMUNDS

£
o

c«
<L>

-1-

-3'

-20246
In Dry Tissue (mg)

"o 0

c -2
^o

*4— I

03
i_

'a, -4

•a -6-

-20246
In Dry Tissue (mg)

Figure 2. Respiration plotted against biomass in small colonies of Siderastrea siderea. (A) Regression of the
log of the respiration rate per coral on the log of dry tissue biomass: regression equation: y = 0.176 x + 1.717,
,• = 0.494. The slope of 0.176 ± 0.031 (±SE. » = 26 corals) deviates significantly from 1 (/ = 26.59. df = 24,
P < 0.0001 ). indicating allometric scaling. (B) Regression of the log of the mass-specific respiration rate on the
log of dry tissue biomass (recalculated from the data in Fig. 2A); regression equation: v = -0.924 .v - 1.9752.
r = 0.986.

were subject to measurement error (Ricker, 1973; Sokal and
Rohlf, 1995). In this technique, the slope (or scaling expo-
nent) is obtained by dividing the standard error of the
dependent variable by the standard error of the independent
variable, which results in a slope greater than that generated
by least-squares linear regression (Sokal and Rohlf, 1995).
The null hypothesis of isometry was tested using a t test
(//0: b =: 1), where a significant deviation (P < 0.05)
indicates an allometric relationship.

Results

Respiration

Respiration rates were estimated in 26 corals ranging in
diameter between 3 and 37 mm. The regression of the log of
respiration rate (micromoles of oxygen per coral per hour)
on the log of colony dry tissue biomass (Fig. 2a) was
significant (F( , 24l = 7.952, P < 0.01 ). and produced a slope
of 0.176 ± 0.031 (±SE, n = 26 corals), which deviated
significantly from 1 (t = 26.59. df = 24. P < 0.0001 ). This
significant departure from a slope of 1 indicates that respi-
ration scaled allometrically on biomass such that respiration
increased disproportionately more slowly than colony size
(biomass). As a result, a doubling of biomass corresponds to
only a 13% increase in the respiration rate per colony (Fig.
2a), and a 47% decline in mass-specific respiration (micro-
moles of oxygen per milligram of tissue per hour) (Fig. 2b).

Snrfiice area on hiomtiss

The scaling of colony surface area with biomass was
quantified in 25 of the 26 corals; 1 coral was excluded as an

outlier due to high leverage (Sokal and Rohlf, 1995). The
regression of the log of surface area on the log of the dry
tissue biomass of the colonies (Fig. 3) also was significant
(F(\ 23, = 294.973, P < 0.0001 ), and the slope of 0.730 ±
0.041 (±SE, n = 25 corals) deviated significantly from 1
(t = 6.60, df = 23, P < 0.0001 ). This indicates that colony
surface area scaled allometrically with biomass such that
surface area increases disproportionately more slowly than

-4-

In Dry Tissue (mg)

Figure 3. Colony surface area plotted against biomass in small colo-
nies of SUIcrastrea siderea. Regression of the log of colony surface area on
the log of dry tissue biomass; regression equation: v = 0.730 .v - 1.356.
/• = 0.963. The slope of 0.730 ± 0.041 (±SE. n = 25 corals) deviates
significantly from 1 (I = 6.60, df = 23. P < 0.0001 ), indicating allometric
scaling,

ALLOMETRIC SCALING IN SMALL CORALS

biomass, and the ratio of surface area to biomass declines
with increasing colony size. As a result, a doubling of
biomass corresponds to only a 66% increase in surface area.
Moreover, the slope of 0.730 for surface area on biomass
does not deviate significantly (: = 1.453, df = 23, P =
0.1597) from the expectation of geometric isometry (b =
0.67). Thus, the modular design of these small corals does
not confer significantly higher ratios of surface area to
biomass than would be expected if geometric similarity was
maintained.

Discussion

Contrary to the isometric scaling predicted for colonial
modular organisms (sensu Hughes and Hughes, 1986), res-
piration and surface area scaled allometrically with biomass
in small colonies of Siderastrea siderea. As a result, both
mass-specific respiration and the surface-area-to-biomass
(SA:B) ratio declined with colony size. Thus, although
respiration scales isometrically in at least one colonial mod-
ular organism — the encrusting bryozoan Electro pilosa
(Hughes and Hughes, 1986) — the present results show that
isometric scaling is not axiomatic with a colonial modular
design. Instead, allometry describes the size-dependency of
two traits in S. siderea, and has been demonstrated previ-
ously for respiration in the octocoral Alcyoiiiiini siderium
(Sebens, 1987b) and predicted on the basis of chemical and
mass-transfer theory (Patterson, 1992a). Although compar-
isons across taxa are difficult due to the wide variation in
intraspecific metabolic scaling exponents, the exponent of
0.176 calculated for small colonies of S. siderea falls within
the observed range (/? == 0.15 -• 1.28) of intraspecific
exponents for metazoans (see Peters, 1983; Patterson,
1992a). Metabolic scaling exponents in unitary anthozoans
range from 0.54 to 0.94 (Patterson, 1992a) and include the
solitary scleractinian Fun^ia scutaria (b = 0.79; Krupp,
1982; exponent calculated in Patterson, I992a). As for
metabolic scaling in small colonies of S. siderea, we posit
that the unusually small scaling exponent (b = 0.176) is a
result of the changes in the SA:B ratio and the developmen-
tal stage of the small colonies investigated.

Maintaining a constant surface-area-to-volume (SA:V)
ratio (and, with invariate biomass density, a constant SA:B
ratio) is the theoretical basis for isometry in colonial mod-
ular organisms (Jackson, 1979). However, isometry can
occur only where colony biomass is restricted to a single
layer of modules with conserved dimensions; this design is
typical of hydroids, scleractinians, and cheilostome bryozo-
ans (Jackson, 1979). However, where there is metabolically
active biomass outside the modules, the SA:B ratio de-
creases as extra-modular biomass increases volumetrically.
Thus, the extra-modular biomass in octocorals (i.e.. the
coenenchyme) and compound ascidians (i.e., the gelatinous
matrix) should favor allometric scaling (Jackson, 1979;

Sebens, 1987a). These predictions are supported by exper-
imental data from the encrusting bryozoan Electra pilosa
and the fleshy octocoral Alcyonium siderium (cited above).
However, although the single layer of uniformly sized pol-
yps in scleractinians also should allow for a constant SA:B
ratio, this is not the case for S. siderea, where colony surface
area scales allometrically on biomass (b = 0.730). Thus, in
small colonies of S. siderea, the SA:B ratio declines with
increasing colony size, such that larger colonies have dis-
proportionately more biomass than their smaller counter-
parts (Fig. 3).

The functional basis for the allometric scaling of the
SA:B ratio is unknown, but it is probably related to calci-
fication (Barnes, 1973) and the selective pressure for rapid
growth in small corals (Jackson, 1977). Thus, the smallest
corals may sustain high rates of linear growth (i.e.. calcifi-
cation) at the expense of tissue growth, so that the existing
tissues are "stretched" thinly over the increasing surface
area. Then, as the colonies become larger, they may con-
centrate resources on tissue growth, thereby increasing bio-
mass and tissue thickness. Support for this hypothesis
comes from two studies. First, reanalysis of the data of
Jokiel and Morrisey ( 1986) for the coral Pocilloponi dami-
cornis demonstrates allometric scaling of surface area with
biomass (b = 0.700 ± 0.057, mean ± SE, n = 6) as well as
respiration (b = 0.840 ± 0.041, mean ± SE, n = 6) (Jokiel
and Morrisey, 1986). Thus, biomass is added more rapidly
than surface area and. as in S. siderea. the resulting allo-
metric scaling of the SA:B ratio provides a possible expla-
nation for the allometric scaling of respiration in P. dami-
cornis. Second, trade-offs in growth between skeleton and
tissue, similar to those proposed for S. siderea (described
above), have been reported for Porites from the Great
Barrier Reef (Barnes and Lough, 1993), as have systematic
differences in tissue thickness for the same species (Barnes
and Lough, 1992). Indeed, the positive relationship between
tissue thickness and colony height in Porites (Barnes and
Lough. 1992), together with the large amount of extra-
modular biomass ( ==90% by thickness, Barnes and Lough,
1992), might be prominent in this genus. Thus, variation in
tissue biomass. thickness, or both with colony size may be
a general feature of scleractinian corals. However, in addi-
tion to putative changes in tissue thickness driving the
observed changes in the SA:B ratio, it is possible that the
SA:B ratio was biased by the use of the aluminum foil
method (Marsh, 1970) to measure the surface area. This
technique is widely used for determining the surface area of
corals with relatively smooth and unconvoluted surfaces
like those in 5. siderea (see Hoegh-Guldberg. 1988. for an
alternative approach), but it is unable to quantify the area
of the expanded polyps. Quantifying the area of expanded
polyps is made difficult by their highly variable morphology
and degree of expansion and, as a result, previous studies
have relied on geometric approximations to obtain polyp or

S. V. VOLLMER AND P. J. EDMUNDS

tentacle area (Sebens, 1981). Regardless of the methodolog-
ical difficulties, currently there is no evidence of systematic
variation in polyp dimensions with colony size (i.e.. allom-
etry): moreover, polyp dimensions may be highly conserved
for mass-transfer purposes (Patterson, 1992a). Thus, given
that the thickness of coral tissues is known to vary (e.g.,
Barnes and Lough. 1992), we believe that changes in the
SA:B ratio are more likely to be driven by tissue thickness
than by the area of expanded polyps. Still, a definitive test
of the hypothesized mechanism of variation in the SA:B
ratio is required, and this will necessitate an analysis of
tissue thickness and skeletal extension as a function of
colony size.

The allometric scaling of the SA:B ratio in S. siderea
could drive the scaling of respiration through indirect ef-
fects on mass transfer of metabolites to the coral tissue.
Mass transfer with the surrounding seawater is determined,
in part, by surface area, which decreases relative to biomass
as S. siderea increases from 3 to 37 mm in diameter. Thus,
all things being equal (i.e., excluding the boundary layer
arguments described below) and within the size range stud-
ied here, small corals should maintain relatively higher
fluxes of metabolites than large corals, which could support
the higher respiration rates observed in the small corals (Fig.
2). Additionally, increases in biomass will be accompanied
by increases in biovolume that probably lengthen diffusion
pathways (i.e., the tissue thickness) and reduce the rates of
solute transport (Patterson. 1992b). For the colony size
range studied, the respiration of large S. siderea therefore
may be depressed by limitations on the delivery of oxygen
to metabolically active tissue. This hypothesis could be
tested by measuring the magnitude of the flow dependency
of respiration (sensu Patterson and Sebens, 1989), with the
expectation of a greater effect in larger colonies than in
smaller ones.

Although the scaling of the SA:B ratio provides a testable
hypothesis to explain the scaling of respiration in S. siderea.
it does not exclude the possibility that other factors might
also be important. Of these, variation in energy expenditure
among developmental phases (i.e., colony sizes) has the
greatest potential to explain, in part (or entirely), the allo-
metric scaling of respiration. In benthic marine inverte-
brates, scaling exponents typically are affected by the size
range and developmental phase of the organisms investi-
gated (Zeuthen, 1953). Lower exponents are characteristic
of early and late developmental phases and of the extremes
of the natural size range. For example, metabolic scaling
exponents (/?) for the mussel Mytilus edulis change from
0.80 in recruits «0.1 mg) to 0.95 in sub-adults (0.1 to I
mg) and to 0.65 in adults (> 1 mg) (Zeuthen, 1953). The low
scaling exponents in the smallest (i.e., youngest) size classes
demonstrate that their metabolic rates are relatively high
compared to those of the larger sub-adults, and are thought
to be a consequence of the elevated energy expenditure

necessary to sustain accelerated growth (Zeuthen, 1953).
Size and age are poorly related in scleractinians (Hughes
and Jackson. 1980), but the colonies of S. siderea used in
the present study (^37 mm diam.) are young relative to the
largest colonies of this species ( = 1 m diameter and > 1 00 y
old; Foster. 1979). and the smallest corals (3 mm diam.)
may be only a few months old (Van Moorsel, 1988). Re-
gardless of age, small corals are probably exposed to selec-
tive pressure for rapid growth (Jackson, 1977), as occurs in
other colonial modular organisms (Jackson, 1977; Sebens,
1982; Karlson, 1988). because of the mortality risks of
being small (Jackson, 1977). Thus, in addition to the SA:B
explanation for allometric scaling in small colonies of S.
siderea (described above), it is possible that the respiration
rate (per coral) in the smallest colonies is elevated by the
high metabolic rate of young tissues or by the costs of
responding to the selective pressure for rapid growth.

Two other hypotheses could account for allometric scal-
ing of respiration in small colonies of S. siderea — namely.
mass transfer effects (sensu Patterson, 1992a) and the pop-
ulations of endosymbiotic zooxanthellae — but these are not
supported by the available data. The "mass transfer hypoth-
esis" focuses on the importance of mass transfer in moving
metabolites between the coral tissue and seawater and driv-
ing coral respiration (Patterson and Sebens, 1989; Patterson
el ul., 1991 ). The boundary layers next to the coral have a
critical role in determining rates of mass transfer (Denny,
1988; Patterson. 1992b) and are a function of the interaction
of the flow regime with the size and shape of the coral
colony. Based on these relationships. Patterson (1992a)
predicted that metabolic scaling in aquatic organisms could
be explained with a mass transfer argument. In short,
changes in organism size and shape can be sufficient to alter
mass transfer and support the allometric scaling of metab-
olism, with exponents similar to published values (Patter-
son. 1992a). For hemispherical objects like the small colo-
nies of S. siderea used in the present study, the mass transfer
explanation for metabolic scaling (sensu Patterson, 1992a)
would predict an exponent (b) of =0.47 (Helmuth et ul..
1997). This is 2.7-fold higher than the allometric scaling
exponent we calculated for respiration in small colonies of
S. siderea that have hemispherical colonies (h = 0.176).
One reason for this discrepancy is that the colonies used
(3-37 mm diam.) were probably too small to establish their
own equilibrium boundary layers (Denny, 1988) and were,
instead, affected by upstream roughness elements in the
respiration chamber (Gardella and Edmunds, unpubl. data).
In other words, small colonies of S. siderea may be an
exception to the mass transfer argument for allometric scal-
ing (sensu Patterson. I992a). because they all are too small
(i.e., £37 mm diam.) to affect their own boundary layers
directly.

The "zooxanthellae hypotheses" focus on the role of the
/ooxanthellae in contributing to the respiration of the col-

ALLOMETRIC SCALING IN SMALL CORALS

ony (i.e., the cnidarian host plus algal symbionts) (Musca-
tine ct til.. 1981; Edmunds and Davies, 1986) to account for
allometric scaling of coral respiration. Thus, changes in the
density or metabolic activity of zooxanthellae should affect
the respiration of the colony and, if these changes are
correlated with size, could affect metabolic scaling. In S.
sidereu, zooxanthellae densities cannot account for allomet-
ric scaling of respiration, because zooxanthellae densities
scaled isometrically with biomass (Vollmer, 1999). It is
possible, however, that the respiration rate per zooxanthella
varied with colony size, but this possibility cannot be ex-
amined experimentally at the current time because zooxan-
thellar respiration can only be measured /;; vitro, and these
rates may be different from those attained in hospite (Gates
ct til.. 1999). In the absence of in hospite determinations of
zooxanthellar respiration, and given that zooxanthellae den-
sities scale isometrically, we conclude tentatively that the
zooxanthellae are unlikely to be a proximal cause of the
allometric scaling of respiration in small S. sidereu.

This study demonstrates allometric scaling in small col-
onies of Siderastrea siderea and underscores two mecha-
nisms that probably drive this scaling — i.e.. disproportion-
ate changes in the SA:B ratio and the developmental stage
of the colonies examined. Because both mechanisms may be
associated with the rapid growth necessary to escape the
risky life-history stage of being small, allometric scaling
probably has strong fitness consequences. While it remains
to be seen whether the present findings are applicable to
other species, or to a larger size range of colonies, further
studies of allometric scaling in scleractinians will be valu-
able.

Acknowledgments

This research was submitted in partial fulfillment of the
Master of Science degree to S. V. Vollmer at California
State University, Northridge (CSUN). We thank R. C. Car-
penter, D. Carlon, and three anonymous reviewers for their
comments on earlier drafts of this paper. Field work was
made possible through the generosity of M. Haley and the
staff of the Discovery Bay Marine Laboratory (DBML), and
numerous dive buddies who assisted in the field, including
G. F. River and S. Tallack. This research was funded with
a grant from the CSUN Office of Graduate Studies and
International Programs (SVV) and a CSUN Research and
Grants Award (PJE). This is DBML contribution number
623.

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Reference: Bio/. Bull. 199: 29-40. (August 2000)

Frog Lim-1-like Protein Is Expressed Predominantly

in the Nervous Tissue, Gonads, and Early Embryos of

the Bivalve Mollusc Mytilus galloprovincialis

MARIO TORRADO AND ALEXANDER T. MIKHAILOV*

Developmental Biology Unit, Institute of Health Sciences, University of La Contna, As Xubias,

La Coruna 15006, Spain

Abstract. In a few well-known cases, the biological con-
sequences of the disruption of liin-1 homeodomain (HD)
genes have demonstrated the important roles of these genes
in vertebrate development, especially in the nervous tissue,
kidney, and gonads. Functional assay approaches require
information not only about lim-l gene organization, but also
about properties and tissue localization of Lim-l proteins.
Although lim-l genes have been identified in certain phyla
of invertebrates, no information is available on Lim-l pro-
teins and genes in bivalve molluscs. Our study represents
the beginning stage of identification of the Lim-l -related
proteins in marine bivalves. Using antibodies against the
C-terminal region of the Xenopus laevis Lim-l protein, we
describe cross-reactive antigen patterns in adults and early
embryos of the mussel Mytilus galloprovincialis, as well as
in sea urchin and chick embryos. In adult mussels, nervous
ganglia and gonads display the most prominent Lim-l im-
munoreactivity. Further, the antibodies verified the predic-
tion that mussel Lim-l antigens, like Lim-l HD proteins in
general, can be localized in the nucleus. Moreover, antibody
detection allowed us to identify the Lim-1-like antigens in
unfertilized mature eggs, as well as in very early embryos of
bivalve molluscs and sea urchins (Strongylocentrotus pur-
pnratns). In mussel eggs and embryos, Lim-l antigens are
expressed in multiple forms (40, 45, and 65 kDa), as de-
tected by SDS-PAGE followed by Western blot. Taken
together, the observations emphasize the conservation of the
Lim-l protein expression pattern in the nervous tissue and

Received 13 December 1999; accepted 8 May 2000.

* To whom correspondence should be addressed: E-mail: margot
@udc.es

Abbreviations: LIM. an abbreviation derived from the names of three
homeodomain transcription factors, containing a cysteine-rich zinc-finger
domain (i.e.. LIM-domain): Lin- 11 of Caenorhabditis elegans, Isl-1 of the
rat, and Mec-3 of C. elegans', MW, molecular weight; SDS-PAGE. sodium
dodecylsulfate polyacrylamide gel electrophoresis; HD. homeodomain.

gonads of different animal groups, and demonstrate that
Lim-1-like polypeptides can be maternally accumulated in
eggs and, therefore, are present in very early embryos
before zygotic expression of the genes begins.

Introduction

A cysteine-rich zinc finger domain, named LIM, was first
identified in the Caenorhabditis elegans homeobox genes
lin-1 1 and mec-3, and in the rat DNA binding factor Isl-1.
Then the LIM domain was found in a variety of proteins
including transcription factors, cytoskeletal proteins, and
LIM kinases (Dawid et ai, 1998; Bach. 2000). LIM do-
mains appear to play a primary role in protein-protein
interactions, through the formation of dimers with identical
or different LIM domains or by binding distinct proteins
(Breen et ai. 1997; Dawid et ai. 1998; Curtis and Heiling,
1998; Hobert and Ruvkun. 1998; Hobert and Westphal,
2000).

Phenotypic analysis of patterns of lim gene expression
reveals that the genes can participate in a number of impor-
tant events in early embryonic development, as well as in
cell fate determination and cell differentiation at advanced
stages of organogenesis. Moreover, some lim genes are
constitutively expressed in adult tissues, where they may
contribute to certain tissue-specific functions (Dawid et ai.
1998; Hobert and Westphal, 2000). The early and late
ontogenetic expression phases of lim genes suggest that they
have multiple and distinct functions at different stages of the
animal life cycle. In the latter context, the LIM containing
homeodomain (HD) lim-l genes have been most exten-
sively studied in a range of animals.

Lim-l encodes a protein with a pair of LIM domains
located N-terminal to the HD. In vertebrates, //';;/ / was
originally identified in the frog, Xenopus laevis. as Xlim 1
(Taira et ai, 1992). In X. laevis, the Xlim-l is expressed in

M. TORRADO AND A. T. MIKHAILOV

the Spemann's organizer during gastrula stages, and in late
embryos primarily in the nervous system and kidney (Taira
etui.. 1992. 1994. 1997: Wallingford et til.. 1998; Carroll et
a!.. 1999a; Carroll and Vize. 1999). Similar patterns of
lim-l expression have been observed in fishes (Toyama et
ul.. 1995; Carroll et ai, 1999b). chickens (Tsuchida et til..
1994). mice (Fujii et ul.. 1994; Shawlot and Behringer,
1995: Li et til.. 1999). rats (Furuyama et til.. 1994; Kara-
vanov et til., 1998). and humans (Dong et ul.. 1997). The
biological consequence of the disruption of lim-l HD genes
or modifications of their expression patterns have demon-
strated the crucial role of these genes in development,
especially in the nervous tissue, kidney, and gonads
(Shawlot and Behringer. 1995; Shawlot el til.. 1999: Taira et
til.. 1997; Carroll and Vize. 1999).

In invertebrates, lim-l related genes have been identified
in nematodes. fruit flies, and sea urchins. The lin-11 gene of
C. elegans (the closest homolog of amphibian and mamma-
lian lim-l) is expressed in different subsets of neurons and
in the vulva, and it is essential for uterine morphogenesis
(Hobert et ai. 1998: Hobert and Westphal. 2000; Newman
et al. 1999). In Drosophila. the gene termed dlim I is
expressed in the head, the brain lobes, and in neurons of the
ventral nerve cord (Lilly et til.. 1999). The ///(/-/-related HD
gene of the sea urchin (Hemicentrotiis pulcherrimns).
HpLiml, is detected in early embryos and involved in the
differentiation of endoderm, mesenchyme, and aboral ecto-
derm (Kawasaki etui., 1999).

Thus, although expression in neural tissues seems to be a
common feature of Lim-l -related HD factors in both ver-
tebrates and invertebrates, most of these factors are also
characterized by their expression in excretory and reproduc-
tive organ systems. These expression phenotypes have been
described mainly at the molecular level by analysis of lim-l
gene transcription patterns. An alternative approach is the
analysis of post-translational Lim-l expression by immuno-
chemical methods. This approach makes it possible to mea-
sure this factor at the protein level in different cell types and
to detect other tissues that express the polypeptide at dif-
ferent stages of development and in the adult state (Kara-
vanov et til., 1996; Brown et til., 1999; Lilly et at.. 1999;
Shimono and Behringer, 1999; Mauch et til., 2000).

To our knowledge, no information is available on Lim-l
proteins (genes) in marine bivalve molluscs, although such
data would be useful for further comparative analysis of
Lim-l expression patterns and functions in invertebrates
and vertebrates (Hobert and Westphal, 2000). Our study
represents the beginning stage of the identification of Lim-
1 -related proteins in bivalve molluscs. We describe the
distribution patterns of immunoreactive Lim-l -like proteins
in adults and early embryos of the mussel Mytilus gallopro-
vincitilis. We also report the first examination of Lim-l
antigen signals in sea urchin (Strongylocentrotus purpiirt/-
lus) embryos, as well as in different compartments of the
chick embryo brain.

Materials and Methods
Animals and embryos

Adult mussels (Mytilus galloprovincialis) and sea urchins
(Strongylocentrotus purpuratus) were purchased during the
spawning season (April-May of 1999) from commercial
suppliers in La Coruiia (Galicia, NW Spain). Published
procedures (Sprung and Bayne. 1984; Holland and Holland.
1993; Mikhailov et ai. 1996) were followed for stripping of
animals to obtain oocytes and sperm and for the subsequent
in vitro fertilization and culture of embryos. At each chro-
nological stage, the bulk of the embryos were re-collected,
placed on ice, and typed morphologically under a dissecting
microscope (Nikon). The embryos, selected according to
morphology, were put into centrifuge tubes containing ster-
ile seawater and permitted to settle to the bottom of tubes or
pelleted by low-speed centrifugation: then the upper solu-
tion was discarded. Fertilized chicken (Callus gullus) eggs
were obtained from the cooperative chick network hatchery
(Ferrol. Province of La Coruna. Galicia) and incubated at
37°C. Different brain regions (forebrain. optic lobes, and
cerebellum) were microsurgically isolated in cold minimum
essential medium (MEM; Gibco) and pelleted by low-speed
centrifugation.

Tissue dissection and processing

Before use, adult mussels (M. galloprovincialis) were
kept in a dry state for 1 h at 4°C. Mussels were opened with
the aid of scalpel and placed on ice; a small portion of gonad
material was microscopically examined to determine the sex
of the individual. Different tissues and organs (gonad, foot,
labial palps, hepatopancreas, gill, and fragments of posterior
adductor muscle) were excised, rinsed in sterile seawater,
and blotted on sterile filter paper. To obtain cell suspensions
of sperm or oocyte, gonad follicle biopsy was performed as
described in Torrado and Mikhailov (1998). Then, follicle
luminal masses were aspirated and resuspended in ice-cold
sterile seawater; the released cell suspension was micro-
scopically tested for the presence of spermatozoa or oo-
cytes. The sperm suspension was then centrifuged ( 100 X i>,
5 min, 2 C). the oocytes were permitted to settle to the
bottom of the tubes, and the rinse solution was discarded.
For some experiments, gonad collecting tubules (with adja-
cent connective tissue) were dissected manually from the
ripe male or female gonad and the excised tissue was
microscopically examined to definitively determine the ab-
sence of gametes (spermatozoa or oocytes) in the tubule
lumen. Isolated tissues were additionally shaken in ice-cold
sterile seawater for 20 min; after settling, rinse solutions
were discarded. Pedal ganglia (see Fig. 4A) were microsur-
gically dissected under a stereomicroscope (Nikon), pooled
in ice-cold sterile seawater. and pelleted by low-speed cen-
trifugation. When the samples were not homogenized im-
mediately, they were stored at -85°C for several days.

MUSSEL LIM-I RELATED PROTEINS

Spent male gonads were sampled in 1998. and the corre-
sponding frozen and Bourn-fixed tissue fragments were
stored in liquid nitrogen and ethanol (70%). respectively.

Sample preparation

Mussel and sea urchin eggs and embryos were resus-
pended in cooled deionized water containing 2 mM EDTA
(Merck). 6 M urea (Merck), and the protease inhibitor
cocktail P2714 (Sigma). The supernatants obtained after
centrifugation (30.000 X g. 8°C, 30 min) were mixed with
SDS sample buffer containing the protease inhibitor cock-
tail, kept for 1 h at room temperature, and stored at — 30°C
until use. All other tissue samples were first homogenized in
1:5 (v/v) ratio in 100 mM Tris (Sigma). 2 mM EDTA
(Merck) solution, containing the protease inhibitor cocktail.
After centrifugation (10.000 X g. 2°C. 30 mm), the super-
natants were discarded, and the pellets were then re-ex-
tracted and assayed as described for embryos.

Antibodies

Anti-XLim-1 antibodies were a generous gift from Dr.
A. A. Karavanov and Prof.. Dr. I. Dawid. These polyclonal
rabbit antibodies produced against the C-terminal region of
the XLim-1 downstream of the HD (amino acids 265-403)
(Taira et a I., 1992) have been characterized and shown to
cross-react with Lim-1 proteins of fishes, mice, rats, and
humans (Karavanov et al., 1996. 1998). Gamma-globulin
fraction was obtained from anti-XLim-1 serum with the aid
of the Mab Trap G II Kit for antibody purification (Phar-
macia), according to the manufacturer's protocol. The frac-
tion was concentrated using concentrator units (Millipore),
supplemented by glycerol (Merck) at a final concentration
of 50%, and stored at — 20°C in aliquots. Chemicon has
recently commercialized these anti-XLim- 1 antibodies.

Protein determination

Protein concentration was measured (Ultrospec 1000E
spectrophotometer. Pharmacia) according to the Bradford
method using rabbit immunoglobulin G (Sigma) or bovine
serum albumin (Sigma) as standards.

SDS -PAGE assays

For all separations, the Mini-Protean II electrophoretic
cell (Bio-Rad) was used. Samples were electrophoresed
using 5% stacking and 10% resolving Tris-glycine SDS-
polyacrylamide gels (Bio-Rad). The gels were stained with
Coomassie blue R250 (Sigma) or electrophoretically trans-
ferred to membranes. The apparent molecular weights of the
bands were determined by comparing low and high molec-
ular weight calibration kits (Pharmacia) in the same gel. A
micro-preparative variant of SDS-PAGE was performed as
previously described (Mikhailov et al., 1997) using a Mini-
Protean II comb with one reference well. After electro-

phoresis, the reference gel strip was stained and used for
isolating the Lim-1 -containing fraction in the remaining gel
slab. Alternatively, whole gel slabs were stained with Coo-
massie solution (0.0004% in 20% methunol and 3% acetic
acid), and fractions of interest were cut out (Mikhailov
ct til., 1996). The protein was eluted from gel fractions
so obtained using a model 442 electro-eluter (Bio-Rad)
in accordance with the manufacturer's recommendation:
eluted solutions were concentrated using microconcentra-
tion units (Amicon, the 30-kDa cut-off membrane).

Blotting assays

Proteins resolved in 10% SDS-PAGE were transferred to
nylon (Nytran. Schleicher and Schuell) or nitrocellulose
(Optitran. Schleicher and Schuell) membranes by routine
methods (Mikhailov et at.. 1997) using the mini Trans-Blot
cell (Bio-Rad). Protein loading and localization of molecu-
lar weight standards was verified by membrane staining
with amido black (Merck) or Ponceau S (Sigma). For im-
munodetection. the blots were incubated in blocking solu-
tion containing 20% of normal horse serum (Sigma) at room
temperature for 1 h and further assayed as described in
Mikhailov and Simirsky (1991). As primary antibodies,
rabbit anti-XLim-1 or rabbit pre-iinmune (negative control)
gamma-globulin fractions were used at appropriate dilutions.
Peroxidase-labeled anti-rabbit immunoglobulins (Sigma)
were used as the second-stage reagent, and diaminoben-
zidine (Sigma) was used to develop the blots. The relative
amounts of antibody-labeled proteins were quantified by
densitometry (GS-700 densitometer, Bio-Rad) and image
software (Molecular Analysis, Bio-Rad). For total carbohy-
drate detection, blots were treated with an Immun-Blot kit
(Bio-Rad) for glycoprotein detection as described (protocol
1A) by the manufacturer: chicken egg ovalbumin (Sigma)
and rabbit liver carboxylesterase (Sigma) were used as
positive controls. For a precise comparison of the position
of the glycoprotein signal with that of Lim-1 immunoreac-
tivity, the blot membrane was cut (along the direction of
electrophoretic separation) at the middle of the run pocket
width; one half was treated with Immun-Blot kit and the
other with anti-XLim-1 antibodies (see Fig. 3C.D).

Deglycosylation ussa\

Extracts and Lim-1 -containing fractions of mussel pedal
ganglia and forebrains of 16-day-old chick embryos were
desalted (using Microcon units), re-dissolved in 250 mM
sodium phosphate (Merck). pH 6.0. and treated with an
enzymatic deglycosylation kit (Bio-Rad) according to the
manufacturer's denaturing protocol. Briefly, both neuro-
minidase (EC 3.2.1.18) and O-glycosidase (EC 3.2.1.97)
were first added to the reaction vials; after the incubation
and denaturation step, Af-glycosidase F (EC 3.5.1.52) was
added to the mixture. To determine deglycosylation effi-
ciently, samples (before and after deglycosylation) were

M. TORRADO AND A. T. MIKHAILOV

subjected to SDS-PAGE followed by Coomassie staining
(to detect the shift in band mobility) or blotting. Blots were
treated with an Immun-Blot kit (Bio-Rad) for glycoprotein
detection to additionally check the efficiency of the degly-
cosylation reaction. Bovine fetuin (Bio-Rad) and rabbit
liver carboxylesterase (Sigma) were used as positive con-
trols.

Ultrafiltration procedures

Lim-1 -containing fractions isolated from mussel pedal
ganglia and forebrains of 16-day-old chick embryos were
subjected to subsequent ultratiltration using Microcon mi-
cro-units with 100-kDa and 50-kDa cut-off YM membranes
( Amicon) according to the manufacturer's recommendation.
Bovine serum albumin (Sigma) and chicken ovalbumin
(Sigma) solutions (1 mg/ml) were used as reference pro-
teins. To prevent aggregation, urea (Merck) and SDS (Bio-
Rad) were added to starting protein solutions at final con-
centrations of 6 M and 1%. respectively. Each sample was
first applied on the Microcon- 100 unit and centrifuged at
2500 X g for 30 min at 10°C. The resulting filtrate was then
introduced into a sample reservoir of the Microcon-50 unit
and centrifuged at 12.000 X g for 20 min at 10°C. The
volume of each retained and filtered fraction so obtained
was adjusted to that of the starting sample, and the solutions
were subjected to SDS-PAGE followed by Western blot
with anti-XLim-1 antibodies.

Immunocytochemistry

Mussel pedal ganglia were fixed in 100 mM 3-(/V-mor-
pholino) propane sulfonic acid (MOPS; Sigma). 2 mM
MgSO4 (Panreac). 2 mM EGTA (Merck), 3.8% formalde-
hyde (Panreac) for 1 h at room temperature. Standard his-
tological techniques were used for sample dehydration, em-
bedding in paraffin, sectioning at 6 jam, deparaffmization,
and rehydration (Mikhailov and Simirsky. 1991). The slides
were precoated with 3-aminopropyltriethoxysilane (Sigma).
Prior to immunostaining. sections were blocked with 20%
normal horse serum in 50 mM Tris-HCl, pH 8.0. Sections
were then incubated with different dilutions of the anti-
XLim-1 immunoglobulin fraction (3 h at room temperature
or overnight at 4°C) and secondary antibodies conjugated to
alkaline phosphatase (Boehringer-Mannheim). Antibody di-
lutions were prepared in blocking solution. All incubations
were followed by six washes (10 min in each) in 50 mM
Tris-HCl. pH 8.0. Staining was developed using 5-bromo-
4-chloro-2-indolyl-phosphate (Sigma) and 4-nitro blue tet-
razolium chloride as substrates (Sigma) as described in
Karavanov et al. (1996). The sections were mounted in
Permount (Fisher) and examined under the Nikon Micro-
phot microscope. Control experiments were included (1)
omitting anti-XLim-1 antibodies, (2) replacing the latter by
normal rabbit immunoglobulins (Sigma), and (3) using anti-
XLim-1 antibodies preadsorbed by fixed pedal ganglia or by

foot tissue of M. galloprovincialis. Fixation was done in
3.8% formaldehyde as above, followed by a methanol wash.
Using micro-forceps, fixed tissues were ground, rehydrated
in 50 mM Tris-HCl, pH 8.0, blocked in blocking solution for
2 h. pelleted by a low-speed centrifugation, resuspended in
anti-XLim-1 antibody solution (at 1/50 or 1/200 dilution),
and incubated overnight at 4°C. For some experiments.
anti-XLim- 1 antibodies were depleted prior to staining by
incubation at 1/50 dilution with hyperfixed Xenopus laevis
embryos (this was performed by Dr. A. A. Karavanov and
Dr. A. T. Mikhailov in the laboratory of Prof. I. Dawid).

Histological analysis

A portion of male ripe and spent gonads of M. gallopro-
vincialis was fixed in Bouin's solution, embedded in paraf-
fin, cut into 6-ju.m sections, and stained with hematoxylin-
eosin; the gonadosomatic index was estimated from the
sections and expressed as the percentage of the gonad
occupied by follicle structures (Mikhailov et al., 1996;
Torrado and Mikhailov. 1998).

Results and Discussion

In this study we used rabbit polyclonal antibodies against
a C-terminal region (as an immunogen) of the Xenopus
laevis Lim-1 protein (i.e., anti-XLim-1 antibodies). It has
been shown that these antibodies detect the Lim- 1 protein in
X. laevis and also cross-react with Lim- 1 polypeptides of the
mouse (Karavanov et al.. 1996; Shimono and Behringer.
1999) and rat (Karavanov et al.. 1998). These studies have
also demonstrated the high specificity of the antibodies to
Lim-1 proteins and the absence of any discrepancy between
the expression patterns of protein and mRNA. Note that the
transcriptional activation domain of Xlim-1 resides in its
carboxyl terminus (Breen et al., 1997).

Although it is generally accepted that the C-terminal
peptide is a good choice for the production of antibodies
specific to a protein of interest (Hancock and Evan, 1992),
we decided to test, additionally, the degree of "specificity"
of the Xlim-1 C-terminal region (used as an immunogen) for
Lim-1 proteins. Using the BLAST program (Altschul el al..
1997), we performed alignments of the C-terminal sequence
of Xlim-1 with all the protein sequences listed in the
SW1SSPROT database (Bairoch and Apweiler, 2000). The
most similar (similarity 80%-90%; Fig. 1) sequences,
which were aligned first, are those of the fish, chick, mouse,
and human Lim-1. Frog and fish Lim 5 factors are charac-
terized by significantly lower primary structural similarity
(47% and 46%. respectively) to the Xlim-1 C-terminus
sequence. It is significant that the regions of homology
reside only in the C-terminus of the sequences mentioned
above. Other sequences returned by the BLAST program
displayed values of similarity with the C-terminus of Xlim-1
(used as a query) that are not distinguishable from those

LIM domain

MUSSEL LIM-1 RELATED PROTEINS

Xenopus laevis Lim-1

LIM domain HOMEOBOX

265

C-terminal region

Immunogen

I •

403

G T P L GB1B3H PQPGHHP S
G T P L GgMD HPIPGHHPS
GT P LG AIW3H PQPGHHPQ

LIM1-XENLA
LIM1 -MOUSE
LIM1 -HUMAN
LIM1-BRARE
1M1-CHICK G E

AQR F T D I
AQR FJT D I

DIMS HflP G I

pITM^H pHc D S P S P E P

;HSMSAEVFG
HSMSAEVFG
H S M sEBlv F G

SPPFSSLSVNGG

YGNHLSHPP - EMNEJTAVW

YGNHLSHPP~

VGNHLSHPP

Y0N H L S H P P

YGNHLSHPP

H QJ3 L L S N U A FJ3o TGLGI I GQGGQ - G V AflT M R V l~GH!KTp S S D L sT

ILIM3-XENLA G I

Figure 1. Multiple alignment of the deduced amino C-terminal sequence of Xlini-l with those identified
from BLAST search comparisons. (A) Schematic structure of the frog Lim-1 deduced from the previously
published Xliin-l sequence (Taira eral.. 1992). The C-terminal region, which was used to generate anti-XLim-1
antibodies (Karavanov el ai, 1996), is shown in black. (B) The complete sequence of the Xennpiix (XENLA)
C-terminus is shown aligned with those of mouse, human, /.ebrafish (BRARE), and chick Lim-ls as well as with
Xenopus Lim-5 and Lim-3 and zebrafish Lim-5. All the protein sequences were obtained from the SWISSPROT
database (Bairoch and Apweiler, 2000). Black — identical amino acid residues. Dash — gaps. Comparison shows
that both the size and the sequence of the XLim-1 C-terminus are highly similar (80%-90% of similarity; extent
internal homology above seven amino acid residues) to that of Lim-1 proteins from other species. At the same
time, the XLim-1 C-terminal sequence reveals no more than 50% of similarity (extent internal homology below
seven amino acid residues) with that of Lim-5 proteins. No significant similarity was observed in the case of the
.\V»i'/'ii.s Lim-3.

expected by chance (Fig. 1. see Xenopus Lim-3 as an
example).

It is generally accepted that short peptides (below about
seven amino acid residues) are of insufficient size to func-
tion as immunogenie and antigenic epitopes (Hancock and
Evan. 1992). Using the CLUSTAL W program (Thompson
i't ul.. 1994), we performed a multiple sequence alignment
of the XLim-1 C-terminal region with that of the Lim-1 and
Lim-5 proteins identified from BLAST searches (see
above). As shown (Fig. 1), the XLim-1 C-terminus shares a
high sequence homology with a number of Lim-1 proteins
but not with Lim-5 factors. Given the above criteria, it is
probable that the XLim-1 C-terminus (used as immunogen)
could generate successful antibodies characterized by a high
cross-reactivity with Lim-1 proteins in other species. At the
same time, it may be predicted that antibodies against the
XLim-1 C-terminus possess a much lower cross-reactivity
with Lim-5 proteins. Note that anti-XLim-1 antibodies used
in this study cross-react with Lim-1 factors from various
species but do not cross-react with the closely similar
XLim-5 protein on tissue sections (Karavanov et til., 1996).

Collectively, the data indicated that the Xlim C-terminal

sequence (used as an immunogen to generate anti-XLim-1
antibodies) is highly conserved among most of other known
Urn- 1 genes and seems to be diagnostic for their protein
products. This would in turn account for the use of the
corresponding antibodies in selective (discriminative) im-
munochemical screening of Lim-1 -related proteins in dif-
ferent species. This suggestion is supported by the results of
application of anti-XLim-1 antibodies for immunocyto-
chemical Lim-1 protein detection in frog, mouse, and
rat tissues (Karavanov ct ul., 1996. 1998; Shimono and
Behringer. 1999).

Anti-Xlim-1 initihoilies cross-react witli mussel, sea
urchin, cind chick tissue antigens

SDS-PAGE followed by immunoblot analysis of a pro-
tein extracted from isolated pedal ganglia of Mytilus gullo-
pnn-inciiilis. mature oocytes of Strongylocentrotus /yiir/ni-
ratus. and brain tissues of chick embryos revealed a single
band with an apparent molecular weight (MW) of approx-
imately 65. 70, and 65 kDa, respectively (Fig. 2). Note that
the open reading frame of the chicken (Tsuchida et ul..

M. TORRADO AND A. T MIKHAILOV

v 1 2[]3 45 6 7 [8 L9

94-
67-

43-

30-

-170
-116

-76
-53

Figure 2. Cross-reactivity of anti-XLim-1 antibodies with mussel, sea
urchin, and chick tissue antigens. Equal amounts (about 100 fig/pocket) of
total protein extracted from different tissues were resolved in a 10%
SDS-PAGE. blotted on membranes, and probed with anti-XLim-1 antibod-
ies at 1/500 dilution. Membrane strips containing electrophoretically sep-
arated molecular weight markers were stained with Ponceau S. Mytiliis
galloprovincialis organs and tissues: Lane 1 — male pedal ganglia; Lane
2 — female pedal ganglia; Lane 3 — male muscle adductor posterior; Lane
4 — male gills; Lane 5 — male foot; Lane 6 — male hepatopancreas; Lane
7 — male labial palps. Lane 8 — Strongylocentrotus purpuratus eggs. Lane
9 — forebrain tissues of 16-day-old chick embryos. LMW and HMW — low
and high molecular weight calibration kit proteins (30-170 kDa), respec-
tively. 65 and 70 — molecular weights of antibody-labeled proteins. kDa.

1994) and sea urchin (Kawasaki ct id.. 1999) lim-1 gene
encodes a protein with a predicted MW about of 45 kDa.
The high MW value of the Lim-1 antigens is apparently not
due to aggregation with other molecules, because it did not
change when the antigens were extracted and electro-
phoretically analyzed in the presence of 6 M urea. In addi-
tion, observed low migration of mussel and chick Lim-1
antigens in SDS-PAGE is not due to their interactions with
non-polymerized products of polyacrylamide gel (data not
shown).

The discrepancy between the theoretical (45 kDa) and
apparent (65 kDa) MWs of the Lim-1 antigens could be due
to post-translation modifications in the protein molecules. In
X. laevis, three Lim-1 bands were detected (by SDS-PAGE
followed by Western blot with anti-XLim-1 antibodies) in
embryos injected with the full-length synthetic Xlini-1
mRNA. The fastest band of the "triplet" co-migrated with
the protein product obtained from the same mRNA in a
cell-free translation system, whereas other fractions were
characterized by a lower electrophoretic migration. The
latter suggested that a portion of the protein could be subject
to post-translational modifications in the embryo (Kara-
vanov et «/., 1996).

Using the ScanProsite tool (Hofmann et til.. 1999), we
found that the Xlim-1 sequence contains three potential sites
for glycosylation. one of which resides in the C-terminus of
the protein. By analogy with the XLim-l protein, we pro-
posed that mussel and chicken Lim-1 antigens run more
slowly than predicted in SDS-PAGE, probably due to a
glycosylation of the corresponding proteins. To investigate

this option, we performed two experiments. First. Lim-1 -
containing fractions isolated from mussel pedal ganglia and
chick embryo brain tissues (Fig. 3A, B) were electropho-
resed on SDS-PAGE, blotted onto nylon membrane, and
treated with the Immun-Blot kit for glycoprotein detection.
Although a portion of each fraction displayed positive stain-
ing, the zones corresponding to Lim-1 antigens were abso-
lutely negative (see Fig. 3C, D). Next, the same fractions
were treated with the Bio-Rad deglycosylation kit, which
enzymatically cleaves all AMinked and most O-linked oli-
gosaccharides from glycoproteins. Treated and untreated
Lim-1 -containing fractions were subjected to SDS-PAGE
followed by Western blot. All comparisons failed to identify
any change in electrophoretic mobility of treated Lim-1
antigens (Fig. 3E, F). Thus, the difference between the
predicted (45 kDa) and the apparent (65 kDa) MW of the
Lim-1 antigens studied is apparently not due to the post-
translational glycosylation of protein products.

To begin characterizing effective size values, Lim-1 -
containing fractions of mussel pedal ganglia and chick
embryo brains were subjected to a subsequent ultratiltration
using 100- and 50-kDa cut-off membranes (Fig. 3G). Since
mussel proteins tend to aggregate during ultrafiltration
(Mikhailov ct al., 1997), the SDS and urea were added to
starting Lim-1 and reference protein (i.e., bovine albumin
and chicken ovalbumin) solutions. Using SDS-PAGE fol-
lowed by Western blot, we found that about 50% of the
Lim-1 immunoreactivity, characteristic of Lim-1 -containing
fractions, is retained by the 100-kDa cut-off membrane,
whereas no more than 40% of the immunoreactivity is
detected in the filtrate. The latter is completely retained by
the 50-kDa cut-off membrane. Such retention and recovery
patterns are more similar to those of bovine serum albumin
(MW 67 kDa) than to those of chicken ovalbumin (MW 43
kDa). In particular, about 60% of the bovine albumin was
retained by the 100-kDa cut-off membrane, whereas more
than 70% of the chicken ovalbumin passed through the
membrane (data not shown). This raises the possibility that
the effective MW (size) of the Lim-1 antigens studied could
be larger than the theoretical one (45 kDa).

Taken together, the results indicate that the apparent
MWs of the Lim-1 polypeptides, immunochemically de-
tected in M. galloprovincialis pedal ganglia. S. r>itrpi<ratiis
embryos, and chick embryo brain tissues, seem to be 40%
higher than those calculated from deduced amino acid se-
quences derived from sea urchin (Kawasaki et ul., 1999).
chick (Tsuchida et al.. 1994). and frog (Taira et <;/.. 1992)
cDNA lini-l clones. We could not find any reference to
apparent MWs of the Lim-1 proteins detected in frog, sea
urchin, and chick tissues. For the other family of the zinc-
finger transcriplional factors, aberrantly high MW values (in
SDS-PAGE) have been found to be due to the particular
amino acid composition of the C- and N-terminal domains
(Klenova et <//., 1997). Examination of amino acid compo-
sition of the chicken, sea urchin, and frog Lim-1 C-terminal

MUSSEL LIM-I RELATED PROTEINS

LMW 1 2 34

94-

67-

-65

43-

mmw

30-

1 2 3

awMMM** *8M*

-65

~—

-65

Figure 3. Characterization of mussel and chick Lim-l-like antigens.
Lim- 1 -containing fractions isolated from mussel and chick tissues were
subjected to SDS-PAGE followed by Western blot with anti-XLim-1
antibodies. Coomassie (A) and antibody (B) staining: Lane 1 and Lane
2 — total extract and Lim- 1 -containing fraction of 16-day-old chick embryo
forebrains. respectively; Lane 3 and Lane 4 — total extract and Lim-1-
containing fraction of Mytilus galloprovincialis pedal ganglia, respec-
tively; LMW — low molecular calibration kit proteins (30-94 kDa). Note
that fractions display the Lim-1 immunoreactivity similar to that of tissue
extracts. In further experiments, chick (C) and mussel (D) Lim-1 -contain-
ing fractions (Lanes 1. 2, and 3. respectively) were resolved in !()'<
SDS-PAGE and blotted on membranes. Each membrane was cut into two
parts (see "Materials and Methods"): one part was treated with an Immun-
Blot kit for glycoprotein detection (Lane 1 and a half of the Lane 2); the
other (a half of the Lane 2 and Lane 3) was treated with anti-XLim-1
antibodies. Note that immunolabeled Lim-1 antigen bands do not reveal
any glycoprotein-specific reaction. Next, chick and mussel fractions were
treated with a deglycosylation kit and subjected to SDS-PAGE followed by
Coomassie staining of the gel (E). or blotted on membranes followed by
antibody staining of the membrane (F): Lane 1 and Lane 2 — untreated and
treated chick Lim-containing fraction, respectively; Lane 3 and Lane
4 — untreated and treated mussel Lim-1-containing fraction, respectively.
Note that the electrophoretic position of antibody-labeled bands in treated
Lim-containing fractions corresponds to the 65-kDa value, as is the case
for untreated fractions (see F; Lane 1 versus Lane 2, and Lane 3 versus
Lane 4). Finally, chick Lim-containing fraction was subjected to subse-
quent ultrariltration. Retained and filtered proteins were subjected to
SDS-PAGE followed by Western blot with anti-XLim-1 antibodies (G).
Immunostaining (signal quantitation, %): Lane 1 — isolated Lim- 1 traction

regions revealed that they are enriched with proline (18%,
20%, and 17%, respectively). Since polypeptides with a
high proline content can give abnormally high MW values
by SDS-PAGE (Hames, 1990), we suggest that the proline-
rich C-terminus of the Lim-1 proteins may be responsible, at
least in part, for their behavior when analyzed by SDS-
PAGE (as could be the case of Lim- 1 antigens studied). It is
unlikely that Lim-l-like tissue antigens detected in this
work are multimeric forms or aggregates that include other
components, because reducing agents were present at all
stages of the separation. Further studies should elucidate the
significance of our findings and explain the discrepancy
between the apparent MW of Lim-1 proteins immuno-
chemically detected in chicken and sea urchin tissues and
that deduced from the coding region of the corresponding
cloned cDNAs.

Mussel pedal ganglia and gonads display the most
prominent Lim- 1 -like immunoreactivity

In adult M. galloprovincialis, the most prominent Lim- 1
immunoreactivity examined by Western blot analysis was
detected in the pedal ganglia and gonads, in both males and
females (Figs. 2, 5, 6). A very weak immunostaining was
observed in gills and muscle tissues but not in hepatopan-
creas, foot, or labial palps (Fig. 2). Pronounced Lim-1
antigen accumulation in mussel nervous tissues and gonads
raises a question about its possible functional importance. In
this respect, it is interesting to note that mice carrying a
disruption of the lim-1 gene (Shawlot and Behringer, 1995)
fail to develop the head and also lack kidneys and gonads.

Immunohistochemical staining was used to further char-
acterize the Lim-1 expression pattern in the mussel nervous
tissue. Figure 4 illustrates the nuclear localization of the
antigen in neurons of pedal ganglia. To avoid the possibility
of nonspecific cross-reactivity, we used anti-XLim-1 anti-
bodies depleted prior to immunostaining by incubation with
hyperfixed X. laevis embryos. This procedure results in the
decrease of background and the enhancement of the signal-
to-noise ratio (Karavanov ct <//.. 1996). The positive im-
muno-signal of mussel nuclei was blocked by adsorption ot
anti-XLim-1 antibodies with fixed pedal ganglia but not
with fixed M. galloprovincialis foot or hyperfixed X. laevis
embryo, providing additional support for tissue-specificity
of immunodetection of the antigen. We reason that obser-
vation of a Lim-l-like positive signal in nuclei of mussel
pedal ganglia is consistent with the generally accepted

(100%). Lane 2— Lim-1 fraction retained by the 100-kDa cut-off mem-
brane (-50%); Lane 3— Lim-1 fraction partially passed through the 100-
kDa cut-off membrane ( -40%); Lane 4 — the latter retained by the 50-kDa
cut-off membrane (-30%). 65 — molecular weight of Lim-l-like antigens.
kDa. Positions of bovine serum albumin (67 kDa) and chicken egg ovalbu-
min (43 kDa) are shown on (A).

M. TORRADO AND A. T. MIKHAILOV

Figure 4. Tissue section visualization of Lim-1 immunoreactivity signal in Mytilii.\ galloprovincialis pedal
ganglia. (A) Localization of the pedal ganglion in the mussel. The anterior end of the animal is to the right;
pg — pedal ganglion; g — gills; hp — hepalopancreas. f — foot; vm — visceral mass; ahrm — anterior byssal retrac-
tor muscle. Ganglia were dissected, fixed, paraffin-embedded, and processed for immunohistochemistry using
primary anti-XLim-l antibodies and secondary antibodies conjugated to alkaline phosphatase. (B) Section
treated with anti-XLim-l antibodies depleted by hyperfixed Xenopus laevis embryos. Note the positive staining
of nuclei (long arrows). (C) Section treated with anti-XLim-l antibodies preadsorbed by fixed foot tissues of M.
Vi///"/"'"1'""'"''"- Short arrows point to a nuclear region (arranged on entire circumference of the ganglion) that
is positive for anti-XLim-l antibody staining. (D) Higher magnification of the section in (B). showing intensive
immunostammg in isolated nuclei. (E) Section treated with anti-XLim-l antibodies preadsorbed by fixed pedal
ganglia of A/, v"""/"""1'""''"'"-' no immunoreactivity is observed in the nuclei (scale bar: A — 5 mm; B — 50 jum;
C— 100 Mm; D and E— 20 /urn).

putative function of Lim-1 HD proteins as transcription
factors.

In the M. galloprovincialis ripe male gonad, Lim-1 pos-

itive signals (i.e., a major 60-kDa and a minor 65-kDa hand)
were detected in somatic gonad tissues, hut not in sperm
cells (Fig. 5A. C). In spent male gonads (i.e.. gonads that do

MUSSEL LIM-I RELATED PROTEINS

not contain sperm cells and consist of gonad tubules and
mantle connective tissue) a very weak 60-kDa immunore-
activity was found. At the histological level, not only sperm
but also mature Sertoli cells were undetectable in seminif-
erous tubules of the spent gonad (Fig. 5B). The results
suggest that in mussel male gonad, the 60-kDa antigen is
mainly associated with Sertoli cells. This finding is consis-
tent with data on Lim- 1 protein cell localization in the fetus
testis of the rat (Karavanov el ai, 1996). The minor 65-kDa
band seems to be also characteristic for gonad somatic
tissue, although its precise cell association remains to be
elucidated.

Next we questioned whether the Lim-1 distribution in the
M. galloprovincialis female gonad was similar to that in the
male gonad. In the female ripe gonad, antibody staining
revealed two Lim-1 -like antigens with MWs of approxi-
mately 65 and 40 kDa (Fig. 6). In the oocyte-free gonad
portion, containing mainly collecting tubules and mantle
mesenchyme cells, only the 65-kDa fraction was detected.
In mature spawned oocytes, both the 65- and the 40-kDa
Lim-1 antigens were found. The 40-kDa band does not

12345

v^nM-

,->;-* ••. F '< F &4

65-
60-

-65
-60

Figure 5. Analysis of Lim- 1 antigen distribution in male gonads of
A/vr;7ii\ galloprovincialis. Histological sections of ripe male gonad before
(Al and after (B) complete spawning (spent gonad). Note that the gonad
samples are characterized by the same patterns of follicle (F) morphogen-
esis and gonadosomatic index values (in both samples, about of 90% of
gonad volume was occupied by follicles) (scale bar — 100 jxm). (C) Ex-
tracts prepared from complementary gonad (Lane I and Lane 3; arrows) of
the same animals, as well as from somatic tissues (Lane 2) and sperm cells
(Lane 4) of the other ripe gonad before spawning, were subjected to
SDS-PAGE followed by Western blot with anti-XLim-l antibodies. Lane
1 — gonad biopsy containing somatic tissue, gonad ducts, fluids, and
sperm; Lane 2 — gonad tube-free sample containing the mantle connective
tissues only; Lane 3 — spent gonad; Lane 4 — mature sperm cells; Lane
5 — optic lobe of 16-day-old chick embryos (reference). 60 and 65 —
apparent molecular weight values of Lim-1 antigens. kDa.

65-

40-

-65

-40

Figure 6. Analysis of Lim-1 antigen distribution in female gonads of
Mvtilus galloprovincialis using SDS-PAGE followed by Western blot with
anti-XLim-l antibodies. Lane 1 — gonad biopsy containing somatic tissues,
gonad ducts, fluids, and oocytes; Lane 2 — gonad collecting ducts; Lane
3 — immature oocytes obtained by biopsy of gonad follicles; Lane
4 — spawned mature eggs; Lane 5 — cerebellum of 16-day-old chick em-
bryos (reference). 40 and 65 — apparent molecular weight values of Lim-1
antigens. kDa.

appear to be an artifact of degradation caused by sample
processing. Detection of the 40-kDa Lim-1 antigenic
polypeptide in the mussel female gonad is perhaps not
surprising, because the X. luevis ovary contains the 2.7-kb
maternal Xlim-1 mRNA that is smaller than the larger
zygotic 3.4-kb transcript found in the adult brain (Taira et
ul.. 1992). It should be noted that in immature oocytes
(obtained by biopsy of female gonad follicles), neither 65-
kDa nor 40-kDa antigens were found (see Fig. 6. Lane 3).
It is clear from these results that the 65-kDa Lim-1 form
in the female is associated with somatic tissues of the gonad
just like the Lim-1 variants detected in the male gonad. At
the same time, in the female gonad the 65-kDa antigen is
also characteristic of mature eggs. The 40-kDa antigen,
despite our uncertainty regarding its precise nature, is spe-
cific to the female germ line. It is likely that neither of the
two antigens are expressed at early phases of oocyte differ-
entiation, but are expressed and accumulated in eggs at
terminal stages of their maturation. Therefore, the M. gal-
loprovinciulis female gonad pattern obtained by Western
immunoblot analysis for Lim-1 antigens may be interpreted
as a compound profile of the 65-kDa variant, which origi-
nates from both somatic tissues and eggs, and of the 40-kDa
form, which seems to be specific to mature oocytes only.

Dynamics of Lint- 1 -like immunoreactivity during
earl\ development

Observations on female gonads have led us to examine
the patterns and timing of maternal expression of the 65-
kDa and 40-kDa Lim-1 variants during early development
of M. galloprovincinlis. Both antigen signals, already seen
in unfertilized and fertilized eggs, persist in embryos during
cleavage. At the beginning of the blastula stage, the inten-

M. TORRADO AND A. T. MIKIIAII.OV

sity of immunostaining of both the 65-kDa and the 40-kDa
antigens decreases. In stereoblastulae, instead of these two
Lim-1 antigenic variants, only 45-kDa immunoreactivity
was observed. The latter was first detected in 8-cell em-
bryos, and its intensity reached a maximum in blastulae
(Fig. 7A).

Data from a variety of sources are consistent with the fact
that zygotic transcription of //;;;-/ genes begins before gas-
trulation at or very shortly after the midblastula transition
(Taira et ai, 1992; Rebbert and Dawid, 1997; Curtiss and
Heiling, 1998; Kawasaki ft ai, 1999). It may be speculated
that in mussels, the 65- and 40-kDa signals are due to
Lim-1 -related maternal molecules stored in the full-grown
oocytes, whereas the 45-kDa protein reflects zygotic activ-
ity of the gene. Such an interpretation may explain why
multiple Lim-1 antigen variants have been detected in em-
bryos of M. galloprovincialis at early blastula stages, but
there is no definitive proof. It remains to be established how
the compound profile of Lim-1 protein variants relates to
maternal and zygotic gene expressions as well as to possible
post-translation modifications of the primary gene product
(Karavanov et a/., 1996) or to the so-called premature
termination of translation processes involving both untrans-
lated and coding regions of the zinc-tinger transcriptional
factors (Klenova el ai. 1997).

In sea urchin embryos, expression of the liin- /-related
gene (Hpliiu-1) has been studied at the transcriptional level
(Kawasaki et ai. 1999), so we decided to examine the
Lim-1 antigen dynamics in the course of sea urchin early
embryogenesis. Levels of Lim-1 immunoreactivity during
S. purpuratus development are shown in Figure 7B. A
relatively high-abundance signal of the Lim-1 antigen, al-

65-

40-

-65

-45
-40

70-

Figure 7. Patterns and timing of Lim-l antigen expression in the
course of earK development of Mylilnx galloprovincialis and StrrwKylu-
centrotus purpiirntii.-. The extracts from eggs and embryos were subjected
to SDS-PAGE followed by Western blot with anti-XLim-l antibodies. (A)
M. t'tilliipriivim'itili', l.ane I -unfcrtili/cd eggs; Lane 2 — fertilized eggs;
Lane 3 — 4- and 8-cell embryos; Lane 4 — 16- and 32-cell embryos; Lane
5 — early blastulae; Lane fi — stereohlustulae. (B) S. />/II/>M/I//».V Lane
I — unfertilized eggs; Lane 2 — fertili/.ed eggs; Lane 3 — 8- and 16-cell
embryos; Lane 4 — unhatched hlastulae; Lane 5 — hatched blastulae; Lane
6 — prism larvae. 40, 45, 65, and 70— apparent molecular weight values of
Lim-1 antigens, kDa.

ready seen in eggs, persists in embryos to the blastula stage,
decreases dramatically in unhatched blastulae, and increases
again in late (post-hatched) blastulae. In more advanced
embryos (i.e., at prism and pluteus stages), trace amounts of
the Lim- 1 antigen were detected. Although the developmen-
tal kinetics of the Lim-1 protein in S. purpuratus is quite
similar to that of the Hplim-1 mRNA in H. pulcherrimus
(Kawasaki et ai, 1999), the most interesting finding re-
vealed by Western blot is that the Lim- 1 antigen is present
at relatively high levels very early during development.
Note that H. pulcherrimus fertilized eggs and cleavage
embryos contain a trace amount of the Hplim-1 mRNA that
becomes abundant only at the blastula stage just after hatch-
ing (Kawasaki et ai. 1999).

Thus, Lim-1 -like polypeptides, which share common
epitopes with the C-terminus of the frog XLim- 1 protein,
have been detected in both M. galloprovincialis and 5.
purpuratus. In these species, characterized by very different
modes of early embryogenesis, the similar developmental
kinetics of the Lim-1 antigens has been demonstrated.
Whether this likeness leads to similar developmental con-
sequences remains to be elucidated. In sea urchin embryos,
ectopic expression of the Hplim-1 inhibits endoderm and
mesoderm differentiation, directing all embryonic cells to
form oral ectoderm (Kawasaki et ai, 1999). It is widely
accepted that maternally expressed gene products, stored in
the egg, establish initial differences within the early embryo
that, in turn, could contribute to further regionalization of
the embryo body (Raff, 1996). The work described here
particularly highlights the fact that in the marine inverte-
brates studied the Lim-1 -like proteins maternally accumu-
lated in the egg could persist after fertilization and be
present in the early embryo long before zygotic expression
of the genes is activated.

As mentioned above, the present study represents the first
step in the identification and characterization of Lim-1 -like
proteins in marine bivalves. If it were accepted that the
antibodies used recognize epitopes of the XLim-1 C-termi-
nal sequence, then their cross-reactivity would appear to be
specific for Lim- 1 -related proteins in many species (see Fig.
I ). The corresponding immunochemical data obtained on
rats (Karavanov et ai, 1996, 1998) and mice (Shimono and
Behringer, 1999) confirm this assumption. Moreover, there
is similarity between Lim-1 antigenic patterns observed in
bivalves and those detected with the aid of the same anti-
bodies in other species. This involves ( 1 ) the immunodetec-
tion of the Lim-1 protein in both ganglia and somatic
gonads (bivalves — this work; rats — Karavanov et ai.
1996), and (2) the nuclear localization of Lim-1 immuno-
reactivity in tissue sections (bivalves — this work; frog
and rats — Karavanov et ai, 1996; mice — Shimono and
Behringer. 1999). In addition, the developmental dynamics
of the Lim-1 antigen (this work) and Hplim-1 mRNA (Ka-
wasaki et ul., 1999) in sea urchin embryos appears to be
very similar.

MUSSEL LIM-1 RELATED PROTEINS

On the basis of the HD sequence similarity, vertebrate
Lim-1 proteins, as well as Lim-5 and Lim-6 factors, have
been included in the so-called LIN- 11 class of LIM-HD
proteins (Hubert and Westphal. 2000). Two lim- /-related
genes have been recently identified in sea urchins (Ka-
wasaki et nl., 1999) and fruit flies (Lilly et til.. 1999), and
we suggest that they may be added to the same LIN- 11
group. To the best of our knowledge, this study is the first
report that describes Lim-1 -like protein patterns in bivalve
mollusc, sea urchin, and chick embryo tissues. Clearly,
much remains to be learned about the corresponding factors
involved, especially in bivalves. Nevertheless, it seems
likely that the results obtained provide precedents for fur-
ther identification of //w-/-related genes and characteriza-
tion of their protein products in bivalve molluscs.

Acknowledgments

The authors thank Dr. A. A. Karavanov and Prof.. Dr.
I. B. Dawid for the gift of anti-XLim- 1 antibodies as well as
for comments and contributions at a time during which
many aspects of this study were discussed and developed.
We also extend our gratitude to two extremely helpful
anonymous referees. The work was supported by the XUGA
grant 16101 A 98 from the Galicia Government to ATM and
by a postdoctoral fellowship from the Institute of Health
Sciences (University of La Coruna, Spain) to MT.

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Morphogenesis During Asexual Reproduction in
Pygospio elegans Claparede (Annelida, Polychaeta)

GLENYS D. GIBSON* AND JENNIFER M. L. HARVEY
Department of Biology. Acadia University; Wolfi'ille, Nova Scotia. Canada BOP 1X0

Abstract. The spionid Pygospio elegans reproduces both
asexually and sexually. Using scanning electron and bright
field microscopy, we examined morphogenesis following
asexual reproduction to determine how "lost" body regions
were regenerated after a worm spontaneously divided.
Asexual reproduction occurred through transverse fission
and divided the parent worm into 2 to 6 fragments (archi-
tomy). All fragments retained their original anterior-poste-
rior polarity. Regeneration in all fragments followed a spe-
cific series of events: wound healing (day 1 ); extension of
the blastema to generate lost body regions — specifically, the
head and thorax for posterior fragments and the tail and
pygidium for anterior fragments (days 2-3); segmentation
(days 3-6); and differentiation of segment- or region-spe-
cific structures (days 4-8). This pattern occurred regardless
of where the original division took place. Subsequent
growth occurred through addition of terminal setigers ante-
rior to the pygidium followed by differentiation of tail
setigers into abdominal setigers, leaving the tail region
about 6 to 10 setigers in size. Division rates were compared
in worms from three populations in Nova Scotia, Canada.
Worms from two populations (Conrad's Beach, Starr's
Point) divided more frequently (about 1.2 and 1.3 weeks
between divisions, respectively) than worms from Bon Por-
tage Island (3.5 weeks between divisions). Fragments con-
taining the original head (original mouth intact, generally
much larger fragment) had a higher survivorship than frag-
ments containing the original tail.

Introduction

Asexual reproduction is the process of forming two or
more offspring from one parent body without involving

Received 23 July 1999; accepted 4 May 2000.

* To whom correspondence should be addressed. E-mail: glenys.
gibson@acadiau.ca

gametes, or cells with a meiotically reduced chromosome
number (Balinsky, 1975; Solomon et al. 1993). Although
polychaetes reproduce sexually, asexual reproduction,
through fission or budding, also occurs in many families
including spionids, cirratulids, syllids, and sabellids (Barnes,
1980). In the spionid Pygospio elegans, asexual reproduc-
tion occurs through transverse fission of the parent body
into fragments, each of which will regenerate "lost" body
regions (Rasmussen, 1953). Asexual reproduction has been
widely reported in P. elegans. with most authors reporting
its occurrence or testing environmental factors that may
influence rates of division (Anger, 1984; Wilson. 1985).
Despite the prevalence of asexual reproduction in this spe-
cies, morphogenesis during post-fission regeneration has
not been described.

Pygospio elegans is a tubiculous polychaete that is com-
mon on mud and sand flats and has a cosmopolitan, tem-
perate distribution (Anger, 1984; Wilson, 1985). Adults
grow to be 12 mm long, and feed on detritus (Wilson. 1985)
and phytoplankton (Anger et al.. 1986). Rasmussen (1953)
first described asexual reproduction in this species. He re-
ported that both females and males could divide anywhere
in the body and generally formed three to four fragments.
Each fragment stayed in the original tube until regeneration
was complete, about 8 d after division (20°C). Subse-
quently, several authors reported asexual reproduction in P.
elegans from populations from the eastern seaboard of the
United States (Hobson and Green, 1968). Washington State
(Wilson, 1985), and the Baltic Sea (Anger, 1984; Gud-
mundsson, 1985). P. elegans also reproduces sexually, and
it exhibits considerable flexibility in reproduction, as both
planktotrophic and adelphophagic (a form of lecithotrophy)
larval development have been reported in worms from dif-
ferent populations (e.g., Thorson, 1946; Hannerz, 1956;
Hobson and Green, 1968; Anger, 1984; Anger et al.. 1986;
Schlotzer-Schrehardt. 1991; Morgan et al.. 1999).

G. D. GIBSON AND J. M. L. HARVEY

Our objective is to describe morphogenesis during post-
fission regeneration in P. elegans. We use bright field and
scanning electron microscopy (SEM) to describe morpho-
genesis after spontaneous divisions to determine ( 1 ) if an-
terior and posterior body regions show similar patterns of
regeneration; and (2) how subsequent growth occurs. We
use the term regeneration to refer to the replacement of lost
body regions (e.g., the head, thorax, tail) and growth as the
addition of setigers to increase size, once the major body
regions have formed. Also, we examine the rates of fission
and fragment mortality in laboratory-maintained worms
originating from three populations. No sexual reproduction
was observed during the present study.

Materials and Methods

Adult specimens of Pygospio elegans were collected be-
tween May and September from three sites: Bon Portage
Island, Starr's Point, and Conrad's Beach, Nova Scotia. At
each site, sediments containing worms were sieved
(500-;u,m mesh), and tubes were brought into the laboratory.
P. elegans was identified following Bromley and Bleakney
(1984). Worms were placed in either 250-ml Pyrex crystal-
lizing dishes or 150-ml custard dishes, with seawater and
defaunated sand. Dishes containing stock cultures were
submerged in larger trays of seawater and aerated. Cultures
were maintained at 20°C on a photoperiod of 16 h light.
Worms were fed a mixture of dehydrated, ground Entero-
morplui and Tetramin fish food suspended in seawater twice
weekly. Seawater was changed once a week.

Stock cultures were sieved daily, and worms were iso-
lated if they could be identified as having divided on that
day (presence of a clean, smooth blastema) or showed signs
that fission was about to occur (constriction of the body
wall). Isolated worms were cultured separately to prevent
movement of worms among culture dishes. Regeneration
was observed with bright field and scanning electron mi-
croscopy. Fragments, anesthetised in 1% MgCl:, were ex-
amined and photographed daily from fission to the comple-
tion of regeneration (8 d post-fission) using bright field
techniques (n =- 25 worms). Fragments at each stage of
regeneration (2 to 3 fragments per stage for both anterior
and posterior fragments) were prepared for SEM by fixation
in 2.5% glutaraldehyde followed by post-fixation in !%•
osmium tetraoxide. both in 0.1 M cacodylate buffer and
seawater (Gibson ft ai. 1999). After fixation, regenerates
were dehydrated in an ascending series of ethanol. critical
point dried with a Bio-Rad E3000 critical point drier, coated
with gold-palladium with a Hummer II sputter coaler, and
observed with a JEOL JSM-25S or JEOL T330A scanning
electron microscope. Growth was followed in additional
worms that had completed the regeneration process (;; =
1 2 worms). After the head and thorax or tail and pygidium

had been regenerated, growth was examined by counting the
number of setigers in each body region for a 17-d period.
Intact worms that showed no signs of a recent asexual
event were cultured in isolation to determine rates of regen-
eration. Worms were observed from Bon Portage Island
(/; =: 15). Starr's Point (;; = 15), and Conrad's Beach
(;; = 10). Dishes were sieved weekly over a 6-week period.
Original worm size was determined as the number of seti-
gers at the beginning of the experimental period. Each
week, the number of fragments per dish was noted, as well
as the size of the fragments (number of setigers) and the
degree of regeneration. Data were compared among the
three study populations using one-way ANOVA in Stat-
works 1.2 (Cricket Software). Where significant differences
were noted, a post-hoc Scheffe comparison was also per-
formed using SPSS 8.0 (SPSS Inc.).

Results

Adult morphology

The overall body plan of Pygospio elegans is divided into
four regions: the head, thorax, abdomen, and tail. The head
is characterized by two ciliated palps, a prostomium with
two or three pairs of eyes and paired nuchal organs (Fig.
la). The thorax contains 10 to 12 abranchiate setigers, each
with a single dorsal ciliary band, capillary notochaetae. and
a lateral tuft of cilia. Neurochaetae are simple capillary on
setigers 1 to 8 and hooded hooks on setigers 9 to 12 (Fig.
Ib). The abdomen is 25 to 35 setigers in length. Each
abdominal segment has paired branchiae and either a single
(first few abdominal setigers) or double ciliary band, with
two closely apposed bands of tufted cilia. Abdominal seti-
gers also have capillary notochaetae, a lateral tuft of cilia,
and neurochaetae that are hooded hooks (Fig. Ic). The tail
contains 6 to 12 abranchiate setigers. Tail setigers have
capillary notochaetae, neurochaetae that are hooded hooks,
and a lateral tuft of cilia. There is a reduced ciliary band on
the first few tail setigers only. The pygidium consists of four
cirri, each with tufts of cilia on the inner surface (Fig. Id).
Male P. flegans have a pair of branchiae on the second
setiger (Fauchald, 1977) and dorsal organs on each setiger
(Schlotzer-Schrehardt, 1991). Only tour males (/; = 200
worms) were observed during the present study. No mor-
phological differences were noted (SEM) between worms
from the three study populations.

Morphogenesis following fission

In all cases, fragments retained their original anterior-
posterior polarity. Posterior fragments regenerated only the
head and thorax, and anterior fragments regenerated only a
new tail and pygidium. Subsequent growth involved elon-
gation of the tail by the addition of terminal setigers. We
based our description on division into two fragments, as that

ASEXUAL REPRODUCTION IN PYGOSP1O

Figure 1. Scanning electron micrographs of adult Pygospio elegans. (A) Head and anterior thorax including
the reduced first setiger. (B) Thorax, showing both anterior setigers with capillary neurochaetae and posterior
setigers with neurochaetae that are hooded hooks. (C) Abdomen, characterized by branchiate setigers and a
double dorsal ciliary band. (D) Tail and pygidium. b = branchus, c = cirrus, d = dorsal ciliary band, h = hooded
hook, n = nuchal organ, cp = capillary chaetae, p = palp, pr = prostomium. t = tuft of cilia. Scale bar =
100 /am.

was the most common form of fission observed in the
present study. The maximum number of fragments observed
per division was six, and regeneration in all fragments
followed the same basic pattern. Table I provides a list of
the structures that were observed during regeneration and
the time at which the regenerated structures were first ob-
served.

On day 1, transverse fission began as a muscular con-
striction in the body wall, usually in the abdominal region
located at a point about two-thirds along the length of the
worm. Constriction of the body wall continued until the gut
separated and the two fragments, each anchored to the
substrate v/a mucous, pulled apart. The anterior fragment
consisted of the head, thorax, and most of the abdomen
(about 25 or more pairs of branchiae), while the posterior
fragment consisted of the tail, pygidium, and usually about
five or fewer branchiate abdominal setigers. The epidermis
healed quickly and formed a smooth surface the same day as
division occurred (Fig. 2a). On day 2, the blastema of both
the anterior and posterior fragments showed a small amount

of new tissue with tiny, scattered tufts of cilia on an other-
wise smooth epidermis (Fig. 2b).

Regeneration on day 3 is characterized by rapid develop-
ment of the blastema and formation of lost body regions
(Table 1). As the anterior blastema increases in size, the
regenerated head and thorax are readily distinguished (Fig.
2c). The head has palp buds, small dorsal depressions indi-
cating formation of the nuchal organs, and a slightly
rounded prostomium. The thorax shows the initial forma-
tion of 3 to 6 setigers, visible with both SEM and bright field
microscopy. The gut, visible with bright field microscopy,
has extended into the thorax near the parental abdomen. The
tail blastema is smaller than the anterior blastema and shows
2-3 slight wrinkles, suggesting early segmentation. Bright
field microscopy also revealed the formation of segments
and as well as the extension of the gut into the tail region.
Cirri buds are also visible (Fig. 2d).

On day 4, regeneration is characterized by further seg-
mentation and early differentiation of region-specific struc-
tures. The anterior blastema has 8 to 12 well-defined seti-

44 G. D. GIBSON AND J. M. L. HARVEY

Table 1
Siimmaiy of morphogenesis during regeneration in Pygospio elegans

Structure Day 1

Day 2 Day 3 Day 4 Day 5

Day 6

Day 7

Day 8

Anterior blastema

Head

Eyes (no. pairs)

1-2

2-3

Mouth

* #*

Nuchal organ

# *# #*

Palps

# ** **

Prostomium

* ** **

Thorax

Setigers (no.)

3-6 8-12 10-12

10-12

Notopodial lobe

Notochaetae

Ciliary tuft

Neuropodial lobe

Neuropodial capillary chaetae

Neuropodial hooded hooks

Posterior blastema

Tail

Setigers (no.)

2-3 3-6 3-6

5-7

Notopodial lobe

Notochaetae

Ciliary tuft

Neuropodial lobe

Neuropodial hooded hooks

Pygidium

Cirri

* #* *#

Ciliary tufts

* *#

+ = body region recognizable, * = structure visible as a bud or rudiment, ** structure well developed but smaller than in parent.

gers in the thorax (Fig. 2e, f), each with two dorsal tufts of
cilia. The mouth and prostomium are visible on the regen-
erating head, and the nuchal organs have small cilia. The gut
has extended from the original abdomen to the head (Fig.
2f). Segments are further developed in the posterior blas-
tema as well, with 3 to 6 well-defined setigers, each with
paired lateral pits in the region of the presumptive noto- and
neurochaetae. Differentiation of the pygidium involves ex-
tension of the cirri and the appearance of small tufts of cilia
on the inner surface (Fig. 2g).

On the fifth day post-fission, the anterior blastema has
regenerated the entire thoracic region and shows early dif-
ferentiation of segment-specific structures. The number of
thoracic setigers (10 to 12) that regenerated in the anterior
blastema is similar in all specimens regardless of where
fission occurred in the parent worm. The head has an
elongate prostomium. The thoracic setigers develop neu-
ropodial and notopodial buds, with a few small capillary
notochaetae and a small tuft of cilia between the neuropo-
dium and the notopoditim (Fig. 3a). The gut extends through
the thorax, and the mouth is complete (Fig. 3b). On the same
day, the 3 to 6 setigers of the posterior blastema also
develop parapodial buds, a few notopodial capillary chaetae
on setigers nearest the abdomen, and small lateral tufts of

cilia. The pygidium has larger cirri with tufts of cilia
(Fig. 3c).

Regeneration on day 6 involves greater differentiation of
segment-specific structures and addition of posterior seti-
gers to restore the parental organization of the tail. The
regenerated head has elongate, ciliated palps, a blunt pros-
tomium (Fig. 3d), and 1 to 2 pairs of subdermal eyes (Fig.
3e). The thorax has dorsal bands of cilia on each setiger and
well-developed notopodial chaetae throughout. Also in the
thorax, the neuropodia exhibit short capillary chaetae on
setigers 1-8 and a single hooded hook per setiger from
setiger 8 posteriorly. The tail blastema has the 5 to 7 setigers
characteristic of this region, with capillary notochaetae and
notopodial hooded hooks that decrease in number from
three on the proximal, earliest-forming setiger. to one on the
later-developing terminal setiger (Fig. 3f). Lateral tufts of
cilia are present on all setigers. The pygidium has cirri that
are mature in size and have well-developed tufts of cilia.

By day 7, the anterior blastema has regenerated a head
and thorax that are identical to those of the parent worm
except in setiger size and number of chaetae (Fig. 3g).
Subsequent development in this region involves an increase
in setiger size but not number. In the tail, setiger size and
chaetae number also increases (Fig. 3h). By day 8, the

ASEXUAL. REPRODUCTION IN PYGOSPIO

Figure 2. Early regeneration in Pygospio elegtinx following spontane-
ous transverse fission. (A) Day 1 post-fission, anterior fragment. (B) Day
2. posterior fragment with blastema. (C) Day 3. anterior blastema, showing
regenerated head, thorax, and evidence of early segmentation. (D) Day 3,
posterior blastema, showing regenerated tail and buds of cirri. (E) Day 4,
regenerated head and thorax with segments. The head has a regenerated
mouth, palp buds, and a rounded prostomium. (F) Day 4, regenerated head
and thorax showing extension of the gut into regenerated tissue. (G) Day
4, regenerated tail and pygidium. A-E. G are scanning electron micro-
graphs. F is a bright field micrograph, bl = blastema, c = cirri bud. n =
nuchal organ, p = palp bud. pr = prostomium. t = tuft of cilia. Arrows
indicate setigers. Scale bar = 100 /j.m for A and E. 50 ^m for B-D and G.

Figure 3. Completion of regeneration in Pygospio elegans. (A) Day 5
post-fission, anterior regenerate. (B) Day 5. anterior regenerate showing
development of the gut. mouth, and setigers. (C ) Day 5. posterior blastema.
(D) Day 6, anterior regenerate. (El Day 6, regenerated head with two pairs
of eyes. (F) Day 6, regenerated tail and pygidium. (G) Day 7, anterior
regenerate. (H) Day 7, posterior regenerate. A. C, D, F-H are scanning
electron micrographs, B and E are bright field micrographs, c = cirrus,
cp = capillary chaetae, d = dorsal ciliary band, e = eyes, g = gut. h =
hooded hooks, m = mouth, ne = neuropodium, no = notopodium. t = tuft
of cilia. Scale bar = 100 /j.m, anterior and posterior fragments for each day
are shown at the same magnification.

regenerated thorax and tail have an increased number of
chaetae, and are similar to the pre-fission organization ex-
cept for setiger size. Also on day 8, the gut extends through
the new tail to the pygidium.

In all fragments, regeneration produces only specific
body regions, regardless of where fission occurred in the
parent. Anterior fragments regenerate only the pygidium
and the 6 to 12 abranchiate setigers of the tail. Posterior
fragments regenerate only the thorax ( 10 to 12 setigers) and
head. Mid-worm fragments concurrently regenerate both
anterior and posterior regions as described above, with the
result that these fragments regenerate the head and thorax
and tail and pygidium but not the abdomen, regardless of the
size of the original fragment (Fig. 4). After regeneration,
worms grow to their pre-fission size by increasing setiger
size and setiger number. During the growth phase, new
setigers will only form immediately anterior to the py-
gidium; new setigers do not form in the thorax or abdomen
once regeneration is complete. Newly formed terminal se-
tigers develop chaetae and parapodial lobes typical of the

Figure 4. Scanning electron micrograph of a specimen of Pygospio
elegcins regenerating from a mid-worm fragment, about 6 days after fission.
The larger, parental setigers originated from the abdominal region and have
branchiae. Both the anterior (head, thorax) and posterior (tail, pygidium)
regions have regenerated. Scale bar = 100 ^im.

G. D. GIBSON AND J. M. L. HARVEY

tail region (Fig. 5). As the tail region increases in setiger
number, anterior tail setigers differentiate into abdominal
setigers by forming dorsal ciliary bands and branchiae buds.
About one-half of a setiger is added each day during the
growth phase (n = 11 worms, mean ± SD 0.52 ± 0.23).
Occasional anomalies were noted in this general pattern.
For example, Figure 6 shows a P. elegtms that regenerated
two thoracic regions and heads, both containing extensions
of the gut. Such anomalies, although rare, reinforced the
general pattern of regeneration described above. For both
heads, the blastema gave rise to a specific number of seti-
gers, and segmentation was followed by differentiation.

Population comparison

Frequency of spontaneous division and mortality were
compared in P. cleganx originating from the three popula-
tions. Specimens from all three populations were roughly
the same size at the start of the experiment and ranged from
25 to 62 setigers overall (Table 2). Worms from Starr's
Point and Conrad's Beach divided about once per week ( 1.3
and 1.2 weeks between divisions, respectively), while
worms from Bon Portage Island divided less frequently (3.6
weeks between divisions; Table 2). although sample sizes
were low for the Starr's Point and Bon Portage Island
worms. Most worms divided into two fragments, but up to
six fragments per division were observed. Conrad's Beach
worms divided at the smallest size (average of 34 setigers),
whereas those from Starr's Point and Bon Portage were, on
average, larger before undergoing fission (42 and 45 seti-
gers, respectively; Table 2).

Mortality was also compared among regenerating frag-

Figure 5. Growth in Pv.i.vi.v/i/n <7ri;<m.v Gruwlh occurs through the
addition of terminal setigers, immediately anterior to the pygidium. New
setigers develop the parapodial lobes and chaetae characteristic of the tail
(tl. Transitional setigers show branchiae buds (h) and tufts of dorsal cilia
(d) as they gradually differentiate into abdominal setters (a). Scale bar =
10(1 /urn

Figure 6. Bright field micrograph of a two-headed individual of P\-
'V"" cU'xuns, formed after a spontaneous asexual event.

ments. Fragments were classified according to the remain-
ing original body region into anterior (containing the orig-
inal head, thorax, and anterior abdomen), mid- worm
(abdominal setigers only), and posterior fragments (original
pygidium. tail, and a few abdominal setigers). Anterior and
mid-worm fragments had a relatively low mortality, approx-
imately 14% for all three populations combined (Table 2),
although few mid-fragments were observed because most
worms spontaneously divided into two fragments (anterior,
posterior) only. Posterior fragments had the highest mor-
tality overall, about 80% among all three populations
(Table 2).

Discussion

Morphogenesis after asexual reproduction in P\gospio
elegans involved two phases: regeneration of lost body
regions (e.g., head, thorax, tail) followed by appositional
growth as terminal setigers were added. During regenera-
tion of the anterior region, the blastema extended to form
the head and thorax, segmented to divide the thorax into
10-12 setigers, and subsequently developed segment-spe-
cific structures (i.e., chaetae). Regeneration of the posterior
blastema was similar and also involved formation of a finite,
though variable, number of setigers (6 to 12). The origin of
blastemal tissues was not examined in the present study, but
probably involves the growth of existing tissues (e.g., epi-
dermis) in combination with the migration of mesodermal —
and possibly endodermal — neoblasts, as occurs in other
annelids (Hill, 1970; Christensen, 1994).

Once the thorax or tail had become reestablished, growth
occurred but was restricted to a growth zone immediately
anterior to the pygidium; new setigers did not appear else-
where in the body. Growth by the formation of terminal
setigers is common in spionid adults and larvae. The abdo-

ASEXUAL REPRODUCTION IN PYGOSPIO
Table 2

Asexual reproduction in Pygospio elegans

Trait

Population

ANOVA

Bon Portage

Starr's Point

Conrad's Beach

Original size (no. setigers)

42.1 ± 3.2

35.4 ± 1.2

41.6 ± 3.0

F(2.39) = 2.2, P =

0.12

n = 15

it = 15

/; = 10

Fission

No. Weeks between divisions

3.6 ± 0.5

1.3 ± 0.3

1 .2 ± 0. 1

F<2.2», = 26, P =

0.000

n = 5

n = 3

n = 21

CB = SP < BP

Size at division (no. setigers)

44.6 ± 3.3

41.5 ± 2.1

34.0 ± 1 .9

F(2.53) = 5.5, P =

0.007

n = 17

n = 1 1

n = 26

CB < BP. SP

No. Fragments per division

2.2 ± O.I

2.2 ± 0.1

2.7 ± 0.2

F — ~> 4 P —

r(2.52l *--^' r

0.10

i, = 17

I! = 1 1

n = 26

Mortality (no. dead/no, fragments per type)

% Mortality

Anterior fragment

6/26

0/11

3/29

13%

Middle fragment

1/2

2/24

14%

Posterior fragment

14/14

7/8

11/18

80%

Data are means, standard errors, and sample sizes (n) for traits indicated, in a comparison between laboratory-maintained worms from three populations.
The final column gives results of a one-way ANOVA among populations and results of a post-hoc Scheffe comparison among populations, where significant
differences were found.

men increased in size only during the growth phase as tail
setigers differentiated into abdominal setigers by develop-
ing branchiae and dorsal cilia. In all fragments, regeneration
produced only specific body regions, regardless of where
fission occurred in the parent. For example, one worm
divided in the original thoracic region and, after fission, had
only a head and nine thoracic setigers. This individual
regenerated only a tail and pygidium; abdominal setigers
redifferentiated from tail setigers as growth proceeded.
Many worms were observed to undergo a second asexual
event before growth was complete (often 8 to 10 days after
fission), and several individuals divided almost immediately
after fission (days 1-3) as evidenced by the presence of
fragments at different stages of regeneration in a single
culture.

Spionids, in general, are not as well known for their
ability to regenerate as are some other polychaetes, such as
sabellids. Asexual reproduction by regeneration is common,
however, in the spionids Polydora tetrabranchia (Camp-
bell, 1955) and throughout the genus Polydorella (Rada-
shevsky, 1996). In Polydorella, unlike P. elegans, new
individuals are formed by paratomy, resulting in a chain of
clones. Otherwise, morphogenesis during an asexual event
in Polydorella dawydoffi is similar to architomy in P. el-
egans: the new individual forms through development of a
growth zone (similar to the blastema reported here), elon-
gation to form specific anterior body regions (i.e., head and
thorax), segmentation resulting in a specific number of
thoracic setigers, and differentiation to form region-specific
structures such as the chaetae, eyes, and branchiae. Once the
new head has formed in Polvdorella. transverse fission

occurs and the two daughter worms separate (Radashevsky,
1996). Although asexual reproduction does not appear to be
widespread in spionids, regeneration as a response to tissue
loss (e.g., palps or the tail) occurs frequently in Polydora
cornuta (Zajac, 1985, 1995), Boccardia proboscidea (Gib-
son, pers. obs.), and Streblospio benedicti (Harvey, pers.
obs.). Further work may reveal whether the restricted po-
tential for asexual reproduction within the spionids could
have arisen by decoupling regeneration and reproduction, as
has been suggested in the oligochaete Paranais litoralis
(Bely, 1999).

Although the mechanisms leading to the restoration and
differentiation of body regions are not known, it seems
likely that the regulatory genes important in embryogenesis
may play a role. For example, distal-less is known to be
important in the development of parapodia in polychaete
embryos (Panganiban et ai. 1997) and possibly is reacti-
vated during regeneration, although this remains to be dem-
onstrated. In an asexual race of Dugesia tigrina (platy hel-
minth), lost body regions are defined during regeneration by
Hox genes that have sequences very similar to those found
in annelids (Bayascas etal., 1998). Interestingly, in this race
of D. tigrina. Hox genes were found to be permanently
expressed in adults, perhaps contributing to the impressive
regenerative capabilities of this species (Bayascas et ai.,
1998).

There were no differences among populations in original
size and number of fragments per asexual event in Pygospio
elegans, although time between divisions and size at divi-
sion did vary. Rasmussen found that rates of division in-
creased at low temperatures, and Wilson ( 1985) found that

G. D. GIBSON AND J. M. L. HARVEY

division rates increased at low worm densities. Anger
(1984) observed that the number of individuals (in an asex-
ual population) increased at low salinity and temperature. In
the present study, fission was observed in isolated worms
that were maintained under constant conditions (34 ppt,
20°C and with an abundance of food); therefore, these
conditions were unlikely to contribute to the differences in
division we observed among laboratory cultures.

Posterior fragments (original tail) had a higher mortality
than did anterior fragments (original head). Posterior frag-
ments were much smaller than anterior fragments; had few
branchiate, abdominal setigers (5-6 on average, vs. 25 for
anterior fragments); and lacked a mouth until day 5 post-
fission and therefore were unable to feed immediately after
division. Differences in mortality could be due to fragment
size (e.g., energy reserves or number of neoblasts available)
or lack of a mouth. However, the few mid-worm fragments
observed during the present study had a high survivorship,
despite their small size. Despite the high mortality of pos-
terior fragments, extensive laboratory culturing by others
indicates a net population growth through asexual reproduc-
tion (Anger, 1984; Wilson. 1985).

Although P. elegans is known to reproduce sexually
(Thorson, 1946; Hannerz, 1956; Anger, 1984; Anger et al.,
1986; Morgan et al., 1999), only asexual reproduction was
noted in the worms observed in the present study (more than
200 in total). This suggests that asexual reproduction is the
dominant reproductive mode in these populations during the
study period (May-September). Anger (1984) reported a
population in the Kiel Bight, Baltic Sea, that reproduces
exclusively through asexual reproduction; two additional
populations were predominantly sexual, although occa-
sional fragmentation was noted. Anger (1984) attempted to
induce specimens of P. elegans from these three populations
to switch between sexual and asexual reproduction by vary-
ing culture conditions (temperature and salinity) but found
that worms retained the reproductive mode of their original
population, leading her to suggest the potential for cryptic
species. Other investigators have reported seasonal differ-
ences in reproductive mode within a single population, with
asexual reproduction being dominant in the spring or sum-
mer, and sexual reproduction prevalent in the fall or winter
(Rasmussen, 1973; Hobson and Green, 1968; Wilson,
1985). Rasmussen (1953) also noted that fission could be
induced in P. elegans by temperatures of 4°-5°C.

In addition to asexual reproduction, P. elegans exhibits
considerable flexibility in sexual reproduction, including
both planktotrophic and adelphophagic larval development
(e.g., Thorson, 1946; Hannerz, 1956; Hobson and Green,
1968; Anger, 1984; Anger et al.. 1986). This suggests the
potential for reports of P. elegans to include cryptic species,
but Morgan et til. (1999) clearly demonstrated that poecil-
ogony does exist in this species, based on a molecular
(allozyme) comparison of populations with planktotrophic

or adelphophagic development. Poecilogony in P. elegans
is, in several regards, similar to that of the spionids Boc-
cardia proboscidea (Blake and Kudenov, 1981; Gibson,
1997) and Polydora connita (Mac Kay and Gibson, 1999),
which also reproduce by means of planktotrophic and adel-
phophagic larval development. Such flexibility makes P.
elegans a valuable model for tests of the ecological conse-
quences of life-history variability, as well as for understand-
ing the developmental mechanisms underlying a change in
development mode.

Acknowledgments

We thank H. Smith for general and field assistance, and
H. Taylor for assistance with the SEM. P. Allen-Wojtas and
S. Carbyn of the Electron Microscopy and Imaging Labo-
ratory, Agriculture and Agri-Food Canada, Kentville, Nova
Scotia, provided facilities and assistance with SEM. The
manuscript has benefited from comments from M. Gibson
and I. Paterson. This research was funded by an NSERC
grant to G. Gibson and Nova Scotia Summer Career Place-
ment to J. Harvey.

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Reference: Biol. Bull. 199: 50-5S. (August 2(1

Factors Influencing Spawning and Pairing in the Scale
Worm Harmothoe imbricata (Annelida: Polychaeta)

G. J. WATSON1-*, F. M. LANGFORD2. S. M. GAUDRON2, AND M. G. BENTLEY2

1 Institute of Marine Sciences, Ferry Road, Eastney, University of Portsmouth, Portsmouth, P04 9LY,

UK; and " Department of Marine Sciences and Coastal Management , Faculty of Agriculture and
Biological Sciences. Ridlev Building. University of Newcastle, Newcastle-upon-Tyne, NE1 7RU, UK

Abstract. Endocrine and environmental factors control
reproduction of the polynoid scale worm Harmothoe imbri-
cata. We confirmed that the rate of vitellogenesis was
greater in winter specimens transferred from ambient re-
gimes of photoperiod and temperature to a light:dark (LD)
photoperiod of 16:8 at 10°C and showed that the number of
females spawning was significantly greater than for those
transferred to LD8:16 at I()°C. The endocrine mediation of
this response was investigated using prostomium implanta-
tions. Significantly more LD8:16 females implanted with
prostomia from LD16:8 conditioned females spawned than
LD8:16 females implanted with LD8:16 prostomia. Fe-
males without prostomia failed to spawn. LD16:8 exposure
may increase levels of a possible "spawning hormone" in
the prostomium. Spawning proceeded in these LD16:8 fe-
males and allowed spawning to occur in LD8:16 females
implanted with LD16:8 prostomia. In LD8:16 prostomia.
liters of the spawning hormone reached the threshold in
significantly fewer individuals, so that significantly fewer
females implanted with LD8:16 prostomia spawned.

Using Y-maze choice chambers, pair formation was
shown to be under pheromonal control, with males being
attracted to mature females but not to females carrying
fertilized oocytes or to LD8:I6 conditioned females. Pro-
duction of this attraction pheromone can, therefore, be ma-
nipulated through photoperiodic control, suggesting a link
between oogenesis. spawning, and pheromone production.

Received 23 November 19W; accepted 31 May 200(1.
* To whom correspondence should he addressed. E-mail: gordon.
watson@port.ac.uk

Introduction

The polynoid scale worm Harmothoe imbricata is a com-
mon inhabitant of temperate intertidal rocky shores, where
it lives under rocks and small stones. It is an active carni-
vore that preys upon other small invertebrates. The repro-
ductive biology of this species is relatively well understood.
It is a dioecious, iteroparous species with an annual cycle of
reproduction (Daly. 1972, 1974; Garwood. 1980). Females
develop two cohorts of oocytes: the first is grown slowly
during the winter months to be spawned in March; the
second is produced rapidly and spawned about 30 days after
the first. During the breeding season individuals pair, a
behavior in which a male lies closely along the dorsal
surface of the female (Daly. 1972). Cohorts of oocytes, once
spawned, are fertilized and earned under the female's dorsal
elytra (scales) during embryogenesis. Embryos are released
as trochophores after about 16 days.

Oogenesis is initiated in late September without any
specific environmental input. During the autumn, the first
cohort grows under conditions of decreasing environmental
temperature, and growth is accelerated by exposure to low
temperatures (Garwood, 1980). The stabilization of oocyte
development also requires exposure to a light-dark (LD)
cycle with a photophase less than 13 h for between 42 and
55 days during the late autumn period. If these photoperiods
are not experienced, oocyte development is aborted (Clark.
1988). Once the winter solstice has passed, natural popula-
tions respond to a second photoperiodic input. An exposure
to LD cycles with a photophase above 10 or 11 h increases
oocyte growth rate and may synchronize oogenesis and
spawning among individuals (Garwood and Olive, 1982;
Clark. 1988).

Although environmental manipulation affects both oo-
genesis and spawning, the endocrine role in mediating these

REPRODUCTION IN HAKMOTHOE IMBRICATA

influences has received comparatively little attention. Bent-
ley et til. ( 1994) and Lawrence ( 1996) confirmed the pres-
ence of a gonadotrophic hormone from the prostomium that
probably mediates oogenesis, and it seems likely that the
hormone is secreted only under the appropriate regimes of
photoperiod and temperature. Nevertheless, the role of the
endocrine system in spawning is unknown in this species. In
a number of other polychaete species, the implantation or
injection of prostomia into individuals has been used to
investigate their endocrine control of reproduction and
spawning (Golding. 1983: Bentley et al., 1984; Pacey and
Bentley, 1992; Bentley et al., 1994; Watson et al., 2000). In
the work reported here, transplantation experiments estab-
lish the role of the endocrine system in mediating photope-
riodic influences, and environmental manipulation is used to
investigate the effect of photoperiod on spawning and oo-
genesis of the first cohort of oocytes.

Pherornones coordinate and control reproduction in a
number of marine invertebrates, including species of the
opisthobranch mollusc Aplysia (Zeeck et al.. 1990, 1996;
Painter et al., 1998). by inducing spawning and by attracting
other individuals towards spawning animals. In this paper
we provide evidence of an attraction pheromone produced
by female Harmothoe imbricata to attract males for mating,
and we examine the influence of environmental manipula-
tion on its production.

The results presented here provide the first link between
environmental conditioning, endocrine activity, and phero-
monal control of attraction for pairing and spawning in a
polychaete species. Harmothoe imbricata is thus likely to
become a model species for the investigation of environ-
mental-endocrine-pheromone interactions.

Materials, Methods, and Results

Photoperiodic control of ooc\te growth and spawning
mediated b\ the endocrine svstem

Collection anil maintenance of animals. Specimens of
Harmothoe imbricata were collected from the intertidal
zone of three rocky shores in E. Scotland, UK: St Andrews
(56°20'N, 2°47'W), Kingsbarns (56°18'N, 2°38'W) and
Fife Ness (56°16'N, 2°35'W). Individuals were maintained
in glass crystallizing dishes containing 100 ml of TFSW
(twice-filtered seawater, pore size 0.34 jam) and provided
with a cleaned Patella vulgata shell for shelter. All worms
were hand-fed pieces of Mytilus edulis muscle once per
week, after which the water was changed. All individuals
were collected in December and January and kept at 10°C
and ambient photoperiods until the experiments commenced
on 9 February 1999.

To assess their state of maturity, individuals were narco-
tized in 5% ethanol in seawater and a small incision was
then made in the lower edge of the 16th setiger. For micro-
scopic analysis, a small sample of coelomic fluid and blood

vessel was removed using a 20-;u.l glass micropipette. The
diameters of about 30 oocytes from five randomly chosen
females from each treatment group were measured using a
compound microscope. Sperm activity was assessed after
dilution.

Experimental protocol. On 9 February 1999, half of the
females were transferred to conditions of 16 h light and 8 h
dark (LD16:8) at 10°C. The individuals that remained in
LD8:16 conditions were termed LD8:16 controls while
those transferred to the LD16:8 were termed LD16:8 con-
trols. Oocyte diameters were measured from five randomly
selected individuals from the two groups on this day and
subsequently once per week for 3 weeks.

Prostomium (PM) transplants were performed on 26 Feb-
ruary 1999, 2 weeks after photoperiod manipulation had
commenced. Individuals from the LD16:8 control and the
LD8:16 control were narcotized as described above to pro-
vide prostomia. They were termed LD16:8 donors and
LD8:16 donors, respectively. The PM was accessed by
removing the first two scales and then excised with iridec-
tomy scissors. Once removed, the PM was trimmed of
excess flesh and tentacles and then placed in seawater on ice
until implanted. Both sets of PM donors were then returned
to seawater to recover from the narcotization.

Individuals designated to received the implanted PM
were also narcotized, and a PM was inserted through an
incision in the 2()th setiger. This was far enough from the
head to prevent any interference with movement. Twenty-
five LD16:8 control females were used as PM donors and
their PMs were implanted into females from the LD8:16
control group. They were termed LD8:16PM(LDI68) females
(the superscript notation refers to the conditions that the
female, from which the prostomium used for implantation
was removed, was exposed to). As a control, eight LD8: 16
control females were also implanted with LD8:16 control
PMs, and these were termed LD8:16PM(LD8:I6'. After im-
plantation, all PM recipients and PM donors were returned
to their respective photoperiod treatments. All individuals
were examined daily for evidence of spawning (the pres-
ence of oocytes under the elytra). The diameters of about 30
oocytes were measured from individuals that had spawned.

Statistical aitalvses. The nature of the oocyte diameter
data (unbalanced nesting and sample sizes) precluded anal-
ysis using a multiple ANOVA for all data. Instead, mean
oocyte diameters for each female were obtained, and these
data were then assessed using one-way and two-way
ANOVAS. Subsequent pairwise comparisons were per-
formed using Tukey tests. The numbers of spawning indi-
v iduals were analyzed for independence by using an R X C
contingency table and the chi-square statistic f^2); pairwise
comparisons were performed subsequently using a modified
Tukey test.

Results: photoperiodic control of oocyte growth. Mean
oocyte diameters of LD8:16 and LD16:8 controls and PM

G. J. WATSON ET AL.

recipients (LD8:16PM|LDI ' and LDS:16PM(L ' are
shown in Figure 1. Analysis, using a two-way ANOVA. of
the mean oocyte diameters of the LD8:16 and LD16:8
controls for weeks 1 to 3 shows that significant differences
were present between weeks (F = 28.64, P < 0.001 ), but
not between treatments. There were also significant inter-
action effects (F == 7.407. 0.05 > P > 0.001). Pairwise
comparisons using a Tukey test confirm that the mean
diameters of LD16:8 and LD8:16 controls were not signif-
icantly different from each other in week 1. By week 2,
LD16:8 control diameters increased significantly when
compared to week 1, whereas LD8:16 control diameters did
not increase. By week 3, LD8:16 control diameters in-
creased significantly when compared to week 2. At the same
time, all mean oocyte diameters of all treatments were not
significantly different from each other when analyzed with
a one-way ANOVA. (F = 0.1 1. P > 0.05). Analysis of all
treatments in week 4 shows that there were significant
differences between the treatments (F = 9.56, 0.05 > P >

0.001 ). However, pairwise comparisons reveal that only the
LD16:8 control mean diameter was significantly greater
than the LD8:16 control and LD8:16PM(LD8 lftl. No other
pairwise comparisons were significantly different.

Results: photoperiodic control of female spawning medi-
ated by the endocrine system. During the experimental
period, all individuals in all treatments were monitored
daily for spawning; the cumulative percentage of individual
spawning females is shown in Figure 2. Over the duration of
the total experimental period (9 February- 14 March), 96%
(24 individuals) of the LD16:8 controls spawned, with the
majority (16 individuals) spawning on 23 February and
another 8 spawning between 24 and 28 February. In com-
parison, only 33% of the LD8:16 control females spawned
during the experimental period, one on 19 February, two on
23 February, and another on 25 February.

During the experimental period for prostomial manipula-
tion (26 February-14 March), 80% of the LD8:16PM(LD168)
treatment group spawned, with 10 individuals spawning on

120-

10-

90 -

n=5

5=? n=5

LD16:8 control
LD8:16 control

LD8:U/N"""""'

LD8:16

PM (U>X- Ih)

n=5

zL?"3

/>vnt

0=3

9-Feb(Wcckl) 16-Feb (Week 2) 23-Feb (Week 3)

Week

2-Mar (Week 4)

Figure 1. Mean weekly oocyte diameter ( ±95% confidence limits) of female Hannothoe imbricata exposed
to various treatments at 10"C: LD16:8 control, females maintained in long-day photoperiod (LD16:8); LD8:16
control, females maintained in short-day photoperiod (LD8:16); LD8:I6PM|LD">I", females maintained in
I '• 16 each implanted with one prostomium from un LDI6:H control female; LD8:16PM|LDS "". females
maintained in LD8:16. each implanted with one prostomium from an LD8:16 control female. All individuals
were collected in December and maintained in ambient photoperiod at 10°C prior to the start of the experiment
on 9 Februar> I'l'W (week 1 ). All transplantations were performed on 26 February 1999 (week 3); n = number
of females sampled, 30 oocytes were counted per female.

REPRODUCTION IN HARMOTHOE IMBKICATA

o
eo

1 00 -,

80-

60-

40-

20-

•— LD16:8 control
o — LD8: 16 control
._LD8: ,„""">"«'
._LD8:16™ <'•""«
o — LD8: 16 donor
x — LD16:8 donor

•B-B(n=S)

c3 a rt
2! 2 S

Date

Figure 2. Cumulative percentage of spawning female Harmothoe imhricata after exposure to various
treatments at 10°C. LD16:8 control, females maintained in long-day photoperiod (LD16:8); LD8:16 control,
females maintained in short-day photoperiod (LD8:16): LD8:16PM|LD168'. females maintained in LD8:lft and
each implanted with one prostomium from an LD16:8 control female; LD8:16PM|LD8 lf", females maintained in
LD8:16 and each implanted with one prostomium from an LD8:16 control female; LD8:16 donor, LD8:16
control females used as prostomial donors; LD16;8 donor. LD16:8 control females used as prostomial donors.
All individuals were collected in December and maintained in ambient photoperiod prior to the start of the
experiment on 9 February 1999, start of photoperiodic conditioning. All transplantations were performed on 26
February 1999. n is the number of females in each treatment group.

27 February and a further 10 spawning between 1-8 March.
In contrast, only 37.5% (3 individuals) of the LD8:
16PM|LDS l6' treatment group spawned, and none of the
LD8:16 or LD16:8 donors spawned.

Statistical analysis using an R X C test of independence
and the \2 statistic reveals highly significant differences
between the total proportion of spawning to nonspawning
females between all treatments (^2 = 68.409, P < 0.001 ).
Statistical analysis of pairwise comparisons, using a modi-
fied Tukey test, shows that all pairwise comparisons were
significantly different from each other, except the following
treatments: LD8:16 control compared with LD8:16PM" Ds 16'.
and LD8:16 donor compared with LD16:8 donor.

Pheromone influence on pairing behavior

Y-maze behavioral bioassays. Six Y-mazes. the dimen-
sions of which are shown in Figure 3A, were constructed
from 5-mm-thick clear acrylic plastic. Each was sealed with
silicon sealant and allowed to cure by soaking in seawater
for several days prior to use. Each arm had a removable
acrylic partition with 0.4-mm-diameter holes drilled
through to allow pheromonal diffusion. All experiments
were performed at 10°C in ambient illumination.

Animals. Animals were collected from the Fife Ness site
during March and April and maintained as described above.
Individuals collected in March and April (classed as "old"
and "new," respectively) were sexed, and their state of
maturity assessed as described above. Of the old individuals
collected, 10 females were carrying fertilized oocytes and 7
females still had fully grown oocytes in the coelomic cavity.
Of the males collected, 18 had active sperm in their coelo-
mic cavity. Of the new individuals collected, 10 females had
not spawned and 10 males had sperm in their coelomic fluid.

Experimental protocol. The basic experimental protocol
is summarized in Figure 3B. Before each set of experiments,
each maze was washed in fresh water before air-drying.
During a run of experiments each maze was washed in
TFSW between tests. After each maze was filled, a potential
stimulus animal was placed in one arm of the maze behind
the partition. After 1 min, a test male was placed in the base
of the maze. To minimize handling, a glass tube was used in
positioning the test animals. Each test was run for 30 min
(except the first directional bias test with old males, see
below), after which the position of the test male was noted.
At the end of the 30 min, a response was considered positive
if the test male was in the arm with the stimulus animal.

G. J. WATSON ET AL.

Wash maze in freshwater and allow to dry

Fill maze to 5cm depth

Place stimulus animal in left or
right arm behind gate (position X)

Leave for 1 minute

I
Place test male at start position (*) of maze

Allow 30 minutes before recording position

onses:

( + ) Animal is in arm with stimulus
(-) Animal is in opposite arm to stimulus
(NC) No choice, in bottom on "Y"

Figure 3. (A) Schematic diagram of Y-maze for the investigation of pheromonal attraction of male
Harmothoe imbricata to various stimuli. Dashed lines represent removable partitions. Depth of seawater in
Y-maze: 5 cm. (B) Summary flow diagram of the experimental protocol.

negative if it was in the opposite arm, and a no choice if it
was in the base of the Y. During an experimental run, all
potential stimulus individuals were alternated between each
arm in consecutive experiments and cross-tested with all
test males.

Tests performed. All tests were performed within 2 weeks
of collection. To assess for any directional bias ("handed-
ness") and for chance levels of attraction in the maze, two
sets of experiments were performed in which males were
placed in the Y-maze with no stimulus animal. In the first
experiment, nine old males were each run three times in the
Y-maze and the position of the animal was recorded after 10
min. From observations of the males, it was suspected that
this time period was not sufficient for the test animals to
complete exploratory behavior before settling. A further six
old males were tested without a stimulus six times each, and
their position was recorded after 30 min. All subsequent
tests were run for 30 min. Old males were tested against
each of the following: old mature females (two sets) and old
females carrying fertilized oocytes. New males were tested
against each of the following: new mature females, new
females maintained in short days, and old males. All statis-
tical significance was assessed using \2 analyses.

Results. The first set of experiments was performed with
old (collected in March) males and females (Fig. 4). To
assess for any directional bias and for chance levels of
attraction, the positions of the males were recorded 10 min
after being introduced into the maze. Fifty-three percent of

Figure 4. Percentage response of old (collected March 1999) male
Hunmiihoe imhricata to various stimuli in a Y-maze run at IO°C. SW. no
stimuli added (number of runs = 36 [6 males x 6 tests]): Mature Fem. I,
mature old females (number of runs = 27 [9 males X 3 females]); Mature
Fem. II, second group of mature old females (number of runs = 63 [7
males X 9 females]); Pert. Females, females carrying fertili/ed oocues
under their elytra (number of runs = 27 [9 males x 3 females]). The
position of each male was recorded 30 min after introduction to the maze.
Responses were classed as positive if the male was in the arm containing
the stimulus (or designated as stimulus if seawater only), negative if in the
arm with no stimulus, or no choice if it moved to neither arm.

REPRODUCTION IN HARMOTHOE IMBRICATA

these males moved to the arbitrarily labeled positive arm
and 47% moved to the negative arm. However, this exper-
iment was omitted from the graph and from statistical
analysis beeause the males had not completed their explor-
atory behavior within the 10-min time frame. The experi-
ment was, therefore, repeated with a further six old males
whose positions were recorded 30 min after introduction. In
the 36 tests performed, 28% of the males moved to the
arbitrarily positive arm and 19% to the negative arm, while
53% made no choice. This result was not significantly
different form an expected value of 33.3% moving to each
arm or making no choice (^2(2) = 2.60. P > 0.05).

These nine old males were then tested against three old
mature females, and a significantly higher level of attraction
(X2(2) = 23.07, P < 0.001) was observed: 81% of the males
moved to the positive arm and the other 1 9% moved to the
negative arm (without the females). To confirm this attrac-
tion response, a further test was performed with 1 1 males
and 7 old mature females. Sixty-one percent of the males
moved to the positive arm. 13% moved to the negative arm,
and 26% made no choice. This level of attraction was also
significantly higher than shown for the seawater 30-min
control (x2(2) = 10.58. 0.05 > P > 0.001).

An additional experimental run was performed using the
nine old males and three old females that had spawned in
the field and were carrying fertilized oocytes under their
elytra. In the 27 tests performed, 48% of the males moved
to the positive stimulus arm, 37% moved to the negative
arm. and 15% made no choice. Statistical analysis of this
data shows that this was a significantly higher level of
attraction than shown for the seawater control (^2(2) =
9.69. 0.05 > P > 0.001). However, if the no choice and
negative results are combined and compared statistically
with the positive stimulus, there was no significant differ-
ence between the treatments (^(1) = 2.74, P > 0.05),
confirming that the differences were due to the decrease in
no choices and not to an increase in positive results. Statis-
tical analysis also shows that there was a significantly lower
level of attraction for fertilized females than for both mature
female I (^2(2) = 6.87. 0.05 > P > 0.001) and mature
female II experiments (\2(2) = 7.97. 0.05 > P > 0.001).

The second set of experiments was performed with new
animals collected in April (Fig. 5). To assess again for
directional bias, six new males were tested with no stimulus,
and their position was recorded after 30 min. Thirty-three
percent and 36% of the males moved to the arbitrary posi-
tive and negative arms respectively, with 31% making no
choice. This was not significantly different from an ex-
pected value of 33.3% moving to each arm or making no
choice ()C(2) = 0.593, P > 0.05).

These new males were then tested against six new mature
females. A significantly higher level of attraction was ob-
served when compared to the seawater test, with 61% of the
males moving to the positive arm, 22% to the negative arm.

LDIS lf> K'nuiks

Stimulus

Figure 5. Percentage response of new (collected April 1999) male
Harmothoe imhricata to various stimuli in a Y-maze run at 10°C. SW. no
stimuli added (number of runs = 36 [6 males x 6 tests]): Mature Females,
mature new females (number of runs = 36 [6 males x 6 females]); Males:
new males tested against themselves (number of runs = 36 [6 males x 6
males]): LD8:16 Females, females maintained in short days (LD8:16) for
3 months (number of runs = 36 [6 males x 6 females]). The position of
each male was recorded 30 min after introduction into the maze. Responses
were classed as positive if the male was in the arm containing the stimulus
(or designated as stimulus if seawater only), negative if in the arm with no
stimulus, or no choice if it moved to neither arm.

and 17% making no choice (/(2) = 12.95. 0.05 > P '-
0.001).

These six new males were also tested against themselves.
Twenty-five percent moved to the positive arm. 33% moved
to the negative arm. and 42% made no choice. Statistical
analysis of these data shows that this was not a significantly
different level of attraction when compared to the seawater
control (,\2(2) = 0.506. P > 0.05), and males were signif-
icantly less attractive than mature females <^2(2) = 12.61,
0.05 > P > 0.01).

These males were also tested against six mature females
that had been maintained in LD8:16 cycles for 3 months.
Twenty-five percent moved to the positive arm, 50% moved
to the negative arm, and 25% made no choice. Levels of
attraction for this test also did not differ significantly from
seawater (^2(2) = 0.7, P > 0.05). and LD8:16 females were
significantly less attractive than mature females (^2(2) =
10.5. 0.05 > P > 0.001).

Discussion
Photoperiodic control of oocylc u

The rate of oogenesis of the first oocyte cohort in Hur-
motlioe imbricata can be altered by manipulation of tem-
perature and photoperiod (Garwood. 1980; Garwood and
Olive. 1982; Clark. 1988). A period of exposure of 42-55
days of less than 13 h (winter conditions) photophase is

G. J. WATSON ET AL

required to prevent the first cohort of oocytes from being
aborted. In experiments in this study, females had been
maintained in ambient photoperiods prior to the experi-
ments commencing in February. They were, therefore, not
exposed to either LD16:8 or LD8:16 conditions until after
the critical minimum number of days had been reached, thus
preventing resorbtion.

In the field, once females have been exposed to this
critical period of LD8:16 cycles, an increasing ambient
photophase (after the winter solstice) allows oogenesis to
proceed normally. Above a critical photoperiod of 10-1 1 h
photophase, oogenesis is accelerated; however, an increased
number of winter condition cycles (between 55 and 73) is
required for this response to be exhibited (Garwood, 1980;
Garwood and Olive. 1982; Clark, 1988). The results pre-
sented in Figure 1 confirm that an increase in photoperiod
(LD16:8 conditions) accelerates oogenesis. The results also
show that the response to LD16:8 conditions was rapid, with
effects occurring within I week from exposure. However,
oocyte growth continued under LD8: 16 conditions, as by 23
February both LD16:8 and LD8:16 conditions had oocytes
that were not significantly different from each other.

Incubating prostomium homogenate with oocytes in vitro
significantly increases their uptake of radiolabeled amino
acid and their subsequent protein synthesis (Bentley et nl.,
1994; Lawrence, 1996). These authors suggested the pres-
ence in the prostomium of a gonadotrophic hormone that
promotes and controls oogenesis. The manipulation of pho-
toperiod and temperature may, therefore, be acting directly
on the levels of this hormone and it is this hormone level
that subsequently mediates oogenesis. Ambient conditions
(increasing photophases) in January and February may sus-
tain oogenesis by increasing the circulating tilers of this
substance. The exposure of females to LD16:8 conditions
may have triggered a burst of secretion of the hormone
(above the levels normally experienced in February), induc-
ing an increase in mean oocyte diameter that occurred in
week 2. Females exposed to continued LD8:16 conditions
had no burst of secretion; instead, oogenesis continued at a
constant rate, resulting in the delayed increase (week 3) in
mean oocyte diameter.

By week 3, oocytes were fully grown — approximately
120 jam (Daly, 1972); data from Figure 1 show that, in these
experiments, oogenesis was complete by 23 February. Data
also show that implantation of prostomia had no effect on
oocyte diameter and did not induce oocyte degeneration.
The diameters of spawned oocytes from females implanted
with LD8:16 or LD16:8 prostomia were not significantly
different from each other, from the LD8:16 controls in
weeks 3 and 4. or from the LD16:8 control in week 3. In
week 4, the mean oocyte diameters of LD 16:8 controls were
significantly higher than those in any other treatment, al-
though this is more likely to be due to the low numbers of
females sampled than to any effect of the treatment.

Photoperiodic control of spawning mediated b\ the
endocrine system

Early transition through the critical photoperiod can ad-
vance the time of spawning; long days or photoperiods with
greater than 1 1 h photophase can cause the first cohort of
oocytes to be spawned about 1 month earlier than the
natural date (Garwood and Olive, 1982). In this study,
females carrying fertilized oocytes were collected from the
field on 3 March 1999. We calculated that their natural
spawning data was late February to early March. From the
results presented in Figure 2, exposure to a LD16:8 photo-
period did not induce notably earlier spawning dates when
compared to a LD8:16 photoperiod, and neither was differ-
ent from the natural date. Instead, when compared to
LD16:8 exposure, LD8:16 exposure actually prevented
spawning in the majority of females. Ninety-six percent of
the LD16:8 females spawned, as opposed to only 33% of the
LD8:16 females.

We conclude that the presence of the prostomium is
required for spawning to occur in H. imbricata, as none of
the females that were used as LD16:8 or LD8:16 donors
spawned. Examination of the oocytes form these donors
also showed that they had not increased in diameter after
prostomium removal and had begun to degenerate (data not
shown). The prostomium is, therefore, required for the
maintenance of oogenesis and for spawning to occur. Nev-
ertheless, a failure to spawn after prostomium removal
cannot, at present, be attributed solely to a loss of endocrine
function because it may also be due to the severance of
nervous connections controlling spawning.

To investigate whether the inhibition of spawning
through exposure to LD8:16 photoperiods is endocrine me-
diated, we implanted LD8:16 control females with prosto-
mia from females maintained in LD16:8 or LD8:16 photo-
periods for 2 weeks. Although the implantation of
prostomia is an established technique for investigating the
role of endocrine substances in polychaetes (see Golding,
1987), this is the first time that it has been used successfully
for H. inihriciiui. Previous attempts with this species re-
sulted in the degeneration of the implanted prostomium
(P. J. W. Olive, University of Newcastle upon Tyne, pers.
comm.). Prostomia implanted during our experiments
showed no obvious degeneration with light microscope
analysis up to one month after implantation (data not
shown). It should be noted that implantation of prostomia is
not an ideal technique for identifying the putative spawning
hormone in this species. Future investigations will focus on
the development of in vitro bioassays to reduce the numbers
of prostomia used and to meet the levels of sensitivity and
reliability that are essential for purification studies.

The results presented in Figure 2 show that 80% of the
LD8: 16PM(I m" Nl females spawned, as opposed to 37.5% of

theLD8:16

I'MlI 1)S:I6) 4.-

" females. These results confirm that the

REPRODUCTION IN HAKMOTHOE IMBRICATA

implanted prostomia were still functioning as endocrine
organs. We suggest the following hypothesis for the endo-
crine control of spawning. A spawning substance present in
the prostomium is required for spawning to occur. The liters
of this substance, as with the gonadotrophic hormone, are
mediated by photoperiod. The exposure of females to
lengthening photophases (ambient conditions in February)
increases the liters of this spawning substance to a level
above which spawning can occur. Exposure of females to
LD16:8 conditions also allows liters of the spawning sub-
stance to reach the threshold level, so that nearly all the
females spawn. In contrast, only a small proportion of the
females exposed to LD8:16 conditions have liters of the
spawning substance that reach the threshold, so significantly
fewer spawn. Implanting the prostomia of an LD16:8 ex-
posed female into an LD8:16 female also provides a source
of higher levels of spawning hormone and thus increases the
total circulating liters, enabling the LD8: 16 females to reach
the threshold required for spawning to occur. Implanting the
prostomia of an LD8:16 exposed female into an LD8:16
female provides a second source of the hormone, but at
lower concentrations. In most females, the combined level
of hormone is lower than the threshold, so most fail to
spawn.

The nature and action of a spawning substance in H.
imbricata may take two forms. It could be a "true" spawn-
ing substance like that found in Nephtys Iwmbergii. In that
species, a hormone released from the supraesophageal gan-
glion induces spawning by acting on the musculature to
allow the release of gametes through the anus (Bentley el
al., 1984). The maturation of the gametes is independent of
the spawning hormone; they mature once released into
seawater (Olive, 1976; Olive and Bentley, 1980).

The other form of spawning substance induces gamete
maturation and subsequent spawning either directly or in-
directly. This form occurs in Arenicola marina. Oocytes
mature through a two-step system involving a substance
from the prostomium and then a second substance in the
coelomic fluid. The latter, termed the coelomic maturation
factor (CMF), induces the oocytes to mature; these oocytes
are subsequently spawned (Watson and Bentley, 1997). It is
unclear whether CMF also acts on the musculature to facil-
itate spawning. However, in male A. marina, the sperm
maturation factor (8, 11, 14 eicosatrienoic acid) not only
induces maturation of the sperm but also produces specific
behavioral changes associated with spawning (Pacey and
Bentley, 1992).

In H. imbricata, oocytes are released from the ovaries at
prophase of the first meiosis a few days before spawning.
They mature to metaphase of meiosis I in the coelomic Huid,
are collected by the nephridia, and spawned (Daly, 1972).
Further experiments are required to elucidate whether the
putative spawning substance from the prostomium can in-

duce spawning of immature oocytes or actually induces the
maturation of the oocytes that are subsequently spawned.

Pheromone influence on /minni; behavior

Initial observations of the test males in the Y-maze indi-
cated that 10 min was not sufficient time for them to
complete their exploratory behavior. After 10 minutes most
of the males were still actively searching. In all subsequent
experiments we allowed 30 min for the males to settle
before their position was recorded. This time period was
sufficient for the males to settle and complete their explor-
atory behavior.

Results presented in Figures 4 and 5 provide the first
evidence that pheromones are involved in the reproductive
behavior of H. imhncata. Specifically, these data indicate
that a mature female with fully grown oocytes in its coelo-
mic cavity releases a waterborne substance or substances
that attracts significantly more mature males than are at-
tracted by seawater. males, or females that are carrying
fertilized oocytes.

Harmothoe imbricata is a solitary species outside the
breeding season, but it reproduces by forming single copu-
lating pairs (Daly, 1972). This method of reproduction
requires mature individuals to locate each other, but at low
population densities, chance encounters may be infrequent.
An attraction pheromone released by a mature female in-
creases the chances that a male will find her and, therefore,
increases the number of successful fertilizations. The pher-
omone may also maintain the pair bond and could suppress
the cannibalistic tendencies of both individuals, allowing
pairing to proceed. Once the female has fertilized oocytes,
she stops releasing the pheromone and becomes unattractive
to the male; this is confirmed by the data shown in Figure 5.

One of the best-studied attraction pheromones in marine
invertebrates is attractin, a peptide found in the egg cordon
of the opisthobranch mollusc Aplysia spp. The function of
this 58-residue peptide is to attract other individuals to the
mating aggregation and to induce mating (Painter et ai,
1991, 1998). Pheromones are also a component of a number
of polychaete reproductive strategies, particularly in some
nereid species (for review, see Zeeck et ai, 1996). How-
ever, these pheromones have been isolated only from spe-
cies such as Platvnereis dumerlii and Nereis succinea that
swarm en masse in the water column (Zeeck et al., 1988.
1996; Hardege et al., 1998). The results presented here are.
therefore, the first report of a waterborne cue being used as
an attraction pheromone from a polychaete that reproduces
following pair formation.

Although the evidence for pheromones in marine inver-
tebrates is steadily growing, the environmental control of
pheromone production has not been investigated. The effect
of environmental manipulation (particularly photoperiod)
on oogenesis is confirmed and the influence of photoperiod

G. J. WATSON ET AL

on spawning in Harmothoe imbrictitei has been described
for the first time (Garwood and Olive. 1982; Clark, 1988).
Establishing a link between the photoperiodic input and the
production of a pheromone that regulates spawning behav-
ior is an important step. Results presented in Figure 5 show
that females maintained in LD8:16 photoperiods were only
as attractive to mature males as seawater or other males. Just
as LD8:16 exposure may prevent the production (or the
attainment of threshold levels) of the putative spawning
hormone and thus prevent spawning, it may also prevent the
production of the attraction pheromone. The relationship
between the spawning hormone and pheromone production
requires further investigation, but the production of the two
may be intricately linked, and they may be the same or
similar substances.

Acknowledgments

The authors acknowledge the support of NERC grant
GR3/ 10521 to MGB. The authors declare that the experi-
ments comply with the current laws of the country in which
the experiments were performed.

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Reference: Biol. Bull. 199: 59-67. (August 2000)

Fertilization in Callochiton castaneus (Mollusca)

JOHN BUCKLAND-NICKS1 * AND ALAN N. HODGSON-

lSt. Francis Xavier University, Department of Biology, Antigonish. Nova Scotia. Canada B2G 2W5: and
^Rhodes University, Department of Zoology and Entomology. Graluumtiwn f>l40. South Africa

Abstract. A fine-structural study of fertilization in Callo-
chiton castaneus has revealed that the mechanism of sperm
penetration into the egg is intermediate between the primi-
tive condition found in members of the order Lepidopleu-
rida and the more derived condition found in the Chitonida.
C. castaneus sperm have the long needlelike nuclear fila-
ment and reduced acrosome that characterizes all Chitonida.
but they have retained several plesiomorphic features such
as an unspecialized mid-piece and a lack of flagellar rein-
forcement. As in some Lepidopleurida but unlike any Chi-
tonida, the egg hull in this species comprises a thick, smooth
jelly coat permeated by pores that permit sperm rapid access
to the vitelline layer. The jelly coat is delicate and quickly
dissolves when a sperm concentrate is used, suggesting that
excess acrosomal enzymes may be responsible. Once the
sperm have penetrated the vitelline layer, the long nuclear
filament bridges the gap to cups in the egg membrane.
However, once the fertilization membrane is raised, the
perivitelline space exceeds the length of the nuclear fila-
ment, preventing other sperm from penetrating the egg. A
fertilization cone forms around the nuclear filament of the
penetrating sperm, but it does not appear to engulf the body
of the sperm. Rather, the nuclear chromatin is injected into
the egg as a long thread. The remaining sperm organelles
are apparently abandoned on the egg surface. If this is the
case, it would be a significant departure from fertilization in
other molluscs and many other metazoans, in which sperm
organelles, such as centrioles and mitochondria, enter the
egg.

New sperm and egg characters, as well as significant
differences in fertilization, indicate that Callochitonidae are
basal to all other members of the order Chitonida and may

Received 7 December 1999; accepted 25 May 2000.
* To whom all correspondence should be addressed.
jbucklan@stfx.ca

E-mail:

warrant separation as the sister taxon to the suborders Chi-
tonina and Acanthochitonina.

Introduction

Sirenko ( 1997) recently classified extant chitons into two
orders. Lepidopleurida (suborders; Lepidopleurina and
Choriplacina) and Chitonida (suborders; Chitonina and
Acanthochitonina). Lepidopleurida are considered basal to
chitons in general, because they possess many primitive
traits. For example, shell valve structure and articulation is
simpler and ties in well with the fossil record (Sirenko.
1997): also they are the only chitons known to have typical
aquasperm with prominent acrosomes, and eggs with
smooth hulls (Hodgson et al., 1988; 1989; Eernisse and
Reynolds. 1994; Buckland-Nicks. 1995: Pashchenko and
Drozdov, 1998). Presumably, in the ancestor of all members
of Chitonida. the sperm acrosome became reduced to a
minute vesicle atop a long needlelike extension of the
nucleus, since this arrangement is found in all extant Chi-
tonida (Buckland-Nicks et al.. 1990) but not in any Lepi-
dopleurida examined to date (Hodgson et al., 1988; Pash-
chenko and Drozdov, 1998). The intermediate condition of
a prominent acrosome and short nuclear filament was re-
cently discovered in the lepidopleurid Deshayesiella cur-
vata (Pashchenko and Drozdov. 1998).

The term egg "hull" is used here instead of "chorion" as
in previous publications (Buckland-Nicks et al.. 1988a. b:
Buckland-Nicks, 1993. 1995), to describe the noncellular
envelope enclosing the egg and its vitelline layer, because
Richter ( 1986) showed that both the vitelline layer and the
hull of chitons are formed by the egg and not by the follicle
cells as in other Metazoa (for review of terminology, see
Eernisse and Reynolds, 1994).

Chiton eggs evolved from having smooth unspecialized
hulls, with fertilization presumably occurring anywhere on
the surface, to having elaborate spinous or cupulous hulls
that focus sperm to specific regions of the egg surface

J. BUCKLAND-NICKS AND A. N. HODGSON

FERTILIZATION IN CALLOCH1TON

(Buckland-Nicks, 1993. 1995). Among Lepidopleurida.
Leptochiton asellus has a smooth hull comprising a thick
homogeneous jelly coat (Hodgson et til., 1988). whereas D.
ciin'ata egg hulls are composed of a jelly coat that is
penetrated by regularly spaced pores (Pashchenko and
Drozdov, 1998). The mechanism of fertilization must be
quite different in these lepidopleurids compared to Chito-
nina such as Stenoplax conspicua (Buckland-Nicks. 1995).
or Acanthochitonina such as Tonicella lineata (Buckland-
Nicks et «/., 1988b). because of basic differences in sperm
acrosomes and egg hull structure. Sperm structure of dif-
ferent species has been found to be an accurate indicator of
phylogenetic relationship and sometimes is used as grounds
for reclassifying species to alternative taxa (Jamieson, 1987;
Healy. 1988; Hodgson et al, 1996).

The family Callochitonidae, although placed within the
order Chitonina and regarded as "evolutionarily advanced"
(Sirenko. 1997). exhibits several plesiomorphic traits
(Buckland-Nicks. 1995) and is therefore one of several
groups that are important to investigate in clarifying the
phylogeny of chitons. This study describes in detail, for the
first time in Callochiton castanens, the structure of the egg
hull and the mechanism of fertilization. The information
gained is discussed in relation to the evolution of mecha-
nisms of fertilization in chitons, as well as to chiton phy-
logeny in general.

Materials and Methods

Specimens of Callochiton castanens Wood, 1815, were
obtained in breeding condition from beneath intertidal rocks
at East London (33° 03' S; 28° 03' E) and Port Alfred (33°
52' S; 26° 53' E), South Africa, from August to October
1999. The animals were brought back to the laboratory and
placed individually in 60-mm petri dishes half-rilled with
0.45-;um filtered seawater (FSW). Some individuals of each
sex had spawned by the second day. Alternatively, eggs or

sperm were obtained by removing the foot and digestive
gland and puncturing the dorsal gonad.

If the specimen was male, the white "dry" sperm concen-
trate would ooze from the punctured testis and could be
easily aspirated into a pipette. For a "concentrated sperm
suspension." one drop of dry sperm was diluted in 5 ml of
FSW. For a "dilute sperm suspension." one drop of the latter
was further diluted in 5 ml of FSW. If the animal was
female any free-spawned eggs were collected from the petri
dish; otherwise the eggs were flushed from the ovary by
aspirating a stream of FSW into the gonad. Eggs were then
pipetted individually into another clean petri dish containing
FSW, to reduce debris. Batches of about 50 eggs were
removed into six small glass vials containing 4 ml of FSW.
Two vials served as unfertilized controls. Two vials re-
ceived three drops of diluted sperm suspension, and in two
vials the FSW was replaced with concentrated sperm sus-
pension. The eggs in one set of vials were transferred to
primary fixative (see below) after 30 s, and the eggs in the
second set of vials were fixed after 10 min.

Light microscopy

A few eggs were removed from control and experimental
vials during the experiment to monitor the progress of
fertilization and to be photographed using an Olympus
BX50F-3 light microscope equipped with bright field and
DIG optics. Slides were made by pipetting a few eggs onto
a clean glass slide, placing pieces of coverslip around to
make a well, then adding a whole coverslip, drying off
excess water, and finally sealing the coverslip with nail
varnish.

Sperm activity was noted to be highly variable. We tested
the effects of serotonin (5-hydroxytryptamine) on sperm
activity by mixing a drop of sperm concentrate with 1 ml of
serotonin solution, to give a final concentration ranging
from 1 to 10 jii/W serotonin. One drop of this sperm solution

Figures 1-6. Micrographs of unfertilized eggs of Callochiton castaneus.

Figure 1. Ripe egg with intact layer of follicle cells (FC) dissected from the ovary. Note regular arrangement
of pores in hull (arrows) visible beneath follicle cells. Scale bar = 100 /urn.

Figure 2. Light micrograph of l-/j.m section of egg removed from the ovary, showing a layer of intact
follicle cells (FC) and regularly spaced pores (P) in the jelly hull; the pores penetrate to the vitelline layer (VL)
overlying the egg membrane cups (MC). Note meshwork of fibers (arrowheads) supporting pore structure. Scale
bar = 10 /im.

Figure 3. Similar to Figure 1 except the egg has been rolled on sticky tape, which removed follicle cells and
pore openings (arrowhead), thus revealing pores in the jelly hull (arrows). Scale bar = 100 /Mm.

Figure 4. Close-up of a spawned egg in which the follicle cells have retracted, revealing the arrangement
of pores in naked jelly hull. Pore entrance (PE) is usually one-third of the diameter of the pore itself (double
arrow), thus restricting sperm entry. A sperm (Sp) is visible at the entrance to one damaged pore. Scale bar =
2 /^m.

Figure 5. Vitelline layer (VL) of the egg has been rolled off on sticky tape, revealing a regular series of
membrane cups (arrows) that match up with pores in the hull. Scale bar = 15 ^m.

Figure 6. Close-up of Figure 5, showing egg membrane cups with microvilli that are prominent on the raised
edges (arrows) but sparse in the bases of cups. Scale bar = 5 /j.m.

J. BUCK.LAND-NICKS AND A. N. HODGSON

was placed on a slide next to a separate drop of control
sperm in filtered seawater, and observed periodically.

were examined and photographed in a JEOL JSM 840
scanning electron microscope.

Electron microscopy

The primary fixative was made by mixing I ml of 25%
glutaraldehyde with 9 ml FSW and adding this to 10 ml of
0.2 M Na cacodylate buffer (pH 7.4). This gave a final
concentration of 2.5% glutaraldehyde in 0.1 M Na cacody-
late buffer and FSW. to which was added 0. 1 M sucrose.
The fixative was refrigerated before use. Samples were fixed
overnight and then washed in two changes of 0.1 M Na
cacodylate buffer in FSW (pH 7.4) before post-fixing for 1 h
in 1.5% osmium tetroxide in the same buffer. Fixed eggs
were rinsed in distilled water and dehydrated in an ethanol
series to 100%. At this stage about half of the eggs in each
vial were removed to a second series of vials containing
100% ethanol for preparation for scanning electron micros-
copy (SEM).

In samples destined for transmission electron micros-
copy, ethanol was replaced with propylene oxide and then
the eggs were transferred through infiltration media consist-
ing of mixtures of propylene oxide and TA AB 8 1 2/Araldite
CY212 resin (75:25. 50:50. 25:75) for 2 h in each mixture
in capped vials (after Cross, 1989). Samples were ex-
changed into pure resin and left overnight in uncapped vials
in a desiccator. The next afternoon, with the aid of a
dissecting microscope, groups of five to six eggs were
aspirated into BEEM capsules half filled with resin. Eggs
were allowed to sink and then were arranged into the center
of each mold using a stainless steel insect pin. Labels were
added, and the BEEM capsules were placed in a 60"C oven
for 40 h. Thick sections were cut with glass knives in an
LK.B 8800 ultratome, transferred to glass slides, and stained
with 1% toluidine blue for about 20 s before rinsing with
distilled water and air drying. Thin sections, with silver/gold
interference color, were cut on a diamond knife (Diatome)
and picked up on naked 150-mesh copper grids. Sections
were reverse stained with aqueous lead citrate for 1 min,
followed by aqueous uranyl acetate for 2 min, after the
method of Daddow ( 1986). Stained sections were examined
and photographed in a JEOL 1210 transmission electron
microscope operated at 80 kV.

Samples destined for SEM were exchanged through an
amyl acetate series to 100% and then aspirated under a
dissecting microscope into Teflon flow-through specimen
vials (Pelco) before capping and critical point drying. Sub-
sequently, individual Teflon vials were uncapped and in-
verted on an SEM stub coated with a carbon sticky tab. The
eggs stuck fast but could be rolled with an insect pin to
remove the egg hull and expose either the vitelline layer or
the egg membrane itself. Some eggs were cut in two with a
Kesei rnicroknife (Japan). Stubs prepared in this way were
coated with gold in a Polaron E5100 sputter coaler. Stubs

Results

Morphology of the egg

The unspawned ripe eggs of Callochiton castaneus are
about 220 ju,m in diameter and surrounded by a vitelline
layer and a smooth, 20-/u,m-thick egg hull enclosed by a
single layer of follicle cells, usually hexagonally disposed
(Figs. 1, 2). The egg hull is made up of a delicate jelly coat
supported by a fibrous matrix that is permeated by a series
of pores spaced at regular intervals of 9 ju,m all over the
surface (Figs. 3. 4). If unspawned eggs are rolled on sticky
tape to remove the follicle cells, the regular array of pores
in the jelly coat becomes visible (Fig. 3). At its entrance,
each pore is about 2 /urn in diameter (Fig. 4), but below this
the diameter enlarges to about 8 /-im (Fig. 2). Furthermore,
there is a network of fibers that criss-cross each pore from
apex to base (Fig. 2). Opposite, but below, the point at
which the pores contact the vitelline layer, the egg mem-
brane is formed into a series of cups (Figs. 5, 6). The
depression in each cup usually coincides with a pore, and
the lip of each cup coincides with the division between two
pores (Fig. 2). The raised edges of the cups are rich in
microvilli that penetrate into the vitelline layer (Figs. 2, 6);
in the base of the cups, microvilli are sparse (Fig. 7).

The mechanism of fertilization

Soon after spawned eggs contact seawater, any remaining
follicle cells retract, thus exposing the pores in the hull
(Figs. 4. 8). In dilute sperm suspensions, sperm quickly
locate the entrance to these pores and swim down to the egg
surface (Fig. 8). If a concentrated sperm suspension is used,
many sperm arrive at the egg surface simultaneously, over-
riding any potential block to polyspermy (Fig. 9). In these
cases the jellylike hull is dissolved in 1 or 2 min, leaving the
exposed vitelline layer of the egg coated with thousands of
penetrating sperm, many of which induce fertilization cones
(Figs. 9, 10). The fragile jelly coat degenerates in about an
hour even under natural conditions, and it was not preserved
intact by routine fixation.

When a fertilizing sperm penetrates the vitelline layer, the
needlelike nuclear filament bridges the perivitelline space
and egg cup to reach and fuse with the egg membrane (Figs.
1 1-13). The distance between the base of each egg cup and
the vitelline layer varies from 1.5 -4 /im (Fig. 2), but the
elongate nuclear filament permits sperm-egg fusion up to a
distance of 6 /urn (Fig. 12). Contact between sperm and egg
results in their fusion and the formation of a narrow tube (<
0. 1 /xm in diameter) through which the threadlike chromatin
is injected into the egg cortex (Fig. 14). This is visible as a
thin white thread when stained with Hoechst's 33358 DNA

FERTILIZATION IN CALLOCHITON

Figures 7-10. Micrographs of fertilized eggs of Callochiton castaneus: SEM = scanning electron micro-
graph: DIC LM = differential interference contrast light micrograph.

Figure 7. SEM of an unfertilized egg split in half with a Kesei microknife to show egg membrane cups
(arrowheads). The vitelline layer has been removed. Scale bar = 10 (nm.

Figure 8. SEM view of broken edge of the jelly hull, showing regular arrangement of pores above
(arrowheads) and penetrating sperm (Sp) on vitelline layer below. Scale bar = 2 ju.ni.

Figure 9. DIC LM of a polyspermic egg showing numerous fertilization cones (arrows) beneath the elevated
vitelline layer (VL). Scale bar = 70 /A in.

Figure 10. SEM of a polyspermic egg showing fertilization cones (arrowheads) beneath the vitelline layer,
which has been partly removed by rolling the egg on sticky tape. Note also numerous sperm (Sp) on region of
intact vitelline layer (VL). Scale bar = 10 /am.

stain and viewed under UV light (Fig. 12 inset). A fertili-
zation cone is raised up around the penetrating nuclear
filament from the surrounding egg cortex, as well as from
fusion of adjacent egg microvilli (Fig. 13). However, the
fertilization cone remains below the hairier of the vitelline

layer and engulfs only the nuclear filament (Figs. 12. 14).
The vitelline membrane raises up and forms the fertilization
membrane (Figs. 9, 12). The raised edges of the egg mem-
brane cups retract from the vitelline layer, creating a larger
(6-7 jam) peri vitelline space that excludes late-arriving

J. BUCKLAND-NICKS AND A. N. HODGSON

Figures 11-17. Micrographs of sperm and fertilized eggs of Ciillix'liiiuii ftiMnni'its: SEM = scanning
electron micrograph; TEM = transmission electron micrograph; DIG LM = differential interference contrast
light micrograph.

Figure 11. SEM of polyspermic egg that has been rolled on sticky (ape. stripping the vitelline layer (VL)
next to a penetrating sperm (Sp) and revealing the fertilization cone (FC) beneath it. Scale bar = 2 jj.ni.

Figure 12. DIG LM of polyspermic egg showing one sperm (Sp) that has penetrated the vitelline layer (VL)
and induced a fertilization cone, and a second, late-arriving sperm that cannot reach the egg membrane with Us
nuclear filament (arrowhead). Note also sperm flagellum (F) exhibiting large amplitude beat. Inset: Same as
Figure 12 except that the penetrating sperm has been labeled with Hoechst's 33358 DNA stain and photographed
under UV epifluorescence. revealing nucleus (N) injecting chromatm (arrowhead) into the egg cortex through the
fertilization cone (FC). Scale bar = 6 /urn.

Figure 13. TEM of a fertilization cone (FC) with part of a penetrating sperm (Sp) visible above the vitelline
layer (VL). Note that the fertilization cone incorporates elevated cytoplasm as well as microvilli (arrowheads).
Scale bar = 2 /am.

Figure 14. TEM of penetrating sperm injecting chromatin (arrowhead) into egg cortex. Note that the
vitelline layer (VL) is intact except for a small pore through it. Sperm mitochondria (M) and remnants of
glycogen (arrow) have collected in the membrane bag posterior to (he nucleus (N). Compare with Figure-Id.
Scale bar = 1 jum.

FERTILIZATION IN CALLOCHITON

sperm (Fig. 12). The sperm organelles — including the cen-
trioles, flagellum, and mitochondria, as well as some resid-
ual glycogen granules — collect in a bag of membranes
above the fertilization membrane and do not appear to enter
the egg cortex (Figs. 11, 14).

Morphology of the sperm

The sperm of Callochiton castaneus (described by Hodg-
son et ul., 1988) has a bullet-like nucleus extending into a
long nuclear filament tipped by a minute acrosome, but it
has retained a relatively unspecialized mid-piece (Figs. 15-
17). Hodgson et al. (1988) believed that C. castaneus sperm
did not possess an acrosome. Careful reexamination of their
sections, however, revealed the presence of the minute
acrosomal vesicle, which is separated from the nuclear
extension by a basal plate (Figs. 15, 17). It was not possible
to discern any subdivision of the acrosomal vesicle in this
species. The main body of the nucleus is 3 /J,m long, and the
nuclear filament is a further 6 /J.m. The mid-piece comprises
five oblong mitochondria arranged fairly symmetrically
around the centrioles (Figs. 15, 16).

Sperm dissected from some males were inactive even
after being placed in seawater. However, all became
active within 1 min following the addition of 1 ju,M
serotonin. The degree of activity increased with increas-
ing concentrations of serotonin, up to the maximum
tested concentration of 10

Discussion

Morphology of the sperm

Except in the suborder Lepidopleurina, the acrosome of
all chitons examined has been reduced to a small vesicle at
the tip of a needlelike nuclear filament, which is an exten-
sion of the main body of the nucleus (see review by Buck-
land-Nicks, 1995). Partial reduction of the acrosome is
noted among species of Lepidopleurina such as De-
shayesiellu cun'iitn (Pashchenko and Drozdov, 1998). How-
ever, sperm of a number of families in this basal suborder
have not been examined; these include Hanleyidae,
Choriplacidae, and Nierstraszellidae. Acrosomes are fully
reduced in Cullochitonidae, although the mid-piece of
sperm in this family has retained the primitive state, in
which mitochondria are symmetrically disposed around the
centrioles and there is no reinforcement of the flasellum

(Buckland-Nicks, 1995). In most Chitonina the mitochon-
dria are asymmetrically distributed around the centrioles
and, in addition, the flagellum is reinforced near the annulus
(Buckland-Nicks, 1995).

The activation of sperm by serotonin, which was ob-
served here for C. castaneus, has not been previously re-
corded in chitons, although this response is well known
from bivalves (Juneja et ul., 1993) and has also been noted
in limpets (Bucklund-Nicks and Howley, 1997; Buckland-
Nicks and Hodgson, unpubl. data). In bivalves, serotonin
has been shown to initiate sperm motility and egg matura-
tion, as well as to improve fertilization success (Juneja et
ul., 1993). This preliminary evidence for a function of
serotonin in chiton reproduction brings hope that induction
of spawning also may be possible. Until now, the inability
to predict spawning has hampered studies of chiton repro-
duction.

Morphology of the egg

In Chitonida the egg hull is resilient, easily preserved,
and elaborated into spines or cupules that not only slow the
sinking rate but direct sperm to specific locations on the egg
surface (Buckland-Nicks, 1993, 1995). The egg hulls of
most Chitonina have elaborate spines with narrow bases and
highly variable tips (Eernisse, 1984; Sirenko, 1993). Eer-
nisse ( 1984) first suggested developing independent charac-
ter sets based on hull spine structure and gill placement to
test the validity of phylogenies based solely on shell valve
morphology (Smith, 1960; Van Belle, 1983). Sirenko's sub-
sequent investigations proved that these characters (1993).
as well as variation in the articulamentum ( 1997) are useful
in the analysis of chiton phylogeny. The new sperm and egg
characters described here for C. castaneus will be important
in future cladistic analyses of the Chitonina because they
indicate that Callochitonidae are basal to Chitonina.

The egg hull of C. castaneus differs in some key respects
from that of other chitons studied; in particular, it is unlike
that of any other Chitonina. The vitelline layer is enclosed
by a fragile, smooth jelly coat that is permeated by large
pores. However, this type of hull may also occur in De-
shayesiel/a cun'uta (Lepidopleurina); drawings of this spe-
cies show a similar jelly coat containing regularly spaced
pores (Pashchenko and Drozdov, 1998), although no micro-
graphs of this feature have been published. In fertilization
experiments with polyspermic eggs of D. cun'ata, it was

Figure 15. SEM of sperm, showing acrosome (A) at tip of nuclear filament (NF). main body of nucleus (N).
mid-piece (MP). and flagellum (F). Scale bar = 1 /urn. Inset: TEM of apex of sperm revealing acrosomal vesicle
(AV) separated from nuclear filament (NF) by basal plate (BP|. Scale bar = 0.7 /j,m.

Figure 16. TEM of sperm nucleus (N) and mid-piece showing mitochondria (M), centrioles (C). and
glycogen granules (G). Note portion of nuclear filament (arrowhead). Scale bar = 0.7 ^im.

Figure 17. TEM of apex of sperm revealing acrosomal vesicle ( AV) separated from nuclear filament (NF)
by basal plate (BP). Scale bar = 0.7 /urn.

J. BUCKLAND-NICKS AND A. N HODGSON

observed that the jelly coat disintegrated, much like that of
C. castaneus (Buckland-Nicks and Sirenko, unpubl. re-
sults). This phenomenon of a fragile jelly coat is a plesi-
omorphy shared between Lepidopleurina and Callochi-
tonidae. which excludes all other Chitonina studied thus far.

In species that have egg hulls with closed cupules. sperm
penetrate the hull exclusively between the cupules, usually
where their hexagonal bases meet (Buckland-Nicks. 1995).
Eggs of some Acanthochitonina. such as Lepidochitona
dentiens and L. femuldi. have micropores in this region.
These permit easier access to the vitelline layer, although
sperm have two granules in the acrosome, suggesting that
the hull may still represent a barrier (Buckland-Nicks et al.,
1990). C. castaneus appears to have a simpler acrosome
structure, which would correlate with the provision of direct
access to the vitelline layer by large pores in the jelly hull.

In summary, C. castaneus gametes and those of the
Lepidopleurina share plesiomorphic characters such as a
simple arrangement of mitochondria in the sperm mid-piece
and a smooth egg hull. Yet C. custuneus shares the derived
characters of nuclear extension and reduction of acrosome
with all other Chitonida. A previous cladistic analysis of
chitons, which was largely based on sperm and egg char-
acters, predicted that Callochitonina was distinct from Chi-
tonina (Buckland-Nicks, 1995). The present study corrobo-
rates this prediction and indicates that C. castaneus is
unique and perhaps should be placed in a sister taxon to
both Chitonina and Acanthochitonina.

The mechanism of fertilization in chitons

Fertilization in C. castaneus and other Chitonida bears
some similarity to that in other molluscs, in the sense that a
sperm acrosome releases enzymes that digest a pore in the
egg envelope, enabling the inner acrosomal membrane to
fuse with the egg membrane (Buckland-Nicks et al.. 1988.
this study). However, in many other respects the mechanism
of fertilization in these chitons is highly derived when
compared with those of other molluscs and of metazoans in
general.

Firstly, there is no extrusion of an acrosomal process or
"perforatorium" by the polymerization of actin, as occurs
throughout molluscs and other metazoan groups (see review
by Tilney. 1985). Rather, in all members of Chitonida the
permanent needlelike nuclear filament has replaced the per-
foratorium (Buckland-Nicks et al.. 198Sb. 1990). The inter-
mediate condition of a short nuclear extension found in
Deshayesiella cunata (Pashehenko and Drozdov, 1998)
suggests that reduction in acrosome size among lepidopleu-
rids may be linked with an increase in the length of the
nuclear filament. Furthermore, all lepidopleurids examined
have a subacrosomal granule, which in other metazoans is
composed of actin for extruding the perforatorium during
fertilization.

A second important difference observed between Chi-
tonida and other molluscs is that the sperm organelles, as
well as most of the nuclear membrane, apparently remain on
the surface of the egg (Buckland-Nicks el al., 1988b; Buck-
land-Nicks. 1995; this study). No chiton sperm has been
observed becoming completely engulfed by a fertilization
cone. The probable reason for this is that the vitelline layer,
disturbed only by a minute pore permitting penetration of
the nuclear filament, remains a barrier to the envelopment of
the sperm by the fertilization cone. In other molluscs such as
bivalves, as well as in many other metazoans, the vitelline
layer is breached and the fertilization cone raises up through
it to engulf the entire sperm, including part of the flagellum
(see reviews by Tilney. 1985: Longo, 1987). In these spe-
cies there is an initial paternal contribution of centrioles and
mitochondria to the egg at fertilization, although, with the
exception of some bivalve molluscs (Hoeh et al.. 1991 ), the
paternal mitochondria degenerate and do not contribute to
the zygote. Furthermore, in sea urchins and some other
metazoans. a sperm centriole contributes to the movement
of the pronuclei as well as to the formation of the mitotic
spindle prior to first cleavage (see review by Gilbert, 1999).
Chitons may be unique among molluscs if. in addition to the
exclusion of paternal mitochondria, the centrioles that form
the mitotic spindle are also maternally derived. Confirma-
tion of this derivation will require appropriate labeling of
sperm centrioles and mitochondria before and after fertili-
zation.

To better understand how the mechanism of fertilization
has evolved in chitons, it will be important to examine
fertilization in a species like Leptocliiton iixellus, which has
a typical molluscan acrosome (Hodgson et al., 1988) and
perhaps a mechanism of sperm entry more similar to that of
limpets or bivalves.

Acknowledgments

We thank Robin Cross and Shirley Pinchuk for assistance
in setting up electron microscopes and ancillary equipment,
as well as Marvin Randall for doing most of the darkroom
work. We are grateful to Doug Eernisse for important and
helpful criticisms. This research was supported by a Hugh
Kelly Fellowship from Rhodes University and an NSERC
of Canada research grant, both to J.B-N.. as well as a
Rhodes University research grant to A.N.H.

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Early Development of Zooxanthella-Containing Eggs

of the Corals Pocillopora verrucosa and P. eydouxi

with Special Reference to the Distribution

of Zooxanthellae

M. HIROSE', R. A. KINZIE III2, AND M. HIDAKA1 *

1 Marine Environmental Science, Department of Chemistry, Biology and Marine Science, University of

the Rviikvus, Nishihara. Okinawa 903-0213, Japan; and 2 Department of Zoology and Hawaii Institute

of Marine Biologv. University of Hawaii, Honolulu. Hawaii 96822

Abstract. Some hermatypic corals spawn eggs that con-
tain zooxanthellae. We followed development of zooxan-
thella-containing eggs of two such species, Pocillopora
verrucosa and P. eydouxi. We also documented changes in
the distribution pattern of zooxanthellae during develop-
ment. Oocytes of both species took up zooxanthellae 3 to 4
days before spawning. At first, zooxanthellae were evenly
distributed in oocytes, but they later moved to the hemi-
sphere that contained the germinal vesicle. After fertiliza-
tion, early cleavage events were holoblastic, progressing by
furrow formation. The first cleavage furrow started at the
hemisphere that contained zooxanthellae. dividing the zoo-
xanthellate complement of the zygote about equally into the
two blastomeres. The second division divided each blas-
tomere into one zooxanthellae-rich cell and one with few
zooxanthellae. With continued cell division, blastomeres
containing zooxanthellae moved into the blastocoel. The
blastocoel disappeared at about 5 h after the first cleavage,
and the central region of the embryo was filled with cells
containing either zooxanthellae or lipid droplets, forming a
stereogastrula. Our results suggest that only blastomeres
that had been determined to develop into gastrodermal cells
receive zooxanthellae during cleavage. This determination
appears to take place, at the latest, by the second cell
division at the four-cell stage.

Received 22 October IWJ; accepted 17 May 2000.
* To whom correspondence should be addressed. E-mail: hidaka
@sci.u-ryukyu.ac.jp

Introduction

Reef-building corals harbor intracellular symbiotic
dinotlagellates, zooxanthellae, in their endodermal cells.
Some hermatypic corals acquire their symbionts from their
mother colony before fertilization (Kojis and Quinn, 1981;
Babcock and Heyward, 1986; Tomascik and Sander, 1987;
Yeemin, 1988; Glynn et al., 1991. 1994; Heyward et til..
1987; Kinzie. 1993, 1996; Sier and Olive, 1994; Kruger and
Schleyer, 1998). It is not known how zooxanthellae are
delivered to oocytes and how their distribution relates to
their eventual restriction to the endodermal cells in adults.
Early development of scleractinian corals has been de-
scribed in various species (e.g.. Szmant-Froelich ct a/..
1980, 1985; Babcock and Heyward, 1986; Harrison and
Wallace, 1990). However, early development of corals with
oocytes containing zooxanthellae has been described only in
the spawning species Montipora effusa (Yeemin, 1988) and
M. verntcoxa (Mate et al., 1998) and the brooding species
Porites porites (Tomascik and Sander, 1987).

Although zooxanthellae are generally restricted to the
gastrodermis of adult corals, they are at least temporarily
observed in the ectoderm of planulae of some corals and
soft corals (Szmant-Froelich, 1985; Benayahu ft al.. 1988;
Benayahu, 1997; Benayahu and Schleyer. 1998; Sehwarz et
al.. 1999). This is probably because infection first occurred
in the ectoderm cells of embryos or early planulae (Szmant-
Froelich ct al.. 1985) or because dividing cells of these
stages transferred the multiplying symbionts to their daugh-
ter cells, including presumptive ectoderm cells (Benayahu.
1997; Benayahu and Schleyer, 1998). In these cases, zoo-
xanthellae were transferred from ectoderm to endoderm

ZOOXANTHELLATE EGGS OF CORALS

across the mesoglea before larvae develop into mature plan-
ulae (Benayahu, 1997; Benayahu and Schleyer. 1998).
Montgomery and Kremer (1995) also found that in the
larvae of a scyphozoan, Linuche ungiiiculata. the algae were
found mostly in the ectodermal cells, and suggested mech-
anisms by which zooxanthellae could be transferred from
ectoderm to endoderm of planulae.

The corals Pocillopora eydonxi and P. verrucosa release
zooxanthellate eggs, which display an uneven distribution
of algal cells (Hirose et <//., unpubl. data). It is likely that, in
these corals, zooxanthellae are not equally delivered to all
daughter cells but go more or less exclusively to presump-
tive endoderm cells. If zooxanthellae become restricted to
endoderm cells during the course of development, the larvae
do not need to transfer the algae from ectoderm to endoderm
as described in the soft corals (Benayahu, 1997).

In the present study, we followed early development of
zooxanthellate eggs of the corals P. eydouxi and P. verrn-
cosa. We studied changes in the distribution pattern of
zooxanthellae during early development of the corals to
determine mechanisms by which the distribution of zoo-
xanthellae becomes localized to the endoderm of planulae.

Materials and Methods

Branches, 7-12 cm long, were collected from colonies of
Pocillopora verrucosa a few days before the new moon and
from P. eydouxi a few days before the full moon in June and
July 1998. Colonies were collected from reefs at Sesoko
Island. Okinawa. The branches were placed separately into
3-1 plastic containers supplied with unfiltered running sea-
water. The hermaphroditic colonies of P. verrucosa and P.
eydouxi spawned gametes for about 30 min in the early
morning a few days after the new moon, and a few days
after the full moon, respectively (Kinzie, 1993; Hirose et ul..
unpubl. data). Both species first released sperm and then
negatively buoyant eggs. To collect gametes, the supply of
seawater was stopped before the expected spawning time,
about 0630 h. After sperm had been shed, they were col-
lected by sucking up seawater from the container in a large
plastic pipette. The pipette was rinsed with a diluted hypo-
chlorite solution and then with seawater to avoid contami-
nation of gametes. Released eggs were collected by pipette
from the bottom of the container and placed in a plastic
beaker. Eggs were fertilized by mixing released gametes
( 100-300 ml suspension each) from two or three colonies in
a plastic beaker. Filtered (0.45 /u,m) seawater was added to
the beaker to make the final volume to 1 or 2 I. Fertilized
eggs were kept in the seawater at a room temperature
28°-30°C. Eggs and embryos were sampled and observed
under a light microscope at intervals of from 30 min to I h,
and photomicrographs were taken with a microscope
equipped with an epi fluorescent system (Nikon Microphot).

Histolog\ and transmission electron microscopy

Eggs and embryos were collected in a microtube and
allowed to settle to the bottom. The supernatant was then
discarded and fixative added. The specimens were fixed in
2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4)
containing 3% NaCl for 2 h or more. The specimens were
rinsed in the same buffer three times and post-fixed in 1 %
osmium tetroxide in the same buffer for 1 h on ice, dehy-
drated in a graded series of acetone, immersed in /(-butyl
glycidyl ether (QY1). and embedded in Spurr's resin. For
light microscopic observation, sections 0.5-1 /im thick
were stained with 1% methylene blue-1% azur II in 1%
borax. For electron microscopy, silver to gold sections were
stained with uranyl acetate and lead citrate and observed
under a JEOL JEM-2000EX electron microscope at an
acceleration voltage of 100 kV.

Results

Oocytes took up zooxanthellae 3 — I days before spawning
in both species. Zooxanthellae were first distributed evenly
in the ooplasm (Fig. 1A, B), but later. 1-2 days before
spawning, the algae became concentrated in the hemisphere
that contained the germinal vesicle. The other hemisphere
contained many lipid droplets of about the same size as the
zooxanthellae. Although the germinal vesicle was no longer
apparent by the time the eggs were spawned, the zooxan-
thellae remained concentrated in the hemisphere of the egg
that contained the nucleus (Fig. 1C, D). At spawning, eggs
of both species were about 140 ju,m in diameter and con-
tained about 130 zooxanthellae (Hirose et ai, unpubl. data).

Cleavage took place by progressive furrow formation
(Fig. IE), at intervals of 30 to 40 min. The first cleavage
furrow started at the hemisphere that contained the zooxan-
thellae and the oocyte nucleus, dividing the zooxanthellae
equally into two blastomeres, each with a roughly equal
complement of zooxanthellae still concentrated in the hemi-
sphere containing the nucleus (Fig. IF). At the second
cleavage, each blastomere was divided into one zooxanthel-
lae-rich blastomere and one with few zooxanthellae (Fig.
2A). By the 64- to 128-cell stage, some blastomeres in the
morula contained one or sometimes more zooxanthellae.
while others contained none (Fig. 2B). As cleavage pro-
ceeded, a blastocoel formed and blastomeres with zooxan-
thellae moved into this space (Fig. 2C, D). The outer layer
of the blastula consisted of relatively large blastomeres
containing no zooxanthellae. At about 5 h after fertilization,
blastomeres containing zooxanthellae, lipid droplets, or
both filled the blastocoel, resulting in a stereogastrula (Fig.
2E, F). Gastrulation appeared to occur by delamination
rather than by invagination.

Blastomeres in the outer layer had microvilli on their
outer surface and characteristic granules just below the cell
membrane (Fig. 3 A). Blastomeres in the outer layer were

M. HIROSE ET AL

Figure 1. Early development of Pocillopora vermcosa: from unfertilized egg to two-cell stage. (A) Oocyte
isolated from the gonad. Zooxanthellae are distributed evenly in the cytoplasm. The germinal vesicle is at the
center of the oocyte. (B) Oocyte viewed under epifluorescence (BV excitation). The red fluorescence is due to
algal chlorophyll. Cytoplasm of the oocyte exhibits blue-green autofluorescence. (C) Spawned egg. Zooxan-
thellae are mainly located in the right hemisphere and lipid droplets in the left hemisphere. (D) The same egg.
observed under epifluorescence (BV excitation). (E) First cleaving stage. Cleavage furrow (arrow) starts at the
hemisphere that contains the /.ooxanthellae. (F) Two-cell stage. Zooxanthellae are divided equally into the two
hl.isii'ineres. Bars = 100 fim.

connected to each other by the contact junctions near the
apical surface (Fig. 3B). In other regions of the interface,
blastomeres were only loosely attached to each other or
were separated by extracellular space. Villi-like cellular
processes were observed in the extracellular space, and

those from neighboring blastomeres were often intermin-
gled (Fig. 3C). When blastomeres in the outer layer con-
tained Zooxanthellae, the /ooxanthellae were usually at the
lower or lateral margin of the blastomere. Zooxanthellae at
the lateral margin of the blastomere bulszed into the extra-

ZOOXANTHELLATE EGGS OF CORALS

Figure 2. Early development of Pocilloptmi verrucosa: four-cell stage to gastrula. (A) Four-cell stage. The
second cleavage plane was normal to the first cleavage plane, thus dividing the blastomere into a zooxanthellae-
rich blastomere and a lipid-droplet-rich blastomere. (B) Morula-stage embryo. Blastomeres are round; some
contain a single zooxanthellae. (Cl Blastula. Zooxanthellae are still restricted to one hemisphere. (D) Section of
a blastula. Blastomeres containing one or more zooxanthellae (arrow) and those containing lipid droplets are
located in the blastocoel (be), while the surface layer is composed of larger blastomeres with no algae. (E)
Gastrula. The center of the gastrula appears dark due to accumulation of zooxanthella-containing blastomeres.
(F) Section of a gastrula. Blastomeres containing zooxanthellae and those containing lipid droplets fill the inner
space of the gastrula. forming a stereogastrula. Bars = 100 /urn.

M. HIROSE ET AL.

Figure 3. Electron micrographs of an early gastrula of Pot-illo/rum vt'iriicuxti. (A) Blastomeres in the outer
layer of an embryo. (B) Higher magnification of the boxed area in (A), showing contact junction near the apical
surface (arrow). (C) Blustomere in the outer layer containing zooxanthellae. Zooxanthellae bulge into the
neighboring blastomere. (D) Two zooxanthellae in a cellular process, which appears to be still connected to the
ouier layer hlastomere. (E) Zooxanthellae surrounded by a small amount of host cytoplasm. (F) Lipid droplet
surrounded by a small amount of cytoplasm, be = blastocoel. bl = blastomere. cp = cytoplasm, li = lipid
droplet, mv = microvilli. zo = zooxanthella. Bars = 5 /J.m except in (Bl, where bar = 1 /nm.

cellular space and sometimes into neighboring blastomeres
(Fig. 3C). Similarly, /ooxunthellae or lipid droplets located
at the lower margin of the hlastomercs bulged into the

blastocoel. In such cases, a constriction was often observed
between the central cytoplasm and the protrusion containing
a /ooxanthella or a lipid droplet (Fig. 3D). Most zooxan-

ZOOXANTHELLATE EGGS OF CORALS

Figure 4. Planula of Pocillnpora verrucosa 24 h after fertilization. (A) Photomicrograph of a fixed planula
taken under differential interference optics. The planula is completely ciliated at this stage. (B) Histological
section of a planula. Zooxanthellae and lipid droplets are in the endodermal cells. (C-D) Photomicrographs of
the body wall of a planula taken under oil immersion. The ectoderm and endoderm are clearly separated by the
mesoglea (arrowhead). The ectoderm consists of columnar cells, c = ectoderm, g = gastrovascular cavity, m =
mouth opening, n = endoderm. Bars = 100 ;xm in (A) and (B), 20 fj.m in (C) and (D).

thellae and lipid droplets in the blastocoel were surrounded
by a small amount of cytoplasm and appeared to be free —
that is, detached — from blastomeres in the outer layer (Fig.
3E, F).

Spherical embryos with a smooth surface as shown in
Figure 2E and F were observed 6 h after fertilization.
Ciliated larvae started to swim 8 h after fertilization. The
embryos became elliptical and swam spirally by 9-10 h
after fertilization. An oral pore was formed by invagination
of the epidermis, and a gastrovascular cavity was formed as
gastrodermal cells became organized 24 h after fertilization
(Fig. 4A, B). At this stage the ectodermal layer — the plan-
ula's epidermis — consisted of characteristic columnar cells,
and the epidermis and gastrodermis were separated by dis-
tinct mesoglea (Fig. 4C, D). Embryos at this stage were
typical planulae. Generally, only gastrodermal cells con-
tained zooxanthellae, though a few zooxanthellae were ob-
served in ectoderm of some planulae. Planulae 48 h old
possessed some nematocysts.

Discussion

Although early development of scleractinians has been
described (e.g., Szmant-Froelich et al., 1980, 1985; Bab-

cock and Hey ward, 1986; Harrison and Wallace, 1990), this
is the first report describing the processes by which zoo-
xanthellae become restricted to the endoderm during the
course of embryogenesis. In the two Pocillopora species
studied, regions of egg cytoplasm are differentiated and cell
fates are apparently decided early in development, possibly
before fertilization. Zooxanthellae moved toward the animal
pole 1-2 days before spawning. The first cleavage appor-
tioned zooxanthellae more or less equally between the first
two blastomeres. At the second cleavage, however, two of
the four blastomeres received almost all the zooxanthellae,
while the other two had few or none. This uneven distribu-
tion of zooxanthellae persisted until the zygotes developed
into gastrulae.

As cleavage progressed, relatively large blastomeres
without zooxanthellae came to occupy the outer layer of the
embryo as the blastocoel opened. Later, blastomeres con-
taining zooxanthellae or lipid droplets detached from the
outer layer and dropped into the blastocoel until it was filled
with blastomeres containing zooxanthellae and lipid drop-
lets. In these two species of Pocillopora, gastrulation may
occur due to delamination rather than invagination, result-
ing in a stereogastrula. Gastrulation through delamination

M. HIROSE ET AL

has been suggested for Astrangia danae (Szmant-Froelich el
til.. 1980). Faviafragum (Szmant-Froelich el a!., 1985), and
Montipora rerrucosa (Mate el at., 1998).

Titlyanov et al. (1996, 1998) observed degraded zooxan-
thellae in planulae as well as in adult polyps of hermatypic
corals and suggested that digestion of zooxanthellae occurs
both in planulae and in adult polyps. We saw no such
degraded zooxanthellae in the surface layer of embryos or
early planulae. If zooxanthellae are not digested during
early development, they must be transferred from blas-
tomeres that are determined to develop into symbiont-free
ectodermal cells to blastomeres that are fated to develop
into algae-bearing endodermal cells. This ontogenetic redis-
tribution of algae might occur in several ways. One possi-
bility is that zooxanthellae move basally within blastomeres
so that subsequent horizontal cell division results in surface
ectodermal cells and centrally located endodermal cells that
contain zooxanthellae. Our observations suggest that zoo-
xanthellae, along with small amounts of cytoplasm, were
separated from surface cells and dropped into the blastocoel.
This process is similar to the "pinching off suggested for
the transfer mechanism of zooxanthellae from follicle cells
to oocytes and from ectoderm to endoderm in some soft
corals (Benayahu, 1997; Benayahu et al., 1992; Benayahu
and Schleyer, 1998). However, the small "blastomeres"
containing zooxanthellae (Fig. 3E, F) could also be pro-
duced by unequal division rather than by pinching off. If
this were the case, there should be animal nuclei in these
structures. These basally derived cells would then develop
into gastrodermal cells. Another possibility is that ectoder-
mal cells expel zooxanthellae by exocytosis and adjacent
endodermal cells take them up by phagocytosis. The obser-
vation that zooxanthellae within vacuoles of the blastomere
in the outer layer often protruded to the intercellular space,
bulging into the neighboring cell, suggests that this possi-
bility cannot be ruled out.

Few if any zooxanthellae were found in the outer layer of
gastrulae or the ectoderm of planulae of the two Pocillopora
species studied. However, zooxanthellae are sometimes
found in the ectoderm of early planulae of some corals
(Faviafragum: Szmant-Froelich el al.. 1985; Fiingiu scii-
taria: Schwarz ct til.. 1999), soft corals (Xeiiiu iimbellata:
Benayahu et al., 1988; Litophytoii tirhoreiim: Benayahu et
al.. 1992; Benayahu. 1997; Ant/ieliu glauca: Benayahu and
Schleyer. 1998), and the scyphozoan Linuche unguiculata
(Montgomery and Kremer, 1995). It has been suggested
that, in the early developmental stages, zooxanthellae show
no specificity towards presumptive endodermal cells
(Benayahu, 1997; Benayahu and Schleyer, 1998). However,
as planulae develop, zooxanthellae are found increasingly in
the endoderm and eventually become restricted to the gas-
trodermis of polyps. Several mechanisms by which the
algae are translocated from ectoderm to endoderm have
been suggested (Montgomery and Kremer, 1995; Benayahu,

1997; Benayahu and Schleyer, 1998). Montgomery and
Kremer (1995) suggested that ectoderm cells infected by
zooxanthellae may migrate to the endoderm of planulae.
Benayahu (1997) and Benayahu and Schleyer (1998) ob-
served that, in the soft corals they studied, zooxanthellae
pass through temporarily opened gaps in the mesoglea to-
wards the endoderm. Throughout the process, each zooxan-
thella resides within a vacuole in the detached ectodermal
cytoplasm. However, we did not observe such a transfer of
zooxanthellae from ectoderm to endoderm in planulae of the
two Pocillopora species. In these corals, zooxanthellae ap-
peared to be transferred more or less exclusively to blas-
tomeres that were fated to develop into endodermal cells.
This suggests that determination of presumptive endoderm
cells and specificity of zooxanthellae towards presumptive
endoderm cells occur earlier in the two Pocillopuni species
than in the soft corals studied.

We described changes in the distribution of zooxanthellae
during early development as well as during final maturation
of oocytes in the corals Pocillopora verrncosa and P. ey-
douxi. Zooxanthellae moved to the hemisphere of the oo-
cyte that contained the germinal vesicle 1 to 2 days before
spawning. Zooxanthellae moved to the lateral or basal mar-
gins of the surface blastomeres and bulged into extracellular
spaces or into the blastocoel. Blastomeres containing zoo-
xanthellae or lipid droplets along with a small amount of
cytoplasm were produced, probably by unequal mitotic di-
vision, and then dropped into the blastocoel and became
endodermal cells. It is not clear whether the presence of
zooxanthellae affects development of the blastomere or if
the fate of a blastomere is determined by the nature or
quantity of its cytoplasm. Further study is necessary to
understand how zooxanthellae move to a region of oocytes
and to certain areas of blastomeres.

Acknowledgments

We thank the staff of Sesoko Station. Tropical Biosphere
Research Center, University of the Ryukyus, where part of
this study was done. Y. Nozawa, N. Takahashi, and W. Diah
Permata kindly helped us. This study was partly supported
by the Grant-in-Aid for Scientific Research No. 08644216
and 11694223 from the Ministry of Education, Science,
Sports and Culture. Japan, and by the Sasagawa Scientific
Research Grant from the Japan Science Society.

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Revised Description of the Fine Structure of in situ
"Zooxanthellae" Genus Symbiodinium

TIMOTHY S. WAKEFIELD1 *, MARK A. FARMER2, AND STEPHEN C. KEMPF1

^Department of Biological Sciences, Auburn University, Alabama 36849-5414: and * Center for
Advanced Ultrastructural Research, University of Georgia, Athens, Georgia 30602

Abstract. The fine structure of the symbiotic dinoflagel-
late genus Symbiodinium has been well described. All of the
published descriptions are based on tissue that was fixed in
standard aldehyde and osmium fixatives and dehydrated in
an ethanol series before embedding. When the technique of
freeze-substitution was used to fix tissue from Cassiopeia
xamachana, Aiptasia pallida, and Phyllactis flosculifera and
prepare it for embedding, thecal vesicles were revealed
within the in situ symbionts of all three species. Although
these structures have been identified in cultured symbionts,
they have never been described in the in situ symbionts. A
review of the literature has revealed several instances where
thecal vesicles were either overlooked or identified incor-
rectly. Thus the formal description of the genus Symbio-
dinium, which describes the in xitu symbionts. contains
information that is based on artifact and should be revised.
A revision of the genus is suggested, and the true nature of
these structures and their significance in the symbiotic as-
sociation are discussed.

Introduction

The symbiotic algae found in the tissues of numerous
marine invertebrates and often called "zooxanthellae" have
included members of the classes Bacillariophyceae, Cryp-
tophyceae. Dinophyceae, and Rhodophyceae (Rowan,
1998). In recent years, common usage has relegated this
term almost exclusively to the description of dinoflagellate
symbionts. Loeblich and Sherley (1979) suggested that
many of the ta.xonomically described dinoflagellate symbi-
onts should be assigned to a new genus they termed Zoo-
Minthella in recognition of very early work on dinoflagellate
symbiosis by Brandt ( 1SSI >. However, continued studies in

Received 27 September 1999; accepted 18 April 2000.
* To whom correspondence should he addressed.

symbiotic dinoflagellate taxonomy have shown that there
are. in fact, many species from several genera including
Amphidiiiiinn (Taylor, 197 la; Trench and Winsor. 1987),
Aureodinium (Anderson and Be, 1976), Gymnodinium
(Spero. 1987). Gyrodiniimi (Spindler and Hemleben, 1980),
Prorocentrum (Yamasu, 1988), Pyrocystis (Alldredge and
Jones, 1973), Scrippsiella and Gloeodinium (Banaszak et
<//.. 1993). The most frequently occurring of these is the
gymnodinioid-like dinoflagellate assigned to the genus Sym-
hiodiiiiiim. the alga commonly found in a variety of marine
invertebrates such as tridacnid clams, certain nudibranch
molluscs, reef-building corals, other anthozoans, and scy-
phozoans.

The first formal description of the dinoflagellate genus
Symbiodinium was published by Hugo D. Freudenthal
( 1962). This work provided a detailed histological descrip-
tion of the species S. microadriaticum that had been ex-
tracted and cultured from a scyphozoan host described as
Cassiopeia sp. In 1968 Taylor published the preliminary
ultrastructural description of dinoflagellate "zooxanthellae"
from Anemonia snlcata. The author stated that the symbi-
onts were "akin" to S. microadriaticum, but that the status
of any relationship would remain unknown until an ultra-
structural study was performed on the symbionts ot Cassio-
peia. Kevin ci al. ( 1969) provided this information in their
ultrastructural examination of S. microadriaticum from the
hosts Cassiopeia sp. (Scyphozoa) and Condylactis sp. (An-
thozoa). They stated that the symbionts from these two
genera were the same species as those seen by Taylor, and
concluded by amending Freudenthal's formal description to
include the ultrastructural data. Taylor agreed with their
conclusions and later identified the dinoflagellates from
several species of Tridacnidae as S. microadriaticum (Tay-
lor, 1969). In 1987, Trench and Blank published the most
recently amended formal description of the genus Symbio-

REVISED DESCRIPTION OF SYMBIODINIUM

dininm, and described three new species in the genus. For
the most part, all four of these papers describe similar
ultrastructural features. This is not surprising considering
that both Kevin and Trench describe S. microadriaticum
from its definitive host Cassiopeia, and that all four used a
standard chemical fixation that included buffered glutaral-
dehyde and osmium followed by a dehydration series in
preparing their tissues for the transmission electron micro-
scope.

In the following paper, we further amend the ultrastruc-
tural description of the genus Symbiodinium on the basis of
the use of freeze-substitution, an alternative fixation tech-
nique that provides better preservation of fine structure. The
symbionts from three hosts were examined: two anthozo-
ans — Aiptasia pallida (S. bermudense) and Plivllactis
flosculifera (Symbiodiniiini sp.) — and the definitive scypho-
zoan host Cassiopeia xamachana (S. microadriaticum).
This has resulted in a more clearly resolved ultrastructure
revealing previously undescribed features in the in situ
symbionts. These results suggest an alternative hypothesis
for the origin of the multilayered "periplast" or "symbio-
some membrane" that is prevalent around the dinoflagellate
symbionts found in invertebrate hosts.

Materials and Methods

Animal collection and care

Specimens of Cassiopea xamachana, Aiptasia pallida.
and a second anthozoan species identified as Phvllactis
flosculifera (Fautin, University of Kansas, pers. comm.)
were collected in the Florida Keys and transported to Au-
burn University, Alabama. These species were maintained
in established 150-gallon aquaria filtered with both under-
gravel and external trickle filtration. Artificial seawater was
prepared with Reef Crystals (Aquarium Systems) and
deionized water at about 30 ppt. The aquaria were main-
tained at 25°-30° C on a 12/12 h light/dark cycle, and the
animals were fed with newly hatched brine shrimp nauplii
every other day.

Fixation and embedding

Antho-oan procedure. Individual specimens of A. pallida
and P. flosculifera were transferred to small glass bowls
containing 0.45-/im Millipore-filtered artificial seawater
(MFAS) and were allowed to acclimate, attach to the glass
substratum, and fully expand. Small pieces of tentacle (<10
mm in length, but very thin) were excised from individuals
of both species and suspended from small lengths of plati-
num wire. The tissues were then plunge-frozen in liquid
propane (Menco, 1986) and transferred to liquid nitrogen.
The frozen tissues were transferred to glass vials containing
a 2% osmium tetroxide-acetone solution that had been pre-
viously cooled to -80° C. The vials were held within a

precooled. methanol-saturated sponge, and the tissue sam-
ples were left at -80° C for 48-72 h before being transferred
to -20° C. Specimens remained at -20" C for 12-24 h and
were then moved to 4° C for an additional 1 2-24 h before
being returned to room temperature. While the specimens
were being transferred from -80° C to room temperature, a
gradual warming was achieved by moving the entire meth-
anol-saturated sponge containing the vials from one envi-
ronment to another. After reaching room temperature, the
tissue samples were removed, and the platinum wire was
discarded. Tissues were washed in 100% acetone and were
either transferred to 100% propylene oxide before infiltra-
tion, or were immediately infiltrated and embedded in Poly-
Bed 812 plastic.

Scyphozoun procedure. Several attempts were made to
fix Cassiopeia xamachana symbionts in situ using the pro-
cedure described above, but all resulted in very poor fixa-
tion. We speculate that the copious amounts of mucus
produced by the scyphozoan tissue acted as an insulator
against the rapid freezing of the tissue. Thus the following,
more successful, procedure was used. Six large specimens
of C. xamachana were transferred to a large bowl of MFAS.
The oral arms of each specimen were excised and discarded.
With a glass slide, the external symbiotic layer of tissue
from the oral side of the specimen's bell was scraped free
from the underlying mesoglea and collected. The remaining
inesoglea and aboral ectoderm was discarded. The collected
symbiotic tissues were homogenized in MFAS using a
Virtis Handishear tissue homogenizer. The tissue homoge-
nate was centrifuged at —250 X g for 3 min to pellet the
separated symbiosomes, and the supernatant was discarded.
Small ( - 1 mm2) samples of the pelleted symbiosomes were
placed on thin strips of filter paper and then immediately
plunge-frozen, fixed, and embedded in the manner de-
scribed above for the anthozoan tissue.

Standard fixation and embedding. As a control for the
ultra-rapid freezing and freeze-substitution fixation process,
a standard aldehyde-osmium fixation at room temperature
was performed on each of the three symbiotic cnidarian
tissues. Briefly, small pieces (<10 mm in length) of tentacle
from symbiotic A. pallida and P. flosculifera, as well as
small portions (<10 mm2) of the bell margin from C.
.xamachana. were removed from living specimens and im-
mersed in 2.5% glutaraldehyde in 20 mA/ Millonigs phos-
phate buffered saline (MPBS), pH 7.6. The tissue was
allowed to fix for 1 h. rinsed twice for 5 min in 20 mM
MPBS. and then postfixed in 2% OsO4 in 1.25% NaHCO,
for 1 h. Residual OsO4 was rinsed away in two 5-min rinses
of 1.25% NaHCO,. Tissues were then dehydrated in an
ethanol series (30%, 50%. 70%, 85%, 90%, 95%, 3 X
100%). followed by three rinses in propylene oxide ( 10 min
in each solution), all at room temperature. Tissues were then
infiltrated and embedded in Poly-Bed 812 plastic in the
same manner as the freeze-substituted tissues.

T. S. WAKEFIELD ET AL.

Sectioning mul .^tuining

Ultrathin sections of each kind of tissue were cut with a
Reichert-Jung Ultracut E ultramicrotome and collected on
Formvar-coated slot grids. Ultrathin sections were stained
with lead citrate and uranyl acetate and were examined on a
Zeiss EM- 10 transmission electron microscope (TEM).

Measurements

Cell wall layers and thecal vesicles were measured di-
rectly from TEM negatives with a hand lens, light box. and
metric ruler. A total of 100 thecal vesicles were measured
for both Symbiodinium bermudense (Aipiasiu pallida) and
Symbiodinium sp. (Phyllactis flosculiferd). In those symbi-
onts that contained more than 10 vesicles, only 10 were
measured. Other symbionts did not contain 10 clearly de-
fined vesicles; thus a total of 1 5 5. bermudense and 1 3
Symbiodinium sp. were used to obtain the 100 total vesicles
measured.

Even using the alternative method, thecal vesicle preser-
vation for S. microadriaticum from Cassiopeia xamachana
was still inferior to that of the symhionis from the other
hosts. In this species, only those thecal vesicles that could
be clearly identified were measured, resulting in a lower ";/"
value from 26 cells for the vesicles measured in S. inicroud-
riaticum. Cell wall layers (S. bermudense) and total cell
wall thickness (all symbiont species) were measured as
described above. Conversions from millimeters to microme-
ters or nanometers were made using a published nornogram
(Ghadially et al, 1481).

Results

Many aspects of the ultrastructure of freeze-substituted in
situ symbionts show good correspondence with previously
published descriptions of the genus Symbiodinium (Taylor,
1968, 1969; Kevin et al.. 1969; Trench and Blank, 1987).
The multi-lobed chloroplast is peripherally located, with
parallel rows of thylakoids usually arranged in groups of
three. A single, stalked pyrenoid may be seen emerging
from the chloroplast. Its membrane appears to be continuous
with the chloroplast envelope with no invading thylakoids,
and a relatively thick starch coat surrounds the entire struc-
ture. A single large, irregularly shaped, granular accumula-
tion body is present in many cells. Typical dictyosomes and
mitochondria also occur, as do many lipid vacuoles and
some calcium oxalate crystals. Large, well-preserved "fi-
brous bodies" are present in many of the symbionts (Dodge.
1967). The nucleus of the symbionts is distinct and recog-
nizable, but the compact spirals of chromatin typically seen
in the mesokaryotic nucleus are not always visible.

The freeze-substitution process did. however, reveal
some previously undescribed features of the in situ symbi-
onts, as well as differences among Symbiodinium species.

The "amorphous layer" of the periplast described by Kevin
et (//. ( 1969) has been correctly identified as the continuous,
vegetative cell wall of the symbiont (Trench and Blank.
1987). However, our results (Fig. 1) indicate that in Sym-
biodiniitm bennudense this structure is in fact composed of
three distinct layers: an outer layer that appears to be mem-
branous (OL), an electron-dense middle layer (EDL). and a
less dense inner layer (IL). As can be seen in Table I, the
greatest variation in layer width was seen in the IL, which
ranged from 30 to 300 nm. This variation is evident in
nonmitotic cells (Fig. 2), but it is most common in recently
divided cells, where the OL and EDL retain similar thick-
ness circumferentiallv around the divided cells, while the IL

ch _

Figure 1. S\nihi<>Jiiunin hciinuJciL'.c within the host Ai/'imui /><////</<(.
The cell «all nl the symbiont shows three distinct regions beneath the
multiple layers of membranous material that surround it. ch = chloroplast;
EDL = electron dense cell wall layer; IL = inner cell wall layer; ml =
multiple layers of symbiosome membrane; OL = membranous outer layer;
pm = algal cell plasma membrane; sm = outer symbiosome membrane;
arrow = thecal plate; arrowheads indicate thecal vesicles. Scale bar =
100 nm

Figure 2. S\mhn>Jininm hcriinuli'ii.w within host Aiptu.iiu pulliilu.
Note the difference in thickness of the cell wall inner layer between points
a and b; ch = chloroplast; arrowheads indicate accumulations of symbio-
some membrane outside of vegetative cell wall. Scale bar = 500 nm.

Figure 3. Dividing Symbiodinium hcnmulfiixc within Aipiasia pa/lulu.
ch = chloroplast; nti = nucleus; arrowheads indicate areas of thickening of
inner layer of the cell wall along the division furrow. Scale bar = 1 /nm.

REVISED DESCRIPTION OF SYMBIODINIUM

Table 1

Cell wall layer thickness, total cell wall thickness, and ihecal vesicle thickness lull measurements in nanometers, mean ± sttl. dev.t

Symbiont species IL EDL

OL Total thickness (in

Thecal vesicle thickness («)

Symbiodinium sp.
S. microadriaticum
S. bermudense 96.7 ± 4S.4 40.4 ± 7.7

137.8 ± 65.5(100)
IOS.4 ± 37.7(100)
10.6 ± 1.4 147.7 ± 49.9(100)

34.3 ± 7.8(100)
38.8 ± 7.3 (50)
34.9 ±8.5(100)

EDL = electron dense layer. IL = inner layer, OL = outer layer; — indicates no visible layers.

becomes increasingly thicker in the region of the division
furrow (Fig. 3). Symbiodinium sp. from P. flosculifera and
S. microadriaticum lacked this three-layered structure, the
entire cell wall being similar in consistency to the IL layer
of S. bermudense.

A typical multilayered symbiosome membrane could be
seen around the symbionts of all three host animals, but was
most prevalent around S. bermudense (Figs. 1, 2, 4). The
membranes that completely surrounded most symbionts
seemed to be distributed evenly, but in some cases a dis-
proportionate number of membranes were located to one
side of the symbiont (Figs. 2. 4).

*•• 9

\\ ' "

Figure 4. Symbiodinium bermudense within Aiptasia pallida. ac =
accumulation body; ch = chloroplast; li = lipid vacuole; nu = nucleus;
PV = pyrenoid; arrowhead indicates large number of membranes on only
one side of symbiont. Scale bar = 2 fj.m.

Figure 5. Thecal vesicles in Symbiodinium bermudense. ch = chloro-
plast; arrowheads indicate individual thecal vesicles. Scale bar = 500 nm.

Figure 6. Symbiodinium sp. within the host Phylactis flosculifera.
ac = accumulation body; ch = chloroplast; nu = nucleus; py = pyrenoid.
Scale bar = 1 jj.ni.

Figure 7. Thecal vesicle in Symbiodinium sp. from Ph\lactis floscu-
lifera. ch = chloroplast; cw = cell wall; arrow = algal cell plasma
membrane; arrowhead = thecal vesicle. Scale bar = 300 nm.

Just inside of the cell wall is the continuous cell mem-
brane. Below this cell membrane, the freeze-substitution
process has revealed distinct thecal vesicles. These vesicles
are most prominent in Symbiodinium bennudense (Figs. 4,
5) and the Symbiodinium sp. (Figs. 6, 7), but they are also
present in S. microadriaticum (Figs. 8, 9). In TEM sections,
each vesicle is membrane bound and has rounded edges at

•\

Figure 8. Symbiodinium microadriaticum from the host Cassiopeia
\iiinachana. ch = chloroplast; nu = nucleus. Scale bar = 500 nm.
Figure 9. Thecal vesicles in Symbiodinium microadriaticum. ch =

chloroplast; co = calcium oxalate crystal; cw = cell wall; arrowheads
indicate thecal vesicles. Scale bar = 100 nm.

Figure 10. SynibioJiiiitim heriiuulcine within host Aiptasia palliJa.
ch = chloroplast: cw = cell wall; ml = multiple layers of symbiosome
membrane; sm = outer symbiosome membrane; arrows identify thecal
vesicles; arrowheads indicate linear array of microtubules beneath thecal
vesicles. Scale bar = 300 nm.

T. S. WAKEFIELD ET AL.

Figure 11. Svmbioilininin bermiulfiixi' within heist .4</>/i/.w'</
Standard glutaraldehyde-osmium tissue fixation. Note scrolled membrane
in region where thecal vesicles should be (arrowheads), ch = chloroplast;
cw = cell wall: ml = multiple layers of symbiosome membrane; arrow-
heads identify area where thecal vesicles should be located. Scale bar =
200 nm. Inset (a) is a higher magnification of an additional symbiont
showing layered membranes internal to the cell wall. Scale bar = 100 nm.

each end, with a distinct cytoplasmic separation between
adjacent vesicles. Internal thecal plates were apparent
within some vesicles of S. benmulense (Fig. 1. arrow). The
enclosed thecal plate lies separate from the vesicle mem-
brane. The overall thickness of the vesicles for each species
is reported in Table 1 . Since serial sections of an entire cell
were not cut, we could not determine whether the differing
widths of the vesicles reflect differences among species,
large and small vesicles, or different vesicle profiles. In
several micrographs, a linear array of microtubules was
present underlying the vesicles (Fig. 10).

Standard aldehyde-osmium fixed tissues of all three spe-
cies of symbiont failed to show many of the fine structures
seen in the freeze-substituted tissue. The multiple-layered
symbiosome membrane was apparent; but only the OL and
the IL of the cell wall were seen in S. benmulense. and all
three species failed to show any thecal vesicles or plates.
Instead, there appeared to be several layers of membrane
beneath the vegetative cell wall (e.g.. Fig. 11, 1 la).

Discussion

A multiple-layered cell wall in Symbiodiniwn
dcnse has been previously described by Palincsar et al.
( 1988; they identified the symbiont of Aiptcixin pulliilii as S.
inifrodtlriiiticiim at 'hat time; however, the species has
subsequently been rcdescribed as S. bermudense [see
Trench. 1993]). The description by Palincsar et al. (1988)

included four layers. From the inside out. the layers were
described as a vesicular layer immediately outside the sym-
biont' s plasma membrane; a thick homogeneous finely
granular layer; and a "line-thin" dark layer, overlaid by an
even thinner membranous layer. They also reported a wide
variation in cell wall structure, with some cells displaying
an almost entirely homogeneous, finely granular wall and
others an almost entirely vesicular wall. The presence of
multiple-layered cell walls is not surprising since similar
cell wall features have been identified in other dinoflagellate
species (Morrill and Loeblich, 1981). Bricheux et al. (1992)
identified a four-layered pellicle in the free-living
dinoflagellate Glenodiniiim foliaceum. The two layers im-
mediately outside of the cell membrane (identified as the
homogeneous layer and the dense layer) strongly resemble
the IL and the EDL we see in species of Symbiodinium. The
homogeneous layer in both G. foliaceum and Peridinium
bdlticum, a symbiont-containing dinoflagellate. has been
experimentally shown to contain cellulose (Morrill and
Loeblich. 1981; Loeblich, 1984). However, the cellulose in
this layer was assumed to be in an amorphous or low
crystalline state, since no cellulose fibers have been identi-
fied with the electron microscope. On the other hand, the
dense layer of Heterocupsa niei and other dinoflagellates
has been suggested to contain sporopollenin. a very resistant
plant terpenoid that has been demonstrated to be insoluble,
even in hot ethanolamine (Loeblich. 1970; Morrill and
Loeblich, 1981 ). Although the exact chemical nature of the
cell wall of Svmbiodininm is unknown, Markell et ul. (1992)
reported that the cell walls of S. microadriaticum, S.
kuwagutii, and S. pilosum did contain cellulose, an obser-
vation based on cellulase digestion of isolated cell walls.
They also reported that in intact symbionts, either living or
glutaraldehyde fixed, cellulase was ineffective in digesting
the cell wall. If a part of Symbiodinium' s cell wall, such as
the EDL. is composed of an extremely insoluble compo-
nent, such as sporopollenin. the IL may have been unaf-
fected by the cellulase simply because the enzyme could not
penetrate to this layer in intact cells.

The current formal description of the genus Symhio-
diniiim (Trench and Blank, 1987) states that the cell wall of
the coccoid stage varies in thickness. It also states that this
variation depends on the life history of the cell. This fact is
supported by Bricheux et til. (1992), who found that the
thickness of the cell wall of the free-living dinoflagellate G.
fiiliticeiim was relatively thin following ecdysis of the thecal
armor; however, as the vegetative stage of the life history
progressed, the cell wall became progressively thicker, with
the largest amount of change seen in the inner homogeneous
layer (IL). Similarly, our study has shown that, although the
OL and EDL certainly contribute to the overall thickness of
the cell wall, the thickness of the IL has the most variation.
The presence of the thecal vesicles within the symbionts
is not in itself surprising, since Loeblich and Sherley (1979)

REVISED DESCRIPTION OF SYMBIODINIUM

described the thecate nature of the motile stage of S. mi-
croadriaticum; but this is the first report that thecal vesicles
are present within in situ coccoid symbionts. The formal
description of the genus Symbiodinium describes the coc-
coid symbionts as having a "continuous cell wall . . . un-
derlain by a series of membranes" (Trench and Blank.
1987). We found no such membranes in our ultrastructural
investigations of freeze-substituted symbionts, but instead
found distinct thecal vesicles in this region. We did see
apparent membranes like those described by Trench and
Blank (1987) when the tissues were fixed with standard
aldehyde-osmium fixations. Since thecal vesicles are well
described in other dinoflagellate species (Dodge and Craw-
ford, 1970; Bricheux et «/., 1992: Hohfeld and Melkonian,
1992). have been reported in /// vitro cultured Symbiodinium
(Taylor, 1971b; Loeblich and Sherley, 1979; Trench and
Blank, 1987), and are not artifactual in nature, we conclude
that the multiple membranes reported in the formal in situ
description of the genus Symbiodinium (Trench and Blank,
1987) were the result of a fixation artifact.

Although this is the first report identifying the presence of
thecal vesicles within the in situ coccoid symbionts, review
of the ultrastructural literature relating to the genus Svmbio-
dinium reveals apparent thecal vesicles in the figures from
various published papers. In Taylor's (1968) original paper
on symbiont ultrastructure. figure 3 clearly shows what
appear to be thecal vesicles beneath the cell wall of the
coccoid stage. However, Taylor states that the vesicles are
formed in older symbionts as two of the multiple membrane
layers undergo a series of infoldings to form these isolated
and discrete vesicles, and he does not associate them with
the thecal covering of the motile stage. Kevin et til. ( 1969)
show, in their figure 5. an in situ symbiont that clearly has
thecal vesicles beneath the plasma membrane; however,
these structures are neither identified nor mentioned in the
text. In 1970, Dodge and Crawford reported that two mem-
branes, "the outer of which appear to be folded over in
places," surrounded the cytoplasm of the symbionts from
Anemonia sulcata. Their plate 7 also shows distinct thecal
vesicles inside the cell wall (Dodge and Crawford, 1970). A
paper by Tripodi and Santisi ( 1982) describes the ultrastruc-
ture of S. microadriaticum within the octocoral Eunicella
stricta. In their figures 4, 5, and 6. the electron micrographs
show distinct thecal vesicles beneath and adjacent to the cell
membrane of the in situ symbionts. Their figure 8, a line
drawing describing the cell covering of the symbionts, also
clearly shows distinct thecal vesicles. However, the figure
captions, as well as the text, describe these vesicles as
profiles of the endoplasmic reticulum and once again do not
associate them with the thecate motile stage.

In all of these reports, the symbiotic tissues underwent
standard fixation in 3%-4% glutaraldehyde followed by
post-fixation in \%-2% osmium tetroxide. Therefore, the
thecal vesicles can be visualized within the in situ symbi-

onts using this type of fixation; but the results in these
papers are the exception rather than the rule. It would appear
that, during a standard chemical fixation, if conditions are
not right (osmolality, temperature, length of fixation, age of
fixative, etc.). the fragile thecal vesicles rupture and com-
bine with the plasma membrane to take on the appearance of
multiple or scrolled layers of membrane beneath the cell
wall. Our results suggest that a more reliable way of visu-
alizing this fine structure is by use of the freeze-substitution
process.

The presence of thecal vesicles beneath the plasma
membrane of the in situ symbionts lends further credence
to an interesting hypothesis. In 1971, Taylor reported the
development of flagellar structures in the in situ symbi-
onts of the cnidarian Velella velella (pi. Ill A) and the
flatworm Amphiscolops langerhansi (pi. IIIB). In 1980.
Schoenberg and Trench also reported the presence of
flagellar structures in in situ Symbiodinium microadriati-
cum from ProtojHilythoa sp. (figure 8. plate 5). They state
that the sporadic appearance of these flagellar structures
could represent the transient production of motile zoo-
spores /'/; situ.

The production of motile gymnodinioid zoospores is a
daily occurrence in log-phase cultures of Symbiodinium
in vitro. Within the cell wall of the vegetative mother
cell, a metamorphosis occurs that results in the develop-
ment of a characteristically gymnodinioid motile cell
with thecae, hypocone, epicone, and longitudinal and
transverse flagellae in their respective grooves or furrows
(Freudenthal, 1962; Loeblich and Sherley, 1979). Motile
stages often develop after the cell has divided into daugh-
ter cells, but they can also develop without any previous
cell division (Schoenberg and Trench, 1980). When ma-
ture, the zoospores escape from the mother cell by some
unknown mechanism to swim freely within the culture
medium (Trench and Blank, 1987). After a relatively
short motile stage the cells spontaneously undergo ec-
dysis, a process by which they shed their thecae and
flagella, again by some unknown mechanism, and settle
to the substratum, where they enter the longer vegetative
phase of their life cycle.

The mitotic rate of in situ symbionts is decreased in
comparison to cultured symbionts. Fitt and Trench ( 1983)
determined that, within cultures of Symbiodinium mi-
croadriaticum. as many as 25% of the cells could be
dividing, but Palincsar et at. (1988) determined the mi-
totic rate of S. bermudense within A. pallida to be as low
as 1.2%. Although the mitotic rate in situ is decreased, it
is not halted. We hypothesize that a similar sort of delay
occurs in the ecdysis cycle and that, on a slower and
perhaps irregular schedule, ecdysis continues in situ. This
would explain the discrepancies between our cell wall
description and that of Palincsar et al. ( 1988). The vari-
ations in cell wall structure that they describe (totally

T. S. WAKEFIELD ET AL

granular, to half granular/half vesicular, to almost com-
pletely vesicular) could be explained by symbionts
caught in varying stages of the ecdysis. Similarly, such a
cycle would also result in the sporadic appearance of
flagella, as reported in some /// situ symbionts (Taylor,
1971a, b; Schoenberg and Trench, 1980). and the pres-
ence of thecal vesicles, as reported in this paper.

We also suggest that the delayed ecdysis cycle could be
responsible for the multiple layers of apparent membranes
that are often found just outside of the vegetative cell wall
of in situ symbionts. Unlike those of cultured symbionts, the
shed thecal plates and outer plasma membrane of in situ
symbionts would not be discarded but would accumulate
inside of the symbiosome membrane (Fig. 12). Such shed
plates would appear as the accumulations of the apparent
membranous material (ml) that has been observed between
the outer symbiosome membrane and the vegetative cell
wall of the symbionts (Taylor, 1968; Kevin ft til., 1969;
Schoenberg and Trench, 1980; Tripodi and Santisi, 1982;

-sm

•KXttWWX^ftiWM^** cwl

• pml

tvl

(pml+tvl)

•cw
- pm

Figure 12. A hypothesis for how multiple layers of membrane could
accumulate within symhiosome membrane: step 1. Vegetative cell
gives rise to motile "thecate" cell; cell wall disappears, step 2. Mo-
tile cell undergoes ecdysis and sheds plasma membrane and thecal
vesicles as the cell wall is reformed and new thecal vesicles form
beneatli ii. ^:p ^ After several cycles, accumulated material from algal
plasma membi .1, >uil ihecal vesicles appear as multiple membranes
between the outei - mbiusnme membrane and the vegetative algal cell
wall, cw = cell wall; ml = multiple layers of membrane; pin T
symbiont plasma mcmlnaue; sm = surface of symbiosome membrane;
tv = thecal vesicles. | figure adapted from Hohfeld and Melkonian.
1992.)

Colley and Trench, 1983; Blank, 1987; Trench and Blank.
1987; Trench and Winsor. 1987; Palincsar et at.. 1988;
Rands et til., 1993).

These accumulations of apparently membranous mate-
rial were first described by Taylor (19681, who ascribed
their origin to both the algal cell and the host, but gave no
specific structures from which they might arise. Later,
Kevin et al. (1969) redefined the location of the mem-
branes surrounding the algal cell, but once again failed to
clearly state their origin. This uncertainty as to the origin
of these membranes has continued throughout the litera-
ture, with some authors attributing it to the host (Tripodi
and Santisi. 1982; Colley and Trench, 1983; Palincsar et
til.. 1988; Rands et al.. 1993), and others to the algal cell
(Schoenberg and Trench. 1980; Trench and Blank, 1987).
Specifically, Trench and Blank (1987) reported that the
outer layer of the cell wall is periodically "sloughed off"
the surface and often produces a "scroll-like" appearance
in sections. However, they do not offer any evidence to
support this process as the origin of the multiple mem-
branous layers, nor do they offer any suggestions about
how this sloughed layer might be regenerated outside of
the continuous cell wall. Our hypothesis of a continuing
in situ ecdysis cycle as the origin of the apparent mem-
branes (see Fig. 12) is based on the presence of symbiont
thecal vesicles in situ, and on a known event within the
life cycle of the symbiont. As such, it does not require the
proposal of another "unknown mechanism" to explain
how additional membranes would be added to those
already present around the symbiont.

There is another question that must be addressed if our
hypothesis is correct. If a delayed ecdysis cycle is continu-
ing within the host cell symbiosome, then in addition to the
shedding of the theca and plasma membrane, there must
also be a shedding of the cell wall. If the thecal vesicles are
retained within the host membrane, what happens to the cell
wall material? Although this is a valid question, it is not
unique to our hypothesis. The same question can be asked of
mitotically active, vegetative cells in situ.

It has been assumed that, at the conclusion of a mitotic
event, the symbiont daughter cells within the same sym-
biosome membrane are separated by invading extensions
of symbiosome membrane and host cytoplasm (Reisser,
1992). Following division, each new daughter cell pro-
duces a new cell wall within the old cell wall of the
parent cell (Taylor. 1968: Kevin ct til.. 1969) Thus, if the
"old" cell wall were not degraded in some way, each host
symbiosome would contain remnants of cell wall mate-
rial from previous mitotic events. Because such remnants
have not been observed, the old cell wall material must
be degraded, perhaps by enzymes released from the sym-
biont itself. As was mentioned previously, the release of
motile zoospores from within the parent cell wall is
controlled by an unknown mechanism, presumably enzy-

REVISED DESCRIPTION OF SYMBIODIN1UM

matic in nature. Perhaps this same mechanism is respon-
sible for the degradation of the cell wall within the host
symbiosome during the mitotic event, with the outer
symbiosome membrane retaining active enzymes in the
vicinity of the discarded cell wall. In the case of a
retarded ecdysis cycle, if the thecal plates differ in com-
position from the cell wall and thus are not subject to
similar enzymatic breakdown, they could accumulate as
the multiple layers of membranous material found be-
tween the symbiosome membrane and symbiont cell wall
in situ.

These membranes contribute to the membranous struc-
ture of the symbiosome and are part of the boundary be-
tween host and symbiont. All "communication" between
host and symbiont. transport of gasses, and translocation of
photosynthetically fixed carbon must occur through, and in
conjunction with, these membranes (Rands et ai. 1993).
Thus their origin and their role in these events is of great
importance.

Acknowledgments

We thank Dr. Daphne Fautin for her aid in identifying the
anthozoan species Phyllactis flosculifera. We also thank Dr.
William Fitt and the Key Largo Marine Research Labora-
tory, Dr. Mike Miller and the Auburn University Biological
Electron Microscope Imaging Facility, and the University
of Georgia's Center for Advanced Ultrastructural Research
for their assistance in this research. This work was sup-
ported by NSF #9018698 (SCK), NSF-BIR #9220230
(SCK), ONR #3231114 (SCK), and a grant from the Ala-
bama Agricultural Experiment Station (SCK) Journal #
6-996072.

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Reference: Binl. Bull. 199: 85-94. (August 21)00)

Overgrowth Competition Between Clades:

Implications for Interpretation of the Fossil Record

and Overgrowth Indices

DAVID K. A. BARNES' * AND MATHEW H. DICK2

1 Department of Zoology and Animal Ecology, University College Cork, Cork, Ireland; and
2 Department of Biology, Middh'hitry College, Middlebury, Vermont 05753

Abstract. Overgrowth interactions (2693 in total) were
observed among three major groups (arguably clades) of
bryozoans — cheilostomatids (57 species), ctenostomutids
(3 species), and cyclostomatids (14 species). The bryozoans
studied here occur in shallow water at high-temperate polar
latitudes where they encrust hard substrata such as rock
piles. The main study site was the intertidal and infralittoral
zones of Kodiak Island, Alaska, but observations were also
made in similar zones of South Georgia Island and the
Falkland Islands in the South Atlantic Ocean. Cheilostoma-
tids dominated the number of species, individuals, and
interactions at all depths. Intraclade interactions formed
73.7% of the encounters for cheilostomatids, 1 .6% for cten-
ostomatids, and 5.7% for cyclostomatids. The competitive
ranking of the three clades was broadly ctenostomatids >
cyclostomatids > cheilostomatids. Significantly, these re-
sults contradict all previous quantitative studies of bryozoan
overgrowth, in which cheilostomatids are reported to over-
grow cyclostomatids at a higher rate. From these studies and
the literature, we calculated win indices to vary from 0 to
0.42 for living cyclostomatids, from 0.08 to 0.9 for living
cheilostomatids, and from 0.25 to 0.75 for living ctenosto-
matids. The win indices of cyclostomatid and cheilostoma-
tid clades show significantly more variation in living assem-
blages than in fossil assemblages. This disparity may be due
to differential preservation (polar and subpolar assemblages
last less than 4 years). The diversity was very high in terms
of both species richness and interaction types (outcomes
between competitor pairs). Comparison with the literature
suggests the possibility that nearshore diversity of bryozo-
ans may be bimodal (have two peaks) between high arctic

Received 21 August 1999; accepted 20 April 2000.

* To whom correspondence should he addressed. E-mail: DKAB@ucc.ie

and antarctic latitudes. Indices of success in overgrowth
competition have been constructed in various ways. For
cheilostomatids, the method of calculation had little in-
fluence on the ranking of representatives. In contrast, the
apparent success of ctenostomatids and cyclostomatids var-
ied hugely with how the index was calculated. This incon-
sistency is due to the use of very different strategies in
overgrowth competition; among the two latter groups, many
interactions involve tied outcomes.

Introduction

Cyclostomatida, Ctenostomatida. and Cheilostomatida
are the major groups of the Phylum Bryozoa (the fourth is
exclusively fresh water). For convenience, we will use the
term "clade" to refer to these major groups of marine
bryozoans, but this is not entirely accurate. Although the
Cyclostomatida and the bulk of the Cheilostomatida prob-
ably represent monophyletic groups, or clades, Ctenostoma-
tida is probably a paraphyletic group (Todd, 2000), better
referred to as a "grade." Representatives of these groups
occur together in most benthic assemblages, where fre-
quently the encrusting members are directly competing for
space and food (e.g., Stebbing, 1973; Sebens, 1986; Lopez
Gappa. 1989). Cheilostomatids generally dominate the
bryozoan component of assemblages in space occupied,
numbers of species, numbers of colonies, and overgrowth
performance. As a result of such dominance, most studies of
competition between encrusting benthos have either docu-
mented cheilostomatid interactions with representatives of
other benthic phyla (Quinn, 1982; Sebens, 1986) or have
been restricted solely to cheilostomatid-cheilostomatid in-
teractions (e.g., Jackson, 1979a; Buss. 1980; Palumbi and

D. K. A. BARNES AND M. H. DICK

Jackson, 1983; Tanaka and Nandakumar. 1994; Barnes and
Rothery, 1996). A few studies have shown that representa-
tives of the Ctenostomatida may be high or mid-ranked in
overgrowth performance against the cheilostomatid repre-
sentatives (Stebbing, 1973; Turner and Todd. 1994). Cy-
clostomatids, in contrast, have been found to be almost
always overgrown by cheilostomatids in the few studies
of Recent (living) competition between the two clades
(Harmelin. 1976; Buss and Jackson, 1979; Lopez Gappa,
1989: McKinney, 1992). Analysis of the fossil record has
shown that competitive performance has been stable for the
last 100 million years, with cheilostomatids overgrowing
cyclostomatids in about 66% of encounters (McKinney,
1995a). Although broad trends have been described, many
factors contribute to the outcome of interactions between
any pair of competitors.

Phylum membership is the principal factor determining
overgrowth ability, with ascidians > sponges > bryozo-
ans > unitary forms such as barnacles, annelids (Buss and
Jackson, 1979; Russ, 1982; Sebens, 1986). Growth form is
also important, with foliaceous forms > encrusting sheets >
stoloniferous types (Buss. 1979; Barnes and Rothery. 1996).
and bryozoans that have the capability of frontal budding
overgrowing those that lack it (Lidgard and Jackson, 1989;
McKinney, 1992; 1995a). To explain the competitive ad-
vantage that cheilostomatids have over cyclostomatids,
McKinney (1992; 1993; 1995a, b) has described a number
of attributes, including higher growth rates and larger col-
onies and feeding structures. The crucial features of chei-
lostomatids, however, are probably ( 1 ) rapid ontogenetic
development resulting in full-sized zooidal skeletons (and
feeding structures) at colony margins, (2) labile morphoge-
netic responses at colony margins (raised growing edges.
frontal budding, stolon production and others), and (3)
water excurrents that leave around the colony margin, po-
tentially into the area of uptake for a cyclostomatid com-
petitor. These features may explain the great radiation that,
since the mid-Cretaceous period, the cheilostomatids have
undergone relative to the cyclostomatids (Lidgard ct ul..
1993), or this may be due to unrelated factors such as the
acquirement of planktotrophic larvae (Taylor, 1988). It
seems likely that an increase in encounters with a superior
competitor would contribute to the decline of cyclostomatid
species richness from the end of the Cretaceous to Recent
periods (Lidgard el ul., 1993; McKinney, 1995a: Sepkoski
et ai, 2000).

Studies involving interpretation of competitive interac-
tions between major groups or clades of the same phylum
are rare for both living and fossil assemblages, and this is
the case with the Bryozoa. Consequently, our knowledge is
biased to the results of the few studies earned out and the
limited distribution of the localities of these studies. Many
studies are based on relatively few interactions between
many species pairs and even fewer between clades, and thus

a synoptic interpretation of overall outcomes is difficult.
Problems of interpretation are compounded by differences
in the way performance is measured (e.g., wins compared to
losses, or wins compared to total interactions) and by the
way contact matrices are analyzed (that is. by using transi-
tivity indices) (see Petraitis, 1979; Rubin, 1982; Tanaka and
Nandakumar, 1994). Perhaps the largest barrier to meaning-
ful comparison, though, is that the three marine bryozoan
clades have not, to date, been evaluated in the same study
(at a single locality).

In this study we investigate intraspecific and interspecific
competitive encounters among representatives of the bryo-
zoan clades Cyclostomatida, Ctenostomatida, and Cheilo-
stomatida from the intertidal and infralittoral zone of Ko-
diak Island, Alaska. The boreal/subpolar region is unusually
diverse with respect to many taxa, but particularly bryozo-
ans (see Barnes and Dick. 2000; Dick and Ross, 1986;
1988). and provides an opportunity for comparing interac-
tions between abundant representatives of the three clades.
We compare the outcomes using different methods of com-
petitive strength calculation, and we evaluate these meth-
ods. We also compare win indices and rankings of the
clades with unpublished work involving interactions among
the clades at two south Atlantic localities: the Falkland
Islands and South Georgia Island. Win indices and rankings
from other localities and time periods were extracted from
the literature for comparisons with our results.

Materials and Methods

Study site and species

Bryozoan overgrowth interactions were analyzed on 110
rocks from 14 sites at Narrow Strait, Kodiak Island (57°
54'N, 152° 27'W) in the Alaskan boreal-Arctic (see Dick
and Ross, 1988, for more detail). Four tidal levels or depths
were represented by a number of rock-pile sites: upper
midlittoral (2), lower midlittoral (4). upper infralittoral (3).
and lower infralittoral (5). Rock surface area was measured
using a nonelastic grid of square centimeters as per Barnes
and Rothery ( 1996), but percent cover and colony size were
not measured. All competitors were identified into the three
orders of bryozoans present and to genus or species level
where possible. Poor taxonomic resolution in the initial
stages of the study led to uncertainty about the particular
species involved in interactions within the genera Caulor-
lutinpluis (5 species), Microporella (4 species), Celleporella
(2 species), and Alcyonidium (2 species). The cyclostoma-
tids from NaiTow Strait have not been worked up taxo-
nomically and here were identified to ordinal level only,
with the exception of a common lichenoporid designated
Lichenopom sp.

COMPETITION BETWEEN BRYOZOAN CLADES

Table 1

Mean number of species per rock and proportion of rocks
(in pari'nihi'scx) ci>li>ni:ril hy species occurring rarely ui
tilt' Miul\ \ites, Altisku

Mean no. species per

rock

Tidal cover/depth

Cyclostomatids

Ctenostomatids

Cheilostomatids

Upper midlittoral
Lower midlittoral

0
0.88(0.31)

0.25(0.13)
0.75 (0.09)

4.88 (0.49)
6.81 (0.49)

Upper infralittoral
Lower infralittoral

1.54(0.72)
4.25 (0.44)

0.54(0.25)
0.73 (0.30)

1(1,7(1.00)
13.0(0.63)

Measurement of interactions

All colony-to-colony interactions between representa-
tives of the three clades of bryozoans were recorded from
each rock and site, along with the number of intraspecific
and interspecific encounters within clades. When the grow-
ing edge of competitor A covered the apertures of compet-
itor B, A was determined to have overgrown B. Only
"frontal" overgrowth interactions between two living com-
petitors, without direct settlement onto one of the compet-
itors, was counted as overgrowth for the purposes of this
study (see Rubin, 1982; Turner and Todd, 1994; Barnes and
Rothery, 1996). The actual scores were tabulated into a
competitor-contact matrix (as Turner and Todd, 1994;
Barnes and Rothery, 1996). Measurements of overgrowth
performance were calculated for each competitor that took
part in more than 20 between-clade interactions. Various
measurements of overgrowth performance were used: a
score system in which a win = 3, a tied outcome = 1 . and
a loss = 0 (wins rated much higher than ties because ties
often prevent further growth and development of colonies,
see Barnes and Clarke [1998]); the number of wins divided
by the total number of interactions for that competitor; the
number of losses divided by the total number of interactions
for that competitor; the number of wins divided by the
number of losses for that competitor; and an aggregate
measure in which the mean of the rankings from all methods
was obtained. The rankings in Table 2 are those calculated
from raw data. These may differ from true population rank-
ings because the number of encounters with each competitor
was not the same for each species, and some potential
competitors did not meet. The rankings were standardized
by multiplying all pairwise interactions such that each had a
total value of 100, then recalculating the total wins, losses,
and ties for each competitor identity.

Results

Between-species interactions

A total of 74 species of bryozoans were recorded during
the study: 57 cheilostomatids, 3 Ctenostomatids, and the

remainder cyclostomatids (later analyzed to be 14 species).
The mean number of cheilostomatid and cyclostomatid spe-
cies per rock increased with depth of rocks (Table 1), the
former dominating the number of species at all depths. The
proportion of ctenostome species was small with respect to
the total number of bryozoan species, and it varied incon-
sistently with depth. The mean number of interactions in-
creased with depth for all clades (Fig. 1 ). Cheilostomatids
were involved in 2653 interactions, of which 73.7% were
within the clade; Ctenostomatids were involved in 367 in-
teractions, of which 1 .67r were within the clade; and cy-
clostomatids were involved in 384 interactions, of which
5.7% were within the clade. All cheilostomatids that en-
countered Ctenostomatids or cyclostomatids on more than
20 occasions are illustrated in a species-contact matrix
(Table 2). The remaining cheilostomatid species are pooled
because fewer than a total of 20 competitive interactions
were not considered to be representative. Certain pairs of
competitors had anomalously higher frequencies of encoun-
ters, such as Alcyonidium spp. and Porella alba. The chei-
lostomatid species Callipora craticiila encountered only
Ctenostomatids and cyclostomatids, despite the overwhelm-
ing numerical dominance of cheilostomatids and being in-
volved in 30 interspecific interactions.

Most (99.3%) of the intra-clade encounters observed
were interspecific interactions. Over 80% (1 14) of the pos-
sible competitor-pair interactions (136 in the matrix Table
2) and 44 of the 45 between-clade interactions were ob-
served (but these represented only a small proportion of the
74 X 74 species interactions theoretically possible). The
proportion of indeterminate outcomes (neither competitor
won all encounters) from competitor pairs was significantly
higher within the clade of cheilostomatids (15.9%) than
between cheilostomatids and other clades (5.1%) (Mann-
Whitney U test, P < 0.01 ). The proportion of tied out-
comes or standoffs in competitor pairs was significantly
higher between clades than within clades (Mann-Whitney,
P < 0.001 ). The proportion of ties was also significantly

Lower mid

°s

= — Upper infra -

0 50 100 150 200

Number of interclade interactions

Figure 1. Mean number of interactions per clade with depth. All data
are presented as mean with standard error.

88 D. K. A. BARNES AND M. H. DICK

Table 2

Matrix of competitive interactions for Alaskan cheilostomatid, ctenostomatid, and cyclostomatid bryozoans (latter nro groups are grav shaded)

F-; i~ § C C 5 t-:

•5;

254321 121011

1 3

•)

1 7

Tegella anmfera 9 16 3 10 1 4 10 13 2 3 2 4

27 31

19 25

0 11 0 0 211 0 0 1

1 2

1 5

Tegella aquilostris 8191 1 471267

16 19

16 24

0 0 251 0 1 3

4 5

0 11

Myrio-oclla plana 3 3 7 14 5 604

34 43

1 1

8 41

024042

5 13

2 9

Caiilorhamphits sp. 1 32639

21 39

1 1

24 77

43131 1

12 14

1 8

Other Cyclostomatids 372624

16 42

7 39

2 1

1 3

2 2

Microporella califomica 6 9

3 7

7 26

3 3

0 5

Tegella urtica

13 19

1 1 1

21
8

29
0

20 21
1 16

Lichenopora sp.

5 17

5 21

11 IS

Other Cheilostomatids

110

148

17 94

6 34

Microporella sp.

102

27 89

3 10

Cribnlina

annit

lain

9 14

24 40

Poi

r//<;

alba

KM) 73

1 34

Alcyt

tnidium sp.

6 39

Celleporella

sp.

Data are displayed in standard form — see Turner and Todd (1994), Barnes and Rothery ( 1996). If row = competitor A and columns = competitor B.
for each cell the top left, top right, and bottom left data give, respectively, the number of ties between species A and B, wins by B (= losses by A) and
wins by A (= losses by B). The number in the bottom right of each cell is the total number of observed interactions for that species pair.

higher in the Ctenostomatids than in the \uin-Liclu'n<>/>oni
cyclostomatids (Mann-Whitney, P < 0.001) but was not
significantly different from that in the total cyclostomatids
(Mann-Whitney. P = 0.053). Both clades had a signifi-
cantly higher proportion of tied outcomes than did the
cheilostomatids (Mann-Whitney, P < 0.001 ).

Ctenostomatids and cyclostomatids met too infrequently
to assess their overgrowth performance against each other.
Both of these clades, however, encountered cheilostomatids
on many occasions anil won more encounters than they lost.
In both the midlittoral to the infralittoral, Ctenostomatids
won about 55% of the encounters that had a decided out-
come, but the proportion of ties increased from from 4% in
the midlittoral to 58% in infralittoral. Cyclostomatids were
better competitors against cheilostomatids, winning 87% of
midlittoral encounters and 62% of infralittoral encounters.

As with ctenostomatid-cheilostomatid encounters, the pro-
portion of tied outcomes increased from the midlittoral to
the infralittoral, but less dramatically, from 31% to 35%.

The overall transitivity of the assemblage measured using
the index of Tanaka and Nandakumar ( 1 994) was 0.62. This
was 25% lower than the value obtained for interactions just
within the clade of cheilostomatids (0.83 — Barnes and
Dick, unpubl. data). This value indicates a generally hier-
archical system (Buss, 1980; Russ, 1982) but, as predicted
by Jackson (1979b). one that is more intransitive between
clades than within the clade of cheilostomatids.

The competitors involved in interactions could be ranked
in a sequence of overgrowth performance from several
typically overgrown by others (cheilostomatids) to several
typically overgrowing others (also cheilostomatids). Over-
growth performance can be and has been measured in a

COMPETITION BETWEEN BRYOZOAN CLADES 89

Table 3

Ranking of competitive ability in Alaskan cheilostomatid, ctenostomatid. and cyclostomatid hryo-oans (latter two clades are in bold), with nutnex listed
in descending order of initial number of wins

Standardized ranking

Taxon identity and coding

Initial

Win-tie scored

WAT

L/T

W/L

Aggregate

Tegella armifera

(Ta)

Tegella aquilirostris

(Tq)

Myriozoella plana

(Mp)

Cauloramphus sp.

(Ca)

p..

Microporella californica

(Me)

v-y

L, d

i r
Li

Tegella arctica

(Tr)

Lichenopora spp.

(Li)

Cheilostomatids

(C)

Microporella sp.

(M)

Crihrilina awutlata

(Cn)

Porella alba

(P)

Aleyonidium spp.

(A)

Cylindroporella tubulosa

(Ct)

Celleporella spp.

(Ce)

Rankings are Initial (number of wins) and Standardized (multiplied up so every competitor meets each other on the same number of occasions).
Standardized rankings are win-tie scored (wins score 3, ties score 1). W/T (proportion of wins over total number of interactions). L/T (proportion of losses
over total number of interactions), W/L (proportion of wins over losses), and aggregate (of W/T, L/T, and W/L).

"Cheilostomatids" in the species-identity column refers to the remainder of the cheilostomatid species present but not listed.

number of ways (Table 3). A ranking based on the number
of wins (actual overgrowth of the competitor) placed some
cyclostomatids as intermediate competitors but others
(Lichenopora sp.) and the ctenostomatids as poor competi-
tors. In contrast, lichenoporid cyclostomatids and. to a lesser
extent, ctenostomatids were good competitors when as-
sessed by the ratio of losses to total interactions (they were
rarely overgrown). Cheilostomatids spanned the whole
range of competitor performance, but those that scored
highly in the win index also scored highly on the loss index
(i.e.. good overgrowers were rarely overgrown; poor over-
growers were usually overgrown). The average ranking
change for a competitor between these different indices was
1.4 for Cheilostomatids, but 6 for ctenostomatids and 6.5 for
cyclostomatids. At the level of species, the performance of
selected Cheilostomatids against the combined representa-
tives of each clade is illustrated in Table 4. All performed
better against other Cheilostomatids than against ctenosto-
matids or cyclostomatids, but most performed better against
cyclostomatids than against ctenostomatids (e.g., Micro-
porella californica), although a few (e.g., Porella alba) did
the converse. A good competitor against one clade was
generally a good competitor against the other, but some
(e.g., Microporella californica) had quite different perfor-
mances against competitors from different clades (Table 4).
The between-clade win index of the three clades varied
(Table 5) between the Alaskan site and others we analyzed
from County Cork (Ireland) and the Falkland Islands and
South Georgia Island (both South Atlantic). Other literature

and unpublished data in Table 5 for which between-clade
win scores have been calculated show the overgrowth
scores of cyclostomatids in Alaska and County Cork to be
the highest recorded.

Table 4

Performance of various Alaskan cheilostomatid species in overgrowth
interactions with other Cheilostomatids. ctenostomatids and
cyclostomatids: values are the probability of a win for
competitor A against competitor B

Competitor B

Competitor A
identity

Cheilostomatids

Ctenostomatids

Cyclostomatids

Tegella aqid/ostris

0.76

0.44

0.55

Tegella armifera

0.71

0.53

0.65

Myrio-oel/a plana

0.66

0.38

0.41

Laganicella

neosocialis

0.60

—

0.29

Caidoramphis spp.

0.52

0.08

0.07

Tegella arcticu

0.51

0.30

0.38

Microporella

californica

0.46

0.00

0.33

Cribrilina anituUitu

0.34

0.09

0.13

Microporella spp.

0.32

0.22

0.25

Porella alba

0.29

0.16

O.I I

Cel/epore/la spp.

0.22

0.14

0.12

Callipora craticn/u

0.06

0.14

D. K. A. BARNES AND M. H. DICK

Table 5

overgrowth competition performance scores and rankings in the three bryo-oan clades (all other rankings are cheilostomatids)

Cyclostomatids vs.

Cheilostomatids vs.

Ctenostomatids vs.

cheilostomatids

Cyclostomatids

cheilostomatids

vs. Ctenostomatids*

Location

Latitude

Win index Ranking

Win index

Ranking

Win index

Alaska (USA)"

57 N

0.24

7/12

0.42 (0.25*)

6/13

0.21 (0.18*)

Scotland

56 N

0.5

5/18

—

(0.32*)

Ireland""0 '

51.5 N

—

0.41

6/8

0.56

Ireland""7 2t

51.5N

0.56

1/6

—

(0.37*)

England

SON

0.75

1/5

—

(0.08*)

Croatia

43 N

—

0.08

—

0.78

Jamaica

18 N

—

Low

10/10

-0.9

Mozambiquet

12 S

—

0.2

13/15

0.60

Australia

38 S

—

0.0

8/8

0.74

Argentina

47 S

—

0.33

9/12

0.62

Falkland Is

52 S

—

0.32

7/10

0.44

South Georgia'!'

54 S

0.4

1/7

0.18 (0.08*)

6/7

0.6 (0.32*)

Signy 1st

60.5 S

—

0.1

~> ">/">">

0.73

Data are taken from Stebbing ( 1973). Buss and Jackson ( 1979). Russ ( 1982), Lopez Gappa (1989). McKinney ( 1992), Turner and Todd ( 1994), Maughan
and Barnes (in press), Barnes and Lehane (unpubl. data), present study (") and unpublished sources (t).

Discussion

Amongst the major groups of the phylum Bryozoa, chei-
lostomatids are generally the major space occupiers, the
most speciose, and the superior overgrowth competitors
(see, for example. Buss and Jackson, 1979). Sometimes they
may even locally dominate the macrobenthic community—
for example, on shells (Kay and Keough, 1981), on reef
rubble (Jackson and Winston. 1982), on floating debris
(Barnes and Sanderson, 2000). in mid-latitude shelf waters
(James et aL. 1992). and on Antarctic shallow-water hard
substratum (Barnes, 1995). When present, the rarer Cteno-
stomatids may effectively compete against cheilostomatids
(Stebbing. 1973), but Cyclostomatids are typically over-
grown in meetings (Buss and Jackson, 1979). Overgrowth
of calcified benthos by soft-bodied forms such as ctenosto-
matid bryozoans or ascidians may not. however, always
prove lethal to the overgrown competitor (Todd and Turner,
1988). In this study, the first to encompass all three
"clades." at the same localities, cheilostomatids were the
major space occupiers, were involved in most interactions,
and were the most speciose of the three clades, as found
elsewhere (Table I ). They were, however, outcompeted by
Ctenostomatids at all three localities, and at Kodiak Island,
Alaska, they were uniquely outcompeted by Cyclostomatids
(Table 5).

McKinney analyzed living and Recent relict (1992;
1995b) and fossil ( 1995a) cyclostomatid and cheilostomatid
interactions and found that living and Recent relict assem-
blages in Rovinj. Croatia, were similar to those over the last

105 million years. The win index of fossil cheilostomatids
oscillated around a value of 0.66 (66% win rate), and that of
Cyclostomatids at around 0.29. In the Alaskan assemblage
studied here the win index of Cyclostomatids was nearly 1.5
times greater and the win index of cheilostomatids 3 times
lower. The win index of clades varied with site (Table 5) or
possibly latitude (Fig. 2). In the living assemblages studied
here (from Alaska, the Falkland Islands, and South Georgia
Island) and other indices taken or calculated from the liter-
ature, the win indices of Cyclostomatids varied from 0 to
0.42. The win indices of living cheilostomatids varied be-

nj
E
2 05 -

g ° 03 -
1.1

-20 0 20

Latitude ("South)

Figure 2. Magnitude of overgrowth C7r I of Cyclostomatids by cheilo-
stomatids in Meso/oic and Ceno/oic periods, adapted from McKinney
(1 995 1.

COMPETITION BETWEEN BRYOZOAN CLADES

tween 0.08 and 0.9 and those of living ctenostomatids from
0.25 to 0.75 from the few studies carried out (Table 5).
There is significantly more variation (Fig. 3) in the living
assemblages that have been studied than in the fossil as-
semblages that have been studied in both the cyclostomatid
clade (homogeneity of variance test. F = 7.6, P < 0.01 )
and the cheilostomatid clade (F = 24.0, P < 0.01 ). The
sample size in both the living and fossil assemblages is
reasonable (though not high in the former). McKinney
(1995b) has shown that, through careful consideration of
differential preservation of overgrower and overgrown,
analysis of fossil assemblages probably gives an accurate
representation of conditions at the time of preservation.
Either there is more variation today than over the past 100
million years or we are getting some information from
living assemblages that we are not getting from fossil as-
semblages. There is some evidence for both of these expla-
nations.

The highest values of cyclostomatid win index and the
lowest values of cheilostomatid win index are from high
(50°-60°) latitude localities (this study and Maughan and
Barnes, unpubl. data). The high values for the cheilostoma-
tid win index are generally from studies in mid to low
latitudes (and typically from more sheltered sites) (e.g.,
Buss and Jackson, 1979; McKinney, 1992). Thus the high
range of modern index values is probably partly due to the
range of exposure of localities. Antarctic (high latitude)

o
'o
c
o

'•e
o

Q.
O

9 Fossil assemblages
0 Living assemblages

.. :?

Cyclostomatids

or-

Cheilostomatids

-100 -80 -60 -40 -20 0

Time (Million yrs)

Figure 3. Non-cheilostomatid (ctenostomatid and cyclostomatid) spe-
cies richness of rock-pile habitats with latitude. Data points are from
Powell and Crowell ( 1 967 ), Gordon ( 1 980 ), Winston 1 1 982 ), Cook ( 1 985 ).
Rao and Ganapati ( 1985). McKinney ( 1992), Barnes el ul. ( 1996). Barnes
and Arnold ( 1999). Maughan and Barnes (unpubl. data), present study, and
unpublished sources.

•60 -40

•20 0 20

Latitude (degrees)

Figure 4. Cyclostomatid win index with latitude. Dala points are from
Buss and Jackson (1979). Russ (1982). Lopez Gappa (1989), McKinney
(1992), Barnes and Rothery (1996). Maughan and Barnes (in press),
present study, and unpublished sources.

values (from data from Barnes and Rothery. 1996; Barnes,
unpubl. data), however, also showed high values for the
cheilostomatid win index (Fig. 4). The geologically recent
succession of glaciation periods, cooling of the poles, and
separation of continents has probably resulted in higher
levels of disturbance through wave action and ice-scour
than before. So the range of nearshore conditions may be
greater than in the past 100 million years. Depth is a
potential confounding factor because most interaction data
that has been recorded from fossil communities is from
deeper water shelf environments (see Taylor and Allison,
1998). whereas most modern data sets of similar nature are
from shallow water (see Buss and Jackson. 1979: Russ.
1982; Turner and Todd. 1994; Barnes and Rothery. 1996).
However, studies of fossil assemblages such as those by
McKinney ( 1992) are largely from mid latitudes, suggesting
that conditions for fossilization of communities seem to be
most prevalent in mid-latitude conditions (Taylor and Alli-
son. 1998). The survival of encrusting communities (and.
more specifically, bryozoan colonies) becomes progres-
sively shorter with increasing latitude within the Southern
Ocean, such that the longest survival time of any individual
(even skeletal material) at 68° S is just 4 years (Barnes and
Arnold, 1999). Additional support is provided by the high
win-index values, similar to those in the fossil record, of
cheilostomatids in mid to low latitudes and in sheltered
localities (Buss and Jackson, 1979; McKinney. 1992). Thus
it is possible, and even probable, that a broad range of index
values have occurred throughout the last 100 million years
but have not been preserved because the very conditions
that yield extreme values prevent preservation (Lescinsky.
1993; McKinney, 1995b). This situation makes judgments
about the evolutionary ecology and historical position of
clades and the non-escalation of competition problematical
(Liddell and Brett, 1982; McKinney (1992; 1995a. b).

D. K. A. BARNES AND M. H. DICK

The diversity of the Alaskan assemblages studied here,
with respect to both species richness and variety of species-
pair interactions, was high — more so than at any compara-
ble site studied to date (Barnes and Dick. 2000). The next
highest value of both non-cheilostomatid and cheilostoma-
tid species richness in intertidal or shallow subtidal rock-
pile habitat is from the temperate zone of the southern
hemisphere (Russ, 1982). James et al. (1992) have also
shown bryozoan-rich carbonates peaking in mid-latitude
shelf environments. This finding raises the possibility of
two peaks in nearshore bryozoan species diversity centered
around temperate shores (Fig. 4). though clearly more data
would be needed to test this hypothesis. If such a pattern is
mediated through frequency of disturbance, one would ex-
pect the peak in the northern hemisphere to be at a higher
latitude than that in the south because the continental effect
of Antarctica increases the range of latitude influenced by
ice scour in the southern hemisphere. Species richness typ-
ically increases towards the tropics (Thorson, 1957; Kendall
and Aschan. 1993), but may peak around subequatorial
levels (Silva, 1992). Bryozoans (along with polychaetes) are
one of the few taxa that have a diversity center in Antarctic
waters (Clarke, 1992; Hayward. 1995).

The definition of a win or tie in overgrowth competition
has undergone some evolution (Jackson, 1979a). A tied
outcome has been found to represent a variety of situations
including a cessation of growth (e.g., Stebbing, 1973), mi-
nor overgrowth (Russ, 1982), mere stalling of a future win
for one of the two competitors (Sebens, 1986), mutual
overgrowth (McKinney, 1992), fusion of colonies (intraspe-
cific meetings), and redirection of growth (Barnes and Roth-
ery, 1996). Tanaka and Nandakumar (1994) argued that a
tied outcome was a result equal in importance to a win or a
loss and should be included in index calculations. The
method of win index tabulation, analysis, and interpretation
has also changed dramatically, but for bryozoans has typi-
cally been based around cheilostomatids (Petraitis. 1979;
Buss and Jackson. 1979; Rubin, 1982; Tanaku and Nanda-
kumar, 1994). In this study, the various methods of ranking
competitors in overgrowth competition indicate that the
type of index of success used has little influence on the
relative or absolute positions of cheilostomatids. In contrast.
the apparent success of ctenostomatids and cyclostomatids
varies hugely with how the index is calculated because they
use a very different strategy in overgrowth competition;
many inici -:iions result in tied outcomes. The strategy is
essentially del .nsive rather than offensive, involving not
many wins but not many losses. Such a strategy may pay off
better where encounters and superior competitors are rare,
because either a win or a tie may result in persistence, hut
a loss can be lethal (though not necessarily to the genet). In
very exposed environments, such as that in the present study
and in Antarctic assemblages, encounters and good compet-
itors are rare due to habitat ephemerality, so even poor

competitors may dominate assemblages (Barnes and Clarke,
1998). The non-lichenoporid cyclostomatids have a higher
number of wins, but also a higher number of losses, than
lichenoporids. Most cheilostomatids also have mostly de-
terminate interactions, which may be a better strategy where
habitats are more stable and competition is more intense. To
tie with one competitor would not ensure persistence, as
sooner or later an even better competitor will arrive. Al-
though the assemblages studied here have not been followed
through time, ties involving cyclostomatids cannot be con-
sidered likely to be delays on eheilostomatid wins (see
Rubin, 1982; Sebens, 1986). This is partly because the
majority of decided outcomes between these clades in-
volved a win for the cyclostomatid competitor and partly
because the majority of tied outcomes observed in other
similarly exposed latitudes (e.g.. South Georgia Island
[Barnes and Arnold, 1999]; Signy Island [Barnes and Roth-
ery. 1996]) had remained as "standoffs" for a period of
years. The disadvantages of cyclostomatids compared to
cheilostomatids. in functional body plan and feeding or
water flow dynamics (McKinney. 1992), may be reasons for
achieving a defensive rather than aggressive strategy in
overgrowth competition. There may, however, be other
explanations, such as differential growth rates or budding
patterns between environments; and other selective forces,
such as the frequency of disturbance, may be more impor-
tant.

Although the cyclostomatids are marginally superior to
the cheilostomatids in overgrowth competition at Kodiak
island, Alaska, the other study locations and literature sug-
gest that this is atypical. The clades on aggregate are ranked
ctenostomatids > cheilostomatids > cyclostomatids, but
all, particularly the cheilostomatids, have a range of com-
petitors with widely varying overgrowth strength and strat-
egy. This study suggests, however, that locality, method of
measurement, and number and identity of clades included in
competition studies have important influences on and im-
plications for the result obtained.

Acknowledgments

We thank those who assisted with data collection: Pat
Chan and Clare Lehane (University College Cork) for the
County Cork and Falkland Islands data; Rod Arnold (British
Antarctic Survey) for the South Georgia Island data; and the
Society for Environmental Exploration, FRONTIER, for the
Mozambique data. We also wish to thank Irene O'Sullivan
for help with preparation of Table 2.

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Marine

Biological

Laboratory

Woods Hole

Massachusetts

One Hundred and Second Report

for the Year 1999
One Hundred and Eleventh Year

Officers of the Corporation

Sheldon J. Segal, Chairman of the Board of Trustees

Frederick Bay. Co-Vice Chair

Mary J. Greer, Co-Vice Chair

John E. Dowling, President of the Corporation

John E. Burris, Director and Chief Executive Officer

Mary B. Conrad. Treasurer

Robert E. Mainer, Clerk of the Corporation

Contents

Report of the Director and CEO Rl

Report of the Treasurer R7

Financial Statements RN

Report of the Library Director R19

Educational Programs

Summer Courses R21

Special Topics Courses R25

Other Programs R32

Summer Research Programs

Principal Investigators R35

Other Research Personnel R3(i

Library Readers R38

Institutions Represented R39

Year-Round Research Programs R43

Honors R55

Board of Trustees and Committees R62

Administrative Support Staff R66

Members of the Corporation

Life Members R69

Members R70

Associate Members R80

Certificate of Organisation R84

Articles of Amendment R84

Bylaws R84

Publications . R89

Photo credits:

B. Armstrong . . . .R5. R21, R22, R23, R24. R25, R27.
R30, R31, R35, R36. R37. R69, R84

J. Atema R44

M. Dobbins R46

M. Dornblaser Rl

L. Eckelbecker R58

L. M. Colder R3 (bottom)

R. Howard R55

T. Kleindinst R19

B. Liles R2

G. G. Lower R7

A. C. Malier R43

P. Presley R4

R. Silver R48 (bottom)

Tsoi/Kobus R3 (top)

L. Amai-al Zettler . R62

Report of the Director
and Chief Executive Officer

I am pleased to share with you this report as I have
each year for the past seven years. 1999 is especially
meaningful as it marks my last full year serving as
Director and CEO of this wonderful institution. In August
of 2000 I will leave the Laboratory to serve as the
President of Beloit College, a small liberal arts institution
in Wisconsin. This is an exciting opportunity for my
family and me, but leaving the MBL. the community that
we call home, and our friends and colleagues, will be
difficult.

I have been proud to serve as Director of the Marine
Biological Laboratory. The MBL is a remarkable and
special place, thanks largely to the dedication and
commitment of its scientists and staff. Curiosity,
camaraderie, and thoughtfulness abound here. Throughout
my tenure I have been impressed by the innovation and
technical expertise at the Laboratory, which enables awe-
inspiring advances in our knowledge.

1999 was a landmark year of growth and prosperity for
the Marine Biological Laboratory. I am pleased to report
that our finances are sound, our educational programs
exceptional and expanding, and our research efforts
increasingly exciting and novel. More and more, public
awareness of the MBL's importance to biology,
biomedicine. and environmental science is growing.

I am excited about what the future holds for the Marine
Biological Laboratory. Thanks to the generosity of the
many donors to the Discovery Campaign, we have
already enhanced our educational program, strengthened
our resident and summer research programs, made major
strides towards building a new research facility for The
Ecosystems Center, and begun shoring up our physical
plant. Although we have work yet to do to complete the
Campaign by December 2000, the Laboratory is now well
positioned to continue its leadership role in the

biomedical and environmental sciences well into the 2P'
Century.

The Marine Resources Center

One of the many gratifying gifts of this campaign was
made recently by a long-time, dear friend of the Marine
Biological Laboratory. Late in 1999. Honorary Trustee
Ellen Grass made a historic gift when she endowed the
director's chair of the Marine Resources Center. This is
the first time in the history of the Laboratory that a
research center director's chair has been endowed. This
far-sighted gift will enable us to expand our research
projects in the Marine Resources Center, while ensuring
the MBL's ability to attract high-quality leadership for
this key facility in perpetuity.

The MRC is one of the world's most advanced
facilities for maintaining and culturing aquatic organisms
essential for biological, biomedical, ecological and
aquacultural research. As I reported last year, the Ryan-
Dowling Program in Scientific Aquaculture has been
established at the Marine Resources Center. We are in the
final stages of conducting a national search for a scientific
aquaculturist who will oversee this exciting effort.

The MRC is already actively culturing organisms for
biomedical research. Currently the MRC is host to a
colony of zebrafish. These tiny freshwater fish have
become an exciting and important research model used by
embryologists, geneticists and developmental
neuroscientists. Interestingly, zebrafish and humans share
much of the same genetic material. These two-inch
natives of India may hold the key to understanding how
all vertebrates — including humans — develop from an
embryo into a whole organism. What we learn from a
zebrafish ultimately may help us understand — and perhaps

R2 Annual Report

treat — a wide range of birth defects, among other
disorders.

MRC Director Roger Hanlon and his colleagues made
substantial progress in 1999 in adapting and applying
DNA fingerprints to test sexual selection processes in
squid. Their results are being used by fishery managers as
they decide whether to continue to allow targeted fishing
on spawning squids off Cape Cod. In another area of
research at the MRC, studies have progressed on
understanding the mechanisms and function of
polarization vision in cephalopods. MRC investigators
recently found that cuttlefish are able to overcome the
counter-shading camouflage of silvery fishes by detecting
polarization patterns that are reflected by fish scales. The
result is that cuttlefish and squid can easily detect and
prey upon species that are otherwise camouflaged to most
predators.

The Ecosystems Center

One of the most crucial objectives remaining to be met
in our Discovery Campaign is building the new
Environmental Sciences Building to house the MBL's
Ecosystems Center. Founded 25 years ago, the Center is
home to an interdisciplinary group of scientists whose
expertise covers the fields of terrestrial and aquatic
ecology, microbiology, chemistry, botany, zoology,
physiology, hydrology, mathematics, and genetics. Their
goal is to study the impact of humankind on the
environment and discover what must be done to sustain
and manage the earth's resources.

Among key environmental issues being examined are
the ecological consequences of global warming, the
effects of tropical deforestation, how trees in northeast
forests are handling excess nitrogen, and how pollution
and habitat destruction are damaging coastal ecosystems.
The problems are global and so are the Center's research
sites. MBL scientists have been conducting more than 30

projects around the world — in Brazil, Alaska, Sweden,
Russia, and East Africa, as well as closer to home in the
woods of northern New England and along coastal
estuarine systems at both Plum Island, north of Boston,
and here in Waquoit Bay on Cape Cod.

This summer, for example. Senior Scientist Bruce
Peterson will travel to Siberia to work on the Russian-
American Initiative on Land-Shelf Environments
sponsored by the National Science Foundation. The goal
of the project is to estimate the flux of nutrients from
Eurasia to the Arctic Ocean.

Nearby on Martha's Vineyard, scientists from The
Ecosystems Center are working on a pilot ecosystem
restoration program. Assistant Scientists Chris Neill and
Mathew Williams have begun collaborating with The
Nature Conservancy on a 10-year study of how a forest
functions. The project involves large-scale cutting,
burning, and restoration on a track of land on the
Vineyard. Pre-treatment monitoring will be conducted in
2000; cutting and burning treatments are scheduled to
begin in 2001. This is a great opportunity to see how a
forest works and to determine how a prior, less forested
landscape functioned. Center scientists will concentrate on
understanding how such treatments influence water
balance, soil nitrogen cycling, and the retention and
movement of nitrogen to adjacent coastal ponds. The
project will also provide The Nature Conservancy with
practical information on restoration design and
techniques, as well as provide a place to bring people
who are interested in biodiversity preservation and its
connection with ecosystem functions. Center scientists see
this as a pilot project for how they might eventually
restore larger areas of fire-adapted ecosystems in the
Vineyard State Forest and at the Massachusetts Military
Reservation.

Because of the need to identify and confront real and
present worldwide threats to the environment, it is not
surprising that The Ecosystems Center has grown so
dramatically in size, scope, and reputation. The staff has
increased six-fold and the budget has more than doubled
in recent years, making the Center's office and laboratory
space inadequate, and forcing researchers to work at
scattered locations around the MBL campus.

Soon, Ecosystems Center scientists will be in the
Environmental Sciences Building on Albatross Street.
This new building will have a cutting-edge geographic
information systems facility, state-of-the-art laboratories
for plant and soil sample analysis, a stable isotope
laboratory, offices, teaching facilities, a
classroom/conference room for the Semester in
Environmental Science Program, ample storage areas for
all equipment, and field staging areas.

To that end, the MBL has received an important
challenge grant of $500.000 from The Kresge Foundation.

Report of the Director and CEO R3

Payment of that grant is conditional upon the MBL
raising an additional $2. 1 million for the project. With
further fundraising success, we hope to break ground for
the $8 million building this spring. What a fitting way to
celebrate The Ecosystem Center's 25th anniversary.

Josephine Bay Paul Center for Comparative
Molecular Biology and Evolution

At the Bay Paul Center, 35 scientists and support staff
continue to explore a number of aspects of molecular
evolution and comparative molecular biology. Their
efforts to sequence the genome of Giardia, a water-borne
human pathogen that attacks the intestinal tract, is now
more than 50 percent complete. Bay Paul Center Director
Mitchell Sogin is the principal investigator on that study,
which is sponsored by a major grant from the National
Institutes of Health.

In 1998 the Center welcomed Dr. Michael Cummings
to the scientific staff. He is currently investigating ways
to accurately predict drug-resistant strains of tuberculosis
by examining specific gene sequences. With a recent
grant from the Alfred P. Sloan Foundation, he is also
developing novel computer-based analytical procedures to
study color vision.

In January 2000, Dr. Jennifer Wernegreen joined the
staff as an Assistant Scientist. Dr. Wernegreen comes to
the MBL from the University of Arizona where she was
an NIH postdoctoral fellow in the Department of Ecology
and Evolutionary Biology. Wernegreen uses comparative
approaches to explore the molecular evolution of certain
species of bacteria that live symbiotically with specific
insects. These bacteria are thought to supplement
nutritionally unbalanced diets of their hosts by providing
essential amino acids, vitamins, and other nutrients.

Other Research Initiatives

Elsewhere at the MBL, scientists are working on a
variety of biological and biomedical problems. Dr. David
Keefe has developed a new method of non-invasively
imaging the meiotic spindle of eggs during human in
vitro fertilization at his clinic at Women and Infants
Hospital in Rhode Island. This technique was developed
at the MBL using the polscope designed by Dr. Rudolf
Oldenbourg. Application of this exciting technology has
doubled the pregnancy rates during intracytoplasmic
sperm injection, and improved clinicians' ability to
predict fertilization. Dr. Keefe has also been working with
Dr. Peter Smith, Director of the BioCurrents Research
Program at the MBL. They have developed a novel
approach to measuring oxygen uptake by individual
mammalian embryos using a non-invasive, self-
referencing oxygen sensor. This work was recognized as
one of the 50 most important biotechnology
breakthroughs at a special program at the National
Institutes of Health. It was also a semi-finalist for the
Christopher Columbus Science Innovation Award
sponsored by Discover magazine.

MBL Distinguished Scientist Shinya Inoue was
awarded two patents in 1999 for the Slit Scan Centrifuge
Microscope and the Centrifuge Microscope Capable of
Realizing Polarized Light Observation. These instruments
were developed in collaboration with Olympus Optical
and Hamamatsu Photonics Co. Dr. Inoue has been using
these and other microscopes that he has developed over
the years to study several unexplored attributes of living
cells. Over the past year he has taken the first
measurements of crawling forces of a cell, shown how
mammalian cells can be separated into fractions that only
contain certain types of organelles, and recorded thin

R4 Annual Report

optical slices of rapid molecular changes in living cells
hitherto unseen. He's been able to trace the assembly of
protein filaments that move chromosomes in yeast cells
and track the movement of individual protein molecules
within those filaments — something thought impossible
only a few years ago.

Two new resident scientists joined the MBL in 1999.
Dr. Paul Colinvaux, an Adjunct Scientist, reconstructs
Pleistocene climatic and environmental histories of the
continents from the sediments of ancient lakes,
particularly those found in the Amazon basin. Research
has shown that the lowland Amazon forests persisted
through glacial cycles, with some re-assortment of species
as temperatures fluctuated from the last glacial maximum
to the present. Although Colinvaux's research now is
concentrated in the Neotropics, he maintains sites in
Alaska and Russia for continued study of the
paleoecology of the Arctic.

Dr. Ayse Dosemeci came to the MBL in October from
the National Institutes of Health as an Adjunct Scientist.
She is a neurobiologist who studies synaptic plasticity, a
biological phenomenon that may be involved in learning
and memory and other central nervous system functions.

Summer Research

Last summer — as has been the case for more than a
century — investigators from around the world came to the
Marine Biological Laboratory to do research. As always,
the excitement of study, scientific exchange, and
discinery was great. The 126 principal investigators came
from 12 countries and represented more than 100
institution--
One of the many highlights from last summer's
research was the ivf-nt publication of a paper in Nature
magazine by Drs. Miuuel Holmgren, Jonathan Wagg.
Francisco Bezanilla, Robert Rakowski, Paul De Weer,
and David Gadsby. In that paper they describe their latest
findings about how a specialized cellular machine, the

sodium/potassium exchange pump, works. Because this
pump is essential to the health of virtually every cell in
all animals, including humans, scientists at the MBL have
spent years studying the molecular mechanisms by which
this pump transports sodium and potassium ions across
cellular membranes. They use the giant nerve cell of the
Woods Hole squid as a model system for their research.
These investigators already knew that this pump, which is
a single protein molecule, transports three sodium ions
across the cell membrane at once. In the Nature paper
they showed that three separate changes in the shape of
the pump protein release the three sodium ions from the
pump one at a time, in a fixed sequence. This new
information will help scientists understand in greater
detail how these, and other, essential ion pumps perform
the crucial work that keeps all our cells alive.

Another important cellular pump, the sarcoplasmic
reticulum-Ca~+ pump, is being studied by Dr. Larry
Rome and his colleagues from the University of
Pennsylvania. They are interested in learning more about
how muscle design influences an organism's behavior.
This past summer at the MBL they developed a new way
to measure, in real time, calcium pumping and
sarcoplasmic reticulum function in muscle fibers — a
necessary first step in understanding the biological basis
of behavior. As their model, they used the swimbladder
muscle of the toadfish, the fastest known of all vertebrate
fast muscle tissues. The muscles that envelop the
swimbladder contract and relax at a remarkable 200 times
per second, creating the animal's distinctive "boatwhistle"
mating call. These muscles operate almost 100 times
faster than the fish's locomotory muscles, which function
just adequately to get the rather sluggish creature where it
needs to go.

Among the principal summer investigators at the
Laboratory last summer, 1 2 were awarded Grass
Fellowships in neurobiology and 19 were awarded other
named MBL Fellowships to conduct research on a variety
of biological topics at the MBL. These scientists come
from around the country and the world to work in Woods
Hole for the summer. For example. Pavel Balaban of the
Russian Academy of Sciences used the mollusc Helix to
study putative command neurons that modulate

Report of the Director and CEO R5

withdrawal behavior and the activities of neurons
underlying this behavior. Elizabeth Jonas of Yale
University School of Medicine measured ionic currents on
membranes of mitochondria during neurotransmission in
squid. Anthony DePass of Long Island University used
sea urchins to study how calcium enters heart and nerve
cells when a cell is stimulated. And David Ogden of the
National Institute for Medical Research in London studied
how the skate senses small electric potentials in
surrounding seawater to locate prey.

Joining the annual gathering of scientists were 19 print
and broadcast journalists who had been awarded MBL
Science Writing Fellowships. This program offers writers
the chance to step into the shoes of people they cover, to
study basic biomedical and environmental science and —
for some — to spend additional time doing course work in
Woods Hole or research at Ecosystems Center field sites
in Alaska and Brazil.

Education

It is my great pleasure to tell you that in 1999 the
Howard Hughes Medical Institute awarded a new grant of
$2.2 million to the MBL. The four-year award will
support many of our advanced laboratory courses for
graduate students, postdoctoral fellows, and university
faculty members. The MBL has received $8.2 million in
HHMI grants since 1988 for which we are extremely
grateful. In that time, more than 4900 students have
participated in courses taught by the best faculty in the
world.

Last summer, the MBL offered 20 courses, involving
579 faculty and guest lecturers and 427 students.
Molecular Biology of Aging and a second session of
Medical Informatics were added to the course list in
1999. Also in 1999 we welcomed David Garbers (HHMI,
University of Texas Southwestern Medical Center) and
Randall Reed (HHMI, Johns Hopkins University School
of Medicine) as new directors of the Physiology course.
This past summer was also the final year in the tenures of
the directors of the Neurobiology, Neural Systems &
Behavior, and Microbial Diversity courses. My special
thanks to retiring directors Gary Banker and Dan
Madison, Janis Weeks and Harold Zakon, and Ed
Leadbetter and Abigail Salyers.

Last fall, the Semester in Environmental Science
Program was held at the MBL for the third time, and
results again were impressive. In 1999 four new
schools — Beloit College, Lawrence University,
Southwestern University, and Trinity University — joined
the consortium of institutions that participate in the
program, bringing the total number of colleges and
universities to 37. Undergraduates from a number of these

small liberal arts colleges and universities around the
country were immersed in a 15-week program of lectures,
laboratory and fieldwork and independent research, all of
this under the sponsorship of the MBL's Ecosystems
Center. Students explored how human activity, such as
deforestation, fisheries exploitation, changes in
biodiversity, eutrophication and fossil fuel combustion
alter ecological processes and ecosystem structure locally,
regionally, and globally.

Trustees

The Board of Trustees elected three new members at
November's meeting. Nobel Laureate Dr. Torsten I.
Wiesel is President Emeritus and Vincent and Brooke
Astor Professor Emeritus of The Rockefeller University.
Dr. George M. Langford is the Ernest Everett Just
Professor of Natural Sciences and Professor of Biological
Sciences at Dartmouth College and Adjunct Professor of
Physiology at Dartmouth Medical School. M. Howard
Jacobson has been a Senior Advisor at Bankers Trust
Private Bank since 1991. Current Board members G.
William Miller, Frank Press, and Christopher M. Weld
were re-appointed to the Board as members of the class
of 2004. Longtime Trustee and stalwart supporter of the
MBL Mary Ellen Cunningham was appointed an honorary
member of the board. The Board also recognized the
efforts of retiring members Alexander W. Clowes. Story
C. Landis, and Irwin B. Levitan.

The Discovery Campaign

The Discovery Campaign, now in its final year, was
remarkably successful in 1999, thanks to the tireless
efforts of our volunteers and the generosity of donors.
More than $9.9 million in private support was raised — the
most ever in a single year. Our Annual Fund surpassed

R6 Annual Report

the half-million dollar mark last year as well, raising
nearly 12 percent more than the year before.

This record support pushed the Discovery Campaign past
its $25 million goal in August of 1999, an astonishing 16
months ahead of schedule. By January 1. 2000, we had
raised $30 million in support of research, education, and
facilities at the MBL. As I write this report, some key
objectives still remain to be funded before the Campaign
ends in December 2000. I am confident that we will meet, if
not exceed, these important goals.

With many important gifts and the efforts of the Board's
Investment Committee, the Laboratory's endowment has
also grown dramatically — from $ 1 6 million when I came in
1992 to $47 million at the end of 1999.

I thank all Campaign donors and volunteers for making
1999 such a successful year. Your strong support is a
testimony to your belief in the special mission of the
Marine Biological Laboratory.

/// Conclusion

As was noted many years ago in Life Science in
Woods Hole, science is more than the accumulation of
facts and findings and more than their interpretation.
Science is, most of all, a grand collective curiosity. At
the MBL, that collective curiosity is alive and well,
and everyone who is touched by this place, who
investigates, studies, learns and wonders is looking into
the new century with hope and great expectations. I
know that this place has touched me, and I will always
be grateful for having been a part of it. I will miss this
special institution and the many friends that I have
made here. Thank you for making my tenure at the
MBL such a pleasure.

— John E. Burris

Report of the Treasurer

During 1999 the Marine Biological Laboratory had an
outstanding year. This was the result of favorable growth
in all the areas of operating support and auspicious
investment results that further strengthened the balance
sheet.

All six areas of Operating Support grew by at least 6%,
with double-digit growth in Government Grants (11.9%),
Private Contracts ( 1 1.7%), and Fees for Conferences and
Services (10.2%). The biggest change was from
Contributions, which increased $3.8 million (78.8%) from
1998 levels as the Discovery Campaign hit full stride.
These increases in Operating Support combined with
restraining expense growth to 8% resulted in an almost
sevenfold increase in the Change in Net Assets before
Non-operating Activity. Also note that this included an
underlying positive change in unrestricted net assets from
operations of $138 thousand, the first surplus since 1994.
This is particularly favorable considering these results
include coverage of $1.56 million in depreciation.

Reviewing our Non-operating Activities further
demonstrates positive trends. New to our presentation this
year is a breakout of the Contributions to Plant, which
almost tripled. We have taken this step in light of our
extensive capital improvement plans for the next few
years. By pulling this out of the ongoing operations we
have a much more informative display that gives a better
indication of how we are doing in both areas. Total
Investment Income and Earnings increased sixfold and
enabled us to reinvest over $4.6 million after using
roughly one-fifth for Operations.

As a result, we reported for the fifth year in a row a
positive Total Change in Net Assets. The increase of

$11.2 million represents a very healthy 16.7% return on
average net assets.

A review of the 1 999 balance sheet further demonstrates
the positive trends and our continued strong liquidity. The
increase in Pledges and Other Receivables reflects $3 million
in increased pledge receivables. The Endowment and Similar
Investments increased 21.4%. Also Property. Plant and
Equipment (net) increased (3%) for the first time in five
years. Total Net Assets increased 18.3% during the year,
with Unrestricted Net Assets increasing 7.8%.

A subsequent event, which will have a major impact on
the balance sheet, occurred on March 8, 2000, when the
Massachusetts Development Finance Agency issued on
behalf of the Laboratory $10.2 million in variable rate
revenue bonds. A portion was used to refinance the
Laboratory's long-term debt at a lower interest cost. The
balance of the proceeds will be used to finance capital
improvements of the Laboratory's educational, research,
and administrative facilities, including the Environmental
Sciences Building. The leverage ratio (unrestricted &
temporarily restricted net assets-to-debt) on a pro forma
basis is an acceptable 521% and represents suitable
leverage of the financial strength of the Laboratory.

In summary, the Laboratory had a very successful year
of operations, fundraising, and investment performance
that has greatly enhanced the financial strength of the
Laboratory. The success of the Discovery Campaign and
the bond financing will facilitate the upgrading of the
MBL's physical plant and the continued expansion of our
research and educational activities in the new millennium.

— Mary B. Conrad

Financial Statements

PricewaterhonseCoopers LLP

One International Place
Boston MA 02 110
Telephone (hi 7) 478 5000
f.ii •.mule ((,17) 478 3900

REPORT OF INDEPENDENT ACCOUNTANTS

To the Board of Trustees of
Marine Biological Laboratory:

In our opinion, the accompanying balance sheet of Marine Biological Laboratory (the "Laboratory") as of
December 31, 1999 and the related statements of activities and of cash flows for the year then ended present
fairly, in all material respects, the financial position of the Laboratory as of December 31, 1999, and the
changes in its net assets and its cash flows for the year then ended in conformity with accounting principles
generally accepted in the United States. These financial statements are the responsibility of the Laboratory's
management; our responsibility is to express an opinion on these financial statements based on our audit. We
conducted our audit in accordance with auditing standards generally accepted in the United States. Those
standards require that we plan and perform the audit to obtain reasonable assurance about whether the
financial statements are free of material misstatement. An audit includes examining, on a test basis, evidence
supporting the amounts and disclosures in the financial statements. An audit also includes assessing the
accounting principles used and significant estimates made by management, as well evaluating the overall
financial statement presentation. We believe that our audit provides a reasonable basis for the opinion
expressed above.

Our audit was conducted for the purpose of forming an opinion on the basic financial statements taken as a
whole. The supplemental schedule of functional expenses as of December 31, 1999 is presented for the
purpose of additional analysis and is not a required part of the basic financial statements. Such information
has been subjected to the auditing procedures applied in the audit of the basic financial statements and. in
our opinion, is fairly stated, in all material respects, in relation to the basic financial statements taken as a
whole.

April 7, 2000

MARINE BIOLOGICAL LABORATORY

BALANCE SHEET

As of December 31. 1999

(with comparative totals as of December 31, 1998)

ASSETS

Cash and cash equivalents

Short-term investments, at market (Note 3)

Accounts receivable, net of allowance for doubtful accounts of $59,978 in 1999 and

$34.195 in 1998

Current portion of pledges receivable (Note 8)
Receivables due for costs incurred on grants and contracts
Other assets

Total current assets

Long-term investments, at market (Notes 3 and 4)
Pledges receivable, net of current portion (Note 8)
Plant assets, net (Notes 2. 5 and 6)

Total long-term assets
Total assets

1999

$ 1.942,285
3.182.537

1.158,073

3.974.385
1,380,766

306.518

I | .944.564

45.001,493

3,498.787

20.118.725

68.619.005
$80.563.569

1998

$ 1.187,954
3.56 1. 544

1,242.530

1.607.664

1.531.083

557,908

9.688.683

37,054.120

2.855.352

19.536.171

59.445.643
$69,134.326

LIABILITIES AND NET ASSETS

Current portion of long-term debt (Note 5)
Accounts payable and accrued expenses
Deferred income and advances on contracts

Total current liabilities

Annuities and unitrusts payable

Long-term debt, net of current portion (Note 5)

Advances on contracts

Total long-term liabilities

Total liabilities
Commitments and contingencies (Notes 5, 7, 9 and 10)

267,404

1.957,508

656.745

2.881,657

1.460.94X
2,056.692
1.574.758

5.0C)2.39S
7.974,055

243.274

2,057.741

462.873

2.763.888

1.412.200
2,324.096
1.272.390

5.008.686

7.772.574

Net assets:
Unrestricted
Temporarily restricted
Permanently restricted

Total net assets (Note 2]

Total liabilities and net assets

19.887.437
33,349,244
19.352.833

72,589.514
$80,563.569

1 8.45 1 ,865
25,635,237
17.274.650

61.361.752
$69,134.326

The accompanying notes are an integral part of the financial \tiiicnifiii.\.

MARINE BIOLOGICAL LABORATORY

STATEMENT OF ACTIVITIES

for the year ended December 31, 1999

(with comparative totals for the year ended December 31, 1998)

Operating support and revenues:
Government grants
Private contracts
Laboratory rental income
Tuition

Fees for conferences and services
Contributions
Investment income
Miscellaneous revenue
Present value adjustment to annuities
Net assets released from restrictions

Total operating support and revenues

Expenses:
Research
Instruction

Conferences and services
Other programs (Note 2)

Total expenses

Change in net assets before nonoperating activity

Nonoperating revenue:
Contributions to plant:
Private
Government
Release from restriction

Invested in plant

Total investment income and earnings

Less: investment earnings used for operations

Reinvested (utilized) investment earnings
Total change in net assets
Net assets, beginning of year

Net assets, end of year

Temporarily Permanently

1999

1998

Unrestricted

Restricted Restricted

Total

SI 2.248.442

$ — $ —

$12.248.442

$10,943.239

1,819,240

— —

1.819.240

1,629,283

1.548.168

— —

1,548.168

1.470.372

537.835

— —

537,835

489,726

3.765,039

— —

3,765.039

3,415,519

1,781.643

4.604.501 2.234.375

8.620.519

4.822,227

705,85 1

1.354.627

2,060.478

1,955,735

466.903

— —

466.903

405,633

—

(22.680) (7,853)

(30,533)

(76,702)

3.705,796

(3.705.796) —

—

26,578,917

2,230,652 2.226,522

31,036,091

25,055.032

14.147.645

12,666,746

4,742,287

— —

4.742.287

4,433.789

2.252.842

— —

2,252,842

1.999.433

5.297,773

— —

5.297.773

5,365,530

26,440,547

26.440.547

24.465.498

138.370

2.230.652 2,226,522

4.595.544

589.534

1,507,319 250,000

1,757.319

515,775

198.443

— —

198,443

—

912,046

(912,046) —

1.1 10.489

595.273 250,000

1,955.762

515,775

220.372

6.116.443 (398.339)

5,938.476

819,705

(33.659)

(1.228.361) —

(1.262.020)

(1,246,913)

186.713

4,888.082 (398.339)

4.676,456

(427,208)

1.435.572

7,714.007 2.078,183

11.227,762

678.101

18,451.865

25.635.237 17,274.650

61,361.752

60.683.651

$19,887,437

$33.349.244 $19.352.833

$72.589.514

$61.361.752

The accompanying notes tire cm integral port of the financial sttttetnents.

RIO

MARINE BIOLOGICAL LABORATORY

STATEMENT OF CASH FLOWS

for the year ended December 31. 1999

(with comparative totals for the year ended December 31, 1998)

1999

Cash flows from operating activities:
Change in net assets
Adjustments to reconcile change in net assets to net cash provided hy

(used in) operating activities:
Depreciation

Unrealized (gain) loss on investments
Realized gain on investments

Present value adjustment to annuities and unitrusts payable
Contributions restricted for long-term investment and annuities
Provision for bad debt
Provision for uncollectible pledges
Change in certain balance sheet accounts:

Accounts receivable

Pledges receivable

Grants and contracts receivable

Other assets

Accounts payable and accrued expenses

Deferred income and advances on contracts

Annuities and unitrusts payable

Advances on contracts

Net cash provided by operating activities

Cash flows from investing activities:
Purchase of property and equipment
Proceeds from sale of investments
Purchase of investments

Net cash used in investing activities

Cash flows from financing activities:

Payments on annuities and unitrusts payable
Receipt of permanently restricted gifts
Annuity and unitrusts donations received
Payments on long-term debt

Net cash provided by financing activities

Net increase in cash and cash equivalents
Cash and cash equivalents at beginning of year

Cash and cash equivalents at end of year

$ 11.227,762

1,562.487
(3,544.380)
(1,639.795)

30.533
(2,485,624)

36,968

47.489

(3,010.156)

150,317

25 1 .390

(100,233)

193.872

68,112

302.368

3,091.110

(2,145.041)
63.101.047
(65.485.238)

(4,529. 232 1

(49.897)
2,438,148

47,476
(243.274)

2.192.453

754.33 1
1.187.954

$ 1.942,285

1998

$ 678,1

1,505,696
2,755.079
(2,805.560)
76,702
(682,817)
15.771
250.000

(36,520)

(255.134)

(373.918)

2.361

562.793

78,615

163.700

(160.818)

1,774,051

(1.015.287)

18,935.050

(19.478.036)

(1.558,273)

(41,785)

653.152

29,665

(229.657)

411.375

627,153
560.801

$ 1,187.954

The accompanying notes are an integral part of the financial statements.

Rll

R12 Annual Report

Marine Biological Laboratory
Notes to Financial Statements

1. Background:

The Marine Biological Laboratory (the "Laboratory") is a private, independent not-for-profit research and educational institution dedicated to
establishing and maintaining a laboratory or station for scientific study and investigation, and a school for instruction in biology and natural history.
The Laboratory was founded in 1888 and is located in Woods Hole. Massachusetts.

2. Significant Accounting Policies:
Basis of Presentation

The accompanying financial statements have been prepared on the accrual basis of accounting and in accordance with the principles outlined in the
American Institute of Certified Public Accountants' Audit Guide, "Not-For-Profit Organizations." The financial statements include certain prior-year
summarized comparative information in total but not by net asset class. Such information does not include sufficient detail to constitute a presentation
in conformity with generally accepted accounting principles. Accordingly, such information should be read in conjunction with the Laboratory's
financial statements for the year ended December 31, 1998, from which the summarized information was derived.

The Laboratory classifies net assets, revenues, and realized and unrealized gains and losses based on the existence or absence ol donor-imposed
restrictions and legal restrictions imposed under Massachusetts State law. Accordingly, net assets and changes therein are classified as follows:

Unrestricted

Unrestricted net assets are not subject to donor-imposed restrictions of a more specific nature than the furtherance of the Laboratory's mission
Revenues from sources other than contributions are generally reported as increases in unrestricted net assets. Expenses are reported as decreases in
unrestricted net assets. Gains and losses on investments and other assets or liabilities are reported as increases or decreases in unrestricted net asset-,
unless their use is restricted by explicit donor stipulations or law. Expirations of temporary restrictions on net assets, that is, the donor-imposed
stipulated purpose has been accomplished and or the stipulated time period has elapsed, are reported as reclassirications between the applicable classes
of net assets and titled "Net assets released from restrictions."

Temporarily Restricted

Temporarily restricted net assets are subject to legal or donor-imposed stipulations that will be satisfied either by the actions of the Laboratory, the
passage of time, or both. These assets include contributions for which the specific, donor-imposed restrictions have not been met and pledges,
annuities, and unitrusts for which the ultimate purpose of the proceeds is not permanently restricted. As the restrictions are met, the assets are released
to unrestricted net assets. Also, rcali/cd/unrealized gains/losses associated with permanently restricted gifts which are not required to be added to
principal by the donor are classified as temporarily restricted but maintain the donor requirements for expenditure.

Permanently Restricted

Permanently restricted net assets are subject to donor-imposed stipulations that they be invested to provide a permanent source of income to the
Laboratory. These assets include contributions, pledges and trusts which require that the corpus be invested in perpetuity and only the income be made
available for program operations in accordance with donor restrictions.

Nonoperating revenues include realized and unrealized gains on investments during the year as well as investment income on the master pooled
investments and revenues that are specifically for the acquisition or construction of plant assets. Investment income from short-tenn investments and
investments held in trust by others are included in operating support and revenues. To the extent that nonoperating investment income and gains are
used for operations as determined by the Laboratory's total return utilization policy (see below), they are reclassilied from nonoperating as "Investment
earnings used for operations" to operating as "Investment income" on the statement of activities. All other activity is classified as operating revenue.
The Laboratory recorded net realized gains of $1.639.795. net unrealized gains of $3.544.380 and dividend and interest income of $1.533.579 in 1999.

Cash anil Cash Equivalents

Cash equivalents consist of resources invested in overnight repurchase agreements and other highly liquid investments with original maturities of three
months or less.

Financial instruments which potentially subject the Laboratory to concentrations of risk consist primarily of cash and investments. The Laboratory
maintains cash accounts with one hankini; institution.

Investments purchased by the Laboratory are carried at market value. Donated investments are recorded at fair market value at the date of the gift. For
closely held non-publicly traded investments, management determines the fair value, based upon the most recent information available from the Limited
Partnership. For determination of gain or loss upon disposal of investments, cost is determined based on the first-in, first-out method. Investments with
an original maturity of three months to one year, or those that are available for operations within the next fiscal year, are classified as short-term. All
other investments are considered long-term. Investments are maintained primarily with three institutions.

In 1424, the Laboratory became the beneficiary of certain investments, included in permanently restricted net assets, which are held in trust by others.
The Laboratory has the continuing rights to the income produced by these funds in perpetuity, subject to the contractual restrictions on the use of such

Financial Statements R13

funds. Accordingly, the trust has established a process to conduct a review every ten years by an independent committee to ensure the Laboratory
continues to perform valuable services in biological research in accordance with the restrictions placed on the funds by the agreement. The committee
met in 1994 and determined that the Laboratory has continued to meet the contractual requirements. The market values of such investments are
$7,275,488 and $7,673,828 at December 31, 1999 and 1998. respectively. The dividend and interest income on these investments, included in
unrestricted support and revenues, totaled $221.882 and $260.80? in 1999 and 1998. respectively.

Investment Income ami Distribution

For me master pooled investments, the Laboratory employs a total return utilization policy that establishes the amount of the investment return made available
for spending each year. The Finance Committee of the Board of Trustees has approved a standing policy that the withdrawal will be based on a percentage
of the 12 quarter average ending market values of the funds. The market value includes the principal plus reinvested income, realized and unrealized gains and
losses. Spending rates in excess of 5%, but not exceeding 7%. can be utilized if approved in advance by the Finance Committee of the Board of Trustees. For
fiscal 1999 and 1998, the Laboratory obtained approval to expend 6% of the latest 12 quarter average ending market values of the investments.

The net appreciation on permanently and temporarily restricted net assets is reported together with temporarily restricted net assets until such time as
all or a portion of the appreciation is distributed for spending in accordance with the total return utilization policy and applicable state law.

Investment income on the pooled investment account is allocated to the participating funds using the market value unit method (Note 4).
Plum Assets

Buildings and equipment are recorded at cost. Donated facility assets are recorded at fair market value at the date of the gift. Depreciation is computed
using the straight-line method over the asset's estimated useful life. Estimated useful lives are generally three to five years for equipment and 20 to 40
years for buildings and improvements. Depreciation is not recorded for those assets classified as construction-in-process as they have not yet been placed
into service. Depreciation expense for the years ended December 31. 1999 and 1998 amounted to $1.562.487 and $1,505.696. respectively, and has been
recorded in the statement of activities in the appropriate functionalized categories. When assets are sold or retired, the cost and accumulated depreciation
are removed from the accounts and any resulting gain or loss is included in unrestricted income for the period.

Annuities and Unitrnsts Payable

Amounts due to donors in connection with gift annuities and unitrusts are determined based on remainder value calculations, with varied assumptions
of rates of return and payout terms.

Deferred Income and Advances on Contracts

Deferred income includes prepayments received on Laboratory publications and advances on contracts to be spent within the next year. Advances on contracts
includes funding received for grants and contracts before it is earned. Long-term advances are invested in the master pooled account until they are expended.

Revenue Recognition

Sources of revenue include grant payments from governmental agencies, contracts from private organizations, and income from the rental of laboratories
and classrooms for research and educational programs. The laboratory recognizes revenue associated with grants and contracts at the time the related
direct costs are incurred or expanded. Recovery of related indirect costs is recorded at predetermined fixed rates negotiated with the government.
Revenue related to conferences and services is recognized at the time the service is provided, while tuition revenue is recognized as classes are offered.
The tuition income is net of student financial aid of $527.258 and $523,190 in 1999 and 1998. respectively. Fees for conferences and other services
include the following activities: housing, dining, library, scientific journals, aquatic resources and research services.

Contributions

Contribution revenue includes gifts and pledges. Gifts are recognized as revenue upon receipt. Pledges are recognized as temporarily or permanently
restricted revenue in the year pledged and are recorded at the present value of expected future cash flows, net of allowance for unfulfilled pledges. Gifts
and pledges, other than cash, are recorded at fair market value at the date of contribution.

Expenses

Expenses are recognized when incurred and charged to the functions to which they are directly related. Expenses that relate to more than one function
are allocated among functions based upon either modified total direct cost or square footage allocations.

Other programs expense consists primarily of fundraising, year-round labs and library room rentals, costs associated with aquatic resource sales and
scientific journals. Total fundraising expense for 1999 and 1998 is $1.008,920 and $1.037.495. respectively.

Use of Estimates

The preparation of financial statements in conformity with generally accepted accounting principles requires management to make estimates and
assumptions that affect the reported amounts of assets and liabilities and disclosure of contingent assets and liabilities at the date of financial statements
and the reported amounts of revenues and expenses during the reporting period. Actual results could differ from those estimates.

Tax-Exempt Status

The Laboratory is exempt from federal income tax under Section 501(c)(3) of the Internal Revenue Code.

Reclassification

Certain prior year balances have been reclassified to conform with the current year presentation.

R14 Annual Report

3. Investments:

value of investments at December 31, 1999 and 1998:
Market

Cost

The following is a summary of the cost and market

Certificates of deposit
Money market securities
U.S. Government securities
Corporate fixed income
Common stocks
Mutual funds
Limited partnerships

Total investments
Investment portfolios for the years ended December

Short-Term Investments

1999

$ 40.000
1,781,128
69, 1 25
2,364,068
15,665.205
26,664,204
1 ,600,300

1998

1999

$ 40,000
1,781,128

69,951
2,536,808
10.608,588
23.851,004
958,982

7998

$ 40.000
1.052,276
1.136.219
2,472.653
4,290.581
26.225,214
958.982

$ 40,00(1
1,052,276
1,397,686
2,504,507
5,033,704
29,548,891
1,038,600

$48, 1 84.030

$40,615.664

$39,846,461

$36,175,925

31, 1999 and 1998 are as follows:
Market

Cost

1999

7998

$ 40,000
559,314
2,955,989
6,241

1999

$ 40,000
233,938
2,965.273
33, 1 1 9

1998

$ 40,000
233,938
2,875,480
33,119

$ 40.000
559,314
2,940.929

6,241

Certificates of deposit
Money market 1 784 Fund
Mutal funds
Common stocks in transit

Total investments
Lang-Term Investments

3,182.537

3,561,544

3,272,330

3,546,484

$35,354,938

5.717,108
1.558.380
2,371.067

$27,057,909

6,038.153
1,635,675
2.322,383

$27,514,505

5,335,721
1 ,448,569
2.275,336

$23,723,343
5,433,574

Pooled investments:
Master pooled investments
Separately invested:
General Chase Trust
Library Chase Trust
Annuity and unitrust investments

Total
Total investments

1,477,462
1,995.062

45.001.493

37,054.120

36.574,131

32,629,441

$48,184,030

$40.615,664

$39,846.461

$36,175,925

Financial Statements R15

4. Accounting for Pooled Investments:

Certain net assets are pooled for investment purposes. Investment income from the pooled investment account is allocated on the market value unit basis,
and each fund subscribes to or disposes of units on the basis of the market value per unit at the beginning of the calendar quarter within which the
transaction takes place. The unit participation of the funds at December 31, 1999 and 1998 is as follows:

Unrestricted
Temporarily restricted
Permanently restricted
Advances on contracts

1999

8.573
42.351
65.789

5.557

122.270

4.001
44.455
65.016

d.437

119,909

Pooled investment activity on a per-unit basis was as follows:

Unit value at beginning of year
Unit value at end of year

Total return on pooled investments

1999

$ 225.51
2X3.37

$ 57.86

1998

$ 220.30

225.51

$ 5.21

5. Long-Term Debt:

Long-term debt consisted of the following at December 31:

Variable rate (6.3% at December 31, 1999) Massachusetts Industrial Finance

Authority Series 1992A Bonds payable in annual installments through 2012
6.63% Massachusetts Industrial Finance Authority Series 1992B Bonds.

payable in annual installments through 2012
5.8% The University Financing Foundation. Inc.. payable in monthly

installments through 2000
5.8% The University Financing Foundation. Inc., payable in monthly

installments through 2002

1999

$ 890.1

1 , 1 75,000
120,929

138.167
$2,324.096

/99.S'

$ 925,000

1,230,000

226,024

186,346

Subsequent to year-end, all existing debt was extinguished and new debt was issued (Note 10).

In 1992, the Laboratory issued $1,100,000 Massachusetts Industrial Finance Authority (MIFA) Series 1992A Bonds with a variable interest rate and
$1,500,000 MIFA Series 1992B with an interest rate of 6.63%'. Interest expense totaled $142,545 for the year ended December 31. 1999. The Series
1992 A and B Bonds mature on December 1. 2012 and are collateralized by a first mortgage on certain Laboratory property.

On March 17. 1998. the Laboratory entered into a ten-year interest rate swap contract in connection with the Series 1992 A Bonds. This contract
effectively fixes the interest rate at 6.30% through December 17, 2008. This contract was canceled as part of the extinguishment of debt and new debt
issuance.

The agreements related to these bonds subject the Laboratory to certain covenants and restrictions. Under the most restrictive covenant of this debt, the
Laboratory's operating surplus, exclusive of interest expense and depreciation expense, must he greater than or equal to 1.2 times all debt service
payments, as defined by the agreement.

In 1996. the Laboratory borrowed $500.000 with an interest rate of 5.8% per annum from the University Financing Foundation, Inc. The interest expense
for the year ended December 31, 1999 was $10.345. The loan matures in 2000 and is collateralized by 50,000 shares of a fixed income fund with a
fair value of $576,000 at December 31, 1999.

In 1997, the MBL borrowed $2.50,000 with an interest rate of 5.8%^ per annum from the University Financing Foundation, Inc. The interest expense
for the year ended December 3 1 , 1999 was $9,541. This loan matures in 2002 and is collateralized by 19,440 shares of a fixed income mutual fund with
a fair value of $223,949 at December 31, 1999.

The Laboratory has a line of credit agreement with a commercial bank from which it may draw up to $1,000,000. This line of credit has an interest
rate of prime plus V? percent. The line has no expiration date but is reviewed periodically by the hank for renewal. No amounts were outstanding under
this agreement as of December 31, 1999 and 1998.

R16 Annual Report

6. Plant Assets:

Plant assets consist of the following at December 3 1 :

Land
Buildings
Equipment
Construction in process

Total
Less: Accumulated depreciation

Plant assets, net

7999

$ 702,908

33,702,485

4,667,026

1.510.821

40,583,240
(20.464,515)

$20.118.725

/99.S'

$ 702,908

33,082.164

4.401.1X4

251.943

38,438,199

(18.902.028)
$19,536.171

7. Retirement Pliin:

The Laboratory participates in the defined contribution pension plan of TIAA-CREF (the "Plan"). The Plan is available to permanent employees who
have completed two years of service. Under the Plan, the Laboratory contributes 10% of total compensation for each participant. Contributions
amounted to $785,509 and $737,156 for the years ended December 31, 1999 and 1998, respectively.

8. Pledges:

Unconditional promises to give are included in the financial statements as pledges receivable and the related revenue is recorded in the appropriate net
asset category. Unconditional promises to give are expected to be realized in the following periods:

In one year or less

Between one year and five years

After five years

1999

$3.974,385

3.632,683

202.948

J99.S

$1,607,664

3.110.354

146.586

Total

7.810.016

4.864.604

Less: discount of $236,844 in 1999 and $301,588 in 1998
and allowance of $100.000 in 1999 and $100,000 in 1998

(336.844)

(401.588)

$7,473.172

$4.463.016

Financial Statements R17

9. Postretiremen! Benefits:

The Laboratory accounts for its postretiremen! benefits under Statement No. 106. "Employers' Accounting for Postretiremen! Benefits Other than
Pensions," which requires employers to accrue, during the years that the employee renders the necessary service, the expected cost of benefits to be
provided during retirement As permitted, the Laboratory has elected to amortize the transition obligation over 20 years commencing on January I. 1994.

The Laboratory's policy is that all current retirees and certain eligible employees who retired prior to June 1. 1994 will continue to receive postretiremen!
health benefits. The remaining current employees will receive benefits; however, those benefits will be limited as defined by the Plan. Employees hired
on or after January 1, 1995 will not be eligible to participate in the postretiremen! medical benefit plan.

The following tables set forth the Plan's funded status as of December 31:

Benefit obligation at December 31

Fair value of plan assets at December 3 1

Funded status
Accrued benefit cost

Weighted-average assumptions as of December 31:

Discount rate

Expected return on plan assets

Compensation increase rate
Benefit cost
Employer contribution
Benefits paid

$ 2,091,057
935.257

$(1.155.8(10)

$ (26.654)

6.75%

7.25%

N/A

209,430

190.090

129,589

799*

$ 2.171.1 19
820,645

S( 1.350.474)

$ (2h.h54i

6.75%

7.25%

N/A

210.339

192,082

109.404

For measurement purposes a 6.75% annual rate of increase in the per capita cost of covered health care benefits was assumed for 2000. The rate was
assumed to decrease by half of 1.00% per year to 4.25% in 2006 and remain at that level thereafter. Pension plan assets consist of investment in a money
market fund.

10. Subsequent Event:

On March 8. 2000, the Massachusetts Development Finance Agency issued on behalf of the Laboratory a series of Variable Rate Revenue Bonds (the
"Bonds") in the amount of $10.200,000. The initial interest rate on the issue was 3.65% and the interest rate will be reset weekly. The bonds are
scheduled to mature on February 1 , 2030. The Laboratory is required to make interest payments only for the first five years. The first principal payment
is due February 1 , 2006 with incremental increases through maturity. The proceeds of these bonds are to be used to finance the capital improvements
of the Laboratory's educational, research and administrative facilities, specifically the construction and equipping of the Environmental Sciences
building. A portion of the proceeds were used to extinguish all of the Laboratory's capital obligations (Note 51.

As collateral for the bonds, the Laboratory has entered into a Letter of Credit Reimbursement Agreement which is set to expire on March 15. 2007.
The Letter of Credit is in an amount sufficient to pay the aggregate principal amount of the bonds and up to forty-six days' interest.

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KI8

Report of the Library
Director

The Library has been on the move since the fall of
1999. thanks to a long-overdue and welcome project to
install air conditioning in the Library offices, reading
rooms, and stacks. During the construction, each volume
in the front stack was moved, alphabetized, and cleaned.
When Library staff and patrons felt the first cool breezes
of air conditioning in the stacks in early March, we
quickly forgot about the inconveniences we encountered
during this complex and time-consuming project. The
construction was well worth the effort, and I am pleased
to report that this summer Library patrons will find a
clean, organized, and cool environment, which will be a
comfort to them as well as a benefit to the collection.

The Traditional Library

At the brink of a new millennium we are focusing on
the Library's traditional mission: acquiring, preserving,
conserving, and distributing volumes in our collection. An
article published recently in the Library Journal titled
"Farewell to Alexandria: Not Yet!" reports on the number
of publications that flow from an individual institution in
relation to its library holdings, including citation
productivity. The latter provides a rough measure of the
scholarship quality of an individual institution. The
statistics support the conclusion that the size of library
holdings and institutional scholarly productivity go hand-
in-hand. In this new era we need to continue to support
acquisitions and preservation and conservation efforts as
well as provide leadership in the creation of the new,
technology-driven, scholarly environment.

Books and journals continue to be printed in numbers
inconceivable even a half century ago. While the MBL/
WHOI Library is providing new services and creating
greater access to digital collections worldwide, we have
not outlived print. Therefore, we must store it, preserve it,
and make it available to our patrons.

Special Collections

The Library completed the inventory of several Special
Collections in 1999. These include Charles Wilkes and
his U.S. Exploring Expedition, Memoirs of the Museum
of Comparative Zoology (Harvard) from 1865 to 1899,
and the Harriman Alaska Expedition, the re-creation of
which will become the basis of a public television
special.

Science historian Dr. Garland Allen has also recently
provided us with the final installment of a much-needed
survey of valuable journals currently stored on the open
shelves of the stacks that require preservation and secure
storage.

The Journals

The Library solicited bids for a new serials vendor in
1999 and awarded the contract to RoweCom/Faxon. Our
electronic resources were improved with the purchase of
Science Direct from Elsevier. This product provides full-
text access to the 107 titles that the Library subscribes to
plus a transactional allowance for staff-limited access to
all 1 100 titles provided by Elsevier. Combined with the

R19

R20 Annual Report

addition of 400 electronic journals published by Springer-
Verlag, our digital library has increased substantially. We
have also recently purchased The Procatyotes. an on-line
version of the book series; Marine Mammal Science,
volumes 1 to 13 on CD-ROM; and Cambridge Scientific
Abstracts Biological Sciences, an interdisciplinary
database offering abstracts and citations to a wide range
of research in biomedicine, biotechnology, zoology,
ecology, and some aspects of agriculture and veterinary
science.

Harvard Depositorv

By early 1999, the space remaining to accommodate
future growth of the journal collection in the stacks had
been exhausted. This problem was further exacerbated by
the installation of air conditioning ductwork in the area.
Therefore, we needed to find remote storage for
approximately 8000 journal volumes. Because room for
collection growth was needed in the active stacks, it was
decided that volumes of cancelled series in that section
were prime candidates for storage. Holding records of the
selected series were created for display in the on-line
catalog, and an in-house inventory of each series and
volume was created. By the end of the year, 5038
volumes had been prepared and shipped to the Harvard
Depository for storage. An additional 3000 volumes,
consisting of series for which we have purchased an
online counteipart, were subsequently sent to the
Depository. Although these volumes are no longer in
Woods Hole, they may be retrieved within 24 hours from
the Harvard Depository.

Document Delivery

A major accomplishment in Document Delivery was
the creation and implementation of the web-based Inter-
Library Loan (ILL) request form. Members of the Woods
Hole scientific community may now request ILLs with
this form rather than using the traditional paper form.
Also, with the addition of full text electronic journals,
desktop delivery of information is now a reality.

Cooperating Libraries

The Boston Library Consortium has received funding
for a virtual catalog and interlibrary loan direct distance-
borrowing project. Our Library is an early participant in
the project, which will eventually make it possible for
patrons to easily ascertain which of the 16 BLC Libraries
has the desired material and then order it directly from
that Library.

The National Library of Medicine's Medical
Informatics course, sponsored by the Library, has
expanded to two sessions, one in June and the other in
October. The course continues to be very popular and
successful with a focus on medical database design.
Internet interfacing, and web page design.

The MBLAVHOI Library hosted the 25th Anniversary
Conference of IAMSLIC (International Association of
Aquatic and Marine Science Libraries and Information
Centers) in October. The group was organized and held
its first meetings in Woods Hole in 1975. Over the years
it has grown from the original 25 East Coast Marine
Science Librarians to the international organization of 295
members it is today.

The Library signed a new five-year contract with
NOAA for the continuing operation and support of the
NMFS Library at the Northeast Fisheries Center in
Woods Hole and support of their serial and monograph
collection held at the MBL.

Volunteers

Once again we thank Carol Winn and Millie and Bob
Huettner for their tireless help and support with Rare
Books and Special Collections. During 1999, more than
150 volumes were sent out for preservation under the
Huettner' s tutelage. Carol has provided cataloging support
for esoteric material in languages from Old German to
19"' century Swedish.

— Catherine Norton

Educational Programs

Summer Courses

Biology of Parasitism: Modern Approaches
(June 10-August 13)

Directors

Pearce, Edward. Cornell University

Tsehudi, Christian. Yale University School of Medicine

Faculty

Phillips, Meg, University of Texas Southwest, Dallas
Russell. David, Washington University Medical School
Scott, Phillip. University of Pennsylvania
Selkirk. Murray. Imperial College of Science, Technology and

Medicine, United Kingdom

Sibley, David. Washington University Medical School
Ullu, Elisabetta. Yale University School of Medicine
Waters. Andrew P., University of Leiden. The Netherlands

Teaching Assistants

Appleby. Todd. Cornell University
Beatty, Wandy, Washington University Medical School
Giddings, Olivia, Washington University Medical School
Hussein. Ayman. Imperial College of Science, Technology and

Medicine. United Kingdom
Kinch, Lisa, University of Texas Southwest
La Flamme, Anne Camille. Cornell University
Mair, Gunnar, Yale University School of Medicine
Mordue, Dana, Washington University Medical School
van der Wei. Annemarie, Biomedical Primate Research Centre,

The Netherlands
Zaph. Colby, University of Pennsylvania

Lecturers

Andrews, Norma, Yale University School of Medicine

Bangs. James, University of Wisconsin, Madison

Beckers. Cornelis, University of Alabama. Birmingham

Beverley, Stephen, Washington University Medical School

Carucci, Daniel. Naval Medical Research Institute

Clark, Theodore. Cornell University

Cully, Dons, Merck & Co.

Day, Karen. Oxford University, United Kingdom

Dell, Anne. Imperial College of Science, Technology and Medicine,

United Kingdom
Doolan, Denise, Naval Medical Research Institute

Englund, Paul, Johns Hopkins University School of Medicine
Finkelman, Fred, Veterans Administration Medical Center
Frevert, Ute, New York University Medical Center
Goldberg, Daniel, Washington University Medical School
Grencis, Richard K., University of Manchester. United Kingdom
Gull. Keith. University of Manchester, United Kingdom
Hajduk, Steve, University of Alabama, Birmingham
Hedstrom, Liz, Brandeis University
Hunter, Christopher, University of Pennsylvania
Johnson, Patricia. University of California, Los Angeles
Komuniecki. Richard, University of Toledo
Kopf, Manfred, Basel Institute for Immunology. Switzerland
Langhorne. Jean. Imperial College of Science, Technology and

Medicine, United Kingdom
Long, Carol, National Institutes of Health
Matthews, Keith. University of Manchester, United Kingdom
Mottram. Jeremy. University of Glasgow. United Kingdom
Pearlman. Eric, Case Western Reserve University
Rathod, Pradip, Catholic University of America
Roos, David, University of Pennsylvania
Sacks, David, National Institutes of Health
Scherf, Artur. Institut Pasteur, France
Sher, Alan, National Institutes of Health

Sollner-Webb, Barbara, Johns Hopkins University School of Medicine
Tarlelon, Rick, University of Georgia
Turco, Sam, University of Kentucky Medical Center
Ullman, Buddy, Oregon Health Sciences University
Wirth, Dyann. Harvard School of Public Health

Workshop Coordinators

Cooper, Peter, National Institutes of Health

Ealich, Steve. Cornell University

Lo Verde, Philip. State University of New York, Buffalo

Course Assistants

Chappie. Taylor, Boston University
Chipperfield, Caitlin Nadine, Cornell University

Students

Angeli, Ve'ronique. Pasteur Institute, France

Aviles, Hernan, Indiana State University

Barragan. Antonio, Karolinska Institute. Sweden

Batchelor, Adrian, Walter and Eliza Hall Institute, Australia

Bishop, Joseph, University of Alabama. Birmingham

Djimde, Abdoulaye, University of Maryland

Dobbin. Caroline, University of Technology, Sydney, Australia

Falcone, Franco, University of Edinburgh. United Kingdom

R21

R22 Annual Report

Gavrilescu. Cristina, Cornell University
Jones, Stacy, University of Virginia

Montgomery, Jacqui, Walter and Eliza Hall Institute, Australia
Santori, Isabel, University of Buenos Aires, Argentina
Sodre, Catia, Universidade Federal do Rio de Janeiro, Brazil
Stem, Leah, University of California, San Francisco
Toe, Laurent, World Health Organization, West Africa
Wang. Zefeng, Johns Hopkins University

Lartillot. Nicholas, Universite Paris-Sud, France
Maduro. Morris, University of California. Santa Barbara
Muriani, Francesca, University of California. Berkeley
Micchelli, Craig. University of Wisconsin, Madison
Ober. Hike. Max-Planck-Institute. Germany
Pepicelli, Carmen. Harvard University
Pizette. Sandrine, Memorial Sloan-Kettering Cancer Center
Trainor, Paul, Medical Research Council, United Kingdom
Wallingford. John, University of California, Berkeley
Walsh, Emily. University of California. San Francisco
Wilson. Valerie, University of Edinburgh, United Kingdom

Lecturers

Davidson. Eric. California Institute of Technology

Heasman-Wylie. Janet, University of Minnesota School of Medicine

Holland. Linda, University of California. San Diego

Hopkins, Nancy, Massachusetts Institute of Technology

Levine, Michael, University of California, Berkeley

Rosenthal, Nadia, Massachusetts General Hospital-East

Rothenberg, Ellen, California Institute of Technology

Soriano. Philippe, Fred Hutchinson Cancer Research Center

Stem. Claudio. Columbia University

Tabin, Clifford, Harvard University Medical School

Wylie, Christopher C., University of Minnesota Medical School

Embryology: Concepts and Techniques in

Modern Developmental Biology

(June 13-July 24)

Directors

Bronner-Fraser. Marianne, California Institute of Technology
Fraser, Scott, California Institute of Technology

Faculty

Adoutte, Andre. University of Paris-Sud, France
Blair, Seth S., University of Wisconsin, Madison
Carroll, Sean, University of Wisconsin, Madison
Collazo, Andres. House Ear Institute
Eltensohn. Charles. Carnegie Mellon University
Harland, Richard, University of California, Berkeley
Hartenstein. Volker, University of California. Los Angeles
Henry, Jonathan. University of Illinois
Krumlauf, Robb, National Institute for Medical Research,

United Kingdom

Martindale, Mark. Kewalo Marine Laboratory
Niswander, Lee, Memorial Sloan-Kettering Cancer Center
Rothman, Joel. University of California. Santa Barbara
Saunders. John Jr.. Marine Biological Laboratory
Shankland. Martin, University of Texas, Austin
Wray, Gregory. State University of New York, Stony Brook
Zeller, Robert, University of California, San Diego

Teaching Assistant.*

Baker, Clare. California Institute of Technology

Baker, Julie. University of California. Berkeley

Georgopoulos, Katia. Harvard University

Hartenstein. Amelia, University of California. Berkeley

Kourakis, Matlhew. University of Chicago

Kuhlman, Julie, Memorial Sloan-Kettering Cancer Center

Lane. Mary Ellen. University of Massachusetts Medical Center

Course Assistant.*

Stringer. Kristen. Marine Biological Laboratory
Wylie, Matthew, Marine Biological Laboratory

Lab Assistant

Wylie, Sara, Marine Biological Laboratory

Students

Basch. Martin, California Institute of Technology

Casanueva, Olivia. University of Chicago

Clements, Wilson, University of Washington

Corson, Laura, Ludwig Institute for Cancer Research

Ewald. Andrew. California Institute of Technology

Freistadt, Marion, Louisiana State University Medical Center

Glavic, Alvaro, University of Chile. Chile

Gould, Thomas. Wake Forest University Medical School

Junghlut, Benno, University of Tubingen, Germany

Li, Dongling. University of Texas. Austin

Lwigale. Peter, Kansas State University

Meyers, Jason, University of Virginia

Mui, Stina, University of California, San Diego

Nance. Jeremy. University of Arizona

Panopoulou. Georgia, Max-Planck-Inslitute, Germany

Paul, Angelika, University of Otago, New Zealand

Pfeiffer, Sven, National Institute for Medical Research.

United Kingdom

Pizer, Margaret. State University of New York, Stony Brook
Ragusa, Maria, Alberto Monroy Foundation. Italy
Robertson. Christie. University of Washington
Saiide, Leonor, National Institute for Medical Research.

United Kingdom

Spengler. Tatjana, Universite Paris. France
Sumanas. Saulius, University of Minnesota
Vukovich. Wolfgang, Max-Planck-Institute, Germany
Zigler. Kirk. Duke University

Educational Programs R23

Microbial Diversity (June 13-july 29)

Directors

Leadbetter, Edward, University of Connecticut
Salyers, Abigail, University of Illinois, Urbana

Faculty

Dawson, Scott, University of California. Berkeley

Hanselmann. Kurt, University of Zurich. Switzerland

Holmes, Dawn, University of Massachusetts, Amherst

Kenyon, Sarah, Forsyth Dental Center

Klappenhach. Joel. Michigan State University

Plugge. Caroline M.. Wageningen Agricultural University,

The Netherlands
Schauder, Rolf, Frankfurt. Germany

Lecturers

Blake. Ruth, Yale University

Emerson. David. ATCC

Farrand. Stephen, University of Illinois, Urbana

Fouke, Bruce, University of Illinois. Urbana

Hayes, John, Woods Hole Oceanographic Institution

Leadbetter, Jared. University of Iowa

Lovely, Derek, University of Massachusetts

Metcalf, William, University of Illinois

Newman. Dianne. Harvard University

O'Neill, Scott, Yale University

Paster, Bruce, Forsyth Dental Center

Ruby. Ned. University of Hawaii

Runimel, John, NASA

Schmidt. Thomas. Michigan State University

Shoemaker, Nadja, University of Illinois, Urbana

Sogin. Mitchell. Marine Biological Laboratory

Stein, Jeffrey, Quorum Pharmaceuticals

Teske, Andreas. Woods Hole Oceanographic Institute

Visscher, Pieter, University of Connecticut. Avery Point

Waterbury, John, Woods Hole Oceanographic Institute

Whitman, William. University of Georgia

Young. Lily, Rutgers University

Course Assistants

Ament, Nell, Marine Biological Laboratory
White. Kalina, University of Connecticut

Students

Aislabie, Jacqueline. Landcare Research, New Zealand
Bedard, Donna, General Electric Corporate Research Center
Casillas. Lilliam. Autonomous University of the State of Puebla.

Mexico

Christner, Brent, Ohio State University
Chyba. Christopher. SETI Institute
Dollhopf, Sherry, Michigan State University
Gaidos. Eric. California Institute of Technology
Gillor. Osnat, The Hebrew University, Israel
Gregory. Kelvin, University of Iowa
Niggemyer. Allison. University of Idaho
Norris, Tracy, University of Oregon
Nyholm, Spencer, University of Hawaii
Pomper, Barbara, Max-Planck-Institute. Germany
Rukayadi, Yaya, Bogor Agricultural University, Indonesia
Salmassi. Tina, California Institute of Technology
Shipman. Joseph, University of Illinois, Urbana
Tuit. Caroline, Massachusetts Institute of Technology
Van Lith. Yvonne. Swiss Federal Institute of Technology, Switzerland
Warren, Lesley, McMaster University, Canada
Zopfi. Jakob, Max-Planck-Institute. Germany

Neural Systems & Behavior (June 13-August 6)

Directors

Weeks, Janis, University of Oregon
Zakon, Harold, University of Texas, Austin

Faculty

Barnes, Carol, University of Arizona, Tucson

Calabrese, Ronald L.. Emory University

Carr, Catherine, University of Maryland

French, Kathleen, University of California, San Diego

Glanzman, David, University of California, Los Angeles

Hooper, Scott, Ohio University

Hyson, Richard, Florida State University

Kristan. William, University of California. San Diego

Levine. Richard, University of Arizona. Tucson

McNaughton. Bruce, University of Arizona, Tucson

Muir, Gillian, University of Saskatchewan, Canada

Nadim, Farzan, Rutgers University

Nusbaum, Michael, University of Pennsylvania School of Medicine

Prusky, Glen, University of Lethbridge, Canada

Roberts. William, University of Oregon

Wenning-Erxleben. Angela, Universitat Konstanz, Germany

Wood, Emma, University of Edinburgh, Scotland

Teaching Assistants
Armstrong. Cecilia, University of Oregon
Blitz, Dawn Marie, University of Chicago
Bower, Mark, University of Arizona. Tucson
Chitwood, Raymond. University of Texas. San Antonio
Few. Preston. University of Texas, Austin
Gamkrelidze. Georgi, Lucent Technology
Gerrard, Jason, University of Arizona, Tucson
Golowasch. Jorge, Brandeis University
Hill, Andrew, Emory University
Lenzi, David, University of Oregon
McAnclly, Lynne, University of Texas
Melville, Johnathan. Oregon State University
Murphy, Geoffrey. Lmiversity of California. Los Angeles

R24 Annual Report

Sandstrom, David, University of Arizona, Tucson

Shaw, Brian, The Neurosciences Institute

Villareal. Greg, University of California, Los Angeles

Yong. Rocio, University of California, Los Angeles

Zee, Michelle. University of Oregon

Zirpel. Lance. University of Utah School of Medicine

Lecturers

Augustine, George, Duke University Medical Center

Barlow, Robert, State University of New York Health Science Center

Beer, Randall, Case Western Reserve University

Bodznick, David, Wesleyan University

Cohen, Avis, University of Maryland

Davis, Graeme, University of California, San Francisco

Katz, Paul, Georgia State University

Scholar-ill -Resilience

Abbott, Lawrence. Brandeis University
Nishikawa, Kiisa C., Northern Arizona University
Wilson. Martin, University of California, Davis

Lab Technician

Stengel. Keith. Neuralynx Inc.

Course Assistants

Aimers, Lucy, Marine Biological Laboratory
Stell. Brandon. Marine Biological Laboratory

Students

Baca, Serapio, University of California, San Diego

Beenhakker. Mark. University of Pennsylvania

Cain. Shaun, University of North Carolina. Chapel Hill

Chance. Frances. Brandeis University

Coddington, Emma, Oregon State University

Crisp, Kevin, University of Minnesota

Franks. Kevin, University of California. San Diego

Greenwood, Anna, Stanford University

Hausrath, Cassandra, University of Virginia

Kao, Mimi, University of California, San Francisco

Knittel, Laura, Oregon Health Sciences University

Kricger. Patrik. Karolinska Institute!, Sweden

Maravall. Miguel. Cold Spring Harbor Laboratory

Maruska, Karen, Florida Institute of Technology

Paradis. Suzanne. University of California, San Francisco

Rao, Shankaranar. National Centre for Biological Sciences, India

Rut, Jason. Boston University

Suadicani, Sylvia, Albert Einstein College of Medicine

Wainger. Brian, Columbia University

Wissman, Anne Marie. University of Washington

Neurobiology (June 13-Angust 14)

Directors

Banker, Gary, Oregon Health Sciences University
Madison, Daniel. Stanford University Medical Center

Section Directors

Greenberg. Michael, Children's Hospital

Smith. Stephen, Stanford University School of Medicine

Faculty

Delaney, Kerry, Simon Fraser University. Canada

Edmonds, Brian, Universily of California. Los Angeles

Feller, Maria, National Institutes of Health

Ginty. David, Johns Hopkins University School of Medicine

Griffith, Leslie. Brandeis University

Hanson, Phyllis, Washington University School of Medicine

Hart, Anne, Massachusetts General Hospital

Haydon, Philip, Iowa State University

Khodakhah, Kamran, University of Colorado School of Medicine

Reese, Thomas, National Institutes of Health

Schweizer. Felix, University of California, Los Angeles

Shamah, Steven, Children's Hospital

Smith, Carolyn. National Institutes of Health

Terasaki. Mark, University of Connecticut Health Center

Thompson, Stuart. Stanford University

Van Vactor, David, Harvard University Medical School

Teaching Assistants

Boies. Sarah, Brandeis University

Brinkhaus, Heike. Friedrich Miescher Institute, Switzerland

Imani, Farzin, University of Colorado School of Medicine

McQuiston, Rory. Duke University Medical Center

Pereda, Alberto. Allegheny University of the Health Sciences

Winters, Christine. National Institutes of Health

Lecturers

Barres, Ben. Stanford University School of Medicine

Birren, Bruce. Massachusetts Institute of Technology

Burden. Steven, New York University

Ehrlich, Barbara. Yale University School of Medicine

Ellisman. Mark. University of California. San Diego

Faber, Donald, Allegheny University of the Health Sciences

Flanagan, John, Harvard University Medical School

Greene. Lloyd, Columbia University College of Physicians and Surgeons

Hanson, Roland, Arizona State University

Heuser, John. Washington University Medical School

Lipscombe. Diane, Brown University

Llinas, Rudolfo, New York University

Nicoll, Roger. University of California, San Francisco

Ogden, David, National Institute tor Medical Research.

LInited Kingdom

Rosenberg, Robert, LIniversity of North Carolina. Chapel Hill
Li-Huei, Tsai. Harvard University Medical School
Ziff, Edward, New York University Medical Center
Zimmerberg. Joshua, National Institutes of Health
Zimmerman, Anita, Brown University

Educational Programs R25

Course Assistants
Baughman, Kenneth, Boston University
Chiu, Delia. Stanford University

Students

Abenavoli, Allesandra, Scientific Institute San Raffael. Italy

Diana, Marco, Max-Planck-Institute, Germany

Haapasalo, Annakaisa, A.I. Virtanen Institute. Finland

Hrahetova, Sahina, New York University Medical Center

Matsui. Ko, University of Tokyo, Japan

Samuel. Aravinthan, Harvard University

Schmolesky, Matthew, University of Utah

Smith, Gregory, Princeton University

Spotts, James, Children's Hospital

Vollrath, Melissa. Baylor College of Medicine

Yoon. Miri. Northwestern University

Yu. Xiang. Medical Research Council. United Kingdom

Physiology: The Biochemical and Molecular
Basis of Cell Signaling (June 13-July 24)

Directors

Garbers. David, University of Texas Southwestern Medical Center
Reed, Randall, Johns Hopkins University School of Medicine

Faculty

Beuve, Annie, University of Texas Southwestern Medical Center
Munger. Steven. Johns Hopkins University School of Medicine
Prasad, Brinda. Johns Hopkins University School of Medicine
Quill. Timothy A., University of Texas Southwestern Medical Center
Robinson, Susan W.. University of Texas Southwestern

Medical Center

Wang, Song S., Johns Hopkins University School of Medicine
Wedel, Barbara, University of Texas Southwestern Medical Center
Zhao, Haiqing, Johns Hopkins University School of Medicine
Zielinski, Raymond. University of Illinois. Urbana

Lecturers

Brady, Scott, University of Texas Southwestern Medical Center

Buck, Linda, Harvard University Medical School

Clapham. David. Harvard University Medical School

Corey. David, University of Texas Southwestern Medical Center

Devreotes, Peter, Johns Hopkins University School of Medicine

Dixon, Jack. University of Michigan Medical School

Flanagan, John, Harvard University Medical School

Furlow. John. University of California. Davis

Ginty. David. Johns Hopkins University School of Medicine

Huganir, Richard. Johns Hopkins University School of Medicine

Hurley, James, National Institutes of Health

Kirschner, Marc. Harvard University Medical School

Li. Min, Johns Hopkins University School of Medicine

Ranganathan. Rama, University of Texas Southwestern

Medical Center
Yanagisawa, Masashi. University of Texas Southwestern

Medical Center

Course Coordinator

Rossi. Kristen. University of Texas Southwestern Medical Center

Course Assistant

Kirby, Melissa, Marine Biological Laboratory

Students

Chen, Lihong, University of North Carolina, Chapel Hill

D'Souza, Jacinta. Tata Institute of Fundamental Research. India

van Drogen. Frank, Swiss Institute for Experimental Cancer Research,

Switzerland

Duncan. Tod, Imperial Cancer Research Fund, United Kingdom
Fort, Alfredo, Albert Einstein College of Medicine
Franco, Peter, Harvard University Medical School
Ganguly, Anindita. University of Utah
Han, Qin. University of California, San Francisco
Holdaway-Clarke. Terena, University of Massachusetts. Amherst
Horn. Erik, University of California. San Francisco
Jessani, Nadim. Scripps Research Institute
Kimbell, Jennifer, University of Hawaii
Macias. Chanda. Howard University
March. Tony, University of Idaho
Mazzatenta, Andrea, University of Pisa, Italy
Narayan. Sujatha, Bryn Mawr College
Nzambi. Eduardo, Howard University
O'Neill, Forest, University of California, Santa Barbara
Purves, Dianne. California State University. Sacramento
Rao. Anita. University of Maryland
Sawai, Satoshi. Tohoku University, Japan
Sutton, Timothy, Indiana University
Tefft, Denise. University of Southern California
Tidwell, Judy, Wake Forest University

Varshney, Anurag, National Centre for Biological Sciences, India
Welman, Arkadiusz. Friedrich Miescher Institute, Switzerland
Wen. Ying, University of North Carolina, Chapel Hill
Woo, Caroline, Albert Einstein College of Medicine

Special Topics Courses

Analytical and Quantitative Light Microscopy
(May 6 -May 14)

Directors

Sluder, Greenfield, University of Massachusetts Medical School
Wolf. David. University of Massachusetts Medical School

Faculty

Amos, William B., Medical Research Council. United Kingdom
Cardullo, Richard, University of California. Riverside
Chaisson, Eric, Tufts University

R26 Annual Report

Gelles, Jeff, Brandeis University

Hinchcliffe, Edward. University of Massachusetts Medical School

Inoue. Shinya, Marine Biological Laboratory

Lippincott-Schwartz. Jennifer. National Institutes of Health

Oldenbourg. Rudolf, Marine Biological Laboratory

Silver, Randi, Cornell University Medical College

Spring. Kenneth. National Institutes of Health

Swedlow, Jason. University of Dundee, Scotland

Tuft, Richard, University of Massachusetts Medical School

Teaching Assistant
Thompson, Christine, University of Massachusetts Medical School

Course Coordinator
Miller, Frederick, University of Massachusetts Medical School

Students

Bearman, Gregory. Jet Propulsion Laboratory

Botvinick. Elliot. University of California, San Diego

Bowden. Emma. Georgetown University

Brooks. John. Bio-Rad Microscience

Bulseco. Dylan. University of Massachusetts Medical School

Carrero. Jenny. Unilever Research U.S., Inc.

Danuser, Gaudenz. Swiss Federal Institute of Technology, Switzerland

Faulkner, Nicole, University of Massachusetts Medical School

Heynen, Susanne, University of California, San Diego

Hochegger, Helfrid, Imperial Cancer Research Fund, United Kingdom

Holbrook, Pamela, National Institutes of Health

Holz. Ronald. University of Michigan

Hughes Fulford, Millie, University of California, San Francisco

Keating, Christine, Penn State University

Koehler. Julia. Whitehead Institute

Kreitzer. Geri. Cornell University Medical College

Kwan. Kristen, Harvard University Medical School

Levin. Max, Wallenberg Laboratory for Cardiovascular Research,

Sweden

Lmdberg, Seth. Procter & Gamble Co.
McDonald, John. Mayo Clinic Scoltsdale
Novoradovskaya, Natalia. Stratagenc
Pfister. Kevin. University of Virginia
Reichelt. Stefanie, University of London. United Kingdom
Roberts, Theresa, National Institutes of Health
Rohatgi, Rajat, Harvard University Medical School
Shirani. Jamshid, Albert Einstein College of Medicine
Shonn, Marion, University of California, San Francisco
Tanphaichitr. Nongnuj, Loeb Health Research Institute, Canada
Tse. William, Children's Hospital
Van Dover, Cindy Lee, College of William and Mary
Yarovoi, Serge, University of Massachusetts Medical School

Frontiers in Reproduction: Molecular and

Cellular Concepts and Applications

(May 24-July 4)

Directors

Hunt, Joan, University of Kansas Medical Center

Mayo, Kelly, Northwestern University

Schatten, Gerald, Oregon Health Sciences University

Faculty

Ascoli. Mario, University of Iowa

Bowen, Jeffery A.. University of Kansas Medical Center

Camper. Sally. University of Michigan Medical School

Croy, Barbara Anne, University of Guelph. Canada

Handel, Mary Ann, University of Tennessee

Herr. John C., University of Virginia School of Medicine

Hunt, Patricia A.. Case Western Reserve University

Jaffe, Laurinda, University of Connecticut Health Center

Petroff, Margaret. University of Kansas Medical Center

Shupnik, Margaret. University of Virginia Health Sciences Center

Simerly. Calvin, Oregon Regional Primate Research Center

Terasaki, Mark, University of Connecticut Health Center

Tnmarchi, James, Marine Biological Laboratory

Weigel, Nancy. Baylor College of Medicine

Teaching Assistants
Aldrich. Carrie, University of Chicago
Berard, Mark, University of Michigan
Cunningham. Meghan, Georgetown University
Diekman. Alan, University of Virginia Health Sciences Center
Giusti, Andrew. University of Connecticut Health Sciences Center
Greenwood. Janice, University of Guelph, Canada
Hinkle, Beth Anne, University of Connecticut Health Sciences Center
Hodees, Craig, Case Western Reserve University
Mukherjee, Abir. Northwestern University
Nakamura, Kazuto, University of Iowa Medical School
Phillips. Teresa. University of Kansas Medical Center
Resnick. Eileen, University of Virginia Health Sciences Center
Rowan. Brian. Baylor College of Medicine
Runt't. Linda, University of Connecticut Health Center
Westbrook. Anne. University of Virginia Health Sciences Center

Lecturers

Behringer. Richard. University of Texas
Campbell. Keith. PPL Therapeutics, Scotland
Carroll. David. University of California. Santa Barbara
Crowley. William. Massachusetts General Hospital
Dominko, Tanja. Oregon Regional Primate Research Center
Fazleabas. Asgi, University of Illinois
Handyside. Alan, St. Thomas' Hospital, United Kingdom
Hennighausen, Lothar, National Institutes of Health
Hewitson. Laura. Oregon Regional Primate Research Center
Johnson. Peter M.. University of Liverpool Medical School,

United Kingdom

Keefe, David, Marine Biological Laboratory
Kopf, Greg, University of Pennsylvania Medical Center
Ober. Carole, University of Chicago
Orth, Joanne, Temple University School of Medicine
Pederson. Roger, University of California, San Francisco
Pollard, Jeffrey W., Albert Einstein College of Medicine
Richards. JoAnne, Baylor College of Medicine
Ruderman. Joan. Harvard University Medical School
Shenker. Andrew. Children's Memorial Hospital
Tilly, Jonathan L., Massachusetts General Hospital
Wessel, Gary, Brown University
Woodruff. Teresa. Northwestern University

Course Administrator

Emme. Michelle, Oregon Health Sciences University

Educational Programs R27

Chief Course Coordinator

Payne, Christopher, Oregon Health Sciences University

Course Coordinators

Daggett, Melissa, University of Kansas Medical Center
McMullen. Michelle. Northwestern University

Students

Akhmedkhanov, Arslan, New York University School of Medicine

Belts, Dean, University of Guelph. Canada

Bos-Mikich, Adriana, Fundaijao Universitaria de Endocrinologia e

Fcrtilidade, Brazil

Buhimschi, Irina, University of Maryland
El Guiziry. Dalai, Alexandria University, Egypt
Johanputra, Vaidehi, All India Institute of Medical Sciences, India
Johnson, Quinton, University of the Western Cape
Lue, Yanhe, Harbor-University of California, Los Angeles,

Medical Center

Man'n Bivens, Carrie, University of Massachusetts, Amherst
Mendeluk, Gabriela, University of Buenos Aires. Argentina
Natesampillai, Sekar, University of Virginia
Ollero, Mario, Harvard University Medical School
Paidas. Michael, New York University School of Medicine
Pritts. Elizabeth, Yale University School of Medicine
Sprague, David, Texas A&M University
Witlin, Andrea, University of Texas Medical Branch. Galveston

Medical Informatics (May 30-june 5)

Director

Masys. Daniel, University of California, San Diego

Students

Adams. Martha. Duke University

Babbitt, Patricia, University of California, San Francisco

Barclay. Donald, Houston Academy of Medicine

Bernhard, Jeffrey, University of Massachusetts Medical School

Finley, Allen, Dalhousie University. Canada

Goldstein, Cynthia, Tulane University Medical Library

Lin. Chen-Tan, University of Colorado Health Sciences Center

Lindherg, Don. Regenstrief Institute

Lyons, Amy, University of Buffalo Health Sciences Library

Mahoney, Diane, Hebrew Rehabilitation Center for Aged

Markovitz, Barry, Washington University

McGrath, St. John, Tufts University School of Medicine

Meyers, Arlen, University of Colorado Health Sciences Center

Mulluly-Quijas, Peggy, University of Missouri, Kansas City

Pelok, Scott, University of Michigan

Pifer, Eric, University of Pennsylvania Health System

Robinson, Judith, Eastern Virginia Medical School

Rosnian. Alan, Bronx Veteran's Administration Medical Center

Sack, Jean, Johns Hopkins University

Sarchet, Patricia, University of Buffalo Health Sciences Library

Seago, Brenda, Virginia Commonwealth University

Siblcy. Deborah. University of Massachusetts Medical School

Stroman, Rosalie. National Institutes of Health Library

Swanson. Sandra. Cook Institute of Research and Education

Thompson, Laurie, State University of New York Health Science

Center. Syracuse

Tomlinson, Louise, Morehouse School of Medicine
Travers. Robin, Boston University School of Medicine
Tunnan, Lynne, Virginia Commonwealth University
Volpp, Bryan, Veterans Affairs Medical Center
Warlick, Becky, Duke University Medical Center

Medical Informatics (October 3-October 9)

Director

Cimino, James, Columbia University

Faculty

Bakken, Suzanne, Columbia University

Canese, Kathi, National Library of Medicine

Cimino, Chris, Albert Einstein College of Medicine

Friedman. Charles. University of Pittsburgh

Hightower, Allen, Centers for Disease Control and Prevention

Jenders, Robert, Columbia University

Lindberg, Donald, National Library of Medicine

Masys. Daniel, University of California, San Diego

Safran, Charles. Center for Clinical Computing

Starren, Justin, Columbia University

Wheeler, David. National Library of Medicine

Faculty

Cimino. James. Columbia University
Friedman, Charles, University of Pittsburgh
Hightower, Allen, Centers for Disease Control and Prevention
Hripcsak, George, Columbia-Presbyterian Medical Center
Kingsland, Lawrence, National Library of Medicine
Landsman, David, National Library of Medicine
Lindberg, Donald D.A.B., National Library of Medicine
Safran, Charles. Center for Clinical Computing
Sengupta. Soumitra, Columbia University
Starren, Justin, Columbia University

Students

Beidas, Sary, Prince George's Hospital Center

Boyle, Marian, University of Florida

Calarco. Pascal, Virginia Commonwealth University

Chong. Lisa, Science magazine

Cohen, Arlene, University of Guam

Coster, Trinka, US Army Medical Research Institute

Delia. Catherine, George Washington University

Dimitroff, Alexandra. University of Wisconsin-Milwaukee

Doyle. Jacqueline, Samaritan Health Systems, Phoenix

Eaton, Elizabeth, Tufts University Health Sciences Library

R28 Annual Report

Feldman, Marc, University of Alabama. Birmingham

Francis, Marcia. Idaho Slate University

Fuller. Howard. University of California. San Francisco

Hogan, Linda, University of Pittsburgh

Hornby. Kathryn. University of British Columbia. Canada

Knight, Barbara, University of North Dakota

Kufreja. Neera. Cook County Hospital

Linton. Anne. George Washington University

Livingston, Jill, University of Connecticut Health Center

Massanari. Mike. Wayne State University

Miller. Stephen, Massachusetts General Hospital/Martha's Vineyard

Hospital

Parada. Jorge. Cook County Hospital
Reilly. James, State University of New York Health Science Center,

Brooklyn

Strassner. Howard, Rush-Presbyterian-St. Luke's Medical Center
Swanton, James, Harlem Hospital Center
Swiatek-Kelley. Janice. Bridgeport Hospital
Teal. Janis, University of New Mexico
Walker. James. Penn State College of Medicine
Wu, Carol, New York University School of Medicine
Wulff, Judith, University of Louisville
Yue, Cheung. MetroHealth Medical Center

Methods in Computational Neuroscience
(August 1 -August 28)

Directors

Bialek, William. NEC Research Institute

van Steveninck. Rob de Ruyter. NEC Research Institute

Faculty

Abbott. Lawrence. Brandeis University

Colby. Carol, University of Pittsburgh

Dan, Yang, University of California. Berkeley

Delaney. Kerry. Simon Fraser University. Canada

Doupe, Allison, University of California, San Francisco

Ermentrout, Bard, University ol Pittsburgh

Hoplield, John, Princeton University

Johnston. Daniel, Baylor College of Medicine

Kelley, Darcy, Columbia University

Klemleld, David, University of California, San Diego

Kopell. Nancy. Boston University

Marder. Eve, Brandeis University

Meister. Markus. Harvard University

Miller. K. D.. University of California. San Francisco

Mitra. Partha. AT&T Bell Laboratories

Rieke. Fred, University of Washington

Seung, H. Sebastian, Massachusetts Institute of Technology

Sigvardt, Karen, University of California, Davis

Solla, Sara A.. Northwestern University

Sompi'linsk\ . Haim. Hebrew University of Jerusalem. Israel

Tank, David. AT&T Bell Laboratories

Tishby. Nattali. Hebrew University of Jerusalem. Israel

Zucker. Sie\ni. " il>- University

Teaching Asv/.vMm.s

Aguera y Areas. B . r,i!u.;-u>ii 1 'niversity

Jensen. Roderick. \Visk-\.,n I HUCIM!)

Koberle. Roland. Universidade ih Sac Paulo, Brazil

Lewen. Geoffrey David. NEC Research Institute

Nemenman. I., Princeton University

White. John. Boston University

Lecturers

Baylor. Denis. Stanford University Medical Center

Laughlin. Simon Barry, University of Cambridge, United Kingdom

Logothetis, Nikos. Max-Planck-Institute for Biological Cybernetics.

Germany
Srinivasan, Mandyam V.. Australian National University, Australia

Course Coordinator

Stogryn. Krista, Marine Biological Laboratory

Students

Borisyuk. Alia, New York University

Buss, Robert. McGill University, Canada

Gang, Jianhua, University of Virginia

Chechik. Gal. Hebrew University of Jerusalem, Israel

Cowen, Stephen, University of Arizona

Dumont. Sophie, Princeton University

Garcia de Polavieja. Gonzulo. University of Cambridge,

United Kingdom

Jacobson, Gilad, Hebrew University of Jerusalem, Israel
Karbowski. Jan. Boston University

Kefalov. Vladimir, Boston University School of Medicine
Kozloski, James. Columbia University
Laing, Carlo, University of Pittsburgh
Mayraz. Guy. University College London. United Kingdom
Mazurek, Mark. University of Washington
Naylor, David, University of California, Los Angeles
Petersen, Ras. International School of Advanced Studies, Italy
Prescott, Steven, McGill University, Canada
Rubin. Jonathan. Ohio State University
Spence. Andrew, Cornell University
Still, Susanne, University of Zurich, Switzerland
Wittenberg. Gayle. Princeton University
Zeddies, David. Northwestern University

Microinjection Techniques in Cell Biology

(May 18-May 25)

Director

Silver. Robert B.. Marine Biological Laboratory

Faculty

Cousins, Susan, Cornell University

Klaessig. Suzanne, Cornell University

Kline, Douglas, Kent State University

Mehlmann. Lisa. University of Connecticut Health Center

Shelden. Eric. University of Michigan

Teaching Assistant

Warnke. Honey, University of Maine

Students

Araujo, Loraine, State University of Rio de Janeiro, Brazil

Cohen. David, Oregon Health Sciences University

Dabrowski, Konrad. Ohio State University

James. Marianne. Massachusetts General Hospital

Koulen. Peter, Yale University

Kozek, Wieslaw, University of Puerto Rico

Kuan, Chia-Yi, Yale University

Lahti. Jill. St. Jude Children's Research Hospital

Larkin. Janet. Barnard College

Educational Programs R29

Li. Bin, University of California. San Francisco

McGowan. Francis. Harvard University Medical School

Nemoto, Yasuo, Yale University

Nusser. Kevin. Oregon Regional Primate Research Center

Pai. Vinay, Florida Slate University

Rueda. Angelica, Centre de Investigation y de Estudios Avanzados

del I.P.N.. Mexico

Wentz-Hunter, Kelly, University of Illinois, Chicago
Yu, Han-Gang, State University of New York. Stony Brook

Molecular Biology of Aging
(August 10 -August 27)

Directors

Guarente, Leonard P.. Massachusetts Institute of Technology
Wallace, Douglas, Emory University School of Medicine

Faculty

Aiken, Judd M., University of Wisconsin-Madison
Austad. Steven. University of Idaho
Bohr, Vilhelm A.. National Institutes of Health
Campisi, Judith, Berkeley National Laboratory
Finch. Celeb. University of Southern California
Grossman. Lawrence, Johns Hopkins University
Harley. Calvin, Geron Corporation
Hekimi. Siegfried, McGill University. Canada
Johnson, Thomas, University of Colorado
Jones. Dean P.. Emory University

Kenyon. Cynthia, University of California. San Francisco
Kim. Stuart. Stanford University of Medicine
Kirkwood. Tomas, University of Manchester. United Kingdom
de Lange. Titia, The Rockefeller University
Lithgow, Gordon J.. University of Manchester, United Kingdom
Longo. Valter, University of Southern California
Martin, George, University of Washington School of Medicine
Melov. Simon. Buck Center for Research in Aging
Richardson, Arlan, University of Texas Health Science Center
Ruvkun, Gary. Massachusetts General Hospital
Sohal, Rajindar. Southern Methodist University
Tanzi, Rudolph E., Harvard University Medical School
Tower. John, University of Southern California
Van Voorhies. Wayne. University of Arizona. Tucson
Wright. Woodring E.. University of Texas Southwestern
Medical Center

Teaching Assistants
Bilger. Johannes, Emory University
Pinar, Elif. Emory University
Cottrell, Barbara. Emory University
Esposito, Luke. Emory University
Jegalian. Beatrice, Massachusetts Institute of Technology
Johnson, Brad. Massachusetts Institute of Technology
Kokoszka. Jason. Emory University
Levy. Shawn, Emory LIniversity
McNabb. David. Massachusetts Institute of Technology
Murdock. Deborah, Emory University

Course Coordinator

Burke, Rhonda E.. Emory University School of Medicine

Course Assistant

Abisla, Richard. University of Chicago

Students

Ayala-Torres. Sylvette. University of Texas Medical Branch

Brown. Jeremy. Roslin Institute, Scotland

Brown-Borg, Holly, University of North Dakota

Chen. Yaohui. Yale University Medical School

Chung. Namjm, Duke University Medical Center

Crawford. Douglas, University of California, San Francisco

de Lacalle, Sonsoles. Beth Israel Deaconess Medical Center

Eshoo, Mark. Buck Center for Research in Aging

Ford. Carolyn. Northwestern University

Goto, Joy, University of California. Los Angeles

Henning. Karla, National Institutes of Health

Kennell, John, Southern Methodist University

Kukull. Walter, University of Washington

Martin, Kareen, Biological Gerontology Group. Manchester,

LInited Kingdom
McChesney, Patricia, University of Texas Southwestern

Medical Center

Merker. Robert, New York University Medical Center
Squier. Thomas. University of Kansas
Torres-Ramos. Carlos. University of Texas Medical Branch

Molecular Mycology: Current Approaches to
Fungal Pathogenesis (August 8-August 27)

Directors

Edwards, John Jr., Harbor-UCLA Medical Center
Magee, Paul T.. University of Minnesota
Mitchell. Aaron P.. Columbia University

Faculty

Casadevall. Arturo, Albert Einstein College of Medicine
Cole, Gary T., Medical College of Ohio
Davidson. Robert, Duke University Medical Center
Davis, Dana, Columbia University
Filler. Scott. Harbor-UCLA Medical Center
Fonzi. William, Georgetown University Medical Center
Heitman, Joseph. Duke University Medical Center
Keath, Elizabeth, St. Louis University School of Medicine
Klein. Bruce. University of Wisconsin-Madison
Kurtz, Myra. Merck Research Lab
Kwon-Chung, June, National Institutes of Health
Lodge. Jennifer. St. Louis University School of Medicine
Murphy. Juneann, University of Oklahoma
Oliver, Brian. University of Cincinnati
Rhodes, Judith, University of Cincinnati
White. Theodore, Seattle Biomedical Research Institute
Whiteway, Malcolm. National Research Council, Canada

Course Assistant

Sandri, Brian. Marine Biological Laboratory

Students

Bammert, Gary, Pharmacia & Upjohn
Cowen. Leah, University of Toronto, Canada
Cruz, Cristina, Duke University Medical Center
de Jesus-Bem'os, Marisol, University of Puerto Rico
Devasamavam, Gina, Wadsworth Center

R30 Annual Report

Efimov, Vladimir, University of Medicine and Dentistry, New Jersey

Giles, Steven, University of Wisconsin-Madison

Goldstein, Alan, Duke University

Inglis, Diane, University of California, San Francisco

Kellog. Christina, Georgetown University

Latouche, Nicholas. Westmead Hospital Sydney University, Australia

Miller, Nancy. Johns Hopkins Medical Institution

Niekerson, Ken, University of Nebraska

Smith, Christina, State University of New York. Buffalo

Warenda, Amy, State University of New York, Stony Brook

Wormley, Floyd. Louisiana State University Medical Center

Neural Development and Genetics of Zebrafish

(August 15-Augiist 27)

Directors

Dowling, John E., Harvard University

Hopkins, Nancy, Massachusetts Institute of Technology

Faculty

Baker, Robert, New York University Medical Center

Collazo, Andres, House Ear Institute

Eisen, Judith S., University of Oregon

Fetcho. Joseph, State University of New York, Stony Brook

Fricke, Cornelia, University of Utah Medical Center

Hanlon, Roger, Marine Biological Laboratory

Kimmel, Charles, University of Oregon

Lin. Shuo. Medical College of Georgia

Neuhauss, Stephan, Max-PIanck-Institut fur Entwicklungsbiologie,

Germany

Talhot. William S., Stanford University
Wilson. Stephen, University College London, United Kingdom

Teaching Assi 'ants

Clarke, .lon.iih.m, ' niversity College London. United Kingdom

Fadool. James Hi l.i State University

Granato. Michiu ' , of Pennsylvania

Kainz, Pamela, Harvard i 'Diversity

Link, Brian, Harvard Uimvrsity

Lorent, Kristin, Um\cisii\ i,l Pennsylvania School of Medicine

Moens, Cecilia, Fred Hulchmson Cancer Research Center

Mullins, Mary, University of Pennsylvania

Sirotkin, Howard. New York Universiiv School of Medicine

Walker-Durchanck, Charline, Universiiv of Oregon

Lecturers

Astrosfky. Keith, Massachusetts Institute of Technology
Fraser, Scott. California Institute of Technology

Laboratory Technicians

Linnon, Beth, Marine Biological Laboratory
Mazanec, April, LIniversity of Oregon

Course Coordinator

Schmitt. Ellen. Harvard University

Course Assistant

Sweeney, Neal, Marine Biological Laboratory

Students

Ashworth. Rachel. University College London, United Kingdom

Belletroid, Enc. Universite Libre de Bruxelles. Belgium

Bishop, Charles, Baylor College of Dentistry

Chan. Joanne. Dana-Farber Cancer Institute

Endres. James, University of California. San Diego

Glanzman, David, University of California, Los Angeles

Levandoski, Mark. Brown University

Lightfoot. Kurt, University of the Witwatersrand, South Africa

Lunde, Karen, University of California, San Diego

Poznanski, Ann, Midwestern University

Rmkwitz, Silke. New York University Medical Center

Tong, Betty. Whitehead Institute

Vlachakis, Nikolaos. University of Massachusetts Medical Center

Waterbury. Julie. University of Pennsylvania

Wiemelt, Anthony, University of Pennsylvania

Williams, Fred, University of Toledo

Optical Microscopy and Imaging in the
Biomedical Sciences (October 6-October 14)

Director

Iz/ard, Colin. State University of New York, Albany

Faculty

DePasquale, Joseph. New York Stale Department of Health
Dunn, Kenneth, Indiana University Medical Center
1 laid, Robert, State University of New York, Buffalo
Herman, Brian, University of Texas Health Science Center
Murray, John, University of Pennsylvania School of Medicine
Piston, David M., Vanderbilt University
Snyder, Kenneth, State University of New York, Buffalo
Spring, Kenneth, National Institutes of Health
Swedlow. Jason. University of Dundee, Scotland

Teaching Assistants

Pierini, Lynda, Cornell University Medical College
Sigurdson, Wade, State University of New York, Buffalo

Lecturers

Hinsch, Jan, Leica, Inc.

Inoue, Shinya. Marine Biological Laboratory

Keller, H. Ernst, Zeiss Optical Systems

Educational Programs R31

Students

Bhalla, Needhi, University of California, San Francisco

Biggins, Sue, University of California. San Francisco

Brotz, Tilmann. National Cancer Institute

Chien, Edward, University of Chicago

Combs, Christopher, National Institutes of Health

Cromey. Douglas. University of Arizona

Duca, Karen, University of Wisconsin

Fletcher, Tara, Albany Medical College

Gustashaw, Karen. Case Western Reserve University

Hoja, Mary-Rose. Karolinska Institute Stockholm, Sweden

Holt. Matthew. Medical Research Council. United Kingdom

Hudson, Emma, University of Dundee, Scotland

Kaplan, David, Food and Drug Administration

Lin, Keng-hui, University of Pennsylvania

Love, Dona, National Institutes of Health

Martini, Lene, University of Copenhagen, Denmark

Murnion. Mairead, University of Dundee. Scotland

Reilly, Thomas, Johns Hopkins LIniversity School of Medicine

Saslowsky. David. Virginia Tech

Shestopalov, Valery, Washington University

Silverman. Michael, Oregon Health Sciences University

Sossick, Alex, University of Cambridge, United Kingdom

Tang, Cha-Mei, Creative MicroTech. Inc.

Wallace, Wes. Brown University

Pathogenesis of Neuroiinmiinologic Diseases
(August 16-August 27)

Directors

Brosnan, Celia F., Albert Einstein College of Medicine
Rosenbluth, Jack. New York University School of Medicine

Faculty

Etty. Benveniste, University of Alabama, Birmingham

Berman. Joan, Albert Einstein College of Medicine

Brightman, Milton W., National Institutes of Health

Burden, Steven, New York University School of Medicine

Coyle, Patricia, State University of New York, Stony Brook

Darnell, Robert, Rockefeller University

Drachman. Daniel, Johns Hopkins University School of Medicine

Felten, David, Loma Linda University School of Medicine

Gould, Robert M., New York State Institute of Basic Research

Griffin. Diane, Johns Hopkins University

Griffin, John, Johns Hopkins University

Hickey, William. Dartmouth-Hitchcock Medical Center

Itescu, Silviu, Columbia/Presbyterian Medical Center

Kaplan. Gilla. Rockefeller University

Knopf. Paul, Brown University

Kocsis. Jeffery D.. Yale University School of Medicine

Kuchroo. Vijay, Brigham and Women's Hospital

Lipton, Stuart, The Burnham Institute

Martiney, James, Picower Institute for Medical Research

McKinnon, Randall D., R. W. Johnson Medical School

Popko. Brian, University of North Carolina, Chapel Hill

Price. Donald L.. Johns Hopkins University School of Medicine

Ransohoff. Richard. Cleveland Clinic Foundation

Ransom. Bruce, University of Washington School of Medicine

Reder, Anthony, University of Chicago

Salzer, James, New York University Medical Center

Saper, Clifford, Beth Israel Hospital

Shin, Moon, University of Maryland School of Medicine

Shrager, Peter. University of Rochester Medical Center
Solimena. Michele, Yale University
Sontheimer, Harald, University of Alabama, Birmingham
Steinberg. Esther. National Institutes of Health
Waksman, Byron. Foundation for Microbiology
Weiner. Howard, Harvard University Medical School

Course Coordinator

Stogryn. Krista, Marine Biological Laboratory

Students

Andjelkovic. Anuska, University of Connecticut

Brundula. Veronika, University of Calgary, Canada

D'Aversa, Teresa, Albert Einstein College of Medicine

DeFeo, Anthony. Mercy College

Dzenko. Kirk, University of Connecticut Health Center

Fischer. Falko. Harvard University Medical School

Hillert. Jan, Karolinska Institute, Sweden

Hjelmstrom, Peter, Yale University

Janson, Christopher, Thomas Jefferson University

Kuljis. Rodrigo, University of Miami

Lu, Weiquan, State University of New York, Stony Brook

Luedtke, Robert. University of North Texas

O'Brien, Niklci. Australian National University, Australia

Odyniec, Artur, Medical Academy of Lodz, Poland

Regardsoe. Emma, University of Oxford, United Kingdom

Reis, Donald, Cornell Medical College

Robichaud, Lillian. Parke-Davis Research

Salzberg, Heather, Rutgers University

Sivakumar, M. R.. Apollo Hospitals. India

Troncoso. Juan, Johns Hopkins University

Vari, Gabor. Brown University

Vijayan, Shrijay. City University of New York

Woodman, Scott. Albert Einstein College of Medicine

Wu, Dona. Alberl Einstein College of Medicine

Yates, Jennifer. University of North Carolina, Chapel Hill

Workshop on Molecular Evolution
(August 1-Aiigust 13)

Directors

Davison, Daniel B.. Bristol-Myers Squibb PRI
Sogin, Mitchell, Marine Biological Laboratory

R32 Annual Report

Faculn

Cummings, Michael, Marine Biological Laboratory
Eddy, Sean, Washington University
Edwards, Scott, University of Washington
Eisen. Jonathan, Institute for Genomic Research
Felsenstein, Joseph. University of Washington
Fitch. David H.A., New York University
Fraser, Claire M.. Institute for Genomic Research
Kuhner. Mary, University of Washington
Maddison. David, University of Arizona, Tucson
Miyamoto, Michael, University of Florida
Muse, Spencer, North Carolina State University
Olsen, Gary, University of Illinois, Urbana
Pace, Norman, University of Colorado, Boulder
Pearson, William, University of Virginia Health Sciences Center
Rice. Ken. SmithKline Beecham Pharmaceuticals
Riley, Margaret. Yale University
Swofford, David, Smithsonian Institution

Teaching Assistants

Edgcomb. Virginia, Marine Biological Laboratory
Mac-Arthur, Andrew, Marine Biological Laboratory
Thompson, Steven, Florida State University

Laboratoiy Technician

Holder. Michael. University of Houston

Course Coordinator

Hams, Marian. Marine Biological Laboratory

Students

Ariey. Frederic, Inslitut Pasteur, France

Babm. Josephine. Louisiana State University

Baumgarlner. Manuela. University Regensburg. Germany

Beati. Lorenza. Centers for Diseases Control and Prevention

Becker, Jennifer. Lehigh University

Best, Aaron, University of Illinois

Blatter. Robert, University of Basel, Switzerland

Bond, Philip, University of Wisconsin

Bouchet. Valerie. Boston University School of Medicine

Bouzat, Juan, University of Illinois

Brazeau, Dan, University of Florida

Brinkmann, Anna, University of Wisconsin-Milwaukee

Bnones. Marcelo. Universidade Federal de Sao Paulo. Brazil

Carrigan. Matthew. University of Florida

Chaturvedi, Vishnu, New York State Department of Health

Chyba, Christopher, SETI Institute

Clark. Ann Marie. University of Florida

Craven, Kelly, University of Kentucky

Dacks, Joel, Dalhousie University, Canada

Dennis, Paige, University of Massachusetts, Boston

Di Meo, Carol, University of Delaware

Dimsoski, Pero, United Slates Environmental Protection Agency

Fitzpatrick, Jennifer, Tufts University School of Medicine

Fleming. Melissa. University of Alaska Museum

Franck, Jens. Occidental College

Freire. Nicole. University of Florida

Gasparich. Gail. Towson University

Gaudier, Eric. University of Florida

Gribaldo, Simonetta. Universita "La Sapienza," Italy

Gueneau-Novoa, Pulchene, Instituto Venezolano Investigaciones

Cientih'cas, Venezuela
Hansen, Jan, Technical University of Denmark, Denmark

Harbmski, Fred, Harvard University

Ho, Hoi Yan, Chinese University of Hong Kong. Hong Kong

Hurtado, Luis. Rutgers University

Inagaki. Yuji. Dalhousie University. Canada

Klingbeil. Michele. Johns Hopkins School of Medicine

Lawrence. Carolyn, University of Georgia

Liebert, Cynthia. University of Georgia

Maiwald. Matthias, Stanford University

McGraw, Beth, Yale University

Mead, Louise, University of Massachusetts

Moore, Jon. National Marine Fisheries Service

Pilcher, Carl. NASA Headquarters

Pineda, Augustin, Florida Slate University

Posada, David. Brigham Young University

Pntham. Ellen, Universily of Massachusetls, Boston

Reed. David. Louisiana State Universin

Richardson. Susan, Yale University/Woods Hole Oceanographic

Institution
Rinke De Wit, Tobias. Ethiopian Health and Nutrition Institute.

The Netherlands
Sabo, Aniko, Purdue University

Salamin, Nicolas. Universite de Lausanne, Switzerland
Schreiber, Edgar. PE Applied Biosystems
Seffemick. Jennifer. University of Minnesota
Sellers. Holly. United States Department of Agriculture
Sinclair, Elizabeth. Brigham Young University
Skirnisdottir, Sigurlaug. IceTherm Inc.. Iceland

Stockley. Bruce. Southampton Oceanography Centre. United Kingdom
Worapong, Jeerapun, Montana State University
Zmasek. Christian, Washington University Medical School

Other Programs

Marine Models in Biological Research
Undergraduate Program (June 8-August 6, 1999)

Directors

Browne. Carole L.. Wake Forest University

Tytell. Michael. Wake Forest University School of Medicine

Course Assistant
Begley. Gail. Marine Biological Laboratory

Faculty

Allen. Nina S., North Carolina State University

Borst, David, Illinois State University

Furie, Barbara. Harvard University

Furie, Bruce. Harvard University

Hanlon. Roger. Marine Biological Laboratory

Jonas, Elizabeth. Yale University

Laufer. Hans, University of Connecticut

Malchow, R. Paul, University of Illinois

Mensinger, Allen. Washington University

Wainwright, Norman. Marine Biological Laboratory

Seminar Speakers

Frank. Tammy. Harbor Branch Oceanographic Institution
Inoue, Shinya, Marine Biological Laboratory
Kuzirian, Alan. Marine Biological Laboratory
Reinisch, Carol. Marine Biological Laboratory
Silver. Robert. Marine Biological Laboratory

Kducutional Programs R33

Students

Baliga, Meghan. Wake Forest University
Clifton. Christine. Mount Holyoke College
Harris!. Alexia. Yale University
Helm, Jessica, Washington and Lee University
Lassen, Kara. Wake Forest University
Mitchell, Michael. Wake Forest University
Peck, Raphaela. Reed College
Price. Nichole, Connecticut College
Ramsey, David, Harvard University
Rankin, Ellen, Colgate University
Tang, Kathleen, Washington University
Taylor, Kevin, Wake Forest University
Vasse, Aimee, Williams College

NASA Planetary Biology Internship
(June-September 1999)

Directors

Margulis, Lynn, University of Massachusetts
Dolan, Michael F., University of Massachusetts

Interns

Caylor, Kelly, University of Virginia

Chacon, Elizabeth. Universidad Nacional Autonoma de Mexico

Franklin. Rima, University of Virginia

French, Jason, University of Alberta. Canada

Gauci. Vincent, The Open University, United Kingdom

Marx, Joseph G., International Space University, France

Omelon, Christopher, McGill University, Canada

de Peyer, Oliver, University of Reading, United Kingdom

Popa. Radu, University of Cincinnati

de Vera Gomez. Alvin. University of the Philippines

Wilson, Cindy, University of Montana

Sponsors

Cady. Sherry L., Portland State University

Des Marais, David, NASA Ames Research Center

Joyce, Gerald, Scripps Research Institute

Knoll, Andrew, Harvard University

Mancmelli. Roco, NASA Ames Research Center

Matthews. Elaine, Goddard Institute for Space Studies

Nealson, Ken. Jet Propulsion Laboratory

Privette, Jeff, NASA Goddard Space Flight Center

Rothschild, Lynn. NASA Ames Research Center

Teske, Andreas, Woods Hole Oceanographic Institution

Wheeler, Raymond, NASA Kennedy Space Center

Semester in Environmental Science

(Septembers-December 17, 1999)

Administration

Hobbie, John E., Director

Foreman, Kenneth H., Associate Director

Moniz, Polly C., Administrative Assistant

Faculty

Deegan, Linda A.
Giblin, Anne E.
Hopkinson. Charles S. Jr.
Hughes, Jeffrey
Liles, George
Nadelhoffer, Knute J.

Neill, Christopher
Peterson. Bruce J.
Rastetter, Edward B.
Shaver. Gams R.
Vallino, Joseph J.
Williams, Mathew

7999 Research and Teaching Assistants
Bahr, Michelle
Kelsey, Sam
Kwiatkowski. Bonnie
Micks. Patricia
Parker, Sophie
Tholke. Kris

7999 SES Students

Arling, Jeremy. Bowdoin College
Avery, Jennifer. Brandeis University
Butman, David. Connecticut College
Glueck, Lara. Claremont McKenna College
Greenbaum, Adena, Wellesley College
Hinckley, Eve-Lyn, Middlebury College
Horowitz. Julie, Hampshire College
Kirkby, Ryan, Harvey Mudd College
Mathrani, Vandana, Scripps College
Mathrani. Varsha, Scripps College
Mifflin. Amanda. Wellesley College
Morrisseau, Sarah, Connecticut College
Peterson, G. Gregory. Wesleyan University
Romagnano. Joseph. Worcester Polytechnic
Sohm, Jill, Harvey Mudd College
Spivak, Amanda, Bryn Mawr College
Williams, Samantha. Mount Holyoke College
Ziemann, Tori. Beloit College

SPINES — Summer Program in Neuroscience,
Ethics and Survival (June 12-July 10)

Directors

Martinez, Joe L. Jr.
Townsel, James

Fellows

Herne. Moss, Boston University School of Medicine
Hubbard. Aida, University of Texas, San Antonio
McCrery. Karen, Texas Women's University
Meadows, Adimika, Boston University
Mohamed, Somaia, University of Iowa
Nelson, Rhonda, Meharry Medical College
Orfila, James, University of Texas, San Antonio
Simples. James. University of Pittsburgh
Villarreal. Julissa, University of Texas. San Antonio
Zayas, Ricardo, Tufts University

Teachers' Workshop: Living in the Microbial
World (August 15-21)

Course Directors

Olendzenski, Lorraine, University of Connecticut, Storrs
Dugas, Jeff, University of Connecticut, Storrs

Curriculum Specialist

Dorritie, Barbara, Cambridge Rindge and Latin School,
Cambridge, MA

R34 Annual Report

Course Assistant
Wier, Andrew. University of Massachusetts, Amherst

Presenters

Margulis, Lynn. University of Massachusetts. Amherst

Guerrero. Ricardo, University of Barcelona. Spain

Knoll. Andrew, Harvard University

Edgcomb. Virginia. Marine Biological Laboratory

Cast. Rebecca. Woods Hole Oceanographic Institution

Runimel. John. National Aeronautics and Space Administration

Teacher Participants

Molyneaux, Leslie, Hanover Middle School, Hanover, MA
Buckley, Kalhryn. Mashpee High School, Mashpee, MA
Henderson, Forest, Bellingham Jr./Sr. High School, Bellinghain, MA

Muscatell. Gina, Bellingham Jr./Sr. High School, Bellingham. MA

Bennett. Tara, Norwell High School, Norwell, MA

Webber. Alan. Norwell High School. Norwell. MA

Yuhas, Joseph. Kennebunk High School. Kennebunk. ME

Johnston, Ross B.. Nauset Regional High School. N. Eastham, MA

Albright, Lori, Nauset Regional High School. N. Eastham, MA

Carotenuto, Sheila, Quashnet River School. Mashpee. MA

Rocio, Zamaria. Horace Mann Middle School. San Diego. CA

Conn, Kathleen. West Chester Area School District, West Chester, PA

Carty, Susan, West Chester Area School District, West Chester, PA

Rutland. Susan, West Chester Area School District. West Chester. PA

Settertield. Elena. King Ethelbert School, Kent, England

Scales, Sacha, King Ethelbert School, Kent, England

Cronin, Maureen. Nonington C.E.P. School, Kent. England

Scott. Nyree. Nonington C.E.P. School. Kent, England

Summer Research Programs

Principal Investigators

Adamo, Shelley, Dalhousie University, Canada
Armstrong, Clay, University of Pennsylvania
Armstrong, Peter B.. University of California, Davis
Augustine, George J., Duke University Medical Center

Balaban, Pavel. Russian Academy of Sciences, Russia

Barlow. Robert B. Jr.. State University of New York Health Science

Center
Beauge, Luis, Institute de Investigation Medica "Mercedes y Martin

Ferreyra," Argentina

Beckman, Matthew, University of Alabama. Birmingham
Ben-Jonathan, Nira, University of Cincinnati
Bennett. Michael V. L., Albert Einstein College of Medicine
Bodznick. David, Wesleyan University
Boron, Walter, Yale University Medical School
Borst, David, Illinois State University
Boyer, Barbara, Union College
Boyle. Richard. Oregon Health Sciences University
Brady. Scott T. The University of Texas Southwestern Medical Center.

Dallas

Brock, Matthew, Stanford University

Browne, Carole, Wake Forest University School of Medicine
Burger, Max M.. Friedrich Miescher Institut, Switzerland

Cardullo, Richard, University of California, Riverside

Carvan, Michael, University of Cincinnati

Chappell. Richard L., Hunter College, City University of New York

Cohen, Lawrence B., Yale University School of Medicine

Cohen, William D., Hunter College, City University of New York

Crespi. Marco, Scientific Institute S. Raffaele, Italy

De Weer, Paul, University of Pennsylvania School of Medicine

DePass. Anthony, Long Island University, Brooklyn

DePina, Ana S., Dartmouth College

DiPolo, Reinaldo. Instituto Venezolano Investigaciones Cientificas,

Venezula

Dodge, Frederick, State University of New York Health Science Center
Doussau, Frederic, Duke University Medical Center

Edds-Walton, Peggy. Parmly Hearing Institute
Ehrlich, Barbara, Yale University School of Medicine

Fay, Richard, Loyola University of Chicago

Field, Christine, Harvard University Medical School

Fishman, Harvey M., University of Texas Medical Branch, Galveston

Flamarique, Inigo Novales, University of Victoria. Canada

Gadsby, David. Rockefeller University

Gerhart, John, University of California. Berkeley

Giuditta. Antonio, University of Naples, Italy
Goldman, Robert D., Northwestern University Medical School
Gould, Robert, New York State Institute for Basic Research
Groden. Joanna, University of Cincinnati

Haimo, Leah, University of California, Riverside

Han. Yi. Baylor College of Medicine

Heck. Diane. Rutgers University

Hershko, Avram, Technion-Israel Institute of Technology, Israel

Highstein, Steven M., Washington University School of Medicine

Hill, Susan Douglas. Michigan State University

Hines, Michael, Yale University School of Medicine

Hoskin. Francis, US Army Natick RD&E Center

Innocenti. Barbara. Iowa State University

Johnston. Daniel, Baylor College of Medicine
Jonas, Elizabeth, Yale University School of Medicine
Jones, Teresa, National Institutes of Health
Joye, Samantha, University of Georgia

Kaczmarek, Leonard, Yale University School of Medicine
Kaplan, Barry, National Institutes of Mental Health
Kaplan, Ilene M., Union College

Kier, William. University of North Carolina, Chapel Hill
Kirschner, Marc, Harvard University Medical School
Koulen, Peter, Yale University School of Medicine
Kuhns, William, The Hospital for Sick Children, Canada

Later. Eileen M., University of Texas Health Science Center

Landowne, David, University of Miami School of Medicine

Langford, George, Dartmouth College

Laskin, Jeffrey, University of Medicine and Dentistry of New Jersey

Laufer, Hans. University of Connecticut

LaVail. Jennifer, University of California, San Francisco

Lipicky, Raymond J.. Food and Drug Administration

Llinas, Rodolfo R., New York University Medical Center

Magee, Jeff, Louisiana State University Medical Center

Major, Guy, Lucent Technologies

Malgaroli, Antonio. University of Milan. Italy

Martinez. Joe, University of Texas. San Antonio

McAllister. A. Kimberly, Salk Institute of Biological Studies

McNeil. Paul, Medical College of Georgia

Mensinger, Allen. Washington University School of Medicine

Metuzals. Janis, University of Ottawa Faculty of Medicine, Canada

Mitchison, Timothy, Harvard University Medical School

Miyakawa. Hiroyoshi, Tokyo University of Pharmacy and Life Science,

Japan

Moore, John W., Duke University Medical Center
Mooseker, Mark, Yale University

R35

R36 Annual Report

Nasi, Ennco. Boston University School of Medicine

Ogden, David. National Institute for Medical Research
Ogunseitan. Oladele A.. University of California, Irvine

Palazzo, Robert, University of Kansas

Pant, Harish, National Institutes of Health

Parysek. Linda, University of Cincinnati

Paydarfar, David, University of Massachusetts Medical School

Quigley, James P., State University of New York, Stony Brook

Rabhitt, Richard, University of Utah

Rakowski. Robert F.. Finch University of Health Sciences/The Chicago

Medical School

Ramus. Seth. Boston University
Ratner. Nancy, University of Cincinnati
Reese, Thomas S., National Institutes of Health
Rieder, Conly, Wadsworth Center

Ripps, Harris, University of Illinois College of Medicine
Rome, Larry, University of Pennsylvania
Russell. John M., Hahnemann University

Salmon, Edward, University of North Carolina, Chapel Hill

Siwicki. Kathleen. Swarthmore College

Sloboda. Roger D.. Dartmouth College

Spiegel, Evelyn, Dartmouth College

Spiegel. Melvin, Dartmouth College

Srinivas, Miduturu. Albert Einstein College of Medicine

Standart, Nancy, University of Cambridge. United Kingdom

Steinacker, Antoinette, University of Puerto Rico

Sugimon, Mutsuyuki, New York University Medical Center

Suszkjw, Janusz, University of Cincinnati

Telzer, Bruce, Pomona College

Tilney, Lewis, University of Pennsylvania

Trinkaus. John P.. Yale University

Troll, Walter, New York University Medical Center

Tytell, Michael. Wake Forest University School of Medicine

Walters. Edgar, University of Texas. Houston
Weidner, Earl. Louisiana State University

Yamaguchi, Ayako. Columbia University

Yamoah, Ebenezer, University of Cincinnati College of Medicine

Zecevic, Dejan P.. Yale University School of Medicine
Zimmerberg, Joshua, National Institutes of Health
Zito, Karen, University of California, Berkeley
Zochowski, Michal. Yale University School of Medicine
Zottoli. Steven, Williams College
Zukin. R. Suzanne. Albert Einstein College of Medicine

Other Research Personnel

Adams, Curt, University of California, Riverside

Akanki. Feyisara, Williams College

Allen. Nina. North Carolina State University

Antic, Srdjan Henry, Yale University School of Medicine

Anton. Roberto, Hunter College

April, liana, Connecticut College

Armstrong, Clara. University of Pennsylvania

Asokan. R., Universitv of California, Davis

Baliga, Meghna. Wake Forest University

Banini, Bubu. Swarthmore College

Bashi, Esther. Yale University

Bearer, Elaine, Brown University

Benjamins. Steven, Groningen University. The Netherlands

Bergamaschi, Andrea, Fondazione Centra San Raffaele del Moute

Tabor. Italy

Berger-Sweeney. Joanne. Wellesley College
Bertetto. Lisa, Wesleyan University

Bezanilla, Francisco. University of California, Los Angeles
Billack. Blase. Rutgers University
Bingham. Eula. University of Cincinnati Medical School
Bonacci. Lisa, Hunter College

Bronner-Fraser, Marianne. California Institute of Technology
Brown. Joel, Albert Einstein College of Medicine
Bucior, Inona, Friedrich Miescher Institute, Switzerland
Burris, Jennifer, Northwestern University Medical School

Chan, Sena. Long Island University
Cho, Myoung-Soon. National Institutes of Health
Clarkson, Melissa, University of Kansas
Clifton. Christine. Mount Holyoke College
Crawford, Karen, St. Mary's College of Maryland

Davis, Bruce. Yale University

Debowy, Owen, New York University School of Medicine

Desai. Arshad. European Molecular Biology Laboratory, Germany

Detrait. Eric. University of Texas Medical Branch

Devlin, Leah. Penn State University

Doherty. Ann, Connecticut College

Dou. Hongwei, University of Cincinnati

Dumollard, Remi, Station Zoologique de Ville-Franche Sur Mer. France

Eddleman. Christopher. University of Texas Medical Branch. Austin
Escalona de Motta. Gladys. University of Puerto Rico
Eyman, Maria. University of Naples, Italy

Fakhrzadeh, Ladan. Rutgers University

Felke. Erin, University of Illinois

Femandez-Busguets. Xavier. Friedrich Miescher Institute. Switzerland

Fraser, Scott. California Institute of Technology

Fukui. Yoshio. Northwestern University Medical School

Gainer. Harold. National Institutes of Health
Galanis, Jennifer. National Institutes of Health
Galbraith. James A., National Institutes of Health
Gallant, Paul E.. National Institutes of Health

Summer Research R37

Gallo. Michael, University of Medicine and Dentistry of New Jersey

Gerosa-Erni, Daniela, Friedrich Miescher Institute. Switzerland

Gioio, Anthony, National Institutes of Mental Health

Gleeson. Richard, University of Florida

Goldman, Anne E., Northwestern University Medical School

Gomez, Maria del Pilar, Boston University School of Medicine

Gonzalez-Lima, Francisco, University of Texas, Austin

Grant, Philip, National Institutes of Health

Grassi, Daniel, Food and Drug Administration

Gyoeva, Fatima K., Institute of Protein Research. Russia

Hagar, Robert, Yale University School of Medicine

Harrington, John, University of South Alabama, Mobile

Harrist, Alexia, Yale University

Harrow, Faith, Hunter College

Harwood, Claire, University of Pennsylvania

Helfand, Brian, Northwestern University Medical School

Helm. Jessica, Washington and Lee University

Hernandez, Carlos, New York University School of Medicine

Herrick, Scott, University of California, Riverside

Hitt, James, State University of New York Health Science Center

Hiza, Nicholas, Williams College

Ho-Sang. Dwight, Williams College

Hogan. Emilia, Yale University Medical School

Holford, Kenneth, Illinois State University

Holmgren, Miguel, Harvard University Medical School

Hoof, Laura, University of Chicago

Nguyen, Michael P., University of Texas Medical Branch

Oegema. Karen, European Molecular Biology Laboratory. Germany

Ogan, Jeff, Illinois State University

Orfila, James, University of Texas, San Antonio

Peck, Raphaela, Reed College

Petersen, Jennifer. National Institutes of Health

Powers, Maureen. Vanderbilt University

Prahcad, Veena, Northwestern University Medical School

Prasad, Kondury. University of Texas Health Science Center

Price, Nichole, Connecticut College

Quinn, Kerry, Yale University School of Medicine

Ingrassia. Rosaria, University of Milan, Italy

Inoue, Masashi, Tokyo University of Pharmacy and Life Science, Japan

Intravaiu. Anthony, NASA Ames Research Center

Janowitz, Tobias, University Hannover, Germany

Ramsey, David, Harvard University

Rankin, Ellen, Colgate University

Reinsch, Sigrid, NASA Ames Research Center

Rhodes, Paul, New York University Medical School

Ring, Sabine, University of Frankfurt. Germany

Rosenstein. Fred, Hunter College

Ruta, Vanessa, Hunter College

Kamino, Kohtaro, Tokyo University School of Medical and Dental,

Japan

Kannenberg, Kai. University of Milan, Italy
Kapoor, Turun, Harvard University Medical School
Kifaieh, Nidal. Long Island University
King. Alison Jane, Dalhousie University, Canada
Klimov, Andrei, University of Pennsylvania
Koroleva, Zoya, Hunter College
Kuner, Thomas, Duke University Medical Center

Lassen, Kara G., Wake Forest University

Lee, Kyeng Gea, Hunter College

Lee, Rosalynn, University of Georgia

Lesher, Sarah. University of Maryland

Leznik, Elena, New York University School of Medicine

Loboda. Andrey, University of Pennsylvania

Lowe. Christopher, University of California, Berkeley

Schuette. Etha, Hunter College

Schwartz. William, University of Massachusetts Medical School

Simpson, Tracy, University of Hartford

Steffen, Walter. University of Rostock, Germany

Steinacker, Antoinette. University of Puerto Rico

Stockbridge, Norman, Food and Drug Administration

Szalisznyo, Krisztina, Hungarian Academy of Science. Hungary

Tamse, Catherine, University of Rhode Island

Tan, Xiao, Williams College

Tang, Kathleen. Washington University

Taylor, Kevin, Wake Forest University

Thorn, George, University of Cambridge. United Kingdom

Tokumaru. Hiroshi. Duke University Medical Center

Tokumaru. Keiko, Duke University Medical Center

Townsel, James G.. Meharry Medical College

Tran. Phong. Columbia University

Twersky. Laura, Saint Peter's College

Maddox, Paul. University of North Carolina, Chapel Hill

Malchow. Robert Paul, University of Illinois, Chicago

Melishchuk. Alexey. University of Pennsylvania

Mitchell, Michael, Wake Forest University

Miyake, Katsuya, Fukushima Medical College, Japan

Mohan, Nishal, Hunter College

Moir, Robert. Northwestern University

Motta. Melissa, Williams College

Vasse. Aimee, Williams College
Villa-Komaro. Lydia. Northwestern University

Wachowiak, Matt, Yale University School of Medicine

Ward, Rita. National Institutes of Health

Watts. Kisha, Williams College

Wen, Huajie, National Institutes of Mental Health

Wisniewski, Rachel. University of Georgia

R38 Annual Report

Woellert, Torsten, University of Rostock, Germany
Wu, Samuel, Baylor College of Medicine

Yancey. Sadiga, Mass Bay Community College
Yang, Stacy. University of California, Irvine
Young, Iain, University of Pennsylvania

Zakevicius, Jane M., University of Illinois College of Medicine
Zavilowitz, Joe, Yale University

Library Readers

Abbott, Jayne, Marine Research, Inc.
Adelberg. Edward, Yale University
Ahmadjian. Vernon, Clark University
Allen. Garland, Washington University
Allen, Nina, North Carolina State University
Alliegro, Mark, Louisiana State University
Alsup, Peggy, Tennessee Department of Health
Anderson. Everett. Harvard Medical School

Barrett, Dennis, University of Denver

Barry. Susan, Mount Holyoke College

Bedard, Andre, York University

Benjamin, Thomas, Harvard Medical School

Bcrnhard, Jeffery, University of Massachusetts Medical School

Bernheimer, Alan, New York University School of Medicine

Borgese, Thomas, Lehman College-CUNY

Boyer. John, Union College

Campos, Ana, McMaster University

Candelas. Graciela. University of Puerto Rico

Chang. Donald, Hong Kong University

Child, Frank. Woods Hole. MA

Clark, Douglas, John Hopkins University

Clarkson, Kenneth, Lucent Technologies

Cobb, Jewel, California State University

Cohen, Seymour. Woods Hole, MA

Colinvaux, Paul. Marine Biological Laboratory

Collier, Marjorie, University of Louisiana

Cooperstein. Sherwin, University of Connecticut Health Center

Copeland, Eugene, Woods Hole, MA

Corwin, Jeffery, University of Virginia

Couch, Ernest, Texas Christian University

Cowling, Vincent, University of Florida

Duncan, Thomas K., Nichols College
Epstein, Herman. Brandeis University

Farmant'armaian, A. Verdi, Rutgers University

Fee, Michale, Bell Laboratories

Frenkel. Krystyna. New York University School of Medicine

Gabriel, Mordecai, Brooklyn College

Galatzer-Levy. Robert. University of Chicago

German, James, Cornell University

Ginsberg, Harold, National Institutes of Health

Goldstein, Moise, Johns Hopkins University

Grossman, Albert, New York University School of Medicine

Gruner, John, Cephalon, Inc.

Guttenplan. Joseph, New York University Dental and Medical School

Haimo. Leah, University of California
Harrington, John, University of South Alabama
Hays, Thomas, University of Minnesota
Hernandez, Mari-Luz, University of Nice
Herskovits. Theodore, Fordham University
Hunter. Robert, Gartnaval Royal Hospital

Inoue, Sadayuki, McGill University

Jacobson, Allan, University of Massachusetts
Josephson, Robert, University of California

Kaltenbach, Jane, Mount Holyoke College

Kamino, Kohtaro, Tokyo Medical and Dental School of Medicine

Karlin, Arthur, Columbia University

King, Kenneth, Falmouth, MA

Klein, Donald, Colorado State University

Kornberg. Hans, Boston University

Krane, Stephen M., Harvard Medical School

Laster, Leonard, University of Massachusetts Medical Center

Lee, John, City College of CUNY

Levy, Arthur, St. Vincents Hospital

Lorand, Laszlo, Northwestern University Medical School

Luckenbill, Louise, Ohio University

MacNichol. Edward, Boston University School of Medicine
Masland. Richard, Massachusetts General Hospital
Mauzerall, David, Rockefeller University
Mitchell. Ralph, Harvard University/DEAS
Mizell, Merle, Tulane University

Nagel, Ronald. Albert Einstein College of Medicine
Narahashi, Toshio, Northwestern University Medical School
Naugle, John. National Aeronautics and Space Administration
Nicaise, Ghislain, University of Nice
Nickerson. Peter. State University of New York. Buffalo

Olds, James, George Mason University

Pappas. George, University of Illinois, Chicago

Pollen, Dan, University of Massachusetts Medical Center

Porter, Mary, University of Minnesota

Schippers, Jay, The HNG Foundation

Schuel, Herbert, University of Buffalo, SUNY

Shepro. David, Boston University

Shriftman, Molly, Woods Hole, MA

Spector, Abraham, Columbia University

Spotte, Stephen, University of Connecticut

Sundquist, Eric, U.S. Geological Survey

Sweet, Frederick, Washington University School of Medicine

Trager, William, Rockefeller University
Tweedell, Kenyon, University of Notre Dame
Tykocinski. Mark. University of Pennsylvania

Van Holde, Kensal, Oregon State University

Walton. Alan, University of Cambridge
Warren, Leonard, Wistar Institute
Wennger, Elora, Pfizer Central Research

1999 Library Room Readers

Dan Alkon

National Institute of Health

Lucio Cariello

Stazione Zoologica A. Dohm

Giuseppe D'Alessio
University of Naples

Robert Goldman

Northwestern University Medical School

Roberto GonzalezPalaza
Northwest Indian College

Harlyn Halvorson

Marine Biological Laboratory

Michael Hines

Yale Univ. School of Medicine

Andres Kanner
Rush University

Alex Keynan

Israel Academy of Science

Kamino Kohtaro

Tokyo Medical and Dental

John W. Moore

Duke University Medical Center

Leyla Morrel
Rush University

Michael Rabinowitz

Marine Biological Laboratory

George Reynolds
Princeton University

Ann Stuart
UNC Chapel Hill

Gerald Weissmann
NYU School of Medicine

Summer Research

Whillaker, J. Richard, University of New Brunswick
Wolken, Jerome J., University of Pittsburgh

Yevick, George, Stevens Institute of Technology

Domestic Institutions Represented

Acorda Therapeutics

Alabama. University of. Birmingham

Alaska Museum, University of

Albany Medical College

Albert Einstein College of Medicine

Allegheny University of the Health Sciences

Arizona State University

Arizona, University of, Tucson

Barnard College

Baylor College of Dentistry

Baylor College of Medicine

Bell Laboratories

Berkeley National Laboratory

Beth Israel Hospital

Beth Israel-Deaconess Medical Center

Boston University

Boston University School of Medicine

Brandeis University

Bridgeport Hospital

Brigham and Women's Hospital

Brigham Young University

Bristol-Myers Squibb PRI

Bronx Veterans Administration Medical Center

Brown University

Bryn Mawr College

Buck Center for Research in Aging

Buffalo, University of

Burnham Institute

California Institute of Technology

California State University. Sacramento

California, University of, Berkeley

California, University of, Davis

California, University of, Irvine

California, University of, Los Angeles

California, University of. Riverside

California, University of, San Diego

California, University of, San Francisco

California, University of, Santa Barbara

Carl Zeiss, Inc.

Carnegie Mellon University

Case Western Reserve University

Catholic University of America

Center for Clinical Computing

Centers for Disease Control and Prevention

Chicago, University of

Children's Hospital, Boston

Cincinnati, University of

Cleveland Clinic Foundation

Cold Spring Harbor Laboratory

Colgate University

Colorado Health Science Center, University of

Colorado School of Medicine, University of

Colorado, University of. Boulder

Columbia University

Columbia-Presbyterian Medical Center

Connecticut College

Connecticut Health Center, University of

Connecticut, University of

Cook County Hospital

Cook Institute for Research and Education

Cornell University

Cornell University Medical Center

Cornell University Medical College

Creative Micro Tech. Inc.

Dana-Farber Cancer Institute

Dartmouth College

Dartmouth-Hitchcock Medical Center

Delaware, University of

Duke University

Duke University Medical Center

Eastern Virginia Medical School

Emory University

Emory University School of Medicine

Finch University of Health Sciences

Florida Institute of Technology

Florida State University

Florida, University of

Food and Drug Administration

Forsyth Dental Center

Foundation of Microbiology

Fred Hutchinson Cancer Research Center

General Electric Corporate Research Center

George Washington University

Georgetown University

Georgetown University Medical Center

Georgia State University

Georgia, University of

Geron Corporation

Guam. University of

Hahnemann University

Harbor-UCLA Medical Center

Harlem Hospital Center

Hartford, University of

Harvard University Medical School

Harvard School of Public Health

Harvard University

Hawaii, University of

Hebrew Rehabilitation Center for Aged

House Ear Institute

Houston Academy of Medicine

Houston, University of

Howard University

Hunter College

Idaho State University

Idaho, University of

Illinois State University

Illinois. University of, Chicago

Illinois, University of, Urbana-Champaign

Indiana State University

Indiana University

Indiana University School of Medicine

Institute for Genomic Research

R40 Annual Report

Iowa State University
Iowa, University of

Jet Propulsion Laboratory

Johns Hopkins University School of Medicine

Kansas Medical Center. University of
Kansas, University of
Kent State University
Kentucky. University of
Kewalo Marine Laboratory

Lehigh University

Leica. Inc.

Loma Linda University School of Medicine

Long Island University

Louisiana State University

Louisiana State University Medical Center

Louisville. University of

Loyola University of Chicago

Lucent Technologies

Ludwig Institute for Cancer Research. San Diego

Marine Biological Laboratory

Maryland School of Medicine, University of

Maryland. University of

Mass Bay Community College

Massachusetts General Hospital

Massachusetts Institute of Technology

Massachusetts Medical School, University of

Massachusetts. University of

Mayo Clinic Scottsdale

Medical College of Georgia

Meharry Medical College

Memorial Sloan-Kettermg Cancer Center

Merck & Co.

Merck Research Laboratory

Mercy College

MetroHealth Medical Center

Miami. University of

Michigan Medical School, University of

Michigan State University

Michigan. University of

Midwestern University

Minnesota School of Medicine, University of

Minnesota, University of

Missouri, University of

Montana State University

Morehouse School of Medicine

Mount Holyoke College

NASA Ames Research Center

National Cancer Institute

National Institutes of Health

National Institutes of Health Library

National Institutes of Mental Health

National Library of Medicine

National Marine Fisheries Service

Naval Medical Research Institute

Nebraska. University of

Neuralynx Inc.

Neurosciences Institute

New Jersey. University of Medicine and Dentistry

New Mexico. University of

New York and Presbyterian Hospital

New York Health Science Center. State University of

New York State Department of Health

New York State Institute for Basic Research

New York University Medical Center

New York University School of Medicine

New York, City University of

New York, State University of, Albany

New York, State University of. Buffalo

New York, State University of. Stony Brook

North Carolina State University

North Carolina, University of, Chapel Hill

North Dakota. University of

North Texas, University of

Northern Arizona University

Northwest Indian College

Northwestern LIniversity

Northwestern University Medical School

Occidental College

Ohio State University

Ohio University

Ohio, Medical College of

Oklahoma, University of

Oregon Health Science University

Oregon Regional Primate Research Center

Oregon State University

Oregon, University of

Parke-Davis Research

Parmly Hearing Institute

PE Applied Biosystems

Penn State University

Pennsylvania Health System, University of

Pennsylvania LIniversity School of Medicine

Pennsylvania. University of

Pharmacia & Upjohn

Pittsburgh, University of

Pomona College

Prince George's Hospital Center

Princeton LIniversity

Procter & Gamble

Puerto Rico, University of

Purdue University

Quorum Pharmaceuticals

Reed College

Rhode Island, University of

Rochester Medical Center, University of

Rockefeller University

Rush-Presbyterian-St. Luke's Medical Center

Rutgers University

Saint Peter's College

Samaritan Health System. Phoenix

Scnpps Institution of Oceanography

Seattle Biomedical Research Institute

SETI Institute

SmithKlme Beecham Pharmaceuticals

Smithsonian Institution

South Alabama, University of. Mobile

Southern California, University of

Southern Methodist University

Summer Research R-ll

St. Jude Children's Research Hospital

St. Louis University School of Medicine

St. Mary's College of Maryland

Stanford University

Stanford University Medical Center

Stratagene

Swarthmore College

Temple University School of Medicine

Tennessee. University of

Texas A&M University

Texas Health Science Center. University of

Texas Medical Branch. University of

Texas Southwestern Medical Center, University of

Texas Southwestern, University of

Texas, University of

Thomas Jefferson University

Toledo, University of

Towson University

Tufts University

Tufts University Health Sciences Library

Tufts University School of Medicine

Tulane University Medical Library

Unilever Research
Union College

United States Army Medical Research Institute
United States Army Natick RD&E Center
United States Department of Agriculture
United States Environmental Protection Agency
Utah Medical Center, University of
Utah, University of

Vanderbilt University

Veterans Administration Medical Center

Virginia Commonwealth University

Virginia Health Sciences Center. University of

Virginia School of Medicine. University of

Virginia Tech

Virginia. University of

Wadsworth Center for Labs and Research

Wake Forest University

Wake Forest University School of Medicine

Washington and Lee University

Washington University School of Medicine

Washington, University of

Wayne State University

Wellesley College

Wesleyan University

Western Cape, University of the

Whitehead Institute for Biomedical Research

William and Mary, College of

Williams College

Wisconsin, University of, Madison

Wisconsin. University of, Milwaukee

Woods Hole Oceanographic Institution

Yale University

Foreign Institutions Represented

Alberto Monroy Foundation Palermo, Italy
Alexandria University, Egypt

All India Institute of Medical Sciences, India
Apollo Hospitals, India
Australian National University. Australia
Autonomous University of the State of Puebla, Mexico

Basel Institute for Immunology. Switzerland
Basel, University of. Switzerland
Bio-Rad Microscience. United Kingdom
Bogor Agricultural University, Indonesia
British Columbia, University of, Canada
Bruxelles, Universite Libre de, Belgium
Buenos Aires, University of, Argentina

Calgary, University of. Canada

Cambridge, University of. United Kingdom

Centre de Investigation y de Estudios Avanzados, Mexico

Chile, University of. Chile

Copenhagen, University of. Denmark

Dalhousie University. Canada

Denmark. Technical University of. Denmark

Dundee. University of. Scotland

Edinburgh. University of. Scotland

Ethiopian Health and Nutrition Institute. The Netherlands

European Molecular Biology Laboratory. Germany

Fondazione Centre San Raffaele del Moute Tabor, Italy

Frankfurt. University of, Germany

Friedrich Meischer Institute. Switzerland

Fukushima Medical College. Japan

Fundacao Universitaria de Endocrinologia e Fertilidade, Brazil

Glasgow, University of. United Kingdom
Gromngen University, The Netherlands
Guelph, University of. Canada

Hannover. University of, Germany

Hebrew University of Jerusalem, Israel

Hong Kong, The Chinese University of. Hong Kong

Hospital for Sick Children. Canada

Hungarian Academy of Science. Hungary

IceTherm Inc., Iceland

Imperial Cancer Research Fund, United Kingdom

Imperial College of Science, Technology and Medicine,

United Kingdom

Institute of Protein Research. Russia
Instituto de Investigacion Medica "Mercedes y Martin Ferreyra.'

Argentina

Instituto Venezolano Investigaciones Cientificas, Venezuela
International School of Advanced Studies. Italy

Karolmska Institute Stockholm. Sweden
Konstanz, University of. Germany
Kuopio. University of, Finland

Landcare Research, New Zealand

Lausanne. University of, Switzerland

Leiden, University of. The Netherlands

Lethhridge, University of, Canada

Liverpool Medical School, University of. United Kingdom

Lodz, Medical Academy of. Poland

R42 Annual Report

Loeh Health Research Institute. Canada
London, University of. United Kingdom

Manchester, University of. United Kingdom

Max-Planck-Institut, Germany

McGill University, Canada

McMaster University, Canada

Medical Research Council. United Kingdom

Melbourne. University of, Australia

Milan, University of, Italy

Naples. University of, Italy

National Centre for Biological Sciences, India

National Institute for Medical Research. United Kingdom

National Research Council, Canada

Otago, University of. New Zealand
Ottawa, University of, Canada
Oxford, University of. United Kingdom

Paris, University of, France

Pasteur Institute-Lille, France

Palerson Institute for Cancer Research, United Kingdom

Pisa. University of, Italy

PPL Therapeutics, Scotland

Regensburg, University of, Germany

Rio de Janeiro, Federal University of, Brazil

Rio de Janeiro, State University of, Brazil

Roslin Institute, Scotland

Rostock. University of, Germany

Russian Academy of Sciences. Russia

Sao Paulo, University of. Brazil

Saskatchewan, University of. Canada

Scientific Institute San Raffaele. Italy

Simon Fraser University, Canada

Southampton Oceanography Centre, United Kingdom

St. Thomas' Hospital London. United Kingdom

Station Zoologique de Ville-Franche Sur Mer. France

Swiss Federal Institute of Technology, Switzerland

Swiss Institute for Experimental Cancer Research. Switzerland

Sydney, University of Technology, Australia

Sydney, University of. Australia

Tata Institute of Fundamental Research, India
Technion-Israel Institute of Technology, Israel
Tohoku University, Japan

Tokyo University of Pharmacy and Life Science, Japan
Tokyo University School of Medical and Dental. Japan
Tokyo. University of, Japan
Toronto, University of, Canada
Tubingen, University of. Germany

Universidad Nacional Autonoma de Mexico, Mexico

Universita "La Sapienza," Italy

Universite Paris-Sud, France

University College London. United Kingdom

Wageningen Agricultural University, The Netherlands
Wallenberg Laboratory for Cardio Research, Sweden
Walter and Eliza Hall Institute, Australia
Witwatersrand, University of the. South Africa
World Health Organization. West Africa

Zurich, University of, Switzerland

Year-Round Research
Programs

Architectural Dynamics in Living Cells
Program

The Josephine Bay Paul Center for
Comparative Molecular Biology and Evolution

Established in 1992, this program focuses on architectural dynamics
in living cells — the timely and coordinated assembly and disassembly of
macromolecular structures essential for the proper functioning, division,
motility. and differentiation of cells; the spatial and temporal
organization of these structures; and their physiological and genetic
control. The program is also devoted to the development and application
of powerful new imaging and manipulation devices that permit such
studies directly in living cells and functional cell-free extracts. The
Architectural Dynamics in Living Cells Program promotes
interdisciplinary research carried out by resident core and visiting
investigators.

Resident Core Investigators

Danuser, Gaudenz, Postdoctoral Fellow
Inoue, Shinya. Distinguished Scientist
Katoh, Kaoru. Postdoctoral Scientist
Oldenbourg, Rudolf. Associate Scientist

Staff

Geer, Thomas. Research Assistant

Knudson, Robert. Instrumental Development Engineer

Baraby. Diane. Laboratory Assistant

MacNeil. Jane. Executive Assistant

Staff

Arimoto. Rieko, Washington University School of Medicine

Biggs. David, AutoQuant Imaging Inc.

Desai. Arshad. EMBL. Heidelburg. Germany

Fukui, Yoshio, Northwestern University Medical School

Goda, Makoto. Kyoto University. Japan

Inoue. Theodore D., Universal Imaging Corporation

Keefe, David, Rhode Island Women and Infants Hospital

Liu. Lin. Rhode Island Women and Infants Hospital

Maddox, Paul, University of North Carolina-Chapel Hill

Matsurnoto, Brian. University of California-Santa Barbara

Milchison, Timothy J.. Harvard Medical School

Murray. John M., University of Pennsylvania

Salmon, Edward D., University of North Carolina-Chapel Hill

Tran. Phong, Columbia University

Major emphasis in the Josephine Bay Paul Center in Comparative
Molecular Biology and Evolution is placed upon comparative/
phylogenetic studies of genes and genomes, molecular microbial
ecology/biodiversity and evolution of host defense mechanisms in
marine invertebrates. The Center encourages studies of genotypic
diversity across all phyla and promotes the use of modern molecular
genetics and phylogeny to gain insights into the evolution of molecular
structure and function. The Josephine Bay Paul Center is a member of
NASA's Virtual Institute for Astrobiology.

Other major research programs include Mitchell Sogin's studies of
molecular evolution in eukaryotes and studies of genome sequences
from parasitic microorganisms, Monica Riley's metabolic database and
evolutionary studies of protein sequences, Neal Cornell's comparative
molecular studies of genes critical to heme biosynthesis, and Michael
Cummings' studies of evolution of pathogenetic microorganisms.

Other collaborative projects include studies of P450 evolution (M.
Sogin and John Stegeman's laboratory at Woods Hole Oceanographic
Institution [WHOI] ). a molecular ecology component of the Long Term
Ecological Research project (M. Sogin's laboratory and John Hobbie of
The Ecosystems Center), and studies of molecular diversity among
marine protists and bacteria (with marine microbiologists at WHOI).
Future recruiting efforts will focus upon molecular evolution in
developmental biology and genome sciences.

The Center has excellent resources for studies of molecular evolution:
automated DNA sequencing, well-equipped research laboratories, and
powerful computational facilities. In addition to participating in the
Parasitology and Microbial Diversity courses, the Center sponsors the
Workshop in Molecular Evolution at the MBL, which has gained an
international reputation for excellence. This Workshop offers 60 students
a series of lectures and minisymposia that are complemented by a state-
of-the-art computational facility.

The Josephine Bay Paul Center in Comparative Molecular Biology
and Evolution includes the laboratories of Neal Cornell. Michael
Cummings. Monica Riley. and Mitchell Sogin.

Resident Core Investigators

Sogin. Mitchell, Director and Senior Scientist
Cornell, Neal, Senior Scientist
Cummings. Michael. Assistant Scientist
Riley. Monica. Senior Scientist
Wainwrieht, Norman, Senior Scientist

R43

R44 Annual Report

Adjunct Scientists

Halanych. Ken, Woods Hole Oceanographic Institution
Teske, Andreas, Woods Hole Oceanographic Institution

Laboratory of Neal Cornell

Research in this laboratory is concerned with the comparative
molecular biology of genes that encode the enzymes for heme
biosynthesis, with particular emphasis on 5-ammolevulinate synthase,
the first enzyme in the pathway. Because the ability to produce heme
from common metabolic materials is a near universal requirement for
living organisms, these genes provide useful indicators of molecular
aspects of evolution. For example, 5-aminolevulinate synthase in
vertebrate animals and simple eukaryotes such as yeast and Plasmodium
ftili'iparum have high sequence similarity to the enzyme from the alpha-
purple subgroup of eubacteria. This supports the suggestion that alpha-
purple bacteria are the closest contemporary relatives of the ancestor of
eukaryotic mitochondria. The analysis also raises the possibility that
plant and animal mitochondria had different origins. Aminolevulinate
synthase genes in mitochondria-containing protists are currently being
analyzed to obtain additional insight into endosymbiotic events. Also,
genes of primitive chordates are being sequenced to gain information
about the large-scale gene duplication that played a very important role
in the evolution of higher vertebrates. Other studies in the laboratory
have been concerned with the effects of environmental pollutants on
heme biosynthesis in marine fish, and it has been shown that
polychlorinated biphenyls (PCBs) enhance the expression of the gene for
aminolevulinate synthase.

Staff

Cornell. Neal W., Senior Scientist
Faggart. Maura A., Research Assistant
Foster, Martin, Laboratory Assistant
Frisbee, Cameran. Laboratory Assistant

Visiting Scientist

Fox, T.O., Harvard Medical School

Laboratory of Michael P. Cummings

The research is in the area of molecular evolutionary genetics and
includes the study of the mechanisms of molecular genetic processes,
and uses methods from molecular biology, statistics, computer science,
molecular systematics, and population genetics. The basis for much of
the research is comparative, across several levels of biological
organization, and involves both computer-based and empirical studies.

The major focus of research is using novel statistical methods to
study relationships between genotype and phenotype. Current
investigations in this area examine how gene sequence data can be used
to understand and predict drug resistance in tuberculosis, variation in
color vision, and basic immune system functions at the molecular level.
For example, using drug resistance in Mycobacterium tuberculosis as a
model system, we are investigating how well phenotype (level of drug
resistance) can be predicted with genotype information (DNA sequence
data). Drug resistance is a major problem in the treatment of infectious
diseases. Understanding evolution of drug resistance, and developing
accurate methods for its prediction using DNA sequence data can help
in assessing potential resistance in a more timely fashion and circumvent
the need for culturing bacteria, which takes several weeks in the case of
tuberculosis. More generally, the relationship of genotype to phenotype
is a fundamental problem in genetics, and through these investigations
we hope to gain insight. The primary empirical work in the laboratory
involves examination of opsins, proteins involved in color vision, from
local species of Odonata (dragonflies and damselflies).

Research on evolution of pathogenic bacteria also examines species
within the genus Mycobacterium. Mycobacterium provides an excellent
model system for studying evolution of pathogenicity and emergent
pathogens; it is a large and widely distributed group that occupies a
range of habitats (e.g., soil, water, skin), and exhibits a broad range of
relationships with other organisms (e.g., free-living, commensal,
parasitic). Importantly, the group contains a number of major human
pathogens (e.g., those that cause tuberculosis and leprosy), including
recently emerged pathogens. We are using phylogenetic analysis of
DNA sequence data to study the evolutionary patterns of pathogenicity
within Mycobacterium to discern patterns in the emergence of new
pathogens. The goal of this work is to understand the origins of new
pathogens and provide information that may aid in diagnosis and
treatment efforts.

Staff

Cummings, Michael P., Assistant Scientist
Mclnerney, Laura A., Research Assistant

Visiting Scientist

Neel, Maile C., University of California. Riverside

Laboratory of Monica Ri/ey

The genome of the bacterium Escheric/iia call contains all of the
information required for a free-living chemoautotrophic organism to
live, adapt, and multiply. The information content of the genome can be
dissected from the point of view of understanding the role of each gene
and gene product in achieving these ends. The many functions of E. coli
have been organized in a hierarchical system representing the complex
physiology and structure of the cell. In collaboration with Dr. Peter
Karp of SRI International, an electronic encyclopedia of information is
being constructed on the genes, enzymes, metabolism, transport

Year-Round Research R45

processes, regulation, and cell structure of E. coli. The interactive
EcoCyc program is now publicly available and has graphical hypertext
displays, including literature citations, on nearly all of E. coli
metabolism, all genes and their locations, a hierarchical system of cell
functions and some regulation processes. This work is continuing.
In addition, the E. coli genome contains valuable information on
molecular evolution. We are analyzing the sequences of proteins of E.
coli in terms of their evolutionary origins. By grouping like sequences
and tracing back to their common ancestors, we learn not only about the
paths of evolution for all contemporary E. coli proteins, but we extend
even further back before E. coli, traversing millennia to the earliest
evolutionary times when a relatively few ancestral proteins served as
ancestors to all contemporary proteins of all living organisms. The
complete genome sequence of E. coli and sophisticated sequence
analysis programs permit us to identify evolutionary related protein
families, determining ultimately what kinds of unique ancestral
sequences generated all of present-day proteins. The data developed in
the work has proven to be valuable to the community of scientists
sequencing microbial genomes. E. coli data serve as needed reference
points.

Staff

Riley. Monica, Senior Scientist
Kerr. Alastair, Postdoctoral Scientist
Liang, Ping, Postdoctoral Scientist
MacGregor, Alicia, Laboratory Clerk
Nalium, Laila, Postdoctoral Scientist
Pelegrini-Toole, Alida, Research Assistant II
Porterfield, Pamela, Laboratory Clerk
Serres. Margerethe, Postdoctoral Scientist

sudden evolutionary radiations that cannot be resolved by rRNA
comparisons and will provide insights into the presence or absence of
important biochemical properties in the earliest ancestors common to all
eukaryotic species.

More recently, we initiated a study of the complete genome of
Giardia lamblia.

Staff

Sogin, Mitchell L., Director and Senior Scientist

Amaral-Zettler, Linda, Postdoctoral Scientist

Beaudoin, David, Research Assistant

Bressoud, Scott, Laboratory Technician

Eakin, Nora, Research Assistant

Edgcomb, Virginia, Postdoctoral Scientist

Fair. Rebecca, Research Assistant

Gao, Lingqui, Research Assistant II

Harris. Marian, Executive Assistant

Holder, Greg, Research Assistant

Kim, Ulandt, Research Assistant

Kysela, David, Research Assistant

Laan. Maris, Research Assistant II

Lim. Pauline. Executive Assistant

Luders, Bruce. Research Assistant

McArthur, Andrew, Postdoctoral Scientist

Medina, Monica, Postdoctoral Scientist

Morrison, Hilary G., Postdoctoral Scientist

Nixon, Julie. Postdoctoral Scientist

Roger, Andrew, Postdoctoral Scientist

Shakir, Muhhamed Afaq, Postdoctoral Scientist

Silberman. Jeffrey. Postdoctoral Scientist

Program in Comparative Molecular Biologv and Evolution:
Laboratory of Mitchell L. Sogin

This laboratory in molecular evolution employs comparative
phylogenetic studies of genes and genomes to define patterns of
evolution that gave rise to contemporary biodiversity on the planet
Earth. The laboratory is especially interested in discerning how the
eukaryotic cell was invented as well as the identity of microbial groups
that were ancestral to animals, plants, and fungi. The lab takes
advantage of the extraordinary conservation of ribosomal RNAs to
define phylogenetic relationships that span the largest of evolutionary
distances. These studies have overhauled traditional eukaryotic microbial
classifications systems. The laboratory has discovered new evolutionary
assemblages that are as genetically diverse and complex as plants, fungi,
and animals. The nearly simultaneous separation of these eukaryotic
groups (described as the eukaryotic "Crown") occurred approximately
one billion years ago and was preceded by a succession of earlier
diverging protist lineages, some as ancient as the separation of the
prokaryotic domains.

At the same time, this data base provides a powerful tool for the
newly emerging discipline of molecular ecology. Using the ribosomal
RNA data base and nucleic acid-based probe technology, it is possible
to detect and monitor microorganisms, including those that cannot be
cultivated in the laboratory. This strategy has uncovered new habitats
and major revelations about geographical distribution of
microorganisms.

The laboratory has initiated a program to sample genomic diversity
from eukaryotic microorganisms that do not have mitochondria. The lab
previously demonstrated that these taxa represent some of the earliest
diverging lineages in the evolutionary history of eukaryotes. The
objective is to develop a set of additional molecular markers for
studying molecular evolution. These will be invaluable in unraveling

Visiting Investigators

Bahr, Michele, The Ecosystems Center
Campbell, Robert, Serono Laboratories, Inc.
Crump. Byron. The Ecosystems Center
Weil. Jennifer. Joslin Diabetes Center

Adjunct Scientists

Halanych. Kenneth, Woods Hole Oceanographic Institution
Teske, Andreas, Woods Hole Oceanographic Institution

R46 Annual Report

BioCurrents Research Center

The Biocurrents Research Center (BRCl. one of the NIH National
Centers for Research Resources, pioneers methods in the study of
transmembrane currents and hosts numerous research pursuits. The
Center provides visiting investigators access to a variety of unique
technologies as well as new approaches to experimentation in the
hiomedical sciences.

Four systems are available at the BRC. All these probe technologies
are based on the principle of a self-referencing electrode, maximizing
sensitivity by noise and drift reduction. All the probes are non-invasive
and generally placed in close proximity to the membrane of cells or
tissues, in some cases at sub-micron distances. The two older techniques
are designed to measure the movement of ions across the membranes ot
living tissues or cells with the minimum of disturbance. The current
probe, developed in 1974, is still available for the study of external
current densities resulting from the general net balance ot ion transport.
Most use is made of the ion-selective probes (Sens), which measure and
follow the transmembrane transport of specific ions such as calcium,
potassium and protons. This system also can detect non-electrogenic
transporters. Two newer techniques are also available: the BioKelvin
probe and the non-invasive electrochemical or polarographic probe
(Serp). The BioKelvin probe measures voltages around living tissues in
air. A radically different approach is being taken to the measurements of
biocurrents using the electrochemical microprobes. Presently applied to
molecular oxygen, such a technique offers opportunity for the study of
molecular transport by using the chemical redox potential. This probe
has been applied to single neurons. |8-pancreatic cells, damaged neural
tissues, developing embryos, and others. We are currently developing
further applications of the Serp probes to measure nitric oxide, ascorbic
acid, and insulin as well as the production of biosensors.

A state-of-the-art system offers non-invasive ion probes coupled with
current and voltage clamp (both single, two electrode, and patch) along
with ratio imaging via a Zeiss Attofluor system, all of which are finding
uses in the hosted biomedical studies, as well as BRC research and
development.

As in previous years, a wide variety of biological and biomedical
subjects have been studied by BRC staff and visitors. In R&D we have
continued developing the application of ion-selective and
electrochemical microsensors. all applicable to single cells with square
micron spatial resolution. We are currently exploring ways to combine
these sensors with a variety of techniques known collectively as near
field optical microscopy. In an experimental context we have advanced
our technology into several fields, including reproductive physiology,
diabetes research, neuroscience, development, gravitropic responses, ion

transport, and homeostasis. Details of our research program and a list of
publications can be found at <www.nibl.edu/BioCurrents>.

MBL year-round laboratories with which BRC is in active
collaboration are the Laboratory of Rudolf Oldenbourg and the
Laboratory of Reproductive Medicine, headed by David Keefe. Dr.
Keefe and Dr. Peter Smith. BRC Director, are Co-Investigators on a
project to support the development of new technology to assess the
developmental potential of preimplantation embryos and to study the
pathophysiology of oocyte dysfunction.

Staff

Smith. Peter J.S., Director and Senior Scientist
Baikie. Iain D., Associate Scientist
Danuser. Gaudenz M.. Postdoctoral Fellow
Hammar. Katherine, Research Assistant 111
McLaughlin. Jane A., Research Assistant III
Porterfield. D. Marshall, Staff Scientist I
Sanger, Richard H., Research Assistant III

Part Time and Temporary Staff'

Jaffe. Lionel F.. Senior Scientist

Moore. Laurel. Science Reference Librarian

Pepperell. John R., Staff Scientist I

Graduate Student

Tamse, Catherine T.. University of Rhode Island

Visiting Scientists and Publications

This year the Research Center hosted 47 visitors. Scientific publications
during the year numbered 25.

Boston University Marine Program

Faculty

Atema, Jelle, Professor of Biology, Director
Dionne, Vincent, Professor of Biology
Golubic, Stjepko. Professor of Biology
Humes, Arthur, Professor of Biology Emeritus
Kaufman, Les, Associate Professor of Biology
Lohel, Phillip. Associate Professor of Biology
Voigt. Rainer, Research Associate Professor
Ward, Nathalie, Lecturer

Staff

Decarie. Linette, Senior Staff Coordinator

DiNunno. Paul, Research Assistant. Dionne Lab

Hall. Sheri, Program Manager

McCafferty. Michelle. Administrative Assistant

Olson, Nancy, Program Assistant

Tomasky. Gabrielle. Research Assistant, Valiela Lab

Wheatlev, MaryJo, Information Officer

Postdoctoral Investigators

Basil. Jenny. Atema Laboratory
Cehrian. Just. Valiela Laboratory

Year-Round Research R47

Grasso, Frank, Atema Laboratory
Trott, Thomas, Atema Laboratory

Visiting Faculty and Investigators

Hanlon, Roger, Marine Biological Laboratory
Hecker. Barbara, Meeker Consulting
Margulis, Lynn, University of Massachusetts, Amherst
McFall-Ngai, Margaret, Kewalo Marine Laboratory
Moore, Michael. Woods Hole Oceanographic Institution
Nowacek, Douglas, Mote Marine Laboratory
Ruby, Edward, Kewalo Marine Laboratory
Simmons, Bill, Sandia National Laboratory
Wainvvright. Norman, Marine Biological Laboratory

Other

Dolan, Mike. Visiting Teaching Assistant
Weir. Andrew. Visiting Teaching Assistant

Graduate Students
PhD Students

Existing
Cole. Marci
Dale. Jonathon
Economakis. Alistair
Hauxwell. Jennifer
Herrold, Ruth
Kroeger, Kevin
Lindholm, James
Ma, Diana
Miller. Carolyn
Oliver, Steven
Sloan. Kevin
Stieve, Erica
Zettler, Erik
Zhao, Jing
New

Dooley, Brad
Tomasky, Gabrielle
York. Joanna

Masters Students

Existing
Allen, Christel
Atkinson. Abby
Barlas. Margaret
Bentis, Christopher
Bowen. Jennifer
Cavanaugh, Joseph
Chichester. Heather
D'Ambrosio. Alison
Evgenidou, Angeliki
Ferland. Amy
Fern. Sophie
Fredland, Inga
Griffin, Martin
Homkow, Laura
Keith, Lucy
Koenig, Eduardo
Konkle, Anne
Lamb. Amy
Lawrence. David

Levine, Michael
McKenna. Ian
Neviackas. Justin
Ramon. Marina
Smith, Spence
Watson. Elise
Wright. Dana

New

Casper, Brandon
Errigo, Michael
Frenz, Christopher
Grable, Melissa
Grebner, Dawn
Kollaros, Maria
Lever, Mark
Malley, Vanessa
Martel, David
Oweke, Ojwang William
Perez, Edmundo
Pugh, Tracy
Ripley, Jennifer
Roycroft, Karen
Stueckle, Todd
Sweeny. Melissa
Tuohy-Sheen, Elizabeth
Weiss, Erica

Undergraduate Students

Spring 99
Champagne, Jaimie
Preto, Luca
Watkins, Cari
Weisbaum. Dolores

Fall 99

Burgess, Robyn
Gottlieb, Jennifer
Griggs, Ryan
Kwong, Grace
Loewensteiner. David
Matsumoto, Rae
Muhlm. Jessica
O'Connell. Timmy
Peyton, Scott
Pytel. Julie
Sarno, Jillian
Silverston, Jennifer
von Kampen. Marie
Walker. Andrew
Williams. Jade
Wingert, Sarah
Woods. Pamela

Summer 1999 Interns

Berkey, Cristin
Cantield, Susannah
Cubbage, Andrea
Hanna. John
Komarow, Sharon
McLaughlin, Leslie
Mijos. Katnn

R48 Annual Report

Walters. Jennifer
Wai M in, Amy
Wolfe. Felisa
Young. Talia

Summer 1W9 Volunteers

Hancock. Amy
Qumn, Elizabeth

Laboratory of J vile Atenui

Many organisms and cellular processes use chemical signals as their
main channel of information about the environment. All environments
are noisy and require some form of filtering to detect important signals
Chemical signals are transported by turbulent currents, viscous flow, and
molecular diffusion. Receptor cells extract chemical signals from the
environment through various filtering processes. In our laboratory, fish,
marine snails, and Crustacea have been investigated for their ability to
use chemical signals under water. Currently, we use the lobster and its
exquisite senses of smell and taste as our major model to study the
signal-filtering capabilities of the whole animal and its narrowly tuned
chemoreceptor cells.

Research in our laboratory focuses on amino acids, which represent
important food signals for the lobster, and on the function and chemistry
of pheromones used in lobster courtship. We examine animal behavior
in the sea and in the lab. This includes social interactions and
chemotaxis. To understand the role of chemical signals in the sea we
use real lobsters and untelhered small robots. Our research includes
measuring and computer modeling odor plumes and the water currents
lobsters generate to send and receive chemical signals. Other research
interests include neurophysiology of receptor cells and anatomical
studies of receptor organs and pheromone glands.

Liibomton' of Vincent Dionne

How does the brain learn about an odor? This simple question frames
a complex problem about how information is transferred into and within
the brain. Odors are powerful stimuli. They can focus the attention,
elicit behaviors, and resurrect forgotten memories. These actions depend
on the initial transduction and encoding of odor signals by olfactory
sensory neurons located deep in the nasal passages. Odor transduction
involves a number of intracellular processes wherein odor receptors on
the surfaces of olfactory receptor neurons are coupled to ion channels in
the neuronal membrane through G proteins and other intracellular
elements. Odors activate the transduction machinery, causing the neuron
to fire a coded message carrying information that the brain is able to
interpret. The information encoded after just one sniff of odor is actually

earned by many olfactory neurons simultaneously, but each neuron
appears to carry only part of the message. Thus encoding of odor
information is a multicellular process, and different olfactory neurons
can carry different pieces of the code.

We are studying the cellular processes that underlie odor transduction
and encoding in aquatic salamanders and in mice. Using
electrophysiological, imaging, and pharmacological tools, our goal is to
learn how these most fundamental actions work, for they represent an
elegant and very ancient solution to a complex problem of neural
function.

Laboratory of Arthur G. Humes

Research interests include systematics, development, host specificity,
and geographical distribution of copepods associated with marine
invertebrates. Current research is on taxonomic studies of copepods
from invertebrates in the tropical Indo-Pacific area, and poecilostomatoid
and siphonostomatoid copepods from deep-sea hydrothemial vents and
cold seeps.

Laboratory of Lex Kuufniuii

Current research projects m the laboratory deal with speciation and
extinction dynamics of haplochromine fishes in Lake Victoria. We are
studying the systematics. evolution, and conservation genetics of a
species flock encompassing approximately 700 very recently evolved
taxa in the dynamic and heavily impacted landscape of northern East
Africa. In the lab we are studying evolutionary morphology, behavior,
and systematics of these small, brightly colored cichlid fishes.

Another area of study is developmental and skeletal plasticity in
fishes. We are studying the diversity of bone tissue types in fishes,
differential response to mineral and mechanical challenge, and
matrophic versus environmental effects in the development of coral reef
fishes.

We also study the biological basis for marine reserves in the New
England fisheries. We are involved in collaborative research with
NURC. NMFS, and others studying the relative impact on groundfish
stocks of juvenile habitat destruction rtr.vii.v fishing pressure.

Luhorator\ of Phi/lip Lobe/

Fishes are the most diverse vertebrate group and provide opportunities
to study many aspects of behavior, ecology and evolution. We primarily
study how fish are adapted to different habitats and behavioral ecology
of species interactions. Current research focuses on fish acoustic
communications.

We are also conducting a long-term study of (he marine biology of

Year-Round Research R49

Johnston Atoll, Central Pacific Ocean. Johnston Atoll has been occupied
continuously by the military since the 1930s and proved a unique
opportunity for assessing the biological impacts of island
industrialization and its effects on reefs. Johnston Atoll is the site of the
US Army's chemical weapons demilitarization facility. JACADS.
Ongoing projects also include fish faunal studies in the African
Congo, Belize Central America, and Wake Atoll, Pacific.

Laboratory of Ivan Valiela

A focus of our work is the link between land use on watersheds and
consequences in the receiving estuarine ecosystems. The work examines
how landscape use and urbanization increase nutrient loading to
groundwater and streams. Nutrients in groundwater are transported to
the sea, and, after biogeochemical transformation, enter coastal waters.
There, increased nutrients bring about a series of changes on the
ecological components. To understand the coupling of land use and
consequences to receiving waters, we study the processes involved,
assess ecological consequences, and define opportunities for coastal
management.

A second long-term research topic is the structure and function of salt
marsh ecosystems, including the processes of predation, herbivory.
decomposition, and nutrient cycles.

Center for Advanced Studies in the
Space Life Sciences

In 1995, the NASA Life Sciences Division and the Marine Biological
Laboratory established a cooperative agreement with the formation of
the Center for Advanced Studies in the Space Life Sciences (CASSLS)
at MBL. The Center's overall goals are to increase awareness of the
NASA Life Sciences Program within the basic science community, and
to examine and discuss potential uses of microgravity and other aspects
of spaceflight as probes to provide new insights to fundamental
processes of basic biology and medicine.

Through symposia, workshops and seminars. CASSLS advises NASA
and the biological science community on a wide variety of topics.
Through fellowships. CASSLS supports summer research for
investigators in areas pertinent to the aims of NASA life sciences.

Since the Center began its operations in July 1995, more than 300
people have attended the seven CASSLS workshops. Typically these
workshops last for two to four days and feature an international array of
scientists and NASA/International space agency staff. In many cases,
workshop chairs have a long-time association with the MBL. Workshop
schedules incorporate many opportunities for interaction and discussion.
A major outcome for workshops is the publication of proceedings in a
peer-reviewed journal. Moreover, our meetings introduce outstanding
biologists to research questions and to prominent scientists involved in
gravitational biology and the NASA Life Sciences Program.

The Center sponsored two workshops in 1999: Microgravity's Effects
on Biological Systems and Behavior: An Integrative Approach, chaired
by Richard Wassersug, Dalhousie University; and Cells in Spaceflight:
Past, Present and Future, chaired by Dihp Kondepudi, Wake Forest
University. The Center sponsored one Fellow during the summer of
1999: Dr. Paul McNeil of the Medical College of Georgia. Dr. McNeil
used sea urchin eggs as a model system to study the subcellular and
molecular basis of the cell's response to a temporary disruption in
plasma membrane integrity.

Staff

Blazis. Diana E.J., Administrator

Oldham. Pamela A., Administrative Assistant

The Ecosystems Center

The Center carries out research and education in ecosystems ecology.
Terrestrial and aquatic scientists work in a wide variety of ecosystems
ranging from the streams, lakes and tundra of the Alaskan Arctic (limits
on plant primary production) to sediments of Massachusetts Bay
(controls of nitrogen cycling), to forests in New England (effects of soil
warming on carbon and nitrogen cycling), and South America (effects
on greenhouse gas fluxes of conversion of rain forest to pasture) and to
large estuaries in the Gulf of Maine (effects on plankton and benthos of
nutrients and organic matter in stream runoff). Many projects, such as
those dealing with carbon and nitrogen cycling in forests, streams, and
estuaries, use the stable isotopes "C and 15N to investigate natural
processes. A mass spectrometer facility is available. Data from field and
laboratory research are used to construct mathematical models of whole-
system responses to change. Some of these models are combined with
geographically referenced data to produce estimates of how
environmental changes affect key ecosystem indexes, such as net
primary productivity and carbon storage, throughout the world's
terrestrial biosphere.

The results of the Center's research are applied, wherever possible, to
the questions of the successful management of the natural resources of
the earth. In addition, the ecological expertise of the staff is made
available to public affairs groups and governmental agencies who deal
with problems such as acid rain, coastal eutrophication, and possible
carbon dioxide-caused climate change.

The Semester in Environmental Science was offered again in Fall
1999. Eighteen students from 14 colleges participated in the program.
There are opportunities for postdoctoral fellows.

Administrative Staff

Hobbie. John E., Co-Director

Melillo, Jerry M., Co-Director

Foreman, Kenneth H.. Associate Director. Semester in Environmental

Studies

Berthel, Dorothy J.. Administrative Assistant
Donovan, Suzanne J., Executive Assistant
Moniz, Priscilla C., Administrative Assistant. Semester in Environmental

Studies

Nunez, Guillermo, Research Administrator
Seifert, Mary Ann, Administrative Assistant
Scanlon, Deborah G.. Executive Assistant, LMER Coordination Office

Scientific Staff

Hobbie, John E.. Senior Scientist
Melillo, Jerry M., Senior Scientist
Deegan, Linda A.. Associate Scientist
Giblin, Anne E., Associate Scientist
Herbert. Darrell A., Staff Scientist
Holmes, Robert M.. Staff Scientist
Hopkinson, Charles S., Senior Scientist
Hughes, Jeffrey E., Staff Scientist
Nadelhoffer. Knute J., Associate Scientist
Neill. Christopher. Assistant Scientist
Peterson, Bruce J., Senior Scientist
Rastetter, Edward B., Associate Scientist
Shaver, Gaius R., Senior Scientist
Steudler, Paul A., Senior Research Specialist
Tian, Hanqin, Staff Scientist
Vallino. Joseph J.. Assistant Scientist
Williams, Mathew, Assistant Scientist

R50 Annual Report

Educational Staff Appointments

Bovard. Brian. Postdoctoral Research Associate
Buzby. Karen. Postdoctoral Research Associate
Cieri. Matthew D.. Postdoctoral Research Associate
Crump, Byron. Postdoctoral Research Associate
Garcia-Montiel. Diana C.. Postdoctoral Research Associate
Hartley, Anne E., Postdoctoral Research Associate
Kappel-Schmidt, Inger, Postdoctoral Research Associate
Nordin, Annika. Postdoctoral Research Associate
Raymond. Peter. Postdoctoral Research Associate
Tobias, Craig R., Postdoctoral Research Associate

Technical Staff

Ahrens, Toby. Research Assistant

Bahr. Michele P., Research Assistant

Bettez, Neil D., Research Assistant

Byun, James P., Research Assistant

Carpino, Elizabeth, Research Assistant

Claessens, Lodevicus H. J. M., Research Assistant

Colman, Ben, Research Assistant

Downs, Martha R.. Research Assistant

Fox, MaryKay, Research Assistant

Garritt. Robert H., Senior Research Assistant

Holland. Keri J.. Research Assistant

Hrywna. Yarek. Research Assistant

Jablonski. Sarah A., Research Assistant

Jillson, Tracy A., Research Assistant

Kelsey, Samuel, Research Assistant

Kicklighter, David W., Senior Research Assistant

Klcmhenz. Andrew, Research Assistant

Kwiatkowski, Bonnie L., Research Assistant

Laundre, James A., Senior Research Assistant

Lux. Heidi, Research Assistant

Micks, Patricia, Research Assistant

Newkirk. Kathleen M., Research Assistant

Nolin, Amy L., Research Assistant

Nowicki. Genevieve, Research Assistant

Pan, Shufen, Research Assistant

Regan. Kathleen M., Research Assistant

Ricca, Andrea, Research Assistant

Schwamb. Carol, Research Assistant

Slavik, Karie A., Research Assistant

Thieler, Kama K., Research Assistant

Tholke, Kristin S., Research Assistant

Thomas, Suzanne M.. Research Assistant

Tucker, Jane, Senior Research Assistant

Vasiliou, David S., Research Assistant

Weston, Nathaniel B., Research Assistant

Wollheim. Wilfred M., Research Assistant

Wright, Amos, Research Assistant

Wyda, Jason C. Research Assistant

Consultants

Bowles, Francis P., Research Systems Consultant
Bowles, Margaret C., Administrative Consultant

Visiting Scientists and Scholars

Banta, Gary. Roskilde University, Roskilde, Denmark
Duncan, Thomas, Nichols College

Fleischer. Dirk. Friedrich-Alexander Universitat Erlangen-Nurnberg.
Germany

Mondrup. Thomas. Roskilde University. Roskilde. Denmark
Moore. Marianne, SES Faculty Fellow, Wellesley College

Laboratory of Aquatic Biomedicine

Work in this laboratory centers on comparative immunopathology and
molecular biology using marine invertebrates as experimental models.
Examples of current research include determining the prevalence of
leukemia in M\a arenaria (the soft shell clam) in Massachusetts.
Monoclonal antibodies developed by this laboratory are being used to
diagnose clam leukemia, identify and characterize a tumor-specific
protein, and differentiate other leukemias in bivalve molluscs.
Development and chemically induced changes in gene expression and
neuronal growth are also being studied in the surf clam. Spisula
solidissima. Work in molecular biology is creating a clearer
understanding of the comparative etiology and pathogenesis of tumors,
particularly in environmentally impacted aquatic animals.

Staff

Reinisch, Carol L.. Senior Scientist
Jessen-Eller, Kathryn, Postdoctoral Scientist
Kreiling. Jill. Postdoctoral Scientist

Visiting Scientists

Stephens. Raymond, Boston University

Walker, Charles, Professor of Zoology, University of New Hampshire

Student

Steiger. Daniel, Tufts University School of Veterinary Medicine

Laboratory of Cell Communication

Established in 1994, this laboratory is devoted to the study of
intercellular communication. The research focuses on the cell-to-cell
channel, a membrane channel built into the junctions between cells. This
channel provides one of the most basic forms of intercellular
communication in organs and tissues. The work is aimed at the
molecular physiology of this channel, in particular, at the mechanisms
that regulate the communication. The channel is the conduit of growth-
regulating signals. It is instrumental in a basic feedback loop whereby
cells in organs and tissues control their number; in a variety of cancer
forms it is crippled.

This laboratory has shown that transformed cells lacking
communication channels lost the characteristics of cancer cells, such as
unregulated growth and Himorigenicity, when their communication was
restored by insertion of a gene that codes for the channel protein. Work
is now in progress to track the channel protein within the cells from its
point of synthesis, the endoplasmic reticulum. to its functional
destination in the plasma membrane, the cell-to-cell junction, by
expressing a fluorescent variant of the channel protein in the cells.
Knowledge about the cellular regulation of this process will aid our
understanding of what goes awry when a cell loses the ability to torm
cell-to-cell channels and thus to communicate with its neighbors,
thereby taking the path towards becoming cancerous.

Another line of work is taking the first steps at applying information
theory to the biology of cell communications. Here, the intercellular
information spoor is tracked to its source: the macromolecular
intracellular information core. The outlines of a coherent information
network inside and between the cells are beginning to emerge.

Year-Round Research R51

Staff

Loewenstein, Werner, Senior Scientist
Rose, Birgit, Senior Scientist
Jillson, Tracy, Research Assistant

Laboratory of Paul Colinvaux

The research of this laboratory reconstructs Pleistocene climatic and
environmental histories of the continents from the sediments of ancient
lakes, particularly in the Amazon basin. The team has raised sediment
cores from lakes in the lowland Amazon forests that span the last
30,000 years, including records of the last glacial maximum (LGM).
Several of these sites were extremely remote. We use pollen analysis to
reconstruct the history of vegetation around the lakes, an undertaking
that required us to produce a pollen taxonomy for the diverse Amazon
forests (published this year as an Amazon Pollen Manual and Alias).
The research has shown that the lowland Amazon forests persisted
through glacial cycles, with some reassortment of species as temperature
fluctuated from the LGM to the present. These data are useful for the
calibration of global climate models and in understanding how the great
diversity of the Amazon biota is maintained. Because of the variety of
analytical techniques other than pollen analysis used, we organize the
research in collaboration with specialized laboratories at other
institutions. Although our research is now concentrated in the
Neotropics we also have a continued interest in the paleoecology of the
arctic, with sites in Alaska and Russia.

Staff

Colinvaux, Paul, Adjunct Scientist

Laboratory of Ayse Dosemeci

The laboratory investigates molecular processes that underlie synaptic
modification. The current project is aimed at clarifying how the
frequency of activation at a synapse can determine whether the synapse
will be potentiated (strengthened) or depressed (weakened) through the
participation of an enzyme called CaM kinase II. Self-regulatory
properties of this enzyme are investigated to prove that it can respond to
the temporal pattern of calcium, the intracellular signal generated upon
synaptic activation. Related projects in collaboration with Dr. Thomas
Reese (NIH, NINDS) involve tracing changes in the distribution of
CaMKII in cultured hippocampal neurons in response to sustained
glutamate receptor activation and investigating the structural plasticity of
the postsynaptic density under these conditions.

primary laboratory is the synthesis and function of y-carboxyglutamic
acid in blood clotting proteins and the role of vitamin K.

Cone snails are obtained from the South Pacific and maintained in the
Marine Resources Center. Until recently, the marine cone snail had been
the sole invertebrate known to synthesize •y-carboxyglutamic acid (Gla).
The venomous cone snail produces neurotoxic conopeptides. some rich
in Gla, which it injects into its prey to immobilize it. To examine the
biosynthetic pathway for Gla, we have studied the Conns carboxylase
which converts glutamic acid to y-carboxyglutamic acid. This activity
has an absolute requirement for vitamin K. The Conus carboxylase
substrates contain a carboxylation recognition site on the conotoxin
precursor. Given the functional similarity of mammalian vitamin In-
dependent carboxylases and (he vitamin K-dependent carboxylase from
Conus textile, we hypothesized that structurally conserved regions would
identify sequences critical to this common functionality. Furthermore,
we examined the diversity of animal species that maintain vitamin K-
dependent carboxylation to generate y-carboxyglutamic acid. We have
cloned carboxylase homologs in full length or partial form from the
beluga whale (Delphinapterus leucas), toadfish (Opsanus tau), chicken
(Callus gallus), hagfish (Myxine glutinosa), horseshoe crab (Limulus
polyphemus) and cone snail (Conns textile} in order to compare these
structures to the known bovine, human, rat and mouse cDNA sequences.
Comparison of the predicted amino acid sequences identified a highly
conserved 32-amino acid residue region in all of these putative
carboxylases. In addition, this amino acid motif is also present in the
Drosophila genome and identified a Drosophila homolog of the y-
carboxylase. Assay of hagfish liver and Drosophila demonstrated
carboxylase activity in these non-vertebrates. These results demonstrate
the broad distribution of the vitamin K-dependent carboxylase gene,
including a highly conserved motif that is likely critical for enzyme
function. The vitamin K-dependent biosynthesis of -y-carboxyglutamic
acid appears to be a highly conserved function in the animal kingdom.

Novel y-carboxyglutamic acid-containing conopeptides have been
isolated from the venom of Conus textile. The amino acid sequence,
amino acid composition, and molecular weights of these peptides have
been determined. For several peptides, the cDNA encoding the precursor
conotoxin has been cloned. The three-dimensional structure of some of
these Gla-containing conopeptides as well as conantokin G have been
determined by 2D NMR spectroscopy. Complete resonance assignments
were made from 2D 'H NMR spectra via identification of intraresidue
spin systems using 'H-'H through-bond connectivities. NOESY spectra
provided daN, dNN and dpN NOE connectivities and vicinal spin-spin
coupling constants 3JHNa were used to calculate <$> torsion angles.
Structure generation based on interproton distance restraints and torsion
angle measurements yield convergent structures generated using distance
geometry and simulated annealing methods. The goal of this project is
to determine the structural role of y-carboxyglutamic acid in the Gla-
containing conotoxins and other y-carboxyglutamic acid-containing
proteins.

Staff

Dosemeci, Ayse, Adjunct Scientist

Laboratory of Barbara Furie and Bruce Furie

y-Carboxyglutamic acid is a calcium-binding amino acid that is found
in the conopeptides of the predatory marine cone snail, Conus. This
laboratory has been investigating the biosynthesis of this amino acid in
Conus and the structural role of y-carboxyglutamic acid in the
conopeptides. This satellite laboratory relates closely to the main
laboratory, the Center for Hemostasis and Thrombosis Research, on the
Harvard Medical School campus in Boston; the main focus of the

Staff

Furie. Barbara C. Adjunct Scientist
Furie. Bruce, Adjunct Scientist
Stenflo, Johan, Visiting Scientist
Czerwiec, Eva, Postdoctoral Fellow
Begley. Gail. Scientist I
Rigby, Alan. Adjunct Scientist

Laboratory of Roger Hanlon

This laboratory investigates the behavior and neurobiology of
cephalopods. Studies of various learning capabilities are currently being

R52 Annual Report

conducted, as are studies on reproductive strategies that include
agonistic behavior, female mate choice, and sperm competition. The
latter studies involve DNA fingerprinting to determine paternity and help
assess alternative mating tactics. Currently we are studying sensory
mechanisms and functions of polarization vision in cephalopods.
Complementary field studies are conducted locally and on coral reefs.
The functional morphology and neurobiology of the chromatophore
system of cephalopods are also studied on a variety of cephalopod
species, and image analysis techniques are being developed to study
crypsis and the mechanisms that enable cryptic body patterns to be
neurally regulated by visual input.

Staff

Hanlon, Roger, Senior Scientist
Buresch, Kendra, Research Assistant
Maxwell. Michael. Postdoctoral Scientist
Rummel, John, Visiting Scientist
Shashar, Nadav, Postdoctoral Scientist
SusMnan. Raquel. Investigator

Visiting Investigators

Adamo, Shelley. Dalhousie University

Baker, Robert, New York University

Benjamins, Steven. Graduate Student, University of Groningen

Boal, Jean, Adjunct Scientist

Cavanaugh. Joseph. Graduate Student. Boston University Marine

Program

Fern. Sophie, Graduate Student. Boston University Marine Program
Hatfield, Emma. Postdoctoral Fellow
Kier. William, University of North Carolina
King, Alison. Graduate Student. Dalhousie University
Milbury, Coren, Research Assistant
Ring. Sabine, Graduate Student, University of Frankfurt
Saidel, William, Rutgers University
Spotte, Stephen, University of Connecticut

Laboratory of Shinya Inoue

Scientists in this laboratory study the molecular mechanism and
control of mitosis, cell division, cell motility, and cell morphogenesis,
with emphasis on biophysical studies made directly on single living
cells, especially developing eggs in marine invertebrates. Development
of biophysical instrumentation and methodology, such as the centrifuge
polarizing microscope, high-extinction polarization optical and video
microscopy, digital image processing techniques including dynamic
stereoscopic imaging, and exploration of their underlying optical theory
are an integral part of the laboratory's efforts.

Staff

Inoue, Shinya, Distinguished Scientist

Coda, Makoto. Visiting Scientist

Baraby, Diane. Laboratory Assistant

Knudson, Robert. Instrument Development Engineer

MacNcil, Jane, Executive Assistant

Laboratory of Alan M. Kuzirian

Research in the laboratory explores the functional morphology and
ultrastructurc of various organ systems in molluscs. The program

includes mariculture of the nudibranch, Hennissenda crassicomis. with
emphasis on developing reliable culture methods for rearing and
maintaining the animal as a research resource. The process of
metamorphic induction by natural and artificial inducers is being
explored in an effort to understand the processes involved and as a
means to increase the yield of cultured animals. Morphologic studies
stress the ontogeny of neural and sensory structures associated with the
photic and vcstibular systems which have been the focus of learning and
memory studies, as well as the spatial and temporal occurrence of
regulatory and transmitter neurochemicals. Concurrent studies detailing
the toxic effects of lead on Hennis.iendti learning and memory, feeding,
and the physiology of cultured neurons are also being conducted. New
studies include cytochemical investigations of the Ca2+/GTP binding
protein, calexcitin, and its modulation with learning and lead exposure.

Collaborative research includes histochemical investigations on
strontium's role in initiating calcification in molluscan embryos (shell
and statoliths). immunocytochemical labelling of cell-surface antigens,
neurosecretory products, second messenger proteins involved with
learning and memory, as well as intracellular transport organelles using
mono- and polyclonal antibodies on squid (Loligo pealei) giant axons
and Herniixxcndu sensory and neurosecretory neurons. Additional
collaborations involve studying neuronal development of myelin,
myelination defects, as well as nerve regeneration and repair in
phylogenetically conserved nervous systems.

Additional collaborative research includes DNA fingerprinting using
RAPD-PCR techniques in preparation for isogenic strain development of
laboratory-reared Hermissenda and hatchery produced bay scallops
(Argopectin irradians) with distinct phenotypic markers for the rapid
field identification and stock assessments. Recently obtained funding
will expand this research to perform population genetic analyses of
currently designated yellowtail flounder (Limanda f'erruginea) stocks
occurring in the Northeast Fisheries Region.

Systematic and taxonomic studies of nudibranch molluscs, to include
molecular phylogenetics, are also of interest.

Scientific Stuff

Ku/inan. Alan M.. Associate Scientist

Visiting Investigators

Chikarmane. Hemant, Investigator

Clay, John R., NINDS/N1H

Gould. Robert, NYS Institute of Basic Research

Laboratory of Rudolf Oldenbourg

The laboratory investigates the molecular architecture of living cells
and of biological model systems using optical methods for imaging and
manipulating these structures. For imaging cell architecture non-
invasively and non-destructively, dynamically and at high resolution, we
have developed a new polarized light microscope (Pol-Scope). The Pol-
Scope combines microscope optics with new electro-optical components,
video, and digital image processing for fast analysis of specimen
birefringence over the entire viewing field. Examples of biological
systems currently investigated with the Pol-Scope are microtubule-based
structures (asters, mitotic spindles, single microtubules); actin-based
structures (acrosomal process, stress fibers, nerve growth cones); zona
pellucida of vertebrate oocytes; and biopolymer liquid crystals.

Year-Round Research R53

Staff

Oldenbourg, Rudolf, Associate Scientist

Knudson, Robert, Instrument Development Engineer

Baraby. Diane, Laboratory Assistant

Laboratory of Michael Rabinowitz

This laboratory investigates environmental geochemistry and
epidemiology. Areas of recent activity include modeling lead
bioavailability, writing a history of lead biokinetic models, performing a
case control survey of tea drinking and oral cancer in Taiwan,
quantifying the transport and fate of various sources of residential lead
exposure, and serving on several advisory boards of Superfund research
projects in Boston and New York. Current activity focuses on
characterizing lead paints and pigments.

Staff

Rabinowitz, Michael, Associate Scientist

Laboratory for Reproductive Medicine, Brown

University and Women and Infants Hospital,

Providence

Work in this laboratory centers on investigating cellular mechanisms
underlying female infertility. Particular emphasis is placed on the
physiology of the oocyte and early embryo, with the aim of assessing
developmental potential and mitochondria dysfunction arising from
mtDNA deletions. The studies taking place at the MBL branch of the
Brown Laboratory use some of the unique instrumentation available
through the resident programs directed by Rudolf Oldenbourg and Peter
J.S. Smith. Most particularly, non-invasive methods for oocyte and
embryo study are being sought. Of several specific aims, one is to use
the Pol-Scope to analyze the dynamic birefringence of meiotic spindles.
An additional aim is to study transmembrane ion transport using non-
invasive electro-physiological techniques available at the BioCurrents
Research Center. The newly developed oxygen probe offers the
possibility of looking directly at abnormalities in the mitochondria
arising from accumulated mtDNA damage. Our laboratory has also
focused on studying the mechanism underlying age-associated infertility
in terms of oocyte quality and has attempted to rescue developmentally
compromised oocytes or embryos through nuclear-cytoplasmic transfer
technology. We have characterized oxidative stress-induced
mitochondria! dysfunctions, developmental arrest and cell death in early
embryos using animal models. Ultimately, this laboratory aims to
produce clinical methods for assessing preimplantation embryo viability,
an advance that will significantly contribute to the health of women and
children.

Staff

Keefe, David, Director
Liu, Lin, Research Scientist
Trimarchi, James, Staff Scientist

Laboratory of Osamu Shimomura

Biochemical mechanisms involved in the biolurrunescence of various
luminescent organisms are investigated. Based on the results obtained.

various improved forms of bioluminescent and chemiluminesccnt probes
are designed and produced for the measurements of intracellular free
calcium and superoxide anion.

Staff

Shimomura, Osamu, Senior Scientist, MBL, and Boston University

School of Medicine
Shimomura, Akemi. Research Assistant

Laboratory of Robert B. Silver

The members of this laboratory study how living cells make
decisions. The focus of the research, typically using marine models, is
on two main areas: the role of calcium in the regulation of mitotic cell
division (sea urchins, sand dollars, etc.) and structure and function
relationships of hair cell stereociliary movements in vestibular
physiology (oyster, toadfish). Other related areas of study, i.e. synaptic
transmission (squid), are also, at times, pursued. Tools include video
light microscopy, multispectral, subwavelength, and very high speed
(sub-millisecond frame rate) photon counting video light microscopy,
telemanipulation of living cells and tissues, and modeling of decision
processes. A cornerstone of the laboratory's analytical efforts is high
performance computational processing and analysis of video light
microscopy images and modeling. With luminescent, fluorescent, and
absorptive probes, both empirical observation and computational
modeling of cellular, biochemical, and biophysical processes permit
interpretation and mapping of space-time patterns of intracellular
chemical reactions and calcium signaling in living cells. A variety of I'M
vitro biochemical, biophysical, and immunological methods are used. In
addition to fundamental biological studies, the staff designs and
fabricates optical hardware, and designs software for large video image
data processing, analysis, and modeling.

Staff

Silver, Robert. Associate Scientist

Visiting Investigators

Crawford, Karen. St. Mary's College
Hummel. John, Argonne National Laboratory
Pearson, John, Los Alamos National Laboratory

Intern

Deming, Nicole A., REU Intern, St. Mary's College

Laboratory of Norman Wainwright

The mission of the laboratory is to understand the molecular defense
mechanisms exhibited by marine invertebrates in response to invasion
by bacteria, fungi, and viruses. The primitive immune systems
demonstrate unique and powerful strategies for survival in diverse
marine environments. The key model has been the horseshoe crab
LJmulus [>ol\phenn/s. Limulus hemocytes exhibit a very sensitive LPS-
triggered protease cascade which results in blood coagulation. Several
proteins found in the hemocyte and hemolymph display microbial
binding proteins that contribute to antimicrobial defense. Commensal or
symbiotic microorganisms may also augment the antimicrobial
mechanisms of macroscopic marine species. Secondary metabolites are
being isolated from diverse marine microbial strains in an attempt to

R54 Annual Report

understand their role. Microhial participation in oxidation of the toxic
gas hydrogen sulfide is also being studied.

Staff

Wainwright, Norman, Senior Scientist
Child, Alice, Research Assistant

Visiting Investigator

Anderson, Porter. University of Rochester

Laboratory of Seymour Zigman

This laboratory is investigating basic mechanisms of photooxidative
stress to the ocular lens due to environmentally compatible UVA
radiation. This type of oxidative stress contributes to human cataract
formation. Other studies are the search for and use of chemical
antioxidants to retard the damage that occurs. Cultured mammalian lens
epithelial cells and whole lenses in vitro are exposed to environmentally
compatible UVA radiation with or without previous antioxidant feeding.
The following parameters of lens damage are examined: molecular
excitation to singlet states via NADPH (the absorber); cell growth
inhibition and cell death; catalase inactivation; cytoskeletal description
(of actin, tubulin. integrins); and cell membrane damage (lipid oxidation,
loss of gap junction integrity and intercellular chemical
communications). Thus far, the most successful antioxidant to reduce
these deficiencies is alpha-tocopherol ( 10 fig/ml) and tea polyphenols
(especially from green tea). The preliminary phases of the research are
usually carried out using marine animal eyes (i.e., smooth dogfish) as
models. Our goal is to provide information that will suggest means to
retard human cataract formation.

Staff

Seymour Zigman, Laboratory Director, Professor of Ophthalmology,

Boston University Medical School

Keen Rafferty, Research Associate, Boston University Medical School
Nancy S. Rafferty, Research Associate, Boston University

Medical School
Bunnie R. Zigman, Laboratory Manager. Boston University

Medical School

The Marine Resources Center

The Marine Resources Center (MRC) is one of the world's most
advanced facilities for maintaining and culturmg aquatic organisms
essential to advanced biological, biomedical, and ecological research.

Service and education also play an important and complementary role in
the modern, 32,000-square-foot facility.

The MRC and its life support systems have already increased the
ability of MBL scientists to conduct research and have inspired new
concepts in scientific experiments. Vigorous research programs focusing
on basic biological and biomedical aquatic models are currently being
developed at the Center. The Program in Scientific Aquaculture was
initiated in 1998.

In addition to research, the MRC provides a variety of services to the
MBL community through its Aquatic Resources Division, the Water
Quality and System Engineering Division, and the Administrative
Division.

Research and educational opportunities are available at the facility to
established investigators, postdoctoral fellows, graduate, and
undergraduate students. Investigators and students will find that the
MRC's unique life support and seawater engineering systems make this
a favorable environment in which to conduct independent research and
masters and doctoral theses using a variety of aquatic organisms and
flexible tank space for customized experimentation on live animals.
Prospective investigators and students should contact the Director of the
MRC for further information.

Staff

Hanlon, Roger, Director and Senior Scientist
Buresch, Kendra, Research Assistant
Kuzirian, Alan, Associate Scientist
Maxwell, Michael, Postdoctoral Scientist
Santore, Gabrielle. Executive Assistant
Shashar, Nadav, Postdoctoral Scientist
Smolowitz, Roxanna, MBL Veterinarian
Sussman, Raquel, Investigator

Visiting Investigators

Adamo, Shelley. Dalhousie University

Baker, Robert, New York University

Benjamins, Steven, Graduate Student. University of Groningen

Boal, Jean, Adjunct Scientist

Cavanaugh, Joseph. Graduate Student. Boston University Marine

Program

Fern, Sophie, Graduate Student, Boston University Marine Program
Gilland. Edwin, Staff Scientist
Hatfield, Emma, Postdoctoral Fellow
Kier, William, University of North Carolina
King, Alison. Graduate Student. Dalhousie University
Milbury. Coren. Research Assistant
Ring, Sabine, Graduate Student, University of Frankfurt
Saidel. William. Rutgers University
Spotte. Stephen, University of Connecticut

Honors

Friday Evening Lectures

June 18
June 25
July 2
July 9
July 16
July 22, 23
July 30
August 6
August 13

Barbara and Bruce Furie, Harvard Medical School

"Of Molluscs and Men: Vitamin K-dependent Synthesis of Gla, a Novel Amino Acid"

Roderick MacKinnon. Rockefeller University

"Potassium Channels"

Nancy Kanwisher, Massachusetts Institute of Technology

"Functional Specialization in Human Visual Cortex: Faces and Places"

Judith Kimble. University of Wisconsin. Madison

"Regulation of Development of C. elegans — Lessons from the Gonad" (Classman Lecture)

Eric Chivian, Harvard Medical School

"The Value of Plants, Animals, and Microbes to Human Health"

Marianne Bronner-Fraser and Scott E. Fraser, California Institute of Technology

1. "Formation of the Neural Crest" 2. "Working with the Wiring of the Developing Brain" (Forbes Lectures)

Dr. Bernd U. Budelmann. Marine Biomedical Institute. University of Texas

"The Sensory World of Cephalopods" (Lang Lecture)

Dr. Gerald Fischbach, National Institute of Neurological Disorders and Stroke

"Neuroscience at the New Millennium"

Dr. Luca Cavalli-Sforza, Stanford University School of Medicine

"Crucial Times in Human Evolution"

Fellowships and Scholarships

In 1999. the MBL swarded research fellowships to 20 scientists from around the world. These fellows' research topics ranged from a study of how calcium
enters heart and nen'e cells when a cell is stimulated to research on how the skate senses small electric potentials in surrounding seawater to locate prey.
The MBL awarded scholarships to 65 students in the MBL's summer courses as well as 1 1 post course research awards.

In 1999, donors provided gifts for endowed and expendable funds amounting to $103.309 in support of the research fellowships program and an
additional $233,884 to provide scholarships to students im MBL courses. The individuals who received fellowships and scholarships are listed
beginning on p. R56.

Robert Day Allen Fellowship Fund

Drs. Joseph and Jean Sanger

American Society for Cell Biology
Scholarships

American Society for Cell Biology

Frederick B. Bang Fellowship Fund

Mrs. Betsy G. Bang

Jean and Katsuma Dan Fellowship
Fund

Drs. Joseph and Jean Sanger
Mrs. Eleanor Steinbach

Bernard Davis Fellowship Fund

Mrs. Elizabeth M. Davis

Fries Fellowship

Trust of Anna B. Fries

Aline D. Gross Scholarship Fund

Dr. and Mrs. Paul R. Gross
Technic, Inc.

Keffer Hartline Fellowship Fund

Dr. Max Snodderly

E. E. Just Research Fellowship
Fund

Ayco Charitable Foundation
William Townsend Porter Foundation

Fred Karush Endowed Library
Readership

Dr. and Mrs. Laszlo Lorand

Dr. and Mrs. Arthur M. Silverstein

S. O. Mast Founders Endowed
Scholarship Fund

Dr. and Mrs. John B. Buck
Mrs. Louise M. Specht

James A. And Faith Miller
Fellowship Fund

Drs. David and Virginia Miller

Frank Morrell Scholarship Fund

Dr. Leyla de Toledo-Morrell

R55

R56 Annual Report

Emily Hartshorne Mudd
Scholarship Fund

World Academy of Art and Science

Mountain Memorial Fund

Dr. and Mrs. Dean C. Allard, Jr.
Dr. and Mrs. R. Walter Schlesinger

Neural Systems & Behavior
Scholarship Fund

Dr. and Mrs. Alan Gelperin

Josephine Bay Paul and C. Michael Paul

Foundation
Drs. Harold Zakon and Lynne McAnelly

Nikon Fellowship Fund

Nikon, Inc.

The Ann Osterhout
Edison/Theodore Miller Edison and

Olga Osterhout Sears/Harold

Bright Sears Endowed Scholarship

Fund

Ms. Nancy Miller Arnn

Mr. and Mrs. Alan K. Karplus

Phillip H. Presley Scholarship
Fund

Carl Zeiss, Inc.

William Townsend Porter

Scholarship Fund for Minority

Students

William Townsend Porter Foundation

The Evelyn and Melvin Spiegel
Fellowship Fund

Drs. Joseph and Jean Sanger
The Sprague Foundation

H. B. Steinbach Fellowship Fund

Mrs. Eleanor Steinhach

Horace W. Stunkard Scholarship
Fund

Dr. Albert Stunkard and Dr. Margaret Maurin

Eva Szent-Gyorgyi Scholarship Fund

Dr. and Mrs. Laszlo Lorand

Dr. Andrew Szent-Gyorgyi and Ms. Ursula Rowan

Selman A. Waksman Endowed
Scholarship in Microbial Diversity

Foundation for Microbiology

Walter L. Wilson Endowed
Scholarship

Dr. Paul N. Chervin

Mrs. Rigaumont

Mr. and Mrs. Leslie J. Wilson

Young Scholars/Fellows Program

Mr. and Mrs. David Bakalar

Mrs. LeRoy Clark

Mrs. George H. A. Clowes

Mr. and Mrs. Daniel D. Federman

Dr. and Mrs. Harold S. Ginsberg

Dr. and Mrs. Robert Haselkorn

Mr. and Mrs. Gary G. Hayward

Dr. and Mrs. John E. Hobbie

Dr. and Mrs. Edward F. MacNichol, Jr.

Mr. and Mrs. William J. Pechilis

Mrs. Atholie K. Rosett

Dr. and Mrs. Edward A. Spiegel

Mr. and Mrs. Leslie J. Wilson

Drs. Jonathan and Beatrice Wittenberg

Fellowships Awarded

MBL Summer Research Fellows

• Pavel Balaban, Ph.D.. is a Professor at the Institute of Higher
Nervous Activity and Neurophysiology of the Russian Academy of
Sciences in Moscow. He is interested in the mechanism of post-
synaptic activation potentials in connection with synaptic plasticity,
using the terrestrial mollusc. Helix, as a model organism. Dr. Balaban
studies the putative command neurons, a group of serotonin-containing
cells, which modulate withdrawal behavior and the activity of neurons
underlying this behavior. Spikes in these neurons do not elicit behavior,
yet behavioral responses evoked by noxious stimuli are changed. Dr.
Balaban was funded by the Herbert W. Rand Fellowship.

• Richard Cardullo, Ph.D., is an Associate Professor in the
Department of Biology at the University of California, Riverside. His
research project was titled: "Microscopic evaluation and functional
analysis of the egg extracellular matrix." Dr. Cardullo is interested in
the molecular determinants in fertilization. He uses advanced imaging
technologies along with biochemical and biophysical methodologies to
determine both the molecules involved in fertilization and the precise
sequence of molecular events ultimately leading to the fusion of
mammalian sperm and egg. Dr. Cardullo was supported by the Lucy B.
Lemann Fellowship Fund, the Robert Day Allen Fellowship, the
Charles R. Crane Fellowship, the John O. Crane Fellowship, and an
MBL Research Fellowship.

• Anthony DePass. Ph.D., is an Assistant Professor in the
Biology Department at Lung Island University in Brooklyn. NY. His
research focuses on how caiaurr enters heart and nerve cells when a
cell is stimulated. He uses Lytechus pictus, Arbacia puncntlata (sea
urchins), and Raja arinacius (skate) as biomedical models in this work.

Specifically, he studies receptors that mediate Ca2+ release from
intracellular stores and the second messenger pathways involved in
signal transduction. Dr. DePass was a Josiah Macy, Jr. Foundation
Research Fellow.

• Ana S. DePina is a graduate student at Dartmouth College. Her
research project this summer was titled "Actin-based movement in clam
oocyte extracts." She studies vesicle transport on actin filaments in
clam oocyte extracts. She wants to determine the types of inyosins that
function as molecular motors for vesicle transport. Ms. DePina was
sponsored by the Mi/ton L. Shifman Endowed Scholarship and the
William Townsend Porter Fellowship.

• Inigo Novales Flamarique, Ph.D., is a post-doctoral fellow in
the Department of Biology at the University of Victoria, British
Columbia. His summer's research project was "Optical recordings of
UV sensitivity in the optic tectum of rainbow trout using voltage
sensitive dyes." His research focuses on the chromatic organization of
neural pathways in the visual systems of vertebrates. The ultraviolet
sensitive cones in the retinas of some fishes disappear and are
reincorporated in the photoreceptor layer at specific stages during the
animal's life. As such, the ultraviolet neural pathway is a good model
to study the cellular mechanisms behind cell apoptosis and
regeneration. Dr. Novales Flamanque was funded by the Stephen W.
Huffier Fellowship.

• Elizabeth A. Jonas, MD. is in the Department of Pharmacology
at the Yale University School of Medicine in New Haven. Connecticut.
Her research project was titled "Activation of conductances on
intracellular organelles during synaptic transmission." She is interested
in measuring ionic currents on membranes of mitochondria during
neurotransmission in squid. She has found that, in addition to their role

Honors R57

in calcium management in cells, mitochondria! ion channels maintain
electrochemical gradients that are essential to mitochondria! function as
well as the regulation of the transport of peptides and metabolites
between the cytosol and the inner mitochondria! matrix. Dr. Jonas was
supported by the Frank R. Li/lie Fellowship Fund.

* Samantha Joye, Ph.D., is an Assistant Professor in the
Department of Marine Sciences at the University of Georgia in Athens.
Her research project was titled "Denitritication of coastal marshes;
relationship to nitrogen loading." Dr. Joye's research focuses on
biogeochemical cycling in coastal environments and on understanding
how humans impact coastal ecosystems. She examines how nitrogen
and phosphorus cycles are altered by human activities, with a focus on
developing critical new tools for evaluating how pristine environments
might be affected by nitrogen loading. Dr. Joye was supported by the
Lucy B. Lemann Fellowship, an MBL Associates Fellowship, and an
MBL Research Fellowship.

* Eileen M. Lafer, Ph.D. is Associate Professor at the Institute of
Biotechnology at the University of Texas Health Science Center in San
Antonio. At the MBL she studied the molecular mechanisms thai
regulate neurotransmission. Her research focuses on the biochemical
studies of various peptides. specifically the significance of the clathrin
pathway at the squid giant synapse in an effort to understand
neurotransmission in synaptic vesicles. Dr. Lafer was funded by the
Ann E. Kammer Memorial Fellowship, the Frederick B. Bang
Fellowship, the Evelyn and Melvin Spiegel Fellowship, and an MBL
Research Fellowship.

* Jennifer LaVail. Ph.D.. is a Professor of Anatomy/Ophthalmology
at the University of California, San Francisco. She investigated the
genetic and molecular regulation of Herpes simplex virus transport
using GFP-labeled virus injected into squid axons. This movement was
monitored by confocal microscopy. It is hypothesized that studying the
virus transport mechanisms will shed light on transport in other classes
of neurotropic viruses, and on organelle trafficking in general. Dr.
LaVail was funded by the Evelyn and Melvin Spiegel Fellowship and
the Frederick B. Bang Fellowship.

* Jeff Magee, Ph.D., is an Assistant Professor at the Department
of Neuroscience at the Louisiana State University in New Orleans.
Louisiana. His research project was titled "Mechanisms of Ca2+ entry
into neurons." He uses optical imaging to study varying concentrations
of calcium ions in hippocampal neurons. Changes in the strength of
synaptic connections are thought to form the basis of memory because
they ultimately lead to changes in the firing patterns of neurons. Dr.
Magee was supported by an MBL Associates Fellowship.

* Guy Major. Ph.D.. is a Research Fellow at Lucent
Technologies/Bell Labs in Murray Hill, NJ. His research project was
titled "Voltage-sensitive dye recordings from cortical neurons." He
studies how single neurons function by means of voltage-sensitive dyes
and imaging. He has been successful in measuring the spread of the
action potential through the axodendntic tree of the injected cell. Dr.
Major was an MBL Associates Fellow.

* Antonio Malgoroii. Ph.D.. is a Professor in the Neurobiology
of Learning Unit in the Department of Biological and Technological
Research at the Scientific Institute of San Raffaele in Milan, Italy. Dr.
Malgoroii studies the cellular and molecular events that form the basis
of synaptic plasticity in the hippocampus, especially as it relates to
learning and memory. He investigates changes in calcium concentration
in the postsynaptic neuron as a function of long-term potentiation
(LTP). Dr. Malgaroli is pursuing research on the nature of pre-
synaptically silent synapses that are recruited into active
neurotransmission during LTP. Dr. Malgoroii was supported by the
Herbert W. Rand Fellowship.

* Paul McNeil, Ph.D.. is a Professor in the Department of
Cellular Biology and Anatomy at the Medical College of Georgia in
Augusta. Dr. McNeil studies resealing mechanisms used in the repair of

large plasma membrane disruptions. He uses the sea urchin egg as a
model system to define the mechanistic basis of this fundamental cell
survival response. Specifically, he investigates the calcium flows that
regulate the fusion of intracellular vesicles that, in turn, fuse with the
plasma membrane to reseal the disruption. Dr. McNeil was sponsored
by the NASA Life Science Program Fellowship and the Baxter
Postdoctoral Fellowship.

• David Ogden. Ph.D.. is a Principal Investigator at the National
Institute for Medical Research in London. His research project was
titled "Central electrosensory processing in the skate." This summer he
studied how the skate senses small electric potentials in surrounding
seawater to locate prey. Specifically, he investigated the area of
electroreceptors in the skin of the skate that generate sensory
information that is relayed to the dorsal nucleus of the brain stem. The
dorsal nucleus shows organization and structural features similar to the
cerebellum and it is likely that mechanisms of plasticity will prove to
be similar to mammalian cerebellar learning mechanisms. Dr. Ogden
was an M.G.F. Fuones Memorial Fellow and an H.B. Steinbach
Fellow.

• Oladele A. Ogunseitan. Ph.D., is Associate Professor in the
Department of Environmental Analysis and Design at the University of
California, Irvine. Dr. Ogunseitan studies bacterial populations in
aquatic systems because they are highly sensitive indicators of the
phyiological consequences of toxic compounds, including trace metals.
Molecular analyses of these natural microbial communities provide
valuable ecotoxicological information, especially when coastal habitats
have been affected by human habitation resulting in dynamic shifts in
chemical speciation and concentration fluxes. Dr. Ogunseitan was
supported by the Jositili Mac\. Jr. Foundation Research Fund.

• David Paydarfar. Ph.D.. is Associate Professor at the
Department of Neurobiology at the University of Massachusetts
medical School in Worcester. The title of his research project was "Can
noise regulate oscillatory state'1 In ni/mero and in vitro analysis of
squid axon membrane." He studies how electrical nerve activity is
controlled and has found that a variety of neural oscillators can exhibit
abrupt and lasting transformation of activity from an oscillatory to an
arrhythmic state. Dr. Paydarfar was funded by the M.G.F. Fuones
Memorial Fellowship Fund and the H. Keller Hanline Fellowship.

• Edward Salmon, Ph.D., is Professor in the Department of
Biology at the University of North Carolina, Chapel Hill. Dr.
Salmon and members of the Cell Division Group investigated the
protein assemblies that achieve accurate chromosome segregation in
cell division using sand dollars and frogs as model systems. Using
advanced imaging technology. Dr. Salmon and his colleagues
studied mechanisms of chromosome segregation during meiosis and
mitosis, pronuclear movement during fertilization, and cytokinesis at
cell division. They were especially interested in developing
experimental approaches to directly measure the magnitude and
direction of forces associated with microtubule flux in mitosis in
living cells. Dr. Salmon and the Cell Division Group were
sponsored by the Nikon Fellowship.

• Edgar T. Walters, Ph.D., is a Professor in the Department of
Integrative Biology at the University of Texas in Houston, Texas. His
research project was titled "Network representation of nociceptive
memory in Aplysia." He studies the primitive neural mechanisms
underlying central memory of peripheral injury, using simple molluscan
preparations. He uses optical recording with voltage-sensitive dyes to
compare spike activity in the neurons in the abdominal ganglion of
Apl\sia californica before, during, and afer intense noxious stimulation
of the siphon. Dr. Walters was funded by the James A. and Faith
Miller Fellowship Fund.

• Ebenezer Yamoah, Ph.D., is Assistant Professor in the Department
of Anatomy and Cell Biology at the University of Cincinnati College of
Medicine in Cincinnati. Ohio. Dr. Yamoah's research focuses on

R58 Annual Report

characterizing the role of the plasma membrane calcium pump in hair cell
calcium homeostasis. This work is important because the perception of
sound and the ability to balance in relation to head position depend on the
proper function of hair cells in the inner ear. A better understanding of the
functional determinants of hair-cell sensitivity will provide rational
strategies for treating hearing and vestibular disorders. Dr. Yamoah was a
Josiah Macy. Jr. Foundation Research Fellow.

Grass Fellows

• Matthew L. Beckman. Ph.D., University of Alabama at
Birmingham. Project: "Analysis of lobster serotonin transporter
expression and function in Homarus americanua."

• Mathew Brock, Hopkins Marine Station, Stanford University.
Project: "Block of squid axon Ik by S-nitrosidithiothreitol."

• Marco Crespie, Scientific Institute S. Raffaele, Italy. Project:
"Expression of LTP at CA3-CA1 hippocampal synapses: A dendritic-
synaptic model to reveal contributions from recruitment of silent
synapses and address spread of changes."

• Frederic Doussau, Ph.D.. Laboratoire de Neurobiologie
Cellulaire, France. Project: "Control of synaptic vesicle traffic by the
action cytoskeleton."

• Yi Han, Ph.D.. Baylor College of Medicine. Project: "Electrophysiology
studies of zebrafish retinal mutants with an abnormal b-wave."

• Barbara Innocenti, Ph.D., Iowa State University. Project:
"Imaging of calcium-dependent glutamate release from Miiller cells."

• Peter Koulen, Ph.D., Yale University School of Medicine.
Project: "Differential localization of ryanodine receptor and inositol

1 ,4,5-trisphospate receptor isofornis in neurons and its relationship to
the regulations of intracellular calcium.

• Seth J. Ramus, Ph.D., Boston University. Project: "Learning in
the Eocene ocean: The first systemic examination of learning and
memory in the Nautilus (Nautilus pompilius)."

' Miduturu Srinivas, Ph.D., Albert Einstein College of Medicine.
Project: "Biophysical characterization of gap junction channels in
marine invertebrates."

• Ayako Yamaguchi, Ph.D.. Columbia University. Project:
"Neuronal coding of sexually differentiated behavior by motoneurons."

• Karen Zito. Ph.D., University of California, Berkeley. Project:
'7;i vitrn analysis of Drosophila neuromuscular development" and
"Role of the adhesion molecule, Fascilin II, in synaptic function."

• Michal Zochowski. Ph.D., Yale University School of Medicine.
Project: "Investigating physiological, functional and dynamical
properties of synchronous oscillatory signal in turtle olfactory system
using optical techniques."

MBL Science Writing Fellowships Program

Fellows

Ballingrud, David, St. Petersburg Times
Bates, Todd, Asburv Park Press

Beeman, Perry, The Des Moines Register

Burns, Michael K., The Baltimore Sun

Cohen, Nancy, Freelance reporter

Cuthbert, Lori, Discovery Channel Online

Eckelbecker, Lisa, Worcester Telegram & Ga-ette

Erickson, James, Ari-ona Daily Star

Grossman. Daniel, NPR's Living on Earth

Hogue, Cheryl, Bureau of National Affairs. Inc.

Lesser, Carolyn. Author of children's books

Miller, John, Freelance medical and science reporter/producer

Moran, Barbara. Freelance science writer/producer

Parks. Noreen, Freelance science writer

Pennybucker. Mindy, The Green Guide

Potera, Carol. Freelance writer/editor

Rogerio, Graciela. WABC-TV Eyewitness News

Schueller. Gretel, Audubon magazine

Witze. Alexandra. The Dallas Morning News

Program Directors

Goldman. Robert D.. Northwestern University
Rensberger. Boyce. Knight Science Journalism Program

Hands-On Laboratory Course Directors

Chisholm. Rex, Director. Northwestern University (Biomedical)
Hobbie, John E., Co-Director. Marine Biological Laboratory

(Environment)
Mclillo, Jerry, Co-Director, Marine Biological Laboratory

(Environment)
Palazzo. Robert, Associate Director, University of Kansas (Biomedical)

Scholarships Awarded

American Society for Cell Biology

Baca. Serapio, University of California, San Diego

Casillas. Lilliam. Autonomous University of the State of Pueblo

Jones. Stacy. University of Virginia

Macias. Chanda. Howard University

Marin Bivens. Carrie. University of Massachusetts. Amherst

Nzambi, Eduardo, Howard University

Purves, Dianne, California State University. Sacramento

Biology Club of the College of the City of New York

Suadicani. Sylvia, Albert Einstein College of Medicine

Honors R59

C. Lalor-Burdick Scholarship

Buhimschi. Irina, University of Maryland at Baltimore
Lwigale. Peter, Kansas State University

Burroughs Wellcome Fund
Biology of Parasitism Course

Angeli, Veronique. Pasteur Institute-Lille

Aviles. Hernan. Indiana State University

Barragan. Antonio, Karolinska Institute

Dobbin, Caroline, University of Technology, Sydney

Falcone, Franco, University of Edinburgh

Gavrilescu. Cristina, Cornell University

Sodre. Catia, Universidade Federal do Rio de Janeiro

Stem, Leah, University of California, San Francisco

Wang, Zefeng, Johns Hopkins University

Burroughs Wellcome Fund
Frontiers in Reproduction Course

Belts, Dean, University of Guelph

Bos-Mich. Adriana. FUEFE-Porto Alegre-Brazil

Buhimschi. Irina, University of Maryland, Baltimore

Jobanputra, Vaidehi, All India Institute of Medical Sciences

Lue. Yanhe, Harbor-UCLA Medical Center

Marin Bivens. Carrie, University of Massachusetts, Amherst

Mendeluk, Gabriela, University of Buenos Aires

Natesampillai. Sekar, University of Virginia

Ollero. Mario, Beth Israel Deaconess Medical Center

Pritts, Elizabeth, Yale University

Sprague, David, Texas A&M

Burroughs Wellcome Fund
Molecular Mycology Course

Cowen, Leah, University of Toronto

Devasamayam. Gina. Wadsworth Center

Giles, Steven. University of Wisconsin. Madison

Goldstein. Alan, Duke University Medical Center

de Jesus-Bem'os, Marisol. University of Puerto Rico

Latouche. Nicholas. Sydney University

Miller. Nancy. The Johns Hopkins Medical Institute

Wormley. Floyd, Louisiana State University Medical Center

Gary N. Calkins Memorial Scholarship Fund

Li, Dongling. University of Texas

Edwin Grant Conklin Memorial Fund

D'Souza, Jacinta. Tata Institute of Fundamental Research

Contraceptive Research and Development Program

Mendeluk, Gabriela, University of Buenos Aires

William F. and Irene C. Diller
Memorial Scholarship Fund

Vukovich, Wolfgang, Max-Planck-Institute of Biophysical Chemistry

Caswell Grave Scholarship Fund

Pfeiffer. Sven, National Institute for Medical Research

Daniel S. Grosch Scholarship Fund

Van Lith, Yvonne, ETH Zurich

Zopfi, Jakob. Max-Planck-Institute for Marine Microbiology

Aline D. Gross Scholarship Fund

Spengler. Tatjana. Universite Paris XI

William Randolph Hearst Foundation Scholarships

Jessani, Nadium, Scripps Research Institute
Kimbell, Jennifer, University of Hawaii
March, Tony, University of Idaho

International Brain Research Organization

Ashworth, Rachel. University College. London
Bellefroid, Eric. Universite Libre de Bruxelles
Lightfoot, Kurt. University of Witwaterstand
Rinkwitz. Silke. New York University Medical Center

Arthur Klorfein Scholarship and Fellowship Fund

Junghlut. Benno. University of Tubingen

Panopoulou. Georgia. Max-Planck-Institute fiir Molekulare Genetik

Paul, Angelika, University of Otago

Saiide, Leonor, National Institute for Medical Research

Frank R. Lillie Fellowship and Scholarship Fund

Chen. Lihong. University of North Carolina. Chapel Hill

March Tony. University of Idaho

van Drogen, Frank. ISREC

Varshney. Anurag. National Centre for Biological Sciences

Jacques Loeb Founders' Scholarship Fund

van Drogen. Frank. ISREC

Massachusetts Space Grant Consortium

Best. Aaron A.. University of Illinois-Urbana
Bouchet, Valerie B.. Boston University
DiMeo. Carol A., University of Delaware
Harbinski, Fred M.. Harvard University
Hurtado, Luis A.. Rutgers University
Mead. Louise A.. University of Massachusetts
Richardson. Susan L.. Yale University
Worapong. Jeerapun. Montana State University

S. O. Mast Memorial Fund

Jungblut, Benno. University of Tubingen
Spengler, Tatjana, Universite Paris XI

MBL Associates Endowed Scholarship Fund

Rao, Anita. University of Maryland, College Park

R60 Annual Report

MBL Pioneers Scholarship Fund

Glavic, Alvaro, University of Chile

Lwigale. Peter, Kansas State University

Pierdra. Elisa. Universidad de Cantabria, Spain

Vukovich, Wolfgang, Max-Planck-Institute of Biophysical Chemistry

Merck & Company, Inc. Scholarships

Batchelor, Adrian, Walter and Eliza Hall Institute

Bishop, Joseph, University of Alabama at Birmingham

Montgomery, Jacqui, University of Melbourne

Santori, Isabel, University of Buenos Aires

Stem, Leah, University of California, San Francisco

Toe, Laurent. World Health Organization

Wang, Zefeng, Johns Hopkins University

Charles Baker Metz and William Metz
Scholarship Fund in Reproductive Biology

Belts, Dean, University of Guelph

Ollero. Mario. Beth Israel Deaconess Medical Center

Sprague. David. Texas A&M

Frank Morrell Endowed Memorial Scholarship

Matsui. Ko, University of Tokyo

Mountain Memorial Fund Scholarship

Chen, Lihong. University of North Carolina, Chapel Hill

Holdaway-Clarke. Teresa. University of Massachusetts, Amherst

Mazzatenta. Andrea, University of Pisa

Tefft, Denise, University of Southern California

Varshney, Anurag, National Centre for Biological Sciences. Bangalore

Ann Osterhout Edison/Theodore Miller Edison

and Olga Osterhout Sears/Harold Bright Sears

Endowed Scholarship Fund

Avery, Jennifer. Brandeis University

Pfizer Inc. Endowed Scholarship Fund

Ragusa. Maria, Alberto Monroy Foundation. Palermo

Planetary Biology Internship Awards

Van Lith, Yvonne, ETH Zurich

Phillip H. Presley Scholarship Award,
Funded by Carl Zeiss, Inc.

Batchelor, Adrian, Walter and Eliza Hall Institute

Dobbin, Caroline, University of Technology. Sydney

Franco, Peter. Harvard Medical School

Krieger. Patrik, Karolinska Institute

Maruska. Karen. Florida Institute of Technology

Welman, Arkadiusz, Fnedneh Miescher Institute

Herbert W. Rand Fellowship and Scholarship Fund

Duncan. Tod. Imperial Cancer Research Laboratory
Fort, Alfredo, Albert Einstein College of Medicine
Franco, Peter, Harvard Medical School
Holdaway-Clarke, Terena. University of Massachusetts
Junghlut, Benno. University of Tubingen

Mazzatenta, Andrea, University of Pisa
Sawai, Satoshi, Tohoku University
Tefft, Denise. University of Southern California
Tidwell, Judy, Wake Forest University

Ruth Sager Memorial Scholarship

Haapasalo, Annakaisa. University of Kuopio

Society for Developmental Biology Scholarships

Clements. Wilson, University of Washington

Mui, Stina, University of California. San Diego

Pizer. Margaret. State University of New York at Stony Brook

Robertson. Christie, University of Washington

Zigler, Kirk. Duke University

Society of General Physiologists' Scholarships

Diana. Marco. Max-Planck-Institute of Biophysical Chemistry
Duncan. Tod, Imperial Cancer Research Laboratory
Greenwood, Anna, Stanford University
Paul, Angelika. University of Otago

Howard A. Schneiderman Endowed Scholarship

Yu, Xiang, MRC Laboratory of Molecular Biology

Moshe Shilo Memorial Scholarship Fund

Gillor. Osnat. The Hebrew University

Marjorie W. Stetten Scholarship Fund

Yoon, Miri. Northwestern University Medical School

Horace W. Stunkard Scholarship Fund

Bos-Mikich. Adriana, FUEFE-Porto Alegre-Brazil

Surdna Foundation Scholarship

Abenavoli, Alessandra, Scientific Institute San Raffaele
Diana. Marco, Max-Planck-Institute of Biophysical Chemistry
Haapasalo. Annakaisa, University of Kuopio
Matsui, Ko, University of Tokyo
Rao, Anita. University of Maryland

William Townsend Porter Fellowship
For Minority Students

Jones. Stacy. University of Virginia
Macias, Chanda, Howard University
Nzambi, Eduardo, Howard University

William Morton Wheeler Family
Founders' Scholarship

Abenavoli, Alessandra. Scientific Institute San Raffaele
Yu, Xiang. MRC Lab of Molecular Biology

Walter L. Wilson Endowed Scholarship Fund

Tidwell. Judy. Wake Forest University

World Academy of Arts and Sciences
Emily Mudd Scholarship

el Guiziry, Dalai, Alexandria University

Honors R61

Post-Course Research Awards

van Drogen. Frank, ISREC (Physiology)
Kimbell. Jennifer, University of Hawaii (Physiology)
Mactas, Chanda, Howard University (Physiology)
Mazzatenta, Andrea, University of Pisa (Physiology)
Meyers. Jason, University of Virginia (Embryology)
Narayan, Sujatha, Bryn Mawr College (Physiology)

Niggemyer. Allison, University of Idaho (Microbiul Diversity)
O'Neill. Forest, University of California. Santa Barbara (Physiology)
Purves, Dianne, California State University, Sacramento (Physiology)
Tidwell. Judy, Wake Forest University (Physiology)
Varshney. Anuray. National Centre for Biological Sciences
(Physiology)

Board of Trustees and
Committees

Corporation Officers and Trustees

Chairman of the Board of Trustees. Sheldon J. Segal, The Population

Council
Co-Vice Chair of the Board of Trustees, Frederick Bay. Josephine Bay

Paul and C. Michael Paul Foundation

Co-Vice Chair of the Board of Trustees, Mary J. Greer. New York, NY
President of the Corporation, John E. Dowling, Harvard University
Director and Chief Executive Officer, John E. Burris, Marine Biological

Laboratory*
Treasurer of the Corporation, Mary B. Conrad, Fiduciary Trust

International*

Clerk of the Corporation. Robert E. Mainer. The Boston Company
Chair of the Science Council, Kerry S. Bloom, University of North

Carolina*

Class of 2003

Kelley, Darcy Brisbane. Columbia University

Landeau, Laurie J.. Marinetics. Inc.

Lee. Burton J. Ill, Vero Beach, FL

O'Hanley, Ronald P.. Mellon Institutional Asset Mgt.

Pierce, Jean, Boca Grande. FL

Ryan, Vincent J., Schooner Capital LLC

Honorary Trustees

Cunningham, Mary Ellen. Grosse Pointe Farms, MI
Ebert, James D., Baltimore, MD
Golden. William T., New York. NY
Grass, Ellen R.. The Grass Foundation

Class of 2000

Clowes, Alexander W., University of Washington School of Medicine

Landis, Story C., Case Western Reserve University

Levitan, Irwin B., Brandeis University

Miller. G. William. G. William Miller and Co., Inc.

Press, Frank, The Washington Advisory Group

Weld, Christopher M.. Sullivan and Worcester, Boston

Class of 2001

Anderson. Porter W., North Miami Beach. FL

Bay, Frederick. Josephine Bay Paul and C. Michael Paul Foundation,

Inc.

Cox, Martha W., Hobe Sound, FL
Greer. Mary J.. New York. NY
Steere. William C. Jr., Pfizer Inc.
Weissmann, Gerald, New York University School of Medicine

Class of 2002

Cone, Sydney M III, Cleary, Gottlieb, Steen and Hamilton

Lakian, John R.. The Fort Hill Group, Inc.

Ruderman, Joan V , lljiuiul Medical School

Segal. Sheldon J., The Population Council

Speck. William T., New York Presbyterian Hospital

Zeien, Alfred M.. The Gillette Company

/ \ i '/In u>

Trustees Emeriti

Adelberg, Edward A., Yale University. New Haven. CT

Buck. John B.. Sykesville, MD

Cohen, Seymour S.. Woods Hole, MA

Colwm, Arthur L., Key Biscayne, FL

Colwin. Laura Hunter, Key Biscayne. FL

Copeland, Donald Eugene, Woods Hole, MA

Crowe!!, Sears Jr.. Indiana University. Bloomington, IN

Hayashi, Teru, Woods Hole, MA

Huhbard. Ruth, Cambridge. MA

Kleinhol/., Lewis, Reed College, Portland, OR

Krahl, Maurice. Tucson, AZ

Prosser, C. Ladd, University of Illinois. Urbana, IL

Russell-Hunter, W.D.. Syracuse University. Syracuse. NY

Saunders. John W., Waquoit, MA

Trigg. D. Thomas. Wellesley. MA

Vincent, Walter S., Woods Hole. MA

Directors Emeriti

Ebert. James D.. Baltimore. MD

Gross, Paul, Falmouth, MA

Halvorson. Harlyn O., Woods Hole, MA

Executive Committee of the
Board of Trustees

Segal, Sheldon J., Chair
Bay, Frederick, Co-Vice Chair
Greer. Mary J.. Co-Vice Chair
Burris. John E.
Bloom. Kerry S.

R62

Trustees and Committees R63

Conrad. Mary B., Treasurer
Mainer, Robert E.
Ruderman, Joan V.
Weissmann. Gerald

Science Council

Bloom, Kerry S., Chair (2000)
Bums. John E.*
Armstrong, Clay M. (2000)

Armstrong, Peter (2000)
Atema. Jelle (2001)
Barlow, Robert B. (2001)
Dawidowicz, E.A.*
Haimo, Leah (2001)
Hopkinson, Charles (2000)
Jaffe, Launnda 12001 1
Smith, Peter J.S. (2001)
Sogin. Mitchell (2000)

Standing Committees of the Board of Trustees

Development

Speck, William. Chair
Anderson, Porter W.
Barlow, Robert
Bay. Frederick
Conrad, Mary B.
Cox, Martha

Cunningham, Mary Ellen
Ebert, James D.
Grant, Philip
Lakian, John R.
Lee, Burton J.
Miller, G. William
Pierce, Jean
Steere. William C.
Weld, Christopher M.

Facilities and Capital Equipment

Ruderman, Joan. Chair
Anderson, Porter W.
Bay, Frederick
Cohen, Lawrence
Cornell, Neal
Landis, Story C.
Levitan, Irwin B.
Pierce. Jean
Press, Frank
Weld. Christopher M.

Investment

Conrad. Mary B., Chair
Cone, Sydney M.
Lakian, John R.

Muiner, Robert E.
Miller. G. William
O'Hanley. Ronald P
Ryan, Vincent J.
Segal, Sheldon J.
Zeien. Alfred M.

Finance

O'Hanley, Ronald, Chair
Clowes, Alexander W.
Cone, Sydney M.
Conrad, Mary B.
DeHart, Donald
Kelley, Darcy Brisbane
Lakian, John R.
Landeau, Laurie J.
Loewenstein, Werner
Mainer, Robert E.
Manz, Robert
Miller, G. William
Ryan, Vincent J.
Zeien, Alfred M.

Nominating

Weissmann. Gerald, Chair
Bloom, Kerry S.
Clowes. Alexander W.
Cox. Martha W.
Greer. Mary J.
Landis. Story C.
Pierce, Jean
Segal. Sheldon J.
Speck, William T.
Steere, William C.

Standing Committees of the Corporation and Science Council

Buildings and Grounds

Cohen. Lawrence B., Chair
Boyer, Barbara C.
Cutler, Richard*
Fleet, Barry*

Hayes, Joe*
McArthur, Andrew
Pelerson, Bruce J.
Tweedell. Kenyon S.
Valiela. Ivan

*£v officio

R64 Annual Report

Education Committee

Dione, Vincent, Chair
Bloom. Kerry S.*
Dawidowicz. E.A.*
Dunlap, Paul
Fink. Rachel
Hanlon, Roger
Jatfe, Laurinda
Mehane. Dori Chrysler*
Mendelsohn, Michael
Venuti, Judith
Wadsworth. Patricia
Zottoli. Steve

MBL/WHOI Library Joint Advisory Committee

Shcpro, David. Chair, MBL
Ashmore, Judy.* MBL
Dow. David, NMFS
Fornari. Daniel. WHOI
Harbison, G. Richard. WHOI
Hobbie, John. MBL
Hurter, Colleen,* WHOI
Kurz, Mark. WHOI Alternate
Norton. Cathy,* MBL
Robb. James. USGS
Rose, Birgit. MBL
Smith, Peter J.S.. MBL
Warren. Bruce, WHOI

Fellowships

Pederson, Thoru, Chair

Dawidowicz, E.A.*

Deegan. Linda

Ehrlich, Barbara

Kaufmann. Sandra* (Recording Secretary)

Lemos, Jose

Sluder. Greenfield

Smith. Peter J.S.

Treistman, Steven (Guest Member)

Housing, Food Service and Child Care

Browne. Carole, Chair
Bloom, Kerry S.*
King, LouAnn*
Malchow. Robert P.
Stuart. Ann E.
Weeks. Janis C.

Assisting

Browne, Robert (representing MBL Child Care)
Goux, Susan (Administrator)
Hinklc, Pamela Clapp (representing the MBL Club)
Cutler. Richard (representing Housing)

Discovery: The Campaign for Science at the Marine Biological Laboratory Steering
Committee

Research Services and Space

Laufer. Hans, Chair
Armstrong. Peter B.
Cornell, Neal
Cutler, Richard*
Dawidowicz, E.A.*
Foreman. Kenneth
Kerr, Louis M.*
Landowne. David
Mattox, Andrew*
Melillo. Jerry
Mizell, Merle
Smith, Peter J.S.
Steudler, Paul
Valiela, Ivan

Bay, Frederick, Campaign Chair
Golden. William T., Honorary Chair
Grass. Ellen R., Honorary Chan
Clowes, Alexander W., Vice-Chair
Cox. Martha W., Vice-Chair
Miller, G. William. Vice-Chair
Weissmann. Gerald. Vice-Chair
Anderson, Porter W.
Barlow, Robert B. Jr.
Bernstein, Norman
Cobb, Jewell Plummer
Conrad. Mary B.
Cunningham, Mary Ellen

*Ex officiti

Dowling, John E.
Ebert. James D.
Fischbach. Gerald D.
Goldman, Robert D.
Greer. Mary J.
Jacobson. M. Howard
Landeau, Laurie J.
Langford. George M.
Lee, Burton J. Ill
Pierce, Jean
Prendergast, Robert A.
Shcpro, David
Speck. William T.
Steere. William C. Jr.
Weld. Christopher M.
Zeien, Alfred M.

Trustees and Committees R65

Council of Visitors

Norman B. Asher, Esq., Hale and Dorr. Counsellors at Law, Boston,
MA

Mr. Donald J. Bainton, Continental Can Co.. Boca Raton, FL

Mr. David Bakalar. Chestnut Hill, MA

Mr. Charles A. Baker, The Liposome Company, Inc., Princeton. NJ

Dr. George P. Baker, Massachusetts General Hospital, Boston, MA

Dr. Sumner A. Barenberg. Bernard Technologies, Chicago, IL

Mr. Mel Burkan, The Barkan Companies, Boston, MA

Mr. Robert P. Beech, Component Software International, Inc.. Mason.

Mr. George Berkowitz, Legal Sea Foods. Allston, MA
Mr. and Mrs. Nathaniel J. Bickford. New York, NY
Dr. Elkan R. Blout, Harvard Medical School, Boston, MA
Mr. and Mrs. Philip Bogdanovitch, Lake Clear, NY
Mr. Malcolm K. Brachman, Northwest Oil Company, Dallas, TX
Dr. Goodwin M. Breinin, NY University Medical Center, New York,

Mr. John Callahan, Carpenter, Sheperd & Warden, New London, NH

Mrs. Elizabeth Campanella, West Falmouth, MA

Thomas S. Crane, Esq., Mint/ Levin Cohen Ferris Glovsky & Popeo,

PC, Boston, MA

Dr. Stephen D. Crocker, Cyber Cash Inc.. Reston, Virginia
Mrs. Lynn W. Piasecki Cunningham, Film and Videomaker, Piasecki

Productions, Brookline, MA
Dr. Anthony J. Cutaia, Anheuser-Busch. Inc., St. Louis, MO

Mrs. Sara Greer Dent, Chevy Chase, MD

Mr. D. H. Douglas-Hamilton, Hamilton Thorne Research, Beverly, MA

Mr. Benjamin F. du Pont. Du Pont Company. Deepwater, NJ

Dr. Sylvia A. Earle, Founder, Deep Ocean Engineering. Oakland, CA

Mr. and Mrs. Hoyt Ecker, Vero Beach. FL

Mr. Anthony B. Evnin, Venrock Associates, New York, NY

Stuart Feiner, Esq.. Inco Limited, Toronto, ON, Canada
Mrs. Hadley Mack French, Edsel & Eleanor Ford House, Grosse Pointe
Farms. MI

Mr. and Mrs. Huib Geerlings. Boston, MA

Mr. William J. Gilbane. Jr.. Gilbane Building Company, Providence, RI
Dr. Michael J. Goldblatt. Intelligent Biocides, Tewksbury, MA
Mr. Maynard Goldman. President, Maynard Goldman & Associates,
Boston, MA

Ms. Charlotte I. Hall, Edgartown, MA

Drs. Linda Hirshman. Brandeis University. Waltham, MA, and David

Forkosh, FMH Foundation
Mr. Thomas J. Hynes, Jr., Meredith & Grew, Inc., Boston, MA

Mr. M. Howard Jacobson. Bankers Trust, Westborough, MA
Mrs. Elizabeth Ford Kontulis. New Canaan, CT

Mr. and Mrs. Robert Lambrecht. Boca Grande. FL

Dr. Catherine C. Lastavica. Tufts University School of Medicine.

Boston. MA

Mr. Joel A. Leavitt, Boston, MA

Mr. Stephen W. Leibholz. TechLabs, Inc., Huntingdon. PA
Mrs. Margarei Lilly, West Falmouth, MA
Mr. Richard Lipkin. ResponseLogic. Inc., New York, NY
Mr. George W. Logan, Valley Financial Corp., Roanoke, VA

Mr. Michael T. Martin. SportsMark, Inc.. New York. NY

Mrs. Christy Swift Maxwell. Grosse Pointe Farms, MI

Dr. Georges de Menil. DM Foundation, New York, NY

Mr. Ambrose Monell, G. Unger Vetlesen Foundation, Palm Beach. FL

Dr. Mark Novilch, Washington, DC

Ms. Julie Packard. Monterey Bay Aquarium. Monterey, CA

Mr. David R. Palmer, David Ross Palmer & Associates, Waquoit, MA

Dr. Roderic B. Park, Richmond. CA

Mr. Santo P. Pasqualucci, Falmouth Co-Operative Bank. Falmouth, MA

Mr. Robert Pierce, Jr.. Pierce Aluminum Co.. Canton, MA

Mr. Richard Reston, Vineyard Ga/ette. Edgartown, MA

Mr. Marius A. Robinson, Fundamental Investors Ltd., Key Biscayne,

FL
John W. Rowe, M.D., Mt. Sinai School of Medicine & Mt. Sinai

Medical Center, New York, NY
Mr. Edward Rowland, Tucker, Anthony, Inc., Boston, MA

Mr. Gregory A. Sandomirsky, Mintz Levin Cohen Ferris Glovsky &

Popeo. PC. Boston. MA
Mrs. Mary Schmidek, Marion. MA
Dr. Cecily C. Selby, New York, NY
Mr. Robert S. Shifman, St. Simon's Island. Georgia
Mr. and Mrs. Gregory Skau. Grosse Pointe Farms. Ml
Mr. Malcolm B. Smith, General American Investors Co.. New York.

Mr. John C. Stegeman, Campus Rentals, Ann Arbor. MI
Mr. Joseph T. Stewart. Jr.. Skillman, NJ

Mr. John W. Stroh. III. The Stroh Brewery Company, Detroit, MI
Mr. Gerard L. Swope, Washington, DC
Mr. John F. Swope. Concord. NH

Mr. and Mrs. Stephen E. Taylor. Milton, MA

Mrs. Donna Vanden Bosch-Flynn. Spring Lake. NJ
Mrs. Carolyn W. Verbeck, Vineyard Haven, MA

Mr. Benjamin S. Warren III, Grosse Pointe Farms, MI

Nancy B. Weinstein, R.N.. The Hospice, Inc., Glen Ridge. NJ

Stephen S. Weinstein. Esq., Morristown, NJ

Mr. Frederick J. Weyerhaeuser. Beverly, MA

Mr. Tony L. White. The Perkin Elmer Corporation, Norwalk, CT

Dr. Torslen N. Wiesel, The Rockefeller University, New York. NY

Administrative Support Staff1

Biological Bulletin

Greenberg, Michael J.. Editor-in-Chief
Hinkle, Pamela Clapp. Managing Editor

Burns. Patricia

Gibson. Victoria R.

Schachinger. Carol H.

Director's Office

Burris. John E.. Director and Chief Executive Officer
Donovan. Marcia H.

Equal Employment Opportunity

MacNeil, Jane L.

Veterinarian Sen'ices
Champagne. Jaimie2
Reynolds, Martha
Smolowitz, Roxanna
Stukey. Jetley

Ecosystems Center Administrative Staff

Berthel. Dorothy J.
Donovan, Suzanne J.
Nunez. Guillermo
Seifert, Mary Ann

External Affairs

Carotenuto, Frank C.. Director
Butcher. Valerie
Faxon. Wendy P.
Johnson. A. Kristine
Martin. Theresa H.
Patch-Wing, Dolores
Quigley. Barbara A.
Shaw. Kathleen L.

Associates Pn H; liim
Bohr, Kendall I!
Zeida, Robert

' Including persons who joined or left the staff during
2 Summer or temporary.

IW9.

Communications Office
Hinkle. Pamela Clapp, Director
Cox. Sarah2
Flynn, Bridget
Furfey. Susan2
Joslin, Susan
Liles. Beth R.
Noonan, Ryan2
Schanhacher, Jennifer2

Financial Sen'ices Office

Lane, Homer W. Jr.. Chief Financial Officer
Bowman, Richard, Controller

Afonso, Janis

Barry, Maureen

Crosby, Kenneth

Eidelman, Dana

Lancaster. Cindy

Livingstone, Suzanne

McLaughlin. Rebecca Jill

Ran/inger. Laura

Stellrecht. Lynette

Stock Room

Schorer. Timothy M.. Supervisor

Brmson. Robert2

Burnette. Donald

O'Connor-Lough, Susan

Purchasing

Hall. Lionel E. Jr., Supervisor
Gannon, Katherine2
Shamon. Lynne R.

Housing and Conferences

King. LouAnn D., Director
Grasso, Deborah
Hanlon. Arlene K.2
Johnson-Horman, Frances N.
Masse, Todd C.
Perito. Diana
Ridley, Alberta2

R66

Administrative Support Staff R67

Human Resources

Goux, Susan P., Director
Houser. Carmen

Josephine Bay Paul Center for Comparative Molecular
Biology and Evolution Administrative Staff

Harris. Marian
Lini. Pauline

Cc/>v Center

Mounttord. Rebecca J., Supervisor

Abisla, Richard L.2

Adams. Jessica2

Churchill, Elizabeth2

Clark. Tamaru L.

Cosgrove, Nancy

Kefeauver, Lee

Mancini. Mary E.

Waterbury. Andrew2

Journal of Membrane Biology

Loewenstein. Werner R.. Editor
Fay, Catherine H.
Howard Isenberg. Linda L.
Lvnch. Kathleen F.

Marine Resources Center

Hanlon. Roger T, Director
Santore. Gabnelle

Aquatic Resources Department

Enos, Jr., Edward G., Superintendent

Bourque, Ryan M.2

Chappell, P. Dreux2

DeGiorgis, Joseph A.2

Grossman, William M.

Gudas. Christopher N.2

Kilpatrick. Brian2

Klimm. Henry W. Ill

Reynolds. Justin2

Sexton. Andrew W.

Smith. Gary2

Sullivan. Daniel A.

Tassinari. Eugene

MRC Life Support System

Mebane, William N., Systems Operator

Carroll, James

Clark. Heidi2

Hanley. Janice S.

Kuzirian, Alan

Till. Geoffrey A.

MBUWHOI Library

Norton. Catherine N.. Director
Ashmore. Judith A.
Buckingham. Andrew2
Costa. Marguerite E.
Crocker, Daniel2
Deveer. Joseph M.
Farrar. Stephen R.L.
Fredericks. Julie2
Medeiros, Melissa
Monahan. A. Jean
Moniz. Kimberly L.
Moore. Laurel E.
Nelson. Heidi
Person, Matthew
Riley. Jacqueline
Shinkle. Chelsea2

Information Systems Division

In/ina, Barbara, Network Manager

Berrios, Kelly2

Cohen, Alex2

Milliard. Channing2

Kokmeyer. Remmert2

Malchow, Robert2

Mountford, Rebecca J.

Moynihan, James V.

Purdy, Heather2

Remsen. David P.

Renna. Denis J.

Space, David B.

Wheeler. Patrick

NASA Center for Advanced Studies in the Space Life
Sciences

Blazis. Diana, Administrator
Amit, Udeni P.
Golden. Catherine
Oldham. Pamela

Research Administration & Educational Programs

Dawidowicz, Eliezar A.. Director
Hamel, Carol C.
Holzworth, Kelly
Kaul'mann, Sandra J.
Kleinhenz, Andrea
Lynn. Rebecca
Mebane, Dorianne C.
White, Laurie

Central Microscopy Facility and General Use Rooms
Kerr. Louis M., Supervisor
Luther, Herbert
Matthews, Douglas2
Peterson. Martha B.

Safety Sen'ices

Mattox, Andrew H.. Environmental. Health, and Safety Manager
Lingler. Nicholas2
Normand. Danielle2

Satellite/Periwinkle Children 's Programs

Robinson, Paulina H.2
Borst, Douglas2
Browne. Jennifer L.2
Curran. Kelly2
David, Amy2

R68 Annual Report

Fitzelle. Annie"
Gallant, Cynthia2
Guiftrida. Beth2
Halter. Sarah2
Hike. Kelly2
Mekelatos, Sharon2
Noonan. Brendan"
Robbins, Sarah2
Shanley, Jennifer2

Sen'ice, Projects and Facilities

Cutler. Richard D.. Director
Enos, Joyce B.
Guarente, Jeffrey

Apparatus

Baptiste, Michael G.
Barnes. Franklin D.
Haskins, William A.

Building .SVrnVc.v & Grounds

Hayes. Joseph H.. Superintendent

Anderson, Lewis B.

Atwood. Paul R.

Baker, Harrison S.

Barnes, Susan M.

Beernik, Daniel

Berthel, Frederick

Billings. Julia2

Boucher. Richard L.

Brereton, Richard S.2

Callahan. John J.

Cameron, Lawrence M.2

Chen, Zhi Xm

Clayton. Daniel

Collins, Paul J.

Cowan. Matthew B.2

Cutler. Matthew D.2

Cutillo, David

Dimond, Jay2

Doherty. Garrett2

Dorris, John .1.

Eldridge. Myles2

Elias, Michael

Fernandez, Peter R.2

Foster, Martin2

Gibbons, Roberto G.

Hannigan. Catherine
Illgen. Robert F.
Joyner. Matthew2
Ledwell, L. Patrick2
Lynch. Henry L.
MacDonald, Cynthia C.
Malchow, Kate2
McCarthy, Michael
McDonnell. Gregory
McNamara. Moreen M,
McQuillan. Jeffrey2
Parker, Karen2
Plant, Stephen W.
Pratt, Barry
Stites, Clint2
Thiele, Tod2
Ware, Lynn M.

Plnnt Openilitms untl Maintenance

Fleet, Barry M., Manager

Cadose, James W.. Maintenance Supervisor

Barnes, John S.

Blunt. Hugh F.

Bourgoin. Lee E.

Callahan, John

Carroll, James R.

Davis, Douglas

Fish. David L. Jr.

Fuglister. Charles K.

Goehl. George

Gonsalves, Walter W. Jr.

Hathaway. Peter J.

Henderson, Jon R.

Kelley. Kevin

L'Abbe, Robert

Langill, Richard

Lochhead, William M.

McAdams, Herbert M. Ill

McHugh, Michael O.

Mills, Stephen A.

Olive, Charles W. Jr.

Rattacasa, Frank2

Rozum. John

Schoepf. Claude

Settlemire, Donald

Shepherd. Denise M.

Toner, Michael

Wetzel. Ernest D.2

Members of the
Corporation

Life Members

Acheson, George H., 25 Quissett Avenue. Woods Hole, MA 02543
Adelberg, Edward A., 204 Prospect Street, New Haven. CT 065 1 1 -

2107
Afzelius, Bjorn, University of Stockholm, Wenner-Gven Institute.

Department of infrastructure Research, Stockholm, Sweden
Amatniek. Ernest, (address unknown)
Arnold, John M., 329 Sippewissett Road. Falmouth, MA 02540

Bang, Betsy G., 76 F. R. Lillie Road. Woods Hole. MA 02543
Bartlett, James H., University of Alabama, Department of Physics, Box

870324, Tuscaloosa, AL 35487-0324
Berne, Robert M., University of Virginia School of Medicine,

Department of Physiology, Box 1116, MR4, Charlottesville, VA

22903
Bernheimer, Alan W., New York University Medical Center.

Department of Microbiology, 550 First Avenue. New York, NY

10016
Bertholf, Lloyd M., Westminster Village. #21 14, 2025 East Lincoln

Street. Bloomington, IL 61701-5995

Bosch, Herman F., 163 Elm Road, Falmouth, MA 02540-2430
Buck, John B., Fairhaven C-020. 7200 Third Avenue, Sykesville, MD

21784

Burbanck, Madeline P., P.O Box 15134, Atlanta, GA 30333
Burbanck, William D., P.O. Box 15134. Atlanta. GA 30333

Clark, Arnold M., 53 Wilson Road, Woods Hole, MA 02543
Clark, James M., 258 Wells Road, Palm Beach, FL 33480-3625
Cohen, Seymour S., 10 Carrot Hill Road, Woods Hole, MA 02543-

1206
Colwin, Arthur L., 320 Woodcrest Road, Key Biscayne, FL 33149-

1322
Colwin, Laura Hunter, 320 Woodcrest Road, Key Biscayne, FL

33149-1322
Cooperstein, Sherwin J., University of Connecticut, School of

Medicine, Department of Anatomy, Farmington, CT 06030-3405
Copeland, D. Eugene, Marine Biological Laboratory, Woods Hole, MA

02543

Corliss, John O., P.O. Box 2729, Bala Cynwyd, PA 19004-2116
Costello, Helen M., Carolina Meadows, Villa 137, Chapel Hill, NC

27514-8512
Crouse, Helen, Rte. 3. Box 213, Hayesville, NC 28904

DeHaan, Robert L., Emory University School of Medicine, Department
of Anatomy and Cell Biology, 1648 Pierce Drive, Room 108, Atlanta.
GA 30322

Dudley, Patricia L., 3200 Alki Avenue SW. #401, Seattle. WA 98116

Edwards, Charles, 3429 Winding Oaks Drive, Longboat Key, FL

34228
Elliott, Gerald F., The Open University Research Unit. Foxcombe Hall,

Berkeley Road, Boars Hill. Oxford OX1 5HR, United Kingdom

Failla, Patricia M., 2149 Loblolly Lane. Johns Island, SC 29455
Ferguson, James K. W., 56 Clarkehaven Street, Thomhill, Ontario L4J
2B4, Canada (deceased 1999)

Gabriel, Mordecai L., Brooklyn College, Department of Biology, 2900

Bedford Avenue, Brooklyn, NY 11210
Glusman, Murray, New York State Psychiatric Institute, 722 W. 168th

St.. Unit #70, New York. NY 10032
Graham, Herbert, 36 Wilson Road, Woods Hole, MA 02543

Hamburger, Viktor, Washington University, Department of Biology,

740 Trinity Avenue, St. Louis, MO 63130
Hamilton, Howard L., University of Virginia, Department of Biology,

238 Gilmer Hall. Charlottesville, VA 22901

Harding, Clifford V. Jr., 54 Two Ponds Road. Falmouth. MA 02540
Haschemeyer, Audrey E. V., 2 1 Glendon Road, Woods Hole, MA

02543-1406
Hauschka, Theodore S., 333 Fogler Road. Bremen. ME (deceased

1999)

Hayashi, Teru. 15 Gardiner Road. Woods Hole, MA 02543-1 1 13
Hisaw, Frederick L., (address unknown)
Hoskin, Francis C. G., c/o Dr. John E. Walker, U.S. Army Natick

RD&E Center, SAT NC-YSM, Kansas Street. Natick. MA 01760-

5020
Hubbard, Ruth, Harvard University, Biological Laboratories,

Cambridge, MA 02138
Humes. Arthur G., Marine Biological Laboratory, Boston University

Marine Program, Woods Hole, MA 02543 (deceased 1999)
Hunter, W. Bruce, 305 Old Sharon Road, Peterborough, NH 03458-

1736
Hurwitz, Charles, Stratton VA Medical Center, Research Service,

Albany, NY 12208

Katz, George, Merck. Sharp and Dohme. Fundamental and

Experimental Research Laboratory. PO Box 2000. Rahway, NJ 07065
Kingsbury, John M., Cornell University, Department of Plant Biology,

Plant Science Building. Ithaca, NY 14853
Kleinholz, Lewis, Reed College. Department of Biology. 3203 SE

Woodstock Boulevard. Portland. OR 97202
Kusano, Kiyoshi, National Institutes of Health, Building 36, Room 4D-

20. Bethesda. MD 20892

Laderman, Ezra, Yale University, New Haven. CT 06520

R69

R70 Annual Report

LaMarche, Paul H., Eastern Maine Medical Center, 489 State Street,

Bangor, ME 04401
Lauffer, Max A., Penn State University Medical Center. Department of

Biophysics and Physiology, Hershey, PA 17033
Lochhead, John H., 49 Woodlawn Road, London SW6 6PS. United

Kingdom
Loewus, Frank A., Washington State University, Institute of Biological

Chemistry. Pullman. WA 99164
Loftfield, Robert B., University of New Mexico, School of Medicine.

915 Stanford Drive. Albuquerque, NM 87131
Lorand, Laszlo, Northwestern University Medical School, CMS

Biology, Searle 4-555, 303 East Chicago Avenue, Chicago. IL 60611-

3008

Malkiel, Saul, 174 Queen Street, #9 A. Falmouth, MA 02540
Marsh, Julian B., 9 Eliot Street, Chestnut Hill, MA 02467-1407
Martin, Lowell V., 10 Buzzards Bay Avenue, Woods Hole. MA 02543
Mathews, Rita W., East Hill Road. P.O. Box 237, Southfield, MA

01259-0237
Moore, John A., University of California, Department of Biology.

Riverside, CA 92521
Moore, John W., Duke University Medical Center, Department of

Neurobiology, Box 3209, Durham, NC 2771(1
Moscona, Aron A., University of Chicago, Department Molecular

Genetics and Cell Biology, Chicago, IL 60637
Musacchia, X. J., P.O. Box 5054, Bella Vista, AR 72714-0054

Nasatir, Maimon, P.O. Box 379, Ojai, CA 93024

Passano, Leonard M., University of Wisconsin, Department of

Zoology, Birge Hall, Madison. WI 53706
Price, Carl A., 20 Maker Lane, Falmouth, MA 02540
Prosser, C. Ladd, University of Illinois, Department of Physiology, 524

Burrill Hall. Urbana, IL 61801
Prytz, Margaret McDonald, (Address unknown)

Ratner, Sarah, Public Health Research Institute, Department of

Biochemistry. 455 First Avenue, New York, NY 10016 (deceased

1999)

Renn, Charles E., (Address unknown)
Reynolds, George T., Princeton University, Department of Physics,

Jadwin Hall. Princeton, NJ 08544

Rice, Robert V., 30 Burnham Drive, Falmouth. MA 02540
Rockstein, Morris, 600 Biltmore Way, Apt. 805, Coral Gables, FL

33134
Ronkin, Raphael R., 3212 McKinley Street. NW. Washington. DC

20015-1635
Roslansky, John D., 57 Buzzards Bay Avenue, Woods Hole, MA

02543
Roslansky, Priscilla F., Associates of Cape Cod. Inc.. P.O. Box 224.

Woods Hole, MA 02543-0224

Sanders, Howard L., Woods Hole Oceanographic Institution. Woods

Hole. MA 02543
Sato, Null . N igova University, 3-24-101, Oakinishi Machi, Toba

Mie 517-0023. Japan

Schlesinger, R. Walter, 7 Langley Road, Falmouth, MA 02540-1809
Scott, Allan C., Colby College, Waterville, ME 04901
Silverstein, Arthur M., Johns Hopkins University. Institute of the

History of Medicine. 1900 E. Monument Street. Baltimore, MD

21205
Sjodin, Raymond A., 3900 N. Charles Street, Apt. #1301. Baltimore,

MD 21218-1719
Smith, Paul F., P.O. Box 264, Woods Hole, MA 02543-0264

Speer, John W., 293 West Main Road. Portsmouth. RI 02871
Sperelakis, Nicholas, University of Cincinnati, Department of

Physiology/Biophysics. 231 Bethesda Avenue. Cincinnati, OH 45267-

0576
Spiegel. Evelyn, Dartmouth College. Department of Biological Sciences,

204 Oilman, Hanover, NH 03755
Spiegel, Melvin, Dartmouth College, Department of Biological

Sciences, 204 Oilman. Hanover, NH 03755
Stephens, Grover C., University of California. School of Biological

Sciences. Department of Ecology and Evolution/Biology, Irvine, CA

92717
Strehler, Bernard L., 42903 Moulton Parkway. #248. Laguna Hills,

CA 92653-6425

Sussman. Maurice, 72 Carey Lane, Falmouth, MA 02540
Sussman, Raquel B., Marine Biological Laboratory. Woods Hole, MA

02543
Szent-Gyorgyi, Gwen P., 45 Nobska Road, Woods Hole, MA 02543

Thorndike, W. Nicholas, Wellington Management Company, 200 State

Street, Boston, MA 02109
Trager, William, The Rockefeller University, 1230 York Avenue. New

York, NY 10021-6399
Trinkaus, J. Philip, 870 Moose Hill Road, Guilford, CT 06437

Villee, Claude A. Jr., Harvard Medical School, Carrel L. Countway

Library, 10 Shattuck Street, Boston, MA 021 15
Vincent, Walter S., 16 F.R. Lillie Road, Woods Hole, MA 02543

Waterman, Talbot H., Yale University. Box 208103, 912 KBT Biology

Department, New Haven. CT 06520-8103
Wigley, Roland L., 35 Wilson Road, Woods Hole, MA 02543

Members

Abt, Donald A., Aquavet, University of Pennsylvania, School of

Veterinary Medicine, 230 Mam Street, Falmouth, MA 02540
Adams. James A., 3481 Paces Ferry Road, Tallahassee, FL 32308
Adelman, William J., 160 Locust Street, Falmouth, MA 02540
Alkon, Daniel L., National Institutes of Health, Laboratory of Adaptive

Systems, 36 Convent Drive, MSC 4124, 36/4A21, Bethesda, MD

20892-4124
Allen, Garland E., Washington University, Department of Biology. Box

1137. One Brookings Drive, St. Louis. MO 63130-4899
Allen, Nina S., North Carolina State University. Department of Botany.

Box 7612, Raleigh, NC 27695
Alliegro, Mark C., Louisiana State University Medical Center,

Department of Cell Biology and Anatomy, 1901 Perdido Street. New

Orleans, LA 70112
Anderson, Everett, Harvard Medical School. Department of Cell

Biology. 240 Longwood Avenue, Boston, MA 021 15-6092
Anderson, John M., 110 Roat Street, Ithaca. NY 14850
Anderson, Porter W., 100 Bayview Drive. #2224. North Miami Beach.

FL 33160
Armett-Kibel, Christine, University of Massachusetts, Dean of Science

Faculty, Boston, MA 02125
Armstrong, Clay M., LIniversity of Pennsylvania School of Medicine.

B701 Richards Building, Department of Physiology, 3700 Hamilton

Walk, Philadelphia. PA 19104-6085

Armstrong, Ellen Prosser, 57 Millfield Street. Woods Hole, MA 02543
Arnold. William A., Oak Ridge National Laboratory, Biology Division,

102 Balsalm Road, Oak Ridge, TN 37830
Ashton, Robert W., Bay Foundation, 1 7 West 94th Street, New York,

NY 10025

Members of the Corporation R71

Atema, Jelle, Boston University Marine Program, Marine Biological
Laboratory. Woods Hole. MA 02543

Baccetti, Baccio, University of Sienna. Institute of Zoology, 53100

Siena, Italy
Baker. Robert G., New York University Medical Center, Department

Physiology and Biophysics, 550 First Avenue. New York, NY 10016
Baldwin, Thomas O., University of Arizona, Department of

Biochemistry, P.O. Box 210088. Tucson, AZ 85721-0088
Baltimore, David, California Institute of Technology. 1200 East

California Boulevard, Pasadena. CA 91 125
Barlow, Robert B., SUNY Health Science Center at Syracuse, 750 East

Adams Street. Center for Vision Research, 3258 Weiskotten Hall,

Syracuse, NY 13210
Barry, Daniel T., National Aeronautics and Space Administration, Lyn

B. Johnson Space Center, 2101 NASA Road 1, Houston, TX 77058
Barry, Susan R., Mount Holyoke College. Department of Biological

Sciences. South Hadley, MA 01075
Bass, Andrew H., Cornell University, Department of Neurobiology and

Behavior, Seely Mudd Hall. Ithaca. NY 14853
Battelle, Barbara-Anne, University of Florida, Whitney Laboratory.

9505 Ocean Shore Boulevard, Augustine, FL 32086
Bay, Frederick, Bay Foundation. 17 W. 94th Street. First Floor, New

York. NY 10025-7116

Baylor, Martha B., P.O. Box 93, Woods Hole, MA 02543
Bearer, Elaine L., Brown University, Division of Biology and

Medicine. Department of Pathology. Box G, Providence, RI 02912
Beatty, John M., University of Minnesota. Department of Ecology and

Behavioral Biology, 1987 Conner, Street Paul, MN 55108
Beauge, Luis Alberto, Instituto de Investigacion Medica, Department of

Biophysics. Casilla de Correo 389. Cordoba 5000. Argentina
Begenisich, Ted, University of Rochester, Medical Center, Box 642,

601 Elmwood Avenue, Rochester. NY 14642
Begg, David A., University of Alberta. Faculty of Medicine,

Department of Cell Biology and Anatomy, Edmonton, Alberta T6G

2H7. Canada

Bell, Eugene, 305 Commonwealth Avenue, Boston, MA 02115
Benjamin, Thomas L., Harvard Medical School, Pathology, D2-230,

200 Longwood Avenue. Boston. MA 021 15
Bennett, Michael V. L., Albert Einstein College of Medicine,

Department of Neuroscience. 1300 Morris Park Avenue, Bronx. NY

10461
Bennett, Miriam F., Colby College. Department of Biology. Waterville.

ME 04901
Bennett, R. Suzanne, Albert Einstein College of Medicine, Department

of Neuroscience. 1300 Morris Park Avenue. Bronx, NY 10461
Berg, Carl J. Jr., P.O. Box 681, Kilauea. Kauai, HI 96754-0681
Berlin. Suzanne T., 5 Highland Street. Gloucester, MA 01930
Bernstein. Norman, Columbia Realty Venture. 5301 Wisconsin

Avenue, NW, #600, Washington, DC 20015-2015
Bezanilla, Francisco, Health Science Center. Department of Physiology,

405 Hilgard Avenue. Los Angeles, CA 90024
Biggers, John D., Harvard Medical School, Department of Physiology,

Boston, MA 02115
Bishop, Stephen H., Iowa State University, Department of Zoology,

Ames, I A 50010
Blaustein, Mordecai P., University of Maryland, School of Medicine.

Department of Physiology, Baltimore, MD 21201
Blennemann, Dieter, 1117 East Putnam Avenue. Apt. #174. Riverside,

CT 06878-1333
Bloom, George S., The University of Texas Southwestern Medical

Center, Department of Cell Biology and Neuroscience, 5323 Harry

Hines Boulevard, Dallas, TX 75235-9039
Bloom, Kerry S., University of North Carolina. Department of Biology.

623 Fordham Hall CB#3280. Chapel Hill. NC 27599-3280

Bodznick, David A., Wesleyan University, Department of Biology.

Lawn Avenue, Middletown, CT 06497-0170
Boettiger, Edward G., 17 Eastwood Road, Storrs, CT 06268-2401
Boolootian, Richard A., Science Software Systems, Inc., 3576

Woodcliff Road, Sherman Oaks, CA 91403
Borgese, Thomas A., Lehman College. CUNY. Department of Biology.

Bedford Park Boulevard, West, Bronx, NY 10468
Borst, David W. Jr., Illinois State University, Department of Biological

Sciences, Normal, IL 61790-4120
Bowles, Francis P., Marine Biological Laboratory, Ecosystems Center.

Woods Hole. MA 02543
Boyer, Barbara C., Union College. Biology Department. Schenectady.

NY 12308
Brandhorst, Bruce P., Simon Fraser University. Institute of Molecular

Biology/Biochemistry. Bamaby, B.C. V5A 1S6, Canada
Brinley, F. J. Jr., N1NCDS/NIH, Neurological Disorders Program.

Room 812 Federal Building, Bethesda, MD 20892
Bronner-Fraser, Marianne, California Institute of Technology.

Beckman Institute Division of Biology. 139-74, Pasadena, CA 91125
Brown, Stephen C., SUNY. Department of Biological Sciences,

Albany, NY 12222

Brown, William L., 80 Black Oak Road, Weston, MA 02193
Browne, Carole L., Wake Forest University. Department of Biology,

Box 7325 Reynolds Station, Winston-Salem, NC 27109
Browne, Robert A., Wake Forest University. Department of Biology,

Box 7325, Winston-Salem. NC 27109
Bucklin, Anne C., University of New Hampshire. Ocean Process

Analysis Laboratory, 142 Morse Hall, Durham, NH 03824
Bullis. Robert A., Oceanic Institute of Applied Aquaculture, 41-202

Kalanianaole Highway, Waimanalo, HI 96795
Burger, Max M., Friedrich Miescher-Institute, P.O. Box 2543. CH-

4002 Basel, Switzerland
Burgess, David R., Boston College, Bourneuf House. 84 College Road.

Chestnut Hill, MA 02467-3838
Burgos, Mario H., IHEM Medical School, UNC Conicet, Casilla de

Correo 56. 5500 Mendoza, Argentina
Burky, Albert, University of Dayton, Department of Biology, Dayton,

OH 45469
Burris, John E., Marine Biological Laboratory, 7 MBL Street, Woods

Hole. MA 02543
Burstyn, Harold Lewis, United States Air Force, Air Force Materiel

Command, Rome Research Site RL/JA, 26 Electronic Parkway,

Rome, NY 13441-4514
Bursztajn, Sherry, LSU Medical Center, 1501 Kings Highway,

Building BRIF 6-13, Shreveport, LA 71 130

Calabrese, Ronald L., Emory University. Department of Biology. 1510

Clifton Road. Atlanta, GA 30322
Callaway, Joseph C., New York Medical College, Department of

Physiology. Basic Sciences Building, Valhalla, NY 10595
Cameron, R. Andrew, California Institute of Technology, Division of

Biology 156-29. Pasadena. CA 91 125
Campbell, Richard H., Bang-Campbell Associates, Eel Pond Place,

Box 402, Woods Hole, MA 02543
Candelas, Graciela C., University of Puerto Rico, Department of

Biology. P.O. Box 23360, UPR Station, San Juan, PR 00931-3360
Cariello, Lucio, Stazione Zoologica "A. Dohrn," Villa Comunale.

80121 Naples. Italy
Case, James F., University of California, Marine Science Institute.

Santa Barbara, CA 93106
Cassidy, Father Joseph D., Providence College, Priory of St. Thomas

Aquinas, Providence, RI 02918-0001
Cavanaugh, Colleen M., Harvard University, Biological Laboratories.

16 Divinity Avenue. Cambridge, MA 02138

R72 Annual Report

Chaet, Alfred B., University of West Florida, Department of Cell and

Molecular Biology, 1 1000 University Parkway, Pensacola. FL 32514
Chambers, Edward L., University of Miami School of Medicine,

Department of Physiology and Biophysics P.O. Box 016430, Miami,

FL 33101
Chang, Donald C., Hong Kong University, Science and Technology,

Department of Biology, Clear Water Bay, Kowloon, Hong Kong
Chappell, Richard L., Hunter College. CUNY. Department of

Biological Sciences, Box 210. 695 Park Avenue, New York, NY

10021
Child, Frank M., 28 Lawrence Farm Road, Woods Hole. MA 02543-

1416
Chisholm, Rex Leslie, Northwestern University, Medical School.

Department of Cell Biology, Chicago, IL 6061 1
Citkowitz, Elena, Hospital of St. Raphael, Lipid Disorders Clinic, 1450

Chapel Street, New Haven. CT 06511
Clark, Eloise E., Bowling Green State University, Biological Sciences

Department, Bowling Green, OH 43403
Clark, Hays, 150 Gomez Road, Hobe Sound, FL 33455
Clark. Wallis H. Jr., 12705 NW 1 12th Avenue, Alachua, FL 32615
Claude, Philippa, University of Wisconsin, Department of Zoology,

Zoology Research Building 125, 1 1 17 W Johnson Street, Madison,

WI 53706
Clay, John R., National Institutes of Health, N1NDS, Building 36,

Room 2-CO2, Bethesda, MD 20892
Clowes, Alexander W., University of Washington, School of Medicine,

Department of Surgery, Box 356410, Seattle. WA 98195-6410
Cobb, Jewel Plummer, California State University, 5151 University

Drive, Health Center 205, Los Angeles, CA 90032-8500
Cohen, Carolyn, Brandeis University, Rosenstiel Basic Medical.

Sciences Research Center, Waltham, MA 02254
Cohen, Lawrence B., Yale University School of Medicine, Department

of Physiology, 333 Cedar Street, New Haven, CT 06520
Cohen, Maynard M., Rush Medical College, Department of

Neurological Sciences, 600 South Paulina, Chicago, IL 60612
Cohen, William D., Hunter College, Department Biological Sciences,

New York, NY 10021
Coleman, Annette W., Brown University, Division of Biology and

Medicine, Providence, Rl 02912
Colinvaux, Paul, Marine Biological Laboratory, Woods Hole, MA

02543

Collier, Jack R., 3431 Highway, #107, P.O. Box 139, Effie, LA 71331
Collier, Marjorie McCann, 3431 Highway 107. P.O. Box 139, Effie.

LA 71331
Cook, Joseph A., Edna McConnell Clark Foundation. 250 Park Avenue.

New York. NY 10177-0026
Cornell, Neal W., Marine Biological Laboratory, Woods Hole, MA

02543 (deceased, 2000)
Cornwall, Melvin C. Jr., Boston University School of Medicine.

Department of Physiology L714, Boston, MA 02118
Corson, D. Wesley Jr., Storm Eye Institute, Room 537, 171 Ashley

Avenue. Charleston. SC 29425
Corwin, Jeffrey T., University of Virginia, School of Medicine,

Department Otolaryngology and Neuroscience, Box 396,

Charlottesville, VA 22908
Couch, Ernest F., Texas Christian University, Department of Biology,

TCU Box 298930, Fort Worth. TX 76129
Cox, Rachel Llanelly, Woods Hole Oceanographic Institute, Biology

Department, Woods Hole, MA 02543

Crane, Sylvia E., 438 Wendover Drive. Princeton, NJ 08540
Cremer-Bartels, Gertrud, Horstmarer Landweg 142, 48149 Muenster.

Germany
Crow, Terry J., University of Texas Medical School, Department of

Neurobiology and Anatomy, Houston, TX 77225

Crowell, Sears, Indiana University. Department of Biology.

Bloomington. IN 47405
Crowther, Robert J., Shriners Hospitals for Children, 51 Blossom

Street, Boston, MA 02114
Cummings, Michael P., Marine Biological Laboratory. Bay Paul

Center, Woods Hole, MA 02543
Cunningham, Mary-Ellen, 62 Cleverly Road. Grosse Pointe Farms. MI

48236-3313
Cutler, Richard D., Marine Biological Laboratory. Woods Hole. MA

02543

Davidson, Eric H., California Institute of Technology, Division of

Biology 156-29, 391 South Holliston, Pasadena. CA 91125
Davison, Daniel B., Bristol-Myers Squibb PRI. Bioinformatics

Department, 5 Research Parkway, Wallingford, CT 06492
Daw, Nigel W., 5 Old Pawson Road, Branford, CT 06405
Dawidowicz, Eliezar A., Marine Biological Laboratory, Office of

Research Administration and Education, Woods Hole, MA 02543
De Weer, Paul J., University of Pennsylvania, B400 Richards Building.

Department of Physiology. 3700 Hamilton Walk, Philadelphia, PA

19104-6085
Deegan, Linda A., Marine Biological Laboratory. The Ecosystems

Center. Woods Hole, MA 02543
DeGroof, Robert C., 145 Water Crest Drive, Doylestown, PA 18901-

3267
Denckla, Martha Bridge, Johns Hopkins University, School of

Medicine, Kennedy-Kneger Institute, 707 North Broadway. Baltimore.

MD 21205
DePhillips, Henry A., Trinity College, Department of Chemistry. 300

Summit Street, Hartford. CT 06106
DeSimone, Douglas W., University of Virginia, Department of Cell

Biology, Box 439, Health Sciences Center, Charlottesville, VA 22908
Dettbarn, Wolf-Dietrich, 4422 Wayland Drive, Nashville. TN 37215
Dionne, Vincent E., Boston University Marine Program, Marine

Biological Laboratory. Woods Hole. MA 02543
Dowling, John E., Harvard University. Biological Laboratories. 16

Divinity Street, Cambridge, MA 02138
Drapeau, Pierre, Montreal General Hospital, Department of Neurology.

1650 Cedar Avenue. Montreal. Quebec H3G 1A4, Canada
DuBois. Arthur Brooks, John B. Pierce Foundation Laboratory, 290

Congress Avenue. New Haven. CT 06519
Duncan, Thomas K., Nichols College, Environmental Sciences

Department. Dudley, MA 01571
Dunham. Philip B., Syracuse University, Department of Biology, 130

College Place. Syracuse. NY 13244-1220
Dunlap. Paul V., University of Maryland Biotechnology Institute,

Center of Marine Biotechnology, Columbus Center. Suite 236. 701

East Pratt Street, Baltimore, MD 2 1 202

Ebert, James D., The Johns Hopkins University, Department of

Biology, Homewood. 3400 North Charles Street, Baltimore, MD

21218-2685
Eckberg, William R., Howard University. Department of Biology, P.O.

Box 887, Administration Building, Washington, DC 20059
Edds, Kenneth T., R & D Systems, Inc., Hematology Division, 614

McKinley Place. NE. Minneapolis, MN 55413
Eder, Howard A., Albert Einstein College of Medicine, 1300 Morris

Park Avenue. Bronx. NY 10461

Edstrom, Joan, 53 Two Ponds Road. Falmouth, MA 02540
Egyud, Laszlo G., Cell Research Corporation, P.O. Box 67209.

Chestnut Hill. MA 02167-0209
Khrlich, Barbara E., Yale University Medical School, Department of

Pharmacology. New Haven, CT 06473

Members of the Corporation R73

Eisen, Arthur Z., Washington University, Division of Dermatology, St.

Louis, MO n3l 10
Eisen, Herman N., Massachusetts Institute of Technology, Center for

Cancer Research, El 7- 1 28. 77 Massachusetts Avenue, Cambridge.

MA 02 1 39-4307
Elder, Hugh Young. University of Glasgow. Institute of Physiology,

Glasgow G 1 2 8QQ, Scotland
Englund, Paul T., Johns Hopkins Medical School. Department of

Biological Chemistry, 725 North Wolfe Street. Baltimore. MD 21205
Epel, David, Stanford University. Hopkins Marine Station. Ocean View

Boulevard, Pacific Grove, CA 93950
Epstein, Herman T., 18 Lawrence Farm Road. Woods Hole. MA

02543
Epstein, Ray L., 701 Winthrop Street. #311. Taunton. MA 02780-2187

Farb, David H., Boston University School of Medicine, Department of

Pharmacology L603, 80 East Concord Street, Boston, MA 021 18
Farmanfarmaian, A. Verdi, Rutgers University. Department of

Biological Sciences. Nelson Biology Laboratory FOB 1059.

Piscataway. NJ 08855
Feldman, Susan C., University of Medicine and Dentistry. New Jersey

Medical School. 100 Bergen Street. Newark, NJ 07103
Festoff, Barry William. VA Medical Center, Neurology Service (151).

4801 Linwood Boulevard. Kansas City. MO 64128
Fink, Rachel D., Mount Holyoke College. Department of Biological

Sciences. Clapp Laboratories. South Hadley, MA 01075
Finkelstein, Alan, Albert Einstein College of Medicine, 1300 Morris

Park Avenue. Bronx, NY 10461
Fischbach, Gerald D., National Institute of Health, Neurological

Disorders and Strokes, 3 1 Center Drive, MSC 2540, Bldg 3 1 , Rm

8A03. Bethesda, MD 20892-2540
Fishman, Harvey M., University of Texas Medical Branch, Department

of Physiology and Biophysics, 301 University Boulevard, Galveston.

TX 77555-0641

Flanagan, Dennis, 12 Gay Street, New York. NY 10014
Fluck, Richard Allen, Franklin and Marshall College. Department of

Biology. Box 3003, Lancaster, PA 17604-3003
Foreman, Kenneth H., Marine Biological Laboratory. Woods Hole.

MA 02543
Fox, Thomas Oren, Harvard Medical School, Division of Medical

Sciences, MEC 435. 260 Longwood Avenue. Boston. MA 021 15
Franzini-Armstrong, Clara, University of Pennsylvania, School of

Medicine. 330 South 46th Street, Philadelphia. PA 19143
Fraser, Scott, California Institute of Technology, Beckman Institute

139-74, 1201 East California Boulevard, Pasadena. CA 91 125
Frazier, Donald T., University of Kentucky Medical Center.

Department of Physiology and Biophysics. MS501 Chandler Medical

Center, Lexington, KY 40536
French, Robert J., University of Calgary, Health Sciences Centre,

Alberta, T2N 4NI, CANADA
Fulton, Chandler M., Brandeis University. Department of Biology. MS

008. Waltham. MA 02454-91 II)
Furie, Barbara C.. Beth Israel Deaconess Medical Center. BIDMC

Cancer Center, Kirstein 1, 330 Brookline Avenue, Boston. MA 02215
Furie, Bruce. Beth Israel Deaconess Medical Center. BIDMC Cancer

Center, Kirstein 1. 330 Brookline Avenue. Boston. MA 02215
Furshpan, Edwin J., Harvard Medical School. Department of

Neurobiology. 220 Longwood Avenue, Boston. MA 021 15
Futrelle, Robert P., Northeastern University. College of Computer

Science, 360 Huntington Avenue. Boston. MA 021 15

Gabr, Howaida, Sue/, Canal University, Department of Marine Science,
Faculty of Science, Ismailia, Egypt

Gadsby, David C., The Rockefeller University, Laboratory of Cardiac-
Physiology. 1230 York Avenue. New York. NY 10021-6399
Gainer, Harold, National Institutes of Health. NINDS, BNP, DIR,

Neurochemistry. Building 36, Room 4D20. Bethesda, MD 20892-

4130

Galatzer-Levy, Robert M., 534 Judson Avenue, Evanston, IL 60202
Gall, Joseph G., Carnegie Institution, I 15 West University Parkway.

Baltimore. MD 21210
Garber, Sarah S.. Allegheny University of the Health Sciences.

Department of Physiology, 2900 Queen Lane, Philadelphia, PA 19129
Gascoyne, Peter, University of Texas. M. D. Anderson Cancer Center.

Experimental Pathology. Box 89, Houston, TX 77030
Gelperin, Alan, Bell Labs Lucent. Department Biology Comp.. Rm

1C464. 600 Mountain Avenue. Murray Hill, NJ 07974
German, James L. Ill, The New York Blood Center, Laboratory of

Human Genetics. 310 East 67th Street. New York. NY 10021
Gibbs, Martin, Brandeis University. Institute for Photobiology of Cells

and Organelles. Waltham, MA 02254
( .ililin. Anne E., Marine Biological Laboratory, The Ecosystems

Center, Woods Hole, MA 02543
Gibson, A. Jane, Cornell University, Department of Biochemistry.

Biotech Building. Ithaca. NY 14850
Gifford, Prosser, Library of Congress, Madison Building LM605,

Washington DC 20540
Gilbert, Daniel L., National Institutes of Health. Biophysics Sec.. BNP,

Building 36. Room 5A-27. Bethesda, MD 20892
Giudice, Giovanni, Universita di Palermo. Dipartimento di Biologia,

Cellulare e Dello Sviluppo, 1-90123 Palermo. Italy
Giuditta, Antonio, University of Naples. Department of General

Physiology. Via Mezzocannone 8. Naples. 80134, Italy
Glynn, Paul, P.O. Box 6083. Brunswick, ME 04011-6083
Golden, William T., Chairman Emeritus, American Museum of Natural

History, 500 Fifth Avenue, 50th Floor, New York, NY 101 10
Goldman. Robert D., Northwestern University Medical School,

Department of Cell and Molecular Biology, 303 E. Chicago Avenue.

Chicago. IL 60611-3008
Goldsmith, Paul K., National Institutes of Health. Building 10. Room

9C-101. Bethesda, MD 20892
Goldsmith, Timothy H., Yale University. Department of Biology, New

Haven. CT06510
Goldstein, Jr., Moise H., The Johns Hopkins University. ECE

Department. Barton Hall. Baltimore, MD 2121X
Gould, Robert Michael, NYS Institute of Basic Research, 1050 Forest

Hill Road. Staten Island, NY 10314-6399
Govind, C. K., Scarborough College, Life Sciences Division. 1 265

Military Trail. West Hill. Ontario MIC IA4, Canada
Grace, Dick, Doreen Grace Fund, The Brain Center, Promontory Point.

New Seabury. MA 02649
Graf, Werner M., College of France, 1 1 Place Marcelin Berthelot.

7523 1 Paris Cedex 05, France
Grant, Philip, National Institutes of Health, NINDS\BN\DIR-

Neurochemistry. Building 36, Room 4D20, Bethesda. MD 20892-

4130
Grass, Ellen R., The Grass Foundation. 77 Reservoir Road. Quincy.

MA 02170-3610
Grassle, Judith P.. Rutgers University. Institute of Marine and Coastal

Studies. Box 231. New Brunswick. NJ 08903
Graubard, Katherine G., University of Washington, Department of

Zoology, NJ-15, Box 351800, Seattle, WA 98195-1800
Greenberg, Everett Peter, University of Iowa, College of Medicine,

Department of Microbiology. Iowa City, IA 52242
Greenberg, Michael J., University of Florida. The Whitney Laboratory.

9505 Ocean Shore Boulevard, St. Augustine, FL 32086-8623

R74 Annual Report

Greer, Mary J., 176 West 87th Street. #12A, New York, NY 10024-

2902
Griffin, Donald R., Harvard University. Concord Field Station. Old

Causeway Road. Bedford, MA 01730

Gross, Paul R., 123 Perkins Street. Jamaica Plain, MA 02130
Grossman, Albert, New York University Medical Center, 550 First

Avenue, New York, NY 10016
Grossman, Lawrence, The Johns Hopkins University, Hygiene

Building, Room W8306, Baltimore, MD 21205
Gruner, John A., Cephalon. Inc., 145 Brandy wine Parkway, West

Chester, PA 19380-4245

Gunning, A. Robert, P. O. Box 165. Falmouth. MA 02541
Gwilliam, G. Francis, Reed College, Department of Biology, Portland,

OR 97202

Haimo, Leah T., University of California, Department of Biology,

Riverside. CA 92521
Hajduk, Stephen L., University of Alabama. School of

Medicine/Dentistry. Department of Biochemistry/Molecular Genetics,

University Station. Birmingham. AL 35294
Hall, Linda M., Shriners Hospital for Children, 2425 Stockton

Boulevard. Sacramento, CA 95X17
Hall, Zach W., University of California, Department of Physiology, San

Francisco, C A 941 14
Halvorson, Harlyn O., University of Massachusetts, Policy Center for

Marine Biosciences and Technology. 100 Morrissey Boulevard,

Boston, MA 02 1 25-3393
Haneji, Tatsuji, The University of Tokushima, Department of Histology

and Oral Histology, School of Dentistry. 18-15. 3 Kuramoto-cho.

Tokushima 770-8504, Japan
Hanlon, Roger T., Marine Biological Laboratory, Woods Hole, MA

02543
Harosi, Ferenc, New College of the USF. Division of Natural Sciences,

5700 North Tamiami Trail. Sarasota, FL 34243-2197
Harrigan, June F., 7415 Makaa Place, Honolulu. HI 96825
Harrington, Glenn W., Weber State University. Department of

Microbiology, Ogden, UT 84408
Harrington, John P., University of South Alabama, Department of

Chemistry, Mobile, AL 36688
Harrison, Stephen C., Harvard University. Department of Molecular

and Cell Biology, 7 Divinity Avenue, Cambridge, MA 02138
Haselkorn, Robert, Llniversity of Chicago, Department of Molecular

Genetics and Cell Biology, Chicago, IL 60637
Hastings, J. Woodland, Harvard University. The Biological

Laboratories. 16 Divinity Avenue. Cambridge. MA 02138-2020
Haydon-Baillie, Wensley G., Porton Institute. 2 Lowndes Place,

London SW1X 8Dd. United Kingdom
Hayes, Raymond L. Jr., Howard University, College of Medicine, 520

W Street. NW. Washington, DC 20059
Heck, Diane E., Rutgers University, Department of

Pharmacology/Toxicology, 681 Frelinghuysen Road, Piscataway, NJ

08855
Henry, Jonathan Joseph, University of Illinois, Department of Cell and

Structural Biology. 601 South Goodwin Avenue #B107, Urbana. IL

61801-3709
Hepler, Peter K , iMiy of Massachusetts. Department of Biology.

Morrill III, Amiu-i M\ 01003
Herndon, Walter K., n isiiy of Tennessee, Department of Botany,

Knoxville. TN >.7'"
Hersliko, Avram, Technion IM:J<-| Institute of Technology, Unit of

Biochemistry. The Bruce Rappaport Faculty of Medicine. Haifa

31096. Israel
Herskovits, Theodore T., Fordham University. Department of

Chemistry. John Mulcahy Hall, Room 638. Bronx. NY 10458

Hiatt, Howard H., Bngham and Women's Hospital, Department of

Medicine, 75 Francis Street. Boston. MA 021 15
Highstein, Stephen M., Washington University. Department of

Otolaryngology. Box 8115, 4566 Scott Avenue, St. Louis. MO 63110
Hildebrand, John G., University of Arizona. ARL Division of

Neurobiology, P.O. Box 210077, Tucson, AZ 85721-0077
Hill, Richard W., Michigan State University. Department of Zoology,

East Lansing. MI 48824
Hill, Susan D., Michigan State University. Department of Zoology. East

Lansing. MI 48824
Hillis, Llewellya W., Marine Biological Laboratory. Woods Hole. MA

02543
Hinchcliffe, Edward H., University of Massachusetts Medical School,

Department of Cell Biology. 377 Plantation Street. Worcester, MA

01605
Hinkle, Gregory J., Bioinformatics Group, Cereon Genomics, One

Kendall Square, Building 200. Cambridge, MA 02139
Hinsch, Gertrude W., University of South Florida, Department of

Biology, Tampa. FL 33620

Hinsch, Jan, Leica. Inc.. 1 10 Commerce Drive, Allendale, NJ 07401
Hobbie, John E., Marine Biological Laboratory, The Ecosystems

Center. Woods Hole, MA 02543

Hodge, Alan J., 3843 Mount Blackburn Avenue. San Diego, CA 921 1 1
Hoffman, Joseph F., Yale University School of Medicine. Cellular and

Molecular Physiology, 333 Cedar Street, New Haven, CT 06520-8026
Hollyneld, Joe G., The Cleveland Clinic, Ophthalmic Research, 9500

Euclid Avenue. Cleveland, OH 44195
Holz, George G. IV, New York Llniversity Medical Center. Medical

Sciences Building. Room 442, 550 First Avenue, New York, NY

10016
Hopkinson, Charles S. Jr., Marine Biological Laboratory, Woods Hole.

MA 02543
Houk. James C., Northwestern University Medical School, 303 East

Chicago Avenue, Ward 5-315. Chicago. IL 60611-3008
Hoy, Ronald R., Cornell University. Section of Neurobiology and

Behavior. 215 Mudd Hall. Ithaca. NY 14853
Huang, Alice S., California Institute of Technology. Mail Code 1-9,

Pasadena, CA 91125
Hufnagel-Zackroff, Linda A., University of Rhode Island, Department

of Microbiology, Kingston, RI 02881
Hummon, William D., Ohio University. Department of Biological

Sciences, Athens, OH 45701
Humphreys, Susie H., Food and Drug Administration. HFS-308. 200 C

Street, SW. Washington. DC 20204-0001
Humphreys, Tom, University of Hawaii. Kewalo Marine Laboratory,

41 Ahui Street. Honolulu. HI 96813
Hunt, Richard T., ICRF. Clare Hall Laboratories. South Mimms

Potter's Bar. Herts EN6-3LD, England
Hunter, Robert D., Oakland University, Department of Biological

Sciences, Rochester, MI 48309-4401
Huxley. Hugh E., Brandeis University. Rosenstiel Center. Biology

Department. Waltham, MA 02154

Ilan, Joseph, Case Western Reserve University. School of Medicine,

Department of Anatomy, Cleveland. OH 44 1 (Id
Ingoglia, Nicholas A., New Jersey Medical School. Department of

Pharmacology/Physiology, 185 South Orange Avenue. Newark. NJ

07103
Inoue. Saduyki, McGill Llniversity. Department of Anatomy. 3640

University Street. Montreal. PQ H3A 2B2, Canada
Inoue, Shinya, Marine Biological Laboratory. Woods Hole, MA 02543
Isselbacher, Kurt J., Massachusetts General Hospital Cancer Center,

Charlestown. MA 02 1 29
Issidorides, Marietta Radovic, University of Athens. Department of

Psychiatry. Monis Petraki 8. Athens. 140. Greece

Members of the Corporation R75

Izzard, Colin S., SUNY-Albany, Department of Biological Sciences,
1400 Washington Avenue, Albany. NY 12222

Jacobs. Neil, Hale and Dorr, 60 State Street. Boston, MA 02109
Jaffe, Laurinda A., University of Connecticut Health Center,

Department of Physiology. Farmington Avenue. Farmington. CT

06032

Jaffe. Lionel, Marine Biological Laboratory. Woods Hole, MA 02543
Jeffery, William R., University of Maryland. Department of Biology,

College Park, MD 20742
Johnston, Daniel, Baylor College of Medicine. Division of

Neuroscience. Baylor Plaza, Houston. TX 77030
Josephson. Robert K., University of California. School of Biological

Science, Department of Psychobiology. Irvine. CA 92697

Kaczmarek, Leonard K., Yale University School of Medicine,

Department of Pharmacology. 333 Cedar Street. New Haven. CT

06520
Kaley, Gabor, New York Medical College. Department of Physiology.

Basic Sciences Building. Valhalla, NY 10595
Kaltenbach. Jane, Mount Holyoke College. Department Biological

Sciences. South Hadley, MA 01075
Kaminer, Benjamin, Boston University Medical School. Physiology

Department, 80 East Concord Street, Boston, MA 02 1 I S
Kaneshiro, Edna S., University of Cincinnati. Biological Sciences

Department. JL 006. Cincinnati, OH 45221-0006
Kaplan, Ehud, Mount Sinai School of Medicine. 1 Gustave Levy Place.

Box 1 1S3. New York, NY 10029
Karakashian, Stephen J., Apartment 16-F. 165 West 91st Street. New

York. NY 10024
Karlin, Arthur, Columbia University, Center for Molecular

Recognition, 630 West 168th Street, Room 11-401. New York. NY

10032
Karnovsky, Morris John, Harvard Medical School, Department of

Pathology. 200 Longwood Avenue. Boston. MA 02 1 1 5
Keller, Hartmut Ernst, Carl Zeiss, Inc.. One Zeiss Drive, Thornwood.

NY 10594
Kelley, Darcy B., Columbia University, Department of Biological

Sciences, 91 1 Fairchild, Mailcode 2432, New York. NY 10027
Kelly, Robert E., 5 Little Harbor Road, Woods Hole. MA 02543
Kemp. Norman E., University of Michigan, Department of Biology.

Ann Arbor. MI 48109
Kendall. John P., Faneuil Hall Associates. 176 Federal Street. 2nd

Floor, Boston. MA 021 10
Kerr, Louis M., Marine Biological Laboratory, Woods Hole. MA

02543
Keynan, Alexander, Israel Academy of Science and Humanity, P.O.

Box 4040, Jerusalem. Israel
Khan, Shahid M. M., Albert Einstein College of Medicine. Department

of Physiology and Biophysics. 1 300 Morris Park Avenue. Room

U273. Bronx, NY 10461
Khodakhah. Kamran, University of Colorado School of Medicine,

Department of Physiology and Biophysics. 4200 East 9th Avenue,

C-240. Denver, CO 80262
Kiehart, Daniel P., Duke University Medical Center. Department of

Cell Biology. Box 3709, 308 Nanaline Duke Building, Durham. NC

27710
Kleinfeld, David, University of California. Department of Physics. 0319

9500 Oilman Drive. La Jolla, CA 92093
Klessen, Rainer, (address unknown)
Klotz, Irving M., Northwestern University. Department of Chemistry,

Evanston. 1L 60201
Knudson, Robert A., Marine Biological Laboratory. Woods Hole. MA

02543

Koide, Samuel S., The Rockefeller University, The Population Council.

1230 York Avenue. New York. NY 10021
Kornberg, Hans, Boston University. The University Professors. 745

Commomveath Avenue, Boston. MA 02215
Kosower, Edward M., Tel-Aviv University. Department of Chemistry.

Ramat-Aviv. Tel Aviv. 69978, Israel

Krahl. Maurice E., 2783 West Casas Circle. Tucson. AZ 85741
Krane, Stephen M., Massachusetts General Hospital, 55 Fruit Street,

Bulf-165. Boston, MA 021 14
Krauss, Robert, P.O. Box 291, Denton, MD 21629
Kravitz, Edward A., Harvard Medical School, Department of

Neurobiology. 220 Longwood Avenue. Boston. MA 021 15
Kriebel. Mahlon E., SUNY Health Science Center. Department of

Physiology. Syracuse. NY 13210
Kristan, William B. Jr., University of California, Department of

Biology 0357, 9500 Oilman Drive. La Jolla. CA 92093-0357
Kropin.ski, Andrew M., Queen's University. Department of

Microbiology/Immunology. Kingston, Ontario K7L 3N6. Canada
Kuffler, Damien P., Institute of Neurobiology. 201 Boulevard del

Valle, San Juan 00901. PR
Kuhns, William J., Hospital for Sick Children. Biochemistry Research,

555 University Avenue, Toronto, Ontario M5G 1X8, Canada
Kunkel, Joseph G., University of Massachusetts, Department of

Biology. Amherst, MA 01003
Kuzirian, Alan M., Marine Biological Laboratory, Woods Hole, MA

02543-1015

Laderman, Ainilee D., Yale University, School of Forestry and

Environmental Studies, 370 Prospect Street, New Haven, CT 0651 1
I .mil. ;m, Laurie J., Listowel, Inc., 2 Park Avenue, Suite 1525, New

York. NY 10016
Landis, Dennis M. D., University Hospital of Cleveland. Department

Neurology, 1 1 100 Euclid Avenue. Cleveland. OH 44106
Landis, Story C.. National Institutes of Health. Building 36. Room

5A05. 36 Convent Drive. Bethesda, MD 20892-4150
Landowne, David, University of Miami Medical School, Department of

Physiology, PO Box 016430, Miami, FL 33101
Langford, George M., Dartmouth College, Department of Biological

Sciences. 6044 Oilman Laboratory. Hanover. NH 03755
Laskin, Jeffrey, University of Medical and Dentistry of New Jersey,

Robert Wood Johnson Medical School. 675 Hoes Lane. Piscataway,

NJ 08854
Lasser-Ross. Nechama, New York Medical College. Department of

Physiology. Valhalla, NY 10595
Laster, Leonard, University of Massachusetts Medical School, 55 Lake

Avenue, North, Worcester. MA 01655
Laties, Alan, Scheie Eye Institute, Myrin Circle, 51 North 39th Street,

Philadelphia. PA 19104
Laufer, Hans, University of Connecticut. Department of Molecular and

Cell Biology. U- 1 25. 75 North Eagleville Road Storrs. CT 06269-

3125
Lazarow, Paul B., Mount Sinai School of Medicine. Department of

Cell Biology and Anatomy. 1190 Fifth Avenue. Box 1007, New

York. NY 10029-6574
Lazarus, Maurice, Federated Department Stores, Sears Crescent, City

Hall Plaza. Boston. MA 02108
Leadbetter, Edward R., University of Connecticut. Department of

Molecular and Cell Biology. U-131, Beach Hall, Room 249, 354

Mansfield Road, Storrs. CT 06269-2131
Lederberg, Joshua, The Rockefeller University, Suite 400 (Founders

Hall). 1230 York Avenue. New York. NY 10021
Lee, John J., City College of CUNY, Department of Biology. Convent

Avenue and 138th Street. New York. NY 10031
Lehv, Donald B., 35 Willow Field Drive. North Falmouth. MA 02556

R76 Annual Report

Leighton, Joseph. Aeron Biotechnology, Inc.. 1933 Davis Street #310,

San Leandro, CA 44577 (deceased 1999)
Leighlon, Stephen B., National Institutes of Health. Building 13. 3W13.

Bethesda, MD 20892
Lemos, Jose Ramon. University of Massachusetts Medical Center.

Worcester Foundation Campus. 222 Maple Avenue. Shrewsbury. MA

01545-2737
Lerner, Aaron B., Yale University School of Medicine, Department of

Dermatology. P.O. Box 3333. New Haven. CT 06510
Levin, Jack, Veterans Administration. Medical Center, 1 1 1 H2. 4150

Clement Street. San Francisco, C A 94121
Levine, Michael, University of California, Department MCB, 401

Barker Hall. Berkeley. CA 94720
Levine, Richard B., University of Arizona, Division of Neurobiology,

Room 61 1. Gould Simpson Building. PO Box 210077. Tucson. AZ

85721-0077
Levinthal. Francoise, Columbia University, Department of Biological

Sciences. Broadway and 1 16th Street, New York, NY 10026
Levitan, Herbert, National Science Foundation, 4201 Wilson

Boulevard. Room 835, Arlington, VA 22230
Levitan, Irwin B., University of Pennsylvania. School of Medicine. 218

Stemmler Hall, 3450 Hamilton Walk. Philadelphia, PA 19104-6074
Linck, Richard VV., University of Minnesota School of Medicine, Cell

Biology and Neuroanatomy Department. 4-135 Jackson Hall. 321

Church Street. Minneapolis. MN 55455
Lipicky, Raymond J., Food and Drug Administration. CDER/ODE1/

HFD-1 10, 5600 Fishers Lane. Rockville. MD 20857
Lisman, John E., Brandeis University, Molecular and Cell Biology. 415

South Street. Waltham. MA 02454-91 10
Liuzzi, Anthony, 180 Beacon Street. #80. Boston. MA 021 16
Llinas, Rodolfo R., New York University Medical Center, Department

of Physiology/Biophysics, 550 First Avenue. Room 442. New York.

NY 10016
Lohel, Phillip S., Boston University Marine Program, Marine Biological

Laboratory, Woods Hole. MA 02543
Loew, Franklin M., Becker College. 61 Sever Street, Worcester, MA

01615-0071
Loewenstein, Birgit Rose, Marine Biological Laboratory. Woods Hole,

MA 02543
Loewenstein, Werner R., Marine Biological Laboratory, Woods Hole,

MA 02543
London, Irving M., Harvard-MIT. Division. E-25-551, Cambridge. MA

02 1 39
Longo, Frank J., University of Iowa, Department of Anatomy, Iowa

City. IA 52442
Luckenhill, Louise M., Ohio University. Department of Biological

Sciences. Irvine Hall. Athens. OH 45701

Macagno, Eduardo R., Columbia University. 109 Low Memorial

Library, Mail Code 4306. New York. NY 10027
MacNichol. Edward F. Jr., Boston University School of Medicine.

Department of Physiology, 80 East Concord Street, Boston, MA

02 1 IS
Maglott-Dultield, Donna R., American Type Culture Collection, 12301

Parklawn Drive, Rockville, MD 20852-1776
Maienschein, Jane Ann, Arizona State University, Department of

Philosophy, PO B. - S72004. Tempe, AZ 85287-2004
Mainer, Robert ¥.., ":\tL Boston Company, Inc.. One Boston Place.

OBP-I5-D. Boston. MA 02108
Malhon, Craig C., SUNY, University Medical Center. Pharmacology-

HSC. Stony Brook. NY I l?'>4-Sf>5l
Malchow, Robert P., UimciMly ol Illinois, Department of

Ophthalmology, 1855 West Taylor Street N/C 648, Chicago. IL

(,0(i I 2

Manalis, Richard S., Indiana-Purdue University, Department of

Biological Science, 2101 Coliseum Boulevard East, Fort Wayne, IN

46805

Manz, Robert D., 304 Adams Street. Milton. MA 02186
Margulis, Lynn, University of Massachusetts. Department of

Geosciences, Morrill Science Center. Box 35820. Amherst, MA

01003-5820

Marinucci, Andrew C., 102 Nancy Drive, Mercerville, NJ 08619
Martinez, Joe L. Jr., The University of Texas, Division of Life

Sciences, 6900 North Loop 1604 West, San Antonio, TX 78249-0662
Martinez-Palomo, Adolfo, CINVESTAV-IPN, Sec. de Patologia

Experimental, 07000 Mexico, D.F.A.P. 140740, Mexico
Mastroianni, Luigi Jr., Hospital of University of Pennsylvania, 106

Dulles, 3400 Spruce Street. Philadelphia, PA 19104-4283
Mauzerall, David, Rockefeller University, 1230 York Avenue, New

York. NY 10021
McAnelly. M. Lynne, University of Texas, Section of Neurobiology,

School of Life Sciences. Austin, TX 78712
McCann, Frances V., Dartmouth Medical School, Department of

Physiology, Lebanon. NH 03756
McLaughlin, Jane A., Marine Biological Laboratory. Woods Hole. MA

022543
McMahon, Robert F., University of Texas. Arlington, Department of

Biology, Box 19498. Arlington. TX 76019
Meedel, Thomas, Rhode Island College, Biology Department. 600

Mount Pleasant Avenue, Providence. RI 02908
Meinertzhagen, Ian A., Dalhousie University. Department of

Psychology, Halifax, NS B3H 4J1, Canada
Meiss, Dennis E., Immunodiagnostic Laboratories, 488 McCormick

Street, San Leandro, CA 94577
Melillo, Jerry M., Marine Biological Laboratory, Ecosystems Center,

Woods Hole. MA 02543
Mellon, DeForest Jr., University of Virginia, Department of Biology,

Gilmer Hall. Charlottesville. VA 22903

Mellon. Richard P., P.O. Box 187, Laughlintown, PA 15655-0187
Mendelsohn, Michael E., New England Medical Center, Molecular

Cardiology Laboratory, NEMC Box 80. 750 Washington Street,

Boston. MA 02 1 1 I
Mensinger, Allen F., Washington University School of Medicine.

Department of Otolarynology. 4566 Scott Avenue. St. Louis, MO

63110
Merriman, Melanie Pratt, 7511 Beach View Drive. North Bay Village.

FL 33141
Meselson, Matthew, Harvard University. Fairchild Biochemistry

Building. 7 Divinity Avenue. Cambridge. MA 02138
Metuzals, Janis, University of Ottawa Faculty of Medicine, Department

of Pathology and Laboratory Medical, 45 1 Smyth Road, Ottawa,

Ontario K1H 8M5, Canada
Miledi, Ricardo, University of California, Irvine. Department of

Psychobiology. 2205 Biology Science II. Irvine, CA 92697-4550
Milkman, Roger D., University of Iowa. Department of Biological

Sciences. Biology Building, Room 318, Iowa City, IA 52242-1324
Miller. Thomas J., Analogic. 8 Centennial Drive, Peabody. MA 01960
Mills. Robert, 10315 44th Avenue, W 12 H Street, Brandenton. FL

34210
Misevic. Gradimir, University Hospital of Basel, Department of

Research, Mebelstr. 20. CH-4031 Basel. Switzerland
Mitchell. Ralph, Harvard University, Division of Applied Sciences, 29

Oxford Street, Cambridge, MA 02138
Miyakawa, Hiroyoshi, Tokyo College of Pharmacy. Laboratory of

Cellular Neurobiology. 1432-1 Horinouchi. Hachiouji, Tokyo 192-03,

Japan
Miyamoto, David M., Drew University, Department of Biology,

Madison, NJ 07940

Members of the Corporation R77

Mi/ell. Merle, Tulane University, Department of Cell and Molecular,

Biology, New Orleans. LA 701 18
Moreira. Jorge E., National Institutes of Health. NICHD. Department

of Cell and Molecular Biol., Bethesda. MD 20852
Morin, James G., Cornell University, Dept. of Ecology & Evolutionary

Biology, G14 Stimson Hall, Ithaca. NY 14853-2801
Morrcll. Leyla de Toledo, Rush-Presbyterian-St. Lukes Medical Center.

1653 West Congress Parkway. Chicago. IL 60612
Morse, M. Patricia, National Science Foundation, Room 885, Esie,

Arlington. VA 22230
Morse, Stephen S., DARPA/DSO, 3701 North Fairfax Dnve, Arlington,

VA 22203-1714
Mote, Michael I., Temple University, Department of Biology,

Philadelphia. PA 19122
Muller, Kenneth J., University of Miami School of Medicine,

Department of Physiology and Biophysics, 1600 NW 10th Avenue.

R-430, Miami, FL 33136
Murray, Andrew W., University of California. Department of

Physiology. Box 0444. 513 Parnassus Avenue. San Francisco. CA

94143-0444

Nabrit. Samuel M., 686 Beckwith Street, SW, Atlanta, GA 30314
Nadelhoffer. Knute J., Marine Biological Laboratory. 7 MBL Street,

Woods Hole. MA 02543
Nagel, Ronald L., Albert Einstein College of Medicine. 1300 Morris

Park Avenue, Bronx. NY 10461

Naka. Ken-ichi, 2-9-2 Tatumi Higashi. Okazaki. 444, Japan
Nakajima, Yasuko, University of Illinois, College of Medicine,

Anatomy and Cell Biology Department, M/C 512, Chicago, IL 60612
Narahashi, Toshio, Northwestern University Medical School,

Department of Pharmacology, 303 East Chicago Avenue, Chicago, IL

60611
Nasi, Enrico, Boston University School of Medical, Department of

Physiology, R-406, 80 East Concord Street. Boston. MA 02 1 1 8
Neill, Christopher, Marine Biological Laboratory, 7 MBL Street.

Woods Hole, MA 02543
Nelson, Leonard, Medical College of Ohio, Department of Physiology,

CS 10008. Toledo. OH 43699 (deceased 1999)
Nelson, Margaret C., Cornell University, Section of Neurobiology and

Behavior, Ithaca, NY 14850

Nicholls, John G., SISSA. Via Beirut 2, 1-34014 Trieste. Italy
Nickerson. Peter A., SUNY at Buffalo. Department of Pathology.

Buffalo. NY 14214
Nicosia, Santo V., University of South Florida, College of Medicine,

Box 1 1, Department of Pathology, Tampa, FL 33612
Noe, Bryan D., Emory University School of Medicine, Department of

Anatomy and Cell Biology, Atlanta, GA 30322
Norton, Catherine N., Marine Biological Laboratory, 7 MBL Street,

Woods Hole, MA 02543
Nusbaum, Michael P., University of Pennsylvania School of Medicine,

Department of Neuroscience, 215 Stemmler Hall, Philadelphia, PA

19104-6074

O'Herron, Jonathan, Lazard Freres and Company, 30 Rockefeller

Plaza, 59th Floor, New York, NY 10020-1900
Obaid, Ana Lia, University of Pennsylvania School of Medicine,

Neuroscience Department, 234 Stemmler Hall, Philadelphia, PA

19104-6074
Ohki, Shinpei, SUNY at Buffalo, Department of Biophysical Sciences,

224 Cary Hall. Buffalo. NY 14214
Oldenbourg, Rudolf, Marine Biological Laboratory, 7 MBL Street.

Woods Hole, MA 02543
Olds, James L., George Mason University. Krasnow Institute for

Advanced Studies, Mail Stop 2A1, Fairfax, VA 22030-4444

Olins, Ada L.. 45 Eastern Promenade. #7-D, Portland. ME 04101
Olins. Donald E.. 45 Eastern Promenade. #7-D. Portland. ME 04101
Oschman, James L., Nature's Own Research Association, P.O. Box
5101. Dover. NH 03820

Palazzo, Robert E., University of Kansas. Department of Physiology

and Cell Biology. Lawrence, KS 66045
Palmer, John D., University of Massachusetts, Department of Zoology,

221 Merrill Science Center, Amherst. MA 01003
Pant, Harish C., National Institutes of Health. NINCDS, Laboratory of

Neurochemistry, Building 36. Room 4D20. Bethesda, MD 20892
Pappas, George D., University of Illinois, College of Medicine,

Department of Anatomy. Chicago, IL 60612
Pardee, Arthur B., Dana-Farber Cancer Institute, D810, 44 Binney

Street, Boston, MA 021 15
Pardy, Rosevelt L., University of Nebraska, School of Life Sciences,

Lincoln, NE 68588
Parmentier, James L., AstraZeneca, 725 Chesterbrook Boulevard,

Wayne, PA 19087-5677
Pederson, Thoru, University of Massachusetts Medical Center.

Worcester Foundation Campus, 222 Maple Avenue, Shrewsbury, MA

01545

Perkins, Courtland D., 400 Hilltop Terrace. Alexandria. VA 22301
Person, Philip, 137-87 75th Road. Flushing. NY 11367
Peterson, Bruce J., Marine Biological Laboratory. 7 MBL Street.

Woods Hole, MA 02543
Pethig. Ronald, University College of North Wales, School of

Electronic Engineering, Bangor, Gwynedd, LL 57 IUT, United

Kingdom
Pfohl, Ronald J., Miami University, Department of Zoology. Oxford,

OH 45056
Pierce, Sidney K. Jr., University of South Florida, 4202 East Fowler

Avenue. Tampa, FL 33620
Pleasure, David E., Children's Hospital. Neurology Research. 5th

Floor, Ambramson Building, Philadelphia. PA 19104
Poindexter. Jeanne S.. Barnard College, Columbia University, 3009

Broadway, New York, NY 10027-6598
Pollard, Harvey B., U.S.U.H.S., 4301 Jones Bridge Road. Bethesda.

MD 20814
Pollard, Thomas D., Salk Institute for Biological Studies, 10010 N.

Torrey Pines Road, La Jolla, CA 92037

Porter, Beverly H., 5542 Windysun Court, Columbia, MD 21045
Porter, Mary E., University of Minnesota, Department of Cell Biology

and Neuroanatomy. 4-135 Jackson Hall, 321 Church Street SE,

Minneapolis, MN 55455
Potter, David D., Harvard Medical School, Department of

Neurobiology, 25 Shattuck Street, Boston. MA 021 15
Potts, William T., University of Lancaster. Department of Biology,

Lancaster, England
Powers, Maureen K., University of California, Department of

Molecular & Cellular Biology. Life Sciences Addition, Berkeley, CA

94720

Prendergast. Robert A., 38 Pondlet Place, Falmouth. MA 02540
Prior, David J., Northern Arizona University. Arts and Sciences Dean's

Office. Box 5621. Flagstaff, AZ 8601 1
Prusch. Robert D., Gonzaga University, Department of Life Sciences.

Spokane, WA 99258
Purves. Dale. Duke University Medical Center. Department of

Neurobiology, Box 3209. 101 -I Bryan Research Building. Durham,

NC 27710

Quigley, James P., The Scnpps Research Institute, Department of
Vascular Biology, 10550 N. Torrey Pines Road VB-1, La Jolla, CA
92037

R78 Annual Report

Rabb, Irving W., 1010 Memorial Drive, Cambridge. MA 02138
Rabin, Harvey, 1 102 Ralston Road. Rockville. MD 20852
Rabinowitz, Michael B., Marine Biological Laboratory, 7 MBL Street.

Woods Hole, MA 02543
Rafferty, Nancy S., Marine Biological Laboratory, 7 MBL Street,

Woods Hole, MA 02543
Rakowski, Robert F., Finch University of Health Sciences, The

Chicago Medical School, Department of Physiology and Biophysics,

3333 Greenbay Road. N. Chicago, 1L 60064
Ramon, Fidel, Universidad Nacional Autonoma de Mexico, Division

EStreet Posgrado E Invest., Facultad de Medicina, 04510, D.F .

Mexico
Rastetter, Edward B., Marine Biological Laboratory, The Ecosystems

Center. Woods Hole. MA 02543
Rebhun, Lionel I., University of Virginia. Department of Biology,

Gilmer Hall 45, Charlottesville, VA 22901
Reddan, John R., Oakland University. Department of Biological

Sciences. Rochester. MI 48309-4401
Reese, Thomas S., National Institutes of Health, NINDS, Building 36,

Room 2A29, Bethesda, MD 20892
Reinisch, Carol L., Marine Biological Laboratory, 7 MBL Street,

Woods Hole, MA 02543

Rickles, Frederick R.. 2633 Danforth Lane. Decatur, GA 30033
Rieder, Conly L., Wadsworth Center. Division of Molecular Medicine.

P.O. Box 509, Albany, NY 12201-0509
Riley, Monica, Marine Biological Laboratory, 7 MBL Street, Woods

Hole, MA 02543
Ripps, Harris, University of Illinois at Chicago, Department of

Ophthalmology/Visual Sciences, 1855 West Taylor Street. Chicago,

IL60612
Rjtchie, J. Murdoch, Yale University School of Medicine, Department

of Pharmacology, 333 Cedar Street, New Haven, CT 06510
Rome, Lawrence C., University of Pennsylvania, Department of

Biology, Philadelphia, PA 19104
Rosenbluth, Jack, New York University School of Medical,

Department of Physiology and Biophysics, RR 714. 400 East 34ih

Street. New York, NY 10016
Rosenbluth, Raja, Simon Fraser University, Institute of Molecular

Biology and Biochemistry, Burnaby, BC V5A 1S6. Canada
Rosenfield, Allan, Columbia University School of Public Health, 600

West 168th Street, New York, NY 10032-3702

Rosenkranz, Herbert S., 130 Desoto Street, Pittsburgh, PA 15213-2535
Ross, William N., New York Medical College, Department of

Physiology, Valhalla, NY 10595
Roth, Jay S., 26 Huettner Road, P. O. Box 692, Woods Hole, MA

02543-0692
Rottenfusser, Rudi, Marine Biological Laboratory, 7 MBL Street,

Woods Hole. MA 02543
Rowland, Lewis P., Neurological Institute. 710 West 168th Street, New

York. NY 10032
Ruderman, Joan V., Harvard Medical School, Department of Cell

Biology, 240 Longwood Avenue, Boston. MA 021 15
Rummel, John D., NASA Headquarters, Office of Space Science,

Washington, D.C. 20546
Rushforth, Norman B., Case Western Reserve University. Department

of Biolo;jy, Ci.-veland, OH 44106
Russell-Hunter, V. illiam D., 711 Howard Street, Easton, MD 21601-

3934

Saffo, Mary Beth, AM ia State University West, Life Science
Department, MC 2352. I'.O. Box 37100, Phoenix, AZ 85069-7100

Salama, Guy, University of Pittsburgh, Department of Physiology,
Pittsburgh. PA 15261

Salmon, Edward D., University of North Carolina, Department of
Biology. CB 3280. Chapel Hill, NC 27514

Salyers, Abigail, University of Illinois. Department of Microbiology,

407 South Goodwin Avenue, Urbana. IL 61801
Salzherg, Brian M., University of Pennsylvania School of Medicine,

Department of Neuroscience, 215 Stemmler Hall. Philadelphia, PA

19104-6074
Sanger, Jean M., University of Pennsylvania School of Medicine.

Department of Anatomy, 36th and Hamilton Walk, Philadelphia. PA

19104
Sanger, Joseph W., University of Pennsylvania Medical Center,

Department of Cell and Developmental Biology. 36th and Hamilton

Walk. Philadelphia, PA 19104-6058

Saunders, John W. Jr.. P.O. Box 3381, Waquoit. MA 02536
Schachman. Howard K., University of California. Molecular and Cell

Biology Department, 229 Stanley Hall, #3206, Berkeley, CA 94720-

3206
Schatten, Gerald P., Oregon Health Sciences University, Oregon

Regional Primate Research Center, 505 N.W. 185th Avenue.

Beaverton, OR 97006
Schatten, Heide, University of Wisconsin, Department of Zoology,

Madison, WI 53706
Schmeer, Arlene C., Mercenene Cancer Research Institute, 790

Prospect Street, New Haven, CT 0651 1
Schuel. Herbert, SUNY at Buffalo. Department of Anatomy/Cell

Biology, Buffalo, NY 14214
Schwartz, James H., New York State Psychiatric Institute, Research

Annex, 722 West 168th Street, 7th floor. New York. NY 10032
Schwartz, Lawrence, University of Massachusetts. Department of

Biology. Morrill Science Center. Amherst, MA 01003
Schweitzer, A. Nicola, Brigham and Women's Hospital. Immunology

Division, Department of Pathology, 221 Longwood Avenue. LMRC

521. Boston. MA 02115
Segal. Sheldon J., The Population Council. One Dag Hammarskjold

Plaza. New York, NY 10036
Senft, Stephen Lamont, Yale University,

Neuroengineering/Neuroscience Center, P.O. Box 208205, New

Haven. CT 06520-8205
Shanklin, Douglas R., University of Tennessee. Department of

Pathology, Room 576. 800 Madison Avenue, Memphis. TN 381 17
Shashar, Nadav, The Interuniversity Institute of Eilat. P.O. Box 469,

Eilat 88103. Israel
Shashoua, Victor E., Harvard Medical School. Ralph Lowell Labs.

McLean Hospital. I 15 Mill Street, Belmont. MA 02178
Shaver, Gaius R., Marine Biological Laboratory. The Ecosystems

Center, Woods Hole, MA 02543
Shaver, John R., Michigan State University, Department of Zoology,

East Lansing, MI 48824
Sheetz, Michael P., Duke University Medical Center, Department of

Cell Biology, Bx 3709, 388 Nanaline Duke Building, Durham. NC

27710
Shepro, David, Boston University, CAS Biology, 5 Cummington Street,

Boston, MA 02215
Shimomura, Osamu, Marine Biological Laboratory. 7 MBL Street,

Woods Hole, MA 02543

Shipley, Alan M., P.O. Box 943. Forestdale. MA 02644
Silver, Robert B., Marine Biological Laboratory. 7 MBL Street. Woods

Hole. MA 02543
Siwicki, Kathleen K., Swarthmore College, Biology Department, 500

College Avenue. Swarthmore, PA 19081-1397
Skinner. Dorothy M., 24 Gray Lane, Falmouth, MA 02540
Sloboda, Roger D., Dartmouth College. Department of Biological

Science, 6044 Oilman, Hanover, NH 03755-1893
Sluder. Greenfield, University of Massachusetts Medical School, Room

324, 377 Plantation Street, Worcester, MA 01605

Members of the Corporation R79

Smith. Peter J.S.. Marine Biological Laboratory, 7 MBL Street, Woods

Hole, MA 02543
Smith, Stephen J., Stanford University School of Medicine. Department

of Molecular and Cellular Physiology. Beckman Center. Stanford. CA

94305
Smolowitz, Roxanna S., Marine Biological Laboratory, 7 MBL Street.

Woods Hole. MA 02543
Sogin. Mitchell L., Marine Biological Laboratory. 7 MBL Street,

Woods Hole. MA 02543
Sorenson, Martha M.. Cidade Universitaria-UFRJ. Department

Bioquimica Medica-ICB, 21941-590 Rio de Janerio. Brazil
Speck, \\illiam T.. Massachusetts General Hospital. 55 Fruit Street.

Boston, MA 021 14
Spector, Abraham, Columbia University. Department of

Ophthalmology. 630 West 168th Street, New York, NY 10032
Speksnijder, Johanna E., University of Groningen. Department of

Genetics, Kerklaan 30, 975 1 NN Haren, The Netherlands
Spray. David C., Albert Einstein College of Medicine. Department of

Neuroscience, 1300 Moms Park Avenue, Bronx, NY 10461
Spring. Kenneth R., National Institutes of Health. 10 Center Drive.

MSC 1598. Building 1(1. Room 6N260. Bethesda, MD 20892-1603
Steele, John H., Woods Hole Oceanographic Institution. Woods Hole.

MA 02543
Steinacker, Antoinette, University of Puerto Rico, Instituet of

Neurobiology. 201 Boulevard Del Valle. San Juan, PR 00901
Steinberg, Malcolm. Princeton University, Department of Molecular

Biology, M-18 Moffett Laboratory, Princeton. NJ 08544-1014
Stemmer, Andreas C., Institut fur Robotik. ETH-Sentrum. 8092 Zurich.

Switzerland
Stenflo, Johan, University of Lund. Department of Clinical Chemistry,

Malmo General Hospital. S-205 02 Malmo. Sweden
Stetten, Jane Lazarow. 4701 Willurd Avenue. #1413, Chevy Chase,

MD 20815-4627
Steudler. Paul A., Marine Biological Laboratory. The Ecosystems

Center. Woods Hole. MA 02543
Stokes, Darrell R., Emory University. Department of Biology, 1510

Clifton Road NE. Atlanta, GA 30322-1 100
Stommel, Elijah W., Darmouth Hitchcock Medical Center, Neurology

Department, Lebanon. NH 03756
Stracher, Alfred, SUNY Health Science Center. Department of

Biochemistry, 450 Clarkson Avenue, Brooklyn, NY 1 1203
Strumwasser, Felix, P.O. Box 2278, East Falmouth, MA 02536-2278
Stuart, Ann E.. 1818 North Lakeshore Drive. Chapel Hill, NC 27514
Sugimori, Mutsuyuki, New York University Medical Center.

Department of Physiology and Neuroscience, Room 442. 550 First

Avenue, New York. NY 10016
Summers, William C., Western Washington University, Huxley College

of Environmental Studies. Bellingham. WA 982259 1 8 1
Suprenant, Kathy A., University of Kansas. Department of Physiology

and Cell Biology, 4010 Haworth Hall, Lawrence, KS 66045
Swenson, Katherine I., Duke University Medical Center, Department of

Molecular Cancer Biology, Box 3686, Durham. NC 27710
Sydlik, Mary Anne, Hope College, Peale Science Center. 35 East 1 2th

St./PO Box 9000, Holland, MI 49422
Szent-Gyorgyi, Andrew G., Brandeis University. Molecular and Cell

Biology, 415 South Street, Waltham, MA 02454-91 10

Tamm, Sidney L., Boston University. CAS Biology, 5 Cummington

Street, Boston, MA 022 1 5
Tanzer, Marvin L., University of Connecticut School of Dental

Medicine, Department of Biostructure and Function, Farmington. CT

06030-3705
Tasaki, Ichiji, National Institutes of Health, NIMH. Laboratory of

Neurobiology, Building 36, Room 2B-16, Bethesda, MD 20892

Taylor, D. Lansing, Carnegie Mellon University. Center for

Flurorescence Research. 4400 Fifth Avenue. Pittsburgh, PA 15213
Taylor, Edwin W., University of Chicago. Department of Molecular

Genetics. 920 E. 58th Street. Chicago. IL 60637
Teal, John M., Woods Hole Oceanographic Institution. Department of

Biology. Woods Hole. MA 02543
Telfer, William H., University of Pennsylvania, Department of Biology,

Philadelphia. PA I1) 1 04
Telzer, Bruce, Pomona College. Department of Biology. Thille

Building. 175 West bin Street. Claremont. CA 91711
Terasaki. Mark, University of Connecticut Health Center. Department

of Physiology. 263 Farmington Avenue, Farmington. CT 06032
Townsel, James G., Meharry Medical College. Department of

Physiology. 1005 DB Todd Boulevard. Nashville. TN 37208
Travis, David M., 19 High Street. Woods Hole, MA 02543-1221
Treistman, Steven N., University of Massachusetts Medical Center,

Department of Pharmacology, 55 Lake Avenue North, Worcester, MA

01655

Trigg. D. Thomas, One Federal Street, 9th Floor. Boston. MA 022 1 1
Troll, \Valter, NYU Medical Center, Department of Environmental

Medicine. 550 First Avenue. New York, NY 10016
Troxler, Robert F., Boston University School of Medicine. Department

of Biochemistry. 80 East Concord Street. Boston. MA 021 18
Tucker, Edward B., Baruch College. CUNY. Department of Natural

Sciences, 17 Lexington Avenue, New York, NY 10010
Turner, Ruth D., Harvard University. Museum of Comparative

Zoology. Mollusk Department, Cambridge. MA 02138
Tweedell, Kenyon S., University of Notre Dame. Department of

Biological Sciences. Notre Dame. IN 46556-0369
Tykocinski, Mark L., Case Western Reserve University, Institute of

Pathology, 2085 Adelbert Road. Cleveland, OH 44106
Tytell, Michael, Wake Forest University. Bowman Gray School of

Medicine. Department of Anatomy and Neurobiology. Winston-

Salem. NC 27157

Ueno, I In osln. Kyoto University, AGR Chemistry, Faculty of
Agriculture, Sakyo, Kyoto 606-8502, Japan

Valiela, Ivan, Boston University Marine Program, Marine Biological

Laboratory, Woods Hole, MA 02543
Vallee, Richard, University of Massachusetts Medical Center.

Worcester Foundation Campus. 222 Maple Avenue. Shrewsbury. MA

01545

Valois, John J., 420 Woods Hole Road, Woods Hole. MA 02543
Van Dover, Cindy Lee, The College of William and Mary, Biology

Department, 328 Millington Hall, Williamsburg, VA 23187
Van Holde, Kensal E., Oregon State University, Biochemistry and

Biophysics Department, Corvallis, OR 97331-7503
Vogl, Thomas P., Environmental Research Institute of Michigan, 1101

Wilson Boulevard. Arlington. VA 22209

Waimvright, Norman R., Marine Biological Laboratory, 7 MBL Street,

Woods Hole, MA 02543
Waksman, Byron H., New York University Medical Center,

Department of Pathology, 550 First Avenue. New York. NY 10016
Wall, Betty, 9 George Street. Woods Hole, MA 02543
Wang, Hsien-Yu, State University of New York, University Medical

Center. Physiology and Biophysics-HSC. Stony Brook. NY 1 1794-

8633
Wangh, Lawrence J., Brandeis University. Department of Biology, 415

South Street. Waltham. MA 02254
Warner, Robert C., 1609 Temple Hills Drive. Laguna Beach. CA

9265 1

R80 Annual Report

Warren, Leonard, Wistar Institute. 36th and Spruce Streets,

Philadelphia. PA 19104
Waterbury, John B., Woods Hole Oceanographic Institution,

Department of Biology. Woods Hole, MA 02543
Waxman, Stephen G., Yale Medical School. Neurology Department,

333 Cedar Street. P.O. Box 208018. New Haven. CT 06510
Webb, H. Marguerite, 184 Chestnut Street. Foxhoro. MA 02035-1548

(deceased 1999)
Weber, Annemarie, University of Pennsylvania School of Medicine.

Department of Biochemistry and Biophysics. Philadelphia, PA 19066
Weeks, Janis C., University of Oregon, Institute of Neuroscience.

Eugene. OR 97403-1254
Weidner, Earl, Louisiana State University, Department of Biological

Sciences, 508 Life Sciences Building, Baton Rouge, LA 70803-1715
Weiss, Alice Sara, 105 University Boulevard West. Silver Spring. MD

20901
Weiss, Dieter G., University of Rostock. Institute of Zoology. D- 18051

Rostock. Germany
Weiss, Leon P., University of Pennsylvania School of Veterinary

Medicine, Department of Animal Biology. Philadelphia, PA 19104
Weiss, Marisa C., Paoli Memorial Hospital, Department of Radiation

Oncology. 255 W. Lancaster Avenue. Paoli. PA 19301
Weissmann, Gerald, New York University Medical Center, Department

of Medicine/Division Rheumatology, 550 First Avenue, New York,

NY 10016
Westerfield, Monte, University of Oregon. Institute of Neuroscience,

Eugene, OR 97403
Whittaker, J. Richard, University of New Brunswick. Department of

Biology, BS 4511, Frederiction, NB E3B 6E1. Canada
Wiesel, Torsten N., Rockefeller University, 1230 York Avenue. New

York. NY 10021
Wilkens, Lon A., University of Missouri, Department of Biology, 8001

Natural Bridge Road, St. Louis, MO 63121-4499
Wilson, Darcy B., Torrey Pines Institute, 3550 General Atomics Court,

Building 2, Room 138, San Diego, C A 92121
Wilson, T. Hastings, Harvard Medical School, Department of

Physiology, 25 Shattuck Street, Boston. MA 021 15
Witkovsky, Paul. NYLI Medical Center, Department of Ophthalmology,

550 First Avenue, New York. NY 10(116

MBL Associates

Wittenberg, Beatrice, Albert Einstein College of Medicine, Department

of Physiology and Biophysics, Bronx, NY 10461
Wittenberg. Jonathan B., Albert Einstein College of Medicine,

Department of Physiology and Biophysics. Bronx, NY 10461
Wolken, Jerome J., Carnegie Mellon University. Department of

Biological Sciences, 440 Fifth Avenue. Pittsburgh, PA 15213

(deceased 1999)
Wonderlin, William ¥., West Virginia University, Pharmacology and

Toxicology Department, Morgantown, WV 26506
Worden, Mary Kate, University of Virginia, Department of

Neuroscience. McKim Hall Box 230, Charlottesville, VA 22908
Worgul, Basil V., Columbia University, Department of Ophthalmology,

630 West 168 Street, New York. NY 10032
Wu, Chau Hsiung, Northwestern University Medical School,

Department of Pharmacology (S215), 303 East Chicago Avenue,

Chicago. IL 60611-3008
Wyttenbach, Charles R., University of Kansas, Biological Sciences

Department. 2045 Haworth Hall. Lawrence. KS 66045-2106

Yen, Jay Z., Northwestern University Medical School, Department of
Pharmacology, Chicago. IL 60611

Zacks, Sumner I., 65 Saconesset Road, Falmouth, MA 02540-1851

(deceased 2000)
Zakon, Harold H., University of Texas, Section of Neurobiology,

School of Life Science, Austin. TX 78712
Zigman, Seymour, Marine Park Condominiums. 174 Queen Street. Unit

10-F. Falmouth. MA 02540
Zigmond, Michael J., University of Pittsburgh, S-526 Biomedical

Science Tower, 3500 Terrace Street, Pittsburgh. PA 15213
Zimmerherg, Joshua J., National Institutes of Health, LCMB. NICHD,

Building 10. Room 10D14, 10 Center Drive, Bethesda. MD 20892
Zottoli, Steven J., Williams College. Department of Biology,

Williamstown, MA 01267
Zucker, Robert S., University of California. Neurohiology Division.

Molecular and Cellular Biology Department, Berkeley, CA 94720

Executive Board

Ruth Ann Laster. President

Jack Pearce. Vice President

Kitty Brown. Treasurer

Molly Cornell, Secretary

Elizabeth Farnham, Membership Chair

Tammy Smith Amon

Duncan Aspinwall

Barbara Atwood

Kitty Brown

Julie Child

Seymour Cohen

Michael Fenlon

Sallie Giffen

Alice Knowles

Rebecca Lash

Cornelia Hanna McMurlne

Joan Pearl man

Virginia R. Reynolds

Volker Ulbrich

John Valois

Associates Liaison/Gift Sliop Coordinator
Kendall B. Bohr

Supporting Associate

Mrs. George H. A. Clowes

Dr. and Mrs. James D. Ebert

Mr. and Mrs. David Fausch

Mrs. Janet F. Gillette

Drs. Luigi and Elaine Mastroianni

Ms. Linda Sallop and Mr. Michael Fenlon

Mrs. Anne W. Sawyer

Dr. John Tochko and Mrs. Christina Myles-Tochko

Mr. and Mrs. John J. Valois

Mr. and Mrs. Leslie J. Wilson

Sustaining Associate

Dr. and Mrs. James J. Ferguson. Jr.
Mr. and Mrs. Lon Hocker

Members of the Corporation R81

Mr. Robert A. Jaye

Dr. and Mrs. Edward F. MacNichol. Jr.

Fainilv Membership

Dr. Frederick W. Ackroyd

Dr. and Mrs. Edward A. Adelberg

Dr. and Mrs. Dean C. Allard, Jr.

Mr. and Mrs. Douglas F. Allison

Drs. Peggy and Fred Alsup

Drs. James and Helene Anderson

Dr. and Mrs. Samuel C. Armstrong

Mr. and Mrs. Duncan P. Aspinwall

Mr. and Mrs. Donald R. Aukamp

Mr. and Mrs. John M. Baitsell

Mr. and Mrs. David Bakalar

Mr. and Mrs. William L. Banks

Dr. and Mrs. Robert B. Barlow, Jr.

Mr. and Mrs. John E. Barnes

Dr. and Mrs. Robert M. Berne

Drs. Harriet and Alan Bernheimer

Mr. and Mrs. Robert O. Bigelow

Dr. and Mrs. Edward G. Boettiger

Mr. and Mrs. Kendall B. Bohr

Dr. and Mrs. Thomas A. Borgese

Mr. and Mrs. Richard M. Bowen

Dr. and Mrs. Francis P. Bowles

Dr. and Mrs. John B. Buck

Dr. and Mrs. John E. Burns

Mr. and Mrs. William O. Burwell

Mr. and Mrs. G. Nathan Calkins, Jr.

Mr. and Mrs. D. Bret Carlson

Prof, and Mrs. James F. Case

Dr. and Mrs. Richard L. Chappell

Dr. and Mrs. Frank M. Child

Mr. and Mrs. Thomas Claflin

Dr. and Mrs. Arnold M. Clark

Mrs. LeRoy Clark

Mr. and Mrs. James Cleary

Dr. and Mrs. Laurence P. Cloud

Mr. and Mrs. Harrington T. Collins

Dr. and Mrs. Neal W. Cornell (Dr. Cornell

deceased, 2000)
Mr. and Mrs. Norman C. Cross
Dr. and Mrs. John M. Cummings
Mr. and Mrs. Bruce G. Daniels
Mr. and Mrs. Joel P. Davis
Mr. and Mrs. Richard C. Dierker
Mr. and Mrs. F. Gerald Douglass
Dr. and Mrs. Arthur Brooks DuBois
Dr. and Mrs. Michael J. Fishbein
Mr. and Mrs. Harold Frank
Mr. and Mrs. Howard G. Freeman
Dr. and Mrs. Robert A. Frosch
Dr. and Mrs. John J. Funkhouser
Dr. and Mrs. Mordecai L. Gabriel
Dr. and Mrs. Sydney Gellis
Dr. and Mrs. James L. German. Ill
Dr. and Mrs. Harold S. Ginsberg
Dr. and Mrs. Murray Glusman
Drs. Alfred and Joan Goldberg
Mrs. Mary L. Goldman
Mr. and Mrs. Charles Goodwin, III
Dr. and Mrs. Philip Grant

Mr. and Mrs. Anthony D. Green

Dr. and Mrs. Thomas C. Gregg

Dr. Newton H. Gresser

Dr. and Mrs. Antoine F. O. Hadamard

Mr. and Mrs. Peter A. Hall

Dr. and Mrs. Harlyn O. Halvorson

Drs. Alexander and Carol Hannenberg

Dr. and Mrs. Richard Bennet Harvey

Dr. and Mrs. J. Woodland Hastings

Mr. and Mrs. Gary G. Hayward

Dr. and Mrs. Howard H. Hiatt

Mr. and Mrs. David Hibbitt

Dr. and Mrs. John E. Hobbie

Drs. Francis C. G. Hoskin and Elizabeth M.

Farnham

Dr. and Mrs. Robert J. Huettner
Dr. and Mrs. Shinya Inoue
Dr. and Mrs. Kurt J. Isselbacher
Dr. and Mrs. Gary Jacobson
Mrs. Mary D. Janney
Mr. and Mrs. DeWitt C. Jones. Ill
Dr. and Mrs. Benjamin Kanuner
Mr. and Mrs. Paul W. Knaplund
Mr. and Mrs. A. Sidney Knowles. Jr.
Sir and Lady Hans Kornberg
Dr. and Mrs. S. Andrew Kulin
Mr. and Mrs. Trevor Lambert
Dr. and Mrs. George M. Langford
Dr. and Mrs. Leonard Laster
Dr. and Mrs. Hans Laufer
Mr. William Lawrence and Mrs. Barbara

Buchanan

Dr. and Mrs. Berton J. Leach
Mr. and Mrs. Stephen R. Levy
Mr. and Mrs. Robert Livingstone. Jr.
Dr. and Mrs. Laszlo Lorand
Mr. and Dr. Bernard Manuel
Mr. and Mrs. Joseph C. Martyna
Mr. and Mrs. Frank J. Mather. Ill
Mr. and Mrs. John E. Matthews
Dr. and Mrs. Robert T. McCluskey
Mr. Paul McGonigle
Dr. and Mrs. Jerry M. Melillo
Mr. and Mrs. Wesley J. Merritt
Mr. and Mrs. Richard Meyers
Mr. and Mrs. Charles A. Mitchell
Dr. and Mrs. Merle Mi/ell
Dr. and Mrs. Charles H. Montgomery
Mr. and Mrs. Stephen A. Moore
Mr. and Mrs. Charles F. Murphy
Dr. and Mrs. John E. Naugle
Dr. Pamela Nelson and Mr. Christopher

Olmsted

Mr. and Mrs. Frank L. Nickerson
Dr. and Mrs. Clifford T. O'Connell
Mr. and Mrs. James J. O'Connor
Mr. and Mrs. David R. Palmer
Mr. and Mrs. Robert Parkinson
Mr. and Mrs. Richard M. Paulson. Jr.
Dr. and Mrs. John B. Pearce
Mr. and Mrs. William J. Pechilis
Mr. and Mrs. John B. Pen
Dr. and Mrs. Courtland D. Perkins
Dr. and Mrs. Philip Person

Mr. and Mrs. Frederick S. Peters

Mr. and Mrs. George H. Plough

Dr. and Mrs. Aubrey Pothier, Jr.

Dr. and Mrs. Carl A. Price

Mr. and Mrs. Allan Putnam

Dr. and Mrs. Lionel I. Rebhun

Dr. and Mrs. George T. Reynolds

Dr. and Mrs. Harris Ripps

Ms. Jean Roberts

Drs. Priscilla and John Roslansky

Mr. and Mrs. John D. Ross

Dr. and Mrs. John W. Saunders, Jr.

Dr. and Mrs. R. Walter Schlesinger

Mr. and Mrs. Harold H. Sears

Mr. John Seder and Ms. Frances Plough

Dr. and Mrs. Sheldon J. Segal

Dr. and Mrs. David Shepro

Mr. and Mrs. Bertram R. Silver

Mr. and Mrs. Jonathan O. Simonds

Drs. Frederick and Marguerite Smith

Dr. and Mrs. William K. Stephenson

Mr. and Mrs. E. Kent Swift, Jr.

Mr. and Mrs. Gerard L. Swope, III

Mr. Norman N. Tolkan

Dr. and Mrs. Walter Troll

Mr. and Mrs. Volker Ulbrich

Ms. Susan Veeder

Drs. Claude and Dorothy Villee

Dr. and Mrs. Gerald Weissmann

Dr. and Mrs. Paul S. Wheeler

Dr. Martin Keister White

Mr. and Mrs. Geoffrey G. Whitney, Jr.

Mr. and Mrs. Lynn H. Wilke

Dr. and Mrs. T. Hastings Wilson

Mr. and Mrs. Dick Yeo

Dr. and Mrs. Sumner I. Zacks (Dr. Zacks

deceased. 2000)
Dr. Linda and Mr. Erik Zettler

Individual Membership

Mrs. Jean E. Ahearn

Mr. Henry Albers

Dr. Nina S. Allen

Mrs. Tammy Amon

Mr. Dean N. Arden

Mrs. Ellen Prosser Armstrong

Mrs. Kimball C. Atwood. Ill

Dr. Serena Baccetti

Mr. Everett E. Bagley

Dr. Millicent Bell

Mr. C. John Berg

Dr. Thomas P. Bleck

Ms. Avis Blomberg

Mr. Theodore A. Bonn

Mr. James V. Bracchitta

Mrs. Jennie P. Brown

Mrs. M. Kathryn S. Brown

Dr. Robert H. Broyles

Ms. Henriete Bull

Dr. Alan H. Burghauser

Mrs. Barbara Gates Burwell

Mr. Bruce E. Buxton

Mr. Patrick J. Calie

R82 Annual Report

Mrs. Anne M. Campbell

Dr. Graciela C. Candelas

Mr. Frank C. Camtenuto

Dr. Roben H. Carrier

Mrs. Patricia A. Case

Ms. Mia D. Champion

Dr. Sallie Chisholm

Mrs. Octavia C. Clement

Mr. Allen W. Clowes

Mrs. Margaret H. Cohurn

Dr. Seymour S. Cohen

Ms. Anne S. Concannon

Prof. D. Eugene Copeland

Dr. Vincent Cowling

Mrs. Marilyn E. Crandall

Ms. Dorothy Crossley

Ms. Helen M. Crossley

Mrs. Villa B. Crowell

Mr. Norman Dana

Dr. Morton Davidson

Mrs. Elizabeth M. Davis

Ms. Carol Reimann DeYoung

Mrs. Shirley Dierolf

Mr. David L. Donovan

Ms. Su/.anne Droban

Mr. Roy A. Duffus

Mrs. Charles Eastman

Dr. Frank Egloff

Mr. Raymond Eliott

Ms. Judy Ernst

Mrs. Ruth Alice Fitz

Mr. Robert Fitzpatrick

Ms. Sylvia M. Flanagan

Mr. John W. Folino, Jr.

Dr. Krystyna Frenkel

Mr. Paul J. Freyheit

Mrs. Ruth E. Fye

Mr. Joseph C. Gallagher

Miss Eleanor Garfield

Mrs. Ruth H. Garland

Mr. John Garnett

Ms. Sallie A. Giffen

Mr. Charles Gifford

Mrs. James R. Glazebrook

Mr. Michael P. Goldring

Mrs. Phyllis Goldstein

Mrs. DeWitt S. Goodman

Ms. Muriel Gould

Mrs. Rose Grant

Mrs. Jeanne B. Griffith

Mrs. Valerie A. Hall

Dr. Peter J. Hamre

Ms. Mary Eli/.aheth Hamstrom

Ms. Elizabeth E. Hathaway

Dr. Robert R. Haiilun h

Mrs. Jane M. Heakl

Mrs. Nathan Hir.schfeld

Mr. Roger W. Hubhell

Miss Elizabeth B. Jackson

Mr. Raymond L. Jewett

Mrs. Barbara W. Jones

Mrs. Megan H. Jones

Mrs. Joan T. Kanwisher

Mrs. Sally Karush

Mrs. Marcella Katz

Ms. Patricia E. Keoughan

Dr. Peter N. Kivy

Dr. Bruno P. Kremer

Mr. Bernard H. Labitt

Mrs. Janet W. Larcom

Dr. Marian E. LeFevre

Dr. Mortimer Levitz

Mr. Edwin M. Libbin

Mr. Lennart Lindberg

Mrs. Barbara C. Little

Mrs. Sarah J. Loessel

Mrs. Ermine W. Lovell

Mr. Richard C. Levering

Mrs. Victoria H. Lowell

Mrs. Margaret M. MacLeish

Ms. Anne Camille Maher

Mrs. Annemarie E. Mahler

Mr. Patrick J. Mahoney

Dr. Saul Malkiel

Ms. Diane Maranchie

Mr. Daniel R. Martin

Dr. Miriam Jacob Mauzerall

Mrs. Mary Hartwell Mavor

Mrs. Nella W. McElroy (deceased 1999)

Dr. Susan Gerbi Mcllwam

Ms. Mary W. McKoan

Ms. Jane A. McLaughlin

Ms. Louise McManus

Ms. Cornelia Hanna McMurtrie

Mrs. Ellen L. Meigs

Mr. Ted Melillo

Martin Mendelson. M.D.. Ph.D.

Ms. Carmen Merryman

Mrs. Grace S. Metz

Mrs. Mary G. Miles

Mrs. Florence E. Mixer

Mr. John T. Moakley

Mr. Lawrence A. Monte

Mrs. Mary E. Montgomery

Ms. Cynthia Moor

Mr. Alan F. Morrison

Dr. M. Patricia Morse

Mrs. Eleanor M. Nace

Mr. William G. Neall

Mrs. Anne Nelson

Mr. Edmund F. Nolan

Ms. Catherine N. Norton

Mr. John J. O'Connor (deceased 1999)

Dr. Renee Bennett O'Sullivan

Miss Carolyn L. Parmenter

Mrs. Dolores Patch-Wing

Ms. Joan Pearlman

Mr. Raymond W. Peterson

Ms. Victoria A. Powell

Ms. Elizabeth T. Price

Ms. Dianne Purves

Mrs. Julia S. Rankin

Mr. Fred J. Ravens. Jr.

Ms. Anecia Kathy Regis

Ms. Mary W. Rianhard

Dr. Renato A. Ricca (deceased 1<W)

Dr. Mary Elizabeth Rice

Dr. Monica Riley

Master Alexander Meigs Rives

Mrs. Lola E. Robertson

Mrs. Ruth J. Robinson

Mrs. Arlene Rogers

Mrs. Wendy E. Rose

Ms. Hilde Rosenthal

Mrs. Atholie K. Rosett

Dr. Virginia F. Ross

Dr. John D. Rummel

Mr. Raymond A. Sanborn

Ms. Elaine Schott

Mrs. Elsie M. Scott

Sea Education Association. Inc.

Dr. Cecily C. Selby

Mrs. Deborah G. Senft

Mrs. Charlotte Shemin

Ms. Enid K. Sichel

Dr. Jeffrey D. Silberman

Mrs. Phyllis J. Silver

Mrs. Cynthia C. Smith

Mrs. Louise M. Specht

Dr. Guy L. Steele, Sr.

Dr. Robert E. Steele

Mrs. Eleanor Steinbach

Mrs. Jane Lazarow Stetten

Dr. Maurice Sussman

Mr. Albert H. Swain

Mr. James K. Taylor

Mr. Emil D. Tietje, Jr.

Mrs. Alice Todd

Mr. Arthur D. Traub

Mr. D. Thomas Trigg

Ms. Natalie Trousof

Mrs. Frances W. Tytell

Ms. Ciona Ulbrich

Dr. Kensal E. van Holde

Ms. Sylvia Vatuk

Mr. Lee D. Vincent

Mr. Arthur D. Voorhis

Mrs. Eve Warren

Mr. John T. Weeks

Ms. Lillian Wendortt

Dr. William M. Wheeler

Ms. Mabel Y. Whelpley

Mrs. Barbara Whitehead

Mrs. Ava Whittemore

Mrs. Joan R. Wickersham

Mrs. Clare M. Wilber

Ms. Nancy Woitkoski

Ms. Marion K. Wright

Mrs. Dorothy M. York

Mrs. Bunnie Rose Zigman

Mrs. Donald J. Zinn

MBL Gift Shop Volunteers

Marion Adelberg
Barabara Atwood
Caroline Banks
Harriet Bernheimer
Avis Blomberg
Gloria Borgese
Kitty Brown
Jewel Cobb

Members of the Corporation R83

Janet Daniels
Carol De Young
Fran Eastman

Helen Murphy
Jack Pearce
Bertha Person

Grace Witzell
Bunnie Rose Zigman

Alma Ebert
Jane Foster

Margareta Pothier
Liz Price

MBL Summer Tour Guides

Becky Glazebrook
Muriel Gould

Julie Rankin
Arlene Rogers

Gloria Borgese
Frank Child

Barbara Grossman

Lil Saunders

Julie Child

Jean Halvorson
Hanna Hastings
Sally Karush
Marcella Katz

Louise Spechl
Cynthia Smith
Peggy Smith
Jane Stetten

Sears Crowell
Henry Dooley
Nancy Fraser
Sallie Giffen

Alice Knowles

Barbara Thomson

Lincoln Kraeuter

Evelyn Laufer

Alice Todd

Barbara Little

Elaine Troll

Steve Oliver

Winnie Mackey

Natalie Trousof

Julie Rankin

Diane Maranchie

Barbara Van Holde

Lola Robertson

Miriam Mauzerall
Mary Mavor
Jane McCormack
Louise McManus
Florence Mixer

Doris Van Keuren
Susan Veeder
Carol Ann Wagner
Mabel Whelpley
Clare Wilber

Arlene Rogers
Pucky Roslansky
Suzanne Thomas
Mary Ulbrich
John Valois

Lorraine Mizell

Betty Wilson

Margery Zinn

Certificate of Organization
Articles of Amendment
Bylaws

Certificate of Organization

Articles of Amendment

(On File in the Office of the Secretary of the Commonwealth)

No. 3170

We, Alpheus Hyatt, President, William Stanford Stevens, Treasurer, and William T.
Sedgwick, Edward G. Gardiner, Susan Mims and Charles Sedgwick Minot being a
majority of the Trustees of the Marine Biological Laboratory in compliance with the
requirements of the fourth section of chapter one hundred and fifteen of the Public
Statutes do hereby certify that the following is a true copy of the agreement of
association to constitute said Corporation, with the names of the subscribers thereto:

We, whose names are hereto subscribed, do. by this agreement, associate ourselves
with the intention to constitute a Corporation according to the provisions of the one
hundred and fifteenth chapter of the Public Statutes of the Commonwealth of Mas-
sachusetts, and the Acts in amendment thereof and in addition thereto.

The name by which the Corporation shall be known is
THE MARINE BIOLOGICAL LABORATORY.

The purpose for which the Corporation is constituted is to establish and maintain a
laboratory or station for scientific study and investigations, and a school for instruc-
tion in biology and natural history.

The place within which the Corporation is established or located is the city of Boston
within said Commonwealth.
The amount of its capital stock is none.

In Witness Whereof, we have hereunto set our hands, this twenty seventh day of
February in the year eighteen hundred and eighty-eight, Alpheus Hyatt, Samuel Mills,
William T- Sedgwick, Edward G. Gardiner, Charles Sedgwick Minot. William G.
Farlow, William Stanford Stevens, Anna D. Phillips, Susan Mims, B. H. Van Vleck.
That the first meeting of the subscribers to said agreemenl was held on the thirteenth
day of March in the year eighteen hundred and eighty-eight.

In Witness Whereof, we have hereunto signed our names, this thirteenth day nl March
in the year eighteen hundred and eighty-eight, Alpheus Hyatt, President, William
Stanford Stevens. Treasurer. Edward G. Gardiner. William T. Sedgwick, Susan Minis,
Charles Sedgwick Minot.
(Approved on March 20, 1SXS as follows.

I hereby certify that it appears upon an examination of the within written certificate
and the records o| ihc t_i']|ioi,iliun Jul\ submitted to my inspection, that the require-
ments of sections one, two and three of chapter one hundred and fifteen, and sections
eighteen, twenty and twenty-one ol chapter one hundred and six, of the Public
Statutes, have been complied with and I hereby approve said certificate this twentieth
day of March A.D. eighteen hundred and eighty-eight.

Charles Endicott
Commissioner of Corporations)

We, James D. Ebert, President, and David Shepro, Clerk of the Marine Biological
Laboratory, located at Woods Hole, Massachusetts 02543, do hereby certify that the
following amendment to the Articles of Organization of the Corporation was duly
adopted at a meeting held on August 15, 1975, as adjourned to August 29, 1975, by
vole of 444 members, being at least two-thirds of its members legally qualified to vote
in the meeting of the corporation:

Voted: That the Certificate of Organization of this corporation be and it hereby is

amended by the addition of the following provisions:

"No Officer, Trustee or Corporate Member of the corporation shall be personally
liable for the payment or satisfaction of any obligation or liabilities incurred as a result
of, or otherwise in connection with, any commitments, agreements, activities or
affairs of the corporation.

"Except as otherwise specifically provided by the Bylaws of the corporation, meet-
ings of the Corporate Members of the corporation may be held anywhere in the United
Slates.

"The Trustees of the corporation may make, amend or repeal the Bylaws of the
corporation in whole or in part, except with respect to any provisions thereof which
shall by law, this Certificate or the bylaws of the corporation, require action by the
Corporate Members."

The foregoing amendment will become effective when these articles of amendment
are filed in accordance with Chapter 180, Section 7 of the General Laws unless these
articles specify, in accordance with the vote adopting the amendment, a later effective
date not more than thirty days after such filing, in which event the amendment will
become effective on such later date-
In Witness whereof and Under the Penalties of Perjury, we have hereto signed our
n.uiiL-s this 2nd day of September, in the year 1975, James D. Ebert, President; David
Shepro, Clerk.

(Approved on October 24. 1975, as follows:

I hereby approve the within articles of amendment and. the filing fee in the amount
of $10 having been paid, said articles are deemed to have been filed with me this 24lh
day of October. 1975.

Paul Guzzi

Secretary of the Commonwealth)

Bylaws

(Revised August 7. 1992 and December 1(1. IW2)
ARTICLE I— THE CORPORATION

A. Name anil Pnrpiise. The name of the Corporation shall he The Marine Biolog-
ical Laboratory. The Corporation's purpose shall be to establish anil maintain a

KS4

Bylaws of the Corporation R85

laboratory or station for scientific study and investigation and a school for instruction
in biology and natural history.

B. Nondiscrimination. The Corporation shall not discriminate on the basis of age,
religion, color, race, national or ethnic origin, sex or sexual preference in its policies
on employment and administration or in its educational and other programs.

ARTICLE II— MEMBERSHIP

A. Members. The Members of the Corporation ("Members") shall consist of
persons elected by the Board of Trustees (the "Board"), upon such terms and
conditions and in accordance with such procedures, not inconsistent with law or these
Bylaws, as may be determined by the Board. At any regular or special meeting of the
Board, the Board may elect new Members. Members shall have no voting or other
rights with respect to the Corporation or its activities except as specified in these
Bylaws, and any Member may vote at any meeting of the Members in person only and
not by proxy. Members shall serve until their death or resignation unless earlier
removed with or without cause by the affirmative vote of two-thirds of the Trustees
then in office. Any Member who has retired from his or her home institution may.
upon written request to the Corporation, be designated a Life Member. Life Members
shall not have the right to vote and shall not be assessed for dues.

B. Meetings. The annual meeting of the Members shall be held on the Friday
following the first Tuesday in August of each year, at the Laboratory of the Corpo-
ration in Woods Hole, Massachusetts, at 9:30 a.m. The Chairperson of the Board shall
preside at meetings of the Corporation. If no annual meeting is held in accordance
with the foregoing provision, a special meeting may be held in lieu thereof with the
same effect as the annual meeting, and in such case all references in these Bylaws,
except in this Article II. B., to the annual meeting of the Members shall be deemed to
refer to such special meeting Members shall transact business as may properly come
before the meeting. Special meetings of the Members may be called by the Chair-
person or the Trustees, and shall be called by the Clerk, or in the case of the death,
absence, incapacity or refusal by the Clerk, by any other officer, upon written
application of Members representing at least ten percent of the smallest quorum of
Members required for a vote upon any matter at the annual meeting of the Members,
to be held at such time and place as may be designated.

C. Quorum. One hundred ( 1 00) Members shall constitute a quorum at any meeting.
Except as otherwise required by law or these Bylaws, the affirmative vote of a
majority of the Members voting in person at a meeting attended by a quorum shall
constitute action on behalf of the Members.

D. Notice of Meetings. Notice of any annual meeting or special meeting of
Members, if necessary, shall be given by the Clerk by mailing notice of the time and
place and purpose of such meeting at least 15 days before such meeting to each
Member at his or her address as shown on the records of the Corporation.

E. Waiver of Notice. Whenever notice of a meeting is required to be given a
Member, under any provision of the Articles or Organization or Bylaws of the
Corporation, a written waiver thereof, executed before or after the Meeting by such
Member, or his or her duly authorized attorney, shall be deemed equivalent to such
notice.

F. Adjournments. Any meeting of the Members may be adjourned to any other
time and place by the vote of a majority of those Members present at the meeting,
whether or not such Members constitute a quorum, or by any officer entitled to preside
at or to act as Clerk of such meeting, if no Member is present or represented. It shall
not be necessary to notify any Members of any adjournment unless no Member is
present or represented at the meeting which is adjourned, in which case, notice of the
adjournment shall be given in accordance with Article II. D. Any business which could
have been transacted at any meeting of the Members as originally called may be
transacted at an adjournment thereof.

ARTICLE III— ASSOCIATES OF THE CORPORATION

Associates of the Corporation. The Associates of the Marine Biological Laboratory
shall be an unincorporated group of persons (including associations and corporations)
interested in the Laboratory and shall be organized and operated under the general
supervision and authority of the Trustees. The Associates of the Marine Biological
Laboratory shall have no voting rights.

ARTICLE IV— BOARD OF TRUSTEES

A. Powers. The Board of Trustees shall have the control and management of the
affairs of the Corporation. The Trustees shall elect a Chairperson of the Board who
shall serve until his or her successor is elected and qualified. They shall annually elect
a President of the Corporation. They shall annually elect a Vice Chairperson of the
Board who shall be Vice Chairperson of the meetings of the Corporation. They shall
annually elect a Treasurer. They shall annually elect a Clerk, who shall be a resident

of Massachusetts. They shall elect Trustees-at-Large as specified in this Article IV.
They shall appoint a Director of the Laboratory for a term not to exceed five years,
provided the term shall not exceed one year if the candidate has attained the age of
65 years prior to the date of the appointment. They shall choose such other officers
and agents as they shall think best. They may fix the compensation of all officers and
agents of me Corporation and may remove them at any time. They may fill vacancies
occurring in any of the offices. The Board shall have the power to choose an
Executive Committee from their own number as provided in Article V, and to
delegate to such Committee such of their own powers as they may deem expedient in
addition to those powers conferred by Article V. They shall, from time to time, elect
Members to the Corporation upon such terms and conditions as they shall have
determined, not inconsistent with law or these Bylaws.

B. Composition and Election.

1 I ) The Board shall include 24 Trustees elected by the Board as provided below:

(a) At least six Trustees ("Corporate Trustees") shall be Members who are
scientists, and the other Trustees ("Trustees-at-Large") shall be individuals who need
not be Members or otherwise affiliated with the Corporation.

(b) The 24 elected Trustees shall be divided into four classes of six Trustees
each, with one class to be elected each year to serve for a term of four years, and with
each such class to include at least one Corporate Trustee. Such classes of Trustees
shall be designated by the year of expiration of their respective terms.

(2) The Board shall also include the Chief Executive Officer, Treasurer and the
Chairperson of the Science Council, who shall be ex officio voting members of the
Board.

(3) Although Members or Trustees may recommend individuals for nomination
as Trustees, nominations for Trustee elections shall be made by the Nominating
Committee in its sole discretion. The Board may also elect Trustees who have not
been nominated by the Nominating Committee.

C. Eligibility: A Corporate Trustee or a Trustee-at-Large who has been elected to
an initial four-year term or remaining portion thereof, of which he/she has served at
least two years, shall be eligible for re-election to a second four-year term, but shall
be ineligible for re-election to any subsequent term until one year has elapsed after
he/she has last served as a Trustee.

D. Removal. Any Trustee may be removed from office at any time with or without
cause, by vote of a majority of the Members entitled to vote in the election of
Trustees; or for cause, by vote of two-thirds of the Trustees then in office. A Trustee
may be removed for cause only if notice of such action shall have been given to all
of the Trustees or Members entitled to vote, as the case may be, prior to the meeting
at which such action is to be taken and if the Trustee to be so removed shall have been
given reasonable notice and opportunity to be heard before the body proposing to
remove him or her.

E. Vacancies. Any vacancy in the Board may be filled by vote of a majority of the
remaining Trustees present at a meeting of Trustees at which a quorum is present. Any
vacancy in the Board resulting from the resignation or removal of a Corporate Trustee
shall be filled by a Member who is a scientist.

F. Meetings. Meetings of the Board shall be held from time to time, not less
frequently than twice annually, as determined by the Board. Special meetings of
Trustees may be called by the Chairperson, or by any seven Trustees, to be held at
such time and place as may be designated. The Chairperson of the Board, when
present, shall preside over all meetings of the Trustees. Written notice shall be sent to
a Trustee's usual or last known place of residence at least two weeks before the
meeting. Notice of a meeting need not be given to any Trustee if a written waiver of
notice executed by such Trustee before or after the meeting is filed wifh the records
of the meeting, or if such Trustee shall attend the meeting without protesting prior
thereto or at its commencement the lack of notice given to him or her.

G. Quorum and Action b\ Trustees. A majority of all Trustees then in office shall
constitute a quorum. Any meeting of Trustees may be adjourned by vote of a majority
of Trustees present, whether or not a quorum is present, and the meeting may be held
as adjourned without further notice. When a quorum is present at any meeting of the
Trustees, a majonty of the Trustees present and voting (excluding abstentions) shall
decide any question, including the election of officers, unless otherwise required by
law, the Articles of Organization or these Bylaws.

H. Transfers of Interests in Land. There shall be no transfer of title nor long-term
lease of real property held by the Corporation without prior approval of not less than
two-thirds of the Trustees. Such real property transactions shall be finally acted upon
at a meeting of the Board only if presented and discussed at a prior meeting of the
Board. Either meeting may be a special meeting and no less than four weeks shall
elapse between the two meetings. Any property acquired by the Corporation after
December 1, 1989 may be sold, any mortgage or pledge of real property (regardless
of when acquired) to secure borrowings by the Corporation may be granted, and any
transfer of title or interest in real property pursuant to the foreclosure or endorsement

R86 Annual Report

of any such mortgage or pledge of real property may be effected by any holder of a
mortgage or pledge of real property of the Corporation, with the prior approval of not
less than two-thirds of the Trustees (other than any Trustee or Trustees with a direct
or indirect financial interest in the transaction being considered for approval) who are
present at a regular or special meeting of the Board at which there is a quorum.

ARTICLE V— COMMITTEES

A. Executive Committee. There shall be an Executive Committee of the Board of
Trustees which shall consist of not more than eleven (11) Trustees, including ex
officio Trustees, elected by the Board.

The Chairperson of the Board shall act as Chairperson of the Executive Committee
and the Vice Chairperson as Vice Chairperson. The Executive Committee shall meet
at such times and places and upon such notice and appoint such subcommittees as the
Committee shall determine

The Executive Committee shall have and may exercise all the powers of the Board
during the intervals between meetings of the Board except those powers specifically
withheld, from time to time, by vote of the Board or by law. The Executive
Committee may also appoint such committees, including persons who are not Trust-
ees, as it may, from time to time, approve to make recommendations with respect to
matters to be acted upon by the Executive Committee or the Board

The Executive Committee shall keep appropriate minutes of its meetings, which
shall be reported to the Board. Any actions taken by the Executive Committee shall
also be reported to the Board.

B. Nominating Committee. There shall be a Nominating Committee which shall
consist of not fewer than four nor more than six Trustees appointed by the Board in
a manner which shall reflect the balance between Corporate Trustees and Trustees-
at-Large on the Board. The Nominating Committee shall nominate persons for
election as Corporate Trustees and Trustees-at-Large, Chairperson of the Board, Vice
Chairperson of the Board, President. Treasurer. Clerk, Director of the Laboratory and
such other officers, if any, as needed, in accordance with the requirements of these
Bylaws. The Nominating Committee shall also be responsible for overseeing the
training of new Trustees. The Chairperson of the Board of Trustees shall appoint the
Chairperson of the Nominating Committee. The Chairperson of the Science Council
shall be an ex officio voting member of the Nominating Committee.

C. Science Council. There shall be a Science Council (the "Council") which shall
consist of Members of the Corporation elected to the Council by vote of the Members
of the Corporation, and which shall advise the Board with respect to matters con-
cerning the Corporation's mission, its scientific and instructional endeavors, and the
appointment and promotions of persons or committees with responsibility for matters
requiring scientific expertise. Unless otherwise approved by a majority of the mem-
bers of the Council, the Chairperson of the Council shall be elected annually by the
Council. The chief executive officer of the Corporation shall be an ex officio voting
member of the Council

D Board of Overseers. There shall be a Board of Overseers which shall consist of
not fewer than rive nor more than eight scientists who have expertise concerning
matters with which the Corporation is involved. Members of the Board of Overseers
may or may not be Members of the Corporation and may be appointed by the Board
of Trustees on the basis of recommendations submitted from scientists and scientific
organizations or societies. The Board of Overseers shall be available to review and
offer recommendations to the officers. Trustees and Science Council regarding
scientific activities conducted or proposed by the Corporation and shall meet from
time to time, not less frequently than annually, as determined by the Board of
Trustees.

E. Board Committees Generullv. The Trustees may elect or appoint one or more
other committees (including, but not limited to, an Investment Committee, a Devel-
opment Committee, an Audit Committee, a Facilities and Capital Equipment Com-
mittee and a Long-Range Planning Committee) and may delegate to any such
committee or committees any or all of their powers, except those which by law, the
Articles of Organization or these Bylaws the Trustees are prohibited from delegating;
provided that any committee to which the powers of the Trustees are delegated shall
consist solely of Trustees. The members of any such committee shall have such tenure
and duties as the Trust a -s .lull determine. The Investment Committee, which shall
oversee the management of the Corporation's endowment funds and marketable
securities shall include as t-.v offi ><> members, the Chairperson of the Board, the
Treasurer and the Chairperson of the Audit Committee, together with such Trustees
as may be required for not less than two-thirds of the Investment Committee to consist
of Trustees. Except as otherwise provided by these Bylaws or determined by the
Trustees, any such committee may make rules for the conduct of its business, but,
unless otherwise provided by the Trustees 01 in such rules, its business shall be
conducted as nearly as possible in the same manner as is provided by these Bylaws
for the Trustees.

F. Actions Without a Meeting. Any action required or permitted to be taken at any
meeting of the Executive Committee or any other committee elected by the Trustees
may be taken without a meeting if all members of such committees consent to the
action in writing and such written consents are filed with the records of meetings.
Members of the Executive Committee or any other committee elected by the Trustees
may also participate in any meeting by means of a telephone conference call, or
otherwise take action in such a manner as may. from time to time, be permitted by
law.

G. Manual of Procedures. The Board of Trustees, on the recommendation of the
Executive Committee, shall establish guidelines and modifications thereof to be
recorded in a Manual of Procedures. Guidelines shall establish procedures for: ( 1 )
Nomination and election of members of the Corporation, Board of Trustees and
Executive Committee; (2) Election of Officers; (3) Formation and Function of
Standing Committees.

ARTICLE VI— OFFICERS

A Enumeration- The officers of the Corporation shall consist of a President, a
Treasurer and a Clerk, and such other officers having the powers of President,
Treasurer and Clerk as the Board may determine, and a Director of the Laboratory
The Corporation may have such other officers and assistant officers as the Board may
determine, including (without limitation) a Chairperson of the Board, Vice Chairper-
son and one or more Vice Presidents, Assistant Treasurers or Assistant Clerks. Any
two or more offices may be held by the same person. The Chairperson and Vice
Chan-person of the Board shall be elected by and from the Trustees, but other officers
of the Corporation need not be Trustees or Members. If required by the Trustees, any
officer shall give the Corporation a bond for the faithful performance of his or her
duties in such amount and with such surety or sureties as shall be satisfactory to the
Trustees.

B. Tenure. Except as otherwise provided by law, by the Articles of Organization
or by these Bylaws, the President, Treasurer, and all other officers shall hold office
until the first meeting of the Board following the annual meeting of Members and
thereafter, until his or her successor is chosen and qualified.

C. Resignation. Any officer may resign by delivering his or her written resignation
to the Corporation at its principal office or to the President or Clerk and such
resignation shall be effective upon receipt unless it is specified to be effective at some
other time or upon the happening of some other event.

D. Removal. The Board may remove any officer with or without cause by a vote
of a majority of the entire number of Trustees then in office, at a meeting of the Board
called for that purpose and for which notice of the purpose thereof has been given,
provided that an officer may be removed for cause only after having an opportunity
to be heard by the Board at a meeting of the Board at which a quorum is personally
present and voting.

E. Vacancy. A vacancy in any office may be filled for the unexpired balance of the
term by vote of a majority of the Trustees present at any meeting of Trustees at which
a quorum is present or by written consent of all of the Trustees, if less than a quorum
of Trustees shall remain in office.

F. Chairperson. The Chairperson shall have such powers and duties as may be
determined by the Board and, unless otherwise determined by the Board, shall serve
in that capacity for a term coterminous with his or her term as Trustee.

G. Vice Chairperson. The Vice Chairperson shall perform the duties and exercise
the powers of the Chairperson in the absence or disability of the Chairperson, and
shall perform such other duties and possess such other powers as may be determined
by the Board. Unless otherwise determined by the Board, the Vice Chairperson shall
serve for a one-year term.

H. Director. The Director shall be the chief operating officer and. unless otherwise
voted by the Trustees, the chief executive officer of the Corporation. The Director
shall, subject to the direction of the Trustees, have general supervision of the
Laboratory and control ot the business of the Corporation. At the annual meeting, the
Director shall submit a report of the operations of the Corporation for such year and
a statement of its affairs, and shall, from time to time, report to the Board all matters
within his or her knowledge which the interests of the Corporation may require to be
brought to its notice.

I. Deputy- Dirt-dor. The Deputy Director, if any, or if there shall be more than one.
the Deputy Directors in the order determined by the Trustees, shall, in the absence or
disability of the Director, perform the duties and exercise the powers of the Director
and shall perform such other duties and shall have such other powers as the Trustees
may. from time to time, prescribe.

J. President. The President shall have the powers and duties as may be vested in
him or her by the Board.

K, Treasurer and Assistant Treasurer. The Treasurer shall, subject to the direction
of the Trustees, have general charge of the financial affairs of the Corporation,

Bylaws of the Corporation R87

including its long-range financial planning, and shall cause to be kept accurate books
of account. The Treasurer shall prepare a yearly report on the financial status of the
Corporation to be delivered al the annual meeting. The Treasurer shall also prepare or
oversee all filings required by the Commonwealth of Massachusetts, the Internal
Revenue Service, or other Federal and State Agencies. The account of the Treasurer
shall be audited annually by a certified public accountant

The Assistant Treasurer, if any, or if there shall be more than one, the Assistant
Treasurers in the order determined by the Trustees, shall, in the absence or disability
of the Treasurer, perform the duties and exercise the powers of the Treasurer, shall
perform such other duties and shall have such other powers as the Trustees may, from
time to time, prescribe.

L. Clerk and Assistant Clerk. The Clerk shall be a resident of the Commonwealth
of Massachusetts, unless the Corporation has designated a resident agent in the
manner provided by law. The minutes or records of all meetings of the Trustees and
Members shall be kept by the Clerk who shall record, upon the record books of the
Corporation, minutes of the proceedings at such meetings. He or she shall have
custody of the record books of the Corporation and shall have such other powers and
shall perform such other duties as the Trustees may, from time to time, prescribe.

The Assistant Clerk, if any, or if there shall be more than one, the Assistant Clerks
in the order determined by the Trustees, shall, in the absence or disability of the Clerk,
perform the duties and exercise the powers of the Clerk and shall perform such other
duties and shall have such other powers as the Trustees may, from time to time,
prescribe.

In the absence of the Clerk and an Assistant Clerk from any meeting, a temporary
Clerk shall be appointed al the meeting.

M. Other Powers and Duties. Each officer shall have in addition to the duties and
powers specifically set forth in these Bylaws, such duties and powers as are custom-
arily incident to his or her office, and such duties and powers as the Trustees may,
from time to time, designate.

ARTICLE VII— AMENDMENTS

These Bylaws may be amended by the affirmative vote of the Members at any
meeting, provided that notice of the substance of the proposed amendment is stated
in the notice of such meeting. As authorized by the Articles of Organization, the
Trustees, by a majority of their number then in office, may also make, amend or repeal
these Bylaws, in whole or in part, except with respect to (a) the provisions of these
Bylaws governing (i) the removal of Trustees and (ii) the amendment of these Bylaws
and (b) any provisions of these Bylaws which by law, the Articles of Organization or
these Bylaws, requires action by the Members.

No later than the time of giving notice of meeting of Members next following the
making, amending or repealing by the Trustees of any Bylaw, notice thereof stating
the substance of such change shall be given to all Members entitled to vote on
amending the Bylaws.

Any Bylaw adopted by the Trustees may be amended or repealed by the Members
entitled to vote on amending the Bylaws.

ARTICLE VIII— INDEMNITY

Except as otherwise provided below, the Corporation shall, to the extent legally
permissible, indemnify each person who is, or shall have been, a Trustee, director or
officer of the Corporation or who is serving, or shall have served at the request of the
Corporation as a Trustee, director or officer of another organization in which the
Corporation directly or indirectly has any interest as a shareholder, creditor or
otherwise, against all liabilities and expenses (including judgments, fines, penalties,
and reasonable attorneys' fees and all amounts paid, other than to the Corporation or
such other organization, in compromise or settlement) imposed upon or incurred by
any such person in connection with, or arising out of, the defense or disposition of any
action, suit or other proceeding, whether civil or criminal, in which he or she may be
a defendant or with which he or she may be threatened or otherwise involved, directly
or indirectly, by reason of his or her being or having been such a Trustee, director or
officer.

The Corporation shall provide no indemnification with respect to any matter as to
which any such Trustee, director or officer shall be finally adjudicated in such action,
suit or proceeding not to have acted in good faith in the reasonable belief that his or
her action was in the best interests of the Corporation. The Corporation shall provide
no indemnification with respect to any matter settled or comprised unless such matter
shall have been approved as in the best interests of the Corporation, after notice that
indemnification is involved, by (i) a disinterested majority of the Board of the
Executive Committee, or (ii) a majority of the Members.

Indemnification may include payment by the Corporation of expenses in defending
a civil or criminal action or proceeding in advance of the final disposition of such
action or proceeding upon receipt of an undertaking by the person indemnified to

repay such payment if it is ultimately determined that such person is not entitled to
indemnification under the provisions of this Article VIII. or under any applicable law

As used in the Article VIII, the terms "Trustee," "director," and "officer"
include their respective heirs, executors, administrators and legal representatives, and
an "interested" Trustee, director or officer is one against whom in such capacity the
proceeding in question or another proceeding on the same or similar grounds is then
pending.

To assure indemnification under this Article VIII of all persons who are determined
by the Corporation or otherwise to be or to have been "fiduciaries" of any employee
benefits plan of the Corporation which may exist, from time to time, this Article VIII
shall be interpreted as follows: (i) "another organization" shall be deemed to include
such an employee benefit plan, including without limitation, any plan of the Corpo-
ration which is governed by the Act of Congress entitled "Employee Retirement
Income Security Act of 1974," as amended, from time to time, ("ERISA"); (ii)
"Trustee" shall be deemed to include any person requested by the Corporation to
serve as such for an employee benefit plan where the performance by such person of
his or her duties to the Corporation also imposes duties on. or otherwise involves
services by, such person to the plan or participants or beneficiaries of the plan; (iii)
"fines" shall be deemed to include any excise tax plan pursuant to ERISA; and (iv)
actions taken or omitted by a person with respect to an employee benefit plan in the
performance of such person's duties tor a purpose reasonably believed by such person
to be in the interest of the participants and beneficiaries of the plan shall be deemed
to be for a purpose which is in the best interests of the Corporation.

The right of indemnification provided in this Article VIII shall not be exclusive of
or affect any other rights to which any Trustee, director or officer may be entitled
under any agreement, statute, vote of Members or otherwise. The Corporation's
obligation to provide indemnification under this Article VIII shall be offset to the
extent of any other source of indemnification of any otherwise applicable insurance
coverage under a policy maintained by the Corporation or any other person. Nothing
contained in the Article shall affect any rights to which employees and corporate
personnel other than Trustees, directors or officers may be entitled by contract, by
vote of the Board or of the Executive Committee or otherwise.

ARTICLE IX— DISSOLUTION

The consent of every Trustee shall be necessary to effect a dissolution of the Marine
Biological Laboratory. In case of dissolution, the property shall be disposed of in such
a manner and upon such terms as shall be determined by the affirmative vote of
two-thirds of the Trustees then in office in accordance with the laws of the Com-
monwealth of Massachusetts.

ARTICLE X— MISCELLANEOUS PROVISIONS

A. Fiscal Year. Except as otherwise determined by the Trustees, the fiscal year of
the Corporation shall end on December 31st of each year.

B. Seal. Unless otherwise determined by the Trustees, the Corporation may have
a seal in such form as the Trustees may determine, from time to time.

C. Execution of Instruments. All checks, deeds, leases, transfers, contracts, bonds,
notes and other obligations authorized to be executed by an officer of the Corporation
in its behalf shall be signed by the Director or the Treasurer except as the Trustees
may generally or in particular cases otherwise determine. A certificate by the Clerk or
an Assistant Clerk, or a temporary Clerk, as to any action taken by the Members.
Board of Trustees or any officer or representative of the Corporation shall as to all
persons who rely thereon in good faith be conclusive evidence of such action.

D. Corporate Records. The original, or attested copies, of the Articles of Organi-
zation, Bylaws and records of all meetings of the Members shall be kept in Massa-
chusetts at the principal office of the Corporation, or at an office of the Corporation's
Clerk or resident agent. Said copies and records need not all be kept in the same office.
They shall be available at all reasonable times for inspection by any Member for any
proper purpose, but not to secure a list of Members for a purpose other than in the
interest of the applicant, as a Member, relative to the affairs of the Corporation.

E. Articles of Organization. All references in these Bylaws to the Articles of
Organization shall be deemed to refer to the Articles of Organization of the Corpo-
ration, as amended and in effect, from time to time.

F. Transactions with Interested Parties. In the absence of fraud, no contract or other
transaction between this Corporation and any other corporation or any firm, association,
partnership or person shall be affected or invalidated by the fact that any Trustee or officer
of this Corporation is pecuniarily or otherwise interested in or is a director, member or
officer of such other corporation or of such firm, association or partnership or in a party
to or is pecuniarily or otherwise interested in such contract or other transaction or is in any
way connected with any person or person, firm, association, partnership, or corporation
pecuniarily or otherwise interested therein; provided that the fact that he or she individ-
ually or as a director, member or officer of such corporation, firm, association or

R88 Annual Report

partnership in such a party or is so interested shall be disclosed to or shall have been authorizing any such contract or transaction with like force and effect as if he/she were not

known by the Board of Trustees or a majority of such Members thereof as shall be present so interested, or were not a director, member or officer of such other corporation, firm,

at a meeting of the Board of Trustees at which action upon any such contract or association or partnership, provided that any vote with respect to such contract or

transaction shall be taken; any Trustee may be counted in determining the existence of a transaction must be adopted by a majority of the Trustees then in office who have no

quorum and may vote at any meeting of the Board of Trustees for the purpose of interest in such contract or transaction.

Publications

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Altamirano, A. A., G. E. Breitwieser, and J. M. Russell. 1999. Acti-
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ions: Evidence for ordered binding. Bitichim. Biophvs. Acta 1416:
145-207.

Antic. S., and D. Zecevic. 1999. Imaging membrane potential changes in
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Antic. S.. C. X. Falk. L. B. Cohen, Y. W. Lam, M. Wachowiak, D.
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Antic, S., G. Major, and D. Zecevic. 1999. Fast optical recording of
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Armstrong, C. M. 1999. Distinguishing surface effects of calcium ion
from pore-occupancy effects in Na+ channels. Proc. Natl. Acad. Sci.
USA 96: 4158-4163.

Armstrong, C. M., and G. Cota. 1999. Calcium block of Na+ channels
and its effect on closing rate. Proc. Natl. Acad. Sci. USA 96: 4154-
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Volume 199

THE

Number

BIOLOGICAL
BULLETIN

OCTOBER 2000

Published by the Marine Biological Laboratory

We walk in the footsteps of visionaries.
And see with the eyes of disciples.

1 Galileo up close: astronomer, physicist,
I mathematician, dethroner of Aristotle, defender
of Copernicus and, yes, credited with making
double convex lenses and the first biological
observations with a microscope. A compound microscope.
In 1624.

Hooke: 43 years later, the compound microscope
reveals living things are composed of cells.

Leeuwenhoek: invents the first practical microscope
and discovers bacteria.

Armed with the knowledge and inspiration of these
and other Olympians, secure in the history of our own
accomplishments and aware that the mind's eye should
always focus on challenging the enigmatic as well as
the undeniable, Olympus has created the BX2 series of
clinical and research microscopes, noteworthy for their
enhancements in optics, imaging and ergonomics.

And each so individual, it deserves the nomenclature
My Microscope.

From possibility to actuality, it is wonderful to behold.
A posse ad esse est mirabile visu.

TheBX4l

The new standard *"»*

in laboratory r
microscopy vis-a-vis " ; '. '
performance, ver-
satility, operation.

Uncanny digitized imaging:

enhanced with the DP11 digital
camera, delivering high-resolution
image recording beyond the nonn.

Unusually i
optics: obse
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Choice of condenser

includes Abbe, swing

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out, pnase-c'

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The tilting, telescoping observation
tube: a marvel of adjustability, it can
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'" ""mm higher

d 70mm
closer to the
operator than
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al tilting tube.
1 And the eye-
re J forward by 45mm
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The new stan-
dard in research
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The words "ne^
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justice. How about
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or "prototypical." Prepare yourself
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Not 4, not 5, but 6, so single and multi-
band imaging of new fluorochromes
and fluorescent proteins is faster,
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ours alone. By cleverly matching
the camera's field of view, only the
area requiring fluorescence excita-
tion is exposed. Which means that
the surrounding areas are protected
from photo
bleaching.

The fluorescence excitation bal-
ancers: another Olympus exclusive.

A continuously variable excitation
bandwidth. Visualization is enhanced,
differentiation of multi-labels is
immeasurably improved, capturing
of images is at an unheard-of level.

Triple Labeled Specimen (1 to r):

nt FITC
with prevalent DAPI and Cy£
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wider mirror aspnencai lens

The aspherical collector lens:

sui generis, in a class by itself.
Specimens appear brighter and more
evenly illuminated; and achromatic
performance now incorporates
wavelengths from UV to IR.

The universal ^
condenser: with
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accept optical inserts
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to encompass prisms for enhanced
contrast and image resolution.

OLYMPUS

FOCUS ON LIFE

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THE

BIOLOGICAL BULLETIN

OCTOBER 2000

Editor
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University of Southern California

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WOODS HOLE, MASSACHUSETTS

»ne Biological Laboratory '

.'3phicinsi,:tuiio,i
Library

NOV

Wooc':

2000

Cover

Fission yeast (Saccharomyces pombe) — in contrast
to budding yeast — is a rod-shaped cell that divides
by cleaving medially. The nucleus is located at the
geometric center of the cell, where it is attached to
multiple bundles of dynamic microtubules that push
on it. As shown on the cover, both the nuclear
membrane and the tubulin bundles can be visual-
ized by fluorescence microscopy in a strain of fis-
sion yeast that expresses a pair of proteins fused to
green fluorescent protein (GFP): nucleoporin-GFP
and GFP-tubulin.

P.T. Tran, V. Doye, F. Chang, and S. Inoue have
used this fluorescent strain of fission yeast to test
the hypothesis that the microtubular bundles deter-
mine the central position of the nucleus which, in
turn, determines the position of the cleavage plane
and the septum that forms between the daughter
cells. The details were reported at the General Sci-
entific Meetings of the Marine Biological Labora-
tory in August, 2000 (see Tran el al., p. 205 in this
issue).

The images on the cover were produced at room
temperature by time-lapse fluorescence micros-
copy. The panel on the left comprises successive
images — taken at intervals of 2 hours — of untreated
cells undergoing cell division. During interphase,
the microtubules span the length of the cell. As the
cells grow, the nucleus comes to lie at the center of
the cell, where subsequent cell division and septa-
tion occurs, creating two daughter cells of approx-
imately equal length. Note that, during mitosis, the
interphase microtubules disappear from the cell cy-
toplasm, whilst the mitotic spindle appears promi-
nently inside the cell nucleus.

The right panel on the cover shows images of cells
treated with MBC, a drug that depolymerizes mi-
crotubules. Without microtubules, the nuclei are
offset, the cell cycle is delayed, and no spindles
form. Subsequent division planes and septa are
formed at the location of the offset nuclei, creating
"cut" nuclei in daughter cells of unequal length.
These treated cells do not survive.

Cover design b\ Beth Liles

CONTENTS

VOLUME 199. No. 2: OCTOBER 2000

RESEARCH NOTE

Hourdez, Stephana, Jason Lamontagne, Pat Peterson,
Roy E. Weber, and Charles R. Fisher

Hemoglobin from a deep-sea hvdrothermal-vent
copepod 95

PHYSIOLOGY

Marsh, Adam G., Patrick K.K. Leong, and Donal T.
Manahan

Gene expression and enzvme activities of the sodium
pump during sea urchin development: implications

for indices of phvsiological state 100

Hill, Richard W., John W.H. Dacey, and Ahser Edward
Dimethylsulfoniopropionate in giant clams (Tridac-
nidae) . 10S

ECOLOGY AND EVOLUTION

Baker, Shirley M., Jeffrey S. Levinton, and J. Evan Ward

Particle transport in the zebra mussel, Dreissena poly-

morpha (Pallas) 116

Lajeimesse, T.C., and R.K. Trench

Biogeography of nvo species of Symbiodinium
(Freudenthal) inhabiting the intertidal sea anemone
Anthoplfura ekgantissima (Brandt) 126

NEUROBIOLOGY AND BEHAVIOR

Rosenthal, Joshua J.C., and Francisco Bezanilla

Seasonal variation in conduction velocity of action
potentials in squid giant axon 135

DEVELOPMENT AND REPRODUCTION

Misamore, M.J., andJ.W. Lynn

Role of the cytoskeleton in sperm entry during
fertilization in the freshwater bivalve Dreissena
polymorpha 144

SHORT REPORTS FROM THE 2000 GENERAL

SCIENTIFIC MEETINGS OF THE MARINE

BIOLOGICAL LABORATORY

/•AM

REPORT

The Editors

Introduction to the featured report. On mapping
odor qualitv ................................ 161

Wachowiak, Matt, Michal Zochowski, Lawrence B.

Cohen, and Chun X. Falk

The spatial representation of odors by olfactory re-
ceptor neuron input to the olfactory bulb is concen-
tration invariant. . 162

NEUROBIOLOGY

Landowne, David

Heavy water (D2O) alters the sodium channel gating
current in squid giant axons 164

White, Thomas H., Harris Ripps, Miduturu Srinivas,

and Roberto Bruzzone

Voltage gating properties of channels formed by a
skate retinal connexin 165

Molina, Anthony J.A., Peter J.S. Smith, and Robert Paul

Malchow

Hydrogen ion fluxes from isolated retinal horizontal
cells: modulation bv glutamate 168

Wang. Jing W.

Odor-induced oscillatory activity in Drtnophila CNS ... 170

Hitt, James M., Frederick A. Dodge, Ehud Kaplan, and

Robert B. Barlow

Orcadian rhythms in the receptive fields of the Limu-

lus lateral eye 171

Fay, Richard R.. and Peggy L. Edds-Walton

Frequency response of auditor)1 brainstem units in
toadfish (Opsanus tau) 173

Yamaguchi, Ayako, Leonard K. Kaczmarek, and Darcy

B. Kelley

Intrinsic membrane properties of laryngeal mo-
toneurons that control sexually differentiated vocal
behavior in African clawed frogs, Xenopus laevis .... 1 75

Atherton, Jillian L., Matthew A. Krntky, James M. Hitt,

Frederick A. Dodge, and Robert B. Barlow

Optic nerve responses of Limulux in its natural habi-
tat at night 1 76

Krutky, Matthew A., Jillian L. Atherton, Spence Smith,
Frederick A. Dodge, and Robert B. Barlow

Do the properties of underwater lighting influence

the visually guided behavior of Limulus? 178

Hale, Melina E.

Startle responses of fish without Mauthner neurons:
escape behavior of the lumpfish (Cyclopterus liimpus) 180

Epstein, David A., Herman T. Epstein, Frank M. Child,

and Alan M. Kuzirian

Memory consolidation in Hermissenda crassicornis ... 182

Abenavoli, A., L. Forti, and A. Malgaroli

Mechanisms of spontaneous miniature activity at
CA3-CA1 synapses: evidence for a divergence from a
random Poisson process 184

PHYSIOLOGY AND BIOCHEMISTRY

Novales Flamarique, Inigo, Kristiina Ovaska, and
Theodore M. Davis

UV-B induced damage to the skin and ocular system

of amphibians 187

Harrington, John M., and Peter B. Armstrong

Initial characterization of a potential anti-fouling sys-
tem in the American horseshoe crab, Limulus
polyphemus 189

Asokan, Rengasamy, Margaret T. Armstrong, and Peter

B. Armstrong

Association of a.,-macroglobulin with the coagulin
clot in the American horseshoe crab, Limulii*
polyphemus: a potential role in stabilization from pro-
teolysis 190

Kuhns, William J., Max M. Burger, Mohan Sarker,

Xavier Fernandez-Busquets, and Tracy Simpson

Enzymatic biosynthesis of N-linked glycan by the ma-
rine sponge Microciona prolifera 192

Armstrong, Peter B., and Rengasamy Asokan

A Ca+2-independent cytolytic system from the blood

of the marine snail Bus^cmi canaliculum 194

Heck, Diane E., Lydia Louis, Michael A. Gallo, and

Jeffrey D. Laskin

Modulation of the development of plutei by nitric
oxide in the sea urchin Arbacia punctuhna 195

Jung, Sung-Kwon, Katherine Hammar, and Peter J.S.

Smith

Development of self-referencing oxygen microsensor
and its application to single pancreatic HIT cells:
effects of aclenylate cyclase activator forskolin on ox-
vgen consumption 197

Hanselmann, Rhea, Roxanna Smolowitz, and Daniel G.

Gibson

Identification of proliferating cells in hard clams. . . 199

Brothers, Christine, Ernest Marks III, and Roxanna

Smolowitz

Conditions affecting the growth and zoospoi illation

of the protistan parasite O_PX in culture 200

CELL BIOLOGY

Sandberg, Leslie, Phillip Stafford, and George M.
Langford

Effects of myosin-II antibody on actin-dependent ves-
icle transport in extracts of clam oocytes 202

Stafford, Phillip, Jeremiah Brown, and George M.

Langford

Interaction of actin- and microtubule-based motors
in squid axoplasm probed with antibodies to myosin
V and kinesin 203

Tran, P.T., V. Doye, F. Chang, and S. Inoue

Microtubule-dependent nuclear positioning and nu-
clear-dependent septum positioning in the fission
yeast, Sacrhammws pombe 205

Crawford, Karen

The role of microtuhules during blastodisc forma-
tion of the squid Loligo pealei 207

Weidner, Earl

Cytoplasmic proteins on the surface of discharged
microsporidian sporoplasms 208

MacKenzie, Roger, David Newman, Max M. Burger,

Rene Roy, and William J. Kuhns

Adhesion of a viral envelope protein to a non-self-
binding domain of the aggregation factor in the
marine sponge Micwriona prolifera 209

Goda, Makoto, Mario H. Burgos, and Shinya Inoue
Fertilization-induced changes in the fine structure of
stratified Arbacia eggs. I. Observations on live cells
with the centrifuge polarizing microscope 212

Burgos, Mario H., Makoto Goda, and Shinya Inoue
Fertilization-induced changes in the fine structure of
stratified Arbacta eggs. II. Observations with electron
microscopy 213

Gould, Robert M., Concetta M. Freund, John Engler,

and Hilary G. Morrison

Optimization of hornogenization conditions used to
isolate mRNAs in processes of myelinating oligoden-
drocytes 215

ECOLOGY, BIOGEOCHEMISTRY, AND POPULATION BIOLOGY

Kirkby, Ryan, Luc Claessens, Charles Hopkinson, Jr.,
Edward Rastetter, and Joseph Vallino

Modeling the effects of land-use change on nitrogen
biogeochemistry in the Ipswich watershed, Massachu-
setts 218

Perring, Anne, Michael Williams, Charles Hopkinson,

Jr., Edward Rastetter, and Joseph Vallino

Solute dynamics in storm flow of the Ipswich River
Basin: effects of land use 219

Westgate, Elizabeth J., Kevin D. Kroeger, Wendy J.

Pabich, and Ivan Valiela

Fate of anthropogenic nitrogen in a nearshore Cape
Cod aquifer 221

Denault, Michelle, Erica Stieve, and Ivan Valiela

Effects of nitrogen load and irradiance on photosyn-
thetic pigment concentrations in Cladophora vaga-
bundaand Gracilaria tikvahiae'm estuaries of Waquoit

Bav 223

Greenbaum, Adena, and Anne Giblin

Differences in properties of salt marsh sediment be-
tween haved and reference sites 225

Chikarmane, Hemant M., Alan M. Ku/irian. Robbin
Kozlowski, Mark Kuzirian, and Tony Lee

Population genetic structure of the goosefish, Lo-
phnif nmmranus 227

ORAL PRESENTATIONS
Published bv title only. .

229

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Reference: Bio/. Bull. 199: 95-99. (October 2000)

Hemoglobin From a Deep-Sea
Hydrothermal-Vent Copepod

STEPHANE HOURDEZ1 *, JASON LAMONTAGNE-, PAT PETERSON2,
ROY E. WEBER3, AND CHARLES R. FISHER1

1 Department of Biology, 208 Mueller Lab, Pennsylvania State University. University Park, Pennsylvania

16802; 2PaIisades High School. 35 Church Hill Road. Kintnersville. Pennsylvania 18930;
and ^Center for Respiratory Adaptation. Department of Zoophysiology, University of Aarhus,

8000 Aarhus C. Denmark

Abstract. Deep-sea hydrothermal-vent fauna live in a
highly variable environment where oxygen levels can be
very low, and carbon dioxide and sulfide can reach high
concentrations ( 1 ). These conditions are harsh for most
aerobic metazoans, yet copepods can be abundant at hydro-
thermal vents. Here we report the structure and functional
properties of hemoglobin extracted from the copepod Ben-
thoxynus spiculifer, which was found in large numbers in a
paralvinellid/gastropod community collection made during
a cruise to the Juan de Fuca Ridge in 1998. Although
hemoglobin has been reported in some littoral copepods (2).
this is the first study of the structure and functional prop-
erties of copepod hemoglobin. Hemoglobin represents
about 60<7r of the total soluble proteins extracted from B.
spiculifer, and although it imparts a red color to the cope-
pod, it does not provide a significant storage pool of oxygen.
It is a 208-kDa protein, composed of 14 globin chains — 7 of
14.3 kDa and 7 of 15.2 kDa. The hemoglobin has a very
high and temperature-sensitive oxygen affinity, with no
cooperativity or Bohr effect. These properties are adaptive
for an animal living in a low-oxygen environment in which
the primary function of the hemoglobin is most likely ox-
ygen acquisition to support aerobic respiration.

Copepods occur in both freshwater and marine environ-
ments that range from pelagic to benthic and littoral to
deep-sea (3). Red copepods have been observed at hydro-
thermal vents of the Mid-Atlantic Ridge, Juan de Fuca
Ridge, and East Pacific Rise (SH, pers. obs.). However, the

Received 19 April 2000; accepted 7 July 2000.

*To whom correspondence should be addressed. E-mail: hourdez@sb-
roscoff.fr. Portions of this work were performed at the Station Biologique
de Roscoff, CNRS-UPMC, BP74. 29682 Roscoff cedex, France.

number of animals collected has previously been too small
for a study of their oxygen-binding protein. On dive 3259 of
the DSRV Alvin, a paralvinellid (worm I/gastropod commu-
nity (similar to Community III described by Sarradin el al.
[4]) was collected from the base of the S & M chimney on
the main field of the Endeavour segment of the Juan de Fuca
Ridge. In that community, the animals are probably exposed
to temperatures ranging from 10° to 25°C (4). The collec-
tion was made using a new device, nicknamed the Chimney
Master, which is a hydraulically actuated net lined with
62-jum mesh and suspended in an aluminum frame. The
30-cm-diameter open end of the device is placed over a
community to be collected, and then the net is drawn closed
by a stainless steel cable while the frame is held firmly
against the substrate by the submersible. In an appropriate
environment, the Chimney Master removes and collects all
attached and associated fauna from the substrate along with
a surface layer of loose rocks and sulfides.

The collection contained many specimens of the copepod
Benthoxymis spiculifer. Examination of the animals re-
vealed that their deep-red color was not due to the gut
content (which consisted of white filamentous material re-
sembling bacteria) but rather to a soluble pigment distrib-
uted throughout the rest of the body. About 6000 specimens
were separated from the collection, using a pipette; these
were rinsed, concentrated by centrifugation. and frozen at
-70°C in several cryovials.

The hemoglobin was purified from an extract of about
4000 animals that were thawed; homogenized in an extrac-
tion buffer containing 1 fiM PMSF (phenylmethanesulfonyl
fluoride) and 1mA/ EDTA in 50 mM Tris, pH 8; and then
centrifuged to remove animal debris. The extract was puri-

S HOURDEZ £T AL.

fied by size exclusion chromatography (see legend of Fig.
I ). The pigment eluted as a single pink band that repre-
sented about 55%! to 60% of the total soluble proteins in the
extract. Using proteins of known molecular weight for cal-
ibration, we estimated the apparent native molecular weight
of the pigment to be 208 kDa. This pure fraction was used
for further studies.

The light absorbance spectrum of the 208-kDa fraction
showed the typical peaks for oxy-hemoglobin: a. ft. and 8
(Soret's band) peaks at 578 and 544 and 414 nm. respec-
tively (Fig. 1 ). The y and the protein peaks were present at
348 nm and 270 nm, respectively. The absence of a methe-
moglobin peak at 630 nm confirmed that little or no hemo-
globin had been oxidized. The ratio a/(3 was 0.83, smaller
than unity, as reported for some other extracellular hemo-
globins (see [5]). The presence of hemoglobin has previ-
ously been reported in other copepods from reduced envi-
ronments by Fox (2), who detected this protein in vivo using
a microspectrophotometer. To determine the subunit struc-
ture of the 208-kDa fraction, it was further fractionated by
SDS polyacrylamide gel electrophoresis in the presence and
absence of |3-mercaptoethanol (Fig. 2). Two bands, of 14.3
and 15.2 kDa, were resolved under both conditions. This
suggests that the native molecule is composed of monodo-
main globin chains that are not linked by disulfide bonds.
The bands were of similar intensity, suggesting that the
intact hemoglobin molecule is composed of 14 chains (7 of
each type), with a calculated mass of 206.5 kDa. This agrees
well with the native mass estimated by gel filtration of 208

B MW

0,8-

0.6-

0,4-

0,2-

Protein

250 300 350 400 450 500 550
Wavelength (nm)

600 650

700

Figure 1. Absorbance spectrum of Bcniliu.\\iiii\ spiciilifer hemoglo-
bin, extracted in 50 mA7 Tns pH X, EDTA 1 mAl. and PMSF 1 /j.M , and
purified by FPLC on a Superose 6 column (5 to 5000 kDa mass separation
range). The elution buffer contained NaCI 400 muiol 1 '. KCI 2.45 mmol
I'1. MgS04 32 mmol 1 '. CaCI, 1 1 mmol 1 '. and HEPES 50 mmol 1 ' at pH
7.0. This spectrum shows the typical Soret band at 414 nm and the a and
)3 peaks of the liganded hemoglobin (at 578 and 544 nm. respectively).

180
116

18.4

14.2
6.5

Figure 2. SDS-PAGE of Bi'iitliu\\nn.\ .\piciitifer hemoglobin. Lane A:
SDS-lreated sample; Lane B: SDS- and /3-mercaptoethanol-treated sample.
The gel was composed of a stacking gel (4% acrylamide) overlying a
gradient separation gel ( 10% to 20% acrylamide) and was then silver-
stained to reveal the presence of the proteins. The molecular weight of each
subnnit ( 14.3 and 15.2 kDa) was estimated using the Silver Staining Wide
Range Molecular Weight Markers (Sigma) as calibration proteins ("MW"
lane). Molecular weights are given in kilodaltons.

kDa. This structure is unusual for an arthropod hemoglobin
(Table 1). Insect hemoglobins are generally much smaller
( 15-30 kDa). whereas those of Crustacea have a high mo-
lecular weight (220-4000 kDa). The mass of B. spiculifer
hemoglobin is at the lower limit of those observed among
crustaceans. The subunits are monodomain globins and not
multidotnain globins as is more normal for crustaceans
(Table 1 ). With regard to subunit mass, B. spiculifer hemo-
globin is similar to that found in Rhizocephala, although the
native mass is 5 to 20 times smaller.

The functional properties of the hemoglobin were studied
using the step-by-step procedure (6) in a modified diffusion
chamber (7). Under the conditions we used, the oxygen
affinity of the hemoglobin is extremely high, with P50
values at pH 7.3 of 0.05. 0.13, and 0.35 mm Hg at 10°, 20°,
and 30°C, respectively (Fig. 3A). These affinities are among
the highest reported for arthropod hemoglobins (8). Among
the arthropods, only the conchostracan C\-icus hierosolymi-
uiiius (9) has hemoglobin with higher affinity (P50 = 0.035
nimHg at 28 °C and pH 7.2. Table 1). Both species. B.

COPEPOD HEMOGLOBIN

Table I
Occurrence tint! structural tintl functional characteristics of hemoglobin in (//7///

SbP C SbC SpO O

Native
mass (kDa)

Suhunit
(kDul

Domains/
Mihimit

Psn (Torr) nsn

Bohr factor AH (kJ/mol)

Reference

Uniramia

Hexapoda

Diplura

15 and 30

0.7 1

-0.1 5 to -0.9 -42.6

17. IS

Crustacea

Branchiopoda

Anostraca

260

130

1.8 to 5. 3 1.6-l.M

(I to -0.21 -22.6 to -54.8

Notostraca

600-800

20 2

0.13 -30.9

Cladocera

420-670

2.1 to 3.5 ?

0 ?

Conchostraca

220-300

0.035 to 5.9 2.3-2.5

? -20.5

9. 22

Maxillopoda

Ostracoda

7 7

Copepoda

Harpacticoida

7 7

Siphonostomatoida

208

14-15

0.05 1

0 -68.7

This study

Cirri pedia

Rhizocephala

1000-4000

7 7

Malacostraca

Eumalacostraca

Peracarida

Amphipoda

1800

175

10?

9 9

7 9

SbP: subphylum; C: class; SbC: subclass: SpO: superorder: O: order. Modified after Terwilliger ( 16). Conchastracan Hb, P,n measured at 28°C. all other
P50 measured at 20°C.

spiciilifer and C. hierosolymitanus, have P5() values 20 to
600 times smaller than the P50 values of other arthropod
hemoglobins. The affinity of B. spiculifer hemoglobin for
oxygen is also higher than that reported for the hemocyanins
of other hydrothermal vent crustaceans, although their he-
mocyanin P50 values are quite low (reviewed in [ 10]).

Benthoxynus spiciilifer hemoglobin lacks cooperativity
(nw = 1.0) over the range of temperature (10° to 30°C) and
pH (6.7 to 8.1) examined. The hemoglobin components of
Chironomus thunimi tluimmi (Insecta) also lack cooperativ-
ity; however, in contrast to B. spiciilifer hemoglobin, they
are monomeric or dimeric (Table 1). The other arthropod
hemoglobins are multimeric and exhibit some cooperativity
(n = 1.6 to 2.3). Like the hemoglobin of most crustaceans,
that of B. spiculifer does not exhibit a significant Bohr effect
(<£ = +0.04) (Table 1). However, the hemocyanins of
hydrothermal vent crustaceans often show substantial Bohr
effects (10), as do the hemoglobin components of the ar-
thropod C. tlnimmi t/niiunii (<J> = -0.9). B. spiciilifer he-
moglobin thus does not show any homotropic or hetero-
tropic interactions and behaves like a myoglobin.
Temperature has a strong effect on B. spiculifer hemoglo-
bin, as shown by the apparent AH value of -69 kJ • mole"'
(Fig. 3B). This value is higher than that of other arthropod
hemoglobins (Table 1 ) and is consistent with the absence of
a Bohr effect (oxygenation-linked proton dissociation that is
endothermic and decreases the overall exothermic heat of
oxygenation) in Benthoxynus hemoglobin (11).

To determine the in vim hemoglobin concentration, a
small group of copepods (about 400 animals) was weighed
and homogenized in a ground-glass tissue homogenizer, and
the hemoglobin content of the homogenate was determined
using the cyan-met-hemoglobin method and a millimolar
absorption coefficient of 11 cm1 at 540 nm (12). The
estimated in vivo concentration of 0.95 mM heme explains
the conspicuous red color of the animals. Large hemoglobin
pools can play an important role in oxygen storage in some
situations. However, assuming a respiratory rate similar to
that of littoral harpacticoid copepods (13) and an abrupt
switch from aerobiosis to anaerobiosis, we estimate that the
quantity of hemoglobin present would support aerobic res-
piration for less than 2 min at 15°C and about 30 s at 25°C.
Thus the hemoglobin pool is insufficient to allow the cope-
pod to make more than short forays into anaerobic micro-
habitats without relying on anaerobic respiration. Another
role of high affinity hemoglobins has been theorized to be
detoxification of free radicals from oxygen or nitrogen
monoxide (14). Although free radicals do form in sulfidic
systems, and some vent animals have detoxification mech-
anisms (15), we consider it more likely that the hemoglobin
of B. spiculifer functions primarily in oxygen acquisition
from the environment. The other hemoglobin-containing
copepods identified by Fox (2) were collected from muddy
and reduced environments with low levels of oxygen and
high levels of sulfide. The adaptive significance of hemo-
globin for acquisition of oxygen in these environments is

S. HOLIRDEZ ET AL.

100

A Benthoxynus spiculifer

3.3 3.4 3.5
1/1000T

-0.5

o
0?

-1.0

-1.5

30°C

10°C

•

0.5

0.1

0.05

_ i^fs *' ' —

•\

• i i i i i i i , i , i i i i . i

6.5 7.0 7.5 8.0

0.5 1.0

PO2 (mmHg)

Figure 3. (A) Oxygen equilibrium curves of Benthoxynus spiculifer hemoglobin at 10°. 20°, and 30°C,
measured as previously described (5), and (inset) arrhenius plot showing calculated values of the apparent
oxygenation enthalpy values (AH). (B) Variation of P5n and n,n values with pH and temperature.

apparent, and the very high affinity of the hemoglobin in B.
spiculifer probably also reflects the very low oxygen ten-
sions this species experiences in its hydrothermal vent mi-
crohabitat. In this context it is relevant that hemoglobins or
hemocyanins with high oxygen affinity characterize many
hydrothermal vent animals (10).

Acknowledgments

We thank the captains, pilots, and crews of the R/V
Atlantis and DSRV Alvin for their assistance at sea; Verena
Tunnicliffe and Maia Tsurumi for identifying B. spiculifer.
Anny Bang for assistance with the oxygen equilibrium
experiments; and Veronique Robigou and the REVEL
Project for providing science teachers with research oppor-
tunities. This project was supported by NSF OCE 9633105
and the REVEL Program (NSF OCE-98 14073 to J.R.
Delaney, with additional support from the Pennsylvania
State University and the American Museum of Natural
History).

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Reference: Biol. Bull. 199: 100-107. (October 2000)

Gene Expression and Enzyme Activities of the Sodium

Pump During Sea Urchin Development: Implications

for Indices of Physiological State

ADAM G. MARSH*. PATRICK K.K. LEONGt, AND DONAL T. MANAHANi

Department of Biological Sciences, University of Southern California,
Los Angeles, California 90089-0371

Abstract. The sodium pump consumes a large portion of

the metabolic energy (407r ) in sea urchin larvae. Under-
standing the developmental regulation of ion pumps is im-
portant for assessing the physiological state of embryos and
larvae. We sequenced a partial cDNA clone (1769 bp) from
the sea urchin Strongylocentrotiis pitrpuratits and found it to
contain the C-terminal portion of an open reading frame
coding for 195 amino acids that exhibited high sequence
similarity (897r) to invertebrate a-subunits of the Na+,K+-
ATPase sodium pump. Northern blots using the 3' untrans-
lated region of this cDNA specifically recognized a 4.6-kbp
transcript under high stringency. During embryonic devel-
opment, a rapid increase in levels of this mRNA transcript
during gastrulation (25 h postfertilization) was paralleled by
a concomitant increase in the total enzymatic activity of
Na+,K+-ATPase. Expression of this subunit during gastru-
lation increased to a maximum at 36 h, followed by a rapid
decline to trace levels by 60 h. The rate of removal of the
transcript from the total RNA pool after 36 h closely fol-
lowed a first-order exponential decay model (r= 0.988),
equivalent to a degradation rate of 7.87r h" . By 83 h.
transcription of the a-subunit gene was low. yet sodium
pump activity remained high. Molecular assays for the
expression of this gene would underestimate sodium pump
activities for assessing physiological state because of the
temporal separation between maximal gene expression in a

Received 29 February 2000; accepted 31 July 2000.

*Present address: College of Marine Studies. University of Delaware.
Lewes, DE 19958.

tPresent address: Department of Physiology and Biophysics. University
of Southern California School ot Medicine. Los Angeles. CA 90033.

tTo whom correspondence should be addressed. E-mail: manahan
@ usc.edu

gastrula and maximal enzyme activities in the later larval
stage. This finding illustrates the difficulty of using molec-
ular probes for assessing the physiological state of inverte-
brate larvae.

Introduction

Maintaining Na+ and Kf ion gradients is one of the most
energetically demanding processes of an organism's main-
tenance physiology. In general, animal cells routinely ex-
pend 20%-30% of their total metabolic energy on the ac-
tivity of a single protein complex, the sodium pump
(Na + ,K + -ATPase; Siems et at., 1982. 1992), and for adult
marine invertebrates, the sodium pump can potentially ac-
count for 30%-70% of tissue metabolism (Baker and Con-
nelly, 1966: Lucu and Pavicic. 1995). The ion gradients
established by the sodium pump are critical for maintaining
a cell's osmotic balance and resting membrane potential, as
well as providing the electrochemical gradient necessary for
the uptake of other ions, sugars, amino acids, and neuro-
transmitters via Na+ coupled co-transporters (Blanco and
Mercer, 1998).

The requirements for ion regulation change rapidly dur-
ing embryonic development. The increase in cell number
during early embryogenesis and the consequent increase in
cellular-membrane surface area necessitates the production
of more sodium pumps to regulate intracellular ion flux.
The in vivo physiological activity of Na+,K+-ATPase
has been characterized during early development in the sea
urchins Stwngylocentrotus purpiiratus and Lytechimis pic-
tns (Leong and Manahan, 1997). Using stlRb+ as a radio-
active tracer for K+ ion transport, Leong and Manahan
(1997) described the ontogenetic changes in activity of

100

SODIUM PUMPS IN SEA URCHIN EMBRYOS

101

Na+,K+-ATPase in living embryos. They ton nil a large
increase in activity — from nondetectable levels prior to fer-
tilization to a high level accounting for 40% of total meta-
bolic energy consumption at the pluteus larval stage (72 h
postfertilization). By the same radiotracer techniques, the
metabolic energy demand of Na+,K+-ATPase activity in
the Antarctic sea urchin Sterechinus neumayeri was found
to be as high as 80% of total metabolism at the pluteus
larval stage at -1.5°C (Leong and Manahan, 1999). In the
sea urchin Hemicentrotus pulcherrimus, the total protein
activity and gene expression of Na+,K+-ATPase increases
rapidly during gastrulation (Mitsunaga-Nakatsubo et til.,
1992a. b). Overall, the physiological importance of
Na+,K+-ATPase activity during embryogenesis in sea ur-
chins has significant implications for metabolic energy con-
sumption during development.

This universal importance of Na^.K^-ATPase in animals
suggests that measurements of this enzyme could be a
useful indicator of physiological state. For larval stages in
which direct enzyme assays are limited by the small amount
of protein in an individual, measurements of gene expres-
sion might provide the sensitivity necessary to assay small
amounts of tissue. Functional Na+.K+-ATPase pumps are a
heterodimer (a, )3 subunits; Jorgensen and Skou. 1969).
with the a-subunit possessing the ATP binding site and
catalytic activity (Kyte, 1971 ). In this study, we describe the
timing between transcription of the a-subunit and the ap-
pearance of functional sodium pumps during the develop-
ment of Strongylocentrotus purpuratus. We also describe
the ontogenetic changes in expression of the a-subunit to
determine the developmental timing between increases in
enzyme activity and the potential for using these measures
as an index of physiological state in embryos and larvae.

Materials and Methods

Embryo cultures

Adult Strongylocentrotus purpuranis were induced to
release gametes (injections of 0.5 M KC1), and fertilized
eggs were divided into six 20-liter culture containers at a
concentration of about 20 individuals per milliliter of fil-
tered seawater (0.2 jum). Culture temperatures were main-
tained at 15°C during development. Embryos were main-
tained in suspension by paddles connected to slow stirring
motors (—30 rpm). For the gene expression analysis, time-
course samples were collected throughout development at
the following times from an egg to a 4-arm pluteus larva: 0,
6, 8, 10. 12, 14. 16. 18, 20, 25, 31. 36, 42, 48, 60, 72, and
83 h postfertilization (n = 17). For each sample, about
100,000 embryos were removed by sieving (80-jtun mesh)
and pelleted by centrifugation ( 1000 X g) into 50-ml screw-
cap tubes. Embryos were immediately dissolved in an acid-
guanidinium buffer (4 M guanidinium isothiocyanate, 25

mM Na-citrate, 0.2% Sarkosyl and 215 mM /3-mercapto-
ethanol: pH 5.2; Chomzinsky and Sacchi, 1987) and frozen
at -80°C.

cDNA clone: sequencing and analvsis

An expressed sequence tag (EST) library from activated
coelomocytes of adult S. purpuratus was prepared by Smith
et al. ( 1996), and a sequence fragment of one cDNA clone
(#020) was found to have a high nucleotide similarity to the
bovine a-subunit of Na+,K+-ATPase. We sequenced this
clone (provided by C. L. Smith and E. H. Davidson) by
random transposon insertion in a modified pBluescript
(Stratagene) plasmid (pMOB: Strathmann et til., 1991 ). The
introduced transposon elements contained defined priming
sites for subsequent manual sequencing of double-stranded
plasmid templates using standard dideoxy termination reac-
tions with "S-labeled dATP (Sequenase Reaction Kit,
USB). Sequencing gels were visualized by autoradiography
on X-ray film (Kodak, XAR 5). Nucleotide sequences were
entered and edited using the software package MacVector
5.0 (Mac OS: Oxford Molecular Group), and contiguous
overlaps between fragments were identified using the soft-
ware package AssemblyLign 2.0 (Mac OS; Oxford Molec-
ular Group). Both strands of the open reading frame (ORF)
were sequenced by overlapping subclones so that most of
the contiguous ORF sequence was assembled from three
independent sequencing reactions. For phylogenetic com-
parisons, nucelotide and putative amino acid sequences
from other animal species were structurally analyzed and
aligned using the OMIGA 2.0 software package (Oxford
Molecular Ltd.). Identity and similarity scores for the de-
duced amino acid alignments were calculated from the
FASTA routine available in the GCG Wisconsin Package
8.0 (UNIX OS).

mRNA analysis: isolation and quantification

Total RNA was extracted from each sample by an acid
guanidinium-phenol method (after Chomzinsky and Sacchi.
1987) and further purified by sequential precipitations in
lithium chloride (4 M LiCl), sodium acetate (3 M NaOAc,
pH 4.2) and ethanol (70% EtOH). After each precipitation,
the RNA pellets were washed in 70% EtOH and dried under
vacuum: before proceeding with the next precipitation, the
pellets were resuspended in RNase-free TEN buffer ( 10 mM
Tris pH 8.0. 1 mM EDTA and 10 mM NaCl). The final RNA
precipitates were resuspended in RNase-free water and
quantified by their optical density at 260 nm. From each
developmental time point, 10 jug of total RNA was size-
separated by formaldehyde gel electrophoresis and blotted
overnight via capillary transfer onto nylon membranes.
RNA on the nylon membranes was UV cross-linked
(Stratalinker), and the membranes were stored dry at room

102

A. G. MARSH ET AL.

temperature. A cDNA probe was generated from the 3'-
untranslated region (UTR) of clone #020. The terminal
1185 bp were PCR amplified (5'- TGG GAT TGA AGO
ACT CAG -3' and T7 oligonucleotide primers) and gel
purified for further use in standard Northern hybridizations
(see general methods in Ausubel et al., 1992). Membranes
were prehybridized for several hours in 40% formamide, 25
mMNa3PO4 (pH 7.2), 5x SSC, 0.1% SDS, 5x Denhardt's,
and 50 jug/ml yeast RNA at 45°C in a hybridization oven.
The 3'-UTR PCR probe (1185 bp) was radiolabeled by
random priming (Promega) with a-32P-dCTP (3000 Ci
mmol ' ), added to the hybridization tube with a fresh 10-ml
aliquot of hybridization buffer (as above), and incubated
overnight at 50°C. The blots were initially washed with
0.1X SSC, 1.0% SDS. and 0.5% Na4P2O7 at 45°C for 1 h.
Additional washes at higher temperatures (max. 55°C) were
performed as necessary to further reduce the background
signal. Autoradiograms (Kodak Biomax X-ray film) were
digitized on a high-resolution scanner (1200 dpi), and grain
densities for the signal bands were quantified using the
image analysis routines in the software program PhotoShop
4.0 (Win95 OS; Adobe).

Na+,K+-ATPase enzyme activity

Total enzyme activity of Na+,K+-ATPase was measured
at short intervals between 20 and 50 h postfertilization, the
period during which enzyme activity increases rapidly dur-
ing development in S. piirpuratus (Leong and Manahan,
1997). Ouabain-sensitive Na+,K+-ATPase activity (details
in Leong and Manahan, 1997) was determined in all sam-
ples on the same day with one set of standards to minimize
the between-sample assay error. Total Na+,K+-ATPase ac-
tivity was measured as the rate of hydrolysis of ATP (Es-
mann, 1988). Briefly, embryo tissues were thawed, soni-
cated, and resuspended in histidine buffer (10% sucrose, 5
mM EDTA and 5 mM histidine, pH 7.7) at a final protein
concentration of 0.5 to 1.0 mg ml"1. In the present study, the
total Na+,K"l"-ATPase activity of the sea urchin embryos
was measured as the difference in ATPase activity in the
presence and absence of 2 mM ouabain at 25°C. A detailed
consideration of the inclusion of detergents in the Na+,K + -
ATPase assay is presented in Leong and Manahan (1997).
In summary, neither deoxycholate (a common detergent
used in Na"l",K+-ATPase assays) nor alamethicin (a mem-
brane-perrneabilizing agent) had any effect on the total
Na+,K+-ATPase activity in homogenates of S. piirpuratus
embryos, suggesting that inside-out and right-side-out ves-
icles are not a significant problem in assaying Na+,K+-
ATPase activity in seu urchin embryos (Leong and Mana-
han, 1997). The protein content of the samples was
determined by the Bradford assay with the modifications of
Jaeckle and Manahan (1989).

Table 1

Comparison of nucleolide and deduced ammo acid sequences for
different a-subunit Na* . K* -ATPase

Amino

acid

GenBank

Nucleotide

accession

identity

Identity

Similarity

Species

number

(%)

Drosophila

AF04494

69.8

73.3

89.2

Caenoi'habditis

U 18546

69.7

72.8

89.2

Xenopus

U49238

67.6

69.2

89.2

Anemia

X56650

65.2

72.3

89.7

Hydra

M75140

64.1

67.7

89.2

Identity and .similarity to the Slrongyloc entrains piirpuratus cDNA open
reading frame (clone #020) are presented as percentages determined from
scoring by the GCG Wisconsin Users Group software program. Scores
include only the terminal portion of the sea urchin gene's ORF: 588 base
pairs (195 amino acid residues and stop codon).

Results

A partial Strongylocentrotus piirpuratus cDNA clone
(#020: Smith et al., 1996) was characterized in this study
and found to contain 1769 bp with the terminal portion of an
ORF coding for 195 amino acids (588 bp with the stop
codon). The remaining sequence (1181 bp) comprised a
putative 3' UTR domain. The clone's ORF was compared to
other tt-subunits of Na+,K + -ATPase, and the S. piirpuratus
nucelotide sequence ranged from 64% to 70% identity to
these terminal ORF domains (Table 1). The deduced amino
acid sequences of these organisms were aligned to the
putative amino acid sequence of the S. piirpuratus clone and
evidenced a high degree of sequence conservation in this
terminal domain (Table 2). When compared to the S. piir-
puratus sequence, the derived amino acid sequence was
68%-73% identical and 89%-90% similar (Table 1 ).

The terminal region of the ORF of known «-Na + ,K + -
ATPases is believed to contain several transmembrane do-
mains; there is some debate over the exact number of these
domains and the extra- vs. intracellular orientation of some
of the intervening regions in a-Na+,K+- ATPase (Shull and
Greeb, 1988; Takeyasu et al.. 1990; Blanco and Mercer,
1998). The hydropathy of the S. piirpuratus sequence was
estimated with Kyte-Doolittle scoring using a grouping of
11 amino acid residues (Fig. 1) and suggests a high proba-
bility of four transmembrane domains in the terminal por-
tion of this ORF. Overlaying these domains on a structure
detailed by Takeyasu et al. ( 1990) indicates that the region
between the seventh and eighth transmembrane domains
could have an extracellular localization. In the absence of
crystallographic data, it is generally believed that most
a-subunits of transmembrane ATPases (both Na+ and
Ca+ + ) are structurally similar, with 10 transmembrane do-

SODIUM PUMPS IN SEA URCHIN EMBRYOS
Table 2

103

Alignment of deduced amino acid sequence for the terminal IV5 residues of the a-subunil of Na+. K+-ATPase in the sea urchin Strongylocentrotus
purpuratus

URCHIN

SDIMKRRPRD

PQNDKLVNER

LISVSYGQIG

MIQRSAGFFA

YFVIMGENGF

FLY

ADIMKRPPRD

PFNDKLVNSR

LISMAYGQIG

MIQAAAGFFV

YFVIMAENGF

SHRIMP

SDIMKRRPRN

PVTDKLVNER

LISLAYGQIG

MIQASAGFFV

YFVIMAECGF

NEMATODE

SDIMKRQPRD

PIRDKLVNER

LISLAYGQIG

MIQASAGFFT

YFWIMADNGF

FROG

SDIMKRQPRN

PKTDKLVNER

LISMAYGQIG

MIQALGGFFT

YFVILAENGF

HYDRA

SDIMKRHPRN

PIRDKLVNER

LISLAYGQIG

MMQATAGFFT

YFIILAENGF

URCHIN

LPNDLIMLRS

KWDDKAVLNV

EDSYGQQWGF

YQRKQLEYTC

HTAFFASIW

FLY

LPKKLFGIRK

MWDSKAVNDL

TDSYGQEWTY

RDRKTLEYTC

HTAFFISIVV

SHRIMP

LPWDLFGLRK

HWDSRAVNDL

TDSYGQEWTY

DARKQLESSC

HTAYFVSIVI

NEMATODE

MPWDLYQLRA

QWDSRAYNNV

LDSYGQEWTY

ANRKILEYTC

QTAYFVSIVV

FROG

LPWTLLGIRV

NWDDRWTNDV

EDSYGQQWTY

EQRKIVEFTC

HTSFFISIVV

HYDRA

LPSYLFGLRS

QWDDMSNNNL

LDSFGSEWTY

FQRKEIELTC

QTAFFTTIVV

•k

•k ~k

101

-"'1150

URCHIN

VQWADVIICK

TRRNSLIHQG

MNNWVLNFGL

FFETALAAFL

SYCPGLENGL

FLY

VQWADLIICK

TRRNSIFQQG

MRNWALNFGL

VFETVLAAFL

SYCPGMEKGL

SHRIMP

VQWADLIISK

TRRNSVFQQG

MRNNILNFAL

VFETCLAAFL

SYTPGMDKGL

NEMATODE

VQWADLIISK

TRRNSLVQQG

MSNWTLNFGL

VFETALAWFM

CYCPGLDNGL

FROG

VQWADLIICK

TRRNSVFQQG

MKNKILIFGL

FEETALAAFL

SYCPGMDVAL

HYDRA

VQWADLIISK

TRRLSLFQQG

MTNWFLNFGL

FFETALAAFL

QYTPGVNTGL

151 |

195

URCHIN

RMYPLRIGWW

FVAFPFSLLI

FVYDECRRFI

LRHNPGGWVE

LETYYJ

FLY

RMYPLKLVWW

FPAIPFALAI

FIYDETRRFY

LRRNPGGWLE

QETYYJ

SHRIMP

RMYPLKINWW

FPALPFSFLI

FVYDEARKFI

LRRNPGGWVE

QETYYJ

NEMATODE

RMYGLRFSWW

FCALPFSILI

FVYDEIRRFL

IRRYPGGWVE

RETYYJ

FROG

RMYPLKPTWW

FCAFPYSLII

FIYDEVRKLI

IRRSPGGWVE

KESYYJ

HYDRA

RLRPMNFTWW

LPGLPFSLLI

FVYDEIRRYL

LRKNPGGWVE

KETYYJ

* * *

* *

* * * *

The shaded blocks and bold lettering indicate the putative transmembrane domains identified by Kyte-Doolittle hydropathy scores in the sea urchin
sequence (bold lettering; see Fig. 1). Genus names for the organisms and GenBank accession numbers for the sequences: urchin = Strongylocentrotus (this
study), fly = Drosophila (AF04494), shrimp = Anemia (X56650), nematode = Caenorhabditis (U18546), frog = Xenopus (U49238), Hydra = Hydra
(M75140); * = amino acid identity for all sequences; J = termination codon.

mains and a large extracellular loop between transmem-
brane domains 7 and 8 (Canfield and Levenson, 1993;
Blanco and Mercer, 1998).

Northern blots using the 3' UTR of clone #020 specifi-
cally recognized a 4.5 to 4.7 kb transcript under high strin-
gency (Fig. 2). In another sea urchin, Hemicentrotus pul-
cherriimts, the full-length «-Na+,K+-ATPase cDNA has
been cloned and has an mRNA transcript size of 4.6 kb
(Mitsunaga-Nakatsubo et a/., 1992a). The a-Na+,K + -ATP-
ase gene is differentially expressed during development in

5. purpuratus (Fig. 2). The level of mRNA transcripts is low
during early cleavage, then rises rapidly around gastrulation
(at 25-36 h postfertilization; Fig. 3). After gastrulation,
mRNA returns to a low level comparable to that initially
found in the egg (Fig. 3). The rapid disappearance of the
a-Na+.K+-ATPase transcript from the total RNA pool after
gastrulation closely followed a first-order exponential decay
model [/lv)= 98.512 eK)l28xl; r = 0.988; Fig. 3], The
decay constant of the regression is equivalent to a degrada-
tion rate of 7.8% h"1 of the transcript. At 83 h, a-subunit

104

A. G. MARSH ET AL.

extracellular

200 150 100 50

AA residues from 3' ORF terminus

TAA

Figure 1. Secondary structure characterization for the last 195 amino
acids of a putative a-Na+,K*-ATPase cDNA cloned from Stront>vloct'ii-
trotus purpuratus. The Kyte-Doolittle hydropathy score suggests the pres-
ence of four transmembrane domains, which match the structure of other
a-Na+,K+-ATPase subumts (Blanco and Mercer. 1998).

transcripts were barely detectable under the conditions we
used for Northern blots of total RNA.

The rapid increase in a-Na + ,K^-ATPase mRNA tran-
scripts during gastrulation in 5. purpuratus was paralleled
by a concomitant increase in the total activity of the sodium
pump (Fig. 4). Activity levels were very low during early
development in S. purpuratus and then increased after 20 h
to a maximum level at the pluteus larval stage (Leong and
Manahan. 1997). The rapid increase in activity between 20
and 40 h of development (Fig. 4) can be described by the
exponential function (fl\) = 1.167(1 + e|lv x""4571)-': r =
0.9664; maximum activity of 1.17 jumol P, h"' mg~' pro-
tein]. The present study resolves the increase in enzyme

f(x)= 98.51 2e('0128x>
r2 = 0.988

I - ' - 1 - ' - 1
20 40 60

Developmental time (hours)

Figure 3. Relative transcript levels of the a-Na*,K*-ATPase cDNA
during development in Strongylocentrotus purpuratus (quantified from
Fig. 2). mRNA levels are presented relative to the maximal expression
obtained at 36 h postfertilization. The rapid decline in mRNA abundance
after 3f> h his a first-order exponential decay function (r = 0.988;
regression line plotted with shaded symbols).

activity at a finer time scale (cf. Leong and Manahan. 1997)
and reveals the close coordination between a-subunit gene
transcription and the assembly of functional sodium pumps
in sea urchin embryos between fertilization and gastrula-
tion.

Discussion

It has long been a general goal of physiological ecologists
to identify a sensitive biochemical indicator of an animal's
physiological state or metabolic activity — for example, the
ratio of RNA to DNA (Westerman and Holt, 1994) or the
glycolytic enzyme activities (Childress and Somero, 1990).
For developmental stages with low biochemical contents,
such assays are often not possible. Molecular biological
techniques have the necessary sensitivity and potentially
offer an alternative for assessing physiological state in lar-
vae and small zooplankton. Because the sodium pump con-
sumes such a large portion of cellular energy metabolism

9.5-

7.5 —

4.4 —

2.4-
14 —

10 12 14

18 20

25 31 36 42 48 60 72 83

<28S
<18S

Figure 2. Northern blot hybridization of total RNA during development in Strongylocentrotus purpuraius
using a radiolabeled probe from the .V-untranslated domain of the «-Na*,K + -ATPase cDNA clone. RNA
samples were collected at short time intervals during embryogenesis as shown by the hours post-fertilization at
the top of each lane. Molecular size (kilobases) is indicated on the left; ribosomal RNA positions are indicated
on the right. The probe recognizes a single transcript that is approximately 4.6 kb in size.

SODIUM PUMPS IN SEA URCHIN EMBRYOS

105

Q- Q."

* E

1 25 -
1.00 -
075 -
0.50 -
0.25 -

000 -> T

- 100%

- 80% £ 6

oj E

h 60% CD g-

- 40% > 1

- 20% a a

- 0%

40 60

Age (hours)

Figure 4. Total enzyme activity of Na+.K+-ATPase during gastrula-
tion in Strongylocentrotus purpuratus. Protein-specific enzyme activity
(closed circles) is plotted on the left axis; mRNA levels from Fig. 3 are
overlaid (dashed line) to illustrate the temporal relationship between a-sub-
unit gene expression and the appearance of functional sodium pump
proteins. Data from Leong and Manahan (1997) are also plotted (open
circles) to show the pattern of relative enzyme activities.

(e.g., 40% in sea urchin larvae, Leong and Manahan 1997),
it would seem to be a good candidate for such an assay, with
the potential to provide sensitive information regarding
rates of energy utilization in a single larva.

Several lines of evidence strongly support the conclusion
that the partial cDNA clone (#020) in Strongylocentrotus
purpuratus is the a-subunit of the sodium pump: ( 1 ) the
putative amino acid sequences show a high similarity to
those of other animals; (2) the 3'-UTR probe recognizes a
4.6-kb transcript, which is the full-length transcript size in
other invertebrate species; (3) the ontogenetic increase in
expression during gastrulation is similar to the expression
pattern in another sea urchin (Mitsunaga-Nakatsubo ct ai,
1992b); (4) total Na+,K+-ATPase enzyme activities show a
concomitant increase as mRNA transcripts of clone #020
accumulate during gastrulation.

In the sea urchin Hemicentrotus pulcherrimus. the ex-
pression of the a-Na+,K+-ATPase gene increases rapidly
during gastrulation (Mitsunaga-Nakatsubo et ai, 1992b). In
5. purpuratus, the expression of the a-Na+,K + -ATPase
gene evidences a similar pattern of ontogenetic regulation,
with a sharp rise during gastrulation followed by a subse-
quent decline to much lower levels. In conjunction with the
total Na+,K+-ATPase enzyme activity that is present during
development (this study. Fig. 4; see also Leong and Mana-
han, 1997). temporal changes in both mRNA transcripts and
protein activity indicate that the enzyme activity is low
during early cleavage. At the point when an embryo ap-
proaches gastrulation, a-subunit gene transcription and sub-
sequent mRNA translation increase greatly, producing a
large increase in sodium pumps (Fig. 3), presumably as a
necessary component of the physiological function of pro-
liferating cells.

Once these pumps have been synthesized, mRNA tran-

scripts for the a-Na+,K + -ATPase are rapidly lost. The
decrease in mRNA levels over time fits a first-order expo-
nential decay model (7.8% h"') so that by 83 h, transcription
of the a-subunit gene was barely detectable (Fig. 2). At
gastrulation. S. purpuratus appears to have synthesized
most of the necessary sodium pumps. Total enzymatic ac-
tivities show little increase after 50 h, further supporting this
observation that the number of Na+,K+-ATPase ion pumps
is set by the rapid transcription during gastrulation. and that
once these transcripts are degraded, an early larva's sodium
pump complement remains unchanged until further growth
occurs, usually after feeding is initiated.

In vertebrates, the a-subunit Na+,K+-ATPase has several
isoforms (Rossier et at., 1987) that differ in many aspects,
including sensitivity to proteases and cross-linking agents
(Sweadner, 1979). electrophoretic mobility (Peterson et ai,
1982), and affinity for ouabain (Lytton et al, 1985). In brine
shrimp (Anemia salina), the a-Na + ,K+-ATPase is present
in two isoforms that are differentially expressed during
early development (Peterson et al.. 1982). In the sea urchin
Hemicentrotus pulcherrimus, two a-subunit isoforms are
expressed during embryogenesis (Yamazaki et al. 1997).
However, these two isoforms are encoded by a single gene
and have identical sequences except for the 5' leader se-
quences (Yamazaki et ai. 1997). If 5. purpuratus, like H.
pulcherrimus, has a similar isoform complement, then the
cDNA probe we used for the present study (from the 3'-
UTR) should hybridize to other a-subunit isoforms ex-
pressed during early development. Regardless of the mech-
anism, the disparity at 83 h postfertilization between the
transcript measurements and the complement of active so-
dium pumps indicates the difficulty in isolating a single
molecular factor to be used as an index for physiological
rate processes.

The observation that Na+,K + -ATPase gene transcription
and translation events are limited to a brief developmental
period is intriguing. The sodium pump is considered to be a
"housekeeping" protein. Consequently, for such an impor-
tant physiological process, we would have expected the
expression of a subunit gene to be constitutive and at a low
level so that there would always be some subunit synthesis
to replace any turnover in functional pump proteins. Such a
continual level of replacement might have offered a sensi-
tive assay for assessing the physiological state of individual
larvae by providing a molecular index of the activity of one
of the most energy-demanding cellular processes. However,
this is not the case. The a-subunit expression is develop-
mentally regulated so that gene expression is initiated rap-
idly at about 20 h. peaks at about 36 h, and is subsequently
"turned-off." Such a temporal pattern of regulation high-
lights the difficulty of using molecular probes as simple
indices of physiological state. Similar difficulties in the
interpretation of physiological activity and expression have

106

A. G. MARSH ET AL.

been found for other specific housekeeping genes (e.g.,
Weinstein el al., 1992; Yang and Somero, 1996). For the
multiple enzymes in metabolic pathways, the control mech-
anisms at the level of genes and proteins are even more
complex (Hochachka el al., 1998).

Ontogenetic changes in the metabolic rates of embryos
have important consequences for subsequent survival be-
cause of the finite quantity of energy reserves in an egg.
During development, metabolic rates increase in embryos as
their cell numbers increase (Marsh et al, 1999), and the
activity of the sodium pump can consume a large fraction of
total metabolism in some sea urchin embryos and larvae
(Leong and Manahan, 1997, 1999). Understanding ontoge-
netic changes in sodium pump activities is important for
assessing the metabolic energy costs of development. In the
pluteus larval stage of S. purpuratus (at 83 h postfertiliza-
tion), the in vivo sodium pump activity consumes 40% of
total metabolism, with a potential reserve activity that could
increase to a maximum of 77% of metabolism (Leong and
Manahan, 1997). However, a-Na+,K+-ATPase gene ex-
pression is barely detectable at this point in larval develop-
ment (Fig. 3). Consequently, molecular assays for expres-
sion of this gene would not be informative for assessing
sodium pump activity as an index of a larva's physiological
state. It is likely that during development and growth many
physiological processes have functional rates of protein
activity that are not strictly paralleled in time by the expres-
sion of their genetic components. A knowledge of the tem-
poral relationship between gene and enzyme activities is
critical to developing a molecular genetic index of physio-
logical state in larval forms.

Acknowledgments

We thank E. Davidson and C. Smith for providing the
clone that we have characterized. D. Pace and M. Moore
assisted with the culture sampling. This project was supported
by California Sea Grant #R/MP-75C, and NSF #9420803.

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Reference: Biol. Bull. 199: 108-115. (October 2000)

Dimethylsulfoniopropionate in Giant Clams

(Tridacnidae)

RICHARD W. HILL1 *, JOHN W. H. DACEY:. AND AHSER EDWARD3

' Department of Zoology, Michigan State University. East Lansing. Michigan 48824; 2 Department of

Biology, Woods Hole Oceanographic Institution. Woods Hole, Massachusetts 02543; and College of

Micronesia-FSM. P. O. Box 159, Kolonia. Pohnpei. Federated States of Micronesia 96941

Abstract. The tridacnid clams maintain symbiotic associ-
ations with certain dinoflagellates (termed zooxanthellae).
Tridacnids are thus candidates to have high tissue concen-
trations of dimethylsulfoniopropionate (DMSP), a tertiary
sulfonium compound that is not synthesized by animals but
is commonly produced by dinoflagellates. This study estab-
lishes that DMSP is about an order of magnitude more
concentrated in the light-exposed and shaded mantle and
gills of Tridacna maxima and T. squamosa than in any other
known animal tissues. The DMSP concentration in the
light-exposed, siphonal mantle — the location of most zoo-
xanthellae— is an inverse function of body size, paralleling
an inverse relation between apparent density of zooxanthel-
lae (measured as pheophytin concentration) and body size.
The shaded mantle and gills are high in DMSP despite
having low densities of zooxanthellae, indicating that high
DMSP concentrations occur in molluscan tissue, not just in
algal cells. DMSP is almost an order of magnitude less
concentrated in the adductor muscle than in other tissues.
The high DMSP concentrations found in tridacnids, by
providing abundant substrate for formation of volatile di-
methylsulfide. probably explain the peculiar tendency of
tridacnids to rapidly develop offensive odors and tastes after
death: a serious problem for their exploitation as food.
Tridacnids are the one group of animals in which DMSP
concentrations are high enough in some tissues to be in the
range capable of perturbing enzyme function at high phys-
iological temperatures. Thus, tridacnids may require en-
zyme forms adapted to DMSP.

Received I December 1999; accepted 16 June 2000.
* To whom correspondence should be addressed. E-mail: hillr@msu.edu
Ahhreviations: DMS. dimelhylsulride; DMSP. dimethylsultomopropi-
onate.

Introduction

Some of the most interesting marine animals are those
that maintain symbiotic associations with dinoflagellates.
The symbiotic dinoflagellates are known as zooxanthellae.
Included are the reef-building scleractinian corals, many
alcyonarians, all of the about eight species of giant clams of
the family Tridacnidae, and a few other bivalves (Ma-
ruyama et al.. 1998). Dinoflagellates, as a group, are note-
worthy for synthesizing relatively large quantities of di-
methylsulfoniopropionate (DMSP), a nonvolatile tertiary
sulfonium compound that is the precursor of volatile di-
methylsulfide (DMS) (Keller ct al., 1989a,b). DMSP is not
synthesized endogenously by animals. However, the wide-
spread synthesis of DMSP by dinoflagellates provides rea-
son to predict accumulations of DMSP and DMS within the
tissues of zooxanthellate animals. This prediction has been
assessed heretofore only in reef-building scleractinians. The
presence of the reduced-sulfur compounds in reef-building
corals was confirmed initially by the observation of DMS
release from damaged reefs (Andreae et al.. 1983). Later,
two of us quantified DMSP and DMS in the tissues of
healthy corals and in free-living dinoflagellates isolated
from corals (Hill et al., 1995b).

In giant clams, the zooxanthellae, which currently are
assigned to two of the major subdivisions of Symbiodiniiim
(Rowan, 1998), occur primarily in the siphonal mantle
tissue (Norton et al., 1992). This expansive tissue faces
upward when the clams are in their natural orientation and
is presented to the sun as a light antenna. The part of the
mantle that is positioned near the downward-facing byssal
opening and hinge, shaded from the sun. contains relatively
few zooxanthellae (documented in this study). Similarly,
zooxanthellae are sparse or absent from the adductor mus-
cle, gills, and other tissues besides the siphonal mantle.

IDS

DMSP IN GIANT CLAMS

109

Recent molecular evidence confirms that the giant clams are
a monophyletic group (Maruyama el ai. 1998). This paper,
based on two species, is the first to look for or quantify
DMSP and DMS in the group. It is also the first to examine
DMSP and DMS in zooxanthellate animals besides reef-
building scleractinians.

DMSP and DMS are of current interest for several rea-
sons. The most prominent is that atmospheric DMS origi-
nating from marine organisms affects cloud cover and cli-
mate over the oceans (Shaw. 1983; Charlson et ai, 1987;
Falkowski et ai, 1992; Andreae and Crutzen. 1997). Coral
reefs are sufficiently extensive that DMS from corals could
be of local climatic significance (Andreae et al., 1983; Hill
et ul.. 1995b). However, DMS from giant clams seems
unlikely to be climatically important except as a minor
component of reef-community contributions, because the
clams are insufficiently abundant, especially in modern
times. On the other hand, DMSP and DMS in giant clams
are likely important in two major ways.

First, DMS is well known to have critical effects on taste
whenever it is present in organisms used for food (Mo-
tohiro, 1962; Ackman et ai. 1966: Levasseur et ui. 1994).
Giant clams have long been important sources of food and
protein throughout much of the Indo-Pacific, so much so
that many clam populations are decimated (Munro, 1989;
Dalzell et ai. 1996). A problem for the indigenous and
commercial exploitation of giant clams is that after death,
the meat often promptly develops a strong, "unquestionably
offensive" odor (Peavey and Riley, 1993, 1994), which is
sometimes described as seaweed- or kelp-like (e.g.. Cowan,
1988). The cause has been unknown. Our experience with
DMS and with the odors of the clams led us to postulate that
the cause is DMS derived from the algal symbionts. If this
hypothesis is confirmed, the stage will be set for a rational
approach to a problem that seriously detracts from the value
of the clams as sources of food in subsistence economies
and as aquacultured species. Giant clams are attractive
animals for aquaculture (Braley, 1988; Munro, 1989) in part
because their symbionts enable them to get most of their
energy for maintenance and growth from sunlight (Klumpp
and Griffiths, 1994); they have been described as "the only
phototrophic, and thus self-feeding, potential farm animals
known to humankind" (Munro, 1989).

Second, recent research (Nishiguchi and Somero, 1992:
Karsten et ai. 1996) has established that DMSP sometimes
negatively perturbs enzyme function at high physiological
temperatures. The enzyme-perturbing effects of DMSP
have heretofore been considered relevant only to plants and
algae, because no animals have been known to have DMSP
concentrations sufficiently high to be influential. We hy-
pothesized that the giant clams might have tissue DMSP
concentrations high enough that they could potentially re-
quire biochemical adaptations to DMSP.

Materials and Methods

Tridacmi maxima and T. sc/namosa were collected near
Pohnpci in the Federated States of Micronesia. Two collec-
tions were made: one of six specimens of T. maxima (1 1-14
cm shell length) and four of T. squamosa ( 10-14 cm) on 13
July 1995. on the northern barrier reef of the main island of
Pohnpei; the second of three specimens of T. maxima
(16-18 cm) and nine of T. si/iuunosa (14-22 cm) on 17
August 1995, in the lagoon of Ant atoll. 27 km from the first
location. Collected animals were taken by boat to Kolonia.
Pohnpei. where they were promptly dissected (3-4 h after
collection). Samples (1-2 g) were cut from four tissues of
each clam: light-exposed, siphonul mantle: shaded mantle
from near the byssal opening: adductor muscle; and gill
(sometimes both right and left gill sets combined). Each
sample of tissue was weighed and placed into 20 ml of
HPLC-grade absolute methanol in a 37-ml glass vial sealed
with a Teflon-faced butyl-rubber septum (Regis Technol-
ogy) secured with a crimped aluminum ring.

Samples prepared in Pohnpei were shipped in light-tight
containers to Woods Hole, Massachusetts, for assay. Ship-
ments required 6-8 days to reach Woods Hole, and assays
were completed 8-10 days after collection. Vials had been
filled with methanol and weighed prior to shipment to
Pohnpei, and they were reweighed on return to Woods Hole
as a check for leakage (none occurred).

To measure DMSP and DMS, 1.0 ml of methanol was
drawn by syringe from each tissue-sample vial and placed in
25 ml of 2 N KOH in a sealed vial. Incubation in cold base
quantitatively converts DMSP to DMS (Dacey and Blough,
1987). Thus, after incubation (20 h. 2°C), DMSP (plus any
DMS present in the initial samples) could be measured by
assaying DMS. For assay, the vials of KOH were brought to
room temperature (ca. 22° C), and DMS was measured in
head-space samples by gas chromatography, using a Chro-
mosil 330 (Supelco) column at 54°C for separation, Sievers
350B sulfur chemiluminescence detector, and Hewlett
Packard 3390A integrator. Standards were prepared in 25
ml of 2 N KOH plus 1 ml methanol using reagent grade
DMS (Fluka). All measures were duplicated.

Chlorophyll in each tissue sample was measured as an
index of the density of zooxanthellae. In fact, because some
chlorophyll could have degraded to pheophytin during sam-
ple preparation and shipment, and because our interest was
not in chlorophyll itself but in an index of relative levels of
zooxanthellae, we degraded all chlorophyll by acidification
(3 niA/ HC1) and used the resulting total pheopigment levels
as our index (Hill et al.. 1995b). Chlorophyll a and pheopig-
ment a were measured using a calibrated Turner model 10
fluorometer and standards of chlorophyll a from spinach
(Sigma) in methanol following procedures recommended by
Holm-Hansen and Reimann (1978). Aliquots of methanol
drawn by syringe from tissue-sample vials were diluted in

110

R. W. HILL ET AL.

600

500

400

§ 200

g 100

.c
o^

V=-17.6X+ 568

10 12 14 16 18 20 22
Length of Shell (cm)

Figure 1. Pheophytin a per gram of siphonal mantle (wet weight) as a
function of shell length in Tridacna maxima (filled symbols) and 7".
squamosa (open symbols). The line and equation are results of linear,
least-squares regression, all data taken together. Two data points for 1 7-cm
clams are shifted laterally for clarity.

absolute methanol to place concentrations on the linear parts
of calibration curves.

In Pohnpei. tissue samples were prepared, weighed, and
inserted in vials as solid blocks of tissue to minimize po-
tential loss of DMS. To assure that extractions of DMSP,
DMS, and chlorophyll from samples into methanol were
complete, tissue samples were removed briefly from sample
vials after completion of the measurements described above
and minced with scissors into small pieces (ca. 1 mm
greatest thickness) that fell back into the vials (the proce-
dure required about 1 min per sample). After 24-48 h, all
measurements were repeated. Chlorophyll concentrations
were not altered by mincing, and concentrations of pooled
DMSP and DMS (measured as earlier described) were al-
tered little, if at all (possibly 3%-5% in the case of mantle
samples). The mincing test demonstrated that extraction
from whole tissue was complete or virtually complete, and
all assays for a sample were averaged to obtain the results
reported. A second check on our technique was to test
whether the high tissue DMSP concentrations we encoun-
tered might be so high as to saturate the methanol. The
highest concentration of DMSP in the methanol in any
tissue-sample vial was 4.3 mA/. Without attempting to de-
termine the absolute solubility of DMSP in methanol, we
ascertained that pure DMSP sufficient to make a solution
three times as concentrated dissolved rapidly in methanol.
Thus, saturation of the methanol in the sample vials did not
occur.

Total amounts of pooled DMSP and DMS (in micro-
moles) and of pheopigment a (in micrograms) in tissue
samples were calculated from concentrations in sample-vial
methanol by multiplying by the volume of methanol (20
ml), then expressed per unit wet-weight of tissue.

Results

For simplicity of language, we express results in terms of
DMSP, although we do not know what proportions of the
DMS analyzed were initially in the form of DMS or DMSP.
Most was probably DMSP (the nonvolatile form known to
be the principal chemical species in algae; see also com-
parative data on molluscs presented later). Results from
Tridacna maxima and 7". squamosa were not statistically
distinguishable and thus are generally pooled.

Pheophytin a per gram of siphonal (light-exposed) mantle
was an inverse function of body size (analyzed by linear
regression, P = 0.008, r = 0.30), as shown in Figure 1.
DMSP per gram of siphonal mantle was likewise an inverse
function of body size (P = 0.015. r = 0.26). as shown in
Figure 2. The concentrations of DMSP and pheophytin in
the siphonal mantle were strongly correlated (P < 0.001 ), as
shown in Figure 3. The ratio of DMSP concentration to
pheophytin concentration in siphonal mantle was quite con-
sistent, the mean and standard error being 0.107 ± 0.0048
(range: 0.070-0.158) /j,mol//u,g.

Pheophytin per gram did not show a systematic relation
to body size in the byssal (shaded) mantle, adductor, or gill.
Table 1 summarizes the pheophytin concentrations in these
tissues. Comparison to Figure 1 shows that the concentra-
tions were far lower than in the siphonal mantle. The simple
mean concentration in the siphonal mantle, 303 ju.g/g, is of
uncertain utility because of the regular relation between
siphonal-mantle concentration and body size, but it helps
bring to light that pheophytin concentrations in the byssal
mantle and gill were only about 7% and 3% as high as those
in siphonal mantle. Concentrations of pheophytin in the
adductor approached zero.

Q.
t/5

X=-1.43X+52.7

10 12 14 16 18

Length of Shell (cm)

Figure 2. Concentration of dimethylsulfomopropionate (DMSP) in
siphonal mantle (wet weight) as a function of shell length in giant clams.
The line and equation are results of linear, least-squares regression, all data
taken together. Filled symbols. Tridacna maxima; open symbols, T. squa-

DMSP IN GIANT CLAMS

Ill

| 20

r=0.74

100 200 300 400 500

[Pheophytm] in Siphonal Mantle (|ig/g)

Figure 3. Correlation between concentrations of dimethylsulfoniopro-
pionate (DMSP) and pheophvtin a in siphonal mantle of giant clams. Filled
symbols. Tridacna maxima; open symbols, T. squamosa. r = correlation
coefficient.

r=0.68 «

• .

0 0 •

~ca

>,
CD

• 0 ° •

*> o •

20 30 40

[DMSP] in Siphonal Mantle (umol/g)

Figure 4. Correlation between concentrations of dimethylsulfoniopro-
pionate (DMSP) in byssal and siphonal mantle in giant clams. Filled
symbols. Tridacna maxima: open symbols. T. si/iuimosa. r = correlation
coefficient.

As shown in Figure 4. the concentration of DMSP in the
byssal mantle tissue was strongly correlated with (P <
0.001 ) and similar to that in the siphonal mantle tissue, even
though densities of zooxanthellae in the byssal tissue, as
inferred from byssal pheophytin concentrations, were a
small fraction of those in the siphonal tissue. Presumably
because of the correlation between siphonal and byssal
concentrations, the byssal DMSP concentration exhibited a
regular relation to body size, similar to that in Figure 2
[linear regression: Kl/imol/g) = — 2.46X(cm) + 74.4; P =
0.001]. Byssal DMSP concentration showed no correlation
with byssal pheophytin concentration.

The DMSP concentrations in gill and adductor were

Table 1

Pheophytm a per gram of tissue of giant clams (wet weight, species
combined) in the three tissues that showed no relation benveen
concentration and body size

Pheophytin a (/ng/g)

Tissue

Mean

Range

Byssal mantle*

Gill

Adductor muscle

22.10
10.00

0.74

1.34-86.90
0.35-31.30
0.05-3.32

Data for siphonal mantle are omitted because they are presented else-
where (Fig. 1) and because the mean is a possibly misleading statistic for
a parameter that varies systematically with body size.

* The four highest values for byssal mantle occurred in four of the
smallest clams (two of each species), suggesting that the effort to keep
tissue-sample size consistent might have led to the inclusion of other types
of tissue in byssal-mantle samples of some small clams. If the four highest
values are excluded, the mean and range for byssal mantle are 12.8 and
1.34-33.1

unrelated to body size and uncorrelated with pheophytin
concentrations in the respective tissues. The DMSP concen-
tration in gill was positively correlated with that in mantle
(/• = 0.61, P < 0.01 for siphonal mantle; r = 0.45. P < 0.05
for byssal mantle), but the DMSP concentration in adductor
was not correlated with that in mantle. Table 2 presents
DMSP concentrations in gill and adductor. For comparison,
the means in siphonal and byssal mantle were 31.2 and 37.4
iiunoi/g (see Fig. 4). Note that gill exhibits DMSP concen-
trations similar to those of mantle. In adductor, however.
DMSP is almost an order of magnitude less concentrated
than in mantle.

Discussion

DMSP in the mantle and gill tissues of Tridacna maxima
and T. squamosa is far more concentrated than in any
animal tissue heretofore known. Most comparative data in
the literature represent pooled concentrations of DMSP and
DMS (similar to the data we collected). In discussing the
literature, we distinguish DMSP and DMS only if the orig-

Table 2

Dimethylsulfoniopropionate (DMSP) per gram of tissue of giant clams
(wet weight, species combined) in the nro tissues that showed no
relation between concentration and bod\ size

DMSP (/imol/g)

Tissue

Mean

Range

Gill

Adductor muscle

33.3
4.4

20.3-46.1
1.8-7.2

Data for mantle are omitted for reasons stated in note to Table 1.

112

R. W. HILL ET AL.

inal investigators did. We also exclude from consideration
tissues (e.g., stomach) that could contain unassimilated
food. Two surveys of DMSP concentrations in molluscs
have been carried out. lida and Tokunaga ( 1986) measured
both DMS and DMSP in 1 1 species of bivalves and 5 of
gastropods from Japanese waters. An average of 8% of the
total molar amount of the two compounds was DMS and
92% was DMSP in the mantle, gill, and adductor tissues of
the bivalves. The sum of the two concentrations was usually
less than 0.2 jumol/g. The single highest sum was 0.9
^mol/g in adductor muscle of oysters (Crassostrea gigas).
Ackman and Hingley (1968) reported 0-1.8 /nmol/g in the
tissues of 10 species of bivalves and gastropods from Ca-
nadian waters. Most values were toward the low end of the
range; the highest concentrations were in adductor muscles
of scallops (Placopecten megullciniciis) and oysters (C. vir-
ginica). The highest concentrations observed in populations
of mussels (Mytilux editlis) at Cape Cod, Massachusetts,
were 2-4 /imol/g (Hill et al., 1995a). In wild-caught fish,
muscle or liver concentrations of 0.2-1.0 /nmol/g (predom-
inantly DMSP in fresh tissue) are high and commercially
problematic because they cause off-flavors (see later) (Mo-
tohiro, 1962; Ackman ft al., 1967; lida et al., 1986; Dacey
ct al.. 1994). Even the highest tissue concentrations in fish
fed DMSP supplements were only 4-8 /imol/g (Ackman et
al., 1966). A survey of DMS and DMSP in several species
of shrimp and krill indicated that tissue concentrations are
very low in most species, although pooled concentrations
(predominantly DMSP) as high as 3 /j,mol/g are sometimes
observed in muscle of Eiiplun/sia superba (Tokunaga ct al.,
1977). In the context of these comparative data, the con-
centrations in the mantle and gill tissues of tridacnids,
averaging 31-37 jumol/g, are extraordinary. The only pub-
lished animal data that are at all in the same range come
from a single report on pteropods (Levasseur et al., 1994) in
which extremes of 30-40 /Ainol/g were observed (calcu-
lated form published data on tissue dry weights assuming
the animals to be 70% water). Such concentrations, how-
ever, are exceptional in the literature on pteropods; other
reports are 0.2-3.7 ju.mol/g (Motohiro. 1962; Ackman and
Hingley, 1968) or lower (Ackman ct al., 1972). Further-
more, the pteropod data are for whole animals, including
digestive-tract contents. In terms of documented evidence
on tissues of animals collected in the wild, the concentra-
tions in tridacnid mantle and gill are extreme: an order of
magnitude higher than the highest concentrations observed
in other bivalve or gastropod molluscs, fish, crustaceans,
and other animals. Other than the tridacnids, the animals
mentioned acquire DMSP strictly from ingested foods.

We hypothesize that the high concentrations in tridacnids
are a consequence of the production of DMSP by their algal
symbionts. Three pieces of evidence support this hypothe-
sis. First, at least in T. M/iianinsa, symbiont photosynthesis
supplies over 90% of all organic carbon acquired by indi-

viduals of the body sizes we studied (Klumpp and Griffiths,
1994). Thus, although the clams ingest food to some extent
and could acquire DMSP from their food, the known par-
titioning between phototrophic and heterotrophic nutrition
makes phototrophic production of DMSP the more likely
source of most DMSP. especially because symbiotic
dinofiagellates are documented to produce DMSP (Hill et
al., 1995b). Second, the sheer magnitude of the DMSP
concentrations in the tridacnids points to their algal symbi-
onts as the source, because in all the many species of studied
molluscs that lack algal symbionts, even the most extreme
DMSP concentrations seen do not come close to the routine
concentrations seen in the tridacnids. Third, there are sev-
eral reports that the density of zooxanthellae in tridacnid
tissues is an inverse function of body size (e.g.. Fisher et al.,
1985; Griffiths and Klumpp. 1996). and we found evidence
of this same relation in siphonal mantle (Fig. 1 ). If the
zooxanthellae are the primary source of DMSP and if the
zooxanthellae become less dense in the siphonal mantle as
individuals grow, one would predict that the DMSP con-
centration would be an inverse function of body size. This
is what we found (Fig. 2). The DMSP and pheophytin
concentrations in the siphonal mantle were well correlated
(Fig. 3). and the ratio of the concentrations was consistent at
0.107 ± 0.0048 (SE) /nmol/^g.

If our hypothesis is correct that the zooxanthellae are the
main source of DMSP in giant clams, then tissues other than
the siphonal mantle must receive DMSP by internal trans-
port from the siphonal mantle because, as our pheophytin
data confirm, the zooxanthellae occur at high densities only
in siphonal mantle (Table 1 ). Internal transport could occur
by circulation of hemolymph or by transport in the zoo-
xanthellal tubular system (Norton et al., 1992). Molluscan
tissues are well known to accumulate DMSP (Ackman and
Hingley, 1968; Hill et al., 1995a). The high concentrations
of DMSP in siphonal mantle could possibly be explained by
high concentrations in the algal cells only. However, the
byssal mantle and gills are so low in algal cells that the high
concentrations of DMSP there almost certainly demonstrate
that the animal cells of giant clams can experience very high
DMSP concentrations. The physiological basis for the dra-
matic difference in concentration between the adductor
muscle and other tissues (Fig. 5) awaits study.

Whatever the cause of the high DMSP concentrations in
tridacnids, the concentrations are likely to be important to
the biology of the clams in two major ways. The first is
taste. One of the principal conclusions of research on com-
mercialization of giant clams is that their meat is exception-
ally perishable because of the rapid development of a "par-
ticularly offensive and pervasive odor" (Peavey and Riley.
1994). Refrigeration (Peavey and Riley, 1994) or freezing in
a domestic free/er (Peavey and Riley, 1993) does little to
prevent this problem, even if the viscera have been re-
moved. Comparing mantle and adductor meal, a disagree-

DMSP IN GIANT CLAMS

113

Kidney

Siphonal
Mantle

Pericardium

Adductor
'Muscle

Hinge
Region

Cilli

Umbo

Byssal Mantle

Figure 5. A giant clam in the natural orientation showing average
pooled concentrations 1/u.mol/g) of dimethylsulfoniopropionate (DMSP)
and dimethylsulhde (DMS) in the four tissues studied (drawing by Jayne
Doucette).

able "seaweed-like" odor has been particularly associated
with the mantle (Cowan, 1988), and the mantle is far less
commercially valuable than the adductor (Braley, 1988;
Tisdell and Tacconi, 1993). These are serious matters in
many parts of the world where giant clams occur. The clams
are sources of food and protein in subsistence economies,
and a number of cash-poor governments are making sub-
stantial investments in the development of giant clam
aquaculture (Lucas, 1994). Partly because of preservation
problems, the mantle meat may actually have a negative
monetary value in commercial aquaculture (Hambrey and
Gervis. 1993).

We believe we have discovered the cause of the unattrac-
tive odor and taste, thereby setting the stage for a rational
approach to improvement. Although DMS has not been
mentioned previously as a likely component of triducnid
tissues, it is well recognized as an important taste constitu-
ent in other seafoods. DMS produced from dietary DMSP is
a negative taste factor in fish. The DMS generated during
certain sorts of processing offish meat containing as little as
0.1-1 ju.mol DMSP/g can cause the meat to smell "like
petroleum" or taste like turnip or radish (Motohiro, 1962;
Ackman et al., 1966) and force catches to be discarded. On
the other hand, very low concentrations of DMS are part of
the valued flavor of some clams and oysters (Ackman and
Hingley. 1968: Brooke et ai, 1968; lida and Tokunaga.
1986). Brooke et al. (1968), for example, found that about
0.02 /xmol/g of DMS helps impart a desirable "clamlike"
odor to A/vfl arenaria, whereas more than 0.3 jumol/g of
DMS is excessive.

Our data reveal that the potential for DMS formation in
tridacnid tissues, particularly mantle, is enormous. We as-

sume that in the fresh clams we studied, most of the sulfur
we measured was in the form of DMSP (lida and Tokunaga,
1986), which has unknown taste effects. After the death of
the clam. DMSP is likely under many circumstances (Mo-
tohiro. 1962: Ackman et ai. 1966) to be broken down to
DMS enzymatically (e.g., by bacterial DMSP lyases; Led-
yard el al., 1993) or nonen/ymatically (e.g.. Dancey and
Blough. 1987). With over 30 jumol/g of DMSP present, the
concentrations of DMS that could readily be formed are far
in excess of ones known to make all other foods inedible. It
is probably no accident that the methods devised by indig-
enous people to preserve tridacnid meat include acidic
washes and drying (Munro, 1989; Hambrey and Gervis,
1993). Acid pHs inhibit the nonenzymatic breakdown of
DMSP to DMS (Motohiro. 1962; Dacey and Blough, 1987),
and because DMS is very volatile, drying would remove it
from tissue. Looking to the future, it might be possible to
inhibit DMS formation in a chemically specific manner or
even to develop strains of zooxanthellae that produce little
DMSP.

The second major way in which DMSP concentrations
are potentially important to tridacnid biology is their bio-
chemical significance. Recognizing that DMSP is employed
by some organisms to help set the colligative properties of
cellular solutions. Nishiguchi and Somero (1992) studied
the effects of DMSP on cellular proteins. They found evi-
dence of temperature dependence. Whereas DMSP exhib-
ited stabilizing effects on proteins at low temperatures, it
could perturb protein function at high physiological temper-
atures. In particular. Nishiguchi and Somero found that
DMSP promotes the denaturation of glutamate dehydroge-
nase at 37°C in a concentration-dependent manner, with
effects evident at the lowest concentration tested, 100 mM
DMSP. Similarly. Karsten et al. ( 1996) observed a concen-
tration-dependent suppression of activity of malate dehy-
drogenase at 30°C: the suppression became evident at con-
centrations between 19 and 75 mM DMSP. Such effects of
DMSP have been tacitly assumed to be relevant just to
plants and algae because only plants and algae have hitherto
been thought to have native DMSP concentrations high
enough to be in the effective range. Our results make clear
that alone among animals, tridacnids can have DMSP con-
centrations within the range shown to have enzyme-perturb-
ing effects. In T. maxima and T. squamosa of the sizes we
studied, the mantle averages about 0.83 ml water per gram
wet weight (our unpublished data). Thus, if we assume that
the DMSP in mantle is entirely dissolved and distributed
evenly in tissue water, the mean concentration of DMSP in
the tissue water is over 40 mM. On the basis of the chemical
structural properties of DMSP and the preferential hydration
model. Nishiguchi and Somero (1992) argue that DMSP.
like dimethylsulfoxide. may be toxic to cells and may de-
nature proteins at high physiological temperatures. In warm
tropical waters and especially in shallows where solar heat-

114

R. W. HILL ET AL.

ing can occur, the high DMSP concentrations of tridacnids
may be stressful by-products of extreme exploitation of
phototrophic nutrition (Klumpp and Griffiths, 1994). Tri-
dacnids may require specializations of metabolic chemistry
to reduce or tolerate enzyme-perturbing and other toxic
effects of their high DMSP concentrations. Interspecific
differences in such specializations might help explain dif-
ferences in growth rates and energetics that have previously
remained enigmatic (Klumpp and Griffiths, 1994).

Acknowledgments

Special thanks to Raouf Kilada for planting the seeds of
this study. Thanks to Dave Kulis, Ishmael Paul, and Eric
Zettler for help with sampling and chlorophyll assays. Sup-
ported by NSF OCE-97 13407 and WHOI Mellon
27005129. This is Woods Hole Oceanographic Institution
Contribution No. 9993.

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Reference: Biol. Bull. 199: 1 16-125. (October 2000)

Particle Transport in the Zebra Mussel,
Dreissena polymorpha (Pallas)

SHIRLEY M. BAKER1 *. JEFFREY S. LEVINTON1. AND J. EVAN WARD2

^Department of Ecolog\ and Evolution. State University of New York. Stony Brook. New York 11794;
and 'Department of Marine Sciences. University of Connecticut. Groton, Connecticut 06340

Abstract. The capture, transport, and sorting of particles by
the gills and labial palps of the freshwater mussel Dreissena
pohmorpha were examined by endoscopy and video image
analysis. More specifically, the morphology of the feeding
organs in living zebra mussels was described; the mode and
speeds of particle transport on the feeding organs was mea-
sured; and the sites of particle selection in the pallial cavity
were identified. Particle velocities (outer demibranch lamellae.
90 jam s~ '; inner demibranch lamellae. 129 jam s~ '; marginal
food groove of inner demibranchs. 1 56 ju,m s '; dorsal ciliated
tracts, 152 /xm s"1). as well as the movement of particles on
the ctenidia and labial palps of D. polymorpha. are consistent
with mucociliary, rather than hydrodynamic. transport. Parti-
cles can be sorted on the ctenidia of zebra mussels, resulting in
a two-layer transport at the marginal food groove of the inner
demibranch. That is: preferred particles are transported inside
the marginal groove proper, whereas particles destined for
rejection are carried superficially in a string of mucus. Sorting
also occurs at the ventral margin of the outer demibranch;
desirable particles are retained on the outer demibranch.
whereas unacceptable particles are transferred to the inner
demibranch and ultimately excluded from ingestion. We sug-
gest that the structure of homorhabdic ctenidia does not pre-
clude particle sorting, and that some ecosystem modifications
attributed to zebra mussels may ultimately be due to ctenidial
sorting mechanisms.

Introduction

Many suspension-feeding organisms, including bivalves,
sort food particles on the basis of size ( Vahl, 1972; Stenton-

Received 27 October 1999: accepted 14 July 2000.

* Present address: Department of Fisheries and Aquatic Sciences. Uni-
versity of Florida. 7922 NW 71st Street. Gainesville. Florida 32653-3071.
E-mail: smbaker@mail.ifas.url.edu

•\hhrc\-uitioits: OIT, optical insertion tube of an endoscope.

Dozey and Brown, 1992; Defossez and Hawkins, 1997) and
quality (MacDonald and Ward, 1994; Arifin and Bendell-
Young. 1997; Ward et al.. 1997). Moreover, endoscopie
examination and video image analysis are now frequently
used to directly observe particle capture, transport, and
sorting in marine bivalves (Ward et ai. 1991, 1998;
Beninger et ai. 1992; Ward. 1996). With this method,
pallial structures can be observed in vivo in relatively un-
disturbed specimens, so that the direction, velocity, fate, and
hydrodynamic mechanisms of ciliary transport of particles
within the pallial cavity can be determined directly (Ward et
al.. 1993). The endoscope does not disturb the morpholog-
ical and hydrodynamic relationships of pallial organs, an
advantage over the usual techniques of excision and surgical
alteration of living bivalves (Nelson, 1960; Galtsoff, 1964;
J0rgensen, 1966).

In previous studies. Ward et al. ( 1998) found that particle
sorting in oysters (subclass Pteriomorphia; psuedolamelli-
branch, heterorhabdic. plicate gills) takes place on the
ctenidia; particles of differing food qualities are partitioned
between the marginal groove and the dorsal ciliated tract. In
contrast, the ctenidia of marine mussels (subclass Pteri-
omorphia; filibranch. homorhabdic, nonplicate gills) play
little role in particle selection. Ward et al. (1998) suggested
that selection by the oyster ctenidia reflects the greater
complexity of those organs. (For review of bivalve gill
anatomy, see Atkins, 1937a,b; Ruppert and Barnes, 1994.)

Zebra mussels [Dreissena polymorpha (Pallas. 1771 )] are
freshwater suspension-feeding bivalves in the subclass Het-
erodonta. Like marine mussels, zebra mussels have homo-
rhabdic. nonplicate ctenidia. But. unlike marine mussels,
they are eulamellibranchs; ctenidial filaments are connected
by interfilamentous tissue junctions. This ctenidial condi-
tion is more similar to that of the pseudolamellibranch
oysters in which the filaments are connected by some.

116

PARTICLE TRANSPORT IN THE ZEBRA MUSSEL

117

although not extensive, interfilamentous tissue junctions.
An examination of zebra mussel ctenidia. therefore, might
indicate whether ctenidial sorting is related to morphology
or to phylogeny. Indeed, recent flow-cytometry studies by
Baker et al. ( 1998) demonstrate that the zebra mussel pallial
organs effectively sort particles. In this investigation, we
use endoscopic examination to observe ( 1 ) the morphology
of feeding organs in living zebra mussels. (2) the mode and
speeds of particle transport on the feeding organs, and (3)
sites of particle selection in the pallial cavity. We compare
our endoscopic examination of zebra mussels with previous
reports of feeding processes in both zebra mussels and
marine bivalves.

Zebra mussels have invaded many freshwater localities in
Europe and North America. In systems where large popu-
lations of these mussels have become established, not only
has phytoplankton biomass declined (Holland, 1993; Fah-
nenstiel et al., 1995; Caraco et ul., 1997), but seston com-
position has changed as well (Vanderploeg et al,, 1996;
Smith et al., 1998; Strayer et al.. 1999). Understanding the
form and function of feeding structures in zebra mussels and
their mechanisms of particle selection will contribute to a
better understanding of these effects on ecosystems.

Materials and Methods

Phytoplankton cultures were obtained from the Univer-
sity of Texas Culture Collection and grown in a freshwater
enrichment medium WCL1 (Guillard, 1983: Guillard and
Hargraves, 1993). Cultures were grown at room tempera-
ture, under a 16:8 h light:dark regime. Species of
phytoplankton that were cultured included Cvclotellu »ie-
neghiniana (LB 2455; barrel-shaped, 18x6 jam) (Bacilla-
riophyceae). Crucigenia tetrapedia (63; disk-shaped, 5 X
11 /nm), Scenedesmus quadricauda (LB 614; four cells
stacked, total 25 X 10 ;am) (Chlorophyceae), and Micro-
cystis aeruginosa (LB 2386; spherical, 4 ju,m) (Cyanophy-
ceae). Cell dimensions were measured with a compound
microscope and calibrated ocular micrometer.

Nonliving particles were also used in endoscopic obser-
vations. Polystyrene beads (Polysciences. Inc., Warrington,
PA) of 1, 10, or 22 /xm were often used as tracer particles.
Dead cattail (Typha sp.) leaves from the previous growing
season were collected for use as detrital material. The leaves
were washed of debris and processed in a blender with
distilled water for 5 min. The resulting suspension was
sieved through a 20-ju.m nylon screen, and the retained
particles (>20 ju,m) were discarded; 90% of the particles in
the remaining suspension were <3.5 jam, as measured by an
electronic particle counter (Coulter Electronics, Multisizer).

Specimens of Dreissena polymorpha, about 20 mm in
length, were collected from the Hudson River at Tivoli,
New York, or from the Huron River, Ann Arbor, Michigan.
Mussels were maintained in 40-1 aquaria at 16°C and fed a

daily ration of cultured phytoplankton plus a mixture of
preserved diatoms (Diet C, Coast Seafoods, Co.. Quilcene.
WA). Partial water changes (ca. 20%) were performed on
alternating days; freshwater was prepared according to
Sprung (1987).

We prepared zebra mussels for endoscopy by drilling a
small hole (<2 mm in diameter) in one valve with a rotary
tool (Dreinel. Racine, WI) and cauterizing the underlying
mantle tissue. The hook side of a piece of hook and loop
fastener (Velcro brand) was cemented with epoxy to the
valve opposite the drilled hole for later use in positioning
the animal for examination. The mussels were allowed to
recover for at least one day. This treatment caused no
apparent adverse change in the behavior of the mussels, and
shell and mantle repair at the site of the drilled hole often
began within several days.

Endoscopic examinations were performed according to
Ward et al. (1991, 1993. 1994). An endoscope (K12-09-15-
53, Olympus Corp., Lake Success, NY), with an optical
insertion tube (OIT) of 1.2 mm diameter, was connected to
an optical zoom-adaptor (Scholly Fiberoptic, Denzlinger,
Germany) and attached to a monochrome or color CCD
camera (4990 or 8280. Cohu Electronics. San Diego, CA).
A halogen (HLS24-0, Welch Allyn. Skaneateles Falls. NY)
or xenon lamp (ALS-6250U, Olympus High Intensity He-
lioid Light Source) provided light to the OIT. The camera
and endoscope were mounted on a macro-focusing rail,
allowing hue adjustments of the OIT. Video was recorded at
30 frames s~' on an sVHS videocassette recorder (VCR)
(AG-1960, Panasonic Industrial Company, Secaucus, NJ).

For endoscopic examination, mussels were placed in a
500-ml plastic container set in a 15-1 water bath with its
temperature maintained between 16° and 18°C. A dome
inside the plastic container had been covered with the loop
side of Velcro-brand fastener, allowing rapid mounting and
precise positioning of mussels. The OIT was inserted into
the pallial cavity of the mussel through the inhalent siphon,
the pedal gape, or the drilled and cauterized hole. Record-
ings were made after the mussel showed active feeding
behavior, as indicated by extension of the mantle and si-
phons and by the intake of particles. Mussels were exposed
to suspensions of one or two particle types at concentrations
of 104, 105, and 106 particles ml~'. Particle suspensions
were delivered to the plastic container by gravity from a 4-1
carboy. The container was frequently flushed to maintain
particle concentration, which was also monitored with a
Coulter Multisizer.

We observed and recorded the positions and movements
of the ctenidia and labial palps, as well as the movement of
particles on these organs. The best observations were made
when the OIT was inserted into the pallial cavity through
the drilled hole in the shell and mantle. In this position, the
mussels fed normally, uninterrupted by movements of the
OIT. Although the pallial cavity could be entered through

118

S. M. BAKER ET AL

the inhalent siphon, any movement of the OIT resulted in
the cessation of feeding. And when the pallial cavity was
entered through the pedal gape, the foot usually touched the
OIT, coating it with mucus. Results were based on the
examination of 21 mussels.

Particle velocities on feeding structures were determined
from the number of video frames required for a particle to
traverse a known distance. Distances were calibrated ac-
cording to Ward (1996): i.e.. the pallial organs were dis-
sected from several mussels, and the widths of the ctenidial
filaments, palp ridges, and marginal grooves were measured
with a compound microscope equipped with a calibrated

Ant

iPL

ocular micrometer. Velocities
means ± 1 standard deviation.

Results

s~') are presented as

When observed by endoscopy, the positions of the
ctenidia within the pallial cavity are different from those
that might be expected from dissected specimens (Fig. 1).
The demibranchs are held curved towards the visceral mass,
and the ventral margin of the outer demibranchs is partic-
ularly bent inward (Fig. 2). These gill postures are main-
tained despite variation in the overall orientation of the
mussels.

Through the relatively transparent ciliated epithelia of the
ctenidia. we observed internal bands of muscular cross-
struts (Medler and Silverman, 1997) that are perpendicular
to the ctenidial filaments and 60-80 /xm apart. Ostia, lo-
cated in the epithelium of the interfilamentary spaces, are
lacking directly above the struts (Medler and Silverman.
1997).

The inhalent flow of suspended particles sometimes
stops, or even reverses momentarily, especially under high
particle concentrations. In addition, the ctenidia often con-
tract during active feeding; the interfilamentary spaces.
where the ostia are located, alternately flare and close at a
rate of 1 cycle s '. The extension of the mantle and siphons.
often used as an indication of steady feeding, does not
change during flow cessation and reversals, or during pul-
sation of the ctenidia.

Particles captured by the ctenidia move smoothly along
the frontal surfaces of the ctenidial filaments, and particles
of different types and sizes maintain their distance from
each other. Mucous strings were observed on the frontal
surfaces of the filaments only when the particle concentra-
tion was extremely high.

Outer demibranchs and their ventral imirt>iii!i

The outer demibranchs and their ventral margins were
observed in seven specimens, on 42 occasions, for 15.5 h of
total observation time and 1.7 h of video recording. The
outer demibranchs are held relatively straight, but with an
inward bend, especially of the ascending lamella, near the

Ventral
dL of oD and iD

oDCT

"aL of iDand oD

Dorsal

Figure 1. Diagram of labial palps and anterior portions of the inner
and outer demibranchs of Dreissena polymorpha as observed through the
endoscope (looking dorso-anteriorly). Palp lamellae are shown slightly
spread apart, with the outer Palp lamella (oPL) curled back. Solid arrows
indicate particle paths after capture. The white arrow indicates movement
of the outer demibranch (oD). Panicles in the medial dorsal ciliated tract
(niDCT) are transported in mucous clumps and strings. Particles are
transported at the ventral margin of the inner demibranch (iD) as both a
groove mucous string (gMS) in the marginal groove proper (mGp) and as
a superficial mucous string (sMS). The palps (iPL and oPL) enclose the inner
demibranch only, drawing in the superficial mucous string (sMSl from the
inner demibranch (iD) (see text for details). (aL = ascending lamella. Ant =
anterior, dL = descending lamella. gMS = groove mucous string, iD = inner
demibranch. iDCT = inner dorsal ciliated tract. iPL = inner palp lamella.
mDCT = medial dorsal ciliated tract, mGp = marginal groove proper. oD =
outer demibranch, oDCT = outer dorsal ciliated tract. oPL = outer palp
lamella, scS = smooth ciliated surface, sG = superficial groove, sMS =
superficial mucous string. VM = ventral margin.) See Video Note. p. 124.

ventral margin (Fig. 2). The position of the outer demi-
branch changes with pumping activity: the outer demi-
branch is positioned near the inner demibranch when inhal-
ent flow speeds are low; as flow speeds increase, the outer
demibranch moves laterally away from the inner demi-
branch (Fig. 1). Particles captured on the descending la-
mella of the outer demibranch are transported dorsally to the

PARTICLE TRANSPORT IN THE ZEBRA MUSSEL

119

Ventral
dL of oO and ID

aL of ID and oD

Dorsal

Figure 2. Diagram of the ventral margin (VM) of the outer demi-
branch (oD) ot Dreissena polymorpha as observed through the endoscope
(looking dorso-anteriorlyl. Solid arrows indicate particle paths after cap-
ture. Particles on the ascending lamella (aL) are transported either dorsally
or ventrally, depending on whether they are above or below a divergence
area (DA) when captured. Upon reaching the ventral margin (VM), parti-
cles traveling ventrally either ( 1 ) move over the ventral margin and proceed
dorsally on the descending lamella (dL): (2) continue moving ventrally.
leaving the surface of the ctenidia and becoming resuspended; or (3) make
a right angle turn and begin moving anteriorly on the ventral margin (VM)
(see text for details). (aL = ascending lamella. Ant = anterior. DA =
divergence area, dL = descending lamella. mDCT = medial dorsal ciliated
tract. oDCT = outer dorsal ciliated tract, VM = ventral margin.) See Video
Note, p. 124.

space between the two demibranchs is small (Fig. 1). Par-
ticles captured ventral to the divergence line on the ascend-
ing lamella of the outer demibranch move ventrally. Upon
reaching the ventral margin, one of the following three
behaviors occurs (Fig. 2): ( 1 ) The particles move over the
ventral margin and proceed dorsally on the descending
lamella of the demibranch. (2) The particles continue mov-
ing ventrally, leave the surface of the ctenidia. and become
resuspended. Most often, these resuspended particles are
then recaptured by the descending lamella of the inner
demibranch and continue moving ventrally. The majority of
particles that leave the ventral margin of the outer demi-
branch are large, like Scenede sinus. (3) The particles make
a right angle turn and begin moving anteriorly on the ventral
margin. Individual particles bounce along the ventral mar-
gin from filament to filament at a mean velocity of 65 p.m
s"' (Table 1). At high concentrations Old6 ml"1), parti-
cles are sometimes earned along the ventral margin in
clumps of mucus. As a result of these three particle trajec-
tories, desirable particles are retained on the outer demi-
branch, while unacceptable particles are transferred to the
inner demibranch and ultimately excluded from ingestion.

Inner demibranchs and their marginal grooves

The inner demibranchs and their marginal grooves were
observed in seven specimens, on 60 occasions, for 29.7 h of
total observation time and 2.9 h of video. Particles captured
on either descending or ascending lamellae of the inner
demibranch are transported toward the ventral margin,
whose mean width is 276 /j,m (n = 7; Fig. 1 ). Mean particle
velocities on the ascending and descending lamellae of the
inner demibranch are 129 /xm s"1 (Table 1).

Material at the ventral margin of the inner demibranch is
transported anteriorly in one of two channels, one deep and
one superficial (Figs. 1. 3). The deep channel, the marginal
groove proper, is nearly enclosed by the projection of the
ventral tips of the filaments over the groove. The superficial

medial dorsal ciliated tract (Fig. 2). On the ascending la-
mella of the outer demibranch, particles are transported
either dorsally or ventrally, depending on whether they were
captured above or below a divergence area located near the
bend of the demibranch (Figs. 1,2). When particles are
captured directly at the divergence area, they oscillate in
place for several seconds before proceeding either ventrally
or dorsally. The position of the divergence area can shift
ventrally or dorsally by about 1 mm. This shift does not
appear to be correlated with any particular stimulus, such as
particle type or concentration. Mean particle velocity on the
frontal surfaces of the ascending lamella of the outer demi-
branch is 90 jam s"1 (Table 1).

Due to the bend of the outer demibranch, the ventral
margin is pointed towards the inner demibranch, and the

Table 1

Particle velocities (22-jiim polystyrene beads) on the riullial organs of
-ebru mussels. Dreissena polymorpha, at 18°C

Location

Mean velocity ± SD Range

l^m s ' ) (/itn s"'

Outer demibranch

Frontal surface

90 ± 22

60-123

Ventral margin

65 ± 23

24-104

Inner demibranch

Frontal surface

129 ± 54

42-25 1

Ventral food groove

156 ± 53

45-354

106

Dorsal ciliated tract

152 ± 62

41-305

Labial palps

Frontal surfaces

94 ± 34

54-150

Ventral margin

54 ± 21

16-1 13

120

S. M. BAKER ET AL

channel or groove is a depression at the center of the ventral
margin, with openings between adjacent and opposite fila-
ment tips leading to the marginal groove proper (Fig. 3).
The superficial groove is entirely exposed to the inhalent
flow.

Particles transported in the superficial groove are embed-
ded in a string of mucus, up to 80 /xm thick (Figs. 1, 3),
which moves at a mean velocity of 156 /xm s~' (Table 1).
The presence of a superficial mucous string, as opposed to
a particle slurry, was confirmed by observing the dislodg-
ment of strings from the ventral margin when the valves
were rapidly adducted, or when a jet of water was pipetted
into the hole through which the OIT was inserted. After
dislodgment, the superficial mucous string remains unbro-
ken and returns to the ventral margin of the demibranch.
Polystyrene spheres (1 /LUII) and the large green alga
Scenedesmns are incorporated into the superficial mucous
string and are eventually rejected as pseudofeces.

Particles transported anteriorly inside the marginal
groove proper are also embedded in a string of mucus ( Figs.
1, 3). These particles appear to be those that are eventually
ingested. This observation was confirmed when specimens
were fed a combination of Microcystis, which is preferen-
tially ingested, and Scenedesmns or Typha detritus, which
are both rejected (Baker el at., 1998). The resulting colors of
the mucous strings, as well as the relative particle sizes,
indicate that Microcystis is incoiporated into the groove
mucous string, whereas Scenedesmns or Typha is incorpo-
rated into the superficial mucous string. Two other obser-
vations suggest that particles moving inside the marginal
groove proper are also embedded in mucus. First, particles
inside the marginal groove proper move at the same velocity
as particles transported in the superficial mucous string.
Second, particles both inside and outside the marginal
groove maintain positions relative to each other as they
move anteriorly. Additional observations suggest that the
deep and superficial mucous strings are not continuous with
each other, but are physically separate. For example, when
the superficial mucous string is dislodged from the superfi-
cial groove, the groove mucous string is not disturbed and
remains within the marginal groove proper.

Particles approaching the ventral margin of the inner
demibranch have three potential fates (Fig. 3): ( 1 ) As par-
ticles round the crest of the ventral margin, some move
anteriorly and diagonally, bouncing from filament to fila-
ment, before being entrained in the superficial mucous
string. These particles join the more ventral portion of the
superficial mucous string, and move in a uniform, smooth
manner (Fig. 3, path 1 ). (2) Other particles move laterally
prior to rounding the crest of the ventral margin, and enter
the interfilamentary space between two adjacent filaments.
Some of these particles stall, oscillate for several seconds,
and then disappear from view, possibly lost to an underlying
water tube through an ostial opening. Many particles, how-

Ventral
dL of oD and ID

. of ID and oD

Dorsal

Figure 3. Diagram of the ventral margin of the inner demibranch of
Dreissena pohmorpha as observed through the endoscope (looking dorso-
anteriorly). The groove mucous string (gMS) and a portion of a superficial
mucous string (sMS) are shown. Solid arrows indicate particle paths after
capture. Shaded arrows indicate particle paths behind a filament. Particles
approaching the ventral margin (VM) of the inner demibranch (iD) enter
the mucous strings in three ways: ( 1 ) As particles round the crest of the
ventral margin (VM), they move anteriorly and diagonally, bouncing from
filament to filament. These particles are entrained in the top (ventral side)
of the superficial mucous string, where they move smoothly towards the
anterior of the ctenidium. (2) Particles move laterally prior to rounding the
crest of the ventral margin and enter the furrow between two adjacent
filaments (dashed arrow). These particles become entrained in the bottom
(dorsal side) of the superficial mucous string and follow the contours of the
filament tips as they move anteriorly. (3) Particles remain on the frontal
MII laces of the filaments until reaching the tips, where they move laterally
and down (dorsally) into the marginal groove proper (see text for details).
These particles move smoothly towards the anterior in the groove string.
(aL = ascending lamella. Ant = anterior, dL = descending lamella.
gMS = groove mucous string. mGp = marginal groove proper. sG =
superficial groove, sMS = superficial mucous string, VM = ventral mar-
gin.) See Video Note. p. 124.

ever, continue moving toward the superficial groove and
became entrained in the more dorsal portion of the super-
ficial mucous string. These particles do not move smoothly
but follow the contours of the filament tips, bouncing as
they move anteriorly (Fig. 3, path 2). (3) Still other particles
appear to remain on the frontal surfaces of the filaments
until reaching the tips, where they move laterally into the

PARTICLE TRANSPORT IN THE ZEBRA MUSSEL

121

marginal groove proper through gaps between adjacent and
opposing filament tips (Fig. 3, path 3). Those particles
transported in the groove proper are alternately seen through
the gaps between adjacent filament tips and, faintly, as they
pass behind the relatively transparent filament tips.

Because the superficial mucous string is sometimes
opaque, we were unable to observe the marginal groove
proper at all particle concentrations and types. Therefore, it
is unclear whether the filament tips forming the marginal
groove proper flare "open" and "closed." Although the gaps
through which particles enter into the marginal groove
proper appear absent at times and large at others, the overall
width of the superficial groove does not change markedly,
ranging from 17 to 38 jam wide (/; = 10).

At high particle concentrations, the ventral margin of the
inner demibranch occasionally presses against the visceral
mass for several seconds. In these cases, the superficial
mucous string is transferred to ciliated tracts on the visceral
mass and is transported posteriorly, presumably to the in-
halent siphon for rejection. Movement of the mucous string
inside the marginal groove proper does not appear to be
interrupted.

Dorsal ciliated tracts

The dorsal ciliated tracts were observed in three speci-
mens, on 12 occasions, for 1 1.4 h of total observation time
and 47 min of video. There are three dorsal tracts on each
side of the visceral mass: at the junction of the viscera and
inner demibranch (inner dorsal ciliated tract), between the
two demibranchs (medial dorsal ciliated tract), and at the
junction of the outer demibranch and the mantle (outer
dorsal ciliated tract) (Fig. 1). Particles enter the medial
dorsal ciliated tract from the descending lamella of the outer
demibranch (Figs. 1.2); they are carried anteriorly as indi-
viduals at low particle concentrations, or in mucous clumps
and discrete strings at higher concentrations (Fig. 1 ). Parti-
cles moving in the medial dorsal ciliated tract sometimes
stop or reverse for several seconds, and this behavior is
associated with extreme flaring of the interfilamentary
spaces on the adjacent demibranchs. In addition, quick
successive contractions by the adjacent demibranchs seem
to make the mucous clumps less cohesive. At high particle
concentrations, the two demibranchs occasionally contract
strongly, and a slurry of particles becomes resuspended in
the pallial cavity. It was not possible to determine whether
these particles are recaptured. Particles in the medial dorsal
ciliated tract move at a mean velocity of 152 /u.rn s~'
(Table 1).

A few particles are also transported anteriorly in the inner
and outer dorsal ciliated tracts (Fig. 1 ). Particles enter these
tracts not only from the demibranchs, but also from the
mantle or body, suggesting that cilia on these surfaces can
trap some particles.

Labial palps

The labial palps were observed in four specimens, on 14
occasions, for 1 1 h of total observation time and 2.2 h of
video. Two pair of palp lamellae lie at the anterior end of the
ctenidia, one pair on each side of the mouth. A pair of palps
forms a functional unit consisting of one inner and one outer
palp lamella (Figs. 1, 4). The apposing surfaces of each pair
of palp lamellae are highly ciliated and folded into deep
grooves and ridges (see Galtsoff, 1964; Ward et ai. 1994).
The labial palps transport material from the ctenidia to the
mouth, control the volume of food ingested, and may also

sMS

pMS

iPL

Ant

mDCT

oDCT

Ventral
dl_ of oO and ID

aL of ID and oD

Dorsal

Figure 4. Diagram of the labial palps and the anterior portions of the
inner and outer demibranchs of Dreissena polvmorpha as observed through
the endoscope (looking medially). Solid arrows indicate particle paths after
capture. Dashed arrows indicate particle paths behind the outer demibranch
(oD) and outer palp lamella (oPL). At the anterior termination of the outer
demibranch (oD). mucous clumps and strings from the medial dorsal
ciliated tract (mDCT) move ventrally onto the inner demibranch (iD) and
enter between the palp lamellae (PL). The superficial mucous string (sMS)
is drawn from the ventral margin (VM) of the inner demibranch (iD) and
between the palp lamellae (PL). The material disperses on the palp lamellae
(PL), and rejected particles move ventrally from the palp grooves (pG).
Rejected particles ire incorporated into a mucous string (pMS) that moves
posteriorly and forms an irregular ball at the palp apices. This hall is
expelled as pseudofeces (PF) (see text for details). (Ant = anterior, DA =
divergence area. Dor = dorsal, iD = inner demibranch. iPL = inner palp
lamella, mDCT = medial dorsal ciliated tract, oD = outer demibranch.
oDCT = outer dorsal ciliary tract, oPL = outer palp lamella, pA = palp apex,
PF = pseudofeces, pG = palp groove, pMS = palp mucous string. sMS =
superficial mucous string. VM = ventral margin.) See Video Note, p. 124.

122

S. M. BAKER ET AL.

sort particles before ingestion (Yonge, 1926; Menzel, 1955;
Newell and Jordan, 1983).

In zebra mussels, the labial palps enclose the inner demi-
branch only. Along the entire free dorsal edge of each palp
lamella is a smooth ciliated surface that rests against the
demibranch lamellae. These smooth ciliated surfaces are
narrow at the distal apices of the palp lamellae and increase
in width anteriorly, up to four palp ridges wide. The distal
apex of the inner palp lamella often sweeps from the as-
cending lamella of the associated demibranch, across the
superficial groove, and back, drawing in the superficial
mucous string (Fig. 1).

Although the superficial mucous string is drawn between
the palp lamellae at their distal apices, the superficial groove
extends the entire length of the inner demibranch, ending at
the oral groove between the palp pair. It was unclear
whether, or at what point, the mucous string is removed
from the marginal groove proper. This material may remain
within the marginal groove proper to be deposited directly
into the oral groove, without processing by the palps.

At the anterior terminus of the outer demibranch, mucous
clumps and discrete strings from the medial dorsal ciliated
tract move ventrally on the filaments of the descending
lamella of the inner demibranch (Figs. 1. 4). The superficial
mucous string is drawn between the palp lamellae at a point
posterior to this location (Fig. 1). Rather than proceeding
anteriorly in the superficial groove, the mucous clumps
from the dorsal tract continue moving in a ventral direction,
entering between the labial palp lamellae.

Mucous strings or clumps from the superficial groove or
the dorsal ciliated tract disperse on the palp lamellae. Indi-
vidual particles bounce anteriorly over the ridges of the
palps, slowing in the grooves and moving more quickly over
the ridges to the next groove (Fig. 1 ); the mean velocity is
94 /j,m s~ ' (Table 1 ). The palp lamellae alternate between
being spread slightly apart and being closely appressed.
When the palp lamellae part slightly, particles remain close
to one or the other lamella and continue their bouncing
motion. Occasionally, the palp lamellae spread wide apart,
and particles can be seen suspended between the apposing
palp surfaces and moving posteriorly. This material may
include particles that are moving ventrally from the dorsal
ciliated tract. When appressed, the lamellae rub together
with an anterior-posterior displacement of the width of one
to two palp ridges (one palp ridge == 125 /xm), and the
smooth outer surfaces of the palp lamellae undulate in
waves from dorsal to ventral.

Rejected particles move ventrally from the palp grooves
(Figs. 1; 4). At the ventral margins of the palp lamellae,
these particles form a mucous string which then moves
posteriorly at a mean velocity of 54 jam s~" (Table 1 ). The
palp mucous string moves toward the distal apices of the
palps, where it forms an irregular ball (Fig. 4). Once the ball
of mucus and particles reaches a particular size, the palps

push or "clap" the ball away. In this manner, the ball is
transferred to ciliated tracts on the mantle, presumably to be
expelled from the inhalent siphon or pedal gape as pseudo-
feces. Sometimes the palp mucous strings are transferred to
the mantle before reaching the palp apices.

During exposure to high particle concentrations ( 1 06
ml"'), the processing of particles by the labial palps
changes according to the acceptability of the particles.
When mussels were fed high concentrations of a combina-
tion of both desirable and unacceptable particles (Microcys-
tis and Scenedesmus), the ball of mucus that forms near the
palp apices was drawn back between the palp lamellae and
once again dispersed. We observed mucous balls being
reprocessed by the palps up to four times before finally
being rejected. In contrast, when mussels were fed
high concentrations of primarily unacceptable particles
(Scenedesmus alone), the superficial mucous string from the
inner demibranch sometimes by-passed processing by the
labial palps. In this case, the superficial mucous string does
not disperse on the ridged surfaces of the palp lamellae but
is transferred from the marginal groove of the inner demi-
branch directly to the palp apices by the extreme posterior
section of the smooth ciliated surface (see Fig 1 ). At the
palp apices, the material is formed into a mucous ball and
rejected.

Discussion

The observations reported here explain the efficient se-
lection of particles measured in our previous work (Baker et
al, 1998) with Dreissena polymorpha. Particles are sorted
on the ctenidia of zebra mussels, and more specifically, at
the marginal food groove of the inner demibranch. We
observed a two-layer transport at the marginal food groove:
desirable particles appear to be transported inside the
groove proper, while unacceptable particles are carried su-
perficially. We also observed sorting at the ventral margin
of the outer demibranch: desirable particles are retained on
the outer demibranch, while unacceptable particles are
transferred to the inner demibranch and ultimately rejected.
Here, we compare and contrast our observations with pre-
vious reports of feeding processes in both zebra mussels and
marine bivalves. We suggest that the structure of homo-
rhabdic ctenidia does not preclude particle sorting, and that
the changes in seston composition attributed to /ebra mus-
sels may ultimately be due to the ctenidial sorting mecha-
nisms observed in this study.

Foster-Smith ( 1975) proposed that three conditions must
be met for particle selection to take place at the marginal
groove of bivalve ctenidia (i.e., in Mytilus edulis, Cerasto-
denna edule. Venerupis pullastra): (1) some particles must
be able to enter the deep area of the marginal groove; (2)
particles in the deep area of the marginal groove must be
physically separate from the superficial material: and (3) the

PARTICLE TRANSPORT IN THE ZEBRA MUSSEL

123

superficial material must be rejected, while the material in
the deeper area of the marginal groove is accepted. The
two-layer transport that we observed at the marginal groove
of zebra mussels meets these requirements for particle se-
lection.

Two-layer transport has previously been described for
filibranchs and pseudolamellibranchs, but does not neces-
sarily indicate the capacity for particle selection. Foster-
Smith (1975) reported two-layer transport in M. edidis (fili-
branch), with the particles in the deep region of the marginal
groove tending to be small, and those in the superficial
material tending to be larger. But the two layers are con-
tiguous, precluding particle selection. In M. editlis. parti-
tioning between the two layers may be temporal, rather than
physical. J0rgensen ( 1975) reported that particles arriving at
the marginal groove might either enter the groove between
the bases of the filament tips or pass outside, depending on
whether the groove is open or closed. We never observed
the marginal groove in zebra mussels to be "open" with
filament tips flared, as J0rgensen (1975) illustrated for M.
edulis, although our observations suggest that there may be
some regulation of the amount of material allowed to enter
the marginal groove proper.

Two-layer transport at the marginal groove, in combina-
tion with particle sorting, has previously been reported only
for pseudolamellibranchs. Atkins (1937a) described both
two-layer transport and the potential for size sorting at the
marginal groove in Pinna fmgilis and several Pinna-like
species (pseudolamellibranchs). In these species, which
have plicate heterorhabdic ctenidia, fine particles trans-
ported by the principal filaments are deposited into the
depth of the marginal groove proper, while coarse particles
transported by the ordinary filaments are deposited outside
the groove and are usually rejected (Atkins, 1937a). Al-
though the mode of particle introduction to the marginal
groove of D. polymorpha differs from that observed in
Pinna sp. due to the nonplicate nature of the zebra mussel
ctenidia, the marginal groove appears to function similarly
in both species.

Previous feeding studies have indicated that, in addition
to selection by particle size in D. polymorpha, a chemical
mechanism of selection is also present (Ten Winkel and
Davids. 1982; Baker et ai, 1998). In the present study, the
disparate sizes of particle types embedded in the superficial
mucous string in D. polymorpha suggest that some factor
other than size is important in the shunting of particles
either to the marginal groove proper or to the superficial
groove. The superficial mucous string is picked up by the
apices of the palps, and much of the material is rejected. The
arrangement of the ctenidium/palp junction suggests that
material within the marginal groove proper may be trans-
ported to the anterior portion of the labial palps or directly
to the oral groove. The differing degree to which the two
mucous strings are processed by the palps suggests that the

material in the superficial mucous string is of lower quality
than that in the groove string. This two-layer transport at the
marginal groove could potentially increase the rate of pro-
cessing and decrease the possibility of sorting mistakes at
the palps. Microscopic examination of the structure and
function of cilia at the marginal groove may help elucidate
the sorting mechanisms.

The labial palps of zebra mussels function very similarly
to those of other bivalves, despite differences in demibranch
structure and function. Zebra mussels have a smooth ciliated
surface along the free dorso-posterior edge of the labial palp
lamellae, similar to that of oysters (Ward et at., 1994). Our
observations of mucous ball formation near the apices of the
labial palps are similar to those described for both oysters
(Menzel, 1955; Galtsoff, 1964; Ward et ai, 1994) and
marine mussels (Beninger and St-Jean, 1997a). As in oys-
ters, D. polymorpha palp lamellae alternate between being
appressed and being slightly separated. When separated, we
observed off-surface posterior movements of particles like
those reported by Galtsoff (1964) and Ward et al. (1994) for
oysters. Ward et al. (1994) speculated that the posterior
movement allows the particles to be cycled through the
palps several times before being rejected or ingested. In
addition to this type of reprocessing, we observed a second
recycling method: the mucous ball forming near the palp
apices is sometimes re-engulfed by the palps up to four
times before finally being rejected.

Video endoscopy allowed us to observe. /// situ, the
position of the feeding organs within the pallial cavity of
living zebra mussels. These observations build on previous
reports of feeding organ functioning based on dissected
specimens of zebra mussels (Atkins. 1937b; Morton, 1969).
For example, like Atkins (1937b) and Morton (1993). we
observed particles passing off the outer demibranch at the
ventral margin and being transferred to the inner demi-
branch. Dissected preparations, however, did not allow the
authors of previous studies to observe the bend in the outer
demibranch and the curvature of the inner demibranch that
occurs under natural feeding conditions. Our observations
suggest that maintenance of the ctenidia in these positions
may facilitate particle recapture; this natural ctenidial mor-
phology enhances the transfer of some particles from the
outer demibranch to the inner demibranch.

Our observations of particle transport in zebra mussels
contradict some previous observations and corroborate oth-
ers. For example, Atkins ( 1937b) described rare filaments of
the descending lamella of the outer demibranch that trans-
port particles ventrally; these particles are then passed to
normal filaments that transport them dorsally. During our
observations of this area (five specimens on 14 occasions,
for 8.2 h total observation time), all filaments of the de-
scending lamella of the outer demibranch transported par-
ticles dorsally. In addition, Atkins (1937b) did not report
any anteriorly directed movement on the ventral margin of

124

S. M. BAKER ET AL

the outer demibranch, such as we occasionally observed.
That anterior movement is. however, similar to that of
mucous-particle masses on the ventral bend of Placopecten
magellanicus ctenidia, which also lack a ventral groove
(Beninger et ai. 1992).

In greater contrast, both Atkins (1937b) and Morton
(1969) reported ventral movement of particles on the as-
cending lamella of the outer demibranch. whereas we ob-
served dorsally directed movement, above a divergence
area. Atkins (1937b) reported dorsally directed currents on
the ascending lamella of the outer demibranch of the Union-
idae, another unrelated group of freshwater bivalves.

The dorsally directed movement on the ascending lamella
of the outer demibranch allows some proportion of material
to be directed to the outer dorsal tract, rather than to the
medial dorsal ciliated tract between the two demibranchs,
perhaps preventing overloading of the latter tract. Both
Atkins (1937b) and Morton (1993) described the anterior
movement in the dorsal tract at the junction of the mantle
and ascending lamella of the outer demibranch as well;
Atkins (1937b) noted that anterior movement in this outer
tract usually occurs only in bivalves with heterorhabdic
ctenidia. Partitioning material between two dorsal tracts
may increase the rate of total particle transport.

Particle velocities, as well as the movement of particles
on the ctenidia and labial palps of D. polymorphic are
consistent with mucociliary, rather than hydrodynamic,
transport (Ward el a!.. 1993; Beninger and St-Jean, 1997b).
The velocities of particles transported on the frontal sur-
faces of the demibranchs overlap the ranges reported for M.
echtlis and for the plical crests of C. virginica (Ward a ai,
1993). In addition, the superficial mucous string at the
marginal groove moves at a rate similar to the mucous
strings observed in C. virginica, M. edulis, M\u arenaria,
and Placopecten magellanicus (Ward et al., 1993, 1994).
Material in the dorsal ciliated tract, however, travels at a
rate many times slower than it does in C. virginica or P.
magellanicus. In D. polymorpha, material at the dorsal
ciliated tract is embedded in mucous clumps and trains, but
in the oyster and scallop, the material is in a slurry (Ward et
al., 1993). Transport rate is generally inversely correlated
with the viscosity of the mucus (Menzel, 1955; Winet and
Blake, 1980), and therefore, material in a slurry moves at a
faster rate than material in more cohesive mucous clumps.
The lack of hydrodynamic transport in zebra mussels may
reflect a dorsal tract that is smaller and less well developed
than that in oysters and scallops.

Zebra mussels have had major impacts on the freshwater
systems in which they have become established. Because of
the high clearance rates of these mussels, phytoplankton
biomass has decreased by more than 609J- in many of the
invaded systems (Morton, 1971; Kryger and Riisgard. 1988;
Holland. 1993; Fahnensteil et al.. 1995). In addition, seston
composition has changed in some systems, including the

Hudson River, New York, where the phytoplankton com-
munity has shifted from a prevalence of cyanobacteria to
diatoms (Vanderploeg et al., 1996; Smith et al., 1998).
Recent studies using flow cytometry (Baker et al., 1998)
have shown that zebra mussels can very effectively sort
particles and preferentially accept the cyanobacterium Mi-
crocystis. In the present study we found that accepted par-
ticles were directed to the inside of the marginal groove of
the inner demibranch and appear to be transported directly
to the mouth for ingestion.

In summary, we observed pallial organ morphology, par-
ticle transport, and particle sorting in zebra mussels by using
video endoscopy. These observations contribute to a grow-
ing body of information on the feeding dynamics of bi-
valves and suspension-feeding invertebrates. More impor-
tantly, our results suggest that particle sorting occurs on
zebra mussel ctenidia, despite their homorhabdic nature and
their lack of adjacent tracks of frontal cilia beating in
opposing directions. Our direct observations of zebra mus-
sel ctenidia provide an explanation for the efficient selection
of particles measured by Baker et al. ( 1998) and, ultimately,
for the role of zebra mussels in ecosystem modification. The
role of ctenidial morphology in particle selection by zebra
mussels exemplifies the direct link between the functioning
of individual bivalves and ecosystem-level processes.

Acknowledgments

This paper is dedicated to the memory of Dr. James H.
Crichton. uncle and mentor (SMB). We thank the following
persons: C. Nieder, New York State Department of Envi-
ronmental Conservation, and H. Roditi, SUNY at Stony
Brook, provided zebra mussels. Amy Dunham. SUNY at
Stony Brook, and Heather Herb, University of Florida, drew
the illustrations. J. Kurdziel and P. Baker, SUNY at Stony
Brook, provided assistance during all aspects of this study.
Comments from two reviewers improved this manuscript.
This study was supported by grants from the Hudson River
Foundation (01096A and 00798A) and the National Science
Foundation (OCE981 1380).

Video Note

Supplementary video clips are available for viewing on
The Biological Bulletin Website at (http://www.mbl.edu/
BiologicalBulletin/VIDEO/BB. video. html).

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Biogeography of Two Species of Symbiodinium

(Freudenthal) Inhabiting the Intertidal Sea Anemone

Anthopleura elegantissima (Brandt)

T. c. LAJEUNESSE* AND R. K. TRENCH

Department of Ecologv, Evolution and Marine Biology, University of California nt Santa Barbara,

Santa Barbara, California 93106

Abstract. We have analyzed the genetic profiles of
dinoflagellate populations obtained from the Pacific coast
sea anemone Anthopleura elegantissima (Brandt) at collec-
tion sites from Washington to California. Genetic dif-
ferences within the symbiont populations of California
anemones have been uncovered by restriction length
polymorphism (RFLP) analysis of the small subunit (SSU)
and large subunit (LSU) ribosomal RNA genes, and by
denaturing gradient gel electrophoresis (DGGE) of the in-
ternal transcribed spacer region 2 (ITS 2). The existence of
two Symbiodinium species is substantiated by sequence
analysis of the variable regions VI, V2. and V3 of the
SSUrDNA. which also establishes their phylogenetic relat-
edness to other members of the genus Symbiodinium.
Anemones from Washington and Oregon harbor a single
dinoflagellaie species, for which we propose the name S.
muscatinei sp. nov. At these northern locations, S. muscati-
nei either exists alone or co-occurs with the Chlorella-\\ke
green algal symbiont. Our results indicate that S. muscatinei
co-occurs with a second dinoflagellate, 5. californium, in
mixed populations in central and southern California. We
suggest that the geographic distribution of these dinoflagel-
lates is related to the temperature cline created by latitude.

Introduction

Intertidal anemones of the genus Anthopleura are abundant
along the Pacific coast of North America (Hand, 1955). A.

Received 2 February 2000; accepted 17 July 2000.

* To whom correspondence should be addressed. E-mail: lajeunes@
lifesci.ucsb.edu

Abbreviations: DGGE, denaturing gradient gel electrophoresis; ITS 2,
internal transcribed spacer region 2; LSU. large subunit; RFLP, restriction
fragment length polymorphism; SSU. small subunit.

elegantissima. the most common and wide ranging species, is
distributed along the rocky intertidal from Alaska to central
Baja California (Hand. 1955; Francis, 1979; McFadden et al.,
1997). Throughout its geographical range this species harbors
intracellular dinoflagellates of the genus Symbiodinium. Anem-
ones in regions north of California may also host a Chlorella-
like (Chlorophyta) alga alone or in mixed populations with the
dinoflagellate (Muscatine, 1971). Whether a particular algal
symbiont occurs in an anemone, and whether it is present alone
or in mixed populations are phenomena believed to be influ-
enced by physical parameters. Relative sensitivities to light and
temperature appear to be most significant in regulating the
distribution of algal populations. Anemones in warm, bright
habitats typically possess the dinoflagellate symbiont, whereas
anemones in cool, shadier habitats contain the green alga
(Secord. 1995; Saunders and Muller-Parker, 1997).

Environmental parameters have also been implicated in
modulating Svmbiodinium populations in some tropical
symbioses. The Caribbean reef-building corals Montastraea
annularis and M. faveolata either host a single algal taxon
or have mixed algal populations with patterns of distribution
and relative abundance that may be in response to irradiance
and temperature (Rowan and Knowlton, 1995; Rowan et al.,
1997). Continued analyses of Symbiodinium populations in
scleractinians are resolving greater diversity within individ-
ual hosts than previously recognized (Baker and Rowan,
1997; Baker et al., 1997; Baker, 1999). Restriction fragment
length polymorphism (RFLP) analysis of the large subunit
(LSU) ribosomal DNA from 107 Pacific and Caribbean
corals (Baker, 1999) identified 69 species that harbored a
single algal taxon; 13 other species harbored more than one
Svmbiodinium taxon, but not in the same colony, and 25
species sometimes harbored more than one algal taxon
within the same colony. Depth (and thus reduced light) was

126

SYMBIODINIUM SPECIES IN ANTHOPLEURA

127

regarded as the most significant factor regulating the distri-
bution of a particular alga.

Previous studies conducted on algae isolated from A.
elegantissima collected in Washington and California
showed marked differences in mitotic indices (Wilkerson el
ill.. 1983) and carbon translocation (Shick and Dykens,
1984; Verde and McCloskey, 1996). The possibility that
these differences reflect different Symbiodinium species has
been suggested (Verde and McCloskey, 1996), but has not
been empirically tested. A limited genetic study was con-
ducted by Rowan and Powers (1991) on symbiont popula-
tions collected from Pacific Grove, California. Their RFLP
and partial small subunit (SSU) ribosomal DNA sequence
analyses from cloned amplification products detected only
one dinoflagellate taxon belonging to lineage B Symbio-
dinium (sensii Rowan and Powers, 1991). It is now recog-
nized that analyses based on the conserved SSUrRNA gene
underestimate genetic diversity (Rowan, 1998). Further-
more, sampling from a single collection site would not
necessarily identify symbiont diversity in a host with a
range of thousands of kilometers.

The extensive geographic range of A. elegantissima, its
occupancy of diverse intertidal habitats, its occurrence in
two different growth forms — solitary and clonal — which
may represent two distinct species (McFadden el ai, 1997).
and its ability to harbor symbionts from different algal
divisions suggest that A. elegantissima may harbor more
than one taxon of symbiotic dinoflagellate. This possibility
is strengthened by the finding that different Symbiodinium
spp. are adapted to different regimes of light (Iglesias-Prieto
and Trench. 1994, 1997b) and temperature (Warner el al..
1996). To test the hypothesis that different dinoflagellate
taxa may co-occur in Anthopleura, anemones were col-
lected from intertidal habitats along a latitudinal gradient
ranging from Puget Sound in Washington to San Diego in
Southern California. A genetic examination, using tradi-
tional RFLP analyses of the SSUrDNA and LSUrDNA, and
analyses of partial SSUrDNA sequences were conducted on
Symbiodinium populations isolated from these anemones.
Denaturing gradient gel electrophoresis (DGGE; Myers el
al., 1985; Abrams and Stanton, 1992) is a technique fre-
quently used to characterize and compare genetic diversity
in complex microbial assemblages from samples collected
over spatial and temporal scales (Muyzer et al.. 1993;
Muyzer and Smalla, 1998). This technique was used here to
analyze the variable internal transcribed spacer region 2
(ITS 2), and thus to visualize and identify the occurrence of
more than one algal taxon within a host.

Materials and Methods

Anemone collections

Specimens of symbiotic Anthopleura elegantissima were
collected from rocky intertidal locations along the Wash-

ington, Oregon, and California coastlines. Between Febru-
ary 1997 and August 1998, collections were conducted
at Anaco Beach, Fidalgo Island. Washington (48°29';
122°42'); Coos Bay, Charleston, Oregon (43°34': 124°33');
Carmel, California (36°55'; 121°92'); Cayucos, California
(35°44'; 120°88'); Ellwood Beach and Campus Point.
Santa Barbara, California (34°43'; 119°83'); and Swami's
Beach, Cardiff-by-the-Sea (Encinitas). California (33°04';
117°29'). Symbiotic A. xanthogrammica were collected
only from Cayucos. Individual anemones were collected
from a range of habitats, from exposed upper intertidal to
shaded lower intertidal locations. Aposymbiotic A. elegan-
tissima were obtained from drainage sluice-ways beneath
the wet lab facilities of the Marine Science Institute of the
University of California, Santa Barbara, California.

Isolation of algal cells from tissues of host anemones

Oral discs and tentacles from anemones were macerated
in a Tenbroek tissue grinder. This step was followed by a
5-min centrifugation at about 800 X g in a Dynac II bench-
top centrifuge. The pellets containing the algal cells were
resuspended and further homogenized with a Tissue Tearor
(Model 985-370) to dissociate most of the remaining animal
cellular debris. After a second centrifugation, the algal
pellets were resuspended in Millipore-filtered (porosity 0.22
/urn) seawater, centrifuged, and resuspended two or three
times to remove most of the animal debris from the algal
cell preparation.

The cultured Symbiodinium californium (#383. Banaszak
et al.. 1993) was originally isolated, in 1989, from a solitary
form of A. elegantissima by using a technique developed by
Polne-Fuller (1991). The isolate was grown in 1 1 of
ASP-8A (Blank, 1987) for 2 months at 17°C, illuminated by
banks of VitaLite fluorescent tubes delivering 80 /zmol
quanta m~2s~' photosynthetically active radiation on a
14:10 (light:dark) photoperiod. Algae were harvested by
centrifugation at 9000 X g in a Sorvall RC-5B centrifuge.

DNA extraction, amplification. RFLP

Symbiodinium populations were isolated from 64 speci-
mens of A. elegantissima (37 clonal and 27 solitary) and
three of A. .\anthogrammica. DNA was extracted from
about 25 mg of algal material by using a proteinase diges-
tion and spin-column separation protocol described in the
QIAamp Tissue kit (Qiagen Corporation, Santa Clarita,
CA). From the spin-column eluate, 1 p,\ of product was used
as a template to amplify the small-subunit ribosomal RNA
gene (SSUrDNA) and part of the large subunit ribosomal
RNA gene (LSUrDNA) (Lenaers et al., 1989). SSUrDNA
was amplified on a Perkin-Elmer thermal cycler 2400 using
primers of Rowan and Powers (1991), and under the fol-
lowing conditions: an initial denaturing step of 3 min at
92°C followed by 35 cycles of 30 s at 92°C. 40 s at 52°C,

128

T. c. LAJEUNESSE AND R K. TRENCH

and 30 s at 72°C, followed by a single cycle of 5 min at
72°C. A fragment corresponding to a region between 28 bp
and 929 bp of the Prorocentnun micans LSU rRNA gene
containing the variable portions D1-D3 (Lenaers el ai,
1989; Wilcox, 1998) was amplified using primers described
by Wilcox (1998) with the same protocol used for the
SSUrDNA, but with an optimal annealing temperature of
48°C.

Restriction digests were performed by incubating ampli-
fied products with 1-5 units of Taq I (New England Bio-
Labs, Beverly, MA) at 65°C for 3 to 5 h or with 1-5 units
of Dpn II (New England BioLabs) for 4 to 5 h at 37°C.
Products of the digests were separated by electrophoresis in
a 2.5% high-melt agarose gel at a constant 70 V for 3 h.

Denaturing gradient gel electrophoresis (DGGE)

Primers for polymerase chain reaction (PCR)-DGGE
analyses were designed to amplify the variable internal
transcribed spacer region II (ITS 2), producing a fragment
size of 330-360 bp. ITS and 5.8S rDNA sequence data
(unpublished) from cultured Symbiodinium spp. isolated
from various cnidarian and molluscan hosts were compared
to identify conserved regions. An internal primer "ITSint-
for2" (5'GAATTGCAGAACTCCGTG-3'( was designed
from this analysis, and it anneals to a conserved region of
the 5.8S rDNA. Primer ITS-Reverse (Coleman et ul.. 1994)
was modified with a 40-bp GC clamp (Sheffield et ai, 1989)
and is referred to as "ITS2CLAMP" (5'CGCCCGCCG-
CGCCCCGCGCCCGTCCCGCCGCCCCCGCCCGGGA-
TCCATATGCTTAAGTTCAGCGGGT-3'). A "touch-
down" amplification (Don et ul., 1991 ) protocol was used to
ensure specificity. Initial annealing conditions began 10°C
above the final annealing temperature of 52°C. Every two
cycles, the annealing temperature was decreased one de-
gree. After 20 cycles the annealing temperature was held,
and remained at 52°C for another 15 cycles. Reaction prod-
ucts were loaded onto an 8% acrylamide denaturing gradi-
ent gel (a 40% to 75% gradient, 100% consists of 7 M urea
and 40% deionized formamide). PCR products were loaded
on the gel with a 2% Ficoll loading buffer (2% Ficoll-400,
10 mM Tris-HCl pH 7.8, 1 mA/ EDTA, 1% bromophenol
blue) and separated by electrophoresis for 22 h at 90 V at a
constant temperature of 60°C. The gel was then stained in a
IX TAB and 5 jug/ml ethidium bromide solution for 15 min,
washed in deionized water for 15 min and photographed.

DNA sequencing, alignment, and anal\sis

Partial sequences of the SSUrDNA and the ITS spacer
(ITS 1 and 2 and 5.8 rRNA gene) were obtained from direct
cycle sequencing of amplified products. Cycle sequencing
of amplified SSUrDNA products was accomplished using
Rowan and Power's ( 1991 ) forward primer and an internal
primer (Anderson et ai, 1993), which permitted sequence

reads spanning the VI, V2, and part of the V3 variable
regions (Sogin and Gunderson, 1987; as described in
Rowan and Knowlton. 1995). Reagents were supplied and
reaction conditions specified in the ABI Prism Big Dye
Terminator Cycle Sequencing Ready Reaction Kit (PE Ap-
plied Biosystems, Foster City, CA). Reaction products were
analyzed on an Applied Biosystems 310 Genetic Analyzer
(Division of Perkin Elmer, Foster City, CA). The resulting
chromatograms were checked and edited using Sequence
Navigator, version 1.0, software (ABI, Division of Perkin
Elmer, Foster City. CA). Sequence composites of the
SSUrDNA were assembled and, including gaps, totaled 375
nucleotides. Partial sequences from the two algal types
identified in our RFLP and DGGE analyses were then
aligned by eye with published S\mbiodinium sequences
obtained from Genbank; they included S. corciilomm, S.
microadriaticum, S. pilosum, S. pulchrorum, Gymnodinum
varians, G. simplex, Porocentrum micans, and four Svmbio-
diniuni spp. symbiotic with the coral Montastraea annu-
laris. Cladistic analyses, by the method of parsimony, were
conducted on the aligned data set using PAUP 4.0b4 soft-
ware under default settings (Swofford, 1993). A bootstrap
analysis was conducted on the most parsimonious tree to
assess relative support for each branch (Felsenstein, 1985).

Results

Anemones collected from high and low intertidal habitats
at each study site contained the same Symbiodinium popu-
lations. We found no indication, based on our molecular
analyses, that the Symbiodinium populations in A. elegan-
tissima are influenced by the local environmental differ-
ences within the littoral zone. However, we found signifi-
cant differences between the algal populations in anemones
collected from Washington and Oregon, and those collected
at lower latitudes in California. Figure 1 summarizes the
findings from our RFLP and DGGE analyses that detected a
single genetic entity in northern anemones and at least two
genetic entities in southern anemones.

Taq I digests of the SSUrDNA amplified from northern
populations of Symbiodinium exhibited a characteristic
"clade B" restriction pattern (Rowan and Power. 1991 ) (Fig.
1 A, lane 1 ). The same analysis conducted on southern
populations produced a mixed pattern consisting of the
"clade B" type with a second undescribed pattern (Fig. 1C,
lane 1 ). We have identified this latter pattern as diagnostic
of a second algal type. It is identical to the restriction pattern
of cultured S. californium, whose SSUrDNA does not yield
a classical "clade A. B, or C" pattern (Fig. 1C. lane 2) (cf.
RFLP type "T6" in Darius et ul., 1998). This new pattern
results from the loss of a restriction site at approximately
position 1500 and the gain of a restriction site at position
870 of the amplified 1785 bp product, as determined from
the entire SSUrDNA sequence of 5. californium (GenBank

SYMBIODINIUM SPECIES IN ANTHOPLEURA

129

NORTHERN SYMBIODINIUM POPULATIONS (WA, OR)

Fidalgo Island, WA

2 3

1018 -
516 -

220 =

• Charleston, OR

220

Taq 1 (SSU) Dpn II (LSU)

DGGE (ITS 2!

SOUTHERN SYMBIODINIUM POPULATIONS (CA)

C 1 2 34 D 1 2 3 4

220

1018 -

516 - Z 516

220

Taq 1 (SSU) Dpn II (LSU)

DGGE (ITS 2)

(Carmel, CA
JCayucos, CA

(Santa Barbara, CA

Figure 1. Pacific coastline of North America depicting collection sites and genetic analyses of northern and
southern Symbiodinium populations. ITS 2, internal transcribed spacer region 2; LSU, large subunit; SSU, small
subunit. (A) RFLP analysis of the SSUrDNA and LSUrDNA from northern algal populations. Lane 1. Taq I
digest of SSUrDNA indicative of lineage B Svmhioilinium (Rowan and Powers, 1991 ); lane 2, Dpn II digest of
the D1-D3 variable region of the LSUrRNA gene: and tor comparison, in lane 3. Dpn II digest of the same
region from 5. californium (#383). (B) DGGE gel of the ITS 2 depicting a single ITS signature (black arrow).
(C) RFLP analysis of the ribosomal repeat from southern algal populations; lane 1, Taq 1 digest of SSUrDNA
showing the lineage B Symbiodinium co-occurring with a second pattern not belonging to any of the described
"clades" (sensu Rowan and Powers, 1991 ); lane 2, Taq 1 digest of SSUrDNA of #383, a pattern identical to the
undescribed RFLP profile in lane 1. Lanes 3 and 4 are Dpn II digest on LSUrRNA gene amplified from natural
populations and the cultured clonal isolate #383 respectively; indicates more than one algal taxon in southern
populations. (D) DGGE gel showing three representative profiles of ITS 2 signatures from natural populations,
lanes 1. 2, and 4; and for comparison, lane 3 depicts the ITS signature for #383 (white arrow). (See text for
further explanation.)

accession #AF225965). The poor amplification of the
SSUrDNA from S. californium in mixed populations may
explain why the smallest fragment (130 bp) appears to be

absent from RFLPs on natural samples (Fig. 1C, lane 1).
Dpn II restriction digests of SSUrDNA from both freshly
isolated northern and southern algal samples and cultured 5.

130

T. c. LAJEUNESSE AND R. K. TRENCH

californium produced a single restriction pattern character-
istic of "clade B" Symbiodinium (Rowan and Powers, 1991 )
(data not shown). The utility of RFLP analyses of the
SSUrDNA in distinguishing sequence variation, especially
among congeneric members, has limitations. The more vari-
able gene regions and more informative techniques were
therefore employed to achieve a better resolution of the
variation detected by the Taq 1 enzyme.

An 850-bp fragment of the LSUrRNA gene was exam-
ined by restriction analysis in an attempt to measure the
extent of the genetic varitation observed in our RFLP anal-
ysis of the small subunit and to uncover possible variation
not resolved by the SSUrRNA gene (Baker el al.. 1997;
Wilcox, 1998). Dpn II restriction digests of LSUrDNA
amplified from all northern Symbiodinium populations pro-
duced the fragment pattern in Figure 1A, lane 2. A restric-
tion site exists at one end of the amplified product and
produces two bands, one 740 bp, the other about 70-80 bp.
A Dpn II digest of the LSUrDNA from S. californium lacks
a restriction site for this enzyme, and a single band is
depicted (Fig. 1A. lane 3, and also 1C. lane 4). RFLPs
conducted on algal populations from southern anemones
always contained a nondigested fragment, as observed for S.
californium, as well as two fragments identical to those
observed in restriction patterns from the northern algal
populations. This indicates the presence of two different
gene sequences in the same amplification product — one
with and one without a restriction site for Dpn II. These data
are consistent with our results from the SSUrDNA digests.
Taq 1 digests of the LSUrDNA (data not shown) also
showed the presence of a single type in northern populations
and two types in southern populations of A. elegantissima.
The same two algal types were also found in A. .\antho-
grammica from Cayucos.

The ITS region has historically been useful in resolving
species-level differences (Gonzalez et ai, 1990; Coleman el
til.. 1994; Goff et ai, 1994; Larsen and Medlin, 1997).
Among dinoflagellates. it has been used to resolve differ-
ences between closely related species within the genus
Alexandrhtm (Adachi el al.. 1996) and to assess intraspe-
cific variation in Gymnodinium catenatiim (Adachi et al.,
1997). Hunter et al. (1997), in their preliminary study.
reported that the ITS is potentially a good marker for
interspecific comparisons between Symbiodinium taxa. In
this study, a region encompassing the ITS 2 and a portion of
the 5.8S was chosen for DGGE analyses.

DGGE separates amplification products by differences in
sequence composition and nucleotide order. The results are
therefore a qualitative assessment of the entire sequence.
Application of DGGE has permitted the rapid assessment of
complex microbial populations and identification of specific
microbes from marine, aquatic, and terrestrial samples
(Muyzer and Smalla, 1998). This analysis, applied to Sym-
hiodiniuin populations, has verified the existence of a single

Symbiodin'nun species in anemones collected from northern
locations (Washington and Oregon) (Fig. IB, black arrow).
In contrast, at least two algal species were identified in
populations of Antliopleura from California.

In southern populations, the algal ITS "signature" found
in northern anemone populations (black arrow) is often
associated with an ITS signature consistent with S. califor-
nium (white arrow; Fig. ID, lanes 1 and 3 respectively). An
unidentified ITS type was observed (light gray arrow; Fig.
ID, lane 2) but is believed to be a variant of S. californium
based on our findings from the RFLP data on the
LSUrDNA. In our analyses of southern populations, a
fourth ITS type (hatched arrow), although sometimes ap-
pearing faint, is always associated with the most common
ITS type (black arrow). This is believed to either be a
DGGE artifact or to represent fixed variation within the
ribosomal repeat.

An inferred phylogeny (Fig. 2) reconstructed from partial
SSUrDNA sequences shows the relationships between the
algae identified in A. elegantissima and those from tropical
hosts. The algal species found throughout northern and
southern anemone populations is a member of the B lineage
found by Rowan and Powers ( 1991 ). Here, we propose the
name Symbiodinium muscatinei sp. nov. to refer to the
dinoflagellate symbiotic with A. elegantissima from Wash-
ington to California. (A morphological description must

Symbiodinium microadriaticum

S . corculonttn

Symbiodinium sp. (M . annularis)

S . pilosurn

S . californium

1_4 I (southern A . elegantissima)

100)

L Gymnodinium variant

Symbiodinium sp.(Marginopora)
Symbiodinium sp.(M. annularis)

Symbiodinium sp. (M. annularis) _
Symbiodinium sp. (M . annularis)
S. pit Ichror urn

— S. muscatineii (A. elegantissima)
- Gymnodinium .simplex

Figure 2. Phylogenetic reconstruction of the Symbiodinium lineage by
the method of parsimony using a 375-bp composite of the SSUrRNA gene
spanning the VI, V2, and V3 variable region. S. californium is closely
related to Gymnodinium varians and forms a separate lineage. 5. nniscan-
nei groups with members of the B lineage and is probably the same taxa
identilied in the study by Rowan and Powers (19911. Numerals above
branch segments indicate number of base pair changes; numbers in paren-
theses below show bootstrap consistency based on 1000 replicates. For
simplicity, the out group, Prorocentrum inicans, is not shown.

SYMBIOD1NIUM SPECIES IN ANTHOPLEURA

131

await achieving axenic culture, but the partial sequence of
the SSUrDNA has been given the GenBank accession num-
bers AF228362 and AF228363 for the VI. and V2 and V3
regions of the SSUrDNA. respectively.) S. californium,
identified in anemones from California only, is closely
related to Gymnodinium various; together they form a lin-
eage separate from the described "clades A, B, and C"
(bracketed). Sequence differences between the SSUrDNA
from S. mnscatinei and 5. californium are significantly
greater than differences observed between described spe-
cies. The entire ITS region was sequenced from each alga
and compared (data not shown). No reliable alignment was
possible due to extreme sequence divergence. The ITS
sequence of S. mnscatinei was aligned and compared with
sequences from several other lineage B Syinbiodinium. Re-
sults indicated a level of divergence many times greater
(12%) than interspecific variation observed among other
dinoflagellates (Adachi el al.. 1997). These data collectively
indicate the presence of two distinct dinoflagellate species.

Animal DNA isolated from aposymbiotic A. elegan-
tissimu was used to determine whether host material would
be a source of contamination in freshly isolated algal sam-
ples. Amplification was never achieved with the primers and
reaction conditions used for the amplification of LSUrDNA
and SSUrDNA. However, the ITS 2 primers designed for
DGGE amplified host DNA. but only rarely when algal
DNA was present.

Cloned S. californium (#383) served as a control, so we
could ascertain the presence of pseudogenes or natural
variation in ribosomal repeats within a single genome. Ri-
bosomal pseudogenes have been observed in some
dinoflagellates (Scholin et al., 1993: Adachi et a!.. 1996).
Although none have been reported in Symbiodinium, their
presence cannot be discounted. The DGGE analysis on S.
californium (#383) repeatedly identified a single type with
no obvious intragenomic variation.

Discussion

The molecular methods employed in this study demon-
strate that there are two distantly related species of S\mbio-
dinium in populations of Anthopleura elegantissima along
the Pacific coast of the United States. Evidence based on
RFLP. DGGE, and sequence analysis indicates the presence
of a single Symbiodinium species, designated here as S.
muscatinei, in northern populations. This is consistent with
previous reports that many hosts harbor a single population
of algae (Schoenberg and Trench, 1980a; Baker and Rowan,
1997;Bythell ff«/.. 1997: Billinghurst et al., 1997; Stochaj
and Grossman, 1997). Our analyses also show that anemo-
nes from southern latitudes in California harbor a mixed
dinoflagellate population consisting of two congeneric spe-
cies: S. mnscatinei identified from northern anemones, and
S. californium.

It has been recognized for some time that some inverte-
brate taxa may simultaneously harbor more than one algal
taxon (Muscatine. 1971; Trench and Winsor. 1987). The
coexistence of two or more Symbiodinium taxa in the same
host was first described by Rowan and Knowlton (1995) in
the Caribbean reef building corals Montastrea unmiluris
and M. faveolata. Since then, more than one Symbiodinium
taxon has been identified in populations of certain other
coral species. Furthermore, some individual colonies have
been shown to harbor mixed symbiont taxa (Baker and
Rowan, 1997; Darius et al.. 1998; Baker, 1999; Carlos et
al.. 1999: Banaszak et al., 2000). From the data of Baker
( 1999), about 23% of the total coral taxa sampled may have
mixed symbiont populations, indicating that the presence of
more than one symbiont simultaneously is not uncommon.

Environmental parameters have been hypothesized to
regulate the distribution and population dynamics of each
symbiont in hosts that harbor more than one algal species
(Rowan et al.. 1997; Baker, 1999). The patterns of these
distributions are specific and correlate closely with environ-
mental gradients. Symbiodinium species examined in cul-
ture and /';; hospite show species-specific physiological
adaptations to photosynthetically active radiation and tem-
perature (Chang et al.. 1983. Iglesias-Prieto and Trench.
1994, 1997a, b; Warner et al.. 1996, 1999). Algal species
better adapted for a particular environment will out-compete
those less suited (Schoenberg and Trench, 1980b; Rowan et
al., 1997; Saunders and Muller-Parker. 1997).

Previous studies have focused primarily on the reef-wide
distribution of symbioses involving more than one algal
taxon. but very little is known about the biogeography of
algal symbionts and about how the distribution of algal
species in geographically widespread hosts may be influ-
enced by differences in environment. Ultimately, the prob-
lem revolves around the determination of the species of
algae involved in the associations. The difficulty in delin-
eating species has been a long-standing problem for ocean-
ographers studying phytoplankton biogeography (Round,
1981). With regard to S\mbiodiniitm, one example is the
coral Plesiastrea versipora, which has an unusually broad
latitudinal distribution along the east coast of Australia. On
the tropical Great Barrier Reef it forms a symbiosis with
S\mbiodinium sp. of the C lineage, while in cooler temper-
ate waters off Sydney it harbors a Symbiodinium sp. from
the B lineage (Baker. 1999).

Temperature and irradiance are the most significant en-
vironmental variables that change predictably with latitude.
Within the tropics, among coral species with more than one
algal taxon. the algae exhibit ecological zonation that cor-
relates with irradiance (Rowan and Knowlton, 1995; Baker,
1999). Irradiance in temperate regions is less consistent and
may not be an important parameter regulating the distribu-
tion of S. californium. When compared to California. Wash-
ington and Oregon have longer periods of daylight during

132

T. c. LAJEUNESSE AND R K. TRENCH

the summer, but experience shorter periods in the winter.
Assessment of the influence of light as an environmental
factor that regulates algal distribution is further complicated
because anemones act to control irradiance levels impinging
on the algae by covering their surfaces with fragments of
rock and shell (Dykens and Shick, 1984).

Temperature is an environmental factor that regulates
species distributions along the coast of California (New-
man, 1979), and it probably governs the distribution of S.
californium. Collection sites from Oregon and Washington
routinely experience colder temperatures than locations in
California (Fig. 3), particularly in winter. In addition, north-
ern anemones experience greater annual temperature fluc-
tuations, which may also be of selective importance. Low
temperature extremes, like high temperatures, may result in
the loss of symbionts from hosts (Muscatine et al.. 1991).
Temperature as a selective force is supported by the obser-
vation that, in northern anemone populations that harbor S.
muscatinei and the green Chlorella-like alga, symbiont
growth rates are more strongly affected by temperature than
by irradiance (Saunders and Muller-Parker, 1997). Temper-
ature changes associated with increased latitude may also
influence the competitive balance between S. californium

G" 18

, ! ,

Z> 16

* A A •
• 1

I —

| * ' * a

LU
Q. 14

• * • ; •

• T a A
• A Q

LU 12
O

A °
D

^ 10

* A A D

LU o
CO 8

a a
a o

i i i i i i i i i i

c -° « ^ S~? ^oS.*- ><.
foQ)™ Q-5D"5 30)ooc

_n "^ -^ *=Z. ^? — **• rr\ r\ —r t-

Figure 3. Average coastal sea surface temperature data from the
Comprehensive Ocean-Atmosphere Data Set (COADS) monthly climatol-
ogy records (1946-1989). Graph compares the seasonal fluctuation of sea
surface temperature in degrees Celsius at collection sites in Washington
(open square). Oregon (open triangle), and from central and southern
California (Carmel, solid circle; Cayucos. solid triangle; Santa Barbara,
solid square; Cardiff-by-the-Sea (Encinitas), solid diamond). The locations
are shown on the map in Figure 1. The southern locations in California
have similar seasonal temperatures and are always 1° to 6°C warmer than
northern locations depending on the time of year. The range in temperature
fluctuation between March (lowest yearly temperature) and September
(highest) is greater for Washington and Oregon (ca. 7°C) than for locations
in California (ca. 4°-5°C).

and 5. muscatinei by affecting their relative growth rates.
We also suggest the possibility that low temperatures found
in the north exclude S. californium; in the south. S. califor-
nium and 5. muscatinei coexist because the latter species
may have a wider temperature tolerance. Because attempts
to culture S. muscatinei were unsuccessful, controlled phys-
iological comparisons were not possible.

The nonrandom sorting or specificity of algal-inverte-
brate symbioses is contingent on the interplay of multiple
factors (Trench et al.. 1981; Trench, 1988. 1997), and is not
expressed as one alga for one host (Schoenberg and Trench.
198()b). Cnidarian hosts are symbiotic with selectively few
microalgal taxa (Trench, 1997), yet they are exposed to
hundreds and possibly thousands of "free-living" microalgal
species. The "complementariness of the dynamically inter-
acting attributes" (Dubos and Kessler, 1963) of both sym-
biont and host substantially limits possible symbiotic com-
binations. The extent to which a host shows specificity for
one or more algal species depends ultimately on the poorly
understood cellular and molecular processes that may take
place during initial intracellular contact, and possibly also
after the association is established (Colley and Trench.
1983; Trench. 1993). In those hosts harboring more than
one algal taxon, symbiont distributions are strongly influ-
enced by changes in the physical environment over both
local (Rowan and Knowlton, 1995; Secord, 1995) and geo-
graphic sales (Baker. 1999). Because the influence of envi-
ronment on host-symbiont dynamics can be variously inter-
preted, further experimental analyses are needed to explain
the mechanisms that result in these observed patterns (Saun-
ders and Muller-Parker, 1997). For example, it is unclear
whether changes in the physical environment have an in-
trinsic or extrinsic effect on the biology of the symbiosis.
Changes in the environment might modify the physiological
integration of the symbiotic partners so that they are no
longer compatible. Finally, differential changes in symbiont
growth rates may, and can, cause competitive exclusion of
one algal species over another (Provasoli et al., 1968).
Progress in elucidating these mechanisms should provide a
more accurate description of host symbiont interactions and
specificity.

Acknowledgments

We thank Drs. Ed Delong and Scott Hodges for use of
their lab facilities. We thank Dr. Gisele Muller-Parker for
collecting specimens from Anacortes. Washington; Dr. Jeff
Goddard for collecting anemones in Coos Bay, Oregon, and
Dr. Shana Goffriedi and Victoria Orphan for collections at
Cardiff-by-the-Sea, near Encinitas. California. Dr. Robert
A. Andersen and Dr. Craig Bailey at the Center for Culture
of Marine Phytoplankton. Bigelow Laboratory for Ocean
Sciences, and Dr. Lynda Goff and Dr. Deb Moon at the
University of California, Santa Cruz, are greatly appreciated

SYMH10DIN1UM SPECIES IN ANTHOPLEURA

133

for their assistance in sequencing. We especially thank Dr.
Alison Murray for her advice, invaluable technical guidance
on DGGE analysis, and comments on an early draft of this
paper.

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Reference: Biol. Hull. 1W: 135-143. (October 2000)

Seasonal Variation in Conduction Velocity of Action
Potentials in Squid Giant Axons

JOSHUA J. C. ROSENTHAL AND FRANCISCO BEZANILLA*

Departments of Physiology and Anesthesiology, UCLA School of Medicine,
Los Angeles, California 90095

Abstract. To determine whether the electrical properties
of the squid giant axon are seasonally acclimated, action
potentials, recorded at different temperatures, were com-
pared between giant axons isolated from Loligo pealei
caught in May, from relatively cold waters (-10°-12°C),
and in August, from relatively warm waters (-20°C). Pa-
rameters relating to the duration of the action potential (e.g.,
maximum rate of rise, maximum rate of fall, and duration at
half-peak) did not change seasonally. The relationship be-
tween conduction velocity and temperature remained con-
stant between seasons as well, in spite of the fact that May
axons were significantly larger than August axons. When
normalized to the fiber diameter, mean May conduction
velocities were 83% of the August values at all temperatures
tested, and analysis of the rise time of the action potential
foot suggested that a change in the axoplasmic resistivity
was responsible for this difference. Direct measurements of
axoplasmic resistance further supported this hypothesis.
Thus seasonal changes in the giant axon's size and resistiv-
ity are not consistent with compensatory thermal acclima-
tion, but instead serve to maintain a constant relationship
between conduction velocity and temperature.

Introduction

Signal transduction in the nervous system is profoundly
temperature sensitive. Acclimation of higher order nervous
function to variation in environmental temperature has been
the subject of many investigations (see Prosser and Nelson,
1981); however, the precise mechanisms of such acclima-

Received 3 March 2000; accepted 7 July 2000. E-mail: fbezanil®
UCLA.edu

* To whom correspondence should he addressed.

Abbreviations: Cm, membrane capacitance: MRF. maximum rate of fall:
MRR. maximum rate of rise; /?,., external resistivity; /?,, internal resistiv-
ity; i\.. external resistance; r,. internal resistance.

tions are not well understood. Do they involve changes at
the level of the action potential, the synapse, or both?
Action potential duration and propagation are strongly in-
fluenced by acute temperature changes (Hodgkin and Katz,
1949), largely due to temperature sensitivity of the under-
lying ion channels (Hodgkin et ai, 1952). If action poten-
tials themselves are a common target for thermal acclima-
tion, which properties are affected? In the giant nerve fibers
of earthworms, cold acclimation speeds the action potential
duration, conduction velocity, and refractory period vs. tem-
perature relationship, but not to the extent that, when mea-
sured at rearing temperatures, the kinetics of cold- and
warm-acclimated worms are equivalent (Lagerspetz and
Talo, 1967; Talo and Lagerspetz, 1967). In goldfish cardiac
muscle cells, the action potential's duration is reduced in
cold-acclimated fish (Ganim et ai, 1998). In certain Aplysia
neurons, however, early potassium current kinetics are not
affected by rearing temperature (Treistman and Grant.
1993).

The squid giant axon, long a model for understanding the
basic physiology of voltage-dependent ion channels, is, for
a variety of reasons, an excellent system for examining
temperature-dependent acclimation of the action potential.
First, its output participates in a known function — the jet-
propelled escape response (Prosser and Young, 1937;
Young, 1938; Otis and Gilly, 1990) — and presumably it is
advantageous for this response to be rapid. Second, the
dimensions of the giant axon permit action potentials, mac-
roscopic ionic currents, gating currents, and single-channel
currents to be measured from the same preparation
(Hodgkin and Huxley, 1952; Armstrong and Bezanilla,
1973; Conti and Neher, 1980; Bezanilla, 1987). Third. Na
and K currents, which underlie the action potential, have
been extensively characterized in this system (see Gilbert et
<//.. 1990). Fourth, giant axon Na and K channels have been

135

136

J. J. C. ROSENTHAL AND F. BE7.ANILLA

defined on a molecular level (Rosenthal and Gilly, 1993;
Rosenthal et it/., 1996). Finally, and perhaps most impor-
tant, squid of the genus Loligo (and other genera that
contain "giant" axons) inhabit a wide variety of thermal
environments.

Squid of the species Loligo pen lei live off the eastern
seaboard of North America, where inshore water tempera-
tures undergo large seasonal fluctuations. By examining the
axons from these squid in both May and August, the present
study seeks to identify those properties of the action poten-
tial that change on a seasonal basis.

Materials and Methods

Squid collection and water temperatures

Adult specimens of Loligo pealei were collected from the
waters surrounding Woods Hole, Massachusetts, in 1997
and 1998 during May and August. In May, specimens were
jigged from the town dock, and in August they were caught
by trawls in Vineyard Sound. During two trawls, water
temperature was measured at the net opening with a data
logger. Daily temperatures, recorded near the Marine Bio-
logical Laboratory (MBL) seawater intake system at a depth
of 15 feet, were kindly furnished by Janice Hanley. MBL
water quality technician. Data used in Figure 2 were re-
corded by the National Oceanic and Atmospheric Admin-
istration (NOAA) at the Woods Hole Oceanographic Insti-
tution pier (tide station number 8447930, available on the
NOAA website). Squid were maintained in flowing seawa-
ter, whose temperature was kept within 1 degree of the
intake temperature, and were used within 2 days of capture.
Animals were killed by rapid decapitation, and hindmost
stellar nerves were removed and carefully cleaned of small
fibers in seawater. All experiments, unless otherwise spec-
ified, were performed in 10 K+ artificial seawater (ASW;
composition in millimoles: 430 NaCl, 10 KC1, 50 MgCl2. 10
Cad,, 10 HEPES. pH 7.5, 970 mOsm).

Action potential measurements

Propagated action potentials were measured by mounting
a freshly dissected axon (4-6 cm) in a long, rectangular
glass chamber filled with 10 K. * ASW. Temperature was
regulated using two Peltier units mounted under the cham-
ber and was measured with a hand-held thermocouple po-
sitioned directly adjacent to the axon. After equilibration of
the chamber, temperatures at all points along the axon were
within ±0.5°C of the recorded temperature. Action poten-
tials were stimulated intracellularly at one end of the axon
using brief current pulses (2-20 juA for 200-400 jus) ad-
ministered through a 0.4-0.6 Mfi micropipette filled with 3
M KC1. and connected to the output of the online D/A
converter. Stimuli, which varied from axon to axon, were
the minimum required to produce a consistent action poten-

tial. Voltage signals were recorded at two points along the
axon using two additional micropipettes (3 M KC1. 1-3
Mil) connected to two high-impedance, capacitance-com-
pensated electrometers. The chamber was grounded using
two Ag+/AgCl coils embedded in 10 K" ASW + 3%
agarose and connected to virtual ground. Signals were col-
lected using software written in-house and a PC44 (Inno-
vative Technologies) signal processor board connected to a
PC compatible computer. Sampling rates varied between
200 kHz and 1 MHz, depending on the temperature, and
signals were filtered at Vw of the sampling rate. Axon
diameters and distances between micropipettes were mea-
sured with an eyepiece micrometer. Data were analyzed
only from electrode impalements with resting potentials
more hyperpolarized than —55 mV.

Capacitance and resistance measurements

Membrane capacitance was measured in the voltage-
clamp configuration as previously described (Bezanilla et
<;/., 1982a. b) with minor modifications. Signals were col-
lected as described in the previous section. A 75-/am plat-
inum wire for passing current and an internal measuring
electrode consisting of an 80-/am glass capillary filled with
0.6 M KC1 and containing a floating 25-/im platinum wire
were inserted into the axon in a piggy-back configuration
( Hodgkin ft <//.. 1 952 ). A wide-aperture glass capillary filled
with 10 K* ASW + 3% agar was used as an external
reference. Capacity transients were generated by brief
pulses from a holding potential of -80 mV to -90 mV. The
average of 10 such records was used for analysis. Data were
collected at 500 kHz and filtered at 50 kHz. For resistance
measurements, axons were carefully cleaned of all adhering
accessory fibers and placed on a platform of clear acrylic-
plastic. The axon ends were then cut and dipped into two
pools containing 500 mM K-glutamate. 10 HEPES. 5
EGTA, pH 7.5. These pools were voltage clamped, and
signals were collected, as described above. In all cases, data
were analyzed using in-house software. Error bars represent
the standard error of the mean (SEM). and a Student's t test
was used where probabilities are indicated in the text.

Results

In the giant axon, action potential duration and conduc-
tion velocity are strongly affected by temperature (Hodgkin
and Katz, 1949; Chapman. 1967). Figure 1A shows three
action potentials, recorded at different temperatures, from a
single electrode inserted into a giant axon. Clearly, the
durations of the rising and falling phase both increase as the
temperature is decreased. The half-width of the action po-
tential recorded at 2()°C is 365 /as. This number increases to
850 /as at 10.2°C. and to 2400 /as at 1.8°C. Furthermore, the
conduction velocity shows a similar temperature depen-
dence. In Figure IB. action potentials, evoked by a single

1 8C

SEASONAL CHANGES IN THE GIANT AXON
B. 25 n

137

40 mV

20.0 C

20 C

20 -

a is -\

e 10
I

5 -

Figure 1. Action potentials recorded al different temperatures. (A)
Propagated action potentials from a single position in a single giant axon
measured at three temperatures. (B) Propagated action potentials recorded
at two positions in a single axon measured at two temperatures. Axons
were bathed in 10 K+ artificial seawater. Brief transients at the beginning
of records are stimulus artifacts.

Figure 2. Seasonal temperature variation in the Woods Hole Passage.
Hourly temperature records from April through September for 1997 (solid
line) and 1998 (dotted linel. Temperatures were taken from NOAA station
number 8447930 located at the Woods Hole Oceanographic Institution at
41° 31.5' N, 70° 40.3' W.

stimulus, are recorded at two points, separated by 19.6 mm,
along the same axon. From this experiment the conduction
velocity is calculated to be 9.56 m/s at 5°C and 18.94 m/s at
20°C. The axons used in this figure were dissected from a
squid captured in May.

Water temperatures in the Woods Hole Passage and in
Vineyard Sound, where squid for these studies were cap-
tured, change dramatically between the spring and summer.
In May, squid were collected by jigging from waters di-
rectly measured to be 10°-12°C. In late August, squid were
captured by trawling, and on two trawls the average tem-
perature at the net opening was recorded to be 20.4°C and
21.2°C. Figure 2 shows hourly water temperatures recorded
by the NOAA tidal monitoring station (adjacent to the
Woods Hole Oceanographic Institution pier in the Woods
Hole Passage) between April and October, 1997 and 1998.
During May, water temperatures vary between about 10°
and 12°C. In August they average slightly greater than
20°C. These values are corroborated by daily temperature
records taken near the MBL seawater intake system at a
depth of 15 feet. In 1998, May and August average temper-
atures (±SD) were 12.4°C ± 1.2 and 21.8°C ± 0.49.
respectively. The few data points available for this site in
1997 are similar to the 1998 values (10.2°C on May 5,
12.8°C on May 28, 22.0°C on August 8, and 21.TC on
August 26). Thus, water temperatures during the study
period in both 1997 and 1998 changed by about 10°C.
Acute temperature changes of this magnitude would clearly
affect the giant axon's electrical properties (see Fig. 1 ). Do
these properties change to compensate for seasonal temper-
ature variation?

To answer this question, action potentials were compared

between squid captured in May and in late August. Figure 3
compares maximal rates of rise (MRR) and fall (MRF) of
the action potential, at various temperatures, for May and
August squid. No significant difference exists for either
measurement between groups. MRRs have a nearly linear

900 -

800 -
700 -

| 600-
g 500 -

5 400 ~

300 -

200

10 15 20

Temperature (-C)

Figure 3. Action potential maximum rates of rise and fall do not
change seasonally. Action potential records from squid in May (filled
symbolsl and August (open symbols) were differentiated numerically, and
maximum values (rates of rise; circles) and minimum values (rales of fall,
absolute values; triangles) were plotted against temperature. Action poten-
tials were recorded at two positions along an axon. Error bars represent the
standard error of the mean; n = 1 0 records (5 axons) for May and 1 1
records (6 axons) for August.

138

J. J. C. ROSENTHAL AND F. BEZANILLA

temperature relationship, while that of the MRFs is expo-
nential. In addition, the MRFs have a higher temperature
coefficient. Q10 values for the MRF are 3.7 and 2.5 for the
temperature ranges 0°-12.5°C and 12.5°-25°C. respec-
tively. For the same intervals, (2lo's for the MRR are 2.4
and 1.5. Other measurements relating to the action potential

duration (e.g., rise time, fall time, and duration at half-peak
amplitude) also showed no seasonal difference.

In contrast, resting potentials and conduction velocities
did exhibit significant seasonal variation. Figure 4A com-
pares the resting potential vs. temperature relationship for
May and August axons. Between 0° and 15°C, both groups

-52-

-54-
-56-

g -58

tin

.« -60

t/1

I
-62

-64

§ g 20
tj ^^
-§

6 .5

5 10 15 20 25
Temperature (C)

o
o

a
o

•a

1600

1400

1200

1000

. 800

600

400

0 5 10 15 20 25 30
Temperature (C)

Figure 4. Resting potentials and conduction velocities change seasonally. (A) Resting potential i'i. temper-
ature relationship for May axons (filled symbols) and August axons (open symbols). Error bars represent the
standard error of the mean (SEM); n = 10 for each season. (B) Conduction velocities vs. temperature for May
and August axons (same symbol convention) as in A. (C) Conduction velocities, normali/ed to the square root
of the diameter, r.v. temperature for May and August axons (same symbol convention). Error bars represent SEM;
» = 6 for each season for both B and C.

SEASONAL CHANGES IN THE GIANT AXON

139

become more hyperpolarized as the temperature is raised. In
this temperature range, the May axons are on average ~3
mV more depolarized. At temperatures greater than 15°C,
May axons become progressively more depolarized, while
August axons continue to hyperpolarize until approximately
20°C, after which they level out. Thus at 25°C the resting
potential disparity reaches —5 mV.

Figures 4B and 4C show the relationship between con-
duction velocity and temperature. Absolute (non-normal-
ized) conduction velocities were equivalent between May
and August squid (4B) at all temperatures tested. Interest-
ingly. May axons had significantly larger diameters (506 ±
26.8 /urn; mean ± SEM) than those in August (383 ± 14.68
jam; mean ± SEM). Conduction velocity in the giant axon
is proportional to the square root of the axon diameter, a
relationship originally established by Hodgkin and Huxley
( 1952) and verified by many other groups (Chapman, 1967;
Taylor, 1963). Taking this into account, normalized con-
duction velocities are plotted vs. temperature in Figure 4C.
Conduction velocities from May axons are relatively slow,
being on average 83% ± 2.5% (SD) of the August values.
In both seasons the (2io values were equivalent (1.5 be-
tween 10° and 20°C).

The fact that normalized conduction velocity values
changed from May to August, but the MRR and MRF did
not, suggested that the passive electrical properties of the
axon had also changed. The action potential's initial rate of
rise, or "foot," can be used to extract information related to
the axon's cable properties by the following relationship:

Cm(r,.+ c,,) = \/r02

where Cm is membrane capacitance, r, is internal resistance,
rf is external resistance, r is the time constant of the rise
time of the action potential foot, and 0 is the conduction
velocity (Taylor, 1963). Figure 5A shows an example of an
exponential fit to the foot of an action potential recorded at
10°C from a May axon. In this case, r was 210 /as, 0 was
12.45 m/s, and therefore C,n(r, + re) was 30.7 fl*F*cm.
Similar analysis was extended to action potentials from May
and August, and all data were normalized to account for
axons of different diameters (Fig. 5B). At all temperatures,
normalized C,,,(r, + re) was greater in May than in August
axons. On average. May values were 31.7% ± 6% (SD)
greater.

The preceding analysis indicated that the product of re-
sistance and capacitance was variable between seasons;
however, it did not identify which property changed. To
accomplish this, capacitance and resistance were measured
independently. Capacitance was measured at a variety of
temperatures using a conventional axial wire voltage clamp.
A <2io of 1.06 was determined for the relationship between
capacitance and temperature in two axons (data not shown).
This number agrees well with previously published data

from squid (Taylor et nl.. 1962) and was used to extrapolate
all experimental values to 15°C. For May and August axons,
mean capacitance was determined to be 1.03 ± 0.039 /xF/
cm2 (SEM, n = 6) and 0.96 ± 0.027 /LiF/cnr (SEM. n =
6), respectively. A statistical difference between these
means is not well supported (P - 0.18). Thus differences
in capacitance are not sufficient to account for the Cln(Rl +
/?,.) data from the previous section.

Axoplasmic resistance was measured directly in dissected
axons. Axons were blotted dry and placed on an acrylic
plastic platform; their ends were cut and dipped into two
reservoirs containing internal solution (Fig. 6A). The reser-
voirs were then voltage clamped, and after transients had
subsided, the current flow through the axon was measured
and normalized to the axon's cross sectional area and
length. All measurements were conducted at room temper-
ature (about 20°C). The results from a typical axon segment
of 404 /xm diameter and 3.65 cm length are shown in Figure
6B. In this case, the voltage between the reservoirs and the
current flow through the axon were 107.3 mV and 1.24 /J.A..
Thus the resistance (r, + r(.) for this axon was calculated to
be 86.5 kfl, and the specific resistance (R, + Rt) was 30.3
fl*cm. As expected, the current voltage relationship at a
variety of test potentials is linear (Fig 6C). A series of axons
from May and August were analyzed in a similar manner
and their specific resistivities were found to differ. Mean
R, + Re was measured to be 35.2 ± 1.3 fl*cm (± SEM,
n = 6) and 28.4 ± 2.5 (± SEM, n = 6; P = 0.05) in May
and August, respectively. Thus on average. May values are
22% greater than August values.

Discussion

The present investigations were initiated to identify sea-
sonal changes in the giant axon's electrical properties, and
to address whether these changes could compensate for
seasonal temperature variability. Implicit in these studies is
that the squid do in fact experience seasonal temperature
variability. Temperature data from all sources, taken at
various depths, all show a temperature profile similar to that
in Figure 2 (i.e., an approximate 10°C difference between
May and August). The recorded spring temperatures are
probably maximum values and thus are a conservative es-
timate of the squid's environment. Summer temperatures
reported in this paper are probably representative for Vine-
yard Sound and the Woods Hole Passage, the locations
where the squid used for these experiments were captured.
Turbulence, created by large tidal flows, prevents the for-
mation of thermoclines in these shallow areas, and temper-
atures are uniform throughout the water column. Other
papers have come to a similar conclusion about mixing and
report summer temperatures as high as 23°C (Summers,
1968; McMahon and Summers, 1971). Outside of the Vine-
yard Sound, temperatures are likely to be significantly

140

J. J. C. ROSENTHAL AND F. BEZANILLA

20 mV

= 210us •'

0.5 ms

(Q-F-cm)

0.005

0.0045

0.004

0.0035

0.003

0.0025

5 10 15 20
Temperature (C)

Figure 5. Product of resistance and capacitance changes seasonally. (A) Example of measurement. Dotted
line is an action potential recorded at 10°C from a May axon; solid line is an exponential tit to the action potential
foot. (B) Values of (/?, + Rt.)Cm vs. temperature for May (tilled triangles) and August (open triangles) axons.
See text for derivation of ( /?, + KelCm. Error bars represent the standard error of the mean; n = 6 for May and
5 for August.

lower. Schopf (1967) reports the presence of thermoclines
and maximum annual bottom-water temperatures of 13°C in
the waters off Nantucket.

The migration patterns of Loligo pealei are not well
understood. On a seasonal basis this species is reported to
winter near the break of the continental shelf where it can
avoid temperatures below 8°C (Summers, 1969). In the
spring these squid move inshore when waters warm past
about 10°C (Summers. 1969; Mesnil, 1977). The first group
to arrive are the 2-year-olds, normally in early May, fol-
lowed by the 1 -year-olds in June (Summers, 1971). This
sequence of events probably explains the larger diameter of
the May axons. Migration on a shorter time scale has not
been reported for this species, and therefore it is unknown
whether these squid migrate to colder oceanic waters on a
daily basis. We consider such a migration unlikely for two
reasons. First a substantial horizontal shift would be re-

quired to reach deep oceanic waters (depending on the point
of departure in Vineyard Sound). Second, the August squid
were routinely captured during the day. In other cephalo-
pods, daily migrations involve a nocturnal shift to shallow
waters (Boyle. 1983; Hanlon and Messenger, 1996). There-
fore it is likely that the squid spend a significant portion of
their time at the water temperatures specified in this report.
Various reports document the presence of L. pealei in yet
warmer waters (e.g.. 22°-29°C in the Gulf of Mexico; see
Boyle. 1983).

This study presents no evidence for a seasonal compen-
sation in the propagated action potential's duration, as there
is no change in the curve of MRR or MRF vs. temperature.
These data also suggest that the properties of the underlying
ionic current do not change, and studies using voltage-
clamped giant axons support this conjecture (data not
shown). Therefore it is predicted that in vivo, the duration of

SEASONAL CHANGES IN THE GIANT AXON

141

I. V

V, I.

Axon

Ou.A-

OmV-

4uA
40 mV

-200 -150 -100 -

2ms

IOiA)

50 100 150 200
V(mV)

Figure 6. Direct resistance measurements. (A) Schematic of experimental setup. A defined length of axon
was placed on a clear plastic platform, and each end was cut and placed in a bath containing internal solution
(in mM: 500 K-glutamate, 10 HEPES, 2.5 EGTA, pH 7.5). The interbath voltage was clamped using a
home-built squid axon apparatus, and the resulting current was measured. (B) An example of the current
resulting from a 100-mV voltage step (May axon). (C) Current-voltage relationship from the same axon. All
experiments performed at temperatures between 18° and 20°C.

the action potential in May squid is over twice as long as it
is in August squid (see Fig. 1 for examples of action
potentials recorded at 10°C and 20°C). Unlike the action
potential duration, the conduction velocity does appear to be
regulated between seasons. However, the direction of the
change is not consistent with a compensatory thermal ac-
climation: despite a seasonal disparity in axon diameter, the
relationship between conduction velocity and temperature
remains constant, due mostly to resistive changes in the
axon. Computer simulations of conduction velocities using
the Hodgkin and Huxley equations (Hodgkin and Huxley,
1952) support this assertion. By substituting the May and
August values for Cm(/?; + /?,.). determined by tits to the
action potential foots (32.4 OF and 42.7 flF, respectively, at
10°C), there is a 14.2% increase in conduction velocity. The
difference determined from direct measurements of conduc-
tion velocities in May and August axons at 10°C was
14.6%.

Fits to the action potential foot predicted that the product
of (/?,. + Re) and C,,, increased by approximately 30%
between August and May. This is in reasonable agreement
with direct measurements of (/?,- + /?t,) for the same sea-
sons, which increased by 22%. Capacitive changes were not

found to be statistically significant. It is probable that resis-
tive changes are due to changes in Rt, as the contribution of
Re to our measurements is expected to be very small. Great
care was taken to blot the axon's external surface prior to
recording, thus the layer of adhering seawater would be
quite small compared to the cross-sectional area of the axon.
Cole and Hodgkin, who employed a similar experimental
setup, came to the same conclusion (Cole and Hodgkin,
1939). In addition, our reported values of/?, + Re, partic-
ularly those for August (28.7 ll*cm), are consistent with
previously reported values of /?,-. Cole and Hodgkin re-
ported 29 fi«cm (Cole and Hodgkin, 1939) and in a separate
report. Cole reported the resistivity of extruded axoplasm to
be 28 fl*cm (Cole, 1975). Using two internal microelec-
trodes. Carpenter el al. (1975) reported it to be 31 O«cm. It
is unclear during which season these studies were con-
ducted.

Seawater. which is isosmotic with axoplasm (Gilbert et
nl., 1990), has a specific resistivity of only 20 fi«cm
(Cole and Hodgkin, 1939). Why is axoplasm a relatively
poor conductor? First, unlike seawater, axoplasm con-
tains mostly large organic anions that have lower mobil-
ities than chloride. In addition, our measurements of

142

J. J. C. ROSENTHAL AND F. BEZANILLA

resistivity consider the axon as a conducting cylinder,
and do not take into account the organelles, which occupy
an unknown percentage of the volume. Finally, axoplasm
contains a good deal of immobile protein. We ob-
served that axoplasm appears gelatinous in May, whereas
in August it is considerably more liquid, possibly due to
a decrease in the order of the underlying cytoskeletal
proteins. Differences in any of these parameters could
underlie the seasonal differences in axoplasmic re-
sistivity.

In the absence of a seasonal acclimation, giant axon
action potentials would travel much faster in August than
in May. The resistive changes discussed in this paper
would not help compensate for temperature changes in
conduction velocity and therefore they do not contribute
to a compensatory acclimation. However, the giant axon
is clearly a part of a larger motor system, the pieces of
which do not have equal temperature sensitivities. Per-
haps thermal acclimation involves maintaining specific
ratios between the rates of processes. For example, the
temperature dependence of the giant synapse is steep
compared to the various properties related to the action
potential discussed in this paper (see Llinas, 1999). Be-
tween 10° and 20°C, synaptic delay at the giant synapse
has a <2io of 3.8 (Llinas el ai, 1987) as compared with the
Q,0 of 1.5 measured for action potential conduction ve-
locity in this work. Therefore the ratio of synaptic trans-
mission to conduction velocity, which may be important
for integration in the nervous system, would be greater in
August than in May. The resistive changes discussed in
this paper would help to maintain a more similar ratio
between seasons. In support of this conjecture it is note-
worthy that the non-normalized relationship between
conduction velocity and temperature did not vary be-
tween May and August, in spite of the fact that August
axons were significantly smaller.

Acknowledgments

We thank Drs. David Gadsby, Paul De Weer, Robert
Rakowski, and Barbara Ehrlich for generously sharing
laboratory space at the Marine Biological Laboratory;
Roger Hanlon for help procuring and maintaining squid:
and Janice Hanley, Dr. George Hampson, and John Va-
lois for providing temperature data. This work was sup-
ported by a National Institute of Health Grant (GM
30376) and a National Institute of Health NRSA post-
doctoral training grant (NS 07101-18-19) for Dr. Joshua
Rosenthal.

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Reference: Biol. Bull 199: 144-156. (October 2000)

Role of the Cytoskeleton in Sperm Entry During

Fertilization in the Freshwater Bivalve

Dreissena polymorpha

M. J. MISAMORE* AND J. W. LYNNt
Department of Biological Sciences, Louisiana State University', Baton Rouge, Louisiana 70803

Abstract. The present study examined the role of the
cytoskeleton in sperm entry and migration through the egg
cytoplasm during fertilization in the zebra mussel, Dreis-
sena polvmorpha (Bivalvia: Veneroida: Dreissenidae). Fer-
tilization in this freshwater bivalve occurs outside the man-
tle cavity, permitting detailed observations of fertilization.
After its initial binding to the egg surface, the sperm is
incorporated in two stages: (1) a gradual incorporation of
the sperm nucleus into the egg cortex, followed by (2) a
more rapid incorporation of the sperm axoneme. and trans-
location of the sperm head through the egg cytoplasm.
Initial incorporation into the egg cortex was shown to be
microfilament dependent. Microfilaments were found in the
sperm's preformed acrosomal filament, the microvilli on the
egg surface, and in an actin-filled insemination cone sur-
rounding the incorporating sperm. Treatment of eggs with
cytochalasin B inhibited sperm entry in a dose- and time-
dependent manner. Microtubule polymerization was not
necessary for initial sperm entry.

Following incorporation of the sperm head, the flagellar
axoneme entered the egg cytoplasm and remained active for
several minutes. Associated with the incorporated axoneme
was a flow of cytoplasmic particles originating near the
proximal end of the flagella. Inhibition of microtubule
polymerization prevented entry of the sperm axoneme, and
the subsequent cytoplasmic current was not observed. After
sperm incorporation into the egg cortex, no appreciable

Received 16 May 2000; accepted 5 July 2000.

*Present address: Dept. Cell Biology, Univ. of Texas-Southwestern
Medical Center, 5323 Hurry Mines Blvd.. Dallas, TX 75235-9039. E-mail:
michael.misamorels'email. swmed.edu

t To whom correspondence should he addressed. E-mail: /olynn
(fplsu.edu

Abbreviations: CB, cytochalasin B; MF. microtilament; MT. microtu-
hule; PI, post insemination; PW, pondwater.

microfilaments were associated with the sperm nucleus. A
diminutive sperm aster was associated with the sperm nu-
cleus during its decondensation, but no obvious extension
toward the female pronucleus was observed. The sperm
aster was significantly smaller than the spindle associated
with the female pronucleus, suggesting a reduced role for
the sperm aster in amphimixis.

Introduction

During fertilization in the freshwater zebra mussel,
Dreissena polymorpha (Bivalvia: Veneroida: Dreissenidae),
gametes are spawned directly into the water column, where
gamete binding occurs. External fertilization is common
among marine bivalves but is unusual among freshwater
bivalves, in which fertilization and larval development typ-
ically occur in the mantle cavity (Burky. 1983). Broadcast
spawning of gametes, the relative ease of inducing spawn-
ing (Ram et al., 1993). and the highly transparent nature of
the egg cytoplasm make zebra mussels a good model system
for studying early fertilization events.

Sperm entry into the egg cytoplasm during fertilization is
a two-step process (Misamore et al., 1996). During the
initial phase, the sperm head and midpiece are gradually ( 10
jim/min) incorporated into the egg cortex. Following sperm
binding, an insemination cone forms on the egg surface and
envelopes the fertilizing sperm head as it passes through the
egg plasma membrane. Once inside the egg cortex, the
sperm rotates 180° and "drifts" laterally along the egg
cortex. During the second phase of sperm entry, most of the
sperm axoneme is incorporated into the egg cytoplasm, and
the sperm head may be rapidly ( 1 jum/s) translocated
through the egg cytoplasm. The amount of translocation
apparently depends on the position of the female genome in
relation to the site of sperm entry (M. Misamore. unpubl.

144

CYTOSKELETON IN ZEBRA MUSSEL FERTILIZATION

145

results). After axonemal incorporation and nuclear translo-
cation. the sperm chromatin decondenses and is encom-
passed by a membrane forming the male pronucleus. Con-
current with sperm fusion, meiosis is reinitiated from
metaphase I arrest, polar bodies form, and the female pro-
nucleus develops. Pronuclear fusion and cleavage occur by
60-70 min postinsemination (PI).

The gradual incorporation of the sperm head in D. pol\-
morpha displays similarities to early sperm entry in other
invertebrate species (Longo and Anderson, 1968; Schatten
and Mazia, 1976; Longo, 1973b). In these systems, micro-
filaments are believed to play a critical role in sperm incor-
poration into the egg cortex (Longo. 1980; Tilney and Jaffe.
1980; Cline et al. 1983; Cline and Schatten. 1986). This
theory is supported by the localization of microfilaments
both in egg microvilli (Burgess and Schroeder, 1977; Spu-
dich and Amos, 1979; Tilney and Jaffe, 1980) and in sperm
acrosomes (Tilney. 1975. 1978; Tilney el al.. 1973); by the
fertilization modifications of microfilament distribution,
particularly in fertilization cones (Longo, 1978b, 1980; Til-
ney and Jaffe, 1980; Schatten and Schatten, 1980; Cline and
Schatten, 1986); and by the blockage of fertilization by the
inhibition of microfilament polymerization using cytocha-
lasins (Gould-Somero et al.. 1977; Longo. 1978a: Byrd and
Perry, 1980; Schatten and Schatten, 1980). Movement of
the sperm nucleus, or subsequent pronucleus, through the
egg cortex in most invertebrate and mammalian systems is
attributed primarily to the formation of a microtubule-con-
taining sperm aster. However, many bivalve species pro-
duce only a rudimentary sperm aster.

The objective of this study was to determine the role of
microfilaments and microtubules in the two-step incorpora-
tion of sperm into the egg cytoplasm in Dreissena polymor-
pha. The initial, gradual incorporation of the sperm into the
egg cortex required microfilament polymerization, whereas
the rapid translocation of the sperm nucleus through the egg
cytoplasm was microfilament independent but could be
inhibited by preventing flagellar incorporation into the egg.
Furthermore, a flow of cytoplasmic particles was observed
to be associated with the sperm axoneme, which remained
active following entry into the egg.

Materials and Methods

Collection of gametes

Specimens of Dreissena polymorpha were collected from
the Mississippi River near Baton Rouge, Louisiana, or from
Portage Lake near Ann Arbor, Michigan, and maintained at
9°C. Animals were individually isolated overnight in arti-
ficial pond water (PW) (Dietz et al., 1994) to avoid cross-
contamination of gametes prior to use. Spawning was in-
duced by external exposure of animals to 2 X 10~4 M
serotonin (5-hydroxytryptamine) for 12 min. Animals were
washed twice with PW. At the start of spawning, females

were transferred to 50-ml crystalizing dishes to complete
spawning. Eggs were inseminated with about 300 sperm per
egg.

Three preparation and fixation protocols were used. For
light microscopy, eggs were fixed in 3.2% paraformalde-
hyde in mussel buffer (5 mM TAPS, 0.8 mM NaCl. 0.145
mM KC1. 1.8 mM Na2SO4, 0.887 mM MgSO4 • 7H:O, 1.32
mM NaHCO.,. 1.19 mM CaCl2 • 7H2O. pH = 7.6)" for 3 h
followed by two washes in mussel buffer. Eggs for antibody
labeling were permeabilized in an extraction buffer ( 1 .0 mM
TAPS, 5 mM KC1. 0.5 mM MgCl:, 1.0 mM EGTA, 0.05%
Nonidet P-4O, 1% glycerol. pH = 7.6) for 5 min. followed
by fixation in 3.2% paraformaldehyde in a low-Ca"+ mussel
buffer (3 mM EGTA. 10 mM TAPS, 3 mM NaCl, 0.01 mM
KC1. 1.5 mM MgSO4. 0.01% sodium azide, pH = 7.6) for
3 h, followed by two washes in phosphate buffered saline
(PBS) (8.1 mM sodium phosphate dibasic, 1.8 mM sodium
phosphate monobasic. 25 mMNaCl, 25 mMKCl, pH = 7.8)
with 0.5% bovine serum albumin. Electron microscopy
samples were fixed in 2.5% glutaraldehyde in mussel buffer,
washed twice with 30 mM sodium cacodylate buffer, post-
fixed in 0.5% osmium tetroxide for 1 h, dehydrated through
a graded acetone series, and embedded in a modified Spurt's
medium (Spurr, 1969).

Microscopy

For real-time observations, either a Nikon Optiphot with
phase contrast and epifluorescence optics or a Nikon Dia-
phot with differential interference contrast (DIC) was used.
Confocal imaging was performed on a Noran Instruments
(Madison, WI) argon laser confocal microscope with In-
tervision Software. A JEOL 100CX transmission electron
microscope was used for high-magnification observations.

Eggs were stained with 1 /xg/ml Hoechst 33342 for 8 min
to label DNA. To distinguish sperm bound to the egg
surface from sperm incorporated into the egg cytoplasm, a
dual-labeling procedure was used. Briefly, inseminated eggs
were fixed and dual-labeled with Hoechst 33342 and FITC-
conjugated wheat germ agglutinin (WGA) (Sigma Chemi-
cals. St. Louis). Hoechst 33342 labeled sperm and egg
DNA; WGA-FITC labeled sperm and egg surfaces (Kreim-
borg and Lynn, 1996). Sperm bound to the surface were
labeled with both fluorochromes; incorporated sperm were
labeled only with Hoechst. To label microfilaments, sam-
ples were stained with 1 jug/ml FITC-conjugated phalloidin
(Sigma Chemicals) for 10 min then washed twice with
mussel buffer. To label egg microtubules (MTs). permeabil-
ized samples were exposed to a monoclonal antibody to
yeast a-tubulin (Accurate Antibodies MAS-077, Westbury,
NY) at 1:20 dilution for 45 min, followed by a FITC-
conjugated secondary antibody. A monoclonal antibody to
acetylated a-tubulin (Sigma Chemicals) at 1:100 dilution
for 1 h was used to selectively label sperm axonemes. This

146

M. J. MISAMORE AND J. W. LYNN

100
66

. 46

_ en
.— O)
O LU

£ E

S. a

co </)

Figure 1. The monoclonal antibody against acetylated a-tubulin rec-
ognizes microtubules associated with the sperm but not the egg. Western
blot analysis against whole-cell sperm (Sp) or Eggs (E) labeled a 55-Kd
band in sperm that was not present in eggs.

antibody selectively labeled sperm axonemal MTs but not
egg MTs (Fig. 1) (L'Hernault and Rosenbaum. 1983;
L"Hernault and Rosenbaum. 1985: Fechter et al., 1996).

Inhibition experiments

Prior to exposure to the inhibitors, eggs were treated with
0.005% sodium periodate for 2 min to increase permeability
through the extracellular coats of the eggs. This pretreat-
ment does not significantly impact sperm binding or fertil-
ization (Misamore, unpubl. results). Eggs from one spawn
were aliquoted into the various treatment groups.

Cytochalasin B (CB) (Sigma Chemicals. St. Louis, MO)
was used to inhibit microfilament (MF) polymerization dur-
ing fertilization. Microtubule polymerization was inhibited
with colchicine and colcemid, which form complexes with
tubulin on MT ends, preventing further polymerization; or
with nocodazole, a synthetic benzimidazole that promotes
MT depolymerization (Bray, 1992).

To determine the dose-dependent effects of the inhibitors
on fertilization, periodate-treated eggs were incubated in the
inhibitors (final concentrations: CB— 6.2, 12.4 ;uM in 0.6%
DMSO; colchicine— 50 to 200 jaM; colcemid— 51 ju,A/ in
0.4% DMSO; nocodazole— 0.1, 50 /mW) or the appropriate
PW control (with or without DMSO) for 10 min prior to
insemination. Eggs were inseminated in the presence of the
inhibitors without washing. Real-time observations were
captured using video microscopy from 30 s to 40 min PI.
Samples were fixed at 10 and 30 min PI and dual-stained
with Hoechst 33342 and WGA-FITC. Stained eggs (;i =
100) were scored to determine sperm entry, number of
bound sperm, meiotic stage of female DN A, and presence of
polar bodies. To determine whether the effects of the inhib-

itors are reversible, eggs were incubated in the inhibitors for
10 min, washed twice with PW. and inseminated.

Based on preliminary findings, a time-exposure series
was also performed to determine the temporal effect of CB
on early sperm entry. Cytochalasin B ( 12.4 /iA/) was added
to periodate-treated eggs at the following time points: 10
min before insemination or 0 min. 2 min, or 4 min PI.
Control eggs were incubated in 0.6% DMSO 10 min prior to
insemination.

Statistical analysis

For each trial, eggs from a single female were divided
into the treatment groups. From each treatment, 100 eggs
were scored for incorporated sperm nuclei and polar body
formation. Replicate trials (n = 5) for the dose-dependent
and time-dependent experiments were performed, and
means of numbers of eggs with incorporated sperm were
calculated for all treatments. A two-way analysis of vari-
ance (ANOVA) blocking for variability between trials (i.e.,
different batches of eggs) as one factor and treatment as the
second factor was performed using SigmaStat software,
version 2.0 (SPSS Science. Chicago. ID. Tukey multiple
comparisons were performed if significant differences (a =
0.05) were found.

Results

Morphology

As background pertinent to the results, an expanded de-
scription of the basic gamete morphology is presented here.
Additional descriptions of Dreissena polymorpha gametes
are given by Franzen (1983). Denson and Wang (1994).
Misamore et al. (1996). and Walker et al. (1996). The
acrosome of D. pnlymoiplia sperm contains four distinct
regions, including an axial rod 1.4 jum in length. The
filaments composing the axial rod are about 6 nm in diam-
eter (Fig. 2) and extend from the apex of the acrosome to the
anterior margin of the nucleus (Misamore et al.. 1996).
FITC-phalloidin labeling was localized to a spike-like struc-
ture in the acrosomal region of both bound and acrosome-
reacted sperm (Fig. 2, inset).

The surface of spawned eggs is uniformly covered in
microvilli at an estimated density of 50 microvilli/jinr over
the entire egg surface. The microvilli are 0.8 jiun in length
and 0.07 /u,m in diameter (Misamore ft al., 1996). The
internal core of the microvilli consists of 5-nm parallel
fibers perpendicular to the egg cortex. The distal tips of the
microvilli have many tufts of filamentous material perpen-
dicular to the long axis of the microvilli (Fig. 3).

Initial sperm entry

Shortly after sperm binding, the sperm acrosome opened,
exposing the membrane-bound axial rod. The preformed

CYTOSKELETON IN ZEBRA MUSSEL FERTILIZATION

147

Figure 2. Transmission electron micrograph ITEM) of a cross section
through the sperm acrosome. The central axial rod (arrowhead) extended
the length of the acrosome and consisted of numerous 6-nm filaments.
Inset: Fluorescent micrograph of a spontaneously acrosome-reacted sperm
stained with the fluorochrome phalloidin-FITC. Intense fluorescent local-
ization occurred with the anterior portion of the sperm nucleus and the
axial rod (arrowhead). Additional localization was observed at the basal
region of the sperm nucleus (arrow). Bar = 0.5 fim.

Figure 3. TEM of the egg cortex exhibiting numerous microvilli
embedded in a 0.7-ju.m-thick extracellular coat. The distal tips of the
microvilli were bulbous and possessed minuscule, fibrous lateral projec-
tions. The microvilli were 0.8 /urn in length. 0.07 /j,m in diameter, and had
an internal core of 5-nm parallel fibers perpendicular to the egg surface.
CV — cortical vesicle. Bar = 1 /xm.

Figure 4. TEM of the sperm axial rod spanning the extracellular
matrix and contacting the egg plasma membrane. The axial rod (arrow-
head) did not elongate following binding with the egg surface. N — sperm
nucleus. Bar = 0.5 /xm.

Figure 5. Numerous bundles 60 nm in diameter consisting of 6-nm
filaments (arrows) extended the length of the insemination cone perpen-
dicular to the plasma membrane in this TEM. Cortical vesicles (CV) were
not exocytosed following egg activation and remained subjacent to the
insemination cone. Bar = 1 jtm.

Figure 6. Epifluorescent micrograph of an inseminated egg labeled
with the actin-specific fluorochrome phalloidin-FITC. A fluorescing region
(arrowhead) corresponded to the insemination cone. Inset: Phase micro-
graph of the insemination cone showing the position of the sperm nucleus
(arrow) within the insemination cone. Dual labeling with the DNA-specific
fluorochrome Hoechst 33342 confirmed the position of the nucleus within
the phalloidin localization (data not shown). Bar = 10 fj.m.

axial rod was long enough to traverse the egg investment
coat to the egg surface (Fig. 4). No additional polymeriza-
tion or elongation of the axial rod was observed. Microvilli
directly subjacent to the open acrosome remained perpen-
dicular to the egg surface. Small fibrous structures posi-
tioned peripheral to bound sperm and angled toward the
bound sperm were observed with scanning electron micros-
copy (Misamore et al., 1996). However, no reorientation of
microvilli toward the sperm was discernible. The filamen-
tous tufts of the distal microvillar tips did not appear to be
associated with a specific region of the acrosome. No
change in microvilli length was observed up to 10 min PI.

About 2-3 min PI, an insemination cone formed on the
egg surface. As the sperm passed through the oolemma, the
cone initially appeared as a conical structure about 1.6 p,m
in diameter and 2.7 /xm in length. Once the sperm head
reached the egg cortex, the distal end of the cone became
pointed as the egg cytoplasm closed around the sperm head.
Filaments 6 nm in diameter extend the length of the fertil-
ization cone in the egg cytoplasm (Fig. 5). FITC-phalloidin
labeled the fertilization cone as a well-defined region on the
oolemma surrounding the sperm nucleus (Fig. 6). After the
sperm had entered the egg cortex, no definitive phalloidin
labeling was associated with the sperm head; however, high
background fluorescence may have obscured minimal label-
ing.

About 5-7 min PI, the first polar body began to form.
There was a clearing of cortical vesicles and a reduction in
microvilli length associated with the site of polar body
extrusion. When the egg stained with phalloidin-FITC, a
brightly fluorescing region was seen on the surface where
the polar body first emerged (Fig. 7). After expulsion of the
female dyad, a cleavage furrow formed, separating the polar
body cytoplasm from the remaining egg cytoplasm. This
polar body furrow corresponds with a fluorescing ring at the
base of the first polar body (Fig. 7).

This initial passage of the sperm through the insemination
cone into the egg cortex was blocked by inhibiting micro-
filament polymerization with CB in a dose-dependent man-
ner (Fig. 8). In the presence of CB, sperm were able to bind
to the egg surface but did not enter into the egg cytoplasm.
When eggs were exposed to 12.4 fj.M CB, sperm entry was
completely inhibited, whereas exposure to 6.2 fiA/ CB sig-
nificantly reduced the number of eggs with incorporated
sperm relative to the control (P < 0.001 ) (Fig. 8; Fig. 9B',
C'). Polar body formation was completely inhibited by both
6.2 and 12.4 juM CB. The female chromosomes were posi-
tioned at the egg periphery and persisted through anaphase

Figure 7. Actin localization with phalloidin-FITC produced a highly
fluorescing region associated with the polar bodies in this fertilized egg.
The actin-containing region was associated with the margins separating the
first and second polar bodies (arrowhead) and the second polar body and
egg surface (arrow). Bar = 25 ju.m.

148

M. J. MISAMORE AND J. W. LYNN

•°

S. 80%

a a

— T

1 1 "0%

S o%
^

PWCtl DMSO 62uMCB 124uMCB
Treatment

tion when the sperm head was immediately (<10 /urn)
subjacent to the cortex, the sperm head frequently pivoted
around the entry point through angles exceeding 90° (Fig.

Figure 8. Dose-dependent effects of cytochalasin B on sperm entry
into the egg cortex during fertilization. Eggs were exposed to two concen-
trations of the microfilament inhibitor cytochalasin B (CB). Unfertilized
eggs were pretreated with periodic acid, then transferred to a solution of
either 6.2 /nM CB or 12.4 y.M CB in O.htf DMSO or to a control of either
artificial pondwater (PW Ctl) or 0.6% DMSO. Eggs were incubated 10 min
and inseminated directly in the egg-inhibitor solution. Eggs were fixed at
10 min PI and scored for number of incorporated sperm (n = 100/trial).
Values are mean ± standard error (n = 5). Letters indicate statistically
significant differences based on a two-way ANOVA and Tukey multiple
comparisons (P < 0.05).

(Fig. 9). Although a brief extrusion of cytoplasm was ob-
served during the period of normal polar body formation (5
min PI), no cytoplasmic separation occurred and the extru-
sion was reabsorbed by 10 min PI.

Sperm entry into the egg cortex was not affected by
inhibition of MT polymerization. In both normal (control)
fertilizations and fertilizations in the presence of MT inhib-
itors, D. polymorpha sperm bound to the egg surface within
30 s of insemination, and an insemination cone formed by 3
min PI. The insemination cones of both control and MT-
inhibited trials were morphologically similar, and the sperm
head and mitochondria passed through the oolemma and
entered the egg cytoplasm within 4 min of binding (Fig. 9D,
D'). Compared to the control, there was no significant
difference (Fig. 10) in the numbers of eggs exhibiting sperm
entry in the samples treated with colchicine (P = 0.935) or
nocodazole (P = 0.774). Colcemid did significantly de-
crease sperm entry relative to controls (P = 0.032); how-
ever, greater than 68% of the eggs had sperm incorporated.
While not affecting sperm entry, the MT inhibitors dis-
rupted the first meiotic spindle, and female chromosomes
appeared scattered and did not reach anaphase I (Fig. 9D,
D'). Subsequent polar body formation was likewise pre-
vented.

Sperm translocation through egg cytoplasm

Following incorporation into the egg cortex, the sperm
head passed through the insemination cone, rotated 180° in
the egg cortex — positioning its basal end toward the egg
center, and moved laterally along the egg cortex (Fig.
I1A-D: Fig. 12A-C). The direction of lateral movement
was towards the quadrant of the egg containing the female
genome. Following rotation, most of the axoneme entered
the egg cytoplasm. During the initial axonemal incorpora-

Figure 9. Complementary phase (A. B, C, D) and epifluorescent (A',
B', C', D') micrographs of eggs incubated in various treatments for 10 min
prior to insemination. (A, A') Control fertilization of eggs incubated in
0.67r DMSO. The female chromatin (f) was positioned subjacent to the
first polar body (pb) following meiosis I. The male chromatin (m) was seen
decondensing in the egg cytoplasm. (B. B') Egg incubated in 6.2 /nAf
cytochalasin B prior to insemination. Sperm entered the egg cytoplasm and
began chromatin dccondensation (m). Egg activation occurred, as evident
by resumption of meiosis. The female chromatin (f) separated in anaphase
I, but polar body formation was inhibited. (C. C') Egg incubated in 12.4
IJ.M cytochalasin B for 10 min prior to insemination. Sperm bound to the
egg surface (arrow) but were not incorporated into the egg cytoplasm.
Female chromatin separated (f), but polar body formation was inhibited.
(D, D') Egg treated with 50 mA7 colcemid. Sperm entered the egg cyto-
plasm and began chromatin decondensation (M). The female chromatin
appeared scattered and not arranged in either the arrested (metaphase) or
activated (anaphase) orientation. Eggs were lived 10 min PI and stained
with the DNA-specitic fluorochrome Hoechst 33342. Bar = 25 /-im.

CYTOSKELETON IN ZEBRA MUSSEL FERTILIZATION

149

100%

80%
60%
40%
20%
0%

50 uM 100uM
Colch Colch

DMSO 100nM
Ctl Noc

DMSO 50 mM
Cll Colce

Figure 10. The effect of the microtubule inhibitors on sperm entry.
Unfertilized eggs were pretreated with periodic acid, then incubated in
various inhibitors or their respective control solutions. Eggs were incubated
10 min and inseminated directly in the egg-inhibitor solution. Eggs were
fixed at 10 min postinsemination and scored for incorporated sperm (n =
100/trial). Values are mean percentage of eggs with incorporated sperm ±
standard error for 5 replicate trials. PW Ctl — eggs incubated with pond-
water, DMSO Ctl — control eggs incubated in 0.6'7r DMSO, Colch — eggs
incubated in either 50 yM or 100 jj.M colchicine. Noc — eggs incubated in
100 mM nocoda/ole in 0.6<7r DMSO. Colcemid — eggs incubated in 50 nM
colcemid in 0.6% DMSO. No statistically significant difference was de-
tected between inhibitor treatments and their respective controls (P >
0.78) except colcemid (P = 0.032). * — indicates significant difference.

13). As more of the axoneme entered the cytoplasm, the
axoneme extended deeper into the egg cortex relative to the
sperm head, which remained near the egg cortex (Fig. 14 A,
A'). Once the bulk of the axoneme passed through the
oolemma, the sperm head frequently moved rapidly through
the egg cytoplasm. This movement did not occur in the
sinusoidal pattern typical of sperm swimming. Rather, a
portion of the axoneme extended ahead of the trailing sperm
head along the direction of movement.

Although they did not impact entry of the sperm head into
the egg cortex. MT inhibitors suppressed the rotation and
lateral movement of the sperm head along the cortex. Fur-
thermore, MT inhibitors prevented the incorporation of the
sperm axoneme into the egg cortex (Figs. 12A-C; 14B, B';
15; 16). Under these conditions, the sperm heads exhibited
rapid, sporadic oscillations immediately subjacent to the
insemination cone for several minutes after passage through
the cone. This sporadic oscillation seen in the presence of
MT inhibitors corresponded temporally to the period of
expected axoneme incorporation in control fertilizations. In
most inhibitor observations, the sperm remained immedi-
ately subjacent to the cone up to 7 min PI and the axoneme
ultimately fractured, allowing the sperm nucleus to move
deeper into the ooplasm — although more slowly than in
controls (Fig. 12). During this period the sperm flagellum
remained outside the egg. extending out through the insem-
ination cone. On occasion, the unincorporated portion of the
flagellum detached from the egg surface and remained ac-
tive for several minutes. In contrast to control observations,
probing MT-inhibited eggs with the flagellar monoclonal
antibody failed to label an axoneme associated with decon-
densing sperm nuclei (Fig. 14B, B').

Once sperm passed through the egg cortex, inhibition of

MF polymerization did not affect sperm head or axoneme
incorporation or subsequent nuclear decondensation. Expo-
sure of eggs to 12.6 p.M CB at various times pre- and
postinsemination showed a temporal effect of CB on insem-
ination (P < 0.001 ) (Fig. 17). Eggs incubated in CB 10
min prior to insemination showed no sperm incorporation or
polar body formation (see Fig. 8). Eggs exposed immedi-
ately prior to insemination (0 min) showed almost complete

4 min

Figure 11. DIC microscopy Time series, in differential interference
contrast (DIC) microscopy, of captured video images showing the incor-
poration of the sperm head during a normal (control) fertilization. The
arrowhead parallels the left side of the sperm and illustrates the orientation
of the sperm head with the apex of the arrowhead corresponding to the
anterior end of the sperm nucleus. (A) Sperm head immediately subjacent
to insemination cone. (B-D) Sperm head rotated and was laterally dis-
placed along the egg cortex. Notice the rotation and lateral displacement of
the sperm nucleus along the egg cortex relative to the insemination cone.
Times indicate elapsed time from initial frame (A). Bar = 20 /xm.

Figure 12. DIC microscopy time series of captured video images
showing the incorporation of the sperm head into eggs exposed to 100 nA/
nocodazole. The arrowhead parallels the left side of the sperm and illus-
trates the orientation of the sperm head, with the apex of the arrowhead
corresponding to the anterior end of the sperm nucleus. (A) Sperm head
immediately subjacent to insemination cone ( * ). ( B ) Sperm head remained
subjacent to insemination cone, did not undergo rotation or lateral dis-
placement. (C) Sperm head separated from the attached flagellum and
moved deeper into the egg. The arrow marks a particle adhering to the
flagellum. Notice that the flagellum remains external to the egg. Times
indicate elapsed time from initial frame (A). Bar = 20 ;um.

150

M. J. MISAMORE AND J. W. LYNN

blockage of sperm entry and were not significantly different
from the preincubution trial (P < 0.05). Addition of CB at
2 min PI significantly reduced sperm entries (P < 0.05)
relative to controls but did not cause complete inhibition
(Fig. 17). Sperm entry was not significantly different from
controls when CB was added 4 min PI. Sperm entry and
rotation in the egg cytoplasm occurred in eggs treated at 4
min PI with CB. At about 3 min PI, sperm-head transloca-
tion and axoneme incorporation were observed. The pres-
ence of the axoneme in the egg cytoplasm was verified
using the monoclonal antibody to acetylated a-tubulin.

At about 8 min PI, sperm nuclei began to decondense in
control (Fig. 18) and MF and MT inhibitor trials. Sperm
mitochondria separated from the nucleus and moved away
from the nucleus concurrent with the decondensation of the
sperm chromatin. At the same time, or immediately afterward,
a small sperm aster started to develop (Fig. 19). The aster
formed at the base of the decondensing sperm head and was
only detectable with the monoclonal yeast a-tubulin antibody,
visualized with confocal microscopy. In contrast, the female
meiotic spindle and astral array were clearly discernible with
light microscopy. In control fertilizations, the sperm aster was
detectable as a diminutive structure as late as 40 min PI.
During this period the female aster and associated bundle were
substantially larger (Fig. 19) (Walker, 1996). Colchicine-
treated eggs exhibited no obvious sperm aster, and the female
meiotic spindle was not observed. Since egg activation was
determined based on the resumption of meiosis as visualized
by movement of chromosomes into anaphase I, disruption of
the meiotic spindle made the status of egg activation in MT
inhibitor treatments difficult to ascertain.

Flow of cytoplasmic particles

Concurrent with axoneme entry, sporadic "vibrations"
were observed in the egg cytoplasm between the sperm head

Figure 13. A diagrammatic representation of the oscillations exhibited
by a single sperm head following its passage through the insemination
cone. Once the sperm head had entered the egg cortex there was a
coordinated flexing of the internal and external portions of the flagellum.
This movement resulted in oscillation of the sperm head 1 1 • • 4) within the
egg cortex.

Figure 14. Complementary epifluorescent micrographs of the incor-
porated sperm dual-labeled for flagella (A, B — a monoclonal antibody to
acetylated tubulin) and sperm nucleus (A'. B' — Hoechst 33342). (A, A')
Control fertilization — The sperm axoneme (arrowhead) extended deeper
into the egg cytoplasm relative to the peripherally positioned sperm nu-
cleus (m). (B, B') Eggs pretreated with colchicine prior to insemination. No
incorporated axoneme (B) was observed in association with fertilizing
sperm nuclei (m) seen in (B'l. These findings support real-time observa-
tions that the sperm axoneme was not incorporated in the presence of MT
inhibitors. 10 min PI. Bar = 25 jam.

Figure 15. Epifluorescent micrograph of an colchicine-treated egg
fertilized with a sperm dual-labeled with FITC-WGA (arrow) and Hoechst
33342 (arrowhead) prior to insemination. The sperm nucleus (arrowhead)
was incorporated into the egg cytoplasm and began decondensing. Colchi-
cine inhibited the incorporation of the sperm flagellum. which was visu-
alized by the FITC-WGA labeling of the sperm plasma membrane (arrow).
Bar = 25 /im.

Figure 16. Epifluorescent micrograph of Hoechst-stained eggs insem-
inated and exposed to 12.4 iiM cytochalasin B 4 min PI. By 10 min PI,
sperm entered into the egg cortex and began decondensing. The incorpo-
rated sperm nucleus (m) was clearly identified by the less intensely fluo-
rescing halo formed by the dispersing chromatin. Polar body formation was
inhibited, f — female chromalin.

and the sperm entry site near the expected position of the
axoneme. A directed flow of cytoplasmic particles was
similarly observed originating near the base of the sperm

CYTOSKELETON IN ZEBRA MUSSEL FERTILIZATION

151

100%

80%
60%
40%
20%

a.c

DMSO Pcelnc O1 CB Z CB
Treatment

Figure 17. Temporal effects of cytochalasin B (CB) on sperm entry
following the addition of the microfilament inhibitor at various time points
pre- and postinsemination (PI). CB was added to sodium periodate-treated
eggs at 12.4 jiA/. Treatments were (DMSO). Control of 0.6% DMSO at 10
min preinsemination; (Prelnc). CB added 10 min preinsemination; (()' CB).
immediately following insemination; (2' CB). 2 min PI; and (4' CB), 4 min
PI. Eggs were fixed at 10 min PI and scored for the number of eggs with
incorporated sperm. Values are mean ± standard error (n = 5). Letters
indicate statistically significant differences based on a two-way ANOVA
and Tukey multiple comparisons (P < 0.05).

head and penetrating as deep as 10 ju.m into the ooplasm
(http://www.mbl.edu/BiologicalBulletin/VIDEO/BB.video.hrml).
The vibrations in the egg cytoplasm began shortly after

Figure 18. Phase-micrograph time series of video-captured images
showing the sperm decondensation and pronuclear formation. Sperm nu-
cleus (arrowhead) was visible in the egg cortex by 4 mm PI. (B) Sperm
chromatin (arrowhead) decondensation occurred by 10 min PI. (C) Sperm
pronuclear (arrowhead) formation began by 30 min PI. Similar results were
observed in eggs incubated in 100 \iM colchicine prior to insemination
(D-G)orin 12.4 \iM cytochalasin B at 4 min PI (not shown). Note multiple
decondensing sperm pronuclei. indicating polyspermic eggs. Bar = 25 ,um.

rotation and lateral displacement of the sperm head, lasted
from 1-3 min. and ceased shortly before the sperm nucleus
began to decondense. Although the extent varied between
fertilizations, this cytoplasmic flow was evident in greater
than 70% of the 20-30 similar filmed observations and on
about 80 more unri lined occasions. These observations are
the norm rather than the exception in the hundreds of
fertilizations observed during several reproductive seasons.
Furthermore, polyspermic eggs exhibited multiple currents
associated with the polynumery sperm. When flagellar in-
corporation was inhibited, no flow of cytoplasmic particles
was observed in eggs. Similarly, the flow of cytoplasmic
particles was also observed in eggs exposed to MT inhibi-
tors followed by washing prior to insemination.

Discussion

Initial sperm entry

The incorporation of the sperm components through the
oolemma into the egg cortex in Dreissena polymorpha
occurs in two morphologically distinct steps (Misamore et
ai, 1996). During the initial incorporation, the sperm head
and midpiece gradually enter into the egg cortex at a rate of
1 jum/min (Misamore et ai. 1996). A distinct, cylindrical
insemination cone encompasses the sperm as it passes
through the oolemma. The insemination cone of D. pol\-
morpha consisted of many 6-nm-thick filaments (Fig. 5) and
labeled with FITC-phalloidin (Fig. 6), suggesting the pres-
ence of microfilaments. The cone assumed a more pyrami-
dal configuration once the sperm entered the egg cortex.
This is similar to morphological changes in the insemination
cone of sea urchins, in which an initially rounded cone
becomes a "spike-like" cone following sperm entry (Tilney
and Jaffe. 1980: Cline and Schatten. 1986). When treated

Figure 19. Laser scanning confocal image of a diminutive sperm aster
in an egg. The 30-min PI egg was labeled with a monoclonal antibody to
yeast a-tubulin. Notice the relatively small size of the male aster (small
arrow) in relation the large microtubule array (large arrow) associated with
the female pronucleus and polar body. The asterisk (*) indicates the
relative position of the sperm nucleus dual-labeled with Hoechst 33342
(not shown). Bar = 25 /urn.

152

M. J. MISAMORE AND J. W. LYNN

with the inhibitor to MF polymerization, cytochalasin B,
this initial sperm entry was blocked (Fig. 9C. C"). and no
fertilization cone formed. These findings suggest that the
initial incorporation of the sperm into the egg cortex was
dependent on the polymerization of MFs.

Although the critical involvement of microfilaments in
sperm incorporation has been reported for many marine
invertebrates (Gould-Somero et ai, 1977; Longo, 1978a,
1980; Byrd and Perry, 1980; Schatten and Schatten, 1980;
Cline and Schatten, 1986; Schatten el ai. 1986) and the
present freshwater model, exactly how sperm pass through
the oolemma is not well understood. Microfilaments are
associated with several processes during fertilization in D.
pol\morpha that could potentially account for the inhibition
of initial sperm entry. The major sites of polymerized MFs
include the sperm acrosome, the egg microvilli. and the
fertilization cone. Furthermore, MFs are critical for cytoki-
nesis during polar body formation (Longo, 1972; Longo et
ai, 1993).

The sperm of several marine bivalves including Spisula
and Mytilus possess preformed acrosomal processes that do
not undergo a polymerization-driven elongation (Hylander
and Summers, 1977; Longo, 1978a). Following activation,
Mytilus sperm extend a preformed acrosomal process with-
out the polymerization of new MFs (Dan, 1967; Longo.
1977, 1983). Like these marine bivalves, D. polymorpha has
sperm that possess a preformed acrosomal process (Fig. 2)
that does not elongate and is apparently insensitive to CB
treatments at the dosages tested. Although it was not pos-
sible to expose only sperm to CB because washing disrupted
the fragile acrosomes, polymerization of sperm MFs is not
believed to be the critical component in sperm entry. This
conclusion is based on several pieces of evidence. First, no
elongation of the acrosomal process was observed during
fertilization in D. polymorphu. Second, several studies in
which washing of sperm was possible have shown that CB
does not affect the fertilization capability of sperm (Sanger
and Sanger, 1975; Longo. 1978a; Byrd and Perry, 1980).
Third, the relative polarity of MFs in sperm acrosomes and
egg microvilli is inappropriate to allow a myosin-actin
ratcheting mechanism to draw the sperm into the egg in
urchins (Tilney, 1978), and this is presumably the case for
D. polymorpha as well. Finally, the addition of CB at 2 min
PI allowed sufficient time for the sperm binding to occur
prior to inhibition; however, sperm entry was still sup-
pressed, suggesting MF involvement at a stage later than
sperm binding.

The involvement of microvilli in sperm entry varies
greatly between species, but a role has been suggested in
hamsters (Yanagimachi and Noda, 1970), urchins (Tilney
and Jaffe, 1980), annelids (Anderson and Eckberg, 1983),
and bivalves (Longo, 1987). Furthermore, Wilson and Snell
(1998) propose that microvillus-like structures may be es-
sential for most types of cell-cell fusion events. Hylander

and Summers (1977) proposed a generalized model of fer-
tilization in Mollusca. According to their model, sperm
binding occurs between an inner acrosomal region of the
sperm and microvilli tufts. Similar microvillar tufts were
observed in D. polymorpha: however, no obvious associa-
tion between these tufts and the inner acrosomal membrane
was observed. Furthermore, microvilli appeared to remain
perpendicular to the egg surface and did not reorient toward
bound sperm as reported for Spisula (Longo and Anderson,
1970; Hylander and Summers, 1977). Misamore et ai
(1996) reported extracellular fibers extending toward at-
tached sperm; however, these fibers are substantially
smaller than the egg microvilli.

Microfilament presence in insemination cones has been
well documented. In urchins, insemination cones may form
from the fusion of microvilli (Schatten and Schatten, 1980),
and MFs in the cones are polymerized into discrete bundles
(Tilney and Jaffe, 1980) from monomeric actin in the egg
cortex (Spudich and Amos. 1979). Molluscan insemination
cones are markedly smaller than urchin cones and MFs are
not consistently reported in the cones (Longo, 1983). As in
Mytilus and Spisula (Longo and Anderson, 1970: Longo,
1983), in D. polymorpha MFs in fertilization cones run the
length of the ooplasmic projection, but not in discrete bun-
dles as observed in urchins (Fig. 5). While the exact mech-
anisms involved remain unclear, insemination cones are
implicated in sperm entry (Longo, 1980). The inhibition of
sperm entry by CB also suppresses the formation of insem-
ination cones (Longo, 1980; Schatten and Schatten, 1980.
1981).

Cytochalasin B was shown to have a reversible, dose-
dependent effect on fertilization: partial inhibition occurred
at 6.2 juA/CB and complete inhibition at 12.4 /u,A/. Byrd and
Perry (1980) reported similar dose-dependent findings in
two urchin species. Strongylocentrotus purpuratiis and
L\techinus pictus. Sperm entry was decreased at 2.5 jug/ml
(5 p.M) CB in the former species and at 5 /Ag/ml (10 ^.M) in
the latter; inhibition was complete at 5 /ng/ml ( 10 |U.A/) and
10 /Ltg/ml (20 H,M) respectively. Gould-Somero et ai ( 1977)
found that slightly lower levels of CB partially (1 /aAf) or
completely (2 ju,A/) blocked sperm entry. That the dose-
dependent responses are similar is somewhat remarkable
considering the variability in the gametes, extracellular
coats, and fertilization mechanisms between these diverse
species.

Sperm entry was effectively blocked when CB addition
preceded or was concomitant with insemination (Fig. 17).
Addition of CB at 2 min PI resulted in fewer eggs exhibiting
sperm penetration, but at 4 min PI sperm entry was not
significantly affected. These findings suggest that CB was
able to rapidly (within 1-2 min) block sperm entry, and that
the period of susceptibility to CB inhibition was completed
by 4 min PI. The first 4 min PI during D. polymorpha
fertilization corresponds to the 1 ju.ni/min gradual-incorpo-

CYTOSKELETON IN ZEBRA MUSSEL FERTILIZATION

153

ration phase into the egg cortex. After 4 min PI, CB was
unable to inhibit sperm-axoneme incorporation, mitochon-
dria detachment, or male-chromatin decondensation and
pronuclear formation. CB impact on fertilization is limited
to the first 6 min PI in several urchin species (Longo, 1980;
Byrd and Perry, 1980: Schatten and Schatten, 1980), and
echiuroid worms (Gould-Somero ft ai, 1977). The restric-
tion of MF involvement in sperm incorporation to the first 6
min following insemination applies across a wide taxo-
nomic range.

Unlike MF polymerization, MT polymerization was not
required for the initial incorporation of the sperm nucleus
into the egg cortex in D. polymorpha. Initial entry of sperm
into eggs in marine invertebrates (Schatten and Schatten,
1981; Schatten er ai, 1982. 1989) and algae (Swope and
Kropf. 1993) also does not require MTs. Sperm were able to
enter the egg cortex in MT inhibitors at a rate ( 1 /xm/min)
similar to that observed in normal fertilizations. MT inhib-
itors were able to penetrate the egg and were effective at
disrupting the meiotic spindle, thereby preventing polar
body formation. Furthermore, no MT array was observed to
be associated with entering sperm nuclei when a-tubulin
monoclonal antibody was used to label fertilized eggs.

Sperm nuclear translocation and flagellar incorporation

After passing through the fertilization cone and entering
the egg cortex, D. polymorpha sperm rotated 180°, posi-
tioning the basal end of the nucleus centrad (Fig. 1 1 ). The
first fluorochrome-detectable MTs associated with entering
sperm were the diminutive sperm asters adjacent to decon-
densing sperm chromatin (Fig. 19). Small sperm asters have
been reported for other bivalve species (Longo and Ander-
son, 1969, 1970; Longo et ai, 1993). In contrast, the sperm
aster is significantly larger in most invertebrate and mam-
malian systems and is believed to be responsible for the
migration of the male and female pronuclei during
syngamy. For example, in sea urchins the sperm aster ex-
tends toward the female pronucleus and is thought to effect
the migration of the two pronuclei (Zimmerman and Zim-
merman, 1967; Longo and Anderson, 1968; Longo, 1976;
Schatten, 1981; Bestor and Schatten, 1981; Sluder et ai.
1985). The role of the sperm aster in D. polymorpha is not
fully understood as it does not extend toward the female
pronucleus.

A markedly larger MT array is associated with the female
chromatin in D. polymorpha (Walker, 1996). A dense bun-
dle of MTs is observed immediately subjacent to the polar
bodies. Emanating from the MT bundle toward and sur-
rounding the female pronucleus is a prominent cone-shaped
array of MTs. This MT bundle is believed to anchor the
female pronucleus and guide its centrad movement into the
egg (Walker, 1996). The large female aster is also believed
to play an important role in movement of the male pronu-

cleus. An analogous structure may also be present in both
Spisula and Mytilus (Longo, 1973a). Furthermore, as has
been found in urchins (Zimmerman and Zimmerman, 1967),
colcemid prevents pronuclear migration in D. polymorpha
(Walker, 1996).

Following sperm rotation, most of the sperm axoneme
was incorporated into the egg cytoplasm and the sperm head
often was rapidly (1 /im/s) translocated through the egg
cytoplasm (Misamore ct til., 1996). Microfilament polymer-
ization appeared to play little or no role in the second stage
of sperm entry. Addition of CB after initial sperm entry (4
min PI trials) failed to prevent the rapid translocation of the
sperm nucleus or the incorporation of the flagella. Further-
more, no obvious association of MFs and incorporated
sperm was observed using epifluorescence or electron mi-
croscopy.

Conversely, MT polymerization played a prominent, yet
somewhat unconventional, role in sperm nuclear transloca-
tion and flagellar incorporation. Following entry of the
sperm head into the cortex, the bulk of the sperm axoneme
was incorporated into the egg cytoplasm in D. polymorpha.
During this incorporation, dramatic movements of the
sperm head were observed, as well as a lateral migration of
the sperm head along the egg cortex. These movements may
be analogous to "jerking" movements exhibited by urchin
sperm during axoneme incorporation (Schatten, 1981).
Schatten (1981) suggests that the continued movement of
the sperm tail may be involved in its movement through the
fertilization cone and into the cytoplasm proper. In D.
polymorpha, there is an obvious correlation between move-
ments of the flagellum as it enters the egg cytoplasm and
movements exhibited by the sperm head in the egg cortex.
The exact mechanisms involved in flagellar incorporation
are not known. Video microscopic observations of both
urchins (Schatten, 1981) and zebra mussels (this study)
suggest that flagellar movement may be involved. Further-
more, flagellar incorporation in D. polymorpha was blocked
by MT inhibitors. Microtubule polymerization appears to be
essential for flagellar incorporation in D. polymorpha. This
finding is in contrast to the results of studies with urchins, in
which nocodazole did not inhibit axoneme incorporation
(Schatten and Schatten, 1981; Fechter et «/., 1996). Further-
more, the exaggerated movement of the sperm head imme-
diately subjacent to the insemination cone during the inhib-
ited axoneme incorporation further supports the concept that
flagellar movement takes part in axoneme incorporation.

In contrast, Epel et at. (1977) reported that deflagellated
sperm heads were able to bind and enter sea urchin eggs.
Attempts to duplicate those experiments with D. polymor-
pha in the present investigations were unsuccessful. Finally,
Schatten and Schatten (1981) reported that MT inhibitors
increased the lateral displacement of the sperm head along
the cortex and that the formation of the sperm aster may
signal the end of this lateral movement (Schatten, 1982). In

154

M. J. MISAMORE AND J. W. LYNN

D. polymorpha, lateral displacement of the sperm head was
restrained by the attached yet unincorporated flagellum.
Once the flagellum was severed, however, lateral displace-
ment and decondensation were observed.

Following axonemal incorporation and quiescence, the
sperm mitochondria separated from the nucleus as it began
to decondense. As in other invertebrate species (Schatten
and Schatten. 1981), sperm decondensation in D. polymor-
pha was not affected by either MF or MT inhibitors. In
zebra mussels, nuclear decondensution and mitochondria!
separation are apparently unaffected by MT inhibitors, sug-
gesting that flagellar detachment is also unaffected. In sea
urchins, microtubules appear to be essential for detachment
of the sperm tail, its migration toward the female pronu-
cleus, and its disassembly (Schatten and Schatten, 1981;
Fechter et al., 1996). Similarly, activation of D. polymorpha
eggs by sperm was unaffected by the presence of either MF
or MT inhibitors. Several studies have shown that early egg
activation occurs during fertilization even in the presence of
CB (Byrd and Perry, 1980: Schatten and Schatten, 1980;
Dale and DeSantis, 1981; Lynn, 1989). In these studies,
initiation of the cortical granule release or an electrophysi-
ological response were used as indicators of egg activation.
Like those of most molluscs (Humphreys, 1967; Longo,
1983), the eggs of D. pol\morpha do not release cortical
granules immediately following egg activation. Since D.
polymorpha eggs are inseminated at metaphase I arrest, the
resumption of meiosis can serve as an indicator of egg
activation (Longo, 1972, 1978a: Longo et al., 1993). In this
study, D. polymorpha sperm readily bound to the egg sur-
face in the presence of MF or MT inhibitors and apparently
induced the resumption of meiosis, since eggs devoid of
bound sperm remained in metaphase arrest.

Flow of cytoplasmic particles

During axoneme incorporation, a significant cytoplasmic
movement was noted in the region of the axoneme. Flows of
cytoplasmic particles were observed in numerous regions
near the site of sperm entry and conspicuously originating at
the basal region of the sperm head. The impetus or func-
tional significance of this flow remains in question. Two
possible mechanisms for generating these currents include
beating by a functional axoneme displacing the particles or
plus-end-directed transport along axonemal MTs by motor
proteins associated with either the cytoplasmic particles or
the sperm axoneme.

There appear to be few. if any. reported instances where
flagella retain dynamic function once incorporated into the
egg cytoplasm (Schatten, 1981; 1982). The last movement
typically associated with flagellar bending occurs shortly
after sperm binding, and movement of the sperm nucleus
once inside the egg is typically associated with cytoskeletal
elements, specifically the sperm aster (Schatten. 1982;

Longo, 1987). Technical limitations make it difficult to
isolate movements attributed to incorporated axonemes
from egg-derived events; nevertheless, several pieces of
evidence support the concept of an active axoneme inside
the egg.

First, the sperm axoneme retains the ability to generate
movement following demembranation during incorporation.
Active movement by isolated, demembraned sperm axon-
emes has been demonstrated in other species (Bray, 1992).
Second, video microscopy of mechanically-ruptured, fertil-
ized D. polymorpha eggs revealed incorporated sperm vig-
orously moving within the collapsing egg membrane (Mis-
amore, pers. obs.). Third, there is an apparent alteration of
the flagellar beat pattern in incorporated sperm. During the
rapid translocation of the sperm through the egg cytoplasm
(Misamore et al., 1996). the sperm head trails the proximal
portion of the axoneme. The proximal third of the axoneme
becomes the leading portion of the moving sperm cell.
Similar types of flagella-driven movement can be seen in
other systems. For example, the single-celled flagellate Eu-
glena moves via a singular flagellum that extends slightly
more anterior than the cell body before bending posteriorly
(Bray, 1992). Helical waves running the length of the fla-
gellum propel the cell forward, resulting in a rotational
movement to the cell body. Hamster sperm exhibit a pro-
nounced change in beat pattern upon entry into the dense
cumulus oophorous surrounding the egg. Penetrating sperm
frequently progress with the proximal portion of the flagel-
lum extending slightly forward, with the head ratcheting
through the dense cumulus oophorous (Yanagimachi,
1994). The change in flagellar beating seen in D. polymor-
pha may be attributable to the greater viscosity of the egg
cytoplasm relative to the external milieu.

A second potential source of the observed cytoplasmic
flow could result from active movement of particles down
the exposed axoneme MTs by molecular motors. The plus-
end-directed flow of particles suggests the presence of a
kinesin or kinesin-like motor. Initial attempts to label in-
corporated axonemes with kinesin antibodies have been
unsuccessful (data not shown); however, support for this
hypothesis is as follows. Microtubule motors are relatively
abundant in the egg cytoplasm. Gilksman and Salmon
(1993) reported substantial MT gliding along surfaces
coated with an ooplasm extract. Scholey et al. ( 1985) have
isolated a kinesin from urchin eggs. Porter et al. (1987)
showed that this egg kinesin exhibited plus-end movement
along isolated axonemes, and kinesin-coated beads translo-
cate along centrosome MTs. Furthermore, Kozminski et al.
(1995) found that a flagellar kinesin. FLAK), facilitated
movement of intraflagellar particles, or rafts, along the
length of the axoneme of Chlamydomonas flagella. Clearly,
more detailed testing of this hypothesis is needed.

Finally, no flow of cytoplasmic particles was observed in
D. p(il\morpha zygotes when axoneme incorporation was

CYTOSKELETON IN ZEBRA MUSSEL FERTILIZATION

155

blocked with MT inhibitors. The observed flow is either
directly, or at least indirectly, related to the incorporated
flagellum. The significance of this particle flow down the
axoneme remains an intriguing question.

In summary, initial sperm entry into the egg cortex is a
gradual. MF-dependent process, while subsequent flagellar
incorporation is MT dependent. Dynamic movement of the
incorporated sperm head and flagella is observed inside the
egg cytoplasm, and a flow of cytoplasmic particles associ-
ated with the incorporated axoneme was observed. D. poly-
morpha serves as a good model for studying fertilization
and exhibits many similarities to other fertilization models.
However, as demonstrated in this study, its remarkably
transparent cytoplasm allows extremely detailed observa-
tions of the intracellular interactions between eggs and their
incorporated sperm, revealing previously undescribed phe-
nomena that warrant further study in both this and other
systems.

Acknowledgments

We gratefully acknowledge the following individuals for
their invaluable contributions to this research: Susan J.
Nichols, J. Rachel Walker, Katie Kreimborg, Steven Smith,
Thomas Dietz, and the staff of the M.D. Socolofsky Mi-
croscopy Center. This research was funded in part by a grant
from Sea Grant of Louisiana NOAA grant #46RG00960,
project R/ZMM-2.

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Reports of Papers Presented at

THE GENERAL SCIENTIFIC MEETINGS

OF THE MARINE BIOLOGICAL LABORATORY,

Woods Hole, Massachusetts
14 to 16 August 2000

Program Chairs:

BARBARA BOYER, Union College

WILLIAM ECKBERG, Howard University

CHARLES HOPKINSON, Ecosystems Center, MBL

ROBERT PAUL MALCHOW, University of Illinois at Chicago

Each of these reports was reviewed by two members of a special editorial board
drawn from the research community of Woods Hole, Massachusetts.

Reviewers included scientists from

THE MARINE BIOLOGICAL LABORATORY,

THE WOODS HOLE OCEANOGRAPHIC INSTITUTION,

AND THE NATIONAL MARINE FISHERIES SERVICE.

SHORT REPORTS FROM THE 2000 GENERAL SCIENTIFIC MEETINGS OF THE

MARINE BIOLOGICAL LABORATORY

FEATURED REPORT

The Editors

Introduction to the featured report. On mapping
odor quality 161

Wachowiak, Matt, Michal Zochowski, Lawrence B.

Cohen, and Chun X. Falk

The spatial representation of odors by olfactory re-
ceptor neuron input to the olfactory bulb is concen-
tration invariant. . 162

Hale, Melina E.

Startle responses of fish without Mauthner neurons:
escape behavior of the lumpfish (C.-yrlnfilmts lumpus) 18(1

Epstein, David A., Herman T. Epstein, Frank M. Child,

and Alan M. Kuzirian

Memory consolidation in llrniiissi'mla crassicornis . . . 182

Abenavoli, A., L. Forti, and A. Malgaroli

Mechanisms of spontaneous miniature activity at
CA3-CA1 synapses: evidence for a divergence from a
random Poisson process 184

NEUROBIOLOGY

Landowne, David

Heavy water (D.,0) alters the sodium channel gating
current in squid giant axons 164

White, Thomas H., Harris Ripps, Miduturu Srinivas,

and Roberto Bruzzone

\'oltage gating properties of channels formed by a
skate retinal connexin 165

Molina, AnthonyJ.A., Peter J.S. Smith, and Robert Paul

Malchow

Hydrogen ion fluxes from isolated retinal horizontal
cells: modulation bv glutamate 168

Wang, Jing W.

Odor-induced oscillatory activity in Dmsitphila CNS ... 17(1

Hitt, James M., Frederick A. Dodge, Ehud Kaplan, and

Robert B. Barlow

C.ircadian rhythms in the receptive fields of the Limu-

lu\ lateral eve 171

Fay, Richard R., and Peggy L. Edds-Walton

Frequency response of auditory brainstem units in
toadfish (O/>siinus Ian) 173

Yamaguchi, Ayako, Leonard K. Kaczmarek, and Darcy

B. Kelley

Intrinsic membrane properties of laryngeal mo-
toneurons that control sexually differentiated vocal
behavior in .African clawed frogs, Xena/ius lamis .... 175

Atherton, Jillian L., Matthew A. Krutky, James M. Hitt,

Frederick A. Dodge, and Robert B. Barlow

Optic nerve responses of Limulus in its natural habi-
tat at night 1 76

Krutky, Matthew A., Jillian L. Atherton, Spence Smith,

Frederick A. Dodge, and Robert B. Barlow

Do the properties of underwater lighting influence

the visually guided behavior of Limulus? 178

PHYSIOLOGY A\D BIOCHEMISTRY

Novales Flamarique, Inigo. Kristiina Ovaska, and
Theodore M. Davis

UV-B induced damage to the skin and ocular system

of amphibians 187

Harrington, John M., and Peter B. Armstrong

Initial characterization of a potential anti-fouling sys-
tem in the American horseshoe crab, Limulus
polyphemus 189

Asokan, Rengasamy. Margaret T. Armstrong, and Peter

B. Armstrong

Association of a.,-macroglobulin with the coagulin
clot in the American horseshoe crab, Limulus
pohphemus: a potential role in stabilization from pro-
teolysis 190

Kuhns, William J., Max M. Burger, Mohan Sarker,

Xavier Fernandez-Busquets, and Tracy Simpson

Enzymatic biosynthesis of N-linked glycan by the ma-
rine sponge Microciniia fmiUJmi 192

Armstrong, Peter B., and Rengasamy Asokan

A Ca + J-independent cytolytic system from the blood

of the marine snail Busyron canaliculum 194

Heck, Diane E., Lydia Louis, Michael A. Gallo, and

Jeffrey D. Laskin

Modulation of the development of plutei by nitric
oxide in the sea urchin Arbaria punctulata 195

Jung, Sung-Kwon, Katherine Hammar, and Peter J.S.

Smith

Development of self-referencing oxygen microsensor
and its application to single pancreatic HIT cells:
effects of adenyiate cyclase activator forskolin on ox-
vgen consumption 197

Hanselmann, Rhea, Roxanna Smolowitz, and Daniel G.

Gibson

Identification of proliferating cells in hard clams. . . 199

159

160

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

Brothers, Christine, Ernest Marks III, and Roxanna
Smolowitz

Conditions affecting the growth and zoospomlation

of the protistan parasite QPX in culture 200

CELL BIOLOGY

Sandberg, Leslie, Phillip Stafford, and George M.
Langford

Effects of myosin-II antibody on actin-dependent ves-
icle transport in extracts of clam oocytes 202

Stafford, Phillip, Jeremiah Brown, and George M.

Langford

Interaction of actin- and microtubnle-based motors
in squid axoplasm probed with antibodies to myosin
V and kinesin 203

Tran, P.T., V. Doye, F. Chang, and S. Inoue

Microtubule-dependem nuclear positioning and un-
clear-dependent septum positioning in the fission
yeast, Saccharomyces pombe 205

Crawford, Karen

The role of microtubules during blastodisc forma-
tion of the squid Loliga pealei 207

Weidner, Earl

Cytoplasmic proteins on the surface of discharged
microsporidian sporoplasms 208

MacKenzie, Roger, David Newman, Max M. Burger,

Rene Roy, and William J. Kuhns

Adhesion of a viral envelope protein to a non-self-
binding domain of the aggregation factor in the
marine sponge Microdona prolifera 209

Goda, Makoto, Mario H. Burgos, and Shinya Inoue
Fertilization-induced changes in tin- fine structure of
stratified Arbacia eggs. I. Observations on live cells
with the centrifuge polarizing microscope 212

Burgos, Mario H., Makoto Goda, and Shinya Inoue
Fertilization-induced changes in the fine structure of
stratified Arbnria eggs. II. Observations with election
microscopy 213

Gould, Robert M., Concetta M. Freund, John Engler,

and Hilary G. Morrison

Optimization of homogenization conditions used to
isolate mRNAs in processes of myelinating oligoden-
drocytes 215

ECOLOGY, BIOGEOCHEMISTRY, AND POPULATION BIOLOGY

Kirkby, Ryan, Luc Claessens, Charles Hopkinson, Jr.,
Edward Rastetter, and Joseph Vallino

Modeling the effects of land-use change on nitrogen
biogeochemistry in the Ipswich watershed, Massachu-
setts 218

Perring, Anne, Michael Williams, Charles Hopkinson,

Jr., Edward Rastetter, and Joseph Vallino

Solute dynamics in storm flow of the Ipswich River
Basin: effects of land use 219

Westgate, Elizabeth J., Kevin D. Kroeger, Wendy J.

Pabich, and Ivan Valiela

Fate of anthropogenic nitrogen in a nearshore Cape
Cod aquifer 221

Denault, Michelle, Erica Stieve, and Ivan Valiela

Effects of nitrogen load and irradiance on photosyn-
thetic pigment concentrations in Clndoplwrn vaga-
bunda and Gniciliiria tikvalriae'm estuaries of Waquoit
Bav. . 223

Greenbaum, Adena, and Anne Giblin

Differences in properties of salt marsh sediment be-
tween hayed and reference sites 225

Chikarmane, Hemant M., Alan M. Kuzirian, Robbin

Kozlowski, Mark Kuzirian, and Tony Lee

Population genetic structure of the goosefish, Lo-
phius ammcanus 227

ORAL PRESENTATIONS
Published bv title onlv. .

Reference: Biol. Bull. 199: 161. (October 2000)

Introduction to the Featured Report
On Mapping Odor Quality

In vertebrate olfactory systems, the coding of odor quality by the brain is constrained by four considerations.
Three of these suggest that the code is specific: i.e.. about 1000 genes encode olfactory receptors; every primary
olfactory receptor neuron expresses only one of those genes; and the axons of all of the neurons expressing a
particular gene project to the same glomerulus (a synaptic exchange site in the olfactory bulb). The fourth
consideration suggests that coding lacks specificity, for single receptor neurons respond, though not always with
the same potency, to a variety of odor molecules. Thus, the recognition of a chemically pure odor cannot be
restricted to its effect on a single specific set of receptor neurons and their common glomerulus. Rather, odor
quality must be identified by some array or pattern of inputs induced in diverse neurons and glomeruli,

Many previous experiments show that these patterns of response or input vary with concentration. On the other
hand, psychophysical experimentation — as well as common experience — tells us that recognition of an odor is not
confounded by even enormous differences in concentration.

Matt Wachowiak and his colleagues point, in their brief report, to a resolution of this paradox. They fill the
olfactory nerve terminals of the three-toed box turtle with a fluorescent dye, apply pulses of odor, and produce a
map representing the location of the glomeruli responding to the odor, as well as the amplitude of the response.
With this preparation, the variation in the pattern of olfactory receptor neuron inputs to the olfactory bulb can be
measured as a function of concentration. In fact, the experiments confirm that the absolute size of the response to
an odor increases with concentration. But if the responses are normalized, the resulting maps are virtually invariant
with concentration. Wachowiak et al. conclude that — if higher olfactory centers receive and can analyze
normalized maps of the input to the olfactory bulb — then odor recognition, independent of concentration, would
be possible.

— The Editors
August 2000

161

162 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

Reference: Biol. Bull. 199: 162-163. (October 2000)

The Spatial Representation of Odors by Olfactory Receptor Neuron Input
to the Olfactory Bulb is Concentration Invariant

Matt Wachowiak, Michal Zochowski, Lawrence B. Cohen, and Chun X. Falk {Department of Cellular ami
Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520)

We wish to understand how odorants are distinguished and how
one odorant is recognized as the same across a concentration range
of several orders of magnitude. To this end we have measured the
spatial pattern of the olfactory receptor neuron input to the olfac-
tory bulb in the three-toed box turtle (Terepene triunguis).

To monitor the input to the bulb we labeled the nerve terminals
of the olfactory receptor neurons with Calcium Green- 1 dextran 10
kD (Molecular Probes) following the method developed by
Friedrich and Korsching (1). We then formed a magnified (4X)
image of the bulb on an 80 X 80 CCD camera (NeuroCCD;
RedShirtlmaging. LLC, Fairfield. CT) and recorded the changes in

fluorescence that resulted from a 2-s odorant pulse delivered to the
nose. The signals we measured had approximately the same time-
course everywhere in the bulb, and we therefore characterized the
response by the amplitude of the signal as a function of its position
on the bulb.

Figure 1 shows three pseudocolor representations of activity in
response to the odorant, hexanone. Red represents a large signal in
each measurement and blue represents a signal 30% as large. The
left-hand image shows the response to hexanone at a concentration
that was 0.3% of saturation. The largest signal in the response was
colored red (normalized scaling). Both right-hand images show the

Concentration-dependence: normalized vs. absolute maps

10% hexanone

normalized scaling
6.2

4.7 —

% dF/F

1.4

0.3% hexanone

normalized scaling

% dF/F

1.9 — to

max=4.7% dF/F

4.7

% dF/F

1.4 —

max=6.2% dF/F
absolute scalinc

max=6.2°b dF/F

Figure 1. Normalized maps of receptor neuron input t(> the turtle olfactory bulh are concentration-invariant. The left panel shows a pseudocolor map
of the response Jo a 0.3f/f dilution of saturated vapor of 2-he\anone. The map is normalised to the maximum signal amplitude for this trial. The right panels
show pseudocolor maps of the response to a 10% dilution of 2-hexanone. The map on the toft is normalised to its maximum signal amplitude. The map
on the bottom (absolute scaling) shows the same data using the same scaling as for the 0.3% hexanone trial. The figure shows a concentration-dependent
increase in the number ofglomeruli activated above a given absolute level, but sho\vs concentration-invartancc in the relative levels of input to all glomeruli
activated b\ an odorani. 4 • image magnification. The field of vie\v i\ approximately 4 mm • 4 mm.

FEATURED ARTICLE 163

response to 10% hexanone using two different scaling procedures. lion could be achieved if higher olfactory centers "read" the
The bottom image shows the response using the same scale as that normalized maps of the input to the olfactory bulb,
used for the response to 0.3% hexanone (absolute scaling). This Supported by NINDS Grant, NS08437 and an NRSA fellow-

image is qualitatively different from the 0.3% image. In contrast, ship, DC 00378.
the top image shows the response using normalized scaling. Again.
the largest signal was colored red. This image is essentially iden-
tical to the image on the left, even though the concentration of

odorant differed by a factor of 30. Thus, normalized maps of input . „. ,

to the olfactory bulb appear to be concentration invariant.

We hypothesize that concentration invariant odorant identifica- I Friedrich, R., and S. Korsching. 1997. Neuron 18: 737-752.

164 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

Reference: Biol. Bull. 199: 164-165. (October 2000)

Heavy Water (D2O) Alters the Sodium Channel Gating Current in Squid Giant Axons

David Landowne (University of Miami, Miami, Florida 33101 )

When nerve axon membranes are abruptly depolarized, a small
outward displacement current precedes the sodium current that
underlies the propagated nerve impulse. This displacement current
is asymmetric; it does not appear in a symmetrical hyperpolariza-
tion. It was named "gating current" by earlier workers because of
its association with the opening of the activation gates of the
sodium channels. The gating currents were isolated by replacing
permeant ions with impermeant ones, thus reducing ionic currents,
subtracting away symmetrical currents and. in most cases, block-
ing the ionic sodium current with tetrodotoxin (1, 2).

Replacing the H,O in solutions with D^O slows many chemical
and biological reactions including the squid axon action potential
(3), ionic currents (4), and the sodium pump (5). However. Meves
(6) reported that D2O had no significant effect on the asymmetry
currents of squid axons. This result was confirmed in Myxicalu (7)
and crayfish (8) axons. Most measurements were made at voltages
less than +20 mV, which would not be expected to open all of the
channels, and in the presence of tetrodotoxin. Both the ionic and
gating currents can be recorded if the experiments are carried out
in solutions with low sodium content (9). When this is done, D-.O
can be seen to reduce the amplitude of the gating currents at more
positive potentials.

Segments of squid axons were bathed in an artificial seawater
containing 44 mM NaCl, 396 mM tetramethylammonium (TMA)

chloride, and 2 mM TMA Hepes. pH 7.4; and the internal Cs
perfusion fluid contained 150 mM Cs glutamate, 50 mM CsF, 750
mM sucrose, and 40 mM Cs Hepes. pH 7.4. Solutions were made
up with either H-.O or 99.8% D^O. The axons were voltage-
clamped at a —70 mV holding potential. Gating currents were
recorded with a p/8 protocol, as follows: the holding potential was

shifted to - 140 mV. and 8 small "subtraction" pulses,

the

amplitude of the test pulse, were applied and their currents
summed. Then the potential was shifted back to —70 mV, and a
single test pulse was applied. This procedure was repeated every
two seconds. Currents were filtered at 40 kHz and sampled at 100
kHz. The records presented are the difference between the test
current and the summed subtraction currents averaged over 64
cycles. Experiments were performed at 3°-4°C.

The figure shows records made with pulses to +25 and +50
mV. The effect of D2O (filled symbols) is to reduce the initial
outward gating current by about 30%, to increase the time to peak
inward current to about 1 .4 times its value in H,O, and to slow the
decline of inward current associated with inactivation of the so-
dium channels. The sodium conductance was reduced by about
35%. The changes in ionic currents are similar to those previously
described (3, 6, 7). In 56 measurements at 0 to +75 mV on 1 1
axons in the absence of tetrodotoxin, D,O reduced the peak of the

LOO 1 6

LI A/cm

250 LIS
2ms

Figure 1. D:O alters gating and ionic currents. Open symbols are currents in H:O-based solutions; filled symbol.i indicate D:O-based solutions. The
records are for a steps from a — 70 mV holding potential. The upper records are to +25 mV; the lower, to +50 mV. The records on the right are shown
at the e.\panded timchase.

NEUROBIOLOGY

165

initial outward gating current to 0.70 ± 0.02 times its value in
H,O.

The simplest interpretation of these results is that D2O slowed
the rate of the conformational change by 30%, thus reducing the
amplitude of the gating current and increasing the time required to
open the channels. This could occur by changing the channels or
changing the environment in which they operate. The viscosity of
D,O is larger than that of H,O. and in fact, the reduction of gating
and the slowing of ionic currents described above are qualitatively
similar to those seen in solutions with a viscosity that has been
increased with non-electrolytes (10). On the other hand, the D2O
effect seems larger than predicted by viscosity alone. Perhaps D,O
alters the gating machinery. About 40% of the amide protons of the
Streptomyces lividans K+ channel exchange within 3 minutes of
D,O exposure accompanied by subtle structural changes (11). To
test between these two possibilities, currents were recorded during
the transition from 0 mM sodium H,O seawater into 44 mM
sodium D^O seawater. In the 0 mM Na H-.O solution, there was no
inward sodium current. By 90 s after beginning the switch into the
44 mM Na D2O solution, the inward current appeared — but in the
H2O pattern, similar to the open symbols in Figure 1 . Over the next
3 min the current pattern switched to the D-.O pattern, similar to

the filled symbols. This suggests that the D2O effect involves
changes in channel structure.

I thank Dr. R. J. Lipicky and the Howard Oilman Foundation for
encouragement and support.

Literature Cited

I Armstrong, C. M., and F. Bezanilla. 1973. Nature {Land.} 242:
459-461.

2. Keynes, R. D., and E. Rojas. 1973. ./. Physiol. {Lund) 233: 28P-
30P.

3. Thies, R. E., and F. I). Carlson. 1955. Biol. Bull. Ill: 295.

4. Conti, F., and G. Palmier!. 1968. Biophvsik. 5: 71-77.

5. Landowne, D. 1987. ./. Mcmhr. Biol. 96: 277-281.

6. Meves, H. 1974. J. Pliyxiol. ll.oihl) 243: 847-867.

7 Schauf, C. L., and J. (). Bullock. 1979. Biophys. J. 21: 193-208.

8. Alicata, D. A., M. D. Rayner, and J. G. Starkus. 1990. Biophys. J.
57: 745-758.

9. Bezanilla, F., and C. M. Armstrong. 1974. Science 183: 753-754.
10. Kukita, F. 1997. J. Physiul. tLtwd.) 498: 109-133.

1 1 Tatulian, S. A., D. M. Cortes, and E. Perozo. 1998. FEBS Leu.
423: 205-212.

Reference: Biol. Bull. 199: 165-168. (October 2000)

Voltage Gating Properties of Channels Formed by a Skate Retinal Connexin

Thomas W. White1, Harris Ripps~, Miduliiru Srinivas , and Roberto Bruzzone
(Marine Biological Laboratory, Woods Hole, Massachusetts 0254:!)

Gap junctions provide pathways for electrical and chemical
communication between networks of coupled cells. They act as
simple electrical synapses, and also synchronize and regulate a
broad range of cellular activities. The structural proteins constitut-
ing gap-junctional channels in vertebrates are the connexins, mem-
bers of a multigene family that exhibit a common topology: four
transmembrane domains separating two well-conserved extracel-
lular loops and three cytoplasmic domains. Six connexin polypep-
tides oligomerize to form a membrane hemichannel or connexon.
clusters of which join with the connexons of adjacent cells to
create the gap junction. Once assembled, the gap-junctional chan-
nel consists of an aqueous pore that allows the cell-to-cell diffu-
sion of ions, second-messenger molecules, and small metabolites.
Different cell types contain connexins that are unique to their
special needs, and variations in the molecular structure of the
individual connexins determine the gating properties, voltage
dependence and cellular interactions of their gap-junctional chan-
nels ( 1 ).

The vertebrate retina is a useful model with which to studv the

1 Department of Neurobiology. Harvard Medical School. Boston. MA
02115.

2 Department of Ophthalmology and Visual Sciences, UIC College of
Medicine, Chicago, IL 60612.

3 Department of Neuroscience, Albert Einstein College of Medicine,
Bronx, NY 10461.

4 Department de Virologie. Institut Pasteur, 75015 Paris, France.

diversity of electrical coupling in nervous tissue. Diverse experi-
mental approaches have shown that virtually every class of retinal
neuron and glial cell makes gap junctions with neighboring cells of
similar type, and in some cases with cells of another type (2, 3).
Moreover, coupling between different cell types appears to be
mediated by gap junctions that exhibit asymmetric dye transfer, as
well as distinct pharmacological properties (4. 5). Although there
is abundant evidence that the electrical synapses formed by gap
junctions affect every aspect of retinal function, relatively little is
known about the connexins mediating these effects. The situation
has been changed by the identification of a distinct subgroup (y) of
the connexin family that shows a pattern of expression restricted to
the retina and the central nervous system (6-1 1 ). The first member
to have been discovered, Cx35, was cloned from a skate retinal
cDNA library (6), and some of its functional characteristics were
examined in the Xenopits oocyte expression system (12). In the
present study, we have extended these observations to analyze
more fully the voltage sensitivity and kinetics of the gap junctions
formed by Cx35 in paired oocytes. In addition, we have investi-
gated the properties of the non-junctional hemichannels formed in
single oocytes. and have compared the effects of quinine on the
kinetics of the tail currents evoked at the termination of voltage
pulses.

The procedures for preparing cRNA, and its analysis in Xenopus
oocytes have been described previously (12). Oocytes were iso-
lated by enzymatic digestion and injected with either an antisense
oligonucleotide (3 ng/cell) to suppress the endogenous Xenopus

166

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

40 nA

2 sec

75
70
65
60

50
45
40
35

V. = 100 mV
= 0.448 sec

0.0 0.4 0.8 1.2 1.6 2.0

time [sec]

control

quinine

1.2-

1.0-

0~ 0.8-

0)
N

"oj

O 0.4
0.2-

0.0-J-r

-120 -80 -40 0 40 80 120

0.8-,

0.6-

0.4-

0.2-

0.0

025-

0.20-

V 0.15^

0
to

M 0.10-1

0.05-

0.00

V [mV]

80 90 100 110 120

V, [mV]

— • — control

n— 100 nM quinine

40 50 60 70 80

Figure 1. Voltage gutinx <>) 'channels in Xenopus oocytes expressing skate Cx35. (A) Gap junctions! currents (lt) were elicited h\ transjunetional
voltage (V :) steps, 4 s in duration, applied in ±20 mV increments from a holding potential of —40 mV. (B) Plotting steady state junctional conductance
(GJS,, normali-eit in the values measured at ±20 mV) vs. VjShowsthat. even at the extremes of &Vj(± 120 mV), the residual conductance is equal to about
half the initial value: data are the means (±SD) of 4 cell pairs. Cunvs drawn through the data were derived from the Bolt-maun equation in which the
parameters for positive values ofV: were A = 0.06; V,, = 95; G,,,,,,, = /; and GJmin = 0.40. For negative values ofVf the corresponding mines were 0.05,

NEUROBIOLOGY

167

Cx38 (13), or a combination of antisense (as above) plus Cx35
RNA (5 ng/cell). To study intercellular channels, oocytes were
stripped of their vitelline membranes and brought into contact at
their vegetal poles for 48 h before electrophysiological analysis.
This step was omitted to explore hemichannel activity, which was
recorded 48-72 h after RNA injection.

Intercellular communication was quantified by dual cell voltage
clamp ( 12, 14). To determine the voltage-gating properties of the
intercellular channels, transjunctional potentials (V,) of opposite
polarity were generated by hyperpolarizing or depolarizing one
cell in 20 mV steps (over a range of ±120 mV). while clamping
the second cell at -40 mV. Currents were measured 4 s after the
onset of the voltage pulse, at which time they approached steady
state (IJSV), and the macroscopic conductance (G|ss) was calculated
by dividing Ilss by Vj. GJSS was then normali/ed to the values
determined at ±20 mV, and plotted against Vr Data describing the
relationship of GJSS as a function of Vj were fit to a Bolt/.mann relation
( 14) of the form: G^ = {<Gjmax - Gjmln)/(l + exp[A(V, - V,,)]) +
Gpmin, where GJSS is the steady-state junctional conductance, G]max
(normalized to unity) is the maximum conductance, Gjmin is the
residual conductance at large values of Vr and V,, is the transjunc-
tional voltage at which GJSS = (G]max — G|min)/2. The constant A
(=iui/kT) represents the voltage sensitivity in terms of gating charge
as the equivalent number (/?) of electron charges (q) moving through
the membrane, k is the Boltzmann constant, and T is the absolute
temperature. The time constants ( T) of voltage-dependent transitions
of junctional conductance were calculated using data-fitting functions
in Microcal Origin. To characterize the hemichannel activity of con-
nexons, non-junctional current recordings were obtained from single
oocytes with a two-electrode voltage-clamp procedure. Cells were
clamped at -40 mV, and whole cell currents recorded in response to
depolarizing voltage steps (from —20 to +80 mV in 20-mV intervals)
imposed for 5 s.

In a previous study (12), we restricted our observations of
voltage gating to transjunctional potentials of ±80 mV and did not
quantify voltage dependence. In the current study, we have ex-
tended this analysis to much larger values of V, and have quanti-
fied the degree and kinetics of voltage gating. Figure 1 A illustrates
a family of gap-junctional currents elicited at transjunctional volt-
age steps up to ±120 mV in oocyte pairs expressing Cx35. Voltage
gating occurred symmetrically at the higher values of Vj (80-120
mV), and the current decay approached steady-state levels by the
end of the imposed step. Quantitative analysis (Fig. IB) indicated
that a large residual conductance, equivalent to approximately half
of the initial value, was still present at the extremes of the voltage
range tested. Fitting the G, vs. Vj relationship to the Bolt/.mann
equation yielded Vu values > 80 mV (see figure legend) confirm-
ing the relatively weak Vj gating described earlier. Thus, voltage is

not likely to be a primary modulator of Cx35-mediated intercel-
lular communication in retinal neurons.

The voltage gating characteristics of Cx35 were further explored
by analyzing the kinetics of channel closure for values of Vj > 80
mV. i.e., sufficient to consistently induce current decay. Figure 1C
illustrates results obtained from one cell pair in response to a
transjunctional voltage step of +100 mV. The time-dependent
decline in Ij was well fit by a single exponential function with a
time constant (ri of 0.448 s. Interestingly, the mean values of r,
obtained both for different values of Vr as well as for positive and
negative voltage steps, hovered about 0.4 s and showed no signif-
icant change as a function of either the polarity or the magnitude
of V, (Fig. ID). These data are in sharp contrast to kinetic analyses
of many other connexins, where T values decreased with increasing
driving force (15, 16). This feature is shared by another y con-
nexin, mouse Cx36 (data not shown), and illustrates further the
unique properties of this subgroup.

The ability of connexins to form hemichannels in Xenopus
oocytes. a property reminiscent of membrane currents observed in
some retinal neurons (17. 18), prompted us to investigate the
kinetics of C.\35 hemichannel closure by analyzing tail currents.
As we showed previously, quinine-sensitive hemichannel currents
can be recorded from oocytes expressing skate Cx35 (12). This is
confirmed in Figure IE. which shows the increase in the outward
(non-junctional) current recorded from a single oocyte in response
to depolarizing voltage increments z 40 mV, and the current
enhancement produced by the addition of 100 juM quinine to the
normal bath solution. To determine whether quinine exerted an
effect on the gating properties of the hemichannels, we measured
the kinetics of the tail currents recorded at the termination of the
voltage step. Figure IF shows that the mean of the time constants
of the single exponential decay functions describing the data for
the return of Vm to -40 mV from values of +40 to +80 mV were
unaffected by quinine. However, the hemichannel time constants
are not directly comparable to the intercellular channel r values, as
the ionic strength of control bath solution is greatly reduced in
comparison to ooplasm, and K+ is replaced by Na+ as the prin-
cipal cation. Further studies are required to determine the precise
relationship between hemichannel and intercellular channel gating,
and to clarify the mechanism whereby quinine markedly increases
Cx35 mediated hemichannel currents.

The authors thank Jane Zakevicius for technical help and Marco
White for assistance with the animal care. This work was sup-
ported by National Eye Institute grants (EY-13163 to TWW and
EY-06516 to HR), by the Association RETINA France (to RB) and
by MBL fellowships (to TWW, MS, and RB).

84, 1, and 0.45, respectively. The slight asymmetry in Bo/l:mann values implies a small degree of dependence of GJa on Vm. 1C) In response to a +100
mV voltage step, the transjunctional current decayed toward a steady state level along a single exponential with a time constant (T) of 0.448 s. (D) The
values of r remained relatively constant for voltage steps ranging from ±80 to ±120 mV and were not dependent on the polarity ofVf data are the means
(±SDj of 8 experiments. (E) Hemichannel activitv of C.\35 recorded from single oocytes expressing Cx35. With the cells clamped at —40 mV,
non-junctional currents were elicited by depolari-ing voltage steps (from —20 to +80 mV in 20 mV intervals! imposed for a duration of 5 s. When bathed
in control solution, progressively larger currents were obtained as the depolarising voltage step exceeded +20 mV. With the addition of 100 fj.M quinine.
activation of hemichannel activity occurred over the same voltage range, but (he magnitudes of the ounvard currents were greatlv enhanced. {F) Tail
currents measured at the end of the voltage pulses were we/I fit bv single exponentials having ^ values of —0.125 s. Quinine had no significant effect on
the gating kinetics of Cx35 hemichannels: data are the means (±SD) of 6 experiments.

168

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS
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16. White, T. W., R. Bruzzone, D. A. Goodenough, and D. L. Paul.
1992. A/o/. Bio/. Cell 3: 711-720.

17 DeVries, S. H., and E. A. Schwartz. 1992. J. Pliysiol. (Lund. ) 445:

201-230.
18. Malchow, R. P., H. Qian, and H. Ripps. 1993. J. Neurosci. Res.

35: 237-245.

Reference: Biol. Bull. 199: 168-170. (October 2000)

Hydrogen Ion Fluxes from Isolated Retinal Horizontal Cells: Modulation by Glutamate

Antlwnv J. A. Molina (Department of Biological Sciences, University of Illinois at Chicago, Chicago,
Illinois 60607), Peter J. S. Smith1, and Robert Paul Malchow2

Retinal horizontal cells are second order neurons that receive
direct input from photoreceptors. These cells are believed to play
a crucial role in the formation of the surround aspect of the classic
center-surround receptive fields of visual neurons. Debate still
persists as to the molecular mechanisms used by horizontal cells to
establish the surround portion of these receptive fields. One hy-
pothesis, promulgated recently by Kamermans and colleagues ( 1 ),
suggests that horizontal cells may exert their lateral inhibitory
actions by modulating the calcium flux into the synaptic terminals
of photoreceptors, thus altering the release of the photoreceptor
neurotransmitter. Hydrogen ions are among several agents that
have been proposed to act in this modulatory role (2). and in fact,
the responses to light by second order retinal neurons are very
sensitive to changes in extracellular pH (3, 4). In an elegant series
of experiments. Barnes and coworkers (5) demonstrated that this
pH-dcpendent modulation of synaptic transmission was due to the
marked sensitivity of calcium channels in the photoreceptors to
extracellular hydrogen ions. These investigators found that ele-
vated concentrations of H f shifted the voltage-dependence of the
calcium current activation curve of the photoreceptors to more
depolarized levels and also reduced the calcium conductance.
Moreover, small light-induced changes in extracellular pH within
the intact retina have been reported by Oakley and Wen (6).

Horizontal cells could thus exert their inhibitory influences by

1 BioCurrents Research Center, Marine Biological Laboratory, Woods
Hole. MA.

2 Departments of Biological Sciences and Ophthalmology, University of
Illinois at Chicago. Chicago. IL.

modifying the concentration of hydrogen ions in the external
milieu. In the present work, we have used pH-selective microelec-
trodes to monitor the flux of hydrogen ions surrounding isolated
retinal horizontal cells. In particular, we examined whether the
amino acid glutamate could alter the flux of hydrogen ions re-
corded from these cells. We reasoned that, if the release of hydro-
gen ions from horizontal cells is indeed a key factor in the creation
of the surround portion of retinal receptive fields, then such a flux
should be modified by glutamate, the neurotransmitter believed to
be released by vertebrate photoreceptors (7).

The pH-selective electrodes were used in a self-referencing
mode (8), which greatly enhances their signal sensitivity and
stability, eliminating much of the electrical noise and drift inherent
in such electrodes. In this format, the electrode is first placed just
adjacent to the membrane of the cell, and a reading taken; the
electrode is then moved a set distance away (typically 30 /am), and
a second reading taken. The difference between the voltage read-
ings at the two positions reflects differences in the free hydrogen
activity at the two locations. This method allowed us to measure
the small hydrogen ion fluxes that would otherwise have been lost
in the noise of the recordings.

pH selective electrodes were prepared by pulling thin-walled
glass capillary tubing (o.d. 1.5 mm) to a tip diameter of 2-4 jum.
The pipettes were silanized and back-filled with 100 mM potas-
sium chloride, and the fluid was forced to the tip of the pipette by
air pressure applied to the back of the pipette from a syringe. The
pipette tip was then filled with a pH-selective resin (hydrogen
ionophore 1 -Cocktail B, Fluka Chemical; the tip was placed in
contact with a source pipette containing the resin, and about 50 /xm

NEUROBIOLOGY

169

of the resin was then drawn up by suction on the back of the
pipette. The resin employed here has a particularly high selectivity
for hydrogen ions, and is reported to be more than 10'' times more
sensitive to hydrogen ions than to either sodium or potassium ions
(9). Isolated retinal horizontal cells were obtained by enzymatic
dissociation of the retina of the skate (Rajti erinacea or R. ocellata )
as described in Malchow ft al. (10). Briefly, the animals were
chilled in ice. cervically transected, and double pithed. The eyes
were removed, and the retinas were isolated and placed for 45 min
under gentle agitation into a skate-modified L-15 culture medium
containing 2 mg/ml papain and 1 mg/ml cysteine. The retinas were
then rinsed 8 times in media lacking papain and cysteine. and then
mechanically agitated through a 5-ml graduated glass pipette.
Single drops of this cellular suspension were placed in 35-mm
plastic culture dishes that had previously been coated with 1%
protamine sulfate and 0.1% concanavalin A. Cells were stored at
14°C for up to 4 days before use. Recordings were made in a skate
Ringer's solution containing 2 mM of the pH buffer HEPES and no
added bicarbonate. A 5 mM glutamate stock solution was prepared
in skate Ringer and adjusted to pH 7.6 with 1 M NaOH. Glutamate
was applied by adding 1 ml of the 5 mM glutamate solution to 4
ml of Ringer already present in the culture dish, resulting in a final
concentration of 1 mM glutamate.

Under these conditions, a steady differential signal was obtained
from horizontal cells indicative of a higher concentration of hy-
drogen ions near the membranes of the cells. The size of this signal
decreased as the concentration of the pH buffer HEPES was
increased, consistent with the hypothesis that the signal detected
indeed reflected hydrogen ions. Moreover, as shown in Figure 1.
the application of 1 mM glutamate resulted in a marked decrease
in the size of the differential signal. A differential signal of
approximately 100 juV was initially recorded from this cell. The

140
120 -
100 -

80 -

40 •

20 •

AAV/1

-20
-40

200

400

600

800

1000

time(s)

Figure 1. Effects of glutamate on the hydrogen ion flux recorded from
a single isolated retinal horizontal cell. The differential voltage recorded
from a pH-selective electrode is plotted as a function of time. Before the
application of glutamate. a differential signal indicative of a higher con-
centration of hydrogen ions near the membrane of the cell is observed. At
the arrow, glutamate was added such that the final concentration in the
dish was 1 mM. A marked decrease in the differential signal recorded hy
the pH-selective electrode \vas obsen'ed.

actual proton flux represented by this differential voltage can be
calculated using an equation derived by D. M. Porterfield [in prep.;
see aKo (12)] as follows:

J= - D(A[FT] + [Buffer] * 0.25A[H+] * K;1) * Ar1

Where J is tlu- flux. D is the diffusion coefficient for hydrogen
ions. A[H + ] the change in hydrogen ion activity between the
two poles of measurement. [Buffer] is the buffer concentration
expressed in moles per cm~\ Ka is the pK., of the buffer
expressed in cm" \ and Ar is the distance in cm between the two
measuring positions of the probe. Taking into account a small
loss of the signal within the electronics of the amplification
system (8). under our experimental conditions the 100 /J.V
signal we observe is then estimated to be indicative of a proton
flux of —75 pM cm"2 s"1. In 6 cells studied in this fashion. 1
mM glutamate reliably reduced the differential signal by an
average of 60%.

We thus conclude that glutamate. the presumed neurotransmitter
from vertebrate photoreceptors. can indeed alter the flux of hydro-
gen ions from horizontal cells. In this context, it is interesting to
note that glutamate has previously been reported to promote an
acidification of the internal milieu of catfish retinal horizontal cells
as measured using the pH-indicator dye BCECF (11). We hypoth-
esize that glutamate may shut down the transport of hydrogen ions
from horizontal cells, thus trapping hydrogen ions in the interior of
the cell. This would account for the increased intracellular acidity
and the alkalinization of the extracellular milieu that we have
observed. The alteration in extracellular pH induced by glutamate
may be important in modifying signaling within the outer plexi-
form layer of the retina. Indeed, extracellular alkalinizations in-
duced by neuronal activity occur in several other regions of the
nervous system (reviewed by Chester (13)), and excitatory amino
acid receptors have been implicated in the generation of these
phenomena. Thus, modulation of extracellular pH within the CNS
by glutamate may be a common means by which synaptic activity
is altered. Future experiments are planned in which specific phar-
macological agents will be used to determine which transporter or
transporters may be involved in the glutamate-induced changes in
extracellular hydrogen ion concentrations.

We are grateful to Kasia Hammar for her generous assistance
with electrode preparation and cell culture, Naomi Rosenkranz
for help preparing isolated cells, and Richard H. Sanger for
electronic and computer assistance. This work was supported by
grants EYO941 1 from the National Eye Institute. P41 RR01394
from the National Center for Research Resources, and a grant
from the Campus Research Board of the University of Illinois at
Chicago.

Literature Cited

1. Venveij. J., M. Kamermans, and H. Spekreijse. 1996. Vision Res.
36: 3943-3953.

2. Kamermans, M., and H. Spekreijse. 1999. Vision Rex. 39: 2449-
2468.

3. Kleinschmidt, J. 1991. Ann. N.Y. Acad. Sci. 635: 468-470.

4. Harsanyi, K., and S. C. Mangel. 1993. Vis. Neurosci. 10: 81-91.
?. Barnes, S., V. Mere-hang, F. Mahmud. 1993. Proc. Natl. Acad. Sci.

U.S.A. 90: 10081-10085.

170

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

6. Oaklev, B. and R. Wen. 1989. ./. Physiol. (LonJ) 419: 353-378.

7. Copenhagen, D. R., and C. E. Jahr. 1989. Nature 341: 536-539.

8. Smith, P. J. S., K. Hammar, D. M. Porterfield, R. H. Sanger, and
J. R. Trimarchi. 1999. Micmsc. Res. Tech. 46: 3W-417.

4. Fluka, 1991. Selectophore, lonophores for Ion-Selective Electrodes
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10 Mali-how, R. P., H. Qian. H. Ripps, and J. E. Dowling. 1990.

J. Gen. Physiol. 95: 177-188.

1 1. Dixon, D. B., K-I. Takahashi, and D. R. Copenhagen. 1993. Neu-
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12. Smith, P. J. S. and J. Trimarchi. 2000. Am. .1. Ph\siol. (in press.)

13. Chester, M. 1990. Progr. Neurohwl. 34: 401-427.

Reference: Biol. Bull. 199: 170-171. (October 2000)

Odor-induced Oscillatory Activity in Drosophila CNS

Jing W. Wang (Department of Biochemistry anil Molecular Biophysics, Howard Hughes Medical Institute,

Columbia Universit\, New York, New York 10032)

In mammals and the fruit fly, the vast array of odors in the
environment is discriminated by a large number of receptor mol-
ecules ( 1, 2, 3). Individual olfactory sensory neurons express only
one of the many receptor genes (1,2, 3). Neurons expressing the
same receptor gene project to the same glomerulus (4, 5, 6),
providing the anatomical evidence for a spatial coding mechanism.
Electrophysiological recordings from olfactory neurons suggest
that the temporal pattern of their responses can also convey infor-
mation about odor quality (7). Odor-induced oscillatory activity,
an indication of synchrony, has been observed in phylogenetically
different species, including molluscs, insects, and mammals (7. 8,
9. 10. 11. 12).

The adult Drosophila antennal lobe, organized in spheroidal
subcompartments termed glomeruli, receives about 1200 olfactory
afferents from the antenna and 120 afferent fibers from the max-
illary palp (13). Although the fly and mammals share the similarity
that receptor neurons expressing the same receptor gene project to

one or two glomeruli in a stereotypic manner (4, 5, 6), there are
only 60 receptor genes and 43 glomeruli in Drosophila, in contrast
to the 1000 receptor genes and 1800 glomeruli within the olfactory
bulb of mammals ( 1, 2, 3). The lower complexity in anatomy and
the rich behavioral repertoire in Drosophila makes it an attractive
system with which to study olfaction. Moreover, sophisticated
genetic tools and behavioral mutants can now also be used to study
the olfactory system in Drosophila. Nevertheless, understanding
mechanisms of odor discrimination in the CNS of the fly has been
difficult due to a lack of physiological tools for functional studies.
Odor-induced oscillations have been observed in several insect
species, including the locust, cockroach, honeybee, bumblebee,
and wasp (7). Local field potential (LFP) recordings show odor-
induced oscillation at —10 Hz. which typically lasts for the dura-
tion of odor stimulation. I have investigated this phenomenon in
the Drosophila CNS. LFPs were recorded with glass electrodes
(tip, 5 /urn) that were filled with Drosophila HL3 saline and

Peppermint

Local Field Potential

Amyl Acetate

Power Spectrum Density

Peppermint

Amyl Acetate

Frequency

Figure 1. Local field potential recordings of odor-induced oscillation in the CNS of Drosophila. The left panel shows five sequential responses to
peppermint stimulation recorded from the same preparation. Responses to ainyl acetate from the same preparation are shown in the middle panel. Averaged
power spectrum density from the five trials is shown in the right panel. The LFP response to peppermint appears to have a higher density at 2 Hz than
the response to amyl acetate.

NEUROBIOLOGY

171

positioned with a motorized manipulator (MP285. Sutler). A patch
clamp amplifier (EPC 7. Heka) was used, and the signal was
filtered (band pass at 0.1 to 20 H/.) with a signal conditioner
(CyberAmp, Axon Instruments) and recorded with software (Axo-
Scope. Axon Instruments) run on a PC. Adult flies (less than a
week after eclosion) were lightly anesthetized with CO2 and de-
capitated. The heads were immobilized with wax on a microscope
slide with the antennae pointing upward. A small opening was
made on the dorsal cuticle for the extracellular recording.

Figure 1 shows LFP recordings from the CNS of the Canton-S
wild-type fly that reveal an odor-induced oscillation. This phenom-
enon was confirmed in 6 preparations. A power spectrum analysis
indicates that the major frequency components are less than 4 Hz
(Fig. 1 ). This LFP oscillation signal appears to be sensitive to the
position of the electrode, and the coordinates taken from the
manipulator suggest that the recordings may have originated in the
antennal lobe. Future experiments with GFP-labeled antennal lobe
may help in identifying the sources of the oscillatory activity. The
patterns of oscillation in response to the same odor appear to be
roughly similar in sequential recordings from the same animal. The
LFP patterns generated in response to peppermint (from McCor-
mick) and amyl acetate (from Sigma) were distinguishable by eye.
Moreover, the power spectrum analysis indicates that peppermint
generates slightly more high frequency components.

This is the first LFP recording from the Drosophila CNS. The
preliminary results presented here show that odor-induced oscil-
lation occurs in Drosophila; this finding suggests that a temporal
coding mechanism may be employed by the fly. and that the power
of genetics may be applied in the future to decipher the physio-
logical significance of the odor-induced oscillation.

I would like to thank Alan Gelperin for his generous support.
Leonardo Belluscio for critical comments on the manuscript, and
Carl Zeiss. Inc., and Axon Instruments, Inc., for providing equip-
ment. This research was carried out in the Grass Laboratory at the
Marine Biological Laboratory, Woods Hole. Massachusetts, and
was supporu d by the Grass Foundation.

Literature Cited

I Buck, L., and K. Axel. 1991. Cell 65: 175-187.

2. Clyne, P. J., C. (,. Warr, M. R. Freeman, D. Lessing, J. Kim, and
J. R. Carlson. 1999. Neuron 22: 327-338.

3. Vosshall, L. B., H. Amrein, P. S. Morozov, A. Rzhetsky, and R.
Axel. 1999. Cell 96: 725-736.

4. Gao, Q., B. Yuan, and A. Chess. 2(100. Nature Neuro.ici. 3: 780-
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5 Vosshall, L. B., A. M. Wong, and R. Axel. 2000. Cell 102: 147
159.

6. Ressler, K. J., S. L. Sullivan, and L. B. Buck. 1994. Cell 79:
1245-1255.

7. Stopfer, M., M. Wehr, K. MacLeod, and G. Laurent. 1999. Pp.
163-180 in Insect Olfaction. B. S. Hunsson, ed. Springer, Berlin.

X. Adrian, E. D. 1942. J. Physio/. Lond, 100: 459-473.

9. Delaney, K. R., and J. B. Hall. 1995. J. Neurosci. Methods 68:

193-202.
10. Gray, C. M. 1994. Neurosci. 1: I 1-38.

1 1 Tank, D. W.. A. Gelperin, and D. Kleinfeld. 1994. Science 265:
1819-1820.

12 Lam, V.-W., L. B. Cohen, M. Waclumiak. and M. R. Zochowski.
2000. J. Neurosci. 20: 749-762.

13. Stacker, R. F., and V. Rodrigues. 1999. Pp. 283-314 in Insect
Olfaction, B. S. Hansson, ed. Springer, Berlin.

Reference: Biol. Bull. 199: 171-173. (October 2000)

Circadian Rhythms in the Receptive Fields of the Liimilus Lateral Eye

James M. Hilt (Marine Biological Laboratory, Woods Hole, Massachusetts 02543),
Frederick A. Dodge, Ehud Kaplan, and Robert B. Barlow

Hartline found that in the frog "a given optic nerve fiber re-
sponds to light only if a particular region of the retina receives
illumination." He called the region the receptive field of that fiber
(1). Continuing Hartline's study of the frog retina. H. B. Barlow
detected an inhibitory influence surrounding the excitatory region
of the receptive field (2). In the lateral eye of the horseshoe crab
Limiilitx polyphemus, the receptive fields of single ommatidia have
both excitatory centers and inhibitory surrounds. The field of view
of a single ommatidium defines the narrow excitatory center,
whereas the neural network connecting neighboring ommatidia
( — 200) generates the wide inhibitory surround. A circadian clock
in the animal's brain transmits signals to the lateral eye at night,
changing its structure and function to increase the retinal sensitiv-
ity (3) so that the animal can detect mates nearly as well at night
as it can during the day (4). Several mechanisms underlying the
remarkable nighttime sensitivity have been identified: they are
increased photoreceptor gain, decreased photoreceptor noise, de-
creased lateral inhibition, and increased photon catch as a conse-
quence of an increased acceptance angle for each ommatidium (3).

High retinal sensitivity at night is associated with highly vari-
able ("noisy") optic nerve responses, which result from random
photon events at low nighttime levels of illumination. Such noisy
neural responses hinder our efforts to measure properties of the
nighttime state of the eye and, thus, our development of an accu-
rate cell-based model of retinal function. Our goal is to understand
the neural code the eye sends to the brain at night as we have
already done for the daytime state of the eye (5, 6). Here we report
an analysis of retinal receptive fields and demonstrate how their
properties change from day to night.

Our method takes advantage of the remarkable linearity of the
responses of the lateral eye to small modulations of the visual
input. We employ time-varying sinusoidal stimuli and linear sys-
tems analysis. Modulated square patterns are presented on a TV
monitor having a grey background, with nighttime stimuli attenu-
ated by a 4.25 log unit neutral density filter. After isolating the
response of a single optic nerve fiber, we align the animal so that
the optic axis of the recorded ommatidium views the center of the
pattern. We use animals that had been entrained to the natural

172

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

lighting cycle, taking daytime measurements before 1730 h and
nighttime measurements from 2200 to 0100. The square pattern is
modulated ±10% relative to the background at 1 Hz. Using a
strategy previously developed for studying retinal receptive fields
in the frog (2), we measured the width of the excitatory center by
illuminating the eye with square stimuli of increasing size. Be-
cause the inhibitory surround extends into the excitatory center,
responses are a mixture of excitatory and inhibitory inputs; but the
responses to the smallest square stimuli are predominantly excita-
tory.

Figure 1A plots the amplitude of modulated optic nerve re-
sponses as a function of the visual angle of the square stimuli
during the day (gray points) and night (black points). For each data
set, the ordinate scale ranges from no response (0%). to the
theoretical maximum excitatory response (100%) that would be
achieved in the absence of inhibition. The points plotted in Figure
1A show an increase in response for increasing stimulus size, up to
24° of visual angle, beyond which responses decrease because
more of the inhibitory surround is illuminated by the square
stimulus. The growth of the responses to expanding stimuli
(smooth curves) are estimated from the responses to the smallest
square stimuli, because the small stimuli minimally activate the
inhibitory surround. Assuming that the excitatory center can be
well represented by a two-dimensional gaussian function (3). the
"pure" excitatory response is proportional to the volume of the
excitatory center surface covered by a stimulus. Using this rela-
tionship, we estimated the size of the excitatory center based on the
recorded responses to the four smallest square stimuli and extrap-
olated the theoretical maximum response of the excitatory center
(100% on the ordinate in Figure 1A: Ep in Equation [1]). The
smooth curves plot the theoretical responses, yielding excitatory
centers with half-maximal width of 12° during the day (gray curve)
and 16° at night (black curve). Because the size of the excitatory
center increased at night, the recorded response reached only 85%'
of its maximum theoretical value before decreasing as a result of
surround inhibition. Although the inhibitory surround overlaps the
excitatory center, the effects of surround inhibition are minimal for
small squares. We therefore attribute the observed changes in
Figure 1A to an expanded excitatory field width at night, arising
from circadian changes in ommatidial structure, that is. a shift of
photoreceptor position and migration of pigmented cells (3).

The vectors in Figure IB plot the modulated optic nerve re-
sponse in terms of its phase and amplitude relation to the stimu-
lus— a sine wave with a direction of 0° (vertical) and a length of
1.0. To measure the strength of the inhibitory surround, we first
determine the maximal excitatory response, as described above,
and plot it as a vector in Figure IB (open circles). We next
determine the response vector for full-field stimulation by modu-
lating the entire TV monitor with a ± 10% contrast at 1 Hz (open
squares); this vector represents the summed response of excitatory
and inhibitory inputs. Finally, we measure the response vector for
inhibition by modulating the surround while holding constant the
stimulus to the center (crosses). Because the Limulus eye responds
linearly to small amplitude stimulation, the effects of excitation
and inhibition superimpose; i.e.. the sum of center and surround
response vectors should equal the full-field response. The vector
sum of excitatory and inhibitory responses during the day is
plotted as a thin line that lies adjacent to the vector for the full-field

100%

80%
60%

1 40%

5 10 15 20 25

Visual Angle (Degrees)

Night

-1

O Center illumination
x Surround illumination
D Full-Field illumination

1 2

Cosine component

Figure 1. A: Plot of the response of a single optic nen'e (ordinate) as
a function of the size of the centrally located visual stimulus (abscissa).
Davtime (gray points) and nighttime (black points) responses are normal-
ized to their respective theoretical maximum excitatory response. As ex-
pected, the response increases with the size of the stimulus. Solid lines
show the growth of the response calculated on the basis of a gaussian-
shaped excitatory center. B: Vector plots of responses to illumination of the
center (unfilled circle), surround (crosses), and full-field (squares) stimu-
lation. Vectors are plotted relative to the sine wave stimulus, which has an
angle ofO° and a length of 1. As explained in the te.\t, responses to center
and surround alone predict with reasonable accuracv the response to
full-field stimulation, which drives both the excitatory center and the
inhihitorv surround. Such vector addition confirms the linear properties of
the Limulus lateral eye. The nighttime vectors show a reduction in the
difference bet\\<een excitation and full-field responses, indicating u de-
crease in the strength of the inhibitory surround.

response (open square) and thus confirms response linearity. We
attribute the discrepancy between the vectors at night to response
noise at low nighttime light levels caused by random photon
events.

NEUROBIOLOGY

173

From the Hartline-Ratliff formulations of lateral inhibitory in-
teractions in the retina (7). the firing rate of the pth ommatidium
(Rp) equals its pure excitatory response (Ep) minus the sum of
inhibitory' influences from its neighbors; the inhibition delivered
from the jth ommatidium equals the strength of its inhibitory
coupling to the p'h ommatidium (Kp,) multiplied by the response of
the j'h ommatidium. These relationships can be expressed as:

Rp = Ep - S

X Rr

For the case of full-field illumination (8), all ommatidia in the eye
receive the same illumination and thus respond at the same rate:
Rp = R! = R. Equation ( 1 ) thus simplifies to:

R — Ep K,0[a| X R,

(2)

where Ktota] is the summed total strength of inhibition in the
surround, and Ep is the theoretical, maximum response of pure
excitation. All responses are measured to stimuli modulated at 1
Hz. Rearranging Equation (2) gives:

Kr,

= (Ep - R)/R.

(31

where R is the magnitude of the vector for full-field illumination
(square), and Ep is the magnitude of the vector for center modu-
lation (circle). From the data in Figure 1, K,ola| = 2.2 during the
day, and KtotaJ = 0.8 at night.

In Figure IB. the nighttime vector set shows a counterclockwise
shift and a reduction in the modulated response amplitudes. The
reduction in nighttime vector lengths does not represent a diminu-
tion of sensitivity to light, but rather a decreased modulated re-
sponse to sinusoidal stimulation at 1 Hz. The reduced amplitude

and phase shift of the nighttime response vectors result from
circadian changes in adaptation and the temporal response prop-
erties of the retina.

We conclude, first, that the excitatory center of the receptive
field in the lateral eye increases at night. Second, we conclude that
the total strength of inhibition in the eye decreases by more than
50% at night. Whether the nighttime decrease in the strength of
inhibition also includes a decrease in the size of the inhibitory
surround is not yet known, but these results point to a circadian
modulation of the synaptic mechanisms that mediate lateral inhi-
bition.

Supported by the National Institute of Mental Health, National
Science Foundation. National Eye Institute. Research to Prevent
Blindness, and the Central Lions of New York.

Literature Cited

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2. Barlow. H. B. 1953. J. Physiol. 119: 69-88.

3. Barlow, R. B., S. C. Chamberlin, and J. Z. Levinson. 1980. Science
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65-66.
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Barlow. 1997. Pmc. Nat/. Acad. Sci. USA 94: 12649-12654.
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physiol. 80: 1800-1815.
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Rockefeller University Press, New York.
S. Barlow, R. B., and D. A. Quarles. 1975. J. Gen. Physiol. 65:

709-730.

Reference: Biol. Bull. 199: 173-174. (October 2000)

Frequency Response of Auditory Brainstem Units in Toadfish (Opsanus tan)

Richard R. Fay and Peggy L. Edds-Walton (Loyola University Chicago. Chicago, Illinois 60611)

Toadfish are vocal teleosts. Both males and females produce a
pulsed grunt in agonistic contexts. In addition, male toadfish call
during courtship, using a "boatwhistle" sound to attract females to
the nest. This sound consists of rapidly repeated pulses having a
fundamental frequency (F0) of about 130 to 220 Hz in the Woods
Hole area, and several higher harmonics up to about 800 Hz (1;
Edds-Walton, Mangiamele. and Rome, unpub. obs.). These and
other sounds are thought to be detected by the sacculi, which
respond to the particle motion component of underwater sound (2).
Previous studies (2. 3) showed that primary saccular afferents
respond in a phase-locked and directional manner to particle
motions as small as 0. 1 nm in the frequency range from below 50
Hz to above 200 Hz. One goal of this work is to understand the
brain's representations of the boatwhistles that these fish use
during the breeding season. The present study focused on the range
of frequencies represented in the brainstem. including the descend-
ing octaval nucleus of the medulla (DON), and the torus semicir-
cularis (TS) of the midbrain. The DON is one of the medullary
nuclei that receives input from saccular afferents and is known
to be a site of auditory processing (4). Previous work has also

shown that toadfish DON cells project to the TS, primarily con-
tralaterally (5).

Extracellular recordings were made from single units of the left
side of the brain, at 20-22°C, as they responded to oscillatory
motion at a range of frequencies between 50 and 303 Hz. Record-
ing electrodes were either metal-filled glass pipettes with tip di-
ameters of about 10 /nm (6), or 3 M NaCl, 47r neurobiotin-filled
pipettes with tip diameters of 3-7 fxm. For medullary recordings,
the DON was sampled between the entrances of cranial nerves
VIII and IX. For midbrain recordings, electrodes were advanced
through the optic tectum to the TS. Stimuli were created with a
three-dimensional shaker system, described previously (2. 7), as
linear oscillations at -30° azimuth (to the left front and right rear)
in the horizontal plane, and at 0° elevation [the best direction for
a majority of saccular afferents (2. 4)]. Tone bursts (500 ms in
duration, including 20-ms rise-fall times) were presented at equal
displacement levels, eight times each, at 50, 65, 84. 100, 141. 185.
244. and 303 Hz. Average spike rate was plotted as a function of
tone frequency to define the frequency response areas. Frequency
response areas were typically determined at four or more stimulus

174

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

50 65 84 100 141 185 244 303
Best Frequency (Hz)

50 65 84 100 141 185 244
Best Frequency (Hz)

303

Figure 1. Frequency distribution.'! of single unit best frequencies (BF)
for units recorded in the descending ochival nucleus (DON. A: n = 921 and
the torus semicircularis (TS. B: n = 24} of the tuadfish. The numbers along
the abscissa give the stimulus tone frequencies used, und the abscissa is
approximately logarithmically scaled.

levels within a unit's dynamic range (generally between —10 and
30 dB re: 1 nanometer, root mean square). Ninety-two units of the
DON (23 animals, 6 of which were females), and 24 units of the
TS ( 12 animals, 3 of which were females) have been successfully
characterized. A unit's best frequency, or BF, was defined as the
stimulus frequency producing the greatest spike rate at the approx-
imate center of the unit's dynamic range. Frequency response
range was defined as the frequency range within which the unit
responded at or above 50% of the maximum spike rate
(at BF).

Figure 1 shows frequency distributions of BF for the DON units
(A-top) and TS units (B-bottom). The lowest BF was 50 Hz (or
less), and the highest was 303 Hz (or more). Most BFs were
between 100 and 185 Hz. The mode for BF was 18? Hz for DON
cells, and 141 H/ for TS cells. There were no differences in the
range of BFs for males versus females. The single animal with a
BF of 303 was male. While the distributions for both recording
sites overlap substantially, relatively fewer units with BFs above
141 Hz were found in the TS. This could be due to sampling error,
or it might have resulted from a nonhomogeneous spatial distri-
bution of BF within the TS. which was not explored as systemat-
ically as the DON. Frequency response area widths averaged 64
Hz for TS and 1 1 7 Hz for DON. This difference probably reflects
the lower mean BF for the TS distribution.

Because the fundamental frequency of the boatwhistles recorded
in the Woods Hole area varies between 130 and 220 Hz, these
results indicate that most, but not all, auditory brainstem cells of
the toadfish represent Fn. Thus, contrary to a previous report (8),
there is a good "match" between the fundamental frequency of the
vocalization and the response of the auditory portions of the brain.
In addition, because the highest BF is about 303 Hz, the higher
harmonics of the boatwhistle (first harmonic at 360 to 440 Hz), so
evident in field recordings (at 19°-22°C; Edds-Walton, Mangia-
mele, and Rome, unpub. obs.), are probably not represented by the
brain, and thus may play no role in boatwhistle perception or other
behaviors. The neural representations of the boatwhistle encode
the fundamental frequency only. Although not demonstrated ex-
perimentally as yet. this frequency is probably equivalent to the
rate at which muscular contractions to the swimbladder occur
during generation of the sound.

Supported by an R01 grant from NIH, NIDCD to R.R.F.

Literature Cited

1. Watkins, W. A. 1967. Pp. 15-43 in Marine Bioacoustics. W. N.
Tavolga. ed. Pergamon Press, New York.

2. Fay, R. R., and P. L. Edds-Walton. 1997. Hear. Res. Ill: 1-21.

3. Fay, R. R., and P. L. Edds-Walton. 1997. Hear. Res. 113: 235-246.

4 Edds-Walton. P. I... R. R. Fay. and S. M. Highstein. 1999. J. Com/'
Neurol. 411: 212-238.

5 Edds-Walton. P. L. 1998. Hear. Res. 123: 41-54

6. Dowben, R. M., and J. D. Rose. 1953. Science 118: 22

7. Fay, R. R. 1984. Science 225: 951-954.

S. Fine, M. 1981. Pp. 257-263 in Hearing and Sound Communication in
Fishes. W. N. Tavolga. A. N. Popper, and R. R. Fay. eds. Springer-
Verlag, New York.

NEUROBIOLOGY

175

Reference: Biol. Bull. 199: 175-176. (October 2000)

Intrinsic Membrane Properties of Laryngeal Motoneurons that Control Sexually Differentiated Vocal

Behavior in African Clawed Frogs. Xenopus laevis

Avako Yamaguchi, Leonard K. Kaczmarek , and Darcy B. Ke!ic\ (Department of Biological Sciences,
Columbia Universin, Sherman Faircluld, MailCode 2430. New York. New York 10027)

Males and females behave differently during reproduction. Al-
though sexually differentiated patterns of behavior in vertebrates
are clearly regulated by the action of gonadal steroids, the neural
mechanisms underlying the expression of sex-specific behavior are
largely unknown.

Male and female African clawed frogs (Xenopus laevis) produce
sexually distinct vocalizations composed of a series of clicks. The
fundamental difference between male and female calls is the rate
at which the clicks are repeated (reviewed in 1 ); male calls cover
a wide range of click repetition rates (8 to 80 Hz), whereas female
calls contain only slow repetition rates (2 to 20 Hz). This behav-
ioral difference can conveniently be reduced to the sexual differ-
ence in contraction rate of laryngeal muscle (2), which, in turn, is
determined by the sexually distinct firing patterns of laryngeal
motoneurons (3). Thus, there is a direct correspondence between
the sexually dimorphic patterns of vocalization and the activity of
motoneurons.

How. then, do male and female laryngeal motoneurons produce
sex-specific patterned activity? While the overall pattern is prob-
ably produced by a pattern generator upstream of the motoneurons,
the motoneurons themselves may have intrinsic membrane prop-
erties that differ between male and female Xenopus. Testing this
possibility was the goal of this study.

Whole-cell patch clamp recordings were used to characterize
the membrane properties of laryngeal motoneurons in n.IX-X of
adult male and female Xenopus. A thick brain slice preparation
of Xenopus hindbrain was developed, and the neurons were
visualized by IR/DIC microscopy. To facilitate identification,
the motoneurons were retrogradely labeled with fluorescent dye
(AlexaFluor 594 biocytin. Molecular Probes, Eugene, OR) be-
fore the brain was sliced. Responses to hyperpolarizing and
depolarizing current steps (200 ms long) by 6 male and 10
female motoneurons (3 male and 6 female frogs), were recorded
in current clamp mode. The resting membrane potential, thresh-
old, spike amplitude, and spike half-width were directly mea-
sured from voltage traces. The membrane time constant was
determined by fitting single exponential curves to hyperpolar-
izing voltage responses. Input resistance was calculated from
the steady-state membrane potential in response to different
hyperpolarizing current pulses. The capacitance of the cell was
calculated from input resistance and time constant. The peak
firing rate was determined by measuring the interval between
the first two action potentials in response to the largest depo-
larizing current (1.5-2nA) applied to each neuron.

' Department of Pharmacology, Yale University School of Medicine,
333 Cedar Street, New Haven. CT 06520.

Membrane properties of male and female motoneurons are sum-
marized in Table 1 . All the properties measured are statistically
similar in the two sexes except for the input resistance and the cell
capacitance; input resistance is significantly lower, and the cell
capacitance is significantly higher in male motoneurons than in
female motoneurons. These differences predict that male motoneu-
rons are larger than female motoneurons. A previous study has
shown that the dendrites of male n.IX-X neurons are longer than
those of female n.IX-X neurons, although the somal size is similar
in the two sexes (4). The difference in the dendritic arborization
may account for the differences in input resistance and cell capac-
itance in the two sexes. Functionally, sexual differences in the
input resistance imply that the male and female motoneurons
exhibit different responsiveness to synaptic input.

At depolarized membrane potentials, all the motoneurons
showed repeated action potential firing that accommodated over
a time course of 100 ms. The peak firing rates, determined by
the first two spikes in response to depolarizing currents, were
well over 100 Hz in both sexes. To determine whether the
neurons could maintain this rapid firing frequency for more than
two spikes, four female and two male motoneurons were stim-
ulated with trains of depolarizing pulses at various rates (0.5
ms, 7-10 V, 10 to 300 Hz). Both male and female motoneurons
could follow the depolarizing pulses at frequencies of at least
90 Hz. Although the maximum click rate of female vocaliza-
tions is 20 Hz. and that of male calls is 80 Hz, the motoneurons
of both sexes can fire at a much higher frequency than is
required for call production.

Taken together, the results suggest that the laryngeal motoneu-
rons of Xenopus do not limit the click repetition rate, and that the
motoneurons may be sexually differentiated in their responsive-
ness to synaptic input.

Table 1

Basic membrane properties of male and female laryngeal motoneurons
of adult Xenopus laevis

Membrane properties

Male
(» = 6)

Female
(n = 10)

P-value

Resting membrane

potential (mV)

-49.1

± 1.2

-48.1

± 2.5

n.s.

Time constant (ms)

9.15

± 0.91

11.46

± 1.44

n.s.

Input resistance (Mfl)

69.4

± 3.9

182.4

± 25.7

P < 0.01

Cell capacitance (nF)

0.148

± 0.017

0.066

± 0.009

P < 0.01

Spike amplitude (mV)

35.56

± 3.81

35.94

± 4.30

n.s.

Spike half-width (ms)

0.69

± 0.10

0.67

±0.11

n.s.

Threshold (mV)

-29.11

± 10.25

-36.14

± 3.12

n.s.

Highest firing

frequency (Hz)

192.17

± 29.47

257.40

± 25.86

n.s.

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

Literature Cited 3. Yamaguchi, A., and I). B. Kelley. 2000. J. Nenrosci. 20: 1559-

1. Kelley, D. B. 1996. Pp. 143-176 in Biology of Xenopus. R. C. 1?67-

Tinsley. and H. R. Kobel, eds. Clarendon Press, Oxford. 4 Kelley, D. B., S. Fenstemaker, P. Hannigan, and S. Shih. 1988.

2. Tobias. M., and D. B. Kelley. 1987. J. Neurosci. 7: 3191-3197. J. Neumhinl. 19: 413-429.

Reference: Binl. Bull. 199: 176-178. (October 2000)

Optic Nerve Responses of Limulus in its Natural Habitat at Night

Jillian L Atherton1, Matthew A. Krutky1, James M. Hin\ Frederick A. Dodge, and Robert B. Barlow
(Marine Biological Laboratory. Woods Hole, Massachusetts 02543)

What information does the eye send to the brain when an animal
sees? We are exploring this question with the relatively simple
visual system of the horseshoe crab. Limulus pohphennts. By
combining cell-based computational models of the retina with
single-cell electrophysiology, we have examined the optic nerve
code underlying Linni/iis vision during the day in the animal's
natural habitat ( 1 ).

Field studies during the animals' mating season show that male
horseshoe crabs use vision to find mates and do so about equally
well day and night (2). We attribute their remarkable nighttime
vision to a circadian modulation of the sensitivity of their lateral
eyes (3). At night, efferent optic nerve fibers carry signals from the
circadian clock in the animal's brain to its eyes, increasing night-
time retinal sensitivity as much as 1 .000,000 times. The increased
sensitivity nearly compensates for the average 1 ,000,000-fold de-
crease in ambient light intensity after sundown. Here we investi-
gate optic nerve activity recorded from the animal in its habitat,
both day and night, with emphasis on signals that convey infor-
mation about potential mates at night.

A convenient method for recording what the horseshoe crab sees
underwater is to mount a miniature video camera, "CrabCam," on
the animal ( 1 ). This documents the crab's eye view during the day,
but not at night when light levels fall below the camera's sensi-
tivity. To investigate the optic nerve responses of an animal in its
natural habitat at night, we used a repetitive, artificial stimulus that
simulates the movement of a potential mate within the animal's
visual field. The stimulus is a rotating grey cylinder (30 cm in
diameter. 15 cm in height) with a black sector (30 cm in width) that
simulates the size of a typical female horseshoe crab. The cylinder
was placed 1 m from the crab and rotated by hand (4-8 rpm),
moving the black sector horizontally at 7-13 cm/s. which simu-
lates the average speed of a horseshoe crab. Our strategy is as
follows: first, during the day and under water, we record the optic
nerve response of a stationary crab to the rotating cylinder while
simultaneously videotaping the eye's input with the shell-mounted
CrabCam. This allows us to document the visual input when
ambient light levels are sufficient for CrabCam operation. We then
leave the animal and cylinder in place underwater until after
sundown, when we repeat the experiment carried out during the
day, but of course without the Crabcam. This method allows us to

1 Allegheny College. Meadville. PA.
~ Syracuse University, Syracuse. NY.
3 Upstate Medical University. Syracuse, NY.

record the response of the eye to a known visual stimulus at night
in the animal's natural habitat.

We recorded the response of single optic nerve fibers following
a procedure developed in this laboratory ( 1 ). In brief, we trephine
a hole in the carapace about 2 cm anterior to the right lateral eye.
expose the optic nerve trunk, and draw it into a chamber that is
then attached to the carapace. We tease away a single active optic
nerve fiber corresponding to a single ommatidium and pull it into
a micro-suction electrode. The chamber is sealed to make it water
tight, and then a small point light source is used to locate the optic
axis of the recorded ommatidium. We then mount the CrabCam on
the carapace and align it in the direction of view of the recorded
ommatidium. The CrabCam (72° by 54° field of view) encom-
passes about a quarter of the hemispheric view of the lateral eye.
which is seen by about 250 ommatidia, each viewing about a 6°
region during the day and about a 12° at night (4). The animal is
firmly attached to a weighted platform that is placed on the sandy
bottom of the animal's habitat and oriented so that the optic axis of
the recorded ommatidium intersects the axis of rotation of the
cylinder located about 1 m away. Experiments were carried out at
depths of 0.5-1 m in estuaries near the Marine Biological Labo-
ratory in Massachusetts. Signals from the microsuction electrode
and the CrabCam are led via shielded cables ( 13 m in length) to a
portable camcorder on shore or in a nearby boat.

Figure 1 (right) shows two video frames taken with the Crab-
Cam during the day. These frames show the rotating cylinder at
two distances (.87 m and 1 m) from the horseshoe crab. On the left
are 14 s samples ("Day") of the responses of a single optic nerve
fiber to rotations of the cylinder at the two distances from the crab.
The video frames were taken 6 s after the beginning of the
response records (arrows), when the grey-black edge of the cylin-
der began to enter the field of view of the recorded ommatidium
from the right. In both cases, the black sector evoked clear de-
creases in response, with the larger decrease recorded when the
cylinder was closer to the animal and water turbidity was minimal.
The top "Day" response was recorded at 1630 h and the second
was recorded at 1800 h. In both cases, the setup was bathed in
direct sunlight. After the second record was recorded, the animal
and cylinder were left underwater as nightfall approached.

The experiment was then repeated several hours later after
sundown (2030-2100 h) but without CrabCam recordings because
of insufficient lighting. Figure 1 ("Night") displays the responses
of the same single optic nerve fiber to nine sequential rotations of
the cylinder (thin black traces). The heavy black trace gives the
average of the nine responses. Note that the individual responses

NEUROBIOLOGY

177

"5
CL

c
o

Day

.i 10

Day

^ft^H ? JMB^vAjiH

Seconds

Figure 1. The responses of a single ommatidium to an undenvater rotating visual stimulus. The records on the left plot the instantaneous frequency
(reciprocal of the interval between adjacent optic nerve impulses) as a function of the rotation of the stimulus shown mi the right. The speed of rotation
is approximated the same for all records. The visual stimulus is aligned so that the optic axis of the recorded ommatidium is centered on the rotating
cvlinder with its held of view lying within the black sector. At a distance of I in the black sector intercepts the optic i/vn of about V ommatidia. The "Day"
responses were recorded benveen 1630 and 1800 h. and the "Night" responses were recorded benveen 2<>jll and 2 I/HI h. The top video frame was taken
in the clear water of Great Harbor, Woods Hole. Massachusetts, where the grey/black sectors of the cylinder had a contrast of 69% /contrast = (LCrfy —
i-Bi:,ct>Ai-cr^ + LB/Mt)]. The second video frame was taken near Stonev Beach. Woods Hole, where turbid water reduced the contrast of the grey/black
sectors to 26%. Arrows indicate the times at which the undenvater scenes to the right were videotaped. At these times the black sector begins to enter the
field of view of the recorded ommatidium, reducing its response rate. The "Day" records are responses to a single rotation of the cvlinder in a right to
left direction (loop with arrow). The "Night" records \liow responses to nine consecutive rotations of the cylinder (thin black traces; period of rotation ~
16 s) and their average (thick black trace). The peaks and valleys of the thin black traces reflect the hig/tly variable rate of discharge of the single optic
nen'e fiber itniler /mr nighttime levels of illumination.

are highly variable relative to those recorded during the day, and
that the average response rate to the grey sector is about 3
impulses/s which is 6-fold lower than the mean daytime response
rate of about 18 impulses/s (middle trace). We attribute the highly
variable response rates to random photon events occurring at the
very low nighttime levels of illumination. The nighttime sky
during this experiment was heavily overcast and lacked moonlight.
From radiometric measurements we estimate that ambient light
decreased by about 106 to 107 relative to daytime levels. The
circadian increase in lateral eye sensitivity cited above nearly
compensates for such large reductions in ambient lighting. Exper-
iments in the laboratory (R. Barlow and F. Dodge, unpub. obs.)

indicate that the average response to the grey sector of -3 im-
pulses/s is about 50<7f lower than expected for the low nighttime
levels of illumination. The surgery performed to isolate the single
optic nerve fiber may have partially damaged the fragile efferent
fibers that carry the circadian clock's signal from the brain to the
eye; as a consequence, the lateral eye may not have received the
normal efferent input and thus the retina may not have shifted
completely to its fully sensitive nighttime state. Nevertheless the
eye's circadian increase in sensitivity was sufficient to detect the
rotating black sector of the cylinder, which mimics a moving mate.
Computational analyses of visual processing in the Limuhm
brain indicate that retinal inputs may sum at the first synaptic level

178

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

(5). Spatial summation across a matrix of 5-10 ommatidia signif-
icantly increases the signal-to-noise properties of responses re-
corded at night. Indeed summing seven sequential optic nerve
responses to the rotating cylinder yielded a relatively noise-free
response.

These experiments represent our first attempts to analyze lateral-
eye responses of Limulus at night in the animal's natural habitat.
The use of a periodic stimulus obviated the need for video docu-
mentation of the visual stimulus, which is not feasible under
nighttime lighting conditions. With this technique, we successfully
recorded visual responses in the animal's habitat and found that the
lateral eye transmits information to the brain about mate-like
objects at night under dark overcast skies. Under such conditions
Lintulus could see what we could not.

Supported by the National Science Foundation. National Insti-
tute of Mental Health, National Eye Institute, Research to Prevent
Blindness, and the Central Lions of New York.

Literature Cited

1 Passaglia, C., F. Dodge, E. Herzog, S. Jackson, and R. Barlow. 1997.

Pn>c. Null. ACM/. Sci. 94: 12649-12654.

2. Barlow, R. B., L. C. Ireland, and L. Kass. 1982. Nature 296:
65-66.

3. Barlow, R. B. 1983. J. Neuruscience 3: 856-870.

4 Barlow, R. B., S. C. Chamberlain, and S. C. Levinson. 1980. Si-i-

encc 210: 1037-1039.
5. Hitt, J.. C. Passaglia, F. Dodge, and R. Barlow. 2000. Ninth Annual

Computational Neitroscience Meeting. Brugge. Belgium, p. 75.

Reference: Bioi Bull. 199: 178-180. (October 2000)

Do the Properties of Underwater Lighting Influence the Visually Guided Behavior of Limulus?

Matthew A. Krutky1, J ilium L. Atherton2, Spence Smith, Frederick A. Dodge and Robert B. Barlow
(Marine Biological Laboratory, Woods Hole, Massachusetts 02543)

In the spring, horseshoe crabs. Limit/us pol\phemus, migrate to
the water's edge along the East coast of the United States to pair
off and build nests ( 1 ). As they enter a nesting area, males use their
lateral eyes to locate mates both day and night (2). They approach
females and objects resembling them, such as rocks, patches of
seaweed, or mate-like objects. What does a male see in a female?
Her size and contrast are two important factors. Males are attracted
to objects that approximate the size of females. They orient toward
mate-like objects at distances up to 1.2 meters, detecting higher
contrast objects better than lower contrast ones (3). How do the
properties of underwater lighting in the animal's natural habitat
influence whether a crab finds a mate during the day or at night?
The approximate 1.000,000-fold reduction in ambient lighting
after sundown has no appreciable effect. Their remarkable visual
performance results in part from a circadian increase in lateral eye
sensitivity of as much as 1.000,000 times at night (4). In this paper
we consider another property of the animal's underwater habitat,
termed "strobic lighting."

In the shallow waters of nesting areas, overhead waves act like
lenses, creating moving beams of sun- and moonlight that reflect
off the sandy bottom and submerged objects. On average, the peak
intensity of these beams is about three times that of ambient
illumination. The fields of view of single ommatidia are wider than
the moving beams of light. Because ommatidia sum the illumina-
tion within their field of view, the amplitude of modulation of the
light beams reaching the underlying photoreceptor cells decreases
to about 70% contrast. The strobic illumination by the beams
strongly modulates the firing rate of an ommatidium, with peak
firing rates reaching three times the mean (5). Such strobic illu-
mination might be expected to enhance the detectability of under-

1 Syracuse University, Syracuse, New York.

2 Allegheny College. Meadville. Pennsylvania.

water objects, such as potential mates. Indeed, an earlier study
suggested that strobic conditions enhance the visibility of low
contrast mate-like objects, and that without strobing. Limulus is
attracted to higher contrast objects (6). We have further explored
the influence of strobic lighting by carrying out more field studies
and combining the results with those collected over the past five
years.

We investigated the visual performance of Limulus during their
springtime mating seasons at Mashnee Dike, Bourne, and North
Monomoy Island. Chatham, both located in Massachusetts. Our
study and those of previous years were carried out day and night
under various weather conditions ranging from dense cloud cover
to clear skies, yielding 105 to 107-fold diurnal changes in the
intensity of ambient illumination, with an average change of about
106. In all our studies, a modified two-alternative forced choice
technique adapted from human psychophysics was used (7). As
shown in Figure 1, we placed on the sandy bottom a clear Plexiglas
chute with a funnel at one end and a narrow chute at the other.
Crabs entered the funnel and, upon exiting the chute, were pre-
sented with the choice of a black or gray female-sized object; these
were located 1 m from the exit of the chute and 1 m from each
other, creating an equilateral triangle. The objects were either a
hemisphere (diameter of 0.3 in) or a cylinder (height of 0.15 m;
diameter of 0.3 m), both approximating the size of an adult female
horseshoe crab. The objects were switched periodically during an
observation period to avoid any effects of directional bias in
behavior. Their black and gray tones represent the greatest range of
contrast of the female carapace (8). The black object has a negative
contrast of 37% against the background of sand and seawater. and
the gray object has a positive contrast of 35%. Animals exiting the
chute either approached and contacted one of the two targets or
swam by them. In 1999 and 2000, about 60% of animals exiting
the chute did not approach or contact either submerged object. The
animals passing by both objects were not recorded in the years

NEUROBIOLOGY

179

Figure 1. Left: Diagram of field experiment. Limulus approaches the Plexiglas chute and exits toward gra\ fG) ami black (B) mate-like objects located
1 mfrom the opening and I in from each other. Middle: Photograph taken under nonstrobic conditions showing an animal (outlined) exiting the chute and
oriented toward the black object. Right: Photograph taken under strobic conditions showing an animal approaching the grn\ object which is highlighted
by bright beams of light. Only the chute and part of the funnel are visible in the photos. Nonstrobic condition?, occurred when cither no sun or moon was
visible, when the wind was calm, or when overhead waves were blocked by a clear-bottom Plexiglas box.

preceding 1999. More than 99% of the animals studied were
males, because animals in amplexus were prevented from entering
the funnel, and <1% of single animals in nesting areas are females
(1). We could not determine whether a specific male crab passed
through the chute more than once in a single observation period,
but since animals in nesting areas are abundant, such events are
unlikely to have occurred. When an animal exited the chute,
observers noted whether the lighting conditions were strobic or
nonstrobic. In 1995 through 1998, nonstrobic conditions occurred
naturally day and night, under cloudy skies or in calm water. In
1999 and 2000, we controlled strobing by placing a clear-bottomed
box on the surface of the water above both targets. The Plexiglas
bottom of the box prevented rippling wave action, thereby elimi-
nating strobic lighting of the underwater scene (See Fig. I ).

Table 1 summarizes the data collected in 2000 and during the
five previous years. Taken together, the data for all six years
("Total" in Table 1) indicate that, under nonstrobic conditions.
there is no significant difference between the number of animals
attracted to the two objects day or night (P-values of 0.76 and
0.077 respectively, as determined by the x* test). Under strobic
conditions, significantly more animals, 69 or 24% more, ap-
proached the gray object during the day (P < 0.00005). The
greater number of animals, 12, attracted to the gray target at night
under strobic conditions, was not significant (P > 0.4).

These field studies show that when the distribution of illu-
mination in the animal's natural habitat is uniform (nonstrobic
conditions), the animals detect black and gray mate-like objects
about equally well day and night. This is understandable be-
cause the black and gray objects have about the same absolute
contrast, 37% and 35% respectively, against the underwater
background. Under strobic conditions, significantly more ani-
mals are attracted to the gray object during the day. but not at
night. This is also understandable because, as described above,
the moving, underwater light beams increase the contrast of the
gray object, but not the black one. Indeed optic nerve recordings
in the animals' natural habitat reveal bursts of activity in
response to gray objects illuminated by strobic light (5). Why
the gray objects are not more attractive under strobic conditions

at night is not understood. The highly variable optic nerve
discharge resulting from random photon events at low levels of
nighttime illumination (9) might be masking the bursts of
activity generated by strobic lighting. We conclude that the
properties of ambient lighting can affect an animal's vision in
its natural habitat, particularly during the day. Limulus is not
unique. Strobic lighting appears to have a prominent role in the
visual performance of other marine animals (10. 11).

Supported by the National Science Foundation, National Insti-
tute of Mental Health, National Eye Institute, Research to Prevent

Table 1

Number of crabs that exited the chute and hit black or grey targets in
the 1995-2000 mating seasons

Strobic

Nonstrobic

Year

Time

Black

Gray

Black

Gray

1995

Day

Night

1996

Day

Night

1997

Day

Night

1998

Day

Night

1999

Day

Night

2000

Day

193

190

Night

Total

Day

107

176

338

346

Night

100

112

139

11 1

Hits were divided into strobic and nonstrobic categories depending on
the underwater lighting conditions when an animal left the chute. Strobic
refers to moving beams of sun- and moonlight that reflect off the sandy
bottom and submerged objects, whereas nonstrobic refers to the absence of
this phenomenon.

180

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

Blindness, and the Central Lions of New York. Special thanks to
Lori Broderick, Hoyt and Deborah Ecker. Elizabeth Flynn. Jason
Friedenfelds. Son Gibson, Carrie McGuinnes. Michelle Parshley.
and Vanessa Ruta, and the Monomoy National Wildlife Refuge.
U.S. Dept. of Interior. Chatham. MA.

Literature Cited

1 Barlow, R. B., M. K. Powers, H. Howard, and L. Kass. 1986.
Bwi Bull. 171: 310-329.

2 Barlow, R. B., L. C. Ireland, and L. Kass. 1982. Nature 296:
65-66.

3. Herzog, E. D., M. K. Powers, and R. B. Barlow. 1996. Visual

Neurosci. 13: 31-41.
4 Barlow, R. B. 1983. J. Neuroscience. 3: 856-870.

5. Passaglia, C. L., F. A. Dodge, E. Herzog, and R. B. Barlow. 1997.
Proc. Nail. Acad. Sci. USA. 94: 12,649-12,654.

6. Passaglia, C. L., M. E. McSweeny, K. M. Stewart, E. Kim, E. J.
Mole, M. K. Powers, and R. B. Barlow. 1997. Biol. Bull. 193:
205-207.

7. Teller, D. Y. 1979. Infant Behav. and Dev. 2: 135-153.

8. Herzog, E. D., and R. B. Barlow. 1992. Visual Neurosci. 9: 571-
580.

9. Atheron, J. L., M. A. Krutky, J. Hitt, F. Dodge, and R. B. Barlow.
2000. Binl. Bull. 199: 176-178.

10. Loew E. R., and W. N. McFarland. 1990. Pp. 1-40 in Visual
System of Fish. R. H. Douglas and M. B. A. Djagmoz. eds. Chapman
and Hall. London.

11. Glantz, R. M., and A. Bartels. 1994. J. Neurophysioi 71: 2168-
2182.

Reference: Biol. Bull. 199: 180-182. (October 2000)

Startle Responses of Fish Without Mauthner Neurons:
Escape Behavior of the Lumpfish (Cycloptenis liimpus)

Melina E. Hale (Dept. of Neurobiology and Behavior, SUNY Stony Brook. Stony Brook,
New York 1 1794-5230 and Grass Foundation Fellow, Marine Biological Laboratory,

Woods Hole, Massachusetts 02543)

Fast start escape responses are the primary behaviors used by
fishes to avoid an attacking predator. Of particular importance is
the C-start type of fast start (reviewed by 1, also see 2. 3). During
a C-start the fish rapidly turns away from a threatening stimulus
into a "C" shaped body bend, called stage 1. Frequently, stage 1 is
followed by u tail stroke to the opposite side of the body, stage 2.
which propels the fish away from the stimulus. The C-sturt is
initiated by the Mauthner cells, a pair of large reticulospinal
interneurons (4. 5 1. Each M-cell has a large axon that crosses the
body midline and extends the length of the spinal cord, exciting
motoneurons that innervate the lateral muscle. In response to a
stimulus from the right side of the body, the right M-cell fires an
action potential that propagates rapidly down the axon to cause
nearly simultaneous contraction of muscle on the opposite side of
the body from the M-cell soma and the "C" bend away from the
stimulus (6, 7).

Although Mauthner cells have been identified in a large number
of taxa broadly representing the phylogenetic diversity of acti-
nopterygian fishes, a few species appear to lack these neurons (8).
This study examines the startle behavior of one such species, the
lumpfish (Cycloptenis liimpus). Two specific questions are ad-
dressed. First, do lumpfish have a startle response that is distinct
from routine swimming? If so. how does the behavioral pattern and
performance compare with the M-cell initiated C-start of other
fishes?

The startle response was examined in larval lumpfish rather than
in mature individuals. The larval lumpfish have a more generali/ed
morphology than mature lumpfish, and so it was thought that the
response ot the larvae to a startle stimulus may be more easily
compared to other species. Additionally, it seemed that if the

lumpfish were to have high performance behavioral responses to
predation. it would be seen in the larvae because of greater
vulnerability to predators due to less developed morphological
defenses. It is possible that M-cells are present in larval lumpfish
and are reduced or lost during development; however, morpholog-
ical examination of the reticulospinal neurons of the larval lump-
fish (n = 30) with retrograde labeling has not identified Mauthner
neurons or homologous cells.

For studies of behavior, lumpfish (n = 12: 6.2 ± 1.0 mm, total
length) were hatched from eggs collected off the coast of Glouces-
ter, Massachusetts, at approximately 6 m depth. Eggs and larvae
were maintained in a 10-gallon aquarium with flow-through sea-
water chilled to 1 1'C. Behavioral trials were conducted within a
week of hatching. A tactile stimulus — touching the head with a
fine gauge wire — was used to elicit startle behavior which was
filmed in a small petri dish (3.5 cm diameter). The responses were
captured on high-speed video (1000 Hz) taken with an EG&G
Reticon digital camera imaging through a Zeiss Stemi SR micro-
scope. Three trials from each fish (36 total trials) were analyzed
with Microsoft Excel 98 and Scion Image 1 .6. Parameters exam-
ined were the angles of head movement during stage 1 and stage
2, the latency between stimulus and response, and the durations of
stages 1 and 2.

The larval lumpfish respond to the stimulus with a C-start
behavior pattern (Fig. 1A). Fish turned tightly away from the
stimulus direction in stage 1 [Fig. I A. left column (0-24 ms)] with
an average stage 1 angle of 146r:' ± 23°C degrees. Stage 1 was
consistently followed by a stage 2 tail stroke [Fig. 1A, right
column (24-56 ms)] and movement away from the stimulus. The
stage 2 angle, generally in the opposite direction of the stage 1

NEUROBIOLOGY

181

Stage I Angle

Stage 2 Angle

Stage 1 Duration

Stage 2 Duration

a. — c
= c '-=
— c
.o

c. —
E c

Figure 1. A. /I typical startle response oflan-al lumpfish (Cyclopterus lumpus). Stage 1. the tight "C" bend away from the stimulus, lusts 24 ms (column
I) and stage 2. the first propulsive tail stroke, follows from 24 10 56 ms (column 2). Data for the angle of movement and kinematic stage durations are
shown in B, with comparative data from brown trout (Salmo truttaj. chinook salmon (Oncorhynchus tshawytscha), coho salmon fOncorhynchus kiisutch)
(10. minimum mines in scaling relationships), zehmftsh (Danio rerio) (91 and herring (Clupea harengusj (12).

turn, was consistently smaller than that of stage 1 (stage 2 angle =
50° ± 30°). The movement angles made by larval lumpfish during
the C-start are comparable to those of other species (Fig. IB; e.g.
9, 10. 12. 13).

Several important fast start performance variables are the
latency of response to the stimulus and the duration of the
kinematic stages. The latency between stimulus and initiation of
movement of an M-cell initiated startle can take less than 4 ms
(9) and the duration of the response is generally less than 100
ms ( 1 ). The latency of the lumpfish, recorded for a subset of the
trials (one from each of 10 individuals) was 9 ± 2.1 ms. It was
considerably longer than that of the larval zebratish (3.9 ± 0.2
ms) (9). The duration of stage 1 of the larval lumpfish was
22.8 ± 5.2 ms. and the duration of stage 2 was 26.3 ± 6.8 ms.
Because the duration of the fast-start stages changes with size
(11) and developmental stage (10), direct comparisons among
species are difficult. Still, the durations of kinematic stages 1
and 2 of the larval lumpfish are in the same range of values as
other immature fishes; all under 5 cm (Fig. IB; 9, 10, 12). The
total duration of the fast start (stages 1 and 2) for the larval

lumpfish is shorter than the fast start duration of most larger
fishes (reviewed in 1).

Although the lumpfish has a longer response latency to a startle
stimulus than zebrafish larvae, the C-start of the larval lump-
fish — in pattern and in the duration of response — has the charac-
teristics of the M-cell initiated C-start. One explanation for the
similarities in the startle response among taxa is that the Mauthner
cell and its homologs are present in the larval lumpfish but have
not yet been identified. Another is that alternative neural circuits
can generate rapid C-start behavior and that the Mauthner cell and
its homologs are most critical for rapid initiation of movement. If
so. such mechanisms may be taxon specific since ablating the
Mauthner cell and its homologs in the larval zebrafish results in a
significant decrease in performance (9). The presence of a rapid
C-start type escape behavior in the lumpfish, a species that appears
to generate the fast start behavior without the Mauthner cell
system, provides an exciting opportunity for comparative exami-
nation of an evolutionarily conserved neural and behavioral system.

I thank S. Van Sant and J. Fetcho for their contributions of fish
and equipment, respectively. Thanks to S. Zottoli and M. Westneat

182

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

for helpful discussion and advice on this paper and to the reviewers
of this paper for their comments. This project was supported by a
Grass Foundation Fellowship and a NIH NRSA fellowship (MH
11861).

Literature Cited

1. Domenici, P., and R. W. Blake. 1997. ./ E.\/>. Biol. 200: 1165-
1178.

2. Weihs, D. 1973. ./. Tlicor. Biol. 106: 1X9-206.

3. Webb. P. W. 1976. J. £.»/>. Biol. 65: 157-177.

4 Hackett, J. T.. and D. S. Faber. 1983. Ncnmseience 8: 317-331

5 Eaton, R. C., R. DiDomenico, and J. Nissanov. 1991. Brain Behav.

Evol. 37: 272-285.
6. Foreman, M. B., and R. C. Eaton. 1993. J. Nenrosei. 13: 4101-

4133.
7 Jayne, B. C'., and G. V. Lauder. 1993. J. G™/> Pli\si<>l. A. 173:

495-508.

8. Zottoli. S. J. 1978. ./. Comp. Neiirosci. 178: 741-758.

9. Liu, K. S., and J. R. Fetcho. 1999. Neuron 23: 325-335.

10. Hale, M. E. 1999. J. £v/>. Biol. 202: 1465-1479.

11. Webb, P. W. 1978. J. £.v/i. Biol. 74: 21 1-226.

12. Blaxter, J. H. S., and R. S. Batty, 1985. J. Mar. Biol. Assoc. U.K.
65: 737-750.

Reference: Biol. Bull 199: 182-183. (October 2000)

Memory Consolidation in Hermissenda crassicornis

David A. Epstein, Herman T. Epstein, Frank M. Child, and Alan M. Kuzirian
(Marine Biological Laboratory, Woods Hole, Massachusetts 02543)

Experiments with shell-less molluscs (Aplysin and Hermissenda)
have revealed a number of processes that underlie learning by these
organisms and also by some vertebrates. Hennissenda, for example,
shows significant Pavlovian conditioning capabilities ( 1 ). Follow-up
investigations on these molluscs dealt with the sensory stimuli needed
for short-term memory (STM) and long-term memory (LTM) (2).
The relationship of the two memories with in vitro changes in exci-
tatory post-synaptie potentials (EPSPs) have also been investigated in
the neural networks of these organisms (3). Many studies of the
molecular aspects of these two different memory regimes have led to
quite detailed descriptions of the events (4. 5. 6).

Both Aplvsiu and Hermissenda have been tested for their recall
of induced behavioral modifications after one, two, or many con-
ditioning events (CEs). In Aplysia, the EPSP component of learn-
ing produced by 1 CE was compared to that produced by 5 CEs
(3). In Hermissenda, the comparison was made between 2 CEs and
9 CEs (2). Five to ten minutes after finishing one or two condi-
tioning events, both animals exhibited significant behavioral recall
(i.e. STM); but there was no recall after an hour or more (i.e. no
LTM). The larger numbers of CEs, however, did induce LTM in
both species.

Since STM and LTM are clearly responding to a different set of
conditions, we focused first on what might inhibit or block STM. This
problem was partially anticipated in 1900, according to McGaugh (7)
who cited Muller and Pilzecker as having found that "memory of
newly learned information was disrupted by the learning of other
information shortly after the original learning" (8).

This concept led us to test, in Hermissenda. whether STM recall
(at 5 min) might be blocked simply by the input of additional
information (i.e. extraneous sensory stimuli) if the latter were
applied within the first 5 nun after conditioning. The initial results
of the blocking experiments, which showed that the simple sensory
inputs blocking STM also blocked LTM. then led to the hypothesis
that temporal consolidation of LTM could be detected by measur-
ing when the blocking sensory input was no longer effective.

Hermissenda (Sea Life Supply, Sand City, CA) were tested with
2 and 9 paired CEs for induction of STM and LTM. Conditioning

events consisted of exposing the animals to 6 s of bright, white
light (CS) explicitly paired with 4 s of strong orbital agitation (US)
following a 2-s onset delay with an inter-trial interval of 1 min.
Recall of the behavioral modification induced by associative con-
ditioning was assessed by recording the animal's change in foot
length when presented with 6 s of light alone. The conditioned
response (CR) was foot contraction, the unconditioned response
(UR) was foot elongation (9). Two paired conditioning events
initiated behavioral recall after 5 min but not after 90 min; the
LTM input of 9 pairings was recalled at both 5 and 90 min (Fig.
1A). The small and non-overlapping S.E.Ms for each point indi-
cate statistical significance (P = <0.01, r = 3.18).

After giving the animals the paired CS and US stimuli leading
to STM. we tested two simple paradigms of blocking sensory
stimuli. The first was a modification of the conditioning stimuli:
dim orange light and very slow orbital rotation. The second block-
ing stimulus tested consisted of rotating the tray containing the
animals upside down and, after 5 s, rotating il upright again
(rotational block). Both experimental paradigms blocked STM and
LTM (Fig. IB).

To determine the temporal specificity of LTM in Hermissenda.
the following experiments were done. Animals were trained with
9 CEs, and the CR was measured at the usual 90 min. However, at
selected time intervals (2. 25, 50, 55, 60, 65 min) post-condition-
ing, the animals were rotationally blocked. Control animals re-
ceived only the 9 paired CEs. When the animals' behavior was
plotted, a clear and decisive LTM consolidation interval in Her-
missenda appeared; consolidation occurred between 55 and 60 min
(Fig. 1C). Presentations of rotational blocking prior to 55 min
totally blocked memory consolidation. However, the stimulus
given after 60 had no blocking effects, and the animals demon-
strated the CR. The consistency of and surprisingly little variability
in the response among the majority of the animals indicated the
robustness of the paradigm. When the data were analyzed with
Most and F-test statistics, they were found to be highly significant,
whether compared between data points or to zero (P = <O.OOI.
/ = 15.24; F-value, inf).

NEUROBIOLOGY

183

Figure 1 A

17,49

- 25,70 0

22,65

0 14,44

2CE5 2CE90 9CE5 9CE90

TREATMENT

Figure IB

M
DC
g 20

J °°
DC
E
M

5 -20
g

•40

•

! A

" I/)

T W
_ 1 ON

i J S

— u '

• y

9CE90i c

S f5 S S 2

in in in m IM

U 0 U S |
M r, * ^ g

9CE90B2

Treatment

Figure 1C

01.
«
5

NO BLOCK

Time of Blocking (mins)

Figure 1. (A) The typical percent changes in foot lengths of Hermis-
senda after 2 and 9 paired conditioning events fCEs) measured 5 min and
90 min after completing training (e.g. 2CE5). A positive value indicates
elongation of the animal's foot, the (non-learned) unconditioned response
( UR). Negative values indicate foot contraction and a positive conditioned

As a secondary result of these experiments, we could assess the
degree to which an animal's training is modified by extraneous
handling. The less than optimal (non-significant) training results of
animals given 9 CEs and tested after 90 min (i.e. 9CE90 animals; Fig.
IB) could i-t- directly attributable to the effects of handling. In future
behavioral experimental protocols, as well as in routine animal train-
ing procedures, the potential for introducing extraneous blocking
stimuli that could confound results must be eliminated.

Memory consolidation has long been recognized to depend
on the species being studied, the nature and the frequency of the
instructional inputs, and the kind of blocking activity used to
determine when the consolidation is completed (7). Our results
thus far apply only to the simplest of conditioning stimuli.
However, if the concept of consolidation time applies to hu-
mans, then awareness of it could help with schooling in two
ways: by alerting teachers to the possible blocking of learning
by presenting unrelated materials too close to the main point, or
by wakening teachers to the need for waiting past the consol-
idation time before asking complex questions about what had
just been presented to the students. Our best estimate, derived
from the literature, is that human consolidation time for simple
inputs is about 6 to 10 min (10).

The authors acknowledge Dr. Daniel Alkon, Laboratory of
Adaptive Systems, NINDS-NIH, Bethesda. MD, for his support
and for providing Hermissenda for these experiments.

Literature Cited

1. Alkon, D. L. 1974. ./. Gen. Physiol. 64: 70-84.

2. Ramirez, R. R., C. C. Gandhi, I. A. Muzzio, and L. D. Matzel.
1998. Learn. Mem. 4: 462-477.

3. Montarolo, P. G., P. Goelet, V. F. Caslellucci, J. Morgan, E. R.
Kandel, and S. A. Schacher. 1986. Science 234: 1249-1254.

4. Bailey, C. H., D. Bartsch, and E. R. Kandel. 1996. Proc. Nat.
Acad. Sci. 93: 13445-13452.

5. Muzzio, I. A., A. C. Talk, and L. D. Matzel. 1998. Neuroreport 11:
1625-1631.

6. Izquierdo, I., J. H. Medina, M. R. M. Vianna, L. A. Izquierdo, and
D. M. Barros. 1999. Belnn: Brain Re.\. 103: 1-11.

7. McGaugh, J. L. 2000. Science 287: 248-251.

8. Muller, G. E., and A. Pilzecker. 1900. Z. Psychol. Suppl. 1-288.

9. Kuzirian, A. M., H. T. Epstein. T. J. Nelson, N. S. Rafferty, and
D. L. Alkon. 1998. BID/. Bull. 195: 198-201.

10. Lechner, H. A., L. R. Squire, and J. H. Byrne. 1999. Learn. Mem.
2: 77-87.

response (CR). The numbers heside the points ( e.g. 25, 70) signify the number
of animals measured, and observational differences averaged. (B) The effects
of the t\vo experimental Hocking stimuli applied during the first 5 min after
training and tested for inliihition of short-term memory (STM) ami long-term
memory (LTM). Designations are as in Figure I A with the addition of B I. first
blocking sensory stimuli (dim light plus slow agitation); B2, second blocking
stimulus consisting of vertically rotating the animals upside down for 5 s, then
re-righting them. Both blocking paradigms interfered with STM and LTM.
Weakened conditioning response of9CE90 was due to the blockade of memory
induced bv handling. (C) Temporal specificity of memory consolidation in
Hermissenda. Percent foot changes measured 90 min after training with 9
CEs, but with the second blocking stimulus (B2, vertical rotation) applied at
designated times after the end i>( training. Memory consolidation appeared to
occur between 55 and 60 min. Statistical analyses using i-tests and F-tests
were highly significant (P = <O.OOI).

184 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

Reference: Biol. Hull. 199: 1X4-1X6. (October 201)0)

Mechanisms of Spontaneous Miniature Activity at CA3-CA1 Synapses: Evidence for a Divergence

From a Random Poisson Process

A. Abenavoli, L Forti1, ami A. Malgaroli (Unit of Neurobiology of Learning, Universita San Raffaelc,
Milano, Italy ami Marine Biological Laborattuy, Woods Hole, Massachusetts 02543)

Most of the CNS synapses investigated so far release quanta
in a spontaneous manner. Since the pioneering work of Fatt and
Katz ( 1 ). spontaneous release of synaptic quanta is considered
a probabilistic process where each quantum is randomly dis-
charged at individual release sites. Release sites are assumed to
behave independently and discharge quanta at a very low and
stable rate. This hypothesis has been continued by a large
number of observations at the neuromuscular junction and at
other peripheral terminals (1-2). Regrettably, it is unclear that
such a description of the release process applies to small CNS
synapses. To investigate this problem, we used whole-cell re-
cordings to study the stochastic properties of spontaneous re-
lease at hippocampal synapses. In these experiments, clustering
of miniature events (minis) was a consistent observation. In
about 60% of the experiments, multiple decaying exponentials
were required for best tit of interval distributions, and this
facilitatory behavior — which could not be accounted for by
random coincidences — was independent of calcium elevations
in the cytosol. For these experiments, postnatal CA3-CA1 hip-
pocampal cultures were prepared from P4-5 neonatal rats es-
sentially as previously described (3). Neurons were used for
synaptic experiments 10-21 days after plating. Whole-cell re-
cordings of miniature currents were also made, as previously
described (3).

Hippocampal neurons were continuously perfused with a
bath solution containing (in niM): NaCl 119, KC1 5. CaCK 2.
MgCK 2. HEPES 25, glucose 30. picrotoxin 100 jjiM (Sigma),
APV (D-2-amino-phosphonovalerate) 25-100 fj-M, tetrodotoxin
0.5 pM. adjusted to 305 mOsm and pH 7.4. Patch electrodes
(2-5 Mil) contained (in mM): Cs-gluconate 110. MgCK 5,
NaCl 10, EGTA or BAPTA 0.6-10, ATP 2, GTP 0.2, HEPES
49, adjusted to pH 7.2 and 290 mOsm. Current traces were
digitized off-line from the magnetic tape at 10-70 KH/ after
low-pass filtering at 3-5 KH/.. Miniature events were detected
semiautomatically (3). Intervals between events were log-
binned and plotted on a log-log scale: the bin content was
normalized for the bin width (4). The resulting histograms were
fitted with nested models made by the sum of exponentials with
a Simplex algorithm and a maximum likelihood estimator. The
minimum number of exponential components was chosen using
the log-likelihood-ratio test. Averaged values are reported as
the mean ± s.e.m., and statistical comparisons were obtained
using an independent Student's / test, unless otherwise indi-
cated. In these conditions, when the occurrence of spontaneous
events was analyzed, clusters of minis (i.e.. very brief episodes
with a few consecutive quanta! releases) were found to be a

1 Present Address: Dipurtimenlu di FiMnlogia. Universita di Pavia. Via
Furlanini 6, 1-27100 Pavia. Italy

very consistent observation. The statistical significance of this
finding can be tested by analyzing the distributions of inter-
event intervals from large data sets. According to predictions
from Poisson's law. a frequency distribution of random inter-
vals should display a mono exponential profile:

F(.\> * cxp( - A.vJ

where F(.v) is the probability of having an interval greater than
.v. and A is the mean mini frequency. Log-binned frequency
distributions of mini-intervals indicated (4) that in most cases
in --'- 14/24; P < 0.05, log-likelihood ratio test), multiple
decaying exponentials are required for optimal fit of interval
distributions. This indicates a clear divergence of spontaneous
exocytosis from a random Poisson process (Fig. 1, A-B). The
best fit of the interval distributions indicated that the area
underneath the fast or bursting component (a,.lsl) could be as
high as 66% or as low as 3% (mean a,as, = 12 ± 4%; n = 14)
(Fig. 1, A-B). Since miniature events arise from a large popu-
lation of independent synapses, could this divergence arise
simply from the temporal averaging? The answer is "No,"
according to the following argument. If each synapse generates
spontaneous events according to a Poisson process, then the
probability of finding k events in the time interval At is:

P(k) =

exp(-ju,- Af) • JU.J1

where /u, is the mean Poisson rate at the ith synapse. With a
population of N independent synapses, the occurrence of minis at
the soma will also be a Poisson process with a single parameter /j.
which is just the sum of the individual parameters:

Therefore, based on these simple mathematical considerations, if
every synapse made onto an individual neuron is releasing in a
random manner, whole-cell mini interval distributions should dis-
play a single exponential component. Moreover, this conclusion
would also be valid in the presence of a large variability in
spontaneous quantal rates at different synapses, as previously
reported in the same system (5). In agreement with these expec-
tations we have used a technique that permits to us record minis
from individual hippocampal synapses and have found that, even
at the level of a single terminal, the generation of quanta diverges
from a random memory-less Poisson process (Abenavoli et al.,
unpub.l.

A transient up-modulation of quantal discharges, such as the

NEUROBIOLOGY

185

70 -

CL>
O

10 J 10

Intervals (s)

Figure 1. Distribution of intervals between minis at hippocampal synapses. A) Minis were acquired in voltage clamp using the Whole-cell recording
configuration. Short trains of minis could be seen consistently under these conditions. In these experiments multiple exponentials were always required for
best fit of mini-interval distributions indicating a divergence from Poisson's statistics. The histogram presented was best fitted b\ the sum {solid line) of
two decaying exponentials {dotted lines). B) Summon' data for the area (af) of the short-inten'al component {range af = 3-66%, mean value = 12 ± 4%;
range rf = 1.56 - 48.64 ms, mean value 20.37 ± 4.07 ins).

one observed in the fast component of mini interval distribu-
tions, might result from some sort of transient change in pre-
synaptic Ca2+ levels (see ref. 6 for review). We have therefore
tested the effects of cadmium (50 juM). a broad spectrum Ca2 +
channel blocker. When cadmium was applied, no effects on
mini frequency and mini amplitude were detected. In 9 cells, the
average mini frequency in control conditions was 2.51 ± 0.75
Hz. and it was 2.56 ± 0.74 Hz after the application of cadmium.
In these experiments, when log-binned distributions of mini-
intervals were constructed, if multiple decaying exponentials
were required for optimal fit in control conditions, they were
also required in the presence of Cd2 + (;i = 4/4; P < 0.01 ). We
also examined the effects of BAPTA, a high affinity, fast-
binding Ca2+ chelator (7). BAPTA was introduced in all syn-
aptic terminals impinging upon a postsynaptic neuron by per-
fusing those neurons with the membrane permeable analog
BAPTA-AM while recording synaptic events. Long-term appli-
cation of BAPTA (>20 mint (in the presence of tetrodotoxin)
produced no significant effect on mini frequency (<fctr> =
2.14 ± 1.30 Hz, <fBapta> = 1.74 ± 1.56, mean ± sd; n = 7).
Importantly, interval-distributions of minis displayed no detect-
able change after the BAPTA treatment (n = 3/3: P < 0.05;

= 8 ± 3%;

= 8 ± 3%). Taken together, these

observations rule out a role in the divergence from a random
Poisson process for a brief elevation in presynaptic Ca2 + ,
whether from an influx through the plasma membrane or release
from internal stores. Our electrophysiological recordings re-
vealed that the dynamics of spontaneous quanta is more com-

plex than previously thought and cannot be simply predicted by
applying the Poisson theorem (1-2). This is because short
epochs of multiple quanta releases were consistently present in
the recordings. The genesis of this phenomenon is independent
of Ca2 + elevation in the presynaptic terminals. Our relative
ignorance about the molecular organization of release sites
precludes any deeper understanding of this synaptic behavior.
Nonetheless, we can speculate that, since minis keep occurring
in the absence of any incoming electrical activity, trophic
support through minis would circumvent requirements for Heb-
bian mechanisms to maintain some forms of synaptic plasticity
(8). In particular, the rapid discharge within a burst would
certainly lead to a temporal summation in the postsynaptic spine
and dramatically increase the probability of calcium influx
through postsynaptic NMDA channels. The input-output prop-
erties of CNS synapses are an additional consideration.

The results presented might also be relevant to the hypothesis
that the release of multiple vesicles is happening under some
conditions and in some neuronal systems during spontaneous and
evoked exocytosis (9-12). Regardless of the frequency of mul-
tivesicular exocytosis, this could certainly have an impact on the
synaptic input-output characteristics of hippocampal synapses.
since glutamate AMPA receptors are not saturated by the content
of a single vesicle (3).

A. Abenavoli was a recipient of an Armenise-Harvard fellow-
ship. This research was supported by a Rand award. Telethon.
Human Frontier and MURST grants to A.M.

186 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

Literature Cited 7. Adler. E. M., G. J. Augustine, S. N. Duffy, and M. P. Charlton.

1991. J. Neiirosci. 11: 1446-1507.
1 Fan, P., and B. Kat,. 1952. ./. Physiol (U>nd.) 117: 109-128.

8 T y and Q L Co|li rid 1993. Nature 361: 31_39.

2 Rotshenker, S.. and R. Rahamimolt. 1970. Sr/«ire 170: 648-649.

9. korn, H., C. Sur, S. C harpier, P. Legendre, and D. S. Faber. 1994.

3. Forti, L., M. Bossi, A. Bergamaschi, A. Villa, and A. Malgaroh. .

„„_ ,„_ 0_0 Pp. 301— 322 in Molecular and Cellular Mechanisms of Neurotrans-

1997. Nature 388: S74-S7a.
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4. McManus, O. B.. A. L. Blatz. and L. Magleby. 1987. Pfliiwrs

Arch 410-530-5^3 l(l Au8er- c-, S. Kondo, and A. Marty. 1998. J. Neiirosci. 18: 4532-

5. Malgaroli, A., A. E. Ting. B. \\endland, A. Bergamaschi, A. Villa, 4347

R. W. Tsien, and R. H. Scheller. 1995. Science 268: ]624-lh2K. ' ' Tong- G- and C. E. Jahr. 1994. Neim<n 12: 51-59

6 Augustine, G. J., M. P. Charlton, and S. J. Smith. 1987. Mm,. 12. Ryan T. A.. H. Reuter, and S. J. Smith. 1997. Nature 388: 478-
Re\: Neumxci. 10: 633-693. 482.

PHYSIOLOGY AND BIOCHEMISTRY

187

Reference: «/<•/. Hull. 199: 187-188. (October 2000)

UV-B Induced Damage to the Skin and Ocular System of Amphibians

Iriigo Novales Flamarique (Department of Biological Sciences. Simon Fraser University, 888 Unirer.sitv
Drive, Burnaby, British Columbia, V5A 1S6, Canada). Kristiiiui Ovaska, and Theodore M. Davis

The depletion of stratospheric ozone and the consequential
increase in ultraviolet (UV-B) radiation reaching the Earth's
surface may be partly responsible for the recent declines in
some amphibian populations (1). Present or predicted UV-B
levels for the next decades in northern latitudes can cause
pronounced mortalities in amphibians during early development
both under laboratory and semi-natural conditions (2-6). How-
ever, except for limb and spinal malformations (7. 8), observ-
able damage at the systems level has been seldom described.
Here, we report on the incidence of skin burns and eye cataracts
in tadpoles exposed to enhanced UV-B levels under semi-
natural conditions.

The tadpoles examined in this study originated from experi-
ments carried out in a forest clearing near Victoria (British Co-
lumbia, Canada) using containers filled with pond water (4). Two
species of frogs (Hyla regilla and Rana aurora) were raised from
eggs under one of three light regimes: (1) ambient sunlight. (2)
sunlight blocked for UV wavelengths less than 450 nm (control),
or (3) sunlight enhanced from 4.8% to 23% UV-B (280-320 nm)
over ambient levels at noon, depending on atmospheric conditions
(from sunny to overcast days, respectively [5]). The enhanced
UV-B treatment was obtained by placing fluorescent UV-B lamps,
with peak output in the range of 280-315 nm, over the appropriate
containers. These lamps were on from 0900-1700 h each day.
resulting in a mean hourly increase of approximately 30% UV-B
radiation per day over ambient levels for the duration of the study.
Tadpoles were collected from each treatment during weeks 3 and
4 after hatching and examined under a dissecting microscope for
damage to the lens (i.e.. presence of lens opacities) and to the skin
(i.e., integument burns). To assess damage to retinal photorecep-
tors. the retinas from five animals collected during week 4 were
prepared for electron microscopy. This procedure involved enu-
cleation of the eyecup and fixation in primary fixative (2.5%
glutaraldehyde, 1 % paraformaldehyde in 0.06 M phosphate buffer.
pH 7.3); post-incubation of extracted retinas in secondary fixative
(1% osmium tetroxide) for 1 h at 4°C; dehydration of the tissue
through a series of ethanol solutions of increasing concentration;
and embedding in Epon plastic. Thin (75 nm) radial sections were
cut. exposing photoreceptors from the centroventral part of the
retina along their lengths. These sections were mounted on copper
grids, stained with uranyl acetate and lead citrate, and examined
with the transmission electron microscope.

The numbers of dead embryos and larvae were monitored
throughout the study (4). Mortalities during the study can be
summarized as follows. Although significantly higher egg mor-
tality was observed in the enhanced UV-B treatment for R.
aurora only, tadpoles of both species suffered high mortality in
the enhanced treatment: only 2.6% of R. aurora and 18.4% of
H. regilla survived 1 month after hatching, compared to 55%

and 51.7% in the ambient treatment, and 43.8% and 65% in the
control.

In the control and ambient treatments, the majority of animals
had clear lenses (Fig. la). Of the controls, only 3 of 10 R. aurora
and 2 of 10 H. regilla tadpoles examined during weeks 3 and 4
after hatching showed signs of lens opacities. The extent of these
opacities was smaller than that presented in Figure Ib. In the
ambient treatment. 3 of 9 R. aurora and 1 of 10 H. regilla showed
similar small lens opacities. In the enhanced UV-B treatment,
however. 16 of 20 R. aurora and 12 of 16 H. regilla tadpoles
showed prominent lens opacities. Thus, for both species, more
tadpoles raised under the enhanced UV-B treatment showed lens
opacities than did those from the control or ambient treatments (R.
aurora \2 = 1 1.4, df = 2. P < 0.005; H. regilla )C = 13.4. df =
2. P < 0.005). Large opacities, a sign of advanced cataracts, were
particularly visible among R. aurora tadpoles (Fig. Ic). Substantial
skin burns were also present in 6 H. regilla and 9 R. aurora
tadpoles from the enhanced UV-B treatment (Fig. Id); however,
this skin damage may have been amplified by fungal infections (9).
No skin sores were found in tadpoles from the ambient and control
treatments.

Electron micrographs revealed single cones that were similar in
all animals irrespective of treatment (Fig. le). The outer segments
of cones (Fig. If), which contain the light-sensitive visual pig-
ments, were not appreciably different from those of fish at early
developmental stages (personal observation). Our data, therefore,
suggests that the damage from enhanced UV-B radiation to the
ocular system of amphibians occurs primarily in the lens. It may be
that the early onset of lens opacities prevents substantial levels of
UV-B radiation from reaching the photoreceptor layer of the retina
and disrupting cell structure. However, other explanations, includ-
ing the involvement of repair mechanisms, may also account for
these results.

The incidence of skin sores and cataracts, as observed in our
UV-B enhanced treatment, can have important consequences
for tadpoles in nature. Animals with cataracts are incapable of
forming a clear image in the retina due to scattering of light
within the lens, hence their foraging and predator avoidance
capabilities are greatly reduced. In addition, the skin sores can
be infected by parasites, thereby further increasing tadpole
mortality (9).

In many regions of the world, the removal of riparian vegetation
and creation of large clearcuts increases the exposure of small
water bodies, used by amphibians for oviposition. to UV-B and
total radiation levels (10). Tadpoles developing in such habitats
might be exposed to UV-B levels sufficient to cause the type of
damage reported here. Monitoring the incidence of cataracts in
clearcuts and forest pools could potentially be used as an indicator
of biological effects of increased UV-B radiation.

188

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

Figure 1. Photographs of (a) Hyla regilla tadpole with clear lens: (b) H. regilla tadpole with lens opacity (arrowhead): (cl Rana aurora tadpole showing
extensive cataracts (arrowhead}: (d) lesion area o/R. aurora tadpole exposing dorsal musculature: (e) R. aurora i -one photoreceptor showing normal inner
and outer segments (is and os respectively): bar = 1.2 ft,ni: and (f) details of two cone outer segments, bar = 0.6 jj.ni. No tluimigi' was readily apparent
in the photoreceptor layer oj 'tadpole retinas from any i if the treatments.

Literature Cited

1 Blaustein, A. R., P. I). Hoffman, D. G. Hokit, J. M. Kiesecker, S. C.
Walls, and J. B. Hays. 1994. Proc. Nail. Acad. Sci. USA 91:
1741-1745.

2 Blaustein. A. R.. B. Kdmond, J. M. Kiesecher, .1. J. l?e;itty, and
D. G. Hokit. 1995. £<•»/. Appl. 5: 740-743.

3. Grant, K. P., and L. E. Licht. 1445. Can. ./. Zool. 73: 2292-2301 .

4. Ovaska. K., T. M. Davis, and I. Novales Kliiinarique. 1997. Can.
J. Zool. 75: HIS I -KISS.

5. Ovaska, K.. T. M. Davis, and 1. Novales I l.nn.ii h|iu . 1998. Can.
J. Zool. 76: 1747.

6. Anzalone, P. G., L. B. Kats. and M. S. Gordon. 1998. Cimsen:
Kiol. 12: 645-653.

7. Blaustein, A. R., J. M. Kiesecker, D. P. Chiversand, and R. G.
Anthony. 1997. Pmc. Nail. Acad. Sci. USA 94: 13.735-13.737.

8. Ankley, G. T., J. E. Tietge, D. K. DeKoe, K. M. Jensen, G. W.
Holcombe, E. J. Durhan, and S. A. Diamond. 1998. Environ.
Toxicol. Chcm. 17: 2530-2542.

4 Kiesecker, J. M., and A. R. Blaustein. 1995. Proc. Nail. Acad. Sci.

USA 92: I 1.044-1 1.052.

10 Novales Flamark|ue, I., and \V. Hammer. 1999. Environ. Bio/.
/•V.v/ic.i 55: 274-243.

PHYSIOLOGY AND BIOCHEMISTRY

189

Reference: Biol. Bull. 199: 189-190. (October 2000)

Initial Characterization of a Potential Anti-fouling System in the

American Horseshoe Crab, Limiilus polyphemus

John M. Harrington (Biological Sciences Department, Uni\\'i:\it\ of South Alabama.
Mobile. Alabama 36688) ami Peter B. Armstrong*

Although the surfaces of most solid objects are rapidly colo-
nized by sessile fouling organisms within a short time after im-
mersion in the ocean, macroscopic fouling organisms are surpris-
ingly sparse on the cuticle of healthy adults of the American
horseshoe crab, Limulus polyphemus. One of the major identified
causes of mortality of adult horseshoe crabs is the erosion of the
cuticle that follows its colonization by green or blue-green algae
( 1 ). Thus it is in the interest of the animal to maintain the cuticle
clear of fouling species. The mechanism(s) responsible for keeping
the cuticle clean in an environment liberally stocked with fouling
species have not been identified. One likely candidate is a mucous
secretion from the hypodermal glands, a system of glands that
discharge onto the surface of the cuticle (2). Here we show that this
secretion has antibiological activities that may contribute to its
ability to deter the colonization of the cuticle of Limulus by fouling
organisms.

The cuticular secretion can be provoked by housing healthy
adults in the presence of decaying fish (3). The secretion from the
hypodermal glands was scraped from the dorsal cuticle of the
Limulus shell with a rubber scraper and stored at 4°C in the
presence of 0.2 mg/ml NaN, to prevent bacterial contamination.
Antibodies to whole cuticular secretion were prepared in rabbits.
Immunostaining utilized a 1:1000 dilution of the primary anti-
serum, a 1:1000 dilution of HRP-anti rabbit IgG, and 4-chlo-
ronaphthol using standard methods for staining Western and dot
blots (4).

Stimulated animals renewed the layer of cuticular secretion
within 1-2 h after the cuticle had been scraped clean. Although
most of the animals in the Marine Resources Center of the Marine
Biological Laboratory, which are collected from the pristine waters
of Pleasant Bay. lacked the thick layer of cuticular secretion of the
pollution-challenged animal, the secretion was present as a thin
layer on their surfaces as well. Anti-cuticular secretion antisera
specifically immunostained swipes of the surface of the cephalo-
thorax of these animals, indicating the presence of the secreted
products of the hypodermal glands. Control swipes immunostained
with non-immune rabbit IgG in place of immune serum failed to
stain.

It has been proposed that the cuticular secretion contains the
secreted products of the Limulus blood cells (2). This apparently is
not true because the principal secreted product of the blood cells,
the clotting protein coagulogen, is absent from the secretion.
Antibodies to coagulogen failed to immunostain dot blots of cu-
ticular secretion, and the anti-cuticular secretion antiserum failed
to stain coagulogen or the collective secretion of the blood cells.
This latter material was prepared as described previously (5, 6).

1 Molecular and Cellular Biology, University of California. One Shields
Avenue. Davis, CA 95616, (pbarmstrong@ucdavis.edu).

The anti-coagulogen antiserum did stain dot blots of coagulogen.
and the anti-cuticular secretion antiserum did stain dot blots of
cuticular secretion. The cuticular secretion showed 1? protein
bands by SDS-PAGE (reducing conditions, silver-staining) in the
molecular mass range of 143-20 kDa.

The anti-biological activity of the cuticular secretion was
tested by its cytolytic actions on target cells. Cuticular secretion
hemolysed sheep red blood cells at a 1:16 dilution in a standard
hemolysis assay (7) (Fig. 1A>. Hemolysis was judged to be
divalent cation-dependent because the divalent cation chelator.
ethylenediaminetetraacetic acid, reduced hemolysis (Fig. 1A).
The macromolecular osmolites dextran-8 (Mr 8-12 kDa) and,
to a lesser extent, dextran-4 (Mr 4-6 kDa) reduced the extent of
hemolysis (Fig. IB). This suggests that hemolysis is the result
of hydrophilic membrane channels established in the plasma
membrane of the target red blood cell by the hemolytic protein
of the cuticular secretion. Protection by the dextrans is sug-
gested to result from their ability to balance the osmotic pres-
sure across the permeabilized cell membrane, which will reduce
the flow of water through the hemolytic pore and into the cell
and will prevent swelling and lysis (8). It is difficult to envision
ways for macromolecular osmolites to protect the cell if the
hemolytic process featured such other possible mechanisms as
phospholipase action or detergent-mediated membrane reorga-
nization.

It is proposed that the cuticular secretion is one agent that
helps maintain the cleanliness of the cuticle of Limulus. Its

80 -,

60
g

<o 40

20 -

E
o

o m

CO l-

Figure 1. Hcmolvlic activity »/ the cuticular secretion from Limulus
polyphemus in rubhil red cells. All samples contain a 1:16 dilution of the
cnticuliir secretion, with the indicated additions. The hemolysis buffer is
0.2? M NaCl. O.»l M Cat:. 0.14 M dextrose. 0.01 M Tris, pH 7.3. Added
EDTA {10 mM) reduces hemolysis (Fig. I A). Dextran-8 and dextran-4
reduce hemolysis (Fig. IB).

190

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

anti-biological activity, exemplified by its ability to lyse foreign
cells such as mammalian erythrocytes. may contribute to this
activity. Under normal conditions, the volume of cuticular
secretion is low, but it can be detected by iminunological
assays. Under conditions of challenge by a polluted environ-
ment, the volume of the secretion is augmented. In addition to
its anti-biological activity, the continuous production of the
cuticular secretion can be expected to exert a mechanical action,
entrapping and sweeping potential fouling organisms away
from the solid surface of the cuticle. Contrary to previous
suggestions (2), the cuticular secretion does not contain secre-
tion products of the blood cells.

Supported by Grant No. MCB-97-26771 from the National
Science Foundation. We thank Ms. Yvonne Coursey for the anti-
coagulogen antiserum.

Literature Cited

1. Leibovitz. L.. and G. A. Lewbart. 1987. Bial. Bull. 173: 430 (abstr.).

2. Stagner. J. I., and J. R. Redmond. 1975. Mm: Fish. Rev. 37: 1 1-14

3. Harrington, J. M.. and P. B. Armstrong. 1999. Biol. Bull. 197:
274-275.

4. Harlon, K., and D. Lane. 1988. Antibodies, A Laboratory Manual.
Cold Spring Laboratory. New York. 726 pp.

5. Armstrong, P. B., and J. P. Quigley. 1985. Biocliiiii. Bio/ihys. Actu
827: 453-459.

6 Armstrong, P. B., J. P. Quigley, and F. R. Rickles. 1990. Bint. Bull.

178: 137-143.
7. Swarnakar, S., R. Asokan, J. P. Quigley, and P. B. Armstrong. 2000.

Biochcm. J. 347: 679-685.
8 Hatakeyama, T., H. Nagatomo, and N. Yamasaki. 1995. J. Biol.

Chem. 270: 3560-3564.

Reference: Bi,<l. Bull. 199: 190-192. (October 2000)

Association of os-Macroglobulin with the Coagulin Clot in the American Horseshoe Crab,

Limulus polyphemus: A Potential Role in Stabilization from Proteolysis

Rengasamy Asokan (Molecular and Cellular Biology, University of California, Davis, California 95616),

Margaret T. Armstrong1, and Peter B. Armstrong'

The blood clot of the horseshoe crab is established by the
proteolytic conversion of the soluble clotting protein, coagulogen,
into the form (coagulin) that polymerizes to establish a fibrillar
extracellular clot ( 1 ). The coagulin clot functions in hemostasis
(2), wound repair (3), and entrapment of bacteria invading through
wounds in the cuticle. Bacteria entrapped in the coagulin clot are
so strongly held that they fail even to show Brownian motion (2).
Entrapment reduces the systemic dissemination of invading bac-
teria to locations far from the wound and its coagulin clot.

Secreted proteases serve important roles in parasitic virulence,
and protease inhibitors of the plasma and blood cells of the host
play a substantial role in immunity by the inuctivation and clear-
ance of the foreign proteases of invading parasites (4). In the
present context, if entrapped bacteria were able to proteolyze the
coagulin clot, then the period of entrapment would be reduced and
the systemic dissemination of the invading bacteria would be
facilitated. The sole protease inhibitor of the plasma of the horse-
shoe crab is a,-macroglobulin (a,M) (5). The blood cells also
secrete a-,M when activated (6). Here we investigate the potential
role of osM in the protection of the coagulin clot from proteolysis.

Limulus «2M was purified as described previously (7). Coagu-
logen was purified as described by Srimal (8). Antibodies to
Limulus osM and coagulogen were produced in rabbits. Western
and dot blotting were conducted using standard methods and
controls (9). Specimens for immunohistochemistry were fixed in
4% paraformaldehyde in seuwater. quenched in 0.1 M glycine,
blocked with 5c/c serum albumin, and stained with rabbit first

1 Molecular and Cellular Biology. University of California, One Shields
Avenue, Davis. CA 95616, (nitarmstrong@ucdavis.edu).

: Molecular and Cellular Biology. University of California, One Shields
Avenue. Davis, CA 95616, (pharmstrong@ucdavis.edu).

antibodies and fluoresceinated goat anti-rabbit IgG. Affinity resins
were prepared by coupling purified protein to CNBr-activated
Sepharose 4B using standard methods (10). Extracts subjected to
affinity chromatography were first exposed to large volumes of
plain Sepharose and were then exposed to Sepharose conjugated to
the desired target protein, washed with high-salt buffer (containing
0.5 M NaCl. 10 mMCa+-, 10 mWTris. pH 7.3). and finally eluted
with a buffer containing the Ca + 2 chelator, ethylenediaminetet-
raacetic acid (EDTA).

Biochemical evidence for the specific binding of coagulogen to
Limulus osM was provided by our ability to isolate coagulogen
from detergent extracts of Limulus blood cells by affinity chroma-
tography over Limulus a2M-Sepharose. The protein that was
eluted following removal of Ca + 2 was identified as coagulogen by
molecular mass (—21 kDa) and reactivity with anti-coagulin anti-
bodies (Western blotting and dot blotting). Coagulogen was by far
the most abundant protein eluted from the osM affinity column.

Inimunohistochemical and biochemical approaches were used to
investigate the binding of a,M to the coagulin clot. Blood clots
were prepared in vitro by collecting 0.3 ml of whole blood into
90-mm petri dishes containing 10 ml of sterile, cell-free Limulus
plasma. The cells that attach to the surface of the petri dish flatten
and degranulate, and the coagulogen that is released from the
secretory granules polymerizes into a fibrillar coagulin clot situ-
ated above the cell layer (11). These fibrils immunostain specifi-
cally with antisera directed against coagulogen (Fig. 1A), showing
their similarity to the fibrils of the blood clot that forms in situ at
the locations of wounds to the cuticle of the animal. When treated
with an affinity-purified antibody to Limulus osM, the clot fibrils
also stain brightly for Limulus a-M (Fig. IB). Treatment of the
clot with 50 mM EDTA prior to fixation and processing for
immunohistochemistry reduced but did not eliminate staining with

PHYSIOLOGY AND BIOCHEMISTRY

191

• ,

;

Figure 1. Immunofluorescenl demonstration of the decoration of the coagulin fibrils of the blood clot h\ Limulus aM. Blood cells were allowed to
attach to microscope coverglasses in numbers sufficient to produce a confluent monolayer. The attached cells degranulate and produce afibril/ar coagulin
clot that lies above the cell layer and stains with antibodies against coagulin (panel A. top left). The clot stains intensely wirh an affinity-purified antibodv
against Limulus a:M (panel B, top right), indicating the presence ofatM attached to the clot fibrils. Most, but mil nil. of the aM is removed by treatment
with 50 mM EDTA prior to fixation (panel C. bottom left). The specimen treated with non-immune serum failed to stain (panel D. bottom right).
Photomicrographs B. C. and D were exposed identically to permit comparison of differing fluorescent intensities. Although most of the blood cells attach
to the glass and degranulate. a few cells migrate to the top of the clot. These fail to degranulate and show as cells with autofluorescent granules in all
pictures. Scale bar (Fig. ID) = SO fj.m.

smli-Limulus a2M antibodies (Fig. 1C). A similar pattern of dec-
oration of clot fibrils by osM was found in clots extracted with
0.5% Triton X-100. Control specimens treated with non-immune
rabbit IgG failed to show staining of the fibrillar elements of the
coagulin clot (Fig. ID). This indicates that a-,M does .indeed
decorate the coagulin fibrils of the clot and that the binding is
largely Ca + 2-dependent. It is possible that the EDTA-insensitive
a2M is covalently linked via transglutaminase-catalyzed isopep-
tide bonds. The Limulus clot does contain such protein-protein
bonds (12) and human osM is a substrate for transglutaminase
crosslinking.

Additional evidence for osM binding was provided by the
characterization of the proteins that co-purify with the blood clot.
For this, the clots formed in vitro were washed exhaustively with

high-salt buffers (0.5 M NaCl. 10 mM Cad,. 10 mM Tris, pH 7.3)
and detergent (0.5% Triton-X 100), and were then scraped from
the petri dish and transferred to microcentrifuge tubes, where they
were compressed by centrifugation. washed further, and finally
eluted by removal of Ca+2 with 0.1 M EDTA. The coagulin clot
was not solubilized by this treatment. The supernatant fraction
contained substantial quantities of a,M. as shown by Western
blotting and dot blotting using an affinity-purified antibody to
Limulus a-,M.

This study provides evidence that osM binds selectively to
coagulogen and coagulin in a Ca + 2-dependent fashion. We suggest
that this serves the important function of positioning osM from the
plasma onto the fibrils of the blood clot, where it is perfectly
situated to protect the clot from microbial proteolysis and the

192

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

consequent compromise of the clot's function as a device that
immobilizes entrapped bacteria.

Supported by Grant No. MCB-97-26771 from the National
Science Foundation. We thank Ms. Yvonne Coursey for the anti-
coagulogen antiserum.

Literature Cited

1. Iwanaga, S., T. Miyata, F. Tokunaga, and T. Muta. 1992.

Thromb. Res. 68: 1-32.

2. Bang, F. B. 1979. Pp. 109-123 in BiomeJical Applications of the
Horseshoe Crab (Limulidae). E. Cohen, ed. Alan R. Liss. New York.

3. Bursey, C. R. 1977. Can. J. Zool 55: 1 158-1 165.

4 Armstrong, P. B., and J. P. Quigley. 20(10. Phylogenetic Perspec-
tives on the Vertebrate Immune System. G. Beck, ed. Plenum Publish-
ing. New York.

5 Quigley, J. P., and P. B. Armstrong. 1983. J. Biol. Chem. 258:
7903-7906.

6. Armstrong, P. B., J. P. Quigley, and F. R. Rickles. 1990. Biol.
Bull. 178: 137-143.

7. Armstrong, P. B.. R. Melchior, and J. P. Quigley. 1996. J. Insect
Physiol. 42: 53-64.

8. Srimal, S., T. Miyata, S. Kawabata, and S. Iwanaga. 1985. J. Bio-
chem. (Tokyo) 98: 305-318.

9. Harlow, E., and D. Lane. 1988. Antibodies, a Laboratory Manual
Cold Spring Laboratory, New York. Pp. 1-726.

10. Pharmacia Fine Chemicals. Affinity Chromatography. 1979. Upp-
sala, Sweden. Pharmacia Fine Chemicals.

1 1. Armstrong, P. B., and F. R. Rickles. 1982. Exp. Cell Res. 140:
15-24.

12 Wilson, J., F. R. Rickles, P. B. Armstrong, and L. Lorand. 1992.
Biochem. Biophvs. Res. Comnnin. 188: 655-661.

Reference: Biol. Bull. 199: 192-194. (October 2000)

Enzymatic Biosynthesis of N-Linked Glycan by the Marine Sponge Microciona prolifera

William J. Kuhns1, Max M. Burger1, Mohan Sarkar1, Xavier Fernandez-Busquets3 , and Tracy Simpson4

(Marine Biological Laboratory, Woods Hole, Massachusetts 02543)

Complex carbohydrates occur in and on practically all cell types
and in cellular secretions. They participate in a multitude of
functions, such as fertilization, development and differentiation,
cell sorting, motility, signal transduction and phagocytosis (I, 1).
The Microciona aggregation factor (MAP) is a highly glycosylated
compound that is involved in such functions. Olycosylation of
carbohydrates proceeds enzymatically by the stepwise addition of
single activated sugars to a growing sugar chain. The products may
consist entirely of sugars, i.e., polysaccharides, or they may be
linked to lipids or proteins. There are two types of protein-sugar
binding structures: O-linked serine or threonine and N-linked
asparagine. Favored N-glycosylation sites possess a characteristic
asn-x-ser/thr tripeptide sequence (3). The protein expressed from
the cDNA that encodes MAF contains many of these tripeptide
motifs (4 1. Moreover, when MAF is treated with specific glycosi-
dases, sugars known to be present in complex N-glycans, i.e.,
mannose, /V-acetylglucosamine (GlcNAc), fucose and galactose
(5) appear as products. Confirmatory studies have demonstrated
GlcNAc, mannose, fucose and galactose lectin binding to sponge
cell membranes (6).

Here we report for the first time that tissues of Microciona
appear to contain an enzyme activity, here named /V-acetyl glu-
cosaminyltransferase I or GnT I, that processes N-linked glycans.
This activity is part of a series of enzymatic events that begin in the
cytoplasm where an oligosaccharide-linked dolichol phosphate is
transported to the lumen of the endoplasmic reticulum whence it
continues to the Golgi. During the process, the sugar is transferred
from the dolichol to a tripeptide (see above) and is modified further

1 Hospital for Sick Children, Toronto. Ontario. Canada.

2 Friedrich Miescher Institute. Basel. Switzerland.

' Department de Bioquimica i Biologia Molecular, Facultat de Farmacia.
Universitat de Barcelona. Barcelona, Spain.

4 Department of Biology, University of Hartford. Hartford, CT.

by processing enzymes (7; Fig. la). N-glycosylation and complex
glycan synthesis can be inhibited by specific compounds such as
tunicamycin and swainsonine (8).

Assays for sponge GnT I were performed using as a source of
enzyme fresh-packed sponge cells extracted in 1% Triton X-100
and buffered in 100 mM Tris at pH 7.4. The substrate was a
synthetic compound, trimannosyl octyl. which has proved useful
as an alternate to natural substrates in which the octyl group is
replaced by two GlcNAc residues (9). The co-substrate and active
glycosyl donor was a 14C-labeled uridine diphosphate A'-acetyl-
glucosoamine (UDP-14C-GlcNAc), with a specific activity of 7.1
mM and 7402 dpm/nmol. Replicate tubes containing these com-
pounds were added to a buffered cocktail that consisted of 25 mM
adenosine monophosphate, 0.5 M A'-acetylglucosamine, 0.2% Tri-
ton X-100. bovine serum albumin 2 mg/ml. 40 mM MnCK. and 0.2
M 2-(N-morpholino) ethanesulfonic acid pH 6.1 in a total volume
of 62.5 /nl. After incubation for I h at 37°C, the reaction was
terminated by the addition of 20 ;ul of 2% sodium borate in 20 mM
EDTA. Separation of unreacted UDP-'4C-GlcNAc from glycosy-
lated radioactive product was accomplished by passing the reac-
tion mixture through an AG1-X8 (BioRad) ion exchange column.
Additional purification of the reaction product could be achieved
by HPLC with CIS matrix that binds the hydrophobic product.
Following a wash with deionized water, the purified radioactive
product was eluted with methanol and the eluate was placed in
vials for determination of counts. The average figures for product
yield, expressed as nanomoles per hour per milligram of protein
(nm/h/mg), were derived from duplicate counts from which
were subtracted nanomoles present in endogenous assays lacking
acceptor.

In a series of assays to determine acceptor-product ratios, the
amount of trimannosyl substrate was increased over a fourfold
range. A straight line relationship was demonstrated over a quan-
tity of acceptor ranging from 2.5 to 10.0 /xl (12.5 to 50 nmol) (Fig.

I'lIYSIOI <><;V AND BlnCHI MISIKY

193

M-M
M-M-M6

\1-Gn-Gn-Asn
Glc-Glc-Glc-M-M-f/S

M-M
v M-M-M6

M-M-M3

M-Gn-Gn-Asn

'M-Gn-Gn-Asn ,
Gn2-M3

M
M-M6

GnT I

M v2

M-M6

M-Gn-Gn-Asn .

M-Gn-Gn-Asn ,

Gn2-M3

GnTII

Gn2-M6

'M-Gn-Gn-Asn

Gn2-M3

A = glucosidases
B = mannosidases
C = mannosidase II
GnT = GlcN Ac-transferase

5 10 15

nmol I mg / h

Figure 1. (a) Biosynthesis of N -linked glycans. Encircled numbers 1-6 designate structural formulae for glycopeptides resulting from sequential
enzyme activities (A-C) and of GnTs (I, II). Briefly, compound I is acted upon by glycosidases (A), which remove glucose (Glc) to produce compound 2.
This is modified by mannosidases (B) to yield the M5 structure (3), which in turn becomes a substrate for steps 4 -5 after the additional removal ofmannose
residues by another mannosidase (C). Compound 6 results from GnT II activity upon the GnT I product. Succeeding structures are enriched, one GlcNAc
at a time, b\ the actions of GnT III. IV. and V. This scheme is practically universal as inferred from many species studied, including lower invertebrates
(7). (Fig. la adapted from ref. 2). (b) The results of trimannosyl substrate-product assays are presented for substrate concentrations ranging from 12.5
to 50 nanomoles. A straight line relationship was observed. The inset (lower right) represents the biosynthelic activity presumed to occur when GnT I
converts trimannosvloctyl substrate into GlcNAc 2-trimannosyloctyl product.

lb). This relationship established GlcNAc-trimannosyl octyl as the
presumed product catalyzed by sponge lysate GnT I activity. The
specificity of this synthetic acceptor substrate has been confirmed
using a wide range of Gn-T I compounds, including cell lysate
from vertebrate and non-vertebrate sources, as well as the cloned
and expressed rabbit, human, and mouse Gn-T I gene product (9).
Final confirmation of the GlcNAc-trimannosyl link requires addi-
tional detailed NMR and spectroscopic analysis of scaled-up pu-
rified product.

The finding of a GnT I activity in the sponge raises the prospect
of manipulating sponge N-glycan structures by the use of specific
GlcNAc enzyme inhibitors. Gray cells, regarded as the immuno-
cytes of the sponge (10), bear the highly N-glycosylated CD44
surface antigen (T. Simpson and W. Kuhns, unpub. datal that may
be an appropriate substrate for such chemical interventions. CD44
is likely to be involved in allograft rejection events (11), and if so,
alterations in its structure by de-N-glycosylation may play a role as
biological response modifiers. In summary. GnT I enzyme activity
appears to function in a manner very much like its counterpart in

higher species. Since the sponges are the most ancient eukaryotes
with a multicellular lineage, the occurrence of cellular GnT I
speaks to its importance in cell functions and to its remarkable
conservation over time.

Literature Cited

Curr. Opin. Struct. Biol. 2:
Glycoproteins and Hu-

1. Drickamer, K., and .). Carver. 1992.
653.

2. Brockhausen, I., and W. J. Kuhns. 1997.

man Disease. R. G. Landes, Austin, TX.

3. Schachter, H., and I. Brockhausen. 1993. Pp. 263-332 in Glyco-
conjugates, Composition, Structure and Function. H. Allen and E.
Kisailus. eds. Marcel Dekker, New York.

4. Jarchow, J., J. Fritz, D. Anselmetti, A. Calabro, V. Hascall. D.
Gerosa, M. Burger, and X. Fernandez-Busquets. 2000. ./ Struct.
Biol. (in press).

5. Misevic, G., J. Finne, and M. Burger. 1987. J. Biol. Chem. 262:
5870-5877.

194

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

d. Kaltenbach, J., K. Hudock, M. Burger, G. Misevic, and W. J.
Kuhns. 1996. Bio/. Bull. 191: 296-297.

7. Kornfeld, S., and R. Kornfeld. 1985. Anim. Rev. Biochem. 54:
631-664.

8. Kaushal, G., and A. Elbein. 1994. Methods En-ymol. 230: 316-
320.

9 Reck, F., M. Springer, H. Paulson, I. Brockhau.sen, M. Sarkar, and
H. Schachter. 1994. Carhohyjr. Res. 259: 93-101

10. Humphreys, T., and E. Reinherz. 1994. Imiminol. Today 15: 316-
320.

11 Fernandez-Busquets, X., and M. Burger. 1999. Microsc. Res.
Tech. 44: 204-218.

Reference: Bio/. Bull. 199: 194-195. (October 2000)

A Ca+2-Independent Cytolytic System from the Blood of the Marine Snail, Busycon caiialiculum

Peter B. Armstrong* and Rengasamy Asokan (Molecular and Cellular Biology,
University of California, Davis. California 95616)

The immune system is the ensemble of cell-based and humoral
agents that protect the organism against parasites that have gained
access to the internal milieu. One of the important immune activ-
ities for a variety of animals is the outright lysis of foreign cells
that contact the blood ( 1 ). Cytolytic systems are frequently ex-
plored as if they were hemolytic systems, where the foreign cell
that is destroyed is the mammalian erythrocyte. The red cell is
convenient for the assay of cytolysis because its hemolysis is
easily quantified by the release of hemoglobin into the bathing
medium. Here we report the presence of a hemolytic system in the
plasma of the marine snail. Busycon.

Blood was obtained as follows. The foot of the adult whelk was
wounded, and the blood flowing from the wound was collected and
then immediately centrifuged to remove the cells. Hemocyanin
was removed from the plasma by centrifugation (40.000 RPM.
4 h). The hemocyanin-free plasma was dialyzed into Tris-buffered
saline (0.15 M NaCl, 50 m/WTris, pH 7.3). Hemolysis was assayed
with rabbit red cells as described previously (2).

The hemolytic activity of hemocyanin-depleted Busycon plasma
is shown in Figure 1 . Hemolysis is progressive, requiring 4 h for
completion. The hemolytic activity is unaffected by the inclusion
of EDTA in the hemolysis assay, and thus is independent of
divalent cations. The lack of a Ca + 2 dependence distinguishes this
hemolytic system from one reported previously in Busycon (3).
Hemolysis is reduced at low ionic strength, showing a broad
activity maximum in buffers containing in excess of 0.2 M NaCl.
The hemolytic activity is inactivated by trypsin treatment of the
plasma, indicating a proteinaceous character to the hemolytic
system. The hemolytic activity of plasma is thermolabile, showing
complete inactivation by treatment of plasma at 40°C for 0.5 h.
The hemolytic protein(s) are retained by a PM30 Amicon filter and
pass through a YM100 filter, indicating a molecular mass between
30 and 100 kDa. The hemocyanin-free plasma shows 13 distinct
protein bands in this interval by SDS-PAGE. with prominent bands
at 78. 55, 43. 35. and 34 kDa.

At least three mechanisms can be envisioned for hemolysis: the
insertion of the hemolytic protein into the lipid phase of the plasma
membrane to create a hydrophilic trans-membrane pore, the enzy-
matic modification of the lipid head groups by a phospholipase,
and the initiation of membrane-lipid phase-transitions by a deter-

1 Molecular and Cellular Biology. University of California, One Shields
Avenue. Davis, CA 95616. (pbarnistrongfe'ucdavis.edu).

gent-like, or a surface-active protein (4). To test the first possibil-
ity, we determined the effects of macromolecular osmolites. The
macromolecular osmolite dextran-4 (Mr 4-6 kDa) reduced hemo-
lysis significantly (from 57.0 ± 2.0% to 8.0 ± 2.3% in one trial).
This suggests that hemolysis in the present system is produced by
insertion of the hemolytic protein into the plasma membrane,
generating hydrophilic channels that allow water to flow into the
cell in response to the high internal concentration of macromolec-
ular osmolites, principally the protein hemoglobin. The presence
of osmolites in the external milieu larger than the channel pore size
(e.g., dextran-4), at concentrations sufficient to balance the os-
motic pressure of hemoglobin in the cell, would protect the cell
from osmotic rupture (5). The molecular size of dextran-4 is
approximately 1.7 nm (6). indicating an effective pore size for the
membrane-associated hemolytic protein as no larger than this
value.

The hemolytic system of Busycon is sensitive to the presence
of lipopolysaccharide (LPS) from the cell wall of gram-negative
bacteria (Fig. IB). The reduction in the hemolytic action at
higher concentrations of LPS may derive from the binding of
the hemolytic agent to this important signature molecule of the
gram-negative bacterium, reflective of an anti-bacterial action

80 n

_ 60 -

40 -

20 -

1 5 dilution of Busycon
plasma in all samples

03 040

Busycon plasma (fraction [Lipopolysaccharide] (mg/ml)
of volume of assay)

Figure 1. Hemolytic activity from the plasma of Busycon canalicula-
tum. Hemocyanin was removed h\ ultracentrifugation, and the supernatant
wax dial\:ed into Tris-hiiffered saline l<>. 15 M NaCl, 51) inM Trix, pH 7.31
Figure I A, dependence of hemolysis on the presence of hemocyanin-free
Busycon plasma. Figure IB, sensitivity of hemolysis to lipopolysaccharide.

PHYSIOLOGY AND BIOCHEMISTRY

195

of the hemolytic agent. Although the concentrations of LPS
used for Figure IB are high, the actual concentration of LPS at
the surface of the bacterium is far higher than the solution
concentrations used in this trial. The possibility that the hemo-
lytic agent does bind to gram-negative bacteria deserves further
investigation.

The ability to destroy foreign cells that come in contact with the
blood is an important defense strategy for a variety of animals. In
mammals, the cytolysis of foreign cells is conducted by the com-
plement system, a multi-component ensemble of plasma proteins
whose membrane attack elements are activated by a proteolytic
cascade that, itself, is initiated by a variety of stimuli indicative of
parasitic invasion (7). The complement system is found only in the
deurostomate animals (i.e., the echinoderms and the chordates) and
is absent from protostomate animals (8, 9). In the latter, the
relatively few cytolytic systems that have been characterized are
less complex than the vertebrate complement system, with some
the province of a single protein that both recognizes and binds to
the foreign cell and mediates its cytolytic destruction (5, 10). Only
a few cytolytic systems have been reported in the plasma of
molluscs (11, 12), and we have not found any reports for gastro-
pods. The systems reported from bivalves (11, 12) are Ca+2
dependent, suggesting that they are based on different effector
molecules than the system described here from Bnsycon.

Supported by Grant No. MCB-97-26771 from the National
Science Foundation.

Literature Cited

1. Canicatti, C. 199(1. Experientia (Basel} 46: 239-244.

2. Swarnakar. S., R. Asokan, J. P. Quigley, and P. B. Armstrong.
200(1. Biticlwm. J. 347: 674-685.

3 Biswas, C.. and P. B. Armstrong. 1999. Biol. Bull. 197: 276-277.

4. Oliver, A. E., F. Tahlin, N. J. Walker, and J. H. Crowe. 1999.
Biochim. fi/Vy./n v Actu 1416: 349-360.

5. Hatakeyama, T., H. Nagatomo, and N. Yamasaki. 1995. ./ Biol.
Client. 270: 3560-3564.

6. Scherrer, R.. and P. (ii-rhardt. 1971. ./. Bucteriol. 107: 718-735.

7. Law, S. K., and K. B. M. Reid. 1988. Complement. IRL Press,
Oxford.

8. Smith, L. C., L. Chang, R. .). Britten, and E. H. Davidson. 1996.
J. IniiiiitiHil. 156: 593-602.

9 Al-Sharif, W. Z., J. O. Sunyer, J. D. Lambris, and L. C. Smith.
1998. J. Immimol. 160: 2983-2997.

10. Armstrong, P. B., S. Swarnakar, S. Srimal, S. Misquith, E. A.
Hahn, R. T. Aimes, and J. P. Quigley. 1996. J. Bioi Chem. 271:
14,717-14.721.

11. Anderson, R. S. 1981. /Vr. Comp. Imiiiunot. 5: 575-585.

12. Yoshino, T., and T.-L. Tuan. 1985. Dev. Camp, liimnniol. 9: 515-
522.

Reference: Biol. Bull. 199: 195-197. (October 2000)

Modulation of the Development of Plutei by Nitric Oxide in the Sea Urchin Arbacia Punctiilata

Diane E. Heck, Lydia Louis. Michael A. Gallo and Jeffrey D. Luskin {Departments of Pharmacology

and Toxicology, Rutgers University, and Environmental and Community Medicine,

UMDNJ Robert Wood Johnson Medical School, Piscatawa\, New Jersev 08854)

Nitric oxide, a reactive free radical, has recently been identified
as a key mediator of intercellular signaling in numerous species
( 1 ). It is produced enzymatically from 1-arginine by the nitric oxide
synthase family of oxidoreductases (2). Nitric oxide regulates a
variety of physiological functions including relaxation of vascular
smooth muscle, long-term potentiation, tumor cell apoptosis, and
cytostasis (3). In addition, inappropriate or excessive production of
nitric oxide has been implicated in tissue injury (4). Nitric oxide is
known to initiate biochemical effects through binding to iron and
iron-sulfur-containing proteins and modulating their activity (5).
Nitric oxide can also modify DNA and is known to alter growth
factor-mediated transcription processes (5). At the present time, no
clear role for this free radical species in regulating development
has been defined.

We have previously reported that nitric oxide synthase inhibi-
tors alter fertilization and differentiation of sea urchin eggs (6). In
the present studies, we examined the direct effects of nitric oxide
on the development of the sea urchin Arbacia punctulata. For these
studies, fertilized eggs were prepared from sea urchins, as de-
scribed by Hinegardner (7), and maintained at 24°C. Embryos

were exposed to S-nitroso-N-acetylpenicillamine (SNAP. Molec-
ular Probes. Eugene, OR I, an agent that spontaneously releases
nitric oxide, for 1 h at various developmental stages. These stages
included: immediately after fertilization, following the first and
fifth divisions, the morula and prism stages, and at two points in
pluteal development, i.e., 24 and 48 h after fertilization. Treated
embryos and untreated controls were further evaluated at regular
intervals for 72 h. Both morphological abnormalities and transient
delays in development were observed, but particular effects were
dependent on the developmental stage at the time of treatment.
Morphological abnormalities were quantified at the morula. prism,
and pluteus stages.

In initial experiments, we determined the effects of a range of
concentrations of SNAP (2 nM-2 /J.M) applied to sea urchin eggs
immediately after fertilization. Embryo mortality with little cell
division was observed when embryos were treated with concen-
trations of SNAP in excess of 200 nM. However, both transient
and permanent developmental changes were found after treatment
with lower concentrations of the nitric oxide releasing agent (see
further below). For our studies, we used 20 nM SNAP; at this

196

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

concentration, prominent morphological alterations occurred with-
out significant toxicity.

Developmental lesions are often induced by temporally re-
stricted biochemical changes that eliminate critical downstream
events or cause them to occur inappropriately. The resulting ab-
errant development reflects a stage-specific sensitivity to a phar-
macological agent or event. To determine the developmental
stages at which sea urchin embryos are sensitive to nitric oxide, we
next exposed fertilized eggs to SNAP during various stages of
development. Following fertilization, sea urchin eggs divide syn-
chronously until the fifth division. At this stage, the embryo is
designated a morula, and the uniformly distributed pigment gran-
ules become unevenly distributed to specific cells, with most going
to the 8 macromere cells. When SNAP was applied after fertili-
zation or first division, we observed no morphological alterations
in the morula. Both drug-treated embryos and controls contained
2-4 darkly pigmented cells.

Sea urchin embryos hatch and become free-swimming shortly
after the initiation of motility. Later, about 18 h after fertilization,
the free-swimming sea urchin embryos become pyramidal, and
enter the prism stage. When such embryos were exposed to SNAP,
no anatomical aberrations were observed. However, embryos
treated with SNAP at any stage prior to hatching exhibited delays
in development (Table 1 ). Thus. 18 h after fertilization, embryos
treated with SNAP prior to the morula stage had not progressed
beyond the blastula stage. In addition, embryos exposed to SNAP
before hatching contained fewer than the 6-8 darkly pigmented
cells observed in controls.

Developing sea urchins generally progress to the pluteus by
about 24 h after fertilization. At this stage, plutei that had been
treated with SNAP immediately after fertilization, or the first
division, exhibited gross morphological alterations. Additionally,
the pigment cells aggregated and were fewer in number (Table 1;
Fig. 1. compare panels A and B; other data not shown). This effect
was associated with fewer animals progressing through develop-

Tahlo 1

Changes in \CLI urchin embryo development in response to treatment
with SNAP

Developmental stage 24 hours after
fertilization (embryos/50 fj.1)

Developmental stage at
treatment

Morula

Prism

Pluteus

Untreated

1 ± 2.3

0 ± 0.6

1 3 ± 3.9

Following fertilization1

24 ± o.l

7 ± 3.2

0 ± 0.3

First Division

27 ± 0.6

14 ± 2.3

1 ± 3.3

Division 5

19 ± 4.0

10 ± 1.4

0 ± 3.4

Morula

1 ± 4.3

4 ± OS

9 ± 2.3

Prism

0 ± 3.4

0 ± 0.9

2 1 ± 4.5

Pluteus

2 ± 3.3

1 ± 0.3

13 ± 2.6

' Approximately I05 embryos were suspended in 10 ml of sea water
supplemented with 20 nA/ SNAP at the indicated developmental stages.
Alter 1 h. embryos were transferred into 100 ml of SNAP-free sea water.
Embryos were then evaluated for progress through development at 24 h
after fertilization. Each point represents the average number of embryos in
cadi stage ot development tor 5 experiments ± SEM.

Figure 1. Effect of nitric o\idc mi the development of sen urchin eggs.
Immediately following fertilization, sea urchin eggx were treated with 20
nM SNAP. After 1 h llie embryos were washed and a/lowed to develop.
Punch A and B are sea urchin embryos 24 h after fertilisation; panels C
and D show embryos 48 h after fertilization. Panels A and C are control
1'inhrvos: panels B and D are embryos that have been treated with SNAP.

merit. Those that did develop further exhibited diminished arm
extension when evaluated 48 h after fertilization (Fig. 1. compare
panels C and D and not shown). As these plutei continued to
develop, the poorly extended arms were often oriented 90° to the
longitudinal axis rather than parallel to it. Aberrant morphology
was less pronounced in embryos exposed to nitric oxide after
division 5. In addition, when evaluated 24 h after fertilization, the
number of pigment cells remained unchanged in embryos treated
with SNAP before division 5, while about 4 times as many darkly
pigmented cells were identified in untreated embryos.

When observed 24 h after fertilization, delayed development
was again evident in embryos treated with SNAP at all stages prior
to hatching. However, following exposure to SNAP at the morula
stage, embryos appeared normal in development by 48-72 h after
fertilization. No effects on morphology or development were ob-
served in embryos exposed to nitric oxide at the prism stage or as
plutei either 24 or 48 h after fertilization (not shown). Anatomical
changes resulting from exposure to nitric oxide were preceded by
an apparent inhibition of pigment cell division. These results
indicate that sea urchin embryos exposed to nitric oxide before
their development into morula show permanent morphological
changes, whereas exposure at later stages has no apparent perma-
nent effects.

Taken together, our results demonstrate that brief exposure of
sea urchin embryos to nitric oxide during early development
causes irreversible abnormalities in plutei. including skeletal

PHYSIOLOGY AND BIOCHEMISTRY

197

aberrations and changes in the proliferation and migration of
pigment cells. The effects of nitric oxide applied at later stages are
reversible. We speculate that nitric oxide may act as a negative
regulator of pigment cell division and skeletal extension in vivo.

Literature Cited

I Hohbs, A. J., A. Higgs, and S. Moncada. 1999. Anini. Rev. Phur-
imicol. To.vicol. 39: 191-220.

2. Hemmens. B., and B. Mayer. 1998. Methods Mol. Bint. 100: 1-32.

3. Moncada, S. 1999. ./. R. Soc. Meil. 92: 164-169.

4. Laskin, J. D., D. E. Heck, and D. L. Laskin. 1994. Tn-mh Emln-
crinol. Melah. 5: 377-3S2.

5. Lane, P., and S. Gross. 1999. Semni. Nephrol. 19: 215-229.

6. Heck, D. E., J. I). Laskin, S. Zigman, and W. Troll. 1994. Biol.
Bull. 187: 24S-249.

7. Hindgardner. R. 1975. Pp. 10-25 in The Sea Urchin Emhr\<>.
Biochenn\ir\ unJ .\l<tr/tlioi;i'm:\i*, G. Czihak. ed. Springer-Verlag.
Berlin.

Reference: Biol. Bull. 199: 197-198. (October 2000)

Development of Self-Referencing Oxygen Microsensor and its Application to Single Pancreatic
HIT Cells: Effects of Adenylate Cyclase Activator Forskolin on Oxygen Consumption

Sung-Kwon Jung*. Katherine Hanimar. and Peter J. S. Smith (BioCitrrcnts Research Center.
Marine Biological Laboratoiy. Woods Hole, Massachusetts 02543)

Information about oxygen dynamics is valuable for evaluating
the metabolic activity of pancreatic ft cells, which contribute to the
regulation of glucose homeostasis in the blood. When glucose, the
key secretagogue. is metabolized in ft cells, intracellular messen-
gers such as ATP are formed. ATP-sensitive potassium channels
(KATP) then close, which depolarizes the cell, leading to Ca2 +
entry and stimulation of insulin secretion by exocytosis (1.2). The
relationships between metabolism, oxygen consumption, insulin
secretion, and other biochemical changes in pancreatic islets have
been examined (3-5). In the present investigation, we focused on
the relationship between altered cAMP levels and oxygen con-
sumption. According to Ammala el ai. insulin release by intra-
cellular cAMP is calcium-independent (6). But exocytosis is met-
abolically driven, and intracellular calcium levels are tightly
correlated with oxygen consumption (3). Therefore, we need to
study the oxygen dynamics of ft cells, but at the level of a single
cell, because pancreatic islets are composed of four distinct cell
types. However, such studies with single ft cells are very difficult,
primarily because single cells generate very small changes in
oxygen level during respiration. Often, therefore, the electrochem-
ical drift of an oxygen sensor dwarfs such small changes, so
interpretation of the data is difficult. We have now developed a
self-referencing oxygen microsensor with a tip diameter < 3 /urn
and have measured the oxygen consumption of single pancreatic
HIT cells.

The self-referencing technique has been successfully used to
measure selected ion and oxygen fluxes at single cell levels by
minimizing the impact of sensor drift and noise (7. 8). The tech-
nique is based on the translational movements of a microprobe
between two points in the concentration gradient that extends
outward from the cell membrane. The difference in the values
measured at two points provides information about the gradient.
The oxygen sensor used in the current study was designed to have
the characteristics required for the self-referencing technique, such

* Corresponding author: sjung@mbl.edu

as minimal convection perturbation and a response time <1 s. The
sensor has an etched Pt wire electrochemically recessed inside
a pulled glass micropipette. which was coated with cellulose
acetate (9).

To validate the applicability of the self-referencing oxygen
sensor to flux measurements, an oxygen gradient was artificially
generated and measured (Fig. 1 A). The apparent oscillation of the
DC trace is the result of sensor relocations in the oxygen gradient.
The translational frequency was optimized for maximal sensitivity
(0.2 Hz). For example, as observed in Figure 1 A. the signal rapidly
equilibrated as the sensor was repeatedly brought to a new position
in the self-referencing mode compared to the stationary mode.
lonView (a product of BioCurrents Research Center. Marine Bio-
logical Laboratory. Woods Hole. MA) automatically subtracts
current values at the far position from those at the near position.
The first half of the values at each position were discarded to
eliminate the artifacts from sensor motion and response time; the
second half were sampled for mathematical subtraction (7). The
resulting data, designated as AC. are used for calculating oxygen
flux according to the Pick equation:

J = -DjACYAr). (1)

where J is flux (mol cm 2 s '), D,, is the diffusion coefficient of
oxygen (2.6 X 10"3 cm2 s"1), and (AC/Ar) is the concentration
difference divided by the translational distance (i.e.. the concen-
tration gradient).

We applied the self-referencing oxygen sensor to single pancre-
atic HIT cells. Oxygen consumption was significantly decreased
upon the injection of rotenone, an inhibitor of mitochondria! res-
piration (Fig. IB). Based on equation 1, the oxygen flux with a Ac
of 30 joim at 25 /Am from the plasma membrane of single cells was
640 ± 40 fmol/cnr/s (mean ± SEM. n = 5) at 1 1 mM glucose,
which is then converted into a consumption (3.2 fmol/cell/min) by
taking the geometric surface area into account. After rotenone
injection, the oxygen flux significantly decreased to 97 ± 15
fmol/cnr/s (mean ± SEM, n = 5), a consumption of 0.4 fmol/

198

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

•40 i

stationary self referencing mode

200 300

time (s)

40 -

30 -

$ 20-

3 10
0

-10

10 |jM rotenone

11 mM glucose

0 100 200 300 400 500 600

time (s)

-" 10

10 nM forskolin

11 mM glucose

100

200

time (s)

300

400

Figure 1. I A) Real time trace (DO unit lonView processed trace (AC)
of oxygen current as a function of distance from an oxygen source (diam-
eter - 10 /nw) measured with a stationary and a self-referencing oxygen
microsensor. In the self-referencing mode, the oxygen sensor was oscil-
lated within a Ill-pun i:\cnrsion a! a frequency of 0.2 Hz. The numbers are
the relative distance from the oxygen source in microns. (B) Influence o)
rotenone on oxygen consumption by a single pancreatic HIT cell. The mice
is a representative of five measurements. (C) Influence of forsknlin on
oxygen consumption hy a single pancreatic HIT cell. The trace is a
representative of four measurements. There is a significant difference in
oxygen consumption after forskolin treatment (P < 0.01). In the above
single cell measurements, the oxygen sensor was positioned 10 u.m from
the plasma membrane of the cell and oscillated between 10 and 40 n/n ui
30-fjjn excursion! at a frequency of 0.2 Hz. A potential of -600 mV vs.
Ag/AgCI iri/.s applied to the o\\gen sensor in a modified HEPES buffer at
37°C for all experiments.

cell/min (P < 0.001 according to two-tailed Student's / tests).
This decrease ( — 88%) is presumably due to the inhibition of
NADH-dependent respiration. We injected forskolin, a specific
activator of adenylate cyclase, to elevate cAMP level and observed
a flux of 0.76 ± 0.07 prnol/cnr/s (mean ± SEM, n =: 4), u
consumption of 4.0 fmol/cell/min (see Fig. 1C). The oxygen con-

sumption after forskolin treatment was significantly higher than
before (P < 0.01 ). This increase (-19%) may account for the
ATP demand in insulin secretion.

Calcium ions may have a role in the response of HIT cells to
adenylate cyclase activation. Oxygen dynamics and intracellular
Ca2 ' ([Ca2+],) are well correlated in islet cells, in that increases in
oxygen consumption closely follow increases in [Ca2+],. accord-
ing to the observation by Jung el al. (3). The activation of ade-
nylate cyclase will lead to a decrease in ATP levels unless ATP is
rapidly replenished. Therefore, we postulate that the forskolin-
induced increase in oxygen consumption reflects the release of
Ca2 + from subcellular organelles, a notion supported by the find-
ing that blockage of sarco-endoplasmic reticulum Ca2+-ATPase
(SERCA) pumps by thapsigargin abolishes transient increase in
[Ca2 + ], induced by forskolin (10). On the other hand. cAMP could
directly deinhibit the activity of phosphofructokinase, a controlling
enzyme of glycolysis. resulting in NADH elevation and then an
increase in oxygen consumption. The above intertwined relation-
ships could be explained by a recent report suggesting that, while
Ca2+ may activate respiration, its entry into /3 cells has a net effect
of decreasing the ATP/ADP ratio resulting from the activation of
numerous ATP consuming processes, such as insulin secretion and
ion-pumping (11).

In conclusion, the development of a self-referencing oxygen
microsensor and its application to single HIT cells hold promise
for the elucidation of the relationship between oxygen dynamics
and the role of cAMP in exocytosis. Further use of the self-
referencing oxygen sensor will include correlated measurements
with [Ca2 + ],, ATP/ADP and insulin secretion at the single cell
level.

We thank Richard Sanger for technical assistance. This work
was supported by the NIH Grant NCRR P41 RR01395 to PJS
Smith.

Literature Cited

1 Asfari, M., D. Janjic. P. Meda, G. Li, P. A. Halban. and C. B.
Wollheim. 1992. Emlocrinol. 130: 167-178.

2. Bergsten, P., E. Grapengiesser, E. Gylfe, A. Tengholm, and B.
Hellman. 1994. J. Biol Client. 269: 8749-8753.

3. Jung, S.-K., L. M. Kauri, W.-J. Qian, and R. T. Kennedy. 2000.
J. Biol. Chem. 275: 6642-6651).

4. Corkey, B. E., K. Tornheim, J. T. Deeney, M. C. Glennon, J. C.
Parker, F. M. Matschinsky, N. B. Ruderman, and M. Prentki.
1988. J. Biol. Chem. 263: 4254-4258.

5 Larsson, O., H. Kindmark, R. Branstrom, B. Fredholm, and P.-O.
Berggren. 1996. Proc. Nail. Acad. Sci. USA 93: 5161-5165.

6 Ammala. C., F. M. Ashcroft, and P. Rorsman. 1993. Nature 363:
356-358.

7. Smith, P. J. S.. K. Hamniar. D. M. Porterfield, R. H. Sanger. and
J. R. Trimarchi. 1999. Microsc. Res. Tech. 46: 398-417.

8. Land, S. C., D. M. Porterfield, R. H. Sanger, and P. J. S. Smith.
1999. J. Exp. Biol. 202: 211-218.

9. Jung, S.-K., YV. Gorski, C. A. Aspinwall, L. M. Kauri, and R. T.
Kennedy. 1999. Anal. Chem. 71: 3642-3649.

10. Liu. Y.-.J., E. Grapengiesser, E. Gylfe, and B. Hellman. 1996.
Arch. Biochem. Biophvs. 334: 295-302.

1 1 . Detimary, P., P. Gilon. and J. C. Henquin. 1998. Biochem. J. 333:
269-274.

PHYSIOLOGY AND BIOCHEMISTRY

Reference: Bio/. Bull. 1«W: 149-200. (October 20(10)

Identification of Proliferating Cells in Hard Clams

Rhea Hanse liiiann ' . Ro.\anna Siuolowitz (Marine Biological Laboratory, Woods Hole, Massachusetts),

and Daniel Gibson?

The origin of hemocytes, the circulating "blood cells" of bivalve
molluscs, including hard clams (Mercenaria mercenaria} has not
been identified (1,2). Proliferation of hemocytes. however, can be
recognized through their increased numbers in diseased animals.
Proliferating cell nuclear antigen (PCNA), or cyclin, is a protein
produced during the late Gl and S phases of the cell cycle (3. 4).
Using antibodies that recognize PCNA in mice, we attempted to
identify the origin of hemocytes in the hard shell clams. Quahog
parasite unknown (QPX) is a protist that causes severe inflamma-
tion and mortality in infected clams (5. 6). We attempted to induce
hemocyte proliferation by exposing clams to QPX in a 10-1 water
column in which 12 ml of undiluted QPX culture (at a concentra-
tion of 7*106 cells/ml) were added every 10 days; by injecting
QPX between the membranous mantles and the right valves, 3 cm
ventral anterior to the siphon and into the pericardial cavities (0.25
ml of undiluted QPX culture): and by injecting an inert particle
(India ink. 1:10 dilution in sterile seawater [7, 8]) into the peri-
cardial cavities. The controls consisted of two groups of clams.
One group was injected with sterile seawater in the pericardial
cavities: the other was untreated. Groups were sampled at 24 h.

Worcester Polytechnic Institute. Worcester. MA.

and at 1.4. and 8 weeks after the start of the experiment. At
sampling, the animals were shucked, fixed in 10% neutral buffered
formalin (NBF) for 24 h. and embedded in paraffin. Sections were
cut (4-6 /xm), mounted onto positively charged slides (Fisher-
brand, Superfrost/Plus and ProbeOn Plus slides) and stained either
with hematoxylin and eosin (H&E) (9) or with anti-PCNA with a
hematoxylin counterstain (Zymed, PCNA Staining Kit).

Clams injected with QPX in the pericardial cavities showed
mild focal inflammation associated with viable and necrotic QPX
organisms. At 2 months post-injection, viable QPX organisms
were no longer identified. QPX organisms and associated inflam-
mation were not observed in clams injected in the mantle cavity.
After 2 months of water column exposure, only very rare infection
by QPX organs with minimal inflammation was observed in man-
tle tissue. India ink injection caused a minimal inflammatory
response. Pools of injected ink in the tissues and vascular spaces
were either engulfed by individual hemocytes or surrounded and
sequestered by hemocytes (encapsulation), forming thin- walled
granulomas (6, 10). Numerous individual hemocytes containing
India ink were eliminated from the clams by diapedesis over
lumenal epithelial surfaces (Fig. 1A). Thick- walled granulomas (6,
10) were also identified in the gills, pericardial sacs, and other

*' ^<

£} ^*S-'

' A *'.

Figure 1. Proliferating epithelial cell.', ami hemoc\tes «/ Mercenanu mercenaria stained with anti-Mouse Proliferating Cell Nuclear Antigen (PCNA).
(A) India ink is phagocytized and eliminated by diapedesis of India ink-filled hemocytes (urrou ; into the renal tubular lumens (H&E. bar = 13 tun). (B)
Nuclei of proliferating resenv cells of the digestive gland's tubular epithelia stained black furrow) with anti-PCNA. PCNA negative nuclei stain blue
(arrowhead) (hematoxylin counterstain. bar = 13 tun). (C) Strong PCNA nuclear staining is present in the proliferating nuclear epithelial cells at the buse
of the gills (arrows). PCNA negative nuclei stain blue (arrowhead) (hematox\lin counterstain. bar = 13 /j.m). (D) Nuclei of some hemocytcs in thick-walled
granulomas in the kidney tissue of clams are positive for PCNA stain (arrows) and present evidence for the proliferation of hemocytes directly at the
inflammatory site (hematoxylin counterstain. bar = 13 (Mm).

200

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

organs of saline-injected animals, indicating that the injection may
not have been sterile.

Using the anti-mouse PCNA, areas of abundant PCNA staining
(black-stained nuclei), indicating areas of marked cell prolifera-
tion, were identified in the reserve cells of the digestive gland (Fig.
IB), the proliferate epithelial cells of the gill base (2) (Fig. 1C),
and in early proliferative phases of reproductive epithelium. As
expected, cells of other tissues throughout the body also stained
positive for PCNA (i.e.. epithelium of the intestine, foot and body
wall, and gill plical epithelium), but in much lower numbers
Proliferating hemocytes were identified in the inflammatory cells
forming the thick-walled granulomas (Fig. ID) and rarely in ad-
jacent non-inflammatory cells. In no other areas examined were
proliferating hemocytes identified.

These results demonstrate that the epitopes associated with
PCNA are conserved between the clam and the mouse, as shown
by the positive staining of known proliferative cells in the clam
body. Previous studies have shown that hemocytes appear to
migrate to areas of infection in bivalves (2, 6). In diseased bi-
valves, hemocyte numbers appear to increase; the site of origin of
these hemocytes has never been determined (2). This study pro-
vides evidence that the hemocytes of the hard clam proliferate
directly at the inflammatory site, as opposed to a possible bone
marrow-like area in the body of the clam, with subsequent migra-
tion of hemocytes to sites of infections, as seen in vertebrates.

This work was supported, in part, by a NOAA Sea Grant Award
to the Woods Hole Oceanographic Institution Sea Grant Program,
Grant No. NA86RG0075. Project Number R/A-39.

Literature Cited

1. Moore. C. A., and A. F. Eble. 1977. Biol. Bull. 152: 105-1 19.

2. Cheng, T. C. 1996. Pp. 631-642 and 299-333 in The Eastern
Oyster, Crassostreu virginica. V. S. Kennedy, R. I. E. Newell, and
A. F. Eble. eds. Maryland Sea Grant College Publication UM-SG-TS-
96-0 1 .

3 Waseem, N. H.. and D. P. Lane. 1990. J. Cell. Sci. 96: 121

4. Ogata, K., Y. Ogata, R. M. Nakamura, and E. M. Tan. 1985.
J. Immunoi 135: 2623.

5. Kleinschuster, S. J., R. Smolowitz, and J. Parent. 1998. J. Slwll-
,/H/I ffc.v. 17: 75-78.

6. Smolowitz, R.. D. Leavitt, and Frank Perkins. 1998. J. Invertebr.
Patlwl. 71: 9-25.

7. Stauber, L. A. 1950. Biol. Bull. 98: 227-241.

8. Tripp, M. R. 1960. Biol. Bull. 119: 273-282.

9. Humanson, G. L. 1997. Pp. 101-1 18 in Htimanson's Animal Tissue
Techniques, 5th ed. J. K. Presnell and M. P. Schreibman, eds. The John
Hopkins University Press. Baltimore. MD.

10. Eble, A. F.. and R. Scro. 1996. Pp. 25-30 in The Eastern Oyster.
Crassostrea virginica. V. S. Kennedy. R. I. E. Newell, and A. F. Eble.
eds. Maryland Sea Grant College Publication UM-SG-TS-96-01.

Reference: Biol. Bull. 199: 200-201. (October 2000)

Conditions Affecting the Growth and Zoosporulation of the Protistan Parasite QPX in Culture

Christine Brothers1, Ernest Marks III2, and Roxanna Smolowitz
(Marine Biological Laboraton; Woods Hole. Massachusetts 02543)

Quahog Parasite Unknown (QPX) is a protistan disease of hard
clams (Mercenaria mercenaria). The QPX organism has been
classified in the phylum Labyrinthomorpha (1. 2). Disease result-
ing from QPX infection has been identified in New Brunswick and
Prince Edward Island, Canada; Barnegat Bay, New Jersey;
Chatham. Duxbury. and Provincetown, Massachusetts; and three
locations in Virginia (1, 2). Mortality from QPX can be severe,
with losses especially high in clams just under market size (about
2 years old). An important clinical sign of infection is the occur-
rence of QPX-infected inflammatory nodules in the mantle.

Whyte et al. (3) isolated QPX cells from infected clams; when
placed in artificial seawater, these cells produced sporangia and
zoospores. Kleinschuster and Smolowitz (2) recently described
continuous in vitro culture of QPX. QPX was isolated from in-
flamed mantle nodules and cultured in modified MEM medium at
pH 7.2 at 22°C. Mature cultures (after 5 to 10 days) showed thalli.
immature sporangia, and mature sporangia containing endospores.
Organisms in these stages ranged from 5 to 120 juni in diameter.

1 Falmouth High School. X74 Gifford St.. Falmouth. Massachusetts
02540.

: Barnstable County AmeriCorps Cape Cod. PO Box 427. Barnstable.
MA 02630.

Endospores released from mature sporangia became the new thalli.
Cultured QPX organisms produced, and were embedded in, a thick
mucoid material that could be removed intact from the remaining
unused culture medium. When placed into sterile seawater, QPX
produced motile zoospores within 4 days.

The effects of different environmental conditions on the occur-
rence of QPX and the resulting disease in the field are unknown.
Determination of how environmental parameters affect cultured
QPX may help in understanding the pathogenesis of the disease in
the field. In this study, the environmental effects of temperature,
pH. and salinity were investigated on QPX cells in culture.

Medium (pH 7.2 and salinity 40 ppt) was prepared using the
standard methods (2). Modified medium (40 ppt) was prepared at
pH 6.0, 7.0, and 8.0 by adjusting pH with 2 M HC1 and 2 M NaOH.
Modified medium (pH 7.2) was also prepared at 20, 28, and 34 ppt
by proportionally reducing the salt content of the medium and
monitoring the resulting solutions with a refractometer. All media
were filter sterilized. To test the effects of pH and salinity on the
proliferation of QPX in culture, 0.4 ml of two QPX subcultures
was placed in a culture flask with 10 ml of each of the three pH
variations or four (including standard) salinity variations. Fourteen
cultures were created (seven of each subculture). These cultures
were incubated for 10 days at 22°C. To test the effects of temper-
ature on QPX growth in culture, the same procedure was followed

PHYSIOLOGY AND BIOCHEMISTRY

201

6.8
6.6
6.4
6.2
6

5.8
5.6
5.4

•I

3 E

1
0

7
pH

- million ceils/ml

-ml of mucus

28 34

salinity ppt

- million cells/ml

-ml of mucus

10 16 24 32
temperature degrees C

- million cells/ml

-ml of mucus

Figure 1. Growth of QPX as a function of pH, salinity, and temper-
ature as measured by cell count (millions of cells per mil and rolnmc of
mucoid containing QPX material produced (ml of mucus).

ganisms were measured by counting the number of cells per
milliliter of a 1:10 saline dilution of the mucus, using a hemocy-
tometer. Initial concentrations of QPX averaged 1.4 million cells
per milliliter (range = 1.1 to 1.8 million). Final concentrations and
volumes are shown in Figure 1 .

QPX concentrations per milliliter and volume of mucus pro-
duced both increased with increasing pH and increasing salinity. In
culture, the modified MEM medium becomes more acidic as the
culture matures. This lower pH may inhibit further growth of the
culture. At low salinities. QPX thalli have previously been ob-
served lysing, which may explain the lower cell concentrations at
20 ppt. The cell concentration was highest at 0°C and decreased
with increasing temperature. This may represent a thinning out and
spreading of the mucus containing the cells with increasing tem-
perature. However, the total number of QPX organisms and total
mucus production was greatest at 24°C. The volume of mucus
containing QPX produced was low from 0° to 16°C, peaked at
24°C. then declined with increasing temperature. Above 32°C.
there was no growth of QPX and no mucus production. Whether
QPX will grow above pH 8.0 and above 40 ppt should be inves-
tigated.

Proliferation of cultured QPX is best at a temperature of 24°C.
pH 7 to 8. and salinities of 28 ppt and above. Such findings are
consistent with the field observation of increased infection in the
summer and occurrence of QPX disease primarily in high-salinity
waters.

Conditions affecting the zoosporulation of QPX were also in-
vestigated. Seawater (pH 8.0, salinity 30 ppt) was adjusted to pHs
of 6.0, 7.0. and 9.0 (using 2 M HC1 and 2 M NaOH) and to
salinities of 20 and 40 ppt (by dilution with distilled water or
addition of NaCl). then filter sterilized. Concentrations of 1% and
10% QPX in seawater of each pH and salinity were placed in
replicates in a 24-well sterile plate. The plates were incubated at
10°. 16°. 22°, 32°, and 38°C and examined daily for 5 days.

Other researchers (2. 3) have reported zoosporulation in QPX:
however, there is reason to believe these may not have been from
pure cultures of QPX. This study attempted to replicate those
findings; however, no zoospores were observed in repeated trials,
even in normal sterile seawater. Whether in fact QPX produces
zoospores, as not all members of the phylum do, and under what
conditions it does so are important both in the further classification
of the organism and in studying transmission of the disease.
Therefore, this question deserves further investigation.

This work was supported, in part, by a NOAA Sea Grant Award
to the Woods Hole Oceanographic Institution Sea Grant Program.
Grant No. NA86RG0075, project number R/A-39.

using 10 ml of standard medium to create a total of 12 cultures.
One flask of each subculture was incubated at six temperatures (0°,
10°, 16°, 22°, 32°. 38°C) for 10 days.

Concentrations of the mucus containing QPX from each of the
four initial subcultures, as well as from the 26 experimental cul-
tures at the end of the 10-day incubation, were determined. The
QPX-containing mucus was extracted from the culture medium
and measured using a 10-ml pipette. Concentrations of QPX or-

Literature Cited

1. Smolowitz, R., D. Leavitt, and F. Perkins. 1998. J. Invert. Path. 71:
9-25.

2. Kleinschuster. S. J., R. Smolowitz, and J. Parent. 1998. J. Slicllp-.li
Res. 17: 75-78.

3 Whyte, S. K., R. J. Ca« thorn, and S. E. McGladdery. 1994. Dis.
Aquat. Org. 19: 129-136.

202 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

Reference: Biol. Bull 199: 202-203. (October 2000)

Effects of Myosin-II Antibody on Actin-Dependent Vesicle Transport in Extracts of Clam Oocytes

Leslie Sandberg, Phillip Stafford and George M. Langford
(Dartmouth College, Hanover, New Hampshire 03755)

The movement of vesicles and organelles in cells has been
shown to occur on both microtubules and actin filaments (1. 2).
The transport of vesicles on microtubules permits movement over
long distances in cells, while the short-range transport of \esicles
on actin filaments produces the fine and precise movements re-
quired to position vesicles at membrane sites for capture, docking,
or fusion (3. 4). The transport of vesicles on actin filaments
requires vesicle-associated myosins. some of which have been
identified. Myosin V, for example, transports endoplasmic reticu-
lum (ER) vesicles (5-7) and synaptic vesicles (8) in neurons and is
involved in the capture of melanosomes at the tips of mouse
melanocytes (9). In addition, myosin V is involved in the transport
of lysosomes in some cells (10), secretory vesicles in yeast (II).
and melanosomes in Xenopus melanophores (12).

Myosin II, also known as conventional myosin. is vesicle-
associated in extracts of clam (Spisula solidissima) oocytes (4. 13).
In addition, myosin II was reported to be associated with Golgi
vesicles (14), but the antibody used in these studies cross-reacts
with coatomer proteins on Golgi-derived vesicles ( 15-17). There-
fore, the role of myosin II in vesicle transport remains controver-
sial. In this study we performed antibody-inhibition experiments
with extracts of clam oocytes to provide additional evidence that
myosin II is a vesicle motor. Clam oocyte extracts are well
characterized and have been used extensively to study cell cycle

events including cyclin synthesis and degradation, as well as
ubiquitin-mediuted proteolysis of cyclins (18).

Extracts were prepared from quiescent oocytes by the method
of Ruderman et a/.. (18) with slight modifications. Fluorescence
microscopy was used to view rhodamine-phalloidin stained
actin filaments in the extracts, and AVEC-DIC microscopy was
used to assay vesicle transport on actin filaments. Motile activ-
ity in extracts was determined by counting the number of
vesicles moving per minute per field (v/m/f). Antibody-inhibi-
tion experiments were performed with an antibody made to
oocyte myosin II. The myosin II in oocyte extracts was precip-
itated with ammonium sulfate, dissolved in buffer (20 mM Tris
pH 7.2, 1 mM EDTA-K, 0.2 mM Cad,, 1 mM DTT), and
dialyzed overnight at 4°C. The pure fraction of the myosin II
was prepared by running the solubilized fractions on SDS-gels
and eluting the myosin II heavy chain band. The eluted protein
was used for antibody production in rabbit. The serum of the
immunized rabbit was collected, and the polyclonal antibody
(ap-205) was purified on protein A beads.

The antibody (ap-205) recognized a single band at 205 kD on
western blots of oocyte extracts (Fig. 1A) and in myosin II-
enriched fractions (Fig. IB). The protein recogni/.ed by ap-205
was also recognized by a myosin II antibody. M2.42 (Fig. 1A),
produced to a peptide in the head domain of Acanthamoeba

^pp^^^^^

Figure 1. Panel A: Lane I i\ a i oomassie blue Gained SOS-PAGE gel of an oocvtc extract. Lane 2 i.\ a western hint of the same i:\truct probed with
M2.42. a myosin II antibody, showing a single hand nt 2115 kit. Lane _•> is a western hlot probed with ap-205 showing « single hand at 21)5 kD. Panel B:
Lane I is cooinassie blue stained SDS-PAGE gel showing a myosin H-enriched fraction prepared from clam oocytes. Lane 2 is the corresponding western
hint of the fraction probed with ap-205 and showing a single hand at 205 kD. Panel C: Lane I is a gel of protein-A purified ap-205 showing til least four
hands: the antibody at 55 kl~> and ihiee sennn proteins that co-pnrif\ with the antibods. Lane 2 shows an immunoprecipitation with ap-205. Six proteins
are seen: the /mo- serum proteins, msosin II at 205 kD. and actin at 45 kD. Lane 3 is the corresponding western hlot probed with ap-205: the antibod\
ret t>gni~cs the four serum proteins unil ms'osiu 11

CELL BIOLOGY

203

myosin II (gift of Donald Kaiser). Therefore, the ap-205 antibody
exhibited high affinity and specificity for clam myosin II. In
immuno-precipitation (IP) experiments with ap-205, proteins of
205 and 45 kD. the respective molecular weights of myosin II and
actin, were present in the precipitate (Fig. 1C, lane 2). A blot of the
ap-205 immune-precipitate probed with ap-205 (Fig. 1C. lane 3)
identified the 205 kD band as myosin II, and a blot with an
antibody to actin identified the 45 kD band as actin (data not
shown). The 4 additional bands seen on both the gel and blot were
present in the protein A-purified antibody (Fig. 1C. lane 1 ); there-
fore, they represent serum proteins rather than proteins in the
oocyte extracts. The IP data provided evidence that the antibody
binds to native myosin II and may serve as a function-blocking
antibody. We therefore examined the effects of the ap-205 anti-
body on actin-dependent vesicle transport.

The protein-A purified ap-205 antibody was concentrated to 3
mg/ml and buffer-exchanged into vesicle motility buffer (T buffer.
pH 7.2) for antibody-inhibition experiments. An antibody
(aQLLQ) made to squid myosin V (5) that does not detect oocyte
proteins on western blots was used as the control. Extracts were
prepared for motility assays with either 0.38 or 1.0 mg/ml of
ap-205, and a control sample was prepared at the same time with
aQLLQ. The motile activity was determined in the control and the
treated samples at regular intervals for a period of 1.5 hours.
Motile activity in the control remained at 412 ± 96 vesicles/
minute/field (v/m/f) for the observation period, while the extracts
treated with 0.38 mg/ml ap-205 decreased to 232 ± 5 1 v/m/f (43%
inhibition). At 1.0 mg/ml ap-205, motile activity decreased to
199 ± 58 v/m/f. while the control remained at 363 ± 71 v/m/f
(45% inhibition). These data showed that ap-205 inhibited 40% to
45% of the motile activity in clam oocyte extracts. We plan to use
affinity-purified ap-205 to determine whether motile activity is
inhibited completely.

In summary, the inhibition of vesicle transport by a myosin
II-specific antibody provides evidence in support of the conclusion
that myosin II in clam oocytes functions as a vesicle motor. The
lack of 100% inhibition by ap-205 may suggest the involvement of
other myosin motors in actin-based vesicle transport in oocytes.
The vesicles in these extracts are probably ER-derived. and myosin

II may therefore be involved in the transport of ER vesicles during
the early events of fertilization and embryonic development.

Supported by NSF grants DCB 9974709 and DBI-9977613 and
an MBL fellowship sponsored by the Josiah Macy. Jr. Foundation.

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7. Takagishi, V.. S. Oda. S. Hayasaka, K. Dekker-Ohno, T. Shikata,
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Reference: Biol. Bull. 199: 203-205. (October 2000)

Interaction of Actin- and Microtubule-Based Motors in Squid Axoplasm Probed
with Antibodies to Myosin V and Kinesin

Phillip Stafford, Jeremiah Brown and George M. Langford
{Dartmouth College, Hanover, New Hampshire 03755)

Myosin V, an actin-dependent molecular motor highly ex-
pressed in neurons, transports ER vesicles on actin filaments in the
giant axon of the longfin squid. Loligo pealei ( I ). The amino acid
sequence of squid brain myosin V is similar to those of mouse and
human myosin Va (2). In a recent study. Huang et al. (3) showed
that the rod-tail domain of ubiquitous kinesin (aa680 to aallOO)
and the AF6/cno globular tail domain of MyoV (aa!643 to aa 1800)

bind to each other. This led to the hypothesis that the two motors
form a complex on vesicles through tail-tail interaction. The direct
interaction of these motors could provide a mechanism by which
vesicles move efficiently from microtubules to actin filaments, as
postulated in the dual filament model of vesicle transport (4-7).
In this study, we used antibodies raised to squid brain myosin
V (aQLLQ) and squid brain kinesin (H2 antibody provided by

204

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

Dr. S. Brady) to determine whether kinesin binds to myosin V
in squid neurons. In addition, we used recombinanl tail frag-
ments of mouse MyoV and mouse kinesin (constructs provided
by Dr. Huang) to test whether these fragments form heterolo-
gous complexes with squid brain kinesin and myosin V, respec-
tively. The plasmids containing the glutathione S-transferase
(GST)-labeled mouse AF6/cno tail-globular-domain (GST-
MyoV-tail aa!569 to aa!768 without the coiled medial tail
domain) and the histidine (His)-labeled, kinesin-tail fragment of
mouse ubiquitous kinesin (aa763-856) were expressed in E.
cnli. The bacterially expressed mouse GST- or His-tagged frag-
ments were purified on affinity columns and used in binding
assays of squid brain extracts.

The specificity of aQLLQ and H2 was determined by probing
blots of squid brain extracts with the two antibodies. aQLLQ
recognized a single protein of 196 kD in blots of squid brain
extracts (Fig. 1A, lane 2), and H2 recognized a single protein of
120 kD (Fig. 1A. lane 3). These antibodies were then used in
immunoprecipitation experiments to determine whether myosin V
and kinesin interact directly. Squid brain extracts were incubated
with the aQLLQ and then with protein-A beads. The precipitate
was collected by centrifugation and run on a SDS-PAGE gel (Fig.
IB, lane 1). Proteins in the 50-55 kD range representing the
antibody were the primary components in the precipitate. A blot of
the immuno-precipitate probed with aQLLQ showed a myosin-V
band at 196 kD, protein bands at 50-55 kD representing the
antibody, and one unknown protein at about 100 kD. The unknown
protein may represent a serum protein that binds to the antibody

Antibodies

2 3

IP:

QLLQ

Ab: QLLQ H2

rf*

Figure 1. Protein si:e murker is listed on the left, dashes indicate the
corresponding vi;c\ on the gels In the right. Pane/ A: Lane I is a
coomassie-stained SDS-PAGE gel showing total st/nid optic lobe homog-
eiuite. lune 2 i\ an immunoblot using an antibod\ to the M\oV tail, QLLQ,
lane 3 is the -.ante him probed with H2, an antibody against the kinesin
heavy chain (provided b\ Dr. Scott Brady). Panel B: Lane I shows a gel
/>/ an immunoprecipitation using QLLQ to /mil down MyoV from si/iiid
opth lobes. Lane 2 is a western probed with QLLQ showing MvoV at 196
kit anil the QLLQ antibody nt —511 kl) and a possible M\o\'-tail break-
down product at — 100 kD. Lane 3 is a western probed with H2. Panel C:
Lane I shows a gel from an immunoprecipitation using HI to pull down
ki/u'\in. Lane 2 is a wc\lcrn prohcd with H2.

during protein-A purification or a breakdown product of myosin V.
A blot of the immunoprecipitate was probed with H2, and a protein
of 120 kD representing kinesin was revealed (Fig. IB. lane 3).
Therefore, these data support the hypothesis that kinesin is a
binding partner of myosin V and the interaction may function to
regulate the motor activity of myosin V.

In corollary experiments, squid brain extracts were incubated
with the kinesin antibody H2 followed by incubation with pro-
tein-A beads. The precipitate was collected by centrifugation and
analyzed by SDS-PAGE. Multiple bands were observed, including
a protein of 120 kD and one of 200 kD (Fig. 1C. lane 1). The
antibody (50 kD) was the other major band on the gel. The 120 kD
protein was shown to be kinesin by probing a blot with H2 (Fig.
1C, lane 2). The H2 antibody recognized several other unknown
proteins. These results complement those obtained with aQLLQ
and support the conclusion that kinesin and myosin V bind to each
other in neurons.

The purified mouse GST-MyoV-tail fragment and the His-
labeled kinesin-tail fragment were analyzed by SDS-PAGE, trans-
ferred to nitrocellulose, and probed with aQLLQ and H2. aQLLQ
and H2, respectively (data not shown), did not detect the GST-
MyoV-tail fragment and the His-kinesin-tail fragment. The
aQLLQ antibody was made to a 14-amino acid synthetic peptide
in the AF6/cno domain of squid myosin V (8). so its failure to
recognize the mouse protein fragment was not surprising. Affinity
isolation experiments were performed to determine whether the
GST-MyoV-tail fragment binds to squid brain kinesin. Squid brain
extracts were incubated with the GST-MyoV-tail fragment and
then with glutathione beads. The beads were washed 5x with PBS
and eluted by the addition of 3 bead-volumes of 1 X glutathione.
then analyzed by SDS-PAGE. A blot of the precipitate probed with
an antibody to squid brain kinesin (H2) did not show kinesin as
one of the proteins in the complex (data not shown). In a similar
experiment, purified mouse His-kinesin-tail fragment was ana-
lyzed for its ability to interact with squid brain myosin V. Squid
brain extracts were incubated with the His-labeled kinesin frag-
ment followed by incubation with the His-antibody. The antibody
kinesin-fragment was precipitated with protein A-Sepharose beads
and analyzed on SDS-gels. Blots of the immuno-precipitate probed
with an antibody to squid brain myosin V (aQLLQ) did not show
myosin V as one of the proteins in the complex. Therefore, these
data suggested that heterologous complexes do not form between
the mouse MyoV-tail and native squid kinesin and between mouse
kinesin-tail and native squid myosin V.

In summary, these experiments demonstrate that squid brain my-
osin V and squid brain kinesin are binding partners in neurons. The
mouse recombinant fragments and squid native proteins did not form
complexes that could be detected on blots. Therefore we plan to
produce squid recombinant proteins to determine whether vesicle
transport is inhibited in axoplasm. The inhibition of vesicle transport
by tail fragments of myosin V and kinesin has been shown in cells
grown in culture (9. 10) but has not been demonstrated in vitro.

Supported by NSF grunts DCB 9974709 and DBI-9977613 and
an MBL fellowship sponsored by the Josiah Macy. Jr. Foundation.

Literature Cited

1 Tabb, J. S., B. J. Molyneaux, D. L. Cohen, S. A. Kuznetsov, and
G. M. Langford. 1998. J. Cell Sci. Ill: 3221-3234.

CELL BIOLOGY

20?

2. Mnlyneaux, B. .1., M. K. Mulcahey, P. Stafford, and G. M. Lang-
ford. 20(10. Cell Motil, Cytoskeleton 46: 108-1 IS.

3. Huang, J. D., S. T. Brady, B. VV. Richards, D. Stenoien, J. H.
Rcsau, N. G. Copeland, and N. A. Jenkins. 1999. Nunirc 397:
267-270.

4. Langford, G. M. 1995. Curr. Opm. Cell Biol. 7: S2-88.

5. Langford, G. M., and B. J. Molyneaux. 1998. Bruin Rex. Rev. 28:
1-8.

6. Langford, G. M. 1999. FASEB J. 13 Suppl 2:5248-250.

7. DePina, A. S., and G. M. Landlord. 1999. Mieroxc. Rex. Tech. 47:
93-106.

8. Molyneaux, B. J., and G. M. Langford. 1997. Biol. Hull 193:
222-223.

9 Wu, X., B. Bowers, K. Rao, Q. Wei, and J. A. Hammer, 3rd. 1998.

J. Cell Biol. 143: 849-418.

10. Wubbolts, K.. M. Fernande/.-Borja, I. Jordens, E. Reits. S. Dus-
seljee, C. Eche\erri, R. B. Vallee, and J. Neefjes. 1999. J. Cell Sci.
112: 785-795.

Reference: Biol. Bull. 199: 205-206. (October 2000)

Microtubule-Dependent Nuclear Positioning and Nuclear-Dependent Septum Positioning

in the Fission Yeast Saccharomyces pombe

P. T. Tran[ , V. Doye , F. Chang , ami S. Inoue
(Marine Biological Laboratory, Woods Hole, Massachusetts 02543)

The correct placement of the cell division plane is important for
cell shape, size, and orientation, and for the proper partition of
cellular determinants for development. The position of the division
plane has been shown to be dependent on mierombules in many
organisms ( 1 ). What molecular mechanism of the fission yeast
ensures that the division plane and septum will be properly posi-
tioned?

Fission yeast is a rod-shaped cell that divides by medial cleav-
age. The nucleus is located at the geometric center of the cell,
attached to multiple bundles of dynamic, anti-parallel microtubules
that push on the nucleus (2-4; our unpublished results). The
position of the division plane and septum coincides with the
position of the interphase nucleus (5). By examining the effect of
depolymerizing microtubules within the cell, we tested the hypoth-
esis that microtubules dictate the central position of the nucleus;
and the position of the nucleus, in turn, dictates the future position
of the septum.

A wild-type fission yeast strain (h~ leul-32 nup!07-GFP nmt-
GFP-atb2), expressing ( 1 ) fusion of a nuclear pore protein to the
green fluorescent protein and (2) fusion of the green fluorescent
protein to tubulin, was used to visualize both the nucleus and the
microtubules. For imaging, cells grown to mid-log phase in liquid
yeast media (EMM + 5 /ng/ml thiamine) at room temperature
(21°-23°C) were mounted between a coverslip- and slide-sealed
chamber containing a thin pad of 2% agarose and yeast media
(YE5S). Methyl-2-benzimidazole-carbamate (MBC), a potent in-
hibitor of microtubule polymerization, was used from fresh stock
(100X in DMSO) at the final concentration of 25 /xg/ml to depo-
lymerize the microtubules. Time-lapsed images (1-h interval. 1-s
exposure time) were digitally acquired at room temperature (21°-
23°C) with Metamorph Software (Universal Imaging Corp.) con-
trolling a CCD digital camera (Orca-1. Hamamatsu Corp.). at-
tached to a Leica DMRX microscope stand equipped with DIC
optics, as well as with a PL Fluotar 100X/1.3NA oil-immersion
objective (Leica Corp.) and a mercury arc lamp for wide-field
epi-fluorescent microscopy.

1 Columbia University. New York. NY 10032.

2 Institute Curie, Pdris, France.

To determine whether microtubules play a role in the placement
of the nucleus at the cell center, we used time-lapse microscopy to
examine the position of the nucleus in intetphase cells treated with
the microtubule-depolymerizing drug MBC. Nuclear position in a
cell can be expressed as the ratio of two lengths: the length from
the center of the nucleus to the shorter cell tip (Lshorl), and the
length from the center of the nucleus to the opposite longer cell tip
(Llong). The ratio Lsh,,rl/Llc,nt, = 1 when the nucleus is exactly at the
cell geometric center, and Lvh,,rl/L1(>nt, < 1 when the nucleus is
off-center. MBC-treated cells, which have no microtubules and
ultimately die, continued to lengthen for several hours at a rate
similar to that of control cells. ~ 1 .5 /j,m/h. During a 2-h period, the
control cells grew from an average length of 9.16 ± 1.04 /urn to
12.16 ± 1.47 jixm (N = 23 cells). Almost all nuclei were posi-
tioned in the middle of the cell, with an average Lshort/Llong ratio
of 0.96 ± 0.03; and -967r of cells had better than 0.90 ratio. In
contrast, MBC-treated cells grew from an average length of 8.73 ±
1.55 fxm to 11.45 ± 2.01 /xm (N = 40 cells), with many offset
nuclei, an average Lshl,n/Llonp ratio of 0.80 ± 0.15: and only
-28% of cells had better than 0.90 ratio (Fig. 1 A). Clearly, while
MBC did not affect the cell growth rate, the proper central posi-
tioning of the nucleus was dependent on microtubules.

To test whether the position of the nucleus dictates the position
of the division plane and septum, we examined the position of the
septum in MBC-treated cells, which have off-center nuclei. Lack-
ing microtubules. MBC-treated cells showed a delay in the cell
cycle, and a curvilinear or "bent" growth pattern (6. 7). Whereas
control cells exhibited a cell cycle time of ~4 h, the cell cycle in
MBC-treated cells was significantly delayed to —6-8 h. However.
MBC-treated cells attempted to divide at the end of the cell cycle
delay, and eventually each cell formed a septum at the site of the
offset nucleus that "cut" the nucleus. The new daughter cells
subsequently died. Figure KB, C) illustrates the formation and
position of the septum in control and MBC-treated cells, respec-
tively. Thus, the septum formed at the position of the nucleus, even
when the nucleus was not at the center of the cell.

Our preliminary results are consistent with our hypothesis that
the central position of the nucleus is dependent on microtubules.
and that the position of the nucleus, in turn, may dictate the

206

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

100

— 80

§ 40

U_ 20

• C ontrol cells

D ME.'! -treated': -His

n. n.

0 4 U 5 0 6 07 08 09

Ratio L short

Figure 1. Cells treated with MBC exhihil a misplaced nucleus and
septum. Fission yeast cells expressing nucleoporin-GFP ami GFP-lnhidin
were imaged at room temperature (21"-23"C) with time-lapse fluorescence
microscopy. lAI Pint ot distribution f'ret/iieney of nuclear positions ex-
pressed as the ratio £.1/l,,r(/L;,,,li;. where /,,,„„, is the length Irom the center
of the nucleus to the short cell tip and /.,„„,. is tin- length from the center of
the nucleus to t/ie opposite long cell lip. (B) Wild-type cells through one
cell cvclc- Lett panels arc I>IC images s/un\ ing septum formation at the
middle of the icll I at 0 h and later ul 4 hi. Right panels arc fluorescent
images of nuclear membrane and microtubules. Microtubules span the
length of the cell during interphase. As the cell grew, nuclei were posi-
tioned h\ Jsnnnn, mi, roinhnles at the center of the cells where siihsci/ucnt
cell division and scptanon o, i iirred. creating two daughter cells of ap-
pro\inui!cl\' cifiuil length. \i>t< ihe disappearance <>/ interphase nncrotu-
hulcs in the cell cytoplasm iind the appearance «/ the nntotic spindle inside
the cell nucleus during nnlosis i at 4 hi. tCl MBC-treated cells through one
cell c\cle. No microtubules weic present in MBC-treated cells. \Vithoui

position of the plane of cell division and the septum. Cells which
have lost their microtubule cytoskeleton can continue to grow, and
can undergo septation and cytokinesis after a cell cycle delay.

Our results are consistent with phenotypes seen in studies of
tuhulin mutants (7). However, our experimental conditions ensure
an almost complete loss of the microtubule cytoskeleton; and our
time-lapse microscopy allows long-term viewing of the develop-
ment of phenotypes.

It has been proposed that cytokinesis factors may be localized to
the nuclear region by association or movements on microtubules
(8). However, our studies suggest that microtubules may not be
strictly required for the assembly or localization of the ring at the
nucleus.

P.T.T. thanks Dr. Rudolf Oldenbourg for generously providing
lab space and equipment during this study. This work was funded
in part by NIH and March of Dimes grants to P.T.T. and F.C.

Literature Cited

1. Field, C., R. Li, and K. Oegema. 1999. Curr. Opin. Cell Biol. 11:
68-80.

2. Drummond, D. R., and R. A. Cross. 2000. Curr. Biol. 10: 766-775

3. Hagan. I. 1998. J. Cell Si i. Ill: 1603-1612.

4. Tran, P. T., P. Maddox, F. Chang, and S. Inoue. 1999. Biol. Bull.
197: 262-263.

5. Chang, F., and P. Nurse. 1996. Cell 84: 141-194.

6. Sawin, K. E.. and P. Nurse. 1998. J. Cell Biol. 142: 457-471.

7 Toda, T., K. Umesomo, A. Hirata, and M. Vanagida. 1983. J. Mol.

Biol. 168: 251-270.
8. Chang, F. 1999. Curr. Biol. 9: 849-852.

microtubules, the nuclei nvrc offset, the cell c\cle was delaved. no spindles
were formed, and subsequent division planes ami septum were also offset,
creating "cut" nuclei and daughter cells of unei/iial length. The DIC
panels from fl-4 h show a cell will] a "hirlh \car. " not to he confused with
the septum. Bar = III p.in.

CELL BIOLOGY

207

Reference: Kin!. Bull. 199: 207-208. (October 2000)

The Role of Microtubules During Blastodisc Formation of the Squid, Loligo pealei

Karen Crawford (Department of Biology, St. Mary's College of Maryland, St. Mary's City. Maryland 20686)

After fertilization, cytoplasm streams from the vegetal region of
the squid egg towards the animal cap to form a blastodisc where
meroblastic cleavage will occur (1. 2). This process begins at
fertilization, accelerates after second polar body formation (90
min, at 20°C), and continues through third cleavage (6.5-7.0 h). A
blastodisc cap is formed, although at a slower rate, in eggs that
have been artificially activated with 10 /u.g/ml A23187 (Molecular
Probes) (3). To explore the role of the cytoskeleton in this process,
/';; vitro fertilized (4) or activated embryos were placed in small
petri dishes lined with 0.2% agarose (Sigma, Type II) and filled
with 20°C Millipore-filtered seawater (MFSW). The dishes were
placed on ice and cooled to 4°C. Exposure to cold was chosen to
perturb cytoplasmic movements targeting microtubules (5). so that
the effect on the embryos could be easily reversed. Cold treatment
periods were selected to include the first and second polar body
meiotic divisions (20 min and 1.5 h respectively), and the first (3.5
h), second (4.0 h) and third (6.5 h) cleavage events. Treatment
periods were 20 min to 3 h. 3 to 4 h. 4 to 5 h. 5 to 6 h and 6 to 7 h
of development. After treatment, dishes of embryos were removed
from the ice and allowed to return to room temperature (20°C).
Embryos were compared to control embryos for blastodisc forma-
tion, the presence of polar bodies, and cleavage pattern. Cleavage
in squid is bilateral (Fig. la). First cleavage occurs along the line
between the polar bodies and the apex of the embryo where the
male pronucleus enters the egg. Second cleavage occurs perpen-
dicular to this, and third cleavage is unequal and distinguishes the
future right and left sides of the developing embryo.

Exposure to cold inhibited blastodisc cap formation in all em-
bryos treated prior to cytoplasmic streaming: it also arrested
streaming in embryos treated after second polar body formation.
Twenty minutes after removal from cold exposure, precleavage

stage embryos develop a blister-like swelling of clear cytoplasm
surrounding the male pronucleus. Activated eggs do not form
blisters of cytoplasm when removed from cold treatment, although
a small crescent of cytoplasm may form over the female pronu-
cleus after 50 minutes. Over this same period of time, the polar
bodies that are present swell to more than 4 times their normal
diameter of 10 /u.m and then slowly return to normal size. Over the
next 20 min the blister of cytoplasm around the male pronucleus
relaxes into a small but growing blastodisc cap that resembles a
normal cap in most (95%) cases. Abnormal cap formation was
observed in about 5% of the embryos examined and included
displacement of the cytoplasm to one side of the animal pole or
splitting of the cap at the apex into two regions. Normal cleavage
did not occur in these cases. In contrast to control squid embryos,
which form two polar bodies, in vitro fertilized embryos treated
during polar body formation possessed one (59/73. 37%) or two
(3/73. 4%) and more frequently no (43/73 or 59%) polar bodies.
Similar results were observed in activated eggs treated with cold
during polar body formation. Fertilized embryos that failed to
complete their meiotic divisions often possessed three nuclei at the
apex of the blastodisc cap prior to cleavage, indicating that cold
shock at this early stage induces polyploidy. These embryos sel-
dom underwent normal cleavage. Interestingly, in contrast to the
2%-10% of control-activated eggs that underwent a cleavage
event, 60% (79/132) of activated eggs treated with cold during
their meiotic divisions possessed cleavage furrows. Embryos
treated with cold from 3 to 4 h, the time when control embryos
undergo first cleavage, possessed two polar bodies (as did all other
embryos treated at later times), formed normal blastodisc caps, and
cleaved normally. In contrast, even though first cleavage begins at
3.5 h, embryos treated from 4 to 5 h of development and returned

Figure 1. Cold exposure inhibits specific cleavage furrows in the squid enibnn. Individual furrows are numbered in order of their appearance, (at
Control third cleavage stage embryo, animal pole view. Note that third cleavage is iniei/iial mid that the polar bodies (pb) lie adjacent to the first cleavage
furrow, which marks the midline body axis, (b) Embryo treated with cold exposure from 4 to 5 h after fertilization. The first cleavage farrow is missing,
anil third cleavage is equalized, (c) Embryo treated with cold exposure from 5 to 6 h after fertilization. The second cleavage furrow is missing in this
embryo, while first and third cleavage furrows are present and normal. Images magnified 650 X.

208

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

to room temperature failed to retain their first cleavage furrow
(Fig. Ib) in 90% of the cases examined (36/40). In these embryos,
because the polar bodies mark the region through which first
cleavage will form, it is possible to determine that second cleavage
occurred normally, while third cleavage — which is normally un-
equal— was equalized to mirror the cells in the future dorsal region
of the embryo. Most embryos treated between 5 and 6 h of
development retained a reduced first cleavage furrow at the center
of the blastodisc and formed normal second and third cleavage
furrows (76/89 or 85%), while other embryos from this group
developed without a second cleavage furrow (Fig. Ic). This pattern
was also observed in embryos treated between 6 and 7 h. Surpris-
ingly, most of the embryos from all treatment groups continue to
develop and at 48 h appeared fairly normal, although they often
possessed clumps of large cells, uneven blastoderm yolk boarders,
and regions where cell layers appeared thicker than controls.

These results suggest that the ordered movement of cytoplasm,
which forms the blastodisc in the squid, is disturbed by cold
treatment. Cold exposure also induced polyploidy and perturbed
cleavage furrows. The failure to retain or create specific cleavage
furrows may be due to the direct action of cold on the microfila-
ments responsible for furrow formation, cell membranes, or spe-
cific factors that regulate mitosis. However, the formation of the
blister-like swellings of cytoplasm around the male pronucleus.
likely initiated by the sperm centriole to form microtubule arrays,
suggests that cytoplasmic movements are rapidly resuming and
may disturb the previously formed or forming microfilaments
responsible for cleavage. That the polar bodies, which are little

more than unwanted chromosomes and microtubules, swell rapidly
during this same period further suggests that microtubules may be
partially responsible for these events, although this does not rule
out the possibility that cold exposure results in destabilization of
membranes in these cells. Microtubules originating from the sperm
pronucleus are crucial for the reorganization of cytoplasm after
fertilization in frog eggs (6). The result that cold exposure can
equalize third cleavage in squid embryos is nearly identical to what
was reported when squid embryos were treated with the micro-
filament inhibitor cytochalasin B. although first cleavage furrows
were still present in some of those embryos (7). To address the
importance of microtubules and microfilaments alone and in con-
cert to blastodisc formation and cleavage in the squid, it will be
necessary to selectively challenge each element with specific in-
hibitors and characterize their appearance over time with immu-
nohistochemistry.

This work was supported by a Research Opportunity Award
from the National Science Foundation to Karen Crawford.

Literature Cited

1. Brooks, W. K. 1881). Anniv. Mem. Roslon Sue. N.H. 1-22.

2. Arnold, J. M. 1968. Dev. Biol. 18: 180-197.

3. Crawford, K. 1985. Biol. Bull. 169: 540.

4. Klein, K. C., and L. A. Jaffe. 1984. Biul. Bull. 167: 518.

5. Yahara, I., and F. Kakimoto-Sameshima. 1978. Cell 15: 251-259.

6 Elinson, R. P., and B. Rowning. 1988. Oct. Biol. 128: 185-197.

7 Arnold, J. M., and L. D. Williams-Arnold. 1974. J. Emhryol. Exp.
Morphol. 31: 1-25.

Reference: Biol. Bull. 199: 208-209. (October 2000)

Cytoplasmic Proteins on the Surface of Discharged Microsporidian Sporoplasms

Earl Weulner (Bioloi>\, Louisiana State University, Baton Rouge, Louisiana)

The spore cell of an intracellular microsporidian parasite is a
missile which, when activated, explosively discharges an invasion
tube. The spore contents (sporoplasm) pass through the invasion
tube and are introduced into a target cell. Evidence reported earlier
indicates that the membrane surrounding the newly discharged
sporoplasm cell is derived in part from the polaroplast organelle of
the spore ( 1 ). An early accepted model for microsporidian sporo-
plasm discharge held that the membrane everts with the cytoplas-
mic face shifting outward during extrusion (2). If this is what
happens, it would seem that cytoplasmic proteins might remain
attached to this membrane and end up on the surface of the
discharged sporoplasm. Evidence presented here indicates that
cytoplasmic tubulin and dynactin proteins are on the surface of
discharged microsporidian Sporoplasms, thus supporting the idea
of membrane eversion during spore extrusion.

In this study, proteins were identified from the sporoplasms
discharged from spores of the microsporidian, Spmxiteu lophii.
The protocol for isolating the sporoplasms was reported earlier (3).
Sporoplasms examined immunocytochemically for surface tubulin
(using IgG monoclonal or polyclonul primary antibody, with fluo-

rescein-coupled secondary antibody) revealed an even, but some-
times patchy labeling (Fig. 1A). Similar results were found when
sporoplasms were tested with fluorescein-labeled colchicine. In a
follow-up experiment, sporoplasms were incubated in tubulin as-
sembly medium with fluorescein-coupled tubulin. The results
showed a preferential bordering of the labeled tubulin around the
sporoplasms (Fig. IB).

The S. lophii sporoplasms were also tested for surface dynactin
proteins by using antibodies for pl50gl"cd. dynein intermediate
chains, and dynein heavy chains. The site of binding was visual-
i/ed with colloidal gold or fluorescein-coupled secondary anti-
body. The results showed some uneven labeling for p!50glued (Fig.
1C) and dynein light chains, but no dynein heavy-chain labeling
was apparent. Western blot analyses revealed substantial levels of
pl50glued and dynein light-chain proteins. The positioning of these
proteins onto the sporoplasm surface suggests that the sporoplasm
membrane at first faces the cytoplasm within the spore, but shifts
to the outside during spore discharge.

Surface dynactin is an important component in the movement of
membranous structures within cells. Recall, moreover, that 5.

CELL BIOLOGY

209

lupliii parasitizes the central nervous system of different species of
angler fish of the genus Luphiits, the infections being particularly
evident in the cranial ganglia, dorsal root ganglia, and the su-
pramedullary neurons. Surface dynactin. therefore, is prob-
ably involved in positioning the microsporidian parasites in neu-
ronal cell bodies within the central nervous system of their piscine
hosts (4).

Literature Cited

1 . Weidner, E., VV. Byrd, A. Scarborough, J. Pleshinger, and D. Sibley.
1984. ./. Pi;>t,>:<>«l. 31: 145-198.

2. Lorn, J., and J. Vavra. 1963. Ada Protocol. 1: 279-283.

3. Weidner, E., and A. Findley. 1999. Bio/. Bull. 197: 270-271

4. Weidner, E., and T. King. 1998. Hi,,/. Bull. 195: 229-228.

Figure 1. Spraguea lophii sporoplusnis with tubulin labeling. (A) Anti-
tubulin fluorescence confined to sporoplasm surface. (B) Time-interval
recordings of fluorescein-coupled tubulin bordering spherical sporo-
plasms. (C) Anti-dynactin plSO*1""' label with patchy positioning on sporo-
plasms. All scale bars represent 5 IJLIII.

Reference: Bio/. Bull. 199: 209-211. (October 2000)

Adhesion of a Viral Envelope Protein to a Non-Self-Binding Domain of the Aggregation Factor

in the Marine Sponge Microciona prolifera

Roger MacKenzie1, David Newman2, Max M. Burger*, Rene Roy4, ami William J. Kuhns5
(Marine Biological Laboratory, Woods Hole, Massachusetts 02543)

Studies of the Microciona aggregation factor (MAP) have thus far
been focused upon its self-binding characteristics. This study demon-
strates for the first time an MAP non-self-binding domain. The pur-
pose of this study has been two-fold: (a) to purify the binding motif as

1 Institute for Biological Sciences. National Research Council of Can-
ada, Ottawa, Ontario.

2 Natural Products Branch. National Cancer Institute, Frederick, MD.

3 Friedrich Miescher Institute, Basel, Switzerland.

4 Department of Chemistry, University of Ottawa, Ottawa, Ontario,
Canada.

5 Hospital for Sick Children, Toronto, Ontario. Canada.

a potential pharmacoactive mierobicide; and (b) to characterize non-
self adhesins as participants in the cross-species binding of microbes
to sponge cells. Cell-cell aggregation in Microciona prolifera is
mediated by its aggregation factor, a species-specific compound ex-
hibitins a unique sunburst structure at high magnifications, with a
molecular weight of 2 x 107 Da (1, 2). There is a bracelet-like protein
core composed of multiple beads, each attached to a protein-carbo-
hydrate arm. Multiple anionic glycans on the arms polymerize to form
a viscous gel in the presence of calcium. A sulfate disaccharide and a
pyruvate trisaccharide mediate self-binding to adjacent arms and to
cell membranes (3, 4). A hyaluronic acid (HA)-like compound stabi-
lizes the core-arm connections (5). Binding inhibition studies of MAP

210

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

120
100

§ 80

| 60

c 40

o
Qj

a. 20

-20

400
300
200
100

3100

I 125

100

-25

• Infected Treated Culture
Q Uninfected Treated Culture
— 100%, 50% Ref. Lines
Viral Cylopathic Effect

0 1 2

Q of Sample Concentration (pg/ml)

200

400

600

200

400

600

110
100
90
80

70
60
50
40
30
20

'ercent of Uninfected Untreated Control Cultur

D Dose (pg/ml)

Percent of
Protection

1.19X 1CT1

56.99

3.76 x 10~1

55.68

1.19X 10°

88.99

3.76X10°

105.80

1.18x10+1

96.46

3.75x10+1

95.03

1.18x10+2

95.21

3.75X 10+2

86.80

200

150

100

-50

400

100 200 300 400 500 600 700

0 100 200 300 400 500 600 700

Time (sec)

Figure 1. Summary of the antiviral propertv of MAF in CEM lymphoblastoid cells infected in//? human immunodeficiency virus (H1V-1} (a, b).
Sensorgrarns indicate MAF inhibition of rgp!20-rCD4 binding bv MAF ft', d), whereas synthetic sitlfated and pyruvylated constructs show no inhibition
(e, f). (a, b) Results of forma-an XTT assays for viral inhibitory properties of MAF derivatives. Round black dots and diamonds depict infected treated
culture and Uninfected treated culture respectively. MAF gave >50% protection at a dose as low as 0. 1 p.g/ml (EC50), while demonstrating little or no
toxic effects. Lower dotted line demonstrates viral pathogenic effect in infected untreated culture. Straight dashes are 50% ami 100% reference lines, (c-f)
Sensorgrarns which schematize the relative binding affinities of self and non-self MAF binding epitopes. The small letters that accompany each set ofcun'es
represent the following: a = rpg!20 — rCD4 binding, b = rpg/20 + putative inhibitor — rCD4 interaction, c = putative inhibitor - rCD4 reaction. The
constants for these reactions are given b\ the terms k:l and kj. where k:l refers to the association constant and ktl describes the dissociation constant. The
values for rpg 1 20 at the stated concentration were 1.2 X Iff1 and 3.3 x 10 4 respectively and indicated a strong binding affinity between rpg/20 and rCD4
(a curves). The reactions did not differ appreciably when gp!20 was in mixture with the sit/fate or pyruvylated compounds (b cun'es). However, both MAF I
and MAF ds were reactive as inhibitory compounds. MAFI a/so possessed a strong affinity for rCD4. as indicated by the c cun'e shown on the left middle
drawing.

fractions were carried out using as a binding model recombinant (r)
gp!20 human immunodeficiency vims (HIV-1) envelope protein and
lymphocyte rCD4 protein receptor.

MAF was prepared from cuttings of sponge branches and pro-
cessed as described elsewhere (6). The clear gel derived from
cesium chloride gradient ultracentrifugation was dialyzed repeat-

edly against 200 X volume de-ionized water and then lyophilized
(MAF-I). The result was a truncated molecule, as shown by elec-
tron microscopy — one having morphologically intact bracelets,
but lacking arms (7). Low molecular weight anionic glycans were
prepared from MAF-1 by ethanol precipitation (8) followed by
recovery and lyophili/ation of the ethanolic supernatant (MAF-ds).

CELL BIOLOGY

Compositional analyses of MAFds demonstrated a high sulfate and
carbohydrate content; spectroscopic analysis showed a major peak
that had a mass/charge ratio of 3 kDa and represented about 60%
of the solids in the dried sample. Neither fraction was active in
aggregation assays at levels above 20 /ng/ml, although freshly
derived MAP was active at a level of 0.5 /j,g/ml.

Binding of the HIV-1 envelope protein gp!20 to target lympho-
cytes via CD4 peptide is necessary for syncytium formation and
viral entry and multiplication in target cells (9). Inhibition of
gp!20-CD4 binding by MAP fractions was evaluated using surface
plasmon resonance as the detection principle for molecular inter-
action analysis (10). Instrumentation provided by BIACORE Com-
pany (Piscataway, New Jersey) permits ligands to be immobilized
on a gold sensor chip upon which a light beam is directed. A
continuous flow system permits injection of binding compounds
alone or in mixture with inhibitors over the ligands. Binding causes
a change in the angle of the light beam, with an association phase
beginning at analyte injection and a dissociation phase at the end
of injection. The changes are recorded as a sensorgram. The
inhibition of HIV-1 by MAP compounds was assayed in infected
lymphoblastoid cells by using a colorimetric method in which a
colorless compound (formazan XTT) is metabolically converted
by healthy cells, but not dead cells, to an orange -colored deriva-
tive (II).

The results of MAP titrations for two MAP fractions in
formazan assays indicated that amounts as low as 0. 1 ;ug conferred
protection on more than 50% of the cells (EC50) while simulta-
neously showing little or no toxic effects toward non-infected cells
(Fig. la, h). Data for the binding of recombinant rgp!20 to rCD4
gave a dissociation constant of 17 nM, which agrees well with the
value of 19 nM previously reported by Wu el til. (12). At concen-
trations of 0.75 mg/ml, both MAP derivatives inhibited binding
(Fig. Ic, d). Tests for binding inhibition using synthetic constructs
and polymers of the MAP self-binding epitopes sulfuted disaccha-
ride and pyruvylated trisaccharide were completely negative (Fig.
le. f). Of the four compounds, only MAF-1 showed any binding to
rCD4 at the end of the injection cycle, while MAF-ds was bound
to rgp!20, but not to rCD4.

In summary, a partially purified MAP derivative (MAFds) in-
hibited the replication of the human immunodeficiency virus
(HIV) by specific gp!20 binding and interfering with syncytium
formation between the viral gp!20 envelope protein and the lym-
phocyte CD4 antigen. Thus, in pure form, it will qualify at a

clinical level as a microbicide or as a microbistatic agent, similar
in its chemical properties to HIV-inhibitory products derived from
some other marine invertebrates (13). Although it seems unlikely
that MicriH-imiti would encounter the HIV virus in its natural
surrounding, we propose that this model typifies cross-species
binding (as demonstrated by a non-self adhesin unrelated to the
known self-binding epitopes), and it may provide the sponge with
a means of immobilizing symbionts or other forms that are re-
quired for nutrition or for disposal by macrophages.

Literature Cited

1. Humphreys, S., T. Humphreys, and J. Sano. 1977. ./. Siiprumol.
Struct. 1: 339-351.

2. Fernandez-Busquets, X., and M. M. Burger. 1999. Microsc. Res.
Tech. 44: 204 -2 IS.

3 Spillmann, D., J. Thomas-Gales, J. van Kuik, J. Vliegenthart, G.
Misevic, M. Burger, and J. Finne. 1995. J. Bid. Cliem. 270:
5089-5097.

4 Spillmann, D., K. Hard, J. Thomas-Gates, J. Vliegenthart, G.
Misevic, M. M. Burger, and J. Finne. 1993. J. Bio/. Cliem. 268:
13378-13387.

5. Jarchow, J., J. Fritz, D. Anselmetti, A. Calabro, V. Hascall, D.
Gerosa, M. M. Burger, and X. Fernandez-Busquets. 2000. ./
Struct. Biol. (In press).

6. Misevic, G., J. Finne, and M. M. Burger. 1987. J. Biol. Cliem. 262:
5870-5877.

7. McLaurin, J., T. Franklin, W. Kuhns, and P. Fraser. 1999. Ainy-
loul. Int. J. Ev/>. din. Invest. 6: 233-243.

8 Cardellina, J., R. Munro. K. Fuller, T. Manfredi, M. McKee, H.
Tischler, K. Bokesch, J. Gustafson, J. Beutler, and M. Boyd. 1993.
J. Nat. Prod. 56: I 1 23- 1 124.
9. Chimule, N., and S. Phawa. 1996. Microbiol. Rev. 60: 386.

10. Inagawa, J., J. Okazaki, K. Morimoto, and S. Hashimoto. 20(10.
Pp. 37-57 in Real Time Analysis of Bionmleciilar Interactions. Appli-
cations of BIOCORE. K. Nagata and H. Handa, eds. Springer Verlag,
Tokyo.

I 1. Weislow, O.. R. Kiser, D. Fine, J. Bader, R. Shoemaker, and M.
Boyd. 1989. J. Nail. Cancer Inst. 81: 577-586.

12. Wu, H., D. Myszka, S. Tendian, C. Brouillette, R. Sweet, I.
Chaiken, and W. Hendrickson. 1996. Proc. Natl. Acad. Sci. USA
93: 15030-15035.

13. Beutler, J., T. McKee, R. Fuller, M. Tischler, J. Cardellina, K.
Snader, T. McCloud, and M. Boyd. 1996. Antivir. Chem. Cliento-
ther. 4: 83-88.

212 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

Reference: Biol. Bull- 199: 212-213. (October 2000)

Fertilization-induced Changes in the Fine Structure of Stratified Arbacia Eggs. I.
Observations on Live Cells with the Centrifuge Polarizing Microscope

Mukoto Coda1, Mario H. Burgos2, and Sliinya I none {Marine Biological Luboratory.

Woods Hole. Massachusetts)

Eggs of the sea urchin (Arbacia punctulata), when suspended in
isopienic seawater and subjected, by centrifugation for several
minutes, to a gravitational field a few thousand times greater than
the earth's, stratify into several layers. As shown earlier by Har-
vey, who used supra vital dyes ( 1 ). the layers are, from top
(centripetal pole) to bottom: oil cap, a large clear zone immediately
below the oil cap containing the nucleus, the mitochondria! layer,
yolk granules, and the pigment granule layer.

Observing this phenomenon with a recently developed cen-
trifuge polarizing microscope (CPM) (2. 3), we find that cen-
trifugation introduces to the upper part of the clear zone a
negatively birefringent curtain of material that drapes down

1 Kyoto University. Japan

2 IHEM. UNC-CONICET, Argentina

from the oil cap and surrounds the isotropic nucleus (Fig. 1A,
B). The negative birefringence increases with both time and
speed of centrifugation. The negative sign of birefringence
(larger refractive index perpendicular to the texture), fluores-
cence staining with brefeldin A, and electron microscopy of
fixed cells (5) suggest that this negatively birefringent material
is a pleated series of endoplasmic reticulur membranes, strati-
fied and oriented by the centrifugation.

When a stratified egg is fertilized, the birefringence, viewed
with the CPM, disappears in a few seconds, a surprising obser-
vation (Fig. 1C-E). Furthermore, the egg concurrently starts
floating up in the Percoll-seawater density gradient (Fig. 1F-H).
As the egg floats up, the fertilization envelope rises and its
positive birefringence increases over the next 3 to 4 min. In the
next 10 or so min. the negative birefringence below the oil cap

16:25:06

16:25:52

16:26:06

16:26:10

16:26:12

16:26:16

16:26:38

^^^^Hill^^^^F

16:27:10

Figure 1. Fertilization of vr« urchin egg observed with the CPM. After the unfertilized Arbacia eggs were centrifugally stratified at 3000 X gfor
iihmil I? nun, the CPM was Mopped, and u drop of sperm suspension was plated mi the wall of the specimen chamber above the Pereo/l seinvitler
containing the egg.s. Upon re-starting the CPM, the sperm suspension crept down and fused with the egg suspension. Sperm then swum and reached the
eggs (at about I'' 2d nut, which were Mill stratified, hut hail roniuletl up fliiring the few minutes that the CPM was stopped. (A. B): Negatively birefringent
curtain uf material sho\\ \ in the upper region of the clear rone .surrounding the nucleus of unfertilized eggs. (C-E): During about the first 10 s after
fertilization, the neganvi birefringence disappears. <F-H): The egg starts to float as the (positively birefringent) fertilization envelope gradually rises. Time
of day in /i. wm v Hgg diiimeter: about 75 fjjn. In 1952. McCulloch observed negative birefringence in the upper region of the clear zone in Arbacia
eggs examined after < cnli i/ii::arion but not the changes we saw utter fertilization l-fl. He attributed the birefringence to cytoplasmic fibrils, and with the
fi.\ati]:cs available in th/>\c </.:\ y, he was unable to discern iinv ultrastructurc in the clear :one using an electron micr(>*copc, the only C-\ccption heing the
annulate liimellae. which lie inu'i i>:> I<:J lo be "coarse fibrous components. "

CELL BIOLOGY

213

gradually returns again, but with a much more complex align-
ment of material.

To test whether these changes reflect a rise in cytosolic Ca2 +
(released from the endoplasmic reticulum [ER], an intracellular
Ca2 + storage organelle). we observed the responses of unfertilized
stratified eggs to the calcium ionophore A-23187 (6). Whether in
normal or Ca2+-free seawater, the eggs indeed responded exactly
as when fertilized. We surmise that elevation of cytosolic Ca2 + in
the seconds immediately following fertilization (7) is correlated
with the transient breakup of the ER (8, 9), and that this breakup
is manifested as the rapid loss of birefringence. As the Ca~+ level
drops again, the ER must re-assemble into large layered sheets
since the negative birefringence reappears. In control experiments,
inactivated eggs, that had received the identical history of centri-
fugation, retain their negative birefringence for more than 30 min.

Following fertilization, the egg may become less dense due to
exocytosis and swelling of the cortical granules, or by uptake of
water by the egg or the egg jelly. While not observed when
stratified eggs were fertilized in normal, Ca2 + -containing seawa-
ter, eggs activated with the Ca2 + ionophore suddenly fall in the
density gradient after steadily rising for several minutes. Since this
fall is accompanied by a sudden release of diffuse material sur-
rounding the fertilization envelope, swelling of the egg jelly may
be primarily responsible for the increased buoyancy of the egg
following its activation.

Support of research by Hamamatsu Photonics KK, Olympus
Optical Company, Kyoto University, and the Marine Biological
Laboratory is gratefully acknowledged. We thank Drs. B. Kaminer
and M. Terasaki for extensive discussions of their work relating to
changes in cytosolic Ca2 f and ER organization following fertili-
zation.

Literature Cited

1. Harvey, E. B. 1941. Bu>l Hull. 81: I 14-118.

2. Inline. S., R. A. Knudson. K. Suzuki, N. Okada, H. Takahashi. M.
lida, and K. Yamanaka. 1997. Microxc. Microanal. 4: 36-37.

3. Inoue, S. 1999. h\\SHH .1, (.Vn/v/J 13: sl85-s!90.

4. McCulloch, D. 1952. ./. /:v/> 7n,'l. 119: 47-65.

5. Burgos, M. H., M. Coda, and S. Inoue. 2000. Biol. Hull. 199:
213-214.

6. Steinhardt, R. A., and 1). Epel. 1974. Prac. Null. Aciul. Sci. USA 71:
1915-1919.

7 Gilkey, J. C., I,. F. Jaffe, E. B. Ridgeway, and G. T. Reynolds. 1978.
J. Cell Bwl. 76: 448-466.

8 Henson, J. H., D. A. Begg, S. M. Bealieu, D. J. Fishkind, E. M.
Bonder, M. Terasaki, I). Leheche, and B. Kaminer. 1989. ./. Cell
Biol. 109: 144-161

4 Terasaki, M., and L. A. Jaffe. 1991. ./. Cell Biol 114: 929-940.

Reference: Biul. Bull- 199: 213-214. (October 2(100)

Fertilization-induced Changes in the Fine Structure of Stratified Arbacia Eggs. II.
Observations with Electron Microscopy

Mario H. Burgos[, Makoto Goda2, and Shin\a Inoue
(Marine Biological Lahoratory, Woods Hole, Massachusetts)

Unfertilized Arbacia eggs are stratified by centrifugation: the
centripetal pole is occupied by an oil cap, which crowns a large
clear zone containing the nucleus ( 1 ). When such eggs are ob-
served with the centrifuge polarizing microscope (CPM). a curtain
of negatively birefringent material, draping down from the oil cap,
is introduced to the upper part of the clear zone (2). When stratified
eggs are fertilized or activated by the Ca2 + ionophore A23 1 87, this
birefringence disappears within a few seconds — even before the
fertilization envelope starts to elevate. Its sign, and the fluorescent
staining by brefeldin A, suggest that the negative birefringence is
due to a stack of membranes, stratified and aligned by centrifuga-
tion, and oriented more or less parallel to the direction of the
centrifugal force.

To evaluate this proposal further, we investigated the birefrin-
gent region of the egg by electron microscopy. We used 2%
glutaraldehyde in phosphate-buffered saline made up into 700-mA/
sucrose to prevent swelling of the Arbacia egg. Eggs placed in
fixative without sucrose swelled up to about eight times the vol-

1 IHEM. UNC-CONICET, Argentina

2 Kyoto University, Japan

ume of the unfixed egg. lost their microvilli. and (reversibly) lost
their negative birefringence.

Thin sections of stratified non-activated eggs, fixed with su-
crose-glutaraldehyde, retained their negative birefringence and
revealed that the birefringent region is occupied by stacks of
smooth and rough endoplasmic reticulum (ER; Fig. 1A). The ER
surrounded the nucleus and was aligned more or less parallel to the
axis of centrifugation. A small number of Golgi membrane stacks
were found amidst the ER. but with random orientation. At the
lower region of the ER. we found stacks of annulate lamellae (3.4).
These are most likely the refractile rod- and plate-like structures
that are seen in centrifuged eggs by light microscopy, especially
clearly in DIC. They tended, at first, to lie parallel to the axis of
centrifugation. but changed their orientation as time elapsed after
the centrifuge was stopped.

In centrifugally stratified eggs fixed about 5 min after fertil-
ization— well after the negative birefringence had disappeared,
but before it re-appeared — the distribution of the Golgi and
annulate-lamellar material was basically unchanged. However,
the ER was no longer in large sheets oriented along the cen-
trifugal axis; rather, the sheets had fragmented into smaller
vesicles (Fig. IB), as was anticipated from their loss of bire-

214

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

f-ER

Figure 1. Ultrastrncnire of upper eleur ;one in centrifugally stratified Arhacia egg. A. Unfertilized: B. fixed uhoitt 5 win after fertilization. After
centrifiigalion on a Percoll-seawater density gradient for about 30 min {A), and after an additional 5 inin centrifugation following fertilization (B}. the eggs
were fixed in 2% glularaldehyde phosphate Imfter made up in 700-inM sucrose solution. After confirming, with 1-p.m sections of the unfertilized eggs, that
the negative birefringence in the upper part of the clear zone remained intact, the cells were post fixed with osmium, dehydrated, embedded in Epon. and
sectioned for electron micro.scnp\. In A. the upper region of the clear zone contains a dense stack of ER, oriented more or less in the direction of the
centrifugal force (long arrow t. In B. onl\ small pieces of ER remain, consistent with the (transient) loss of the negative birefringence, at: annulate lamellae.
ER: endoplasmic reticiilum. f-ER: fragmented ER. ne: nuclear envelope. Scale bars 0.5 urn.

fringence. Because the birefringence of activated live eggs does
return in the upper half of the clear zone after about 10 min,
albeit with less ordered alignment of the birefringence axes, the
EM of cells fixed at that stage would be expected to again show
stacks of large ER membrane sheets, but with the stacks ori-
ented along less uniform axes.

These observations suggest that the birefringence observed in
live eggs with the CPM is a good indicator of membrane aniso-
tropy, distribution, and especially their dynamic changes. In addi-
tion, centrifugally fragmented mini-cells could well prove to be a
useful source for several isolated membrane components of the
cells.

We thank Hamamatsu Photonics KK, Olympus Optical Com-

pany, Kyoto University, and the Marine Biological Laboratory for
support of this project. We also thank Louis Kerr and Christina
Stamper of the MBL Central Microscope Facility for their coop-
erative help with electron microscopy. M.B. was supported by an
MBL Chairman of the Board Fellowship.

Literature Cited

1. Harvey, E. B. 1941. Biol. Bull. 81: 114-118.

2. Goda, M., M. H. Burgos, and S. Inoue. 2000. Biol. Bull. 199:
212-213.

3. Afzelius, B. 1955. E\p. Cell Re.s. 8: 147-15S.

4. Swift, H. 1956. J. Biophys. Biochem. Cytol. iSnppl.t 2: 4 1 5-4 IS and
4 plates.

CELL BIOLOGY

215

Reference: Bio/. Hull. 1"W: 215-217. (October 2(100)

Optimization of Homogenization Conditions Used to Isolate mRNAs
in Processes of Myelinating Oligodendrocytes

Robert M. Gould1, Concetto M. Freund1, John Engler2, and Milan- G. Morrison3

Many of us visualize the myelinated nervous system from
light microscope (LM) images. Almost all white matter space is
occupied by myelinated fibers, each sheath having a thick-
ness that relates to the caliber of its axon. Myelin sheaths are
made by oligodendrocytes during nervous system development.
The enormous space occupied by myelinated fibers leaves oli-
godendrocytes so far apart that it is impossible to see connec-
tions between cell bodies and myelin sheaths in adult tissue
sections.

A picture of how myelin sheaths form comes from reconstruc-
tions of morphological images. Oligodendrocyte precursors mi-
grate to each of the regions where axons are developing. When
they arrive, they replicate and send out processes, which select,
ensheath, and myelinate axons that have reached a certain size.
Each process must successfully compete for the axon segment that
it myelinates and then produce and maintain a sheath of identical
dimensions to its neighbors. This morphological picture represents
a multitude of complex process that requires coordinated expres-
sion of many gene products. At present only a very small number
of these genes are known. The immediate goal of our research
program is to identify new genes involved in myelination and
determine their contributions.

As a first step we developed a method to identify a population
of mRNAs that are important for myelination in rat brain. This
mRNA population is selectively translated near sites where
myelin sheaths assemble. Myelin basic protein, a dominant
myelin protein, is selectively synthesized in these sites, for
unlike other myelin proteins, MBP enters myelin within min-
utes of its synthesis. For MBP to enter myelin so rapidly, not
only MBP mRNA, but also all other components needed for its
translation, must be transported from the oligodendrocyte soma
to each myelin sheath assembly site. We reasoned that the
capacity to synthesize proteins at sites distant from the oligo-
dendrocyte soma would not be limited to a single protein.
Furthermore, as we identified other proteins synthesized near to
where myelin basic protein was incorporated into myelin. we
would broaden our understanding of how myelin sheaths are
assembled.

David Colman and his collaborators (1,2) provided a starting
point for our studies. They showed that MBP mRNA behaves
differently from mRNAs for other myelin proteins when brain
samples are subjected to subcellular fractionation. MBP mRNA
purifies in myelin vesicles whereas mRNAs for other known
myelin-related proteins do not. We used rat brain myelin

1 N. Y. S. Institute for Basic Research in Developmental Disabilities,
Staten Island, NY.

: Dept. of Biology, North Carolina State University, Raleigh, NC.

' Bay Paul Center for Molecular Biology and Evolution. Marine Bio-
logical Laboratory, Woods Hole, MA.

mRNA as starting material and suppression subtractive hybrid-
ization to isolate cDNAs that represent mRNAs which co-
localize with MBP mRNA in myelin sheath assembly sites. In
the initial study (3), we compared two different homogenization
media, one isoosmotic (0.32 M sucrose) and one hypertonic
(0.85 M sucrose). Although we obtained more myelin RNA
from samples homogenized in hypertonic sucrose, we worried
that this RNA had higher levels of contaminating RNA than
samples obtained from tissue homogenized in isoosmotic su-
crose. We thus conducted most (2 of 3) suppression subtractive
hybridization studies with myelin RNA prepared from isoos-
motic homogenates (4). However, when we counted the novel
mRNAs obtained in screens with samples prepared under each
condition, we found a far more diverse population was obtained
when samples homogenized in 0.85 M sucrose were used (4).
Some of the mRNAs with particular relevance to myelin sheath
biogenesis, SH3pl3 or endophilin 3 (5) and dynein light inter-
mediate chain (6) were obtained from the screen with the
hypertonic sample (4).

During the summer of 2000, we analyzed 90 (25 were se-
quenced) subtraction products prepared from samples homog-
enized in 0.32 M sucrose and 90 subtraction products (46 were
sequenced) prepared from samples homogenized in 0.85 M
sucrose. We used colony hybridization and hybridization of
inserts prepared from mini-prep samples to identify cDNAs
derived from MBP and MOBP (myelin-associated oligodendro-
cytic basic protein) mRNAs. These RNAs are known to be
located in oligodendrocyte processes (7), since these were
highly enriched in myelin (3; Fig. 1). The remaining cDNAs
were sequenced in the Bay Paul Center sequencing facility at
the Marine Biological Laboratory in Woods Hole, Massachu-
setts. Confirming results from our recent study (4), we found
that far more (30 versus 13) novel cDNAs were obtained from
myelin prepared in 0.85 M sucrose. Most of these cDNAs relate
to known mRNAs (Table I). A significant portion of the
mRNAs generate proteins involved in regulating protein syn-
thesis, namely eukaryotic translation elongation factors alpha
and delta and ribosornal proteins L7a and L21. A few, kinesin
light chain, rab7 and evectin. increase the number of proteins
with recognized functions in membrane trafficking and biogen-
esis. We have analyzed four cDNAs from the 0.32 M sucrose
subtraction product and six cDNAs from the 0.85 M sucrose
subtraction product by northern blot comparisons (starting ma-
terial RNA versus myelin RNA). All of them have mRNAs that
are highly enriched in myelin. Among the known mRNAs
analyzed so far were ferritin light chain, eukaryotic elongation
factor alpha- 1 and kinesin light chain.

Future studies aim to locate the mRNAs and proteins in myeli-
nating tissue. In addition, we will use this approach to identify
mRNAs located in myelin sheath assembly sites in spiny dogfish.

216

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

MBP

MtO

Know n

EST

Unknow n

Distribution of cDNA sequences

Figure 1. Number and distribution ofcDNAs obtained from subtractive hybridization studies with rat brain samples homogenized in 0.85 M sucrose
(black bars) or 0.32 M sucrose (gra\ bars). Clearly larger numbers of novel (30 vs. 13) cDNAs, many of which are related to known mRNAs (see Table
I}, are obtained in the sample homogenized in 0.85 M sucrose. This difference is mainly due to the higher numbers (58 vs. 33) of cDNAs derived from
niYc/in basic protein (MBP) or myelin-associated oligodendrocytic basic protein (MOBP) mRNAs in the 0.32 M sucrose sample. Other abbreviations: Mito,
sequences related to the mitochondria! genome: Known, sequences in the non-redundant GenBank database: KR, sequences related to known GenBank
sequences but with less than 50% of the set/uence matching the known: EST, sequences in the GenBank EST database; Unknown, sequences unrelated to
sequences in either the non-redundant or EST databases.

The results of this study will, in particular, help us to select
appropriate conditions to homogenize dogfish brain, which exists
in an environment of far higher tonicity (approximately I M) than
mammalian brain.

This work was funded by the National Multiple Sclerosis Soci-
ety grant RG2944 (RMG) and the G. Linger Vetlesen Foundation
(HGM). Funds for John Engler were from the Marine Models in
Biological Research Proaram (NSF grant, DBI-99 12287). We

Identities of "known" mRNAs obtained in this study

Table 1

Name

Accession*

Size (region )t

Homogenization

Astrocytic phosphoprotein

AJ243949

1565-2073(2341)

0.85 M

Phosphodiesterase I

D28560

2282-2898(3216)

0.85 M

Ferritin light chain

NM_008064

605-886(886)**

0.85 M

Glial maturation factor

NMJI04124

6-291 (4131)

0.85 M

Eukaryotic translation elongation factor « 1

X63561

584-1449(1714)

0.85 M

Eukaryotic translation elongation factor 81

NM_001960

37-281 (991)

0.85 M

Ran?

NM_009005

1179-1361 (2089)

0.85 M

Ribosomul protein L7a

XI 50 13

417-838(851)**

0.85 M

Ribosomal protein L21

X15212

268-543 (554)**

0.85 M

RANP- 1

D50559

1237-1540(1712)

0.85 M

Zinc finger homeodomain enhancer protein

U51583

1172-2094(3403)

0.85 M

Kinesin lighl chain A, B, C

M75146

1322-1705(2308)

0.32 M

KPL-i. evectin

AF081582

933-1411 (1903)

0.32 M

Lens epithelial protein

U20525

660-705(2308)

0.32 M

* GenBank accession number.

t Size refers to the nucleotide sequence in the known that matches the cDNA sequence we obtained. The size of the known is in parentheses.

** Highly related to known mRNAs — there are differences evident in comparison of aligned sequences.

CELL BIOLOGY 2 I 7

would like to thank Dr. Mitchell Sogin for the generous use of the 3. Gould, R. M., C. M. Freund, and E. Barbarese. 1999. J. Neitro-
Bay Paul sequencing facility. chem. 73: 1913-1924.

4 Gould, R. M., C. M. Freund, F. Palmer, and D. L. Feinstein. 20(10.

Literature Cited J' Neurochem- <in Press>

5. Ringstad, N., Y. Nemoto, and P. De Camilli. 1997. Proc. Null.

1. Colman, D. R., G. Kreibich, A. B. Frey. and D. D. Sabatini. 1982. Acad. Sci. USA 94: 8569-8574.

J. Cell Biol. 95: 598-608. 6. Hughes, S. M., K. T. Vaughan, J. S. Herskovits, and R. B. Vallee.
2 Gillespie, C. S., L. Bernier, P. S. Brophy, and D. R. Colman. 199(1. 1995. / Cell Sci. 108: 17-24.

J. Neurochem. 54: 656-661. 7. Holz, A., et al. 1996. J. Neurosci. 16: 467-477.

218 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

Reference: Bint. Bull. 199: 218-219. (October 2000)

Modeling the Effects of Land-Use Change on Nitrogen Biogeochemistry
in the Ipswich Watershed, Massachusetts

R\an Kirkbv, Luc Claessens, Charles Hopkinson Jr., Edward Rastetter, and Joseph Vallino
(The Ecosystems Center, Marine Biological Laboratory, Woods Hole, Massachusetts 02543)

The Ipswich River Basin, which is located in northern Massa-
chusetts and drains into the Plum Island Sound Estuary, covers a
400-km2 area composed of forest, wetlands, open and agricultural
land, and a gradient of low- to high-density residential and com-
mercial land ( 1 ). Over the last century, population growth and
land-use changes in the basin have altered the land cover of the
watershed. The United States Geological Survey (USGS) has
recently modeled the hydrology of the Ipswich River Basin using
a precipitation-runoff model called Hydrological Simulation Pro-
gram-Fortran (HSPF) (2). Their intent was to develop a better
understanding of the effects of water withdrawal on the water
budget of the river basin (1 ). In addition to the hydrological data
that has been collected by the USGS, we have monitored nutrient
loading to build a better picture of the effects of land-use change
on nutrient biogeochemistry in the Ipswich river basin. To further
investigate nutrient processing in the watershed, the USGS HSPF
model ( 1 ) was modified to include simulation of nutrient process-
ing on land and in the Ipswich River and tributaries. Considering
the projections that have been made for urban development in the
Ipswich watershed, the ability to accurately model the resulting
changes in nutrient processing may be an important tool in under-
standing the health of the Ipswich ecosystem. It could also become
an important aid in planning future development that minimizes
harmful effects to the watershed.

HSPF, in addition to simulating hydrology, is capable of simu-
lating nutrient processing, sediment transport, pH and gasses,
phytoplankton, and algae dynamics in a watershed. Nitrate-pro-
cessing components for stream reaches and land areas were added
to the HSPF model. Initial values required by the model were
obtained from a database (3) which contains parameter values used
in similar HSPF projects in the northeastern United States. Nitrate
processing and output from different land types was further cali-
brated using an empirical relationship between fractional cover of
agricultural and forested land in small catchments \v/™\ nitrate
concentration in the streams into which they drain (Fig. 1A, B). In
this calibration, the only nitrate input was atmospheric deposition;
so the differences in nitrate output between the two land types (Fig.
1 B ) represent the different values chosen for constants in equations
governing the simulation of nutrients in the two land-use types. A
more rigorous calibration of the model, which is in progress, will
include comparing simulated data on nitrate concentration in the
Ipswich River with data we are collecting. All simulations run on
the model were driven by meteorological input for the years 19H9
to 1993, but future work on the model will include adding more
recent meteorological data. The base simulation was run using
1991 land-use data for the watershed; other land-use change sce-
narios were run by modifying the areas of different land types in
certain parts of the river basin.

Calibration of the model resulted in simulated nitrate output

from forest and from open plus agricultural land (Fig. IB) that
coincided with the empirical relationship for fractional cover ver-
sus nitrate concentration (Fig. 1A). The base concentration of
nitrate in first-order streams draining only forested lands was
approximately 10 /u,M, whereas the value for streams fed by
agricultural and open pasture land was closer to 70 p,M (Fig. IB).
Modeled nitrate transects along the main stem of the Ipswich River
show a strong trend of decreasing concentration near the head of
the river, followed by a slowly decreasing concentration toward
the Ipswich dam (Fig. 1C). This same general trend is seen in data
collected for the same month, although in a different year (Fig.
1C). Stream-flow data along the Ipswich River reveal the opposite
trend: a quick increase in flow near the head of the river, followed
by a slower increase moving towards the mouth of the Ipswich
River (Fig. 1C). Seasonally, nitrate concentrations at the mouth of
the Ipswich River reach a peak during winter and spring (Fig. ID).
Similarly, river flow at the mouth has its highest peak in the spring
and another, smaller peak in the winter (Fig. ID). Data we have
collected show similar correlation between peak discharge and
peak nitrate concentrations (4).

The opposite trends in stream flow and nitrate concentration
along transects from the head to the mouth of the Ipswich River
suggest that the decreasing nitrate concentration may be due, at
least partially, to a dilution effect. The other factors contributing to
diminishing nitrate concentration in the river are in-stream pro-
cesses, such as denitrification and uptake by plants and algae, that
can be examined using the model. One purpose of continuing to
examine nutrient processing with this model is to help determine
what processes are the most important contributors to the trends
that have been observed and modeled.

The HSPF model can be used to examine different scenarios for
land use by modifying the areas of different land types in the basin.
A 12-knr residential development was modeled at different loca-
tions in the watershed. The results from those scenarios indicated
that nitrate concentration would increase at the mouth of the river,
and that the increase would be greater the closer the development
is to the mouth of the river. The model predicts that urbanization
in the watershed will have a smaller effect if it occurs farther
upstream and on tributaries, as opposed to farther downstream and
on the main stem of the river. One simulation of two different
scenarios showed that a 12-knr residential area built in the lower
watershed on the main stem would increase nitrate concentrations
at the mouth of the river by approximately 5 (U/W, but an identical
development on a tributary feeding into the main stem at the same
location would produce roughly baseline conditions at the mouth.
The model can be used to look more closely at the sources and
sinks of nitrate in the river basin to better characterize the pro-
cessing of nitrogen and other nutrients in the watershed.

ECOLOGY, BIOGEOCHEMISTRY, AND POPULATION BIOLOGY

219

0-0 0.2 04 06 0.8 1 .0

Land-Use Fractional Cover (% urban +
agriculture)

40 _. 30 , 20
River km

200
150

100

140
120

_ 100

3 80

2 60

* 40

- Forest
Open/ Agriculture

JlJ

FMAMJ JASOND

Month

15 J

u.
10 a

FMAMJJASOND

Month

Figure 1. (A) An empirical relationship ben\-een the forest cover in a catchment and the nitrate concentration in streams draining the catchment. (B)
Modeled nitrate concentration timeseries in first-order streams with all forest and all open plus agriculture contributing land area. (C) Modeled nitrate
concentration and stream flow transect along the main stem of the Ipswich River in July 1993. and observed nitrate data from July 1998. (D) Modeled
nitrate concentration and river flow at the mouth of the Ipswich River over the course of 1993.

This research was funded through the NSF-EPA Water and
Watersheds program DEB-9726862.

Literature Cited

1. Zarriello. P., and K. Rics. 2000. A Precipitation-Runoff Model for
Analysis of the Effects of Water Withdrawals on Streamflow, Ipswich
River Basin, Massachusetts. United States Geological Survey, Denver,
CO.

2. Bicknell, B. R., J. C. Imhof'f, J. L. Kittle, A. S. Donigian, and
R. C. Johanson. 1993. HyJmloxic Simulation Program— FORTRAN
(HSPF): User's manual for release 10.0. EPA 600/3-84-066. U.S.
Environmental Protection Agency, Environmental Research Labora-
tory, Athens. GA.

3. EPA HSPFParm site. http://www.epa.gov/docs/ostwater/BASINS/
support.htm [21 Aug. 2000],

4. The Ecosystems Center Plum Island Estuary data, hup://
ecosystems.mbl.edu/pie |2I Aug. 2000].

Reference: Bio/. Bull. 199: 219-221. (October 2000)

Solute Dynamics in Storm Flow of the Ipswich River Basin: Effects of Land Use

Anne Perring, Michael Williams, Charles Hopkinson Jr., Edward Rastetter, and Joseph Vallino
(The Ecosystems Center, Marine Biological Laboratory; Woods Hole, Massachusetts 02543)

The Ipswich River in northeastern Massachusetts has supplied
surrounding suburban communities with water since the 1800s.
With current projections of increased urbanization in the water-
shed ( 1 ), solute fluxes from developed areas may have an impact
on the ecology of the Ipswich River. Solute fluxes from storm flow
are particularly important since storms commonly flush solutes
from storage reservoirs, thereby increasing the mass transfer of
solutes to the aquatic system (2). The objectives of this study were
to observe solute dynamics in storm flow in three first-order

catchments of the Ipswich River basin to infer how increased
development will affect the aquatic system.

The three catchments were selected to represent the end-mem-
bers of different land-use areas commonly found in the Ipswich
River basin. The catchments represent predominately urban
(URB), agricultural (AG) and forested (FOR) areas. The baseline
discharges were 100, 0.4 and 1 0 1/s at the URB, AG and FOR sites,
respectively. Rain volume at each site was measured using manual
rain gauges, and samples for chemical analyses were collected.

220

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

Baseline samples of stream water were collected before and after
a storm that occurred 15-16 June, 2000. Each hour during the
storm, filtered and unrtltered samples of stream water were col-
lected and stage measurements were taken. Discharge was esti-
mated from stage measurements. Stream water and rain samples
were filtered immediately with glass-fiber filters, stored on ice in
the field, and refrigerated at the laboratory until analysis. All
filtered samples were analyzed for NH4 colorimetrically, for Cl,
NO,, and SO4 using ion chromatography, and for Na, K, Ca, and
Mg by atomic absorption. Unfiltered samples were analyzed for
acid neutralizing capacity (ANC) and pH.

Total rainfall at the sites ranged from 19 to 46 mm. Maximum
stage observed was 25, 12, and 3 cm above base flow at the URB,
AG and FOR sites, respectively; maximum discharges were 400,
140, and 70 L/s. Solute concentrations in rain were similar among
all sites and much lower than those found in stream water. Base
flow values for all solutes were lower at the FOR site than at the
other, more developed sites. Sodium and Cl concentrations were
high at the URB site, probably because of salting roadways in
winter months. Calcium concentrations were high in the AG site,
perhaps due to the addition of lime to agricultural fields. During
the storm, solute concentrations in stream water of the FOR site
were relatively invariant compared to the URB and AG sites (Fig.
la-c). Concentrations of NH4 and NO, increased at the beginning
of the storm, and trends were similar at the AG and URB sites (Fig.
la). Concentrations of other solutes at the URB and AG sites
decreased with the onset of the storm (Figs. Ib, c). After the storm,
solute concentrations at the AG site increased rapidly toward base
flow values (Figs. Ib, c). Stream water discharge from the URB
site was the highest of the three catchments, as was the net flux of
solutes measured (Fig. Id).

The variations in solute concentrations observed are primarily
due to site-specific differences in the relative proportions of
groundwater and overland flow inputs to the stream. The propor-
tion of these inputs is commonly regulated by the type and amount
of ground cover in a particular catchment. The lack of forest cover
in agricultural areas and impermeable surfaces in urban settings
increase overland flow inputs to streams during storms (3). In
forested catchments, runoff is typically smaller than in more de-
veloped catchments because soil and vegetation allow much of the
precipitation to percolate slowly to the groundwater table. In
contrast, soils in predominately agricultural catchments can be-
come quickly saturated during storms, causing larger inputs of
water to enter a stream in the form of overland flow and diluting
solute concentrations. As a storm subsides, solute concentrations
in stream water will typically return to base flow levels as the ratio
of groundwater inputs to overland flow increases. Urban settings
characteristically have large amounts of impervious ground cover
preventing rain from percolating to groundwater reservoirs,
thereby increasing the proportion of overland flow (urban runoff)
to the stream. Hence, the large decreases in solute concentrations
observed at the AG and URB sites during a storm are probably due
to a larger overland flow component in these catchment streams. In
contrast, the increases of NH4 and NO, concentrations at these
sites must be due to strong sources of nitrogen in overland flow
and groundwater at the beginning of a storm that may be linked to
the application of fertilizers in developed settings.

Our results show that there are marked differences in the solute

~ 6000

5 Time(h) 10

~ 4000 -

'§ 2000

0 n

• • • • --*

V *~^r

0 5 10
Time (h)

5 Time(h) 10

| 3000 -,
H 2000 -
1 1000

•K n 4

D Net Flux URB
D Net Flux AG
• Net Flux FOR

[NH4]

[CIJ/10
Solute

Figure 1. Concentrations of ammonium (A), chloride (Bl, and calcium
(C) plotted against time. Relative fluxes of ammonium, chloride (divided by
10) and calcium tit the three stiu1\ sites (D).

dynamics of storm flow among streams in areas characterized by
different land uses. Because anthropogenic inputs of nitrogen are
associated with the eutrophication of receiving waters, further
study is required to determine the impact of increased NH4 and
NO, export from urban and agricultural catchments on the aquatic
ecology of the Ipswich River.

Literature Cited

I Pontius, R. G. Jr., L. Claessens, C. S. Hopkinson, Jr., A. Mar-
y.ouk, K. B. Rastetter, L. C. Schneider, and J. Vallino. 2(1(10. In

ECOLOGY. BIOGEOCHEMISTRY. AND POPULATION BIOLOGY

Proceedings of the 4'1' International Conference on Integrating CIS
and Environmental Mode/ing (GIS/EM4): Problems, Prospects anil
Research Needs. Banff, Alberta. Canada. 2-8 September 2000. (In
press).

l. Wood, E. F., M. Sivapalan, and K. Beven. 1990.

physics. 28: 1-18.
3. Jordan, T. E., D. L. Cornell, and D. E. Weller. 1997.

Qual. 26: 836-.S4S.

221

Reviews of Gt-o-

J. Environ.

Reference: «;>>/. Bull. 199: 221-223. (October 2000)

Fate of Anthropogenic Nitrogen in a Nearshore Cape Cod Aquifer

Elizabeth J. Westgate1, Kevin D. Kroeger, Wendy J. Pabich, am/ Iran Valiela
(Boston University Marine Program, Marine Biological Laboratory, Woods Hole, Massachusetts 02543)

Nitrogen loading from land is a principal cause of eutrophica-
tion of shallow estuaries ( 1, 2. 3). In regions such as Cape Cod,
Massachusetts, which are underlain by unconsolidated sands, the
major mechanism that transports nitrogen to estuaries is ground-
water flow, and the major nitrogen source (primarily in the form of
nitrate. NO,) is often wastewater from septic systems (1, 2. 3).
Wastewater nitrate concentrations decrease during travel in
groundwater due to dilution with clean groundwater and to loss by
denitrification (4). The loss of nitrogen during flow between a
septic tank and receiving estuary can be calculated by determining
the reduction in concentration of dissolved inorganic nitrogen
relative to the change in concentration of a passive tracer that
accounts for dilution.

We investigated losses of nitrate for a domestic septic system in
the watershed of Quashnet River, Cape Cod. Effluent from septic
systems moves downgradient within plumes containing high con-
centrations of nitrate. In addition, the study area has plumes
derived from fertilized turf or fields. To sort out the different
plumes, we measured boron (B, a passive tracer derived from
laundry detergents and associated with wastewater sources [5, 6.
7]) and potassium (K, associated with both wastewater and fertil-
izer sources [8, 9]) in the samples of groundwater.

To calculate loss of nitrate along the plumes, we collected samples
from nine wells downgradient from the septic system. Each well was
furnished with 14 ports that allowed us to sample groundwater at
intervals of 1-2 m. We collected 300 ml of water from 129 ports
during June and July 2000 and measured concentrations of nitrate
(NO, + NO2) and ammonium (NH4) using colorimetric and fluoro-
metric techniques, respectively. We selected samples with nitrate
concentrations above 8 \iM and conductivities less than 4.000 /j,S/cm
for measurements of B and K. These samples were analyzed by Ward
Laboratories (Kearney, NE).

Examination of vertical and horizontal profiles of nitrate and am-
monium suggested that there were three distinct plumes within our
well field (Fig. 1 ). The upper plume moved along near the surface of
the water table and contained the highest nitrate concentration of the
three plumes; at nearly 3000 \j.M, it was similar to literature values (8)
for septic effluent that has just left the leaching field. The nitrate, B.
and K concentrations in this plume differed considerably from those
of the other plumes (Fig. 2, A and B).

In contrast, the lower plume showed no increase in nitrate
relative to increase in B (Fig. 2, A). It did, however, show a
positive relationship to K, and at a given K concentration had a

1 Lafayette College, Easton, PA 18042.

much higher nitrate concentration than did the upper plume (Fig.
2, B ). This evidence suggests that the lower plume might be due to
fertilizer use upgradient of our septic system.

The middle plume had no significant relationships between
nitrate and B or K, perhaps because of the small number of
samples and the low concentrations. The concentrations of nitrate.
B, and K from the middle plume do, however, fit on the lower
portions of the curves for the upper plume (Fig. 2, A and B). These
circumstances lead us to think that the middle plume was probably
the leading edge of a plume from a septic system located farther
upgradient from our septic system. We therefore used data for the
upper and middle plumes in our examination of the fate of septic-
system nitrogen in this watershed.

Concentrations of nitrate and B diminished as water parcels
aged (age. Fig. 2, C and D, calculated from Vogel equations [10]
that predict time since recharge as a function of depth in aquifer).
To allow for dilution, we normalized the data by expressing
concentrations as NO3/B (Fig. 2. E). We estimated the NO,/B in
the effluent that had just left the septic system (age 0) by using a
literature value (8) (Fig. 2, E, upper dashed line). The NO,/B
values we used came from a Cape Cod site near our study area, and
the data dated from 1992. only a 7-8 year difference from our date
of collection. We presume that differences in B were therefore a
reasonable proxy for those in our study system. We calculated
losses of NO, as the difference between the age 0 nitrate concen-
tration, allowing for dilution, and the measured nitrate concentra-
tion.

Losses of nitrate in excess of dilution were quite rapid, with
rates reaching 50% loss at 0.2 years (Fig. 2, F). The loss rates
diminished with time, which suggests that, if these data are rep-
resentative of losses elsewhere, N losses by denitrification and
retention take place primarily near the septic system source. Ex-
trapolating the curve of Figure 2 (F), we find that near-complete
losses may be reached at 4.8 years, which is equivalent to 480-730
m from the septic system, assuming a travel rate of 100-150 m per
year (11).

As a minimum estimate of loss, we also calculated loss relative
to our highest measured NO,/B ratio (Fig. 2, E, lower dashed line).
If our initial NO,/B ratio were closer to this measured value, our
estimate of time to 50% NO, loss would increase to 0.6 years; but
the estimate of time to 100% loss was not affected. The extrapo-
lation to 100% loss assumes that the relationship between percent
loss NO, and age continues to hold beyond our oldest sample. This
would not be the case if the availability of labile organic carbon
were to limit NO, loss before 100% loss is achieved.

222

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

-8-

Soil surfac

20 40

Distance from shore (m)

. water table, and aauifer throin'h our field ofmiiltml,

Figure 1. Vertical cross section from the soil surface, water table, and aquifer through our field of multiple sampling wells (elevation relative to mean
tow water [MLW]). The numbers are concentrations of NO, (fxMJ/oc water samples collected from each of the 14 ports in each of the 9 wells. Although
the wells were not all in one plane, for simplicity they are shown as if they were. Contour lines are drawn to indicate NO, concentrations of 32, 128, 512,
and 2048 ;uM. Position of salty water determined from salinity of water samples.

— 3000-,
12000 -
g 1000-

0.2
B (ppm)

— 3000 -

a 2000 -

g 1000-
Z 0

§-3000-

., B

52000-

o 1000 •

Z 0-

^f •

ddlt -9-^^"^^

0 10 20
K (ppm)

~ 0.4-i
I 0.3-
3 0.2-

D
• .

CQ 0.1 -

n .

•
• . • • • •••

12000 -
8000
4000-
0

1 2
Age (y)

Age (y)

120-

-2 80-
co

i 40 H

1
Age (y)

Figure 2. A: NO, concentration versus B concentration for samples collected from upper (•), middle • and lower (A) plumes. B: NO, concentration
versus K concentration for all three plumes. C: NO, concentration versus age for upper and middle plumes. D: B concentration versus age for upper and
middle plumes. E: NO, to B ratio versus age for upper and middle plumes. F: Percent loss of NO, versus age for upper and middle plumes.

ECOLOGY, BIOGEOCHEMISTRY, AND POPULATION BIOLOGY

223

If coastal zone managers wish to regulate septic nitrogen loads,
they could concentrate on management of septic systems that lie
within 480-730 m of the shore, since these appeared to be the
major contributors of nitrate to receiving estuaries. Septic sources
farther upgradient probably contribute less significantly.

This research was supported by an internship from the Woods
Hole Marine Science Consortium to Elizabeth Westgate, an MIT
Sea Grant (#65591) awarded to Ivan Valiela and Harold Hemond,
and National Estuarine Research Reserve Fellowships to Kevin
Kroeger and Wendy Pabich.

Literature Cited

1 Valiela, I., et al. 1992.
2. Valiela, I., et al. 1997.

Estuaries 15: 443-457.
Eco/. Appl. 7: 358-380.

3. Valiela, I., el al. 200(1. Biogeochemistry 49: 277-293.

4. Wilhelm, S. R., S. L. Schiff, and J. A. Cherry. 1994. Ground
Watt, 32: 905-91(1.

5. Barf:. S. 1998. Water Res. 32: 685-690.

6. LeBlanc, D. R. 1984. U.S. Geological Survey Water-Supply Paper
22IS

7. Barber, L. B. II, E. M. Thurman, M. P. Schroeder, and D. R.
LeBlanc. 19X8. Environ. Sci. Technol. 22: 205-211.

8. DeSimone, L. A.. P. M. Barlow, and B. L. Howes. Open-File
Rep.-U.S. Geol. Surv. 1995, No. 95-290.

9 Bjerg, P. L., and T. H. Christensen. 1992. ./. Hydrol. 131: 133-
149.

10. Vogel, J. C. 1967. Pp. 355-369 in Isotopes in Hydrology. Proceed-
ings of IAEA-SM-83/24, Vienna.

11. LeBlanc, D. R., et al. 1991. Wat. Res. 27: 895-910.

Reference: Biol. Bull., 199: 223-225. (October 2000)

Effects of Nitrogen Load and Irradiance on Photosynthetic Pigment Concentrations in
Cladophora vagabitnda and Gracilaria tikvahiae in Estuaries of Waquoit Bay

Michelle Denault , Erica Stieve, and Ivan Valiela
(Boston University Marine Program. Marine Biological Laboratory, Woods Hole, Massachusetts 02543)

Two major controls of activity and standing crop in macroalgae
are nitrogen supply and irradiance ( 1 ). Increased nitrogen loads
increase production (2) and biomass of macroalgae such as
Cladophora vagabitnda and Gracilaria tikvahiae (E. Stieve. un-
pub. data). Lower light availability lowers growth rates of mac-
roalgae, although this effect varies among species (3). Because of
exponential attenuation and self shading within algal mats, the
irradiance available for benthic algae depends on water depth ( 1 ).
Supply as well as storage of nitrogen and photons affect the
concentration of photosynthetic components in macroalgae (1,4).
Photosynthetic pigments such as phycoerythrin also act as nitrogen
pools, and macroalgae acclimate to different irradiance regimes by
changing pigment concentrations (5).

To examine the effects of different nutrient supplies on
photosynthetic pigment concentrations in a green and a red
alga, we collected samples of Cladophora vagabunda (L.) van
den Hoek and Gracilaria tikvahiae McLachlan from five estu-
aries within Waquoit Bay (Childs River. Eel River, Quashnet
River, Sage Lot Pond, and Timms Pond) that are exposed to
different nitrogen loads (6). To study the effect of irradiance on
pigment concentration we collected samples at a range of
depths (80 to 210 cm). Irradiance at each sampling depth was
measured using a spherical underwater sensor attached to a
Li-Cor DataLogger LI-1000. Samples were collected during
one day in early June.

At each site, six samples of benthic macroalgal material were
collected and sorted to isolate fronds of C. vagabunda and
G. tikvahiae. The samples were sorted by species. Chlorophyll a,
b, and carotenoids were extracted as described by Figueroa et al.

1 Brown University, Providence. RI 02912.

(7); phycobiliproteins were extracted as described by Beer and
Eshel (8). Pigments were extracted within 36 h of collecting and
were kept at 5°C until extraction to avoid pigment degradation.
Concentrations of pigments were determined by use of a Perkin
Elmer UV/VIS spectrophotometer (8. 9. 10). Chlorophyll a and
carotenoid concentrations were measured in both C. vagabitnda
and G. tik\'ahiae. Chlorophyll b concentrations were measured in
C. vagabunda. Phycoerythrin concentrations were measured in
G. tikvahiae. To further ascertain the internal storage of nitrogen
and carbon under different nitrogen and irradiance regimes, we
dried macroalgal samples and measured percent nitrogen and per-
cent carbon in a Perkin Elmer elemental analyzer according to the
manufacturer's instructions.

Concentrations of chlorophyll a (Fig. 1A) and carotenoids (Fig
1 B ) in both species of macroalgae increased as nitrogen load to the
estuaries increased. There was no consistent difference between
upstream and downstream sites within the estuaries, and there was
no apparent effect of different salinities at the sites of collection
(range of 10%<? to 32%o) on pigment concentrations (data not
shown), so data were pooled within each estuary. Concentrations
of chlorophyll a and carotenoids in C. vagabunda and G. tikvahiae
are within the range found in other published literature (5); more
importantly, the nitrogen loads increase pigment concentrations
from values characteristic of nitrogen-poor waters to those
of nitrogen-rich estuaries (Fig. 1A. B) (5). The response of
C. vagabunda to nitrogen supply was more pronounced than that
of G. tikvahiae (Fig. 1A, B).

These results suggest that nitrogen supply has important effects
on pigment concentrations and nitrogen content of fronds, and that
the response depends on the species. Surprisingly, concentrations
of phycoerythrin in G. tikvahiae did not increase as nitrogen load

224

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

CHLOROPHYLL a

C. v.

CAROTENOIDS

0.8n

"D
O)

S 0.4-

T3
O)

PHYCOERYTHRIN

T3
O)

' A

400

800

Nitrogen load
(kgha-1 y1)

0 I

500 1500

Irradiance
(//mole photon m'2 s~1)

increased (Fig. IE), even though this pigment is known to function
as a nitrogen reserve (11). C. tikvahiae may not be nitrogen-
limited in these estuaries, an inference that we base on both the
relatively low response of chlorophyll a (Fig. 1A) and carotenoid
(Fig. IB) concentrations, and the lack of response of phycoerythrin

Figure 1. Milligrams dry weigh! of pigments per gram dry weight of
macroalgae are shown versus nitrogen load (left column ) and versus
irradiance (right column). (A) Chlorophyll a (C. vagabunda. F = 32.83. P
< 0.001: G. tikvahiae, F = 6.86. P < 0.05). (B) carotenoid (C. vagabunda.
F = 26.88. P < 0.001; G. tikvahiae, F = 6.67. P < 0.05), and (C)
phycoerythrin (ns) concentrations versus nitrogen load. The dotted lines
show published (5) pigment concentrations ranges for macroalgae in
nitrogen-poor waters (lower dotted lines) and nitrogen-rich water (upper
dotted lines). (D) Chlorophyll a (C. vagabunda, F = 8.52, P < 0.01;
G. tikvahiae. F = 7.50, P < 0.05). (E) carotenoids (C. vagabunda, F =
6. 71. P < 0.05; G. tikvahiae, F = /0.60, P < 0.01 ) and (F) phycoerythrin
(ns) concentrations versus irradiance.

concentrations (Fig. 1C) to nitrogen load. We also measured
concentrations of chlorophyll /;. which showed a positive response
to nitrogen load (data not shown). Curiously, concentrations of
chlorophyll b responded more strongly to increased nitrogen sup-
ply than did chlorophyll a concentrations (Fig. 2A). We have no
explanation for this response.

Concentrations of chlorophyll a (Fig. ID) and carotenoids (Fig.
IE) in both species of algae decreased similarly and significantly
at lower irradiance. These results suggest that the mechanism of
response to irradiance is similar for both species of algae. Phyco-
erythrin concentrations (Fig. IF) did not change with differences in
irradiance. The percent N content of C. vagabunda, but not of
G. tikvahiae. increased in parallel to nitrogen load (Fig. 2B, C).
The increase in nitrogen content in C. vagabunda is even more
striking in view of the decrease of carbon content in C. vagabunda
as nitrogen load increased (Fig. 2B). Percent nitrogen in C. tikva-
hiae fronds did not increase significantly with nitrogen load, in
agreement with the results of Figure 2C.

Increased nitrogen loads were paralleled by significant
increases in photosynthetic pigments in C. vagabunda and
G. tikvahiae as well as by an increase in percent N in
C. vagabunda. but not in G. tikvahiae. These results suggest
that the supply of nitrogen may be sufficient to support growth
in G. tikvahiae, but that C. vagabunda is nitrogen-limited in
those estuaries of Waquoit Bay that receive the lowest nitrogen
load from land. This conclusion is verified by biomass data (E.
Stieve, unpub. data) that show that the response of standing
crop of C. vagabunda is a function of nitrogen load, and that

0 0.5 1 1.5 2
Chlorophyll a
(mgdw gdw1 )

0 400 800

Nitrogen load

(kgha'V1)

0 400 800

Nitrogen load

(kgha'V1)

Figure 2. (A) Chlorophyll b concentration versus chlorophyll a concentration (r = 0.785). (B) Percent carbon (C. vagabunda, F = 19.17. P < 0.01;
G. tikvahiae, F = 3.00. ns) and 1C) percent nitrogen (C. vagabunda. F = 34.56, P < 0.001 : G. tikvahiae, F = 0.99, ns) versus nitrogen load.

ECOLOGY, BIOGEOCHEM1STRY. AND POPULATION BIOLOGY

22.5

standing crop of G. tikvahiae responds less to nitrogen supply
than to seasonal changes in light availability. The increase of
chlorophyll a and carotenoid concentrations in C. vagabunda
and G. tikvahiac in response to high irradiance is paralleled
by biomass data taken from the estuaries (E. Stieve, unpub.
data). The biomass of G. tikvahiae was greater than that of
C. vagabunda in low-nitrogen estuaries that also furnished high
irradiance to algae.

The physiological changes in photosynthetic pigments and ni-
trogen concentrations created by increased loads suggest increased
growth of at least the green alga C. vagabunda. Increased nitrogen
load may also increase phytoplankton standing crop, increasing
light attenuation in the water column and therefore decreasing the
growth of light-limited benthic algae. On balance, the growth-
stimulating effect of increased nutrients seems to more than com-
pensate for the detrimental effect of light attenuation from the
influence of phytoplankton shading at the time of sampling; and
C. vagahiuula, in particular, proliferates and causes macroalgal
blooms in nitrogen enriched estuaries.

This research was supported by the Woods Hole Marine Science
Consortium. Special thanks to Jennifer Hauxwell for help with
algae and Jennifer Bowen for her endless patience.

Literature Cited

Ruecker, J., .1. Kohl, and K. Kaiser. 1995. Arch. Hnlrohiol.

(Sii/v/.) 108: 51-65.

Peckul, P., and J. Rivers. 1996. Estuarine Coaxial Shelf Sci. 43:

311-32?.

Peckol. P., and J. Rivers. 1995. / Phycnl. 31: 229-232.

Peckol, P., B. DeMeo-Anderson, J. Rivers, I. Valiela, M. Maldo-

nado, and J. Yates. 1994. Mm: Biol. 121: 175-185.

Bird, K., C. Hahig. and T. DeBusk. 1982. J. Phycol. 18: 344 -34S.

Valiela, 1., K. Foreman, M. LaMontagne, D. Hersh, J. Costa, P.

Peckol. B. DeMeo-Anderson, C. D'Avanzo, M. Babione, C.-H.

Sham, J. Brawle>, and K. Lajtha. 1992. Extiiaricx 15: 443-457.

Figueroa, F., S. Soluna, .1. Aguilera, C. Jimenez, J. Mercado, B.

Vinegla, A. Flores-Mova, and M. Altamirano. 1997. Mm: Ecol.

Prog. Ser. 151: 81-40.

Beer, S., and A. Eshel. 1985. Aust. J. Mm: Fre.ilm: Rex. 36:

785-792.

Jeffrey, S., and G. Humphrey. 1975. Biochcm. Phyxiol. Pftan:..

167: 191-144.

Dawes, C. 1998. Pp. 380-382 in Marine Botany . John Wiley. New

York.

Lapointe, B. 1985. ./. Exp. Mai: Bit,!. Ecol. 93: 21 1-222.

Reference: Biol. Bull. 199: 225-226. (October 2000)

Differences in Properties of Salt Marsh Sediment Between Hayed and Reference Sites

Adena Greenhaum (Wellesley College) and Anne Giblin:

The practice of haying salt marsh grasses began in colonial
times. Early settlers began harvesting marsh grasses for fodder,
and the practice has continued to the present ( I ). Current haying
techniques remove more than 90% of aboveground plant biomass,
and could have a number of effects on processes within the marsh.
Salt marsh food webs are based on detritus, so it can be hypoth-
esized that removal of plant biomass could alter food webs. Nu-
trient cycles, benthic algal biomass. microbial processes, and spe-
cies composition could also be affected (2, 3). This study
examined the effect of detritus removal on several sediment prop-
erties to assess the long-term effects of haying.

Sediment cores were taken from Plum Island Sound intertidal
marsh, a long-term ecological research site located in northeastern
Massachusetts. To study the effects of detritus removal, we mea-
sured several characteristics of sediment in areas where the marsh
grass is hayed by commercial farmers. We compared the results to
those of reference areas, which are not hayed. We sampled two
areas that are hayed every other year, a practice the commercial
farmers recognized produced the highest hay yield.

Duplicate cores were taken from each of two hayed and refer-
ence sites. All cores were taken from high marsh areas that were
heavily dominated by Spartina patens. Measurements of the fol-
lowing sediment characteristics were taken at 2 cm intervals above
10 cm and 5 cm intervals below 10 cm, to a depth of at least 25 cm.
Bulk density, a measure of soil density, was expressed as ratio

1 Ecosystems Center, Marine Biological Laboratory, Woods Hole. MA.

between the weight and volume of sediment; percent organic-
matter was measured by loss of ignition; total sulfur was measured
using a LECO sulfur analyzer; sedimentation rates were calculated
using Pb profiles (4); and total phosphorus was measured using the
technique of Krom and Berner (5). These properties were mea-
sured to examine sediment composition.

There was no significant difference in bulk density between
surface sediments in the hayed and reference sites (Fig. 1). Bulk
densities for both the hayed and reference areas decreased from a
range of 0.37 to 0.34 g cm ' at the surface to 0.22 g cm""3 at 12.5
cm. Below this depth, there was a slight difference in bulk density
values. The hayed areas increased to a maximum of 0.34 g cm '
at 23 cm. However, the bulk density of the reference sites re-
mained around 0.23 g cm 3. Judging from surface values for bulk
density, current haying practices apparently do not compact the
sediments.

Percent organic matter was similar between the managed and
natural areas to a depth of 12.5 cm. Values ranged from 31.7% to
44.5% organic matter. Deeper sediment samples of reference plots
had a slightly higher percent organic matter than the hayed sites,
but the difference between them was not significant. This indicated
that the removal of biomass from the hayed sites does not affect
organic matter content.

Haying did not appear to affect the total sulfur content in the
sediment. In both hayed and reference areas, total sulfur in-
creased from about 0.65% at the surface to a maximum of 2.2%
between 17 and 22 cm, and then decreased to about 1.55% at a
depth of 30 cm.

226

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

Bulk Density (g/cm )
00 0.2 04 0.6

Organic Matter (%)
20 40

Total Sulfur (%)
1 2

5 -

_ 10 •
~ 15
20 •
25 •
30

a
1

5 i

_ 10

c. 15

20 •

15 •
20 •
25
30

Pb (ug/g)
0 20 40 60 80 100

Total P (urrVg)
0 10 20 30 40 50

~ 10
o
S 15

D 20
25-

- - -A - - Reference Sites
— | Hayed Sites

Figure 1. Measures of bulk density, percent organic matter, total sulfur, sedimentation rate, and total phosphorus in ha\ed and reference sites of salt
marsh sediment in Plum Island Estuary.

Values between hayed and reference sites for percent total
sulfur, along with bulk density and percent organic matter deviated
slightly below 12.5 cm. This sediment was deposited more than 25
years ago, and since land practices before this time are unknown,
it is difficult to hypothesize reasons for the deviation.

Sedimentation rates can be estimated by using stable lead pro-
files to approximate dates of deposition. The lead profiles for
hayed and reference areas were not significantly different. Al-
though the cores were not deep enough to reach pre-industrial
background values, we used a previous study to establish back-
ground levels (Schmitt. unpub. data). Using both sets of data, we
calculated a sedimentation rate of 0.54 cm y~' for both the
reference and the hayed areas. The lack of differentiation in
sedimentation between the hayed and natural marsh is surprising
given that the haying process removes a large portion of the
aboveground biomass. One possible explanation is that most of the
organic matter making up the peat comes from other sources.
Belowground biomass could contribute significant amounts of
organic matter to detritus, especially since a large percent of
biomass in a salt marsh could be below ground. Organic matter
washed in with the tide could settle as detritus as well. The profile
for the hayed sites was more variable than that of the reference site.
Perhaps the tractor and trailer used for haying disturbs the surface
sediment as it travels over the marsh.

There was a significant difference between total phosphorus
measured in hayed and reference sites. The hayed areas had less
total phosphorus than the reference sites from the surface until 22.5

cm deep in the sediment, where the values for the two areas
converged. One explanation is that the input of phosphorus to the
marshes of the area is very low. and the periodic removal of
biomass from the system eventually leads to a measurable loss in
phosphorus.

Of the sediment properties we examined — bulk density, percent
organic matter, total sulfur, sedimentation rate, and total phospho-
rus— only the last was affected by harvesting the aboveground
biomass every other year. Current haying practices on the marsh
did not significantly alter most of the properties we measured.
However, other processes in the marsh could be affected by the
decrease in phosphorus, and other systems could respond differ-
ently to comparable practices.

This research was supported by the Plum Island Sound LTER
and a Research Experience for Undergraduates NSF fellowship.
Thanks to Linda Deegan, Hap Garritt, and Nat Weston for advice
and assistance with sampling.

Literature Cited

1 . Teal, J., and M. Teal. 1969. Lift- ami Death of the Suit Marsh. Pp. 2 1 ,
183.

2. Knapp, A. K.. and T. R. Seastedt. 1986. Bioscience 36: 662-668.
3 Bouchard, V., and J. Lefeuvre. 2000. Aquat. Bot. 67: 23-42.

4. Ng, A., and C. Patterson. 1982. Geochem. Cosmochim. Ada 48:

2307-2321.
5 Kroni, M. D., and R. A. Berner. 1981. Geochem. Cosmochim. Acta

45: 207-216.

ECOLOGY, BIOGEOCHEMISTRY, AND POPULATION BIOLOGY
Reference: Bio/. Bull. 199: 227-228. (October 2000)

227

Population Genetic Structure of the Goosefish, Lophius ainericanus

Hemant M. Chikarmane, Alan M. Ku-irian {Marine Biological Laboratory, Woods Hole, Massachusetts),

Robbin Kozlowski1, Mark Kuzirian,2 and Tony Lee3

Lophius ainericanus Cuvier & Valenciennes 1837 (1). the
goosefish, anglerfish, or monkfish, is common in coastal waters of
the northeastern United States. Its geographic range extends from
the northern Gulf of St. Lawrence south to Cape Hatteras, North
Carolina (2, 3). The highest fish concentrations are found along the
shallower depths of the shelf from 70 to 100 m, but there is also a
significant deep-water population below 190 m. Adult fish migrate
seasonally in response to spawning, food availability, and optimal
temperatures (3°-9°C) (2). The species is also dispersed through
the drifting of egg rafts. Total dispersal time from embryonic
development through larval and juvenile stages can extend to
several months until benthic recruitment occurs. Sexual maturity is
reached between 3 and 4 years of age (3).

Goosefish is the fourth largest commercial species in the U.S.
fishery, and number one in demersal species landings. Goosefish
landings have risen steeply through the 1980s, reaching approxi-
mately 28,800 mt ($35 million) for 1997 (4). Since the 1980s, the
Canadian contribution to the fishery has declined precipitously,
and now the major landings occur in the southern regions of the
species range. In their autumn survey data, the Northeast Fisheries
Science Center, Woods Hole, Massachusetts, has documented
recent sharp declines in goosefish abundance, from 2.24 kg/tow in
1986 to 0.74 kg/tow in 1996. The New England and Mid-Atlantic
Fishery Management Councils (NEFMC and MAFMC) conse-
quently designated goosefish as overexploited and at low abun-
dance (5). The 23rd Stock Assessment Workshop at the Northeast
Fisheries Science Center concluded that it was not possible to
delineate the stock structure for goosefish because of the lack of
genetic, tagging, or migration studies. Nevertheless, the Councils
divided the coastal population into northern and southern stocks
(41°N latitude) for stock management purposes. This formula led
to fishing restrictions being placed geographically, and made cer-
tain areas uneconomical to fish. Because of the lack of definitive
stock data for goosefish (5), we undertook a population genetic-
study of goosefish in eastern waters from the Canadian border to
North Carolina. We used random amplification of polymorphic
DNA and PCR (RAPD-PCR) (6) to analyze the genetic structure
of the sampled populations.

Eight representative sampling sites were chosen, extending from
Maine (42°40' N, 68°20' W) to North Carolina (35°40', 75°00'),
from depths to about 300 m. Fish were collected from September
1999 to June 2000. Up to 45 fish were sampled at each location.
Tissue samples were collected in tissue preservation buffer (7).
Genomic DNA was purified by standard phenol-chloroform pro-
cedures, and was finally dissolved in Tris-EDTA (TE) buffer (8).
DNA fingerprinting was performed by RAPD-PCR (6), using 10

1 Cape Cod Community College, W. Barnstable, MA.

2 University of Rhode Island, Kingston, RI.

3 Duke University. Durham, NC.

/nl per reaction. Amplification products were separated by electro-
phoresis on 1.2% agarose gels in 0.5x TBE (8). Gels were stained
with ethidium bromide and photographed under UV light. The
presence or absence of amplification products was scored manu-
ally. Cluster analysis was performed with the RAPDistance pack-
age (9).

Six fish, three each from Georges Bank and New York/New
Jersey sites were first screened with the seven primers shown in
Table I. As expected, the number of amplification products per
primer varied, ranging from 2 to 9. and very few bands were
polymorphic (Table 1 1. On the basis of the initial screening, a
subset of 6-8 DNA samples from each site was analyzed, using
primers 101 and 103; Figure 1 shows data for primer 103.

There appeared to be no significant differences between indi-
viduals or between populations, with either primer. Polymorphic
bands were present in a minority of individuals, usually one or
two. A set of eight fish collected off Martha's Vineyard, Massa-
chusetts, by the Marine Biological Laboratory, was examined with
an additional set of primers (115, 119. 130. and 143). Again, the
band distribution was very homogeneous (data not shown). Of the
22 identifiable bands produced by these primers, 21 were present
at a frequency of 100%. Band 22 was present at a frequency of
58%. All the MBL samples were clustered as one group by the
RAPDistance package. These results taken together imply that the
fish populations are relatively homogeneous genetically across all
geographic sampling sites, the level of polymorphism within pop-
ulations being as low as that between populations. Fish caught at
shallower (<200 m) depths could not be differentiated from those
at lower (>200 m) depths, neither could those collected north or
south of the 41°N line. For the primers tested, there was no
amplification product (or the absence of one) that uniquely char-
acterized a particular population. The trend in the data is clear even
though only a subset of samples was analyzed with two primers.
We are currently examining the entire sample set with more
primers to reinforce the validity of our results.

Table I

RAPD primer sequences and polymorphic bands for Lophius americanus

Primer Sequence

Number of bands Polymorphic bands

101

GCGGCTGGAG

103

GTGACGCCGC

104

GGGCAATGAT

105

CTCGGGTGGG

106

CGTCTGCCCG

107

CTGTCCCTTT

108

GTATTGCCCT

The primer numbering system and sequence is from the University of
British Columbia RAPD primer kits ( 10).

22X

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

NY/NJ

Figure 1. Representative RAPD-PCR profile* with Primer 103 fur individual goosefish samples from indicated locations. GB-Georges Bank,
NY/NJ-New York/New Jersey, NC-North Carolina. ME-Maine.

The homogeneity of the goosefish populations off the eastern
coastline of the United States suggests that there is unrestricted
gene flow across the region. This is very plausible considering the
preferred temperature profile and migratory patterns of the adults,
and the long dispersal times of the embryos, larvae, and postlarval
juveniles (2. 3). These data will have serious implications for
management of the goosefish fishery. The study results run counter
to the current NEFMC/MAFMC policy of dividing the fishery into
northern and southern stocks. Any management plan will be dif-
ficult to implement because the spawning stock biomass is un-
known. More data is also needed to determine the location of the
standing reproductive population, and to assemble specific tempo-
ral data on when spawning occurs over the fish's geographic range.
Integration of the published data on seasonal abundances (NMFS
Spring/Autumn Bottom Trawl Surveys) with yearly temperature
profiles along the coastlines might suggest some possible avenues
to pursue these answers. Such data will assist in defining the
natural and fishing mortality rates (F) and what the Flhreshold should
realistically be for this commercially important species.

This work was supported in part by the Monkfish Defense Fund.
H.M.C. and A.M.K. are indebted to Kathy Downey of the MDF for
acquainting them with the problems of the goosefish fishery, and
for organizing the fishermen for sample collection. We thank the
Aquatic Resources Division, MBL, for collecting some goosefish
used in this study.

Literature Cited

Histoire Naturelle des

1. Cuvier, G., and A. Valenciennes. 1837.

Poissons. 12. Bertrand. Paris.

2. Grosslein, M. D., and T. R. Azarovitz. 1982. MESA New York
Bight Atlas Monograph. N.Y. Sea Grant Institute. Albany, NY.

3 Bigelow, H. B., and W. C. Schroeder. 1953. Fish. Bull. 74: 53.

4. NOAA technical memo. 1999. Our Living Oceans. Report on the
Status of U.S. Living Marine Resources. U.S. Department of Com-
merce, National Oceanic and Atmospheric Administration, National
Marine Fisheries Service. Washington, DC.

5. Idoine, J. 1998. Pp. 88-89 in Status of Fisher,' Resources off the
Northeastern United Stales for /W,V. S. H. Clark, ed. U.S. Department of
Commerce, National Marine Fisheries Service, Woods Hole, MA.

f.. Williams. J. G. K., A. R. Kubelik. K. J. IJvak, J. A. Rafalski, and
S. V. Tingey. 1990. Nucl. Acids Res. 18: 6531-6535.

7. Asahida. T., T. Kobayashi, K. Saitoh, and I. Nakayama. 1996.
Fish. Sci. 62: 727-730.

8. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular
Cloning. A Laboratory Manual. Cold Spring Harbor Press. Cold
Spring Harbor, NY.

9 Armstrong J. S., A. J. Gibbs, R. Peakall, and G. Weiller. 1994.
The RAPDistance Package. ftp://life.anu.edu.au/pub/RAPDistance [21
Aug. 2000).

K). Primer Kits. NAPS Unit, University of British Columbia Biotechnol-
ogy Laboratory, http://www.biotech.ubc.ca/services/naps/primers.html
[21 Aug. 2000].

PUBLISHED BY TITLE ONLY

229

Published by Title Only

Bewley, Arnaud

Variations in white and black oaks as a function of their
distance from the coast.

Chiao, Chuan-Chin, and Roger Hanlon

Visual mechanism of body patterning in young cuttlefish,
Sepia pharaonis.

Dayel, Mark

Analysis of keratocyte motility and fine structure.

Haines, Jamie, Matthew Cieri, and Linda Deegan

Food choice convergence of benthic and pelagic fishes
along an estuarine gradient.

Hinckley, Eve-Lyn

From forest to shore: the path of total dissolved nitrogen
in a pristine coastal system, Martha's Vineyard, Massa-
chusetts.

Horowitz, Julie, Linda Deegan, and Robert Garritt

Stable isotope analysis of detrital food webs in hayed and
imhayed salt marsh.

Janowitz, Tobias, P. Koulen, and B. E. Ehrlich

Characterization of ryanodine receptors from zebrafish
skeletal muscle.

Kreitzer, Matthew, Naomi Rosenkranz, and Robert
Paul Malchow

Effects of extracellular ATP on skate retinal horizontal
cells.

Schwartz, Jesse, and Les Kaufman

Mechanistic mass-balance models and community ecol-
ogy.

Sergeant, Kara, Matthew Cieri, and Linda Deegan

Bivalve grazing pressure on primary producers in a New
England estuary.

Silver, Robert B., and Steven A. Farber

Calcium-independent perinuclear phospholipase A2 and
leukotriene B4 regulate the calcium signal required for
nuclear envelope breakdown in sand dollar (Echinarac-
nius parma) cells.

Wolf, Amelia, Craig Tobias, and Bruce Peterson

Dissolved inorganic nitrogen uptake kinetics and relative
preference indices for estuarine primary producers.

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BIOLOGICAL
BULLETIN

DECEMBER 2000

Published I >\ I IK \l.u iuc I'm .1. ,-i« .il I .ibi >i .11. .1 \

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THE

BIOLOGICAL BULLETIN

DECEMBER 2000

Editor
Associate Editors

Section Editor
Online Editors

Editorial Board

Editorial Office

MICHAEL J. GREEKBERG

Louis E. BURNETT
R. ANDREW CAMERON
CHARLES D. DERBY
MICHAEL LABARBERA

SHINYA INDUE, Imaging and Microscopy

JAMES A. BLAKE, Keys to Marine
Invertebrates of the Woods Hole Region
WILLIAM D. COHEN, Marine Models
Electronic Record and Compendia

PAMELA CLAPP HINKLE
VICTORIA R. GIBSON
CAROL SCHACHINGER
CAROL MARRAMA

The Whitney Laboratory. University of Florida

Grice Marine Biological Laboratory, College of Charleston
California Institute of Technology
Georgia State University
University of Chicago

Marine Biological Laboratory

ENSR Marine & Coastal Center. Woods Hole

Hunter College. City University of New York

University of California, Davis

Bodega Marine Lab., University of California, Davis

Louisiana State University

Oregon Institute of Marine Biology, Univ. of Oregon

Hopkins Marine Station. Stanford University

Cereon Genomics, Cambridge, Massachusetts

Hiroshima University of Economics, Japan

University of North Carolina Greensboro

University of Southern California

Kewalo Marine Laboratory. University of Hawaii

Institute of Neurobiology, University of Puerto Rico

Tokyo Institute of Technology. Japan

National Institute for Basic Biology. Japan

Marine Biomed. Inst.. Univ. of Texas Medical Branch

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Published by

MARINE BIOLOGICAL LABORATORY
WOODS HOLE, MASSACHUSETTS

Library

JAN 5 2001

Cover

The sacculinid rhizocephalans are parasitic crusta-
ceans related to barnacles (class Cirripedia): their
hosts are primarily decapod crustaceans. The image
on the cover shows a portunid crab (Charybdis
longicollis, Leene) on its back; and protruding into
the space under the crab's ventrally recurved abdo-
men are six young externae of the sacculinid Het-
erosaccus tlollfusi Boschma. For such a large num-
ber of parasites to inhabit a single host is unusual,
since a mature externa can be 2 cm long, and the
maximum width of the crab is 6.6 cm. The speci-
men was collected off the Mediterranean coast of
Israel at a depth of 35 m, but both species - the host
and the parasite - are invaders from the Red Sea.
The photograph was taken by B. Galil and D. Fried-
man, both affiliated with the National Institute of
Oceanography in Haifa.

The protruding externa is the only externally visible
part of the parasite and is its reproductive compo-
nent; when young, it contains the female gonad and
two empty sperm sacs. The externa is produced by
the interna. a mass of rootlets that ramify through-
out the body of the host, obtaining nutrition for the
parasite. The adult sacculinid has virtually no ar-
thropod-like features, and its relationship with free-
living barnacles is based largely on a common
larval stage - the cypris, which develops from a
pelagic nauplius larva. If she settles on a crab, a
female cypris larva metamorphoses into a device

that injects a mass of undifferentiated cells into the
body of the host; the interna develops from these
cells. Once the externa appears, two male cypris
larvae may settle on it, each entering a sperm sac to
form a testis, enabling fertilization. The ramifying
rootlets of the interna also modify the morphology,
physiology, and behavior of the host. In particular,
the abdomen of male crabs becomes broader like
that of a female, providing protection for the ex-
terna; and both male and female crabs are sterilized.
A parasitized crab of either sex ventilates and
grooms the externae under its abdomen as it would
its own eggs, and assists in the dispersion of the
sacculinid' s nauplius larvae when they are released
after fertilization.

The extravagant life history of the sacculinid rhizo-
cephalans is one of a series of case studies that
illustrate B. Rinkevich's reevaluation of the "unit of
selection" (UOS) in this issue (p. 231 ). The concept
that natural selection operates upon a particular
biological entity or entities - the UOS - is straight-
forward, but attempts to identify these entities un-
ambiguously have led to controversy. In his essay,
Rinkevich adopts the principle that any biological
entity, regardless of its morphological level of or-
ganization, can constitute all, or even a part, of a
UOS. The sacculinids, comprising three allogeneic
units of selection (two males and a female), are used
to argue that multiple conspecific UOSs can, to-
gether, constitute a new morphological unit.

CONTENTS

VOLUME 199. No. 3: DECEMBER 2000

EVOLUTION

Rinkevich, B.

A critical approach to the definition of Darwinian
units of selection -31

NEUROBIOLOGY AND BEHAVIOR

Leise, Esther M., and Michael G. Hadfield

An inducer of molluscan metamorphosis transforms
activity patterns in a larval nervous system 241

Finley, Luke, and David Macmillan

The structure and growth of the statocyst in the
Australian crayfish Chemx drstructor 251

Jury, Steven H., and Winsor H. Watson HI

Thermosensitivity of the lobster, Homariis americamu,

as determined by cardiac assay 257

Cromarty, S. I., J. Mello, and G. Kass-Simon

Molt-related and size-dependent differences in the
escape response and post-threat behavior of the
American lobster, Homarus americanus 265

CELL BIOLOGY

Baghdasarian, Garen, and Leonard Muscatine

Preferential expulsion of dividing algal cells as a mech-
anism for regulating algal-cnidarian symbiosis 278

DEVELOPMENT AND REPRODUCTION

Komm.imi. Tetsuya, and Hiromi Takata

Cellular basis of gasti illation in (he sand dollar
Scaphechinus mimhili\ 287

Vickery, Minako S., and James B. McClintock

Effects of food concentration and availability on the
incidence of cloning in planktotrophic larvae of the
sea star Pinaster iirhmceus 298

Dickinson, Amanda J. G., Roger P. Croll, and Elena E.

Voronezhskaya

Development of embryonic cells containing seroto-
nin, catecholamines, and FMRFamide-related pep-
tides in AfilyMii califarnica 305

RESEARCH NOTE

Takeda, Naokuni

Development of a penis from the vestigial penis in

the female apple snail, Pomacea canaliculata 316

Index for Volume 199 321

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CONTENTS

for Volume 199

No. 1 . AUGUST 2000

RESEARCH NOTE

Bolton, Toby F., Florence I. M. Thomas, and Celere N.
Leonard

Maternal energy investment in eggs and jelly coats sur-
rounding eggs of the echinoid Arbaria punctulata .... 1

PHYSIOLOGY

Johnson, M. L., P. M. J. Shelton, E. Gaten, and P. J.
Herring

Relationship of dorsoventral eyeshine distributions
id habitat depth and animal size in mesopelagic de-
capods I'

Dietz, T. H., A. S. Udoetok, J. S. Cherry, H. Silvernian,

and R. A. Byrne

Kidney function and sulfate uptake and loss in ihc
freshwater bivalve Toxolasma texasensis 14

Vollmer, Steven V., and Peter J. Edmunds

Allometric scaling in small colonies of the scleraiiin-

ian coral Sitlrmslmi siderm (Ellis and Solander) .... 21

DEVELOPMENT AND REPRODUCTION

Torrado, Mario, and Alexander T. Mikhailov

Frog Lim-1-like protein is expressed predominantly
in the nervous tissue, gonads, and early embryos of

the bivalve mollusc M\tilit\ galloprovincialii 29

Gibson, Glenys D., and Jennifer M. L. Harvey

Morphogenesis during asexual reproduction in I\-

gosf>i<i r/rtwjs Claparede (Annelida, Polychaeta) ....

Watson, G. J., F. M. Langford, S. M. Gaudron, and
M. G. Bentley

Factors influencing spawning and pairing in the scale
worm Hannothoe imbricala (Annelida: Polychaeta) . . .

Bucldand-Nicks, John, and Alan N. Hodgson

Fertilization in Callochiton insi/nii'iis (Mollusca)

Hirose, M., R. A. Kinzie III, and M. Hidaka

Early development of zooxanthella-containing eggs
of the corals Pixi/ln/i/nii ivrruioMi and P. eydouxi with
special reference to the distribution of zooxanthellae

CELL BIOLOGY

Wakefield, Timothy S., Mark A. Farmer, and Stephen C.
Kempf

Revised description ol the fine structure of in situ
"Zooxantheilae" genus Symbiodinium 7l>

ECOLOGY AND EVOLUTION

Barnes, David K. A., and Mathew H. Dick

Overgrowth competition between clades: implica-
tions for interpretation of the fossil record and over-
growth indices 85

41 Annual Report of the Marine Biological Laboratory. .

No. 2, OCTOBER 2000

RESEARCH NOTE

PHYSIOLOGY

Hourdez, Stephane, Jason Lamontagne, Pat Peterson,
Roy E. Weber, and Charles R. Fisher

Hemoglobin from a deep-sea hydrolhei mal-vent
copepod 95

Marsh, Adam G., Patrick K.K. Leong, and Donal T.
Manahan

Gene expression and enzyme activities of the sodium
pump during sea urchin development: implications

for indices of physiological state 10(1

Hill, Richard W., John W.H. Dacey, and Ahser Edward
Dimethylsulfoniopropionate in giant clams (Tridac-
nidae) 108

CONTENTS: VOLUME 199

ECOLOGY AND EVOLUTION

Baker, Shirley M., Jeffrey S. Levinton, and J. Evan Ward

Particle transport in the zebra mussel, Dreissena puly-

morpha (Pallas) 116

Lajeunesse, T.C., and R.K. Trench

Biogeography of two species of Symhindinium
(Freudenthal) inhabiting the intertidal sea anemone
Aiithopltura elrgniittwmii (Branch) 12h

NEUROBIOLOGY AND BEHAVIOR

Rosendial, Joshua J.C., and Francisco Bezanilla

Seasonal variation in conduction velocity of action
potentials in squid giant axon 135

DEVELOPMENT AND REPRODUCTION

Misamore, M.J., and J.W. Lynn

Role of the cvtoskeleton in sperm entry during
fertilization in the freshwater bivalve Drmsena
polymorpha ... 144

SHORT REPORTS FROM THE 2000 GENERAL

SCIENTIFIC MEETINGS OF THE MARINE

BIOLOGICAL LABORATORY

Hitt, James M., Frederick A. Dodge, Ehud Kaplan, and
Robert B. Barlow

C.ircadian rhythms in the receptive fields of the Litnu-

ln\ lateral eye 171

Fay, Richard R., and Peggy L. Edds-Walton

Frequency response of auditory brainstem units in
toadfish (O(u>anuf ttiu) 173

Yamagnchi, Ayako, Leonard K. Kaczmarek, and Darcy

B. Kelley

Intrinsic membrane properties of laryngeal 1110-
toneurons that control sexually differentiated vocal
behavior in African clawed frogs, Xenopiu laevis .... 175

Atherton, Jillian L., Matthew A. Krutky, James M. Hitt,

Frederick A. Dodge, and Robert B. Barlow

Optic nerve responses of Limului in its natural habi-
tat at night 176

Krutky, Matthew A., Jillian L. Atherton, Spence Smith,

Frederick A. Dodge, and Robert B. Barlow

Do the properties of underwater lighting influence

the visually guided behavior of Limulm? 178

Hale, Melina E.

Startle responses of fish without Mauthner neurons:
escape behavior of the Inmpfish (Cydopterus lii>njni\) ISO

Epstein, David A., Herman T. Epstein, Frank M. Child,

and Alan M. Kuzirian

Memory consolidation in Hermissenda crassicornis . . . 182

Abenavoli, A., L. Forti. and A. Malgaroli

Mechanisms of spontaneous miniature activity at
CA3-CA1 synapses: evidence for a divergence from a
random Poisson process 184

/•/..I I'I'RKU

i,} A\/> BIOCHEMISTRY

The Editors

Introduction to the featured report. On mapping
odor quality' 161

Wachowiak, Matt, Michal Zochowski, Lawrence B.

Cohen, and Chun X. Falk

The spatial representation of odors by olfactory re-
ceptor neuron input to the olfactory bulb is concen-
tration invariant 162

NEUROBIOLOGI

Landowne, David

Heavy water (D.,O) alters the sodium channel gating
current in squid giant axons 164

White, Thomas H., Harris Ripps, Midiituru Srinivas,

and Roberto Bruzzone

Voltage gating properties of channels formed by a
skate retinal connexin 165

Molina, Anthony J.A., Peter J.S. Smith, and Robert Paul

Malchow

Hydrogen ion fluxes from isolated retinal horizontal
cells: modulation bv glutamate 168

Wang, Jing W.

Odor-induced oscillatoiy activity in l)i»\npliil<i (INS ... 170

Novales Flamarique, Inigo, Kristiina Ovaska, and
Theodore M. Davis

IT-B induced damage to the skin and ocular system

of amphibians 187

Harrington, John M., and Peter B. Armstrong

Initial characleri/ation of a potential ami-fouling sys-
tem in the- American horseshoe crab, l.nnii/ii\
t,,,lyl>l,>;n,n 1*9

Asokan, Rengasamy, Margaret T. Armstrong, and Peter

B. Armstrong

Association of a.,-macroglobulin with the coagulin
clot in (he American horseshoe crab, I.iimilin
pnlyplii'iinn. a potential role in stabilization Irom pro-
teolvsis 190

Kuhns, William J., Max M. Burger, Mohan Sarker,

Xavier Fernandez-Busquets, and Tracy Simpson

Enzymatic biosynthesis of N-linked glycan bv the' ma-
rine sponge M/inii/inir/ /nnli/mi 192

Armstrong, Peter B., and Rengasamy Asokan

A Ca4 "'-independent cytolytic system from the blood

of the marine snail liii\\n»i canaliculum 194

Heck, Diane E., Lydia Louis, Michael A. Gallo, and

Jeffrey D. Laskin

Modulalion <>l the development of plutei bv nitric
oxide in the1 sea urchin .\ilnmn /niiirliilnln 195

CONTENTS: VOLUME 199

Jung, Sung-Kwon, Katherine Hammar, and Peter J.S.
Smith

Development of self-referencing oxygen microsensor
and its application to single pancreatic HIT cells:
effects of adenylate cyclase acdvator forskolin on ox-
ygen consumption

Hanselmann, Rhea, Roxanna Smolowitz, and Daniel G.

Gibson

Identification of proliferating cells in hard clams. . .

Brothers, Christine, Ernest Marks III, and Roxanna

Smolowitz

Conditions affecting the growth and zoosporulation
of the protistan parasite QPX in culture

199

200

Burgos, Mario H., Makoto Goda, and Shinya Inoue
Fertilization-induced changes in the fine structure of
stratified Arbariti eggs. II. Observations with electron
microscopy 213

Gould, Robert M., Concetta M. Freund, John Engler,

and Milan G. Morrison

Optimization of homogenization conditions used to
isolate mRNAs in processes of myelinating oligoden-
drocytes 9)5

ECOLOGY, BIOGEOCHEMISTRY, A\D POPULATION BIOLOGY

CELL BIOLOGY

Sandberg, Leslie, Phillip Stafford, and George M.
Langford

Effects of myosin-II antibody on actin-dependent ves-
icle transport in extracts of clam oocytes 202

Stafford, Phillip, Jeremiah Broun, and George M.

Langford

Interaction of actin- and microtubule-based motors
in squid axoplasm probed with antibodies to myosin
V and kinesin 203

Tran, P.T., V. Doye, F. Chang, and S. Inoue

Microtubule-dependent nuclear positioning and nu-
clear-dependent septum positioning in the fission
yeast, Saccharomyces pombe 205

Crawford. Karen

The role of microtubules during blastodisc forma-
tion of the squid Loligo pealei 207

Weidner, Earl

Cytoplasmic proteins on the surface of discharged
microsporidian sporoplasms 208

Mackenzie, Roger, David Newman, Max M. Burger,

Rene Roy, and William J. Kiihns

Adhesion of a viral envelope protein to a non-self-
binding domain of the aggregation factor in the
marine sponge Mnrorionn fm>lifii~ri 209

Kirkby, Ryan, Luc Claessens, Charles Hopkinson, Jr.,
Edward Rastetter, and Joseph Vallino

Modeling the effects of land-use change on nitrogen
biogeochemistry in the Ipswich watershed, Massachu-
setts 218

Perring, Anne. Michael Williams, Charles Hopkinson,

Jr., Edward Rastetter, and Joseph Vallino

Solute dynamics in storm flow of the Ipswich River
Basin: effects of land use 219

Westgate, Elizabeth J., Kevin D. Kroeger. Wendy J.

Pabich, and Ivan Valiela

Fate of anthropogenic nitrogen in a nearshore Cape
Cod aquifer 221

Denault, Michelle. Erica Stieve. and Ivan Valiela

Effects of nitrogen load and irradiance on photosyn-
thetic pigment concentrations in Cladophora vaga-
lni>idaa.nd Gmcilaria tikvahiaem estuaries of Waquoit
Bav 223

Greenbaum, Adena, and Anne Giblin

Differences in properties of salt marsh sediment be-
tween hayed and reference sites 225

Chikarmane, Hemant M., Alan M. Kuzirian, Robbin

Kozlowski, Mark Kuzirian, and Tony Lee

Population genetic structure of the goosefish, Lo-
pluus amrricanus 227

OIUL PRESENTATIONS
Published bv tide onlv. . .

No. 3, DECEMBER 2000

EVOLUTION

NEUROBIOLOGY AND BEHAVIOR

Rinkevich, B.

A critical approach to the definition of Darwinian
units of selection 931

Leise. Esther M., and Michael G. Hadfield

An inducer of molluscan metamorphosis transforms

activity patterns in a larval nervous system 241

Finley, Luke, and David Macmillan

The structure and growth of the statocyst in the
Australian crayfish Cherax destructor 251

CONTENTS: VOLUME 199

Jury, Steven H., and Winsor H. Watson III

Thermosensitivity of the lobster, Homarus americanus,

as determined bv cardiac assay 257

Cromarty, S. I., J. Mello, and G. Kass-Simon

Molt-related and size-dependent differences in the
escape response and post-threat behavior of the
American lobster, Homarus americanus 265

CELL BIOLOGY

Baghdasarian, Garen, and Leonard Muscatine

Preferential expulsion of dividing algal cells as a mech-
anism for regulating algal-cnidarian symbiosis 273

DEVELOPMENT AND REPRODUCTION

Vickery, Minako S., and James B. McClintock

Effects of food concentration and availability on the
incidence of cloning in planktotrophic larvae of the
sea star Pisaster ochraceus 298

Dickinson, Amanda J. G., Roger P. Croll, and Elena E.

Voronezhskaya

Development of embryonic cells containing seroto-
nin, catecholamines, and FMRFamide-related pep-
tides in Aplysia californica 305

RESEARCH NOTE

Takeda, Naokuni

Development of a penis from the vestigial penis in

the female apple snail, Pomacea canaliculata 316

Kominami, Tetsuya, and Hiromi Takata

Cellular basis of gastruladon in the sand dollar
Scaphechmus mirabilis 287

Index for Volume 199 321

xii

ERRATA

The Biological Bulletin Volume 199, Number 2

In the article by P. T. Tran, V. Doye, F. Chang, and S. Inoue, which appeared on pages 205 to 206, the
genus name Saccharomyces is in error. The correct genus name, in all instances, is Schizosacchromyces.
Cover legend: The organism described as Saccharomyces pornbe should be Schizosacchromyces pombe.
The editors regret these errors.

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Reference: Biol. Bull. 199: 231-240. (December 2000)

A Critical Approach to the Definition of Darwinian

Units of Selection

B. RINKEVICH

Israel Oceanographic and Limnological Research, National Institute of Oceanography,
P.O. Box H030. Haifa 31080, Israel

Abstract. What are the biological units of selection? In
fact, the notion of "unit of selection" (UOS) is blurred by
ambiguity and controversy. To further evaluate the biolog-
ical entities that are the objects of natural selection, three
novel conceptual criteria (holism, minimalism, functional-
ism) are critically applied: they reveal, in addition to the
self-evident case of the "individual," at least six distinct
types of UOSs. These UOSs do not always have a defined
structural organization; they can be parts of a living organ-
ism, a cohesive group of conspecifics, a multiunit entity, a
totipotent cell, a DNA fragment, or a whole organism. UOS
types diversify by amalgamation or parcelation processes of
apparent entities. Therefore, previous attempts to character-
ize the UOSs solely on some morphological levels (gene,
individual, group) without applying stringent criteria have
failed to cope with the structural variations of natural phe-
nomena and have led to the ambiguity of terms used.

Introduction

Much of the ambiguity, confusion, and controversy en-
gendered by the concept of the "unit of selection" (UOS)
seem to arise from a failure to identify the biological entities
upon which natural selection operates (Sober and Wilson,
1994; Mayr. 1997; Gould and Lloyd, 1999, and literature
therein). Along with the debates about the three to four
possible organizational levels of selection (gene, individual,
group, and metapopulation). the objections to the hierarchi-
cal theory of selection (Wilson and Sober, 1994; Michod,
1997; Gould. 1998; Gould and Lloyd. 1999), and the dis-
tinction between transmitted units and those which transmit
(Wynne Edwards, 1962; Lewontin. 1970; Mayr. 1970.
1997: Dawkins. 1976; Hull. 1980; Gliddon and Gouyon,

Received 17 August 1999; accepted 11 September 2000.
E-mail: buki@ocean.org.il

1989; Sober and Wilson. 1994; Wilson and Sober. 1994;
Williams. 1996; Gould. 1998). "metaphors have replaced
the empirical world as foci for discussion while precise
meanings and derivatives have been forgotten in the pro-
cess" (Slobodkin. 1986). Even the basic term "unit of se-
lection" is under dispute (Wilson and Sober, 1994), bearing
polemic aspects (Mayr, 1997) as do other terms in this
discipline (Gould and Lloyd, 1999).

One approach to clarifying such an ambiguous field is a
critical evaluation of the arguments and definitions used
(Hull, 1980; Sober and Wilson. 1994; Mayr, 1997; Gould
and Lloyd. 1999). Such a reevaluation process might ger-
minate a novel idea or might help dispel excessive ambigu-
ity. On the other hand, anathematized concepts could reap-
pear, revealing further ill-considered definitions (Gould and
Lloyd, 1999) or adding additional ambiguities. An alterna-
tive approach is to envisage the main controversial issues
through an untraditional analysis. In this essay, such an
untraditional approach is used to examine the biological
entities that are the objects of natural selection. By adapting
the unbiased principle that any living thing can be all or part
of a potential UOS, we can critically evaluate organisms —
regardless of their level of morphological organization — on
the basis of a few conceptual criteria.

Criteria for Analysis of UOSs

Three conceptual criteria guide this examination:

Holism

Genes and soma are not necessarily independent. The
distinction between the terms "interactor" and "vehicle," as
opposed to "replicator" and "gene" (Dawkins, 1976; Hull,
1980) is central in the debate over UOSs (Hull. 1980; Sober
and Wilson. 1994; Mayr. 1997). The use of these terms to

231

232

B. RINKEVICH

identify different units of selection evolved from the a
priori rationale that living organisms are made of at least
two distinct types of evolutionarily selected units. Addition-
ally, the notion of the UOS has become ambiguous because
it was used to refer to either replicators or vehicles, depend-
ing on the choice of the author (Wilson and Sober, 1994). I
suggest that this rationale is false and misleading, that it
artificially distinguishes between "genes," "information,"
and "replication" on the one hand, and "soma," "vehicle."
and "interactor" on the other (Lewontin. 1970; Dawkins,
1976: Hull. 1980; Buss, 1982; Gliddon and Gouyon, 1989;
Sober and Wilson. 1994; Mayr. 1997). The genes in any
organism have a fate in common with their amalgamated
soma (Sober and Wilson, 1994, and literature therein). They
are part of a whole; they are not completely independent
(with the exception of specific cases as outlined in the next
section), but rather functionally integrated within the soma.
In physics, light and mass are regarded as two facets of
energetic matter. Similarly, in biology, genes and soma
should be regarded as two facets of an organic entity that
constitutes a living organism. Even the term "unit" (Oxford
Dictionary) embraces this metaphysical concept of holism.
A unit is a thing (individual, person, group, etc.) that is
complete or distinctive and that has the characteristics of the
complex whole. Following this rationale, the so-called rep-
licators and interactors of each entity are intermingled to
form, tor each UOS, its idiographic (its own peculiar) entity,
which is presented to natural selection as a coherent whole.
This is in contrast to the acknowledgement of recent years
that interactors, not replicators, constitute the causal unit of
selection" (Gould and Lloyd. 1999).

Minimalism

Ignore complex cases; choose the simplest ones. Addi-
tional ambiguity is caused by different hypotheses for the
UOS that deliver opposing predictions about the traits that
have evolved (Sober and Wilson, 1994; Wilson and Sober.
1994). In such cases, a search for the simplest manifestation
of the system, the minimalist approach (Slobodkin, 1986),
has been suggested to be the most useful in maintaining
clarity. This approach has been characterized as "the pro-
cess of deliberately choosing to work in the simplest pos-
sible mode that is still recognizable as part of an existing
professional field." Slobodkin ( 1986) has also discussed the
main objection against this approach as the claim for un-
critical acceptance of standards. However, this objection
may not be the case in the controversy over the UOS, where
metaphors, rather than empirical themes, dominate the sci-
entific discipline (Wilson and Sober. 1994; Gould and
Lloyd, 1999). When employing the minimalist approach
(Slobodkin, 1986), or the very similar "back to basics"
(Sober and Wilson, 1994) treatment, complex cases (such as
the situations illustrated in Wynne Edwards, 1962) are left

aside for future analyses when the field will presumably be
more formally organized. Therefore, we must accept the
idea that the UOS theory, almost three decades after it was
first elaborated (Lewontin, 1970), should still be conceptu-
alized through the clearest examples.

Functionalism

UOSs function in vivo. A unit of biological organization
upon which selection might act should be both an autono-
mous functional entity and physically and structurally co-
herent, even if it is in the form of a gene. It cannot be in the
form of "information" or "avatar" (Gliddon and Gouyon,
1989: Tuonii and Vuorisalo. 1989a) or "anything in the
universe of which copies are made" (Dawkins, 1989). A
UOS must function, because functionalism is the primary
focus of natural selection. Functionalism, therefore, does
not rest upon an active maintenance of distinctive properties
(Gould, 1998), but evaluates the general sum of independent
activities presented by a UOS. At this point, the existence of
only a single functional level or of several functional levels
(in hierarchical order. Tuomi and Vuorisalo, 1989a: Gould
and Lloyd, 1999; or not) will not be discussed. Only a
holistic unit (possessing cohesive structural and information
properties) may reveal the capacity for functionalism.
Therefore, previously distinguished UOSs such as replica-
tors, interactors. vehicles, memes. etc.. that are literally not
holistic, are excluded from being real UOSs. They remain as
highly justified theoretical paradigms that characterize only
components of holistic and functional units of selection.

The three conceptual criteria (holism, minimalism, func-
tionalism) provide enormous flexibility for analysis and
circumvent the use of ill-defined issues and debatable argu-
ments. These criteria have been used to scrutinize different
potential types of UOSs that are presented by a variety of
organismal entities. The term "organism" refers here to "any
biological entity whose parts have evolved to function in a
harmonious and coordinated fashion" (Wilson and Sober,
1994). This analysis has revealed several types of UOSs; of
these, one traditional and six new characteristic types (Table
1. Nos. 1-7) are briefly described below.

Seven Types of Units of Selection

/ inn — n/hl /n/rt of me ;'.v //

Molecular sequences may themselves be UOSs. "Doctor
there is a fly in my genome" was the title chosen by the
journal New Scientist (Vol. 149, p. 16, 1996) for an article
about a tiny fragment of an insect genome (called mariner.
a jumping gene first discovered in the fruit fly Dro.wphila)
that is embedded in human chromosome 17. This location
directly coincides with a recombination hot spot and has
been associated with distinct hereditary neurological syn-
dromes (Reiter et <//., 1996). This is only one of an enor-

Ine Biological Bulletin

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233

espeare. King Lear, Act I , Scene 4

Examples

ic DNA in eukaryotic and
lomes

i and mosaicism

tism

e-body chimerism and invertebrate

:>ed organisms, monozygotic twins.

ripedia
anisms

mous number of documentations that eukaryotic and pro-
karyotic cells carry foreign DNA molecules of various types
(plasmids, B chromosomes, t haplotypes. retroviruses, and
more), as well as diverse mobile DNA sequences (such as
transposons, retrotransposons, LINEs, SINEs. mobile in-
trons) that are transmitted vertically or horizontally within
genomes (Zeyl and Bell, 1996; Flavell. 1999) and may be
regarded as real UOSs. These DNA sequences have func-
tional and holistic properties: they are characteri/.ed by a
discrete organismal realm, function in a coordinated fash-
ion, and are clearly subject to natural selection forces. Many
examples now point to real UOSs situated within the ge-
nomes of other UOSs. A few will be outlined below.

One well-studied group is the B-chromosomes. a variety
of germ-line parasites described from more than a thousand
species of plants and animals. These small chromosomes do
not contribute to the regular functions of the host, and their
numbers per cell vary even within the same host organism.
More important, although they share the same nucleus with
regular chromosomes, they have evolved peculiar charac-
teristics of their own. By various non-Mendelian systems of
biased transmission and by their ability to move specifically
to one of the two products of the first meiosis division (such
as by avoiding penetration into the polar body during
oogenesis), they increase their representation in the germ-
line nuclei. The B-chromosomes in the wasp Nasonia.
which are transmitted solely through sperm, are a represen-
tative case. The entire parental set of chromosomes in an
infected zygote becomes condensed and is lost, leaving a
haploidized animal that develops as a male, transmitting the
B-chromosome to all its gametes (citations in Bell and Burt,
1990). Such functionalism of the parasitic entity reveals
distinct host and parasitic units of selection. Within this

context, I am reluctant to consider the B-chromosomes as
selfish chromosomes. They are distinct molecular UOSs.

The mouse t haplotypes (each extending over the proxi-
mal half of chromosome 17) also have developed the ability
to propagate at the expense of the wild-type homolog from
heterozygous males. These entities probably evolved from a
wild-type form of chromosome 17. Genes that were re-
cruited later on, together with the addition of accompanying
inversions, all increased the survival rates of the t haplo-
types, until finally these entities started "taking on a life of
their own" (reviewed in Silver, 1993).

Not only a whole piece of chromosome may be counted
as a UOS; even transposable genetic elements, gene size
segments of DNA. may be so considered. This field is too
broad to be even partially covered here, so only the most
relevant features of these mobile elements will be discussed.
Many transposable elements have the ability to jump from
place to place on the chromosomes; they can behave like
new introns creating novel intron processing patterns; they
may spread vertically and horizontally within host organ-
isms; and they can promote their own replication (the func-
tionalism component). With time, the mobile elements be-
come domesticated through full integration into the host's
genome. A good example is the mariner which, by being
functional in both germ lines and somatic cell lines, could
infect many organisms, crossing several phyletic borders
(arthropods, platyhelminths, nematodes, chordates), proba-
bly by splicing into viral or other pathogenic genomes.
During each introduction into a new host species, the mar-
ine'/- transposon was probably highly mobile and signifi-
cantly disruptive. With time, more and more defecting
transposons with mutations that disabled the cut-and-paste
enzyme were accumulated, littering eukaryotic genomes

232

identify different units of
priori rationale that living
two distinct types of evolutic
ally, the notion of the UOS
it was used to refer to either
ing on the choice of the autl
suggest that this rationale
artificially distinguishes be
and "replication" on the on
and "interactor" on the otl
1976; Hull. 1980; Buss, 19!
Sober and Wilson, 1994; I>
organism have a fate in cc
soma (Sober and Wilson, IS
are part of a whole; they ;
(with the exception of speci
section), but rather function
In physics, light and mass

energetic matter. Similarly, .„ ,,_,, ^ „

should be regarded as two facets of an organic entity that
constitutes a living organism. Even the term "unit" (Oxford
Dictionary) embraces this metaphysical concept of holism.
A unit is a thing (individual, person, group, etc.) that is
complete or distinctive and that has the characteristics of the
complex whole. Following this rationale, the so-called rep-
licators and interactors of each entity are intermingled to
form, for each UOS, its idiographic (its own peculiar) entity,
which is presented to natural selection as a coherent whole.
This is in contrast to the acknowledgement of recent years
that interactors, not replicators, constitute the causal unit of
selection" (Gould and Lloyd, 1999).

Minimalism

Ignore complex cases; choose the simplest ones. Addi-
tional ambiguity is caused by different hypotheses for the
UOS that deliver opposing predictions about the traits that
have evolved (Sober and Wilson, 1994: Wilson and Sober.
1994). In such cases, a search for the simplest manifestation
of the system, the minimalist approach (Slobodkin, 1986),
has been suggested to be the most useful in maintaining
clarity. This approach has been characterized as "the pro-
cess of deliberately choosing to work in the simplest pos-
sible mode that is still recognizable as part of an existing
professional field." Slobodkin (1986) has also discussed the
main objection against this approach as the claim tor un-
critical acceptance of standards. However, this objection
may not be the case in the controversy over the UOS, where
metaphors, rather than empirical themes, dominate the sci-
entific discipline (Wilson and Sober. 1994; Gould and
Lloyd. 1999). When employing the minimalist approach
(Slobodkin. 1986). or the very similar "hack to basics"
(Sober and Wilson, 1994) treatment, complex cases (such as
the situations illustrated in Wynne Edwards, 1962) are left

activities presented by a UOS. At this point, the existence of
only a single functional level or of several functional levels
(in hierarchical order. Tuomi and Vuorisalo, 1989a; Gould
and Lloyd. 1999; or not) will not be discussed. Only a
holistic unit (possessing cohesive structural and information
properties) may reveal the capacity for functionalism.
Therefore, previously distinguished UOSs such as replica-
tors, interactors. vehicles, memes. etc.. that are literally not
holistic, are excluded from being real UOSs. They remain as
highly justified theoretical paradigms that characterize only
components of holistic and functional units of selection.

Seven Types of Units of Selection

I am — and pan of me is it

Molecular sequences may themselves be UOSs. "Doctor
there is a fly in my genome" was the title chosen by the
journal New Scientist (Vol. 149. p. 16. 1996) for an article
about a tiny fragment of an insect genome (called manner,
a jumping gene first discovered in the fruit fly Droso/'lii/u)
that is embedded in human chromosome 17. This location
directly coincides with a recombination hot spot and has
been associated with distinct hereditary neurological syn-
dromes (Reiter el <//., 1996). This is only one of an enor-

DEFINING UNITS OF SELECTION
Table 1

233

— Shakespeare, King Lear. Act I. Scene 4

No.

Type of unit of selection

Organizational level on which
selection acts

Examples

I am — and part of me is it

I am — and part of me is he
I am — and this is actually he
I am — and this is actually we

I am — and this is actually only part of me

We are — and this is actually me
I am — that I am (Exodus 3:14|

On a molecular level, a piece of
DNA, usually not larger than a
single chromosome (B-
chromosomes, however, can pan
among themselves to form a
chiasmata)

On a whole organismal level

On a cellular level

On groups of conspecifics that
intermingled together

On different ramets of the same genet

On multiunit entities

On the whole organismic level

Symbiotic/parasitic DNA in eukaryotic and
prokaryotic genomes

Natural chimerism and mosaicism

Germ cells parasitism

Mammalian whole-body chimerism and invertebrate

multichimerism
Asexually developed organisms, monozygotic twins,

polyembryony
Rhizocephalen cirri pedia
Many unitary organisms

mous number of documentations that eukaryotic and pro-
karyotic cells carry foreign DNA molecules of various types
(plasmids, B chromosomes, t haplotypes, retroviruses, and
more), as well as diverse mobile DNA sequences (such as
transposons, retrotransposons, LINEs, SINEs, mobile in-
trons) that are transmitted vertically or horizontally within
genomes (Zeyl and Bell, 1996; Flavell, 1999) and may be
regarded as real UOSs. These DNA sequences have func-
tional and holistic properties; they are characterized by a
discrete organismal realm, function in a coordinated fash-
ion, and are clearly subject to natural selection forces. Many
examples now point to real UOSs situated within the ge-
nomes of other UOSs. A few will be outlined below.

One well-studied group is the B-chromosomes. a variety
of germ-line parasites described from more than a thousand
species of plants and animals. These small chromosomes do
not contribute to the regular functions of the host, and their
numbers per cell vary even within the same host organism.
More important, although they share the same nucleus with
regular chromosomes, they have evolved peculiar charac-
teristics of their own. By various non-Mendelian systems of
biased transmission and by their ability to move specifically
to one of the two products of the first meiosis division (such
as by avoiding penetration into the polar body during
oogenesis), they increase their representation in the germ-
line nuclei. The B-chromosomes in the wasp Naxonia.
which are transmitted solely through sperm, are a represen-
tative case. The entire parental set of chromosomes in an
infected zygote becomes condensed and is lost, leaving a
haploidized animal that develops as a male, transmitting the
B-chromosome to all its gametes (citations in Bell and Burt.
1990). Such functionalism of the parasitic entity reveals
distinct host and parasitic units of selection. Within this

context, I am reluctant to consider the B-chromosomes as
selfish chromosomes. They are distinct molecular UOSs.

Not only a whole piece of chromosome may be counted
as a UOS; even transposable genetic elements, gene size
segments of DNA, may be so considered. This field is too
broad to be even partially covered here, so only the most
relevant features of these mobile elements will be discussed.
Many transposable elements have the ability to jump from
place to place on the chromosomes; they can behave like
new introns creating novel intron processing patterns; they
may spread vertically and horizontally within host organ-
isms; and they can promote their own replication (the func-
tionalism component). With time, the mobile elements be-
come domesticated through full integration into the host's
genome. A good example is the mariner which, by being
functional in both germ lines and somatic cell lines, could
infect many organisms, crossing several phyletic borders
(arthropods, platy helminths, nematodes, chordates). proba-
bly by splicing into viral or other pathogenic genomes.
During each introduction into a new host species, the mar-
iner transposon was probably highly mobile and signifi-
cantly disruptive. With time, more and more defecting
transposons with mutations that disabled the cut-and-paste
enzyme were accumulated, littering eukaryotic genomes

234

B. RINKKVICH

with foreign elements in varying stages of decay (Zeyl and
Bell. 1996: Flavell. 1999). and blurring the boundaries
(Dawkins. 1990) between two distinct units of selection.
Many of the mobile elements constitute a significant portion
of host genomes. The Alii elements (the largest family of
SINEs in humans) represent in excess of 5 X 106 copies per
haploid genome, and constitute about 5c/c of the human
genome. The chloroplast genome of Enxleiui gracilix pos-
sesses at least 155 mobile introns, making up 39% of the
genome by forming complex nested structures of introns
within introns (literature cited in Zeyl and Bell. 1996).

From highly functional to nonfunctional: natural selec-
tion has shaped foreign DNA elements between these two
extreme levels of activity. With respect to the UOS para-
digm, elements with well-distinguished sequences and with
high activity levels of their own (even if they are the si/.e of
a single gene) can be regarded as units of selection. Natural
selection may act on them independent of their host, and
may especially act on those elements that move between
different organisms (Flavell, 1999). Other elements that are
completely integrated in the host's genome, replicating
when the entire collective of genes reproduce and contrib-
uting to basic functions and processes derived by the host
cells (such as the LINE elements that preserve the telomeres
of Drosophila; literature cited in Flavell. 1999), are clearly
not UOSs.

/ am — niul part of me is he

In chimeras or mosaics, two or more UOSs amalgamated
to form a single distinct UOS. Genetically nonhomogenous
entities can be established by chimerism (a situation where
an organism possesses cells simultaneously derived from at
least two genetically distinct conspecifics) or by a inosa-
icism (production of an organism with genetically different
cells that derived from a single zygote lineage). Both phe-
nomena have been widely documented: chimeric entities in
nature have been recorded from a variety of protists, plants,
and animals, distributed over nine phyla (Buss, 1982): and
a number of factors may produce mosaicism in almost any
living organism (Benirshke, 1981; Hall, 1988: Gill et ui.
1995). Clear distinctions between chimeras and mosaics are
often not available in reported cases because an insufficient
number of genetic characters were employed (Benirshke.
1981 ). Although, in many cases, a chimera or mosaic seems
to represent a single UOS, this "single organism" actually
consists of two or more distinct embedded units of selection
upon which natural selection acts. This type of "blurring of
the boundaries" between the interacting entities (sensu
Dawkins, 1990) obliges us to develop epistemological tools
with which we may distinguish between false UOSs and
real ones whose existence does not depend upon the re-
searcher's perception.

Mosaic, sectorial, and cytomictial (mixed-cell) chimeras

often occur after allogeneic encounters in a variety of co-
lonial marine invertebrates (Rinkevich. 1996a). Participants
in such chimeras are sometimes so intermingled that the
death of one of them (e.g., from senescence) results in
chimeric death (Rinkevich and Weissman, 1989; Rinkevich
ct nl.. 1992). The evolutionary significance of chimerism
has been evaluated by comparing (Buss, 1982; Grosberg
and Quinn. 1986; Rinkevich and Weissman. I987a; Rinke-
vich. 1996a) the fitness cost-benefit ratio of the chimera
with that of the genetically homogenous UOS. Several
classes of benefits, including the increase of genetic vari-
ability, improvements in growth rates, reproduction or sur-
vivorship, and developmental synergism (citations in Buss.
19S2: Rinkevich and Weissman. 1987a; Rinkevich. 1996a),
have been attributed to chimeric states. Costs are the threats
of somatic and germ-cell parasitism (next section) and.
within chimerical selection, towards the more heteroge-
neous partner (Rinkevich, 1996b). If the outcome is a state
of chimerical improvement, each UOS participating in it
gains. Biological and environmental factors may directly
affect just one UOS within a chimerical entity or may
influence the chimera as a whole.

Vertebrates also exhibit a variety of naturally occurring
chimeras, mostly in the form of di/ygotic twin bone-mar-
row transplantation and as diseases like choriocarcinomas
(Benirshke, 1 981: Tippet. 1984; Benirshke and Kaufman.
1990). These and other types (whole body and germ cell
chimerism, which will be discussed in the next two sections)
are much more common than is usually believed.

Studies on cattle, sheep, goats, pigs, horses, humans,
rodents, deer. mink, birds, and other vertebrates (Benirshke,
1981; Benirshke and Kaufman, 1990) have unequivocally
established the occurrence of placental (when applicable) or
vascular anastomoses between di/ygotic twins. Hematopoi-
etic precursor cells are then frequently exchanged during
early embryonic periods; and by virtue of acquired toler-
ance, they may continue to propagate throughout life in the
new host. The new UOS. thus formed at one higher level
entity, also bears evolutionary relevance in at least two
types of phenomena. The first type comprises resultant costs
such as freemartinism (rnasculini/.ation of the female twin,
resulting in sexual reproductive sterility; Benirshke, 1981)
and a high frequency of malignancy (Picus et ai. 1 985). The
second type — more interesting, but sporadically ob-
served— is gonadal chimerism (literature cited in Benirshke.
1981). In this phenomenon, primordial germ cells may
reach the gonads of the other partner through early vascular
anastomoses. Since a mammalian XY germ cell, for exam-
ple, has the capacity to develop into an oocyte (Evans et <//..
1977), it is possible that even in twins of different sexes
moving germ cells may become functional, giving an evo-
lutionary significance to both genotypes in the chimera.

Genetic heterogeneities are also frequently developed as
sinule-gene. chromosomal, and sierm-cell mosaicisms (Be-

DEFINING UNITS OF SELECTION

235

nirshke. 1981; Hall, 1988; Gill el til.. 1995), and are also
recorded in human monozygotic twins (Ford, 1969). This
scientific field is too broad to be even partly covered here.
As UOSs, however, many mosaic cases have evolutionary
relevance because they are hereditarily transmitted and may
manifest a variety of costs (Benirshke. 1981; Hall, 1988;
Gill et nl.. 1995; Rinkevich. 1996a). Studies of human
syndromes in offspring have shown that somatic mutations
of the germ line may occur in phenotypically normal parents
(Hall. 1988). These mutations probably evolved from a
germ-line cell or its precursors, before the meiotic event.
The same holds for plants and for at least nine different
animal phyla in which a variety of organisms develop by
somatic embryogenesis (where at least one cell lineage
remains totipotent throughout the whole life cycle) or epi-
genetic development (where sequestration of germ cells is
made late in the life span; refs. in Buss, 1982; Gill et nl..
1995). Somatic mutations in those organisms not only pro-
vide the variation necessary to deal with fluctuating envi-
ronments (Gill et a!.. 1995). they also create new entities
that may maintain and inherit the genetic heterogeneities
through the colonial expansion of viable mutated cells.

/ ci/ii — and thi\ /,v netmilly he

Phenotypically expressed entities can serve as "incuba-
tors" for the germ line of other conspecific entities. For
example, a detailed and very thorough study (Mayr et til..
1979) reported the case of a human female chimera detect-
able only by investigation of her progeny. None of the four
children fitted genetically with their mother, and none of the
21 unique genetic markers found in the children could be
detected in the woman. The possibility of any type of
somatic mutation was ruled out, as was the least probable
hypothesis that all four children had been interchanged. The
conclusion of this study was that this female possessed two
populations of allogeneic cells, one in the soma and the
second in her gonads. An extreme somatic clearance process
was suggested for this case, occurring either in a dispermic
chimera or after the fusion of two embryos into one entity
(see next section), with only the germ line to be left from
one partner.

Colonies of the cosmopolitan urochordate Botnilus
schlosseri may undergo natural transplantation reactions
upon allogenic contacts between their peripheral blood ves-
sels. They may develop cytotoxic lesions in contact zones or
form vascular parabionts (review in Weissman et at.. 1990;
Rinkevich. 1992). This histocompatibility discrimination
resides in a single highly polymorphic fusibility-histocom-
patibility (Fu/HC) locus (Weissman et al.. 1990). Alloge-
neic fusions occur between colonies that share at least one
Fu/HC allele; rejecting partners share no Fu/HC allele. After
fusion, all modular units (zooids) from one partner in the
chimera are resorbed by massive phagocytosis, leaving the

zooid of the other colony intact, a phenomenon called
colony resorption (Rinkevich and Weissman, 1987b). In
three clear, independent studies (Pancer et al.. 1995; Stoner
and Weissman, 1996; Stoner et ai, 1999). polymorphic
molecular markers were used to demonstrate somatic and
germ-cell parasitism of the inferior partners in the resorp-
tion phenomenon. Of special interest are the cases where the
soma were cleared of foreign cells, but the only foreign
partner's cells were found in the gonads. This unilateral
germ-cell parasitism (Pancer et al., 1995; Stoner and Weiss-
man. 1996; Stoner etui. 1999) documents another example
of an incubator that carries and successfully delivers the
genetic material of an allogeneic partner to the next gener-
ation (Stoner et al.. 1999).

Incubated entities, as in the above cases, are the evolu-
tionarily successful UOSs, whereas the incubator entities
are those with the role of directly interacting with the
environment. In such unique cases, natural selection there-
fore operates with consequences that do not fit the accepted
dogma (Lewontin, 1970). because the positively selected
organisms inherit different, nonrelated sets of genetic ma-
terial. The intimate relationships between the incubator en-
tities (which cannot be regarded as valid UOSs and better fit
the notion of the "extended phenotype"; Dawkins. 1989)
and the incubated UOSs are still unknown. Moreover, with-
out discussing, at this point, the conflicts of interests be-
tween the genes of the incubated and the incubator entities,
it is evident that the physically blended incubated entities
blur the conventional practical divisions between one or-
ganism and the other. The perception of a UOS as a group
of dispersed stem cells raises the conceptual dilemma of a
physically noncoherent UOS.

/ am — and this is actitallv we

Whole body chimerism — a complete integration of two
or more genetically different conspecifics into a single uni-
fied entity, with a shared participation in the soma and the
germ line — creates another type of self-maintaining UOS.
Such a new form may bear specific properties, different
from those expressed by each of the components. Natural
selection may act simultaneously on each component and on
each of the chimeric entities as a whole. In some chimeric
entities, the physical boundaries between the different units
are so blurred that a morphological separation between the
components is not possible. The literature reveals instances
where such a blending is beneficial to the original compo-
nents, and others that are characterized by malformations or
a variety of costs (such as higher rates of malignancy and
other pernicious phenomena). Both situations will be dis-
cussed here, since successful sexual reproduction has been
recorded even by malformed entities.

Colonies of Botryllus schlosseri may also form natural
muitichimeras (multiple partners; more than two fused ge-

236

B. RINKEVICH

notypes) that result from an aggregated co-settlement of
Fu/HC compatible colonies (Rinkevich. 1996b). When
compared with bichimeras, multichimeras grow faster;
reach larger sizes: do not fragment: have lower frequencies
of colony resorption cases; and like more equilibrated en-
tities, show other features that increase robustness (Rinkev-
ich and Shapira, 1999). In these "monsters," the various
costly intraspecific conflicts between the participant geno-
types neutralize each other, generating an improved entity.
In such an instance, natural selection may act on the "group"
level (the chimera as a whole; Rinkevich, 1996b; Rinkevich
and Shapira, 1999). The increase in fitness of the multichi-
meric entity, a new higher level of UOS, eventually in-
creases the individual fitness of each UOS within this chi-
meric alliance. Therefore, even less adapted genotypes may
survive and propagate.

A whole-body chimerisin in mammals is a state in which
the entire body consists of cells with at least two genetic
lineages that are derived from separate fertilization products
(Benirshke and Kaufman. 1990). Two types of genetic
chimerism are of interest here: the early fusion of two
embryos into one entity and the case of dispermic-chimer-
ism. simultaneous fertilization of an ovum and the polar
body by two spermatoza (Bernishke. 1981; Tippet, 1984;
Bernishke and Kaufman, 1990). Both conditions are char-
acterized by uniform dissemination, throughout the chime-
ral body, of the different cell lineages in the admixture, and
they are found frequently in a variety of animals (Benirshke,
1981). including humans (Tippet. 1984; Benirshke and
Kaufman. 1990). In some cases, due either to limited back-
ground information or complexity, the two conditions can-
not be easily distinguished. One such example (summarized
in Tippet. 1984) is a case of a monozygous pair of male
twins identical in chromosome markers. HLA, isozymes
and serum proteins, both XX/XY in the blood, but differing
in other organs sampled such as skin and secretory tissues.
One explanation for the unusual chimerism was that two
embryos started to develop as XY monozygotic twins. One
continued in the normal way, whereas the second fused with
a dead XX triplet embryo which was completely adsorbed.
In humans, many of such whole-body chimerisms are char-
acterized by sexual reproductive sterility and a variety of
tumors, but some of them are fertile (Tippet, 1984). One of
the most interesting examples is a report (Talerman ci «/.,
1990) of a 29-year-old phenotypic female, a true hermaph-
rodite with bilateral ovotestes. a 46XX/46XY karyotype.
and a successful pregnancy (before the development of a
dysgerminoma. a germ-cell tumor). Several XX/XY male
phenotypic dispermic chimeras have been recorded as sex-
ually normal by having children (Tippet. 1984). but there is
yet no study that analyzes the possible activation of both
germ lines in the gonads, or the genetic constituents of the
offspring in fertile cases.

Whole-body chimerism, and even true hermaphroclitism.

were recorded in a variety of vertebrates, most commonly in
cats, but also in dogs. mink, horses, pigs, cattle, sheep,
goats, deer, rabbits, rodents, chickens, and primates. In
humans, as in other animals. XX/XY dispermic chimeras
tend to be phenotypically males (Tippet. 1984). a phenom-
enon which further simplifies sexual reproduction. It is also
possible that many cases of dispermic chimeras, even
XX/XY ones, may remain undisclosed (Tippet, 1984) as
long as they are healthy and remain fit.

/ inn — and this is iictnallv onlv pun of me

The same UOS may replicate endlessly to produce mul-
tiple identical copies. When addressing the issue of the unit
at which selection acts, most biologists take into consider-
ation only a simple list of basic biological organizations
(e.g.. genes, cells, organisms, group). Most discussions (but
see Tuomi and Vuorisale. 1989a, b) eschew conceptually
challenged phenomena, such as modular organisms (which
consist of repeated morphological units) and organisms that
propagate similar, but morphologically independent, struc-
tures through a variety of processes, wrongly subsumed
under the title of "asexual reproduction." The fuzzy bound-
aries of terms like "individual." "colony." and "clonal or-
ganism" (Michod, 1997) become even more apparent when
they emerge in evolutionary concepts, in our case the con-
cept UOS. For example, what are the levels of structural
organization and what is the UOS of a stand of 47.000 aspen
trees covering 100 ha of land, all produced from a single
founder tree by "asexual" reproduction process (Gill ct ai.
1995)'.' Or of a large branching coral colony that, during an
episodic storm, is broken into fragments which are "replant-
ed" and grow separately in different microhabitats? Or
where a larva of an ophiuroid echinoderm produces second-
ary larval clones (Balser. 1998)? Numerous sessile marine
organisms can generate detached fragments that by different
mechanisms are dispersed before establishing themselves as
independent colonies (Highsmith, 1982; Wulff. 1991). Fol-
lowing that, even the analysis for fragment size may reveal
a whole range of controversial aspects, since a variety of life
history patterns — such as growth rates, partial or whole
fragment mortalities, and fecundity — are directly correlated
with size rather than, for example, with the classical eval-
uated trait of "age" in unitary organisms ( Hughes and Con-
nell, 1987).

For this consideration of the UOS issue and evaluation of
organismal body constructions, we shall deliberately treat
"asexual reproduction" and "modularity" in the wider sense.
No consideration will be given to the order of integration in
modular organisms, to the physiological or morphological
aspects, or to life history parameters. Consequently, it is not
important for this discussion whether modules emerge spon-
taneously by self-organization, are developmentally con-
trolled by a genetic mechanism, or are the products of

DKFINING UNITS OF SELECTION

237

environmental or biological causes that affect different con-
specifics at random. All that matters is that when separation
occurs, the original organism and the fragments continue to
survive.

Three classes of "modularity," in which independent sep-
arated units (Harper. 1977) are produced, if taken together,
may characterize another UOS prototype: a single entity
that occurs simultaneously in several places, all distant from
each other. The first class includes numerous colonial and
clonal organisms (such as plants or marine invertebrates)
that divide by fission (spontaneously, or under genetic con-
trol) to produce autonomous ramets. The second class in-
cludes unitary and clonal organisms (invertebrates, plants)
that can, by budding, produce many similar modules that
separate from their point of origin upon morphological
completion. A well-known example is the freshwater hydra,
a small carnivorous organism that, under normal conditions,
shows no evidence of aging and continuously buds off
unlimited numbers of "copies" of entirely comparable units
(Slobodkin. 1986). Bosch et al. (1989) further described a
dramatic mode of cloning by fission in the planktotrophic
larvae of a sea star. The great multiplicative potential of this
species prolongs the pelagic life of a genet and enhances its
chances for recruitment into benthic adult populations. The
third class includes mammalian monozygotic twins (two
normally developed organisms that share the same genetic
constituents) and polyembryony, in which the division of a
single fertilized egg produces several to hundreds of similar
genetic larvae. Polyembryony occurs in invertebrates and
vertebrates and appears to be a paradox of evolution be-
cause it clones more of an unproven genotype at the expense
of genetic diversity in a clutch of eggs (Craig et al.. 1997).

The above three classes of modular organisms share one
basic life history trait, the production and dispersal of so-
matic individuals, the ramets. Each single genotype is there-
fore represented by more than one ramet. In ecological
terms, each ramet could be regarded as an individual
(Harper, 1977); from the perspective of the UOS. the whole
genet constitutes a single unit of selection (assuming that no
somatic mutation or any other type of somatic mosaicism is
taking place). Among modular organisms, each unit of
selection may be found simultaneously under different en-
vironmental conditions and exposed to a variety of selection
pressures that sometimes oppose each other. Under these
conditions, some ramets will die, while others will survive,
which provides the option for each specific genet to "exer-
cise" its phenotypic potentiality.

We are — and this i.\ actually me

Several genets may form one coherent whole. The situ-
ation wherein several conspecific UOSs combine to form a
morphologically new structure is best represented by certain
primitive crustaceans (order Rhizocephala in the subclass

Cirripedia, the barnacles). The rhizocephalans are mostly
known for the genera Sticciiliim and Peltogcister (Hoeg and
Rybakov. 1992; Glenner and Hoeg, 1995). which are para-
sitic, almost exclusively on decapod crustaceans, and are
structurally unique. The "adults" have neither appendages
nor segmentation, in contrast to all other arthropods, and
their massive body is fastened to the host by a stalk from
which "roots" proceed into the host tissues. These creatures
also have neither an alimentary canal nor a mouth.

The life history of these parasitic crustaceans (Hoeg and
Rybakov. 1992; Glenner and Hoeg, 1995) reveals a unique
type of UOS. The cypris larva develops from a nauplius
stage (both larval types are characteristic of primitive crus-
taceans). When the cypris is attached to the host crab,
remarkable changes occur: the whole trunk of the parasite is
discarded and a hollow, dart-like organ is formed. This
organ is thrust into the crab's body cavity and the remnant
of the cypris. a mass of undifferentiated cells enclosed
within a thin ectodermal layer, is injected. The cell mass
travels through the host's body cavity, attaches itself to the
intestine, and anchors there by rootlets. Recent studies
(Glenner and Hoeg. 1995) have further documented that the
injected parasite has the form of a motile vermiform body
that splits up into a number of naked, motile amoeboid cells.
Each cell has the potential to develop into an adult parasite.
A globular mass begins to develop. This structure will
develop only the female gonads. Meanwhile, other cypris
larvae attach themselves to the body of the juvenile parasite
and inject their cellular contents into its mantle cavity. Only
the first two will be successful in this enterprise. The cells
from each such larva migrate and eventually enter one of the
two "testes" (a better term would be spermatheca); there
they develop into spermatozoa. Additional larvae attached
to the parasite will be rejected. Reproduction is internal and
within each parasitic unit.

Each single rhizocephalen organism is therefore an amal-
gamated structure, consisting of three distinct conspecific
UOSs (two form only spermatozoa, one the soma and eggs).
Together they participate in forming a different adult struc-
tural organism and a new unit of selection at a higher level.
Selection acts only on this adult structure.

Epilogue

Thompsonia. another rhizocephalan parasite, is an ex-
treme case; this crustacean has degenerated to the level of a
fungus with rootlets that diffuse throughout the host crab.
The rootlets branch off numerous sacs on small stalks, each
sac contains one ovum per sac. The structureless parasite
has no testes. ganglia, alimentary canal, or appendages, and
there is no evidence of segmentation. It is believed that ova
develop into cypris larvae by parthenogenesis, escaping the
sacs through small openings (Li.it/en. 1992). although recent
studies have challenged this hypothesis.

238

B. RINKEVICH

What is the unit of selection in this example? ("selection
of?" sen.sit Sober. 1984). It is only one out of many cases
where the data are insufficient for such analysis. However,
the six types of UOSs characterized in this essay, in addition
to the whole organismic level as a UOS (No. 7 in Table 1 ;
not discussed here), indicate that a multiplicity of patterns
are shaped by selective forces. The examples raised here
symboli/e the failure of many biologists and theoreticians to
grasp the rich diversity of UOSs imposed upon the endless
variety of adaptive structures found among living organ-
isms. That many of the UOSs described in this essay are
unconventional was therefore to have been expected, when
the three novel conceptual criteria were applied to the
analysis.

The concept of UOS is variously defined by different
authors. Former attempts to identify the particular entities
that are the targets of natural selection (Wynne-Edwards,
1962; Lewontin, 1970; Mayr. 1970, 1997; Dawkins, 1976,
1989; Hull, 1980; Buss, 1982; Gliddon and Gouyon, 1989;
Sober and Wilson. 1994; Wilson and Sober. 1994; Wil-
liams, 1996; Michod, 1997; Gould. 1998; Gould and Lloyd,
1999) have suggested three or four potentially "structural"
UOSs — the gene, the individual, the group, and the meta-
population — but there has been no consensus. Some
(Kitcher et a/., 1990) have even argued that there are no
"things" like UOSs, stating that "asking about the real unit
of selection is an exercise in muddled metaphysics." How-
ever, I completely agree with the notion that "if selection is
real, then so are units of selection" (Shanahan. 1997).

Kitcher et al. (1990), on the other hand, have correctly
pointed to a major pitfall in the concept of the UOS by
advocating that biologists "assume that for each selection
episode, there is a unique account that will identify the level
of selection." When the descriptions of UOSs in the litera-
ture are aligned with the organizational levels, they fail, in
many cases, to grasp the structural comprehensiveness of
other UOSs and no consistency emerges (Hull, 1980;
Kitcher et al., 1990; Sober and Wilson. 1994; Mayr. 1997;
Shanahan, 1997). For example, the argument for the "gene,"
allegedly the most appropriate UOS (the reductionist ap-
proach), does not hold if we consider the changes that genes
may go through during development (structurally and func-
tionally). One such change is gene methylation. A methyl-
ated gene must be demethylated before it can be transcribed
(Cedar, 1988). Another example is the changes that occur in
the maturation of the mammalian immune system: the T and
B cell genome rearrangement, the reshuffling of DNA frag-
ments like a kaleidoscope to generate enormous genetic
recombination patterns. Within a single individual, no two
B cells, of more than 10s produced, are alike. In such
situations, a single gene on its own may be regarded as only
a tiny information fragment, a fraction within the organis-
mal machinery that cannot produce anything unless it is in
the risiht internal environment. With all its biological im-

portance, a single gene cannot be termed a UOS (except in
UOS type 1 ; Table 1 ).

In this essay, I have focused on the argument that real
UOSs should evince a kind of holism and should possess the
properties of independent functionalism. I have also elimi-
nated cases that fail to comply with Slobodkin's (1986)
minimalistic approach; thus I have omitted symbiosis (Nar-
don. 1999) and complicated cases such as symbiotic-para-
sitic relationships between a virus, an algal chloroplast. and
a sea slug (Pierce et al.. 1999). Following from this analysis,
six new types of UOSs were discussed (Table I , Nos. 1-6),
in addition to the self-evident case (Table 1. no. 7) of the
"individual" (but see the search for several kinds of indi-
viduals based on characterizations of genetic uniqueness,
genetic homogeneity, and autonomy; Santelices, 1999),
which was not discussed here.

All UOSs differ from each other in substantial ways, and
the characteristic properties of any one of them cannot be
imposed on others. The analysis further revealed that neither
the morphology nor the structural organization of a UOS is
always orthodox. UOSs can also be blended morphologi-
cally into the somatic background of other conspecitics or
different organisms. The blurred boundaries between organ-
isms and colonies may raise a new theoretical question
about the definition of "an organism." We find here that
UOSs are associated with a variety of structural organiza-
tions, ranging from a DNA fragment (No. I in Table 1 ). to
cells (No. 3), part of an organism (No. 5), whole organisms
(Nos. 2. 7; that differ in the contents of the entity), a group
of conspecifics (No. 4), and finally to a multiunit level entity
(No. 6). The UOSs discussed here are, variously, based on
one or a mixture of conspecific entities (Nos. 2-7), or on an
association between several biological species (No. 1 ).
There are probably other UOSs belonging to other biolog-
ical organizations, even where the dividing line between
components is not blurred; one good example is the exis-
tence of symbiotic unicellular algae within animal cells.
Since these types of UOSs are more complicated, they were
not analyzed here. In any event, all of the above UOSs bear
in common their holistic character and their functionalism.
All multiply through a variety of reproductive activities.

This essay reveals that a unit of selection can be a part of
a biological organization, or can be an integration of several
such organizations. It is not necessarily related to any con-
ventional biological organization. Different selective forces
operating on different levels of biological organizations
may account for the diversification of UOSs by processes of
integration (Nos. 1-4, 6; Table I) or parcelation (No. 5).
The simple characterization of the UOS on the basis of pure
morphological level (gene, individual, group) may lead to
unsatisfactory results. An entity like a single "individual"
organism may represent a group of conspecifics that are
intermingled (No. 4 in Table 1 ). only a part of a larger UOS
(No. 5), an entity that possesses other types of UOSs (No.

DEFINING UNITS OF SELECTION

239

1), another conspecific UOS (No. 3), a conglomerate of two
units (No. 2), more than the sum of several conspecific
UOSs (No. 6), or simply the traditional "individual" as the
unit of selection (No. 7). Using an unprejudiced analysis on
biological phenomena, we seem able to slip from the biased
thinking of UOSs as being fixed entities, into an understand-
ing that a UOS is the existence at a specific time point of a
holistic and functional entity. Points of disagreement with
traditional opinions always arise from the plurality in na-
ture.

Acknowledgments

This study is part of the research carried out in the
Minerva Center for Marine Invertebrate Immunology and
Developmental Biology. Thanks are due to T. Newberry, K.
Wasson, and three anonymous referees for critically reading
the manuscript.

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An Inducer of Molluscan Metamorphosis Transforms
Activity Patterns in a Larval Nervous System

ESTHER M. LEISE1-2'* AND MICHAEL G. HADFIELD1

lKewalo Marine Laboratory, Pacific Biomedical Research Center, University of Hawaii, 41 Ahui Street,
Honolulu. Hawaii 96813: and Department of Biology. University of North Carolina Greensboro,

Greensboro, North Carolina 27402-6 J 74

Abstract. Larvae of the nudibranch mollusc Phestilla si-
bogae metamorphose in response to a small organic com-
pound released into seawater by their adult prey, the scler-
actinian coral Porites compressa. The transformations that
occur during metamorphosis, including loss of the ciliated
velum (swimming organ), evacuation of the shell, and
bodily elongation, are thought to be controlled by a combi-
nation of neuronal and neuroendocrine activities. Activation
of peripheral chemosensory neurons by the metamorphosis-
inducing compound should therefore elicit changes within
the central nervous system. We used extracellular recording
techniques in an attempt to detect responses of neurons
within the larval central ganglia to seawater conditioned by
P. compressa, to seawater conditioned by the weakly induc-
tive coral Pocillopora damicomis. and to non-inductive
seawater controls. The activity patterns within the nervous
systems of semi-intact larvae changed in response to both
types of coral exudates. Changes took place in two size
classes of action potentials, one of which is known to be
associated with velar ciliary arrests.

Introduction

For a number of molluscan larvae, specific chemical
compounds from the juvenile environment can act as che-
mosensory cues and trigger metamorphosis. For example,
inductive compounds may be given off by the adult prey

Received 18 January 2000; accepted 7 September 2000.

* To whom correspondence should be addressed. Present address: De-
partment of Biology. University of North Carolina Greensboro, Greens-
boro, NC 27402-6174. E-mail: Esther_Leise@uncg.edu

Abbreviations: ASW. artificial seawater; CAS, ciliary arrest spike; CNS,
central nervous system; FSW. 0.2-jM.m-filtered natural seawater; ISW.
Pon'to-conditioned seawater; PSW. PonV/o/wra-conditioned seawater:
SU. smaller units.

(Hadfield and Karlson. 1969; Hadfield. 1977, 1978; Chia
and Koss, 1978, 1988; Lambert and Todd, 1994; Avila et
al, 1996; Lambert e tal., 1997), by adult conspecifics (Pech-
enik, 1980: McGee and Targett. 1989; Pechenik and Gee,
1993), by bacteria associated with adult conspecifics (Fitt et
al.. 1990; Tamburri et al., 1992), and by the algal food of
the juveniles (Scheltema, 1961; Kriegstein et al., 1974;
Switzer-Dunlap and Hadfield, 1977; Morse et a!., 1979;
Levantine and Bonar, 1986; Morse, 1990; Boettcher and
Targett, 1996; Leise et al.. 1996). In gastropods, sensory
neurons that may mediate the induction of settlement and
metamorphosis occur on the head, between the ciliated velar
lobes (Bonar, 1978; Chia and Koss, 1982, 1984; Wodicka
and Morse. 1991; Baxter and Morse, 1992; Uthe. 1995;
Marois and Carew, 1997; Kempf et al.. 1997), and on the
foot (Chia and Koss, 1989). Our understanding of how these
neurons function is still limited. Observations of Morse and
colleagues (Trapido-Rosenthal and Morse, 1985; Baxter
and Morse, 1987, 1992; Morse, 1990; Wodicka and Morse,
1991 ) strongly imply that receptors for lysine, an amino acid
that modifies inducer reception, lie on chemosensory cilia in
the apical sensory organ of larval abalone. If pre-competent
nudibranch and abalone larvae are exposed to an inducer
substance, they display habituation — that is, decreased rates
of metamorphosis — when they reach competency (Hadfield,
1980; Hadfield and Scheuer, 1985; Trapido-Rosenthal and
Morse. 1986; Avila et al., 1996). Habituation is thus a
phenomenon associated with the morphogenetic pathway
that directly initiates metamorphosis.

More recent studies are beginning to elucidate further
internal mechanisms that are downstream from the chemo-
sensory processes. These include changes in gene expres-
sion (Degnan and Morse. 1993, 1995; Degnan et al.. 1997),
protein synthesis, and second messenger levels (Inestrosa et

241

242

E. M. LEISE AND M. G. HADFIELD

al.. 1993). Although the cellular circuitry that actually
drives metamorphosis is still unknown, recent pharmaco-
logical studies have revealed some attributes of this path-
way. Serotonin, which occurs widely in larval molluscan
nervous systems (Goldberg and Kater. 1989; Marois and
Carew, 1997: Kempf et til., 1997). apparently acts as a
neurotransmitter or neuromodulator that promotes meta-
morphosis in the mud snail Ilytnuissa obsolete! (Couper and
Leise, 1996). The neurotransmitter dopamine appears to be
necessary for metamorphorphosis in the nudibranch Phes-
tilla sibogae and the slipper limpet Crepidula fornicata,
whereas norepinephrine may endogenously inhibit this pro-
cess in Crepidula (Pires ct til., 1996, 2000). Nitric oxide
appears to be yet another endogenous inhibitor of metamor-
phosis, as shown by studies on Ilytnuissa (Froggett and
Leise, 1999). Yet, even with these recent advances, we still
have much to learn about the integrative mechanisms that
follow the reception of chemosensory information to pro-
duce, ultimately, a juvenile organism.

Electrophysiological studies conducted on a variety of
molluscan veligers have also provided some insight into
their neural activities. Rapid and coordinated velum-wide
ciliary arrests are driven by action potentials in the ciliated
cells of the preoral band (Mackie et til., 1976; Arkett et til.,
1987), and ramp depolarizations can slow ciliary beating on
a more localized level (Arkett et al., 1987). Thus, metachro-
nal beating appears to be controlled by the relative depo-
larization of the ciliated cells and is modulated by excitatory
neuronal input, presumably from the brain ganglia (Carter,
1926; Mackie et til., 1976; Arkett et til., 1987). These
mechanisms are likely to be involved in the cessation of
ciliary beating that accompanies larval settlement and
crawling, behaviors that often precede metamorphosis. Bar-
low (1990) demonstrated that the ciliated velar cells in
abalone larvae change their spiking activity only as an
indirect response to the presence of the inducer substance.
They do not act as sensory receptor cells. Arkett et al.
(1989) recorded depolarizing receptor potentials from sen-
sory neurons in nudibranch larvae in response to a settle-
ment-inducing substance, although the use of cobalt anes-
thetic in their experiments limits the conclusions that can be
drawn from their electrophysiological traces. Larvae of sev-
eral molluscan species can be induced to metamorphose by
an increase in external potassium ion concentration (Baloun
and Morse, 1984; Yool et til., 1986; Pechenik and Heyman,
1987; Todd et til., 1991 ; Inestrosa et til.. 1992; Pechenik and
Gee, 1993), a classical method for depolarizing nerve cells
(Nicholls et al.. 1992), which again suggests that the pe-
ripheral nervous system, the larval central nervous system
(CNS), or both are active during the initial phases of meta-
morphosis. If so, changes in the activity of central neurons,
as well as in peripheral sensory receptors, should be detect-
able as they respond to a natural inducing substance.

The full range of metamorphic phenomena will most

likely be controlled by neuroendocrine products as well as
by classical synaptic interactions (Scheltema, 1974;
Schacher et al., 1979). but molluscan metamorphosis in-
cludes at least two relatively rapid events that may be under
direct neuronal control. These are loss of the velum, a
process common to all molluscan veliger larvae, and shell
dehiscence, which occurs in many opisthobranchs (Bonar
and Hadtield. 1974; Hadfield, 1978). These events, in ad-
dition to the chemosensory initiation of metamorphosis,
could involve neuronal networks within the CNS that drive
appropriate effector organs. Indeed, Hadtield (1978) sum-
marized data in support of the hypothesis that the nervous
system was the most likely and sufficient regulatory system
underlying all facets of metamorphosis in molluscs.

To learn more about the role played by the nervous
system during the metamorphosis of marine invertebrates,
we used larvae of a nudibranch mollusc, Phestillti sibogae,
to study the response of the CNS to a natural metamorpho-
sis-inducing compound. The scleractinian coral Porites
compressa is the major prey for adult P. sibogae in Hawaii.
A small organic compound that is a natural exudate from
live P. compressa induces metamorphosis in developmen-
tally competent larvae (Hadfield and Karlson, 1969; Had-
field, 1977; Hadfield and Pennington, 1990). Our extracel-
lular recordings from the exposed dorsal surface of the brain
ganglia provide evidence that activity patterns in the CNS
change in the presence of the coral extract. We propose that
the electrical changes we observed are associated with the
initiation of metamorphosis, and that some of them are
specific responses to larval exposure to P. compressa.

Materials and Methods

Veliger larvae of the nudibranch Phestillti sibogae Bergh
were cultured in the laboratory in 0.2-/xm-filtered natural
seawater (FSW) using previously described methods (Miller
and Hadfield, 1986; Pires and Hadfield. 1991). During ini-
tial experiments, insufficient electrical activity was recorded
from the epidermal surfaces of intact larvae, so we used an
//; vitro reduced preparation to maximize our ability to
record spiking activity. To facilitate access to the larval
brain, larvae without shells were used in all electrophysio-
logical experiments. Deshelled larvae settle and metamor-
phose normally, although they do not undergo shell dehis-
cence (Pennington and Hadfield, 1989). Larval shells were
decalcified by culturing about 100 larvae in a slender dish in
30 ml of artificial seawater (ASW) (Cavanaugh, 1956) lack-
ing the usual 2.14 X 10~3 M sodium bicarbonate and
buffered instead with 0.01 M Tris to pH 7.0 (Pires and
Hadfield. 1993). Nine-day-old larvae were kept in ASW
overnight so that metamorphically competent, shell-less,
10-day-old larvae were available as experimental subjects.
About 70% of larvae cultured in this fashion had no shells
14 h after immersion. Deshelled larvae were rinsed in six

ACTIVITY CHANGES IN A LARVAL CNS

243

changes of FSW over the following 2 h to reacclimate them
to normal seawater (pH 8.3) before experimentation began.

Isolated larval heads (Fig. 1) were produced by chilling
20-25 individuals in FSW in a small petri dish in an ice
water bath. Larvae became immobile as the FSW tempera-
ture approached 0°C. Small knives made from broken razor
blades (Pires and Hadfield. 1993) were used to remove the
visceral mass and foot from these cold, anesthetized larvae.
This cut (line A in Fig. 1 A) exposed the dorsal surface of the
brain for extracellular recording, although it may have also
eliminated pan of the pedal ganglia. The eyes and statocysts
remained in this isolated head preparation.

We also conducted experiments on animals from which
only the visceral mass was removed (head-foot prepara-
tions). Results were similar, but we have chosen to leave
those data unreported because fewer controls were con-
ducted. Initial activity patterns in all experiments were

Figure 1. (A) Drawing of a deshelled larva (after Rasmussen, 1444)
showing approximate location of the cut used to remove the visceral mass
and foot from the head. Grey area represents approximate extent of the
brain. The upper lobe containing the eyespot is likely to be a fusion product
of the cerebral and pleural ganglia and may also contain elements of the
parietal and buccal ganglia (Tardy. 1970). The region below the statocyst
corresponds to the pedal ganglion. (B) Isolated head on the end of a suction
electrode. The micropipette tip shown here is smaller than that typically
used for recording purposes, to make the head more visible. The left
eyespot is at arrow; right eyespot is visible through the transparent neural
tissue within the open tip of the electrode. Velum is at arrowhead. For
recording purposes, micropipettes were sized appropriately so that the
entire cut surface could be contained by the electrode. >' 1 7X

somewhat varied (Fig. 3A. C. E, G). so data from different
dissected veliger heads were not pooled.

Immediately after being cut, the chilled, isolated heads
were transferred to fresh FSW at room temperature, where-
upon they recovered normal metachronal beating of the
velar cilia. Electrical recordings were made with a fire-
polished glass micropipette suction electrode with an inner
tip diameter of 40 to 50 ju.ni. The suction electrode was
appressed to ihe exposed dorsal surface of the brain and
gentle suction was applied to maintain contact between the
electrode and the larval tissue.

Larvae were exposed to one of three experimental solu-
tions: FSW. FSW containing the natural metamotphosis-
inducing compound produced by Porites compressa Dana
(ISW), or a similar exudate from the relatively non-induc-
tive coral Pocilloponi Jciiiucornis (PSW). PSW induces less
than 30% metamorphosis compared to 90% induced by ISW
(Hadfield. 1977). Adult P. siho<>ae do not use Pocilloponi
as prey (Hadfield, 1977). ISW and PSW were prepared by
placing about 22 g of living coral into 250 ml of aerated
seawater in a covered beaker. Coral tips were used to
maximize the ratio of living tissue to skeleton. The coral
was removed after 48 h and the resulting conditioned sea-
water passed through a 1.2-/j,m filter. ISW and PSW were
stored in the refrigerator and used within 48 h of production.
Freshly made ISW normally induces more than 92% of
10-day-old intact larvae to metamorphose within 24 h. If the
coral showed signs of ill health during preparation of ISW
or PSW, the coral and solutions were discarded. Assays for
the metamorphosis-inducing capabilities of ISW and PSW
were compared to FSW controls and conducted with intact
larvae as previously described (Pennington and Hadfield,
1989). Assays were examined at 24 and 48 h and scored for
number of larvae, juveniles, and empty shells. We also
tested 34 isolated heads for their ability to metamorphose.
These heads were cultured under sterile conditions for 48 h
as previously described (Pires and Hadfield. 1993). then
examined for loss of ciliated velar cells.

Electrophysiological data were recorded for 5-10 mm
before and after the addition of experimental solutions. The
decision to expose each head to control or experimental
solutions was made before recordings were initiated. Ex-
periments were conducted in 35 X 10 mm plastic petri
dishes in about 6 ml of FSW. Changes to bath solutions
were made manually: 4 ml of the bath solution were ex-
changed four times over the course of 1-3 min. during
which time recording continued. Solution changes some-
times introduced mechanical artifacts, so results are re-
ported for spiking activity occurring after solution changes
were complete. Changes in spiking activity typically began
2-3 min after solutions first contacted the larval head. Data
were collected from a new isolated head for each experi-
ment, amplified through a differential AC amplifier (A-M
Systems, Inc.), and recorded in digital format on videocas-

244

E. M. LEISE AND M. G. HADFIELD

Figure 2. Representative trace from an isolated head in FSW. Large spikes are truncated and correlate with
spontaneous velar ciliary arrest. No stimulus was used to elicit these large ciliary arrest spikes. Bottom trace is
manually controlled cue (event marker) on the PCM data recorder. Cue was depressed, yielding an upward
deflection, whenever spontaneous velar cilia were observed to cease beating. Audio monitor was turned off to
avoid biasing the observer. Cessation of ciliary beating coincides with the largest spikes.

sette tape through an Instrutech VR-100 PCM (pulse code
modulation) device. This device has a manually operated
event marker, or "cue" switch. When depressed, a positive
2.5-V deflection from ground is recorded on a separate
channel on the videotape. Data were played back directly
onto a Western Graphtek thermal chart recorder or. alterna-
tively, collected on a 486 Insight computer and analyzed
with the Enhanced Graphics Acquisition and Analysis
(EGAA) software programs, ver. 3.50.02 (RC Electronics,
Goleta, CA). Action potentials of different magnitudes were
identified and counted using the EGAA Waveshape Recog-
nition program, which stores start and stop times in digital
data tiles. As necessary, files were converted to standard
ASCII text format and analyzed further with Microsoft
Excel 97 (Microsoft Corp.). Traces with relatively few
spikes were analyzed directly from chart recorder records or
the EGAA display screens. Two-sample analyses (two-
tailed ; tests) were conducted with Statgraphics Plus ver. 7.1
(Manugistics, Inc.. Rockville, MD) or GB-STAT 6.0 (Dy-

namic Microsystems. Silver Spring. MD). Results were
graphed with DeltaGraph 4.0 (SPSS, San Francisco, CA).

Results

Extracellular recordings from the dorsal surfaces of
brains in isolated heads of competent veliger larvae dis-
played two general sizes of spiking units in FSW (Fig. 2).
Continuous recordings were made while the preparations
were exposed to the various experimental solutions. The
largest spikes, between 200 and 500 /xV, were associated
with partial or velum-wide ciliary arrests that occurred
spontaneously in all preparations (Figs. 2. 3; Mackie ct til..
1976; Arkett ct til., 1987). No stimulation was needed to
elicit this activity. Initial patterns of activity in FSW were
varied, but we recorded spontaneous ciliary arrest spikes
(CASs) in all preparations (Fig. 3A. C. E. G). CAS activity
typically occurred tonically. as relatively regular trains of
single action potentials at 1 Hz or less. Spikes from smaller

Figure 3. Representative 64-s traces, taken about 3 min before (A. C. E. G) and I mm after (B. D. F, H)
addition of experimental solutions, demonstrate induced changes in spiking activity. Traces A. C. E, and G.
under the heading "before." all illustrate activity in tillered seawater (FSW). Trace B. a sham experiment, shows
activity after the addition of FSW. Traces D and F show activity in seawater conditioned by the presence of the
inductive coral l'i>rnc\ . .™/>n -w/ (ISW), while trace H shows activity after the addition of seawater conditioned
by the presence of /Ji>c///<>/>»™ iltiniicunux (PSW). In all traces, most velar ciliary arrest spikes (CASs) are
truncated and were maximally 200 ^V in C and D and 500 ,uV in all oilier traces. Traces A and B from Expt.
90-60b, iraces C and D from expt. 41-21, traces E and F from e\pt. 40-M. (races G and H from e\pt. 41-22. (A)
Note relative lack of activity in small units (SU). (B) Addition of FSW did not significanlly change ihe firing
rates of CASs when averaged over 5 min (]|r| = 0.80] < f,, „.„,,.„ = 2.31 ). Low activity levels in SUs were
likewise unaffected (Figs. 4A. 5A|. (C) Note the variable firing patterns of SUs in FSW. (D) Addition of ISW

larrowl significantly increased activity of SUs ([\t

= 3.14] > r.,,,5,,,.,4 = 2.15, Fig. 5B). but did nol affect
aclmty of \elar arrest spikes (Fig. 4B|. (E) Note variable firing pattern of SUs. (F) In this experiment, addition
of ISW did not significantly change activity in large or small units (Figs. 4B. 5B), but produced a qualitative
change in the firing paltern of CASs. We recorded short bursts of 2 — t spikes during ihe 10 min after ISW
addition. Longer hursts, with spike frequencies at or above I H/ (asterisks I. coincided with a contraction ot the
velar lobes and cessation ol ciliary beating. (G) Note variable firing patterns of SUs. (H) Addition of PSW again
produced no significant changes in average number of spikes/minute in large or small units, but induced an
increased variability m the firing pattern of CASs (Figs. 4C, 5C).

ACTIVITY CHANGES IN A LARVAL CNS

245

before

FSW

50
10s

ISW

20
10s

after

ISW

50
10s

PSW

50 n
10s

* *

246

E. M. LEISE AND M. G. HADFIELD

units (20-100 jiiV) also occurred spontaneously, but with
less regularity (Fig. 3 A, C, E, G).

Ciliary arrest was often accompanied by a contraction of
the entire velar lobe; during prolonged arrest periods the
cilia and velar tissue were held in an upright position. At
CAS frequencies below 1 Hz. velar cilia resumed beating
between arrest spikes (Figs. 2; 3A, B). During spiking
activity at frequencies above 1 Hz. cilia remained relatively
motionless (Fig. 3F).

We compared firing rates of CASs and the smaller units
(SUs) before and after addition of experimental and control
solutions to 13 isolated heads. In one experiment, addition
of FSW elicited statistically significant changes in firing
frequencies of both CASs and small spikes (Figs. 4A, 5A).
In the remaining two experiments, as expected, no statisti-
cally significant differences were seen in spiking activity
after the addition of FSW (Figs. 3 A. B; 4A; 5A).

In contrast to larval heads that were exposed to FSW.
those exposed to ISW exhibited some type of statistically
significant change in firing pattern, in either CASs, SUs, or
both, in 6 of 7 experiments (Figs. 4B, 5B). In only one
experiment. #90-61 (Fig. 3E. F), did we fail to observe any
statistically significant differences in spiking activity in
response to ISW. However, in this experiment, after the
addition of ISW, CASs tended to occur in short bursts of
2-4 spikes (Fig. 3F). Short bursts of spikes elicited longer
periods of ciliary arrest than did single CASs, and were
often accompanied by contractions of the velar lobes. We
observed similar results from preparations with an intact
foot on several occasions (data not shown). In 4 of the 7
experiments, addition of ISW elicited a significant decrease
in the frequency of CASs (Fig. 4B) and a change in the
spiking activity of SUs (Fig. 5B).

The addition of PSW to isolated heads elicited no statis-
tically significant changes in firing rates (Figs. 4C, 5C), but
in all cases, PSW elicited a qualitative change in CAS
activity. With PSW. the firing pattern of the CASs became
irregular (Fig. 3H). which accounted for the significant
increase in variance that occurred in all experiments (Fig.
4C). No such increase in variance was detected for the firing
rates of small spikes.

Finally, we tested 34 isolated heads for their ability to
metamorphose. The results were equivocal: four (12%) lost
velar cilia, suggesting that isolated heads may be able to
detect and respond to ISW. depending, perhaps, upon the
amount of intact central nervous tissue. Because a large
proportion (56%) died within 48 h, we cannot make a
definitive conclusion about the metamorphic capabilities of
isolated heads.

Discussion

Metamorphosis in the nudibranch Pliestillu siho^ue is
triggered by a chemosensory event, namely, the perception

by a competent larva of a small organic compound given off
by its adult prey, the coral Porites compressa (Hadfield and
Scheuer, 1985; Hadfield and Pennington, 1990). In 6 of 7
experiments, we recorded statistically significant changes in
electrical activity from in vitro heads of larval P. sibogae
shortly after the addition of a metamorphic inducer. In 3 of
the 4 experiments in which spiking activity in small units
changed, activity increased. In 4 of the 7 experiments with
ISW. firing rates of velar ciliary arrest spikes decreased.
Although we did not record consistent responses from all
preparations, it is clear that long-lasting changes in electri-
cal activity are initiated within minutes of initial exposure to
the coral inducer.

Competent larvae of P. sibogae display a rapid behav-
ioral response to ISW that can be reliably observed under
laboratory conditions (Koehl and Hadfield. unpubl. obs.).
These larvae, which are negatively buoyant, stop swimming
and rapidly sink when encountering ISW (Hadfield, unpubl.
data). In the field, such a response would increase the
chances of a larva contacting its adult food source. External
signs of metamorphosis occur only 18-20 h after larvae
have been exposed to an inducer substance for at least 4-6
h (Hadfield. 1977; Hadfield and Pennington, 1990). During
this delay period, crucial physiological transformations and
biochemical pathways must be activated as a prelude to the
more obvious morphological transformations of metamor-
phosis.

The reduced preparation that we used may have produced
neural activity different from that which occurs in an intact
organism. The isolated heads retained most of the brain
ganglia as well as intact velar lobes, eyespots, and stato-
cysts. However, central circuits may have been damaged by
a loss of gangliunic tissue, resulting in decreased connec-
tivity and insufficient afferent information. This in turn may
have led to unusual patterns of activity. Because we are
reporting results from a relatively small number of experi-
ments with a limited number of controls, we cannot fully
explain the variability in endogenous activity, nor the vari-
ability in our results. The responses to Porites compressa
that we recorded in four experiments would lead to an
increase in larval sinking, but not to a complete cessation of
ciliary beating, as seen in the behavioral responses men-
tioned above. This suggests that the isolated heads are not
responding in a completely normal fashion.

Larval Phestilla can apparently differentiate between
their adult prey and at least one other coral species in their
reef habitat. In addition to positive metamorphic responses,
negative responses to unfavorable or even potentially lethal
juvenile environments have been reported for other inver-
tebrates, including several polychaete species (Woodin,
1986. 1991; Woodin el al., 1993; Walters el ai, 1996),
bryozoan larvae (Walters et al.. 1996), and veligers of the
gastropod ll\anassa obsoleta (Leise et al.. 1996). The abil-
ity of Plu'stilla larvae to respond differentially to species of

ACTIVITY CHANGES IN A LARVAL CNS

247

Porites and Poci/lo/xirn is thus not without precedent. How
many coral species these small larvae can distinguish re-
mains to he investigated.

Beat frequency of the velar cilia is modulated hy excita-
tory neural input in veliger larvae of the snails Mangelia
nchiila (Mackie ct ai. 1976) and Callioswnm ligutiim (Ar-
kett et til.. 1987) and the abalone Haliotis rufescenx (Bar-
low. 1990). Velum-wide ciliary aiTests are caused hy an
action potential that propagates throughout the velar ciliated
cells. The large action potentials we recorded were always

FSW

n=6

Q.
CO

90-60a 90-60b 90-6 1a
Experiment number

r n=7

I 60

tfl

40
20

90-58 90-60d 90-61 91-05 91-11 91-18 91-21
Experiment number

PSW

91-02 91-19 91-22

Experiment number

Figure 4. Mean number of velar ciliary arrest spikes recorded per
minute before and after addition of experimental solutions. ± one standard
deviation. Asterisks (*) indicate mean firing rate is significantly different
from initial conditions (P < 0.05) after addition of control or experimen-
tal solution. Experiments 90-60 (a. b) incorporated different isolated heads.
Means were averaged from 10 min of continuous recordings whenever
possible. Exceptions are noted on graphs as n = .\ number of minutes. (A)
In one experiment, addition of FSW elicited a significantly slower rate of
firing of CASs ([|f | = 4.85] > r,,,,<,:,.,: = 2.18). (B) Addition of ISW
elicited a significant decrease in the firing rate of CASs by 40% or more in
4 ol the 7 experiments (e.g.. expt 91-18. [|r| = 4.16] > /„„..;, 2 >.,„ =
2.10). (C) No change in mean number of arrest spikes per minute was
recorded from isolated heads after addition of PSW (e.g., expt 91-19, [|r|

= 1.96] < /„

= 2.30). However, addition of PSW elicited a

significant increase in the variance in all experiments (e.g.. expt 91-22,
[F = 11.4] > Fo.05,2,.9.9 = 4-03).

FSW

• before
D after

90-60a

90-60b
Experiment number

ISW

1 600

1 400

i 200

Mean Spikes/Mir

60-
40-
20-
0-

90-58 90-60d 90-61 91-05 91-11 91-18 91-21

Experiment number
PSW
n=5

91-02 91-19 91-22

Experiment number

Figure 5. Mean number of spikes per min recorded from smaller units
before and after addition of experimental solutions, ± standard deviation.
Means calculated from 10 min before and after addition of experimental
solutions, except as indicated on graph (H = A number of minutes).
Asterisks (*) indicate that mean hring rates before and after addition of
experimental solution were significantly different. (A) Addition of FSW in
one experiment elicited a significant increase in the number ot SUs ([\t\ =

4.16] > rn

- 2.10). (B) Activity levels of SUs were highly

variable both before and after addition of ISW. Firing rate of SUs increased
significantly after addition of ISW in three experiments {e.g., expt 91-21.
[|f = 3.13] > ',,05,1, ,4 = 2.15). but decreased in one experiment. (C)
Addition of PSW elicited no change in tiring rates of SUs. Variances were
similar in all of these experiments, both before and after PSW addition (cf.
Fig. 4C).

associated with ciliary arrests and were smaller than, hut
similar to. the signals recorded from the velum of Mangelia
and Calliostomti (Mackie ct til., 1976: Arkett et ai. 1987).
The exact origin of the large spikes in Phestilla is unclear:
they may be the propagated action potentials of the ciliated
cells, or a combination of these spikes plus the summed
output of central activity that drives ciliary arrests. In her
work with larval abalone. Barlow (1990) found that expo-
sure to an inducer substance increased the likelihood and
duration of ciliary arrests. In our experiments, we mostly
observed a decrease in firing frequency of the CASs, which

248

E. M. LEISE AND M. G. HADFIELD

would lead to fewer, not more, ciliary arrests. Only the
qualitative change to short bursts ot'CASs, as seen in some
experiments (e.g., #90-61) would lead to longer ciliary
arrests.

The behavioral relevance of the spiking activity in the
smaller-sized units is unknown. We do not know if their
activity arises from circuits that detect environmental odor-
ants or drive motor activities, such as crawling or changes in
swimming speed or direction. As elicited by ISW. the bursts
of smaller action potentials are irregular, unlike bursts from
any of the well-known molluscan motor systems (e.g.. Get-
ting and Dekin, 1985) or recently described olfactory cir-
cuits (Gelperin and Tank, 1990; Gelperin ft at., 1993, 1996;
Laurent and Davidowitz, 1994; Laurent et ai, 1996;
Delaney et ai, 1994). Activity in the smaller larval units
was also quite variable, with firing rates ranging from a few
spikes per minute to hundreds per minute. We have no
explanation for such variability, beyond suggesting that the
amount of SU activity may reflect the amount of tissue lost
during dissection. We also have no explanation for the
increase in SU activity seen in one control experiment (Fig.
5 A). Extracellular recordings from distal stumps of either
the rhinophoral or oral-tentacle nerves of adult P. sibogae
display changes in firing activity of small units in response
to Porites compressa that are similar to the changes we
record from SUs in response to ISW ( Boudko and Hadricld,
unpubl. data). We can only speculate that the SUs recorded
from larval P. sibogae might indicate olfactory activity.

The high mortality rate that occurred in experiments on
the metamorphic capabilities of isolated heads does not
allow us to make a definitive statement about their ability to
metamorphose. Isolated velar lobes do not metamorphose —
that is, they retain their ciliated velar cells in the presence of
ISW — but such lobes lack the neural apparatus that can
respond to a metamorphic inducer (Pires and Hadfield,
1993). Although our results support the idea that larval
perception of an inducer substance depends upon peripheral
chemosensory neurons and central processing circuitry, an
additional caveat is warranted. Suction electrodes do not
provide a tight seal against passage of fluid between the
bathing medium and the core of the electrode. Thus, in our
experiments, ISW in the bath seawater could have been
interacting directly with neurons of the CNS as well as with
epidermal sensory neurons. Thus, the neural activity we
recorded in response to ISW may or may not duplicate
neural activity occurring within intact larvae at the initiation
of metamorphosis. Still, the responses we recorded suggest
that the beginning of this process in Phestilla sibogae is
accompanied by lasting changes in central neural activity.

Acknowledgments

We would like to acknowledge the late Dr. Robert Kane
for many constructive discussions. We also thank Dr. An-

thony Pires for advice, important technical suggestions, and
help in conducting the in vitro induction experiments. Dr.
Stephen Kempt" for his critique of an earlier version of this
manuscript. Dr. Louise Page for neuroanatomical insights,
and Bryan Turner for technical assistance. This work was
supported by ONR grant NOOO 14-9 1-J- 1533 and NSF grants
DCB-8903800 to M.G.H. and IBN-9604516 to E.M.L. We
are also grateful to UNCG for providing equipment and a
Research Assignment award to E.M.L.

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The Structure and Growth of the Statocyst in the
Australian Crayfish Cherax destructor

LUKE FINLEY AND DAVID MACMILLAN*
Department of Zoolog\, University of Melbourne, Pcirkville, Victoria 3052, Australia

Abstract. The morphology of the statocyst of the Austra-
lian crayfish Cherax destructor was examined using scan-
ning electron microscopy. It resembles in general structure,
size, and position the statocysts of crayfish described pre-
viously, and the size and distribution of the fields of setae on
the floor of the capsule are similar but not the same. Over
the size range examined, the relationship between the car-
apace length, the length of the basal antennular segment, the
diameter of the statocyst capsule, and the total number of
setae are all linear. The number and position of setae on the
floor of the statocyst capsule were mapped for animals in
two size classes (small, ca. 20 mm; large, ca. 50 mm) to test
for changes in their arrangement during growth. The change
in the ratio of setal number to statocyst size between the two
size classes was about three times greater for the anterior
setal field than for the other fields. We propose that differ-
ential development of the setal fields may be related to
changes in the force-monitoring requirements of the animals
as they increase in size, but this remains to be experimen-
tally tested.

Introduction

Many decapod crustaceans have paired equilibrium or-
gans called statocysts in the basal segment of each anten-
nule. Statocysts monitor spatial orientation and movement
(Cohen, 1955; Schone and Neil. 1977; Sekiguchi and Tera-
zawa, 1997). Each statocyst is a sac-like epidermal invagi-
nation of cuticle with a number of mechanosensory setae
inside, mainly on the ventral floor. These are typically
associated with a dense mass of sand, the statolith. The setae
can be adjacent to the statolith and free to move, adjacent
and touching, or cemented to the sand grains of the statolith.

Received 14 October 1999; accepted 31 August 2000.
* To whom correspondence should be addressed.
d.macmillan@zoology.unimelb.edu.au

E-mail:

When the statolith deflects a seta it stimulates the neurons
innervating it, and setae can differ in their physiological
responses to stimulation (Cohen. 1955. 1960; Breithaupt
and Tautz. 1988; Gate and Roye, 1997). The position and
movement of the animal determine the pattern of setal
stimulation, which in turn determines the form of compen-
satory movements made by the appendages and body (San-
deman and Okajima. 1972; Schone and Neil. 1977; Patton
and Grove, 1992b).

The morphology and spatial arrangement of setae within
the statocyst vary between species (Cohen, 1955; Kovalev
and Kharkeevich, 1993: Sekiguchi and Terazawa, 1997).
and it has been suggested that groups of features may be
associated with higher taxonomic groupings (Sekiguchi and
Terazawa, 1997). In the statocyst of the crayfish Orconectes
limosits, Hertwig et al. ( 1991 ) identified four separate fields
of innervated setae: a lateral group of two semicircles, an
approximately fusiform medial group with its axis roughly
parallel to the long axis of the statocyst, and a single row of
proximal setae. These setae appeared to be morphologically
identical internally, but they differed in length and diameter
in different parts of the field. Whether they differ in their
physiological responses has not been tested.

Although both structure and function of crustacean sta-
tocysts are well understood, their growth has not been
described as it has for other cuticular sensors on the crayfish
and lobster tailfan (Letourneau. 1976; Schmitz. 1992: Stuart
and Macmillan. 1997) and other appendages (Sandeman
and Sandeman. 1996; Macmillan et al.. 1998; Steullet et al.,
2000). Growth in crustaceans occurs by periodic shedding
of the cuticle, a process known as ecdysis. or molting, the
body increasing in size with each molt. As the body grows,
the sensory representation from the integument may need to
change to maintain appropriate sensory input and function.
As new sensory structures can only be added to the cuticle
when the animal molts, a comparison of sensory structures

251

252

L. FINLEY AND D. MACMILLAN

in sequential molt stages reveals the order in which ele-
ments develop. Because of their accessibility, setae on the
telson have been the subject of a number of developmental
studies based on this principle. Letourneau (19761 found.
for example, that the order of addition of sensory setae to
the telson of Procambarus clarkii is a function of the
growth of the animal. Schmit/. 1 1992) described four func-
tionally distinct setal types that are added at different rates.
"Short smooth hairs" and "guard hairs" increase rapidly in
number throughout development, whereas the number of
two types of "feathered hydrodynamic hairs" remains rela-
tively constant.

We describe here the basic structure of the statocyst in the
Australian crayfish C. destructor, and the relationship be-
tween body size, basal antennal segment size, and statocyst
capsule size over the size range of animals examined. We
report the first data on the pattern of addition of setae within
the capsule as the animal grows by comparing the statocysts
from small and larse individuals.

large foreign particles, effectively forming a closed capsule
(Fig. IB). The cavity itself is oval and slightly pointed
posteriorly (Figs. 1C. 2 A). The ventral floor of the cavity
has an oval depression (Fig. 2 A, B). and setae project
dorsally through the cuticle adjacent to this. A statolith
composed of fused sand grains sits in the depression (Fig.
20.

Relationships between size of animal and size of
antennule and ca\'it\

The length of the basal segment of the antennule corre-
lates closely with the carapace length (n -- 39; R2
0.971 1; P < 0.001; Fig. 3A), so we were able to collect
data on both body size and statocyst parameters from scan-
ning micrographs of the local area. The length of the stato-
cyst capsule increases linearly as a function of the size of
the basal segment of the antennule (n = 26, R~ ~ 0.9546:
P < 0.001: Fie. 3A) and hence of the size of the animal.

Materials and Methods

Individuals of Che rax destructor were obtained from a
commercial hatchery at Bendigo, Victoria, Australia. They
were kept in 50 X 20 X 120 cm aquaria under constant
temperature with a normal 12-hour light/dark cycle, and
were fed dried pellet food weekly.

Specimens with carapace lengths from 20 to 50 mm were
examined. The animals were anesthetized by chilling in
crushed ice for 30 min and were then decapitated. Statocysts
were dissected from the dorsal surface of the basal segment
of the antennules, and any extraneous tissue or adhesions
were removed from around the cuticle of the statocyst with
a fine paintbrush. The preparations were dehydrated in a
series of ethanol solutions before being transferred to 1009r
ethanol for 12 h. After an additional 24 h in a desiccator,
conducting graphite paint was used to glue the preparations
to a scanning electron microscope stub. They were sputter
coated with gold, and examined with a Phillips 505 scan-
ning electron microscope. The images were processed using
Adobe Photoshop Version 4.0. Measurements of carapace,
basal segment of the antennule, and statocyst diameter were
recorded for body index relationships, and comparisons
were made using SYSTAT 6.0 for Windows.

Results

Location and general structure of the statoc\st

The statocysts of Che rax destructor are in the dorsal
region of the basal segment within the antennules (= first
antennae; Fig. 1 A, B). The statocyst is a cup-like invagina-
tion of the cuticle forming a cavity with a triangular, ante-
riorly facing opening on the dorsal surface. The opening is
covered with a dense mat of setae that prevents entry of

Arrangement of setae and changes in distribution during
growth

All of the setae on the base of the statocyst capsule of C.
destructor, except those in the anterior part of the anterior
setal field, are bound to the statolith (Fig. 2C). All setae that
could be seen in scanning micrographs, because they were
not obscured by the statolith. appeared to have the same
external morphology (Fig. 2E), even though they varied in
size. Because of the close association between the setae and
the statolith, the process of removing it to examine the base
of the capsule usually removed not only the setae but all
associated tissues, including the tissues passing through the
holes in the floor of the capsule. Remnants of these re-
mained in a number of our preparations, however: these
demonstrated that at least some of the setae are innervated
through the holes in the base of the capsule (Fig. 2F). The
presumption is that the holes represent innervation chan-
nels, as they do in other species (Hertwig et al., 1991 ). The
holes indicate the precise position of each seta on the floor
of the capsule (Fig. 2A. B). Their disposition around the
depression that normally holds the statolith resembles that
in Orcont'ctc'fi liniosim (Hertwig et al., 1991). and direct
correspondence with three of the four setal fields they
described and named is apparent. A curved field made up of
an inner double row and an outer single row forms a
semicircle around the medial and posterior rim of the central
depression. On the lateral side, this merges into the narrow
end of a large triangle of setae occupying the area lateral to
the rim of the depression. Opposite this large field, on the
medial side of the depression, is a smaller triangular field. In
an adult animal of around 50-mm carapace length, these
fields are composed of about 68. 135. and 36 setae, respec-
tively (Fig. 2D, Fig. 4) The total number of setae increases

CRAYFISH STATOCYST GROWTH

253

5mm

2 mm

Figure 1. Morphology of antennular region and statocyst of the crayfish Clicnn tlcMiiiflnr. (A) Dorsal view
of the basal segment (BS) of the antennule. and the locution of the statocyst opening (SO). The rostrum and eyes
have been removed. The position occupied by the rostrum is indicated by dotted lines. (B) Higher magnification
of the basal segment (BS) of the antennule showing the dense screen of setae (H) that covers the statocyst
opening (SO). (C) The statocyst capsule viewed through a window cut in the dorsal cuticle of the basal segment
(BS) of the antennule to reveal the setae (SS) projecting upwards from the ventral floor (F) of the capsule. The
statolith. with which all but the anterior setae make contact, has been removed.

linearly with the size of the animal (n = 24: R~ - 0.8663;
P < 0.005: Fig. 3B).

To examine the way in which this increase occurs, we
counted the number of setae in a group of animals with a
basal antennule length of 1.97 mm (SD = 0.19) ("small")
and compared the result with a sample of animals with a
basal antennule length of 5.75 mm (SD = 0.27) ("large").

The results of the survey are shown in Figure 4. A two-
factor analysis of variance on the data testing for setal field
type and size of animals showed that the large animals have
significantly more setae in each field than the small animals
(F -- 322.6. P < 0.01 ). the number of setae in the

three fields is significantly different (F( 2 50) — 848.9. P •
0.01 ). and the si/.e of the difference varies between fields

0.1mit.20.1 kU 166«m 6094X01

Figure 2. Scanning electron micrographs showing the statocyst of the crayfish Cherax destructor. (A) Dorsal
view of the right antennule of an animal from the "small" group with part of the dorsal cuticle (C) cut away to reveal
the floor of the capsule ot the statocyst (S). The basal segment of this animal was 1.99 mm long. (B) Dorsal view of
the floor of the statocyst (S) from the left antennule of an animal from the "large" group. The basal segment of this
animal was 5.7 mm long. The magnification is the same in A and B so that the large increase in the number of setae
in the anterior lield is readily apparent. (C) Dorsal view of the statocyst capsule with part of the dorsal cuticle (Cl
removed to reveal the sensory setae (SS) in contact with the statolith (SL). Note that many of the setae in the anterior
field do not contact the statolith. (Dl Dorsal view of the ventral floor of the statocyst showing position of setae. The
fields have been marked to correspond with the classification used previously in Onuih-ck^ /HHO.WI.V A Large anterior
field 1 134 setae); curved field (69 setae): O, outer row (29 setae); + inner rows (40 setae); X. small field (4f> setae).
(E) High magnification view of base of a seta from the outer curved held viewed from the dorsal aspect. (F) High
magnification view of the ventral surface of the same statocyst base as in E, showing holes beneath each seta and
remnants of the mechanical and neural connections broken during the statocyst removal and preparation process.

CRAYFISH STATOCYST GROWTH

255

Carapace: FT = 0.9711
Statocyst: R2 = 0.9546

2345678
Basal segment of antennule (mm)

300

250
200

150

100

50-

= 24

01234567
Basal segment of antennule (mm)

Figure 3. Statocyst size relationships. (A) Relationship between sta-
tocyst diameter, carapace length, and length of the basal segment of the
antennule. The bold and dotted lines are the linear regression lines. Note
the high correlation for both body measurement indices. (B) Relationship
between length of basal segment of antennule and total number of setae
within the statocysts. Note the high level of correlation between the base of
the antennule (and hence body size) and the number of setae.

(F,-, 5()) = 69.2, P < 0.01). Tukey-Kramer pairwise
comparisons between the three fields in both large and small
animals showed that the number of setae is different in the
three fields at the P < 0.01 significance level.

Discussion

The outcome of this work is straightforward. The result is
a description of Statocyst morphology in a previously un-
described crayfish species which permits some species com-
parisons to be made. In addition, this is the first report on
changes in the size and setal arrangements of the Statocyst
with changing body size. The results therefore have impli-
cations for comparative and developmental questions.

In a mini-review, Sekiguchi and Terazawa (1997) com-
pared information on statocysts across a range of crustacean
species and found considerable morphological variation be-
tween taxonomic groupings but some evidence of consis-

tency within them. The number of examples available,
however, is probably not yet sufficient for a firm conclusion
on this issue. The general morphology of the Statocyst of
Cheni.\ destructor does appear, however, to be closely
similar to that of other crayfish species examined (Proctim-
barus clitrkii: Takahata and Hisada, 1979; Orconectes li-
mosus: Hertwig et <//., 1991 ). Because they used transmis-
sion electron microscopy as well as scanning electron
microscopy, Hertwig et ul. ( 1991 ) were able to show that all
the setae on the floor of the Statocyst capsule in O. limosits
are morphologically identical. The external morphology of
the setae in C. dextructor suggests that they too may be of
one type and probably even are closely similar to those in O.
limosus. This does not, of course, mean that they are uni-
form in their physiological responses, because setae that
appear closely similar may differ in their responses (Patton
and Grove, 1992a). Irrespective of the way in which they
transduce the detected forces into electrical signals, the
positioning of different setae relative to the statolith must
reflect the displacement forces that they can monitor (Co-
hen, 1955. 1960): thus the results suggest that further com-
parison of the arrangement of these elements across a range
of species with differing lifestyles has the potential to reveal
principles of Statocyst structure and function.

Fortuitously, we were able to select specimens with a
mean basal antennal segment length of 5.75 mm, which is
close to the 5-mm length of O. limosus used by Hertwig et
ul. (1991), making comparisons between the fields in the
two species less likely to be confounded by a size factor.
They found four distinct groups of setae in O. limosus. Of
these, three are clearly present in C. destructor and, in two
cases, in comparable numbers: the curved field (O. limosus:
C. destructor 60:68), the large anterolateral field
(135:<60), and the smaller medial field (30:36). The pos-
terior line of 8 setae is not evident in C. destructor, but it is
possible that they are part of the outer curved group but less

160

140

120

100

• S

CURVED FIELDS

LARGE FIELD

Figure 4. Comparison of the number of setae ( N) in the different fields
(curved, large, small) in a sample of small animals (S, mean basal antennal
segment of 1.97 mm) and large animals (L, mean basal antennal segment
of 5.75 mm).

256

L. FINLEY AND D. MACMILLAN

distinctly separate than they are in O. limosus. If this were
the case, the two species would both have about the same
number of setae (ca. 68) in the posterolateral complex
formed by these adjacent groups. In other decapod species
studied, behavioral responses to stimulation of setae were
found to correlate with the spatial location within the sta-
tocyst (Ozeki etal.. 1978: Kovalev and Kharkeevich, 1993).
The curved and small fields occupy roughly the same posi-
tion relative to the statolith in O. limosus and C. destructor
and have approximately the same number of setae, all of
which are attached to the statolith, an arrangement Cohen
( 1955) suggested as indicative of a prescribed output. It is
therefore probable that they serve similar functions in terms
of the requirements of the two species for positional infor-
mation during behavior. In the lobster, setae in the lateral,
posterior field respond to body roll, whereas setae anteriorly
respond mostly to acceleration (Cohen, 1960). Although the
argument rests on a body of cross-species data, it is likely
that the body roll monitoring systems of O. limosus and C.
destructor are similar. This then raises the interesting ques-
tion of why the number of setae in the large anterior field
differs so significantly between the two species.

In both O. limosus and C. destructor, the large anterior
field differs from the other fields because many of the setae
do not make contact with the statolith. Hertwig et al. ( 1991 )
argue that setae that are free of the statolith are most suited
for detecting angular accelerations, an observation sup-
ported by behavioral observations in other species (Cohen,
1960; Patton and Grove, 1992a). The comparison of setal
numbers in the different fields in the two size classes ex-
amined in our experiments showed that the ratio of change
in the anterior free-field was about three times that seen in
the small medial and the curved groups. Their rapid differ-
ential growth is therefore likely to correlate with the inter-
action between increasing body size and particular behav-
ioral activities that involve a high degree of body
mobility — activities such as three-dimensional movements
in the water column or escape. The precise relationship
between these behavioral considerations and the develop-
ment of the statocyst remains to be determined.

Acknowledgments

We thank Dr. Jeremy Sullivan for suggesting this project
and for advice on statocyst dissection and preparation. We
thank Ms. Joan Clark for advice and assistance with the
scanning electron microscopy. Dr. Zen Faulkes for assis-
tance in preparing the figures, and Dr. Jo Drummond for
suggestions and critical advice on late drafts of the manu-
script. Supported by an Australian Research Council Grant
to D. Macmillan.

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Kovalev, V. A., and T. A. Kharkeevich. 1993. Studies on morpholog-
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Letourneau, .1. G. 1976. Addition of sensory structures and associated
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Macmillan, D. L., T. Stuart, and M. Thomas. 1998. Development of a
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Reference: Biol. Bull. 199: 2?7-264. (December 2000)

Thermosensitivity of the Lobster, Homarus
americanus, as Determined by Cardiac Assay

STEVEN H. JURY* AND WINSOR H. WATSON 111

Zoology Department and Center for Marine Biology, University of New Hampshire.
Durham, New Hampshire 03824

Abstract. It is generally accepted that crustaceans detect,
and respond to, changes in water temperature, yet few
studies have directly addressed their thermosensitivity. In
this investigation a cardiac assay was used as an indicator
that lobsters (Hoinarus americantts) sensed a change in
temperature. The typical cardiac response of lobsters to a
1 -min application of a thermal stimulus, either warmer (;; =
19) or colder (n = 17) than the holding temperature of 15
°C, consisted of a short bradycardia (39.5 ± 8.0 s) followed
by a prolonged tachycardia (188.2 ± 10.7 s). Lobsters
exposed to a range of rates of temperature change (0.7, 1 .4,
2.6, 5.0 °C/min) responded in a dose-dependent manner,
with fewer lobsters responding at slower rates of tempera-
ture change. The location of temperature receptors could not
be determined, but lesioning of the cardioregulatory nerves
eliminated the cardiac response. Although the absolute de-
tection threshold is not known, it is conservatively esti-
mated that lobsters can detect temperature changes of
greater than 1 °C, and probably as small as 0.15 °C. A
comparison of winter and summer lobsters, both held at 15
°C for more than 4 weeks, revealed that although their
responses to temperature changes were similar, winter lob-
sters (n - 18) had a significantly lower baseline heart rate
(34.8 ± 4.4 bpm) and a shorter duration cardiac response
(174 s) than summer lobsters (n = 18: 49.9 ± 5.0 bpm, and
320 s respectively). This suggests that some temperature-
independent seasonal modulation of cardiac activity may be
occurring.

Received 22 March 1999; accepted 14 August 2000.
*Present address: Caribbean Marine Research Center. Tequesta, FL
33469. E-mail: sjury@cmrc.org

Introduction

Temperature is one of the most important and pervasive
environmental influences on the American lobster, Homa-
rus americanus (Cobb and Phillips, 1980; Aiken and
Waddy, 1986; Factor. 1995). It is generally accepted that
locomotory activity in this species is temperature dependent
(McLeese and Wilder. 1958: Reynolds and Casterlin, 1979;
Haakonsen and Anoruo. 1994) and that it carries out sea-
sonal inshore to offshore migrations to gain the develop-
mental benefits of warmer coastal temperatures in the spring
and summer (Cooper and Uzmann, 1971; Pezzack and Dug-
gan, 1986; Karnofsky el al.. 1989; Haakonsen and Anoruo,
1994; Factor, 1995; Watson el al, 1999). Laboratory studies
have demonstrated that H. americanus has a thermal pref-
erence of about 16 °C (Reynolds and Casterlin. 1979; Cros-
sin et al., 1998). and it has been proposed that behavioral
thermoregulation may allow members of the species to
occupy thermal niches which maximize their metabolic or
behavioral efficiency. The behavioral responses of lobsters
to thermal gradients suggest they have some mechanism to
sense temperature so that they may effectively respond to
the thermal properties of their environment.

Thermosensitivity in lobsters may be mediated by distinct
thermoreceptors or thermosensitive neurons as in some
other invertebrates (Prosser and Nelson, 1981; Mori and
Ohshima, 1995). Although behavioral studies strongly sug-
gest that H. americanus can sense temperature (Reynolds
and Casterlin. 1979; Crossin et al., 1998), to our knowledge
only one study has addressed how neurons respond to
changes in temperature in this species. In that study, firing
of cells associated with thoracic ganglia connectives gener-
ally showed no spontaneous activity below 14 °C, but most
became spontaneously active above this temperature. Inter-
estingly, these cells "cycle reversibly from silent to contin-
uously active to bursting and back as the temperature is

257

258

S. H. JURY AND W. H. WATSON III

increased and decreased" (Konishi and Kravitz, 1978).
Other than these cells, which may or may not play a role in
thermally guided behaviors, we know little about the loca-
tion of putative thermoreceptors, or the mechanisms used to
detect temperature, in lobsters and most other crustaceans
(Dorai Raj and Murray. 1962; Ache, 1982).

In situations where the precise receptors have not been
identified, or are not readily accessible to electrophysiolog-
ical investigation, cardiac assays are a valuable tool for
preliminary investigations of sensory capabilities (Larimer,
1964; Offutt, 1970; Florey and Kriebel. 1974; Dufort.
1997). For example, many crustaceans exhibit a drop in
heart rate in response to novel stimuli (Maynard, 1960;
Larimer, 1964; McMahon and Wilkens, 1972; DeWachter
and McMahon, 1996). This cardiac response has been used
to measure the ability of H. americanus to detect sound
(Offutt, 1970) and salinity (Dufort, 1997). Although a num-
ber of studies have addressed the effect of temperature on
decapod heart rates at time scales ranging from hours to
days (Ahsanullah and Newell. 1971; Florey and Kriebel,
1974; DeFur and Magnum, 1979; DeWachter and McMa-
hon. 1996; DeWachter and Wilkens. 1996; Hokkanen and
Demont. 1997). few have characterized the initial response
(i.e., <5 min.) to brief changes in water temperature. The
present study used a cardiac assay to demonstrate that
American lobsters are consistently capable of sensing in-
creases or decreases in temperature that are greater than 1
°C. The typical response elicited by both cold and warm
stimuli was a brief slowing of the heart rate, followed by
prolonged cardioacceleration. Winter and summer lobsters
responded somewhat differently to thermal stimuli, suggest-
ing some type of seasonal temperature-independent modu-
lation of their responsiveness to thermal stimuli.

Materials and Methods

Animals

Adult (82-92 mm carapace length), intermolt lobsters
were held at 15 ± 1 °C (salinity 30 ± 1 ppt) for more than
4 weeks prior to use, and experiments were initiated at this
temperature. All lobsters were captured from coastal New
Hampshire waters, and experiments were conducted at the
University of New Hampshire, Durham, New Hampshire.
Experiments were carried out in both summer and winter
under ambient light conditions. In the summer, the thermo-
sensitivity of 18 lobsters was determined (cold stimuli, n =
9; warm stimuli, n = 9); in the winter, lobsters kept at the
same temperature ( 15 °C) as summer lobsters were used in
identical experiments (cold stimuli, n = 8; warm stimuli, ;;
= 10).

Recording of temperature and heart aciivit\

Small wire electrodes were inserted through the dorsal
carapace above the heart and used with a UFI impedance

converter (model #2991) to record heart rate (Dyer and
Uglow, 1970). Because the impedance recording technique
can be sensitive to temperature, the method was verified by
using a second pair of electrodes and a Grass model 7D
polygraph to simultaneously monitor the electrical activity
associated with lobster heart contractions (see Watson and
Wyse, 1978; Watson, 1980). External temperature was re-
corded using a small (3 mm X 1 mm) thermistor (C & B
Sciences/iWorx, Inc., Dover, NH) placed on the dorsal
carapace. The thermistor was calibrated weekly over the
range of temperatures used in the experiments. The time
constant of the thermistor was 2.0 s (time to achieve 67% of
the final response). The absolute resolution of the thermistor
was ±0.15 °C, but it could accurately detect changes in
temperature as small as 0.01 °C. However, because of
turbulent mixing within the recording chamber, the slight
time delay due to the time constant of the thermistor (Fig.
1 ). and the unknown location of temperature-sensitive neu-
ral elements relative to the location of the thermistor, it was
not possible to assess the thermal detection threshold with
great accuracy. All temperatures presented are those re-
corded by the externally located thermistor above the dorsal
carapace. These should be interpreted conservatively, in the
context of the methods used and the unknown location of
the sensory receptors.

Experimental chamber

After insertion of the electrodes, lobsters were placed in
a recording chamber consisting of an 18-cm-diameter PVC
pipe covered on the top and bottom by perforated plates
through which seawater (temperature 15 ± 1 °C) continu-
ously flowed (Fig. 1). This arrangement kept lobsters rela-
tively immobile and ensured that changes in temperature
within the recording chamber were rapid and relatively
homogeneous. The chamber was placed in an acrylic plastic
insert (30 X 30 X 30 cm) that was immersed in a temper-
ature-controlled 120-1 aquarium (the ambient bath). Ambi-
ent seawater was continuously pumped (2 1/min) from the
aquarium through the recording chamber, into the insert,
and back to the aquarium. Thermal stimuli were delivered
by switching the source of seawater from the ambient bath
to the stimulus bath. This switching was accomplished by
turning a stopcock and was considered the initiation of the
stimulus (see arrows in Fig. 2). The stimulus bath was filled
from the ambient bath to minimize novel chamber chemo-
sensory cues (Fig. 1 ) and brought to the appropriate exper-
imental temperature using aquarium heaters or cooling
coils. The recording chamber was covered with black plastic
to minimize visual disturbance, and the lobster was left in
the experimental apparatus overnight before an experiment.
Lobsters are much more sensitive to stimuli if allowed to
recover from electrode insertion and become accustomed to
the recording chamber (Larimer. 1964; Dufort, 1997).

THF.RMOSHNS1TIVITY OF LOBSTERS

259

Heat/Cool Stopcock

Animal
i Chamber

Heart
Electrodes

Stimulus ^V Ambient Balh

Bath Pumps

50 100 150 200 250
Time (s)

-1

1 23456
Time (s)

Figure 1. Experimental apparatus used to record lobster cardiac re-
sponses to changes in temperature. (A) Seawater (I? °C> flows continu-
ously from the ambient bath into the animal chamber through perforated
plates located above and below the lobster (direction of flow indicated by
dark arrows). Heart rate is recorded before, during, and after exposure to a
temperature stimulus. Switching the stopcock changes the source of sea-
water from the ambient bath to seawater from the stimulus bath (direction
of flow indicated by white arrows). A thermistor on the dorsal carapace is
used to monitor temperature during each trial. (B) Rates of temperature
change in a typical experiment in response to I min stimuli (turned on al
time = 0 and off at arrow) of ± 0.7. 1.4. 2.6. and 5.0 °C warmer or colder
than the ambient temperature. (C) Time constant of the thermistor when
exposed to a step change in temperature of ± 2 °C (dotted line). The
estimated time to achieve 67% of final temperature is 2.0 s.

The following day, after basal heart rate was measured
for at least 30 min, each animal was exposed for 1 min to a
warm or cold stimulus that changed the temperature in the
recording chamber at a rate of ±0.7 °C/min. This was
followed by stimuli delivered at targeted rates of ±1.5
°C/min, ±2.5 °C/min. and ±5.0 °C/min for 1 min. Tem-
perature was allowed to return to ambient (Fig. 2) between
each treatment. Treatments were separated by at least 30
min. The temperature in the recording chamber was moni-
tored with the dorsally located thermistor, and the actual
mean rates achieved were 0.72 ± 0.04. 1.37 ± 0.06. 2.61 ±
0.10. and 4.95 ±0.16 °C/min. Thus, the average maximum
warm stimuli after 60 s were 15.7. 16.4. 17.6. and 20.0 °C,
and the maximum cold stimuli were 14.3. 13.6. 12.4. and
10.0 °C.

Which stimulus (warm or cold) was tested on the first day
was assigned randomly, and the other set of stimuli (warm

or cold) were tested on the following day. A 25% change in
heart rate — bradycardia (decrease) or tachycardia (in-
crease)— was used as an indicator that lobsters sensed a
change in water temperature (Offutt, 1970; Dufort. 1997).
All records were digitized using a MacLab system (C & B
Sciences/i Worx. Inc.) and were analyzed to determine the
following: (1) delay to a response: (2) duration of brady-
cardia, tachycardia, or both: (3) heart rate (bpm) during
bradycardia: and (4) heart rate (bpm) during tachycardia. In
addition, thermosensitivity thresholds were estimated from
the water temperature measured above the dorsal carapace
at the time of the initial cardiac response. Controls were
conducted before any thermal stimuli were applied; the
same protocol described above was followed, but without
changing the temperature in the stimulus bath.

Localization of punitive temperature receptors

In an attempt to localize regions with putative tempera-
ture receptors, lobsters missing antennae (n = 4) or missing
antennae and antennules (n = 4) were tested for a response
to a temperature change of +2.5 °C/min. Antennae or
antennules were removed bilaterally at their base, the
wounds were sealed with wax to prevent blood loss, and the
lobsters were allowed more than 24 h to recover.

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

Time (s)

Figure 2. Typical cardiac response to a change in temperature. (A)
The top trace shows the typical response to a + 1 .4 °C/min stimulus: the
lower trace is a plot of the temperature change during the 60-s trial. The
dark closed arrow shows when the stimulus flow was turned on. and the
white open arrow shows when it was turned off. (B) An enlargement of the
hiahliahted area from (A), showing the time course of the bradycardia and
associated temperature change. Note that the rapid response may be a result
of the combination of the location of the thermistor relative to the location
of the unknown temperature sensitive receptors and the slight delay due to
the time constant of the thermistor. There was no response to controls when
the flow was switched but the temperature was not changed.

260

S. H, JURY AND W. H. WATSON III

To determine whether changes in cardiac activity were
mediated by the cardioregulatory nerves, responses to ther-
mal stimuli were measured before and after nerve lesions (n
= 5). Changes in heart rate were initially recorded in
response to thermal stimuli of +1.5 °C/min and -1.5 °C/
min. Then the cardioregulatory nerves were cut. and lob-
sters were allowed at least 2 days to recover. Finally their
cardiac responses were measured again in response to the
same stimuli that were applied before the lesions. Lesions
were made as described in Guirguis and Wilkens ( 1995). A
small (3-cnr) rectangular piece of dorsal carapace just
above the heart was removed, and superficial cuts were
made with fine scissors through the connective tissue along
the border of the opening. The shell was then replaced and
fastened in place with tape. Sham-operated control animals
(/; = 4) were treated in the same manner except that no cuts
were made in the connective tissue.

Statistical analysis

Throughout the text, variation is presented as standard
error of the mean (i.e.. mean ± SEM). A P value of <0.05
was considered to be significant for all statistical tests.

Results

Typical response to a change in temperature

The typical cardiac response to both warm and cold
stimuli consisted of a short bradycardia (39.5 ± 8.0 s).
followed by a significantly (paired t test) longer tachycardia
(188.2 ± 10.7 s; Fig. 2). In general, changes in heart activity
were similar in response to both warm (n = 19) and cold (n
- 17) stimuli. Although the intensity and duration of car-
diac responses were similar for all temperatures tested
(ANOVA, P > 0.05). some lobsters did not respond to
slower rates of change (0.7 and 1.4 C/min). whereas almost
all lobsters responded to the maximum rate of change (5.0
°C/min; Fig. 3). There was no cardiac response in control
trials (H = 36). where temperature was not changed but
ambient water was pumped through the chamber from the
stimulus bath (Fig. 4).

Sensitivity to warm and cult/ stimuli

Lobsters were extremely sensitive to both warm and
cold stimuli (Fig. 3). For example, when subjected to a
+ 2.6 °C/min stimulus, lobsters responded after just
3.8 ± 0.5 s, when the temperature in the chamber had
changed by only 0.09 ± 0.04 °C. Lobsters exposed to the
-2.6 °C/min stimulus responded after a drop of only
0.13 ± 0.09 °C. and the latency to respond (4.6 ± 1.8 s)
was not significantly different (paired t test) than during
a warm stimulus (Fig. 3).

Temperature change measured al the initiation of a
cardiac response by individual lobsters ranged from 0.01

0.50

0 1.0 2.0 3.0 4.0 5.0

Rate of Change (°C/min)

Figure 3. Responses to thermal stimuli at different rates of change. (A)
The thermal detection threshold, or the amount of temperature change
required to elicit a cardiac response, was similar even when hot and cold
stimuli were applied at different rates. (B) When thermal stimuli were
applied at slow rates of change, the delay to respond was longer, especially
in the case of cold stimuli. (C) Although lobsters responded similarly to
thermal stimuli applied at fast and slow rates of change, some animals did
not respond at all to slow rates of change, while all animals responded to
hisiher rates ol chansic.

to 0.79 °C. There were no significant differences (un-
paired t test) between the sexes in the temperature change
at initial response; when lobsters responded, they exhib-
ited comparable thresholds, at all measured rates of
change (Fig. 3. Kruskal-Wallis test). The average tem-
perature-detection threshold, for all trials in which ani-
mals responded, was 0.15 ± 0.03 °C. This is considered
to be only an estimate because of the inherent time
constant and resolution of the thermistor, the How of
water in the chamber, and the location of the thermistor
relative to the still unknown location of the receptors
mediating the response. Nonetheless, this assay demon-
strates that lobsters are sensitive to very small changes in
temperature.

THERMOSENSITIVITY OF LOBSTERS

261

D response latency
S bradycardia
D tachycardia

100

150

200 250 300 350

Time (s)

80 -

' T

fl Control rate

D Initial rate

£ 60 -

£t

T _

D Bradycardia
D Tachycardia

(I,

rt to-

4-J

(

= 2°-

—

cold warm
stimulus stimulus

Summer Winter

Figure 4. Responses to cold and warm stimuli by summer and winter
animalv (A) The mean response latency, duration of bradycardia. and
duration of tachycardia in response to ± 2.6 °C/min stimuli in both
summer and winter lobsters. While the latency to respond, and thus
thresholds, were similar between seasons, the duration of both bradycardia
and tachycardia responses tended to be longer in summer animals. IB)
Differences in the responsiveness of summer and winter lobsters. For all
treatments, an application of control 15 °C stimuli to lobsters held at 15 C
did not cause a statistically significant change in heart rate. Lobsters in both
seasons responded to ± 2.6 °C/min stimuli with a short bradycardia
followed by a longer tachycardia. The major difference between summer
and winter lobsters, other than the duration of responses shown in A. was
that animals in the winter had significantly lower initial heart rates. Thus,
although all animals were held at 15 °C, there appears to be some temper-
ature-independent seasonal modulation of heart rate.

Localization of receptors

All lobsters with ablated antennae (n - 4) or ablated
antennae and antennules (n = 4) showed typical responses
to a stimulus of +2.5 °C/min. There was no significant
difference (unpaired t tests) in the duration of bradycardia
and tachycardia, the thermosensitivity threshold, or the
baseline heart rate when compared to intact lobsters (/; =
18).

Prior to cardioregulatory nerve lesions, lobsters (n = 5}

had a mean heart rate of 41 ± 2.9 bpm. Following recovery,
their heart rate increased to 70 ± 5.3 bpm. which was
significantly higher (paired t test) than the basal rate. This
increase may not have been due solely to the lesion ot the
cardioregulatory nerves: sham-operated controls (;i = 4)
also had heart rates (48 ± 9.4 bpm) that were slightly higher
than their pre-operation baselines (37 ± 5.4 bpm). although
that difference was not significant (paired t test). In response
to a hot or cold stimulus, all lobsters exhibited typical
bradycardia and tachycardia responses before cardioregula-
tory nerve lesioning. However, after lesioning none of the
lobsters showed an increase or decrease in heart rate in
response to thermal stimuli. In contrast, all of the sham-
operated lobsters showed typical responses (100% showed
both bradycardia and tachycardia) when exposed to identi-
cal thermal stimuli. These data indicate that the change in
heart rate elicited by warm and cold stimuli is mediated by
the cardioregulatory nerves and not by the direct influence
of temperature on the heart itself.

The influence of season

The characteristic cardiac response of lobsters to a
change in temperature (a short bradycardia followed by a
longer tachycardia) was similar for lobsters tested in the
winter and those tested in the summer. However, the fol-
lowing differences were apparent: ( 1 ) the mean baseline
heart rate was significantly lower (unpaired t test) in winter
lobsters (34.8 ± 4.4 bpm) than in summer lobsters (49.9 ±
5.0 bpm: Fig. 4): (2) the duration of bradycardia and tachy-
cardia responses tended to be shorter in the winter lobsters
(unpaired t tests. P < 0. 1 ); and (3) winter lobsters tended to
respond to temperature changes with both tachycardia
(83%) and bradycardia (72%). whereas only 50% of the
summer lobsters responded with tachycardia, but 94%
showed bradycardia. Thus, even though winter and summer
lobsters were both held at 15 °C for at least one month and
tested with identical warm and cold stimuli, they responded
differently. This difference may be related to the observed
seasonal differences in basal physiological state (Fig. 4).

Discussion

This study supports the findings of two previous behav-
ioral studies which infer that American lobsters can sense
changes in temperature (Reynolds and Casterlin. 1979:
Crossin et ai. 1998). Assuming that the initiation of a
cardiac response indicates detection of an environmental
change, our conservative estimate is that lobsters can sense
both increases and decreases in water temperature of greater
than 1 °C (Fig. 3) and probably as small as 0.15 °C. Similar
thermal sensitivity has been documented in a number of
terrestrial arthropods (Murphy and Heath, 1983; Altner and
Loftus. 1985). For example, the spider Cupieniws salei has
a warm receptor with a detection threshold between 0.08

262

S. H. JURY AND W. H. WATSON III

and 0.6 °C (Ehn and Tichy. 19%). Studies of thermorecep-
tion in aquatic species are fewer, but are consistent with our
findings. For example. Forward (1990) found that crab
larvae (Rhithropanopeus Imrrinii and Neopanope sayi) as-
cend or descend in a water column in response to absolute
temperature changes of 0.29-0.49 °C, as long as the rate of
change is fast enough (0.06-0.24 °C/min, depending on
larval stage and species). Thus, the American lobster is
probably not unusual in its ability to detect small changes in
temperature, although the extent to which this level of
thermosensitivity exists in other crustaceans remains to be
investigated.

Although several behavioral studies indicate that crusta-
ceans are quite sensitive to changes in temperature, little is
known about thermosensitivity in this large group of pri-
marily aquatic invertebrates. A study of the thermal sensi-
tivity of the dactyl receptors of Cancer imtewuirhts, C.
antlwnvi. and Panidinis iiiterntptus strongly suggests that
they possess a thermal sensory system capable of integrat-
ing temperature information for use in thermally cued be-
havior (Cook, 1984). However, the actual thermoreceptors
have not been identified in these species. In lobsters, a
number of neurons change their rate of firing in response to
shifts in temperature, but it is not clear if these cells are
actually serving the function of thermoreceptors. For exam-
ple, intracellular recordings from cells of the thoracic gan-
glia connectives of H. americanus show firing patterns that
reversibly change from silent to continuously active to
bursting over the range of 10-17 "C (Konishi and Kravitz,
1978). This is within the normal ecological range for this
species, and while it is unknown what physiological or
motor output results from this neuronal property, the corre-
spondence to the behaviorally determined preferred temper-
ature ( 16 "C; Crossin et til.. 1998) for this species is intrigu-
ing. In the spiny lobster, Puniilinix japonicns, ligamental
nerves innervating the pericardia! organ have also been
reported to increase their firing in response to cold stimu-
lation (Kuramoto and Tani. 1994). Once again, temperature
stimuli were shown to have a direct physiological effect in
viim. but it is unknown how. or if. this effect is related to the
existence of thermoreceptors or behavioral thermoregula-
tion. Ablation studies indicate that lobsters missing anten-
nae or antennules respond to temperature just like intact
animals, suggesting that while these appendages may or
may not contain thermosensitive elements, they are not
necessary in order for lobsters to exhibit a cardiac response
to temperature. Thus, while localisation of receptors and
mechanisms of thermoreception are beginning to be eluci-
dated in some invertebrates (Mori and Ohshima. 1995;
Komatsu el ai. 1996: McCleskey, 1997), it remains unclear
exactly where and how crustaceans are sensing temperature
(Ache. 1982).

Although gradual changes in temperature have a pro-
found, well-documented inlHicnce on the metabolism and

cardiovascular function of lobsters (Mercaldo- Allen and
Thurberg. 1987: McMahon. 1995: Whiteley et <//., 1995;
DeWachter and McMahon. 1996). brief decreases in heart
rate following acute temperature changes are also common
in lobsters and many crustaceans (McMahon and Wilkens.
1972; McMahon. 1995; DeWachter and McMahon. 1996).
It is unlikely that these acute responses are due to a direct
impact of temperature on the heart for the following rea-
sons: ( 1 ) lobsters with cut cardioregulatory nerves do not
change their heart rate in response to acute temperature
changes; (2) QM, values for heart rates of intact lobsters
generally range from 1.5 to 2.5 (Mercaldo-Allen and Thur-
berg. 1987; Schreiber et «/., 1998), but excised lobster
hearts are not very sensitive to changes in temperature over
the range of 12 to 19 °C, and they generally have lower QM1
values than intact lobsters over the same range (Schreiber et
i//.. 1998; Jury, unpublished data); (3) the initiation of a
cardiac response is immediate and robust, and the response
extends well beyond the duration of a temperature stimulus
(Fig. 2): and (4) the response is similar (i.e., bradycardia
followed by tachycardia) whether warm or cool stimuli are
applied (Fig. 3). Therefore, although temperature can have
a long-term, direct influence on heart rate and cause release
of modulatory substances from the pericardia! organs of
lobsters (Kuramoto and Tani, 1994), all current data
strongly suggest that in lobsters some type of thermosensi-
tive mechanism senses a change in temperature, and this
leads to a change in heart rate through activation of inhib-
itory or excitatory cardioregulatory nerves.

Lobsters do not appear to have seasonal differences in
their ability lo detect temperature. However, winter lobsters
have lower basal heart rates and respond to temperature
somewhat differently than summer lobsters. These differ-
ences suggest the presence of some type of seasonal mod-
ulation of the lobster cardiovascular system, similar to the
actions of thyroid hormones in frogs (Miller and Mizell.
1972). Biogenic amines, such as serotonin and octopamine.
have been shown to increase cardiac output. Seasonal
changes in circulating levels of these, or similar, neuro-
modulators might increase basal heart rates in the summer
or alter how the heart responds to input from cardioregula-
tory nerves (Fingerman et a/.. 1994; Wood et ai. 1995;
Weiger, 1997). This type of seasonal modulation is likely to
influence responses to a variety of stimuli, in addition to
temperature. The precise mechanisms underlying these sea-
sonal changes in the cardiovascular system ot crustaceans
remain to be resolved. A recent study of blue crabs, Culli-
ncctcx scipitliis, which documented seasonal differences in
their behavioral responses to injected biogenic amines and
proctolin (Wood et <//., 1995), suggests that seasonal vari-
ability in the expression of receptors may be the mechanism.

The rate of temperature change may be an important
variable in detection of thermal shifts in the environment.
The lobsters in this study responded to rates as low as 0.7

THERMOSENSITIVITY OF LOBSTERS

263

°C/min. and there was no statistically significant difference
between the thermal-detection thresholds obtained over the
range of rates tested. We did not attempt to determine the
slowest rate of change they were able to detect, but we did
find that some lobsters did not respond at all to the slower
rates of change used in our experiments (0.7-1.4 ~C/min).
Florey and Kriebel ( 1974) found that in Cancer species, the
rate of change must be greater than 0.33 °C/min to "avoid
hysteresis effects." This is interpreted as meaning that acute
bradycardias or tachycardias were seen at rates of change
faster than this, but only long-term changes in heart rate
were observed at slower rates. Crab larvae (R. hurrisii)
descend in the water column when the temperature is ele-
vated at rates ranging from 0.07 to 0.24 °C/min. and they
ascend when the temperature decreases at rates of 0.06 to
0.1 °C/min. However, "the average absolute amounts of
temperature change needed to evoke a response was inde-
pendent of the rate of change at rates above threshold and
ranged from 0.29 to 0.49 C" (Forward. 1990). Seasonal
changes in water temperature in some lobster habitats (e.g.,
coastal New Hampshire) can range from 0 to 25 °C. but
rates of change may be too slow to directly stimulate puta-
tive lobster thermoreceptors. However, tidal changes or
thermoclines may change fast enough to be detected. We
have measured rates of temperature change as high as 0.33
°C/min in the Great Bay estuary, although average rates are
about 0.004 °C/min (based upon hourly Licor CTD read-
ings, R. Langan, University of New Hampshire. Durham,
unpublished data). Some studies have also found lobsters to
aggregate at thermoclines (Ennis. 1984: Estrella and Mor-
rissey. 1997). Movement of a lobster (or an appendage)
within a thermally heterogeneous habitat may increase the
realized rate of change to the point at which temperatures
fall within the detection range. In addition, movement of
water (i.e.. a current) past a stationary or moving lobster
could also increase the rate of change if the water was
thermally heterogeneous. Thus, under appropriate condi-
tions, the combination of mobility and sensitivity to small
temperature changes may provide lobsters with sufficient
information to behaviorally thermoregulate in their natural
habitat in the same manner observed in small thermal gra-
dient tanks (Crossin et <//.. 1998). Nonetheless, although
lobsters can apparently use temperature as a cue in habitat
selection (Reynolds and Casterlin, 1979: Crossin et ul..
1998). the neural mechanisms giving rise to thermally
guided movements are unknown.

In this study we used changes in heart rate to determine
the sensitivity of lobsters to thermal stimuli. While this
assay is useful for determining an animal's ability to sense
small changes in temperature, it is not known whether
lobsters exhibit similar changes in heart rate when they
encounter thermal shifts in their natural habitat. Crustaceans
held for more than 12 h in a state of "sensory deprivation"
have been reported to be much more responsive to a variety

of environmental stimuli (Offutt. 1970: Florey and Kriebel.
1974); thus, the sensitivity of lobsters to temperature may
have been enhanced in our experiments. In contrast, lobsters
that are not given time to recover from handling have high
basal heart rates and often do not exhibit typical responses
to environmental stimuli. Thus, although lobsters in their
natural habitat can probably sense very small changes in
water temperature, these thermal stimuli may not always
lead to the types of cardiac responses observed in quiescent
laboratory animals. This hypothesis is being tested by re-
cording from freely moving lobsters subjected to acute
changes in temperature as they move spontaneously through
thermal gradients.

Acknowledgments

We thank the following people for helping to make this
project possible: Heather Jury. Chris Dufort. Jim Newcomb.
Paul Bartell, Christina Rockel. and Glen Crossin. Special
thanks to Dan O'Grady for conducting the cardioregulatory
nerve lesion experiments: Hunt Howell at the UNH Coastal
Marine Lab for supplying animals: Rich Langan at the UNH
Jackson Estuarine Lab for housing animals; Dan Reves for
drawings; and Cliff Bredneberg and Ashish More of C & B
Sciences. Inc.. for technical assistance. This work was sup-
ported by USD A (Hatch) and NO A A (Sea Grant) grants to
WHW and by UNH Graduate student enhancement and
Center for Marine Biology Grants to SHJ. This study was
part of the doctoral dissertation research of SHJ. This manu-
script was greatly improved by the comments of three
anonymous reviewers. This is contribution #355 ot the
UNH Center for Marine Biology Series.

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Reference: Biol. Bull. 199: 265-277. (December 2000)

Molt-Related and Size-Dependent Differences in the

Escape Response and Post-Threat Behavior of the

American Lobster, Homarus americanus

S. I. CROMARTY,* J. MELLO, AND G. KASS-SIMONt
Biological Sciences Department, University of Rhode Island, Kingston. Rhode Island 02881

Abstract. Videotaped recordings of adult lobsters of dif-
ferent molt stages were analyzed. The escape response of
adults was compared with that of juveniles recorded in an
earlier study.

Juvenile lobsters always respond to a threat with escape
behavior irrespective of their molt stage, but in adults the
probability of eliciting a response was a function of molt
stage: more hard-shelled (intermolt stage C) and (premolt
stage D) animals tailflipped than did soft-shelled (postmolt
stages A and B) animals.

The number, frequency, and duration of tailflips, and the
average distance swum by animals in each molt stage were
measured for the entire escape response, for the initial
power swim, and for the subsequent swims. These measure-
ments were used to compute several parameters: velocity,
acceleration, force, and work; average distance traveled in a
tailflip for each kilogram of body weight (distance/kg/tail-
flip): and average distance traveled for each bodylength
(distance/bodylength).

Among adults, intermolt (stage C) lobsters traveled sig-
nificantly farther and faster than postmolt animals (stages A
and B). Among juveniles, late postmolt (stage B) animals
traveled farther. Among adults, although the total number of
tailflips and the duration of the response were not signifi-
cantly different among molt stages, the number of tailflips/
second (frequency) and distance traveled/kg/tailflip were
greater for intermolt animals. In juvenile intermolts. how-
ever, frequency and distance/kg/tailflip were markedly

Received 22 October 1999; accepted 7 July 2000.

* Present address: Department of Natural Sciences, Assumption Col-
lege, 500 Salisbury Street, Worcester. MA 01609-1296. E-mail:
scromart@eve.assumption.edu

t To whom correspondence should be addressed. Biological Sciences
Department. University of Rhode Island, 100 Flagg Rd.. Kingston, RI
02881-0816. E-mail: Kass.Simon@uri.edu

lower than in the premolt stages. Although values were
lower than intermolts and premolts, postmolt adults sus-
tained their swimming frequency, distance/kg/tailflip, and
distance/bodylength for the entire escape distance (as did
postmolt juveniles). These parameters then dropped off
sharply for both adult and juvenile intermolt and premolt
animals in the second half of the escape distance.

Post-threat behaviors reveal that stage D animals have the
highest aggression index and often attack the presented
stimulus, whereas stage A animals are the least likely to
approach the stimulus and typically back away in a non-
aggressive posture.

Thus, although effects of the molt cycle on adult and
juvenile escape behavior are similar in some ways, other
physical characteristics of adults, such as weight, allometry.
and physiology, seem to become important in determining
the likelihood of escape behavior and the characteristics of
the escape swim in each molt stage.

Introduction

The behavior of the American lobster, Homarus ameri-
canus, varies (both in the laboratory and in the field) with
sex and reproductive state (Cowan and Atema, 1990; Figler
et al.. 1997, 1998: Cromarty et al, 1998; Mello et ai,
1999), relative size (Scrivener, 1971; Lang et al., 1977),
time in residence (O'Neill and Cobb, 1979; Peeke et al.,
1998; Cromarty et al., 1999), and dominance (Karnofsky
and Price. 1989; Huber and Kravitz, 1995). However, molt-
cycle-related behaviors have been rarely been studied —
probably because the long-term approaches and experimen-
tal designs needed are complex.

The physiological transformations that occur in decapod
crustaceans over the molt cycle are clearly profound; they
include a variety of metabolic, neuroendocrine. and neuro-

265

266

S. I. CROMARTY ET AL.

physiological changes (Knowles and Carlisle, 1956; Pas-
sano, 1960; Kleinholz and Keller. 1979; Quackenbush,
1986) that could manifest themselves in distinctive molt-
stage-related behavioral modifications. Specifically, the es-
cape response behavior in decapod crustaceans is ideally
suited for modulation because it is composed of multiple
tailflips, or swims. The escape response consists of an initial
power swim followed by a series of subsequent swims; in
crayfish, the initial power swim is elicited by visual and
tactile excitation in the front of the animal which is medi-
ated by the medial giant neuronal system, while tactile
excitation of the telson at the rear of the animal is mediated
by the lateral giant system. The subsequent swims immedi-
ately following the power swim are mediated by the non-
giant system, which innervates the phasic flexor abdominal
musculature (Wine and Krasne, 1972, 1982). Although the
physiology of tailflip escape circuits has not yet been fully
described in lobsters, the close similarity between the two
species suggests that the innervation is similar.

While studying confrontations between juvenile Ameri-
can lobsters, Tamm and Cobb ( 1978) identified an increased
probability- of eliciting an escape response in early postmolt
stages (stages A and B). In contrast, the frequency of
aggressive behaviors, in particular the meral spread, in-
creased during mid-premolt stages (stages D, and D2).
Hard-shelled lobsters tend to be aggressive, but soft-shelled
lobsters tend to avoid confrontations. Stomatopods exhibit
similar behavioral differences (Steger and Caldwell, 1983).
These differences are understandable in view of the fact that
in their postmolt, soft-shelled state, these animals are much
more vulnerable to predation by predators and conspecifics
than are hard-shelled animals, and they are less able to fend
off attacks with aggressive behaviors, so that when threat-
ened, they are forced to try to escape instead of mounting a
defense (Tamm and Cobb. 1978; Atema and Cobb. 1980;
Atema and Voigt. 1995).

One might expect that a newly molted animal would have
difficulty doing much of anything until the exoskeleton
hardens. Although this is true for lobsters in very early stage
A. our studies revealed subtle differences in escape behavior
among juvenile lobsters over the molt cycle (Cromarty et
a/., 1991; Cromarty. 1995). We found that overall, postmolt
lobsters produced the more effective escape behavior. Soft-
shelled, postmolt juvenile lobsters (stage B) traveled farther,
produced more tailflips, and swam longer, at sustained
velocity, than did premolt lobsters. Earliest postmolt (stage
A) juveniles swam at a higher frequency. In contrast, pre-
molt juveniles produced a quick, forceful initial power
swim, followed by subsequent swims that rapidly decreased
in velocity, acceleration, force, and work output (Cromarty
et al.. 1991).

The above studies focused on juvenile lobsters; even less
information exists on molt-related changes in escape behav-
ior in adult or larger animals. We know that the escape

response occurs more frequently among juveniles and
smaller adults than among large adults (Lang et al., 1977).
and that the conduction time of medial giant impulses from
the brain to the sixth abdominal ganglion increases greatly,
causing an increase in the latency of the response. The
relative ratio of abdomen length to carapace length de-
creases with increased size, forcing the abdominal flexing
muscles to propel a larger body mass (mostly claws). As
with crayfish (Krasne and Wine, 1975), removal of the
claws of a large lobster increases its propensity to tailflip
(Lang ct nl., 1977), as we have also observed in these
experiments. Since large lobsters are less apt to be preyed
upon than small ones (Atema and Voigt, 1995), it is ex-
pected that large soft- and hard-shelled adult lobsters would
exhibit different but unique escape behaviors from one
another.

Because of the physical and behavioral differences be-
tween adults and juveniles, on the one hand, and the phys-
iological and behavioral differences among animals of dif-
ferent molt stages, on the other, we wished to investigate
whether adults and juveniles in the same molt stage differed
in the measurable characteristics of the escape response. We
therefore examined the escape response of adult male lob-
sters of different molt stages in an experiment similar to the
one we had designed for juvenile lobsters (Cromarty ct al..
1991 ). We measured distance traveled (m); number of tail-
flips (TO; duration of the response (s); frequency of tailflips
(Tf/s); velocity (m/s), acceleration (m/s/s). force (N; kg •
m/s/s) and work (J) of each tailflip; distance traveled in each
tailflip for each unit of body weight (m/kg/Tf); distance
traveled in each tailflip for each unit of bodylength (dis-
tance/bodylength); and distance traveled in each tailflip for
each unit of body weight (m/kg). In addition, we compared
the escape thresholds of juveniles and adults.

Our earlier work indicated that lobsters could show sig-
nificant differences in post-stimulus behaviors towards the
threatening object, as well as in the characteristics ot the
escape behavior itself (Cromarty et al., 1999). Thus we also
analyzed post-stimulus agonistic behaviors and now present
evidence that these behaviors — like escape behavior — dif-
fer significantly from one molt stage to another: premolt
lobsters are more likely than postmolt animals to attack a
threatening stimulus, and postmolt animals are more likely
to back away from a stimulus with no display of aggression.

Materials and Methods

Procedures and experimental protocols are essentially the
same as those described elsewhere (Cromarty et al.. 1991,
1998, 1999), but are summarized again here with relevant
differences included.

MOLT-RELATED DIFFERENCES IN ESCAPE BEHAVIOR

267

Animals

Adult American lobsters (carapace length 74 to 90 mm)
were obtained and housed as described previously
(Cromarty el a!., 1999). Twenty-four hours prior to an
experiment, an animal was moved to the Kingston campus
of the University of Rhode Island, where it was placed in a
holding tank (30 cnv1) and was not fed during this acclima-
tion and experimental period. Isolation periods in the hold-
ing tank were identical for all experimental animals. The
tank had its own air supply. To avoid possible sex-related
effects, only males were used in this study. Ten lobsters
from each molt stage (A. B, C, and D) were randomly
selected as they entered the stage. Lobsters weighed (in
grams) an average of 451.4 ± 69.6 (mean ± SD) and had an
average carapace length (in millimeters) of 81.3 ± 4.7
(mean ± SD).

The experiments were performed randomly so that no
molt-stage clustering occurred. A correlation statistic was
run to check for molt stage and date of experiment. No
correlation was found between the animals' molt stage, the
time between the animals' capture and their use in the
experiment, and the sequence of experiments (R2 < 0.18,
F > 0.05).

Lobsters were presented with the stimulus only once and
were immediately sacrificed for identification of possible
molt-related differences in the phasic flexor musculature
system that is responsible for the escape response behavior.
This required that over 200 animals be individually housed
so that lobsters entering different molt stages could be
selected.

Freshly caught lobsters were continually added to the
holding population to reduce "inactivity" and potential for
increasing aggressive behavior (Cromarty et ai, 1999). Be-
cause of possible seasonal differences in physiology and
morphology such as those described in crayfish (Lnenicka
and Zhao, 1991 ), experiments were conducted between June
and October when Rhode Island waters maintain tempera-
tures between 18°C and 23°C and similar conditions can be
maintained in the indoor holding tanks. Again, no correla-
tion was found between any of the significant parameters
and the date of experiment.

Experiments

Each experiment was run between 1 200 and 1 500 hours
in a 4000-1 tank filled with filtered recirculated seawater.
The large amount of seawater held in the experimental tank
made it impractical to drain the tank after each experiment,
but carbon filters were continuously used throughout this
experimental period to remove possible recognition odors
originating from the lobsters' urine. Nevertheless, a corre-
lation statistic was run to check for success of tailflipping
and lobster order. No correlation was found between the

order of experiments and the animals' success and failure of
tailflipping (R2 C 0.13, F > 0.05).

Salinity was kept between 29(7cr and 33^c. and adjust-
ments (if any) were made before each experiment. One hour
before an experiment, the physical condition of each animal
was checked. Animals were used only if they moved around
the tank or exhibited antennule flicking.

Water temperature in the experimental tank was main-
tained between 18°C and 20°C by a chiller. The experimen-
tal area consisted of an open-ended tank ( 1.0-m L X 0.3-m
W X 0.3-m H) immersed in a larger main tank (2.2-m L X
0.75-m W X 0.91-m H). A weighted wooden partition with
a pulley acted as a blind (and separation to the main tank) at
the open side of the experimental tank (Fig. 1 A).

The experimental tank was designed with an open end so
that a threatening stimulus could be introduced at that end.
To ensure that lobsters were initially at the closed, non-
stimulus end, a light was placed at the open end of the tank.
The partition was raised once the lobster had reached the
closed end. The light was then placed over the closed.
non-stimulus end. This served to "push" the animal back
towards the open (stimulus) end. Because adult lobsters did
not respond to the stimulus that was used to induce an
escape response in juveniles (a flat shiny and reflective
mirror. 0.1 m2, housed in a wooden frame attached to a
dowel stick), a piece of PVC tubing ( 15-cm L X 10-cm W)
weighted with pebbles weighing 1.45 kg served as the
threatening stimulus. The stimulus was raised above the
open end. as depicted in Figure 1A. and was released into
the water at a preset distance of 10 cm (measured from the
open edge of the tank to the lobster's rostrum) whenever a
lobster approached the open end of the tank.

Cameras were placed in horizontal and vertical positions
so that the experiments were simultaneously recorded on
two video recording systems. Video recordings of each
lobster were analyzed frame-by-frame. To measure distance
traveled, a metric grid divided into 0.5-cm units was painted
onto the side of the experimental tank. Distance traveled
along the length of the tank was measured using the position
of the tip of the lobster's rostrum as the point of reference.
Time was automatically recorded on the videotape, and
numbers of tailflips were counted in subsequent viewing of
the recordings.

After each experiment, the animal's molt stage was de-
termined by examining cuticular and setal development in
the pleopods (Aiken. 1973). Because animals become pro-
gressively harder after ecdysis (stage E), we also determined
postmolt periods by testing various carapace areas for ri-
gidity (Aiken. 1980). Experimental animals were placed in
the following categories: intermolt (stage C); premolt
(stages D0, D,, D:. or D,); and postmolt (stage A up to 48 h
following ecdysis and stage B from 48 to 96 h after ecdysis).

Probabilities of an escape response were determined for
each molt stage (n == 10) and statistically compared. The

268

S. [. CROMARTY ET AL

Figure 1. (A) Diagram of the experimental tank (E). The stimulus was
a weighted piece of PVC tubing filled with pebbles (A); the screen (B) was
lifted while the lobster (Fl was at the opposite end of the tank. A light (D)
at the closed end of the tank caused the animal to move towards the darker
open end (K). The escape behavior of each lobster was recorded against a
0.5-cm metrically divided grid (G). The recording system consisted of
vertical (C) and horizontal (H) cameras, a monitor (1). and a time-lapse
VCR (J). (B) Schematic breakdown of a single tailflip as it was seen in the
video analysis: (a) = beginning of swim; (f) = end of a single tailflip.
(Drawings by K. Davignon, Graphics specialist. LIRI.) Previously pub-
lished in The Biological Bulk-tin (Cromarty t-l til.. 1998).

other characteristics of the escape response of animals that
escaped were analyzed as in our earlier study of juvenile
escape behavior (Cromarty et <//.. 1991, 1998. 1999).

Videotape dimly*!.'*

Each of the escape parameters was analyzed for ( 1 ) the
entire escape response; (2) the initial power swim; (3) the
subsequent swims over the entire subsequent swimming dis-
tance; and (4) the subsequent swims in each half of that

distance, since earlier experiments showed that there were
differences in the total subsequent swimming distance traveled
by lobsters. We therefore divided the distance traveled in the
subsequent swims by half and analyzed each half (Cromarty et
til.. 1991. 1998. 1999). Because the distances differed and
because each distance was divided equally in half for each
escape sequence for each animal, no data are available to
compare distance traveled between the two halves of the sub-
sequent swims for each molt stage. (A complete tailflip, or
swim, is defined as beginning immediately after the start of
abdominal flexion and ending at abdominal extension [Fig. 1 B:
sequence a through f].) The following characteristics of the
escape response were analyzed for each lobster; distance trav-
eled (m). number of tailflips (Tf). duration of the response (s),
frequency of tailflips (Tf/s). velocity (m/s), acceleration (m/s/
s), force [N; (kg • m/s/s)]. work (J). distance traveled/weight/
tailflip (m/kg/Tf). distance traveled/weight (m/kg). and dis-
tance traveled/lobster bodylength. The latter two parameters
were calculated to determine whether individual lobster vari-
ability in weight and size altered the relative significance of a
parameter. Velocity, acceleration, force, and work are all ini-
tially calculated from the distance that the individual tailflipped
divided by the length of time the animal spent tailflipping.
Calculations were based on the distance measured on the video
records for the total escape response, the power stroke, and the
subsequent swims. Therefore, because of small differences in
each measurement due to the finite resolution of the number of
frames per second of the video camera, the added mean values
of the power stroke and subsequent swims are slightly different
from the mean values of the total escape response. (The anal-
ysis of the escape response is meant to reflect relative changes
in lobster escape behavior and not kinematic relationships such
as those investigated by other researchers [Batchelor. 1967;
Daniel and Meyhofer, 1989; Nauen and Shadwick 1999].)

To quantify the degree of "aggression" in the post-stim-
ulus behavior of each animal, we ordered the behavior
towards the stimulus and then subjectively ranked an ani-
mal's post-stimulus threat behavior on a scale of 0 to 6
(Cromarty et til., 1999):

0 = back away, never approach

1 = approach but remain more than one bodylength away

2 = approach within one bodylength

3 = approach, touch

4 = approach, touch, grasp

5 = approach, touch, grasp, and tug or pull

6 = approach, touch and grasp, tug or pull and. execute an

offensive tailflip

Statixtictil i/iidlysis

Molt stiifte versus /irohtihilitv of escupe: Fisher's exact
probability tests (FEPs) were used to determine differences
in probabilities of an escape response over the molt stages.
Because of the small number of staee A and B lobsters (4

MOLT-RELATED DIFFERENCES IN ESCAPE BEHAVIOR

269

out of our original sample of 20 animals) that tailHipped
(n = 4). the two molt stages were collapsed into a single
sample to represent postmolt. soft-shelled lobsters. Stage C
animals were classified as intermolts, and stage D lobsters
were classified as premolts.

Comparison of weight and carapace length versus molt
stage: Weight and carapace length among the molt stages
were compared in a one-way analysis of variance
( ANOVA) with a post-hoc Scheffe test to compare means of
the planned comparisons. Weights of animals that escaped
within each molt stage were compared to those that did not
in a Mann-Whitney U test (MW).

Characteristics of the escape respon.se: Linear regres-
sions were performed on all escape parameters against
weight to double-check weight influence. Due to a non-
normal distribution of data. Kruskal-Wallis tests (KWs)
were used for all the escape parameters except for the
comparison of the first and second halves of the subsequent
swims, where a multiple analysis of variance (M ANOVA)
with a one-way repeated measures follow-up test was used
to compare the two halves of the subsequent swims.

Post-threat behaviors: Post-threat behaviors were quan-
tified according to the "aggression index" and were com-
pared in a one-way analysis of variance (ANOVA) with a
post-hoc Scheffe test used to compare means of the planned
comparisons.

KWs, ANOVAs, MANOVAs, and Scheffe tests were run
using the University of Rhode Island mainframe computer
(IBM ES/9000) and SPSS 6.1 software (SPSS Inc.. Chi-

cago) for the Macintosh G3 computer. Values for all tests
were considered significant at P < 0.05. while trends were
considered at (0.05 < P < 0.10).

Results

Comparison of adult and juvenile escape probabilities

The data are summarized in Figure 2. In contrast to our
earlier studies on small juveniles (±14 g) in which 100% of
the 36 juvenile lobsters tailtlipped (9 in each of the four
stages; Cromarty et a/., 1991), none of the larger juvenile
(S150 g) or adult (>450 g) lobsters responded to the stick
stimulus, regardless of si/.e class or molt stage (see Fig. 2).
Various stimuli (water injection over the lobster, a larger
conspecific lobster, a predator (tautog), and bubbles blown
over the lobster) also failed to elicit an escape response.
However, a 15-cm-long piece of PVC tubing weighted with
pebbles, dropped suddenly from above as the lobster ap-
proached, caused both larger juvenile (±150 g) and adult
(±450 g) lobsters to tailflip (Fig. 2). In the large-juvenile
size class, 23 out of 34 tailflipped (stage A: 4 of 8; stage B:

1 of 8: stage C: 9 of 9: stage D: 9 of 9). In the large-adult
size class. 15 out of 40 tailflipped (stage A: 2 of 10: stage B:

2 of 10; stage C; 5 of 10: stage D; 6 of 10) or 20% of
soft-shelled (stages A and B) and 55% of hard-shelled
lobsters (stages C and D: FEP. P = 0.01 ). When compar-
ing the probability of eliciting an escape response for soft-
shelled (stages A and B) versus hard-shelled (stages C and
D) lobsters, soft-shelled postmolt lobsters were significantly

(A)

(B)

°>

)

•

VN°

pa
o «

j 2S~
£

^ ^

^i*

^* ^*

Q V

A B C D A B C D A B C D
14 gram 150 gram 450 gram

\BCDABCDABCD

14 gram 150 gram 450 gram

BCDABCD

>600 gram >600 gram

STICK STIMULUS

15CMPVC STIMULUS

15CM PVC

30 CM PVC

Figure 2. Percentage (%) of lobsters escaping at each weight and molt stage in response to stimuli of
different sizes. The stimulus was (Ala stick to which a mirror was attached: (B) a 15-cm length of PVC weighted
with stone weighing 1.45 kg: (C) two pieces of PVC tubing, one was 15 cm in length and weighing 1.45 kg. and
the other 30 cm in length and weighing 2.45 kg (animals heavier than 600 g did not respond to the two stimuli ).
Molt stages, weights of animals, and types of stimuli are below the .Y-axis. Ratios at top of each bar are the
number of lobsters escaping to the total number of animals presented with the stimulus.

270

S. I. CROMARTY ET AL

less likely to tailflip than hard-shelled premolt lobsters: 20%
(4 of 20) compared to 55% ( 1 I of 20) (FEP. P = 0.01 ).
Adult lobsters larger than 600 g did not tailflip, even when
the size of the PVC tubing was increased from 15-cm L X
10-cm W. 1.45 kg, to 30-cm L X 10-cm W. 2.45 kg.

Analvsis of escape behavior in adults

A. Effect of weight on escape response in the various
molt stages

As had been shown by Lang ct al. ( 1977). size and weight
have significant effects on a lobster's propensity to exhibit
an escape response.

Among the groups of animals tested, there were signifi-
cant differences in weights (Table la). Stage B lobsters
weighed significantly more than either stages C or D
(ANOVA, F(3, 36) = 7.42, P == 0.0005). Stage B
lobsters also had significantly larger carapace lengths than
either stages C or D (ANOVA. F(3. 36) == 15.69. P =
0.0001).

To determine whether any of the characteristics of the
escape response were correlated with weight, linear regres-
sions were calculated in which each of the following seven
parameters were evaluated against weight, irrespective of
molt stage: (1) probability of tailflipping: (2) duration of
escape swimming: (3) tailflip frequency, (4) velocity, and
(5) acceleration of the total escape swim; (6) force exerted
during the swim; and (7) work performed. No correlation
was found between the animals' weights and any of the
parameters tested (R2 < 0.20; F > 0.05).

Of the animals tested, only 15 out of 40 tailflipped. When
the weights of animals that tailflipped (Table Ib) were
compared, there were no significant differences among the
four molt stages (ANOVA. F(3. 1 1 ) = 2.61 , P = 0.1 1 ).
These animals were therefore subsequently used to analyze
the characteristics of the escape response with respect to
weight and molt stage.

Among the animals that did not tailflip (Table Ic), there
were significant molt-stage differences in the weights of the
animals. Stages A and B weighed significantly more than
stages C and D( ANOVA, F(3. 21) = 4.22, P = 0.002).
More soft-shelled lobsters ( Id out of 20) than hard-shelled
animals (9 out of 20) did not tailflip, suggesting that size in
this weight class could determine whether an animal will
tailflip; possible reasons for this are detailed in the discus-
sion.

B. Parameters of the athtlt escape response

A summary of all the parameters tested, with means and
standard deviations for each molt stage, are summarized in
Table 2. Precision of measurements is a function of the
number of video frames per second; therefore, since the
power stroke and subsequent swims were separately ana-
Ivzed, their mean values are not additive.

Table 1

Weight (in grains) for adult lobsters in the four molt stages; values are
mean ± standard error of the mean

Softshelled

premolt

Hardshelled
mtermolt

Premolt

STAGE A

STAGE B

STAGE C

STAGE D

(a) Combined wei

ghts of all lobsters

irrespective of escape behavior

514.3

595.1

420.3

372.1

474,2

512.1

421.0

403.4

470.0

582.0

373.0

440.2

592.2

358.6

456.0

326.2

401.0

544.2

435.6

424.5

497.0

441.4

417.4

484.9

400.0

487.6

438.7

396.0

535.1

438.0

390.6

360.4

476.2

513.4

433.5

378.0

420.8

596.3

369.0

4r,5.l

478.1 ± 19.3

506.9 ± 24.6

415.5 ± 9.2

405,1 ± 15.5

(b) Animals that tailflipped

470.0

441.4

420.3

403.4

420.8

513.4

421.0

326.2

456.0

484.9

435.6

396.0

438.7

360.4

378.0

445.4 ± 24.6

477.4 ± 36.0

434.3 ± 6.6

391.5 ± 21.8

did not escape

514.3

595,1

373.0

372,1

474.2

512.1

417.4

440.2

592.2

358.6

390.6

424.5

401.0

544.2

433.5

465.1

497.0

582.0

369.0

400.0

487.6

535.1

438.0

476.2

596.3

486.3 ± 22.4

514.2 ± 29.7

396.7 ± 12.6

425.5 ± 19.7

/. Total escape response (initial power swim plus sub-
sequent swims)

Intermolt (stage C) animals tailflipped farther than either
postmolt (stages AB) or premolt (stage D) animals (KW,
X2 = 5.42, P = 0.046; Fig. 3A). Although the total time
spent in the escape response was not significantly different
among the three molt stages (KW, x2 = 2.58, P = 0.28;
Fig. 3B), the velocity (distance/time) of the swim was also
significantly higher for intermolt animals than for postmolt
lobsters (KW, X2 = 5.94, P = 0.041; Fig. 3C). Although
the apparently shorter duration of the swim for premolts was
not significant, when time was used to calculate acceleration
(velocity/time), the resulting value became significantly
greater for premolt (stage D) lobsters (KW, x2 ' 6.76,
P = 0.034; Fig. 3D). Neither the force (weight X accel-
eration) exerted nor the work (force X distance) performed
proved to be significantly different for the three molt stages

MOLT-RELATED DIFFERENCES IN ESCAPE BEHAVIOR

Table 2

«/ .\ixnitiiwtr differences iiiiii>iit> all components analyzed over the escape response

271

Molt
Component stage

Total escape
response

Initial power
swim

Total
subsequent

SU HI!

Subsequent
swims 1 (SSI )

Subsequent
swims 2 (SS2)

SSI vs.
SS2

Distance (m)

C > (AB = D)

0.29 ± 0.27

0.06 ± 0.01

0.25 ± 0.23

0.71 ± 0.10

0.17 ± 0.02

0.64 ± 0.08

0.39 ± 0.05

0.08 ± 0.02

0.35 ± 0.23

Duration (s)

1 .08 ± 0.53

0.29 ± 0.23

1.03 ± 0.47

0.35 ± 0.24

0.70 ± 0.39

1.32 ±0.29

0.13 ± 0.04

1.08 ± 0.39

0.38 ± 0.10

0.86 ±0.16

0.81 ±0.58

0.21 ± 0.13

0.75 ± 0.62

0.29 ± 0.25

0.61 ± 0.38

Velocity (m/s)

C > (AB = D)

0.28 ± 0.22

0.63 ± 0.24

0.65 ± 0.26

0.90 ± 0.29

0.38 ± 0.08

0.56 ±0.14

0.33 ± 0.26

0.28 ± 0.28

0.51 ± 0.33

0.25 ±0.18

0.15 + 0.12

0.44 ± 0.46

0.37 ± 0.09

0.64 ± 0.29

0.27 ± 0.04

Acceleration (m/s/s)

D > (AB) = C

0.27 ± 0.19

2.87 ± 3.06

0.22 ±0.16

1.77 ± 1.00

0.38 ± 0.26

0.46 ± 0.20

5.64 ± 3.16

0.36 ±0.12

2.66 ± 1.42

0.47 ±0.18

0.55 ± 0.20

4.12 ± 4.1

0.50 ±0.18

3.27 ± 1.79

0.66 ± 0.49

Force (N; (kg • m/s/s)

0.14 ± 0.11

1 .56 ± 1 .66

0.10 ± 0.06

1.02 ± 0.61

0.22 ± 0.16

0.17 ± 0.07

2.00 ± 0.88

0.14 ±0.04

0.95 ± 0.39

0.18 ± 0.07

0.22 ± 0.09

1.51 ± 1.39

0.20 ± 0.06

1.23 ± 0.69

0.25 ± 0.20

Work (J)

0.06 ± 0.09

0.16 ± 0.12

0.05 ± 0.04

0.04 ± 0.02

0.02 ± 0.01

0.12 ± 0.05

0.20 ± 0.06

0.11 ± 0.06

0.09 ± 0.04

0.02 ± 0.03

0.05 ± 0.03

0.15 ± 0.07

0.04 ± 0.07

0.03 ± 0.06

0.01 ± 0.03

Number of tailflips (Tf)

3.5 ± 1.9

2.5 ± 1.5

1.8 ± 0.9

0.9 ± 0.3

5.8 ± 1.8

4.8 ± 1.8

3.6 ± 1.0

1.3 ± 0.4

3.5 ± 3.1

2.5 ± 2.7

2.0 ± 2.0

1.6 ± 0.8

Frequency (Tf/s)

(C = D) > AB

Cl > C2

3.05 ± 1.08

6.96 ± 1.69

3.63 ±3.17

3.33 ± 2.59

Dl > D2

4.52 ± 1.34

10.52 ±4.17

8.40 ± 3.54

2.12 ±0.91

3.94 ± 1 .02

10.78 ± 1.02

9.13 ± 2.40

1 .65 ± 1 .94

Distance/Weight (rn/kg)

0.52 ± 0.46

0.10 ± 0.03

0.41 ±0.50

1.67 ± 0.16

0.17 ± 0.03

1.52 ± 0.11

0.84 ± 0.72

0.21 ± 0.07

0.61 ± 0.72

DistanceAVeight/Tailflip

(m/kg/Tf)

C > (AB = D)

0.14 ± 0.07

0.10 ± 0.03

0.12 ±0.05

0.41 ± 0.09

0.17 ± 0.03

0.38 ± 0.06

0.23 * 0.07

0.21 ± 0.07

0.19 ± 0.05

Distance/Bodylength

C >(AB = D)

C > (AB = D)

3.5 ± 1.9

0.13 ± 0.03

3.1 ± 1.6

5.8 ± 1.8

0.10 ± 0.02

5.4 ± 1.7

3.5 ± 3.1

0.15 ±0.03

3.1 ± 2.8

A. B. C. and D represent the four molt stages. Mean ± SD of all components analyzed for three molt stages. Significant differences are indicated in boxes
at the top of each column. Stages equated with those in the parentheses are not significantly different from them.
AB. results of experiments with stages A and B were pooled due to only 4 animals that tailflipped.
SSI. first half of the subsequent swimming distance; SS2. second half of the subsequent swimming distance.
SSI versus SS2 compares the component in the two halves of the subsequent swimming distance.
NA, not analyzed due to the experimental design (see methods).
NS. no significant difference.

272

S. I. CROMARTY ET AL

|A
D

C -
A/B-

0.00 0.25 0.50 0.75

Total Distance (m)

1.00

D-

C -

A/B-

0.0 0.5 1.0 1.5

Total Time (s)

2.0

D-

C -

A/B-

0.0 0.2 0.4 0.6

Total Velocity (m/s)

0.8

D-
C -

A/B-
0

D-
C -
A/B-

0 0.2 0.4 0.6 0.
Total Acceleration (m/s/s)

1 I :: '

1 '

D-

C -

A/B-

0.1 0.2 0.3

Total Force (kgm/s/s)

0.4

0.00 0.05 0.10 0.15

Total Work (J)

0.20

Figure 3. Parameters of the total escape response (initial power swim plus subsequent swims) for adult
lobsters in all three molt stages. An asterisk (*) indicates significant differences. (A) Distance traveled in meters
(in). (B) Time spent escaping in seconds (s). (Cl Velocity of tailflips in meters/second (m/s). (D) Acceleration
in meters/second/second (m/s/s). (E) Force of tailflips in newtons (kg • m/s/s). (F) Work produced (force x
distance) is measured in joules (J).

(KW, x2 = 4.98. P = 0.083 and ,Y: = 2.15. P = 0.16:
Fig. 3E and 3F, respectively).

Although the total number of tailflips and the total time
were not significantly different (KW. x2 z 4.20, P =
0.123 and x2 = 2.58, P = 0.275, respectively), swim
frequency was significantly higher for intermolt and premolt
lobsters, with stage C and D lobsters performing more
tailflips per second than AB animals (KW, x2 = 6.93. P =
0.048; Fig. 4A). Distance traveled per lobster weight per
tailflip was greater for intermolt (stage C) animals than for
the other molt stages (C > (AB = D); KW, x2 = 5-98,
P = 0.046, Fig. 4D), and distance traveled per bodylength
was also greater for intermolt animals than for the other
molt stages (stages (C > (AB = D); KW. x2 = 5.36, P =
0.047, Fig. 4F).

2. Initial power swim

Except for distance traveled, none of the parameters were
significantly different for the three molt stages at P < 0.05,
no doubt because of the large variability among the animals
that exhibited an escape response. However, trends in the
tests suggest that intermolt and premolt lobsters executed a
faster, more accelerating, and more forceful power swim
than postmolt animals (0.05 < P < 0.10; KW).

3. Subsequent swiin\

a. Entire subsequent swim

Of the original 15 animals that responded to the stimulus
with escape swimming, only 12 executed subsequent
swims. Of these. 3 were postmolts (stages A and B), 5 were
intermolts (stage C). and 4 were premolts (stage D). The
following parameters were statistically different: the fre-

MOLT-RELATED DIFFERENCES IN ESCAPE BEHAVIOR

273

D-

C-

A/B-

—

D-
C-
A/B-

H *

• '

-H

i i i

01234567 !

Total Frequency (TF/s) . —

~ET] E

A/B

5 10 15 20 o 5 10 15

Subsequent Swim Frequency (TF/s) ss Frequency j vs ss Frequency 2

D-

C-

A/B-

D-

C-

A/B-

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 «•» 0.1 0.2 O.J 0.4 0.5 0.6

Total Distance/Weight/Tailflip Subsequent Swim Distance/Weight/Tailflip

D-

C-

A/B-

D-

C-

A/B-

01234567
Total Distance/Bodylength

02468
Subsequent Swim Distance/Bodylength

Figure 4. Mean frequency of tailflips (Tf/s) for adult lobsters for (A) total swim sequence; (B) the total
subsequent swims frequency, and (C) the subsequent swims of both the first and second halves. Mean distance
per weight per tailrlip (m/kg/Tf) for the total swim sequence (D) and for the total subsequent swims — that is.
minus the power stroke (E). Mean distance per bodylength for total distance of swim sequence per bodylength
(F) and for the total subsequent swims distance per bodylength (G). Stage AB (n = 3), stage C (;i = 5), and
stage D (n = 4). An asterisk (*) indicates significant differences among the molt stages; a triangle (A) indicates
significant differences between the two halves of the subsequent swims.

quency of swimming was higher for stages C and D (KW
X2 = 7.92, P = 0.037; Fig. 4B); the distance swum per
weight per tailflip was higher in intermolt (stage C) animals
(KW, x2 = 8.01, P = 0.028, Fig. 4E); and distance per
bodylength was greater for intermolt (Stage C) animals than
for the other molt stages (KW, x2 = 6.46, P = 0.046, Fig.
3G).

b. Comparisons of SSI and SS2

As in the previous study (Cromarty et ai, 1991 ), the total
distance traveled by each animal during the entire subse-
quent escape swims was divided in half and then the swim-
ming parameters were compared for each of the two halves
across the molt stages and between the two halves of the
swimming distance within each molt stage. SSI = the first
half of the distance; SS2 = the second half.

Comparison of SSI across molt stages. In the first half of

the subsequent swims, stage C and D lobsters swam at a
higher frequency than stages A and B (MANOVA. F( 1 ,
9) = 23.18, P = 0.014; Fig. 4C).

Comparison of SS2 across molt stages. No significant
differences were found in any of the parameters for the
second half of the subsequent swims among the molt stages
(Table 2).

Comparison of SSI and SS2 (SSj vs. SS,J within each
mult stage. For the following parameters — subsequent
swimming distance, duration, velocity, acceleration, force,
work output, number of tailflips — no significant differences
were found between the two halves of the subsequent swims
within each molt stage. There was a significant drop-off in
the frequency of swims between the first to the second
halves of the subsequent swims for hard-shelled (stages C
and D), while no differences between SSI and SS2 were

274

S. I. CROMARTY ET AL

observed for soft-shelled (stages A and B) lobsters
(MANOVA F(\. 9), P < 0.003; Fig. 4C).

Comparison of SSI and SS2 across molt stages. Fre-
quency of swimming was significantly different among the
three molt stages (MANOVA. P < 0.026). Among hard-
shelled lobsters (stages C and D). the frequency of swim-
ming was greatly reduced in the latter half of the escape
swims (Fig. 4C).

C. Post-threat behavior

There was a gradual increase in the aggression index of
all lobsters in the experiment, such that stage A had an index
value of 0.3 ± 0.5. while stage D had a value of 2.2 ± 1.4.
The values for stage D were significantly greater than for
molt stages A. B, and C (ANOVA F(3, 39), P < 0.0012:
Fig. 5A). This is especially interesting given that stage B
animals were significantly larger than stages C and D (see
section A above), yet the post-threat aggression of the
smaller hard-shelled lobsters was significantly higher.

Among the animals that tailflipped, there were significant
differences in the aggression index among the four molt
stages (ANOVA F(3. 1 1 ). P < 0.02): soft-shelled lobsters
(stages A and B) had very low or zero aggression towards
the stimulus, whereas hard-shelled lobsters (stages C and D)
had an overall aggression index of 1.4 ± .09 (Fig. 5B).
Importantly, no weight differences were observed among
the molt stages for the animals that did tailflip.

When the post-threat behaviors of lobsters that did not
tailflip were compared over the molt stages, a progression in
the index was observed: starting with a value of 0.4 ± 0.5
for stage A lobsters, the index gradually increased until the
index for stage D animals was 3.3 ± 0.5. Stage D lobsters
had a significantly higher aggression index than molt stages
A, B, and C (ANOVA F(3, 21). P < 0.0001: Fig. 5C).
Although the soft-shelled animals were significantly larger
than the hard-shelled ones (see section A), the smaller
hard-shelled lobsters were more aggressive in their post-
threat behaviors.

Discussion

In this study, we show that, like juveniles, adult male
lobsters display significant molt-related differences in es-
cape behavior. However, the escape behavior of adults,
unlike that of juveniles (weight less than 100 g), is also
influenced by physical factors.

Thus, we have found that among animals that did not
respond to a threat with an escape response, soft-shelled
adults weighed significantly more than hard-shelled adults.
This suggests that an animal's weight begins to modify the
molt-dependent swimming response to threat.

In our earlier experiments, all juvenile animals (both soft
and hard-shelled; ±14 g) responded to a stimulus threat
with escape swimming (Cromarty et «/.. 1991). No adults
responded to the same stimulus that induced 14-g juveniles

•o

(10)

do) T

Stage A Stage B Stage C Stage D

(6)

(5)

(2) (2)

Stage A Stage B Stage C Stage D

D>(A=B=C)

(4)T

(5)

(8)

1 -

(8)

Stage A Stage B Stage C Stage D

Figure 5. Mean aggression index for post-threat behavior of adult
lobsters for (A) all lobsters regardless of tailflipping; (B) lobsters that did
tailflip; and (C) lobsters that did not tailflip. The numbers in parentheses
represent the number of individuals in each molt stage; the statistical
differences are displayed in a box in the upper left-hand corner of each
graph.

to swim. Indeed, adults failed to respond to a number of
other stimuli (such as air bubbles, water injection, larger
conspecifics) that were presented to them and ultimately
responded only to a heavy weight (PVC tubing filled with
pebbles and weighing 1.45 kg) dropped suddenly in their
paths.

There seems to be an inverse relationship between

MOLT-RELATED DIFFERENCES IN ESCAPE BEHAVIOR

275

the probability of eliciting an escape response and the
weight of an animal: 14-g juveniles tailflipped with a 100%
probability; 450-g adults tailflipped with a probability of
50%, and 600-g adults failed to tailflip even when the
stimulus size was doubled. Other workers have shown that
lobsters weighing more than 600 g could be induced to
tailflip only if their claws were autotomized (Lang et al.,
1977), as we also have observed.

It appears, therefore, that the effects of molt stage — that
is, an animal's physiological condition, characterized by the
hardness of its shell (Aiken, 1973: 1980). the flaccidity of its
muscles (Passano, 1960), and the tilers of its hormones
(Stevenson et al., 1979; Fadool et al., 1989; Snyder and
Chang, 199 la, b) — begin to be modified by size and weight.

That size and weight begin to modulate the molt-deter-
mined characteristics of escape swimming can be seen if all
our findings are taken into account. Among adults, as
among juveniles, there was a significant drop off in the
frequency and distance traveled/weight/tailflip during the
second half of the subsequent swims for premolt hard-
shelled animals, but not for postmolt animals. This suggests
that escape swimming may have evolved as the primary
survival strategy among soft-shelled juvenile animals, and
that this strategy is retained in adults even as they become
heavier; however, fewer large animals were likely to tailflip.
perhaps because swimming becomes less energy-efficient
(the heavier the animal, the more work is involved).

Although size and weight appear to modulate the effects
of molt stage on escape swimming, with larger adult soft-
shelled animals not tailflipping, an inverse relationship to
weight became apparent in the post-threat behaviors of our
experimental animals. Regardless of whether the animals
had tailflipped — and even when weight was taken into ac-
count— the indices of aggression of the post-threat behav-
iors increased incrementally from stages A and B (the
largest animals) to stages C and D (the smallest animals).
Weight and size appears to be of secondary importance in
post-threat aggression; indeed, changes in aggression over
the molt stages were the deciding factor, with the lobsters
responding to a threat in accordance with the expected
molt-related changes in aggressive behaviors (Tamm and
Cobb. 1978). Undue significance should not be given to the
inverse relationship of weight and size on aggression in
general, however. In confrontations between lobsters in the
same molt stage, the size of an opponent significantly af-
fected the outcome of a bout (Scrivener, 1971 ; Mello et al.,
1999; Bolingbroke and Kass-Simon, 2000).

Overall, we found that among juveniles, soft-shelled an-
imals were better swimmers than their hard-shelled coun-
terparts, but among adults, hard-shelled premolt and inter-
molt lobsters were the best swimmers. Thus juveniles of
stage B outperformed stage C and D animals in the follow-
ing parameters: distance traveled, number of tailflips pro-
duced, distance/tailflip. time spent swimming, and velocity

(Cromarty et al.. 1991). In contrast, among adults, hard-
shelled premolt and intermolt animals outperformed soft-
shelled animals in distance traveled, velocity, acceleration,
frequency and distance traveled/lobster weight/tailflip. This
would suggest that molt stage is the predominant determi-
nant of the characteristics of escape behavior in smaller
animals, while other physical factors such as weight and
claw size may begin to dominate among adults.

The physiological bases for the differences in adult and
juvenile escape behavior over the molt cycle are likely to be
manifold and varied. In addition to probable differences
within the central nervous system, differences in endocrine,
sensory, and motor systems are certain to exist.

With regard to sensory systems (Watson. 1992). synaptic
modulation has been described for mechanoreceptors (Pasz-
tor and Bush. 1987) and stretch receptors (El Manira et al..
1991). Studies by Coulter (1988) indicate that lobsters in
stages C and D responded (with a meral spread) at different
speeds to the presentation of an expanding black disc.
Increases in lobster size have been correlated with a de-
crease in the speed of an action potential traveling from the
sensory system to the central nerve cord (Lang et al., 1977).
It is possible that juvenile and adult lobsters perceive and
respond to stimuli differently due to inherent age-related
differences in sensory functioning.

Other factors such as central (Kravitz et al., 1984; Krav-
itz. 1988; Yeh et al., 1996. 1997; Horner et al.. 1997) and
peripheral modulations (Kravitz et al., 1980; Kravitz, 1990;
Schwanke et al., 1990) are likely to affect molt-cycle be-
havior. We have recently found that 20-hydroxyecdysone
(20-HE). the active steroid regulating the molt, also alters
the neuromuscular properties of the claw-opener and phasic
flexor systems in intermolt animals (Cromarty. 1995;
Cromarty and Kass-Simon. 1998). in a way that is consis-
tent with molt-determined behavioral differences (Tamm
and Cobb. 1978; Cromarty et al., 1991). Our findings are
consistent with the rise in the blood liter of 20-HE (Snyder
and Chang, 1991a, b) when lobster aggression is beginning
to peak. In our 20-HE experiments we found that 20-HE
increases the size of the excitatory junctiona] potential (EJP)
in the claw opener muscle, which is used in threat displays,
and decreases the EJP amplitude in the abdominal phasic
flexor, which is used in escape behavior. In crayfish. Cooper
and Ruffner ( 1998) have found that EJP amplitude in the
opener muscle of the walking legs is reduced; this keeps the
dactyl from splaying and allows the animal to stand tall, as
has been observed in dominant lobster displays (for recent
review on modulation of aggressive behavior, see Kravitz,
2000). The effects of 20-HE on these above-mentioned
tissues are consistent with the functions attributed to them
during agonistic behavior. Our recent studies also show that
when 20-HE is directly injected into the lobsters' hemo-
lymph, aggressive behavior increases dramatically during
agonistic encounters, although the probability of eliciting

276

S. I. CROMARTY ET AL

escape swimming is unaltered (Bolingbroke and Kass-Si-
mon. 2000).

The sexual status of an American lobster may also alter
its use of escape behavior. Intermolt gravid females that are
presented with a startle stimulus do not tailflip. whereas
intermolt males and non-gravid females tailflip readily
(Cromarty et id., 1998): gravidity did not appear to affect
escape behavior during a confrontation, but caused an in-
crease in aggressive tail flipping (Mello et ai. 1999). It
remains to be seen whether neuromuscular properties are
modulated to reflect these sex-related behavioral differ-
ences.

Among juveniles, we have found that EJPs in the distal
region of the muscle in soft-shelled stage B animals are
larger and have a greater amplitude-duration integral than
those of hard-shelled D stage animals (Cromarty et ul.,
1995). Earlier studies by Schwanke ct ul. ( 1990) also found
molt-related differences in the dactyl opener muscle. These
findings correspond with the fact that stage B juveniles
swim greater distances by covering more distance in each
swim than do hard-shelled (stage C and D) juveniles. Fur-
ther, although EJPs continue to be produced at frequencies
up to 6 Hz in stage A and B juveniles, they begin to fail at
4 Hz in stage C and D animals. This also correlates with the
fact that stage B juveniles swim longer and cover more
ground, and that stage A animals are able to sustain swim-
ming longer and at a higher frequency than stage C or D
animals (Cromarty et ul., 1991: Cromarty et al.. 1995).

Among adults, EJPs were found to fail at 4 Hz in soft-
shelled postmolts but continued to 6 H/ in intermolt and
premolt animals (Cromarty and Kass-Simon. 1994). As in
juveniles, in adults, EJPs were largest and longest lasting in
the distal region of soft-shelled postmolts. We have also
found that EJPs in the proximal region of the abdominal
phasic flexor muscles, the anchorage or insertion region of
the contracting muscle, are significantly greater in adult C
and D stages than in juvenile C and D stages (Cromarty and
Kass-Simon. 1994). This coincides with their greater swim-
ming ability and supports our present finding that escape
swimming is used less as the animal grows, although hard-
shelled animals retain it longer than soft-shelled animals do.
This might be because escape swimming would be less
effective in large soft-shelled animals — not only because of
the large mass that would need to be propelled by the
still-flaccid muscles, but also because the large transmitter
output required by these muscles might not be sustainable
for long periods at higher frequencies.

Acknowledgments

The authors thank Bill MacElroy for allowing us to
collect animals while he was fishing offshore, and Tom
Angell of the Rhode Island Department of Environmental
Management for supplying us with additional lobsters.

Thanks to Dr. Mike Clancy and Kathy Castro for help in
lobster collection and maintenance. Drs. Stanley Cobb and
Frank Heppner kindly provided laboratory space and equip-
ment. We also thank Malia Schwartz for critiquing an
earlier draft of the manuscript. This research was supported
by a Whitehall Foundation grant to G.K-S. Grant-in Aid of
Research from Sigma Xi and Lerner Gray Grants for Marine
Research to S.I.C.

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Preferential Expulsion of Dividing Algal Cells as a
Mechanism for Regulating Algal-Cnidarian Symbiosis

GAREN BAGHDASARIAN* AND LEONARD MUSCATINE

Department of Organismic Biologv, Ecology, and Evolution, University of California, Los Angeles,

Los Angeles, California 90095-1606

Abstract. A wide range of both intrinsic and environmen-
tal factors can influence the population dynamics of algae in
symbiosis with marine cnidarians. The present study shows
that loss of algae by expulsion from cnidarian hosts is one
of the primary regulators of symbiont population density.
Because there is a significant linear con-elation between the
rate of algal expulsion and the rate of algal division, factors
that increase division rates (e.g.. elevated temperature) also
increase expulsion rates. Additionally, ?H-thymidine is
taken up to a greater extent by algae destined to be expelled
than by algae retained in the host cnidarians. Taken to-
gether, data for rates of expulsion, rates of division at
different temperatures, and uptake of H-thymidine suggest
that dividing algal cells are preferentially expelled from
their hosts. The preferential expulsion of dividing cells may
be a mechanism for regulation of algal population density,
where the rate of expulsion of algae may be an inverse
function of the ability of host cells to accommodate new
algal daughter cells. This kind of regulation is present in
some cnidarian species (e.g.. Aiptasia pulchella. Pocillo-
pura ilamicornis), but not in all (e.g., Montipora verrucosa,
Porites compressa, and Fungia xctituriii).

Introduction

Algal-cnidarian symbioses are characterized by long-
term stability wherein neither partner outgrows the other,
and where algal population densities remain relatively con-
stant (Drew, 1972; Pardy, 1974; Davies. 1984). During
repopulation of aposymbiotic sea anemones (recovery from
bleaching), symbiotic dinoflagellates grow at relatively high
rates normally associated with log phase growth in culture
(Berner et ai. 1993). If these high growth rates are sus-

Received 2 August 1999; accepted 25 September 2000.
* To whom correspondence should be addressed.

tained. host fitness is reduced (Smith, 1992), leading to the
eventual breakdown of the symbiosis (Neckelmann and
Muscatine, 1983; Taylor et al.. 1989). Instead, as the size of
the algal population reaches some optimum level, its growth
rate decreases by a factor of 20 (Kinzie. 1974; Kinzie and
Chee. 1979; Berner et al. 1993), and a "steady state" is
achieved, in which the growth rates of the algae and the host
cells are in dynamic equilibrium. Clearly, regulation of
symbiont population density is essential in maintaining a
stable symbiosis, yet little is known of the cellular mecha-
nisms involved.

A number of intrinsic and environmental factors can
potentially regulate algal cell division and population
growth. These factors could act pre- or post-mitotically
(Hoegh-Guldberg and Smith, 1989); that is. population
growth rate could be regulated either before or after algal
cell division. Algal numbers could be regulated pre-mitoti-
cally by limited nutrient availability (see, for example.
Blank and Muscatine, 1987; Kolber et al., 1988; Falkowski
ct ul.. 1993; Hoegh-Guldberg, 1994; Muller-Parker ct al..
1994; Snidvongs and Kinzie. 1994); by density-dependent
negative feedback by the algae themselves (Muscatine and
Pool, 1979; McAuley and Darrah, 1990); by host-induced
release of photosynthate from the algae (Muscatine, 1967;
Cook, 1983; Douglas and Smith, 1984; Sutton and Hoegh-
Guldberg, 1990; McAuley. 1992; Gates ct al.. 1995); or by
factors manifested by the host cells that inhibit the algal cell
cycle (Smith and Muscatine. 1999). Algal numbers could
also be regulated postmitotically by degradation of algae in
.situ (Muscatine and Pool. 1979; Titlyanov et ai, 1996;
Jones and Yellowlees, 1997), by direct expulsion of excess
algae (Hoegh-Guldberg and Smith, 1989: Stimson and Kin-
zie, 1991; McCloskey et al.. 1996; Jones and Yellowlees,
1997), or by accommodation of excess algae by division of
host cells (Muscatine and Pool, 1979; Neckelmann and

278

REGULATION OF ALGAL-CNIDARIAN SYMBIOSIS

279

Muscatine, 1983; Smith and Muscatine. 1986; Titlyanov et
«/., 1996).

Whereas previous studies have addressed pre-mitotic
control of algae in symbiotic cnidarians. there is little in-
formation on mechanisms involved in post-mitotic regula-
tion, especially relating to loss of algae by expulsion. Jones
and Yellowlees (1997) showed that the combined effect of
changes in rates of algal division and loss are involved in
repopulation of bleached corals and in the eventual regula-
tion of steady-state algal-cnidarian symbioses. In a previous
study, we observed that the Hawaiian sea anemone Aiptaxhi
pulchellii maintained in the laboratory expels algae at a rate
of about 0.046 d ' (Baghdasarian and Muscatine, unpubl.
data), a value high enough to be a major factor in regulating
algal densities. Studies of other cnidarians report not only
that algae are lost by expulsion, but also that the expelled
algae have higher mitotic indices than algae retained by the
host (Suharsono and Brown, 1992; McCloskey etui., 1996).
It has been hypothesized that the higher mitotic indices of
expelled algae are due to release from some regulatory
constraint by the hosts. In this study we test an alternative
hypothesis, that higher mitotic indices of expelled algae are
the result of preferential expulsion of dividing cells. It is
important to note here that the release mechanism probably
entails detachment of host cells (Gates et «/.. 1992), their
disintegration, and release of algae. Whereas the mechanism
of preferential detachment of host cells remains to be ad-
dressed, here we focus on algal parameters.

Materials and Methods

Collection and maintenance of organisms

Specimens of the sea anemone Aiptasia pulchella Carl-
gren (1943) symbiotic with the dinoflagellate Symbiodinium
pulchrorum were collected from Kaneohe Bay, Oahu, Ha-
waii (spring and fall 1995/1996). The animals were trans-
ferred by air to the University of California, Los Angeles,
and maintained in natural seawater in 1 .5-liter glass bowls
in a Percival model 1-35 VL incubator at 25°C on a 12-h
light/dark cycle at irradiance levels of 80-100 jumole pho-
tons m~2 s"1, from two Rainbow Lifeguard 40- watt
Primetinic and two General Electric 40-watt Cool White
light sources. The animals were fed twice a week in the
evenings on Anemia sp. The morning after every feeding,
the bowls were cleaned using cotton swabs, and the seawa-
ter, collected from Redondo Beach, California, was re-
placed. Prior to the experiments, sea anemones were starved
for 24 h under the light and temperature conditions de-
scribed above.

The symbiotic corals Porites compressa Dana 1846,
Montipora verrucosa Lamarck 1816, Pocillopora damicor-
nis Linnaeus 1758, and Fungia scutaria Lamarck 1801 were
collected from Kaneohe Bay, Oahu, Hawaii (October/No-
vember 1996). The corals were transferred to the laboratory

of the Hawaii Institute of Marine Biology, and were main-
tained in running seawater and exposed to natural light
levels. All experiments were performed within 24 h of the
collections.

Mitotic iin!c\ of algae

Five A. pnlclwlUi were homogenized individually and
their symbiotic algae isolated by repetitive centrifugation
and resuspension (Steen, 1987). The technique was applied
rigorously to eliminate the possibility of inclusion of algae
still within host cells. The algal mitotic index (MI), defined
as the percentage of cells with division plates (Wilkerson et
ai, 1983) was determined by examination of at least 1000
algal cells under 400 X magnification using an Olympus
BH-2 microscope. All additional microscopic observations
were made under the same specifications.

Incorporation of3H-thymidine by algae in situ

Each of five A. pulchella specimens was incubated indi-
vidually in 4 ml of a 2-^Ci/ml solution of 3H-thymidine
(Sigma Chemical Company; Sp. act. 50 Ci per mmol) in
filtered seawater (FSW) for 24 h under the same mainte-
nance conditions as the stock animals. The sea anemones
were then washed serially 10 times in FSW to remove
unincorporated 3H-thymidine. Each wash consisted of add-
ing fresh FSW, irrigating the coelenteron with a Pasteur
pipette, and then waiting 3 min before changing the water
again. Seven to nine washes were sufficient to remove
unincorporated 3H-thymidine from the sea anemones and
the incubation medium. Algae expelled during the labeling
period were discarded. 3H-thymidine-labeled sea anemones
were then incubated in FSW for an additional 1 5.5 h at 25°C
in the light. At the end of the incubation period, both the
algae released and those remaining in the hosts were iso-
lated (using techniques described above on ice), adjusted to
known volumes, and counted with a hemacytometer (Fisher
Scientific). One-hundred-microliter samples of algae, along
with 5 ml of Bio-Safe II biodegradable counting cocktail,
were then added to plastic scintillation vials, and the incor-
poration of 3H-thymidine by the algae was determined using
an LKB Wallac 1214 Rackbeta liquid scintillation counter.
Results were expressed as DPM * (106 algae)"1. To deter-
mine whether the incorporation of 3H-thymidine was by the
algae or by host cells that contain the algae and may have
been released along with them, techniques described in
Gates and Muscatine (1992) were used to stain cells with
Hoechst 33258 to check for the occurrence of host cell
nuclei.

Rates of expulsion of algae

Fourteen sea anemones were allowed to settle in 4 ml of
FSW in 15-ml test tubes. Six of the sea anemones were

280

G. BAGHDASARIAN AND L. MUSCAT1NE

incubated for 15.5 h at 25°C in the light, and the other eight
were incubated for 15.5 h at 27.5°C in the light. After the
incubation, algae in the incubation medium were recovered
by centrifugation and set aside for analysis. Algae remain-
ing in the animals were isolated by homogenization and
centrifugation (Steen. 1987). Both the algae expelled into
the medium and those retained by the hosts were counted,
and the MI of the expelled algae was determined (Hoegh-
Guldberg er al., 1987).

Mitotic index and rate of expulsion of algae in corals

Mitotic indices and rates of algal expulsion were also estab-
lished for four species of scleractinian corals commonly found
in Kaneohe Bay, Oahu, Hawaii. Six pieces of each coral type
from different colonies (P. compressa, M. verrucosa, P. dami-
comis, and in the case of F. scutaria, entire corals) were
incubated in 30 ml of FSW at 27°C (seawater temperature in
Kaneohe Bay) for 15.5 h. Next, to determine the effect of slight
elevations of temperature on MI values and algal expulsion
rates, six additional pieces from each coral type were incubated
at 29.5°C for the same length of time. At the end of the
incubation period, the corals were removed from the seawater.
The released algae were collected by high-speed centrifugation
of the incubation medium using a Damon IEC clinical centri-
fuge, followed by resuspension in 5 ml of fresh FSW. Algae
remaining in the corals were removed with a toothbrush. The
product was collected in FSW. The algae were then cleaned of
mucus and animal tissue by centrifugation and resuspension in
20 ml of FSW, and MI values and rates of algal expulsion were
determined using the techniques described above.

Results

Mitotic index of algae: natural expulsion vs. mechanical
isolation

Algae naturally expelled from A. pulchella have a higher
MI than the algae remaining in the hosts (Fig. 1; Wilcoxon
signed rank nonparametric test: P = 0.001 ). To determine
if the higher MI of the expelled algae is due to the absence
of host-related regulation, algae were mechanically isolated
from the sea anemones, and the MI was measured immedi-
ately and after a 15.5-h incubation at 25°C in the light. No
significant changes in MI were observed following the
15.5-h incubation period (Wilcoxon signed rank nonpara-
metric test: P = 0.593). These data suggest that the higher
MI of algae expelled by A. pulchella is not necessarily due
to release from putative host-related regulation.

Incorporation of H-thymidine b\ algae

in situ

To determine if the higher MI of expelled algal cells is
due to preferential expulsion of dividing algae, sea anemo-
nes were incubated with ^H-thymidine for 24 h, rinsed free
of unincorporated 'H-thymidine, and then incubated in

Figure 1. Mitotic index of the symbiotic alga Symbiodinium pulchro-
nim after a 15.5-h incubation of the host sea anemone. Aiptasia pulchella,
at 25°C. Comparison between expelled algae (H) and algae retained by the
host (•). Error bars represent standard deviations of the mean.

FSW for 15.5 h. Algae expelled and algae retained in the
hosts were then assayed for incorporation of 3H-thyrnidine.
Expelled algae had incorporated significantly higher levels
of ?H-thymidine than cells remaining in the hosts (Fig. 2;
Wilcoxon signed rank nonparametric test: P -- 0.043).
Because released algae are often contained within host cells
(Gates et al.. 1992), it was important to determine whether
the ^H was associated with the algae or with the nuclei of
host cells. This question was investigated by staining sam-
ples of retained and released algae with Hoechst 33582 to
detect host cell nuclei that might be associated with the
algae. Using epifluorescence microscopy to analyze the
cells (Gates et al.. 1992), we found no evidence of host cell
nuclei (i.e.. host nuclear DNA contamination). Taken to-
gether, these data suggest that the host preferentially expels
algal cells that have entered S-phase of the cell cycle.

Mitotic index and rate of expulsion of algae

The correlation between the MI of the expelled algae and the
rate of expulsion of these cells from the host could distinguish
between expulsion of algae in random phases of the cell cycle
YITMIX expulsion of algae in a preferred phase of the cell cycle.
If expulsion of algae from the host is random, then there should
be no correlation between division rate of the expelled algae
(i.e., MI) and rate of expulsion. If. however, expulsion of algae
is a function of cell cycle phase (more specifically, preferential
release during late G-, or M phases), then a positive correlation
between the two parameters would be expected, as illustrated

REGULATION OF ALGAL-CNIDARIAN SYMBIOSIS

281

6000-

5000-

4000-

3000

2000-

1000-

•c
c.

Figure 2. 'H-Thymidine incorporation by the symbiotic alga Symhio-
Jimum piilclirnniiii after a 15.5-h incubation of the host sea anemone.
Ai/nusui pulchella. in tillered seawater. Comparison between expelled
algae (M) and algae retained by the host (•). Sea anemones were initially
incubated in a 2-/j,Ci/ml solution of 'H-thymidine for 24 h. Error bars
represent standard deviations of the mean.

theoretically in Figure 3. Figure 4 shows that there is a positive
linear correlation between expulsion rate of algae and their MI.

Effect of temperature on rate of algal expulsion

If expulsion of algae is affected by algal division rate,
then environmental factors (such as slight increases in sea-
water temperature) that increase algal division rate (and
hence MI) should also increase rate of expulsion of algae.
Slightly elevated temperatures resulted in higher MI and
expulsion rates (Fig. 5; Wilcoxon signed rank nonparamet-
ric test; Expulsion rate: P == 0.034, MI: P == 0.050).
Further, the ratio of algal expulsion to MI at 25°C = 2.2,
and at 27.5°C = 2.4. The similarity of these two ratios
suggests that the higher MI values due to slight elevations in
temperature are concomitant with higher algal expulsion
rates. Finally, at 27.5°C, the observed changes in rates of
algal division and expulsion follow the same positive linear
correlation associated with preferential algal expulsion as a
function of their MI (Fig. 6).

Rates of algal expulsion in corals

The relation between algal expulsion rate and division
rate was investigated in four species of Hawaiian corals.
In general, the released algae had a higher MI than did
the algae remaining in the hosts (Fig. 7; Wilcoxon signed

Algal Expulsion Rate

Figure 3. Hypothetical correlation between mitotic index of expelled
symbiotic algae and their rate of expulsion from the hosts under conditions
of preferential versus random cell expulsion.

rank nonparametric test; significant differences seen in
Pocillopora damicornis, Montipora vcrrucosa, Fungia
scutaria: P = 0.028. but not in Porites compressa: P =
0.249). However, the linear correlation between algal

2.0-1

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1.6-

T3
o

= 1-4

o 1.2

i.o-

<*• 0.81

0.6

0.00 0.02 0.04 0.06 0.08 0.10

-1.

Algal Expulsion Rate (d )

Figure 4. Correlation between mitotic index and rate of expulsion of
Synihiiiiliiiimn r>iilclir,iri<in. Y = 0.69971 + 1I.185A'; R2 = 0.711.

282

8.00

7.00

6.00

5.00

4.00

3.00

2.00

1.00

0.00

G. BAGHDASARIAN AND L. MUSCATINE
hO.08 30 -

-0.07
-0.06

-0.00

27.5 °C

Figure 5. Effect of a 2.5°C temperature increase on rate of expulsion
(H) and mitotic index (•) of S\iiihii>diiiiuni imlchrnniin released from the
symbiotic system. Error bars represent standard deviations of the mean.

expulsion rate and MI, observed in A. pulchella, ap-
pears to hold only for P. dainicornis (Fig. 8a): it does not
hold for P. compressa. M. rernicosu. or F. xcutaria (Fig.
8b, c, d).

4.0n

o
re

Q.
X

3.0-

2.0-

•-S 1-0-1

0.0

0.00

0.05

0.10

0.15

0.20

-1

Algal Expulsion Rate (d )

Figure 6. Correlation between mitotic index and rate of expulsion of
S\inhn ijinium pulchrorum under control and elevated temperatures. 25°C
control (D). 27.5°C experimental (A). )' = -0.53328 + I7.244.V: R~ =
0.771.

Figure 7. Mitotic indices of expelled algae (O) and algae remaining
within host tissues (•) of different corals, following a 15.5-h incubation at
27°C. Error bars represent standard deviations of the mean.

Discussion

In algal-cnidarian symbioses, regulation of algal numbers is
an essential part of the symbiotic relationship, both during
"steady state" (Muscatine et id., 1975a, b; Trench, 1987) and
during recovery of cnidarians from "bleaching events" (Gates,
1990; Hayes and Bush. 1990; Fitt et al.. 1993; Jones and
Yellowlees, 1997). The present study has established that pref-
erential expulsion of dividing algae contributes to regulation of
algal-cnidarian symbiosis. If dividing algal cells are more
likely to be expelled from the host, net algal population growth
(within their hosts) will be effectively regulated.

Mitotic index of algae expelled naturally versus isolated
mechanically

Algae naturally expelled from Aiptasia pulchella and other
Hawaiian marine cnidarians have a higher MI than the algae
remaining in their hosts (Figs. 1,7). Suharsono and Brown
(1992) and McCloskey et al. (1996) have also observed this
phenomenon in other cnidarian species. Citing the most parsi-
monious explanation, these studies suggested that the increase
in algal division rates is perhaps due to a lack of host regulation
in the released algae. The present study tests this hypothesis by
addressing an alternative hypothesis — that the higher MI of the
released algae could be explained by preferential expulsion of
dividing algal cells. These two possibilities were tested by
separating the algae from the host and observing any changes
in the algal MI. If the increase in MI is simply a function of a
lack of host regulation, then artificially releasing the algae
should result in an increase in MI. However, algae artificially

5.0-1

C 4.0
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.£ 3.0-

D.
X

2 2.0

•o

- 1.0
2

REGULATION OF ALGAL-CN1DARIAN SYMBIOSIS

4.0T

283

Pocillopora damicornis

o.o-

0.00

0.02

0.04

0.06

Algal Expulsion Rate (d )

oo 3.0-

T3
111

2.01

•O

£ 1.0-

0.0

^orites compressa

0.00

0.05

0.10

0.15

Algal Expulsion Rate (d )

3.0-

01
r:
CO

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Montipora verrucosa

0.00 0.02 0.04 0.06 0.08 0.10
Algal Expulsion Rate (d '^ )

3.0-

2.0-

•u

£ 1.0-

0.0

Fungia scuta

0.0 0.1 0.2 0.3

Algal Expulsion Rate (d )

Figure 8. Correlation between mitotic index and rates of expulsion for various hermatypic corals from
Kaneohe Bay, Oahu. Hawaii. (A) Pocillopora Jamicornis (Y = 0.57457 + 54.019.Y: R~ = 0.869). (B)
Ponies compressa (Y = 2.0435 - 3.7634X; R2 = 0.023). (C) Montipora verrucosa (Y = 1.5287 +
0.12587.Y: R2 = 0.000). (D) Fungia stntariu (Y = 1.3582 + 0.111')2X: R2 = 0.000).

released from their host sea anemones (A. pitlchella) in this
study showed no increase in MI.

Incorporation of H-thymidine b\ algae in situ

Uptake and incorporation of 3H-thymidine is an indicator
of cells advancing through S-phase of the cell cycle. In A.
piilchella. cells naturally lost from the symbiosis (following

a preincubation in 'H-thymidine) incorporate more 3H-thy-
midine (just prior to release) than did cells remaining in the
hosts (Fig. 2). These data suggest that the released algae had
been in S-phase during the 3H-thymidine incubation and, by
inference, were growing and dividing. Therefore, it can be
concluded that dividing cells are preferentially expelled
from the population.

284

G. BAGHDASARIAN AND L. MUSCATINB

This is the first study to successfully employ 3H-thymi-
dine as an indicator of algal division in symbiotic
dinoflagellates. Cheney ( 1974) observed uptake of ?H-thy-
midine by the host cells of the coral Pocillopora damicor-
nis, but not by the symbiotic algae. Absence of H-thymi-
dine in the algae may have been due to ( 1 ) very low growth
rates of the resident algae, resulting in minimal uptake of the
3H-thymidine; (2) uptake and retention of the 3H-thymidine
by the host, thus minimizing availability of H-thymidine to
the algae; or (3) a high percentage of substitution of the base
thy mine by 5-hydroxymethyluracil in the algal DNA (Blank
er nl.. 1988: Taylor, 1990), resulting in low affinity for the
3H-thymidine molecules in P. damicornis. The success of
the present experiment may be attributed, in part, to the
application of longer incubation times or higher doses of
3H-thymidine. 3H-thymidine is a tool that, in parallel with
more classic approaches, may be useful in studying regula-
tion of algal-cnidarian symbiosis; in this case, it has pro-
vided evidence for preferential loss of dividing algae from
symbiotic cnidarians.

Mitotic index, algal expulsion, and effect of temperature

Figure 4 shows a direct linear correlation between expul-
sion and division rates of algae. That is. higher rates of
division translate into greater expulsion of algae, supporting
our suggestion of preferential loss of dividing cells (Fig. 3).
Further, small increases in temperature, which result in
slightly higher rates of division, also result in greater ex-
pulsion of algae (Fig. 5). This increased expulsion follows
the same linear correlation patterns (relative to division rate)
observed previously (Fig. 6). The data therefore strongly
support the interpretation that expulsion of algae is prefer-
ential and related to the position of the algal cells within the
cell cycle.

Expulsion of algae as a mechanism for regulating algal-
cnidarian symbiosis

The mechanism involved in preferential expulsion of
dividing cells is not clear, but appears to be related to the
host's ability to accommodate algal growth. Whereas the
expulsion rate of algae from /\. pnlchella in "steady-state"
symbiosis is about 0.046 d ', expulsion of algae from
re-infected aposymbiotic anemones during log phase re-
population is negligible (pers. ohs.), confirming a previous
study on repopulation of bleached individuals of the coral
Acroporaformosa (Jones and Yellowlees, 1997). Therefore,
it appears that algal cells are primarily expelled from the
system when the host cells can no longer accommodate
them. This process can act as a "fine tuning" mechanism for
regulating a steady-state symbiosis, where expulsion of
algae may be viewed as an inverse function of the host's
ability to accommodate new algal cells. Further, this mech-
anism could explain the differential expulsion of algae from

tentacle versus body regions of the cnidarians, where dif-
ferences in MI and algal densities would require different
rates of expulsion from those regions (Muller-Parker and
Pardy. 1987).

Preferential expulsion of dividing cells can also play a
stabili/ing role in algal-cnidarian symbiosis by dampening
the effects of environmental conditions that can influence
algal division rates. For example, whereas large increases in
temperature can lower algal photosynthetic capacities, small
increases in temperature may increase algal photosynthesis,
metabolism, and thus growth and rates of division (Iglesias-
Prieto ct al.. 1992). However, if rates of algal expulsion
vary as a function of the environmentally induced changes
in rates of division (Fig. 6), then effectively, by releasing the
"excess" cells, the total number of algae within the host will
be regulated.

Preferential expulsion of dividing cells is not, however,
the only means of regulating algal-cnidarian symbiosis. In
case of the green hydra symbiosis, studies have already
shown that algal ejection is not the normal mechanism for
regulating population densities (McAuley. 1982). probably
because of the higher numbers and growth rates of algae per
host cell. Among Hawaiian anthozoans. although A. pulch-
clla and P. damicornis do use preferential expulsion of
dividing cells as a regulatory mechanism, other cnidarian
species, such as P. compressa, M. vermcosa. and F. xcu-
taria. may not (Fig. 8). This interspecific variability could
be a function of the magnitude of "normal" algal growth
rates at steady-state for each of the different species con-
sidered. Species with higher steady-state growth rates might
be more likely to depend on a system of preferential expul-
sion of dividing cells for regulating their symbiosis. In
contrast, among species that have very low daily rates of
algal expulsion (such as Xenia macroxpiculata, Heteroxenia
fiiscescens, Stylophora pistillata. and Millepora dichotoma;
Hoegh-Guldberg et al.. 1987), the incidence of expulsion of
algae would not significantly affect the regulation of popu-
lation dynamics. Another possibility is that cnidarians with
different algal clades may have evolved different mecha-
nisms of regulation based on sensitivity to environmental
factors or on the physiology of their hosts (Rowan et «/.,
1997). In the case of M. vermcosa, P. compressa. and F.
scutaria. the higher Ml values of the expelled algae relative
to those remaining in the hosts (Fig. 7) may simply be due
to a lack of host regulation of division following algal
release (Suharsono and Brown. 1992; McCloskey et al..
1996).

A complete understanding of the role of expulsion of
algal cells in regulating algal-cnidarian symbiosis requires
further research into the release mechanisms involved. This
study has shown that at least some symbiotic cnidarians
preferentially expel dividing algal cells. In combination
with other regulatory factors, this pattern of cell loss can

REGULATION OF ALGAL-CNIDARIAN SYMBIOSIS

2S5

play a major role in regulating ulgal-cnidarian symbiosis in
steady state.

Acknowledgments

This research was partially funded by various National
Science Foundation grants to L.M. We thank two anony-
mous reviewers for their comments on this work.

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Reference: BioL Bull- 1<W: 2X7-297. < December 2()t)()i

Cellular Basis of Gastrulation in the Sand Dollar
Scaphechinus mirabilis

TETSUYA KOMINAMI* AND HIROMI TAKATA

Department of Biology and Earth Sciences, Faculty of Science. Ehime University, 2-5 Bunkyo-Cho,

Matsuvama. 790-8577, Japan

Abstract. The processes of gastrulation in the sand dollar
Scaphechinus mirabilis are quite different from those in
regular echinoids. In this study, we explored the cellular
basis of gastrulation in this species with several methods.
Cell-tracing experiments revealed that the prospective
endodermal cells were convoluted throughout the invagina-
tion processes. Histological observation showed that the
ectodermal layer remained thickened, and the vegetal cells
retained an elongated shape until the last step of invagina-
tion. Further, most of the vegetal ectodermal cells were
skewed or distorted. Wedge-shaped cells were common in
the vegetal ectoderm, especially at the subequatorial region.
In these embryos, unlike the embryos of regular echinoids,
secondary mesenchyme cells did not seem to exert the force
to pull up the archenteron toward the inner surface of the
apical plate. In fact, the archenteron cells were not stretched
along the axis of elongation and were in close contact with
each other. Here we found that gastrulation was completely
blocked when the embryos were attached to a glass dish
coated with poly-L-lysine, in which the movement of the
ectodermal layer was inhibited. These results suggest that a
force generated by the thickened ectoderm, rather than
rearrangement of the archenteron cells, may play a key role
in the archenteron elongation in 5. mirabilis embryos.

Introduction

The processes of gastrulation in sea urchin embryos have
been divided into two phases, known as primary and sec-
ondary invagination (Dan and Okazaki, 1956; Gustafson
and Kinnander, 1 956 1. During primary invagination, the
thickened vegetal plate buckles into the blastocoel and gives

Received 9 September 1999; accepted 10 August 2000.
* To whom correspondence should be addressed. E-mail: tkomte1
sci.ehime-u.ac.jp

rise to a short stub-like gut rudiment. After a couple of
hours, the gut rudiment begins to elongate, and its tip
reaches the inner wall of the apical plate. After secondary
invagination, the gut rudiment results in a slender tube-like
archenteron.

Primary invagination is autonomous, because the excised
vegetal plate can still undergo morphological changes sim-
ilar to those in the intact embryo (Moore and Burt, 1939;
Ettensohn. 1984). One of the driving forces for primary
invagination seems to be generated by bottle cells (Naka-
jima and Burke, 1996; Kimberly and Hardin, 1998).
Wedge-shaped cells, which are frequently observed in the
vegetal ectodermal layer, may produce another motive force
for primary invagination (Burke et ai. 1991). During sec-
ondary invagination, a population of secondary mesen-
chyme cells (SMCs) connect the archenteron tip to the inner
surface of the apical plate and exert the force to pull up the
archenteron (Dan and Oka/.aki. 1956; Hardin, 1988). Re-
arrangement of the archenteron cells is an important cellular
basis for elongation of the gut rudiment (Ettensohn, 1985;
Hardin and Cheng, 1986; Hardin. 1989).

However, these mechanisms apply mainly to the regular
echinoids and cannot fully explain the processes of gastru-
lation in a variety of sea urchins (Ettensohn, 1999). The
embryos of a primitive sea urchin, Encidaris tribuloides,
show a different manner of gastrulation (Schroeder, 1981;
Hardin, 1989); SMCs are not formed, but the embryos
gastrulate. Even in the so-called "Regularia," some species
of embryos do not show a typical manner of gastrulation
(Amemiya et al.. 1982a, b). In a previous study, we indi-
cated that the processes of gastrulation in the sand dollar
Scapliechinns mirabilis (Clypeasteroida) were different
from those reported in regular echinoids (Kominami and
Masui. 1996). The processes of gastrulation could not be
divided into two phases, because invagination proceeded at

287

288

T. KOMINAMI AND H. TAKATA

a constant rate from beginning to end. Unlike the SMCs of
regular echinoids, those of 5. inircibilis did not form long
filopodia. Moreover, the number of archenteron cells ob-
served in cross sections remained unchanged, suggesting
the absence of cell rearrangement in the archenteron.

One of the ways to a full understanding of the mecha-
nisms of gastrulation in a species is to study the processes of
gastrulation in a variety of related species. A less important
factor in one species may be crucial in another species. The
purpose of this study is to elucidate the cellular basis of
gastrulation in S. minibilis embryos. Changes in the mor-
phology of gastrulating embryos were quantified. To clarify
the movements of vegetal cells during gastrulation. cell-
tracing experiment was undertaken. The shapes of constit-
uent cells were observed on scanning electron micrographs
and in immunostained specimens. To examine whether the
ectodermal layer plays a role in archenteron elongation, the
movement of the layer was inhibited by attaching the em-
bryos to a glass dish coated with poly-L-lysine. Some of the
results are compared with those obtained in the sea urchin
Hemicentrotus pulcherrimus (Echinida), which shows a
typical pattern of sea urchin gastrulation.

Materials and Methods

Animals

Adults of the sand dollar Scaphechinus mirabilis and the
sea urchin Hemicentrotus pulcherrimus were collected and
kept in aquaria supplied with circulating cold seawater
(18°C for S. mirabilis and 15°C for H. pulcherrimus).
Gametes of both species were handled as previously de-
scribed (Kominami and Masui. 1996). Millipore-filtered
seawater (MFSW) supplemented with 100 units/ml penicil-
lin and 50 /xg/ml streptomycin (Meiji Seika. Tokyo) was
used throughout the experiments. Embryos of both species
were cultured at 18 ± 1°C.

Histological observation

Gastrulating embryos were fixed with 1% glutaraldehyde
dissolved in MFSW at room temperature for 2 h. After two
rinses with MFSW, an aliquot of fixed embryos was dehy-
drated with an acetone series and mixed with Spurr resin. A
drop of the mixture was mounted on a glass slide, sealed
with coverslips, and polymerized. These specimens were for
measurements of the thickness of the blastocoel wall, which
cannot be obtained accurately in living embryos, because of
optical reflections through the ectodermal layer.

Another aliquot of specimens was post-fixed with 1%
OsO4 dissolved in MFSW for 1 hr. The embryos were
dehydrated with an ethanol series and critical-point dried
(Hitachi, HCP-I, Tokyo). The specimens were adhered to
the stub with a piece of double-sided transparent tape, and
were fractured with a fine tungsten needle under a dissecting

microscope. These specimens were coated with gold and
platinum (Eiko, IB-3, Tokyo), and observed under a scan-
ning electron microscope (Hitachi. S-450DX).

Cell contours were traced on scanning electron micro-
graphs using transparent sheets. Shapes of cells were clas-
sified into four types according to the criteria described in
Burke et al. (1991): columnar, cells longer than wide and
rectangular in outline; wedge-shaped, apical surface broader
than basal surface; skewed, the apex of the cell deviates
from the apical basal axis; and others. The frequency of the
appearance of these types of cells was obtained by exam-
ining between 4 and 19 SEM images at each observation
point. Degree of archenteron invagination was obtained
according to the method described in the previous report
(Kominami and Masui, 1996). More than 20 embryos were
measured at each observation point.

Cell tracing

The vegetal blastomeres of the 8-cell-stage embryos of S.
minibilis were somewhat smaller than those in the animal
hemisphere. This morphological characteristic was used to
orient the 8-cell-stage embryos so that Lucifer yellow CH
(Sigma, St. Louis, MO) could be injected into one of the
vegetal blastomeres. An experimental setup for ionto-
phoretic injection of the dye was previously described
(Kominami, 1988). The dye-injected embryos were ob-
served under an epifluorescence microscope (Olympus, BH-
RFL, Tokyo), and photographed.

Immunofluorescence

Gastrulating embryos were fixed successively with cold
methanol and ethanol (— 20°C), 20 min in each. Fixed
embryos were embedded in polyester wax (BDH Labora-
tory Supplies, Poole, England) and sectioned (6 /u,m). After
removal of the wax with absolute ethanol, specimens were
rinsed twice with phosphate-buffered saline (PBS), and
were reacted with a monoclonal antibody (VE10) that rec-
ognizes the carbohydrates at the cell surface. After the
primary reaction, the specimens were rinsed twice with
PBS, and then reacted with FITC-conjugated goat anti-
mouse IgG (Sigma) for 1 h. The immunostained sections
were mounted on a glass slide with a small amount of
glycerol.

Attaching the embryos to the glass dish

The cleaned glass dish was soaked overnight in I mg/ml
poly-L-lysine (Sigma) dissolved in distilled water. After the
solution was removed, the glass dish was rinsed three times
with distilled water and air-dried. A micropipette was used
to attach the gastrulating embryos to the bottom of the glass
dish; in some cases, the embryos were pressed against the
bottom of the dish with a fine glass needle, to ensure

GASTRULATION IN SAND DOLLAR

289

F ^zZ&

Figure 1. Processes of gastiulation in Hcmii'cntn >ni.i pulchenimus and Scuphirhiiiiu mimhilis. Embryos of both
species were cultured at 18°C and observed hourly. Embryos were embedded in Spurr resin. (A-F) H. pulcherrimus,
17-22 h. (G-L) S. mimhilis, 14-19 h. In H. pulcherrimus embryos, the primary and secondary invagination is clearly
distinguished by the presence of a pause in the archenteron elongation (C-D. 1-2 hours). After the occurrence of
the secondary invagination. the archenteron became slender. In S. mimbilis embryos, the archenteron invaginated contin-
uously, and the diameter of the archenteron remained unchanged during gastrulation. The scale bar indicates 50 /xm.

attachment. The processes of gastrulation were photo-
graphed at intervals of 10 min.

Results
Morphological changes during gastrulation

Figure 1 shows the processes of gastrulation in embryos
of H. pulcherrimus (A-F) and S. mirabilis (G-L) kept at
18°C. In a regular echinoid, H. pulcherrimus, primary (Fig.
1 A-C) and secondary invagination (Fig. 1 D-F) were clearly
distinguished by the presence of a time lag in archenteron
elongation (1-2 h. Fig. 1C-D). On the other hand, the
archenteron of S. mirabilis embryos elongated at a constant
rate during the course of invagination (Fig. 1G-L).

Besides archenteron elongation, several differences were
noticed in the morphology of the embryos of these two
species. In H. pulcherrimus, the height of the embryo in-
creased to some extent during primary invagination. After
the onset of secondary invagination, the embryos were
shortened along the animal-vegetal axis (Fig. 2A). The
width of the embryos increased as gastrulation progressed
(Fig. 2B). This was caused by the expansion of the ecto-
dermal layer, especially at the lateral blastocoel wall (Fig.
2C). In contrast, S. mirabilis embryos became shorter as
invagination progressed (Fig. 2D). Though their width in-
creased to some extent (Fig. 2E), the expansion of the
ectodermal layer was not so conspicuous as in H. pulcher-
rimus (Fig. 2F).

Tracing of the vegetal blastomeres

The dye-injected S. miruhilis embryos were examined to
learn how vegetal cells move toward the blastopore during
gastrulation (Fig. 3). Before the initiation of invagination,
the boundary between labeled and nonlabeled cells was at
about 50% of the distance from the vegetal pole to the

animal pole (Fig. 3A, A'). The boundary gradually shifted
toward the vegetal pole side as invagination progressed
(Fig. 3B, B', C. C', D, D'). At the end of invagination, the

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Time after fertilization (hour)

Figure 2. Change in the height and width of the embryos, and in the
thickness of the blastocoel wall (the lateral part of the embryo) during
gastrulation. (A-C) Hemicentrotits pulcherrimus. (D-F) Scaphechinus
mimhilis. Single arrows indicate the time of the initiation of gastrulation.
Double arrows indicate the time of the onset of the secondary invagination
in //. pulcherrimus embryos. Changes in the height of the embryo (A, D)
show different patterns. In both species, the width of the embryo increased
as invagination progressed (B, E). This is more evident in H. pulcherrimus
than in S. mimhilis. The thickness of the blastocoel wall decreases during
gastrulation. but the change is not conspicuous in S. mirabilis (C. F).

290

T. KOMINAMI AND H. TAKATA

Figure 3. Movement of vegetal cells during gastrulation in Scaplifcliinnx mirabilis embryos: bright-field
images (A-EK fluorescence images (A'-E1). (A. A') 14 h. (B. B'l 16 h. (C. C') 17 h. (D. D'l 18 h. (E. E'l 20 h.
Distribution of labeled cells in invaginating gastrulae, which had been injected with Lucifer yellow CH into one
of vegetal blastomeres at the 8-cell stage, was examined. As gastrulation proceeds, the boundary between labeled
and nonlabeled cells moves downward. Arrowheads in A-E demarcate the boundary observed on fluorescent
imaaes. The scale bar indicates 50 jxm.

boundary was located at 10%- 15% of the embryo length
from the vegetal pole (Fig. 3E. E'). This value corresponds
to the thickness of the anal plate ectoderm. The relationship
between the degree of invagination and the position of the
boundary is shown in Figure 4. During the early stages of
invagination. the position of the boundary shifted rapidly to
the vegetal pole; during the later stages, the rate of the
movement decreased gradually. The result clearly indicates
that the involution of cells through the blastopore continues
until the archenteron tip reaches the apical plate.

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Degree of imagination (B/A, %)

Figure 4. Change in the position of the boundary between animal and
vegetal ectoderm during gastrulation. The degree of invagination (%) and
the position of the boundary along the embryo axis (%) were sought using
the parameters shown in the inset. The most fitting hyperbolic curve is also
shown. The movement of the vegetal cells toward the vegetal pole con-
tinues throughout the invagination processes, though the rate of the move-
ment changes.

Shapes of the ectodermal cells during early stages of
gastrulation

The shapes of the ectodermal cells during early stages of
gastrulation were examined with SEM (Fig. 5). The cells
were classified as columnar, skewed, wedge-shaped, and
other, as described in the Materials and Methods. Among
these types, wedge-shaped cells showed differences in their
distribution between the two species of embryos. In H.
pitlcherriiiins. two to three wedge-shaped cells were ob-
served just at the bending point of the ectodermal epithe-
lium (Fig. 5A-D, arrowheads). In S. mirabilis, such wedge-
shaped cells were distributed more broadly apart from the
blastopore (Fig. 5E-H. arrowheads). In addition, the num-
ber of wedge-shaped cells was larger than in H. pulchcrri-
mus. At the beginning of invagination, bottle-shaped (api-
cally constricted and basally rounded-up) cells were
frequently observed in the bending vegetal plate (arrows
indicate bottle cells in Fig. 5D \H. /nilclicrriiinix] and 5G [S.
mirabilis]').

Figure 6 shows the change in the ratio of these three types
of cells during early stages of gastrulation. In both species,
it takes 3 — 1 h to give rise to a short, stub-like gut rudiment
after the first sign of invagination. These stages were di-
vided hourly and designated Stages I-IV. In the animal
halves, columnar and skewed cells were abundant (Fig. 6A,
H. piilclieiriiiin.'i; 6C, S. mirabilis). In both species, the ratio
of columnar cells increased as gastrulation progressed. In
contrast, most cells in the vegetal half were skewed or
distorted (Fig. 6B. H. piilcherrimus; 6D, 5. inimbilis). In H.
pitlchernmns. wedge-shaped cells occupied nearly 40% of
the total at the initial stage of invagination. but decreased to

GASTRULATION IN SAND DOLLAR

291

Figure 5. Scanning electron micrographs of the cells in the ectoderm and invaginated archenteron during
early stages of gastrulation: Hemicentrotus pulclierrimus (A-D): Scaphechinus mirabilis (E-H). In A-D,
arrowheads indicate the bending point: in E-H they indicate the boundary between animal and vegetal cells.
Arrows in D and G indicate bottle cells. Ectodermal cells of 5. mirabilis (F-H) were more elongated in the
apico-basal direction than those of H. piilcherrimus (B-D). Columnar and skewed cells were frequently observed
in both species. Wedge-shaped cells were also observed in both species, especially in the vegetal half. In H.
pulcherrimus. two to three wedge-shaped cells were observed just at the bending point of the epithelium (B. C).
Such wedge-shaped cells are distributed more broadly apart from the blastopore in S. mirabilis (F, G). The scale
bar indicates 10 jum.

about 209r as invagination progressed; in S. mirabilis, the
ratio remained constant at a rather higher level. Columnar
cells were barely observed in S. mirabilis (Fig. 6D), whereas
this type of cell increased in H. pulclierrimus at the end of
primary invagination (Fig. 6B).

Secondary mesenchyme cells at the archenteron tip

Figure 7 shows the secondary mesenchyme cells ob-
served at the archenteron tip of the midgastrulae. In H.
pulcherrimus, these cells were globular and formed long
thin filopodia. Several SMCs were located between the
archenteron tip and the future oral opening region. In 5.
mirabilis, SMCs were flattened to some extent and formed
broad ruffled membranes. No cells were observed between
the archenteron tip and the future oral opening region.
Although more than 200 gastrulating S. mirabilis embryos
were examined, an image that showed direct contact be-
tween the filopodia of the SMCs and the inner surface of the
apical plate could not be obtained.

Shape of archenteron cells during later stages of
invagination

Figure 8 shows cross fractures of the archenteron at later
gastrula stages. SEM images of the archenteron at three levels
along its axis (top, middle and bottom) are shown. The cells in
the archenteron of H. pulcherrimus were cuboid and loosely in
contact with each other (Fig. 8A-C). The numbers of cells
observed in cross fractures increased from top (6-7) to bottom
(about 12) of the archenteron. The archenteron cells had a
rounded basal surface. In contrast, cells in the archenteron of S.
mirabilis embryos were elongated along the apico-basal direc-
tion (Fig. 8D-F). The numbers of cells observed in cross
fractures were almost the same at the top ( 14 - 1 5 ), middle (13),
and bottom (13) levels of the archenteron.

As gastrulation proceeded, it became difficult to crack the
archenteron along its long axis. The shapes of cells in the
embryos at later stages of invagination were examined on
histological sections. The stages shown in Figure 9 corre-
spond to the secondary invagination in H. pulcherrimus
embryos. As clearly shown, the cells in the archenteron of

292

T. KOMINAMI AND H. TAKATA

60 1

St. 1 Si. 2

SI. .1 SI. 4 SI. 1 SI. 2 Si. 3

Stage in early phase of imagination

St. 4

Figure 6. Frequency of the appearance of columnar, skewed, and
wedge-shaped cells during gastrulation: Hemicentrotus pit/cherriinus (A.
B); Scai'hi'cliinin mirabilis (C, D). (A. C) Animal hemisphere. IB. D)
Vegetal hemisphere. Columnar cells (solid lines) are more abundant in the
animal hemisphere. Skewed cells (dotted lines) were observed more fre-
quently in S. mirabilis. In both species, the population of columnar cells
increased as the gastrulation proceeded (A. C). Wedge-shaped cells (tiro-
ken lines) appear sparsely in the animal hemisphere. In contrast, the most
abundant type of cells are wedge-shaped cells in the vegetal halves (B. D).
Columnar cells were rarely observed in S. mirtihilis. but such cells in-
creased in H. pulclierriinii* after the secondary imagination had started.

H. /ntlclwrrinnis embryos were stretched along the axis of
the archenteron (Fig. 9A-D). After the completion of the
secondary invagination. the cells resumed a cuboid shape
(Fig. 9E-F). In contrast, the cells in the archenteron of 5.
mirabilis embryos were not stretched at any stage of later
invagination (Fig. 9G-L). It should be noted that the cells
near the blastopore were elongated along their apico-basal
direction through all the stages examined.

These changes in cell shape were quantified according to the
methods described by Hardin (1988); two ratios, YIX (ratio of
lengths along and perpendicular to the axis of the archenteron)
and L/W (ratio of cell length and width) were obtained (Fig.
10). Both YIX and L/W increased during the secondary invag-
ination in H. pulcherrimus embryos, and decreased to the
initial level at the end of secondary invagination (Fig. 10A).
On the other hand, the ratios did not change in S. mirabilis
embryos through these stages of invagination (Fig. 10B). The
result clearly shows that the archenteron cells in S. mirabilis
embryos were not stretched along the axis of the archenteron.

Attaching embryos to a glass disli coated with poly-L-
Ivsine

The obtained results suggest the ectodermal layer plays a
role in the invagination process in S. mirabilis embryos. If

Figure 7. Scanning electron micrographs of the secondary mesenchyme cells at the archenteron tip.
Hemicciitronix puUlii'rriiiui\ (A): Scaphechiints mirahili.i (B). Insets in A and B show whole view of the
mid-gastrula. Secondary mesenchyme cells in H. fiulchcrriimt* are globular in shape and form long thin
filopodia. Several secondary mesenchyme cells (SMCs) are located between the archenteron tip and the inner
surface of the future oral opening region. In contrast. SMCs are flattened and form ruffled membranes in S.
minihili* gastrulae. No SMCs were observed between the archenteron tip and the future oral opening region. The
scale bar indicates 10 /.<m.

GASTRULAT1ON IN SAND DOLLAR

293

Figure 8. Scanning electron micrographs of the cells in the arch-
enteron after the completion of invagination: Hemicentrotux pulcherrimus
(A-C); Scaphechinus mirabilis (D-F). Top (A. D). middle (B. E). and
bottom (C, F) levels of the archenteron are shown. The cells in the
archenteron of H. pulcherrimus are cuboid, while those in S. mirabilis are
elongated along the apico-basal direction. Note that the wall of the arch-
enteron is thicker in S. mirahilis than in H. pulcherrimus. The scale bar
indicates 10 jim.

this is the case, immobilization of the ectodermal layer
should affect the invagination process. To test this possibil-
ity, the change in the length of the archenteron was moni-
tored in gastrulating embryos attached to a glass dish coated
with poly-L-lysine.

H. pulcherrimus gastrulae attached to the poly-L-lysine
coated glass dish (Fig. 1 1 A). If the embryos had been under-
going primary invagination when attached, the invagination
was slowed and tip of the archenteron could not reach the
apical plate. However, the embryos gastrulated almost nor-
mally if they had been attached after primary invagination. In
these cases, the rate of archenteron elongation was not different
from that in control embryos (Fig. 1 IB).

In contrast, the invagination process was greatly affected
in 5. mirtibilis embryos. If the embryos were not pressed
usuinst the bottom of the 2lass dish usina a class needle.

they did not firmly attach to the glass dish. Embryo II shown
in Figure 12 loosely attached to the glass dish, so that its
position changed during observation. In this embryo, invag-
ination occurred almost normally. On the other hand, em-
bryos I, III. and IV were rather firmly attached to the glass
dish. In these embryos, invagination of the gut rudiment was
considerably delayed. Nonetheless, embryos III and IV re-
started invagination when they detached from the glass dish
(Fig. 12F and H. respectively). Embryos that were firmly
attached to the glass dish could not gastrulate at all, irre-
spective of the degree of invagination at attachment (Fig.
13A and B). In addition, the contour of the embryos was
distorted during prolonged observation.

Discussion

Involution of the vegetal cells continues during
gastrulation

Endodermal tissue in sea urchin embryos had been
thought to be exclusively derived from the veg: tier of
blastomeres formed at the 60-cell stage (Horstadius, 1973;
Cameron et til.. 1987. 1991; Ruffins and Ettensohn. 1996).
More recently, it has been shown that the descendants of the
veg, tier of blastomeres also participate in the formation of
the digestive tract (McClay and Logan. 1996; Logan and
McClay. 1997). The recruitment of the veg,-derived cells
seems to occur only after the tip of the archenteron reaches
the apical plate (Martins et ai, 1998; Piston et ai. 1998;
Ransick and Davidson, 1998). The mechanisms of such late
ingression of endodermal cells are, however, unknown.
Here we focus on the invagination processes that occur
before and around the time that the archenteron tip reaches
the inner surface of the apical plate.

In embryos of an irregular echinoid, 5. mirabilis, some
unique aspects of gastrulation were elucidated from the
measurement of the size of the gastrulating embryos. As is
well known, the embryos of regular echinoids expand con-
siderably during gastrulation (Fig. 1A-F). This expansion
was caused by thinning of the ectoderm (Fig. 2C). In con-
trast, the thinning of the blastocoel wall was not so conspic-
uous in S. mirabilis (Fig. 2F). Although the width of the
embryos became somewhat larger, their height became
smaller as invagination progressed (Fig. 1G-L). This im-
plies a physical continuity between endodermal and ecto-
dermal epithelia during invagination. In fact, convolution of
the vegetal cells continued until the archenteron tip reached
the apical plate (Figs. 3, 4), whereas such convolution
terminates at the end of primary invagination in regular
echinoids (Burke et ai, 1991 ).

In H. pulcherrimus embryos, invagination was par-
tially inhibited if the embryos just undergoing primary
invagination were attached to a glass dish coated with
poly-L-lysine (Fig. 1 1 , embryos III and IV). However, the
archenteron elongated almost normally if the embryos

294

T. KOMINAMI AND H. TAKATA

Figure 9. Change in the comour of cells during later stages of invagination as seen in immunostained
histological sections. (A-F) Hemicentrotus pulcherrimus embryos at 19, 20. 22. 24, 26, and 28 h. respectively.
(G-L) Scaphechinus mirabilis embryos at 17. 18, I1), 20. 21. and 22 h, respectively. The ectodermal cells in H.
/ink hi'rriiniis embryos were initially elongated along the apico-basal direction (A), and became cuboid with the
progress of gastrulation. The archenteron cells were stretched along the axis of the archenteron (B-D or E), and
resumed a cuboid shape (F). In contrast, both the ectodermal and endodermal cells in 5. mirabilis embryos
remained elongated along the apico-basal direction during the later stages of invagination (G-L).

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Figure 10. Change in the shape of the archenteron cells during later
stages of gastrulation: Heinicciiiiniii\ />iilflifrriiiiii.\ (A): Scaphechinus
mimhilis (B). Shape was expressed as two ratios: )7.V (length along the
archenteron axis to length perpendicular to the axis) and L/W (cell length
to width I. In H. pulcherrimus embryos, both ratios increased as secondary
invagination progressed, up to at least 22 h. Then the ratios decreased to
about 1.0. In S. mirabilis embryos, the ratios did not change significantly.
though the degree of invagination increased. The Y/X ratio remained about
1.0, which indicates that the archenteron cells are not stretched along the
axis of the archenteron.

had finished primary invagination when attached (Fig.
11. embryos I and II). This suggests that the vegetal
ectodermal layer moves toward the blastopore during
primary invagination, and that the layer loses physical
continuity from the gut rudiment after the completion of
primary invagination. On the other hand, elongation of
the archenteron was completely blocked in S. mirubilis if
the embryos were attached to a coated glass dish, irre-
spective of the degree of invagination (Fig. 13). This
inhibitory effect of poly-L-lysine cannot be ascribed
solely to the chemical toxicity of the drug, because the
embryos restarted gastrulation soon after they detached
from the glass dish (Fig. 12). We suppose that the ecto-
dermal epithelium and the invaginated archenteron are
physically continuous during the invagination processes
and that the blockage of invagination is mainly due to
physical constraint of the ectodermal layer attached to the
glass dish.

The precise mechanism by which elongation of the gut
rudiment is blocked in S. niirnhilis embryos is unknown.
The ectodermal layer seems to be more rigid in S. mimhilis
embryos than in those of H. ptilclierrinn/s. because the
former is thicker (Fig. 2F). In addition. S. ininibilis embryos
retained normal configuration after they were fixed with
10% formaline, while the ectodermal layer of H. pulcher-
riinus embryos was severely distorted when the fixative was
applied. The ectodermal cells in S. mirabilis embryos are
probably tightly connected with each other, forming a rigid
structure over the entire vegetal ectoderm. Even if embryos
are attached to the glass dish on one side of the body, such
a rigid structure may be destroyed totally, resulting in a
blockage of archenteron elongation.

GASTRULATION IN SAND DOLLAR

295

1:10

3 70

0 60

| 5°
"S 40

01 30

. IV o

J 30 40 50 60 70 80 90 100

Time after the attachment (minute)

Figure 11. Adhesion of Hemicentrotus pulcherrimus embryos to the
glass dish coated with poly-L-lysine. The numeral at the top right corner in
each photograph in A indicates the time after the attachment to the glass
dish. Roman numerals (I-IV) in A and B indicate the same embryos. The
embryos gastrulated almost normally if they had been attached after the
primary invagination (embryo I and II). If the embryos had been just in the
primary invagination when attached, the rate of elongation of the arch-
enteron was slowed and the archenteron could not reach the apical plate
(embryo III and IV). The scale bar indicates 100 /j.m.

The initial phase of gastrulation

In both species of embryos, bottle cells (Nakajima and
Burke, 1996) were observed in the vegetal plate (Fig. 5D, G,
arrows). The appearance of bottle cells in the vegetal plate
may lead to the first step of invagination. if the archenteron
cells retain the monolayer arrangement (Gustafson and

Wolpert, 1963, 1967). Unlike the archenteron cells in H.
puk-lieirimux embryos (Fig. 5C. D), those in S. mirabilis
embryos were variable in shape and were not organized into
a complete monolayer sheet (Fig. 5G, H). As a result, the
force produced by bottle cells does not necessarily cause the
bending of the vegetal plate. Other forces seem to be nec-
essary to produce the invagination of the vegetal plate cells
in S. iniruhilis embryos.

In this study, several types of cells were observed on SEM
images. The role of each type of cell is unknown. If cells are
pulled apically or basally, they should become skewed, be-
cause cells are connected with extracellular matrix ( Wessel and
McClay, 1987; Burke et «/.. 1991: Berg et ai. 1996). If a
monolayer cell sheet is bent, wedge-shaped cells should appear
at the bending point. Thus, the shapes of cells are signs of the
existence of the forces generated by surrounding tissues or by
the cells themselves. In both species of embryos, the ratio of
columnar cells in the animal hemisphere increased as invagi-
nation progressed (Fig. 6A, C). On the other hand, most cells
in the vegetal hemisphere were distorted (Fig. 6B, D). Espe-
cially in 5. mimhilix embryos, columnar cells were barely
observed through the stages examined. These results imply that

Figure 12. Adhesion of the Sctiphechiiius mirabilis embryos to the
glass dish coated with poly-L-lysine. Embryo II was loosely attached to the
glass dish, because its position changed during observation. In this embryo,
invagination occurred almost normally. On the other hand, embryos I. 111.
and IV were rather firmly attached to the glass dish and invagination of the
2ut rudiment were considerably delayed. Nonetheless, embryos III and IV
reinitiated invagination when they detached from the glass dish. The scale
bar indicates 100 /nm.

296

T. KOMINAMI AND H. TAKATA

1:0(1

I II HI

V ..' .< 'i 0:20

3- 70

£ 60
u

C 50
S

1 40
<u 30
? 20

10 20 30 40 50 60 70 80 MO 100

Time after the attachment (minute)

Figure 13. Adhesion of the Scaphechinus mimhilis embryos to the
glass dish coated with poly-L-lysine. The numeral at the top right corner in
each photograph in A indicates the time after the attachment to the glass
dish. Roman numerals (I-IV) in A and B indicate the same embryos. 5.
ininthilis embryos could not gastrulate at all. irrespective of the degree of
invagination. The scale bar indicates 101) uin.

some forces continue to operate in the vegetal hemisphere of 5.
mimhilis embryos.

Elongation of the archenteron

It is well-established that SMCs pull up the gut rudiment
during secondary invagination in some sea urchins (Dan and
Okazaki. 1956; Gustafson and Kinnander. 1956; Hard in.

1988; Hardin and McClay, 1990). In H. /nilchcrrimus em-
bryos, a chain of SMCs was observed to connect the arch-
enteron tip and the apical plate (Fig. 7A). The height of the
embryos became shortened after the onset of secondary
invagination (Fig. 2A). This shortening could be caused by
contraction of the SMCs' pseudopodia. which connect the
archenteron tip and the apical plate across the blastocoel.
The contraction of pseudopodia should stretch the arch-
enteron. In fact, the archenteron cells in H. piilclierrimus
embryos were stretched along the axis of the archenteron
during secondary invagination (Fig. 9B-D, Fig. 10A). On
the other hand, the SMCs of 5. mirabilis embryos did not
connect the archenteron tip and the site of the future oral
opening (Fig. 7B). Although it is possible that the connect-
ing filopodia were broken by fixation, the archenteron cells
in S. mirabilis embryos were not stretched throughout the
invagination processes (Fig. 9G-L, Fig. 10B). Thus, unlike
the SMCs in regular echinoids, those in S. mirabilis em-
bryos may not pull up the archenteron.

Rearrangement of archenteron cells is another cellular
basis of the extension of the gut rudiment during secondary
invagination (Ettensohn, 1985; Hardin and Cheng. 1986;
Hardin. 1989). The archenteron cells of H. piilcherrimiis
embryos were cuboid, with rounded basal surfaces (Fig.
8A-C). In contrast, the archenteron cells in S. mirabilis
embryos were elongated — a configuration that should be
brought about by close contact between cells. In fact, the
archenteron cells of S. mirabilis embryos looked closely
compacted (Fig. 7B). It is unlikely that these cells change
their position freely in the archenteron. Moreover, the num-
bers of cells observed in the cross fractures of the arch-
enteron were almost the same at any level along the axis of
the archenteron (Fig. 8D-F). Thus, rearrangement of the
archenteron cells is not a major cellular basis of archenteron
elongation in S. mirabilis.

Motive force for the elongation of the archenteron

During the early stages of invagination. prospective
endodermal cells are elongated and concentrated in the
vegetal plate in both species of embryos. This configuration
of cells may be brought about by an increase in cell adhe-
siveness or by lateral pressure generated by the surrounding
tissues. In S. mirabilis embryos, cells near the blastopore
were elongated throughout the invagination processes (Fig.
9G-L). On the other hand, vegetal cells regained cuboid
shape after the completion of primary invagination in H.
piilcherrimiis (Fig. 9A-F). The archenteron cells in S. mira-
bilis embryos were also elongated along the apico-basal
direction (Fig. 8, 9). These cell shapes suggest the existence
of the lateral pressure in the thickened vegetal plate and the
archenteron.

Wedge-shaped cells observed in the thickened vegetal
ectoderm are one possible source of such pressure. Appear-

GASTRULATION IN SAND DOLLAR

297

ance of the wedge-shaped cell in a monolayer cell sheet
should produce a bending force, although such a shape of
cells is merely a result of a bending of the cell sheet. In both
species of embryos, wedge-shaped cells appeared in the
thickened vegetal plate prior to invagination (Fig. 5A. E). It
should be noted that a mass of wedge-shaped cells is found
at the subequatorial region (corresponds to the boundary
between the descendants of an2 and veg,) in 5. mirabilis
embryos, but is located just at the bending point (boundary
between veg, and veg2) around the vegetal plate in H.
/nilchi-irimus. Wedge-shaped cells should be formed by
constriction of the basally distributed microfilaments. Mi-
crofilaments play a key role in generating the motive force
for invagination (Lane et ai, 1993; Nakajima and Burke,
1996), whereas the elongation of the archenteron is inde-
pendent of microtubules (Hardin, 1987). The precise distri-
bution of microfilaments, especially in wedge-shaped cells,
remains to be examined in S. mirabilis embryos.

Acknowledgments

We thank the staff of Tateyama Marine Biological Lab-
oratory for collecting animals and for their hospitality.

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Reference: Biul. Bull. 199: 24S-304. (December 201)0)

Effects of Food Concentration and Availability on the

Incidence of Cloning in Planktotrophic Larvae of the

Sea Star Pisaster ochraceus

MINAKO S. VICKERY* AND JAMES B. MrCLINTOCK

Department of Biology. University of Alabama at Birmingham, Birmingham, Alabama 35294-1 170

Abstract. A decade ago, cloning was first observed in the
planktotrophic larvae of sea stars obtained from plankton
tows. However, no controlled experimental studies have
investigated what factors may regulate this remarkable phe-
nomenon. In the present study we offer the first documen-
tation of cloning in the planktotrophic larvae of Pisastcr
ochraceus from the northern Pacific coast. This species was
used as a model system to investigate three factors that may
influence the incidence of asexual reproduction (cloning) in
planktotrophic sea star larvae. In an initial experiment,
larvae were reared under nine combinations of three tem-
peratures and three food (phytoplankton) concentrations.
Larvae reared at 12-15°C and fed the highest food concen-
trations grew larger than the other larvae and produced
significantly more clones. In a second experiment, qualita-
tively different algal diets were fed to larvae reared under
the conditions found to be optimal in the initial experiment.
Up to 24% of the larvae consuming a mixed phytoplankton
diet of Isochrysis galbanu, Cluietocerons calcitrans, and
Dunaliella tertiolecta cloned, and significantly more clones
were produced by these larvae than by those fed monospe-
cific diets. Our experiments indicate that cloning generally
occurs after larvae have attained asymptotic body length
and only when food is abundant and of high quality. Since
larval mortality is considered to be extremely high for
marine invertebrates with planktotrophic larvae, production
of clones under optimal conditions of temperature and food
may serve to increase larval populations when the environ-
ment is most conducive to larval growth.

Received 29 February 2000; accepted 21 August 2000.
•' To whom correspondence should be addressed:
vickerym@uab.edu

I -m.ii

Introduction

Asexual reproduction and regeneration of missing body
parts are well-known phenomena in adult sea stars (Ander-
son. 1956: Emson and Wilkie. 1980: Shirai and Walker.
1988: Mladenov et at.. 1989; Mladenov and Burke, 1994).
The capacity for asexual reproduction (cloning) in sea star
larvae was first suggested over 60 years ago from observa-
tions of the bipinnaria larvae of Luidia sarsi (Tattersall and
Sheppard, 1934). but subsequent laboratory experiments
indicated that these larvae were incapable of asexual repro-
duction (Wilson, 1978). Only recently has cloning and
regeneration of missing body parts been confirmed in plank-
totrophic larvae of sea stars (Bosch et ai, 1989; Rao et al..
1993: Jaeckle. 1994; Vickery and McClintock. 1998;
Kitazawa and Komatsu, 2000).

Cloning in the bipinnaria larvae of the sea star Luidia sp.
was first documented from plankton samples collected in
the Sargasso Sea in which clones were observed to develop
from buds that formed on the larval arms (Bosch, 1988;
Bosch et al., 1989). Shortly thereafter, two additional re-
ports confirmed the occurrence of cloning in the natural
environment in two unidentified species of planktotrophic
sea star larvae also obtained from plankton samples (Rao et
til.. 1993; Jaeckle, 1994). In addition to the budding process
described above (Bosch, 1988; Bosch et ill.. 1989), autot-
omy of the anterior portions of larvae was also observed in
one of these studies (Jaeckle, 1994). Recently, brachiolaria
larvae of the sea star Distolasterias brucei have been re-
ported to undergo cloning in laboratory cultures (Kitazawa
and Komatsu. 2000) in a manner identical to that described
in previous reports (budding and autotomy). Moreover,
cloning in laboratory cultures of the planktotrophic larvae of
the brittle star Ophiopholis aculeata from the northern Pa-
cific has been reported (Balser, 1998). We reported regen-

SEA STAR LARVAL CLONING

299

eration in planktotrophic larvae of the sea stars Luidia
foliolata and Pisaster ochraceus (Vickery and McClintock,
1998). In addition, we observed similar regenerative capac-
ity in planktotrophic echinopluteus larvae of the sea urchins
Dendraster excentricus and Lytechinus variegatus (Vickery
and McClintock, 1999). Thus, among Echinodermata, re-
generative capacity in planktotrophic larvae has been dem-
onstrated in sea stars, brittle stars, and sea urchins.

The existence of cloning and regenerative capacity
among echinoderms with planktotrophic modes of repro-
duction suggests that there may be selective and adaptive
advantages associated with such life history traits. These
processes presumably operate under a suite of energetic
constraints and trade-offs, whereby only larvae exposed to
the most appropriate conditions would be expected to un-
dergo clonal and regenerative events.

To date, no experimental studies have investigated the
factors that regulate cloning in planktotrophic marine inver-
tebrate larvae. Larval development, growth, and survivor-
ship are particularly influenced by temperature and food
availability (e.g., George, 1994; Fenaux et at., 1994). These
may also be important factors affecting rates of larval clon-
ing (Levitan. 1995; Morgan. 1995). In the present study we
offer the first confirmed report of cloning in the planktotro-
phic larvae of the sea star P. ochraceus. We also examined
the effects of temperature and both food concentration and
availability on growth and cloning in P. ochraceus larvae.

Materials and Methods
Lan'al culturing

Pisasler ochraceus is commonly found in intertidal and
shallow subtidal habitats of the U.S. Pacific Northwest. The
breeding season of P. ochraceus is in the late spring and
early summer months in the vicinity of Puget Sound, Wash-
ington (Strathmann. 1987). Adult specimens were collected
during late spring months in 1997 (for temperature-food
experiment) and 1998 (for food availability experiment)
from rocky substrates along the shore of East Sound, Orcas
Island, and transported to Friday Harbor Laboratories, San
Juan Island, Washington. Ovaries and testes were dissected
from sexually mature specimens (a single female and a
single male). Fertilizable ova were obtained by treating
excised ovaries with 1-methyladenine (10~4 M) (Kanatani.
1969), and sperm were diluted in filtered seawater prior to
fertilization. During fertilization, ova were rinsed in filtered
seawater to remove excess sperm. Embryos and larvae were
reared in 2.5-liter glass jars. The cultures were gently stirred
and the seawater was changed every 3 days following the
methods outlined by Strathmann (1987). The single-celled
algae Chaetocerous calcitrans, Diinaliella tertiolecta, and
Isochrysis galbana were selected for larval diets (Strath-
mann, 1987). Observations and photographs of larvae were

made with both a Wild M-5 dissecting microscope and a
Nikon Optiphot-2 compound microscope.

Combined temperature and food-level experiments

Once the bipinnaria larvae developed functional digestive
systems they were separated into nine experimental treat-
ments (3x3 factorial design) to examine the effects of
temperature and food level on rates of cloning. Each exper-
imental treatment combining temperature and food level
was replicated three times, and each consisted of about 2400
larvae held in 2.5 1 of seawater in a glass jar (approx. 1
larva/ml). Ambient spring seawater temperatures (12-15°C)
were bracketed in increments of about 5°C to yield treat-
ment groups of low (7-10°C). medium ( 12-15°C), and high
(17-20°C) temperature. Ambient seawater temperatures
along the northern Pacific coast generally fall within the
middle temperature range (12-15°C) throughout the year,
but it is likely that larvae encounter lower temperatures
(7-10°C) at the northern limit of their biogeographic range
and in deeper water, or during the late winter months
(Cannon. 1978). Similarly, larvae may encounter higher
temperatures ( 17-20°C) at the southern limits of their bio-
geographic distribution, or in surface waters during the early
summer months (Cannon, 1978; Strathmann. 1987).

Three levels of food composed of equal cell numbers (as
determined using a hemocytometer) of the phytoplankton
species Chaetocerous calcitrans, Diinaliella tertiolecta, and
Isochiysis galbana were proffered to larvae in each treat-
ment. The three concentrations of mixed algal cells were
5 X 10-, 5 X 10\ and 5 X 104 cells/ml (modified after
Basch, 1996; Fenaux et al., 1994). The nine experimental
treatments combining temperature and food level were
therefore as follows: high temperature and high food (HT-
HF). high temperature and medium food (HT-MF). high
temperature and low food (HT-LF), medium temperature
and high food (MT-HF). medium temperature and medium
food (MT-MF). medium temperature and low food (MT-
LF). low temperature and high food (LT-HF). low temper-
ature and medium food (LT-MF), and low temperature and
low food (LT-LF). Subsamples of larvae in each experi-
mental treatment (n = 200) were examined every 3 days
under a dissecting microscope. The numbers of clones and
of larvae undergoing clonal reproduction as evidenced by
budding were recorded. The lengths of the larvae in each
subsample of each experimental treatment and of all larvae
in the process of cloning were measured along the larval
axis (George. 1994). Clones and larvae undergoing cloning
were placed in separate containers and monitored every 3
days to determine whether clones successfully developed
into normal functional larvae and metamorphosed into ju-
veniles. Larval lengths were compared within temperature
and food-level experiments using analysis of variance. Only

300

M. S. VICKERY AND J. B. McCLlNTOCK

probability levels where P < 0.05 were considered statis-
tically significant.

Food-avuilahility experiment

To examine the effects of food availability (phytoplank-
ton type) on the rate of cloning, simulating conditions in
which nutrient diversity might be limited, larvae of Pisaster
ochraceux were obtained as described above using ova and
sperm from a different set of parent sea stars than those used
for the combined temperature and food-level experiment.
The larvae were cultured at 12-15°C at a density of about
2400 larvae per 2.5-1 of seawater. Larvae were fed 5 X 104
cells/ml of either a monospecific diet of Chaetocerous cal-
citrans, Diinuliellu tertiolecta. or Isochrysis galbana or a
mixed diet composed of an equal cell number of these three
algae. Each of the four experimental treatments was repli-
cated three times. Subsamples of larvae (n = 200) were
examined every 3 days under a dissecting microscope and
analyzed using the same methods as for the combined
temperature and food-level experiment. Larval lengths were
compared within food availability experiments using anal-
ysis of variance. Only probability levels where P ^ 0.05
were considered statistically significant.

Results

Combined temperature and food-level experiments

Bipinnaria larvae exposed to seawater temperatures of
12-15°C and fed the highest food level (MT-HF) attained
lengths significantly greater than those of larvae in any other
experimental treatment (Fig. 1). Moreover, cloning oc-
curred only in this experimental treatment (Fig. 2). Thirty-

1350

-A- High food
-D- Med food
• •» - Low food

Larval death

20 25 30 35 40 45 50 55 60 65 70
Age (days)

Figure 1. Growth of larvae of Pi\u.\lei nc/iraceiis (measured as
changes in length) reared at 12-15°C and fed low. medium, and high levels
of mixed phytoplankton. M indicates stage at which brachiolaria larvae
developed an adult rudiment and a decrease in length of the larval body
(indicating metumorphic competence). Larvae that reached metamorphic
competence were later observed to complete the metamorphosis to juve-
niles. Error bars represent mean values ± I SD.

40 45 50

Age (days)

Figure 2. Percentages of larvae of Pisaster ochraceus undergoing
cloning when reared at 12-15°C and fed high levels of mixed phytoplank-
ton. Diagram indicates approximate onset of brachiolaria stage. Error bars
represent mean values ± 1 SD.

four days after fertilization, larval clones were first observed
in the MT-HF bipinnaria culture (1.2%, Fig. 2). Subse-
quently, small numbers of additional larval clones were
observed. Once bipinnaria in this treatment had doubled in
length while developing into brachiolaria larvae (at about
day 45), a fivefold increase was observed in the incidence of
cloning (6%, Fig. 2).

Larval clones obtained from the MT-HF cultures were
isolated and their development was followed through meta-
morphosis. The clones produced resulted from the regener-
ation of anterior and posterior portions of bipinnaria and
brachiolaria larvae (Fig. 3A, B) by processes that closely
resembled those described in detail by Vickery and Mc-
Clintock (1998). After about 2 weeks, fully developed
clonal larvae were functionally and morphologically indis-
tinguishable from larvae in the cultures from which they
were originally isolated. A number of bipinnaria and bra-
chiolaria larvae in the MT-HF cultures had missing larval
arms or were missing small fragments of the larval body.
Some larvae with missing larval arms were in the process of
cloning, as evidenced by the development of projections or
buds, which later became functional larvae, at the site of the
missing fragment (Fig. 3C). However, some larvae with
missing larval arms did not form projections or buds, but
instead regenerated the larval arm. In addition, some small
fragments of larval body parts, including severed larval
arms, were observed in the MT-HF cultures, presumably the
result of damage incurred when the water in the larval
culture was changed. A number of these fragments, includ-
ing severed arms, were separated from the cultures and
observed for 2 weeks. During this time the fragments nei-
ther grew nor formed clones, although no mortality was
observed.

Those bipinnaria larvae exposed to the highest tempera-
ture treatments (HT-LF. HT-MF, and HT-HF) all died

SEA STAR LARVAL CLONING

301

Figure 3. Light photomicrographs of clonal larvae of Pisaster ochraceiix including the anterior (A) and
posterior (B) portion of bipinnaria larvae. (C) Bipinnaria larva in the process of budding (see arrow). The bud
later formed an early bipinnaria stage larva and subsequently detached (similar to that described by Bosch et ul.,
1989). (D) Bipinnaria larva in the process of autotomization by fission. This larva was observed for about 2
weeks, during which the secondary larva (see arrow) detached and became functionally and morphologically
indistinguishable from the primary larva in a manner similar to that described by Jaeckle (1994). We have
observed this type of autotomy in several other species of planktotrophic larvae (pers. obs.. M. Vickery). Scale
bars = 200 /urn.

302

M. S. VICKERY AND J. B. McCLINTOCK

475

425

- •»• -Low food
-O- Med food
-A— High food

20 25 30 35 40 45 50 55 60 65 70
Age(days)

Figure 4. Growth of larvae of Pisasrer ochracens (measured as
changes in length) reared at 7-10°C on low, medium, and high levels of
mixed phytoplankton. Error bars represent mean values ± 1 SD.

within one week; no cloning occurred in the short period
before mortality. Unlike larvae held at mid-range tempera-
tures, bipinnaria larvae reared at low temperatures (7-10°C)
and fed any of the three levels of food (LT-LF, LT-MF, and
LT-HF) never developed into brachiolaria larvae over a
70-day period. They remained morphologically identical to
early-stage bipinnariae. Moreover, no clones were pro-
duced. Larvae presented any food level at low temperature
did not increase in length. In fact, they were slightly reduced
in length by the end of the 70-day observation period (Fig.
4). Considerable larval mortality occurred throughout the
experiment in all low-temperature treatment groups.

Food availability experiment

Bipinnaria larvae in experimental treatments fed a mono-
specific diet of /. galbana or an equivalent density of a
mixture of equal cell numbers of C. calcitrans. D. terti-
olecta, and /. galbana grew significantly larger than larvae
fed monospecific diets of C. calcitrans or D. tertiolecta
(Fig. 5). The incidence of cloning was greatest (24%) on a
mixed diet during and after the transformation from the
bipinnaria to the brachiolaria stage (Fig. 6). Although tem-
perature and food concentration were similar in this treat-
ment and in the MT-HF treatment group of the combined
temperature and food-level experiment (Figs. 1, 2), growth
was more rapid, and cloning began earlier and was more
frequent. This might be attributed to the high variability in
larval development rate in batches of larvae of P. ochnicens
(Strathmann. 1978). Also, larvae used for this experiment
were the offspring of a different set of parents than those
used in the previous experiments.

Discussion

Cloning and regenerative capacity in echinoderm larvae
has only recently been documented (Bosch, 1988; Bosch et

1400
1200
1000

- 800
•

" 600
400
200

D tertiolecta
I galbana
C. calcitrans
Mixed

15 20

Age (days)

*• Larval dealh
25 30 35

Figure 5. Growth of larvae of Pisaster ochracens (measured as
changes in length) reared at 12-15°C and fed high levels of four different
phytoplankton diets. M indicates stage at which brachiolaria larvae devel-
oped an adult rudiment and a decrease in length of the larval body
(indicating metamorphic competence). Larvae that reached metamorphic
competence were later observed to complete the metamorphosis to juve-
niles. Error bars represent mean values ± 1 SD.

«/.. 1989; Rao et ai. 1993; Jaeckle, 1994; Balser, 1998;
Vickery and McClintock, 1998, 1999; Kitazawa and Kom-
atsu, 2000). In the present study we provide the first docu-
mentation of cloning in the planktotrophic larvae of Pisaster
ochracens. The results of our bifactorial analysis indicated
that both seawater temperature and food level are important
factors affecting growth and survival — and therefore clon-
ing— of P. ochracens larvae. Simulated environmental con-
ditions that produced normal development and optimal lar-
val growth generated the greatest number of clones. Larvae
reared at temperatures (12-15°C) with the most abundant
food exhibited normal development and positive growth,
which resulted in the highest survival, with the greatest
incidence of cloning. Although we observed rapid larval
growth in the high-temperature treatment (15-17°C), none
of the larvae survived beyond one week. One possible

o 10
I

•o 30

I 20
*o

a 10

D terliolectz
I. galbana
D Mixed

20 25

Age (days)

Figure 6. Percentages of larvae of Pisaster ochraceus undergoing
cloning when reared at 12-15°C and fed high levels of four different
phytoplankton diets. Error bars represent mean values ± 1 SD.

SEA STAR LARVAL CLONING

303

explanation for the high mortality is that the higher temper-
atures triggered an increase in bacterial and microalgal
growth in the cultures.

In contrast, sea star larvae reared in the low-temperature
treatments (7-10°C) showed no net positive growth, and in
most cases, decreased in length, regardless of food avail-
ability. These larvae also failed to attain the brachiolaria
stage of development. It is unlikely these larvae would
eventually become clonal because they continued to shrink
in length over the course of the experiment. Decreased rates
of growth and development at low temperatures may be
related to decreased rates of larval metabolism (Boidron-
Metairon, 1995). While low seawater temperatures have
been suggested as an indirect cause of mortality in marine
invertebrate larvae (Thorson, 1950). no studies of larval
culturing have shown that low temperature can actually lead
to a decrease in larval length as seen in the present study.

The production of larval clones was greatest during
phases of rapid larval growth in MT-HF condition. As P.
ochraceus in the North Pacific spawns in the late spring,
larvae typically encounter moderate seawater temperatures
(12-15°C) and high phytoplankton availability (Cannon,
1978). Such conditions could be expected to enhance in situ
rates of larval cloning. Further analysis indicated that pre-
senting larvae with different levels and types of food under
an optimal regime of seawater temperature had a pro-
nounced effect on the initiation and rate of larval clone
production.

The greatest numbers of clones were produced by larvae
in cultures presented a mixture of three single-celled algae.
Although monospecific patches of single-celled algae are
unlikely to exist in the natural environment, our use of
monospecific algal diets simulated conditions in which nu-
trient diversity might be limited. Thus some of the observed
differences in growth (and cloning) rates among the larvae
fed monoalgal diets may have resulted from differences in
the nutrient content of the food rather than in the type of
food, since the larvae were fed equal cell numbers of algae,
not an equal nutritional content (Pechenik and Fisher.
1979). However, the amount of nutrients actually consumed
by the larvae does not necessarily have any correlation with
the nutrient content of the food presented, as some food
types may be more palatable to the larvae than others.
Future studies may shed more light on this subject. The
important information gained from the food-availability ex-
periment is that nutrient availability may be an important
factor affecting larval growth and therefore the rate of
cloning, as evidenced by the fact that growth rates among
larvae fed a monoalgal diet of Isocluysis galbana were
similar to those fed a diet of mixed algae, yet the larvae fed
the mixed diet produced far more clones.

In adult echinoderms. cloning (fission) is common and
has been well described (Emson and Wilkie. 1980). Sea-
sonal fluctuations in the incidence of cloning in adult sea

stars, especially a high incidence in summer months, have
been related to periods of maximum growth (Emson and
Wilkie. 1980). This suggests that suitable biotic and envi-
ronmental conditions such as abundant food and moderate
temperatures may trigger cloning processes in adults just as
they did in the larvae studied here. In some instances, more
than 50% of the adults in a population were observed
undergoing cloning (fission) when conditions were optimal
(Emson and Wilkie, 1980).

Cloning may serve as a mechanism to enhance recruit-
ment in P. ochraceus and perhaps in other marine inverte-
brates with planktotrophic modes of reproduction. Larvae
dispersed across significant distances are likely to encounter
a variety of environmental and biotic conditions, and our
results suggest that those larvae encountering favorable
conditions may be stimulated to reproduce by cloning,
thereby possibly increasing the probability of successful
larval metamorphosis and juvenile recruitment. Future stud-
ies of the effects of larval cloning on larval survivalship and
recruitment will provide more insight into the true impact of
this phenomenon on the life history of sea stars with plank-
totrophic larvae.

Acknowledgments

We thank Dennis Willows and Richard Strathmann of
Friday Harbor Laboratories, University of Washington, for
use of laboratory facilities and technical support. We are
grateful to Al Schuetz of Johns Hopkins University for
providing ovaries from Pisaster ochraceus. We thank Mi-
chael Vickery for his assistance in collection and mainte-
nance of larval cultures. Our appreciation is also extended to
Asim Bej, Thane Wibbels. and Daniel Jones for their sup-
port. Funding for this research was provided by Sigma-Xi,
The Alabama Academy of Science, and The University of
Alabama at Birmingham.

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Reference: Biol. Bull. 199: 305-315. (December 2000)

Development of Embryonic Cells Containing

Serotonin, Catecholamines, and FMRFamide-Related

Peptides in Aplysia calif ornica

AMANDA J. G. DICKINSON1'*, ROGER P. CROLL1. AND ELENA E. VORONEZHSKAYA'

1 Department of Physiology and Biophysics, Faculty of Medicine, Dallwusic Universitv, Halifax,

Nova Scotia. B3H 4H7, Canada; and ' Institute of Developmental Biology,

Russian Academy of Sciences, 117808 Moscow, Russia

Abstract. This study demonstrates the presence of a rel-
atively extensive but previously unrecognized nervous sys-
tem in embryonic stages of the opisthobranch mollusc Aply-
sia californica. During the trochophore stage, two pairs of
cells were observed to be reactive to antibodies raised
against the neuropeptides FMRFamide and EFLRIamide.
These cells were located in the posterior region of the
embryo, and their anterior projections terminated under the
apical tuft. As the embryos developed into veliger stages,
serotonin-like immunoreactive (LIR) cells appeared in the
apical organ and were later observed to innervate the velum.
Also, aldehyde-induced fluorescence indicative of cat-
echolamines was present in cells in the foot, oral, and
possibly apical regions during late embryonic veliger
stages. Just before the embryo hatches as a free-swimming
veliger. additional FMRFamide-LIR and catecholamine-

Received 24 January 2000; accepted 2 October 2000.

* To whom correspondance should be addressed. E-mail: ajdickin®
is2.dal.ca

Abbreviarions: ACP, 35 amino acid acidic peptide; CC-1. catecholamin-
ergic central cell one; CC-2. catecholaminergic central cell two; CC-3,
catecholaminergic central cell three; CNS. central nervous system;
EFLRIamide, Glu-Phe-Leu-Arg-Ile-NH,; EDTA. ethylenediaminetet-
raacetic acid; FC-1, FMRFamide central cell one; FC-2, FMRFamide
central cell two; FC-3. FMRFamide central cell three; FITC, fluorescein
isothiocyanate; F-/1, left FMRFamide-LIR posterior cell one; F-/2, left
FMRFamide-LIR posterior cell two; F-H, right FMRFamide-LIR posterior
cell one; F-r2, right FMRFamide-LIR posterior cell two; FMRFamide.
Phe-Met-Arg-Phe-NH2; LIR, like-immunoreactive; PBS, phosphate-buff-
ered saline; SEEPLY, 22 amino acid peptide SEQPDVDDYLRDVVLQ-
SEEPLY; S-/1, left serotonin-LIR bilateral cell one. S-/2, left serotonin-
LIR bilateral cell two. S-cl. right serotonin-LIR bilateral cell one; S-/-2,
right serotonin-LIR bilateral cell two; SUM, serotonin-LIR unpaired me-
dian cell.

containing cells appeared in regions that correspond to the
ganglia of what will become the adult central nervous
system (CNS). Neurons and connectives that will contribute
to the adult CNS appear to develop along the pathways that
are pioneered by the earliest posterior FMRFamide-LIR
cells. These observations are consistent with the hypothesis
that, besides their presumed roles in the control of embry-
onic behaviors, some elements may also guide the develop-
ment of the CNS. Embryonic nervous systems that develop
prior to and outside of the adult CNS have also been
reported in pulmonate and prosobranch species of molluscs.
Therefore, the demonstration of early developing neurons
and their transmitter phenotypes in A. californica presents
new opportunities for a better understanding of the ontog-
eny and phytogeny of both behavioral and neuronal function
in this important model species.

Introduction

The opisthobranch gastropod Aplysia californica. which
has been studied extensively as a model for understanding
the neuronal underpinnings of behavior (for review see
Kandel [1979]), has also become an important model in the
study of molluscan neurodevelopment. The development of
the ganglia that constitute the central nervous system (CNS)
has been studied in detail (Kriegstein, 1977; Schacher et ai,
1979; Kandel et al., 1981; Jacob, 1984). but more recent
studies also report the presence of nerve cells that exist
outside the boundaries of the developing ganglia that will
constitute the adult CNS. For example, in an early embry-
onic veliger stage, three serotonin-like immunoreactive
(LIR) cells exist in the anterior apical organ: an unpaired
median cell and a bilateral pair of cells (Croll and Voron-

305

306

A. J. G. DICKINSON ET AL

ezhskaya, 1995; Croll and Voronezhskaya, 1996b; Marois
and Carew, 1997a, b, c). Soon afterwards, these three cells
are joined by another more lateral pair of apical serotonin-
LIR cells. By the end of the embryonic period, as the veliger
is about to hatch as a free-swimming larva, serotonin-LIR
projections extend into the velar lobes, foot, and abdominal
and visceral regions (Marois and Carew, 1997a, b, c).
Kempt" el al. (1997) showed that such apical cells and
processes appear to be general features of opisthobranch
larvae. Similarly shaped and positioned apical cells have
also been reported in larvae of other molluscan species
(Bonar. 1978: Kulakovskiy and Flyachinskaya. 1994; Rai-
neri and Ospovat, 1994; Raineri, 1995; Leise, 1996; Lin and
Leise, 1996a, b; Dickinson el al, 1999; Voronezhskaya el
al., 1999; Fames, 2000).

In addition to the cells of an apical organ, other neuronal
elements have been observed outside the boundaries of the
developing adult CNS. Croll and Voronezhskaya (1996b)
reported preliminary observations of elements containing
peptides related to Phe-Met-Arg-Phe-NH2 (FMRFamide) in
posterior regions of embryonic A. californica. Recent stud-
ies also indicate the presence of similar neuronal elements
in other molluscan species. For example, Croll and Voron-
ezhskaya (1995; 1996a) identified neuronal elements in
what corresponds to the trochophore or early veliger stage
(Mescheryakov, 1990) of the pulmonate Lymnaea stagnalis.
using antibodies raised against FMRFamide. These FMRF-
amide-LIR cells develop in posterior regions of the embryo
and send anterior projections that terminate in the regions of
the future cerebral and pedal ganglia. A posterior FMRF-
amide-LIR cell has also been observed in the early devel-
opmental stages of the prosobranch Cre/'ithila fornicata
(Dickinson et cil.. 1999). As in L sta^iuilis. this posterior
FMRFamide-LIR cell also sends anterior projections that
terminate in the region of the future cerebral and pedal
ganglia. Therefore, cells expressing FMRFamide-like ini-
munoreactivity appear to develop in a posterior-to-anterior
sequence rather than the anterior-to-posterior development
of the ganglia. In addition, these FMRFamide-LIR cells and
their fibers seem to mark the pathways along which the
adult ganglia and connectives develop, and therefore they
may be involved in guiding the developing CNS.

Additional peripherally located neurons in the foot and
surrounding the mouth were revealed in the gastropods L.
stn^nali.\ (Voronezhskaya et al.. 1999), C. fornicata (Dick-
inson et al.. 1999), and Pliestilla sihoxae (Pires et al., 2000)
and the bivalve Mytilus ednlis (Croll el til.. 1997), using
techniques to localize catecholamines.

The above descriptions of neurodevelopment in represen-
tative species suggest the presence of a primary larval
nervous system that appears earlier than and outside of the
developing adult CNS. Morphological descriptions in other
species also indicate that components of such primary larval
nervous systems may either be incorporated into the adult

ganglia or disappear. The present study investigated the
early development of neurons that may compose a primary
larval nervous system in A. califonuca. starting at the tro-
chophore stage and continuing until the embryo hatches as
a free-swimming veliger. We used immunocytochemical
techniques to study the first cells expressing FMRFamide
and related peptides, and we provide details of the morphol-
ogy of these cells and the timing of their appearance, with
comparisons to the earliest cells exhibiting serotonin-like
immunoreactivity. We additionally used aldehyde-induced
fluorescence, which has been previously applied to mollus-
can tissues (Croll et til., 1997, 1999; Smith et al.. 1998), to
examine cells containing catecholamines. This study shows
that, as in these other molluscs, A. californica also has a
nervous system first present in early embryonic stages.
Furthermore, these observations suggest several new hy-
potheses regarding the mechanism shaping the ontogeny of
the nervous system in this well-studied species.

Materials and Methods

Aninuilx

Adult specimens of Aplysiu californica were purchased
from the Aplysia Resource Facility of the University of
Miami and maintained in a salt-water aquarium. Egg masses
were collected soon after oviposition and kept in separate
containers of artificial salt water (Crystal Sea. Baltimore,
MD) at 20°-22°C. Under these conditions, embryos re-
quired about 9-10 days to develop from first cleavage to
hatching. The hatched veliger required another few weeks
before becoming competent to metamorphose into juvenile
sea slugs (Kandel, 1979; Kandel et al.. 1981: Marois and
Croll, 1992; Marois and Carew, 1997b).

The developmental stages of A. californica were de-
scribed previously (Kriegstein, 1977; Kandel, 1979; Kandel
et til.. 1981; Marois and Croll. 1992; Marois and Carew,
1997b). In the present study, morphological and behavioral
features were examined on each day from first cleavage to
hatching, and the embryonic development was divided into
three stages. During the trochophore stage (days 2.5-4) the
embryo had a distinct apical tuft and a shell gland and began
to move using the prototrochal cilia. On day 4 the body
began to change shape as the rudiments of a velum, foot,
and shell were observed. During the early embryonic veliger
stage (days 5-7) the velum became bilobate and possessed
long cilia along its edge. During the late embryonic veliger
stage (days 8-10) the velum, foot, and shell enlarged and
differentiated.

Immunocytochemistry

Immunohistological procedures were performed accord-
ing to Marois and Croll (1992) and Marois and Carew
( 1997b). Egg ribbons were fixed in 47c paraformaldehyde in

NEURODEVELOPMENT IN APU'SIA CALIFORNIA

307

phosphate-buffered saline (PBS; 50 mM Na:HPO4 • 7H2O
and 140 mA/ NaCl in distilled water adjusted to pH 7.2) for
1 — I h at room temperature. Then embryos were removed
from the capsules, washed in PBS, and stored in 70%
ethanol at -18°C until further processing. For immunohis-
tochemical processing, the stored embryos were first given
two to three 5-min washes in PBS. The shells of older
embryos (>day 4) were then decalcified with 10% ethyl-
enediaminetetraacetic acid (EDTA) (Sigma Chemical Co.,
Mississauga. ON) in PBS for 30-45 min. Embryos were
next washed for 2-3 h in 47r Triton X-100 in PBS. The
embryos were then incubated in antibodies raised against
FMRFamide, serotonin (both obtained from Diasorin, Still-
water, MI), or antibodies (gifts from Dr. P. R. Benjamin,
University of Sussex) against three FMRFamide gene en-
coding peptides: the pentapeptide Glu-Phe-Leu-Arg-Ile-
NH2 (EFLRIamide), the 22-amino-acid peptide SEQPDVD-
DYLRDVVLQSEEPLY (SEEPLY), and a 35-amino-acid
acidic peptide, SDPFFRFGKQQVATDDSGELDDEILSR-
VSDDDKNI (ACP) (Santama et ul.. 1996). All these anti-
bodies except anti-SEEPLY were diluted 1:500-1:1000 in
PBS with the addition of 1.0% normal goat serum and 1.0%
Triton X-100. The SEEPLY antibody was diluted 1:200 in
a solution of 50 mM Tris base, 150 mM NaCl, pH 7.6.
containing 0.25% w/v gelatin and 1% v/v Trition X-100
(Santama et al.. 1993). Incubation periods lasting 48 h at
4°C or 12 h at room temperature gave comparable results.
The embryos were next rinsed three times (5 min each) with
PBS and given a final wash for 1 h before incubating for
24-48 h in goat anti-rabbit antibodies conjugated to fluo-
rescein isothiocyanate (FITC) or rhodamine (Bio/Can Sci-
entific, Mississauga. Ontario) and diluted 1:50 in PBS with
the addition of 1.0% Triton X-100.

To localize FMRFamide-like immunoreactivity relative
to external morphological structures at the trochophore
stage, some embryos were double-labeled with monoclonal
antibodies against *-tubulin (DM1 A clone from Sigma
Chemical Co., Mississauga, ON) (Jackson et al., 1995).
These embryos were first labeled, as described above, for
FMRFamide-like immunoreactivity, then rinsed three times
(5 min each) in PBS. Next, the embryos were incubated in
anti-atubulin (diluted 1:500 in PBS) for 12 h at room
temperature. The embryos were washed again three times in
PBS before incubating in sheep anti-mouse serum conju-
gated to FITC or rhodamine for 12 h at room temperature.
These secondary antibodies were diluted 1:50 in PBS and
1% Triton X-100.

Embryos processed for immunocytochemistry were
mounted on glass slides in a 3: 1 mixture of glycerol to PBS
for viewing on a Leitz Aristoplan microscope equipped for
epifluorescence. FITC fluorescence was viewed using a
450-490-nm excitation filter and a 525/20-nm barrier filter;
rhodamine fluorescence was viewed using a 530-560-nm
excitation filter and 580-nm long-pass barrier filter. Em-

bryos processed for FMRFamide- and *-tubulin-like immu-
noreactivity were also viewed on a Zeiss Axiovert micro-
scope equipped for confocal laser scanning (model LSM
410).

As negative controls, embryos were processed without
incubation in primary antibody; such specimens exhibited
no detectable fluorescence. Positive controls involved par-
allel processing of embryonic L. stagnalis that exhibited
typical staining, as described elsewhere for serotonin and
FMRFamide (Marois and Croll, 1992; Croll and Voron-
ezhskaya, 1995; Croll and Voronezhskaya, 1996a).

Catecholanune histo fluorescence

The formaldehyde glutaraldehyde technique of Furness et
al. (1977) was used to localize catecholamines. Embryos
were incubated for at least 12 h in a solution consisting of
4% paraformaldehyde. 0.5% glutaraldehyde. and 35% su-
crose in PBS. Similar results were also obtained when the
embryos were stored in this solution for several weeks. The
fixed embryos were decalcified in 10% EDTA in PBS for 45
min. Embryos were then placed on glass slides, air dried for
several hours, and then mounted in a 3: 1 mixture of glycerol
and PBS. These embryos were viewed and photographed
through the Leitz compound microscope equipped with a
355-425-nm excitation filter and 460-nm long-pass barrier
filter. Positive controls involved parallel processing of em-
bryonic L. stagnalis that exhibited typical blue-green fluo-
rescent staining, as described elsewhere for catecholamines
(Voronezhskaya et til., 1999). Negative controls were per-
formed by omitting the glutaraldehyde from the formalde-
hyde glutaraldehyde solution, thus eliminating the charac-
teristic fluorescent staining.

Photography

Most histological preparations were photographed on the
Leitz compound microscope using Kodak TMAX 100 film:
the negatives were digitally scanned. Photographs from the
Zeiss confocal microscope were produced by superposition-
ing stacks of 10-15 images obtained through stepped se-
quences of focal planes at intervals of 1-2 ju.m. All the
images were then assembled into plates and labeled using
Photoshop 5.0 (Adobe Systems, Inc.. San Jose. CA). Con-
trast and brightness of the images were adjusted to provide
consistency within plates.

Results

Trochophore stage (days 2.5-4)

Halfway through day 2, two bilaterally symmetrical pairs
of FMRFamide-LIR posterior cells were observed (Figs.
1A. 2A). Fibers projected ipsilaterally and anteriorly from
each cell on the right (F-rl and F-r2) and the left (F-/1 and
F-/2) (Fig. 1 A). By day 3 these fibers terminated in a plexus

308

A. J. G. DICKINSON ET AL.

A DAY 2.5

apical tuft

B DAY 4

C DAYS

DDAY9

prototroch

'telotroch
Right Lateral Views

prototroch

Trochophore

foot

Veliger

Figure 1. Schematic representations of FMRFamide-LIR cells and fibers in embryos of Aplysia californica.
Top row: views from the right side and slightly superior to give a three-dimensional perspective; bottom row:
dorsal views. Anterior is to the right in each figure. (A) Two pairs of posteriorly located FMRFamide-LIR cells
with anterior projections observed on day 2.5. (B) FMRFamide-LIR cells and their processes observed on day
4. The FMRFamide-LIR processes reached the anterior region, where they formed a plexus under the apical tuft.
(C) FMRFamide-LIR cells were no longer symmetrical by day 5; the cells (/I and 12} on the left appeared in a
ventral position, and the cells on the right (/•! and i2) appeared more dorsally. (D) On day 9 additional
FMRFamide-LIR cells (FC-1, FC-2, FC-3) appeared in the anterior region.

of FMRFamide-LIR processes in the region beneath the
apical tuft (see day 4, Figs. IB. 2B, D). Also by day 3, one
to two additional FMRFamide-LIR fibers extended across
the midline of the body just anterior to the somata of the
FMRFamide-LIR posterior cells (See day 4, Figs. IB, 2D).
Initially the pairs of FMRFamide-LIR cells were positioned
symmetrically within the embryo, but they gradually be-
came displaced and by the end of day 4 had all moved to the
right side of the body (Fig. 1C). The FMRFamide-LIR cells
and processes were also identified using antibodies against
EFLRIamide (Fig. 2C). No immtmoreactivity was detected
during the trochophore stage or any later stages with anti-
bodies against SEEPLY and ACP.

Etirly embryonic veliger staifc ( duyx 5-7)

By day 5 the FMRFamide-LIR cells had assumed more
anterior positions in the embryo. F-/-1 and F-/-2 moved apart
from each other, with F-H occupying a more dorsal loca-
tion. F-/1 and F-/2 remained close to each other and together
assumed a central and ventral position (Figs. 1C, 3 A). Their
anteriorly projecting fibers crossed the midline and formed
a commissure in the anterior region. All cells and processes

listed above were also identified using antibodies against
EFLRIamide (Fig. 3B).

Also by day 5, a serotonin-LIR unpaired median cell
(SUM; see Marois and Carew [1997b]) appeared beneath
the apical tuft. Soon afterwards, a pair of vase-shaped
serotonin-LIR cells (S-rl and S-/1 ) were observed to the left
and right of SUM. Short serotonin-LIR fibers projected
ventrally from the SUM. S-rl, and S-/1 to form a plexus in
the same region as the FMRFamide-LIR commissure (see
day 9, Figs. 4A, 5A, B, also see Marois and Carew [1997b]).

By day 7 the three serotonin-LIR cells (SUM, S-rl, and
S-/1 ) were joined by a new pair of serotonin-LIR cells (S-r2
and S-/2) located slightly posteroventrally to S-rl and S-/1
on either side of the plexus (see day 9, Figs. 4A, 5A, B).

Late embn-tmic veliger on prehutching stage (days 8-10)

By day 8 the posterior FMRFamide-LIR cells and their
fibers appeared just anterior to the midpoint along the an-
teroposterior axis. F-/1 and F-/2 together assumed a ventral
and central position, while F-rl and F-r2 each assumed
more dorsal positions than previously (see day 9, Figs. ID,
3C). FMRFamide-LIR fibers extended ventrally from the

NEURODEVELOPMENT IN APLYS1A CAUFORNICA

309

Figure 2. Aplvsia californica during trochophore stages. Anterior is to the right in each figure. (A) Dorsal
view of an embryo on day 2.5 showing the two pairs of posteriorly located FMRFumide-LIR cells with anterior
projections. Scale bar = 20 /mm. (B) Right lateral view of an embryo showing immunoreactivity for '^-tubulin
on day 3. Displays the locations of the apical tuft, prototroch. telotroch, and anal cell in the trochophore. Scale
bar = 25 /xm. (C) EFLRIamide-like immunoreactivity observed on day 3. showing F-rl, F-/1 and -12 (which are
not in focus), and the plexus under the apical tuft (arrow). Scale bar = 25 ju.ni. (D) FMRFamide-like
immunoreactivity observed in the same embryo as 2B showing F-rl, -r2, -1\ and -12, and plexus under the apical
tuft. Scale bar = 25 /am. C and D demonstrate the similar pattern of immunoreactivity for EFLRlamide and
FMRFamide.

apical commissure toward the foot. Also by day 8, formal-
dehyde glutaraldehyde-induced fluorescence, indicative of
catecholamine-containing cells, was observed in the foot
region. The catecholamine-containing foot cells were vase-
shaped and appeared in two bilaterally symmetric groups of
two to three cells on each side (see day 9, Figs. 4B, 6A, B).
By day 9 additional FMRFamide-LIR cells were ob-
served in positions consistent with the locations previously
identified as the developing cerebral, pedal, and pleural
ganglia of the future adult CNS in A. califoniicii (Krieg-

stein, 1977; Marois and Carew, 1990; Marois and Carew,
1997b) and other opisthobranchs (Kempf et al.. 1997a). One
of these FMRFamide central cells (FC-1) appeared near
F-rl, another (FC-2) was located near F-r2, and a third
(FC-3) was observed to the left of the apical commissure
(Figs. ID, 3C). Also by day 9, the number of catechol-
amine-containing cells increased to four to five cells on each
side of the foot (Figs. 4B, 6A, 6B). Another pair of cate-
cholamine-containing cells was located in the oral region.
Catecholamine-containing fibers also extended from each

310

A. J. G. DICKINSON ET AL.

A 5-HT

B CA's

Figure 3. FMRFamide- and EFLRIamide-LIR cells and fibers in repre-
sentative embryonic veliger stages of Aplyxiu ciilifnrnica. Anterior is to the
right in each figure. Arrows indicate the FMRFamide-LIR plexus. (A) FMRF-
amide-like imnumoreactivity observed on day 5, showing the asymmetry of
F-/1, F-/2, F-i-l, and F-cl. Scale bar = 15 /urn. (B) Montage of two photo-
graphs showing the EFLRIamide-LIR cells and libers on day 7. Scale bar =
28 fim. (C) FMRFamide-like immunoreactivity on day 9. Two additional
cells, FC-1 and FC-3, are shown in this focus. Scale bar = 2X /uni.

Right Lateral View

Dorsal View

Ventral View

Figure 4. Schematic representations of serotonin-LIR and catechol-
amine-containing neurons in Aplysia califomica during day 9. Top row:
views from the right side and slightly superior to give a three-dimensional
perspective. Anterior is to the right in each figure. (A) Serotonin-LIR cells
(S-/1, S-/2. S-/'l, S-i'2, SUMl and fibers in the apical organ. Bottom row:
dorsal view. (Bl Cells and fibers containing catecholamines were located in
the foot (arrow heads), oral region (arrow), and the region of the future
CNS (CC-1, CC-2, CC-3). Bottom row: ventral view.

group of foot cells toward the region below the apical tuft
where the FMRFamide-LIR apical commissure and seroto-
nin-LIR plexus were located. In this region, three cate-
cholamine-containing central cells were observed (Fig. 6B);
two of these cells (CC-1, CC-2) were located on the right
and another cell (CC-3) on the left. Unfortunately, the
formaldehyde glutaraldehyde technique resulted in high
background fluorescence, making it difficult to determine
whether these cells were located in the apical organ or the
developing cerebral or pedal ganglia of the future adult
CNS. Also by this time, serotonin-LIR fibers could be seen
projecting toward the velum, foot, and posterior region
(Figs. 4A, 5 A).

Discussion

The current study offers evidence that a relatively exten-
sive nervous system forms during embryonic development
of the opisthobranch gastropod Aplysiti califomica. The
early nervous system includes posterior FMRFamide-LIR
cells that first appear during the trochophore stage. By the
veliger stage, serotonin-LIR cells appear in the apical organ
(Marois and Carew, 1997a, b, c), and shortly before hatch-
ing catecholamine-containing cells appear around the mouth
and in the foot. The first neurons within the developing

NEURODEVELOPMENT IN APLYSIA CALIh'OKNICA

311

Figure 5. Serolonin-LIR cells and fibers in Aplyxia ciilifornicii during
days 8-9. Anterior is to the right in each figure. (A) Right lateral view of
an embryo on day 9 showing serotonin-LIR cells in the apical organ (S-/1,
S-/2, S-rl. SUMl and fibers projecting into the foot (arrowhead) and velum
(arrow). Scale bar = 30 /xm. (B) Dorsal view of an embryo on day S
showing three of the serotonin-LIR cells (S-/I. S-rl. SUM) of the apical
organ. The apical commissure is indicated by the arrowhead. Scale bar =
28 /urn.

ganglia, which will eventually constitute the adult CNS,
only begin to appear during late embryonic stages (Schacher
ft nl., 1979). Such an arrangement of neuronal cells and
fibers is similar to that found in representative pulmonate
and prosobranch gastropod species (Croll and Voronezh-
skaya. 1996a; Dickinson et al., 1999).

Posterior FMRFumide-LlR cells

Two pairs of FMRFamide-LIR cells appear in posterior
regions and project anteriorly directed fibers in early tro-
chophore stages of A. califarnica. FMRFamide-like immu-
noreactivity has also been observed posteriorly in early
embryos of the other gastropod molluscs; however, differ-
ences were observed in the number and precise positions of
these cells. Lyiwuiea stagwili.? (Croll and Voronezhskaya,
1995, 1996a) and Crepidula fornicata (Dickinson el <//.,
1999) each possess a single medial FMRFamide-LIR cell in

posterior regions near the shell gland. No such cell was
observed during embryonic development in A. culifoniica.
Nevertheless, it could have been missed if it was present
only for a very brief period of development and if it did not
exhibit reactivity to the anti-FMRFamide antibodies used in
this study. During early veliger stages of L. stagnalis, ad-
ditional FMRFamide-LIR posterior cells exist on the left
and right libers projecting from the most posterior medial
cell (Croll and Voronezhskaya, 1995, 1996a). The location
of these left and right cells in L. staifnalis may correspond
to the left and right pairs of FMRFamide-LIR posterior cells
in A. califarnicn. FMRFamide-LIR cells in such lateral
regions were not detected in C. fornicata (Dickinson et til..
1999).

Despite variations in the number and position of posterior
FMRFamide-LIR cells in the different species, they all
share certain features. In all three species, the posterior

Figure 6. Catecholamine-containing cells and libers in Aplvsiii cali-
fornica during day 9. Anterior is to the right in each figure. (A) Right
lateral view of an embryo showing catecholamine-containing cells in the
foot (arrowheads), oral region (arrows), and apical region or region corre-
sponding to the future ganglia of the adult CNS (CC-3). Scale bar = 28
pun. (B) Ventral view montage showing cells containing catecholammes.
Cells are shown in the foot (arrowheads), oral region (arrows), and apical
region or region corresponding to the future ganglia of the adult CNS
(CC-1, CC-2. CC-3). Scale bar = 28 /im.

312

A. .1 G, DICKINSON ET AL.

FMRFamide-LIR cells appear before any other nerve cells
are detected. Also, the posterior FMRFamide-LIR cells all
extend anteriorly directed axons that pass through the region
in which the cerebral ganglia will later develop, and they
eventually terminate in a region of the future pedal ganglia.
FMRFamide-LIR fibers also appear under the apical tuft
during trochophore stages of both A. californica and C.
fornicata. This region later develops into the apical sensory
organ, the underlying cerebral commissure, or both (Marois
and Carew, 1997a. c). The plexus of immunoreactive fibers
is extensive in this region, even at very early developmental
stages. Although no other immunoreactive somata were
detected at these stages, we cannot exclude the possibility
that at least some fibers may derive from sources other than
the posterior cells. In fact, FMRFamide-LIR fibers also
branched repeatedly under the apical plate in early embry-
onic stages of L. stagnalis, but these fibers originate from
nearby somata that exhibit little or no immunoreactivity
(Croll and Voronezhskaya, 1996a).

Similarity also exists in the patterns of expression of the
FMRFamide-related peptides in two species of molluscs. In
both L. stagnalis and A. californica, the cells and fibers
expressing FMRFamide- and EFLRIamide-like immunore-
activity are similar (Voronezhskaya and Elekes, 1997). The
FMRFamide antiserum is immunoreactive to several
FMRFamide-related peptides (Gaus et al., 1993), whereas
the EFLRIamide antiserum is immunoreactive only to
EFLRIamide and FMRFamide itself (Santama et al., 1995a,
b, 1996). Our results thus indicate that immunoreactivity in
early stages of A. californica is due to the presence of
FMRFamide and EFLRIamide and not to the exclusive
presence of other FMRFamide-related peptides. Antibodies
against SEEPLY and the acidic peptide (ACP) that are
processed from the FMRFamide precursor protein of L.
stagnalis are not immunoreactive in embryonic A. califor-
nica. The sequence of SEEPLY is identical in L. stagnalis
and A. californica (Greenberg and Price, 1992), but consis-
tent with our findings in A. californica, Voronezhskaya and
Elekes (1997) reported no detectable occurrence of this
peptide in FMRFamide-LIR cells during embryonic stages
of L. stagnalis. Conversely, ACP has not been isolated in A.
californica. Therefore, the lack of positive immunoreaction
for this latter peptide may be attributed to species-specificity
of the amino acid sequence.

The aim of the present study was to examine the ontog-
eny of the nervous system during embryonic development,
thus representing times much earlier than typically exam-
ined in A. californica. Since we did not examine later stages,
the fates of the FMRFamide-LIR cells are unclear. It seems
possible, however, that at least some of these FMRFamide-
LIR cells and fibers later become incorporated in the ganglia
and connectives of the CNS. The regions where the ganglia
of the adult CNS are located have been previously identified
in later veliger stages of A. californica ( Kriegstein, 1977:

Marois and Carew, 1990, 1997b). In the present study,
FMRFamide-LIR cells can be observed in positions that
correspond to these regions. For example. F-/1 and F-/2
seem to lie in positions that correlate to the future site of the
left abdominal ganglia. F-r2 appears in the region where the
osphradium will develop, whereas FC-2 seems to lie in a
position that corresponds to the location of the right abdom-
inal ganglia. F-H. FC-1, and FC-3 appear in regions where
the pleural or pedal ganglia will develop. Confirmation of
the locations of these FMRFamide-LIR cells within the
CNS must, however, await additional histological examina-
tion to identify the boundaries of the developing central
ganglia. Although our work may have tentatively identified
individual central neurons at earlier stages than previously
demonstrated, the findings are still consistent with other
descriptions of gangliogenesis. The presence ot cerebral and
pedal cells has been previously reported in hatchlings of A.
californica (Kriegstein, 1977; Marois and Carew. 1997b)
and other gastropods (D'Asaro, 1969; Page, 1992a. b; Lin
and Leise, 1996b; Dickinson et al.. 1999). Anlagen of the
visceral loop ganglia have also been identified in hatching
stages of A. californica (Schacher et al.. 1979).

It is also possible that at least some posterior FMRF-
amide-LIR cells lie outside the developing ganglia and
transiently express their transmitter phenotype during a
short phase of embryogenesis. Such a fate for the early
posterior FMRFamide-LIR cells has been shown in the
developing embryos of L stagnalis (Croll and Voronezh-
skaya, 1995; Voronezhskaya and Elekes, 1996).

Whatever the exact locations and fates of the various
somata, FMRFamide-LIR fibers clearly mark pathways that
span the length and breadth of the embryos in A. californica.
and they may play a role in pioneering the various commis-
sures and connectives of the adult nervous system. Such a
role for early FMRFamide-LIR cells and fibers has been
suggested in other molluscs such as L. stagnalis (Croll and
Voronezhskaya, 1995. 1996a) and C. fornicata (Dickinson
ft al.. 1999) and is consistent with hypothesized roles of
early developing fibers in other invertebrate groups such as
insects (Bate. 1976; Caudy and Bentley. 1986; Goodman
and Shatz, 1993) and annelids (Lacalli, 1981. 1982). The
necessity of such pioneering fibers for the normal develop-
ment of the CNS in gastropods must be tested in future
experiments.

Serotonergic apical cells

The serotonin-LIR cells in the apical organ of A. califor-
nica have been previously described in detail (Croll and
Voronezhskaya, 1995: Marois and Carew, 1997a, b, c). A
similar arrangement of serotonin-LIR cells was also re-
ported in other opisthobranchs (Kempf et al., 1997a), proso-
branchs (Dickinson et al., 1999). and bivalves (Croll ct al.,
1997).

\l 1 KOD1 \ II Ol'MI-M IN U7 )S/l ( \//l IIRMi \

313

The apical organ in molluscs and other invertebrate lar-
vae may control larval behaviors such as swimming, feed-
ing, and crawling (Leise, 1996; Lin and Leise. 1996a, b).
Morphological evidence supports such hypothesized func-
tions since serotonin-LIR cells of the apical organ innervate
the velum in A. californica (Marois and Carew. 1997H. c)
and other molluscs (Kulakovskiy and Flyachinskaya, 1994;
Croll et al., 1997; Kempt' et al.. 1997; Dickinson el ai,
1999). Such velar innervation probably controls locomotion
and feeding currents generated by cilia, which are respon-
sive to serotonin in both adult forms (Audesirk et al.. 1979;
Murakami, 1987; Syed and Winlow, 1989) and larval stages
(Koshtoyants et al., 1961; Beiras and Widdows, 1995). The
apical organ is also thought to control the transduction of
the metamorphic signal (Hirata and Hadtield, 1986; Couper
and Leise, 1996; Hadtield et al., 2000) and possibly to
influence subsequent development of the adult CNS
(Lacalli. 1981. 1994: Marois and Carew. 1997c).

Although non-serotonergic apical cells were not identi-
fied in the present study, other neurons in the apical organ
of A. californica were previously identified using electron
microscopy (Marois and Carew. 1997b). Flask-shaped.
FMRFamide-LIR cells have also been observed in the api-
cal region of the pulmonate L. stagnalis (Croll and Voron-
ezhskaya, 1996a), the prosobranch C. fornicata (Dickinson
et al., 1999). and the opisthobranch P/iestilla sibogae
( Kempt" et ai. 1992). It is possible that apical somata exhibit
other transmitters or peptides in later stages of development
in A. californica.

Anterior catecholaminergic cells

Catecholamine-containing cells exist in the foot region of
A. californica. Similar cells also appeared in the foot of the
gastropods L stagnalis (Voronezhskaya et al.. 1999) and C.
fornicata (Dickinson et al.. 1999), and in the bivalves Myti-
lus edulis and Placopecten magellanicus (Croll et al.,
1997). Such cells may be involved in metamorphosis, be-
cause catecholamines were found to modulate or induce
metamorphosis in both gastropods (Pires et al.. 2000) and
bivalves (Coon and Bonar, 1986). Similarly located cells
have also been found to be responsive to the chemical cues
inducing metamorposis in the opisthobranch Onchidoris
hilaniellata (Arkett et al., 1989).

Catecholamine-containing cells are also found near the
mouth of embryonic A. californica, consistent with similar
cells reported in pulmonates, prosobranchs, and bivalves
(Croll et al., 1997; Dickinson et al.. 1999; Voronezhskaya et
al.. 1999). The role for these cells is also unknown, although
it is tempting to suggest that they might influence feeding
behavior, which already displays a significant degree of
sophistication within larval stages of molluscs (Baldwin and
Newell, 1995).

We suggest that at least some other catecholarnine-con-

taining cells found in later larval stages might lie within the
developing central ganglia, consistent with previous discus-
sions of other central neurons in this paper. Confirmation of
the locations of these cells must, however, await further
experimentation to identify the boundaries of the develop-
ing central ganglia during embryonic stages.

Torsion and neurodevelopment

It was previously hypothesized that the opisthobranch
nervous system lacks chiastoneury, or the twisting of the
visceral loop into a figure-eight pattern, because neurode-
velopment begins after torsion (Kandel et al.. 1981). How-
ever, the present study shows posterior neurons and fibers
before the onset of torsion in A. californica, and if such
neurons are later incorporated in the developing ganglia, a
reevaluation of this hypothesis may be necessary. In early
stages of the prosobranch C. fornicata. chiastoneury was
observed in a figure-eight pattern of the FMRFamide-LIR
fibers (Dickinson et al.. 1999). In corresponding stages of A.
californica, such a figure-eight pattern did not exist, prob-
ably due to the different degrees of torsion in the two
groups: 1 20-degree rotation in the opisthobranchs compared
with the 180-degree rotation in the prosobranchs (Kandel.
1979: Kandel et a!., 1981). Cells and fibers may also be
positioned more anteriorly and the connectives between
ganglia may be shorter in opisthobranchs than in proso-
branchs. Detorsion in opisthobranchs (Kandel. 1979; Kan-
del et al., 1981 ) presumably eliminates much of the remain-
ing evidence of twisting of the neural pathways.

Conclusion

The present study provides morphological evidence of
neuronal elements that appear earlier in development than
any previously identified neural structure, including the
apical organ, in A. californica. Many catecholamine-con-
taining cells are also identified in anterior regions of early
embryonic stages. Together these elements (posterior cells,
apical organ, and anterior catecholaminergic cells) may
compose a distinct, primary larval nervous system that may
be responsible for the control of several embryonic and
larval behaviors such as swimming, feeding, and metamor-
phosis. The fate of such a primary larval nervous system is
unknown, but evidence suggests that some neurons and
connectives may be incorporated into the adult CNS while
other cells disappear. Other invertebrate larvae from taxa
such as polychaetes. nemertines. echinoderms. and pho-
ronids have embryonic nervous systems that are very sim-
ilar to those of molluscs (Hay-Schmidt. 1990a. b. c; 1995).

This report also suggests the possibility that the larval
nervous system may form a scaffold along which the adult
nervous system develops. Such a hypothesis is consistent
with the suggested role of FMRFamide-LIR cells and pro-
cesses in corresponding stages of other gastropods and other

314

A. J. G. DICKINSON ET AL.

invertebrate groups. The demonstration of early developing
neurons and their transmitter phenotypes in A. californicti
opens new opportunities for a deeper understanding of the
ontogeny and phylogeny of both behavior and neuronal
function in this important model species.

Acknowledgments

We thank J. Nason for technical assistance and L. Nezlin
for critical readings of this manuscript. Funding was pro-
vided by grants from the Natural Sciences and Engineering
Research Council of Canada (NSERC) to R.P.C. (grant
number: OGP0038863) and from the Russian Fund for
Basic Research (RFBR) to E.E.V. (grant number 99-04-
48415).

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Development of a Penis from the Vestigial Penis in the
Female Apple Snail, Pomacea canaliculata

NAOKUNI TAKEDA

Brainway Group, Brain Science Institute. The Institute of Physical and Chemical Research,

2-1 Wako. Saitama 351-0198, Japan

In the apple snail (Pomacea canaliculata), females have
an undifferentiated mass of tissue near the anus. Although
this mass is called the vestigial penis, there are no signs of
a hermaphroditic gonad or any structure that represents a
transition from one se.x to the other. Based on consider-
ations of the steroid hormone theory of reproduction and in
view of disruption of endocrine s\slems in molluscs by
organotins, a study was made of the effects of tributyltin on
female snails. Exposure to tributyltin resulted in the so-
called imposex phenomenon, and both a penis and a penis
sheath were newly generated from the so-called vestigial
penis. The same phenomenon was also induced b\ testos-
terone. Thus the vestigial penis, named more than one
hundred vears ago, has been demonstrated for the first time
to be a rudiment of the penis itself.

The Ampullariidae (Gastropoda, Prosobranchia, Archi-
taenioglossa) is a family of freshwater prosobranchs that are
widely distributed in Asia, Africa, and South America ( 1 ). It
is well known that some molluscs exhibit unusual sexual
diversity and hermaphroditism (2, 3). One of the most
interesting features of members of the Ampullariidae is that
females in 5 of the 10 genera — namely, Pila (4, 5, 6. 7. 8.
9, 10, 11, 12), Pomacea (13, 14. 15. 16), Lunixtes (13, 17,
18), Afropomus (19), and Turbinicola (13) — have a so-
called vestigial penis in addition to the normal reproductive
system (Fig. 1). The vestigial penis was first described by
Bouvier (4, 5), a skilled neuroanatomist. in Ampullaria
(Pila) polita more than 100 years ago. Since then, many
molluscan researchers have used that term for this tissue
without further explanation. The question examined here is
this: Is the organ historically described as a vestigial penis
a remnant of a penis — a degenerate structure that lacks the

Received 7 December 1999; accepted 10 August 2000.
E-mail: takeda@brainway.riken.go.jp

capacity to develop further — or is it a rudiment, or precur-
sor— an incipient structure that, under the proper conditions,
could develop into a penis?

The vestigial penis is a tongue-like structure lying inside
the elevated mantle skirt, near the anus (Fig. 2 A). Histolog-
ically, it consists of connective tissue, and no differentiation
of the structure is apparent during the life cycle of the
female (Fig. 2B).

To my knowledge, no experimental evidence has been pre-
sented to justify the designation of the elevated tissue near the
anus in some female snails as a "vestigial penis" (4, 5). No
clear evidence of hermaphroditism has yet been shown in any
extant species of Ampullariidae. In Pomacea canaliculata, the
apple snail, the positions of the gonads are basically different:
the testis is located at the tip of the spiral, and the ovary is
spread over the surface of the hepatopancreas at a location
similar to that of the testis. It has also been confirmed that there
is no apparent precursor or vestige of a hermaphroditic condi-
tion in any part of the reproductive system throughout the life
history. As Andrews (13) stated, the copulatory apparatus
appears to develop at the same rate in both sexes until the
gonad becomes active, when its growth is arrested in the
female. Andrews hypothesized that the gonad might produce a
hormone responsible for the cessation of growth, but in the
early 1960s, when this work was published, the chemical
nature of reproductive hormones in molluscs had not yet been
established. In 1991, Berthold (20) proposed that the so-called
vestigial organ be designated an "oriment" — a term implying
that the tissue is a precursor with the potential to develop
into an adult organ. The question then arose as to whether
such a designation might be appropriate. As a basis for such
a designation, at the very least, some experiments involving
implantation of testes into females should be performed to
determine whether a true penis might develop from the
tissue mass.

316

IMPOSEX IN THE APPLE SNAIL. P. CANALICULATA
.7

317

Figure 1. General appearance of the vestigia] penis in a female apple snail (Pomacea canaliculata). The
shell was removed and the region to the left of the head was dissected to reveal the vestigial penis ( 1 ), rectum
(2), and oviduct (3), which are enclosed in a square. The siphon (4). pulmonary sac (5), albumen gland (6), and
ovary (7) are also indicated. Scale bar represents 1 cm.

As an approach to this problem, I examined the effects of
an endocrine disruptor that appears to be associated with as
yet unresolved environmental problems. Organotins, and in
particular tributyltin, which is a component of some anti-
fouling paints, induce a condition known as "imposex" in
prosobranch gastropods. Imposex, in which a penis and vas
deferens develop in females (21 ), has been widely observed
in marine snails that belong to the Caenogastropoda; Nu-
cella and Littorina are common examples (22). Females of
these species lack a vestigial penis, but the capacity exists
for induction of a penis and vas deferens. Pomacea canal-
iculata has been proposed as a potential bioindicator for
tributyltin (22), which is used as a biocidal agent against
molluscs, in fungicides (23) and in anti-fouling paints in
freshwater environments. Anticipating possible endocrine
disruption by tributyltin, I examined its effects to see
whether the vestigial penis in this species might develop

further after female snails were exposed to this compound
and, if such a penis did develop, how would it differentiate?

Female specimens off. canaliculata were reared in water
that contained 30 ng/1 tributyltin. About 3 months after the
start of treatment, the outside of the elevated tissue of the
vestigial penis began to form a long process that resembled
a penis, and its interior developed as a thick mass. These
structures grew gradually and reached a maximum size after
about 6 months of treatment with tributyltin (Fig. 3A).
Histological staining revealed that the inside tissue mass
contained a penis; the cross section of a penis was also
found within the tissue mass (Fig. 3B). Thus, the outside
structure appeared to be a penis sheath. Within about one
further month, a complete penis had developed from the
tissue of the vestigial penis (Fig. 4).

In P. canaliculata, the copulatory system of the male
consists of a stout penis sheath and a long, slender penis

318

N. TAKEDA

Figure 2. (A) General appearance of the vestigial penis in a female
apple snail (Pomacea canaliculata: control) reared by artificial mass cul-
ture. Scale bar represents 1 mm. (B) Histological appearance of the
vestigial penis in A. The regions containing a vestigial penis were fixed in
Bouin's fluid and embedded in paraffin wax by the standard method.
Sections were stained with hematoxylin and eosin. Scale bar represents 200
;im. A. anus; C, ctenidium; OD, oviduct; VP. vestigial penis.

within it (24). The penis and the penis sheath are located
together to the left of the extreme right margin of the mantle
cavity. In treated females, the arrangement of these male
copulatory organs was similar but differed in the distance
between the penis sheath and the penis: the penis sheath in
females was located at the edge of the ctenidium at the
mantle skirt, at a distance from the penis.

It has been suggested that tributyltin inhibits cytochrome
P450 aromatase, which converts testosterone to estradiol in
females (25, 26). Inhibition of aromatase activity thus in-
creases levels of testosterone which induces imposex, with
the development of male copulatory organs. Development
of the imposex phenomenon in P. canaliculata was also
confirmed by direct treatment with testosterone. Female
snails reared in water that contained 500 ng/1 testosterone
exhibited changes similar to those induced by tributyltin,
including the development of a penis sheath and a penis.

Therefore, these observations support the proposed mecha-
nism of action of tributyltin.

It is difficult to explain the unusual phenomenon of a
rudimentary penis in females; however. I propose the fol-
lowing hypothesis. In the early stages of development, both
sexual rudiments develop as an undifferentiated tissue mass.
Once the sex of the snail is determined genetically (27),
however, these rudiments differentiate in response to the
secretion of specific sex steroid hormones. The undifferen-
tiated tissue mass that develops into a penis in males is left
as an arrested rudiment in females. The vestigial penis
develops into a complete copulatory organ only if the anlage
of the gonad becomes a testis.

Figure 3. (A) Morphology of the imposex induced by trihutyltin in a
female apple snail (Pumacea canaliculata). Female snails (n = 100) for
experiments were reared in a freshwater tank that contained tributyltin
(Tokyo Kasei, Co. Ltd.. Tokyo, Japan) at 30 ng/l for about f> months. The
state of imposex was checked at weekly intervals. Scale bar represents 1
mm. Similar results were also obtained in female snails (n = 100) reared
with testosterone (Wako. Co. Ltd., Osaka. Japan) at 500 ng/1 for about 7
months. (B) Histological appearance of the vestigial penis in A. The
arrowhead indicates the cross section of a penis. Hematoxylin and eosin
stain. See legend to Fig. 2B for methods. Scale bar represents 200 JLUTI. P.
penis; PS. penis sheath; VD. vas deferens (see legend to Fig. 2 for other
abbreviations).

1MPOSEX IN THE APPLE SNAIL. P. CANALICULATA

319

Figure 4. (A) A penis that arose from the vestigial penis of a female apple snail (Pomacca canaliculate!)
after treatment with tributyltin for about 7 months. The same phenomenon was also seen in female snails reared
with testosterone for about 8 months. See legend to Fig. 3A for methods. (B) The extirpated penis from the
vestigial penis. See legends to Fig. 2 and 3 for other abbreviations. Scale represents 1 mm.

The "steroid hormone theory," which I proposed previously
(28, 29) for the reproduction of terrestrial pulmonates, appar-
ently also applies to prosobranch snails. This theory states
basically that the development of accessory sex organs is
controlled by steroid hormones secreted by the gonad. This
concept of the effects of hormones on snail reproduction,
together with the effects of endocrine disruption in molluscs,
allowed me to demonstrate, for the first time, that the so-called
"vestigial penis," named more than one hundred years ago (4.
5), is a rudiment of the penis itself.

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reproductive system in the apple snail, Pomucea canaliculate. J. Anal.
Biosci. 22: 425-432.

25. Spooner, N., P. E. Gibbs, G. W. Bryan, and L. J. Goad. 1991. The
effects of tributyltin upon steroid litres in the female dogwhelk, Nu-
cella lapi/lus. and the development of imposex. Mar. Environ. Res. 32:
37-49.

26 Bettin, C.. J. Oehlmann, and E. Stroben. 1996. TBT-induced
imposex in marine neogastropods is mediated by an increasing andro-
gen level. Helgol. Meeresunters. 50: 299-317.

27. Brand, E., T. Yokosawa, and Y. Fujio. 1990. Chromosome anal-
ysis of apple snail Pomacea canaliculate. Tohokn J. Agric. Res. 40:
81-89.

2X. Takeda, N. 1983. Endocrine regulation of reproduction in the snail,
Euhadra peliomphala. Pp. 106-110 in Molluscan Neuro-Endocrinol-
ogy, }. Lever and H. H. Boer, eds. North-Holland, Amsterdam.

29. Takeda, N. 1989. Hormonal control of reproduction in land snails.
Venus 4»: 99-139.

INDEX

ABENAVOLI, A., L. FORTI, AND A. MALGAROLI, Mechanisms of spontaneous

miniature activity at CA3-CA1 synapses: evidence for a divergence

from a random Poisson process, 1 84
Actin, 202, 203
Action potential, 135
Adenylate cyclase, 197
Adhesion of a viral envelope protein to a non-self binding domain of the

aggregation factor in the marine sponge Microciona prolifera, 209
Adhesive proteoglycans, 192
Aggregation factor, 192, 209
Aggression, 265
Aiptasia, 76
Allometric scaling in small colonies of the scleractinian coral Siderastrea

siderea (Ellis and Solander), 21
Allometry, 21
cAMP. 197
Amphibian. 187
An inducer of molluscan metamorphosis transforms activity patterns in a

larval nervous system, 241

Annual Report of the Marine Biological Laboratory, v. 199 ( 1 ), R 1
Anuran, 187
Apical organ, 305
Aplysia, 305
Arbacia eggs, 212, 213

ARMSTRONG. MARGARET T., see Rengasamy Asokan. 190
ARMSTRONG, PETER B., AND RENGASAMY ASOKAN. A Ca+:-independent

cytolytic system from the blood of the marine snail Busycon cana-

liciilum. 194
ARMSTRONG. PETER B., see John M. Harrington, 189; Rengasamy Asokan,

190

ASOKAN, RENGASAMY, MARGARET T. ARMSTRONG, AND PETER B. ARM-
STRONG, Association of a,-macroglobulin with the coagulin clot in the

American horseshoe crab. Limulus polyphemus: a potential role in

stabilization from proteolysis, 190
ASOKAN, RENGASAMY, see Peter B. Armstrong, 194
Association of a-,-macroglobulin with the coagulin clot in the American

horseshoe crab, Limulus polyphemus: a potential role in stabilization

from proteolysis, 190
Asteroidea. 298
ATHERTON, JILLIAN L., MATTHEW A. KRUTKY, JAMES M. HITT, FREDERICK

A. DODGE, AND ROBERT B. BARLOW, Optic nerve responses of Limulus

in its natural habitat at night, 176
ATHERTON, JILLIAN L., see Matthew A. Krutky, 178
Axonal transport, 202, 203
Axoplasm, 203

BAGHDASARIAN, GAREN, AND LEONARD MLISCATINE, Preferential expulsion

of dividing algal cells as a mechanism for regulating algal-cnidarian

symbiosis, 278
BAKER, SHIRLEY M., JEFFREY S. LEVINTON, AND J. EVAN WARD. Particle

transport in the zebra mussel, Dreissena polymorpha (Pallas), 1 16
BARLOW, ROBERT B.. see James M. Hitt, 171; Jillian L. Atherton, 176;

Matthew A. Krutky, 178
BARNES, DAVID K. A., AND MATTHEW H. DICK, Overgrowth competition

between clades: implications for interpretation of the fossil record and

overgrowth indices, 85
BENTLEY, M. G., see G. J. Watson, 50
BEZANILLA, FRANCISCO, see Joshua J. C. Rosenthal, 135

Biogeochemistry, 218

Biogeography, 126

Biogeography of two species of Symbiodinium (Freudenthal) inhabiting the
intertidal sea anemone Anthopleura elegantissima (Brandt), 126

Biosynthesis, 192

Birefringence, 212

Bivalve, 29, 116, 144, 199

Blood clotting, 190

Boatwhistle, 173

BOLTON. TOBY F., FLORENCE I. M. THOMAS, AND CELERE N. LEONARD,
Maternal energy investment in eggs and jelly coats surrounding eggs
of the echinoid Arbacia punctulata, 1

Boron, 221

BROTHERS, CHRISTINE, ERNEST MARKS in, AND ROXANNA SMOLOWITZ, Con-
ditions affecting the growth and zoosporulation of the protistan par-
asite QPX in culture, 200

BROWN. JEREMIAH, see Phillip Stafford, 203

BRUZZONE, ROBERTO, see Thomas H. White. 165

Bryozoan. 85

BucKLAND-NlCKS, JOHN, AND ALAN N. HODGSON, Fertilization in Callo-
chiton castaneus (Mollusca), 59

BURGER, MAX M., see William J. Kuhns, 192; Roger MacKenzie, 209

BURGOS, MARIO H., MAKOTO GODA, AND SHINYA INOUE, Fertilization-
induced changes in the fine structure of stratified Arbacia eggs. II.
Observations with electron microscopy ,213

BURGOS, MARIO H., see Makoto Goda, 212

BYRNE, R. A., see T. H. Diet?., 14

Ca2 + , 212

A Ca+2-independent cytolytic system from the blood of the marine snail

Busvcon canaliculwn, 194
Cable properties, 135
Calcium imaging, 162
Camouflage, 6
Carotenoid, 223
Cassiopeia. 76
Cataract, 187
Catecholamines, 305
Cell

contact, 287

motility, 202, 203

movement, 287

proliferating, 199

Cellular basis of gastrulation in the sand dollar Scaphechinus mirabilis, 287
Centrifugation, 212, 213
Centrifuge polarizing microscope, 212
CHANG, F., see P. T. Tran, 205
Channel, 165
Cherax destructor, 25 1
CHERRY, J. S., see T. H. Dietz. 14

CHIKARMANE, HEMANT M., ALAN M. KUZIRIAN. ROBBIN KOZLOWSKI, MARK
KUZIRIAN, AND TONY LEE, Population genetic structure of the goose-
fish, Lophius americamis, 227
CHILD. FRANK M., see David A. Epstein, 182
Chimerism, 231
Chlorophyll, 223

Circadian rhythms in the receptive fields of the Limulus lateral eye, 171
Clades, 85

Cladophora vagabunda, 223
CLAESSENS, Luc, see Ryan Kirkby, 218

321

322

INDEX TO VOLUME 199

Cleavage. 207

Cnidaria, 68, 278

COHEN, LAWRENCE B.. see Matt Wachowiak. \(i2

Colony. 231

Conditions affecting the growth and z.oosporulation of the protistan para-
site QPX in culture, 200

Conduction velocity, 135

Connexin, 165

Copepod, 95

Coral, 21.68

CRAWFORD, KAREN. The role of microtubules during blastodisc formation
of the squid Loligo pealei. 207

Crayfish. 251

A critical approach to the definition of Darwinian units of selection. 231

CROLL. ROGER P.. see Amanda J. G. Dickinson, 305

CROMARTY. S. I., J. MELLO. AND G. KASS-SIMON, Molt-related and size-
dependent differences in the escape response and post-threat behavior
of the American lobster, Hoinarus americanus, 265

Crustacea, 265

Cytoplasmic streaming. 207

Cytoskeleton. 144. 202. 203

DACEY, JOHN W. H.. see Richard W. Hill. 108

DAVIS, THEODORE M.. see Inigo Novales Flamarique. 187

Denaturing gradient gel electrophoresis. 126

DENAULT, MICHELLE. ERICA STIEVE, AND IVAN VALIELA. Effects of nitrogen
load and irradiance on photosynthetic pigment concentrations in
Cladophora vagabiinJn and Gmcilaria tikvahiiie in estuaries of Wa-
quoit Bay. 223

Denitrification, 221

Descending octaval nucleus. 173

Detritus. 225

Development. 68. 195

Development of a penis from the vestigial penis in the female apple snail,
Ponutcea canaliculata, 316

Development of embryonic cells containing serotonin, catecholamines. and
FMFRamide-related peptides in Aplysia califomica, 305

Development of self-referencing oxygen microsensor and its application to
single pancreatic HIT cells; effects of adenylate cyclase activator
forskolin on oxygen consumption. 197

DICK, MATTHEW H., see David K. Barnes. 85

DICKINSON, AMANDA J. G., ROGER P. CROLL, AND ELENA E. VORONEZH-
SKAYA, Development of embryonic cells containing serotonin, cat-
echolamines. and FMFRamide-related peptides in Aplysia califurnk n.
305

DIETZ, T. H.. A. S. UDOETOK. J. S. CHERRY, H. SILVERMAN, AND R. A.
BRYNE, Kidney function and sulfate uptake and loss in the freshwater
bivalve Toxolastna texasensist 14

Differences in properties of salt marsh sediment between hayed and ref-
erence sites, 223

Differentiation. 41

Dimethylsulfoniopropionate, 1 1 18

Dimethylsulfoniopropionate in giant clams (Tridacnidae), 108

Disease, 199

Do the properties of underwater lighting inlluence the visually guided
behavior of Linnilu\'!. 178

DODGE, FREDERICK A., see James M. Hut, 171; Jillian L. Atherton, 176;
Matthew A. Krutky, 178

DOYE, V., see P. T. Tran, 205

Dreissenn, 116, 144

Drosopliila. 170

Early development of zooxanthella-containing eggs of the corals Pocillo-
pora verrucosa and P. eydtnixi with special reference to the distribu-
tion of zooxanthellae, 68

Echinodermata. 298

Echinoid. 1

EDDS-WALTON, PEGGY L., see Richard R. Fay. 173

EDMUNDS, PETER J.. see Steven V. Vollmer. 21

EDWARD. AHS[ R. see Richard W. Hill. 108

Effects of food concentration and availability on the incidence of cloning
in planktotrophic larvae of the sea star Pisaster ochraceus, 298

Effects of myosin-II antibody on actin-dependent vesicle transport in
extracts of clam oocytes, 202

KttC'.ts of nitrogen load and irradiance on photosynthetic pigment concen-
trations in Clutloptun'ii vagabunda and Grticiltiriu tikviihiti? in estu-
aries of Waquoit Bay. 223

Electron microscopy. 213

Embryo, 305

Embryogenesis, 29

Endocrine disruption, 316

Endoplasmic reticulum, 212, 213

Endoscopy, 1 16

ENGLER, JOHN, see Robert M. Gould, 2 1 5

Enzymatic biosynthesis of N-linked glycan by the marine sponge Micro-
fioiui prolifera, 192

Enzyme activity, 100

EPSTEIN. DAVID A.. HERMAN T. EPSTEIN, FRANK M. CHILD. AND ALAN M.
KUZIRIAN. Memory consolidation in Hermissenda crassicornis, 182

EPSTEIN. HERMAN T., see David A. Epstein, 182

ER transport, 202, 203

Escape response. 265

Eye. 171. 176

Eyeshine, 6

Factors influencing spawning and pairing in the scale worm Hannothoe

inihriiiiki, 50

FALK. CHUN X., see Matt Wachowiak, 162
FARMER, MARK A., see Timothy S. Wakefield, 76
Fate of anthropogenic nitrogen in a nearshore Cape Cod aquifer, 221
FAY, RICHARD R., AND PEGGY L. EDDS-WALTON, Frequency response of

auditory brainstem units in toadfish Opsamm tan. 1 73
FERNANDEZ-BUSQUETS. XAVIER, see William J. Kuhns. 192
Fertilization, 59. 144. 212, 213
Fertilization in Callochilon castaneus (Mollusca). 59
Fertilization-induced changes in the fine structure of stratified Arbacia

eggs. I. Observations on live cells with the centrifuge polarizing

microscope. 212
Fertilization-induced changes in the fine structure of stratified Arbacia

eggs. II. Observations with electron microscopy. 213
FINLEY, LUKE, AND DAVID MACMILLAN. The structure and growth of the

statocyst in the Australian crayfish Cherax destructor. 251
Fish, startle response. 180
FISHER, CHARLES R., see Stephane Hourdez, 95
FMRFamide, 305
Forskolin. 197

FORTI. L., see A. Abenavoli. 184
Fouling organism, 189
Freeze substitution, 76
Frequency response of auditory brainstem units in toadfish Opsannx tan.

173"

FREUND, CONCETTA M., see Robert M. Gould. 215
Frog Lim-1-like protein is expressed predominantly in the nervous tissue.

gonads, and early embryos of the bivalve mollusc Mytilus gallopro-

yiitciiili.i. 29

GALLO. MICHAEL A., see Diane E. Heck. 195

Gastropod. 241

Gastrulation, 287

GATEN. E.. see M. L. Johnson. 6

Gating current, 164

GALIDRON, S. M.. see G. J. Watson, 50

Gene, 231

activity, 100

expression, 215

INDEX TO VOLUME 199

323

Gene expression and enzyme activities of the sodium pump during sea
urchin development: implications for indices of physiological state,
100

Giant axon. squid. 135

GIBLIN. ANNE, see Adena Greenbaum. 223

GIBSON. DANIEL G., see Rhea Hanselmann. 199

GIBSON. GLENYS D.. AND JENNIFER M. L. HARVEY. Morphogenesis during
asexual reproduction in Pygospio elegans Claparede (Annelida.
Polychaeta). 41

GODA, MAKOTO. MARIO H. BURGOS, AND SHINYA INOUE. Fertilization-
induced changes in the fine structure of stratified Arbacia eggs. I.
Observations on live cells with the centrifuge polarizing microscope,
212

GODA. MAKOTO, see Mario H. Burgos. 213

Gonad, 29

GOULD. ROBERT M., CONCETTA M. FREUND, JOHN ENOLER, AND HILARY G.
MORRISON, Optimization of homogenization conditions used to isolate
niRNAs in processes of myelinating oligodendrocytes, 215

Gracilaria tikvahiae. 223

GREENBAUM, ADENA. AND ANNE GIBLIN. Differences in properties of salt
marsh sediment between hayed and reference sites. 223

Groundwater. 221

Growth. 251

HADFIELD. MICHAEL G.. see Esther M. Leise. 24 1

HALE. MELINA E.. Startle responses of fish without Mauthner neurons:

escape behavior of the lumpfish (Cyclopterus lumpus), ISO
HAMMAR. KATHERINE. see Sung-Kwong Jung. 197
HANSELMANN, RHEA. ROXANNA SMOLOWITZ. AND DANIEL G. GIBSON. Iden-
tification of proliferating cells in hard clams, 199
Hard clam. 199
Hannothoe imbricate, 50
HARRINGTON, JOHN M., AND PETER B. ARMSTRONG, Initial characterization

of a potential anti-fouling system in the American horseshoe crab,

Limiiliis polyphemus, 189

HARVEY. JENNIFER M. L., see Glenys D. Gibson. 41
Haying. 225
Hearing. 173
Heart rate. 257
Heavy water. 164
Heavy water (D,0) alters the sodium channel gating current in squid giant

axons. 164
HECK. DIANE E.. LYDIA Louis, MICHAEL A. GALLO, AND JEFFREY D.

LASKIN. Modulation of the development of plutei by nitric oxide in the

sea urchin Arbacia punctulata, 195
Hemocyte proliferation, 199
Hemoglobin. 95

functional properties. 95
structure. 95

Hemoglobin from a deep-sea hydrothermal-vent copepod. 95
Hemolysis. 194
Htrinissenda, 182

HERRING, P. J.. see M. L. Johnson, 6
HIDAKA, M., see M. Hirose, 68
HILL, RICHARD W.. JOHN W. H. DACEY. AND AHSER EDWARD, Dimethyl-

sulfoniopropionate in giant clams (Tridacnidae), 108
Hippocampus. 184
HIROSE. M.. R. A. KINZIE III, AND M. HIDAKA, Early development of

zooxanthella-containing eggs of the corals Pocillopora verrucosa and

P. evdouxi with special reference to the distribution of zooxanthellae.

68 "

HITT. JAMES M.. see Jillian L. Atherton, 176
HITT. JAMES M., FREDERICK A. DODGE. EHUD KAPLAN. AND ROBERT B.

BARLOW. Circadian rhythms in the receptive fields of the Limulus

lateral eye. 171

HODGSON. ALAN N.. see John Buckland-Nicks. 59
Hoimmis. 257. 265

HOPKINSON. CHARLES. JR., see Ryan Kirkby, 218: Anne Perring, 219
Horizontal cell. 168

HOURDEZ, STEPHANE, JASON LAMONTAGNE, PAT PETERSON, ROY E. WEBER.
AND CHARLES R. FISHER. Hemoglobin from a deep-sea hydrothermal-
vent copepod. 95

Human immunodeficiency virus. 209

Hydrogen ion fluxes from isolated retinal horizontal cells, modulation by
alutamate. 168

Identification of proliferating cells in hard clams. 199

Immunity, innate. 189. 190. 194

Imposex, 316

Individual, 231

Initial characterization of a potential anti-fouling system in the American
horseshoe crab, Limulus polyphemus, 189

INOUE. SHINYA. see P. T. Tran, 205; Makoto Goda. 212; Mario H. Burgos.
213

Interaction of actin- and microtubule-based motors in squid axoplasm
probed with antibodies to myosin V and kinesis. 203

Intercellular communication. 165

Intrinsic membrane properties of laryngeal motoneurons that control sex-
ually differentiated vocal behavior in African clawed frogs, Xenopus
leavis, 175

Ipswich. 218

Irradiance. 223

Jelly coat. 1

JOHNSON. J. L., P. M. J. SHELTON, E. GATEN. AND P. J. HERRING. Relation-
ship of dorsoventral eyeshine distributions to habitat depth and animal
size in mesopelagic decapods. 6

JUNG, SUNG-KWON, KATHERINE HAMMAR, AND PETER J. S. SMITH. Devel-
opment of self-referencing oxygen microsensor and its application to
single pancreatic HIT cells; effects of adenylate cyclase activator
forskolin on oxygen consumption, 197

JURY, STEVEN H., AND WINSOR H. WATSON III. Thermosensitivity of the
lobster, Homarus americamis, as determined by cardiac assay. 257

KACZMAREK, LEONARD K.. see Ayako Yamaguchi. 175

KAPLAN, EHUD, see James M. Hitt, 1 7 1

KASS-SIMON. G.. see S. I. Cromarty. 265

KELLEY. DARCY B.. see Ayako Yamaguchi. 175

KEMPF, STEPHEN C., see Timothy S. Wakefield. 76

Kidney function. 14

Kidney function and sulfate uptake and loss in the freshwater bivalve
Toxolasma te.wsensis. 14

Kinesin, 203

KINZIE, R. A.. Ill, see M. Hirose. 68

KIRKBY. RYAN, Luc CLAESSENS. CHARLES HOPKINSON, JR., EDWARD RAS-
TETTER, AND JOSEPH VALLINO, Modeling the effects of land-use
changes on nitrogen biogeochemistry in the Ipswich watershed, Mas-
sachusetts. 218

KOMINAMI, TETSUYA. AND HIROMI TAKATA, Cellular basis of gastrulation in
the sand dollar Scaphechinus mirabilis, 287

KOZLOWSKI, ROBBIN. see Hemant M. Chikarmane, 227

KROEGER, KEVIN D., see Elizabeth J. Westgate. 221

KRUTKY, MATTHEW A.. JILLIAN L. ATHERTON. SPENCE SMITH. FREDERICK A.
DODGE. AND ROBERT B. BARLOW. Do the properties of underwater
lighting influence the visually guided behavior of Limulus?, 178

KRUTKY. MATTHEW A., see Jillian L. Atherton. 176

KUHNS. WILLIAM J., MAX M. BURGER. MOHAN SARKER, XAVIER FERNAN-
DEZ-BuSQUETS, AND TRACY SIMPSON. Enzymatic biosynthesis of N-
linked glycan by the marine sponge Microciona prolifera. 1 92

KUHNS, WILLIAM J.. see Roger MacKenzie, 209

KUZIRIAN. ALAN M.. see David A. Epstein. 182; Hemant M. Chikarmane.
227

KUZIRIAN, MARK, see Hemant M. Chikarmane. 227

324

INDEX TO VOLUME 199

Labynnthomorpha, 200

LAJEUNESSE. R. C, AND R. K. TRENCH, Biogeography of two species of

Symbiodinium (Freudenthal) inhabiting the intertidal sea anemone

Anthopleura elegantissima (Brandt), 126
LAMONTAONE, JASON, see Stephane Hourdez, 95
LANDOWNE, DAVID, Heavy water (D2O) alters the sodium channel gating

current in squid giant axons, 164
LANOFORD, F. M., see G. J. Watson. 50

LANOFORD, GEORGE M.. see Leslie Sandberg, 202; Phillip Stafford, 203
LASKIN, JEFFREY D., see Diane E. Heck, 195
Learning, human, 182

LEE, TONY, see Hemant M. Chikarmane. 227
LEISE, ESTHER M., AND MICHAEL G. HADFIELD, An inducer of molluscan

metamorphosis transforms activity patterns in a larval nervous system,

241

LEONARD, CELERE N., see Toby F. Bolton. 1
LEONG, PATRICK K. K., see Adam G. Marsh, 100
LEVINTON, JEFFREY S., see Shirley M. Baker, 1 16
Lim-1 protein, 29
Limulus, 171, 176. 178. 189, 190
Lobster, 257, 265
Loligo, 135

Lophius americanus, 227
Louis, LYDIA, see Diane E. Heck, 195
LYNN, J. W., see M. J. Misamore, 144

MACKENZIE, ROGER, DAVID NEWMAN, MAX M. BURGER, RENE ROY. AND
WILLIAM J. KUHNS, Adhesion of a viral envelope protein to a non-self-
binding domain of the aggregation factor in the marine sponge Mi-
crociona prolifera, 209
MACMILLAN, DAVID, see Luke Finley, 251
Macroalgae, 223
a:macroglobulin, 190

MALCHOW, ROBERT PAUL, see Anthony J. A. Molina, 168
MALGAROLI, A., see A. Abenavoli, 184
MANAHAN, DONAL T., see Adam G. Marsh, 100
Marine Biological Laboratory

Annual Report, v. 199 (1), Rl

General Scientific Meetings, Short Reports. 157
Marine invertebrate reproduction. 1
MARKS, ERNEST, III, see Christine Brothers, 200

MARSH, ADAM G., PATRICK K. K. LEONG, AND DONAL T. MANAHAN, Gene

expression and enzyme activities of the sodium pump during sea

urchin development: implications for indices of physiological state.

100

Maternal energy investment in eggs and jelly coats surrounding eggs of the

echinoid Arbacia punclulahi. 1
Maternal investment, 1
Mauthner neuron, 180
MBL

Annual Report, v. 199 (1), R I

General Scientific Meetings, Short Reports, 157
McCLiNTOCK, JAMES B., see Minako S. Vickery, 298
Mechanisms of spontaneous miniature activity at CA3-CA1 synapses:

evidence for a divergence from a random Poisson process, 184
MELLO, J., see S. I. Cromarty, 265
Memory

blocking, 182

long-term, 182

short-term, 182

consolidation, 182

Memory consolidation in Hermissenda crassicomis, 182
MEPSC, 184
Mesopelagic ecology, 6
Microciona, 192, 209
Microsporidian, 208
Microtubule. 205, 207

Microtubule-dependent nuclear positioning and nuclear-dependent septum

positioning in the fission yeast, S. pombe, 205
MIKHAILOV, ALEXANDER T., see Mario Torrado. 29
MISAMORE, M. J., AND J. W. LYNN, Role of the cytoskeleton in sperm entry

during fertilization in the freshwater bivalve Dreissena polymorpha,

144
Modeling the effects of land-use changes on nitrogen biogeochemistry in

the Ipswich watershed, Massachusetts, 218
Modular organisms, 21
Modulation of the development of plutei by nitric oxide in the sea urchin

Arbacia punclulala, 195
MOLINA, ANTHONY J. A., PETER J. S. SMITH, AND ROBERT PAUL MALCHOW.

Hydrogen ion fluxes from isolated retinal horizontal cells, modulation

by glutamate, 168
Mollusc, 59, 194, 241
Molt cycle. 265
Molt-related and size-dependent differences in the escape response and

post-threat behavior of the American lobster, Homarus americanus,

265

Monkfish, 227
Morphogenesis, 287
Morphogenesis during asexual reproduction in Pygospio elegans Clapa-

rede (Annelida, Polychaeta), 41
Morphology, 251

MORRISON, HILARY G., see Robert M. Gould, 215
Mosaic, 231
Motoneuron, 175
mRNA transport. 215

MUSCATINE, LEONARD, see Garen Baghdasarian, 278
Myelination, 215
Myosin, 202. 203

Nervous ganglion, 29

NEWMAN. DAVID, see Roger MacKenzie, 209

Nitrate, 221

Nitric oxide. 195

Nitrogen, 223

processing, 218
NOVALES FLAMARIQUE, INIGO, KRISTIINA OVASKA, AND THEODORE M.

DAVIS. UV-B-induced damage to the skin and ocular system of

amphibians, 187
Nuclear positioning, 205
Nudibranch. 241

Odor recognition, 162

Odor-induced oscillatory activity in Drosophila CNS. 1 70

Olfaction, 170

Olfactory bulb, input to, 162

Oocyte, 202

Opisthobranch. 241

Optic nerve, 1 76

Optic nerve responses of Limulus in its natural habitat at night, 1 76

Optimization of homogenization conditions used to isolate mRNAs in

processes of myelinating oligodendrocytes, 215
Organotin, 316
Oscillation, 170

OVASKA, KRISTIINA, see Inigo Novales Flamanque, 187
Overgrowth competition, 85
Overgrowth competition between clades: implications for interpretation of

the fossil record and overgrowth indices, 85
Oxygen

consumption, 197

microsensor. 197

PABLICH, WENDY J., see Elizabeth J. Westgate
PAH clearance. 14

INDEX TO VOLUME 199

325

Pancreatic HIT cell. 197

Particle selection, 1 Id

Particle transport in the zebra mussel, Dreissena polymorpha (Pallas). I Id

Passive tracer, 221

PCNA. 199

PEG clearance, 14

PERRING, ANNE, MICHAEL WILLIAMS, CHARLES HOPKINSON, JR.. EDWARD

RASTETTER. AND JOSEPH VALLINO, Solute dynamics in storm flow of

the Ipswich River Basin: effects of land use, 219
PETERSON, PAT, see Stephane Hourdez. 95
pH. 200
Pheromone, 50
Phycoerythrin, 223
Pigment. 223
Pigment cell, 195
Planula, 68
Plume, 221
Polychaete, 41, 50
Polyplacophora, 59
Pomticeii ctiniilicitUihi, 316
Population, 227

Population genetic structure of the goosefish, Lophius americanus, 227
Preferential expulsion of dividing algal cells as a mechanism for regulating

algal-cnidarian symbiosis. 278
Protease inhibitor, 190
Protein synthesis, 215
Proteoglycans, 192, 209

QPX. 199, 200

Quanta, spontaneous release. 184

RAPD. 227

RASTETTER. EDWARD, see Ryan Kirkby, 218; Anne Perring, 219

Regeneration. 41

Relationship of dorsoventral eyeshine distributions to habitat depth and
animal size in mesopelagic decapods, 6

Renal clearance. 14

Reproduction, 50

Retina, 165, 168

Revised description of the fine structure of in situ "Zooxanthellae" genus
Symbiodinium, 76

RINKEVICH, B., A critical approach to the definition of Darwinian units of
selection, 231

RIPPS, HARRIS, see Thomas H. White. 165

The role of microtubules during blastodisc formation of the squid Lo/igo
pealei, 207

Role of the cytoskeleton in sperm entry during fertilization in the fresh-
water bivalve Dreissena polvmorpha, 144

ROSENTHAL, JOSHUA J. C., AND FRANCISCO BEZANILLA. Seasonal variation
in conduction velocity of action potentials in squid giant axon, 135

ROY. RENE, see Roger MacKenzie, 209

Salinity, 200

Salt marsh, 225

SANDBERG. LESLIE, PHILLIP STAFFORD, AND GEORGE M. LANGFORD, Effects

of myosin-II antibody on actin-dependent vesicle transport in extracts

of clam oocytes, 202

SARKER, MOHAN, see William J. Kuhns, 192
Scaling, 21

Sea star larval cloning, 298
Sea urchin, 195, 287
development, 100
Seasonal variation in conduction velocity of action potentials in squid giant

axon. 135
Segmentation, 41

Self-referencing, 197

Septic, 221

Septum positioning, 205

Serotonin, 305

Sex steroid hormone. 316

Sexual differences. 175

SHELTON. P. M. J.. see M. L. Johnson. 6

SILVERMAN, H., see T. H. Dietz, 14

SIMPSON. TRACY, see William J. Kuhns, 192

Size-dependent behavior, 265

Skate. 168

SMITH. PETEK J. S.. see Anthony J. A. Molina, 168; Sung-Kwong Jung, 197

SMITH, SPENCE, see Matthew A. Krutky, 178

SMOLOWITZ, ROXANNA, see Rhea Hanselmann. 199; Christine Brothers, 200

SO4 clearance. 14

Sodium
channel, 164
pump, 100

Solute dynamics, 219

Solute dynamics in storm flow of the Ipswich River Basin: effects of land
use, 219

The spatial representation of odors by olfactory receptor neuron input to
the olfactory bulb is concentration invariant, 162

Spawning, 50

Species diversity, 126

Sponge. 192, 209

Sporoplasm, 208

Squid. 135, 164. 207

SRINIVAS, MIDUTURU, see Thomas H. White. 165

STAFFORD, PHILLIP. JEREMIAN BROWN, AND GEORGE M. LANGFORD, Inter-
action of actin- and microtubule-based motors in squid axoplasm
probed with antibodies to myosin V and kinesis, 203

STAFFORD, PHILLIP, see Leslie Sandberg, 202

Startle responses of fish without Mauthner neurons: escape behavior of the
lumpfish (Cyclopterus lum/nts), ISO

Statocyst, 251

Stem cell, 231

STIEVE, ERICA, see Michelle Denault, 223

The structure and growth of the statocyst in the Australian crayfish Cherax
destructor, 251

Sulfate transport. 14

Suppression subtractive hybridization. 215

Surface motor proteins, 192

Symbiodinium, 76, 108, 126

Symbiosis, 76, 126. 278

Symbiotic dinoflagellate, 278

Synapse, 184

Synchrony, 170

Tail flip, 265

TAKATA, HIROMI, see Tetsuya Kominami, 287

TAKEDA, NAOKUNI, Development of a penis from the vestigial penis in the

female apple snail, Pomacea canaliculate, 316
Temperature. 200, 257

acclimation, 135
Thermosensitivity. 257
Thermosensitivity of the lobster. Homarus americanus, as determined by

cardiac assay, 257

THOMAS, FLORENCE I. M., see Toby F. Bolton, 1
TORRADO, MARIO. AND ALEXANDER T. MIKHAILOV, Frog Lim-1-like protein

is expressed predominantly in the nervous tissue, gonads, and early

embryos of the bivalve mollusc Mytilus ga/loprovincialis, 29
Torus semicircularis, 173
TRAN, P. T., V. DOYE, F. CHANG, AND S. INOUE, Microtubule-dependent

nuclear positioning and nuclear-dependent septum positioning in the

fission yeast, S. pombe, 205
Transitivity index, 85
TRENCH, R. K., see R. C. LaJeunesse, 126
Tridacnidae, 108

326

INDEX TO VOLUME 199

UDOETOK. A. S., see T. H. Diet/., 14

infrastructure. 76

Unionid, 14

Unit of selection, 23 1

UV-B radiation. 187

UV-B-induced damage to the skin and ocular system of amphibians. 187

VALIELA, IVAN, see Elizabeth J. Westgate. 221; Michelle Denault, 223

VALLINO, JOSEPH, see Ryan Kirkby. 218; Anne Perring. 219

Veliger. 241, 305

Vesicle transport, 202, 203

Vestigial penis, 316

VICKERY, MINAKO S., AND JAMES B. McCuNTOCK, Effects of food con-
centration and availability on the incidence of cloning in planktotro-
phic larvae of the sea star Pisasrer ochraceus, 298

Viral envelope, 209

Vision. 6, 171, 176, 178

Vocal behavior, 175

VOLLMER, STEVEN V., AND PETER J. EDMUNDS, Allometric scaling in small
colonies of the scleractinian coral Siderastrea siderea (Ellis and
Solander), 21

Voltage gating properties of channels formed by a skate retinal connexin,
165

VORONEZHSKAYA, ELENA E., see Amanda J. G. Dickinson, 305

Revised description of the fine structure of in situ "Zooxanthellae"

genus Symbiodinium, 76

WANG, JIN W., Odor-induced oscillatory activity in Drosophila CNS, 170
WARD. J. EVAN, see Shirley M. Baker, 1 16
Wastewater, 221
Watershed modeling. 218
WATSON, G. J., F. M. LANOFORD. S. M. GUADRON, AND M. G. BENTLEY,

Factors influencing spawning and pairing in the scale worm Har-

mothoe imbricata, 50

WATSON, WINSOR H., Ill, see Steven H. Jury. 257
WEBER. ROY E.. see Stephane Hourdez, 95
WEIDNER. EARL, Cytoplasmic proteins on the surface of discharged mi-

crospondian sporoplasms. 208
WESTGATE, ELIZABETH J.. DEVIN D. KROEGER, WENDY J. PALICH, AND IVAN

VALIELA, Fate of anthropogenic nitrogen in a nearshore Cape Cod

aquifer, 221
WHITE, THOMAS H., HARRIS RIPPS, MIDUTURLI SRINIVAS. AND ROBERTO

BRUZZONE, Voltage gating properties of channels formed by a skate

retinal connexin, 165
WILLIAMS, MICHAEL, see Anne Perring, 219

YAMAGUCHI. AYAKO, LEONARD K. KACZMAREK. AND DARCY B. K.ELLEY,
Intrinsic membrane properties of laryngeal motoneurons that control
sexually differentiated vocal behavior in African clawed frogs. Xeno-
pus leavis. 175

WACHOWIAK, MATT. MICHAL ZOCHOWSKI, LAWRENCE B. COHEN, AND CHUN
X. FALK, The spatial representation of odors by olfactory receptor
neuron input to the olfactory bulb is concentration invariant. 162

WAKEFIELD, TIMOTHY S., MARK A. FARMER, AND STEPHEN C. KEMPF,

Zebra mussel, 116. 144

ZOCHOWSKI. MICHAL. see Matt Wachowiak, 162

Zooxanthella, 68, 76, 108, 126, 278

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