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Number I 

GT/^i A ih 

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. 


The new standard 
in laboratory 
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. 

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 
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 matcnm s 
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 

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 


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. 



Visit us at 
or call 1-800-455-8236 

Olympus America Inc. 



f *VOOnc w~i . ^ u a 'CSl i ah 

AUGUST 2000 

Associate Editors 

Section Editor 
Online Editors 

Editorial Board 

Editorial Office 




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 





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

Marine Biological Laboratory 

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Louisiana State University 

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Cereon Genomics, Cambridge, Massachusetts 

Hiroshima University of Economics, Japan 

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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 

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Published by 



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. 


VOLUME 199. No. 1: AUGUST 2000 


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

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


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

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

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 Txt>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) .... 


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 


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

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


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 


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 


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(T 4 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 mm 3 ). 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 mm 3 ). 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 
mm 3 , and the volume of the egg alone was 1.71 0.19 X 
1()~ 4 mm 3 (Table 2). The volume of the jelly coat alone was 
0.96 0.48 X 1() 4 mm 3 (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 mm 3 (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) 






3.15 (0.13) 

























3.05 (0.44) 


10. N 



2.47 (0.46) 





3.11 (0.07) 









3.97 (0.79) 




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%). 


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 



Egg and jelly coat 

Jelly coal 

Egg and jelly coat 







10.61 (3.08) 









.73 (0.79) 

2.61 (0.35) 

















.73 (0.79) 




1 1.04 


.73 (0.79) 


















.49 (0.52) 

2.56(061 ) 





.71 (0.14) 

2.67 (0.30) 





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 


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 





jelly coat (^m) 


Strongylocentrotus purpuraius 





1 .50 

Strongylocentrotus franciscanus 






Strongylocentrotus droebachiensis 





1.61 (0.16) 

A rhticia punctulata 






Lytechinus variegalus 






DenJra.iter excentricns 






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. 


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 


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. 


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: 

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- 

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 


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 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 

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. 


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 


<i - 


Day Depth (m) 



T ; 

T* l 

I ; 

T T 


Depth (m) 




T x ; 











1 1 







O 1 

1 1 


o 1 -1- 




J- o 



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 <//.. 


Table 1 

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




Eyeshine intensity by size class 

(carapace length. 


0-5 5-10 



Acanthephyra gracilipes 



84.0 5.5 

85.8 7.3 

Acanthephyra pelagica 




68.7 17.5 

104.5 7.8 


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 




31.1 1.1 


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 


51.3 3.2 

33.5 0.8 


94.8 9.1 

Sergia robustus 



31.4 0.2 


35.5 0.5 


Syste/laspis cristata 









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 


a) Non-migrants b) Migrants 


S 100- 


? 75 



= 50- 

~* Acanthephyra pelagica 

"* Acanthephyra stvloroslratis 

50 100 150 200 
Degrees from dorsal 

Parapandalus richardi 


125 - 


* Plesionika martia 






25 - 

*^i^_ r *^J- -L 


D - 

50 100 150 200 
Degrees from dorsal 



c ) Oplophorus spinosits 

Cl = 4 mm 
Cl = 16mm 

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) Systellaspis debilis 



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). 



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 log lo depth and log lo 
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). 


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 

5 1.8- 


.1 1.7- 

1 1.6- 



b) Night depth v. Dorsal eyeshine intensity 


1.x - 
1 .6 - 

Depth (m) 

Depth (m) 



c) Day depth v. Ventral eyeshine intensity 


2.5 3 3.5 

Depth (m) 



d ) Night depth v. Ventral eyeshine intensity 

I 9- 




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. 




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- 


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 

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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 


^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 SO 4 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 35 SO 4 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 SO 4 reabsorbed was 
significantly reduced relative to mussels acclimated to PW. 
T. texasensis conserved SO 4 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 

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

* To whom correspondence should he addressed. E-mail: 

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


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 




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 SO 4 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 2C. The pondwater 
composition (in millimoles per liter) was 0.5 NaCl. 0.4 
CaCl 2 , 0.2 NaHCO,. 0.2 MgSO 4 , 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 MgSO 4 , 24.4 MgCU, 9.9 CaCl 2 , 
6.6 KC1. 2.4 KHCO,, 0.8 KBr, 0.4 H,BO 3 , "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 La 2 O_,/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 SO 4 was precipitated as BaSO 4 crystals 
by the addition of 100 ju.1 of 1 mol 1~' BaCl 2 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^SO 4 . 

Sulfate uptake 

Sulfate uptake was measured by the appearance of the 
radiolabeled SO 4 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~' SO 4 ), 
and trace amounts of 35 SO 4 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 (90C) to 
determine dry mass. The amount of 35 SO 4 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, SO 4 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 [ 3 H-polyethylene glycol (PEG, 4 
kDa), and Na 2 35 SO 4 ] 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 
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 90C 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 



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 35 SO 4 in the 
blood, the quantity of 35 SO 4 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 35 SO 4 
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 




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 

SO 4 . mmol 1 ' ' 

1.8 0.2 

1.7 0.1 

^SO 4 , 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). 


Solute balance and SO 4 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, SO 4 is concentrated to the same level as 
Cl (Table 1). 

The SO 4 influx is slow in unionids (Dietz, 1978); thus we 
did not measure the unidirectional influx of SO 4 by the 
disappearance of isotope from the bathing medium. Less 
than 1000 dpm ml"' of 35 SO 4 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 1S SO 4 that 
accumulated in the blood over the incubation interval was 
small, but significantly greater than zero. The uptake of 
' S SO 4 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). 



Table 2 

Volume of blood cleared of polyethylene gl\col (PEG] and " SO 4 , 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 


Clearance, ml g ' dry tissue h 

Sulfate, jiimol g ' dry tissue h 






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 


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 35 SO 4 administered simultaneously (0.09 
0.03 ml g" 1 dry tissue h~') was 12% of the clearance of 
3 H-PEG, suggesting that filtered SO 4 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 35 SO 4 and 3 H-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 "SO 4 and ^H-PEG. 
The SO 4 clearance in 10% SW-acclimated animals ap- 
peared to be unchanged relative to the PW-acclimated mus- 
sels. However, because the clearances of SO 4 and PEG were 
similar, these data suggest that 10% SW-acclimated animals 
had reduced their reabsorption of SO 4 (Table 2). The SO 4 
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~' SO 4 ). 
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 


mosm kg ' 

Ion concentration, mmol I ' 

Total solute 






S0 4 

10% SW 

110 If 

45.1 0.7e 









2.8 0.2c 

1 h PW 

102 2e 

41.0 0.7d 








I. Id 

2.1 0.4ab 


79 Id 

29.6 0.7c 








2.4 0.1 he 

8 h PW 

67 Ic 

27.7 0.5c 








1.4 0.2a 

24 h PW 

55 Ib 

20.1 0.7b 









1.4 0.2a 

48 h PW 

45 3a 








1.4 0.2a 

72 h PW 

46 la 

16.6 0.2a 









2.1 O.labc 


44 2a 

19.1 0.9b 









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). 



PW. PEG and "SO 4 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 35 SO 4 clearance remained elevated for 
8 h and then returned to PW control levels by 24 h. The 
elevation in SO 4 excretion was due to a significant increase 
in filtration. During the first hour after transfer to PW, the 
SO 4 clearance remained statistically the same as the PEG 
clearance. By 4 h, the SO 4 clearance was significantly less 
(P < 0.05) than the corresponding PEG clearance. The 
reduction in SO 4 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. 


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 ' SO 4 . 

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 SO 4 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 
SO 4 in the blood increased to about 2.8 mmol 1~ ' (equal to 
the bathing medium). These data contrast with the some- 
what more rapid SO 4 transport rates observed in Dreissena 
polymorphtt, and with the apparent cessation of SO 4 reab- 
sorption in that species when acclimated to 10% SW (Dietz 
and Byrne. 1999). The low blood SO 4 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 

In the sulfate-rich 10% SW environment. T. texasensis 
became isosmotic and isoionic for SO 4 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- 



vated filtration, it was 24 h before SO 4 clearance returned to 
PW control levels, even though SO 4 reabsorption was im- 
mediately reestablished. D. polymorpha also required about 
24 h to reestablish SO 4 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~' SO 4 , using an anion exchange mech- 
anism (Larsen and Simonsen, 1988). The anion transport 
inhibitor. DIDS. significantly decreased the amount of 
SO 4 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 

Sulfate reabsorption is subject to regulatory mechanisms 
in freshwater bivalves. When mussels were acclimated to 
10<7r SW, the concentration of SO 4 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 SO 4 environment, but trans- 
port systems and conservation mechanisms both allow them 
to maintain a SO 4 concentration almost 10 times higher than 
that in the medium. 


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

Allometric Scaling in Small Colonies of 

the Scleractinian Coral Siderastrea siderea 

(Ellis and Solander) 


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. 

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. 


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- 




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 


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 



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 (26C) 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 




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 (r 2 > 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% 
HNO 3 . and drying the resulting tissue tunic at 60C 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 













In Dry Tissue (mg) 



c -2 

*4 I 


'a, -4 

a -6- 

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 
(// : b = : 1), where a significant deviation (P < 0.05) 
indicates an allometric relationship. 



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 







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 



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 


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 



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 

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



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- 


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 

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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 


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 

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. 


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, 

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 




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 
37C. Different brain regions (forebrain. optic lobes, and 
cerebellum) were microsurgically isolated in cold minimum 
essential medium (MEM; Gibco) and pelleted by low-speed 

Tissue dissection and processing 

Before use, adult mussels (M. galloprovincialis) were 
kept in a dry state for 1 h at 4C. 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 -85C for several days. 



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. 8C, 30 min) were mixed with 
SDS sample buffer containing the protease inhibitor cock- 
tail, kept for 1 h at room temperature, and stored at 30C 
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. 2C. 30 mm), the super- 
natants were discarded, and the pellets were then re-ex- 
tracted and assayed as described for embryos. 


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 20C 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 and O-glycosidase (EC 
were first added to the reaction vials; after the incubation 
and denaturation step, Af-glycosidase F (EC was 
added to the mixture. To determine deglycosylation effi- 
ciently, samples (before and after deglycosylation) were 



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- 

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 10C. 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 10C. 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. 


Mussel pedal ganglia were fixed in 100 mM 3-(/V-mor- 
pholino) propane sulfonic acid (MOPS; Sigma). 2 mM 
MgSO 4 (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 4C) 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 4C. 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 


Xenopus laevis Lim-1 



C-terminal region 










pITM^H pHc D S P S P E P 

H S M sEBlv F G 





Y0N H L S H P P 




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 
.\Vi'/'ii.s Lim-3. 

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

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.. 



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








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 

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 



LMW 1 2 34 









1 2 3 

awMMM** *8M* 








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). 



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 M m; D and E 20 /urn). 

putative function of Lim-1 HD proteins as transcription 

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 



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 

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 



,->;-* . F '< F &4 




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. 





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- 



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- 








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. 



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. 


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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Reference: Biol. Bull 199: 41-49. (Auaust 2000) 

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

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. 


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. 

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 (20C). 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). 




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 20C 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.). 


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 



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- 

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- 


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 




Eyes (no. pairs) 





* #* 




Nuchal organ 

# *# #* 





# ** ** 





* ** ** 






Setigers (no.) 

3-6 8-12 10-12 




Notopodial lobe 










Ciliary tuft 





Neuropodial lobe 





Neuropodial capillary chaetae 




Neuropodial hooded hooks 




Posterior blastema 




Setigers (no.) 

2-3 3-6 3-6 




Notopodial lobe 










Ciliary tuft 





Neuropodial lobe 





Neuropodial hooded hooks 







* #* *# 




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 



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. 



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 <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). 


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- 

Table 2 


Asexual reproduction in Pygospio elegans 




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 = 


n = 15 

it = 15 

/; = 10 


No. Weeks between divisions 

3.6 0.5 

1.3 0.3 

1 .2 0. 1 

F<2.2, = 26, P = 


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 = 


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 


i, = 17 

I! = 1 1 

n = 26 

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

% Mortality 

Anterior fragment 





Middle fragment 





Posterior fragment 





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 

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., 

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 



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, 
20C 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 4-5C. 

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. 


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) 


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 10C 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. 


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 




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 
(5620'N, 247'W), Kingsbarns (5618'N, 238'W) and 
Fife Ness (5616'N, 235'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 10C 
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 

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 10C. 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 LD8:16 PM(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 LD8:16 PM(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 



recipients (LD8:16 PM|LDI ' and LDS:16 PM(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 LD8:16 PM(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 LD8:16 PM(LD168) 
treatment group spawned, with 10 individuals spawning on 






90 - 



5=? n=5 

LD16:8 control 
LD8:16 control 

LD8:U/ N """""' 


PM (U>X- Ih) 


zL?" 3 



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


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:I6 PM|LD " >I ", females maintained in 
I ' 16 each implanted with one prostomium from un LDI6:H control female; LD8:16 PM|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 10C 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. 








1 00 -, 






LD16:8 control 
o LD8: 16 control 
._LD8: ,""">"' 
._ LD8:16 <'"" 
o LD8: 16 donor 
x LD16:8 donor 




c3 a rt 
2! 2 S 


Figure 2. Cumulative percentage of spawning female Harmothoe imhricata after exposure to various 
treatments at 10C. LD16:8 control, females maintained in long-day photoperiod (LD16:8); LD8:16 control, 
females maintained in short-day photoperiod (LD8:16): LD8:16 PM|LD168 '. females maintained in LD8:lft and 
each implanted with one prostomium from an LD16:8 control female; LD8:16 PM|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: 
16 PM|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 LD8:16 PM " 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 10C 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. 



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 

Place test male at start position (*) of maze 


Allow 30 minutes before recording position 


( + ) 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 IOC. 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. 



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 
(X 2 (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 (x 2 (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 


Figure 5. Percentage response of new (collected April 1999) male 
Harmothoe imhricata to various stimuli in a Y-maze run at 10C. 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 '- 

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). 

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 



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: 16 PM(I m " Nl females spawned, as opposed to 37.5% of 


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

" females. These results confirm that the 



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 

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 



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. 


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) 


l St. 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 

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. 


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


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 







(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 

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. 



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 


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 



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 

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 




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 

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. 




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 


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- 

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. 



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 

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 

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- 

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. 


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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Reference: Bwl. Bull. 199: 68-75. (August 2000) 

Early Development of Zooxanthella-Containing Eggs 

of the Corals Pocillopora verrucosa and P. eydouxi 

with Special Reference to the Distribution 

of Zooxanthellae 


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 


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 




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 
28-30C. 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. 


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 



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- 




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. 



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- 



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. 


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 



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. 


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 


^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. 


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- 

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- 




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- 

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 mm 2 ) 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 mm 2 ) 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% OsO 4 in 1.25% NaHCO, 
for 1 h. Residual OsO 4 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. 



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). 


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 

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). 


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. 


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 

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. 



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). 


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) 



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 

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 



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; 


KXttWWX^ftiWM^** cwl 





- 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. 

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- 



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 


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 # 

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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 


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 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: 

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. 


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 




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. 



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 


Tidal cover/depth 




Upper midlittoral 
Lower midlittoral 


0.75 (0.09) 

4.88 (0.49) 
6.81 (0.49) 

Upper infralittoral 
Lower infralittoral 

4.25 (0.44) 

0.73 (0.30) 


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 = (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. 


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 


= Upper infra - 

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. 


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-: 






254321 121011 


1 3 







1 7 

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



27 31 








19 25 

11 211 1 


1 2 






1 5 

Tegella aquilostris 8191 1 471267 



16 19 








16 24 

251 1 3 


4 5 







Myrio-oclla plana 3 3 7 14 5 604 


34 43 



1 1 

1 1 




8 41 




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 







Tegella urtica 


13 19 

1 1 1 










20 21 
1 16 

Lichenopora sp. 

5 17 







5 21 








11 IS 

Other Cheilostomatids 








17 94 






6 34 

Microporella sp. 






27 89 




3 10 







9 14 


24 40 





KM) 73 


1 34 


tnidium sp. 


6 39 



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 


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 


Win-tie scored 





Tegella armifera 








Tegella aquilirostris 








Myriozoella plana 








Cauloramphus sp. 












Microporella californica 






L, d 



i r 

Tegella arctica 








Lichenopora spp. 
















Microporella sp. 








Crihrilina awutlata 








Porella alba 








Aleyonidium spp. 








Cylindroporella tubulosa 








Celleporella spp. 








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 




Tegella aqid/ostris 




Tegella armifera 




Myrio-oel/a plana 








Caidoramphis spp. 




Tegella arcticu 









Cribrilina anituUitu 




Microporella spp. 




Porella alba 



O.I I 

Cel/epore/la spp. 




Callipora craticn/u 





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. 




vs. Ctenostomatids* 

vs. Ctenostomatids* 



Win index Ranking 

Win index 


Win index 

Alaska (USA)" 

57 N 



0.42 (0.25*) 


0.21 (0.18*) 


56 N 




Ireland"" ' 

51.5 N 




Ireland"" 7 2 t 











43 N 




18 N 





12 S 





38 S 





47 S 




Falkland Is 

52 S 




South Georgia'!' 

54 S 



0.18 (0.08*) 


0.6 (0.32*) 

Signy 1st 

60.5 S 


~> ">/">"> 


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). 


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 to 
0.42. The win indices of living cheilostomatids varied be- 


2 05 - 

g 03 - 

-20 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. 



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- 

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) 







9 Fossil assemblages 
Living assemblages 

.. :? 





-100 -80 -60 -40 -20 

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 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). 



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- 

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. 


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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Woods Hole 


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 


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 

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' 

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 

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 

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 

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 
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. 


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 

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. 


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 

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 


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 

April 7, 2000 




As of December 31. 1999 

(with comparative totals as of December 31, 1998) 


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 


$ 1.942,285 




I | .944.564 






$ 1.187,954 
3.56 1. 544 











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) 






5.0 C )2.39S 








Net assets: 
Temporarily restricted 
Permanently restricted 

Total net assets (Note 2] 

Total liabilities and net assets 



1 8.45 1 ,865 


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




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 

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

Total operating support and revenues 


Conferences and services 
Other programs (Note 2) 

Total expenses 

Change in net assets before nonoperating activity 

Nonoperating revenue: 
Contributions to plant: 
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 




Restricted Restricted 



SI 2.248.442 

$ $ 
















4.604.501 2.234.375 



705,85 1 







(22.680) (7,853) 






2,230,652 2.226,522 



















2.230.652 2,226,522 



1,507,319 250,000 







1.1 10.489 

595.273 250,000 




6.116.443 (398.339) 








4,888.082 (398.339) 




7,714.007 2.078,183 




25.635.237 17,274.650 




$33.349.244 $19.352.833 



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




for the year ended December 31. 1999 

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


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: 

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 







25 1 .390 







(4,529. 232 1 




754.33 1 

$ 1.942,285 


$ 678,1 





















$ 1,187.954 

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


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 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. 


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 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. 


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: 


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 


$ 40.000 
69, 1 25 
1 ,600,300 



$ 40,000 



$ 40.000 

$ 40,00(1 

$48, 1 84.030 




31, 1999 and 1998 are as follows: 




$ 40,000 


$ 40,000 
33, 1 1 9 


$ 40,000 

$ 40.000 


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

Total investments 
Lang-Term Investments 










1 ,448,569 


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

Total investments 










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: 

Temporarily restricted 
Permanently restricted 
Advances on contracts 








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 


$ 225.51 

$ 57.86 


$ 220.30 


$ 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 


$ 890.1 

1 , 1 75,000 



$ 925,000 




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 

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 : 

Construction in process 

Less: Accumulated depreciation 

Plant assets, net 


$ 702,908 







$ 702,908 






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 












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





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 


$ (26.654) 








$ 2.171.1 19 

S( 1.350.474) 

$ (2h.h54i 







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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Report of the Library 

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 

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 

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 


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 

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. 


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) 


Pearce, Edward. Cornell University 

Tsehudi, Christian. Yale University School of Medicine 


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 


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 


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 


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 


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) 


Bronner-Fraser. Marianne, California Institute of Technology 
Fraser, Scott, California Institute of Technology 


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 


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) 


Leadbetter, Edward, University of Connecticut 
Salyers, Abigail, University of Illinois, Urbana 


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 


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 


Aislabie, Jacqueline. Landcare Research, New Zealand 
Bedard, Donna, General Electric Corporate Research Center 
Casillas. Lilliam. Autonomous University of the State of Puebla. 


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) 


Weeks, Janis, University of Oregon 
Zakon, Harold, University of Texas, Austin 


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 


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 


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) 


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 


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 


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 


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) 


Garbers. David, University of Texas Southwestern Medical Center 
Reed, Randall, Johns Hopkins University School of Medicine 


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 


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 


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, 


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) 


Sluder, Greenfield, University of Massachusetts Medical School 
Wolf. David. University of Massachusetts Medical School 


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 


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, 


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) 


Hunt, Joan, University of Kansas Medical Center 

Mayo, Kelly, Northwestern University 

Schatten, Gerald, Oregon Health Sciences University 


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 


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 


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) 


Masys. Daniel, University of California, San Diego 


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) 


Cimino, James, Columbia University 


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 


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 


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 


Parada. Jorge. Cook County Hospital 
Reilly. James, State University of New York Health Science Center, 


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) 


Bialek, William. NEC Research Institute 

van Steveninck. Rob de Ruyter. NEC Research Institute 


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 


Baylor. Denis. Stanford University Medical Center 

Laughlin. Simon Barry, University of Cambridge, United Kingdom 

Logothetis, Nikos. Max-Planck-Institute for Biological Cybernetics. 

Srinivasan, Mandyam V.. Australian National University, Australia 

Course Coordinator 

Stogryn. Krista, Marine Biological Laboratory 


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) 


Silver. Robert B.. Marine Biological Laboratory 


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 


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) 


Guarente, Leonard P.. Massachusetts Institute of Technology 
Wallace, Douglas, Emory University School of Medicine 


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 


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) 


Edwards, John Jr., Harbor-UCLA Medical Center 
Magee, Paul T.. University of Minnesota 
Mitchell. Aaron P.. Columbia University 


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 


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) 


Dowling, John E., Harvard University 

Hopkins, Nancy, Massachusetts Institute of Technology 


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, 


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 


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 


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) 


Iz/ard, Colin. State University of New York, Albany 


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 


Hinsch, Jan, Leica, Inc. 

Inoue, Shinya. Marine Biological Laboratory 

Keller, H. Ernst, Zeiss Optical Systems 

Educational Programs R31 


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) 


Brosnan, Celia F., Albert Einstein College of Medicine 
Rosenbluth, Jack. New York University School of Medicine 


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 


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) 


Davison, Daniel B.. Bristol-Myers Squibb PRI 
Sogin, Mitchell, Marine Biological Laboratory 

R32 Annual Report 


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 


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 

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) 


Browne. Carole L.. Wake Forest University 

Tytell. Michael. Wake Forest University School of Medicine 

Course Assistant 
Begley. Gail. Marine Biological Laboratory 


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 


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) 


Margulis, Lynn, University of Massachusetts 
Dolan, Michael F., University of Massachusetts 


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 


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) 


Hobbie, John E., Director 

Foreman, Kenneth H., Associate Director 

Moniz, Polly C., Administrative Assistant 


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) 


Martinez, Joe L. Jr. 
Townsel, James 


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 


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 

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. 


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, 


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, 


Moore, John W., Duke University Medical Center 
Mooseker, Mark, Yale University 


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, 


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 


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.' 


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 

Architectural Dynamics in Living Cells 

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 

Resident Core Investigators 

Danuser, Gaudenz, Postdoctoral Fellow 
Inoue, Shinya. Distinguished Scientist 
Katoh, Kaoru. Postdoctoral Scientist 
Oldenbourg, Rudolf. Associate Scientist 


Geer, Thomas. Research Assistant 

Knudson, Robert. Instrumental Development Engineer 

Baraby. Diane. Laboratory Assistant 

MacNeil. Jane. Executive Assistant 


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 


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. 


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. 


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 


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. 


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 

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 

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 <>. 

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. 


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 


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 


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 


Dolan, Mike. Visiting Teaching Assistant 
Weir. Andrew. Visiting Teaching Assistant 

Graduate Students 
PhD Students 

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 

Dooley, Brad 
Tomasky, Gabrielle 
York. Joanna 

Masters Students 

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 


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 

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 

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 

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 

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. 


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 15 N 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 


Berthel, Dorothy J.. Administrative Assistant 
Donovan, Suzanne J., Executive Assistant 
Moniz, Priscilla C., Administrative Assistant. Semester in Environmental 


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 


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. 

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. 


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 


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 


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. 


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 d aN , d NN and d pN NOE connectivities and vicinal spin-spin 
coupling constants 3 J HNa 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 


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 


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. 


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 


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. 


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 Ca 2+ /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 


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. 


Rabinowitz, Michael, Associate Scientist 

Laboratory for Reproductive Medicine, Brown 

University and Women and Infants Hospital, 


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 


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. 


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. 


Silver, Robert. Associate Scientist 

Visiting Investigators 

Crawford, Karen. St. Mary's College 
Hummel. John, Argonne National Laboratory 
Pearson, John, Los Alamos National Laboratory 


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. 


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. 


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 

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. 


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 


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 


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 

American Society for Cell Biology 

Frederick B. Bang Fellowship Fund 

Mrs. Betsy G. Bang 

Jean and Katsuma Dan Fellowship 

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 

Ayco Charitable Foundation 
William Townsend Porter Foundation 

Fred Karush Endowed Library 

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 


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 

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 


Ms. Nancy Miller Arnn 

Mr. and Mrs. Alan K. Karplus 

Phillip H. Presley Scholarship 

Carl Zeiss, Inc. 

William Townsend Porter 

Scholarship Fund for Minority 


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 

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 

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 Ca 2+ 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 Ca 2+ 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 

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 


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 

Mclillo, Jerry, Co-Director, Marine Biological Laboratory 

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 

Board of Trustees and 

Corporation Officers and Trustees 

Chairman of the Board of Trustees. Sheldon J. Segal, The Population 

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 

Treasurer of the Corporation, Mary B. Conrad, Fiduciary Trust 


Clerk of the Corporation. Robert E. Mainer. The Boston Company 
Chair of the Science Council, Kerry S. Bloom, University of North 


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, 


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. 


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 


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. 


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. 


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. 


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 


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. 


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 

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, 

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, 

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 Staff 1 

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. Jaimie 2 
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. 


Communications Office 
Hinkle. Pamela Clapp, Director 
Cox. Sarah 2 
Flynn, Bridget 
Furfey. Susan 2 
Joslin, Susan 
Liles. Beth R. 
Noonan, Ryan 2 
Schanhacher, Jennifer 2 

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. Robert 2 

Burnette. Donald 

O'Connor-Lough, Susan 


Hall. Lionel E. Jr., Supervisor 
Gannon, Katherine 2 
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, Alberta 2 


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. Jessica 2 

Churchill, Elizabeth 2 

Clark. Tamaru L. 

Cosgrove, Nancy 

Kefeauver, Lee 

Mancini. Mary E. 

Waterbury. Andrew 2 

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. Dreux 2 

DeGiorgis, Joseph A. 2 

Grossman, William M. 

Gudas. Christopher N. 2 

Kilpatrick. Brian 2 

Klimm. Henry W. Ill 

Reynolds. Justin 2 

Sexton. Andrew W. 

Smith. Gary 2 

Sullivan. Daniel A. 

Tassinari. Eugene 

MRC Life Support System 

Mebane, William N., Systems Operator 

Carroll, James 

Clark. Heidi 2 

Hanley. Janice S. 

Kuzirian, Alan 

Till. Geoffrey A. 

MBUWHOI Library 

Norton. Catherine N.. Director 
Ashmore. Judith A. 
Buckingham. Andrew 2 
Costa. Marguerite E. 
Crocker, Daniel 2 
Deveer. Joseph M. 
Farrar. Stephen R.L. 
Fredericks. Julie 2 
Medeiros, Melissa 
Monahan. A. Jean 
Moniz. Kimberly L. 
Moore. Laurel E. 
Nelson. Heidi 
Person, Matthew 
Riley. Jacqueline 
Shinkle. Chelsea 2 

Information Systems Division 

In/ina, Barbara, Network Manager 

Berrios, Kelly 2 

Cohen, Alex 2 

Milliard. Channing 2 

Kokmeyer. Remmert 2 

Malchow, Robert 2 

Mountford, Rebecca J. 

Moynihan, James V. 

Purdy, Heather 2 

Remsen. David P. 

Renna. Denis J. 

Space, David B. 

Wheeler. Patrick 

NASA Center for Advanced Studies in the Space Life 

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, Douglas 2 
Peterson. Martha B. 

Safety Sen'ices 

Mattox, Andrew H.. Environmental. Health, and Safety Manager 
Lingler. Nicholas 2 
Normand. Danielle 2 

Satellite/Periwinkle Children 's Programs 

Robinson, Paulina H. 2 
Borst, Douglas 2 
Browne. Jennifer L. 2 
Curran. Kelly 2 
David, Amy 2 

R68 Annual Report 

Fitzelle. Annie" 
Gallant, Cynthia 2 
Guiftrida. Beth 2 
Halter. Sarah 2 
Hike. Kelly 2 
Mekelatos, Sharon 2 
Noonan. Brendan" 
Robbins, Sarah 2 
Shanley, Jennifer 2 

Sen'ice, Projects and Facilities 

Cutler. Richard D.. Director 
Enos, Joyce B. 
Guarente, Jeffrey 


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. Julia 2 

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, Jay 2 

Doherty. Garrett 2 

Dorris, John .1. 

Eldridge. Myles 2 

Elias, Michael 

Fernandez, Peter R. 2 

Foster, Martin 2 

Gibbons, Roberto G. 

Hannigan. Catherine 
Illgen. Robert F. 
Joyner. Matthew 2 
Ledwell, L. Patrick 2 
Lynch. Henry L. 
MacDonald, Cynthia C. 
Malchow, Kate 2 
McCarthy, Michael 
McDonnell. Gregory 
McNamara. Moreen M, 
McQuillan. Jeffrey 2 
Parker, Karen 2 
Plant, Stephen W. 
Pratt, Barry 
Stites, Clint 2 
Thiele, Tod 2 
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, Frank 2 

Rozum. John 

Schoepf. Claude 

Settlemire, Donald 

Shepherd. Denise M. 

Toner, Michael 

Wetzel. Ernest D. 2 

Members of the 

Life Members 

Acheson, George H., 25 Quissett Avenue. Woods Hole, MA 02543 
Adelberg, Edward A., 204 Prospect Street, New Haven. CT 065 1 1 - 

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 

Bernheimer, Alan W., New York University Medical Center. 

Department of Microbiology, 550 First Avenue. New York, NY 

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 


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- 

Colwin, Arthur L., 320 Woodcrest Road, Key Biscayne, FL 33149- 

Colwin, Laura Hunter, 320 Woodcrest Road, Key Biscayne, FL 

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 


Corliss, John O., P.O. Box 2729, Bala Cynwyd, PA 19004-2116 
Costello, Helen M., Carolina Meadows, Villa 137, Chapel Hill, NC 

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 

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 

Hauschka, Theodore S., 333 Fogler Road. Bremen. ME (deceased 


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- 

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- 

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 


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 

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- 


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 

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 


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 

Ronkin, Raphael R., 3212 McKinley Street. NW. Washington. DC 

Roslansky, John D., 57 Buzzards Bay Avenue, Woods Hole, MA 

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 

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- 

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 

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 

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 


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 

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 

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 

Child, Frank M., 28 Lawrence Farm Road, Woods Hole. MA 02543- 

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 


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. 

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 

Cutler, Richard D., Marine Biological Laboratory. Woods Hole. MA 


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 

Deegan, Linda A., Marine Biological Laboratory. The Ecosystems 

Center. Woods Hole, MA 02543 
DeGroof, Robert C., 145 Water Crest Drive, Doylestown, PA 18901- 

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 

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 

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- 


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- 

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- 

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 

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 

Henry, Jonathan Joseph, University of Illinois, Department of Cell and 

Structural Biology. 601 South Goodwin Avenue #B107, Urbana. IL 

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 

Hinchcliffe, Edward H., University of Massachusetts Medical School, 

Department of Cell Biology. 377 Plantation Street. Worcester, MA 

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 

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 

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 


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 

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 

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 

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 

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 


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, 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 


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- 

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 

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 

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 

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 .., ":\t L 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 


Manz, Robert D., 304 Adams Street. Milton. MA 02186 
Margulis, Lynn, University of Massachusetts. Department of 

Geosciences, Morrill Science Center. Box 35820. Amherst, MA 


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 

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 

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 

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, 

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 


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 

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 


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 

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 


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 

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 


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 

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 . 

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, 

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 

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- 


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 

Sanger, Jean M., University of Pennsylvania School of Medicine. 

Department of Anatomy, 36th and Hamilton Walk, Philadelphia. PA 

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- 

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 

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 

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. 

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 

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 I 1 ) 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 


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 


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- 

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 

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. 


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 


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 


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 

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 

Certificate of Organization 

Articles of Amendment 

(On File in the Office of the Secretary of the Commonwealth) 

(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 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 

"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) 


(Revised August 7. 1992 and December 1(1. IW2) 

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 


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. 


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 

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. 


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. 


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 

(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. 


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 

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 

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. 


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 

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, 

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. 


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. 


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 

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 

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. 


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. 


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. 


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Volume 199 





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 
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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 

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 


VOLUME 199. No. 2: OCTOBER 2000 


Hourdez, Stephana, Jason Lamontagne, Pat Peterson, 
Roy E. Weber, and Charles R. Fisher 

Hemoglobin from a deep-sea hvdrothermal-vent 
copepod 95 


Marsh, Adam G., Patrick K.K. Leong, and Donal T. 

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 


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 


Rosenthal, Joshua J.C., and Francisco Bezanilla 

Seasonal variation in conduction velocity of action 
potentials in squid giant axon 135 


Misamore, M.J., andJ.W. Lynn 

Role of the cytoskeleton in sperm entry during 
fertilization in the freshwater bivalve Dreissena 
polymorpha 144 






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 


Landowne, David 

Heavy water (D 2 O) 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 


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 


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. 


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. 


Identification of proliferating cells in hard clams. . . 199 

Brothers, Christine, Ernest Marks III, and Roxanna 


Conditions affecting the growth and zoospoi illation 

of the protistan parasite O_PX in culture 200 


Sandberg, Leslie, Phillip Stafford, and George M. 

Effects of myosin-II antibody on actin-dependent ves- 
icle transport in extracts of clam oocytes 202 

Stafford, Phillip, Jeremiah Brown, and George M. 


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 


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 

Published bv title only. . 



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Reference: Bio/. Bull. 199: 95-99. (October 2000) 

Hemoglobin From a Deep-Sea 
Hydrothermal-Vent Copepod 


1 Department of Biology, 208 Mueller Lab, Pennsylvania State University. University Park, Pennsylvania 

16802; 2 PaIisades 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- 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 25C (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 
-70C 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- 




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 







250 300 350 400 450 500 550 
Wavelength (nm) 

600 650 


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 . MgS0 4 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). 





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 P 50 
values at pH 7.3 of 0.05. 0.13, and 0.35 mm Hg at 10, 20, 
and 30C, 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 (P 50 = 0.035 
nimHg at 28 C and pH 7.2. Table 1). Both species. B. 


Table I 
Occurrence tint! structural tintl functional characteristics of hemoglobin in (//7/// 

SbP C SbC SpO O 

mass (kDa) 



P sn (Torr) n sn 

Bohr factor AH (kJ/mol) 





15 and 30 



0.7 1 

-0.1 5 to -0.9 -42.6 

17. IS 







1.8 to 5. 3 1.6-l.M 

(I to -0.21 -22.6 to -54.8 






20 2 

0.13 -30.9 






2.1 to 3.5 ? 







0.035 to 5.9 2.3-2.5 

? -20.5 

9. 22 






7 7 

7 7 







7 7 

7 7 






0.05 1 


This study 

Cirri pedia 





7 7 

7 7 









9 9 

7 9 


SbP: subphylum; C: class; SbC: subclass: SpO: superorder: O: order. Modified after Terwilliger ( 16). Conchastracan Hb, P, n measured at 28C. all other 
P 50 measured at 20C. 

spiciilifer and C. hierosolymitanus, have P 5() values 20 to 
600 times smaller than the P 50 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 P 50 values are quite low (reviewed in [ 10]). 

Benthoxynus spiciilifer hemoglobin lacks cooperativity 
(n w = 1.0) over the range of temperature (10 to 30C) 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 cm 1 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 15C and about 30 s at 25C. 
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 



A Benthoxynus spiculifer 

3.3 3.4 3.5 




















_ 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 

PO 2 (mmHg) 

Figure 3. (A) Oxygen equilibrium curves of Benthoxynus spiculifer hemoglobin at 10. 20, and 30C, 
measured as previously described (5), and (inset) arrhenius plot showing calculated values of the apparent 
oxygenation enthalpy values (AH). (B) Variation of P 5n 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). 


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 

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14. Minning, D. M., A. J. Gow, J. Bonaventura, R. Braun, M. 
Dewhirst, D. E. Goldberg, and J. S. Stamler. 1999. Ascaris hae- 
moglobin is a nitric oxide-activated 'deoxygenase'. Nature 401: 497- 

15. Tapley, D. W., J. M. Shick, and G. R. Buettner. 1992. Free radical 
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16. Terwilliger, N. B. 1992. Molecular structure of the extracellular 
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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 


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 

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. 


Maintaining Na + and K f 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 stl Rb + as a radio- 
active tracer for K + ion transport, Leong and Manahan 
(1997) described the ontogenetic changes in activity of 




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 -1.5C (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 15C 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 -80C. 

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 



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 
mMNa 3 PO 4 (pH 7.2), 5x SSC, 0.1% SDS, 5x Denhardt's, 
and 50 jug/ml yeast RNA at 45C in a hybridization oven. 
The 3'-UTR PCR probe (1185 bp) was radiolabeled by 
random priming (Promega) with a- 32 P-dCTP (3000 Ci 
mmol ' ), added to the hybridization tube with a fresh 10-ml 
aliquot of hybridization buffer (as above), and incubated 
overnight at 50C. The blots were initially washed with 
0.1X SSC, 1.0% SDS. and 0.5% Na 4 P 2 O 7 at 45C for 1 h. 
Additional washes at higher temperatures (max. 55C) 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 25C. 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 




















U 18546 



















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). 


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- 

Table 2 


Alignment of deduced amino acid sequence for the terminal IV5 residues of the a-subunil of Na + . K + -ATPase in the sea urchin Strongylocentrotus 
















































































k ~k 









































151 | 







































* * * 

* * 

* * * * 

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 e K)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 




200 150 100 50 

AA residues from 3' ORF terminus 


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 > 
r 2 = 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- 


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 





10 12 14 


18 20 

25 31 36 42 48 60 72 83 


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. 



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 



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. 


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 



' 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: 
Ahhreviations: DMS. dimelhylsulride; DMSP. dimethylsultomopropi- 


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. 




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. 2C), 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 54C 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 









g 100 


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 

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. 


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. 












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- 












| 20 





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 


















*> 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 

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) 




Byssal mantle* 


Adductor muscle 




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 

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. 


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) 





Adductor muscle 



Data for mantle are omitted for reasons stated in note to Table 1. 



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- 










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 

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 

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 37C 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 30C: 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- 



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). 


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) 


^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. 


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. 

\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. 




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 dec