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Full text of "The Biological bulletin"

August 2003 



Volume 205 Number 1 



BIOLOGICAL 
BULLETIN 




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THE BIOLOGICAL BULLETIN 



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SEEKERS 
THE SOCIETY 
OF CELLS. 



At Dr. Simon Watkins' lab, they look at cells 
the way anthropologists look at human culture: 
as communities of good guys and bad guys, 
of traders and communicators, of connections 
and relationships. "We are the observers," 
Simon says. "We never jump to conclusions. We let the conclusions jump 
to us." His mantra? "Imaging is everything." Which is why the best and 
the brightest of tomorrow's seekers and solvers find their way to Pittsburgh 
and the Watkins Lab. 

ROCKET SCIENCE me ca o. - p 8 " ' 

OLYMPUS* 



(From L to K) 

Ana Bursick - Research Specialist 
Stuart Shand - Research Specialist 
Simon C. Watkins, Ph.D. - Director 
Glenn Popworth - Research Associate 
Romesh Draviam Graduate Student 
Center for Biologic Imaging, 
University of Pittsburgh Medical School, 
Pittsburgh, PA 



AUG 2 5 2003 




Cover 



The deep sea is, in general, sparsely occupied; but 
in restricted areas and under unusual conditions, 
such as cold seeps, vents, and seamounts, dense 
communities do exist and persist for generations. 
Sparse populations also aggregate temporarily to 
facilitate mating, breeding, and brooding, and such 
reproductive aggregations are well known in vari- 
ous habitats but not in the deep sea, where only 
three such aggregations have previously been doc- 
umented. 

In this issue of The Biological Bulletin (p. 1 ), Jef- 
frey C. Drazen and colleagues at the Monterey Bay 
Aquarium Research Institute (MBARI, California) 
describe, for the first time in the deep sea, a multi- 
species reproductive aggregation or reproductive 
not S p 0t w ith an unusually high population den- 
sity and biomass. This aggregation is featured on 
the cover; it is located in 1500-1600 meters of 
water on the Gorda Escarpment, a submarine pla- 
teau off Cape Mendocino in northern California. 
The site was discovered in the course of 1 5 explor- 
atory dives by MBARI's remotely operated vehicle 
(ROV) Tiburon (top left image on the cover); the 
vehicle's two main cameras are identifiable by the 
white protective collars around their glass domes. 
The map on the cover locates the hot spot (red 
circle). Cape Mendocino (red dot), and the ROV 
dives (the line of small, irregular black areas ex- 
tending westward). 

Reproductive aggregations of two species an oc- 
topus (Graneledone sp.), and a fish, the blob sculpin 
(Psychrolutes phrictus) co-occurred at this site. 
The bottom left image shows three octopuses (body 
width, -16 cm) in a characteristic brooding posi- 
tion; their eggs are underneath them, attached to the 
rock outcrop. Also attached are several anemones of 



various species; the crab is Chionocetes sp. The 
image at the top right shows octopus eggs (length, 
40 mm) being sampled by the suction sampler on 
the ROV. Many of the eggs hatched during sam- 
pling; one hatchling appears in the sampler tube, 
and another is swimming away.' In the lower right 
image watching from behind a rock, which is 
covered in brisingid sea stars and anemones is a 
blob sculpin (length, ~60 cm) with a nest of large, 
pinkish eggs behind it. Another fish is just visible in 
the upper left corner of the image. Most blob scul- 
pins were seen attending to their egg masses (e.g., 
Fig. 3A, p. 4). the first direct observations of pa- 
rental care by an oviparous deep-sea fish. 

The particular location of this reproductive hot spot 
could be due to environmental heterogeneity; that 
is, the animals were concentrated at the crest of the 
local topography and near cold seeps. In these sit- 
uations, they might benefit from enhanced current 
flow and local productivity, critical resources for 
reproductive success in the deep sea. where oxygen 
tension is very low and food is in short supply. 
Thus, for some deep-sea species, the fortuitous oc- 
currence of critical environmental features may be 
essential for reproduction. 

The four images are frames selected from videos 
taken during dives in 2001 and 2002. The videos 
were produced collaboratively by the crew of the 
support ship R/V Western Flyer, the ROV Tiburon 
pilots, and the scientists. Photo credit is to MBARI. 
Jeffrey C. Drazen contributed to the cover and 
legend. The final cover was designed by Beth Liles 
(Marine Biological Laboratory, Woods Hole, Mas- 
sachusetts). 



1 The octopus hatchlings are being described by Janet Voight (Chi- 
cago Field Museum), an MBARI collaborator. 



THE 

BIOLOGICAL BULLETIN 

AUGUST 2003 



Editor 



Associate Editors 



Section Editor 
Online Editors 



Editorial Board 



Editorial Office 



MICHAEL J. GREENBERG 

Louis E. BURNETT 
R. ANDREW CAMERON 
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MICHAEL LABARBERA 

SHINYA INOUE, Imaging and Microscopv 

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

PETER B. ARMSTRONG 
JOAN CERDA 
ERNEST S. CHANG 
THOMAS H. DIETZ 
RICHARD B. EMLET 
DAVID EPEL 

KENNETH M. HALANYCH 
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NANCY KNOWLTON 
MAKOTO KOBAYASHI 
ESTHER M. LEISE 
DONAL T. MANAHAN 
MARGARET MCFALL-NGAI 
MARK W. MILLER 
TATSUO MOTOKAWA 
YOSHITAKA NAGAHAMA 
SHERRY D. PAINTER 
J. HERBERT WAITE 
RICHARD K. ZIMMER 

PAMELA CLAPP HINKLE 
VICTORIA R. GIBSON 
CAROL SCHACHINGER 
WENDY CHILD 



The Whitney Laboratory, University of Florida 

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

Marine Biological Laboratory 

ENSR Marine & Coastal Center, Woods Hole 

Hunter College, City University of New York 



University of California, Davis 

Center of Aquaculture-IRTA, Spain 

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Hopkins Marine Station, Stanford University 

Auburn University, Alabama 

Millennium Pharmaceuticals, Cambridge, Massachusetts 

Scripps Inst. Oceanography & Smithsonian Tropical Res. Inst. 

Hiroshima University of Economics, Japan 

University of North Carolina Greensboro 

University of Southern California 

Kewalo Marine Laboratory, University of Hawaii 

Institute of Neurobiology, University of Puerto Rico 

Tokyo Institute of Technology, Japan 

National Institute for Basic Biology, Japan 

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

University of California, Santa Barbara 

University of California, Los Angeles 

Managing Editor 

Staff Editor 

Editorial Associate 

Subscription & Advertising Administrator 



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MARINE BIOLOGICAL LABORATORY 
WOODS HOLE, MASSACHUSETTS 



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CONTENTS 



VOLUME 205, No. 1: AUGUST 2003 



RESEARCH NOTE 

Drazen, Jeffrey C., Shaiia K. Goffredi, Brian Schlining, 
and Debra S. Stakes 

Aggregations of egg-brooding deep-sea fish and 
cephalopods on the Gorda Escarpment: a reproduc- 
tive hot spot 

EVOLUTION 

Zigler, Kirk S., and H. A. Lessios 

250 million years of bindin evolution 



NEUROBIOLOGY AND BEHAVIOR 

Painter, Sherry- D., Bret Clough, Sara Black, and Gregg 

T. Nagle 

Behavioral characterization of attractin, a water- 
borne peptide pheromone in the genus Aplysifi . . . 

Bergman, Daniel A., and Paul A. Moore 

Field observations of intraspecific agonistic behavior 
of two crayfish species, Orconectes nisticus and Or- 
conectes i>i>ilis, in different habitats .............. 



PHYSIOLOGY AND BIOMECHANICS 

Etnier, Shelley A. 

Twisting and bending of biological beams: distri- 
bution of biological beams in a stiffness mechano- 
space ..................................... 



26 



36 



Eyster, L. S., and L. M. van Camp 

Extracellular lipid droplets in Idiosepiiu nutoides, the 
Southern pygmy squid 47 

Christensen, Ana Beardsley, James M. Colacino, and 

Celia Bonaventura 

Functional and biochemical properties of the hemo- 
globins of the burrowing brittle star Hemipholis elon- 
frtiiii Say (Echinodermata, Ophiuroidea) 54 

SYMBIOSIS AND PARASITOLOGY 

Davy, Simon K,, and John R. Turner 

Early development and acquisition of zooxanthellae 
in the temperate symbiotic sea anemone Anthopleura 
ballii (Cocks) 66 

DEVELOPMENT AND REPRODUCTION 

Neumann, Dietrich, and Heike Kappes 

On the growth of bivalve gills initiated from a lobule- 
producing budding zone 73 

Beninger, Peter G., Gael Le Pennec, and Marcel Le 

Pennec 

Demonstration of nutrient pathway from the diges- 
tive system to oocytes in the gonad intestinal loop of 
the scallop Pecten maximus L 83 



Annual Report of the Marine Biological Laboratory ... Rl 



THE BIOLOGICAL BULLETIN 

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Reference: Bi,>l. Bull. 205: 1-7. (August 2003) 
2003 Marine Biological Laboratory 



Aggregations of Egg-Brooding Deep-Sea Fish and 

Cephalopods on the Gorda Escarpment: 

a Reproductive Hot Spot 



JEFFREY C. DRAZEN*. SHANA K. GOFFREDI, BRIAN SCHLINING. AND 

DEBRA S. STAKES 

Monterey Ba\ Aquarium Research Institute, 7700 Sandholdt Road. 
Moss Landing, California 95039-9644 



Localized areas of intense biological activity, or hot 
spots, in the deep sea are infrequent but important features 
in an otherwise sparsely occupied habitat (1). Hydrother- 
mal vents, methane cold seeps, and the tops of seamounts 
are well documented areas where dense communities per- 
sist for generations (2-5). Reproductive aggregations 
where conspecifics concentrate for the purposes of spawn- 
ing or egg brooding could be thought of as transient hot 
spots. It is likely that they occur in populations with low 
densities to maximize mate location and increase reproduc- 
tive success (6). However, only a few deep-sea reproductive 
aggregations have ever been documented (7-9). demon- 
strating the paucity of present-day information regarding 
reproductive behavior of deep-sea animals. In this paper we 
describe a unique mitltispecies reproductive aggregation 
located on the Gorda Escarpment, California. We document 
some of the highest fish and octopus densities ever reported 
in the deep sea, with most individuals of both species 
brooding eggs. We describe the nesting behavior of the blob 
sculpin, Psychrolutes phrictus, and the egg-brooding behav- 
ior of an octopus, Graneledone sp. observed during annual 
dives of a remotely operated vehicle (ROV) on the Gorda 
Escarpment. The animals are concentrated at the crest of 
the local topography and near cold seeps where they may 
benefit from enhanced current flow and local productivity. 
These findings provide new information on the reproductive 
behaviors of deep-sea animals. More importantly, they 
highlight how physical and bathymetric heterogeneity in the 
environment can result in reproductive hot spots, which 



Received 14 February 2003; accepted 12 May 2003. 
* To whom correspondence should be addressed. 
jdrazen@mban.org 



E-mail: 



may be a critical resource for reproductive success in some 
deep-sea species. 

Fifteen ROV dives were conducted on the Gorda Escarp- 
ment and Mendocino Ridge during three visits in August 
2000, August 2001. and July 2002 (Fig. 1). The Gorda 
Escarpment is a submarine plateau offshore of northern 
California. The Mendocino Ridge extends westward from 
its northern edge at 40.35 N. The Escarpment's northern 
side is characterized by steep topography, frequent rocky 
outcrops and talus fields, sediment slumps, and drainage 
channels ( 10). The depth of investigation ranged from 1300 
to 3000 m. 

Reproductive aggregations of both blob sculpin and oc- 
topus were present at Site 1 (Fig. 1 ). The biomass of P. 
phrictus alone at this site was equivalent to the average total 
biomass of fishes on the continental slope. Likewise, the 
density of Graneledone sp. was considerably greater than 
previously published estimates (Fig. 2). Eighty-four indi- 
viduals off. phrictus and 64 nests (Fig. 3A) were observed. 
They were present at two sites, with the highest density 
occurring at Site 1 in both August 2000 and August 2001 
(Fig. 1). The fish were found over the steepest topography 
and at a topographic break between the steep northern side 
of the ridge and the more gently sloping top (Fig. 4). P. 
phrictus and associated nests were absent in July 2002. Two 
hundred and thirty-two individuals of Graneledone sp. (Fig. 
3B) were observed across all locations, with the highest 
densities observed at Site 1 during all three visits (Fig. 1 ). 
The octopus co-occurred with the blob sculpin, with 51% of 
the octopus observed within 5 m of sculpin adults or nests 
in 2001. Smaller aggregations of brooding blob sculpin and 
octopus were observed at Site 2. 

Site 1 (depth 1547-1603 m; dives T208, T349, T448) was 



J. C. DRAZEN ET AL 

12600'W 12500'W 





Density (# ha ') 
0-10 

11-20 



21-30 



31 + 





25 



50 



75 



100 



Km 



o 

O 



o 

o 

O 
O 



Figure 1. Balhymetric map of the Mendocino Ridge and Gorda Escarpment, showing all dive sites. Depths 
are in meters. One hundred and fourteen hours of video from ROV bottom time was recorded, annotated, and 
analyzed. Annotations of all occurrences of discernible animals and geologic features were stored in a searchable 
database with corresponding environmental (CTDO), observational (time, position), and system (camera zoom) 
data. Bathymetry is derived from a hull-mounted EM300 sonar system with 20-m pixel resolution. Ultrashort 
baseline Transponders (Sonardyne. Houston. TX) mounted on the ROV and the ship determine position. 
Tracklines are derived in a real-time ArcView-based (Environmental Systems Research Institute) navigation 
system. Closed circles, open circles, and hatched circles are densities (# ha~'l of blob sculpin (yellow) and 
octopus (red) from dives in 2000, 2001. and 2002 respectively. For each dive the densities reflect the number 
of animals observed over the surveyed area of seafloor. Areas for density estimates were calculated using the 
navigation to determine track length and assuming an average observational width of 4 m. Overlap of the dive 
track was accounted for in the calculations. 



characterized by small rocky cliffs and bouldered slopes 
that shoaled to a sloping talus field in which the gravel and 
boulders were interspersed with sediment. Site 2 (depth 
1534-1583 m; dive T351; Fig. 1) was on a shallowly 
sloping mud and sand bottom interspersed by boulders, 
talus, and small rock outcrops. Diffuse cold seeps at the 
base of several bouldered slopes at both sites were evident 
by the presence of small patches of vestimentiferan tube 
worms and vesicomyid clams (10). Sites 1 and 2 were 
characterized by an average bottom water temperature of 
2.4 C (range = 2.3 - 2.7 C) and very low oxygen con- 
centration (mean = 1.07 ml 1"'; range = 0.73-1.46 ml 
1 ~ ' ). The temperature at Site 1 was slightly elevated above 



ambient (0.1-0.2 C) due to local subsurface fluid seep- 
age from the substrate (10). 

Blob sculpin attended nests of large (4.0 0.6 mm; n = 
50) pinkish eggs (Fig. 3A). The majority of the nests had 
fish in close attendance (within 3 m). often sitting directly 
on or touching the eggs. Some nests and fish were observed 
by themselves primarily in the roughest terrain where it was 
difficult to see behind nearby rocks and ledges. Eggs were 
free of sediment, suggesting that the adults cleaned or 
fanned their nest sites. Brooding fish were almost always 
found very close to each other, and nests were often on 
neighboring boulders separated by only 1-2 m. Generally 
the parent fish did not move when the ROV approached; 



DEEP-SEA REPRODUCTIVE HOT SPOT 



A) 



17- 

-- 10 ' 

3 8 " 


Ps 


ychrolutes 


phi 


ictus 








6 ' 












1 4 

n 2 

n - 













all fishes 




Gorda Gorda N. Pacific* N. Pacific* N.Atlantic* 

2000 2001 



B) 



" 45 

40 - 
_~ 35 - 
^ 30 - 
* 25 - 

S 15- 
" 10 - 












Graneledone sp. 










5 - 
n - 













all octopuses 



Gorda 2000 Gorda 2001 Gorda 2002 N. Pacific* N. Atlantic* 

Figure 2. Biomass of blob sculpin (Psychrolutes phrictus) and density 
of octopus (Graneledone sp.) at Site 1 (grey bars) compared to "back- 
ground" averages for total fishes and total octopuses at similar depths 
elsewhere (black bars). (A) Biomass (g m~~: the metric typically used for 
fish) of blob sculpin was estimated by assuming an average adult size of 4.5 
kg (26). (B) Density of octopuses (#ha "'; typically used for cephalopods). 
Data from other locations are from trawling* (16, 27. 28; J. R. Voight, pers. 
comm.) or camera surveys^ (29). 



however, this was also true for fish without eggs, which 
precluded any conclusions about nest-guarding behavior. 
The sex of the fish could not be determined from video 
observations. Fecundity was estimated for four egg masses 
and ranged from 9375 to 108,125 eggs. The eggs were 
generally laid on the flat exposed surfaces of large boulders 
and rock outcrops. Of the 64 egg masses, 57 (89%) had been 
laid on single rocks; the other seven were each strewn 
across as many as three neighboring rocks or across large 
fissures in a flat rock face. 

This study presents the first direct evidence of parental 
care (11) in an oviparous deep-sea fish. It is likely that 
members of the families Zoarcidae and Liparidae also ex- 
hibit parental care, but this has not been confirmed. The 
zoarcid Melanostigma atlanticitm was captured in a burrow 
with its eggs by a box core at 350 m depth (12). The 
developmental stage of the eggs was not determined; thus, 
it is unknown whether the parents were in the process of egg 
laying (one female still retained her eggs) or whether they 
were staying to guard and ventilate the eggs. Many liparids 
have large eggs and very low fecundities, suggesting some 
form of parental care, but no direct evidence has been 
described (13). Reviews of reproduction in other diverse 
deep-sea fishes make no note of parental care (1, 14-16). 

Individuals of Graneledone sp. were observed brooding 



eggs under their bodies while sitting vertical and motionless 
with arms curled outwards on boulders, rock outcrops, and 
ledges (Fig. 3C). An adult specimen of Graneledone boreo- 
pacifica was collected in July 2002 with 51 eggs. The eggs 
were 40 mm in length, and many began hatching prema- 
turely when collected; these juveniles still retained egg sacs. 
Site 1 was revisited in July 2002, and 63 individuals of 
Graneledone sp. were observed in the curled position. Nine 
of ten individuals in this position were confirmed to be in 
the process of brooding eggs. In August 2001, 43 eggs of a 
specimen of Benthoctopus sp. were collected from Site 2. 
This octopus, unlike the others observed, was underneath a 
small rock with its eggs out of plain view; it was also much 
smaller than the Graneledone sp. (mantle length 10 cm), 
and its eggs were only 16 mm long. It was only observed 
during the course of collecting the rock for geologic exam- 
ination. 

Our observations provide the first evidence of a multi- 
species reproductive aggregation in the deep sea. To our 
knowledge, the only other reproductive aggregations de- 
scribed from deep-sea environments are for spawning ag- 
gregations of orange roughy (7); another brooding aggrega- 
tion of octopuses, including a species of Graneledone, in the 
North Pacific (8); and small (2-6 individuals) aggregations 
of an echinoid (9). In addition, there are reports of aggre- 
gations of two echinoderm species in the North Atlantic (17, 
18) that may have been for the purpose of reproduction, and 
pair formation has been documented in a holothuroid (19). 
Observations over 3 years indicate that the aggregation at 
Site 1 is either long-lived or recurs at the same location 
perhaps every year. The aggregations of blob sculpin and 
octopus exhibit densities and biomass among the highest 
recorded in the deep sea (Fig. 2). Localized aggregations of 
this magnitude could have profound influences on local 
food webs and fauna. 

There are several possible explanations for the presence 
of the dense aggregations of animals on the Gorda Escarp- 
ment. For instance, the presence of brooding aggregations 
of Benthoctopus sp. and Graneledone sp. in the North 
Pacific have been explained previously by the availability of 
both rocky substrate for egg attachment and bivalve prey 
from nearby cold seeps (8). Rocky substrate for egg attach- 
ment is an obvious requisite for spawning by both sculpin 
and octopus. Rocky substrate, however, occurred at all dive 
locations yet reproductive aggregations were present at only 
two locations, suggesting that substrate is not the only 
criterion involved in site selection for brooding. Further- 
more, aggregations of sculpin or octopuses have not been 
observed on other rocky features, including some within our 
dive areas on the Gorda Escarpment and Mendocino Ridge 
and on the Davidson, Guide, and Gumdrop seamounts in the 
North Pacific. These seamounts have been observed using 
the Monterey Bay Aquarium Research Institute's ROV at 
various times of the year, but no aggregations such as we 



J. C. DRAZEN ET AL. 




Figure 3. Egg-brooding fish and octopus. (Al Three blob sculpin. Psychrolutes phrictus. attending nests. 
The fish on the left has a nest just outside of the field of view. Size-calibrated images were used to determine 
fish egg size and fecundity. When the camera had zoomed such that the plane of focus was narrow, then the 
horizontal dimension of the field of view (field widthl could be determined (30). From the resulting calibrated 
images. Optimas image analysis software (ver. 6) (Optimas Corporation. Bothell. WAl was used to measure fish 
egg diameters. Occasionally when field width could be used to calibrate the size of objects in the video, the 
Optimas software was used to calculate the area of fish egg masses. The eggs appeared to be laid in a thin layer 
across the rocks, and in a few cases they were piled on top of each other near the center of the mass. 
Consequently, egg numbers were estimated by assuming that a single layer of eggs was placed across the nest 
area as closely together as possible. (B) Eight egg-brooding individuals of Graneledone sp. on a rock outcrop. 
(C) A specimen of Graneledone sp. showing eggs protected under arms and mantle. 



describe have been found (J. Drazen. unpubl. data). Like- 
wise, on more than 200 dives in the Monterey Bay area at 
depths greater than 1000 m and often in areas of rocky 
substrate (i.e., canyon walls and slopes), no brooding octo- 
puses were observed (although octopuses are common) and 
only 13 blob sculpin were seen, none with eggs. 

The presence of cold seeps can dramatically influence the 
local productivity of surrounding deep-sea communities by 
transfer of organic nutrients (2). Diffuse cold seeps were 
observed at both sites of sculpin and octopus aggregations, 
suggesting that enhanced local productivity from cold seeps 
on the Gorda Escarpment may also influence the aggrega- 
tions. This is unconfirmed, however, because only six oc- 
topus were seen in the immediate vicinity of seep organisms 



and the distribution of nesting blob sculpin was much 
broader than that of the seeps (Fig. 4). 

Cold seeps are related to the upward flow of warm, 
methane-rich pore fluids from depth; this flow has also 
generated slight increases in temperature (0.1-0.2 C above 
ambient) at Site 1 (10). Increases in temperature could 
shorten egg development times, which would be an advan- 
tage to species that invest parental care. Assuming a Q w of 
2, an increase of 1.5 C would be required for a 10% 
reduction in incubation time. Similar conclusions were 
drawn for benthic octopus brooding near cold seeps at the 
Baby Bare site off of Washington State (8). However, 
temperature elevations of this magnitude around cold seeps 
are very unlikely. Furthermore, animal occurrences did not 



DEEP-SEA REPRODUCTIVE HOT SPOT 



iveT342 

,-^~ 

T448 




Figure 4. Three-dimensional sunshaded map of dive tracks and locations of all sightings of blob sculpin, 
octopus, and cold seeps at Site 1. Contours are in meters. Mapping information was generated as for Figure 1. 
The compass is also a scale bar with each arm equivalent to 500 m. Note that, due to the typical perspective of 
a three-dimensional rendering, the apparent distances for each axis are not equal. 



correlate with the highest temperature anomalies. Therefore, 
we conclude that cold seeps do not benefit these animals 
physically, but they may provide a food source that could 
play a role in the location of the animal aggregations. 

In addition, elevated currents may influence site selection 
by brooding aggregations. All blob sculpin and most octo- 
pus were observed near the ridge crest where exposure to 
elevated currents is likely (1,3, 20). As on seamount crests, 
abundant suspension feeders such as brisingid sea stars, 
tunicates, gorgonians, and venus flytrap anemones were 
found at the crest of the Gorda Escarpment, providing 
evidence of accelerated current speeds. Some shallow-liv- 
ing sculpins have a strong preference for nesting sites that 
are exposed to the current: this exposure aids in gas ex- 
change and waste removal and accelerates embryogenesis 
(21. 22). At Site 1, where oxygen concentrations are very 
low, enhanced water movement may be required to deliver 



adequate oxygen for embryogenesis. A reduction in the 
need to ventilate or fan the eggs could be an energetic 
benefit to the adults. In addition, benthic egg brooding and 
hatching implies a demersal larval/juvenile phase (23). Bot- 
tom currents in the deep sea are generally low, so these 
organisms may take advantage of intensified currents at this 
site to enhance the dispersal of larvae or juveniles within the 
demersal habitat. 

At one time the deep sea was thought to be a sparsely 
populated and homogenous environment ( 1 ). Today, dense 
localized communities such as the chemosynthetic commu- 
nities of hydrothermal vents and methane cold seeps (2) and 
the suspension-feeding communities of seamounts (3) are 
well known. Our study site on the Gorda Escarpment is 
another unique type of biological hot spot in the deep sea. 
The site is connected to the continental margin but topo- 
graphically exhibits characteristics of a seamount environ- 



J. C. DRAZEN ET AL. 



merit. In addition, small cold seeps are present. We hypoth- 
esize that the local topography interacting with the physical 
and geologic setting has created a localized reproductive hot 
spot in the deep sea utilized by at least two very different 
animals. 

This information has several important implications. The 
reproductive hot spot on the Gorda Escarpment (and future 
sites determined to be similar) might qualify as an area to be 
protected from fishing. The protection of habitats associated 
with vulnerable life stages, notably spawning aggregations, 
is a main objective of marine reserves (24). Our study site 
could be threatened by commercial trawling and long-lining 
operations. In the last two decades, the world has seen a 
rapid development of deep-sea fisheries to depths of 
2000 m, and currently fishers regularly operate at depths of 
1000 m off of the west coast of the United States (25). From 
an ecological perspective, our findings contribute to our 
understanding of habitat heterogeneity within the broader 
deep-sea ecosystem as well as providing sites where scien- 
tists can predictably observe reproductive biology in deep- 
sea animals, a prospect that is exciting for the study of these 
elusive species. 

Acknowledgments 

Special thanks to Linda Kuhnz. Kyra Schlining, Susan 
Von Thun, and Kris Walz for video annotation. Dan Davis 
provided helpful advice and software for measuring egg and 
nest sizes from video framegrabs. We are indebted to Dave 
Clague, Robert Young, and Jenny Paduan for their assis- 
tance with dive T448. Janet Voight also provided assistance 
on that dive and helped to confirm the octopus identity. Bob 
Vrijenhoek was the principal investigator on dives T349 and 
T351. Thanks to the pilots of the ROV Tiburon and the crew 
of the RV Western Flyer. Thanks to Jim Barry, Brad Seibel. 
Bruce Robison, and Greg Cailliet for discussion and com- 
ments. Waldo Wakefield, Eric Hochberg, and an anony- 
mous reviewer provided valuable insight and revisions. 
Dives T348, T350, and T352 were funded by a grant from 
the National Undersea Research Program awarded to Robert 
Duncan (Oregon State University). J. C. Drazen was sup- 
ported by a postdoctoral fellowship from MBARI. 



Literature Cited 



1. Gage, J. D., and P. A. Tyler. 1991. Deep-Sea Biology: a Natural 
History of Organisms at the Deep-Sea Floor. Cambridge University 
Press. Cambridge. 

2. Tunnicliffe, V., A. G. McArthur, and D. McHugh. 1998. A bio- 
geographical perspective of the deep-sea hydrothermal vent fauna. 
Adv. Mar. Biol. 34: 353-442. 

3. Genin, A., P. K. Dayton, P. F. Lonsdale, and F. N. Spiess. 1986. 
Corals on seamount peaks provide evidence of current acceleration 
over deep-sea topography. Nature 322: 59-61. 

4. Van Dover, C. L., C. R. German, K. G. Speer, L. M. Parson, and 
R. C. Vrijenhoek. 2002. Evolution and biogeography of deep-sea 
vent and seep invertebrates. Science 295: 1253-1258. 



5. Wilson, R. R., Jr., and R. S. Kaufmann. 1987. Seamount biota and 
biogeography. Pp. 355-377 in Seamounts. Islands, and Atolls. B.H. 
Keating. P. Fryer, R. Batiza, and G.W. Boehlert, eds. Geophysical 
Monograph 43, American Geophysical Union, Washington DC. 

6. Domeier, M. L., and P. L. Colin. 1997. Tropical reef fish spawning 
aggregations: defined and reviewed. Bull. Mar. Sci. 60: 698-726. 

7. Pankhurst, N. W. 1988. Spawning dynamics of orange roughy, 
Hoplostethus atlanticus, in mid-slope waters of New Zealand. Envi- 
ron. Biol. Fishes 21: 101-116. 

8. Voight, J. R., and A. J. Grehan. 2000. Egg brooding by deep-sea 
octopuses in the North Pacific Ocean. Biol. Bull. 198: 94-100. 

9. Young, C. M., P. A. Tyler, J. L. Cameron, and S. G. Rumrill. 1992. 
Seasonal breeding aggregations in low-density populations of the 
bathyal echinoid Stylocidaris lineata. Mar. Biol 113: 603-612. 

10. Stakes, D. S., A. M. Trehu, S. K. Goffredi, T. H. Naehr, and R. A. 
Duncan. 2002. Mass wasting, methane venting, and biological com- 
munities on the Mendocino transform fault. Geology 30: 407-410. 

1 1. Smith, C., and R. J. Wootton. 1995. The costs of parental care in 
teleost fishes. Rev. Fish Biol. Fish. 5: 7-22. 

12. Silverberg, N., H. M. Edenborn, G. Ouellet, and P. Beland. 1987. 
Direct evidence of a mesopelagic fish, Melanostigma atlanticum (Zo- 
arcidae) spawning within bottom sediments. Environ. Biol. Fishes 20: 
195-202. 

13. Stein, D. L. 1980. Aspects of reproduction of liparid fishes from the 
continental slope and abyssal plain off Oregon, with notes on growth. 
Copeia 687-699. 

14. Mead, G. W., E. Bertelsen, and D. M. Cohen. 1964. Reproduction 
among deep-sea fishes. Deep-Sea Res. 11: 569-596. 

15. Merrett, N., and R. L. Haedrich. 1997. Deep-Sea Demersal Fish 
and Fisheries. Chapman and Hall. London. 

16. Haedrich, R. L. 1997. Distribution and population ecology. Pp. 
79-114 in Deep-Sea Fishes. D. J. Randall and A. P. Farrell. eds. 
Academic Press, San Diego. 

17 Grassle, J. F., H. L. Sanders, R. R. Messier, G. T. Rowe, and T. 
McLellan. 1975. Pattern and zonation: a study of the bathyal 
megafauna using the research submersible Alvin. Deep-Sea Res. 22: 
457-481. 

18. Billett, D. S. M., and B. Hansen. 1982. Abyssal aggregations of 
Kolga hya/inu Danielssen and Koren (Echinodermata: Holothurioidea) 
in the Northeast Atlantic Ocean: a preliminary report. Deep-Sea Res. 
29: 799-818. 

19 Tyler, P. A., C. M. Young. D. S. M. Billett, and L. A. Giles. 1992. 
Pairing behaviour, reproduction and diet in the deep-sea holothurian 
genus Parori-a (Holothuroidea: Synallactidae). J. Mar. Biol. Assoc. 
UK 72: 447-462. 

20. Fock, H., F. Uiblein, F. Roster, and H. von Westernhagen. 2002. 
Biodiversity and species-environment relationships of the demersal 
fish assemblage at the Great Meteor Seamount (subtropical NE Atlan- 
tic), sampled by different trawls. Mar. Biol. 141: 185-199. 

21. DeMartini, E. E. 1978. Spatial aspects of reproduction in buffalo 
sculpin. Enophrys bison. Environ. Biol. Fishes 3: 331-336. 

22. DeMartini, E. E., and B. G. Patten. 1979. Egg guarding and 
reproductive biology of the red Irish lord. Hemi/epidotiis hemi/epido- 
tus (Tilesius). Syesis 12: 41-55. 

23. Hochberg, F. G.. M. Nixon, and R. B. Toll. 1992. Order Octopoda 
Leach. 1818. Pp. 213-280 in "Larval" ami Juvenile Cephalopods: A 
Manual for Their Identification. M. J. Sweeney. C. F. E. Roper. K. M. 
Mangold. M. R. Clarke, and S. v. Boletzky. eds. Smithsonian Contri- 
butions to Zoology 513. Smithsonian Institution Press, Washington, 
DC 

24 Roberts, C. M., S. Andelman, G. Branch, R. H. Bustamante, J. C. 
Castilla, J. Dugan, B. S. Halpern, K. D. Lafferty, H. Leslie, and J. 
Lubchenco. 2003. Ecological criteria for evaluating candidate sites 
for marine reserves. Ecol. Appl. 13: SI 99 -2 14. 



DEEP-SEA REPRODUCTIVE HOT SPOT 



25. National Research Council. 2002. Effects of Trawling and Dredg- 
ing on Seafloor Habitat. National Academy Press, Washington, DC. 

26. Matarese, A. C'.. and D. L. Stein. 1980. Additional records of the 
sculpin Psychmlutes phrictus in the eastern Bering Sea and off Ore- 
gon. Fish. Bull. 78: 169-171. 

27. Alton, M. S. 1972. Characteristics of the demersal fish fauna inhab- 
iting the outer continental shelf and slope off the northern Oregon 
coast. Pp. 583-634 in The Columbia River Estuan- and Adjacent 
Ocean Waters. A. T. Pruter and D. L. Alverson. eds. University of 
Washington Press, Seattle. 

28. Collins, M. A., C. Yau, L. Allcock, and M. H. Thurston. 2001. 



Distribution of deep-water benthic and bentho-pelagic cephalopods 
from the north-east Atlantic. J. Mar. Biol. Assoc. UK 81: 105- 
117. 

29. Wakefield, VV. W. 1990. Patterns in the distribution of demersal 
fishes on the upper continental slope off central California with studies 
on the role of ontogenetic vertical migration in particle flux. Ph.D. 
dissertation, Scripps Institution of Oceanography. University of Cali- 
fornia, San Diego, La Jolla, CA. 

30. Davis, D. L., and C. H. Pilskaln. 1993. Measurements with under- 
water video: camera field width calibration and structured light. Mar. 
Techno!. Soc. J. 26: 13-19. 



Reference: Bio/. Bull. 205: 8-15. (August 2003) 
2003 Marine Biological Laboratory 



250 Million Years of Bindin Evolution 



KIRK S. ZIGLER 1 - 2 * AND H. A. LESSIOS 1 

1 Smithsonian Tropical Research Institute, Balboa, Panama: and 2 Department of Biology, 
Duke University; Durham, North Carolina 



Abstract. Bindin plays a central role in sperm-egg attach- 
ment and fusion in sea urchins (echinoids). Previous studies 
determined the DNA sequence of bindin in two orders of the 
class Echinoidea, representing \Q9c of all echinoid species. 
We report sequences of mature bindin from five additional 
genera, representing four new orders, including the distantly 
related sand dollars, heart urchins, and pencil urchins. The 
six orders in which bindin is now known include 70% of all 
echinoids, and indicate that bindin was present in the com- 
mon ancestor of all extant sea urchins more than 250 million 
years ago. Over this span of evolutionary time there has 
been ( 1 ) remarkable conservation in the core region of 
bindin, particularly in a stretch of 29 amino acids that has 
not changed at all; (2) conservation of a motif of basic 
amino acids at the cleavage site between preprobindin and 
mature bindin; (3) more than a twofold change in length of 
mature bindin; and (4) emergence of high variation in the 
sequences outside the core, including the insertion of gly- 
cine-rich repeats in the bindins of some orders, but not 
others. 

Introduction 

Various studies have shown that molecules involved in 
reproduction (and particularly in gamete interactions) 
evolve rapidly, often under the influence of positive selec- 
tion (reviewed in Swanson and Vacquier, 2002). Among 
these proteins there are examples of both high (Metz and 
Palumbi, 1996) and low (Metz et al., 1998b) levels of 
intraspecific variation. In some cases a single molecule 
displays domains that are highly conserved and other do- 
mains that are highly variable (Vacquier et ai, 1995). Vari- 
ation in such proteins is usually studied at a low taxonomic 



Received 25 February 2003; accepted 3 June 2003. 

* To whom correspondence should be addressed. Current address: Fri- 
day Harbor Laboratories. University of Washington. 620 University Road, 
Friday Harbor. WA 98250. E-mail: zilerk@u. washinaton.edu 



level, often within species, sometimes within genera, but 
rarely across an entire class. There are good reasons for this 
focus: such studies are likely to uncover mutational changes 
that are important in mate recognition and in speciation. 
However, comparisons across broad taxonomic levels can 
offer insights into the evolution of such molecules. They can 
reveal which features of these molecules are conserved (and 
are thus essential for basic functions) and which features are 
free to vary. For the parts that do vary, such comparisons 
can determine common features of evolution. Most of all, 
the comparisons can address the question of the universality 
of a particular molecule by asking how far back in evolution 
one needs to search to find the point at which a completely 
different molecule has taken over the essential functions 
involved in gamete binding and fusion. 

Echinoids (sea urchins, heart urchins, and sand dollars), 
with their readily obtainable gametes, have long been model 
organisms for fertilization studies. Because fertilization is 
external, the molecules involved in gamete recognition and 
fusion are associated exclusively with the gametes. Bio- 
chemical studies in sea urchins identified the first "gamete 
recognition protein," bindin (Vacquier and Moy, 1977). 
Bindin is the major insoluble component of the sperm 
acrosomal vesicle and has been implicated in three molec- 
ular interactions (Hofmann and Glabe, 1994). First, after the 
acrosomal reaction, bindin self-associates, coating the acro- 
somal process. Second, it functions in sperm-egg attach- 
ment by binding to carbohydrates in the vitelline layer on 
the egg surface. Third, it is involved in the fusion of sperm 
and egg membranes (Ulrich et ai, 1998. 1999). 

Bindin is translated as a larger precursor, from which the 
N-terminal preprobindin portion is subsequently cleaved to 
produce mature bindin (Gao et al., 1986). The mature bindin 
molecule contains an amino acid core of about 55 residues 
that is highly conserved among all bindins characterized to 
date (Vacquier et al.. 1995). An 18 amino acid section of 
this conserved core (B18) has been shown to fuse lipid 



EVOLUTION OF BINDIN 



vesicles in vitro, suggesting that this region functions in 
sperm-egg membrane fusion (Ulrich ft til.. 1998. 1999). 
Thus far, bindin is known only from echinoids; no homol- 
ogous molecules have been identified in any other organism 
(Vacquier. 1998). 

To date, the nucleotide sequence of bindin has been 
determined in six genera of sea urchins. In Echinometra 
(Metz and Palmnbi. 1996). Strongylocentrotus (Gao el nl.. 
1986; Minor etui., 1991; Biermann. 1998; Debenham et nl.. 
2000). and Heliocidaris (Zigler et al.. 2003), there are many 
sequence rearrangements among individuals and species, 
and indications of positive selection in regions on either side 
of the core. In Arhacia (Glabe and Clark. 1991; Metz et al.. 
1998a) and Tripneustes (Zigler and Lessios. 2003), there are 
fewer sequence rearrangements and no evidence for positive 
selection. In Lvtechinus, only one sequence has been pub- 
lished (Minor et al.. 1991 ), so the mode of evolution of the 
molecule remains unknown. 

The five genera in which bindin was previously se- 
quenced belong to two echinoid orders, the Echinoida and 
the Arbacioida. These two orders contain only 10% of all 
extant echinoid species (Kier. 1977; Smith. 1984: Little- 
wood and Smith. 1995). The molecular structure of bindin 
in the other 13 orders of the class Echinoida has not been 
studied. The only evidence that bindin is present outside the 
Echinoida and Arbacioida comes from Moy and Vacquier 
( 1979). who reported that an antibody to bindin of Strongy- 
locentrotus purpuratus reacted with sperm from one species 
of the order Phymosomatoida and two species of the order 
Clypeasteroida. As Vacquier ( 1998) has pointed out. mole- 
cules that mediate fertilization in contrast to those central 
to other basic life processes often differ between taxa. For 
example, in the molluscan class Bivalvia. completely dif- 
ferent proteins are involved in gamete recognition of oysters 
(Brandriff >//., 1978) and of mussels (Takagi et al., 1994). 
It is. therefore, not safe to assume without empirical evi- 
dence that bindin is present in all orders of echinoids. or that 
it has the same general structure as in the taxa in which it 
has already been characterized. 

As a first step in determining which orders of echinoids 
possess bindin and, if they do. how its structure varies, we 
cloned and sequenced mature bindin from five genera of sea 
urchins, four of which belong to orders in which bindin was 
previously unknown. We combined our data with those of 
previous studies of bindin in genera belonging to the orders 
Echinoida and Arbacioida. The final data set includes bindin 
from 10 genera of sea urchins, pencil urchins, sand dollars, 
and heart urchins, and the results indicate that the molecule 
was present in the common ancestor of all extant echinoids 
that diverged from each other over 250 million years ago. 
The core sequence has remained remarkably unchanged 
over this period of time, whereas the areas flanking the core 
have undergone substantial modification, resulting in great 



differences in molecular size, amino acid sequence, and 
number of repeats. 



Materials and Methods 



Samples 



The pencil urchins (order Cidaroida) were represented in 
our study by Eucidaris tribuloides, collected on the Atlantic 
coast of Panama; the order Diadematoida by Diadema an- 
ti/lcinnn. also from the Atlantic coast of Panama. The sand 
dollars (order Clypeasteroida) were represented by Encope 
stokesii from the Pacific coast of Panama; the heart urchins 
(order Spatangoida) by Moira clotho collected at the Perlas 
Islands in the Bay of Panama. Heliocidaris erythrogramma 
(order Echinoida) was collected near Sydney, Australia. 

DNA isolation and sequencing 

We injected various individuals of each species with 0.5 
M KC1 until we encountered one that produced sperm. The 
testes of this ripe male were removed and used either 
directly for mRNA extraction, or after preservation in either 
RNALater (Ambion Inc.) or in liquid nitrogen. The methods 
for mRNA isolation, reverse transcription reactions, initial 
polymerase chain reactions. 3' and 5' rapid amplification of 
cDNA ends (RACE) reactions, and DNA sequencing were 
as described in Zigler and Lessios (2003). with the follow- 
ing modifications. ( 1 ) A fragment of the core region of 
bindin was amplified from the reverse transcriptase reac- 
tion product or from genomic DNA. using primers 
MB1 130+ (5'-TGCTSGGTGCSACSAAGATTGA-3') and 
either core200- (5'-TCYTCYTCYTCYTGCATIGC-3') or 
core 157- (5'-CIGGRTCICCHATRTTIGC-3'). These prim- 
ers correspond to amino acids VLGATKID. ANIGDP, and 
AMQEEEE. respectively (Vacquier et al., 1995). (2) When 
complete 5' mature bindin sequences were not obtained 
during the first round of 5' RACE, new primers were 
designed at the 5' end of the obtained sequence; then a 
second round of RACE amplification was conducted. (3) A 
5' preprobindin primer was designed based on a comparison 
of preprobindin sequences of Moira clotho (this study) to 
preprobindin sequences of Arbacia (Glabe and Clark. 1991). 
Strong\locentrotus (Gao et al.. 1986; Minor et al.. 1991), and 
Lvtechinus (Minor et al., 1991). This primer. prolSO (5'- 
AAGMGIKCIAGYSCIMGIAAGGG-3'). which corresponds 
to the conserved amino acids KR(A/S)S(A/P)RKG of the 
preprobindin, was used in combination with exact primers 
from the bindin core to amplify mature bindin sequences 5' 
of the core from Eucidaris tribuloides testis cDNA. (4) 
Bindin sequences obtained from RACE were subsequently 
confirmed by amplification, cloning, and sequencing of full 
mature bindin sequences from testis cDNA. 

Sequencing of both DNA strands was performed on 
an ABI 377 automated sequencer, and sequences were 



10 



K. S. ZIGLER AND H. A. LESSIOS 



edited using Sequencher 4.1 (Gene Codes Corp.). Se- 
quences have been deposited in GenBank (Accession num- 
bers AY126482-AY126485. AF530406). Published mature 
bindin sequences from a single exemplar from each of 
the five genera in which bindin had been previously se- 
quenced were taken from GenBank. These representatives 
were Strongylocentrotus purpuratus (Accession number: 
M14487, Gao et aL 1986), Lytechimts variegatus (M59489, 
Minor et ul., 1991), Arbacia punctitlata (X54155, Glabe and 
Clark, 1991), Echinometra oblonga (U39503, Metz and 
Palumbi, 1996), and Tripneustes ventricosus (AF520222, 
Zigler and Lessios, 2003). Three amino acids of the core 
region of the bindin of Lytechinus variegatus [numbers 367 
(N), 368 (L), and 385 (Y) in the alignment of Vacquier et 
al.. (1995)] were changed to A, V, and D, respectively, 
based on our own sequence data of Lytechinus bindin from 
25 individuals representing 5 species; all 25 sequences had 
these amino acids at the 3 sites (Zigler and Lessios. unpub.). 
In Echinometra oblonga, sequences for the extreme 3' end 
of preprobindin are not in GenBank. They were inferred 
from the primer sequences used by Metz and Palumbi 
(1996) to amplify mature bindin sequences. 

Sequence analysis 

We aligned the mature bindin amino acid sequences with 
ClustalXver. 1.81 (Thompson et al. . 1997), and adjusted the 
alignment by eye in Se-Al (ver. 2.0a5, Rambaut, 1996). We 
characterized the amino acid changes observed in the core 
region of bindin as either radical or conservative with re- 
spect to charge and polarity (Taylor, 1986; Hughes et al., 
1990). The PROTPARAM tool of the EXPASY proteomics 
server of the Swiss Institute for Bioinformatics (http://www. 
expasy.org) was used to calculate Kyte and Doolittle ( 1982) 
hydrophobicity plots (window size = 11 amino acids) for 
each mature bindin sequence. The PROTSCALE tool of the 
same server was used to calculate amino acid composition 



for the mature bindins both for the core region (10 se- 
quences, 55 amino acids per sequence) and for mature 
bindin sequences outside the core ( 10 sequences of varying 
length for a total of 1909 amino acids). The program 
CODONS (Lloyd and Sharp, 1992) was used to calculate 
the effective number of codons (ENC), a measure of codon 
usage bias (Wright, 1990), for each sequence. ENC values 
can range from 20 to 61, with 61 indicating that all synon- 
ymous codons are used in equal frequency (no codon bias), 
and 20 indicating that only a single codon is used for each 
amino acid (maximum codon bias). The statistical analysis 
of protein sequences (SAPS, http://www.isrec.isb-sib.ch/ 
software/SAPS_form.html) program was used to identify 
separated repeats, simple tandem repeats, and periodic re- 
peats in each mature bindin sequence (Brendel et al.. 1992). 

Results and Discussion 

Figure 1 shows the phylogenetic relationships among the 
echinoid orders from which bindin was sequenced, as they 
have been reconstructed from molecular, morphological, 
and fossil evidence (Littlewood and Smith. 1995; Smith et 
al., 1995). As the figure indicates, bindin is present not only 
in the Echinoida and the Arbacioida (from which it was 
previously known), but also in the sand dollars (Clypeas- 
teroida) and the heart urchins (Spatangoida), as well as the 
phylogenetically much more distant Diadematoida and Ci- 
daroida. Along with the sequence of Heliocidaris, reported 
in this paper, and the previously known sequences from 
Arbacia, Strongylocentrotus, Tripneustes, Lytechinus, and 
Echinometra. the data set covers orders that contain more 
than 70% of all extant echinoid species (Kier, 1977). The 
Cidaroida, the only extant order of the subclass Perischo- 
echinoidea. is the lineage most divergent from all other 
echinoids. It was separated from the Euechinoidea approx- 
imately 250 mya. Bindin's presence in both extant sub- 
classes of the Echinoidea indicates that it was present in 



Millions of years ago 



Species 



Order 



Source 




Eucidaris tributoides 

Diadema antillarum 

Encope stokesii 

Moira clotho 

Arbacia punctulata Arbacioida 

Strongylocentrotus purpuratus Echinoida 

Tripneustes ventricosus Echinoida 

Lytechinus variegatus Echinoida 

Heliocidaris erythrogramma Echinoida 

Echinometra oblonga Echinoida 



Cidaroida this study 

Diadematoida this study 

Clypeasteroida this study 

Spatangoida this study 

Glabe and Clark. 1991 
Gaoetal.. 1986 
Zigler and Lessios, 2003 
Minorca/., 1991 
this study 
Metz and Palumbi. 1996 



Figure 1. Phylogenetic relationships, divergence times, and systematic position of genera in which bindin 
has been sequenced. Echinoid phylogeny and divergence times are from Smith ( 1988) and Smith el al. ( 1995). 
Source of bindin sequence data is also indicated. 



EVOLUTION OF BINDIN 



11 



their common ancestor and that it has been evolving along 
each of the branches of the sea urchin phylogenetic tree for 
more than 250 my. Whether bindin is present in other 
echinoderms remains uncertain. Moy and Vacquier (1979) 
found that their antibody to Strongylocentrotus purpiirutiis 
bindin did not react with sperm from three species of sea stars. 
and "zoo blots" using S. purpiininis bindin sequences to 
probe genomic DNAs of a sea cucumber and a sea star were 
negative (Minor et at., 1991). No attempt has been made to 
determine bindin's presence in the ophiuroids or crinoids. 
Figure 2 indicates that the aligned mature bindin se- 
quences are a mosaic of highly conserved and highly diver- 



gent regions. Over the past 250 my, the 55 residues of the 
core (ami no acids 155-209) have been remarkably con- 
served. This region does not contain any insertions or de- 
letions in any echinoid lineage. Of the 55 amino acids, 45 
are conserved across all of the 10 exemplars, including a 
stretch of 29 residues in a row (amino acids 164-192). The 
B18 sequence of 18 amino acids implicated in membrane 
fusion (Ulrich et ill., 1998. 1999) is part of this perfectly 
conserved section. Seven amino acid sites in the core region 
exhibit a singleton amino acid change (i.e.. a change found 
in only one of the sequences). Four of these changes are 
conservative with respect to charge and polarity (amino 



Eucidaris trihulaides 
Diadema antillantm 
Encope stokesu 
Moira clolho 
Arbacia punctulala 
SlronKvlocenlroms purpuratus 
Tripneustes venlncosus 
Lvlechinus \-ariegalus 
Helioctdaris en-lhrogramma 
Echtnomelra oblonea 



RC F 

K Q R R 

RV 

RG 

RG FJP 

RK 

RK 



YV A 



GIT --- YT RGGGHC 

PT GN V GRA 

Y PMMM - - - PN 

A AVMD 

AQGA - - - GGMOGGYG 
V N T M - --C 

G N R - - 
GNM - - - - - - N 

GNMM - 
YJG ............. N YPQ 



T RPGE l[(fT 

GAOOGG|GTFAAYPPAQSGRPNYY|GPR 
A A PS P Y^N R 
GMPGD V|GG 

A GGAQY 



Q A P 
QGL 
Y P Q 
YPC 



|AMSPQW 



1NOQM C 
rtN Q P M C 
fN POM GGJG 
AMN P PMGGG 



QEV 
I P V 



A NN POPA YAG 
- . - MP 

POMGLPVQGYOGNQ|L 
MN Q G - - - - p PM 

GQPA .... 

PGQ- - - 

PGQP - - - - - P 

PGPG -AMI 

PVPGOAPMGOPAddG 



OC 


A 


V Y R 


GL 


P 


Q 




GPGP 


A A P 


RE 


Y 


GA P 


GA 


F 


Q 


QO YMEN PS L PSG 


RANG 


A A P 


MP 


M 


M P P 


GH 





N 




- GGP 


V G V 


C L 


P 


AVV 


PC 


C 


Q 


L PSGGL AGGGL P 


V GGL 


A GG 


C F 


P 


A A A 


GP 


A 


C 




GFG 


A PO 


OG 


Y 


ANQ 


GK 


G 


G 


C 


V GGG 


S - - 




1 
















OC 


Y 


A A P 


GM 


C 


G 


P 


V GGG 


GGG 



E.I. 
Da, 
.1. 
M.c. 
A.p 
Sp 
T.Y 
i.v. 
He 
E.O. 



13 




P D IS 
1 PG 
ADS 
GFP 
E A 


GGG 
PAY 
A A E 
GGG 
GGG 


100 125 




TO SO I -VAEDDG 






50 




16. 


G PRQGA PSG 
G P MN N 1 P S 1 
G L PVGGLAG 
GQP PV GOP 1 


h 
A 
G 
C 


GPSYGPAPAGPV I PPAEGAGGDFDSVSRTMESDQPLHE 


SL 

SN 

ss 


T SA 
T NA 
T N A 
T N A 
T SA 
T SA 
T SA 
L SD 
T SA 


D 
E 
D 
E 

F! 
R 
K 
R 
K 


V L E 

TME 
MME 
VME 
VMD 
V MQ 
V I Q 
V L E 
VMD 


K 
K 

F 
K 

D 
D 
D 
N 
N 


KA V LGAT K D L 
KAV LGATKVD L 
RA 1 LGATK D L 
KA VLGAT K D L 
KAV L GAT K D L 
K A V L GA T K D L 
KAV LGATK D L 
KAV LGAT K D L 
KAV L GAT K D L 






AEITADLETGGE 






GS 
S S 
SS 


SSSS - - V DGGDT 
AST---.EEGET 


NRG 
GAVGAGAMG 


G 
C 


P G 
P G 


GGG 

GGG 










G - GAMA 


R 


PFG 


GGG 


AMAGPVGGGGAGPPEFGGMPAAE- - -GAEGEGDED 


S S 


SV EEET 



Et 
Da. 
E! 
Me 
A.p, 
Sp 
T.v. 
Lv. 
He 
E.o. 



El 
D.a. 
E.s 
M.c. 

A.p 
S.p 



H.e. 
E.o. 



PV D 
PVD 
PV D 
PVD 
PV D 
PVD 
PV D 
PVD 
PVD 


ND P 
N D P 
N D P 
ND P 
ND P 
NDPl 
NO PI 
ND f\ 
NDPl 


DLGLLLRHLRHHSNLLAN 
DLGLLLRHLRHHSNLLAN 
DLGLLLHHLHHHSNLLAN 
DLGLLLRHLRHHSNLLAN 
DLGLLLRHLHHHSNLLAN 
'DLGLLLRHLHHHSNLLAN 
DLGLLLRHLRHHSNLLAN 
'DLGL LLRHLHHHSNLLAN 
'DLGLLLRHLRHHSNLLAN 


GD PD 
DOPE 
GDPE 
GDPE 
GD PA 
GDPE 
GDPE 
GD PA 
GDPE 


/RGQVLTAMQEDEYEEERDA 
REQVLAAMQEEE- - -EQDA 
RNQVLSAMQEEEQEEEODA 
RNQVLTAMQEEEEEEEQDA 
REQVLSAMQEEEEEEEEDA 
REQVLSAMQEEEEEEEQDA 
REQVLSAMQEEEEEEENDA 
REQVLTAMQEEEEEEQODA 
REQVLSAMQEEEEEEEQDA 


V T GA F 
N - - F 
N A V F 
N GV F 
TGAC 
NGA F 
N GV F 
N GV F 
N GV F 


E E 
PD 
NE 

DN 
QG 
DN 
EN 
N N 
DN 


LMA L 
ETLT 
V L N N 
V L N N 


N 
G 
V 
L 


A 
P 

N 

N 


GGGEGAVEHRHI RANQDYSEQEEEES 








V L NN 
V L N N 
V L N N 
V L N N 


L 

L 
L 
1 


N 
N 
N 
N 








AN 


RR-R'-RR'-R'-'-C' 





D I NQDSQEEPSDYPONGElG|vaASOYa|NN 
A 



A 

G 

G 



PIAOGC 
GQGC 

OlOJFGGC 
GRAG 



A GA G 
SO Y 

M F N 



- MG 

i F P 



GGWQ 



G- 
GJGGGlG 



qooqc 

RSDFDOE|s|aOVNRFRSP|SE 
G A A 

GJGJGPAPlFGGJOGOFGGNMGMV 
P GA G A V A G - - A A M A 
APJGQAGFGGGGGGGAWMSP 



QPQL GMGG Y 

MP P YPGGAO GGJMRV 

QPQGM I GQP 

T A - - 

AG- - 



GGMGMAGGGGGGCGGM 



YTNC 

NDRNAGFPFDDDYEAOFGDGJGN 



GGJMGAPVGMGNAGRYNNYAOGC 
- GGQPQN 
MGFPHEGMGGPPQGMGMPH 
GGJL GR - - - - MGN 
MGN 



G|G|GPMGGGGPM,GG|GGM MGFQGMGCOPP- 

'GMGC QV 



MGL P 



YSG 
A F 
PMGG 



345 
RGGY 

EOHRSGFENEFAQDRDND 



'PQGMGMPP 
IAPG 



El. 
Da 
E.s. 
M.c. 
A.p. 
Sp 
T.v 
L.V. 
H.e 
E.o. 



EOEDDYVDDDSDYDDEDEPEYQQEARRYGQRPQ 


V '; 

FC 


G - P 
G - Y 


A Y 
P H 




NGQ 


EDNYHRQETRNNPYGQRQARAGRQGGSRAGVSQRRGR' 






















































>' 














YN 

Y- N 


- - P 
- - P 


GF 
GY 


R - 
R - 


PG ' 
QG- 




N A 





Figure 2. Mature bindin amino acid alignment. The first four amino acids are the presumed cleavage site 
from preprohindin. Sites at which amino acids are identical in more than 50% of the genera are enclosed in 
boxes. Conservation across all 10 genera is indicated by an asterisk below the site. Dashes indicate deletions. The 
site for which an intron is known to exist in Echinometra, Arbacia, Strongylocentrotus, Lytechinus. Tripneustes. 
Heliocidaris, and Diadema is indicated by an "I" under the alignment. The core region is shaded. The B 1 8 region 
of the core is indicated by a bar beneath the alignment. Sites in the core where radical amino acid changes have 
occurred are marked with an 'R' under the alignment; sites in the core where only conservative amino acid 
changes have occurred are marked with a 'C'. Stop codons are indicated by an asterisk after the last amino acid. 



12 



K. S. ZIGLER AND H. A. LESSIOS 



acids at positions 155, 157, 164. and 208), and three are 
radical (positions 193, 194, and 200). Each of positions 196, 
199, and 203 contain three amino acids across the 10 
genera, indicating that there have been at least two changes 
at each of these sites. At least one of the changes at each site 
must have been a radical change. Thus, radical changes are 
observed in only six amino acid positions of the core region, 
all of them concentrated in a small portion of the core close 
to the C terminus (amino acids 193, 194, 196, 199, 200, and 
203). The rest of the core (amino acids 155 through 192 and 
204 through 209) contains only four conservative singleton 
amino acid substitutions. 

A second conserved region is the cleavage site at the 
border between preprobindin and mature bindin (Fig. 2). In 
Strongylocentrotus piirpuratus, the cleavage site is marked 
by a motif of four basic amino acids (RKKR) (Gao et al., 
1986). Multibasic motifs are also present in the other nine 
genera (Fig. 2). Such multibasic motifs typically mark the 
cleavage sites of proproteins from the mature molecule 
during the secretory process through the action of propro- 
tein convertases (Steiner, 1998; Seidah and Chretien, 1999). 
The conservation of this multibasic motif in bindin rein- 
forces the idea that it functions as a signal for the cleavage 
of preprobindin from mature bindin in all echinoids. 

In contrast to the core and to the cleavage site, the rest of 
the molecule is so variable between orders that we have 
little confidence that the alignment of these regions depicted 
in Figure 2 is correct. There is a great amount of variation 
in the length of mature bindin both on the 5' and on the 3' 
side of the molecule (Table 1 ). This study identifies both the 
longest and the shortest bindins described to date. Bindin in 
Diadema antillarum (418 amino acids) is more than twice 
as long as bindin in Encope stokesii (193 amino acids). 
Bindin length 5' of the core ranges from 78 to 148 amino 
acids, while bindin length 3' of the core ranges from 56 to 
215 amino acids. There seems to be no discernible evolu- 
tionary trend in bindin length. Closely related orders do not 

Table 1 

Number of amino acids in three regions of I he mature bindin in 10 
genera 



Core 



Total 



Eucidaris 


101 


55 


60 


216 


Diadema 


148 


55 


215 


418 


Encope 


82 


55 


56 


193 


Moira 


138 


55 


94 


287 


Arbacia 


105 


55 


73 


233 


Lytechinus 


103 


55 


60 


218 


Tripneustes 


88 


55 


68 


211 


Strongylocentrotus 


82 


55 


99 


236 


Heliocidaris 


78 


55 


73 


206 


Echinometra 


111 


55 


75 


241 



5' and 3' regions are defined relative to the conserved core. 



tend to have bindins that are of similar length. Indeed, it 
cannot be assumed that the genera we have included in the 
study are representative of their orders in this respect. The 
regions on either side of the core were found to confer 
species-specificity in Strongylocentrotus (Lopez et al., 
1993). If their variation reflects the requirements of this 
function, they can be expected to vary in a phylogenetically 
unpredictable fashion. 

An intron located at a conserved position just 5' of the 
core region has been identified in the mature bindins of 
Echinometra (Metz and Palumbi, 1996), Arbacia (Metz et 
al., 1998a), Strongylocentrotus (Biermann, 1998), and Tri- 
pneustes (Zigler and Lessios, 2003). In each of these genera, 
the intron is located at a conserved valine (amino acid 150 
in Fig. 2). Comparison of sequences derived from both 
cDNA and from genomic DNA in Heliocidaris (Zigler et 
al., 2003), Lytechinus, and Diadema revealed that in these 
genera the intron also exists at the same location and that its 
point of insertion is also a valine. We have made no attempt 
to amplify bindin from genomic DNA in Eucidaris, Moira, 
and Encope, so we do not know whether this intron is a 
universal feature of all bindins. These three genera do not 
have a valine in the site at which the intron is known to exist 
in the others, but the significance of this pattern cannot be 
evaluated with the present data. 

Previous studies have identified both bindins with gly- 
cine-rich repeat structures and bindins that lack such struc- 
ture. Glycine-rich repeats were found in the bindins of 
Lytechinus (Minor et al., 1991), Strongylocentrotus (Bier- 
mann, 1998), Echinometra (Metz and Palumbi, 1996), and 
Tripneustes (Zigler and Lessios, 2003), all members of the 
order Echinoida. Consistent with the phylogenetic position 
of Heliocidaris, its bindin also contains glycine-rich repeat 
sequences, with MGGGN and VGGGGP on the 5' side of 
its core, and the series MGGG-MGGGGP-MGGGGP- 
MGGGGM-MGFQG-MGGQPP on the 3' side. Although 
Moira belongs to a different order, its bindin also contains 
extensive glycine-rich repeats, with the sequence PGGGL- 
PSGGL-AGGGL-PVGGL-AGGGL-PVGGL-AGGGF- 
PGGGL-QGGGF-QGGGL-PGGGG found 5' of the core. 
Glabe and Clark (1991) noted that bindin from Arbacia 
punctulata lacked significant repeat structure, and this ob- 
servation was extended to three other species of Arbacia 
(Metz et al., 1998a). Eucidaris, Encope, and Diadema re- 
semble Arbacia in containing only minimal tandem or sep- 
arated repeats, the longest of which is PAAP-PPAP-PAAP 
in the region flanking the 5' side of the core in Eucidaris. 
Thus, glycine-rich repeat structure remains a common trait 
of the bindin of the Echinoida. although, as the data from 
the spatangoid Moira indicate, it is not a characteristic 
limited to this order or even to a closely aligned clade. 

There are no cysteine or tryptophan residues in any 
mature bindin. Disulfide bonds formed between cysteine 
residues are often critical for protein structure, and in rap- 



EVOLUTION OF BINDIN 



13 



idly evolving proteins such as toxins of cone snails (Duda 
and Palumhi. 1999) and pheromones of the marine ciliate 
Euplotefi (Luporini et al., 1995) cysteine residues are of- 
ten among the most conserved amino acids, serving as 
guides for aligning sequences. Thus, the lack of cysteine 
residues in bindin may have important structural conse- 
quences. When all sequences are pooled, glycine is by far 
the most common amino acid outside the core, constituting 
nearly a quarter of all residues. If the orders that possess 
glycine-rich repeats (Echinoida and Spatangoidu) are sepa- 
rated from those that do not. glycine remains the most 
common amino acid in both categories, constituting 29.6% 
of the non-core amino acids in the former and 16.4% of 
non-core residues in the latter. The six most common resi- 
dues outside the core (G, A, P, Q, N. and E) compose 63.9% 
of all non-core residues. Leucine is the most common amino 
acid in the core, present in 10 completely conserved amino 
acid positions, including 6 of the 18 amino acids in the B 18 
region. There is a much higher proportion of charged resi- 
dues in the core (31.8%) than in the rest of the molecule 
( 15.6%). Each of the five charged amino acids (E, D. R. H, 
and K) is more common in the core. 

Another common feature of all bindins is their lack of 
codon usage bias. ENC values among the 10 genera range 
from 61 (for Eucidaris and Diadenui) to 48.1 (for Arbacia), 
with an average of 56.4. Low levels of codon usage bias 
have also been observed in sex-related genes in Drosopliila 
(Civetta and Singh. 1998) and in the Chlamvdomonas mat- 
ing-type locus genes Mid and Fusl (Ferris et al., 2002). 

Given the large divergence in amino acid sequence and 
length (and the uncertainties in alignments), it is not sur- 
prising that hydrophobicity plots (Fig. 3) from these bindins 
are diverse. The conserved amino acid sequence of the core 
and its flanking regions causes all plots to be similar through 
the middle of the molecule. Plots of the closely related 
Tripneustes ventricosus, Lytechinus variegatus. Helioci- 
daris erythro gramma, and Echinometra oblonga bindins are 
similar throughout their lengths. The rest of the hydropho- 
bicity plots are not clearly similar. One particularly distinct 
region is the long hydrophilic stretches in Diadema bindin 
along its extended length. A second is the highly hydropho- 
bic region 3' of the core of Arbacia bindin. noted by Glabe 
and Clark (1991). 

The only other gamete recognition protein that has been 
studied in marine invertebrates separated for as long as 250 
my is the gastropod sperm protein lysin. Lysin opens a hole 
in the vitelline envelope of free-spawning snails and thus 
enables sperm to penetrate to the plasma membrane of the 
egg. It has been studied in the abalones (Hciliotis) (Lee and 
Vacquier, 1992; Lee et al., 1995: Yang et al., 2000; re- 
viewed in Kresge et al., 2001 ) and in two genera of turban 
snails, Tegula and Norrisin (Hellberg and Vacquier. 1999). 
Abalones and turban snails diverged 250 mya. roughly the 
same time the cidaroids separated from the euechinoids. The 



E.i. 

D.a. 

E.s. 

M.c. 
A.p. 

S.p. 
T.v. 
L. v. 
H.e. 
E.o. 






"/V 



~^\/ 



uy f 



<Y\?Vv jyvW^ 





Figure 3. Hydrophobicity plots of mature bindin in 10 genera. The 
genera are presented in the same order as in Figure 1 and abbreviated as 
follows: E.t.: Eucidaris tribuloides, D.a.: Diadema aniilliinini, E.s.: En- 
cope stokesii, M.c.: Moira clotho. A.p.: Arbacia punciiilata. S.p.: Stmngv- 
locentrotus pnrpn ranis, T.v.: Tripneustes ventricosus. L.r.: Lytechinus 
variegatus, H.e.: Heliocidaris etythrogramma, and E.o.: Echinomelra ob- 
longu. A box encloses the core, and a bracket indicates the hydrophobic 
region in Arbacia. The scale bars above and below each zero line mark +1 
and 1, respectively. 



additional bindin sequences reported here reinforce the con- 
clusions of Hellberg and Vacquier ( 1999) from comparisons 
between the modes of evolution of these two proteins. 
Although they are both involved in gamete recognition and 
both lack cysteine residues, they evolve in different fash- 
ions. There is no equivalent of a bindin core region in lysin: 
amino acid substitutions are spread throughout the mole- 
cule, with only three amino acids conserved between all 
Haliotis species and the two teguline genera. Instead of 
conserving a section of the molecule, lysin has maintained 
its function by conserving secondary structure through con- 
servative amino acid substitutions (Hellberg and Vacquier. 
1999). Length variation is another obvious difference. Ma- 
ture bindin length varies from 193 to 418 amino acids, but 
lysin length (at least in the two groups studied to date) only 
from 126 to 138 amino acids. 



14 



K. S. ZIGLER AND H. A. LESSIOS 



Conclusions 

The comparisons of bindin from 10 genera of echinoids 
reveal the results of long-term evolution under two oppos- 
ing selective forces acting on gamete recognition molecules. 
The sections of the molecule involved in the basic functions 
of gamete fusion and post-translational cleaving of the 
preprobindin have been remarkably conserved over 250 my 
of evolution, presumably through purifying selection. The 
sections involved in species recognition have been evolving 
rapidly in seemingly unpredictable directions, presumably 
under diversifying selection; such changes are likely to be 
specific to each species. 

A number of features identified by these comparisons are 
in need of functional explanations. Among the conserved 
features, the lack of change in the core region is the only one 
that can be easily explained. We do not yet know whether 
there is a particular reason for the low codon usage bias of 
all bindins, for the absence of tryptophan or cysteine resi- 
dues, or for the absence of major hydrophobic regions in all 
bindins except that of Arbacia. The differences between the 
orders are equally puzzling. Is there a functional reason for 
the length variation of the regions outside the core? Why do 
the Echinoida and the Spatangoida have glycine-rich repeats 
in the regions flanking the core, while other orders do not? 
Comparisons alone cannot provide answers to these ques- 
tions; but they can identify features of the molecule that are 
worthy of functional study. 

Acknowledgments 

We are grateful to A. and L. Calderon for providing 
support in the laboratory, to M. McCartney for primer 
design and advice on the RACE technique, to T. Duda for 
collecting Moira clotho, and to E. Popodi for providing 
testis RNA from Heliocidaris ery thro gramma. Comments 
from C. Cunningham, D. McClay, R. Sponer. W. Swanson, 
V. Vacquier, and two anonymous reviewers improved the 
manuscript. This work was supported by National Science 
Foundation and Smithsonian predoctoral fellowships to 
KSZ, by the Duke University Department of Zoology, and 
by the Smithsonian Molecular Evolution Program. 

Literature Cited 

Biermann, C. H. 1998. The molecular evolution of sperm bindin in six 
species of sea urchins (Echinoida: Strongylocentrotidae). Mol. Biol. 
Evol. 15: 1761-1771. 

Brandriff, B., G. W. Moy, and V. D. Vacquier. 1978. Isolation of 
sperm bindin from the oyster ( Crassostrea gigas). Gamete Res. 89: 
89-99. 

Brendel, V., P. Bucher, I. Nourbakhsh, B. E. Blaisdell, and S. Karlin. 
1992. Methods and algorithms for statistical analysis of protein se- 
quences. Proc. Natl. Acad. Sci. USA 89: 2002-2006. 

Civetta, A., and R. S. Singh. 1998. Sex-related genes, directional sexual 
selection, and' speciation. Mol. Biol. Evol. 15: 901-909. 

Debenham, P., M. A. Brzezinski, and K. R. Foltz. 2000. Evaluation of 



sequence variation and selection in the hindin locus of the red sea 
urchin, Strongylocentrotus franciscanus. J. Mol. Evol. 51: 481-490. 

Duda, T. F., and S. R. Palumbi. 1999. Molecular genetics of ecological 
diversification: duplication and rapid evolution of toxin genes of the 
venomous gastropod Conns. Proc. Natl. Acad. Sci. USA 96: 6820- 
6823. 

Ferris, P. J., E. V. Armbrust, and U. W. Goodenough. 2002. Genetic 
structure of the mating-type locus of Chlamydomonas reinhardtii. 
Genetics 160: 181-200. 

Gao, B., L. E. Klein, R. J. Britten, and E. H. Davidson. 1986. Se- 
quence of mRNA coding for bindin, a species-specific sperm protein 
required for fertilization. Proc. Natl. Acad. Sci. USA 83: 8634-8638. 

Glabe, C. G., and D. Clark. 1991. The sequence of the Arbacia punclu- 
lata bindin cDNA and implications for the structural basis of species- 
specific sperm adhesion and fertilization. Dev. Biol. 143: 282-288. 

Hellberg, M. E., and V. D. Vacquier. 1999. Rapid evolution of fertil- 
ization selectivity and lysin cDNA sequences in teguline gastropods. 
Mol. Biol. Evol. 16: 839-848. 

Hofmann. A., and C. G. Glabe. 1994. Bindin, a multifunctional sperm 
ligand and the evolution of new species. Semin. Dev. Biol. 5: 233-242. 

Hughes, A. L., T. Ota, and M. Nei. 1990. Positive Darwinian selection 
promotes charge profile diversity in the antigen-binding cleft of class I 
major-histocompatibility-complex molecules. Mol. Biol. Evol. 7: 515- 
524. 

Kier, P. M. 1977. The poor fossil record of the regular echinoid. Paleo- 
biology 3: 168-174. 

Kresge, N., V. D. Vacquier, and C. D. Stout. 2001. Abalone lysin: the 
dissolving and evolving sperm protein. Bioessays 23: 95-103. 

Kyte, J., and R. F. Doolittle. 1982. A simple method for displaying the 
hydrophobic character of a protein. J. Mol. Biol. 157: 105-132. 

Lee, Y.-H., and V. D. Vacquier. 1992. The divergence of species- 
specific abalone sperm lysins is promoted by positive Darwinian se- 
lection. Biol. Bull. 182: 97-104. 

Lee, Y.-H., T. Ota, and V. D. Vacquier. 1995. Positive selection is a 
general phenomenon in the evolution of abalone sperm lysin. Mol. Biol. 
Evol. 12: 231-238. 

Littlewood, D. T. J., and A. B. Smith. 1995. A combined morphological 
and molecular phylogeny for sea urchins (Echinoidea: Echinodermata). 
Philns. Trans. R. Soc. Land. B 347: 213-234. 

Lloyd, A. T., and P. M. Sharp. 1992. CODONS: A microcomputer 
program for codon usage analysis. J. Hered. 83: 239-240. 

Lopez, A., S. J. Miraglia, and C. G. Glabe. 1993. Structure/function 
analysis of the sea-urchin sperm adhesive protein bindin. Dev. Biol. 
156: 24-33. 

Luporini, P., A. Vallesi, C. Miceli, and R. A. Bradshaw. 1995. Chem- 
ical signaling in ciliates. J. Eukaryot. Microbiol. 42: 208-212. 

Metz, E. C., and S. R. Palumbi. 1996. Positive selection and sequence 
rearrangements generate extensive polymorphism in the gamete recog- 
nition protein bindin. Mol. Biol. Evol. 13: 397-406. 

Metz, E. C., G. Gomez-Gutierez, and V. D. Vacquier. 1998a. Mito- 
chondria! DNA and bindin gene sequence evolution among allopatric 
species of the sea urchin genus Arbacia. Mol. Biol. Evol. 15: 1 85-195. 

Metz, E. C., R. Robles-Sikisaka, and V. D. Vacquier. 1998b. Nonsyn- 
onymous substitution in abalone sperm fertilization genes exceeds 
substitutions in introns and mitochondrial DNA. Proc. Natl. Acad. Sci. 
USA 95: 10,676-10.681. 

Minor, J. E.. D. R. Fronison, R. J. Britten, and E. H. Davidson. 1991. 
Comparison of the bindin proteins of Strongylocentrotus franciscanus. 
S. inirpiiratus, and Lytechinus variegatus: sequences involved in the 
species specificity of fertilization. Mol. Biol. Evol. 8: 781-795. 

Moy, G. W., and V. D. Vacquier. 1979. Immunoperoxidase localization 
of bindin during the adhesion of sperm to sea urchin eggs. Curr. Top. 
Dev. Biol. 13: 31-44. 

Rambaut, A. 1996. Se-Al: Sequence Alignment Editor. University of 



EVOLUTION OF BINDIN 



15 



Oxford. Oxford [Online]. Available: http://evolve.zoo.ox.ac.uk/ (ac- 
cessed June 2003]. 

Seidah, N. G., and M. Chretien. 1999. Proprotein and prohormone 
convertases: a family of subtilases generating diverse bioactive polypep- 
tides. Brain Hi-,. 848: 45-62. 

Smith, A. B. 1984. Echinoid Paleobiology. George Allen and Unwin, 
London. 190 pp. 

Smith, A. B. 1988. Phylogenetic relationship, divergence times, and 
rates of molecular evolution for camarodont sea urchins. Mai. Bio/. 
Evol. 5: 345-365. 

Smith. A. B., D. T. J. Littlewood. and G. A. Wray. 1995. Comparing 
patterns of evolution: larval and adult life history stages and ribosomal 
RNA of post-Paleozoic echinoids. Philos. Trims. R. Sm: Lond. B 349: 
11-18. 

Steiner, D. F. 1998. The proprotein convertases. Ciirr. Opin. Chem. Bil. 
2: 31-39. 

Swanson. \V. J., and V. D. Vacquier. 2002. Reproductive protein 
evolution. Annii. Rci. Ecoi Sysr. 33: 161-179. 

Takagi. T., A. N'akamura. R. Deguchi. and K. Kyozuka. 1994. Isola- 
tion, characterization, and primary structure of three major proteins 
obtained from Mytitus etlulis sperm. J. Biochem. 116: 598-605. 

Taylor. \V. R. 1986. The classification of amino acid conservation. J. 
Theor. Biol. 119: 205-218. 

Thompson. J. D.. T. J. Gibson, F. Plewniak, F. Jeanmougin. and D. G. 
Higgins. 1997. The ClustalX windows interface: flexible strategies 
for multiple sequence alignment aided by quality analysis tools. Nu- 
cleic Acids Res. 24: 4876-4882. 



Ulrich. A. S., M. Otter, C. G. Glabe, and D. Hoekstra. 1998. Mem- 
brane fusion is induced by a distinct peptide sequence of the sea urchin 
fertilization protein bindin. J. Biol. Chem. 273: 16.748-16,755. 

Ulrich, A. S., W. Tichelaar, G. Forster, O. Zschornig, S. Weinkauf, and 
H. W. Meyer. 1999. Ultrastructural characterization of peptide-in- 
duced membrane fusion and peptide self-assembly in the lipid bilayer. 
B//'/i.v.v. J. 77: 829-841. 

Vacquier, V. I). 1998. Evolution of gamete recognition proteins. Science 
281: 1995-1998. 

Vacquier, V. D., and G. W. Moy. 1977. Isolation of bindin: the protein 
responsible for adhesion of sperm to sea urchin eggs. Proc. Nail. Acad. 
Sci. USA 74: 2456-2460. 

Vacquier, V. D., VV. J. Swanson. and M. E. Hellberg. 1995. What have 
we learned about sea urchin sperm bindin' 1 Dev. Growth Differ. 37: 
1-10. 

Wright, F. 1990. The "effective number of codons" used in a gene. Gene 
87: 23-29. 

Vang, Z., W. J. Swanson. and V. D. Vacquier. 2000. Maximum like- 
lihood analysis of molecular adaptation in abalone sperm lysin reveals 
variable selective pressures among lineages and sites. Mol. Biol. Evol. 
17: 1446-1455. 

Zigler, K. S., and H. A. Lessios. 2003. Evolution of bindin in the 
pantropical sea urchin Tripneustes: comparisons to bindin of other 
genera. Mol. Biol. Evol. 20: 220-231. 

Zigler, K. S., E. C. Raff, E. Popodi, R. A. Raff, and H. A. Lessios. 2003. 
Adaptive evolution of bindin is correlated with the shift to direct 
development in the genus Heliocidaris. Evolution (In press). 



Reference: Biol Bull 205: 16-25. (August 2003) 
2003 Marine Biological Laboratory 



Behavioral Characterization of Attractin, a Water- 
Borne Peptide Pheromone in the Genus Aplysia 

SHERRY D. PAINTER*. BRET CLOUGH, SARA BLACK, AND GREGG T. NAGLE 

The Marine Biomedical Institute and the Department of Anatomy and Neitrosciences, 
University of Texas Medical Branch, Galveston. Texas 77555-1069 



Abstract. Pheromones play a significant role in coordi- 
nating reproductive activity in many animals, including 
opisthobranch molluscs of the genus Aplysia. Although 
solitary during most of the year, these simultaneous her- 
maphrodites gather into breeding aggregations during the 
reproductive season. The aggregations contain both mating 
and egg-laying animals and are associated with masses of 
egg cordons. The egg cordons are a source of pheromones 
that attract other Aplysia to the area, reduce their latency to 
mating, and induce egg laying. One of these water-borne 
egg cordon pheromones ("attractin") has been characterized 
and shown to be attractive in T-maze assays. Attractin is the 
first water-borne peptide pheromone characterized in inver- 
tebrates. 

In the current studies, behavioral assays were used to 
better characterize the attraction, and to examine whether 
attractin can induce mating. Although the two activities 
could be related (i.e., attraction occurring because animals 
were looking for a partner), this was not tested. T-maze 
assays showed that attractin works as part of a bouquet of 
odors: the peptide is attractive only when Aplysia brasiliana 
is part of the stimulus. The animal does not need to be a 
conspecific, perhaps explaining why multiple species may 
be associated with one aggregation. Native and recombinant 
attractin are equally attractive, verifying that /V-glycosyla- 
tion at residue 8 is not required for attraction. 

Mating studies showed that both native and recombinant 



Received 8 October 2002: accepted 16 April 2003. 

* To whom correspondence should be addressed. Marine Biomedical 
Institute. 2.138 Medical Research Building. University of Texas Medical 
Branch, 301 University Blvd., Galveston, Texas 77555-1069. E-mail: 
sdpainter@houston.rr.com 

Abbreviations: ASW, artificial seawater; Att. attractin; CH,CN, aceto- 
nitrile; HFBA, heptafluorobutyric acid; M-REP. Marine Research and 
Educational Products; RP-HPLC, reversed-phase high performance liquid 
chromatography. 



attractin reduce the latency to mating. The effects are larger 
when hermaphroditic mating is considered: in addition to 
reducing latency, attractin doubles the number of pairs 
mating as hermaphrodites. The effect may result from at- 
tractin stimulating both animals to mate as males and would 
be consistent with behaviors previously seen in the T-maze. 
Attractin may thus be contributing to the formation of 
copulatory chains and rings seen in aggregations in the field. 
These results may be interpreted in two ways: ( 1 ) attrac- 
tin has multiple activities that contribute to the establish- 
ment and maintenance of the aggregation; or (2) the induced 
desire to mate may make attractin attractive when it is 
presented in conjunction with an animal. In either case, the 
results open the door for cellular and molecular studies of 
mechanism of action. 

Introduction 

Chemical communication is the most ancient form of 
communication and is used by most, if not all, animals 
examined. The organisms include, for example, ciliated 
protozoans (Luporini et al.. 1995), yeast (Kodama et ai, 
2003), insects (Monsma and Wolfner, 1988; Roelofs et al., 
2002; Saudan et al.. 2002), molluscs (Painter et al., 1998), 
worms (Ram el al.. 1999), fish (Li et al., 2002), amphibians 
(Kikuyama et al.. 1995; Rollmann et al., 1999; Wabnitz et 
al., 1999), rodents (Stowers et ai, 2002; Novotny, 2003) 
and humans (Savic et al.. 2001.). The number of phero- 
mones characterized in each species depends, at least in 
part, on the chemical nature of the pheromones and on 
whether the pheromones are water-borne. 

Opisthobranch molluscs of the genus Aplysia are simul- 
taneous hermaphrodites that do not normally fertilize their 
own eggs. Field studies (Kupfermann and Carew, 1974; 
Audesirk, 1979; Susswein et al.. 1983, 1984) have shown 
that they are solitary animals that move into breeding ag- 



16 



APLYSIA PHEROMONAL ATTRACTANT 



17 




76 aa 



iQNCDIGNITSQCQMQHKNCEDANGCDTIIEECKTSMVERCQNQEFESAAGSTTLGPQ 
QNCD I GN I TSOCQMQH!lNc3DANGCDTI I E E C KT S MVE RC Q NQE FE S A 



Figure 1. I A) Schematic diagram of the precursor to the uttractin pherotnone from the albumen gland of 
.A/>/v.v/'(i californica. Cleavage of the signal sequence (arrow) generates the 58-residue pheromone attractin. The 
disulfide-bonding pattern of cyxleine residue-. (S) is I-IV. II-V. and 11I-1V, where the Roman numeral indicates 
the order of occurrence in the primary sequence (Schein el al.. 2001). Unlike attractin. the precursors for 
pheromones that act as part of a group of scents often contain sequences of more than one scent. (B) The amino 
acid sequences of attractin from the two species of Aplysia used in the current studies, A. californicu and A. 
briisiliiinu (Painter el al.. 1998, 2000). Amino acid residues that are identical to those in A. californica attractin 
are indicated h\ the black background. 



gregations during the reproductive season. The aggregations 
typically contain both mating and egg-laying animals and 
are associated with masses of recently deposited egg cor- 
dons, often deposited one on top of another. Most of the 
egg-laying animals mate simultaneously as females even 
though mating does not cause reflex ovulation (Blankenship 
ft al., 1983), suggesting that egg laying precedes mating in 
the aggregation and that egg laying may release pheromones 
that establish and maintain the aggregation. 

Similar observations have been made in the laboratory 
when animals were not individually caged (Audesirk. 1979; 
Blankenship et al.. 1983; Susswein et al.. 1983, 1984). and 
behavioral studies have shown that egg-laying animals with 
cordons are more attractive than sexually mature but non- 
laying conspecifics (Aspey and Blankenship, 1976; Jahan- 
Parwar. 1976; Audesirk. 1977; Painter et al.. 1989). T-maze 
assays show that at least some of the attractants derive from 
the egg cordon and are waterborne: (1) recent egg-layers 
without egg cordons are no more attractive than non-laying 
conspecifics; (2) recently deposited egg cordons are attrac- 
tive, with or without a non-laying conspecific, but sham egg 
cordons are not; and (3) both recently deposited egg cordons 
and their eluates increase the attractiveness of non-laying 
conspecifics when placed in the surrounding seawater 
(Painter et al.. 1991: Painter, 1992). 

One of the water-borne pheromonal attractants has been 
isolated from eluates of the egg cordon and characterized. 
Named attractin, it is a 58-residue peptide that has six 
cysteines that form three intramolecular disulfide bonds 
(Fig. 1; Painter et al.. 1998; Schein et al.. 2001). Attractin 
was isolated from a Pacific Coast species (A. californica) 
and bioassayed in a species from the Gulf of Mexico (A. 
brasiliana). This was done because individuals of .4. cali- 
fornica tend to crawl out of T-maze chambers before they 
are exposed to the stimulus. A. californica attractin was 
attractive to A. brasiliana and produced behaviors that were 
suggestive of mating (Painter et al.. 1998), but these be- 
haviors were not further analyzed. The amount of attrac- 



tin that was attractive to conspecifics and induced the 
potential mating behaviors (1-10 pmol in 6 1 artificial sea- 
water) was in the range of concentrations normally observed 
with pheromones, demonstrating that attractin has phero- 
monal activity. 

There is no geographical overlap between the distribu- 
tions of the two species, suggesting that attractin or an 
attractin-related peptide is a pheromonal attractant in A. 
brasiliana. A peptide was subsequently isolated from the A. 
brasiliana albumen gland and sequenced. It is 58 amino 
acids in length and differs from A. californica attractin at 
only 3 amino acids (Fig. 1; Painter et al. 2000). It is 
deposited on the egg cordon and elutes into the seawater 
following deposition. It could thus serve a pheromonal 
function in A. brasiliana, but its pheromonal activities have 
yet to be tested. 

In the present study, behavioral assays were used to better 
characterize the attraction and to examine whether mating is 
induced. The current T-maze assays showed that attractin 
works as part of a bouquet of water-borne odors: the peptide 
is attractive only when individuals of A. brasiliana or A. 
californica are part of the stimulus. The animal does not 
need to be a conspecific, perhaps explaining why multiple 
species of Aplysia may be associated with one aggrega- 
tion for example, A. vaccaria with A. californica aggre- 
gations (Kupfermann and Carew. 1974; Pennings, 1991); A. 
californica with A. vaccaria aggregations (S. LePage, Ma- 
rine Research and Educational Products (M-REP), pers. 
comm.); and A. depilans with A. fasciata aggregations 
(Achituv and Susswein, 1985). Recombinant attractin was 
also tested for two reasons: ( 1 ) to see whether W-glycosyl- 
ation at Asn 8 is necessary for attraction (native attractin is 
glycosylated and recombinant attractin is not); and (2) to see 
whether the two are equally attractive, so that recombinant 
uttractin could be used in 3D nuclear magnetic resonance 
solution structure studies (Garimella et al., 2003) and for 
future studies of mechanism of action at the receptor level. 

A series of mating assays was performed because behav- 



18 



S. D. PAINTER ET AL 



iors in earlier T-maze assays suggested that both the stim- 
ulus and test animals wanted to mate (Painter et at.. 1998). 
The assays showed that attractin reduces the latency to 
mating at concentrations consistent with a pheromone. At- 
tractin also reduces the latency to hermaphroditic mating 
and doubles the number of pairs mating as hermaphrodites. 
This effect may result from attractin stimulating both ani- 
mals to mate as males and would be consistent with behav- 
iors seen in earlier T-maze assays (Painter et al., 1998). 
These results suggest that attractin, acting in an aggregation 
where there are more animals, could be at least partially 
responsible for the copulatory chains and rings that have 
been observed. Recombinant attractin also induces mating, 
both one-way and as a hermaphrodite, showing that N- 
glycosylation is not required for the induction of either type 
of mating. The attraction and mating data demonstrate that 
attractin may contribute to the establishment and mainte- 
nance of breeding aggregations, and to successful reproduc- 
tion. 

Materials, Methods, and Results 

Animals 

Specimens of Aplysia brasiliana (Rang), ranging in 
weight from 100 to 500 g, were collected from South Padre 
Island, Texas, and were used in experiments between June 
and September. A. brasiliana was used as the experimental 
animal in the T-maze and mating experiments because it is 
more reproductively active than A. califomica (see fig. 2 in 
Painter et al., 1998), does not crawl out of T-mazes, makes 
fewer false choices, and can be collected in large numbers 
from the south Texas coast during the reproductive season. 
Previous T-maze assays (Painter et al., 1998) showed that 
an individual of A. brasiliana is attracted to a non-laying 
conspecific and displays behaviors suggestive of mating 
when 10 pmol of attractin is placed in the adjacent artificial 
seawater. even though attractin is a product of the A. cali- 
fornica albumen gland. 

The animals were housed in individual plastic cages in 
one of five aquaria containing recirculating artificial seawa- 
ter (ASW; Instant Ocean Marine Salt, Longhorn Pet Supply, 
Houston, Texas). Water was maintained at room tempera- 
ture (20 2C); the salinity ranged from 30 to 32 ppt. A 
14:10 light:dark cycle was maintained in the aquarium 
facility, with the light period starting at 0600. Animals were 
fed dried laver in the late afternoon (1600-1800) after 
experiments were completed. 

Egg-laying activity was checked twice every day (0800- 
0900, 1600-1800), egg-laying activity was recorded, and 
egg cordons were removed. All animals used in assays were 
sexually mature, as defined by the ability to lay eggs spon- 
taneously or in response to injection of atrial gland extracts 
(made as described in Painter et al., 1991). 

Specimens of A. califomica (Cooper) were obtained from 



Alacrity Marine Biological Services (Redondo Beach. Cal- 
ifornia) and M-REP (Escondido, California). They were 
maintained as described above, except that the water tem- 
perature was 14 2C. This species was used as a stim- 
ulus animal in one set of T-maze assays and as the source of 
albumen glands for purification of native attractin. 

Purification of native and recombinant attractin 

Procedures. Attractin from the albumen gland of A. cali- 
fomica was purified by analytical CIS reversed-phase high 
performance liquid chromatography (RP-HPLC) as previ- 
ously described (Painter et al.. 1998). To prepare recombi- 
nant attractin. the A. califomica albumen-gland attractin 
cDNA (Fan et al., 1997) was subcloned into the baculovirus 
expression vector pFastBac 1, and recombinant virus was 
generated using the Bac-to-Bac Baculovirus Expression 
System (Invitrogen). Attractin was expressed in Sf9 insect 
cells grown at 27-28 C in Sf-900 II serum-free medium 
(Invitrogen). 

Expressing Sf9 cells were centrifuged, and the pellet was 
resuspended in 20 ml of ice-cold 0.1% heptafluorobutyric 
acid (HFBA) and sonicated. The resulting lysates were 
purified on CIS Sep-Pak Vac cartridges (5 g; Waters Corp.) 
that were pretreated with 10 ml of 100% acetonitrile 
(CH,CN) containing 0.1% HFBA and rinsed with 20 ml of 
0.1% HFBA. The peptides were loaded, eluted with 15 ml 
of 50% CH 3 CN containing 0.1% HFBA, and lyophilized. 
The lyophilizate was resuspended in 2.5 ml of 0.1% HFBA 
and applied to a Vydac analytical CIS RP-HPLC column 
(4.6 X 250 mm). 

The column was eluted with a two-step linear gradient of 
0.1% HFBA in water and 100% CH 3 CN containing 0.1% 
HFBA. The first step was 0%-10% CH 3 CN in 5 min, 
followed by a shallower gradient from 10% to 34% CH 3 CN 
in 85 min. The column eluate was monitored at 215 nm, and 
1-min ( 1 ml) fractions were collected. The attractin-contain- 
ing fractions were combined, lyophilized, and repurified by 
Vydac C18 RP-HPLC. The same gradient conditions were 
used as described above, except that 0.1% trifluoroacetic 
acid was the counterion. 

Results. The RP-HPLC peak fractions containing A. cali- 
fomica recombinant attractin, identified by comparison to 
the elution time of native attractin, were characterized by 
amino acid compositional and microsequence analyses; the 
58-residue peptide sequence was identical to A. califomica 
albumen-gland attractin except that, according to matrix- 
assisted laser desorption/ionization mass spectrometry, the 
native peptide is /V-glycosylated at Asn 8 and the recombi- 
nant peptide is not. 

Pheromonal attraction 

Procedures. The T-maze, and its associated cages, is 
illustrated in Figure 2. Before each assay, 6 1 of ASW was 



APLYSIA PHEROMONAL ATTRACTANT 



19 



101 cm 



12.7 cm 



10.2 cm 



30.5 cm 



10.2 cm 

Figure 2. Schematic diagram of the T-maze with removable stimulus 
cages (dashed outlines) in place. T-maze depth: 10.2 cm. 

put into the maze; the ASW was stationary during experi- 
ments. To minimize the amount of stress experienced by the 
animals during transfer to the maze, the ASW was similar in 
temperature and salinity to that in the aquarium from which 
the animals were taken. The ASW placed in the maze had 
not previously contacted A. californica or A. brusiliana, 
because there are animal-derived factors that make a non- 
laying conspecific attractive (Painter er al., 1991 ). 

A non-laying conspecific was placed in one of the stim- 
ulus cages and a potential attractant added to the adjacent 
ASW; this is the stimulus animal. After 5 min, a non-laying 
animal, known as the test animal, was placed in the base of 
the maze and watched for up to 20 min. In most cases, the 
test animal moved directly to the top of the maze and 
exhibited one of two patterns of behavior. ( I ) It stopped, 
moved its head from side to side, then either moved into one 
arm or returned to the base of the maze and remained there. 
(2) It swam around in the maze, often visiting both cages 
before deciding where to stop. A response was considered to 
be positive if the test animal traveled to the stimulus within 
20 min, and then maintained contact with the stimulus cage 
for 5 min. It was negative if the test animal traveled to the 
cage in the opposite arm and maintained contact for 5 min. 
The response was considered to be no choice if the test 
animal did neither. Ten assays were performed for every 
potential attractant. and the attractant was alternated be- 
tween arms in consecutive assays. Statistical significance 
was assessed by chi-square analysis. In each case, test 
animals were choosing between a stimulus in one arm and 
no stimulus in the other. 

Animals for each assay were selected on the basis of three 
criteria. First, the animals must have been sexually mature 
but not have laid eggs or been used in a bioassay during the 
preceding 24 h. Second, the test animal must not have been 
exposed previously to the fraction being tested. Third, stim- 
ulus and test animals must have been housed in the same 
aquarium (Painter et al.. 1 998). An exception was made to 



the third criterion in one set of assays, when A. califoniica 
was used as the stimulus animal. A. brasi liana was always 
used as the test animal (Painter et al.. 1 998). 

Several series of experiments were performed. The first 
compared the attractiveness of a non-laying specimen of A 
hnuilitimi with I pmol of either native or recombinant 
attractin in the adjacent ASW to a non-laying conspecific 
with nothing added. We were asking several questions. Are 
smaller amounts of native attractin attractive? Is recombi- 
nant attractin as attractive? Would it be feasible to use 
recombinant attractin in future behavioral, molecular, or 3D 
structural studies? The second series examined whether a 
non-laying conspecific was needed for I pmol attractin to be 
attractive. We were asking: does attractin function alone or 
as part of a "bouquet of scents," as other pheromones do in 
many systems? The third series examined whether the non- 
laying animal must be a conspecific. We were asking: could 
the presence of multiple species at one breeding aggregation 
be due. partially or completely, to attractin? If animal- 
derived factors are necessary, do they differ among species? 

Results. The results of the experiments comparing the 
attractiveness of native and recombinant attractin are shown 
in Figure 3. In the negative control (non-laying conspecific 
with nothing placed in the adjacent ASW). two animals 
(20%) traveled to the right arm and remained, two (20%) 
traveled to the left arm and remained, and six (60%) did 
neither. Of the four animals making a choice, only two went 
to the stimulus animal, one of which was in the right arm 
and the other of which was in the left arm of the maze. 
These bioassays verify that there is no directional bias in the 
maze and establish chance levels of attraction at two ani- 
mals. 

The response pattern changed when I pmol of either 
native or recombinant attractin was placed in the seawater 
adjacent to the stimulus animal (Fig. 3): 9 of 10 animals 
(90%) were attracted to recombinant attractin, and 8 of 10 
animals (80%) were attracted to native attractin; in both 
cases, fewer animals went to the opposite arm and fewer 
failed to make a choice. The response patterns for each 
differed significantly from that for a non-laying conspecific 
alone [recombinant: ^ 2 (2) = : 13.75: P < 0.005; native: 
X 2 (2) = 10.44. 0.05 < P < 0.1]. but did not differ signif- 
icantly from each other L\ 2 (2) = 2.1. 0.25 < P < 0.5]. 

The results of the experiment examining whether an 
animal is needed for attraction are shown in Figure 3. When 
1 pmol recombinant attractin was placed in the seawater 
without a stimulus animal. 3 of 10 animals (30%) were 
attracted to recombinant attractin. two animals (20%) went 
to the opposite arm. and five animals (50%) did neither (Fig. 
3). The response pattern to 1 pmol recombinant attractin 
alone differed significantly from that to 1 pmol recombinant 
attractin with an animal ],v : (2) = 6.00: 0.025 < P < 0.05]. 
but did not differ from that to a non-laying conspecific alone 
[X 2 (2) = 0.277: 0.95 < P < 0.975]. 



20 



S. D. PAINTER ET AL. 




Positive 
Negative 

No Choice 



Nonlaycr Konlayer Nonlayer No Animal A brasiliana A. californica 
Native Att RccombAn Recomb Alt Recomb AR RecombAn 

Material Added 

Figure 3. Both native and recombinant attractin are attractive; attractin acts in conjunction with other odors; 
and the animal-derived factor is not species-specific. The number of Aplysia brasiliana individuals attracted to 
a non-laying conspecific (Nonlayer) was increased by placing I pmol of either native attractin (Nonlayer Native 
Att) or recombinant attractin (Nonlayer Recomb Att) in the adjacent seawater. In each assay, animals chose 
between a stimulus in one arm and no stimulus in the other. Fewer A. brasiliana individuals were attracted to 
recombinant attractin when the stimulus did not contain a non-laying conspecific (No Animal Recomb Att; 1 
pmol). About the same number of A. brasiliana individuals were attracted to the specimen of A. californica with 
recombinant attractin (A. californica Recomb Att; 1 pmol) as were attracted to the specimen of A. brasiliana with 
recombinant attractin (A. brasiliana Recomb Att; 1 pmol). 



Animals do not release attractin unless they are laying 
eggs; therefore, the combined odor of a non-laying animal 
and attractin produces a qualitatively different stimulus 
from attractin alone. The data confirm that attractin func- 
tions as part of a bouquet of scents and led us to ask. Does 
the animal-derived pheromone have to come from a con- 
specific or can it come from a different species of Aplysia, 
perhaps accounting for the presence of multiple species at 
an aggregation? This would also be consistent with reports 
of multiple species showing up at one aggregation in the 
field. 

The results of experiments examining whether the stim- 
ulus animal needs to be a conspecific in order for attractin to 
be attractive are shown in Figure 3. When 1 pmol of 
recombinant attractin was placed in the seawater adjacent to 
A. brasiliana. 8 of 10/4. brasiliana (80%) were attracted to 
the non-laying conspecific. When 1 pmol of recombinant 
attractin was placed in the seawater adjacent to A. califor- 
nica, 1 of 10 A. brasiliana (70%) were attracted to the 
non-laying A. californica. The response patterns for the two 
species did not differ significantly from each other [^(2) = 
0.265; 0.75 < P < 0.9], but each differed significantly from 



that for a non-laying A. brasiliana alone [A. brasiliana, 
X 2 (2) = 10.44; 0.005 < P < 0.01; A. californica, * 2 (2) = 
7.50; 0.005 < P < 0.01]. 



Pheromonal induction of mating activity 

Procedures. As in the T-maze bioassays, three criteria 
were used to select animals for each experiment. First, the 
animals must have been sexually mature but not have laid 
eggs or been used in a bioassay during the previous 24 h. 
Second, the animals must not have been exposed previously 
to the fraction being tested or have been paired with the 
same animal twice. Third, both animals must have been 
housed in the same aquarium (Painter et ai, 1998). 

Each assay was performed in 3 1 of aerated ASW in a 4-1 
plastic beaker. The ASW had approximately the same os- 
molarity and temperature as the ASW in the aquarium from 
which the animals were removed, and had not previously 
contacted A. brasiliana. Animal-conditioned ASW not only 
increases the attractiveness of a non-laying cospecific, but 
also reduces the latency to mating (Painter et al., 1991). 



APLYSIA PHEROMONAL ATTRACTANT 



21 



A 



tn 
a 



t 
I 



o> 

I 






t 
I 



100 
80 
60 
40 
20 




100 
80 
60 
40 
20 




Native Attractin 



Native Attractin 




- i 




40 80 120 160 200 240 
Time (min) 

Recombinant Attractin 



D 



ASW 1 pmol 10 pmol 

Material Added 

Recombinant Attractin 



a- a- a- ft- ft- ft- a- e 




150 



1 pmol i 



g 120 
O) 



a) 



-o- ASW 



90 



60 



0) 

W 30 





40 80 120 160 200 240 
Time (min) 



ASW 1 pmol 

Material Added 



Figure 4. Both native and recombinant attractin reduce the latency to mating in Aplysia brasiliana. (A) The 
percentage of animals mating at early time periods was increased when native attractin was placed in the adjacent 
seawater. (B) The latency to mating was reduced by placing either 1 pmol or 10 pmol native attractin in the 
seawater. (C) The percentage of animals mating at early time periods was increased when recombinant attractin 
was placed in the adjacent seawater. (D) The latency to mating was reduced by placing 1 pmol recombinant 
attractin in the seawater. 



Animals were rinsed in fresh non-conditioned ASW before 
being introduced into the experimental beaker. 

Two individuals of A. brasiliana and a test sample were 
added to a beaker, and behaviors were assessed at 10-min 
intervals for 270 min. Three categories of behavior were 
identified: ( 1 ) mating as a female or male (one-way mating), 
(2) mating as a hermaphrodite, and (3) laying eggs. Since an 
egg cordon is a source of multiple contact and water-borne 
pheromones that modify reproductive behaviors, egg-laying 
activity was noted and the bioassay stopped; the bioassay 
for that sample was repeated with other animals. Egg laying 
occurred rarely with any stimulus. 

Test samples included ASW with nothing added (nega- 
tive control), ASW with 1 or 10 pmol native attractin added, 
and ASW with 1 pmol recombinant attractin added. The 
statistical significance of the differences between time 
points was determined by chi-square analysis; the statistical 



significance of differences in mean latency was determined 
by one-way analysis of variance. The same number of 
assays was performed for each treatment. 

Results (native attractin). When 1 or 10 pmol of native 
attractin was placed in ASW containing two non-laying 
specimens of A. brasiliana, the percentage of animals mat- 
ing at each time point ( 10-min intervals) was recorded. The 
percentage of animals mating was significantly increased 
for 10 pmol attractin at 10, 20, 30, and 40 min, and there 
was a nonsignificant trend in this direction for 1 pmol 
attractin (Fig. 4 A). The mean latency to mating was signif- 
icantly reduced for 10 pmol attractin (% 1 ( 1 ) > 3.84 for each; 
P < 0.05; n = 10), and there was a nonsignificant trend in 
this direction for 1 pmol (Fig. 4B). Although the latency to 
mating was reduced, the overall percentage of animal pairs 
mating during the 270-min period was not affected (nega- 
tive controls: 90% mated; native attractin: 100% mated). 



22 



S. D. PAINTER ET AL. 



perhaps reflecting the long duration of the assay or animal 
housing in individual cages. In these experiments, nearly all 
animal pairs eventually mated during the 270-min time 
period, regardless of whether attractin was present. Never- 
theless, the results suggest that attractin facilitates, but does 
not induce, mating 

Results (recombinant attractin). When 1 pmolofrecom- 
binant attractin was placed in ASW containing two non- 
laying specimens of A. brasiliana. the percentage of animals 
mating at 10, 20, 170, and 240 min was significantly in- 
creased compared to negative controls [^(l) > 3.84 for 
each; P < 0.05; n = 10; Fig. 4C]. The mean latency to 
mating was significantly reduced for 1 pmol recombinant 
attractin (P < 0.05; one-way analysis of variance; Fig. 4D). 
Although the latency to mating was reduced, the total per- 
centage of animal pairs that mated during the entire 270-min 
period was similar (negative controls: 90% mated; recom- 
binant attractin: 100% mated), suggesting again that attrac- 
tin facilitates, rather than induces, mating. 



Pheromonal induction of hermaphroditic mating 

Procedure. This is a re-analysis of the data collected in 
the mating assays, focusing on whether attractin can induce 
or facilitate hermaphroditic mating. As noted above, her- 
maphroditic mating was recorded during the mating assays. 

Results (native attractin). When native attractin was 
placed in the ASW surrounding two non-laying specimens 
of A. brasiliana, the percentage of animal pairs mating as 
hermaphrodites was significantly increased for 10 pmol 
attractin at every time point between 20 and 170 min and for 
190, 200, 230, and 250 min (^ 2 (1) > 3.84 for each; P < 
0.05; n = 10); the same was true for 1 pmol attractin at 230, 
240, and 250 min (\ 2 ( 1 ) > 3.84 for each; P < 0.05; n= 10) 
(Fig. 5A). The mean latency to hermaphroditic mating was 
significantly reduced for 10 pmol attractin (P < 0.05; one- 
way analysis of variance), and there was a nonsignificant 
trend in this direction for 1 pmol attractin (Fig. 5B). Com- 
pared to control assays, the percentage of animal pairs that 
mated as hermaphrodites during the 270-min period was 
about doubled (negative controls: 40%: 1 pmol: 70%; 10 
pmol: 80%). This suggests that attractin induces, rather than 
facilitates, hermaphroditic mating, perhaps by stimulating 
both animals to mate as males. This induction could be 
responsible for copulatory rings and chains in the field, 
which may result because there are usually more than two 
animals in an aggregation. 

Results (recombinant attracting The percentage of ani- 
mals mating as hermaphrodites at any given time point and 
the latency to hermaphroditic mating were not significantly 
increased upon addition of 1 pmol of recombinant attractin. 
although there were trends in this direction (Fig. 5 C, D). 
Although the percentage of animals mating as hermaphro- 



dites was not significantly increased at any particular time 
point, the percentage of animal pairs that mated as hermaph- 
rodites at some point during the assay did increase (negative 
controls: 40% mated as hermaphrodites; recombinant attrac- 
tin: 70% mated as hermaphrodites). A dose of 10 pmol was 
not tested, which may account for the lack of statistical 
significance. 

Discussion 

We purified native attractin from extracts of Aplysia 
californica albumen gland (Painter et al., 1998) and recom- 
binant attractin from insect cells to better characterize the 
biological activity of the peptide and to see whether recom- 
binant attractin could be used in future molecular studies. 

Pheromonal attraction 

In the T-maze, the attractiveness of a stimulus animal was 
significantly increased when 1 pmol of either native or 
recombinant attractin was placed in the adjacent seawater, 
verifying that both peptides are attractive in amounts con- 
sistent with pheromonal activity, and confirming that N- 
glycosylation is not required for attraction. The response 
patterns for the two peptides do not differ significantly from 
each other, demonstrating that either could be used in future 
studies. Recombinant attractin was therefore used in subse- 
quent T-maze bioassays. Since it was not W-glycosylated, 
recombinant attractin was also used to determine the solu- 
tion structure of the pheromone by 3D nuclear magnetic 
resonance (Garimella et al., 2003). 

Fewer individuals of A. brasiliana were attracted to re- 
combinant attractin when the stimulus did not contain a 
non-laying conspecific. demonstrating that attractin acts in 
concert with other unidentified odors to stimulate attraction. 
These results, combined with earlier observations (the egg 
cordon is attractive without a stimulus animal. Painter et al., 
1991: attractin elutes from the egg cordon, Painter et al., 
1 998 ). suggest that the composition of the bouquet of scents 
can vary. To identify other attractive chemical factors in the 
egg-cordon bouquet of scents, we have begun isolating 
other peptides/proteins that elute from the cordon for bio- 
assay. 

To begin looking for animal-derived attractants, we 
tested whether the stimulus animal needs to be a conspe- 
cific. It does not. A. californica with attractin and A. bra- 
siliana with attractin each attracted a similar number of A. 
brasiliana. This pairing may seem inappropriate since the 
two species do not overlap in their geographic distributions 
(A. californica, Pacific Coast; A. brasiliana. Gulf of Mex- 
ico), but it may help explain why multiple Aplysia species 
are sometimes associated with one aggregation. For exam- 
ple, A. californica and A. vaccaria have been observed in 
the same breeding aggregations off the coast of California 
(Kupfermann and Carew, 1974; S. LePage, M-REP, pers. 



APLYSIA PHEROMONAL ATTRACTANT 



23 



A 



Native Attractin 



S2 



90 
80 
70 



jg 60 
50 
40 
30 
20 
10 




C 




+ 10 pmol 
-&-- 1 pmol 
-o- ASW 



40 80 120 160 200 240 

Time (min) 

Recombinant Attractin 




40 80 120 160 200 240 

Time (min) 



B 



Native Attractin 



, 250 



200 



0> 

I 



I 

.C 
0. 
CO 

E 

I 
o 



D 



., 300 



S 200 



.c 

Q. 

CO 

100 

X 

o 

I 

B o 




ASW 1 pmol 10pmol 

Material Added 



Recombinant Attractin 




ASW 1 pmol 

Material Added 



Figure 5. Both native and recombinant attractin induce hermaphroditic mating in Aplysia brasiliana. (A) 
The percentage of animals mating as hermaphrodites was increased when native attractin was placed in the 
adjacent seawater. (B) The latency to hermaphroditic mating was reduced by placing either 1 pmol or 10 pmol 
native attractin in the seawater. (C and D) The percentage of animal pairs mating as hermaphrodites was 
increased when 1 pmol recombinant attractin was placed in the adjacent seawater. The mean latency to 
hermaphroditic mating was also reduced. 



comm.), and have been seen mating with each other in the 
aggregation (S. LePage, M-REP, pers. comm.). A. fasciata 
and A. depilans have also been seen associated with the 
same aggregation (Achituv and Susswein, 1985), but mating 
has not been observed because their reproductive cycles are 
not entirely synchronized. Audesirk (1977) previously 
found that A. californica was not attracted to conspecifics in 
Y-maze assays, and Audesirk and Audesirk (1977) showed 
that there was no seasonal effect on the sensitivity to con- 
specifics. Furthermore, experimental perfusion of the A. 
californica rhinophore nerve with seawater that had bathed 
A. californica, A. vaccaria, or Pleurobranchia californica 
evoked about the same increase in afferent activity, suggest- 
ing that aggregations of Aplysia species in the field are not 
determined by species-specific chemical cues (Chase, 
1979). 



Pheromonal induction of mating 

Mating assays were performed because behaviors seen in 
earlier T-maze assays suggested that exposure to attractin 
could stimulate behaviors suggestive of mating as a male 
(Painter er /., 1998). The current studies showed that when 
attractin is added to the seawater adjacent to a pair of A. 
brasiliana, the latency to mating is reduced relative to 
controls. However, the overall percentage of animal pairs 
mating during the prolonged assay period was not signifi- 
cantly different, suggesting that attractin facilitates, but does 
not induce, mating. 

Attractin also significantly reduces the latency to her- 
maphroditic mating when added to the seawater surround- 
ing a pair of A. brasiliana. The percentage of animal pairs 
mating as hermaphrodites during the assay period was about 
doubled, suggesting that attractin induces hermaphroditic 



24 



S. D. PAINTER ET AL. 



mating. This effect may result from attractin stimulating 
both animals to mate as males, as suggested by T-maze 
behaviors. Overall, these data suggest that attractin contrib- 
utes to the establishment and maintenance of breeding ag- 
gregations. 

Attractin does not stimulate species-specific attraction 

The attracting appear to be a structurally diverse family of 
peptides, each of which is sequence-specific for a given 
species. Attractin has recently been characterized from A. 
brasiliana, A. fasciata, A. vaccaria, A. depiluns, and Bur- 
satella leachii and found to be 95%, 91%, 43%, 41%, and 
21% identical to A. California! attractin, respectively (Paint- 
er et al, 2000, and unpubl. data). Nevertheless, attractin is 
attractive to all aquatic gastropods tested to date: ( 1 ) A. 
californica attractin is attractive to A. brasiliana (Painter et 
al., 1998); (2) A. vaccaria attractin is attractive to A. bra- 
siliana (unpubl. data); and (3) A. californica attractin is 
attractive to the freshwater pulmonate Lymnaea stagnalis 
(A. ter Maat, Free University, Amsterdam, pers. comm.). 
Although the primary structures of attractin-related peptides 
are divergent, their 3D structures may be similar to A. 
californica attractin (Garimella et al.. 2003). To our knowl- 
edge, the attractins are the first peptide pheromone family in 
invertebrates that is not species-specific. In contrast, water- 
borne peptide pheromonal attractants in amphibians are 
species-specific (Kikuyama et al.. 2002). 

There may be advantages to attracting multiple species to 
the same breeding aggregation. If members of a second 
species lay eggs on those of a different species, the mixed 
egg mass becomes larger, which might in some way protect 
the eggs of both species. Another possibility is that egg 
laying by one Aplysia species attracts a second species that 
then lays eggs and releases attractin. which may eventually 
attract members of the first species. Because attractin is 
continuously degraded from the C-terminus after its release 
(Painter et al.. 1998, and unpubl. obs.). it may be advanta- 
geous to attract as many individual Aplvsia as possible, 
regardless of species, to lay eggs and maintain the elevated 
attractin concentrations needed to recruit new individuals to 
the breeding aggregation. 

Chemical communication frequently involves the use of 
blends of pheromones rather than single-compound phero- 
mones. Blends of airborne pheromones are important for 
species-specific signaling in many organisms, including ar- 
thropods. Mate finding in most moth species, for example, 
involves the release of long-distance airborne sex phero- 
mones, which are produced in specialized female abdominal 
glands, generally via unsaturated fatty-acid precursors pro- 
duced by desaturases (Roelofs et al.. 2002). A great diver- 
sity of pheromone structures is used throughout the Lepi- 
doptera, even among closely related species, and the blend 
ratio is important for species-specific signaling. There is 



strong selection pressure against novel blends and response 
preferences (Roelofs et al., 2002). Although airborne sex 
pheromones capable of inducing spatial orientation of con- 
specifics "downwind" are well established in insects (Carde 
and Minks, 1996), this is not the case in vertebrates, whose 
identified sex pheromones tend to have a small range of 
effectiveness; in fish, the known sex pheromones are go- 
nadal steroids, prostaglandins, or bile acids (Li et al., 2002). 
Mate attraction in the genus Aplysia, and perhaps in other 
gastropods, appears to involve long-distance signaling via 
waterborne pheromone blends. Attractin by itself is not 
attractive to Aplysia species, but egg cordons alone are 
sufficient to attract Aplysia species "downstream," indicat- 
ing that the cordons themselves contain a blend of phero- 
mones. Once aggregations of multiple Aplysia species form, 
appropriate intraspecific mating may be achieved through 
the use of specific proximal cues involving contact chemo- 
reception and mechanoreception (Chase, 1979). 

Acknowledgments 

We thank Drs. A. Susswein, A. ter Maat. and M. Miller 
for their interesting comments and acknowledge the Uni- 
versity of Texas Medical Branch (UTMB) Protein Chemis- 
try Laboratory, which is supported by the UTMB Educa- 
tional Cancer Center, for compositional and microsequence 
analyses. This work was supported by NSF Grant IBN- 
9985778 (S.D.P.). and John Sealy Memorial Endowment 
Fund for Biomedical Research Development Grant 2579- 
02R (G.T.N.). 

Literature Cited 

Achituv, Y., and A. Susswein. 1985. Habitat selection by two Mediter- 
ranean species of Aplvsia: A. fasciata Poiret and A. tiepilans Gmelin 
(Mollusca: Opisthobranchia). J. Exp. Mar. Biol Ecol. 85: 113-122. 

Aspey, W. P.. and J. E. Blankenship. 1976. Aplysia behavioral biology. 
II Induced burrowing in swimming A. brasiliana by burrowed con- 
specifics. Behav. Biol. 17: 301-312. 

Audesirk, T. E. 1977. Chemoreception in Aplysia californica. III. Evi- 
dence for pheromones influencing reproductive behavior. Behav. Biol. 
20: 235-243. 

Audesirk, T. E. 1979. A field study of growth and reproduction in 
Apl\sia californica. Biol. Bull. 157: 407-421. 

Audesirk, T. E., and G. J. Audesirk. 1977. Chemoreception in Aplysia 
californica. II. Electrophysiological evidence for detection of the odor 
of conspecifics. Coinp. Biochem. Ph\siol. 56A: 267270. 

Blankenship, J. E., M. K. Rock, L. C. Robbins, C. A. Livingston, and 
H. K. Lehman. 1983. Aspects of copulatory behavior and peptide 
control of egg laying in Aplysia. Fed. Proc. 42: 96-100. 

Carde, R. T., and A. K. Minks. 1996. Molecular mechanisms of pher- 
omone reception in insect antennae. Pp. 1 15-130 in Insect Pheromone 
Research. H. Breer, ed. Chapman and Hall. New York. 

Chase, R. 1979. An electrophysiological search for pheromones of Aply- 
sia californica. Can. J. Zool. 57: 781-784. 

Fan, X., B. Wu, G. T. Nagle, and S. D. Painter. 1997. Molecular 
cloning of u cDNA encoding a potential water-borne pheromonal 
attractant released during Aplysia egg laying. Mol. Brain Res. 48: 
167-170. 



APLYSIA PHEROMONAL ATTRACTANT 



25 



Garimella. R., Y. Xu, C. H. Schein, K. Rajarathnam, G. T. Nagle, S. D. 
Painter, and W. Braun. 2003. NMR solution structure of attractin, 
a water-borne protein pheromone from the mollusk Aplysia culifornica. 
Biochemistry. (In press). 

Jahan-Parwar, B. 1976. Aggregation pheromone from the egg-mass of 

Aplysia califomica. Physiologist 19: 240. 

Kikuyama, S., F. Toyoda, Y. Ohmiya, K. Matsuda, S. Tanaka. and H. 
Hayashi. 1995. Sodefrin: a female-attracting peptide pheromone in 
newt cloacal glands. Science 267: 1643-1645. 

Kikuyama, S., K. Yamamoto, T. Iwata, and F. Toyoda. 2002. Peptide 
and protein pheromones in amphibians. Camp. Biochem. Physiol. B 
132: 69-74. 

Kodama, T., T. Hisatomi, T. Kanemura, K. Mokubo, and M. Tsuboi. 
2003. Molecular cloning and DNA analysis of a gene encoding alpha 
mating pheromone from the yeast Saccharomyces naganishii. Yeast 20: 
109-115. 

Kupfermann, I., and T. Carew. 1974. Behavior patterns of Aplysia 
culifornica in its natural environment. Behav. Biol. 12: 317-337. 

Li, W., A. P. Scott, M. J. Siefkes, H. Yan, Q. Liu, S.-S. Yun, and D. A. 
Gage. 2002. Bile acid secreted by male sea lamprey that acts as a sex 
pheromone. Science 296: 138-140. 

Luporini, P., A. Vallesi. C. Miceli, and R. A. Bradshaw. 1995. Chem- 
ical signaling in ciliates. / Eukaryot. Microbiol. 42: 208-212. 

Monsma, S. A., and M. F. Wolfner. 1988. Structure and expression of 
a Drosophilii male accessory gland gene whose product resembles a 
peptide pheromone precursor. Genes Dev. 2: 1063-1073. 

Novotny, M. V. 2003. Pheromones. binding proteins and receptor re- 
sponses in rodents. Biochem. Soc. Trans. 31: 1 17-122. 

Painter, S. D. 1992. Coordination of reproductive activity in Aplvsia: 
peptide neurohormones, neurotransmitters, and pheromones encoded 
by the egg-laying hormone family of genes. Biol. Bull. 183: 165-172. 

Painter, S. D., A. R. Gustavson, V. K. Kalman, G. T. Nagle, R. A. 
Zuckerman. and J. E. Blankenship. 1989. Induction of copulatory 
behavior in Aplysia: atrial gland factors mimic the excitatory effects of 
recently deposited egg cordons. Behav. Neural Biol. 51: 222-236. 

Painter. S. D., M. G. Chong, M. A. Wong, A. Gray, J. G. Cormier, and 
G. T. Nagle. 1991. Relative contributions of the egg layer and egg 
cordon to pheromonal attraction and the induction of mating and 
egg-laying behavior in Aplysia. Biol. Bull. 181: 81-94. 

Painter, S. D., B. Clough, R. W. Garden, J. V. Sweedler, and G. T. 
Nagle. 1998. Characterization of Aplysia attractin. the first water- 
borne peptide pheromone in invertebrates. Biol. Bull. 194: 120-131. 



Painter, S. D., D.-B. G. Akalal. B. Clough, A. J. Susswein, M. Levy, and 
G. T. Nagle. 2000. Characterization of four new members of the 
attractin family of peptide pheromones in Aplysia. Soc. Neurosci. 
Abstr. 26: 1166. 

Pennings, S. C. 1991. Reproductive behavior of Aplvsia califomica 
Cooper: diel patterns, sexual roles and maling aggregations. J. Exp. 
Mar. Biol. Ecol. 149: 249-266. 

Ram, J. L., C. T. Muller, M. Beckmann, and J. D. Hardege. 1999. The 
spawning pheromone cysteine-glutathione disulfide Cnereithione'l 
arouses a multicomponent nuptial behavior and electrophysiological 
activity in Nereis succinea males. FASEB J. 13: 945-952. 

Roelofs, W. L., W. Liu, G. Hao, H. Jiao, A. P. Rooney, and C. E. Linn, 
Jr. 2002. Evolution of moth sex pheromones via ancestral genes. 
Proc. Natl. Acad. Sci. USA 99: 13621-13626. 

Rollmann, S. M., L. D. Houck, and R. C. Feldhoff. 1999. Protein- 
aceous pheromone affecting female receptivity in a terrestrial 
salamander. Science 285: 1907-1909. 

Saudan, P., K. Hauck, M. Soller, Y. Choffat, M. Ottiger, M. Sporri, Z. 
Ding, D. Hess, P. M. Gehrig, S. Klauser, P. Hunziker, and E. Kubli. 
2002. Ductus ejaculatonus peptide 99B (DUP99B). a novel Drosoph- 
ila melanogaster sex-peptide pheromone. Eur. J. Biochem. 269: 989- 
997. 

Savic, I., H. Berglund, B. Gulyas, and P. Roland. 2001. Smelling of 
odorous sex hormone-like compounds causes sex-differentiated hypo- 
thalamic activations in humans. Neuron 31: 661-668. 

Schein. C. H., G. T. Nagle, J. S. Page, J. V. Sweedler, Y. Xu, S. D. 
Painter, and W. Braun. 2001. Aplysia attractin: biophysical char- 
acterization and modeling of a water-borne pheromone. Biophvsical J. 
81: 463-472. 

Stowers, L., T. E. Holy, M. Meister, C. Dulac, and G. Koentges. 2002. 
Loss of sex discrimination and male-male aggression in mice deficient 
forTRP2. Science 295: 1493-1500. 

Susswein, A. J., S. Gev, E. Feldman, and S. Markov-itch. 1983. Ac- 
tivity patterns and time budgeting of Aplysia fasciata in field and 
laboratory conditions. Behav. Neural Biol. 39: 203-220. 

Susswein, A. J., S. Gev, Y. Achituv, and S. Markovitch. 1984. Behav 
ioral patterns of Aplysia fasciata along the Mediterranean coast of 
Israel. Behav. Neural Biol. 41: 203-220. 

Wabnitz. P. A., J. H. Bowie, M. J. Tyler, J. C. Wallace, and B. P. 
Smith. 1999. Aquatic sex pheromone from a male tree frog. Nature 
401: 444-445. 



Reference: Biol. Bull. 205: 26-35. (August 2003) 
2003 Marine Biological Laboratory 



Field Observations of Intraspecific Agonistic Behavior 

of Two Crayfish Species, Orconectes rusticus and 

Orconectes viriliSj in Different Habitats 

DANIEL A. BERGMAN AND PAUL A. MOORE* 

Laboratory for Sensory Ecology, Department of Biological Sciences itiul the J. P. Scott Center for 

Neuroscience, Mind, and Behavior, Bowling Green State University, Bowling Green, Ohio 43403; 

and University of Michigan Biological Station. 9008 Biological Road. Pellxtun, Michigan 49769 



Abstract. Agonistic behavior is a fundamental aspect of 
ecological theories on resource acquisition and sexual se- 
lection. Crustaceans are exemplary models for agonistic 
behavior within the laboratory, but agonistic behavior in 
natural habitats is often neglected. Laboratory studies do not 
achieve the same ecological realism as field studies. In an 
attempt to connect laboratory results to field data and in- 
vestigate how habitat structure affects agonistic interac- 
tions, the nocturnal behavior of two crayfish species was 
observed by scuba diving and snorkeling in two northern 
Michigan lakes. Intraspecific agonistic interactions were 
analyzed in three habitats: two food resources macro- 
phytes and detritus and one sheltered habitat. The overall 
observations reinforce the concept that resources influence 
agonistic bouts. Fights in the presence of shelters were 
longer and more intense, suggesting that shelters have a 
higher perceived value than food resources. Fights in the 
presence of detritus patches had higher average intensities 
and ended with more tailflips away from an opponent, 
suggesting that detritus was a more valuable food resource 
than macrophytes. In addition, observations of aggressive 
behavior within a natural setting can add validity to labo- 
ratory studies. When fights in nature are compared with 
laboratory fights, those in nature are shorter, less intense, 
and less likely to end with a tailflip. but do show the 
fundamental fight dynamics associated with laboratory stud- 
ies. Extrinsic and intrinsic factors affect intraspecific ag- 
gression in many ways, and both should always be recog- 
nized as having the potential to alter agonistic behavior. 



Received 16 December 2002: accepted 19 May 2003. 
* To whom correspondence should he addressed. E-mail: pmoore@ 
bgnet. bjjsu.edu 



Introduction 

Many observations of crayfish behavior have been made 
under controlled laboratory conditions. These studies gen- 
erally focus on intraspecific aggressive behavior in terms of 
shelter acquisition (Capelli and Hamilton, 1984; Peeke et 
nl.. 1995; Figler et al.. 1999). chemical communication 
(Bovbjerg, 1956; Zulandt Schneider et al.. 1999, 2001), 
mating (Hill and Lodge, 1999), food preferences (Capelli 
and Munjal. 1982). and starvation (Hazlett et al., 1975; 
Stocker and Huber. 2001). Laboratory experiments have 
been invaluable in clarifying the extrinsic and intrinsic 
factors that affect agonistic interactions. Intrinsic factors 
that have been shown to affect aggression are size, sex, 
reproductive state, hunger state, and social experience, 
while extrinsic factors are status and individual recognition, 
resource availability, prior residence, and shelter presence. 

Asymmetries in fighting ability may be produced by 
some intrinsic features or extrinsic circumstances that favor 
one contestant (Parker, 1974; Maynard Smith and Parker, 
1976). Intrinsic asymmetries are accurate predictors of dom- 
inance during interactions between pairs of decapod crus- 
taceans; they include physical body size (Bovbjerg, 1953, 
1970; Rubenstein and Hazlett, 1974; Berrill and Arsenault, 
1984; Pavey and Fielder. 1996). chelae size (Garvey and 
Stein. 1993: Rutherford et al.. 1995). and sex (Stein. 1976; 
Peeke et al., 1995. 1998). Extrinsic asymmetries such as 
prior residence (Peeke et al.. 1995, 1998), differing fight 
strategies (Guiasu and Dunham, 1997). and previous history 
in agonistic encounters (Rubenstein and Hazlett, 1974; 
Daws et al.. 2002; Bergman et al., 2003) contribute to the 
outcome of agonistic interactions. Seasonal variations in 
food availability can also increase activity levels that lead to 



26 



FIELD STUDY OF CRAYFISH AGONISM 



27 



increased social contact and consequently to increased ag- 
gressive interactions (Hazlett et al.. 1975). Laboratory ex- 
periments are an invaluable aid to understanding behavioral 
mechanisms, but they have limitations in their applicability 
to natural ecosystems (Bovbjerg, 1953. 1956; Peeke et al., 
1995). One severe constraint on laboratory studies of ag- 
gression is the restriction of space, which reduces an ani- 
mal's ability to escape from an opponent. 

Dominance hierarchies, territorial defense, mate selec- 
tion, substrate preferences, and escalation of fight behavior 
observed under laboratory conditions may not be represen- 
tative of behaviors in a natural setting (Karnofsky et al., 
1989). These changes in agonistic behavior observed within 
the laboratory may largely be caused by an inability to 
escape an agonistic conflict (Hediger, 1950). To circumvent 
this artifact, studies have been conducted in artificial ponds 
or streams that are less restrictive than the aquaria used in 
standard laboratory experiments. By increasing the com- 
plexity of the experimental environment, studies in these 
semi-natural settings attempt to obtain a more natural rep- 
ertoire of behavior. They provide useful information about 
agonistic interactions, foraging, mating, orientation, shelter 
acquisition, and molting (Abrahamsson, 1966; Ranta and 
Lindstrom, 1992; Tomba et al., 2001 ). However, even stud- 
ies in semi-natural environments cannot illustrate the "true" 
behavioral ecology of the crayfish. Because of this short- 
coming, field studies are invaluable to the understanding of 
crayfish behavior. They minimize laboratory bias and allow 
for an integration of behaviors observed in laboratories with 
those in a natural setting. 

Crustaceans, particularly crayfish, have been used as a 
model system to study aggression (Dingle, 1983; Hyatt, 
1983) because of the ritualized nature of their agonistic 
bouts (Bruski and Dunham. 1987), the presence of formi- 
dable chelipeds (Garvey and Stein, 1993; Schroeder and 
Huber, 2001), and the use of sensory information during 
such encounters (Zulandt Schneider et al, 1999, 2001; 
Bergman et al., 2003). The ultimate goal of any aggressive 
encounter is to obtain an elevated social status that gives an 
individual an advantage in obtaining a resource, such as 
food, mates, and shelters (Wilson, 1975; Atema, 1986). 
Conversely, a subordinate individual may lose access to 
resources through unsuccessful bouts, but may obtain a net 
benefit by avoiding costs such as increased energy expen- 
diture, injury from a conspecific, or increased predation risk 
(Wilson, 1975; Edsman and Jonsson, 1996). If a subordinate 
does not gain a benefit, then the lower status will have a 
negative effect on fitness. Consequently, a subordinate will 
have less food and shelter and fewer mating opportunities. 

Extrinsic environmental factors can have a profound ef- 
fect on aggressive activities; thus a connection between 
extrinsic factors in the laboratory and their effects in nature 
need further validation. Agonistic behavior has been studied 
extensively in the laboratory and in semi-natural conditions. 



but less emphasis has been placed on agonistic behavior in 
a natural setting. For this reason, we examined agonistic 
behavior under natural nocturnal conditions in two northern 
Michigan lakes. The study was conducted in three different 
habitats within the lakes to provide a global view of in- 
traspecific agonistic behavior in nature that could be corre- 
lated to laboratory results on aggression. The results of this 
study also allowed us to examine differences in agonistic 
behavior that may be correlated to differing extrinsic factors 
in the laboratory and nature. 



Materials and Methods 



Stud\ site 



The study was sited in two remnant glacial lakes in the 
northern part of the lower peninsula of Michigan: Douglas 
Lake (lat. 4533' N. long. 8457' W) and Burt Lake (lat. 
4528' N, long. 8440' W). The Burt Lake substrate is 
predominantly sand and small gravel. Water depth ranges 
from 0.4 (shallow) to 2.0 m (deep). A mixture of sand and 
gravel containing intermittent patches of detritus dominates 
the shallow-water substrates. The deep water contains a 
sand substrate with a population of macrophytes (dominated 
by Potamogeton sp. and Vallisneria sp.) and their associated 
epiphytes. Water temperatures range from 14 to 23 C. 
Observation points were accessed by snorkeling. The Doug- 
las Lake substrate is sand that contains a small band of iron 
substrata forming natural holes that crayfish use as shelters 
(burrows). This site ranges from 7.5 to 18.0 m in depth and 
is devoid of macrophytes. The water temperature ranges 
from 10 to 15 C. Observation points were accessed through 
scuba diving. 

Studv animals 

Both the Bun Lake and Douglas Lake sites contained two 
species of crayfish, Orconectes rusticus and Orconectes 
virilis. Crayfish species were determined by the color of the 
periopods (chelae and legs), which are bright blue in O. 
virilis and brownish-green in O. rusticus. The determination 
of species allowed for an analysis of conspecific fights. In 
Douglas Lake, only O. rusticus conspecific fights were 
observed in the shelter habitat. In the Burt Lake population 
conspecific interactions for O. virilis were observed only on 
the macrophyte beds and not on the detritus patches, even 
although both species were present in the two regions. The 
observers took care to avoid physically disturbing any of the 
animals; they remained as motionless as possible by using 
intermittent kick strokes to drift over the observation areas 
(Karnofsky et al.. 1989). None of the animals were handled 
before or captured after behavioral observations. Conse- 
quently, male and female crayfish could not be distin- 
guished when aggressive interactions were analyzed, but the 
relative size difference between crayfish was determined on 



28 



D. A. BERGMAN AND P. A. MOORE 



a video screen (Sony Trinitron color monitor; model 

# PVM- 1 3 1 5Q) by calcu ' '-''it size difference of 
the opponents. 

Behavioral oh'-' 

Observations v. ; , .ade during July and August between 
the hours of 2 ) .inu 0100 (nocturnal activity period) from 
1996 to 2002 (no observations were made in the summer of 
1999) All observations were made on clear, calm nights 
when the water surf was below 8.0 cm. Interactions were 
recorded on a video camera (Sony Hi-8 Handycam; model 

# CCD-TR700) that was illuminated with white lights 
mounted on an underwater housing (Stingray video hous- 
ing; model # SR-700) that contained the camera. Animals 
were filmed from a minimum distance of 0.4 m. Slow 
swimming motions were made to follow animals, and when 
the lights on the underwater housing noticeably disturbed an 
animal, the interaction was removed from the analysis. 
Crayfish are primarily nocturnal animals, and any behav- 
ioral alterations caused by the sudden exposure to white 
light could not be determined from this study. For this 
reason, any animal that tailflipped away or used a meral 
spread in the absence of an interaction was removed from 
the data analysis; however, this does not take into account 
any unnoticeable changes in behavior in response to the 
artificial light. Crayfish do appear to alter their behavior 
when light intensities are altered (Bruski and Dunham, 
1987); however, since uniform white lighting was used in 
all observations, there should be no differential effects on 
the behavior. 

Two sampling techniques were used. The first technique 
was to follow a single crayfish until it had an agonistic 
interaction with a conspecific. The second method was to 
scan detritus patches (Burt Luke), macrophyte beds (Burt 
Lake), and the shelter areas (Douglas Lake) for two crayfish 
that were within two body lengths of one another. When 
agonistic interactions were observed with either of these 
sampling techniques, the encounter was videotaped from 
initiation to termination of the fight and the interactions 
were later analyzed by playing the tape on a Panasonic VHS 
recorder (model # AG-7530-P) onto the Sony Trinitron 
monitor. 

Analvsis of fight heluivinr 

All videotaped fight trials were analyzed using an etho- 
gram modified from Bui :.i and Dunham (1987) (Table 1). 
An agonistic encounter in a laboratory setting with no 
resources available typically begins when an individual 
approaches a potential opponent (intensity 1). The encoun- 
ter may then progress to a series of agonistic threat displays 
using a meral spread (intensity 2). If neither individual 
retreats, the bout gradually increases in fight intensity, start- 
ing with chelae contact and progressing to pushing with 



Table 1 



Crayfish ellmgram codes 



Intensity 
Level 



Description 



Tailflip away from opponent or fast retreat 

Slowly back away from opponent 

lanore opponent with no response or threat display 

Approach without a threat display 

Approach with threat display using meral spread and/or 

antennal whip 
Initial claw use by boxing, pushing, or touching with 

closed claws 

Active claw use by grabbing opponent with open claws 
Unrestrained fighting by grasping and pulling opponent's 

claws or appendages 



closed chelae (intensity 3). When the chelae are opened and 
used to grab an opponent, a new intensity level is reached 
(intensity 4). The most intense interactions have periods of 
unrestrained fighting in which an individual appears to 
attempt to injure an opponent by grasping at chelae, legs, or 
antennae (intensity 5). A conflict is concluded when one 
individual retreats (intensity -1). usually signified by a 
tailflip away from the opponent (intensity -2), and usually 
followed by a submissive posture (Bruski and Dunham, 
1987). A subordinate will retreat consistently and assume a 
posture in which the cephalothorax. abdomen, and claws are 
near the substrate. Typically, crayfish did not respond to 
each other when separated by greater than two body lengths 
(intensity 0). The temporal dynamics of these changes in 
behavior were recorded to include the total duration of the 
encounter and the time it took to reach the different intensity 
levels. Duration, time to different intensities, maximum 
intensity level reached, and average maximum intensity 
levels were analyzed using a one-way MANOVA and a 
Tukey honestly significant difference (hsd) post hoc test. 
The retreating animals (tailflip away) and maximum inten- 
sity achieved during an encounter were recorded and ana- 
lyzed using a multiple comparisons for proportions contin- 
gency table (90.05.-.4 :: 3 - 633 > that allows for testing 
analogous to the Tukey or Student-Newman-Keuls tests 
(Zar. 1999). Significant results are represented by giving a 
<7oo5*4 value > 3 - 314 from the mult 'P le comparisons test 
and a P value < 0.05. An additional power analysis (Power 
= 1 -- |3) was included for the ANOVA and multiple 
comparisons for proportions contingency table tests. The 
size differences of agonistic opponents were obtained in 1 1 7 
of the fights. Size differences are presented as a percentage 
of the larger animal in the pairing. Thus, a value of 20% 
means that the smaller animal is 20% smaller than the larger 
animal. A regression analysis between size difference in 
percentage and fight duration was analyzed using an expo- 
nential regression using the least-squares method. 



FIELD STUDY OF CRAYFISH AGONISM 



29 



Results 

Qualitative description of fight dynamics 

In general, as in the laboratory, crayfish quickly ap- 
proached one another and immediately began to interact 
(Bruski and Dunham, 1987; Bergman et at. 2003). In most 
instances, fights, unlike those in the laboratory, were rela- 
tively short and did not always show a stepwise progression 
in intensities (Stocker and Huber, 2001; Zulandt Schneider 
et at, 2001; Bergman et at. 2003). Crayfish retreated 
quickly from opponents by moving away in a different 
direction. Fights rarely progressed to the high intensities 
seen in the laboratory (Stocker and Huber, 2001; Bergman 
et at, 2003), but did seem to include many of the stereo- 
typical agonistic behaviors (Huber and Kravitz, 1995). Sur- 
prisingly, the number of fights ending in tailflips was low 
(45%) compared to fights in a laboratory (>90% for labo- 
ratory fights; Moore, pers. obs.). In addition, multiple inter- 
actions between the same opponents within a short time 
were virtually nonexistent, which may be due to social 
recognition (Daws et at, 2002). 

Quantitative description of all fights 

Two hundred and forty-six encounters were included in 
the data analysis. Statistical tests were performed on con- 
specific fights for O. rusticus for the three habitat types. 
Conspecific fights for O. virilis were observed only in the 
macrophyte habitat, and all statistical tests were done on 
these animals. Within the macrophyte bed habitat, no sig- 
nificant differences were found for any of the following 
statistical tests. For this reason, the data for the macrophyte 
habitat fights were pooled to provide a more global descrip- 
tion of the parameters of average agonistic encounters in 
nature. In the subsequent statistical tests, encounters were 
separated on the basis of the habitat in which the encounter 
occurred. The mean duration of all encounters was 5.3 
0.4 s (mean SE); (n = 246; Fig. 1A), and 0.45 (111 of 
246 encounters) of the conflicts ended with the behavior 
"tailflips away from an opponent" (Fig. 2). Intensity 2 was 
reached in 0.49 of the encounters (121 of 246; Fig. 3 A). 
intensity 3 was reached in 0.39 (95 of 246; Fig. 3A), and 
intensity 4 was reached in 0.12 (29 of 246; Fig. 3A). The 
average maximum intensity of all encounters was 2.6 
0.04 on the crayfish ethogram scale (Table 1; Fig. 3B). The 
rate of escalation is a measure of time to different levels of 
intensity and averaged 1.5 0.1 s for escalation to intensity 
2 (246 of 246 encounters; Fig. 4), 3.9 0.2 s to intensity 3 
(124 of 246 encounters; Fig. 4), and 9.5 0.9 s to intensity 
4 (28 of 246 encounters). 

Fight duration 

The overall fight duration in the three habitats for the 
collective pool of crayfish showed a significant difference 



A) 



= 6- 



2- 



B) 





09 
08 
07 
06 
05 
04 
0.3 
0.2 
1 
0.0 





l^B Shelter 
I I Detritus 

i':':?'!l Macrophyte 



N= 118 



Habitat 



I I 



1-3 4-6 7-9 10-12 13-15 16-18 19-21 22-24 25-28 29-31 

Fight Duration (s) 

Figure I. (A) The mean (SEM) tight duration of all fights (hatched), 
fights near shelters (black), fights on detritus patches (white), and fights 
among macrophytes beds (Crosshatch). Values above bars (N =) indicate 
numbers used for the statistical calculations. The letters above the bars 
denote a significant difference between the habitat types (one-way 
ANOVA, Tukey-hsd post hoc test; P < 0.05). (Note: Nine interactions 
were not categorized into a habitat type and are only included in the "All" 
category). (B) Frequency histogram showing the proportion of fight dura- 
tions in the shelter, macrophyte, and detritus habitats in 3-s bins. 

using a one-way MANOVA with a Tukey post hoc analysis 
(Fig. 1A). The fight duration in the shelter habitat (11.1 
0.7 s; n = 85) significantly differed from both the detritus 
patch (2.9 0.3 s; n = 33) and macrophyte bed interactions 
(1.80.1s;n = 1 18; Power = 1.00) (P < 0.05). There was 
no significant difference in fight duration between the con- 
flicts occurring on detritus patches and on macrophyte beds 
(P > 0.05). Fight durations for encounters in the shelter 
habitat ranged between 1 and 3 1 s, whereas the duration of 
encounters on macrophyte beds and detritus patches did not 
exceed 6 s (Fig. IB). 

Tailflip-away 

A contingency table for multiple comparisons of propor- 
tions demonstrated that agonistic encounters ended in a 
tailflip significantly more often when the fight was in the 
shelter (61/85 == 0.72; q = = 19.01; Power == 0.84) and 
detritus patch habitats (20/33 = 0.61; q = 10.36; Power = 
0.14) than when in macrophyte bed habitats (30/1 18 = 0.25; 
Power = 0.98) (P < 0.05; Fig. 2). No significant difference 
was found between conflicts in the shelter and detritus 
habitats (q = 3.29; P > 0.05). 

Fight intensity 

A significantly greater proportion of agonistic encounters 
on macrophyte beds (0.85; Power = 1.00) reached a max- 



30 



D. A. BERGMAN AND P. A. MOORE 





All 

Shelter 
Detritus 
Macrophyte 



Habitat 

Figure 2. Frequency histogram showing the proportion of lights that 
ended in a tailliip for all fights (hatched), fights near shelters (black), fights 
on detritus patches (white), and tights among macrophytes beds (cross- 
hatch). The letters above the bars denote a significant difference between 
the habitat types (contingency table for multiple comparisons of propor- 
tions; P < 0.05). 



imum intensity level of 2 (meral spread display) than either 
encounters in the shelter (0.0: q = 44.31; Power = 1.00) or 
detritus habitat (0.36; q = 14.86 Power = 0.21 ) (P < 0.05; 
Fig. 3A). A significantly greater proportion of encounters on 
detritus patches reached the maximum intensity of 2 than 
did encounters in the shelter habitat (q = 16.57; P < 0.05; 
Fig. 3A). A maximum intensity of 3 (pushing with chelae) 
was reached in a significantly greater proportion of fights 
when in the shelter (0.67; q = 22.01: Power = 0.62) and 
detritus habitats (0.61; q = 13.99; Power = 0.11) than in 
macrophyte beds (0.15; Power = 0.62) (P < 0.05; Fig. 3A). 
There was no significant difference between fights in the 
detritus and macrophyte habitats (q = 1.89; P > 0.05). In 
addition, maximum intensity 4 (open chelae use by grab- 
bing) was reached by a greater proportion of conflicts in the 
shelter habitat (0.33; Pov\u 0.24) than by interactions on 
detritus patches (0.03: q = \ 1.23: Power = 0.15) or mac- 
rophyte beds (0.0; q = 20.0) (P < 0.05; Fig. 3 A). There was 
no significant difference between the detritus and macro- 
phyte fights (q = 2.83; P > 0.05). No fights in any habitat 
achieved intensity 5 (unrestrained fighting). Encounters in 
the shelter habitat had a significantly higher average maxi- 



All 

Shelter 
Detritus 

[ : : : : : >: : : : : : l Macrophyte 




Intensity 2 



B) 



Intensity 3 
Maximum Intensity 



Intensity 4 



4-] 

I 3 - 
1 2 ~ 
I 1 - 

00 

0- 



a 



< Habitat 

Figure 3. (A) Frequency histogram showing the proportion of fights 
that achieved each maximum intensity level for all fights (hatched), fights 
near shelters (black), fights on detritus patches (white), and fights among 
macrophytes beds (Crosshatch). The letters above the bars denote a signif- 
icant difference between the habitat intensities (contingency table for 
multiple comparisons of proportions; P < 0.05). (B) The average maxi- 
mum fight intensity level achieved per habitat type. The letters above the 
bars denote a significant difference between the average maximum inten- 
sity per habitat (P < 0.05). 



mum intensity (3.33 0.05) than encounters in either of the 
other two habitats (P < 0.05; Fig. 3B). Interactions on 
detritus patches had a significantly higher average maxi- 
mum intensity (2.67 0.09) than encounters on macro- 
phyte beds (average maximum intensity of 2.16 0.03) 
(P < 0.05; Fi2. 3B). 



I Shelter 
] Detritus 
] Macrophyte 



4 - 



2- 




Intensity 2 



Intensin 



Figure 4. The mean (SEM) time to intensity levels of all fights 
(hatched), fights near shelters (black), fights on detritus patches (white), 
and fights among macrophytes beds (Crosshatch). The letters above the bars 
denote a significant difference for the time to reach intensity levels for each 
habitat (one-way ANOVA Tukey hsd post hoc test; P < 0.05). 



FIELD STUDY OF CRAYFISH AGONISM 



31 



Rate of escalation 

The average time to intensity 3 was significantly longer in 
the shelter habitat (4.3 0.5 s) than on the detritus patches 
(3.0 0.2 s) or macrophyte beds (3.1 0.2 s) (P < 0.05; 
Power = 1.00; Fig. 4). Intensity 2 showed no significant 
difference among the habitats, whereas intensity 4 was 
primarily achieved in the shelter habitat; however, no sta- 
tistical test could be performed because of the lack of fights 
in the macrophyte (n = 0) and detritus (n = 1) habitats. 

Effect of size differential on fight duration 

A significant exponential regression analysis using the 
least-squares method demonstrated that the duration of ag- 
onistic interactions (n = 117) was longer when the size 
differential between opponents was smaller (P < 0.05; Fig. 
5). Encounters were longer when opponents were size- 
matched within 10%, whereas fights with a size difference 
greater than 10% did not exceed 4 s. 

Discussion 

Extrinsic and intrinsic factors of agonistic behavior 

Crayfish agonistic interactions were longer (Fig. 1A), 
more intense (Fig. 3A, 3B, 4), and more likely to end with 
a tailflip (Fig. 2) when the interaction took place near a 
shelter (burrow) than on or near food-resource habitats 
(detritus and macrophytes). Interactions in the shelter hab- 
itat were more likely to reach higher intensities, but they 
also took longer to reach those intensities (Fig. 4). These 
results indicate that shelters were more valuable than either 



25- 



20- 



15-t 



Q 10- 



5- 



0- 



~ , , ~, fc *- , (267948-0218825i7edifTcrcnccl 

Duration = 1 .2 1 656 + exp 
r 2 =0.85586 

A Detritus 
O Macrophyte 
Shelter 




5 10 15 20 25 

% Size Difference of Combatants 



30 



35 



Figure 5. The percentage size differences of agonistic opponents an- 
alyzed with an exponential regression using the least-squares method. 
Size-matched fights lasted longer than fights between unevenly sized 
opponents (P < 0.05). 



macrophytes or detritus patches. Shelters may be alluring 
because of their use to attract mates or in defense from 
predators (Hill and Lodge, 1999). Conflicts were more 
intense (Fig. 3A, B) and ended more often with a tailflip 
(Fig. 2) when they occurred on detritus patches as opposed 
to macrophyte beds. 

Extrinsic factors, such as the availability of a shelter or a 
food resource, seem to influence aggressive fighting behav- 
ior in crayfish. With reference to food resources, when 
crayfish are fed a strictly macrophyte diet (Anmicola sp. and 
Lymnaea sp.) they have slower growth rates and higher 
levels of mortality than crayfish fed detritus (Hill et al., 
1993). Physiologically, it appears as if detritus is more 
nutritious and thus a more valuable resource than macro- 
phytes. Moreover, crayfish have been observed foraging on 
both species of macrophyte (Potamogeton sp. and Vallisne- 
ria sp.) and on detritus, suggesting that all three are viable 
food resources (Lodge and Lorman, 1987; Hill et al., 1993; 
Cronin et al., 2002). Among these food resources, detritus 
was located in distinct patches, whereas the macrophytes 
and their associated epiphytes were far more abundant and 
consistently distributed in Burt Lake. Moreover, shelters 
and detritus patches are limited resources, hence more easily 
defended. Conversely, macrophyte beds are usually an eas- 
ily accessible and abundant food source (Capelli, 1982), and 
defense becomes difficult and unnecessary when they are 
widely available. Given the increased nutritional value and 
limited distribution of detrital food sources, we predict that 
intraspecific encounters on detritus patches would be more 
intense and longer than fights in a macrophyte habitat. 
Indeed, in our sample, intraspecific fights lasted longer, 
reached a higher average maximum intensity, and ended 
more often with a tailflip. These results may be caused by 
the relative scarcity and temporal unpredictability of detri- 
tus patches within Burt Lake. Patches are often destroyed or 
moved overnight by physical wave action. Detrital patch 
heterogeneity may limit this potential nutritional resource, 
and when a crayfish finds a rare patch, the interactions 
become more intense in defense of it. In contrast, the 
macrophyte beds and their associated epiphytes had a more 
homogeneous distribution and greater temporal stability 
than detrital patches. As a result, macrophytes interactions 
were the least intense of the habitat types. 

Our results for the crayfish interactions in the macrophyte 
and detritus habitats are consistent with the idea that detritus 
is more valuable than macrophytes because of its increased 
nutritional value (Hill et al., 1993). However, no definitive 
conclusion about the relative merits of detritus and macro- 
phyte diets can be drawn from our study due to the unknown 
and varying composition of the detritus. Nevertheless, both 
macrophyte and detritus food resources appear to be less 
valuable than shelters. Shelters have been shown to have an 
effect on agonistic outcomes in that the previous owner is 
more likely to retain a shelter and initiate more interactions 



32 



D. A. BERGMAN AND P. A. MOORE 



(Peeks eta!.. 1995: Edsman and Jonsson, 1996). Capelli and 
Hamilton ( 1984) have shown that fond and prior residencies 
affect agonistic behavior in a ip d laboratory environ- 
ment. They reported thai activity decreases with 
the increased availabilir both shelters and food. In 
addition, they shov ncrease in shelter availability 
reduces aggression mou :un an increase in food availabil- 
ity. Thus, high food availability, more macrophytes than 
detrital patches, and low shelter availability would lead to 
more intense conflict over shelters, followed by detritus 
patches, and then macrophyte beds. 

Conspecific conflicts can usually be thought of as a 
"limited war." in which serious injury is avoided (Maynard 
Smith and Price, 1973). However, conspecific conflicts be- 
tween crayfish involve potentially lethal chelae that allow 
for an "unlimited war" with the possibility for more intense 
and lethal fights. High-intensity fights are common in a 
laboratory environment, largely because the opponents have 
been closely matched for size of carapace and chelae (Huber 
and Kravitz, 1995; Karavanich and Ateina, 1998a. b). In 
nature, an advantage in size directly confers an advantage in 
resource holding power (RHP) to the larger individual. 
Parker (1974) noted that as RHP disparity (size difference) 
increases, conflicts become less intense and shorter. Both 
combatants may increase their overall fitness by minimizing 
the chance for injury and reducing energy expenditure from 
long and intense fights. The winners of such interactions 
gain access to more valuable resources such as mates, food, 
or shelters, while the losers reduce their risk of predation, 
minimize energy costs, and emigrate to find a new resource. 

Our results are typical for asymmetric contests (Maynard 
Smith and Parker, 1976) in which a larger individual holds 
more valuable resources (shelters), and conflicts are longer 
when the opponents are size-matched. Moreover, when re- 
source availability is asymmetrical, conflicts will generally 
be shorter when the least valuable resource macrophytes 
in this study is in dispute. The shelter habitat appears to 
have some significance tied to it because the longest fights 
were in this habitat, and these fights were the most closely 
size-matched (Fig. 5). The longest fights in all three habitats 
occurred when the opponents were within 10% of each 
other in size (Bruski and Dunham. 1987; Schroeder and 
Huber, 2001; Stocker and Huber. 2001; Bergman ct al., 
2003). However, the shelter habitat appears to be more 
closely matched than iln food resource habitats; conse- 
quently, the valuable resource (shelter) may attract larger 
individuals, which causes smaller individuals to move to the 
periphery or into other habitats (detritus and macrophyte). 
Moreover, a hierarchy has likely been established in the 
stable shelter habitat, whereas the macrophyte and detritus 
habitats do not provide the same temporal stability and do 
not function to decrease predation. The recognition of hier- 
archical status is probably reinforced by visual or chemical 



social or individual recognition of conspecifics (Bruski and 
Dunham, 1987; Karavanich and Atema, 1998a, b). Intrinsic 
factors, such as size and recognition, and extrinsic factors, 
such as environmental surroundings, are important in deter- 
mining intraspecific agonistic outcomes. However, the ex- 
tent of the role each intrinsic and extrinsic factor plays is yet 
to be conclusively determined. 

Cursory review of laboratory- studies in relation to field 
observations 

Intraspecific aggressive behavior between decapod crus- 
taceans can be influenced by a myriad of extrinsic factors. 
For example, an extrinsic factor such as small aquarium size 
will sometimes elicit a "critical reaction" (Hediger. 1950). 
A critical reaction occurs when antagonists are crowded 
together in an aquarium with no possibility of escape. The 
inability to escape a competitor can cause changes in fight 
duration, retreat behavior, and intensity levels reached in 
fights (Peeke ct al., 2000). The presence of a defendable 
extrinsic resource can also cause an escalation in fight 
intensities in small aquaria. When shelters are present, fights 
will be more intense than when they are absent (Peeke ct al., 
1995). Intrinsic factors such as size. sex. and social expe- 
rience can also affect aggressive activities. Size-matched 
large crayfish escalate more slowly to high intensities and 
have longer fight durations than size-matched small crayfish 
(Schroeder and Huber, 2001). Generally, male crayfish are 
more aggressive than females (Bruski and Dunham. 1987), 
and social experiences in the form of winner and loser 
effects influence the likelihood of success in subsequent 
fights (Daws et ai, 2002; Bergman ct al., 2003). These 
extrinsic and intrinsic factors change the dynamics of fights 
in the laboratory so that they do not necessarily show the 
same characteristics as fights in a natural setting. 

In general, fights were shorter (5.3 0.4 s; Fig. 1) and 
had lower average maximum intensities (2.6; Fig. 3B) in the 
field than in laboratory studies (Table 2). The average 
maximum fight intensity in the field was lower than in 
laboratory fights seen by both Schroeder and Huber (2001 ) 
(2.7 and 2.8) and Bergman et al.. (2003) (4.2 and 3.5) 
(Table 2). In addition, the time to different intensity levels 
has been used as a measure of the rate of escalation in 
violence during fights and was considerably shorter for all 
intensities in the field than in the laboratory fights of Stocker 
and Huber (2001 ) and Bergman et al. (2003) (Table 2). 

Within a laboratory environment, all aspects of a con- 
frontation can be controlled to lengthen conflicts or increase 
fight intensities. Sex, species, size of opponents, size of 
aquarium, reproductive state, status/individual recognition, 
social experience, and hierarchy establishment can all be 
controlled in the laboratory. An example of a controlled 
variable is size-matched opponents (Bruski and Dunham, 
1987; Rutherford et al., 1995; Stocker and Huber, 2001: 



FIELD STUDY OF CRAYFISH AGONISM 
Table 2 



33 



Ciir.\ory review of crustacean agonistic experiments in the laboratory 



Reference 


Animal 


Treatment 


Avg. 
Duration Intensity Time to Time to Time to 
(s) Value Intensity 2 Intensity 3 Intensity 4 


Bergman and Moore 


Crayfish 


Field observations 


5.3 2.6 1.5 3.9 9.5 


(This study) 








Bergman et al.. 2003 


Male crayfish 


Previous win experience vs. 


127.0 4.2 4.6 8.6 18.0 






size-matched opponent 






Male crayfish 


Previous win experience vs. 


452.0 3.5 87.0 72.0 336.0 






size-matched anosmic 








opponent 




Stocker and Huher. 2001 


Satiated male 


Food odor present; Size-matched 


85.0 135.0 210.0 




crayfish 








Starved male 


Food odor present; Size-matched 


60.0 90.0 125.0 




crayfish 






Zulandt et al., 2001 


Familiar male 


Urine present; Fight I/Fight 2 


80/70 1.9/1.8 




crayfish 








Familiar male 


Urine absent; Fight I/Fight 2; 


230/80 2.4/2.0 




crayfish 


Size-matched 




Schroeder and Huber. 2001 


Male crayfish 


Small; Size-matched 


16.7 2.7 




Male crayfish 


Large; Size-matched 


30.6 2.8 


Guiasu and Dunham, 1998 


Male crayfish 


First fight/Last fight; 


95.3/46.2 






Size-matched 




Guiasu and Dunham. 1997 


Male crayfish 


First fight/Last fight; 


115.6/26.2 






Size-matched 




Bruski and Dunham, 1987 


Male crayfish 


Dark; Size-matched 


42.0 




Male crayfish 


Light; Size-matched 


17.0 




Female crayfish 


Dark; Size-matched 


29.0 




Female crayfish 


Light; Size-matched 


11.0 


Karavanich and Atema, 1998a 


Male lobster 


Control; Day 1 Size-matched 


510.0 




Male lobster 


Control; Day 2 Size-matched 


150.0 




Male lobster 


Anosmic; Day 1 Size-matched 


350.0 




(anosmic) 








Male lobster 


Anosmic; Day 2 Size-matched 


525.0 




(anosmic) 






Huber and Kravitz, 1995 


Male and 


Day 1; Size-matched; 


568.0 




female 


Laboratory-raised juveniles 






lobster 








Male and 


Day 2; Size-matched; 


365.0 




female 


Laboratory-raised juveniles 






lobster 







Bergman et al.. 2003). Size matching increases the likeli- 
hood that rights will be longer and more intense than usually 
observed in the field. Field encounters had an average fight 
duration of 5.3 s (Fig. 1 ), whereas crayfish fight durations in 
the laboratory ranged from an average of 1 1 .0 to 452.0 s 
(Bruski and Dunham, 1987; Bergman et at.. 2003) and 
lobster interactions took longer yet, ranging from 350.0 to 
568.0 s (Huber and Kravitz, 1995; Karavanich and Atema, 
1998a) (Table 2). This study does show that the fights of 
closely size-matched individuals are longer than those of 
unmatched opponents (Fig. 5), but they are not as long as 
fights seen in the laboratory. A possible extrinsic influence 
on this increased duration of fights is confinement of ani- 
mals within an aquarium. Within the laboratory, a push to 
use larger aquaria will reduce the "critical reaction" effect 



on fights by providing space for a possible escape that 
signifies the end of a conflict. Generally, the dynamics of 
laboratory fights tends to mimic field observations. How- 
ever, Guiasu and Dunham (1997, 1998), using relatively 
large aquaria, showed average fight durations of 115.6 and 
95.3 s, times that are considerably longer than those seen in 
this study (Table 2). The light regime also affects the 
duration of crayfish fights. Crayfish fights are shorter in the 
light than in the dark (Bruski and Dunham, 1987; Table 2). 
However, under different circumstances, fights can reach 
very long durations under lighted conditions, as was ob- 
served by Zulandt Schneider et al. (2001) and Bergman et 
ul. (2003) (Table 2). One cannot discount the fact that 
laboratory conditions may have an unknown effect on ag- 
onistic behavior. 



34 



D. A. BERGMAN AND P. A. MOORE 



Summary 

These field observations sugge ihe environmental 

surroundings have a signihY i on intraspecific ago- 

nistic bouts in crayfish >ted by Parker (1974), 

asymmetries in resoiiR 1 ' <ower can be an important 

factor in fight prop- Conflicts in the presence of 

shelters were longei ;e intense, suggesting that shel- 

ters have a higher nn . ;s value than detritus or macrophyte 
food resources. A shelter's protective value may outweigh 
the value of the food sources when the threat of predation is 
especially high. Detrital food sources are likely more valu- 
able than macrophyte food sources because of their patchy 
distribution and the nutritional inadequacy of macrophytes 
(Hill etui., 1993). It is quite evident from this study's results 
that extrinsic resources are an intricate influence on the 
agonistic interactions of crayfish. 

Moreover, we conclude that aggressive behavior must be 
examined both in the laboratory and in the field to better 
understand the factors that influence crayfish aggression. 
Each experimental environment has unique benefits and 
problems. Observations in nature contribute to an under- 
standing of habitat usage, movement patterns, shelter occu- 
pation, and food availability. Laboratory experiments are 
invaluable in elucidating the behavioral mechanisms and the 
environmental components that affect aggression. By con- 
trolling different aspects of agonistic interactions, such as 
size, sex, food preferences, and shelter accessibility, a re- 
searcher can test facets of agonistic behavior that are not 
easily controlled in a natural setting. However, such inves- 
tigations do not answer the question of whether the behavior 
is an artifact of laboratory confinement or a behavior that is 
displayed in nature. Consequently, one must be hesitant 
when using laboratory results to explain agonistic behaviors 
in the wild. Laboratory and field observations show consid- 
erable differences in fight dynamics. A combination of the 
two is needed to develop a realistic picture of aggressive 
behavior. 

Acknowledgments 

The authors thank the Laboratory for Sensory Ecology 
for comments on the manuscript and the University of 
Michigan Biological Station for the use of their facilities. 
Thanks also to Robert Schneider, Rebecca Zulandt 
Schneider, and Mary Wolf for help in collecting the data. 
Support for this research was supplied by the National 
Science Foundation (DAB 9874608, IBN-95 14492. and 
IBN-0131320). 

Literature Cited 

Abrahamsson, S. A. A. 1966. Dynamics of an isolated population of the 

crayfish Asiacus astacus Linne. Oikos 17: 96-107. 
Atema, .1. 1986. Review of sexual selection and chemical communica- 



tion in the lobster. Homarus americanus. Can. J. Fish. Ac/uat. Sci. 43: 
2283-2290. 

Bergman, D. A., C. P. Kozlowski, J. C. Mclntyre, R. Huber, A. G. 
Daws, and P. A. Moore. 2003. Temporal dynamics and communi- 
cation of winner-effects in the crayfish. Orconectes nisticus. Behaviour 
(In Press). 

Berrill, M., and M. Arsenault. 1984. The breeding behaviour of the 
northern temperate orconectid crayfish. Orconectes nisticus. Anim. 
Belmv. 32: 333-339. 

Bovbjerg, R. V. 1953. Dominance order in the crayfish Orconectes 
virilis (Hagan). Physiol. Zool. 26: 173-178. 

Bovbjerg, R. V. 1956. Some factors affecting aggressive behavior in 
crayfish. Physiol. Zool. 29: 127-136. 

Bovbjerg, R. V. 1970. Ecological isolation and competitive exclusion in 
two crayfish (Orconectes virilis and Orconectes immunis). Ecology 51: 
225-236. 

Bruski, C. A., and D. \V. Dunham. 1987. The importance of vision in 
agonistic communication of the crayfish Orconectes rusticus. Behav- 
iour 103: 83-107. 

Capelli, G. M. 1982. Displacement of northern Wisconsin crayfish by 
Orconectes rusticus (Girard). Limnol. Oceanogr. 27: 741-745. 

Capelli, G. M., and P. A. Hamilton. 1984. Effects of food and shelter 
on aggressive activity in the crayfish Orconectes rusticus (Girard). J. 
Cmstac. Bio/. 4: 252-260. 

Capelli, G. M., and B. L. Munjal. 1982. Aggressive interactions and 
resource competition in relation to species displacement among cray- 
fish of the genus Orconectes. J. Crustac. Biol. 2: 486-492. 

Cronin, G.. D. M. Lodge, M. E. Hay, M. Miller, A. M. Hill, T. Hovath, 
R. C. Bolser, N. Lindquist, and M. Wahl. 2002. Crayfish feeding 
preferences for freshwater macrophytes: the influence of plant structure 
and chemistry. J. Crustac. Biol. 22: 708-718. 

Daws, A. G., J. L. Grills, K. Konzen, and P. A. Moore. 2002. Previous 
experiences alter the outcome of aggressive interactions between males 
in the crayfish. Procambarus clarkii. Mar. Freslm: Behav. Physiol. 35: 
139-148. 

Dingle, H. 1983. Strategies of agonistic behavior in Crustacea. Pp. 85- 
1 1 1 in Studies in Adaptation: The Behavior of Higher Crustacea, S. 
Rebach and D. W. Dunham, eds. John Wiley and Sons. New York. 

Edsman, L., and A. Jonsson. 1996. The effect of size, antennal injury, 
ownership, and ownership duration on fighting success in male signal 
crayfish. Pacifastactts /eniusculits (Dana). Nord. J. Freshw. Res. 72: 
80-87. 

Figler, M. H., H. M. Cheverton, and G. S. Blank. 1999. Shelter 
competition in juvenile red swamp crayfish (Procambarus clarkii): the 
influences of sex differences, relative size, and prior residence. Aaua- 
ciiltun' 178: 63-75. 

Garvey, J.. and R. A. Stein. 1993. Evaluating how chela size influences 
the invasion potential of an introduced crayfish (Orconectes rusticus}. 
Am. Nat. 129: 172-181. 

Guiasu, R. C., and D. VV. Dunham. 1997. Initiation and outcome of 
agonistic contests in male Form I Cambarus robustus Girard, 1852 
crayfish (Decapoda. Cambaridae). Crustaceana 70: 480-496. 

Guiasu, R. C., and D. \V. Dunham. 1998. Inter-form agonistic contests 
in male crayfishes, Cambarus robustus (Decapoda. Cambaridae). In- 
vertehr. Biol. 117: 144-154. 

Hazlett, B., D. Rubenstein, and D. Rittschof. 1975. Starvation, energy 
reserves, and aggression in the crayfish Orconectes virilis (Hagen, 
1870) (Decapoda. Cambaridae). Crustaceana 28: 11-16. 

Hediger, H. 1950. Wild Animals in Captivity. Butterworths, London. 

Hill, A. M., and D. M. Lodge. 1999. Replacement of resident crayfishes 
by an exotic crayfish: the roles of competition and predation. Ecol. 
April. 9: 678-690. 

Hill, A. M., D. M. Sinars, and D. M. Lodge. 1993. Invasion of an 



FIELD STUDY OF CRAYFISH AGONISM 



35 



occupied niche by the crayfish Orconecles rusticus: potential impor- 
tance of growth and mortality. Oecologia 94: 303-306. 

Huber. R., and E. A. Kravitz. 1995. A quantitative analysis of agonistic- 
behavior in juvenile American lobsters (Homarus americanus L.). 
Brain Beha\: Evol. 46: 72-83. 

Hyatt, G. W. 1983. Qualitative and quantitative dimensions of crusta- 
cean aggression. Pp. 1 13-139 in Studies in Adaptation: The Behavior 
of Higher Crustacea, S. Rebach and D. W. Dunham, eds. John Wiley 
and Sons. New York. 

Karavanich. C., and J. Atema. 1998a. Olfactory recognition of urine 
signals in dominance fights between male lobster. Homarus america- 
nus. Behaviour 135: 719-730. 

Karavanich, C., and J. Atema. 1998b. Individual recognition and mem- 
ory in lobster dominance. Anim. Behav. 56: 1553-1560. 

Karnofskv, E. B., J. Atema, and R. H. Elgin. 1989. Field observations 
of social behavior, shelter use. and foraging in the lobster, Homarus 
americanus. Biol. Bull. 176: 239-246. 

Lodge, D. M., and J. G. Lorman. 1987. Reductions in submersed 
macrophyte biomass and species richness by the crayfish Orconectes 
rusticus. Can. J. Fish Aquat. Sci. 44: 591-597. 

Maynard Smith. J., and G. A. Parker. 1976. The logic of asymmetric 
contests. Anim. Behav. 24: 159-175. 

Maynard Smith. J.. and G. R. Price. 1973. The logic of animal conflict. 
Nature 246: 15-18. 

Parker, G. A. 1974. Assessment strategy and the evolution of fighting 
behavior. J. Theor. Biol. 47: 223-243. 

Pavey, C. R., and D. R. Fielder. 1996. The influence of size differential 
on agonistic behaviour in the freshwater crayfish. Cherax cuspidams. J. 
Zool. 238: 445-457. 

Peeke, H. V. S., J. Sippel, and M. H. Figler. 1995. Prior residence 
effects in shelter defense in adult signal crayfish (Pacifastacus lenius- 
culus) results in same- and mixed-sex dyads. Crustaceana 68: 873- 
881. 

Peeke, H. V. S., M. H. Figler, and E. S. Chang. 1998. Sex differences 
and prior residence effects in shelter competition in juvenile lobsters, 
Homarus americanus Milne-Edwards. / Exp. Mar. Biol. Ecol. 229: 
149-156. 

Peeke, H. V. S., G. S. Blank, M. H. Figler, and E. S. Chang. 2000. 



Effects of exogenous serotonin on a motor behavior and shelter com- 
petition in juvenile lobsters (Homarus americanus). J. Comp. Ph\\ml. 
.4 186: 575-582. 

Ranta, E., and K. Lindstriim. 1992. Power to hold sheltering burrows 
by juveniles of the signal crayfish. Pacifastacus leniuscu/its. Etholog\ 
92: 217-226. 

Ruben.stein, D. I., and B. A. Hazlett. 1974. Examination of the agonis- 
tic behaviour of the crayfish Orconectes virilis by character analysis. 
Behaviour SO: 193-216. 

Rutherford, P. L., D. W. Dunham, and V. Allison. 1995. Winning 
agonistic encounters by male crayfish Orconectes rusticus (Girard) 
(Decapoda. Cambaridae): chela size matters but chela symmetry does 
not. Crustaceana 68: 526-529. 

Schroeder, L., and R. Huber. 2001. Fight strategies differ with size and 
allometric growth of claws in crayfish, Orconectes rusticus. Behaviour 
138: 1437-1444. 

Steele, C.. C. Skinner, P. Alberstadt, and J. Antonelli. 1997. Impor- 
tance of adequate shelters for crayfishes maintained in aquaria. At/uar. 
Sci. Consen: 1: 189-192. 

Stein, R. A. 1976. Sexual dimorphism in crayfish chelae: functional 
significance linked to reproductive activities. Can. J. Zool. 54: 220- 
227. 

Stacker, A. M., and R. Huber. 2001. Fighting strategies in crayfish 
Orconectes rusticus (Decapoda. Cambaridae) differ with hunger state 
and the presence of food cues. Etho/ogv 107: 727-736. 

Tomba, A. M., T. A. Keller, and P. A. Moore, 2001. Foraging in 
complex odor landscapes: chemical orientation strategies during stim- 
ulation by conflicting chemical cues. J. North Am. Bemhol. Soc. 20: 
211-222. 

Wilson, E. O. 1975. Sociobiology. Belknap Press. Cambridge, MA. 

Zar, J. H. 1999. Multiple comparisons for proportions. Pp. 563-565 in 
Biostatistical Analysis. 4' h ed. Prentice Hall. Englewood Cliffs, NJ. 

Zulandt Schneider, R. A., R. W. S. Schneider, and P. A. Moore. 1999. 
Recognition of dominance status by chemoreception in the red swamp 
crayfish, Procamharus ciarkii. J. Chem. Ecol. 25: 781-794. 

Zulandt Schneider, R. A., R. Huber. and P. A. Moore. 2001. Individ- 
ual and status recognition in the crayfish. Orconectes rusticus: the 
effects of urine release on fight dynamics. Behaviour 138: 137-153. 



Reference: Biol. Bull. 205: 36-46. (August 20031 
2003 Marine Biological Laboratory 



Twisting and Bending of Biological Beams: 

Distribution of Biological Beams in 

a Stiffness Mechanospace 

SHELLEY A. ETNIER* 
Department of Biologv, Duke University, Di/iiuiin, North Carolina 27708-0338 



Abstract. Most biological beams bend and twist rela- 
tively easily compared to human-made structures. This pa- 
per investigates flexibility in 57 diverse biological beams in 
an effort to identify common patterns in the relationship 
between flexural stiffness and torsional stiffness. The pat- 
terns are investigated by mapping both ideal and biological 
beams into a mechanospace defined by flexural and tor- 
sional stiffness. The distribution of biological beams is not 
random, but is generally limited to particular regions of the 
mechanospace. Biological beams that are stiff in bending 
are stiff in torsion, while those that bend easily also twist 
easily. Unoccupied regions of the mechanospace represent 
rare combinations of mechanical properties, without prov- 
ing that they are impossible. The mechanical properties of 
biological beams closely resemble theoretical expectations 
for ideal beams. Both distributions are potentially being 
driven by the interdependence of the material and structural 
properties determining stiffness. The mechanospace can be 
used as a broadly comparative tool to highlight systems that 
fall outside the general pattern observed in this study. These 
outlying beams may be of particular interest to both biolo- 
gists and engineers due to either material or structural 
innovations. 

Introduction 

Flexibility, or the ability to deform in response to a load, 
is a property of mosl Mnlogical beams (Vogel, 1984; 
Denny, 1988), yet the bii.l :>1 consequences of flexibility 
vary widely. In motile organisms, flexibility permits the 
relative movements of structural elements in response to 



Received 14 February 2003; accepted 21 May 2003. 
*Present address: University of North Carolina at Wilmington. 601 S. 
College Road, Wilmington. NC 28403. E-mail: etniers@uncwil.edu 



internal forces generated by muscular contractions or hy- 
drostatic pressures. The flexibility of a fish backbone influ- 
ences the mechanical behavior of the body during undula- 
tory swimming (McHenry et al., 1995; Long and Nipper. 
1996), while the flexibility of mammalian backbones has 
been implicated in locomotor differences between species 
(Gal, 1993). In many sessile organisms, flexibility allows 
structures to passively adjust their posture relative to the 
forces experienced (Wainwright et al.. 1976; Vogel, 1984). 
Leaf petioles (Vogel, 1989; Niklas, 1991) and herbaceous 
plants (Ennos, 1993; Etnier and Vogel, 2000) reduce flow- 
induced drag by bending or twisting in response to wind, 
and similar drag-reducing mechanisms have been found in 
hydroid colonies (Harvell and LaBarbera, 1985) and anem- 
ones (Koehl, 1977a). Other flexible organisms take advan- 
tage of external forces to passively orient their filter-feeding 
structures in response to ever-changing flow (Wainwright 
and Dillon, 1969; Koehl, 1977b; Harvell and LaBarbera, 
1985; Best, 1988). Thus, the ability to deform in response to 
loads is observed in both motile and sessile organisms living 
in either an aquatic or a terrestrial environment, apparently 
independent of phylogenetic affiliations. Such convergence 
may be viewed as a red flag indicating the tremendous 
importance of flexibility in the design of biological organ- 
isms (Lauder, 1982; Vogel, 1998). 

Flexibility is measured in terms of stiffness, where flex- 
ural stiffness (El in N nr) represents the resistance of a 
beam (a structure that is long relative to its width) to 
bending, and torsional stiffness (GJ in N nr) represents the 
resistance of a beam to twisting. Flexural stiffness and 
torsional stiffness are composite variables that are influ- 
enced both by material and structural properties (Wain- 
wright et al.. 1976). Every beam is characterized by a 
combination of flexural stiffness and torsional stiffness, and 
the relationship between these two variables determines 



36 



TWISTING AND BENDING BIOLOGICAL BEAMS 



37 



how the beam responds to a given load. The ratio of flexural 
stiffness to torsional stiffness, commonly termed the twist- 
to-bend ratio, has been used as a dimensionless (and, thus, 
size invariant) index describing the relationship between 
these two variables (Niklas. 1992; Vogel, 1992, 1995; Et- 
nier and Vogel, 2000). The twist-to-bend ratio indicates the 
relative resistance of a beam to bending versus twisting. 
More intuitively, a higher twist-to-bend ratio indicates a 
structure that twists more readily than it bends, without 
reference to the magnitude of either variable. 

While flexibility is a common property of a phylogeneti- 
cally diverse group of organisms, are there any common 
patterns or trends in the relationship between flexural stiff- 
ness and torsional stiffness in biological beams? This paper 
investigates such patterns with a mechanospace defined by 
values of flexural and torsional stiffness. The mechano- 
space, similar to Raup's (1966) classic morphospace, is a 
broadly comparative tool used to visualize the relationships 
between mechanical variables in biological beams. The 
concept is based on the premise that the mechanical prop- 
erties of flexural and torsional stiffness are common to all 
biological beams. Three variations of this mechanospace 
will be used to compare the patterns of flexibility seen in a 
large diversity of biological structures. First, material and 
structural properties will be used in combination to predict, 
on the basis of principles of engineering beam theory, the 
theoretical relationships between bending and twisting in 
ideal beams. Second, experimentally measured values of 
flexural stiffness and torsional stiffness for biological struc- 
tures will be examined within the context of the theoretical 
distribution. Finally, the relative contribution of overall size 
to the mechanical properties of biological beams will be 
explored. The results suggest that the distribution of bio- 
logical beams within the mechanospace is not random, due 
to the interdependence of material and structural properties 
determining stiffness. 

Materials and Methods 

Distribution of ideal beams 

The distribution of ideal beams was determined using 
principles from engineering beam theory. Importantly, this 
distribution is limited to structures built of a single, isotropic 
material (i.e., the material properties are not directional ly 
variable), with precise specifications for the cross-sectional 
shape of the beam in question (Roark, 1943). Additionally, 
engineering beam theory stipulates that the material is lin- 
early elastic, and that the beam is straight and does not vary 
in size or shape along its length, nor does the beam undergo 
deflections greater than 10% of total length (Roark. 1943). 
More complex solutions are required for beams that un- 
dergo larger deflections (e.g., Morgan and Cannell, 1987; 
Morgan, 1989). 



Material properties. The material properties influencing 
flexural stiffness and torsional stiffness are Young's mod- 
ulus (E in N m 2 ) and the shear modulus (G in N m~ 2 ), 
respectively. Young's modulus and the shear modulus are 
related to one another by Poisson's ratio (v), which is the 
dimensionless ratio of the induced strain, causing lateral 
contraction of the specimen, to the applied strain, causing 
the specimen to elongate (Vincent, 1990). Poisson's ratios 
can vary from to 0.50 for naturally occurring isotropic 
materials. Mollusc shell has a Poisson's ratio of about 0.10, 
while rubber has a ratio closer to 0.50 (Denny, 1988) and 
materials such as cornstalks have moderate ratios around 
0.23 (Prince and Bradway. 1969). Commonly occurring 
metals have Poisson's ratios between 0.25 and 0.30 (Niklas, 
1992). 

For isotropic materials, the shear modulus is related to 
Young's modulus (Roark. 1943) by: 



G = 



2(1 + v) 



(I) 



Thus, the Young's modulus for a typical isotropic material 
will range from 2 to 3 times its shear modulus as Poisson's 
ratio varies from to 0.50 (Wainwright et ai, 1976: Niklas, 
1992). 

Structural properties. The structural variables influencing 
flexural stiffness and torsional stiffness are the second mo- 
ment of area (/ in m 4 ) and the polar moment of area (J in 
m 4 ), respectively. These variables reflect the geometry of a 
cross section of a beam and are influenced by size, shape, 
and orientation (Roark, 1943). The relationship between 7 
and J depends upon the cross-sectional shape of the struc- 
ture in question (Roark. 1943). Formulas for the calculation 
of / and J for most simple shapes can be found in any basic 
engineering handbook (e.g., Gere and Timoshenko, 1984). / 
and J are both proportional to radius to the fourth power, 
hence radius is a very strong determinant of stiffness 
(Roark, 1943). For example, for a beam with a circular 
cross-sectional area 



/ = 



and J = 



77T 



(2) 



the value of II J is 0.50. Small changes in the cross-sectional 
shape of a beam can greatly influence the values of / and J; 
thus, the value I/J for noncircular cross sections can be 
much higher (Table 1). Note that there may be several 
values of II J for a beam with an asymmetric cross-sectional 
shape, depending on its orientation with respect to the 
applied load (Table 1 ). 

Relationship benveen flexural and torsional stiffness. 
El and GJ are dependent variables, with Poisson's ratio 
linking the material properties (E and G), and geometry 
linking the structural properties (/ and /). Theoretically, the 
relationship between flexural stiffness and torsional stiff- 



38 



S. A. ETNIER 

Table 1 

Theoretical re/tin. ^ tv/; flexural and torsional stiffness 



Ratio of El/GJ 



onal shape 


111 


then EIG = 2 


then EIG = 3 





fe . 0.50 


1.00 


1.50 


kl major:minor axis ....^ 


m^^. 0.27 


0.54 


0.81 



Ellipse 4:1 majorminor axis 



I 



4.25 



8.50 



12.75 



The ratio of II J was determined from beam-theory equations (Niklas, 1992) and represents the structural 
contributions to stiffness in these beams. For the ellipses, / is calculated about the axis indicated by the dashed 
line. The ratio of EIC represents the material contribution to beam stiffness as Poisson's ratio was allowed to 
vary from to 0.50. The chosen cross-sectional shapes are broadly representative of biological beams 
(Wainwright, 1988). 



ness can vary only slightly, based on this interdependence. 
The relationships between flexural stiffness and torsional 
stiffness can be calculated for beams of different shapes and 
materials (Table 1 ). The ratio of El/GJ for a given circular 
beam can range between 1 and 1.5 as Poisson's ratio varies 
from to 0.5. In contrast, an elliptical beam with a major to 
minor axis ratio of 4.1 has a range of El/GJ (with El 
measured about the minor axis) from 8.50 to 12.75 for the 
same range in Poisson's ratio (Niklas, 1992). These partic- 
ular cross-sectional shapes are presented because they are 
broadly representative of biological beams (Wainwright, 
1988). Note that two beams can have the same El/GJ ratio 
despite vast differences in size, shape, and material because 
the ratio is determined by the relative magnitudes of flexural 
stiffness to torsional stiffness, not by their absolute values. 
The relationship between El and GJ for ideal beams can 
be mapped into a mechanospace defined by these variables 
(Fig. 1). Each quadrant of the mechanospace represents 
different combinations of flexural stiffness and torsional 
stiffness, ranging from the upper left, where beams twist 
easily but do not bend, to the lower right, where beams bend 
easily but do not twist. The dashed line in Figure 1 repre- 
sents a circular beam with a moderate Poisson's ratio of 
0.25. The solid lines in Figure 1 represent the two orienta- 
tions of a beam with a 4: 1 elliptical cross-sectional shape as 
Poisson's ratio is maximized (v = 0.5) or minimized (v = 
0). An elliptical beam of this form was chosen because such 
an ellipse has a large l/J ratio and likely represents an 
extreme shape for biological structures. Thus, the area be- 
tween the two solid lines represents the range of values for 
beams with moderate to extreme material values, with 
shapes that are broadly representative of biological beams 
(Wainwright, 1988). Importantly, the solid lines do not 
represent absolute theoretical limits, but rather identify the 
expected extremes for ideal beams composed of a single. 



isotropic material. Greater shape modification will slightly 
alter these limits. 



Distribution of biological beams 

Although biological beams are commonly modeled using 
beam theory (e.g., Koehl, 1977b; Carrier, 1983; Vogel, 
1992; Baumiller, 1993; Ennos, 1993; Vogel, 1995; Niklas, 
1998; Etnier and Vogel, 2000; Etnier, 2001), they are rarely, 
if ever, made up of a single, isotropic material. More typi- 
cally, they are made up of multiple materials, whose distri- 
bution varies both across the cross section of the beam and 
along its length. Yet. in practice, flexural stiffness and 
torsional stiffness are measured experimentally using basic 
engineering formulas for beams. In general, a known load is 
applied to a beam, causing it to deform, either by bending or 
by twisting. The exact equation used is dependent on how 
the beam is loaded. For example, for end-loaded cantilever 
beams (one end fixed and the other end free to deform), 
flexural stiffness is calculated as: 



El = 



3y 



(3) 



where F is the applied force, L is the length of the specimen, 
and v is the deflection of the free end of the specimen (Gere 
and Timoshenko, 1984). Similarly, torsional stiffness is 
calculated using the following formula: 



Fd 



(4) 



where F is the force applied at a moment arm d, L is the 
length of the specimen, and 6 is the rotation of the free end 
in radians (Gere and Timoshenko, 1984). 

Data were compiled from the literature and from the 



TWISTING AND BENDING BIOLOGICAL BEAMS 



39 



E 

Z 



<U 


C/3 

2 

3 
X 



OJD 
O 



-3 



-6 



Elliptical beam 
v = 0.50 



Daffodil 



Sedge 





Circular beam 
v = 0.25 m 



-3 

log Torsional Stiffness (Nm ) 



o antennae 
n crinoid arms 
o horsetails 
A stems 
petioles 
+ root 
A vines 

gorgonians 

tree branches 



Figure 1. The mechanospace defined by values of flexural stiffness and torsional stiffness. The lines 
represent the distribution of ideal beams based upon an assumed cross-sectional shape and Poisson's ratio. The 
symbols represent the nine groups (Table 2), with each individual point representing an average value for a 
species (appendix). The upper line represents an elliptical beam (4:1 major to minor axis) with /// calculated 
about the short axis and a Poisson's ratio of v = 0.50. The lower line represents that same beam with I/J 
calculated about the long axis and a Poisson's ratio of v = 0. The dashed line represents a circular beam with 
a Poisson's ratio of v = 0.25. 



author's research to obtain average flexural stiffness and 
torsional stiffness measures for 25 species of plants and 
animals (see appendix). These data were obtained following 
protocols similar to those discussed above. In other cases 
(n = 32), published data consisted only of measures of E 
and G. In these cases, the researchers experimentally deter- 
mined El and GJ, but then factored out / and J based on the 
cross-sectional size and shape of the structures. But note 
that the experimental data were still based on the overall 
mechanical behavior of the beam (i.e., deformation due to a 
load), rather than direct measures of material properties. In 
cases where published data consisted only of measures of 
and G, both size and shape were estimated to determine 
values for / and J (appendix). Flexural stiffness and tor- 
sional stiffness were then calculated, based on these esti- 
mates. The estimates of beam diameter were deliberately 
conservative, while cross-sectional shape was always as- 
sumed to be circular. For example, for 14 of the 16 vines 
and 5 of the 1 1 tree branches included in the mechanospace. 
the beam was assumed to have a circular cross section with 
a diameter of 0.02 m. The reported diameters for these 
species were 0.02-0.05 m (Putz and Holbrook. 1991). Sim- 



ilar assumptions were made for the gorgonian corals, with 
an estimated diameter of 0.002 m (Jeyasuria and Lewis, 
1987). The assumption of a relatively small diameter will 
affect the overall magnitudes of flexural stiffness and tor- 
sional stiffness, but will not affect the ratio of the two. The 
assumption of a circular cross-sectional shape will result in 
lower estimated values of I/J than would be seen in noncir- 
cular beams (Table 1 ). 

The species (total /; = 57) were divided into nine groups 
based upon broad morphological similarities (Table 2). 
Structures varied greatly in size, with diameters ranging 
from 0.001 m (red maple petioles) to 0.05 m (small tree 
branches). Average values for log GJ versus log El for all 
species (n = 57) were plotted in the mechanospace (Fig. 1 ). 
Note that each point represents an average value for a 
species, and thus does not reflect individual variation in size 
and material properties. For asymmetric beams, such as the 
daffodils, crinoid arms, and crustacean antennae, flexural 
stiffness is reported as the average of the different orienta- 
tions. Coefficients of variation for these organisms varied 
between 357r and 196% for flexural stiffness, and from 36% 
to 110% for torsional stiffness (Etnier and Vogel, 2000; 



40 



S. A. ETNIER 



Table 2 

Basic group characteristics for beams ui\il,-;t'd m this study 



Groups 



K characteristics 



Crustacean antennae 

Cnnoid arms 
Horsetails 
Herbaceous stems 
Leaf petioles 

Tree roots 
Vines 
Gorgonian corals 

Tree branches 



Mil beam. Antennae used for sensory 
ution and in some cases, for 
jicssive interactions. Not loaded 
gravitationally. Aquatic animal. 

Multi-jointed beam. Arms used for passive 
filter feeding. Not loaded gravitationally. 
Aquatic animal. 

Multi-jointed beam. Stem must maintain 
upright position for photosynthesis, so self- 
supporting. Terrestrial plant. 

Continuous beam. Stem must maintain upright 
position for photosynthesis, so self- 
supporting. Terrestrial plant. 

Continuous beam. Petiole must support leaf 
against gravity and withstand wind, so self- 
supporting. Terrestrial plant. 

Continuous beam. Root is not self-supporting. 
Loaded in tension. Terrestrial plant. 

Continuous beam. Vine is not self-supporting. 
Loaded in tension. Terrestrial plant. 

Continuous beam. Support individual polyps 
against water flow. Not loaded 
gravitationally. Aquatic animal. 

Continuous beam. Tree must maintain upright 
position for photosynthesis, so self- 
supporting. Terrestrial plant. 



The biological beams investigated were divided into nine groups, based 
on broad differences in morphology and function. 



Etnier. 2001), reflecting the individual variation noted 
above. 

The twist-to-bend ratio for each species was calculated 
and compared to the predicted values as an additional de- 
scriptor of flexibility in biological beams (Fig. 2). Standard 
deviations for the twist-to-bend ratios, when available, are 
reported in the appendix. 



Size-normalized mechanospace 

Size greatly influences the stiffness of a biological 
beam, both in twisting and in bending. The structural 
variables, / and J. are both proportional to radius to the 
fourth power; thus, small increases in size will greatly 
increase beam stiffness, regardless of cross-sectional 
shape or material composition. Values of El and GJ were 
normalized for size by dividing by radius to the fourth 
power to determine if size alone was driving the observed 
patterns. These values were then mapped into a size- 
normalized mechanospace (Fig. 3). This normalization 
accounts for size alone, rather than equating to calcula- 
tions of E or G for a given beam. 



Results 

The lines shown in Figure 1 are based upon the assump- 
tions of engineering beam theory and represent values for 
ideal beams. The distribution of biological beams closely 
matched that of the ideal beams, with 93% (53 of 57) of the 
points falling within the bounded region (Fig. 1 ). The 
boundaries reflect possible extremes for biological beams, 
based on assumed material values and chosen cross-sec- 
tional shapes. Flexural stiffness and torsional stiffness 
changed concurrently in the biological beams. Thus, beams 
that bent easily also twisted easily, while those that were 
hard to bend were also hard to twist. Overall, flexural 
stiffness and torsional stiffness each varied over 9 orders of 
magnitude. Species within the defined groups occupied sim- 
ilar regions of the mechanospace, implying that the flexural 
stiffness and torsional stiffness of group members were of 
similar magnitude. 

Due to the finite nature of the data set, the unoccupied 
regions of the mechanospace suggest that other combina- 
tions of mechanical values are rare without proving that 
they are impossible. Biological beams may exist within 
these unoccupied regions, given sufficient variation in ma- 
terial or structural design. The four samples falling outside 
of the predicted boundaries for ideal circular and elliptical 
beams included two herbaceous stems (daffodils Narcissus 
pseudonarcissus and sedges Carex acutiformis), a tree 
branch (Dendropanex arboreus), and a tropical vine (Marc- 
gravia rectiflora). These systems were all characterized by 
high flexural stiffness relative to torsional stiffness that is, 
they twisted more easily than they bent (Fig. 2). 

Twist-to-bend ratios varied dramatically among different 
species (Fig. 2), with both the maximum (52.9) and the 
minimum (1.4) occurring in tropical vines. The average 
twist-to-bend ratio for all groups combined was 7.2, which 
falls between the predicted values for ideal beams that are 
circular or elliptical in cross-sectional shape. 

When the data were normalized for size (Fig. 3), the 
observed distribution changed slightly. In particular, the 
relative position of the antennae, horsetail rushes, and gor- 
gonian corals shifted up and to the right relative to the other 
beams. This result suggests that these structures are all 
relatively stiff for their size. While the position of groups 
changed slightly, structures that were stiffer in bending were 
also stiffer in torsion, regardless of their overall size. 

Discussion 

The close resemblance in the distribution of ideal and 
biological beams suggests that both are limited by the 
interdependence between the material properties, E and G, 
and between the geometric properties, / and J. Yet, keep in 
mind the assumptions of beam theory, which are violated 
almost universally by biological beams. The theoretical 
relationships apply only to beams consisting of a single. 



TWISTING AND BENDING BIOLOGICAL BEAMS 



41 



Circular beam v = 0.0 



Elliptical beam v = 0.50 




4J 5/1 

a g 

4* 

s "o 



i 

5? 



o 

I 






e 
> 



o 

DC 
O 



Figure 2. Twist-to-bend ratios for the nine groups. The horizontal lines indicate the predicted values for 
ideal beams with circular (El/GJ = 1.00. v = 0) and elliptical (EI/GJ = 12.75. v = 0.50) cross sections. The 
beams are arranged by size within each group, with the smallest diameter beam to the left. Standard deviations 
for the twist-to-bend ratios, when available, are reported in the appendix. 



isotropic material (Roark, 1943). Biological materials are 
more typically anisotropic, and values for Poisson's ratio 
can vary greatly from the theoretical expectations (Vincent, 
1990). More importantly, biological beams are almost al- 
ways composite structures built of multiple materials that 
differ greatly in their mechanical properties. Thus, it is not 
obvious that El and GJ would be highly interdependent in 
biological beams, particularly in the beams that deviate 
most from the theoretical assumptions, such as the jointed 
crinoid arms or crustacean antennae. 

The four samples falling outside of the predicted bound- 
aries are all characterized by high flexural stiffness relative 
to torsional stiffness that is, they will twist more easily 
than bend (Fig. 2). The two herbaceous stems have flowers 
or seed heads that extend perpendicular to the long axis of 
the stem, potentially causing the stem to bend or twist when 
the wind blows. Rather than resisting this load with a high 
torsional stiffness, daffodils (Narcissus pseudonarcissus) 
and sedges (Carex acntifonnis) have a low torsional stiff- 
ness, which allows them to twist in the wind. Daffodils 
reduce flow-induced drag with this action (Etnier and Vo- 



gel, 2000), and a similar function has been suggested for 
sedges (Ennos, 1993). The functional relevance of the high 
ratios of flexural stiffness to torsional stiffness in the tree 
trunk and vine has not been explored. 

Conservative estimates of size and shape were made for 
many of the tree branches, vines, and gorgonian corals 
(appendix). Beam size affects El and GJ equally; thus, 
errors in the size estimates will not affect the position of 
data points relative to the predicted boundaries (Fig. 4). 
Rather, an increase or decrease in diameter will cause these 
points to shift parallel to the boundaries. In contrast, shape 
estimates will differentially affect El and GJ, causing data 
points to shift relative to the predicted boundaries (Fig. 4). 
Although branches and vines are fairly circular in cross 
section, this assumption may not be valid for the gorgonian 
corals, whose cross-sectional shape can be circular, ellipti- 
cal, or even triangular (Jeyasuria and Lewis, 1987). The 
assumption of a circular cross section potentially underes- 
timates the range of values seen in the gorgonian corals. For 
example, if the cross-sectional shape of the gorgonians is 
assumed to be a 4: 1 ellipse rather than circular, then all of 



42 



S. A. ETNIER 



H 

c 



1Z.UU 




















O 










* * 










A 


** A** 










* A A A A 








o.OO 




A 












D 













A 




















4.00 6.00 8.00 10.00 12.00 


log GJ / r 4 



o antennae 
n crinoid arms 
o horsetails 
A stems 

petioles 
+ root 

A vines 

gorgonians 

tree branches 



Figure 3. Flexural and torsional stiffness normalized by size for all groups (Table 2). The data points were 
normalized by dividing by radius to the fourth power. 



these beams will fall outside of the predicted boundaries. 
Future studies that include the cross-sectional shape of the 
gorgonian corals may identify group members that are of 
particular mechanical and functional interest within a bio- 
logical context. 

Flexural stiffness and torsional stiffness are both highly 
dependent on the size of the structure in question. While the 
pattern seen in the stiffness mechanospace is driven in part 
by size increases alone, the size-normalized mechanospace 
(Fig. 3) suggests a robust pattern. Even with differences in 
size removed from the comparison, there is still a strong 
relationship between how a beam bends and how it twists. 
The size-normalized mechanospace suggests some interest- 
ing comparisons between the different groups. For example, 
the antennae have a size-normalized flexural stiffness sim- 
ilar to that of tree branches while exhibiting a higher 
size-normalized torsional stiffness. Again, the biological 
implications of such variation have not been sufficiently 
explored in the literature. 

The size, shape, and function of a beam may change 
during development (Wainwright and Dillon, 1969; Carrier, 
1983; Katz and Gosline, 1992; Gallenmiiller et al.. 2001). 
Such changes potentially influence both flexural stiffness 
and torsional stiffness, thus affecting how the beam re- 



sponds to a given load. The mechanospace introduced in 
this study is well suited for investigations relating the mor- 
phology, mechanics, and functional demands of biological 
beams during ontogeny. A systematic study of the relation- 
ship between El and GJ over a developmental series can 
reveal clues to the changing functional demands on a system 
during growth (Gallenmuller et al.. 2001 ). Concurrent mor- 
phological studies could determine whether mechanical 
changes are due to variation in material or structural prop- 
erties, potentially identifying the mechanisms that different 
systems use to modulate mechanical properties. 

Species within a group occupy a similar region of the 
mechanospace, signifying that the flexural stiffness and 
torsional stiffness of group members are of similar magni- 
tude. The same pattern is true for the size-normalized mech- 
anospace. While group members tend to occupy similar 
regions of the mechanospace, there is still variation within 
each group. Interspecific variation in shape and material 
may permit different combinations of flexural and torsional 
stiffness that may subtly influence how each species func- 
tions in its environment. 

Despite clear differences in design, no obvious mechan- 
ical differences distinguish jointed beams from continuous 
beams. In continuous biological beams, the observed me- 



TWISTING AND BENDING BIOLOGICAL BEAMS 



43 




-3 



-2 



-1 



log Torsional Stiffness (Nm ) 



small circular beam 
r = 0.002 m 



O large circular beam 
r = 0.004 m 



X elliptical beam 
rmax= 0.004 m, 
rmin = 0.001 m 



Figure 4. Changes in the assumptions of size and shape of beams will affect their distribution in the 
mechanospace. The small filled circles are the gorgonian values graphed in Figure 1 and were based on an 
assumed radius of 0.002 m. The lines represent the predicted values for ideal beams discussed previously. 
Changes in radius will affect the magnitude of / and J equally; thus, an increase or decrease in radius will cause 
these points to shift parallel to the predictions. For example, increasing the radius from r = 0.002 m to r = 0.004 
m shifts the distribution up and to the right (large circles). In contrast, shape estimates differentially affect / and 
J. If the cross-sectional shape is assumed to be a 4: 1 ellipse (r max = 0.004 m, r mn = 0.001 m) rather than a circle, 
and / is calculated about the short axis, the data points move outside of the upper boundary (stars). Note that the 
cross-sectional area of the elliptical beams is equivalent to that of the small, circular beams; thus, these changes 
do not reflect an increase in the total amount of material, but rather a change in its distribution. 



chanical properties may be attributed to materials that 
extend the entire length of the beam, but this explanation 
is inadequate for the jointed beams, which have no materi- 
als extending uninterruptedly along their entire length. 
The similarity in values for flexural stiffness and torsional 
stiffness for both jointed and continuous systems sug- 
gests that these beams may represent alternative designs to 
meet the functional need for flexibility in biological struc- 
tures. Two of the jointed systems, horsetails and antennae, 
are relatively stiff for their size, suggesting that the presence 
of joints does not necessarily equate with increased flexi- 
bility. 

Neither the ideal beams nor the biological beams are 
distributed uniformly throughout the mechanospace (Fig. 
1). Unoccupied regions of the mechanospace correspond to 
beams that bend but do not twist and beams that twist but do 
not bend. The distribution within the mechanospace may be 



determined by inherent principles governing the relation- 
ships between E and /, as well as G and J. Conversely, 
empty areas within the distribution may not be an indication 
of physical impossibility, but of evolutionary history. There 
may be an absence of environmental patterns of change 
causing natural selection for particular combinations of me- 
chanical properties (Raup and Stanley, 1971 ). Alternatively, 
once an evolutionary pathway has been initiated, phyloge- 
netic canalization may limit future options for change 
(Lauder, 1982). Finally, empty spaces within the mechano- 
space may reflect temporal, rather than physical, limitations 
to those areas (Raup, 1966). The empty spaces will even- 
tually be occupied, given enough time. Although the iden- 
tification of boundaries within this mechanospace may not 
reveal their ultimate source, the boundaries do identify 
factors that may influence the observed pattern (Lessa and 
Patton, 1989). 



44 



S. A. ETNIER 



Despite the structural diversity of the samples used in 
this study, they are merely a subset of the biological 
possibilities. A notable limitati n is the absence of fiber- 
wrapped beams in the mei .ham- pace. Internally pressur- 
ized, hydrostatic skeletons are > ; pically wrapped with 
reinforcing fibers (Wai:v,<.n;_s<; n al., 1978). The fibers 
may be arranged orthogonally with the fibers parallel and 
perpendicular to the long axis of the structure, or they 
may be in a helical array with fibers running in right- and 
left-handed helices around the long axis. Orthogonal 
arrays offer little resistance to twisting, while being rel- 
atively stiff in bending (Wainwright et al., 1978). Thus, 
these beams would potentially fall into the upper left- 
hand corner of the mechanospace. In contrast, helical 
fiber arrays allow pressurized beams to bend smoothly 
without kinking, while resisting torsional deforma- 
tions (Wainwright et al., 1978), potentially positioning 
these beams in the lower right of the mechanospace. 
Fibrous support systems may decouple the relation- 
ship between El and GJ. permitting novel combinations 
of mechanical properties. Thus, their inclusion in the 
mechanospace may greatly expand the observed distribu- 
tion. 

The mechanospace presented here is a useful approach 
for investigating patterns of flexibility in biological beams. 
Importantly, the mechanospace does not imply that flexi- 
bility has critical functional relevance in each system. 
Rather, it should be used as a broadly comparative tool to 
highlight systems in which flexibility may be biologically 
important. Biological beams that do not follow the basic 
pattern seen in the mechanospace may be of particular 
interest to both biologists and engineers, either due to ma- 
terial or structural innovation. 

Acknowledgments 

I am grateful for the comments of Dr. Steve Vogel, Dr. 
Steve Wainwright, and Dr. Bill Hoese on early drafts of this 
manuscript, as well as informative and invaluable discus- 
sions with Dr. John Gosline and Dr. D. A. Pabst. This 
manuscript was greatly improved by the comments of the 
reviewers. 

Literature Cited 

Baumiller, T. K. 1993. Crinoid stalks ;>s cantilever beams and the nature 
of the stalk ligament. Neues Juhrh. Geol I'alaeontol. Abh. 190: 279- 
297. 

Best, B. A. 1988. Passive suspension feeding in a sea pen: effects of 
ambient flow on volume flow rate and filtering efficiency. Biol. Bull. 
175: 332-342. 

Carrier, D. R. 1983. Postnatal ontogeny of the musculo-skeletal sys- 
tem in the black-tailed jack rabbit (Lepus californicus). J. Zoo/. 201: 
27-55. 

Denny, M. W. 1988. Biology and the Mechanics of the Ware-swept 
Environment. Princeton University Press. Princeton, NJ. 



Ennos, A. R. 1993. The mechanics of the flower stem of the sedge Carex 
acutifonnis. Ann. Bot. 72: 123-127. 

Etnier, S. A. 1999. Flexural and torsional stiffness in biological beams: 
the morphology and mechanics of multi-jointed structures. Ph.D. dis- 
sertation. Duke University. Durham. NC. 147 pp. 

Etnier, S. A. 2001. Flexural and torsional stiffness in multi-jointed 
biological beams. Biol. Bull. 200: 1-8. 

Etnier, S. A., and S. Vogel. 2000. Reonentation of daffodil (Narcissus: 
Amaryllidaceae) flowers in wind: drag reduction and torsional flexi- 
bility. Am. J. Bot. 87: 29-32. 

Gal, J. 1993. Mammalian spinal biomechanics. II. Intervertebral lesion 
experiments and mechanisms of bending resistance. J. Exp. Biol. 174: 
281-297. 

Gallenmuller, F., U. Miller, N. Rowe, and T. Speck. 2001. The growth 
form of Crown pullei (Euphorbiaceae) functional morphology and 
biomechanics of a neotropical liana. Plant Biol. 3: 50-61. 

Gere, J. M.. and S. P. Timoshenko. 1984. Mechanics of Materials. 
Brooks/Cole Engineering Division, Monterey. CA. 

Harvell, C. D., and M. LaBarbera. 1985. Flexibility: a mechanism for 
control of local velocities in hydroid colonies. Biol. Bull. 168: 312 
320. 

Jeyasuria, P., and J. C. Lewis. 1987. Mechanical properties of the axial 
skeleton in gorgonians. Coral Reefs 5: 213-219. 

Katz, S. L., and J. M. Gosline. 1992. Ontogenetic scaling and mechan- 
ical behaviour of the tibiae of the African desert locust (Schistocerca 
gregaria). J. Exp. Biol. 168: 125-150. 

Koehl, M. A. R. 1977a. Effects of sea anemones on the flow forces they 
encounter. J. Exp. Biol. 69: 87-105. 

Koehl, M. A. R. 1977b. Mechanical organization of cantilever-like 
sessile organisms: sea anemones. J. Exp. Biol. 69: 127-142. 

Lauder, G. V. 1982. Historical biology and the problem of design. J. 
Theor. Biol. 97: 57-67. 

Lessa, E. P., and J. L. Patton. 1989. Structural constraints, recurrent 
shapes and allometry in pocket gophers genus Thomomys. Biol. J. Linn. 
Soc. 36: 349-364. 

Long. J. H., Jr., and K. S. Nipper. 1996. The importance of body 
stiffness in undulatory propulsion. Am. Zoo/. 36: 678-694. 

McHenry, M. J., C. A. Pell, and J. H. Long, Jr. 1995. Mechanical 
control of swimming speed: stiffness and axial wave form in undulating 
fish models. J. Exp. Biol 198: 2293-2305. 

Morgan, J. 1989. Analysis of beams subject to large deflections. Aero- 
nautical J. 93: 356-360. 

Morgan, J., and M. G. R. Cannel. 1987. Structural analysis of tree 
trunks and branches: tapered cantilever beams subject to large deflec- 
tions under complex loading. Tree Physiol. 3: 365-374. 

Niklas, K. J. 1991. The elastic moduli and mechanics of Populus tremu- 
loides (Salicaceae) petioles in bending and torsion. Am. J. Bot. 78: 
989-996. 

Niklas, K. J. 1992. Plant Biomechanics: An Engineering Approach to 
Plant Form and Function. University of Chicago Press. Chicago. 

Niklas, K. J. 1998. The mechanical roles of clasping leaf sheaths: 
evidence from Arundinaria tecta (Poaceae) shoots subjected to bending 
and twisting forces. Ann. Bot. 81: 23-34. 

Prince, R. P., and D. W. Bradway. 1969. Shear stress and modulus of 
selected forages. Am. Soc. Agr. Eng. Trans. 12: 426-428. 

Putz, F. E., and N. M. Holbrook. 1991. Biomechanical studies of vines. 
Pp. 73-97 in The Biology of Vines. F. E. Putz and H. A. Mooney, eds. 
Cambridge University Press. New York. 

Raup. D. M. 1966. Geometric analysis of shell coiling: general prob- 
lems. J. Paleontoi 40: 1178-1190. 

Raup, D. M., and S. M. Stanley. 1971. Principles of Paleontology. 
W. H. Freeman, San Francisco. 

Roark. R. J. 1943. Formulas for Stress and Strain. 2nd ed. McGraw- 
Hill. New York. 



TWISTING AND BENDING BIOLOGICAL BEAMS 45 

Vincent, J. 1990. Structural Biomateriuls. Princeton University Press, Symmorphosis Debate. E. R. Weibel, C. R. Taylor, and L. Bolis, eds. 

Princeton. NJ. Cambridge University Press, Cambridge. 

Vogel, S. 1984. Drag and flexibility in sessile organisms. Am. Zooi 24: Wainwright, S. A. 1988. Axis and Circumference: The Cylindrical 

37-44. Shape of Plants and Animals. Harvard University Press. Cambridge. 

Vogel, S. 1989. Drag and reconfiguration of broad leaves in high winds. MA. 

J. Exp. Bot. 40: 941-948. Wainwright, S. A., and J. R. Dillon. 1969. On the orientation of sea 

Vogel, S. 1992. Twist-to-bend ratios and cross-sectional shapes of peti- fans. Bio/. Bull. 136: 130-139. 

oles and stems. J. Exp. Bot. 43: 1527-1532. Wainwright, S. A., W. D. Biggs, J. D. Currey, and J. M. Gosline. 1976. 

Vogel, S. 1995. Twist-to-bend ratios of woody structures. J. Exp. Bot. 46: Mechanical Design in Organisms. Princeton University Press, Prince- 

981-985. ton. NJ. 

Vogel, S. 1998. Convergence as an analytical tool in evaluating design. Wainwright, S. A., F. Vosburgh, and J. H. Hebrank. 1978. Shark skin: 

Pp. 13-20 in Principles of Animal Design: The Optimization tine/ function in locomotion. Science 202: 747-749. 



46 S. A ETNIER 

Appendix 

Species (n = 57) used in the mechanpsp^v ;ilong with their source, diameter, flexural stiffness (7), torsional stiffness (G/), and the ratio EI/GJ. 
Species for which assumptions were m i iboul their size and shape are marked with an asterisk. The standard deviations for EI/GJ are given in 
parenthesis, when available 






Diameter 


El 


GJ 






Group 


Species 


(m) 


(Mm 2 ) 


(Nitr) 


EI/GJ 


Source 


Crustacean antennae n = 2 


Procambarus sp. Crayfish 


0.0004 


2.50E-05 


6.00E-06 


4.5 (3.7) 


Etnier, 2001 




I'unulirus argus Lobster 


0.002 


5.83E-03 


2.04E-03 


1.8(1.3) 


Etnier, 2001 


Crinoid arms n = 1 


Comactinia echinoptera 


0.002 


5.00E-05 


1.50E-05 


4.3 (3.8) 


Etnier, 2001 




Florometra serratissitna 


0.002 


3.92E-04 


5.10E-05 


6.6(2.7) 


Etnier, 2001 


Horsetails n = 1 


Equisetum hyema/e 


0.005 


1.87E-02 


3.83E-03 


4.1 (1.8) 


Etnier, 1999 


Herbaceous stems n = 6 


Cucumis sativus Cucumber 


0.004 


8.73E-03 


1.83E-03 


5.4(1.2) 


Vogel, 1992 




Helianrhus annuus Sunflower 


0.005 


7.30E-05 


5.30E-05 


1.4(0.4) 


Vogel, 1992 




Lvcopersicon esculeniwn Tomato 


0.005 


1.20E-02 


3.46E-03 


3.9(1.1) 


Vogel, 1992 




Carex acutiformis sedge 


0.005 


7.70E-03 


2.34E-04 


36.0(11.3) 


Ennos, 1993 




Tulips 


0.006 


2.02E-02 


2.40E-03 


8.3 (3.2) 


Etnier and Vogel, 2000 




Narcissus pseudonarcissus Daffodil 


0.007 


1.19E-02 


8.90E-04 


13.3 (1.0) 


Etnier and Vogel, 2000 


Leaf petioles n = 5 


Acer rubnim Red maple 


0.001 


1.94E-04 


7.10E-05 


2.8(1.2) 


Vogel, 1992 




Liqitidambar styracifiuct Sweet gum 


0.002 


9.84E-04 


1.99E-04 


5.1(1.3) 


Vogel, 1992 




Phaseolus vulgaris Green bean 


0.002 


6.77E-04 


1.43E-04 


4.9(2.1) 


Vogel, 1992 




Populus alba White popular 


0.003 


7.75E-05 


1.50E-05 


4.95 


Vogel. 1992 




Populus Iremuloides 


0.003 


l.OOE-05 


l.OOE-06 


4 


Niklas, 1991 


Tree roots n = 1 


Pinus taeda Loblolly pine 


0.05 


1.72E + 02 


7.90E+01 


2.3(0.8) 


Vogel, 1995 


Vines n = 16 


Crolon piillei juvenile 


0.01* 


3.30E+00 


3.04E-01 


10.9 


Gallenmuller et al. 2001 




Cisscunpelos pareira 


0.02* 


3.63E+00 


4.90E-01 


7.4 


Putz and Holbrook, 1991 




Cissus sicyoides 


0.02* 


1.15E + 00 


1.30E-01 


8.8 


Putz and Holbrook, 1991 




Forsteronia portoricesis 


0.02* 


1.67E+00 


8.30E-01 


2.0 


Putz and Holbrook, 1991 




Heleropteris laurifolia 


0.02* 


1.07E+01 


4.60E + 00 


2.3 


Putz and Holbrook, 1991 




Hippocratea vo/ubilis 


0.02* 


4.38E+00 


6.80E-01 


6.4 


Putz and Holbrook, 1991 




Ipomoea repanda 


0.02* 


3.41E+00 


3.10E-01 


11.0 


Putz and Holbrook, 1991 




Marcgravia rectiflora 


0.02* 


1.32E + 01 


2.50E-01 


52.8 


Putz and Holbrook. 1991 




Mikania fragilis 


0.02* 


1.48E+00 


2.00E-01 


7.4 


Putz and Holbrook, 1991 




Paullinia pinnata 


0.02* 


4.71E+00 


6.30E-01 


7.5 


Putz and Holbrook. 1991 




Rourea surinamensis 


0.02* 


6.48E+00 


1.23E+00 


5.3 


Putz and Holbrook, 1991 




Schlegelia brach\anthii 


0.02* 


4.34E+00 


9.60E-01 


4.5 


Putz and Holbrook, 1991 




Securidaca virgala 


0.02* 


1.59E+00 


3.60E-01 


4.4 


Putz and Holbrook. 1991 




Crolon piillei mature 


0.02* 


4.87E+00 


3.61E+00 


1.4 


Gallenmuller el al., 2001 




Vitus rotundifolia Grape 


0.046 


3.08E+02 


1.I4E+02 


2.7(0.3) 


Vogel, 1995 




Wisteria sinensis Wisteria 


0.051 


2.85E+02 


6.70E+01 


4.5(2.1) 


Vogel, 1995 


Gorgonian corals n = 13 


Ellisella barbadensis 


0.004* 


1.14E-01 


2.16E-02 


5.3 


Jeyasuria and Lewis, 1987 




Eunicea calyculata 


0.004* 


9.07E-03 


1.76E-03 


5.1 


Jeyasuria and Lewis, 1987 




Eunicea clavigera 


0.004* 


7.94E-03 


2.26E-03 


3.5 


Jeyasuria and Lewis, 1987 




Gorgonia ventalina 


0.004* 


1.66E-02 


1.05E-02 


1.6 


Jeyasuria and Lewis, 1987 




Leptogorgia virgulata 


0.004* 


4.47E-02 


1.33E-02 


3.3 


Jeyasuria and Lewis, 1987 




Lophorgogia cardinally 


0.004* 


6.35E-02 


1.46E-02 


4.3 


Jeyasuria and Lewis, 1987 




Muhceopsis flavida 


0.004* 


8.82E-03 


3.51E-03 


2.5 


Jeyasuria and Lewis, 1987 




Plexaura flexuosa 


0.004* 


1.25E-02 


2.01E-03 


6.2 


Jeyasuria and Lewis, 1987 




Plexaurella grisea 


0.004* 


5.66E-02 


9.54E-03 


5.9 


Jeyasuria and Lewis, 1987 




Pseudoplexaura crucis 


0.004* 


7.31E-03 


2.01E-03 


3.6 


Jeyasuria and Lewis, 1987 




Pseudopterogorgia bipinnata 


0.004* 


4.17E-02 


9.04E-03 


4.6 


Jeyasuria and Lewis, 1987 




Plerogorgia citrina 


0.004* 


1.95E-02 


3.01E-03 


6.5 


Jeyasuria and Lewis, 1987 




Swiftiu exserta 


0.004* 


8.82E-03 


1.76E-03 


5.0 


Jeyasuria and Lewis, 1987 


Tree branches n = 1 1 


Brunellia comocladifolia 


0.02* 


2.08E+01 


1.65E+00 


12.6 


Putz and Holbrook, 1991 




Dendropanax urboreus 


0.02* 


9.42E+01 


2.81E+00 


33.5 


Putz and Holbrook. 1991 




Guarea trichilioides 


0.02* 


1.92E+01 


1.95E+00 


9.9 


Putz and Holbrook, 1991 




Inga veru 


0.02* 


3.74E+01 


5.47E+00 


6.8 


Putz and Holbrook, 1991 




Ocoteti floribunda 


0.02* 


1.31E+01 


4.23E+00 


3.1 


Putz and Holbrook, 1991 




Juniperus virginiana E. red cedar 


0.04 


7.44E+02 


1.68E+02 


4.4(0.7) 


Vogel, 1995 




Linodendron tulipifera Tulip poplar 


0.045 


1.16E+03 


1.30E+02 


8.9(1.4) 


Vogel, 1995 





Acer rubrurn Red maple 


0.047 


2.07E+03 


2.50E+02 


8.3 (0.9) 


Vogel, 1995 




Phyllostachys sp. Bamboo 


0.05 


8.03E+03 


9.50E+02 


8.6(1.3) 


Vogel, 1995 




Pinus taeda Loblolly pine 


0.051 


2.06E+03 


3.19E+02 


6.1 (0.9) 


Vogel, 1995 




Liquidambar styraciflua Sweet gum 


0.054 


2.38E + 03 


2.60E+02 


8.9(1.6) 


Vogel, 1995 



Reference: Biol. Bull. 205: 47-53. (August 2003) 
2003 Marine Biological Laboratory 



Extracellular Lipid Droplets in Idiosepius notoides, the 

Southern Pygmy Squid 

L. S. EYSTER* AND L. M. VAN CAMP 
School of Biological Sciences, Flinders Universitv, Adelaide, South Australia 5001, Australia 



Abstract. Cephalopod metabolism typically involves car- 
bohydrates and proteins, so that the lipid content of the 
mantle and all internal organs except the digestive gland is 
very low. Despite clear evidence of nonlipoid metabolic 
trends in cephalopods, we observed extracellular spheres, or 
droplets, in the cecum and digestive gland of newly col- 
lected juvenile, male, and female individuals of Idiosepius 
notoides. the southern pygmy squid. Prior to staining, the 
droplets were various shades of yellow and were often large 
enough to detect at 7X magnification. The droplets were 
less dense than water, hydrophobic, and sudanophilic, stain- 
ing positively with Sudan III. Sudan IV, and Sudan Black B. 
We conclude that these spheres are lipid and that they derive 
from the squid's normal field diet. When newly collected 
squid were starved in the laboratory, the droplets disap- 
peared in 7-8 d and then reappeared in the cecum about 3 h 
after feeding. 

Introduction 

Although cephalopods require considerable energy for 
rapid movement, growth, and reproduction, they apparently 
have limited capacity to metabolize or store lipids 
(Hochachka et ai, 1975; Storey and Storey, 1983; O'Dor 
and Webber. 1986; Moltschaniwskyj and Semmens. 2000). 
In 1975, cephalopod metabolism was described as poorly 
understood, and squid storage substrates were "a well 
known mystery in marine biochemistry" (Hochachka et ai, 
1975). That mystery arises because lipids, efficient energy 
storage molecules due to their high per-gram energy con- 
tent, are typically not abundant in cephalopods (Hochachka 
et ai, 1975; Castro et ai, 1992: Clarke et al., 1994). 

Received 24 September 2002; accepted 12 June 2003. 

* To whom correspondence should be addressed. Current address: Sci- 
ence Department, Milton Academy, Milton, Massachusetts, 02186. E-mail: 
Linda_Eyster@ Milton.edu 

Ahhreviation: ML = mantle length. 



Despite evidence of limited lipid metabolism in cephalo- 
pods, lipid storage has been reported for the digestive gland, 
the only cephalopod organ consisting of more than a few 
percent lipid. The cecum is not reported to be a typical lipid 
storage site, although cecal cells have been ascribed a va- 
riety of functions, including fat absorption in several species 
(Bidder. 1966; Boucaud-Camou and Boucher-Rodoni, 
1983; O'Dor et al., 1984: Westermann and Schipp. 1998). 

Idiosepius notoides Berry 1921 is a small sepioid found 
in seagrass beds in southern Australian waters, from Cock- 
burn Sound in Western Australia to Morton Bay. Queens- 
land (Shepard and Thomas, 1989). We report the existence 
and retention of yellow spheres within the cecal lumen and 
digestive gland of field-collected specimens of /. notoides 
subjected to starvation, and explore the possible lipoid na- 
ture of these droplets. We also speculate on the origin, 
function, and fate of the droplets. 

Materials and Methods 

Idiosepius notoides was collected by seining over sea- 
grass beds ( 1 8 C, 39 ppt salinity ) near the mouth of the Port 
River, Adelaide, South Australia, on 29 March 2002 (Day 
0). All squid were recognized by their small size (about 10 
mm dorsal mantle length), by a pair of rounded fins near the 
rear of the body, and by their attachment to seagrasses and 
Ulva sp. using dorsal duoadhesive glands (Norman and 
Reid, 2000). Because idiosepiids are morphologically un- 
usual, they historically have been placed with the teuthids 
but are currently classified as sepiids (Berry. 1932; Hylle- 
berg and Nateewathana, 1991); despite their cuttlefish alli- 
ances, they are commonly called pygmy "squid" and will be 
referred to as squid in this paper. 

Squid that died on collection (/; = 2) were examined on 
Day 0. Live squid were kept unfed in an aquarium con- 
nected to the recirculating seawater system ( 16 C, 40 ppt 
salinity. 12 h light:12 h dark cycle) until natural death or 



47 



48 



L. S. EYSTER AND L. M. VAN CAMP 



sacrifice on Days 4-15. To investigate whether "starved" 
squid ate small organisms (f.;; >ds present in the 

recirculating seawaten. trmv were videotaped indi- 

vidually (2.5 h total. per second) in a tank 

measuring 19.3 X 7 cm ( mg one liter of unfiltered 

seawater from the re. tig system). 

Before sacrifice, endi squid (chosen at random) was 
observed at about 10X magnification in a small, flat dish for 
15 min in an attempt to locate droplets prior to anesthesia or 
dissection. The dark-pigmented mantle of active, stressed 
animals often hid droplets. However, during the squids' 
occasional flashes of transparency, we could see through the 
mantle tissue and determine droplet location. 

At sacrifice, squid were transferred (in seawater) to a 
freezer for terminal anesthesia, measured (dorsal mantle 
length = ML), and then decapitated (Boyle. 1991). (Al- 
though cold water may be analgesic rather than anesthetic 
[Boyle, 1991], in this paper the treatment is referred to as 
cold anesthesia.) The mantle was cut open to expose but not 
damage internal organs. We recorded gender, stage of sex- 
ual maturation (based on Lipinski's maturation scale as 
interpreted by Moltschaniwskyj, 1995), droplet presence, 
and sometimes droplet diameter. Droplets to be tested for 
lipid content were obtained by puncturing the cecum. Be- 
cause it is unknown if handling of squid may break drops 
into smaller droplets (and we wanted larger drops for stain 
testing), we minimized handling of squid prior to anesthesia 
and during dissection. 

Droplets were tested for lipid content using three stains: 
Sudan III, Sudan IV, and Sudan Black B (one stain per 
squid). Because these stains have very low water solubilities 
(ranging from <0.1 mg/ml for Sudan III to 0.7 mg/ml for 
Sudan IV; Green, 1990) compared to solubility in ethanol. 
staining solutions were prepared as saturated solutions in 
70% ethanol. Staining solutions were prepared only a few 
days before use to avoid the deterioration that can occur in 
Sudan/alcohol solutions (Gurr, 1962). 

We tried three approaches to cecal droplet staining (one 
method per squid). For ceca cut open unsubinerged, stain 
was pipetted directly onto the body. For ceca punctured 
submerged, filtered stain was added to the seawater (i.e., 
drops were stained floating on the water surface). Additional 
droplets collected from the water surface by patting with 
strips of filter paper were flooded with stain for about 10 
min, followed by ethanol rinses to de-stain the paper. Con- 
trol tests were conducted using food-grade vegetable oil. 
Positive stain for lipid includes yellow-orange for Sudan 111. 
orange-red for Sudan IV, ancJ blue-black for Sudan Black B 
(Conn. 1961; Gurr, 1962, 1065). 

To determine if changes in extracellular lipid volume or 
location could be detected after feeding, we fed one squid 
and then repeatedly examined it for droplets. This squid (see 
9.7-mm-ML male on Day 7, Table I ) was chosen because it 
seemed less stressed by handling: compared to most of our 



other squid, its movements were less vigorous in the small 
observation dish, and it seemed to have a transparent mantle 
more frequently. No anesthesia was used on the day of 
feeding, but it had to be used on the second and third days 
after feeding. Before feeding, the squid had no cecal drops 
but did have two small, equal-sized drops at the anterior end 
of the digestive gland, one on the left and one on the right 
side. After it caught the live shrimp provided (Hippolyte sp., 
-19 mm long), the squid was left undisturbed until it 
discarded the intact but almost empty exoskeleton 45 min 
later. No effort was made to locate oil droplets during 
feeding because preliminary work showed that disturbed 
squid tended to abandon their prey. We examined the squid 
for droplets immediately after its meal, at hourly intervals 
for the next 7 h, and then up to twice daily until no drops 
were detected at a magnification of about 30X. We mea- 
sured the cecal droplets if the squid remained stationary and 
was transparent during observations. 

To address the possibility of droplet expulsion, we kept 
four immature squid (4.4-6.9 mm ML) in individual glass 
bowls (colorless) until their oil droplets were undetectable. 
We used small bowls ( 100 ml of filtered seawater, 16 C) to 
decrease the surface area we would have to search for oil. 
These squid, from a separate collection made in late April 
2002 near Noarlunga (south of Adelaide), all had oil drops 
when collected and when placed in the glass bowls. Prior to 
use in this experiment they were maintained in an aquarium 
connected to the recirculating seawater system and fed 
field-collected mysid shrimp for 1 week. After placement in 
individual dishes, these squid were kept without food and 
were examined with a dissecting microscope once per day 
to determine whether droplets were present in the digestive 
system. Prior to the daily cleaning and refilling of the bowls, 
the water surface was examined with a dissecting micro- 
scope for floating droplets and then was patted with white 
paper toweling, which was also examined for droplets. 

Results 

Of the 16 squid collected in March, 11 were males, 3 
were juveniles, and 2 were females. Dorsal mantle length 
ranged from 6.5 to 16.5 mm. Females tended to be larger 
than males, as previously recorded (Norman and Reid, 
2000); mantle length was 15.4 1.1 mm (mean SD) in 
females, 8.4 0.9 mm in males, and 6.8 0.3 mm in 
juvenile squid. 

Droplets were easiest to locate in dissected, fresh squid or 
in healthy, stationary squid in transparent phase. Opaque 
mantle tissue in preserved and moribund squid hid the 
droplets. For active squid, it was difficult or impossible to 
obtain accurate droplet counts or measurements. 

In the first week after collection, we examined one juve- 
nile, nine male, and two female squid. Of these 12 unfed 
squid. 1 1 had droplets (Table 1 ). Droplets were not detected 



EXTRACELLULAR LIPID IN PYGMY SQUID 



49 



Table I 

'r. reproductive mutiiritv singe, utjti ilorstil tminlle length of 
stanvd Idiosepius notoides individuals examined Days 0-15 post- 
collection for extracellular droplets in the i/i^omr rruc! 



Days 
(post-collection) 


Gender 
maturity s 


& 
tage 


Mantle length Droplets 
(mm) seen? 





J 


1 


6.5 + 





M 


3 


9.0 + 


4 


M 


3 


9.1 + 


4 


M 


3 


8.7 + 


4 


M 


3 


S.I + 


4 


F 


5 


16.5 


5 


M 


3 


8.0 + 


5 


M 


3 


8.0 + 


5 


M 


3 


8.0 + 


7 


M 


5 


9.8 + 


7 


F 


nd 


14.3 + 


7 


M h 


nd 


9.7 + 


9 


J 


1 


nd 


9 


F 


1 


nd 


10 


F a 


nd 


13.7 


10 


i< 


1 


7.0 


14 


M 


3 


8.6 


15 


M 


3 


6.5 



J, juvenile: M, male; F, female; nd, no data. 

a Same squid examined Days 7 and 10. 

b This squid was used in a feeding experiment beginning later on Day 7. 

c Same squid examined Days 9 and 10. 



in the largest squid, a female. During Days 9-15 after 
collection, we found no droplets in any of the unfed squid 
(Table 1). 

Drops were most obvious in the cecum (Fig. la), a 
digestive sac near the rear of the body. The cecum wall was 
transparent, and the lumen of the organ contained a clear 
greenish or bluish fluid without obvious particles (Fig. la, 
b), all of which made the droplets conspicuous. We did not 
notice any distinct progression of color changes in cecal 
fluid (Lipiriski, 1990). Cecal droplets moved around, appar- 
ently due to ciliary currents and cecal contractions. Because 
the drops floated, they were easily detectable in the cecum 
whether we removed a dorsal or ventral piece of mantle 
(Fig. la, b). Unlike the lumenal oil droplets described in the 
loliginid squids Sepioteuthis lessoniana and Photololigo sp. 
(Semmens, 1998), the droplets in Idiosepius notoides ap- 
parently are not membrane-bound; when pushed together 
with a microprobe, droplets could readily fuse. 

We also saw droplets in the anterior end of the digestive 
gland, just under the edge of the uncut mantle, but these 
were often harder to detect than cecal drops. Drops were 
seen in the left, right, or both sides of the digestive gland 
simultaneously. We never found drops outside the cecum or 
digestive gland when we carefully cut only mantle tissue. 

Droplet color varied among squid but typically looked 
like some shade of yellow. It was difficult to be sure of color 




Figure 1. Cecal lipid droplets in IJioxepias notoide.i. Tissue was cut from dorsal (a) or ventral (b) mantle 
to expose the cecum and its yellow droplets. All oil drops shown are from starved male squid sacrificed Days 
4-5 after collection (8-9 mm ML). The floating drops in c and d are from a cecum punctured under water. 
Filtered Sudan IV (in 70% ethanol) was added to the seawater. Photographs were taken every 5 min for 1 h on 
an Olympus DP 10 digital camera attached to an Olympus SZH microscope. Shown are droplets at 5 min (c) and 
60 min Id] staining, c = cecum. dg = digestive gland, e = eye. od = oil droplets, t = testis 



50 



L. S. EYSTER AND L. M. VAN CAMP 



comparisons for cecal droplets beoaise the variable blue- 
green hues of the cecal flui ; . rig. la vs. Ib) probably 
altered our color pen.- er drops of yellow food 

oils (almond, olive, si' < < .-getable) all appeared col- 

orless floating in unuer the same lighting condi- 

tions. Unstained collected onto white filter paper 

from two Port Riv_ j i ,.;iud were conspicuous at 10X due to 
their brilliant !> on vellow color, while droplets from three 
of the four Noarlunga squid looked colorless in the cecum. 

Number and size of droplets varied among squid. For 
example, one squid had a huge cecal drop (-0.9 mm in 
diameter; Fig. la), and another had dozens of tiny droplets 
(Fig. Ib). However, on Days 0-7 after collection, most 
starved squid had 2-14 droplets with a typical diameter of 
0.05-0.2 mm per droplet. 

Evidence supporting the lipid nature of the droplets is 
summarized in Table 2 and in Figure Ic and d. The yellow 
droplets were hydrophobic. typically forming small spheres 
in the cecum. When we cut a submerged cecum, droplets 
rapidly escaped and floated to the water surface. On hitting 
the air-water interface, larger drops "popped" from a sphere 
into a thin disk on the water surface. One large drop pipetted 
onto a piece of thin brown-paper bag and pressed onto the 
paper using Parafilm left a translucent window ( 1 1 mm in 
diameter) in the paper for a 48-h observation period, 
whereas other random fluids from the same squid did not. 
Floating droplets collected onto filter paper changed from 
yellow (before staining) to orange with Sudan III, orange- 
red with Sudan IV, and blue-black with Sudan Black B. 
Floating droplets exposed to Sudan IV changed from yellow 
(Fig. Ic) to orange (Fig. Id). 

There was no evidence of feeding in any "starved" squid. 
All squid were accounted for, so no cannibalism occurred. 
Two of the videotaped squid explored the aquarium walls 
with their arms for about 10% of the filmed time, and they 
sometimes made motions as if catching something, but 
frame-by-frame viewing of those times gave no conclusive 
evidence of feeding. 

In the squid fed the large shrimp, no cecal droplets were 
seen until 3 h after feeding ended (Table 3). Cecal droplet 
volume increased on the day of feeding, then decreased, 
becoming negligible by 3 d later. When cecal drop total 

Table 2 

Summary of evidri*:. -Hire of cecal droplets in southern pygmy 

l, Idiosepiux is 







Consistent 


Criterion 


flicin 


with lipid' 1 


Color 


yellow 


+ 


Solubilily 


insoluble in water 


+ 


Density 


less than water 


+ 


Paper bag test 


translucent mark 


+ 


Sudan stains 


sudanophilic 


+ 



volume was largest (6 h to 2 d after feeding), no drops were 
seen on either side of the digestive gland (Table 3). Diges- 
tive gland droplets were seen more often on the left side (12 
times) than on the right side (6 times) (Table 3). Droplets 
persisted for about 5 d after the squid ate that one shrimp. 
No droplets were detectable 7 d after feeding (Table 3). 

No evidence of droplet expulsion was obtained from 
squid held in individual bowls. We saw no oil on the water 
surface, or on paper toweling that was used to wipe the 
water surface. Droplets disappeared in all four squid some- 
time between the third and fourth day of starvation. 

Discussion 

Despite the limited storage or usage of lipids in cepha- 
lopods, we show in this work that droplets in the cecal 
lumen of Idiosepius notoides are lipoid. To our knowledge, 
these extracellular droplets have not been reported in any 
other cephalopod. We believe these droplets are common in 
this species and that droplets persist for up to 7 d in starved 
squid as a result of slow absorption, slow expulsion, or both. 
Our preliminary evidence suggests that expulsion of large 
drops was not occurring in the laboratory, but expulsion of 
small droplets could have been undetected. 

Because droplets appear to be free and not membrane- 
bound in the cecal lumen, lipases could have ready access to 
them. Perhaps droplets can persist in the cecal sac for a 
week after a meal because no, or few, lipases are consis- 
tently present or active there. The digestive gland and ce- 
cum both produce various digestive enzymes (Boucaud- 
Camou and Boucher-Rodoni, 1983), but few studies have 
demonstrated lipase activity in any cephalopod digestive 
tract fluids or extracts (Bidder, 1966). Lipases probably 
occur in cephalopod digestive organs, including those of 
paralarvae (Boucaud-Camou and Roper, 1995), but oily 
feces in animals that were fed a diet high in lipids, and the 
accessibility of lipids in Octopus vulgaris for several days 
after feeding support the conclusion that cephalopod lipid 
metabolism is "slow and inefficient" (O'Dor et al, 1984). A 
study of lipase activity in various regions of the digestive 
tract of Idiosepius notoides before, during, and after meals 
should be informative. 

Droplets seemed easier to see in the cecum than in the 
digestive gland, probably because of the opaque brownish 
color and tubular structure of the latter organ. In addition, 
larger droplets may form more readily in the cecum because 
small droplets might meet and coalesce into larger spheres 
more easily in the cecal lumen than in the digestive gland, 
whose structure may interfere with contact between 
droplets. 

We observed movement of small droplets along the length 
of both the left and right halves of the digestive gland in live 
squid, but never saw movement of droplets between the two 
sides. The digestive gland of the congener /. pygmaeiis is a 



EXTRACELLULAR LIPID IN PYGMY SQUID 
Table 3 



51 



after feeding: the 


squid was starved in the laboratory for one week 


before feeding 






Cecum 


Digestive Gland 




Totals 


Time 


Number of Diameter of cecal drops No. of drops No. of drops Location of 


Total number 


Total cecal drop volume 


(after feeding)* 


cecal drops 


(mm)t on left side on right side largest dropt 


of drops 


(mm 3 x 10~ 2 ) 


-1 h 










1 dg 


2 


0.0 


Oh 










1 dg 


2 


0.0 


1 h 










1 dg 


2 


0.0 


2h 










1 Idg 


n 


0.0 


3h 


2 


nd 


1 Idg 


4 


> 0.0 


4h 


2 


nd 


Idg 


3 


>0.0 


5h 


2 





2. 0.3 


(I Idg 


3 


1.8 


oh 


4 





2. 0.25. 0.35. 0.45 cecum 


4 


8.3 


7h 


4 





1,0.1.0.2.0.55 cecum 


4 


9.2 


2d 


2 


0.1.0.5 1) cecum 


~> 


6.6 


3d 








6 Idg 


6 


0.0 


4 d a.m. 


3 


nd 


1 dg 


5 


>0.0 


4 d p.m. 


1 





1 


Idg 


2 


0.05 


5 d a.m. 










Idg 


1 


0.0 


5 d p.m. 


1 


0.05 


Idg 


T 


0.007 


7d 














0.0 



* h signifies the end of the meal. 

t nd = no data. 

t dg = digestive gland: Idg = left side of digestive gland. 

Total volume of cecal drops was calculated from the number of drops and their diameter. Digestive gland drops were not measured. 



"single unilobed organ" that is "ventrally bilobed with an 
incomplete dorsal septum" (Semmens et ai, 1995). It a 
digestive gland septum occurs in /. notoides, it might ex- 
plain this apparent separation and separate movement of left 
and right drops. It does not explain why we saw droplets 
more frequently on the left side of the digestive gland in the 
one squid examined repeatedly over a 7-d period (Table 3). 

Mantle length seemed irrelevant to either the presence or 
size of oil droplets, but we could rind only two large squid 
for our study. The fact that the only squid without droplets 
in Week 1 of starvation was also the only gravid female 
suggests that further records of reproductive stage versus 
droplets are warranted. 

Cephalopods are active carnivores, typically catching 
crustaceans, molluscs, and fish that are relatively large 
often as long as two-thirds of the predators' mantle length 
(Bidder. 1966; Wells and Clarke, 1996). However, members 
of Idiosepius are the world's smallest cephalopods (Norman 
and Reid. 2000) and may also be able to feed on small 
organisms that are not readily apparent to the unaided eye. 
We observed "nibbling" behavior (Moynihan, 1983) in our 
squid but no definite feeding during nibbling. Although our 
"unfed" squid may have fed on small organisms like cope- 
pods that were present in the recirculating seawater system. 
we consider it unlikely that this potential feeding affects any 
of our conclusions. 

Most of the carbon in cephalopod meals is in protein 



molecules, and growth in cephalopods is primarily through 
protein formation (O'Dor et al., 1984). A typical squid body 
is about 80% muscle, but only 1%-1.5% lipid, and less than 
0.4% carbohydrate (O'Dor and Webber, 1986). In line with 
their proteo-metabolic capabilities, all cephalopod organs, 
except the digestive gland, are also high in protein and low 
in lipid (e.g., 10%-17% protein versus l%-2% lipid dry 
mass in squid mantle, head, testis, or spermatophoric com- 
plex [Hochachka er al., 1975; Clarke et al.. 1994], and 3% 
lipid in cuttlefish gonad [Blanchier and Boucaud-Camou. 
1984 1 ). The only cephalopod organ containing abundant 
lipid is the digestive gland (called liver or hepatopancreas in 
older literature). Content of that organ varies from one 
species to another: reported values for lipid content are 
4%-6% in Photololigo sp. (Semmens, 1998; Moltschaniw- 
skyj and Semmens, 2000), 8%-ll% in Sepia officinulis 
(Blanchier and Boucaud-Camou. 1984), about 30% in lllex 
argentinus (Clarke et al.. 1994), and 27%-56% in Moro- 
teuthis ingens (Brachi, 1953; Phillips et al.. 2001). 

Food in a typical cephalopod travels through the buccal 
mass and down the esophagus to the stomach for initial 
extracellular digestion, probably aided by salivary and di- 
gestive gland enzymes (Boucaud-Camou and Boucher- 
Rodoni, 1983). Smaller food materials move to the digestive 
gland or the cecum for further digestion, and wastes travel 
the intestine to the anus (Bidder, 1966). The cecum. con- 
nected to the digestive tract between the stomach and anus. 



52 



L. S. EYSTER AND L. M. VAN CAMP 



receives and processes fine particles, has its own sphincter 
to isolate contents, and is the primary site of absorption 
from food fluids (Bidder, 1966: Boucaud-Camou and 
Boucher-Rodoni, 1983; O'Dor and Webber, 1986). Perhaps 
the cecum of /. noioiilcs can absorb dietary lipids, as re- 
ported for Octopus. Loligo, Sepia, and Nautilus (Bidder, 
1966: Boucaud-Camou and Boucher-Rodoni, 1983; O'Dor 
et al.. 19S4: Westermann and Schipp, 1998). 

In this study, we saw extracellular lipid in two organs: the 
cecum and the digestive gland. In other cephalopods, the 
only organ with significant lipid (reported as cellular depos- 
its) is the digestive gland; therefore, it has been considered 
the only storage site for lipid molecules. These lipid mole- 
cules might be oxidized during reproductive maturation or 
starvation, as in other marine organisms (Voogt, 1983; 
Boucaud-Camou, 1971. cited in Blanchier and Boucaud- 
Camou, 1984; Kreuzer, 1984, cited in Castro et al.. 1992; 
Clarke et al., 1994). Besides storing lipids, the digestive 
gland may be absorptive in some but not all species (Bidder. 
1966; Boucaud-Camou and Boucher-Rodoni, 1983). In fact, 
lipid seen inside digestive gland cells of two squid species 
appears to be packaged for expulsion, not storage (Sem- 
mens, 1998); because expulsion would be energetically 
wasteful and would make the squid denser, perhaps some 
lipid is moved to the cecum rather than expelled from the 
organism. Although very few cephalopods produce and 
store lipids for buoyancy (Clarke. 1988; O'Dor, 2002). 
perhaps the cecal lipid in /. notoides aides in the support of 
this small but negatively buoyant cephalopod. 

It is clear from our study that retention of extracellular 
lipid occurs in /. notoides; it is less clear whether retention 
for 7 to 8 days qualifies as "storage." The term storage is 
used in the literature without reference to length of reten- 
tion, without evidence of retention versus replacement of 
molecules, and with the implication of future use. Labeling 
studies may be useful in determining if "storage" of extra- 
cellular lipid in the cecum and digestive gland of /. notoides 
is due to slow utilization and/or slow elimination, either or 
both coupled with the addition of new molecules from the 
next meal. 

During our laboratory study with /. notnides, lumenal oil 
droplets disappeared slowly, over a period of days (7-8 
days in our field-fed squid; 3-4 days in our mysid-fed 
squid), not hours or minutes. This slow disappearance of 
lipid suggests slow absorption, slow expulsion, or both in 
starved squid. We cunnoi nil'.- out rapid expulsion of large 
drops in the field. It is also unknown if this species makes 
rapid vertical movements in the field. In the laboratory, 
these squid spent most of iheir time sitting on aquarium 
walls or on the undersurface of plastic plants, and move- 
ments were mostly horizontal. Anesthetized squid sank to 
the bottom, indicating that even with lipid droplets in the 
cecum, these squid are negatively buoyant. Rapid expulsion 
of large lipid drops by these small cephalopods might pro- 



vide a quick increase in negative buoyancy during a dive, 
and new drops could apparently be formed at the next meal. 
Our squid could not deep dive in our expulsion study 
because the water was only a couple of centimeters deep; 
the water level in our holding tank was about 10 cm deep, 
and the tank contained no predators that might induce 
swimming up and down in the water column. 

Extracellular lipid droplets have not been previously re- 
ported in Idiosepius species. Perhaps the drops are specific 
to /. notoides, which is not a well-researched species. Per- 
haps droplet presence and color vary with lipid content of 
the field diet; less fatty prey might not lead to droplets, and 
some prey might lead to paler droplets that are harder to see. 
Perhaps droplets were overlooked in previous studies of /. 
notoides because tiny droplets in live squid can resemble the 
yellow chromatophores in size and color, or because the 
droplets were obscured or extracted by preservatives (e.g., 
alcohol can turn the cecum opaque white). 

We considered using chemical anesthesia on /. notoides 
to help us locate and measure droplets in live squid, but this 
can move material from organ to organ in the digestive tract 
of cephalopods (Bidder, 1966). Decapitation and dissection 
can also cause movement of material between organs (Bid- 
der, 1966). Although movement or breakage of drops due to 
handling cannot be ruled out in our study, we minimized 
post-collection handling and confirmed for some squid that 
droplets were in the cecum before chilling. 

We provide preliminary evidence that cecal oil droplets 
originate from food a few hours after consumption. Al- 
though droplets were admittedly less obvious in the diges- 
tive gland than in the cecum, the amount of lipid in the 
cecum at 3-7 h after feeding far exceeded the amount 
detected in the digestive gland before feeding. Thus, the 
"new" cecal lipid probably derived from the latest meal. 

Cephalopod digestion times (defined as time from food 
capture to return of stomach and cecum to "hunger condi- 
tion") include 15-20 h in Octopus and Sepia and 4-12 h in 
Loligo (Bidder. 1966; Lipiriski. 1990). Although cephalo- 
pods "digest quickly, convert efficiently, and grow but do 
not store energy during their 'live fast, die young' lives" 
(O'Dor and Webber, 1986), we cannot say with certainty 
that the digestive gland of /. notoides was empty after 7 d of 
starvation. However, because digestive gland lipid in Octo- 
pus dropped from 0.3% to 0.06% of body weight with a 6-d 
starvation (O'Dor et al.. 1984), 7-d starvation in /. notoides 
may deplete most non-membrane lipid from its digestive 
gland. The large volume of "new" cecal lipid after feeding, 
coupled with a week of prior starvation, leads us to conclude 
that the post-meal cecal lipid seen in /. notoides was pro- 
duced in a few hours from the recent meal. Both the reap- 
pearance of lipid drops in the squid fed after a 7-d starvation 
and the continued absence of drops in all squid starved more 
than 7 d support this conclusion. 

This study describes extracellular lipid droplets in /. 



EXTRACELLULAR LIPID IN PYGMY SQUID 



53 



notoides but leaves unanswered whether these tiny cepha- 
lopods expel the material over time, whether they are met- 
abolically capable of obtaining energy from the lipid. and 
whether the drops confer a buoyant advantage. The fact that 
lipid droplets did not disappear until the eighth or ninth day 
of starvation in field-fed animals suggests that these squid 
may use the droplets as an energy source. However, slow 
expulsion of the lipid as a dietary waste cannot yet be ruled 
out. 

Acknowledgments 

We are grateful to Jon N. Havenhand for use of his 
laboratory facilities, to J. Robertson and A. R. Dyer for the 
use of their boats, to O. A. Pechenik for typing and data 
entry, and to Cat Darrow for technical help in preparing the 
digital color plate. Research was supported by a sabbatical 
leave and research grant from Milton Academy to L. S. 
Eyster. 

Literature Cited 

Berry, S. S. 1932. Cephalopods of the genera Sepio/oidea. Sepiailariuni, 
and Idiosepius. Rec. South Austral. Mus. 47: 39-55. 

Bidder, A. M. 1966. Feeding and digestion in cephalopods. Pp. 97-124 
in Physiology ofMolhisca. Vol II. K. M. Wilbur and C. M. Y'onge. eds. 
Academic Press, New York. 

Blanchier, B., and E. Boucaud-Camou. 1984. Lipids in the digesiiM.- 
gland and the gonad of immature and mature Sepia ofjicianalis (Mol- 
lusca: Cephalopoda). Mar. Biol. 80: 39-43. 

Boucaud-Camou, E. 1971. Constituunts lipidiques du foie de Sepia 
officianalis. Mar. Biol. 8: 66-69. (Cited in Blanchier and Boucaud- 
Camou. 1984.) 

Boucaud-Camou, E., and R. Boucher-Rodoni. 1983. Feeding and di- 
gestion in cephalopods. Pp. 149-187 in The Mollitsca, P. R. Boyle, ed. 
Academic Press. New York. 

Boucaud-Camou, E., and C. Roper. 1995. Digestive enzymes in 
paralarval cephalopods. Bull. Mar. Sci. 57: 313-327. 

Boyle, P. R. 1991. The UFAW Handbook on the Care and Management 
of Cephalopods in the Laboratory. Universities Federation for Animal 
Welfare, Potters Bar. UK. 62 pp. 

I'.I.H In. R. M. 1953. Examination of some components of cephulopod 
and sperm-whale liver oils by the chromatographic method. Biochem. 
J. 54: 459-465. 

Castro. B. G.. J. L. Garrido, and C. G. Sotelo. 1992. Changes in 
composition of digestive gland and mantle muscle of the cuttlefish 
Sepia officianalis during starvation. Mar. Biol. 114: 11-20. 

Clarke, A., P. G. Rodhouse, and D. J. Gore. 1994. Biochemical com- 
position in relation to the energetics of growth and sexual maturation in 
the ommastrephid squid lllex argentinus. Philos. Trans. R. Soc. Land. 
B. 344: 201-212. 

Clarke. M. R. 1988. Evolution of buoyancy and locomotion in recent 
cephalopods. Pp. 203-213 in The Mollusca. M. R. Clarke and E. R. 
Trueman. eds. Academic Press, New York. 

Conn, H. J. 1961. Biological Stains: a Handbook on the Nature and 
Uses of the Dyes Employed in the Biological Laboratory. Williams and 
Wilkins. Baltimore. MD. 350 pp. 



Green, F. J. 1990. The Sigma-A/drich Handbook of Stains. Dyes, and 
Indicators. Aldrich Chem. Co.. Milwaukee, WI. 776 pp. 

(iurr, E. 1962. Staining Animal Tissues: Practical and Theoretical. 
Leonard Hill Ltd.. London. 631 pp. 

Gurr. E. 1965. The Rational Use of Dyes in Biology and General 
Siainiii.x Methods. Leonard Hill Ltd.. London. 422 pp. 

Hochachka, P. VV., T. W. Moon, T. Mustafa, and T. W. Storey. 1975. 
Metabolic sources of power for mantle muscle of a fast swimming 
squid. Comp. Biochem. Physiol. 52B: 151-158. 

Hylleberg, J., and A. Nateewathana. 1991. Redescription of IJiosepius 
p\Xiaeus Steenstrup. 1881 (Cephalopoda: Idiosepiidae), with mention 
of additional morphological characters. Phuket Mar. Biol. Cent. Res. 
Bull. 55: 33-42. 

Kreuzer, R. 1984. Cephalopods: handling, processing and products. 
FAO Fish. Tech. Pap. 254: 1-108. (Cited in Castro et al. 1992.) 

l.ipinski. M. R. 1990. Changes in pH in the caecum of Loligo vulgaris 
reynaudii during digestion. 5. Afr. J. Mar. Sci. 9: 43-5 1 . 

Moltschaniwskyj, N. A. 1995. Multiple spawning in the tropical squid 
Photololigo sp.: What is the cost in somatic growth? Mar. Biol. 124: 
127-135. 

Moltschaniwskyj, N. A., and J. M. Semmens. 2000. Limited use of 
stored energy reserves for reproduction by the tropical loliginid squid 
Photololigo sp. J. Zoo/. Lond. 251: 307-313. 

Moynihan, M. 1983. Notes on the behaviour of Idiosepius pygmaeus 
(Cephalopoda: Idiosepiidae). Behaviour 85: 42-57. 

Norman, M.D., and A. Reid. 2000. A Guide to Squid. Cuttlefishes and 
Octopuses of Australasia. CSIRO Publishing and the Gould League of 
Victoria. Melbourne. Australia. 96 pp. 

O'Dor, R. K. 2002. Telemetered cephalopod energetics: swimming, 
soaring, and blimping. Integ. Comp. Biol. 42: 1065-1070. 

O'Dor, R. K., and D. M. Webber. 1986. The constraints on cephalo- 
pods: Why squid aren't fish. Can. J. Zoo/. 64: 1591-1605. 

O'Dor, R. K., K. Mangold. R. Boucher-Rodoni, M. J. Wells, and J. 
Wells. 1984. Nutrient absorption, storage and remobilization in Oc- 
topus vulxaris. Mar. Behav. Physiol. 11: 239-258. 

Phillips. K. L., G. D. Jackson, and P. D. Nichols. 2001. Predation on 
myctophids by the squid Moroteuthis ingens around Macquarie and 
Heard Islands: stomach contents and fatty acid analyses. Mar. Ecol. 
Prog. Series 215: 179-189. 

Semmens, J. M. 1998. An examination of the role of the digestive gland 
of two loliginid squids, with respect to lipid: storage or excretion? 
Proc. R. Soc. Loud. B. 265: 1685-1690. 

Semmens, J. M.. N. A. Moltschaniwskyj, and C. G. Alexander. 1995. 
Effect of feeding on the structure of the digestive gland of the tropical 
sepioid Idiosepius pygmaeus. J. Mar. Biol. Assoc. UK 75: 885-897. 

Shepard, S. A., and I. M. Thomas. 1989. Marine Invertebrates of 
Southern Australia, Part II. SA Government Printing Division, Ad- 
elaide. 

Storey, K. B., and J. M. Storey. 1983. Carbohydrate metabolism in 
cephalopod molluscs. Pp. 92-137 in The Mollusca I. Metabolic Bio- 
chemistry and Molecular Biomechanics. P. W. Hochachka, ed. Aca- 
demic Press. London. 

Voogt, P. A. 1983. Lipids: their distribution and metabolism. Pp. 329- 
370 in The Mollusca I. Metabolic Biochemistry and Molecular Biome- 
chanics. P. W. Hochachka. ed. Academic Press. London. 

Wells, M. J., and A. Clarke. 1996. Energetics: the costs of living and 
reproducing for an individual cephalopod. Philos. Trans. R. Soc. Loud. 
351: 1083-1 104. 

Westermann, B.. and R. Schipp. 1998. Cytological and enzyme-histo- 
chemical investigations on the digestive organs of Nautilus pompilius 
(Cephalopoda, Tetrabranchiata). Cell Tissue Res. 293: 327-336. 



Reference: Biol. Bull. 205: 54-65. (August 2003) 
2003 Marine Biological Laboratory 



Functional and Biochemical Properties of the 

Hemoglobins of the Burrowing Brittle Star Hemipholis 

elongata Say (Echinodermata, Ophiuroidea) 



ANA BEARDSLEY CHRISTENSEN 1 '*, JAMES M. COLACINO 2 , AND 
CELIA BONAVENTURA 3 

^Biology Department. Lainar University, PO Box 10037. Beaumont. Texas 77710; 'Department of 

Biology, Clemson University. 132 Long Hall, Clemson, South Carolina 29634; and ^Marine Biomedical 

Laboratory, Duke Marine Laboratory. Duke University, 135 Duke Marine Lab Road. 

Beaufort, North Carolina 28516 



Abstract: The burrowing brittle star Hemipholis elongata 
(Say) possesses hemoglobin-containing coelomocytes 
(RBCs) in its water vascular system. The RBCs, which 
circulate between the arms and body, are thought to play a 
role in oxygen transport. The hemoglobin of adult animals 
has a moderate affinity for oxygen (P 5U = 1 1.4 mm Hg at 
pH 8, 20 C, measured in cellulo) and exhibits cooperativity 
(Hill coefficient > 1.7). The hemoglobin of juveniles has a 
higher affinity (/%, = 2.3 minHg at pH 8.0. 20 C) and also 
exhibits cooperativity. The oxygen-binding properties of the 
hemoglobin are relatively insensitive to pH, temperature, 
and hydrogen sulfide. Adult hemoglobin is a heterogeneous 
mixture composed of three major fractions. The combined 
results of electrospray mass spectrometry and oxygen-bind- 
ing experiments performed on purified fractions indicate 
that the native hemoglobin is in the form of homopolymers. 
A partial amino acid sequence (about 40 amino acids) of 
adult hemoglobin reveals little homology with holothurian 
hemoglobins. 

Introduction 

The hemoglobin of the burrowing brittle star Hemipholis 
elongata Say (Echinodermata, Ophiuroidea) is contained in 
anucleate coelomocytes (red blood cells, RBCs) present in 
the water vascular system (WVS) (Hajduk and Cosgrove, 



Received 4 September 2002; accepted 27 May 2003. 

* To whom correspondence should be addressed. E-mail: 
christenubls 1 hal.lamar.edu 

List of abbreviations: RBCs. red blood cells; WVS. water vascular 
system. 



1975; Hajduk, 1992: unpubl. data). The presence of RBCs 
in the WVS imparts a bright red color to the tube feet, which 
are external projections of the WVS, and readily distin- 
guishes H. elongata from other burrowing ophiuroids (fam- 
ily Amphiuridae) occurring in the same locations. RBCs 
and the fluid of the WVS are circulated throughout the body 
by a series of synchronous contractions of the tube feet 
(Beardsley and Colacino, 1998). H. elongata does not ven- 
tilate its burrow, and it has been hypothesized that hemo- 
globin in the WVS transports oxygen from arms extended 
into the water column to buried body parts (Beardsley and 
Colacino, 1998). 

H. elongata is often found in the lower intertidal zone in 
protected, low-energy areas of the southeast coast of the 
United States (Hendler et a!.. 1995), and the sediments it 
inhabits are often soft, poorly oxygenated and may contain 
hydrogen sulfide (Windom and Kendall, 1979; Camargo, 
1982; unpubl. data). The distribution of H. elongata is 
sporadic, but densities in a given area may be as high as 
2000/nr (Valentine. 1991a). The juveniles of H. elongata 
often settle out onto the arms of the adults (adult disc 
diameter 5 to 12 mm) (Mortensen, 1920; Valentine, 199 la, 
b), crawl down the arms of the adult, and grow in the 
burrow until they reach a size at which they can establish 
their own burrows. Recently settled juveniles (disc diame- 
ter > 0.48 mm) also possess RBCs. Although juveniles 
smaller than 0.43 mm apparently do not have RBCs (un- 
publ. data), the stage at which they start producing RBCs is 
unknown. 

Only three of the approximately 2000 species of brittle 
stars possess hemoglobin; Ophiactis virens (Foettinger, 



54 



HEMOGLOBINS OF A BURROWING BRITTLE STAR 



55 



1880: Cuenot, 1891), Hemipholis elimgata (Hajduk and 
Cosgrove, 1975; Heatwole. 1981; Beardsley et ai. 1993), 
and Ophiactis simplex (Christensen. 1998). Of these three, 
only H. elongata burrows; the other species inhabit the 
fouling communities of rock jetties and pilings. As for the 
functional and biochemical properties of ophiuroid hemo- 
globins, very little has been reported in the literature 
(Hajduk and Cosgrove, 1975; Beardsley et al.. 1993: Chris- 
tensen. 1998; Weber and Vinogradov, 2001). Hajduk and 
Cosgrove (1975) reported that the hemoglobin of H. elon- 
gata has a high affinity for oxygen (P w = 9 mmHg) and is 
composed of five components separable by acrylamide gel 
electrophoresis. They also reported two components with 
molecular weights of 19,000 and 23,000 Da separable by 
SDS gel electrophoresis. The hemoglobins of the holothu- 
rians, the other group of extant echinoderms possessing the 
iron-containing pigment, have been more thoroughly inves- 
tigated (Terwilliger and Terwilliger, 1988 [review]; Suzuki. 
1989; Mauri et al.. 1991; McDonald et ai. 1992: Baker and 
Terwilliger. 1993; Kitto et ai. 1998). 

The goal of the present study was to characterize the 
functional, biochemical, and structural properties of the 
hemoglobins of Hemipholis elongata in more detail. Com- 
parisons were made with the findings of Hajduk and Cos- 
grove (1975), and the functional and structural properties 
observed in this study were compared to those of the ho- 
lothurian hemoglobins. 

Materials and Methods 

Collection and care of animals 

Animals were collected with a shovel and sieve during 
low tide from Johnson Creek. Hunting Island. South Caro- 
lina. Animals were transported to the laboratory and kept at 
room temperature (24 C) in an aquarium containing sedi- 
ments from the collection site and aerated natural seawater. 
All experiments using whole animals or RBCs were con- 
ducted within 6 weeks of collection. Unless otherwise 
noted, all experiments used hemoglobin or RBCs from adult 
animals. 

Preparation of hemolysates and determination of total 
hemoglobin per animal 

RBCs were extracted by cutting the animal into small 
fragments in a small quantity of buffered seawater (50 mM 
TRIS. pH 8.0 at 20 C) and then rinsing the fragments until 
no red color was visible. The body fragments were removed 
and the cell/buffer mixture was placed on ice. The cells 
were washed three times in fresh buffer. After a final wash, 
the supernatant was removed and the cell pellet frozen and 
thawed to lyse the cells. The thawed pellet was resuspended 
in about 1 ml of 50 mM TRIS, pH 8 at 20 C in distilled 
water and centrifuged at 14.000 X g for 5 min to remove 



cell remnants and debris. The hemolysate was diluted with 
appropriate buffer to an absorbance of 0.4 to 0.5 OD units 
at 540 nm to minimize photometric error (van Assendelft. 
1970). 

For determination of total hemoglobin, the absorption 
spectrum of the solution was recorded in a Beckman DU-65 
spectrophotometer. The hemoglobin concentration, as 
heme, was calculated using the Beer-Lambert law and the 
extinction coefficient for human hemoglobin at 542 nm (van 
Assendelft, 1970). This concentration was multiplied by the 
total sample volume to give total hemoglobin (as heme) in 
millimoles, and then divided by animal wet weight to obtain 
total hemoglobin per gram of wet weight. 

Hemolysates used in the oxygen-binding equilibria and 
the sulfide sensitivity experiments were prepared as above, 
but with RBCs taken only from excised arms with the aim 
of avoiding potential interfering effects due to enzymes 
released from the gut and gonads. Such material may have 
increased the formation of methemoglobin in the whole 
animal hemolysates (see Discussion for methemoglobin ef- 
fects). 

Intracellular heme concentration 

RBCs collected from an animal as described above were 
resuspended in isotonic filtered seawater buffered to pH 8.0 
at 20 C with 50 mM TRIS. A few drops of a suspension of 
polystyrene microbeads (Poly sciences. Inc.) of 5.85 /u.m 
0.13 jam diameter were added to the cell suspension. A 
small drop of this mixture was placed on a glass microscope 
slide, and a glass coverslip was placed on top of the drop. 
Excess water was wicked away with a tissue until the 
coverslip was resting on the microbeads. and the cells were 
flattened between the coverslip and the slide. This technique 
serves to set the pathlength for the subsequent absorbance 
measurements (Colacino and Kraus. 1984). The slide was 
then placed on the stage of the microspectrophotometer 
(Mangum et al.. 1989). The light transmission spectrum 
through an area of the slide containing only buffer was 
collected as a reference. Cells were picked at random on the 
slide, and the transmission spectrum was recorded through 
each. The absorbance at 540 nm. 560 nm, and 577 nm was 
computed from light transmission data. The intracellular 
hemoglobin concentration was calculated as heme concen- 
tration using the Beer-Lambert Law and the extinction 
coefficients for human hemoglobin at the chosen wave- 
lengths (van Assendelft, 1970). Concentrations calculated at 
the three wavelengths were averaged for each cell. A total of 
48 cells were measured (12 cells on each of four slides 
prepared from the same cell suspension). 

Separation of hemoglobins 

The presence of multiple hemoglobins was determined 
using a Pharmacia fast protein liquid chromatography 



56 



A. B. CHRISTENSEN ET AL. 



(FPLC) system. Crude hemolysates were collected as pre- 
viously described and resuspended in 50 mM TRIS, pH 8.0, 
at 20 C. Hemolysates IP n 7-10 individuals were pooled 
due to the small qu;iv . or hemoglobin collected from 
each animal. The p( d samples were equilibrated with 50 
mM TRIS. pH N.< y dialysis. The hemolysates were then 
loaded onto a D!AE Q Sepharose Hi-Load 2610 column, 
60-ml column solume. The resin had been equilibrated with 
four volumes of 50 mM, pH 8.0, TRIS buffer. A linear salt 
gradient (50 mM TRIS to 0.075 M NaCl + 50 mM TRIS) 
was used for elution. The total gradient volume was 300 ml. 
Fractions were collected at a rate of 6 min per fraction, and 
absorbance was read at 280 nm (for protein) and 415 nm 
(hemoglobin peak). 

The fractions showing peak absorbance were electropho- 
resed on a Pharmacia Phast system using a native gel. 
Samples were run on a 10% acrylamide gel with pH 8.3 
electrophoresis buffer and stained with Coomassie blue. A 
low-porosity stacking gel (upper fraction) was used to 
sharpen the banding pattern. Samples contained 5-25 /ag of 
protein. Crude hemolysate and human hemoglobin A were 
electrophoresed as references. 

Determination of molecular weight 

The molecular weights of the purified hemoglobin frac- 
tions were determined by electrospray ionization mass spec- 
trometry. Samples were prepared by the method described 
by Stevens et al. (1994). Measurements were made on a 
Fissons-VG BIO-Q triple quadrupole mass spectrometer 
equipped with a pneumatically assisted electrospray ioniza- 
tion source operating at atmospheric pressure (supplied by 
VG Biotech. Altrincham. UK). 



at 100 ml/min. A total of 60 cells taken from 47 animals 
were used in these experiments. 

To examine the equilibrium oxygen-binding characteris- 
tics of the hemoglobin in cellulo, cells were exposed to 
gases at nine oxygen tensions, ranging from to > 150 
mmHg (room air). Fractional saturation values were com- 
puted from transmitted light intensities (540 nm, 560 nm, 
and 580 nm) using a two-wavelength modification of a 
standard analysis (Rossi-Fanelli and Antonini. 1958). The 
effect of pH on oxygen affinity was determined from oxy- 
gen equilibrium experiments conducted at pH 7.0, 8.0, and 
9.0. Temperature effects on oxygen binding were deter- 
mined from oxygen affinity experiments conducted at 10 
C, pH 8.0. and 20 C, pH 8.0. The heat of oxygenation 
(AH) was calculated from the van't Hoff equation. 



Oxygen-binding equilibria in vitro 

For measurements on crude hemolysates, about 50 /nl of 
the hemolysate was placed in the working chamber of the 
gas slide along with a 0.5-cm 2 piece of monofilament nylon 
mesh (105-ju.m mesh opening) (Small Parts, Inc.). A freshly 
prepared hemolysate solution was used in each experiment 
because repeated freezing and thawing increased methemo- 
globin formation (see Discussion for methemoglobin ef- 
fects). The nylon mesh served to ensure a stable pathlength 
for light transmission measurements. Oxygen-binding equi- 
libria were measured as previously described for in cellulo 
measurements at 20 C. Measurements on purified FPLC 
fractions were made using standard tonometric techniques 
at pH 7 at 20 C. 



Hemoglobin spectra and oxygen-binding equilibria in 
cellulo 

A small portion (< 5 mm) of the distal end of an arm was 
excised from an unanesthetized animal and rinsed in buffer 
to remove any adhering mud. The arm tip was placed in a 
large depression slide containing a small amount of 
0.45-ju.m filtered isotonic seawater buffered with 50 mM 
TRIS of the desired experimental pH (7. 8, or 9). The tube 
feet were manually stimulated to contract, forcing the RBCs 
out of the cut end of the radial canal. About 50 p.\ of the 
buffer/RBC mixture was transferred to a specially designed 
gas slide (Colacino and Kraus, 1984). The gas slide was 
placed on the microscope stage of a diode array microspec- 
trophotometer (Mangum et al., 1989). The slide was main- 
tained at a constant temperature (20 0.5 C) with water 
pumped from a refrigerated water bath (Forma Scientific). 
The internal gas tension of the slide was controlled by a 
gas-mixing flowmeter (Cameron Instrument Co.). The gases 
were humidified and brought to experimental temperature 
before flowing through the sample chamber of the gas slide 



Oxygen-binding equilibria of juvenile hemoglobin 

Oxygen-binding equilibria for hemoglobins of juvenile 
H. elongata were measured on both isolated cells and intact 
animals. For the isolated-cell measurements, cells were col- 
lected from individuals of disc diameter < 1 mm (// = 5) by 
excising a small portion of an arm in a small quantity of 
buffer, 50 mM TRIS. pH 8.0, at 20 C. The cells were 
loaded onto the gas slide, and the oxygen binding was 
measured by the method previously described for the adult 
cells. 

For the whole-animal measurements, an intact juvenile 
was loaded onto the gas slide. The juvenile was anesthetized 
by placing it in a small quantity of buffer (< 200 /nl) 
containing a few drops of 7% MgCU. The individual was 
then placed centrally in the gas slide chamber in a small 
drop of buffer/MgCK. Hemoglobin spectra were taken 
through a tube foot with RBCs in it. Oxygen-binding ex- 
periments were conducted as before. Four individuals were 
measured in this manner. 



HEMOGLOBINS OF A BURROWING BRITTLE STAR 



57 



Hemoglobin sensitivity to sulfide 

The effects of hydrogen sulfide on oxygen-binding equi- 
libria were examined using hemolysates prepared as de- 
scribed earlier (hemoglobin 50 mA/ TRIS in distilled water, 
pH 8.0 at 20 C, adjusted to give an absorbance reading of 
0.4-0.5 at 540 nm in an 0.5-cm cuvette). Four milliliters of 
this solution were placed into each of two glass tonometers 
equipped with 0.5-cm pathlength cuvettes. The initial oxy- 
genated spectra were recorded from 650 nm to 400 nm on a 
Beckman DU 65 spectrophotometer. The samples were 
deoxygenated with 99.999% N 2 , and a deoxygenated spec- 
trum was taken. Then 100 ju,l of 10 mM Na 2 S solution was 
injected into one tonometer with a syringe; 100 p.] of 
deoxygenated buffer was added to the control tonometer. 
The spectrum was again recorded. Oxygen was gradually 
introduced in a stepwise fashion by the injection of room air 
samples after an equal volume had been removed from the 
tonometers, and the spectra were measured after a 10-min 
equilibration. This experiment was repeated three times 
with freshly prepared hemolysates pooled from three to five 
individuals. 

Stopped-flow measurements of ligand kinetics 

Kinetics of the hemoglobin-oxygen reaction were esti- 
mated using the stopped-flow technique on the crude cell 
hemolysates suspended in 50 mM HEPES. pH 7, at 20 C; 
the crude hemolysates represent pooled samples. Measure- 
ments were made for the O 2 "off reaction (dissociation) 
and the CO "on" reaction (association). The CO "on" reac- 
tion was also examined using flash photolysis. The presence 
of modulator effects was determined by performing the 
above reactions in the presence of ATP. 

Experiments were performed on a Gibson-Durrum 
stopped-flow apparatus that consists of a Durrum model 
13000 light source and monochromator and Durrum model 
110 stopped-flow instrument with pneumatic drive. The 
dissociation constant. A;,,,, was determined by reacting oxy- 
hemoglobin, from crude hemolysates, with sodium dithio- 
nite (Na 2 S 2 O 4 ) (about 0.5%). The time course of the reac- 
tion was monitored by measuring transmitted light 
intensities. 

Light intensity data were collected by a DASAR data 
acquisition, storage, and retrieval system with a DW-2 
interface to a Tektronix 4052 computer. Initial data analyses 
were carried out with the ASYST program (Macmillan 
Software Co.) prior to curve-fitting analysis by a nonlinear 
least-squares program (Johnson et at, 1981 ). 

The carbon monoxide (CO) association reaction was 
monitored by the stopped-flow technique and flash photol- 
ysis. Flash photolysis was performed with dual fast extin- 
guishing (approximately 30 /us) flash tubes and a Xenon 
Corp. model B micropulser. The subsequent association of 
CO and the hemoglobin was then monitored as before. 



Aniino acid seanence of hemoglobin fractions 

Partial amino acid sequences of each purified hemoglobin 
fraction were determined by the automated Edman method 
using a Porton Instruments PI 2090 integrated micro-se- 
quencing system. The amino acids were identified by visual 
inspection of hardcopy plots, using retention times from a 
standard of PTH amino acids. 

Proteins were not digested with proteases prior to se- 
quencing. Each analysis used 150-200 pmol of protein. 
Samples of fractions 1 and 2 were processed for 40 cycles, 
at which time it became difficult to distinguish the amino 
acid peaks from the background noise. Fraction 3 was run 
for 21 cycles. 

The partial sequence for fraction 1 was compared to other 
protein sequences using the MacVector sequence analysis 
software (Oxford Molecular Group PLC) and the Entrez 
database (National Center for Biotechnology Information). 

Results 

Total hemoglobin per individual 

The average wet weight of the adult animals used for this 
measurement was 0.39 g 0.16 g (mean SD, n = 9). 
These individuals contained 8.5 X 10~ 5 mmol 4.4 X 
10~ 5 mmol of hemoglobin, measured as heme. Using a 
hemoglobin subunit molecular mass of 16,000 Da, this 
figure translates into 0.35% of the total body mass ac- 
counted for by the hemoelobin. 



Intracellnlar hemoglobin concentration and estimation of 
hematocrit 

The RBC hemoglobin concentration, as heme, was 
19.5 5.0 mM (mean SD, n = 48 cells). Using a mean 
cell diameter of 9 /LUTI (Hajduk and Cosgrove, 1975; Heat- 
wole, 1981; unpubl. data) and an intracellular hemoglobin 
concentration of 19.5 mM, the volume of a single cell is 3.8 
X 10~ 7 mm 3 and contains 7.4 X 10" 12 mmol hemoglobin. 
If an animal has a total of 8.5 X 10~ 5 mmol of hemoglobin, 
it has approximately 1 X 10 7 cells. The total cell volume is 
equal to 4.3 mm 3 ; and for an animal with a total WVS 
volume of 18.1 mm 3 (Beardsley and Colacino. 1994). the 
fraction of the WVS taken up by cells is 0.24. 

Separation of the hemoglobins 

The hemoglobin is a heterogeneous mixture. Separation 
of oxygenated crude cell hemolysates by DEAE Q Sepha- 
rose FPLC resulted in three fractions that absorb at 280 and 
415 nm (Fig. 1). Native gel electrophoresis of the crude 
hemolysates yielded five bands, two of the major bands 
corresponding to FPLC fractions 1 and 2, a third major band 
of uncertain identity, and two minor bands (Fig. 2). Lack of 



A. B. CHRISTENSEN ET AL. 




Figure 1. Cliromatogram of crude hemolysates from Ht'mipholis elun- 
gata, separated by FPLC ion exchange chromatography. Separation was 
achieved using a DEAE Q Sepharose Hi-Load 2610 column. 60-ml column 
volume. A linear salt gradient was established from 50 mM Tris to 0.75 M 
NaCl + 50 mM Tris; gradient volume was 300 ml. The profile represents 
absorbance at 280 nm and 415 nm. 



banding in lane 5 (FPLC fraction 3) is attributed to low 
concentration applied to the gel. 

As the sample placed on the column was a pooled sample, 
it is unknown if all of the FPLC fractions are found in a 
single individual or in separate individuals. However, the 
separation was performed on two occasions using animals 
from different collections, and both experiments yielded the 
same results. 

Molecular weight of hemoglobin subunits 

The technique used to determine molecular weight, elec- 
trospray mass spectrometry, breaks polymeric hemoglobins 
into monomeric subunits and causes the heme to dissociate 
from the protein. The signals for FPLC fractions 1 and 2 
indicated that each was composed of a single protein and a 
heme peak. The molecular weights of the subunits were as 
follows: fraction 1 16,080, fraction 2 16,1 19. and frac- 
tion 3 16,143. The hemes all had a molecular weight of 
616. The differences in the protein weight indicate differ- 
ences in amino acid composition for the three subunits. 

The mass spectrum generated by the third FPLC fraction 
indicated the possibility of more than one subunit. However, 
the low concentration of this sample resulted in high-noise 
mass spectrometry data, making this conclusion uncertain. 

Oxygen-binding equilibria in cellulo and in vitro 

The hemoglobin of Hemipholis elongata has a moderate 
affinity for oxygen (P 5I , = 11.4 mmHg at pH 8.0, 20 C) 
(Table 1 ). The P 50 at pH 8.0 is similar to those reported for 
holothurian hemoglobins (Table 1). The Hill numbers (n) 
were greater than 1 for both in cellulo and in vitro measure- 
ments, indicating cooperativity and functional hemoglobin 
composed of at least two subunits (a dimer) (Table 1 ). Then 
n values are greater for the //; cellulo measurements 0; = 
2.81) than for the crude hemolysates (n = 1.91). The 
difference in Hill coefficients may be due to concentration 
differences (19.5 mM /;; cellulo v.v. 0.1 mM in vitro) (see 
Discussion for concentration effects). 



The P 50 values measured on the FPLC fractions are lower 
than those measured on the crude hemolysates and in cel- 
lulo. P 50 at pH 7.0 at 20 C for fraction 1 is 6. 1 mmHg and 
that for fraction 2 is 2.5 mmHg. These low values may be 
due to the formation of methemoglobin (see Discussion). 
The Hill numbers for the purified FPLC fractions 1 and 2 
are greater (about 1.8) (Table I ). 

Values for P 5{) measured at the three pHs are significantly 
different from one another (Student's r test, P = 0.05) 
(Table 1 ). Oxygen affinity increases slightly as pH de- 
creases. This is opposite to the usual pH effect. The slope of 
the Bohr plot is 0.072. The temperature dependence of the 
oxygen affinity is small (P S() = 8.9 mmHg at 10 C v.v. 1 1.4 
mmHg at 20 C). The heat of oxygenation (AH) is -4.1 
kcal/mol. 

Oxygen-binding equilibria of juvenile hemoglobin 

The hemoglobin of juveniles (disc diameter 0.48 to 0.8 
mm) has a higher affinity (P ?(l = 2.3 mmHg in cellulo and 
4.0 mmHg in tube feet of intact animals, pH 8.0 at 20 C) 
for oxygen than the adult hemoglobin (P 50 =11.4 mmHg, 
disc diameter 5 to 12 mm) (see Table 1). This was true for 
both isolated RBCs and intact animal measurements. The 
greater apparent P 50 for measurements using intact animals 
may be explained by oxygen consumption of the animal. 
The Po-, within the tissues is lower than the external Po 2 due 
to oxygen consumption by the tissues. The difference in Po 2 
can lead to an overestimation of the P 50 . Even with the 



12345 



Figure 2. Native gel eletrophoresis of crude hemolysates and purified 
FPLC fractions of Hemiplmlis elimgata hemoglobin. Samples were run on 
a W7c acrylamide gel with pH 8.3 electrophoresis buffer and stained with 
Coomassie blue. A low-porosity stacking gel (upper fraction) was used to 
sharpen the banding pattern. Lack of banding in lane 5 is attributed to low 
concentration applied to the gel, since mass spectrometry results showed 
fraction 3 to contain hemoglobin. Lane 1 : Human hemoglobin A: lane 2: 
Crude hemolysate; lane 3: FPLC fraction 1; lane 4: FPLC fraction 2: lane 
5: FPLC fraction 3. 



HEMOGLOBINS OF A BURROWING BRITTLE STAR 



59 



Table 1 
Oxygen P S( , values and Hill numbers ofechinoderm hemoglobins (mean SE) 



Species 


PSO 

mmHg 


Hill number 
(") 


# of measurements 
(ft of individuals)* 


Study 


Ophiuroids 










HemiphoHs elongatu 










in cellulo 








Present study 


pH 7.0. 20 C 


9.5 1.2 


2.35 0.07 


15(11) 




pH S.O, 10 C 


8.9 1.1 


3.20 0.10 


12(8) 




pH 8.0. 20 C 


11.4 1.2 


2.81 0.08 


17(14) 




pH 9.0. 20 C 


13.1 1.2 


2.67 0.09 


16(14) 




i>i vitro crude hemolysates 








Present study 


pH 7.0, 20 C 


8.2 1.1 


1.78 0.04 


6 




pH 8.0. 20 C 


10.5 1.1 


1.91 0.05 


4 




pH 9.0, 20 C 


11.3 1.2 


1.73 0.06 


4 




I'H n'/ro purified 








Present study 


fraction 1 . pH 7.0. 20 C 


6.1 


1.83 


2 




fraction 2. pH 7.0. 20 C 


2.5 


1.41 


2 




juvenile 








Present study 


in vitro, pH 8.0. 20 C 


4.0 1.2 


3.28 0.37 


4 




in cellulo, pH 8.0, 20 C 


2.3 1.5 


1.76 0.51 


5(4) 




O/ilmictis simplex in cellulo, pH 8.0. 20 C 


22.3 1.2 


3.04 0.18 


15(12) 


Christensen. 1998 


Holothuroidst 










Cucumaria miniata 


8.0 1.5 


1.86 0.07 




Terwilliger, 1975 


Cucumaria curala 


7.1 


1.6 




Roberts et at, 1984 


Thyonetla gemmata 


2.6 


1.4 




Steinmeier & Parkhurst. 1979 


Molpadia oolitica 


4.0 


1.6 




Terwilliger & Read, 1972 


Molptidici intermedia 


2.0 


<1.0 




Manwell, 1966 


Ciiitdina arenicola 


3.5 


1.5 




Bonaventura et a/., 1976 


Sclerodactyla (Thyone) briareus 


8.1 


1.08 




Colacino, 1973 


Paracaudina chilensis 


1.5 


1.3 




Baker & Terwilliger, 1993 



* The numbers in # of measurements reflect the number of cells measured; the number in parentheses reflects the number of individuals represented in 
the experiment. No animal was used more than twice. 
t All holothurian P 5n values were measured I'M vitro. 



overestimation of the juvenile P 50 , the value is significantly 
less than that of the adult, indicating distinct functional 
differences. 

Hemoglobin sensitivity to sulfide 

Exposure to sulfide caused no changes in the absorption 
spectrum, and there was no peak at 620 nm; this peak is 
characteristic of human sulfhemoglobin in the visible region 
of the spectrum (van Assendelft, 1970; Carricoef /., 1978). 
There was also no change in the oxygen affinity of the 
hemolysates in the presence of sulfide (Fig. 3). 

Kinetics of ligand binding 

Both of the ligand reactions with the crude hemolysates 
were biphasic, with the two components in each reaction 
accounting for about 50% of the reacting species (134.6 
2.2 (SD) s~' and 22.3 0.3 s" 1 for the O 2 "off reaction 
and 5.2 x If) 4 A/~'s~' and 2.8 X 10 4 M~'s~' for the CO 
"on" reaction). These data are consistent with the heteroge- 



neity of the crude hemolysate. Given that FPLC reveals 
three distinct hemoglobins with two (fractions 1 and 2) 
accounting for the majority of the protein, one might expect 
to see two distinct reaction components in ligand-binding 
experiments. A third kinetic component representing the 
third FPLC fraction was not seen, either due to the small 
amount of it present in the crude hemolysates or to a lack of 
difference in rate constants. 

There was no effect of ATP on the CO "on" reaction. 
However, the presence of ATP caused a small, but statisti- 
cally significant (P = 0.05) increase in the dissociation rate 
constant for both phases of the biphasic oxygen "off reac- 
tion of H. elongata hemoglobin (144.3 2.4 s^ 1 and 
24.2 0.2 s~'). 



Amino acid sequence of the protein 

Figure 4 shows the partial amino acid sequences for the 
three FPLC fractions of the hemoglobin. The sequences for 



60 



A. B. CHR1STENSEN ET AL 



06 

04 - 

02 - 

00 - 

> - 2 - 

00 
.2 -06 

-08 - 
-1 - 
-1 2 - 
-14 




0.0 



02 



04 06 08 

LogPO, (mmHg) 



10 




Figure 3. Hill plots for the oxygen-binding equilibria of Hemipholis 
elongate hemoglobin in the absence () and presence of Na 2 S (O). 
Hemoglobin is crude hemolysate suspended in 50 mAf Tris. pH 8.0. at 20 



fractions 1 and 2 differ by eight amino acids. The partial 
sequence for fraction 3 was identical to that of fraction 2. 
Neither hemoglobin fraction appears to be blocked at the 
N-terminus of the globin, unlike the hemoglobins of many 
holothurians (Terwilliger and Terwilliger, 1988). Proteins 
blocked at the N-terminus are resistant to the Edman reac- 
tion used in protein sequencing (Kitto et ai, 1976). No 
modification (e.g., digestion with proteases) of the H. elon- 
gata protein was necessary to obtain amino acid sequences. 

Discussion 

Intracellular hemoglobin concentrations and hematocrit 

The intracellular heme concentration of Hemipholis elon- 
gata (19.5 mM) is comparable to the values reported for 



holothurian RBCs (12.5 mM for Sclerodactyla (Thyone) 
briareus [Colacino, 1973] and 15.8 mM for Cucumaria 
miniata [from data in Man well. 1959, and Terwilliger and 
Read, 1972]). These values are similar to those of human 
RBCs (20 mM heme) (calculated from Guyton, 1991) and 
phoronid (Phoronis architecta) RBCs ( 14 mM heme) ( Van- 
dergon and Colacino, 1989). If the hemoglobin of//, elon- 
gata exists as dimers, this would mean that the intracellular 
hemoglobin concentration is 9.8 mM. 

The computed hematocrit value of H. elongata (0.24) is 
comparable to the tube foot hematocrits of the holothurian 
S. briareus (0.24, by direct measurement) (Colacino, 1973). 
These values are greater than the reported hematocrits of the 
perivisceral fluids of the holothurians Cucumaria pseudocu- 
rata (0.032) (Roberts et al., 1984) and Paracaudina chilen- 
sis (0.015) (Baker and Terwilliger, 1993), but less than 
those for humans (0.4) (Guyton, 1991) and other mammals 
(> 0.4) (Schmidt-Nielsen, 1990). 

Structural properties of the hemoglobin 

The hemoglobins of H. elongata are a heterogeneous 
mixture of three components, as evidenced by FPLC. Be- 
cause the samples were pooled from several individuals, it 
is not known whether hemoglobin heterogeneity is a normal 
characteristic of H. elongata blood or an artifact of sample 
mixing. However, the identical results (e.g., same ratio of 
fractions) obtained on two separate occasions suggest that 
all three fractions are present within an individual. Hajduk 
and Cosgrove (1975) reported only two components sepa- 
rable by gel filtration, one apparently composed of mono- 
mers and the other of dimers. The difference in number of 
fractions may be attributable to difference in separation 
techniques. Gel filtration separates on the basis of size and 
shape; DEAE Q Sepharose FPLC (ion exchange) separates 
molecules on the basis of charge. 

Both the present study and Hajduk and Cosgrove (1975) 
obtained five bands by acrylamide gel electrophoresis. Two 
of the major bands reported here correspond to FPLC frac- 
tions 1 and 2. Because this sample was a crude hemolysate, 
the two minor bands may have been due to proteins other 



Fl: val ile ser ala gly glu lys thr leu ile arg asp ser trp ala pro val tyr ala gly asp 
F2. val ile ser ala asp glu lys asn leu ile arg ser 1 trp phe thr val tyr ser gly asp 
F3: val ile ser ala asp glu lys asn leu ile arg ser 1 trp phe thr val tyr ser 



Fractions, continued 

Fl: arg phe gin ile gly val asn val phe thr asn phe ? 

F2: arg phe gin val gly val asp val phe thr asn phe 1 



ala tyr pro ala 
ala tyr 



Figure 4. Partial amino acid sequences for the three FPLC fractions of Hemipholis elongata hemoglobin. 
Amino acids in bold type represent differences in primary sequence;? represents amino acids that could not be 
definitively identified. Fl = fraction 1. F2 = fraction 2. F3 = fraction 3. 



HEMOGLOBINS OF A BURROWING BRITTLE STAR 



61 



than hemoglobin, found within or associated with the RBCs. 
Due to the presence of herne at high relative concentration, 
two of the major bands from the crude hemolysate were 
visible on the gel prior to staining with Coomassie blue. The 
two minor bands were not visible. If the minor bands do 
represent hemoglobins, the low concentration, as evidenced 
by the faintness of the bands on the gel. could explain why 
these fractions were not isolated by FPLC. 

The molecular masses of the various fractions (approxi- 
mately 16.000 Da) are comparable to human )3 chain 
(15.860 Da) (Dickerson and Geis. 1980) and holothurian 
hemoglobin monomers (17.000-18.000 Da) (Terwilliger 
and Terwilliger. 1988). These values are smaller than those 
reported by Hajduk and Cosgrove (1975) (19.000 and 
23.000 Da). Differences between the previously reported 
weights and those of the present study may be attributed to 
differences in techniques. Mangum (1992) remarked that 
many of the early studies on holothurian hemoglobin re- 
ported similarly large molecular weights that were later 
found to be smaller (-17,000 Da). She attributed these 
differences to refinement of molecular techniques. 

The cooperative binding of oxygen, both in cellulo and in 
vitro (Hill number > 1), indicates that the functional he- 
moglobin exists as a polymer. Many of the holothurian 
hemoglobins are known to exist as dimers (Terwilliger and 
Terwilliger. 1988) and possibly tetramers (Baker and Ter- 
williger. 1993). The purified fractions, 1 and 2, of H. elon- 
gata hemoglobin have only one globin chain type but ex- 
hibit cooperativity in oxygen binding, suggesting that both 
are able to form cooperative homodimers. The Hill numbers 
typically observed at low concentrations, somewhat less 
than 2. are consistent with the formation of cooperative 
homodimers. Larger assemblies may form under some cir- 
cumstances, as evidenced by Hill numbers greater than 3 
that were observed under some conditions (e.g., in cellulo). 

The normally existing homopolymers of several inverte- 
brate hemoglobins exhibit cooperativity (Scapharca inae- 
i/iiivul\-i\ [Chiancone et ai, 1981; Royer el ai, 1985]; 
holothurians [Terwilliger and Read. 1972; Bonaventura et 
ill.. 1976]). although homodimers and homotetramers of 
human (and other vertebrate) hemoglobins do not. In fact, 
the mechanism for cooperativity in invertebrates is thought 
to be different from that utilized by vertebrates (Riggs, 
1998). Cooperativity is thought to be due to interactions 
between the E and F helices of the hemoglobin subunits, 
first described in the arcid clam Scapharca inaequivalvis 
(Royer et ai, 1985, 1990). This same association has been 
described for the innkeeper worm, Urechis caupo (Kolatkar 
ct /.il.. 1994) and a sea cucumber. Caiidina arenicola (Mitch- 
ell etui.. 1995). However, Kitto m//. ( 1998) believe that the 
cooperativity mechanism in C. arenicola differs from that in 
S. inaequivalvis as the residues involved at the crucial 
contact points are different for the two species. Further 



structural studies on H. elongata hemoglobin are needed to 
investigate the nature of the interactions of its subunits. 

Comparison of the amino acid sequence of fraction 1 
from H. elongata with sequences reported for the globins of 
the holothurians Caiidina arenicola (Mauri ct al.. 1991; 
McDonald et al., 1992) and Paracaudina chilensis (Suzuki, 
1989) reveals little homology. The lack of homology be- 
tween the ophiuroid and holothurian globins has contributed 
to the inability to identify the brittle star globin gene by 
using holothurian primers (Kitto, pers. comm.). Further- 
more, no successful primers for the hemoglobin gene have 
been generated based on the 39 amino acid sequence of the 
ophiuroid (Kitto, pers. comm.). 

Oxygen-binding characteristics of the hemoglobin 

The hemoglobin of H. elongata has a moderate affinity 
for oxygen, both //; cellulo and in vitro. It is not certain at 
this time whether all of the FPLC fractions exist in the same 
cell, in separate cell populations, or even within the same 
animal. However, a large number of cells from many indi- 
viduals were examined microspectrophotometrically and lit- 
tle variation was seen within the measured P 50 values ob- 
served within each treatment. This suggests that the 
different hemoglobins represented by the purified FPLC 
fractions are present within a single cell. The ratio of this 
mixture is unknown. 

The oxygen affinity results of the present study differ 
greatly from those reported by Hajduk and Cosgrove ( 1975) 
f 90 = 9 mmHg). On the basis of the Hill numbers from the 
present study, a hemoglobin with a P w of 9 mmHg (pH 7.2) 
should have a P w of about 3 mmHg. The in cellulo and in 
vitro analysis in the present study demonstrated a P 50 be- 
tween 8 and 9 mmHg at pH 7.0. 20 C. This difference in 
oxygen affinities may be attributable to the formation of 
methemoglobin. The oxygen affinity of mammalian hemo- 
globin increases with increasing percentages of methemo- 
globin in the sample (Darling and Roughton, 1942) 

Several of the holothurian hemoglobins are prone to 
oxidization and denaturation at pH 7.0 (Terwilliger and 
Read, 1972; Bonaventura et al., 1976; Steinmeier and 
Parkhurst, 1979). When the crude hemolysates of H. elon- 
gata were repeatedly frozen and thawed, the P ?0 decreased. 
Tests for the presence of methemoglobin showed an in- 
crease in the amount present in the sample. Freshly prepared 
H. elongata hemolysates were typically 5%-l% methemo- 
globin as determined by the ferrocyanide method (van As- 
sendelft, 1970). In one experiment, the methemoglobin frac- 
tion was 13% initially and rose to 45% by the end. The P so 
calculated for this run was 3.6 mmHg. This corresponds to 
the P 5( j value estimated from the data of Hajduk and Cos- 
grove (1975). 

The formation of methemoglobin may explain the low 
P 50 of the purified fractions. Although it is not unusual for 



62 



A. B. CHRISTENSEN ET AL. 



different hemoglobins within an individual to have different 
binding affinities, the apparent P 50 of the mixture usually 
lies between those of the oarate fractions, not above them. 
During oxygen-bim 1 L \periments on the purified frac- 
tions, methemogiob.!; rose from 10% to 16% of the total in 
fraction 1 and from 32% to 42% in fraction 2. While the 
large proportion of methemoglobin, particularly in fraction 
2, makes comparisons of P 50 difficult, the importance of this 
experiment is the demonstration of the cooperative binding 
of homopolymers. 

Another explanation for the difference in oxygen affini- 
ties may be the presence of intracellular modulators that 
would have been removed during the purification of the 
fractions. However, many invertebrate hemoglobins, in- 
cluding the holothurian hemoglobins, are insensitive to or- 
ganic phosphates (Terwilliger and Terwilliger. 1988; 
Scholnick and Mangum. 1991; Baker and Terwilliger, 
1993). The rate constant for the oxygen "off reaction of H. 
elongata crude hemolysates exhibited a small, but signifi- 
cant, increase in the presence of ATP. 

The differences in apparent cooperativity between the in 
cellule) and in vitro measurements may be due to concen- 
tration effects. Dilute preparations may be less aggregated 
than concentrated ones, with the consequence that the Hill 
coefficient increases due to subunit interactions as the con- 
centration of hemoglobin increases. This may account for 
the fact that the hemoglobins of capitellid worms were 
reported to have greater cooperativity in cellulo than in vitro 
(Mangum et at., 1992). 

Other than those reported here, no data on ligand-binding 
kinetics are available for the brittle star hemoglobins. The 
smaller of the two dissociation rate constants for H. elon- 
gata hemoglobin is similar to those of human (40 s~', 
Antonini and Brunori. 1971) and holothurian hemoglobin 
(S. briareus, 4.8 s~' [Colacino, 1973]; Thyonella gemmata, 
3.6 to 8 s~ ' [Steinmeier and Parkhurst, 1979]). The larger of 
the two dissociation rate constants (134.6 s~') is among the 
largest reported. The CO association rate constant of H. 
elongata is between those of holothurian hemoglobin (S. 



briareus. ~10 3 M 's ' [Colacino, 1973]; T. gemmata, 10 3 to 
10 4 M~'s~' [Steinmeier and Parkhurst, 1979]) and human 
hemoglobins (30 X 10 5 A/~'s~' [Antonini and Brunori, 1971]). 
Most hemoglobins exhibit a decrease in oxygen affinity 
as the pH decreases (Weber, 1980). It is generally accepted 
that most holothurian hemoglobins are insensitive to pH 
(Terwilliger and Terwilliger, 1988). A weak pH dependence 
(normal direction) on the oxygen affinity of hemoglobin has 
only been reported for two holothurians, Paracauding chil- 
ensis (Baker and Terwilliger, 1993) and Molpadia arenicola 
(Bonaventura et ai, 1976). The hemoglobin of H. elongata, 
however, shows a slight increase in its oxygen affinity, both 
in cellulo and in vitro, with a decrease in pH (Table 1 ). The 
magnitude of the Bohr shift of H. elongata hemoglobin. 



however, is very small and may not have any adaptive 
significance under physiological conditions. 

There is not a large temperature dependence, as evi- 
denced by the small change in affinity with a change in 
temperature. The heat of oxygenation. AH = -4.1 kcal/mol, is 
smaller than that reported for other hemoglobins, 6 kcal/ 
mol to 8 kcal/mol (Antonini and Brunori, 1971; Colacino, 
1973). The possession of a hemoglobin with a small tem- 
perature dependence may be adaptive to an intertidal organ- 
ism, because it would make it relatively insensitive to the 
large temperature changes that can occur in the intertidal 
environment (Cochran and Burnett, 1996; unpubl. data). 

The high affinity of the hemoglobin of juvenile brittle 
stars is clearly different from that of the adults. This func- 
tional difference could be adaptive in their normal habitat. 
The planktonic larvae often settle out onto the extended 
arms of the adults. The juveniles then crawl down the arm 
into the burrow of the adult, where they move around within 
the burrow (pers. obs). Small juveniles (disc diameter < 1 
mm) do not extend their arms into the water column as the 
adults do; therefore, they must obtain oxygen from the 
burrow environment or from the adult. The measured Po 2 of 
the burrow water is very low (undetectable with FOXY 
system [Ocean Optics, Inc.]; unpubl. data), but some oxy- 
gen must escape into the environment from the adult, as 
evidenced by the fact that the sediments lining the burrow 
are oxidized. The possession of high-affinity hemoglobin by 
the juveniles would aid them in the acquisition of oxygen 
from this low-oxygen environment. The switch from high- 
affinity hemoglobin in the fetus/juvenile to lower affinity 
hemoglobin in the adult has been documented in many 
animals (Barcroft, 1935; Riggs, 1951). The time in devel- 
opment in H. elongata when the switch from high-affinity to 
low-affinity hemoglobin occurs is not known. Also un- 
known is whether the hemoglobin present in the juvenile 
RBCs is one of the hemoglobins found in the adults or an 
entirely different one. Due to the small size of the juveniles 
and the small numbers collected, insufficient hemoglobin 
has been isolated to make comparisons with adult hemoglo- 
bin by gel electrophoresis. 

The hemoglobins of H. elongata have a higher affinity for 
oxygen than those of another ophiuroid species. Ophiactis 
simplex (Christensen. 1998) (see Table 1). The differences 
in the oxygen-binding properties could be related to differ- 
ences in their lifestyles. H. elongata burrows in anoxic mud, 
does not ventilate its burrow, and lacks genital bursae, 
structures known to serve as sites of gas exchange in ophiu- 
roids. O. simplex is epibenthic and commonly occurs in 
fouling communities associated with rock jetties and wharf 
pilings. The oxygen levels in these environments may not be 
as limiting as in the mud. The function of hemoglobin in O. 
simplex is not known, but is under investigation. 



HEMOGLOBINS OF A BURROWING BRITTLE STAR 



63 



Effects of sill fide on oxygen binding 

Vertebrate and many invertebrate hemoglobins bind with 
sulfide to form sulfhemoglobin. In human hemoglobin A, 
the binding of sulfide takes place at the heme. but not to the 
iron, and is effectively irreversible (Berzofsky et cil., 1971; 
Carrico et til.. 1978). This also decreases oxygen affinity so 
much as to render the hemoglobin functionless under nor- 
mal conditions (Cameo et ai. 1978). Many invertebrate 
hemoglobins show reversible binding with sulfide (Arp and 
Childress. 1983: Childress et ai, 1984; Doeller et ai, 1988; 
Somero et ai, 1989). In many cases the binding of sulfide is 
vital to the organism because it harbors endosymbionts that 
require sulfide as an energy source (Felbeck, 1983; Hand 
and Somero. 1983; Powell and Somero. 1986). No such 
endosymbionts have been observed in H. elongata (unpubl. 
data.) 

No abnormal spectral changes were observed during the 
oxygen equilibrium experiments on H. elongata hemoglo- 
bin exposed to sulfide. This indicates that either the hemo- 
globin is insensitive to sulfide or there are detoxifying 
enzymes present. However, due to the nature of the hemo- 
lysate solution, any enzymes present would be greatly di- 
luted, thus decreasing their efficiency. The lack of abnormal 
spectra points toward a hemoglobin that is insensitive to 
sulfide. The hemoglobins of the phoronid Phoronis arclii- 
tecta (Vandergon and Colacino, 1991) and the polychaete 
Abarenicola affinis (Wells and Pankhurst. 1980) also appear 
to be insensitive to sulfide. If sulfide does bind to the 
hemoglobin of H. elongata. it does so in a way that does not 
alter oxygen affinity or the visible absorption spectra. For 
organisms that live in sulfide-rich environments but do not 
rely on sulfide-requiring symbionts. it would be highly 
adaptive to possess hemoglobin whose function is not dis- 
rupted by exposure to sulfide. 

Summary 

It appears that the hemoglobins of Hemipholis elongata 
are well suited to the habitat and lifestyle of the animal. H. 
elongata does not ventilate its burrow, and its aerobic me- 
tabolism must be supported by circulation of the WVS fluid 
and RBCs between arms exposed to the water column and 
buried body pans. The moderate /%<> ( I 1.4 mmHg, pH 8.0, 
at 20 C) and cooperativity (Hill number ^ 1.7) of the 
hemoglobin would allow it to extract oxygen from the 
overlying water column and deliver it to buried body parts 
over a wide range of external and internal Po 2 values. The 
relative insensitivity of the hemoglobins to changes in tem- 
perature and pH preserve hemoglobin function when con- 
ditions change, as they frequently do in an intertidal envi- 
ronment. The insensitivity to hydrogen sulfide ensures that 
the hemoglobins continue to function below the sediment 
surface where the animal is situated and sulfide levels may 
be high. 



Acknowledgments 

We thank the following people for their contributions to 
this research: Ed Ruppert for his aid in animal collections 
and discussions on brittle stars; Barry Kitto and Pei Thomas 
for their work in trying to identify the brittle star hemoglo- 
bin gene; Robert Cashon for his analysis of the hemoglobin 
kinetics; Bob Stevens for the electrospray mass spectrome- 
try work; Ellen Beedle for operation of the protein se- 
quencer; Gerald Godette and Shirley Tesh for their help in 
purification of the hemoglobins and oxygen binding of 
purified fractions; Steve Stancyk for his discussions on H. 
elongata: John Wourms for his help in DAPI staining and 
fluorescence microscopy to look for the presence of nuclei 
in the RBCs; Will Johnson for his measurements made at 10 
C; William C. Bridges for his help in the statistical anal- 
ysis; and Jim Schindler and Stuart Mellinchamp for their 
instruction and use of the Coulter Counter to determine 
RBC size. 

Literature Cited 

Antonini, K., and M. Brunori. 1971. Hemoglobin and Myoglobin in 
Their Reaction.', with Ligands: Frontiers of Biology, Vol. 21. North- 
Holkind Publishing, Amsterdam. 436 pp. 

Arp, A. J., and J. J. Childress. 1983. Sulfide binding by the blood of the 
hydrothermal vent tube worm Riftiu pachyptila. Science 219: 295-297. 

Baker, S. M., and N. B. Terwilliger. 1993. Hemoglobin structure and 
function in the rat-tailed sea cucumber. Paracaudina chilensis. Biol. 
Bull. 185: 115-122. 

Barcroft, J., 1935. Foetal respiration. Proc. R. Soc. Lomi B. 118: 
242-263. 

Beardsley, A. M., and J. M. Colacino. 1994. Red blood cell circulation 
and oxygen transport in Hemipholis elongata (Ophiuroidea. Echino- 
dermata). Am. Zool. 34: 35A. 

Beardsley, A. M., and J. M. Colacino. 1998. System-wide oxygen 
transport by the water vascular system of the burrowing ophiuroid. 
Hemipholis elongata (Say). Pp. 323-328 in Echinoderms: San Fran- 
cisco. Proceedings of the 9th International Echinoderm Conference, R. 
Mooi, and M. Telford. eds. Balkema, Rotterdam. 

Beardsley, A. M., J. M. Colacino, R. Cashon, and C. J. Bonaventura. 
1993. The hemoglobins of the brittlestar, Hemiphulix elonxutu (Say) 
(Ophiuroidea, Echinodermata): biochemical and ligand binding prop- 
erties. Am. Zool. 33: 43A. 

Berzofsky, J. A., J. Peisach, and W. E. Blumberg. 1971. Sulfheme 
proteins. II. The reversible oxygenation of ferrous sulfmyoglobin. 
J. Biol. Chem. 246: 7366-7372. 

Bonaventura, C., J. Bonaventura, G. B. Kitto, M. Brunori, and E. 
Antonini. 1976. Functional consequences of ligand-linked dissocia- 
tion in hemoglobin from the sea cucumber Mnlpadia arenicola. Bio- 
chun. Binphys. Ada 428: 779-786. 

Carrico, R. J., W. E. Blumberg, and J. Peisach. 1978. The reversible 
binding of oxygen to sulfhemoglobin. J. Biol. Chcm. 253: 7212-7215. 

Chiancone, E., P. Vecchini, D. Verzili, F. Ascoli, and E. Antonini. 1981. 
Dimeric and tetrameric hemoglobins from the mollusc Scapharca 
iiKict/tiinilvis. J. Mot. Biol. 152: 577-592. 

Childress, J. J., A. J. Arp, and C. R. Fisher, Jr. 1984. Metabolic and 
blond characteristics of the hydrothermal vent tube-worm Riftiu pachyp- 
tila. Mar. Biol. 83: 109-124. 

Christensen, A. B. 1998. The properties of the hemoglobins of Ophiactis 
simplex (Echinodermata. Ophiuroidea). Am. Zool. 38: 120A. 



64 



A. B. CHRISTENSEN ET AL. 



Cochran, R. E., and L. E. Burnett. !996. Respiratory responses of the 

salt marsh animals. Finidiiliis hsri'm litus, Leiostomiis xanhurus, and 

Paleomonetes pugio to env , >>-.entaI hypoxia and hypercapnia and to 

the organophosphate . azinphosmethyl. J. Exp. Mar. Bin/. 

Ecol. 195: 125-144 
Colacino, J. M. 197 A mathematical model of oxygen transport in the 

tube fool-. in: -tern of the sea-cucumber. Thyone briareus L. 

Ph.D. disscn.. IL State University of New York at Buffalo. 143 pp. 
Colacino, .1. M., and D. W. Kraus. 1984. Hemoglobin-containing cells 

of Neodasvs (Gastrotricha. Chaetonotida) II. Respiratory significance. 

Comp. Hiochem. Physiol. 79A: 363-369. 
Cuenol. L. 1891. Etudes sur le sang et les glandes lymphatiques dans la 

serie animale. II. Invertebrates. Arch. Zool. Exp. Gen. 9: 595-670. 
Darling, R. C., and F. J. VV. Roughton. 1942. The effect of methemo- 

globin on the equilibrium between oxygen and hemoglobin. Am. J. 

Physiol. 137: 56-68. 
de Camargo, T. M. 1982. Changes in the echinoderm fauna in a polluted 

area on the coast of Brazil. Papers from the Echinoderm Conference. 

The Australian Museum Sydney. 1978. F. W. E. Rowe, ed. Ausl. Mus. 

Syd. Men,. 16: 165-173. 
Dickerson, R. E., and I. Geis. 1980. Hemoglobin: Structure. Function. 

Evolution and Pathology. Benjamin/Cummings. Menlo Park, NJ. 176 

PP 
Doeller, J. E., D. W. Kraus, J. M. Colacino, and J. B. Wittenberg. 1988. 

Gill hemoglobin may deliver sulhde to bacterial symbionts of Sotemya 

vellum (Bivalva, Mollusca). Biol. Bull. 175: 3XX-396. 
Felbeck, J. 1983. Sullide oxidation and carbon fixation by the gutless 

clam Soleniya reidi: an animal-bacteria symbiosis. 7. Com/). Physiol. 

152: 3-11. 
Foettinger, A. 1880. Sur 1'existence de 1'hemoglobine chez les echino- 

dermes. Arch. Biol. Paris 1: 405-415. 
Guyton, A. C. 1991. Textbook of Medical Physiology. 8th ed. W.B. 

Saunders. Philadephia. 1(114 pp. 
Hajduk. S. I,. 1992. Ultrastructure of the tube-foot of an ophitiroid 

echinoderm. Hemipholis elongaia. Tissue Cell 24: 1 1 1-120. 
Hajduk. S. L., and \V. B. Cosgrove. 1975. Hemoglobin in an ophiuroid. 

Hemipholis elongata. Am. Zool. 15: 808. 
Hand, S. C., and G. N. Somero. 1983. Energy metabolism pathways of 

hydrothermal vent animals: adaptations to a food-rich and sulhde-rich 

deep-sea environment. Biol. Bull. 165: 167-181. 
Heatvvole, D. W. 1981. Spawning and respiratory adaptations of the 

ophiuroid. Hemipholis eltmgatu (Say) (Echinodermata). M.S. thesis. 

University of South Carolina. 84 pp. 
Hendler, G. U J. E. Miller, D. L. Pawson, and P. M. Kier. 1995. Sea 

Stars. Sea Urchins, and Allies: Ec/nnodcrms of Florida and the Ca- 

nhhean. Smithsonian Institution Press. Washington. DC. 390 pp. 
Johnson, M. L., J. J. Correia, D. A. Yphantis, and H. R. Halvorson. 

1981. Analysis of data from the analytical ultracentrifuge by nonlin- 
ear least-squares technique. Biophys. J. 36: 575-588. 
Kitto, G. B., D. Erwin. R. West, and J. Omnaas. 1976. N terminal 

substitution of some sea cucumber hemoglobin. Comp. Biochcm. 

Physiol. 55B: 105-107. 
Kitto, G. B., P. W. Thomas, and M. L. Hackert. 1998. Evolution of 

cooperativity in hemoglobins: what can invertebrate hemoglobins tell 

us? 7. Exp. Zool. 282: 120-126. 
Kolatkar, P. R., M. L. Hackert, and A. F. Riggs. 1994. Structural 

analysis of Urechis canpo hemoglobin. 7. Mol. Biol. 237: 87-97. 
Manguni, C. P. 1992. Respiratory function of the red blood cell hemo- 
globins .i| six animal phyla. Pp. 1 17-149 in Blood and Tissue Oxygen 

Carriers. Adv. Comp. Env. Physiol.. vol. 13. C. P. Mangum. ed. 

Springer-Verlag, Heidelberg. 
Mangum, C. P., J. M. Colacino, and T. L. Vandergon. 1989. Oxygen 

binding of single red blood cells of the annelid bloodworm Glycera 

dihranchiata. 7. Exp. Zool. 249: 144-149. 



Mangum, C. P., J. M. Colacino, and J. P. Grassle. 1992. Red blood 
cell oxygen binding in capitellid polychaetes. Biol. Bull. 182: 129-134. 

Manwell, C. 1959. Oxygen equilibrium of Cuciimaria miniata hemoglo- 
bin and the absence of the Bohr effect. 7. Cell Comp. Phvsiol. 53: 
75-83. 

Manwell, C. 1966. Sea cucumber sibling species: polypeptide chain 
types and oxygen equilibrium of hemoglobin. Science 152: 1393-1395. 

Mauri, F., J. Omnaas, L. Davidson, C. Whitfill, and G. B. Kitto. 1991. 
Amino acid sequence of a globin from the sea cucumber Caudina 
(Molpadia) arenicola. Biochim. Bioph\s. Ada. 1078: 6367. 

McDonald. G. D., L. Davidson, and G. B. Kitto. 1992. Amino acid 
sequence of the coelomic C globin from the sea cucumber Caudina 
(Molpadia) arenicola. 7. Protein Chem. 11: 29-37. 

Mitchell, D. T., G. B. Kitto, and M. L. Hackert. 1995. Structural 
analysis of monomeric hemichrome and dimeric cyanomet hemoglo- 
bins from Caudina arenicola. 7. Mol. Biol. 251: 412-431. 

Mortensen, T. 1920. On hermaphroditism in viviparous ophiuroids. Acta 
Zool. 1: 1-19. 

Powell, M. A., and G. N. Somero. 1986. Adaptations to sulfide by 
hydrothermal vent animals: sites and mechanisms of detoxification and 
metabolism. Biol. Bull. 171: 274-290. 

Riggs, A. 1951. The metamorphosis of hemoglobin in the bullfrog. 
7. Gen. Physiol. 35: 23-44. 

Riggs, A. 1998. Self-association, cooperativity and supercooperativity of 
oxygen binding by hemoglobins. 7. Exp. Biol. 201: 1073-1084. 

Roberts, M. S., R. C. Terwilliger, and N. B. Terwilliger. 1984. Com- 
parison of sea cucumber hemoglobin structures. Comp. Biochem. 
Physiol. 77B: 237-243. 

Rossi-Fanelli, A., and E. Antonini. 1958. Studies on the oxygen and 
carbon monoxide equilibria of human hemoglobin. Arch. Biochem. 
Biophys. 77: 478-492. 

Royer, W. E., W. E. Love, and F. F. Fenderson. 1985. Cooperative 
dimeric and tetramenc clam hemoglobins are novel assemblages of 
myoglobm folds. Nature 316: 277-280. 

Royer, W. E., W. A. Hendrickson, and E. Chiancone. 1990. Structural 
transitions upon ligand binding in a cooperative dimeric hemoglobin. 
Science 249: 518-521. 

Schmidt-Neilsen, K. 1990. Animal Physiology: Adaptation and Envi- 
ronment. Cambridge University Press, Cambridge. 602 pp. 

Scholnick, D. A., and C. P. Mangum. 1991. Sensitivity of hemoglobins 
to intracellular effectors: primitive and derived features. 7. Exp. Zool. 
259: 32-42. 

Somero, G. N., J. J. Childress, and A. E. Anderson. 1989. Transport, 
metabolism, and detoxification of hydrogen sulfide in animals from 
sulride-rich marine environments. Rev. Aquat. Sci. 1: 591-614. 

Steinmeier, R. C., and L. Parkhurst. 1979. Oxygen and carbon mon- 
oxide equilibria and the kinetics of oxygen binding by the cooperative 
dimeric hemoglobin of Thvonella gemmata. Biochemistry 18: 4645- 
4656. 

Stevens, R. D., J. Bonaventura, C. Bonaventura, T. R. Fennel, and 
D. S. Millington. 1994. Application of electrospray ionization mass 
spectrometry for analysis of hemoglobin adducts with acetonitrile. 
Biochem. Soc. Trans. 22: 543-548. 

Su/uki, T. 1989. Amino acid sequences of a major globin from the sea 
cucumber Paracaudina chilen.sis. Biochim. Biophys. Acta 998: 292- 
296. 

Terwilliger, R. C. 1975. Oxygen equilibrium and subunit aggregation of 
a holothurian hemoglobin. Biochim. Biophys. Acta 386: 62-68. 

Terwilliger, R. C., and K. Read. 1972. Hemoglobin of the holothurian 
echinoderm, Molpadia oolilica Pourtales. Comp. Biochem. Physiol. 42: 
65-72. 

Terwilliger, R. C., and N. B. Terwilliger. 1988. Structure and function 
of hololhurian hemoglobins. Pp. 589-595 in Echinoderm Biology: 
Proceedings from the Sixth International Echinoderm Conference, 



HEMOGLOBINS OF A BURROWING BRITTLE STAR 



65 



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

Valentine, J. F. 1991a. Temporal variation in populations of the brittle- 
stars Hemipholis elongaia (Say, 1825) and Microphiopholis alra 
(Stimpson, 1852) (Echinodermata: Ophiuroidea) in eastern Mississippi 
Sound. Bull Mar. Sci. 48: 597-605. 

Valentine, J. F. 1991b. The reproductive periodicity of Microphiopholis 
ami (Stimpson, 1852) and Hemipholis e/ongala (Say, 1825) (Echino- 
dermata: Ophiuroidea) in eastern Mississippi Sound. Ophelia 33: 121- 
129. 

van Assendelft, O. \V. 1970. Speclrophotometry of Hemti\>lobin Deriv- 
atives. Charles C. Thomas, Spnngtield. MA. 152 pp. 

Vandergon, T. L., and J. M. Colacino. 1989. Characterization of he- 
moglobin from Phoronis architects (Phoronida). Comp. Biochem. 
Physiol. B. 94: 31-39. 

Vandergon. T. L., and J. M. Colacino. 1991. Hemoglobin function in 



the lophophorate Phoronis architecta (Phoronida). Physiol. Zoo/. 64: 
1561-1577. 

Weber, R. K. 1980. Functions of invertebrate hemoglobins with some 
special reference to adaptation to environmental hypoxia. Am. Zoo/. 20: 
79-101. 

Weber, R. E., and S. N. Vinogradov. 2001. Nonvertebrate hemoglo- 
bins: functions and molecular adaptations. Physiol. Rev. 81: 569- 
628. 

Wells, R. M. G., and N. W. Pankhurst. 1980. An investigation into the 
formation of sulfide and oxidation compounds from the haemoglobin of 
the lugworm Aharenicola affinis (Ashworth). Comp. Biochem. Physiol. 
66C: 255-259. 

Windom. H. L., and D. R. Kendall. 1979. Accumulation and biotrans- 
formation of mercury in coastal and marine biota. Pp. 303-323 in The 
Biogeochemislry of Mercury in the Environment. J. O. Nriagu, ed. 
Elsevier/North-Holland Biomedical Press, Amsterdam. 



Reference: Biol. Bull. 205: 66-72. (August 2003) 
2003 Marine Biological Laboratory 



Early Development and Acquisition of Zooxanthellae 

in the Temperate Symbiotic Sea Anemone 

Anthopleura ballii (Cocks) 

SIMON K. DAVY 1 '* AND JOHN R. TURNER 2 

' Institute of Marine Studies, University' of Plvinouth, Drake Circus, Plvmouth PL4 8AA, UK; and 

~ School of Ocean Sciences, University of Wales - Bangor, Marine Science Laboratories, 

Menai Bridge, Anglesey LL59 5EY, UK 



Abstract. The ova of Anthopleura ballii become infected 
with zooxanthellae (endosymbiotic dinoflagellates) of ma- 
ternal origin just prior to spawning. After fertilization, the 
zygotes undergo radial, holoblastic cleavage, and then gas- 
trulate by invagination to form ciliated planulae. Because 
the zooxanthellae are localized on one side of the ovum 
and later, within the blastomeres at one end of the embryo 
invagination leads to the zooxanthellae being restricted to 
the planular endoderm and hence to the gastrodermal cells 
of the adult anemone. We propose that maternal inheritance 
of zooxanthellae plays an important part in the success of 
these temperate sea anemones, which live in regions where 
potential sources of zooxanthellae are scarce. 

Introduction 

Associations between marine invertebrates and endosym- 
biotic dinoflagellates (zooxanthellae) are abundant in nutri- 
ent-poor tropical seas, where the zooxanthellae supply pho- 
tosynthetically fixed carbon to their hosts and facilitate the 
conservation and recycling of essential nutrients (Musca- 
tine. 1990; Davies, 1992). These nutritional advantages are 
not immediately obvious in temperate waters, which show 
marked seasonal fluctuations in irradiance, high levels of 
nutrients, and seasonal abundance of planktonic food (Davy 
et al., 1996, 1997; Muller-Parker and Davy, 2001). Indeed, 
associations between invertebrates and zooxanthellae are 
uncommon at temperate latitudes (Turner, 1988; Davy et 
a/.. 1996; Muller-Parker and Davy, 2001). 



Received 7 August 2002; accepted 1 1 May 2003. 

* Author for correspondence and current address: School of Biological 
Sciences, Victoria University of Wellington, PO Box 600, Wellington, 
New Zealand. 



Hosts may acquire zooxanthellae either by maternal in- 
heritance or from the surrounding seawater. Maternal inher- 
itance is probably the rarer mechanism in the tropics. For 
example, while some reef corals inherit their symbionts 
(Lewis, 1974; Kojis and Quinn, 1981; Richmond, 1981; 
Babcock et at., 1986; Glynn et ai, 1991), the vast majority 
of coral species spawn gametes that lack zooxanthellae 
(Babcock et al.. 1986). 

In contrast to tropical symbioses, for temperate symbio- 
ses transmission modes have been identified in only a few 
cases. These include the soft coral Capnella gaboensis, 
which inherits zooxanthellae from the parent colony (Far- 
rant, 1986); the scleractinian coral Astrangia danae. which 
spawns zooxanthella-free gametes (Szmant-Froelich et al., 
1 980); and a small number of sea anemones, the majority of 
whose ova contain algal symbionts (reviewed by Shick, 
1991; Muller-Parker and Davy, 2001). Moreover, the cel- 
lular events leading to the acquisition of zooxanthellae and 
their eventual restriction to the host's endodermal cells have 
been reported for tropical scleractinian corals (Hirose et al., 
2000, 2001), soft corals (Benayahu et al., 1988, 1992; 
Benayahu and Schleyer, 1998), and jellyfish (Montgomery 
and Kremer, 1995), but not for temperate corals or sea 
anemones. 

The sea anemone Anthopleura ballii (Cocks) is locally 
abundant along the southwestern coasts of Europe, where it 
is found from intertidal regions to depths of about 25 m 
(Manuel, 1988; Turner. 1988; Davy et al., 1996, 1997). The 
zooxanthellae harbored by A. ballii belong to the genus 
Symbiodinium (Davy et al., 1997), though they have yet to 
be subjected to molecular characterization. In this study, we 
documented cellular events from gametogenesis through to 
planula development in A. ballii. paying particular attention 



66 



EARLY DEVELOPMENT IN ANTHOPLEURA KALLII 



67 



to the transmission and distribution of zooxanthellae within 
the host's tissues. 

Materials and Methods 

Experimental organisms 

Specimens of the zooxanthellate sea anemone Antho- 
pleura bnllii were collected, from between and 25 in 
depth, in the Lough Hyne Marine Nature Reserve, Eire. 
Anemones were then maintained for up to one year in 30-1 
recirculating seawater tanks at 10-15 C. Irradiance of 96 
/nmol photons m~ 2 s~' was provided on a 12-h light: 12-h 
dark regime, and the anemones were fed twice weekly with 
Anemia sp. nauplii. 

Microscopical examination 

Spawning of A. bnllii, which is dioecious, was induced 
during summer. This was done by exposing anemones to air 
for between 3 and 5 h. The expelled gametes were collected 
by pipette and maintained in 100-ml sterile flasks containing 
artificial seawater at 15 C. Fertilization occurred within 
hours and, every second day. the embryos were pipetted into 
new flasks, which also contained artificial seawater. This 
procedure ensured that the only possible source of zooxan- 
thellae was the adult anemone. 

Gametes, fertilization, and subsequent early development 
were examined by taking samples, first at hourly intervals 
and later once daily, for microscopical observation. A care- 
ful search for zooxanthellae was made, using interference 
contrast microscopy, by optical sectioning at each develop- 
mental stage. A Leitz Dialux 20 microscope with Vario- 
orthomat photographic system was employed, and a photo- 
graphic record of early development was produced. In 
addition, cellular events occurring during gametogenesis 
were documented, again using interference contrast micros- 
copy. This was made possible by anesthetizing anemones in 
equal parts artificial seawater and 7.5% MgCU 6H 2 O for 
12 to 24 h, and then teasing gametes out of the gonads. 

Results 

Gametogenesis and gametes 

Dissection occasionally revealed germ cells in the go- 
nadal tissue on the mesentery. The mesenterial tissue was 
densely packed with zooxanthellae, and the tissues around 
the oocytes contained many zooxanthellae, but the oocytes 
themselves were never observed to contain zooxanthellae 
(Fig. 1A). 

Unfertilized ova, examined by interference phase micros- 
copy immediately upon release from the adult anemone, 
were spherical, yellow-brown, and 300 /xm in diameter 
(Figs. IB, 2A). The surface of each ovum was covered in 
fine translucent cytospines (stiffened bundles of long mac- 



rovilli that are characteristic of actiniarians; Larkman, 
1980), about 23 jum in length. The cytoplasm was hetero- 
geneous, dense and granular, and no nuclei were visible 
under low power in unstained preparations. When the ova 
were optically sectioned by interference contrast micros- 
copy, the zooxanthellae could be observed in the cytoplasm, 
just inside the cell membrane. Moreover, the zooxanthellae 
were concentrated in one hemisphere of the ovum (Fig. IB). 
Out of a total of 380 ova examined, only one aposymbiotic 
ovum was observed. 

Spermatozoa were examined under high power ( X 1000) 
in a live preparation. The head was rectangular, 3.5 /j.m in 
length and 2.3 ;am across, with dense cytoplasm and a large, 
dark nucleus. No basal body was visible. The tail was about 
50 ;u.m long. 

Fertilization 

The gametes were shed into open water, where fertiliza- 
tion occurred. Each released ovum was surrounded by nu- 
merous sperm, which were aggregated between the cyto- 
spines. Fertilization usually occurred within 3.5 h of 
spawning, and unfertilized ova disintegrated after about 7 h. 
liberating their zooxanthellae. Spermatozoa were still active 
at this stage and became inactive after 20-32 h. 

Cleavage 

About 3.5 h after spawning. 2-, 4-. 8-. and 16-cell 
blastulas were observed (4- to 16-cell stages shown in 
Fig. 1C-1E; 2-cell stage not shown). Cleavage was equal, 
radial, complete (i.e.. holoblastic). and rapid, dividing the 
embryo into a ball of cells (blastomeres). Due to the 
initial localization of the zooxanthellae, symbionts were 
distributed unevenly in the blastomeres, being concen- 
trated in those cells at only one end of the embryo. The 
zooxanthellae remained just inside the cell membrane of 
each blastomere. Blastulas of 32 cells were observed 
after about 5.5 h, and blastulas of 64 cells or more were 
apparent after 6 h. The blastomeres became ever smaller 
due to repeated cleavage, and after 8 h. a coeloblastula 
consisting of many cells and one cell layer was formed 
(Figs. IF, 2B). The blastomeres were now 20-30 jim in 
diameter, even in size, and rarely contained more than 
one zooxanthella each (Fig. 1G). The cytospines were 
resorbed and replaced by cilia, which soon began to 
exhibit the characteristic metachronal rhythm that ren- 
dered the coeloblastula motile. 

Gastrulation 

Few gastrulae (Figs. 1H-I. 2C-E) were seen, suggesting 
that this developmental stage is very short. Twenty hours 
after spawning, the motile coeloblastulas began to show a 
slight depression at the pole about which the algae were 



68 



S. K. DAVY AND J. R. TURNER 








i 



H 

Jtef 

G 





B 





* 

-^ 




Figure 1. Larval development and zooxanthella acquisition in the temperate sea 
anemone Anthoplcnni hullii. (A) Developing oocyte. The oocyte contains no zooxanthel- 
lae, which are. however, scattered throughout the surrounding tissues. (B) Released ovum. 
The zooxanthellae are concentrated within the left hemisphere of the ovum, and the ovum 
has cytospines on its surface. (Cl 4-cell hlastula. (D) S-cell blastula. (E) 16-cell blastula. 
(F) Coelohlastula. Zooxanthellae continue to be localized on one side of the embryo. (Gl 
Blastomeres at coeloblastula stage. Many blastomeres contain only a single zooxanthella 
cell, with the remaining blastomeres not being infected at this stage. (H) Mid-gastrula. 
Note the blastopore on the left-hand side. (I) Late gastrula. The blastopore and blastocoel 
are indicated by the clear central region. Zooxanthellae are largely restricted to the 
endoderm, while the ectoderm is largely zooxanthella-free. (J) Cell layers of gastrula, 
showing distribution of zooxanthellae. Most zooxanthellae are in the endoderm, but two 
can be seen in the ectoderm. One of these zooxanthellae (see arrow) appears to be 
degenerating. (K) Early planula. (L) Late planula. Zooxanthellae are distributed along the 
mesenteries. (M) Aposymbiotic mid-planula. Scale bar in A applies to all images except 
G and J and represents about 100 jum; scale bars for G and J represent about 50 /.ini. 



EARLY DEVELOPMENT IN ANTHOPLEURA BAl.l.ll 



69 



cytospine 
cytoplasm 
symbiotic alga 



blastocoel 




tentacle 
rudiment 



mouth 



cilia 



archenteron 




actinopharynx 



coelenteron 



mesentery 



t 



blastopore 

ectoderm 

endoderm 




SOOum 
i 1 

Figure 2. Localization of zooxanthellae during early development of Anthopleura ballii. (A) Ovum. 
showing localization of zooxanthellae. (B) Coeloblastula. (C) Early gastrula, showing invagination. (D) Mid- 
gastrula. showing localization of zooxanthellae in the endoderm. (E) Late gastrula-early planula. (F) Mid- 
planula. showing zooxanthellae distributed along the mesenteries. (G) Late planula, prior to settlement. 



concentrated. Gastrulation by invagination (and perhaps 
epiboly) followed (Figs. 1H. 2C). with the blastomeres 
aggregated, at first, around the blastopore. Gastrulation led 
to the formation of an embryo with two cell layers encom- 
passing a central cavity the archenteron (Figs. II, 2D). 
During gastrulation, almost all of the blastomeres contain- 
ing zooxanthellae moved into the endoderm from around 
the blastopore region. Only very occasionally were zooxan- 



thellae seen in the ectoderm, and many of these cells ap- 
peared to disintegrate (Fig. 1J). 

Planulation 

After 27 h, most embryos had become late gastrulae or 
early planulae (Figs. IK, 2E). By this stage, the developing 
larvae had shown no growth, remaining about 300 ^im in 



70 



S. K. DAVY AND J. R. TURNER 



diameter. However, after 2 days, most planulae began to 
elongate along their vertical axis, tapering slightly towards 
the posterior end. The zeoxanthellae were clearly visible, 
aggregated in striatinn- i -ining the length of the endoderm. 
The surface of ea<. ^ arva was completely ciliated, and an 
apical tuft of longer cilia was visible. After 3 days, the 
larvae began !; exhibit signs of differentiation (Figs. 1L, 
2F), with (he development of nematocysts, and a ciliated 
actinopharynx, which replaced the blastopore. Between 3 
and 5 days, the planulae began to grow to about 400-600 
/urn in length and 300 /j,m in diameter, even though they 
were not fed. The number of zooxanthellae also increased 
(not quantified), and dividing zooxanthellae were seen fre- 
quently. As the mesenteries developed, it became clear that 
most zooxanthellae were located along these structures. 
Interestingly, only one aposymbiotic planula was observed 
throughout the course of this work (Fig. 1M), which is 
consistent with the absence of aposymbiotic A. ballii at the 
field site (Lough Hyne). Tentacle rudiments were seen very 
occasionally in some planulae (Fig. 2G). Although care was 
taken to isolate the surviving planulae, they could not be 
kept alive for more than 7 days and so settlement was not 
observed. 

Discussion 

Gametogenesis, spawning, and early development in An- 
thopleura ballii follows the pattern exemplified by many 
anemone species (Siebert, 1973; Chia, 1976; Jennison, 
1979, 1981 ). All these species are dioecious, shedding their 
gametes into open water where fertilization occurs. The 
zygote then undergoes radial, holoblastic cleavage and 
forms a hollow coeloblastula. Gastrulation follows by in- 
vagination to form a ciliated, pelagic planula larva. This 
mode of development is notably different from that shown 
by the larger, yolky, meroblastic ova of the anemones 
Tealia crassicornis (Chia and Spaulding, 1972) and Cri- 
brinopsis femaldi (Siebert and Spaulding, 1976), in which 
cleavage is incomplete, unequal, and relatively slow. The 
sequence and timing of events in A. ballii were very similar 
to those described for the temperate zooxanthellate or zoo- 
chlorellate anemones Anthopleura elegantissima and An- 
thopleura xanthogrammica (Siebert, 1973). However, un- 
like these anemones, A. ballii spawned ova that contained 
zooxanthellae. In A. ballii, concentration of the zooxanthel- 
lae in one hemisphere of the ovum, and invagination (and 
perhaps epiboly) during the gastrula stage, led to the local- 
ization of symbionts within the host's endoderm; this same 
process occurs in the temperate anemone Anemonia viridis 
(Turner, 1988). 

Gametogenesis and zoo.\anthella acquisition 

In A. ballii. the endodermal tissue surrounding the devel- 
oping oocytes was heavily laden with zooxanthellae, though 



infected oocytes were never observed. In contrast, spawned 
ova almost always harbored zooxanthellae, indicating that 
infection must occur at, or just prior to, release. We could 
not ascertain whether infection occurs in the gonadal tissue 
or after the ova have been released into the coelenteron. But, 
as the anemones were kept in artificial (and so zooxanthella- 
free) seawater in sterile flasks, and as spawning occurred in 
air, we can be certain that the zooxanthellae were of ma- 
ternal origin, and that infection occurs prior to release into 
the surrounding seawater and hence prior to fertilization. 

While the mode of zooxanthella acquisition has been 
determined in relatively few species of cnidarians, early 
indications are that infection prior to fertilization is quite 
uncommon. For example, the vast majority of scleractinian 
corals investigated do not harbor zooxanthellae in their eggs 
(Szmant-Froelich et ai. 1980; Babcock el al, 1986; Harri- 
son and Wallace, 1990), though some species of Pocillo- 
pora and Montipora do release zooxanthellate ova (Bab- 
cock et al., 1986; Harrison and Wallace, 1990; Glynn el al., 
1991; Himseetai, 2001). Of note, the eggs of the hard coral 
Montipora digitata become infected just 24 h prior to spawn- 
ing (Harrison and Wallace, 1990), suggesting that the delayed 
infection seen in A. ballii eggs also occurs in some other hosts. 
Furthermore, in brooding species like the soft corals Xenia 
itmbellata and Anthelia glauca. where zooxanthellae are trans- 
mitted maternally, infection does not occur until the later 
stages of embryogenesis or larval development (Benayahu et 
al., 1988; Benayahu and Schleyer. 1998). 

The mechanism of entry into the ovum is unknown, but 
may well be similar to that described for the soft coral 
Litophyton arboreum (Benayahu et al.. 1992). In L ar- 
boreum, zooxanthellae pass through gaps in the mesogloeal 
covering of the oocytes, accumulate in the perioocytic zone, 
and ultimately bulge through the oolema and enter the 
mature oocyte. A similar "phagocytosis" of algal symbionts 
has been reported for the oocytes of several scleractinian 
corals (Hirose et al.. 2001), as well as for the freshwater 
Hydra viridissima (Campbell, 1990). 

Spawning, early development, and the localization 
of zooxanthellae 

The sperm of A. ballii are similar to those of other 
Anthopleura spp. (Siebert, 1973). Moreover, as in other 
symbiotic Anthozoa, the heads are too small (3.5 X 2.3 /urn) 
to act as vectors for paternal transmission of zooxanthellae; 
zooxanthellae in A. ballii are about 10 /urn in diameter 
(Turner, 1988; Davy et ai. 1996). 

During the early stages of development, and throughout 
cleavage, the zooxanthellae remain localized at one end of 
the embryo. By the time a coeloblastula forms, most zoo- 
xanthellae are located in individual blastomeres, at one end 
of the coeloblastula. That this positioning is of paramount 
importance for the ultimate localization of the zooxanthellae 



EARLY DEVELOPMENT IN ANTHOPLEURA BALl.ll 



71 



becomes evident during gastrulation, when zooxanthellae 
are situated within invaginating blastomeres and so become 
localized within the endoderm. Indeed, the mechanism is so 
successful that "stray" zooxanthellae, which end up in the 
ectoderm, are rare (Fig. 1J). 

The initial localization of zooxanthellae seen here is 
similar to that seen in the corals Pocillopora vernicosa and 
P. eydou.\i (Hirose et ai, 2000). However, as in some other 
coral species (Szmant-Froelich et al., 1980, 1985). gastru- 
lation in P. vernicosa and P. eydouxi occurs via delamina- 
tion rather than imagination. This means that, in marked 
contrast to events observed in A. ballii. blastomeres con- 
taining zooxanthellae move into the blastocoel of develop- 
ing embryos, eventually filling the space and forming a 
stereogastrula (Hirose et al.. 2000). The precise mechanism 
by which the zooxanthellae move into the blastocoel is 
unknown. 

Planulation 

As stated above, the position of zooxanthellae in the 
embryo, and the subsequent localization of zooxanthellae in 
the endodermis by invagination, means that "stray" zoo- 
xanthellae in the epidermal cells of planulae are very rare. A 
similar paucity of stray zooxanthellae was also reported for 
the reef corals Pocillopora vernicosa and P. eydouxi (Hi- 
rose et al., 2000). However, the planulae of some sclerac- 
tinian corals (Szmant-Froelich et ai, 1985; Schwarz et al., 
1999), soft corals (Farrant, 1986; Benayahu et al., 1988, 
1992; Benayahu and Schleyer, 1998), and jellyfish (Mont- 
gomery and Kremer. 1995) may contain zooxanthellae in 
their epidermal cells more frequently. In these cases, the 
zooxanthellae infect either the planulae or, as in the jellyfish 
Linuche unguiculata, both the embryos and planulae (Mont- 
gomery and Kremer, 1995), as opposed to the gametes. The 
zooxanthellae may then be transferred to the endodermal 
tissue via cell migration (Montgomery and Kremer, 1995) 
or trans-mesogloeal passages (Benayahu, 1997; Benayahu 
and Schleyer, 1998). Alternatively, stray zooxanthellae may 
degrade in the host or be expelled as a result of being 
harbored by an inappropriate cell type. Degrading zooxan- 
thellae have been observed in planulae of the scleractinian 
corals Stylophora pistillata. Sehatopora caliendrum, and 
Pocillopora verrucosa, though always in the endodermis, 
rather than the epidermis (Titlyanov et al., 1998). 

Mode of transmission as a function of latitude 

Symbiotic invertebrates are abundant in tropical seas and 
regularly release zooxanthellae into the surrounding seawa- 
ter (Hoegh-Guldberg et al., 1987); viable zooxanthellae are 
also released in the feces of numerous corallivorous preda- 
tors (Muller-Parker, 1984). This may result in low selective 
pressure for the evolution of maternal inheritance in tropical 
regions, as zooxanthellae are readily available from exoge- 



nous sources to infect potential hosts (Buddemeier and 
Fautin, 1993; Kinzie et al., 2001). 

In contrast, while transmission mechanisms have been 
investigated in relatively few species of zooxanthellate in- 
vertebrate, initial observations (including those presented 
here) suggest that maternal transmission of zooxanthellae is 
more likely to occur in temperate regions than in the tropics 
(reviewed by Muller-Parker and Davy, 2001). A predomi- 
nance of maternal transmission mechanisms at high lati- 
tudes would not be surprising, as it could be related to a 
scarcity of exogenous sources of zooxanthellae and, there- 
fore, selection against hosts that acquire their symbionts 
from exogenous supplies (Muller-Parker and Davy, 2001). 
Indeed, a scarcity of sources of zooxanthellae could explain 
why the temperate coral Astrangia danae, which does not 
acquire its zooxanthellae maternally, is sometimes found 
devoid of these symbionts (Szmant-Froelich et al., 1980). In 
addition, maternal transmission, combined with the ability 
of temperate algal-invertebrate symbioses to tolerate a wide 
range of environmental variables (Kevin and Hudson, 1979; 
Squire, 2000; Howe and Marshall, 2001), could explain the 
persistence of zooxanthellate organisms at high latitudes 
(Davy et al.. 1997; Muller-Parker and Davy, 2001). More 
analyses of zooxanthellar transmission mechanisms at dif- 
ferent latitudes, and of the ecological advantages conveyed 
by symbioses in nutrient-rich temperate waters, will help 
resolve this matter. 

Acknowledgments 

This work was funded by a NERC award to JRT and was 
conducted, in part, at the University of Oxford. JRT thanks 
Professor Sir David Smith FRS FRSE for support. SKD 
thanks the Institute of Marine Studies, University of Ply- 
mouth for financial assistance. 

Literature Cited 

Babcock, R. C., G. D. Bull, P. L. Harrison, A. J. Hey ward, J. K. Oliver, 
C. C. Wallace, and B. L. Willis. 1986. Synchronous spawnings of 
105 scleractinian coral species on the Great Barrier Reef. Mar. Binl. 90: 
379_394. 

Benayahu. Y. 1997. Developmental episodes in reef soft corals: ecolog- 
ical and cellular determinants. Proc. fi' 1 ' Int. Coral Reef Svmri. 2: 
1213-1218. 

Benayahu, Y., and M. H. Schleyer. 1998. Reproduction in Anilie/ia 
glauca (Octocorallia: Xeniidae). II. Transmission of algal symbionls 
during planular brooding. Mar. Biol. 131: 433-442. 

Benayahu, Y., Y. Achituv, and T. Berner. 1988. Embryogenesis and 
acquisition of algal symbionts by planulae of Xenia umbellata (Octo- 
corallia: Alcyonacea). Mar. Binl. 100: 93-101. 

Benayahu, Y., D. Weil, and Z. Malik. 1992. Entry of algal symbionts 
into oocytes of the coral Litophwon urborewn. Tissue Ceil 24: 473- 
482. 

Buddemeier, R. W., and D. G. Fautin, 1993. Coral bleaching as an 
adaptive mechanism: a testable hypothesis. Bioscience 43: 320-325. 

Campbell, R. D. 1990. Transmission of symbiotic algae through sexual 



72 



S. K. DAVY AND J. R. TURNER 



reproduction in hydra: movement of algae into the oocyte. Tissue Cell 

22: 137-147. 

Chia, F.-S. 1976. Sea anemone reproduction: patterns and adaptive ra- 
diations. Pp. 261-270 in C.'fif'tieraie Ecology and Behaviour, D.D. 

Mackie. ed. Plenum Press. I. ondon. 
Chia, F.-S., and .1. ' ./;u.Iding. 1972. Development and juvenile 

growth of the sea ; .rmone, Tealia crassicornis. Bio/. Bull. 142: 206- 

218. 
Davies, P. S. 1992. Endosymbiosis in marine cmdarians. Pp. 51 1-540 in 

Plant-Animal Interactions in the Marine Benthos, D. M John. S. J. 

Hawkins, and J. H. Price, eds. Clarendon Press. Oxford. 
Davy, S. K., I. A. N. Lucas, and J. R. Turner. 1996. Carbon budgets in 

temperate anthozoan-dinoflagellate symbioses. Mar. Biol. 126: 773- 

783. 
Davy, S. K., J. R. Turner, and I. A. N. Lucas. 1997. The nature of 

temperate anthozoan-dinoflagellate symbioses. Proc. Slh Int. Coral 

ReefSymp. 2: 1307-1312. 

Farrant, P. A. 1986. Gonad development and the planulae of the tem- 
perate Australian soft coral Capne/la gaboensis. Mar. Biol. 92: 381- 

392. 
Glynn, P. W., N. J. Gassman. C. M. Eakin, J. Cortes, D. B. Smith, and 

H. M. Guzman. 1991. Reef coral reproduction in the eastern Pacific: 

Costa Rica. Panama, and Galapagos Islands (Ecuador). I. Pocillopori- 

dae. Mar. Biol. 109: 355-368. 
Harrison, P. L., and C. C. Wallace. 1990. Reproduction, dispersal and 

recruitment of scleractinian corals. Pp. 133-207 in Ecosystems of the 

World: Coral Reefs. Vol. 25. Z. Dubinsky, ed. Elsevier, Amsterdam. 
Hirose, M., R. A. Kinzie III, and M. Hidaka. 2000. Early development 

of zooxanthella-containing eggs of the corals Pocillopora verrucosa 

and P. eydouxi with special reference to the distribution of zooxanthel- 

lae. Biol. Bull 199: 68-75. 
Hirose, M., R. A. Kinzie III, and M. Hidaka. 2001. Timing and process 

of entry of zooxanthellae into oocytes of hermatypic corals. Coral 

Reefs 20: 273-280. 
Hoegh-Guldberg, O., L. R. McCloskey, and L. Muscatine. 1987. Ex 

pulsion of zooxanthellae by symbiotic cnidarians from the Red Sea. 

Coral Reefs 5: 201-204. 
Howe, S. A., and A. T. Marshall. 2001. Thermal compensation of 

metabolism in the temperate coral. Plesiastrea versipora (Lamarck, 

1816). J. Exp. Mar. Biol. Ecol. 259: 231-248. 
Jennison, B. L. 1979. Gametogenesis and reproductive cycles in the sea 

anemone Anthopleura elegantissima (Brandt, 1835). Can. J. tool. 57: 

403-411. 
Jennison. B. L. 1981. Reproduction in three species of sea anemones 

from Key West, Florida. Can. J. Zooi 59: 1708-1719. 
Kevin, K. M., and R. C. L. Hudson. 1979. The role of zooxanthellae in 

the hermatypic coral Plesiastrea versipora (Milne Edwards and Haime) 

from cold waters. J. Exp. Mar. Biol. Ecol. 36: 157-170. 
Kinzie, R. A. Ill, M. Takayama, S. R. Santos, and M. A. Coffroth. 



2001. The adaptive bleaching hypothesis: experimental tests of crit- 
ical assumptions. Biol. Bull. 200: 51-58. 

Kojis, B. L., and N. J. Quinn. 1981. Reproductive strategies in four 
species of Porites (Scleractinia). Proc. 4th Int. Coral Reef Symp. 2: 
145-152. 

Larkman, A. U. 1980. Ultrastructural aspects of gametogenesis in Ac- 
tinia equina. Pp. 61-66 in Development and Cellular Biology of 
Coelenterates, P. Tardent and R. Tardent, eds. Elsevier, Amsterdam. 

Lewis, J. B. 1974. The settlement behaviour of planulae larvae of the 
hermatypic coral Favia fragum (Esper). J. Exp. Mar. Biol. Ecol. 15: 
165-172. 

Manuel, R. L. 1988. British Antho-oa. Academic Press, London. 

Montgomery, M. K., and P. M. Kremer. 1995. Transmission of sym- 
biotic dinoflagellates through the sexual cycle of the host scyphozoan 
Liiniche unguiculata. Mar. Biol. 124: 147-155. 

Muller-Parker, G. 1984. Dispersal of zooxanthellae on coral reefs by 
predators on cnidarians. Biol. Bull. 167: 159-167. 

Muller-Parker, G., and S. K. Davy. 2001. Temperate and tropical 
algal-sea anemone symbioses. Invertebr. Biol. 120: 104-123. 

Muscatine, L. 1990. The role of symbiotic algae in carbon and energy 
flux in reef corals. Pp. 75- 87 in Ecosystems of the World: Coral Reefs. 
Vol. 25, Z. Dubinsky, ed. Elsevier, Amsterdam. 

Richmond, R. 1981. Energetic considerations in the dispersal of Pocil- 
lopora damicomis (Linnaeus) planulae. Proc. 4th Int. Coral ReefSymp. 
2: 153-156. 

Schwarz, J. A., D. A. Krupp. and V. M. Weis. 1999. Late larval 
development and onset of symbiosis in the scleractinian coral Fungia 
scutariu. Biol. Bull. 196: 70-79. 

Shick. J. M. 1991. A Functional Biology of Sea Anemones. Chapman and 
Hall. New York. 

Siebert, A. E. 1973. A description of the embryology, larval develop- 
ment, and feeding of the sea anemones Anthop/eura elegantissima and 
A. xanthogrammica. Can. J. Zool. 52: 1383-1388. 

Siebert, A. E., and J. G. Spaulding. 1976. The taxonomy, development 
and brooding behavior of the anemone, Cribrinopsis ferna/di sp. nov. 
Biol. Bull. 150: 128-138. 

Squire, L. R. 2000. Natural variations in the zooxanthellae of temperate 
symbiotic Anthozoa. Ph.D. thesis. University of Wales. Bangor. 

Szmant-Froelich, A., P. Yevich, and M. E. Q. Pilson. 1980. Gameto- 
genesis and early development of the temperate coral Astrangia danae 
(Anthozoa: Scleractinia). Biol Bull. 158: 257-269. 

Szmant-Froelich, A., M. Reutter, and L. Riggs. 1985. Sexual repro- 
duction of Favia fragum (Esper): lunar patterns of gametogenesis, 
embryogenesis and planulation in Puerto Rico. Bull. Mar. Sci. 37: 
880-892. 

Titlyanov, E. A.. T. V. Titlyanova, Y. Loya, and K. Yamazato. 1998. 
Degradation and proliferation of zooxanthellae in pianulae of the 
hermatypic coral Stylophora pistil/ata. Mar. Biol. 130: 471-477. 

Turner, J. R. 1988. The ecology of temperate symbiotic Anthozoa. D. 
Phil, thesis. University of Oxford. 



Reference: Bio/. Bull. 205: 73-82. (August 2003) 
2003 Marine Biological Laboratory 



On the Growth of Bivalve Gills Initiated From a 
Lobule-Producing Budding Zone 

DIETRICH NEUMANN* AND HEIKE KAPPES 
Zoological Institute, University of Cologne. D-50923 Koln, Germany 



Abstract. The growth of bivalve gills proceeds at the 
posterior end of the gill from a meristem-like budding zone, 
that is, an undifferentiated terminal organ, which continu- 
ously proliferates new gill elements in growing bivalves. In 
representatives of protobranch, filibranch, and eulamelli- 
branch gills (13 species from Protobranchia, Pteriomorphia, 
Palaeoheterodonta, and Heterodonta), the first growth steps 
demonstrate a uniform basic pattern. The budding zone 
produces either transverse folds that split after a transition 
zone into parallel pairs of lobules (which themselves later 
differentiate into the inner and outer demibranchs), or it 
produces the lobules directly, without first forming a tran- 
sition zone. The lobules elongate, differentiate into lobes, 
and transform into leaflet-like structures (protobranchs) or 
into filaments (filibranchs and eulamellibranchs). The fila- 
ments represent the differentiated outer margins of each 
lobe, of which the central tissue (interlamellar septum) 
becomes incised or fenestrated, or transformed by tissue 
junctions. A distally located main growth zone for each lobe 
is suggested. With regard to the delayed onset of the dif- 
ferentiation of the outer demibranch in juvenile unionids. an 
additional temporary growth zone for filaments is suggested 
to exist at the anterior end of the outer demibranch. 

Introduction 

Bivalve gills are unique organs that show a continuous 
terminal growth by adding new elements in correlation with 
the lifelong increase in shell size. The gills consist of two 
plate-like demibranchs that are extended anterior-posteri- 
orly on each side of the visceral mass (the only exception: 
Lucinidae with only one demibranch; Ridewood, 1903). In 
the case of the phylogenetically primitive protobranch gill, 
the demibranchs are comparatively small and consist of a 



Received 29 August 2002; accepted 30 April 2003. 
* To whom correspondence should be addressed. E-mail: dietrich. 
neumann@uni-koeln.de 



series of ciliated leaf-like discs. In filibranch gills, the 
demibranchs are considerably longer and consist of ex- 
tended parallel structures the filaments rather than par- 
allel disks. The filament structure also appears on the sur- 
face of the demibranch in eulamellibranch gills; however, 
their demibranchs are much more complex organs, because 
the filaments are connected by various tissue junctions (see 
Ridewood. 1903). 

These gills all share two functional elements: a peripheral 
ciliary pump that creates a flow of oxygenated water over 
and through the demibranchs, and an internal circulatory 
system that carries the oxygenated hemolymph to the heart. 
During the evolution of filibranch and eulamellibranch bi- 
valves, the size of the gills increased in relation to body 
mass and mantle cavity, and two additional functions 
evolved: feeding on inhaled particles, facilitated by mucous 
secretion and followed by food-string transport along food 
grooves; and in some taxa, brood care within the interla- 
mellar spaces (for reviews, see Purchon. 1968; Bayne et a/.. 
1976; Morton, 1996). 

Bivalve gills develop new elements from their posterior 
end as they grow (Wasserloos, 1911; Ansell, 1962; Kor- 
niushin. 1997). In the past, however, studies on gill growth 
processes focused only on the early organogenesis of the 
gill during the postlarval development (for review of the 
older literature, see Raven, 1966). In all subclasses except 
Protobranchia, these early stages start with a short row of 
unidirectional slender filaments of only the inner demi- 
branch (Jackson, 1890; Drew. 1901; Wasserloos, 1911; 
Ansell, 1962; Waller. 1981; Gros et ai, 1998; Chaparro et 
al.. 2001). The row of filaments extends in anterior-poste- 
rior sequence. Detailed studies of postlarval stages of the 
eulamellibranch Veneridae revealed that the unidirectional 
filaments first display knob-like thickenings at their distal 
ends and then transform to widened, roughly V-shaped 
filaments; no bending or reflexion was involved in this 
process (Ansell, 1962; Moueza et al.. 1999). At the end of 



73 



74 



D. NEUMANN AND H. KAPPES 



this postlarval development, the new filaments of the inner 
demibranch arise from the posterior end of the gill base in 
the form of V-shaped elc; . nts (Ansell, 1962). 

In contrast to these .opmental results on postlarvae 
and early juveniles ^tudy is focused on the continual 
growth of the i\'<" .ntiated bivalve gill from its posterior 
growth zone, > .re new filaments are added. Bivalves of 
different subclasses were examined. For juvenile unionids, 
we further describe the beginning of the outer demibranch, 
which lags behind the early formation of the inner demi- 
branch. The results offer new insights into the increase in 
filament number, the differentiation of the filaments, and 
bivalve gill growth in general. 



Materials and Methods 



Material 



To examine gill development in bivalves possessing pro- 
tobranch gills, we studied three Nucula species (subclass 
Protobranchia) preserved in ethanol (Museum Senckenberg. 
Frankfurt a.M, FRG): Nucula nucleus Linnaeus (Helgoland 
ex Wolf/2, Coll.-No. SMF 320968/2). N. sulcata Bronn 
(Me5/51 Ku/3. Coll.-No. SMF 320966/3). and N. tennis 
(Montagu) (Gauss-St. 101 Ku/6. Coll.-No. SMF 320967/6). 

As examples of filibranch gills, we examined species 
belonging to the subclass Pteriomorphia: Mytilus ednlis 
Linnaeus (sampled at Gromitz on the shore of the Baltic 
Sea, Schleswig-Holstein, Germany), M. galloprovincialis 
Lamarck (Ria de Vigo, Galicia, Spain), and Anadara sp. 
(Kakinada Bay, Andhra Pradesh, India; soft body directly 
preserved in Bouin's fluid). 

Eulamellibranch gills from the subclasses Palaeoheter- 
odonta [Unio pictorum (Linnaeus), U. tumidus (Philipsson)] 
and Heterodonta [Dreissena polymorpha (Pallas), Cor- 
biculci fluminea (O.F. Miiller), and Pisidium casertanum 
(Poli)] were studied. Corbiciilu was collected from the 
Rhine River near Cologne (Rh.-km 683); the other species 
from waters of the flood plain of the Lower Rhine (Haf- 
fensche Landwehr near Rees, Rh.-km 840). We also in- 
spected Mya arenaria (L.) from Gromitz and Venempis 
decussatu (L. ) from Ria de Vigo. 



Scanning electron microscopy 

Fresh gills (posterior sections) of U. pictorum. C. flumi- 
nea. D. polymorpha, and P. casertanum were fixed in 2% 
glutaraldehyde in 0.133 mol phosphate buffer (pH 7.2) for 2 
h. This material, as well as prefixed gills from Anadara sp., 
N. sulcata. and./V. tennis, were then dehydrated in ethanol. 
After two rinses in pure acetone for 2 h each, the gills were 
stored overnight in pure acetone, then dried with COS, 
mounted, and sputte r ed (ca. 140-nm gold layer). 



Histology of subtidult Unio gills 

For histological analysis of the budding zone, two spec- 
imens of U. pictorum (shell lengths 4.85 mm and 20.1 mm) 
were fixed in Bouin-Allen's fluid (2 h, 37 C). After rinsing 
in 70% ethanol followed by standard dehydration, the tis- 
sues were embedded via Rotihistol (15 h) and Rotihistol- 
Rotiplast 1:1 ( 1.5 h at 61 C) in Rotiplast (paraffin, melting 
point 58 C). Serial 10-/u,m microtome sections were stained 
with Domagk's stain (Romeis, 1968). 

Dissection of juvenile gills 

Early juvenile eulamellibranchs possess only slender fil- 
aments of the inner demibranch. We estimated the shell size 
at which the outer demibranchs first developed. Specimens 
of U. pictorum and U. tumidus (shell length between 3.5 and 
16.95 mm) preserved in ethanol were dissected for this 
purpose. The gills were placed on slides (after dehydration 
in ethanol) and embedded in Rotihistokitt (Roth, Karlsruhe- 
FRG). The longest filament of each demibranch was then 
measured from its dorsal base to its ventral tip with an 
image analyzing system attached to a CCD-camera linked to 
a Leitz microscope. With a pointer on the monitor, the 
length could be measured to the nearest 0.01 mm. Linear 
regression lines were calculated using SPSS 7.5 and Stat- 
graphics 4.0. 

Terms used for gill structures 

Various anatomical terms have been used for the descrip- 
tion of bivalve gills (Mitsukuri, 1881; Ridewood, 1903; 
Yonge, 1947; Kilias, 1956; Beninger et al.. 1988; and 
others). To avoid terminological confusion, we summarize 
most of these terms and mark (by single quotation marks 
and italics) those that we will use in this study. In most 
aspects we follow Ridewood (1903). However, with regard 
to the posterior growth zone of the gill, we will introduce 
new terms. 

When juvenile filibranch and eulamellibranch bivalves 
have passed the early period of gill differentiation, they 
possess two 'demibranchs' (also gill plates) in an anterior- 
posterior extension on each side of the foot. These two 
demibranchs, that is, the 'inner and 'outer' ones (Fig. Ib, 
right side: id and od), are attached by a 'gill base' (also gill 
axis, gill root) on the dorsal side of the 'mantle cavity' (also 
pallial cavity) between the visceral mass and the mantle. 
Each dorsoventrally lengthened demibranch is formed into 
two 'lamella' -like structures (also membrane plates, leaves) 
consisting of vertically ciliated 'filaments' in parallel (also 
gill bars, ciliated discs) (Fig. la). The terms 'descending 
limb' of the filament (also descending portion of the fila- 
ment, i.e.. that part of the filament connected to the gill 
base) and 'ascending limb' (the other part of the filament 
dorsally unattached or fused with foot or mantle) will be 
circumvented as far as possible because of developmental 



ON THE GROWTH OF BIVALVE GILLS 



75 





m 



Figure 1. (a) Schematic view of shell shape and the relative position 
of the two adductor muscles (aa: anterior adductor, pa: posterior adductor) 
and the gill in Unio tumidus. The budding zone (bz) of the successively 
increasing number of filaments (fi) is located at the posterior end. The 
filaments lengthen during shell growth, (b) Schematic view of a transverse 
section showing the age-dependent difference in gill organization between 
juvenile unionids of shell length 3.5 mm (left half) and 8.1 mm (right half), 
id: inner demibranch, showing early differentiation and later differentiation 
on the left and right sides, respectively (in most parts of the gill, not yet 
attached to the foot), od: outer demibranch, gb: gill base, ilj: interlamellar 
junction, ils: interlamellar space, m: mantle, ft: foot. 



and functional connotations. 'Interlamellar junctions' may 
stabilize the elongated filibranch filaments, and adjacent 
filaments are held together by 'ciliated knobs' (also ciliated 
discs), which are arranged dorsoventrally, and more or less 
equidistantly (Fig. 2). Eulamellibranchs possess two types 
of tissue bridges: 'interlamellar junctions' (also septa) be- 
tween the descending and ascending limbs of the filaments, 
and 'interfilamentar junctions' between adjacent filaments. 
The variety of tissue junctions increases the complexity of 
the branchial architecture, with 'interlamellar spaces' or 
'gaps' (also suprabranchial chambers, vertical water tubes, 
interlamellar cavity) and 'interfilamentar pores' (also ostia, 



slits) through which the inhaled water passes. The filaments 
of each demibranch are strengthened by skeletal rods and 
are joined at their ventrodistal margins, thus forming the 
ciliated 'food groove' (also marginal groove). As the central 
structures of the lamellae increase in complexity, two gen- 
eral types of gills homorhabdic and heterorhabdic be- 
come evident in different species. Homorhabdic gills con- 
tain only 'ordinary filaments' , whereas heterorhabdic gills 
contain both 'ordinary' and 'principal' filaments (Ride- 
wood, 1903). 

Gills of protobranch bivalves are smaller, restricted to the 
posterior part of the mantle cavity, and characterized by a 
simple anatomy. However, the gross design is the same as 
that of the filibranch and eulamellibranch types, that is, two 
demibranchs on each side. Each consists of a series of 
extended leaflets (also discs), ciliated and attached to each 
other by ciliated knobs. 

Results 

Budding zones of protobranch and filibranch gills 

On the basis of our material, the posterior growth zone of 
these two gill types can be demonstrated best in the fili- 
branch gill of Anadara (Pteriomorphia) (Fig. 2a). In this 
species, the posterior part of the gill base ends in a small, 
rounded projection of undifferentiated cells, from which the 
separation of new filaments starts (Fig. 2b). We name this 
meristem-like cell complex the 'budding zone' . 

As was observed in all dissections, the budding zone of 
Anadara is not attached to the mantle but projects into the 
mantle cavity. The budding zone of the specimen presented 
(Fig. 2b) is already marked on its ventral side by a fine 
medial line. This is the onset of the deep longitudinal groove 
that separates the inner and outer demibranchs. The second 
step of early differentiation is the appearance of transverse 
folds which form undifferentiated 'lobules' of the inner and 
the outer demibranchs in a characteristic 1 : 1 relationship. 

We were able to confirm the Ill-ratio of demibranch 
lobules in the filibranch mussel Mytilus (not shown). How- 
ever, the undifferentiated budding zone of Mytilus is fol- 
lowed by a 'transition zone' characterized by a number of 
transverse folds that have not yet split medially into the 
lobules of the two demibranchs (as in Unio, compare Fig. 
4a). The length of the transition zone differed among spec- 
imens. In M. galloprovincialis from Vigo (/; = 12), small 
specimens (0.5-0.7 cm) and larger ones (1.0-3.3 cm) re- 
vealed 4-6 and 8-12 transverse folds, respectively (excep- 
tion: one 3.7-cm specimen with only four folds). In M. 
edulis (n = 7) from Gromitz, the length of the transition 
zone had no relation to shell length (2 folds in specimens of 
1.7 and 2.1 cm shell length. 5 folds for sizes of 1.8 and 2.1 
cm. 7 for a 1.2-cm specimen, 9 and 10 folds for sizes of 3.2 
cm and 2.0 cm, respectively). It is possible that such vari- 
ations are correlated with different rates of gill increase. 

Similar to Anadara. in Nucula tennis (Protobranchia) the 



76 



D. NEUMANN AND H. KAPPES 




Tr J^ IkJ -r-R >-- : 

i?4 JhW'i ' * ; ^ v 

'^r^*/i . I .'^,' 

I 14 : >; i 



tiny budding zone of the gill is represented by the posterior 
apex of the gill base and has no contact to the mantle (Fig. 
2c). The same was observed in dissected specimens of the 
other two Nucula species. In Nuculu, both the transverse 
folds and the separation of inner and outer demibranchs 
occur simultaneously. Thus, the lobules appear from the 
very beginning. 

Lobule differentiation in protohranchs and filibranchs 

In the protobranch Nucula. the lobules of the demi- 
branchs extend laterally and dorsoventrally and form the 
leaf-like lobes. Part of the margin of each lobe becomes 
thickened and ciliated, whereas the expanded inner portion 
of the lobe its 'lamina' (also interlamellar septum) re- 
mains unchanged. These are then the differentiated leaflets. 

The lobules in the filibranchs Anadara and Mytilus 
mainly increase dorsoventrally and form elongated lobes. 
Their margins differentiate into descending and ascending 
limbs of the filament, as already described for Arcidae and 
Mytilidae (Ridewood, 1903). Simultaneously, the lamina of 
each lobe becomes transformed. In Anadara, a gap (inter- 
lamellar space) occurs within the lamina and separates the 
filament's margins (Fig. 3a). The length of the gap may be 
as little as 50% or as much as 90% of the filament's length. 
The outer margins of the filament now resemble descending 
and ascending limbs. Adjacent filaments are attached to 
each other by nearly equidistant ciliated knobs that are 
arranged in vertical rows, one on either side of the two 
ciliated margins. Lateral views of the lamellae reveal that 
the equidistant knobs on adjacent filaments are aligned, and 
that the number of equidistant lines of knobs increases as 
the filaments elongate. It was obvious that, during elonga- 
tion, new lines of knobs appeared stepwise near the fila- 
ment's distal end. Thus, a main growth zone of each fila- 
ment must be localized in this distal area (Fig. 3a). 
However, it can also be seen that a new line of knobs 
becomes inserted in a few areas along the length of the 
lamellae after the distance between two rows has increased 
(not shown). This fact suggests that, in Anadara gills, some 
incremental elongation of the filaments also occurs all along 
the dorsoventral length of the lamina. 

In Mytilus, the lobules occur after the medial splitting of 
the transition zone into inner and outer demibranchs (see 
above). Each lobule elongates and forms a lobe. Then, its 
lamina becomes transformed. A few interlamellar junctions 



Figure 2. Scanning electron micrographs of the differentiating demi- 
branchs in the filibranch Anailara sp. {Asp; full-grown specimen) and the 
protobranch Nuculu r?iutis (Nt; 6.5 mm), (a) Overview of the posterior end 
of the left gill of an adult Anadara specimen showing the budding zone 
(bz). (b) Budding zone of Anadara differentiating into lobules of the inner 
and outer dennhranch (id. od). (c) Budding zone and separating inner and 
outer demibranchs of Nucula', the lobules are partly covered by mucus and 
cilia. 



ON THE GROWTH OF BIVALVE GILLS 



77 




Figure 3. Gill filaments of two filibranch bivalves demonstrating the 
lengthened lobules with the equidistantly arranged ciliated knobs (kn), 
which increase in number from the distal end. The proposed distal growth 
zone (gz) is indicated, (al Anadara sp. (shell size about 5 cm); (b) \f\tilnx 
edulis (shell size of 2.2 cm), fg: food groove, gb: gill base, ilj: interlamellar 
junction, ils: interlamellar space. 



remain and stabilize the small distance between the outer 
margins (Fig. 3b); the first ones appeared at about the 30th 
filament (counted anteriorly from the budding zone). Again, 
the margins strongly resemble filamentary structures. All 
adjacent filaments are attached to each other by equidistant 
ciliated knobs (Fig. 3b). In dissections of the gills of adult 
Mytilus, we observed that the ciliated knobs form equidis- 
tant lines, which are arranged parallel to the gill base along 
the whole gill. The number of lines increased with the size 
of the demibranchs and the length of their filaments. In 
contrast to Anadara, Mvtilus shows new lines of knobs only 
near the ventral end of the filaments. No inserted lines of 
knobs were detected in lateral views of the lamina. Thus, 
unlike growth in Anadara, the main growth zone responsi- 
ble for elongation must be restricted to this area. 

Budding -ones of eulamellibranch gills 

The four representatives of the Palaeoheterodonta (Unio) 
and Heterodonta (Dreissena, Corbicula, and Pisidiinn) stud- 
ied possess eulamellibranch gills. Again, an undifferentiated 
budding zone lies at the posterior end of each species' gills 
(Fig. 4). In each example, the transverse folds form in a way 



similar to that of protobranchs and filibranchs: the folds split 
into inner and outer demibranchs. Apart from these com- 
mon differentiation events, species-specific differences ex- 
ist in the relative size of the budding zone and in the 
extension of a segmented transition zone before the two 
parallel rows of lobules begin to emerge (Fig. 4). Cilia 
already occur on the early lobules. 

Unio (Fig. 4a): Only the growing left gill and the adjacent 
mantle epithelium are visible; the right gill is obscured. The 
budding zone is located on the posterior process of the gill 
base, which is curved inwards. At least six transverse folds 
extend from it; this is the transition zone. These folds then 
become separated by the prospective dorsal food groove 
into two parallel rows of further enlarged lobules. The 
lobules become ciliated as they grow ventrally. As can be 
seen in the outer demibranch (Fig. 4a), the outer edges of 
the lobules (later forming the outer lamellae) are attached to 
the mantle tissue from the very beginning. The correspond- 
ing processes occur on the other side of the foot, on the 
growing right gill. 

Dreissena (Fig. 4b): In this view from the ventral side, 
only the left gill is horizontally positioned, so that the details 
of the transitions between the budding zone and the trans- 
verse folds can be seen. The budding zone is smaller than in 
Unio and is followed by a short transition zone. No excep- 
tions to the 1:1 relationship in lobule number between 
developing inner and outer demibranchs was found. As was 
demonstrated during dissections, the budding zone is not 
attached to the mantle. 

Corbicula (Fig. 4c): The separation of the transverse 
folds follows the same principles as described above. The 
budding zones of the left and right gills lie close together 
and seem to be attached to the mantle. The dorsal margins 
of the outer demibranch lobes are also attached to the 
mantle from the beginning on. However, two peculiarities 
can be noticed in this species. Firstly, the first transverse 
fold starts at the inner demibranch of each gill. Secondly, a 
transition zone, such as is found in Unio and Dreissena, 
does not exist. 

Pisidiinn (Fig. 4d): The convergent budding zones were 
as short as in Corbicula. The separation of lobules and their 
early differentiation appear to be somewhat advanced in the 
most recent section of the inner demibranch, without dis- 
turbing the 1:1 relationship of the older inner and outer 
lobules (compare idl and odl in Fig. 4d). 

Dissection of two other lamellibranchs revealed a differ- 
entiation of new lobules more or less identical to that shown 
in Figure 4. In adult Venerupis decussata specimens, neither 
the budding zone nor the transition zone was fused to the 
mantle; and the transition zone extended over only two or 
three transverse folds. When the demibranchs were sepa- 
rated, the most posterior lobules of both demibranchs were 
still solid, lacking any transformation. A small specimen of 
Mya arenaria (shell length 3.8 mm) showed a similar pat- 



78 



i 



D. NEUMANN AND H. KAPPES 
8- - -, 




Figure 4. Scanning electron micrographs of the budding zones and the start of both lobule and demibranch 
differentiation in four eulamellibranch bivalves, (a) Unio piclorum (Up; shell length 1.7 cm); (b) Dreissena 
polymorplui (Dp; 1.8 cm); (c) Corhicula fluminea (Cf; 0.9 cm); and (d) Pisidium casertanum (Pc; 2.1 mm). The 
budding zones of the left and right gills (bzl, bzr) may lie next to each other, as in Dp. Cf. and PC. Each is 
differentiating into the lobules of the separating inner and outer demihranchs (idl, odl. and idr, odr). In Unio, a 
transition zone (trz) of about six transverse folds is shown, m: mantle. 



ON THE GROWTH OF BIVALVE GILLS 



79 



tern. The transition zone consisted of three folds at this 
stage. 

Lobule differentiation in euliimellihranchs 

Histological sections of a suhadult specimen of Unii> 
(Fig. 5) were examined to follow the differentiation of the 
lobules into the filaments of eulamellibranch gills. The 
posterior end of the gill, that is, the budding zone, reveals 
the character of an undifferentiated tissue with a high den- 
sity of nuclei (Fig. 5a). New transverse folds are added to 
the already differentiated gill from the budding zone. The 
longitudinal splitting of the folds into two rows of lobules 
(representing the inner and outer demibranch) is clearly 
visible in Figure 5b. The development of these lobules into 
extended lobes (still unfenestrated) is followed by those 
differentiations that continuously transform the lengthening 
lobes into the complexly structured filaments of the eula- 
mellibranchs. Three processes can be distinguished during 
this development. As they cannot be followed in only one 
frontal section, we present sections of different levels (Fia 
5a-c). 

The first skeletal rods (grayish opaque, no nuclei) are 
formed in the late transition zone during the beginning of 
demibranch formation. At first, these rods seem to be con- 
fined to the gill base between the inner and outer derni- 
branchs (Fig. 5a); later they extend into the filaments. 

The other processes of lobule differentiation can be seen 
in Figure 5c. Tissue bridges representing interfilament junc- 
tions occur between adjacent lobes (ifj in Fig. 5c). As 
documented further, interlamellar spaces appear in the lam- 
inae of most lobes (ils in Fig. 5c). In some lobes, the lamina 
remains unchanged and constitutes an interlamellar junction 
(ilj in Fig. 5c). When the demibranchs of eulamellibranchs 
are observed in situ, the lamellae appear filamentous be- 
cause the outer ciliated margins of the lobes have differen- 
tiated into the descending and ascending limbs of the so- 
called filament. However, this filamentary appearance is due 
to no more than the outermost 50 /xm of the tissue, which 
bears the ciliary machinery and the vertical hemolymph 
vessels of the gill. 

Outer demibranch formation in juvenile unionids 

The dorsal mantle cavity of a 3.5-mm-long specimen of 
Unit) pictoruni revealed no indication of filaments of the 
outer demibranch (Fig. Ib). However, six short filaments of 
the outer demibranch were identified in frontal sections of a 
U. pictonun specimen of 4.9-mm shell length. Apart from 
the anterior one, these were already differentiated in that 
they were fused at their outer margins with the mantle 
epithelium. 

Because the development of the outer demibranch lags 
behind that of the inner, the two demibranchs differ in 
filament number, in gill-base length, and in demibranch 
height (as expressed by the length of the longest filament in 



the row; Fig. la). In Figure 6 the height of the demibranch 
in juveniles is plotted against shell length, up to 17 mm 
(years 1-3, based on the number of growth rings). The data 
for both Unio species were pooled because no species- 
specific deviation was found when they were tested sepa- 
rately (P > O.I). The linear regressions of maximum fila- 
ment length versus shell length were significantly different 
with respect to the intercepts for the inner and outer demi- 
branchs (P < 0.001). The slopes differed only marginally 
(P = 0.051). 

In the case of the smallest Unio specimen in which an 
outer demibranch was observed (shell length 4.9 mm), the 
anteriormost filament of the outer demibranch was located 
next to the 27th filament of the inner demibranch. In larger 
specimens, we never found such a large difference in fila- 
ment number at the anterior margins of inner and outer 
demibranchs; usually the difference was 10-12 filaments 
(n= 10 specimens with shell lengths between 6.9 and 14.6 
mm). Along the whole gill axis, the skeletal rods of parallel 
filaments of inner and outer demibranchs touched each other 
at the gill base, resulting in the strict 1 : 1 -arrangement of 
filaments already shown in Figure 5a. 

Discussion 

The terminal growth zone of bivalve gills was described 
based on dissections, scanning electron micrographs, and 
histological sections. Despite the anatomical differences of 
the three main gill types (protobranchs, filibranchs, and 
eulamellibranchs), gill formation in juveniles and adults of 
13 species shows a common and uniform pattern. The 
increase in the number of leaflets in protobranchs, and of 
filaments in filibranchs and eulamellibranchs, starts from an 
undifferentiated cell complex that we termed the 'budding 
zone'. This growth zone generates a series of transverse, 
paired lobules that constitute, in a Ill-relationship, both the 
inner and outer demibranch. The lobules grow into extended 
and elongated lobes that become transformed into leaflets in 
protobranch gills and into filaments in filibranch and eula- 
mellibranch gills. 

The budding zone should be seen as a specific, undiffer- 
entiated complex of dividing cells that is active in growing 
bivalves. This terminal zone can be characterized as meris- 
tem-like because it produces new gill elements during the 
whole life of these animals, similar to the formation of new 
leaves from a shoot apical meristem in higher plants, or the 
development of new polyps from a terminal cell complex in 
the elongating stems or stolons of thecate hydrozoans 
(Berking et al.. 2002). 

One may assume that this terminal growth zone is a 
projection of the postlarval gill axis composed of peripheral 
ectodermic and internal mesodermic cells. The budding 
zone either first produces transversal folds (in cases of 
delayed splitting into inner and outer demibranch lobules, as 
in Mytilus. Unio, Dreissena, Venerupis. A/v) or it directly 



80 



D. NEUMANN AND H. KAPPES 



forms lobules (in cases of simultaneous splitting of the 
demibranchs: Nucula, Anailuni. Crbicula, Pisidium). The 
segregation may resemble the first steps of somitic segmen- 
tation in the early embryology of segmented animals (Wol- 
pert et <//.. 1998). accompanied by a change in cell adhesion 
between distinct blocl- -> of ectodermic cells. 




"^ 

m :" - 



.' 






demibranch 
inner 
outer 




5 10 15 

shell length (mm) 



20 



Figure 6. Length of the longest filaments of the inner and outer 
demibranchs (solid and open triangles, respectively) in Unio pictorum and 
U. tumidus specimens of different shell lengths (pooled data of both 
species). The regression lines are y jd = 0.22(0.01) A + 0.17(O.I5), (; 
= 0.97) in the case of the inner demibranchs: and y^ = 0. 19(0.01) .v - 
0.93(0. 13), (r = 0.97) in the case of the outer demibranchs. 



The conformity of initial lobule formation in all bivalves 
tested supports the monophyly of this class. In Nucula, the 
protobranch gill closely resembles the ctenidia of proso- 
branch gastropods, because both consist of a series of leaf- 
lets along a gill axis (Yonge, 1947). This simple gill struc- 
ture is distinct from the more complex gills in the rest of the 
bivalves. Based on morphological and molecular data sets, 
the Protobranchia are therefore considered to be a sister 
group to the other bivalves, which are grouped as Auto- 
branchia (Hoeh et ai, 1998; Giribet and Wheeler, 2002). 

In the Autobranchia, the gills are adapted to additional 
functions, such as feeding and breeding. The decisive evo- 
lutionary step was the strong elongation of the lobe. The 
lobe's transformation into filibranch or eulamellibranch fil- 
aments can be understood as a series of developmental steps 
correlated with increasing efficiencies of the gill's various 
functions. Fossil records (Cope, 1996) as well as morpho- 
logical and molecular data (Hoeh et ai, 1998; Giribet and 
Wheeler, 2002) indicate that the filibranch gill represents 



Figure 5. Frontal (horizontal) sections (slightly slanted) through the 
posterior end of the left gill of a subadult Unio pictorum (shell-length 20 
mm), (a) Section near the gill base with the undifferentiated budding zone 
(bz), and with a transition zone (trz) and the beginning of lobe differenti- 
ation ria lobules, (b) The same section (about 30 /j.m ventral of section a), 
showing the increasing gap between inner and outer demibranch (id, od). 
(c) Section of the outer demibranch, ventral to the left part of section b, 
revealing further details of the differentiation of lobules into early filaments 
with interfilamentar junctions (ifj), interlamellar spaces (ils), and interla- 
rnellar junctions (ilj). 'af: ascending limb of filament, 'df: descending 
limb of filament, m: mantle tissue, r: rod structure inside the gill base 
connecting the inner and outer demibranchs. 



ON THE GROWTH OF BIVALVE GILLS 



81 



the plesiomorphic type, and that the eulamellibranch gill 
characters evolved polyphyletically. 

Because lobules and lobes are the primary structural 
elements, it is interesting to follow their successive trans- 
formation into the so-called filaments. The present study 
confirms that neither bending nor folding of filamentary 
structures occurs in juvenile and adult bivalves. The final 
V-shape of the filaments results from the continuous trans- 
formation of a lobular anlage via lobes into filaments by a 
dominating ventral growth zone near the tip of the filaments. 
Evidence of high mitotic activities in this zone was ob- 
served in adult filibranchs (Crenomytilus, Mytilus) by H - 
thymidine autoradiographical labeling (Leibson and 
Movchan, 1975). This result perfectly correlates with our 
conclusion, which is based on the pattern of equidistant 
lines of ciliated knobs. Leibson and Movchan (1975) de- 
tected two additional areas of DNA-synthesizing activities 
in Mvtilus. Both were situated close to the dorsal food 
grooves, one at the gill base, the other one at the dorsal apex 
of the filament's ascending limb. As these authors already 
stated, further studies are needed to investigate whether 
these two areas represent additional growth zones of the 
filaments or a higher renewal rate of epithelium cells. In any 
event, during the transformation of lobes into filaments and 
the succeeding elongation, no bending or reflection occurs; 
as declared by Yonge ( 1947. p. 501 ): "this mode of origin is 
impossible." The convenient terms 'descending limb' and 
'ascending limh' are referring neither to the direction of 
growth of the filaments, nor to the direction of hemolymph 
flows, because both arterial and venous lacunae (separated 
by an intrafilamentar septum; Ridewood. 1903) are located 
inside the limbs (Yonge. 1947; Kilias, 1956; our observa- 
tions on Anadara). 

In the early development of the unidirectional slender 
filaments in postlarvae. developmental processes similar to 
those described above seem to occur. One could term them 
'pro-filaments' because they strongly differ from the fila- 
ments in adults. Based on scanning electron microscopy 
figures of juveniles of the pseudolamellibranch Ostreu chit- 
ensis (Chaparro et a!., 2001) it may be inferred that growth 
occurs without bending; the gill rudiments (e.g., pp. 201- 
203: Fig. Ic, Fig. 2a) perfectly correspond to compact 
transverse structures, i.e.. lobules. During postlarval devel- 
opment of the eulamellibranch Veneridae, the unidirectional 
filaments of the inner demibranch display a thickened distal 
end and transform into V-shaped filaments without any 
bending (Ansell. 1962; Moueza etui. 1999). This thickened 
end may be recognized as a kind of lobule. Corresponding 
conclusions may be derived from the thickened ends of the 
filaments presented in figures of Pecten (Beninger et <//., 
1994). freshly metamorphosed Unio (Herbers. 1913) and 
juvenile Sphaerium (Wasserloos, 1911. figs. K. L). 

The onset of the outer demibranch formation and its delay 
in the Autobranchia reveals two further interesting devel- 
opmental aspects, as shown in Unio pictarum and U. tu- 



inidus. Firstly, a certain body size must be reached before 
outer demibranch development is initialized. In the Unio 
population we studied, differentiation of the outer demi- 
branch started at shell lengths of about 4-4.5 mm. Speci- 
mens of this size showed one growth ring, indicating the 
cessation of growth during the first winter. In contrast, 
Korniushin ( 1997) found the first filaments in 2.4-mm Unio 
specimens. Whether such a difference in the start of outer 
demibranch formation is due to environmental or genetic 
factors remains unclear. Secondly, at the anterior end of the 
gill axis, the number of filaments in inner and outer demi- 
branchs differs. The difference is size-dependent and de- 
creases in larger juveniles. Such a decrease in the difference 
of anterior filament number was also observed in several 
other eulamellibranch species (Komiushin, 1997). A reduc- 
tion of the foremost filaments of the inner demibranch 
seems to be unlikely, because all filaments were completely 
differentiated at the 4.9-mm stage; i.e., they were fused to 
the foot and were functionally integrated. We hypothesize 
that the outer demibranch also extends its range at its 
anterior end where during a short developmental peri- 
od a limited number of lobules differentiate from the gill 
axis in parallel to the filaments of the inner demibranch. 

In summary, the generation of simply structured lobules 
from the posterior budding zone and their differentiation 
into protobranch leaflets, filibranch filaments (interlamellar 
junctions and ciliated knobs between adjacent filaments), or 
more intricate structures (with complex interfilamentar 
junctions, as in pseudolamellibranchs and eulamellibranchs) 
may be an interesting model for further developmental 
studies, which may also offer insight into the evolution of 
the various gill types that occurred during the phylogeny of 
bivalves. 

Acknowledgments 

Our sincere thanks are due to Dr. Ronald Janssen (For- 
schungsinstitut und Naturmuseum Senckenberg, Frankfurt 
a. M.) for lending Nucula specimens from the museum 
collection; to Prof. Gerard Van de Velde (Dept. of Ecology, 
University of Nijmengen) for acquiring the Anadara spec- 
imens from India; to Hans-Peter Bollhagen, Helmut Wratil 
(University of Cologne), and Dr. Markus Weitere (Dept. of 
Biology, Free University of Berlin) for SEM assistance; to 
Frederic Bartlett for linguistic comments; and to Prof. M. J. 
Greenberg and anonymous referees for valuable suggestions 
for improving the text. 

Literature Cited 

Ansell, A. I). 1962. The functional morphology of the larva, and the 
postlarval development of Venus striatula (Da Costa). /. Mar. Bio/. 
Assoi: UK 42: 419-443. 

Bayne, B. L., J. Widdows, and R. J. Thompson. 1976. Physiology: 
feeding and digestion. Pp. 121-206 in Marine Mussels: Their Ecology 
and Ph\\ii>li>g\. B. L. Bayne. ed. Cambridge University Press, Cam- 
bridge. 



82 



D. NEUMANN AND H. KAPPES 



Beninger, P. G., M. Le Pennec, and M. Salaiin. 1988. New observa- 
tions of the gills of P/acopecten mazclltiniciis (Mollusca: Bivalvial, 
and implications for nutrition. I. General anatomy and surface micro- 
anatomy. Mar. Biol. 98: 61-70. 

Beninger, P. G., S. A. P. Dwiono, and M. Le Pennec. 1994. Early 
development of the gill and implications for feeding in Pecten maximus 
(Bivalvia: Pectenidue) Mar. Biol. 119:405-412. 

Berking, S., M. Hesse, and K. Herrmann. 2002. A shoot meristem-like 
organ in animals: monopodial and sympodial growth in Hydrozoa. Int. 
J. Dev. Biol. 46: 301-308. 

Chaparro, O. R., J. A. Videla, and R. J. Thompson. 2001. Gill 
morphogenesis in the oyster Ostrea chilensis. Mar. Biol. 138: 199- 
207. 

Cope, J. C. W. 1996. The early evolution of the Bivalvia. Pp. 361-370 
in Origin and Evolutionary Radiation of the Mollusca, J. Taylor, ed. 
Oxford University Press, Oxford. 

Drew, G. A. 1901. The life-history of Nucula delphinodonta (Mighels). 
Q. J. Microsc. Sci. 44: 313-391. 

Giribet, G., and W. Wheeler. 2002. On bivalve phylogeny: a high-level 
analysis of the Bivalvia (Mollusca) based on combined morphology 
and DNA sequence data. Invertebr. Biol. 121: 271-324. 

Gros, O., L. Frenkiel, and M. Moueza. 1998. Gill filament differenti- 
ation and experimental colonization by symbiotic bacteria in aposym- 
biotic juveniles of Codakia orbicu/aris (Bivalvia: Lucinidae). Inver- 
tebr. Reprod. Dev. 34: 219-231. 

Herbers, K. 1913. Entwicklungsgeschichte von Anodonta ce/lensis 
Schrot. Z. Wiss. Zoo/. 108: 1-174. 

Hoeh, W. R., M. B. Black, R. Gustafson, A. E. Bogan, R. A. Lutz, and 
R. C. Vrijenhoek. 1998. Testing alternative hypotheses of Neotrigo- 
nia (Bivalvia: Trigonioida) phylogenetic relationships using cyto- 
chrome c oxydase subunit I DNA sequences. Malacologia 40: 267- 
278. 

Jackson, R. T. 1890. Phylogeny of the Pelecypoda: the Aviculidae and 
their allies. Mem. Bost. Soc. Nat. Hist. 4: 277-394. 



Kilias, R. 1956. Uber die Kieme der Teichmuschel (Anodonta Lam.) 

(Ein Beitrag zur Anatomic). Mitt. Zoo/. Mus. Berl. 32: 151-174. 
Korniushin, A. V. 1997. Patterns of gill structure and development as 

taxonomic characters in bivalve molluscs (Mollusca Bivalvia). Ann. 

Zoohgici 46: 245-254. 
Leibson, N. L., and O. T. Movchan. 1975. Cambial zones in gills of 

Bivalvia. Mar. Biol. 31: 175-180. 
Mitsukuri, K. 1881. On the structure and significance of some aberrant 

forms of lamellibranch gills. Q. J. Microsc. Sci. 21: 595-608. 
Morton, B. 1996. The evolutionary history of the Bivalvia. Pp. 337-359 

in Origin and Evolutionary Radiation of the Mollusca, ). D. Taylor, ed. 

Oxford University Press, Oxford. 
Moueza, M., O. Gros, and L. Frenkiel. 1999. Embryonic, larval and 

postlarval development of the tropical clam, Anomalocardia brasiliana 

(Bivalvia, Veneridae). J. Molluscan Stud. 65: 73-88. 
Purchon, R. D. 1968. The Biology of the Mollusca. Pergamon Press, 

Oxford. 

Raven, Chr. P. 1966. Morphogenesis: The Analysis of Molluscan De- 
velopment. Pergamon Press, Oxford. 
Ridewood, W.G. 1903. On the structure of the gills of the Lamelli- 

branchia. Philos. Trans. K. Soc. Land. B 1905: 147-284. 
Romeis, B. 1968. Mikroskopische Technik. Oldenburg, Munich. 
Waller, T. R. 1981. Functional morphology and development of the 

veliger larvae of the european oyster, Ostrea edu/is Linne. Smithson. 

Contrib. Zoo/. 328: 1-70. 
Wasserloos, E. 1911. Die Entwicklung der Kiemen bei Cyclas cornea 

und anderen Acephalen des siiBen Wassers. Zoo/. Jahrb. Anal. 31: 

171-284. 
Wolpert, L., R. Beddington, J. Brookes, Th. Jessell, P. Lawrence, and 

E. Meyerowitz. 1998. Principles of Development. Current Biology, 

London. 
Yonge, C. M. 1947. The pallial organs in the aspidobranch gastropoda 

and their evolution throughout the mollusca. Philos. Trans. R. Soc. 

Land. B 232: 443-518. 



Reference: Biol. Bull. 205: 83-92. (August 2003) 
2003 Marine Biological Laboratory 



Demonstration of Nutrient Pathway From the 

Digestive System to Oocytes in the Gonad Intestinal 

Loop of the Scallop Pecten maximus L. 

PETER G. BENINGER' *, GAEL LE PENNEC 2 , AND MARCEL LE PENNEC 2 

1 Laboratoire de Biologie Marine, Faculte des Sciences, Universite de Nantes, 44322 Nantes Cedex, 

France; and 2 Institut Universitaire Europeen de la Mer, Universite de Bretagne Occidentale, Site 

Technopole Brest-Iroise. 29280 Plouzane, France 



Abstract. The mechanism of nutrient transfer from the 
digestive system to the gonad acini and developing oocytes 
was investigated in the gonad-intestinal loop system of the 
queen scallop Pecten maximus L. Ferritin was injected 
directly into the purged intestine of specimens from the 
wild. Subsequently, a histochemical reaction and transmis- 
sion electron microscopy were used to localize ferritin in 
various cell types. Ferritin was rapidly absorbed by the 
intestinal epithelium, and then appeared in hemocytes in the 
surrounding connective tissue. In the hemocytes, territin 
was stored in variously sized inclusions, as well as in the 
general cytoplasm. In all sections examined for the 12 
experimental individuals, hemocytes were always found in 
association with connective tissue fibers extending from the 
base of the intestinal epithelium to gonad acini. After 30- 
min incubation, ferritin appeared inside the acini of all 
individuals. Ferritin-bearing cells were rarely found in as- 
sociation with male acini or gametes, nor with mature 
female gametes, but often with developing female gametes. 
Not all individuals showed the same temporal dynamics of 
ferritin transport, suggesting that nutrient transfer to oocytes 
is either not a continuous process, or that among individu- 
als, transfer is not synchronized on short time scales. This is 
the first demonstration of a pathway of nutrient transfer 
from the intestine, and more generally the digestive system, 
to developing oocytes in the Bivalvia. 



Received 21 November 2000; accepted 4 June 2003. 
* To whom correspondence should be addressed. E-mail: Peter. 
Beninger@Isomer.univ-nantes.fr 



Introduction 

In the Bivalvia, the digestive and reproductive systems 
are closely situated and often intertwined, either within the 
visceral mass (the majority of bivalves), or more distinctly 
separated from the visceral mass, as in the Pectinidae (Galt- 
soff, 1964; Morales- Alamo and Mann, 1989; Beninger and 
Le Pennec, 1991; Morse and Zardus, 1997). The ultrastruc- 
tural characteristics of gametogenesis have only recently 
begun to be elucidated in this class (Pipe, 1987a, b; Dorange 
and Le Pennec, 1989; Eckelbarger and Davis, 1996a, b). 
However, gametogenesis must rely on the transfer of nutri- 
ents, which are acquired almost exclusively by other tissues 
or organs and transferred to the gonad. 

Transfer of nutrients from storage or digestive sites to the 
gonad has been inferred or demonstrated in a number of 
bivalve species (Goddard and Martin, 1966; Vassallo, 1973; 
Ansell, 1974; Comely, 1974; Gabbott, 1975, 1983; Adachi, 
1979; Zaba, 1981; Lubet el ai, 1987; Le Pennec el al, 
1991a, b). Although successful gamete production relies on 
such transfers, very little is known about the underlying 
pathways and mechanisms. The elaboration of oocyte re- 
serves has been the subject of considerable research in many 
invertebrates, but is largely lacking in bivalves (see Eckel- 
barger and Davis, 1996a, for review). Regardless of whether 
bivalve gametes ultimately elaborate vitelline reserves using 
autosynthetic (Suzuki el al., 1992) or heterosynthetic path- 
ways (as suggested by Eckelbarger and Davis, 1996a), or 
both, it is clear that nutrients must be made available, 
largely from the diet, for the synthesis of the gametes and 
their reserves. 

A summary of known or inferred pathways that transfer 
nutrients to the gonad acini and gametes has been outlined 



83 



84 



P. G. BEN1NGER ET AL 



for the queen scallop. Pecten imuiniitx, based on anatomi- 
cal, ultrastructural, and histoch -iiical observations (Le Pen- 
nec el ul.. 199 la). In p:; :; - transfer of nutrients from 
the intestine to the A ig gametes was proposed. Al- 

though it has long h. nown that both extracellular and 
intracellular di ike place in the intestine of bivalves 

(Zacks. 1955: Reid, 1966; Payne et /., 1972: Mathers, 
1973; Teo and Sabapathy. 1990). the persistence of the 
"conventional wisdom" that the intestine merely serves as a 
conduit for undigested matter prompted Purchon (1971) to 
call for a reexamination of the role of this organ. In the 
family Pectinidae, the intestine loops within the otherwise 
anatomically distinct gonad. and indeed Le Pennec et til. 
(199 la) provided data suggesting that nutrients are trans- 
ferred from this structure to developing gametes. They also 
proposed a transfer mechanism and pathway involving he- 
mocytes. Enzymatic and detailed ultrastructural studies sub- 
sequently showed that the scallop intestinal loop is capable 
of digestion and assimilation (Le Pennec et til., 1991b). This 
research provided a framework for the demonstration of 
transfer pathways using direct physiological techniques 
such as labeling. In this study, therefore, we have used 
ferritin as a marker to examine the proposed transfer path- 
way from the gonad intestinal loop to gonadal tissue in the 
scallop Pecten maximus. 

Ferritin is an iron-containing transfer protein, consisting 
of a core of up to 4300 iron cations in the form of ferric 
oxyhydroxide and ferric phosphate, and a protein shell of 
approximately 450,000 Da (Miksys and Saleuddin. 1986). 
In the specific tissues of living organisms which contain 
ferritin, the molecules are often grouped into variously sized 
clusters, with a near-crystalline appearance (Bottke and 
Sinha. 1979; Miksys and Saleuddin. 1986). A specific stain 
for iron can therefore be used to distinguish it from other 
proteins (Bockman and Winbom. 1966; Heneine et til.. 
1969; Block et ah. 1981: Bottke et til.. 1982; Boucher- 
Rodoni and Boucaud-Camou, 1987; Paar et til.. 1992; Ito et 
til., 1992). Ferritin is also visible in uncontrasted transmis- 
sion electron microscopy (TEM) sections as small, vari- 
ously sized electron-dense clusters (Bottke and Sinha. 1979; 
Miksys and Saleuddin. 1986). Ferritin has been used both to 
demonstrate intestinal absorption mechanisms (Bockman 
and Winborn, 1966; Boucher-Rodoni and Boucaud-Camou. 
1987) and to study mechanisms of uptake into the ferritin- 
rich yolk of snail oocytes (Bottke et til.. 1982). In this study 
we use ferritin as a substrate model with which to follow the 
transfer of nutrient molecules from the intestine to the 
gonadal tissue of Pecten maximus. Although hemoglobin is 
present in the hemocytes of some bivalve families (see 
reviews by Reid. 1966; Bonaventura and Bonaventura. 
1983), none has yet been reported in the pectinids. and in 
any event, this substance cannot confound histochemical 
detection of injected ferritin since the iron of hemoglobin 
cannot be demonstrated histochemically without total de- 



struction of histochemical sections (Kiernan, 1990). Control 
for the eventual presence of naturally occuring ferritin can 
be accomplished through the use of control subjects. 

Pectinids are ideal candidates for such experiments, be- 
cause the gonad-intestinal complex is well-separated from 
the other organs. Pecten maximus was chosen in part be- 
cause it is a simultaneous hermaphrodite, thus allowing 
investigation of both male and female components within 
the same individual under identical experimental condi- 
tions. The gonad intestinal loop of pectinids also presents 
the advantage of being easily visible throughout most of the 
reproductive cycle. No respiratory function has yet been 
ascribed to bivalve hemocytes, and bivalve plasma gener- 
ally lacks circulating respiratory pigments (Booth and Man- 
gum. 1978). obviating possible artifacts. 

Materials and Methods 

Twelve specimens of Pecten maximus (size range 9-10 
cm shell length, antero-posterior axis) were collected from 
the Bay of Brest (Finistere, France). The valves of each 
scallop were kept open with a wedge in the posterior dorsal 
region, and the proximal part of the descending intestinal 
loop was located by directing a cold light source at the male 
portion of the translucent gonad (see Fig. 1 ). Into this 
portion of the intestine in each scallop. 1 ml of a 4 mg ml 
solution of cadmium-free ferritin (Sigma horse spleen Type 




9 



PO 



Figure 1. AYr/cii ;<I.V/HI.V. Schematic diagram to show planes of 
section in anterior (A), median (M). and posterior (PO) gonad levels. 
Histological sections were performed on (hese planes, such that the region 
of the median level surrounding the descending branch of the intestinal 
loop (DB) contained predominantly male acini, whereas the region of the 
median level surrounding the ascending branch of intestinal loop (AB) 
contained predominantly female acini. AM, adductor muscle; DC. diges- 
tive gland: FT. fool: I., lips; R. rectum: S. stomach; 8. male, and 9. female 
parts of gonad. 



PECTEN NUTRIENT PATHWAY TO OOCYTES 



85 



1 




N 




N 



0.4 pm 





Figure 2. Transmission electron micrographs of uncontrasted oocytes and hemocytes in posterior (female) 
region of gonad. from individuals which had not been injected with ferritin. 2.1. 2.2 Details of early-developing 
oocytes at two magnifications (20.000 and 40.000 x. respectively). 2.3 Detail of rounded hemocyte. showing 
non-ferritin-containing inclusions (I); 2.4 Detail of pseudopod-bearing hemocyte. Note absence of ferritin 
clusters in all micrographs. N. nucleus. 



1, 10-12 nm diameter molecules) was slowly injected. 
Complete distribution of ferritin throughout the length of 
the intestinal lumen was monitored visually by the appear- 
ance of the colored solution in the ascending branch. The 
scallops were divided into four groups of three individuals 
each, corresponding to 10. 30. 120. and 300 min of exposure 
to the ferritin solution. Exposure was carried out in 15C 
filtered seawater. which was aerated with a pump and air- 
stone. Following the designated period of exposure, the 
scallops were immediately dissected. Transverse slices of 
the gonad (about 1-2 mm thick) were removed at three 
levels along the antero-posterior axis of the gonad as shown 
in Figure 1. and immediately processed for histology and 
electron microscopy. To control for the eventual presence of 
clusters of endogenic ferritin molecules or other electron- 
dense particles, a control group of three individuals was 



injected with 0.8 /urn filtered seawater, and processed for 
histology as detailed below. 



Histology 

Slices of gonad were fixed for histology in aqueous 
Bouin's solution (24 h), dehydrated in an ascending etha- 
nol-xylene series, and embedded in paraffin. Transverse 
sections corresponding to each exposure time and gonad 
level were cut at 5 p.m. and each exposure series for a given 
level were positioned on a single microscope slide. The 
slides were then immersed in 35% ammonium sulfite for 2 h 
and rinsed with Milli-Q-filtered ultrapure water (all glass- 
ware at this stage was also prerinsed with ultrapure water). 
Sections were stained using the Turnbull blue protocol. 



86 



P. G. BENINGER ET AL. 



Exposure time 
(min) 

Distribution star? 


10 

345 


30 
12345 


120 
12345 


300 
12345 


<$ A 
AB 
MDB 




= 


zz 


= 






^^^^^^^^" 






O AR 










PO HR 










AR 





















Figure 3. Dynamics of ferritin distribution in the Pecten maximus gonad for the three individuals at each 
exposure time. A, anterior region of gonad; M, median region of gonad; PO. posterior region of gonad; <J. male 
portion of gonad; 9 . female portion of gonad; AB, tissues of ascending branch of intestine-gonad complex; DB, 
tissues of descending branch of intestine-gonad complex; 1. uptake into intestinal epithelium; 2, appearance in 
hemocytes within connective tissue surrounding intestine; 3, appearance in hemocytes at the outer faces of the 
acini; 4, appearance in hemocytes within the acini; 5, appearance in hemocytes/follicle cells appressed to oocytes. Thin 
line represents one individual; medium line represents two individuals; thick line represents three individuals. 



counterstained with nuclear red (Gabe, 1968; Vacca, 1985), 
dehydrated, and mounted under coverslips for photography. 

Transmission electron microscopv 

For transmission electron microscopy (TEM), the gonad 
slices were fixed for 10 h in cold (4C) 2.5% glutaraldehyde 
-0.1 M sodium cacodylate buffer at pH 7.3 and 1100 
mOsm. They were then cut into pieces of about 1 mm 3 , 
rinsed twice with the sodium cacodylate buffer solution, and 
post-fixed in cold 1% osmium tetroxide in buffer for 1 h. 
The tissue pieces were then rinsed with 70% ethanol, de- 
hydrated in an ascending ethanol-xylene series, and embed- 
ded in Epon 812 resin. Transverse sections (400-600 nm) 



were cut with a Reichert ultracut-S ultramicrotome and 
examined without further contrast using a JEOL 100CX 
transmission electron microscope. The lack of further con- 
trasting allowed ferritin granules to be identified without 
ambiguity in the resulting micrographs. However, lack of 
contrast and the very small size of the free ferritin molecules 
and masses render observation via the fluorescent TEM 
screen somewhat challenging. 

Results 

As will be shown below, it is important to distinguish 
between oocytes in different states. Oocytes may be in 
development (previtellogenic and vitellogenic oocytes), or 



Figure 4. Pecten maximus. Histological sections showing the presence of ferritin in the intestine and gonad. 
Turnbull blue-neutral red staining protocol. 4.1 Low-magnification view, after 120-min incubation, of ciliated 
intestinal epithelium (IE) in the anterior (male) gonad level with assimilated ferritin (F). surrounding connective 
tissue (CT), and adjacent acini (A) in the male region of the gonad. BL. basal lamina; L, lumen of intestine. 4.2 
Basal region of intestine, in median (male + female) level of gonad after 10-min incubation, showing 
femtin-containing hemocytes (HE) associated with connective tissue (CT) surrounding basal lamina (BL) of 
intestine. 4.3 Ferritin-containing hemocytes (HF) associated with connective tissue fibers (CF) leading from the 
basal lamina of the intestine to gonad acinus (A) in median (male + female) level of gonad. Incubation time: 
120 min. 4.4 Ferritin-containing hemocytes (HF) positioned both at the base ( >)andon the inside of gonad acini 
(^), in posterior (female) level of gonad. Note proximity and association of hemocytes with developing (DO) 
and vitellogenic oocytes (VO) within the acinus (A). Incubation time: 30 min. 4.5 Ferritin-containing hemocytes 
(HF) within acinus (A) of posterior (female) level of gonad. DO, developing oocyte; RMO, residual mature 
oocyte. Incubation time: 30 min. 4.6 Ferritin-containing hemocytes (HF) attached to developing oocyte (DO) 
within acinus (A) of posterior (female) level of gonad. RMO. residual mature oocyte. Incubation time: 30 min. 
4.7 Multiple large, round, ferritin-containmg hemocytes (HF) attached to developing oocytes (DO) within acinus 
(A) in posterior (female) level of gonad. RMO, residual mature oocyte. Incubation time: 30 min. 4.8 Residual 
vitellogenic oocytes (RVO. late-developing stage) within an acinus (A) in posterior (female) level of gonad. Note 
attached large, round, ferritin-containing hemocytes/follicle cells (HF). CT, inter-acinal connective tissue. 
Incubation time: 120 mm. 



PECTEN NUTRIENT PATHWAY TO OOCYTES 



87 



BL 



IE 



BL 



CT .^p 

' ' ^11 



CT 



HF -% 



A 



HF 



CF 



CF 



VO 



HF 



HF 

\ 



DO 



HF 



RMO 



DO 









' A 



8 




HF 



RVO 




P. G. BENINGER ET AL 




Figure 5. Pecten maximus. Uncontrasted transmission electron micrographs of cells in the intestine-gonad 
complex following ferritin injection in the intestinal lumen. 5.1 Detail of cytoplasm of absorptive cell from 
intestirul epithelium, in median (male + female) level of gonad showing ferriting clusters (F) in inclusions 
(FCI). 5.2 Enlargement of indicated region in Fig. 4.1. Note presence of ferritin both within the inclusions and 
distributed I'reel;. in the cytoplasm ( >). 5.3 Detail of a large, rounded hemocyte in the connective tissue 
surrounding the acini in median (male + female) level of gonad. Note presence of ferritin (F) in variously sized 
inclusions. 5.4 Enlargement of region indicated in Fig. 4.3.. showing ferritin (F) in inclusions. 5.5, 5.6 Portion 



PECTF.N NUTRIENT PATHWAY TO OOCYTES 



89 



they may be mature (detached from the acinal wall). Fol- 
lowing spawning, some oocytes of both types may remain in 
the acinus; these are termed residual oocytes. We will thus 
adopt this terminology in the present paper. 

No ferritin was detected using either the Turnbull method 
or TEM observation (Fig. 2) in any of the control individ- 
uals; we may thus conclude that the ferritin observed in our 
histological and electron microscopical sections was in- 
jected. 

The histological observations of the entire set of individ- 
uals and gonad levels revealed that the distribution of fer- 
ritin in the sampled tissues could be divided into 5 sequen- 
tial steps: (1) uptake into intestinal epithelium. (2) 
appearance in hemocytes among the connective tissue sur- 
rounding the intestine, (3) appearance in hemocytes at the 
exterior faces of acini, (4) appearance in hemocytes within 
the acini, and (5) appearance in hemocytes/follicle cells 
appressed to oocytes. It is not possible to ascertain whether 
these cells were hemocytes or follicle cells in the histolog- 
ical sections (only TEM profiles can distinguish these cell 
types). The TEM profiles of these cells described below do 
not correspond to follicle cells, which are rich in rough 
endoplasmic reticulum (Dorange and Le Pennec. 1989). 
absent in the two pectinid hemocyte types (Beninger and Le 
Pennec, 1991) and in the cells observed appressed to the 
oocytes; however, as the TEM sections were uncontrasted, 
it is not possible to distinguish these cells with certainty. 
Although ferritin-containing hemocytes were very rarely 
observed associated with male spermatogonia. they were 
never observed appressed to developing male gametes. The 
distribution of ferritin among the sections is summarized in 
Figure 3, which presents each step in the sequence, for each 
gonad level, and each intestinal branch (ascending and 
descending), for each exposure time. 

Light microscopy of the histological sections showed that 
ferritin was distributed in cells of the intestinal epithelium at 
all three antero-posterior levels of the gonad-intestinal com- 
plex, from the apex to the basal region of the columnar 
intestinal cells (Fig. 4). Ferritin appeared very rapidly in the 
intestinal cells, and was observed in all preparations, even 
after 10 min of exposure. TEM micrographs revealed that 
ferritin appeared predominantly in variously sized inclu- 
sions of the intestinal cells, although isolated granules could 
also be found in the cytoplasm (Fig. 5. 1. 5.2). Large, round, 
ferritin-containing hemocytes were also detected beneath 
the intestinal basal lamina within the surrounding connec- 
tive tissue after only a 10-min exposure (Fig. 4.2). Ferritin 



appeared in these hemocytes in variously sized inclusions, 
as well as being generally distributed within the cytoplasm 
(Fig. 5.3-5.6). In all sections examined for the 12 experi- 
mental individuals, ferritin-containing hemocytes were al- 
ways observed in close association with connective tissue 
fibers extending from the basal lamina of the intestinal 
epithelium and the acini (Fig. 4.2, 4.3). 

Further transport of ferritin to the gonad acini and oocytes 
appeared to be somewhat independent of exposure time 
(Fig. 3). Specimens in which further ferritin transport was 
observed showed ferritin-containing hemocytes/follicle 
cells both at the outside faces of gonad acini (including 
those which were remote from the intestine) and inside the 
gonad acini (Fig. 4.3-4.8). These hemocytes/follicle cells 
were typically found appressed to developing oocytes (Fig. 
4.3-4.8). Although no positive Turnbull blue reaction was 
observed in female gametes, transmission electron micro- 
graphs revealed dispersed ferritin clusters within the cyto- 
plasm of oocytes to which ferritin-containing hemocytes/ 
follicle cells were appressed (Fig. 5.7, 5.8). 

The localization and distribution of ferritin within the 
various cell types involved in the transfer sequence pre- 
sented notable differences, which also explains the absence 
of a visibly positive Turnbull reaction in some cell types. 
Both the intestinal cells and the transport hemocytes pos- 
sessed variously sized inclusions with considerable concen- 
trations of ferritin, and these cells presented visibly positive 
Turnbull reactions. 

Upon histological examination, 3 of the 12 experimental 
scallops were observed to be mature and ready to spawn, 
while the remaining 9 had already spawned and had begun 
producing a new cohort of female gametes. Although fer- 
ritin appeared in the intestinal epithelium and surrounding 
connective tissue of the mature scallops, ferritin-containing 
hemocytes were virtually never observed, either in the acini, 
or appressed to the oocytes of these individuals. Moreover, 
despite the presence of mature residual oocytes in the acini 
of the individuals which had spawned previously, ferritin- 
containing hemocytes/follicle cells were never observed 
appressed to them. Ferritin-containing hemocytes/follicle 
cells were, however, observed appressed to late-developing 
residual oocytes in these individuals (Fig. 4.8). 

Discussion 

The uptake of ferritin in the intestinal epithelium of 
Pecten imi.\innis. observed in the present study, demon- 



of a large, rounded hemocyte within an acinus in posterior (female) level of gonad. showing ferritin-containing 
inclusions (FCI). as well as ferritin molecules and clusters distributed freely in the cytoplasm ( >). CM. cell 
membrane; MT. mitochondria. 5.7, 5.8 Detail of association between early developing oocyte (EO) and 
pseudopods of hemocyte/follicle cell (HP), in posterior (female) level of gonad. Note ferritin freely distributed 
in cytoplasm of both cells ( >). N. nucleus. 



90 



P. G. BENINGER ET AL. 



strates that proteinaceous substrates are absorbed by the 
scallop intestinal epithelii' ! least some of which subse- 
quently appear in heir at the cell bases. The rapidity 
and ubiquity of this u viserved in the present study, as 

well as the cyi< id enzymatic equipment of the 

intestinal epitHe' Pennec el ai, 1991b), suggest that 

the scallop intestine is well-adapted for both a digestive and 
a transfer fiMCiinn. This result is consistent with the view of 
the intestine as a digestive organ in bivalves (Zacks, 1955; 
Reid. 1966; Payne ft al.. 1972; Mathers, 1973; Teo and 
Sabapathy, 1990). The development of the gonad around the 
intestine optimizes the potential for the transfer of nutrients 
to developing gametes. 

The results of the present study allow us to identify the 
various cell categories and pathways that mediate the en- 
tero-gonadal transfer system in bivalves: intestine epithelial 
cells; large hemocytes which concentrate ferritin in cyto- 
plasmic inclusions, in addition to that present freely in the 
cytoplasm; and connective fibers which are often associated 
with the hemocytes. Previous studies have shown that he- 
mocytes may move across the intestinal epithelium: those 
containing material of little or no nutritional value move 
toward the lumen, while those containing nutritionally valu- 
able material move from the lumen to the tissues surround- 
ing the intestinal epithelium (see Cheng, 1996, for review of 
bivalve hemocyte types and functions). The present study 
shows that the scallop intestinal cells may, themselves, 
move nutrients from the lumen to the basal lamina; hemo- 
cytes subsequently act as transport vectors to the surround- 
ing gonad tissue. This line of investigation does not seem to 
have been pursued previously, despite long-held anatomical 
knowledge of the scallop intestine-gonad relationship. 

The pathway of intestine-oocyte transfer seems to con- 
form largely to that postulated by Le Pennec et ai, based on 
detailed histological observations (1991a). These authors 
proposed that nutrients assimilated by the intestinal epithe- 
lium are transferred to hemocytes at the base of the basal 
lamina, as observed in the present study. They further 
proposed that the efficiency of this transport relied upon 
connective tissue fibers linking the basal lamina to the acini, 
such that incorporated nutrients could be directed specifi- 
cally to acini. In the present study, ferritin-containing he- 
mocytes were always observed in association with connec- 
tive tissue fibers between the base of the intestinal 
epithelium and the bases of the acini. 

The asymmetry in ferritin distribution between the male 
and female parts of the simultaneous hermaphroditic gonad 
is consistent with the difference in the composition and 
consequent energetic demand of male and female gametes. 
While ferritin-containing hemocytes/follicle cells were 
readily observed appressed to developing oocytes, which 
elaborate substantial vitelline reserves, they were rarely 
observed within male gonad acini, which produce small 



spermatozoa with few energy reserves (see Beninger and Le 
Pennec, 1991, for the sizes of spermatozoa and oocytes in 
pectinids. and Beninger and Le Pennec, 1997, for the sizes 
of spermatozoa and oocytes in bivalves generally); and 
ferritin-containing hemocytes were never observed ap- 
pressed to developing male gametes. This asymmetry sug- 
gests that the entero-gonadal pathway is specific to female 
gametes. Another such possible distinction is described 
below. 

The fact that ferritin-containing hemocytes/follicle cells 
were always found in association with developing oocytes, 
and never in association with mature oocytes, suggests that 
the ferritin-containing cells might be able to distinguish 
between these two states, and can supply nutrients to the 
oocytes most in need, i.e., developing oocytes. It should be 
noted that the follicle cells detach from the mature oocytes 
of Pecten maximus (Dorange and Le Pennec, 1989). Al- 
though both residual mature and residual developing oo- 
cytes were observed in individuals that had recently 
spawned, ferritin-containing cells were only observed ap- 
pressed to the residual developing oocytes, and never to the 
residual mature oocytes. This finding suggests that the 
spawning status (i.e., prespawning or postspawning) does 
not influence nutrient transport; rather, the oocyte develop- 
mental stage appears to be the determining feature of such 
transport, even when these gametes are destined for atresia 
and metabolic recycling (Pipe, 1987b; Dorange and Le 
Pennec, 1989; Le Pennec et a!., 199 la). The data of Figure 
3 show that ferritin transfer to developing oocytes does not 
occur at a uniform rate for all individuals; indeed, after 300 
min, some individuals had no ferritin-containing cells ap- 
pressed to developing oocytes. While this could be due to 
the stress induced by the experimental procedure, it could 
also indicate that nutrient transfer from the intestine is not a 
continuous physiological activity, or that among individu- 
als, transfer is not synchronous on such short time scales. 
We are unaware of any studies that present the dynamics of 
oogenesis on such short time scales, but this is an interesting 
physiological question. 

While the particular scallop gonad-intestine anatomical 
relationship is not common in bivalves, the digestive system 
and gonad are generally closely associated and intertwined, 
with loose connective tissue containing abundant fibers 
between these epithelia (Galtsoff, 1964; Morales-Alamo 
and Mann, 1989; Morse and Zardus, 1997). These similar- 
ities to the pectinid system suggest that transfer from diges- 
tive epithelia to developing oocytes via a pathway similar to 
that described above may be a general feature of bivalve 
physiology. 

Acknowledgments 

The authors wish to thank Dr. Ita Widowati, Diponegoro 
University, Semarang, Indonesia, for having carried out 



PECTEN NUTRIENT PATHWAY TO OOCYTES 



91 



previous unpublished experiments on this subject, which 
guided the methodology of the present work. We are in- 
debted to the staff of The Biological Bulletin for their very 
rigorous editorial work on this paper, which allowed us to 
substantially improve earlier versions of the manuscript. As 
a study of no direct economic or medical impact on humans, 
it is not possible to acknowledge funding from any of the 
French fundina aaencies. 



Literature Cited 

Adachi. K. 1979. Seasonal changes of the protein level in the adductor 
muscle of the clam. Tapes philippinarum (Adams and Reeves) with 
reference to the reproductive season. Comp. Biochem. Physiol. 64A: 
85-89. 

Ansell, A. D. 1974. Seasonal changes in the biochemical composition of 
the bivalve Chlamys septemradiata from the Clyde Sea area. Mar. Biol. 
25: 85-99. 

Beninger, P. G., and M. Le Pennec. 1991. Functional anatomy of 
scallops. Pp. 133-223 in Scallops: Biology. Ecology and Aquaculture, 
S. E. Shumway. ed. Elsevier Science Publishers B. V., Amsterdam. 

Beninger, P. G., and M. Le Pennec. 1997. Reproductive characteristics 
of a primitive bivalve from a deep-sea reducing environment: giant 
gametes and their significance in Acharax aline (Cryptodonta: Sole- 
myidae). Mar. Ecol. Prog. Ser. 157: 195-206. 

Block, J., A. A. Mulder-Stapel, L. A. Ginsel, and W. T. Daems. 1981. 
Endocytosis in absorptive cells of cultured human small-intestinal 
tissue: horse-radish peroxidase, lactoperoxidase and ferritin as markers. 
Cell Tissue Res. 216: 1-13. 

Bockman, D. E., and W. B. Winborn. 1966. Light and electron mi- 
croscopy of intestinal ferritin absorption. Observations in sensitized 
and non-sensitized hamsters (Mesocricetus auratus). Ana:. Rec. 155: 
603-622. 

Bonaventura, C., and J. Bonaventura. 1983. Respiratory pigments: 
structure and function. Pp. 1-50 in The Mollusca. Vol. 2: Environmen- 
tal Biochemistry and Physiology, P. W. Hochachka, ed. Academic 
Press. New York. 

Booth, C. E., and C. P. Mangum. 1978. Oxygen uptake and transport in 
the lamellibranch mollusc Modiolus demissus. Physiol. Zoo/. 51: 17- 
32. 

Bottke, \V., and I. Sinha. 1979. Ferritin as an exogenous yolk protein in 
snails. Wilhelm Roux's Arch. 186: 71-75. 

Bottke, W., I. Sinha, and I. Kiel. 1982. Coated vesicle-mediated trans- 
port and deposition of vitellogenic ferritin in the rapid growth phase of 
snail oocytes. J. Cell Sci. 53: 173-191. 

Boucher-Rodoni, R., and E. Boucaud-Camou. 1987. Fine structure 
and absorption of ferritin in the digestive organ of Loligo vulgaris and 
L forbesi (Cephalopoda. Teuthoidea). /. Morphol. 193: 173-184. 

Cheng, T. C. 1996. Hemocytes: forms and functions. Pp. 299-333 in 
The Eastern Oyster Crassostrea virginica. V. S. Kennedy. R. I. E. 
Newell, and A. F. Eble, eds. Maryland Sea Grant College, College 
Park. MD. 

Comely, C. A. 1974. Seasonal variations in the flesh weights and bio- 
chemical content of the scallop Pecten maximus L. in the Clyde Sea 
area. J. Cons. Cons. Int. Explor. Mer 35: 281-295. 

Dorange, G., and M. Le Pennec. 1989. Ultrastructural study of oogen- 
esis and oocytic degeneration in Pecten maximus from the Bay of St 
Brieuc. Mar. Biol. 103: 339-348. 

Eckelbarger, K. J., and C. V. Davis. 1996a. Ultrastructure of the gonad 
and gametogenesis in the eastern oyster, Crassostrea virginica. I. 
Ovary and oogenesis. Mar. Biol. 127: 79-87. 



Eckelbarger, K. J., and C. V. Davis. 1996b. Ultrastructure of the gonad 
and gametogenesis in the eastern oyster, Crassostrea virginica. II. 
Testis and spermatogenesis. Mar. Biol. 127: 89-96. 

Gabbott, P. A. 1975. Storage cycles in marine bivalve molluscs: a 
hypothesis concerning the relationship between glycogen metabolism 
and gametogenesis. Pp. 191-21 1 in Proceedings of the 9"" European 
Marine Biological Symposium. H. Barnes, ed. Aberdeen University 
Press. Aberdeen, Scotland. 

Gabbott, P. A. 1983. Development and seasonal metabolic activities in 
marine molluscs. Pp. 165-217 in The Mollusca, Vol. 2, P. W. 
Hochachka. ed. Academic Press. London. 

Gabe, M. 1968. Techniques Histologiques. Masson & Cie. Paris. 

Galtsoff, P. S. 1964. The American oyster Crassostrea virginica Gmelin. 
Fish. Bull. 64: 1-480. 

Goddard, C. K., and A. W. Martin. 1966. Carbohydrate metabolism. 
Pp. 275-308 in Physiology of Mollusca, Vol. 2. K. M. Wilbur and 
C. M. Yonge. eds. Academic Press, New York. 

Heneine, I. F., G. Gazzinelli. and W. L. Tafuri. 1969. Iron metabolism 
in the snail Biomphalaria glabatra: uptake, storage and transfer. Comp. 
Biochem. Physiol. 28: 391-399. 

Ito, T., H. Kitamura, Y. Inayama. A. Nazawa, and M. Kanisawa. 1992. 
Uptake and mtracellular transport of cationic ferritin in the brachiolar 
and alveolar epithelia of the rat. Cell Tissue Res. 268: 335-340. 

kiernan, J. A. 1990. Histological and Histochemical Methods: Theory 
am! Practice. P. 226. Academic Press. New York. 

Le Pennec, M., P. G. Beninger, G. Dorange, and Y. M. Paulet. 1991a. 
Trophic sources and pathways to the developing gametes of Pecten 
maximus (Bivalvia Pectinidae). J. Mar. Biol. Assoc. UK 71: 451-463. 

Le Pennec, M., G. Dorange, P. G. Beninger, A. Donval, and I. Wido- 
wati. 1991b. Les relations trophiques anse intestinale-gonade chez 
Pecten maximus (Mollusque, Bivalve). Haliotis 21: 57-69. 

Lubet, P., J. Y. Besnard, R. Faveris, and I. Robbins. 1987. Physiolo- 
gic de la reproduction de la coquille Saint Jacques (Pecten maximus). 
Oceanis 13: 265-290. 

Mathers, N. F. 1973. A comparative histochemical survey of enzymes 
associated with the processes of digestion in Ostrea edulis and Cras- 
sostrea angulata (Mollusca: Bivalvia). J. Zoo/. Loud. 169: 169-179. 

Miksys, S., and A. S. M Saleuddin. 1986. Ferritin as an exogenously 
derived yolk protein in Heliosoma duryi (Mollusca: Pulmonata). Can. 
J. Zoo/. 64: 2678-2682. 

Morales-Alamo, R., and R. Mann. 1989. Anatomical features in histo- 
logical sections of Crassostrea virginica (Gmelin, 1791), as an aid in 
measurements of gonad area for reproductive assessment. J. Shellfish 
Res. 8: 71-82. 

Morse, M. P., and J. D. Zardus. 1997. Bivalvia. Pp. 7-118 in Micro- 
scopic Anatomy of Invertebrates, Vol. 6A. F. W. Harrison and A. J. 
Kohn. eds. Wiley-Liss, New York. 

Paar, M., E. M. Liebler, and J. F. Pohlenz. 1992. Uptake of ferritin by 
follicle-associated epithelium in the colon of calves. Vet. Pathol. 29: 
120-128. 

Payne, D. W., N. A. Thorpe, and E. Donaldson. 1972. Cellulolytic 
activity and a study of the bacterial population in the digestive tract of 
Scrobicularia plana (Da Costa). Proc. Malacol. Soc. Loud. 40: 147- 
160. 

Pipe. R. K. 1987a. Ultrastructural and cytochemical study on interaction 
between nutrient storage cells and gametogenesis in mussel Mytilus 
edulis. Mar. Biol. 96: 519-528. 

Pipe, R. K. 1987b. Oogenesis in the marine mussel Mytilus edulis: an 
Ultrastructural study. Mar. Biol. 95: 405-414. 

Purchon, R. D. 1971. Digestion in filter feeding bivalves a new con- 
cept. Proc. Malacol. Soc. Loud. 39: 253-262. 

Read, K. R. H. 1983. Molluscan hemoglobin and myoglobin. Pp. 209- 
232 in Physiology of Mollusca, Vol. 2. K. M. Wilbur and C. M. Yonge, 
eds. Academic Press, New York. 



92 P. G. BENINGER ET AL. 

Reid. R. G. B. 1966. Digestive trai :' enzymes in the bivalve Lima Vacca, L. 1985. Laboratory Manual of Histochemistry. Raven Press, 

hians Gmelin and M\a aren -. Co/up. Biochem. Physiol. 17: New York. 578 pp. 

417-433. Vassallo, M. T. 1973. Lipid storage and transfer in the scallop Chlamvs 

Suzuki, T., A. Hara, K. V .iic-i.i, and K. Mori. 1992. Purification hericia Gould. Comp. Biochem. Plnxiol. 44A: 1 169-1 175. 

and immunoliK: ' i . itellin-like protein from the Pacific Zaba. B. M. 1981. Glycogenolytic pathways in the mantle tissue of 

oyster CrasM^-- <lar. Biol. 113: 239-245. Mytitiix eJiilix L. Mar. Biol. Lerr. 2: 67-74. 

Teo, L. H., and I . - f. 1990. Preliminary report on the digestive Zacks. S. I. 1955. The cytochemistry of the amoebocytes and intestinal 

enzymes pre-. . digestive gland of Perna viridis. Mar. Biol. 106: epithelium of Venn* niercenariii (Lamellibranchiata), with remarks on 

403-407. a pigment resembling ceroid. Q. J. Microsc. Sci. 96: 57-71. 



MBL 



annual report 2002 




MARINE BIOLOGICAL LABORATORY 
Woods Hole, Massachusetts 



The Marine Biological Laboratory does not discriminate in employment or in access to any of its activities or 
programs on the basis of race, color, religion, sex, sexual orientation, national origin, ancestry, age, marital status, 
pregnancy, physical or mental disability, or veteran status. In addition, the MBL is committed to the prevention and 
elimination of sexual harassment, as well as other forms of unlawful harassment, in the workplace. Through training 
programs and disseminated information, MBL strives to educate its employees, students, faculty, and visitors on 
these important issues. 



! ON THE COVER: Alpha ganglion cell retina, Peter Koulen 



The MBL Annual Report 2002 is published by the Marine Biological Laboratory. Although the greatest possible care 
has been taken in the preparation of this record, the Marine Biological Laboratory recognizes the possibility of 
omissions or inaccuracies. If any are noted, please accept our apology and advise us of any corrections to be made. 



Office of Communications and Public Affa 

Marine Biological Laboratory 

7 MBL Street 

Woods Hole, MA 02543 

Phone: (508) 289-7423 

Fax (508) 289-7934 

www mbl.edu 



rs 



Rl 




TABLE OF CONTENTS 



Dogfish thrombocytes, 
Kyeng-Gea Lee 



R2 REPORT OF THE DIRECTORS CEO 

R7 RESEARCH 

R44 EDUCATION 

R63 MBL/WHOI LIBRARY 

R66 FINANCIAL INFORMATION 

R69 GIFTS 

R85 GOVERNANCE & ADMINISTRATION 



R2 



report of the director and CEO 




I am pleased to share with you 
highlights from the Marine 
Biological Laboratory for the 
year 2002. I've now served as 
Director and CEO of the MBL 
for nearly three years. I 
continue to be impressed with 
the breadth of the science 
being done here, the impact that our basic 
research continues to have on human health 
as well as the environment, and the remark- 
able commitment our scientists, faculty, and 
staff have to their work and to the Marine 
Biological Laboratory itself. 

(Developing a Strategic Plan 

Much of my time in 2002 was devoted to 
directing, with MBL President John Dowling 
and the aid of the consulting firm McKinsey & 
Company, a planning effort aimed at devel- 
oping a strategic roadmap for the Laboratory 
over the next five to ten years. This process, 
and the recommendations emerging to date, 
are outlined in detail on our web site 
(www.mbl.edu/inside/what/planning/ 
index.html). Although we continue to fine- 
tune the plan, the initial phases of the 
process are largely complete. 

As a result of this effort, we are already 
undertaking a number of key initiatives. 
These include developing a graduate 
program with Brown University, hiring a Chief 
Academic and Scientific Officer, establishing 
a new resident research program in Cellular 
Dynamics and Imaging, expanding the Board 
of Trustees, preparing for major renovations 
of the Whitman Building for summer and 
visiting research, and working with a site 
planner to help us further reconfigure and 
renovate space to meet the emerging needs 
of the strategic plan. 



Developing the Laboratory's first strategic plan 
has been an exciting and illuminating process 
involving participation at all levels of the 
organization. I'm most grateful to everyone 
who has contributed to the experience. 



Establishing the Program in Global 
Infectious Diseases 

One of the highlights of the year was establish 
ing our newest resident research program in 
Global Infectious Diseases. Funded by a 
$5 million grant from the Ellison Medical 
Foundation, the Program links scientists who 
study disease-causing organisms with experts 
in molecular biology, phylogenetics, and 
environmental microbiology and creates a 
one-of-a-kind international center for research 
and training dedicated to studying pathogens 
and the complex relationships they have with 
their hosts. 

We were fortunate to recruit leading parasi- 
tologist and molecular biologist Stephen L. 
Hajduk to direct the Program. Steve comes to 
the MBL from the University of Alabama at 
Birmingham where he was a Professor in the 
Department of Biochemistry and Molecular 
Genetics and Senior Scientist in the AIDS 
Center and the Comprehensive Cancer Center. 
Steve brings with him a number of students, 
postdocs, and technicians. In addition, we 
welcomed two new Assistant Scientists 
Robert Sabatini and Andrew McArthur to the 
Program in early 2003. 

Steve's research program is broadly based in 
the area of molecular and biochemical basis of 
pathogenesis. Many of his studies focus on 
African trypanosomes, which cause human 
sleeping sickness, a fatal disease that has 
reemerged as a major health problem in 
sub-Saharan Africa. 



R3 



The importance of research in infectious disease 
cannot be overstated. No single reason can 
explain our inability to eradicate or minimize 
the global impact of infectious agents on 
human health. In total, 25% of all deaths 
worldwide are caused by bacterial, viral, fungal, 
and parasitic pathogens. Every year, one to 
three million people die from malaria, a disease 
caused by the organism Plasmodium. Tubercu- 
losis infects one person every second, and over 
the coming decade, at least 30 million will die 
from the disease. 

The Global Infectious Disease Program is part 
of the MBL's Josephine Bay Paul Center for 
Comparative Molecular Biology and Evolution. 
Directed by Mitchell Sogin, the Bay Paul Center 
has an active research program with strong ties 
to infectious disease. Bay Paul Center scientists 
have contributed many important insights 
about the evolution of parasitic protists using 
modern genomic approaches. Recently they 
embarked upon the sequence analysis of the 
G/ard/a genome. Excluding bacterial patho- 
gens, Giardia is a principal cause of diarrheal 
disease in children and adults and therapeutic 
treatments for the parasite are almost as 
devastating as the disease itself. The availabil- 
ity of the Giardia genome may lead to the 
identification of novel treatments that have 
minimal side effects. 

The technology necessary to sequence entire 
genomes is found at very few institutions. 
The MBL's Global Infectious Diseases Program 
will build on the unique expertise of the Bay 
Paul Center to provide more traditional 
parasitologists the opportunity to expand their 
research and better apply genomics to their 
research areas. This blending of genomics 
and parasitology will create a uniquely produc- 
tive research environment and provide the 
catalyst for exciting new discoveries on the 
important pathogens. 



The MBL has a rich history in studying the basic 
science of parasitism and infectious disease. 
Twenty years ago, the laboratory launched the 
field of molecular parasitology with the estab- 
lishment of its Biology of Parasitism course, 
which continuously reinvents itself as it trains 
new investigators in this ever-expanding field. 
The Global Infectious Diseases Program 
includes a strong training component that will 
allow tropical health and infectious disease 
scientists from the world to conduct research 
they would be unable to do at their home 
institutions. The Program's unique strengths lie 
within its capability to integrate visiting 
scientists into the lab and give them access to 
instruments and technology that would 
otherwise be unavailable to them. The broad 
impact that the training component will have on 
the field of infectious diseases globally is very 
exciting. 



| Other Resident Research Highlights 

The MBL's resident research program grew in 
other areas during 2002 as well. We welcomed 
Frederick Goetz in January, when he joined the 
staff of the Marine Resources Center (MRC) as 
Director of the Program in Scientific Aquacul- 
ture. Through exploring factors such as an 
organism's nutritional and water quality 
requirements, physiological characteristics, 
reproductive biology, diseases, and genetic 
background, the MBL's Scientific Aquaculture 
Program is developing novel research tech- 
niques and addressing problems faced by both 
scientific and commercial aquaculture interests. 
Rick is spearheading the Program's efforts to 
use DNA technology as a tool to understand 
the growth, reproduction, and disease resis- 
tance in commercially important fish and 
shellfish. He is also focusing on enhancing 
the MRC's culture and husbandry of biomedical 
models such as squid, clams, toadfish, 
and zebrafish. 

Continued... 




Heliozoan, Linda Amaral 
Zettler and Erik Zettler 




Clam egg, Robert Palazzo 



R4 




Woods Ho/e aerial, Doug Weisman 

Pigment cells on the surface of the cunner spinal cord, Steven Zottoli 

Gabriele Gerlach also joined the staff of the MRC 
in 2002 as an Associate Scientist. A specialist in 
behavioral ecology and population genetics, 
Gabby is using the zebrafish facilities at the MRC 
to explore the genetic basis of behavior. 

Scientists at The Ecosystems Center received 
strong support from the National Science 
Foundation through the competitive peer-review 
grant process again this year. Of special note is 
the $2.7 million, multi-year award in support of 
the Long-Term Ecological Research project at 
Plum Island Sound, a research site located north 
of Boston. Here scientists are studying the 
effects of land-use change on watersheds. The 
Center also received $850,000 from the Andrew 
W. Mellon Foundation to support research on 
nitrogen transformation in terrestrial landscapes. 



[Summer and Visiting Research 

During a typical Woods Hole summer, MBL 
researchers look for basic principles of life in 
organisms from squid to surf clams to zebrafish. 
They ask how nerve cells communicate, how cells 
regulate their complex processes, and how they 
proliferate. They explore how organisms repro- 
duce and develop, how they fight disease, how 
sense organs gather information, and how brains 
cess it. The investigators who gather each 
iner bring a diversity of approaches and 
questions to their research. In 2002 we wel- 
comed 129 principal investigators and 237 other 
researchers from 124 institutions representing 12 
countries. 



The MBL's summer and visiting research 
program was further strengthened in 2002 with 
a remarkable and farsighted gift of $2.3 million 
from long-time summer investigators Laura and 
Arthur Colwin. Their gift established the Laura 
and Arthur Colwin Endowed Summer Research 
Fellowship Fund, which, when mature, will 
provide full support for approximately 10 
independent investigators conducting research 
in the fields of cell and developmental biology 
at the MBL for a minimum of two months during 
the summer. The MBL is committed to ensuring 
that the very best scientists have the opportu- 
nity to conduct research here each summer. We 
can help do this by providing financial support 
in the form of fellowships like these. 

On the recommendation of the Science 
Council, the Laboratory reinstated the MBL 
Awards for outstanding presentations at the 
annual General Scientific Meetings in 2002. 
During the meeting, which was held in the Lillie 
Auditorium August 12 to 14, 56 presentations 
were made. After peer-review of all papers and 
talks, four awards and two honorable mentions 
were presented in the categories of Senior 
Investigator (Peter Armstrong), Junior Investiga- 
tor (Michael Smotherman), Graduate Student 
(Beate Mittman), and Undergraduate Student 
(Jane La Du). 



R5 



| Education 

The 2002 Education Program provided 499 
graduate and advanced-level students from 
288 institutions and 31 countries an opportu- 
nity to study a range of biological topics with 
some of the leading scientists in the world 
serving as course faculty and lecturers. The 
Laboratory welcomed 554 faculty members 
and staff and 203 lecturers to the courses in 
2002. They represented 175 institutions and 
33 countries. 

The MBL offered two new courses in 2002. 
"Advances in Genome Technology and 
Bioinformatics," was co-directed by Mitchell 
Sogin, director of the MBL's Bay Paul Center, 
and Claire M. Fraser of The Institute of 
Genomic Research. Twenty-four students from 
the U.S. and Europe participated in 
the inaugural course, held October 6 to 
November 1, 2002. The course integrates 
bioinformatics with the latest laboratory 
techniques for genome sequencing, genome 
analysis, and high-throughput gene 
expression. 

The second course, "Neuroinformatics," 
was directed by Partha Mitra of Lucent 
Technologies and Emery Brown of Massachu- 
setts General Hospital. Twenty-six students 
participated in that course, which was held 
August 17 to September 1. 

In addition to our programs for graduate 
students, the MBL also offers a "Semester in 
Environmental Science" program for under- 
graduates each fall. In 2002, 19 undergradu- 
ates from 14 colleges and small universities 
around the country participated in the pro- 
gram, which is hosted by The Ecosystems 
Center. The goal of the SES program, now 
in its 6 th year, is to help prepare the next 
generation of leaders in environmental 
science and policymaking. 



I MBL/WHOI Library 

The MBL/WHOI Library continues to grow in non- 
traditional directions. More and more informa- 
tion is being delivered directly to scientists' 
desktops via the Internet. In early 2002, the 
Library was delivering approximately 52% of its 
serials and 100% of its databases electronically, 
extending the Library's reach beyond its walls to 
wherever its patrons may be at any time of the 
day or night. 

In addition, the Library is actively involved in the 
development of new electronic research tools. 
Thanks to a grant of $500,000 from the Andrew 
W. Mellon Foundation, Library and IT staff are 
developing the Universal Biological Indexer and 
Organizer (uBio), an exciting new database and 
Internet tool that provides an innovative means 
of accessing, sorting, and collating taxonomic 
information contained in databases distributed 
throughout the Internet. 

A portion of the Library's archival collection was 
also featured during the summer of 2002 in the 
"Wall Charts as Art" exhibit in the Meigs Room. 
The exhibit showcased 1 5 life-size reproductions 
of the 1 16 charts produced by Rudolf Leukart 
between 1 877 and 1 892. Noted for their detail, 
these charts were used as teaching aids in 
universities around the world. The Archives has 
one of the few remaining complete collections 
of the original chromolithographs. We are 
grateful to Ann Weissmann for curating this 
outstanding exhibit. 



| Outreach 

During the fall of 2002, the MBL worked with 
Avari Studios to reorganize and redesign the 
MBL's web site. The new site was launched in 
January 2003 to very positive reviews. Early data 
show that the site is being visited by nearly 
50,000 unique users a month. We are continuing 
to build and enhance the site, which is becoming 
an increasingly important tool for institutional 
advancement, marketing, communications, and 
outreach. If you haven't seen the new site, I 
invite you to visit it at <www.mbl.edu>. 




Continued. . 



R6 




Eel Pond through the 
front doors of Ebert Hall 



[Construction and Facilities 

We were fortunate in 2002 to be able to 
Continue to fund depreciation and upgrade 
our facilities. Lab and office space in both the 
Marine Resources Center and the Crane Wing 
of the Lillie building were substantially 
renovated for new Marine Resources and 
Global Infectious Disease program staff. The 
Grass Fellows also enjoyed 1400 square feet 
of newly renovated laboratory space in the 
Whitman Building during the summer of 
2002. This fully modernized space was 
designed to meet the Fellows' research needs 
and also foster the cooperative and collegial 
nature of the program. Educational programs 
displaced by this renovation, including 
SPINES and a program for undergraduates 
from Williams College, were moved to the 
Loeb teaching building. 

The MBL also purchased a five-acre farm in 
Newbury, MA. The property will enable the 
expansion of the MBL's Plum Island Research 
Program, which focuses on understanding 
how coastal ecosystems are affected by 
changing land cover, climate, and sea level. 
Upon the departure of Ecosystems Center 
staff to the C.V. Starr Building, the Homestead 
Building was gutted and renovated for use by 
the administrative departments of Financial 
Services, Human Resources, Education, and 
The Biological Bulletin. The Candle House, 
now home to the Director's Office, Develop- 
ment Office, Associates Office, and Commu- 
nications and Public Affairs Office, also 
received a modest facelift in 2002. 



[Trustees 

The Board of Trustees elected five new 
members whose terms began in 2002. 
Margaret C. Bowles of Woods Hole, MA; 
Martha W. Cox of Hobe Sound, FL; Walter E. 
Massey of Atlanta, GA; Marcia C. Morris of 
Boston, MA; and Gerald Weissmann, M.D., of 
New York, NY, are all serving four-year terms 
as members of the Class of 2006. In addition, 
John E. Dowling, Mary B. Conrad, and 



Thomas S. Crane were elected to serve as 
President of the Corporation, Treasurer, 
and Clerk, respectively. Sheldon Segal was 
also elected to serve a final year as Chair of 
the Board. 

On November 8, 2002, Shelly stepped down as 
Chair of the Board and handed his virtual gavel 
to Al Zeien, the former Chairman and CEO of 
the Gillette Company, who assumed the duties 
of Chair at the February 2003 meeting of the 
Board. Shelly has served as a member of the 
Board for 20 years, 10 as its Chair. He will now 
serve as an Honorary Trustee. 

Shelly has been, and will continue to be, a vital 
force at the MBL. On behalf of the MBL 
community, I thank Shelly for all his efforts over 
these many years. I know we can count on his 
guidance and wisdom in the future. 



[ Conclusion 

We are living in challenging times, and the 
Marine Biological Laboratory is not immune to 
the impact that the downturn in the national 
economy is having on businesses, foundations, 
and state and local governments. Like every- 
one, we've had to tighten our belts and 
carefully consider the impact new initiatives 
may have on our budget. So far, the MBL has 
been able to weather the economic storm and 
continue to work in positive ways towards 
achieving the goals we've begun to establish 
through the strategic planning process. 

This is an exciting time for the Marine Biological 
Laboratory. I look forward to working with you 
as we further build upon the strengths of this 
vital institution to ensure that the Marine 
Biological Laboratory continues to have a 
disproportionate impact on the biological, 
biomedical, and environmental sciences long 
into the future. 

William T. Speck 



R7 




research 




Zebrafish cardiovascular 
system, Jonathan 
Muyskens 



Throughout its history, the MBL has been a place where the world's top biologists can focus on 
their research, not distracted by departmental affairs, committee work, or other aspects of 
university life. The MBL provides both the resource support and the intellectual environment 
that enables many scientists to do their best work. 

Today 47 principal investigators and and their staff conduct research at the Laboratory year- 
round in areas such as cellular, developmental, and reproductive biology; molecular biology and 
evolution; neurobiology and sensory physiology; ecology and ecosystems studies; global 
infectious diseases; and marine biotechnology and aquaculture. 

The population of investigators grows dramatically each summer when hundreds of distin- 
guished scientists from around the world gather here to do research. During a typical MBL 
summer, researchers look for basic principles of life in organisms from squid to surf clams to 
zebrafish. They ask how nerve cells communicate, how cells regulate their complex processes, 
and how they proliferate. They explore how organisms reproduce and develop, how they fight 
disease, how sense organs gather information, and how brains process it. The investigators who 
gather each summer bring a diversity of approaches and questions. Along with the large 
number of faculty associated with the summer courses, they make the MBL the largest and most 
exciting biological laboratory in the world. 



Top photo, Elizabeth Armstrong 



R8 



Resident Research 



THE ECOSYSTT 5 CENTER 



The Ecosystems Center, founded in 1975, is a collegia! association of 
scientists led by co-directors John Hobbie and Jerry Melillo. Its 
mission is to understand how ecosystems are structured and how they 
function, to predict their response to changing environments, to apply 
the best scientific knowledge to the preservation and management of 
natural resources, and to educate scientists and citizens of the future. 

In 2002, the Center continued 
the Semester in Environmental 
Science. This program brings 
undergraduates from a consor- 
tium of nearly 60 small liberal 
arts colleges and universities to 
the MBL campus for an inten- 
sive introduction to environmen- 
tal sciences. 

The complex nature of modern 
ecosystems research requires a 
multi-disciplinary collaborative 
approach to address a variety of 
questions. Accordingly, Center 
scientists collaborate on more 
than 60 projects. We conduct 
our field studies in many 
locations, from the North 

American and European Arctic to Brazil, from the temperate forests of 

New England to the estuaries of the eastern U.S. 

One question addressed in 2002 was the effect that a warmer climate 
will have on the high amounts of organic matter accumulated in forest 
soils over the centuries. If microbes decompose most of the organic 
matter, the forests would switch from a global sink of carbon dioxide 
gas to a source causing an acceleration of global warming. Jerry 
Melillo and Paul Steudler of the Center have collaborated with Univer- 
sity of New Hampshire scientists in a decade-long experiment in which 
the soil of 6 x 6 m plots was heated 5 above the temperature of similar 
control plots. They found that soil warming did accelerate soil organic 
matter decay and carbon dioxide fluxes to the atmosphere but that the 
response was small and short-lived because microbes were able to 
decompose only a small proportion of the total organic matter. 




The Ecosystems Centers C V. Starr Building 




Eel grass. Rick Crawford 



CO-DIRECTORS 
John E. Hobbie 
Jerry M. Melillo 

SENIOR SCIENTISTS 
John E Hobbie 
Charles S. Hopkinson 
Jerry M. Melillo 
Knute J. Nadelhoffer 
Bruce J. Peterson 
Edward B Rastetter 
Gaius R Shaver 

ASSOCIATE SCIENTISTS 
Linda A- Deegan 
Anne E. Giblin 

ASSISTANT SCIENTISTS 
Christopher Neill 
Joseph J. Vallino 

SENIOR RESEARCH 
SPECIALIST 
Paul A Steudler 



R9 




Robert Holmes at the Yem'sey River, Russia 



International Study Shows River Discharge 
in Arctic Ocean is Increasing 



Another question investigated microbes in nature. 
These are responsible for many of the transformations in 
the carbon and nitrogen cycles that control important 
ecological processes such as carbon storage and nutrient 
recycling. This study looked at the link between the 
structure of the microbial populations in an Arctic lake and 
seasonal changes. This research, carried out by Byron 

Crump and John Hobbie 
of the Center, could only 
be done with the assis- 
tance in molecular tech- 
niques from Mitch Sogin of 
the Bay Paul Center. New 
techniques allowed the 
identification of microbes 
and species changes, 
which is the first step 
towards the long-term goal 
of linking species to 

ecological function. The study, the first of its kind for 
lakes, revealed that there was a resident population of 
bacterial species throughout the year. However, a 
distinctly different group of species appeared when large 
amounts of organic matter entered the lake during the 
spring meltwater runoff. This organic matter, and perhaps 
the bacteria, came from the plant litter and soil in the 
surrounding watershed. 




An Ecosystems scientist 
collects samples from the 
Kuparuk River in Alaska 



Researchers from The Ecosystems Center, along with an interna- 
tional team of hydrologists and oceanographers, have docu- 
mented that the flow of freshwater from Arctic rivers into the 
Arctic Ocean has increased significantly over recent decades. 
If the trend continues, some scientists predict this could impact 
the global climate, perhaps leading to the cooling of 
Northern Europe. 

Ecosystems Center researchers Bruce Peterson, Robert (Max) 
Holmes, and James McClelland led the team of scientists from 
the United States, Russia, and Germany. They analy2ed discharge 
data from the six largest Eurasian rivers that drain into the Arctic 
Ocean. These rivers, all located in Russia, account for more than 
40% of total riverine freshwater inputs to the Arctic Ocean. 

Peterson and his colleagues found that combined annual 
discharge from the Russian rivers increased by 7% from 1936 to 
1999. They contend that this measured increase in runoff is an 
observed confirmation of what climatologists have been saying 
for years that freshwater inputs to the Arctic Ocean and North 
Atlantic will increase with global warming. "If the observed 
positive relationship between global temperature and river 
discharge continues into the future, Arctic river discharge may 
increase to levels that impact Atlantic Ocean circulation and 
climate within the 21st century," says Peterson. 

Continued. . . 



This project was funded by the Arctic System Science Program of the 
National Science Foundation. 



RIO 



Arctic River Discharge, continued 



A significant increase v.vater flow to 

the Arctic Ocear : }wn or shut 

off the formation :3ntic Deep 

Water. The drivir ! -ehind the great 

underwater " -sit" current known 

as thermoh.- Nation. Thermohaline 

circuL risible for moving great 

amounts c mal energy around the 
globe, influencing the planet's climate. 
One of the potential effects could be 
cooling of Northern Europe. 

Data analyzed in this study, published in 
the December 13, 2002, issue of Science 
magazine, is important because it 
represents net precipitation (precipitation 
minus evapotranspiration) over a vast area, 
in contrast to point measurements of 
precipitation and evapotranspiration which 
are difficult to extrapolate to a large area. 

"These data are a unique measure of an 
environmental trend both in terms of how 
long the time series is and in that it 
integrates over a vast area rather than just 
measuring a precipitation trend at a few 
locations," says co-author Stefan 
Rahmstorf of the Potsdam Institute for 
Climate Impact Research. 

Project scientists are hopeful that this 
study, which links the work of hydrologists 
and oceanographers, will stimulate the two 
fields of science to better communicate 
their scientific findings with each other. The 
group will focus their future work on the 
links between the atmospheric, continen- 
tal, and oceanic components of the Arctic 
hydrologic cycle and on the biogeochemi- 
cal tracers that allow scientists to follow 
the circulation of riverine freshwater 
throughout the northern oceans. This 
research is needed to better understand 
the current functioning of the linked land- 
ocean-atmosphere hydrologic system and 
improve confidence in predictions of the 
future behavior of the vvstem. 



A researcher from . .';: . osystems 
Center collects water from a stream in 
the Ipswich River watershed in the Plum 
Island Ecosystem Long Term Ecological 
Research site in northeastern 
Massachusetts. Investigators study 
different land uses and their impacts on 
nutrient loading into the estuaries of 
Plum Island Sound. 



| Staff 



RESEARCH STAFF 
Toby Ahrens, Research Assistant 
Michele P. Bahr. Research Assistant 
Jonathan P. Benstead, Postdoctoral Scientist 
Neil D. Bettez. Research Assistant 
Zy F. Biesinger, Research Assistant 
Mary S. Booth, Postdoctoral Scientist 
Laura C. Broughton, Postdoctoral Scientist 
Donald W. Burnette, Research Assistant 
Elizabeth H. Burrows, Research Assistant 
Alvarus S. K. Chan. Postdoctoral Scientist 
Benjamin P. Colman, Research Assistant 
Christopher P. Crockett, Research Assistant 
Byron Crump, Postdoctoral Scientist 
Jeffrey A. Evans, Research Assistant 
Benjamin Felzer, Research Associate 
Solange Filoso, Postdoctoral Scientist 
Robert H Garritt, Research Assistant 
Marcus O. Gay, Research Assistant 
Joshua H. Goldstein, Research Assistant 
Adrian C- Green, Research Assistant 
Heather Haas, Postdoctoral Scientist 
Diana C Garcia-Montiel, Staff Scientist 
Darrell A. Herbert, Staff Scientist 
Robert M. Holmes, Staff Scientist 
Shaomin Hu, Research Assistant 
Jeffrey E. Hughes, Staff Scientist 
Samuel Kelsey, Research Assistant 
David W. Kicklighter, Research Associate 
Bonnie L- Kwiatkowski, Research Assistant 
James A Laundre, Research Assistant 
Corey R. Lawrence, Research Assistant 
John M. Logan, Research Assistant 
Ann L. Lezberg, Research Assistant 
Heidi Lux, Research Assistant 
Roxanne Marino, Staff Scientist 
James W McClelland, Postdoctoral Scientist 
Patricia Micks, Research Assistant 
Sarah Morrisseau. Research Assistant 
Marshall L Otter. Research Assistant 
Suzanne Randazzo, Research Assistant 
Heather M. Rueth, Postdoctoral Scientist 
Diane M Sanzone, Postdoctoral Scientist 
Carol Schwamb, Laboratory Assistant 
Karie A. Slavik, Research Assistant 



Martin Sommerkom, Postdoctoral Scientist 
Erica L. Stieve, Research Assistant 
Kristin S. Tholke, Research Assistant 
Suzanne M Thomas, Research Assistant 
Craig R. Tobias, Postdoctoral Scientist 
Jane Tucker. Research Assistant 
Zhenwen Wan, Postdoctoral Scientist 
Ian J. Washboume, Research Assistant 
Michael R Williams, Postdoctoral Scientist 
Yuriko Yano, Postdoctoral Scientist 
Qianlai Zhuang, Postdoctoral Scientist 

ADJUNCT SCIENTISTS 
Robert W. Howarth, Cornell University 
Paul A. Colinvaux. Smithsonian Tropical 
Research Institute (retired) 

VISITING SCIENTISTS 

James Galloway, University of Virginia 

Robert Naiman, University of Washington 

ADMINISTRATIVE STAFF 

Kenneth H. Foreman, Associate Director, 

Semester in Environmental Science Program 
Dorothy J Berthel, Administrative Assistant 
Anthony J Cave, Research Administrator 
Suzanne J Donovan, Executive Assistant 
Frances Johnson-Horman, Administrative 

Assistant 

Guillermo Nunez. Research Administrator 
Deborah G Scanlon, Executive Assistant 
Mary Ann Seifert, Administrative Assistant 

CONSULTANTS 

Francis P. Bowles, Research Designs 

Margaret C. Bowles 



Boardwalks protect vegetation from distur- 
bance at research sites in the Arctic heath and 
mountain birch ecosystem around the Abisko 
Naturvetenskapliga Station in northern 
Sweden. Plots shown here are part of a pilot 
soil-warming project begun dunng 1 993 at 
Abisko, Rose Crabtree 




Rll 




Publications 



Barren, S., C. F. Weber, R. Marino, E. A. 
Davidson, G. Tomasky, and R. W. Howarth. 
2002. Effects of varying salinity on phytoplank- 
ton growth in a low-salinity coastal pond under 
two nutrient conditions. Bfol. Bull. 203: 260-261 . 

Bashkln, V. N., and R. W. Howarth. 2002. 
Modern Biogeochemistry. Kluwer, Dordrecht. 
S61 pp. 

Bettez, N., P. Rublee, W. J. O'Brien, and M. C. 
Miller. 2002. Changes in abundance, composi- 
tion and cont rols within the plankton of a 
fertilized arctic lake. Freshw. Bio/. 47: 303-311. 

Boyer, E. W., and R. W. Howarth, eds. 2002. 
Global and Regional Synthesis of the Nitrogen 
Cycle. Kluwer, Dordrecht. 561 pp. 

Boyer, E. W., C. L Goodale, N. A. Jaworski, and 
R. W. Howarth. 2002. Anthropogenic nitrogen 
sources and relationships to riverine nitrogen 
export in the northeastern U.SA Biogeochem- 
istry 57/58: 137-169. 

Bret-Harte, M. S., G. R. Shaver, and F. S. 
Chapin, III. 2002. Primary and secondary stem 
growth in arctic shrubs: implications for 
community response to environmental change. 
J.Ecol. 90:251-267. 

Bush, M. B., M. C. Miller, P. E. De Oliveira, and 
P. A. Colinvaux. 2002. Orbital forcing signal in 
sediments of two Amazonian lakes. J. 
Paleolimnol . 27: 341-352. 

Clein, J. S., A. D. McGuire, X. Zhang, D. W. 
Kicklighter, J. M. Mellllo, S. C. Wofsy, P. G. 
Jarvis, and J. M. Massheder. 2002. Historical 
and projected carbon balance of mature black 
spruce ecosystems across North America: the 
role of carbon-nitrogen interactions. Plant Soil 
242:15-32. 



Currle, W. S., and K. J. Nadelhoffer. 2002. The 
Imprint of land use history: patterns of carbon 
and nitrogen in downed woody debris at the 
Harvard Forest. Ecosystems 5:446-460. 

Currie, W. S., K. J. Nadelhoffer, and B. Colman. 
2002. Long-term movement of 1 5N tracers Into 
fine woody debris under chronically elevated N 
inputs. Plant Soil 238: 31 3-323. 

Dargaville, R. J., M. Heimann, A. D. McGuire, I. 
C. Prentice, D. W. Kicklighter, F. Joos, J. S. 
Clein, G. Esser, J. Foley, J. Kaplan, R. A. Meier, 
J. M. Melillo, B. Moore III, N. Ramankutty, T. 
Reichenau, A. Schloss, S. Sitch, H. Tian, L. J. 
Williams, and U. Wittenberg. 2002. Evaluation 
of terrestrial carbon cycle models with 
atmospheric C0 2 measurements: results from 
transient simulations considering increasing 
CO , climate and land-use effects. Global 
Biogeochem. Cycles 16: 1092. DOI:10.1029/ 
2001 GM001 426. 

Deegan, L. A. 2002. Lessons learned: the effects 
of nutrient enrichment on the support of nekton 
by seagrass and salt marsh ecosystems. Special 
SCOR Volume. Estuaries 25: 
727-742. 

Deegan, L. A., A. Wright, S. G. Ayvazian, J. T. 
Finn, H. Golden, R. R. Merson, and J. Harrison. 
2002. Nitrogen loading alters seagrass 
ecosystem structure and support 
of higher trophic levels. Aquat. Conserv. Mar. 
Freshw. Ecosyst. 12: 193-212. 

Driscoll, C, D. Whitall, J. Aber, E. Boyer, M. 
Castro, C. Cronan, C. Goodale, P. Groffman, K. 
Lambert, G. Lawrence, C. Hopkinson, and S. 
OHinger. 2002. Nitrogen Pollution: From the 
Sources to the Sea. Science Links - Hubbard 
Brook Research Foundation, Hanover, NH. 



The Ecosystems Center's 
Long-Term Ecological 
Research site at Toolik 
Lake, Alaska, Knute 
Nadelhoffer 



Felzer, B. S., D. W. Kicklighter, J. M. Mellllo, C. 
Wang, Q. Zhuang, and R. G. Prlnn. 2002. Ozone 
Effects on Net Primary Production and Carbon 
Sequestration In the Conterminous United States 
Using a Biogeochemistry Model. Report No. 90, 
MIT Joint Program on the Science and Policy of 
Global Change, Cambridge, MA. 

Garcia-Montiel, D. C. 2002. Legacy of human 
activity in the present neotropical forests. Pp. 97- 
1 16 in Ecology and Conservation of Neotropical 
Forests, M.R. Guariguata and G.H. Kaftan, eds. 
Libro Universitario Regional, Cartago, Costa Rica, 
(In Spanish) . 

Garcia-Montiel, D. C., J. Melillo, P. A. Steudler, 
and C. Neill. 2002. Relationship between N 2 O and 
C0 ; emissions from the Amazon Basin. Geophys. 
Res. Lett. 29: 14.1 -14.3. 

Goodale, C. L., K, Lajtha, K. J. Nadelhoffer, E. W. 
Boyer, and N. A. Jaworski. 2002. Forest nitrogen 
sinks in large eastern U. S. watersheds: inventory 
and modeled estimates. Biogeochemistry 57/58: 
239-266. 

Gough, L., P. A. Wookey, and G. R. Shaver. 2002. 
Dry heath arctic tundra responses to long-term 
nutrient and light manipulation. Arct. Antarct. Alp. 
Res. 34:211-218. 

Grams, T. E. E., A. R. Kozovits, I. M. Reiter, J. B. 
Winkler, M. Sommerkorn, H. Blaschke, K.-H. 
Haberle, and R. Matyssek. 2002. Quantifying 
competitiveness in woody plants. Plant Biol. 4: 
153-158. 

Hobbie, S. E., K. J. Nadelhoffer, and P. Hogberg. 
2002. A synthesis: the role of nutrients as 
constraints on carbon balances in boreal and arctic 
regions. Plant Soil 242: 163-170. 

Continued... 



R12 



Holmes. R. M.. J W. McClelland. B. J Peterson. I. 
A Shiklomanov, A. I Shiklomanov, A. V. Zhulidov, 
V. V. Gordeev. and N. N. Bobrovitskaya. 20C2. A 
circumpolar perspective on fluvial sedimen: flux to 
the Arctic Ocean Global B/o-jeochem. 
10 1029/2001 GB001849 

Hopkinson, C. S , J. J. V?'! 

2002 Decomposition of 3 ganic matter 

from the contine - -a Res. II 49: 

4461-4478 

Howarth. R _ nitrogen cycle. 

Pp. 429-435 . edia of Global Environ- 

mental Change. Vol. 2, The Earth System: 
Biologies! and Ecological Dimensions of Global 
Environmental Change, H. A. Mooney and J.G 
Canadell, eds. Wiley, Chichester. 

Howarth. R. W. 2002. Nutrient Over-Enrichment 
of Coastal Waters in the United States. Steps 
Toward a Solution. Pew Oceans Commission, 
Washington. DC. 

Howarth, R. W., E. W Boyer, W. J. Pabich, and J. 
N. Galloway. 2002. Nitrogen use in the United 
States from 1961-2000 and potential future 
trends Ambio 31 : 88-96 

Howarth, R. W., and D. M. Rielinger. 2002. 
Nitrogen From the Atmosphere. Understanding 
and Reducing a Major Cause of Degradation of 
our Coastal Waters. Science and Policy Bulletin 
No 8, Waquoit Bay National Estuarine Research 
Reserve, Waquoit. MA. 

Howarth, R. W., D. Scavia, and R. Marino. 2002. 
Nutrient Pollution in Coastal Waters: Priority 
Topics for an Integrated National Research 
Program for the United States. National 
Oceanic and Atmospheric Administration, 
Silver Springs, MD. 



Howarth. R. W.. D Walker, and A Sharpley 2002 
Sources of nitrogen pollution to coastal waters of 
the United States Estuaries 25: 656-676. 

Hughes. J. E,. L A. Deegan, J C Wyda. and A. 
Wright. 2002. An index of biotic integnty based on 
fish community structure applied to Rhode Island 
and Connecticut estuaries. Pp. 49-58 in Proceed- 
ings of the Fifth Biennial Long Island Sound 
Research Conference 2000, M Van Patten, ed. 
Connecticut Sea Grant Publication CTSG-01-02. 
Stamford, CT. 

Hughes, J. E., L. A Deegan, M. J. Weaver, and J 
E. Costa. 2002 Regional application of an index of 
estuarine biotic integrity based on fish 
communities. Estuaries 25: 250-263. 

Hughes, J. E.. L A Deegan, J C Wyda, M J 
Weaver, and A. Wright 2002. The effects of 
eelgrass habitat loss on estuarine fish communities 
of southern New England Estuaries 25: 
235-249 

Hunter- Thomson, K.. J. Hughes, and B. Williams. 
2002. Estuarine-open-water comparison offish 
community structure in eelgrass (Zostera marina L.) 
habitats of Cape Cod Biol. Bull. 203: 247-248. 

Koop-Jakobsen, K., and A. Giblin. 2002. Tidal 
flushing of ammonium from intertidal salt marsh 
sediments: The relative importance of adsorbed 
ammonium. 810). Bull. 203: 258-259. 

LaMontagne, M. G., A. E Giblin, and I. Valiela. 
2002. Denitrification and the stoichiometry of 
nutrient regeneration in Waquoit Bay. MA. 
Estuaries 25: 272-281. 

Levine, U. Y., and B. C Crump 2002. 
Bacterioplankton community composition in 
flowing waters of the Ipswich River watershed. 
Biol. Bull. 203:251-252 




Marino, R., F. Chan, R. W Howarth. M Pace, and 
G. E Ukens 2002. Ecological and biogeochemi- 
cal interactions constrain planlctonic nitrogen 
fixation in estuaries. Ecosystems 5: 719-725. 

Mayer. B , E. W. Boyer. C. Goodale. N. A. 
Jaworski, N van Breemen, R. W. Howarth, S P 
Seitzmger, G Billen, K Lajtha, K. Nadelhoffer, D. 
van Dam, L. J Hetlmg, M. Nosil, K. Paustian, and 
R. Alexander. 2002 Sources of nitrate in rivers 
draining sixteen watersheds in the northeastern 
US isotopic constraints Siogeochemistry 57/58: 
171-197. 

Mayer, B., N. Jaworksi, E Boyer, R. Howarth, C 
Goodale, L Hetling, S. Seitzinger, G. Billen, R 
Alexander. N. van Breemen. K Paustian. D. van 
Dam, K Lajtha, and K Nadelhoffer 2002 On 
the feasibility of using the nitrogen and oxygen 
isotope ratios of nitrate for describing the origin 
of riverine nitrate and N transformations in large 
watersheds. Siogeochemistry 57/58: 238-266. 

McClelland, J W., and J P. Montoya 2002. 
Trophic relationships and the nitrogen isotopic 
composition of amino acids in plankton. Ecology 
83:2173-2180. 

McGuire. A D., C Wirth, M. Apps, J Bennger. J 
Clein. H. Epstein, D W Kicklighter, J. Bhatti, F. 
S. Chapin III, B de Groot, D Efremov, W. 
Eugster, M Fukuda, T Gower. L. Hinzman, B 
Huntley. G J Jia, E. Kasischke, J Melillo, V. 
Romanovsky, A Shvidenko, E Vaganov, and D. 
Walker 2002 Environmental variation, 
vegetation distribution, carbon dynamics and 
water/energy exchange at high latitudes J Veg. 
Sci. 13 301-314 

McKane, R. B., L. C. Johnson, G. R Shaver, K J. 
Nadelhoffer, E. B Rastetter, B. Fry, A. E. Giblin, 
K. Kielland, B. L Kwiatkowski, J. A Laundre, and 
G Murray 2002 Resource-based niches provide 
a basis for plant species diversity and dominance 
in arctic tundra Nature 415: 68-71. 

Melillo. J M. 2002 How earnest thou in this 
pickle? Pp. 23-31 in Engineering and Environ- 
mental Challenges: Technical Symposium on 
Earth Systems Engineering. National Academy 
Press, Washington. DC. 

Melillo, J. M , and E. B. Cowling 2002 Reactive 
nitrogen and public policies for environmental 
protection. Ambio 31 : 1 50-1 58 

Melillo, J M..O. Sala, eta/. 2002 Ecosystem 
services. Pp. 13-18 in Biodiversity: Its Importance 
to Human Health, E. Chivian, ed. Center for 
Health and the Global Environment, Harvard 
Medical School. Boston, MA 



Bruce Peterson looks for 
bryophytes and filamentous algae 
in the mountain stream at the 
Ivishak IS N addition site in the 
Arctic National Wildlife Refuge, 
Laura Broughton 



R13 



Mehllo, J M , P. A. Steudler, J. D Aber, K Newkirk. H. Lux, 
F P. Bowles, C Catricala, A. Magill, T. Ahrens, and S. 
Mornsseau 2002. Soil warming and carbon-cycle feedbacks 
to the climate system. Science 298: 2173-2176 

Mulholland, P J.J L Tank, J R Webster, W. B. Bowden, 
W. K Dodds, S V Gregory. N. B. Grimm, S K Hamilton, S. 
L. Johnson, E. Marti, W. H. McDowell, J L. Merriam, J. L. 
Meyer. B. J. Peterson. H. M. Valett, and W M. Wollheim 
2002 Can uptake length in streams be determined by 
nutrient addition experiments 7 Results from an interbiome 
comparison study J. North Am Benthol. Soc. 21: 544-560 

Nadelhoffer K J., L Johnson, J. Laundre, A. E. Giblin, and 
G R Shaver. 2002 Fine root production and nutrient use in 
wet and moist arctic tundras as influenced by chronic 
fertilization. Plant Soil 242: 107-113. 

Novak, J M., and A. S. K. Chan 2002. Development of P- 
hyperaccumulator plant strategies to remediate soils with 
excess P concentrations. Grit. Rev. Plant So. 21 : 493-509 

Pan, Y , A. D. McGuire, J. M. Melillo, D W. Kicklighter, S. 
Sitch, and I. C. Prentice. 2002. A biogeochemistry-based 
successional model and its application along a moisture 
gradient in the continental United States. J. Veg. Sci. 13: 
369-382 

Peterson, B J., R. M. Holmes, J. W McClelland, C. J. 
Vorosmarty. I. A Shiklomanov, A. I Shiklomanov, R. B 
Lammers, and S Rahmstorf 2002. Increasing river discharge 
to the Arctic Ocean. Science 298: 2171-2173. 

Rastetter, E B., and G. I. Agren 2002. Changes in individual 
allometry can lead to species coexistence without niche 
separation Ecosystems 5: 789-801. 

Rueth, H. H., and J. S Baron. 2002. Differences in 
Englemann spruce forest biogeochemistry east and west 
of the Continental Divide in Colorado, USA. Ecosystems 
5 45-57 

Rueth, H. M., J. S. Baron, and L. A. Joyce. 2002. Natural 
resource extraction: past, present and future Pp 85-112 in 
Rocky Mountain Futures. An Ecological Perspective, J. S 
Baron, ed Island Press, Washington, DC 

Scavia, D., J C. Field, D. Boesch, R. Buddemeier, V. Burkett, 
D. Canyan, M Fogarty, M. A. Harwell. R. W. Howarth, C. 
Mason, D. J Reed, T. C. Royer, A H. Sallenger, and J. G. 
Titus 2002. Climate change impacts on U.S. coastal and 
marine ecosystems Estuaries 25: 149-164. 

Schmidt, I. K., S. Jonasson, G Shaver, A Michelsen, 
and A Nordin. 2002 Mineralization and allocation of 
nutrients by plants and microbes in four arctic ecosystems: 
responses to warming Plant Soil 242: 93-106 

Seitzinger, S. P., R V. Styles, E W Boyer, R. Alexander, G. 
Billen, R. Howarth, B. Mayer, and N. van Breemen, 2002. 
Nitrogen retention in rivers: model development and 
application to watersheds in the northeastern U.S. 
Biogeochemistry 57/58: 199-237 



Steudler, P. A , D C Garcia-Montiel, M C Piccolo, C. 
Neill, J. M. Melillo, B. J Feigl, and C. C. Cerri. Trace gas 
responses of tropical forest and pasture soils to N and P 
fertilization. Global Biogeochem Cycles 16: 10 1029/ 
2001 GB001 394. 

Twichell, S., S. Sheldon, L. Deegan, and R. Garritt. 2002. 
Nutrient and freshwater inputs from sewage effluent 
discharge alter benthic algal and infaunaf communities in a 
tidal salt marsh creek. Blot. Bull. 203 256-258 

van Breemen, N , E W Boyer, C. L. Goodale, N A 
Jaworski, K. Paustian, S P. Seitzinger, K Lajtha, B. Mayer. 

D. van Dam, R. W. Howarth, K, J Nadelhoffer, M. Eve, and 
G. Billen Where did all the nitrogen go? Fate of nitrogen 
inputs to large watersheds in the northeastern U.S A. 
Siogeochemistry 57/58: 267-293 

Vitousek, P. M., K. Cassman, C. Cleveland, T. Crews, C. B. 
Field, N. B. Grimm. R. W. Howarth, R Marino, L. Martinelli. 

E. B. Rastetter, and J. I. Sprent. 2002. Towards an 
ecological understanding of biological nitrogen fixation. 
Biogeochemistry 57-58: 1-45. 

Weber, C. F., S. Barron. R. Marino, R W. Howarth, G. 
Tomasky, and E. A. Davidson. 2002. Nutrient limitation of 
phytoplankton growth in Vineyard Sound and Oyster 
Pond, Falmouth, Massachusetts. Bio/. Bull. 203: 261-263 

Williams, B. S., J. E. Hughes, and K. Hunter-Thomson. 
2002. Influence of epiphytic algal coverage on fish 
predation rates in simulated eelgrass habitats Biol. Bull. 
203: 248-249 

Williams, M.. Y. Shimabokuro, D. A. Herbert, S. Pardi- 
Lacruz, C. Renno, and E. B. Rastetter. 2002 
Heterogeneity of soils and vegetation in eastern 
Amazonian rain forest: implications for scaling up biomass 
and production. Ecosystems 5: 692-704. 

Wyda. J C . L A. Deegan, J. E. Hughes, and M. J. 
Weaver. 2002. The response of fishes to submerged 
aquatic vegetation complexity in two ecoregions of the 
mid-Atlantic Bight: Buzzards Bay and Chesapeake Bay 
Estuaries 25: 86-100 

Zhuang. Q., A. D. McGuire, J. Harden, K. P. O'Neill, and J. 
Yane 2002. Modeling the soil thermal and carbon 
dynamics of a fire chronosequence in interior Alaska. J. 
Geophys. Res. 107: 8147. DOI: 10.1029/2001JD001244. 

Zwart, G-, B C. Crump, M. Agterveld, F. Hagen, and S. K 
Han. 2002. Typical freshwater bacteria an analysis of 
available 1 6S rRNA gene sequences from plankton of 
lakes and rivers Aquat. Microb. Ecol. 28: 141-155. 




Scientists collect samples 
at the spring stream at the 
Ivishafc IS N addition site in 
the Arctic National 
Wildlife Refuge, 
Laura Brougnton 



R14 



JOSEPHINE BAY F CENTER FOR 
MOLECULAR E OGY AND EVOLUTION 



DIRECTOR 
Mitchell Sogin 

SENIOR SCIENTISTS 
Stephen Hajduk 
Monica Riley 

ASSISTANT SCIENTISTS 
Michael Cuirtmings 
Robert Sabatmi 
Jennifer Wernegreen 




Ocean-dwelling ancantnanan, 
Linda Amaral Zettler 



The underlying theme of the Josephine 
Bay Paul Center is to explore the 
evolution and interaction of genomes 
of diverse organisms that play signifi- 
cant roles in environmental biology and 
human health. This dynamic research 
program integrates the powerful tools 
of genome science, molecular 
phylogenetics, and molecular ecology 
to advance our understanding of how 
living organisms are related to each 
other, to provide the tools to quantify and 
assess biodiversity, and to identify genes and 
underlying mechanisms of biomedical 
importance. 

Three interlocking programs define the scope 
of research in the Bay Paul Center. They are 
the Program in Global Infectious Diseases, the 
Program in Molecular Evolution, and the 
Program in Molecular Microbial Diversity. This 
past year has marked significant growth in the 
Bay Paul Center. We attracted Mat Meselson's 
molecular evolution program to the MBL. 
Meselson is an esteemed member of the 
National Academy of Science and in collabo- 
ration with David Mark Welch and Jessica 
Mark Welch, he has established a molecular 
evolution group in the Bay Paul Center that 
explores genome evolution in asexual rotifers. 

A generous award by the Ellison Medical 
Foundation allowed us to move forward with a 
dramatically expanded program in Global 
Infectious Diseases. This grant provided 
support for a major renovation that accommo- 
dates 24 scientists and visitors to the Bay Paul 
Center. Dr. Steve Hajduk is the director of this 
new initiative and has brought six graduate 
and post doctoral fellows to the MBL. His 
research emphasizes the post transcriptional 
processing of RNA in African trypanosomes, 
the cause of human sleeping sickness. RNA 




Giardia lamblia. Barb Davids (UCSD) 



Life at the Extremes Molecular Technology 
Uncovers Astonishing Diversity in Spain's 
"River of Fire" 

Living conditions are tough for bacteria, algae, and 
other microscopic organisms in the Rio Tinto, the 
highly acidic, vividly crimson river that flows through 
the countryside of southwestern Spain. Mined 
since 3000 B.C., the Rio Tinto contains heavy metal 
concentrations that are several orders of magnitude 
higher than those of typical fresh water. New 
findings from the Rio Tinto, published in the May 9, 
2002, issue of the journal Nature, present the first 
molecular description of eukaryotes in a highly 
acidic, high metal environment and reveal the 
River's incredible eukaryotic diversity. The results 
show that adaptation to extreme conditions is much 
more widespread than originally expected and 
provide a new understanding of the range of 
organisms capable of living at life's extremes and 
perhaps on other planets. 

Eukaryota describes those organisms whose genetic 
material is contained within a membrane-bound 
nucleus. This includes plants, animals, and humans. 
Previous studies of the Rio Tinto relied on morphol- 
ogy to describe the river's diversity and alerted 
scientists to only a few of the evolutionary similari- 
ties between its eukaryotic organisms. By examin- 
ing the DNA of organisms extracted from the Rio 
Tmto's sediment and biofilm, the slimy substance 
that coats the surface of the River's water and rocks, 
scientists have uncovered new eukaryotic lineages 



R15 



editing results in the post-transcriptional insertion or deletion of 
nucleotides into mRNA, at specific sites, creating functional open 
reading frames. It has been suggested that the enzymes involved in 
RNA editing might be potential targets for drug development. As part 
of this major expansion we have recruited two other new investigators 
to the GID program. Dr. Andrew McArthur has been a Staff Scientist at 
the MBL since 2000. Andrew directs NIH-funded programs to explore 
gene expression during different stages of the life cycle of the parasite 
Giardia lamblia and in Trypanosoma brucei. Robert Sabatini studies 
the role of unusual base modifications in trypanosomes referred to as 
"X-base." The enzymes involved in these genetic alterations may be 
unique to Trypanosomes and therefore may serve as valuable targets 
for drug design. 

Finally, we offered a new course titled Advances in Genome Sciences 
and Bioinformatics, which is co-directed by Mitchell Sogin of the MBL 
and Claire Fraser from The Institute of Genome Research (TIGR). 
Important research publications this past year include descriptions of 
eukaryotic microbial populations that thrive in very acidic environments 
(pH levels between 1 .7 and 2.2) in the presence of iron concentrations 
that can exceed 20 milligrams/ml; descriptions of eukaryotic diversity in 
warm, anoxic sediments that are proximal to hydrothermal vents; the 
discovery of introns in the primitive eukaryote, Giardia lamblia; and 
evidence of lateral transfer of genes from several different bacteria into 
the genome of Giardia lamblia. 



Camponotus nearcticus, collected in Fa/mouth, MA. Adam Lazarus 





Rio Tinto, J. L. de Lope, J. M. Sanchez 



Rio Tinto, continued 

that escaped detection by traditional methods. 

The work, led by Linda Amaral Zettler and Mitchell 

Sogin of the Josephine Bay Paul Center for 

Comparative Molecular Biology and Evolution, has 

also revealed 

completely new 

eukaryotes as well 

as others which 

have never been 

seen before in such 

a highly-acidic 

environment. 

In mapping out the 
evolutionary family 
tree (or phylogeny) 
for the Rio Tinto, 

Amaral Zettler, Sogin, and their colleagues have 
detected a close relationship between the River's 
acid-loving eukaryotes and other species that 
prefer neutral environments. The short evolutionary 
distance between the two tells the scientists that 
adaptations are, in addition to being widespread, 
occurring rapidly when measured on an evolution- 
ary time scale. 



The project was funded by the National Science 
Foundation's Life in Extreme Environments (or LexEn) 
Program and NASA's Astrobiology Institute. 




Linda Amaral Zettler. Erik Zettler 



R16 



Staff 



I Publications 



RESEARCH STAFF 

Linda Amaral Zettler, Staff c 

David Beaudom, Research A: 

Steven J. Biller, Resean. 

Amy Crump, Resear 

Patrick Degnan, : - Distant I 

Ashita Dhillon .. Scientist 

Daniel Golde :., ate Student 

Sulip Gosv : -earch Assistant II 

Josh Herb ...itdoctoral Scientist 

Jennifer > , ih, Research Assistant I 

Ulandt Kim, Research Assistant II 

Abby Laatsch, Research Assistant I 

Erica Lasek-Nesselquist, Research Assistant I 

Adam Lazarus, Research Assistant I 

Bruce Luders, Research Assistant II 

Jessica Mark Welch, Postdoctoral Scientist 

David Mark Welch, Staff Scientist II 

Andrew McArthur, Assistant Scientist 

Hilary Morrison, Staff Scientist II 

Maria Murray, Research Assistant I 

Daniel Myers, Lab Assistant 

Laila Nahum, Postdoctoral Scientist 

Lorraine Olendzenski, Postdoctoral Scientist 

Bertil Olsson, Senior Research Assistant 

Sarah Pacochal, Research Assistant II 

Carmen Palacios, Postdoctoral Scientist 

Gretta Serres, Staff Scientist I 

Lynn Sherrer, Graduate Student 

Jillian Ward, Research Assistant I 

VISITING SCIENTISTS 

Maristela Camargo, University of Sao Paolo, 

Brazil 

Robert Campbell, Ares Pharmaceutical 
Lynne McAnelly, University of Texas, Austin 

ADJUNCT SCIENTISTS 

Matthew Meselson, Harvard University 

Roger Milkman, Professor Emeritus, University 

of Iowa 
Harold Zakon, University of Texas, Austin 

HIGH SCHOOL INTERN 

Alexandria Papa 

VISITING UNDERGRADUATE 
Alissa Cohen 

HHMI SUMMER UNDERGRADUATES 
Sarah Biber 
Marsha Wheeler 

NATIONAL SCIENCE FOUNDATION 
RESEARCH EXPERIENCE FOR 
UNDERGRADUATE STUDENTS 
Chad Brock 
Andrew Magis 
Amy McCurley 
John Sander 

ADMINISTRATIVE STAFF 
Pauline Lim 
Tara Nihill 



Cross, M , R Kieft, R. Sabatini, A Dirks-Mulder, 
I Chaves, and P Borst 2002 J-binding protein 
increases the level and retention of the unusual 
base J in trypanosome DNA Moi Microbiol 
46: 37-47 

Dehal, P., Y Satou, R. K. Campbell, J. Chapman, 
B Degnan, eta). 2002. The draft genome of 
Ciona intestina/is insights into chordate and 
vertebrate origins. Science 298 2157-2167. 

Edgcomb, V P., D T Kysela, A. Teske, A. 
de Vera GUmez, and M. L. Sogin 2002 Benthic 
eukaryotic diversity in the Guaymas Basin 
hydrothermal vent environment Proc Nat/. 
Acad. Sci. USA 99: 7658-7662. 

Edgcomb, V. P., A. G. B. Simpson, L. A. Amaral 
Zettler, T A. Nerad, D J Patterson, M E 
Holder, and M. L Sogin 2002 Pelobionts are 
degenerate protists: insights from molecules and 
morphology Mol Bio/. Evol. 19 978-982 

Garcla-Verela, M , M. P. Cummings, G Perez- 
Ponce de Leon, S L Gardner, and J. P Laclette 
2002 Phylogenetic analysis based on 18S 
nbosomal RNA gene sequences supports the 
existence of class Polyacanthocephala 
(Acanthocephala) Mol Phy/ogenet. Evol 
23: 288-292. 

Laan, M., H. Richmond, C. He, and R K 
Campbell 2002 Zebrafish as a model for 
vertebrate reproduction characterization of 
the first functional zebrafish (Danio reno) 
gonadotropin receptor Gen. Comp. Endocnnol 
125: 349-364. 

Langford, T D , J D. Silberman, M E -L 
Weiland, S G Svard, J.M McCaffery, M L 
Sogin, and F D Gillin 2002 Giardia lamblia 
identification and characterization of Rab 
and GDI proteins in a genome survey of the 
ER to Golgi endomembrane system. Exp 
Parasite/ 101: 13-24 

Morrison, H G , G Zamora, R K. Campbell, 
and M. L. Sogin. 2002 Inferring protein function 
from genomic sequence Giardia lamblia 
expresses a phosphatidyhnositol kinase-related 
kinase similar to yeast and mammalian TOR. 
Comp. Siochem Physio/. B Siochem Mol Biol. 
133:477-491 

Nixon, J E J,A Wang, J. Field, H G Morrison, 
A G McArthur, M. L Sogin, B J Loftus, and J 
Samuelson. 2002 Evidence for lateral transfer of 
genes encoding ferredoxins, nitroreductases, 
NADH oxidase, and alcohol dehydrogenase 3 
from anaerobic prokaryotes to Giardia lamblia 
and Entamoeba histolytica Eukaryotic Cell 
1 181-190 

Nixon, J E J , A Wang, H G Morrison, A. G. 
McArthur, M. L Sogin, B Loftus, and J 
Samuelson 2002 A spliceosomal mtron in 
Giardia intestina/is. Proc. Nat/. Acad. Sci. USA 
99 3701-3705 



Palacios, C , and J. J Wernegreen 2002. 
A strong effect of AT mutational bias on 
ammo acid usage in Buchnera is mitigated at 
high-expression genes. Mol. Biol. Evol 19 
1575-1584 

Podar, M , L Mullmeaux, H -R Huang, P S 
Perlman, and M L Sogin 2002 Bacterial 
Group II introns in a deep-sea hydrothermal 
vent environment App/ Environ. Microbio/ 
68: 6392-6398 

Sabatini, R , N Meeuwenoord, J H van Boom, 
and P. Borst 2002 Recognition of base J in 
duplex DNA by J-bmding protein J Biol Chem 
277 958-966. 

Sabatini, R , N Meeuwenoord, J. H van Boom, 
and P. Borst. 2002 Site-specific interactions of 
JBP with base and sugar moieties in duplex 
J-DNA. J. Bio/. Chem. 277 28,150-28,156 

Simpson, A. G- B., L A. Amaral Zettler, F. 
Gomez, E Zettler, B G Keenan, R Amils, and 
M L Sogin 2002 Heavy-metal, acid-loving 
eukaryotes from Spain's "River of Fire " Nature 

417 137 

Simpson, A G. B , A J Roger, J D Silberman, 
D D Leipe, V. P. Edgcomb, L. S Jermnn, D J 
Patterson, and M L Sogin 2002. Evolutionary 
history of "early diverging" eukaryotes. the 
excavate taxon Carpediemonas is a close relative 
of Giardia. Mol Biol. Evol. 19: 1782-1791 

Sun, C.-H., D. Palm, A. G. McArthur, S. G. Svard, 
and F G Gillin 2002 A novel Myb-related 
protein involved in transcnptional activation of 
encystation genes in Giardta lamblia Mo/. 
Microbio/- 46: 97 1-984 

Tamas, I , L Klasson, B Canback, A K, Naslund, 
A S Eriksson, J Wernegreen, J P. Sandstrom, 
N A Moran, and S G Andersson 2002 50 
million years of genomic stasis in endosymbtotic 
bacteria Science 296- 2376-2379 

Teske, A., K -U Hmrichs, V Edgcomb, A de 
Vera Gomez, D. Kysela, S P. Sylva, M. L Sogin, 
and H W Jannasch 2002 Microbial diversity 
of hydrothermal sediments in the Guaymas 
Basin: evidence for anaerobic methanotrophic 
communities App/. Environ. Microbiol. 
68: 1994-2007 

Wernegreen, J J 2002. Genome evolution in 
bacterial endosymbionts of insects Nat Rev. 
Genet 3: 850-861 

Wernegreen, J J , A. B Lazarus, and P. H 
Degnan 2002 Small genome of Candiclatus 
b/ochmannia, the bacterial endosymbiont of 
Camponotus, implies irreversible specialization 
to an mtracellular lifestyle Microb/o/ogy 148: 
2551-2556. 



R17 




ARCHITECTURAL DYNAMICS IN LIVING CELLS PROGRAM 




M/crotubu/es radiating in 
all directions from a 
centrosome, Rudolf 
Oldenbourg, with Robert 
E. Palazzo 



The Architectural Dynamics in Living Cells Program, established 
at the MBL by Dr. Shinya Inoue in 1992, continues the pioneering 
research and educational activities in biophysical inquiries directly 
in living cells that Dr. Inoue started at Princeton University in 
1949. The Program focuses on architectural dynamics in living 
cells the timely and coordinated assembly and disassembly of 
macromolecular structures essential for the proper functioning 
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 tools that permit such studies directly in 
living cells and functional cell-free extracts. Program members 
have special expertise in the use of polarized light for analyzing 
the local arrangement of molecular bonds and fine structure in 
biological specimens. Unique instrumentation developed by 
Program members include the universal light microscope, 
centrifuge polarizing microscope, the liquid-crystal based Pol- 
Scope, and related technology. Biological phenomena currently 
under investigation include mitosis/meiosis and related motility, 
amoeboid movement, microtubule-centrosome interaction, and 
optical properties of green fluorescent protein. The Architectural 
Dynamics in Living Cells Program is an active component of the 
MBL's resident cell research group and promotes interdisciplinary 
research and training among its resident core researchers, visiting 
investigators, and collaborating manufacturers. 



DISTINGUISHED SCIENTISTS 
Shinya Inoue 

SENIOR SCIENTIST 
Rudolf Oldenbourg 

STAFF SCIENTIST II 
Michael Shribak 



Meiosis II in spermatocyte of 
the crane fly, recorded with the 
new Pol-Scope, James R. 
Lafountain (U. at Buffalo) and 
Rudolf Oldenbourg 



R18 



Polarized fluorescence of single crystal of GfP rotated 
between parallel polarizers, Shinya Inoue 




I Staff 



RESEARCH STAFF 

Diane Baraby, Research Assistant 

Grant Harris, Software Engineer 

Robert Knudson, Instrument Development Engineer and Research Associate 

Gwen Szent-Gyorgyi, Research Assistant 

VISITING INVESTIGATORS 

Michael Bennett, Albert Einstein College of Medicine 

Michael Braun, Woods Hole Oceanographic Institution 

Robert Campbell, Serono 

Larry Cohen, Yale University School of Medicine 

Makoto Goda, Japan Biological Informatics Consortium, Tokyo, Japan 

Peter Hepler, University of Massachusetts at Amherst 

Joseph Hoffman, Yale University School of Medicine 

David Keefe. Rhode Island Women & Infants Hospital 

James R LaFountain, University at Buffalo 

Un Uu, Rhode Island Women & Infants Hospital 

Jessica Mark Welch, Bay Paul Center, MBL 

Timothy Mitchison, Harvard University 

Robert Palazzo, Rensselaer Polytechnic Institute 

Prem Ponka, McGill University, Montreal 

Edward D Salmon, University of North Carolina at Chapel Hill 

Orion Vanderlinde, Florida State University 

INDUSTRIAL COLLABORATORS 

Cambridge Research and Instrumentation, Inc , Wobum, MA 

Nikon, Japan 

Technical Video, Woods Hole, MA 

Yokogawa Electric, Japan 

Universal Imaging Corporation, Downingtown, PA 



|Pub//cat/ons 



Inoue, S. 2002. Polarization microscopy. In 
Current Protocols in Cell Biology, Suppl. 13, J. 
Lippincott-Schwartz, ed. John Wiley & Sons, 
pp. 4.9.1-4.9.27. 

Inoue, S., O. Shimomura, M Goda, M. Shribak, 
and P.T. Tran. 2002. Fluorescence polarization 
of green fluorescent protein (GFP) Proc. Nat/. 
Acad. So. USA 99(7): 4272-4277. 

Inoue, S., and T. Inoue. 2002. Direct-view high- 
speed confocal scanner theCSU-10. In 
Cell Biological Applications of Confocal 
Microscopy, 2nd Edition, B. Matsumoto, ed. 
Academic Press, pp. 87-123. 

Oldenbourg, R 2002. Retardance measurement 
method. US Patent, Number 6,501,548. USA, 
Assignee: Cambridge Research & Instrumenta- 
tion Inc. (Woburn, MA). 

Shribak, M., S. Inoue, and R. Oldenbourg 2002 
Polarization aberrations caused by differential 
transmission and phase shift in high NA lenses 
theory, measurement and rectification. Opt. 
Eng. 41(5):943-954. 

Shribak, M. I., and R. Oldenbourg. 2002 
Scanning aperture polarized light microscope: 
observation of small calcite crystals using 
oblique illumination. In Three-Dimensional and 
Multidimensional Microscopy: Image Acquisition 
and Processing IX, J.-A. Conchello, C. J. 
Cogswell and T. Wilson, eds. San Jose, 
Proceedings of SPIE 4621: 104-109. 

Shribak. M., and R. Oldenbourg. 2002. Sensitive 
measurements of two-dimensional birefringence 
distributions using near-circularly polarized 
beam. In Polarization Analysis, Measurement 
and Remote Sensing V, D H. Goldstein, D. B. 
Chenault, W Egan and M Duggin, eds Seattle, 
Proceedings of SP/E 4819: 56-67 

Wang, W H., L Meng, R. J. Hackett, R. 
Oldenbourg, and D. L Keefe. 2002 Rigorous 
thermal control during intracytoplasmic sperm 
injection stabilizes the meiotic spindle and 
improves fertilization and pregnancy rates. 
Fertil. Steril. 77: 1274-1277. 



ADMINISTRATIVE STAFF 
Jane MacNeil 



R19 




BOSTON UNIVERSITY MARINE PROGRAM 



I staff 



DIRECTOR 

Jelle Atema, Professor of Biology 

FACULTY 

Paul Barber, Assistant Professor of Biology 
John Finnerty, Assistant Professor of Biology 
Stjepko Golubic, Professor of Biology 
Les Kaufman, Associate Professor of Biology 
Phillip Lobel, Associate Professor of Biology 
Gil Rosenthal, Assistant Professor of Biology 
Ivan Valiela, Professor of Biology 

ADJUNCT FACULTY 
Roger Hanlon, MBL 
Gabriele Gerlach, MBL 
Anne Giblin, MBL 
Frederick Goetz, MBL 
Norman Wainwright, MBL 

SENIOR STAFF COORDINATORS 
Sharifa Gulamhussein 
Jennifer Ripley 

ADMINISTRATIVE STAFF 

Sheri Hall, Program Manager 

Stefano Mazzilli, Research Assistant, Valiela Lab 

Michelle McCafferty, Program Coordinator 



Continued . . 



Cuttlefish embryos. Lisa Kerr Lobel 



In 2002, the Boston University Marine Program realized its vision for an 
expanded research focus in behavioral ecology and population genetics. 
We appointed two new assistant professors, Paul Barber (Berkely, PhD; 
Harvard, postdoc) and Gil Rosenthal (University of Texas PhD; UC San 
Diego, postdoc). In addition, joint planning and advertising with the MBL's 
Marine Resources Center resulted in additional strength in this area with 
the appointment of MBL associate scientist Gabi Gerlach (University of 
Konstanz). Their scientific perspectives complement the sensory and 
behavioral ecology strengths in the labs of senior faculty Phil Lobel and 
Jelle Atema and MRC director, Roger Hanlon. Newly appointed MRC 
senior scientist Rick Goetz further enhances the scientific goals of this 
research focus. Gerlach and Goetz are both BUMP adjunct professors. 

The coastal ecology program headed by Ivan Valiela flourished with a 
continuous stream of graduate students, postdocs, and international 
visiting scientists. Its Research Experience for Undergraduates Program 
continues to bring 10 outstanding undergraduates to Woods Hole doing 
research that ends up being published regularly. Anne Giblin of the MBL's 
Ecosystems Center has an adjunct appointment at BUMP. 

With the new faculty, the Program immediately took in a graduate class of 
17 highly competitive students for both PhD and Masters degrees, 
continuing its mission to provide exceptional educational opportunities to 
students in Marine Biology. Several of our current 35 students work 
directly with scientists at the Woods Hole Oceanographic Institution, and 
the National Marine Fisheries Service. 

The undergraduate program also continued its mission successfully 
by providing eight challenging research-based courses to 16 students 
primarily from Boston University. Here too, student research has led 
to several publications. 



R20 



| Publications 




Ruth Carmichael, BUMP 



Staff, continued 

RESEARCH TECHNICIANS 
Sarah Boyce 
Devin Drown 
Janelle Morano 
David Portnoy 

VISITING FACULTY 
Bill Simmons, Sandia 
National Laboratory 
Nathalie Ward, Lecturer 

PhD STUDENTS 
Brendan Anrrett 
Jennifer Bowen 
Ruth Carmichael 
Marci Cole 
Eric Crandall 
Heidi Fisher 
Sara Grady 
Kevin Kroeger 
Carolyn Miller 
Vanessa Miller-Sims 
Elizabeth Neeley 
Jason Philibotte 
Mindy Richlen 
Gregory Skomal 
Molly Steinbach 
Mirta Teichberg 
Gabrielle Tomasky 
Joanna York 
Erik Zettler 

MASTERS STUDENTS 

Abby Atkinson 

Andrea Bogomotni 

Lisa Bonacci 

Chelsea Bouchard-Harnish 

Jessica Buckingham 

Michael Cermak 

Pieter deHart 

James Estrada 

Christopher Florio 

Sophia Fox 

Sarah Good 

Dawn Grebner 

Andrea Hsu 

Alison Leschen 

Mark Lever 

Vanessa Malley 

David Martel 



Amee Mehta 
Karen Morschauser 
Catherine O'Keefe 
Mollie Oremland 
Aaron Rice 
Deborah Rutecki 
Andrea Shriver 
Elizabeth Soule 
Todd Stueckle 
Erin Summers 
Melissa Sweeny 

UNDERGRADUATE 
STUDENTS 
Alaina Avery 
Colleen Butler 
Sean Dixon 
Yael Erlitz 
Deirdre Grant 
Alfred Huang 
Laura Kloepper 
Heather Marlow 
Melissa Penn 
William Rowan 
Elisse Ruiz 
James Saenz 
Josiah Sewell 
Jami Whitney 
Kristen Wright 
Julia Young 
Chandra Ziegler 

SUMMER REU INTERNS 
Stacy Barron 
Emily Gaines 
Kristin Hunter-Thompson 
Jane La Du 
Melissa Millman 
Sarah Rohrkasse 
Dianne Suggs 
Jeremy Testa 
Carolyn Weber 
Bradley Williams 

SUMMER VOLUNTEER 
Jillian Barber 

SUMMER STUDENT 
RESEARCHERS 
Rachel Allen 
Jillian Barber 
Margaret Johnson 



Armstrong, Peter 8., Margaret T. Armstrong, 
R. L Pardy, Alice Child, and Norman 
Wainwright- 2002. Immunohistochemical 
demonstration of a lipopolysaccharide in the 
cell wall of a eukan/ote. the green alga, 
Ch/ore/la. Biol. Bull. 203: 203-204. 

Atema. J , M. K Kingsford, and G Gerlach 
2002. Larval reef fish could use odour for 
detection, retention and orientation to reefs. 
Mar. Ecol. Prog. Ser. 241: 151-160 

Barber, P. H., M. K. Moosa, and S. R. 
Palumbi. 2002. Rapid recovery of genetic 
diversity on coral reefs and the temporal and 
spatial scale of larval dispersal: examples 
from Krakatau. Proc R. Soc. Lond. B 269: 
1591-1597. 

Barber, P. H , S. R. Palumbi, M. V. Erdmann. 
and M. K. Moosa. 2002. Sharp genetic 
breaks among populations of a benthic 
marine crustacean indicate limited oceanic 
larval transport: patterns, cause, and 
consequence. Mol. Ecol. 1 1 : 659-674 

Barron, Stacy, Carolyn Weber, Roxanne 
Marino, Eric Davidson, Gabrielle Tomasky, 
and Robert Howarth. 2002. Effects of varying 
salinity on phytoplankton growth in a low- 
salinity coastal pond under two nutrient 
conditions. Biol. Bull. 203: 260-261 

Evgenidou, A., and I. Valiela. 2002. 
Response of growth and density of a 
population of Geukensia demissa to 
land-derived nitrogen loading, in Waquoit 
Bay, Massachusetts Estuar. Coast. Shelf So. 
55. 125-138 

Gaines, Emily F., Ruth H. Carmichael, 
Sara P. Grady, and Ivan Valiela 2002. 
Stable isotopic evidence for changing 
nutritional sources of juvenile horseshoe 
crabs Biol. Bull. 203: 228-230 

Grasso, F. W., and J. Atema. 2002. 
Integration of flow and chemical sensing 
for guidance of autonomous marine robots 
in turbulent flows. Environ. Fluid Mech. 
2:95-114. 

Hunter-Thomson, Kristin, Jeffrey Hughes, 
and Bradley Williams, 2002. Estuarine- 
open-water comparison of fish community 
structure in eelgrass (Zostera marina L.) 
habitats of Cape Cod Biol. Bull. 203: 
247-248. 

Millman, Melissa, Mirta Teichberg, 
Paulina Martinetto, and Ivan Valiela 2002 
Response of shrimp populations to land- 
derived nitrogen in Waquoit Bay, Massachu- 
setts Biol. Bull. 203: 263-264. 

Philibotte, Jason. 2002 Pelagic larval 
duration of the Caribbean wrasse, 
Thalassoma bifasciatum Biol. Bull. 
203: 245-246 



Rohrkasse, Sarah M., and Jelle Atema 2002. 
Tracking behavior of Busyconinae whelks. 
Bio). Bull. 203: 235-236 

Rosenthal, G. G , M. J. Ryan, and W. E. 
Wagner, Jr 2002 Secondary loss of 
preference for swords in the pygmy 
swordtail Xiphophorus nigrensis (Pisces: 
Poeciliidae). Anim. Behav. 63: 37-45. 

Shady, S., D. I. A. MacLeod, H. S. Fisher, 
and J. Y. Liang. 2002. Adaptation from 
invisible luminance and chromatic flicker. 
J. Vision 2: 68. 

Shriver, A. C., R- H. Carmichael, and I. Valiela. 
2002. Growth, condition, reproductive 
potential, and mortality of bay scallops. 
Argopecten irradians, in response to 
eutrophic-driven changes in food resources. 
J. Exp. Mar. Biol. Ecol. 279: 21-40. 

Suggs, Dianne N.. Ruth H. Carmichael, Sara P. 
Grady, and Ivan Valiela- 2002. Effects of 
individual size on pairing in horseshoe crabs 
Biol. Bull 203: 225-227. 

Testa, Jeremy M., Matthew A. Charette, 
Edward R. Sholkovitz, Matt C Allen, Adarn 
Rago, and Craig W. Herbold. 2002 Dissolved 
iron cycling in the subterranean estuary of a 
coastal bay: Waquoit Bay, Massachusetts 
Biol. Bull. 203: 255-256. 

Valiela, I., and J. L Bowen. 2002, 
Nitrogen sources to watersheds and estuaries: 
Role of land cover mosaics and losses within 
watersheds. Environ. Pollut. 118: 239-248. 

Valiela, I., J. L. Bowen, and K. D Kroeger. 
2002. Assessment of models for estimation of 
land-denved nitrogen loads to shallow 
estuaries. Appl, Geochem. 17: 935-953. 

Valiela, I., and M. L. Cole 2002. Comparative 
evidence that salt marshes and mangroves 
may protect seagrass meadows from land- 
derived nitrogen loads Ecosystems 5: 92-102. 

Weber, Carolyn F., Stacy Barron, Roxanne 
Marino, Robert W. Howarth, Gabrielle 
Tomasky, and Eric A. Davidson. 2002 
Nutrient limitation of phytoplankton growth in 
Vineyard Sound and Oyster Pond, Falmouth, 
Massachusetts. Biol. Bull. 203: 261-263 

Weiss, E. T., R. H Carmichael, and I. Valiela 
2002. The effect of nitrogen loading on the 
growth rates of quahogs (Mercenaria 
mercenaria) and soft-shell clams (Mya arenaria) 
through changes in food supply. Aquacu/ture 
211: 1-4. 

Williams, Bradley S., Jeffrey E. Hughes, and 
Kristin Hunter-Thomson. 2002. Influence of 
epiphytic algal coverage on fish predation 
rates in simulated eelgrass habitats. Biol 
Bull. 203: 248-249 



R21 




MARINE RESOURCES CENTER PROGRAMS 



Staff 



DIRECTOR/SENIOR 
SCIENTIST 
Roger Hanlon 

SENIOR SCIENTIST 
Frederick Goetz 

ASSOCIATE SCIENTISTS 
Gabriele Gerlach 
Alan Kuzirian 

ASSOCIATE SCIENTIST/ 
VETERINARIAN 
Roxanna Smolowitz 



The Marine Resources Center (MRC) is a national center for the develop- 
ment and use of aquatic organisms in basic biological research, biomedi- 
cal research, aquaculture, and fisheries science. The research programs 
focus on biological processes integrated at the level of the whole 
organism. 



We made three primary faculty hires in the 
research ranks during 2002: Frederick Goetz, 
Senior Scientist; Gabriele Gerlach, Associate 
Scientist; and Scott Lindell, Research 
Specialist. In addition, two Assistant 
Professors affiliated with the MRC were hired 
by the Boston University Marine Program: 
Gil Rosenthal and Paul Barber. Collectively, 
these hires help establish a critical mass of 
year-round researchers in the MRC. 




Marine Resources Center photos, Elizabeth Armstrong 



R22 




MRC Staff, continued 



James Carroll, Life Support Technical Assistant 

Edward Enos, Aquatic Resources Division Superintendent 

William Grossman, Marine Specimen Collector/Diving 

Safety Officer 

Janice Hanley, Water Quality and Animal Health Technician 
William Klimm, Licensed Boat Captain - R/V Gemma 
Scott Lindell, Manager, Aquatic Resource Services and 

Aquaculture Research Specialist 
Beth Linnon, Special Projects Coordinator 
William Mebane, Aquaculture and Engineering Division 

Superintendent 

Gabrielle Santore, Executive Assistant 
Andrew Sexton, Marine Organism Shipper 
Daniel Sullivan, Boat Captain 
Eugene Tassinari, Senior Biological Collector 
Sean Whelan, Diver/Marine Specimen Collector 

SEASONAL EMPLOYEES AND VOLUNTEERS 

Amanda Carroll, Intern, Lawrence School 
Brianne Como, Intern, University of Massachusetts 

at Dartmouth 

Jay Dimond, Diver/Collector 
George Gannon, Intern, Massachusetts Bay Community 

College 

Andrew Sterling, Diver/Coilector 
Christian Sterling, Diver/Collector 
Monica Weedo.i, Intern, Pratt Institute 



Program in Sensory Biology, Behavioral Ecology 
& Population Genetics 

Our studies of the physiological, sensory and genetic mechanisms of 
behavior bridge neuroscience, behavior, and ecology. Such an ap- 
proach allows us (1) to study evolutionary processes of natural and 
sexual selection that shape the lives of animals and humans, and (2) to 
investigate the genetic consequences of behavioral interactions in an 
ecological context, including the population level. 

In 2002, using DNA fingerprinting, we discovered that there are five 
genetic stocks of squids in the northwest Atlantic, and that each stock 
tends to return to certain spawning grounds each summer. This finding 
will not only revise the federal fishery management plan, but also 
highlights the sophistication of the squids' sensory and behavioral 
abilities. 

Social signals such as pheromones regulate reproductive behavior in 
fish as well as in other vertebrates including humans. Using zebrafish, 
we found that male and female pheromones released during complex 
behavioral interactions greatly influenced female reproduction. This 
research will lay the foundation for future analysis of the genetic 
background of fertility and reproduction in vertebrates. 

Learning and memory experiments centered upon defining the 
different stages of memory formation. Using the marine mollusc 
Hermissenda, we were able to correlate memory acquisition and 
retention with the animals' responses to an accessory sensory stimulus. 
By quantifying the immunocytochemistry, we demonstrated that levels 
of calexcitin increase and persist during the formation stages of long- 
term memory. Calexcitin is a key element for regulating internal 
calcium release in memory acquisition. 



Photos by Elizabeth Armstrong 




R23 




| Program in Scientific Aquaculture 

This program focuses on biotechnology research, applied research on 
biomedical and commercial organisms, and policy development in 
both of those areas. The biotechnology research is aimed at basic 
mechanisms that control growth, behavior, reproduction, and disease 
in commercially important finfish and shellfish. This includes studies on 
novel regulators of growth and reproduction in fish and shellfish, 
pathogen-regulated genes in fish, and the development of molecular- 
based diagnostic techniques. 

In 2002, with collaborators in Spain, we established for the first time 
in any fish species a primary cell culture technique to obtain differen- 
tiated trout macrophages. We then demonstrated that only these 
differentiated macrophages can respond to pathogenic antigens by 
upregulating early response genes such as tumor necrosis factors and 
interleukins. It will now be possible to use global genomic techniques 
to obtain all of the macrophage genes that are regulated by antigen 
exposure and this is being pursued. 

We received funding from the Southeastern Massachusetts Aquacul- 
ture Center and developed new technology to help commercial 
shellfish growers overwinter quahog clam seed, which will circumvent 
the 60-80% "winter-kill" common in field-planted seed. 

The Policy Center for Marine Biosciences and Technology, directed by 
former MBL Director Harlyn Halvorson, has now been aligned jointly 
with the University of Massachusetts (Boston) and the Marine 
Resources Center. The Policy Center defines major problems in the 
fields of marine aquaculture and biotechnology, and conducts 
international workshops to address these important societal issues. 



Photos by Elizabeth Armstrong 



AMERICORPS VOLUNTEERS (SENIOR MEMBERS) 

Pat Kosky 

Joan Lemieux 

Haskell Maude 

Birgit Nelson 

Joseph Sheeny 

Judith Sheehy 

Joyce Wynne 



Laboratory of Roger Han/on 

STAFF 

Roger Hanlon, Senior Scientist 

Jean Boal, Adjunct Scientist 

Kendra Buresch, Research Assistant 

Martha Delaney, Research Assistant 

Chris Florio, Graduate Student, Boston University 

Nicole Gilles, Research Assistant 

Mary Beth Saffo, Adjunct Scientist 

Nadav Shashar, Adjunct Scientist 

Mollie Tubbs. Research Assistant 

VISITING INVESTIGATORS 

Chuan-Chin Chiao, Postdoc, Howard Hughes 

Medical Institute 
Melissa Grable, Graduate Student, Boston University 

Marine Program 

Nuutti Kangas. Postdoc, Academy of Finland 
Miranda Karson, Graduate Student, Michigan 

State University 

Allen Mensinger, University of Minnesota at Duluth 
Marie-Jose Naud. Graduate Student, Flinders University 
Andrew Simpson, MMBR Student, University of 

California, Santa Barbara 

INTERNS 

Angela Abbott, Massachusetts Maritime Academy 

Melissa Cox, Purdue University 

Robert Nobuhara, Colorado State University 

Reshma Patel, Emory University 

Camille Riviere, EN. S.A.I. A. 

Eric Stone, University of Massachusetts, Dartmouth 

Kate Sweeney, Colby College 

Continued. . . 



R24 




Marine models, 
JoAnna DeNobi/e, 
Irina Chaikhoutdmov, 
and Lydia Louis 



Laboratory of Alan Kuzirian 

STAFF 

Alan Ku:inan, Associate Scientist 
Hemant Chikarmane, Investigator 
Herman Epstein, Investigator 

VISITING INVESTIGATORS 

Frank Child 

John Clay, National Institutes of Health 

Robert Gould, New York State Institute for Basic Research 

INTERNS 

Kimberly Borley, Ohio University 

Alex Hangsterfer, Roger Williams University 

Justin Walker, Massachusetts Maritime Academy 



Laboratory of Roxanna Smolowitz 

STAFF 

Roxanna Smolowitz, Veterinarian 

Kevin Uhlinger, Research Assistant 

INTERNS 

Krystal Baird, AmeriCorps Member 

Amy Hancock, Summer Veterinary Intern 

Jen Hsieh, AmeriCorps Member 

Andrea Hsu, Graduate Student, Boston University 

Kyle Hunt, Mashpee High School 



Laboratory of Frederick Coetz 

STAFF 

Frederick Goetz, Senior Scientist 

Scott Lindell, Manager, Aquatic Resource Services 

and Aquaculture Research Specialist 
Linda McCauley, Research Assistant 
Steven Roberts, Postdoctoral Researcher 
Raquel Sussman 
Andrew Sweetman, Graduate Student, University 

of Bergen 
Dimitar Iliev, Graduate Student, University of Notre Dame 



Laboratory of Gabhele Gerlach 

STAFF 

Gabriele Gerlach, Associate Scientist 

Jenny Lusk-Yablick, Research Assistant 

VISITING INVESTIGATORS 

Thomas Breithaupt, Konstanz University 

INTERNS 

Martha Delaney, University of Massachusetts at Amherst 

Chris Follett, Acton Boxborough Regional High School 

Nick Hanney, Bishop Stang High School 

Nick Ryan, Thayer Academy 



| Publications 



Atema. J., M. J Kingsford. and G. Gerlach. 2002 Larval reef 
fish could use odour for detection, retention and orientation 
to reefs. Mar. Ecol. Prog. Ser. 241: 151-160. 

Basil, J. A., G. 8. Lazenby, L. Nakanuku, and R T. Hanlon. 
2002 Female Nautilus are attracted to male conspecific 
odor. Bull. Mar. Sd. 70: 217-225 

Borley, K. A., H. T Epstein, and A. M. Kuzirian. 2002 Effects 
of a sensory block on calexcitin levels in the photoreceptors 
of Hermissenda crassicornis. Biol. Bull. 203: 197-198 

Clay, John R , and Alan M. Kuzirian 2002. Trafficking of 
axonal K' channels potential role of Hsc70. J. Neurosci. Res. 
67: 745-752. 

Delaney, M., C Follet, N. Ryan, N. Hanney, J. Lusk-Yablick, 
and G. Gerlach. 2002 Social interaction and distribution of 
female zebrafish (Danio rerio) in a large aquarium Biol. Bull. 
203: 240-241. 

Gerlach, G., and S Bartmann. 2002 Reproductive skew, 
costs and benefits of cooperative breeding in female wood 
mice (Apodemus sylvaticus). Behav. Ecol. 13 408-418 

Grable, M M , N. Shashar, N. L. Gilles, C.-C. Chiao, and R 
T. Hanlon. 2002 Cuttlefish body patterns as a behavioral 
assay to determine polarization perception. Biol. Bull. 203: 
232-234 

Hall, K. C , and R. T. Hanlon 2002 Principal features of the 
mating system of a large spawning aggregation of the giant 
Australian cuttlefish Sepia apama (Mollusca: Cephalopoda) 
Mar. Biol. 140: 533-545. 

Hsieh, J. L.. H. M. Chikarmane, R. Smolowitz, K. R. Uhlinger, 
W. Mebane, and A. M Kuzirian 2002 Microbial analysis of 
ozone disinfection in a recirculating seawater system Biol. 
Bull 203: 266-267. 

Kusakabe, M., T, Todo, H. J. McQuillan, F. W. Goetz, and G. 
Young 2002. Characterization and expression of ste- 
roidogenic acute regulatory protein and MLN64 cDNAs in 
trout. Endocrmology 143: 2062-2070. 

Roberts, S. B 2002 Charactenzation of growth hormone in 
yellow perch and myostatin in several teleost species Oiss. 
Abst. Int. B Sci. Eng 63: 1710 

Saffo, M B. 2002 Themes from variation: probing the 
commonalities of symbiotic associations. Integr Comp. Biol. 
42: 291-294 

Shashar, N , C A Milbury, and R T Hanlon 2002 
Polarization vision in cephalopods: neuroanatomical and 
behavioral features that illustrate aspects of form and 
function. Mar. Freshwat. Behav. Physio/. 35 57-68 

Smolowitz, R , J. Hanley, and H. Richmond. 2002 A three- 
year retrospective study of abdominal tumors in zebrafish 
maintained in an aquatic laboratory animal facility Biol. Bull. 
203: 265-266. 

Sunila, I.. N A. Stokes, R Smolowitz, R C. Karney, and E. M. 
Burreson. 2002. Haplosporidium costale (seaside organism), 
a parasite of the eastern oyster, is present in Long Island 
Sound J. Shellfish Res. 21 113-118 



R25 




Rat cardiac muscle cell. Peter J. 5. Smith 



PROGRAM IN MOLECULAR PHYSIOLOGY 



The Program in Molecular Physiology (PMP) brings together a group of 
resident and visiting scientists who share common interests in the 
molecular bases of cellular physiology. The several laboratories making 
up the PMP focus on cellular plasticity and the properties of molecular 
transport mechanisms. A variety of experimental approaches are used 
ranging from molecular and biochemical methodologies, through 
biophysics, to advanced optical and electrochemical imaging tech- 
niques. An example of a research area spanning the independent 
laboratories within PMP is the role of metabolism and the mitochon- 
drion in health and disease. How, for example, does the mitochondrion 
contribute to insulin secretion, heme synthesis, or channel modulation? 
Does the aging process, targeting metabolic disorders, contribute to 
reproductive and neural malfunction, degeneration, and apoptosis? 

In addition to our interests in basic biology the laboratories of the PMP 
carry on a strong tradition within the Marine Biological Laboratory 
resident programs for instrumentation development. The BioCurrents 
Research Center a national bioengineering resource of the National 
Institutes of Health (NCRR) has pioneered the use of electrochemical 
sensors to define cellular activity through monitoring conditions in the 
extended boundary layer. 

A notable characteristic of the PMP is the extensive year-round collabo- 
rative outreach to regional universities and hospitals. Members contrib- 
ute to three Boston based NIH Program Project Grants in protein 
trafficking, diabetes, and anemia. Collaboration also allows the group to 
rapidly advance in areas of topical interest as with an ongoing 
initiative to study the molecular physiology of the multi-drug resistant 
transporters, players of critical interest to cancer research and our 
understanding of infectious diseases. Annually, the member laboratories 
host more than 40 national and international visitors, taking advantage 
of the unique combination of scientific and technical expertise concen- 
trated at the Marine Biological Laboratory. Access is provided to 
experimental platforms, cutting edge imaging techniques, and a diverse 
array of marine models suitable for studying both basic and biomedical 
problems. Strong collaborative and joint research projects are also 
underway with other members of the resident MBL community 
notably the Architectural Dynamics Program and the Bay Paul Center. 



(Staff 

BioCurrents Research Center (NIH: 

Transport of Bioactive Molecules; Development of 

Electrochemical and Optical Sensors 

DIRECTOR/SENIOR SCIENTIST 
Peter J. S Smith 

RESEARCH ASSISTANTS 
Katharine Hammar 
Laurel Moore 
Richard Sanger 

TECHNICIAN 
Robert Lewis 



Laboratory of Peter J.5. Smith: Molecular Physiology 
of Transport and Sensor Development 

SENIOR SCIENTIST 
Peter J. S. Smith 

STAFF SCIENTIST 
Mark Messerli 

POSTDOCTORAL RESEARCHERS 
Abdoullah Diarra 

VISITING SCIENTIST 
Radwan Khawaled 

ADJUNCT SCIENTIST 
George Holz 



Laboratory of Stefan McDonough: Channel Biophysics 

ASSISTANT SCIENTIST 
Stefan McDonough 



Laboratory of Orian Shirihai: Molecular Physiology 
of Mitochondria 

ASSISTANT SCIENTIST 
Orian Shirihai 

POSTDOCTORAL RESEARCHERS 
Sarah Haigh 
Shana Katzman 

Continued... 



R26 



Staff, continued 



Publications 



RESEARCH ASSISTANTS 
Erica Corson 
Solomon Graf 
Gil Palchik 



Laboratory for Reprouu /e Medicine: 
Molecular Physiology of Reproduction 

DIRECTOR 

David L. Keefe, Adjunct Scientist, Brown University 

ADJUNCT SCIENTISTS 

Lin Liu, Brown University 

James Trimarchc, Brown University 

POSTDOCTORAL RESEARCHER 
Eva Czerwiec, Brown University 



Laboratory of Ayse Dosemeci: Synaptic Plasticity 

ADJUNCT SCIENTIST 
Ayse Dosemeci 



Cooper, R A , and S Jung. 2002 Single cell electrochemistry. In Encyclopedia of 
Electrochemistry: 9 (Biochemistry), G S Wilson, ed Wiley & Sons, New York 

Kennedy, R. T., L. M. Kauri. G. M. Dahlgren, and S -K Jung 2002 Metabolic oscillations 
inp-cells Diabetes 51 (suppl. 1): S152-S161 

Liu, L., and David L. Keefe. 2002 Aging-associated aberration in meiosis of oocytes from 
Senescence-Accelerated Mouse (SAM). Hum. Reprod. 17: 2678-2685. 

Liu, L.. Maria A. Blasco, James R Trimarchi, and David L. Keefe. 2002. An essential role for 
functional telomeres in mouse germ cells during fertilization and early development Dev. 
Biol. 249: 74-84. 

Liu L, J R. Trimarchi, P. J. S. Smith, and D. L. Keefe. 2002. Checkpoint for DNA integrity at 
the first mitosis after oocyte activation. Mo/. Reprod. Dev. 62 277-288 

Liu, L, Maria A Blasco, and David L Keefe 2002 Requirement of functional telomeres for 
metaphase chromosome alignments and integrity of meiotic spindles EMBO Reports 3: 
230-234 

Liu L, J R. Trimarchi, and D L Keefe 2002 Haploidy but not parthenogenetic activation 
leads to increased incidence of apoptosis in mouse embryos. Biol. Reprod. 66: 204-210 

Liu, I I. R Trimarchi. P. J. S. Smith, and D. L Keefe 2002. Mitochondnal dysfunction 

leads to telomere attrition and genomic instability Aging Cell 1 40-46. 

McDonough, S. I., L. M. Boland. I. M. Mintz, and B. P Bean. 2002. Interactions among 
toxins that inhibit N-type and P-type calcium channels J. Gen. Physio/. 
119 313-328 




Robinson. K. R , and M A. Messerli 2002. 
Pulsating ion fluxes and growth at the pollen 
tube tip. (Online]. Science's STKE 2002 (162): 
PE51. 

Trimarchi, J. R , L. Liu, P, J. S. Smith, and D. L. 
Keefe 2002 Apoptosis recruits two-pore 
domain potassium channels used for 
homeostatic volume regulation. Am J. Physio/. 
Cell Physio/. 282: C588-C594. 

Twig. G , R. P. Malchow, K Hammar. P J S. 
Smith, H Levy, and I Perlman 2002 A novel 
turtle retinal preparation for simultaneously 
measuring light-induced electrical activity and 
changes in metabolite levels Biol. Bull. 203 
198-200 



Representative immunofluorescence images of spindles (green!, actin filaments (red), and chromosomes (blue) of 
oocytes from young and old mice. Lin Liu 



R27 



LABORATORY OF AQUATIC BIOMEDICINE 



(Staff 



SENIOR SCIENTIST 
Carol L Reinisch 

ADJUNCT SCIENTIST 
Raymond Stephens. Boston 
University 

POSTDOCTORAL 
RESEARCHERS 
Rachel Cox 
Jill Kreiling 

STUDENT 
Daniel Kabat 

VISITING SCIENTISTS 
Sylvie St Jean. Division of 
Fisheries and Oceans, 
Moncton, Canada 
Greg McCallum, Atlantic 
Veterinary College, 
Chartottetown, Prince 
Edward Island, Canada 




This laboratory is dedicated to using 
marine invertebrates as biomedical 

models to study issues of health at %. \ IBM 

the molecular level. In an embryo 

model, we are examining how industrial chemicals influence neural 
development, plasticity, and function. Specifically, polychlorinated 
biphenyls (PCBs) or chemicals found in the wells of Brick, NJ, a site of 
autism in children, are added to developing clam embryos and 
neuronal development assessed. We evaluate how chemicals pinpoint 
molecular targets such as the p53 gene family. We are dissecting how 
p53 gene expression and function are altered by chemical exposure. 
Currently we are focusing on the p73 gene, which is critically impor- 
tant in regulating neuronal development. 

The second line of research examines the induction of leukemia in 
clams or mussels at industrially polluted sites. We have developed in 
vitro technology to grow the tumor cells for genetic analyses. 
(Supported by the Alternatives Research and Development.) In 
collaboration with the Division of Fisheries and Oceans (DFO) Canada, 
we are examining the rate of induction of leukemia in Myt/7us edulis, 
the blue mussel. Mussels are placed in both clean and dirty sites in 
Canadian harbors. Five to six months later, the animals are retrieved 
and assessed for cancer using a leukemia-specific monoclonal anti- 
body generated by this laboratory. Thus far we have determined that 
exposure of mussels to PAHs, PCBs, heavy metals, and other industrial 
compounds increases both the rate and severity of leukemia. This 
research is funded by DFO, Canada. 



A non-adhenng dam 
(Mya arenaria) 
leukemia cell, stained 
reddish via the 
monoclonal antibody 
"1E10", atop a mat 
of spreading, normal 
clam hemocytes, 
Carol Reinisch 



Publication 

Jessen-Eller, eta/., 2002. 
A new invertebrate 
member of the p53 gene 
family is developm en tally 
expressed and responds to 
the polychlorinated 
biphenyls(PCBs) Environ. 
Health Perspect. 110: 3 
77-385. 




A 72-hour-old Spisula 
solidissima embryo, 
stained with an 
antibody to clam 
neurofilament/ 
intermediate filament 
protein (NF/IF). Jilt 
Kreiling 



Photo by Elizabeth Armstrong 



R28 




LABORATORY OF BARBARA FURIE AND BRUCE FURIE 



|Pub/ications 



Brown, M., B. Hambe, B. 
Furie, B. C. Furie, J. 
Stenflo, and L M 
Stenberg. 2002. Detection 
of vitamin K-dependent 
proteins in venoms with a 
monoclonal antibody 
specific for p- 
carboxyglutamic acid, 
lexicon 40: 447-453, 

Czerwiec, E., G. S. Begley, 
J. Stenflo, K Taylor, B. C. 
Furie, and B Furie. 2002. 
Structural similarity and 
functional differences 
between invertebrate and 
vertebrate carboxylases: 
expression and character- 
ization of recombinant 
vitamin K-dependent (J- 
glutamyl carboxylase from 
Conus textile. ur. J. 
Biochem. 269:6162-6172. 



Cone snails, Volker Steger 



P-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 p-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, whose main focus is the 
synthesis and function of p-carboxyglutamic acid in blood clotting 
proteins and the role of vitamin K. 



[Staff 



ADJUNCT SCIENTISTS 
Barbara C. Furie. Harvard 

Medical School 
Bruce Furie, Harvard 

Medical School 
Alan Rigby, Harvard 

Medical School 

VISITING SCIENTIST 

Johan Stenflo, University of Lund 

STAFF SCIENTIST II 
Eva Czerwiec 



Until recently, the marine cone snail had been the sole invertebrate 
known to synthesize the vitamin K-dependent amino acid, p-- 
carboxyglutamic acid (Gla), but the work of this laboratory and others 

has shown that this synthetic pathway has been preserved in most animal phyla. The 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 Conus carboxylase which 
converts glutamic acid to p-carboxyglutamic acid in the presence of vitamin K. We examined 
the diversity of animal species that maintain vitamin K-dependent carboxylation to generate 
Gla. We have cloned full length carboxylase from the beluga whale (De/ph/napterus /eucas), the 
toadfish (Opsanus tau), and the cone snail (Conus text//e) to compare these structures to the 
known bovine, human, rat, and mouse cDNA sequences. In addition, we have partially cloned 
the carboxylase gene from chicken (Ga//us gallus), hagfish (Myxine g/ut/nosa), and horseshoe 
crab (L/mu/us po/yphemus). In addition, the Drosophi/a genome contains the p-carboxylase 
gene. The predicted amino acid sequence of the carboxylase cDNA from Conus textile shows 
most regions are nearly identical to the mammalian sequence, and that there is about 40% 
sequence similarity. This protein has been expressed, and the recombinant enzyme identified 
as a carboxylase and epoxidase. These results demonstrate the broad distribution of the vitamin 

-dependent carboxylase gene, including a highly conserved motif that is likely critical for 
enzyme function. The vitamin K-dependent biosynthesis of Gla is a highly conserved function in 
the animal kingdom and we are now searching for a novel Gla containing protein that is critical 
for survival of animal species. 



LABORATORY OF NORMAN WAINWRIGHT 



R29 




The mission of this laboratory is to understand the molecu- 
lar defense mechanisms exhibited by marine invertebrates 
in response to invasion by bacteria, fungi, and viruses. 
Their primitive immune systems demonstrate unique and 
powerful strategies for survival in diverse marine environ- 
ments. The key model has been the horseshoe crab 
Limulus polyphemus. Limulus hemocytes exhibit a very 
sensitive LPS-triggered protease cascade that results in 
blood coagulation. Several proteins found in the hemocyte 
and hemolymph display microbial binding properties that 
contribute to antimicrobial defense. Commensal or 
symbiotic microorganisms may also augment the antimi- 
crobial mechanisms of macroscopic marine species. 
Secondary metabolites are being isolated from diverse 
marine microbial strains in an attempt to understand their 
role. Microbial participation in oxidation of the toxic gas 
hydrogen sulfide is also being studied. 



Photo by Volker Sieger 



I Staff 



DIRECTOR/SENIOR SCIENTIST 
Norman Wainwright 

RESEARCH ASSISTANTS 
Alice Child 
Kendra Williams 

VISITING SCIENTIST 
Porter Anderson 



| Publication 



Armstrong, Peter B., Margaret T. Armstrong, R. L Pardy, 
Alice Child, and Norman Wainwright. 2002- Immunohis- 
tochemical demonstration of a lipopolysaccharide in the 
cell wall of a eukaryote. the green alga. CMore/la. Bio/. Bull. 
203: 203-204. 




Limulus, Volker Steger 



Limulus trilobites, Beate Mittmann 



R30 




Mertidal mats, John Spear 



DSS section. Jack D Farmer 



Crystalline gypsum, John Spear 



CENTER FOR ADVANCED STUDIES IN THE SPACE LIFE SCIENCES 



Meeting proceed- 
ings published in 
The Biological 
Bulletin: 

"Limits to Self-Organiza- 
tion in Biological Systems " 
Includes 12 peer-reviewed 
articles ranging from 
computational to 
behavioral studies of self- 
organizing phenomena. 
Bio/. Bull. 202. 243-320. 
June 2002. 



In 1995, NASA's life sciences programs and the MBL established a 
cooperative agreement with the formation of the Center for Advanced 
Studies in the Space Life Sciences (CASSLS at MBL). CASSLS strives to 
increase awareness of NASA's life sciences interests and to expand 
NASA's interactions with talented biologists. In support of these goals 
in 2002, CASSLS had its busiest year ever with the presentation of 
several meetings and workshops. Scientific meetings ranged in content 
from information technology to evolutionary biology, and served more 
than 125 participants. Additionally, in another workshop, 17 East Coast 
teachers spent four days learning about astrobiology and space 
life sciences. 

Three Scientific Conferences were held at the Erik Jonsson Center for 
the National Academy of Sciences: 

April 22-24, 2002. "Combating Uncertainty with Fusion," 
presented in collaboration with meeting Chair Misha Pavel, 
Ph.D., of the Oregon Graduate Institute 

May 1-3, 2002. "Outcomes of genome-genome interactions," 
presented in collaboration with meeting Chair Mitchell Sogin, 
Ph.D., of the Marine Biological Laboratory 

September 22-24, 2002. "Understanding Mechanisms of 
Evolution," presented in collaboration with meeting Chair Eric 
Davidson, Ph.D., of the California Institute of Technology 

Teacher Enhancement Workshop held at the MBL: 

November 22-24, 2002: "Life and Living in Space," co-directed 
by Diana Jennings and Lorraine Olendzenski 



Staff 



DIRECTOR 
Diana E Jennings 

ADMINISTRATIVE 
ASSISTANT 
Heather K Farrell 



R3I 



SUMMER AND VISITING RESEARCHERS 




Sea urchin cell, Philip Presley 



Many visiting MBL investigators use marine organisms as models for 
studying basic biological processes. Research using squids, sea 
urchins, horseshoe crabs, dogfish, clams, toadfish, and sea slugs, for 
example, has increased our fundamental understanding of a broad 
range of diseases and medical conditions including cancer, diabetes, 
epilepsy, hypertension, multiple sclerosis, arthritis, and neurological 
disorders. 

During 2002, the MBL welcomed 129 Principal Investigators and 237 
other researchers from 124 institutions, representing 12 countries. 
Members of the summer community come from Harvard and Howard, 
from the University of Alabama and the Universitat de Barcelona, from 
the Food and Drug Administration and the National Institutes of 
Health, from Canada, Argentina, England, and Switzerland, among 
many other institutions, universities, agencies and countries. 

MBL summer researchers find an infrastructure and an informal, 
interactive scientific community that allows them to launch into 
research almost immediately upon their arrival. Advice and equipment 
always seem available from other researchers or from the summer 
courses. Free from academic duties at their home institutions, some 
veteran summer scientists report they do more hands-on research in 
three months at the MBL than they do during the rest of the year at 
their home institutions. 



Spider crab embryo 
Gundrun Aspoek 




R32 




Spisub :. - Jissima nuclei, Anne Goldman 



2002 Summer Investigators 



Elizabeth Armstrong 



Armstrong, Clay 
University of Pennsylvania 

Armstrong, Peter B 
University of California, Davis 

Augustine, George J 

Duke University Medical Center 

Baker, Robert 

New York University Medical Center 

Barlow, Jr., Robert B. 

State University of New York 

Upstate Medical University 

Barry, Susan 

Mount Ho/yoke College 

Beauge, Luis 

Institute de Investigacion Medica 

"Mercedes y Martin Ferreyra," 

Argentina 

Bennett, Michael V. L. 

Albert Einstein College of Medicine 

Bodznick, David 
Wesleyan University 

Botto, Florencia 

Universidad Nacional de Mar del 

Plata, Argentina 

Boyer, Barbara 
Union College 

Brady, Scott T. 

The University of Texas Southwest- 
ern Medical Center, Dallas 

Brown, Joel 

Albert Einstein College of Medicine 

Browne. Carole 

Wake Forest University School of 

Medicine 

Burbach, Peter 

Rudolf Magnus Institute for 

Neurosciences, The Netherlands 

Burger, Max M 

Novartis International AG, 

Switzerland 



Burgess, David 
Boston College 

Camargo, Maristela 
University of Sao Paulo, Brazil 

Canessa, Cecilia 
Vale University 

Chang, Fred 
Columbia University 

Chappell, Richard L 

Hunter College, City University of 

New York 

Clay, John 

National Institutes of Health 

Cohen, Lawrence B. 

Yale University School of Medicine 

Cohen, William D. 

Hunter College, City University of 

New York 

Crawford, Karen 

St. Mary's College of Maryland 

De Polavieja, Gonzalo 
University of Cambridge, United 
Kingdom 

De Weer, Paul 

University of Pennsylvania School 

of Medicine 

Denk, Winfried 

Max-Planck-lnstitute for Medical 
Research, Germany 

Desai, Rooma 

Yale University School of Medicine 

Dickinson, Bonny 
Children's Hospital 

DiPolo, Reinaldo 
Institute Venezolano 
/nvestigaciones Cientificas, 
Venezula 

Dodge, Frederick 

State University of New York 

Upstate Medical University 



Douglas, John K. 
University of Arizona 

Eckberg, William 
Howard University 

Edds-Walton, Peggy 
Parmly Hearing Institute of 
Loyola University 

Ellenberg, Jan 

European Molecular Biology 

Laboratory, Germany 

fay, Richard 

Loyola University of Chicago 

Field, Christine 

Harvard University Medical School 

Fields, Douglas 

National Institutes of Health 

Fishman, Harvey M. 

University of Texas Medical Branch, 

Galveston 

Gadsby, David 

The Rockefeller University 

Galione, Antony 

Oxford University, United Kingdom 

Gandhi, Sunil 
The Salt Institute 

Garber, Sarah 

The Chicago Medical School 

Gerhart, John 

University of California, Berkeley 

Giuditta, Antonio 

Universifa di Napoli "Federico II, " 

Italy 

Goldman, Robert D. 
Northwestern University Medical 
School 

Gould, Robert 

New York State Institute for Basic 

Research 



R33 



Groden, Joanna 
University of Cincinnati 



Koonce, Micnael 
Wadsworth Center 



Pant, Hansh 

National Institutes of Health 



Sugimon, Mutsuyuki 

New York University Medical Center 



Gruenbaum, Yosef 


Kuhns, William 


Parysek, Linda 


Tank, David 


The Hebrew University of Jerusalem, 


The Hospital for Sick Children, 


University of Cincinnati 


Princeton University 


Israel 


Canada 










Perlman, (do 


Telzer, Bruce 


Gruhn, Matthias 


Lafer, Eileen M., University of Texas 


Technion Israel 


Pomona College 


Cornell University 


Health Science Center, San Antonio 










Ponka, Prem 


Tilney, Lewis 


Haimo, Leah 


Lambert, Nevin 


McGill University, Canada 


University of Pennsylvania 


University of California, Riverside 


Medical College of Georgia 










Rakowski, Robert F. 


Treistman, Steven 


Hardege, Jorg 


Landowne, David 


Ohio University 


University of Massachusetts Medical 


Hull University, United Kingdom 


University of Miami School of 




School 




Medicine 


Ratner, Nancy 




Harper, Mary-Ellen 
University of Ottawa, Canada 


Langford, George 


University of Cincinnati 


Tytell, Michael 
Wake Forest University School of 




Dartmouth College 


Reese, Thomas S 


Medicine 


Heck. Diane 




National Institutes of Health 




Rutgers University 


Laskin, Jeffrey 




. fi^^^ML-ld^^Kf^^H 




University of Medicine and 


Rieder, Conly 


'.< '& 


Hershko, Avram 


Dentistry of New Jersey 


Wadsworth Center 


k "J Ea 


Technion-lsrael Institute of 








Technology, Israel 


Laufer, Hans 


Rinberg, Dima 






University of Connecticut 


Bell Laboratories 


Efl &i 



Highstein, Steven M 
Washington University School of 
Medicine 

Hines, Michael 

Yale University School of Medicine 

Holmgren, Miguel 

Harvard University Medical School 

Iribame, Oscar 

Universidad Nacional de Mar del 

Plata, Argentina 

Johnston, Daniel 

Baylor College of Medicine 

Jonas, Elizabeth 

Yale University School of Medicine 

Kaczmarek, Leonard 

Yale University School of Medicine 

Kaplan. Barry 

National Institutes of Mental Health 

Kaplan, llene M. 
Union College 

Kauer, Julie 
Brown University 

Kaupp, U.B 

Institut fur Siologische 

Informationsverarbeitung, Germany 

Khodakhah, Kamran 

University of Colorado School of 

Medicine 

Khodjakov, Alexey 
Wadsworth Center 

Kirschner, Marc 

Harvard University Medical School 



LeBaron, Richard 

University of Texas, San Antonio 

Lipicky, Raymond J 

Food and Drug Administration 

Lipscombe, Diane 
Brown University 

Llinas, Rodolfo R 

New York University Medical Center 

Magee, Jeff 

Louisiana State University Medical 

Center 

Malchow, Robert Paul 
University of Illinois, Chicago 

Martinez, Joe 

University of Texas, San Antonio 

McNeil, Paul 

Medical College of Georgia 

Mensinger, Allen 

University of Minnesota, Duluth 

Mitchison, Timothy 

Harvard University Medical School 

Mittmann. Beate 

Institute fur Biologle, Germany 

Moore, John W. 

Duke University Medical Center 

Mooseker, Mark 
Yale University 

Nasi, Enrico 

Boston University School of 

Medicine 

Palazzo, Robert 

Rensselaer Polytechnic Institute 



Ripps, Harris 

University of Illinois College of 

Medicine 

Rodnguez-Contreras, Adrian 
University of California, Davis 

Rome, Larry 

University of Pennsy/vania 

Russell, James 

National Institutes of Health 

Salmon. Edward 

University of North Carolina, 

Chapel Hill 

Silver, Robert 

Wayne State University School 

of Medicine 

Sloboda, Roger D 
Dartmouth College 

Sluder, Greenfield 
University of Massachusetts 
Medical School 

Smotherman, Michael 
University of California, Los 
Angeles 

Spiegel, Evelyn 
Dartmouth College 

Spiegel, Melvin 
Dartmouth College 

Stemacker, Antoinette 
University of Puerto Rico 

Stockbridge, Norman 

Food and Drug Administration 




Squid vesicles, Harvey Fishman 



Vollrath, Melissa Ann 
Baylor College of Medicine 

Weidner, Earl 

Louisiana State University 

Wheeler, Damian 
McGill University, Canada 

Whittaker. J. Richard 
University of new Brunswick, 
Canada 

Zecevic, Dejan P. 

Yale University School of Medicine 

Zimmerberg, Joshua 
National Institutes of Health 

Zottoli. Steven 
Williams College 

Zukin-Bennett. R. Suzanne 

Albert Einstein College of Medicine 



R34 




Mitochondria/ dysfunction 
and oxidative stress lead to 
te/omere attrition and 
chromosomal end-to-end 
fusions (indicated by 
arrows) in mouse embryos, 
Lin Liu 



MBL Research Fellows 



Twenty-two 
scientists received 
awards to conduct 
research at the 
MBL in 2002. 



Peter Armstrong, Ph.D. 

University of California, Davis 

His research focused on immune defense proteins and 

defense processes of arthropods that show evolutionary 

conservation. Dr. Armstrong was funded by The Laura and 

Arthur Colwin Endowed Summer Research Fellowship Fund. 



Florencia Botto, Ph.D 

Universidad Nacional de Mar del Plata, Mar del Plata, 

Argentina 

"The role of intertidal burrowing species (e.g., crabs) on the 

dynamics of organic matter in estuarine environments." 

Dr. Botto was funded by the MBL Associates, The Catherine 

Filene Shouse Foundation, and the Lucy B. Lemann 

Fellowship Fund. 



Cecilia M. Canessa, M.D. 

Yale University, New Haven, Connecticut 

"Cloning and characterization of ASIC channels in marine 

vertebrates " Dr Canessa was funded by The Erik B Fries 

Endowed Fellowship, the M.G F. Fuortes Memorial 

Fellowship Fund, The Stephen W. Kuffler Fellowship Fund, 

an MBL Research Fellowship, and the Ann E Kammer 

Memorial Fellowship Fund 



Fred Chang, M.D., Ph.D. 

Columbia University College of Physicians and Surgeons, 

New York, New York 

"Placement of the cell division plane " Dr. Chang 

was funded by The Universal Imaging Corporation 

Fellowship Fund. 



J. Peter H. Burbach, Ph.D. 

Rudolf Magnus Institute for Neurosciences University 

Medical Center, Utrecht, The Netherlands 

"The stellate ganglion of the squid as a model for 

neurodevelopment gene cascades." Dr, Burbach was 

funded by The Stephen W. Kuffler Fellowship Fund and the 

Baxter Postdoctoral Fellowship Fund. 



David Burgess, Ph.D. 

Boston College, Chestnut Hill, Massachusetts 

"Cytokinesis in embryonic cells." Dr. Burgess was funded by 

the Josiah Macy, Jr Foundation, the Robert Day Allen 

Fellowship Fund, and the William Townsend Porter 

Foundation. 



Maristela Camargo, D.V.M., Ph.D. 

University Sao Paulo, Sao Paulo, Brazil 

"An evolutive study of Th1/Th2 differentiation." Dr. 

Camargo was funded by The Catherine Filene Shouse 

Foundation, The Frederik B Bang Fellowship Fund, and an 

MBL Research Fellowship 



Karen Crawford, Ph.D. 

St. Mary's College of Maryland, St. Mary's City, Maryland 
"Molecular analysis of B-catenin expression, axes formation 
and early embryogenesis in the squid, Loligo pealei, 
insights into evolution." Dr Crawford was funded by 
the Evelyn and Melvin Spiegel Fellowship Fund, the 
MBL Associates, and the James A and Faith Miller 
Fellowship Fund 



Bonny Dickinson, Ph.D. 

Harvard Medical School and Children's Hospital, Boston 
"Calmodulm and the unconventional myosins play key roles 
in FcRn trafficking by mediating interaction with the actin 
cytoskeleton" Dr Dickinson was funded by The Laura and 
Arthur Colwin Endowed Summer Research Fellowship, The 
Frederik B Bang Fellowship Fund, the MBL Associates, and 
an MBL Research Fellowship. 



R35 



John K. Douglass, Ph.D. 

University of Arizona, Tucson 

"An electrophysiological and anatomical study of central 

visual pathways in Limulus po/yphemus." Dr. Douglass was 

funded by the H. Keffer Hartline Fellowship Fund, the Plum 

Foundation, John E. Dowling Fellowship Fund, and the 

Herbert W. Rand Fellowship. 



Jan Ellenberg, Ph.D. 

European Molecular Biology Laboratory, 

Heidelberg, Germany 

"Mechanism of nuclear envelope breakdown (NEBD) in 

echinoderm oocytes and embryos." Dr. Ellenberg was the 

2002 Nikon Fellow, funded by Nikon Instruments, Inc. 



Sarah Garber, Ph.D. 

Chicago Medical School, North Chicago, Illinois 

"Correlation of ion flux and regulation of cell volume." 

Dr. Garber was funded by The Erik 8. Fries Endowed 

Fellowship. 



Yosef Gruenbaum, Ph.D. 

Institute of Life Sciences at The Hebrew University of 

Jerusalem, Jerusalem, Israel 

"Molecular and functional dissection of the nuclear lamina 

in the surf clam." Dr. Gruenbaum was funded by The Gruss 

Upper Foundation, The Frank R. Lillie Fund, The Erik B. 

Fries Endowed Fellowship, the Robert Day Allen 

Fellowship Fund, and the H. Burr Steinbach Memorial 

Fellowship Fund- 



Leah Haimo, Ph.D. 

University of California, Riverside 

Her research focused on how molecular motors are 

regulated to control organelle transport. Dr. Haimo was 

funded by The Laura and Arthur Colwin Endowed Summer 

Research Fellowship Fund. 



Jorg Hardege, Ph.D. 

Hull University, Hull, United Kingdom 

"Do sex pheromone differences in Nereidid polychaetes 

lead to reproductive isolation?" Dr. Hardege was funded 

by the Lucy B. Lemann Fellowship Fund, The Charles R. 

Crane Fellowship Fund and The John O. Crane Fellowship 

Fund. 



Mary-Ellen Harper, Ph.D. 
University of Ottawa, Ontario, Canada 
"Use and construction of self-referencing microelectro- 
chemical probes for studies into the role of Uncoupling 
Protein-3 (UCP3) in myocellular energy metabolism." 
Dr. Harper was funded by The Laura and Arthur Colwin 
Endowed Summer Research Fellowship and the H. Burr 
Steinbach Memorial Fellowship Fund. 



Oscar Iribarne, Ph.D. 

Universidad Nacional de Mar del Plata, Argentina 
"The role of the SW Atlantic intertidal burrowing crab 
Chasmagnathus granu/ata in the dynamics of nutrients." 
Dr. Iribame was funded by the Lucy B. Lemann 
Fellowship Fund. 



Diane Lipscombe, Ph.D. 

Brown University, Providence, Rhode Island 

"The identification of novel conus toxins to discriminate 

among voltage-gated calcium channels and their splice 

variants." Dr. Lipscombe was funded by The Catherine 

Filene Shouse Foundation and the MBL Associates 



Ido Perlman, Ph.D. 
Technion-lsrael Institute of 
Technology, Haifa, Israel 
"Nitric oxide synthesis in the 
vertebrate retina and its 
physiological and cellular 
functions " Dr. Perlman was 
funded by The Gruss Upper 
Foundation. 



Prem Ponka, M.D., Ph.D. 

McGi/l University, Montreal, 

Canada 

"Iron Trafficking in Erythroid Cells: 

A Collaborative Program " 

Dr. Ponka was funded by the Frank 

R. Lillie Fund. 



Nancy Ratner, Ph.D. 
University of Cincinnati College of 
Medicine, Cincinnati, Ohio 
The title of her research project 
was "Cyclin-dependent kinases in 
fast axonal transport." Dr. Ratner 
was funded by the Frank R. Lillie 
Fund and The Herbert W Rand 
Fellowship Fund. 



J. Richard Whittaker, Ph.D. 

University of New Brunswick in Fredericton, New 

Brunswick, Canada 

"The Sea Squirt's Secret: How We Discovered Our 

Chordate Ancestry." Dr. Whittaker was funded by the 

Frank A. Brown, Jr. Readership Fund 




Kevin Begos 



R36 




Grass Fellows 

Nine scientists participated in the 2002 Grass Fellowship Program in Neuroscience at the Marine Biological 
Laboratory. The program is sponsored by The Grass Foundation and offers independent research opportuni- 
ties to young neuroscientists. The 2002 program was directed by Dr. Susan R. Barry of Mount Holyoke 
College. Dr. Melissa Ann Vollrath of Baylor College of Medicine was the program's Associate Director. 



Rooma Desai, Ph.D., Yale School of Medicine, 
"Isolation of K' Currents Underlying the 'Chopper 
Response' of the Principal Cells of Lateral Superior 
Olive (LSO)" 

Sunil Gandhi, The Salk Institute, "Evanescent Wave 
Microscopy of Single Vesicle Recycling in Goldfish 
Retinal Bipolar Terminals" 

Matthias Gruhn, Ph.D., Cornell University, 
"Correlation of Extracellular Nerve Recordings 
and Behavioral Activity in Live Crayfish Using 
Implantable Electrodes and High-Speed 
Video Technology" 

Beate Mittmann, Institut fur Biologic, "The 
Development of the Nervous System in the 
Horseshoe Crab Limulus po/yphemus (Chemicerata, 
Ziphosura) and its Implication for Arthropod 
Relationships" 



Gonzalo Garcia de Polavieja, Ph.D., UCLA School 
of Medicine, "Behavioral Algorithm and Circuitry 
for Visual Motion Detection in the Leech" 

Dima Rinberg, Ph.D., Bell Laboratories Lucent 
Technologies, "Optical Recording of Multineuron 
Activity Using Ballistic Delivery of Voltage Sensitive 
Dyes" 

Adrian Rodriguez-Contreras, Ph.D., University of 
California, Davis, "Intrinsic Properties, Distribution 
and Morphology of Inhibitory Neurons in the 
Midbrain Auditory Pathway of Chicken" 

Michael S. Smotherman, Ph.D., UCLA, "Descend- 
ing Control of Chromatophore Motoneurons in the 
Cephalopod Brain" 

Damian G. Wheeler, McGill University, 
"Multiprotein Complex Signaling from Synapse to 
Nucleus" 



Limulus centra/ nervous system, Beate Mittmann 



Other Research Personnel 



R37 



Abe. Teruo, Niigata University, Japan 
Adams, Christina, Williams College 
Akingbade, Kathenne, University of Oxford, 

United Kingdom 

Alber, Merryl, University of Georgia 
Alimi, Mariam, Wake Forest University 
Alliegro, Mark, Louisiana State University 

Health Sciences Center 
Arnolds, David. Williams College 
Artigas, Pablo. Rockefeller University 
Asomoah, Nikiya, Williams College 
Ayliffe, Harold, University of Utah 

Banta, Gary, Roskilde University, Denmark 

Bartels-Hardege. H , Hull University, United Kingdom 

Beach, Rebecca, Hollins University 

Bearer, Elaine, Brown University 

Berbenan, Graciela, Institute de Investigacion Medica 

"Mercedes y Martin Ferreyra," Argentina 
Bertetto, Lisa, Wesleyan University 
Biber, Sarah, Earlham College 
Binion, Samantha, Emory University 
Bodily, Jill, Stanford University 
Bordenstem, Seth, Marine Biological Laboratory 
Borley, Kimberly. Ohio University 
Bernstein, Gil, Technion, Israel 
Braun, Alexander, Hunter College 
Brewton, Luke, University of Texas Medical Branch 
Brown, Jeremiah, Dartmouth College 
Bucior, Iwona, Friedrich Miescher Institut, Switzerland 

Cameron, Lisa, Univesity of North Carolina, Chapel Hill 

Cameron, Luiz, University of Rio de Janeiro, Brazil 

Carroll, Amanda, Marine Biology Laboratory 

Cefaliello, Carolina, University of Naples, Italy 

Chaikhoutdin, Irina, Hunter College 

Chang, Donald, Kong Kong University, Kong Kong 

Chen, Xiaobing, National Institutes of Health 

Chiao, Chuan-Chin, Massachusetts General Hospital 

Chludzinski, John, National Institutes of Health 

Churchill, Grant, University of Oxford, United Kingdon 

Clark, Michael, Medical College of Georgia 

Collis, Leon, University of Rhode Island 

Conrad, Mara, Hunter College 

Coric, Tatjana, Yale University School of Medicine 

Corson, Erica, Mt Holyoke College 

Couch, Ernest, Texas Christian University 

Cox, Melissa, Marine Biological Laboratory 

De Stefano, Rosanna, University of Naples, Italy 

Delaney, Martha, Marine Biological Laboratory 

DeNobile, JoAnna, Hunter College 

Dmeen, Shauna, Williams College 

Djunsic, Maja, Yale University School of Medicine 

Ehsanian, Reza, NASA Ames Research Center 

Evans. Louise. Harvard Medical School 

Eyman, Maria, Universita di Napoli "Fedenco II," Italy 

Fernandez-Busquets, Xavier, Universitat de Barcelona, Spam 
Ferrara, Eugenia, Unversita di Napoli "Federico II," Italy 
Fevrier, Salem, Williams College 
Follett, Christopher. Marine Biological Laboratory 
Franzmi-Armstrong, Clara, University of Pennsylvania 

School of Medicine 
Frick, Andreas, Baylor College of Medicine 

Gainer, Harold, National Institutes of Health 
Garnham, Give, University of Oxford, United Kingdom 
Garza, John, University of Texas at San Antonio 
Gaszewska, Anna, Medical College of Georgia 



Ge, Lan, University of California, Riverside 

Gifford, Raeann, University of Kansas 

Gilland, Edwin, New York University School of Medicine 

Gioio, Anthony, National Institutes of Health 

Goda, Makoto, Japan Biological Information Research Center, Japan 

Goldman, Anne, Northwestern University Medical School 

Gomez, Maria del Pilar, Boston University School of Medicine 

Grant, Philip, National Institutes of Health 

Gratton, Michael Anne, University of Pennsylvania 

Greer, Jonathan, Brown University 

Guo, YiFan, Williams College 

Gyoeva, Fatima, Institute of Protein Research, Russia 

Hangsterfer, Alexandra, Marine Biological Laboratory 

Hanney, Nicholas, Marine Biological Laboratory 

Harrington, John, University of California, Davis 

Harwood, Claire, University of Pennsylvania 

Hatoum, Nagi, New York University Medical School 

Helbig, Anmka, Institut fur Biologische Informationswerarbeltung, 

Germany 
Hellemons, Anita, Rudolf Magnus Institute for Neuroscience, 

The Netherlands 

Helm, Jessica, Yale University School of Medicine 
Hepler, Peter, University of Massachusetts 
Hernandez, Carlos, New York University School of Medicine 
Hernandez, Ruben, University of Texas 
Hess, Sam, National Institutes of Health 
Hoffman, Mathew, Boston College 
Holtz, Scott, Northwestern University Medical School 
Homsi, Sara, Wake Forest University 
Horseman, Nelson, University of Cincinnati 

Iliev, Dimitar, University of Notre Dame 

Jackson, Ticaria, Howard University 
Johnson, Michael, University of Connecticut 
Jurkovicova, Dana, National Institutes of Health 

Katar, Mazkhan, Wayne State University 
Khavandgar, Simin, Albert Einstein College 
King, Curtis, University of Utah 
Knowles, James, Colgate University 
Koester, Helmut, Baylor College of Medicine 
Konnerth, Arthur, University of Munich, Germany 
Koop-Jabobsen, Ketil, Roskilde University, Denmark 

Lee, Kyeng-Gea, Hunter College 

Levy, Hanna, Technion, Israel 

Li, YuLong, Duke University Medical Center 

Lober, Robert, Medical Collegte of Georgia 

Lockard, Jon, National Institutes of Health 

Louis, Lydia, Rutgers, The State University of New Jersey 

Lowe, Chris, University of California, Berkeley 

Maddox, Paul, University of North Carolina 

Marangoni, Maria Natalia, University of Buenos Aires, Argentina 

Martinez, Gabnela, University of New Hampshire 

Masgrau, Roser, Oxford University, United Kingdom 

Maude, Haskell, Marine Biological Laboratory 

Mbanu, Chijioke, Wayne State University 

McAnelly, Lynne. University of Texas, Austin 

McCurley. Amy, Richmond University 

Molina, Anthony, University of Illinois at Chicago 

Momose-Sato, Yoko, Tokyo Medical and Dental University, Japan 

Montanez, Marlena, Mt Holyoke College 

Montgomery, John, University of Auckland, New Zealand 

Moran, Kimberly, New York University School of Medicine 

Moreira, Jorge, University of Sao Paulo, Brazil 

Morfini, Gerardo, University of Texas Southwestern Medical Center 

Morgan, Anthony, University of Oxford, United Kingdom 

Morse, Thomas, Yale University 



Continued. 



R38 



Najera, Julia, University of Texas 

Nedoluzhko, Aleksey, Wadsworth Cent ,- 

Ng, Michelle, Boston College 

Nobuhara, Robert, Colorado S* ersity 

Nonaka, Mio, Kyoto Univere^ 

Normand, Danielle, Unive': Hampshire 

Nuccitelli, Richard. Unive' Connecticut Health Center 

O'Neal, Jessie;, scleston 

Obata, Shuictv s City University, Japan 

Olsen, Gary, Ur.ivei : Illinois, Urbana 

Ortiz, Christopi:e., University of California, Irvine 

Palmer, Luc/, University of Minnesota 

Pascal, Akil, Williams College 

Passianoto, Caio, Marine Biological Laboratory 

Patel, Reshma, Marine Biological Laboratory 

Peacock-Villa, Elizabeth, Dartmouth College 

Pelletier, Cory, Brown University 

Petersen, Jennifer, National Institutes of Health 

Pollema, Sarah, University of Minnesota, Duluth 

Prasad, Kondury, University of Texas Health Science Center 

Quigley, James, Scripps Research Institute 

Rabbitt, Richard, University of Utah 

Radojicic, Mihailo, Yale University 

Redenti, Stephen, Hunter College 

Remick, Kathenne. University of Texas Medical Branch 

Rhodes, Paul, New York University Medical School 

Richmond, Hazel, University of Minnesota 

Ridings, Corey, Occidental College 

Rieder, Leila, Albany High School 

Rinkwitz, Silke, New York University Medical School 

Ripps, Jeff, Towson University 

Rummel, John, NASA 

Sanchez, Carlos, University of Texas 

Sato, Katsushige, Tokyo Medical and Dental University, Japan 

Satpute, Prasanna, Brown University 

Schnackenberg, Bradley, University of Carolina, Chapel Hill 

Scotto Lavina, Zeno, National Institutes of Health 

Short, Michelle, Marine Biological Laboratory 

Shumaker, Dale, Northwestern University Medical School 

Simpson, Andrew, University of California, Santa Barbara 

Smillie, Darren, University of Edinburgh, United Kingdom 

Smith, Kalmia, Cornell University 

Steeds, Craig. Kansas University 

Stone, Eric, Marine Biological Laboratory 

Sweeney, Catherine, Marine Biological Laboratory 

Takahashi, Hajime, Olympus Optical Co , Ltd., Japan 

Tanner, Geoffrey, Wesleyan University 

Terasaki. Mark, University of Connecticut Health Center 

Thompson, Reid, Dartmouth College 

Tokumaru, Hiroshi, Duke University Medical Center 

Tokumaru, Keiko, Duke University Medical Center 

Twig, Gilad, Technion, Israel 

Tzur, Yonatan, Hebrew University, Israel 

Vautrin, Jean, University of Montpellier, France 
Vetrano, Anna, Rutgers University 
Villalba-Galea, Carlos, Duke University Medical Center 
Vucinic, Dejan, Yale University 

Wachowiak, Matt, Yale University School of Medicine 
Weedon, Monica, Marine Biological Laboratory 
Wetherington, Jonathan. Medical College of Georgia 
Weyand, Ingo, Institut fur Biologische Informationswerarbeitung, 

Germany 

Wheeler. N'sreha, Earlham College 
Williams, Keuuirsh. Howard University 
Wollert. Torsten. Universitat Rostock, Germany 

Yamasaki, Michiko. jnivc-rsity of Oxford, United Kingdom 
Young, lain, University of Pennsylvania 



Domestic Institutions Represented 



Albert Einstein College of Medicine 
Arizona State University 

Barnard College 

Baylor College of Medicine 

Beth Israel Deaconess Medical Center 

Boston College 

Boston University School of Medicine 

Brown University 

California Institute of Technology 
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 Francisco 
Cincinnati, University of 
Colorado School of Medicine, University of 
Columbia University 
Connecticut, University of 
Cornell University 
Courant Institute 

Dartmouth College 

Duke University 

Duke University Medical Center 




Emory University 

Federal Department of Agriculture 

Flower Garden Banks National Marine Sanctuary 

Food and Drug Administration 

Gladstone Institute of Neurological Disease 

Hartford, University of 

Harvard University 

Harvard University Medical School 

Hawaii, University of 

Howard University 

Hunter College 

Illinois, University of 



Zakevicius, Jane, University of Illinois College of Medicine 
Zakon. Harold, University of Texas, Austin 



R39 



Kansas. University of 

Louisiana State University 
Loyola University of Chicago 

Maryland, University of 

Massachusetts, University of 

Medical College of Georgia 

Miami School of Medicine, University of 

Michigan State University 

Millersville University 

Minnesota, University of 

NASA 

National Institutes of Health 

National Institutes of Mental Health 

New York State Institute for Basic Research 

New York University 

New York University School of Medicine 

North Carolina State University 

North Carolina, University of 

Northwestern University Medical School 

Ohio University 

Penn State University 
Pennsylvania, University of 
Pomona College 
Providence College 
Puerto Rico, University of 

Rutgers, the State University of New Jersey 

Scripps Research Institute 

South Carolina, University of 

St Mary's College of Maryland 

Stanford University 

State University of New York Upstate Medical University 

Syracuse University 

Texas Health Science Center, University of 
Texas Southwestern Medical Center, University of 
Texas, University of, Austin 
Texas, University of, San Antonio 
The Rockefeller University 

Union College 
Utah, University of 

Virginia, University of 

Wadsworth Center 

Wake Forest University 

Washington University School of Medicine 

Wayne State University School of Medicine 

Wesleyan University 

Williams College 

Women and Infants Hospital 



Foreign Institutions Represented 



Auckland, University of, New Zealand 

Barcelona, Universitat de, Spain 
Buenos Aires, University of, Argentina 

Cambridge, University of. United Kingdom 

Edinburgh, University of, United Kingdom 
European Molecular Biology Laboratory, Germany 

Friedrich Miescher Institute, Switzerland 

Hebrew University of Jerusalem, Israel 
Hong Kong University, Hong Kong 
Hospital for Sick Children, Canada 
Hull University, United Kingdom 

Institut fur Biologische Informationswerarbeitung, Germany 

Institute of Protein Research, Russia 

Institute de Investigacion Medica "Mercedes y Martin Ferreyra, 

Argentina 
Institute Venezolano Investigaciones Cientificas, Venezuela 

Japan Biological Information Research Center, Japan 
Kyoto University, Japan 

Max-Planck-lnstitute for Medical Research, Germany 
McGill University, Canada 
Montpellier, University of, France 
Munich, University of, Germany 

Napoli "Federico II", Universita di, Italy 
New Brunswick, University of, Canada 
Nugata University, Japan 
Novartis International AG, Switzerland 

Olympus Optical Co , Ltd . Japan 
Ottawa, University of, Canada 
Oxford, University of, United Kingdom 

Rio de Janeiro, University of, Brazil 

Roskilde University, Denmark 

Rostock, Universitat, Germany 

Rudolf Magnus Institute for Neuroscience, The Netherlands 

Sao Paulo, University of, Brazil 
Technion-lsrael Institute of Technology, Israel 
Tokyo Medical and Dental University, Japan 

Universidad Nacional de Mar del Plata, Argentina 
University of College London, United Kingdom 
Utrecht, University of, The Netherlands 

Yokohama City University, Japan 



Yale University 

Yale University School of Medicine 



R40 





| General Scientific Meetings Awards 



On the recommenda- 
tion of the Science 
Council, the MBL 
reinstated the MBL 
Award for outstand- 
ing presentations at 
the Laboratory's 
annual General 
Scientific Meetings. 
The award in each 
category consists is 
a crystal clock and 
a $300 cash prize. 

Fifty-six presentations 
were given during 
the Meetings, which 
were held August 72 
to 14 in the Lillie 
Auditorium. After 
peer-review of all 
papers and talks, four 
awards and two 
honorable mentions 
were p; -sented. 



Senior Investigator: 

Peter Armstrong, Margaret Armstrong, R. L. Pardy, 
Alice Child, and Norman Wainwright, "Histochemi- 
cal demonstration of lipopolysaccharide in the cell 
wall of a eukaryote, the green alga Chlorella" 

Junior Investigator: 

Michael Smotherman, "Acetylcholine mediates 
excitatory input to chromatophore motoneurons in 
the squid, Loligo pealei" 

Graduate Student: 

Beate Mittmann, "Early neurogenesis in the 
horseshoe crab Limu/us po/yphemus and its 
implication for arthropod relationships" 

Undergraduate Student: 

Jane La Du, Deana Erdner, Sonya Dyhrman, and 
Don Anderson, "Molecular approaches to under- 
standing population dynamics of the toxic 
dinoflagellate A/exandrium fundyense" 

Student Honorable Mentions: 

Jeremy M. Testa, Matt Charette, Edward Sholkovitz, 
Matt Allen, Adam Rago, and Craig Herbold, 
"Dissolved iron cycling in the subterranean estuary 
of a coastal bay: Waquoit Bay, Massachusetts" 

D. E. Arnolds, 5. J. Zottoli, C. E. Adams, S. M. 
Dineen, S. Fevrier, Y. Quo, and A. J. Pascal, 
"Physiological effects of tricaine on the 
supramedullary/dorsal neurons of the cunner, 
Tautogo/abrus adspersus" 



2002 
FRIDAY EVENING LECTURES 



June 21 

Barry Bloom, Harvard School of Public Health 

"Economic and Political Implications of 

Global Infectious Diseases" 

June 28 
R. Alan B. Ezekowitz, Massachusetts Gen 

Hospital for Children 
"Fighting Infections from Flies to Man" 

JulyS 

Lang Lecture 

Michael J. Ryan, University of Texas, AL 
"Sexual Selection and The Brain" 

July 12 

Gail K. Naughton, Advanced Tissue Sciences 

"Stem Cells and Tissue Engineering: 

From Science Fiction to Medical Fact" 

July 18, 19 

Forbes Lectures 

William Newsome, Stanford University 

I.Thursday, July 18 

"Making Decisions: The Brain's Link Between 

Perception and Action" 

II. Friday, July 19 

"Seeing Motion: Linking Neurophysiology 
to Perceptual Psychology" 

July 26 

Michael Brown, University of Texas 

Southwestern Medical Center at Dallas 

"Genetic Defenses Against Heart Attacks" 



August 2 

Steven Hyman, Harvard University 

"Reflections on Behavior in 

the Postgenomic Era" 

August 9 

Dan Barry, NASA 

"Sensations of Space Flight" 

August 16 
Tim Hunt, Cancer Research UK, 

Clare Hall Laboratories 

"What is the Cell Cycle and 

How is it Controlled?" 




Nerve branches and synaptic endings in a musc/e of a transgenic 
mouse, Jeff Lichtman 



Pub/ications 



R41 



Alliegro, M C 2002. Coiled body heterogeneity induced 
by G1 arrest with amilonde + bumetanide. Exp. Cell Res. 
279 111-117 

Alhegro, M C , and M A Alliegro 2002 Nuclear injection 
of anti-pigpen antibodies inhibits endothelial cell division 
J Bio/. Chem. 277: 19,037-19,041 

Armstrong, Peter B , Margaret T Armstrong, R L Pardy, 
Alice Child, and Norman Wainwright 2002 Immunohis- 
tochemical demonstration of a lipopolysacchande in the 
cell wall of a eukaryote, the green alga, Ch/orella Biol Bull 
203 203-204 

Arnolds, D E W.. S J Zottoli, C E Adams, S. M. Dineen, 
S Fevrier, Y Guo, and A J Pascal 2002. Physiological 
effects of tncaine on the supramedullary/dorsal neurons 
of the cunner, Tautogolabrus adspersus Biol Bull 203: 
188-189 

Bearer, E L , and P. Satpute-Krishnan 2002 The role of 
the cytoskeleton in the life cycle of viruses and mtracellular 
bacteria tracks, motors, and polymerization machines. 
Curr. Drug Targets Infect. Disord 2 247-264 

Borst, Douglas, and Robert Barlow. 2002 Orcadian 
rhythms in locomotor activity of juvenile horseshoe crabs. 
Bio/. Bull. 203 227-228 

Boyle R , S M Highstem, J P Carey, and J P Xu 2002 
Functional recovery of anterior semicircular canal afferents 
following hair cell regeneration in birds JARO 3 149-166 

Brown, J R , E M Peacock-Villada, and G. M. Langford 
2002 Globular tail fragment of myosin-V displaces vesicle- 
associated motor and blocks vesicle transport in squid 
nerve cell extracts Biol Bull 2 210-211 

Cermak, Michael J 2002 Caranx /atus (Carangidae) 
chooses dock pilings to attack silverside schools a tactic to 
interfere with stereotyped escape behavior of prey? Bio/. 
Bull. 203. 241-243. 

Chappell, R L., E Schuette, R. Anton, and H Ripps 2002 
GABAc receptors modulate the rod-driven ERG b-wave of 
the skate retina Doc. Ophtha/mol 105: 179-188. 

Clay, John R , and Alan Kuzinan 2002 Trafficking of axonal 
K+ channels potential role of Hsc70 J. Neurosci. Res. 67 
745-752 

Clay, John R , and Alvin Shner 2002 Temperature 
dependence of bistability in squid giant axons with alkaline 
mtracellular pH J Membr. Biol 187 213-223 

Claypool S M , B L. Dickinson, M Yoshida, W I Lencer, 
and R S Blumberg 2002 Functional reconstitution of 
human FcRn in Madm-Darby canine kidney cells requires 
co-expressed human beta 2-microglobulin J. Biol- Chem 
277 28,038-28,050 



Cox, B. L., Popa, R., Bazylinski, D A , Lanoil B , Douglas, 
S , Belz, A , Engler, D L and Nealson, K.H 2002 
Organization and elemental analysis of P-, S-, and Fe-rich 
inclusions in a population of freshwater magnetococci. 
Geomicrobio/. J. 19. 387-406 

Crawford, Karen 2002 Culture method for in vitro 
fertilization to hatching of the squid, Loligo pealeit 
Bio/. Bull- 203 216-217, 

DeGiorgis, J A , T S Reese, and E L Bearer 2002. 
Association of myosin II with axoplasmic organelles 
implications for axonal transport Mol. Biol Cell 13 
1046-1057 

Eddleman, C S , G D Bittner. and H M Fishman 2002 
SEM comparison of severed ends of giant axons isolated 
from squid (Loligo pea/en) and crayfish (Procambarus 
darkii) B.ol. Bull. 203: 219-220 

Edds-Walton, P L . and R R. Fay 2002 Directional 
auditory processing by the oyster toadfish, Opsanus tau. 
Bioacoustics 1 2 202-204 

Edds-Walton, P L , L A Mangiamele, and L C Rome 
2002 Variations of pulse repetition rate in boatwhistle 
sounds from oyster toadfish (Opsanus tau) Bioacoustics 
13. 153-173 

Fay, R R 2002 The sense of hearing in fishes. Bioacous- 
tics 12 167-169 

Fay. R R , and P L Edds-Walton 2002 Preliminary 
evidence for interpulse interval selectivity of cells in the 
torus semicircularis of the oyster toadfish (Opsanus tau). 
Bio/. Bull. 203 195-196 

Femandez-Busquets, X , W J Kuhns, T L Simpson, M 
Ho, D. Gerosa, M. Grab, and M M Burger 2002 Cell- 
adhesion-related proteins as specific markers of sponge 
cell types involved in allogeneic recognition Dev. Comp 
Immunol. 26: 313-323 

Garber, Sarah S , and Mary M Hoffman 2002. Cl and 
glutamate" competition for a volume-regulated anion 
channel Biol. Bull. 203 194-195 

Giuditta, A., M Eyman, and B B Kaplan 2002 Gene 
expression in the squid giant axon neurotransmitter 
modulation of RNA transfer from penaxonal glia to the 
axon Bio/. Bull. 203 189-190 

Giuditta, A , B B Kaplan, J. van Mmnen, J Alvarez, and E 
Koenig 2002. Axonal and presynaptic protein synthesis 
new insights into the biology of the neuron. Trends 
Neurosci. 25: 400-404 



Continued 



R42 



Publications, continued 




Stained hippocamal 
pyramidal neuron fratj, 
Dan Johnston 



Golan. A., Y. Yudkovsky, and A. Hershko 2002 The cyclm- 
ubiquitin ligase activity of the cyclosome/APC is jointly 
activated by protein kinases Cdkl-cyclin B and Plk. 
J Biol. Chem. 277: 15,552-15,557. 

Graf, Werner, Edwin Gilland, Matt McFarlane, Laura Knott, 
and Robert Baker 2002 Central pathways mediating 
oculomotor reflexes in an elasmobranch, Scyltorhinus 
canicu/a. Biol. Bull. 203: 236-238. 

Hinkle, B . B Slepchenko, M M. Rolls, T. C. Walther, P. A 
Stem, L M. Mehlmann, J Ellenberg, and M. Terasaki 2002 
Chromosomal association of Ran during meiotic and mitotic 
divisions J. CellSd. 115: 4685-4693 

Huffaker, Diana, and R. Gil Pontius, Jr. 2002 Reconstruc- 
tion of historical land cover in the Ipswich Watershed Biol. 
Bull. 203: 253-254 

Husted L. B., E. S. Sorensen, P. B. Armstrong, J. P. Quigley, 
L Kristensen, L Sottnjp- Jensen 2002. Localization of 
carbohydrate attachment sites and disulfide bridges in 
Umu/us a2-macroglobulin. Evidence for two forms differing 
primarily in their bait region sequences. J. Biol. Chem. 277: 
43,698-43,706. 

Islas-Flores, I., S. Corrales-Villamar, E L. Bearer, J C. Raya, 
and M.-A Villanueva 2002. Isolation of lipoxygenase 
isoforms from Glycine max embryo axes based on cross- 
reactivity with anti-myosin antibodies Bioch/m. Biophys 
Acta 1571: 64-70. 

Jimenez C J., M. Eyman, 2 Scotto Lavina, A. E. Gioio, K. 
W. Li. R van der Schors, W. P. M. Geraerts , A Giuditta, B 
B. Kaplan, and J. van Minnen. 2002. Protein synthesis in 
synaptosomes: a proteomics analysis. J. Neurochem. 81 : 
735-744 

Kuner, T , H. Tokumaru, and G J Augustine. 2002. 
Peptides as probes of protein-protein interactions involved 
in neurotransmitter release Pp 552-570 in Peptide-lipid 
Interactions, S. A. Simon and T. J. Mclntosh, eds Academic 
Press, San Diego. 

La Du, Jane, Deana Erdner, Sonya Dyhrman, and Don 
Anderson 2002. Molecular approaches to understanding 
population dynamics of the toxic dinoflagellate 
A/exandrium fundyense Biol. Bull. 203: 244-245 

Landowne, D. 2002. Perchlorate prevents sodium channel 
gating and sodium protects in the squid giant axon. Bio/. 
Bull. 203: 190-191. 

Langford, G. M. 2002. Myosin-V, a versatile motor for short- 
range vesicle transport. Traffic 3: 859-865 

Lee, K-G , A. Braun, I. Chaikhoutdinov, J. DeNobile, M. 
Conrad, and W. Cohen. 2002. Rapid visualization of 
microtubules in blood cells and other cell types in marine 
model organisms, Biol. Bull. 203: 204-206. 



Ludlam, John P., David H. Shull, and Robert Buchsbaum 
2002. Effects of haying on salt-marsh surface invertebrates 
Bio/. Bull 203:250-251. 

Ma. W.-L., and R R Fay 2002 Neural representations of 
the axis of acoustic particle motion in the auditory midbrain 
of the goldfish, Carassius auratus J. Comp. Physio/ 188: 
301-313. 

Maddox, P., A. Desai, K. Oegema, T. J. Mitchison, and E 
D. Salmon. 2002. Poleward microtubule flux is a major 
component of spindle dynamics and anaphase A in mitotic 
Drosophi/a embryos. Curr. Biol 12: 1670-1674. 

Megela-Simmons, A , R R Fay, and A N Popper, eds. 
2002 Springer Handbook of Auditory Research, Vol. 14, 
Acoustic Communication. Springer-Verlag, New York. 

Mehlmann, L M , T L. 2. Jones, and L. A. Jaffe 2002. 
Meiotic arrest in the mouse follicle maintained by a Gs 
protein in the oocyte Science. 297: 1343-1345 

Mensinger, A. F., and M Deffenbaugh 2002. Acoustical 
neural telemetry from free-swimming fish Bioacoustics 12: 
333-334. 

Mikhailov, A . R W. Cole, and C L. Rieder 2002 DNA 
damage during mitosis in human cells delays the 
metaphase/anaphase transition via the spindle assembly 
checkpont. Curr. Biol. 12: 1797-1806. 

Mittmann, Beatte 2002 Early neurogenesis in the 
horseshoe crab Limulus po/yphemus and its implication for 
arthropod relationships Bio). Bull. 203: 221-222 

Montgomery, John, Guy Carton, and David Bodznick 2002. 
Error-driven motor learning in fish. Biol. Bull. 203: 238-239. 

Morgan, J R., G J Augustine, and E M Lafer 2002 
Synaptic vesicle endocytosis: the races, places, and 
molecular faces Neuromolecular Med 2: 101-114. 

Pahl, Nicholas, Sara Homsi, Hilary G Mornson, and Robert 
M Gould 2002 mRNAs located in Squalus acanthias 
(Spiny Dogfish) oligodendrocyte processes Bio'. Bu//. 203: 
217-218 

Piccoli, G., M. Gomez, and E. Nasi 2002 Role of protein 
kmase C in light adaptation of molluscan microvillar 
photoreceptors J. Physio/. 543 481-494. 

Rakowski, R F . D C Gadsby. and P. De Weer 2002. 
Single ion occupancy and steady-state gating of Na 
channels in squid giant axon. J. Gen. Physio/ 1 19: 235-249. 

Redenti, S., and R. L. Chappell. 2002. Zinc chelation 
enhances the zebrafish retinal ERG b-wave Biol. Bull. 203: 
200-202 



R43 



Ridings, C , D Borst, K. Smith, F Dodge, and R B Barlow. 
2002. Visual behavior of juvenile Ltmulus in their natural 
habitat and in captivity Bio/. Bull. 203 224-225. 

Rieder, C L , and R W Cole. 2002. Cold shock and the 
mammalian cell cycle Cell Cycle 1 169-175 

Ripps, H , H Qian, and J Zakevicius. 2002 Blockade of an 
inward sodium current facilitates pharmacological study of 
hemi-gap-junctional currents in Xenopus oocytes Biol. Bull. 
203: 192-194 

Ripps, H., H Qian, and J Zakevicius 2002 Pharmacologi- 
cal enhancement of hemi-gap-junctional currents in 
Xenopus oocytes J Neurosci Methods 121: 81-92, 

Ripps, H 2002 Cell death in retinitis pigmentosa gap 
junctions and the 'bystander' effect Exp Eye Res 74 
327-336 

Shuster, C B., and D R Burgess 2002 Targeted new 
membrane addition in the cleavage furrow is a late, 
separate event in cytokinesis Proc. Nat/. Acad. So. USA. 
99: 3633-3638 

Shuster, C B , and D. R Burgess 2002 Transitions 
regulating the timing of cytokinesis in embryonic cells. 
Curr.Biol. 12.854-858. 

Silver, Robert B., and Marc Bartman 2002 A metronome- 
like control of the calcium signal leading to nuclear 
envelope breakdown and mitosis in sand dollar 
(Echinaracmus parma) cells Biol. Bull. 203 213-215 

Smith, K , C. Ridings, F A Dodge, and R B Barlow 2002 
Development of the lateral eye of juvenile Limu/us Biol. 
Bull. 203 222-223 

Smotherman, Michael 2002 Acetylcholme mediates 
excitatory input to chromatophore motoneurons in the 
squid, Lo/igo pea/eii Bio). Bull. 203 231-232 

Sommers, M.G , Dollhopf, M.E. and Douglas, S 2002 
Freshwater ferromanganese stromatolites from Lake 
Vermilion, Minnesota microbial culturing and environmen- 
tal scanning electron microscopy investigations 
Geomicrobio/. J. 19: 407-427 

Thompson R F , and G M Langford 2002 Myosin 
superfamily evolutionary history Anat Rec 268 276-289 

Tirnauer, J , Julie C. Canman, E D Salmon, and Timothy J 
Mitchison 2002 EB1 targets to kinetochores with attached, 
polymerizing microtubules Mo/ Biol. Cell 13 4308-4316 



Tirnauer J. S., S. Grego, E D Salmon, and T J Mitchison. 
2002 EB1-microtubule interactions in Xenopus egg 
extracts: role of EB1 in microtubule stabilization and 
mechanisms of targeting to microtubules Mo/ Biol. Cell 
13: 3614-3626. 

Twig, G , R P. Malchow, K. Hammar, P J S Smith, H. 
Levy, and I. Perlman 2002 A novel turtle retinal prepara- 
tion for simultaneously measuring light-induced electrical 
activity and changes in metabolite levels Biol Bull 203: 
198-200 

Wachowiak, M., L B Cohen, and M Zochowski. 2002 
Distributed and concentration invariant spatial representa- 
tions of odorants by receptor neuron input to the turtle 
olfactory bulb J. Neurophysio/ 87 1035-1045 

Weeg, M , R. R. Fay, and A. Bass 2002 Directional 
response and frequency tuning in saccular nerve fibers of 
a vocal fish, Ponchthys notatus J Comp. Physio/. 188: 
631-641. 

Weidner, Earl, and Ann Findley 2002 Peroxisomal catalase 
in extrusion apparatus posterior vacuole of microspondian 
spores. Biol. Bull 203 212 

Wen, H , D Jurkovicova, V. M Pickel, A E Gioio, and 
B. B. Kaplan 2002 Identification of a novel membrane- 
associated protein expressed in neurons of the squid and 
rodent nervous system Ps/euroscience 1 14: 995-1004 

White, T.W., M. Srinivas, H Ripps, A. Trovato-Salmaro, 
D. F. Condorelli, and R Bruzzone 2002 Virtual cloning, 
functional expression and gating analysis of human 
connexin31 9 Am. J. Physio/. Ce/( Physiol. 283: C960- 
C970 

WollertT., A. S DePina, R F Thompson, and G M 
Langford 2002 Ca 2 ' effects on myosm-ll-mediated 
contraction of pseudo-contractile rings and transport of 
vesicles in extracts of clam oocytes Biol. Bull 203 
206-208 

WollertT, A S DePina, R F Thompson, and G M 
Langford. 2002 GTPase Rho is involved in myosin-ll- 
mediated contraction of pseudo-contractile rings and 
transport of vesicles in extracts of clam oocytes Biol Bull. 
203 208-210 



R44 




education 



The 2002 Education Program provided 499 students from 288 institutions and 30 countries an 
opportunity to study a range of biological topics with some of the best and brightest scientists 
in the world serving as course faculty and lecturers. The Laboratory welcomed 554 faculty 
members and staff and 203 lecturers to the courses in 2002. They represented 175 institutions 
and 31 countries. Among the many outstanding lecturers last summer, we were especially 
pleased to host two Nobel Laureates, Michael Brown and Tim Hunt, who gave the Arthur K. 
Parpart and the Irvin Isenberg Lectures, respectively, in the Physiology course. 

In addition to the MBL's six major summer courses, we offered 14 special topics courses 
throughout the year, including two exciting new courses: Advances in Genome Technology and 
Bioinformatics, directed by Claire M. Fraser, TIGR, and Mitchell Sogin, MBL; and 
Neuroinformatics, directed by Partha Mitra of Lucent Technologies, Emery Brown of Massachu- 
setts General Hospital, and David Kleinfeld of the University of California, San Diego. 

At the end of the 2002 season, we bid farewell to Chris Tschudi and Elisabetta Ullu, directors of 
the Biology of Parasitism course. Jay Bangs of the University of Wisconsin, Madison, will take 
the helm of that course in 2003. We also said goodbye to Bill Bialek and Rob de Ruyter Van 
Steveninck of the Computational Neuroscience course. Bard Ermentrout of the University of 
Pittsburgh and John White of Boston University will assume the directorship of the course in 
2003. In addition, Sandra Masur, of Mount Sinai School of Medicine, joined David Papermaster 
as co-director of the Vision Research course in 2002. 

The MBL's educational program once again received a stamp of approval from the National 
Institutes of Health's competitive peer review process with renewed funding for the Embryol- 
ogy, Neural Systems & Behavior, and Neurobiology courses, and new funding for the inaugural 
Neuroinformatics course. 



R45 



SUMMER COURSES 



Biology of Parasitism: Modern 
Approaches 

June 13- August 10, 2002 

DIRECTORS 

Tschudi, Christian, Yale University Medical School 

Ullu, Elisabetta, Yale University Medical School 

FACULTY 

Bangs. James, University of Wisconsin-Madison 
Grencis, Richard, University of Manchester 
Hajduk, Stephen. University of Alabama- 
Birmingham 

Matthews. Keith, University of Manchester 
McFadden, Geoff, University of Melbourne 
Parsons. Marilyn. Seattle Biomedical Research 

Institute 

Rathod, Pradip, University of Washington 
Reiner. Steven, University of Pennsylvania 

Tarleton. Rick. University of Georgia 

LECTURERS 

Allen, Judith, University of Edinburgh 
Cross, George, Rockefeller University 
Deitsch. Kirk, Weill Medical College of 

Cornell University 

Englund, Paul, Johns Hopkins Medical School 
Garside, Paul, University of Glasgow 
Hunter, Christopher, University of Pennsylvania 
Muller. Miklos. The Rockefeller University 
Nutman, Thomas. National Institutes of Health 
Panigrahi, Aswini, Seattle Biomedical Research 

Institute 

Pearlman, Eric. Case Western Reserve University 
Phillips. Meg, UT Southwestern Medical Center 
Riley. Eleanor, London School of Hygiene & 

Tropical Medicine 

Rudenko, Gloria. University of Oxford 
Sacks. David. NIAID, NIH 
Scherf, Artur, Institut Pasteur 
Stanley. Sam, Washington University 
Striepen. Bons, University of Georgia 
Waters, Andrew. Leiden University. The 

Netherlands 

White. Michael, Montana State University 
Wilson, lain. National Institute for Medical 

Research 

Wynn, Thomas. National Institutes of Health 
Dobbelaere. Dirk, University of Bern 
Roditi, Isabel, University of Bern 
Soldati, Dominique, Imperial College of Science 

& Technology 

TEACHING ASSISTANTS 
Jiang, Lei, University of Washington 
Karthikeyan, Ganesan, University of Washington 
Mollard, Vanessa, University of Melbourne 
Artis, David, University of Pennsylvania 
Cummings, Kara. University of Georgia 
Jensen, Bryan, Seattle Biomedical Research 

Institute 

Mair, Gunnar, Queen's University Belfast 
Martin, Diana, University of Georgia 
Pennock, Joanne, University of Manchester 
Ralph, Stuart, University of Melbourne 
Triggs, Veronica, University of Wisconsin-Madison 
van Deursen, Fredenck, The University 

of Manchester 




Kevin Begos 



COURSE ASSISTANTS 

Bridegam, Patrick, Texas A&M University 

McKmnon, Nicole. University of Victoria. 8-C 

STUDENTS 

Avila, Andrea, Inst de Biol Molecular do 

Parana-IBMP 

Chamond, Nathalie, Institut Pasteur 
Cockburn, Ian, University of Edinburgh 
Fenn, Katelyn, University of Edinburgh 
Green, Heather , New York University 
Karnataki, Anuradha, University of Washington 
Klotz, Christian, Humboldt-University-Berlin 
KOOIJ, Taco, Leiden University Medical Centre 
Lee, SooHee. Johns Hopkins School of Medicine 
Li, Hongiie, Yale University 
Meissner, Markus. Imperial College of Science, 

Technology & Medicine, UK 
Mueller, Ann-Kristin. University School of 

Medicine, Heidelberg 
Nkmin, Wuyika, University of Yaounde 
Slavin, lleana, Universidad Nacional de Cordoba 
Stubbs. Janine, Royal Melbourne Hospital 



Embryology: Concepts and 
Techniques in Modern 
Developmental Biology 

June 16 -July 28, 2002 

DIRECTORS 

Richard Harland, University of California. Berkeley 
Joel Rothman, University of California, 
Santa Barbara 

FACULTY 

Fraser, Scon, California Institute of Technology 

Levine, Michael. University of California. Berkeley 

Rokhsar, Dan. University of California. Berkeley 

Tabm. Clifford, Harvard University 

Blair, Seth, University of Wisconsin-Madison 

Bronner-Fraser. Marianne. California Institute 

of Technology 

Collazo. Andres. House Ear Institute 
Ettensohn, Charles, Carnegie Mellon University 
Halpern, Marnie, Carnegie Institution of 

Washington 

Henry, Jonathan, University of Illinois 
Krumlauf, Robb, Stowers Institute for Medical 

Research 



Martmdale. Mark, University of Hawaii 
Niswander, Lee, Sloan-Kettermg Institute 
Patel, Nipam, University of Chicago 
Saunders, John, Retired 
Sherwood, David, California Institute of 

Technology 
Zeller, Robert, San Diego State University 

LECTURERS 

Davidson, Eric. California Institute of Technology 

Keller, Ray, University of Virginia 

Greenwald. Iva, Columbia University 

Joyner, Alexandra, New York Umversity/HHMI 

McGmnis. William, University of California. 

San Diego 

Robertson, Elizabeth, Harvard University 
Sanes, Joshua, Washington University 
Shankland, Martin. University of Texas at Austin 
Struhl. Gary. Columbia University 
Tramor. Paul, Stowers Institute for Medical 

Research 

Wieschaus. Eric, Princeton University 
Wray, Gregory, Duke University 
Yelon, Deborah, New York University School 

of Medicine 

S MERYL ROSE LECTURER 

Gerhart, John, University of California. Berkeley 

TEACHING ASSISTANTS 
Baker. Clare. University of Cambridge 
Cheeks, Rebecca, University of North Carolina- 
Chapel Hill 
Gamse, Joshua, Carnegie Institution of 

Washington 

Gerberdmg, Matthias, University of Chicago 
Gross, Jeffrey. Duke University 
Khokha. Mustafa, University of California, 

Berkeley/MCB 

Kuhlman, Julie, University of Oregon 
Lartillot. Nicolas, Centre Genetique Moleculaire 
Liu. Karen. University of California, Berkeley 
Maduro, Morris, University of California, 

Santa Barbara 

Nederbragt, Alexander, Unversity of Hawaii/PBRC 
Solomon, Keely, Emory University 
Tobey. Allison, Memorial Sloan Kettenng 

Cancer Center 
Wallmgford, John, University of California, 

Berkeley 
Walsh, Emily. Whitehead Institute for Biomedical 

Research 
Weatherbee. Scott. Memorial Sloan Kettenng 

Cancer Center 
Wiellette, Elizabeth, Whitehead Institute for 

Biomedical Research 

Wilson. Valene. Centre for Genome Research 
Wolfe. Adam. University of Illinois. Urbana 

COURSE ASSISTANTS 

Balligan, Sarah, University of Missouri-Columbia 

McCluskey. Kathryn, Marine Biological 

Laboratory 
Tai, Phillip, University of California, Berkeley 



Continued. 



R46 



STUDENTS 

Berry, Katy, University of Sheffield 
Brown, Ann, Medgar Evers College 
Caracino, Diana, Emory University School 

of Medicine 

Copf, Tijana, University of Crete 
Crotwell, Patricia, University of South Dakota 
Dash, Satya, University of East Anglia 
Delalande, Jean-Marie, University College 

London 

Drago, Grazia, Universita Degli Studi di Palermo 
Extavour, Cassandra, University of Cambridge 
Guest, Jennifer, National Institute for Medical 

Research 

Kee, Yun, California Institute of Technology 
Kerney, Ryan, Harvard University 
Koziel, Lydia, Max-Planctc-lnst. for Molecular 

Genetics 
Livi, Carolina, University of Texas Health 

Science Center, San Antonio 
Malartre, Marianne, University of Portsmouth 
Maslakova, Svetlana, Smithsonian Institution 
Matus, David, University of Hawaii 
Mitchell, Tracy, University of Wisconsin-Madison 
Muyskens, Jonathan, University of Oregon 
Nouri, Ali, Princeton University 
Orsborn, Apnl, University of Missouri-Columbia 
Primus, Alexander, University of Texas, Austin 
Roche, Daniel, University of California, Berkeley 
Su, Yi-Hsien, Scripps Inst. of Oceanography, 

MBRD 
Van Stry, Melante, Boston University School of 

Medicine 



Microbial Diversity 

June 16- August 2, 2002 

DIRECTORS 

Harwood, Caroline, University of Iowa 

Spormann, Alfred, Stanford University 

FACULTY 

Buckley, Daniel, University of Connecticut 
Gibson. Jane. Cornell University (Emerita) 
Marsh, Terence, Michigan State University 

LECTURERS 

Boetius, Antje, MPI fur Marine Mikrobiologie 
Breznak, John, Michigan State University 
Delong, Edward, Monterey Bay Aquarium 
Elhai, Jeff, Virginia Commonwealth University 
Giovannoni, Stephen, Oregon State University 
Gottschalk, Gerhard, Institut fur Mikrobiologie 

u Genet 

Handelsman, Jo, University of Wisconsin 
Larimer, Frank, Oak Ridge National Laboratory 
Lory. Stephen, Harvard Medical School 
Loveley, Derek, University of Massachusetts 
Meeks, John, University of California 
Metcalf. Wii:.. ' '-.vr'sity of Illinois 
Rocap, Gabnelle, University of Washington 
Strous. Marc, b rsil , :>f Nymegen 
Thauer, Rudolf, MHI fur Terrestr. Mikro 
Wackett, Lawrence, University of Minnesota 
Wolfe. Ralph, University of Illinois 




Kevin Begos 



TEACHING ASSISTANTS 

Behrens, Sebastian, MPI for Marine Microbiology 
Martiny, Adam, Danmark Tekniske Universitet 
Mueller, Jochen, Stanford University 
Schaefer, Amy, University of Iowa 

COURSE COORDINATOR 
Hawkins, Andrew, University of Iowa 

LAB ASSISTANT 

Waterbury, Matthew, Marine Biological Laboratory 

STUDENTS 

Boucher, Yan, Dalhousie University 

Case, Rebecca, University of New South Wales 

Clement, Barbara, Doane College 

Denef, Vincent, Michigan State University 

Dethlefsen, Les, Michigan State University 

Dick, Gregory, Scripps Institute of 

Oceanography 
Erbs, Marianne, Swiss Fed. Inst. for 

Environmental Science & Technology 
Gentile, Margaret, Stanford University 
Ginder-Vogel, Matthew, Stanford University 
Graco, Michelle, University of Pierre et Marie 

Curie 

Harrison, Faith, University of Iowa 
Koren, Omry, Tel Aviv University 
Lostroh, Phoebe, University of Iowa College 

of Medicine 

Maresca, Julia, Pennsylvania State University 
Pinel, Nicolas, University of Washington 
Rajagopal, Soumitra, University of Nebraska 
Remold, Susanna, Michigan State University 
Sharp, Katherine, Scripps Institute of 

Oceanography 

Spain, Jim, United States Air Force 
Walker, Jeffrey. University of Colorado 



Neural Systems & Behavior 

June 16 - August 10, 2002 

DIRECTORS 

Carr, Catherine, University of Maryland 

Levine, Richard, University of Arizona 

FACULTY 

Baines, Richard, University of Warwick 

Calabrese, Ronald. Emory University 

Chitwood, Raymond, Baylor College of Medicine 

Davis, Graeme, University of California, 

San Francisco 

French, Kathleen, University of California, San Diego 
Glanzman, David, University of California, 

Los Angeles 

Golowasch, Jorge, Rutgers University 
Kristan, William, University of California, San Diego 
Mooney, Richard, Duke University 
Nadim, Farzan, Rutgers University 
Philpot, Ben, Brown University 
Pnjsky, Glen, University of Lethbridge 
Reyes, Alex, New York University 
Ribera, Angeles, University of Colorado Health 

Sciences Center 

Roberts, William, University of Oregon 
Simon, Jonathan, University of Maryland 
Stein, Wolfgang, Universitaet Ulm 
Szczupak, Lidia, Universidad de Buenos Aires 
Weeks. Jams, University of Oregon 
Wenning-Erxleben. Angela, Emory University 
Wessel, Ralf, Washington University 
Wilson, Richard. University of Calgary 
Wood, Debra, Case Western Reserve University 
Wood, Emma, University of Edinburgh 
Zhang, Bing, University of Texas at Austin 

LECTURERS 

Augustine, George, Duke Medical Center 
Bate. Michael, University of Cambridge 
Feldman, Daniel, University of California, San Diego 
Finger, Thomas. University of Colorado Health 
Sciences Center 



R47 




Stewart, Bryan, University of Toronto 
Trussell, Larry. Oregon Health & Science University 
White, Stephanie, University of California, Los 
Angeles 

TEACHING ASSISTANTS 
Beenhakker, Mark, University of Pennsylvania 
Bradford, Yvonne, University of Oregon 
Chen, Shanping, House Ear Institute 
Coleman, Melissa, Duke University 
Ezzeddine, Youssef, University of California, Los 

Angeles 

Fairies, Michael, University of Pennsylvania 
Heiser, Ryan, University of Colorado Health 

Sciences 
MacLeod, Katnna, University of Maryland, 

College Park 

Miller, Julie. University of Arizona 
Roberts, Adam. University of California, 

Los Angeles 

Roy. Arani, Duke University 
Siegel, Jennifer, Bowling Green State University 
Scares, Daphne, University of Maryland 
Svoboda. Kurt, University of Colorado Health 

Sciences Center 
Villareal, Greg, University of California. 

Los Angeles 
Zee. M Jade. University of Oregon 

COURSE ASSISTANTS 
Cardon, Aaron, Texas A & M University 
Rodrigues, Elizabeth, University of Oregon 
Shaw, Abigail, Stanford University 

STUDENTS 

Chang, Andrew, Oregon State University 

De Labra. Carmen, University College London 

Dellen. Babette, Washington University, St. Louis 

Doiron, Brent, University of Ottawa 

Dulcis, Davide, University of Arizona. Tucson 

Higley, Michael. University of Pennsylvania 

Hughes, Cynthia, Indiana University 

Julian, Glennis, University of Arizona 

Khalil, Mona. Columbia University 

Miranda, Jason, University of Texas at Austin 

Pfeiffer, Keram, Philipps-Umversitat Marburg 

Renart, Alfonso, Brandeis University 

Rutherford. Mark, University of Oregon 

Sebe, Joy. University of Washington 

Spitzer, Nadja, Georgia State University 

Steinberg, Rebecca, University of Texas 

Sternson, Scott, Rockefeller University 

Witney. Alice. University of Birmingham 

Medical School 
Wohlgemuth, Sandra, Humboldt-Universitat 

zu Berlin 
Zomik, Erik, Columbia University 



Vestibular spinal neurons in the goldfish brain, 
Steven Zottoli 



Neurobiology 

June 16- August 17. 2002 

DIRECTORS 

Faber. Donald, Albert Einstein College of 

Medicine 
Lichtman, Jeff, Washington University School 

of Medicine 

SECTION DIRECTOR 

DeFranco, Donald. University of Pittsburgh 
School of Medicine 

FACULTY 

Buchanan, JoAnn, Stanford University 

Conchello. Jose-Angel, Washington University 

Medical School 

Coyle, Joseph, Harvard University 
Denk, Winfried, MPI fur Medical Research 
Gan, Wenbiao, New York University 
Hart, Anne, Massachusetts General Hospital 
Heuser, John, Washington University 
Jacob, Michele, Tufts University 
Kaprielian. Zaven. Albert Einstein College 

of Medicine 
Khodakhah, Kamran, Albert Einstein College 

of Medicine 

Lambert, Nevin, Medical College of Georgia 
Levinthal, David, University of Pittsburgh 
Lin, Jen-Wei, Boston University 
Littleton, J. Troy, Massachusetts Institute 

of Technology 

Logothetis. Nikos, MPI for Biological Cybernetics 
McMahon, Lori, University of Alabama 
Pimenta, Aurea, University of Pittsburgh School 

of Medicine 
Preuss, Thomas, Albert Einstein College of 

Medicine 

Price, Donald, Johns Hopkins Universtiy 
Reese, Tom, NIH 
Schweizer, Felix, University of California. 

Los Angeles 

Smith, Stephen, Stanford University 
Thompson. Wesley, University of Texas 
Wong, Rachel, Washington University 

LECTURERS 

Aimers. Wolfhard, Vollum Institute 

Auerbach, Anthony, SUNY at Buffalo 

Bear, Mark, HHMI/Brown University 

Brodin, Lennart. Karolinska Institute! 

Chapman, Ed, University of Wisconsin-Madison 

Harlow, Mark, Stanford University 

Harris, Ken Rutgers University 

Hoh. Jan, Johns Hopkins School of Medicine 

Hopkins, Nancy, Massachusetts Institute of 

Technology 

Huguenard, John, Stanford University 
Kernan, Maurice, SUNY at Stony Brook 
Lipscombe, Diane, Brown University 
Magee, Jeffrey, Louisiana State University Health 

Science Center 

McMahan, Del J , Stanford University 
Nedivi, Elly, Massachusetts Institute of 

Technology 

Nicolelis, Miguel, Duke University 
Ogden, David, National Institute for Medical 

Research 

Ryan, Tim, Weill Medical Cornell 
Sigworth, Fred, Yale University 
Svoboda. Karel. Cold Spring Harbor Laboratory 
Tsien, Roger. University of California. San Diego 
Tully. Tim, Cold Spring Harbor Laboratory 
Yellen, Gary, Harvard Medical School 



TEACHING ASSISTANTS 

Allana, Tariq. Boston University 

Chang, Paul, New York University 

Godinho, Leanne, Washington University School 

of Medicine 
Guan, Zhuo, Massachusetts Institute of 

Technology 
Misgeld, Thomas, Washington University School 

of Medicine 

Rosenberg, Madelaine, Tufts University 
Szabo, Theresa. Albert Einstein College 

of Medicine 

COURSE ASSISTANTS 

Hall. David. Marine Biological Laboratory 

Satterlee, Danielle, Texas A & M University 

STUDENTS 

Boassa, Daniela, University of Anzona College 

of Medicine 
Campbell, Susan, University of Alabama, 

Birmingham 

Ewald, Rebecca, Cold Spring Harbor Laboratory 
Hobbs, Steven, University of Colorado 
Hu, Hailan, University of California, Berkeley 
Ihring. Alexandra, Max-Planck-Institute of 

Neurobiology 

Koirala, Samir, University of Southern California 
Kozhevnikov, Alexay. Bell Labs/Lucent 

Technologies 
Montana, Enrico, Massachusetts Institute of 

Technology 
Petersen, Rasmus, International School for 

Advanced Study (SISSA) 

Ryan, Amy, University of Virginia Health Systems 
Zhou. Zhaolan (Joe), Harvard Medical School 



Physiology: The Biochemical 
and Molecular Basis of Cell 
Signaling 

June 16- July 27, 2002 

DIRECTORS 

Garbers, David, UT Southwestern Medical 

Center/HHMI 
Reed, Randall, Johns Hopkins University/HHMI 

FACULTY 

Duncan, Tod, Imperial Cancer Research Fund 

Ehrlich, Barbara, Yale University 

Franco, Peter, University of Minnesota 

Furlow, David. University of California, Davis 

Kao, Ling-Rong, University of Texas 

Southwestern Medical Center 
Lim, Wayland. University of California, Davis 
Megraw, Timothy, University of Texas 

Southwestern Medical Center 
Schultz, Nikolaus, University of Texas 

Southwestern Medical Center 

LECTURERS 

Bennett, Anton, Yale Medical School 
Clapham, David, Harvard Medical School 
Comerford, Sarah, University of Texas 

Southwestern Medical Center 
Devreotes. Peter. Johns Hopkins University 

School of Medicine 

Dietnch, William, Harvard Medical School 
Gardner, Kevin. University of Texas Southwestern 

Medical Center 

Continued 



R48 



2002 SPECIAL LECTURE SERIES 



Hammer, R< , of Texas 

South --cdical Center 

Hepler "rsity of Massachusetts 

Mang*: 3 .J^vid, University of Texas 

Southwestern Medical Center 
Nambu, John, University of Massachusetts 
Stock, Ann, University of Medicine & Dentistry, 

New Jersey-RW Johnson Medical School/HHMI 
Tilney, Lewis, University of Pennsylvania 
Welsh, Michael, University of Iowa 

IRVIN ISEN8ERG LECTURER 
Hunt, Timothy, International Cancer Research 
Fund, Clare Hall Laboratories 

GERTRUDE FORKOSH WAXLER LECTURER 
Weinberg, Robert, Whitehead Institute 

ARTHUR K. PARPART LECTURER 
Brown, Michael, University of Texas 
Southwestern Medical Center 

TERU HAYASHI LECTURER 
Cobb, Melanie, University of Texas 
Southwestern Medical Center 

TEACHING ASSISTANTS 
Anyatonwu, Georgia, Yale University 
Rengifo, Juliana, Yale University 

COURSE COORDINATOR 
Rossi, Kristen. University of Texas Southwestern 
Medical Center 

COURSE ASSISTANTS 

Grellhesl, Dana, University of Texas Southwestern 

Medical Center 
Swaney, Sara-Love, University of Texas 

Southwestern Medical Center 

STUDENTS 

Aguilar, Arturo, Institute Politecnico National, 

Mexico 

Amarie, Dragos, University of Notre Dame 
Chen, Yen-Chin, National Cheng Kung University 

Medical College 

Chong, Curtis, Johns Hopkins School of Medicine 
Davis, Kevin, University of Pittsburgh 
Dojcinovic, Danijel, Arizona State University 
Gadea, Bedrick, Harvard Medical School 
Ge, Lan, University of California, Riverside 
Goentoro, Lea, Princeton University 
Kaynar, Murat, Beth Israel Deaconess 

Medical Center 
Kelly, Melissa, University of Kentucky College 

of Medicine 

Kydd, Alison, University of Calgary 
LaPointe, Nichole, Northwestern University 
McVaugh, Cheryl, University of Pennsylvania 
Oh, Ji-Eun, University of Illinois at Chicago 
Pace, Margaret, University of Texas 
Pignatelli, Vincenzo, University of Pisa 
Pineda, Gabn'el, University of Texas 

Southwestern Medical Center 
Ramos, Arnolt, Children's Hospital, Boston 
Rossi, Chiara, University of Pisa 
Sha, Edward, Indiana University Medical Center 
Thamatrakoln, Kimberlee, University of California, 

San Diego 
Vega, Rebecca, Stanford University 




Meryl S. Rose Lecture (June 17) 
John Gerhart, University of California, Berkeley 

"Cells. Embryos, and Evolution. Toward a Cellular and Developmental Understanding of 
Phenotypic Variation and Evolutionary Adaptability" 

/rv;n /senberg Lecture (June 21) 

Timothy Hunt, Nobel Laureate, International Cancer Research Fund, Clare Hall Lab 
"Protein Synthesis and the Control of the Cell Cycle" 

Gertrude Forfcosh Waxier Lecture (July 1 7) 

Robert A. Weinberg, Massachusetts Institute of Technology 

"Rules for Making Human Tumor Cells" 

Teru Ha/ashi Lecture (July 23) 

Melanie H. Cobb, University of Texas Southwestern Medical Center 
"Information Flow in MAP Kinase Cascades" 

Arthur K. Parpart Lecture (July 26) 

Michael Brown, Nobel Laureate, University of Texas Southwestern Medical Cente 
"The SREBP Pathway: How the Membrane Tells the Nucleus What it Needs" 

Ruth Sager Lecture in Genetr'cs (August 30) 

Mark Fishman, Massachusetts Hospital and Harvard Medical School 
"Genetic Modules: Fashioning Organs in Zebrafish" 



SPECIAL TOPICS COURSES 

Advances in Genome Technology 
& Bioinformatics 

October 6 - November 2, 2002 

DIRECTORS 

Fraser, Claire, The Institute for Genomic Research 

Sogin, Mitchell, Marine Biological Laboratory 

FACULTY 

Bateman, Alex, Sanger Institue 

Blake, Judith, Jackson Laboratory 

Churchill, Gary, Jackson Laboratory 

Cummings, Michael, Marine Biological Laboratory 

deJong, Pieter, Children's Hospital Oakland 

Research Institute 

DeLong, Edward, Monterey Bay Aquarium 
Eisen, Jonathan, The Institute for Genomic 

Research 

Eisen, Michael. Lawrence Berkeley 
Feldblyum, Tamara, The Institute for Genomic 

Research 

Felsenstein, Joe, University of Washington 
Florens, Laurence. Scripps Research Institute 
Gill, Steven, The Institute for Genomic Research 
Gray, Michael, Dalhousie Institute 
Heidelberg, John, The Institute for Genomic 

Research 
Jaffe, David, Whitehead Institute for Biomedical 

Research 

Kent, Jim, University of California, Santa Cnjz 
Kim, Ulandt, Marine Biological Laboratory 
Lee, Norman, The Institute for Genomic Research 
Mann, Barbara, University of Virginia Health 

System 

McArthur, Andrew, Marine Biological Laboratory 
Mesirov, Jill, Whitehead Institute for Biomedical 

Research 

Morrison, Hilary, Marine Biological Laboratory 
Myers, Eugene, Celera Genomics 
Nelson, Karen, The Institute for Genomic 

Research 
Nierman, William, The Institute for Genomic 

Research 
Nusbaum, Chad, Whitehead Institute for 

Biomedical Research 
Ochman, Howard, University of Arizona 
Olsen, Gary, University of Illinois 



Palmer, Jeffrey, Indiana University 
Pearson, William, University of Virginia 
Peterson, Scott, The Institute for Genomic Research 
Pop, Mihai, The Institute for Genomic Research 
Quackenbush. John, The Institute for Genomic 

Research 

Reich, Claudia, University of Illinois 
Reysenbach, Anna-Louise, Portland State University 
Ringwald, Martin, Jackson Laboratory 
Roos, David, University of Pennsylvania 
Salzberg, Steven, The Institute for Genomic Research 
Smith, Hamilton, Celera Genomics 
Tettelin, Herve, The Institute for Genomic Research 
Venter, Craig, The Institute for Genomic Research 
Wernegreen, Jennifer, Marine Biological Laboratory 
White, Owen, The Institute for Genomic Research 

TEACHING ASSISTANTS 
Tallon, Luke, The Institute for Genomic Research 
Radune, Diana, The Institute for Genomic Research 
Vamathevan, Jessica, The Institute for Genomic 

Research 

Davidsen, Tanja, The Institute for Genomic Research 
Gill, John, The Institute for Genomic Research 
Bhagabati, Nirmal, The Institute for Genomic Research 
Saeed, Alexander, The Institute for Genomic Research 
White, Joseph, The Institute for Genomic Research 
Thiagarajan, Mathangi, The Institute for Genomic 

Research 

Wang, Hong-Ying, The Institute for Genomic Research 
Gaspard, Renee, The Institute for Genomic Research 
Frank, Bryan, The Institute for Genomic Research 
Hasseman, Jeremy, The Institute for Genomic Research 

STUDENTS 

Bebout, Brad, NASA Ames Research Center 

Brodhagen, Marion, Oregon State University 

Bundy, Becky, University of Georgia 

Chen, Lishan, University of Washington 

da Silva, Alexandre, Centers for Disease Control 

and Prevention 

Doak, Thomas, University of Utah 
Duvefelt, Kristina, Karolinska Institute! 
Fenwick, Brad. Kansas State University 
Francis, Susan, University of Washington 
Gilchrist, George, College of William & Mary 
Golden, Daniel, University of Alabama, Birmingham 



R49 



Handley, Heather. Woods Hole Oceanographic 

Institution 
Hildebrandt, John. Medical University of South 

Carolina 

Kanzok, Stefan, Yale University School of Medicine 
Kiesling, Traci, University of Miami 
Kimbell. Jennifer, University of Hawaii 
Kinnersley, Margie, University of Montana 
Neumann, Tobias, Carl Zeiss 
Jena-Pineda, Fernando, Johns Hopkins 

Bloomberg School of Public Health 
Radniecki, Tyler, Yale University 
Ranson, Hilary. Liverpool School of Tropical 

Medicine 

Sawyer, Sara, Cornell University 
Thomas, Bolaji, Tufts University 
Williams, David, Illinois State University 



Analytical and Quantitative Light 
Microscopy 

May 9- May 17, 2002 

DIRECTORS 

Sluder, Greenfield, University of Massachusetts 

Medical Center 
Wolf, David, BioHybrid Technologies 

FACULTY 

Amos, William, MRC Lab of Molecular Biology 

Bulseco, Dylan, University of Massachusetts 

Medical School 

Cardullo. Richard. University of California 
Gelles, Jeff, Brandeis University 
Hinchcliffe, Edward, University of Notre Dame 
Inoue, Shinya, Marine Biological Laboratory 
Moomaw, Butch, Hamamatsu Photonic Systems 
Reichelt, Stefanie, MRC Lab of Molecular 

Biology 
Salmon, Edward, University of North Carolina, 

Chapel Hill 
Silver, Randi, Weill Medical College Cornell 

University 

Spring, Kenneth, National Institutes of Health 
Swedlow, Jason, University of Dundee 
Waters Shuler, Jennifer, Harvard Medical School 
Sears, Kathryn, Sensor Technologies 

LECTURERS 

Straight, Aaron, Harvard Medical School 

Wachowiak, Mel, Smithsonian Institution 

TEACHING ASSISTANT 
Ehrhardt. Anka, University of Massachusetts 
Medical School 

COURSE ASSISTANT 

Nordberg, Joshua, University of Massachusetts 
Medical School 

STUDENTS 

Chatterjee, Samit, Weill Medical College of 

Cornell University 
Cooke, Emma-Louise, AstraZeneca R&D, 

Charnwood. UK 
Counterman, Anne, Pennsylvania State 

University 
Espinosa Tanguma, Ricardo, University of 

San Lui Potosi 
Fischer, Robert, The Scnpps Research Institute 



Fitzpatrick, John, Yale University School 

of Medicine 

Fleming, Shawna, Brown University 
Fu, Lianwu, University of Alabama, Birminghan 
Galko, Michael, Stanford University School 

of Medicine 
Glover, Greta. Oregon Health and Sciences 

University 

Green, Heather, New York University 
Gruenbaum. Lore, Boehringer Ingelheim 

Pharmaceuticals 
Habdas, Piotr, Emory University 



Molecular Biology of Aging 

August 5 - August 24, 2002 

DIRECTORS 

Guarente, Lenny, Massachusetts Institute of 

Technology 
Wallace. Douglas, University of California, Irvine 

FACULTY 

Culotta, Valeria, Johns Hopkins University 
Kirkwood, Tom, University of Manchester 
Lambeth, Dave, Emory University 




Drosophila, April Orsborn 

Hidalgo, Carlos, Institute Venezolano de 

Investig- Cientificas 

Iszard, Melissa, Raytheon Polar Services 
Jungnickel, Melissa, University of Massachusetts 

Medical School 

Liu, Songtao. Fox Chase Cancer Center 
Luna, Elizabeth, University of Massachusetts 

Medical School 

McKeown, Caroline, University of Utah 
Paladino, Simona. University of Naples 
Panetti, Tracee, Temple University School of 

Medicine 
Poskanzer, Kira, University of California. San 

Francisco 

Powers, Maureen, Emory University 
Raphael, Marc, The Naval Research Laboratory 
Rusan. Nasser, University of Massachusetts, 

Amherst 
Smyth. Jeremy, University of Massachusetts, 

Amherst 

Tarn, Jenny, Tufts University 
Wagener, Michael. Carl Zeiss, Inc. 
Welch, Jeffrey, Duke University Medical Center 
Yamamoto, Akihiro. RIKEN, Japan 
Zandbank-Zuker, Keren, Hebrew Universtiy 
Zhang. LingLi, University of Pennsylvania 



Weindruch, Richard, University of Wisconsin 
Bishop, Nicholas, Massachusetts Institute 

of Technology 
Helfand, Stephen, University of Connecticut 

Health Center 

Price, Donald, Johns Hopkins University 
Ruvkun, Gary, Massachusetts General Hospital 
Wright, Woodring, University of Texas 

Southwestern Medical Center 

LECTURERS 

Pelicci. Pier Giuseppe, European Institute of 

Oncology 

Austad, Steven, University of Idaho 
Bohr, Vilhelm, National Institute on Aging, NIH 
Campisi, Judith, Lawrence Berkeley National 

Laboratory 

Davenport, John, AAAS 

Donehower, Larry, Baylor College of Medicine 
Goldberg, Alfred, Harvard Medical School 
Hanawalt, Philip, Stanford University 
Harley, Calvin, Geron Corporation 
Hekimi, Siegfried, McGill University 
Jones, Dean. Emory University 
Kim, Stuart, Stanford University 
Martin, George. University of Washington 

Continued... 



R50 



Partridge, Linda, University College London 
Richardson, Arlan, University of Texas Health 

Science Center at San Antonio 
Tatar, Marc, Brown Unive'j'ty 
Tower, John, Universe ?'n California 

TEACHING ASSIST, 

Coskun, Elif Pi ;ry University 

Kaeberlein. ' jchusetts Institute of 

Technology 

Kerstani-,. Keilh, Emory University 
Kokoszka, Jason, Emory University 
Visithawan. Mo, Massachusetts Institute 

of Technology 
Liszt, Gregory, Massachusetts Institute of 

Technology 
Subramaniam, Vaidya, Emory University 

COURSE COORDINATOR 
Burke, Rhonda, Emory University 

COURSE ASSISTANT 

Wylie, Michael, University of Michigan 

STUDENTS 

Almeida, Claudia, Weill Medical College 

of Cornell University 

Bishop, Glenda, Case Western Reserve University 
Bokov, Alex. University of Texas Health Science 

Center, San Antonio 
Chen, Lishan, University of Washington 
Dunaief, Joshua, University of Pennsylvania 
Fuller, Kattiryn, University of Minnesota 
Harvey, Sarah, Medical College of Virginia 
Hong, Eun-Jm (Erica), Yale University 
Johnston, Janet, Queen's University Belfast 
Lledias, Fernando, Institute de Biotecnologia, 

UNAM 
Lu, Xiangdong, University of North Carolina, 

Chapel Hill 

Moynihan, Kathryn, Washington University 
Ogle, William, Stanford University 
Powers. Ralph, The University of Washington 
Proctor, Carole, University of Newcastle 
Rea, Shane, University of Colorado 
Rutten, Bart. University of Maastricht 
Shirasawa, Takuji. Tokyo Metropolitan Institute 

of Gerontology 

Tong, Liqi, University of California, Irvine 
Zou, Ymg, University of Texas Southwestern 

Medical Center 



Frontiers in Reproduction: 
Molecular and Cellular Concepts 
and Applications 

May 1 9 - June 29, 2002 

DIRECTORS 

Fazleabas, Asgerally, University of Illinois at 

Chicago 

Hunt, Patricia, Case Western Reserve University 
Woodruff, Teresa, Northwestern University 

FACULTY 

Albertini, David, Tufts University School of 

Medicine 

Ascoli, Mario. The University of Iowa 
Behnnger, Richard, University of Texas 
Cross, James C, University of Calgary 
Croy, B Anne, University of Guelph 
DeMayo. Francesco, Baylor College of Medicine 



Ducibella, Thomas, New England Medical Center 

Hunt, Joan, University of Kansas 

Jaffe, Launnda, University of Connecticut 

Health Center 

Mayo. Kelly. Northwestern University 
Moore, Karen, University of Florida 
Overstrom, Eric, Tufts University School 

of Veterinary Medicine 
Schatten, Gerald, University of Pittsburgh 
Shupnik, Margaret, University of Virginia 

Medical Center 
Terasaki, Mark, University of Connecticut 

Health Center 
Weigel, Nancy, Baylor College of Medicine 

Lecturers 

Gosden, Roger, Eastern Virginia Medical School 
Guillette, Louis, University of Florida 
Hassold, Terry, Case Western Reserve University 
Miele, Lucio, University of Illinois at Chicago 
Nilson, John, Case Western Reserve University 
Ober, Carole, University of Chicago 
Richards, JoAnne. Baylor College of Medicine 
Sarras Jr, Michael, University of Kansas 

Medical Center 

Schultz. Richard, University of Pennsylvania 
Seminara, Stephanie, Massachusetts General 

Hospital 
Shenker, Andrew, Northwestern University 

Medical School 

Simerly, Calvin, Pittsburgh Development Center 
Suarez-Quian, Carlos, Georgetown University 

Medical School 
Tasca, Richard, NIH 

Wells, Dagan, St Barnabas Medical Center 
Welt, Cornne, Massachusetts General Hospital 
Yin, Hang, McGill University 

Teaching Assistants 

Agoulnik, Irina, Baylor College of Medicine 

Aldrich, Carrie, Roche Molecular Systems 

Berard, Mark, University of Michigan 

Britton, Chad, Case Western Reserve University 

Brudney, Allison, University of Illinois at Chicago 

Carroll, David, Florida Institute of Technology 

Combelles, Catherine, Tufts University 

Curtin, Denis, University of Virginia 

Galet, Colette, University of Iowa 

Hadsell. Louise, Baylor College of Medicine 

Huntress, Victoria, Tufts University School of 

Veterinary Medicine 

Jackson, Jodi, Case Western Reserve University 
Kalinowski, Rebecca, University of Connecticut 

Health Center 

Kenny, Hilary, Northwestern University 
Kim, Julie, University of Illinois at Chicago 
Klemhenz, Andrew, Case Western Reserve 

University 
Payne, Christopher, Magee-Womens Research 

Institute 
Petroff, Margaret, University of Kansas 

Medical Center 
Runft, Linda, University of California, Santa 

Barbara 

Santiago. Jose, Northwestern University 
Stein. Paula. University of Pennsylvania 
Susiarjo. Martha, Case Western Reserve University 
Suszko, Magdalena, Northwestern University 
Wang, Min-Kang, Tufts University Veterinary 

School 
Wang, Jie, Baylor College of Medicine 



COURSE COORDINATOR 

Roberts, Sheila. Bridgewater State College 

COURSE ASSISTANT 

Harrison, Emily, Bridgewater State College 

STUDENTS 

Akcali, Kamil, Bilkent University, Turkey 

Bachman, Katherine, Case Western Reserve 

University 

Busso, Dolores, Buenos Aires University 
Gill, Ryan, University of Kansas Medical Center 
Gonsalves, Joanna, University of California, 

San Francisco 

Hallikas, Outi, University of Helsinki 
Ka, Hakhyun, University of Kansas Medical Center 
Kayisli, Umit, Yale University School of Medicine 
Kreeger, Pamela, Northwestern University 
Perez. Christian, University of Pennsylvania 
Prosen, Tracy, University of Pittsburgh 
Sachdeva, Geetanjali, Institute for Research 

in Reproduction 
Torrens. Javier, New Jersey Medical School, 

UMDNJ 

Tou, Janet. NASA Ames Research Center 
Wang, Eileen, Northwestern Medical School 



Fundamental Issues in Vision 
Research 

August 1 1 - August 24, 2002 

DIRECTORS 

Masur, Sandra, Mount Sinai School of Medicine 
Papermaster, David, University of Connecticut 
Health Center 




Limulus eye, Robert Barlow 



FACULTY 

Beebe, David, Washington University 

Bok, Dean, Univensty of California, Los Angeles 

Born, Richard, Harvard Medical School 

Colley, Nansi. University of Wisconsin - Madison 

Gordon, Marion, Rutgers University 

Horwitz, Joseph, Jules Stein Eye Institute, UCLA 

Masland, Richard, Harvard/MGH 

Sugrue. Stephen, University of Florida 



R51 



LECTURERS 

Barlow, Robert, SUNY Upstate Medical University 

Barres, Ben. Stanford Medical School 

Berson, Elliot., Harvard Medical School 

Besharse, Joseph. Medical College of Wisconsin 

Birk, David, Jefferson Medical College 

Bok, Dean, University of California, Los Angeles 

Colley, Nansi, University of Wisconsin-Madison 

Dowling, John, Harvard University 

Green, Carla, University of Virginia 

Griep, Ann, University of Wisconsin Medical 

School 
Hauswirth, William, University of Florida College 

of Medicine 
Hernandez, Rosano, Washington University 

School of Medicine 
Horton, Jonathan, University of California, San 

Fransico 

Hunter, Chyren, National Eye Institute/NIH 
Lang, Richard, Children's Hospital, Cincinnati 
Liberman, Ellen, National Eye Institute/NIH 
LaVail, Jennifer, University of California, San 

Fransico 

Moses, Marsha, Harvard Medical School 
Niederkorn, Jerry, University of Texas 

Southwestern Medical Center 
Piatigorsky, Joram, NIH/NEI 
Raviola, Elio, Harvard Medical School 
Stepp, Mary Ann, GWU Medical Center 
Wasson, Paul, Eye Health Services, Weymouth, 

MA 
Wiggs, Janey, Massachusetts Eye & Ear Infirmary 

COURSE COORDINATOR 

Zekaria, Dania, Mount Sinai School of Medicine 

STUDENTS 

Bensinger, Steven, University of Pennsylvania 

Cusato, Karen, Albert Einstein College of 

Medicine 

Dominy, Nathaniel, University of Chicago 
Ehrhch, Jason, Stanford University School of 

Medicine 

Ensslen, Sonya, Case Western Reserve University 
Katz, Elizabeth, University of Maryland 
Levy, Hanna, Technion Israel Instituterof 

Technology 

Lishko, Polina, Massachusetts Eye & Ear Infirmary 
McCabe, Sarah, Brown University 
Ortega, Nathalie, University of California, San 

Francisco 

Prasad, Dipti, University of California, San Diego 
Ramsey, David, University of Illinois, Chicago 
Reiter, Chad, Pennsylvania State University 

College of Medicine 
Shepard, Laura, University of Oklahoma Health 

Science Center 
Steinle, Jena, Texas A&M University Health 

Science Center 

Wei, Echo Shiyi, University of Southern California 
Wu, David, University of Michigan 
Yang, Ellen, Mount Sinai School of Medicine 
Zamora. David, Oregon Health & Science 

University 
Zandy, Anna, Washington University in St. Louis 



Medical Informatics I 

May 26 - June 2, 2002 

DIRECTOR 

Cimmo, James, Columbia University 

FACULTY 

Ackerman, Michael, National Library of Medicine 
Bakken, Suzanne, Columbia University 
Friedman, Charles, University of Pittsburgh 
Hammond, William, Duke University 

Medical Center 

Kingsland, Lawrence, National Library of Medicine 
Kohane, Issac, Children's Hospital 
Lindberg, Donald, National Library of Medicine 
Miller, Perry, Yale University 
Starren, Justin, Columbia University 

LECTURERS 

Canese, Kathi, National Library of Medicine 

Cimmo, Christopher, Albert Eistein College 

of Medicine 
McCray, Alexa, National Library of Medicine 

STUDENTS 

Alverson, Dale, University of New Mexico 
Anderson, Karen, University of North Dakota 
Bergman, Dale, Alberta Research Council 
Binstock, Mark, Ohio Permanente Medical Group 
Bowles, Kathy, University of Pennsylvania 
Bragdon, Lynn, Veterans Affairs Medical Center 
Brock, Tina, University of North Carolina, 

Chapel Hill 

Cheng, Grace, Hospital Authority 
Chuma, Chomba, Avenue Healthcare Ltd 
Cowen, Janet, Maine Medical Center 
Dee, Cheryl, University of South Florida 
Dhara, Rosaline, Centers for Disease Control 

& Prevention 

Erdley, William, University of Buffalo 
Hayes, Barrie, University of North Carolina, 

Chapel Hill 

Henry, Nancy, Pennsylvania State University 
Kovach, Christine, Kaiser Permanente Northwest 
Lambert-Lannmg, Anita, Toronto West Hospital- 
University Health Network 
Luberti, Anthony, The Children's Hospital of 

Philadelphia 

Marazzo, Donald, Family Health Council 
Markland, Mary, University of North Dakota 
McCabe, Jennifer, James Madison University 
Pond, Fred, Dartmouth College 
Prendergast, Neville, Washington University 

School of Medicine 
Raglow, Gregory, Good Samaritan Family 

Practice Center 
Rangappa, Shantaram, Virginia Commonwealth 

University 

Robertson, Nan, Kaiser Permanente-Northwest 
Ruiz, Jorge, University of Miami School of 

Medicine 

Smha, Sunil, VA Maryland Health Care System 
Smith, Scott, University of North Carolina 
Suzewits, Jeffrey, Southern Illinois University 

School of Medicine 



I Medical Informatics II 
September 29 - October 6, 2002 

DIRECTOR 

Cimino, James, Columbia University 

FACULTY 

Friedman, Charles, University of Pittsburgh 

Kingsland, Lawrence, National Library of Medicine 

Lindberg, Donald, National Library of Medicine 

Miller, Perry, Yale University 

Miller, Randolph, Vanderbilt University Medical 

Center 

Nahin, Annette, National Library of Medicine 
Nesbitt, Thomas, University of California, Davis 
Ozbolt, Judy, Vanderbilt University 
Shortliffe, Edward, Columbia University 
Stead, William, Vanderbilt University 

LECTURERS 

McCray, Alexa, National Library of Medicine 

Ash, Joan, Oregon Health and Sciences University 

STUDENTS 

Aarstad, Robert, Louisiana State University 

Medical School 

Beaudoin, Denise, Utah Department of Health 
Beyea, Suzanne, AORN 

Bird, Geoffrey, Children's Hospital of Philadelphia 
Bowen, Elizabeth, Morehouse School of Medicine 
Contini, Janice, University of California, 

Los Angeles 
Cuddy, Colleen, New York University School 

of Medicine 

Dnebeek, Mary, Duke University 
Hughes, Christopher, Monongahela Valley 

Hospital 
James, Veronica, Georgetown University 

Medical Center Library 
Just, Melissa, Childrens Hospital Los Angeles 
Long, Susan, Virginia Mason Medical Center 
Mays, Brynn, Georgetown University Medical 

Center 

Meisel, Jim, Massachusetts General Hospital 
Mittal, Richa, University of Toronto 
Nagle, Ellen, University of Minnesota 
Perley, Cathy, Empona State University 
Pimental, Sara, Kaiser Permanente 
Reavie, Keir, University of California, San 

Francisco 
Reismger, Curtis, North Shore Long Island 

Jewish Health System 
Schardt, Connie, Duke University 
Schilling, Lisa, University of Colorado Health 

Sciences Center 
Schmidt, Heidi, University of California, San 

Francisco 

Sennett, Cary, American College of Cardiology 
Silverman, Howard, Banner Health System 
Tnmarchi, Michaeleen, The Scripps Research 

Institute 

Wax, Diane, University of New Mexico 
White, Mary, Kaiser Permanente 
Williams, Annette, Vanderbilt University 

Medical Center 
Zumga, Miguel, Texas A&M University Health 

Sciences Center 



Continued.. 



R52 



Methods in Computational 
Neuroscience 

August 4 - September 1, 2002 

DIRECTORS 

Bialek, William, F ^rsity 

de Ruyter van V . .ob, Princeton 

University 

FACULTV 

Abbott. 3, Brandets University 

Aslin, Rich; , University of Rochester, New York 

Ermeni ut, Bard, University of Pittsburgh 

Faimali, Adrienne, Princeton University 

Gelpenn, Alan, Monell Chemical Senses Center 

Jensen, Roderick, Wesleyan University 

Koberle, Roland, University of Sao Paulo 

Kopell, Nancy, Boston University 

Lewen, Geoffrey, NEC Research Institute, Inc 

Remagel, Pamela, Harvard Medical School 

Saffran, Jenny, University of Wisconsin-Madison 

Schneidman, Elad, Princeton University 

Shadmehr, Reza, Johns Hopkins University 

Solla, Sara, Northwestern University 

Sompolinsky, Haim, Hebrew University 

Tank, David, Princeton University 

Tishby, Naftali, The Hebrew University 

White, John, Boston University 

LECTURERS 

Alon, Uri, Weizmann Institute 
Ayuera y Areas, Blaise, Princeton University 
Doupe, Alhson, UCSF 

Fettiplace, Robert, University of Wisconsin- 
Madison 

Hopfield, John, Princeton University 
Johnston, Dan, Baylor College of Medicine 
Kennedy, Mary, Cal Tech 
Logothetis, Nikos, Max-Planck-lnstitute 
Menzel, Randolf, Freie Universitaet Berlin 
Rieke, Fred , University of Washington 
Saffran, Jenny, University of Wisconsin-Madison 
Seung, Sebastian, Massachusetts Institute of 

Technology 

Srinivasan, MV, Australian National University 
Zucker, Steven, Yale University 

TEACHING ASSISTANT 

Still, Susanne, Princeton University 

COURSE ASSISTANT 

Jensen, Kate, Princeton University 

STUDENTS 

Barreto, Ernest, George Mason University 

Beck, James, New York University School 

of Medicine 
Chakraborty, Santanu, Cold Spring Harbor 

Laboratory 

Chiappe, Eugenia, The Rockefeller University 
Connell, Michael, Harvard University 
Dobbins, Heather, University of Maryland 

at Baltimore 

Farries, Michael, University of Pennsylvania 
Gaertner, Tara, University of Texas Health 

Sciences Center 

Herrera-Vakj--z. Marco, University of Arizona 
Huys, Quen- ~.-,mbndge University 
Lien, Cheng-C- 3 3. Physiology Institute of 

University h. , 

Loebel, Alex. The \A : .-mann Institute of Science 
Ma, Whee Ky, Califor .nstitute of Technology 
Miller, Robyn, Cornei \ersity 
Mirny, Leonid, Massachusetts Institute of 

Technology 



Montgomery, Kimberly, Northwestern University 
Nasir, Sazzad, University of California, San 

Francisco 
Pinto, Reynaldo, Institute de Fisica da Univ of 

Sao Paulo 

Raffi, Milena, Rutgers University 
Shahrezaei, Vahid, Simon Fraser University 
Stephens, Greg, Los Alamos National Laboratory 
Werner-Reiss, Uri, Dartmouth College 
Wright, Geraldme, Ohio State University 
Zhou, Yi, Boston University 



Molecular Mycology: Current 
Approaches to Fungal 
Pathogenesis 

August 12 - August 30, 2002 

DIRECTORS 

Edwards, John, Harbor-UCLA Medical Center 

Mitchell, Aaron, Columbia University 

FACULTY 

Calderone, Richard, Georgetown University 

Medical Center 
Casadevall, Arturo, Albert Einstein College 

of Medicine 

Cole, Gary, Medical College of Ohio 
Filler, Scott, Harbor-UCLA Medical Center 
Heitman, Joseph, Duke University 
Magee, Paul, University of Minnesota 
Rhodes, Judith, University of Cincinnati 
Sanglard, Dominique, University Hospital 

Lausanne 

LECTURERS 

Alspaugh, Andrew, Duke University Medical 

Center 

Brown, Alistair, University of Aberdeen 
Gale, Cheryl, University of Minnesota 
Goldman, William, Washington University 
Kronstad, Jim, University of British Columbia 
Kumamoto, Carol, Tufts University 
White, Ted, Seattle Biomedical Res. Inst 
Yeaman, Michael, UCLA-Harbor Medical Center 

COURSE COORDINATOR 

Rafkin, Wendy, Harbor-UCLA Medical Center 

COURSE ASSISTANT 

Mitchell, Hannah, Glen Ridge High School 

STUDENTS 

Andes, David, University of Wisconsin 

Berbes, Carlos, Virginia Commonwealth 

University 

Frank, Charlotte, Yale University 
Heung, Lena, Medical University of South 

Carolina 
Jabra-Rizk, Man/Ann, University of Maryland, 

Baltimore 

MacCallum, Donna, University of Aberdeen 
Mayorga, Maria, Microbia Inc 
Morais, Flavia, Umversidade Federal de Sao 

Paulo 
Nielsen, Kirsten, Duke University Medical 

Center, HHMI 
Noble, Suzanne, University of California, San 

Francisco 
Ramon, Ana, Georgetown University Medical 

Center 



Reese, Amy, Washington University School 

of Medicine 

Stembach, William, Duke University 
Sturtevant, Joy, Louisiana State University Health 

Sciences Center 

Vallim, Marcelo, Duke University Medical Center 
Van Dijck, Patrick, Flemish Interuniversity Inst. for 

Biotechnology 
Wozniak, Karen, Louisiana State University 

Health Sciences Center 



Neural Development and 
Genetics of Zebrafish 

August 18 -August 31, 2002 

DIRECTORS 

Moens, Cecilia, Fred Hutchmson Cancer 

Research Center/HHMI 
Talbot, William, Stanford University 

FACULTY 

Chien, Chi-Bm, University of Utah 

Collazo, Andres, House Ear Institute 

Dowlmg, John, Harvard University 

Fetcho, Joseph, SUNY at Stony Brook 

Granato, Michael, University of Pennsylvania 

Hanlon, Roger, Marine Biological Laboratory 

Kimmel, Charles, University of Oregon 

Lin, Shuo, University of California, Los Angeles 

Link, Brian, Medical College of Wisconsin 

Linnon, Beth, Marine Biological Laboratory 

Mullins, Mary, University of Pennsylvania 

Neuhauss. Stephan, ETH Zurich 

Raible, David, University of Washington 

Wilson, Carole, University College London 

LECTURERS 

Astrofsky, Keith, Praecis Pharmaceutical 
Fadool, James, Florida State University 
Hopkins, Nancy, Massachusetts Institute of 
Technology 

TEACHING ASSISTANTS 

Cooke, Julie, Fred Hutchmson Cancer Research 

Center 

Downes, Gerald, University of Pennsylvania 
Durchanek, Charlme, University of Oregon 
Hutson, Lara, University of Utah 
Schumacher, Jennifer, University of Pennsylvania 
Ungos, Josette, University of Washington 

COURSE COORDINATORS 
Lawrence, Christian, Harvard University 
Lesko, Suzanna, Case Western Reserve 

University 

Perkins, Brian, Harvard University 
Wilson, Steven, University College London 

STUDENTS 

Ahlgren, Sara, California Institute of Technology 
Campbell, Douglas, University of Cambridge 
Dambly-Chaudiere, Christine, Universite 

Montpellier2 

Durr, Katnn, University of Freiburg 
Gerlach, Gabnele, Marine Biological Laboratory 
Hammonds-Odie, Latanya, Spelman College 
Leach, Steven, Johns Hopkins University 
Malaga-Tnllo, Edward, University of Konstanz 
Masino, Marie, SUNY, Stony Brook 
Morns, Jacqueline, Cleveland Clinic Foundation 
Panzer, Jessica, University of Pennsylvania 



R53 



Prober, David, University of Washington 
Renier, Corinne, Stanford University 
Stewart. Rodney, Dana Farber Cancer Institute 
Wang, Weiyi, University of Pennsylvania 
Westerlund, Johanna, University of Helsinki 
Young, Rodrigo. Universidad de Chile 



Neuroinformatics 

August 17 - September 1, 2002 

DIRECTORS 

Brown, Emery, Massachusetts General Hospital 

Klemfeld, David, University of California, San 

Diego 
Mitra, Partha, Bell Laboratories 

Faculty 

Bnllinger, David, University of California, Berkeley 
Bokil, Hemant. California Institute of Technology 
Fee, Michael, Bell Labs. Lucent Technologies 
lyengar, Satish, University of Pittsburgh 
Mehta, Mayank. Massachusetts Institute of 

Technology 

Pesaran, Bijan, California Institute of Techology 
Purpura, Ketih, Cornell Medical School 
Richmond, Barry. National Institute of Mental 

Health 
Victor, Jonathan, Weill Medical College of 

Cornell University 

LECTURERS 

Dale, Anders, Harvard University 

Fitch, Tecumseh, Harvard University 

Gardener, Dan, Cornell Medical School 

Hu, Xiaoping, University of Minnesota 

Jacobs, Gwen, Montana State University 

King, Wayne, Ohio State University 

Loader, Catherine. Bell Labs. Lucent Technologies 

Margoliash, Daniel, University of Chicago 

Miller, John, Montana State University 

Sch.ff, Nicholas. Weill Medical College of 

Cornell University 

Schmidt, Marc, University of Pennsylvania 
Strothers, Steven, University of Minnesota 
Tchernikovski, Ofer, City College of New York 
Vicano. David. Rutgers University 

STUDENTS 

Aldworth, Zane, Montana State University 

Anderson, Bntt, Brown University 

Baron, Jerome. Max-Planck-Institute for Brain 

Research 

Bauer, Markus, University of Nijmegen 
Bodelon, Clara. Boston University 
Boloori. Alireza, Harvard University 
Buffalo, Elizabeth, National Institutes of Health, 

NIMH 
Cimenser, Aylin, Bell Laboratories/Lucent 

Technologies 
Diogo, Antonia, Federal University of Rio de 

Janeiro 

Fanselow, Enka. Brown University 
Froud, Karen, Massachusetts Institute of 

Technology 
Grun, Sonja, Max Planck Institute for Brain 

Research 
Hudson, Andrew, Weill Medical College of 

Cornell University 
Jones. Lauren, University of Maryland at 

Baltimore 




Herb Luther 

Jones, Matthew, Massachusetts Institute of 

Technology 

Knutsen, Per, Weizmann Institute of Science 
Kronhaus, Dma, University of Edinburgh 
Litvak, Vladimir. Technion-lsrael Institute of 

Technology 

McKeehan, Troy, Montana State University 
Moravcikova, Gabriela, University of Pittsburgh 
Nicolaou, Nicoletta. University of Reading 
Reddy, Leila, California Institute of Technology 
Sanjana, Neville, Massachusetts Institute of 

Technology 
Smith, Spencer, University of California, Los 

Angeles 

Sripati, Arun, Johns Hopkins University 
Yokoo, Takeshi, Mount Sinai School of Medicine 



Optical Microscopy 

October 9 - October 1 8, 2002 

DIRECTOR 

Izzard, Colin, University at Albany 

FACULTY 

DePasquale. Joseph. New York State 

Department of Health 
Hard. Robert. SUNY. Buffalo 
Keller, H Ernst, Carl Zeiss. Inc 
Maxfield, Frederick. Cornell University 

Medical College 
Murray, John, University of Pennsylvania School 

of Medicine 

North, Allison, The Rockefeller University 
Pierini, Lynda, Cornell University Medical 

College 

Piston, David, Vanderbilt University 
Spring, Kenneth, National Institutes of Health 
Swedlow, Jason, The University of Dundee 

TEACHING ASSISTANTS 

Hao, Mmgming, Cornell University Medical 

College 

Oberski, Danial, University of Buffalo 
Platani, Melpomeni, The University of Dundee 
Sigurdson. Wade, SUNY, Buffalo 
Snyder, Kenneth, University at Buffalo 

STUDENTS 

Ahir, Alpa, University College London 
Bauer, Christoph, Universiy of Geneva 
Boes, Marianne, Harvard Medical School 
Boxem, Mike, Dana Farber Cancer Institute 



Bruce. Ashley. University of Chicago 
Davenne. Marc, Cold Spring Harbor Laboratory 
Dorman, Jennie, University of Washington 
Ferris. Matthew. Los Alamos National Laboratory 
Gray. Annette, Brown University 
Hooper. John, The Scripps Research Institute 
Jenik, Pablo, Carnegie Institution of Washington 
Kateneva, Anna, Oklahoma Medial Research 

Foundation 

Kolb, Robert, Case Western Reserve University 
McCauley, Anita, Wake Forest University 
McDonough, Stefan, Marine Biological Laboratory 
Morgan, Jeffrey, Brown University 
Murthy, Vmit, Rice University 
Pfeifer, Andrea, National Institutes of Health 
Popoola, Joyce. King's College London 
Prigozhma, Natalie. The Scripps Research Institute 
Seth. Abhinav, University of Texas Southwestern 

Medical Center at Dallas 
Vaishnava, Shipra, University of Georgia 
Veklich, Yuri, University of Pennsylvania School 

of Medicine 
Walters, Katherine, University of Iowa 



Rapid Electrochemical 
Measurements 

May 9- May 13, 2002 

DIRECTOR 

Gerhardt. Greg, University of Kentucky Medical 
Center 

FACULTY 

Allen, Jennifer. University of Kentucky 

Medical Center 
Apparsundaram, Subu, University of Kentucky 

Medical Center 

Burmeister. Jason, Center For Sensor Technology 
Currier, Theresa, University of Kentucky 

Medical Center 
Davis, Heather. University of Kentucky 

Medical Center 
Daws, Lynette, University of Texas Health 

Sciences Center at San Antonio 
Hoffman, Alex, National Institute on Drug Abuse 
Huettl, Peter. University of Kentucky Medical 

Center 
Palmer, Michael, University of Colorado Health 

Sciences Center 
Pomerleau, Francois, University of Kentucky 

Medical Center 
Porterfield. D Marshall, University of 

Missouri-Rolla 
Surgener, Stewart, University of Kentucky 

Medical Center 

TEACHING ASSISTANTS 

Caulder, Tara, NIH/NIDA 

Knight, Tim, University of Kentucky 

Parnsh, J Michael. University of Kentucky 

Medical Center 
Robinson, Scott, University of Kentucky 

Medical Center 



COURSE COORDINATOR 
Lindsay. Robin, University of Kentucky Medical 
Center 



Coniim : 



R54 



STUDENTS 

Almeida, Catanna, Umversidade Avem Portugal 
Baccei, Christopher, Merck Research Labs 
Caldwell, Ray, University of i Carolina, 

Chapel Hill 
Cao, Bo-Jin, University of I ; lealth Sciences 

Center 

Cavus, Idil, Yale I . , 

Concur, John, Ur . ^rsity of Illinois at Urbana- 

Champaign 

Douglas, Christopher, University of Michigan 
French, Kristen, Medical University of South 

Carolina 

Galvan, Adnana, Emory University 
Herzog, Chris, Ohio State University 
Inglis, Fiona, Tulane University 
Korean, Wayne, University of South Dakota 
Li, Guichu, East Carolina University School of 

Medicine 

Martin, Joshua, The Ohio State University 
Noll, Elizabeth, Bngham & Women's & Children's 

Hospitals 

Oldenziel, Weite, University Centre for Pharmacy 
Overh, Oyvind, University of South Dakota 
Sarter, Martin, The Ohio State University 
Schad, Christina, Chicago Medical School 
Sokoloski, Joshua, University of Pittsburgh 
Stephens, Jr, Robert, Ohio State University 
vanHorne, Craig, Brigham & Women's Hospital 
Wagner, Amy , University of Pittsburgh 
Willis, Lauren, Medical University of South 

Carolina 
Zapata, Agustin, National Institutes of Health 



Summer Program in 
Neuroscience, Ethics, and 
Survival (SPINES) 

June 15 -July 13,2002 

DIRECTORS 

Martinez, Joseph, University of Texas at San 

Antonio 
Townsel, James, Meharry Medical College 

FACULTY 

Hernandez, Ruben, University of Texas, San 

Antonio 
LeBaron, Richard, University of Texas, San 

Antonio 

Zottoli, Steven, Williams College 
Berger-Sweeney, Joanne, Wellesley College 
Castaneda, Edward, Arizona State University 
Etgen, Anne, Albert Einstein College of Medicine 
Fox, Tom, Harvard Medical School 
Gonzalez-Lima, Erika, University of Texas 

at Austin 
Gonzalez-Lima, Francisco, University of Texas 

at Austin 

Hildebrand, John, University of Arizona 
Jones, James M , American Psychological 

Association 
Nickerson, Kim, American Psychological 

Association 

LECTURERS 

Burgess, David, Boston College 

Kaplan, Barry, NIH/IVi\ 

Langford, George, D.; '_,:h College 

Mensmger, Allen, Univi .( Minnesota 

Palazzo, Robert, University, ' Kansas 

Zakon, Harold, University oi as, Austin 

Kravitz, Edward A , Harvard Medical School 



TEACHING ASSISTANT 

Orfila. James, University of Texas, San Antonio 

STUDENTS 

Black, Carlita, University of Virginia 

Cruz, Nelson, Brandeis University 

Diaz, Manuel, University of Puerto Rico 

Dieguez, Jr , Dario, University of Texas at San 

Antonio 

Gallegos, Diana, San Jose State University 
Gutierrez, Tannia, University of Georgia 
Hyde, Rhonda, Harvard University 
Jones, Floretta, University of Texas at San Antonio 
Martinez, Veronica, Texas A&M University 
Mendes, Shannon, University of Miami 
Padilla, Mayra, University of California, Berkeley 
Prather, Richard, Massachusetts Institute of 

Technology 

Salas-Ramirez, Kalins, Michigan State University 
Sanchez, Javier, Baylor College of Medicine 
Torres-Reveron, Annelyn, Ponce School of 

Medicine 
Wrubel, Kathryn, University of Texas 



I Workshop on Molecular Evolution 
July 28 - August 9, 2002 

DIRECTOR 

Cummings, Michael, Marine Biological 
Laboratory 

FACULTY 

Beerli, Peter, University of Washington 
Edwards, Scott, University of Washington 
Eisen, Jonathan, The Institute for Genomic 

Research 

Felsenstein, Joseph, University of Washington 
Kuhner, Mary, University of Washington 
Lewis, Paul, University of Connecticut 
Meyer, Axel, University of Konstanz 
Rand, David, Brown University 
Swofford, David, Florida State University 
Thompson, Steven, Florida State University 
Yang, Ziheng, University College London 
Yoder, Anne, Yale University 

LECTURERS 

Fraser, Claire, The Institute for Genomic Research 
Pearson, William, University of Virginia 
Sanderson, Michael, University of California, Davis 
Voytas. Daniel, Iowa State University 
Yokoyama, Shozo, Syracuse University 

TEACHING ASSISTANTS 

Bowie, Rauri C K , University of Cape Town 

Mead, Louise, Oregon State University 

Rokas, Antonis, University of Wisconsin-Madison 

Winka, Katarina, Umea University 

STUDENTS 

Banks, Michael, Oregon State University 
Behrmann, Jasminca, University of Constance 
Benavides, Edgar, Brigham Young University 
Borchardt, Mark, Marshfield Medical Research 

Foundation 

Boudreau, Ellen, Dalhousie University 
Boyce, Sarah Lyn, Natural History Museum of 

Los Angeles County 
Brown, Joseph, Queen's University 
Budd, Aidan, European Molecular Biology 

Laboratory 
Bumbaugh, Alyssa, Michigan State University 



Burk, Robert, Albert Einstein College of Medicine 
Burleigh, Gordon, University of Missouri- 
Columbia 
Buschbom, Jutta, Field Museum of Natural 

History 

Caufield, Page, New York University 
Conley, Catharine, NASA-Ames Research Center 
Countway, Peter, University of Southern California 
Dopman, Erik, Cornell University 
Fehling, Johanna, Dunstaffnage Marine Laboratory 
Flores-Ramirez, Sergio, Autonomous University 

of Baja California Sur 
Gmzel, Matthew, University of Illinois at Urbana- 

Champaign 

Goetze, Erica, University of California, San Diego 
Gonzalez, Francisco, Universidad Autonoma del 

Estado de Morelos 

Gopal, Shuba, The Rockefeller University 
Gormley, Joseph, Massachusetts Biomedical 

Initiative 

Hall, Paula, University of New Mexico 
Hallam, Steven, Monterey Bay Aquarium 

Research Institute 

Holder, Mark, University of Connecticut 
Hudelot, Cendnne, University of Manchester 
Jennings, Bryan, University of Texas at Austin 
Jolly, Marc, Universite Pierre et Mane Curie 
Kiontke, Karin, New York University 
Komadina, Naomi, WHO Influenza Center 
Lange, Martin, Staatliche Lehr-und 

Forschungsanstalt fur Landwirtschaft 
Langeland, James, Kalamazoo College 
Lee, Dan, The Institute for Genomic Research 
Leonard, Jennifer, University of California, 

Los Angeles 

Lim, Grace, Scnpps Institution of Oceanography 
Linse, Katrin, British Antarctic Survey 
Martmello, Rick, Yale Universty School of Medicine 
Merson, Rebeka, Woods Hole Oceanographic 

Institution 

Opazo, Juan, P Universidad Catolica de Chile 
Pearman, Peter, University of Zurich 
Pie, Marcio, Boston University 
Presa, Pablo, University of Vigo 
Raes, Jeroen, University of Gent 
Rodriguez, Carmina, Universidad Complutense 

Madrid 

Rubicz, Rohma, University of Kansas 
Sakwa, James, University of Pretoria 
Sievert, Stefan, Woods Hole Oceanographic 

Institution 

Sipe, Tavis, Wake Forest University 
Smith, Catherine, Centers for Disease Control 

& Prevention 

Smith, Una, Los Alamos National Laboratory 
Stahls-Makela, Gunilla, University of Helsinki 
Storz, Jay, University of Arizona 
Stuart, Gary, Indiana State University 
Terry, Rebecca, University of Leeds 
Thomas, Bolaji, Tufts University 
Tilley, Stephen, Smith College 
Wang, Zhenshan, University of Washington 
Whitford, Tracy, East Stroudsburg University 
Wojciechowski, Martin, Arizona State University 
Wu, Martin, The Institute for Genomic Research 
Zmser, Erik, Massachusetts Institute of Technology 
Zwickl, Derrick, University of Texas, Austin 



R55 



OTHER EDUCATIONAL 
PROGRAMS 



Marine Models in Biological 
Research Undergraduate Program 

DIRECTORS 

Browne. Carole, Wake Forest University 
Tytell, Michael. Wake Forest University School 
of Medicine 

FACULTY 

Augustine. George. Duke University 
Eckberg. William. Howard University 
Fune. Barbara. Harvard School of Medicine 
Furie, Bruce. Harvard School of Medicine 
Gould, Robert New York State Institute for 

Basic Research 

Hanlon, Roger. Marine Biological Laboratory 
Jonas. Elizabeth, Yale University 
Laufer, Hans, University of Connecticut 
Malchow, R, Paul, University of Illinois, Chicago 
Mensinger, Al, University of Minnesota-Duluth 
Palazzo, Robert, Rensselaer Polytechnic Institute 
Rome, Larry, University of Pennsylvania 
Silver, Robert, Wayne State University 
Wainwright, Norman, Marine Biological 

Laboratory 

STUDENTS 

Alimi, Manam, Wake Forest University 
Bodily, Jill. Stanford University 
Borely, Kimberly, Ohto University 
Homsi, Sara, Wake Forest University 
Jackson, Ticana, Howard University 
Montanez, Marlena, Mount Holyoke College 
Najera. Julia, Univ of Texas, El Paso 
Normand, Danielle, University of New 

Hampshire 

O'Neal, Jessica, College of Charleston 
Simpson, Andrew, University of California, 

Santa Barbara 
Steeds. Craig. University of Kansas 




NASA Planetary Biology 
Internship Program 

DIRECTORS 

Dolan, Michael F, University of Massachusetts 

Amherst 
Margulis, Lynn, University of Massachusetts 

Amherst 

INTERNS 

Allen, Michelle, The University of New South 

Wales, Australia 
Chichon Garcia, Francisco J , Universidad 

Autonoma de Madrid 
Fike, David, University of Cambridge 
Guarin. Alejandro, The Pennsylvania State 

University 
Navio, Ruben Peco, Universitatsklinikum 

Hamburg-Eppendorf 
Rosenfeld, Ane. University of Haifa 
Vaisanen, Katarina, University of Aberdeen 
Wier. Andrew. University of Wisconsin- 

Milwaukee 

SPONSORS 

Cabrol, Nathalie A . NASA Ames Research 

Center 

Garland, Jay. NASA Kennedy Space Center 
Hagan. William, College of St. Rose 
Hinkle, C Ross. NASA Kennedy Space Center 
Margulis. Lynn, University of Massachusetts 

Amherst 
Summons, Roger, Massachusetts Institute of 

Technology 

Trent, Jonathan. NASA Ames Research Center 
Wofsy. Steven C , Harvard University 



| Science Journalism Program 

FELLOWS 

Bellmghim, Ruth Helena. Science Reporter. Brazil 

Berrcby, David, Freelance 

Biskup, Agnes, Freelance 

Bogo, Jennifer, Audubon Magazine 

Carter, Kandice, AAAS Science Update 

Dempsey, Dale, Freelance 

Griffin, Katherme, Freelance 

Hosteller. A J , Richmond Times-Dispatch 

King, Robert. The Pa'm Beach Post 

Manier. Jeremy. Chicago Tribune 

Omfade. Diran, Nigerian Television Authority 

Perry. Rebecca. Los Angeles Times 

Reker, Mary Lou. Library of Congress 

Valentine, Vikki, National Public Radio Online 

Wisby. Gary, Chicago Sun-Times 

BIOMEDICAL FACULTY 

Beach, Dale, UNC Chapel Hill 

Bloom, Kerry. Dale Beach, UNC Chapel Hill 

Palazzo. Robert. University of Kansas 

Pearson. Chad, UNC Chapel Hill 

Schnackenberg. Brad, UNC Chapel Hill 

ENVIRONMENT FACULTY 

Foreman, Kenneth, Marine Biological Laboratory 
Neill, Chnstoper, Marine Biological Laboratory 
Tholke. Kris. Marine Biological Laboratory 

CO-DIRECTORS 

Goldman. Robert D , Northwestern University 

Rensberger. Boyce. Director, Knight Science 

Journalism Fellowships, Massachusetts 

Institute of Technology 

ADMINISTRATIVE DIRECTOR 
Hinkle, Pamela Clapp, Marine Biological 
Laboratory 



R56 



Semester in Environmental 
Science 

September 2 - December 16, 20C ' 

DIRECTOR 
Hobbie, John E 

ASSOCIATE DIR': 
Foreman, Kenne n 

FACULTY 
Deegan. Linda A. 
Foreman, Kenneth H. 
Giblin, Anne E 
Hobbie. John E 
Hopkmson, Charles S., Jr 
Liles, George 
Melillo, Jerry M 
Neill, Christopher 
Peterson, Bruce J. 
Rastetter, Edward B 
Shaver, Gaius R 
Steudler, Paul A 
Vallmo, Joseph J 

RESEARCH AND TEACHING ASSISTANTS 

Bahr, Michele 

Bowen, Jennifer 

Creswell, Joel 

Kwiatkowski, Bonnie 

Micks, Patricia 

Tholke. Kris 

Ziemann, Tori 

Administrat've Assistant 
Johnson-Horman, Frances 

Students 

Adams, Jacqueline M , Ripon College 
Burce, Allison E., Harvey Mudd College 
Copeland, Maureen T, Allegheny College 
Dean, Mary D,, Ripon College 
Engelhart, Gabnella J , Lafayette College 
Fila, Laurie A , Mount Holyoke College 
Franklin, Jennifer M , Wheaton College 
Freeman, Christopher J , Connecticut College 
Havassy, Joshua I , Haverford College 
Kang, MoonKoo Simon, Clark University 
Kennedy, Jenny L , Clarkson University 
Leahy, Sarah E , Wheaton College 
Leamy, Claire A , Wellesley College 
Lmdell, Joshua S , Dickinson College 
Roberts, Rachael A , Skidmore College 
Shea, Alexandra E , Earlham College 
Stern, Stephanie B., Wellesley College 
Webster, William K., Trinity University 
Wright, Julie A , Wellesley College 



Teachers' Workshop: Living in the Microbial World 

August 10-16, 2002 



DIRECTORS 

Dorntie, Barbara, Cambridge Rindge and Latin 

School, Cambridge, Massachusetts 
Olendzenski, Lorraine, University of Connecticut, 

Storrs 

FACULTY 

Gunnard, Jessie, University of Massachusetts 

Offerdahl, Enka, University of Arizona 

COURSE ASSISTANT: 

Waksman, George, Massachusetts Institute of 
Technology 

PRESENTERS 

Bermudes, David, Vion Pharmaceuticals, New 

Haven, Connecticut 
Dyer, Betsey, Wheaton College 
Edgcomb, Virginia, Woods Hole Oceanographic 

Institution 

Guerrero, Ricardo, University of Barcelona, Spain 
Margulis, Lynn, University of Massachusetts, 

Amherst 
Rogers, Dan, Woods Hole Oceanographic 

Institute 



Eliot, Judith, Middle Years Alternative School 

for the Humanities, Philadelphia, Pennsylvania 
Freitas, Caroline. Cape Cod Regional Technical 

High School, Massachusetts 
Golet, Gerie, Salem School, Connecticut 
Goodding, Debbie, Kraemer Middle School, 

Placentia, California 
Hammond, Christine, Clarendon House 

Grammar School, Ramsgate, Kent. United 

Kingdom 
Howie, Charles, Old Rochester Regional High 

School, Massachusetts 
Knox, Carol, Northfield Mount Hermon School, 

Massachusetts 
Lee, Marge, Harrington School, Cambridge, 

Massachusetts 
Lichtenstein, Leslie, Massasoit Community 

College, Massachusetts 
Lincoln, Peter, Hmgham Public Schools, 

Massachusetts 
Natoli, Therese, Ledyard Middle School, Gales 

Ferry, Connecticut 
Potrafka, Renee, Father Gabriel Richard Catholic 

High School. Ann Arbor, Michigan 




Teacher Participants 

Barren, Melanie, Cambridge Public Schools, 

Massachusetts 
Berrick. Steve, Cape Cod Regional Technical 

High School, Massachusetts 
Connor, Lynn, Old Rochester Regional High 

School, Massachusetts 
Crook, Jolene, East Lyme High School, 

Connecticut 
Diehl, Penny, Hempfield High School, 

Landisville, Pennsylvania 



Pullan, Mary, Fitch Middle School, Groton, 

Connecticut 
Roark, Eileen, Nathan Hale-Ray High School, 

Moodus, Connecticut 
Ruston, Steve, King Etherbert School, 

Birchmgton, Kent, United Kingdom 
Tanigawa, Joy. El Rancho High School, Pico 

Rivera, California 



Microbes, Donna Bedard 



SCHOLARSHIP AWARDS 



R57 




E/izabeth Armstrong 



I THE BRUCE AND BETTY ALBERTS ENDOWED 

I SCHOLARSHIP IN PHYSIOLOGY 
Agutlar, Arturo, Av Institute PolitEcnico National 
Kelly, Melissa, University of Kentucky College 
of Medicine 

| AMERICAN SOCIETY FOR CELL BIOLOGY 
Brown, Ann, Medgar Evers College 
Campbell, Susan, University of Alabama, 

Birmingham 

Davis, Kevin, University of Pittsburgh 
Gadea. Bedrick, Harvard Medical School 
Gonsalves, Joanna, University of California, 

San Francisco 

Green, Heather, New York University 
Harrison, Faith, University of Iowa 
Livi, Carolina, University of Texas Health Sciences 

Center, San Antonio 

Miranda, Jason, University of Texas at Austin 
Pineda, Gabriel. University of Texas Health 

Sciences Center, Dallas 
TorrEns, Javier, New Jersey Medical School, 

UMDNJ 
Van Stry, Melanie, Boston University School 

of Medicine 
Vega, Rebecca, Stanford University 

I BIOLOGY CLUB OF THE COLLEGE OF 
I THE CITY OF NEW YORK 
Zornik, Erik Columbia University 

JJOHN AND ELISABETH BUCK SCHOLARSHIP 
Thamatrakoln, Kimberlee, University of California. 
San Diego 



|C LALOR BURDICK SCHOLARSHIP 
Dash, Satya, University of East Anglia 
Gonsalves, Joanna, University of California, 

San Francisco 

Hallikas. Outi, University of Helsinki 
Prosen, Tracy, University of Pittsburgh 

I BURROUGHS WELLCOME FUND - 
BIOLOGY OF PARASITISM COURSE 
Chamond, Nathalie, Institut Pasteur 
Cockburn, Ian, University of Edinburgh 
Karnataki. Anuradha, University of Washington 
Klotz. Christian, Humbo'dt-University-Berlin 
Kooij, Taco Leiden, University Medical Centre 
Lee, SooHee, Johns Hopkins School of Medicine 
Li, Hongjie, Yale University 
Mueller. Ann-Kristin, University School of 
Medicine, Heidelberg 

I BURROUGHS WELLCOME FUND - FRONTIERS 
IN REPRODUCTION COURSE 
Akcali, Kamil. Bilkent University 
Bachman, Katherme, Case Western Reserve 

University 

Gill. Ryan. University of Kansas Medical Center 
Gonsalves. Joanna. University of California. San 

Francisco 

Hallikas. Outi, University of Helsinki 
Ka, Hakhyun, University of Kansas Medical 

Center 

Kayisli, Umit, Yale University School of Medicine 
Kreeger, Pamela, Northwestern University 
Perez, Christian, University of Pennsylvania 
Prosen, Tracy, University of Pittsburgh 
TorrEns, Javier, New Jersey Medical School, 

UMDNJ 
Wang, Eileen, Northwestern Medical School 



I BURROUGHS WELLCOME FUND-MOLECULAR 
| MYCOLOGY COURSE 
Berbes, Carlos, Virginia Commonwealth 

University 

Brown, Constance, Howard University 
Heung, Lena, Medical University of South 

Carolina 

MacCallum, Donna, University of Aberdeen 
Morais, Flavia, Universidade Federal de 

Sao Paulo 
Noble, Suzanne. University of California. 

San Francisco 
Ramon, Ana. Georgetown University Medical 

Center 
Reese. Amy. Washington University School 

of Medicine 

Stembach, William, Duke University 
Sturtevant, Joy, Louisiana State University 
Health Science Center 

Vallim, Marcelo, Duke University Medical Center 
Van Dijck, Patrick. Flemish Interuniversity 

Institute for Biotechnology 
Wozniak, Karen. Louisiana State University 

Heath Science Center 

GARY N CALKINS MEMORIAL 
SCHOLARSHIP FUND 
Berry, Katy, University of Sheffield 
Copf, Ti|ana. University of Crete 

| CONRAD 

Sachdeva, Geetanjali, Institute for Research in 
Reproduction, India 

| EDWIN GRANT CONKLIN MEMORIAL FUND 
Caracino, Diana, Emory University School 

of Medicine 
Copf, Ti|ana, University of Crete 

| BERNARD DAVIS FUND 
Boucher, Yart, Dalhousie University 
Denef, Vincent. Michigan State University 
Erbs, Marianne, Swiss Fed Institute for 
Environmental Science & Technology 
Pmel, Nicolas, University of Washington 

I WILLIAM F AND IRENE C DILLER MEMORIAL 
SCHOLARSHIP FUND 
Davis, Kevin, University of Pittsburgh 
Vega, Rebecca, Stanford University 

I THE ELLISON MEDICAL FOUNDATION - 
I BIOLOGY OF PARASITISM COURSE 
Chamond, Nathalie, Institut Pasteur 
Cockburn, Ian, University of Edinburgh 
Karnataki, Anuradha, University of Washington 
Klotz, Christian, Humboldt-University-Bcrlm 
KOOIJ, Taco Leiden, University Medical Centre 
Mueller, Ann-Kristin, University School of 
Medicine. Heidelberg 

THE ELLISON MEDICAL FOUNDATION - 
MOLECULAR BIOLOGY OF AGING COURSE 
Almeida, Claudia, Weill Medical College of 

Cornell University 
Bishop, Glenda, Case Western Reserve 

University 

continued 



R58 



Bokov, Alex, University of Texas Health Science 

Center, San Antonio 
Chen, Lishan, University of Washington 
Dunaief, Joshua, University o< a 

Fuller, Kathryn, University of f 1 esota 
Harvey, Sarah, Medi< Virginia 

Hong, Eun-Jin (Eric <!ty 

Johnston, Janet, C versity Belfast 

Lledias, Ferpjr to de Biotecnologia, 

UNAM 
Lu, XiangcV ;versity of North Carolina, 

Chapel H 

Moynihdn Kathryn, Washington University 
Ogle, William, Stanford University 
Powers, Ralph, The University of Washington 
Proctor, Carole, University of Newcastle 
Rea, Shane, University of Colorado 
Rutten, Bart, University of Maastricht 
Shirasawa, Takuji, Tokyo Metropolitan Institute 

of Gerontology 

Tong, Liqi, University of California, Irvine 
Zou, Ying, University of Texas Southwestern 

Medical Center 

ICASWELL GRAVE SCHOLARSHIP FUND 

Berry, Katy, University of Sheffield 
Chen, Yen-Chin, National Cheng Kung 

University Medical College 
Copf, Tijana, University of Crete 
Delalande, Jean-Mane, University College 

London 
Extavour, Cassandra, University of Cambridge 

| DANIEL S- GROSCH SCHOLARSHIP FUND 
Dethlefsen, Les, Michigan State University 
Ge, Lan, University of California, Riverside 
McVaugh, Cheryl, University of Pennsylvania 

(WILLIAM RANDOLPH HEARST FOUNDATION 
Chong, Curtis, Johns Hopkins School of 

Medicine 
Kelly, Melissa, University of Kentucky College 

of Medicine 

LaPomte, Nichole, Northwestern University 
McVaugh, Cheryl, University of Pennsylvania 
Pace, Margaret, University of Texas 
Pignatelli, Vmcenzo, University of Pisa 
Sha, Edward, Indiana University Medical Center 

HOWARD HUGHES MEDICAL INSTITUTE - 
BIOLOGY OF PARASITISM COURSE 
Meissner, Markus, Imperial College of Science, 

Technology & Medicine, UK 
Slavin, lleana, Universidad Nacional de Cordoba 
Stubbs, Janine, Royal Melbourne Hospital 

IHOWARD HUGHES MEDICAL INSTITUTE 

Boassa, Daniela, University of Arizona College 

of Medicine 

Dash, Satya, University of East Anglia 
Dellen, Babette, Washington University in 

St Louis 

Denef, Vincent, Michigan State University 
Doiron, Brent, University of Ottawa 
Dulcis, Davide, University of Arizona, Tucson 
Graco, Michelle, University of Pierre et 

Mane Curie 



Guest, Jennifer, National Institute for Medical 

Research 
Ihnng, Alexandra, Max-Planck-lnstitute of 

Neurobiology 

Malartre, Marianne, University of Portsmouth 
Nkinm, Wuyika, University of Yaounde 
Petersen, Rasmus, International School for 

Advanced Study (SISSA) 
Pfeiffer, Keram, Philipps-Universitat Marburg 
Pinel, Nicolas, University of Washington 
Rajagopal, Soumitra, University of Nebraska 
Renart, Alfonso, Brandeis University 
Sharp, Kathenne, Scnpps Institute of 

Oceanography 

Werner-Reiss, Un, Dartmouth College 
Witney, Alice, University of Birmingham 

Medical School 
Wright, Geraldme, Ohio State University 

IICRO-UNESCO 

Akcali, Kamil, Bilkent University 
Hallikas, Outi, University of Helsinki 

INTERNATIONAL BRAIN RESEARCH 

ORGANIZATION 

Chakraborty, Santanu, Cold Spring Harbor 

Laboratory 

Chiappe, Eugenia, The Rockefeller University 
Diogo, Antonia, Federal University of Rio 

de Janeiro 

Herrera-Valdez, Marco, University of Arizona 
Hu, Hailan, University of California, Berkeley 
Koirala, Samir, University of Southern California 
Kozhevnikov, Alexay, Bell Laboratories 
Nasir, Sazzad, Unversity of California, San 

Francisco 
Pinto, Reynaaldo, Institute de Flsica da 

University of Sao Paulo 

Reddy. Leila, California Institute of Techology 
Shahrezaei, Yahid, Simon Fraser University 
Snpati, Arun, Johns Hopkins University 
Wang, Weiyi, University of Pennsylvania 
Young, Rodngo, University of Chile 
Zhou, Yi, Boston University 

ARTHUR KLORFEIN SCHOLARSHIP AND 
FELLOWSHIP FUND 

Drago, Grazia, Universita Degli Studi di Palermo 
Extavour, Cassandra, University of Cambridge 
Kee, Yun, California Institute of Technology 
Koziel, Lydia, Max-Planck-lnstitute for Molecular 

Genetics 

Malartre, Marianne, University of Portsmouth 
Maslakova, Svetlana, Smithsonian Institution 
Su, Yi-Hsien, Scripps Institute of Oceanography, 

MBRD 

IFRANK R LILLIE FELLOWSHIP AND 
(SCHOLARSHIP FUND 

Aguilar, Arturo, Av Institute Politecnico 

National 

Amane, Dragos, University of Notre Dame 
Do|Cinovic, Danijel, Arizona State University 
Ge, Lan, University of California, Riverside 
Goentoro, Lea, Princeton University 
Kaynar, Murat, Beth Israel Deaconess Medical 

Center 



Kydd, Alison, University of Calgary 
Oh, Ji-Eun, University of Illinois at Chicago 
Pace, Margaret, University of Texas 
Ramos, Arnolt, Children's Hospital 
Rossi, Chiara, University of Pisa 

I THE GRUSS LIPPER FOUNDATION 
I SCHOLARSHIP 

Keren, Zandbank 

Koren, Omry, Tel Aviv University 

Litvak, Vladimir, Technion-lsrael Institute of 
Technology 

Loebel, Alex, The Weizmann Institute of Science 

JACQUES LOEB FOUNDERS' 
SCHOLARSHIP FUND 
Amane, Dragos, University of Notre Dame 
Dojcmovic, Danijel, Arizona State University 
Pace, Margaret, University of Texas 

(SO MAST MEMORIAL FUND 
Ihnng, Alexandra, Max-Planck-lnstitute of 

Neurobiology 
Zhou, Zhaolan (Joe), Harvard Medical School 

I MBL ASSOCIATES ENDOWED 
I SCHOLARSHIP FUND 
Amane, Dragos, University of Notre Dame 

| MBL PIONEERS SCHOLARSHIP FUND 
Brown, Ann, Medgar Evers College 
Berry, Katy, University of Sheffield 
Caracino, Diana, Emory University School of 

Medicine 

Copf, Tijana, University of Crete 
Dash, Satya, University of East Anglia 
Delalande, Jean-Mane University College, London 
Extavour, Cassandra, University of Cambridge 
Koziel, Lydia, Max-Planck-lnstitute for Molecular 

Genetics 
Livi, Carolina, University of Texas Health Science 

Center, San Antonio 

CHARLES BAKER METZ AND WILLIAM METZ 

SCHOLARSHIP FUND IN REPRODUCTIVE 

BIOLOGY 

Akcali, Kamil, Bilkent University 

Hallikas, Outi, University of Helsinki 

Kreeger, Pamela, Northwestern University 

Prosen, Tracy, University of Pittsburgh 

IFRANK MORRELL ENDOWED MEMORIAL 
SCHOLARSHIP 

Petersen, Rasmus, International School for 
Advanced Study (SISSA) 

(MOUNTAIN MEMORIAL FUND SCHOLARSHIP 
Chong, Curtis, Johns Hopkins School of Medicine 
Kelly, Melissa, University of Kentucky College 

of Medicine 

LaPomte, Nichole, Northwestern University 
Oh, Ji-Eun, University of Illinois at Chicago 
Pignatelli, Vincenzo, University of Pisa 
Ramos, Arnolt, Children's Hospital 



R59 



IALBERTO MONROY FOUNDATION 

Drago, Grazia. Universita Degli Studi di 
Palermo 

| PFIZER INC. ENDOWED SCHOLARSHIP 
Aguilar, Arturo, Av Institute PolitEcnico 
National 
Chong. Curtis, Johns Hopkins School of 

Medicine 
Pignatelli, Vmcenzo, University of Pisa 

I PLANETARY BIOLOGY INTERNSHIP 
SCHOLARSHIPS 

Maresca, Julia, Pennsylvania State University 
Remold, Susanna, Michigan State University 
Walker. Jeffrey, University of Colorado 

I WILLIAM TOWNSEND PORTER FELLOWSHIP 
IAND SCHOLARSHIP FUND 

Brown, Ann, Medgar Evers College 
Campbell, Susan, University of Alabama, 

Birmingham 

Davis, Kevin, University of Pittsburgh 
Gadea, Bedrick. Harvard Medical School 
Gonsalves, Joanna, University of California, 

San Francisco 

Green, Heather, New York University 
Harrison, Faith, University of Iowa 
Livi, Carolina, University of Texas Health 

Science Center, San Antonio 
Miranda, Jason, University of Texas at Austin 
Pineda, Gabriel, University of Texas 

Southwestern Medical Center at Dallas 



POST-COURSE RESEARCH AWARDS 

Extavour, Cassandra, University of Cambridge (Embryology) 
Chong, Curtis, Johns Hopkins School of Medicine (Physiology) 

Davis, Kevin, University of Pittsburgh (Physiology) 
Ge, Lan, University of California, Riverside (Physiology) 
LaPointe, Nichole, Northwestern University (Physiology) 



Torrens. Javier, New Jersey Medical School, 

UMDNJ 
Van Stry, Melanie, Boston University School 

of Medicine 
Vega, Rebecca, Stanford University 

[HERBERT W. RAND FELLOWSHIP AND 
(SCHOLARSHIP FUND 

Ahlgren, Sara, California Institute of Technology 
Amarie, Dragos, University of Notre Dame 
Campbell. Douglas. University of Cambridge 
Chen. Yen-Chin, National Cheng Kung 

University Medical College 
Dojcmovic, Danijel, Arizona State University 
Durr, Katrin, University of Freiburg 
Gaertner, Tara, University of Texas Health 

Science Center 

Goentoro. Lea. Princeton University 
Hammonds-Odie, Latanya, Spelman College 
Kaynar, Murat, Beth Israel Deaconess 

Medical Center 




Marine diatom, 
Ka/ma White 



Lien, Cheng-Chang, Physiology Institute of 

University Freiburg 

Ma, Whee Ky, California Institute of Technology 
Malaga-Trillo, Edward, University of Konstanz 
Masmo, Mark. State University of New York 

at Stony Brook 

Prober, David, University of Washington 
Raffi, Milena, Rutgers University 
Ramos, Arnolt, Children's Hospital 
Rossi. Chiara, University of Pisa 
Sha, Edward, Indiana University Medical Center 

FLORENCE C ROSE AND S MERYL ROSE 
ENDOWED SCHOLARSHIP FUND 
Caracino. Diana, Emory University School 
of Medicine 

| RUTH SAGER MEMORIAL SCHOLARSHIP 
Guest, Jennifer, National Institute for Medical 

Research 
Mitchell, Tracy, University of Wisconsin-Madison 

I HOWARD A SCHNEIDERMAN ENDOWED 
(SCHOLARSHIP 

Boassa, Damela, University of Arizona College 

of Medicine 

Ewald, Rebecca, Cold Spring Harbor Lab 
De Labra, Carmen, University College London 
Doiron, Brent, University of Ottawa 

I MILTON L. SHIFMAN ENDOWED 
I SCHOLARSHIP 

Khali!. Mona, Columbia University 

Kerney, Ryan, Harvard University 

I MOSHE SHILO MEMORIAL 
I SCHOLARSHIP FUND 
Koren, Omry, Tel Aviv University 

(CATHERINE FILENE SHOUSE SCHOLARSHIP 
Avila, Andrea, Institute de Biologia Molecular 

do Paran -IBMP 
Boassa, Daniela, University of Arizona College 

of Medicine 

Brown, Ann, Medgar Evers College 
Caracino. Diana, Emory University School 

of Medicine 

Ewald, Rebecca, Cold Spring Harbor Lab 
Fenn, Katelyn, University of Edinburgh 
Graco, Michelle, University of Pierre et 

Mane Curie 

Kreeger, Pamela, Northwestern University 
Orsborn, April, University of Missouri-Columbia 
Sharp. Katherme, Scripps Institute of 

Oceanography 



Spitzer. Nadja. Georgia State University 
Wohlgemuth, Sandra. Humboldt-Universit zu 
Berlin 

IMARJORIE w STETTEN SCHOLARSHIP FUND 

Witney. Alice, University of Birmingham 
Medical School 

| HORACE W STUNKARD SCHOLARSHIP FUND 
Akcali, Kamil, Bilkent University 
Gill, Ryan, University of Kansas Medical Center 

ISOCIETY FOR GENERAL PHYSIOLOGY 

Brown, Ann. Medgar Evers College 
Dojcmovic. Danijel. Fumio Mekata Scholar, 

Arizona State University 
Hu, Hailan, University of California. Berkeley 
Wohlgemuth. Sandra, Humboldt-Universit zu 

Berlin 

ISURDNA FOUNDATION SCHOLARSHIP 

De Labra. Carmen, University College London 
Dellen, Babette, Washington University in St Louis 
Doiron, Brent, University of Ottawa 
Hobbs. Steven, University of Colorado 
Montana, Enrico, Massachusetts Institute 

of Technology 

Ryan, Amy, University of Virginia Health Systems 
Zhou. Zhaolan (Joe). Harvard Medical School 

| IRVING WEINSTEIN ENDOWED SCHOLARSHIP 
Berry, Katy, University of Sheffield 
Su, Yi-Hsien, Scripps Institute of Oceanography, 
MBRD 

I WILLIAM MORTON WHEELER FAMILY 
I FOUNDERS' SCHOLARSHIP 
Ihnng, Alexandra, Max-Planck-lnstitute of 
Neurobiology 

I WALTER L WILSON ENDOWED 
SCHOLARSHIP FUND 
Dojcmovic, Danijel, Arizona State University 
LaPointe, Nichole, Northwestern University 

WORLD ACADEMY OF ARTS AND SCIENCES 

EMILY MUDD SCHOLARSHIP 

Akcali, Kamil, Bilkent University 

Bachman, Katherme. Case Western Reserve 

University 

Hallikas, Outi. University of Helsinki 
Perez, Christian, University of Pennsylvania 
Wang, Eileen, Northwestern Medical Sch>" 

| WORLD HEALTH ORGANIZATION 
Busso, Dolores, Buenos Aires Un:v. 



R60 



INSTITUTIONS REPRESENTED (students) 



Albert Einstein Colleqp o Medicine 

Alberta Research Cc-.ncil 

American College c' Carjtology 

AORN 

Arizona State University 

AstraZeneca R&D Charnwood, UK 

Autonomous University of Baja California Sur 

Av Institute Politecmco National #2508 

Avenue Healthcare Ltd. 

Banner Health System 

Baylor College of Medicine 

Bell Laboratories/Lucent Technologies 

Beth Israel Deaconess Medical Center 

Bilkent University 

Boehringer Ingelheim Pharmaceuticals 

Boston University 

Boston University School of Medicine 

Brandeis University 

Brigham & Women's Hospital 

Bngham Young University 

British Antarctic Survey 

Brown University 

Buenos Aires University 

California Institute of Technology 

Cambridge University 

Carl Zeiss 

Carnegie Institution of Washington 

Case Western Reserve University 

Centers for Disease Control & Prevention 

Chicago Medical School 

Children's Hospital Boston 

Children's Hospital of Philadelphia 

Children's Hospital Los Angeles 

Cleveland Clinic Foundation 

Cold Spring Harbor Lab 

College of William & Mary 

Columbia University 

Dalhousie University 

Dana Farber Cancer Institute 

Dartmouth College 

Doane College 

Duke University 

Duke University Medical Center 

Dunstaffnage Marine Laboratory 

East Carolina University School of Medicine 

East Stroudsburg University 

Emory University 

Emory University School of Medicine 

Empona State University 

European Molecular Biology Laboratory 

Family Health Council 
Federal University of Rio de Janeiro 
Field Museum of Natural History 
Flemish Interumvo^ity Institute for 

Biotechnology 
Fox Chase Cancer 

George Mason University 
Georgetown University Me iical Center 
Georgia State University 
Good Samaritan Family Practic-= Center 



Harvard Medical School 
Harvard University 
Hebrew Universtiy 
Hospital Authority 
Howard University 
Humboldt-Universitat zu Berlin 

Illinois State University 

Imperial College of Science, Technology 

& Medicine 

Indiana State University 
Indiana University 
Indiana University Medical Center 
Institute Venezolano de Investig Cientificas 
Institut Pasteur 

Institute de Fisica da Univ of Sao Paulo 
Institute for Research in Reproduction 
Institute de Biologia Molecular do Parana-IBMP 
Institute de Biotecnologia, UNAM 
International School for Advanced Study (SISSA) 

James Madison University 

Johns Hopkins Bloomberg School of Public 

Health 

Johns Hopkins School of Medicine 
Johns Hopkins University 

Kaiser Permanente 
Kalamazoo College 
Kansas State University 
Karolmska Institute! 
King's College London 

Leiden University Medical Centre 

Liverpool School of Tropical Medicine 

Los Alamos National Laboratory 

Louisiana State University Health Sciences Center 

Louisiana State University Medical School 

Maine Medical Center 

Marine Biological Laboratory 

Marshfield Medical Research Foundation 

Massachusetts Biomedical Initiative 

Massachusetts Eye & Ear Infirmary 

Massachusetts General Hospital 

Massachusetts Institute of Technology 

Max-Planck-Institute for Brain Research 

Max-Planck-lnstitute for Molecular Genetics 

Max-Planck-lnstitute of Neurobiology 

Medgar Evers College 

Medical College of Virginia 

Medical University of South Carolina 

MerckResearch Labs 

Michigan State University 

Microbia Inc 

Monongahela Valley Hospital 

Montana State University 

Monterey Bay Aquarium Research Institute 

Morehouse School of Medicine 

Mount Sinai School of Medicine 

NASA-Ames Research Center 

National Cheng Kung University Medical College 

National Institute for Medical Research 

National Institutes of Health 

Natural History Museum of Los Angeles County 



New Jersey Medical School, UMDNJ 

New York University 

New York University School of Medicine 

North Shore Long Island Jewish Health System 

Northwestern Medical School 

Northwestern University 

Ohio Permanente Medical Group 
Ohio State University 
Oklahoma Medical Research Foundation 
Oregon Health & Science University 
Oregon State University 

P Universidad Catolica de Chile 
Pennsylvania State University College 

of Medicine 

Pennsylvania State University 
Philipps-Universitat Marburg 
Physiology Institute of University Freiburg 
Ponce School of Medicine 
Princeton University 

Queen's University Belfast 

Raytheon Polar Services 
Rice University 
RIKEN, Japan 
Rockefeller University 
Royal Melbourne Hospital 
Rutgers University 

San Jose State University 

Scnpps Institute of Oceanography 

Simon Fraser University 

Smith College 

Smithsonian Institution 

Southern Illinois University School of Medicine 

Spelman College 

Staatliche Lehr-und Forschungsanstaltfur 

Landwirtschaft 
Stanford University 

Stanford University School of Medicine 
State University of New York at Stony Brook 
Swiss Federal Institute for Environmental Science 

& Technology 

Technion Israel Institute of Technology 

Tel Aviv University 

Temple University School of Medicine 

Texas ASM University 

Texas A&M University Health Science Center 

The Institute for Genomic Research 

The Naval Research Laboratory 

The Scnpps Research Institute 

Tokyo Metropolitan Institute of Gerontology 

Toronto West Hospital-Univ Health Network 

Tufts University 

UMass Medical School 
United States Air Force 
University of Massachusetts, Amherst 
Universidad Autonoma del Estado de Morelos 
Universidad Complutense Madrid 
Universidad de Chile 
Universidad Nacional de Cordoba 
Universidade Aveiro 



R61 




Cyanobactena, Rolf Schauder 

Universidade Federal de Sao Paulo 

Umversita Degli Studi di Palermo 

Universite Montpellier 2 

Umversite Pierre et Mane Curie-Pans 6 

University Centre for Pharmacy 

University College London 

University of Aberdeen 

University of Alabama, Birmingham 

University of Arizona 

University of Arizona College of Medicine 

University of Birmingham Medical School 

University of Buffalo 

University of Calgary 

University of California, Los Angeles 

University of California, Berkeley 

University of California, Irvine 

University of California, Riverside 

University of California, San Diego 

University of California, San Francisco 

University of Cambridge 

University of Chicago 

University of Colorado 

University of Colorado Health Sciences Center 

University of Connecticut 

University of Constance 

University of Crete 

University of East Anglia 

University of Edinburgh 

University of Freiburg 

University of Gent 

University of Georgia 

University of Hawaii 

University of Helsinki 

University of Illinois at Chicago 

University of Illinois at Urbana-Champaign 

University of Iowa 

University of Iowa College of Medicine 

University of Kansas 

University of Kansas Medical Center 

University of Kentucky College of Medicine 

University of Konstanz 

University of Leeds 

University of Maastricht 

University of Manchester 

University of Maryland 

University of Massachusetts Medtcal School 

University of Miami 

University of Miami School of Medicine 

University of Michigan 

University of Minnesota 

University of Missouri-Columbia 

University of Montana 

University of Naples 

University of Nebraska 

University of New Mexico 

University of New South Wales 

University of Newcastle 



University of Nijmegen 

University of North Carolina 

University of North Dakota 

University of Notre Dame 

University of Oklahoma Health Sciences Center 

University of Oregon 

University of Ottawa 

University of Pennsylvania 

University of Pennsylvania School of Medicine 

University of Pierre et Mane Curie 

University of Pisa 

University of Pittsburgh 

University of Portsmouth 

University of Pretoria 

University of Puerto Rico 

University of Reading 

University of San Lui Potosf 

University of Sheffield 

University of South Dakota 

University of South Florida 

University of Southern California 

University of Texas at Austin 

University of Texas Health Science Center, 

San Antonio 
University of Texas Southwestern Medical 

Center at Dallas 
University of Toronto 
University of Utah 
University of Vigo 
University of Virginia 
University of Washington 
University of Wisconsin 
University of Wisconsin-Madison 
University of Yaounde 
University of Zurich 

University School of Medicine, Heidelberg 
University of Geneva 
Utah Department of Health 

VA Maryland Health Care System 
Vanderbilt University Medical Center 
Veterans Affairs Medical Center 
Virginia Commonwealth University 
Virginia Mason Medical Center 

Wake Forest University 

Washington University in St Louis 

Weil! Medical College of Cornell University 

Weizmann Institute of Science 

WHO Influenza Center 

Woods Hole Oceanographic Institution 

Yale University 

Yale University School of Medicine 



COUNTRIES REPRESENTED 

Argentina 

Australia 

Belgium 

Brazil 

Cameroon 

Canada 

Ch.le 

China 

Finland 

France 

Germany 

Greece 

India 

Israel 

Italy 

Japan 

Kenya 

Mexico 

Netherlands 

Portugal 

Scotland 

South Africa 

Spam 

Sweden 

Switzerland 

Taiwan 

Turkey 

United Kingdom 

United States of America 

Venezuela 



INSTITUTIONS REPRESENTED 
(faculty) 



Albert Einstein College of Medicine 
American Association for the Advancement 

of Science 

American Psychological Association 
Arizona State University 

Baylor College of Medicine 
Bell Labs, Lucent Technologies 
BioHybnd Technologies 
Boston College 
Boston University 
Bowlrng Green State University 
Brandeis University 
Bndgewater State College 
Brown University 

California Institute of Technology 
Carnegie Institution of Washington 
Carnegie Mellon University 
Case Western Reserve University 
CCNY 

Center For Sensor Technology 
Centre for Genome Research 
Centre Genetique Moleculaire 
Cold Spring Harbor Laboratory 
Columbia University 
Cornell Medical School 
Cornell University 



Contmued 



R62 



Dartmouth College 
Danmark Tekniske Universitet 
Duke University Medical Center 
Duke University 

Eastern Virginia Medical ? : 
Emory University 
ETH Zurich 

European Institute o\ , 

Florida Institute J| ogy 

Florida State Ui 

Fred Hutchir, , ancer Research Center 

Freie Universitaet Berlin 

Georgetown University Medical School 
Geron 

Hamamatsu Photonic Systems 

Harbor-UCLA Medical Center 

Harvard Medical School 

Harvard University 

Hebrew University 

HHMI/Brown University 

HHMI/Fred Hutchmson Cancer Research Center 

HHMI/UMDNJ-RW Johnson Medical School 

HHMI/UT Southwestern Medical Center 

HHMI/Johns Hopkins University 

HHMI/NYU 

House Ear Institute 

ICRF Clare Hall Laboratories 
Imperial Cancer Research Fund 
Imperial College of Science Technology 
Institut Pasteur 
Iowa State University 

Johns Hopkins School of Medicine 
Johns Hopkins Universtiy 
Jules Stem Eye Institute, UCLA 

Karolinska Institutet 
Kent State University 
King's College, London 

Lawrence Berkeley National Laboratory 
London School of Hygiene and Trop Med 
Louisiana State University Health Science Center 
Leiden University, The Netherlands 

Magee-Womens Research Institute 

Marine Biological Laboratory 

Massachusetts General Hospital 

Massachusetts Institute of Technology 

McGill University 

Medical College of Georgia 

Medical College of Ohio 

Medical College of Wisconsin 

Meharry Medical College 

Michigan State University 

Monell Chemical Senses Center 

Montana State University 

Mount Sinai School of Medicine 

MPI for Medical Research 

MPl for Biological Cybernetics 

MPI for Manne Microbiology 

MRC Lab of Molecular Biology 

Memorial Sloan Kenenng Cancer Center 

National Institute on Aqing, NIH 
National Institute for M.-J.ca! Research 
National Institute of Me";, : '^alth 
National Institute on Druq 
National Institutes of Health 
National Library of Medicine 
NEC Research Institute, Inc 
New England Medical Center 
New York University 



New York University School of Medicine 

Northwestern University 

Northwestern University Medical School 

Ohio State University 

Oregon Health & Science University 

Oregon State University 

Praecis Pharmaceuticals 
Princeton University 

Queen's University Belfast 

Roche Molecular Systems 
Rockefeller University 
Rutgers University 

San Diego State University 

Seattle Biomededical Research Institute 

Sensor Technologies 

Smithsonian Institution 

St Barnabas Medical Center 

Stanford Medical School 

Stanford University 

Stowers Institute for Medical Research 

SUNY at Buffalo 

SU NY at Stony Brook 

Syracuse University 

Texas A&M University 

The Hebrew University 

The Institute for Genomic Research 

The University of Iowa 

The University of Manchester 

Tufts University 

Tufts University School of Medicine 

Tufts University School of Veterinary Medicine 

Umea University 

University of Pittsburgh 

University of Texas Southwestern Medical 

Center 

University of Virginia 
University of Texas at Austin 
University of California, Davis 
University of California, San Diego 
University of California, Santa Barbara 
University of California, Berkeley 
University of California, Los Angeles 
University of California, San Francisco 
University of North Caroline, Chapel Hill 
University of Illinois, Chicago 
University of Kentucky Medical Center 
University of Maryland, College Park 
University of Rochester (NY) 
University of Pittsburgh 

University of Colorado Health Sciences Center 
University of British Columbia 
University of Colorado Health Sciences Center 
University of Kansas Medical Center 
University of Oregon 
University of Pennsylvania 
University of Sao Paulo 
University of Southern California 
University of Pittsburgh School of Medicine 
Universidad de Buenos Aires 
Universitaet Ulm 
University College London 
University Hospital Lausanne 
University of Aberdeen 
University of Alabama 
University of Arizona 
University of Bern 
University of Calgary 
University of Cambridge 
University of Cape Town 
University of Chicago 
University of Cincinnati 



University of Connecticut 

University of Connecticut Health Center 

University of Dundee 

University of Edinburgh 

University of Florida 

University of Georgia 

University of Glasgow 

University of Guelph 

University of Hawaii 

University of Idaho 

University of Illinois 

University of Illinois, Chicago 

University of Illinois, Urbana 

University of Iowa 

University of Kansas 

University of Kentucky 

University of Kentucky Medical Center 

University of Konstanz 

University of Lethbridge 

University of Manchester 

University of Maryland 

University of Massachusetts 

University of Massachusetts Medical School 

University of Melbourne 

University of Michigan 

University of Minnesota 

University of Missoun-Columbia 

University of Missouri-Rolla 

University of North Carolina, Chapel Hill 

University of Notre Dame 

University of Oregon 

University of Oxford 

University of Texas 

University of Texas at Austin 

University of Toronto 

University of Utah 

University of Victoria, B C 

University of Virginia 

University of Warwick 

University of Washington 

University of Wisconsin 

University of Wisconsin - Madison 

UT Health Science Center, San Antonio 

Vollum Institute 

Washington University 

Washington University School of Medicine 

Weill Medical College Cornell University 

Weizmann Institute 

Wellesley College 

Wesleyan University 

Whitehead Institute for Biomedical Research 

Williams College 

Yale Medical School 
Yale University 



COUNTRIES REPRESENTED 



Argentina 

Australia 

Austria 

Brazil 

Canada 

China 

Columbia 

Denmark 

France 

Germany 

Greece 

India 

Indonesia 

Ireland 

Israel 

Italy 

Jamaica 



Lebanon 

Mexico 

New Zealand 

Russia 

South Africa 

Sweden 

Switzerland 

Taiwan 

The Netherlands 

Turkey 

United Kingdom 

United States of Americ 



mbl/whoi library 



R63 




The MBL/WHOI Library maintains one of the 
world's largest print and electronic collec- 
tions of biomedical, oceanographic, and 
marine biological literature. It is jointly 
operated with the Woods Hole Oceano- 
graphic Institution. The main library is 
located in the MBL's Lillie Building, with 
branches on the campuses of the Woods 
Hole Oceanographic and the National 
Marine Fisheries Service in Woods Hole. 




REPORT OF THE LIBRARIAN 

Libraries are the computer-age doorways to information systems 
worldwide. Today the MBL/WHOI Library uses an integrated set of 
resources for online cataloging, resource sharing and reference 
services, with local and regional systems linking Woods Hole with 
large universities and museums all over the world. Libraries like ours 
are now essentially borderless. With increases in journal pricing and 
shrinking budgets, libraries must collaborate with other institutions 
more and more to deliver information that their users require. 

Digitization technology which enables the one-time transformation 
of information from print to electronic format will only increase 
collaborations and encourage the sharing of information over time. 
Thanks to this technology, important articles, books, and images once 
at risk of being lost forever on a dusty shelf can be stored and 
delivered electronically to users around the world. The MBL/WHOI 
Library has shown this to be true with the successful digitization of the 
Leuckart charts, which have been available on the Library's web site 
since 1 996. Over the years, hundred of thousands of people from 
around the world have rediscovered these 19 th century teaching 
charts, which are still very useful today. 

The MBL/WHOI Library continues to grow in non-traditional direc- 
tions. We are developing innovative digitization services and 
important new research tools, including X-ID, an online taxonomic key, 
funded by the Jewett Foundation, and uBio, a network taxonomic 
name server funded by the Andrew W. Mellon Foundation. Through 
these two pilot projects we have established global alliances with 
GBIF (Global Biodiversity Information Facility) and ITIS (Information 
Taxonomic Information Service) and bolstered our relationships with 
museums. Currently, we are creating a digital archive for the MBL 
Herbarium of local fauna and marine algae. A very enthusiastic group 
of MBL Associates is scanning the specimens for the project, which is 
funded by SeaGrant. 

Continued 



R64 



In 2002, we aggressively moved more Library content and 
supporting services to the web for direct delivery to the 
patron. By the end of 2002, the MBL/WHOI Library was 
delivering appro-r -ly 52% of our serials and 100% of 
our database? ' outed, electronic form extending 

the Library' ;rh beyond our walls to wherever our 
patrons " - ) be twenty-four hours a day, seven days a 
week. We continued to improve existing services with the 
enhancement of our web site (www.mblwhoilibrary.org/). 
We also continued to invest in the Library's future through 
major expenditures, projects, technological platforms, 
support systems and replacement of current infrastructure 
and computer technology that support existing library 
services. 

The Library hosted 1 1 9 Readers taking sabbaticals, 
working on writing projects, or conducting long-term 
research projects. The wireless network installed in 2002 
brought Internet access to the stacks and reading rooms. 
Ironically, we have declared the Grass Reading Room to 
be a "technology free" zone to maintain a quiet, contem- 
plative space in a Library that has resolutely embraced 
electronic information delivery. 




| Serials 

We made a smooth transition to a new serials vendor, 
EBSCO, and escaped the turmoil our former vendor 
created when it declared bankruptcy and stranded major 
academic libraries with loss of access to journals. The 
Library realigned serial holdings and services to better 
support current scientific research interests. We analyzed 



serial and database usage statistics and surveyed the 
scientific community about how current subscriptions and 
exchange programs were meeting their needs. Discus- 
sions resulted in substantive changes to the 2003 serial 
collection, including a cut back of long-standing ex- 
change programs at the MBL and WHOL These changes 
reflect input from librarians and members of the commu- 
nity on a number of factors: survey responses; the 
community's request that we subscribe to the Web of 
Science database; rising serials prices; expiring ejournal 
contracts being replaced with higher-cost alternatives; 
space constraints in the Lillie Building; and a 4% decrease 
in the MBL-serials budget line. 



Monographs 

We continued to acquire titles for the book and special/ 
named collections using our general budget and specific 
funds including the Atwood, Aron, and Mullin gift funds. 
The WHOI ship libraries were updated with the addition 
of new reference as well as recreational reading materials. 
The Clark Reading Room was completely dismantled to 
reorganize staff workspace. Non-duplicate Clark materi- 
als were filed with the mam book collection (3rd floor 
Lillie), which was completely shifted. In addition, all 
stacks were relabeled. 



Special Collections 

Dedicated staff and volunteers continued to make great 
strides in Special Collections. The MBL Archives pro- 
cessed the reprint collection of Viktor Hamburger, John 
Burris' Director's papers, James Ebert's scientific and 
Director's papers, and the Arthur Humes collection of 
glass plates, slides, and photographs. We also restored 
and rebound 130 items from Rare Books/Special Collec- 
tions with the support of the Florence Gould Foundation. 
The WHOI Data Library & Archives (DLA) continued to 
process and catalog their extensive collection of technical 
reports, maps, scientist's papers, ship cruise reports and 
logs, etc. They spent a considerable amount of time 
migrating Alvm and other data from old media to newer 
formats. Both MBL and WHOI Archivists are raising 
awareness for the need of institutional records manage- 
ment plans. 



LIBRARY READERS 



R65 



Courses 

Library staff gave 37 general library 
orientations or department-oriented 
sessions and taught 25 courses in 
2002. Trainers were brought in from 
several specific databases to do in- 
depth training sessions. In conjunc- 
tion with our Spring and Fall Medical 
Informatics courses, new interactive 
student web pages were designed, 
and course packs were made avail- 
able over the web. 

| Our staff actively participated in 
professional development activities 
and contributed to the profession as 
\a whole. This serves to promote 
MBL/WHOI Library projects and it 
exposes our staff to new ideas and 
methods. DLA staff hosted a meeting 
of the Marine Technology Society 
concerning scientific instruments. 
The Library Director and Associate 
Director also served on various task 
forces involved in developing a 
strategic plan for the MBL. 

The MBL/WHOI Library continues to 
serve as an electronic bridge between 
Woods Hole and the world providing 
access to collections at universities, 
and in public, corporate, private, 
museum, and laboratory libraries 
around the globe. 

Catherine N. Norton 



Abbott, Jayne, Marine Research. Inc 
Ahmad|ian, Vernon, Clark University 
Allen. Garland. Washington University 
Anderson, Everett, Harvard University 

Baccetti. Bacoo, NRC of Siena. Italy 
Benjamin. Thomas Harvard Medical School 
Bernhard. Jeffery, University of Massachusetts 

Medical Center 

Borgese, Thomas, Leaman College, CUNY 
Bower. James, University of Texas, San Antonio 
Boyer, John, Union College 

Candelas, Graciela, University of Puerto Rico 
Cariello, Lucio, Stanzione Zoologica "A Dohrn" 
Chang, Donald, Hong Kong University 
Child, Frank, Trinity College 
Clarkson. Kenneth, Bell Labs 
Cohen, Seymour, American Cancer Society 
Cook. Erik, Howard Hughes Medical Center 
Cooperstein, Sherwin, University of 

Connecticut 

Copeland. Eugene. Woods Hole, MA 
Corwin, Jeffery, University of Virginia 
Couch, Ernest, Texas Christian University 

D'Alessio, Giuseppe, Universita di Napoli 

Fedenco II 

Davis, Jonathan, Lexigen Pharmaceuticals 
Donavan, Erin, Auburn University 
Dube, Francois. St. Luc, Canada 
Duncan. Thomas, Nichols College 

Epstein, Herman, Brandeis University 

Fmkelstein, Alan. Albert Einstein College of 
Medicine 

Fraenkel, Dan, Harvard Medical School 
Frenkel, Krystyna, New York University 
School of Medicine 

Galatzer-Levy, Robert, University of Chicago 
Gancia-Blanco, Mariano. Duke University 

Medical School 

German, James, Cornell University 
Grossman, Albert, New York University Medical 

School 
Gruner. John, Cephalon, Inc 

Halvorson, Harlyn, University of Massachusetts, 

Boston 

Harrington, John, SUNY New Paltz 
Herskovits. Theodore, Fordham University 

Inoue, Sadayuki. McGill University 

Jaye, Robert, Boston 

Jacobson, Allan, University of Massachusetts 

Medical Center 
Josephson, Robert, University of California 

Kaltenbach, Jane, Mount Holyoke College 

Karlin, Arthur, Columbia University 

Kelly, Robert. Woods Hole, MA 

Keynan, Alexander. Israel Academy of Sciences 

& Humanities 

King. Kenneth, Falmouth, MA 
Knox, Carol, Northfield Mount Herman 
Kornberg. Hans. Boston University 
Krane. Stephen, Harvard Medical School 



Laczko, Jozsef. New York University 
Laderman, Aimlee. Yale University 
Lisman, John. Brandeis University 
Linck, Richard, Umersity of Minnesota 
Llinas. Rudolfo, New York University 
Loewenstem, Werner. Journal of Membrane 

Biology 
Lorand. Laszlo, Northwestern University 

Medical School 
Luckenbill, Louise, Ohio University 

Menmi, Anna, CNR-SISSA 

Milkman, Roger, University of Iowa 

Miller, Andra, NIH 

Mitchell, Ralph, Harvard University 

Morrell, Leyla, Rush Presbyterian St Lukes 

Nagel. Ronald. Albert Einstein College of Medicine 
Narahashi, Toshio. Northwestern University 
Naugle. John. NASA 

Plummer Cobb, Jewel, California State University 
Prendergast, Robert, Johns Hopkins University 




Rabmowiu. Michael, Harvard University 
Rafferty, Nancy, Falmouth. MA 
Reynolds, George, Princeton University 
Rome, Larry, University of Pennsylvania 
Ruderman, Joan, Harvard Medical School 

Segal, Sheldon, The Population Council 
Sheng, Morgan, Massachusetts Institute 

of Technology 

Shephard, Frank, Woods Hole Data Base 
Shimomura, Osamu, Falmouth, MA 
Smith. Tim, Northeast Fisheries Science Center 
Solomon, Dennis, Yarmouth Port. MA 
Spector, Abraham, College of Physicians 

& Surgeons 

Spotte, Stephen, Mote Marine Lab 
Steinberg. Martin. Boston University School 

of Medicine 

Stuart, Ann. University of North Carolina 
Sullivan, Gerald, Savio Prep-Boston 

Tnnkhaus, John, Yale University 

Tweedell. Kenyon. University of Notre Dame 

Tycosmski, Mark. University of Pennsylvania 

Walton, Alan, Cavendish Laboratory 
Warren. Leonard, University of Pennsylvania 
Weissmann, Gerald. New York University 

Medical Center 

Whittaker. J Richard. University of New Brunswick 
Woods Hole Research Center 



R66 



financials 




REPORT OF THE TREASURER 

The financial results for 2002 reflected the difficult operating environ- 
ment in a year where all non-profits were adversely affected by two 
major trends: the continued decline in the investment markets and a 
softening of philanthropic support. 

The Unrestricted Operating results showed a loss of $1.76 million, 
which was a vast improvement over the $2.9 million loss reported in 
2001. This was due to a more rapid increase (14.4%) in Unrestricted 
Operating Support when compared to Operating Expense growth 
(9.6%). On the revenue side, Government Grants increased by 8.2%, 
Lab Rental & Net Tuition increased by 12%, Fees for Conferences and 
Services rebounded 21 .7% from depressed levels in 2001 as a result of 
cancelled events due to September 1 1"\ and Investment and Other 
Revenues increased by 30%. 

On the expense side. Research activities represented two-thirds of the 
increase, growing by approximately $2 million (9.7%) as the Laboratory 
added more than two dozen scientists mainly to gear up for expanded 
research programs in Scientific Aquaculture and Global Infectious 
Disease. As a result, grant applications hit an all-time high and grant 
dollars awarded increased almost 30% over 2001 . Looking at the 
underlying components, double-digit increases were experienced in 
Salaries (13.6%), Fringe Benefits (16.2%), Supplies (19.4%), Utilities 
(12.1%), and Depreciation (16.8%). 

Philanthropic support fell dramatically from the unusually strong levels 
the Laboratory had experienced in the three previous years when the 
MBL received gifts exceeding $10 million each year. Total Contribu- 
tions including those to Plant were $4.6 million, the lowest level since 
1995. The long-term investment portfolio, however, performed quite 
competitively in a year when the average common stock mutual fund 
declined by 28%. The 3.9% decline in market values was in the top 
quartile when compared to how a universe of 1 22 foundations and 
endowments performed in 2002. The MBL had realized and unrealized 
investment losses, which totaled $2 million in 2002. 

Taking into account the challenging near-term market environment, in 
2002 the Laboratory experienced its first decline in Total Net Assets 
since 1994. MBL's Net Assets declined $6.2 million. 



R67 



The MBL's Balance Sheet Assets reflected this impact, 
declining by $7.1 million. The entire decline was concen- 
trated in Short Term Investments and Pledge Receivables. 
The Endowment held up due to the receipt of new 
permanently restricted gifts. Property, Plant & Equipment 
also held steady as $2.4 million in improvements more 
than offset the accrued depreciation. Liabilities declined 
by approximately $1 million, principally due to the relin- 
quishment of a Unitrust to the benefit of the Laboratory. 

Considering some financial performance ratios, our Return 
on Average Net Assets was a negative 6.7%, which is in 
line with most non-profits during this period. The MBL's 
Leverage Ratio (Unrestricted & Temporarily Restricted Net 
Assets/ Debt) remains sound at 4.48X. Also, both our Debt 
Service Coverage ratio of 1 .72X for 2002 and our non- 
permanently restricted Cash & Investments of $25.6 million 
at year-end are well in excess of the financial covenants of 
the Letter of Credit supporting the MBL's Long Term Debt. 



In summary, it was a challenging year financially mainly 
due to the effect the third consecutive year of investment 
market declines had on MBL's philanthropic support and 
investment portfolio. Coming out of our strategic 
planning effort we have already started implementing 
steps that will position the Laboratory for a strong 
rebound in our Government Grants and to ultimately 
gear-up for a new capital campaign that should improve 
our philanthropic support. Our education, summer/ 
visiting scientist, and conference activities remained 
strong and when combined with the expanded resident 
research programs should help us continue to improve 
our operating results in the 
near future. 

Mary B. Conrad 




Lobster eyes, Diane Heck. David Ramsey, Lydia Louis, and Jeff Laskin 



R68 



FINANCIAL STATEMENTS 



Operating History and Balance Sheet as of December 31 , 2002 and 2001 . 



BALANCE SHEET (In Thousands) 



2002 



2001 




The financial statements of the 
Marine Biological Laboratory, for 
the fiscal year ending December 
31. 2002. were audited by 
Pr/cewaterhouseCoopers. LLP 

Complete financial statements are 
available upon request from: 

Mr Homer Lane 
Chief Financial Officer 
Marine Biological Laboratory 
7 M8L Street 
Woods Hole. MA 
02543-1015 



ASSETS: 

Cash and Short Term Investments 

Pledges and Other Receivables 

Assets Held by Bond Trustee 

Other Assets 

Endowment and Similar Investments 

Property Plant and Equipment (Net) 

TOTAL ASSETS: 

LIABILITIES. 

Accounts Payable 

Annuities and Unitrusts Payable 

Deferred Revenue and Other Liabilities 

Long Term Debt 

Total Liabilities: 

NET ASSETS- 
Unrestricted 
Temporary Restricted 
Permanently Restricted 

Total Net Assets: 

TOTAL LIA8ILITES AND NET ASSETS: 

OPERATING HISTORY (In Thousands) 

OPERATING SUPPORT: 

Government Grants 

Private Contracts 

Lab Rental and Net Tuition 

Fees for Conferences and Services 

Contributions 

Investment and Other Revenues 

Total Operating Support 

EXPENSES: 

Research 

Instruction 

Conferences and Services 

Other Programs 

Total Expenses: 



$4.357 
8.794 

631 

42.290 
31.729 

87.801 



2,797 

535 

2.557 

10,200 

16.089 



20,381 
25.278 
26,053 

71.712 
$87.801 



$15.849 
1.495 
2,188 
5.333 
4.522 
3,321 
32,708 



22,371 
5.998 

1.460 
5.027 

34.856 



$8.993 

11,265 

269 

711 

42.181 
31,519 

94.938 



3,133 

1,383 

2,318 

10,200 

17,034 



22.261 
29.941 
25,702 

77.904 
$94,938 



$14,648 

1,675 

1,954 

4.383 

10.886 

2.555 

36,101 



20.399 
5.637 

1.133 

4.643 

31.812 



CHANGE IN NET ASSETS BEFORE 
NON. OPERATING ACTIVITY. 



(2,148) 



4.239 



Non-Operating Activities. 

Contributions to Plant and Other Expenses, Net (149) 

Total Investment Income and Earnings (1,994) 

Less Investment Earnings Used for Operations (1,901) 

Reinvested (Utilized) Investment Earning (3.895) 

TOTAL CHANGE IN NET ASSETS: $(6.192) 



2,244 

(2.930) 
(1.475) 
(4,405) 

$2.128 



R69 



gifts 




P/anlcromc sarcodme The Host 
DNA is heated within the dark. 
centra/ nuc'ear capsule. The 
symbionts are the sma// bright 
specks radfatmg outward along the 
host spines, David Caron 



REPORT OF THE DEVELOPMENT COMMITTEE 



As the MBL engaged in institution-wide strategic planning during this past year as described by 
Dr. Speck earlier in this report, 2002 was a pivotal year for Development efforts. Moving forward 
after our spectacularly successful Discovery Campaign with new research and educational pro- 
grams now in place, we turned our attention to broadening our outreach and education activities 
through a series of events and communications. 

Last summer, we held an enjoyable and informative Day of Science attended by 92 guests from 
nearby communities that included presentations by MBL scientists and lab tours. The Council of 
Visitors meeting drew 80 friends from across the country to learn about Modem Molecular 
Approaches to Global Infectious Diseases and to tour the totally refurbished suite of laboratories 
for the newly established research program in this area. As in past years, our summer lectures and 
childrens' programs in Nantucket and Martha's Vineyard spread the word about the MBL. We 
branched out in 2002 and held our first-ever alumni reception in Chapel Hill, North Carolina, 
attended by 65 course alumni and faculty, MBL Corporation Members, and friends of the Lab. 
And, turning to the Internet, Development information is now featured on MBL's web site so we 
can effectively extend our message to a world-wide community. 

In 2002, MBL raised $4,944,803 in private support. This included $1 ,350,000 from the Andrew W. 
Mellon Foundation in renewed support for terrestrial forest research and support for a new pilot 
project designed to index and organize information about organisms that is distributed on the 
Internet. MBL Council of Visitors member, Robert Shifman, provided an additional gift of 
$564,502 to the Milton Shifman Endowed Scholarship The G. Linger Vetlesen Foundation 
continued its generous and long-standing support for the Bay Paul Center and unrestricted 
support. The Ellison Medical Foundation, the Grass Foundation, and the Burroughs Wellcome 
Fund all provided renewed support for MBL's advanced courses in biology and biomedicme. 

The 2002 Annual Fund had another strong year with $553,620 raised from 898 donors both new 
records in annual fund giving at the MBL. The amount raised and numbers of donors are both up 
by 9% over last year. As in past years, the Whitman Society, comprised of donors whose gifts of 
$1,000 or more accounted for much of this success. I would like to thank Dr. Peter Armstrong for 
serving as Annual Fund Chair and Mr. Michael Fenlon for leading the Annual Fund drive for the 
Associates. We are enormously grateful for this unrestricted support for the Laboratory. 

On behalf of the Development Committee, the Board of Trustees, and the entire MBL community, 
I would like to express my appreciation to the donors whose names appear on the following 
pages, and to those who requested anonymity. We are all most grateful for their generous 
support for the Marine Biological Laboratory's research and educational programs. 



Christopher M. Weld, Chair, Development Committee 



R70 



HIGHLIGHT! 



During 2002, 
the following 
foundations and 
individuals 
provided major 
support for the 
Laboratory. 




Ecosystems Center Staff 
members map out a 
; research plan, 

Elizabeth Armstrong 



Burroughs Wellcome Fund awarded $170,000 
for continuing support of the Molecular 
Mycology course. 

The Ellison Medical Foundation awarded 
$282,670 for continuing support of the 
Molecular Biology of Aging Colloquium for 
the period 2002 through 2004. 

The Grass Foundation awarded $226,500 in 
renewed funding for the Neurobiology and 
Neural Systems and Behavior courses. 

Andrew W. Mellon Foundation awarded two 
grants totaling $1,350,000: $850,000 to 
support research on nitrogen transformation 
in terrestrial landscapes conducted by The 
Ecosystems Center and $500,000 to launch 
the Universal Biological Indexer and Orga- 
nizer (uBio), a database and internet tool to 
provide up-to-date biological information. 



I Mr. Robert Shifman made an additional gift of 
$564,502 to support the Milton L. Shifman 
Endowed Scholarship. Mr. Shifman was a 
member of the Council of Visitors. 

G. Linger Vetlesen Foundation provided 
$150,000 for the Josephine Bay Paul Center 
in Comparative Molecular Biology and 
Evolution; $100,000 in support of the 
program to develop marine models for 
biomedical research; and $100,000 to 
underwrite veterinary services in the Marine 
Resources Center. 



Neura/ system cone mossaic, Inigo Nova'<s F/amanque 




SPECIAL GIFTS 



R7I 



Other Gifts 




A variety of foundations, individuals, and companies provided 
funds for special projects and programs at the Marine Biological 
Laboratory. We gratefully acknowledge their support. 



Major Gifts 

Anonymous (2) 

American Society for Cell Biology 

American Society for Reproductive Medicine 

Applied Biosystems 

Cox Foundation, Inc 

Estate of Emily Ann Cramer 

Environmental Data Research Institute 

ExxonMobil Foundation 

Georgia-Pacific Corporation 

Mr William Golden and Dr Jean Taylor 

Howard Hughes Medical Institute 

Dr and Mrs Kurt J Isselbacher 

Mr and Mrs George W Logan 

The Mantia Family Trust 

Massachusetts Environmental Trust 

The Honorable and Mrs G William Miller 

New York Times Company Foundation 

Nikon Instruments Inc 

The Pfizer Foundation 

Mrs Robert W Pierce 

William Townsend Porter Foundation 

Dr and Mrs John W Rowe 

The Schooner Foundation 

Sholley Foundation, Inc 

The Catherine Filene Shouse Foundation 

Alfred P Sloan Foundation 

Society for Neuroscience 

The Seth Sprague Education & Charitable Foundation 

Drs William Speck and Evelyn Upper 

The Sprague Foundation 

Drs Albert Stunkard and Margaret Maurin 

Mr and Mrs Gerard I Swope 

Universal Imaging Corporation 

Mr Sidney J Wemberg, Jr 

The Irving Wemstein Foundation, Inc. 

Mr and Mrs Christopher M Weld 



American Society for Biochemistry & Molecular Biology 

Mrs KimballC Atwood, III 

Aquatic Eco-systems, Inc 

Ms Susan M Barnes 

Josephine Bay Paul & C Michael Paul Foundation 

Bio-Rad 

Dr Barry R Bloom 

Mr and Mrs Thomas C Bolton 

Mrs Carolyn S Carlson 

Dr Eloise E Clark 

Drs William and Marion Cohen 

Dr Seymour S Cohen 

Elsevier Science Ireland Ltd 

Drs Lawrence Cohen and Barbara Ehrlich 

Federation of American Societies for Experimental Biology 

Fluke Corporation 

Friendship Fund 

Dr and Mrs Harold Gainer 

Drs Robert and Anne Goldman 

Dr and Mrs J Woodland Hastings 

Dr Robert R Haubnch 

Mr and Mrs Irwm Herskowitz 

Mrs Carmela J Huettner 

Mr and Mrs Richard A Huettner 

Dr and Mrs Shmya Inoue 

Mr David Isenberg and Ms Paula Blumenthal 

Invitrogen 

Dr Wallace Ip 

Mr Daniel Isenberg 

Dr. and Mrs Benjamin Kaminer 

Dr Andrew Kropmski and Dr Peggy Pntchard 

Dr Kiyoshi Kusano 

Dr Hans Laufer 

Mr Richard Mawe 

Merck Research Laboratories 

Ms Natalie Miller 

Dr Betty C Moore 

Ms Mary Musacchia and Dr James Faber 

Mr Telephone, Inc 

New England Biolabs, Inc 

Ms Catherine N Norton 

Novartis 

Dr and Mrs Philip Person 

Pharmacia & Upjohn Company 

PhotoArk 

Estate of Madelene E Pierce 

Princeton Day School 

Promega Corporation 

Qiagen, Inc 

Dr and Mrs Harris Ripps 

Dr Raja Rosenbluth 

Mr and Mrs William Rugh 

Schenng-Plough Research Institute 

Smauer Associates Inc . Publishers 

Dr Sol Sepsenwol 

Dr Abraham Spector and Ms Marguerite Filson 

Drs Melvn and Evelyn Spiegel 

Mrs Eleanor Stembach 

Syngenta 

Dr and Mrs Andrew G Szent-Gyorgyi 

Ms Natalie Trousof 

United States Geological Survey 

Dr Dale A Webster 

Dr and Mrs. Gerald Weissmann 

Mrs Clare M Wilber 

World Health Organization 

World Precision Instruments 

The Worthmgton Family Foundation, Inc 



R72 



THE WHITMAN SOCIETY 



Mem.' 1 " j in the Whitman Society is open each year to individual donors who 
' . (e $1,000 or more to the MBL Annual or Alumni Funds. 



| Director's Circle 

Anonymous 

Dr. Porter W. Anderson, Jr. 
Key Largo, Florida 

Mr. and Mrs William C Cox, Jr 
Hobe Sound, Florida 

Dr. and Mrs. Kurt J. Isselbacher 
Newton Center, Massachusetts 

Mr. and Mrs. George W. Logan 
Salem, Virginia 

The Honorable G William Miller 
Washington, DC 

Mrs. Robert W. Pierce 
Boca Grande, Florida 

Mr. Vin Ryan and Ms. Carla Meyer 
Boston, Massachusetts 

Dr. and Mrs. John W. Rowe 
New York, New York 

Drs. William Speck and Evelyn Upper 
West Fa/mouth, Massachusetts 

Mr. Sidney J. Wemberg, Jr. 
New York, New York 

Dr and Mrs Gerald Weissmann 
New York, New York 

Mr. and Mrs. Christopher M Weld 
Essex, Massachusetts 

Mr. and Mrs Alfred M Zeien 
Boston, Massachusetts 



] Benefactor 

Diane and Norman Bernstein 
Washington, DC 

Mr, and Mrs. Jonathan Conrad 
New York, New York 

Mr. and Mrs. Diarmaid Douglas-Hamilton 
Beverly, Massachusetts 



Mr William T. Golden 

American Museum of Natural History, 

New York, New York 

Mr. and Mrs. Amos B. Hosteller Jr. 
Boston, Massachusetts 

Dr. Laurie J. Landeau 
Northport, New York 

Robert A. Prendergast 
Fa/mouth, Massachusetts 

Mr. and Mrs. Richard Reiss 
New York, New York 

Harriet and Sheldon Segal 
Hartsc/ale, New York 



| Patron 

Anonymous 

Frank and Mardi Bowles 
Woods Hofe, Massachusetts 

Mr. and Mrs. Malcolm K. Brachman 
Dallas, Texas 

Mr. and Mrs. Murray H Bring 
East Hampton, New York 

Drs Claire Fraser and J. Craig Venter 
Potomac, Maryland 

Frances and Howard Jacobson 
Westborough, Massachusetts 

Dr. and Mrs. Stephen M. Krane 
Waban, Massachusetts 

Catherine C. Lastavica, M.D. 
Manchester, Massachusetts 

Dr. and Mrs. Leonard Laster 
Woods Hole, Massachusetts 

Dr. Anna Logan Lawson 
Daleville, Virginia 

Birgit Rose and Werner K Loewenstem 
Fa/mouth, Massachusetts 



Gmny and Pete Nicholas 
Boston, Massachusetts 

Arthur B. Pardee and Ann B. Goodman 
Cambridge, Massachusetts 

Mr. Manus A. Robinson 
Key Biscayne, Florida 

Drs Mitchell Sogin and Laurel Miller 
Fa/mouth, Massachusetts 

Mr. and Mrs. John C. Stegeman 
Ann Arbor, Michigan 

Drs. Ann E. Stuart and John W. Moore 
Chapel Hill, North Carolina 



| Member 



Edward and Marion Adelberg 
New Haven, Connecticut 

Dr Louise Adler 
Fa/mouth, Massachusetts 

Dr Garland E Allen 
Saint Louis, Missouri 

Mr and Mrs Douglas F Allison 
Bloomfield Hills, Michigan 

Drs Clay and Clara Armstrong 
Philadelphia, Pennsylvania 

Peter and Margaret Armstrong 
Davis, California 

Dr Samuel C Armstrong 
Everett, Washington 

Jean and Bob Ashton 
New York, New York 

Mr and Mrs David Bakalar 
Chestnut Hill, Massachusetts 

Mr and Mrs Charles A Baker 
Princeton, New Jersey 

Drs Robert and Harriet Baker 
White Plains, New York 

David Baltimore and Alice S Huang 
Pasadena, California 



R73 



Mrs Fredenk B Bang 
Woods Hole, Massachusetts 

Hope and Mel Barkan 
Boston, Massachusetts 

Dr and Mrs Robert B Barlow 
Jamesville, New York 

Fred and Christine Bay 
Landgrove, Vermont 

Bruce Anthony Beal 
Woods Hole, Massachusetts 

Drs Eugene and Millicent Bell 
Boston, Massachusetts 

Drs Michael and R Suzanne Bennett 
New Rochelle, New York 

Mrs. Beth Berne 

Woods Hole, Massachusetts 

Jewelle and Nathaniel Bickford 
New Yoric, New Yorlc 

Mr and Mrs Eric F Billings 
Potomac, Maryland 

Dr and Mrs Elkan R Blout 
Cambridge, Massachusetts 

Mr Allen Bragdon 

South Yarmouth, Massachusetts 

Dr and Mrs Goodwin M Breinm 
New Yorlc, New York 

Dr and Mrs. John B Buck 
Sykesvtlle, Maryland 

Mr and Mrs N Harrison Buck 
Princeton, New Jersey 

Dr Max M Burger 
Basel, Switzerland 

Dr and Mrs Mario Burgos 
Mendoza, Argentina 

Rick and Nonnie Burnes 
Butler's Hole Fund 
Boston, Massachusetts 

Mr and Mrs Malcolm Campbell 
Upper Montcla/r, New Jersey 
Vineyard Haven, Massachusetts 

Dr Graciela C Candelas 
San Juan, Puerto Rico 



Mr and Mrs Frank C. Carotenuto 
East Fa/mouth, Massachusetts 

Dr and Mrs Richard L Chappell 
New York, New York 

Frank and Julie Child 
Woods Hole. Massachusetts 

Dr Eloise E Clark 
Bowling Green, Ohio 

Mr. and Mrs Hays Clark 
Hobe Sound, Florida 

Mr and Mrs James M Clark 
Palm Beach, Florida 

Arnold and Connie Clark 
Woods Hole, Massachusetts 

Drs Alexander W Clowes and Susan E Detweiler 
Seattle, Washington 

Mrs George H A. Clowes 
Dover, Massachusetts 

Dr Jewel PlummerCobb 
Los Angeles, California 

Drs. Arthur L, and Laura H Colwin 
Key Biscayne, Florida 

Dr and Mrs D Eugene Copeland 
Woods Hole, Massachusetts 

Molly N Cornell 
Falmouth, Massachusetts 

Dr William B Cosgrove 
Pittsboro, North Carolina 

Dr Joseph T Coyle 
Belmont, Massachusetts 

Thomas and Geraldine Crane 
Weston, Massachusetts 

Dr and Mrs Stephen D Crocker 
Bethesda, Maryland 

Mrs Sally Cross 
Falmouth, Massachusetts 

Dr and Mrs Anthony J Cutaia 
Ballwin, Missouri 

Mr and Mrs Bruce G Daniels 
Lincoln, Massachusetts 

Dr Eric H Davidson 
Pasadena, California 



Mr and Mrs David L Douglass 
Woodside, California 

Judith and John Dowhng 
Boston, Massachusetts 

The Jane and Allan Dragone Foundation 
Vero Beach, Florida 

Mr and Mrs George Edmonds 
Osterville, Massachusetts 

Mr and Mrs Hans A Ege 
Mahwah, New Jersey 

Drs Herman N. Eisen and Natalie Aronson 
Waban, Massachusetts 

Mrs Ariana Fairbanks 
Honolulu, Hawaii 

Dr. A Verdi Farmanfarmaian 
Princeton, New Jersey 

Ms Linda Sallop and Mr Michael Fenlon 
Newton, Massachusetts 

Mrs James J Ferguson Jr 
Chevy Chase, Maryland 

Mr and Mrs Harold E Foreman, Jr 
Northbroolc, Illinois 

Drs Bruce and Barbara C Furie 
Wellesley, Massachusetts 

Dr and Mrs David C Gadsby 
New York, New Yorlc 

Ms SalheGiffen 
Falmouth, Massachusetts 

Rebeckah DuBois Glazebrook 
Osprey, Florida 

Dr and Mrs Murray Glusman 
Woods Hole, Massachusetts 

Suzanne and Maynard Goldman 
Boston, Massachusetts 

Drs Robert and Anne Goldman 
Chicago, Illinois 

Drs Timothy and Mary Helen Goldsmith 
Northford, Connecticut 

Dr and Mrs Moise H Goldstein, Jr 
Woods Hole, Massachusetts 

Susan and Tom Goux 
Teahclcet, Massachusetts 



Continued 



R74 



Dick and Eleanor Grace 
The Brain Center 

Mew Seabury, Massachusetts 

Philip Grant 
Washington, DC 

Dr Michael J and Mr < :a H Greenberg 

St Augustine, Florida 

Dr Mary J Greer 
New York, New York 

Mr. and Mrs William H Greer, Jr 
Chevy Chase, Maryland 

Dr and Mrs Thomas C Gregg 
Fa/mouth, Massachusetts 

Paul R and Mona Gross 
Jamaica Plain, Massachusetts 

Dr and Mrs Lawrence Grossman 
Baltimore, Maryland 

Dr. and Mrs Harlyn O Halvorson 
Woods Hole, Massachusetts 

Ms Penelope Hare 

West Fa/mouth, Massachusetts 

Dr. and Mrs. Robert Haselkorn 
Chicago, Illinois 

Woody and Hanna Hastings 
Cambridge, Massachusetts 

Dr Robert R Haubnch 
Granville, Ohio 

Synnova Hayes 
Princeton, West Virginia 

Dr Diane E Heck 
Rumson, New Jersey 

Dr. and Mrs Thomas Hedges, Jr 
Moorestown, New Jersey 

Dons and Howard Hiatt 
Cambridge, Massachusetts 

Mr and Mrs David Hibbitt 
Bristol, Rhode Island 

Dr Llewellya Hilhs and Dr Paul Colmvaux 
Woods Hole, Massachusetts 

Mr Timothy T Hilton 
Cambridge, Massachusetts 

Gregory J and Pamela Clapp Hmkle 
Plymouth, Massachusetts 



Dr Gertrude W Hinsch 
Thonotosassa, Florida 

John and Ohvann Hobbie 
Fa/mouth, Massachusetts 

Drs Joseph F Hoffman and Elena Gtkowitz 

New Haven, Connect/cut 

Dr. Francis C G Hoskin and 

Elizabeth M Farnham 
Canton, Massachusetts 

Mrs. Carmela J Huettner 
Woods Hole, Massachusetts 

Mr and Mrs Charles Hunter 
Kansas City, Missouri 

Dr. and Mrs Hugh E Huxley 
Concord, Massachusetts 

Dr and Mrs Shmya Inoue 
Fa/mouth, Massachusetts 

Mary D Janney 
Washington, DC 

Diane and Robert Jaye 
Newton, Massachusetts 

Dr William R Jeffery 
Silver Spring, Maryland 

Ms Barbara W Jones 
Fa/mouth, Massachusetts 

Freda and Benjamin Kaminer 
Woods Hole, Massachusetts 

Drs Darcy B. Kelley and Richard S Bockman 
Sag Harbor, New York 

Anastasia and Tom Kelly 
River Vale, New Jersey 

Dr and Mrs Alexander Keynan 
Jerusalem, Israel 

Mr and Mrs A Sidney Knowles, Jr 
Raleigh, North Carolina 

Sir Hans and Lady Kornberg 
Fa/mouth, Massachusetts 

Ms Ellyn V Korzun 
Chatham, New Jersey 

Dr William J Kuhns 
Toronto, Ontario 



Mr and Mrs Robert P Lambrecht 
Boca Grande, Florida 

Mr and Mrs Rudy Landry 
Pocasset, Massachusetts 

Homer W and Mary Cronan Lane 
North Fa/mouth, Massachusetts 

Mrs Nancy Norman Lassalle 
New York, New York 

Dr Hans Laufer 
Storrs, Connecticut 

Mr Joel A Leavitt 
Boston, Massachusetts 

Mrs J K Lilly, III 

West Fa/mouth, Massachusetts 

Dr and Mrs Anthony Liuzzi 
Boston, Massachusetts 

Laszlo and Joyce Lorand 
Glencoe, Illinois 

Dr and Mrs Robert E Mamer 
Way/and, Massachusetts 

Walter and Shirley Massey 
Atlanta, Georgia 

Mr and Mrs Robert Mastroianni 
Fa/mouth, Massachusetts 

Luigi Mastroianni, M D. and 

Elaine Pierson-Mastroianni, M D , Ph D 
Haverfbrd, Pennsylvania 

Mr. Andrew Mattox and Ms Sandee Parmelee 
Woods Hole, Massachusetts 

Dr and Mrs Robert T McCluskey 
Brooklme, Massachusetts 

Mr and Mrs Michael Meehan 
Wi//iamstown, Massachusetts 

Dr and Mrs Jerry M Melillo 
Falmouth, Massachusetts 

Mr Richard P Mellon 
LaugMmtown, Pennsy/vanja 

Dr and Mrs Roger D Milkman 
Fa/mouth, Massachusetts 

Mr Gernsh H. Milliken 
New York, New York 



R75 



Ralph and Muriel Mitchell 
Cambridge, Massachusetts 

Dr and Mrs Merle Mizell 
New Orleans, Louisiana 

Dr Leyla deToledo-Morrell 
Chicago, Illinois 

Ms Marcia Morris 
Boston, Massachusetts 

Professor and Mrs Toshio Narahashi 
Chicago, ///<nois 

Mr and Mrs Frank L Nickerson 
Fa/mouth. Massachusetts 




Andrew Hawkins 



Ronald P and Karen E O'Hanley 
Ipswich, Massachusetts 

David and Anastasia Palmer 
Waquoif. Massachusetts 

Dr and Mrs George D Pappas 
Chicago, Illinois 

Drs Thoru and Judith Pederson 
Worcester, Massachusetts 

Mr and Mrs Joseph Pellegnno 
Boston, Massachusetts 

Mrs Nancy Pendleton 
Fa/mouth, Massachusetts 

Bertha and Philip Person 
Flushing. New York 

Mr and Mrs Frederick H Pierce 
Amherst, New Hampshire 

Mr and Mrs Robert W Pierce. Jr 
Boston, Massachusetts 



Tom and Patty Pollard 
Branrbrd, Connecticut 

Drs Frank and Billie Press 
Washington, DC 

Mr and Mrs John S Price 
Bryn Mawr, Pennsylvania 

Charlotte and Irving W Rabb 
Cambridge, Massachusetts 

Lionel and Mildred Rebhun 
Char/o tteswl/e, Virginia 

Mr and Mrs Peter W Renaghan 
North Fa/mouth, Massachusetts 

Mr and Mrs Richard Rhoads 
Boston, Massachusetts 

Dr Monica Riley 
Fa/mouth, Massachusetts 

Dr and Mrs Harris Ripps 
Chicago, Illinois 

Dr and Mrs Jack Rosenbluth 
New Rochelle. New York 

Allan and Clare Rosenfield 
Hartsda/e, New York 

Mr and Mrs Edward S Rowland 
Harm/ton, Massachusetts 

Drs Joan and Gerald Ruderman 
Wellesley, Massachusetts 

Mr Andrew Sabm 

East Hampton, New York 

Dr and Mrs Edward D Salmon 
Chapel Hill. North Caro//na 

Dr. and Mrs Hidemi Sato 
Toba M/e, Japan 

Dr and Mrs John W Saunders, Jr 
Waquo/f, Massachusetts 

Mr Morton T Saunders 
G/adwyne, Pennsylvania 

Dr Cecily Cannan Selby 
New York, New York 

Dr and Mrs Douglas R Shanklm 
Memphis, Tennessee 

Marilyn and David Sheprow 
Woods Hole, Massachusetts 

Mrs Jacqueline N Simpkins 
Barnstab'e, Massachusetts 



Drs Dorothy M Skinner and John S Cook 
Falmoufh, Massachusetts 

Drs Melvm and Evelyn Spiegel 
Hanover, New Hampshire 

Mrs Connnc Steel 
Little Neck, New York 

Dr and Mrs Malcolm S Steinberg 
Princeton, New Jersey 

Mr and Mrs James M Stewart 
Nanrucket. Massachusetts 

Carol and Joseph T Stewart, Jr 
Skillman. New Jersey 

Mr Richard H Stowc 
New York, New York 

Drs Alfred and Dorothy Suacher 
Ros'yn, New York 

Gerard and Mary Swope 
Washrngton, DC 

John and Malory Swope 
Concord, New Hampshire 

Andrew G and Ursula Szent-Gyorgyi 
Wa'tham, Massachusetts 

Mr and Mrs Samuel Thome 

Manchester, Massachusetts 

Mr and Mrs Tom Tierney 
Wellesley, Massachusetts 

Mr D Thomas Trigg 
Westwood, Massachusetts 

Elaine and Walter Troll 
New York. New York 

Mr and Mrs Edward Tsoi 
Arlington, Massachusetts 

Drs Walter Vincent and Dore Butler 
Woods Ho'c. Massachusetts 

Dr and Mrs Byron H Waksman 
New York, New York 

Leonard and Eve Warren 
Bato Cynwyd, Pennsylvania 

Mr and Mrs Henry Wheeler 
Boston. Massachusetts 

Ms Rosalind C Wh.tehead 
New York, New York 

Mrs Annette L Williamson 
Fort Worth, Texas 

Dr Matthew Wmkler 
Austin, Texas 



R76 



ANNUAL FUND 2002 




Gifts to the Annual Fund are unrestricted and used immediately where they will best benefit 
the current activities of the MBL research community. 



I Corporate and Foundation Donors 

Aetna Inc 

Amengroup Foundation 

Bay Foundation 

George Botelho, Inc 

The Commonwealth Fund 

Cox Foundation, Inc 

ExxonMobil Foundation, Inc 

Gilbane Building Company 

The Gillette Company 

Doreen Grace Fund 

Harken Foundation 

Johnson & Johnson 

The Henry J Kaiser Family Foundation 

The Gruss Lipper Foundation 

McDonald's Corporation 

National Grid 

Oracle Corporation 

The David and Lucile Packard Foundation 

Josephine Bay Paul and C Michael Paul 

Foundation 

Ralph F Peo Foundation, Inc 
Saint-Gobain Corporation Foundation 
The Schooner Foundation 
Esther Simon Charitable Trust 



The 7888 Club 
$500 - $999 

Drs Ted Begenisich and Sherrill Spires 

Mr and Mrs Robert O Bigelow 

Drs Colleen Cavanaugh and Philip Gschwend 

Mr and Mrs Gorham L Cross 

Drs Robert and Ellen DeGroof 

Dr Philip Dunham and Ms Gudrun Bjarnarson 

Mrs Ruth E Fye 

Dr Joseph Gall and Ms Diane Dwyer 

Mr and Mrs Charles M Ganson, Jr 

Mr and Mrs William Gilbane, Jr 

Mr and Mrs John G McMillian 

Mr Andrew E Norman 

Mr and Mrs Jonathan O'Herron 

Dr Michael Rabinowitz and Ms Diane Hoxmeier 

Dr Edward B. Rastetter 

Dr and Mrs Frederick R, Rickles 

Mr Michael C Ruettgers 

Prof, and Mrs Howard K Schachman 

Mr. and Mrs Hans L Schlesmger 

Dr and Mrs William Trager 

Mr and Mrs John J Valois 

Dr Dyann an M- Peter Wirth 



Fertilized sea urchin egg, Hakhyun Ka 



The Sponsors Club 
$250 - $499 

Dr and Mrs Daniel L Alkon 

Drs James and Helene Anderson 

Mr and Mrs Michael Angelini 

Dr and Mrs George P. Baker, Jr 

Dr Andrew Bass and Ms Margaret Marchaterre 

Dr Martha B Baylor 

Dr Miriam F Bennett 

Mr and Mrs Thomas C Bolton 

Drs Harry Conner and Carol Scott-Conner 

Mr John Cromie and Dr Gloria Gallo-Cromie 

Dr and Mrs. Paul J De Weer 

Drs Lmda Deegan and Christopher Neill 

Prof and Mrs Frederick A Dodge 

Mr and Mrs David Fausch 

Dr Rachel D Fink 

Dr and Mrs Harold Gainer 

Mrs Mary L Goldman 

Dr Stephen L Hajduk 

Dr and Mrs John P Harrington 

Mr William A Raskins 

Mr and Mrs Gary G Hayward 

Mrs Elizabeth Heald 

Dr Yukio Hiramoto 

Mr and Mrs Lon Hocker 

Dr and Mrs Daniel Johnston 

Dr and Mrs Morns John Karnovsky 

Mr and Mrs Gilbert King 

Dr and Mrs Edward A Kravitz 

Dr and Mrs Alan M Kuzman 

Attorney and Mrs William K Mackey 

Ms Lynn Snyder Mackler 

Drs Richard and Hermme Makman 

Dr and Mrs Julian B Marsh 

Ms Jane A McLaughlm 

Dr DeForest Mellon, Jr 

Drs Timothy Mitchison and Christine Field 

Dr Peter A N.ckerson 

Dr and Mrs Santo V Nicosia 

Mr and Mrs William J Pechilis 

Dr Khela Ransier 

Rev Michael Robertson and 

Dr Emmy Robertson 
Mr and Mrs Phillip S Robertson 
Mr Kenneth Rosenthal 
Dr Cynthia V Stauffacher 
Dr Dusan Stefoski 
Dr Andreas C Stemmer 
Drs Maurice and Raquel Sussman 
Mr and Mrs Stephen E Taylor 
Drs William and Mary Telfer 
Mr and Mrs Richard Verney 
Mr and Mrs. Lynn H Wilke 
Mr and Mrs Leslie J Wilson 
Dr Charles Yanofsky 
Mr John F Zietlow, Jr 
Dr Michael J Zigmond 
Mr and Mrs Hans Zimmer 



The Century Club 
$1 00- $249 

Mr Thomas H Aal 

Dr and Mrs Donald A Abt 

Dr Nina Stromgren Allen 

Mr and Mrs David S Ament 

Mr and Mrs Duncan P Aspmwall 

Mr and Mrs Richard Asthalter 

Mrs Kimball C Atwood, III 

Mr Nathaniel Atwood and Ms Susan Parkes 

Mr and Mrs Donald R Aukamp 

Dr. and Mrs David S Babm 

Mr and Mrs John M Baitsell 

Drs Barbara-Anne Battelle and James Alligood 

Dr and Mrs Thomas L Benjamin 

Mr and Mrs Maks Birnbach 

Dr, and Mrs. Thomas P Bleck 

Dr and Mrs David A Bodznick 

George Botelho, Inc 

Drs Barbara and John Boyer 

Mr and Mrs Peter Boyer 

Mrs Eleanor D Bronson 

Ms Sarah Dixwell Brown 

Mr and Mrs Darryl A Buckingham 

Bufftree Building Co , Inc 

Drs William and Madeline Burbanck 

Mrs Barbara Gates Burv/ell 

Mr and Mrs William O Burwell 

Mr and Mrs Bruce E Buxton 

Mr and Mrs John L Callahan, Jr 

Mr and Mrs David J Campbell 

Mr and Mrs D Bret Carlson 

Mr Robert J. Carney 

Father Joseph D Cassidy. O P , Ph D 

Dr and Mrs Estill L Caudill 

Ms Martha Chen 

Mrs Vera S Clark 

Dr Mary Clutter 

Dr Carolyn Cohen 

Dr and Mrs Maynard M Cohen 

Mr and Mrs Alexander Colby 

Mr and Mrs Franz Colloredo-Mansfeld 

Dr. LeBaron C. Colt, Jr 

Mr and Mrs Nathaniel S Coolidge 

Dr and Mrs Sherwm J Cooperstem 

Dr and Mrs John O Corliss 

Dr Helen M Costello 

Mr and Mrs William G Coughlin 

Mr Charles Crane and Ms Wendy Breuer 

Dr Karen Crawford 

Mr and Mrs Richard D Cutler 

Dr and Mrs Giuseppe D'Alessio 

Dr and Mrs Nigel Daw 

Dr Martha Bridge Denckla 

Dr Wolf-Dietrich Dettbarn 

Dr William A Dickson 

Mr and Mrs David L Donovan 

Ms Dorothy L Drummey 



R77 



Annual Fund Volunteers 

Chairman 

Dr Peter B Armstrong 

Associates Chairman 
Mr Michael Fenlon 

Volunteers 

Dr Robert B Barlow. Jr 

Dr A Verdi Farmanfarmaian 

Mr Homer W Lane. Jr 

Dr. Hans Laufer 

Dr Anthony Liuzzi 

Dr Luigi Mastroianni. Jr 

Dr Merle Mizell 

Dr Thoru Pederson 

Dr Robert A Prendergast 

Dr Cecily C Selby 

Dr Byron H Waksman 



Annual Fund, continued 

Dr and Mrs Thomas K Duncan 

Mr and Mrs Hoyt Ecker 

Dr Frank Egloff 

Drs Paul Englund and Christine Schneyer 

Dr and Mrs Herman T Epstein 

Mr Gordon C Estabrooks 

Dr and Mrs Thomas Evans 

Dr Alan Ezekowitz and Ms Debbie Lunder 

Dr Patricia M Failla 

Mr and Mrs Daniel D Federman 

Mr and Mrs Howard G Freeman 

Dr Larry Jay Friedman 

Dr and Mrs John J Funkhouser 

Dr and Mrs Robert M Galatzer-Levy 

Dr and Mrs Frank Gallagher 

Miss Eleanor Garfield 

Ms Margaret A Geist 

Mr and Mrs Vincent Geoffrey 

Dr and Mrs Martin Gibbs 

Mrs Janet F. Gillette 

Drs Alfred and Joan Goldberg 

Dr. and Mrs Herbert Graham 

Dr and Mrs Philip Green 

Mr Noah Greenberg 

Dr Roger L Greif 

Dr and Mrs Newton H Gresser 

Dr June Harngan and Mr Arnold Lum 

Dr Glenn W Harnngton 

Dr and Mrs Neils Haugaard 

Mr and Mrs Edmund Hazzard 

Dr and Mrs Peter K Hepler 

Dr and Mrs Theodore T Herskovits 

Mr and Mrs William Hobart 

Dr Edward G Horn 

Dr and Mrs James C Houk 

Prof Ruth Hubbard 

Dr and Mrs W Bruce Hunter 

Dr and Mrs Kurt J Isselbacher 

Dr Marietta Radovic Issidorides 

Dr Launnda A Jaffe 

Mr and Mrs Ernest G Jaworski 

Mrs Sally S Joslm 

Drs Jane Kaltenbach and Robert Townsend 

Mrs Sally Karush 

Mr and Mrs H Ernst Keller 

Mr Louis M Kerr 

Mr and Mrs Arthur King 




Dr David Klein 

Mr Mark Koide 

Dr Kiyoshi Kusano 

Drs Eugene and Mitsuko Laforet 

Ms. Judith E Laster 

Mr William Lawrence and Mrs Barbara Buchanan 

Dr and Mrs John J Lee 

Ms Alice C Leech 

Dr Marian E LeFevre 

Dr David P Lenzi 

Dr and Mrs Aaron B Lerner 

Dr and Mrs Jack Levin 

Dr. and Mrs Richard B Levine 

Mr and Mrs David W Lillie 

Dr and Mrs Daniel M Lilly 

Dr. and Mrs Richard W. Linck 

Dr Raymond J Lipicky 

Dr Stephen J Lipson 

Dr. and Mrs. John E Lisman 

Dr. Robert B Loftfield 

Dr and Mrs. Irving M London 

Dr Louise M Luckenbill 

Dr Richard Lum 

Mr Gerald Lynch 

Drs Richard and Sylvia Manalis 

Mr and Mrs Ronald Marcks 

Dr Andrew C Mannucci 

Mr and Mrs Lowell V Martin 

Dr and Mrs William M McDermott 

Mr Paul McGonigle 

Dr and Mrs David McGrath 

Mr and Mrs James McSherry 

Dr Martin Mendelson 

Dr Melanie and Mr Klein Merriman 

Dr Nancy S Milburn 

Dr and Mrs Rtcardo Miledi 

Drs David and Virginia Miller 

Mr. and Mrs Robert W Morey. Jr 

Mr and Mrs Samuel Murphy 

Dr. and Mrs X J Musacchia 

Dr. and Mrs John E Naugle 

Messrs William and David Newton 



Elizabeth Armstrong 



Mr and Mrs Brian Nickerson 

Mr. and Mrs Michael O'Rand 

Dr and Mrs Rudolf Oldenbourg 

Dr Janice and Mr Albert Olszowka 

Mr James G Ottos 

Dr and Mrs Ray D Owen 

Mr Malcolm A Park 

Ms Marina H Park 

Dr and Mrs Rodenc B Park 

Dr and Mrs John B Pearce 

Mr and Mrs David Pearson 

Dr and Mrs Courtland D Perkins 

Dr Bruce J Peterson 

Drs David and Jeanette Pleasure 

Dr Jeanne S Pomdexter 

Dr and Mrs Harvey B Pollard 

Drs Mary Porter and Tom Hays 

Dr Dale Purves and Ms Shannon Ravenel 

Mr and Mrs George Putnam, III 

Ms Kathenne E Putnam 

Dr and Mrs James P Quigley 

Dr and Mrs Robert F Rakowski 

Dr Carol Reinisch and Mr Robert Suitor 

Dr Judith Rhodes 

Drs Paul Rmgel and Michele Lorand 

Mr and Mrs John Ripple 

Dr Hope Rmer 

Mr Dana Rodin 

Dr and Mrs Lawrence C Rome 

Dr Joel L Rosenbaum 

Drs William Ross and Nechama Lasser-Ross 

Dr Uldis Roze 

Dr William Devme Russell-Hunter 

Mrs Anne W Sawyer 

Mrs Lilian M Scherp 

Dr Herbert Schuel 

Dr and Mrs Robert Seidler 

Ms Barbara Park Shapiro 

Mr and Mrs John J Sheridan 

Drs Roy Silverstem and Jacquelyn 

Joseph-Silverstein 
Dr Maxine F Singer 

Continued 



R78 



Mr and Mrs Alain Singer 

Mrs Cynthia C Smith 

Dr and Mrs. Paul F Smith 

Drs Roxanna and Ronald L ; >icwitz 

Mr David Space ar ' in/is 

Dr and Mrs Alan 

Dr. and Mrs W I 1 '. phenson 

Mr and Mrs ! " : ewart 

Dr Elijah Ste~ Ms Jasmin Bihler 

Drs. Alber , u rd and Margaret Maurin 

i.^utsuyuki Sugimori 

Mr and Mrs. E. Kent Swift, Jr 

Dr Margaret W Taft 

Drs. Bruce Telzer and Leah T Haimo 

Mr. and Mrs W Nicholas Thorndike 

John F Towle, DOS 

Mr and Mrs William Traver 

Dr and Mrs David M. Travis 

Dr and Mrs Steven N Treistman 

Miss Ruth Tucker 

Dr. and Mrs. Kenyon S Tweedell 

Mr and Mrs Volker Ulbrich 

Dr and Mrs. Ivan Valiela 

Drs. Claude and Dorothy Villee 

Mr and Mrs Samuel Vincent 

Dr Talbot H Waterman 

Dr Annemarie Weber 

Dr and Mrs George Weiffenbach 

Dr Gary Wessel 

Mrs RoseT Wheeler 

Dr and Mrs Martin Keister White 

Dr and Mrs Roland L Wigley 

Drs Jonathan and Beatrice Wittenberg 

Drs Joshua Zimmerberg and Teresa Jones 



| Other 

Mr and Mrs David C Ahearn 

Mr and Mrs Scott M Allard 

Mr and Mrs John J Aziz 

Dr and Mrs Richard H Backus 

Mr and Mrs Joe Barnngton 

Ms Jane Berger and Mr Roger Gittmes 

Ms Pauline F Blanchard 

Ms Casey Bliss 

Mr and Mrs Anthony Briana 

Mrs Jennie P Brown 

Mr and Mrs Thomas A Brown 

Dr Roberto Bruzzone 

Mr and Mrs Arnold H Burrough 

Mr and Mrs E Brewster Buxton 

Mr. and Mrs George Cadwalader 

Mr Arthur D Calfee 

Dr Peter Clark and Ms Ellen Barol 

Mr and Mrs James M deary 

Mrs Elizabeth Ann Cohen 

Mr and Mrs Peter Connolly 

Mr and Mrs J Sterling Crandall 

Mrs Marcia Donovan 

Dr Patricia L Dudley 

Dr Quan-Yang Duh and Ms Ann Comer 

Mrs Frances E Eastman 

Dr Hugh Young Elder 

Mrs Helen M Erickson 

Dr Aian Scott Fanning 

Mrs Ruth Alice Fitz 

Mr John H. Ford 



Mr and Mrs Alvin Fossner 

Dr Krystyna Frenkel 

Drs Patricia Garrett and Oliver Woshmsky 

Dr Stephen E Gellis 

Mr. and Mrs James E Gifford 

Mrs Barbara B Glade 

Dr Joel S Gordon 

Dr Martin Gorovsky 

Mrs Enka A Green 

Dr Nancy Carole Greep 

Mr and Mrs Joseph Guttenplan 

Mr and Mrs Peter A Hall 

Mr and Mrs Robert Hall 

Drs Clifford and Drusilla Harding 

Dr, and Mrs Richard Bennet Harvey 

Dr Audrey E V Haschemeyer 

Ms Elizabeth E Hathaway 

Mr John Hay 

Dr Teru Hayashi 

Dr David S. Hays 

Mrs Jane M Heald 

Dr Simone Helluy 

Drs Richard and Susan Hill 

Dr. and Mrs Robert B Hill 

Mr and Mrs Gerald J Holtz 

Dr Linda A Hufnagel-Zackroff 

Mr and Mrs Felix Inigo 

Mr and Mrs Charles A Johnson 

Dr and Mrs James E Johnson 

Dr Harry S Kahn 

Dr Edna S Kaneshiro 

Mr. and Mrs Alan K Karplus 

Prof Mark D Kirk 

Dr Peter Kivy and Ms Joan Pearlman 

Mr and Mrs Paul W Knaplund 

Mrs Phyllis Kuffler 

Mr and Mrs Ted Kulesza 

Mr and Mrs Ray La Ranger 

Mr and Mrs Steven V Launa 

Ms Corinne Le Bovit 

Dr and Mrs Stephen B Leighton 

Mr and Mrs Edwin M Libbm 

Dr. and Mrs John H. Lochhead 

Mr and Mrs Edward Loessel 

Dr Irene Loewenfeld 

Dr. and Mrs Frank J Longo 

Ms Lena T Lord 

Mr Fred E Lux 

Mrs Priscilla M, Makay 

Dr Robert P Malchow 

Dr Dawn Morin Manck 

Mr and Mrs Joseph S Maranchie 

Dr and Mrs Joe L Martinez, Jr 

Mr, and Mrs Frank J Mather, III 

Dr Rita W Mathews 

Mrs Dorothea J Mautner 

Mrs Polly Miles 

Dr and Mrs Daniel G Miller 

Cdr and Mrs Lloyd C Morris U5N (RET) 

Mr and Mrs Dana S. Morse 

Mr Thomas A Mulholland 



RADM Paul J Mulloy USN (Ret) 

Ms Ins Nelson 

Ms Kathryn Paine 

Mr and Mrs Nicholas Pantazis 

Mr David Parker, Jr 

Dr and Mrs Charles Parmenter 

Dr Leonard M Passano 

Ms Joan Pearlman and Dr Peter Kivy 

Ms Joyce S Pendery 

Dr and Mrs Murray E Pendleton 

Dr and Mrs Ronald J Pfohl 

Mr and Mrs Harold Pilskaln 

Mr and Mrs Kenneth Poehls 

Dr and Mrs Richard A Polin 

Dr. and Mrs Daniel A. Pollen 

Mr and Mrs Abraham I Pressman 

Mr and Mrs Gerald B Reynolds 

Dr Robert V Rice 

Dr Morris Rockstem 

Mr and Mrs Peter J Romano 

Mr Dorothy C Ryder 

Mr and Mrs Herbert Shanker 

Dr and Mrs Gaius R Shaver 

Ms Kathleen Lake Shaw 

Mr Richard W Shrmer 

Dr Jeffrey D Silberman 

Mrs. Louise M Specht 

Rev and Mrs William A Spurrier, III 

Mrs Eleanor Steinbach 

Dr Raymond G Stross 

Mrs. Belle K Taylor 

Dr Leana and Mr Joby Topper 

Prof and Mrs Michael Tytell 

Mrs Alice H van Buren 

Mr James Ware and Ms Sharon McCarthy 

Mr Michael S Weinstein 

Dr and Mrs Charles R Wyttenbach 

Mrs Marilyn J Young 

Mr and Mrs Kenneth H Zimble 

Mrs Margery P Zinn 

Dr and Mrs Steven J Zottoli 



R79 



ALUMNI FUND 2002 




Oihs to the Alumni Fund are used to meet the basic needs of 
MBL courses, including tuition support for students. 



Anonymous 

Dr Joan Abbott 

Prof and Mrs Laurence F Abbott 

Dr William Agnew 

Drs Dianne and Thomas Allen 

Dr C Ronald Anderson 

Dr William DeWitt Andrus, Jr 

Drs Robert and Lynne Angerer 

Dr Carolina V Arancibia 

Drs Carol Arnosti and Andreas Teste 

Dr Michael 5 Ascher 

Dr and Mrs Robert Barker. Jr 

Dr Edward Joseph Behrman 

Dr William H Bergstrom 

Dr. Gerald Bergtrom 

Dr Ari Berkowitz 

Dr Emmanuel C Besa 

Mr Edward M Blumenthal 

Dr Mark Boothby 

Dr Richard T Born 

Dr. Elayne Bornslaeger-Bednar and 

Mr Michael Bednar 
Dr Emil Borysko 
Dr Peter Brodfuehrer 
Dr and Mrs Donald D Brown 
Dr. Harley P Brown 
Dr Richard R Burgess 

Mr and Mrs Patrick J Calie 

Dr David Campbell 

Dr James R Campi 

T. Joiner Cartwnght. Jr, Ph D 

Dr Clarissa M. Cheney 

Dr Jonathan Chernoff 

Dr Chi-Bin Chien 

Dr Carson C Chow 

Dr Ka Hou Chu 

Dr Carl Cyrus Clark 

Dr. William T Clusin 

Dr and Mrs R John Collier 

Dr and Mrs Marc D Coltrera 

Dr Clark E. Corliss 

Dr Jeffrey T Corwin 

Mr John Cromie and Dr Gloria Gallo-Cromie 

Dr Alice M Curry 

Dr. Stephen C Dahl 

Dr and Mrs Harry W Dickerson 

Dr William J Dickinson 

Dr Bruce A Diner 

Mr. Jonathan S and Dr Thelma Dixon 

Mr and Mrs Richard Drucker 

Dr Catherine Asleson Dundon 

Dr Kathleen Dunlap 

Dr and Mrs David Durica 



Ms Joan Edstrom 

Ms Marcia Edwards 

Dr. Manlynn E Etzler 

Dr and Mrs Arnold G Eversole 

Prof. Donald Faber 

Dr. and Mrs Richard R Fay 

Dr. Marta Feldmesser 

Dr Cyril V Fmnegan, Jr 

Dr Thomas R Flanagan 

Dr. Karl W Flessa 

Ms Christine M Foreman 

Dr. Elizabeth Fowler and Dr James Parmentier 

Dr Kenneth I Freedman 

Drs Hugo and Anita Freudenthal 

Dr, Marvin J Fritzler 

Dr Anne E Fry 

Dr. Theresa Gaasterland 

Dr Paul E. Gallant 

Dr. Helen W Gjessing 

Prof James A Glazier 

Dr Joel S Gordon 

Dr. Joan Eiger Gottlieb 

Dr Esther M Goudsmit 

Dr and Mrs Eugene Grebner 

Dr Lewis J. Greene 

Dr Warren M Grill 

Dr Jerome Gross 

Dr Mamie E Halpern 

Dr Lisa M Halvorson 

Dr Cadet Hammond Hand, Jr 

Dr. Susan M Harding 

Dr. Robert D Harvey 

Dr. Norman B Hecht 

Dr. Joseph Heitman 

Drs. Joseph and Barbara Hichar 

Dr Raymond W Holton 

Dr and Mrs Seymour Holtzman 

Mr Timothy E Holy 

Dr Xudong Huang 

Drs Deborah Hursh and Mark Mortin 

Dr Jerard Hurwitz 

Dr Richard Intres 
Dr Allen Isaacson 
Dr and Mrs Stephen K Itaya 

Dr. Jon W Jacklet 

Dr Nancy A Johnson 

Dr Leslie and Mr James Jolly 



Continued 



ALUMNI RELATIONS 
ADVISORY BOARD 



Dr. Robert B Barlow, Jr. (Physiology. 1963) 
SUNY Upstate Medical University 

Dr. Thomas L. Benjamin (Physiology. 1959/ 
Neurophysiology. 1966). Harvard Medical School 

Dr. Richard T. Born (Neural Systems & Behavior, 1987) 
Harvard Medical School 

ephen C. Cannon (Neurobiology, 1988) 
sity of Texas Southwestern Medical Center 

Dr. Eloise E. Clark (Physiology, 1956) 
Bow/ing Green State University 

Dr. Jeffrey T. Corwin (Neurobiology, 1975) 
University of Virginia, School of Medicine 

Dr. Joseph T. Coyle (Neurobiology, 2001) 
Harvard Medical School 

Dr. Joseph R. Fetcho (Neural Systems & Behavior, 
1984), State University of New York 

Dr. Elizabeth Fowler (Physiology, 1 966) 
Millennium Pharmaceutica/s 

Dr. Leah T. Haimo (Physiology, 1974), 
University of California, Riverside 

Dr. Marnie E. Halpern (Neurobiology. 1986) 
Carnegie Institution of Washington 

Dr. Alexander Keynan (Physiology, 1961) 
Israel Academy of Sciences and Humanities 

Dr. Daniel P. Kiehart (Physiology, 1975) 
Dufce University Medical Center 

Dr. George M, Langford (Physiology, 1972), 
Dartmouth College 

Dr William M. McDermott (Invertebrate Zoology. 
1950), U.S. Navy (retired) 

Dr. Melanie Pratt Merriman (Embryology, 1975/ 
Physiology, 1976), Touchstone Consulting 

Dr. Thomas D. Pollard (Physiology, Director 1989- 
1 993), Yale University 

Dr Joshua R. Sanes (Neurobiology, 1971) 
Washington University Medical Center 

Dr. Wise Young (Neurobiology, 1972) 
Rutgers University 



Ex-Officio 

Dr Peter B. Armstrong (Invertebrate Zoology, 1961) 

Chairman, MBL Annua!/Alumni Fund 

University of California, Davis 

Dr. John E. Dowling (Neurobiology, 1970) 

President. MBL Corporation 

Harvard University 



R80 




Students get 
dockside instruction 
from Marine 
Resources 
Center staff, 
Elizabeth Armstrong 



Dr. and Mrs James W Kalat 

Or Mananna M Kane 

Dr Alvm M. Kaye 

Or Gordon I Kaye 

Drs Thomas and Laura Keller 

Dr and Mrs R Emmett Kenney 

Dr Richard G Kessel 

Dr Lynda A Kiefer 

Dr Elizabeth L Kimberly 

Dr Donald W King 

Dr Leonard B- Kirschner 

Dr and Mrs Paul M Knopf 

Dr Robert E Knowlton 

Dr Carol A Koenigsberger 

Dr WieslawJ Kozek 

Dr Ajit Kumar 

Dr Michael J Landzberg 

Dr Holly A Lavoie 

Dr Matthew K Lee 

Dr William J Lehman 

Dr Ellen K. LeMosy 

Dr Ethan Lerner 

Drs Brian Link and Vivian Lee 

Ms Anne M Linton 

Drs Joan and James Lisak 

Mr and Mrs Edward K Lobenhofer 

Dr and Mrs Robert J Loeffler 

Dr Ann M Lohof 

Dr Jane Lubchenco 

Dr Stephen E Malawista 

Dr David E Mann, Jr 

Dr and Mrs. Phillip 6 Maples 

Dr Junko Munakata Marr 

Dr Michael Massanan 

Dr. Jon M McCormick 

Dr Duane P. McPherson 

Dr Anne Messer 

Dr Laurie Miller 

Dr Maria I Morasso 

Dr Yasuhiro Morita 

Mr Stephen h Munroe 

Dr David P Nagle, Jr 

Drs Angus Nairn and Marina Picciotto 

Dr Lee Niswander 



Dr Dana T Nojima 

Mrs Phyllis Norris 

Drs Victor and Ruth Nussenzweig 

Dr Michael D. Oberdorfer 

Dr Catherine L Olsen 

Dr Ingnth Deyrup Olsen 

Prof. John M. Olson 

Dr and Mrs Brett A Oxberry 

Mrs Mary G. Pacifici 

Drs Kimberly Paul and Charles Thomas 

Mr Matthew Person and Ms Jill Enckson 

Dr Norman J Pieniazek 

Dr Louis Pierro 

Dr Carl B PNcher 

Dr and Mrs Anthony Pires 

Dr Sabrina and Mr Bradford Powell 

Dr Esther L Racoosm 

Dr Jennifer L Raymond 

Dr Jean and Mr Ronald Regal 

Dr and Mrs Robert Alan Resnik 

Dr Kristin Lester Revill 

Dr. Randall W Reyer 

Ms Marian C Rice 

Dr Austen F Riggs 

Dr Regma M Robbins 

Drs Duncan and Grace Saunders Rollason 

Brian K Romias, MD, MS 

Dr James T Russell 

Ms Carol Ann Ryder 

Dr. David R Samols 
Dr Noriyuki Satoh 
Dr Richard L Saunders 
Dr Charles H Sawyer 
Dr RolfSchauder 
Dr. Paul R. Schloerb 
Dr James H Schwartz 
Dr Carla Shatz 
Dr and Mrs Alan Sher 
Dr David R Sherwood 
Dr Joyce M Simpson 
Dr Max Snodderly 
Dr Sandra C Souza 
Mr Nelson Spruston 



Dr Joel P Stafstrom 
Dr Gary R Strichartz 
Dr David T Sullivan 
Dr Frank J Swartz 
Ms Hyla C Sweet 
Dr. Ben G Szaro 

Ms Penny A Tavormma 

Mrs Vera A Taylor 

Dr Saul Teichberg 

Dr Wesley J Thompson 

Dr Barbara Holland Toomey 

Dr Jeanine A Ursitti 

Dr Raghunath Virkar 
Ms Melissa A Vollrath 
Dr Susan Volman 

Dr William G Wadsworth 

Dr Brant G Wang 

Dr Deborah Williams Ward 

Drs Gary E Ward and Zail Berry 

Mr and Mrs Herman Ward 

Dr and Mrs Samuel Ward 

Dr Susanna H Weerth 

Ms Kay E Wellik 

Dr Harold Bancroft White 

Dr Clayton Wiley 

Dr Ulrike Wille 

Dr Kathenne Wilson 

Dr Andrea G Witlin 

Dr Anne M Wood 

Drs Lawrence Wysocki and Judith Spiegel 

Dr Ayako Yamaguchi 
Dr Naoyuki Yamamoto 
Ms Judith L Yanowitz 
Dr. Karen P York 
Dr Lin Yue 

Dr and Mrs Cheng Zhu 
Dr Richard E Zigmond 



R81 



FELLOWSHIPS AND SCHOLARSHIPS 




Endowed and expendable funds for scholarships and fellowships are an integral part of the MBL's successful research 
and education programs. In 2002, twenty-two scientists from the U.S. and abroad were awarded fellowship grants that 
allowed them to work in our uniquely collaborative research environment. The Laboratory also awarded scholarships to 
97 highly qualified students, enabling them to participate in our total-immersion courses. 

We gratefully acknowledge the donors listed below, who provided $245,270 for research fellowships and $726,558 for 
scholarships in 2002. 



American Society of Cell Biology 
Summer Research Awards 
American Society of Cell Biology 

I Robert Day Allen Fellowship Fund 
Drs. Joseph and Jean Sanger 

I MBL Associates Endowed Scholarship Fund 
MBL Associates 
Mrs. Nawne Meigs-Brown 

I Frederik B. Bang Fellowship Fund 
Dr and Mrs Jack Levin 

I Charles R. Crane Fellowship Fund 
Friendship Fund 

I John O. Crane Fellowship Fund 
Friendship Fund 

I Jean and Katsuma Dan Fellowship Fund 
Mr William A Haskms 
Mrs Eleanor Steinbach 
Drs Joseph and Jean Sanger 

| Bernard Davis Fellowship Fund 
Mrs Elizabeth M Davis 

I Daniel S. and Edith T. Grosch Scholarship Fund 
Ms Laura Grosch and Mr Herb Jackson 



Mouse oocyte, Umit Ali Kayisli 



I Aline D. Gross Scholarship Fund 
Dr and Mrs Paul R Gross 
Dr and Mrs Benjamin Kaminer 
Dr and Mrs Lewis P Rowland 
Technic, Inc 

I E. E- Just Endowed Research Fellowship Fund 
The Cole Memorial Family Fund 
William Townsend Porter Foundation 
Georgia-Pacific Corporation 

I Fred Karush Endowed Library Readership 
Dr and Mrs Laszlo Lorand 
Dr and Mrs Arthur M Silverstein 

I H Keffer Hartline Fellowship Fund 
Dr. Peter Hartline 
Mr and Mrs Frederick F Hartline 
Dr and Mrs Thomas R Hedges, Jr 
Ms Rebecca Kucera 
Dr Earl Weidner 

I Kuffler Fellowship Fund 
Dr and Mrs Edward A Kravitz 

| Frank R. Lillie Fellowship and Scholarship 
Estate of Emily Ann Cramer 

I Mountain Memorial Fund 
Dr and Mrs Dean C Allard. Jr 
Ms Brenda J Bodian 
Mr and Mrs Amos L Roberts 
Mr and Mrs Thomas H Roberts 

Continued 



R82 




Mouse cell tnvas'cn pores 
and membrane, mouse 
b'astosist, Umit Ali Kayislt 



James A and Faith Miller Fellowship Fund 
Drs David and Virginia Miller 

Frank Morrell Endowed Memorial Scholarship 
Dr Leyla de Toledo Morrell 

| Emily Hartshorne Mudd Scholarship Fund 
World Academy of Art and Science 

I Neural Systems and Behavior Scholarship Fund 
Dr and Mrs Alan Gelpenn 
Dr Warren M Gnll 

Drs William Knstan and Kathleen French 
Dr and Mrs Richard B Levine 
Dr Mark W Miller 

Drs. Jams C Weeks and William M Roberts 
Drs Harold Zakon and M Lynne McAnetly 
Ms M Jade Zee 

Nikon Fellowship 
Nikon Instruments Inc 

The Plum Foundation John E. Dowlmg 
Fellowship Fund 
The Plum Foundation 

I William Townsend Porter Scholarship Fund 
' for Minority Students 

William Townsend Porter Foundation 

I Florence C Rose and S. Meryl Rose Endowed 
I Scholarship Fund 

Dr Gwynn Akin Bowers 

Dr and Mrs D Eugene Copeland 

Mrs Edna B Hill 

Ms Kaaren Janssen 

Dr and Mrs Benjamin Kammer 

W K Kellogg Foundation 

Dr Hans Laufer 

Dr and Mrs Anthony Liuzzi 

Dr and Mrs Roger D Milkman 

Mrs Eleanor M, Nace 

Drs Keen A and Nancy S. Rafferty 

Mrs Florence C Rose 

Dr and Mrs John W Saunders. Jr 

Dr and Mrs J P Trrnkaus 

| Milton L Shifman Endowed Scholarship 
Milton L Shifman Scholarship Trust 



I The Catherine Filene Shouse SES Scholarship Fund 
The Catherine Filene Shouse Foundation 

(The Catherine Filene Shouse Scholarship Fund 
The Catherine Filene Shouse Foundation 

| The Catherine Filene Shouse Fellowship Fund 
The Catherine Filene Shouse Foundation 

| The Gruss Upper Fund 
The Gruss Upper Foundation 

i The Bill and Phoebe Speck Fund 
Dr William T Speck 

| The Evelyn and Melvm Spiegel Fellowship Fund 
Dr and Mrs Jack Levin 
The Sprague Foundation 
Drs Joseph and Jean Sanger 

I H. B. Stembach Fellowship Fund 
Mrs Eleanor Stembach 

| Horace W Stunkard Scholarship Fund 
Drs Albert Stunkard and Margaret Maunn 

| Eva Szent-Gyorgyi Scholarship Fund 
Dr and Mrs Benjamin Kammer 
Dr and Mrs Laszlo Lorand 
Drs Joseph and Jean Sanger 
Dr Andrew and Ms Ursula Szent-Gyorgyi 

I Universal Imaging Fellowship Fund 

Universal Imaging Corporation 

| Walter L. Wilson Endowed Scholarship 
Dr Paul N Chervm 
Dr Jean R Wilson 

I Young Scholars/Fellows Program 
Drs James and Helene Anderson 
Drs Harry Conner and Carol Scott-Conner 
Or and Mrs James L German 
Dr and Mrs Anthony LIUZZI 
Drs Luigi and Elaine Mastroianm 
Drs David and Miriam Mauzerall 
Drs Matthew and Jeanne Meselson 
Drs Dorothy Skinner and John Cook 
Dr and Mrs Edward A Spiegel 
Dr Annemarie Weber 
Drs Jonathan and Beatrice Wittenberg 
Mr and Mrs Kenneth H Zimble 



MEMORIAL AND TRIBUTE GIFTS 



The following donors have chosen to support the Marine Biological 
Laboratory as a special way to remember or honor a relative or friend. 



R83 




Bequests 



Estate of Emily Ann Cramer 
Estate of Madelene E Pierce 



|ln Honor of Drs. Clay and Clara Armstrong 
Ms Lynn Snyder Mackler 

| In Memory of Dr. Kimball C. Atwood, III 
Mrs Eleanor Steinbach 

| In Memory of Dr. Frank A. Brown, Jr. 
Mrs Jennie P. Brown 

| In Memory of Dr. Francis D. Carlson 
Mrs Carolyn S. Carlson 

| In Honor of Mr. Richard D. Cutler 
Gilbane Building Company 

|ln Memory of Eugene Floyd DuBois 
Mrs James R Glazebrook 

| In Memory of Dr. and Mrs. James D. Ebert 
Dr and Mrs Charles R Wyttenbach 

I In Memory of Morris Goldman 
Dr and Mrs Harris Ripps 

| In Memory of Elizabeth K. Hartline 
Dr and Mrs Thomas R Hedges, Jr 



| In Memory of John Helfnch 

Drs. Colleen Cavanaugh and Philip Gschwend 

Drs Bruce and Teresa Corliss 

Harken Foundation 

Ms Ken Holland 

Dr Max Holmes 

Drs Knute Nadelhoffer and Barbara Billings 

Ms Andrea Ricca 

Dr and Mrs Gaius R Shaver 

Mr Jeffrey Shelkey and Ms Joanne Willey 

Ms Jane Tucker 

Dr. and Mrs Lawrence J Wangh 

Dr Xiangming Xiao 

| In Memory of Mr. Robert Huettner 
Mrs Carmela J Huertner 
Mr and Mrs Richard A Huettner 
Ms Catherine N Norton 

| In Memory of Dr. Arthur G. Humes 
Dr Patricia L Dudley 

| In Honor of Macy Lawrence 
Dr and Mrs Gerald Weissmann 

| In Memory of Dr. Frank Morrell 
Dr Thomas P. Bleck 

| In Honor of Cooper Neely 
Mr and Mrs Joe Barnngton 

| In Memory of Dr. Lionel I. Rebhun 
Drs Robert and Anne Goldman 

| In Memory of Elizabeth Russell (Tibby) 
Dr and Mrs Ray D Owen 

| In Honor of Marjone Salmon 

Drs David Forkosh and Linda Hirshman 

| In Honor of Mr. Arthur D. Traub 
Ms Natalie Miller 

| In Memory of Dr. George E Wheeler 
Dr Khela Ransier 
Mrs Rose T Wheeler 

| In Memory of Dr Charles G. Wilber 
Mrs Clare M Wilber 

| In Honor of Alfred and Joyce Zeien 
Mr Michael C Ruettgers 



R84 



OTHER GIFTS AND PROGRAMS 



The quality and success of the MBL educational program is maintained 
through the loan of research equipment, reagents, and computers by: 



Equipment Lenders 




Elizabeth Armstrong 



Matching Gifts 



Aetna Inc 

BP Foundation, Inc 

The Commonwealth Fund 

ExxonMobil Foundation, Inc 

Johnson & Johnson 

The Henry J Kaiser Family 

Foundation 

W K Kellogg Foundation 
McDonald's Corporation 
National Grid 
Oracle Corporation 
The David & Lucile Packard 

Foundation 
Saint-Gobain Corporation 

Foundation 



Accelrys Inc 

Adams & List Associates 

Agilent Technologies. Inc. 

Amersham Pharmacia Biotech Inc 

A.M P.I 

Apogent Discoveries/ Robbins 

Scientific 

Applied Precision, Inc 
Aquatic Eco-Systems. Inc 
Aquatic Habitats 
Arcturus Engineering, Inc 
Atto Bioscience 
AutoQua,nt Imaging, Inc 
Axon Instruments, Inc 

Barnstead/Thermolyne 
Beckman Coulter Instruments. Inc 
Becton Dickinson IS 
Biocare Medical 
BD Biosciences 
Biophotonics international 
Bioptechs 
Bio-Rad Confocat 
Bio-Rad Laboratories 
Bnnkmann Instruments 
Brownlee Precision Co 
Burleigh Instruments, Inc 

Cairn Research LTD 
Cambridge Electronic Design 
Cambridge Research 
Instrumentation, Inc 
CBS Scientific Company, Inc 
Chroma Technology Corporation 
Coherent Inc 

COMPAC Computer Corporation 
Compix Inc . Imaging Systems 
Crest Technologies 
CyBio, Inc 

Dagan Corporation 
Dako Corporation 
David Kopf Instruments 
Del Imaging Systems, LLC 
Diagnostic Instruments, Inc 
DNA Star, Inc 
DVC Co Inc 

Eastman Kodak 

Edinburgh Biocomputing Systems 

Eppendorf Scientific Inc. 

FHC 

Fine Science Tools 

Fisher Scientific/Zylux Corp. 

Fluke Corporation 



General Valve Corporation 
Genetronics, Inc / BTX Instrument 
Division Genomic Solutions 
GlaxoSmithKlme 
Grass Instrument Company/ 
Astro-Med, Inc GrassTelefactor 

Hamamatsu Photonic Systems 
Harvard Apparatus, Inc 

Improvision Inc 

Instrutech Corporation 

Intelligent Imaging Innovations, Inc 

Invitrogen 

Jackson ImmunoResearch 
Laboratories, Inc 

Kepco Power Co 
Kinetic Systems, Inc 
Kipp & Zonen/Division of SCI-TEC 
Instalments, Inc 

Lab Line 

Laser Science 

Leica Confocal 

Leica Microsystems Inc 

Ludl Electronic Products, Ltd 

Ludlum Measurements, Inc 

MatTek Corporation 
McCrone Microscopes 
Micro Video Instruments, Inc 
Microway 

Miltenyi Biotec, Inc 
MJ Research 
Molecular Dynamics 
Molecular Probes 
MWG Biotech 

Nanodrop Technologies 
Nanshige USA, Inc 
National Instruments 
Nikon Confocal 
Nikon. Inc 

Olympus Confocal 
Olympus Corporation 
Omega Optical, Inc 
Ophthalmic Instrument Co 
Optiquip 
Optronics 



PCS Assembly 

Peak Performance Technologies. Inc 
Perkm Elmer Applied Biosystems 
Perkm Elmer Life Sciences 
Photon Technology International 
Photonic Instruments 
Platform Computing Inc 
Promega Corporation 

Quantitative Imaging Corporation 

Research Precision Instruments, Co 
Roche Applied Science 
Roper Scientific 

San Diego Instruments 

Santa Cruz Biotechnology 

Scanalytics 

Scion Corporation 

SD Instruments 

Sigma 

Stnauer Associates, Inc 

Soma Scientific Instruments 

SONY Medical Systems 

Stoelting 

Sutler Instrument Company 

Synaptosoft, Inc 

Technical Manufacturing 

Corporation 

The Company of Biologists, Ltd 
ThermoElectron 
ThermolEC 
ThermoNeslab 
ThermoSavant 
ThermoShandon 
ThermoSpectronics 

Universal Imaging Corporation 

Vashaw Scientific 
Vibratome 
Vincent Associates 

VWR 

Warner Instrument Corporation 

World Precision Instalments 

Carl Zeiss, Inc 
Carl Zeiss Confocal 
Carl Zeiss Imaging 



R85 



governance & administration 




BOARD OF TRUSTEES 



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

Vice Chair of the Board of Trustees 
George W Logan. Pine Street Partners 

President of the Corporation 
John E Dowlmg, Harvard University 

Director and Chief Executive Officer 
William T Speck, Marine Biological Laboratory* 

Treasurer of the Corporation 

Mary B Conrad. Fiduciary Trust International" 

Clerk of the Corporation, 
Thomas S Crane. Mintz. Levin. Cohn. Ferris, Glovsky & 
Popeo, PC* 

Chair of the Science Council 

Robert E Palazzo, Rensselaer Polytechnic Institute' 

| Class of 2003 

Darcy Brisbane Kelley. Columbia University 

Laurie J Landeau, Marmetics, Inc 

Burton J Lee, III. Vero Beach. FL 

Ronald P O'Hanley, Mellon Institutional Asset Mgt 

Jean Pierce, Boca Grande. FL 

Vincent J Ryan. Schooner Capital LLC 

\Classof2004 

M Howard Jacobson, Bankers Trust 
George M Langford, Dartmouth College 
G William Miller, G William Miller & Co . Inc 
Frank Press, The Washington Advisory Group 
Christopher M Weld, Sullivan and Worcester. Boston 
Torsten N Wiesel. The Rockefeller University 

| Class of 2005 

Porter W Anderson. Key Largo. FL 

Claire M Fraser, The Institute for Genomic Research 

George W Logan. Salem, VA 

Robert Prendergast, Falmouth, MA 

John W Rowe, Aetna U S Healthcare 

| Class of 2006 

Margaret C Bowles, Woods Hole, MA 

Martha W Cox, Hobe Sound, FL 

Walter E Massey, Morehouse College 

Marcia C Morris, Boston, MA 

Gerald Weissmann. New York University School of Medicine 



I Ex Officio Trustees 

William T Speck, Director and Chief Executive Officer 
Robert E Palazzo, Chair of the Science Council 
Mary B Conrad, Fiduciary Trust International 



Executive Committee 
of the Board of Trustees 

Margaret C Bowles 
Mary B Conrad' 
George W Logan 
Marcia Morris 
Ronald P O'Hanley 
Robert E Palazzo' 
Sheldon J Segal 
William T Speck- 
Chnstopher M Weld 



| Honorary Trustees 



William T Golden. New York. NY 
Robert E Mainer, Wayland, MA 



| Trustees Emeriti 



Edward A Adelberg, Yale University. New Haven. CT 

John B Buck, Sykesville, MD 

Seymour S Cohen, Woods Hole, MA 

Arthur L Colwm. Key Biscayne. FL 

Laura Hunter Colwm, Key Biscayne. FL 

Donald Eugene Copeland, Woods Hole, MA 

Sears Crowell, Jr. Indiana University. Bloomington, IN 

Teru Hayashi. Woods Hole. MA 

Ruth Hubbard, Cambridge. MA 

C Ladd Prosser, University of Illinois. Urbana, IL 

W D Russell-Hunter. Syracuse University. Syracuse. NY 

John W Saunders. Waquoit. MA 

David Sheprow. Boston, MA 

D Thomas Tngg Wellesley, MA 

Walter S Vincent. Woods Hole. MA 



iDirectors Emeriti 

Paul Gross. Falmouth, MA 

Harlyn O Halvorson. Woods Hole. MA 



'Ex officio 

As of April I 2002 



R86 



STANDING 
COMMITTEES 



| Development Committee 

Christopher Weld, Chair 

Porter W Anderson 

Peter Armstrong 

Robert Barlow 

Mardi Bowles 

Martha Cox 

John Dowling 

Claire Fraser 

M Howard Jacobson 

Burton Lee 

G William Miller 

Jean Pierce 

Robert Prendergast 

John W Rowe 

Torsten Wiesel 



Facilities & Capital 
Equipment Committee 

Mardi Bowles, Chair 
Porter Anderson 
George Langford 
Walter Massey 
Andrew McArthur 
Frank Press 
Christopher Weld 



| Nominating Committee 

John W. Rowe, Chair 

Martha Cox 

'John E Dowling, President of 

the Corporation 
Claire Fraser 
'Robert Palazzo, Science Council 

Chair 

Jean Pierce 
Vincent J Ryan 

William T Speck, Director & CEO 
Gerry Weissmann 



| Finance Committee 

Ronald P O'Hanley, Chair 
Mary B Conrad 
Thomas S Crane 
Maynard Goldman 
M Howard Jacobson 
Darcy Kelley 
Laurie Landeau 
George W Logan 
Robert Prendergast 

I Investment Committee 

Mary B Conrad. Chair 
Thomas S Crane 
George W Logan 
G William Miller 
Marcia Morris 
Ronald O'Hanley 
Vincent J Ryan 



STRATEGIC PLANNING COMMITTEES 



| Steering Committee 


ATTRACTING THE NEXT 


FACILITIES/HOUSING/SUPPORT 


Fred Bay 


GENERATION OF VISITING 


SERVICES 


Kerry Bloom 


SCIENTISTS 


Co-Chairs George Langford and 


Mardi Bowles 


Co-Chairs Joan Ruderman and 


John Lakian 


Ron Calabrese 


Vin Ryan 


Mardi Bowles 


John Dowling, Co-Chair 


Clay Armstrong 


Carole Browne 


M Howard Jacobson 


Barbara Fune 


Bob Goldman 


John Lakian 


Roger Hanlon 


Steve Hajduk 


George Langford 


Pam Clapp Hinkle 


Roger Hanlon 


George Logan 


Marc Kirschner 


Andrew McArthur 


Jerry Melillo 


Andy Mattox 


Joan Ruderman 


Robert Palazzo 


Jerry Melillo 


Onan Shinhai 


Torn Pollard 


Onan Shirihai 


Harold Zakon 


Joan Ruderman 


Mitch Sogin 


Mary Beckwith 


Vin Ryan 


Gerry Weissmann 


Rich Cutler, staff chair 


Sheldon Segal 




Louis Kerr 


Mitchell Sogm 




Bill Mebane 


William T Speck. Co-Chair 


| Phase /// Task Forces 


Eleanor Uhlmger 


Gerald Weissmann 


AFFILIATION 


Rudi Rottenfusser 


Al Zeien 


Co-Cha/rs Gerry Weissmann 






and Al Zeien 




| Phase // Task Forces 


Dick Chappell 
Linda Deegan 


FINANCE/FUNDING OPTIONS 
Co-Chairs Mitche" Sogm and 


ENSURING DISTINCTIVENESS 


Claire Fraser 


Vm Ryan 


AND COLLABORATION IN 


Rick Goetz 


Peter Armstrong 


RESIDENT RESEARCH 


Harlyn Halvorson 


George Augustine 


Co-Chairs John Dowling and 


John Hobbie 


David Burgess 


George Logan 


Marcia Morris 


Bibi Conrad 


Barbara Ehrlich 


Tom Pollard 


Barbara Fune 


Claire Fraser 


Frank Press 


Bob Mamer 


Rick Goetz 


Peter Smith 


Ed Rastetter 


Bob Goldman 


Diana Blazis 


Jack Rowe 


Nancy Hopkins 


Dick Mullen 


Tony Cave 


Matthew Meselson 


Cathy Norton, staff chair 


Wendy Faxon 


Marcia Morris 




Homer Lane, staff chair 


Dick Mullen 




David McLean 


Cathy Norton 


GOVERNANCE/ADMINISTRATIVE 


Becky Mountford 


Ron O'Hanley 


STRUCTURE 




Thonj Pederson 


Co-Chairs Robert Palazzo and 




Bruce Peterson 


George Logan 




Peter Smith 


Tom Crane 




Jennifer Wernegreen 


John Dowling 






Barbara Ehrlich 




ENSURING CONTINUED 


Leah Haimo 
M Howard Jacobson 




EXCELLENCE IN EDUCATION 


Jerry Melillo 




Co-Chairs. Kerry Bloom and 


Shelly Segal 




Howard Jacobson 


Steve Zottoli 




Jelle Atema 


Pamela Clapp Hinkle 


Palazzo. Robert E., Chair 


Bob Barlow 


Susan Goux, staff chair 




Mary Beckwith 


Kate Shaw 


Armstrong. Clay M- 


Ron Calabrese 




Armstrong, Peter 


Lenny Dawidowici 




Atema, Jelle 


Susan Goux 




De Weer, Paul 


Lenny Guarante 




Fraser, Scott 


John Hobbie 




Hadjuk, Steven 


Laurinda Jaffe 




Haimo, Leah 


Bill Miller 




Hopkinson, Charles 


Kip Sluder 




Smith, Peter J.S. 


Steve Zottoli 




Sogin, Mitchell 






Weeks, Janis C. 






Dawidowicz, E A.* 






Speck, William T.' 


*Ex officio 






As of April I 2002 







CORPORATION 



I Life Members 



R87 



Dr Edward A Adelberg, New Haven, CT 
Dr Bjorn Afzelius. Stockholm University 
Dr Ernest Amatniek, (address unknown) 
Dr John M Arnold, Falmouth, MA 

Mrs Betsy G Bang, Woods Hole, MA 

Dr Alan W Bernheimer, New York University 

Medical Center 

Dr Lloyd M Bertholf, Bloomington, IL 
Dr Herman F Bosch, Falmouth, MA 
Dr F J- Brmley, Jr , National Institutes of Health 
Dr John B Buck, Sykesville, MD 
Dr Madeline P Burbanck, Atlanta, GA 
Dr William D Burbanck, Atlanta, GA 

Dr Alfred B Chaet, Maitland, FL 

Dr Arnold M Clark, Woods Hole, MA 

Mr James M Clark, Palm Beach, FL 

Dr Maynard M Cohen, Rush Medical College 

Dr Seymour S Cohen, Woods Hole, MA 

Dr Jack R Collier, Effie, LA 

Dr Marjone McCann Collier, Effie, LA 

Dr Arthur L Colwin, Key Biscayne, FL 

Dr Laura Hunter Colwin, Key Biscayne, FL 

Dr Sherwin J Cooperstein, University of 

Connecticut 

Dr D Eugene Copeland, Woods Hole, MA 
Dr John O Corliss, Bala Cynwyd, PA 
Dr Helen M. Costello, Chapel Hill, NC 
Dr Helen Crouse, Hayesville, NC 

Dr Nigel W Daw, Branford, CT 

Dr Robert L DeHaan, Emory University School 

of Medicine 
Dr Patncia L Dudley, Seattle, WA 

Dr Charles Edwards, Longboat Key, FL 
Dr Gerald F Elliott, The Open University 
Research Unit 

Dr Patricia M Failla, Johns Island, SC 
Dr Donald T Frazier, University of Kentucky 
Medical Center 

Dr Mordecai L Gabriel, Brooklyn College 
Dr Murray Glusman, New York State 

Psychiatric Institute 
Dr Herbert Graham, Woods Hole, MA 

Dr Howard L Hamilton, University of Virginia 

Dr. Clifford V Harding, Jr, Falmouth, MA 

Dr Audrey E V Haschemeyer, Woods Hole, MA 

Dr Teru Hayashi, Woods Hole, MA 

Dr Frederick L Hisaw, McMinnville, OR 

Dr. Francis C G Hoskin, Canton, MA 

Prof Ruth Hubbard, Harvard University 

Dr W Bruce Hunter, Peterborough, NH 

Dr Charles Hurwitz, Stratton VA Medical Center 

Dr George Katz, Sarasota, FL 

Dr John M Kingsbury, Cornell University 

Dr Kiyoshi Kusano, National Institutes of Health 

Mr Ezra Laderman, Yale University 

Dr Paul H LaMarche, Eastern Maine Medical 

Center 
Dr Max A Lauffer, Penn State University 

Medical Center 



Dr Herbert Levitan, National Science Foundation 
Dr John H Lochhead, London, England 
Dr Birgit Rose Loewenstein, Falmouth, MA 
Dr Frank A Loewus, Washington State University 
Dr Robert B Loftfield, University of New Mexico 
Dr Laszlo Lorand, Northwestern University 
Medical School 

Dr Robert E Mainer, The Boston Company, Inc 

Dr Julian B Marsh, Chestnut Hill, MA 

Mr Lowell V Martin, Woods Hole, MA 

Dr Rita W Mathews, Southfield, MA 

Dr, Jams Metuzals, Ontario, Canada 

Dr. John A Moore, University of California 

(deceased 2002) 
Dr. John W Moore, Duke University Medical 

Center 

Dr Aron A Moscona, New York, NY 
Dr X J Musacchia, Bella Vista, AR 
Dr Maimon Nasatir, Ojai, CA 

Dr Leonard M Passano, University of Wisconsin 
Dr. William T W Potts, University of Lancaster 
Dr Carl A Price, Falmouth, MA 
Dr Margaret McDonald Prytz (address unknown) 

Dr Charles E Renn (address unknown) 

Dr George T Reynolds, Princeton University 

Dr Robert V Rice, Falmouth, MA 

Dr Morris Rockstem, Coral Gables, FL 

Dr Raphael R Ronkin, Washington, DC 

Dr John D Roslansky, Woods Hole, MA 

(deceased 2003) 
Dr Priscilla F Roslansky, Associates of 

Cape Cod, Inc. 
Dr Jay S Roth, Woods Hole, MA 

Dr Hidemi Sato, Nagoya University 

Dr R Walter Schlesmger, North Falmouth, MA 

Dr. Allan C Scott, Colby College 

Dr Arthur M Silverstein, Johns Hopkins 

University 

Dr Raymond A Sjodin, Baltimore, MD 
Dr. Paul F. Smith, Woods Hole, MA 
Mr John W Speer, Portsmouth, Rl 
Dr Nicholas Sperelakis, University of Cincinnati 
Dr Evelyn Spiegel, Dartmouth College 
Dr Melvm Spiegel, Dartmouth College 
Dr Graver C Stephens, University of California 
Mrs Jane Lazarow Stetten, Chevy Chase, MD 
Dr Bernard L Strehler, Laguna Niguel, CA 
Dr Maurice Sussman, Falmouth, MA 
Dr Raquel B Sussman, Marine Biological 

Laboratory 
Mrs Gwen P Szent-Gyorgyi, Woods Hole, MA 

Mr W Nicholas Thorndike, Wellington 

Management Company 
Dr William Trager, The Rockefeller University 
Dr J P Tnnkaus, Yale University (deceased 2003) 

Dr Claude A Villee, Jr , Harvard Medical School 
Dr Walter S Vincent, Woods Hole, MA 

Dr Talbot H Waterman, Yale University 
Dr Roland L Wigley, Woods Hole, MA 
Dr Lon A Wilkens, University of Missouri 
Dr Paul Witkovsky, NYU Medical Center 



RSS 



Members 



Dr Donald A Abt, Univer;ity of Pennsylvania 

School of Veterinary Medicine 
Dr James A Adams. Tallai assee, FL 
Dr William J A. .-?i'iian. Jr. Falmouth, MA 
Dr Daniel L Alkon, Rockefeller Neuroscience 

Institute 

Dr. Garland E Allen, Washington University 
Dr Nina Stromgren Allen, North Carolina 

State University 
Dr Mark C Alhegro, Louisiana State University 

Medical Center 

Dr Everett Anderson, Harvard Medical School 
Dr John M Anderson, Ithaca, NY 
Dr Porter W Anderson, Jr, Key Largo, FL 
Dr Christine Armett-Kibel, University of 

Massachusetts, Boston 
Prof Clay M Armstrong, University of 

Pennsylvania School of Medicine 
Mrs Ellen Prosser Armstrong, Woods Hole, MA 
Dr Peter B Armstrong, University of California 
Mr Robert W Ashton, Bay Foundation 
Dr Jelle Atema, Boston University Marine 

Program 

Dr Baccio Baccetti, University of Sienna, Italy 
Dr Robert G Baker, New York University 

Medical Center 
Dr David Baltimore, California Institute 

of Technology 
Dr. Robert B Barlow, Jr, SUNY Upstate 

Medical University 

Dr Daniel T Barry, South Hadley, MA 
Dr. Susan R Barry, Mount Holyoke College 
Dr Andrew H Bass, Cornell University 
Dr. Barbara-Anne Battelle, University of Florida 
Mr Frederick Bay, Bay Foundation 
Dr. Elaine L Bearer, Brown University 
Dr. John M. Beatty, University of Minnesota 
Dr. Luis Alberto Beauge, Institute de 

Investigacion Medica, Argentina 
Dr. Ted Begenisich, University of Rochester 
Dr. David A Begg, University of Alberta, Canada 
Dr. Eugene Bell, TEI Biosciences Inc 
Dr. Thomas L Benjamin, Harvard Medical School 
Dr. Michael V L Bennett, Albert Einstein College 

of Medicine 

Dr Miriam F Bennett, Colby College 
Dr. R Suzanne Bennett, Albert Einstein College 

of Medicine 

Dr. Suzanne T Berlin, York, ME 
Mr Norman Bernstein, Columbia Realty Venture 
Dr Francisco Bezanilla, Health Science Center 
Dr. John D Biggers, Harvard Medical School 
Dr. Stephen H Bishop, Ames, IA 
Dr Dieter Blennemann, Riverside, CT 
Dr George S Bloom, University of Texas 

Southwestern Medical Center 
Dr Kerry S Bloom, University of North Carolina 
Dr. David A Bodznick, Wesleyan University 
Dr Edward G Boettiger, Rochester, VT 
Dr Richard A Boolootian, Sherman Oaks, CA 
Dr. Thomas A Borgese, Lehman College, CUNY 
Dr David W Borst, Jr, Illinois State University 
Dr. Francis P. Bowles. Marine Biological 

Laboratory 



Dr Barbara C Boyer, Union College 

Dr. Bruce P Brandhorst, Simon Fraser University 

Dr Marianne Bronner-Fraser, California Institute 

of Technology 

Dr Stephen C Brown, SUNY at Albany 
Mr William L Brown, Weston, MA 
Dr. Carole L Browne, Wake Forest University 

School of Medicine 

Dr Robert A Browne, Wake Forest University 
Dr Anne C Bucklin, University of New 

Hampshire 

Dr Max M Burger, Novartis International AG 
Dr David R Burgess, Boston College 
Dr Mario H Burgos, IHEM Medical School 
Dr John E Burns, Beloit College 
Dr. Harold L Burstyn, Syracuse University 
Dr Sherry Burszta|n, Dartmouth Medical School 

Dr Ronald L Calabrese, Emory University 
Dr. R Andrew Cameron, California Institute 

of Technology 
Mr Richard H Campbell, Bang-Campbell 

Associates 
Dr Graciela C Candelas, University of 

Puerto Rico 
Dr Lucio Canello, Stazione Zoologica "A 

Dohrn," Italy 

Dr Catherine Emily Carr, University of Maryland 
Prof James F Case, University of California 
Father Joseph D Cassidy, O P, Ph D , 

Providence College 

Dr Colleen M Cavanaugh, Harvard University 
Dr Edward L Chambers, University of Miami 

School of Medicine 
Dr Donald C Chang, Hong Kong University of 

Science and Technology 
Dr Richard L Chappell, Hunter College 
Dr Frank M Child, Woods Hole, MA 
Dr Rex Leslie Chisholm, Northwestern University 
Dr Elena Citkowitz, Hospital of St Raphael 
Dr David E Clapham, Children's Hospital 
Dr Eloise E Clark, Bowling Green 

State University 

Mr Hays Clark, Hobe Sound, FL 
Prof Walhs H Clark, Jr., Madison, ME 
Dr John R Clay, National Institutes of Health 
Dr Alexander W Clowes, University of 

Washington 
Dr Jewel Plummer Cobb, California 

State University 

Dr Carolyn Cohen, Brandeis University 
Dr Lawrence B Cohen, Yale University School 

of Medicine 

Dr William D Cohen, Hunter College 
Dr Annette W Coleman, Brown University 
Dr Paul Colmvaux, Marine Biological Laboratory 
Dr R John Collier, Harvard Medical School 
Dr James P Collins, Arizona State University 
Dr D. Wesley Corson, Jr, Storm Eye Institute 
Dr Jeffrey T Corwm, University of Virginia, 

School of Medicine 

Dr Ernest F Couch, Texas Christian University 
Dr Rachel Llanelly Cox, Marine Biological 

Laboratory 
Thomas S Crane, Esq , Mintz, Levin, Cohn, 

Ferns, Glovsky & Popeo, P C 



Dr Karen Crawford, St Mary's College 

of Maryland 

Dr Gertrud Cremer-Bartels, Muenster, Germany 
Dr Terry J Crow, University of Texas 

Medical School 
Mr Robert J Crowther, Shriners Hospitals 

for Children 
Dr Michael P Cummings, Marine Biological 

Laboratory 
Mr Richard D Cutler, Marine Biological 

Laboratory 

Dr Eric H Davidson, California Institute 

of Technology 

Dr Daniel B Davison, Bristol-Myers Squibb PRI 
Dr EliezarA Dawidowicz, Marine Biological 

Laboratory 

Dr Paul J De Weer, University of Pennsylvania 
Dr Linda A Deegan, Marine Biological 

Laboratory 

Dr Robert C DeGroof, Doylestown, PA 
Dr Martha Bridge Denckla, Johns 

Hopkins University 

Dr Henry A DePhillips, Jr , Trinity College 
Dr Douglas W DeSimone, University of Virginia 
Dr Wolf-Dietrich Dettbarn, Nashville, TN 
Dr. Vincent E Dionne, Boston University Marine 

Program 

Dr John E Dowling, Harvard University 
Dr Arthur Brooks DuBois, John B, Pierce 

Foundation Laboratory 
Dr Thomas K Duncan, Nichols College 
Dr Philip B Dunham, Syracuse University 
Dr Paul V Dunlap, University of Michigan 

Dr William R Eckberg, Howard University 

Dr Kenneth T Edds, Bayer Diagnostics 

Dr Howard A Eder, Albert Einstein College 

of Medicine 

Ms Joan Edstrom, Falmouth, MA 
Dr Barbara E. Ehrlich, Yale University 
Dr Arthur Z Eisen, Washington University 
Dr Herman N Eisen, Massachusetts Institute 

of Technology 
Dr Hugh Young Elder, University of Glasgow, 

Scotland 
Dr. Paul T. Englund, Johns Hopkins 

Medical School 

Dr David Epel, Stanford University 
Dr. Herman T. Epstein, Woods Hole, MA 
Mr Ray L Epstein, Taunton, MA 

Prof Donald Faber, Albert Einstein College 

of Medicine 
Dr David H Farb, Boston University School 

of Medicine 

Dr A Verdi Farmanfarmaian, Rutgers University 
Dr Barry William Festoff, VA Medical Center 
Dr Rachel D Fink, Mount Holyoke College 
Dr Alan Finkelstem, Albert Einstein College 

of Medicine 

Dr Gerald D Fischbach, Columbia University 
Dr Harvey M. Fishman, University of Texas 

Medical Branch 
Mr Dennis Flanagan, New York, NY 



R89 



Dr Richard Allen Fluck, Franklin and 

Marshall College 
Dr Kenneth H Foreman, Marine Biological 

Laboratory 

Dr Thomas O Fox, Harvard Medical School 
Dr Clara Franzim-Armstrong, University of 

Pennsylvania 
Dr Scott Fraser, California Institute 

of Technology 
Dr. Kathleen A French, University of California. 

San Diego 

Dr Robert J French, University of Calgary 
Dr Chandler M Fulton, Brandeis University 
Dr Barbara C Fune, Beth Israel Deaconess 

Medical Center 
Dr Bruce Fune, Beth Israel Deaconess 

Medical Center 

Dr Edwin J Furshpan, Harvard Medical School 
Dr Robert P Futrelle, Northeastern University 

Dr. Howaida Gabr, Suez Canal University 

Dr David C Gadsby, The Rockefeller University 

Dr Harold Gainer, National Institutes of Health 

Dr Robert M Galatzer-Levy, Chicago, IL 

Dr Joseph G Gall, Carnegie Institution 

Dr Michael A Gallo, UMDNJ-Robert Wood 

Johnson Medical School 
Dr Alan Gelperm, Monell Chemical 

Senses Center 
Dr James L German, III, Weill Medical College 

of Cornell University 
Dr Martin Gibbs, Brandeis University 
Dr Anne E Giblin, Marine Biological Laboratory 
Dr A. Jane Gibson, Enta, NH 
Dr Prosser Gifford, Library of Congress 
Prof Giovanni Giudice, Universita di 

Palermo, Italy 
Dr Antonio Giuditta, Universita di Napoli 

"Fedenco II," Italy 
Dr Paul Glynn, Brunswick, ME 
Mr William T Golden, Chairman Emeritus, 

American Museum 

of Natural History 
Dr. Robert D Goldman, Northwestern University 

Medical School 
Dr Paul K Goldsmith, National Institutes 

of Health 

Dr Timothy H Goldsmith, Yale University 
Dr Moise H Goldstein, Jr, The Johns Hopkins 

University 
Dr Robert Michael Gould, NYS Institute of 

Basic Research 

Mr Dick Grace, Doreen Grace Fund 
Dr Werner M Graf, College of France, France 
Dr Philip Grant, National Institutes of Health 
Dr Judith P Grassle, Rutgers University 
Dr Katherine G. Graubard, University of 

Washington 

Dr E. Peter Greenberg, University of Iowa 
Dr Michael J Greenberg, University of Florida 
Dr. Mary J Greer, New York, NY 
Dr Donald R Griffin, Harvard University 
Dr Albert Grossman, New York University 

Medical Center 
Prof Lawrence Grossman, The Johns 

Hopkins University 
Dr. Yosef Gruenbaum, The Hebrew University 

of Jerusalem 
Dr John A Gruner, Cephalon, Inc 



Mr A Robert Gunning, Falmouth, MA 
Dr G. Francis Gwilham, Reed College 

Prof Leah T Haimo, University of California 
Dr Stephen L. Hajduk, Marine Biological 

Laboratory 
Dr Linda M Hall, Functional Insect Genomics 

Institute 
Dr Tatsuji Haneji, The University of 

Tokushima, Japan 
Dr Roger T Hanlon, Marine Biological 

Laboratory 

Dr Ferenc Harosi, New College of the 
University of South Florida 

June F Harngan, Ph D , Honolulu, HI 
Dr John P Harrington, SUNY - New Paltz 
Dr Stephen C. Harrison, Harvard University 
Dr Robert Haselkorn, University of Chicago 
Dr J Woodland Hastings, Harvard University 
Dr Raymond L Hayes, Jr, Howard University 
Dr Diane E Heck, Rutgers University 
Dr Jonathan Joseph Henry, University of Illinois 
Dr Peter K Hepler, University of Massachusetts 
Dr Walter R Herndon, University of Tennessee 
Prof Avram Hershko, Technion-lsrael Institute of 

Technology, Israel 

Dr Theodore T Herskovits, Fordham University 
Dr Howard H Hiatt, Bngham and Women's 

Hospital 
Dr Stephen M Highstem, Washington 

University School of Medicine 
Dr John G Hildebrand, University of Arizona 
Dr Richard W Hill, Michigan State University 
Dr Robert B. Hill, University of Rhode Island 
Dr Susan D Hill, Michigan State University 
Dr Llewellya W Hillis, Marine Biological 

Laboratory 
Dr Edward H Hinchcliffe, University of 

Massachusetts Medical School 
Dr Michael Hmes, Yale University School 

of Medicine 
Dr Gregory J Hinkle, Millenium 

Pharmaceuticals 
Dr Gertrude W. Hinsch, University of 

South Florida 
Dr Jan Hinsch, Leica, Inc 
Dr John E Hobbie, Marine Biological 

Laboratory 
Dr Alan J Hodge, San Diego, CA 

(deceased 2002) 
Dr Joseph F Hoffman, Yale University School 

of Medicine 
Dr George G Holz, IV, New York University 

Medical Center 
Dr Charles S Hopkinson, Jr, Marine 

Biological Laboratory 
Dr James C Houk, Northwestern University 

Medical School 

Dr Ronald R Hoy, Cornell University 
Dr Alice S Huang, California Institute of 

Technology 
Dr Linda A Hufnagel-Zackroff, University of 

Rhode Island 

Dr William D Hummon, Ohio University 
Dr Susie H Humphreys, Food and Drug 

Administration 

Dr. Tom Humphreys, University of Hawaii 
Dr Tim Hunt, ICRF Clare Hall Labs, England 



Dr Robert D. Hunter, Oakland University 
Dr Hugh E Huxley, Brandeis University 

Dr Joseph llan, Case Western Reserve 

University 
Dr Nicholas A Ingoglia, New Jersey 

Medical School 

Dr Saduyki Inoue, McGill University, Canada 
Dr Shinya Inoue, Marine Biological Laboratory 
Dr KurtJ Isselbacher, Massachusetts General 

Hospital Cancer Center 
Dr Marietta Radovic Issidondes, Theodor 

Theohan Cozzika Foundation 
Dr Colin S Izzard, SUNY - Albany 

Dr Laurmda A Jaffe, University of Connecticut 

Health Center 

Dr Lionel Jaffe, Marine Biological Laboratory 
Dr William R Jeffery, University of Maryland 
Dr Diana E. J Jennings, Marine Biological 

Laboratory 

Dr Daniel Johnston, Baylor College of Medicine 
Dr Teresa L Z Jones, National Institutes 

of Health 

Dr Robert K Josephson, University of California 
Dr Leonard K Kaczmarek, Yale University School 

of Medicine 

Dr Gabor Kaley, New York Medical College 
Dr Jane C Kaltenbach, Mount Holyoke College 
Dr Benjamin Kaminer, Boston University 

Medical School 

Dr Edna S Kaneshiro, University of Cincinnati 
Dr Ehud Kaplan, Mount Sinai School of 

Medicine 

Dr Stephen J Karakashian, Milwaukie, OR 
Dr Arthur Karlin, Columbia University 
Dr Morns John Karnovsky, Harvard Medical 

School 

Mr H Ernst Keller, Carl Zeiss, Inc. 
Dr. Darcy B. Kelley, Columbia University 
Dr Robert E Kelly, Woods Hole, MA 
Dr Norman E Kemp, University of Michigan 
Mr John P Kendall, Faneuil Hall Associates 
Mr Louis M Kerr, Marine Biological Laboratory 
Dr Alexander Keynan, Israel Academy of 

Sciences/Humanities, Israel 
Dr Shahid M M Khan, SUNY Upstate 

Medical University 
Dr Kamran Khodakhah, Albert Einstein College 

of Medicine 
Dr Daniel P Kiehart, Duke University 

Medical Center 

Dr Irving M Klotz, Northwestern University 
Mr Robert A Knudson, Marine Biological 

Laboratory 

Dr Samuel S Koide, The Rockefeller University 
Sir Hans Kornberg, Boston University 
Dr Edward M Kosower, Tel-Aviv University, Israel 
Dr Stephen M Krane, Massachusetts 

General Hospital 
Dr Robert Krauss, Denton, MD 
Dr Edward A Kravitz, Harvard Medical School 
Dr William B Knstan, Jr., University of California, 

San Diego 
Dr Andrew M Kropmski, Queen's University, 

Canada 

Dr Damien P Kuffler, Institute of Neurobiology 
Dr. William J Kuhns, The Hospital for Sick 

Children, Canada 



R90 



Dr Joseph G. Kunkel, University of Massachusetts 
Dr Alan M, Kuzinan, Marine Biological Laboratory 

Dr Aimlee D laderman. Vale University 

Dr Laurie J. Landeau, Li ,tov. '!, ' c 

Dr Dennis M D La-"-' -y Hospital 

of Cleveland 

Dr. Story C La", is, National Institutes of Health 
Dr David L,v >.vnr, University of Miami 
Dr. George M. Langford, Dartmouth College 
Dr Jeffrey Laskin, University of Medicine and 

Dentistry of New Jersey 
Dr, Nechama Lasser-Ross, New York 

Medical College 
Dr Leonard Laster, University of Massachusetts 

Medical School 

Dr Alan Laties, Scheie Eye Institute 
Dr Hans Laufer, University of Connecticut 
Dr Paul B Lazarow, Mount Sinai - School 

of Medicine 
Mr Maurice Lazarus, Federated Department 

Stores 

Dr Edward R Leadbetter, University of 
Connecticut 

Dr Joshua Lederberg, The Rockefeller University 
Dr John J Lee, City College of CUNY 
Mr Donald B Lehy, North Falmouth, MA 
Dr. Stephen B Leighton, Beecher Instruments 
Dr Aaron B. Lerner, Yale University School 

of Medicine 
Dr Jack Levin, University of California School 

of Medicine 

Dr Michael S Levine, University of California 
Dr Richard B. Levine, University of Arizona 
Dr Francoise Levmthal, Columbia University 
Prof Irwin B. Levitan, University of Pennsylvania 

School of Medicine 
Dr Richard W Linck, University of Minnesota 

School of Medicine 
Dr Raymond J Lipicky, Food and Drug 

Administration 

Dr. John E Lisman, Brandeis University 
Dr. Anthony Liuzzi, Boston, MA 
Dr Rodolfo R Llinas, New York Unversity 

Medical Center 
Dr Phillip S Lobel, Boston University Marine 

Program, Marine Biological Laboratory 
Dr Werner R Loewenstein, Falmouth, MA 
Dr Irving M London, Harvard-MIT 
Dr Frank J. Longo, University of Iowa 
Dr Louise M Luckenbill, Falmouth, MA 

Dr Edward F MacNichol, Jr, Boston University 

School of Medicine 
Dr Jane Ann Maienschein, Arizona State 

University 

Dr Craig C Malbon, State University of New York 
Dr Robert P Malchow, University of Illinois, 

Chicago 

Dr. Richard S Manalis, Indiana-Purdue University 
Dr Lynn Margulis, University of Massachusetts 
Dr Andrew C Marmucci, Mercerville, NJ 
Dr Joe L Martinez, Jr , University of Texas 
Dr Luigi Mastroianni, Jr, Hospital of University 

of Pennsylvania 

Dr David Mauzerail, Rockefeller University 
Dr M Lynne McAnelly, University of Texas 
Dr Andrew G McArthur, Marine Biological 

Laboratory 



Dr Frances V McCann Murray, Dartmouth 

Medical School 
Ms. Jane A McLaughlin, Marine Biological 

Laboratory 

Dr Robert F McMahon, University of Texas 
Dr. Thomas Meedel, Rhode Island College 
Prof Ian A Meinertzhagen, Dalhousie 

University, Canada 
Dr Dennis E Meiss, Immunodiagnostic 

Laboratories 

Dr Jerry M Melillo, Marine Biological Laboratory 
Dr DeForest Mellon, Jr, University of Virginia 
Mr. Richard P Mellon, Laughlmtown, PA 
Dr Michael E Mendelsohn, New England 

Medical Center 

Dr Allen F Mensmger, University of Minnesota 
Dr Melanie Pratt Mernman, Touchstone 

Consulting 

Dr Matthew Meselson, Harvard University 
Dr Ricardo Miledi, University of California, Irvine 
Dr Roger D Milkman, University of Iowa 
Dr Andrew L Miller, Hong Kong University 

of Science and Technology 
Mr Thomas J. Miller, Concord, MA 
Dr Gradimir Misevic, University Hospital 

of Basel, Switzerland 
Dr Ralph Mitchell, Harvard University 
Dr Timothy Mitchison, Harvard University 

Medical School 
Dr Hiroyoshi Miyakawa, Tokyo College of 

Pharmacy, Japan 

Dr David M Miyamoto, Drew University 
Dr. Merle Mizell, Tulane University 
Dr Jorge E Moreira, National Institutes of Health 
Dr. James G Morin, Cornell University 
Dr. Leyla de Toledo Worrell, Rush-Presbyterian- 

St Lukes 

Dr Stephen S Morse, Columbia University 
Dr Andrew W Murray, Harvard University 

Dr Samuel M Nabrit, Atlanta, GA 

Dr Knute J Nadelhoffer, National Science 

Foundation 
Dr. Ronald L Nagel, Albert Einstein College 

of Medicine 
Dr Yasuko Nakajima, University of Illinois, 

College of Medicine 
Prof Toshio Narahashi, Northwestern University 

Medical School 
Dr Enrico Nasi, Boston University School 

of Medicine 
Dr. Christopher Neill, Marine Biological 

Laboratory 

Margaret C Nelson, Ph.D., Cornell University 
Dr. Peter A. Nickerson, SUNY, Buffalo 
Dr Santo V Nicosia, University of South Florida 
Ms Catherine N Norton, Marine Biological 

Laboratory 
Dr Michael P Nusbaurn, University of 

Pennsylvania School of Medicine 

Mr Jonathan O'Herron, Lazard Freres & Company 
Dr Ana Lia Obaid, University of Pennsylvania 

School of Medicine 
Dr Shinpei Ohki, SUNY at Buffalo 
Dr Rudolf Oldenbourg, Marine Biological 

Laboratory 
Dr James L Olds, George Mason University 



Dr Ada L. Olins, Foundation for Blood Research 
Dr Donald E. Olins, Foundation for Blood Research 
Dr James L Oschman, Dover, NH 

Dr, Robert E Palazzo, Rensselaer Polytechnic 

Institute 

Dr John D Palmer, University of Massachusetts 
Dr. Harish C Pant, National Institutes of Health 
Dr George D Pappas, University of Illinois 
Dr Arthur B Pardee, Dana-Farber Cancer Institute 
Dr Rosevelt L Pardy, University of Nebraska 
Dr James L Parmentier, International Health 

Organization 
Dr Thoru Pederson, University of Massachusetts 

Medical Center 

Dr Courtland D Perkins, Alexandria, VA 
Dr Philip Person, Flushing, NY 
Dr Bruce J Peterson, Marine Biological Laboratory 
Dr Ronald Pethig, University College of North 

Wales, UK 

Dr Ronald J Pfohl, Miami University 
Dr Sidney K Pierce, Jr, University of South Florida 
Dr David E Pleasure, Children's Hospital 
Dr Jeanne S. Poindexter, Barnard College 
Dr Harvey B Pollard, U S U H S 
Dr Thomas D Pollard, Yale University 
Dr Beverly H Porter, Columbia, MD 
Dr Mary E Porter, University of Minnesota 
Dr. David D. Potter, Harvard Medical School 
Dr Maureen K Powers, San Pablo, CA 
Dr Robert A Prendergast, Johns Hopkins University 
Dr David J. Prior, Northern Arizona University 
Dr Robert D. Prusch, Gonzaga University 
Dr Dale Purves, Duke University Medical Center 

Dr. James P Quigley, The Scripps Research Institute 

Mr Irving W Rabb, Cambridge, MA 

Dr. Harvey Rabin, Rockville, MD 

Dr. Michael B Rabinowitz, Boston, MA 

Dr Nancy S Rafferty, Marine Biological Laboratory 

Dr Robert F Rakowski, Ohio University 

Dr Fidel Ramon, Universidad Nacional Autonoma 

de Mexico, Mexico 
Dr Edward B Rastetter, Marine Biological 

Laboratory 
Dr Lionel I Rebhun, University of Virginia 

(deceased 2002} 

Dr John R Reddan, Oakland University 
Dr. Thomas S Reese, National Institutes of Health 
Dr Carol L Reinisch, Marine Biological Laboratory 

Dr Frederick R Rickles, George Washington 

University 

Dr Conly L Rieder, Wadsworth Center 
Dr. Monica Riley, Marine Biological Laboratory 
Dr Harris Ripps, University of Ilinois at Chicago 
Dr J Murdoch Ritchie, Yale University School 

of Medicine 
Dr Lawrence C Rome, University of 

Pennsylvania 
Dr Jack Rosenbluth, New York University 

School of Medicine 

Dr Raja Rosenbluth, Simon Fraser University 
Dr Allan Rosenfield, Columbia University School 

of Public Health 
Dr Herbert S Rosenkranz, Florida Atlantic 

University 



R9I 



Dr William N Ross, New York Medical College 
Mr Rudi Rottenfusser, Carl Zeiss Inc 
Dr Lewis P Rowland, Neurological Institute 
Dr Joan V Ruderman, Harvard Medical School 
Dr John D Rummel, NASA Headquarters 
Dr Norman B. Rushforth, Case Western 

Reserve University 
Dr William Devine Russell-Hunter, Easton, MD 

Dr Mary Beth Saffo, Harvard University 
Dr Guy Salama, University of Pittsburgh 
Dr Edward D Salmon, University of 

North Carolina 

Dr Abigail Salyers, University of Illinois 
Prof Brian M Salzberg, University of 

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

School of Medicine 
Dr Joseph W. Sanger, University of Pennsylvania 

Medical Center 

Dr. John W Saunders, Jr. Waquoit, MA 
Prof- Howard K Schachman, University of 

California 

Dr. Gerald P Schatten, University of Pittsburgh 
Dr Arlene C Schmeer, Mercenene Cancer 

Research Institute 

Dr. Herbert Schuel, SUNY at Buffalo 
Dr Lawrence Schwartz, University of 

Massachusetts 

Dr A Nicola Schweitzer, Brooklme, MA 
Dr Felix E Schweizer, University of California, 

Los Angeles 

Dr Sheldon J Segal, The Population Council 
Dr Stephen Lament Senft, Woods Hole, MA 
Dr Douglas R Shanklin, University of Tennessee 
Dr Nadav Shashar, The Interuniversity Institute 

of Eilat, Israel 

Dr Victor E. Shashoua, Harvard Medical School 
Dr Gaius R Shaver, Marine Biological 

Laboratory 
Dr. John R. Shaver, Michigan State University 

(deceased 2003) 

Dr Michael P Sheetz, Columbia University 
Dr David Sheprow, Boston University 
Dr Irwin W Sherman, University of California 
Dr Osamu Shimomura, Falmouth, MA 
Mr Alan M Shipley, Forestdale, MA 
Dr Robert B Silver, Wayne State University 
Dr Kathleen K Siwicki, Swarthmore College 
Dr Dorothy M Skinner, Falmouth, MA 
Dr Roger D Sloboda, Dartmouth College 
Dr Greenfield Sluder, University of 

Massachusetts Medical Center 
Dr Peter J S Smith, Marine Biological 

Laboratory 
Dr Stephen J Smith, Stanford University School 

of Medicine 
Dr Roxanna S Smolowitz, Marine Biological 

Laboratory 
Dr Mitchell L Sogm, Marine Biological 

Laboratory 
Dr Martha M Sorenson, Cidade Universitana- 

UFRJ, Brazil 
Dr William T Speck, Marine Biological 

Laboratory 



Dr Abraham Spector, Columbia University 
Dr Johanna E Speksnijder, Odikj, The 

Netherlands 
Dr David C Spray, Albert Einstein College 

of Medicine 
Dr Kenneth R Spring, National Institutes 

of Health 
Dr John H Steele, Woods Hole Oceanographic 

Institution 
Dr Antoinette Stemacker, University of 

Puerto Rico 

Dr Malcolm S Steinberg, Princeton University 
Dr Andreas C Stemmer, Institut fuer Robotik, 

Switzerland 
Prof Johan Stenflo, M D , Ph D , University of 

Lund, Sweden 

Mr Paul A Steudler, Marine Biological Laboratory 
Dr Darrell R. Stokes, Emory University 
Dr Elijah W Stommel, Dartmouth Hitchcock 

Medical Center 
Dr. Alfred Stracher, SUNY Health Science Center 

at Brooklyn 

Dr Felix Strumwasser, East Falmouth, MA 
Dr Ann E Stuart, University of North Carolina at 

Chapel Hill 
Dr Mutsuyuki Sugimon, New York University 

Medical Center 
Dr William C Summers, Western Washington 

University 

Dr Kathy A Suprenant, University of Kansas 
Prof Mary Anne Sydlik, Hope College 
Dr Andrew G Szent-Gyorgyi, Brandeis University 

Dr Marvin L Tanzer, University of Connecticut, 

School of Dental Medicine 
Dr Ichiji Tasaki, National Institutes of Health 
Dr Edwin W Taylor, University of Chicago 
Dr William H Telfer, University of Pennsylvania 
Dr Bruce Telzer, Pomona College 
Prof Mark Terasaki, University of Connecticut 

Health Center 

Dr James G Townsel, Meharry Medical College 
Dr David M Travis, Woods Hole, MA 
Dr Steven N Treistman, University of 

Massachusetts Medical Center 
Dr Walter Troll, NYU Medical Center 
Dr Robert F. Troxler, Boston University School 

of Medicine 

Dr Kenyon S Tweedell, University of Notre Dame 
Dr Mark L Tykocinski, Case Western Reserve 

University 
Prof Michael Tytell, Wake Forest University 

School of Medicine 

Dr. Hiroshi Ueno, Kyoto University, Japan 

Dr Ivan Valiela, Boston University Marine Program 
Dr Richard Vallee, University College of 

Physicians & Surgeons 
Mr. John J Valois, Woods Hole, MA 
Dr Cindy Lee Van Dover, The College of William 

and Mary 
Dr Kensal E Van Holde, Oregon State University 



Dr Patricia Wadsworth, University of 

Massachusetts 
Dr Norman R Wamwnght, Marine Biological 

Laboratory 
Dr Byron H Waksman, New York University 

Medical Center 

Dr Betty Wall, Woods Hole, MA 
Dr Lawrence J Wangh, Brandeis University 
Dr Robert C Warner, Laguna Beach, CA 
Dr Leonard Warren, Wistar Institute 
Dr. John B Waterbury, Woods Hole 

Oceanographic Institution 
Dr Stephen G Waxman, Yale Medical School 
Dr Annemane Weber, University of Pennsylvania 

School of Medicine 

Dr Janis C Weeks, University of Oregon 
Dr. Earl Weidner, Louisiana State University 
Dr Alice Sara Weiss, Silver Spring, MD 
Dr. Dieter G Weiss, University of Rostock, 

Germany 
Dr Leon P Weiss, University of Pennsylvania 

School of Veterinary Medicine 
Dr Mansa C Weiss, Paoli Memorial Hospital 
Dr Gerald Weissmann, New York University 

Medical Center 
Dr Jennifer J Wernegreen, Marine Biological 

Laboratory 

Dr Monte Westerfield, University of Oregon 
Dr J Richard Whittaker, University of New 

Brunswick 

Dr Torsten N Wiesel, The Rockefeller University 
Dr Darcy B. Wilson, Torrey Pines Institute 
Dr T Hastings Wilson, Harvard Medical School 
Dr Beatrice Wittenberg, Albert Einstein College 

of Medicine 
Dr Jonathan B Wittenberg, Albert Einstein 

College of Medicine 

Dr William F Wonderlin, West Virginia University 
Dr Mary Kate Worden, University of Virginia 
Dr Basil V Worgul, Columbia University 
Dr Chau Hsiung Wu, Northwestern University 

Medical School 
Dr Charles R Wyttenbach, University of Kansas 

Dr. Harold H. Zakon, University of Texas 

Dr Seymour Zigman, Falmouth, MA 

Dr Michael J Zigmond, University of Pittsburgh 

Dr Joshua J. Zimmerberg, National Institutes 

of Health 
Dr Steven J Zottoli, Williams College 



R92 



ASSOCIATES 



I Patron 

Mr and Mrs. MaicoIm Campbell 
The Honorable and Mrs. John S Langford 
Dr and Mrs Edward F MacNichol, Jr 
Mrs William O. (Miles. II 

[Sustaining Associate 

Mr and Mrs. David Bakalar 

Mr and Mrs Norman Bernstein 

Mr Bnan Bragmton-Smith 

Bufftree Building Co , Inc. 

Mrs Martha Saunders Ferguson 

Dr David Forkosh and Dr Linda Hirshman 

Dr and Mrs. Leonard Laster 

Dr and Mrs John W Rowe 



| Supporting Associate 



Mrs Margaret Clowes 
Mrs Sally Cross 
Ms Anne R DuBois 
Mr Clifton H Eaton 
Dr and Mrs Harold S Ginsberg 
Mrs Rebeckah DuBois Glazebrook 
Drs Alfred and Joan Goldberg 
Ms Penelope Hare 
Mr and Mrs. Gary G Hayward 
Mr and Mrs William K Mackey, Esq 
Dr. and Mrs William M. McDermott 
Mr and Mrs Robert Parkinson 
Mr. and Mrs. William J. Pechilis 
Dr and Mrs Alan D Perlmutter 
Mr and Mrs Walter J Salmon 
Mrs Anne W Sawyer 
Dr John Tochko and 
Mrs. Christina Myles-Tochko 



[Family Membership 



Dr and Mrs David E Adelberg 

Dr and Mrs Dean C Allard, Jr 

Dr Peggy Alsup 

Mr and Mrs Douglas Amon 

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 

Dr and Mrs Robert B Barlow, Jr 

Mr and Mrs John Barnes 

Dr and Mrs Harriet P Bemheimer 

Mr and Mrs Robert O Bigelow 

Dr and Mrs Edward G Boettiger 

Mr and Mrs Kendall 8 Bohr 

Dr and Mrs Thomas A Borgese 

Dr and Mrs Francis P Bowles 

Mr and Mrs Peter Boyer 

Mr and Mrs Thomas A. Brown 

Dr and Mrs John B Buck 

Mr and Mrs William O Burwell 

Mr and M's Bruce E. Buxton 

Dr and Mr, Richard L Chappell 



Dr. and Mrs Frank M, Child 

Dr and Mrs Arnold M Clark 

Mr and Mrs James M. Clean/ 

Dr and Mrs Laurence P Cloud 

Drs Harry Conner and Carol Scott-Conner 

Mr and Mrs Donald B Cook 

Mr and Mrs Theron S Curtis, Jr 

Mr and Mrs Joel Davis 

Mr and Mrs Joseph L Dixon 

Mr and Mrs F, Gerald Douglass 

Dr and Mrs John E. Dowlmg 

Mr and Mrs W J Doyle 

Dr and Mrs Arthur Brooks DuBois 

Mr and Mrs Robert Elias 

Mr and Mrs Jerome Fanger 

Dr and Mrs Michael J Fishbein 

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 David Garber 

Miss Eleanor Garfield 

Dr and Mrs James L German, HI 

Dr and Mrs Prosser Gifford 

Dr and Mrs Murray Glusman 

Dr. and Mrs Moise H Goldstein 

Mrs Ann Goodman and Dr. Arthur Pardee 

Mr and Mrs Charles Goodwin, III 

Mr and Mrs Frederic Greenman 

Dr and Mrs Thomas C Gregg 

Dr and Mrs Newton H Gresser 

Ms Kathryn Hackett 

Dr and Mrs Harlyn O Halvorson 

Mr and Mrs Benjamin Handelman 



Dr and Mrs Richard Bennet Harvey 

Dr and Mrs J Woodland Hastings 

Dr Robert R Haubnch 

Mrs Elizabeth Heald 

Mr and Mrs Edward S Heard 

Dr and Mrs Howard H. Hiatt 

Mr and Mrs David Hibbitt 

Dr Llewellya Hillis and Dr Paul Colmvaux 

Dr and Mrs John E Hobbie 

Dr Peter A Hoenig 

Mr and Mrs Gerald J Holtz 

Drs Francis C Hoskm and Elizabeth Farnhan 

Mrs Carmela J Huettner 

Ms Susan A Huettner 

Dr and Mrs Shmya Inoue 

Dr and Mrs Kurt J Isselbacher 

Mrs Mary D Janney 

Dr and Mrs James E. Johnson 

Mrs Sally S, Joslin 

Dr and Mrs Benjamin Kammer 

Dr and Mrs Morns John Karnovsky 

Dr and Mrs George Katz 

Mr and Mrs Arthur King 

Mr and Mrs Paul W Knaplund 

Mr and Mrs. A. Sidney Knowles, Jr 

Mr and Mrs Walter E Knox 

Sir Hans and Lady Kornberg 

Dr. and Mrs S. Andrew Kulin 

Mr Ezra Laderman 

Dr and Mrs George M Langford 

Dr Hans Laufer 

Dr and Mrs John J Lee 

Mr Russ Lemcke 

Mr. and Mrs James E Lloyd 



THE 2001-2002 FALMOUTH FORUM SERIES 
sponsored by the MBi Associates 



October 5, 2001 

"Why I Sing in the Shower" 

Keith Lockhart, Conductor, The Boston Pops Orchestra 

October 19, 2001 

"Memoirs of a Geisha: The Making of a Novel" 
Arthur Golden, Author, Memoirs of a Geisha 

December 7, 2001 

"An Evening with Norm Abram" 

Norm Abram, Master Carpenter and host of The New Yankee Workshop 

January 18, 2002 

"The Great Powers and the East Mediterranean World" 

Erik Goldstein, Chairman, Department of International Relations; 

Professor of International Relations, Boston University 

February 1 , 2002 
"Around the Other Round Stone Barn: The History, Restoration, 

and Relevance of the Hancock Shaker Community" 
Mary Rentz, Past-President, Board of Trustees, Hancock Shaker Village 

Friday, March 1, 2002 
"Osteoporosis: The Research Frontier - Hopes for a Cure" 

Bjorn R. Olsen, Hersey Professor of Cell Biology; 

Harvard-Forsyth Professor of Oral Biology; Chairman, 

Harvard-Forsyth Department of Oral Biology, Harvard Medical School 



ASSOCIATES EXECUTIVE BOARD 



R93 






Ruth Ann taster, President 
Sallle Giffen, Vice-President 

Kitty Brown, Treasurer 
Ruth Shephard. Secretary 

Tammy Amon 
Helen Barnes 
Peter Boyer 
Bruce Button 

Julie Child 

Martha Ferguson 

Thomas Gregg 

James Johnson 

Alice Knowles 

Hans Kornberg 

Rebecca Lash 

Susan Loucks 

Jack Pearce 

Alan Perlmutter 

Virginia Reynolds 

Marjorie Salmon 

Volker Ulbrich 

Ex-officio members 

William T. Speck, Director & CEO, MBL 
John E. Dowling, President of the 

Corporation, MBL 

Sheldon J. Segal, Chairman of the Board 
of Trustees, MBL 

Associates Administrator 
Susan Joslin 



Associates, continued 

Dr and Mrs Laszlo Lorand 

Mr Richard C Levering 

Mr and Mrs Francis C Lowell, Jr. 

Mrs Phyllis M MacNeil 

Mr and Mrs Joseph Martyna 

Drs Luigi and Elaine Mastroiann: 

Dr and Mrs Robert T McCluskey 

Mr and Mrs Derek J McDonald 

Mr and Mrs James McSherry 

Mrs Nawrie Meigs-Brown 

Dr. and Mrs Jerry M Melillo 

Dr Martin Mendelson 

Mr and Mrs Richard Meyers 

Mr. and Mrs Charles A Mitchell 

Dr and Mrs Merle Mizell 

Dr. and Mrs Charles H Montgomery 

Mr and Mrs Stephen A Moore 

Mr James V Moynihan 

Mr. and Mrs. Lewis Nassikas 

Dr. and Mrs John E Naugle 

Dr. Pamela Nelson and Mr Christopher Olmsted 

Mr. and Mrs Frank L Nickerson 

Dr. and Mrs Clifford T O'Connell 

Mr and Mrs. James J O'Connor 

Mr and Mrs Daniel O'Grady 

Mr and Ms David R Palmer 

Dr and Mrs Clement E Papazian 



Mr. and Mrs Richard M Paulson, Jr 

Dr. and Mrs John B Pearce 

Mrs Nancy Pendleton 

Mr and Mrs John 8 Pen 

Dr and Mrs Courtland D. Perkins 

Dr and Mrs Philip Person 

Mr Frederick S Peters 

Mrs. Grace M Peters 

Mr and Mrs E Joel Peterson 

Mr. and Mrs Harold Pilskaln 

Mr and Mrs Andrew H Plevin 

Mr. and Mrs George H Plough 

Dr and Mrs Aubrey Pothier, Jr. 

Mr and Mrs Allan Ray Putnam 

Mrs Lionel I Rebhun 

Dr and Mrs George T Reynolds 

Dr and Mrs Harns Ripps 

Ms Jean Roberts 

Rev Michael Robertson and Dr. Emmy Robertson 

Drs. Priscilla and John Roslansky 

Mr and Mrs John D Ross 

Dr and Mrs John W Saunders, Jr 

Mr and Mrs Savely Schuster 

Mr and Mrs Harold H Sears 

Dr and Mrs Sheldon J Segal 

Dr and Mrs Robert Seidler 

Mr and Mrs Daniel Shearer 

Dr and Mrs David Sheprow 

Dr. and Mrs Irwm Sherman 

Mr and Mrs Bertram R Silver 

Mr and Mrs Jonathan O Simonds 

Mr and Mrs John A Simounan 

Drs Frederick and Marguerite Smith 

Drs William Speck and Evelyn Lipper 

Dr and Mrs Guy L. Steele, Sr 

Dr and Mrs Alan B Steinbach 

Dr and Mrs William K Stephenson 

Dr and Mrs Thomas R Stetson 

Mr and Mrs E Kent Swift, Jr 

Mr and Mrs Gerard L Swope 

Mr and Mrs Emil D Tietje, Jr 

Mr D. Thomas Trigg 

Dr. and Mrs Walter Troll 

Prof and Mrs Michael Tytell 

Mr. and Mrs Volker Ulbrich 

Mr and Mrs. John J Valois 

Drs. Claude and Dorothy Villee 

Dr John Waterbury and Ms Vicky Cullen 

Mr and Mrs John T. Weeks 

Dr and Mrs Gerald Weissmann 

Dr. and Mrs. Paul S Wheeler 

Ms Mabel Whelpley and Mr George Rollins 

Mrs. Geoffrey Whitney 

Mr. and Mrs. Lynn H Wilke 

Mr and Mrs. Leslie J Wilson 

Dr and Mrs T Hastings Wilson 

Mr and Mrs. Leonard M. Wilson 

Mr and Mrs Richard Yoder 

Mrs Manlyn G Zacks 

Mr and Mrs Bruce Zimmerli 



I Individual Membership 

Mr Richard A Ahern 

Dr Nina Stromgren Allen 

Mrs Fredenca Z Alpert 

Mr Dean N Arden 

Mrs. Ellen Prosser Armstrong 

Mr Garfield M Arthur 

Mrs Barbara Atwood 

Mrs June Atwood 

Mr. Everett E Bagley 

Ms. Megan E Barrasso 

Ms Patricia M. Barry 

Mr Mike Barry 

Dr Millicent Bell 

Mr C John Berg 

Ms Olive C Beverly 

Mr George Billings 

Mrs Ellen F Binda 

Ms. Avis Blomberg 

Dr. Robert H Broyles 

Mr. Joseph W Burke 

Mrs Barbara Gates Burwell 

J G. Butch 

Dr Graciela C Candelas 

Mr Frank C Carotenuto 

Dr Robert H Carner 

Dr Chia-Yen Chen 

Dr Sallie Chisholm 

Ms Paula Ciara 

Mrs Octavia C Clement 

Dr Jewel Plummer Cobb 

Mrs Margaret H Coburn 

Dr Seymour S Cohen 

Ms Genevieve Coleman 

Ms Anne S Concannon 

Ms Margaret S Cooper 

Dr D Eugene Copeland 

Mrs. Molly N. Cornell 

Dr Vincent Cowling 

Mrs Marilyn E Crandall 

Ms Cathleen Creedon 

Ms Helen M. Crossley 

Ms Dorothy Crossley 

Mrs Villa B Crowell 

Mrs Dons M Curran 

Mrs Elizabeth M Davis 

Ms Maureen Davis 

Ms Carol Reimann DeYoung 

Mrs Virginia A Dierker 

Mr David L Donovan 

Ms Suzanne Droban 

Ms Maureen J. Ougan 

Ms. Deborah Dunn 

Mrs Frances E Eastman 

Dr Frank Egloff 

Ms. Elizabeth Egloff 

Mrs Eleanor B Faithorn 

Ms Helen C Farnngton 

Mrs Ruth Alice Fitz 

Ms Sylvia M Flanagan 

Mr John W Folmo, Jr 



R94 



GIFT SHOP VOLUNTEERS 



Marion Adeiburg 
Beth Berne 
Avis Blomberg 
Gloria Borgese 
Julie Child 
Janet Daniels 
Carol De Young 
Frances Eastman 
Pat Ferguson 
Barbara Grossman 
Jean Halvorson 
Hanna Hastings 
Marcella Katz 
Donna Kornberg 
Barbara Little 
Florence Mixer 
Bertha Person 
Julie Ranlcin 
Arlene Rogers 
Atholie Rosen 
Cynthia Smith 
Alice Todd 
Natalie Trousof 
Clare Wilber 
Betty Wilson 
Grace Witzell 
Bunnie Zigman 



TOUR GUIDES 



Gloria Borgese 
Frank Child 
Nancy Fraser 
Sallie Giffen 
Ronald Glantz 
Charles Mahoney 
Haskell Maude 
Andrew McArthur 
Vivagean Merz 
Carl Ollmer 
William Philips 
Julie Rankin 
Howard Redpath 
Sheila Silverberg 
Mary Ulbrich 
John Valois 




Neuron. Peter Koulen 



Associates, continued 

Mr Paul J. Freyheit 

Mrs Ruth E Fye 

Mr Joseph C Gallagher 

Mrs Lois E Galvin 

Miss Lan Ge 

Mrs Matilda L Gellis 

Mrs Dons D Gerace 

Ms Sallie Giffen 

Mrs. Janet F Gillette 

Mr Michael P Goldring 

Ms Muriel Gould 

Mrs Winifred M Green 

Ms Janet M Gregg 

Mrs Jeanne B Griffith 

Mrs Barbara Grossman 

Mrs Valerie A Hall 

Ms Mary Elizabeth Hamstrom 

Mrs Jane M Heald 

Mr Mark Hollander 

Mrs Betsy Honey 

Mr Roger W Hubbell 

Ms Alexandra Izzo 

Dr Diana E Jennings 

Mrs Megan H. Jones 

Ms Barbara W Jones 

Mrs Joan T Kanwisher 

Mrs Sally Karush 

Ms Patricia E Keoughan 

Dr Peter N Kivy 

Ms Kathryn M Kleekamp 

Ms Margaret D Lakis 

Ms Meryl Langbort 

Ms Rebecca Lash 

Mr William Lawrence 

Dr Marian E LeFevre 

Mr Edwin M Libbin 

Mr Lennart Lindberg 

Mrs. Barbara C Little 

Mrs. Sarah J Loessel 

Ms Susan Loucks 

Mr Jeremy Loyd 

Dr Zella Luria 

Ms Sallie G Lyon 

Mrs Margaret M MacLeish 

Mrs Annemarie E Mahler 

Mrs. Nancy R Malkiel 

Mr Brett Mandel and 

Ms Laura Wembaum 
Mrs Diane B Maranchie 
Mrs Marjorie Marshall 
Mrs Mary Hartwell Mavor 
Mr Bill McGoey 
Mr Pauf McGonigle 
Dr Susan G Mcllwain 



Ms Mary W McKoan 

Ms Jane A McLaughlm 

Ms Louise McManus 

Ms Cornelia Hanna McMurtne 

Ms Paula A Mealy 

Dr Carmen Merryman 

Ms Vivagean V. Merz 

Mrs Marianne F Milkman 

Mrs Florence E Mixer 

Mr Ken Moffitt 

Mr Lawrence A Monte 

Ms Cynthia Moor 

RADM Paul J Mulloy USN (Ret) 

Mrs Carol Murray 

Mrs Eleanor M Nace 

Mrs Anne Nelson 

Dr Eliot H Nierman 

Mr Edmund F Nolan 

Ms Peggy Schiffer Noland 

Ms Catherine N Norton 

Dr Renee Bennett O'Sullivan 

Dr Jack S Parker 

Mr David Parker. Jr 

Ms Carolyn L Parmenter 

Ms Joan Pearlman 

Dr Daniel A Pollen 

Mr Barry Pratt 

Ms Elizabeth T Price 

Ms Dianne Purves 

Mrs Julia S Rankin 

Dr Magaret M Rappaport 

Mrs Carol V Rasic 

Mr Fred J Ravens, Jr 

Ms Mary W Rianhard 

Ms Andrea Ricca 

Dr Mary Elizabeth Rice 

Ms Sandy L Richardson 

Dr Monica Riley 

Mrs- Lola E Robertson 

Dr Steven R Rodermel 

Mrs Arlene Rogers 

Mrs Wendy E Rose 

Mrs Atholie K Rosett 

Dr Virginia F Ross 

Dr John D Rummel 

Mrs Rosalind Russell 

Dr Albert Samuel 

Mr Raymond A Sanborn 

Dr Thomas Sbarra 

Ms Mary M Scanlan 

Mr Claude Schoepf 

Mr Samuel C Schon 

Mrs Elsie M Scott 

Dr Cecily Cannan Selby 

Mrs Deborah G Senft 

Ms Dorothy Sgarzi 

Mrs Charlotte Shemin 



Mrs Ruth Shephard 

Mr Frank Shephard 

Ms Dorothy Shiebler 

Ms Enid K Sichel 

Dr Jeffrey D Silberman 

Mrs Phyllis J Silver 

Mrs. Cynthia C Smith 

Mrs Louise M Specht 

Dr Evelyn Spiegel 

Mrs Helene E Spurrier 

Dr Robert E Steele 

Mrs Eleanor Stembach 

Dr Malcolm S Steinberg 

Ms Eleanor Sterling 

Mrs. Jane Lazarow Stetten 

Mr Edward Stimpson, III 

Mrs Elizabeth Stommel 

Ms Maren Studlien 

Mr Albert H Swam 

Mr Dorman J Swartz 

Mr James K Taylor 

Mrs Alice Todd 

Mr Arthur D Traub 

Ms Natalie Trousof 

Mr Louis C Turner 

Ms Eleanor S Uhlmger 

Ms Ciona Ulbrich 

Ms Sylvia Vatuk 

Ms Dawn Vaughn 

Ms Susan Veeder 

Mr Lee D Vincent 

Mr Arthur D Voorhis 

Mrs Ann Wadsworth 

Mrs Eve Warren 

Mr. Michael S Wemstein 

Ms Lillian Wendonf 

Dr Gary Wessel 

Mr George R Wezntak 

Mr Gerry White 

Mrs Barbara Whitehead 

Mrs Clare M Wilber 

Mrs Helen Wilson 

Mrs Grace Witzell 

Ms Nancy Woitkoski 

Mr Dale Wolfgram 

Mrs Dorothy M York 

Dr Linda Amaral Zettler 

Mrs Bunnie Rose Zigman 

Mrs Margery P Zinn 



R95 




COUNCIL OF VISITORS 



The purpose of the Council of Visitors is to increase awareness of the Marine Biological Laboratory and to inform 
members about the broad range of activities in research and educational programs. COV members serve as 
ambassadores of the Laboratory, and thus raise visibility of fhe MBL. 



Mr Robert W Ashton 
Bay Foundation 
New York. New York 

Mr Donald J Bainton 
Continental Can Co 
Boca Raton, Florida 

Mr David Bakalar 

Chestnut Hi/I, Massachusetts 

Dr George P Baker 
Massachusetts General Hospital 
Boston, Massachusetts 

Dr Sumner A Barenberg 
Bernard Technologies 
Chicago, Illinois 

Mr. Mel Barkan 

The Barkan Companies 

Boston, Massachusetts 

Mr Frederick Bay 

Josephine Say Paul & C Michael Paul 

Foundation, Inc 

New York, New York 

Mr Robert P Beech 

Component Software International, Inc 

Mason, Ohio 

Mr George Berkowitz 
Legal Sea Foods 
Al/ston. Massachusetts 

Jewelle and Nathaniel Bickford 
New York, New York 

Dr Elkan R Blout 
Harvard Medical School 
Boston. Massachusetts 

Mr Malcolm K Brachman 
Northwest Oil Company 
Dallas. Texas 



Mitch Sogm, Elizabeth Armstrong 

Nosema locustae spores, Linda Amaral Zertler 



Dr Goodwin M Breinin 

New York University Medical Center 

New York, New Yorlt 

Mr Murray H Bring 
New York, New York 

Mr John Callahan 
Carpenter, Sheperd & Warden 
New Lone/on, New Hampshire 

Mrs. Elizabeth Campanella 
West Fafmouth, Massachusetts 

Dr R John Collier 
Harvard Medical School 
Boston, Massachusetts 

Dr Stephen D Crocker 
Bethesda, Mary/and 

Mr Michael J Cronin 
Cognition Corporation 
Bedford, Massachusetts 

Mrs. Lynn W Piasecki Cunningham 

Film and Videomaker, Piasecki Productions 

New York, New York 

Dr Anthony J Cutaia 
Anheuser-Busch, Inc 
St Louis, Missouri 

Dr, Lorenzo DiCarlo 

Mrs. Sally Stegeman DiCarlo 

Ann Arbor. Michigan 

Dr Charles Di Cecca 
Medford, Massachusetts 

Mr Diarmaid H Douglas-Hamilton 
Hamilton Thorne Research 
Beverly, Massachusetts 

Mr Benjamin F du Pont 
Du Pont Company 
Deepwater, New Jersey 



Dr Sylvia A Earle 

Deep Ocean Engineering 

Oakland. California 

Mr & Mrs Hoyt Ecker 
Vero Beach, Florida 

Mr Anthony B Evnin 
Venrock Associates 
New York, New York 

Mr Michael Fenlon 

Nathan Sallop Insurance Agency. Inc 

Boston, Massachusetts 

Judah Folkman, M D 
Children's Hospital 
Boston, Massachusetts 

Mrs Hadley Mack French 
Edsel & Eleanor Ford House 
Grosse Pomte Farms. Michigan 

Mr and Mrs Huib Geerlings 
Boston, Massachusetts 

Mr Maynard Goldman 
Maynard Goldman & Associates 
Boston, Massachusetts 

Ms Charlotte I Hall 
Edgartown, Massachusetts 

Ms Penelope S Hare 

West Falmouth, Massachusetts 

Mrs Elizabeth Heald 

West Falmouth, Massachusetts 

Dr Thomas R Hedges, Jr 

Neurological Institute, Pennsylvania Hospital 

Philadelphia. Pennsylvania 

Drs Linda Hirshman and David Forkosh 
Brandeis University & FMH Foundation 
Waltham, Massachusetts 



Continued 



R96 



COUNCILOR VISITORS MEETING 
JUNE 27, 28, 2002 



Modern Molecular Approaches to Global Infectious Diseases 
John R. David, M-D., Moderator 
Harvard School of Public Health 

A 21st Century Challenge: Using Genomics, Proteomics, and Molecular 
Immunology to Develop Vaccines for Malaria and Lung Cancer 

Stephen L. Hoffman, M.D. 

Senior Vice President, Biologies 

Celera Genomics 



AIDS in Africa: New Epidemics, New Viruses 

Max Essex, D.V.M., Ph.D. 

Chairman, Department of Immunology and Infectious Diseases 
Harvard School of Public Health 

The Conundrum of Tuberculosis and HIV/AIDS 

Jerrold J. Eilner, M.D. 

Director, Center for Emerging and Re-Emerging Pathogens 
New Jersey School of Medicine 




Mr Robert S Shifman 

St Simon's Island, Georgia 

Mr and Mrs Gregory Skau 
Grosse Pomte Farms, Michigan 

Mr John C Stegeman 
Campus Rentals 
Ann Arbor, Michigan 

Mr Joseph T Stewart, Jr 
Skillman, New Jersey 

Mr Richard H Stowe 
Capital Counsel LLC 
New York, New York 

Mr Gerard L Swope 
Washington, DC 

Mr John F Swope 
Concord, New Hampshire 

Mr and Mrs Stephen E Taylor 
Mi/ton Massachusetts 



Mrs Barbara W Hostetter 
Barr Foundation 
Boston, Massachusetts 

Mr Charles Hunter 
Kessinger Hunter & Co 
Kansas City, Missouri 

Mr Thomas J, Hynes, Jr 
Meredith & Grew, Inc 
Boston, Massachusetts 

Mrs Mary D Janney 
Washington, D.C. 

Dr Morris J Karnovsky 
Harvard Medical School 
Boston, Massachusetts 

Ms Ellyn V Korzun 
Goldman, Sachs & Co 
New York, New York 

Mr and Mrs Robert Lambrecht 
Boca Grande, Florida 

Mr Rudy Landry 
Pocasset. Massachusetts 

Catherine C Lastavica, M D 
Tufts University School of Medicine 
Boston, Massachusetts 

Dr Anna Logan Lawson 
Daleville, Virginia 

Mr Joel A. Leavitt 
Boston, Massachusetts 

Mrs. Margaret Lilly 

West Falmouth, Massachusetts 

Mr. Richard Lipkin 
Shipan Capital 
New York. New York 



Mr Michael T Martin 
SportsMark, Inc 
New York, New York 

Mrs Christy Swift Maxwell 
Grosse Pomte Farms, Michigan 

Mr Ambrose Monell 

G Linger Vetlesen Foundation 

Palm Beach, Florida 

Dr Mark Novitch 
Washington, DC 

Mr David R Palmer 

David Ross Palmer & Associates 

Waquoit, Massachusetts 

Mr and Mrs Joseph P. Pellegnno 
Boston, Massachusetts 

Mr Robert Pierce, Jr 
Pierce Aluminum Co 
Franklin, Massachusetts 

Mr Manus A Robinson 
Fundamental Investors Ltd 
Key Biscayne, Florida 

Mr Edward Rowland 
Tucker Anthony. Inc. 
Boston, Massachusetts 

Mr Andrew E Sabin 
Sabin Metal Corporation 
East Hampton, New York 

Ms Linda Sallop 

Nathan Sallop Insurance Agency, Inc. 

Boston, Massachusetts 

Mr Gregory A Sandomirsky 

Mintz Levin Cohen Ferns Glovsky & Popeo, PC 

Boston, Massachusetts 

Dr Cecily C Selby 
New York. New York 



Mr Samuel Thome 

Thorne Trading Campany, LLC 

Manchester, Massachusetts 

Mrs Karen Tierney 
Wellesley, Massachusetts 

Mrs Donna Vanden Bosch-Flynn 
Spring Lake, New Jersey 

Mr Benjamin S Warren III 
Grosse Pomte Farms, Michigan 

Nancy B Wemstein. R.N 

The Hospice, Inc 

Glen Ridge, New Jersey 

Stephen S Wemstein, Esq 
Mornstown, New Jersey 

John C West. M D 
Danville, Pennsylvania 

Mr Frederick J Weyerhaeuser 
Beverly, Massachusetts 

Mrs Robin Wheeler 

West Falmouth, Massachusetts 

Ms Rosalind C Whitehead 
New York. New York 

Mrs Annette Williamson 
Forth Worth. Texas 



ADMINISTRATIVE SUPPORT STAFF 1 



R97 



[Biological Bulletin 
Greenberg, Michael J , 

Editor-in -Chief 
Hinkle. Pamela Clapp, 

Managing Editor 
Child. Wendy 
Gibson, Victoria R 
Schachmger, Carol H 



I Director's Office 
Speck. William T.. Director 

and Chief Executive Officer 
Donovan. Marcia H 

Equal Employment Opportunity 
MacNeil. Jane L 

Veterinarian Services 
Smolowitz, Roxanna. Campus 

Veterinarian 
Allen, Taylor 
Bonacci, Lisa' 
Dalpe. Heather 
Ehlen. Jill- 
Hancock, Amy 
Stukey. Jetley 



|Educational Programs 
Dawidowicz. Eliezar A , Director 
Hamel, Carol C 
Holzworth, Kelly 

Central Microscopy Facility and 
General Use Rooms 
Kerr, Louis M . Supervisor 
Histen, Gavin 7 
Inzina, Jessica- 
Luther, Herbert 
Ogomo. Christopher On' 
Parmenter, Marjone : 
Peterson, Martha B 
Scanlon, John ? 



(External Affairs 
Carotenuto, Frank C . Director 
Butcher, Valerie 
Faxon, Wendy P 
George, Mary 
Johnson, A Knstme 
Patch-Wing, Dolores 
Quigley. Barbara A 
Shaw, Kathleen L 

Associates Program 
Joslin, Susan 
Sgarzi. Dorothy J. 

Communications Office 
Hinkle. Pamela Clapp Director 
Hemmerdinger, Catherine 
Hartmann. Kelley' 
Hlista. Laurel' 
Langill, Christine' 
Liles, Beth R 
Mansfield, Samantha 
Mock-Munoz De Luna, Dana- 
Rullo. Gina 



[Financial Services Office 
Lane. Jr. Homer W. Chief 

Financial Officer 
McLean, David. Controller 
Mullen, Richard J , Manager, 

Research Administration 
Adams, Taryn 
Aguiar, Deborah 
Bliss, Casey M 
Brady, Caroline 
Crosby, Kenneth 
Griffin, Bonnie Jean 
Lancaster, Cindy 
Newman, Melissa 
Nunes, Kenda 
Solchenberger, Carolyn 
Stellrecht. Lynette 

Stock Room 

Schorer. Timothy M , Supervisor 

Galatzer-Levy. David : 

Olive, Jr, Charles W 

Purchasing 

Hall Jr, Lionel E., Supervisor 

Hunt, Lisa M 

Widdiss, Brittany' 



[Housing and Conferences 
Beckwith, Mary M , Director 
Fuglister. Charles K 
Grasso, Deborah 
Knjger, Sally J 
Livingstone. Suzanne 
Oldham. Pamela 
Pento, Diana 
Stackhouse. Barbara 
Wagner, Carol 

Housekeeping 
Bailey. Jeffrey Jr ; 
Barnes, Susan M 
Barron, Laura : 
Bernos. Jessica 
Chen, Zhi Xm 
Doherty. Bryant-' 
Johnston, David-" 
Hannigan, Catherine 
MacDonald. Cynthia C 
McNamara, Noreen M 
Santiago. Crystal 
Shum, Mei Wah 
Waterbury. Matthew-" 



|Human Resources 
Goux, Susan P, Director 
Damery, Angela" 
Houser, Carmen 
Snow, Linda 



| J. Erik Jonsson Center, NAS 
Carlisle, Ann-' 
Doherty. Joanne 
Ehchalt, Donald 
Shurtleff, Joan : 




Photo by Volker Steger 




Photo by Elizabeth Armstrong 



I Senior Staff 

Mary Beckwith, Director of Housing and Conferences 

Frank Carotenuto, Director of External Affairs 

Richard Cutler, Director of Facilities and Services 

Lenny Dawidowicz, Director of Education 

Susan Goux, Director of Human Resources 

Roger Hanlon, Director of the Marine Resources Center 

Pamela Clapp Hinkle. Director of Communications 

Homer Lane. Chief Financial Officer 

Andrew Mattox, Environmental Health and Safety Manager 

Richard Mullen. Manager of Research Administration 

Cathy Norton. Director of the MBL/WHOI Library 

William T Speck, Director and Chief Executive Officer 



Including persons who joined or left the staff during 2001 
Summer or temporary 



R98 



Support staff, continued 



Josephine Bay Paul Cents 
Comparative Molecular 
Biology and Evolution 
Lim, Pauline 
Nih.ll, Tara 



| Marine Resources Center 
Hanlon, Roger T, Director 
Maddux, Betty L S - 
Santore, Gabnelle 

Aquatic Resources Department 
Enos, Jr., Edward G , 

Superintendent 
Dimond, James L 2 
Grossman, William M 
Klimm III, Henry W 
Sexton, Andrew W 
Sterling, Andrew 2 
Sterling, Christian 2 
Sullivan, Daniel A. 
Tassman, Eugene 
Whelan, Sean P 

MRC Life Support System 
Mebane, William N , Systems 

Operator 
Carroll, James 
Hanley, Janice S 
Kuzirian, Alan 
Linnon, Beth 



IMBL/WHOI LIBRARY 

Norton, Catherine N , Director 
Uhlmger, Eleanor, Assistant 

Director, MBL Library 
Deveer, Joseph M 
Monahan, A Jean 
Nelson, Heidi 
Person, Matthew 
Reuter, Laura 
Riley, Jacqueline 
Stafford, Nancy 
Stout, Amy 
Walton, Jennifer 

Digital Processing Center 
Clark, Sarah 
Hadway, Nancy 
Mannix, Jessica 2 
Reuter, Laura 
Westburg, Joanne 



for Information Technology Division 

Inzina, Barbara, Network Manager 
Callahan, Michael- 
Cohen, Alex 2 
Dematos, Christopher 
Fournier, Pamela 
Jones, Patricia L 
Leary, Patrick 2 
Lowell, Gregory 
Mountford, Rebecca J 
Remsen, David P, 
Renna, Denis J 
Space, David B. 
Wagner, Paul 2 



NASA Center for Advanced 

Studies in the Space Life 

Sciences 

Blazis, Diana, Administrator 

Farrell, Heather 



(Safety Services 

Mattox, Andrew H , Environmental, 

Health, and Safety Manager 
Elder, Kristopher 2 

Research Space Administration 
Kaufmann, Sandra J 

Satellite/Periwinkle Children's 

Programs 

Robinson, Paulina H 2 

Butler, Meredith 2 

Camire, Aaron 2 

Guiffrida, Beth 2 

Halter, Sarah 2 

Karalekas, Nina 2 

Langill, Susan 2 

Leveque, Rachel 2 

Noonan, Brendan 2 

Noonan, Patrick 2 

O'Connor, Caitlin 2 

Orfila, Cecelia 2 

Plourde, Anna 2 

Sturbaum, Sonja 2 

Swetish, Margaret 2 

Swiniarski, Kathryn 2 

Thamm, Jennifer 

Urciuoli, Karen 2 

Vachon, Julia 2 



| Services, Projects and Facilities 
Cutler, Richard D , Director 
Enos, Joyce B 
Stackhouse, Aaron 2 

Apparatus 
Atwood, Paul 
Baptiste, Michael G 
Barnes, Franklin D 
Haskins, William A 
Pratt, Barry 

Transportation Services & Grounds 

Hayes, Joseph H., Supervisor 

Bailey, Jeffrey B 

Boucher, Richard L. 

Brereton, Richard S 2 

Clayton, Daniel 

Collins, Paul J 

Cutler, Matthew 2 

Duane, James 2 

Illgen, Robert F 

Malchow, Robert 2 

McHugh, Clare 2 

Mendoza, Guy 2 

Rogers, William 2 

Santoro, John 2 

Plant Operations and Maintenance 

Abbott, Thomas E., Supervisor 

Anderson, Lewis B 

Atwood, Paul R 

Auclair, Donald 2 

Bailey, Jeffrey B 

Barnes, John S 

Bryant, Horace 2 

Cadose, James W. 

Callahan, John J 

Cormier, Garrett 2 

Elias, Michael 

Ficher, Jason 

Goehl, George 

Gonsalves, Jr, Walter W. 

Henderson, Jon R 

House, James 

Howell, Robert 

Langill, Richard 

Laurmo, Frank 

Mancevice, Connne 2 

McAdams III, Herbert M 

McHugh, Michael O 

McQuillan, Jeffrey 2 



Mendoza, Duke 
Mills, Stephen A 
Pratt, Barry 
Rattacasa, Frank 2 
Settlemire, Donald 
Shepherd, Denise M 
Sullivan, Brendon 
Toner, Michael 
Ware, Lynn M 

Security and Projects 
Fleet, Barry M , Manager 
Blunt, Hugh F 
Fish Jr, David L 
Hathaway, Peter J. 
Kelley, Kevin 
Lochhead, William M 
Rozum, John 
Scanlan, Melanie 



| The Ecosystems Center 
Cave, Anthony, Center Research 

Administrator 
Nunez, Guillermo, Center 
Research Administrator 
Berthel, Dorothy J. 
Donovan, Suzanne J 
Seifert, Mary Ann 



ARINE IXESOURCES CENTER 



MARINE BIOLOGICAL LABORATORY WOODS HOLE. MA 02543 (508)289-7700 
WWW.MBL.EDU/SERVICES/MRC/INDEX.HTML 




Animal and Tissue Supply for 
Education & Research 

150 aquatic species available for shipment via 
online catalog: <http://vwwv.mbl.edu/animals/ 
index.htmb; phone: (508)289-7375; or 
e-mail: specimens@mbl.edu 

zebrafish colony containing limited mutant strains 

custom dissection and furnishing of specific organ 
and tissue samples 



zebrafish facilities 



MRC Services Available 

basic water Quality analysis 

veterinary services (clinical, histopathologic, 
microbial services, health certificates, etc.) 

aquatic systems design (mechanical, biological, 
engineering, etc.) 

educational tours and collecting trips aboard 
the R/V Gemma 



Using the MRC for Your Research 

capability for advanced animal husbandry (temperature, light control, etc.) 

availability of_year-round, developmental life stages 

adaptability of tank system design for live marine animal experimentation 





New 




The evolution in fluorescence microscopy 




ApoTome. Nothing less than a minor 
revolution in fluorescence microscopy. 

In z-direction, the ApoTome 
increases visible resolution by a 
factor of 2. Now you can display 2D 
& 3D images of optical sections 



^ 



collected from the full sample with 
maximum contrast & optimal image 
quality. Even with the thick 
specimens of cell research. 

For more information, 
call 800.233.2343. 



Carl Zeiss Microlmaging, Inc. Thornwood, NY 10594 micro@zeiss.com zeiss.com/micro 



ZEISS 



We make it visible. 



October 200:? 



Volume 205 Number 2 



BIOLOGICAL 
BULLETIN 



Published by the Marine Biological Laboratory 



www.biolbuW.org 




THE BIOLOGICAL BULLETIN 

ONLINE 

The Marine Biological Laboratory is pleased beginning with the October 1976 issue 

to announce that the full text of The Biological (Volume 151, Number 2), and some Tables of 

Bulletin is available online at Contents are online beginning with the 

October 1965 issue (Volume 129, Number 2). 



http://www.biolbull.org 

The Biological Bulletin publishes outstanding 
experimental research on the full range 
of biological topics and organisms, from the 
fields of Neurobiology, Behavior, Physiology, 
Ecology, Evolution, Development and 
Reproduction, Cell Biology, Biomechanics, 
Symbiosis, and Systematics. 

Published since 1897 by the Marine 
Biological Laboratory (MBL) in Woods Hole, 
Massachusetts, The Biological Bulletin is one 
of America's oldest peer-reviewed scientific 
journals. 

The journal is aimed at a general readership, 
and especially invites articles about those 
novel phenomena and contexts characteristic 
of intersecting fields. 

The Biological Bulletin Online contains the 
full content of each issue of the journal, 
including all figures and tables, beginning 
with the February 2001 issue (Volume 200, 
Number 1). The full text is searchable by- 
keyword, and the cited references include 
hyperlinks to Meclline. PDF files are available 
beginning in February 1990 (Volume 178, 
Number 1), some abstracts are available 



Each issue will be placed online 
approximately on the date it is mailed to 
subscribers; therefore the online site will be 
available prior to receipt of your paper copy. 
Online readers may want to sign up for the 
eTOC (electronic Table of Contents) service, 
which will deliver each new issue's, table of 
contents via e-mail. The web site also 
provides access to information about the 
journal (such as Instructions to Authors, the 
Editorial Board, and subscription 
information), as well as access to the Marine 
Biological Laboratory's web site and other 
Biological Bulletin electronic publications. 

The free trial period for access to The 
Biological Bulletin online has ended. 
Individuals and institutions who are 
subscribers to the journal in print or are 
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SEEKERS 
THE SOCIETY 
OF CELLS. 



At Dr. Simon Watkins' lab, they look at cells 
the way anthropologists look at human culture: 
as communities of good guys and bad guys, 
of traders and communicators, of connections 
and relationships. "We are the observers," 
Simon says. "We never jump to conclusions. We let the conclusions jump 
to us." His mantra? "Imaging is everything." Which is why the best and 
the brightest of tomorrow's seekers and solvers find their way to Pittsburgh 
and the Watkins Lab. 



ROCKET SCIENCE 



.CO 



^/microscope 



800455-8236 



OLYMPUS 



{From L to R) 

Ana Bursick - Research Specialist 
Stuart Shond Research Specialist 
Simon C. Watkins, Ph.D. - Director 
Glenn Papworth - Research Associate 
Romesh Droviam - Graduate Student 
Center lor Biologic Imaging, 
University of Pittsburgh Medical School, 
Pittsburgh, PA 




Cover 



Mature eggs of the surf clam Spisula solidissima are 
arrested in prophase of the first meiotic division. 
When the eggs are activated by sperm or KC1, the 
first meiotic division is completed, the second mei- 
otic division follows, and then (with sperm) embry- 
onic development ensues. As in all meioses, the 
cleavages are very eccentric, and each produces a 
small polar body. Because very large numbers of 
Spisula eggs can be activated simultaneously, and 
thus form their polar bodies in near synchrony, 
these eggs are an excellent model with which to 
study, not only the usual embryonic cell division, 
but also polar body formation an example of ex- 
tremely asymmetrical cytokinesis. 

In this issue of The Biological Bulletin (pp. 192- 
193), Rafal Pielak, Valeriya Gaysinskaya, and 
William D. Cohen report on the organization of 
F-actin and microtubules in meiotic stages that 
immediately precede the formation of polar bod- 
ies in Spisula eggs. The movements and locations 
of these structures were revealed by confocal 
fluorescence microscopy after appropriate stain- 
ing (F-actin, red-orange; microtubules. green; 
chromosomes, blue-violet). Four images from the 
report set upon a background of diagrammatic 
surf clams appear on the cover (see scale bars in 
Fig. p. 193). 

At aboi t 13 min after activation (23C), the meta- 
phasc ,s| e of the first meiotic division is already 
fully form, and eccentrically positioned; it then 
moves toward the cell surface. In the upper left 



image on the cover, microtubules of the peripheral 
aster curve outward along the F-actin-containing 
cortex, away from a microtubule-poor central re- 
gion. At about 20 min post-activation, with the aster 
diminishing, the chromosomes are now arranged in 
anaphase (upper-right image), and a "bulls-eye" 
F-actin ring the cytokinetic ring appears on the 
cortex (side view, upper right; computer-generated 
face view, lower left). Finally, at about 26 min 
post-activation, the peripheral nucleus and its re- 
maining centrosomal material enter the F-actin- 
poor center of the ring to produce the first polar 
body (lower- right image). 

These stages include critical activities particu- 
larly, docking of the spindle with the cell cortex, 
and signaling to generate the cytokinetic contractile 
ring that occur in all sexually reproducing animals 
by mechanisms yet unknown. But note that, at 
metaphase, the pattern and dimensions of the con- 
tact between the astral rays and the egg cortex 
approximate those of the F-actin ring at anaphase. 
This correspondence suggests that generation of the 
contractile ring is triggered by signals from the 
astral microtubule-cortex contact. 

Rafal Pielak and Valeriya Gaysinskaya were sum- 
mer 2003 research interns in Hunter College- 
Howard Hughes Medical Institute (HHM1) Under- 
graduate Biological Science Education Program at 
the Marine Biological Laboratory. The surf clam 
pattern on the cover was designed by William D. 
Cohen. The cover was designed by Beth Liles (Ma- 
rine Biological Laboratory). 



THE 

BIOLOGICAL BULLETIN 

OCTOBER 2003 



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Associate Editors 



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Online Editors 



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Invertebrates of the Woods Hole Region 
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RESEARCH NOTE 



CONTENTS 

VOLUME 205, No. 2: OCTOBER 2003 

PHYSIOLOGY AND BIOMECHANICS 



Seibel, Brad A., and Heidi M. Dierssen 

Cascading trophic impacts of reduced biomass in tin 1 
Ross Sea, Antarctica: |usl the tip of the iceberg? . . . 93 
Lee, Raymond W. 

Thermal tolerances of deep-sea hydrothermal vent 
animals from the Northeast Pacific. 98 



Hamdoun, Amro M., Daniel P. Cheney, and Gary N. 
Cherr 

Phenotvpic plasticity of HSP70 and HSP70 gene ex- 
pression in the Pacific oyster (Crassostrea gigas): impli- 
cations for thermal limits and induction of thermal 
tolerance . . 160 



NEUROBIOLOGY AND BEHAVIOR 



Robison, Bruce H., Kim R. Reisenbichler, James C. 
Hunt, and Steven H. D. Haddock 

Light production by the arm tips of the deep-sea 
cephalopod Vampyroteuthu iii/midli* 1(11.' 



SHORT REPORTS FROM THE 2003 GENERAL 

SCIENTIFIC MEETINGS OF THE MARINE 

BIOLOGICAL LABORATORY 



The Editor 

The MBL Awards for y 103 



I>l \l I nl'MI M U BlOl.OC.Y 



175 



ECOLOGY OF PARASITES 

Fingerut, Jonathan T., Cheryl Ann Zimmer, and Rich- 
ard K. Zimmer 

Patterns and processes of larval emergence in an 
estuarine parasite system 1 



DEVELOPMENT AND REPRODUCTION 

Gibson, Glenys D. 

Larval development and metamorphosis in I'l/'iixi- 
branchaea maculata, with a review of development in 
the Notaspidea (Opisthobranchia) PJ1 



ECOLOGY AND EVOLUTION 

Chadwick-Furman. Nanette E., and Irving L. Weissman 

Effects of allogeneic contact on life-history traits of 
the colonial ascidian Botryllm schlossen in Monterey 

Bav 133 

BeU, J. J., and D. K. A. Barnes 

Effect of disturbance on assemblages: an example 
using Porifera 144 



Gilland. Edwin, Robert Baker, and Winfried Denk 

Long duration three-dimensional imaging of calcium 
waves in zebrafish using mnltiphoton fluorescence 
microscopy 1 7(> 

Gileadi, Opher, and Alon Sabban 

Squid sperm to clam eggs: imaging wet samples in a 
scanning electron microscope 177 

Wadeson, P. H., and K. Crawford 

Formation of the blastoderm and yolk syncytial layer 

in early squid development 179 

Crawford, K. 

Lithium chloride inhibits development along the an- 
imal vegetal axis and anterior midline of the squid 
embryo 181 

Hill, Susan D., and Barbara C. Boyer 

HXK-1 N-CAM immunoreactivitv correlates with cil- 
iary patterns during development of the polychaete 
Capitella sp. I 182 



('.ill 



Heck, D. E., and J. D. Laskin 

Rvanodine-sensitive calcium llux regulates motility of 
Arbaciri punctulata sperm ...................... 185 

Gallant, P. E. 

Axotomv inhibits the slow axonal transport of tubulin 

in the squid giant axon ....................... 187 



Delacruz, John, Jeremiah R. Brown, and George M. 
Langford 

Interactions br-t' i ombinant conventional 
squid kinesin . 'c myosin-V 188 

DeSelm, Carl f vaiiah R. Brown, Renne Lu, and 

George M. Lai >id 

Ral>GDI i! its invosin \'-dependent vesicle trans- 
port in. ^q.i,a giant axon 190 

Pielak, R, M., V. A. Gaysinskaya, and W. D. Cohen 
Cytoskeletal events preceding polar bodv formation 
in activated Spisula eggs 192 

Shribak, Michael, and Rudolf Oldenbourg 

Three-dimensional birefringence distribution in re- 
constituted asters of Spisula oocytes revealed by 
scanned aperture polarized light microscopy 194 

Wollert, Torsten, Ana S. DePina, Carl J. DeSelm, and 

George M. Langford 

Rho-kinase is required for myosin-II-mediated vesicle 
transport during M-phase in extracts of clam oo- 
cytes 195 

Cusato, K., J. Zakevicius, and H. Ripps 

An experimental approach to the study of gap-junc- 
tion-mediated cell death 197 

Tepsuporn, S., J. C. Kaltenbach, W. J. Kuhns, M. M. 

Burger, and X. Fernandez-Busquets 

Apoptosis in Microrinna prolifrra allografts 199 

Armstrong, Peter B., and Margaret T. Armstrong 
The decorated clot: binding of agents of the innate 
immune system to the fibrils of the Limuhis blood 
clot 1201 

Isakova, Victoria, and Peter B. Armstrong 

Imprisonment in a death-row cell: the fates of mi- 
crobes entrapped in the Limit/in blood clot 203 

Harrington, John M., and Peter B. Armstrong 

A liposome-permeating activity from the surface of 
the carapace of the American horeshoe crab, Limuhis 
jjolyphemus 205 



NEUROBIOLOGY AMI BEHAVIOR 



Bogorff, Daniel J., Mark A. Messerli, Robert P. Mai- 
chow, and Peter J. S. Smith 

Development and characterization of a self-referenc- 
ing glutamate-selective micro-biosensor 207 

Chappell, R. L., J. Zakevicius, and H. Ripps 

Zinc modulation of hemichannel currents in Xenopits 
oocytes 209 

Zottoli, S. J., O. T. Burton, J. A. Chambers, R. Eseh, 

L. M. Gutierrez, and M. M. Kron 

Transient use of tricaine to remove the telencepha- 
lon has no residual effects on physiological record- 
ings of supramedullary/dorsal neurons of the cun- 
ner, Tautogplabrui adspersus 211 

Redenti, S., and R. L. Chappell 

Zinc chelation enhances the sensitivity of the ERG 
b-wave in dark-adapted skate retina 213 



Molina, Anthony J. A., Katherine Hammar, Richard 
Sanger, Peter J. S. Smith, and Robert P. Malchow 

Intracellular release of caged calcium in skate hori- 
zontal cells using fine optical fibers 215 

Palmer, L. M., B. A. Giuffrida, and A. F. Mensinger 
Neural recordings from the lateral line in free-swim- 
ming toadfish, Opsriiiu\ inu 21(i 

Child, F. M., H. T. Epstein, A. M. Kuzirian, and D. L. 

Alkon 

Memorv reconsolidation in Hermssenda 218 

Kuzirian, A. M., F. M. Child, H. T. Epstein, M. E. Motta, 

C. E. Oldenburg, and D. L. Alkon 

Training alone, not the tripeptide RGD, modulates 
calexcitin in Hennissenda 220 

Savage, Anna, and Jelle Atema 

Neurochemical modulation of behavioral response 

to chemical stimuli in Hoinants americanus 222 

Mann, K. D., E. R. Turnell, J. Atema, and G. Gerlach 
Kin recognition in juvenile zebrafish (Danio rerio) 
based on olfactory cues 224 

Turnell, E. R., K. D. Mann, G. G. Rosenthal, and G. 

Gerlach 

Mate choice in zebrafish (Danio rerio) analyzed with 
video-stimulus techniques 225 

MOLECULAR BIOLOGY, PATHOLOGY, AND MICROBIOLOGY 

Roberts, S. B., and F. W. Goetz 

Expressed sequence tag analysis of genes expressed 

in the bay scallop, Argopecten irradians 227 

Hsu, A. C., and R. M. Smolowitz 

Scanning electron microscopy investigation of 
epizootic lobster shell disease in Homarus ameri- 
canus 228 

Orchard, Elizabeth, Eric Webb, and Sonya Dyhrman 
Characterization of phosphorus-regulated genes in 
Trichodesmium spp 230 

Galac, Madeline, Deana Erdner, Donald M. Anderson, 

and Sonya Dyhnnan 

Molecular quantification ot toxic Alexundriiim fundf- 
I'IIM- in the Gull of Maine 231 

Sangster, C. R., and R. M. Smolowitz 

Description of Vibrio alginolyticus infection in ml- 
tured Sepia officinalis, Sepin tipamn, and Si'pi/i phara- 
onis. 233 

Baird, Krystal D., Hemant M. Chikarmane, Roxanna 

Smolowitz, and Kevin R. Uhliiiger 

Detection of Edwardsiella infections in Opsanus tauby 
polvmerase chain reaction 235 

Weidner, Earl, and Ann Findley 

Catalase in microsporidian spores before and during 
discharge 236 

ECOLOGY A.\<D POPULATION BIOLOGY 

Agnew, A. M., D. H. Shull, and R. Buchsbaum 

Growth of a salt marsh invertebrate on several species 

of marsh grass detritus 238 



Cavatorta, Jason R., Morgan Johnston, Charles Hopkin- 
son, and Vinton Valentine 

Patterns of sedimentation in a salt marsh-dominated 
estuary 239 

Thonis, T., A. E. Giblin, K. H. Foreman 

Multiple approaches to tracing nitrogen loss in the 
\\Vst Falmouth wastewater plume 242 

Talbot, J. M., K. D. Kroeger, A. Rago, M. C. Allen, and 

M. A. Charette 

Nitrogen flux and speciation through the subterra- 
nean estuary of Waquoit Bay, Massachusetts 244 

Abraham, D. M., M. A. Charette, M. C. Allen, A. Rago, 

and K. D. Kroeger 

Radiocheniical estimates of submarine groundwater 
discharge to Waquoit Bay, Massachusetts 245 

Johnston, M. E., J. R. Cavatorta, C. S. Hopkinson, and 

V. Valentine 

Importance of metabolism in the development of salt 
marsh ponds 248 

Agniar, A. B., J. A. Morgan, M. Teichberg, S. Fox, and 

I. Valiela 

Transplantation and isotopic evidence of the relative 
effects of ambient and internal nutrient supply on 
the growth of L'lva lactuca 250 



Morgan, J. A., A. B. Aguiar, S. Fox, M. Teichberg, and 
I. Valiela 

Relative influence of grazing and nutrient supply on 
growth of the green macroalga Ulva lactuca in estu- 
aries of Waquoit Bay, Massachusetts 252 

O'Connell, C. W., S. P. Grady, A. S. Leschen, R. H. 

Carmichael, and I. Valiela 

Stable isotopic assessment of site loyalty and relation- 
ships between size and trophic position of the Atlan- 
tic horseshoe crab, I./i/iii/u\ /mly/ilti'iiius, within Cape 
Cod estuaries 254 

Walker, C., E. Davidson, W. Kingerlee, and K. Savage 
Incubation conditions of forest soil yielding maxi- 
mum dissolved organic nitrogen concentrations and 
minimal residual nitrate 256 

Holden, M. T., C. Lippitt, R. G. Pontius, Jr., and C. 

Williams 

Building a database of historic land cover to detect 
landscape change 257 



ORAL PRESENTATIONS 
Published bv title onlv. . 



259 



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Reference: Bid/. Bull. 205: 93-97. (October 2003 1 
2003 Marine Biological Laboratory 



Cascading Trophic Impacts of Reduced Biomass in the 
Ross Sea, Antarctica: Just the Tip of the Iceberg? 

BRAD A. SEIBEL* AND HEIDI M. DIERSSENt 
Montere\ Ba\ Aquarium Research Institute, Moss Landing, California 95039 



A significant reduction in phytoplankton biontass in the 
Ross Sea was reported in the austral summer of 2000-2001, 
a possible consequence of a disruption in sea-ice retreat 
due to the presence of an immense iceberg, B15 (1) (Fig. 1 }. 
Our obsen'ations in McMurdo Sound suggest temporally 
and trophically cascading impacts of that depression in 
productivitv. Reduced phytoplankton stocks clearly affected 
the pteropod Limacina helicina (Phipps. 1774} (Gastro- 
poda: Mollusca), an abundant primary consumer in the 
region (2, 3), as indicated by depressed metabolic rates in 
2000-2001. The following season, for the first time on 
record. L. helicina was absent from McMurdo Sound. Many- 
important predators, including whales and fishes, rely- 
heavily on L. helicina for food (3, 4). However, most obvi- 
ouslv impacted by its absence was Clione antarctica (Smith, 
1902). an abundant pteropod mollusc (Gastropoda) that 
feeds exclusively on L. helicina (5). Metabolic rates ofC. 
antarctica were depressed by 50% in 2001-2002. Both L. 
helicina and C. antarctica are important components of 
polar ecosystems and may be good indicators of overall 
ecosvstem "health " in McMurdo Sound and perhaps in the 
Ross Sea. In this last austral summer. 2002-2003, sea-ice 
extent was much higher and phytoplankton stocks were 
dramaticall\ lower than any reported previously, effects 
possiblv associated with El Nirio conditions, and we hypoth- 
esize that pteropods and their consumers may be further 
impacted. 

In the Southern Ocean, phytoplankton production is 
linked strongly to the seasonal oscillations in the extent of 
the sea ice (6. 7) and survival of higher trophic levels is 



Received 20 November 2002; accepted 21 July 2003. 

* To whom correspondence should be addressed. Current address: 100 
Flagg Road. Biological Sciences Center. Biological Sciences Department. 
University of Rhode Island. Kingston. RI 02881. E-mail: seibel@uri.edu 

t Current address: Department of Marine Sciences. University of Con- 
necticut at Averv Point. 1080 Shennecosset Road, Groton. CT 06340. 



dependent on reproductive cycles that are synchronous with 
phytoplankton blooms. This is especially true of the direct 
food link between L. helicina and C. antarctica. L helicina 
lives and feeds in the water column by extending a web of 
mucus that traps phytoplankton and. to a lesser extent, small 
zooplankton (3). L. helicina is the exclusive food source of 
C. antarctica throughout the life cycle, and the two species 
have parallel life histories. They grow in concert, with the 
preferred prey size increasing with predator size (3). Such 
specificity within the context of a highly seasonal environ- 
ment requires precise timing to ensure that predator and 
prey coexist. The coevolved predator-prey relationship be- 
tween L helicina and C. antarctica provides a unique 
opportunity to study the ecological and trophic conse- 
quences of a depression in primary productivity in the Ross 
Sea. 

A 50<7r to 75% reduction in phytoplankton biomass, es- 
timated as chlorophyll a (Chi) concentrations, and high 
sea-ice cover was observed in December 2000-2001 rela- 
tive to previous years (Table 1; Fig. 2: 8). A limited bloom 
did form by February, but annual primary production was 
still only 60% of the previous year ( 1 ). We believe that the 
reduced phytoplankton stocks in 2000-2001 had pro- 
nounced impacts on the condition of primary consumers in 
the region, causing cascading effects through higher trophic 
levels in the following year. This assertion is supported here 
by a series of metabolic measurements made on L. helicina 
and C. antarctica between 1999 and 2002. 

Nutritional state is known to be among the primary de- 
terminants of metabolism in all organisms, including ptero- 
pods (3). and is especially important in the highly seasonal 
Antarctic environment (9. 10). Food availability will influ- 
ence, among other things, the rates of protein synthesis, 
oxygen consumption, growth, and reproduction (9-1 1). We 
collected L. helicina and C. antarctica at four sampling 
stations along Ross Island (Fig. 1 ) and measured the oxygen 



93 



94 



B. A. SEIBEL AND H. M. DIERSSEN 




Figure 1. True-color imagery of McMurdo Sound and the iceberg 
B ISA in the Ross Sea. Antarctica, on 26 December 2001. Imagery is from 
the Moderate Resolution Imaging Spectroradiometer (MODIS) (33) at 
250-m resolution. Sites on Ross Island where pteropod specimens were 
collected are marked 1. McMurdo Station; 2. Cape Royds; 3, Cape Bird: 
and 4. ice edge. 



consumption rates of both species in January of 1999. 2001. 
and 2002. using end-point analysis as described previously 
(12, 13). The measurement temperature in all analyses was 



1.86 C. which is the year-round ambient temperature in 
McMurdo Sound. The oxygen consumption rates of L. 
helicina in 2001 were reduced by more than 30% relative to 
those measured in 1999 (Table 1). This reduction was 
presumably a result of food deprivation due to reduced 
phytoplankton stocks, although we cannot rule out a possi- 
ble additional influence of changes in food quality (i.e., 
species composition may also have changed from 1999 to 
2001). The following season, phytoplankton stocks were 
elevated; but for the first time on record (see below), L. 
helicina was not found at any station sampled. 

As a monophagous predator, C. antarcticu was heavily 
impacted by the absence of its prey in McMurdo Sound. The 
oxygen consumption rates measured for this species in 2002 
are only 50% of those measured in previous years (Table 1; 
Fig. 3). We also conducted laboratory experiments in 2001 
in which specimens of C. cmiarctica were deprived of food 
for 3 weeks. Over the first 14 days, metabolic rates declined 
gradually to about 50% of control (wild-caught and labora- 
tory-fed animals) levels. The 2002 rates correspond closely 
to those of individuals deprived of food in 2001, strongly 
supporting the suggestion that the depressed rates resulted 
from the extended absence of L. helicina in the region. 

C. antarctica. like many polar zooplankton (14. 15). 
accumulates large lipid stores (5% wet mass) during the 
productive spring and summer months, presumably for sur- 
vival through the winter and production of eggs that are 
released the following spring (16). With a depressed meta- 
bolic rate of 0.99 /Limol (0.022 ml )O 2 g~' h~' (Table 1 ), an 
oxy-calorific conversion of 4.7 kcal 1~' O 2 , and an energy 
content of 9.4 kcal g~ ' lipid, a 100-mg animal could survive 
nearly 6 months on lipid alone, but at the expense of 



Impacts of reduced hiotnass tin trophic dynamit N 



Table 1 



I997-199X 1998-1999 1999-2000 



2000-2001 



2001-2002 2002-2003 



Mean chlorophyl a ling m" ') in the Western Ross Sea 

(See Fig. 2 for details) 
December 
January 

Fraction of the Western Ross Sea covered with sea ice 

[See Fig. 2 for details) 1 
December 
January 

Oxygen consumption rate (jumol O 2 g~'h~'), mean SE(); 

data pooled from all collection sites (Fig. 1 ) : 
Limacina 
Clione 
Clmne starved 



2.1 
1.6 



0.72 
0.52 



3.9 
1.5 



0.50 
0.29 



n.d. 5.51 0.4(12) 
n.d. 1.93 0.21 (10) 



3.4 
3.1 



0.31 
0.16 



n.d. 
n.d. 



1.0 

2.2 



0.66 

0.57 



5.4 
3.4 



0.30 
0.20 



0.56 
0.56 



0.88 
0.78 



3.78 0.20(22)* absent present 

2.04 0.12 (31) 0.99 0.05 (30)* present 
0.96 0.10(7)* 



1 Sea ice cover determined as the fraction of Western Ross Sea not covered by open water, as shown in Fig. 2. 

2 n.d., no data; "indicates that oxygen consumption rates were significantly different from those in 1998-1999 (P < 0.01). 



TROPHIC IMPACTS OF REDUCED B1OMASS IN THE ROSS SEA 



95 



A) Dec. 1997 



B) Dec. 1998 



C) Dec. 1999 

1 70 e 180 E <7n " W 




-1 -0.5 0.5 1 

log chlorophyll a (mg m 3 ) 



1.5 



Figure 2. Ross Sea chlorophyll a (Chi) concentrations, representing the monthly mean of sea-ice-free pixels 
at 9-km resolution, derived from satellite ocean color imagery obtained from Sea-viewing Wide Field-of-view 
Sensor (SeaWiFS; Level 3 Standard Mapped Image, Reprocessing #4) (33) for December 1997 (Al-2002 (F). 
Gray areas designate land and white areas indicate the presence of sea ice. The dashed magenta line represents 
Ihe average extent of sea ice determined from passive microwave satellite data (SSM/I NASA Team Algorithm). 
The sea ice extent and Chi data reported in Table 1 were determined from the area within this line. The location 
of the B15A iceberg is shown as a solid magenta shape. 



reproduction. A positive correlation between egg produc- 
tion and availability of food (i.e.. Limacina) has been dem- 
onstrated in the laboratory for C. limacina (3). 

L. helicina is typically abundant throughout the Southern 
Ocean, sometimes displacing krill as the dominant zoo- 
plankton (17). In McMurdo Sound. L. helicina may consti- 
tute more than 20% of the zooplankton biomass and reach 
concentrations exceeding 300 individuals per cubic meter 
along the ice edge ( 18, 19). L. helicina is also an important 
prey item for a number of other species in the Antarctic, 
including whales and myctophid and notothenioid fishes (4, 
20), themselves important components in the diet of pen- 
guins and mammals (21, 22). Although Clione limacina, the 
northern hemisphere congener of C. antarctica, has also 
been reported in the diet of fishes and whales (3), C. 
antarctica may have limited importance for higher trophic 
levels in McMurdo Sound because it produces a novel 
"anti-feedant" compound (19). However, both L. helicina 
and C. antarctica are functionally important components of 
the ecosystem with the potential to influence phytoplankton 
stocks (18), carbon flux (23). and dimethyl sulfide (DMS) 
levels (24) that, in turn, influence global climate through 



ocean-atmosphere feedback loops. The state of pteropod 
populations is almost certainly indicative of overall ecosys- 
tem "health" in McMurdo Sound, and perhaps throughout 
the Ross Sea. 

Large aggregations of both pteropod species were found 
at all four sampling stations (Fig. 1 ) in January of 1999 and 
2001. Equally large aggregations of both species have been 
reported in McMurdo Sound in every systematic zooplank- 
ton sampling study to date (2, 5, 18. 19, 25, 26). The 
Antarctic Biology Training Course sponsored by the U.S. 
National Science Foundation also confirmed an abundance 
of L. helicina in McMurdo Sound every year of its operation 
(1994-1996. 1999-2001; D. Karentz. University of San 
Francisco. California, pers. comm.). Thus, the absence of L. 
helicina in 2001-2002 appears to be unprecedented in Mc- 
Murdo Sound, although we cannot rule out the possibility 
that L. helicina was recruited from other parts of the Ross 
Sea later in the year. 

The absence of L. helicina in 2001-2002 may have re- 
sulted from food limitation. In the Arctic. L. helicina has a 
life cycle of 1 .5 to 2 years, and veliger larvae are most 
abundant in late summer to early fall (27). Assuming a 



96 



B. A. SEIBEL AND H. M. DIERSSEN 



O 
o 

o 



f 

o 
U 
c 



1 - 




-H 

0.01 



-H- 



Mass (g) 



Figure 3. Oxygen consumption rates of Clione antarctica plotted as a 
function of wet body mass. All rates were measured at 1 .86 C, the 
year-round ambient temperature in McMurdo Sound. The rates from ani- 
mals captured in 2002 (open circles, y = 0.43.v~ 028 ) were significantly 
lower than those measured in 2001 (black circles, y = 0.93*"-*) or 1999 
(grey circles) (ANCOVA; P < 0.01). Consumption rates of animals 
deprived of food in the laboratory in 2001 ( + ) are similar to those 
measured in 2002, supporting the suggestion that animals captured in 2002 
were suffering food deprivation due to the apparent absence of Liimicinu 
helicina in the region. 



similar life history for L. helicina in the Ross Sea, veligers 
there may not have metamorphosed and grown to adult sizes 
by summer 2001-2002. Relatively short delays in food 
availability are known to lead to failed metamorphosis of 
larval zooplankton (28). Unfortunately, we have no data 
outside of McMurdo Sound in 2002. An alternative hypoth- 
esis is that L. helicina was simply excluded from McMurdo 
Sound by changes in the local currents due to an immense 
iceberg, B15. a large fragment of which ran aground along 
the eastern edge of Ross Island in austral spring 2000-2001 
(Fig. 1 ). The iceberg and associated ice cover in 2001-2002 
may have prevented the typical flow of water from the Ross 
Sea gyre around Cape Bird and southward into McMurdo 
Sound (29). and this may have caused a more localized 
absence of L. helicina. This current typically carries the 
phytoplankton bloom, and presumably, pteropod popula- 
tions into McMurdo Sound. This explanation is consistent 
with the change in the position of the iceberg between 
2000-2001 and 2001-2002, but it is not supported by more 
recent observations. Substantial populations of both C. cint- 
arctica and L. helicina were found in McMurdo Sound in 
2002-2003 (Luke Hunt. Hopkins Marine Station, pers. 
comm.) even, though the iceberg continues to block the 
mouth of McMurdo Sound. 

A number of factors may have influenced the sea-ice 



conditions and thus contributed to the low biomass observed 
in 2000-2001. Among the most compelling is that the 
immense iceberg B15 prevented the retreat of pack ice out 
of the Ross Sea, causing a reduction in open water and a 
shortened growing season that delayed and stunted the 
phytoplankton bloom (1). However, substantial interannual 
variability exists in both sea-ice extent and phytoplankton 
production. For example, both 1997 and 2002 had high ice 
cover (Fig. 2; Table 1 ) even though the iceberg was no 
longer preventing the retreat of pack ice in those years. 
Phytoplankton biomass was reduced somewhat in 1997, but 
was dramatically reduced in 2002 (mean chlorophyll con- 
centration of 0.56 mg m 3 ). Interestingly, both 1997 and 
2002 experienced El Nino events that are known to influ- 
ence Antarctic waters (30). 

Continued monitoring is required to assess the causes of 
variability in Ross Sea phytoplankton stocks, the role of sea 
ice in the Southern Ocean ecosystem, and the resulting 
impacts on trophic interactions. Climate variations may 
further disrupt the timing of sea-ice formation and retreat 
(31, 32), and thus primary productivity, with consequences 
for entire food webs, as observed here. 

Acknowledgments 

We thank J. Rosenthal, F. Bezanilla, R. Dudley, J. Barry, 
B. Robison, J. Drazen. A. DeVries, L. Hunt, D. Karentz, W. 
Smith, and S. Kim for helpful discussions, comments on the 
manuscript, assistance in the field and laboratory, or both. 
The constructive comments of two anonymous reviewers 
greatly improved this manuscript. We thank Raytheon Polar 
Services and the National Science Foundation Office of 
Polar Programs for facilitating and funding this work. This 
work was additionally supported by the Monterey Bay 
Aquarium Research Institute and the NSF-sponsored Ant- 
arctic Biology Training Course and its instructors, including 
A. Marsh. D. Karentz. G. Somero, G. Hoffman, C. Mar- 
shall, L. Goff, and D. Manahan. 

Literature Cited 

1 Arrigo, K. R., G. L. van Dijken, I). G. Ainley, M. A. Fahnestock, 
and R. Markus. 2(102. Ecological impact of a large Antarctic 
iceberg. Geophys. Res. Lett. 29: 1-4. 

2. Foster, B. A. 1989. Time and depth comparisons of sub-ice zoo- 
plankton in McMurdo Sound. Antarctica. Polar Biol. 9: 431-435. 

3. Lalli, C. M., and R. W. Gilmer. 1989. Pelagic Snails: The Biology 
of Holoplanktonic Gastropod Molliixks, Stanford University Press. 
Stanford, CA. 

4. Foster, B. A., and J. C. Montgomery. 1993. Planktivory in benthic 
nototheniid fish in McMurdo Sound. Antarctica. Environ. Biol. Fishes 
36: 3 13-3 IS. 

5. Gilmer, R. W., and C. M. Lalli. 1990. Bipolar variation in Clione. 
a gymnosomatous pteropod. Am. Muliicol. Bull. 81: 67-75. 

6. Brierley, A. S., and D. N. Thomas. 2002. Ecology of southern 
ocean pack ice. Atl\: Mm. Biol. 43: 171-277. 

7. Smith, R. C., K. S. Baker, H. M. Dierssen, S. E. Stainmerjohn, and 



TROPHIC IMPACTS OF REDUCED BIOMASS IN THE ROSS SEA 



97 



M. Vernet. 2001. Variability of primary production in an Antarctic 
marine ecosystem as estimated using a multi-scale sampling strategy. 
.Am. Zoo/. 41: 40-56. 21. 

8. Gow, A. J., S. F. Acklev, J. W. Govoni, and W. F. Weeks. 1998. 
Physical and structural properties of land-fast sea ice in McMurdo 
Sound. Antarctica. Pp. 355-374 in Antarctic Sea Ice: Physical Pro- 
cesses, Interactions and Variability. Vol. 74. M.O. Jeffries, ed. Amer- 22. 
ican Geophysical Union. Washington D.C. 

9. Brockington. S., and A. Clarke. 2001. The relative influence of 23. 
temperature and food on the metabolism of a marine invertebrate. J. 

Exp. Mar. Biol. Ecnl. 258: 87-99. 
10. Peck, L. S. 1998. Feeding, metabolism and metabolic scope in 

Antarctic marine ectotherms. Pp. 365-390 in Cold Ocean Physiology. 

H. O. Former, and R. C. Playle. eds. Cambridge University Press. 24. 

Cambridge. 
1 I Ross, R. M.. L. B. Quetin, K. S. Baker, M. Vernet, and R. C. Smith. 

2000. Growth limitation in young Eitphausia superba under field 

conditions. Limnol. Oceangr. 45: 31-43. 25. 

12. Marsh. A. G., and D. T. Manahan. 1999. A method for accurate 

measurements of the respiration rates of marine invertebrate embryos 

and larvae. Mar. Ecol. Prog. Ser. 18-4: 1-10. 

1 3 Seibel, B. A., E. V. Thuesen, J. J. Childress, and L. A. Gorodezky. 26 
1997. Decline in pelagic cephalopod metabolism with habitat depth 
reflects differences in locomotory efficiency. Biol. Bull. 192: 262-278. 

14 Geiger, S. P., H. G. Kawall, and J. J. Torres. 2001. The effect of 27. 
the receding ice edge on the condition of copepods in the northwestern 
Weddell Sea: results from biochemical assays. Hydrobiologia 453- 

454: 79-90. 28. 

1 5 Hagen. W., E. S. Van Vleet, and G. Kattner. 1996. Seasonal lipid 
storage as overwintering strategy of Antarctic krill. Mar. Ecol. Prog. 

Ser. 134: 85-89. 29. 

16 Phleger, C. F., P. D. Nichols, and P. Virtue. 1997. Lipids and 
buoyancy in Southern Ocean pteropods. Lipids 32: 1093-1 100. 

17. Cabal, J. A., F. Alvarez-Marques, J. L. Acuna, M. Quevedo, R. 30. 
Gonzalez-Quiros, 1. Huskin, D. Fernandez, C. R. Del Valle, and R. 
Anadon. 2002. Mesozooplankton distribution and grazing during 

the productive season in the Northwest Antarctic Peninsula (FRUELA 
cruises). Deep-sea Res. II 49: 869-882. 31. 

18. Hopkins, T. L. 1987. Midwater food web in McMurdo Sound. Ross 
Sea. Antarctica. Mar. Biol. 96: 93-106. 

19 Bryan, P. J., VV. Y. Yoshida. J. B. McClintock, and B. J. Baker. 32 
1995. Ecological role for pteroenone, a novel antifeedant from the 
conspicuous antarctic pteropod Clione antarctica (Gymnosomata: 
Gastropoda). Mar. Biol. 122: 271-277. 33. 

20. Pakhomov, E. A., R. Perissinotto, and C. D. McQuaid. 1996. 



Prey composition and daily rations of myctophid fishes in the Southern 
Ocean. Mar. Ecol. Prog. Ser. 134: 1-14. 

Davis, R. W., L. A. Fuiman, T. M. Williams, S. O. Collier, W. P. 
Hagey, S. B. Kanatous, S. Kohin, and M. Horning. 1999. Hunting 
behavior of a marine mammal beneath the Antarctic fast ice. Science 
283: 993-996. 

Eastman, J. T. 1993. Antarctic Fish Biology: Evolution in a 
Unique. Environment. Academic Press, San Diego, CA. 
Noji, T. T., U. V. Bathmann, B. von Bodungen, M. Voss, A. Antia, 
M. Krumbholz, B. Klein, I. Peeken, C. I.-M. Noji, and F. Rev. 
1997. Clearance of picoplankton-sized particles and formation of 
rapidly sinking aggregates by the pteropod. Limacina retroversa. J. 
Plankton Res. 19: 863-875. 

Levasseur, M., M. D. Keller, E. Bonneau, D. D'Amours, and W. K. 
Bellows. 1994. Oceanographic basis of a DMS-related Atlantic cod 
(Gadus morluia) fishery problem: blackberry feed. Can. J. Fish. Aquat. 
Sci. 51: 881-889. 

Whitehead, K., D. Karentz, and J. I. Hedges. 2001. Mycosporine- 
like amino acids (MAAs) in phytoplankton, a herbivorous pteropod 
(Limacina helicina) and its pteropod predator (Clione antarctica) in 
McMurdo Bay, Antarctica. Mar. Biol. 139: 1013-1019. 
Knox, G. A., E. J. Waghorn, and P. H. Ensor. 1996. Summer 
plankton beneath the McMurdo ice shelf at White Island. McMurdo 
Sound, Antarctica. Polar Biol. 16: 87-94. 

Kobayashi, H. A. 1974. Growth cycle and related vertical distri- 
bution of the thecosomatous pteropod Spiratella ("Limacina") helicina 
in the central Arctic Ocean. Mar. Biol. 26: 295-301. 
Ross, R. M., and L. B. Quetin. 1989. Energetic cost to develop to 
the first feeding stage of Euphausia stiperba Dana and the effect of 
delays in food availability. J. Exp. Mar. Biol. Ecol. 133: 103-127. 
Barry, J. P., and P. K. Dayton. 1988. Current patterns in McMurdo 
Sound, Antarctica and their relationship to local biotic communities. 
Polar Biol. 8: 367-376. 

Comiso, J. C., C. R. McClain, C. W. Sullivan, J. P. Ryan, and C. L. 
Leonard. 1993. Coastal Zone Color Scanner pigment concentra- 
tions in the Southern Ocean and relationships to geophysical surface 
features. J. Geophys. Res. 98(C2):2419-2451. 
Scambos, T. A., C. Hulbe, M. Fahnestock, and J. Bohlander. 2000. 
The link between climate warming and break-up of ice shelves in the 
Antarctic Peninsula. J. G/aciol. 46: 516-530. 

Dierssen, H. M., R. C. Smith, and M. Vernet. 2002. Glacial 
meltwater dynamics in coastal waters west of the Antarctic Peninsula. 
Pmc. Natl. Acad. Sci. USA 99: 1790-1795. 

Yoder, J. A. 2000. Terra's view of the sea. Science 288: 1978- 
1980. 



Reference: Biol. Bull. 205: 98-101. (October 2003) 
2003 Marine Biological Laboratory 



Thermal Tolerances of Deep-Sea Hydrothermal Vent 
Animals From the Northeast Pacific 

RAYMOND W. LEE 
School of Biological Sciences, Washington State University. Pullman, Washington 99164 



Dense biological communities on suljide structures at 
deep-sea hvdrothermal vents survive in one of Earth's most 
extreme environments. The thermotolerance of vent animals 
dwelling on siilfide chimneys in the Northeast Pacific was 
determined b\ maintaining them in pressurized chambers 
under controlled temperature and chemical conditions. Ob- 
sen>ations indicated that lethal temperature correlates 
strongly with distributions observed in nature. One species 
studied, the alvinellid siilfide worm Paralvinella sulfincola, 
exhibited a thermal limit of 50-56 C. Since observations of 
survival under controlled conditions are the only unambig- 
uous means of demonstrating that an animal can tolerate a 
given environmental condition, the documented thermal 
limit for metazoan life at hydrothermal vents should be 
considered to be above 45 C, but less than 60 C. 

Although the biology of hydrothermal vents has been 
actively investigated over the past 20 years, delineating 
linkages between the physical environment and the biota 
has been difficult. Gradients and temporal changes are pro- 
nounced. Therefore, to know what conditions a vent organ- 
ism routinely encounters, measurements would ideally be 
conducted with spatial resolution at the sub-centimeter 
level, with temporal resolution over the course of days to 
weeks, and without modifying fluid flow by the presence of 
the sensor or submersible. Consequently, investigators are 
generally cautious in inferring physiological tolerance from 
environmental measurements. A recent study presents en- 
vironmental data suggesting that Alvinel/a pompejana. a 
vent-chimney alvinellid worm, lives under sustained tem- 
peratures of 60 C, which could make it the Earth's most 
thermotolerant metazoan ( 1 ). However, steep thermal gra- 
dients and the difficulty of sampling fragile alvinellid tubes 
from a submersible have raised questions about the validity 

Received 3 March 2003; accepted 24 June 2003. 



of these conclusions (2). For animals inhabiting less remote 
environments, corroborative evidence has often come from 
laboratory investigations of live animals. Such an approach 
has not been extensively used in studies of vent animals due 
to the requirement that experiments be conducted at high 
pressure. However, this kind of evidence is necessary to 
determine actual physiological limits. Documented survival 
under controlled conditions provides unambiguous evidence 
for thermotolerance. In the present study, this type of direct 
approach was taken to investigate the thermal tolerance of 
several species of vent animals. 

Some of the sulride-chimney assemblages at the Juan de 
Fuca and Explorer ridges in the Northeast Pacific are ideal 
for investigation of environmental tolerance since the dom- 
inant invertebrates, unlike those from other vent systems, 
are small and can fit in relatively inexpensive pressure 
vessels. Pressures at these sites (depths 1500-1800 m) are 
moderate, making it easier to maintain in situ pressure in 
experiments, and most organisms are motile, allowing be- 
havioral investigations. In the present study, thermal limits 
were investigated for four abundant species of chimney 
invertebrates: the paralvinellids Paralvinella sulfincola and 
P. pahnifonms, the limpet Lepetodrilus fucensis, and the 
snail Depressigyra globulus. 

The distributions of these organisms on vent chimneys 
were described by Sarrazin et al. (3) and exhibit a "zona- 
tion" pattern of distinct invertebrate assemblages (named I 
through V) that differ in temperature and flow characteris- 
tics (3). Assemblage I. closest to hot vent fluids, consists 
almost entirely of P. sulfincola, which suggests that this 
species may be the most thermotolerant metazoan at North- 
east Pacific vents. The second warmest assemblage, assem- 
blage II. is dominated by P. sulfincola and P. palmiformis. 
The gastropods L. fucensis and D. globulus are also found in 
assemblage II. but are more common, and dominant, where 



Illl RMAL TOLERANCES OF VENT ANIMALS 



99 



the influence of venting is weaker (assemblages III-V). It is 
not clear what factors govern the distribution of organisms 
on .sulride structures. It has been postulated that tolerance to 
abiotic factors such as high temperature or hydrogen sultide 
may be important regulators, but analysis of available en- 
vironmental data and faunal distributions indicates that less 
than 3()9r of the variance in species distribution can be 
accounted for by abiotic factors that have been measured so 
far (4). If thermal tolerance is a factor governing distribu- 
tions, then thermotolerance should be highest in P. sulfin- 
cola. followed by P. palmifimnis, then L. fucensis and D. 
globulus. Specimens of P. sulfincola were collected from 
assemblage I, P. palmiformis from assemblage II and III, 
and gastropods from assemblage III. Differences in thermal 
tolerance among collections were not tested for. It is likely 
that further study could reveal acclimation to microhabitat 
conditions. 

Observations of mixed assemblages of these species in 
pressurized chambers subjected to temperature increases are 
summarized in Figure 1. Each data point shown represents 
the outcome of a single experiment in one pressure cham- 
ber. The following endpoints were measured: ( 1 ) activity at 
experimental temperature by one or more individuals, with 
continued activity following return to low temperatures; (2) 
obvious reduction or cessation of activity, with restoration 
of activity following return to low temperatures; (3) activity 
at experimental temperature, but no activity at higher tem- 
peratures and no activity after return to low temperature in 
all individuals; or (4) no activity in all individuals, with no 
return of activity at lower temperatures. In some cases, 
category 2 was not readily observable; i.e., D. global us 
appeared to exhibit high activity or none at all. In some 
trials, the threshold temperature at which activity ceases was 
not monitored. In these cases, animals exhibited no activity 
at a given temperature, but the threshold may have been 
lower. These instances were designated as category 4. Ex- 
periments consisted of a temperature increase (10 C per 
hour) followed by return of temperature to 10-15 C. In 
some cases, multiple experiments (at sequentially higher 
maximum temperatures) were conducted on the same sets of 
individuals (for D. globulus. L. fucensis. and P. pabnifor- 
mis, 9 of 23, 8 of 17, and 2 of 8 experiments were from 
individuals exposed to two or three experimental tempera- 
ture increases; all other experiments and all P. siilfincola 
experiments consisted of a single elevated temperature ex- 
posure). It is possible that exposure of animals to more than 
one temperature increase could have resulted in lower ther- 
mal limits for activity in those experiments. 

Thermal tolerance was correlated with distributions ob- 
served in nature. The temperature above which activity 
ceased (with no recovery at lower temperatures) was inter- 
preted to be the thermal limit. L fucensis was the least 
thermotolerant, with reduction or cessation of activity be- 



o 1. active 

A 3. active/not active after 



d 2. reduced activity 
* 4. not active 



P. sulfincola 
P. palmiformis 
D. globulus 
L. fucensis 



O O AA 



O O OOO D D A 



O O 



10 20 30 40 50 

degrees Celsius 



60 



Figure 1. Effects of experimental temperature on activity of chimney 
invertebrates. The ROPOS submersible was used to collect animals from 
sultide structures on the Explorer Ridge at a depth of 1800 ms. Immediately 
upon arrival at the surface, animals were placed in 30-ml pressure cham- 
bers and returned to an in situ pressure of 2600 psi. Depressurization during 
recovery was unavoidable since pressurized recovery systems are only in 
the developmental stages. Within minutes after animals were placed in 
pressure chambers, activity was generally observed. Animals not repres- 
surized and kept at I aim also exhibited activity, but appeared to be less 
active than pressurized animals. For experiments, high-pressure liquid 
chromatography pumps were used to continuously perfuse (0.3 ml/min) the 
chambers with filtered seawater equilibrated with 20% oxygen at pH 8. 
Sulride was metered in to give concentrations of 100-200 micromoles 
liter" '. Chamber temperature was controlled using a programmable recir- 
culating waterbath. Temperature was maintained initially at 10-15 C for 
2-3 h. then ramped at a rate of 10 C/h to experimental temperatures. 
Chamber temperature was determined by monitoring an identical unpres- 
surized control chamber, perfused at the same rate as experimental cham- 
bers, using a Yellow Springs Instruments temperature probe (calibrated 
against a NIST-traceable digital thermometer). Activity was monitored 
through 1.25-cm diameter viewports, either by using a video camera or by 
direct observation. Each data point represents the outcome of a single 
experiment in one pressure chamber. Behavior categories 1 to 4 are 
described in the text. Experiments consisted of a temperature increase 
followed by return of temperature to 10-15 C. 



tween 30 and 35 C. D. globulus and P. palmiformis exhib- 
ited reduction or cessation of activity in the ranges of 35-40 
C and 40 C respectively. The sultide worm P. sulfincola 
was clearly highly thermotolerant. Activity did not cease 
until temperatures of 50-56 C were achieved (Fig. 2; 7/20, 
7/28 expts.). P. sulfincola survived sustained exposure to 45 
C on the order of 1 h or longer (Fig. 2; 8/2 expt.). This 
indicates that temperatures of 50 C or above, rather than 
exposure to lower temperatures, accounted for cessation of 
activity at 50-56 C (Fig. 2; 7/20, 7/28 expts.). Time-lapse 
video from experiments can be found at (http://www.wsu. 
edu/~rlee/sulfideworm/psulf.html). 

These findings are consistent with, and may account for, 
the distributions observed in nature, and indicate that these 
organisms inhabit microenvironments close to their thermal 



100 

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R 

7/20exptj 








I, 


' . 












bU - 

= sn 






X 


n 










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movement 
1 stopped 


.5 3U " 
I/I 

<S 40- 

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y 


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^ 


A~^ 


\ 




/ 




/ 


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5 


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-/ 


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o 


2 3 4 5 6 7 I 
hours 


3 9 10 



Figure 2. Temperature conditions from three experiments in pressure 
vessels containing Paralvinella sulfincola. Arrows denote time point at 
which cessation of activity was observed. 7/20 experiment observation 
of two animals: both ceased activity when temperature was 5 1 C. 7/28 
experiment observation of two animals: one stopped activity when tem- 
perature was 52 C; the second when temperature was 58 C. Gaps in 
temperature data indicate periods when temperature sensor was turned off. 
8/2 experiment observation of three animals: temperature increased to 45 
C, then held. Activity persisted for the duration of the experiment. 

limits. The mean temperature of 42 C (3) observed in the 
sulfide worm habitat is above or at the limit of temperatures 
tolerated by the other species and would explain why they 
are excluded from these areas. Average temperatures mea- 
sured in assemblage II, where all four species are encoun- 
tered, ranged from 17 to 37 C (4), which is within the 
experimental range tolerated by palm worms and can be at 
the limits tolerated by gastropods. Habitat temperature may 
explain why gastropods are more dominant in cooler assem- 
blages. 

It is possible that experimentally determined temperature 
limits underestimate the tolerance shown in situ. This pos- 
sibility is difficult to assess, but will be addressed in future 
tests of different conditions in pressure incubations (e.g.. 
low pH, elevated pCO 2 ) and experiments with animals 
recovered under pressure in pressurized recovery devices. In 
addition, trials of paralvinellid worms were conducted with 
their mucus tubes removed. It is possible that these tubes, 
which also contain mineral deposits, may provide some 
thermoprotective benefit. When animals are brought to the 
surface and repressurized, survival appears to be indefinite. 
At 1 atm, survival was a few days to weeks depending on 
species, indicating that pressure is required for long-term 
maintenance. Experiments in my laboratory and elsewhere 
have shown that vent animals collected from these and other 
sites survive for months in pressure chambers, even with 
periodic depressurization and repressurization to clean 
chambers or remove specimens. Additional study is under- 
way to determine how long animals can tolerate sustained 
(several days) and transient (several minutes) exposure to 



experimental treatments as well as behaviorally preferred 
temperatures. 

The limit of aquatic metazoan life is generally thought to 
be around 45 C. The results presented here for P. sulfincola 
represent the first conclusive evidence that a vent animal 
tolerates temperatures that exceed 40 C. The alvinellid 
Ah'inella pompejana had previously been reported to be the 
Earth's "hottest" living metazoan. based on a finding of 
sustained temperatures of 60 C in occupied A. pompejana 
tubes ( 1 ). In addition, a single individual of A. pompejana 
had been observed to survive brief exposure to 105 C when 
it crawled onto a submersible' s high-temperature probe (5). 
These findings are at odds with biochemical evidence indi- 
cating that the structure and function of enzymes and other 
macromolecules of A. pompejana are perturbed at temper- 
atures of 50 C or below (2, 6-10). The only previous study 
to directly investigate the thermal limits of live vent 
polychaetes shows that Hesiolyra bergi, which lives in the 
A. pompejana environment, does not tolerate temperatures 
above 40 C (11). Thus the thermal limit of alvinellids 
remains a contentious issue. While extreme temperatures 
may be present in the alvinellid environment, alvinellids 
may inhabit cooler microenvironments or receive only tran- 
sient pulses of extreme temperature, perhaps on the order of 
seconds. This disagreement may never be resolved. Unlike 
P. sulfincola, A. pompejana has not yet survived collection 
and recovery. 

P. sulfincola may be as thermotolerant as A. pompejana 
since it exhibits properties of high enzyme thermostability 
(12) and inhabits a similar niche on sulfide structures. Tem- 
peratures at P. sulfincola tube openings range as high as 
80-90 C (S.K. Juniper, University of Quebec at Montreal, 
pers. comm.). Thus the data presented here are probably 
representative of tolerances exhibited by animals living at 
the limits for metazoan life at deep-sea vents. Pressurized 
experiments are the only unambiguous means of testing for 
survival under documented temperature. A combination of 
detailed environmental measurements, biochemical studies, 
and observation under controlled conditions is needed to 
reliably assess the thermal limits of vent fauna. The inves- 
tigation of highly thermotolerant metazoans at deep-sea 
vents will remain an exciting area of investigation. The 
results shown here place the upper limit of aquatic animal 
life in the range of 45-55 C. 

Acknowledgments 

This work benefited greatly from the captain and crew of 
the R.V. Thompson: ROPOS submersible group; Bob Em- 
bley (expedition chief scientist); Amanda Bates (at-sea help 
and discussions); Ray Romjue, George Henry, and John 
Rutherford (vessel design and machining). Funding was 
provided by the National Science Foundation and the West 



THERMAL TOLERANCES OF VENT ANIMALS 



101 



Coast and Polar Regions National Undersea Research 
Center. 

Literature Cited 

1 . Cary, S. C.. T. Shank, and J. Stein. 1998. Worms bask in extreme 
temperatures- \ainrc 391: 545-546. 

2. Choaldonnr. P., C. R. Fisher. J. J. Childress, D. Desbruveres, D. 
Jnllivet. K. Zal. and A. Toulmond. 200(1. Thermotolerance and the 

"Pompeii worms." Mm: Ecol. Prog. Sri: 208: 243-245. 

3. Sarrazin. J., V. Rohigou, S. K. Juniper, and J. R. Delaney. 1997. 
Biological and geological dynamics over four years on a high-temper- 
ature sulride structure at the Juan de Fuca Ridge hydrothermal obser- 
vatory. Mar. Ecol. Prog. Sei: 153: 5-24. 

4. Sarrazin. J., S. K. Juniper, G. Massoth, and P. Legendre. 1999. 
Physical and chemical factors influencing species distributions on 
hydrothermal sulride edifices of the Juan de Fuca Ridge, northeast 
Pacific. Mar. Ecol. Prog. Ser. 190: 89-112. 

5. Chevaldonne, P., D. Desbruyeres, and J. J. Childress. 1992. 
. . . and some even hotter. Nature 359: 593-594. 

6. Dahlhoff, E., J. O'Brien, G. N. Somero, and R. D. Vetter. 1991. 
Temperature effects on mitochondria from hydrothermal vent inverte- 
brates: evidence for adaptation to elevated and variable habitat tem- 
peratures. Ph\s. Zool. 64: 1490-1508. 



7. Dahlhotf, E., and G. N. Somero. 1991. Pressure and temperature 
adaptation of cytosolic malate dehydrogenases of shallow- and deep- 
living marine invertebrates: evidence for high body temperatures in 
hydrothermal vent animals. J. Exp. Biol. 159: 473-487. 

8. Terwilliger, N. B., and R. C. Terwilliger. 1984. Hemoglobin from 
the "Pompeii worm." Alvinella itom/'ejuna. an annelid from a deep sea 
hot hydrothermal vent environment. Mar. Biol. Lett. S: 191-201. 

9. Toulmond, A.. F. El Idrissi Slitine, J. De Frescheville, and C. 
Jouin. 1990. Extracellular hemoglobins of hydrothermal vent an- 
nelids: structural and functional characteristics in three alvinellid spe- 
cies. Biol. Bull. 179: 366-373. 

10. Gaill, F., H. Wiedemann, K. Mann, K. Kiihn, R. Timpl, and J. 
Engel. 1991. Molecular characterization of cuticle and interstitial 
collagens from worms collected at deep sea hydrothermal vents. J. 
Mot. Biol. 221: 209-223. 

11. Shillito. B., D. Jollivet, P. M. Sarradin, P. Rodier, F. Lallier, D. 
Desbruyeres, and F. Gaill. 2001. Temperature resistance of Hesio- 
lyru bergi, a polychaetous annelid living on deep-sea vent smoker 
walls. Mar. Ecol. Prog. Sei: 216: 141-149. 

12. Jollivet, D., D. Desbruyeres. C. Ladrat, and L. Laubier. 1995. 
Evidence for differences in allozyme thermostability of deep-sea hy- 
drothermal vent polychaetes (Alvinellidae): a possible selection by 
habitat. Mar. Ecol. Prog. Sei: 123: 125-136. 



Reference: Bio/. Bull. 205: 102-109. (Month 2003) 
2003 Marine Biological Laboratr: v 



Light Production by the Arm Tips of the Deep-Sea 
Cephalopod Vampyroteuthis infernalis 



BRUCE H. ROBISON*, KIM R. REISENBICHLER, JAMES C. HUNT 1 , 
AND STEVEN H. D. HADDOCK 

Monterey Bay Aquarium Research Institute, 7700 SamihoUlt /?</.. Moss Landing, California 95039 



Abstract. The archaic, deep-sea cephalopod Vampyroteu- 
this infernalis occurs in dark, oxygen-poor waters below 
600 m off Monterey Bay. California. Living specimens, 
collected gently with a remotely operated vehicle (ROV) 
and quickly transported to a laboratory ashore, have re- 
vealed two hitherto undescribed means of bioluminescent 
expression for the species. In the first, light is produced by 
a new type of organ located at the tips of all eight arms. In 
the second, a viscous fluid containing microscopic luminous 
particles is released from the arm tips to form a glowing 
cloud around the animal. Both modes of light production are 
apparently linked to anti-predation strategies. Use of the 
tip-lights is readily educed by contact stimuli, while fluid 
expulsion has a much higher triggering threshold. Coelen- 
terazine and luciferase are the chemical precursors of light 
production. This paper presents observations on the struc- 
ture and operation of the arm-tip light organs, the character 
of the luminous cloud, and how the light they produce is 
incorporated into behavioral patterns. 

Introduction 

Vampyroteuthis infernalis Chun, 1903 (Fig. 1 ) is the lone 
occupant of the cephalopod order Vampyromorpha. Its 
unique morphological characteristics, combining features of 
both the octopodiformes and decapodiformes, suggest that it 
represents an evolutionary position intermediate between 
the two groups (Young, 1977: Healy, 1989); and it may be 
a relic from an ancestral cephalopod line (Pickford, 1946). 
This phylogenetic issue has not yet been clearly resolved. 



Received 10 February 2003; accepted 15 July 2003. 

* To whom correspondence should he addressed. E-mail: robr@ 
mbari.org 

1 Current address: University of New England. 1 1 Hills Beach Rd.. 
Biddeford, ME 04005. 



which only adds to the enigmatic status of the species 
(Young et al, 1998). Vampyroteuthis inhabits temperate 
and tropical waters of the Pacific. Atlantic, and Indian 
Oceans, typically between 600 and 1200 m. At these depths, 
sunlight is dim or absent altogether, oxygen content is low, 
and temperatures range from about 2 to 6C (Pickford, 
1946). In waters over the Monterey Submarine Canyon, we 
have found Vampyroteuthis throughout the year at depths 
between 600 and 900 m and at oxygen concentrations 
centered around 0.4 ml/1. 

When observed in its natural habitat, Vampyroteuthis has 
the appearance of a robust and substantial animal, but this 
impression is somewhat misleading. Manipulation in situ 
and in the laboratory reveals that its body is very soft, with 
watery tissues and little dense musculature. It has a very low 
metabolic rate and lives at extremely low oxygen concen- 
trations, yet it is capable of relatively high swimming 
speeds, relying on its fins rather than jet propulsion (Hunt, 
1996: Seibel et al., 1997. 1998. 1999). 

Vampyroteuthis has been reported to feed upon copepods, 
prawns, and cnidarians (Young. 1977: Nixon. 1987). but 
dietary evidence is very scarce. In the laboratory, members 
of this species will take euphausiids and pieces of fish when 
the food is placed in contact with the oral surface of the 
arms, although this is hardly natural feeding. In turn. 
\'amp\n>teuthix beaks have been reported from the stom- 
achs of large, deep-diving fishes, pinnipeds, and whales, and 
from benthopelagic fishes (e.g., Pearcy and Ambler, 1974; 
Antonelis et al., 1987; Fiscus et al., 1989; Clarke et al.. 
1996; Clarke and Young. 1998; Drazen et al.. 2001 ). All but 
the whales are visually cued predators with large eyes that 
function effectively in dim. monochromatic light. 

Cephalopods. particularly deep-water squids, employ a 
diverse suite of light-producing organs that can occur on the 
mantle, fins, arms, tentacles, head, eyes, viscera, or else- 



102 



\:\MPYROTEVTHIS BIOLUMINESCENCE 



103 




Figure 1. Vampyroteuthis infenhili.\. frame grab from high-definition video footage shot at a depth of 717 m 
in Monterey Bay. California. Mantle length = 10.2 cm. The fin-base photophore is located behind the dark patch 
of skin, posterior to (to the right of) the fin. The composite organ is the small, elongate white patch dorsal to and 
on a line just behind the eye. Minute epidermal organs are scattered over the surface of the mantle and the arms, 
but not on the web. The new arm-tip light organs described here are located on the oral surface of the filamentous 
portion of each arm, beyond the margin of the web. 



where, depending on the species (Herring, 1977). The light 
they produce is used for attracting prey, deterring predators, 
and presumably for intraspecific communication. Luminous 
secretions are found in a number of deep-living inverte- 
brates but are rare among cephalopods and fishes (Herring, 
1977. 1988). 

Three types of light-emitting organs have been described 
in Vampyroteuthis infenuilis: large, paired, complex photo- 
phores at the bases of the fins; small, simple, epidermal 
organs scattered over the surface of the animal; and com- 
posite organs two clusters of small, pale nodules located 
dorsally on a line just behind the eyes (Pickford, 1949). 
Light production has been observed only from the fin-base 
photophores; emission spectra of these organs were mea- 
sured at 460 nm by Herring (1983) and 461-466 nm by 
Widder et al. (1983). Herring et al. (1994) examined all 
three organ types and, based on detailed histological evi- 
dence, concluded that the composite organs are probably 
extraocular photoreceptors. while the epidermal organs are 
most likely light producers. They also found that the reflec- 
tive surfaces in the light organs were collagen instead of the 
iridisomal platelets found in other modern cephalopods. To 
date, no one has observed light from the epidermal organs, 
nor has there been any behavioral evidence of light sensi- 
tivity by the composite organs. We have discovered two 
new forms of bioluminescent expression in Vampyroteuthis: 



light produced by organs at the tips of all eight arms, and 
luminous fluid released by the arm tips. 

Materials and Methods 

In situ behavioral observations and quantitative video 
surveys of meso- and bathypelagic cephalopods have been a 
component of MBARI's midwater research program since 
1991 (Hunt, 1996). The program is based on the use of 
remotely operated vehicles, or ROVs. Over a 10-year time 
span we have carefully observed 57 individuals of Vampy- 
roteuthis in situ and have collected 18 to establish in labo- 
ratory aquaria. Specimens in this study included adult males 
and females with mantle lengths ranging from 7.9 to 12.1 
cm. All were gently collected with the ROV Ventana (Ro- 
bison, 1993) at a time-series station 1600 m deep over the 
axis of the Monterey Submarine Canyon. Field observations 
and collections occurred under full illumination from the 
ROVs four 500-W, broad-spectrum lights. Once the ROV 
was recovered, the animals were placed in darkened con- 
tainers and were quickly transferred to our laboratory 
ashore. In the shoreside facility they were maintained in the 
dark, at 4 to 6 C, in circular, 260-1 kreisel tanks (Hamner, 
1990) for as long as 2 months. 

Most of the specimens appeared to be temporarily 
blinded by the vehicle's lights during capture. After several 
hours in the dark, they responded to point sources of white 



104 



B. H. ROBISON ET AL 



light by moving away, and by contracting the iris-like 
sphincter muscle (Pickford. 1949) that surrounds the front 
of the eye. Laboratory observations were made both under 
red light and in il .ark, often with an image intensitier 
classified as Ge: ,1+ according to the U.S. Army Night 
Vision Laboratory's criteria. Light production was recorded 
with a variety of low-light video cameras. 

For chemical assays of arm-tip light organs, we removed 
the distal portions of arms from several specimens and used 
them either fresh or after they had been frozen in liquid 
nitrogen. Light output from each assay listed below was 
measured with a Hamamatsu HC-124 photomultiplier tube, 
in a custom-built integrating sphere, for at least 20 s. 

Coelenterazine assay: To test for the presence of coelen- 
terazine, we homogenized individual arm tips in 500 /tl of 
methanol (approximately 10:1 by volume). One milliliterof 
purified Oplophorus luciferase in a solution of 20 mM Tris 
and 100 mM NaCl was injected into 200 jul of the sample 
solution. Mantle tissue with epidermal light organs and web 
tissue (which lacks the epidermal light organs) were also 
assayed for the presence of this luciferin. 

Luciferase assay: Sample arm tips to be tested for lucif- 
erase activity were extracted in an aqueous solution of 100 
mM Tris pH 8.1 and 50 mM EDTA. Calcium chloride 
addition caused no light output, indicating that a calcium- 
activated photoprotein was not involved. The test solution 
was added to 20 /A! of coelenterazine in 0.5 /Ag/jul MeOH, 
and the light production was measured. For negative con- 
trols, tissue from the web was homogenized, and the ex- 
tracts were added to methanol. 

Bacterial luciferase assay: Assays for luminous bacteria 



in the arm-tip light organs, the ejecta from the organs, and 
the surrounding water followed the reduced flavin assay 
described in Hastings et al. (1978), with the flavin reduced 
by bubbling with H 2 gas in the presence of platinized 
carbon. Cultured Vibrio han'eyi were used as positive con- 
trols for this assay. We also tested for the presence of 
luminous bacteria in samples of arm-tip exudate that were 
streaked on seawater agar plates kept at 4C for 2 weeks. 

Fluorescence microscopy: Autofluorescence images of 
arm-tip light organs and ejecta were obtained with a Zeiss 
Axioplan microscope using 10X and 40x Neofluar objec- 
tives, under DAPI illumination. 

Electron microscopy: Material was fixed in 2% glutaral- 
dehyde with 0.1 M cacodylate buffer. Samples were post- 
fixed using osmium tetroxide and embedded in Epon. Thick 
( 1-2 /Am) sections through the light-producing region of the 
arm tip were stained with toluidine blue. Thin sections from 
the same region were stained with uranyl acetate and lead 
citrate. 

Results 

In the laboratory we observed that the tips of all eight 
arms often glowed when an animal was handled (Fig. 2). 
The bright blue lights usually appeared as a tight chain of 4 
to 6 small discs, tapering in size distally along the oral 
surface of each arm tip. Occasionally there was a different 
pattern, in which the light appeared as two parallel lines 
separated by a dark gap. With a mild contact stimulus, the 
arms and web flared outward, with the arm tips glowing. 
With stronger prodding, the arms were curled, writhing up 




Figure 2. Frame grab from a low-light video recording, showing the glowing arm tips of Vampyroteulhis 
infernulis. The animal is oriented such that Us head and beak are directed toward the camera, with the arms and 
web beginning to flare outward. 



VAMPYROTEUTHIS B1OLUMINESCENCE 



105 



over the head to the apex of the mantle, exposing the 
suckers and cirri and placing the glowing arm tips in a 
cluster at the top. When an animal rolled the arms and 
mantle back down to their normal position, it frequently 
tucked the arm tips within the web, where they were 
shielded from view. This behavior, which was observed 
both in the field and in the laboratory, is similar to a 
nonluminous pattern seen in octopuses attacked by moray 
eels (Hanlon and Messenger. 1996). The eight arm-tip light 
organs of Vaiiiiiyniteiithis always glowed and dimmed si- 
multaneously. They flashed 1 to 3 times per second, or 
glowed steadily, but rarely for longer than one minute. The 
pulsing could include complete extinction of the light, or 
just dimming, before returning to the previous level of 
intensity. There is a dark, densely pigmented layer of skin 
on the aboral surface and on the sides of each arm tip, but 
the oral surface is generally unpigmented. 

The structure of the oral surface of the arm tips continues 
the basic pattern found along the entire length of the arm a 
series of central plates alternating with paired lateral plates 
(Fig. 3). In the proximal and medial portions of the arm, the 
lateral plates support cirri, while the central plates are the 
bases for suckers (Pickford. 1949, plate VI. fig. 20). Plates, 
cirri, and suckers get smaller toward the distal end; near the 
tip. the plates bear mere rudiments. Proximally. the plates 
are pale and opaque, but as they approach the distal tip they 
become translucent. Within this window at the tip of the arm 
are subdermal clusters of particles that impart an iridescent 
green and yellow sheen to the plates. When the arm is 
viewed from the side, the central plates appear bulbous and 
extend outward beyond the lateral plates (Pickford, 1949, 
plate VI, fig. 19). Light expressed from the central plates 
alone may be the source of the pattern that appears as a 
chain of discs, while light coming from just the lateral plates 
would show as parallel lines. The light-producing area of 
the arm tip can be occluded by the edges of the dark skin 
along both sides, which close together along the midline of 
the oral surface. This means of controlling light output is 
similar to that described for the arm-tip photophores of 
Tuningiu danae by Herring et al. ( 1992). 

Given a strong contact stimulus to the arms or body, the 
arm tips exuded a viscous fluid containing small glowing 
particles. As the arms swept up over the head and mantle, 
the particles dispersed, enveloping the animal in a luminous 
cloud (Fig. 4). To all observers, the light from the cloud was 
much dimmer than that of the fin lights and arm tips, but we 
were unable to measure its intensity. The number of parti- 
cles released varied from a few dozen to several hundred, 
usually related to the strength of the stimulus. Cloud lumi- 
nescence persisted for 2 to 3 min. and individual particles 
glowed for as long as 9 min (Hunt. 1996). Once the particles 
had gone dark, stirring the water did not re-initiate lumi- 
nescence. After several such displays, production of the 
luminous fluid ceased, and while the arm tips could still be 




Figure 3. Arm tip of a living specimen of Vampyroteuthis infemalis, 
showing the distal light-producing region, and patches of small, green 
particles in the lateral plates that are associated with the luminous ejecta. 
cp = central plate; Ip = lateral plate; scale bar = 1 mm; the arrows point 
to patches of iridescent particles that are not present after an extensive 
luminous cloud has been created. 



stimulated to glow, the dense clusters of particles in the arm 
tips were gone. The fluid matrix that bears the luminous 
particles is viscous and somewhat sticky. Arm tips that 
brushed across the inner surface of a kreisel during a biolu- 
minescent display usually left behind a lingering streak of 
light. The release of luminous particles often preceded an 
escape response by the animal. 

The chemical assays provided clear evidence of the pres- 
ence of coelenterazine (luciferin) and luciferase in the arm- 
tip light organs of Vampyroteuthis (Fig. 5), which indicates 
that these compounds are the basis for light production. No 
calcium-activated photoprotein activity was detected in any 
assay. Small amounts of coelenterazine were found in the 
mantle epidermal tissue. These results support the conclu- 
sion by Herring et al. (1994) that the epidermal organs 
produce light. Assays for luciferin and luciferase in the web 
tissue were negative. The assav for bacterial luciferase in 



106 



B. H. ROBISON ET AL. 




Figure 4. Frame grab from a low-light video recording showing the release of glowing particles from the 
arm tips of Vampyroteuthis infernalis. The head of the specimen is directed toward the camera. Particles in the 
cloud are swirled by movements of the arms and web. and by water jetting through the siphon. "Tails" on the 
glowing particles are caused by electronic lag in the camera's image intensifier. 



the tip lights was negative, as were the culturing efforts to 
demonstrate the presence of luminous bacteria in the arm 
tips and their exudate. 

Microscopic examination of the iridescent clusters in the 
arm tips of animals that had not yet secreted luminous 
material revealed extensive patches of rounded yellow par- 
ticles that glowed blue-green under fluorescent illumination 
(Fig. 6). No pores that might release the fluid were evident 
on the arm tips, although the rudimentary suckers are likely 
sites. The particles matched, in size and configuration, par- 
ticles culled from the arm-tip exudate and from the water in 
which a luminous cloud had been produced. Sections of the 
arm tips showed a low-density central core with prominent 
nuclei on the oral side and sparse muscle tissue on the 
aboral. We saw no evidence of an iridosomal reflective layer 
nor of layered collagen fibers like those found by Herring et 
al. ( 1994) in the fin-base photophores. 

A comparison of our specimens with others collected by 
trawling in Monterey Bay and elsewhere in the North Pa- 
cific revealed that, in almost every case, the arm-tip light 
organs had broken off the trawl-caught specimens. This 
observation is similar to that made on Octopoteuthis (Her- 
ring et al., 1992) and may explain why the arm-tip light 
organs of Vampyroteuthis were not discovered until we 
could collect the animals in perfect condition. On two of our 
ROV-caught specimens, we found a short, apparently re- 
generated arm, each with what appeared to be a small light 
source at its tip. 

Over a gradient of stimuli, the fin lights were the most 
readily illuminated, and although this pair always worked 



together, they could operate independently of the other two 
light sources. Light emission from the fin lights was regu- 
lated by chromatophores and by iris-like skin closures sim- 
ilar to those that shield the eyes. The arm-tip lights seldom 
glowed without the fin lights also being on, and all 10 could 
pulse in concert. The luminous ejecta was never observed 
without the tip lights glowing as well. 

On one occasion, male and female specimens were col- 
lected on the same day and were then placed in separate 
kreisels less than a meter apart, in the darkened laboratory 
ashore. When the female was disturbed and began to flash 
her arm-tip lights, the undisturbed male quickly and vigor- 
ously responded with tip-light flashes. This reaction was 
repeated twice (Hunt. 1996). We saw no evidence of dif- 
ferential light production by females and males. In the 
Cranchiidae and Lycoteuthidae, arm-tip photophores de- 
velop as secondary sexual characters (Herring et al., 1992). 
We detected no sexual dimorphism in the light organs of 
Vampyroteuthis. Luminous suckers on the deep-sea octopus 
Suini-ntciitlii.'i syrtensis may be used for intraspecific com- 
munication (Johnsen et al.. 1999a. b). but the structure of 
the light organs in this species is not at all like the arm-tip 
lights of Vampyroteuthis. Although animals in kreisels re- 
acted to point sources of artificial light by shading their eyes 
with their arms and web, or by moving away from the light, 
the response of Vampyroteuthis to artificial light never 
included luminescence. 

Supplemental images (in situ video, laboratory low-light 
video, digital stills, and electron micrographs) are available 
online at http://www.mbari.org/midwater/vamp. 



VAMPYROTEUTHIS BIOLUMINESCENCE 



107 



o 



5 2 

<u 

at 



A 




-A-NwJWV-^ 



1 
Time (s) 



1 5 



20 



3 5 

g. 

1 4 




12 



16 



Time (s) 



Figure 5. Luciferin and luciferase assays for arm-tip light organs of 
Vampyroteuthis infernalis. (A) Light produced by methanolic extracts upon 
the addition of Oplophorus luciferase indicates the presence of the luciferin 
coelenterazine. (B) Addition of coelenterazine to aqueous extracts shows 
high luciferase activity. Negative controls for both assays (omitted here for 
clarity) did not deviate measurably from the pre-injection baseline. The 
higher noise associated with the coelenterazine assay is due to the higher 
gain setting required to detect the presence of that molecule. 



Discussion 

The effect of arm-tip luminescent displays on the dark- 
adapted human eye is striking; coupled with the bright blue 
light emitted by the two fin-base photophores, these dis- 
plays produce a complex and dynamic visual field. The 
fundamental question they raise is, how does Vampyroteu- 
this use the light? Because these responses can be elicited 
by mechanical stimuli, we assume that production of light 
from the arm-tip organs and the cloud of luminous particles 
are elements of an anti-predation strategy based on startling 
or distracting a potential predator, thus allowing for escape 
(Young, 1983). The visual predators that we know about are 
all better swimmers than Vampyroteuthis, so its escape 
strategy must rely on more than speed. Deceptive, deimatic 



behavior, such as chromatophore displays and unpredictable 
protean behavior, is often coupled with locomotion in 
cephalopod escape strategies (Hanlon and Messenger, 
1996). In the darkness of its habitat, Vampyroteuthis may 
substitute luminescence for chromatophore displays in an 
otherwise familiar cephalopod behavior pattern of decep- 
tion, diversion, and flight. 

Arm-tip light organs, which can be bitten or broken off 
and then regenerated, may serve as sacrificial diversions for 
predators (Herring, 1977). Tip lights are found in several 
deep-living squids such as Chiroteuthis and Octopoteuthis, 
where they may also serve as lures for prey, thus function- 
ing like the escae of anglerfish and the barbels of stomiid 
fishes (Herring, 1977; Young, 1983). Our observations of 
apparently regenerated light organs at the ends of shortened 
arms in Vampyroteuthis may be evidence of their potential 
as sacrificial structures. The characteristics of the arm-tip 
displays indicate that there is direct neural control of their 
luminescence. 

The production of luminous clouds is common among 
other deep-living pelagic invertebrates but rare in cephalo- 
pods. Anecdotal evidence for the production of luminous 
clouds by squids was summarized by Young et al. ( 1979), 
who suggested that renal fluid might be the luminous sub- 
strate. The only well-documented case is the sepiolid Het- 
eroteuthis, which ejects a cloud of luminous particles when 
it is disturbed, presumably as a distraction to predators 
(Herring, 1977). The ejecta is produced by glands within the 
mantle that contain dense populations of light-producing 
bacteria, which are combined with ink and mucus during 
release through the siphon (Herring. 1988, 2002). The 
glands themselves emit light and have complex internal 
reflectors, which suggests that they have multiple uses (Her- 
ring, 1988). Structurally and operationally, the release of 
luminous fluids appears to be a completely different process 
in Vampyroteuthis than it is in Heteroteuthis. 

Visual trickery is common within the depth range and 
light regime that Vampyroteuthis occupies (Robison, 1995, 
1999; Herring, 2002), and our observations suggest some 
additional ways that its luminescence may be employed. 
Because the luminous fluid released by Vampyroteuthis is 
sticky, it would adhere to a potential predator and might 
initiate a "burglar alarm" consequence by painting it with 
bioluminescence that cannot be turned off or readily re- 
moved, thus making the attacker vulnerable to secondary 
predators. A similar behavior has been described for the 
bathypelagic holothurian Enypniastes eximia (Robison. 
1992). Glowing particles in the ejecta might be used to 
attract smaller prey such as copepods. which would then 
become trapped by the viscous matrix. This function has 
been proposed for the twinkling bioluminescent suckers and 
mucous glands of the cirrate octopus Stauroteuthis syrtensis 
(Johnsen et til., 199%). It is tempting to correlate the size 
and abundance of light sources in the cloud with the epi- 



108 



B. H. ROBISON ET AL. 




Figure 6. Epitluore.sence micrograph of the oral surface of an arm tip of Vampyroteuthis !ntfrihili.\, showing 
patches of clustered particles. The dark areas along the midline are in the central plates, which bear suckers or 
their rudiments. Scale bar = 1 mm. Individual particles ranged from III to 15 /nm in greatest dimension. 



dermal organs of Vampyroteuthis. but we have never seen 
the latter luminesce. 

The luminous secretion from the arm tips of Vampyro- 
teuthis is unique among the known cephalopod biolumines- 
cent systems. Likewise, the arm-tip light organs are struc- 
turally distinct from all others. Predator avoidance seems 
the most likely function of the luminous behavior we have 
seen, but clearly, much is yet to be learned from observing 
these animals in their natural habitat. 

Acknowledgments 

R. E. Sherlock provided invaluable support both at sea 
and ashore. We gratefully acknowledge the skills and ded- 



ication of the ROV Ventana's pilots and the crew of the R/V 
Point Lobo.s. Coelenterazine, synthesized by S. Inoue, and 
Oplophorus luciferase, purified by Y. Haneda, were kindly 
provided by O. Shimomura. M. Haygood and J. W. Hastings 
gave us advice on the bacterial luciferase assays. We thank 
J. F. Case for a helpful review of the paper. Supported by 
the David and Lucile Packard Foundation. 

Literature Cited 

Antonelis, G. A., M. S. Lowr.v, D. P. DeMaster. and C. H. Fiscus. 1987. 

Assessing northern elephant seal feeding by stomach lavage. Mar. 
Mamm. Sci. 3: 308-322. 
Clarke, M. R., and R. K. Young. 19M8. Description and analysis of 



VAMPYROI'h.l'I'HIS BIOLUM1NESCENCE 



cephalopod beaks from stomachs of six species of odontocete cetaceans 
stranded on Hawaiian shores. J. Mar. Biol. Assnc. t'A' 78: 623-641. 

Clarke, M. R., D. C. Clark, H. R. Martins, and H. M. Da Silva. 19%. 
The diet of the blue shark (Prionace g/auca L.) in Azorean waters. 
An/mpt'laxo 14: 41-56. 

Drazen, J. C., T. W. Buckley, and G. R. Hoft. 2001. The feeding 
habits of slope dwelling macrourid fishes in the eastern North Pacific. 
Deep-Sea Res. 1 48: 909-93?. 

Fiscus, C. H.. D. \V. Rice, and A. A. \Volman. 1989. Cephalopods 
from the stomachs of sperm whales taken off California. NOAA Tech. 
Rep. Nat. Mar. Fish. Sen: 83: i-12. 

Hamner, \V. M. 1990. Design developments in the planktonkreisel, a 
plankton aquarium for ships at sea. J. Plankton Res. 12: 397-41)2. 

Hanlon, R. T., and J. B. Messenger. 1996. Cephulopod Behaviour. 
Cambridge University Press. Cambridge. 

Hastings. J. W., T. O. Baldwin, and M. Z. Nicoli. 1978. Bacterial 
luciferase: assay, purification and properties. Methods En-ymot. 57: 
135-152. 

Heal>. J. M. 1989. Spermatozoa of the deep-sea cephalopod Vampyro- 
tfiirhis infernalis Chun: ultrastructure and possible phylogenetic sig- 
nificance. Philos. Trans. R. Soc. Land. B 323: 589-600. 

Herring, P. J. 1977. Luminescence in cephalopods and fish. Symp. Zoo/. 
Soi: Land. 38: 127-159. 

Herring, P. J. 1983. The spectral characteristics of luminous marine 
organisms. Proc. R. Soc. Loud. B 220: 183-217. 

Herring, P. J. 1988. Luminescent organs. Pp. 449-489 in The Mol- 
lusca. II. Form and Function. E. R. Trueman and M. R. Clarke, eds. 
Academic Press, San Diego. 

Herring, P. J. 2002. The Biology of the Deep Ocean. Oxford University 
Press. Oxford. 

Herring, P. J., P. N. Dilly, and C. Cope. 1992. Different types of 
photophores in the oceanic squids Oclopotenthis and Taningia (Cepha- 
lopoda: Octopoteuthidae). J. Zooi. Loud. Ill: 479-491. 

Herring, P. J., P. N. Dilly, and C. Cope. 1994. The bioluminescent 
organs of the deep-sea cephalopod Vampymteuthis infernalis (Cepha- 
lopoda: Vampyromorpha). J. Zoo/. Land. 233: 45-55. 

Hunt, J. C. 1996. The behavior and ecology of midwater cephalopods 
from Monterey Bay: submersible and laboratory observations. Ph.D. 
dissertation. University of California, Los Angeles. 

Johnsen, S., E. J. Balser, and E. A. Widder. 1999a. Light-emitting 
suckers in an octopus. Nature 398: 113-114. 

Johnsen. S.. E. J. Balser, E. C. Fisher, and E. A. Widder. 1999h. 



Biolummescence in the deep-sea cirrate octopod Slauroteuthis svrten- 

sis Verrill (Mollusca: Cephalopoda). Biol. Bull. 197: 26-39. 
Nixon, M. 1987. Cephalopod diets. Pp 201-219 in Cephalopod Life 

Cycles. II. Comparative Reviews. P. R. Boyle, ed. Academic Press. 

Orlando, FL. 
Pearcy, VV. G., and .1. W. Ambler. 1974. Food habits of deep-sea 

macrourid fishes off the Oregon coast. Deep-Sea Res. 21: 745-759. 
Pickford, G. E. 1946. \\imp\roteiahis infernalis (Chun) an archaic 

dibranchiate cephalopod. I. Natural history and ecology. Dana Rep. 29: 

1-40. 
Pickford, G. E. 1949. \\unp\ioleuthis infernalis (Chun) an archaic 

dibranchiate cephalopod. II. External anatomy. Dana Rep. 32: 1-132. 
Robison. B. H. 1992. Bioluminescence in the benthopelagic holothuriaii 

Enypniastes eximia. J. Mar. Biol. Assoc. UK 72: 463 172. 
Robison, B. H. 1993. Midwater research methods with MBARl's ROV. 

Mar. Teclmol. Soc. J. 26: 32-39. 
Robison, B. H. 1995. Light in the ocean's midwaters. Sci. Am. 273: 

60-64. 
Robison, B. H. 1999. Shape-change behavior by mesopelagic animals. 

Mar. Fres/nv. Behav. Physiol. 32: 17-25. 
Seibel, B. A., E. V. Thuesen. J. J. Childress, and L. A. Gorodezky. 

1997. Decline in pelagic cephalopod metabolism with habitat depth 

reflects differences in locomotory efficiency. Biol. Bull. 192: 262-278. 
Seibel, B. A., E. V. Thuesen, and J. J. Childress. 1998. Flight of the 

vampire: ontogenetic gait-transition in Vampyroleuthis infernalis 

(Cephalopoda: Vampyromorpha). J. E.\p. Biol. 201: 2413-2424. 
Seibel, B. A., F. Chausson, F. H. Lallier, F. Zal, and J. J. Childress. 

1999. Vampire blood: respiratory physiology of the vampire squid 

(Cephalopoda: Vampyromorpha) in relation to the oxygen minimum 

layer. Ev/>. Biol. Online 4: 1-10. 
Widder, E. A., M. I. Latz, and J. F. Case. 1983. Marine biolumines- 

cence spectra measured with an optical multichannel detection system. 

Biol. Bull. 165: 791-810. 
Young, J. Z. 1977. Brain, behaviour and evolution of cephalopods. 

Symp. Zoo/. Sue. Loud. 38: 377-434 
Young, R. E. 1983. Oceanic bioluminescence: an overview of general 

functions. Bull. Mar. Sci. 33: 829-845. 
Young. R. E., C. F. E. Roper. K. Mangold, G. Leisman, and F. G. 

Hochberg. 1979. Luminescence from non-bioluminescent tissues in 

oceanic cephalopods. Mar. Biol. 53: 69-77. 
Young, R. E., M. Vecchione, and D. T. Donovan. 1998. The evolution 

of coleoid cephalopods and their present biodiversity and ecology. S. 

Afr. J. Mar. Sci. 20: 393-420. 



Reference: Biol. Bui/. 205: I 10-120. (October 2003) 
O 2003 Marine Biological Laboraton 



Patterns and Processes of Larval Emergence in an 
Estuarine Parasite System 

JONATHAN T. FINGERUT, 1 '* CHERYL ANN ZIMMER. 1 t AND RICHARD K. ZIMMER 1 2 ' 

1 Department of Biology, 2 Neurosciences Program and Brain Research Institute, University of California, 

Los Angeles. California 90095-1606 



Abstract. Trematode parasites in intertidal estuaries ex- 
perience constantly varying conditions, with the presence or 
absence of water potentially limiting larval transport be- 
tween hosts. Given the short life spans (<24 h) of cercariae, 
emergence timing should be optimized to enhance the prob- 
ability of successful transmission. In the present study, field 
measurements and laboratory experiments identified pro- 
cesses that regulate the emergence of cercariae from their 
first intermediate snail hosts in an intertidal marsh. Larvae 
emerged over species-specific temperature ranges, exclu- 
sively during daylight hours, and only when snails were 
submerged. The three factors operate over different tempo- 
ral scales: temperature monthly, light diurnally (24-h pe- 
riod), and water depth tidally (12-h period). Each stimulus 
creates a necessary condition for the next, forming a hier- 
archy of environmental cues. Emergence as the tide floods 
would favor transport within the estuary, and light may 
trigger direct (downward or upward) swimming toward host 
habitats. Abbreviated dispersal would retain asexually re- 
produced cercariae within the marsh, and local mixing 
would diversify the gene pool of larvae encysting on sub- 
sequent hosts. In contrast to the timing of cercarial release, 
emergence duration was under endogenous control. Dura- 
tion of emergence decreased from sunrise to sunset, perhaps 
in response to the diminishing lighted interval as the day 
progresses. Circadian rhythms that control cercarial emer- 
gence of freshwater species (including schistosomes) are 
often set by the activity patterns of subsequent hosts. In this 
estuary, however, the synchronizing agent is the tides. To- 



Received 12 February 2003; accepted 16 June 2003. 

* Current address: Patrick Center for Environmental Research, Academy 
of Natural Sciences. 1900 Benjamin Franklin Parkway, Philadelphia. PA 
19103-1 19? 

t Formerl) Cheryl Ann Butman. 

Correspond i,"j author. E-mail: z@biology.ucla.edu 



gether. exogenous and endogenous factors control emer- 
gence of trematode cercariae, mitigating the vagaries of an 
intertidal environment. 

Introduction 

Parasite larvae typically disperse prior to finding and 
infecting a host. As with propagules of free-living organ- 
isms, such as crabs (Forward ct ai. 1986; Morgan, 1996), 
sponges (Amano. 1988), and plants (Horn ct al., 2001; 
Sehauber ct al., 2002), external cues may direct emergence 
of parasite larvae under favorable conditions. Because dis- 
persal stages of digenetic trematodes have life spans of 24 h 
or less (McCarthy, 1999; Toledo et al.. 1999), timing emer- 
gence to correspond with host availability would be espe- 
cially advantageous (Combes et ai, 1994; Pechenik and 
Fried, 1995). Moreover, the widespread distribution of 
trematodes in fresh water (Pages and Theron, 1990; Gerard, 
2001) and saltwater (Martin, 1972; Bartoli and Combes, 
1986; Jonsson and Andre. 1992; Curtis, 1997) environments 
allows for cross-habitat comparisons of emergence charac- 
teristics. 

Trematode emergence has been studied largely in fresh- 
water systems, with much of this research addressing med- 
ical and agricultural concerns (Bergquist, 2002; McKerrow 
and Salter. 2002). In common parasites, such as schisto- 
somes, larval emergence from intermediate host snails var- 
ies on a circadian cycle and is synchronized with definitive 
host availability (Pages and Theron, 1990; N'Goran et al., 
1997). Circadian rhythms are usually entrained by photope- 
riod or thermoperiod (Theron, 1984; Mouchet ct al.. 1992; 
Combes ct al.. 1994). Freshwater parasite larvae moving 
from aquatic to terrestrial vertebrate hosts time their emer- 
gence to coincide with waterfront activities of the hosts, on 
scales of hours (Theron, 1989; Raymond and Probert, 
1991). 



no 



PATTERNS AND PROCESSES OF LARVAL EMERGENCE 



111 



In southern California intertidal marshes, there is a guild 
of more than 18 digenetic trematode species (Martin. 1972). 
Sexual reproduction occurs in definitive shorebird hosts, 
which defecate parasite embryos into the marsh. Free-living 
miracidia hatch and infect the California horn snail, Cer- 
ithideu California.! (Haldeman), causing castration and other 
sublethal effects (Sousa, 1983: Sousa and Gleason, 1989). 
Asexual reproduction ensues, producing tens to thousands 
of cercariae per snail per day. The cercariae are produced in 
the area previously filled by the snail gonad. and the larvae 
then crawl within the snail hindgut to emerge from tissues in 
the rectum. Once released into the environment, cercariae 
encyst on second intermediate hosts, such as benthic snails 
(including C. culifomica). crabs, and fishes. Ingestion of 
these intermediate hosts by birds completes the parasite life 
cycle. Swimming cercariae are short-lived, so they must 
move toward or remain near host habitat for effective trans- 
mission. In marshes, tidally varying water depth and cur- 
rents may limit transport. 

The purpose of the present study was to identify factors 
controlling cercarial emergence in an intertidal estuary, as 
compared to freshwater systems. Two hypotheses were 
tested. ( 1 ) Timing of cercarial release varies over the day, as 
in freshwater trematodes. Light (24-h period) may impose a 
similar diel periodicity in both systems, but tidal (12-h 
period) effects (presence or absence of water, currents) 
would be unique to the estuary. (2) Exogenous factors 
control cercarial emergence in intertidal estuarine habitats, 
whereas in many freshwater species, larval release is con- 
trolled endogenously. Circadian rhythms in freshwater cer- 
cariae are often associated with innate activity patterns of 
intermediate or definitive hosts (Combes et al., 1994). Such 
predictable host signals may not be important in an estua- 
rine system, where submergence is limited by the tidal 
cycle. 

Our research was conducted in two stages. Field studies 
measured emerging parasite larvae as a function of external 
variables (temperature, salinity, tidal height). Then, labora- 
tory experiments identified the role of the specific factors in 
controlling the onset and duration of emergence. We herein 
define "emergence" as the shedding of cercariae from in- 
termediate host snails. This study does not distinguish be- 
tween the effects of larval behavior and rates of asexual 
reproduction on cercarial emergence patterns. 

Materials and Methods 
Field observations 

In the field, we collected emerging cercariae of all species 
from snails (Cerhhidea culifornica) while simultaneously 
measuring environmental variables. Data were collected on 
38 days from 1999 to 2002. Measurement intervals were 
selected to span all hours of the day and months of the year. 
Moreover, six night collections (> 1 h after sundown) were 



paired with a day collection on the preceding or proceeding 
tide to compare emergence within the same group of snails. 

The field site (tidal channel) was located in Carpinteria 
Salt Marsh Reserve (CSMR), east of Santa Barbara. Cali- 
fornia (3424'16" N, 11931'3()" W). Detailed physical 
characteristics are given in Fingerut et al. (2003). Biological 
measurements took place along the centerline of a channel 
400 m long, 5 m wide, and 0.6 m deep (at high tide). A 
centrally located 3-m-wide mudflat was carpeted (460 
16/nv) with C. culifomica, the first intermediate host for the 
trematodes studied here. The snail population extended hun- 
dreds of meters in the along-channel direction. The hydro- 
dynamic regime in this and similar southern California 
marshes (e.g.. Mission Bay and Newport Bay) was domi- 
nated by slow flows (< 5 cm/s), with shear velocities (//*) 
of 0.02 cm/s or less occurring more than 80% of the time 
(Fingerut et al., 2003). Rarer, storm-driven currents (H* > 
0.8 cm/s) occurred only about 1% of the time. Intertidal 
mudflats in channels were located 1.3 m above MLLW 
(mean lower low water) and were inundated twice a day by 
the semidiurnal tide. Water depth exceeded this height for 
an average of 4 h during each 12-h tidal cycle. Daily 
variations in salinity were 28-34 psu, consistent across 
seasons (Fingerut et al., 2003). Salinity and temperature 
were relatively constant throughout the water column, indi- 
cating well-mixed conditions in the shallow channel. 

Collections of emerged cercariae. A specially designed 
larval collector was used to measure (non-intrusively) cer- 
carial emergence from a specified group of snail hosts. The 
main body of the collector was a flat, clear acrylic chamber 
(50 cm wide by 50 cm deep by 2.5 cm tall) with 34-jii,m- 
mesh side panels and a solid bottom. Penetrating the top of 
the chamber was a 4 by 4 array of 8-cm-diameter holes 
(covering 32% of the surface area), each fitted with an 
inverted funnel (i.e., small end pointing away from the 
chamber) intake. Contents of all funnels were united into a 
single 12.5-mm (ID) tube that fed into a peristaltic pump 
(Masterflex IP variable speed). The pump continuously fil- 
tered water from the chamber at a rate of about 5 liters/min 
over a 34-|iun mesh. The chamber volume was flushed 8 
times during each 10-min sampling interval. 

During each sampling event, 200 snails from the largest 
(=2.5-cm-long) size class in the marsh were placed in the 
collector. Once on the mudflat, the chamber was gradually 
inundated by the incoming tide at the same time as the 
free-ranging snail population. There was no movement of 
cercariae into or out of the chamber. Captives were provided 
with oxygenated water at ambient temperature. Thus, cer- 
cariae collected on the filter could be ascribed solely to the 
enclosed host population. Size ranges of marsh cercariae are 
50-100 /im (width) and 200-1000 p.m (length) (Martin, 
1950; Adams and Martin, 1963), so the mesh retained all 
species. 

The system self-primed when the water level reached the 



112 



J.T. FINGERUT ET AL 



bottom of the funnels (5 cm water depth), usually 5-10 min 
after the tide first rose > the level of the mudflat. Every 10 
min the filter was ! i^oved (and replaced), dipped in 90% 
ethyl alcohol. . placed in a sealed petri dish for later 
staining (0.5' Lugol's solution) and counting. Samples 
were also examined to identify numerically dominant spe- 
cies throughout the year and day. Each collection lasted 4 h, 
matching the average period of snail inundation. A cumu- 
lative frequency distribution was assembled from the total 
number of emerged cercariae during each 10-min collec- 
tion. The time for 95% of all cercariae to emerge was 
compared between collections. 

Environmental measurements. Properties of the physical 
environment during tidal inundation (4-h interval during 
flood or ebb) were measured on the 38 periods of quantified 
cercarial emergence. The mudflat was exposed to air for 
about 8 h between each tidal inundation. Maximal water 
depth was 60 cm during each tide. Using an ORION model 
140 probe, seawater temperature and salinity were recorded 
1 cm above the bed every 10 min. An underwater quantum 
sensor (LICOR model 190SA) was flush-mounted at the 
mudflat surface before tidal inundation to register light 
intensity at 1 Hz over the duration of the flood and ebb tides. 

Laboratory- experiments 

Field measurements (see Results] indicated that the fol- 
lowing factors may control cercarial emergence: tempera- 
ture, host inundation (water depth), light, and time of day. 
Because we used only infected snails (rather than a random 
sample of the host population) in laboratory experiments, 
numbers of cercariae emerging were 10-100 times higher 
than those reported for field observations. With one excep- 
tion, all laboratory experiments were conducted during 
summer months (June-September). Trials to evaluate cer- 
carial emergence as a function of water temperature were 
performed during the spring (March-April). 

Effects of temperature. Two time series were conducted 
in the laboratory, each using the same 53 snails (C. califor- 
nica). The animals were placed individually in chambers 
(27 cm 3 ) filled with seawater. In the first 7-day series, snails 
were held at a higher ( + 1 C) water temperature on con- 
secutive days, over the range 13-19 C. Water was changed 
to the new temperature at 1000 h each day, and cercariae 
were collected 4 h later. (All times are reported as Pacific 
daylight time). Snails were kept dry at a constant 18 C 
between runs (average spring air temperature for daylight 
hours). The second 7-day series followed the same protocol, 
except that exposure temperature was decreased 1C per 
day, from 19 to 1 3 C. At the end of a 4-h incubation period, 
contents of individual chambers were processed separately 
to determine species (Martin, 1972) and number of emerged 
cercariae. 

Effects of host inundation. Two groups of 100 snails each 



were randomly assigned to one of two treatments, and were 
placed in trays (21 cm long by 1 1 cm wide by 3 cm deep), 
at 20 snails per tray. Both groups were then situated in an 
environmental chamber at 21C (average summer water 
temperature) with constant light (40 jumol/nr/s). For one 
group, trays were kept dry for 4 h; for the other group, trays 
were filled with water from CSMR. At the end of 4 h the dry 
trays were briefly filled to suspend any emerged cercariae. 
and the water from both groups was filtered through a 
34-/j,m mesh. The number of emerged cercariae from each 
treatment was determined using the same processing meth- 
ods as for the field samples. The following day, the treat- 
ments were reversed for the two snail groups. This 2-day 
experiment (one 4-h test period each day) was replicated 
three times, with a new batch of snails each time. 

Effects of light intensity'. Snails were exposed to a midday 
intensity (2000 /imol/nr/s ) or to simulated dawn/dusk 
intensity (40 jumol/nr/s), created by reducing midday 
intensity with a neutral density filter. Snails (100 per group) 
placed in water-filled trays (see Effects of host inundation) 
were randomly assigned to one of the light conditions. 
Every 15 min, trays were overturned onto a 1-mm-mesh 
panel. Snails remained in the trays, but water and cercariae 
passed through. Trays were immediately refilled. Water and 
larvae were filtered over 34-/xm mesh and processed as in 
the field study. To control for the warming effect of the 
stronger sunlight, snail trays were held in a water bath 
maintained at 21C (average for summer months). Trials 
lasted 2 d, with each group exposed to both conditions on 
consecutive days, and was replicated three times, each with 
different snails. 

Effects of time of day. Two series of experiments were 
performed, one testing time of day (TOD) on emergence 
duration and the other testing for the existence of an endog- 
enous rhythm. In the first series, 100 snails were placed in 
water-filled trays (see Effects of host inundation) at constant 
temperature (21C) and light (40 /j,mol/m 2 /s) for 4 h, start- 
ing at a different time each day (0900, 1200. 1500. and 
1800). During each 4-h interval, emergence duration was 
determined as in the light experiments. Three replicate 
trials, each with new snails, were run at all four start times. 

The goal of the second series of experiments was to 
determine if an endogenous rhythm, stimulated by light, 
might control emergence duration. This research is neces- 
sary but not sufficient to establish a circadian rhythm 
(Aschoff. 1960; Pittendrigh, 1993; Dunlap. 1999). The ex- 
periment held lightidark ratio constant while varying the 
onset of daylight. The prediction was that emergence dura- 
tion would track with an internal clock set by incipient 
dawn, independent of the absolute time of day. 

Two temperature- and light-controlled chambers were set 
at 21 C and 40 /j,mol/nr/s. The control chamber was set on 
a light:dark (14:10) cycle, with the natural (0600) sunrise. 
The experimental chamber used the same light:dark ratio 



PATTERNS AND PROCESSES OF LARVAL EMERGENCE 



13 



hul with sunrise shifted either 8 h forward to 1400 or 
backward to 2200. In the first experiment, 100 snails were 
placed in the control and another 100 in the forward-shifted 
light chamber for 1 week before the trial began. After this 
acclimation period, emerging cercariae in both chambers 
were monitored over 4 h, beginning at 1 800, as in the light 
experiments. In the second experiment, new snails were 
acclimated to the control and backward-shifted light regime 
for 1 week, and snails were monitored for 4 h beginning at 
1000. In both experiments, the control and light-shifted 
snails were tested on sequential days. Moreover, as a check 
on the repeatability of the results, the same set of snails was 
monitored over an additional 4 d. Both experimental series 
were conducted once in 2000 and once in 2001. 



Results 



Field observations 



The magnitude of cercarial emergence correlated signif- 
icantly with water temperature, but not with salinity or total 
irradiance (Table 1 and Fig. 1). Snails (Cerithicleti califor- 
nicu) first appeared in tidal channels in February, when 
temperatures rarely exceeded 13 C (Fig. 2 A). Emergence 
did not begin until March, however, when seawater warmed 
to 15 C and above (Fig. 1). Despite continued warming 
during April and May, emergence was moderate until June. 
A relatively close correspondence between temperature and 
emergence is evident for the spring cercariae. which vacated 
their hosts at about the same temperature threshold ( 15 C) 
during each tidal cycle (Fig. 2B). As cool ocean water 
flooded the marsh, the shallow water mass was warmed first 
from contact with the mudflat and then by the sun. Cercarial 
emergence following snail inundation was typically delayed 
up to 2 h, until water temperature exceeded 15 C. 

During the warm summer months, June to September, 
when water temperature was above 18 C (average of 
21 C), the number of emerged cercariae increased substan- 
tially (Fig. 1). Larvae left snails as soon as they were 
inundated (Fig. 2C). Despite similarity in temperature (1-2 
C difference) between paired day/night collections, few or 
no cercariae were collected at night (Table 2). Seawater 
cooled by about 3 C in October, but emergence remained 



Table 1 

Stepwise multiple regression analysis of environmental cues tt.\ source* 
of variation in the number of emerged tc/< LIIUK 











% of variation 


Source 


F-value 


df 


P 


explained 


Temperature 


37.14 


1/31 


<0.0001 


68.3 


Total irradiance 


0.05 


1/31 


0.82 


<1.0 


Salinity 


0.34 


1/31 


0.56 


<I.O 




B 



D. 



.;:> 




1 r 






20 






If 


|T 


t 




S 3 


EI 










& j. 


15 


















n 


34.0 




330 


j 


ft F 




i 


i 


a P 


i j 


d 


: [3 


i f 


i 


f 


* 


320 
11 





























D 



v 2 fe 

fffiifi 



m 4 A 


^ 




t 




1 




t 




1 




* ro lii y 


* 




* 




* 




* 




* 





J FMAMJ J ASOND 


Month 



Figure 1. Variation throughout the year in average monthly (A) daily 
irradiance (histograms) and day length (diamonds), (Bl water temperature. 
(C) salinity. (D) cercarial emergence and snail host presence on the mudflat 
( * ). Histograms represent monthly means, and vertical bars plot the ranges. 
For day length, ranges are smaller than the data points. Irradiance data were 
taken from the California Irrigation Management System website (www. 
ctmis.water.ca.gov) and day length from the United States Naval Obser- 
vatory website (http://aa.usno.navy.mil). 



high until November. Once temperature dipped below 
18 C, only the cool-water species emerged. 

Observations were made on the appearance of dominant 
species in the samples. Himasthla rhigedana (Dietz) and 
Parorchis acanthus (Nicoll) were the first species collected 
during the cool spring months (March to May), making up 
about 90% of the cercarial population. Other species, such 
as Renicola biicluintini (Cohn), Euhaplorchis californensis 
(Martin), and Microphallid sp. (Martin), first appeared in 
late May or early June. Due to its high prevalence (Kuris. 
1990) and fecundity, E. californensis numerically domi- 
nated the June to September samples. Together. H. rhige- 
dana and E. californensis composed more than 75% of all 
cercariae collected throughout the day during the warm 
summer months. 

Whereas cercarial emergence was high and relatively 
constant from June through September, duration of emer- 
gence events varied considerably among the 21 summer 
daylight samples. A stepwise multiple regression analysis of 



114 



J.T. FINGERUT ET AL. 



A 



B 



100 

50- 

25 

0- 



aary-Febmary 



March-May 



-10/99 



Temperature 
Cercanae 



16 



- 14 



4/16/99 



7 




GO 



0) 



w 



U 



18 
16 



03 .S 










-^ i nn ^ 




-18 




r 18 


Oj 
O O 


2/8/00 




3/3/00 / 




O !- 7S- 




. 


j 




o> r i 
- !^ 50- 




- 16 

- 14 


^ J 


1(1 
14 


.; GO 25- 


.yOO-C- ^ 00 ' '^ 




I 




-2 n . 


. - 
... ............... 




^ y 


L 1 ~> 



iou 


2/23/01 




5/2/01 1 




7<; 






/ 




50 




-16 


J 


- 16 




ojy^oo^" 1 ** 00 * 


- 14 




- 14 


25 - 






.../ 




n - 




- n - 




- P 



50 100 150 200 250 



50 100 150 200 250 



c 



June-September 



9/4/99 




7/1/00 



6/12/01 



Time Since Inundation (min) 

Figure 2. Three representative cercarial emergence patterns for each of three seasons of the year: (A) 
January-February (winter). (B) March-May (spring), and (C) June-September (summer). Water temperature 
(open circles) and cumulative percentages of emerged cercariae (closed squares) are from samples taken every 
10 min. In (B). arrows correspond to the point in time when water temperature was first 15 C. 



30 

27 

-24 

21 
18 



30 

L 27 

. 24 

-21 

18 



u 

o 
0> 



03 



<U 



30 

27 

24 

21 

18 



50 100 150 200 250 



emergence duration against temperature, salinity, time of 
year, and time of day yielded only one significant factor: 
time of day (Table 3). Duration of cercarial emergence 
decreased significantly throughout the day (Fig. 3 A). Emer- 
gence intervals ranged from 4 h in the morning to 1 h or less 
by dusk (Figs. 3A and 4). This trend was present regardless 
of the numerical threshold for emergence duration (i.e., 
95%. as used here, versus 15% or 50%). Decreasing dura- 
tion of cercarial emergence over the day resulted from an 
increase in the rate of emergence (number cercariae/h) (Fig. 
3A, and least squares regression: F = 27.36, df = 1/19, P < 
0.0001; r 2 = 0.73). not from a decrease in the total number 
of emerging cercariae (Fig. 3B. and P = 0.68). Long (~4 h) 
emergence durations during the early morning resulted from 
a relatively small but constant larval delivery over the entire 
period ol .nup.dation. As the day progressed, more cercariae 
emerged per unit time, but over a contracted interval. Ap- 
proaching dus!:, large numbers of cercariae emerged in a 
quick burst as inundation commenced. 



Table 2 

Number of cercariae shed during paired day/night collections 



Date 


Day or 
night 


Average 
temperature (C) 


Number emerged 
per 4 h collection 


1 Aug 2000 


Day 


21.4 


1043 




Night 


20.1 


22 


1 8 Aug 2000 


Day 


20.9 


1543 




Night 


19.9 


11 


24 July 2001 


Day 


22.3 


1684 




Night 


20.5 


28 


12 Aug 2002 


Day 


19.3 


537 




Night 


18.2 


3 


22 Aug 2002 


Day 


22.1 


832 




Night 


21.7 


1 


6 Sept 2002 


Day 


19.3 


349 




Night 


17.9 






Wtite r temperature is averaged over the entire 4-h collection. Mean 
intensity during nighttime collections, even under full moon conditions, 
was a/ways < 0.01 fjjnol/m 2 /s, compared to > 1035 jjjnoUm'/s during 
daylight collections. 



PATTHRNS AND PROCESSES OF LARVAL EMERGENCE 



115 



Table 3 



viiriiitiim in fmcr.ifr/iir iliiriitinn 



Source 



/ -\alue 



' i nl variation 
ill P explained 



Time oUl.ii 47.96 1/19 <0.0()()l 

Time ol vc.n iinoiuhl 11(13 1/19 0.86 <1.0 

Temperature <O.C)1 1/19 (1.45 <I.O 

Salinil\ 1 .7S 1/19 0.19 S.I 



Laboratory experiments 

Hn-,1 inundation. Throughout all laboratory studies, cer- 
cariae emerged onl\ if snails were totally submerged. The 
average number of cercariae to emerge in paired treatments 
with submerged (3861 599 SEM) and dry (0 SEM) 
snails showed unequivocally that cercariae would not leave 
the host unless it was underwater (Student's / test: / = 
8.497. df = 5, P < 0.0001 ). The muddy channel containing 
the highest snail densities was at a tidal height of about 



250 



e 

00 

u 
W 



200- 



100 - 



50- 



3 * 




B 



10 12 14 16 18 20 



' ' ' Zi 

"J ^ 



y "3 
-^ O 

Ef- 



-= ua 
H 



10 12 14 16 

Time of Day (h) 



18 20 



Figure 3. Effect of time of day (TOD) on cercarial emergence duration 
in the tield. (A) The amount of time for 95% of all cercariae to emerge 
during a given event (4 h) as a function of TOD. (B) The total number of 
cercariae emerged per snail during a given event as a function of TOD. 



2.0 


9:00 


1.5 




1.0 




0.5 
n 





a 

c 

C/3 

I 

g 

Of) 



8 

'i 



u 

^-i 
O 

I 



z 



2.0 
1.5 
1.0 
0.5 
n 


.Illn 


12:00 
il...i 1 



2.0 


15:00 


1.5 




1 






0.5 

n 


h 


il.... . 



2.0 
1.5 
1.0 
0.5 


18:00 

IL 


9 < _ 




2.0 
1.5 
1.0 
0.5 
n 


20:00 
1... 



120 240 

Time Since Inundation (min) 

Figure 4. Representative series of histograms throughout the day. 
showing number of emerged cercariae per snail (10-min samples) as a 
function of time since tidal inundation. Times indicate when snail hosts 
were first submerged. Because snail inundation resulted from the natural 
high tide, each histogram \\as constructed on a different day. 

1.3 m MLLW. so hosts spent 16 h/day out of water. Thus, 
inundation was a necessary condition for emergence. 

Temperature. Cercariae of five trematode species 
emerged at three temperature thresholds (Fig. 5). Emer- 
gence of H. rhit>edana and P. acanthus began in 15 C 
water and continued for the maximal temperature (19 C) 
tested. Renicola huclianani emerged at an intermediate 
threshold of 17 C through 19 C. The two warm-water 
species, E. californensis and Microphallid sp.. vacated their 
hosts only in the warmest waters tested (18-19 C). Two- 
way nested ANOVAs were performed for each species 
separately. The numbers of emerged cercariae were signif- 
icantly different among temperatures (P < 0.0001 for all 
species) and individual snails (P < 0.04) for all species 
except R. biicluinani (P = 0.17). Because the order (ascend- 
ing, descending) of temperature change had no significant 
(P > 0.1 1) effect on the outcome. Scheffe post hoc com- 
parisons were done for both series. Either way. there were 
two significantly (P ^ 0.005) different temperature group- 
ings for all species: one within their emergence range and 



116 



J.T. FINGERUT ET AL 



4000 


Eulinr forniensii -r 


2000 


n = 14 










A 


a 


a 


a 


a a b 


b 



U 

"+i 


a a a a a ] b| b 




c 

CO 
o 2000 


Microphallid sp. 
a a a a a | b | | b | 


M 
u 




)f Cercariae Ei 

K- U 
>. Ui C 

3 O O C 


Renicola buchanani pj-, rr~| T 
n = 6 

a a a a b b b 




Average Number i 

UJ CTs tO J 

o o o o o c 


Parorchis acanthus T 
a a |b| |b| b| |b| |b 




Hiinasthlti rlu^cilana T 
a a |b| |b| |b| b |b| 


13 14 15 16 17 18 I 1 * 



Temperature (C) 

Figure 5. Number of cercariae emerged per snail over the temperature 
range 13-19 C. Data are shown lor rive common irematode species. 
Histograms are means, and vertical bars are standard errors: n = number 
of snail hosts for a given tremalode species. Means differ significantly 
when highlighted by a different letter lone-way ANOVA with p<^t Inn 
Scheffe test: P < 0.001 1. 



the other for the lower temperatures that prohibited emer- 
gence (Fig. 5). 

Lixht. Although daylight is required for carcarial emer- 
gence (Table 2). even low intensities approximating those at 
dawn and dusk (40 /imol/nr/s) were sufficient to trigger 
emergence under both field and laboratory settings. There 
was no significant difference, however, in the number of 
emerged cercariae or the duration of emergence at maximal 
midday intensity (2000 jumol/nr/s) relative to a minimal 
dawn/dusk intensity (40 /xmol/nr/s) (/ test: t= 1.021. df = 
10, P = 0.36 for duration: / = 0.475. df = 10, P = 0.65 for 
number emerged). 

Time of day. As in the held collections, the duration of 
cercarial emergence decreased over the course of the day 
(Fig. (S A), varying from a modest, protracted stream ot 
cercariae at dawn to a contracted burst at dusk. There was a 
significant relationship between emergence duration and 
TOD (least squares regression: F = 199.78. df = 1/10. P < 
0.0001: r = 0.93). but not between number of emerged 
cercariae and TOD (P = 0.28) (Fig. 6B). Moreover, there 



was a noticeable change in the duration of emergence when 
the host light:dark cycle was shifted forward or back (Fig. 
7). As predicted, emergence duration decreased when sun- 
rise was shifted forward, implying that snails perceived a 
later time of day. Likewise, emergence duration increased 
when sunrise was shifted back. These changes agree qual- 
itatively with the diel pattern observed in both the field and 
laboratory. Shifting the light:dark cycle had no apparent 
effect on the number of cercariae emerging (data not 
shown). 

Discussion 

All digenetic trematodes have a free-swimming cercarial 
stage that transmits infection from the first intermediate host 
to the second intermediate or definitive host. Given the short 
life span of cercariae. emergence timing may be optimized 
to enhance the probability of successful transmission. In this 
study, larvae emerged from Ceritliidea californica over 
species-specific temperature ranges, exclusively during day- 



A 

300 



combo o 



o 



3 

Q 



UJ 



250- 
200 
150 
100 



8 10 



12 14 16 18 20 



B 



80 



SI 60 

U ^ 
2 ^ 40 

P oa 
S3 

20 



10 12 14 16 
Time of Day (h) 



18 20 



Figure 6. Effect of time of day (TOD) on emergence duration in the 
laboratory. (A) The amount of time for 95% of all cercariae to emerge 
during a niveu event (4 h) as a function of TOD. (Bl The total number ot 
cercariae emeiged per snail during a given event as a liiiiction of TOD. 



I'.\ITI-:RNS AND PROCESSES OF LARVAL EMERGENCE 



17 







Q 



C 
u 

E? 
<u 

UJ 



225 - 


I 


Shifted 
Backward 


200- 


D 


I 


175- 


Shifted 


I 


i sn 


Forward 





8 10 12 



14 



16 



18 20 



Time of Day (h) 



Figure 7. Effect on emergence duration of shifting sunrise forward or 
backward S h. compared to mishitted control groups. Filled symbols 
indicate cuntiol \alues b\ \eai (2(100 . 21)01 ). and open symbols 
indicate shifted regimes (200O . 2(101 D). 



light hours, and only when snails were submerged (Fig. 8). 
The three determinants operated over different time scales: 
temperature monthly, light diurnally (24-h period), and wa- 
ter depth tidally (12-h period). Light/dark and tidal cycles 
also varied daily, according to the solar and lunar cycles, 
respectively. Each stimulus creates a necessary condition 
for the next, forming a hierarchy of environmental cues. 
Duration of cercarial emergence varied over the day and 
\sas under endogenous control. Although many studies have 



identified two or more factors that affect propagule release. 
e\ idcncc for a cue hierarchy is relatively rare (some excep- 
tions include Levy et ai, 2000; Watson ct ill., 2000). 

The timing and length of the transmission season was 
controlled, at least in part, by water temperature. Host snails 
(C. culifaniicii) occupied the muddy surface of the channel 
when water was 12 C or above. Thus, the much narrower 
temperature ranges for emergence of the five trematode 
species may forecast the presence of second intermediate 
hosts. For example, cercariae of Hiimixthlu rhi\>edami and 
Parorchis iii-iinthus emerged over the broadest temperature 
range (> 15 C) and use the eurythermal C. califonuca as 
a second intermediate host. In contrast. Eulniplorchis cali- 
fornicnxix, which emerged only in > 18 C water, infects 
several fish species that increase in abundance over the 
wanning summer (Fritz. 1975; Brooks. 1999; Madon ct <//.. 
2001). 

Temperature thresholds for cercarial emergence occur in 
both estuarine and freshwater habitats (Lo and Lee. 1996; 
McCarthy, 1999). The number of emerged cercariae also 
varies with temperature in freshwater species (Shostak and 
Esch, 1990; Lyholt and Buehmann. 1996). Temperature- 
dependent emergence in freshwater species has been as- 
cribed to a trade-off between the number emerged (posi- 
tively related to temperature) and functional (infective), and 
the total life span (both negatively related to temperature) 
(McCarthy, 1999: Toledo ci ai. 1999). 

A characteristic common to estuarine and freshwater sys- 
tems is diel variation in cercarial emergence, supporting our 
first hypothesis (see Introiliicridii). In our study, cercariae 



Time 
Scale 



Cue 



= Conditions suitable for emergence 



Monthly Temperature 



Daily 
(24 h) 

Tidal 
(12 h) 



Light 



Inundation 



2 1456789 10 II 12 13 14 15 16 17 18 19:021 222324 



23456.789101 12 13 14 15 16 1718 19 2(1 21 222324 




Figure 8. Diagram of the hierarchy of cues controlling cercarial emergence al monthly (temperature), daily 
(light), and tidal (host inundation) time scales. Dotted line represents tidal height (13 m above ML.LW) of 
nnidllat where sampling took place. 



18 



J.T. FINGERUT ET AL. 



left submerged snails only during daylight hours. In inter- 
tidal estuaries, tidal currents are a predictable signal that 
determines both submersion time of the hosts and aquatic 
transport of the larvae. Thus, emergence of cercariae 
tracked u ith the daytime flood tide, which changes daily 
according to the lunar cycle. The daylight requirement may 
indicate that light is a critical cue. In laboratory Hume 
studies, photo-triggered downward swimming of H. rhi^c- 
tltiiui was effective in slow flows typical of CSMR (Fingerut 
el ill.. 2003). This activity quickly brings larvae to the bed 
for contact with benthic hosts. Moreover, we observed that 
/:. culifomienxis cercariae swam upward in response to light 
(authors' unpubl. data). Such behavior could increase con- 
tact with host fish in the water column. Likewise, most 
freshwater cercariae are shed on the bottom, and light or 
gravity directs swimming up or down, depending on the 
location of the next host (Combes el <//.. 1994). Within the 
CSMR estuary, cercarial emergence during flood tides 
would transport larvae 100-300 m/h (Fingerut, 2003). Yet. 
emerged H. rhigedana were dispersed only about 1-2 in 
before encystment (Fingerut, 2003). Directed swimming by 
larvae thus may have greatly shortened their transport dis- 
tances. 

Abbreviated dispersal would retain asexually reproduced 
larvae within the marsh, where local mixing could diversify 
the gene pool of cercariae encysting on subsequent hosts. 
All except 2 of 18 species within the southern California 
estuarine trematode guild have benthic second intermediate 
hosts (snails and crabs) that are interspersed within first 
intermediate host populations. In fact, some trematodes 
(e.g., H. rhigedana, P. acanthus) use the same first and 
second host species. Turbulent mixing could commingle 
larval genotypes throughout the marsh, enhancing genetic 
diversity of the multiple cercariae that encyst each second 
host. Ensuing sexual reproduction between dissimilar ge- 
netic parasites within the definitive shorebird host may 
enhance fitness of the trematode population (e.g., Schel- 
tema, 1971; Jablonski, 1986; Pechenik, 1999, for marine 
invertebrates). 

As in digenetic trematodes, larval release in tree-living 
estuarine invertebrates is contingent on tidal flows. For 
example, estuarine mud-dwelling isopods (e.g., Parag- 
luitliia formica [Hess]) release larvae only when high tides 
reach their burrows, facilitating transport (Tinsley and 
Reilly, 2002). Shore crabs living in high-intertidal refuges 
limit larval release to nighttime spring tides (Morgan and 
Christy, 1995; Hovel and Morgan, 1997). Darkness reduces 
predation on spawners, whereas large-amplitude tides flush 
I away from diurnal predators. Similarly, in tidally 

influenced riverine habitats, adult terrestrial crabs, such as 
Sesannn huematochcir (de Haan), release larvae on a night- 
time semilunar cycle that minimizes predation on adults 
while providing optimal conditions for survival and dis- 
persal of larvae (Saigusa, 1982). 



Endogenously regulated emergence differs between the 
estuarine intertidal trematodes studied here and many fresh- 
water species. This result was not predicted by our second 
hypothesis (see Introduction), and may be explained by the 
major synchronizing agents in these systems. In freshwater, 
emergence timing is often under endogenous control and is 
directly linked to presence of the subsequent host. For 
freshwater schistosomes, definitive vertebrate hosts fre- 
quent the waterfront on a predictable innate cycle. Circadian 
rhythms of cercarial emergence are tuned to the biological 
clocks and activity patterns of these vertebrates (Combes. 
1991; Combes et al.. 1994). For example, maximal emer- 
gence of some schistosome cercariae corresponds with the 
proximity of their bovine hosts. Cercariae emerge and swim 
to the surface in the morning, thus infecting the animals 
while they drink at the waterfront (Mouahid et ill., 1991; 
Raymond and Probert. 1991). Likewise, afternoon emer- 
gence peaks occur in trematode species infecting human 
hosts that wash, play, or drink at midday (Theron, 1984; 
Pages and Theron. 1990). Emergence in non-schistosome 
species, such as Proterometra edne\i (Uglem) also occurs 
on a circadian cycle timed to the presence of fish that are 
their second intermediate hosts (Lewis el <//.. 1989). There 
is no emergence rhythm in cercariae of Fasciolti liepaticu 
(Linnaeus), however, because second intermediate host 
plants are always available (Bouix-Busson et <//., 1985). 
making synchronization unnecessary. 

In the CSMR intertidal estuary, duration rather than tim- 
ing of emergence was apparently under endogenous control, 
and the tide was the synchronizing agent. There is a finite, 
tidally determined period of sufficient immersion for larval 
transport in lighted hours. Emergence duration decreased 
throughout the day in response to dwindling daylight. An 
endogenous rhythm associated with the light:dark cycle 
may be responsible for the changing emergence rate over 
the course of a suitable flood tide. Apportioning larvae over 
the entire lighted submerged interval maximizes the dis- 
persal envelope. Contraction of emergence duration 
throughout the day may optimize use of remaining daylight 
hours. As the day progresses, larger pulses of larvae must 
emerge over a shorter interval to take advantage of the 
vestigial light. Such diel adjustments to the emergence 
period are unnecessary in freshwater systems, where water 
depth is relatively stable. Thus driven by the submergence 
constraints of their natal habitat, the emergence strategies of 
estuarine cercariae involve both exogenous and endogenous 
factors that optimize transport to tidally accessible hosts. 

Acknowledgments 

This study was supported by awards from the National 
Science Foundation (OCE 97-22972 and OCE 02-47834) 
and the UCLA Council on Research. We thank M. Grattan 
and E. Maldonado for their tireless help in the field. A. 



PATTERNS AND PROCESSES OF LARVAL EMERGENCE 



119 



Kuris contributed valuable insights on trematode parasites 
and comments that substantially improved earlier drafts of 
this manuscript. 

Literature Cited 

Adams, J. E., and \V. E. Martin. 1963. Lite cycle of Himaxihla 
rhigcilana Dietz 1909 (Trematoda: Echinostomatidae). Trans, Am. 
Microsc. Sue. 82: 1-6. 
\ MLIII.I. S. 1988. Morning release of larvae controlled by the light in an 

intertidal sponge. Callyspangia ramosa. Bin/. Bull. 175: 181-1S4. 
Aschoff. .1. 1960. Exogenous and endogenous components in circadian 

i In thins. Colil Spring Harbor Symp. Quant. Biol. 25: 11-28. 
Bartoli. P., and C. Combes. 1986. Dissemination strategies of trema- 
tode cercariae in a coastal marine ecosystem. Ada Oecoi Oecol. Gen. 
7: 101-1 14. 
Bergquist, N. R. 2002. Schistosomiasis: from risk assessment to control. 

Trends Parasitol. 18: 309-314. 

Bouix-Busson, D., D. Kondelaud. and C. Combes. 1985. L' infestation 
de Lymnaea glabra Muller par Fasciola hepatica L: les caracteristiques 
des emissions cercariennes. Ann. Parasiiol. Hum. Comp. 60: 11-21. 
Brooks. A. J. 1999. Factors influencing the structure of an estuarine tish 
community: the role of interspecific competition (Gil/icht/ivs mirahilis, 
Leptocottiu arinatus). Ph.D. dissertation. University of California. 
Santa Barbara. 
Combes, C. 1991. Ethological aspects of parasite transmission. .4/71. Nut. 

138: 866-880. 

Combes, C., A. Fournier, H. Mone, and A. Theron. 1994. Behaviors 
in trematode cercariae that enhance parasite transmission: patterns and 
processes. Parasitology 109: S3-S13. 
Curtis, L. A. 1997. llyanassa obsoleta (Gastropoda) as a host for 

trematodes in Delaware estuaries. J. Parasitol. 83: 793-803. 
Dunlap, J. C. 1999. Molecular basis for circadian clocks. Cell 96: 

271-290. 

Fingerut. J. T. 2003. From host to host: interaction of environmental 
conditions and larval behavior in transmission of an estuarine parasite. 
Ph.D. dissertation. University of California. Los Angeles. 
Fingerut. J. T., C. A. Zimmer, and R. K. Zimmer. 2003. Larval 
swimming overpowers turbulent mixing and facilitates transmission of 
a marine parasite. Ect>log\ 84: 2502-2515. 

Forward. R. B. Jr., J. K. Douglass, and B. E. Kenney. 1986. Entrain- 
ment of the larval release rhythm of the crab Rhithropanopeus harissi 
(Brachyura: Xanthidae) by cycles in salinity change. Mar. Biol. 90: 
537-544. 

Fritz, E. S. 1975. The life history of the California killih'sh Fiindiiliix 

parvipinnis in Anaheim Bay. California, USA. Fish. Bull. 165: 91-106. 

Gerard, C. 2001. Structure and temporal variation of trematode and 

gastropod communities in a freshwater ecosystem. Parasite 8: 275- 

287. 

Horn, H. S., R. Nathan, and S. R. Kaplan. 2001. Long-distance 

dispersal of tree seeds by wind. Ecol. Res. 16: 877-885. 
Hovel. K. A., and S. G. Morgan. 1997. Planktivory as a selective force 
for reproductive synchrony and larval migration. Mar. Ecol. Prog. Ser. 
157: 79-95. 
Jahlonski. I). 1986. Larval ecology and macroevolution in marine 

invertebrates. Bull. Mar. Sci. 39: 565-587. 

Jonsson. P. R.. and C. Andre. 1992. Mass mortality of the bivalve 
Cerastoderina edule on the Swedish west coast caused by infestation 
with the digenean trematode Cercurui ceraxtuaennae I. Ophelia 36: 
151-157. 

Kuris, A. 1990. Guild structure of larval trematodes in molluscan hosts: 
prevalences, dominance and significance of competition. Pp. 69-100 in 
Parasite Communities: Patterns and Processes. G.W. Esch. ed. Chap- 
man and Hall. London. 



Levy, O., L. Mizrahi, N. E. Chadwick-Furman, and Y. Achituv. 2001. 

Factors controlling the expansion behavior of Favia turns (Cnideria: 
Scleractinial: effects of light, flow, and planktonic prey. Biol. Bull 200: 
118-126. 

Lewis, M. C.. I. G. Welsford. and G. L. Uglem. 1989. Cercarial 
emergence of Proterometra macroxtoma and P. ednevi (Digenea: Azy- 
giidae): contrasting responses to lighcdark cycling. Parasitolo^v 41: 
201-208. 

Lo, C. T., and K. M. Lee. 1996. Pattern of emergence and the effects 
of temperature and light on the emergence and survival of heterophyid 
cercariae (Centrocestus fitrinuxanus and Haplorchis pumilia). J. Para- 
sitol. 82: 347-350. 

Lyholt, H. C. K., and K. Kuchmann. 1996. Diplostomum spathaceiini: 
effects of temperature and light on cercarial shedding and infection of 
rainbow trout. Dis. Aaiiat. Org. 25: 169-173. 

Madon, S. P., G. D. Williams. J. M. West, and J. B. Zedler. 2001. The 
importance of marsh access to growth of the California killifish. Fun- 
dulus parvipinnis. evaluated through bioenergetics modeling. Ecol. 
Model. 136: 149-165. 

Martin, \V. E. 1950. Enhaplorchn mli/orniensis N.G.. N. Sp.. Hetero- 
phydae, Trematoda. with notes on its life cycle. Trans. Am. Microsc. 
Soc. 69: 194-209. 

Martin, \V. E. 1972. An annotated key to the cercariae that develop in 
the snail CerithiJea calitornica. Bull. S. C. Acad. Sci. 79: 39 13. 

McCarthy, A. M. 1999. The influence of temperature on the survival 
and infectivity of the cercariae of Echinuparyphium recurvatiim (Di- 
genea: Echinostomatidae). Parasito/ogv 118: 383-388. 

McKerrow, J. H., and J. Salter. 2002. Invasion of skin by Schistusoma 
cercariae. Trends Paraxiinl. 18: 193-195. 

Morgan, S. G. 1996. Influence of tidal variation on reproductive timing. 
J. Exp. Mar. Biol. Ecol. 206: 237-251. 

Morgan, S. G., and J. H. Christy. 1995. Adaptive significance of the 
timing of larval release by crabs. Am. Nat. 145: 457 179. 

Mouahid, A., H. Mone, A. Chaib, and A. Theron. 1991. Cercarial 
shedding patterns of Schistosoma hovis and Schistosoma haematobium 
from single and mixed infections of Bulinus truncates. J. Helmintliol. 
65: 8-14. 

Mouchet, F., A. Theron, P. Bremond, E. Sellin, and B. Sellin. 1992. 
Pattern of cercarial emergence of Schistosoma curassoni from Niger 
and comparison w ith three sympatric species of schistosomes. J. Para- 
sitol. 78: 61-63. 

N'Goran, E., P. Bremond, and E. Sellin. 1997. Intraspecific diversity 
of Schistosoma haematobium in West Africa: chronobiology of cer- 
carial emergence. Ada Trap. 66: 35 14. 

Pages, J. R., and A. Theron. 1990. Analysis and comparison of 
cercarial emergence rhythms of Schistosoma haematobium. Schisto- 
soma intercatatum. Schi.sto.xuina hovis. and their hybrid progeny. Int. J 
Parasitol. 20: 193-198. 

Pechenik, J. 1999. On the advantages and disadvantages of larval stages 
in benthic marine invertebrate life cycles. Mar. Ecol. Prog. Ser. 177: 
269-297. 

Pechenik. J. A., and B. Fried. 1995. Effect of temperature on survival 
and infectivity of Echino.sioma trivolvis cercariae: a test of the energy 
limitation hypothesis. Parasitology 111: 373-378. 

Pittendrigh, C. S. 1993. Temporal organization: reflections of a dar- 
winian clock-watcher. Annti. Rev. Phvsiol. 55: 16-54. 

Raymond, K.. and A. J. Prohert. 1991. The daily cercarial emission 
rhythm of Schistosoma margrebowiei with particular reference to dark 
period stimuli. J. Helmintliol. 65: 159-168. 

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

Schauber. E. M., D. Kelly, P. Turchin, C. Simon. W. G. Lee, R. B. 
Allen. I. J. Payton, P. R. Wilson, P. E. Cowan, and R. E. Brockie. 



120 



J.T. FINGERUT ET AL. 



2(1(12. Masting h> . igl New Zealand plant species: the role of 

temperature as a s 1 ' . >ng cue. Ecology 83: 1214-1225. 

Scheltema, R. S. 197 \al dispersal as a means of genetic exchange 

between gen >\ separated populations of shallow-water benthic 

marine gasi- Biol. Bull. 140: 2X4-322. 

Shostak. A. .uid G. W. Esch. 1990. Photocycle-dependent emer- 
gence (. ^(.-rcariae of Halipegus occidualis from Helisoma anceps, 
with special reference to cercarial emergence patterns as adaptations 
tor transmission. J. Parasitol. 76: 790-795. 

Sousa. \V. P. 1983. Host life history and effect of parasitic castration on 
growth: a field study ot" Cerithidea califomicu Haldeman (Gastropoda: 
Prosobrachia) and its trematode parasites. J. Exp. Mar. Biol. Ecol. 73: 
273-296. 

Sousa, VV. P., and M. Gleason. 1989. Does parasitic infection compro- 
mise host survival under extreme environmental conditions'? The case 
tor Cerithidea californica (Gastropoda: Prosobranchia). Oecologia 80: 
456-464. 



Theron, A. 1984. Early and late shedding patterns of Schistosoma 
mansoni cercaria: ecological significance in transmission to human and 
marine hosts. J. Parasitol. 70: 652-665. 

Theron, A. 1989. Hybrids between Schistosoma mansoni and Schisto- 
soma rodhuini: characterization by cercarial emergence rhythms. Par- 
asitology 99: 225-228. 

Tinsley M. C., and S. D. Reilly. 2002. Reproductive ecology of the 
saltmarsh-dwelling marine ectoparasite Paragnathict formica (Crusta- 
cea: Isopoda). 7. Mar. Biol. Assoc. UK 82: 79-S4. 

Toledo, R., C. Munoz-Antoli, and J. G. Esteban. 1999. Production and 
chronobiology of emergence of the cercariae of Euparyphium albufe- 
rensis (Trematoda: Echinostomatidae). 7. Parasitol. 85: 263-267. 

Watson G. J., M. E. Williams, and M. G. Bentley. 2000. Can syn- 
chronous spawning be predicted from environmental parameters'? A 
case study of the lugworm Arenicola marina. Mar. Biol. 136: 1003- 
1017. 



Reference: Biol. Bull. 205: 121-132. (October 2003) 
2003 Marine Biological Laboratory 



Larval Development and Metamorphosis in 

Pleurobranchaea maculata, With a Review of 

Development in the Notaspidea (Opisthobranchia) 

GLENYS D. GIBSON 

Department of Biology. Actulin University. Wolfi'ille, Nova Scotia. Canada B4P 2R6 



Abstract. Pleurobranchaea maculata is a carnivorous no- 
taspidean that is common in New Zealand. This species 
produces small eggs (diameter 100 p.m) and planktotrophic 
veligers that hatch in 8 d and are planktonic for 3 weeks 
before settling on biofilmed surfaces ( 14 C). Larval devel- 
opment is known in detail for only two other notaspidean 
species. P. japonica and Beithellina citrina. In all three 
species of pleurobranchids. mantle and shell growth show 
striking differences from veligers of other opisthobranch 
taxa. In young veligers of pleurobranchids, the shell is 
overgrown by the mantle, new shell is added by cells other 
than those of the mantle fold, and an operculum does not 
form. Thus some "adult" traits (e.g.. notum differentiation, 
mechanism of shell growth, lack of operculum) are ex- 
pressed early in larval development. This suggests that 
apomorphies characteristic of adult pleurobranchids 
evolved through heterochrony. with expression in larvae of 
traits typical of adults of other clades. The protoconch is 
dissolved post-settlement and not cast off as occurs in other 
opisthobranch orders, indicating that shell loss is apomor- 
phic. P. maculata veligers are atypical of opisthobranchs in 
having a field of highly folded cells on the lower velar 
surface, a mouth that is posterior to the metatroch. and a 
richly glandular, possibly chemodefensive mantle. These 
data indicate that notaspidean larvae are highly derived in 
terms of the novel traits and the timing of morphogenic 
events. Phylogenetic analysis must consider embryological 
origins before assuming homology. as morphological simi- 
larities (e.g.. shell loss) may have developed through dis- 
tinct mechanisms. 



Received 27 August 2002: accepted 23 June 2003 
E-mail: glenys.gibson@acadiau.ca 



Introduction 

The Notaspidea is a small, specialized order of opistho- 
branchs that are considered to be phylogenetically interme- 
diate between the highly derived Nudibranchia and the more 
basal Cephalaspidea (Schmekel, 1985; Willan. 1987; 
Mikkelsen, 1998) or monophyletic with the Nudibranchia to 
form the Nudipleura (Wa'gele and Willan, 2000). Compar- 
ative embryological studies of the Notaspidea are therefore 
significant for phylogenetic analyses but also for under- 
standing morphological evolution in the Opisthobranchia, a 
clade rich in homoplastic similarities (Gosliner, 1991, 1994; 
Mikkelsen, 1998). Unfortunately, little is known about no- 
taspidean development. My goal is to describe larval devel- 
opment in Pleurobranchaea maculata (Quoy and Gaimard, 
1832) and to provide a preliminary analysis of development 
in Notaspidea. 

Adult notaspideans are carnivores and opportunistic scav- 
engers. They are characterized by a single, external 
ctenidium on the right side, rolled rhinophores, and a flat- 
tened shell (Willan, 1983: Schmekel, 1985; Willan. 1987). 
The order traditionally includes the Umbraculomorpha 
(families Tylodinidae and Umbraculidae) with large limpet- 
like external shells and a small mantle, and the Pleuro- 
branchomorpha (Pleurobranchidae) with a prominent man- 
tle and shells that are internal and reduced or lost in adults 
(Thompson. 1976; Willan. 1983. 1987). Mantle secretions 
provide chemical defense in many species, both shelled and 
shell-less, through the release of acid (Thompson and Slinn. 
1959; Thompson. 1988), secondary metabolites (Ciavatta et 
til.. 1993, 1995: Spmella et ai. 1997). or dietary alkaloids 
(Ebel et ai. 1999). On the basis of adult anatomy, some 
investigators suggest that the Notaspidea are polyphyletic 
(Schmekel, 1985). while others argue that they are 



122 



G. D. GIBSON 



paraphyletic and re- uire inclusion of the Nudibranchia 
(Wagele and Will 00). 

Phylogenetic . ;. lysis promotes an understanding of the 
relatedness ai laxa. and of morphological evolution, for 
which coi: .utive developmental data are essential. Al- 
though tl re is no reason to assume that larval and adult 
traits have coevolved, inclusion of larval traits strengthens 
analyses by increasing the data set, but more importantly, by 
revealing homologies in traits with similar embryological 
origins. While we have an abundance of data on nudi- 
branchs, development is poorly known for some of the less 
speciose orders. For the Notaspidea in particular, detailed 
descriptions of larval development are limited to two spe- 
cies of Pleurobranchidae: Berthellina citrina Rueppell and 
Leuckart, 1828 (Gohar and Abul-Ela, 1957: Usuki, 1969), 
and Pleitrobranchaea japonica Thiele, 1925 (Tsubokawa 
and Okutani. 1991). Partial information on larvae is avail- 
able for less than a half-dozen additional species. Even these 
limited data suggest that larval pleurobranchids are diverse 
and can be planktotrophic, lecithotrophic, or direct devel- 
oping (Gohar and Abul-Ela, 1957; Usuki, 1969; Thompson, 
1976; Tsubokawa and Okutani. 1991; Wagele, 1996; God- 
dard, 20()lb). We lack descriptions of development for all 
species of the Tylodinidae and Umbraculidae. 

My objectives are to describe development of Pleiiro- 
branchaea maculata, and to compare development of nota- 
spideans with other opisthobranchs. P. maculata is an op- 
portunistic carnivore found in New Zealand, southeastern 
Australia. China, Sri Lanka, and Japan (Willan. 1983; Mar- 
cus and Gosliner, 1984). Notes by Willan (1983) indicate 
that P . maculata produces long, cylindrical egg masses that 
release planktotrophic veligers. The present study extends 
this record to include embryology, larval structure, and 
metamorphosis using bright field and scanning electron 
microscopy (SEM). 



Materials and Methods 

Specimens of Pleurobranchaea maculata were collected 
from intertidal sandflats at the Tahuna Torea Reserve, Ta- 
maki Estuary. Auckland. New Zealand, in August 2001. 
Adults were maintained at ambient conditions (seawater 
temperature 14 C) and were fed cockles (Austrcivcmts 
stiitclibinyi Wood, 1828) or oysters (Ostrea lutaria Hutton, 
1873) daily. Egg masses were cultured in flowing seawater 
(14 C). and small sections were removed for observation. 
Larvae were cultured in 1-1 jars of 1 -jam-filtered seawater at 
17-19 C. Larvae were fed a 1:1:1 mixture of Dunalit'lla 
saliiui i idoresco. 1905. Isocluysis galbana Parke, 1949, 
and Pin It- ''itheri Green, 1975 three times weekly. Set- 
tlement occun 1 in pediveligers that were cultured in a 1-1 
jar ( 17-19 C) v n!i a 1-week growth of biofilm on the glass 
surface. 



Live embryos, larvae, and juveniles were observed, 
sketched, and photographed with a Nikon AFX photomi- 
croscope. Measurements were taken to the nearest 5 /urn. 
Pediveligers and early juveniles were fixed for SEM in 2.5% 
glutaraldehyde followed by post-fixation in 1% osmium 
tetraoxide, both in filtered seawater. After fixation, speci- 
mens were dehydrated in ethanol, critical-point-dried with a 
Polaron B3000 Series II critical-point drier, and coated with 
gold-paladium with a Polaron SC7640 sputter coaler. Spec- 
imens were observed with a Phillips XL 30S SEM at 5 kV. 
Plates were composed in Corel Photo Paint 9.0 and Corel 
Draw 9.0, using both scanned negatives (light micrographs) 
and digital images (electron micrographs). 

Results 

The cylindrical egg masses were 171.60 59.61 mm in 
length (X SD; n = 5; range 88-240 mm) and about 8-12 
mm in width, as produced by two adults 1 10 and 145 mm in 
length. P. maculata spawned frequently, and these two 
individuals produced 15 egg masses in about 5 weeks of 
laboratory culture. Usually adults deposited the loosely 
coiled egg strings on the side walls of the tank near the 
air-water interface. The eggs were white, 100.91 2.18 ju,m 
in diameter (n = 45). and deposited in egg capsules housed 
within long strings that formed a double spiral at the pe- 
riphery of the jelly mass. Capsules were 259.50 10.92 by 
250.00 10.54 /urn in size (;i = 20). and contained an 
average of 3.97 1 .02 eggs per capsule (range 2-6; n = 30 
capsules in each of 7 egg masses). Small globules of yolk 
(filled with lipid droplets and several times larger than polar 
bodies) detached from developing young in about 18% of 
the egg capsules observed (n = 244 capsules, from a total of 
8 egg masses). Early veligers ingested this extra-embryonic 
yolk before hatching. Fecundity was estimated to be 
318.417 110,602 embryos per egg mass (/; = 5). Egg 
masses were as figured by Willan ( 1983). 

Spawning generally occurred over a period of 2-3 h in 
the early and mid-morning. A chronology of early embry- 
ological events is summarized in Table 1. Elongate tro- 
chophores, with a flat velar field and small pedal rudiment, 
developed by 4 d. The shell was visible by 4.5 d and had 
grown to cover the visceropallial mass by 5 d. Also at 5 d. 
the statocysts. each with a single statolith, were visible, and 
embryos were capable of ingesting extra-embryonic yolk. 
By 5.5 d, the dark red pigmented mantle organ was visible 
and the internal organs, although still yolky. were better 
defined. The elongate foot lacked an operculum. and velar 
cilia were capable of metachronic beating and reversals. By 
ft d. the internal organs, including the pigmented mantle 
organ, were sharply defined. The larval retractor muscles, 
visible at 6 d. were functional by 7 d although the embryos 
were capable of incomplete retraction only, leaving the 
velar lobes and foot partially exposed. 



DEVELOPMENT OF PLEUROBRANCHAEA 



123 



Table 1 

SiimiiHirv of the major events in emhrynnic development of 
Pleurnhranchaea maculata m 14 C 



Time id. h) 



Event 



Oh Spawning 

2.5 h 1" polar hod\ 

4.5 h 2'"' polar hod\ 

8.5 h P' cleavage 

l ).15h 2 nd cleavage 

9.45 h 3 rd cleavage 

22 h Morula 

2448 h Blastula formation 

48-72 h Gastrulation 

4 d Trochophores: pedal rudiment and velar field present. 

embryo elongate 
4.5 d Cap-like shell visible 

5 d Early veligers: larval shell complete, pigmented mantle 

organ, statocysts, right and left digestive gland 
present, toot visible but lacks an operculum 
o d Pigmented mantle organ well developed, retractor 

muscle present but not functional, digestive glands 
becoming more distinct 

7 d Retractor muscle functional, velar lobes large. 

digestive glands still yolky but yolk depleted from 
stomach, mantle fold well developed 

8 d Hatching: subvelar ridge prominent, kidney rudiment 

present, pigmented mantle organ, mantle edge 
rounded, shell surface granulated. Eyes and 
operculum lacking 

Spawning occurred over a 2-3-h period. Timing of early events is 
relative to the onset of oviposition. 

Veligers hatched at 8 d, with a shell length of 135 5.5 
jum (n = 20), and began feeding on phytoplankton imme- 
diately (Table 1, Fig. 1A). The digestive glands were still 
yolky and the ciliated stomach had hyaline rods in the 
posterior wall, as described in nudibranchs (Thompson, 
1959). Just above the pigmented mantle organ was a small, 
transparent organ, presumably the rudiment of the definitive 
kidney (Gohar and Abul-Ela. 1957). The kidney rudiment 
remained in close association with the pigmented mantle 
organ and anus throughout larval development. Larvae 
lacked eyes at hatching, and the finely ciliated foot lacked 
an operculum throughout development. The Type 1 shell 
(types by Thompson. 1961) lacked pigment and was finely 
granulated on the surface; ridges, as occur in P. japonica 
(Tsubokawa and Okutani. 1991), were not observed. 

One week after hatching, the larval shell was about 190 
15 /Jim {n = 20) in length, and the velar lobes had increased 
substantially in size (Fig. IB. C, D). New sensory structures 
had formed, including a pair of black eyes and the pedal 
tuft, a prominent cluster of elongate cilia on the tip of the 
foot. The stomach was greatly enlarged relative to shell size, 
and the hyaline rods were more numerous in the posterior 
stomach wall. The larval heart was also present and beating. 
The pigmented mantle organ, now black, was larger (Fig. 



IB. D), and the kidney rudiment was much larger, slightly 
bilobed. and lacking any visible contents. Buds of the rhi- 
nophores were present on the anterior velar field (Fig. ID). 

Also in the first week of larval life, the mantle began to 
envelope the larval shell by growing up and over the dorsal 
shell aperture (Fig. IB). The larval shell continued to grow, 
concurrent with overgrowth by the mantle, during larval 
development. The veliger was unable to completely retract 
into the shell, possibly because of the size of the velar lobes 
and also because the growth of the mantle reduced the 
effective shell aperture. 

About 3 weeks after hatching, the first pediveligers set- 
tled on the biofilmed surface of the culture container. The 
shell, 480 23 p.m in length (;/ = 20), was almost com- 
pletely covered by the thick, glandular mantle (Fig. 2A-F). 
The growth zone of the mantle was thin as it extended over 
the shell, while older mantle tissue became thickened as the 
mantle glands developed (Fig. 2C). The mantle glands in- 
vaginated from the epidermis to the shell margin, forming 
elongate, simple tubular glands (Fig. 2D, E). Small tufts of 
cilia were scattered over the mantle surface, between the 
openings of the mantle glands (Fig. 2E). A lateral ciliary 
tract was present externally from the opening of the pre- 
branchial aperture on the right side of the "neck" and along 
the upper right side of the foot (Fig. 2F). The densely 
ciliated mouth was located ventral and posterior to the 
subvelar ridge (Fig. 2F). The foot was well developed and 
covered with fine cilia ventrally and with tufts of cilia on the 
lateral and upper surfaces (Fig. 2F). The pedal tuft, as 
described for veligers I week after hatching, was absent. 

In late-stage larvae and pediveligers, the velum showed 
additional structures on both the upper and lower surfaces. 
The rhinophores extended from the upper velar surface as 
two curved ridges of tissue that were covered with fine cilia 
(Fig. 2 A, F). As the rhinophores developed, they grew 
anteriorly, then laterally; but they remained open on the 
lateral surface, thus giving rise to the scroll-like morphology 
that is typical of adults (Willan, 1983). The oral veil was 
also visible as a broad, ciliated ridge on the upper surface of 
the velum, located immediately ventral to the rhinophores 
but not connected to them (Fig. 2F). The lower velar surface 
was covered by large (-15 /im in diameter) rounded cells 
with a highly folded, microvillar surface (Fig. 2F. G). These 
post-velar cells were tightly packed together and covered 
the lower surface of each velar lobe from the subvelar ridge 
to the body wall. 

Some internal organs were difficult to observe in live 
pediveligers because of the thick mantle. The eyes and 
statocysts were well developed, and the buccal mass was 
prominent in the anterior digestive tract (Fig. 2 A). The 
pigmented mantle organ was darker, and the enlarged, trans- 
parent kidney was easily observed though the thick mantle 
tissue (Fig. 2B. D). 

Acquisition of a juvenile morphology occurred gradually 



124 



G. D. GIBSON 



m svr 




rdg 



Idg a f 



100 |um 




D 



rh 





vl 



Idg f 




pmo 



Figure 1. Early veligers of Pleurobranchaea maculata. (A) Veliger on the first day of hatching, drawn from 
life. (B) Veliger 7 d after hatching, drawn from life. (C. D) Bright field micrographs of veligers 7 d after 
hatching. Scale bar is the same for all four illustrations, a. anus; e. eye; f, foot; hr. hyaline rods; i. intestine; k, 
kidney rudiment; Idg, left digestive gland; Ih, larval heart; m, mantle; pmo. pigmented mantle organ; pt. pedal 
tuft; rdg. right digestive gland; rh. rhinophore bud; rm. retractor muscle; rvl. right velar lobe; s. stomach: st. 
statocvst; svr. suhvelar nde; vl, velar lobes. 



and involved development of the mantle throughout most of 
larval life. The final stage of metamorphosis primarily in- 
volved loss of the velum. As the velar and subvelar cilia 



were shed, the highly folded surface of post-velar cells 
became smooth and the cells were gradually resorbed into 
the lower velar surface, followed by resorption of the velar 



Figure 2. Pediveligers of Pleurobranchaea imicitlatu. 3 weeks after hatching. (A. B) Drawing and bright 
field micrograph of live pediveligers. (C) Scanning electron micrograph of the overgrowth of the larval shell by 
the mantle. (D. E) Mantle glands in bright field and scanning electron micrographs. (F) Scanning electron 
micrographs of a pediveliger. (G) Scanning electron micrograph of the post-velar cells on the lower velar 
surface; the subvelar ridge is shown in the upper right. (H) Scanning electron micrograph of the partially 
i Mirhed velar lobes during metamorphosis, bm. buccal mass; cso, cephalic sensory organ; ctr. lateral ciliary 
tract; f. foot: glz, glandular zone; grz. growth zone; k. kidney rudiment; Ih. larval heart; m. mantle; mg. mantle 
gland; mo, mouth; ov. oral veil; pmo, pigmented mantle organ; pvc, post-velar cells; rh. rhinophore; sh, shell; 
svr. Mihxd.u ridge: vr. velar ridge. 



DEVELOPMENT OF PLEUROBRANCHAEA 



125 




126 



G. D. GIBSON 



lobes into the head (Fi Early juveniles retained lateral 

remnants of the \ es for several days (Fig. 3A-D). 

Loss of the ve! .; scvealed the cephalic sensory organ, 

a prominent .ated organ located dorsally between the 

two velar ii s (Fig. 2H). The rhinophores extended ante- 
riorly fiv; uie remnant of the velar field (Fig. 3A, C), and 
the oral veil projected as a broad ridge to cover the mouth 
and anterior foot (Fig. 3A-C). The prebranchial aperture, 
open on the right side, led to the lateral ciliary tract. The gill 
had not yet formed (Fig. 3D). The opaque mantle, both 
glandular and also with a scattering of red pigment, made it 
difficult to determine when the shell was dissolved, al- 
though the buccal mass and digestive glands were easily 
observed through the ventral body wall (Fig. 3B). The 
pigmented mantle organ appeared to be lost during late 
metamorphosis. The kidney remained next to the pre- 
branchial aperture. 



Discussion 

Morphogenesis of Pleurobranchaea maculata 

Development of Pleurobranchaea maculata is similar to 
that of P. ja/xinica (Tsubokawa and Okutani, 1991 ) in terms 
of egg mass characteristics and overall larval morphology. 
However, the present study revealed several additional traits 
that warrant discussion, including the mantle glands, post- 
velar cells, sensory organs, and the position of the mouth. 

The mantle of P. maculata becomes richly glandular as it 
grows back over the larval shell. These simple, tubular 
glands project through the entire thickness of the mantle, 
appear fairly early in ontogeny, and persist through meta- 
morphosis. Thompson and Slinn (1959) and Thompson 
(1988) reported the secretion of sulfuric acid from the 
mantle of adult Pleurobranchidae from both simple co- 
lumnar cells and flask-shaped glands. Adults of Pleuro- 




Figure 3. Early juveniles of Pleurnhraiichai'ii nnuiilahi. (A) Newly settled juvenile, dorsal aspect, drawn 

oni life. (B) Bright field micrograph of a newly settled juvenile, ventral aspect. (C) Bright field micrograph of 

load. (D) Scanning electron micrograph of a newly settled juvenile, a. anus; bm, buccal mass; ctr. lateral 

tiact; dg. digestive gland; e. eye; es, esophagus; f. foot; k. kidney; m. mantle; mp. mantle pigment; ov, 

01 al >nl: pha, prebranchial aperture; pvc, post-velar cells; rh, rhmophore; s, stomach; v, remnant of velar lobe. 



DI-V1 I OI'MI-NT 01- /'// / KOHK \\CII\I \ 



127 



hranchaea and Pleurobranchus also synthesize a variety of 
defensive compounds (Ciavatta et ai, 1995; Spinella ct <//.. 
1997). The abundance and differentiation of mantle glands 
in P. inacidata pediveligers suggest that they function in 
late-stage larvae and likely confer chemical protection to 
planktonic pediveligers and newly settled juveniles. The 
capacity of larvae to synthesize their own chemical defense 
is unusual. Chemical defense occurs in eggs or egg masses 
of some opisthobranchs; however, in these other taxa. the 
defensive compounds are maternally derived, as occurs in 
the Anaspidea (Pennings. 1994). Ascoglossa (Paul and Van 
Alstyne. 1988). Nudibranchia (Pawlik et at., 1988). and 
tylodinid Notaspidea (Ebel et ul.. 1999). 

The post-velar cells are also unusual and. to my knowl- 
edge, have not been reported elsewhere. These cells develop 
in larvae, persist through early metamorphosis, and are 
resorbed with the rest of the velum. The post-velar cells 
greatly increase the surface area of the lower velar surface 
through their abundance and highly folded apical surface. 
While the function of these cells is unknown, it seems 
reasonable to suggest that they are secretory because of their 
size, number, and morphology. If secretory, they may pro- 
tect the head and velum by neutralizing secretions of the 
mantle glands. 

The rhinophores of P. macitlata originate from the center 
of the upper velar surface; appear fairly early in larval 
development; and grow anteriorly as curved, ciliated ridges 
of tissue. In contrast, the rhinophores of P. japonica arise 
from a pair of depressions at the lateral edges of the oral veil 
(Tsubokawa and Okutani. 1991 ). The intravelar location of 
the rhinophores in P. niucnlata is similar to that of the 
nudibranch Rostanga piilchra MacFarland, 1905 (Chia and 
Koss, 1982. 1991 ). although the definitive morphology dif- 
fers in that the rhinophores of nudibranchs are solid and lack 
a lateral groove. A cephalic organ, as observed in P. inacu- 
lata, was not reported in P. japonica (Tsubokawa and 
Okutani. 1991) but would be difficult to observe without 
SEM. However, a cephalic organ is present in some cepha- 
laspids (Schaeffer and Ruthensteiner, 2001) and nudi- 
branchs (Chia and Koss. 1984). 

Also of interest is the position of the mouth posterior to 
the subvelar ridge in pediveligers. Veligers typically have a 
mouth positioned between the velar cilia (i.e.. the pre-oral 
cilia, or prototroch) and the subvelar ridge (i.e.. the post-oral 
cilia, or metatroch). Whether this is the original embryolog- 
ical location of the mouth (e.g.. protostomal) or represents a 
secondary mouth opening is not known. Comparative work 
is needed to clarify the embryological origins and potential 
migration of the definitive mouth from the protostome. 

The ciliary tract on the right lateral foot has not been 
reported elsewhere in notaspideans, but is reported for nudi- 
branchs (e.g.. Bonar and Hadfield. 1974; Goddard, 1996). 
The ciliary tract in P. maculata. not present in adults, 
occupies the position of the adult gill (Willan. 1983). The 



tract may serve to generate water currents directed away 
from the prebranchial aperture, which houses the anus and 
nephroproct: this function is likely taken over by the gill 
cilia once the gill has formed. Gill formation was not 
observed in the present study, although Tsubokawa and 
Okutani (1991) describe the gill in P. japonica in early 
juveniles. 

Pattern and process in notaspidean development 

Egg mass structure. Egg masses have been described for 
several species of Notaspidea (Table 2). As is typical of 
opisthobranchs. eggs are located in capsules within an elon- 
gate string that runs in a double spiral around the periphery 
of a jelly mass, although in some notaspideans the string is 
poorly defined (Milieu, unpubl. obs. cited in Strathmann. 
1987) or highly modified (Bandel. 1976). In some species, 
capsules contain a single egg; in others, as many as 37 eggs 
per capsule are common (Table 2). Egg size is variable 
among species and. although the sample size is small, 
appears correlated with developmental mode (Table 2), as 
expected for opisthobranchs (Hadfield and Miller. 1987). 
However, in some Notaspidea, egg size can vary consider- 
ably within one species; in the lecithotrophic Berthellina 
citrina, for example, variation in egg size is reported to span 
140 urn, ranging from 270 to 410 ^.m over 15 egg masses 
(Usuki, 1969). 

Lan'al morpholog\. Morphogenesis in notaspidean velig- 
ers is modified from the pattern characteristic of benthic 
opisthobranchs that is observed in the Cephalaspidea, As- 
coglossa. Anaspidea. and Nudibranchia (e.g.. Thompson, 
1976). Major differences include shell growth and loss, 
notum formation, and lack of an operculum. Otherwise, 
morphogenesis appears similar to that of other opistho- 
branchs. 

In most notaspideans (Table 2). larval shells are Type 1 
( whorled; typical of most classes of opisthobranchs) or less 
commonly. Type 2 (inflated and cuplike; occurs in some 
nudibranchs). However, once the protoconch has formed, 
growth of the larval shell in pleurobranchids is atypical of 
other opisthobranchs in mechanism and timing. In other 
opisthobranchs. the larval shell grows via secretions by the 
mantle fold, a ridge of tissue next to the shell aperture 
(Tardy. 1991 ). In pleurobranchids. the mantle fold is lost in 
early veligers at the same time as the mantle extends past 
the aperture to ultimately cover the entire larval shell. This 
occurs after hatching for planktotrophic species of Pleuro- 
hranchaca (Tsubokawa and Okutani. 1991; this study) or 
within the egg capsule for lecithotrophic species of Ber- 
tliellina (Usuki. 1969) and direct-developing Bathyberthella 
(Wa'gele, 1996). Thus larval shell growth is concurrent with 
mantle overgrowth and occurs despite the migration of the 
mantle edge away from the aperture; presumably, the region 
of mantle adjacent to the aperture retains shell-secretion 



128 



G. D. GIBSON 







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Trait 



Shell Growth: 

Cephalaspidca 
Notaspidea 

- Umbraculidae 

- Tylodinidae 

- Pleurobranchidae 
Nudibranchia 

Protoconch: 

Cephalaspidea 
Notaspidea 

- Umbraculidae 

- Tylodinidae 

- Pleurobranchidae 
Nudibranchia 

Growth of Notum: 

Cephalaspidea 
Notaspidea 

- Umbraculidae 

- Tylodinidae 

- Pleurobranchidae 
Nudibranchia 

Operculum: 

Cephalaspidea 
Notaspidea 

- Umbraculidae 

- Tylodinidae 

- Pleurobranchidae 
Nudibranchia 



DEVELOPMENT OF PLEUROBR.\NCHAE.\ 

Ontogenetic Stage 

Embryo Larva Metamorphosis Adult 



129 






(growth from mantle fold) 




(shell +/- in adults) 



(larval shell retained) 



I (dissolved/ internalized) 
(shell cast off) 



(operculum +/- in adults) 



sj(does not develop ?) 



Figure 4. Heterochrony and pleurobranchid development. Data summarize the time of onset and offset of 
shell growth, protoconch (formation/loss), notum growth, and operculum (formation/loss). Timing is generalized 
to ontogenetic stage (embryo, larva, and adult) for Cephalaspidea (light gray bars). Notaspidea (white bars), and 
Nudibranchia (dark gray bars). Different patterns within a bar indicate differences in the developmental process 
underlying a particular trait; for example, shell growth occurs at the mantle fold in early stages and the mantle 
gland in later stages, and shell loss occurs through dissolution (some pleurobranchids) or being cast off 
(Nudibranchia). * Denotes a change in time of onset in pleurobranchids, relative to other indicated groups. +/ 
Indicates that the trait is present in the adults of only some species in each family or order. ? Indicates that time 
of onset is not known. References are provided in the text and in Table 2. 



activity in larvae. Migration of the mantle edge away from 
the aperture suggests that shell growth in pleurobranchids is 
modified in several ways. First, shell growth in larvae 
occurs by means of the early onset of an ontogenetic process 
found in juveniles and adults of species that retain their 
shells (Fig. 4). Second, morphogenesis of the shell and 
mantle are decoupled from other morphological changes 
that occur during metamorphosis. Third, shell-less nota- 
spideans have retained the plesiomorphic mechanism of 
adult shell overgrowth found in shelled notaspideans. This 
mechanism of shell overgrowth is also distinct from the 



growth of epipodial (e.g., Anaspidea) or parapodial (e.g., 
Cephalaspidea) lobes in juveniles of other shelled opistho- 
branchs (Tardy. 1991). 

In shell-less notaspideans, the mechanism of loss of the 
larval shell is also atypical of opisthobranchs (Fig. 4). In 
other opisthobranch orders, the retractor muscles are sev- 
ered and the shell cast off at metamorphosis (e.g., Asco- 
glossa, Nudibranchia. Gymnosomata). In contrast, the shell- 
less Pleurobranchidae lose the larval shell through 
overgrowth and dissolution by the mantle during or shortly 
after metamorphosis (Tsubokawa and Okutani, 1991). This 



130 



G. D. GIBSON 



distinctive mechanism of shell loss supports the hypothesis 
that in the Notaspk!< i -as has been suggested throughout 
the opisthobranci - liner, 1994) shell loss is apomor- 
phic. It add i' suggests that similarities among adults 

in shell los- ,! notum morphology have evolved through 
homoplasy in Notaspidea and Nudibranchia. 

Mantle growth in notaspideans is also atypical. In most 
opisthobranchs the mantle remains small until after meta- 
morphosis. In contrast, the mantle in Notaspidea begins to 
take on adult characteristics at an early larval stage. This 
early development includes formation of the notum (Gohar 
and Abul-Ela. 1957; Thompson, 1976; Tardy, 1991; Tsu- 
bokawa and Okutani. 1991; Wa'gele, 1996) and also the 
differentiation of cilia and glands (present study). Early 
onset of notum differentiation may provide the larva and 
newly settled juvenile with an effective means of defense 
(e.g., potential acid cells), increased sensory perception 
(e.g., mantle ciliary tufts), and a larger size, with the asso- 
ciated advantages of predator deterrence and increased 
buoyancy. 

Larvae of pleurobranchid notaspideans lack an opercu- 
lum. The single recorded exception is Willan's ( 1983) note 
of an operculum in P. maculata; however, an operculum 
was not observed in the present study of this species. Lack 
of an operculum may be characteristic only of pleuro- 
branchids, however, as larvae of the tylodinid Umbracitliun 
sinicum (Gmelin, 1791) is described as having an opercu- 
lum (Ostergaard, 1950). Larvae of all other opisthobranch 
orders have an operculum. with individual exceptions such 
as the nudibranch Aegires alhopunctatus MacFarland, 1905 
(Goddard, 2()()la). The operculum is lost at metamorphosis 
in most opisthobranchs. except for a few species of Cepha- 
laspidea (Thompson, 1976). This suggests that loss of an 
operculum in pleurobranchid larvae is an apomorphic trait 
that represents an earlier (i.e., embryonic) onset of a con- 
dition common to the adults of other orders (Fig. 4). 

As with other opisthobranchs, the mantle cavity of nota- 
spidean veligers contains several cell clusters that have been 
referred to as "larval kidneys" and "anal cells" (Gohar and 
Abul-Ela. 1957; Usuki, 1969; Tsubokawa and Okutani, 
1991). The function and ultrastructure of these organs are 
unknown, and the terms are used inconsistently across gas- 
tropod taxa. Thus, it is more appropriate to use the descrip- 
tor "pigmented mantle organ" to refer to the darkly pig- 
mented structure associated with the anus. A pigmented 
mantle organ is found in the planktotrophic veligers of 
several notaspideans, including P. californica MacFarland, 
1966 (Goddard. 2001a), P. japonica (Tsubokawa and Oku- 
tani, 1991), P. maculata (present study), Berthella califor- 
nicci (Dall, 1900) (Goddard, 1984), B. xtnmgi (MacFarland, 
1966) (Goddard, pers. comm.), Berthellina engeli Gardiner, 
1936 (Goddard, pers. comm.) and Umbraculum xinicnni 
(Ostergaard. 1950). A similar organ is found in planktotro- 
phic species of Cephalaspidea. A pigmented mantle organ is 



lacking in non-planktotrophic notaspideans, including Ber- 
thellina citrinn (Usuki, 1969), the probably lecithotrophic 
Berthella plumula (Montagu, 1803) (Thompson, 1976) and 
the direct-developing Bathyberthella antarctica Willan and 
Bertsch. 1987 (Wa'gele, 1996). Although the pigmented 
mantle organ appears similar in the Notaspidea and Cepha- 
laspidea, ultrastructural and detailed embryological studies 
are needed to determine if they are homologous. All three 
studied species of Pleurobranchaea (P. japonica. P. cali- 
fornica, and P. maculata: Tsubokawa and Okutani, 1991; 
Goddard, 200 Ib; present study) also have a large, transpar- 
ent organ positioned adjacent and dorsal to the pigmented 
mantle organ, which is here considered to be the rudiment of 
the kidney (following Gohar and Abul-Ela, 1957). 

Settlement and metamorphosis. Pediveligers of Ber- 
thellina citrina, Pleurobranchaea japonica, and P. macu- 
lata all settle on biofilmed culture dishes (Gohar and Abul- 
Ela, 1957; Usuki, 1969; Tsubokawa and Okutani, 1991: 
present study). Preferences for specific substrates were not 
tested, but a nonspecific cue is probable as all three species 
are opportunistic carnivores. 

Settlement and metamorphosis are also decoupled events 
in pleurobranchids. Whereas settlement (acquisition of a 
benthic lifestyle) occurs over a relatively short time span, 
metamorphosis (the acquisition of an adult morphology) 
begins early in larval life with an accelerated onset of some 
processes of differentiation typical of adults (e.g.. shell and 
notum growth), while differentiation of other organ systems 
(e.g., velum, foot) appears similar to that of other opistho- 
branch orders. For example, the larval shell is produced by 
a mantle region other than the mantle fold; thus we see an 
early onset of a shell-growth mechanism common in juve- 
niles and adults of shelled species (Fig. 4). Also, in the 
mantle of early larvae, "adult" structures such as glands and 
external cilia undergo rapid growth and differentiation pro- 
cesses that also are associated with juvenile development in 
other orders. Absence of an operculum in pleurobranchid 
larvae is also a trait typical of adult opisthobranchs. Collec- 
tively, these observations suggest that in the Pleurobranchi- 
dae, aspects of the specialized morphology of the adult have 
evolved through heterochronic changes in specific morpho- 
genetic events associated with metamorphosis. 

Phylogenetic implications 

Development in the Notaspidea is relatively poorly 
known, and the data we have primarily includes the Pleu- 
robranchidae, while details are lacking for the Umbraculi- 
dae and Tylodinidae. The descriptions of development that 
are available for the Pleurobranchidae support the current 
hypothesis that the Notaspidea are phylogenetically closely 
linked with the Nudibranchia (Schmekel. 1985; Gosliner. 
1994; Wa'gele and Willan, 2000) yet share some, possibly 
plesiomorphic. traits with the Cephalaspidea. Potentially 



DEVELOPMENT OF PLEUROBRANCHAEA 



131 



plesiomorphic larval traits include the pigmented mantle 
organ (common to planktotrophic species of the Cephu- 
laspidea and Notaspidea), the cephalic sensory organ (found 
in Cephalaspidea and Nudibranchia), and a type 1 larval 
shell (common to all orders). 

Synapomorphies with the Nudibranchia include the shape 
of the egg mass and the presence of rhinophores that arise 
from the upper velar field. This agrees with Wa'gele and 
Willan (2000). who suggest that similarities in innerva- 
tion of the rhinophores support homology in the Pleuro- 
branchoidea and Nudibranchia. despite differences in mor- 
phology (rolled versus solid structure, respectively). 
Apomorphies in the Pleurobranchidae include lack of the 
larval operculum, the mechanism of shell loss or internal- 
ization, and the pattern of notum formation. These apomor- 
phies have evolved through heterochrony. manifest as an 
early (i.e., embryonic or larval) onset of developmental 
processes that typically occur in juveniles of other orders 
(Fig. 4). Understanding the phylogenetic relevance of novel 
traits shown by P. nuicnlatci (e.g., post-velar cells, position 
of the mouth I awaits further, comparative embryological 
and ultrastructural work. 

Acknowledgments 

This research was conducted at the Leigh Marine Labo- 
ratory. University of Auckland. New Zealand. I thank the 
Leigh staff, particularly B. Dobson. for their support. Spec- 
imens were examined with SEM at the Research Centre for 
Surface and Materials Science at the University of Auck- 
land. I especially thank C. Hobson (School of Engineering), 
A. Turner (School of Biological Sciences), and B. James 
(School of Engineering) for their kind assistance and pro- 
viding access to their lab facilities. J. Goddard generously 
provided unpublished observations and insight into opistho- 
branchs. which have considerably added to the manuscript. 
This manuscript benefited from the comments of an anon- 
ymous reviewer and of M. Gibson and I. Paterson. J. Ha- 
venhand and L. Page provided insight on veliger structure 
and physiology. This research was supported by the Natural 
Science and Engineering Research Council of Canada 
(NSERC). 

Literature Cited 

Bandel. K. 1976. Egg masses of 27 Caribbean opisthobranchs 
from Santa Maria. Columbia. Stud. Neotrop. Fauna Environ. 11: 87- 
118. 

Behrens. I). 1980. Pacific Coast Nudibranchs: A Guide to ilic Opi.ftho- 
hruiich.\ of llu- Northeast Pacific. Sea Challengers, Los Osos, CA. 

Bonar, I)., and M. Hadfield. 1974. Metamorphosis of the marine gas- 
tropod Phcstilla sihogae Bergh (Nudibranchia: Aeolidacea): I. Light 
and electron microscopic analysis of larval and metamorphic stages. ./. 
Exp. Mar. Bwl. Ecol. 16: 227-255. 

Chia. F.-S., and R. Koss. 1982. Fine structure of the larval rhinophores 
of the nudibranch. Rt/stant>a pulchra, with emphasis on the sensory 
receptor cells. Cell Tissue Res. 225: 235-248. 



Chia. F.-S.. and R. Koss. 1984. Fine structure of the cephalic sensory 
organ in the larva of the nudibranch Rnstiinga pulchra (Mollusca. 
Opisthobranchia. Nudihranchia). Zoomorphology 104: 131-139. 

Chia, F.-S.. and R. Koss. 1991. Sensory structures and behaviour in 
opisthobranch veliger larvae. Bull. lust. Zool. Acad. Sin. Monograph 
16: 455-4S4. 

Ciavatta, M., E. Trivellone, G. Villani, and G. Cimino. 1993. Mem- 
brenones: New polypropionates from the skin of the mediterranean 
mollusc Pleurobrunchiis membranaceus. Tetrahedron Lett. 34: 6791- 
6794. 

Ciavatta, M., G. Villani, E. Trivellone, and G. Cimino. 1995. Two 
new labdane aldehydes from the skin of the notaspidean P/euro- 
hmnchaea meckelii. Tetrahedron Lett. 36: X673-8676. 

Kbel, R., A. Marin, and P. Proksch. 1999. Organ-specific distribution 
of dietary alkaloids in the marine opisthobranch Tylnilina pervemi. 
Biochem. Sysl. Ecol. 27: 769-777. 

Goddard, .1. 1984. The opisthobranchs of Cape Arago, Oregon, with 
notes on their biology and a summary of benthic opisthobranchs known 
from Oregon. Velit>er21: 143-163. 

Goddard, J. 1996. Lecithotrophic development in Onto amyru (Nudi- 
branchia: Dendronotaceal. with a review of developmental mode in the 
genus. Veliger 39: 43-54. 

Goddard. J. 2001a. The early veliger larvae of Aegires albopunclalus 
(Nudibranchia: Aegiridae). with morphological comparisons to mem- 
bers of the Notaspidea. Ve lige r 44: 398-406. 

Goddard, J. 2001b. Mollusca: Gastropoda. P. 314 in An Identification 
Guide to the Lan l al Marine Invertebrates of the Pacific Northwest, A. 
Shanks, ed. Oregon State University Press, Corvallis, OR. 

Gohar, H., and I. Ahul-Ela. 1957. The development of Benliclliiiu 
citrina (Mollusca: Opisthohranchiata). Pub/. Mai: Biol. Stii., Al 
Ghanlauu, Egypt 9: 69-84. 

Gosliner, T., 1991. Morphological parallelism in opisthobranch gastro- 
pods. Malacologia 32: 313-327. 

Gosliner, T., 1994. Gastropoda: Opisthobranchia. Pp. 253-355 in Mi- 
croscopic Anatom\ of the Invertebrates. F. Harrison and A. Kohn, eds. 
Wiley-Liss. New York. 

Hadlield, M., and S. Miller. 1987. On developmental patterns of opis- 
thobranchs. Am. Ma/acol. Bull. 5: 197-214. 

Hurst, A., 1967. The egg masses and veligers of thirty northeast Pacific 
opisthobranchs. Veliger 9: 255-287. 

Marcus, E., and T. Gosliner. 1984. Review of the family Pleuro- 
branchaeidae (Mollusca, Opisthobranchia). Ann. S. Afr. Mils. 93: 1-52. 

Mikkelsen, P. 1998. Review of shell reduction and loss in traditional and 
phylogenetic molluscan systematics, with experimental manipulation 
of a negative gain character. Am. Malacol. Bull. 14: 201-218. 

Ostergaard, J. 1950. Spawning and development of some Hawaiian 
marine gastropods. Pac. Sci. 4: 75-1 15. 

Paul, V., and K. Van Alstyne. 1988. The use of ingested algal diterpe- 
noids by Elysia halimedae MacNae (Opisthobranchia: Ascoglossa) as 
antipr